Improved assays for potency of human retinal pigment epithelium (RPE) cells and photoreceptor progenitors

Abstract

To provide retinal pigment epithelium (RPE) cell populations produced by in vitro differentiation of pluripotent stem cells.SOLUTION: The cell population has been confirmed to have increased phagocytosis of photoreceptor outer segments (POS) and / or bacterial fragments at 25 to 40°C, 34 to 40°C, or 37°C compared to a control population of RPE cells at 10 to 16°C or 12 to 15°C. The RPE cell population has one or more characteristics selected from: (a) decreased expression of one or more markers indicative of embryonic stem cell identity in the cell population compared to pluripotent stem cells prior to in vitro differentiation; (b) increased expression of one or more markers indicative of RPE identity in the cell population compared to pluripotent stem cells prior to in vitro differentiation; and / or (c) bestrophin-positive expression in at least 70% of cells within the RPE cell population.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 62 / 136,660, entitled "IMPROVED ASSAYS FOR POTENCY OF HUMAN RPE CELLS AND PHOTORECEPTOR PROGENITORS," filed March 23, 2015, which is incorporated by reference in its entirety. Technical Field

[0002] The present invention relates to the use of an in vitro cell-based method to measure phagocytosis of photoreceptor rod outer segments. [Background technology]

[0003] The retinal pigment epithelium (RPE) is the outer pigmented cell layer of the neurosensory retina, located between the underlying choroid (the vascular layer behind the retina) and the overlying retinal visual cells (e.g., rod and cone photoreceptors). The RPE is important for the function and health of photoreceptors and the retina. The RPE maintains photoreceptor function by recycling photopigments, delivering, metabolizing, and storing vitamin A, phagocytosing rod photoreceptor outer segments, transporting iron and small molecules between the retina and choroid, maintaining Bruch's membrane, and absorbing stray light, allowing for better image resolution. For example, WO2009 / 051671;Engelmann and Valtink (2004)"RPE Cell Cultivation." Graefe's Archive for Clinical and Experimental Ophthalmology 242(1):65-67;Irina Klimanskaya, Retinal Pigment Epithelium Derived From Embryonic Stem Cells, STEM CELL ANTHOLOGY 335-346 (Bruce Carlson ed., 2009).

[0004] Degeneration of the RPE can lead to retinal detachment, retinal dysplasia, or retinal atrophy, which are associated with a number of vision-altering diseases that result in photoreceptor damage and blindness, including congenital choroideremia, diabetic retinopathy, macular degeneration (including age-related macular degeneration, AMD), and Stargardt macular degeneration (SMD), the latter two being the top two causes of adult and early-onset blindness worldwide, respectively. Neither condition is currently treatable, but in preclinical models of macular degeneration, transplantation of hESC-derived RPE has been shown to rescue photoreceptors and prevent vision loss (Lund RD, Wang S, Klimanskaya I, et al. Human embryonic stem cell-derived cells rescue visual function in dystrophic rats. Cloning and Stem Cells 2006; 8, 189-199; Lu B, Malcuit C, Wang S, et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 2009; 21, 2125-2135).

[0005] In addition, the further loss of postmitotic neurons is also involved in some of the above diseases. These retinal diseases include rod or cone dystrophy, retinal degeneration, retinitis pigmentosa (RP), diabetic retinopathy, macular degeneration, Leber's congenital amaurosis, and Stargardt's disease (fundus flava). In many cases of retinal degeneration, cell loss occurs mainly in the outer nuclear layer (ONL), which contains rod and cone photoreceptors.

