CD45<Superscript>−</Superscript>CD14<Superscript>+</Superscript>CD34<Superscript>+</Superscript> murine bone marrow low-adherent mesenchymal primitive cells preserve multilineage differentiation potential in long-term <Emphasis Type="Italic">in vitro</Emphasis> culture

Bone marrow-derived cells have been postulated as a source of multipotent mesenchymal stem cells (MSC). However, the whole fraction of MSC remains heterogeneous and the expansion of primitive subset of these cells is still not well established. Here, we optimized the protocol for propagating the low-adherent subfraction of MSC which results in long-term expansion of population characterized by CD45⁻CD14⁺CD34⁺ phenotype along with expression of common MSC markers. We established that the expanded MSC are capable of differentiating into endothelial cells highly expressing angiogenic markers and exhibiting functional properties of endothelium. Moreover, we found these cells to be multipotent and capable of giving rise into cells from neuronal lineages. Interestingly, the expanded MSC form characteristic cellular spheres in vitro indicating primitive features of these cells. In sum, we isolated the novel multipotent subpopulation of CD45⁻CD14⁺ CD34⁺ bone marrow-derived cells that could be maintained in long-term culture without losing this potential.     

Perivascular cells for regenerative medicine

Mesenchymal stem/stromal cells (MSC) are currently the best candidate therapeutic cells for regenerative medicine related to osteoarticular, muscular, vascular and inflammatory diseases, although these cells remain heterogeneous and necessitate a better biological characterization. We and others recently described that MSC originate from two types of perivascular cells, namely pericytes and adventitial cells and contain the in situ counterpart of MSC in developing and adult human organs, which can be prospectively purified using well defined cell surface markers. Pericytes encircle endothelial cells of capillaries and microvessels and express the adhesion molecule CD146 and the PDGFRβ, but lack endothelial and haematopoietic markers such as CD34, CD31, vWF (von Willebrand factor), the ligand for Ulex europaeus 1 (UEA1) and CD45 respectively. The proteoglycan NG2 is a pericyte marker exclusively associated with the arterial system. Besides its expression in smooth muscle cells, smooth muscle actin (αSMA) is also detected in subsets of pericytes. Adventitial cells surround the largest vessels and, opposite to pericytes, are not closely associated to endothelial cells. Adventitial cells express CD34 and lack αSMA and all endothelial and haematopoietic cell markers, as for pericytes. Altogether, pericytes and adventitial perivascular cells express in situ and in culture markers of MSC and display capacities to differentiate towards osteogenic, adipogenic and chondrogenic cell lineages. Importantly, adventitial cells can differentiate into pericyte‐like cells under inductive conditions in vitro. Altogether, using purified perivascular cells instead of MSC may bring higher benefits to regenerative medicine, including the possibility, for the first time, to use these cells uncultured.     

Collaboration between WNT and BMP signaling promotes hemoangiogenic cell development from human fibroblast-derived iPS cells

Induced pluripotent stem (iPS) cells are generated by nuclear reprogramming of mature cells to a pluripotent state, and show biological properties of embryonic stem (ES) cells. The observation that human (h)ES cells generate hemoangiogenic progeny, defined by their high-level expression of KDR and low-level expression of PDGFRα (KDR⁺PDGFRα^lo) via WNT and BMP signaling during 5-8 days of differentiation in a serum-free environment led us to address how hiPS cells give rise to hemoangiogenic progeny. In the presence of WNT3a, four hiPS cell lines derived from human skin fibroblasts commonly generated KDR⁺ and/or PDGFRα⁺ progeny by day 8 of differentiation. Endogenous BMP signaling was required for the WNT3a-directed upregulation of hemogenic cell development and the hemoangiogenic activity was confined in all cases to the KDR⁺PDGFRα^lo fraction. Thus, iPS cells derived from human skin fibroblasts resemble hES cells in the generation of hematopoietic and endothelial cells in vitro.     

