Reconstitution of human adrenocortical specification and steroidogenesis using induced pluripotent stem cells [scRNA-seq]

The adrenal cortex produces vital steroid hormones that maintain homeostasis. While steroid hormones produced from the fetal zone adrenal cortex are essential for both fetal development and maintenance of pregnancy, the molecular mechanisms leading to human adrenal cortex development and steroid synthesis remain poorly understood due to the paucity of model systems. Through progressive generation of fetal zone adrenal cortex-like cells (FZLCs) from human induced pluripotent stem cells through posterior intermediate mesoderm-like, adrenogenic coelomic epithelium and adrenal primordium-like states, we provide the first in vitro reconstitution of human adrenocortical fetal specification. Generation of FZLCs faithfully recapitulates human embryonic adrenal cortex specification as evidenced by histomorphological and ultrastructural analysis, transcriptional profiles and delta-5 steroid biosynthesis and occurs in an adrenocorticotropic hormone (ACTH)-independent manner, consistent with clinical observations. Furthermore, we demonstrate that FZLC generation is promoted by SHH and inhibited by NOTCH, ACTIVIN and WNT signaling and that steroid synthesis is amplified by ACTH/PKA signaling and blocked by pharmacologic inhibitors of delta 5 steroid synthesis enzymes. Finally, NR5A1 appear to self-stabilize its promoter activity and promote FZLC survival and steroidogenesis. Together, these findings provide a framework for understanding and reconstituting human adrenocortical development in vitro and pave the way for future cell-based therapies of adrenal insufficiency. Overall design: scRNA-seq

肾上腺皮质（adrenal cortex）可分泌维持机体稳态的关键类固醇激素（steroid hormones）。尽管胎儿带肾上腺皮质（fetal zone adrenal cortex）产生的类固醇激素对胎儿发育与妊娠维持均不可或缺，但由于缺乏合适的模型系统，目前学界对人类肾上腺皮质发育及类固醇合成的分子机制仍知之甚少。本研究通过诱导人类多能干细胞（human induced pluripotent stem cells）依次分化为后中间中胚层样、肾上腺源性体腔上皮及肾上腺原基样细胞，最终获得胎儿带肾上腺皮质样细胞（FZLCs, fetal zone adrenal cortex-like cells），首次在体外重建了人类肾上腺皮质的胎儿定向分化过程。FZLC的生成可精准复刻人类胚胎期肾上腺皮质的定向分化程序，这一结论得到了组织形态学、超微结构分析、转录组特征及δ5类固醇生物合成实验的验证，且该过程不依赖促肾上腺皮质激素（adrenocorticotropic hormone, ACTH），与临床观察结果相符。进一步研究表明，SHH信号可促进FZLC的生成，而NOTCH、ACTIVIN及WNT信号则对该过程产生抑制作用；类固醇合成可被ACTH/PKA信号通路放大，且可被δ5类固醇合成酶的药理学抑制剂阻断。最后，NR5A1似乎可自主稳定其启动子活性，并促进FZLC存活与类固醇生成。综上，本研究成果为理解并体外重建人类肾上腺皮质发育提供了研究框架，也为未来肾上腺功能不全的细胞替代疗法开辟了道路。整体实验设计：单细胞RNA测序（scRNA-seq）