Abstract
<title>Abstract</title> <p> Background Syringomyelia (SM) is a fluid-associated disorder characterized by a fluid-filled cavity, known as a syrinx, inside the spinal cord caused by cerebrospinal fluid (CSF) displacement. The clinical standard for treating SM is surgery, as molecular signatures are not fully understood. Human choroid plexus (ChP) brain organoids harness the ability to produce CSF-like fluid pockets that could serve as an answer to understanding human SM pathophysiology as a lack of <italic>in vitro</italic> models exists to investigate mechanisms of human syrinx formation/expansion to unlock molecular understanding for syrinx shrinking. Methods Human ChP brain organoids were created from human induced pluripotent stem cells (hiPSCs) and the CSF-like fluid osmolality was characterized. Under different osmotic stress conditions – isotonic, hypertonic, and hypotonic – the regulation of betaine contributing factors, water and ion transporters was investigated in response to the osmotic stress. Immunohistochemistry (IHC), transcriptomics, and osmolality analysis were performed on all human ChP brain organoids. Results We demonstrated that human ChP brain organoids could be formed from hiPSCs and develop CSF-like fluid producing pockets, shown by IHC (TTR, CLIC6). The osmolality of the CSF-like fluid showed physiological relevance to human CSF, indicating this organoid system as a viable <italic>in vitro</italic> SM model. After osmotic stress was induced, IHC and transcriptomics showed upregulation of betaine contributing factors (BGT-1/SLC6A12, CHDH), water transporters (AQP1, AQP4), and ion transport (KCC4/SLC12A7) for hypertonic or hypotonic conditions compared to isotonic. Bulk RNA-sequencing transcriptomics indicated upregulation in pathways related to betaine, osmotic, transport, and CSF-like production for human ChP brain organoids in response to osmotic stress, a possible narrowing down of molecular etiology on how to manipulate the syrinx. Conclusions In summary, these results suggest the human brain ChP organoid system can be an <italic>in vitro</italic> model for studying SM and potential therapies as these results highlight key molecular players that could be targeted for non-surgical ( <italic>i.e.</italic> , pharmaceutical) interventions for syrinx removal. </p>