All ETDs from UAB

Advisory Committee Chair

Lawrence J Delucas

Advisory Committee Members

Alecia Gross

Kejin Hu

Ho-Wook Jun

Shu-Zen Wang

Document Type

Thesis

Date of Award

2016

Degree Name by School

Master of Science in Biomedical Engineering (MSBME) School of Engineering

Abstract

Membrane proteins imperative for cell signal transduction, G-protein coupled receptors (GPCRs) are critical for drug discovery. Unfortunately, sufficient amounts of quantifiable protein cannot be produced using existing expression systems. Nature has an ideal solution—rhodopsin; the GPCR rhodopsin is efficiently and abundantly expressed in rod outer segments (ROS) of photoreceptor cells. As part of this study, human embryonic stem cells (hESC) were cultured on extracellular matrix (ECM) derived substrates and differentiated towards a photoreceptor lineage using conventional basic fibroblast growth factor (bFGF) and novel NM23 growth factor methods. NM23 is the natural ligand of MUC1*, which is surface accessible on stem cells, and has been shown to increase growth and inhibit differentiation without use of bFGF. In accordance with retinal developmental cues, traditional bFGF-grown cells expressed Rx and Pax6 by differentiation day 9, Mitf and Chx10 by differentiation day 27, and Crx and Red-green opsin by differentiation day 75. However, the bFGF-grown retinal pigmented epithelial cells (RPE) began to detach from the poly-d- lysine (PDL) substrate around differentiation day 70 and the novel NM23 growth factor line could not be completed due to production issues. This necessitated the investigation shift towards elucidating the RPE-substrate attachment mechanism. In nature, developing RPE cells attach to the basement membrane layer of the Bruch’s membrane. Due to the incongruence of the negatively charged basement membrane and positively charged surfaces used in other protocols, attachment via charge was addressed. In addition to charge, integrin interactions with substrate ligands were investigated as a possible cause of RPE detachment. In this study, hESCs were grown on Bruch’s membrane basement layer-derived substrates and biomimetic surfaces to determine the role of substrate charge and/or composition and integrin expression in RPE development. Carboxymethlated (CM) dextran and dextrin coatings significantly augmented onset of pigmentation and final yield of pigmented cells when compared to PDL surfaces, suggesting RPE prefer negatively charged substrates. Near RPE detachment from PDL surfaces, the greatest differences in expression were seen in α11, αv, β1, and β5 integrins at differentiation day 60. Overall, these studies contribute to the selection of an ideal RPE differentiation material.

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