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  • v.35(2); 2015

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Human mesenchymal stem cells - current trends and future prospective

Imran ullah.

*Department of Theriogenology and Biotechnology, College of Veterinary Medicine, Gyeongsang National University, Jinju 660-701, Republic of Korea

Raghavendra Baregundi Subbarao

Gyu jin rho.

†Research institute of life sciences, Gyeongsang National University, Jinju 660-701, Republic of Korea

Stem cells are cells specialized cell, capable of renewing themselves through cell division and can differentiate into multi-lineage cells. These cells are categorized as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) and adult stem cells. Mesenchymal stem cells (MSCs) are adult stem cells which can be isolated from human and animal sources. Human MSCs (hMSCs) are the non-haematopoietic, multipotent stem cells with the capacity to differentiate into mesodermal lineage such as osteocytes, adipocytes and chondrocytes as well ectodermal (neurocytes) and endodermal lineages (hepatocytes). MSCs express cell surface markers like cluster of differentiation (CD)29, CD44, CD73, CD90, CD105 and lack the expression of CD14, CD34, CD45 and HLA (human leucocyte antigen)-DR. hMSCs for the first time were reported in the bone marrow and till now they have been isolated from various tissues, including adipose tissue, amniotic fluid, endometrium, dental tissues, umbilical cord and Wharton's jelly which harbours potential MSCs. hMSCs have been cultured long-term in specific media without any severe abnormalities. Furthermore, MSCs have immunomodulatory features, secrete cytokines and immune-receptors which regulate the microenvironment in the host tissue. Multilineage potential, immunomodulation and secretion of anti-inflammatory molecules makes MSCs an effective tool in the treatment of chronic diseases. In the present review, we have highlighted recent research findings in the area of hMSCs sources, expression of cell surface markers, long-term in vitro culturing, in vitro differentiation potential, immunomodulatory features, its homing capacity, banking and cryopreservation, its application in the treatment of chronic diseases and its use in clinical trials.

Short abstract

In this review, we highlighted recent research findings in the area of human mesenchymal stem cells, its application in the treatment of chronic diseases and its use in human clinical trials.


Stem cells are the cells with a specific function with the ability of self-renewal, possess varied potency and differentiate into multilineages [ 1 ]. Because of clinical applications and biological importance, stem cells become a prominent subject in modern research era. On the basis of origin, stem cells are divided into different categories.

Embryonic stem cells (ESCs) are pluripotent stem cells, isolated originally from the inner cell mass (ICM) of mouse early pre-implantation blastocyst, having the capacity to generate into any mature cell of the three germ lines [ 2 ]. Later on, Thomson et al. [ 3 ] also isolated ESCs from ICM of human blastocyst, but until now as compared with humans, only mouse ESCs have been investigated in depth. ESCs possess distinctive self-renewal capacity, pluripotency and genomic stability [ 4 ] and can give rise to almost all lineages and are promising cells for cellular therapy [ 1 ]. From the very first derivation of human ESCs, scientists are keenly interested in the use of ESCs for drug discovery, immunotherapy and regenerative medicine, but their use has been restricted due to ethical issues and also because of difficulty in obtaining quality human oocytes.

Induced pluripotent stem cells (iPSCs) are generated from adult cells by the overexpression of four transcription factors Oct4/3 (octamer-binding transcription factor 4/3), Sox2 (sex determining region Y), Klf4 (kruppel-like factor 4) and c-Myc (Avian Myelocytomatosis virus oncogene cellular homologue) [ 5 ]. The iPSCs at cellular level are almost similar to ESCs as they are having the capacity of self-renewal, differentiation potential and the ability to produce germ line competent-chimeras. After these findings, two groups Takahashi et al. [ 6 ] and Nakagawa et al. [ 7 ] have generated the iPSCs from adult human fibroblasts. Though iPSCs possess great potential for cell therapy, but their genomic stability is still questionable.

Around the world, scientists are researching for stable, safe and highly accessible stem cells source with great potential for regenerative medicine. The cells isolated from mouse bone marrow upon culture exhibited the plastic adherence properties and formed spindle-shaped colonies were referred as colony forming unit fibroblasts [ 8 ]. Due to their ability to differentiate into specialized cells developing from mesoderm, they were named as mesenchymal stem cells (MSCs). MSCs, also known as multipotent cells, exist in adult tissues of different sources, ranging from murine to humans. They are self-renewable, multipotent, easily accessible and culturally expandable in vitro with exceptional genomic stability and few ethical issues, marking its importance in cell therapy, regenerative medicine and tissue repairment [ 9 ].

The current review highlights recent findings in the areas of hMSCs (human MSCs) sources, its ex vivo differentiation ability, immunogenicity, homing ability, banking and cryopreservation, its role in the treatment of chronic diseases and its use in human clinical trials.


Since the first description of hMSCs derived from bone marrow [ 10 ], they have been isolated from almost all tissues including perivascular area [ 11 ]. Still there is neither a single definition nor a quantitative assay to help in the identification of MSCs in mixed population of cells [ 9 ]. However, the International Society for Cellular Therapy has proposed minimum criteria to define MSCs. These cells (a) should exhibits plastic adherence (b) possess specific set of cell surface markers, i.e. cluster of differentiation (CD)73, D90, CD105 and lack expression of CD14, CD34, CD45 and human leucocyte antigen-DR (HLA-DR) and (c) have the ability to differentiate in vitro into adipocyte, chondrocyte and osteoblast [ 12 ]. These characteristics are valid for all MSCs, although few differences exist in MSCs isolated from various tissue origins.

MSCs are present not only in fetal tissues but also in many adult tissues with few exceptions. Efficient population of MSCs has been reported from bone marrow [ 10 ]. Cells which exhibits characteristics of MSCs were isolated from adipose tissue [ 13 , 14 ], amniotic fluid [ 15 , 16 ], amniotic membrane [ 17 ], dental tissues [ 18 , 19 ], endometrium [ 20 ], limb bud [ 21 ], menstrual blood [ 22 ], peripheral blood [ 23 ], placenta and fetal membrane [ 24 ], salivary gland [ 25 ], skin and foreskin [ 26 , 27 ], sub-amniotic umbilical cord lining membrane [ 28 ], synovial fluid [ 29 ] and Wharton's jelly [ 30 , 31 ] ( Table 1 ).

Isolation and initial culturing

There are different protocols reported previously in terms of isolation, characterization and expansion of MSCs, but all MSCs (despite of protocol) exhibits the minimum criteria proposed by International Society for Cellular Therapy.

hMSCs were isolated based on their ability to adhere to plastic surface, but this method resulted in the formation of heterogeneous cells (stem cells along with their progenitor cells) [ 32 ]. Bone marrow-derived MSCs (BM-MSCs) are considered the best cell source and taken as a standard for the comparison of MSCs from other sources.

Establishment of a comprehensive procedure for the isolation, characterization and expansion of MSCs is the key to success for the use of these cells as a good source for regenerative medicine [ 33 ]. Unlike bone marrow, MSCs from other tissues can be easily obtained by non-invasive methods and its proliferation can be maintained up to many passages [ 34 , 35 ]. MSCs from bone marrow, peripheral blood and synovial fluid were isolated by using Ficoll density gradient method with small modifications [ 24 , 30 , 36 ] and seeded into culture plates. While isolating MSCs from bone marrow, some haematopoietic cells also adhere to the plastic plate but during sub-culturing these cells are washed away, leaving only adherent fibroblast like cells [ 37 ]. MSCs from various tissue sources (adipose, dental, endometrium, foreskin, placenta, Wharton's Jelly) were isolated after digestion with collagenase and then cultured at varying densities [ 20 , 25 , 33 ]. Recently an efficient method to isolate BM-MSCs using novel marrow filter device is explored [ 38 ], which is less time consuming and avoids the risk of external contamination. MSCs isolated from different sources were cultured using condition media such as Dulbecco's modified Eagle's media (DMEM) [ 25 , 33 ], DMEM-F12 [ 17 , 20 , 26 ], αMEM [ 19 , 23 , 29 ], DMEM-LG [ 21 , 24 ], DMED-HG [ 27 , 28 ] and RPMI (Roswell Park Memorial Institute medium) [ 39 ]. The primary culture media was supplemented with 10% FBS [ 25 , 33 ], new-born calf serum (NBCS) [ 23 ] or fetal calf serum (FCS) [ 25 ] ( Table 1 ). Besides the culture media and supplementation, the oxygen concentration also affects the expansion and proliferation of MSCs [ 40 ]. MSCs expansion is also documented when cultured in DMEM with low glucose supplemented with growth factors like fibroblast growth factor (FGF), epidermal growth factor (EGF) and B27 [ 27 ]. But most commonly DMEM with 10% FBS is vastly employed in culturing and expanding MSCs in vitro ; however, the use of exogenous FBS is highly debated.

Expression of cell surface markers

According to the International Society for Cellular Therapy standard criteria, expression of specific set of cell surface markers is one of the essential characteristics of hMSCs. Those cells which are positive for CD73, D90, CD105 whereas negative expression of CD14, CD34, CD45 and HLA-DR are considered as MSCs. However, the most characterized and promising markers with highest specificities for MSCs are describe in the present study ( Table 1 ). MSCs have been reported from various human tissues, which exhibit the expression of above mentioned cell surface markers along with positive expression of CD29, CD44, CD146, CD140b specific to tissue origin. The expression of CD34, which is a negative marker, is still controversial [ 41 ]. A number of studies have also reported that stage-specific embryonic antigen (SSEA)-4 [ 13 , 42 ], CD146 [ 43 , 44 ] and stromal precursor antigen-1 (Stro-1) [ 45 ] are the stemnes markers for MSCs. The human amniotic fluid-derived MSCs exhibits the expression of CD29, CD44, CD90, CD105, HLA-ABC [major histocompatibility complex class I (MHC I)] along with SH2 (Src homology 2), SH3 (Src homology 3), SH4 (Src homology 4) but lack the expression of HLA-DR (MHC II) [ 16 ]. Stro-1, which is consider as stemnes marker for MSCs, is reported positive in dental [ 46 ] and bone marrow [ 47 , 48 ] whereas negative in human adipose-derived MSCs (AD-MSCs) [ 49 ].

Long-term in vitro culturing capacity

Although MSCs have great advantages over other stem cells, their clinical applications are hindered by many research barriers. One of the major challenges is to obtain adequate number of cells as these cells were found to lose their potency during sub-culturing and at higher passages. One of the reasons behind the senescence and aging of MSCs during in vitro expansion is the decrease in telomerase activity [ 50 ]. It has been reported that human BM-MSCs become senescent during long-term culture, manifested by decline in differentiation potential, shortening of the telomere length and morphological alterations [ 51 ]. Similar results are also reported when MSCs derived from bone marrow and adipose tissues were progressively cultured at higher passages. The actual age of the cells in culture is usually determined by population doublings (PDs) time and MSCs colonies derived from a single cell has shown up to 50 PDs in 10 weeks [ 52 ], whereas others have reported 30 PDs in approximately 18 weeks [ 51 ]. However, culturing MSCs for a long time resulted in an increase in the probability of malignant transformation [ 53 ] and also showed decline in their multipotency. Early MSCs have proved higher differentiation ability to chondrocytes, adipocytes and osteocytes; however, at higher passages and on long-term culture, this differentiation property declines [ 54 ]. There are two vital compounds which influence MSCs’ properties during in vitro culturing, serum and growth factors, which are associated with malignant transformation of MSCs at higher passages [ 54 ]. In minimal media condition, MSCs culturing requires 10% heat-inactivated FCS, but in such culture conditions the MSCs retain some FCS proteins, which may evoke immunologic response in vivo [ 55 ]. Expanding MSCs in serum-free culture media showed a gradual decrease in differentiation potential and telomerase activity, but cells were resistant to spontaneous transformation and could be expanded at higher passages without any chromosomal alteration [ 54 ]. However, due to variation in culture media and growth factors used, the comparison of data is difficult.

In vitro differentiation potential

hMSCs have the capacity to differentiate into all the three lineages, i.e. ectoderm, mesoderm and endoderm, with various potency by employing suitable media and growth supplements which initiate lineage differentiation ( Table 2 ).

Mesodermal lineages

In addition to multipotency and expressions of cell surface markers, one of the determining properties of MSCs is to differentiate into mesodermal lineages. The in vitro differentiation into adipocytes, osteocytes and chondrocytes, confirmed by production of oil droplet, formation of mineralized matrices and expression of type II collagen respectively, has been evaluated by immunocytochemical, histochemical and PCR analysis [ 10 , 56 – 58 ]. Differentiation of MSCs into adipocytes is induced by proper media supplementations, which activate transcription factors (genes) responsible for adipogenesis. For adipogenesis, MSCs were cultured in growth medium supplemented with dexamethasone, indomethacine, insulin and isobutyl methyl xanthine for 3 weeks and the cells were analysed by accumulation of lipid droplets and expression of adipocytes-specific genes peroxisome proliferator-activated receptor γ (PPARγ), adipocyte protein 2 (ap2) and lipoprotein lipase (LPL) genes [ 10 , 59 ]. Induction of adipogenesis is characterized by two phases: determination phase and terminal differentiation phase [ 60 ]. During determination phase, the cells committed towards pre-adipocytes show similar morphology to fibroblasts and cannot be distinguished from their MSCs precursors; however, at terminal phase the pre-adipocytes become mature adipocytes and formed lipid droplets and express adipocytes-specific proteins [ 59 ]. Overall, adipogenesis is an ordered process, involving multiple signalling cascades which are further discussed later in the present review.

The classical method to differentiate MSCs into osteocytes is by culturing the cells with ascorbic acid, β-glyceralphosphate and dexamethasone for 3 weeks in growth conditioned media. The osteogenic induction of MSCs initiated mineral aggregation and showed increase in alkaline phosphatase activity at final week of differentiation [ 10 ]. These mineralized nodules were found positive for Alizarin Red and von Kossa staining. The process of osteogenesis starts with assurance of osteoprogenitor which first differentiate into pre-osteocytes and then finally differentiate into mature osteoblasts [ 61 ]. One of the most important indicating factors for osteogenesis is the expression of runt-related transcription factor 2 (Runx2) [ 61 ]; however, other transcription factors like osteonectin, bone morphogenic protein 2 (BMP2) and extracellular signal molecules along with Runx2 expression, are involved in this process. In the whole process of bone formation, first osteoblasts synthesize the bone matrix and then help in bone remodelling and mineral deposition.

The differentiation of MSCs into mesenchymal lineage is known to be controlled by diverse transcription factors and signalling cascades. Many investigators have reported that a correlation exists between adipogenesis and osteogenesis [ 62 , 63 ]. It was reported that a converse relationship exists between adipogenesis and osteogenesis during culturing with different media supplements. [ 64 ]. Several signalling pathways such as Hedgehog [ 65 , 66 ], NEL-like protein 1 (NELL-1) [ 63 ] and β catenin-dependent Wnt [ 67 , 68 ] are well manifested for pro-osteogenic and anti-adipogenic stimulations in MSCs, although there are various signalling cascades which demonstrate positive regulation of both adipo- and osteogenesis. Among them, one of the most familiar clinically-relevant molecule is BMP, which promotes MSCs differentiation and its osteogenic commitment [ 69 , 70 ] and also induce pro-adipogenic effects [ 71 ]. PPARγ and Runx2 are the key transcription factors which control the adipogenic and osteogenic signalling cascades and the expression of one transcription factor counteracts expression of other transcription factor [ 14 , 72 ].

Like the adipogenesis and osteogenesis, hMSCs have the potential to differentiate into mature chondrocytes. The first standard protocol for chondrocytes differentiation was established for MSCs derived from human bone marrow [ 73 ]. According to the standard protocol for chondrogenesis, cells were cultured in DMEM media supplemented with insulin transferrin selenium, linoleic acid, selenious acid, pyruvate, ascorbate 2-phosphate, dexamethasone and transforming growth factor-β III (TGF-βIII). The pre-induction stage of chondrogenic differentiation of MSCs resulted in the formation of pre-chondrocytes and expresses type I and type II collagens [ 74 ]. The expression of these genes and other adhesion molecules depends on the presence of soluble factors, i.e. TGF-β family (TGF-β1, TGF-β2 and TGF-β3) [ 75 ]. In the final step, pre-chondrocytes differentiate into mature chondrocytes and express chondrogenic transcription factors like Sox9, L-Sox5 and Sox6 [ 76 , 77 ]. In association with TGF-β1, other growth factors such as, insulin like growth factor-I (IGF-I) and BMP-2 were known to induce the differentiation of MSCs into chondrocytes [ 78 ]. In hMSCs, TGF-β1 interacts with Wnt/β-catenin pathways inhibits osteoblast differentiation and induce chondrogenesis [ 79 ]. When human AD-MSCs were treated with BMP-2, they differentiated into chondrocytes and expressed mature cartilage markers (type II collagen/GAG) [ 80 ]. Besides these growth factors, other hormones such as parathyroid hormone-related peptide (PTHrp) [ 81 , 82 ] and triiodothyronine (T3) also influenced chondrogenesis.

Like cardiomyocytes, MSCs can differentiate into other mesodermal lineages. Twenty years ago, the rat BM-MSCs were cultured with 5-azacytidine which resulted in the differentiation of these cells into multinucleated myotubes [ 83 ]. Later Xu et al. [ 84 ] treated human BM-MSCs with the same chemical and demonstrated that the cells differentiate into myocytes and were expressing myocyte-related genes, β-myocin heavy chain, α-cardiac actin and desmin with additional calcium–potassium-induced calcium fluxes. Human BM-MSCs also differentiate into skeletal muscles and smooth muscles when transfected with notch intracellular domain (NICD) [ 85 ] followed by treatment with TGF-β [ 86 ]. Yet the exact in vivo signalling mechanism which initiates the differentiation of hMSCs into myocytes is not completely understood and under investigation.

Ectodermal lineages

Despite the mesodermal origin, hMSCs have displayed the capacity of trans-differentiation into ectodermal lineages. The hMSCs isolated from different sources have demonstrated trans-differentiation into neuronal cells upon exposure to neural induction media supplemented with cocktails of growth factors. Several growth factors like hepatocyte growth factor (HGF), FGF and EGF were used in neuronal induction media cocktail and successfully obtained neuronal specific phenotypes, i.e. oligodendrocytes, cholinergic and dopaminergic neurons [ 87 – 91 ]. Barzilay et al. [ 89 ] reported that a transcription factor neurogenin-1 was found effective in the trans-differentiation of MSCs into neuronal protein expressing cells. In another study, a LIM homoeobox transcription factor 1 α (LMX1a) expression into human BM-MSCs resulted in differentiation to dopaminergic neurons [ 89 ]. When BM-MSCs were cultured in serum-free media with forskolin and cAMP, cells attained neuronal morphology and elevated the expression of neuronal-specific markers [ 92 ]. β-Mercaptoethanol (BME)- and nerve growth factor (NGF)-treated MSCs also differentiated into cholinergic neuronal cells [ 87 ]. Many studies have shown that factors like insulin, retinoic acid, bFGF, EGF, valproic acid, BME and hydrocortisone support neuronal differentiation of AD-MSCs [ 93 , 94 ]. Glial cell line-derived neurotrophic growth factors (GNDF), brain-derived neurotrophic factors (BDNF), retinoic acid, 5-azacytidine, isobutylmethylxanthine (IBMX) and indomethacin enhanced the MSCs differentiation into mature neuronal cells [ 95 ]. Gangliosides are glycosphingolipids which interact with EGF receptor (EGFR) and enhance osteoblast formation. However, reduction in gangliosides biosynthesis leads to inhibition of neuronal differentiation [ 96 ]. Human umbilical cord blood-derived MSCs (UCB-MSCs) co-transfected with telomerase reverse transcriptase (TERT) and BDNF revealed a longer life span and maintained neuronal differentiation which was effective in recovery of hypoxic ischaemic brain damage (HIBD) [ 97 ]. The dental derived MSCs, which originate from neural crest, successfully differentiated into mature neuronal cells [ 98 , 99 ]. hMSCs originate from mesoderm but have the potential to transdifferentiate into neural cells which can revolutionize the regenerative cell therapy in treating many neurological disorders.

Endodermal lineages

It was believed that hepatocytes could only be derived from the cells originating from endoderm and their progenitor cells. However, MSCs have revealed the capacity of trans-differentiation into hepatocytes and pancreocytes upon induction with their corresponding conditioned media. Human BM-MSCs were trans-differentiated into hepatocyte by using two steps protocol: differentiation step followed by maturation step. In differentiation step, cells were cultured in Iscove's modified Dulbecco's medium (IMDM) supplemented with EGF, bFGF and nicotinamide for a week. Finally during maturation step, differentiated human BM-MSCs were cultured with IMDM supplemented oncostatin M, dexamethasone and ITS + (insulin, transferrin, selenium) premix which resulted in mature hepatocytes [ 100 , 101 ]. The hepatocyte-differentiated cells expressed liver-specific transcription markers, i.e. albumin, α-fetoprotein, nuclear factor 4 α (HNF-4α); however, the differentiation capacity remains inadequate for clinical application. Among these transcription factors, HNF-4α is an essential transcription factor for the morphological and functional differentiation towards hepatocytes [ 102 , 103 ]. When human UCB-MSCs were transduced with HNF-4α, it enhanced the differentiation capacity of the cells and increased expression of liver-specific markers [ 104 ]. In other studies, it was shown that valproic acid, which is histone deacetylase inhibitor, up-regulate the expression of hepatic marker through activation of protein kinase B (AKT) and extracellular signal-regulated kinases (ERK) [ 105 ].

Human BM-MSCs have been successfully differentiated into insulin producing β-cells in vitro and transplanted to streptozotocin-induced diabetic mice which corrected the hyperglycaemic condition [ 106 , 107 ]. The paracrine factors increase the differentiation and maturation of human BM-MSCs into pancreatic lineage without any genetic manipulation [ 108 ]. Human dental pulp stem cells also differentiated into insulin producing cells by induction with growth factors, i.e. acitvin A, sodium butyrate, taurine and nicotinamide [ 109 ]. Till now hMSCs derived from adipose, dental, umbilical cord, amnion, Wharton jelly and placental tissues have successfully differentiated into insulin producing β-cells [ 110 – 112 ]. These studies have revealed that hMSCs can differentiate into endodermal lineages which can transform the current traditional drug therapies to a future promising cell based therapies.

Immunomodulatory features

Regarding clinical research on cellular therapy, it is very important to know about the immunomodulatory capabilities of MSCs. In the current era of cell therapy and transplantation, the infusion of MSCs and host compatibility is the main subject of interest. Due to low expression of MHC I and lack expression of MHC class II along with co-stimulatory molecules, like CD80, CD40 and CD86, MSCs are unable to bring substantial alloreactivity and these features protects MSCs from natural killer (NK) cells lysis [ 113 ]. The MSCs therapy might alleviate disease response by increasing the conversion from Th2 (T helper cells) response to Th1 cellular immune response through modulation of interleukin (IL)-4 and interferon (IFN)-γ levels in effector T-cells [ 114 ]. MSCs have the ability to inhibit the NK cells and cytotoxic T-cells by means of different pathways. The secretion of human leucocytes antigen G5 was also found helpful in the suppression of T lymphocytes and NK cells [ 115 ]. By the secretion of suppressors of T-cells development [ 116 ], inhibitory factors i.e. leukaemia inhibitory factor (LIF) [ 117 ] and IFN-γ [ 118 ] enhance immunomodulatory properties of MSCs. Moreover, it is observed that human BM-MSCs were not recognized by NK cells, as they expressed HLA-DR molecules [ 119 ]. When allogenic hMSCs were transplanted into patients, there was no production of anti-allogeneic antibody nor T-cell priming [ 120 ], but the cytotoxic immune factors were found to be involved in the lysis of MSCs [ 114 , 121 ]. In this situation, the IFN-γ act as antagonist of NK cells, i.e. IL-2-treated NKs are recognized to destroy MSCs whereas IFN-γ helps the MSCs to keep it safe from NKs [ 122 ]. In the same report, Jewett et al. [ 122 ] mentioned that along with the protection of MSCs from cytotoxic factors, IFN-γ also enhances the differentiation of these cells by nuclear factor kappa β (NFκB)-dependent and -independent pathway. Toll-like receptors (TLRs) are the key components of innate immune system, which is critically involved in the initiation of adaptive immune system responses. MSCs have the expression of TLRs that elevate their cytokines secretions as well as proliferation [ 123 ]. MHC class I chain-like gene A (MICA) together with TLR3 ligand and other immunoregulatory proteins kept the MSCs safe from NKs invasion [ 123 ]. Together with other properties, these immunomodulatory features makes MSCs one of the feasible stem-cells source for performing cell transplantation experiments.

Human mesenchymal stem cells and chronic diseases

Considering the homing ability, multilineage potential, secretion of anti-inflammatory molecules and immunoregulatory effects, MSCs are considered as promising cell source for treatment of autoimmune, inflammatory and degenerative diseases. Efforts have been made to discuss the role of MSCs in treating chronic diseases in animal disease model ( Table 3 ).

Neurodegenerative diseases

Amylotrophic lateral sclerosis.

We previously discussed that MSCs have the ability to differentiate into neurons [ 87 – 99 ]. The first MSCs transplantation for neurodegenerative disorder was conducted in acid sphingomyelinase mouse model. After the injection of MSCs, there was a decrease in disease abnormalities and improvement in the overall survivability of the mouse [ 124 ]. Based on this experiment, a new study was designed to ascertain the potency of MSC transplantation into amylotrophic lateral sclerosis (ALS), a neurodegenerative disease that particularly degenerate the motor neurons and disturb muscle functionality [ 124 ]. The MSCs were isolated from the bone marrow of patients and then injected into the spinal cord of the same patients, followed by tracking of MSCs using MRI at 3 and 6 months. As a result, neither structural changes in the spinal cord nor abnormal cells proliferation was observed. However, the patients were suffering from mild adverse effects, i.e. intercostal pain irradiation and leg sensory dysesthesia which were reversed in few weeks duration. In another study, the AD-MSCs were genetically modified to express GDNF and then transplanted in rat model of ALS which improved the pathological phenotype and increased the number of neuromuscular connections [ 125 ].

Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder, characterized by substantial loss of dopaminergic neurons. The MSCs enhanced tyrosine hydroxylase level after transplantation in PD mice model [ 126 ]. MSCs by secretion of trophic factors like vascular endothelial growth factor (VEGF), FGF-2, EGF, neurotrophin-3 (NT3), HGF and BDNF contribute to neuroprotection without differentiating into neurocytes [ 127 , 128 ]. Now new strategies are being adopted like genetic modifications of hMSCs, which induce the secretions of specific factors or increase the dopamine (DA) cell differentiation. BM-MSCs were transduced with lentivirus carrying LMX1a gene and the resulted cells were similar to mesodiencephalic neurons with high DA cell differentiation [ 89 ]. Research group from the university hospital of Tubingen in Germany first time delivered MSCs through nose to treat neurodegenerative patients. The experiments were performed on Parkinson diseased rat with nasal administration of BM-MSCs [ 129 ]. After 4.5 months of administration, MSCs were found in different brain regions like hippocampus, cerebral, brain stem, olfactory lobe and cortex, suggesting that MSCs could survive and proliferate in vivo successfully [ 129 ]. Additionally, it was observed that this type of administration increased the level of tyrosine hydroxylase and decreased the toxin 6-hydroxydopamine in the lesions of ipsilateral striatum and substantia nigra. This novel delivery method of MSCs administration could change the face of MSCs transplantation in future.

Alzheimer disease

Alzheimer disease (AD) is one of the most common neurodegenerative disease. Its common symptoms are dementia, memory loss and intellectual disabilities. Till now no treatment has been established to stop or slow down the progression of AD [ 130 ]. Recently, researchers are in the search to reduce the neuropathological deficits by using stem cell therapy in AD animal model. It was demonstrated that human AD-MSCs modulate the inflammatory environment, particularly by activating the alternate microglia which increases the expression of Aβ-degradation enzymes and decreases the expression of pro-inflammatory cytokines [ 131 ]. Furthermore, it was observed that MSCs modulate the inflammatory environment of AD and inadequacy of regulatory T-cells (Tregs) [ 132 ] and later on it was reported that they could modulate microglia activation [ 133 ]. It was previously demonstrated that human UCB-MSCs activate Tregs which in turn regulated microglia activation and increased the neuronal survival in AD mice model [ 134 ]. Most recently, it was evidenced that MSCs enhanced the cell autophagy pathway, causing to clear the amyloid plaque and increased the neuronal survivability both in vitro and in vivo [ 135 ].

Autoimmune diseases

MSCs are also used to assuage immune disorders because MSCs have the capacity of regulating immune responses [ 1 ]. After revealing the facts that human BM-MSCs could protect the haematopoietic precursor from inflammatory damage [ 136 ], other hMSCs can be used for the treatment of autoimmune diseases.

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a joint inflammatory disease which is caused due to loss of immunological self-tolerance. In preclinical studies on animal models, MSCs were found helpful in the disease recovery and decreasing the disease progression. The injections of human AD-MSCs into DBA/1 mice model resulted in the elevation of inflammatory response in the animal [ 137 ]. They further demonstrated that following the injections of AD-MSCs, the Th1/Th17 antigen-specific cells expansion took place due to which the levels of inflammatory chemokines and cytokines reduced, whereas this treatment increased the secretion of IL-10 [ 138 ]. Along with its anti-inflammatory function, IL-10 is an important factor in the activation of Tregs that controls self-reactive T-cells and motivates peripheral tolerance in vivo [ 138 ]. Similar to this, human BM-MSCs demonstrated the same results in the collagen-induced arthritis model in DBA/1 mice [ 139 ]. These studies suggest that MSCs can improve the RA pathogenesis in DBA/1 mice model by activating Treg cells and suppressing the production of inflammatory cytokines. However, some contradictions were reported in adjuvant-induced and spontaneous arthritis model, showing that MSCs were only effective if administered at the onset of disease, which suggests that on exposing to inflammatory microenvironment MSCs lost their immunoregulatory properties [ 140 ].

Type 1 diabetes

Type 1 diabetes is an autoimmune disease caused by the destruction of β-cells due the production of auto antibody directed against these cells. As a result, the quantity of insulin production reduces to a level which is not sufficient to control the blood insulin. It has been demonstrated that MSCs can differentiate into insulin producing cells and have the capacity to regulate the immunomodulatory effects [ 118 ]. For the first time, nestin positive cells were isolated from rat pancreatic islets and differentiated into pancreatic endocrine cells [ 141 ]. Nestin positive cells were isolated from human pancreas and transplanted to diabetic nonobese diabetic/severe combined immunodeficiency (NOD-SCID) mice, which helped in the improvement of hyperglycaemic condition [ 142 ]. However, these studies were found controversial and it was suggested that besides pancreatic tissues, other tissues can be used as an alternative for MSCs isolation to treat type 1 diabetes. Human BM-MSCs were found effective in differentiating into glucose competent pancreatic endocrine cells in vitro as well as in vivo [ 108 ]. Studies on UCB-MSCs presented a fascinating option for the use of these cells for insulin producing cells. It was demonstrated that UCB-MSCs behave like human ESCs, following similar steps to form the differentiated β-cells [ 143 ]. The most recent findings of Unsal et al. [ 144 ] showed that MSCs when transplanted together with islets cells into streptozotocin treated diabetic rat model enhance the survival rate of engrafted islets and are found beneficial for treating non-insulin-dependent patients in type 1 diabetes.

Cardiovascular diseases

For myocardial repair, cardiac cells transplantation is a new strategy which is now applied in animal models. MSCs are considered as good source for cardiomyocytes differentiation. However, in vivo occurrence of cardiomyocytes differentiation is very rare and in vitro differentiation is found effective only from young cell sources [ 145 , 146 ]. MSCs trans-differentiated into cardiomyocytes with cocktail of growth factors [ 84 ] were used to treat myocardial infarction and heart failure secondary to left ventricular injury [ 147 ]. The systematic injection of BM-MSCs into diseased rodent models partially recompensed the infarcted myocardium [ 148 , 149 ]. Furthermore Katrisis et al. [ 150 ] transplanted autologous MSCs along with endothelial progenitor cells and evidenced the improvement in myocardial contractibility, but they did not decrypt the mechanism which brought out these changes. Although MSCs are effective in myocardial infarction and related problems, but still cell retentivity in the heart is rapidly decreased, after 4 h of cells injection only 10% and after 24 h it was found approximately 1% cell retention [ 151 , 152 ]. Following this study, Roura et al. [ 153 ] reported that UCB-MSCs retained for several weeks in acute myocardial infarction mice, proliferated early and then differentiated into endothelial lineage. Most recently, transplantation of UCB-MSCs into myocardial infarction animal model along with fibronectin-immobilized polycaprolactone nanofibres were found very effective [ 154 ]. All these studies collectively indicate the role of hMSCs in cellular therapy of cardiac infarction and currently there are approximately 70 registered trials investigating the effect of MSCs therapy for cardiac diseases (clinicaltrials.gov).

Homing of MSCs

Homing is the term used when cells are delivered to the site of injury, which is still challenging for cell-based therapies. Most of the time local delivery and homing of cells are found beneficial due to interaction with the host tissues, accompanied by the secretion of trophic factors [ 114 ]. There are a number of factors, like cells age, culturing conditions, cell passage number and the delivery method, which influence the homing ability of MSCs to the injured site.

Higher passage number decreases the engraftment efficiency of MSCs and it has been shown that freshly isolated MSCs had greater homing efficiency than the cultured cells. Besides this, the source from which MSCs are being isolated also influences the homing capacity of MSCs. While culturing MSCs, it was shown that oxygen condition, availability of cytokines and growth factors supplements in the culture media triggers important factors which are helpful in the homing of MSCs. Matrix metallo-proteases (MMPs), the important proteases which are involve in the cell migration, also plays important role in the MSCs migration [ 155 ]. The higher cell numbers and hypoxic condition of the culturing environment influence the expression of these MMPs [ 156 ]. The inflammatory cytokines, i.e. IL-1β, TNF-α and TGF-1 β, enhance the migration of MSCs by up-regulating the level of MMPs [ 155 ]. The next important factor is delivery method via which the MSCs are administered to the desired tissue. Intravenous infusion was the most commonly administered route [ 157 ], because if MSCs were administered systemically it will trap in the capillaries sheet of various tissues, especially in lungs [ 158 ]. That is the reason why most of the time intra-arterial injections of MSCs has been advised, but the most convenient and feasible way of MSCs transplantation is local injection to the site of injury or near the site of injury which provides more number of cells and increases its functional capacity.

