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

> **NIH NIH R01** · UNIVERSITY OF PENNSYLVANIA · 2024 · $471,752

## Abstract

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 h...

## Key facts

- **NIH application ID:** 10909354
- **Project number:** 5R01DK134493-02
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Kotaro Sasaki
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $471,752
- **Award type:** 5
- **Project period:** 2023-08-18 → 2028-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10909354

## Citation

> US National Institutes of Health, RePORTER application 10909354, Stem Cell-Based Models for Elucidating Human Adrenocortical Development and Dysfunction (5R01DK134493-02). Retrieved via AI Analytics 2026-05-21 from https://api.ai-analytics.org/grant/nih/10909354. Licensed CC0.

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