Differentiation Capacity Of Mesenchymal Stem Cells Research Articles

Characterization of Spontaneous Bone Marrow Recovery after Sublethal Total Body Irradiation: Importance of the Osteoblastic/Adipocytic Balance

Many studies have already examined the hematopoietic recovery after irradiation but paid with very little attention to the bone marrow microenvironment. Nonetheless previous studies in a murine model of reversible radio-induced bone marrow aplasia have shown a significant increase in alkaline phosphatase activity (ALP) prior to hematopoietic regeneration. This increase in ALP activity was not due to cell proliferation but could be attributed to modifications of the properties of mesenchymal stem cells (MSC). We thus undertook a study to assess the kinetics of the evolution of MSC correlated to their hematopoietic supportive capacities in mice treated with sub lethal total body irradiation. In our study, colony-forming units – fibroblasts (CFU-Fs) assay showed a significant MSC rate increase in irradiated bone marrows. CFU-Fs colonies still possessed differentiation capacities of MSC but colonies from mice sacrificed 3 days after irradiation displayed high rates of ALP activity and a transient increase in osteoblastic markers expression while pparγ and neuropilin-1 decreased. Hematopoietic supportive capacities of CFU-Fs were also modified: as compared to controls, irradiated CFU-Fs significantly increased the proliferation rate of hematopoietic precursors and accelerated the differentiation toward the granulocytic lineage. Our data provide the first evidence of the key role exerted by the balance between osteoblasts and adipocytes in spontaneous bone marrow regeneration. First, (pre)osteoblast differentiation from MSC stimulated hematopoietic precursor's proliferation and granulopoietic regeneration. Then, in a second time (pre)osteoblasts progressively disappeared in favour of adipocytic cells which down regulated the proliferation and granulocytic differentiation and then contributed to a return to pre-irradiation conditions.

Finasteride as a potential tool to improve Mesenchymal stem cell transplantation for myocardial infarction

Mesenchymal stem cells (MSCs) therapy has emerged as a potent therapeutic strategy to improve myocardial infarction. However MSCs therapy encounters a few obstacles regarding the poor viability of the transplanted cells. Therefore, it is important to explore a strategy to enhance post-transplanted MSC viability. To overcome this problem, several protocols were suggested mainly by activating PI3K/Akt pathway. The PI3K/Akt cascade regulates several cellular processes such as proliferation and apoptosis. Finasteride is a specific inhibitor of type II 5α-reductase; the enzyme converts testosterone (T) to the more potent androgen receptor agonist dihydrotestosterone (DHT). Testosterone is found to stimulate rapid phosphorylation of Akt, and thereby activate the PI3K/Akt pathway. This pathway could lead to decreased apoptosis of the MSCs via increasing the expression of Bcl-2 and reducing Bax expression. It has been also reported that DHT would confine the differentiation capacity of MSCs so that a reduction in DHT levels caused by Finasteride would be accompanied by increased facilitation in differentiation of MSCs to cardiomyocyte by means of the signals originating from the injured cardiac tissue. These mechanisms could propose the potential role for Finasteride to improve the MSCs therapy for myocardial infarction.

Lentiviral vector mediated modification of mesenchymal stem cells & enhanced survival in an in vitro model of ischaemia

IntroductionA combination of gene and cell therapies has the potential to significantly enhance the therapeutic value of mesenchymal stem cells (MSCs). The development of efficient gene delivery methods is essential if MSCs are to be of benefit using such an approach. Achieving high levels of transgene expression for the required period of time, without adversely affecting cell viability and differentiation capacity, is crucial. In the present study, we investigate lentiviral vector-mediated genetic modification of rat bone-marrow derived MSCs and examine any functional effect of such genetic modification in an in vitro model of ischaemia.MethodsTransduction efficiency and transgene persistence of second and third generation rHIV-1 based lentiviral vectors were tested using reporter gene constructs. Use of the rHIV-pWPT-EF1-α-GFP-W vector was optimised in terms of dose, toxicity, cell species, and storage. The in vivo condition of ischaemia was modelled in vitro by separation into its associated constituent parts i.e. hypoxia, serum and glucose deprivation, in which the effect of therapeutic gene over-expression on MSC survival was investigated.ResultsThe second generation lentiviral vector rHIV-pWPT-EF1-α-GFP-W, was the most efficient and provided the most durable transgene expression of the vectors tested. Transduction with this vector did not adversely affect MSC morphology, viability or differentiation potential, and transgene expression levels were unaffected by cryopreservation of transduced cells. Over-expression of HSP70 resulted in enhanced MSC survival and increased resistance to apoptosis in conditions of hypoxia and ischaemia. MSC differentiation capacity was significantly reduced after oxygen deprivation, but was preserved with HSP70 over-expression.ConclusionsCollectively, these data validate the use of lentiviral vectors for efficient in vitro gene delivery to MSCs and suggest that lentiviral vector transduction can facilitate sustained therapeutic gene expression, providing an efficient tool for ex vivo MSC modification. Furthermore, lentiviral mediated over-expression of therapeutic genes in MSCs may provide protection in an ischaemic environment and enable MSCs to function in a regenerative manner, in part through maintaining the ability to differentiate. This finding may have considerable significance in improving the efficacy of MSC-based therapies.

