Project-3: Modeling EA/TEF in human organoids

项目 3：在人体类器官中模拟 EA/TEF

• 批准号: 10647838
• 负责人: James M Wells
• 金额: $ 39.75万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-08-15 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10647838
• 关键词: Achalasia; Acids; Address; Affect; Autologous; Biopsy; Cells; ChIP-seq; Colon; Computer Analysis; Congenital Abnormality; Data; Defect; Development; Distal; Dorsal; Endoderm; Engineering; Enteric Nervous System; Enzymes; Epithelium; Esophageal Atresia; Esophageal Tissue; Esophagus; FOXF1 gene; Fanconi Anemia Complementation Group A Protein; Food; Funding; Future; Gastrointestinal tract structure; Gene Expression Profiling; Gene Mutation; Genes; Genetic Transcription; Genomics; Germ Layers; HMG-Box Domains; Hormone secretion; Human; Human Engineering; Impairment; Ingestion; Injury; MADH6 gene; Malignant neoplasm of esophagus; Megaesophagus; Mesenchymal; Mesenchyme; Mesoderm; Microbe; Modeling; Morphogenesis; Movement; Mus; Muscle; Muscle Contraction; Mutation; Neurons; Oral cavity; Organoids; Patients; Pluripotent Stem Cells; Primitive foregut structure; Publishing; Reconstructive Surgical Procedures; Repression; Role; Smooth Muscle; Source; Stomach; Stratified Epithelium; System; Testing; Tissues; Toxin; Trachea; Tracheoesophageal Fistula; antagonist; cell motility; cell type; constriction; embryo tissue; gene function; human pluripotent stem cell; induced pluripotent stem cell; loss of function; motility disorder; nervous system development; neural; novel; nutrient absorption; programs; reconstruction; response; risk variant; single-cell RNA sequencing; transcription factor

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.

项目 3 |摘要：在人类 PSC 衍生的胚胎组织中对 EA 进行建模 食道由一层复层上皮组成，周围被神经支配的肌肉层包围 它调节食物从口腔到胃的单向运动。食管闭锁 (EA) 或不伴有气管食管瘘（TEF）是最常见的气管食管出生缺陷 (TED)，但大多数导致 EA 的突变尚未确定。该提案旨在利用人类多能干细胞 (PSC) 来源的食管组织既可以理解 EA，也可以作为未来重建的来源。我们 开发了分层的人类食管类器官（HEO）和食管器官筏（HEOR） 食管上皮与人类食管高度相似。我们用这些来研究 SOX2（一种 HMG-box 转录因子对于小鼠食道发育至关重要，可能会导致人类 EA。 类器官在不同分化阶段的转录谱，类似于细胞分化的关键阶段 食管发育发现 SOX2 调节背侧 SFRP2 等 Wnt 拮抗剂的表达 前肠内胚层产生食道。 SOX2表达的丧失导致食管命运的丧失 和形态发生受损，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 可能会影响食道平滑肌或肠神经系统（ENS）的发育。项目 1 已鉴定出新的 TED 相关突变，我们预测这些突变可能作用于特定胚层，从而导致 EA。 已发表的初步数据表明转录因子 RFX6 和 SOX2 在上皮细胞中发挥作用 而 FOXF1 和 SMAD6 在间充质中起作用。由于食管类器官仅含有上皮细胞， 它们对于研究影响间充质或 ENS 的突变没有用处。因此我们设计了一些新的 含有间充质和 ENS 细胞类型的类器官系统均源自 PSC。我们建议使用这些 新型人类食管系统来测试 EA 相关基因影响不同胚层的假设 成分，并可能导致食道随后的发育和功能缺陷。 在这里，我们建议使用 PSC 衍生的 HEO 和包含所有三个胚层的 HEOR 来识别如何 引起 EA 的突变会影响上皮细胞、间充质细胞和 ENS 的发育。然后我们将确定 EA 如何- 引起的突变会影响人类工程食管组织的功能。最后我们将使用 EA 引起的 突变来识别发育中的人类上皮和间充质特异性转录程序 食管。