The exact mechanism via which MSCs migrate and home to the injured site is still unknown, although it is believed that certain chemokine and its receptors are involved in the migration and homing of MSCs to the tissue of interest. MSCs express many receptors and adhesion molecules which assist in its migration process. The chemokine receptor type 4 (CXCR4) and its binding protein stromal-derived factor 1-α (SDF-1α) play a vital role in this process [ 159 ]. In order to know the homing capacity and to monitor the therapeutic efficiency of MSCs, in vivo tracking by non-invasive method are pre-requisite. Some advance techniques, i.e. single photon emission CT (SPECT), bioluminescence imaging (BLI), positron emission tomography (PET) were being applied for tracking the MSCs.

As we discussed earlier that MSCs have higher trans-differentiation potential and exhibits immunomodulatory features, but their off target homing, especially lodging in the lungs, is a major obstacle. There is need for in-depth study of MSCs homing mechanism and finding appropriate tracking without any negative effect on the cells and host.

Cryopreservation and banking

From all the previous studies, it is obvious that the use of hMSCs for clinical applications will increase in future. For clinical applications, a large number of MSCs in an ‘off the shelf’ format are required. For this purpose, a proper set up of in vitro MSCs expansion and subsequent cryopreservation and banking are necessary to be established. This will provide unique opportunities to bring forward the potential uses and widespread implementation of these cells in research and clinical applications. Keeping in mind its use in future clinical and therapeutic applications, there is a need to ensure the safety and efficacy of these cells while cryopreserving and banking. For the selection of optimal cryopreservation media, uniform change in temperature during freezing and thawing, employed freezing device and long-term storage in liquid nitrogen are the indispensable factors to consider.

First considerable factor is the optimal cryopreservation media in which cells can maintain their stem cells abilities for long time. In the cryopreservation media, the cells require the animal base reagent, like FBS, as a source of their nutrients, but previous studies have suggested that animal proteins are difficult to remove from the hMSCs and that these resident protein may enhance adverse reactions in the patients who receive these cells for treatment [ 35 ]. Therefore, a serum-free media is substantial for the cryopreservation of MSCs and researchers have successfully used the serum-free media for cryopreservation of MSCs [ 160 , 161 ]. Most recently, human albumin and neuropeptide were used instead of FBS and MSCs maintained their cell survival and proliferation potential in the culture conditions. Additionally, cryoprotective agents (CPAs) are required for the cryopreservation media to prevent any freezing damage to cells. A large number of CPAs are available [ 162 ] among which DMSO is the most common CPAs used in cryopreservation of MSCs. However, DMSO is toxic to both humans and animals which make it complicated in the use of MSCs freezing for clinical applications and it has been showed that DMSO has bad effects in both animals and humans [ 163 ]. On the infusion of MSCs frozen in DMSO, patients develop mild complications like nausea, vomiting, headache, hypertension, diarrhoea and hypotension [ 164 ] and also severe effects like cardiovascular and respiratory issues were reported [ 165 ]. Due to these toxic effects, it is necessary to remove (washing with isotonic solutions) or replace DMSO with an alternate CPA. There are several methods along with the introduction of automated cells washing for the removal of DMSO from the frozen thawed cells [ 166 ]. Most recently for tissue cryopreservation, a new method was introduced using the mixture of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol in phosphate buffer saline [ 167 ], shown successful isolation and characterization of MSCs after 3 months of cryopreservation of the tissue. Hence, this method is without any DMSO and animal serum, but it is not yet applied for MSCs cryopreservation. From these findings, it is clear that for clinical grade cells, there is a need of a cryopreservation protocol either with low concentration of DMSO or to replace DMSO with non-toxic alternative.

For cryopreservation of MSCs, the second important factor is the freezing temperature rate. Mostly slow freezing at the rate of 1°C/min is the optimum rate for MSCs preservation [ 168 ]. For this purpose, current controlled rate freezers (CRFs) are suitable for controlling temperature, maintaining the rate of temperature during cryopreservation. These CRFs can be programmed to find out the exact temperature which the sample is experiencing during freezing [ 169 ]. Despite of these benefits, these CRFs lack the uniformity of temperature to all vials during large-scale banking of MSCs [ 170 ], so for large-scale banking, the development of advance CRFs are mandatory. Recently more advanced CRF, which provides unidirectional flow of cryogen to each sample, were created by Praxair Inc. On large-scale MSCs banking, along with the safe and efficient cryopreservation, the regulatory guidelines are also important. Like in the U.S.A., Food and Drug Administration (FDA) is responsible whereas in Europe, European Medicines Agency is responsible in Europe for supervising MSCs based cell therapy products.

MSCs in clinical trials

MSCs have a promising future in the world of clinical medicine and the number of clinical trials has been rising since the last decade. Along with preclinical studies, MSCs have been found to be persuasive in the treatment of many diseases [ 1 ]. A large number of clinical trials have been conducted and this trend is gradually increasing ( Figure 1 ). Currently, there are 463 registered clinical trials in different clinical phases (phase I, II etc.), evaluating the potential of MSC-based cell therapy throughout the world (ClinicalTrials.gov). Most of these trials are phase I/II studies and combination of phase II/III studies, whereas very small numbers of these trials are in phase IV or phase III/IV. Among 463 registered trials, 264 trials are in open status which is open for recruitment whereas 199 trials are closed; out of which 106 studies are completed whereas the rest are in active phases. Clinical trials conducted with MSCs showed very less detrimental effects; however, few of them showed mild adverse effects. Due to immunomodulatory properties, MSCs have been used in many human autoimmune disease clinical trials. However, the exact mechanism by which MSCs regulate the immune response is unclear [ 171 ]. To date, 45 autoimmune-disease clinical trials have been registered, out of which seven are completed, 22 are open for recruitment whereas the rest are in active phases (ClinicalTrials.gov). Similarly 70 trials are registered for cardiovascular diseases, 37 for osteoarthritis, 32 for liver disorders, 29 for graft versus host disease (GvHD), 21 for respiratory disorders, 15 for spinal cord injury, 15 for kidney failure, 13 for skin diseases, seven for muscular dystrophy, five for aplastic anaemia, four for Osteogenesis imperfecta, four for AD, two for PD, two for ulcerative colitis and rest are for other diseases ( Figure 2 ). Although the progress of clinical studies so far registered is slow (only seven studies with final results), but the efficient use of MSCs in large clinical trials with upcoming promising results have proven MSCs as boon for regenerative medicine.

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Recent breakthrough discoveries in engineering MSCs have made it an ideal source for future cell therapy in regenerative medicine. MSCs adaptability to the exposed environment has made them an impressive source for disease treatment, though the full understanding of MSCs mechanism is still in their preliminary stages. After performing a large number of preclinical trials, the human clinical trials of MSCs are now on its way to success and many trials have been successfully accomplished (clinicaltrials.gov). During the last decade, many experimental and clinical assays had been developed; however, a number of questions related to MSCs biology are unsolved. These are related to MSCs survival and homing capacity after transplantation, the relationship between the host immunity and MSCs, the route of administration (local or systemic) and whether the properties like proliferation, differentiation and trans-differentiation are maintained after in vivo transplantation. Several reports have documented the successful transplantation, differentiation and homing of hMSCs but their effect in the concerned disease is due to secretion of cytokines rather than direct effect of MSCs. Furthermore the mechanism underlying migration of MSCs remains to be clarified, although evidence suggests that both chemokines and their receptors and adhesion molecules are involved in this process [ 37 ]. The future MSCs research should focus on finding more suitable markers to isolate the source-specific MSCs, basic understanding of growth regulators in differentiation and trans-differentiation and site-specific homing that can revolutionize the cell regeneration therapy. Moreover, to reduce the risk of oncogenic transformation special attention should be paid to the genetic safety of cell preparation. Nevertheless, an active research should focus on bio-banking in a large scale to use them in the future by developing a novel CPA/protocol without hampering their basic characteristics.

hMSCs are not only easy to isolate but they also retain their ability to expand for long period of time without losing its characteristics. However, apart from mesodermal lineages, they have the capacity to trans-differentiate into ectodermal and endodermal lineages. Moreover, hMSCs have the immunomodulatory properties as they secrete certain cytokines and immune relevant receptors to modify the host immune environment. All these properties of MSCs make them distinct from other stem cells and can be used in future cell replacement therapy. Many preclinical and clinical studies were performed using hMSCs in treatment of chronic diseases like neurodegenerative diseases, autoimmune and cardiovascular diseases, but still there are questions that have to be answered before using hMSCs on large clinical scale. Firstly, the safety issues of MSCs should be solved, because after MSCs administration, mild adverse effects were observed and the most severe is that unfortunately long-term cultured MSCs promote tumour growth and metastasis. Secondly, quality control: before directly applying MSCs for in vivo transplantation, additional tests are needed to perform, like cell viability, endotoxin assays and oncogenic tests. Depending upon the severity of disease, an optimal dose and specific administration time is needed to be decided. The third and most important is clinical grade production of MSCs, because for clinical use of MSCs a large number of cells are required, for which in vitro expansion is vital, but MSCs at higher passages could lead to cell transformation. To conclude, though adult-derived hMSCs are a favourite choice, but before hMSCs can be used on large-scale clinical applications for cell therapy, there is a need for completely understanding the underlying mechanisms that regulate and modulate these MSCs.

This work was supported by the Ministry of Food and Drug Safety, Republic of Korea [grant number 12182MFDS666 ].

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The terms MSC and MSCs have become the preferred acronym to describe a cell and a cell population of multipotential stem/progenitor cells commonly referred to as mesenchymal stem cells, multipotential stromal cells, mesenchymal stromal cells, and mesenchymal progenitor cells. The MSCs can differentiate to important lineages under defined conditions in vitro and in limited situations after implantation in vivo. MSCs were isolated and described about 30 years ago and now there are over 55,000 publications on MSCs readily available. Here, we have focused on human MSCs whenever possible. The MSCs have broad anti-inflammatory and immune-modulatory properties. At present, these provide the greatest focus of human MSCs in clinical testing; however, the properties of cultured MSCs in vitro suggest they can have broader applications. The medical utility of MSCs continues to be investigated in over 950 clinical trials. There has been much progress in understanding MSCs over the years, and there is a strong foundation for future scientific research and clinical applications, but also some important questions remain to be answered. Developing further methods to understand and unlock MSC potential through intracellular and intercellular signaling, biomedical engineering, delivery methods and patient selection should all provide substantial advancements in the coming years and greater clinical opportunities. The expansive and growing field of MSC research is teaching us basic human cell biology as well as how to use this type of cell for cellular therapy in a variety of clinical settings, and while much promise is evident, careful new work is still needed.


MSCs have become widely studied over the past ~30 years for their interesting cell biology, broad-ranging clinical potential, and as a central building block in the rapidly growing field of tissue engineering. MSCs grow readily in the culture dish, have intrinsic differentiation potentials not found previously in other cells, and produce an abundance of useful growth factors and cytokines. The isolation of MSCs from various tissues and their re-implantation at other sites raises questions about the natural in vivo MSCs and their ability to normally repair endogenous tissues, a process that clearly diminishes with age. Mesenchymal cell replacement in the large numbers needed to treat significant tissue injury requires engraftment, structural organization and cellular differentiation—a complex process that has made much progress but remains unperfected. Friedenstein was first to culture bone-forming cells from guinea pig and Owen re-energized this inquiry by expanding such work to rats. 1 , 2 The isolation and culture expansion of human bone marrow MSCs were reported in 1992 3 and their infusion into patients was begun as early as 1993 as reported in 1995. 4 Over the past 25 years the infusion procedures have exhibited an excellent safety profile, so much so that there are now over 950 registered MSC clinical trials listed with the FDA. There have been over 10,000 patients treated in a controlled clinical setting, of which 188 early trials (phase 1 or phase 2) have been completed and ten studies have advanced to phase 3 (Mesenchymal stem cells search at www.clinicaltrials.gov and https://celltrials.org/public-cells-data/msc-trials-2011-2018/65 ). Worldwide, for the years 2011−2018, there were 1043 MSC trials planned with a targeted enrollment of 47,548 patients (Mesenchymal stem cells search at www.clinicaltrials.gov and https://celltrials.org/public-cells-data/msc-trials-2011-2018/65 ). For comparison, bone marrow and hematopoietic stem cell (HSC) transplantations have been practiced since 1957, and through 1983, the first 25 years, about 9000 patients were treated. 5

The most common and longest utilized adult source tissues for human MSCs are bone marrow 3 , 6 and the adipose tissue stromal vascular fraction 7 , 8 and these sources form the foundation for most of the data in this field (Fig. 1a ). These are harvestable human tissues that are thought to be renewable (bone marrow) or unwanted (adipose). There are also two young “adult” tissues, umbilical cord tissue 9 and placenta, 10 , 11 that are excellent sources of human MSCs, and these tissues are normally discarded at birth. The decision to use autologous MSCs from bone marrow or adipose, or an allogeneic source tissue to isolate MSCs is a fundamental clinical decision, but both have shown success producing large numbers of MSCs. 6 , 8 For example, a target dose of 100–150 million human MSCs can be produced from 25 ml of bone marrow by cell culturing in about 3 weeks and this number of packed cells has a volume of about 0.4−0.5 ml. 12 There are still surprisingly few animal research reports or clinical studies that use autologous MSCs and most studies use allogeneic MSCs. In humans, there is also a recognized drop-off with age in the number of isolatable MSCs found in bone marrow, suggesting a very different set of circumstances in the aging population with their more injury-prone tissues than in young adults 13 (Fig. 1b ). In the sections below, we highlight developments and understanding in the cell biology of MSCs, a paradigm shift in their mechanism of action, and improvements in the clinical use of MSCs.

figure 1

Characteristics of MSCs. a MSCs can be readily isolated from bone marrow and adipose tissue but all tissues harbor MSC-like cells as part of the microvasculature. b The number of MSCs, indicated here as colony-forming units (CFU-F), isolated from bone marrow drops off after 15–20 yrs of age and continues to decrease. 13 c MSCs are rare in bone marrow and are culture-expanded to achieve high numbers for research or therapeutic use. However, there is a decrease in the clonal complexity with increased passaging, 23 but the effect of this process on MSC uses is unclear. d The MSCs are known to produce a large number of soluble or vesicle-bound growth factors and cytokines, as well as microRNAs, that can signal to other cells and tissues. e The culture-expanded MSCs can differentiate to multiple cell lineages under separate specific in vitro conditions. The standard chrondro-, osteo- and adipo- differentiation conditions 6 are widely used but additional in vitro conditions promote smooth muscle and striated muscle gene expression; changing medium conditions can induce expression of cardiac and liver genes. Once differentiated, the MSCs express virtually all the hallmark genes of the differentiated cell types. Currently, the more prominent MSC therapeutic uses take advantage of the MSC’s production of factors and the responsiveness of other interacting cells, such as cells of the immune system (see Table 1 ).

A prominent question is whether in vitro cultured MSCs represent any stage of natural in vivo MSCs, or similar cells found during development. 14 It is useful to remember that the embryological development of mesenchymal tissues is complex and proceeds from both trunk and head (neural crest-derived) mesenchyme, and these two cell sources are interwoven in some tissues such as the heart. Within a developing organism, cellular differentiation would appear to be deterministic: we can predict the fate of similar cells in the next offspring or all offspring of future generations, or even in distant or unrelated species. However, we also know from many studies in embryology that cells from one presumptive tissue can be implanted in another tissue and acquire a different fate. Their fate is locally regulated by the new environment and the further development of the implanted cells is selective and not directive. This is a feature and not a flaw of stem/progenitor cells such as MSCs. The early human developmental biology of mesenchymal tissues represents a very specific series of temporal events 14 and is far removed from the tissue repair that occurs in the adult 15−80 years later, and while early development might have something to teach us about using cultured MSCs for repairing and regenerating adult tissues, at this time it is unknown exactly what that will be.

The unfortunate rise of unregulated stem cell clinics

MSCs and other stem cells offer remarkable potential but our understanding of their science and medical applications are not ready for unregulated, widespread use. The complexity of tissue repair and cell replacement makes it clear that the proliferation of questionable “stem cell clinics” and off-shore medical tourism offices promoting their autologous “stem cell treatments” of unknown and unproven efficacy will not solve patient maladies in a meaningful way. The divergence between reputable clinical trials and the premature marketing of stem cell products to the public has broadened the gap, and led to confusion in the press as highlighted by Galipeau et al. 15 There are over 700 clinics offering direct-to-consumer marketing of “stem cell” treatments. 16 We cannot support or recommend any treatment utilizing MSCs that does not use characterized cell product, maintain accurate records, measure intermediate parameters, predetermined surrogate endpoints and track and report final patient outcome(s). These steps are common practice in FDA registered trials, but too demanding for under- or unregulated clinics. A recent study (see Murray et al. 16 reference for details) has offered a consensus report on the parameters needed to improve cell therapy outcomes for both patients and practitioners using the acronym DOSES: D—Donor, O—Origin tissue, S—Separation Method, E—Exhibited Characteristics, S—Site of Delivery.

MSC cell biology—microheterogeneity, temporal stochasticity and diversity at the single cell level

The defining characteristics of a vertebrate stem cell are the ability to divide (symmetrically or asymmetrically), to be motile, to differentiate to multiple lineages and to become organized into multifunctional groupings. To become functionally organized, stem cells need a permissive and instructive environment. Hence, phenotypic reprogramming of stem cells is dependent on the cellular environment and the temporal application of instructive agents and their persistence. This attribute is exemplified in MSCs by their osteo-, adipo-, or chondro-genic differentiation 6 over 1−3 weeks (Fig. 1e ) but further illustrated by the stepwise acquisition of cardiomyocyte properties by sequentially changing the culture conditions over 3−4 weeks as shown by Terzic and colleagues. 17 These population differentiation outcomes of MSCs are a composite result and reflect properties at the single-cell level, but the timing of events for each cell may vary somewhat. It has been recognized that stem cell populations are not homogeneous but rather the cells therein often behave as individual cells 18 —even if they are clonally derived. 19 This temporal stochasticity is a common feature of stem/progenitor cells and occurs throughout development. 20 The stochastic events and processes of stem/progenitor cells are perhaps the most difficult to model or approach experimentally, but we can see similar events in vitro. 21 , 22 In the case of MSCs, a single cell may enter a phase of repeated cell division to create a population containing millions of cells, or die by apoptosis in response to nutrient deprivation, DNA damage, membrane injury etc. For example, when MSCs in culture are labeled with lentivirus vectors encoding individual tags to trace the fate of daughter cells, stochastic processes cause the loss of some clones and the proliferation of others, such that a cultured MSC population with an initial complexity of 70 is reduced to a complexity of 3 to 4 surviving clones, and these resulting clones do not represent the most abundant clones at the start 23 (see Fig. 1c ). While troubling our view of culture-expanded MSCs as a homogeneous population, if all the remaining cell clones have the same stereotypical behavior or “abilities” as the starting cells, this reduction in MSC population complexity may not result in the loss of potential or utility. Studies to understand how these events alter adult MSCs and MSC population composition and function, with respect to both their stem/progenitor and paracrine activities, in vitro and in vivo, are important for our biological and clinical potential understanding. Studies of clonal activities of HSCs have long indicated the rise and fall of individual clones, but this may not impact their functional or clinical outcomes. 24 , 25 For in vivo intestinal epithelial stem cells too, the clonal expansion/extinction process has strong experimental evidence. 26 Recent studies indicate that growth factors, cytokines, and other bioactive factors produced by MSCs may be contained in exosomes and microvesicles that function in a paracrine manner. 27 , 28 , 29 , 30 Although the role of exosomes and microvesicles in normal MSC physiology and as therapeutic entities is emerging, how the exosome/microvesicle production and composition are influenced by stochastic processes, clonal expansion and culture complexity or MSC differentiation remains largely unexplored.

MSC transcriptome and phenomics

MSCs are still largely defined by their in vitro expression of a restricted subset of cell surface proteins, and their capacity for stimulus-induced tri-lineage differentiation. 6 , 31 While this minimalistic definition has sufficed for almost two decades and is still widely used today, the field should benefit from the large repository of gene expression-based data (GEO Datasets) that interrogates the MSC transcriptome, and the important expression changes that occur following culture expansion, hypoxia preconditioning, stimulus-directed differentiation, trans-differentiation, exposure to biologics, and coculture with other cell types. The genome-wide gene expression studies can provide insight into the biological nature of MSCs, their expected physiological function, role in disease pathophysiology, and probable therapeutic mode of action. Understanding MSC gene expression data holds promise for refining the operational definition of MSCs, clarifying their native physiological function, and informing how culture conditions and clinical manufacturing protocols can best characterize their composition and function prior to patient administration.

MSC gene expression studies were initially focused on establishing an identity for bone marrow-derived MSCs (BM-MSCs) in vitro that could be shared across laboratories. Toward this goal, the transcriptome of human and mouse BM-MSCs was cataloged via serial analysis of gene expression (SAGE), 32 and the nature of the catalogued transcripts were shown to reflect their stem/progenitor properties and paracrine activities related to hematopoiesis support and skeletal homeostasis. Along with related cellular studies, the gene expression data support the skeletogenic, angiogenic, anti-inflammatory and immunomodulatory activities of MSC populations that are widely utilized for clinical therapies today. Additionally, studies have shown that MSCs isolated and cultured from different tissues/organs are more closely related to each other than other mesodermal lineages, and that their phenotypic signature is similar to that of perivascular cells, 33 , 34 providing a physiological basis for the widespread anatomical distribution of MSCs or MSC-like cells in vivo (see below.) The extent to which the MSC gene expression is influenced by their culture conditions and can be manipulated remains important for their clinical utility.

Recent RNA-seq studies have furthered our understanding of how MSCs respond at the cellular level to differentiation-inducing stimuli. For example, one such study identified prominent changes in the MSC transcriptome following differentiation to the adipogenic vs. osteogenic lineage, and ChIP-Seq studies revealed that the epigenome of MSC-derived osteoblasts, but not adipocytes, more closely resembled that of naïve cultured MSCs. 35 The MSC genome was also shown to contain a high degree of overlap for binding sites of master transcriptional regulators, such as RUNX2 and C/EBPβ, that are epigenetically reduced in size following differentiation, and these promoter regions exhibited high plasticity that enabled MSCs to trans-differentiate from adipocytes to osteoblasts and vice versa. 36 These transcriptional pathways may be relevant to the in vivo differentiation fate of MSCs. 37 The Wnt intracellular signaling protein (WISP-1 or CCN4) was recently shown to modulate the osteo- and adipogenic lineages. 38 These findings broaden the concept of in vitro lineage priming used initially to provide the molecular basis for MSC multi-potency 39 and may yield improved therapies.

In addition to differentiation-inducing stimuli, other treatments have also been identified that alter the biological activity of MSCs by altering gene expression. For example, it was established early on that rodent MSCs exhibit enhanced growth and osteogenic potential under low oxygen (5%) levels, 40 , 41 , 42 which mimics conditions in the bone marrow niche. Subsequently, it was shown that transient exposure of MSCs to hypoxic conditions (<2% oxygen saturation) enhances their proangiogenic activity in vitro 43 , 44 , 45 and in vivo 46 , 47 , 48 and positively impacts growth and survival. 49 , 50 , 51 Profiling studies have revealed that hypoxic preconditioning significantly alters expression of a small subset of genes linked to cell proliferation and survival, glycolysis, and vasculogenesis/angiogenesis in MSCs, the majority of which were upregulated. 52 , 53 A similar approach is being used to interrogate how MSCs respond to inflammatory stimuli to gain further insight into their anti-inflammatory and immunomodulatory activities. For example, in vitro stimulation of human MSCs with lipopolysaccharides, a ligand for TRL4, induced expression of transcripts involved in chemotaxis and inflammatory responses that were principally orchestrated by interferon regulatory factor (IRF1) and nuclear factor kappa B (NF-κB). 54 Alternatively, exposure to interferon (IFN)-gamma licenses the immunosuppressive activity of MSCs by inducing expression of indoleamine 2,3-dioxygenase (IDO1), an enzyme in the kynurenin pathway that consumes tryptophan 55 , 56 resulting in reduced inflammation, and stimulation by TNF upregulates expression of the anti-inflammatory protein TSG-6. 57 Importantly, while TNF- and IFN-gamma-stimulated MSCs expressed distinct sets of proinflammatory factors, these proteins were shown to function synergistically to uniformly polarize MSCs toward a Th1 phenotype characterized by expression of the immunosuppressive factors IL-4, IL-10, CD274/PD-L1 and IDO. 58 This finding is also significant because at the population level, hierarchical clustering of gene expression data from MSC donors revealed that nonstimulated populations exhibited a significantly greater degree of inter-donor heterogeneity. 59 Therefore, treating MSCs with potent stimuli has a normalizing effect on the population and may largely erase interdonor differences in MSC function, and this “cytokine priming” should be tested in animal and clinical studies.

It is important to point out that in addition to enhancing paracrine signaling, exposure to inflammatory stimuli such as TNF and IFN-gamma produces other notable effects on MSCs. For example, IFN-gamma upregulates expression of genes associated with programmed cell death and cellular apoptosis, reflecting its profound negative impact on MSC growth and survival. 60 Such gene expression responses of MSCs to IFN-gamma treatment are also accompanied by alterations in gross morphology. 61 MSCs induced toward the osteogenic lineage have been shown to exhibit upregulation of IFN-gamma inducible genes 62 , 63 and a concomitant impairment in angiogenic activity. 64 Similarly, analysis of MSCs from TSG-6 knockout mice revealed profound alterations in cell morphology, impaired growth, and loss of tri-lineage differentiation potential. 65 In this study, RNA-seq analysis identified 1537 downregulated and 1487 upregulated genes in TSG-6 null MSCs as compared to wild-type cells, and these genes mapped to biological processes including cell cycle, cell death and survival, cell morphology, cellular movement, DNA replication and repair. Notably, the expression of several transcription factors involved in regulating cell division, stem cell differentiation, and Wnt signaling was found to be inhibited upon TSG-6 deletion. These data are consistent with other studies showing that pathways controlling cell growth, differentiation, and paracrine signaling are mechanistically linked, and in some cases may be mutually exclusive. For example, a recent study showed that treatment of equine adipose-derived stromal cells with interleukin-1β and/or TNF compromised their tenogenic properties in part by reducing expression of the tenogenic transcription factor scleraxis. 66 Consequently, more studies are needed to predict which culture conditions are best suited for a desired cellular response or particular clinical indication.

Is there a profile of expressed genes that defines MSCs and can be used to predict success in their therapeutic use? A large integrative analysis of existing MSC genomic datasets was used to establish an “MSC classifier” that accurately distinguishes MSC from non-MSC samples with over 97% accuracy. 67 The gene expression and protein data that contribute to the MSC classifier can add rigor to the current definition of MSCs. Similarly, a comparative genomics approach was recently used to develop a “Clinical Indications Prediction” (CLIP) scale based on in vitro TWIST1 expression levels that reveals differences in the biological activity of different donor MSCs. The CLIP scale may have utility for matching the biological activity of MSC donor populations to specific disease indications and may thereby improve outcomes of MSC-based preclinical models of disease and subsequent clinical studies. This approach could also interrogate in real time how preconditioning regimens, cytokine priming and manufacturing processes may improve the predictability of MSC biological activity and clinical outcomes. The reproducibility of MSC isolation and gene expression is further supported by examining multiple isolations of bone marrow MSCs and comparing results to HSCs, NSCs and ESCs, wherein it was found that the MSCs clustered together more than the other stem cell types tested. 68 The role(s) of microRNAs and circular RNAs in sustaining MSC identity are likely to be another important distinguishing indicator of phenotype. 69 A broad analysis of expressed genes by RNA deep sequencing and translated proteins by nano-liquid chromatography MS/MS for both BM-MSCs and ESC-derived MSCs found many similarities and suggested new membrane surface proteins that may be useful for phenotypic identification in future studies. 70

Keep in mind that for therapeutic MSC products, a release assay related to the clinical proposed function of the MSCs in vivo is requested by the FDA at phase 1 and is required by phase 3. Consequently, these RNA-Seq and ChIP databases and related resources provide a tool kit that should be more thoroughly tested to match appropriate donor MSCs, manufacturing protocols, and patients to improve response rates. This means that MSCs will be produced as a focused product for each therapeutic application and require more specific release criteria and likely include a population gene expression measure such as the TWIST-based CLIP assay as well as an individual cell measure such as flow cytometry. For MSCs for the treatment of graft vs. host disease (GVHD), the release assay should reflect the anti-inflammatory activity of the MSC product 71 and an inexpensive and accessible flow cytometry assay such as that of Ribeiro et al. 72 that can quickly test >10,000 individual MSCs in minutes gives a readout of both the single-cell analysis and a population (product) assay. But if, however, the therapeutic mode is found to be not the cell, but a secreted cytokine(s), exosome enclosed miRNA or factor(s), then more specific assay(s) will be required. Recently, Kaushal and colleagues were able to demonstrate that, following injection into heart tissues, the expanded cardiac-derived cell population (cardiac progenitor cells or CPCs) that includes an MSC-like population, alter their beneficial exosome expression in the in vivo setting. 30 Further understanding of the localized response(s) of ex vivo expanded progenitor cells placed into the in vivo damaged tissue setting is needed for the therapeutic development of cellular therapies.

MSCs and related vascular cell types

Despite extensive efforts to characterize MSCs, these remain, fundamentally, a product of their extended cell culture conditions. They originate from tissue but are they “real” stem cells in vivo? Does the tissue dissociation, adhesion to tissue culture plastic and growth in serum-supplemented medium isolate and drive the sustained proliferation of a rare, elusive tissue-residing progenitor cell(s), or is it that our tissue culture acumen has produced a valuable “artifact” of the process? Some studies may have equated the MSC to a previous histologically identified cell in bone marrow such as the reticulocyte, Weston-Bainton cell, a stromal cell, a fibroblast, etc., but the rare nature of MSCs makes this unlikely and the in vivo identity(s) of MSCs remains obscure—despite the now broad use of MSCs in tissue engineering and regenerative medicine. Further, although bone marrow was first used for MSC isolation and considered a renewable source, MSC-like cells have been isolated from many tissue sources including harvested adipose tissue, 7 , 8 umbilical Wharton’s jelly, 9 placenta, 10 , 11 skin 73 and the roots of shed teeth. 74 The studies of microvascular pericytes soon overlapped with attempts to uncover the innate identity of in vivo MSCs and indicated phenotypic similarities between the two, 75 , 76 , 77 and Crisan et al. 78 showed that pericytes purified by flow cytometry from diverse human organs and cultured for several passages are indistinguishable from conventional, bone marrow-derived MSCs in terms of morphology, proliferation kinetics, surface antigen expression, and differentiation potential, in vitro and in vivo. Moreover, the canonical MSC surface marker combination of CD44 + /CD73 + /CD90 + /CD105 + is detectable on pericytes in situ 78 . This paradigm was later extended to perivascular spaces around larger arteries and veins, in which the outermost tunica adventitia contains a population of fibroblast like presumptive MSCs. 79 The presence of similar cells around the microvasculature is being presently investigated. This affiliation with the vasculature puts MSCs in position to respond quickly to tissue damage. To further understand the respective potential of pericytes and adventitial progenitors, the transcriptome of single cells sorted from human adipose tissue was determined and the differential gene expression, principal component and clustering analysis, as well as the construction of gene coregulation networks showed that adventitial cells constitute a more “primitive” population, expressing genes associated with “stemness”, such as nanog, c-myc, klf2, -4, -6, and osteogenic commitment and differentiation (runx2, nox4, notch2). 80 Conversely, the pericytes appeared overall as more differentiated cells, expressing genes involved in angiogenesis and smooth muscle cell function (angpt2, acta2), in agreement with the in vivo function of these cells.

Accordingly, recent studies have revealed that in the course of osseous regeneration in vivo, pericytes principally stimulate neoangiogenesis, while adventitial cells are more directly involved in bone formation. 81 When cultured in vitro, both perivascular cell types clearly establish an MSC population, but the respective contributions of pericytes and adventitial cells to the multipassage cultured MSC remain to be elucidated.

A largely unanswered question raised by the prospective identification of perivascular cells as innate MSC forerunners is whether these cells play the same progenitor role in their in vivo environment. Not surprisingly, RNA-Seq studies performed on human pericytes and adventitial perivascular cells before and after culture revealed dramatic differences in gene expression associated with their establishment in culture and the transition to the in vitro MSC phenotype, with up to one third of all expressed genes being significantly up- or downregulated. (Hardy et al., manuscript in preparation). This may suggest that perivascular cell -derived MSCs are profoundly modified, or even entirely initiated, by cell culture; however, cell lineage tracking in reporter transgenic mice has uncovered roles for pericytes as mesenchymal progenitors, in the adult, for white adipocytes, 82 myoblasts, 83 follicular dendritic cells, 84 and profibrotic myofibroblasts, 85 , 86 , 87 , 88 , 89 and both pericytes and adventitial progenitor cells are involved in the turnover and repair of dental tissues. 90 A recent study confirming the MSC potential of mouse perivascular adventitial cells also uncovered a pathologic correlation and demonstrated that adventitial cells directly contribute to atheroma formation and calcification in remodeling large vessels, by differentiating into smooth muscle cells and osteoblasts, respectively. 91 Thus, the modern version of embryological tissue transplantation suggests the MSCs have multipotentiality in vivo as well as in vitro.

