Abstract: Objective To establish and optimize the induction system for differentiation of human adipose-derived mesenchymal stem cells (hADSCs) into definitive endodermal (DE) cells. Methods Based on a well-developed protocol inducing ESCs/iPS towards DEs，the effects of activin A, WNT activators, BMP4 and bFGF, which are essential for inducing of ESCs/iPS to DE, were evaluated during the differentiation of hADSCs towards DEs. RT-qPCR was used to detect mRNA levels of DE marker genes FOXA2 and SOX17. Western blot was used to measure protein levels of FOXA2 and SOX17. Immunofluorescence staining was used to identify the expression of FOXA2 and SOX17, which demonstrated the feature of DE cells. Results Low concentration of activin A was more suitable to induce the expression of DE markers than high concentration (p<0.001). The expression of FOXA2 and SOX17 were higher in differentiation medium supplemented with Chir99021 than Wnt3a (p<0.01). BMP4 and bFGF showed no or harmful effects in the differentiation of hADSC to DE (p<0.001). Conclusions Different pathways are needed during the DE differentiation from hADSC compared with ESCs/iPS.

Key words: definitive endoderm, mesenchymal stem cells, differentiation protocol.