THE PERSISTENCE OF NEUROEPITHELIAL PROGENITORS IN NEOCORTICAL ORGANOIDS: IMPEDING ACCURATE MODELS FOR DRUG SCREENING IN NEURODEVELOPMENTAL DISORDERS

Human induced pluripotent stem cell (hiPSC)-derived brain organoids represent a human, druggable model system with growing potential for neuropsychiatric disease modeling and pharmacologic screening. Because organoids can be generated in high throughput, they offer a scalable platform to test compounds across diverse genetic backgrounds. However, the fidelity of drug responses depends critically on whether organoids accurately reproduce the cellular composition and maturation trajectories of the human brain. In organoids and also in fetal development, neocortical neurons arise from neuroepithelial progenitors, which differentiate into radial glia (stem cells of the neural cortex) before taking on terminal neuronal cell fates of the laminar cortex. Using hiPSCs from 5 unique donors, we differentiated our stem cells using a 3D neocortical organoid protocol and using snRNA-sequencing to profile the cells at 5 points in time (i.e. Day, 10, Day 20, Day 35, Day 50, Day 90) spanning early neuroepithelial induction to neuronal differentiation. Unexpectedly, we identified a large population of early neuroepithelial progenitors that persist through Day 90—well beyond their disappearance in the corresponding in vivo developmental window, when radial glia should dominate the progenitor pool. Using palantir trajectory analysis, we computed fate transition probabilities for each cell and defined a “stalling probability” representing the likelihood of maintaining one’s progenitor identity. Across multiple cell classes, we identified distinct “stalling” and “non-stalling” subpopulations, each with unique molecular signatures. Then, using differential gene expression testing within each cell class, we identified key molecular regulators that drive “stalling” or that permit differentiation. Consistent with prior literature, MAPK signaling emerged as a regulator of stalling in radial glia. Our analysis implicated previously undescribed roles for canonical and non-canonical Wnt pathway in early and mature neuroepithelium stalling. These findings highlight intrinsic regulatory mechanisms that may limit maturation in brain organoids, thereby constraining their translational utility for pharmacologic testing. Ongoing experiments are assessing how pharmacologic modulation of Wnt signaling alters early and mature neuroepithelial persistence and overall cell-type diversity. By improving the fidelity of cortical organoid differentiation, we aim to generate models that better recapitulate in vivo developmental trajectories and enhance the reliability of high-throughput drug screening for psychiatric and neurologic disorders.