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.

Cattle In Vitro Induced Pluripotent Stem Cells Generated and Maintained in 5 or 20% Oxygen and Different Supplementation

“…De Castro et al [ 35 ] demonstrated that hypoxic conditions did not improve the reprogramming efficiency in equine iPSCs, as has previously been described in humans and mice; however, they also described different profiles of eiPSCs generated in hypoxic compared to normoxia. Indeed, the reprogramming of iPSC cells in a low oxygen atmosphere has not yet been studied in many species, including cattle.…”

“…Additionally, in in vitro hypoxia conditions (2% oxygen), GLUT1 expression was similar to that of in vivo-derived embryos. Controversially, hypoxia conditions (5% oxygen) decreased GLUT3 expression in equine iPSCs and human embryonic stem cells [ 35 , 42 ]. These controversial results demonstrate the different effects of hypoxia on metabolism pathways between species.…”

Induced pluripotent stem cells in companion animals: how can we move the field forward?

“…Direct comparison of iPSC generation from cells of adult and fetal origin has not been performed in horses. However, when comparing fibroblasts from young and old individuals, it was suggested that the derivation of equine iPSCs is not impaired by aging ( 65 ).…”

“…Only a few equine iPSC studies have reported the use of kinase inhibitors ( 31 , 97 ) or HDAC inhibitors ( 65 ) during reprogramming. However, these studies have not compared different combinations and neither have explored in detail the specific changes elicited by the inhibitors.…”

Influence of Cell Type in In Vitro Induced Reprogramming in Cattle

“…Induced pluripotent stem cells (iPSCs) were first reported from murine, followed by human fibroblasts, transduced with a retroviral vector containing the transcription factors OCT4, SOX2, KLF4, and c-MYC (OSKM) [ 9 , 10 ]. The reprogramming of somatic cells has become a valuable source of pluripotent cells and has been established for several species of domestic and wild animals, including large animals such as swine [ 11 , 12 , 13 , 14 ], equine [ 15 , 16 ], buffalo [ 17 ], and bovine [ 4 , 18 , 19 , 20 ].…”