Multipotent mesenchymal stem cells from amniotic fluid originate neural precursors with functional voltage-gated sodium channels

Background aimsAmniotic fluid (AF) contains stem cells with high proliferative and differentiative potential that might be an attractive source of multipotent stem cells. We investigated whether human AF contains mesenchymal stem cells (MSC) and evaluated their phenotypic characteristics and differentiation potential in vitro.MethodsAF was harvested during routine pre-natal amniocentesis at 14–16 weeks of pregnancy. AF sample pellets were plated in α-minimum essential medium (MEM) with 10% fetal bovine serum (FBS). We evaluated cellular growth, immunophenotype, stemness markers and differentiative potential during in vitro expansion. Neural progenitor maintenance medium (NPMM), a medium normally used for the growth and maintenance of neural stem cells, containing hFGF, hEGF and NSF-1, was used for neural induction.ResultsTwenty-seven AF samples were collected and primary cells, obtained from samples containing more than 6 mL AF, had MSC characteristics. AF MSC showed high proliferative potential, were positive for CD90, CD105, CD29, CD44, CD73 and CD166, showed Oct-4 and Nanog molecular and protein expression, and differentiated into osteoblasts, adypocytes and chondrocytes. The NPMM-cultured cells expressed neural markers and increased Na⁺ channel density and channel inactivation rate, making the tetrodotoxin (TTX)-sensitive channels more kinetically similar to native neuronal voltage-gated Na⁺ channels.ConclusionsThese data suggest that AF is an important multipotent stem cell source with a high proliferative potential able to originate potential precursors of functional neurons.     

Is CD34 truly a negative marker for mesenchymal stromal cells?

The prevailing school of thought is that mesenchymal stromal cells (MSC) do not express CD34, and this sets MSC apart from hematopoietic stem cells (HSC), which do express CD34. However, the evidence for MSC being CD34^? is largely based on cultured MSC, not tissue-resident MSC, and the existence of CD34^? HSC is in fact well documented. Furthermore, the Stro-1 antibody, which has been used extensively for the identification/isolation of MSC, was generated by using CD34⁺ bone marrow cells as immunogen. Thus, neither MSC being CD34^? nor HSC being CD34⁺ is entirely correct. In particular, two studies that analyzed CD34 expression in uncultured human bone marrow nucleated cells found that MSC (BMSC) existed in the CD34⁺ fraction. Several studies have also found that freshly isolated adipose-derived MSC (ADSC) express CD34. In addition, all of these ADSC studies and several other MSC studies have observed a disappearance of CD34 expression when the cells are propagated in culture. Thus the available evidence points to CD34 being expressed in tissue-resident MSC, and its negative finding being a consequence of cell culturing.     

Directed differentiation of mouse P19 embryonal carcinoma cells to neural cells in a serum- and retinoic acid-free culture medium

P19 embryonal carcinoma cells (EC-cells) provide a simple and robust culture system for studying neural development. Most protocols developed so far for directing neural differentiation of P19 cells depend on the use of culture medium supplemented with retinoic acid (RA) and serum, which has an undefined composition. Hence, such protocols are not suitable for many molecular studies. In this study, we achieved neural differentiation of P19 cells in a serum- and RA-free culture medium by employing the knockout serum replacement (KSR) supplement. In the KSR-containing medium, P19 cells underwent predominant differentiation into neural lineage and by day 12 of culture, neural cells were present in 100% of P19-derived embryoid bodies (EBs). This was consistently accompanied by the increased expression of various neural lineage-associated markers during the course of differentiation. P19-derived neural cells comprised of NES⁺ neural progenitors (~?46%), TUBB3⁺ immature neurons (~?6%), MAP2⁺ mature neurons (~?2%), and GFAP⁺ astrocytes (~?50%). A heterogeneous neuronal population consisting of glutamatergic, GABAergic, serotonergic, and dopaminergic neurons was generated. Taken together, our study shows that the KSR medium is suitable for the differentiation of P19 cells to neural lineage without requiring additional (serum and RA) supplements. This stem cell differentiation system could be utilized for gaining mechanistic insights into neural differentiation and for identifying potential neuroactive compounds.     

Relative percentage and zonal distribution of mesenchymal progenitor cells in human osteoarthritic and normal cartilage

Introduction  

Mesenchymal stem cells (MSC) are highly attractive for use in cartilage regeneration. To date, MSC are usually recruited from subchondral bone marrow using microfracture. Recent data suggest that isolated cells from adult human articular cartilage, which express the combination of the cell-surface markers CD105 and CD166, are multi-potent mesenchymal progenitor cells (MPC) with characteristics similar to MSC. MPC within the cartilage matrix, the target of tissue regeneration, may provide the basis for in situ regeneration of focal cartilage defects. However, there is only limited information concerning the presence/abundance of CD105⁺/CD166⁺ MPC in human articular cartilage. The present study therefore assessed the relative percentage and particularly the zonal distribution of cartilage MPC using the markers CD105/CD166.     

