Severe hypoxia exerts parallel and cell-specific regulation of gene expression and alternative splicing in human mesenchymal stem cells

Abstract

BackgroundThe endosteum of the bone marrow provides a specialized hypoxic niche that may serve to preserve the integrity, pluripotency, longevity and stemness of resident mesenchymal stem cells (MSCs). To explore the molecular genetic consequences of such a niche we subjected human (h) MSCs to a pO2 of 4 mmHg and analyzed global gene expression and alternative splicing (AS) by genome-exon microarray and RT-qPCR, and phenotype by western blot and immunostaining.ResultsOut of 446 genes differentially regulated by >2.5-fold, down-regulated genes outnumbered up-regulated genes by 243:203. Exon analyses revealed 60 hypoxia-regulated AS events with splice indices (SI) >1.0 from 53 genes and a correlation between high SI and degree of transcript regulation. Parallel analyses of a publicly available AS study on human umbilical vein endothelial cells (HUVECs) showed that there was a strong cell-specific component with only 11 genes commonly regulated in hMSCs and HUVECs and 17 common differentially spliced genes. Only 3 genes were differentially responsive to hypoxia at the gene (>2.0) and AS levels in both cell types. Functional assignments revealed unique profiles of gene expression with complex regulation of differentiation, extracellular matrix, intermediate filament and metabolic marker genes. Antioxidant genes, striated muscle genes and insulin/IGF-1 signaling intermediates were down-regulated. There was a coordinate induction of 9 out of 12 acidic keratins that along with other epithelial and cell adhesion markers implies a partial mesenchymal to epithelial transition.ConclusionsWe conclude that severe hypoxia confers a quiescent phenotype in hMSCs that is reflected by both the transcriptome profile and gene-specific changes of splicosome actions. The results reveal that severe hypoxia imposes markedly different patterns of gene regulation of MSCs compared with more moderate hypoxia. This is the first study to report hypoxia-regulation of AS in stem/progenitor cells and the first molecular genetic characterization of MSC in a hypoxia-induced quiescent immobile state.

Leptin Signaling Is Required for Augmented Therapeutic Properties of Mesenchymal Stem Cells Conferred by Hypoxia Preconditioning

“…As one approach to investigate mechanism of HPC, we compared global gene expression profiles of normoxic versus hypoxia preconditioned hMSCs by microarray and found that leptin was the most strongly hypoxia‐induced gene . This is in agreement with another study that found leptin to be similarly induced by hypoxia in adipose tissue‐derived stem cells as well as with the known presence of active hypoxia‐induced factor 1α (HIF‐1α) binding elements in the leptin gene promoter .…”

Transcriptomics of human multipotent mesenchymal stromal cells: Retrospective analysis and future prospects

“…Comparative transcriptome analysis has been employed to investigate the cell response, particularly MSCs, in such 3D culture systems (Gatta et al, 2013;Hu et al, 2014;Kim et al, 2014;Pedersen et al, 2013;Roobrouck et al, 2011;Wu et al, 2012;Han et al, 2013; A C C E P T E D M A N U S C R I P T Jakobsen et al, 2014;Neuss et al, 2011). For example, a study by Han et on the scaffolds made with poly(lactic acid) than those made with poly(ɛ-caprolactone) (Neuss et al, 2011).…”

Navigating the bone marrow niche: translational insights and cancer-driven dysfunction

“…The endosteum also contains pre-osteoblastic cadherin-2-expressing cells, and has nerve and blood supplies, 49 although tese features are difficult to detect with plain-light microscopy. The heterogeneous environment of the endosteum contains scattered sites of microvascular infiltration associated with other regions of significant hypoxia, 50 which are highly attractive regions for HSC-homing. 51–53 Interestingly, quiescent HSCs tend to reside in areas of very low blood perfusion, whereas more proliferative HSCs with a lower reconstitution potential tend to prefer areas with greater blood flow.…”