216 Oxygen levels and pluripotency maintenance supplements affect cellular reprogramming of bovine fibroblasts
B. Bessi A , R. Botigelli B A , K. Recchia C A , N. Pieri C A , G. Barbosa A , R. de Castro D A , A. de Souza A , F. Meirelles A and F. Bressan AA Department of Veterinary Medicine, School of Animal Science and Food Engineering (FZEA), University of São Paulo (USP), Pirassununga, SP, Brazil;
B Department of Pharmacology, Institute of Biosciences (IBB), São Paulo State University (UNESP), Botucatu, SP, Brazil;
C Department of Animal Reproduction, School of Veterinary Medicine and Animal Science (FMVZ), University of São Paulo (USP), Pirassununga, SP, Brazil;
D Department of Preventive Veterinary Medicine and Animal Reproduction, Faculty of Agricultural and Veterinary Sciences (FCAV), São Paulo State University (UNESP), Jaboticabal, SP, Brazil
Reproduction, Fertility and Development 32(2) 236-236 https://doi.org/10.1071/RDv32n2Ab216
Published: 2 December 2019
Abstract
Different supplements are used during in vitro cellular reprogramming, usually acting on pluripotency maintenance and/or differentiation inhibition, such as basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), and 2i (MEK inhibitor: PD0325901 + GSK3 inhibitor: CHIR99021). Another important factor affecting the reprogramming process is the oxygen (O2) tension because O2 levels can modify cellular metabolism and epigenetic markers, which are known to modulate pluripotency. Our objective was to evaluate the efficiency of reprogramming bovine fibroblasts in combination with different oxygen tensions (high O2, hO2 × low O2, lO2) in different cell differentiation inhibitors: bFGF and bFGF + LIF + 2i (FL2i). Bovine fibroblasts were transduced with lentivirus harbouring mouse OSKM transcription factors (OCT4, SOX2, KLF4, and cMYC). Three clonal lineages were analysed for each experimental group. Pluripotency was characterised by morphology, detection of alkaline phosphatase, formation of embryoid bodies, and analysis of gene expression. As an initial pluripotency test, all colonies were positive for alkaline phosphatase (AP) activity in passages 5 to 6. Colonies were cultured for at least 15 passages (±140 days) with the exception of bFL2i colonies cultured in lO2, which did not grow beyond 7 to 8 passages. For gene expression analysis, samples of each colony in passages 5, 10, and 15 were used. When gene expression was analysed, both endogenous NANOG and OCT4 were increased in the bFGF group when cultured in hO2, and bFGF cultured in lO2 was higher than in the FL2i group (P < 0.05). Also, NANOG was increased in early passages compared with late passages (P < 0.05); SOX2 and FGF5 were increased in lO2 groups (P < 0.05). The bFGF treatment increased STELLA expression compared with bFL2i (P < 0.05) at both oxygen tensions. Interestingly, exogenous vector expression increased in the bFGF group compared with bFL2i (P < 0.05) but was not affected by oxygen tension (P > 0.05). All colonies tested were able to form embryoid bodies. In conclusion, it was not possible to maintain bovine induced pluripotent stem cells (biPS) in bFL2i treatment cultured in lO2 because these colonies were not able to remain viable after 8 passages. Moreover, small molecule supplementation strongly affected pluripotency gene expression. Further analysis on epigenetic changes, metabolism, and self-renewal is necessary to understand the pluripotent state in biPS under our experimental conditions.
We acknowledge FAPESP for funding (grant 2015/26816-5 and fellowship 2018/24520-7).