However, considering the hundreds of billions of pericytes associated with the 50,000 miles of capillaries present in the human body—plus the other blood vessels—and even though no more than one in ten perivascular cells yields MSCs in culture, the global efficacy of the system to repair/regenerate tissues in the living adult organism would appear to be surprisingly low. 92 , 93 The observed discrepancy between the robust potential exhibited by in vitro cultured MSCs from perivascular tissue and the modest endogenous role evidenced in vivo calls for investigations regarding the clonal selection and gene expression alterations that accompany their establishment in vitro to perhaps understand and facilitate the molecular control of their reprogramming into stem-like reparative cells in situ. It may also be that the perivascular MSCs are engaged in an important tissue function and are not available for mobilization. Once relieved of this obligation by tissue harvest and in vitro culture, the vascular derived MSCs seem free to pursue other roles. In the kidney, pericytes play diverse roles as mesangial cells in glomeruli and renin secreting cells in afferent arterioles. Yet, these specialized pericytes yield “MSCs” when purified, cultured and evaluated. 94

MSC responses—outside-in signaling on hard vs. soft substrates

The bone marrow MSCs reside in their in vivo niches where cell−cell interactions involving N-cadherins are thought to be key to maintaining the stem cell state with the essential interacting domains involving the peptide His-Ala-Val-Asp. As MSCs move away from this nurturing niche environment, they may encounter fewer cell−cell interactions and more extracellular matrix interactions. Most types of in vitro cultured adherent cells, including MSCs, assemble integrin-based focal adhesions that engage extracellular matrix molecules (fibronectin, laminins and collagens, initially supplied in vitro from serum) and form extensive cytoskeletal networks on rigid plastic or glass surfaces but not flexible substrates. 95 In vitro cell culture typically utilizes negatively charged polystyrene dishes or flasks that aid attachment of extracellular matrix proteins and cells. However, for multipotential cells such as MSCs, these hard plastics may not be ideal for deciphering cell lineage potentials. MSCs grown on the rigid polystyrene culture surface can be directed to specific lineages—osteo-, chrondro- and adipogenic but only limited myogenic differentiation occurs (~1–2% positive for desmin and myosin heavy chain proteins). However, several days in culture on compliant substrates caused MSCs to exhibit additional characteristics consistent with the soft-tissue myo- and neuro- lineages. 96

Tissues exhibit a range of stiffness in vivo, as quantified by the elastic modulus (Young’s modulus) measured over a range of 0.1–100 kPa. Brain and marrow tissue stiffness is about ~0.3 kPa, fat ~3 kPa, muscle ~10 kPa, and precalcified bone ~100 kPa, whereas tissue culture plastic or glass is much harder at »1000 kPa. MSCs in bone marrow can exhibit dendritic shapes and express some neuro-typical markers such as CD271, TRK A, B and C mRNAs, and a moderate fraction of MSCs cultured on soft gels that mimic the softness of marrow or neural tissue favor expression of nestin, β3 tubulin, and other neuro-typic traits. 96 , 97 However, adipogenesis is a definitive soft-tissue differentiation pathway of MSCs and soft gels have been repeatedly found to be conducive to adipogenesis when compared to differentiation on stiff substrates, and stiff 2D substrates strongly favor osteogenesis, which is relevant to the epitaxial growth of bone, and similar findings have been reported for 3D gels. 98 , 99 , 100 , 101 , 102 , 103 , 104 Growing MSCs for a longer time on either a soft or a stiff substrate progressively commits the cells to the corresponding lineages, making them refractory to rapid lineage switching by both soluble factors and substrate changes. 96 , 99 The molecular basis for this commitment—be it DNA methylation, microRNA, soluble factors, or other—remains to be identified. With regards to the perivascular localization of in vivo MSCs discussed previously, it was recently demonstrated that purified human pericytes also can be induced to differentiate in culture into either osteocytes, chondrocytes or even neuron-like cells by modifying the stiffness of a hydrogel substrate. 101

The molecular mechanisms that MSCs use to transduce the compliance of a matrix to lineage differentiation cues continue to be studied, with certain reproducible pathways emerging (Fig. 2 ). For MSCs, acto-myosin motility is more prominent on stiffer substrates when substrate adhesion is not limiting (too little or too much). Cytoskeletal contraction forces clearly contribute to differentiation, with the higher forces exerted on stiffer substrates favoring a stiff tissue (bone) lineage. 96 , 100 Smooth muscle actin (SMA) assembles into high tension stress fibers and is understandably upregulated in MSCs on stiff substrates, and although SMA expression can be quite variable between MSCs even on a homogeneously stiff substrate, 102 SMA does contribute to osteogenesis of MSCs. 105 At the nuclear membrane, the structural protein lamin-A engages cytoskeletal stress fibers via linkage proteins that span the nuclear envelope, and high levels of lamin-A in MSCs favor osteogenesis whereas low levels favor adipogenesis, consistent with observations that lamin-A is high in stiff tissues but relatively low in soft tissues. 102 , 104 The transcriptional co-activators YAP and TAZ primarily translocate (see Fig. 2b ) into the nucleus in cells on stiff substrates to promote expression of differentiation genes for “stiff lineages”. 98 , 106

figure 2

MSC interactions with cytoskeletal elements, cell−cell contacts, extracellular matrix and topography can have profound effects on multipotential MSCs. a Harvesting MSCs from a bone marrow niche with its condensed cell-rich environment and culturing them in vitro removes the cell−cell cadherin and connexin connections and replaces them with cell−substrate and cell−matrix interactions, as the cells produce more extracellular matrix. b The stiffness of the MSC culture surface and the nature of the environment have significant input to alter gene transcription and biological responsiveness of the MSCs through nuclear Lamin-A 104 and YAP1 98 ( b ) and the surface curvature can transduce cytoskeletal influence over MSC potential. 107 Also, dynamic stretching and 3D matrix materials can provide new approaches to understanding MSC responses and potential therapeutic applications.

Further, MSCs respond to the surface curvature of their substrate. When present on a rigid convex curvature of ~500 micron radius, the MSC’s long axis (~200 micron) is extended, the nucleus is flattened/deformed by stress fibers, there is more nuclear lamin-A and the cell is prone to osteogenic differentiation, whereas when the MSC is present on a concave surface of the same radius, the cell is more motile, has fewer stress fibers, the nucleus has greater curvature, less lamin-A and is “suspended” by the cytoskeleton. 107 The foregoing description suggests a clonal group of MSCs may be induced to differentiate to several lineages in response to their migration over substrates of different compliance or curvature as may be found during development. Additionally, Fisher and colleagues recently demonstrated that MSC attachment followed by dynamic substrate movement (stretching) provides one more step in the “education” that MSCs may experience in vivo. 108 The growth of MSCs on soft materials may also preserve the regenerative properties of the cells into later passages or allow them to recover from tissue culture-related aging. 109 The biomaterials-MSC field is actively producing new data, especially in relation to 3D tissue mimetics which may have a profound effect on MSCs, and these data are needed for many growing in vivo applications. 110 , 111 Adoption of reproducible methods for culturing MSCs on soft materials or dynamic substrates may provide greater therapeutic potential and effectivity. Can we then use the knowledge of MSCs responsiveness to culture substrate compliance, curvature, and micromovement to better prepare them for clinical applications?

MSC paradigm shift—cell replacement to paracrine provider

The early demonstrated multipotential differentiation of MSCs fueled prospects for cell replacement where damaged tissue could be readily renewed. However, resolution of adult tissue damage wherein ounces of complex tissue must be dissolved, resorbed, renewed and remodeled, is a complex process not likely solved by the MSC itself. Over the past decade the emphasis has shifted toward harnessing the MSCs’ ability to produce factors and cytokines that stimulate innate tissue repair and modulate inflammation and immune responses (Table 1 ). Many MSC clinical trials are testing how the paracrine activity of these cells can be utilized, not the cells ability to differentiate to mesenchymal lineages. This is a very different mode of action from that seen with HSCs and their transplantation, a model that perhaps has hampered more than helped our understanding of MSCs. To date, the clinical trials with MSCs have established a strong safety profile and some success in patient subgroups has been evident (see below), and the MSC paracrine activities have fostered their examination in many diverse therapeutic applications. The immune modulation and anti-inflammation applications of MSCs are broadly applicable in damaged tissue, and the shift in emphasis from cell replacement to modifying the body’s cell and tissue responses from a clinical perspective reflects our progress in understanding the available ex vivo expanded MSCs. To what extent the culture-expanded MSCs reflect the endogenous adult tissue-resident MSCs is not yet clear, as discussed above.

MSC modulation of the immune system

Initially, early studies on MSCs envisioned autologous cell therapy for the orthopedic applications of bone and cartilage repair, and separate studies sought to provide “stromal” cytokine enhancement of bone marrow transplantation in cancer patients. The orthopedic studies initially flourished and animal studies looked very promising with autologous MSCs, and human clinical trials were planned. However, at least two findings prompted the testing of allogeneic MSCs in orthopedics, bone marrow transplantation, and also cardiac infarcts: (1) the costs to produce autologous MSCs for injection were substantial when it was understood that each patient’s culture-expanded MSCs would need to undergo extensive safety testing before infusion to assure the expansion process did not introduce any bacteria, viruses, etc. and (2) many patients previously treated for hematopoietic malignancies had diminished MSC numbers in their bone marrow, and the required autologous MSC dose could not be achieved as quickly as needed (2−3 weeks) to treat these patients. 112 To address the second problem, allogeneic MSCs were isolated from an immunologically matched donor, a family member. These donors were not identical matches and it was anticipated that the use of these allo-MSCs would produce greater graft vs. host disease in the recipients—especially when the third-party HSC treatment was not a perfect match either. However, the investigators found LESS graft vs. host disease in the recipients, not more. 112 , 113 This unexpected important medical benefit of allogeneic MSC treatment has been explored extensively to date. This also appeared to “solve” the other problem of cost by allowing large numbers of MSCs to be grown from a donor, and extensively tested, and then used to treat many patients, thereby reducing treatment costs.

Contemporaneous with early MSC/hematopoietic stem cell transplant clinical studies, in vitro studies tested allo-MSCs in mixed donor lymphocyte reactions and revealed the MSCs prevented lymphocyte proliferation, and do not cause apoptosis of T cells, rather the T cells will respond to subsequent lymphocyte challenge when the MSCs are removed. 114 , 115 Many subsequent studies confirmed these findings. For cell−cell interaction, it was found that MSCs normally express major histocompatibility (MHC) Class I antigens on their surface and not Class II, but Class II antigens are upregulated by inflammatory agents. The intensive search for soluble factors secreted from MSCs that cause them to be immune-modulatory (Fig. 3 ) identified multiple factors 113 , 116 , 117 , 118 , 119 , 120 , 121 , 122 , 123 , 124 that limit immune cell responses including transforming growth factor β, hepatocyte growth factor, prostaglandin E2, interleukin-10, interleukin-1 receptor antagonist, interleukin-6, human leukocyte antigen-G, leukocyte inhibitory factor, indoleamine-2, 3-dioxygenase, nitric oxide, galectins-1 and -9, and TNFα stimulated gene 6 (TSG-6). Further, MSCs skewed maturing immune cell populations resulting in increased regulatory T cells (T Reg ), anti-inflammatory T H 2 cells, and dendritic DC2 cells while fewer proinflammatory T H 1 cells, dendritic DC1 cells, and fewer NK cells were found. MSCs also induced M1 macrophages to the anti-inflammatory M2 form and reduced IgG production from B cells. While many of these identified factors have been used individually to inhibit immune responses, the MSCs produce a more complete immune modulation owing to the multiple factors acting in unison.

figure 3

MSC—Immune cell interactions. Initial studies envisioned autologous use of MSCs. However, studies with immune cells demonstrated that MSCs are not immediately rejected by T cells and other immune cells, prompting the study of allogeneic MSCs in mutiple therapies. MSCs produce at least 11 factors known to affect immune cells. When interacting with T cells (pathways 1 and 5) MSCs cause a reduction in inflammatory T H1 and an increase in T Regs and T H2 cells with the concomitant decrease in IFNγ, increase in IL-10, IL-4 and IL-5. When MSCs interact with dendritic cells (pathways 2, 3,and 4) there is a decrease in proinflammatory mature DC1 with a decrease in TNF-α and IL-12, and an increase in immature DC and DC2, with increased expression of IL-10. When MSCs interact with natural killer cells (pathway 6) there is a decrease in the expression of IFNγ. When macrophages interact with MSCs (pathway 7), there is a decrease in the proinflammatory M1 phenotype and an increase in the anti-inflammatory M2 phenotype, with increased PGE2, TSG-6 and IL-1RA. MSCs can also reduce the secretion of antibodies from B cells (pathway 8) and inhibit bacterial growth by a direct or indirect mechanism (pathway 9). This figure is used with permission from Blood/Aggarwal and Pittenger 116 and has been updated/modified from its original form.

This downmodulation of immune cell proliferation would seem to put the recipient at risk for higher infection rates but this is not seen in vivo when patients receive MSC infusions. The MSC’s production of antibacterial agents PGE2 116 , 125 and LL-37 peptide, 126 that may work in vivo through effects on hematopoietic cells, are at least part of the reason. Thus, the MSCs have been shown capable of modulating immune responses in situations where T, DC, macrophage and NK cell proliferation could lead to a runaway cytokine storm. This property of MSCs is highly desirable and is reflected in the many clinical trials which are testing the immune-modulatory and anti-inflammatory properties of MSCs.

Graft versus host disease (GVHD), a common complication following bone marrow or cord cell transplantation, represents a response of the developing new hematopoietic and immune system against the recipient host and can result in life threatening tissue damage. Promising early clinical trials to use MSCs to treat GVHD patients still lack definitive, successful phase 3 trials. Notably, the Osiris Therapeutics Inc. sponsored phase 3 trial of MSC therapy for GVHD following hematopoietic stem cell transplantation did not meet its proposed endpoints across all ages but showed life-saving benefit in the pediatric patients. 127 , 128 The results did lead to the first approvals for a culture-expanded MSC product for cell therapy against GVHD in Canada and New Zealand but did not achieve the studies endpoints necessary for US FDA approval. Instead, the MSC drug—“Prochymal” or “Remestemcel-L”—was made available in seven countries under the Expanded Access Program. Mesoblast Inc. acquired a license from Osiris to pursue culture-expanded MSCs in 2013 and the phase 3 trial for pediatric GVHD has recently completed enrollment with results expected soon. Several meta-analysis studies each comprising ~300 patients indicate the MSC treatments were effective in certain subpopulations but not all patients, and the reasons for this are unclear. 129 , 130 , 131 However, given the diverse patient populations, varied MSC preparation methods, timing of first MSC infusion, dosing and heterogeneous pharmacological patient treatments, many possible improvements should be considered.

Treating tissue injuries with MSCs

Mature adult tissues regularly perform maintenance and replace cells that have half-lives of hours to days, or from months to years, and in rare cases decades, e.g. brain neurons, chondrocytes, and cardiomyocytes where the cell half-life is on the order of ~50 years. 132 When a tissue is damaged by trauma or disease, these regenerative repair processes may be accelerated, but this capacity diminishes with age, and each tissue ages somewhat differently.

Currently, a typical therapeutic dose of MSCs is 100 million cells and this number of packed cells occupies only ~400 μl. Most bodily injuries of this size are not a problem and clearly this “therapeutic” MSC dose is meant to initiate or augment a repair response from the body rather than serve as “cell for cell” replacement. For example, the adult heart is about the size of two hands clasped together and a “heart attack” may destroy a tissue volume similar to one, two or three fingers. Clearly, the current “therapeutic dose” of MSCs represents only a small portion of the total damaged cells in the tissue but this dose can produce clinically beneficial effects (see below). Further, although multiple dosing with MSCs is possible and many clinical trials now include this provision, it is well known that few transplanted MSCs engraft and survive and as few as <1% may be detectable later. This limited engraftment of transplanted cells is a major problem and not unique to MSCs, being well known in other cellular therapy fields including hematopoietic stem cell transplantation, CAR T-cell therapy, etc.

Most in vitro culture conditions do not prepare the MSCs for the in vivo setting, and there is little evidence for in vivo proliferation of delivered MSCs—likely due to their strong cell−cell contact inhibition of cell division and the lack of a ready “MSC-friendly” niche. Perhaps an exception to the rapid loss of transplanted MSCs is when they are implanted attached to a matrix such as for repairing a bone injury. Their increased survival when attached is likely due to intracellular signaling pathways, including focal adhesion signaling. Facilitating MSC homing to favorable sites of engraftment such as by improving MSC binding to sites of injury through drug pretreatment for attachment to ICAM-1 rich areas 133 should improve in vivo survival. Additionally, most laboratories culture MSCs in atmospheric oxygen (20%)—out of convenience more than designing for cell optimization—but once implanted the MSCs must adapt quite quickly to much lower tissue oxygen levels. There are many studies that have used low oxygen cultivation (1−5% O 2 ) of MSCs with good success and increased survival (see Pezzi et al. 134 and references therein). Therefore, greater effort to prepare MSCs metabolically for the in vivo environment, including low oxygen cultivation, priming for glycolysis and cell attachment, may pay big dividends with respect to enhancing clinical efficacy.

Further, every tissue is complex and composed of several cell types, and the functional parenchyma of any tissue is accompanied by cells of the structural connective tissue. There are blood vessels (to deliver nutrients, remove wastes), sympathetic neurons (for central control), and lymphatic ducts (additional waste product removal) present in every cm 3 of tissue with few exceptions. Due to a tissue’s metabolic needs, it is estimated that every cell in the body is no more than 2−3 cell diameters from a blood vessel, except in cartilage and cornea. Therefore, the repair of damaged functional tissue requires replacing several cell types, and, if using only MSCs for cell replacement therapy, requires the body to supply any missing cells. Although most studies use MSCs alone to attempt tissue repair/regeneration, most investigators agree that additional cell type(s) participate. For example, human endothelial colony forming cells (hECFCs) circulate in the peripheral blood and are capable of vasculogenesis, and when co-transplanted with MSCs, the hECFCs increase the engraftment and differentiation of MSCs in a PDGF-BB-dependent manner. 135 Three-dimensional matrices and decellularized tissue scaffolds have also become a new proving ground for investigation of MSC potential and interaction with other cell types. 136 , 137 More effort to understand the MSC interactions with endothelial progenitor cells, and epithelial cell types in various tissues, may provide important insight and opportunity for more effective tissue repair and clinical treatments.

Clinical progress with MSCs for cardiac injuries, immunologic diseases and aging frailty

Over the past decade numerous advances have been made in the development of allogeneic MSCs as a therapy for a highly diverse group of diseases, including cardiac diseases. As described already, MSCs were considered a multipotential cell envisioned to differentiate into a limited repertoire of mesodermal tissues—bone, tendon, cartilage, muscle, and fat. However, it is now appreciated that MSCs produce many bioactive factors. This can provide a multiplexed approach and is likely effective for both post-infarct and nonischemic left ventricular failure. In this regard, human MSC delivery into the injured heart has demonstrated four potent mechanisms of action that work in concert: reduction of fibrosis, stimulation of neovascularization, immunomodulation, and stimulation of endogenous tissue regeneration. 138 , 139 , 140

These four combined actions are particularly powerful at avoiding the negative remodeling of organs damaged by ischemic injury. For example, post-myocardial infarction remodeling is the cause of substantial morbidity and mortality. The underlying driver of this disease process is ischemic injury to the heart leading to a loss of contractile cardiomyocytes and their replacement by a large area of fibrotic scar tissue. Numerous preclinical and clinical trials have demonstrated that injection of MSCs into the border zone between infarcted and viable cardiac tissue results in a powerful antifibrotic effect, reduced tissue injury and augmentation of viable and perfused tissue. 141 , 142 , 143 , 144 , 145 The improved contractile cardiac muscle results predominantly from enhanced endogenous regeneration mechanisms rather than engraftment and differentiation of the injected MSCs. 146 , 147 This conclusion derives from observations that relatively few MSCs are found engrafted at the site of injury relative to the degree of functional recovery, and that endogenous precursor cells and myocyte mitosis is upregulated with MSC treatment (Fig. 4 ). Recently, there is much interest in defining the molecular pathways and signaling modes for MSC activation of endogenous cell-cycling; for example, it has been shown that cell therapy may activate endogenous cardiac repair mechanisms by dual inactivation of the retinoblastoma and CDKN2a pathways. 148

figure 4

MSCs implanted in vivo in the infarcted left ventricle wall improve cardiac recovery in preclinical models and patient studies. A diagram of cellular therapy approaches tested with MSCs is shown. 1—Peripheral veinous infusion. 2—Endomyocardial delivery via injection catheter. 3—Direct myocardial injection during open chest surgery such as for coronary artery bypass grafting. 4—Delivery via intracoronary arteries. MSCs release anti-inflammatory factors and interact with endogenous cells to improve physiological outcome despite limited engraftment. Panels ( a − d ) 145 — a Porcine female heart receiving male allo-MSC injection show greater repair processes with active stimulation of endogenous cardiomyocyte cell-cycle activity (phospho H3 staining) which are associated with greater functional recovery. b Following direct injection of male MSCs near the infarct border, there are increased phospho-H3 detected at 8 weeks in the infarct (IZ) and border zones (BZ) compared to the remote zones (RZ) away from the infarct. The error bars indicate the mean ± SEM. c , d Immunohistology of data in ( a ) and ( b ). Results of clinical delivery of MSCs ( e ) are shown with MRI cross-section of hearts from patients receiving standard of care or standard of care plus MSCs in the PROMETHEUS trial. 141 The MSC-treated hearts showed smaller infarcts at 12 and 18 months. The MSC-treated patients also had greater heart function (ejection fraction) and stamina (6 min walk test). 141 The error bars indicate the mean ± SEM. Figures reproduced with permission of Kluwers Wolter/Circulation Research/Hare. 141 , 145

Also, the MSCs’ powerful immunomodulatory effects reduce levels of inflammatory cytokines such as TNFα at the injury site. As described earlier, the MSCs act by modulating inflammatory T-, B-, and NK-cell subsets, either at the injury site or by trafficking to the spleen to reduce its inflammatory responses, and/or lodging in the lungs and releasing soluble TSG-6 (see Fig. 3 ). The immuno-privileged and immunomodulatory MSCs appear safe for allogeneic therapy in the heart and the potent immunomodulatory properties of MSCs have also led to their widespread testing in immunologic disorders ranging from multiple sclerosis to aging frailty. 149 , 150 Recently, several groups have become interested in repeat dosing regimens after cardiac infarct to bolster the effects of MSC therapy. 151 , 152 , 153 Despite the MSCs’ diverse secretory repertoire, it is also apparent that MSCs themselves render cell autonomous effects through hetero-cellular coupling mechanisms including connexins in tissues such as the heart and spleen. 153

Several exciting new approaches with MSCs are being tested to enhance therapeutic responses in cardiac damage. First, observations that MSCs interact with endogenous repair pathways led to the hypothesis that cell mixtures (i.e., MSCs plus cardiac stem cells (CSCs)) might enhance cardiac repair mechanisms (Fig. 4 ). This approach is borne out in animal models 146 , 147 , 151 and presently being tested in an NHLBI clinical trial. 154 Other strategies include seeding MSCs on tissue scaffolds to enhance their retention, and the repeat dosing regimen mentioned earlier. Together these approaches should enhance the effects of MSC therapy and hopefully be clinically relevant. While much work remains to understand the full mechanistic underpinnings and therapeutic potential of MSCs in the heart, the clinical testing to date provides an important aspect to cardiac medicine where living cells become a key therapeutic approach.

Optimizing MSCs for therapeutic purposes: tuning their output

As outlined above, MSCs can provide therapy for several clinical situations but the MSCs may exhibit different functional properties depending on how they are produced, handled and administered. The clinical benefits of MSC treatment involve the modulation of the immune system and the improved functionality of the damaged tissue. However, not all patients respond and the MSCs may provide potent effects in 40–50% of patients, meaning there is much more to understand about MSC therapy in patients. This may not be so different from the published clinical trial results from any cellular therapy under development but deserves careful investigation. The nonresponders among patients receiving MSCs appear to be a reflection of a combination of factors; the MSC production method for therapy, the cells’ metabolic activity, the delivered dose, the stage of the disease, and the status and/or genetic receptivity of the patient. 155 , 156 Given this complexity, it has become apparent to many researchers that the MSCs used for therapy must be carefully produced and properly “tuned” for the intended therapy. The tuned MSCs should be optimized for the required medicinal response and for the patients’ capacity to respond, as best this is understood. For example, currently, a single production method may be used to produce doses of MSCs administered for treatment of conditions as diverse as graft-versus-host disease, acute myocardial infarct or lung injury. Clearly, the same MSC production process is not optimized (i.e., tuned) to provide the “best” therapeutic benefit for very different clinical indications. Therefore, while the MSC field has often adopted a reliance on the MSCs “knowing what to do”, today a more sophisticated approach is needed to enhance the cell production, delivery and efficacy of MSC-based therapies.

Optimizing the production of MSCs for a particular medical indication should improve outcomes, and this may involve identifying the marrow donors whose culture-expanded MSCs exhibit an optimized response in a relevant assay that addresses the clinical situation, such as the aforementioned CLIP assay. 67 For a current example, the sponsors Case Western Reserve University, the National Center for Regenerative Medicine and University Hospitals Rainbow Babies and Children’s Hospital in their investigator-sponsored Phase I clinical trial to treat individuals with cystic fibrosis (CF) who have lung infections, have chosen MSC donors based on selected criteria. In this case, donor marrow aspirates were used to isolate and culture MSCs and then each donor’s MSCs were tested by exposure to Pseudomonas aeruginosa or Staphylococcu s bacteria in culture and the culture medium analyzed for the antibiotic protein LL37 and inflammatory mediators. From this test, one donor’s MSCs had a very high expression of these bioactive molecules compared to all the other donor MSCs. Thus, from the in vitro assay, an MSC donor was selected that exhibited an optimized response to the bacteria that are medically relevant for CF-patients with lung infections. The clinical trial is underway and should establish safety and, hopefully, some efficacy of these selected MSCs. Outcomes of such trials are critical to assess the nature and type of surrogate potency assays that may be needed to predict efficacy of MSC-based therapies in patients.

To prepare MSCs for a harsh in vivo environment, selected agents may be added to the in vitro MSC-media to sensitize and adapt the MSCs to the destructive microenvironment that they next encounter. For example, in the case of patients newly diagnosed with rheumatoid arthritis (RA), the production of MSCs for this therapy should expose cells to a strong inflammatory mediator like IL-1. MSCs exposed to IL-1 mount an anti-inflammatory response within 24−48 h that can be monitored by analyzing the culture medium and identifying the most effective donor MSCs to provide greater treatment efficacy. 157 Preconditioning the MSCs in culture to an inflammatory environment will optimize the MSCs to the microenvironment they will experience when infused into an RA patient. These approaches represent progress toward achieving therapies tailored for specific disease indications, and represent a form of “personalized medicine”, that could potentially result in cost−benefit returns and may accelerate the FDA approval process. An important scientific question is how to optimize the responsiveness of MSCs for greater therapeutic effects and clinical benefit. If the untuned MSCs provide potent effects in about half of all patients, perhaps tuned MSCs and selected patients will have much better outcomes and provide a rational basis for further improvements. In this regard, current production MSCs are also a fine starting point for further manipulation to enhance their multilineage potential to not only tune them but guide them towards desired cell types as seen in guided cardiopoiesis 17 or induction of hepatocyte function 158 by physical as well as chemical stimulation. Such optimization requires unique and collegial interactions between academics, industry, doctors, patients and administrators.

The MSC process is the MSC product

How can the MSC in vitro expansion process be refined to produce the best therapeutic MSCs in large amounts in a reproducible and reliable manner? For MSCs, the “process is the product” refers to the quality by design concept that all critical sources of variability are identified and explained, and the product quality attributes can be accurately and reliably predicted over the design space established for the materials used, the process parameters, the manufacturing environment and any other pertinent conditions. When the process is accurate and followed precisely, the product will be the same each time. If the product does not meet its release criteria, a review of the procedures is needed to understand what is happening at critical steps, and thereby seek continuous improvement. 159 This is where we are today. Listed in Table 2 are the topics and areas for improvement for MSC studies. With MSCs and their variable nature—a feature not a flaw—it is necessary to control each step in their cultivation for research and therapeutic use in order to have reproducible results. Critical steps along the cell production process should have an assay that can evaluate progress along the desired path. For the academic laboratory, assay reproducibility is essential, and the notion of continuous improvement should be embraced, while for the cell therapy facility and commercial producers this is a primary and legal responsibility. As discussed earlier, the MSC isolation, establishment in culture, and final expansion are complicated by the clonal expansion/extinction that needs further investigation. Still, we believe there are process steps that can be controlled and consistently met to achieve reproducible results across different laboratories and geographic locations that will result in reproducible MSC cell therapy outcomes. However, this is not an automatic endpoint from the current knowledge base and will require vigilance, persistent effort, and collegial communications.


We have discussed important aspects of MSCs and MSC-like cells based on our understanding of the field’s nearly three decades of development, the current state of adult stem/progenitor cell science, and cellular therapy technologies. The MSCs continue to undergo testing in a broad spectrum of clinical trials, some of which have strong support from animal model studies, but other areas too where the patient need is great and some MSC attributes suggest they would provide benefit. We have pointed out in Table 2 areas where further studies could advance the understanding and the utility of MSCs. The MSCs have properties not found in other stem/progenitor cells and these can be harnessed in many ways as the progress to date demonstrates. In retrospect, MSCs may have started as “a riddle wrapped in a mystery, inside an enigma” (W. Churchill on a separate topic during WWII), but years of research have shown that MSCs are a powerful cellular entity that interacts with their immediate surroundings and neighboring cells to provide cell-based responses that can be therapeutic. There remains much to be gained in terms of scientific knowledge and clinical benefit as the complex biology and therapeutic potential of MSCs are more fully understood.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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The authors thank Frances Verter of CellTrials.org and Jon Rowley of RoosterBio, LLC for discussion of MSC clinical trials. Portions of this work were supported by the National Institute of Biomedical Imaging and Bioengineering under award number P41 EB021911 to A.I.C.

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Mark F. Pittenger

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Dennis E. Discher

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Bruno M. Péault

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M.F.P. and A.I.C. conceived this manuscript. D.E.D., B.M.P., D.G.P., J.M.H., A.I.C. and M.F.P. contributed to the writing and final review of the manuscript.

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M.F.P. is a founder of Longevity Therapeutics, Inc. J.M.H. reported having a patent for cardiac cell-based therapy. He holds equity in Vestion Inc. and maintains a professional relationship with Vestion Inc. as a consultant and member of the Board of Directors and Scientific Advisory Board. J.M.H. is the Chief Scientific Officer, a compensated consultant and advisory board member for Longeveron and holds equity in Longeveron. He is also the co-inventor of intellectual property licensed to Longeveron. BMP is co-inventor of intellectual property on native MSCs for therapeutic use, held by the University of California, Los Angeles. The other authors declare no competing interests.

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Pittenger, M.F., Discher, D.E., Péault, B.M. et al. Mesenchymal stem cell perspective: cell biology to clinical progress. npj Regen Med 4 , 22 (2019). https://doi.org/10.1038/s41536-019-0083-6

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Review article, the therapeutic potential of mesenchymal stromal cells for regenerative medicine: current knowledge and future understandings.

research article on mesenchymal stem cells

In recent decades, research on the therapeutic potential of progenitor cells has advanced considerably. Among progenitor cells, mesenchymal stromal cells (MSCs) have attracted significant interest and have proven to be a promising tool for regenerative medicine. MSCs are isolated from various anatomical sites, including bone marrow, adipose tissue, and umbilical cord. Advances in separation, culture, and expansion techniques for MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine. During tissue healing, MSCs may exhibit several therapeutic functions to support the repair and regeneration of injured tissue. The process underlying these effects likely involves the migration and homing of MSCs, as well as their immunotropic functions. The direct differentiation of MSCs as a cell replacement therapeutic mechanism is discussed. The fate and behavior of MSCs are further regulated by their microenvironment, which may consequently influence their repair potential. A paracrine pathway based on the release of different messengers, including regulatory factors, chemokines, cytokines, growth factors, and nucleic acids that can be secreted or packaged into extracellular vesicles, is also implicated in the therapeutic properties of MSCs. In this review, we will discuss relevant outcomes regarding the properties and roles of MSCs during tissue repair and regeneration. We will critically examine the influence of the local microenvironment, especially immunological and inflammatory signals, as well as the mechanisms underlying these therapeutic effects. Importantly, we will describe the interactions of local progenitor and immune cells with MSCs and their modulation during tissue injury. We will also highlight the crucial role of paracrine pathways, including the role of extracellular vesicles, in this healing process. Moreover, we will discuss the therapeutic potential of MSCs and MSC-derived extracellular vesicles in the treatment of COVID-19 (coronavirus disease 2019) patients. Overall, this review will provide a better understanding of MSC-based therapies as a novel immunoregenerative strategy.


Graphical Abstract. The road map of MSC review.


Mesenchymal stromal cells (MSCs) are currently one of the most extensively investigated therapeutic cellular products for clinical applications. MSCs have several characteristics, such as homing to injured tissue sites, immunotropic functions, and paracrine signaling, which allow their use in various conditions, such as tissue regeneration or immunologic/inflammation-related disorders. MSCs were first discovered by Alexander Friedenstein in the late 1960s. They are self-renewable cells with a high ability to proliferate ( Bagno et al., 2018 ). Advances in the techniques for the separation, culture, and expansion of MSCs have enabled their large-scale therapeutic application. This progress accompanied by the rapid improvement of transplantation practices has enhanced the utilization of MSCs in regenerative medicine ( Han Y. et al., 2019 ). This review is organized as follows. Section “Origin and Characteristics of MSCs” discusses the origin and characteristics of MSCs. Section “Therapeutic Applications of MSCs” covers the main therapeutic applications and clinical uses of MSCs, including tissue repair and wound healing, immunomodulatory effects, and diverse therapeutic applications of MSCs. Section “Cellular and Molecular Therapeutic Mechanisms of MSCs” summarizes the cellular and molecular therapeutic mechanisms of MSCs starting from their pleiotropic effects, paracrine action, direct cell–cell contact, and finally mitochondrial transfer. Section “The Secretome of MSCs” presents MSC-derived extracellular vesicles (EVs) as a new therapeutic option and discusses how MSC-secreted EVs also carry several immunomodulatory, antiapoptotic, angiogenic, and antioxidative factors. Section “Outlook on MSCs and MSC-Derived EVs for the Treatment of COVID-19” provides an outlook on the potential therapeutic application of MSCs and MSC-derived EVs in the treatment of coronavirus disease 2019 (COVID-19) patients. Section “Conclusion” presents the conclusion of this review.

Origin and Characteristics of MSCs

Msc discovery.

Alexander Friedenstein originally identified colony-forming unit fibroblasts and osteogenic stem cells. Since this time, a number of terms have been used and proposed to describe MSCs. In 1988, Maureen Owen suggested using “stromal stem cells” to indicate that these cells reside in the stromal rather than the hematopoietic compartment ( Wilson et al., 2019 ). Rather than highlighting the cells’ compartmental origin, Arnold Caplan proposed the term “mesenchymal stem cells” in 1991 to emphasize the self-renewal property and differentiation potential of the cells. However, this nomenclature was challenged by James Dennis, who suggested that the cells may be progenitors rather than stem cells. As a result, the term “mesenchymal progenitor cells” was proposed. In 2000, Paolo Bianco and Pamela Gehron Robey coined “skeletal stem cells” to specify that the cells give rise to components of the skeletal system, while only 2 years later, the term “multipotent adult progenitor cells” (MAPCs) was proposed by Yuehua Jiang to describe the multipotent nature and potential progenitor status of the cells ( Caplan, 2017 ). As no direct evidence demonstrated the ability of MSCs to self-renew and differentiate in vivo , in 2006, the International Society for Cell and Gene Therapy (ISCT) proposed the term “multipotent mesenchymal stromal cells.” In 2010, Arnold Caplan suggested that the acronym “MSCs” should stand for “medicinal signaling cells” to reflect that the primary therapeutic benefit of MSCs may be attributed to the secretion of bioactive molecules rather than direct cell replacement ( Viswanathan et al., 2019 ). It has been suggested that all multipotent, clonal, and fibroblastoid cells that express MSC markers have a common primary origin, but they adopt different roles during embryogenesis ( Figure 1 ; Brown et al., 2019 ).


