Hypoxia boosts pluripotent-like muse cell ratio in mesenchymal stromal cells and upregulates the pluripotency gene expression

Abstract

Muse cells are SSEA-3-positive pluripotent-like endogenous stem cells found in various tissues, including peripheral blood and organ connective tissue. Their reserve is considered the hypoxic bone marrow. In mesenchymal stromal cell (MSC) cultures, Muse cells comprise several percent of the population. Clinical trials using intravenous administration of Muse cells without genetic modification or differentiation induction have shown significant therapeutic potential. Since Muse cells are a small fraction of MSCs, developing efficient culture methods to increase their proportion while maintaining their stemness is crucial for enhancing efficiency and reducing costs in clinical research. In this study, we investigated the effects of hypoxia on Muse cell proportions, pluripotency gene expression, and metabolism. Hypoxia increased the Muse cell proportion around twofold, driven by HIF2α rather than HIF1α, and enhanced pluripotency gene expression, potentially via microRNA let-7 upregulation. Hypoxia also shifted metabolism from oxidative phosphorylation to glycolysis, linked to maintaining stem cell properties. These findings suggest that hypoxia represents a cost-effective strategy for expanding Muse cells, offering promising potential for clinical applications.

Keywords: Hypoxia; Hypoxia-inducible factor; Mesenchymal stromal cells; Muse cells; Pluripotency genes.

Fig. 1

1% O₂ hypoxia increased the proportion of Muse cells within BM-MSCs. Experimental design for comparing the Muse cell proportion within BM-MSCs. Flow cytometry analysis of Muse cell ratio within BM-MSCs under normoxia and 1% O₂ hypoxia. An isotype antibody was used for gate-setting control. The average and statistical analysis of three replicates (bar plot). Only one representative set of flow cytometry data is shown. **p < 0.01.

Fig. 2

The HIF dynamics in Muse cells under 1% O₂ hypoxia culture. qPCR comparison of HIF1 A, HIF2 A, and HIF3 A expression in Muse cells under normoxia (n = 3 for each). qPCR analysis of HIF1 A expression before and after HIF1 A knockdown under 1% O₂ hypoxia (n = 3). qPCR analysis of HIF2 A expression before and after HIF2 A knockdown under 1% O₂ hypoxia (n = 3). Western blot analysis of HIF1α and HIF2α expression in NC-siRNA-transfected Muse cells over time. β-actin was used as an endogenous control. Western blot analysis of HIF1α and HIF2α expression in HIF1 A-siRNA-transfected Muse cells over time. β-actin was used as an endogenous control. Western blot analysis of HIF1α and HIF2α expression in HIF2 A-siRNA-transfected Muse cells over time. β-actin was used as an endogenous control. Quantification analysis of Western blot comparing HIF1α expression among NC-siRNA-, HIF1 A-siRNA-, and HIF2 A-siRNA-transfected Muse cells. Quantification analysis of Western blot comparing HIF2α expression among NC-siRNA-, HIF1 A-siRNA-, and HIF2 A-siRNA-transfected Muse cells. Comparison of pluripotency gene expression in Muse cells under normoxia and hypoxia (n = 3). Comparison of pluripotency gene expression in non-Muse cells under normoxia and hypoxia (n = 3). ACTB was used as an endogenous control for normalisation. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 3

HIF2α regulated Muse cell ratio within BM-MSCs. (A-D) Flow cytometry analysis comparing the Muse cell ratio within BM-MSCs. Only one representative set of flow cytometry data is shown on A-D. (E) Comparison of Muse cell proportion in BM-MSCs among normoxia NC-siRNA, hypoxia NC-siRNA, hypoxia HIF1 A-siRNA, and HIF2 A-siRNA group (n = 3). *p < 0.05, **p < 0.01, ns: no significant.

Fig. 4

Hypoxia mimicked by CoCl₂ treatment in Muse cells. (A-C) Western blot analysis of HIF1α and HIF2α expression in Muse cells over time (up). Quantification analysis of Western blot (down). β-actin was used as an endogenous control. Comparison of OCR before and after 200 µM CoCl₂ treatment (n = 5). Comparison of ECAR before and after 200 µM CoCl₂ treatment (n = 5). Comparison of OCR before and after HIF1 A knockdown in Muse cells (n = 15). Comparison of ECAR before and after HIF1 A knockdown in Muse cells (n = 5).

Fig. 5

CoCl₂ increased pluripotency gene expression through upregulating let-7 expression. The effect of CoCl₂ treatment on pluripotency gene expression (n = 3). The effect of CoCl₂ treatment on let-7a expression. The effect of CoCl₂ treatment on let-7b expression. The effect of CoCl₂ treatment on let-7e expression. The effect of CoCl₂ treatment on let-7i expression. Schematic summary of this research. **p < 0.01, ***p < 0.001.