Engraftment Of Mesenchymal Stem Cells Research Articles

Therapeutic Effects of Bone Marrow-Derived Mesenchymal Stem Cells Engraftment on Bleomycin-Induced Lung Injury in Rats

Previous studies have demonstrated that bone marrow-derived mesenchymal stem cell (MSC) engraftment attenuated lung injury in a model induced by bleomycin in mice. However, the mechanisms are not completely understood. The primary objective of the present study was to determine whether MSC engraftment can also protect lungs against bleomycin-induced injury in rats and to observe any beneficial effects of cytokines. Twelve hours after bleomycin (5 mg/kg) or phosphate-buffered saline was perfused into the trachea, 5×10 6 DAPI-labeled MSCs or DMEM-F 12 were injected into the tail vein of rats. Two weeks later, MSCs labeled with DAPI were detected by pan-cytokeratin staining. The level of laminin and hyaluronan in bronchoalveolar lavage fluid was measured by radioimmunoassay. Collagen content in lung tissue was calculated by the hydroxyproline assay. TGF-β 1, PDGF-A, B, and IGF-I were measured by real-time PCR. It was observed that some MSCs positive for pan-cytokeratin staining, an indicator of alveolar epithelial cells, were present in injured lung tissue. Bleomycin injection increased the content of hydroxyproline in lung tissue, as well as laminin and hyaluronan in bronchoalveolar lavage fluid, markers for lung injury and fibrosis. However, these effects were attenuated by MSC treatment. Furthermore, the increased mRNA levels of TGF-β 1, PDGF-A, PDGF-B, and IGF-I following bleomycin injection were also significantly decreased by MSC treatment. These observations provided evidence that MSCs are still present in the lung 2 weeks after the initial MSC treatment in rats, as well as documented the beneficial effects of MSC engraftment against bleomycin-induced lung injury associated with changes in TGF-β 1, PDGF-A, PDGF-B, and IGF-I. These results may provide an experimental base for clinical therapy of pulmonary fibrosis in the future.

A perspective: engineering periosteum for structural bone graft healing.

Autograft is superior to both allograft and synthetic bone graft in repair of large structural bone defect largely due to the presence of multipotent mesenchymal stem cells in periosteum. Recent studies have provided further evidence that activation, expansion and differentiation of the donor periosteal progenitor cells are essential for the initiation of osteogenesis and angiogenesis of donor bone graft healing. The formation of donor cell-derived periosteal callus enables efficient host-dependent graft repair and remodeling at the later stage of healing. Removal of periosteum from bone autograft markedly impairs healing whereas engraftment of multipotent mesenchymal stem cells on bone allograft improves healing and graft incorporation. These studies provide rationale for fabrication of a biomimetic periosteum substitute that could fit bone of any size and shape for enhanced allograft healing and repair. The success of such an approach will depend on further understanding of the molecular signals that control inflammation, cellular recruitment as well as mesenchymal stem cell differentiation and expansion during the early phase of the repair process. It will also depend on multidisciplinary collaborations between biologists, material scientists and bioengineers to address issues of material selection and modification, biological and biomechanical parameters for functional evaluation of bone allograft healing.

Distribution of Murine Adipose-Derived Mesenchymal Stem Cells in vivo Following Transplantation in Developing Mice

Systemic delivery of mesenchymal stem cells (MSCs) or stromal cells in vivo is attractive because it offers means of disseminating therapeutic cells to various tissues and organs in vivo. In the present study, we investigated the distribution and engraftment of the murine adipose-derived mesenchymal stem cells (ADSCs) without exposure to or exposed to bone microenvironment or transforming growth factor-beta1 (TGF-beta1) prior to transplantation into developing mice. The ADSCs harvested from the murine inguinal fat pad exhibited potential for differentiation toward osteogenic and adipogenic cell lineages in vitro. Fourteen days after systemic transplantation of the ADSCs marked with enhanced green fluorescent protein (EGFP) into developing mice, minimal donor GFP(+) cells were detected in the skeletal tissues in a limited number of the recipient mice. Exposure of the ADSCs to bone microenvironment for 7 or 14 days prior to transplantation into developing mice enhanced their migration and survival in the bones of the recipient mice. Exposure of ADSCs to TGF-beta1 prior to systemic transplantation exerted similar effects on cell migration and engraftment in various tissues, including the bones of the recipient developing mice. At 28 days following systemic transplantation, the ADSCs exposed to bone microenvironment were restricted mostly to the skeletal tissues of the recipient mice. Donor cells retrieved from the bones of the recipient mice at 28 days following cell transplantation expressed the differentiation markers Runx2 and Osterix (Osx). These data suggest that exposure of ADSCs to bone microenvironment or to TGF-beta1 prior to transplantation enhances their survival in the skeletal tissues following transplantation.

