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.
Highlights
The 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)
We report for the first time a coordinate increase in expression of acidic keratins perhaps indicating a partial mesenchymal to epithelial transition (MET), a decrease in insulin/IGF-1 signaling with lower phosphor-Akt, and decreased expression of anti-oxidant-related genes that suggests lower metabolism and growth compared with aerobic culture
Isolation and characterization of human MSCs Human bone marrow MSCs were isolated as described in Methods and used at passage 8
Summary
The 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). The stem cell niche refers to a well-defined physiological compartment that includes cellular and acellular components and serves to integrate systemic and local signals to regulate the biology of stem cells (reviewed in [1,2]). Like other such niches, the bone marrow provides highly specialized and heterogeneous microenvironments that determine the self-renewal, multipotency, survival and migration of residing hematopoietic and progenitor cells including mesenchymal stem cells (MSCs). Studies on bone marrow or adipose derived MSCs have shown that moderate hypoxic culture equivalent to the central BM sinusoidal niche enhances proliferation and protects against senescence while more severe hypoxia may block proliferation and induce cell death [9,10,11,12,13,14,15,16]
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