[0006] Potential alternative sources of photoreceptor cells include stem cells. Early studies evaluated heterogeneous populations of mouse cells, mouse stem cells, or retinal progenitor cells as possible sources of replacement cells for lost photoreceptor cells. These early studies included transplantation of photoreceptor precursor cells from postnatal day 1 mouse retinas (Maclaren et al. Nature 444(9):203-207, 2006), generation of retinal progenitor cells in vitro from mouse embryonic stem cells (Ikeda et al. Proc. Natl. Acad. Sci. 102(32):11331-11336, 2005), generation of retinal progenitor cells from postnatal day 1 mouse retinas (Klassen et al. Invest. Ophthal. Vis. Sci. 45(11):4167-4175, 2004), and transplantation of bone marrow mesenchymal stem cells in a rat model of retinal degeneration (RCS) (Inoue et al. Exp. Eye Res. 8(2):234-241, 2005). 2007), the production of retinal progenitor cells from the H1 human embryonic stem cell line, including ganglion cells, amacrine cells, photoreceptors in which 0.01% of the total cells express S-opsin or rhodopsin, bipolar cells, and horizontal cells (Lamba et al. Proc. Natl. Acad. Sci. 10(34):12769-12774, 2006), and the induction of pluripotent stem cells (iPS) derived from human fibroblasts to produce retinal progenitor cells (Lamba et al. PLoS ONE 5(1):e8763. doi:10.1371 / journal.pone.0008763) have been described.

[0007] None of these approaches have produced a homogeneous population of photoreceptor precursor cells or photoreceptors for transplantation. None of these approaches have produced a homogeneous population of photoreceptor precursor cells or photoreceptors that exhibit rod or cone function in vivo (e.g., as detectable by resulting in improved visual acuity). The supply of donor-derived tissue (e.g., cadaveric or fetal tissue and live animals) from which photoreceptors or photoreceptor precursor cells can be isolated is limited.

[0008] Stem cells can be propagated and expanded indefinitely in vitro, providing a potentially non-exhaustible source of non-donor-derived cells for human therapy. Differentiation of stem cells into homogenous populations of photoreceptor progenitor cells and photoreceptors may provide an ample supply of non-donor-derived cells for transplantation and treatment of retinal diseases. Photoreceptor progenitor cells may have phagocytic activity.

[0009] Certain subject matter, including methods of making RPE cells, RPE cell compositions, and release assays (including phagocytosis assays) for RPE cells, is disclosed in commonly owned U.S. Patent Application No. 13 / 510,426, filed November 17, 2010, and PCT Application No. US2012 / 65091, the teachings of which are incorporated herein by reference. Certain subject matter, including methods of making photoreceptor progenitor cells and methods of testing photoreceptor progenitor cell composition and function (including phagocytosis assays), is disclosed in commonly owned PCT Application No. US2014 / 029790, the teachings of which are incorporated herein by reference. Summary of the Invention

[0010] summary FITC-labeled photoreceptor outer segments (OS) (usually bovine or porcine) have been used to study phagocytosis by retinal pigment epithelium (RPE) in vitro. However, many quantitative methods used to assess phagocytosis (FACS, fluorescent plate readers) do not distinguish between surface-bound and internalized particles, preventing specific addressing of the mechanisms involved in the surface receptor binding and internalization stages of phagocytosis. In addition, while the pH of lysosomes and lysosome-fused phagosomes is below 5, FITC fluorescence is pH-sensitive and significantly decreases below pH 6. Therefore, FITC-labeled OS may not accurately represent the amount of internalized OS.

[0011] Provided herein is a more sensitive and accurate assay for detecting phagocytosis of photoreceptor outer segments, a key functional measure of RPE cells and photoreceptor progenitor cells, and a key release criterion for RPE cells and photoreceptor progenitor cells that can be used to treat retinal diseases such as rod or cone dystrophies, retinal degeneration, retinitis pigmentosa, congenital choroideremia, diabetic retinopathy, macular degeneration (including age-related macular degeneration and myopic macular degeneration), Leber's congenital amaurosis, and Stargardt's disease (fundus flava). See, e.g., WO2009 / 051671.

[0012] The RPE cells described herein remain functional after transplantation.To achieve this goal, RPE cells form a monolayer between the neurosensory retina and the choroid in the subject (or patient) who receives the transplanted cells.RPE cells can also provide nutrients to adjacent photoreceptor cells and process shed photoreceptor outer segments by phagocytosis.

