Department of Biochemical Engineering, The Advanced Centre for Biochemical Engineering, University College London, London, UK.
Stem Cells Dev. 2012 May 20;21(8):1344-55. doi: 10.1089/scd.2011.0225. Epub 2011 Oct 27.
The efficient differentiation of retinal cells from human pluripotent stem cells remains a major challenge for the development of successful and cost-effective cellular therapies for various forms of blindness. Current differentiation strategies rely on exposing pluripotent stem cells to soluble growth factors that play key roles during early development (such as DKK-1, Noggin, and IGF-1) at 20% oxygen (O(2)). This O(2) tension is, however, considerably higher than O(2) levels during organogenesis and may impair the differentiation process. In this study, we examined the effect of mimicking the physiological O(2) tension (2%) on the generation of retinal progenitor cells (RPCs) from human induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs). Both cell types were induced to differentiate into RPCs at 20% and 2% O(2). After 3 days in suspension culture as embryoid bodies (EBs), 2% O(2) caused the activation of hypoxia inducible factor responsive genes VEGF and LDHA and was accompanied by elevated expression levels of the early eye field genes Six3 and Lhx2. Twenty-one days after plating EBs in an adherent culture, we observed more RPCs co-expressing Pax6 and Chx10 at 2% O(2). Quantitative polymerase chain reaction analysis confirmed that lowering O(2) tension had caused a rise in the expression of both genes compared with 20% O(2). Our results indicate that mimicking physiological O(2) is a favorable condition for the efficient generation of RPCs from both hiPSCs and hESCs.
从人类多能干细胞高效分化出视网膜细胞仍然是开发各种形式致盲的有效且经济的细胞疗法的主要挑战。目前的分化策略依赖于将多能干细胞暴露于在早期发育过程中发挥关键作用的可溶性生长因子（如 DKK-1、Noggin 和 IGF-1），并在 20%的氧气（O2）中培养。然而，这种氧张力比器官发生过程中的氧水平高得多，可能会损害分化过程。在这项研究中，我们研究了模拟生理氧张力（2%）对人诱导多能干细胞（iPSCs）和人胚胎干细胞（hESCs）生成视网膜祖细胞（RPCs）的影响。这两种细胞类型均在 20%和 2%O2 下诱导分化为 RPCs。在悬浮培养形成类胚体（EBs）3 天后，2%O2 会激活缺氧诱导因子反应基因 VEGF 和 LDHA，并伴有早期眼场基因 Six3 和 Lhx2 的表达水平升高。在将 EBs 贴壁培养 21 天后，我们观察到在 2%O2 下有更多共同表达 Pax6 和 Chx10 的 RPCs。定量聚合酶链反应分析证实，与 20%O2 相比，降低氧张力会导致这两个基因的表达增加。我们的结果表明，模拟生理氧条件有利于从 hiPSCs 和 hESCs 高效生成 RPCs。