Granulocyte-Colony Stimulating Factor Increases Donor Mesenchymal Stem Cells in Bone Marrow and Their Mobilization Into Peripheral Circulation but Does Not Repair Dystrophic Heart After Bone Marrow Transplantation

Hereditary disordered cardiac muscle could be replaced with intact cardiomyocytes derived from genetically intact bone marrow (BM)-derived stem cells. Cardiomyopathic mice with targeted mutation of delta-sarcoglycan gene underwent intra-BM-BM transplantation (IBM-BMT) from transgenic mice expressing green fluorescence protein. The host BM and the peripheral blood were completely reconstituted by donor-derived hematopoietic cells by IBM-BMT. Treatment with granulocyte-colony stimulating factor (G-CSF) markedly increased donor-derived mesenchymal stem cells (MSC) in the BM and their mobilization into the peripheral blood after IBM-BMT. Treatment with isoproterenol (iso) for 7 days caused myocardial damage and left ventricular (LV) dysfunction in the cardiomyopathic mice. Co-treatment with iso and G-CSF increased donor BM cell recruitment to the heart and temporarily improved LV function in the cardiomyopathic mice with or without IBM-BMT. However, the cardiac muscle was not replaced with donor BM-derived cardiomyocytes in the cardiomyopathic mice with or without IBM-BMT, and this was associated with no improvement of LV function of mice aged 20 weeks. These results suggest that G-CSF enhances engraftment of donor MSC in the BM and their mobilization into the peripheral circulation after IBM-BMT but MSC recruited to the heart do not differentiate into cardiomyocytes and do not repair the dystrophic heart.

Effect of Pax3 Overexpression on the Engraftment of Mesenchymal Stem Cells in Dystrophic Mice.

Mesenchymal stem cells (MSCs) residing within the bone marrow (BM) not only support hematopoiesis, but also differentiate into multiple lineages, including fat, bone, and cartilage. Because MSCs are multipotent and have a great expansion capacity in vitro, these cells are attractive candidates for clinical applications to repair or regenerate damaged tissues of mesenchymal origin. However, treatment of muscle degenerative diseases with MSCs has been hampered by the poor differentiation of MSCs into the muscle lineage. To date most in vitro methods require the presence of strong non-physiological agents, such as azacytidine. Accordingly, limited engraftment has been observed following the transplantation of BM into dystrophic mice (less than 4%). In the present study we explored the potential of Pax3, the master regulator of the embryonic myogenic program, to promote myogenic differentiation from human BM-derived MSCs. Upon genetic modification with lentiviral vectors encoding Pax3-GFP or GFP only (vector control), MSCs were evaluated for their potential to differentiate towards muscle in vitro. Real time PCR analyses revealed up-regulation of several myogenic regulatory factors in Pax3-transduced MSC cultures in various differentiation conditions, whereas control GFP-MSCs showed undetectable levels. When transplanted into cardiotoxin-injured tibialis anterior (TA) muscles of Rag2−/− γc−/− immunodeficient as well as dystrophic mice, superior engraftment was observed with Pax3-transduced MSCs (31% vs. 12% and 12% vs. 6.5%, respectively). To assess whether such levels of engraftment are therapeutically relevant, functional analyses of transplanted muscles are required. We are currently undertaking these experiments.

Expression of HLA-G1 and G5 Enables Human Mesenchymal Stem Cells To Engraft at High Levels across Xenogeneic Barriers Following Transplantation into Immunocompetent Recipients.

