Date of Degree
Access restricted until 02/23/2018
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Budd A. Tucker
Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the Western world. Although, the majority of stem cell research to date has focused on production of RPE and photoreceptor cells for the purpose of evaluating disease pathophysiology and cell replacement, there is strong evidence that the choroidal endothelial cells (CECs) that form the choriocapillaris vessels are the first to be affected in this disease. As such, to accurately evaluate disease pathophysiology and develop an effective treatment, production of patient-specific stem cell-derived CECs will be required.
During the first stage of my Ph.D work, represented in Chapter 1 of this dissertation, I developed a co-culture system to differentiate mouse stem cells into CECs. I reprogrammed dermal fibroblasts from the Tie2-GFP mouse into two independent iPSC lines. TheTie2-GFP iPSCs were differentiated into CECs using a co-culture method with either the monkey RF/6A CEC line or primary mouse CECs. IPSC-derived CECs were characterized via rt-PCR and immunocytochemistry (ICC) for EC- and CEC-specific markers. The mouse iPSC-derived CECs described in Chapter 1 expressed the CEC-specific marker carbonic anhydrase IV (CA4), eNOS, FOXA2, PLVAP, CD31, CD34, ICAM-1, Tie2, TTR, VE-cadherin, and vWF. These Tie2-GFP iPSC-derived CECs paved the way for the rest of my Ph.D, in which I transitioned into using human iPSCs to generate patient-specific CECs.
During the second phase of my graduate work, presented in Chapter 3, I developed a novel stepwise differentiation protocol suitable for generating human iPSC-derived CECs. I used previously published RNA-seq data of the monkey CEC line, RF/6A and two statistical screens to develop media comprised of various protein combinations. In both screens, I identified connective tissue growth factor (CTGF) as the key component required for driving CEC development. I also found that a second factor, called TWEAKR, promoted iPSC to CEC differentiation by inducing endogenous CTGF secretion. CTGF-driven iPSC-derived CECs formed capillary tube-like vascular networks, and expressed the EC-specific markers CD31, ICAM1, PLVAP, vWF, and the CEC-restricted marker CA4. These patient-specific iPSC-derived CECs made it possible for me to proceed into the next phase of my Ph.D work, in which I started working with AMD patient-specific iPSC-derived CECs to evaluate AMD pathophysiology.
In the final stage of my Ph.D, represented in Chapter 4, I used the novel CEC differentiation method I developed to generate AMD iPSC-derived CECs and use these cells for AMD disease modeling. In line with previous studies that the membrane attack complex (MAC) forms in the AMD choriocapillaris, I showed that the AMD iPSC-derived CECs were much more susceptible to MAC formation and cell death when the cells were antagonized with complement components. I also demonstrated that, unlike the control CECs, the AMD CECs lost their capillary tube-like structures when the cells were cultured for over ten days, indicating that the AMD CECs may also exhibit other disease phenotypes other than susceptibility to MAC and cytolysis.
Overall, the work I present in this dissertation will help push the AMD research field forward by providing a way to directly study AMD patient-specific iPSC-derived CECs and how they differ from healthy iPSC-derived CECs. In combination with RPE and photoreceptor cells, these patient-specific iPSC-CECs will make it possible to study AMD patient-specific CECs in vitro to better understand AMD pathogenesis and to develop autologous cell replacement therapies to replenish patients’ damaged choroids with healthy CECs.
Copyright © 2016 Allison Elaine Songstad