Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.
Modeling early stages of endoderm development in epiblast stem cell aggregates with supply of extracellular matrices.
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在提供细胞外基质的情况下模拟外胚层干细胞聚集体内胚层发育的早期阶段。
DOI：
10.1111/dgd.12663
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Kondoh H.
中科院分区：
文献类型：
--
作者：
Inamori S;Fujii M;Satake S;Iida H;Teramoto M;Sumi T;Meno C;Ishii Y;Kondoh H.
表达FoxA 2和Sox 17的内胚层前体细胞从上胚层通过原肠胚形成过程发育而来。在这项研究中，我们开发了一个实验系统，使用外胚层干细胞（EpiSC）模拟内胚层生成原肠胚形成过程。为此，我们建立了EpiSC系i22，其中增强型绿色荧光蛋白与Foxa 2共表达。将i22 EpiSC作为聚集体培养几天足以启动Foxa 2表达，并且在Matrigel中进一步培养聚集体促进了参与内胚层前体发育的转录因子基因的顺序激活，例如，Eomes、Gsc和Sox 17。在i22细胞的聚集培养3天中，所有细胞均表达POU 5 F1、SOX 2和E-钙粘蛋白（上胚层的特征），而GATA 4和SOX 17的表达在分散的细胞中也被中度激活，表明这些细胞启动了内胚层发育。将聚集体包埋在Matrigel中另外3天引起细胞迁移到覆盖聚集体的富含层粘连蛋白的基质的腔中，其中FOXA 2和SOX 17以高水平表达，伴随着E-钙粘蛋白的损失，表明内胚层前体的迁移阶段。对聚集体的长期培养产生了原肠胚形成后阶段胚胎中发现的三种分离细胞群：（1）共表达高水平SOX 17、GATA 4和E-钙粘蛋白的定形内胚层，（2）表达低水平GATA 4且缺乏E-钙粘蛋白的中胚层细胞，以及（3）表达POU 5 F1、SOX 2且不含E-钙粘蛋白的致敏上胚层细胞。因此，EpiSC聚集，然后将聚集体包埋在富含层粘连蛋白的基质中，模拟原肠胚形成依赖性内胚层前体发育。
Endoderm precursors expressingFoxA2andSox17develop from the epiblast through the gastrulation process. In this study, we developed an experimental system to model the endoderm‐generating gastrulation process using epiblast stem cells (EpiSCs). To this end, we established an EpiSC line i22, in which enhanced green fluorescent protein is coexpressed withFoxa2. Culturing i22 EpiSCs as aggregates for a few days was sufficient to initiateFoxa2expression, and further culturing of the aggregates in Matrigel promoted the sequential activation of transcription factor genes involved in endoderm precursor development, e.g.,Eomes,Gsc, andSox17. In aggregation culture of i22 cells for 3 days, all cells expressed POU5F1, SOX2, and E‐cadherin, a signature of the epiblast, whereas expression of GATA4 and SOX17 was also activated moderately in dispersed cells, suggesting priming of these cells to endodermal development. Embedding the aggregates in Matrigel for further 3 days elicited migration of the cells into the lumen of laminin‐rich matrices covering the aggregates, in which FOXA2 and SOX17 were expressed at a high level with the concomitant loss of E‐cadherin, indicating the migratory phase of endodermal precursors. Prolonged culturing of the aggregates generated three segregating cell populations found in post‐gastrulation stage embryos: (1) definitive endoderm co‐expressing high SOX17, GATA4, and E‐cadherin, (2) mesodermal cells expressing a low level of GATA4 and lacking E‐cadherin, and (3) primed epiblast cells expressing POU5F1, SOX2 without E‐cadherin. Thus, aggregation of EpiSCs followed by embedding of aggregates in the laminin‐rich matrix models the gastrulation‐dependent endoderm precursor development.