Motor neurons differentiation of encapsulated human endometrial stem cells in collagen without HLA-DR expression
Background: Human endometrial stem cells (hEnSCs) are attractive cells in regenerative medicine and neurons derived from EnSCs are one of the best targets for cell therapy due to the fact that they do not stimulate immune system. Small molecules as useful chemical tools can effectively change cell fate. Thus, these small molecules provide a promising strategy for differentiation of cells in regenerative medicine. Purmorphamine (PMA) is a small molecule that possesses certain differentiation effects by activating SHH signaling pathway. In this study, we used PMA as a small molecule to differentiate motor neurons from hEnSCs by using collagen polymer.
Methods and Materials: hEnSCs were cultured in differentiation medium containing 1µM PMA in 2D and 3D environments. Scanning electron microscope (SEM) was used for cell attachment and cell viability was assessed by and 3-(4,5-dimethylthiazoyl-2-yl)2,5-diphenyltetrazolium bromide (MTT). Immunocytochemistry was performed to confirm the expression of islet-1 and Acetylcholine transferase (Chat). Real-time PCR also confirmed for expression of neural markers such as NF, Chat, islet-1, HB9.
Results: Therefore, our data revealed that induced hEnSCs with PMA could significantly express motor neuron markers in RNA and protein levels. Further, flow cytometry was done for the expression of the cell surface antigen HLA-DR in endometrial stem cells and motor neurons derived from these cells. The results confirmed that hEnSCs and motor neurons derived from them do not express HLA-DR. Our motor neurons differentiation protocol provides an accessible system to provide motor neurons that do not stimulate immune system.
Conclusion: In conclusion, encapsulation of hEnSCs in collagen along with induction media containing PMA have potential for being used in neural tissue engineering through activation of the SHH signaling pathway.
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