Stouffer, D. G. | bioRxiv Science

bioRxiv2025

Reconstitution of adrenocortical functional zonation from human pluripotent stem cells

The adrenal cortex produces essential steroid hormones through a concentric zonal architecture, established by the centripetal trans-differentiation of subcapsular progenitors within a capsule-derived niche. To capture this complexity, we establish a human pluripotent stem cell-derived adrenal organoid system that faithfully recapitulates this process. RSPO3/WNT signaling from the capsule specifies definitive zone (DZ) progenitors from the adrenal primordium, which then differentiate into a cortisol-producing transitional zone and an androgen-producing fetal zone under the influence of RSPO3 and ACTH. Loss of NR0B1 impairs DZ specification and triggers direct adrenal primordium-to-fetal zone conversion, mirroring the mechanism of X-linked adrenal hypoplasia congenita. When DZ cells are encapsulated with capsule cells separately derived from pluripotent stem cells, they reconstitute zonation in vivo, forming ACTH-responsive tissue that produces both cortisol and androgens. This organoid platform offers a powerful tool to dissect human adrenal development and establishes a foundation for regenerative therapies targeting adrenal diseases.

developmental biology↗

bioRxiv2022

Functional Recovery from Human Induced Pluripotent Stem Cell-Derived Dopamine Neuron Grafts is Dependent on Neurite Outgrowth

Transplantation of human pluripotent stem cell-derived dopaminergic (DA) neurons is an emerging therapeutic strategy for Parkinsons disease (PD). In this study, we demonstrate, for the first time, that that functional recovery after DA neuron transplant is critically dependent on graft-host integration, and not dependent on graft size or DA neuron content. Specifically, in anticipation of an autologous DA neuron transplant strategy, we studied two human induced pluripotent stem cell lines derived from people with idiopathic PD. We confirmed the cells ability to differentiate into mature DA neurons in vitro by assessing electrophysiology and depolarization-induced neurotransmitter release. To evaluate efficacy, we transplanted DA neuron precursors into a hemiparkinsonian rat model. We differentiated the cell lines for either 18 (d18) or 25 days (d25) to investigate the effect of cell maturity on efficacy. We found comparable graft survival in d18 and d25 groups from both cell lines, but behavioral analysis revealed that only d18 preparations, from both cell lines, resulted in recovery of motor impairments. Immunohistochemical analysis did not reveal any DA neuron-associated markers that correlated with efficacy. However, we found striking and consistent differences in graft neurite outgrowth between the two culture timepoints. The functional grafts from d18 cells had more outgrowth than non-functional d25 grafts. A time course of gene expression during differentiation of the cell lines revealed differences in genes associated with DA neuron development and neurite plasticity. These results are the first to demonstrate that graft-host integration, and not DA neuron content, is key to graft-induced functional recovery. The gene expression profiling may offer insight into the optimal developmental stage for graft efficacy.

neuroscience↗