[0013] RPE cells suitable for transplantation may be selected based on a number of functional and / or phenotypic characteristics, including, but not limited to, phagocytic activity. For example, RPE cells suitable for transplantation may be evaluated according to their phagocytic activity and their proliferation potential. For example, RPE cells may have greater proliferation potential than cells derived from an eye donor (e.g., RPE cells are "younger" than RPE cells from an eye donor). This allows the RPE cells described herein to have a longer useful lifespan than cells derived from an eye donor.

[0014] One of the key parameters for the efficacy of PRE cells in clinical settings is the quantitative measurement of photoreceptor outer segment phagocytosis activity in pharmaceutical preparations of RPE cells. Outer segment phagocytosis leads to the accumulation of phagocytosed cell fragments in low-pH compartments in RPE cells. The present invention provides photoreceptor outer segments that are spectrophotometrically detectable (i.e., covalently or non-covalently linked) to a detectable marker that is selective to provide a first spectrophotometric signal when present at neutral or physiological pH, i.e., pH 7-7.5, and a second spectrophotometric signal when present in intracellular compartments with low-pH environments, such as lysosomes, phagosomes, and endosomes. The difference between the first and second spectrophotometric signals may be one or more of the magnitude of fluorescence emission (increased intensity at low pH relative to neutral pH), a change in fluorescence emission wavelength between neutral and low pH, a change in fluorescence excitation wavelength between neutral and low pH, etc.

[0015] In certain embodiments, the detectable marker can be a fluorescent pH sensor, such as a fluorescent dye. A typical fluorescent dye that can be used in the present invention can be a fluorescent dye moiety having an amino group (aliphatic or aromatic) as a pH-sensitive indicator moiety, i.e., an amine that is unprotonated at the pH of the culture medium in which RPE cells and outer segments are incubated together (i.e., neutral or physiological pH) and protonated at the pH of the intracellular compartment where the outer segments are taken up by cells, such as RPE cells, by phagocytosis. When such a dye absorbs a photon, it generates an excited electronic state, and the lone electron pair of the amino group transfers to the orbital vacated by the excitation. This electron transfer, called photoinduced electron transfer (PET), prevents the excited molecule from undergoing a radiative transition, thus quenching the fluorescence of the dye. Protonation of the amino group changes the nature and energy of the electron pair orbital, terminating the PET. As a result, the fluorescent reporter moiety responds to changes in pH. Protonation of the amino groups counteracts the quenching, so that PET-based sensors become more fluorescent as the pH decreases.

[0016] In certain embodiments, the fluorescent dye is a rhodamine-based pH-sensitive dye, as described in WO2005 / 098437. Such dyes have a benzene ring substituted with -OH or -SH (or their deprotonated forms) at the ortho position to the xanthene moiety. Such dyes exhibit pH dependence similar to amine PET indicators, but are designed to have pKa values ​​less than 6, based on the recognized need for pH sensors targeting cellular compartments where the pH is less than 6.

[0017] In another exemplary embodiment, the fluorescent marker is a pH-sensitive fluorescent nanoparticle. pH-sensitive fluorescent nanoparticles mainly employ polymers linked to small molecule pH-sensitive dyes (Srikun, D., J. Chem. Sci. 2011, 2, 1156; Benjaminsen, RV, ACS Nano 2011, 5, 5864; Albertazzi, L., J. Am. Chem. Soc. 2010, 132, 18158; Urano, Y., Nat. Med. 2009, 15, 104), or the use of pH-sensitive linkers linked to pH-insensitive dyes (Li, C, Adv. Fund. Mater. 2010, 20, 2222; Almutairi, J. Am. Chem. Soc. 2007, 130, 444). To further illustrate, WO2013152059 describes a pH-tunable, highly activatable, multicolor fluorescent nanoplatform that is applicable for use in the present assay.

