Influence of human acellular amniotic membrance on the differentiation of mesenchymal stem cells into neural lineage cells

Background: The method of adult stem cell differentiation into neural cells by extracellular matrix (ECM) and cytokine have not been reported. Objective: Based on theory of stem cell niche, to investigate the conditions for induction of amniotic-derived mesenchymal stem cells (ADMSCs) into neural lineage cells, and the neural markers' expression during differentiation. Design, Time and Setting: The cell morphology observation and protein molecular level detected were performed at the Experimental Center, Medical College, Zhengzhou University from December 2007 to April 2008. Materials: Amnion donate by puerperant was obtained from the Department of Obstetrics, First Affiliated Hospital, Zhengzhou University. Basic fibroblast growth factor and retinoic (Sigma, USA) were used in this study. Methods: ADMSCs were isolated from amnion in vitro. Membrane-like ECM was made by enzyme, chemical method, at a cell density of 4×10⁷/L. Cells in the induction group were treated with basal medium for pre-inducton. Cells at 4×10⁴/L were seeded on the ECM and cultured by basal neuro-medium for two days, and then replaced it with DMEM/F12 neuro-inducted medium, supplemented with 10^-3 mmol/L retinoic acid and 20 μ g/L β-nerve growth factor, and cultured for 24 hours, last cultured in the basal medium for 3 days. Cells in the control group were treated without ECM. Main Outcome Measures: Morphologic changes of differentiated cells, and phenotype of neural cells were measured. Results: At hour 24, cells in the induction group were seeded on the ECM, round and sticked on ECM tightly, with processes stretched out, strong refraction. At day 3, most cell processes extended and connected each other. At days 4-6, processes connected each other into a net and arranged with directionality. At day 3, cells in the control group stretched out bipolar or pluripolar processes. At days 4-6, cells returned to fibroblast-like. After pre-induction, Nestin, neuron specific enolase expresstion of both groups increased significantly. Synaptophysin, glial fibrillary acidic protein expresstion did not change. At day 6, compared with the control group, no significant difference was found in neuron specific enolase expresstion (P > 0.05), while synaptophysin expression increased significantly (P < 0.01), Nestin and glial fibrillary acidic protein expresstion decreased significantly (P < 0.01). Conclusion: On the membrane-like matrix, differentiated cells displayed the morphologic characteristics and markers of a more ripe neuron-like precursor cells. This program induced by extracellular matrix and cytokine could obtain neuronic precursor cells from ADMSCs in high efficiency, and inhibit the differentiation toward glial cells.