Mesenchymal stem cells and progenitor cells in connective tissue engineering and regenerative medicine: is there a future for transplantation?

Transplantation surgery suffers from a shortage of donor organs worldwide. Cell injection and tissue engineering (TE), thus emerge as alternative therapy options. The purpose of this article is to review the progress of TE technology, focusing on mesenchymal stem cells (MSC) as a cell source for artificial functional tissue. MSC from many different sources can be minimally invasively harvested: peripheral blood, fat tissue, bone marrow, amniotic fluid, cord blood. In comparison to embryonic stem cells (ESC), there are no ethical concerns; MSC can be extracted from autologous or allogenic tissue and cause an immune modulatory effect by suppressing the graft-versus-host reaction (GvHD). Furthermore, MSC do not develop into teratomas when transplanted, a consequence observed with ESC and iPS cells. MSC as multipotent cells are capable of differentiating into mesodermal and non-mesodermal lineages. However, further studies must be performed to elucidate the differentiation capacity of MSC from different sources, and to understand the involved pathways and processes. Already, MSC have been successfully applied in clinical trials, e.g., to heal large bone defects, cartilage lesions, spinal cord injuries, cardiovascular diseases, hematological pathologies, osteogenesis imperfecta, and GvHD. A detailed understanding of the behavior and homing of MSC is desirable to enlarge the clinical application spectrum of MSC towards the in vitro generation of functional tissue for implantation, for example, resilient cartilage, contractile myocardial replacement tissue, and bioartificial heart valves.

Human mesenchymal stem cell proliferation and osteogenic differentiation during long-term ex vivo cultivation is not age dependent

Mesenchymal stem cells (MSCs) are of major clinical interest for the development of cell-based strategies to treat musculoskeletal diseases including critical-size bone defects caused by trauma, degenerative disorders, or infections. Elderly people mainly suffer from critical-size bone defects from the rising incidence of trauma, osteoporosis, and arthroplasties. In this study we investigated the influence of donor age on proliferation and osteogenic differentiation in long-term ex vivo cultures of primary human MSCs from patients in different age groups. Fifteen patients (8 men/7 women) comprised three age groups: (I) <50 years, (II) 50-65 years, and (III) >65 years. MSCs harvested from bone marrow derived from routine surgical procedures were isolated and cultured in standard medium over eight passages. Osteogenic differentiation was induced by dexamethasone (10 nM), ascorbic acid (300 μM), and β-glycerophosphate (3.5 mM). Osteogenic differentiation capacity of MSCs was quantified by alkaline phosphatase (ALP) activity, fluorescence-activated cell sorting (FACS) analysis of the surface markers CD9, CD90, CD54, CD166, CD105, CD44, and CD73, and RT-PCR for Coll I and II, Cbfa 1, ALP, OC, BSP1, and GAPDH genes characterized the phenotypic changes during monolayer expansion. In vitro chondrogenic differentiation was analyzed by immunohistochemistry and RT-PCR. Progenitor cells could be expanded in the long term from all bone marrow donations. FACS single staining analysis from MSCs showed no significant difference between the age groups. The surface antigen CD166 was predominantly found in all cell cultures independently of differentiation stage. Comparison of expanded and differentiated MSCs within a single age group showed that undifferentiated MSCs had higher CD44 levels. Osteogenic stimulation of MSCs was confirmed by measuring ALP activity. The highest ALP activity was found in probands of the age group >65 years. Additionally, we observed a tendency toward male-specific ALP increase during differentiation. Osteogenic marker gene expression in MSCs was detected by RT-PCR. No significant expression differences were detected between the three donor age groups. Micromass culture of MSCs resulted histologically and immunohistologically in a chondrogenic phenotype. Elderly osteoprogenitor cell donors are a highly clinically relevant patient population. In summary, cultivation leads to a reduced osteogenic differentiation capacity regardless of age. Because donor age does not affect osteogenic differentiation potential, it should not be used as an exclusion criterion for autologous transplantation of human adult MSCs.