Figure 1. MSCs are isolated from several sources (neonatal, fetal, and adult tissues) and can “in theory” differentiate into different types of cells.

MSC Product Diversification

More than 50 years of research on MSCs has enabled their isolation from various tissues, such as adipose tissue ( Xia et al., 2018 ), skin, dental pulp, corneal limbus ( Shih and Burnouf, 2015 ), peripheral blood ( Tozetti et al., 2017 ), umbilical cord (UC) tissue ( Beeravolu et al., 2017 ), muscles ( Teng et al., 2017 ), lungs ( Pouya et al., 2018 ), menstrual blood, placental tissues ( Macrin et al., 2017 ; Teng et al., 2017 ; Aboushady et al., 2018 ), breast milk, and neonatal tissues ( Nakamura et al., 2015 ; Tozetti et al., 2017 ). Craniofacial MSCs have high differentiation capability and can rapidly proliferate. As they are easily extracted with minor pain during tooth extraction, craniofacial MSCs may represent another alternative for tissue regeneration, although their specific markers have not yet been well characterized ( Zhang et al., 2020 ).

Although the frequency of MSCs in blood from healthy individuals is extremely low, it may increase under challenging conditions, thus supporting the notion that MSCs can be transiently found “circulating” in blood ( Moll et al., 2020a , b ). While these diverse sources of MSCs may solve some issues linked to bone marrow (BM), they can display varying levels of highly procoagulant tissue factor and may adversely trigger the instant blood-mediated inflammatory reaction ( Witkowski et al., 2016 ). The former is considered a main trigger for coagulation, whereas the latter has been recognized as a critical threat to graft survival ( Moll et al., 2015 ; Shiratsuki et al., 2015 ; Christy et al., 2017 ; George et al., 2018 ). Moreover, new clinical standards are crucial to complement the minimal criteria for MSC product description ( Galipeau et al., 2016 ; Galipeau and Sensébé, 2018 ). In this context, Moll et al. (2019) proposed exploring new strategies for screening and monitoring hemocompatibility, as well as developing optimal delivery procedures to guarantee a safe and efficient therapeutic outcome.

Therefore, MSCs can be characterized by the following outstanding properties: (a) can be easily isolated from nearly any tissue, (b) can be differentiated into any cell lineage at its end stage, and (c) can make potential contributions to the management of disease because of their immunological properties ( Gao et al., 2016 ).

MSC Definition

In 2005, the ISCT determined the minimum benchmark criteria for defining in vitro human MSCs: (a) MSCs must be plastic-adherent and display fibroblastoid morphology while preserved in optimal culture conditions; (b) MSCs must present immunophenotypic expression of CD105, CD90, and CD73 and absence of expression of CD34, CD45, CD14, CD19, CD11b, CD79a, and HLA-DR surface indicators; and (c) MSCs must be at least capable of differentiating into osteoblasts, chondroblasts, and adipocytes in vitro ( Du et al., 2016 ; Brown et al., 2019 ). These standards aim to distinguish between mesenchymal stem cells and MSCs, which are not identical. Thus, in addition to their progenitor self-renewal and multilineage differentiation ability, MSCs must possess secretory, homing, and immunomodulatory characteristics ( Table 1 ). Although the basic phenotypic profile must be retained, the International Society for Cellular Therapy (ISCT) committee recommended in 2016 that the following topics be considered: (a) the specific characteristics of each MSC population according to their tissue origin must be determined; (b) the stemness of MSCs should be confirmed in vivo and in vitro ; and (c) robust assays must be implemented to specify the therapeutic action of MSCs ( Galipeau et al., 2016 ).


Table 1. Criteria to identify MSCs ( Dominici et al., 2006 ).

In the early 1970s, Dexter et al. found that BM-derived MSCs could sustain the growth and viability of hematopoietic cells with growth factor deficiency by secreting trophic factors and cytokines ( Han Y. et al., 2019 ). These findings resulted in significant attention placed on the use of MSCs to repair connective tissue wounds resulting from diseases or trauma. They also introduced the concept of possible regulatory effects on different sides of the immune response ( Spees et al., 2016 ). Despite the similar phenotypes of MSCs, they display heterogeneous biological and functional features. This heterogeneity is due to their different growth and proliferation abilities, multilineage diversity prospects, immunomodulatory potential, and proangiogenic characteristics ( Han et al., 2017 ). For example, higher proliferation rate and less immunogenicity have been reported for MSCs isolated from fetal tissues compared to those obtained from adult BM and adipose (A) tissues. In contrast, placental and BM-MSCs present better proangiogenic competences than MSCs isolated from A and UC tissues ( Du et al., 2016 ).

Although MSCs can be easily differentiated into several end-stage lineages, such as osteogenic, adipogenic, neurogenic, and chondrogenic lineages ( Brown et al., 2019 ; Han Y. et al., 2019 ), several reasons have hindered their therapeutic application. First, the procedure to obtain MSCs frequently causes pain and discomfort and can lead to donor morbidity. Second, while progressing to the in vitro stage, MSC differentiation capability is lessened. Third, the differentiation properties of MSCs are highly affected by environmental factors, such as age, stress, and genetic differences ( Mattiucci et al., 2018 ; Russell et al., 2018 ). These factors prompted the identification of other favorable sources of MSCs and led to their isolation from the UC and its blood, placenta, and fetal tissues ( Beeravolu et al., 2016 , 2017 ). Intravenous infusion is considered the most common route of delivery for various MSC products and has generated a mixed clinical outcome ( Ankrum et al., 2014 ; Galipeau and Sensébé, 2018 ). BM-MSC infusion proved to be the safest and exclusive source of MSC clinical products until 2008 according to the Food and Drug Administration ( Mendicino et al., 2014 ).

Therapeutic Applications of MSCs

The trophic and immunomodulatory properties of MSCs have made these cellular products one of the most promising and intensely pursued cellular therapies.

Tissue Trophic Effect of MSCs

Several properties have made MSCs appealing in the field of regenerative medicine ( Hu et al., 2018 ). Many studies have indicated the ability of MSCs to migrate, engraft, and functionally influence the repair process within the site of injury and damage ( Wang Y. et al., 2018 ; Shojaei et al., 2019 ). Following injury, anti-inflammatory activities are essential to offset injury, remove dead tissue, and facilitate migration and proliferation of reparative cell types, as well as to increase vascularization and nutrient supply ( Toh et al., 2018 ). In the presence of MSCs, the healing process is accelerated, and the inflammatory reaction is reduced ( Li Y. et al., 2019 ). According to Kim et al. (2019) , paracrine signaling and differentiation have both been linked to wound healing process. The potential application of MSCs in tissue repair can take three forms: (1) systemically administered stem cells migrate and home to the injured tissue due to chemical gradients; chemoattraction is mediated by a set of chemokines and their corresponding cell surface receptors; MSCs may migrate to tissues under the action of PDGF, SDF-1 (stromal-derived factor 1), CCL5, CCR2, and CCR3 ( Brown et al., 2019 ); the exact mechanism of stem cell–endothelial interactions at the target site is not well established; however, integrins and selectins facilitate these interactions ( Han et al., 2017 ); (2) differentiation and replacement, in which stem cells engraft and then differentiate into diverse cell types; and (3) the secretion of several factors that influence distinct physiological mechanisms locally and systematically ( Fu et al., 2019 ). It was shown that MSCs release cathelicidin peptide−18, which has an antibacterial effect by slowing down the growth of some bacteria, thus preventing wound infections that impair the healing process ( Park et al., 2018 ). Overall, MSCs promote a proregenerative microenvironment that promotes the tissue local repair and regeneration ( Hu et al., 2018 ). MSCs effectively participate in the tissue repair process through their immunomodulatory, trophic antibacterial, antifibrotic, and proangiogenic functions ( Huayllani et al., 2020 ). MSCs also play a central role during the wound-healing process by coordinating between local cells/progenitors, cytokines, chemokines, and extracellular matrix proteins ( Zahorec et al., 2015 , Merimi et al., 2021a ). Under specific conditions, BM-MSCs may directly or indirectly favor the generation and proliferation of local progenitors, such as endothelial cells and fibroblasts ( Hu and Li, 2018 ; Kucharzewski et al., 2019 ; Shojaei et al., 2019 ). The proliferation and functions of keratinocytes, endothelial cells, and fibroblasts are stimulated by molecules present in the secretome of MSCs ( Keshtkar et al., 2018 ). This secretome includes several molecules and cytokines involved in tissue regeneration and immunomodulation ( Fu et al., 2019 ; Huayllani et al., 2020 ). Several studies have found that the conditioned medium of MSCs (MSC-CM) enhances wound healing and increases the number of dermal fibroblasts and blood vessels and collagen density. MSC-CM enhances the migration and formation of fibroblasts and the presence of several important mediators of wound healing ( Pittenger et al., 2019 ). Several growth factors, such as vascular endothelial growth factor (VEGF) and epidermal growth factor, are released by MSCs, which elevates the recruitment of endogenous cells into the wound. MSCs also control several matrix metalloproteinases (MMPs), such as MMP-1 and MMP-9, which contribute to fibroblast regeneration ( Aboushady et al., 2018 ).

Immune-Modulating Effect of MSCs

In the context of wound management, MSCs have been acknowledged to have an immunomodulatory effect, which confers them the potential to promote wound repair and decrease inflammation ( Praveen Kumar et al., 2019 ). Because of their immunological features, MSCs play a major role during the tissue repair process ( Fu et al., 2019 ; Praveen Kumar et al., 2019 ). MSCs display strong immunomodulatory effects mainly mediated by cell–cell contact and secretion of several molecules ( Figure 2 ). These molecules comprise transforming growth factor β (TGF-β), prostaglandin E2 (PGE2), interleukin-10 (IL-10), human leukocyte antigen class I molecule (HLA)-G5, inducible nitric oxide synthase (NOS2), CD39, and CD73 molecules. These factors prevent the proliferation of several immune cells and the secretion of cytokines [IL-1, IL-6, IL-8, IL-12, tumor necrosis factor α (TNF-α), interferon-γ (IFN-γ), TNF-α] and chemokines (CCL2, CCL5) ( Vladimirovna et al., 2016 ; Jiang and Xu, 2020 ). MSCs inhibit the activation and proliferation of CD4 + and CD8 + T cells and decrease the production of immunoglobulin by B cells, which makes them appropriate for allogeneic transplantation ( Fan et al., 2020 ). Furthermore, it was demonstrated that MSCs inhibit the allogeneic T lymphocyte response, thus promoting the persistence of skin grafts ( Fan et al., 2020 ). According to Han Y. et al. (2019) , MSCs are unlikely to be detected by immune surveillance as they lack significant immune-stimulating antigens (decreased expression of HLA-DR, CD40, and CD86). Thus, they can be adopted in biomedical applications and tissue engineering where no graft rejection after transplantation takes place ( Han Y. et al., 2019 ). MSCs can modulate the function of lymphocytes and macrophages through PGE2 and IL-10 secretion ( Hu et al., 2018 ; Li Y. et al., 2019 ). On the one hand, PGE2 plays an important role in regulating the shift of T H 1 cells into T H 2 cells and thus reduces the activation and proliferation of proinflammatory lymphocytes within the injured tissue ( Du et al., 2016 ). On the other hand, IL-10 contributes to the inhibition of scar formation by preventing the accumulation of collagen I and III and the release of reactive oxygen species (ROS) into the wound area ( Honarpardaz et al., 2019 ). It was suggested that the suppression of allogeneic activated lymphocytes is accompanied by the enhancement of regulatory T (Treg) cells. The inhibition of peripheral monocytes and CD34 + progenitor cells from differentiating into antigen presenting cells (APCs), as well as the activation of the cytotoxicity of natural killer (NK) cells, leads to further anti-inflammatory effects. The modulation of the innate and adaptive immune response enables MSCs to suppress fibrosis progression ( Ti et al., 2016 ; Julier et al., 2017 ; Najar et al., 2019c ). Table 2 summarizes many surface markers, secreted proteins, immune-modulating factors, and microRNAs by which MSCs interact with other tissues and cells and may be induced under certain conditions ( Merimi et al., 2021b ). It has also been proven that chemokines and cytokines that are produced by MSCs contribute to the efficiency and effectiveness of autoimmune disease treatment ( Wu Y. et al., 2018 ).


Figure 2. The immunomodulatory effects of MSCs. Various secreted soluble factors (PGE2, TGF-β, HLA-G5, TSG-6, CCL2, IL-1Ra, and IL-10) can activate, suppress, differentiate, and proliferate different immune cell subgroups, including macrophages, mast cells, DC, NK cells, Treg cells, T cells, B cells, and neutrophils. Thus, MCSs will suppress the local inflammation after inhibiting the immune response.


Table 2. Markers, factors, and microRNAs to discriminate MSCs ( Lv et al., 2014 ; Camilleri et al., 2016 ; Oeller et al., 2018 ; Pittenger et al., 2019 ; Brinkhof et al., 2020 ; Jingqiu et al., 2021 ).

Upon examining the ability of adipose stem cells (ASCs) to regulate the T H 17 lymphocyte pathway, a thorough understanding of the biological correlation between T H 17 lymphocytes and ASCs considering both the cell ratio and the inflammatory environment must be considered ( Najar et al., 2019b ). Furthermore, it was suggested that the cell ratio and inflammatory primed BM-MSCs significantly affected the production of T H 17 lymphocytes ( Najar et al., 2019a ). Zhang Y. et al. (2017) suggested that galectin-1 inhibits the function of DCs by controlling the mitogen-activated protein kinase (MAPK) signaling pathway. MSCs can act either as a suppressor or an enhancer of the immune system by relying on the level of soluble factors in the microenvironment. In this context, Li et al. (2018) demonstrated that when inducible NOS is blocked, MSCs act as immune enhancers by stimulating T-cell proliferation. In contrast, Cuerquis et al. demonstrated that MSCs generate a temporary increase in IFN-γ and IL-2 levels by activating T cells before exerting an immunosuppressive effect ( Wang D. et al., 2018 ). Moreover, MSCs induced with IFN-γ suppressed T-cell proliferation by secreting indoleamine 2,3-dioxygenase (IDO), which catalyzes the conversion of tryptophan to kynurenine. The secretion of programmed death 1 ligand 1 (PD-L1) also contributes to the immunosuppressive effect and thus can be used in the treatment of autoimmune diseases ( Figure 3 ). As such, the microenvironment, especially soluble factor levels along with inflammatory levels, plays an important role in the application of MSC-based therapy ( Fan et al., 2020 ).


Figure 3. Fundamental mechanisms of MSC-based therapy. These mechanisms differ in their repair activity, depending on various local microenvironments where MSCs can adjust their therapeutic effects accordingly. The systemic administration of MSCs can activate distal (endocrine) or local (paracrine) effects that include cell-mediated actions, which can take different forms, including (1) stimulation of angiogenesis, (2) stem cell growth and differentiation, (3) fibrosis inhibition, (4) apoptosis inhibition, (5) T- and B-cell suppression, (6) initiation of Treg differentiation, (7) NK cell inhibition, and (8) dendritic cell (DC) maturation inhibition.

Clinical Indications and Considerations of MSCs

MSCs have been investigated, in both animal and human models, as a therapeutic product to manage various diseases ( Harris et al., 2018 ; Xu, 2018 ). MSCs are thus indicated for the treatment of degenerative disorders and diseases by displaying antioxidative, antiapoptotic, and immunomodulatory effects ( de Witte et al., 2018 ). Several studies have investigated the potential therapeutic applications of MSCs in Parkinson disease ( Hong et al., 2018 ), multiple sclerosis ( Harris et al., 2018 ; Nasri et al., 2018 ), degenerative disc disease ( Ahn et al., 2015 ; Beeravolu et al., 2017 ; Perez-Cruet et al., 2019 ), Alzheimer disease ( Cui et al., 2017 ; Han et al., 2018 ), myocardial infarction (MI) ( Selvasandran et al., 2018 ), retinal degenerative disease ( Zhang M. et al., 2017 ; Wang Y. et al., 2018 ), Crohn disease (CD) ( Jahanbazi Jahan-Abad et al., 2018 ; Brown et al., 2019 ), and type 1 diabetes mellitus ( Evangelista et al., 2018 ). Moreover, studies have shown that dental MSCs can be used as a complementary source for the regeneration of nerves and have the capability to treat several diseases, such as diabetes, bone deficiency, and neural disorders ( Dave and Tomar, 2018 ).

Several animal models have described a tissue repair capacity following the transplantation of MSCs. In a rat model, BM-MSCs released several mediators, such as fibroblast growth factor 2 (FGF-2), VEGF-1, angiopoietin-2, and TGF-β, which contributed to the healing of MI ( Selvasandran et al., 2018 ). In a mouse model of burn injury, high levels of VEGF and TGF-β1 were suggested to assist burn wound healing by MSCs ( Oh et al., 2018 ). In a rat model, MSCs enhanced fibroblast and keratinocyte differentiation, leading to accelerated wound healing ( Xia et al., 2018 ; Kim et al., 2019 ). Mouse model of hind limb ischemia revealed that a subset of paracrine factors are efficient biomarkers for predicting vascular regenerative efficacy by Wharton’s jelly-derived MSCs ( Kim et al., 2019 ). In rat periodontal defect model, the implantation of MSC-CM promoted periodontal regeneration by enhancing the mobilization and osteogenesis of local periodontal ligament cells ( Kawai et al., 2015 ). Interestingly, conditioned media (mixed with cosmetic base) from human UC blood-derived MSCs (USC-CM) increased dermal density and decreased skin wrinkle during in vivo test with 22 women volunteers ( Kim et al., 2018 ).

The number of registered clinical studies for MSC therapies has exceeded 1,000 worldwide ( Moll et al., 2019 ; Pittenger et al., 2019 ). Although a meta-analysis of MSC clinical trials has confirmed their safety, the therapeutic efficiency (including the mechanisms of action) of such cellular products formulations should be more scrutinized ( Ankrum et al., 2014 ; Martin et al., 2019 ). Of all clinical trials using MSCs, the main indications are musculoskeletal diseases with 203 registered studies, 146 trials for central nervous system diseases, 146 trials for immune system diseases, 139 for wounds and injuries, 130 for collagen diseases, 130 for rheumatic diseases, 128 for joint diseases, 127 for arthritis, 127 for vascular diseases, 123 for ischemia, 118 for respiratory tract diseases, 112 for digestive system diseases, and 112 for gastrointestinal diseases. There are 10 globally approved MSC therapies including Alofisel for CD (approved in Europe); Prochymal for GvHD (approved in Canada and New Zealand); Temcell HS injection for graft-vs.-host disease (approved in Japan); Queencell for subcutaneous tissue defects, Cupistem for Crohn fistula, Neuronata-R for amyotrophic lateral sclerosis and Cartistem for knee articular cartilage defects (all approved in South Korea); Stemirac for spinal cord injury (approved in Japan); Stempeucel for critical limb ischemia (approved in India); and Cellgram-AMI for acute MI (approved in South Korea). One of the rare clinical trials in phase III involves the use of allogeneic adipose tissue–derived MSCs for complex perianal fistulas in CD (clinical trial no. NCT01541579). The TiGenix/Takeda phase 3 clinical trial that studied the use of MSCs for complex perianal fistulas in CD is arguably the most successful late-stage MSC trial to date (NCT01541579). Results of this study indicated an effective and safe treatment for perianal fistulas in patients with CD ( Panés et al., 2016 ). Another clinical trial using Alofisel under NCT03706456 is also being actively evaluated for CD management.

Although meta-analysis of clinical trials with first-generation MSC products has demonstrated their safety, their clinical efficiency still needs to be improved. A better understanding of the underlying mechanism of action of MSCs as well as potency assessments pretreatment and posttreatment is key to yield an optimal short- and long-term therapeutic benefit. Therefore, a thorough understanding of patient parameters and complementary treatment protocols are crucial in determining the optimal therapeutic pharmacokinetics ( Galipeau and Sensébé, 2018 ; Aijaz et al., 2019 ; Hoogduijn and Lombardo, 2019 ). Efforts should be also developed to improve product design, dosing, and delivery to reach individual clinical needs of patients ( Moll et al., 2020b ).

Functionally Improved MSCs by Using Scaffolds

Although stem cells show considerable promise in regenerative medicine, low cell engraftment and survival of the transplanted cells within the target tissue remain key limitation to the successful application of cell-based therapy in the clinic. Indeed, local injection is often associated with poor cell survival and low engraftment due to the harsh and hostile environment at the site of damaged tissue. To ameliorate cell viability/survival and engraftment after injection, stem cells can be combined with biomaterial scaffolds. One of the most widely used biomaterials for the fabrication of scaffolds is hyaluronic acid (HA). HA is the major component of the extracellular matrix of connective tissues ( Fraser et al., 1997 ). It is also abundantly present in UC and synovial and vitreous fluids ( Gupta et al., 2019 ). HA hydrogels can be designed as cell-free therapies through stimulating natural healing processes through the recruitment of endogenous cells ( Highley et al., 2016 ). The combination of HA-based scaffolds and stem cells has been extensively used in cartilage repair. Chung et al. (2014) demonstrated that treatment with a composite of HA and human UC blood-derived mesenchymal stem cells (hUCB-MSCs) led to a superior degree of cartilage regeneration in rat, rabbit ( Park et al., 2017 ), and minipig ( Ha et al., 2015 ) models of disease. Intra-articular injection of a combination of HA and adipose-derived MSCs in a sheep osteoarthritis (OA) model has efficiently blocked OA progression and promoted cartilage regeneration ( Feng et al., 2018 ). Using adult minipig with cartilage defect, the intra-articular injection of MSCs from iliac crest marrow suspended in HA has shown improved cartilage healing both histologically and morphologically ( Lee et al., 2007 ).

Similarly, coadministration of BM-MSCs and HA produced higher regenerative benefit in small and large models of OA, including dogs ( Li et al., 2018 ), and the Hartley guinea pig model of naturally occurring OA ( Sato et al., 2012 ). The combination of HA and stem cells has also been investigated in different models of osteogenesis. The applicability of adipose-derived MSCs and HA showed higher means of bone regeneration in rat model of bone defects ( Boeckel et al., 2019 ). The combination of BM-MSCs and HA successfully indicated bone regeneration in rat calvarial defect model ( Kim et al., 2007 ).

The effect of HA on the therapeutic efficiency of MSCs was further studied in wound healing. Cerqueira et al. (2014) showed that adipose (AD)-MSCs encapsulated within an HA–base hydrogel demonstrated accelerated wound closure, higher re-epithelialization, and neovascularization in a model of skin full-thickness excisional wounds in mice. Comparable results were reported in a separate study using a mouse model of full-thickness (skin) excision wounds in streptozotocin-induced diabetes ( da Silva et al., 2017 ). The results from a clinical trial for safety and proof of concept indicated cartilage regeneration in osteoarthritic patients following the use of a composite of hUCB-MSC. Recently, scaffolds and exosomes from mice BM-MSCs were developed as a combinatorial cell-free system to initiate synergistic tissue immunotrophic effects. Indeed, exosome-laden scaffolds (fibrous polyester materials) proactively facilitated tissue repair in mice skin injury models by favoring M2/T H 2/Treg responses ( Su et al., 2021 ). Together, all these findings indicate that combined HA and MSCs may constitute an effective strategy in regenerative medicine.

Cellular and Molecular Therapeutic Mechanisms of MSCs

Two main facets exemplify the therapeutic capabilities of MSCs: the replacement of injured tissue and immunomodulatory activity. The main core mechanism underlying MSC therapy is the pleiotropic effect. This effect allows the release of various soluble factors that display immunomodulatory, antiapoptotic, angiogenic, and antioxidant activities ( Figure 3 ; Fan et al., 2020 ). The immunosuppressive effect and cell sustainability are regulated by MSCs through cell–cell contact and transfer of mitochondria by tunneling nanotubes (TNTs) to targeted cells ( Li H. et al., 2019 ). Moreover, an anti-inflammatory effect was noted through the release of exosomes, which include numerous microRNAs that enhance cell proliferation throughout tissue regeneration ( Huayllani et al., 2020 ).

Pleiotropic Therapeutic Effects of MSCs

MSCs play an important role in tissue repair and offer numerous therapeutic applications due to their pleiotropic effects ( Hmadcha et al., 2020 ). Anti-inflammatory and immunoregulatory activities are considered the major pleiotropic contributors to the therapeutic potential of MSCs. Responding to inflammation, MSCs secrete soluble factors, such as TGF-β, TNF-α, IFN-γ, IL-10, and IDO, which alter the inflammatory environment and obstruct the immune system ( Kaundal et al., 2018 ). It was demonstrated that this alteration of immune action triggers a crucial inflammatory mechanism that considerably enhances tissue repair and regeneration by expediting healing and fibrosis ( Julier et al., 2017 ). These pleiotropic effects are also suggested to confer protumor activity to cells. For example, several pivotal studies have shown that MSCs can prevent apoptosis in carcinogenic cells through the release of VEGF and FGF, which are considered soluble prosurvival factors. Numerous studies have agreed on the immunosuppressive effect of MSCs through the secretion of inflammatory factors ( Hmadcha et al., 2020 ). Although MSCs are widely recommended in cell and tissue repair, the engraftment process into the target injured tissue might be influenced by several factors ( Lin et al., 2017 ; Liu et al., 2017 ). One of these main chemical growth factors is hepatocyte growth factor (HGF), which is a pleiotropic factor that is derived from MSCs. The pleiotropic effect is mediated through enhancing the motility, propagation, and sustainability of cells ( Fu et al., 2019 ). In vitro , trafficking of MSCs was linked to significant c-met expression in the presence of HGF concentration gradients. The rat MSC migration process was enhanced through stimulation of the Akt and focal adhesion kinase (FAK) pathways due to the HGF pleiotropic factor ( Zhu et al., 2016 ).

The pleiotropic effect was also mediated by the Abi3bp protein, which acts as an autocrine modulator by significantly enhancing the differentiation of cardiac c-Kit + progenitors ( Mori et al., 2018 ). Moreover, several paracrine factors, such as VEGF, insulin-like growth factor (IGF-1), and FGF, also have pleiotropic characteristics that contribute to the treatment of myocardial injury through different mechanisms. They can affect post–myocardial injury processes such as fibrosis, inflammation, the formation of cardiomyocytes, and neovascularization ( Hodgkinson et al., 2016 ). It has been suggested that directly after MI, the anti-inflammatory reaction is activated through the overexpression of IL-6, which adjusts the paracrine activity of MSCs through the release of VEGF, which enhances the vascularization process. In addition, numerous cytokines, such as IL-1, TNF-α, and IFN-γ, have shown the same inflammatory response as IL-6 through the release of various growth factors that contribute to the regeneration of the myocardium through new capillary formation, cardiomyocyte propagation, and the differentiation of progenitor cells ( Bagno et al., 2018 ). In line with this observation, it was reported that paracrine factors exert pleiotropic actions on repair and regeneration processes through two distinct mechanisms ( Hodgkinson et al., 2016 ). Frizzled-related protein 2 (SFRP2) and hypoxia- and Akt-induced stem cell factor (HASF) are two major paracrine factors that play important roles in cardiac injury by enhancing cardiomyocyte proliferation. While Sfrp2 is linked to the proapoptotic protein Wnt3a in their protective effect, HASF inhibits the death of cardiomyocytes via ε isoform of protein kinase C (PKCε). In addition to their cytoprotective role, SFRP2 inhibits Bmp1 and Sca-1 CPC proliferation, limits fibrosis, and promotes cell differentiation. It was shown that the differentiation process enhanced non-canonical Wnt/planar cell polarity signaling via JNK after Sfrp2 attachment to Wnt6 ( Schmeckpeper et al., 2015 ).

In studying the potential strategies to enhance the therapeutic function of transplanted MSCs in the treatment of damaging neonatal disorders, it was found that the pleiotropic effects are related to paracrine activity and not to regenerative ability. MSCs can detect the microenvironment of the injured area and release various paracrine soluble factors that conduct numerous functions (such as anti-inflammatory, antiapoptotic, antifibrotic, antibacterial, and antioxidant effects) to promote the regeneration and repair of the injured tissue. As such, the efficiency of MSC therapeutic application relies on pleiotropic protection under proper MSC sources, microenvironments, and pharmacokinetics ( Park et al., 2018 ).

Paracrine Action of MSCs

Although MSCs are widely recommended for cell and tissue repair, the engraftment process into the target injured tissue might be influenced by several factors ( Lin et al., 2017 ; Liu et al., 2017 ). One of these main chemical growth factors is HGF, which is a pleiotropic factor that is derived from MSCs. The pleiotropic effect is mediated through enhancing the motility, propagation, and sustainability of cells ( Fu et al., 2019 ). In vitro , trafficking of MSCs was linked to significant c-met expression in the presence of HGF concentration gradients. The rat MSC migration process was enhanced through stimulation of the Akt and FAK pathways due to the pleiotropic factor HGF (A. Zhu et al., 2016 ). While investigating the impact of MSCs on myocardial injury, it was reported that paracrine factors exert pleiotropic actions on repair and regeneration processes ( Hodgkinson et al., 2016 ). SFRP2 and HASF are two major paracrine factors acting through two distinct mechanisms that play important roles beyond only a protective one in the case of cardiac injury by enhancing cardiomyocyte proliferation. While Sfrp2 is linked to the proapoptotic protein Wnt3a in their protective effect, HASF inhibits the death of cardiomyocytes via PKCε. In addition to its cytoprotective role, SFRP2 inhibits Bmp1 and Sca-1 CPC proliferation, limits fibrosis, and promotes cell differentiation. It was shown that the differentiation process enhanced non-canonical Wnt/planar cell polarity signaling via JNK after Sfrp2 attachment to Wnt6 ( Schmeckpeper et al., 2015 ).

The pleiotropic effect was also mediated by the Abi3bp protein, which acts as an autocrine modulator by significantly enhancing the differentiation of cardiac c-Kit + progenitors ( Mori et al., 2018 ). Moreover, several paracrine factors, such as VEGF, IGF-1, and FGF, also have pleiotropic characteristics that contribute to the treatment of myocardial injury through different mechanisms. They can affect post–myocardial injury processes such as fibrosis, inflammation, the formation of cardiomyocytes, and neovascularization ( Hodgkinson et al., 2016 ). It has been suggested that directly after MI, the anti-inflammatory reaction is activated through the overexpression of IL-6, which adjusts the paracrine activity of MSCs through the release of VEGF, which enhances the vascularization process. In addition, numerous cytokines, such as IL-1, TNF-α, and IFN-γ, have exhibited the same inflammatory response as IL-6 through the release of various growth factors that contribute to the regeneration of the myocardium through new capillary formation, cardiomyocyte propagation, and the differentiation of progenitor cells ( Bagno et al., 2018 ).

MSCs play an important role in tissue repair and offer numerous therapeutic applications due to their pleiotropic effects ( Hmadcha et al., 2020 ). Their anti-inflammatory and immunoregulatory activities are considered the major pleiotropic contributors to the therapeutic potential of MSCs. Responding to inflammation, MSCs secrete soluble factors such as TGF-β, TNF-α, IFN-γ, IL-10, and IDO, which alter the inflammatory environment and obstruct the immune system ( Kaundal et al., 2018 ). It was demonstrated that this alteration of immune action triggers a crucial inflammatory mechanism that considerably enhances tissue repair and regeneration by expediting healing and fibrosis ( Julier et al., 2017 ). These pleiotropic effects are also suggested to confer protumor activity to cells. For example, several pivotal studies have shown that MSCs can prevent apoptosis in carcinogenic cells through the release of VEGF and FGF, which are considered soluble prosurvival factors. Numerous studies have agreed on the immunosuppressive effect of MSCs through the secretion of inflammatory factors ( Hmadcha et al., 2020 ). In studying potential strategies to enhance the therapeutic function of transplanted MSCs during the treatment of damaging neonatal disorders, it was found that pleiotropic effects are related to paracrine activity and not to regenerative ability. MSCs are able to detect the microenvironment of the injured area and release various paracrine soluble factors that conduct numerous functions to promote the regeneration and repair of the injured tissue, such as anti-inflammatory, antiapoptotic, antifibrotic, antibacterial, and antioxidant effects. As such, the efficiency of MSC therapeutic application relies on pleiotropic protection under proper MSC sources, microenvironments, and pharmacokinetics ( Park et al., 2018 ).

Direct Cell–Cell Contact

The immunomodulatory effects of MSCs that are applied on the injured sites are either exerted through paracrine mechanisms or via direct cell–cell contact. The cell–cell contact mechanism is crucial for MSCs to stimulate Treg cells and can be adopted for allergic diseases. Furthermore, the immunomodulatory impact of MSCs on T cells and macrophages can be magnified by TSG-6 release through direct cell–cell contact in a proinflammatory environment. Moreover, it was proven that this direct contact decreases the cytotoxicity of NK cells ( Gavin et al., 2019 ). In the context of bone engrafting and cell-based therapeutic applications, MSCs have been differentiated into phenotypes that are similar to pericytes, which promote angiogenesis through direct cell–cell contact ( Julier et al., 2017 ). The interaction with target cells has proven to be one of the key mechanisms in MSC-based therapy. MSCs exert their immunomodulatory effects by promoting Treg cells, inhibiting T cells, and regulating macrophages for numerous inflammatory diseases ( Carty et al., 2017 ). It was proven that T cells are regulated by MSCs through the Fas ligand–Fas relation, B7-H4 molecule, or PD-L1 pathways ( Consentius et al., 2015 ). PD-1 ligand expression, which is present on the MSC membrane, is important for inhibiting the differentiation of allogeneic T H 17 cells, which depends on direct cell–cell contact. In addition, the inhibition of CD4 + and CD8 + T-cell propagation occurs via galectin-1 and 3 ( Li Y. et al., 2019 ). A synergy was found between MSCs and Treg cells, where Treg cells promote the release of IDO by MSCs, which in turn inhibits TNF-α and promotes IL-10 in Treg cells. The relationship between MSCs and macrophages cannot be summarized as a simple anti-inflammatory relationship. After direct cell–cell contact, macrophages can phagocytose MSCs and modify their signature to an M2 suppressive phenotype, which clarifies the long-lasting MSC therapeutic effect ( Braza et al., 2016 ). Intriguingly, in some models and under specific conditions, it appears that dead or dying cells or subcellular particles derived from MSCs may contribute to their therapeutic properties. Understanding the necrobiology of MSCs during their therapeutic functions is essential to promote their efficiency and safety ( Weiss et al., 2019 ). Infused MSCs are rapidly phagocytosed by monocytes, which subsequently migrate from the lungs to other body sites. Phagocytosis of MSCs induces phenotypical and functional changes in monocytes, which subsequently modulate cells of the adaptive immune system ( de Witte et al., 2018 ). More specifically, phagocytic clearance of apoptotic MSCs (efferocytosis) by phagocytes is a crucial step in MSC immunosuppression. Efferocytosis could affect the polarization of macrophages and promote M2 anti-inflammatory and regulatory phenotype and function. Such observation may explain how short-lived MSCs mediate therapeutic effects that persist beyond their survival in vivo ( Ghahremani Piraghaj et al., 2018 ). This theory is supported by the observation that transfusion of MSCs leads to the prompt phagocytosis of nearly half of lung entrapped MSCs by lung resident macrophages, triggering an IL-10–suppressive efferocytotic response ( Galipeau, 2021 ).

