Studies On Buffalo Induced Pluripotent Stem Cells

Ectopically expressing of transcription factors could reprogram somatic cells into induced pluripotent stem cells (iPSCs), which displayed all characteristics of pluripotent stem cells, and this method initiated a new strategy to establish livestock stem cells. Chinese swamp buffalo is one of the most important domestic animals, which mainly distributes in tropical and subtropical regions, and provides milk, meat, and draft for agriculture. Especially, the buffalo milk is famous for its nutrient-rich, but the milk yield is lower than that of cow, and is in urgent need of improving their production traits by genetic manipulation technology. The ESCs based gene targeting is considered to be one of the promising way to generate site-specific gene targeting in large animals. The main reason which limited the research of buffalo ESCs may include lack of knowledge on the signal pathways and culture conditions. It is hard for establishment buffalo ESCs line and for gene targeting research, the study of buffalo iPSCs (biPSCs) generation is necessary for buffalo stem cells research. In this study, buffalo transcription factors:Oct4(O), Sox2(S), Klf4(K), c-Myc (M), Nanog (N) and Lin28(L) were cloned into the retrovirus vector, and were used for the biPSCs genenration. The different transcription factors combinations, the mechanism of promotion biPSCs generation, the biologic characterization of biPSCs and epigenetic status of biPSCs were studied in this research. On the other hand, based on the high functional conservation of the transcription factors between buffalo and other different species, we tried to induce rabbit fibroblast cells (RFFs) into rabbit induced stem cells (RiPSCs) using the buffalo transcription factors. RiPSCs were seemed as the animal model for human regenerative medicine and will also be used in the rabbit genetic improvement.Part Ⅰ Cloning and analysis of the buffalo iPSCs-related transcription factorsAfter multiple sequence comparison, primers were designed to amplify the coding sequences (CDS) of buffalo transcription factors: Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28. The total RNA were extracted from fetus germ ridge (for Oct4, Sox2, Nanog and Lin28), gut (for Klf4and c-Myc), and293T cells (for SV40large T antigen, T). The results revealed that the CDS length of OSKMNLT were1083bp,966bp,1434bp,1320bp,903bp,618bp and2130bp respectively. The amino acids of buffalo OSKMNL exhibited high homology with bovine, pig, human and mouse, the percentage of conservatism displayed:Oct4(98%,96%,91%and81%), Sox2(96%,95%,94%and94%), Klf4(99%,97%,92%and92%), c-Myc (98%,96%,91%and86%), Nanog (90%,81%,69%and47%) and Lin28(98%,97%,89%and91%). At the same time, genomic DNA sequences of Oct4and Nanog were amplified separately by PCR. Sequence analysis revealed that the total length of buffalo Oct4was4508bp, containing5exons and4introns; the total length of buffalo Nanog was4473bp, and containing4exons and3introns.Part Ⅱ Retrovirus vectors construction and the choice of retrovirus system for biPSCs generationTwo retrovirus systems (pMSCV and pMX) were employed to investigate the biPSCs generation. Two different reconstructed retrovirus vectors with green fluorescent protein (GFP) report gene were used to package retrovirus and infect the BFFs, the one which can infected BFFs with high efficiency was used for the study of biPSCs generation. The retrovirus vector carried the buffalo transcription factors were constructed respectively. And the mouse iPSCs were generated using the buffalo defined factors, which provided as a mouse model to study the biPSCs generation. The results revealed that the infection efficiency of pMX retrovirus system was higher than that of the pMSCV retrovirus system, pMX retrovirus system can used to generate biPSCs. After ultracentrifugation, the concentrated retrovirus can infect the BFFs with high efficiency. The buffalo transcription factors can reprogram mouse somatic cells into iPSCs, and the mouse iPSCs displayed positive staining of alkaline phosphatase (AP). The reconstructed vectors used for biPSCs generation were pMSCV-EGFP, pMX-EGFP, pMX-Oct4-IRES-GFP, pMX-Oct4, pMX-Sox2, pMX-Klf4, pMX-c-Myc, pMX-Nanog, pMX-Lin28, pMX-LT, pMX-LT-IRES-GFP, pMX-hTERT-IRES-GFP (LT, SV40large T antigen; hTERT, human telomerase reverse transcriptase, hT).Part III Optimize the system of buffalo iPSCs generation and investigation the efficiency of buffalo iPSCs generation by inhibit p53expressionBased on the combinations of different transcription factors commonly used for iPSCs generation, we tried to find out the best transcription factors combination to induce buffalo iPSCs. Using the best transcription factors combination, the feasibility of SV40large T antigen and p53inhibitor to increase the efficiency of biPSCs generation was investigated. The biPSCs colonies having heavy staining of AP were counted as positive colonies. The results showed that21positive colonies were obtained from OSKM combination, but the number of the positive colonies could reach to38in the OSKMNL combination. Although the expression of Klf4and c-Myc were detected in BFFs, no biPSCs were obtained when Klf4and c-Myc were withdrawed from the two cocktails, including the common combination OSNL. When SV40large T antigen was added to the