Abstract
Purpose :
Retinal organoids derived from stem cells are valuable tools for studying human retinal development, modeling retinal disease, and testing putative therapeutic agents. The model is hindered by a spherical geometry that limits experimental access to the lumen of the organoid and prevents co-culture with RPE. We describe a scaffold that supports the differentiation of a planar retinoid.
Methods :
The scaffold was manufactured from gelatin, chondroitin sulfate, and hyaluronic acid (GCH), which was then decorated with laminin 521 (GCH-521). Retinal progenitor cells were differentiated from human induced pluripotent cells and seeded on the scaffold before retinal cell types began to differentiate. In some experiments, the RPC cultures were layered on a sheet of RPE that was also derived from stem cells. Cultures were monitored by confocal microscopy and quantitative, real-time RT-PCR. For the co-culture experiments the transepithelial electrical resistance (TER) was measured.
Results :
Laminin-521 promoted the adherence of RPC to the scaffold and proliferation prior to differentiation. Laminin-521 promoted differentiation and provided a polarity cue to create a laminated sheet with ganglion-like cells (HuC/D, BRN3 positive) near the scaffold and recoverin positive cells on the free surface. Some cells at the surface expressed red-green opsin with long, tapering processes that resembled cone outer segments. Although rhodopsin positive cells were also detected, the cells exhibited long, thin extensions that did not resemble rod outer segments. The ganglion-like cells survived up to 10 months in culture. Co-culture with RPE failed to produce retinal lamina in the absence of laminin-521. RPE increased the thickness of the recoverin-positive layer and sharpened the boundary between layers. Co-culture also increased the TER and altered the transcriptome for both the RPE and “neurosensory retina” portions of the culture.
Conclusions :
Laminin-521 provided polarity cues to organize the differentiating RPC into layers, while RPE promoted the growth of retinal layers. RPE and neurosensory retinal cells influenced the maturation of each other.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.