The effects of short-term hypoxia on human mesenchymal stem cell proliferation, viability and p16INK4A mRNA expression: Investigation using a simple hypoxic culture system with a deoxidizing agent

Akira Ito ,Tomoki Aoyama ,Xiangkai Zhang ,Masaya Nagai ,Junichi Tajino ,Makoto Yoshizawa ,Hirotaka Iijima ,Satoshi Yamaguchi ,Hiroshi Kuroki

doi:10.46582/jsrm.1101005

Abstract

A hypoxic environment is thought to be important for the maintenance of stemness and suppressing cell senescence, in stem cells. Therefore, a hypoxic condition is induced during cell expansion and/or induction of intended differentiation. However, the induction of these conditions requires a specially equipped hypoxia chamber and expensive gas mixtures, which are expensive and space-consuming. Owing to these restrictions, appropriate hypoxic conditions cannot be provided during cell transportation, which is increasingly required for regenerative medicine. Hence, a simple and economical culture system is required. The purpose of this study was to investigate the effects of short-term hypoxic conditions on human mesenchymal stem cell (MSC) proliferation, viability, and senescence, utilizing the CulturePal system (CulturePal-Zero and CulturePal-Five), a novel and simple hypoxic culture system with a built-in deoxidizing agent. The O2 concentration in the CulturePal-Zero was observed to reduce to <0.1% within 1 h, and to 5% within 24h in the CulturePal-Five system. Cell proliferation under these hypoxic conditions showed a sharp increase at 5% O2 concentration, and no noticeable cell death was observed even at severe hypoxic conditions (<0.1% O2) up to 72h. The p16(INK4A) (cell senescence marker) mRNA expression was retained under hypoxic conditions up to 72h, but it was up-regulated under normoxic conditions. Interestingly, the p16(INK4A) expression altered proportionately to the O2 concentration. These results indicated that the short-term hypoxic condition, at an approximate O2 concentration of 5%, would be suitable for promoting cell proliferation and repressing cell senescence, without aggravating the MSC viability. Therefore, the CulturePal systems may be suitable for providing an appropriate hypoxic condition in stem cell research and transportation.

Highlights

Under in vivo conditions, stem cells exist in a hypoxic environment, which is thought to be an important factor for the maintenance of its phenotype[1]
In order to assess the timelapse changes in O2 and CO2 concentration under hypoxia conditions induced by the CulturePal-Zero and CulturePalFive systems, the reagents were placed in a 2.5-L airtight jar (Mitsubishi Gas Chemical Company Inc.) each
The O2 concentration in the CulturePal-Zero system was observed to drop to less than 0.1% within 1h, and was maintained up to 168h after hypoxia induction (Figure 2A)

Summary

Introduction

Stem cells exist in a hypoxic environment, which is thought to be an important factor for the maintenance of its phenotype[1]. To maintain stemness and avoid oxidative stress, the use of a hypoxic culture, resembling in vivo environments, has been recommended[3, 4]. Hypoxic conditions have been previously reported to promote MSC proliferation[7,8,9], maintain the undifferentiated state of the cell[9,10,11], and induce differentiated state when cultivated under differentiative culture conditions[9]. There have been contrasting reports stating that the hypoxic conditions exert little effect on cell proliferation or induction of differentiation[11, 13, 14]. It is clear that the regulation of gas concentrations, such as that of oxygen (O2) and carbon dioxide (CO2), is an essential parameter for the modulation of cell phenotype and metabolism

Objectives

Methods

Results