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose–derived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose-derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2(-/-)gammaC(-/-) mice resulted in a substantial increase in the number of hMADS cell-derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.

Expansion of umbilical cord blood mesenchymal stem cells

7103 Background: Umbilical cord blood (UCB) is known to harbor 2 major types of stem cells, the hematopoietic stem cells (HSC) &amp; the non-hematopoietic or mesenchymal stem cells (MSC). Under appropriate conditions, MSCs can give rise to cells of bone, fat, hepatic lineages, etc. Based on this potential, MSC hold promise for clinical applications in regenerative medicine. Methods: Stroma-free liquid culture: UCB cryopreserved mononuclear cells (MNC) were cultured in the presence of early growth factors: Flt-3 &amp; SCF (25ng/ml), MGDF (10ng/ml) &amp; human serum (10%). MNC derived adherent MSC were passaged at day 14 during HSC expansion &amp; after enriching in MesenCult medium. Results: We developed a technology to generate &amp; expand HSC &amp; stromal/ MSC from all UCB units (5/5) at the same time using one culture system (stroma-free liquid culture). Following repeated passages, MSC count increased 357- 600-folds &amp; CFU-Fibroblasts colonies (CFU-F) increased too (61–513 &amp; 648–697) after 10 and 20 passages respectively. We used the CFU-F assay to demonstrate MSC activity in stromal cell formation in vitro. Phenotypically, MSC were negative for hematopoietic antigens (CD45, CD34 &amp; CD14) &amp; MHC class-II but &gt;95% + for CD73, CD105, CD29, CD44 &amp; MHC class I. To demonstrate MSC differentiation capacity in vitro, cells were incubated in various induction media to differentiate into adipocytes (fat)), osteoblasts (bone) and hepatocytes (liver) at passage 5. Following induction, positive staining with oil red O for cells of adipocyte and with alkaline phosphatase for cells of osteoblsts lineages was observed. The identity of hepatocytes was verified by the characteristic hexagonal hepatocytic shape as well as albumin, cytokeratin (CK) 18 and CK14 expression, as assessed by flow cytometry. Our data were corroborated by RT-PCR analysis. Conclusions: MSC described herein exhibit in vitro properties of multipotent stem cells. The established, stroma-free culture system facilitates expansion of MSC from all tested UCB units. Our data underline that it will be possible in the future to substitutes properly differentiated hepatocytes which might lead to efficient applications in patients suffering from various end stage liver disease. No significant financial relationships to disclose.

Susceptibility of Human Mesenchymal Stem Cells to Tacrolimus, Mycophenolic Acid, and Rapamycin

Mesenchymal stem cells (MSC) have multilineage differentiation and immunomodulatory capacities and are potentially useful for therapeutic applications, such as tissue regeneration and control of alloreactivity. MSC are present in most tissues including the transplantable organs. It is therefore unavoidable that MSC will be exposed to immunosuppressive drugs in a clinical transplantation setting. The molecular targets of these drugs are expressed in MSC, but the effect of their inhibition on MSC functioning is unknown. MSC were isolated and expanded from heart tissue and the effects of the calcineurin inhibitor tacrolimus, the cell cycle inhibitor mycophenolic acid (MPA), and the mammalian target of rapamycin inhibitor on MSC survival, proliferation, differentiation, and immunosuppressive capacity were examined. Short-term exposure to the immunosuppressants did not induce toxicity or apoptosis in MSC, but high-dose tacrolimus induced toxicity after 7 days. MPA and rapamycin inhibited MSC proliferation at therapeutic doses. The immunosuppressants had differential effects on the differentiation capacity of MSC. Tacrolimus reduced the expression of troponin T type 2 and desmin during cardiomyogenic differentiation of MSC, whereas MPA decreased the deposition of calcified minerals during osteogenic differentiation. Rapamycin stimulated lipid production during adipogenic differentiation. Unexpectedly, MSC had adverse effects on the immunosuppressive efficacy of tacrolimus and rapamycin. There was no such effect of MSC on the function of MPA. Preincubation of MSC with tacrolimus increased the immunosuppressive capacity of MSC. This study demonstrates that therapeutic concentrations of immunosuppressive drugs affect MSC function. MSC affect the efficacy of immunosuppressive medication. These findings are important for potential clinical use of MSC in combination with immunosuppressants.

