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

由于胎儿肾上腺（FAd）发育缺陷可能导致危及生命的原发性肾上腺皮质功能不全（PAI）， 了解控制这一过程的细胞和基因调控机制至关重要。虽然很多有 尽管已经了解了啮齿类动物模型中 FAd 的发育情况，但物种特异性的发育差异限制了我们的研究 了解人类的这一过程。由于人类胚胎的机械评估是站不住脚的，我们最近 开发了第一个人类诱导多能干细胞 (iPSC) 衍生的 FAd 类器官系统 概括了正常的功能发育和类固醇生成。利用该系统，我们将进行 首次对人类 FAd 发育进行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，在提供 对 PAI 驱动机制的重要洞察。人类肾上腺皮质的发育始于规范 从体腔上皮（CE）分离肾上腺原基（AP），然后建立最终的 具有假定的干细胞/祖细胞潜力的区域（DZ）和具有类固醇生成潜力的胎儿区域（FZ）。我们的 最近对人类 FAd 皮层的单细胞 RNA-seq 分析支持 DZ 和 FAd 皮质的 FZ 补充 观察到位于外周的胶囊 (Cap) 中 Wnt 配体/激活剂的表达表明存在生态位 功能。机械地评估人类 FAd 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 类器官系统。虽然转录因子 NR5A1 影响 小鼠早期 FAd 细胞命运并以基因剂量依赖性方式促进类固醇生成，其在人类中的作用 FAd 显得更加复杂。我们的初步研究表明，诱导的 NR5A1 无效突变类器官未能 分化为 AP 样细胞 (APLC)，表现出存活率下降，无法上调受体 促肾上腺皮质激素（ACTH），并且其余细胞的类固醇生成潜力有限。在 在野生型 FAd 类器官中，我们发现 Wnt 的去除增强了 DZ 样细胞（DZLC）的形成 抑制剂/添加Wnt激动剂。我们还发现 WNT4 和 RSPO3 在人类 Cap 中高表达。 体内和 FAd 类器官中的帽状细胞 (CapLC)。有趣的是，我们发现 DZLC 的形成通过 声刺猬（SHH），鼠帽的一种营养因子，表明潜在的 SHH 驱动的利基功能 观察到的人类 Cap 中 SHH 靶基因的上调进一步支持了 Cap 的观点。最后，我们发现 FACS 分选的 DZ 和 DZLC 在 ACTH 刺激下很容易分化为 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，但囊下 DZ 仍然存在，这表明 Cap 衍生的 Wnt 信号传导拮抗 ACTH- DZLC 介导的分化。使用我们的类器官系统，我们将测试中心假设，即剂量- 敏感的 NR5A1 活性促进 AP 的发育，并且 SHH- 提供的 Wnt 信号传导 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.