[0018] Thus, in one aspect, the present specification provides a method for assessing phagocytosis activity, comprising incubating cells with photoreceptor outer segments (POS) for a time and at a temperature sufficient for the cells to phagocytose the POS, wherein the POS fluoresces more strongly at an acidic pH than at a higher pH, and detecting the fluorescence intensity of the cells after incubation, wherein an increase in fluorescence compared to a control indicates phagocytosis of the POS by the cells.

[0019] In some embodiments, the cells are incubated with the POS at a temperature ranging from about room temperature to about 37° C., or from about room temperature to about 40° C. In some embodiments, the cells are incubated with the POS at about room temperature, about physiological temperature, or about 37° C. In some embodiments, the control is cells incubated with POS at below room temperature, hi some embodiments, the control is cells incubated with POS at 4°C.

[0020] Also provided herein is a method for assessing the phagocytic activity of an adherent cell population, comprising incubating the adherent cell population with photoreceptor outer segments (POS) for a time and at a temperature sufficient for cells in the cell population to phagocytose the POS, wherein the POS fluoresces more strongly at an acidic pH than at a higher pH, and detecting the fluorescence intensity of the cell population after incubation, wherein an increase in fluorescence compared to a control indicates phagocytosis of the POS by the cells.

[0021] In some embodiments, the adherent cell population is incubated with POS at a temperature ranging from about 17-40° C., or about 25-40° C., or about 34-40° C., or at a temperature of about 37° C. In some embodiments, the control is a cell population incubated with POS at a temperature of about 10-16° C. In some embodiments, the control is a cell population incubated with POS at a temperature of about 12-15° C.

[0022] Also provided herein is a method for assessing phagocytic activity, comprising providing photoreceptor outer segments (POS) labeled with a fluorescent label that, when phagocytosed into a low pH compartment within the cell, has an altered fluorescence signal relative to the fluorescence signal when present outside the cell; incubating test cells with the labeled POS under conditions that allow phagocytosis of the labeled POS; and detecting any altered fluorescence in the test cells after incubation with the labeled POS, and quantifying the phagocytic activity of the test cells therefrom.

[0023] In some embodiments, the altered fluorescent signal (when the label is phagocytosed into a low pH compartment) is an increase in fluorescent signal intensity relative to when the fluorescent label is extracellular. In some embodiments, the altered fluorescent signal (when the label is phagocytosed into a low pH compartment) is detectable by flow cytometry. In some embodiments, the altered fluorescent signal distinguishes between labeled POS that has been phagocytosed and labeled POS that is bound to the surface of the test cell but not internalized. In some embodiments, the altered fluorescent signal detected in the test cell is compared to a control cell population incubated with labeled POS to quantify the phagocytic activity of the test cell.

[0024] In some embodiments, the test cells are incubated with the labeled POS at about room temperature, at about physiological temperature, at about 37°C, at about 15-40°C, or between room temperature and 37°C, or between room temperature and 40°C. In some embodiments, the control cell population is incubated with the labeled POS below room temperature, including about 4°C.

[0025] Also provided is a method for assessing phagocytic activity, comprising providing fluorescently labeled photoreceptor outer segments (POS) that have a fluorescent signal that changes relative to the fluorescent signal when present outside the cell when phagocytosed into a low pH compartment within the cell; incubating adherent test cells with the labeled POS under conditions that allow phagocytosis of the labeled POS; and detecting any change in fluorescence in the adherent test cells after incubation with the labeled POS, and quantifying the phagocytic activity of the adherent test cells therefrom.

[0026] In some embodiments, the test cells are incubated with POS at a temperature ranging from about 17-40°C, or about 25-40°C, or about 34-40°C, or at a temperature of about 37°C. In some embodiments, the altered fluorescent signal detected in the adherent test cells is compared to a control cell population incubated with labeled POS at a temperature that maintains cell viability but induces little or no phagocytosis (optionally, such a temperature may be in the range of about 12-15°C) to quantify the phagocytic activity of the test cells. In some embodiments, the control cell population is incubated with POS at a temperature of about 10-16°C.