Effect of K+- and kainate-mediated depolarization on survival and functional maturation of GABAergic and glutamatergic neurons in cultures of dissociated mouse cerebellum

The effect of the depolarizing agents, an elevated potassium concentration (25 mM) or kainic acid (50 μM) on neuronal survival and differentiation was investigated in cultures of dissociated neurons from cerebella of 7-day-old mice. When maintained in the presence of an antimitotic agent such cultures consist primarily of glutamatergic and GABAergic neurons. Cell survival was monitored by measurement of DNA, and differentiation by determining uptake and depolarization coupled release of glutamate (D-aspartate as label) and GABA. The depolarizing agents were added separately or together either from the start of the culture period (7–8 days) or at day 5 in culture. The main findings are that K⁺ depolarization is important for differentiation of glutamatergic neurons but not for GABAergic neurons. This depolarizing signal is important during the early phase of development in culture. For glutamatergic neurons, kainate may replace K⁺ as a depolarizing signal whereas in case of the GABAergic neurons, kainate was toxic particularly during the late phase of development. It was further observed that the glutamatergic neurons when maintained in a medium with 5 mM K⁺ during the first 5 days in culture became sensitive to kainate toxicity when this amino acid was added at day 5. This was not the case when the medium contained 25 mM K⁺ from the start of the culture period. Special issue dedicated to Dr. Kinya Kuriyama.     

The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood

Many studies have drawn attention to the emerging role of MSC (mesenchymal stem cells) as a promising population supporting new clinical concepts in cellular therapy. However, the sources from which these cells can be isolated are still under discussion. Whereas BM (bone marrow) is presented as the main source of MSC, despite the invasive procedure related to this source, the possibility of isolating sufficient numbers of these cells from UCB (umbilical cord blood) remains controversial. Here, we present the results of experiments aimed at isolating MSC from UCB, BM and UCM (umbilical cord matrix) using different methods of isolation and various culture media that summarize the main procedures and criteria reported in the literature. Whereas isolation of MSC were successful from BM (10:10) and (UCM) (8:8), only one cord blood sample (1:15) gave rise to MSC using various culture media [DMEM (Dulbecco's modified Eagle's medium) +5% platelet lysate, DMEM+10% FBS (fetal bovine serum), DMEM+10% human UCB serum, MSCGM®] and different isolation methods [plastic adherence of total MNC (mononuclear cells), CD3⁺/CD19⁺/CD14⁺/CD38⁺‐depleted MNC and CD133⁺‐ or LNGFR⁺‐enriched MNC]. MSC from UCM and BM were able to differentiate into adipocytes, osteocytes and hepatocytes. The expansion potential was highest for MSC from UCM. The two cell populations had CD90⁺/CD73⁺/CD105⁺ phenotype with the additional expression of SSEA4 and LNGFR for BM MSC. These results clearly exclude UCB from the list of MSC sources for clinical use and propose instead UCM as a rich, non‐invasive and abundant source of MSC.     

Differentiation and migration properties of human foetal umbilical cord perivascular cells: potential for lung repair

Mesenchymal stem cells (MSC) have been derived from different cultured human tissues, including bone marrow, adipose tissue, amniotic fluid and umbilical cord blood. Only recently it was suggested that MSC descended from perivascular cells, the latter being defined as CD146⁺ neuro‐glial proteoglycan (NG)2⁺ platelet‐derived growth factor‐Rβ⁺ ALP⁺ CD34^– CD45^– von Willebrand factor (vWF)^– CD144^–. Herein we studied the properties of perivascular cells from a novel source, the foetal human umbilical cord (HUC) collected from pre‐term newborns. By immunohistochemistry and flow cytometry we show that pre‐term/foetal HUCs contain more perivascular cells than their full‐term counterparts (2.5%versus 0.15%). Moreover, foetal HUC perivascular cells (HUCPC) express the embryonic cell markers specific embryonic antigen‐4, Runx1 and Oct‐4 and can be cultured over the long term. To further confirm the MSC identity of these cultured perivascular cells, we also showed their expression at different passages of antigens that typify MSC. The multilineage differentiative capacity of HUCPC into osteogenic, adipogenic and myogenic cell lineages was demonstrated in culture. In the perspective of a therapeutic application in chronic lung disease of pre‐term newborns, we demonstrated the in vitro ability of HUCPC to migrate towards an alveolar type II cell line damaged with bleomycin, an anti‐cancer agent with known pulmonary toxicity. The secretory profile exhibited by foetal HUCPC in the migration assay suggested a paracrine effect that could be exploited in various clinical conditions including lung disorders.     