Chondrogenic Potential of Human Adult Mesenchymal Stem Cells Is Independent of Age or Osteoarthritis Etiology

Osteoarthritis (OA) is a multifactorial disease strongly correlated with history of joint trauma, joint dysplasia, and advanced age. Mesenchymal stem cells (MSCs) are promising cells for biological cartilage regeneration. Conflicting data have been published concerning the availability of MSCs from the iliac crest, depending on age and overall physical fitness. Here, we analyzed whether the availability and chondrogenic differentiation capacity of MSCs isolated from the femoral shaft as an alternative source is age- or OA etiology-dependent. MSCs were isolated from the bone marrow (BM) of 98 patients, categorized into three OA-etiology groups (age-related, joint trauma, joint dysplasia) at the time of total hip replacement. All BM samples were characterized for cell yield, proliferation capacity, and phenotype. Chondrogenic differentiation was studied using micromass culture and analyzed by histology, immunohistochemistry, and quantitative reverse transcriptase-polymerase chain reaction. Significant volumes of viable BM (up to 25 ml) could be harvested from the femoral shaft without observing donor-site morbidity, typically containing >10(7) mononuclear cells per milliliter. No correlation of age or OA etiology with the number of mononuclear cells in BM, MSC yield, or cell size was found. Proliferative capacity and cellular spectrum of the harvested cells were independent of age and cause of OA. From all tested donors, MSCs could be differentiated into the chondrogenic lineage. We conclude that, irrespective of age and OA etiology, sufficient numbers of MSCs can be isolated and that these cells possess an adequate chondrogenic differentiation potential. Therefore, a therapeutic application of MSCs for cartilage regeneration of OA lesions seems feasible. Disclosure of potential conflicts of interest is found at the end of this article.

The Effects of Anticholinergic Insecticides on Human Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are located primarily in the bone marrow and are characterized by their capacity to differentiate into mesenchymal lineages such as bone, fat, and cartilage in response to appropriate signals. Several signaling mechanisms act to control MSC survival, proliferation, and differentiation, and failure or disruption of these signaling pathways can lead to degenerative disease or neoplasia. Organophosphate (OP) and carbamate pesticides, which are used in large amounts in agriculture to control insects, are designed to disrupt acetylcholine signaling by inhibiting the enzyme acetylcholinesterase (AChE). Effects of OP and carbamate pesticides on the human central nervous system have been well documented. However, AChE is broadly distributed, and the effects of anticholinergic insecticides on nonnervous tissue have received little attention. In the present study we found that human MSCs express AChE, which makes these cells potential targets for AChE inhibiting agents. We therefore examined the effects of an OP pesticide, chlorpyrifos, and a carbamate, carbofuran, on MSC characteristics. It was found that micromolar concentrations of these anticholinergic insecticides had no effect on MSC survival or proliferation but limited MSC differentiation capacity by inhibiting osteogenic differentiation. These results demonstrate that exposure to micromolar concentrations of OP and carbamate pesticides may affect tissue turnover and pathophysiology by interfering with MSC regulation.

Labelling of human mesenchymal stem cells with indium-111 for SPECT imaging: effect on cell proliferation and differentiation

