Responses to altered oxygen tension are distinct between human stem cells of high and low chondrogenic capacity

Abstract

Background: Lowering oxygen from atmospheric level (hyperoxia) to the physiological level (physioxia) of articular cartilage promotes mesenchymal stem cell (MSC) chondrogenesis. However, the literature is equivocal regarding the benefits of physioxic culture on preventing hypertrophy of MSC-derived chondrocytes. Articular cartilage progenitors (ACPs) undergo chondrogenic differentiation with reduced hypertrophy marker expression in hyperoxia but have not been studied in physioxia. This study sought to delineate the effects of physioxic culture on both cell types undergoing chondrogenesis.

Methods: MSCs were isolated from human bone marrow aspirates and ACP clones were isolated from healthy human cartilage. Cells were differentiated in pellet culture in physioxia (2 % oxygen) or hyperoxia (20 % oxygen) over 14 days. Chondrogenesis was characterized by biochemical assays and gene and protein expression analysis.

Results: MSC preparations and ACP clones of high intrinsic chondrogenicity (termed high-GAG) produced abundant matrix in hyperoxia and physioxia. Poorly chondrogenic cells (low-GAG) demonstrated a significant fold-change matrix increase in physioxia. Both high-GAG and low-GAG groups of MSCs and ACPs significantly upregulated chondrogenic genes; however, only high-GAG groups had a concomitant decrease in hypertrophy-related genes. High-GAG MSCs upregulated many common hypoxia-responsive genes in physioxia while low-GAG cells downregulated most of these genes. In physioxia, high-GAG MSCs and ACPs produced comparable type II collagen but less type I collagen than those in hyperoxia. Type X collagen was detectable in some ACP pellets in hyperoxia but reduced or absent in physioxia. In contrast, type X collagen was detectable in all MSC preparations in hyperoxia and physioxia.

Conclusions: MSC preparations and ACP clones had a wide range of chondrogenicity between donors. Physioxia significantly enhanced the chondrogenic potential of both ACPs and MSCs compared with hyperoxia, but the magnitude of response was inversely related to intrinsic chondrogenic potential. Discrepancies in the literature regarding MSC hypertrophy in physioxia can be explained by the use of low numbers of preparations of variable chondrogenicity. Physioxic differentiation of MSC preparations of high chondrogenicity significantly decreased hypertrophy-related genes but still produced type X collagen protein. Highly chondrogenic ACP clones had significantly lower hypertrophic gene levels, and there was little to no type X collagen protein in physioxia, emphasizing the potential advantage of these cells.

Keywords: Articular cartilage progenitor cell; Chondrogenesis; Hypertrophy; Hypoxia; Mesenchymal stem cell; Pellet culture; Physioxia.

Fig. 1

a Total glycosaminoglycan (GAG) production per pellet for each MSC preparation and ACP clone indicated variation among human donors. A threshold (dashed line) defined as two standard deviations from the total GAG production during pellet chondrogenesis for healthy human chondrocytes was set for each cell type, and low-GAG and high-GAG groups were categorized. b Mean (± SD) fold-change GAG production in physioxia relative to hyperoxia was significant for all groups other than high-GAG ACPs, and a significant difference existed between the fold-change for low-GAG and high-GAG groups of each cell type. c Mean (± SD) fold-change DNA content in physioxia relative to hyperoxia was no different between oxygen level, GAG level, nor cell type. Statistical significance defined as *p < 0.05, ***p < 0.001. ACP articular cartilage progenitor, MSC mesenchymal stem cell

Fig. 2

a Representative toluidine blue stain for total proteoglycans demonstrates smaller pellets with less metachromasia for low-GAG MSC preparations and ACP clones in both hyperoxia and physioxia relative to paired high-GAG MSC preparations and ACP clones at the respective oxygen levels. Images were acquired with bright-field microscopy, scale bars = 100 μm. b Measurement of pellet diameter revealed a statistically significant difference in pellet size between both MSCs and ACPs of high or low chondrogenicity and between high-GAG MSCs at physioxia or hyperoxia. Statistical significance defined as *p < 0.05, **p < 0.01, ****p < 0.0001. ACP articular cartilage progenitor, GAG glycosaminoglycan, MSC mesenchymal stem cell

Fig. 3

Gene expression analysis for fold-change of chondrogenic markers of the articular cartilage phenotype (COL2A1, ACAN), the fibrocartilaginous phenotype (COL1A1), and the hypertrophic phenotype (COL10A1, MMP13) demonstrates varied chondrogenic responses by high-GAG and low-GAG groups of each cell type, MSCs and ACPs, during pellet culture in physioxic relative to hyperoxic conditions. Data are mean ± standard deviation of fold-change for each group (n = 6–10). Statistical significance defined as *p < 0.05 by a paired or unpaired t test where appropriate. # p < 0.05 between low and high GAG pellets. ACP articular cartilage progenitor, GAG glycosaminoglycan, MSC mesenchymal stem cell

Fig. 4

Clustering of oxygen-dependent gene expression based on z-score demonstrates that groups of MSCs and ACPs are more similar between GAG level than within cell type in response to culture in physioxia relative to culture in hyperoxia. ACP articular cartilage progenitor, GAG glycosaminoglycan, MSC mesenchymal stem cell

Fig. 5

Gene expression analysis for fold-change of chondrogenic markers in physioxia relative to hyperoxia demonstrates that high-GAG groups of both MSCs and ACPs are highly responsive to oxygen level and upregulate a majority of genes representative of the articular cartilage phenotype in low-oxygen environments. Data are mean ± standard deviation of fold-change in gene expression for each group (n = 8). Statistical significance defined as *p < 0.05 by a paired t test for normal data and a Wilcoxon matched-pairs signed rank test for nonnormal data. ACP articular cartilage progenitor, MSC mesenchymal stem cell

Fig. 6

Representative immunohistochemistry demonstrates that cartilage pellets cultured in hyperoxia (20 % oxygen) and physioxia (2 % oxygen) exhibit oxygen-dependent expression of extracellular collagen protein, including type II collagen (a–d) of the articular cartilage phenotype, type I collagen (e–h) of the fibrocartilaginous phenotype, and type X collagen (i–n) of the hypertrophic phenotype. Type X collagen expression was variable among ACP clones differentiated in hyperoxia (k, m). Nuclei were counterstained with DAPI, and composite images of collagen staining and nuclei were digitally merged using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Negative controls with isotype-matched antibodies were used for background correction (images not shown). Scale bars = 100 μm. ACP articular cartilage progenitor, MSC mesenchymal stem cell