Although mesenchymal stem cells (MSC) have been shown to be fairly non-immunogenic, recent studies demonstrated that MSC are capable of activating and becoming targets of NK-mediated lysis. Furthermore, the ability of IFN-γ to induce MSC expression of HLA-II molecules shows that MSC are not completely immuno-inert. Expression of HLA-G has been associated with the maintenance of fetomaternal tolerance during pregnancy through its inhibition of the cytolytic functions of NK and cytotoxic T cells and of dendritic cell maturation. Here we investigated whether transduction of human MSC with a retroviral vector encoding HLA-G1 (MSC-G1) or G5 (MSC-G5) would render these cells and their differentiated progeny undetectable by the recipient's immune system, and thereby allow us to efficiently transplant these cells into immuno-competent xenogeneic recipients. First, we performed mixed lymphocyte reactions using unmodified MSC (unMSC), MSC-G1 or MSC-G5 as stimulators, and lymphocytes from allogeneic human or sheep donors as responders. While neither unMSC nor MSC-G1 or -G5 elicited a significant proliferative response from human lymphocytes, sheep lymphocytes proliferated 2-6 fold more when cultured with unMSC than with MSC-G1 or MSC-G5. Next, we tested whether HLA-G1 and HLA-G5 expression would enable the engraftment of human MSC in fetal sheep later in gestation when donor cells are normally rejected due to the presence of a competent immune system. To this end, 105 unMSC, MSC-G1, or MSC-G5 were transplanted (Tx) into fetal sheep recipients during the pre-immune period (55 days; n=9), or after immunocompetence was achieved, at 82 days (n=10) or 104 days (n=8) of gestation. Evaluation of the recipients' hematopoietic system at 42 days post-transplant for the presence of human cell engraftment, using a panel of antibodies specific to human blood cells, revealed that Tx at 55 days resulted in similar levels of engraftment for all cell types (MSC:4±0.9; MSC-G1:6±0.3; MSC-G5:5±0.3%). In contrast, while unMSC engrafted at very low levels at 82d and 104d, Tx of MSC-G1 and-G5 at these later time points not only overcame the immune barriers, but resulted in levels of engraftment that were considerably higher than those achieved during the pre-immune period (82d:7±1;18±2.% and 104d:12±2;16±2%). A similar outcome was seen with liver engraftment and hepatic differentiation with MSG-1 and -G5 giving rise to 3–5 times more hepatocytes than unMSC at later time points. However, even at the early Tx time point of 55days, MSC-G1 and -G5 gave rise to, respectively, 5 times and 2 times more donor-derived hepatocytes than their unMSC counterpart. Our studies demonstrate that the forced expression of HLA-G1 or G5 enables MSC to evade a competent recipient immune system and engraft at significant levels at times in gestation when donor cells are normally rejected. These studies may allow the broadening of the use of MSC to diseases in which an underlying MSC defect precludes the use of the patient's own MSC.

Stem cell factor and mesenchymal and neural stem cell transplantation in a rat model of Huntington's disease

Neural and mesenchymal stem cells have been proposed as alternative sources of cells for transplantation into the brain in neurodegenerative disorders. However, the endogenous factors controlling their engraftment within the injured parenchyma remain ill-defined. Here, we demonstrate significant engraftment of undifferentiated exogenous mesenchymal or neural stem cells throughout the lesioned area in a rat model for Huntington's disease, as late as 8 weeks post-transplantation. We show that stem cell factor (SCF), strongly up-regulated within host cells in the damaged striatum, is able to activate the SCF receptor c-kit and its signaling pathway and to promote the migration and proliferation of mesenchymal and neural stem cells in vitro. Furthermore, c-kit receptor blockade alters neural stem cell distribution within the lesioned striatum. Host SCF expression is observed in atypical cells expressing glial fibrillary acidic protein and doublecortin in the lesioned striatum and in migrating doublecortin-positive progenitors. Taken together, these data demonstrate that SCF produced in situ in the lesioned striatum is an important factor in promoting the engraftment of stem cells within the lesioned brain.

Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium

We hypothesized that preconditioning (PC) with stromal-derived factor 1 alpha (SDF-1) significantly enhances cell survival, proliferation, and engraftment of bone marrow-derived mesenchymal stem cells (MSCs) via SDF-1/CXCR4 signaling. MSCs were cultured and then incubated in medium for 60 min without SDF-1 (control group) or with SDF-1 0.05 microg/mL (SDF-1 group) or CXCR4-selective antagonist, AMD 3100 (AMD) (5 microg/mL, AMD group) or SDF-1 and AMD (0.05 microg/mL, 5 microg/mL, respectively, SDF-1+AMD group). MSCs were treated for 60 min, washed in normal medium, and then exposed to H2O2 (100 micromol/L) for 60 min to determine the effects of various treatments on cell injury, viability, and proliferation. For in vivo studies, rats were grouped (n = 6) after left anterior descending coronary artery ligation to receive 20 microL Dulbecco's modified Eagle's medium without cells or with 5 x 10(5) non-preconditioned MSCs (control group), SDF-1 preconditioned MSCs (SDF-1 group), AMD (AMD group), or MSCs treated with SDF-1 plus AMD (SDF-1+AMD group). Heart function, infarct size, fibrosis, and MSC proliferation and differentiation in infarcted myocardium were determined after 4 weeks. In vitro data showed a marked increase in cell viability and proliferation following SDF-1 PC. In vivo data in preconditioned group showed a robust cell proliferation, reduction in infarct size and fibrosis, and significant improvement in cardiac function. Effects of SDF-1 PC were abrogated by CXCR4 antagonist. We conclude that PC with the chemokine SDF-1 suppresses MSCs apoptosis, enhances their survival, engraftment, and vascular density, and improves myocardial function via SDF/CXCR4 signaling. Chemokine PC is a novel approach for enhancing stem cell survival and regeneration of infarcted myocardium.

Monocyte Chemotactic Protein-1 Secreted by Primary Breast Tumors Stimulates Migration of Mesenchymal Stem Cells

Major barriers to effective adenovirus-based gene therapy include induction of an immune response and tumor-specific targeting of vectors. The use of mesenchymal stem cells (MSC) as systemic delivery vehicles for therapeutic genes has been proposed as a result of their combined ability to home in on the tumor site and evade the host immune response. This study is aimed at investigating factors mediating homing of human MSCs to breast cancer primary cultures and cell lines in vitro and in vivo. Fluorescently labeled MSCs were given to mice bearing breast cancer xenografts, and tumor tissue was harvested to detect MSC engraftment. MSC migration in response to primary breast tumors in vitro was quantified, and chemokines secreted by tumor cells were identified. The role of monocyte chemotactic protein-1 (MCP-1) in cell migration was investigated using antibodies and standards of the chemokine. Serum MCP-1 was measured in 125 breast cancer patients and 86 healthy controls. Engrafted MSCs were detected in metastatic breast tumors in mice after systemic administration. There was a significant increase in MSC migration in response to primary breast tumor cells in vitro (6-fold to 11-fold increase). Tumor explants secreted a variety of chemokines including GROalpha, MCP-1, and stromal cell-derived factor-1alpha. An MCP-1 antibody caused a significant decrease (37-42%) in MSC migration to tumors. Serum MCP-1 levels were significantly higher in postmenopausal breast cancer patients than age-matched controls (P < 0.05). These results highlight a role for tumor-secreted MCP-1 in stimulating MSC migration and support the potential of these cells as tumor-targeted delivery vehicles for therapeutic agents.

Injured microenvironment directly guides the differentiation of engrafted Flk-1 + mesenchymal stem cell in lung

Time window is a key factor in the treatment of lung injury by mesenchymal stem cells (MSC) transplantation. This study was aimed to analyze the engraftment and differentiation behavior of MSC transplanted at different time points after lung irradiation, and the possible mechanisms were discussed. The thorax of C57BL/6 mice was exposed to 1400 cGy, then Flk-1(+)MSCs from enhanced green fluorescent protein C57BL/6 mice were systemically injected into C57BL/6 mice at 4 hours, 60 days, and 120 days post thoracic exposure, respectively. The engraftment and differentiation of Flk-1(+)MSC transplanted at different time points were evaluated. Lung tissue was collected and analyzed for fibrosis. Expression of transforming growth factor (TGF)-beta1 in the lung was qualified by semi-quantitative real-time reverse transcription polymerase chain reaction. In vitro, Flk-1(+)MSCs were cultured in epithelium induction media, together with damaged primary lung cells, supernatants of radiation-injured lung cells, or TGF-beta1 to find the possible factors that might effect Flk-1(+)MSC differentiation. Cells injected immediately after injury were shown to differentiate into functional lung cells, such as epithelial and endothelial cells. Cells injected 2 months later were mostly located in the interstitial area and appeared as myofibrocyte. The in vivo lung microenvironments at different time points after injury were different from each other, especially TGF-beta1 expression. We demonstrated that cytokines secreted by irradiated lung cells could inhibit differentiation of Flk-1(+)MSCs into epithelial cells in vitro. Flk-1(+)MSCs injected into the lung immediately after irradiation could differentiate into functional lung cells, while those injected at later stage after irradiation would be involved in fibrosis development. Thus our in vivo and in vitro studies demonstrated that differentiation of Flk-1(+)MSCs is controlled by the microenvironment.