OSKM,77AP positive colonies were obtained. And62AP positive colonies were obtained when OSKM infected BFFs were treated with PFT-α (20μM) for one week after transduction. The biPSCs colonies derived from OSKMT combination could be expanded more than10passages and exhibited normal karyotypes, but biPSCs colonies derived from other combinations could not expand more than6passages. Quantitative PCR (QPCR) was applied to test the p53relative expression level during the process of biPSCs generation in different combinations. The expression level of p53in BFFs was higher than biPSCs colonies and embryonic germ stem cells (EG-like cells). On the6th day after transduction with OSKM, OSKM+PFT and OSKMT, the p53expression of BFFs decreased, and the decrease extent in BFFs transduced with OSKMT and OSKM+PFT was higher than the BFFs transduced with OSKM. So the OSKM combination can reprogram BFFs into biPSCs, and SV40large T antigen and PFT-a can promote the reprogramming process through inhibiting p53expression.Part Ⅳ Biology characterization of biPSCs and methylation status analysis of pluripotent marker genes in the reprogramming processbiPSCs displayed the special biology characteristics of the stem cells, and was feasible to employed as donor cells for reconstruction nuclear transfer embryos. The morphology of biPSCs colonies were similar to human and pig iPSCs. biPSCs exhibited high AP activity by AP staining and expressed ES cell-related markers:Oct4, Sox2, Nanog, SSEA-1, SSEA-4, TRA-1-81and E-Cadherin by immunofluorescence. RT-PCR also revealed that biPSCs expressed buffalo endogenous pluripotency-related genes, including Oct4, Sox2, Nanog, STAT3, GP130, FOXD3, E-Cadherin, bFGF2and p53. The expression level of endogenous Oct4, Sox2, and Nanog in biPSCs were similar to the inner cell mass (ICM) and EG-like cells by QPCR. The expression of exogenous GFP in biPSCs was silenced after expanded for4-6passages. RT-PCR detected the silencing of exogenous genes precisely, the results showed no exogenous genes expression was detected in the most of colonies after passage10, but some exogenous transgenes expressed continuously in some colonies, such as biPS3. Methylation assay revealed that, the promoters of Oct4and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs (partially reprogrammed biPSCs), while the promoters of Sox2and E-Cadherin were hypomethylated in both BFFs, pre-biPSCs and biPSCs. The differentiation ability of biPSCs was tested in vitro and in vivo analysis. biPSCs could form embryoid bodies (EBs) after suspension culture in vitro, and three germ layers marker genes were detected in EBs:AFP, GATA4(endoderm), ACTA2(mesoderm) and TUBB3(ectoderm). biPSCs could form teratomas after injected into the nude mice. The teratomas contained various tissues of the three germ layers, including epidermis, neural tissues (ectoderm), muscle, cartilage (mesoderm), gut-like epithelium, respiratory epithelium (endoderm) by histological examination. The specific PCR amplification confirmed that these teratomas were from buffalo but not mice. At last, nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts when biPSCs were used as nuclear transfer donors.Part V Buffalo transcription factors induced rabbit fibroblast cells into induced pluripotent stem cellsThe iPSCs-related transcription factors showed high functional conservation between buffalo and other species. In this investigation, we tried to induce RFFs into RiPSCs with the pMX retrovirus vector-mediated four buffalo transcription factors Oct4, Sox2, Klf4and c-Myc (OSKM), and SV40large T antigen (LT). The result revealed that buffalo defined factors OSKM co-expression with LT could reprogram RFFs into RiPSCs. Biology characterization of RiPSCs revealed that RiPSCs displayed the special characteristics of stem cells. The morphology of RiPSCs colonies were similar to human iPSCs and ESCs, and can be expanded more than15passages with normal karyotypes in bFGF and LIF containing medium. RiPSCs exhibited high AP activity by AP staining and expressed ES cell-related markers: UTF-1, Oct4, Nanog, SSEA-1, SSEA-4, TRA-1-81, c-Myc and Klf4by immunofluorescence and RT-PCR. The results of exogenous transgenes silence analysis showed that, exogenous GFP began to silence in the RiPSCs after expanded for7passages, and no exogenous genes expression was detected in RiPS1, RiPS3and RiPS6. But the exogenous transgenes could not silence fully in all colonies, Klf4and LT expressed continuously in RiPS7. Methylation assay revealed that, the promoter of Nanog was hypomethylated in the RiPSCs of15passages, compared with RFFs. This is consisted with the activation of Nanog in RiPSCs. The differentiation ability of RiPSCs was tested in vitro and in vivo analysis. RiPSCs can form embryoid bodies (EBs) after suspension culture in vitro, and three germ layers marker genes were detected in EBs:AFP (endoderm), BMP4, Desmin (mesoderm) and PAX6, MAP2(ectoderm). biPSCs also can form teratomas after injected into the nude mice. The teratomas contained various tissues of the three germ layers, including epidermis tissues (ectoderm), cartilage (mesoderm), gut-like epithelium (endoderm) by histological examination. These efforts indicated that RiPSCs were reprogrammed fully, and obtained the special characteristics of stem cells, including pluripotency and differentiation ability.