Separated Effect Of Activin A And BMP4 On Pluripotency Of Mouse Embryonic Stem Cells

Since mouse embryonic stem cells(ESCs)were built in 1981,researcher continuously explored chemical defined medium to replace the original medium with feeder and fetal bovine serum.Nearly 30 years passed,the study showed that mouse ESCs(2i/LIF-ESCs)maintained a na?ve pluripotent state in chemical defined medium with adding the inhibitors of Mek1/2(PD)and GSK3b(CH),and the leukemia inhibitory factor(LIF).However,two papers published in Nature in 2017 indicate that mouse ESCs cultured with 2i/LIF for long-term can cause irreversible epigenetic changes and genomic instability,the main reason is PD impaired the development of mouse ESCs.In here,we firstly use Activin A and BMP4 to replace the one inhibitor PD of 2i/LIF medium to culture 2i/LIF-ESCs,and induced ACL-ESCs and BCL-ESCs.In second,we test the stem cell characteristics,gene expression pattern and in vivo development potential of ACL-ESCs and BCL-ESCs.The results show below:(1)ACL-ESCs and BCL-ESCs have the same morphological characteristics,and also showed strong positive with alkaline phosphatase dyeing as 2i/LIF-ESCs.However,the cell proliferation rates of ACL-ESCs and BCL-ESCs were different from that of 2i/LIF-ESCs.(2)The expression of pluripotency-related genes such as Oct4,Sox2 and Klf4 in ACL-ESCs and BCL-ESCs was not significantly different with 2i/LIF-ESCs.The DNA methylation-related gene Dnmt3 b and Dnmt3 l,and mesoderm-related genes Hand1,Evx1,Eomes and T were significantly higher expression in BCL-ESCs than 2i/LIF-ESCs.Most notably,the expression level of the oncogene-associated c-Myc in the induced both mouse ESCs were extremely significantly higher to compared with the control cells.(3)The RNA sequencing and analysis results showed the genes highly expressed in 2i/LIF-ESCs are related to skeletal system development,chordate embryonic development and sensory organ development;the genes associated with organic hydroxy compound metabolic process,inorganic molecular entity transmembrane transporter activity andinorganic ion homeostasis were highly expression in ACL-ESCs group;the genes highly expressed in BCL-ESCs are mainly genes of tissue morphogenesis,productive structure development and embryonic morphogenesis.These results suggest that ACL-ESCs and BCL-ESCs have unique gene expression patterns.(4)The results of chimeric experiment showed that while a single ACL-ESCs and BCL-ESCs was injected into 8-cell embryos and then transferred,it was found that ACL-ESCs and BCL-ESCs contribute not only to the fetal tissue,but also to the placenta and yolk sac,however,2i/LIf-ESCs co?Ld only contribute to the part of feta in E10.5 chimares.The res?Lt showed with the 2i/LIf-ESCs cultured in the ACL and BCL culture system,enable expended pluripotency of mouse ESCs.These results,take together,show that Activin A and BMP4 replace of PD,enable maintained morphology and specific features of mouse ESCs in ACL and BCL medium,and also have a function to expand the pluripotency in ACL-ESCs and BCL-ESCs.We propose that ACL-ESCs and BCL-ESCs may serve as cell culture platform for researching the biology of early embryogenesis.