The Role Of Different Biological Factors In Establishment Of Mouse And Dairy Goat Pluripotent Stem Cells

Embryonic stem cells(ESCs)depended on the origin,emerge na(?)ve and primed state,which have different features in epigenetics,transcriptional regulation,and developmental potential.Homogeneity and stability of ESCs is important on its application,however it is hard to achieve.To explore the signaling pathway of regulation and maintenance of pluripotent ESCs and research on the chemical defined medium become a new field for application of ESCs.Our previous study showed combined four factors in ESCs culture(Activin A,BMP4,CHIR99021,LIF: ABC/L)could converse 2i/L-ESCs to advanced ESCs(es ASCs),which have expended pluripotency compared to 2i/L-ESCs.We also found Epi SCs could reverse to epi ASCs in ABC/L-medium.However,ABC/L-medium support to derive ASCs from blastocysts still unclear.In here,we firstly established ASCs from mouse blastocysts;second,we explored combination of different biological factors on culture of dairy goat induced pluripotent stem cells(iPSCs).The results are show in below.1.Established novel ESCs from blastocysts in ABC/L-mediumIn here,we collected blastocysts(E3.5)from Oct4-ΔPE-GFP(GOF/GFP,mixed background of MF1,129/sv,and C57BL/6J strains)×129/sv F1 mice to derived novel ESCs(blASCs)in ABCL-medium.The blASCs in undifferentiated pluripotent state with stable growth over 40 passages,and high alkaline phosphatase(AP)activity,have normal karyotype and express pluripotent markers Oct4,Sox2,and Nanog similar with ESCs in serum medium(S/L-ESCs).Next,we found blASCs in a stable state between 2i/L-ESCs and Epi SCs,and were equal to E5.5 blastocysts based on their molecular features as revealed by the RNA-seq analysis.Finally,we tested the in vivo developmental potential of blASCs in chimeric embryos.We injected single blASCs into 8-cell stage embryos,and noticed that blASCs successfully integrated into E10.5 chimeras.Notably,blASCs robustly to the embryo proper,yolk sac,and placental labyrinth.Chimeric efficient rate was similar to es ASCs.2.Established induced pluripotent stem cell(iPSCs)lines from dairy goat fibroblastsWe expressed Dox-inducible eight exogenous reprogramming factors,b OMSK(bovine Oct4,c-Myc,Sox2 and Klf4),p Nh L(porcine Nanog and human Lin28)and h RL(human Rarg and Lrh1),in dairy goat fibroblasts,delivered via piggy Bac transposition.The picked colonies were passaged in single cell suspension in a serum containing medium(M15)in the presence of Dox,and they were thus named dairy goat iPSCs.The passaged cells expressed high levels of the endogenous pluripotency genes,such as Nanog,Oct4(Pou5f1)and Sox2,and could be maintained undifferentiated in Dox for at least 30 passages with normal karyotype and high or heterogenetic alkaline phosphatase(AP)activity.These dairy goat iPSCs thus provided a platform for identifying culture conditions.We choose five groups contained different cytokines to culture dairy goat iPSCs,the results showed except Activin A+CHIR99021 group,other groups could maintaine clones morphology and self-renew,and two of them,Activin A+b FGF and LIF+b FGF groups show AP staining positive.3.Established iPSCs which be independent of the Dox-induced exogenous factor expressionUpon Dox removal,dairy goat iPSCs were differentiated in 2-3 days.The pluripotency in these iPSCs thus depended on Dox-induced exogenous factor expression in the serum containing medium.We tested culture conditions for mouse ESCs,human ESCs,and bovine ESCs including ABCL,2i/LIF,CTFR,FAC et al,total round 50 kinds of medium to culture dairy goat iPSCs without Dox.We only got one iPSCs line on b FGF+IWR1 group,dairy goat iPSCs formed compact colonies,and expressed high levels of endogenous pluripotency genes,could be maintained for at least 20 passages without noticeable differentiation.In RT-q PCR analysis,the b FGF +IWR1 group cells showed higher expression of Oct4,Sox2 and Nanog than goat fibroblast.In addition,we found some clones developed a blastocysts-like structure when iPSCs culture in some group remove of Dox.The central cells of clones firstly grew separated from feeder,and became a flat monolayer cell,as culture days increased,the clones blastocysts-like directly leave from feeder,and float on the medium.These floating structures could attach feeder again and cell size became bigger,the morphology look like trophoblast stem cells(TSCs).The floating structure and attached clones both showed AP staining positive.Immunostaining results reveal the pluripotent genes OCT4,SOX2 co-express with EOMES,CDX2 and GATA4 in floating structure.We name these floating structures as dairy goat induced TSC-like cells(gi TSLCs),detail features needs further research.Summary,we derived novel pluripotent stem cell(blASCs)from blastocysts in ABC/L-medium.Depend on mouse culture system work,we also established induced dairy goat pluripotent stem cells(iPSCs),and build up independent of the Dox-induced exogenous iPSCs,dairy goat induced TSC-like cells(gi TSLCs).