Establishment Of Novel Mouse Pluripotent Stem Cells

Since derivation of mouse embryonic stem cells(ESCs),a lot of research focus on the transformation between na(?)ve and primed state of ESCs.Recently,Austin Smith suggested there is a formative state resides between na(?)ve and primed state,which named FS cells.In here,depended on our previously published,we proposed AL medium that consisted of Activin A that be required in primed state and leukemia inhibitor factor(LIF)that support self-renewal na(?)ve ESCs.We demonstrated that AL culture system could support to establish and maintain the novel ESCs(ALSCs).The detailed results are shown below:1.Firstly,ALSCs was derived from post-implantation gastrulas(E6.5)under AL culture condition,and possessed similar with mouse epiblast stem cells(Epi SCs)in clone formation process and morphology.The establishment efficiency of ALSCs(26.9%)is lower than Epi SCs(58%).The ALSCs has normal karyotype,and exhibited positive of alkaline phosphatase staining.The results of cell growth curve and single cell establishment were also simliar to Epi SCs.RT-q PCR results indicated that the expression of pluripotent genes,PGC marked genes,DNA methylation related genes and Epi SCs marked gene in ALSCs exhibited intermediate level between ESCs and Epi SCs.The expression of pluripotent genes in ALSCs was also confirmed by immunofluorescence assay and ALSCs possessed one inactive X chromosome.In addition,we also established b ALSCs derived from blastocysts under AL culture condition.b ALSCs showed the similar alkaline phosphatase staining with ALSCs and exhibited GOF/GFP negative stem cell lines derived from GOF/GFP positive blastocysts.The establishment efficiency of b ALSCs is 35.7%.RT-q PCR results of b ALSCs were similar with that of ALSCs,but DNA methylation related genes in b ALSCs showed higher than both Epi SCs and ESCs.2.Second,we test the pluripotency of ALSCs in vivo and in vitro.The results of teratoma test showed the ALSCs have an ability to differentiated into three germ layers in vivo.However,td Tomato~+ALSCs same as Epi SCs were not detected in chimera E6.5gastrulas in vivo.Next,we injected td Tomato~+ALSCs into 8-cell embryos and showed significantly reduced after cultured for 48h in vitro,and suggested ALSCs did not contributed to chimera.In addition,the results of differentiation test in vitro showed ALSCs exhibited similar developmental potential with Epi SCs in vitro.Test of single cytokine addition in culture medium suggested that Activin A was more important than LIF for maintaining self-renew and pluripotency of ALSCs.In the condition of Activin A alone,ALSCs could sustained itself morphology and self-renew.3.Then,we performed RNAseq to analyze the transcriptome of ALSCs.Compared with Epi SCs,gene ontology analysis showed the differential genes were enriched on cell differentiation and tissue development of biological process.KEGG enrichment analysis showed differential genes were most related to Wnt signaling pathway and signaling pathways regulating pluripotency of stem cells.When compared to FS cells established by Austin Smith lab,gene ontology enriched differential genes on cell differentiation and development process of biological process.Then,we obtained the intersection of differential genes of ALSCs vs Epi SCs and ALSCs vs FS cells.Gene ontology analysis showed that the overlaps genes enriched on organismal development of biology process,and organelle of cellular component.KEGG analysis enriched differential genes on Hippo pathway,Wnt pathway and signaling pathways regulating pluripotency of stem cells.4.Final,we successfully reversed ALSCs to r ESCs in na(?)ve state.Even used low cell concentration or single ALSC could be converted to r ESCs.During the induction process,we observed X chromosome reactivation occurred prior to GOF/GFP activation.Epi SCs could also converted to r ESCs through two-step induction system,including cultured in AL condition for over 10 passages(epi ALSCs),and then transferred to CL condition(AF-AL;AL-CL).The conversion from ALSCs to r ESCs showed not affected by addition of inhibitor of BMP4 or Activin A in induction system.However,Epi SCs could not convert to r ESCs when added inhibitor of BMP4 in two-step induction system.Together,we demonstrated that Activin A and LIF supported to established a novel mouse embryonic stem cell line,which could maintain self-renewal and pluripotency,as well as converted into na(?)ve r ESCs.Also,Epi SCs could converted to r ESCs through two-step induction system.Our research provides more material and basis knowledge to explore mouse embryonic stem cells in different states,and the effect of cytokines to mouse embryonic stem cells.