Assays for potency of human retinal pigment epithelium (RPE) cells and photoreceptor progenitors
This disclosure provides a new phagocytosis assay to test the function of RPE cells and photoreceptor progenitors using a pH sensitive fluorescent label.
1. A method for assessing phagocytosis activity comprising
incubating test cells with photoreceptor outer segments (POS) for a time and temperature sufficient for the cells 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 test cells after incubation, wherein an increase in fluorescence intensity compared to a control indicates phagocytosis of the POS by the cells,
wherein the cells are 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 control cells incubated with the POS at a temperature ranging from 10-16° C. or 12-15° C.
2. The method of claim 1 , wherein the test cells are incubated with the POS at a temperature ranging from 34-40° C. or at about 37° C.
3. The method of claim 1 , wherein the test and control cells are incubated with the POS for about 16-20 hours.
4. The method of claim 1 , wherein the test and control cells are retinal pigment epithelium (RPE) cells.
5. The method of claim 1 , wherein the test and control cells are photoreceptor progenitor cells.
6. The method of claim 1 , wherein the POS are labeled with pHrodo® Red dye.
7. The method of claim 1 , wherein the control is control cells incubated with the POS at a temperature ranging from 12-15° C.
8. The method of claim 1 , wherein the POS 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 the fluorescence signal when present extracellularly, and
wherein the method comprises detecting an increased fluorescence, if any, in the test cells after incubation with the labeled POS relative to control, and quantifying the phagocytosis activity of the test cells therefrom.
9. The method of claim 1 , wherein the test and control cells are produced by in vitro differentiation of pluripotent stem cells.
10. The method of claim 1 , wherein the cells are human RPE cells or human photoreceptor progenitor cells.
11. The method of claim 1 , wherein the POS are fragmented POS or sonicated POS.
12. A method for measuring phagocytosis activity comprising
(1) measuring a test fluorescence in a first aliquot of the cells incubated with pHrodo® Red labeled photoreceptor outer segments (POS) and/or with pHrodo® Red E. coli BioParticles, at a temperature ranging from 25-40° C., or 34-40° C., or about 37° C. to obtain a test fluorescence, and
(2) measuring a control fluorescence in a second aliquot of the cells incubated with pHrodo® Red labeled photoreceptor outer segments (POS) and/or with pHrodo® Red E. coli BioParticles, at a temperature ranging from 10-16° C. or 12-15° C. to obtain a control fluorescence,
wherein a test fluorescence that is greater than a control fluorescence indicates phagocytosis activity of the cells.
13. The method of claim 12 , wherein the first aliquot of cells is incubated at a temperature ranging from 34-40° C., or about 37° C.
14. The method of claim 12 , wherein the second aliquot of cells is incubated at a temperature of about 12-15° C.
15. The method of claim 12 , wherein the cells are human RPE cells or human photoreceptor progenitor cells.
16. A method for measuring phagocytosis activity comprising
(1) measuring a test fluorescence in a first aliquot of cells incubated with pHrodo® Red labeled photoreceptor outer segments (POS) and/or pHrodo® Red labeled bacterial fragments, at a temperature ranging from 25-40° C., or 34-40° C., or about 37° C. to obtain a test fluorescence, and
(2) measuring a control fluorescence in a second aliquot of the cells incubated with pHrodo® Red labeled POS and/or pHrodo® Red labeled bacterial fragments, at a temperature ranging from 10-16° C. or 12-15° C. to obtain a control fluorescence,
wherein a test fluorescence that is greater than a control fluorescence indicates phagocytosis activity of the cells,
wherein the cells are human RPE cells or human photoreceptor progenitor cells.
17. The method of claim 16 , wherein the first and second aliquot of cells are incubated with pHrodo® Red labeled POS.
18. The method of claim 16 , wherein the first and second aliquot of cells are incubated with pHrodo® Red labeled bacterial fragments.
19. The method of claim 18 , wherein the pHrodo® Red labeled bacterial fragments are pHrodo® Red labeled E. coli Bioparticles.
20. The method of claim 19 , wherein the first aliquot of cells is incubated at a temperature of about 37° C. and the second aliquot of the cells is incubated at a temperature ranging from 12-15° C.
21. The method of claim 20 , wherein the cells are human RPE cells.
22. The method of claim 20 , wherein the cells are human photoreceptor progenitor cells.