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

Abstract

BackgroundLowering 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.MethodsMSCs 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.ResultsMSC 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.ConclusionsMSC 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0419-8) contains supplementary material, which is available to authorized users.

Differential Production of Cartilage ECM in 3D Agarose Constructs by Equine Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells

“…Another positive effect of hypoxia was the significant reduction in ALP activity in MSC and co-culture constructs, thereby further increasing the quality of the cartilage tissue formed by MSCs. A downregulation of hypertrophic markers and terminal differentiation of MSCs by hypoxia has also been documented in previous studies [ 52 , 55 , 56 , 59 , 60 ]. In the present study, furthermore, PRG4 gene expression was significantly upregulated in ACPCs under hypoxia on day 14 and day 28; however, it was significantly downregulated in co-culture constructs under hypoxia on both days.…”

“…It seemed as if hypoxia enabled MSCs to suppress PRG4 expression of ACPCs in co-cultures, which might not be a desirable effect, but is still worthwhile investigating in further studies. With regard to ACPCs, only a few studies have investigated chondrogenic differentiation under hypoxia before [ 25 , 59 ]. When cultured in self-organized constructs on fibronectin-coated membranes, human ACPCs, in contrast to articular chondrocytes, showed only minor responses to reduced oxygen tension (5 %) [ 25 ].…”

Physioxia stimulates extra-cellular matrix deposition and increases mechanical properties of human chondrocyte-derived tissue-engineered cartilage

“…The advantages of a well characterized allogenic cell bank are clear given the range of GAG and collagen contents due to donor variability. In chondrocyte progenitor experiments, this has been further focused in on showing clonal variability [40] . Interestingly, while there was a wide range of extracellular matrix component concentrations detected, the distribution in mechanical properties was actually relatively narrow.…”

Physiological Hypoxia (Physioxia) Impairs the Early Adhesion of Single Lymphoma Cell to Marrow Stromal Cell and Extracellular Matrix. Optical Tweezers Study

“…However, recently observed rapid development of three-dimensional (3D) cancer models re-emphasizes the need to mimic the tumor microenvironment, including the oxygen tension, which has been considered as the limiting nutrient for 3D cell cultures [ 43 , 44 ]. Another important research trend on physioxia observed lately is human fibroblast [ 45 ] and steam cells biology [ 46 , 47 , 48 ].…”