Safitri Erma
Department of Veterinary Reproduction, Faculty of Veterinary Medicine, Universitas Airlangga, Surabaya 60115, Indonesia.
Stem Cells Research Division, Institute Tropical Disease, Universitas Airlangga, Surabaya 60115, Indonesia.
Vet World. 2020 Nov;13(11):2469-2476. doi: 10.14202/vetworld.2020.2469-2476. Epub 2020 Nov 18.
Octamer-binding transcription factor 4 (OCT4) and sex-determining region Y-box 2 (SOX2) are transcription factors whose functions are essential to maintain the pluripotency of embryonic stem cells. The purpose of this study was to derive stem cells for culture and to maintain their viability and pluripotency, with the goal to obtain a cell line for transplantation in patients with degenerative diseases or injuries. This research focused on examining the effect of low oxygen tension on the ability of bone marrow-derived mesenchymal stem cells (BM-MSCs) to express OCT4 and SOX2 .
BM-MSCs were obtained from femurs of 2000 to 3000 g New Zealand male rabbits. BM-MSCs were divided into three groups to test different culture conditions: A control group under hyperoxia condition (21% O) and two treatment groups with low oxygen tension (1% and 3% O). We characterized the BM-MSCs using flow cytometric measurement of cluster differentiation 44 (CD44) and cluster differentiation 90 (CD90) expression. The expression of OCT4 and SOX2 was measured by immunofluorescence staining after 48 h of incubation in chambers with normal or low oxygen tension with controlled internal atmosphere consisting of 95% N, 5% CO, and 1% O (T1) and 3% O (T2). We considered OCT4 and SOX2 as two markers of pluripotency induction. All immunofluorescence data were subjected to a post hoc normality Tukey's honestly significant difference test; all differences with p<5% were considered significant.
BM-MSCs were positive for CD44 and CD90 expression after isolation. Oxygen tension culture conditions of 1% and 3% O led to OCT4 and SOX2 expression on culture days 2 and 4 (p<0.05), respectively, as compared to the hyperoxia condition (21% O).
Based on the OCT4 and SOX2 immunofluorescence data, we conclude that the stem cells were pluripotent at low O tension (at 1% O on day 2 and at 3% O on day 4), whereas under 21% O the OCT4 and SOX2 were not expressed.
八聚体结合转录因子4（OCT4）和性别决定区Y框蛋白2（SOX2）是转录因子，其功能对于维持胚胎干细胞的多能性至关重要。本研究的目的是获取用于培养的干细胞，并维持其活力和多能性，目标是获得一种可用于移植治疗退行性疾病或损伤患者的细胞系。本研究重点考察低氧张力对骨髓间充质干细胞（BM-MSCs）表达OCT4和SOX2能力的影响。
从体重2000至3000克的新西兰雄性兔股骨中获取BM-MSCs。将BM-MSCs分为三组以测试不同培养条件：高氧条件（21% O₂）下的对照组以及低氧张力（1%和3% O₂）的两个处理组。我们通过流式细胞术检测细胞分化簇44（CD44）和细胞分化簇90（CD90）的表达来对BM-MSCs进行表征。在由95% N₂、5% CO₂和1% O₂（T1）以及3% O₂（T2）组成的可控内部气氛的正常或低氧张力培养箱中孵育48小时后，通过免疫荧光染色检测OCT4和SOX2的表达。我们将OCT4和SOX2视为多能性诱导的两个标志物。所有免疫荧光数据均进行事后正态性Tukey真实显著差异检验；所有p<5%的差异均被视为显著。
分离后的BM-MSCs CD44和CD90表达呈阳性。与高氧条件（21% O₂）相比，1%和3% O₂的氧张力培养条件分别在培养第2天和第4天导致OCT4和SOX2表达（p<0.05）。
基于OCT4和SOX2免疫荧光数据，我们得出结论，干细胞在低氧张力下（第2天1% O₂和第4天3% O₂）具有多能性，而在21% O₂条件下OCT4和SOX2未表达。
