Effect of low oxygen tension on transcriptional factor OCT4 and SOX2 expression in New Zealand rabbit bone marrow-derived mesenchymal stem cells.

Erma Safitri

doi:10.14202/vetworld.2020.2469-2476

Abstract

Background and Aim: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 in vitro 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 in vitro.Materials and Methods: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% O2) and two treatment groups with low oxygen tension (1% and 3% O2). 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% N2, 5% CO2, and 1% O2 (T1) and 3% O2 (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.Results:BM-MSCs were positive for CD44 and CD90 expression after isolation. Oxygen tension culture conditions of 1% and 3% O2 led to OCT4 and SOX2 expression on culture days 2 and 4 (p<0.05), respectively, as compared to the hyperoxia condition (21% O2).Conclusion:Based on the OCT4 and SOX2 immunofluorescence data, we conclude that the stem cells were pluripotent at low O2 tension (at 1% O2 on day 2 and at 3% O2 on day 4), whereas under 21% O2 the OCT4 and SOX2 were not expressed.

Highlights

Pluripotency is the ability of stem cells to differentiate into all cell types that constitute the adult body
Based on the octamer-binding transcription factor 4 (OCT4) and sex-determining region Y-box 2 (SOX2) immunofluorescence data, we conclude that the stem cells were pluripotent at low O2 tension, whereas under 21% O2 the OCT4 and SOX2 were not expressed
After being cultured under hypoxic (1% or 3% O2) or normoxic (21% O2) conditions, we characterized the OCT4 and SOX2 expression in bone marrow-derived mesenchymal stem cells (BM-MSCs) by immunofluorescence

Summary

Introduction

Pluripotency is the ability of stem cells to differentiate into all cell types that constitute the adult body. Multipotent of rabbit’s bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to become pluripotent in vitro when cultured under low O2 tension conditions [1]. The low O2 tension in the BM helps to maintain stem cells adjusted to the body’s normal physiological condition [4]. This maintenance would likely occur if the stem cells are in the G0 phase and not in a cycling state. 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 in vitro

Objectives

Methods

Results

Discussion

Conclusion