Date of Award

January 2015

Document Type

Open Access Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Lawrence J. Rizzolo

Second Advisor

Ron A. Adelman

Subject Area(s)

Ophthalmology, Cellular biology

Abstract

In order to restore vision in the late stages of retinal degeneration, it is necessary to address loss of cells from both the retinal pigment epithelium (RPE) and the neural retina. As a step towards generating neural retinal tissue for translational studies, we evaluated a biodegradable polymer scaffold as a three-dimensional vehicle for the directed differentiation of H9 human embryonic stem cells (H9-hESC) into neural retina. Polymer scaffolds were electrospun from 14% w/v polycaprolactone (PCL), approximated the thickness of native neural retina, and consisted of loose, randomly oriented fibers of subcellular diameter. H9-hESC were seeded to PCL scaffolds, cultured in retinal differentiation media, and compared by immunofluorescence and quantitative real-time RT-PCR (qRT2-PCR) to H9-hESC differentiated on porous Transwell filters. H9-hESC cultured on PCL scaffolds migrated up to 40 microns into the scaffold and expressed markers consistent with cellular proliferation and differentiation into neural retina. Cultures on PCL scaffolds showed equivalent or increased expression of neural retinal markers compared to those grown on Transwell filters. We then co-cultured H9-hESC differentiated on PCL scaffolds with monolayers of RPE and found that each tissue affects the other's maturation. Co-cultured neural retina expressed higher levels of retinal maturation markers, and the transepithelial resistance (TER) of co-cultured RPE was more consistent with physiologic values. Finally, we found that TRP channels expressed in RPE localize to various subdomains of the RPE apical membrane, where they potentially mediate RPE-retinal interactions by sensing the ionic composition of the subretinal space.

Comments

This is an Open Access Thesis.

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