The Multipotency Of Mesenchymal Stem Cells And Therapeutic Benefits On Denervation Muscle Atrophy In Rats

PartⅠThe multipotency of mesenchymal stem cells in rats.Objective To investigate multipotentcy of MSCs differentiation into osteoblasts,skeletal muscle, neuron-like cells and astrocyte-like cells in vitro and evaluate the proliferation and survival capacity of MSCs-differentiated neural progenitor cells, neuron-like cells and astrocyte-like cells in vitro.Methods Adult rat MSCs were isolated and cultured for 5 passages in vitro. Osteogenic differentiation was induced by plating the cells at 1×10~5cells/ml in DMEM medium containing 10%FBS,supplenmented with 0.1μmol/l dexamethasone,50ng/ml ascorbic acid, and 2.16mg/mlβ–glycerophosphate. Cells were cultured for 14days and the medium was changed twice a week. Skeletal muscle differentiation was induced at 1×10~5cells/ml by DMEM/20%FBS supplenmented with 3μmol/l 5-azayttidine for 24h, then cultured for 2~3weeks.Neural differentiation were selectively and progressively induced into neuron-like cells and astrocyte-like cells.by multi-cytokines, first DMEM/F12/2%B27 with EGF(20μg/L) and bFGF(40μg/L) for 10～20days, then DMEM/5%FBS with retinoic acid (0.5μmol/L) or PDGF-BB(10μg/L) for another 10～20days. The differentiated neuron-like cells and astrocyte-like cells were co-cultured at 1×10~5cells/ml at the ratio of 1:1 in DMEM medium containing 10%FBS. Immunohistochemistry stainings were selectively carried out for CD44,CD45,nestin,NF200 and GFAP. The cell proliferation and survival assays were made using cell counting Kit-8.Results The MSCs showed a fibroblast-like morphology, and could self-renew and survive at least 15 passages. In the presence of selected cytokines MSCs were effectively differentiated into osteoblasts for 4 weeks. The cells morphology changed after treatment with 5-azacytidine for 1 week. MSCs connected with adjoining cells after one week, formed myotube-like structures, began spontaneously beating after two weeks, and beat synchronously after three weeks. MSCs were differentiated into neurospheres -like cells with EGF and bFGF for 7 days. Then neurospheres-like cells were differentiated into neuron-like cells with retinoic acid and astrocyte-like cells with PDGF-BB for 10～14 days, respectively positive for nestin, NF200 and GFAP immunohistochemistry stainings. The differentiated neuron-like cells and astrocyte-like cells could survive the next co-culture procedure.The CCK-8 assays demonstrated that the differentiated neuron-like cells could proliferate and survive until the next 13～15 days. However the astrocyte-like cells proliferated and survived better and could be cultured for at least 3 passages.Conclusions MSCs were self-renewing and mutipotent cells which maintain the capacity of differentiation into osteoblasts,skeletal muscle-like cells,neuron-like cells and astrocyte-like cells in vitro by multi-cytokines induction protocols. And the MSCs-derived astrocyte-like cells had a better proliferation and survival capacity than the neuron-like cells. PartⅡTherapeutic benefits of intravenous administration of mesenchymal stem cells on peripheral nerve regeneration in ratsObjective To investigate the migration and distribution of rat bone mesenchymal stem cells in vivo after peripheral nerve injury and evaluate the beneficial effects on peripheral nerve regeneration and delaying denervation muscle atrophy.Methods Eighty female Sprague-Dawley rats were randomly divided into 2 groups, with 40 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. The transplanted group received an infusion of BrdU-labelled MSCs at 2×10~7cells/ml cells/ml through the tail vein. Every week until 12 weeks after surgery the sciatic function index (SFI) was assessed by walking tract test. Sections were carried out for HE staining, BrdU and TUNEL immunohistochemical staining.Results BrdU-labeled cells were detected housing the nerve stumps and denervation muscles at 1 week and 8 weeks. MSCs transplantation group had a promotion of sciatic nerve functional recovery from weeks 4 to 12 and the number of denervation muscle apoptotic cells significantly decreased compared with that of control group at 8 