Inducing Differentiation Of Human Amnion Cells Into Neural Cells In Vitro

Objectives:Somatic stem cells (SSCs) transplantation has received more attention as a new strategy of theray for nervous system diseases. Human amnion-derived stem cells (hAD-SCs) may be a potential cell source. In this study, we induced hAD-SC including human amnion-derived mesenchymal stem cells (hAD-MSCs) and human amniotic epithelial cells (hAECs) to differentiate into Neural Cells (NCs) in vitro to explore a potential new therapy for nervous system diseases.Methods:①Isolation and identification:hAECs and hAD-MSCs were isolated from amniotic tissue with trypsin and collagenase, and the phenotype of the isolated cells was identified by flow cytometry and immunocytochemical staining.②Induced culture conditions:hAECs and hAD-MSCs were divided into induction and non-induction. The induction was treated in serum-free HG-DMEM with200mM L-Glutamine and1μmol/L All-trans-retinoic acid(ATRA) to investigate their differentiation into NCs. The medium used in non-induction with200mM L-Glutamine and10%fetal bovine serum HG-DMEM.③Positive rate of NSE and neuronal markers detection:The hAECs and hAD-MSCs were cultured in a humidified atmosphere of95%air and5%CO2at37℃respectively for48hours and96hours, the expressions of neuron-specific enolase (NSE), neuro filament (NF), glial fibrillary acidic protein (GFAP), microtubule-associated protein-2(MAP-2), Neuronal Nuclei (NeuN) and β-tubulin-III were determined by immunocytochemical staining and Immunofluorescence staining, The content of DA in the induced supernatant of culture hAECs and hAD-MSCs was detected by enzyme-linked immunosorbent assay (ELISA), and the expressions of NSE, NF, GFAP, β-tubulin-Ⅲ, MAP-2and NeuN mRNA were detected by real time polymerase chain reaction (RT-PCR)Results:①Freshly isolated hAECs showed low expression for CD44, did not express CD71, CD34, CD45and were positive for CK19, but hAD-MSCs showed higher expression for CD44, CD29, CD90, CD73, CD105, did not express CD34, CD45, CD19, CD14and HLA-DR and were positive for vimentin.②The percentages of NSE in hAECs and hAD-MSCs induction were respectively61.16±19.8%and47.03±19.2%by flow cytometry, but in non-induction were negative.③The results of IC and IF showed that hAECs and hAD-MSCs induction were expressed NSE, NF, GFAP,(3-tubulin-Ⅲ, MAP-2and NeuN respectively, But not express in all non-induction.④The results of ELISA showed that contents of DA in supernatant from induced hAECs and hAD-MSCs were79.71±11.94ng/L and74.03±9.46ng/L respectively. while low expressed in all non-induction (P<0.05).⑤The results of RT-PCR showed that NSE, NF, GFAP, β-tubulin-Ⅲ, MAP-2and NeuN mRNA was highly expressed the hAECs and hAD-MSCs induction, low expressed in all non-induction (P<0.05)Conclusions:Both hAECs and hAD-MSCs could differentiate into Neural Cells, suggesting that hAD-SC might become a new cell source of therapy for nervous system diseases.