Tissue Engineering Using Mouse Retinal Progenitor Cells and a Novel Biodegradable Poly(e-caprolactone) Nanofiber Scaffold

Abstract

Purpose: : To evaluate the survival and differentiation of mouse retinal progenitor cells (mRPCs) cultured on smooth and nanowire poly(e-caprolactone) (PCL) scaffolds and to analyze the behavior of mRPCs on these scaffolds when placed in explant co-culture.

Methods: : GFP-positive mRPCs were cultured on smooth, 2.5µm and 27µm nanowire template synthesized PCL scaffolds for up to 7 days. Cell adhesion, proliferation and viability on, PCL scaffolds were determined by quantitative fluorometric and luminescence analysis. Differentiation was analyzed using immunohistochemistry and RT-PCR. Smooth 2.5µm and 27µm nanowire length scaffolds with adherent mRPCs were then co-cultured with C57bl/6 and rho -/- mouse retinal explants for one week or transplanted into C57bl/6 and rho -/- mice for 30 days. Retinal explants and transplants were cryosectioned in 12µm sections for analysis. Retinal sections containing GFP-positive mRPCs were imaged to measure survival, migration, and differentiation.

Results: : mRPC adhesion, proliferation and viability were similar in culture on each type of PCL scaffold. Immunohistochemistry revealed that PCL scaffold topology influenced protein expression levels in mRPCs. mRPCs cultured on nanowire scaffolds showed increased expression of PKC-alpha and recoverin. RT-PCR revealed clearly down-regulated Pax6, Hes1, beta-3-tubulin, DCX and partially down-regulated nestin and Sox2. The primary up-regulated gene was GFAP. Scaffolds seeded with mRPCs and co-cultured on retinal explants or transplanted into the subretinal space allowed for the delivery and migration of mRPCs to each retinal layer.

Conclusions: : Cultured progenitor cells can be combined with template-synthesized PCL substrates for the generation of tissue equivalents in culture. Growth on laminin-coated PCL enhances adherence and promotes differentiation. mRPCs delivered using these polymers migrate into the host retina and extend processes. PCL nanowire scaffolds provide a biomimetic environment for tissue engineering and transplantation into the diseased eye.

Keywords: regeneration • photoreceptors • transplantation