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

由于胎儿肾上腺（FAd）发育缺陷可导致危及生命的原发性肾上腺功能不全（派）， 理解控制这一过程的细胞和基因调节机制是至关重要的。虽然许多 在啮齿动物模型中了解到FAd的发展，种属特异性发育差异限制了我们的研究。 了解人类的这一过程。由于在人类胚胎中的机械评价是站不住脚的，我们最近 开发了第一个人诱导多能干细胞（iPSC）衍生的FAd类器官系统， 再现正常的功能发育和类固醇生成。利用这一系统，我们将 首次对人类FAd发育进行了扰动和反向遗传评估，使我们能够阐明 人类肾上腺皮质发育的分子机制，这在提供广泛的意义， 对派驱动机制的重要见解。人类肾上腺皮质的发育始于特化 从体腔上皮（CE）中分离肾上腺原基（AP），然后建立确定的 具有推定的干细胞/祖细胞潜能的区（DZ）和具有类固醇生成潜能的胎儿区（FZ）。我们 最近对人FAd皮质的单细胞RNA-seq分析支持DZ和FZ补充FZ。 观察到的Wnt配体/激活剂在外周定位囊（Cap）中的表达提示了小生境 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， WNT 4突变）驱动派时，我们利用了我们的FAd类器官系统。而转录因子NR 5A 1影响 早期FAd细胞的命运，并促进类固醇激素的基因剂量依赖性的方式在小鼠中，其作用，在人类 FAd看起来更复杂。我们的初步研究表明，诱导的NR 5A 1无效突变体类器官不能 分化为AP样细胞（APLC），表现出降低的存活率，不能上调 促肾上腺皮质激素（ACTH），并且剩余细胞的类固醇生成潜力有限。在 野生型FAd类器官，我们发现DZ样细胞（DZLC）的形成通过去除Wnt而增强 抑制剂/添加Wnt激动剂。我们还发现WNT 4和RSPO 3在人Cap和人Cap中均高度表达， 在体内和在FAd类器官中的帽样细胞（CapLC）。有趣的是，我们发现DZLC的形成是由 音刺猬（SHH），一种鼠Cap的营养因子，提示了一种潜在的SHH驱动的小生境功能， Cap中观察到的SHH靶基因的上调进一步支持了这一点。最后，我们发现 FACS分选的DZ和DZLC在ACTH刺激后容易分化成FZ样细胞（FZLC）， 支持DZLC补充FZLC的能力。然而，由于Wnt-靶基因表达维持在 尽管暴露于ACTH，但被膜下DZ，这表明Cap衍生的Wnt信号传导拮抗ACTH- 介导的DZLCs分化。使用我们的类器官系统，我们将测试中心假设，剂量- 敏感的NR 5A 1活性促进AP的发展，并且SHH-1提供的Wnt信号传导促进AP的发展。 Cap中的依赖性反应既诱导DZ发育又拮抗ACTH驱动的FZ形成。

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.