Expression data from bovine bone-marrow derived MSCs following preconditioning (hypoxia and/or transforming growth factor-beta (TGF-β))

The innate repair and regeneration potential of skeletal tissues such as the intervertebral disc and articular cartilage is extremely limited, in part due to their avascularity and low cell density. Despite recent advances in MSC-based disc and cartilage regeneration, key challenges remain, including the sensitivity of these cells to in vivo microenvironmental stress such as low oxygen and nutrient levels. The objective of this study was to investigate whether preconditioning with hypoxia and/or transforming growth factor-beta (TGF-β) can enhance MSC survival and extracellular matrix production in a low oxygen and nutrient-limited microenvironment. Secondarily, the effects of donor variability on the response of MSCs to preconditioning was examined. MSCs from multiple bovine donors were preconditioned in monolayer in normoxia or hypoxia, with or without TGF-β. The effects of preconditioning on MSC gene expression during monolayer expansion were examined using microarrays. Bone marrow-derived MSCs were isolated from juvenile (age < 6 months) bovine femurs and tibiae. Following the initial isolation, MSCs were expanded to confluence through a single initial passage in standard tissue culture conditions with normal oxygen tension (atmospheric, 21% O2), 5% CO2, in basal medium comprised of high glucose (4.5 g/L) Dulbecco’s Modified Eagle medium (DMEM; ThermoFisher Scientific; Waltham, MA), 10% fetal bovine serum (FBS; ThermoFisher Scientific; Waltham, MA), and 1% penicillin, streptomycin, and fungizone (PSF; ThermoFisher Scientific; Waltham, MA). Following primary expansion and preconditioning (n = 3 donors/biological replicates x 4 expansion conditions for a total of 12 samples), MSCs were harvested and RNA was isolated from each samples. The quality of each RNA sample (RIN > 9) was verified using an Agilent BioAnalyzer and RNA 6000 Pico Kit (Agilent; Santa Clara, CA). Global gene expression was measured using the WTPlus Bovine Gene Chip (Affymetrix, Santa Clara, CA). Gene expression data were normalized using Robust Multi-array Average (Irizarry et al., 2003) and adjusted for false discovery rate to generate adjusted p-values.