Open Access Research article

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

Xinyang Hu1, Rongrong Wu1, Lina A Shehadeh234, Qing Zhou6, Cizhong Jiang6, Xin Huang1, Ling Zhang1, Feng Gao1, Xianbao Liu1, Hong Yu14, Keith A Webster345* and Jian’an Wang1*

Author Affiliations

1 Cardiovascular Key Lab of Zhejiang Province, Department of Cardiology, The Second Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou 310009, P.R. China

2 Department of Medicine, Division of Cardiology, University of Miami Leonard M. Miller School of Medicine, Miami, Florida, USA

3 Interdisciplinary Stem Cell Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida, USA

4 Vascular Biology Institute, University of Miami Leonard M. Miller School of Medicine, Miami, Florida, USA

5 Department of Molecular and Cellular Pharmacology, University of Miami Leonard M. Miller School of Medicine, Miami, FL 33101, USA

6 School of Life Sciences and Technology, Tongji University, Shanghai 200092, PR China

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BMC Genomics 2014, 15:303  doi:10.1186/1471-2164-15-303

Published: 23 April 2014

Abstract

Background

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). 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.

Results

Out 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.

Conclusions

We 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.

Keywords:
Hypoxia; Microarray; Alternative splicing; Stem cell Niche