Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.
Hypoxia enhances differentiation of mouse embryonic stem cells into definitive endoderm and distal lung cells.
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DOI：
10.1089/scd.2014.0343
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发表时间：
2015-03
影响因子：
4
通讯作者：
P. Pimton;S. Lecht;Collin T. Stabler;G. Johannes;E. Schulman;P. Lelkes
中科院分区：
文献类型：
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作者：
P. Pimton;S. Lecht;Collin T. Stabler;G. Johannes;E. Schulman;P. Lelkes
我们研究了缺氧对小鼠胚胎干细胞 (mESC) 自发 (SP) 和激活素 A (AA) 诱导的定形内胚层 (DE) 分化及其随后分化为远端肺上皮细胞的影响。在 1% O2 缺氧条件下，mESC 向内胚层进行 6 天的 SP 分化，但在 3% 或 21%（常氧）条件下则不然，Sox17 和 Foxa2 的表达分别比维持培养物增加了 31 倍和 63 倍。在缺氧条件下用 20 ng/mL AA 处理 mESC 6 天，DE 标记基因 Sox17、Foxa2 和 Cxcr4 的表达分别比维持培养物高 501、1,483 和 126 倍。短至 24 小时的短暂缺氧足以增强 AA 诱导的内胚层形成。使用 HIF-1α(-/-) mESC 和 ROS 清除剂 N-乙酰半胱氨酸 (NAC) 评估缺氧诱导因子 (HIF)-1α 和活性氧 (ROS) 在 AA 诱导的内胚层富集中的参与。在SP条件下，HIF-1α(-/-) mESCs未能增加内胚层标记基因的表达，而是向外胚层转移。缺氧仅诱导 HIF-1α(-/-) mESC 中 AA 诱导的内胚层分化的边际增强。用 AA 和 NAC 处理 mESC 会导致 Sox17 和 Foxa2 表达呈剂量依赖性下降。此外，在最近报道的肺分化方案过程中暴露于缺氧的持续时间导致I型、II型和俱乐部细胞的远端肺上皮细胞标记基因水通道蛋白5 (Aqp5)、表面活性剂蛋白C (Sftpc)和分泌球蛋白1a1 (Scgb1a1)的表达分别差异增强。我们的研究首次展示了体外缺氧对 DE 和肺谱系有效形成的影响。我们认为，缺氧程度和仔细的时机可能是体外分化生物过程的重要组成部分，用于从多能祖细胞分化产生远端肺上皮细胞。
We investigated the effects of hypoxia on spontaneous (SP)- and activin A (AA)-induced definitive endoderm (DE) differentiation of mouse embryonic stem cells (mESCs) and their subsequent differentiation into distal pulmonary epithelial cells. SP differentiation for 6 days of mESCs toward endoderm at hypoxia of 1% O2, but not at 3% or 21% (normoxia), increased the expression of Sox17 and Foxa2 by 31- and 63-fold above maintenance culture, respectively. Treatment of mESCs with 20 ng/mL AA for 6 days under hypoxia further increased the expression of DE marker genes Sox17, Foxa2, and Cxcr4 by 501-, 1,483-, and 126-fold above maintenance cultures, respectively. Transient exposure to hypoxia, as short as 24 h, was sufficient to enhance AA-induced endoderm formation. The involvement of hypoxia-inducible factor (HIF)-1α and reactive oxygen species (ROS) in the AA-induced endoderm enrichment was assessed using HIF-1α(-/-) mESCs and the ROS scavenger N-acetylcysteine (NAC). Under SP conditions, HIF-1α(-/-) mESCs failed to increase the expression of endodermal marker genes but rather shifted toward ectoderm. Hypoxia induced only a marginal potentiation of AA-induced endoderm differentiation in HIF-1α(-/-) mESCs. Treatment of mESCs with AA and NAC led to a dose-dependent decrease in Sox17 and Foxa2 expression. In addition, the duration of exposure to hypoxia in the course of a recently reported lung differentiation protocol resulted in differentially enhanced expression of distal lung epithelial cell marker genes aquaporin 5 (Aqp5), surfactant protein C (Sftpc), and secretoglobin 1a1 (Scgb1a1) for alveolar epithelium type I, type II, and club cells, respectively. Our study is the first to show the effects of in vitro hypoxia on efficient formation of DE and lung lineages. We suggest that the extent of hypoxia and careful timing may be important components of in vitro differentiation bioprocesses for the differential generation of distal lung epithelial cells from pluripotent progenitors.