title: Generation of induced pluripotent stem cells from buffalo (Bubalus bubalis) fetal fibroblasts with buffalo defined factors.
abstract: Ectopically, expression of defined factors could reprogram mammalian somatic cells into induced pluripotent stem cells (iPSCs), which initiates a new strategy to obtain pluripotent stem cell lines. Attempts have been made to generate buffalo pluripotent stem cells by culturing primary germ cells or inner cell mass, but the efficiency is extremely low. Here, we report a successful method to reprogram buffalo fetal fibroblasts (BFFs) into pluripotent stem cells [buffalo induced pluripotent stem cell (biPSCs)] by transduction of buffalo defined factors (Oct4, Sox2, Klf4, and c-Myc) using retroviral vectors. The established biPSCs displayed typical morphological characteristics of pluripotent stem cells, normal karyotype, positive staining of alkaline phosphatase, and expressed pluripotent markers including Oct4, Sox2, Nanog, Lin28, E-Cadherin, SSEA-1, SSEA-4, TRA-1-81, STAT3, and FOXD3. They could form embryoid bodies (EBs) in vitro and teratomas after injecting into the nude BALB/C mice, and 3 germ layers were identified in the EBs and teratomas. Methylation assay revealed that the promoters of Oct4 and Nanog were hypomethylated in biPSCs compared with BFFs and pre-biPSCs, while the promoters of Sox2 and E-Cadherin were hypomethylated in both BFFs and biPSCs. Further, inhibiting p53 expression by coexpression of SV40 large T antigen and buffalo defined factors in BFFs or treating BFFs with p53 inhibitor pifithrin-a (PFT) could increase the efficiency of biPSCs generation up to 3-fold, and nuclear transfer embryos reconstructed with biPSCs could develop to blastocysts. These results indicate that BFFs can be reprogrammed into biPSCs by buffalo defined factors, and the generation efficiency of biPSCs can be increased by inhibition of p53 expression. These efforts will provide a feasible approach for investigating buffalo stem cell signal pathways, establishing buffalo stem cell lines, and producing genetic modification buffaloes in the future.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources,  Guangxi University , Nanning,  People's Republic of China .
authors.forename: Yanfei
authors.initials: Y
authors.lastname: Deng
authors.forename: Qingyou
authors.initials: Q
authors.lastname: Liu
authors.forename: Chan
authors.initials: C
authors.lastname: Luo
authors.forename: Shibei
authors.initials: S
authors.lastname: Chen
authors.forename: Xiangping
authors.initials: X
authors.lastname: Li
authors.forename: Caizhu
authors.initials: C
authors.lastname: Wang
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Liu
authors.forename: Xiaocan
authors.initials: X
authors.lastname: Lei
authors.forename: Huina
authors.initials: H
authors.lastname: Zhang
authors.forename: Hongliang
authors.initials: H
authors.lastname: Sun
authors.forename: Fenghua
authors.initials: F
authors.lastname: Lu
authors.forename: Jianrong
authors.initials: J
authors.lastname: Jiang
authors.forename: Deshun
authors.initials: D
authors.lastname: Shi
mesh_terms: D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics / Q000378:metabolism; D052160:Benzothiazoles / Q000494:pharmacology; D015415:Biomarkers / Q000378:metabolism; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D002020:Buffaloes / Q000196:embryology*; D015820:Cadherins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D019175:DNA Methylation; D058732:Embryoid Bodies / Q000166:cytology / Q000378:metabolism; D044127:Epigenesis, Genetic; D005333:Fetus / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D005822:Genetic Vectors; D005855:Germ Layers / Q000166:cytology / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D053652:Nuclear Transfer Techniques; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013539:Simian virus 40 / Q000235:genetics / Q000378:metabolism; D013724:Teratoma / Q000378:metabolism; D014050:Toluene / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism
chemical_list: D000952:Antigens, Polyomavirus Transforming; D052160:Benzothiazoles; D015415:Biomarkers; D015820:Cadherins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; D016159:Tumor Suppressor Protein p53; D014050:Toluene; C121565:pifithrin