DEVELOPMENT OF A FREE EMBEDDING PROTOCOL FOR CHOROID PLEXUS-CORTICAL ORGANOIDS

Abstract

The choroid plexus (ChP) is a specialized tissue in the brain responsible for the production and secretion of CSF, the maintenance of the blood-CSF barrier, and immune surveillance. While it has been reported to be involved in several neurological disorders, it remains poorly studied because of a lack of physiologically relevant human brain models. Brain organoids have emerged as powerful in vitro systems, but most protocols rely heavily on Matrigel embedding, an undefined extracellular matrix that introduces variability and limits reproducibility. In this study, hPSCs were driven towards neuroectodermal fate, followed by the formation of neural spheroids. These were matured under varying soluble Matrigel concentrations and patterning conditions to induce forebrain and hindbrain ChP. The role of BMP4, CHIR, and SHH signaling in ChP specification was evaluated using morphological assessment, immunostaining, and gene expression analysis. The ChP generation was achieved under optimized patterning conditions, as shown by expression of TTR, where 1% Matrigel provided the most favorable environment. In addition, in an attempt to generate ChP hindbrain, organoids treated with increased SHH concentrations resulted in spatially distinct regions within the organoids, suggesting an interaction between SHH and Wnt signaling. Overall, this study developed a free embedding protocol for generating ChP in cortical organoids. This model provides a physiologically relevant platform with potential various applications such including but not limited to studying human brain development, neurological disease mechanisms, and drug testing.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences