Molecular and cellular pathways driving competency for human vagal neural crest specification

驱动人类迷走神经嵴规范能力的分子和细胞途径

• 批准号: 10727766
• 负责人: LORENZ P. STUDER
• 金额: $ 48.68万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-19 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727766
• 关键词: Adopted; Anterior; Appearance; Automobile Driving; CRISPR screen; Candidate Disease Gene; Cell Cycle; Cell Differentiation process; Cell Line; Cell Therapy; Cells; Cephalic; Clustered Regularly Interspaced Short Palindromic Repeats; Competence; Congenital Megacolon; Data; Development; Disease; Disease model; Ectoderm; Embryo; Embryonic Development; Enteral; Enteric Nervous System; Event; Exhibits; Gene Expression; Gene Expression Profile; Generations; Genetic; Genetic Transcription; Genomics; Heterogeneity; Hour; Human; Improve Access; In Vitro; Link; Mediating; Molecular; Neural Crest; Neural Crest Cell; Pathway interactions; Pattern; Phenotype; Population; Process; Reporter; Signal Pathway; Signal Transduction; Specific qualifier value; System; Technology; Testing; Time; Tissues; Tretinoin; candidate identification; cohort; design; directed differentiation; embryo tissue; experimental study; hindbrain; human pluripotent stem cell; improved; in vivo; multiple omics; programs; response; segregation; stem cell differentiation; transcriptomics

Abstract/Summary In vitro neural crest-like cells (NCCs) can be generated from human pluripotent stem cells (hPSCs). In response to retinoic acid (RA), NCCs can be patterned towards vagal neural crest identity based on gene expression and based on lineage differentiation capacity. Exciting preliminary data from the lab, acquired through the use of CellTag lineage tracing technology, indicate that the process of vagal identity acquisition actually begins at a very early differentiation stage, prior to RA exposure. Our data suggest the spontaneous appearance of vagal- competent versus non-competent populations during those early differentiation time points. Furthermore, we observed that the competent population exhibits a gene expression pattern that matches cells of the primordial ectoderm in the region that becomes the hindbrain. Understanding the process by which these early vagal- competent precursors are established, as well as the mechanism that maintains and executes vagal competence has broad implications. For NCC specification, an improved understanding of spatial patterning in the cranial region may greatly enhance our ability to generate vagal NCCs for the treatment of diseases such as Hirschsprung’s Disease. Beyond NCC patterning, our study may reveal general mechanisms of axial patterning that impact many other developing embryonic tissues including the CNS. In Aim 1 we propose to use scRNAseq to fully characterize the population that exhibits vagal competence in this system before identifying signals that selectively enrich for these cells using a double reporter hPSC line. In Aim 2 we propose to dissect the mechanism of competence with the ambitious combination of (1) parallel scATAC and scRNA sequencing in a multiomics approach to uncover regions of differential accessibility that account for distinct RA response mechanisms, and (2) application of CRISPR-a and CRISPR-i to test the ability of candidate genes to respectively program or disrupt competence in populations with distinct spatial identities.

摘要/概要 体外神经嵴样细胞（NCC）可以由人多能干细胞（hPSC）产生。响应 对于视黄酸（RA），NCC可以基于基因表达朝向迷走神经嵴身份形成模式， 基于谱系分化能力。令人兴奋的初步数据，从实验室，获得通过使用 CellTag谱系追踪技术，表明迷走神经身份获得的过程实际上是从一个 非常早期的分化阶段，在RA暴露之前。我们的数据表明迷走神经的自发出现- 在那些早期分化时间点期间，感受态与非感受态群体。而且我们 观察到感受态群体表现出与原始细胞相匹配的基因表达模式， 后脑区域的外胚层。了解这些早期迷走神经- 建立了主管前体，以及维持和执行迷走神经能力的机制 具有广泛的影响。对于NCC规范，提高了对颅骨空间模式的理解 该区域可能会大大增强我们产生迷走神经NCC的能力，以治疗疾病，例如 先天性巨结肠除了NCC模式，我们的研究可能揭示轴向模式的一般机制 影响许多其他发育中的胚胎组织包括中枢神经系统。 在目的1中，我们提出使用scRNAseq来完全表征在这种情况下表现出迷走神经能力的群体。 系统，然后使用双报告基因hPSC系鉴定选择性富集这些细胞的信号。在Aim中 2.我们建议用以下雄心勃勃的组合来剖析胜任力机制：（1）平行scATAC 和scRNA测序的多组学方法，以揭示区域的差异可及性，占 不同的RA应答机制，和（2）CRISPR-a和CRISPR-i的应用以测试候选RA应答机制的能力。 基因分别编程或破坏具有不同空间身份的群体中的能力。