Date of Award

January 2012

Document Type

Thesis

Degree Name

Medical Doctor (MD)

Department

Medicine

First Advisor

Larry J. Rizzolo

Second Advisor

Ron A. Adelman

Subject Area(s)

Cellular biology, Biomedical engineering

Abstract

As a step towards engineering an outer retina suitable for transplantation, we designed a biocompatible, biodegradable scaffold that allows retinal progenitor cells (RPCs) to form flat, laminar structures. The scaffold minimizes the exposure of retinal progenitors to extracellular matrix components that are not found in the retina, and enables co-culture with the retinal pigment epithelium. Scaffolds were formed from electrospun fibers of polycaprolactone (PCL). Scaffold thickness was varied by increasing the duration of collection during electrospinning; porosity and pore size distribution was adjusted by varying polymer concentration in solution. The porosity and thickness of the sheets were varied to optimize cell to cell contact formation and PCL degradation rate, respectively. RPCs derived from human embryonic stem cells (hESC-derived RPCs) were cultured to form neurospheres. Dissociated neurospheres were seeded onto +/- laminin coated PCL sheets in normal or low O2 incubators, and maintained in two types of serum free medium. Fluorescence labeling and confocal microscopy were used to assess tissue morphology. Individual PCL fibers were ~3 ìm thick, sheets varied from 20-200 ìm in thickness, and pores ranged from 5-100 ìm in diameter. The scaffolds degraded over 4-7 weeks. Scaffolds with pores 25-50 ìm in diameter were subsequently chosen for culture experiments. Biomechanical strength testing estimated a Young's modulus of 0.071 megapascals (MPa). Confocal imaging confirmed that the RPCs penetrated the thickness of the scaffold irrespective of laminin coating and O2 level, and continued to express retinal markers such as Pax6, recoverin, and N-cadherin, with heightened expression in the differentiation medium. Cells adhered uniformly to laminin-coated PCL, but formed aggregates on uncoated PCL. Staining for the proliferative marker, Ki67, indicated active cell division in both medium types and O2 levels. Preliminary RPE: RPC co-culture experiments with laminin coated PCL in low oxygen conditions revealed polarization of N-cadherin, with concomitant stress fiber formation in the RPE monolayer. Overall, electrospun PCL polymers sustained the differentiated properties acquired by neurospheres and appear to be suitable scaffolds for reconstructing an outer retinal layer.

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