The cellular reprogramming into pluripotency is influenced by external and internal cellular factors, such as in vitro 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% O₂, 5% O₂, or 20% to 5% O₂) on these cells. Fibroblasts were reprogrammed, and putative eiPSCs were positive for positive alkaline phosphatase detection; they were positive for pluripotency-related genes OCT4, REX1, and NANOG; immunofluorescence-positive staining was presented for OCT4 and NANOG (all groups), SOX2 (groups 5% O₂ and 20% to 5% O₂), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O₂); 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% O₂ presented a more compact morphology with a well-defined border than cells cultured in 5% O₂ and 20% to 5% O₂. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1α, and HIF2α), 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.

中文翻译：

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在20％或5％的氧气中从马成纤维细胞体外诱导多能性

细胞重编程为多能性受到外部和内部细胞因素的影响，例如体外培养条件（例如环境氧气浓度）和老化过程。在本文中，我们的目的是产生和维持从马的成纤维细胞衍生的马的iPSC（eiPSCs）20岁以上，并评价不同水平的氧张力（大气20％的O的效果₂，5％氧气₂，或20％至这些电池上的5％O ₂）。重新设置成纤维细胞，假定的eiPSC对碱性磷酸酶检测呈阳性。他们对多能性相关基因OCT4，REX1和NANOG呈阳性; 对OCT4和NANOG（所有组），SOX2（5％O ₂和20％至5％O _2组）和TRA-1-60，TRA-1-81和SSEA-1（所有组）进行了免疫荧光阳性染色。仅在20％O _2中）；它们形成胚状体；并且中胚层，内胚层和外胚层的胚芽层自发分化。除了免疫荧光分析结果的差异外，与在5％O ₂和20％到5％O _2中培养的细胞相比，在20％O ₂处产生的eiPSC集落呈现出更加紧凑的形态，边界清晰。在与葡萄糖代谢，线粒体裂变和低氧有关的基因表达中也观察到了显着差异（GAPDH，GLUT3，MFN1，HIF1α和HIF2α），经过重新编程。我们的结果表明，eiPSCs的推导不受老化的影响。此外，这项研究是第一个比较eiPSC的高氧和低氧培养物的研究，显示了具有不同特征的多能细胞的生成。在测试条件下，较低的氧气张力不利于eiPSC的多能性。这项研究表明，培养eiPSC时必须考虑氧气气氛的影响，因为这种情况会影响多能性特征。