Stem cell therapy seems to be a new treatment option within cardiac diseases to improve myocardial perfusion and function. However, the delivery and traceability of the cells represent a problem. Radioactive labelling with 111In could be a method for tracking mesenchymal stem cells (MSCs). However, 111In could influence the viability and differentiation capacity of MSCs, which would limit its use. Therefore, the aim of this study was to evaluate the influence of 111In labelling in doses relevant for SPECT imaging in humans on the viability and differentiation capacity of human MSCs. Human MSCs isolated from bone marrow were incubated with 111In-tropolone (15-800 Bq/cell). The labelling efficiency was approximately 25% with 30 Bq/cell 111In. The MSC doubling time was 1.04+/-0.1 days and was not influenced by 111In within the range 15-260 Bq/cell. Using 30 Bq 111In/cell it was possible to label MSCs to a level relevant for clinical scintigraphic use. With this dose, 111In had no effect on characteristic surface and intracellular markers of cultured MSCs analysed both by flow cytometry and by real-time polymerase chain reaction. Further, the labelled MSCs differentiated towards endothelial cells and formed vascular structures. It is possible to label human MSCs with 111In for scintigraphic tracking of stem cells delivered to the heart in clinical trials without affecting the viability and differentiation capacity of the MSCs. This creates an important tool for the control of stem cell delivery and dose response in clinical cardiovascular trials.

Cellular Iron Metabolism Studies Demonstate Safety of Magnetic Tracking of Mesenchymal Stem Cells.

Background: Ferumoxides-protamine sulfate (Fe-Pro) complexes are used for intracellular magnetic labeling of mesenchymal stem cells (MSCs) to non invasively monitor cell trafficking and tissue distribution by in vivo magnetic resonance imaging (MRI). It has been previously shown that there was no short or long- term toxic effects on cell's viability, proliferation, reactive oxygen species and biological functions including differentiation capacity of MSCs, however the effects of dextran coated superparamagnetic iron oxide (SPIO) nanoparticles labeling on cellular iron metabolism and storage is unknown.Objective: Evaluate the effect of using Fe-Pro complexes for magnetic cell labeling in MSCs on the expression of transferrin receptor (TfR-1) and ferritin, proteins involved in intracellular iron metabolism and storage.Methods: Confluent MSCs were labeled with Fe-Pro complexes at the ratio of 100 ug/mL to 6 ug/mL. After labeling, cells were washed and cultured for 2 months. TfR-1 and ferritin expression were evaluated at the gene and protein levels in total cell lysates, on day 1, 3, 7, 14, 28 and 35 days post-labeling, by real-time polymerase chain reaction (RT-PCR) and Western blotting. MSCs were evaluated for the presence of iron by Prussian blue stain and light microscopy. In addition, TfR-1 and ferritin levels were determined for Fe-Pro labeled and unlabeled MSCs, allowed to slowly multiply, as a result of dividing the cells in half when they reached confluence.Results: All cells were Prussian blue positive on light microscopy following labeling with Fe-Pro. Labeling of MSCs grown to confluence resulted in short-term increase of TfR-1 mRNA (day 1 and day 3, p= 0.0056) without changes in TfR-1 protein levels. Fe- Pro labeled MSC demonstrated an increase in ferritin gene expression on day 7 and 14 (p=0.0003) compared to unlabeled cells, while protein levels were higher compared to unlabeled MSC at each time point (p=0.005). However, ferritin protein levels from Fe-Pro labeled MSCs were not significantly increased after 7 passages.Conclusions: Magnetic labeling of cells with FDA approved ferumoxides complexed to protamine sulfate elicited short-term changes in cellular iron metabolism and storage that was dependent on ability to divide. Fe-Pro labeling did not have any long-term implications validating the safety of Fe-Pro labeling technique for MSCs trafficking studies by MRI.

Mesenchymal Stem Cell Deficiencies in Myeloma Patients.