Regulation of CXCR4 expression in human mesenchymal stem cells by cytokine treatment: role in homing efficiency in NOD/SCID mice

The use of mesenchymal stem cells (MSC) for cell therapy relies on the capacity of these cells to home and engraft long-term into the appropriate target tissue(s). Homing of MSC to bone marrow (BM) post-transplantation can occur, but does so with only poor efficiency. This study was designed to evaluate the role of the SDF-1/CXCR4 axis in the homing of Flk1+ MSC derived from human fetal BM. We investigated the expression of CXCR4 in Flk1+ MSC stimulated with a cytokine cocktail and explored their homing ability 24 hours after intravenous infusion into sublethally irradiated NOD/SCID mice. The peripheral blood was analyzed and human cells in recipients' BM were quantified from 2 to 6 months after transplantation. We found that Flk1+ MSC harbored intracellular CXCR4 which can be rapidly induced to the cell surface within a few hours. Short-term (24 hours) stimulation with the cocktail of cytokines resulted in up-regulation of both cell surface and intracellular CXCR4, increasing in vitro migration capacity to SDF-1 and homing to the BM of irradiated NOD/SCID mice. Moreover, compared to non-treated cells, transplantation of cytokine-treated Flk1+ MSC resulted in faster hematologic recovery and higher levels of donor chimerism in BM. Neutralization of CXCR4 significantly reduced homing and engraftment of Flk1+ MSCs in murine BM. These results suggest that the SDF-1/CXCR4 axis plays an important role in the regulation of motility of Flk1+ MSC. Increasing CXCR4 expression might be a potential strategy to improve engraftment of MSC in BM and accelerate the recovery of hematopoiesis.

Patient and Cellular Characteristics Determine Efficacy of Cell Therapy

See related article, pages 1234–1241 Recent clinical trials have demonstrated the safety and potential benefits of the stem cell based therapies for the preservation and treatment of cardiac dysfunction following acute myocardial infarction.1,2 More recently Assmus and colleagues published the TOPCARE-CHD trial in which they compared the effects of infusing circulating progenitor cells to bone marrow mononuclear cells in patients at a time remote from acute myocardial infarction (average time from AMI to infusion, 6 to7 years).3 Using a crossover clinical trial design that study demonstrated that circulating progenitor cells offered no benefit, where as infusion of whole bone marrow mononuclear cells resulted in an increase of 2.9% in the ejection fraction compared with a loss of 1.2% in control patients.3 The observation that circulating progenitor cells and bone marrow mononuclear cells lead to improved function in acute myocardial infarction,4 but only bone marrow derived progenitor cells led to improvement at times significantly remote from AMI suggest that different cell populations or delivery strategies may be efficacious in one setting but not another. For example, CD34+ cells are present in both circulating progenitor cells and whole bone marrow mononuclear preparations, mesenchymal stem cells are only present in the bone derived progenitor cell preparations. Similarly, the literature to date has demonstrated critical differences between myocardial tissue immediately after and that at times remote from an acute ischemic event with respect to the expression of stem cell homing factors, inflammatory infiltrates and putative differentiation factors.5,6,7 Thus, although CD34+ cells may be able to home to myocardial tissue in the periinfarct period,5,6 CD34+ cells may require direct injection into the myocardial tissue at times …

Functional Expression ofN-Formyl Peptide Receptors in Human Bone Marrow-Derived Mesenchymal Stem Cells

Tissue injury enhances homing and engraftment of mesenchymal stem cells (MSCs). However, the mechanisms by which MSCs sense the signals released by injured tissues and migrate toward injury sites have not been fully defined. In the current report, we investigated whether human MSCs express the N-formyl peptide receptor (FPR) and the formyl peptide receptor-like-1 (FPRL1). These receptors bind to N-formylated peptides by which phagocytes migrate to inflammatory sites and fibroblasts repopulate wounds to remodel the damaged tissues. Reverse-transcription polymerase chain reaction (PCR) demonstrated that MSCs express both FPR and FPRL1 at the transcriptional level. Flow cytometric analyses revealed expression of both receptors at the protein level. Fusion of the enhanced green fluorescence protein (eGFP) to the C terminus of each receptor showed localization to the cell surface. Moreover, MSCs responded to stimulation by N-formyl methionyl leucyl phenylalanine (fMLP), a prototypic N-formyl peptide, demonstrating rapid intracellular calcium mobilization that can be blocked by pertussis toxin or cyclosporin H. It is noteworthy that the fMLP-stimulated MSCs had an enhanced adhesion to extracellular matrix protein-coated surfaces. In addition, MSCs migrated toward gradients of increasing fMLP concentration, indicating that the receptors were functionally involved in positive chemotaxis to formylated peptides. Therefore, the N-formyl peptide receptors present in MSCs may play an important role in signaling stem cell adhesion, migration, and homing to injured and inflamed tissue for repair. Such a mechanism could potentially be exploited to direct the stem cells to target specific tissue sites, such as cystic fibrosis lungs, for therapy. Disclosure of potential conflicts of interest is found at the end of this article.

