| Embryonic stem cells(ESCs)are derived from the inner cell mass(ICM)of the developing blastocyst and have the abilities of self-renewal and pluripotency to reconstitute embryonic lineages.It has been reported that culture conditions could greatly affect genome stability,transcriptome,epigenome,proliferation and differentiation capacity of ESCs.Traditionally,ESCs are cultured based on fetal bovine serum(FBS).FBS is considered important,but there are some inevitable problems in the serum-based culture system,such as unstable batches of FBS,undefined molecules,and more serious,many conflicting signaling.Therefore,m ESCs cultured in the serumbased condition are in a metastable status and exhibit functional heterogeneity.Based on the above problems,a chemically defined serum-free culture condition is established and widely used for human or mouse ESCs culture.At present,the most widely used for mouse ESCs(m ESCs)is the culture system established by Austin Smith’s laboratory in 2008 consisting of N2,B27 and two small molecule inhibitors GSK3 i and MEKi(defined as N/2i system in this paper).It is generally believed that m ESCs exhibit a ground state more similar to inner cell mass in blastocyst under this serum-free condition,and therefore have better developmental potential.However,in recent years,this chemically defined serum-free condition has been revealed to damage telomeres,and cause irreversible methylation erosion and genomic instability.As a result,the m ESCs cultured for a long time under this system lose their developmental potential,which is manifested in the failure to obtain normal surviving tetraploid mice developed from cells.Changing the MEKi in the N/2i system to a weak inhibitor SRCi or reducing its concentration can effectively improve the DNA hypomethylation and in vivo developmental ability of m ESCs,which proves that the MEKi added in the serum-free system is responsible for theses defects.In this study,it was found that replacing N2 and B27 with serum without changing the 2i small molecule can also effectively improve these defects in m ESCs.The above results suggest that MEKi may not be the main cause of abnormal culture in serum-free system,and there may be some substances in serum that help to improve serum-free culture defects.Therefore,by systematically comparing the differences between m ESCs in serum-based and serum-free systems,potential targets are sought to repair the defects of serum-free culture systems.This research will help to further optimize the in vitro culture system of stem cells and improve the quality to support clinical applications such as stem cell therapy.In this study,by comparing the serum-based system and the serum-free culture system,we observed that m ESCs presented the slow proliferation,disordered cell cycle,chromosomal aneuploidy and other defects in self-renewal capacity cultured in N/2i system.More seriously,the differentiation potential of stem cells,especially the in vivo differentiation,was severely affected under N/2i condition.This was manifested in low skin and germline chimerism rates in chimera experiments,as well as failure to obtain fully cell-developed mice through tetraploid complementation experiments.We further compared the transcriptomes of the N/2i system and the serum-based system supplemented with 2i(defined as S/2i system in this paper),and found that the m ESCs in the N/2i system showed global inactive transcription and low expression of important genes and signaling pathways related to pluripotency maintenance.When using growth factors to activate the down-regulated BMP,TGFβ,AKT signaling pathways,it was found that only the activation of the BMP signaling pathway can significantly maintain the self-renewal and pluripotency of m ESCs in N/2i system.Among the growth factors BMP2,BMP4 and BMP7 used,BMP4 had the best effect.In order to explore the underlying mechanism of BMP4 improving cell defects in serum-free conditions,this study conducted analysis of differentially expressed genes by focusing on aneuploidy and slow proliferation.The results showed that the expression of Ube2s(ubiquitin-conjugating enzyme E2S)and Chmp4b(charged multivesicular body protein 4B)in N/2i system was significantly lower,and could be effectively activated in the S/2i system and the N/2i system supplemented with BMP4.Subsequently,knockdown(or short-term induced knockdown)of Ube2 s or Chmp4 b can cause defective phenotype similar to m ESCs cultured in N/2i condition,and these defects cannot be improved by activation of BMP signaling pathway.Surprisingly,overexpression of Ube2 s or Chmp4 b can restore the self-renewal and differentiation potential of m ESCs cultured in N/2i condition.The above results demonstrated that Ube2 s and Chmp4 b,which regulated chromosome segregation and cell cycle,were target genes of BMP4 during maintaining self-renewal of m ESCs.So how does BMP4 activate these two genes? Ch IP-seq data showed that Zbtb7a(zinc finger and BTB domain containing 7a),a downstream target of the BMP-SMAD signaling pathway,had significant signal enrichment in the promoter regions of Ube2 s and Chmp4 b.When Zbtb7 a was overexpression,the expression of Ube2 s and Chmp4 b could be up regulated.Moreover,BMP4 can also activate the expression of Ube2 s and Chmp4 b by recruiting H3K4me3 modification and removing H3K9me3 and H3K27me3 modification.Besides,the amelioration of BMP4 in serum-free systems differed from that reported by manipulating the ERK pathway(lowering MEKi concentrations or employing SRCi),as it did not significantly affect the hypomethylation of m ESCs,while significantly activated the cellular transcriptome in serum-free systems,promoting the expression of pluripotency maintenance-related genes and BMP,TGFβ,AKT signaling pathways.Compared with SRCi,the developmental potential of m ESCs treated with BMP4 had a more significant improvement.In conclusion,this study reveals that m ESCs have weak activation of BMP signaling pathway in serum-free system.When the BMP signaling pathway is activated with the bone morphogenetic protein BMP4,the chromosomal integrity and proliferation ability of m ESCs can be protected by up-regulating the downstream targets Ube2 s and Chmp4 b,thereby maintaining the long-term developmental potential of m ESCs in vitro and in vivo. |
