Iskandar, B. J. | Search bioRxiv

bioRxiv2024

RosetteArray Platform for Quantitative High-Throughput Screening of Human Neurodevelopmental Risk

Neural organoids have revolutionized how human neurodevelopmental disorders (NDDs) are studied. Yet, their utility for screening chemical hazards and prospective therapeutics for NDDs is limited by a lack of morphological reproducibility and cost-effective scalability. Here, we describe the RosetteArray platform, which can be used as an off-the-shelf, 96-well plate assay that standardizes incipient forebrain and spinal cord organoid morphogenesis as adherent, micropatterned, 3-D, singularly polarized neural rosette tissues ([~]200 and [~]800 per well, respectively). Seeded directly from cryopreserved human pluripotent stem cells, RosetteArrays are cultured over 6-8 days and fixed, immunostained and imaged in situ to enable artificial intelligence-based quantitative analysis. By screening the inception of [~]75,000 neural organoids throughout this manuscript, we provide proof-of-concept demonstrations of the platforms utility for detecting developmental neurotoxicity hazard and screening genetic and environmental factors known to cause clinical Neural Tube Defect risk. Given the documented perturbation of rosette morphogenesis in neural organoid models of several NDDs, the RosetteArray platform could enable quantitative high-throughput screening (qHTS) of human neurodevelopmental risk across regulatory and precision medicine applications.

neuroscience↗

bioRxiv2023

Genetic dysregulation of an endothelial Ras signaling network in vein of Galen malformations

To elucidate the pathogenesis of vein of Galen malformations (VOGMs), the most common and severe congenital brain arteriovenous malformation, we performed an integrated analysis of 310 VOGM proband-family exomes and 336,326 human cerebrovasculature single-cell transcriptomes. We found the Ras suppressor p120 RasGAP (RASA1) harbored a genome-wide significant burden of loss-of-function de novo variants (p=4.79x10-7). Rare, damaging transmitted variants were enriched in Ephrin receptor-B4 (EPHB4) (p=1.22x10-5), which cooperates with p120 RasGAP to limit Ras activation. Other probands had pathogenic variants in ACVRL1, NOTCH1, ITGB1, and PTPN11. ACVRL1 variants were also identified in a multi-generational VOGM pedigree. Integrative genomics defined developing endothelial cells as a key spatio-temporal locus of VOGM pathophysiology. Mice expressing a VOGM-specific EPHB4 kinase-domain missense variant exhibited constitutive endothelial Ras/ERK/MAPK activation and impaired hierarchical development of angiogenesis-regulated arterial-capillary-venous networks, but only when carrying a "second-hit" allele. These results illuminate human arterio-venous development and VOGM pathobiology and have clinical implications.

genomics↗