Periodic harvesting of embryonic stem cells from a hollow‐fiber membrane based four‐compartment bioreactor

Different types of stem cells have been investigated for applications in drug screening and toxicity testing. In order to provide sufficient numbers of cells for such in vitro applications a scale‐up of stem cell culture is necessary. Bioreactors for dynamic three‐dimensional (3D) culture of growing cells offer the option for culturing large amounts of stem cells at high densities in a closed system. We describe a method for periodic harvesting of pluripotent stem cells (PSC) during expansion in a perfused 3D hollow‐fiber membrane bioreactor, using mouse embryonic stem cells (mESC) as a model cell line. A number of 100 × 10⁶ mESC were seeded in bioreactors in the presence of mouse embryonic fibroblasts (MEF) as feeder cells. Over a cultivation interval of nine days cells were harvested by trypsin perfusion and mechanical agitation every second to third culture day. A mean of 380 × 10⁶ mESC could be removed with every harvest. Subsequent to harvesting, cells continued growing in the bioreactor, as determined by increasing glucose consumption and lactate production. Immunocytochemical staining and mRNA expression analysis of markers for pluripotency and the three germ layers showed a similar expression of most markers in the harvested cells and in mESC control cultures. In conclusion, successful expansion and harvesting of viable mESC from bioreactor cultures with preservation of sterility was shown. The present study is the first one showing the feasibility of periodic harvesting of adherent cells from a continuously perfused four‐compartment bioreactor including further cultivation of remaining cells. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 32:141–151, 2016     

Rapid clinical‐scale propagation of mesenchymal stem cells using cultures initiated with immunoselected bone marrow CD105+ cells

Current clinical protocols used for isolation and purification of mesenchymal stem cells (MSC) are based on long-term cultures starting with bone marrow (BM) mononuclear cells. Using a commercially available immunoselection kit for enrichment of MSC, we investigated whether culture of enriched BM-CD105⁺ cells could provide an adequate number of pure MSC in a short time for clinical use in the context of graft versus host disease and graft failure/rejection. We isolated a mean of 5.4 × 10⁵ ± 0.9 × 10⁵ CD105⁺ cells from 10 small volume (10–25 ml) BM samples achieving an enrichment >100-fold in MSC. Seeding 2 × 10³ immunoselected cells/cm² we were able to produce 2.5 × 10⁸ ± 0.7 × 10⁸ MSC from cultures with autologous serum enriched medium within 3 weeks. Neither haematopoietic nor endothelial cells were detectable even in the primary culture cell product. Expanded cells fulfilled both phenotypic and functional current criteria for MSC; they were CD29⁺, CD90⁺, CD73⁺, CD105⁺, CD45⁻; they suppressed allogeneic T-cell reaction in mixed lymphocyte cultures and retained in vitro differentiation potential. Moreover, comparative genomic hybridization analysis revealed chromosomal stability of the cultured MSC. Our data indicate that adequate numbers of pure MSC suitable for clinical applications can be generated within a short time using enriched BM-CD105⁺ cells.     

Human placenta and bone marrow derived MSC cultured in serum-free, b-FGF-containing medium express cell surface frizzled-9 and SSEA-4 and give rise to multilineage differentiation

