Abstract
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. Although the inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging. To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. We show that these markers can be used to isolate RPE cells during in vitro differentiation and to track, quantify and improve differentiation efficiency. Finally, these surface markers aided to develop a robust, direct and scalable monolayer differentiation protocol on human recombinant laminin-111 and −521 without the need for manual isolation.
Highlights
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration
After 3 weeks of culture, optical vesicles emerge from the embryoid body (EB) containing the pigmented RPE cells mixed with other cell types
The second screen identified additional markers that labeled the more mature and pure human pluripotent stem cells (hPSCs)-RPE cells, such as CD10419, but neither of these markers were detected in a significant fraction of the RPE in the early optical vesicle stage, suggesting that these would be less useful to track emergence of RPE cells
Summary
In vitro differentiation of human pluripotent stem cells into functional retinal pigment epithelial (RPE) cells provides a potentially unlimited source for cell based reparative therapy of age-related macular degeneration. The inherent pigmentation of the RPE cells have been useful to grossly evaluate differentiation efficiency and allowed manual isolation of pigmented structures, accurate quantification and automated isolation has been challenging To address this issue, here we perform a comprehensive antibody screening and identify cell surface markers for RPE cells. Such manual selection makes large-scale production of hPSC-RPE cells cumbersome and carries a potential risk of tumorigenicity, if residual undifferentiated cells remain undetected in the final product From this perspective, it would be useful to have cell surface markers, which would allow both prospective isolation of hPSC-RPE in an automated manner and quantitative analysis of RPE purity and absence of unwanted cell types, such as undifferentiated cells and alternative lineages that could emerge during the differentiation process. Using these markers together with single-cell RNA-sequencing to evaluate the differentiation process, we have established an efficient xeno-free and defined monolayer differentiation methodology, where culture on supportive human recombinant laminin (hrLN) eliminates the need for manual selection, allowing large-scale production of pure hPSC-RPE
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