[0027] Also provided herein is a labeled photoreceptor outer segment (POS) preparation for assessing phagocytic activity of a test cell population, wherein the POS is labeled with a fluorescent label, and the fluorescent label has a fluorescent signal that, when phagocytosed into a low pH compartment within a cell, is altered relative to the fluorescent signal when the fluorescent label is extracellular. In some embodiments, the altered fluorescent signal (when the label is phagocytosed into the low pH compartment) is an increase in fluorescent signal intensity relative to when the label is extracellular. In some embodiments, the altered fluorescent signal (when the label is phagocytosed into the low pH compartment) is detectable by flow cytometry. In some embodiments, the fluorescent label is pHrodo® Red. In some embodiments, the POS is labeled with pHrodo® Red and pHrodo® Red E. coli BioParticles.

[0028] Also provided herein is a method for measuring phagocytic activity in a cell population, comprising measuring test fluorescence in a test cell population contacted with non-FITC fluorescently labeled photoreceptor outer segments (POS) and comparing the measured test fluorescence to control fluorescence, wherein the non-FITC fluorescently labeled POS fluoresces at acidic pH but fluoresces or fluoresces minimally at higher pH.

[0029] In some embodiments, the test cell population is contacted with the non-FITC fluorescently labeled POS at a temperature ranging from about room temperature to about physiological temperature (i.e., from about room temperature to about 37°C), including, for example, 37°C, or from about room temperature to about 40°C. In some embodiments, the test cell population is contacted with the non-FITC fluorescently labeled POS at a temperature between about 15°C and 40°C, or about physiological temperature, including, for example, about 37°C. In some embodiments, the control fluorescence is the fluorescence of a cell population contacted with the non-FITC fluorescently labeled POS at below room temperature. In some embodiments, the control fluorescence is the fluorescence of a cell population contacted with the non-FITC fluorescently labeled POS at 4°C.

[0030] Also provided herein is a method for measuring phagocytic activity in an adherent cell population, comprising measuring test fluorescence in an adherent test cell population contacted with non-FITC fluorescently labeled photoreceptor outer segments (POS) and comparing the measured test fluorescence to control fluorescence, wherein the non-FITC fluorescently labeled POS fluoresce at acidic pH but fluoresce non- or minimally at higher pH.

[0031] In some embodiments, the test cell population is contacted with non-FITC fluorescently labeled POS at a temperature ranging from about 17-40° C., or about 25-40° C., or about 34-40° C., or at a temperature of about 37° C. In some embodiments, the control fluorescence is the fluorescence of an adherent cell population contacted with non-FITC fluorescently labeled POS at a temperature of about 12-15° C. In some embodiments, the control fluorescence is the fluorescence of an adherent cell population contacted with non-FITC fluorescently labeled POS at a temperature of about 10-16° C.

[0032] Also provided herein is a method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of a cell population incubated with fluorescently labeled photoreceptor outer segments (POS) labeled with pHrodo® Red dye at a temperature ranging from about room temperature to about physiological temperature, including, for example, 37°C, or from about room temperature to about 40°C; and (2) measuring control fluorescence in a second aliquot of a cell population incubated with fluorescently labeled POS labeled with pHrodo® Red dye at a temperature below room temperature, wherein test fluorescence greater than the control fluorescence indicates phagocytic activity of the cell population.

[0033] Also provided herein is a method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of an adherent cell population incubated with fluorescently labeled photoreceptor outer segments (POS) labeled with pHrodo® Red dye; and (2) measuring control fluorescence in a second aliquot of the adherent cell population incubated with fluorescently labeled POS labeled with pHrodo® Red dye, wherein test fluorescence greater than the control fluorescence indicates phagocytic activity of the cell population.

[0034] In some embodiments, the first aliquot of the adherent cell population is contacted with the labeled POS at a temperature ranging from about 17-40° C., or about 25-40° C., or about 34-40° C., or at a temperature of about 37° C. In some embodiments, the second aliquot of the adherent cell population is incubated with the labeled POS at a temperature ranging from about 10-16° C. or about 12-15° C.