Conventionally, mesenchymal stem cells (MSC) are generated by plating cells from bone marrow (BM) or other sources into culture flasks and selecting plastic-adherent cells with fibroblastoid morphology. These cells express CD9, CD10, CD13, CD73, CD105, CD166, and other markers but show only a weak or no expression of the embryonic markers stage-specific embryonic antigen-4 (SSEA-4), Oct-4 and nanog-3. Using a novel protocol we prepared MSC from BM and non-amniotic placenta (PL) by culture of Ficoll-selected cells in gelatin-coated flasks in the presence of a serum-free, basic fibroblast growth factor (b-FGF)-containing medium that was originally designed for the expansion of human embryonic stem cells (ESC). MSC generated in gelatin-coated flasks in the presence of ESC medium revealed a four-to fivefold higher proliferation rate than conventionally prepared MSC which were grown in uncoated flasks in serum-containing medium. In contrast, the colony forming unit fibroblast number was only 1.5- to twofold increased in PL-MSC and not affected in BM-MSC. PL-MSC grown in ESC medium showed an increased surface expression of SSEA-4 and frizzled-9 (FZD-9), an increased Oct-4 and nestin mRNA expression, and an induced expression of nanog-3. BM-MSC showed an induced expression of FZD-9, nanog-3, and Oct-4. In contrast to PL-MSC, only BM-MSC expressed the MSC-specific W8B2 antigen. When cultured under appropriate conditions, these MSC gave rise to functional adipocytes and osteoblast-like cells (mesoderm), glucagon and insulin expressing pancreatic-like cells (endoderm), as well as cells expressing the neuronal markers neuron-specific enolase, glutamic acid decarboxylase-67 (GAD), or class III beta-tubulin, and the astrocyte marker glial fibrillary acidic protein (ectoderm). In conclusion, using a novel protocol we demonstrate that adult BM-and neonatal PL-derived MSC can be induced to express high levels of FZD-9, Oct-4, nanog-3, and nestin and are able of multi-lineage differentiation.     

Efficient generation of multipotent mesenchymal stem cells from umbilical cord blood in stroma-free liquid culture

Background

Haematopoiesis is sustained by haematopoietic (HSC) and mesenchymal stem cells (MSC). HSC are the precursors for blood cells, whereas marrow, stroma, bone, cartilage, muscle and connective tissues derive from MSC. The generation of MSC from umbilical cord blood (UCB) is possible, but with low and unpredictable success. Here we describe a novel, robust stroma-free dual cell culture system for long-term expansion of primitive UCB-derived MSC.

Methods and Findings

UCB-derived mononuclear cells (MNC) or selected CD34⁺ cells were grown in liquid culture in the presence of serum and cytokines. Out of 32 different culture conditions that have been tested for the efficient expansion of HSC, we identified one condition (DMEM, pooled human AB serum, Flt-3 ligand, SCF, MGDF and IL-6; further denoted as D7) which, besides supporting HSC expansion, successfully enabled long-term expansion of stromal/MSC from 8 out of 8 UCB units (5 MNC-derived and 3 CD34⁺ selected cells). Expanded MSC displayed a fibroblast-like morphology, expressed several stromal/MSC-related antigens (CD105, CD73, CD29, CD44, CD133 and Nestin) but were negative for haematopoietic cell markers (CD45, CD34 and CD14). MSC stemness phenotype and their differentiation capacity in vitro before and after high dilution were preserved throughout long-term culture. Even at passage 24 cells remained Nestin⁺, CD133⁺ and >95% were positive for CD105, CD73, CD29 and CD44 with the capacity to differentiate into mesodermal lineages. Similarly we show that UCB derived MSC express pluripotency stem cell markers despite differences in cell confluency and culture passages.Further, we generated MSC from peripheral blood (PB) MNC of 8 healthy volunteers. In all cases, the resulting MSC expressed MSC-related antigens and showed the capacity to form CFU-F colonies.

Conclusions

This novel stroma-free liquid culture overcomes the existing limitation in obtaining MSC from UCB and PB enabling so far unmet therapeutic applications, which might substantially affect clinical practice.     

Multidistrict human mesenchymal vascular cells: pluripotency and stemness characteristics

Background aimsThe presence of ectopic tissues in the pathologic artery wall raises the issue of whether multipotent stem cells may reside in the vasculature itself. Recently mesenchymal stromal cells (MSC) have been isolated from different human vascular segments (VW MSC), belying the previous view that the vessel wall is a relatively quiescent tissue.MethodsResident multipotent cells were recovered from fresh arterial segments (aortic arches, thoracic and femoral arteries) collected in a tissue-banking facility and used to establish an in situ and in vitro study of the stemness features and multipotency of these multidistrict MSC populations.ResultsNotch-1⁺, Stro-1⁺, Sca-1⁺ and Oct-4⁺ cells were distributed along an arterial wall vasculogenic niche. Multidistrict VW MSC homogeneously expressed markers of stemness (Stro-1, Notch-1 and Oct-4) and MSC lineages (CD44, CD90, CD105, CD73, CD29 and CD166) whilst they were negative for hematopoietic and endothelial markers (CD34, CD45, CD31 and vWF). Each VW MSC population had characteristics of stem cells, i.e. a high efflux capability for Hoechst 33342 dye and the ability to form spheroids when grown in suspension and generate colonies when seeded at low density. Again, VW MSC cultured in induction media exhibited adipogenic, chondrogenic and leiomyogenic potential but less propensity to osteogenic differentiation, as documented by histochemical, immunohistochemical, molecular and electron microscopy analysis.ConclusionsOverall, these findings may enlighten the physiopathologic mechanisms of vascular wall diseases as well as having potential implications for cellular, genetic and tissue engineering approaches to treating vascular pathologies when these are unresponsive to medical and surgical therapies.     

