Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles

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

Pluripotent and Multipotent Stem Cells Display Distinct Hypoxic miRNA Expression Profiles

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

Transcriptional regulation of HRMs.

<p>ChIP assay was performed using antibodies against HIF-α or IgG to confirm the binding of HIF-1 α on the putative HREs in the promoters of specific HRMs. qPCR data showing fold enrichment of bound chromatin compared with input in hMSCs grown in <b>(a)</b> hypoxic or <b>(c)</b> normoxic conditions and hESCs in <b>(b)</b> hypoxic and <b>(d)</b> normoxic conditions. <b>(e)</b> Putative hypoxia signal transduction feedback loop in hESC and hMSCs.</p

HREs location in HRM promoters.

<p>The 5kb upstream region of hypoxia induced miRNAs was screened for the presence of HREs in their promoter using PROMO prediction program. Figure showing relative positions of predicted HREs in the promoter region.</p

Co-regulation of miRNA clusters by hypoxia.

<p>Tables representing hypoxia up-regulated and down-regulated miRNA clusters in <b>(a)</b> SHEF1 cells, <b>(b)</b> bone marrow derived MSCs, and <b>(c)</b> preferential induction of the distinct members of the same cluster by hypoxia.</p

Target identification of hypoxia regulated miRNAs.

<p><b>(a)</b> Venn diagram of up and down-regulated genes in response to hypoxia in hESC and hMSCs. <b>(b, c)</b> Putative targets HRMs based on the inverse correlation of their expression with previously published hypoxia-induced differential gene expression of hESCs and MSCs, respectively, under identical conditions.</p

The miRNA signature of hypoxia in hMSCs.

<p>Hierarchical clustering of hypoxia <b>(a)</b> induced and <b>(b)</b> down-regulated miRNAs (> 2 fold, p<0.05) in response to hypoxia (2% O₂) in human bone marrow derived MSCs. Quantitative RT-PCR data of upregulated <b>(c)</b> mature miRNAs and <b>(e)</b> pre-miRNAs and downregulated <b>(d)</b> mature miRNAs and <b>(f)</b> pre-miRs of HRMs in human bone-marrow derived MSCs. The graphical data points in c, d, e and f represent mean ± S.D. of at least three independent experiments. (*P>0.01 and <0.05, **P<0.01).</p

The miRNA signature of hypoxia in hESCs.

<p>Hierarchical clustering of hypoxia <b>(a)</b> induced and <b>(b)</b> down-regulated miRNAs (> 2 fold, p<0.05) in response to hypoxia (2% O₂) in SHEF1 hESC. Quantitative RT-PCR data of upregulated <b>(c)</b> mature miRNAs & <b>(e)</b> pre-miRNAs and downregulated <b>(d)</b> mature miRNAs & <b>(f)</b> pre-miRs of HRMs in SHEF1 cell line. Graphical data points in c, d, e and f represent mean ± S.D. of a minimum of three independent experiments. (*P>0.01 and <0.05, **P<0.01).</p

Pathway analysis of hypoxia regulated miRNAs.

<p>Pathway association of up and down-regulated HRM in hESC and hMSCs, <b>(a, b)</b> respectively. A figure showing miRNA:target gene interaction network for cytokine:cytokine receptor pathway of MSCs drawn using Cytoscape software <b>(c).</b> The green colour indicates up-regulation whereas the red colour refers to down-regulation.</p