Assays for potency of human retinal pigment epithelium (rpe) cells and photoreceptor progenitors

Abstract

This disclosure provides a new phagocytosis assay to test the function of RPE cells and photoreceptor progenitors using a pH sensitive fluorescent label.

1 - 73 . (canceled) 
     
     
         74 . A cell population produced by in vitro differentiation of pluripotent stem cells, wherein the cell population:
 (i) has a rate of phagocytosis of photoreceptor outer segments (POS) and/or bacterial fragments that is at least 50 percent greater than the rate of phagocytosis of POS and/or bacterial fragments by an equivalent number of retinal pigment epithelial (RPE) or photoreceptor progenitor cells isolated from adult eyes, and/or   (ii) phagocytoses at least 20 percent of a total concentration of POS and/or bacterial fragments after 24 hours,   wherein the cell population is a RPE cell population or a photoreceptor progenitor cell population.   
     
     
         75 . The cell population of  claim 74 , wherein the rate of phagocytosis of the POS and/or bacterial fragments is at least 75, 100, 150 or 200 percent greater than the rate of phagocytosis of POS and/or bacterial fragments for an equivalent number of RPE cells or photoreceptor progenitor cells from isolated adult eyes. 
     
     
         76 . The cell population of  claim 74 , wherein the cell population phagocytoses at least 25, 30, 40 or 50 percent of the total concentration of POS and/or bacterial fragments after 24 hours. 
     
     
         77 . The cell population of  claim 74 , wherein the cell population is a human RPE cell population. 
     
     
         78 . The cell population of  claim 74 , wherein the cell population is a human photoreceptor progenitor cell population. 
     
     
         79 . The cell population of  claim 74 , wherein the cell population lacks expression of one or more embryonic stem cell markers selected from the group consisting of OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81. 
     
     
         80 . The cell population of  claim 74 , wherein the cell population is an RPE cell population that expresses one or more RPE cell markers selected from the group consisting of RPE65, CRALBP, PEDF, Bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and tyrosinase. 
     
     
         81 . The cell population of  claim 74 , wherein phagocytosis by the cell population is measured by
 incubating the cell population with the POS and/or bacterial fragments for a time and at a temperature sufficient for cells in the population to phagocytose the POS and/or bacterial fragments, wherein the POS and/or bacterial fragments are labeled with a fluorescent label that fluoresces more at an acidic pH than at a higher pH, and   detecting fluorescence intensity of the cell population after incubation, wherein an increase in fluorescence intensity compared to a control indicates phagocytosis of the POS and/or bacterial fragments by the cell population,   wherein the cell population is incubated with the POS and/or bacterial fragments at a temperature ranging from 25-40° C., or 34-40° C., or at about 37° C., and   wherein the control is cells incubated with the POS and/or bacterial fragments at a temperature ranging from 10-16° C. or 12-15° C.   
     
     
         82 . The cell population of  claim 74 , wherein phagocytosis by the cell population is measured by
 incubating the cell population with the POS for a time and at a temperature sufficient for cells in the population to phagocytose the POS, wherein the POS are labeled with a fluorescent label that fluoresces more at an acidic pH than at a higher pH, and   detecting fluorescence intensity of the cell population after incubation, wherein an increase in fluorescence intensity compared to a control indicates phagocytosis of the POS by the cell population,   wherein the cell population is incubated with the POS at a temperature ranging from 25-40° C., or 34-40° C., or at about 37° C., and   wherein the control is cells incubated with the POS at a temperature ranging from 10-16° C. or 12-15° C.   
     
     
         83 . The cell population of  claim 81 , wherein the POS and/or bacterial fragments are labeled with a fluorescent label having an increased fluorescence signal when internalized by phagocytosis into a low pH compartment in a cell relative to its fluorescence signal when present extracellularly. 
     
     
         84 . The cell population of  claim 81 , wherein the fluorescence is detected by flow cytometry or using a plate reader. 
     
     
         85 . The cell population of  claim 81 , wherein the cell population is incubated with the POS and/or bacterial fragments at a temperature ranging from 34-40° C. or at about 37° C. 
     
     
         86 . The cell population of  claim 81 , wherein the control is cells incubated with the POS and/or bacterial fragments at a temperature ranging from 12-15° C. 
     
     
         87 . The cell population of  claim 81 , wherein the cell population and the control cells are incubated with the POS and/or bacterial fragments for about 16-20 hours. 
     
     
         88 . The cell population of  claim 81 , wherein the bacterial fragments are bioparticles. 
     
     
         89 . The cell population of  claim 81 , wherein the POS are fragmented POS or sonicated POS. 
     
     
         90 . The cell population of  claim 81 , wherein the POS and/or bacterial fragments are labeled with pH-sensitive rhodamine-based dye. 
     
     
         91 . The cell population of  claim 74 , wherein the cell population is suitable for treating retinal degeneration disease. 
     
     
         92 . The cell population of  claim 91 , wherein the retinal degeneration disease is selected from the group consisting of retinal detachment, retinal dysplasia, angioid streaks, retinal atrophy, choroideremia, diabetic retinopathy, macular degeneration, age related macular degeneration, myopic macular degeneration, retinitis pigmentosa, and Stargardt's Disease (fundus flavimaculatus). 
     
     
         93 . A composition comprising the cell population of  claim 74 .