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Petr Baranov, Gary Brooke, Sara Patel, Michael Young, John Sinden; Human retinal progenitor cells as a tool for retinal repair: establishing cell lines for clinical study. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4684.
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Loss of photoreceptors due to retinitis pigmentosa, age-related macular degeneration and other age, trauma and genetic-related retinal degenerative disorders leads to progressive loss of vision, and in some cases complete blindness. The successful application of allotransplantation of mouse photoreceptor precursors, isolated from developing retina or differentiated from pluripotent cell lines demonstrates the feasibility of restoration of retinal function through cell therapy. However, time- and resource-consuming protocols for photoreceptor differentiation from pluripotent cells, as well as the potential for tumor formation, currently limits the translational potential of this approach. An alternative strategy is to isolate mitotically active human retinal progenitor cells (hRPC) from developing retina and expand them in vitro to the quantities required for both preclinical characterization and clinical application.
We isolated hRPC from two separate sources (week 18 fetal retinae), expanded them under defined serum-free conditions in a low-oxygen environment, and established two cell lines. The phenotype of the hRPCs were profiled by flow cytometry, functional properties assessed by the calcium imaging and the ability to differentiate into photoreceptors on polycaprolactone and on pig retinal explants ex vivo evaluated with immunocytochemistry. In addition to flow cytometry, the equivalence of cell lines was determined by microarray analysis (Affymetrix).
We found that both lines are mitotically active (Ki67 and PCNA positive), express stemness/eye field (Klf4, Sox2, Recoverin, SSEA4, Otx2, Pax6) and photoreceptor-precursor specific (Nrl, Crx, CD73) markers and can form photoreceptors (ROM-1, rhodopsin, opsin red/green) both in vitro and ex vivo. We also have established a panel of positive (CD73, Sox2, Pax6, HLA-ABC) and negative (CD133, CD38) markers as release criteria.
hRPCs can be isolated from the developing retina, with different cell lines and different passages of these cell lines demonstrating equivalence. This is an important prerequisite to clinical application of this technology.
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