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Lisa Ryne Conti, Monte J Radeke, Carolyn Radeke, Tyler Jean, Stephanie LaBouve, Pete Coffey; A flow cytometry sorting method for the production of high fidelity retinal pigmented epithelial cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):1137.
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© ARVO (1962-2015); The Authors (2016-present)
Establishing renewable sources of retinal pigmented epithelial cells (RPE) and creating patient-specific cell lines that produce high fidelity RPE is valuable to the field of degenerative eye research. Prevailing techniques for iPSC-derived RPE (iPSC-RPE) production rely on manual dissection or passive enrichment by passage. These methods are often labor intensive, variable and restricted to cell lines with a propensity to produce high quantities of pigmented cells. This study sought to determine whether granularity, conferred by pigment, is sufficient to purify iPSC-RPE cells from mixed differentiated cultures via flow cytometry.
Five iPSC lines were established from fRPE donor lines. Following differentiation using three methods, cells were dissociated and sorted based on side and forward scatter-properties. Pigmented cells were collected and, following an expansion step, transcriptome profiles were compared to their fRPE counterparts. Cultures were further subject to immunocytochemistry (ICC) characterization.
A single round of sorting yielded cell populations that formed uniform pigmented epithelial monolayers. Intra-donor specific transcriptome comparison between iPSC-RPE and fRPE cell lines revealed gene markers consistent with strong RPE cell signatures. In the few cases where sorted cultures displayed heterogeneous pigment patterns, contaminant cell marker signatures, confirmed with ICC, coincided. In these cases, a second round of sorting established culture purity. High fidelity, high purity RPE cultures were expandable up to 1000 fold. To date, we have applied this enrichment method to generate seven iPSC-RPE lines, producing banks that range from 3 to 55 million cells per line
Sorting based on pigment is an effective technique for enriching iPSC-RPE cell lines. Notably, sorting is applicable to iPSC lines with limited pigmented cell production, providing a resource where samples are scarce. Further, this technique can eliminate the need for cell line termination following failed enrichment attempts. In all, we have established a flow cytometry pigment based method for the reliable, user-independent production of high fidelity RPE.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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