Abstract
MicroRNAs are reported to have a crucial role in the regulation of self-renewal and differentiation of stem cells. Hypoxia has been identified as a key biophysical element of the stem cell culture milieu however, the link between hypoxia and miRNA expression in stem cells remains poorly understood. We therefore explored miRNA expression in hypoxic human embryonic and mesenchymal stem cells (hESCs and hMSCs). A total of 50 and 76 miRNAs were differentially regulated by hypoxia (2% O2) in hESCs and hMSCs, respectively, with a negligible overlap of only three miRNAs. We found coordinate regulation of precursor and mature miRNAs under hypoxia suggesting their regulation mainly at transcriptional level. Hypoxia response elements were located upstream of 97% of upregulated hypoxia regulated miRNAs (HRMs) suggesting hypoxia-inducible-factor (HIF) driven transcription. HIF binding to the candidate cis-elements of specific miRNAs under hypoxia was confirmed by Chromatin immunoprecipitation coupled with qPCR. Role analysis of a subset of upregulated HRMs identified linkage to reported inhibition of differentiation while a downregulated subset of HRMs had a putative role in the promotion of differentiation. MiRNA-target prediction correlation with published hypoxic hESC and hMSC gene expression profiles revealed HRM target genes enriched in the cytokine:cytokine receptor, HIF signalling and pathways in cancer. Overall, our study reveals, novel and distinct hypoxia-driven miRNA signatures in hESCs and hMSCs with the potential for application in optimised culture and differentiation models for both therapeutic application and improved understanding of stem cell biology.
Highlights
Human embryonic and mesenchymal stem cell precursors are thought to reside in physiologically hypoxic environments [1, 2]
To explore uniformity of hypoxic miRNA expression modulation in stem cell populations we explored hMSC isolated from bone marrow
We found that levels of both miRNA biogenesis factors are downregulated under hypoxia in hESCs and hMSCs (S1c and S2c Figs)
Summary
Human embryonic and mesenchymal stem cell (hESCs and hMSCs, respectively) precursors are thought to reside in physiologically hypoxic environments [1, 2]. In vitro experimentation has established that hypoxic culture of hESC correlates closely with increased clonogenicity, reduced spontaneous differentiation, increased genetic stability, and transcriptional homogeneity alongside improved epigenetic profiles [4, 5, 6, 7] Coupled to this it is reported that the utilisation of hypoxic culture conditions in the recovery of hMSC from bone marrow, and other tissues e.g. fat, results in dramatic improvements in individual stem cell yield (via Colony-Forming Unit- Fibroblastic quantification), enhanced scale-up, and reduced transcriptional alteration (vs normoxic cultured (21% O2) cells) [8]. Of clinical relevance recent reports have suggested that ex-vivo hypoxic pre-conditioning of MSCs results in enhanced survival post-transplant via resistance to intrinsic and extrinsic death signals [3] Taken together these investigations suggest that either control of modulation of hypoxic signalling, or hypoxia-regulated genes could be of benefit to the regenerative medicine industry. This identifies a need to identify novel players in hypoxic signalling that may serve as candidates for the enhancement of stem cell based therapies
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