Figure 1. Protocol Schematic for the STEMdiff™ Choroid Plexus Organoid Differentiation and Maturation Kits

Choroid plexus organoids can be generated from human embryonic stem (ES) or induced pluripotent stem (iPS) cells in 30 days. The protocol begins with embryoid body (EB) formation, followed by expansion of neuroepithelia and patterning to choroid plexus-like epithelium. After a period of epithelial maturation including extensive bubbling, the organoids develop cystic structures surrounded by an ependymal epithelial layer and filled with a fluid resembling cerebrospinal fluid (CSF).

Figure 2. Organoids Generated Using the STEMdiff™ Choroid Plexus Organoid Differentiation and Maturation Kits Exhibit Fluid-Filled Cysts and Ependymal Epithelial Morphology

(A) Phase-contrast image of a whole choroid plexus organoid at day 40 using the STEMdiff™ Choroid Plexus Organoid Differentiation and Maturation Kits. Organoids exhibit translucent cysts containing a CSF-like fluid. (B) Epithelia on choroid plexus organoids exhibit a tightly packed and cuboidal morphology typical of ependymal cells that line the brain ventricles in vivo.

Figure 3. Choroid Plexus Organoids Express Markers of Ependymal Epithelium and Show Limited Expression of Neuroepithelial Markers

Immunohistological analysis on whole cleared and stained (Masselink et al. 2019) choroid plexus organoids at day 40 reveals that their cystic structures are (A) TTR- (green) and (B) CLIC6- (red) positive. (C) These ependymal cell markers are highly co-localized. (D) Bundles of MAP2-positive (magenta) neurons are observed but are not co-localized with the ependymal cell markers.

Figure 4. Fluid Extracted from Cysts in Choroid Plexus Organoids is Enriched with Clusterin Protein, A Marker of Cerebrospinal Fluid (CSF)

CSF-like fluid was extracted from cysts contained in day 40 choroid plexus organoids using a 28G syringe. A western blot was performed on the extracted fluid to detect Clusterin and shows a band between the 37 and 50 kDa molecular weight marker. Clusterin is a soluble secreted chaperone protein found in high abundance in CSF.