Low physiologic oxygen tensions reduce proliferation and differentiation of human multipotent mesenchymal stromal cells

Abstract

Background: Human multipotent mesenchymal stromal cells (MSC) can be isolated from various tissues including bone marrow. Here, MSC participate as bone lining cells in the formation of the hematopoietic stem cell niche. In this compartment, the oxygen tension is low and oxygen partial pressure is estimated to range from 1% to 7%. We analyzed the effect of low oxygen tensions on human MSC cultured with platelet-lysate supplemented media and assessed proliferation, morphology, chromosomal stability, immunophenotype and plasticity.

Results: After transferring MSC from atmospheric oxygen levels of 21% to 1%, HIF-1alpha expression was induced, indicating efficient oxygen reduction. Simultaneously, MSC exhibited a significantly different morphology with shorter extensions and broader cell bodies. MSC did not proliferate as rapidly as under 21% oxygen and accumulated in G1 phase. The immunophenotype, however, was unaffected. Hypoxic stress as well as free oxygen radicals may affect chromosomal stability. However, no chromosomal abnormalities in human MSC under either culture condition were detected using high-resolution matrix-based comparative genomic hybridization. Reduced oxygen tension severely impaired adipogenic and osteogenic differentiation of human MSC. Elevation of oxygen from 1% to 3% restored osteogenic differentiation.

Conclusion: Physiologic oxygen tension during in vitro culture of human MSC slows down cell cycle progression and differentiation. Under physiological conditions this may keep a proportion of MSC in a resting state. Further studies are needed to analyze these aspects of MSC in tissue regeneration.

Figure 1

Morphological evaluation of MSC under different oxygen conditions. Photomicrographs of two representative MSC cultures from separate donors. In one culture no significant morphological difference after 3 weeks between 21% O₂ (A) and 1% O₂ (B) were found. In a second culture, MSC under 1% O₂ did not adapt the typical triangular shape and became plastic-adherent, but died after 1 week (D), while the control cells maintained under 21% O₂ (C) exhibited the characteristic MSC morphology. Obviously, donor-dependent factor greatly impact the growth and differentiation behavior of MSC under these conditions. Out of 10 cultures 3 showed growth under both oxygen tensions, whereas 7 displayed less tolerance to low oxygen tension of 1%.

Figure 2

Immunophenotyping profile of MSC under 21% O₂ (A) and 1% O₂ (B) by flow cytometric analysis of cell surface marker expression after 14 days showed no changes in immunophenotype of MSC due to varying oxygen concentrations (n = 3).

Figure 3

HIF-1α upregulation in response to low oxygen tension. Exposure of MSC to hypoxia resulted in a more than 3-fold increase of the HIF-1α expression as detected by semiquantitative RT-PCR. The relative index for HIF-1α was normalized against GAPDH relative intensity. Effect of reduced oxygen tension on MSC proliferation kinetics (A). Cell proliferation was measured by an increase in OD using the MTS assay after an incubation period of 7 days under 21% O₂, 5% O₂ (B), 3% O₂ (C), and 1% O₂ (D), respectively. Values indicate the mean and standard deviation for 3 cultures of each condition. Significance for all analysis was set at *** p < 0,001, ** p < 0,01, * p < 0,05 using student's t-test.

Figure 4

Flow cytometric cell cycle analysis of MSC under atmospheric and reduced oxygen conditions. Cells were permeabilized and stained with propidium iodide. DNA content-related cell cycle distribution of MSC after 7 days of incubation under 21% O₂ and 1% O₂ (n = 3).

Figure 5

Effect of hypoxia on chromosomal stability. Chromosomal aberrations were not detectable by high-resolution matrix-based comparative genomic hybridization after 4 weeks of MSC culture at 21% O₂ and 1% O₂. Except for typical polymorphisms, the DNA of MSC did not show any chromosomal abnormalities compared to freshly isolated DNA from PBMC of healthy volunteers (n = 3).

Figure 6

Effect of hypoxia on MSC plasticity. Oil-Red-O stain of MSC in adipogenic induction medium at atmospheric and reduced oxygen concentrations after 14 days of culture: (A) 21% O₂. control; (B) 21% O₂. adipogenic induction; (C) 1% O₂. control; (D) 1% O₂. adipogenic induction. Alizarin Red-S staining for the deposition of calcium precipitates is an indicator for the osteogenic differentiation. Viability and osteogenic differentiation of MSC from most donors was drastically reduced in this medium under 1% O₂ as compared to 21% O₂: (E) 21% O₂. control; (F) 21% O₂. osteogenic induction; (G) 1% O₂. control; (H) 1% O₂. osteogenic induction. However, a slight increase of oxygen to 3% allowed for efficient differentiation of MSC into osteoblasts: (I) 3% O₂. control; (J) 3% O₂. osteogenic induction. Three independent experiments of adipogenic and osteogenic differentiation were performed.