Date of Degree
PhD (Doctor of Philosophy)
Molecular and Cell Biology
Tucker, Budd A.
First Committee Member
Drack, Arlene V.
Second Committee Member
McNamara, James O.
Third Committee Member
Fingert, John H.
Fourth Committee Member
McCray, Paul B.
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.
Age-related macular degeneration (AMD) is the leading cause of incurable blindness in western countries. In the United States alone, there are two million people with advanced AMD and an additional seven million with early AMD. Individuals with advanced AMD have severely impaired central vision, making everyday activities very difficult. Although AMD is a common blinding disease, it is not well understood how the disease progresses. There is strong evidence showing that the vasculature bed behind the retina is the first tissue to degenerate in AMD. Therefore, it is crucial to study these special blood vessels involved in AMD at a cellular level to identify its cause and develop cures to help patients regain their lost vision.
The work described in this dissertation used stem cell technology to make the special vascular tissue behind the retina. To our knowledge, this work is the first in the field to show this vascular tissue regenerated from stem cells of healthy individuals and AMD patients. The vascular tissue made from patient AMD stem cells also showed disease-like signs, suggesting that these AMD cells are able to recapitulate the disease in a laboratory setting. Therefore, this work provides an invaluable tool for researchers to perform in depth studies on these unique blood vessels that trigger AMD development. The groundbreaking approaches explained in this thesis have helped paved the way for researchers to build healthy retinas for AMD patients and to closely study AMD in the laboratory setting.
xxv, 147 pages
Includes bibliographical references (pages 130-147).
Copyright © 2016 Allison Elaine Songstad
Songstad, Allison Elaine. "Generating patient-specific induced pluripotent stem cell-derived choroirdal endothelium to study and treat macular degeneration." PhD (Doctor of Philosophy) thesis, University of Iowa, 2016.