Expression of motor neuron markers in endometrial stem cell derived neurons differentiated with purmorphamin small molecule

Introduction: Human endometrial stem cells (hEnSCs) are being applied in treatment of neurological disorders.EnSCs derived motor neurons are one of the best target for transplantation which is not stimulate immunesystem. NF, Chat and B tubulin are the special proteins of motor neurons. Expression of these gene during celldifferentiation shows that the cells have become motor neurons. Purmurphamin (PMA) is the agonist of a 7-passprotein in shh signaling pathway that causes neural differentiation progressing. The purpose of this study is toinvestigate the expression of neural specific gene on motor neurons that differentiate by PMA.Methods: In this study endometrial stem cells were enzymatically extracted from endometrial tissue. After thirdpassage, the flow cytometry was done for CD90, CD105 and CD146. In the first step of differentiation, the cellswere treated with differentiating medium containing B27, FGF2, IBMX and 2ME for one day. In the second step,the differentiating medium con to N2/B27 and PMA added to the cells for three days. In the next stagedifferentiating medium had Retinoic acid (RA) and PMA. This process prolonged 8 days. In the last stage cellswere treated with medium containing N2/B27 and BDNF and cell morphology were investigated for 14 days.Then immunocytochemistry analysis was done for B tubulin, NF (neurofilament) and Chat markers. Then RealtimePCR was performed in RNA stage for expression of neural markers such as NF, Chat, Nestin and GFAP.Results: The flow cytometry analysis showed that hEnSCs were positive for CD90, CD105 and CD146. Theimmunocytochemistry results showed the cells were expressed B tubulin, NF and Chat markers. Real-time PCRresults demonstrated that the cell treated with PMA expressing neural markers NF and Chat.Conclusion: Expression of neural specific gene in the endometrial derived motor neurons suggests that we canutilize various small molecules to stimulate gene expression. Our motor neurons differentiation protocol providesaccessible system to gaining motor neurons for treatment of neurological disorders.

Keywords:

Motor neuron markers , hEnSCs , PMA