人脂肪间充质干细胞向限定性内胚层细胞分化体系的优化

Optimization of differentiation protocols of directing human adipose derived mesenchymal stem cells into definitive endoderm cells

目的探讨activin A、WNT通路激活剂、BMP4以及bFGF信号对人脂肪间充质干细胞(hADSCs)向限定性内胚层(DE)分化的影响。方法基于胚胎干细胞(ESCs)或多潜能干细胞(iPS)向DE分化的诱导体系,建立并优化hADSCs向DE细胞分化的诱导体系。在hADSCs向DE诱导体系中,分别比较不同浓度的activin A、Chir99021替代Wnt3a、添加/去除bFGF以及BMP4等对分化效率的影响。用RT-qPCR检测诱导前后限定性内胚层标志基因FOXA2和SOX17的表达;Western blot检测FOXA2和SOX17蛋白水平;用免疫荧光检测FOXA2和SOX17的表达,鉴定DE分化。结果与ESCs或iPS向DE分化的诱导体系相比,低浓度的activin A,GSK3抑制剂Chir99021替代Wnt3a,去除BMP信号和bFGF信号的诱导方案可以明显促进hADSCs向DE分化的影响(P<0.01)。结论hADSCs向DE分化与hESC/hiPS相比,需要激活不同信号通路,因此最适诱导体系不同。

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 level 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 Wnt3 a(P<0.01).BMP4 and bFGF showed no effects or even negative 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.