Mitochondrial Transfer

Mitochondrial transfer has been proposed as one of the original approaches used to restore the respiratory function of injured cells and thus can be adopted in regenerative medicine. This mitochondrial transfer can take different forms, such as microvesicles (MVs), TNTs, gap junctions, and cell fusion mitochondrial transfer ( Babenko et al., 2018 ; Jiang et al., 2019 ). Mitochondrial transfer from MSCs exerts a protective outcome in the lung, kidney, cornea, bronchoepithelium, and spinal cord ( Jiang et al., 2019 ; Li H. et al., 2019 ).

The Secretome of MSCs

Despite being a powerful tool for clinical applications, MSCs have limitations in terms of delivery, safety, and variability of the therapeutic response. Interestingly, the secretome of MSCs was identified as a potential alternative to the cellular product. The secretome is mainly composed of cytokines, chemokines, growth factors, regulatory proteins, and EVs ( Eleuteri and Fierabracci, 2019 ). Despite the similarity in their origin, the secretome of MSCs appears to vary significantly, depending on the age of the donor and tissue sources from which they were isolated. Understanding and profiling the secretome of MSCs will enable the use of the secretome as a new cell-free therapeutic option ( Praveen Kumar et al., 2019 ).

Extracellular Vesicles

MSC-derived EVs are promising candidates for cell-based and cell-free regenerative medicine, respectively. It has been reported that MSC-derived EVs may be therapeutically more efficient and safer than their cell of origin. EVs have shown stability in circulation, good biocompatibility, and low toxicity and immunogenicity ( Shi et al., 2021 ). These EVs could support the dynamic immunomodulatory activities during tissue repair and regeneration. EVs are likely carriers of lipid, protein, growth factor, cytokines, chemokines, and nucleic acid. They were identified as components of the MSC secretome and propagated the key regenerative and immunoregulatory characteristics of parental MSCs ( Wang and Thomsen, 2021 ). EVs are signaling vehicles in intercellular communication in normal or pathological conditions. EVs convey their functional contents to adjacent cells or distant cells through the circulatory system ( Toh et al., 2018 ). Thus, MSC-derived EVs demonstrate promising cell-free therapy application potential to cure several diseases after monitoring their isolation, dosage, and storage ( Zhao et al., 2020 ). Despite the substantial increase in the number of publications concerning the pathological and physiological properties of EVs, it is still difficult to purify a specific EV population. Such preparations may include heterogeneous exosomes, MVs, microparticles, ectosomes, oncosomes, and other membranous cell–released structures. In view of this, the International Society for Extracellular Vesicles (ISEV) suggested Minimal Information for Studies of Extracellular Vesicles in 2014 (MISEV2014). New guidelines were published in 2018 by the ISEV, which recommends the use of a collective term of EVs unless the biogenesis pathway is demonstrated ( Théry et al., 2018 ). The main objective of MISEV2018 is to develop and improve the EV preparation field; thus, it offers guidelines for proposed protocols to verify specific EV functional activities. Later on, members of four societies (SOCRATES, ISEV, ISCT, and the International Society of Blood Transfusion) proposed to develop new reliable metrics that harmonize the evaluation of the MSC-EV biology and their therapeutic potency. For each EV preparation, the determination of their cell-origin, size, degree of physical and biochemical integrity, composition, and use of a well-characterized MSC-EV biological reference should be performed to guarantee quality and reproducibility ( Witwer et al., 2019 ).

EVs are secreted by numerous cells, including MSCs, where the most important ones are exosomes and MVs. These EVs are crucial in the communication process between cells, where they contribute to both pathological and physiological environments ( Konala et al., 2016 ). Membrane-bound EVs are secreted by somatic cells and contribute to tissue repair, reproduction, and immunomodulatory functions ( Lai et al., 2016 ; Dostert et al., 2017 ). The main EV markers are CD9, CD44, CD63, CD73, CD80, CD90, and CD105 proteins and antigens; heat-shock protein 60, 70 and 90; and ALG-2–interacting protein X ( Li et al., 2018 ). Microvesicles are produced by various cells through cell membrane budding, which includes cytoskeletal restructuring and depends on the concentration of intercellular calcium ( Konala et al., 2016 ). MVs consist of large quantities of phosphatidylserine proteins, sphingomyelin, ceramide, cholesterol, and CD40 markers. Thus, they contain a load of microRNAs, proteins, and lipids where they bind through receptor–ligand interactions. MVs may either facilitate genetic transmission to the targeted cells, or they may boost angiogenesis by transferring growth factors that will alter the physiological function of the target cell ( Merino-González et al., 2016 ).

It has been recently identified that MVs are the main contributors to tissue regeneration, acting by utilizing biological activity and transmitting information to injured cells ( Rani et al., 2015 ). However, it was recently suggested that MSC exosomes isolated from BM stimulate numerous signaling pathways, mainly STAT3 expression, which participates in its phosphorylation and in the formation of keloid fibroblasts and elevates the expression of growth factors that are mainly related to wound healing, such as IL-8 and C-X-C motif chemokine ligand 1 (CXCL1), nerve growth factor, HGF, IGF-1, and SDF-1 ( Shabbir et al., 2015 ). In the same context, it was demonstrated that STAT3 phosphorylation inhibition reduces the production of collagen in keloid scars. It has been shown that the secretion of exosomes at the wound site plays an immunomodulatory role by preparing a favorable microenvironment through the transfer of microRNAs ( Fang et al., 2016 ; Ti et al., 2016 ). These exosomal miRNAs inhibit TGF-β2/Smad2 signaling and lessen the development of scars by suppressing myofibroblast construction throughout the wound healing process ( Fang et al., 2016 ).

Recently, MSC-derived EVs have been investigated in numerous clinical applications for their therapeutic potential ( Akyurekli et al., 2015 ). The efficiency of EVs isolated from MSCs efficiency has been associated with their role as antiapoptotic and tubular cell proliferation enhancers in the treatment of acute kidney disease. MSC-derived EVs are involved in the treatment of various neurological diseases, such as Alzheimer disease and multiple sclerosis ( Clark et al., 2019 ; Reza-Zaldivar et al., 2019 ), by inhibiting the degradation and demyelination of oligodendroglia, which results in motor function progression ( Reza-Zaldivar et al., 2019 ). Moreover, it was demonstrated that MSC-derived EVs have the potential to lessen MI by enhancing angiogenesis, inhibiting apoptosis, supporting proliferation, and regulating the microenvironment. In the context of cartilage repair, MSC-derived EVs have been examined for chondrocyte survival by stimulating matrix formation, preventing apoptosis, and immunomodulatory reactions ( Zhang et al., 2018 ).

EVs that are extracted from MSCs alter the immune system by stimulating Treg cells and the secretion of anti-inflammatory cytokines, controlling macrophages, reducing B lymphocytes, and recruiting neutrophils ( Dostert et al., 2017 ). On the one hand, exosomes enhance the production of monocytes, which differentiate into macrophages through MYD88 (myeloid differentiation gene 88). These macrophages enhance the release of IL-10, which leads to the growth of Treg cells. On the other hand, it was found that macrophage polarization is boosted by miR-146a, turning them to anti-inflammatory ones ( Song et al., 2017 ). Furthermore, the immunosuppressive impact of EVs on B, T, and NK cells, which is facilitated by PD-L1 expression, has been investigated. In addition, galectin-1 and 5′-ectonucleotidase (CD73) exert immunosuppressive effects on T lymphocytes and the production of adenosine, respectively ( Del Fattore et al., 2015 ; Kerkelä et al., 2016 ). Moreover, miR-16 and miR-100 have been detected and found to exert an antiangiogenic effect in breast cancer by encountering VEGF cells ( Pakravan et al., 2017 ).

In addition, MSC exosomes isolated from the UC have revealed their suppressive function in myofibroblast creation by deterring the TGF-β/SMAD2 pathway and enhancing the presence of some microRNAs, such as miR-21, miR-23a, miR-125b, and miR-145. Consequently, it has been shown that UC exosomes lessen the accumulation of myofibroblasts and scar development ( Fang et al., 2016 ). Moreover, these exosomes have demonstrated an improvement in the re-epithelialization process and cytokeratin 19 and collagen I expression, which contribute to the rejuvenation of skin burns ( Zhang et al., 2015a ). EVs have an exclusive ability to cross the blood–brain barrier, which contributes to some neurological disorder treatments. This feature is considered superior to traditional MSC-based therapies, which may face some limitations, such as incomplete cell differentiation, immune rejection, malignant alteration, and genetic mutation accompanied by cell transplantation in the treatment of neurological disorders ( Li et al., 2018 ). As such, EVs are considered excellent candidates in regenerative medicine ( Fan et al., 2020 ). Moreover, exosomes that are extracted from MSCs have demonstrated enhanced muscle regeneration by fostering myogenesis, as well as angiogenesis ( Nakamura et al., 2015 ). Several MSC-exosomal microRNAs (miR-19a, miR-22, miR-223) have shown antiapoptotic effects and cardioprotective activity by targeting methyl CpG binding protein 2, transcription 3 (Stat3), and (Mecp2) semaphorin-3A (Sema3A). MSC exosomes can also contribute to renal cell prolongation and growth by enhancing proximal tubular cell sensitivity to IGF-1 by transferring mRNA for the IGF-1 receptor ( Zhang et al., 2015a ; Song et al., 2017 ). Finally, MSC-EVs exhibited mixed results in the context of tumor cells. They can act as suppressors or promoters for these cells, depending on their isolation source, stage and type of tumor, and genotype ( Lopatina et al., 2016 ; Whiteside, 2018 ). As such, EV-based therapy must be cautiously assessed in the treatment of cancer ( Fan et al., 2020 ).

Antiapoptotic Factors

The inhibition of apoptosis and enhancement of homeostasis can be mediated through the secretion of BCL-2 by MSCs. The elevation of BCL-2 to BAX levels will lead to a decrease in the pathological sensitivity of cells. Moreover, MSCs can produce and release VEGF, HGF, FGF, survivin, IGF-I, stanniocalcin-1 (STC1), and TGF-β, which play similar roles ( Ono et al., 2015 ). In the same context, Zhang et al. indicated that phosphoinositide-3-kinase (PI3K)/Akt contributes to the BCL-2 signaling pathway in terms of antiapoptotic function, thus enabling MSCs to be used in the treatment of ischemia ( Zhang Y. et al., 2019 ). Antiapoptosis activity due to paracrine function under ischemic conditions was exhibited by MSC-conditioned microenvironment where BAX, FAS, TNF receptor, and CASP3 levels are downregulated ( Park et al., 2018 ). In addition to the direct antiapoptotic effect, soluble factors that are secreted and elevated by MSCs, such as VEGF, HGF, FGF, and IGF-I, under hypoxia have been proven to boost cell survival. In particular, VEGF has been proven to upregulate BCL-2 expression, which leads to vascular endothelial cell antiapoptosis, and to stimulate the activating phosphorylation of FAK, which inhibits p53-mediated apoptosis. Therefore, these soluble factors are crucial for cell survival (A. Zhu et al., 2016 ).

Angiogenic Factors

Angiogenesis mediates the generation of a new blood vessel network through a complicated process associated with several growth factors, such as HGF, VEGF, and FGF. Numerous studies have demonstrated the ability of MSCs to intensify capillary and blood vessel formation ( Merino-González et al., 2016 ). It has been verified that MSCs exert angiogenic effects that contribute mainly to the regeneration of injured skin, MI, and the treatment of ischemia ( Chen et al., 2015 ; Zhang et al., 2015b ). Hung et al. (2007) showed that angiogenesis is stimulated by soluble factors such as monocyte chemotactic protein 1 (MCP-1), IL-6, and VEGF. While MCP-1 is a vital chemoattractant, IL-6 enhances angiogenesis and contributes to the persistence of endothelial cells ( Hung et al., 2007 ). VEGF plays an important role in mediation, migration, and differentiation of endothelial cells through the stimulation of MAPK, PI3K/AKT, and other pathways ( Zhu et al., 2018 ). Moreover, MSCs can enhance angiogenesis through multiple factors, such as SDF-1 and HGF, which facilitate MI repair via SDF-1/C-X-C chemokine receptor type 4 (CXCR4). Additionally, soluble factors in MSCs can be used in the treatment of ischemia because of their angiogenic effects ( Zhang Y. et al., 2019 ).

Antioxidative Factors

It was demonstrated that there is a significant correlation between ROS levels and chronic diseases such as cancer, immune disorders, and neurological diseases ( Kreuz and Fischle, 2016 ). MSCs, through the secretion of STC1, can decrease apoptosis induced by ROS and regulate oxidation reduction. STC1 inhibits angiotensin II–enhanced superoxide formation in cardiomyocytes and stimulates uncoupling proteins 2 and 3 (UCP2 and UCP3), which promote mitochondrial respiration and alveolar epithelial cell persistence ( Ono et al., 2015 ). Moreover, it was shown that STC1 suppresses the NLRP3 inflammasome, which lowers the release of mitochondrial ROS. Furthermore, Chen et al. (2010) verified that HO-1 enhances the paracrine effect, which decreases inflammation and oxidation induced by LPS. As such, MSCs are capable of secreting numerous antioxidative factors in different microenvironments ( Fan et al., 2020 ).

Outlook on MSCs and MSC-Derived EVs for the Treatment of COVID-19

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a global pandemic present in more than 150 countries and has highlighted the multifactorial and complex syndrome named sepsis ( Wu et al., 2020 ). SARS-CoV-2 enters host cells via the cell surface angiotensin-converting enzyme 2 receptor present on many cells, such as alveolar type 2 and blood vessel cells ( Hoffmann et al., 2020 ). In approximately 20% of patients, SARS-CoV-2 leads to an excessive and aberrant host immune response, resulting in severe lung disease characterized by acute respiratory distress syndrome (ARDS) and multiorgan dysfunction. In COVID-19 patients, the immune system produces large amounts of inflammatory factors (IL-2, IL-6, IL-7, MCP-1, TNF-α, etc.), causing a cytokine storm responsible for ARDS, organ failure, and secondary infections ( Mehta et al., 2020 ). Several therapeutics are being evaluated, and because of their anti-inflammatory and immunomodulatory properties, allogeneic MSC therapy has been proposed. The ISCT and ISEV recognized the therapeutic potential of MSCs and their derived EVs as treatments for COVID-19. Efforts should be focused on the generation of appropriate manufacturing and quality control provisions, preclinical safety and efficacy data, rational clinical trial design, and proper regulatory oversight ( Börger et al., 2020 ).

In line, several preclinical studies have reported the protective effect of MSCs in sepsis murine models and septic shock ( Laroye et al., 2017 ). Recent studies have evaluated the efficiency of MSCs for ARDS treatment. A phase I trial reported good tolerance and the absence of major adverse effects ( Zheng et al., 2014 ; Wilson et al., 2015 ). The START study (phase IIa) compared a single intravenous dose of cryopreserved BM-MSCs with placebo in patients with moderate to severe ARDS and reported a significant improvement in oxygenation in the MSC group but without improvement in survival ( Matthay et al., 2019 ). A single-center prospective randomized Russian clinical trial of BM-MSCs in neutropenic patients with septic shock reported hemodynamic stabilization, vasopressor withdrawal, attenuation of respiratory failure, and shortening of the neutropenia duration period ( Galstyan et al., 2018 ). A preliminary analysis of a phase 1 and 2 study using a good manufacturing practice product of allogeneic BM-derived MAPCs in ARDS (MUST-ARDS) demonstrated improvement of oxygenation, reduced lung edema, and decreased proinflammatory cytokines ( Bellingan et al., 2019 ). Two reports from China have shown initial results from MSC therapy in COVID-19 patients. Compassionate use of UC-MSCs (three doses) in a 65-year-old patient requiring mechanical ventilation and with multiple organ failures led to clinical improvement in vital signs and the cessation of mechanical ventilation after the second dose ( Liang et al., 2020 ). A second study reported the use of MSCs from undefined sources to treat seven patients with ARDS. All patients showed clinical improvement after 2 days and remarkable improvements in inflammation markers and in the immune cell repertoire ( Leng et al., 2020 ). Many other clinical trials utilizing MSCs have been initiated for the treatment of COVID-19 (>80 studies declared on the clinical trial.gov website). Most of the trials use allogeneic MSCs, predominantly BM- and UC-MSCs, and perform repeated infusions. Interestingly, few trials use MSC-CM or EVs able to exert similar functions to MSCs ( Sengupta et al., 2020 ).

By exerting their immunomodulatory effects, MSCs may induce tissue repair and organ protection for patients with a confirmed infection. While the need for MSC-based therapy in COVID-19 is apparent, integrating both preclinical and clinical strategies into the current guidelines is critical for safe and effective therapies ( Moll et al., 2020a ). Future randomized controlled trials are also needed to confirm the therapeutic potential of MSCs to treat COVID-19 patients.

MSCs have generated significant interest over the past decade as a novel therapeutic strategy for a variety of diseases. In this review, we discussed the therapeutic properties of MSCs during tissue repair and regeneration. MSCs interact and modulate the local progenitor and immune cells that are involved in tissue homeostasis. Moreover, several immunological and inflammatory signals may critically influence the effects and properties of MSCs. It is essential to understand the impact of the tissue environment on the fate and functions of MSCs. Understanding the paracrine pathway involved in the healing process governed by MSCs is also important to obtain efficient and safe regenerative medicine applications.

Author Contributions

MM and MN conceived and designed the review. All authors listed have made a substantial, direct and intellectual contribution to the work and contributed to manuscript writing, revision, reading, and approval of the submitted version.

This study has received support from the Generation Life Foundation, “Fonds National de la Recherche Scientifique (FNRS),” “Télévie,” “Les Amis de l’Institut Jules Bordet”, “La Chaire en Arthrose de l’Université de Montréal”, The Arthritis Society (SOG-20-0000000046), and The Canadian Institutes of Health Research (PJT 175-1110).

Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We would like to thank the Cell Therapy Unit team for their helpful discussions.

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Keywords : mesenchymal stromal cells, cell therapy, regenerative medicine, trophic function, immunomodulation, paracrine mechanisms

Citation: Merimi M, El-Majzoub R, Lagneaux L, Moussa Agha D, Bouhtit F, Meuleman N, Fahmi H, Lewalle P, Fayyad-Kazan M and Najar M (2021) The Therapeutic Potential of Mesenchymal Stromal Cells for Regenerative Medicine: Current Knowledge and Future Understandings. Front. Cell Dev. Biol. 9:661532. doi: 10.3389/fcell.2021.661532

Received: 30 January 2021; Accepted: 28 May 2021; Published: 18 August 2021.

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Copyright © 2021 Merimi, El-Majzoub, Lagneaux, Moussa Agha, Bouhtit, Meuleman, Fahmi, Lewalle, Fayyad-Kazan and Najar. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Mehdi Najar, [email protected]

† These authors share first authorship

‡ These authors share senior authorship

This article is part of the Research Topic

Mesenchymal Stromal Cell Therapy for Regenerative Medicine

Clinical application of mesenchymal stem cell in regenerative medicine: a narrative review

Stem Cell Research & Therapy volume  13 , Article number:  366 ( 2022 ) Cite this article

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The multipotency property of mesenchymal stem cells (MSCs) has attained worldwide consideration because of their immense potential for immunomodulation and their therapeutic function in tissue regeneration. MSCs can migrate to tissue injury areas to contribute to immune modulation, secrete anti-inflammatory cytokines and hide themselves from the immune system. Certainly, various investigations have revealed anti-inflammatory, anti-aging, reconstruction, and wound healing potentials of MSCs in many in vitro and in vivo models. Moreover, current progresses in the field of MSCs biology have facilitated the progress of particular guidelines and quality control approaches, which eventually lead to clinical application of MSCs. In this literature, we provided a brief overview of immunoregulatory characteristics and immunosuppressive activities of MSCs. In addition, we discussed the enhancement, utilization, and therapeutic responses of MSCs in neural, liver, kidney, bone, heart diseases, and wound healing.


In the last decade, stem cells are increasingly applied as a therapeutic method for numerous disorders. Stem cell therapy, traditionally applied for hematopoietic disorders, nonetheless, is now established for the treatment of non-hematologic disorders [ 1 , 2 ].

Accumulating evidence has shown that mesenchymal stem cells (MSCs) offer an encouraging option for cell treatment and reconstruction of human tissues because of their differentiation multipotency, self‐renewal capacity, long‐term ex vivo proliferation, paracrine potentials, and immunoregulatory effect [ 3 ]. Furthermore, MSCs have the capability to support the progression and differentiation of other stem cells. They can release bioactive molecules, which is a key benefit in tissue regeneration [ 4 , 5 ]. These properties result in progression of treatments for a wide range of diseases, such as diseases affecting the bone, neuron, lung, liver, heart, kidney, etc. [ 4 ]. Due to these features, it is obvious that MSCs will hold a major therapeutic role in clinical trials. Because of these properties, we provided a general overview of the latest trials that studied the effectiveness of MSCs in several diseases such as neural, liver, kidney, bone, heart diseases, and wound healing.

Stem cells in regenerative medicine

In the last years, numerous studies have demonstrated that cellular therapy has exhibited great development in both in vitro and in vivo researches. Stem cells have the capability to self-renew, and also to differentiate into all cell types and are involved in physiological regeneration [ 6 ]. There are multiple stem cell sources of adult and pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) for tissue regeneration. PSCs have a high potential for pluripotency and self-renewal, which makes these cells an important option for treatment of diseases. However, there are ethical issues when using these cells, in which ESCs are separated from blastocyst-stage embryos, requiring destruction of the embryo [ 7 , 8 , 9 ]. The results of studies have revealed the regenerative ability of iPSCs in preclinical setting and conducted the first clinical study for treatment of age-associated with macular deterioration [ 10 , 11 ]. Nonetheless, the tumorigenicity risk remains unsolved. Because of these limitations, researchers began to investigate adult stem cells, the multipotent stem cells found in tissues and organs of adults. Various investigations have reported that stem cell therapy can regenerate and repair injured organs in vivo, including bone repair, cutaneous wound, pulpitis, and ischemic cardiac tissue through stem cell differentiation and production of new particular cells [ 12 , 13 , 14 , 15 ]. Moreover, some investigations have demonstrated that cultured adult stem cells release many molecular factors with anti-apoptotic, immunoregulatory, angiogenic, and chemoattractant features that stimulate regeneration [ 16 , 17 , 18 ]. Hematopoietic stem cells (HSCs) and MSCs are part of adult stem cells, which are the most widely used, generally because they can be isolated from individuals in diseased conditions.

Mesenchymal stem cell

In the late 1960s, Friedenstein and colleagues discovered MSCs as multipotent stem cells for the first time [ 19 ]. MSCs are non-hematopoietic cells and have the capability to differentiate into various lineage including mesodermal (adipocytes, osteocytes, and chondrocytes), ectodermal (neurocytes), and endodermal lineage (hepatocytes) [ 20 , 21 ]. At the beginning, it was thought that MSCs are “stromal” cells instead of stem cells [ 22 ]. Several investigators tried to alter the name of MSCs to medicinal signaling cells due to their function in secretion of some metabolites molecules in the sites of diseases, injuries, and inflammations [ 23 , 24 ]. After that, some studies have stated that MSCs can release prostaglandin E2 (PGE2), which plays a major role in the self-renewal ability, immunomodulation of MSCs, and generating a cascade of events, that demonstrates the stemness of MSCs [ 25 ]. Therefore, the term mesenchymal stem cells is justified.

MSCs chiefly found in the bone marrow (BM) possess the ability of self-renewal and also display multilineage differentiation [ 8 , 26 , 27 ]. They were obtained from various tissues and organs including BM, adipose tissue, Wharton’s jelly, peripheral blood, umbilical cord, placenta, amniotic fluid, and dental pulp [ 3 , 28 , 29 , 30 ]. MSCs can express a wide range of surface markers and cytokine profiles according to the origin of isolation [ 31 ]. Nevertheless, the common characterization markers of MSCs are CD73, CD105, CD90 and lacking expression of CD45, CD34, CD14 or CD11b, CD79α or CD19, and HLA-DR [ 32 , 33 , 34 ]. During the last decades, MSCs have shown various biological roles such as multilineage differentiation, immunomodulation, angiogenesis, anti-apoptotic and anti-fibrotic activity, chemo-attraction, and tissue repair development [ 35 , 36 , 37 ]. The MSCs have broad properties that make them a suitable source for cell therapy, such as stemness potency, easily isolation from different sources, they can be rapidly expanded in a large scale for clinical use, have less ethical issues as compared to ESCs, unlike iPSCs, MSCs transport a lower risk of teratoma formation, and they are beneficial for a wide scale of therapeutic applications due to their capability to migrate to injured tissue through chemo-attraction [ 38 , 39 , 40 ]. In addition, MSCs can release a variety of bioactive components including proteins, growth factors chemokines, microRNAs (miRNAs), and cytokines which can suggest their acceptable application [ 41 ].

The biological roles of MSCs

MSCs have the ability to inhibit the immune response in inflammatory cytokine-rich situations, including infections, wounds, or immune-mediated disorders. These immunomodulatory properties were discovered in preclinical and clinical trials, where MSCs effectively suppressed T cell activation and proliferation along with stimulation of macrophages shift from M1 to M2 [ 42 , 43 , 44 ]. This specific performance of MSCs in the presence and absence of inflammatory mediators is termed MSC polarization. MSCs have the ability to migrate to damaged areas after systemic infusion and consequently exert a beneficial effect by various mechanisms, chiefly immunoregulation, and angiogenesis [ 45 , 46 ]. Although the related mechanism-mediated MSC immunosuppression has not been entirely clear, it appears that cellular interaction, accompanied by many factors, performs the principal function in this process. In the presence of high levels of inflammatory cytokines, e.g., TNF-α and IFN-γ, MSCs release several cytokines including TGF-β and hepatocyte growth factor (HGF) and produce soluble factors including indoleamine 2,3-dioxygenase (IDO), PGE2, and nitric oxide (NO). These mediators suppress T effector cells and enhance the expression of FOXP3, CTLA4, and GITR in regulatory T cells (Tregs) to increase their immunomodulation effects [ 47 , 48 , 49 ]. Moreover, cell-to-cell communication facilitates the stimulation of Tregs by cytokine-primed MSCs [ 50 ]. Overexpression of inducible co-stimulator ligands (ICOSL) induces the stimulation of efficient Tregs [ 51 ].

In addition, MSCs can enhance the generation of Treg cells indirectly. According to the literature, MSCs stimulate M2 macrophage and alter the phenotype through secretion of extracellular vesicles in an in vitro study [ 52 ]. Also, M2 cells that are activated by MSCs express CCL-18 and induce Treg cells [ 53 ]. Moreover, MSCs increase the expression of cyclooxygenase 2 (COX2) and IDO, resulting in expression of CD206 and CD163 in M2 cells, as well as enhance the expression of IL-6 and IL-10 in the microenvironment [ 54 ]. The overexpression of IL-10 that is produced by dendritic cells (DCs) and M2 cells upon MSCs co-culture leads to further immunomodulation via inhibition of effector T cells [ 55 , 56 ]. Furthermore, the secretion of IDO from MSCs can induce the proliferation, activation, and IgG releasing of B cells, thereby suppressing T effector cells [ 57 , 58 ].

One of the typical properties of MSCs is their multipotency capacity in which these stem cells are able to differentiate into a number of tissues in vitro [ 59 ]. Chondrogenic differentiation of MSCs in vitro occurs commonly via culturing them in the existence of TGF-β1 or TGF-β3, IGF-1, FGF-2, or BMP-2 [ 60 , 61 , 62 , 63 ]. MSC differentiation into chondroblasts is characterized by the increasing of various genes such as collagen type II, IX, aggrecan, and proliferation of chondroblast cell morphology. During the process of chondrogenesis, FGF-2 promotes the MSCs induced with TGF-β1 or TGF-β3 and/ or IGF-1 [ 64 ]. According to the literature works, several molecular pathways such as hedgehog, Wnt/β-catenin, TGF-βs, BMPs, and FGFs can regulate chondrogenesis [ 65 ]. In addition, MSCs can exert the osteogenesis function by inducing MSCs with ascorbic acid, β-glycerophosphate, vitamin D3, and/or BMP-2, BMP-4, BMP-6, and BMP-7 [ 66 ].

One of the major abilities of MSCs is anti-fibrotic activity. These cells can differentiate into various cell lineages such as hepatocytes, both in vivo and in vitro [ 67 ]. MSCs contain multiple trophic factors which induce cells and matrix remodeling to stimulate progenitor cells and the recovery of damaged cells. MSCs can decrease myofibroblasts and reverse the fibrotic activity of injured tissues [ 68 ]. Furthermore, these cells release pro-angiogenic factors including VEGF, IGF-1, and anti-inflammatory factors that participate in the recovery of tissue function. For instance, MSCs can increase neovascularization of ischemic myocardium through VEGF in a mice model of heart disease [ 69 ]; also, IGF-1 exerts an advantageous effect on the survival and proliferation of cardiomyocytes [ 70 ].

Bone marrow mesenchymal stem cell-based regenerative medicine

So far, increasing data have lately studied the effects of MSCs in the treatment or regeneration of various disorders (Table 1 ). In this section, we reviewed the latest clinical studies that investigate the potential contribution of MSCs in the regenerative medicine, as shown in Fig.  1 .

figure 1

Effect of bone marrow mesenchymal stem cell-based regenerative medicine

Neural regeneration

The application of BMSCs has demonstrated promising therapeutic results in the treatment of neurological diseases. Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease, is a neurodegenerative disorder that leads to degeneration of the motor neurons that causes paralysis and muscle weakness [ 138 , 139 ]. Syková et al. [ 71 ] carried out a study that intrathecally injected 15 ± 4.5 × 10 6 autologous BMSCs into 26 patients with ALS. After mesenchymal stem cells transplantation (MSCT), ALS functional rating scale (ALSFRS) significantly reduced, forced vital capacity (FVC) remained stable or above 70%, and weakness scales (WSs) were stable in 75% of patients. They have shown that the intrathecal BMSCs intervention in ALS patients is a safe method and it can slow down the development of the disease. There were no significant adverse events related to the trial during and after transplantation of BMSCs. Barczewska and colleagues indicated that three intrathecal injections of 30 × 10 6 Wharton’s jelly-MSCs (WJ-MSCs) improved ALSFRS [ 77 ]. They showed that WJ-MSCs are safe and effective in individuals that suffer from ALS. However, one other group found that intrathecal injection of autologous adipose MSCs does not improve clinical symptoms of ALS patients [ 76 ]. Their results indicated that the levels of CSF protein and nucleated cells were increased and ALSFRS-R showed development of disease in all treated patients. In the trial by OH et al., autologous BMSCs were injected to treat seven participants that suffer from ALS [ 75 ]. The participants were injected twice with autologous BMSCs (one million cells per kg) and followed up for 12 months. No serious adverse events were reported during the follow-up period. Furthermore, during the 12-month follow-up, there was no acceleration in the decrease in the ALSFRS-Revised (ALSFRS-R) score, Appel ALS score, and FVC. Moreover, CSF analysis showed that the levels of TGF-β and IL-10 were evaluated, while MCP-1, which is chemokine-related and exacerbates the motor neuron damage in ALS, was decreased. Their results exhibited that two repeated MSC infusions have safety and feasibility for at least 1 year in seven individuals; nevertheless, the study has some limitations such as low number of participants and short-time follow-up. In another study [ 73 ], 15 ALS patients were transplanted with autologous BMSCs. These 15 patients were divided into two groups (group 1: patients who had ALS with an inherently slow course, group 2: individuals who had ALS with an inherently rapid course) and received three intrathecal infusions of MSCs. There were no significant adverse events in the course of multiple intrathecal injections of MSCs. In group 1, there were no major changes in the rate of disease development and in group 2 ameliorating of the disease was indicated following MSCs therapy. According to their observation, the response of patients with ALS to treatment with MSCs was variable. Also, the authors indicated that due to the small number of patients, less subgroups were available for statistical analysis, limiting their ability to draw conclusions from the data.

Spinal cord injury (SCI) is usually related to devastating results. The damage to the spinal cord leads to injury to the motor, sensory, and autonomic roles of the spinal cord that affects patients’ well-being such as their physical and psychological state [ 140 , 141 ]. In a phase I, nonrandomized, uncontrolled study by Mendonça et al. [ 84 ], 15 SCI patients were administered 1 × 10 7 cells/ml MSCs. The results of the investigation revealed that SCI symptoms were meaningfully decreased by MSCT, all participants showed variable improvements in tactile sensitivity, and eight participants improved lower limb motor functional gains, chiefly in the hip flexors. Seven patients revealed sacral sparing and developed American Spinal Injury Association impairment scale (AIS) grades B or C – partial damage. Nine participants had developments in urologic function and one patient showed alterations in somatosensory evoked potentials (SSEP) 3 and 6 months after MSCT. These results stated that treatment with MSCs ameliorated the organ malfunction in people with SCI and has clinical safety, because no serious adverse effects were reported. The authors indicated that their results should be confirmed in larger and controlled clinical trials. Albu and colleagues have been demonstrated that intrathecal administration of WJ-MSCs considerably improved the pinprick sensation in the dermatomes below the level of damage [ 88 ]. Further results showed that bladder maximum capacity was elevated and bladder neurogenic hyperactivity and external sphincter dyssynergy were reduced. In another study [ 85 ], ten SCI subjects received four subarachnoid injections of 30 × 10 6 autologous BMSCs, maintained in autologous plasma, at weeks 1, 16, 28, and 40 of the trial and followed up for 12 months. There were no adverse events and all participants tolerated the therapy. Vaquero et al. [ 86 ] demonstrated that MSCT is safe and improves sensitivity, motor power, spasms, spasticity, neuropathic pain, sexual function, or sphincter dysfunction in the SCI patients. The results of their study have shown that 55.5% of patients improved in SSEP and 44.4% of patients ameliorated in voluntary muscle contraction together with intralesional active muscle reinnervation. Hur et al. carried out a study in which 14 patients with SCI were administered intrathecally 9 × 10 7 adipose MSCs [ 87 ]. Their observations showed mild progresses in neurological function. No serious adverse events were observed. In a phase 2 study, 13 patients with SCI were intravenously administered a single dose of autologous MSCs cultured in auto-serum [ 82 ]. The results of this trial revealed that SCI symptoms were considerably declined by MSC therapy, ASI, International Standards for Neurological and Functional Classification of Spinal Cord (ISCSCI-92), and Spinal Cord Independence Measure (SCIM-III) demonstrated functional improvements after MSC injection. No severe adverse effects were related to MSC administration.

