In Vitro Induction of Pluripotency from Equine Fibroblasts in 20% or 5% Oxygen

Abstract

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.

Figure 1

Alkaline phosphatase staining and different colony morphologies in passages 3, 16, and 30: (a, d, g) group H; (b, e, h) group HL; (c, f, i) group L. Scale bars: 200 μm.

Figure 2

Immunocytochemistry of eiPSC colonies showing positive and negative results for pluripotency markers SOX2, OCT4, NANOG, TRA-1-60, TRA-1-81, and SSEA-1 for groups H, HL, and L in passages 16 (a) and 30 (b). Scale bars: 100 μm, 200 μm, and 400 μm.

Figure 3

(a–c) Embryoid body (EB) formation from groups H, HL, and L, respectively. (d, e) RT-PCR results for ectoderm gene βIII-tubulin (βIII Tub), endoderm gene alpha-fetoprotein (AFP), and mesoderm gene bone morphogenetic protein (BMP4) and for the expression of the lentiviral vector (hOSKM) and housekeeping gene PPIA in embryoid bodies (EB, d, left) and spontaneous differentiation (SD, d, right), from groups H, L, and HL; in eiPSC cells from groups H, HL (e, first row left and right, respectively), and L (second row) and fibroblast (third row) cultured in high (FH, left) and low (FL, right) oxygen. (f–h) Immunocytochemistry of spontaneous differentiation for mesoderm marker vimentin, ectoderm markers nestin and neurofilament, and endoderm marker GATA6, in groups H (f), HL (g), and L (h). Scale bars: 100 μm and 200 μm.