Study On Induced Differentiation Of Human Umbilical Cord Blood Mesenchymal Stem Cells For The Treatment Of Spinal Cord Injury

Objective:To study the isolation method and culture conditions of mesenchymal stem cells (MSCs)derived from human umbilical cord blood(HUCB),and to explore the optimal condition of inducing the MSCs to differentiated into neural cells in vitro.To observe the function recovery of spinal cord in rat spinal cord injury(SCI)models after transplantation of neural cells derived from the MSCs,in order to provide a kind of valuable stem cell resource for healing central nerve injury.Methods:1.Isolation,culture and amplification of HUCB-derived MSCs in vitro: Mononuclear cells(MNCs)of HUCB were isolated by density gradient centrifugation with Ficoll separating medium,then the cells were cultured in DMEM containing 10%FBS.After 3 passages,the adherent cells were displayed with antibodies and analyzed with flow cytometry.2. Inducation and differentiation of MSCs:Then MSCs were induced with DMEM/F12 containing 20%FBS,BDNF 10ng/ml+RA 0.5μM+bFGF 20ng/ml.After seven days' culture, the expression of astrocytic specific protein GFAP and neuron marker MAP2 of the induced cells were detected by immunofluorescence staining.3.In vivo experiment:Thirty adult rats were randomly divided into three groups.Complete transection of spinal cord at the level of T9-T11 was produced before transplantation.One group was transplanted with the cultured cells labeled by bromodeoxyuridine(Brdu),PBS and nontreatment groups were used as control.Hindlimb motor function was assessed by using the Basso-Beattie-Bresnahan(BBB)locomotor rating scale once a week.The survival and differentiation of transplanted cells in rat spinal cord in vivo were evaluated by histological and immunohistochemistry.Results:It was found that the adherent cells were CD11b~-CD34~-CD45~-CD44~-at the third passage detected by flow cytometry.After induction,most of these cells exhibited typical neuron-like phenotype.The differentiation ratio of glial cells were lower than that of neurons. From 2 weeks to 5 weeks after the transplantation,the locomotor function recovery of rats was significantly improved in the experimental group than that in the control group,(P＜0.05).The transplanted cells were found survived and extended in the surrounding spinal cord tissue for a long time in the transplantation group.Conclusions:UCB-derived MSCs could be induced to neural cells in vitro.Our findings showed that transplantation of induced MSCs into the injured rat spinal cord could survive, migrate to the lesion site.And also could improve neurological function recovery in rats.Neural cells derived from human umbilical cord blood MSCs would be a kind of valuable cell resource for the therapy of central nerve injury.