Plasticity Of Neurogenic Differentiation Of Bone Marrow Mesenchymal Stem Cells

Loss of neurons and preduction of the inhibitory environment at the lesion site represent the main events after injury to the central nervous system(CNS).Bone marrow mesenchymal stem cells(BMSCs),which can be obtained easily and more importantly display a low immunosuppression property,are multipotential.BMSCs can differentiate into several types of mesenchymal cells,including osteocytes,chondrocytes,and adipocytes,but can also differentiate into non-mesenchymal cells,such as neural cells, under appropriate experimental conditions.Therefore,they are expected to be used in cell replacement therapy for the treatment of nervous system diseases.While a number of experiments proved that BMSCs could differentiate into neuron-like cells and these cells could repair the damaged nervous system upon transplantation,it was still unknown whether these neuron-like cells could develop to mature neurons and accordingly display the proper function of these cells.Furthermore, the underlying molecular mechanism of the neurogenic differentiation of BMSCs remains elusive.A mixed culture was established from newborn rat cerebral cortex.After confluence, the culture was changed to serum-free medium and the retrieved medium after 48 hours was used to induce BMSCs to differentiate into neuron-like cells.After the induction for 24 hours,the neuron-like cells derived from BMSCs owned the typical morphological characteristics of mature neurons.The immunofluorescence staining showed that cells were positive for Nestin(15.07%±1.86%),β-Ⅲ-Tubulin(46.29%±2.58%),GFAP(9.54%±1.42%),NCAM(49.82%±3.46%),CD106(96.86%±1.82%),CD90(33.62%±2.48%), CD44(32.44%±2.45%),CD29(14.24%±1.84%)and negative for NSE,CD45,CD71, CD34.High percentage of the neuron-like cells were positive forβ-Ⅲ-Tubulin,a marker widely used for early neurons,while all of them were negative for NSE,a marker for mature neurons,suggesting that these neuron-like cells might be in the transition state during the differentiation from BMSCs to mature neurons.Moreover,the expression of CD106,CD90,CD44,CD29,but not CD45,CD71,CD34,was detected in the BMSCs- derived neuron-like cells,indicating that these neuron-like cells still owned the phenotype of BMSCs.It also confirmed that neuron-like cells might be in the transition state of the differentiation from BMSCs to mature neurons.In this study,we also used the traditional method to generate neurogenic differentiation of BMSCs.Twenty-four hours prior to neuronal induction,medium was replaced with preinduction medium consisting of DMEM(Low glucose),10 ng/ml basic fibroblast growth factor(bFGF)and 10%NCS.To initiate neuronal differentiation,the preinduction medium was removed,and the cells were washed twice with PBS solution and transferred into neuronal induction medium composed of DMEM(Low glucose),2% imethyl dsulphoxide(DMSO),200μmol/L butyl hydroxy anisol(BHA).Cells were fixed for immunocytochemistry at 5h postinduction when the neuron-like cells owned the typical morphological characteristics of mature neurons.The immunofluorescence staining showed that cells were positive for Nestin(21.12%±2.28%),β-Ⅲ-Tubulin(78.26%±3.52%),NCAM(79.86%±2.88%),NSE(70.82%±2.46%),CD106(96.88%±2.75%),CD29 (64.94%±2.16%)and negative for GFAP,CD90,CD44,CD45,CD71,CD34.The neuron-like cells derived from BMSCs not only owned the phenotype of the neural cells, but also owned the phenotype of BMSCs.These results further confimed that BMSCs-derived neuron-like cells might be in the transition state of the neurogenic differentiation of BMSCs.The neuron-like cells from differentiated BMSCs by two kinds of induction methods were then maintained in serum-containing medium.After 24 hours,we found that these cells morphologically changed to BMSCs.Immunofluorescence staining of these cells back from neuron-like cells showed that cells were positive for CD106,CD90,CD44, CD29 and negative for CD45,CD71,CD34,which was very similar with the phenotype of BMSCs.Moreover,they still could differentiate into neuron-like cells,osteocytes and adipocytes.It is well known that mature neurons are terminally differentiated cells and are theoretically difficult to return to the state of stem cells.Results from this study showed that neuron-like cells derived from BMSCs are different from the real neurons.Cells were just going through the transition state of the neurogenic differentiation of BMSCs. BMSCs-derived neuron-like cells morphologically changed to BMSCs after maintained in serum-containing medium for 24 hours and these cells owned the same phenotype and multipotential with BMSCs,confirming the plasticity of neurogenic differentiation of BMSCs.These results would provide certain reference for the treatment of nervous system disease by using the transplant of BMSCs and offer some important experimental information for basic research about the neurogenic differentiation of BMSCs.