Effects Of Activin A And BMP4 On The Developmental Potential Of Mouse Embryonic Stem Cells

Since the beginning of 1980 s,the researchers discovered mouse embryonic stem cells,also called mESCs from the inner cell mass of blastocysts after fertilization 3 days(E3.5)in the co-culture system of feeder and serum,there has been kinds of mESCs obtained constantly with self-renewal capacity,distinct epigenetic characteristics as well as transcriptome map in the past forty years.Here,we first use chimerical defined medium ABC/L(Activin A,BMP4,CHIR99021,and mLif)which established in our lab,to culture 2i/L-ESCs(2i: PD0325901 and CHIR99021,Lif: mLif),in fact,Activin A and BMP4 replaced one inhibitor PD0325901 of 2i/Lif culture medium,induced ESCs named as ABC/L-ESCs.Second,pluripotency,transcriptome characteristics and development potential of ABC/L-ESCs are investigated for the further analysis and identification.The results are shown below:1.The results of cloning morphology and alkaline phosphatase staining showed there was no significant difference between ABC/L-ESCs and 2i/L-ESCs,and both of which present three-dimensional dome-shaped cloning.Also,alkaline phosphatase staining results of them were positive.2.The karyotype analysis showed that the proportion of ABC/L-ESCs with normal chromosome number was significantly higher than that of 2i/L-ESCs.3.Immunofluorescence staining results showed that both of ABC/L-ESCs and 2i/L-ESCs expressed pluripotent stem cell specific proteins as Oct4,Sox2 and Nanog without any significant differences.4.RNA sequencing analysis results showed that there were 989 differentially expressed genes compared with 2i/L-ESCs,and 362 among which were up-regulated.These up-regulated genes in ABC/L-ESCs were similar to S/L-ESCs which were cultured in serum and feeder,and mainly related to MAPK cascade,developmental growth,SMAD protein signal transduction,DNA binding site regulation and DNA methylation related to gamete generation and so on.Meanwhile,the developmental stage of ABC/L-ESCs in vivo was closer to the embryonic stage from E4.5 to E5.5.5.Methylation results showed that the methylation level of ABC/L-ESCs was significantly higher than that of 2i/L-ESCs and close to that of S/L-ESCs.At the same time,mRNA expression levels of methylated transferase Dnmt3 a,Dnmt3b and related cofactor Dnmt3 l were detected to investigate the hypermethylation level mechanism of ABC/L-ESCs.The results showed that the expression levels of these three genes in ABC/L-ESCs were significantly higher than that in 2i/L-ESCs.On the contrary,the expression levels of Prdm14 and Nanog,both genes that relate to DNA methylation in ABC/L-ESCs were significantly lower than 2i/L-ESCs.6.The results of chimeric experiment showed that while a single ABC/L-ESCs or 2i/L-ESCs was injected into 8-cell embryos,the chimeric ability was significantly higher than that of S/L-ESCs.When collecting embryos of E10.5,it was found that 2i/L-ESCs could only contribute to the part of fetal but not the extra-embryonic tissue.As for S/L-ESCs,the chimera could not be collected when a single cell was injected,and the chimeric ability was similar to that of 2i/L-ESCs while about 15-20 cells were injected.In comparison,the chimeric ability of ABC/L-ESCs was significantly higher than that of the two control groups,a single cell contributes not only to the fetal tissue,but also to the placenta and yolk sac.With the prolonged culture in the ABC/L culture system,contribution to the placenta and yolk sac was gradually enhanced.Genital ridge was collected at E12.5 days,and ABC/L-ESCs with GOF/GFP+ green fluorescence could be clearly found.7.Tetraploid compensation experiment results showed that ABC/L-ESCs could give rise to full-term mice by tetraploid compensation technology.In conclusion,a novel embryonic stem cell line of ABC/L-ESCs has been derived from 2i/L-ESCs in this study,which can be passaged stably for a long term in a chemical defined culture system.ABC/L-ESCs not only possesses similar morphology and ability of self-renewal to 2i/L-ESCs,but also own unique transcriptome characteristic and higher DNA methylation level to maintain stable genomic characteristics.In terms of developmental potential,ABC/L-ESCs has a higher developmental potential that contribute to the fetus,PGCs,as well as extra-embryonic tissues.The molecular regulation mechanism of ABC/L culture system on ESCs will be further explored in the following studies.