Background

Under hypoxia, mouse embryonic stem cells (mESCs) lose the ability to self-renew and begin to differentiate through down-regulation of LIFR-STAT3 pathway via hypoxia-inducible factor-1α (HIF-1α). However, it remains largely unknown what kinds of factors are involved in hypoxia-induced differentiation of mESCs.

Purpose

This study aims to identify the differentially expressed genes (DEGs) in early differentiation of mESCs under hypoxia.

Methods

Here we utilized a Genefishing technique^TM to discover the new DEGs during hypoxia-induced early differentiation in CCE mESCs. Next, we investigated the role of DEGs using morphological observation, alkaline phosphatase (ALP) assay, STAT3 activation analysis, and biomarkers analysis for stemness.

Results

We detected 19 DEGs under hypoxia and performed cloning with sequencing in six genes. We confirmed the expression patterns of five DEGs including H2afz and GOT1 by realtime PCR assay. Among them, H2afz was significantly decreased under hypoxia, depending on HIF-1α. H2afz-overexpressing CCE mESCs maintained their ALP activity and stem cell markers (Nanog and Rex1), even in hypoxic condition. On the other hand, the early differentiation markers such as FGF5 and STAT5a, which had been increased in hypoxic conditions, were reduced by H2afz overexpression.

Conclusion

We discovered that H2afz could be a new target gene that functions in hypoxia-induced differentiation in mESCs and have revealed that it is involved in maintaining the pluripotency of mESCs in the early stages of differentiation. These findings will provide insights into mechanisms of hypoxia-mediated differentiation of mESCs during early development.

中文翻译：

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缺氧条件下小鼠胚胎干细胞差异表达基因的鉴定

背景

在缺氧条件下，小鼠胚胎干细胞（mESCs）失去自我更新的能力，并通过缺氧诱导因子-1α（HIF-1α）下调LIFR-STAT3通路开始分化。然而，在缺氧诱导的 mESCs 分化中涉及哪些因素仍然很大程度上未知。

目的

本研究旨在鉴定低氧条件下mESCs早期分化的差异表达基因（DEGs）。

方法

在这里，我们利用 Genefishing 技术^TM在缺氧诱导的 CCE mESCs 早期分化过程中发现了新的 DEG。接下来，我们使用形态学观察、碱性磷酸酶 (ALP) 测定、STAT3 激活分析和干性生物标志物分析研究了 DEG 的作用。

结果

我们在缺氧条件下检测到 19 个 DEG，并通过测序对 6 个基因进行了克隆。我们通过实时 PCR 分析确认了包括 H2afz 和 GOT1 在内的五个 DEG 的表达模式。其中，H2afz在缺氧条件下显着降低，这取决于HIF-1α。即使在缺氧条件下，过表达 H2afz 的 CCE mESC 也能保持其 ALP 活性和干细胞标志物（Nanog 和 Rex1）。另一方面，在缺氧条件下增加的早期分化标志物如 FGF5 和 STAT5a 因 H2afz 过表达而减少。

结论

我们发现 H2afz 可能是一个新的靶基因，在 mESCs 的缺氧诱导分化中起作用，并揭示它在分化的早期参与维持 mESCs 的多能性。这些发现将提供对早期发育过程中缺氧介导的 mESCs 分化机制的见解。