Diseases leading to photoreceptor (PR) degeneration constitute a leading cause of untreatable blindness worldwide. Human pluripotent stem cells (hPSCs) hold promise as tools for the development of novel therapies for these conditions. However, harnessing hPSC technology, either for transplantation or disease modeling, requires detailed knowledge of the cell populations they produce. Studies supported by our present R01 grant demonstrated that hPSC-PR lineage cells can be generated from 3D optic vesicle-like structures (OVs) using a scalable, GMP-compatible culture method. Additional reports defined roles for key transcription factors and explored mechanisms involved in neural retinal progenitor cell (NRPC) production, maintenance, and differentiation. These studies helped support the authenticity of hPSC-NRPCs, an important first step in the examination of their PR progeny. We seek to extend these findings by examining hPSC-PR heterogeneity in culture, investigating mechanisms governing their differentiation, and probing their authenticity. The primary objectives of the current proposal are to a) define and enrich for PRs and cones over time in hPSC-OV cultures, and b) direct cone fate via genetic manipulation of selected factors. We hypothesize that hPSC-OVs employ conserved PR differentiation mechanisms and possess early cone-selective markers, which together can be exploited to reduce culture complexity and to bias toward the production of cones. To test this theory, we will pursue the following aims: 1. Identify, quantify, and analyze neural retinal (NR) and Cone Rod Homeobox (CRX)-expressing cell populations generated over time from differentiating hPSC-OVs. Using a GMP-compatible hPSC line and a gene-targeted CRX-tdTomato hESC reporter line, we will perform the first high content imaging and single cell RNAseq analyses of differentiating hPSC-OVs and post-mortem human NR tissue to monitor and compare temporal changes in NR and CRX+ cell composition and maturation states. 2. Enrich for hPSC-derived cones using cell-selective markers. Single cell data from differentiating hPSC- OVs will be mined for known and novel gene and cell surface markers that can be used to define, monitor, and selectively enrich for cones at multiple stages of differentiation. 3. Utilize patient-specific and gene-edited hiPSCs to selectively overproduce cone subtypes and examine mechanisms underlying PR differentiation. Using information and tools generated from Aims 1 and 2, null NRL hiPSCs and CRISPR/Cas9-modified hPSCs will be tested to ascertain their suitability to produce S or M/L cone-enriched populations to study PR differentiation and/or serve as robust cell sources for future cone replacement strategies. Successful completion of these aims will improve the utility of hPSC-PRs for research and clinical applications and initiate production of a human cone ?toolkit? for widespread use by the scientific community.
The purpose of this proposal is to advance understanding of photoreceptor production in human pluripotent stem cell-derived 3D optic vesicle (or retinal organoid) structures, with a particular emphasis on the derivation, enrichment, and interrogation of cone subtypes. By rigorously probing the authenticity of these critical retinal cells and developing novel tools for use by the scientific community, we aim to improve the utility of hPSC-PRs and cones in basic and translational research. Successful completion of our aims will support efforts to employ this powerful technology to study, model, and treat human photoreceptor degenerative diseases.
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