Project-3: Modeling EA/TEF in human organoids

项目3|总结：在人类PSC来源的胚胎组织中建立EA模型 食道由一层被神经支配的肌肉包围的复层上皮层组成。 这调节了食物从嘴到胃的单向运动。食道闭锁(EA)伴 是否合并气管食管瘘(TEF)是最常见的气管食道出生缺陷 (Ted)，然而大多数导致EA的突变尚不清楚。这项提议旨在利用人类多能干细胞 (PSC)来源的食道组织既可以理解电针，也可以作为未来重建的来源。我们 已开发的人食道器官(HEO)和食道器官型筏(HEOR)分层 与人类食道高度相似的食道上皮。我们用这些来研究SOX2，一个 HMG-box转录因子对小鼠的食道发育至关重要，可能会引起人类的EA。 不同分化阶段的有机化合物的转录图谱，类似于 食道发育研究表明SOX2调节Wnt拮抗剂如SFRP2在背侧的表达 形成食道的前肠内胚层。SOX2表达缺失导致食道命运丧失 并损害形态发生，导致EA。 在人类中，EA患者可能存在持续性运动缺陷，这表明EA相关基因 可能会影响食道的平滑肌肉或肠道神经系统(ENS)的发育。项目 1已经发现了新的与TED相关的突变，我们预测这些突变可能作用于特定的生殖层，导致EA。 已发表的和初步的数据表明，转录因子RFX6和SOX2在上皮中起作用 而FOXF1和Smad6在间充质中起作用。由于食道器官只含有上皮细胞， 它们对研究影响间充质细胞或ENS的突变没有用处。因此，我们设计了几个新的 含有间充质细胞和ENS细胞类型的类器官系统均来源于PSCs。我们建议使用这些 用于验证EA相关基因影响不同生殖层的假说的新型人类食道系统 并可导致以后的食道发育和功能缺陷。 在这里，我们建议使用PSC派生的HEO和包含所有三个生殖层的HEO来识别 EA引起的突变影响上皮、间充质和ENS的发育。然后我们将确定EA如何- 导致突变影响人类工程食道组织的功能。最后，我们将使用EA引发 在发育中的人类中识别上皮和间充质特异性转录程序的突变 食管。

PROJECT 3 | SUMMARY: MODELING EA IN HUMAN PSC-DERIVED EMBRYONIC TISSUES The esophagus is composed of a layer of stratified epithelium that is surrounded by layers of innervated muscle that regulates the unidirectional movement of food from the mouth to the stomach. Esophageal atresia (EA) with or without associated tracheoesophageal fistula (TEF) is the most common Tracheoesophageal birth defect (TED), yet most mutations causing EA are unidentified. This proposal aims to use human pluripotent stem cell (PSC)-derived esophageal tissues both to understand EA and as a future source for reconstruction. We developed human esophageal organoids (HEOs) and esophageal organotypic rafts (HEORs) with stratified esophageal epithelium that is highly similar to human esophagus. We used these to investigate how SOX2, an HMG-box transcription factor that is essential for esophageal development in mice, might cause EA in humans. Transcriptional profiling of organoids at different stages of differentiation that are analogous to critical stages of esophageal development identified that SOX2 regulates expression of Wnt antagonists like SFRP2 in the dorsal foregut endoderm that gives rise to the esophagus. Loss of SOX2 expression resulted in loss of esophageal fate and impaired morphogenesis, resulting in EA. In humans, patients with EA can have persistent motility defects suggesting that EA-associated genes may affect development of the smooth muscles or the enteric nervous system (ENS) of the esophagus. Project 1 has identified new TED-associated mutations that we predict may act in specific germ layers to cause EA. Published and preliminary data indicate that the transcription factors RFX6 and SOX2 act in the epithelium whereas FOXF1 and SMAD6 act in the mesenchyme. Since esophageal organoids only contained epithelium, they were not useful to study mutations impacting mesenchyme or ENS. We therefore engineered several new organoid systems containing mesenchymal and ENS cell types all derived from PSCs. We propose to use these novel human esophageal systems to test the hypothesis that EA-associated genes impact different germ layer components and can cause later developmental and functional deficits in the esophagus. Here we propose using PSC-derived HEOs and HEORs that contain all three germ layers to identify how EA-causing mutations impact epithelial, mesenchymal and ENS development. We will then determine how EA- causing mutations impact the function of human engineered esophageal tissue. Lastly we will use EA-causing mutations to identify epithelial- and mesenchyme-specific transcriptional programs in the developing human esophagus.