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

Autor(es)

de Castro, Raquel V. G. ^{[1, 2]} ; Pieri, Naira C. G. ^{[1, 3]} ; Fantinato Neto, Paulo ^[1] ; Grizendi, Bianca M. ^[1] ; Doria, Renata G. S. ^[1] ; Meirelles, Flavio V. ^[1] ; Smith, Lawrence C. ^[4] ; Garcia, Joaquim M. ^[2] ; Bressan, Fabiana F. ^[1]

Afiliação do(s) autor(es)

^[1] Univ Sao Paulo FZEA USP, Fac Anim Sci & Food Engn, Dept Vet Med, BR-13635900 Sao Paulo - Brazil

^[2] Sao Paulo State Univ, Fac Agr & Vet Sci, Dept Pathol Reprod & One Hlth, BR-14884900 Sao Paulo - Brazil

^[3] Univ Sao Paulo FMVZ USP, Sch Vet Med & Anim Sci, Dept Anim Reprod, BR-13635900 Sao Paulo - Brazil

^[4] Univ Montreal, Fac Vet Med, Ctr Rech Reprod & Fertilite, Montreal, PQ J2S 2M2 - Canada

• Tipo de documento: Artigo Científico
• Fonte: STEM CELLS INTERNATIONAL; v. 2020, NOV 26 2020.
• Citações Web of Science: 0

Resumo

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% O-2, 5% O-2, or 20% to 5% O-2) 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% O-2 and 20% to 5% O-2), and TRA-1-60, TRA-1-81, and SSEA-1 (only in 20% O-2); 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-2 presented a more compact morphology with a well-defined border than cells cultured in 5% O-2 and 20% to 5% O-2. Significant differences were also observed in the expression of genes related to glucose metabolism, mitochondrial fission, and hypoxia (GAPDH, GLUT3, MFN1, HIF1 alpha, and HIF2 alpha), 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. (AU)

• Processo FAPESP: 13/08135-2 - CTC - Centro de Terapia Celular
• Beneficiário: Dimas Tadeu Covas
• Modalidade de apoio: Auxílio à Pesquisa - Centros de Pesquisa, Inovação e Difusão - CEPIDs
• Processo FAPESP: 18/04009-6 - Efeitos de baixa tensão de oxigênio na eficiência da reprogramação e manutenção de células-tronco pluripotentes induzidas de equino
• Beneficiário: Raquel Vasconcelos Guimarães de Castro
• Modalidade de apoio: Bolsas no Brasil - Doutorado
• Processo FAPESP: 15/26818-5 - Investigação de mecanismos celulares e moleculares da aquisição da toti- e pluripotência induzida in vitro - modelo translacional
• Beneficiário: Fabiana Fernandes Bressan
• Modalidade de apoio: Auxílio à Pesquisa - Jovens Pesquisadores