Parkinson’s disease (PD) is a neurological disorder principally characterized by the deterioration of motor activities due to the impairment of the dopaminergic nigrostriatal system [ 142 , 143 ]. It has been indicated that MSCs improved the symptoms of PD. In a phase I controlled, randomized clinical study, patients that suffer from progressive supranuclear palsy were administered autologous BMSCs via intra-arterial injection [ 78 ]. The results of the study exhibited that autologous BMSCs are safe and reduce disease progression. Canesi et al. [ 79 ] have demonstrated that injection of MSCs into cerebral arteries of PD patients led to positive results in 17 PD participants: all treated participants were alive and motor function rating scales remained stable for at least 6 months during the 12-month follow-up period. One patient died 9 months after the injection for reasons not associated with cell infusion or to disease development.

In a study conducted by Jaillard and colleagues in 2019 [ 89 ], 31 individuals with subacute stroke were administered the intravenous injections of autologous BMSCs. The results of the trial exhibited significant improvements in motor-National Institute of the Health Stroke Scale (NIHSS) score, motor-Fugl-Meyer scores, and task-related functional MRI activity in motor cortex-4a. However, there was no remarkable progress in Barthel Index, NIHSS, and modified Rankin scores. In general, their results suggested that BMSCs improved motor recovery via sensorimotor neuroplasticity. In another study, 17 patients with subacute middle cerebral artery infarct received two million cells/kg autologous BMSCs [ 92 ]. During the follow-up process, NIHSS score, modified Rankin Scale or Barthel Index did not improve after the transplantation. Nonetheless, there was a significant improvement in absolute change in median infarct volume, but no treatment-related adverse effects were observed.

In sum, these outcomes suppose that BMSCs can safely and efficiently treat neural diseases, inhibit disease development, and considerably ameliorate the quality of life and clinical manifestations of patients. Consequently, BMSCs can become a new option for the clinical treatment of neural diseases.

Liver regeneration

The potential of BMSCs to differentiate into the endodermal lineage, such as hepatocyte‐like cells, makes them an attractive alternative for the treatment of liver diseases [ 144 ]. Some clinical studies have demonstrated the efficacy and feasibility of BMSC therapy in patients with liver diseases. The effect of BMSCs has been studied in individuals suffering from liver cirrhosis by Suk et al. [ 98 ]. Seventy-two patients were enrolled in this trial and randomly classified into three groups: one control group and two autologous BMSC groups that received one-time or two-time hepatic arterial administrations of fifty million autologous BMSCs 30 days after BM aspiration. Fibrosis quantification exhibited that in one-time and two-time BMSC groups there are a reduction of 25% and 37% in the proportion of collagen, respectively. In addition, the Child–Pugh (CP) scores of both test groups were meaningfully improved following BMSC administration in comparison with the control group. No serious adverse events were associated with MSC injection during the 12-month follow-up. Wang and coworkers have found that intravenous injection of UC-MSCs (0.5 × 10 6 cells/kg) is feasible and well tolerated in patients with primary biliary cirrhosis (PBC) [ 93 ]. They exhibited that MSCs significantly decreased the level of ALP and GGT; however, there were no considerable changes in serum AST, ALT, total bilirubin, albumin, prothrombin time activity, or immunoglobulin M levels. Similarly, Zhang et al. [ 94 ] have demonstrated that intravenous administration of 1.0 × 10 6 cells/kg UC-MSCs is safe and efficient for patients with ischemic-type biliary lesions after liver transplantation. According to their results, MSCs therapy reduced the serum ALP, GGT, and total bilirubin. In a randomized placebo-controlled phase I–II single-center study, nine patients that suffer from acute-on-chronic liver failure (ACLF) grades 2 and 3 were enrolled [ 95 ]. The experiment group (n = 4) received standard medical therapy along with five injections of 1 × 10 6 cells/kg of BMSC for 3 weeks. There were no transplant-related adverse events; however, one patient in the experiment group showed hypernatremia and a gastric ulcer, after the third and fifth administrations, respectively. Furthermore, MSCT revealed a considerable improvement in CP, model for end-stage liver disease (MELD), and ACLF (grade 3 to 0). Thus, MSCT is safe and viable in individuals with ACLF. In an open-label non-blinded randomized controlled study conducted by Lin et al. [ 96 ], 110 patients with hepatitis B virus (HBV)-related ACLF were enrolled in this trial. These patients were divided into two groups: control group (N = 54) was treated with standard medical therapy only and the intervention group (N = 56) was injected four times with 1.0–10 × 10 5 cells/kg allogeneic BMSCs, and then followed up for 6 months. There were no serious adverse events associated with transplantation. The results of that study demonstrated that MSCT significantly improved clinical laboratory measurements, such as serum total bilirubin, and MELD scores in comparison with control group. In addition, mortality from multiple organ failure and prevalence rate of serious infection in the intervention group was lower than that in the control group. Their results clearly established the safety and feasibility of the clinical use of peripheral administration of allogeneic BMSCs for subjects with HBV-associated ACLF, and markedly enhanced the survival rate through enhancing liver function and reducing the prevalence of severe infections.

In summary, MSCT can meaningfully ameliorate the clinical manifestations of these patients, reduce the liver fibrosis, and inhibit the development of disease.

Kidney regeneration

Hurt to renal cells can occur because of a wide range of ischemic and toxic insults and results in inflammation and cell death, which can lead to kidney damage. Inflammation has a significant role in the damage of renal cells, as well as following cellular regeneration processes [ 3 , 145 ]. Various investigations have consistently demonstrated a supportive effect of MSC on acute and chronic renal injury [ 146 ]. Makhlough et al. declared that intravenous administration of 1–2 × 10 6 cells/kg into seven patients with chronic kidney disease failed to induce remission [ 101 ]. They indicated that variations in estimated glomerular filtration rate (eGFR) and serum creatinine during the 18-month follow-up were not statistically significant. Nonetheless, no severe adverse events were reported, and they could not assess the efficacy because of their study design. Authors postulated that limited sample size and lack of a control group led to the lack of success. A study conducted by Swaminathan et al. in 2021, has displayed the effect of allogeneic BMSCs in acute kidney injury patients. They have shown that treatment of MSCs with SBI-101 stimulated an immunotherapeutic response that initiated an enhanced phenotypic alteration from tissue injury to tissue repair [ 102 ]. In a single-arm phase I clinical trial carried out by Makhlough et al. [ 100 ], six patients with autosomal dominant polycystic kidney disease (ADPKD) were intravenously injected 2 × 10 6 cells/kg autologous BMSCs. The results of the study showed that the mean eGFR value declined and the level of serum creatinine enhanced during the 1-year follow-up. Moreover, no remarkable modifications in renal function parameters and blood pressure were observed during the year after intervention. However, there were no severe adverse events after 1-year follow-up. In addition, the authors indicated that there are some reasons for the lack of success, including small number of patients, absence of a comparison group, limited follow-up period, single dose administration, and they did not utilize htTKV as a surrogate endpoint. Abumoawad and colleagues have established that adipose MSCs enhanced blood flow, GFR and reduced inflammatory injury in poststenotic kidneys of individuals that suffer from atherosclerotic renovascular disease (ARVD) [ 99 ]. Their results illustrated that mean renal blood flow was considerably enhanced, and hypoxia, renal vein inflammatory cytokines, and angiogenic factors were considerably attenuated.

Heart regeneration

Heart disease is the first and most frequently diagnosed disease and the leading cause of disease death [ 147 ]. When cardiomyocytes are damaged via ischemic and other factors, the remaining viable cardiomyocytes have a restricted ability to proliferate and dead cardiomyocytes are changed by non-contractile fibrous tissue, leading to functional impairment that elicits the progression of heart failure. According to the developing number of patients with heart disease, there is a vital need to expand an innovative remedy to rescue deteriorating hearts. Regenerative medicine and cell therapy are the upcoming therapeutic opportunities for heart diseases. According to the literature, the transplantation of BM-derived cells and cardiac stem cells into deteriorating hearts appeared to provide functional benefits [ 148 , 149 ].

In a study by Yagyu et al. [ 110 ], 8 individuals with symptomatic heart failure were infused with BMSCs. During the follow-up period, no serious adverse events were observed. There were no major differences in B-type natriuretic peptide, left ventricular ejection fraction (LVEF), and peak oxygen uptake at 2 months. The results of this study recommend further research regarding the feasibility and efficacy of MSCs. In a study by Gao et al. [ 107 ], 116 patients with acute myocardial infarction randomly received an intracoronary injection of WJ-MSCs. They indicated that MSCs therapy elevated the myocardial viability and perfusion within the infarcted territory. In addition, the LVEF was elevated and LV end-systolic volumes and end-diastolic volumes were decreased in the WJ-MSCs group.

Chan et al. demonstrated that intramyocardial infusion of autologous BMSCs in conjunction with transmyocardial revascularization or coronary artery bypass graft surgery was technically feasible and could be performed safely. The results showed that regional contractility in the cell-treated regions improved during the 1-year follow-up; also, the quality of life was improved along with a substantial decrease in angina scores at 12 month post-treatment [ 104 ]. In a study by Kaushal et al. [ 113 ], 12 participants with hypoplastic left heart syndrome were transplanted with allogeneic human MSCs (2.5 × 10 5 cells/kg). This study determined the safety, feasibility, and usefulness of MSC administration into the left ventricular myocardium. No serious adverse effects were reported during the trial. Mathiasen et al. observed that after BM-MSCT, left ventricular end-systolic volume was significantly reduced, also LVEF, stroke volume, and myocardial mass remarkably improved [ 103 ]. In addition, a major decrease in the amount of scar tissue and quality of life score was observed. No side effects were identified. In a randomized, double-blind, placebo-controlled, multicenter, phase II study, 100 patients with anterior ST elevation myocardial infarction received autologous BMSCs and atorvastatin (ATV) treatment. The results of that study represented the absolute change of LEVF within 12 months, improvement in cardiac function, induction of remodeling and regeneration, and improvement in quality of life [ 108 ]. Recently, Celis-Ruiz and coworkers conducted a study in which intravenous administration of adipose MSCs within the first 2 weeks of ischemic stroke onset is safe at 24 months of follow-up [ 106 ]. In a study conducted by Hare et al. [ 112 ], 37 non-ischemic dilated cardiomyopathy patients were divided into two groups and received 10 × 10 7 allogeneic and autologous BMSCs. Minnesota Living with Heart Failure Questionnaire score decreased in both groups. The major adverse cardiac event rate was lower in allo vs. auto. Also, TNF-α decreased, to a greater extent in allo vs. auto at 6 months. These results suggested the clinically meaningful efficacy of allogeneic vs. autologous BMSCs in non-ischemic dilated cardiomyopathy patients. Qayyum et al. have found that intra‑myocardial injections of autologous adipose MSCs ameliorated cardiac functions and unchanged exercise capacity, in contrast to deterioration in the placebo group [ 115 ].

Levy et al. indicated that after allogeneic BMSCs in patients with chronic stroke, Barthel Index scores increased. Moreover, electrocardiograms, laboratory tests, and computed tomography scans of chest/abdomen/pelvis suggest that BMSCs could alleviate the clinical symptoms in patients with stroke [ 90 ].

In sum, BMSC therapy can be an effective, achievable, and safe process that remarkably improves cardiac function and promotes patients’ quality of life.

Bone regeneration

Bone regeneration is a hot topic of research in clinical studies. Bone regeneration is a crucial problem in numerous cases, including bone fracture, defect, osteoarthritis, and osteoporosis, which should be resolved [ 150 , 151 , 152 ]. Autogenous bone grafts are considered the standard approach for bone formation by means of the participants’ own cells that stimulate osteoinductive, bone conductivity, and histocompatibility in bone diseases [ 153 ]. Nevertheless, there are some shortcomings of this procedure such as unpredictable absorption, extended recovery time, and patients commonly experience pain and nerve injury at the harvest area [ 154 , 155 , 156 ]. With the development of understanding bone tissue biology as well as recent approaches in the improvement in tissue regeneration, the application of MSC has become an attractive subject in augmenting bone tissue forming [ 157 , 158 ].

In a pilot study by Jayankura and coworkers, allogeneic BMSCs were applied to treat 22 participants with bone fractures [ 128 ]. All participants received percutaneous implantation of autologous BMSCs (5 to 10 × 10 7 cells) into the fracture area. After intervention, Tomographic Union Score (TUS) and Global Disease Evaluation (GDE) score were improved, and pain at palpation at the fracture site was reduced. In addition, the ratio of blood samples comprising donor-specific anti-HLA antibodies enhanced at 6 months post-intervention. Three serious cell-related adverse events were reported. In another study by Shim and coworkers [ 129 ], intramedullary (4 × 10 7 cells) and intravenous (2 × 10 8 cells) infusion of WJ-MSCs in combination with teriparatide showed beneficial results in individuals with osteoporotic vertebral compression fractures. Their observation displayed that the mean visual analog scale, Oswestry Disability Index, and Short Form-36 scores meaningfully improved. They stated that WJ-MSCs in combination with teriparatide are viable and have a clinical profit for fracture healing by stimulating bone architecture.

Several studies investigated the effect of BMSCs in osteoarthritis (OA) patients. Chahal et al. carried out a clinical phase I/IIa trial that involved 12 individuals with late-stage Kellgren–Lawrence knee OA. These 12 patients were injected with a single intra-articular of 1 × 10 6 , 10 × 10 6 , and 50 × 10 6 BMSCs. The results showed that patients had improved Knee Injury and Osteoarthritis Outcome Score (KOOS) pain, symptoms, quality of life, and Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) stiffness relative to baseline. Moreover, cartilage catabolic biomarkers and MRI synovitis were meaningfully lower at higher doses and the levels of pro-inflammatory monocytes/macrophages and IL-2 reduced in the synovial fluid after intervention. No serious events had occurred [ 116 ]. Dilogo et al. have reported that UC-MSCs (10 × 10 6 cells) significantly decreased the WOMAC and could be a potentially new regenerative treatment for patients with knee OA [ 127 ]. In a study conducted by Hernigou et al. [ 117 ], 140 patients with OA received a subchondral infusion of BMSCs on one side and received total knee arthroplasty (TKA) on the contralateral knee. They demonstrated that subchondral MSCs had a significant effect on pain to postpone or avoid the TKA in the contralateral joint of patients with OA. In a phase II multicenter randomized controlled clinical trial, 60 OA patients received 10 × 10 7 cells of autologous BMSCs along with platelet-rich plasma and followed up for 12 months [ 119 ]. No serious adverse effects were observed after MSCs injection or during follow‑up. According to the observations, treatment with BMSC related to platelet-rich plasma was demonstrated to be a feasible alternative treatment for individuals with OA, along with clinical development at the end of follow-up. Similarly, Bastos et al. have reported that MSCs alone or in combination with platelet-rich plasma are safe and have an advantageous effect on symptoms in OA individuals [ 121 ]. They found that MSCs group and MSCs + platelet-rich plasma group can improve the pain, function and daily living activities, and quality of life subscales. Ten adverse events were reported in three participants in the MSCs group and in two of the MSCs + platelet-rich plasma group. PERS and colleagues reported another clinical phase Ia study that involved 19 individuals suffering from knee OA [ 123 ]. These 18 individuals were classified into three groups and received a single intra-articular administration of 2 × 10 6 , 10 × 10 6 , and 50 × 10 6 adipose MSCs. According to their results, individuals had experienced significant improvement in pain levels and function. There were no severe adverse events; however, 4 individuals experienced transient knee joint pain and swelling after local administration. In a long-term follow-up of a multicenter randomized controlled clinical trial by Espinosa et al. [ 120 ], 30 OA patients were administered the intra-articular infusion of two diverse doses of autologous BMSCs cells (10 × 10 6 or 10 × 10 7 ) versus hyaluronic acid in the treatment of OA. No adverse effects occurred after MSCT or during the 4-year follow‑up. Their results showed that intra-articular infusion of BMSCs together with hyaluronic acid is a safe and viable process that leads to a clinical and functional improvement in knee OA.

Overall, these data display that BMSCs can be a promising, safe and effective alternative for bone regeneration, significantly improve the clinical manifestation of patients, and inhibit development of diseases.

Wound regeneration

The skin has several layers along with different compounds and roles that work together to support internal organs and serve various biological roles. It has three main layers, the epidermis, the dermis, and the subcutaneous layer [ 159 ]. Generally, skin wound healing, triggered by tissue injury, includes four stages: hemostasis, inflammation, proliferation, and maturation. MSCs can assist in all stages of the wound healing process. The use of MSCs for the treatment of skin can improve the regeneration of skin and reduce scarring. MSCs exert their functions through migration into the skin damage site, suppressing inflammation, and increasing the growth and differentiation ability of fibroblasts, epidermal cells, and endothelial cells [ 160 , 161 ]. As MSCs have exhibited wound healing in many preclinical studies, the application of MSCs for chronic wounds contributes to progress toward clinical trials. Falanga et al. have demonstrated that autologous BMSCs are an impressive and safe treatment method for wound healing [ 131 ]. The results of the study indicated a trend toward a reduction in ulcer size or complete wound closure by 4–5 months. No adverse events were noted. In a study by Zhou et al., 346 patients with skin wounds were administered adipose MSCs [ 132 ]. There were no adverse events during the trial. They reported that the granulation tissue coverage rate and thickness of granulation tissue were considerably ameliorated. In an open-label phase I/II study, sixteen participants with vocal fold scarring were administered a single dose of 0.5–2 × 10 6 cells autologous MSCs [ 137 ]. Video ratings of vocal fold vibrations and digitized analysis of high-speed laryngoscopy and phonation pressure threshold were considerably enhanced for 62–75% of the participants. Voice Handicap Index was meaningfully enhanced in eight participants, with the remaining experiencing no remarkable alteration. No serious adverse events or minor side effects were reported. Lonardi et al. observed that micro-fragmented adipose tissue improved skin tropism in patients with diabetic foot ulcer [ 135 ]. Furthermore, the results of studies have shown that adipose-derived stem cells had a beneficial effect on the full-thickness foot dorsal skin wound in diabetic mice with a considerably decreased ulcer area [ 162 ]. Recently, Huang et al. carried out a clinical study in which six subjects with intrauterine adhesion and four with cesarean scar diverticulum enrolled in this trial [ 136 ]. They found that intrauterine injection of UC-MSCs improved the endometrial thickness, cesarean scar diverticulum, and the volume of the uterus.

In the last decades, optimizations of isolation, culture, and differentiation procedures have permitted MSCs to improve closer to clinical uses for improving disorders and various tissue regeneration. MSCs have some important characteristics that make them preferred candidates to use for regenerative medicine: immunomodulatory capability valuable to improve immune system abnormalities, paracrine or autocrine roles that produce growth factors, and the vital potential to differentiate into various cells. Several clinical trials have reported that both autologous and allogeneic MSCs are valuable sources for tissue forming. Particularly, autologous MSCs signify the chief sources examined safe for administration and minimization of immunological threat, regardless of the lack of reported grievances concerning allogeneic MSC-based therapy. According to the studies described in this literature, administration of MSCs appear to be more effective and the usefulness of MSC therapy in bone and heart disorders has been broadly established. In terms of safety, no significant relationship was found between the MSC therapy and incidence of cancer and infection. Intravenous injection of MSCs is the most widely used form of administration and the dosage commonly fluctuates between 1 × 10 6 cells/kg and 2 × 10 8 cells/kg. According to the literature works mentioned in this review, the repeated administration of MSCs suggests being more beneficial than a single injection. In addition, the effectiveness of MSCs therapy in osteoarthritis disorder has been widely established. Long-term follow-up studies exhibited that serum tumor markers did not enhance before and 3 years after MSCs therapy. Nevertheless, there is still a lack of reliable scientific data on the mechanisms whereby the MSC therapy improves the numerous disorders that can develop the MSC modification and increase their prospective clinical application.

Availability of data and materials

Not applicable.


Amyotrophic lateral sclerosis

Association impairment scale

ALS functional rating scale


Acute-on-chronic liver failure

Autosomal dominant polycystic kidney disease


Bone marrow

Bone marrow mesenchymal stem cells

Cyclooxygenase 2

Dendritic cells

Embryonic stem cells

Estimated glomerular filtration rate

Forced vital capacity

Global Disease Evaluation

Hematopoietic stem cells

Hepatitis B virus

Hepatocyte growth factor

Induced pluripotent stem cells

Indoleamine 2,3-dioxygenase

Inducible co-stimulator ligands

International Standards for Neurological and Functional Classification of Spinal Cord

Knee injury and osteoarthritis outcome score

Left ventricular ejection fraction

Mesenchymal stem cells

Mesenchymal stem cells transplantation

Model for end-stage liver disease

Nitric oxide


Pluripotent stem cells

Prostaglandin E2

Spinal cord injury

Somatosensory evoked potentials

Spinal cord independence measure

Regulatory T cells

Tomographic Union Score

Total knee arthroplasty

Weakness scales

Western Ontario and McMaster Universities Osteoarthritis Index

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The authors express their gratitude to the Deanship of Scientific Research at King Khalid University for funding this work through the Research Group Program under grant number RGP. 2/122/43.

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Ria Margiana

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Tyumen State Medical University, Tyumen, Russian Federation

Alexander Markov

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Margiana, R., Markov, A., Zekiy, A.O. et al. Clinical application of mesenchymal stem cell in regenerative medicine: a narrative review. Stem Cell Res Ther 13 , 366 (2022). https://doi.org/10.1186/s13287-022-03054-0

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Challenges and advances in clinical applications of mesenchymal stromal cells

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Mesenchymal stromal cells (MSCs), also known as mesenchymal stem cells, have been intensely investigated for clinical applications within the last decades. However, the majority of registered clinical trials applying MSC therapy for diverse human diseases have fallen short of expectations, despite the encouraging pre-clinical outcomes in varied animal disease models. This can be attributable to inconsistent criteria for MSCs identity across studies and their inherited heterogeneity. Nowadays, with the emergence of advanced biological techniques and substantial improvements in bio-engineered materials, strategies have been developed to overcome clinical challenges in MSC application. Here in this review, we will discuss the major challenges of MSC therapies in clinical application, the factors impacting the diversity of MSCs, the potential approaches that modify MSC products with the highest therapeutic potential, and finally the usage of MSCs for COVID-19 pandemic disease.

Mesenchymal stromal cells (MSCs) are pluripotent non-hematopoietic stem cells with self-renewal capability [ 1 ] and being intensively investigated in clinical trials. Since the discovery of MSCs from bone marrow by Friedenstein in 1970s, MSCs have been isolated from various sources including muscle, umbilical cord, liver, placenta, skin, amniotic fluid, synovial membrane, and tooth root [ 2 , 3 ], and tested in amounts of preclinical and clinical studies (Fig.  1 ). It is now understood that MSCs have wide-ranging physiological effects including the maintenance of tissue homeostasis and regeneration [ 4 , 5 ], as well as the immunomodulatory activities suitable for therapeutic application [ 6 ]. So their indications have been expanded to graft-versus-host disease (GVHD), multiple sclerosis (MS), Crohn’s disease (CD), amyotrophic lateral sclerosis (ALS), myocardial infarction (MI), and acute respiratory distress syndrome (ARDS) [ 7 , 8 , 9 ].

figure 1

Various sources of MSCs used in the registered clinical trials. MSCs isolated from bone marrow are most widely applied in clinical trials, followed by those from umbilical cord and adipose. MSCs from muscles, tooth are also used

Over 300 clinical trials of MSC therapies have been completed in patients including but not limited to degenerative or autoimmune diseases (Table 1 lists some of the representative completed studies). Overall, MSCs have exhibited tolerable safety profile and demonstrated promising therapeutic benefits in some clinical settings, which led to regulatory approvals of MSCs in a few countries. In 2011, the Ministry of Food and Drug Safety (Korea FDA) approved Cartistem®, a MSC product derived from umbilical cord blood and developed by Medipost for the treatment of traumatic or degenerative osteoarthritis [ 10 ]. Thereafter, more MSC products including HeartiCellgram®, Mesoblast, TiGenix, and Stempeutics, were approved by regulatory authorities worldwide for the treatment of a variety of diseases. In the USA, Ryoncil (remestemcel-L) is promising to be the first FDA-approved GVHD treatment for children younger than 12, but is still in the stage of safety verification. The amount of clinics offering exogenous stem cell therapies has doubled from 2009 to 2014 in the USA. This boom in stem cell clinics with 351 companies putting stem cells for sale in 570 clinics in 2016 indicated the mal-practice of the MSC therapies [ 11 ]. Considering the fact that many of the applied exogenous stem cell therapies lack confirmation on safety and effectiveness from large-scale clinical trials and are even illegal, these medical mal-practices do threaten the development of MSC therapies [ 12 ].

In this review, we will focus on the major challenges of MSC therapies and the underlying factors leading to the failure of clinical trials. Recent advances and prospects concerning the translation of MSC techniques into clinical practices will also be discussed.

Challenges in technology transfer of MSCs from bench to bedside

Although transferring MSCs from bench to bedside is theoretically achievable, substantial failures have been reported in many early- or late-stage clinical trials, which account for the disapproval of many products by FDA [ 13 ]. Factors contributing to the failure of MSC clinical development include but not limited to the poor-quality control and inconsistent characteristics of MSCs in terms of immunocompatibility, stability, heterogeneity, differentiation, and migratory capacity [ 14 , 15 ] (Fig.  2 ).

figure 2

The main challenges in clinical applications of MSCs. During preparation of the MSC products, the main challenges include: (1) heterogeneity of MSCs resulted from donor variations such as the health status, genetics, gender, and age. (2) The varying degree of stability of stemness and differentiation capacities between MSCs isolated from different sources, such as bone marrow, adipose tissue, umbilical cord, or muscles. (3) The varying level of expansion capacities under different culture conditions, including confluence, culture surface, oxygen levels, flasks/bioreactors, passage number, and cell surface modifications. At the state of application, challenges remain due to the influence of (1) the homing or migratory capacity of MSCs under different administration route (local/systemic), injection site, infusion time, and cell carrier materials. (2) The immune compatibility between donors and recipients is the key to reduce the risk of rejection, but is affected by environmental inflammatory molecules which could induce distinct expression of MHC-II in MSCs. (3) The complex effective components released by MSCs depending on the host microenvironment (inflammation status, hypoxia, and ECM), which can result in highly variable factors shaping distinct functions of MSCs

Immunocompatibility of MSCs

MSCs were immune privileged due to the low expression of MHC-I and HLA-I, and no expression of HLA-II or costimulatory factors such as CD40, CD80 and CD86. MSCs can be transplanted as allogeneic cells with a low risk of rejection. Generally, the original MSCs are believed to have low immunogenicity [ 16 ]. Most MSC products are manufactured by amplifying a small number of cells obtained from donors, which can increase MSC immunogenicity caused by inappropriate processes and culture conditions. After MSCs infusion, the in vivo inflammatory molecules in turn increase MSC immunogenicity and further decrease MSCs viability and differentiation capacity, particularly when administrating xenogenic MSCs including human MSCs in animal models [ 17 ]. Although the primary immunogenicity of MSCs derived from in vitro experiments might be minimal, the secondary immunogenicity induced by in vivo positive feedback loops can cause the absence of efficacy reported in most clinical trials.

Studies have shown that inflammatory molecules (such as interferon-γ), increased cell density, and/or serum deprivation can induce high expression of MHC-II in MSCs, while TGF-β suppresses MHC-II expression [ 18 ]. The immune compatibility between donors and recipients is the key to reduce the risk of rejection in the event of long-term treatments with repeated infusions, in conditions requiring promotion of transplanted bone marrow integration, or post-renal transplantation rejection treatments [ 19 ]. It has been reported that repeated intra-articular injection of allogeneic MSCs is more likely to cause an adverse reaction than autologous cells when administered in the same manner [ 20 ]. The same observations were reported in horses treated with intracellular xenogen-contaminated autologous MSCs (such as FBS) or non-xenogen-contaminated allogeneic MSCs [ 21 ].

MSCs of high quality is the first step to ensure the safety and efficacy in clinical trials. Understanding the molecular and cellular mechanisms underlying the immune incompatibility of MSCs will help to improve the manufacture of MSC products.

Stemness stability and differentiation of MSCs

MSCs have mesodermal lineage differentiation potential and the potential to regulate tissue regeneration by mediating tissue and organ repair, as well as replacing damaged cells [ 22 ]. Different tissue-derived MSCs exhibit tendencies to differentiate into different end-stage lineage cells [ 23 , 24 ], and such regeneration and differentiation contribute to distinctive clinical efficacy.

Several laboratories have analyzed the proteome modifications associated with MSCs differentiation [ 25 , 26 ]. They indicated that ‘‘stemness’’ genes were highly expressed in undifferentiated and de-differentiated MSCs [ 27 , 28 ]. These highly stemness-related gene clusters in MSCs have been found to be mainly involved in the proliferation, differentiation, and migration [ 29 ]. When MSCs differentiated into osteoblasts, chondrocytes, and adipocytes, expressions of these genes significantly decreased, underlining their unique characteristics. Table 2 lists typical stemness genes of MSCs.

Serial passaging in long-term culture could negatively affect the expression of stemness genes [ 48 , 49 ]. A previous study indicated that CD13, CD29, CD44, CD73, CD90, CD105, and CD106 in MSCs are down-regulated during culture expansion compared to MSCs in the stromal fraction [ 50 ]. The senescence-related proteins p53, p21, and p16 expressed under different conditions [ 51 ]. Rene et al. reported that after short-term in vitro culture, wild-type MSCs became senescent, and p21(−/−)p53(+/+) MSCs showed an elevated spontaneous apoptosis rate but no sign of tumoral transformation [ 52 ]. On the other hand, Mclean et al. discovered cancer-associated MSCs (CA-MSCs), which are determined by the expression of CD44, CD73, and CD90, exhibited the upregulation of the TGF-β superfamily/bone morphogenetic protein (BMP) family [ 53 ], and MSCs harbored the potential to differentiate into cancer-associated fibroblasts (CAFs) at latter passages [ 54 , 55 , 56 , 57 ]. The malignant phenotypes of MSCs associated with CAFs could express Meflin, which is also a marker of MSCs maintaining their undifferentiated state [ 57 , 58 , 59 ].

To provide sufficient MSCs for clinical trials, MSCs need to be amplified in a large scale, which will inevitably face the issue of MSCs senescence and subsequent modifications of gene expressions [ 60 ]. Therefore, the long-term culture of MSCs often results in decreased proliferation and differentiation capacities and shortened life expectancy [ 61 ]. A standardized manufacturing process is essential for the success of clinical trials. Though the above molecules have been found to mediate the stemness of MSCs and regulate their differentiation, it remains challenging to control the fate of MSCs in a complex in vivo environment.

Heterogeneity of MSCs

Heterogeneity of MSCs is determined by multiple factors including but not limited to donors and tissue sources, cell populations, culture conditions, cell isolation techniques, cryoprotective and thawing protocols [ 62 , 63 , 64 ] (Fig.  3 ).

figure 3

MSCs exhibit heterogeneity at multiple levels. Heterogeneity of MSCs is determined by factors at multiple levels. (1) Donors at different health status, genetics, gender, and age may result in variations. (2) Tissue from different sources exhibits distinct characteristics, therefore leading to heterogeneity. (3) Cell isolation techniques may lead to distinct purity and sub-populations. (4) Cell culture environment and preservation conditions could affect the expansion and states of MSCs, therefore also affecting the heterogeneity

MSCs were defined as adherent cells with a spindle-shaped morphology in standard culture conditions according to the minimal criteria developed by the International Society of Cell Therapy in 2006 [ 65 ]. They were characterized by the following features: (1) expression of CD105, CD73, and CD90, but no expression of CD45, CD34, CD14 or CD11b, CD79a, CD19, or HLA-DR; (2) capacity to differentiate into osteoblasts, adipocytes, and chondroblasts in vitro. However, these criteria were insufficient to define MSCs as variations exist at multiple levels. First, MSCs from different donors have distinct functions due to differences in age, health condition, and other individual characteristics. Second, MSCs from different tissues ranging from adipose tissue to bone marrow could be distinct in terms of surface markers and differentiation capacities. This variation probably results from different biological, chemical, and mechanical stresses in stem cell niches, though the culture conditions are similar in vitro. Moreover, MSCs form clones, and cell heterogeneity exists both inter-clonally and intra-clonally. Extracellular matrix genes and osteogenic transcription factor-related genes show increased expression in highly osteogenic clones compared to poor osteogenic clones. Cell morphology and differentiation ability within one clone can also be remarkably different. For instance, cells located at the outer periphery express higher levels of genes related to cell proliferation (MKI67 and PODXL), while extracellular matrix genes (VCAM1) tend to be expressed in interior MSCs [ 66 ].

To identify specific cell subsets in heterogeneous MSCs, researchers have been continuously exploring characteristic cell surface markers and molecular signatures. Single cell-derived colony with rapidly dividing cells shows high colony-forming efficiency. STRO-1, CD146, and CD271 have been identified as cell surface markers for this subset [ 67 ]. However, cell subsets sharing similar surface markers would exhibit different chondrogenic differentiation capacities even under the same culture conditions [ 68 ]. RNA sequencing and microarray analysis have showed transcriptional signals predicting differentiation potential. Osterix and distal-less homeobox5 are the main transcription factors involved in osteoblast differentiation, while peroxisome proliferator-activated receptor gamma (PPAR-γ) and CCAAT/enhancer-binding protein alpha are associated with adipogenic potential [ 69 ]. In addition, MSCs with specific surface markers of differentiation potential may present various physiological functions [ 70 ]. For example, CD105 + MSCs exhibited myogenic potential assisting the repairment of the infarcted myocardium [ 71 ], while CD106 + MSCs showed enhanced multipotency and immunosuppressive ability [ 72 ]. Increasing evidence shows that MSCs comprise multiple subsets with specific surface markers. More work is needed to define these subpopulations based on biomarkers and biological functions.

