Monosialotetrahexosyl Ganglioside GM1 Induces Human Bone Marrow Stromal Cells To Differerntiate Into Neuron-like Cells In Vitro

Objective : Recently,lots of finding shows that bone marrow stromal cells, BMSCs can be induced into nerve cells, and the BMSCs can be used to treat the nervous system disease.But fixing differentiation rate is low ,and survival rate of induced neuron-like cell is too short to meet requirement of transplantation. As we know that searching for an effective,stable inducing methods and elevating survival rate of induced neuron-like cell is a key problem. Along the study going on, it shows that monosialotetrahexosyl ganglioside GM1 plays an important role in the process of nerve cells , development,differentiation,repair and signal transduction. Study shows that BMSCs can be induced into nerve cells by monosialotetrahexosyl ganglioside GM1. We investigate that monosialotetrahexosyl ganglioside GM1 induces human bone marrow stromal cells to differerntiate into neuron-like cells in vitro.Methods : BMSCs harvested from adult normal human marrow, then were passaged and purified in culture medium. We observed the growth status of BMSCs,and identity the immunological characteristic phenotype of CD44,CD45,CD90 and CD105 by using FACS .BMSCs were passaged five times before experiment,then purified BMSCs were induced by 10μg/ml,60μg/ml,90μg/ml monosialotetrahexosyl ganglioside GM1 . 6 hours later, BMSCs were detected with immunocytochemisty staining for micmtubule associated protein-2 (MAP-2) , neuron specific enolase(NSE). The survival of neuron-like cells was detected by means of MTT at 0.5,6,24 hours and 3days after inducement.Results : 2 days After cultivation and washed by PBS there are several colonies of adherent cells . 8 days there are uniform spindle cells . BMSCs which passaged five times are uniform . Detecting the cell surface marker with flow cytometry shows CD44(+),CD90 (+),CD105(+),CD45(-),which reveal the cells we cultured were BMSCs. After 30 - 40 minutes of induction,morphous of cells are changed.After 4 hours of induction , morphous of cells are changed markedly. some BMSCs exhibited immunoreactivity.After 6 hours of induction more cells have monopole or mutiploar neurite ,some neuritis connected as networks. After 5 days of inductions some cells die and the most neurites connected as networks. After 7 days of inductions the situation is likely the 5 days but more cells were die. Control group cell were uniform spindle cell, but growth velocity slowed down . MAP-2 and NSE positive cells existed in all groups,while MAP-2 and NSE positive cells increased markedly in BMSCs treated with 60μg/ml,90μg/ml GM1 after induction of 6h. MAP-2 and NSE positive cells in group 10μg/ml,60μg/ml,90μg/ml GM1 are more than the negative control group,there were significant differences (P<0.05).MAP-2 and NSE positive cells in group60μg/ml,90μg/ml GM1 are more than group 10μg/ml GM(1P>0.05). MAP-2 and NSE positive cells in group 60μg/ml GM1and group 90μg/ml GM1 are no significant differences(P>0.05). The MTT shows neuron-like cells survived better after induction by GM1 than the negative control group.Conclusion : After being induced by 10μg/ml,60μg/ml,90μg/ml GM1, BMSCs could effectively differentiate into neural-like cells with the transformation of morphology and the expression of NSE and MAP-2 in vitro, the same characters which the true neuron cells have. Percentage is higher in 60μg/ml,90μg/ml GM1,moreover the growth status of induced neuron-like cells is better. Differentiation ratio is highest when BMSCs were induced 6h .