[0035] Also provided herein is a method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of a cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at a temperature ranging from about room temperature to about physiological temperature, including 37°C, or from about room temperature to about 40°C; and (2) measuring control fluorescence in a second aliquot of a cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at below room temperature, including 4°C, wherein test fluorescence greater than the control fluorescence indicates phagocytic activity of the cell population.

[0036] Also provided herein is a method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of an adherent cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at a temperature ranging from about 17-40°C, or about 25-40°C, or about 34-40°C, or at a temperature of about 37°C; and (2) measuring test fluorescence in a first aliquot of an adherent cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles. Also provided is a method comprising measuring control fluorescence in a second aliquot of the adherent cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) together with BioParticles at a temperature in the range of about 10-16°C or about 12-15°C, wherein test fluorescence greater than the control fluorescence indicates phagocytic activity of the cell population.

[0037] Various embodiments apply equally to any and all of the above aspects, which are listed below. In some embodiments, the cells, cell populations, test cells, or test cell populations are incubated with the POS at about room temperature, at about physiological temperature, at about 37° C., or from about room temperature to 40° C., or from about room temperature to 37° C., or at about 15-40° C. In some embodiments, the control cell population is incubated with the labeled POS below room temperature, including about 4° C. In some embodiments, the cells, cell populations, test cells, or test cell populations are incubated with the labeled POS at a temperature in the range of about 17-40° C., or from about 25-40° C., or from about 34-40° C., or at a temperature of about 37° C.

[0038] In some embodiments, the control cell, control cell population, control test cell, or control test cell population is incubated with the POS at a temperature below room temperature, in the range of about 10-16°C, or about 12-15°C, or at a temperature of about 4°C.

[0039] In some embodiments, the cell, cell population, test cell, or test cell population comprises a retinal pigment epithelial (RPE) cell. In some embodiments, the cell, cell population, test cell, or test cell population comprises a photoreceptor progenitor cell. In some embodiments, the cell, cell population, test cell, or test cell population is a human cell. In some embodiments, the cell, cell population, test cell, or test cell population is produced by in vitro differentiation of pluripotent stem cells. In some embodiments, the cells, cell populations, test cells, or test cell populations are cryopreserved and thawed prior to use.

[0040] In some embodiments, the cell, cell population, test cell, or test cell population is provided as a confluent monolayer. In some embodiments, the cell, cell population, test cell, or test cell population is digested with an enzyme prior to use. In some embodiments, the cell, cell population, test cell, or test cell population is provided as an adherent cell population. In some embodiments, the POS is fragmented POS. In some embodiments, the POS is sonicated POS.

[0041] In some embodiments, the fluorescent label is pHrodo® Red. Thus, in some embodiments, the POS is labeled with pHrodo® Red dye. In some embodiments, the POS is labeled with pHrodo® Red and pHrodo® Red E. coli BioParticles. In some embodiments, cells are incubated with pHrodo® Red-labeled POS and pHrodo® Red E. coli BioParticles.

[0042] In some embodiments, the fluorescence is detected by flow cytometry. In some embodiments, the fluorescence is detected using a plate reader. In some embodiments, the cells are incubated with the POS for about 15-30 hours, or 16-20 hours, or 20-28 hours. In some embodiments, the cells are provided as a cell culture. In some embodiments, the cells are a confluent cell culture. It should be understood that the test and control cells can be different aliquots of the same cell population.

[0043] In another aspect, the present disclosure provides an isolated cell population characterized by having a photoreceptor outer segment (POS) phagocytosis rate that is at least 50% higher than the POS phagocytosis rate of an equivalent number of primary cells.In some embodiments, the cell population is an RPE cell population.In some embodiments, the cell population is an RPE cell population obtained by in vitro differentiation of pluripotent stem cells, and the primary cells are isolated RPE cells from adult eyeballs.In some embodiments, the cell population is photoreceptor progenitor cells. These and various other aspects and embodiments are described in greater detail herein. [Brief explanation of the drawings]

[0044] [Figure 1A-C]FACS analysis of phagocytosis by RPE. (A) FITC-labeled ROS. (B) pHrodo®-labeled ROS. Plots are shown for a control where no ROS was added, a control where ROS was added and cells were incubated at 4°C, and a test where ROS was added and cells were incubated at 37°C. (C) pHrodo®-labeled BioParticles (bacterial fragments). Plots are shown for a control where no particles were added, a control where particles were added and cells were incubated at 4°C, and a test where particles were added and cells were incubated at 37°C. It should be understood that POS and ROS are used interchangeably herein to refer to photoreceptor rod outer segments.

