Inherited and acquired diseases of the neural retina (NR) and/or retinal pigment epithelium (RPE) are a significant issue in human health and quality of life. Stepwise retinal differentiation of human pluripotent stem cells (hPSCs) can provide a model system to study human retinal development and supply cells for the potential treatment of debilitating retinal diseases. We have shown that two types of hPSCs, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), can differentiate along the retinal lineage in a manner that closely parallels normal human retinogenesis. However, little is known about the factors and events that influence key steps in the differentiation of these cell types from hPSCs. Our long-term mission is to define molecular mechanisms of retinal cell fate determination from hPSCs and apply this knowledge to the study and treatment of human developmental and degenerative retinal disorders. An important step in vertebrate retinogenesis occurs during the optic vesicle (OV) stage, when cells make the seminal decision to develop either as a neural retinal progenitor cell (NRPC) or an RPE cell. VSX2 is the earliest known marker of NRPCs and is hypothesized to pattern the naove OV into the NR and RPE domains by repressing expression of the early RPE-associated gene MITF. Disruption of Vsx2 expression in animal models by various means causes severe defects of the eye and retina, and humans with mutations in this gene exhibit microphthalmia and malformed retinas. Despite the critical roles attributed to VSX2 during retinal development, there is scant information available on its mechanisms of action and regulation in humans. Our unique capability to culture human cell populations from the earliest stages of retinogenesis and to isolate OV- like structures provides a pertinent in vitro model system to study VSX2 function in human retinal cell development. The primary objectives of this proposal are to a) determine the purpose and necessity of VSX2 in the initial production of retinal cell types from hPSCs and b) identify endogenous hPSC signaling mechanisms that control VSX2 expression during differentiation. Experiments are designed to investigate the overall hypothesis that VSX2 expression in hPSC-derived retinal cultures leads to maintenance of a proliferating pool of NRPCs at the expense of RPE. To test this theory, we will pursue the following specific aims: 1. Determine the gene regulatory roles and mechanisms of VSX2 during the early production and proliferation of NRPCs in differentiating hPSCs. 2. Define the ligands and pathways that mediate the reciprocal effects of FGF and TGF2 signaling in the regulation of early VSX2 expression in differentiating hPSCs. 3. Determine the effects of a naturally occurring, human VSX2 mutation on the establishment and expansion of NRPC vs. RPE cell populations using patient-derived hiPSCs.
The purpose of this proposal is to advance understanding of the development of the human retina. By using our stepwise method to coax stem cells to become a full range of retinal cell types in a culture dish, we can model how the building blocks of the human retina are produced, beginning at their most primitive stages. Such a model will have great value in the study of basic mechanisms of retinal development and the causes of inherited eye diseases, and will complement our efforts to develop cell-based therapies for degenerative retinal disorders.
|Capowski, Elizabeth E; Wright, Lynda S; Liang, Kun et al. (2016) Regulation of WNT Signaling by VSX2 During Optic Vesicle Patterning in Human Induced Pluripotent Stem Cells. Stem Cells :|
|Wright, Lynda S; Pinilla, Isabel; Saha, Jishnu et al. (2015) VSX2 and ASCL1 Are Indicators of Neurogenic Competence in Human Retinal Progenitor Cultures. PLoS One 10:e0135830|
|Singh, Ruchira; Kuai, David; Guziewicz, Karina E et al. (2015) Pharmacological Modulation of Photoreceptor Outer Segment Degradation in a Human iPS Cell Model of Inherited Macular Degeneration. Mol Ther 23:1700-11|
|Phillips, M Joseph; Perez, Enio T; Martin, Jessica M et al. (2014) Modeling human retinal development with patient-specific induced pluripotent stem cells reveals multiple roles for visual system homeobox 2. Stem Cells 32:1480-92|
|Zhong, Xiufeng; Gutierrez, Christian; Xue, Tian et al. (2014) Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun 5:4047|
|Capowski, Elizabeth E; Simonett, Joseph M; Clark, Eric M et al. (2014) Loss of MITF expression during human embryonic stem cell differentiation disrupts retinal pigment epithelium development and optic vesicle cell proliferation. Hum Mol Genet 23:6332-44|
|Wright, Lynda S; Phillips, M Joseph; Pinilla, Isabel et al. (2014) Induced pluripotent stem cells as custom therapeutics for retinal repair: progress and rationale. Exp Eye Res 123:161-72|
|Singh, Ruchira; Phillips, M Joseph; Kuai, David et al. (2013) Functional analysis of serially expanded human iPS cell-derived RPE cultures. Invest Ophthalmol Vis Sci 54:6767-78|
|Singh, Ruchira; Shen, Wei; Kuai, David et al. (2013) iPS cell modeling of Best disease: insights into the pathophysiology of an inherited macular degeneration. Hum Mol Genet 22:593-607|
|Gamm, David M; Wright, Lynda S (2013) From embryonic stem cells to mature photoreceptors. Nat Biotechnol 31:712-3|
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