Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.
Hypoxia inducible factors regulate pluripotency and proliferation in human embryonic stem cells cultured at reduced oxygen tensions.
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DOI：
10.1530/rep-09-0300
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发表时间：
2010-01
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影响因子：
--
通讯作者：
Houghton FD
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文献类型：
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作者：
Forristal CE;Wright KL;Hanley NA;Oreffo RO;Houghton FD
人胚胎干细胞（hES）通常在大气、20%氧张力下培养，但来源于存在于3-5%氧（低氧）环境中的胚胎。维持氧稳态对于确保氧依赖性过程的足够水平至关重要。本研究探讨了特异性缺氧诱导因子（HIF）在调节hES细胞缺氧反应中的重要性。我们报告，在20%的氧气培养降低hES细胞增殖，并导致SOX 2，NANOG和POU 5 F1（OCT 4）mRNA的表达显着减少，以及POU 5 F1蛋白相比，缺氧条件。HIF 1A蛋白在20%氧下不表达，在5%氧下仅显示短暂的核定位。HIF 2A（EPAS 1）和HIF 3A显示在初始缺氧培养过程中的细胞质定位，但在5%氧下长期培养后易位到细胞核，并且与在20%氧下培养的细胞相比显著上调。HIF 2A的沉默导致hES细胞增殖和POU 5 F1、SOX 2和NANOG蛋白表达的显著降低，而早期分化标志物SSEA 1伴随增加。HIF 3A上调HIF 2A并抑制HIF 1A的表达，而HIF 3A的敲低导致HIF 1A蛋白的重新出现。总之，这些数据表明低氧张力对于维持高度增殖的多能hES细胞群是优先的。虽然发现HIF 3A调节HIF 1A和HIF 2A的表达，但HIF 2A调节hES细胞多能性以及缺氧条件下的增殖。
Human embryonic stem (hES) cells are routinely cultured under atmospheric, 20% oxygen tensions but are derived from embryos which reside in a 3–5% oxygen (hypoxic) environment. Maintenance of oxygen homeostasis is critical to ensure sufficient levels for oxygen-dependent processes. This study investigates the importance of specific hypoxia inducible factors (HIFs) in regulating the hypoxic responses of hES cells. We report that culture at 20% oxygen decreased hES cell proliferation and resulted in a significantly reduced expression of SOX2, NANOG and POU5F1 (OCT4) mRNA as well as POU5F1 protein compared with hypoxic conditions. HIF1A protein was not expressed at 20% oxygen and displayed only a transient, nuclear localisation at 5% oxygen. HIF2A (EPAS1) and HIF3A displayed a cytoplasmic localisation during initial hypoxic culture but translocated to the nucleus following long-term culture at 5% oxygen and were significantly upregulated compared with cells cultured at 20% oxygen. Silencing of HIF2A resulted in a significant decrease in both hES cell proliferation and POU5F1, SOX2 and NANOG protein expression while the early differentiation marker, SSEA1, was concomitantly increased. HIF3A upregulated HIF2A and prevented HIF1A expression with the knockdown of HIF3A resulting in the reappearance of HIF1A protein. In summary, these data demonstrate that a low oxygen tension is preferential for the maintenance of a highly proliferative, pluripotent population of hES cells. While HIF3A was found to regulate the expression of both HIF1A and HIF2A, it is HIF2A which regulates hES cell pluripotency as well as proliferation under hypoxic conditions.