weeks(P<0.05, P<0.01).Conclusions MSCs is capable in vivo of homing to the nerve stumps and denervation muscles after peripheral nerve injury via the blood circulation, and had therapeutic benefits on promoting peripheral nerve regeneration and delaying denervation muscle atrophy.PartⅢThe effect of neural progenitor cells differentiated from bone marrow mesenchymal stem cells on denervation muscle atrophy Objective To investigate the effect on delaying denervation muscle atrophy after peripheral nerve injury by transplantion of neural progenitor cells differentiated from bone marrow mesenchymal stem cells.Methods Isolated and purified MSCs were induced into neural progenitor cells by EGF (20ng/ml) and bFGF (40ng/ml) for 10～20d. Differentiated cells were analyzed by single cell clone technique and immunohistochemistry staining for nestin. Ninety female Sprague-Dawley rats were randomly divided into 2 groups, with 45 in each. All the sciatic nerves were transected and sutured with 8-0 nylon suture. Before transplantation the neural progenitor cells were labeled with BrdU for 48h and then were injected into the sciatic nerve stumps at 5×10~6cells/ml. At weeks 4, 8 the nerve stump frozen sections were carried out for BrdU immunofluorescence staining, and muscle paraffin sections were carried out for HE staining and TUNEL immunohistochemical staining. Muscle cross-sectional areas and apoptotic cells were measured by an image analyzer.Results MSCs were positive for CD44, however negative for CD45 and nestin. After 10～14d differentiation, neurospheres were observed and mostly of them were positive for nestin. The rate of label of BrdU was 87.9% and the single-clone formed rate was 2.7%.At weeks 4 and 8 after surgery BrdU positive cells were detected at the nerve stumps. Muscle cross-sectional areas of cell transplantation group significantly increased and the number of the denervation muscle apoptotic cells significantly decreased compared with that of control group (P<0.05, P<0.01).Conclusions MSCs can be efficiently differentiated into neural progenitor cells with EGF and bFGF. Differentiated neural stem cell-like cells can survive the next transplantation into nerve stumps and had beneficial effects on delaying denervation muscle atrophy after injury. PartⅣThe effect of differentiated neuron-like cells and astrocyte-like cells from bone marrow mesenchymal stem cells on denervation muscle atrophyObjective To investigate the survive of MSCs-differentiated neuron-like cells and astrocyte-like cells after co-transplantation in vivo and evaluate the beneficial effects on delaying denervation atrophy.Methods Isolated and purified rMSCs were selectively and progressively induced into neuron-like cells and astrocyte-like cells by multi-cytokine, EGF and bFGF first, followed by retinoic acid or PDGF. Immunohistochemistry staining was carried out for CD44, CD45, nestin, NF200 and GFAP. Sixty female Sprague-Dawley rat sciatic nerve transection models were made and then randomly divided into 2 groups, with 25 in each. In the experimental group, differentiated neuron-like cells and astrocyte-like cells were co-injected into the denervated skeletal muscle at 5×10~6cells/ml. At weeks 4 and 8 after cell transplantation muscle frozen sections were carried out for NF200 and GFAP immunofluorescence staining and muscle paraffin sections were carried out for HE staining Muscle cross-sectional areas were measured by an image analyzer.Results MSCs were effectively differentiated into neurospheres -like cells , then neuron-like cells and astrocyte-like cells ,respectively positive for nestin , NF200 and GFAP. At 4 ,8 weeks after transplantation NF200 and GFAP -positive cells were detected in denervated skeletal muscle .Muscle cross-sectional areas of cell transplantation group were significantly increased,compared with that of the control group. There was a statistical significance (P < 0.05, P < 0.01). Conclusions Differentiated neuron-like cells and astrocyte-like cells can be acquired in vitro by effective induction protocols. After co-transplantation into the denervated skeletal muscle cells can survive in vivo and have therapeutic benefits on delaying denervation atrophy.