Study On Pluripotency Maintenance Of Goat IPSC-like Cell Line By Combination Of Small Molecule Compounds Vc And RG108

In this study,goat-induced pluripotent stem cells(giPSCs-like)were used as experimental materials to screen small molecule compounds that promote the maintenance of giPSCs pluripotent state,and to optimize the culture system.Then,a series of biological characteristics were detected in the optimized giPSCs,combined with RNA-seq technology and bioinformatics analysis to explore the molecular mechanisms involved in promoting pluripotency maintenance.1.Screening of culture conditions for promoting pluripotency of giPSC-like cells.(1)By comparing the effects of adding a single small molecule drug acting on epigenetics on giPSC-like cells the morphology,the identification indicators are clonal morphology,alkaline phosphatase activity and expression of related genes.The results showed that the Vc group and the RG108 group promote the growth rate and maintenance morphology of the clones significantly.In addition,the clones are translucent and the alkaline phosphatase(AP)activity is enhanced.At the same time,the addition of Vc group significantly increased the expression of histone demethylase KDM2B and DNA demethylase TET1,TET3.Addition of RG108 group reduced the expression of DNA methyltransferase inhibitor(DNMT1).(2)Further screening of small molecules combination revealed that AP was more active when Vc was combined with RG108(ie,VR)and significantly up-regulated TET1,TET3 and significantly down-regulated DNMT1 expression.In addition,the expression levels of the pluripotency genes OCT4,SOX2 and Nanog were also significantly increased and the maintenance of pluripotency was promoted.(3)Screening of basic media experiments found that DMEM/F12+10%FBS+10%KSR(DFK)basal medium significantly increased the growth rate and quality of the clones.The results of QRT-PCR showed that the expression of OCT4,KLF4,Nanog and TET1 was up-regulated most significantly when medium was combined with VR cultured cells.Finally,the experiment of perfecting the medium showed that adding basic fibroblast growth factor(bFGF)further promoted the clonal proliferation rate and significantly up-regulated the expression of related genes.Finally determining "DFK-VR+bFGF"(DMEM/F12+10%KSR++10%FBS+ Dox+ P-mercaptoethanol + L-glutamine +NEAA)is the optimized culture system.(4)The giPSCs obtained under the "DFK-VR+bFGF" culture system was removal.The experimental result shows that the giPSCs clones are round and translucent and the morphology was gathered to showing normal karyotype of goats,which are 60 chromosomes.Compared with the control group the clone growth rate was fast and the AP activity was strong.The relative fluorescence intensity results showed that fluorescence intensity of H3K9me3 decreased significantly and the fluorescence intensity of the pluripotency genes SOX2 and NANOG were significantly increased.Under the condition of DOX-free culture,the clonal morphology can be maintained to some extent in the "DFK-VR+bFGF" culture system.2.Comparison of transcriptome sequencing of giPSC-like cells in "DFK-VR+bFGF" and control(GIPS)culture systems.According to the sequencing results,analysis of KEGG pathway with significant enrichment of differential genes in DFK-VR+bFGF and GIPS systems.The key signaling pathways that cause the difference in pluripotency between the two groups are the Wnt signaling pathway,the Hippo signaling pathway and the PI3K-AKT signaling pathway.It is found that activating the Wnt signaling pathway is beneficial to the improvement of pluripotency.Activation of the PIP3-AKT signaling pathway promotes cell proliferation,inhibits apoptosis and regulates the glycolysis process.The Hippo signaling pathway is critical for the maintanance of proliferation,survival and pluripotency of giPSCs.In summary,we screened the culture system "DFK-VR+bFGF" which promote the maintenance of pluripotent state of giPSCs.It is speculated that this system may reduce the overall methylation of cells through small molecule compounds Vc and RG108,there by promoting the increase of pluripotency and at the same time activating Wnt signaling pathway,Hippo signaling pathway and PI3K-AKT signaling pathway play an important role.The above results provide a theoretical basis for further optimization of goat pluripotent stem cell culture conditions and also lay a foundation for promoting the production of transgenic animals and development of animal husbandry.