de Castro Raquel V G, Pieri Naira C G, Fantinato Neto Paulo, Grizendi Bianca M, Dória Renata G S, Meirelles Flavio V, Smith Lawrence C, Garcia Joaquim M, Bressan Fabiana F
Department of Pathology, Reproduction and One Health, Faculty of Agricultural and Veterinary Sciences, São Paulo State University, 14884-900, Brazil.
Department of Veterinary Medicine, Faculty of Animal Science and Food Engineering, University of São Paulo (FZEA/USP), 13635-900, Brazil.
Stem Cells Int. 2020 Nov 26;2020:8814989. doi: 10.1155/2020/8814989. eCollection 2020.
The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as culture conditions (e.g., environmental oxygen concentration), and the aging process. Herein, we aimed to generate and maintain equine iPSCs (eiPSCs) derived from fibroblasts of a horse older than 20 years and to evaluate the effect of different levels of oxygen tension (atmospheric 20% O, 5% O, or 20% to 5% O) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes , , and ; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O and 20% to 5% O), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O); they formed embryoid bodies; and there is spontaneous differentiation in mesoderm, endoderm, and ectoderm embryonic germ layers. In addition to the differences in immunofluorescence analysis results, the eiPSC colonies generated at 20% O presented a more compact morphology with a well-defined border than cells cultured in 5% O and 20% to 5% O. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (, , , , and ), after reprogramming. Our results show that the derivation of eiPSCs was not impaired by aging. Additionally, this study is the first to compare high and low oxygen cultures of eiPSCs, showing the generation of pluripotent cells with different profiles. Under the tested conditions, the lower oxygen tension did not favor the pluripotency of eiPSCs. This study shows that the impact of oxygen atmosphere has to be considered when culturing eiPSCs, as this condition influences the pluripotency characteristics.
细胞重编程为多能性受到外部和内部细胞因素的影响，如培养条件（如环境氧浓度）和衰老过程。在此，我们旨在生成并维持源自一匹20岁以上马的成纤维细胞的马诱导多能干细胞（eiPSCs），并评估不同水平的氧张力（大气氧浓度20%、5%或20%至5%）对这些细胞的影响。成纤维细胞被重编程，推定的eiPSCs碱性磷酸酶检测呈阳性；它们多能性相关基因、和呈阳性；OCT4和NANOG（所有组）、SOX2（5% O组和20%至5% O组）以及TRA-1-60、TRA-1-81和SSEA-1（仅在20% O组）免疫荧光染色呈阳性；它们形成胚状体；并且在中胚层、内胚层和外胚层胚胎胚层中有自发分化。除了免疫荧光分析结果存在差异外，20% O条件下产生的eiPSC集落比在5% O和20%至5% O条件下培养的细胞呈现出更紧密的形态，边界更清晰。重编程后，在与葡萄糖代谢、线粒体分裂和缺氧相关的基因（、、、和）表达方面也观察到显著差异。我们的结果表明，衰老不会损害eiPSCs的产生。此外，本研究首次比较了eiPSCs的高氧和低氧培养，显示出具有不同特征的多能细胞的产生。在测试条件下，较低的氧张力不利于eiPSCs的多能性。本研究表明，培养eiPSCs时必须考虑氧气氛的影响，因为这种条件会影响多能性特征。