Engraftment and myogenic differentiation of cord blood mesenchymal stem cells after in vivo transplantation

Muscular dystrophies and neuromuscular disorders are characterized by progressive muscle wasting, and are significant health issues. We used mesenchymal stem cells (MSCs) isolated from human umbilical cord blood (UCB) capable of differentiating into various connective tissue lineages, and studied their myogenic potential. We injected 106 UCB-MSCs into a rat tibialis anterior model of muscle injury, caused by bupivacaine hydrochloride injection. The MSCs, labeled with green fluorescent protein (GFP), engrafted into the muscle after 1 week and stained positive for the early myogenic regulatory factors Myf-5 and MyoD. After 2 weeks, we noted striations of A and I bands showing the presence of sarcomeres in fibers containing UCB-MSCs. The skeletal muscle appeared intact by histological analysis, and the presence of MSCs was confirmed by immunostaining with HLA-A2 antibody, a human specific class I histocompatibility molecule. Expression of Myf-5, MyoD, and sarcomeric tropomyosin, a protein of actin filament was detected. There was no immunological response against the engrafted cells, and no immunosuppressive therapy was used. These results suggest that human UCB-MSCs are able to differentiate towards the myogenic lineage and to play a role in vivo during the muscle regenerative process. Supported by University of Padova, Cariparo Foundation, Italy, and the Coriell Institute.

Engraftment of Human Mesenchymal Stem Cells upon Transplantation into Newborn Rag2−/−gc−/− Mice.

We recently demonstrated human T cell, B cell, dendritic cell, and natural interferon producing cell development and consecutive formation of primary and secondary lymphoid organs in Rag2−/−gc−/− mice, transplanted as newborns intra-hepatically (i.h.) with human CD34+ cord blood cells (Traggiai et al., Science 2004). Although these mice support high levels of human cell engraftment and continuous T and B cell formation as well as CD34+ cell maintenance in bone marrow over at least six month, the frequency of secondary recipient reconstituting human hematopoietic stem and progenitor cells within the CD34+ pool declines over time. Also, although some human immune responses are detectable upon vaccination with tetanus toxoid, or infection with human lymphotropic viruses such as EBV and HIV, these responses are somewhat weak compared to primary human responses, and are inconsistent in frequency. Thus, some factors sustaining human hematopoietic stem cells in bone marrow and immune responses in lymphoid tissues are either missing in the mouse environment, or are not cross-reactive on human cells.Human mesenchymal stem cells (MSCs) replicate as undifferentiated cells and are capable to differentiate to multiple mesenchymal tissues such as bone, cartilage, fat, muscle, tendon, as well as marrow and lymphoid organ stroma cells, at least in vitro (e.g. Pittenger et al., Science 1999). Moreover, it was shown that MSCs maintain CD34+ cells to some extend in vitro, and engraft at low frequency upon transplantation into adult immunodeficient mice or fetal sheep as detected by gene transcripts. We thus postulated that co-transplantation of cord blood CD34+ cells and MSCs into newborn mice might lead to engraftment of both cell types, and to provision of factors supporting CD34+ maintenance and immune system function. MSCs were isolated and expanded by plastic adherence in IMDM, supplemented with FCS and cortisone (first 3 weeks) from adult bone marrow, cord blood, and umbilical vein. To test their potential to support hemato-lymphopoiesis, MSCs were analyzed for human hemato-lymphotropic cytokine transcription and production by RT-PCR and ELISA, respectively. MSCs from all sources expressed gene-transcripts for IL-6, IL-7, IL-11, IL-15, SCF, TPO, FLT3L, M-CSF, GM-CSF, LIF, and SDF-1. Consistently, respective cytokines were detected in supernatants at the following, declining levels (pg/ml): IL-6 (10000-10E6) > SDF-1 > IL-11 > M-CSF > IL-7 > LIF > SCF > GM-CSF (0–450), while FLT3L and TPO were not detectable by ELISA. Upon i.h. transplantation of same passage MSCs (1X10E6) into sublethally irradiated (2x2 Gy) newborn Rag2−/−gc−/− mice, 2-week engraftment was demonstrated by species specific b2m-RT-PCR in thymus, spleen, lung, liver and heart in n=7 and additionally in thymus in n=3 out of 13 animals analyzed. Equally, GFP-RNA transcripts were detectable in the thymus for up to 6 weeks, the longest time followed, upon co-transplantation of same source CD34+ cells and retrovirally GFP-transduced MSCs in n=2 out of 4 animals. Further engraftment analysis of ongoing experiments will be presented.Overall, these results demonstrate that human MSC produce hemato-lymphoid cytokines and engraft in newborn transplanted Rag2−/−gc−/− mice, at least at early time-points analyzed. This model thus might allow studying hematopoietic cell and MSC-derived cell interaction, and might serve as a testing system for MSC delivered gene therapy in vivo.

