Osmolarity Responses of ChP Brain Organoids as an in vitro Model of Syringomyelia

Introduction: Syringomyelia (SM), a chronic neurological disorder that impacts the spinal cord, occurs when a syrinx, or a cyst filled with cerebrospinal fluid (CSF), forms within the spinal cord.1 CSF, which surrounds and protects the brain and spinal cord, assists the brain by providing protection, nourishment, and waste removal.2 The syrinx becomes the focal point for therapeutic action because the shrinking and/or removal of the syrinx could prevent long term neurological damage. The Leipzig lab has recently found via in vitro and in vivo models that a primary response to osmotic disturbances is to upregulate betaine syntheses via choline dehydrogenase (CHDH) alongside extracellular segregation via betaine transporter BGT-1.3-5 However, an unmet need exists to create a relevant human in vitro model to better investigate, the mechanisms and treatments of SM. 3D cell culture utilizing human stem cells, like embryoid bodies (EBs) and organoids, have become methods to create physiologically relevant models for various human diseases. CSF-like fluid-producing choroid plexus (ChP)-specific brain organoids have been developed to investigate the role of CSF in fluid associated disorders in the CNS, however, applications to SM are needed. The objective of this research was to develop a CSF-like fluid-producing ChP brain organoid model from human induced pluripotent stem cells (hiPSCs) and to create an in vitro model of SM to enable study of fluid osmoregulation related responses. Our primary hypothesis was that hyperosmotic conditions would upregulate CHDH and BGT-1 expression alongside water channels (AQP1/4) to reverse imposed osmotic imbalances.3-5

Materials and

Methods: hiPSCs (iPS(IMR90)-4) were cultured and maintained in mTeSRTM on Matrigel® coated 6-well plates until ChP Organoid formation.

ChP organoid formation was initiated by seeding iPSCs into a round bottom low-attachment 96 well-plate at 2,500 cells/well to form EBs. (Figure 1A) Various media formulations were used over the course of the ChP Organoid formation. ChP organoids were evaluated for ChP markers (CLIC6 and TTR) on day 45. (Figure 1A)

Osmolality analysis was performed on day 60 and day 90. The osmolality of the pocket fluid and surrounding culture media compared to fresh media was performed on a vapor pressure osmometer (Vapro 5600). Osmotic stress was induced on ChP organoids with visible cystic structures. Three osmotic conditions – isotonic (CDM+A media), hypotonic (1: 1: CDM+A media:water), and hypertonic (0.5% NaCl + CDM+A media) were selected. ChP organoids were placed in the respective medias overnight then fixed, embedded, sectioned, stained, and imaged to investigate the expression of CLIC6, TTR, BGT-1, CHDH, AQP1, and AQP4.

RNA sequencing was performed following the osmotic stress conditioning. ChP organoids with visible cysts were stored in RNA later at -80°C until RNA retrieval and purification. RNA from ChP organoids (n=6/osmotic condition) was purified via the Qiagen RNeasy® mini kit. Purified RNA was then transcribed into cDNA, which was then amplified, barcoded, and purified via the Oxford Nanopore cDNA-PCR Barcoding Kit V14 before running all cDNA samples on the Nanopore MinIon flow cell.

Results, Conclusions, and Discussions: ChP brain organoids with CSF-like producing cysts were generated from hiPSCs. At various stages during the process, the formation and growth of cysts were visually observed. This was further confirmed via IHC as the ChP organoids were positive for choroidal epithelial-specific markers CLIC6 and TTR by day 45 (Figure 1B). CLIC6 and TTR were highly expressed in both the ChP organoid body and on the edges of the fluid pockets confirming previous reports.6 Osmolality analysis of the fluid from the ChP organoids was measured at ~307 mmol/kg on day 60 and ~313 mmol/kg on day 90, no significance was found for the osmolality of the pocket fluid, spent media, and fresh media (Figure 1C). These values were like human CSF, which ranges from 280 to 300 mmol/kg.7,8 IHC also showed the expression of BGT-1 and CHDH following osmotic stress conditions (Figure 1D). Interestingly, both BGT-1 and CHDH were upregulated with hypertonic conditions and localized to the epithelial layer, as compared to the isotonic and hypotonic conditions. BGT-1 upregulation indicated that the channel would be working to equalize the osmolality by transporting the betaine from the pocket and into the surrounding media. Similarly, expression of the water channels AQP1 and AQP4 were investigated via IHC (Figure 1E). AQP1, has an active role in water movement through the transepithelium in the ChP, and was upregulated after hypertonic conditions as compared to isotonic and hypotonic conditions. AQP4 was only upregulated after hypertonic conditioning, with no expression was seen in the isotonic and hypotonic conditions. Upregulation of both AQP1 and AQP4 indicates an increase in water transport across the cystic membranes due to an osmotic disturbance. Interestingly, these responses are like our previous studies with SM animal models3-5 and highlight the need to further investigate the mechanisms of betaine in osmoregulatory responses.

Overall, ChP brain organoids with cysts were synthesized from hiPSCs and used to investigate how different osmotic conditions impacted the physiology of organoids as an in vitro model for SM. Continuing work involves performing transcriptomics analysis on the ChP brain organoids after osmotic conditioning to further understand the mechanistic actions of SM.

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