Directed migratory capacity of MSCs

The therapeutic efficacy of MSCs is highly dependent on their in vivo migration and homing capacities. The migrating direction is determined by chemokine receptors expressed on MSCs and chemokines in tissues [ 73 ]. Freshly isolated MSCs have a good homing effect, which is decreased after somatic expansion. For example, the chemokine receptor CXCR4 is highly expressed on primary bone marrow MSCs, but gradually lost with passages, resulting in the less recognition of its ligand CXCL12 (also known as SDF-1α) [ 74 , 75 ]. Together, the primary MSCs are expected to have a better therapeutic efficacy due to more potent migration capacity.

However, the expression profile of chemokines in damaged tissues is often not compatible with that of receptors on MSCs. For instance, CXCL1, CXCL2 and CCL7 increased in infarcted myocardium, while expression of corresponding receptors (CCR1 and CXCR2) on MSCs was very low, resulting in low efficiency in the migration of MSCs to infarct sites [ 76 ]. To improve the migration rate, MSCs are genetically modified to express specific chemokine receptors [ 73 ]. For example, CCR7-modified MSCs efficiently migrated to secondary lymphoid organs and demonstrated significant clinical efficacy in the GVHD mouse model [ 77 , 78 ]. CXCR5-modified MSCs migrated to the damaged sites by binding to CXCL13, which was highly expressed in damaged tissues [ 79 ]. Taken together, genetically modified MSCs are an independent treatment entity and could be used as targeted therapy.

The delivery of MSCs emerges as a prerequisite to the unfoldment of their full therapeutic potential. Different delivery routes could affect cell homing, survival, and paracrine function. Systemic delivery is considered a reasonable approach. However, the reported effect in terms of homing rate, survival rate, and maintenance of cellular function was modest and transient [ 80 ] for reasons including poor migration rate from vessels to tissues and high retention rate in the liver, lungs, and spleen [ 81 ]. In contrast to intravenous delivery, intra-tissue or intra-organ delivery showed higher delivery retention and efficiency, as evidenced by a large body of studies [ 82 ]. However, clustering of MSCs and occlusions in microvasculature has been reported in some disease models such as myocardial infarction [ 83 ]. Walczak et al. reported that only cells with a diameter between 20 and 50 μm could avoid intracerebral entrapment [ 84 ]. Therefore, to maximize therapeutic efficacy, both the migratory capacity of MSCs and appropriate delivery methods should be considered.

Limited expansion of MSCs

Theoretically, MSCs can be expanded in vitro in traditional culture plates and flasks to any amount that meets experimental purpose. However, with prolonged culture duration and increased passage numbers, MSCs reach the Hayflick limit, exhibiting a marked decrease in proliferation with a transformation in morphology from a thin spindle shape to a flattened square shape. The cell density seeded in the culture containers also plays a role in the senescence of MSCs. Neuhuber et al. found the optimal cell growth of rat MSCs at 200 cells per cm 2 compared with 20 cells or 2000 cells per cm 2 [ 85 ]. In other studies, a relatively low density (~ 1.5–200 cells per cm 2 ) was suggested to support better proliferation [ 86 ]. Alterations in autocrine secretion and contact inhibition may contribute to the slow growth at high density.

Large-scale expansion in 2D plates over long term also impacts stem cell characteristics of MSCs. According to Zhao et al., hUC-MSCs at various passages have multiple mutation spectra on signatures and functions, and cells at high passage showed declined therapeutic effect in aGVHD mouse model [ 87 ]. It has been shown that chondrogenic differentiation of MSCs in 2D culture is less efficient than that of MSCs in 3D culture [ 88 ]. Therefore, 3D expansion of MSCs was developed to prevent phenotypic changes caused by monolayers, where a broad and flattened morphology upon passaging was well preserved.

Moreover, MSCs have shown the capacity to differentiate into numerous cell types such as neural cells, hepatocyte-like cells, and pancreatic islet-like cells [ 89 , 90 ]. The transient differentiation of MSCs into neural precursor-like cells may experience de-differentiation during extended culture [ 91 ]. Therefore, in vitro induction is often insufficient to yield pure functionally competent cells.

Taken together, developing the technique that can produce a huge number of cells rapidly and cost-effectively with guaranteed cell quality is paramount for the clinical progress of MSCs.

Effective components of MSC treatments

The secretion of cytoprotective factors by MSCs was first reported by Gnecchi and colleagues. They observed that Akt-MSCs (MSCs overexpressing Akt) prevented ventricular remodeling and improved the heart function following surgical myocardial infarction (MI). Since cell transplantation and myogenic pathways would be ineffective over such a brief interval, a new mechanism was proposed that the injected MSCs might act through releasing trophic factors that contribute to myocardial protection following an ischemic insult. This hypothesis was then confirmed by evident improvements in cardiac performance following injection of conditioned medium (CM) collected from hypoxic Akt-MSCs into an induced MI model, which protected ventricular cardiomyocytes with less apoptosis when subjected to a hypoxic condition [ 92 ].

In 2007, Dai et al. observed that MSCs-CM had a similar, albeit less intense, effect of MSCs in myocardial infarction, indicating that at least part of the effect observed following MSCs injection could be attributed to soluble factors [ 93 ]. In the context of neuronal damage, it has been established that the presence of BDNF, GDNF, NGF, and IGF in the MSCs secretome is necessary for the neuronal survival in vitro and in vivo [ 94 , 95 ]. MSCs-CM has demonstrated therapeutic efficacy in some other disease models including chronic kidney disease, certain lung, and liver diseases [ 96 , 97 ].

The paracrine effects of MSCs as an initial mechanism of action inspired further biological analysis of MSCs secretome [ 98 ]. Subsequent studies found more paracrine effectors, including soluble cytokines, growth factors, hormones, miRNAs, or lncRNAs that targeting a variety of cells such as immune cells and injured tissue cells [ 99 ]. In addition, the paracrine effectors could be loaded in extracellular vesicles (EVs) and exerted long-term effects [ 100 ]. In accordance, many studies have shown that MSC-derived EVs retain the biological activity of parental MSCs. It has been demonstrated that EVs showed a similar therapeutic effect as MSCs in selected animal models [ 101 ]. However, different studies found various effective components of MSCs in specific animal models and human diseases, and the interactions and functional differences between effectors remain elusive. Therefore, novel in-depth analytical techniques and platforms are warranted to investigate the MSCs secretome in the future.

Attempts to improve the therapeutic outcomes of MSCs

Although there were no attributable serious adverse events after MSC therapy, fever within 24 h and temporary pain at the injection sites are commonly occurred. Here we summarize four strategies to limit adverse events related to MSC treatments and improve the therapeutic outcomes, including genetic modifications or priming strategies to change the inherent characteristics of MSCs, and biomaterial strategies to modify the outside circumstances, and the usage of MSCs secretome (Fig.  4 ).

figure 4

Current attempts to improve MSC treatment. To improve the therapeutic efficiency of MSCs treatment, modification was made mainly in the following aspects: (1) genetic modification of MSCs by viral transduction or CRISPR/Cas9 techniques to engineer MSCs with enhanced homing, potency, or expansion capacities; (2) priming MSCs with small molecules, hypoxia, or structural stimulations by biomaterials to improve MSC function, survival, and therapeutic efficacy, thus boosting their therapeutic efficacy; (3) biomaterial strategies to improve the survival and function of MSCs by offering a scaffold for MSCs adherence, including modifications on dimensionality, stiffness, topographical cues, surface chemistry, and microstructure of biomaterials. (4) Utilize the MSCs secretome as a drug delivery platform for treatment

Biomaterial strategies to maintain more homogeneous MSCs

Biomaterials for delivering MSCs have been extensively investigated. These materials showed advantages in offering a scaffold for the adherence and survival of MSCs, as well as preserving the functional components MSCs secreted, thus elongating the effective durations in clinical treatment. However, the implantation of biomaterials could induce the foreign-body responses (FBR) in the host immune system, which can potentially result in fibrosis and failure of the implantation. Therefore, biomaterials suitable for MSCs were constructed to ameliorate the FBR and subsequent fibrotic encapsulation [ 102 ]. For example, loading MSCs with small-molecule encapsulating microparticles (MPs) can boost the duration of the products. MPs are composed of biocompatible materials that can be therapeutically tuned according to their composition, polymer molecular weight, drug loading, and release capacities [ 103 ]. MSCs loaded with degradable budesonide-containing MPs exhibited fourfold increase in IDO activity in vitro compared to MSCs without being pre-treated with budesonide [ 104 ]. This led to a twofold improvement in the suppression of peripheral blood mononuclear cells (PBMCs) activation following IFN-γ stimulation [ 105 ].

MSCs are typically delivered to a graft site using a decellularized extracellular matrix (ECM) scaffold. The advent of synthetic polymers has revolutionized tissue engineering. These polymers are highly tunable, homogenous, and cell-free materials and have a high batch-to-batch consistency taking the form of porous hydrogels, sponges, plates, or membranes [ 106 , 107 ]. However, their unique properties could exert different influences on MSCs function. Table 3 summarizes the influence of biomaterials properties on the function of MSCs, including dimensionality, stiffness, topographical cues, surface chemistry, and microstructure of biomaterials.

Genetic modification to produce MSCs with desired biologic function

Viral dna transduction and mrna/dna transfection.

To further optimize the therapeutic efficacy of MSCs, MSCs have been genetically engineered to produce trophic cytokines or other beneficial gene products in numerous preclinical models by transfecting MSCs with viral or non-viral vectors. Over the last few decades, these MSCs have successfully been engineered to express therapeutic peptides and proteins in animal models [ 119 ]. For instance, MSCs expressing thioredoxin-1 (Trx1, a powerful antioxidant, transcription factor and growth factor regulator) improved cardiac function in post-myocardial infarction rat models [ 120 ]. MSCs expressing IL-12 showed potent anticancer activity against melanoma, breast cancer, and hepatoma [ 121 , 122 ]. And MSCs expressing interferon-γ inhibited tumor growth in mouse neuroblastoma and lung carcinoma models [ 123 , 124 ]. In line with these advances achieved in animal models, several MSCs-based therapies are under clinical development (Table 4 ).

However, both viral and non-viral vectors have some limitations. Non-viral vectors present transient gene expression and low-transfection efficiency, while viral transduction is associated with a higher risk of chromosomal instability, insertional mutagenesis, and proto-oncogene activation despite the inherent high transfection efficiency [ 125 ]. The adverse immune reactions induced by viral transduction were reported to impair the stability of transgenes [ 126 , 127 ]. Therefore, the limitations and adverse responses should be valued when modifying MSCs by transfection.

Some studies made attempts on human-induced pluripotent stem cell (iPSC)-derived MSCs to obtain improved expandability. Actually, therapeutic transgenes could be inserted into iPSC-derived MSCs before MSCs derivation. This strategy could eliminate insertional mutation as well as guarantee stable expression of transgenes during prolonged expansion [ 128 ]. So iPSC-derived MSCs may be a candidate of MSCs for usage.

CRISPR-Cas9 technology to obtain highly homogeneous MSCs

With CRISPR/Cas9 technology, genetic modification of MSCs can be done with higher efficiency and specificity [ 129 ]. Compared to transcription activator like effector nuclease (TALEN) and the zinc-finger nucleases (ZFNs), CRISPR/Cas9 technology is faster, more economically efficient, and user-friendly [ 130 ]. CRISPR/Cas9-based gene manipulation has been widely employed in stem cell field particularly MSCs research, including gene knock-in, knock-out, activation or silence, etc.

CRISPR/Cas9-mediated gene knockdown in MSCs has been proved effective in treating diseases such as myocardial infarction [ 131 ]. Targeted gene knock-in promoted the differentiation capacity of MSCs and, in turn, ameliorated the insufficiency of functional cells in local sites [ 132 ]. Genetically modified MSCs have been evaluated in clinical trials. The TREAT-ME-1 study, an open-label, multicenter, and first-in-human Phase 1/2 trial, evaluated the safety, tolerability, and efficacy of genetically modified autologous MSC-apceth-101 treatment in patients with advanced gastrointestinal adenocarcinoma [ 133 ]. Further investigations are still needed to obtain unequivocal evidence on the differentiation and regeneration potentials of MSCs in vivo. Moreover, next-generation sequencing and genotypic techniques might serve as a new paradigm to improve the efficacy on targeting specific cell types for personalized medicine. CRISPR gene-engineered MSCs studies are illustrated in Table 5 .

Despite the specificity of CRISPR/Cas technology in gene delivery [ 143 ], only one clinical trial of MSCs modified with CRISPR/Cas9 has been registered (NCT03855631).

“Priming” MSCs with small molecules to exogenously boost their therapeutic function

Given current manufacture of MSCs cannot meet the requirement for clinical trials in terms of production scale, the alternative is to boost the function of limited cells through priming MSCs. Priming has also been referred to as licensing or preconditioning, which is a concept commonly used in the field of immunology, and it has been adapted to the scope of stem cells [ 144 , 145 ]. One of the commonly used strategies is priming MSCs with pro-inflammatory mediators, including IFN-γ, TNF-α, IL-1α, and IL-1β, and more priming approaches are being proposed to improve the function, survival, and therapeutic efficacy of MSCs [ 146 , 147 ]. The priming approaches could be divided into three categories based on the stimulations: (a) MSCs priming with small molecules, (b) MSCs priming with hypoxia, (c) MSCs priming with biomaterials. Table 6 summarizes some representative priming MSCs.

“Priming” MSCs resulted in exogenously boosted therapeutic function in comparison with original state. Several “primed” MSC products have been applied clinically, with the most notable being NurOwn from Brainstorm Cell Therapeutics Company. NurOwn boosted the expression of multiple neurotrophic factors (NTFs) including GDNF, BDNF, VEGF, and HGF [ 173 ]. When administered to patients with neurodegenerative diseases, NurOwn delivered multiple NTFs as well as the immunomodulatory components secreted by MSCs. This combination demonstrated impressive therapeutic efficacy in a phase 2 clinical trial (NCT02017912), in which ALS patients got reduced ALS progression 24 months after NurOwn infusion compared to the controls [ 174 ]. So the indication of NurOwn has been expanded to include multiple sclerosis.

However, priming approaches of MSCs still have many limitations in clinical translation, such as induction of immunogenicity, high costs, variable effects, and lack of good manufacturing practices (GMP) suitable for clinical application [ 175 ]. Moreover, the long-term effect of priming MSCs has not been evaluated yet. Further studies are needed to evaluate (1) the effects of different priming approaches in clinic; (2) the best sources for MSCs isolation; (3) the epigenetic modifications, immunogenicity, and tumorigenicity of primed and non-primed MSCs; and (4) the appropriate GMP standards for quality control of MSC products, including quality of cryopreserved primed-MSCs at different passages.

Utilize the MSCs secretome as a drug delivery platform for treatment

The “secretome” of MSCs, including secretory proteins such as growth factors, cytokines, and chemokines and EVs such as microvesicles (MVs; 100–1000 nm diameter) and exosomes (40–150 nm diameter), has been shown to exhibit many of the therapeutic properties of MSCs. For example, MSC-derived EVs have demonstrated similar or even superior therapeutic capacity for autoimmune diseases and neurodegenerative disorders compared with their parental MSCs [ 176 , 177 ]. They also have better safety profiles due to their better immunocompatibility. In addition, they can bypass the endothelial layers in the blood–brain barrier or blood-retinal barrier, providing an ideal cargo to deliver biomolecules to the central nervous system [ 178 ].

Several studies have demonstrated the clinical effectiveness of MSC-EVs. For example, hBMMSC-EVs revealed significant improvements in patients suffering from refractory graft-versus-host disease [ 179 ]. In another study, administration of hUCMSC-EVs resulted in overall improvement in patients with grade III-IV chronic kidney disease [ 180 ]. Nassar et al. conducted a clinical trial to assess the effects of hUCMSC-EVs on pancreatic islet beta cell mass in Type-1 diabetic patients (NCT02138331). And there are other ongoing trials conducted to determine the safety and efficacy of human MSC-EVs in ocular diseases such as promoting the healing of large and refractory macular holes (NCT03437759) and relieving dry eye symptoms in oGVHD patients (NCT04213248). Moreover, MSC-EVs have been modified to load small molecules. For example, miR-124 was loaded in exosomes to treat patients with acute ischemic stroke (NCT03384433).

Advances and perspectives to overcome challenges in MSC clinical application

Artificial intelligence (ai) in msc treatment.

Digital technology and AI are driving the revolution of healthcare industry [ 181 ]. The drug research and development became an important application field of AI technology [ 182 ]. AI in de novo design has successfully produced biologically active molecules with desired properties [ 183 ]. The discovery of drug molecules by AI has been selected as one of the "top ten global breakthrough technologies" by MIT Technology Review in 2020. The advances of AI are likewise expected to boost the understanding of MSCs therapies and help identifying the essential elements of MSCs.

AI can find new molecular compounds and emerging drug targets much faster than traditional methods, thus speeding up the progress of drug development [ 184 , 185 ]. At the same time, AI can more accurately predict the follow-up experimental results of new drugs, so as to improve the accuracy at each stage of drug development [ 186 ]. Computer-aided drug design techniques are thus revolutionizing MSCs therapies.

To understand the essential elements in MSCs treatment, AI may recognize the dynamic molecular characteristics of essential elements, which include different protein sequences, molecular structures, as well as the binding forces and stabilities between targeted molecules and cell receptors. These data could be used to train a predictive model to the utmost accuracy [ 187 ]. Predicted elements may also be produced under AI guidance. Powered by a robotic platform, a system developed by MIT researchers partially automates the production of small molecules that could be used in medicine, solar energy, and polymer chemistry. Reportedly, the new system combines three main steps. First, software guided by AI proposes a route for synthesizing a molecule, then chemical experts review this route and refine it into a chemical "recipe," and lastly, the recipe is sent to a robotic platform that automatically assembles the hardware and performs the reactions that build the molecule [ 188 ].

At present, the pharmaceutical world is increasingly engaged in technologies to shorten the time required to identify new drugs and repurpose current drugs. Since MSC therapies showed beneficial effects with complex undetermined components, AI may be well-suited to analyzing and revealing essential elements. Companies such as Merck, GSK, and Roche have developed partnerships with AI companies to construct suitable platforms [ 189 , 190 ]. However, the drug discovery process with AI is a long shot, which need to be verified in clinical trials.

Engineered MSC-EVs for treatment

Paracrine effect was discovered to mediate MSCs therapeutic efficacy in previous studies [ 191 , 192 , 193 ]. EVs are one of the major paracrine effectors, which are bilayer membrane structures transferring bioactive components [ 194 ]. The best-studied EVs can be classified into exosomes and microvesicles according to their sizes, shapes, biogenesis, origins, and compositions [ 195 , 196 ]. Due to their liposome-like structures reflecting biophysical characteristics of the parental cells, EVs are stable in vivo compared to other foreign particles [ 197 ]. Moreover, it is relatively easy to modify and/or improve the contents of EVs and their surface properties to enhance the therapeutic potential or to act as a drug delivery system [ 198 ]. These advantages make EVs promising for clinical treatment. Currently, there are 15 clinical trials registered in ClinicalTrial.gov (Table 7 ). However, none has been completed and challenges remained for the practical application of EVs.

First of all, the manufacture of large scales of MSC-EVs with high purity is difficult. MSC-EVs are isolated from MSC culture media, of which conditions including the seeding cell number, media volume, and isolation method and time of EVs can influence both the quantity and quality of EVs [ 199 ]. Therefore, optimization of culture methods (e.g., hypoxia, sheer stress, and bioreactor) combining with intensive evaluation of the pros and cons of the different EVs isolation methods is prerequisites for MSC-EVs to yield improvements. These procedures should be regulated and controlled to ensure the clinical-grade EVs production [ 200 ]. Recently, Mendt et al. reported using a bioreactor system in the GMP facility to obtain sterile, clinical-grade EVs from BM-MSCs. In that instance, the therapeutic effects of BM-MSCs on pancreatic cancer xenograft mouse models were evaluated, and feasible directions for clinical application of MSC-EVs were provided [ 201 ].

Safety and efficacy of MSC-EVs in various disease conditions need to be ensured in further preclinical and clinical evaluation. In vivo distribution analysis of fluorescence-labeled EVs has shown that MSC-EVs might have homing capacity for injured or tumor-bearing sites comparable as MSCs [ 202 ]. Long-term toxicity and immunogenicity of repetitive EVs administration using hematological examination, histopathological analysis, and immunotyping test should also be performed to find whether MSC-EVs might trigger immune responses or toxic reactions [ 203 ].

After the disclosure of precise mechanisms of action or key therapeutic factors in MSC-EVs therapy, targeted-EVs could be expanded in uniform proliferative cells such as fibroblasts via gene modification technology. Therefore, with big data-based analysis of transcriptome and proteome, engineered EVs may be manufactured with desired elements. For instance, Thomas C. Roberts et al. engineered EVs to express IL6 signal transducer (IL6ST) decoy receptors to selectively inhibit the IL6 trans-signaling pathway. Treatment in the Duchenne muscular dystrophy mouse model with these IL6ST decoy receptor EVs resulted in a reduced phosphorylation of STAT3 in muscles; further functional studies verified the in vivo activity of the decoy receptor EVs as a potential therapy [ 204 ]. Similarly, CXCR4/TRAIL-enriched exosomes were successfully obtained from MSCs overexpressing both CXCR4 and TRAIL. These exosomes exerted activity as a cooperative agent with carboplatin against brain metastasis of breast cancer in vivo, improving the efficacy of chemotherapy and highlighting a novel synergistic protocol with anticancer agents to treat brain diseases [ 205 , 206 ]. Moreover, in a Phase 1 clinical trial, IL-12 was engineered to express on the exosome surface using Codiak’s proprietary engEx Platform. This product could enhance the dose control of IL-12 and limit systemic exposure and associated toxicity. EVs can overcome the reported limitations of parental cells on various aspects, including safety, reproducibility, and cost-effectiveness related to storage and maintenance. Engineered EVs might be novel promising therapeutics for clinical application. Furthermore, to resolve current hurdles in EVs-based therapeutics, the production of EVs should be standardized and optimized, and its underlying mechanisms need further investigation.

MSC usage for pandemic diseases such as COVID-19

Pandemic diseases like 2019 novel coronavirus disease (COVID-19) have dramatically increased the number of sickness and death worldwide. Though vaccines have been developed recently, the viruses are still rapidly mutating and expanding, and the available specific and effective treatment options are currently very limited [ 207 ]. For severe or critical COVID-19 patients requiring hospitalization, acute lung injures (ALI)/acute respiratory distress syndrome (ARDS) was the main pathologic features, characterized by immunopathological complications with cellular fibromyxoid exudates, extensive pulmonary inflammation, pulmonary edema, and hyaline membrane formation [ 208 ]. Besides, inflammation and sepsis are also the leading causes of mortality in COVID-19 patients [ 209 ]. In all these cases, any treatment that could hasten recovery would be in substantial demand. MSC therapy may be one such treatment.

MSC therapeutics may be the ideal candidates for handling the broad spectrum of COVID-19 symptoms due to their multifactorial mode-of-action [ 210 ]. They can release various factors including keratinocyte growth factor, prostaglandin E2, granulocyte–macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-13 to facilitate the phagocytosis and alternative activation of alveolar macrophages, alter the cytokine secretion profile of dendritic cell subsets, and decrease the release of interferon γ from natural killer cells [ 211 ]. For example, IL-10, TGF-β, and tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase secreted from them were reported to suppress the proliferation of T cells and change the cytokine secretion profile of T cell subsets [ 212 ]. Moreover, the proliferation, differentiation, and chemotactic properties of B cells were impaired by MSCs as well. Except for the immune regulatory effects, MSCs can enhance the restoration of capillary barriers, inhibit bacterial growth, and restore alveolar ATP. All these functions mentioned above might also be effective in COVID-19 infection.

COVID-19 has been the top priority of global healthcare systems since its emergence. There have been more than 160 vaccines in development and more than 60 clinical trials are ongoing, and now, only a few vaccines have been approved [ 213 ]. The representative clinical trials of MSC therapy in COVID-19 disease were listed in Table 8 . But the rapid mutation of SARS-CoV-2 virus leads to challenges on the effect of the available vaccine. It is an urgent need to develop more universal and stable therapy to reverse or combat. Though no evidence has showed that coronavirus was eliminated completely after stem cell treatments, preliminary results were promising. Diseased patients were more likely to survive the infection after the treatment. The specific primed MSCs were also investigated for COVID-19 treatment [ 212 , 214 ]. The results will provide a strong foundation for future scientific research and clinical applications for a variety of diseases including pandemic crisis and pulmonary complications. Hopefully, the approaches utilizing MSCs particularly the primed MSCs could be vital for the success of cell therapy in treating COVID-19.


Although MSCs therapies have achieved tremendous advancements over the past decades, substantial challenges remain to be overcome. The main challenges include the immunocompatibility, stability, heterogeneity, differentiation, and migratory capacity. More and more studies are focusing on the attempts to overcome these shortcomings. Although the detailed mechanism of MSCs immunomodulatory effects is still elusive and any attempts to improve MSCs efficacy are still lack of evidence, the preclinical studies are developing rapidly and more standardized clinical trials are wildly carried out. It might be expected that the conversion to canonically registered MSC therapies will flourish with time. The lessons from the current MSCs investigations may provide critical guidance for investigators pursuing further translational processes. With the clarification of MSCs effectors and the emergences of new technologies assisting in-depth studies, MSCs are promising to be proved as effective treatment options for a variety of devastating conditions.

Availability of data and materials

The material supporting the conclusions of this review is included within the article.


Artificial intelligence

Acute lung injures

Amyotrophic lateral sclerosis

Acute respiratory distress syndrome

Bone morphogenetic protein

Brain-derived neurotrophic factor

Crohn’s disease

Conditioned medium

Extracellular matrix


Foreign-body response

Food and Drug Administration

Glial cell line-derived neurotrophic factor

Granulocyte–macrophage colony-stimulating factor

Good manufacturing practices

Graft-versus-host disease

Extracellular vesicles from human bone marrow-derived mesenchymal stromal cells

Hepatocyte growth factor

Human leukocyte antigen

Extracellular vesicles from human umbilical cord-derived mesenchymal stromal cells

Indoleamine 2,3-dioxygenase

Insulin-like growth factor

IL6 signal transducer

Major histocompatibility complex

Myocardial infarction

Massachusetts Institute of Technology


Multiple sclerosis


Nerve growth factor

Neurotrophic factors

Ocular graft-versus-host disease

Peripheral blood mononuclear cells

Transcription activator like effector nuclease


Vascular cell adhesion molecule-1

Vascular endothelial growth factor

Zinc-finger nucleases

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The authors regret that it was not possible to include many interesting studies in the field due to limited space.

This work was supported by the National Key R&D Program of China (No. 2017YFE0131600), National Natural Science Foundation of China (Nos. 81870121, 81700825, 81671585, 82070176), Natural Science Foundation of Guangdong Province, China (Nos. 2019B020236004, 2019B151502006), Science and Technology Planning Project of Guangdong Province, China (No. 2017B020230004), and Science and Technology Program of Guangzhou, China (Nos. 201906010076, 201803040005).

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Department of Hematology, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou, 510080, People’s Republic of China

Tian Zhou, Jianyu Weng, Xin Du & Peilong Lai

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, 510060, People’s Republic of China

Tian Zhou & Chang He

Department of Hepatic Surgery and Liver Transplantation Center, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, 510630, People’s Republic of China

Guangdong Provincial Key Laboratory of Stem Cell and Regenerative Medicine, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Guangzhou, 510530, People’s Republic of China

Duanqing Pei

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PLL, CH, and XD designed and wrote the review. TZ, ZNY, and JYW drafted the manuscript and prepared the figures. DQP helped to modify the manuscript. All authors read and approved the final manuscript.

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Zhou, T., Yuan, Z., Weng, J. et al. Challenges and advances in clinical applications of mesenchymal stromal cells. J Hematol Oncol 14 , 24 (2021). https://doi.org/10.1186/s13045-021-01037-x

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Received : 14 December 2020

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Published : 12 February 2021

DOI : https://doi.org/10.1186/s13045-021-01037-x

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research article on mesenchymal stem cells

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Archives of Medical Research

Epidemiological mesenchymal stem cells current clinical applications: a systematic review, introduction.

Human Mesenchymal Stem Cells (hMSCs) are multipotent stem cells capable of renewing themselves and differentiation in vitro into different kinds of tissues. In vivo hMSCs are sources of trophic factors modulating the immune system and inducing intrinsic stem cells to repair damaged tissues. Currently, there are multiple clinical trials (CT) using hMSCs for therapeutic purposes in a large number of clinical settings.

Material and Methods

The search strategy on clinicaltrials.gov has focused on the key term “Mesenchymal Stem Cells”, and the inclusion and exclusion criteria were separated into two stages. Stage 1, CT on phases 1–4: location, the field of application, phase, and status. For stage 2, CT that have published outcome results: field of application, treatment, intervention model, source, preparation methods, and results.

By July 2020, there were a total of 1,138 registered CT. Most studies belong to either phase 2 (61.0%) or phase 1 (30.8%); most of them focused in the fields of traumatology, neurology, cardiology, and immunology. Only 18 clinical trials had published results: the most common source of isolation was bone marrow; the treatment varied from 1–200 M hMSCs; all of them have similar preparation methods; all of them have positive results with no serious adverse effects.


There appears to be a broad potential for the clinical use of hMSCs with no reported serious adverse events. There are many trials in progress, their future results will help to explore the therapeutic potential of these promising cellular sources of medicinal signals.

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An overview of mesenchymal stem cells and their potential therapeutic benefits in cancer therapy (Review)

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The human body contains numerous different cell types, which make up tissues and organs with specific functions that play a role in ensuring sustainability. It was discovered long ago that differentiated cells in some tissues, e.g., skin, intestinal epithelium and blood, have a short lifecycle and are incapable of self-renewal ( 1 ). Stem cells are able to self-renew and possess developmental potency to differentiate into numerous cell types of an organism. This finding led to the concept of stem cells as small unspecialized cells in the human body devoid of a number of phenotypic traits commonly found in cells from adult tissues for maintaining static and transient cell types ( 2 ). Potency with each differentiation step classifies stem cells into totipotent, pluripotent, multipotent, oligopotent and unipotent stem cells ( 3 ). As potency decreases, the possible cell types that stem cells can differentiate into also decrease accordingly.

Stem cells are generally categorized into two main groups: embryonic and nonembryonic (somatic stem cells). Embryonic stem cells (ESCs) are pluripotent, while somatic stem cells, e.g., mesenchymal stem cells (MSCs), are multipotent ( 4 , 5 ). ESCs were first isolated from mouse embryos ( 6 ), while MSCs were discovered in monolayer cultures of guinea pig bone marrow ( 7 ). Following their initial discovery, human stem cells were isolated and cultured, whereby ESCs were derived from human blastocysts ( 8 ) while MSCs were derived from human bone marrow ( 9 ). These achievements in isolating and culturing human stem cells opened new possibilities to better understand the basic molecular mechanisms behind human development and differentiation, leading to potential new treatments for various diseases. While the potential benefits of research on human ESCs are immense, there is a major ethical issue to address, e.g., the derivation of human ESCs results in the destruction of an embryo. In addition, reliance on human embryos may also lead to the commodification and exploitation of women ( 10 – 12 ). Indeed, the potential exploitation of women involving the donation or sales of oocytes or embryos for research and the purposeful creation of embryos for research remain huge ethical issues that need to be addressed. This ethical dilemma negatively impacts the benefit-to-risk ratio, and hence, research has moved towards somatic stem cells instead. Despite the focus on ESCs, MSCs have been extensively researched in clinical settings during the past decade ( 13 – 24 ) because MSCs can be easily obtained and cultured for clinical use from multiple tissue sources that are easily accessible using minimally intrusive methods, reducing the ethical dilemmas surrounding human stem cell research ( 25 ). Additionally, MSCs can differentiate into a variety of cell types that confer pleiotropic effects when used for therapeutic purposes ( 26 ). MSCs were initially discovered in bone marrow, and studies have reported that these stem cells can also be found in other postnatal organs and tissues, e.g., brain, kidney, liver, lung, spleen, adipose tissue, muscle, hair follicles, teeth, placenta, and umbilical cord ( 27 , 28 ). The International Society for Cellular Therapy (ISCT) defines three minimal criteria that need to be fulfilled for MSCs to overcome the issue of different characteristics due to isolation from different tissue types ( 29 ):

1. MSCs must adhere to plastic surfaces when cultured in vitro .

2. The surface anti-genes CD73, CD90, and CD105 must be expressed by MSCs, while CD34, CD45, CD14 or CD11b, CD79α or CD19, and HLA-DR surface molecules should be absent.

3. MSCs must be able to differentiate into different mesodermal cell types, e.g., adipocytes, chondrocytes, and osteoblasts, when cultured in vitro under certain conditions.

In addition to these criteria, the ISCT recommended three additional conditions in 2019 to further clarify the nomenclature of MSCs to avoid confusion between mesenchymal stem cells and mesenchymal stromal cells ( 30 ). The tissue-source origin of MSCs should be documented to highlight tissue-specific properties, e.g., phenotypic, functional and secretome behaviour. Comprehensive in vitro and in vivo data demonstrate the stemness of MSCs associated with a robust matrix of functional assays that test the functionality of MSCs in vitro and in vivo based on their proposed utility.

Previous studies have reported that MSCs are multipotent and capable of differentiating into cells of mesodermal, ectodermal, and endodermal lineages ( 29 , 31 – 33 ). This plasticity of MSCs and their self-renewal capacity make these cells promising therapeutic targets for various diseases, including cancer treatment and tissue regeneration. MSCs undeniably offer immense potential in the field of medicine; however, the cells also present potential danger due to their ability to differentiate into tumour-associated fibroblasts ( 34 – 36 ), which support tumour growth through their secretome ( 37 , 38 ) and resistance to apoptosis ( 39 ). Due to their conflicting role in cancer progression and regression, efforts to utilize MSCs in anticancer therapies have been unsuccessful. Therefore, it is important to understand the underlying molecular mechanisms of MSCs to fully utilize their therapeutic potential.