Enhanced Engraftment of Mesenchymal Stem Cells in a Cutaneous Wound Model by Culture in Allogenic Species‐Specific Serum and Administration in Fibrin Constructs

Human mesenchymal stem cells (hMSCs), also referred to as multipotent stromal cells, are currently being applied in clinical trials for bone diseases, graft versus host disease, and myocardial infarction. However, the standard growth medium for hMSCs contains 10%-20% fetal calf serum (FCS), and FCS is strongly immunogenic in both rodents and humans. Previously, we reported that by a sensitive fluorescence-based assay, 7-30 mg of internalized FCS is associated with 10(8) hMSCs, a dosage that will probably be needed for most therapies. We also found that a brief culture in medium containing autologous 20% adult human serum (AHS) or autologous 10% AHS supplemented with growth factors (AHS(+)) reduced the contamination by more than 99.9%. We have now extensively characterized the culture conditions and shown that hMSC expansion is possible using heterologous 20% AHS or heterologous 10% AHS(+). The uptake of FCS is an active process that acts to concentrate contamination in the cells even under low serum conditions (2% FCS) but can be actively displaced by incubation of the cells in medium with AHS. Rat MSCs (rMSCs) can be expanded under similar conditions using supplemented heterologous adult rat serum (ARS(+)). After expansion in FCS, a further 8 days of culture with ARS(+) significantly improves the viability of the rMSCs in vivo after encapsulation in fibrin followed by subcutaneous implantation in rats. Our results have the potential to dramatically improve cellular and genetic therapies using hMSCs.

Selective migration and engraftment of bone marrow mesenchymal stem cells in rat lumbar dorsal root ganglia after sciatic nerve constriction

Bone marrow mesenchymal stem cells (MSCs) preferentially migrate to the injured hemisphere when administered intravenously to rats with traumatic or ischemic brain injuries. In this study, we have investigated the localization of MSCs injected into the lumbar-4 dorsal root ganglion (L4-DRG) of rats with a sciatic nerve single ligature nerve constriction (SLNC). MSCs were isolated by their adherence to plastic, cultured until confluence and labelled with Hoechst. Animals with a unilateral injection of MSCs were subjected to an ipsilateral, bilateral or contralateral SLNC. After 9 days, they were perfused and the lumbar DRGs were dissected out, cut in a cryostat and observed with a fluorescence microscope. Large numbers of Hoechst-positive cells were observed in the injected L4-DRG, distributed around primary afferent neurons, resembling the anatomical localization of glial cells. In animals with an ipsilateral SLNC, some cells were detected in the ipsilateral L3, L5 or L6-DRGs but not in the contralateral ganglia. In animals with a bilateral lesion, MSCs migrated to both the ipsilateral and contralateral DRGs whereas in animals with a contralateral ligature, MSCs migrated to the contralateral DRGs. These results suggest that MSCs preferentially engraft in DRGs hosting primary sensory neurons affected by a lesion of their peripheral branches. Further studies should be carried out in order to elucidate the molecular mechanisms involved in this migration and homing, in order to evaluate the possible use of MSCs as a new therapeutic strategy for the treatment of peripheral nerve neuropathies.

