Division of Developmental Biology, Cincinnati, Ohio, USA.
Nat Protoc. 2011 Nov 10;6(12):1920-8. doi: 10.1038/nprot.2011.410.
Here we describe a protocol for generating 3D human intestinal tissues (called organoids) in vitro from human pluripotent stem cells (hPSCs). To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2(+)SOX17(+) endoderm by treating the cells with activin A for 3 d. After endoderm induction, the pluripotent stem cells are patterned into CDX2(+) mid- and hindgut tissue using FGF4 and WNT3a. During this patterning step, 3D mid- or hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3D spheroids are further cultured in Matrigel along with prointestinal growth factors, and they proliferate and expand over 1-3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium comprising all of the major intestinal cell types. To date, this is the only method for efficiently directing the differentiation of hPSCs into 3D human intestinal tissue in vitro.
在这里，我们描述了一种从人类多能干细胞（hPSCs）体外生成 3D 人肠道组织（称为类器官）的方案。为了生成肠道类器官，首先通过用激活素 A 处理细胞 3 天，将多能干细胞分化为 FOXA2(+)SOX17(+)内胚层。内胚层诱导后，使用 FGF4 和 WNT3a 将多能干细胞图案化成 CDX2(+)中肠和后肠组织。在这个图案化步骤中，3D 中肠或后肠球体从附着在组织培养皿上的单层上皮细胞中发芽。将 3D 球体与肠前体生长因子一起进一步在 Matrigel 中培养，并在 1-3 个月内增殖和扩张，形成具有包括所有主要肠道细胞类型的肠道间充质和上皮的肠道组织。迄今为止，这是唯一一种有效地将 hPSCs 体外定向分化为 3D 人肠道组织的方法。
