Activin A's promotion of definitive endoderm differentiation from human embryonic stem cells

SUN Yi1,2,ZHOU Jing1,LIN Ge1,2,LU Guang-xiu1,2(1 Institute of Reproductive and Stem Cell Engineering,Central South University,Changsha,Hu'nan 410078,China; 2 National Research and Engineering Center of Human Stem Cells,Changsha,Hu'nan 410078,China)

stem cell engineering;human embryonic stem cell;definitive endoderm;Activin A

【Objective】 The study was conducted to confirm the affection and the signaling pathway molecules of Activin in promotion of the definitive endoderm(DE) differentiation from human embryonic stem cells(hESCs),which will provide reference for induction optimization of DE differentiation from hESC.【Method】 Cells were cultured on human embryonic fibroblast cells(HEF).Real-time quantitative RT-PCR were preformed to detect the expression of prime streak related genes(Gsc,Mixl1),mesoendoderm precursor related gene Brachyury,endodermal genes(Foxa2,Sox17),mesodermal marker Flk1,ectoderm gene Pax6,pluripotent genes(Oct4,Nanog).Brachyury and Sox17 protein expression was further confirmed by immunofluorescence staining on time 0,6,12,24,48,72,96 and 120 h respectively,with the treatment of 100 ng/mL Activin A.【Result】 After Activin A's induction,the endoderm and mesoderm genes' expressions were upregulated more quickly and dominantly.Immunofluorescence analysis showed that with Activin A's induction,Brachyury and Sox17 proteins expression significantly increased after 12 and 48 h,and reached the top after 48 and 96 h,respectively,in which the DE ratio was up to(81.7±5.4)%.Futhermore,endoderm differentiation process in vitro followed from the prime streak,mesoendoderm precursor,then to the endoderm development process,which was similar to the vivo developmental law.【Conclusion】 The data indicate that Activin A induction in DE differentiation of hESCs can regulate developmental events through transcriptional modulators and promote Brachyury and Sox17 proteins expression.