Genetic regulators for multipotency of MSCs

Significant advancements in DNA sequencing, computational biology, and bioinformatics have been made to identify transcriptional processes associated with the multipotency of MSCs. Based on previous studies, cyclin L2 (CCNL2), stromal cell-derived factor 1 (CXCL12), podocalyxin-like protein (PODXL), and ubiquitin carboxyl-terminal hydrolase 1 (USP1) were identified as four genes responsible for maintaining multipotency, chromosomal integrity, and MSC functions ( 40 – 42 ). CCNL2 was reported to inhibit proliferation and cell specialization while promoting apoptosis upon upregulation in mouse embryonic carcinoma P19 cells. In the same study, CCNL2-overexpressing P19 cells had a remarkably decreased S phase and reduced expression levels of myocardial cell differentiation-related genes, e.g., cardiac actin, GATA binding protein 4 (GATA4), myocyte-specific enhancer factor 2C (Mef2C), homeobox protein Nkx-2.5 (Nkx2.5), and B-type natriuretic peptide (BNP) ( 43 ). On the other hand, CXCL12 is a chemokine protein that induces the migration of stem cells. It functions by binding to CXC chemokine receptor (CXCR) 4, CXCR7 and atypical chemokine receptor 3 (ACKR3) ( 44 , 45 ). CXCL12 has been reported to be responsible for cell survival, growth and migration during tissue/organ development ( 46 ). While the exact mechanism by which CXCL12 helps maintain the stemness of MSCs has not been elucidated, there are numerous reports on its function in other stem cells. The CXCL12-CXCR4 axis was found to be responsible for cell migration, while the CXCL12-CXCR7 axis promotes cell adhesion in cardiac stem cells. Similar findings also reported the importance of CXCL12-mediated CXCR4 signalling in controlling the position of haematopoietic stem cells in bone marrow niches, which contain limiting lymphoid-instructive cytokines that are responsible for the multipotency of HSCs and their maintenance ( 47 ). A study confirmed that CXCL12-mediated CXCR4 signalling promotes the proliferation, survival, and migration of mesenchymal stromal cells in vitro ( 48 ). It is also likely that CXCL12 acts through a similar mechanism to help MSCs maintain their stemness.

PODXL is mainly involved in cell proliferation and oncosphere formation ( 49 ). However, the exact mechanism of action in maintaining the multipotency of MSCs is currently not well understood. A previous study reported that higher expression of PODXL and CD49f in MSCs increased the clonogenic potential, viability, and differentiation capabilities of MSCs ( 41 ). There may also be an interaction between PODXL and CCNL2, whereby both genes work together to help maintain the multipotency of MSCs. Nonetheless, further studies are warranted before this phenomenon can reach a suitable conclusion. USP1 encodes a deubiquitinating enzyme. USP1 was also found to stabilize inhibitors of DNA binding, which play a role in inhibiting cell specialization while enhancing proliferation ( 42 ). As interest in using MSCs for therapeutic purposes grows. Moreover, previous studies have reported other genes and novel mechanisms by which the stemness of MSCs is maintained ( 50 – 52 ). The therapeutic potential of MSCs mostly stems from their ability to self-renew and differentiate. The exact mechanism by which MSC multiplicity is maintained remains ambiguous, and likely, these genes work together in a balancing act to ensure the renewal and stemness of MSCs. Therefore, a clearer understanding should be made available to ensure the safety and efficacy of treatments using MSCs. After all, both the potential therapeutic benefits and danger come from the self-renewal ability, migration, and stemness of MSCs.

Extrinsic regulators for multipotency of MSCs

The niche microenvironment strongly influences the behaviour of stem cells. As mentioned, CXCL12 maintains multipotency by directing MSCs to specific niches, where secreted factors influence their self-renewal and stemness ( 53 ). This phenomenon indicates that the behaviour of MSCs is determined by the interaction between intrinsic transcriptional genes and extrinsic factors of the environment. It has been established that the protein kinase B (Akt) and extracellular-signal-regulated kinase (Erk) signalling pathways control both stem cell proliferation and survival, while the Wnt, Notch, and Sonic hedgehog (Shh) signalling pathways regulate stem cell renewal and differentiation ( 54 – 57 ). A study also proposed two novel mechanisms that help to maintain the stemness of MSCs via the scrapie responsive gene 1 (SCRG1)/bone marrow stromal cell anti-gene 1 (BST1) ligand-receptor combination and cell-cell adhesion through N-cadherin ( 52 ). An improved understanding of the underlying mechanism involved in stem cell renewal and differentiation is important because the original abilities are lost at a high rate during long-term in vitro culture ( 58 , 59 ). Therefore, current work should develop novel techniques to ensure that MSCs maintain their multipotency despite long-term in vitro culture. This would, in turn, maintain the potential of MSCs to be used in regenerative medicine and cell therapy.

Epigenetic factors influence the differential gene expression in MSCs that causes cell differentiation. Hence, the DNA sequences of MSCs and their specialized cell types are similar, with almost no difference. Commonly studied epigenetic modifications include DNA methylation and histone modification, e.g., methylation, acetylation, ubiquitylation, and microRNAs. Once epigenetic modifications occur, gene expression can be influenced by changing the availability of gene promoters, thus affecting the recruitment of supplementary chromatin-modifying enzymes or transcriptional regulators that drive stem cell differentiation ( 60 ). For example, runt-related transcription factor 2 (Runx2) regulates most osteoblast-specific genes by working together with numerous coactivators and corepressors that alter the binding of Runx2 to the osteocalcin promoter. This binding modification occurs through DNA methylation and acetylation of histones H3 and H4 ( 61 ). Additionally, Runx2 changes the expression of its target in response to other signals, e.g., transforming growth factor-beta (TGF-β), bone morphogenetic protein (BMP) and Wnt signalling pathways ( 60 ), is responsible for the osteogenic lineage. MSCs can also undergo adipogenic differentiation, whereby hypomethylation of the genes encoding peroxisome proliferator-activated receptors gamma-2 (PPARγ2), fatty acid-binding protein 4 (FABP4), leptin (lep) and lipoprotein lipase (lpl) was reported to be responsible for these mechanisms ( 61 , 62 ).

In addition to secreted factors, the cyclic tensile strain that can alter cell behaviour should be considered another microenvironmental factor. MSCs have been observed to lose multipotency and spontaneously differentiate after prolonged passaging in vitro ( 25 , 63 ). Therefore, in vitro culture conditions must be optimized to maintain the multipotency of MSCs for their therapeutic potential in clinical settings. A study found that low actomyosin contractility induced by restricting the cells to small islands during initial culture is necessary to ensure the stemness of MSCs ( 64 ). A disparity in differential gene expression when MSCs are cultured in 2D and 3D culture systems is likely due to the interaction between the cells in an intricate 3D structure compared to that in a monolayer 2D culture ( 65 ). Recent studies have also found that cyclic tensile strain promotes bone marrow-derived MSCs (BMSCs) to differentiate into cardiomyocyte-like cells ( 66 ) and adipose stem cells to differentiate into the osteogenic lineage ( 67 ). However, the regulatory pathways and epigenetic factors that might be involved seem to depend on the source of MSCs and the desired cell lineage.

Clinical applications of MSCs

MSCs have been the subject of clinical trials for the past decade, but the outcomes have fallen short of expectations despite promising data in animal models. Studies continue to emerge, as there is no denying the potential of MSCs to treat a wide variety of human afflictions, e.g., neurodegeneration, ageing, blindness, diabetes, and cancers ( 1 ). It is crucial to realistically assess the time and effort required to establish new clinical settings for numerous therapeutic applications. The same concern regarding the efficacy and safety of treatment must also always be at the forefront when considering the usage of MSCs, as there are crucial biological and pharmacological discrepancies in preclinical and clinical studies. The first clinical trial using MSCs as a therapeutic agent was in 1995 ( 68 ). Since then, MSCs have become the most widely clinically studied cell-based therapy worldwide ( 69 ). MSCs are currently classified as advanced therapy medicinal products (ATMPs), which follow the Good Manufacturing Practices (GMP) guidelines of the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) to authenticate and ensure the quality of cells before their administration to patients ( 70 ). This compliance with GMP includes the sources of MSCs, reagents, equipment, packaging materials, procedures, laboratory staff, environment, and final cellular medicine ( 71 ).

It is of the utmost importance that GMP conditions are maintained according to the international and national medicinal governing framework. This act ensures the quality of the administered MSCs and prevents possible contamination issues that may cause adverse reactions in patients and even death. However, there is currently a lack of unified and standard criteria for manufacturing MSCs as a therapeutic agent due to some differences over specific issues depending on the USA, Europe, Canada, Singapore, Japan and so forth. Despite this challenge, consistent physical and microbiological testing of the MSC production laboratory and cleanrooms to ensure the sterility of the production process is also warranted ( 72 ). This act fulfils the requirement of International Standard Organization (ISO) standard 14644.

Currently, 1,088 studies registered as clinical trials list MSCs as a clinical intervention. The majority of these trials, whether ongoing or completed, are phase 1 or 2 studies that evaluate the safety and efficacy of MSCs in humans. Despite the most promising results, MSC-based therapies still have significant limitations due to the nature of the stem cells, e.g., MSCs markedly differ in gene expression profile, cell differentiation ability, growth rate, and therapeutic capacity, depending on their tissue source ( 63 ). Therefore, it may be vital to isolate and culture homogenous populations of MSCs to improve the efficacy and safety of the treatment. The method of transplanting MSCs isolated and grown in large batches from unrelated donor tissues is known as allogeneic transplantation; in contrast to autologous therapy, MSCs are extracted and grown from treated patients. The benefits of allogeneic transplantation include:

1. Efficiency, such as the isolation, expansion, and validation of MSCs from the patient, is not required.

2. The therapeutic functions of allogenic MSCs remain the same, unlike autologous MSCs, which have been reported to have impaired functions when isolated from elderly individuals ( 73 , 74 ).

3. A well-established stock of MSCs following strict GMP requirements reduces the variability of donors and improves the success rate of the treatment.

Allogeneic transplantation, however, may induce an immunogenic response ( 75 ), especially when administered repeatedly at the same site ( 76 ). This phenomenon makes allogeneic therapy less desirable, especially when it needs to be administered for an extended period. At the same time, in vitro studies have reported on the hypoimmunogenic properties (immune-privileged) of MSCs, while the findings of in vivo studies were less conclusive ( 77 ). It was theorized that MSCs lose their hypoimmunogenic properties upon differentiation, which triggers the immune response and rejection after implantation into the host ( 77 , 78 ). A study also reported that different transplantation routes and microenvironments could influence the immunogenicity of implanted MSCs ( 79 ). Because of such inconclusive in vivo results, a paper suggested the term immune evasive be used instead of immune-privileged to describe the immunogenicity of MSCs. It was also reported that while MSCs may not be truly immune-privileged, the rejection of allogeneic MSCs occurs at a slower rate than that of other cell types ( 80 ). This phenomenon means that future studies should also examine strategies to maintain or prolong the immunogenicity of allogeneic MSCs to maximize the therapeutic benefits.

In contrast, autologous transplantation, which triggers less risk of immunogenic response, is an alternative. Autologous MSCs are easily available without identifying a suitable donor ( 81 ). Autologous MSCs also overcome the limitation of long-term in vitro culture for allogeneic MSCs, leading to loss of multipotency, morphological changes, and an increased risk of malignancy ( 25 , 74 ). Nonetheless, the challenge and reliance on autologous MSC transplantation mean that a well-optimized and established protocol for the isolation and ex vivo preparation of MSCs will be required. Such precise standardization may be difficult, as several exogenous factors greatly affect the biological properties of MSCs ( 70 ). Autologous MSCs may not be suitable for treating certain genetic diseases due to the mutations present in stem cells. Flaws in the genetic sequence hinder both the immunomodulatory function and regenerative traits of MSCs. For example, MSCs isolated from patients suffering from systemic lupus erythaematosus have a senescent phenotype with diminished capabilities to differentiate, migrate and regulate the immune system ( 79 , 82 – 84 ). Therefore, more preclinical and clinical studies are required to obtain more information related to the utility of MSCs as a therapeutic approach. Supplementary studies on the basic biology of MSC maintenance and the regulators of MSC differentiation would also provide a clearer picture of how to better administer MSCs as therapeutic agents in the future.

Most of the published clinical studies employing MSCs for diseases have specific treatments with positive outcomes. In neurology, ischaemic stroke patients treated with MSCs yielded positive results, whereby the patients showed significantly improved neurological and motor functions ( 85 – 88 ). Among all of the studies conducted, serious adverse events that were reported included transient ischaemic attack, seizure, asymptomatic subdural haematoma/hygroma, urinary tract infection, sepsis, pneumonia, hyperglycaemia, neutrophilia, shingles, ischaemic stroke, cellulitis, muscle cramps, fracture neck femur, and peripheral vascular disease ( 89 ). However, these side effects were attributed to the procedure rather than cell therapy. The study also reported promising results in the field of cardiology. Studies have shown that diseases, e.g., dilated cardiomyopathy and ischaemic or nonischaemic heart failure, have had clinical and pathophysiological improvements; no serious adverse effects were reported, demonstrating the treatment's safety profile ( 19 , 90 – 92 ). Patients suffering from cartilage lesions and/or osteoarthritis, especially in the knee, were reported to have a clinical improvement in pain, stiffness, and functionality when treated with MSCs. These results show the broad potential of MSCs for clinical usage with no serious adverse effects linked to cell therapy.

Therapeutic potential of MSCs

Interest in developing MSCs as therapeutic agents has not waned in the slightest, despite the obstacles faced, largely due to their immense therapeutic potential. In addition to being multipotent with self-renewing capabilities, MSCs also have the added benefits of migrating to the injury site and promoting tissue regeneration ( 26 ). This phenomenon means that MSCs can be a form of personalized therapy (when opting for autologous therapy) that is site-directed, promotes tissue restoration, and replaces damaged cells through differentiation. It is, therefore, unsurprising that scientists are so invested in advancing this field of research since the therapeutic agent reaches the targeted tissue for effective disease treatment. As MSCs have a natural tendency to be attracted towards damaged sites and the tumour microenvironment, the cells are a prime candidate for further investigation, as MSCs seem to be independent of the type of tumour, immunocompetence and delivery route ( 93 ).

Insight into the mechanism underlying the mobilization of MSCs to the injury site is still limited, but CXCL12-mediated CXCR4 signalling is most likely involved as a pathway that mediates cell migration ( 94 ). Secreted chemokines can mediate inflammation in the tumour microenvironment, and wounds are responsible for attracting MSCs ( 95 ). As the chemotactic properties of MSCs seem to be similar to those of other immune cells, the established model of leukocyte migration can be used as a template to study the factors involved in MSC migration ( 95 ). Other chemokine receptors that react to signals from the injury site or tumour microenvironment induce CCR1-2, CXCR1-2, CCR4, CXCR4-6, CCR7-10, and CX3R1 expression in MSCs ( 95 ). In addition, cell adhesion molecules expressed by MSCs, e.g., CD44, CD49d, CD54, CD102 and CD106, are thought to be involved in MSC migration to injury sites ( 26 , 96 ).

A wide variety of trophic mediators and growth factors are secreted to initiate tissue regeneration once MSCs arrive at the injury site. The pleiotropic effects conferred by MSCs towards damaged tissues include anti-inflammation, immunomodulation, and enhanced cell survival and angiogenesis ( 97 , 98 ). Among these therapeutic effects, anti-inflammation and immunomodulation are key elements that make MSCs an attractive target to study because the immune system plays an integral role in regulating tissue repair and regeneration through healing, scarring and fibrosis ( 99 ). The immunomodulatory process of MSCs occurs through the secretion of several soluble factors that interfere with the immune system, and the inflammation process takes place through cell-cell interactions ( 100 , 101 ). The immunosuppressive effect of MSCs was enhanced by increasing the binding between MSCs and T-cells through intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) ( 102 ). A similar phenomenon was reported when MSCs were shown to heighten the suppressive regulation of T-cells and macrophages regarding proinflammatory macrophages ( 103 ).

The flexibility of multipotent MSCs to differentiate into a wide variety of cells would then allow the cells to replace damaged or dead cells. However, reports on this mechanism are inconclusive, as the engraftment of MSCs is transient, and instead, MSCs secrete specific factors that grow and differentiate into local precursor cells ( 26 ). The potential of MSCs in tissue repair and regeneration is undeniable, regardless of the exact mechanisms.

MSCs in cancer therapy

Over the years, multiple reports have been published that strongly suggest the mechanism of action of MSCs. These actions are mainly attributed to the ability to migrate to the injury site ( 104 – 106 ), the paracrine effect of the secretome ( 107 , 108 ), and the immunomodulatory ability ( 109 , 110 ). The benefits of MSCs are enticing, and it is important to consider the potential side effects and major risk factors that are often associated with stem cell transplantation. There have been contradictory results in describing the anti- and pro-tumour effects of MSCs. As mentioned above, the therapeutic role of MSCs in cancer therapy is similar to that in other diseases; tumours secrete similar chemoattractants to damaged tissues, which initiate the migration of MSCs to the target site through the CXCL12-CXCR4 signalling pathway ( 111 – 114 ). MSCs have also been reported to interact with cancer cells, directly and indirectly, affecting tumour development ( 26 ). Moreover, MSCs secrete various cytokines and growth factors, which alter cellular activities, e.g., cell proliferation (cell cycle), angiogenesis, cell survival, and immunomodulation, to indirectly influence tumour growth. For example, BMSCs were described to enhance the proliferation of B16-LacZ cells and increase tumour size when both cell lines were coinjected into syngeneic mice via enhanced angiogenesis ( 115 ). In contrast, BMSCs were also reported to inhibit proliferation, migration, and invasion and induce cell cycle arrest, which led to apoptosis of human glioma U251 cells by downregulating the PI3K/Akt pathway ( 116 ).

Indeed, such paradoxical results are not uncommon, as divergent effects on cell growth, invasion, and migration have been reported when MSCs sourced from the human umbilical cord were cocultured with glioblastoma cancer stem cells, e.g., direct contact between both cell lines caused an inhibitory response ( 117 ). At the same time, the release of soluble factors triggered a stimulatory reaction ( 117 ). Similar opposing effects were observed during an in vivo study investigating whether coinjection and distant injection of MSCs with breast tumour 4T1 cells exerted different effects on tumour growth ( 118 ). Coinjection supported tumour growth, while in the distant injection model, it inhibited tumour growth by promoting host antitumour immunity ( 118 ). Likewise, MSCs derived from umbilical cord blood and adipose tissue also had divergent effects on the proliferation of glioblastoma multiforme. The former inhibited and promoted the proliferation process ( 119 ).

Several studies have found that upon being recruited to tumour sites, the multipotency of MSCs enables their self-differentiation into carcinoma-associated fibroblasts, which directly contribute to cancer progression ( 120 – 122 ). In addition, MSCs were reported to promote tumour growth and angiogenesis through the secretion of proangiogenic cytokines, e.g., interleukin (IL)-6, vascular endothelial growth factor (VEGF), and transforming growth factor-β (TGF-β) ( 123 – 125 ) ( Fig. 1 ). MSCs also enhanced the metastasis of human breast cancer cells by promoting de novo production of lysyl oxidase (LOX) by cancer cells ( 126 ). In addition, MSCs are able to modulate the production of regulatory T-cells and inhibit the activity of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs), protecting breast cancer cells from the immune system ( 127 ). Similar immunosuppressive effects were observed when MSCs were reported to promote lung cancer metastasis ( 128 ). It was suggested that MSCs have the ability to form a cancer stem cell niche in vivo where tumour cells can preserve the potential to proliferate, thus sustaining the malignant process ( 129 ).

In contrast, MSCs increased the sensitivity of breast cancer cells to radiotherapy and impeded tumour progression by downregulating the signal transducer and activator of transcription 3 (Stat3) signalling pathway ( 130 ). Another study found that MSCs hampered hepatic cancer growth through the secretion of paracrine factors that lowered the insulin-like growth factor 1 receptor (IGF-1R), phosphatidylinositol 3-kinase (PI3K) and Akt signalling pathways ( 131 ). In addition, microRNA-4461 isolated from BMSCs was reported to inhibit tumour pathogenesis in colorectal cell lines and tissues by downregulating the expression of COPB2 ( 132 ). MSCs also inhibited vascular growth in glioma cells by downregulating the platelet-derived growth factor (PDGF)/PDGFR axis ( 133 ). Antiproliferative effects and apoptosis were observed when ovarian cancer cell lines were cocultured with conditioned media of MSCs derived from human bone marrow, adipose tissue, and umbilical cord ( 134 ). The study found that the conditioned media of MSCs showed an increase in IL-4 and IL-10 but a decrease in granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-6, and IL-9. It is undeniable that anti-inflammatory cytokines play an important role in cancers ( 135 – 137 ). However, controversial findings have been reported regarding whether cytokines support or hinder tumour progression ( 138 – 141 ). Regardless, MSCs have been shown to modulate the immune response through the balanced secretion of proinflammatory and anti-inflammatory cytokines ( 142 ). Therefore, this duality of function found in the secretome of MSCs and the complex cell-to-cell interaction between MSCs and cancer cells might be the reason for the conflicting reports regarding the role of MSCs in cancers.

Although the underlying mechanisms are not yet fully understood, there is a consensus that the differences in experimental design, e.g., tumour models used, route of cell administration, control group, tissue source, dosage use, and timing of the treatment that may affect the final results, should be considered ( 37 , 117 – 119 , 143 , 144 ). Research should not make conclusions about the utility of MSCs in cancer therapy based on a single study. Instead, standardized protocols should be established to ensure that the data obtained are more comparable to understand the interaction of MSCs with cancer cells. Additionally, precautions should be taken before the clinical introduction of MSCs for treating cancers since the heterogeneous characteristics of MSCs are easily susceptible to different pathological conditions present in patients, which can hinder the therapeutic mechanisms.

Potential strategy in utilizing MSCs for cancer therapy

MSCs are recognized for their ability to migrate towards tumour sites ( 145 , 146 ), but the literature to support the direct use of MSCs to treat cancer patients remains insufficient. MSCs can play a prominent role in reducing cancer progression since efficient intracellular tracking and directed delivery to the targeted site improve the pharmacological properties of anticancer drugs ( 147 , 148 ). One of the earliest studies developing MSCs for the delivery of biological agents found that MSCs genetically modified to express interferon-β (IFN-β) lowered tumour growth and doubled the survival rate of mice compared to the control group ( 149 ). In addition, IFN-β-transfected MSCs administered cisplatin triggered a high level of apoptosis in a melanoma xenograft mouse model ( 150 ). IFN-β-modified MSCs derived from the human umbilical cord were also reported to induce apoptosis in MDA-MB-231 cells ( 151 ). Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising target that selectively induces apoptosis in cancer cells. TRAIL-modified MSCs have been reported to exert antitumour effects in different cancer cell lines and a mouse melanoma model ( 152 – 156 ). In addition, MSCs have been genetically modified to deliver other cytokines, e.g., IFN-γ ( 156 ), IL-2 ( 157 ), IL-12 ( 158 ), and IL-24 ( 159 ), for antitumour effects.

Numerous studies have been conducted to explore the possibility of enhancing the inherent therapeutic properties of MSCs using genetic engineering. These studies mainly focused on four crucial points: improving migration, adhesion, and survivability while reducing the cell senescence of transplanted MSCs ( 160 – 162 ). This phenomenon is accomplished by inserting a vector loaded with a constructed genetic cassette into MSCs; the cassette expresses certain genes constantly or can be controlled with a gene switch ( 163 ). For example, adipose-derived MSCs (AdMSCs) were transduced with a retroviral vector to upregulate the expression of CXCR4. The study reported that the transduced MSCs showed increased motility, invasion, and placement in the bone marrow when injected into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice ( 164 ). In addition to CXCR4, other genes involved in MSC migration, e.g., aquaporin-1, can be modified. It was reported that the overexpression of aquaporin-1 and CXCR4 promoted the migratory ability of MSCs via the Akt and Erk pathways ( 165 ). MSCs have also been genetically engineered to overexpress integrin-linked kinase (ILK). The study found that genetically modified MSCs had 1.5-fold higher survivability and a 32.3% higher adhesion rate when engrafted into an ischaemic myocardium model, with a higher retention rate of ~4-fold ( 166 ). In addition, BMSCs and AdMSCs were reported to have increased proliferation and differentiation potential when engineered to overexpress Oct4 and Sox2 ( 167 , 168 ). Genetic engineering has the potential to circumvent the current problems that limit the application of MSCs in clinical settings and improve their potential therapeutic properties. Despite the immense benefits, this technique also has potential drawbacks, e.g., the risk of insertional oncogenesis due to viral vectors to introduce plasmid DNA, adverse immune reactions, and high production costs ( 169 ). Great precautions should be taken when considering the use of genetically modified MSCs for cancer therapy.

In addition, previous studies have established a connection between specific Toll-like receptors (TLRs) and the immunomodulatory properties of MSCs ( 170 – 172 ). Interestingly, a study reported that TLR-4-primed MSCs (MSC1) exhibited a proinflammatory phenotype, while TLR-3-primed MSCs (MSC2) secreted immunosuppressive mediators ( 173 ). Indeed, the polarization of MSCs into specific immunomodulatory phenotypes is a promising strategy as well. For example, macrophages cocultured with MSCs showed evidence of alternatively activated macrophages with high levels of CD206 and IL-10 but low levels of IL-12, which displayed a higher level of phagocytic activity ( 174 ). Studies have also reported that TL-3- and TL-4-primed MSCs preserved and enhanced the function of neutrophils through the combined action of IL-6, IFN-β, and GM-CSF ( 175 , 176 ). Furthermore, MSC1 was observed to recruit lymphocytes by activating T-cells and secreting macrophage inflammatory protein-1 (MIP-1), CCL5, CXCL9, and CXCL9 ( 177 ). In contrast, MSCs can change macrophages from a TNFα-secreting MSC1 phenotype to an immunosuppressive IL-10-expressing phenotype through a prostaglandin-(PGE-)2-based mechanism ( 178 ). MSCs have also been reported to inhibit IL-2-induced NK cell proliferation and prevent the initiation of effector functions, e.g., cytotoxic activity and cytokine production, with the production of the soluble factors indoleamine 2,3-dioxygenase (IDO) and prostaglandin E2 (PGE2) ( 179 ). MSCs influence tumour growth through immunomodulation, and as discussed earlier, the polarization of MSCs for cancer treatment warrants further investigation. After all, it is widely accepted that chronic inflammation is a critical hallmark of cancer that elevates the risk of malignancy ( 180 ). The anti-inflammatory cytokines secreted by MSCs can circumvent these effects. On the other hand, tumour cells evade the immune system by avoiding immune recognition and developing an immunosuppressive microenvironment ( 181 ), which can be overcome with the help of MSCs boosting the innate immune system. Therefore, careful and purposeful polarization will benefit the field of cancer therapy and facilitate manipulation of the immunomodulatory capacity of MSCs.

Studies have also investigated the potential of MSCs to act as vectors for oncolytic viruses. For example, MSCs were used as vectors to deliver oncolytic herpes simplex virus to human brain melanoma metastasis models grown in immunodeficient and immunocompetent mice. This study reported that the intervention significantly prolonged the life of the mice through immunomodulatory actions compared to the control group ( 182 ). A recent in vivo study also explored the possibility of using MSCs derived from menstrual blood as a vector for CRAd5/F11 chimaeric oncolytic adenovirus to treat colorectal cancer. It was reported that the chimaeric oncolytic adenovirus was successfully delivered and accumulated at the tumour site, and it inhibited tumour growth ( 183 ). A mathematical model to quantitatively predict the efficacy of MSCs acting as vectors for virotherapeutic agents in vivo has been developed, indicating that MSCs are a promising strategy that improves the efficacy and safety profile of the treatment ( 184 ).

MSCs can also be primed with anticancer drugs for targeted delivery due to their preferential migration towards the tumour site and relative resistance to cytostatic and cytotoxic drugs ( 185 – 187 ). For example, MSCs acquire strong antitumour activity after packaging and delivering paclitaxel (PTX) through extracellular vesicles ( 188 ). The same study also demonstrated that it is possible to produce drugs with higher cell-target specificity by utilizing MSCs as a factory to package the drugs. Similar studies reported that MSCs isolated from different sources were primed with PTX and tested against different cancer cell lines ( 187 , 189 – 191 ). Other drugs were also tested for priming MSCs, e.g., doxorubicin and gemcitabine. A study reported similar results whereby MSCs effectively incorporated the active form of the drugs and released sufficient quantities to produce a significant inhibition of squamous cell carcinoma growth in vitro ( 192 ). Researchers have explored the possibility of using nanoparticles to improve the payload and delivery capacity of MSCs ( 193 , 194 ). All of these studies indicate that MSCs are able to take up and subsequently release drugs in a targeted and gradual manner, which improves the efficacy of anticancer drugs.

Due to the short half-life of most anticancer drugs in the body and their high toxicity to healthy cells, direct administration of these drugs is often associated with unwanted side effects. For example, nausea and vomiting, tiredness, changes in taste, dry mouth, loss of appetite, constipation, and hair loss are common side effects faced by chemotherapy patients ( 195 ). Thus, using MSCs as vectors to deliver therapeutic proteins or anticancer drugs can help to solve this issue advantageously. MSCs can exert therapeutic effects locally due to selective migration and accumulation in tumour sites, increasing treatment efficacy and reducing systemic toxicity. Currently, divergent drugs are being investigated for different cancer therapeutic purposes. For example, MSCs were reported to enhance the therapeutic capabilities of tendon repair when pretreated with pioglitazone ( 196 ). Other studies using pioglitazone as the priming agent also found similar results, where pretreated MSCs had greater therapeutic effects on lung regeneration in an emphysema mouse model ( 197 , 198 ). Pioglitazone has been administered indirectly to breast cancer cells via stem-and-cancer cell interaction ( 199 ). Through this process, modified and viable pretreated stem cells are subsequently administered to patients, and pretreated stem cells are allowed to interact with cancer cells in the patients' bodies. Considering that pioglitazone has been reported to possess anticancer effects ( 200 – 202 ), it may be beneficial to examine the possibility of priming MSCs with pioglitazone for cancer therapy. After all, using MSCs pretreated with pioglitazone as a strategy to improve the overall therapeutic effects, as reported in our study ( 199 ), remains rare. Despite the study on cardiomyogenic transdifferentiation and cardiac function ( 203 ), as mentioned above, MSCs pretreated with pioglitazone for cancer therapy remain to be characterized. A similar strategy was conducted using AdMSCs pretreated with a peroxisome proliferator-activated receptor gamma (PPARγ) agonist to improve the regeneration effects in an elastase-induced emphysema mouse model ( 197 ). Indeed, human umbilical cord-derived mesenchymal stem cells pretreated with IL-6 were also found to abolish the stem cell growth-promoting effect on gastric cancer cells ( 204 ). The potential therapeutic strategies of MSCs in cancer therapy are summarized in Fig. 2 .

Although the potential benefit is undeniable, there are potential risks in using MSCs for cancer treatments. These risks can be categorized as acute issues, e.g., inflammatory reaction or embolic phenomenon, intermediate issues, e.g., graft-versus-host disease (GVHD) or secondary infection, or long-term issues, e.g., risk of tumour growth ( 142 ). It was reported in a clinical study that patients treated with MSCs commonly died due to infection ( 205 ). This phenomenon, coupled with the fact that MSCs can potentially promote tumour growth instead of inhibiting it, as previously discussed, makes it a risky treatment option. However, more studies must be conducted to provide future evidence and improve the therapeutic effects of modified MSCs in cancer treatments. These cells hold great potential to revolutionize the current cancer therapies that are available.

Concluding remarks and future perspectives

It is undeniable that stem cells are promising therapeutic alternatives for numerous human diseases. While the motivation to benefit human health is noble, researchers should take precautions in this field to prevent the potential exploitation of vulnerable groups. Efforts should also be directed towards using MSCs in autologous and allogeneic transplantation, as they do not raise the same ethical concerns as ESCs. In addition, MSCs benefit from their ability to carry anticancer payloads through genetic manipulation or pretreatment of the cells, leading to use in regenerative medicine and potentially oncology. Therefore, it is important to obtain as much information as possible to ensure that stem cell-based therapy is reliable, effective, efficient, safe, and affordable. It should be developed with the physiological condition of the patients in mind to truly benefit humanity.


Not applicable.

The present project was funded by the Exploratory Research Grant Scheme Fasa 1/2013 (grant no. 203/CIPPM/6730098).

Availability of data and materials

Authors' contributions.

SKL and BYK contributed to the conception and design of the study. SKL drafted the manuscript and BYK revised the manuscript. Both authors have read and approved the final manuscript. Data sharing is not applicable.

Ethics approval and consent to participate

Patient consent for publication, competing interests.

The authors declare that they have no competing interests.


November-2021 Volume 22 Issue 5

Print ISSN: 1792-1074 Online ISSN: 1792-1082

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research article on mesenchymal stem cells



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Role of human mesenchymal stem cells in regenerative therapy.

research article on mesenchymal stem cells

1. Introduction

1.1. regenerative medicine—an overview, 1.2. stem cells in regenerative medicine, 1.3. adult mscs in regenerative medicine, 2. mechanism of actions of adult mscs, 2.1. trans-differentiation, 2.2. cell fusion, 2.3. mitochondrial transfer, 2.4. extracellular vesicles or microvesicles, 3. required characteristics of mscs for their application in regenerative medicine, 3.1. colony formation, 3.2. surface phenotypes, 3.3. plasticity and differentiation potential, 3.4. telomerase activity, 4. application of mscs in tissue engineering, 4.1. bioprinting technology, 4.2. scaffolds, 4.3. organoid technology, 5. induced pluripotent stem cell-derived mscs (imscs), 6. challenges associated with mscs for regenerative medicine and tissue engineering, 7. conclusions, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

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Vasanthan, J.; Gurusamy, N.; Rajasingh, S.; Sigamani, V.; Kirankumar, S.; Thomas, E.L.; Rajasingh, J. Role of Human Mesenchymal Stem Cells in Regenerative Therapy. Cells 2021 , 10 , 54. https://doi.org/10.3390/cells10010054

Vasanthan J, Gurusamy N, Rajasingh S, Sigamani V, Kirankumar S, Thomas EL, Rajasingh J. Role of Human Mesenchymal Stem Cells in Regenerative Therapy. Cells . 2021; 10(1):54. https://doi.org/10.3390/cells10010054

Vasanthan, Jayavardini, Narasimman Gurusamy, Sheeja Rajasingh, Vinoth Sigamani, Shivaani Kirankumar, Edwin L. Thomas, and Johnson Rajasingh. 2021. "Role of Human Mesenchymal Stem Cells in Regenerative Therapy" Cells 10, no. 1: 54. https://doi.org/10.3390/cells10010054

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