Int J Stem Cells.  2021 May;14(2):221-228. 10.15283/ijsc20096.

Optimal Hypoxic Preconditioning of Human Embryonic Stem Cell-Derived Mesenchymal Stem Cells (hES-MSCs) and Their Characteristics

• Lee SM
• Jun DW
• Kang HT
• Oh JH
• Saeed WK
• Ahn SB

• KMID: 2515998
• DOI: http://doi.org/10.15283/ijsc20096

Abstract

Background and Objectives
Hypoxia is frequently used to enhance stem cell function. However, the optimal level of hypoxia for growth and function of human embryonic stem cell-derived mesenchymal stem cells (hES-MSCs) is yet to be determined. The purpose of this study was to find the optimal level of hypoxia for hES-MSCs and characteristics of hES-MSCs cultured under these optimal hypoxic conditions.
Methods and Results
Cell viability and changes in the morphology of hES-MSCs were determined through cell proliferation and CCK-8 assay. The hES-MSCs were preconditioned under various hypoxic conditions (0.5∼5% O₂ and 24∼72 h). The expression of cytokines in each culture condition was compared using cytokine array analysis. The morphology of hES-MSCs did not change under various hypoxic culture conditions. hES-MSCs viability after 48 h incubation in 2% O₂condition was higher than that in normoxic condition. HIF1α expression was increased up to six folds after 48 h of hypoxic preconditioning. HIF1α expression in hES-MSCs peaked after 48 h of incubation in 1% O₂ condition. The expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin were increased after hES-MSCs were incubated for 48 h in 2% O₂ condition.
Conclusions
The hES-MSCs viability and expressions of PDGF-BB, IGFBP-6, VEGF-A, and angiogenin increased after 48 h incubation in 2% O₂ condition.

Keyword

Hypoxia; Stem cell; Function; VEGF

Figure

• 

  Fig. 1 Morphology and viability of hES-MSCs under normal and hypoxic conditions. (A) Morphological analysis of hES-MSCs cultured under normal (21% O2) and various hypoxic incubation conditions (0.5%, 1%, 2%, and 5% O2) for 24, 48 and 72 h. The morphology was observed using an optical microscope; Magni-fication: 400×. (B) Cell viability was assessed using CCK-8 cell viability assay; (n=6, mean±SEM; ns, *p< 0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs. normal condition using One-way ANOVA-Dunnett’s multiple comparison test).

• 

  Fig. 2 Correlation between HIF1α protein expression and cell viability of hES-MSCs under various hypoxic conditions. (A) HIF1α protein expression under normal and hypoxic conditions. (B) HIF1α protein quantification using densitometry. (C) Correlation between cell viability and HIF1α relative expression. (D) HIF1α protein expression at various low O2 partial pressures. (E) Cell viability comparison with or without HIF1α inhibitor in hypoxia; (n=6, mean±SEM; ns, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001 vs. normal condition using One-way ANOVA-Dunnett’s multiple comparison test).

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  Fig. 3 Comparison of hES-MSCs cytokines expressions under various hypoxic conditions. (A) Membrane after Ray Bio human cytokine antibody array. (B) Heat map demonstrating relative expressions of biomarkers from screening cytokine antibody array in normal vs. various hypoxic conditions. (C, D) Comparison of cytokines expressions in various hypoxic culture conditions.

Reference

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