The time course of engraftment of human mesenchymal stem cells in fetal heart demonstrates that Purkinje fiber aggregates derive from a single cell and not multi-cell homing

To study the early time course of engraftment of human mesenchymal stem cells in fetal sheep heart and determine the relative roles of proliferation and homing in formation of aggregates of human Purkinje fiber cells. The human sheep xenograft model was utilized for these studies. Prior to injection in the preimmune fetus, human cells were labeled with fluorescent dyes to be able to track human cells at early times of engraftment. Human stem cells were detected in fetal hearts between 29 and 39 hours after intraperitoneal injection. Engraftment was primarily in the Purkinje fiber system. By 45 hours engrafted human cells had a cardiac phenotype. When two groups of human mesenchymal stem cells, each labeled with a different fluorescent dye, were combined prior to injection, aggregates of human Purkinje fiber cells contained cells labeled with either one dye or the other, no aggregate contained cells labeled with both dyes. Human mesenchymal stem cells introduced into fetal sheep rapidly enter the myocardium. The swift differentiation into a cardiac phenotype indicates that the cardiac milieu has a strong influence on the fate of engrafting human mesenchymal stem cells. The absence of any aggregates of human Purkinje fiber cells containing both fluorescent dyes demonstrates that each aggregate of human Purkinje fiber cells is derived from a single mesenchymal stem cell and not from homing of multiple cells to a hotspot.

Murine Mesenchymal Stem Cells Transplanted to the Central Nervous System of Neonatal Versus Adult Mice Exhibit Distinct Engraftment Kinetics and Express Receptors That Guide Neuronal Cell Migration

Mesenchymal stem cells (MSCs) have demonstrated efficacy as cellular vectors for treating a variety of nervous system disorders. Nevertheless, few studies have quantified MSC engraftment levels or explored the mechanisms that promote their survival and migration in nervous tissue. In this study, we compared the engraftment kinetics and anatomical distribution of murine, male MSCs injected intracranially into neonatal versus adult female mice using a real-time PCR assay that targets the mouse SRY gene. These analyses revealed that MSCs exhibited low but equivalent engraftment levels in the central nervous system (CNS) of neonatal and adult transplant recipients at 12 days post-injection. However, MSC engraftment levels were significantly greater at 60 and 150 days post-transplantation in neonates as compared to adults. Despite these differences, engrafted MSCs were widely distributed along the neuraxis of the CNS in both transplant groups. Collectively, these data indicate that proliferation, but not engraftment and migration, of MSCs in brain are regulated by the host microenvironment. Using a genomics approach, we also identified MSC subpopulations that express neural adhesion proteins and receptors that regulate neuronal cell migration in brain, including cadherin 2, neurexin 1, ninjurin 1, neogenin 1, neuropilin 2, and roundabout homolog 1 and 4. Functional studies indicate these proteins confer cell adhesion and migration of MSCs in response to the appropriate chemoattractant. On the basis of these findings, we conclude that the unique molecular composition of MSC subpopulations imparts to them an inherent capacity to engraft and migrate in brain. These subpopulations may represent more potent cellular vectors for treating CNS disorders.

Effect of reduced culture temperature on antioxidant defences of mesenchymal stem cells

Mesenchymal stem cells (MSC) promise to be valuable therapeutic tools but, due to their low numbers, require considerable in vitro expansion before use. This leads to in vitro aging, the accumulation of intracellular oxidative damage, and subsequently a decreased potential for proliferation and differentiation. Optimised culture conditions might help to reduce oxidative damage in MSC in vitro, and therefore, as reduced temperature is known to reduce oxidative stress in other somatic cells, we have investigated the effect of reduced temperature on rat MSC viability, differentiation, and oxidative damage. Temperature reduction did not affect MSC viability but increased differentiation and reduced apoptosis. Oxidative-damage-related indices were improved; reactive oxide species, nitric oxide, thiobarbituric acid reactive substances, carbonyl, and lipofuscin levels were reduced and glutathione peroxidase and superoxide dimutase levels increased. Levels of antiapoptotic heat shock proteins (HSP-27, -70, and -90) were raised and levels of the proapoptotic HSP-60 reduced. These data demonstrate that culturing MSC at reduced temperature decreases the accumulation of oxidative damage and therefore would probably improve long-term viability and successful engraftment of MSC used for tissue engineering or cell therapeutic purposes.