Relationship between interstitial cells of Cajal, fibroblast-like cells and inhibitory motor nerves in the internal anal sphincter

Interstitial cells of Cajal (ICC) have been shown to participate in nitrergic neurotransmission in various regions of the gastrointestinal (GI) tract. Recently, fibroblast-like cells, which are positive for platelet-derived growth factor receptor ?? (PDGFR??⁺), have been suggested to participate additionally in inhibitory neurotransmission in the GI tract. The distribution of ICC and PDGFR??⁺ cell populations and their relationship to inhibitory nerves within the mouse internal anal sphincter (IAS) are unknown. Immunohistochemical techniques and confocal microscopy were therefore used to examine the density and arrangement of ICC, PDGFR??⁺ cells and neuronal nitric-oxide-synthase-positive (nNOS⁺) nerve fibers in the IAS of wild-type (WT) and W/W ^v mice. Of the total tissue volume within the IAS circular muscle layer, 18% consisted in highly branched PDGFR??⁺ cells (PDGFR??⁺-IM). Other populations of PDGFR??⁺ cells were observed within the submucosa and along the serosal and myenteric surfaces. Spindle-shaped intramuscular ICC (ICC-IM) were present in the WT mouse IAS but were largely absent from the W/W ^v IAS. The ICC-IM volume (5% of tissue volume) in the WT mouse IAS was significantly smaller than that of PDGFR??⁺-IM. Stellate-shaped submucosal ICC (ICC-SM) were observed in the WT and W/W ^v IAS. Minimum surface distance analysis revealed that nNOS⁺ nerve fibers were closely aligned with both ICC-IM and PDGFR??⁺-IM. An even closer association was seen between ICC-IM and PDGFR??⁺-IM. Thus, a close morphological arrangement exists between inhibitory motor neurons, ICC-IM and PDGFR??⁺-IM suggesting that some functional interaction occurs between them contributing to inhibitory neurotransmission in the IAS.     

The immunosuppressive effects of human bone marrow-derived mesenchymal stem cells target T cell proliferation but not its effector function

Mesenchymal stem cells (MSC) are non-haematopoietic stem cells that are capable of differentiating into tissues of mesodermal origin. MSC play an important role in supporting the development of fetal and adult haematopoiesis. More recently, MSC have also been found to exhibit inhibitory effect on T cell responses. However, there is little information on the mechanism of this immunosuppression and our study addresses this issue by targeting T cell functions at various level of immune responses. We have generated MSC from human adult bone marrow (BM) and investigated their immunoregulatory function at different phases of T cell responses. MSC showed the ability to inhibit mitogen (CD3/CD28 microbeads)-activated T cell proliferation in a dose-dependent manner. In order to evaluate the specificity of this immunosuppression, the proliferation of CD4⁺ and CD8⁺ cells were measured. MSC equally inhibit CD4⁺ and CD8⁺ subpopulations of T cells in response to PHA stimulation. However, the antiproliferative effect of MSC is not due to the inhibition of T cell activation. The expression of early activation markers of T cells, namely CD25 and CD69 were not significantly altered by MSC at 24, 48 and 72 h. Furthermore, the immunosuppressive effect of MSC mainly targets T cell proliferation rather than their effector function since cytotoxicity of T cells is not affected. This work demonstrates that the immunosuppressive effect of MSC is exclusively a consequence of an anti-proliferative activity, which targets T cells of different subpopulations. For this reason, they have the potential to be exploited in the control of unwanted immune responses such as graft versus host disease (GVHD) and autoimmunity.