The Study Of Induced Goat Fibroblasts Into Pluripotent Stem Cells-like And Trans-differentiation By Small-molecule Compounds

The generation of desired cell types is the long-term goal of regenerative medicine,which has enormous influence on the development of biomedical science and animal breeding,such as cell therapy,drug research,disease models and organ regeneration in vivo,production of transgenic animal,bioreactor and so on.Cellular reprogramming and differentiation technology provide desired cellular resources.However,the classical methods of generation pluripotent stem cells and different lineages cells was almost by transfected exogenous genes,which hinders the clinical application on account of the low efficiency and integrating exogenous genes.Luckily,as a novel approach,small-molecule compounds can not only improve the induction efficiency,but also replace the exogenous genes.To further investigate the effects of small-molecule on reprogramming and differentiation,this study preliminary discussed the following contents for the first time,which included the generation of goat CiPSCs-like,study on the molecular mechanism of chemical induction system,optimization of the chemical induction system and investigation the effect of small-molecule on the cell differentiation.Then we could obtain safe and effective cell types,which could provide theoretical basis for the study of goat chemical induction system and the related mechanism.Moreover,the study may lay the foundation of cell model,animal breeding and production of transgenic animal.1.The study of generation of goat CiPSCs-like by completely small-molecule compounds.Generation the non-virus and non-integrated goat induced pluripotent stem cells by induced goat ear fibroblasts using completely small-molecule compounds system.The results showed that goat CiPSCs-like colonies were dome or island shape and positive of AP staining.RT-PCR results showed the CiPSCs-like colonies expressed pluripotent marker genes,immunofluorescence showed that the colonies expressed OCT4,SOX2,E-CADHERIN and surface specific antigen SSEA-1.Differentiation experiments in vitro indicated that goat CiPSC-like cells could form embryoid bodies(EBs)and cells of three germ layers.Embryo chimeric potential detection showed that porcine embryo injected GFP-CiPSCs colonies by micro-injection developed into chimeric blastocyst expressed GFP,while there was no blastocyst expressed GFP by injected GFP-GEFs.In conclusion,the results proved that we have firstly established the techinal platform for generation goat CiPSCs-like colonies by completely small-molecule compounds and verified partical pluripotency of the colonies,which would lay a foundation for the further research.2.Discussion the effects of genes associated with cellular energy metabolism and epigenetic modification on the generation goat CiPSCs-like colonies by chemical induction system.In order to preliminary explore the possible mechanism for the formation of goat CiPSCs-like,the study investigated the changes of cellular energy metabolism and epigenetic modification during the chemical induced system.According the generation of goat CiPSCs-like or not inducing different goat cell lines by chemical induced system,we called CiPSCs-like colonies group and non-colony group.The results showed CiPSC-like colonies group had stonger AP positive;the expression of glycolysis genes about PGAM1,PKM2 and HXK2 in CiPSC-like colonies were significantly higher than their parental fibroblasts,and the lactate level was higher than control.While non-colony group only showed several positive cells on AP staining,the expression of epigenetic modification of related gene,KDM5B,was significantly up-regulated,but not in the CiPSC-like colonies.In conclusion,glycolysis related genes PGAM1,PKM2 and HXK2 and epigenetic modification related gene KDM5B might be closely related to the formation of goat CiPSCs-like colonies.3.Optimization of goat CiPSC-like reprogramming system by small-molecule compounds.The study established the screening library for small molecular compounds that may affect the efficiency and quality of goat CiPSCs-like colonies.Then the single small-molecule was removed from the original chemical induction system"VCPTFVD",the role of single small-molecule was judged and we could further optimize the unduced system.The results showed that the reprogramming efficiency could be increased by adding some small-molecule under the condition of "VCPTFVD".The adding of AMI-5 and A-83-01 could significantly enhance the reprogramming efficiency.When reduced the small-molecule CHR98014 under the condition of "VCPTFVD",we could observe the morphological changes similar with human iPSCs colonies under the condition of "VPTFND".In addition,adding WNT-C59 or IWP-2,as inhibitors of WNT signaling pathway,which caused the colonies smaller compared to the condition of "VPTFND".And the rate of positive AP staining colonies was significantly descended.The above results indicated AMI-5 and A-83-01 could significantly enhance the reprogramming efficiency.While inhibiting the WNT signal pathway would hinder the goat chemical induced efficiency.4.The study of differentiation of goat fibroblasts by small-molecule compounds.The study explored the status of colonies and the process under the chemical induction system "VPTFN".The results showed the colonies were generated by "VPTFN",which were similar with human ES/iPS,expressed SOX2,E-CADHERIN and different germ layer genes.Moreover,the fluorescence intensity of H3K4me3,H4K8ac and H3K9ac were significantly higher than control group.And the fluorescence intensity of mitochondrial specific staining and the ROS content were significantly lower than control group.We also primarily analyzed the colonies generated under the condition of"VPTFN" by RNA-seq.Functional clustering analysis of differential genes revealed gene changes were mainly focused on system development,layer development and organ development.RNA-seq analysis results showed that the expression of fibroblasts markers genes was significantly d’ecreased in cells after 8d induction.While there was no expression of pluripotent genes.However,JARID2 and ID2 were significantly increased as key genes of regulated embryonic development and lineage fate,respectively.Neuro-progenitor marker genes SOX2 and PAX6,hematopoietic stem cell marker genes THY1 and mammary progenitor cell marker genes LTF and CD44 were significantly upregulated.TFAP2C,SOX7,TNNT1,AREG and AKAP6,which were related to the development of nerves,heart muscle and skeletal muscle,which were also significantly upregulated.QRT-PCR results showed that the relative expression of fibroblasts markers genes,pluripotent genes,key genes were basically consistent with the analysis of RNA-seq.The above results suggested that goat fibroblasts could be differentiated into an activated or intermediate cell population.