The Study Of The Pluripotent Stem Cells And Their Culture System From Pig And Goat

Pluripotent stem cells are characterized by the ability to self-renewal and the potential todifferentiate into three germ layers. Pluripotent stem cells from domestic ainimals would beuseful in various applications, such as human disease modelling, tissue transplantation, aswell as biopharming. Efforts have been made to obtain pluripotent stem cells from domesticanimals, such as pig, bovine and goat. However, questions still remain in the derivation ofthese cells. There is no commonly accepted cell line as the putative pluripotent stem cells aredifficult to matian the undifferentiated state in vitro, and unable to produce chimeras or thechimerism is poor. This may due to inappropriate culture conditions and the undefinedpluripotent signaling network. In this study, we mainly focused on the pluripotent stem cellsfrom pig and goat. For pig pluripotent stem cells, we optimized the culture condition bycomparing effects of different growth factors and small molecular compounds on pig EGCs.In optimized condition, we established two pig iPS cell lines at last. After characterized thepluripotency, we test the effects of SB431542and BMP4during the cell reprogrammingprocess. In addition, we found that the pig iPS cells showd distinct transcriptome profiles bytranscriptome sequencing. For goat pluripotent stem cells, we attempted to isolate goat ESCsin various culture conditions, and then to establish gota iPS cell lines. In this process, weconstruct a reporter systerm to monitor the cell pluripotency.1. Isolation and cultivation of pig embryonic germ cells (EGCs)The cultural condition for pig EGCs was optimized mainly through comparing the effectof different feeder layers and small molecular compounds. Three signal pathway inhibitors(PD0325901, CHIR99021and SB431542) were used in this study. The best combination wasdetermined by the number of AP positive EG cell clones. In the optimized culture medium,we compared the effect of three different feeder layers (MEF, PEF and stromal cells frommesonephron) and confirmed that MEF was appropriate.2. Establishment of pig iPS cell lines and the mechanism during cell reprogrammingPig embryonic fibroblasts were induced by the “Yamanaka factors”(OCT4, SOX2, KLF4and C-MYC). On the basis of the culture condition for pig EGCs, we further optimized theculture medium for pig iPSCs and established two cell lines. The cell colonies were resembled to human ESCs on morphology with high nuclear to cytoplasmic ratio and were AP positive.The integration of ectopic transcription factors were confirmed by genomic PCR and all ofthem was silenced. The pig iPSCs expressed pluripotent transcription factors and stem cellsurface markers and had the ability to different into three germ layers in vivo an in vitro.The influenced factors in the culture medium might affect MET (mesenchymal toepithelial transition) were screened by flow cytometry, calculating the percentage ofE-Cadherin positive cells. Combined with Real time RT-PCR and Western Blotting wedemonstrated SB and BMP4synergistically promote MET during cell reprogramming. Inaddition, the combination of SB and BMP4could promote the cell proliferation rate.3. Transcriptome analysis of pig iPSCsThe mRNA and miRNA sequencing were carried out and the data was compared withPEF and FGF2-piPSCs. The mRNA sequencing results showed that LFB2i-piPSCs expressedhigh level of SOX2, L-MYC and ESRRB, and relatively low levels of POU5F1, KLF4andNANOG. miRNA sequencing showed that members of pluripotency associated miRNAclusters like miR-17/92, miR-302b/367, miR-106a/363and miR-290were up-regulated andmembers of let-7family were down-regulated.4. Construction of goat NANOG promoter repoter systerm and its application for monitoringcell pluripotencyWe attempt to derive ESCs and iPSCs of goat. In this process, the NANOG promoter wascloned, and then, the pGNP-EGFP reporter plasmid was constructed. The recombinantconstruct of pGNP-EGFP was solely activated in pluripotent cells. The construct was thenused to monitor the cell reprogramming and embryo development process.