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建模 食道由一层分层上皮组成，该上皮被神经支配的肌肉所包围 这可以调节食物从口腔到胃的单向运动。食管闭锁（EA） 或没有相关的气管食管瘘（TEF）是最常见的气管食管 （TED），但大多数引起EA的突变都是未识别的。该建议旨在使用人类多能干细胞 （PSC）衍生的食管组织都可以理解EA和将来的重建来源。我们 开发了人类食管器官（HEOS）和食管器官筏（Heors） 食管上皮，与人类食管高度相似。我们用这些调查了Sox2的方式 对于小鼠食管发育至关重要的HMG-box转录因子可能会导致人类EA。 在不同阶段的分化阶段的器官的转录分析类似于关键阶段 食管发育确定SOX2调节背面中Wnt拮抗剂的表达 前胚层引起食道。 SOX2表达的丧失导致食管命运的丧失 形态发生受损，导致EA。 在人类中，EA患者可能存在持续的运动缺陷，表明与EA相关基因 可能会影响食管平滑肌肉或肠神经系统（ENS）的发育。项目 1已经确定了我们预测的新的TED相关突变可能会在特定的细菌层中起作用，从而引起EA。 已发布和初步数据表明上皮的转录因子RFX6和SOX2作用 而FOXF1和SMAD6在间充质中作用。由于食管类器官仅包含上皮，所以 它们对于研究影响间质或ENS的突变没有用。因此，我们设计了几个新的 含有间充质和ENS细胞类型的器官系统均来自PSC。我们建议使用这些 新型人类食管系统，以测试EA相关基因影响不同生殖层的假设 成分并可能导致食道中后来的发育和功能缺陷。 在这里，我们建议使用包含所有三个细菌层的PSC衍生的HEOS和HEOR，以确定如何 引起EA的突变会影响上皮，间质和ENS发育。然后，我们将确定如何 导致突变影响人类工程食管组织的功能。最后我们将使用EA引起 突变以识别发展中的人类的上皮和间质特异性转录程序 食管。