Comparing atmospheric and hypoxic cultured mesenchymal stem cell transcriptome: Implication for stem cell therapies targeting intervertebral discs

Abstract

Background: Mesenchymal stem cells (MSCs) represent an attractive avenue for cellular therapies targeting degenerative diseases. MSC in vitro expansion is required in order to obtain therapeutic numbers during the manufacturing process. It is known that culture conditions impact cellular properties and behavior after in vivo transplantation. In this study, we aimed at evaluating the benefit of hypoxic culturing of human bone marrow derived mesenchymal stem cells on cell fitness and whole genome expression and discussed its implication on cellular therapies targeting orthopedic diseases such as chronic lower back pain. Methods: Human bone marrow mesenchymal stem cells (hBMMSCs) were isolated from fresh human anticoagulated whole bone marrow and were cultured side by side in atmospheric (20% O 2 ) and hypoxic (5% O 2 ) oxygen partial pressure for up to 3 passages. Stem cell fitness was assessed by clonogenic assay, cell surface marker expression and differentiation potential. Whole genome expression was performed by mRNA sequencing. Data from clonogenic assays, cell surface marker by flow cytometry and gene expression by quantitative PCR were analyzed by two-tailed paired Student's t-test. Data from mRNA sequencing were aligned to hg19 using Tophat-2.0.13 and analyzed using Cufflinks-2.1.1. Results: Hypoxic culturing of hBMMSCs had positive effects on cell fitness, as evidenced by an increased clonogenicity and improved differentiation potential towards adipocyte and chondrocyte lineages. No difference in osteoblast differentiation or in cell surface markers were observed. Only a small subset of genes (34) were identified by mRNA sequencing to be significantly dysregulated by hypoxia. When clustered by biological function, these genes were associated with chondrogenesis and cartilage metabolism, inflammation and immunomodulation, cellular survival, migration and proliferation, vasculogenesis and angiogenesis. Conclusions: Hypoxic culturing positively impacted hBMMSCs fitness and transcriptome, potentially improving inherent properties of these cells that are critical for the development of successful cellular therapies. Hypoxic culturing should be considered for the in vitro expansion of hBMMSCs during manufacturing of cellular therapies targeting orthopedic disorders such as lower back pain.