Stem Cell-Based Models for Elucidating Human Adrenocortical Development and Dysfunction

用于阐明人类肾上腺皮质发育和功能障碍的干细胞模型

• 批准号: 10735100
• 负责人: Kotaro Sasaki
• 金额: $ 50.78万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-18 至 2028-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10735100
• 关键词: Addison' s disease; Adrenal Cortex; Adrenal Glands; Agonist; Automobile Driving; Biological Models; Candidate Disease Gene; Cell Line; Cells; Coculture Techniques; Complex; Corticotropin; Corticotropin Receptors; Defect; Development; Disease; Dose; Elements; Embryo; Epithelium; Evaluation; Excision; Exhibits; Exposure to; Functional disorder; Gene Dosage; Gene Expression; Genes; Heterozygote; Homeostasis; Hormone Responsive; Human; Learning; Life; Ligands; Mediating; Molecular; Mus; Mutation; Organoids; Patients; Peripheral; Physiological; Primordium; Process; Proteins; Regulator Genes; Rodent Model; Role; SHH gene; Signal Transduction; Signaling Molecule; Sorting; Source; Specific qualifier value; Steroid biosynthesis; Steroids; Supplementation; System; Testing; Up-Regulation; WNT Signaling Pathway; WNT4 gene; androgen excess; capsule; experimental study; fetal; gene regulatory network; in vivo; induced pluripotent stem cell; inhibitor; innovation; insight; loss of function; mutant; preservation; progenitor; promoter; regenerative therapy; response; reverse genetics; single-cell RNA sequencing; stem cell function; stem cell model; stem cells; steroid hormone; transcription factor; transcriptome sequencing

As defects in fetal adrenal (FAd) development can result in life threatening primary adrenal insufficiency (PAI), understanding the cellular and gene regulatory mechanisms governing this process is essential. While much has been learned about FAd development in rodent models, species-specific developmental differences limit our understanding of this process in humans. As mechanistic evaluation in human embryos is untenable, we recently developed the first human induced pluripotent stem cells (iPSCs)-derived FAd organoid system that recapitulates normal functional development and steroidogenesis. Using this system, we will undertake the first perturbative and reverse genetic assessment of human FAd development, allowing us to elucidate the molecular mechanisms of human adrenocortical development, which has broad implications in providing essential insight into mechanisms driving PAI. Development of the human adrenal cortex starts with specification of the adrenal primordium (AP) from the coelomic epithelium (CE), followed by establishment of the definitive zone (DZ) with putative stem cell/progenitor potential, and the fetal zone (FZ) with steroidogenic potential. Our recent single cell RNA-seq analysis of the human FAd cortex supports FZ replenishment by the DZ and the observed expression of Wnt ligands/activators in the peripherally located capsule (Cap) is suggestive of niche function. To mechanistically assess human FAd development and understand genetic defects (e.g., NR5A1, WNT4 mutations) driving PAI, we utilized our FAd organoid system. While the transcription factor NR5A1 impacts early FAd cell fate and promotes steroidogenesis in a gene-dose dependent manner in mice, its role in human FAd appears more complex. Our preliminary studies show that induced NR5A1 null mutant organoids fail to differentiate into AP-like cells (APLCs), exhibit decreased survival, fail to upregulate the receptor for adrenocorticotropic hormone (ACTH), and that the steroidogenic potential of the remaining cells is limited. In wild-type FAd organoids, we find that formation of DZ-like cells (DZLCs) is enhanced by the removal of Wnt inhibitor/addition of Wnt agonist. We also find that WNT4 and RSPO3 are highly expressed in both human Cap in vivo and Cap-like cells (CapLCs) in FAd organoids. Intriguingly, we find that DZLC formation is enhanced by sonic hedgehog (SHH), a trophic factor for murine Cap, suggesting a potential SHH-driven niche function for the Cap that is further supported by the observed upregulation of SHH target genes in human Cap. Finally, we find that both FACS-sorted DZ and DZLCs readily differentiate into FZ-like cells (FZLCs) upon ACTH stimulation, supporting the ability of DZLCs to replenish FZLCs. However, as Wnt-target gene expression is maintained in the subcapsular DZ despite exposure to ACTH, it suggests that Cap-derived Wnt signaling antagonizes ACTH- mediated differentiation of DZLCs. Using our organoid system, we will test the central hypothesis that dose- sensitive NR5A1 activity promotes development of the AP and that Wnt signaling provided by SHH- dependent responses in Cap both induces DZ development and antagonizes ACTH-driven FZ formation.

由于胎儿肾上腺发育缺陷(FAD)可能导致威胁生命的原发性肾上腺功能不全(PAI)， 了解控制这一过程的细胞和基因调控机制是至关重要的。虽然很多人都有 已经了解到啮齿动物模型中FAD的发展，物种特定的发育差异限制了我们的 对人类这一过程的理解。由于对人类胚胎的机械性评估是站不住脚的，我们最近 开发了第一个由人类诱导多能干细胞(IPSCs)衍生的FAD类器官系统 概述了正常的功能发育和类固醇的生成。使用这个系统，我们将承担 人类FAD发育的第一次微扰和反向遗传评估，使我们能够阐明 人类肾上腺皮质发育的分子机制，这在提供 对推动PAI的机制的基本洞察力。人类肾上腺皮质的发育始于规范 从体腔上皮(CE)中提取肾上腺原基(AP)，然后建立最终的 可能具有干细胞/祖细胞潜能的区域(DZ)和具有类固醇生成潜能的胎儿区(FZ)。我们的 最近对人类FAD皮质的单细胞RNA-SEQ分析支持DZ和DZ对FZ的补充 观察到Wnt配体/激活物在外周定位的囊膜(Cap)中的表达提示有小生境 功能。为了机械地评估人类FAD的发育并了解遗传缺陷(例如，NR5A1， WNT4突变)驱动PAI，我们利用了我们的FAD有机系统。当转录因子NR5A1影响 小鼠早期FAD细胞命运并以基因剂量依赖的方式促进类固醇合成，其在人类中的作用 时尚似乎更为复杂。我们的初步研究表明，诱导的NR5A1缺失突变类有机物未能 分化为AP样细胞(APLC)，存活率下降，未能上调受体 促肾上腺皮质激素(ACTH)，其余细胞的类固醇生成潜力有限。在……里面 野生型FAD类有机物，我们发现Wnt的去除促进了DZ样细胞(DZLCs)的形成 WnT激动剂的抑制剂/添加。我们还发现WNT4和RSP3在两个人的帽子中都有高表达 FAD类器官中的活体和帽样细胞(CapLCs)。有趣的是，我们发现DZLC的形成被 Sonic Hedgehog(SHH)，一种对小鼠帽子的营养因子，暗示了SHH驱动的潜在的生态位功能 观察到的SHH靶基因在人类CAP中的上调进一步支持了CAP。最后，我们发现 FACS分选的DZ和DZLCs在ACTH刺激下都容易分化为FZ样细胞(FZLCs)， 支持DZLCs补充FZLCs的能力。然而，由于Wnt-靶基因的表达在 被膜下DZ尽管暴露于ACTH，但表明Cap来源的Wnt信号拮抗ACTH- 介导DZLCs的分化。使用我们的有机系统，我们将检验中心假说- 敏感的NR5A1活性促进AP的发展，SHH- Cap中的依赖反应既可诱导DZ的发育，又可拮抗ACTH驱动的FZ的形成。