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|总结：在人PSC衍生的胚胎组织中建模EA 食管是由一层复层上皮组成，复层上皮被神经支配的肌肉层所包围 调节食物从口腔到胃的单向运动。食管闭锁（EA）伴 气管食管瘘（TEF）是最常见的出生缺陷， (TED)然而，大多数导致EA的突变是未知的。这项提案旨在利用人类多能干细胞 （PSC）衍生的食管组织，以了解EA和作为重建的未来来源。我们 开发了人类食管类器官（HEO）和食管器官型筏（HEOR）， 与人类食管高度相似的食管上皮。我们用这些来研究SOX 2， HMG盒转录因子是小鼠食管发育所必需的，可能导致人类EA。 类器官在不同分化阶段的转录谱分析，这些分化阶段类似于细胞分化的关键阶段。 食管发育发现SOX 2调节背侧中SFRP 2等Wnt拮抗剂的表达 形成食道的前肠内胚层。SOX 2表达缺失导致食管命运丧失 和受损的形态发生导致EA 在人类中，EA患者可以有持续的运动缺陷，这表明EA相关基因 可能影响食管平滑肌或肠神经系统（ENS）的发育。项目 1已经确定了新的TED相关突变，我们预测这些突变可能作用于特定的胚层，导致EA。 已发表的和初步的数据表明，转录因子RFX 6和SOX 2在上皮细胞中起作用， 而FOXF 1和SMAD 6在间充质中起作用。由于食管类器官仅含有上皮， 它们对于研究影响间充质或ENS的突变没有用处。因此，我们设计了几种新的 含有间充质和ENS细胞类型的类器官系统，所有细胞类型均来源于PSC。我们建议使用这些 新型人类食道系统来测试EA相关基因影响不同种系的假设 成分，并可能导致后来的发育和功能缺陷的食管。 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来确定如何 引起EA的突变影响上皮、间充质和ENS发育。我们将决定如何- 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，以确定上皮和间充质特异性转录程序在发展中的人类 食道