Generation of Gastrointestinal Organoids from Human Pluripotent Stem Cells
Generation of Gastrointestinal Organoids from Human Pluripotent Stem Cells
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DOI：
10.1007/978-1-4939-6949-4_12
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发表时间：
2017-01-01
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影响因子：
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通讯作者：
Wells, James M.
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作者：
Munera, Jorge O.;Wells, James M.
在过去的几十年里，发育生物学家已经发现了器官在胚胎发育中形成的基本机制。有了这些信息，现在可以通过使用重演器官发育的过程逐步分化人类多能干细胞来产生人类“类器官”。对于胃肠道，第一个关键步骤之一是定形内胚层和中胚层的形成，这是一个依赖于TGF β分子Nodal的过程。然后内胚层沿着前-后轴沿着形成图案，其中前内胚层形成前肠，后内胚层形成中肠和后肠。内胚层的A-P模式化通过Wnt、BMP和FGF的组合活性来完成。高Wnt和BMP促进后命运，而抑制这些途径促进前内胚层命运。胃来源于后前肠，视黄酸信号传导是促进后前肠命运所必需的。小肠和大肠分别起源于中肠和后肠，这些胃肠发育阶段可以通过上述途径的时间激活和抑制来精确操纵。例如，用模拟激活素A（另一种TGF-β超家族成员）刺激Nodal途径可以触发多能干细胞分化成定形内胚层（D 'Amour et al. Nat Biotechnol 23：1534-1541，2005）。定形内胚层暴露于高水平的Wnt和FGF促进表达CDX 2的后内胚层和中/后肠组织的形成。在三维基质中培养的中后肠球状体形成人肠类器官（HIO），其本质上是小肠Spence等人，Nature 2011.相反，在BMP抑制剂头蛋白存在下FGF和Wnt的活化促进表达SOX 2的前内胚层和前肠组织的形成。这些表达SOX 2的前肠球状体可以通过添加视黄酸进一步图案化到后前肠中。一旦形成，这些后前肠球状体就可以在具有胃的窦部的所有细胞类型的三维人胃类器官（HISK）中生长（Mc Cracken等人，2014）。在这里，我们描述了从人多能干细胞产生胃/人胃类器官（HISK）和人肠类器官（HIO）的详细方法。我们首先提出了一种从多能干细胞产生定形内胚层的方法，然后将定形内胚层分化成后前肠球状体或中后肠球状体。然后，我们描述了如何三维培养这些球状体的结果，分别在形成的HIOs和HIOs。
Over the past several decades, developmental biologists have discovered fundamental mechanisms by which organs form in developing embryos. With this information it is now possible to generate human "organoids" by the stepwise differentiation of human pluripotent stem cells using a process that recapitulates organ development. For the gastrointestinal tract, one of the first key steps is the formation of definitive endoderm and mesoderm, a process that relies on the TGFb molecule Nodal. Endoderm is then patterned along the anterior-posterior axis, with anterior endoderm forming the foregut and posterior endoderm forming the mid and hindgut. A-P patterning of the endoderm is accomplished by the combined activities of Wnt, BMP, and FGF. High Wnt and BMP promote a posterior fate, whereas repressing these pathways promotes an anterior endoderm fate. The stomach derives from the posterior foregut and retinoic acid signaling is required for promoting a posterior foregut fate. The small and large intestine derive from the mid and hindgut, respectively.These stages of gastrointestinal development can be precisely manipulated through the temporal activation and repression of the pathways mentioned above. For example, stimulation of the Nodal pathway with the mimetic Activin A, another TGF-beta superfamily member, can trigger the differentiation of pluripotent stem cells into definitive endoderm (D'Amour et al., Nat Biotechnol 23:1534-1541, 2005). Exposure of definitive endoderm to high levels of Wnt and FGF promotes the formation of posterior endoderm and mid/hindgut tissue that expresses CDX2. Mid-hindgut spheroids that are cultured in a three-dimensional matrix form human intestinal organoids (HIOs) that are small intestinal in nature Spence et al., Nature 2011. In contrast, activation of FGF and Wnt in the presence of the BMP inhibitor Noggin promotes the formation of anterior endoderm and foregut tissues that express SOX2. These SOX2-expressing foregut spheroids can be further patterned into posterior foregut by addition of retinoic acid. Once formed, these posterior foregut spheroids can be grown in three-dimensional human gastric organoids (HGOs) that have all of the cell types of antral part of the stomach (Mc Cracken et al. 2014).Here, we describe the detailed methods for generating stomach/human gastric organoids (HGOs) and human intestinal organoids (HIOs) from human pluripotent stem cells. We first present a method for generating definitive endoderm from pluripotent stem cells followed by differentiation of definitive endoderm into either posterior foregut spheroids or mid-hindgut spheroids. We then describe how three-dimensional culturing of these spheroids results in the formation of HGOs and HIOs, respectively.