The retinal pigment epithelium (RPE) is a monolayer of cuboidal shaped cells that provide essential support to photoreceptors in the neural retina. RPE functions include the vectorial transport of ions and water between the choroid circulation and retina, light absorption, and phagocytosis of light damaged photoreceptor outer segments. Loss or dysfunction of RPE cells induces devastating secondary effects on photoreceptors and is responsible for inducing some forms of age related macular degeneration (AMD), a progressive blinding disorder that is the leading cause of blindness in industrialized countries. The transplantation of RPE in rat AMD models and in human AMD patients has been shown to stem vision loss and inhibit further degeneration. RPE can be generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) although current methods are slow and the yields are low. If derivation times could be accelerated, and if other concerns about iPSCs such as the oncogenic potential of the transcription factors used to induce pluripotency could be alleviated, they would serve as an excellent source of patient-matched RPE cells. Clues for how to enhance iPSC-RPE derivation can be obtained from developmental studies, and critical steps of RPE development can be recapitulated in vitro by activating key signaling pathways that regulate RPE-promoting gene networks. The Wnt/ss-catenin pathway is an excellent iPSC-RPE enhancing candidate. I have shown that Wnt/ss-catenin signaling is required for RPE cell-fate maintenance and that ss-catenin can directly regulate the key transcription factors Mitf and Otx2. I hypothesize that activating the Wnt/ss-catenin pathway in iPSCs will greatly enhance RPE differentiation. We have derived RPE from hESCs and iPSCs induced to pluripotency using all 4 Thomson factors and with only two (Klf4 and Oct4) and biomicking small molecules. I have preliminary evidence that 1-factor iPSC (Oct4 only) is also competent to generate RPE. I will use well-established assays to characterize the 1-factor iPSC-RPE cells and determine if they are correctly polarized and electrically coupled, and if they express correct gene profiles. I will also transplant the derived cell into rat disease models and monitor rescue. This project offers me the unique opportunity of extending my thesis work into a study of translation medicine and to enhance my understanding of retinal physiology.

Public Health Relevance

The derivation of retinal pigment epithelium (RPE), a critical supporting tissue for the neural retina, from stem-cells and transplantation into human patients with diseases such as age-related macular degeneration (AMD) is a promising therapeutic intervention to halt or slow photoreceptor degeneration. I am working to accelerate derivation times of RPE from induced pluripotent stem-cells and increase the yield by recapitulating embryonic development and activating the Wnt/ss-catenin pathway, which I have shown in my thesis project to be required to maintain RPE fate. I will also characterize the derived RPE cells to ensure that they are properly differentiated and transplant them into RPE-mediated rat photoreceptor degeneration models to determine if symptoms can be halted or slowed.

Agency
National Institute of Health (NIH)
Institute
National Eye Institute (NEI)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
5F32EY021416-03
Application #
8389544
Study Section
Special Emphasis Panel (ZRG1-F05-C (20))
Program Officer
Agarwal, Neeraj
Project Start
2010-12-15
Project End
2013-12-16
Budget Start
2012-12-17
Budget End
2013-12-16
Support Year
3
Fiscal Year
2013
Total Cost
$52,190
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Westenskow, Peter D; Kurihara, Toshihide; Friedlander, Martin (2014) Utilizing stem cell-derived RPE cells as a therapeutic intervention for age-related macular degeneration. Adv Exp Med Biol 801:323-9
Kurihara, Toshihide; Westenskow, Peter D; Friedlander, Martin (2014) Hypoxia-inducible factor (HIF)/vascular endothelial growth factor (VEGF) signaling in the retina. Adv Exp Med Biol 801:275-81
Westenskow, Peter D; Kurihara, Toshihide; Aguilar, Edith et al. (2013) Ras pathway inhibition prevents neovascularization by repressing endothelial cell sprouting. J Clin Invest 123:4900-8
Ukrohne, Tim U; Westenskow, Peter D; Kurihara, Toshihide et al. (2012) Generation of retinal pigment epithelial cells from small molecules and OCT4 reprogrammed human induced pluripotent stem cells. Stem Cells Transl Med 1:96-109
Kurihara, Toshihide; Westenskow, Peter D; Krohne, Tim U et al. (2011) Astrocyte pVHL and HIF-* isoforms are required for embryonic-to-adult vascular transition in the eye. J Cell Biol 195:689-701