Progression of Multiple Myeloma (MM) is associated with disrupted bone remodelling resulting from increased osteoclast activity and reduced osteoblast number in involved bones. The aim of this study was to investigate Mesenchymal Stem Cells (MSCs) properties in MM patients to find out whether disorders exist within the Bone Marrow (BM) microenvironment and during disease progression. We examined MSCs derived from 6 MM patients at diagnosis, 3 patients with Bone Lesions (BL-MSCs) and 3 patients Without Lesions (WL-MSCs), and 3 Normal Donors (ND-MSCs). We focused on mesenchymal phenotype, clonogenic and proliferative capacities, Growth Factor Receptors (GF-R) expression, as well as osteogenic differentiation. BM adherent mononuclear cells were used to initiate MSC cultures and 2 passages were performed when confluency was reached. Adherent MSCs were numbered after each passage and CFU-F was counted (at 11 day culture). Analysis of MSC immunophenotype and GF-R expression were performed by flow cytometry. Osteoblastic differentiation assay was performed in inducible specific medium by analysing alkaline phosphatase activity and matrix mineralisation deposition (Von Kossa method). MSCs represented 0.1 to 1% of BM nuclear cells from all sources. The characteristic MSC phenotype (CD45−/CD90+/CD73+/CD105+) was observed in all sources of cells (MM- and ND-MSCs). The clonogenic capacity of BL-MSCs was 2 fold lower than that of WL-MSCs (44 ± 21 vs 89 ± 23, p = 0.05) and their proliferative capacity was 7.5 fold decreased (16,5 x 106 ± 15 vs 124 x 106 ± 68, p = 0.02, total cells after 2 passages). Expression level of PDGFa & b, IGF-1, EGF and NGF Receptors was lower in MM-MSCs than in ND-MSCs (185 ± 39 vs 413 ± 50 UA, p = 0.0004, 212 ± 63 vs 383 ± 143 UA, p = 0.01, 227 ± 50 vs 534 ± 67 UA, p = 0.0007, 229 ± 34 vs 535 ± 84 UA, p = 0.0001 and 179 ± 26 vs 441 ± 98 UA, p = 0.001, respectively), while the percentage of FGF receptor-expressing cells was dramatically decreased in MM as compared to ND (59 ± 31.5 vs 90 ± 6 %, p = 0.01). Interestingly, the osteogenic differentiation capacity of MSCs was maintained in MM-MSCs whatever the progression status of the disease. This study demonstrates that impaired bone formation in MM could be related to a deficient proliferation capacity of MSCs associated with a low growth factor receptors expression. The lack of expansion capacity increases with disease progression. Therapeutic approaches using normal expanded MSCs grafts may improve bone reconstruction in myeloma patients.

Blood from Mesenchymal Stem Cell.

Mesenchymal stem cells (MSC) have been described to support the maintainance and expansion of clonogenic hematopoietic cells in vitro and as progenitors for nonhematopoietic tissues. They clearly can be distinguished from hematopoietic cells by the surface markers CD45 and CD34. We investigated the potential of human MSC to form blood and endothelial cells. Human MSC in expansion cultures expressed RNA for CD117 and EpoR but not for other hematopoietic antigens. Many transcription factors essential for blood cell differentiation, e.g. c-mpl, Notch-1, Gata-2 and -3, Runx-1 and SCL, were detected in these cells. We used different differentiation strategies: A) Hematopoietic differentiation: 1. Prestimulation in IMDM + fetal calf serum (FCS) + horse serum (HS), mixed with methylcellulose (MC, containing cytokines SCF, IL-3, IL-6, G-CSF, GM-CSF and Epo) and differentiation in MC; 2. Prestimulation in serum free medium supplemented with SCF, TPO, GM-CSF and flt.- 3 ligand and differentiation in serum free MC; B) Endothelial differentiation: Prestimulation in serum free medium supplemented with SCF, TPO, and flt-3L and differentiation in medium + VEGF + bFGF + IGF. Using FACS-analysis, we detected up to 1.8 % CD34-positive cells within 2 weeks. Immunochemically we observed cells positive for CD45, CD34, CD133, CD14, CD16 and CD41. Although hMSC do not form colonies after seeding in MC, after prestimulation we observed colonies with the typical appearance of BFU-E which were GlycophorinA positive. Under endothelial conditions we detected cells positive for CD34 and KDR or CD31 and vWF. However, cell numbers of positive cells were low and therefore RT-PCR results were not very reliable. To confirm the differentiation capacity of MSC we generated CD45.2 mouse MSC (P9 what took about 6 month) and transplanted into lethally irradiated CD45.1 recipient mice. Four weeks after transplantation mice showed normal white blood cell counts but still decreased platelet counts. Analysis of donor chimerism 29 weeks after transplantation revealed up to 36 % (14.3±7.9%) donor cells in peripheral blood, mainly in the CD11b positive myeloid population but not in lymphoid cells. In thymus and bone marrow, marginal donor chimerism (3±0.8%) was detected, but the number in c-kit+ cells reached up to 28.2% (7.9±6.8%). In B-cells and erythroid progenitors chimerism was low but stable, but not in CD3+ cells. Taken together, we have shown that under the right conditions MSC are able to form blood and endothelial cells and reconstitute animals. We suggest that MSC could be an yet unrecognised precursor for both cell types and may contain the long searched hemangioblast in adult organisms.