Int J Stem Cells.  2018 Jun;11(1):61-67. 10.15283/ijsc17054.

Improvement of Cell Cycle Lifespan and Genetic Damage Susceptibility of Human Mesenchymal Stem Cells by Hypoxic Priming

• Lee CW
• Kang D
• Kim AK
• Kim DY
• Kim DI

• KMID: 2413501
• DOI: http://doi.org/10.15283/ijsc17054

Abstract

Hypoxic culture is widely recognized as a method to efficiently expand human mesenchymal stem cells (MSCs) without loss of stem cell properties. However, the molecular basis of how hypoxia priming benefits MSC expansion remains unclear. We report that hypoxic priming markedly extends the cell cycle lifespan rather than augmenting the multipotency of MSC differentiation lineage. Hypoxic priming does not affect to chromosome damage but significantly attenuates the susceptibility of chromosome damage. Our results provide important evidence that multipotency of human MSCs by hypoxic priming is determined by cell cycle lifespan.

Keyword

Mesenchymal stem cells; Hypoxia; Cell cycle; Senescence; Chromosome damage; Multipotency

MeSH Terms

Aging
Anoxia
Cell Cycle*
Humans*
Mesenchymal Stromal Cells*
Methods
Stem Cells

Figure

• 

  Fig. 1 Comparison of potentialities of proliferation, multipotency, and senescence between normoxic and hypoxic conditioning. (A) Phase contrast images of human umbilical cord blood-derived MSCs (MSCs) were cultured in normoxic (21% O2) or hypoxic (1% O2) conditions through multiple passages (passages 3, 5 and 7, respectively). (B) Several different passages of MSCs derived from the same source of MSCs were further cultured in normoxic (21% O2) and hypoxic (1% O2) conditions, and cell numbers were counted at designated times. (C) The graph shows the relative comparison of proliferating cell number following normoxic or hypoxic priming at several passages of MSCs culture. (D) The graph shows the relative incidence of senescence associated β-galactosidase positive MSCs. Data (mean±SEM) are representative of 3 independent experiments.

• 

  Fig. 2 Effect of hypoxic priming on the DNA and chromosome damage and senescence of MSCs. (A) Three different passages of MSCs grown in normoxic or hypoxic condition were analyzed by senescence associated β-galactosidase (SA-β-gal) staining. The graph shows the relative percentage of SA-β-gal positive cells. (B) MSCs were cultured in normoxic or hypoxic condition, or in normoxic condition in combination with 0.5 mM doxorubicin (Doxo) treatment, and stained with anti-γH2AX (a marker for DNA double-strand breaks) and DAPI for DNA. Graph shows the percentage of γH2AX-positive cells at three different passages. (C) Three different passages of MSCs were grown in normoxic or hypoxic condition, and harvested for metaphase chromosome spreading analysis. Arrows indicate the break chromosome. The graphs show the percentage of metaphase MSCs containing the break chromosome. (D) Total number of chromosome in each MSC was examined at passages 4, 8, and 12, respectively. Data (mean±SEM) are representative of 3 independent experiments.

• 

  Fig. 3 Application of hypoxia to MSCs stimulates their proliferating potential and the potential of multi-lineage differentiation. (A) Multi-passaged MSCs were cultured in normoxic or hypoxic condition, labeled with antibodies against specific surface antigens, CD44, CD73, CD90, and CD105 for MSC positive markers and CD31, CD34, and CD45 for MSC negative markers, and analyzed by counting 10,000 cells at passages 4, 7, and 9, respectively. The surface antigen phenotype was characterized by FACS. Immunoglobulin isotype was used as a negative control for FACS analysis. Negative indicates the labelling of cells with CD34, CD45, and CD11b antibodies. Red-colored histograms illustrate the control immunoglobulin, and blue-colored histograms represent the staining against each specified antibodies as indicated. (B) The graph shows the positive percentages of each MSC marker expression. (C) The graph shows the potency of adipogenic differentiation rate at indicated passages. Data (mean±SEM) are representative of 3 independent experiments.

Reference

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