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Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF.

ABSTRACT: Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed 'hypoxic-priming') efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB spheroids even under normoxic conditions. Indeed, hypoxia-primed EBs more efficiently differentiated into cells of vascular-lineage, than normoxic EBs did. We found that hypoxia suppressed Oct4 expression via direct binding of HIF-1 to reverse hypoxia-responsive elements (rHREs) in the Oct4 promoter. Furthermore, vascular endothelial growth factor (VEGF) was highly upregulated in hypoxia-primed EBs, which differentiated towards endothelial cells in the absence of exogenous VEGF. Interestingly, this differentiation was abolished by the HIF-1 or VEGF blocking. In vivo transplantation of hypoxia-primed EBs into mice ischemic limb elicited enhanced vessel differentiation. Collectively, our findings identify that hypoxia enhanced ESC differentiation by HIF-1-mediated inverse regulation of Oct4 and VEGF, which is a novel pathway to promote vascular-lineage differentiation.

SUBMITTER: Lee SW

PROVIDER: S-EPMC3491825 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF.

Lee Sae-Won SW Jeong Han-Kyul HK Lee Ji-Young JY Yang Jimin J Lee Eun Ju EJ Kim Su-Yeon SY Youn Seock-Won SW Lee Jaewon J Kim Woo Jean WJ Kim Kyu-Won KW Lim Jeong Mook JM Park Jong-Wan JW Park Young-Bae YB Kim Hyo-Soo HS

EMBO molecular medicine 20120723 9

Hypoxic microenvironment plays an important role in determining stem cell fates. However, it is controversial to which direction between self-renewal and differentiation the hypoxia drives the stem cells. Here, we investigated whether a short exposure to hypoxia (termed 'hypoxic-priming') efficiently directed and promoted mouse embryonic stem cells (mESCs) to differentiate into vascular-lineage. During spontaneous differentiation of embryoid bodies (EBs), hypoxic region was observed inside EB sp ...[more]

PMID: 22821840

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Project description:To study the mechanisms of pluripotency induction, we compared gene expression in pluripotent embryonic germ cells (EGCs) and unipotent primordial germ cells (PGCs). We found 11 genes ≥1.5-fold overexpressed in EGCs. None of the genes identified were Yamanaka's genes, but instead related to glycolytic metabolism. The prospect of pluripotency induction by cell metabolism manipulation was investigated by hypoxic culturing. We demonstrate that hypoxia alone induces reprogramming in PGCs, giving rise to hypoxia-induced EGC-like cells (hiEGL), which differentiate into cells of the three germ layers in vitro, and contribute to the ICM in vivo, demonstrating pluripotency. The mechanism of hypoxia induction involves HIF1? stabilization and Oct4 deregulation. However, hiEGL cannot be passaged long term. Self-renewal capacity is not achieved by hypoxia likely due to lack of upregulation of c-Myc and Klf4. Gene expression analysis of hypoxia signalling suggests that hiEGLs have not reached the stabilization phase of cell reprogramming. The data suggests that the properties of stemness, pluripotency and selfrenewal, are differentially regulated in primordial germ cell reprogramming induced by hypoxia. 8.5 dpc Oct4-GFP PGCs cultured in hypoxia conditions for 6 days were isolated by FACS. To eliminate the possibility of differences due solely to anaerobic respiration, TGC-10 EGC line was exposed to the hypoxia-mimetic agent Cl2Co at 150 microM for four hours prior to RNA isolation. RNA samples to be analyzed by microarrays were prepared using Qiagen RNeasy columns with on-column DNA digestion. 300 ng of total RNA per sample was used as input into a linear amplification protocol (Tetu-Bio), which involved synthesis of T7-linked double-stranded cDNA and 12 hours of in vitro transcription incorporating biotin-labelled nucleotides. Purified and labeled cRNA was then hybridized for 18h onto MouseRef-8 v2 expression BeadChips (Illumina) following the manufacturer's instructions. After washing, chips were stained with streptavidin-Cy3 (GE Healthcare) and scanned using the iScan reader (Illumina) and accompanying software. Samples were exclusively hybridized as biological replicates. The bead intensities were mapped to gene information using BeadStudio 3.2 (Illumina). Background correction was performed using the Affymetrix Robust Multi-array Analysis (RMA) background correction model. Variance stabilization was performed using the log2 scaling and gene expression normalization was calculated with the method implemented in the lumi package of RBioconductor. Data post-processing and graphics were performed with in-house developed functions in Matlab. Hierarchical clustering of genes and samples was performed with one minus correlation metric and the unweighted average distance (UPGMA) (also known as group average) linkage method.

Dataset Information

Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF.

Publications

Hypoxic priming of mESCs accelerates vascular-lineage differentiation through HIF1-mediated inverse regulation of Oct4 and VEGF.

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