[0045] [Figure 2A-B] pH dependence of fluorescence of FITC (A) and pHrodo® (B) labeled ROS. Fluorescence at neutral and acidic pH is plotted.

[0046] [Figure 3A-F]FACS analysis of phagocytosis of fluorescently labeled ROS by ARPE-19 cell monolayers. No dose-dependent phagocytosis was observed when ARPE-19 cells were incubated with different concentrations of fluorescently labeled ROS in cell monolayers for 24 hours. (A) ARPE-19 incubated with 6 x 106 pHrodo® Red-labeled ROS at 37°C. (B) ARPE-19 incubated with 3 x 106 pHrodo® Red-labeled ROS at 37°C. (C) ARPE-19 incubated with 1.5 x 106 pHrodo® Red-labeled ROS at 37°C. (D) ARPE-19 incubated with 6 x 106 pHrodo® Red-labeled ROS at 15°C. (E) ARPE-19 incubated with 3 x 106 pHrodo® Red-labeled ROS at 15°C. (F) ARPE-19 incubated with 1.5 x 10 pHrodo® Red-labeled ROS at 15° C. Plots for cells incubated without and with ROS are shown, respectively.

[0047] [Figure 4A-F]FACS analysis of phagocytosis of fluorescently labeled ROS by hESC-derived RPE cells. No dose-dependent phagocytosis was observed when RPE cells were incubated with different concentrations of fluorescently labeled ROS in cell monolayers at 37°C for 24 hours. Pulse sonication of fluorescently labeled ROS during reconstitution increased phagocytosis by approximately 10%. (A) RPE cells incubated with 3.75 x 106 pHrodo® Red-labeled ROS reconstituted without sonication. (B) RPE cells incubated with 5 x 106 pHrodo® Red-labeled ROS reconstituted without sonication. (C) RPE cells incubated with 7.5 x 106 pHrodo® Red-labeled ROS reconstituted without sonication. (D) RPE cells incubated with 10x106 pHrodo® Red-labeled ROS reconstituted without sonication, (E) RPE cells incubated with 10x106 pHrodo® Red-labeled ROS reconstituted with sonication, and (F) RPE cells incubated with 13.5x106 pHrodo® Red-labeled ROS reconstituted with sonication.

[0048] Detailed Description Phagocytosis (capacity assay) may be assessed by quantitative fluorescence-activated cell sorting (FACS) analysis of RPE cultures exposed to photoreceptor outer segments (POS) labeled with pHrodo® Red dye (Life Technologies, Molecular Probes). Phagocytosis may be assessed by a FACS-based assay using POS labeled with pHrodo® Red dye (Life Technologies, Molecular Probes). Such labeled dyes and POS fluoresce when incorporated into the reduced pH environment of intracellular phagosomes. POS may be labeled as described herein.

[0049] In some embodiments, the RPE cell culture is confluent. For example, confluent RPE cells may be cultured in a multi-well plate and incubated with POS labeled with pHrodo® Red dye, optionally in the presence of CO2-independent medium (Invitrogen). The incubation may be performed for any time sufficient for the RPE cells to phagocytose the POS. For example, the incubation may be performed for 16-20 hours. The assay is performed at a temperature sufficient for the RPE cells to phagocytose the POS. In some embodiments, the assay is performed at approximately physiological temperature or about 37°C. In some embodiments, the assay is performed at room temperature. In some embodiments, a control (or negative control) plate is incubated at 4°C. Cells may be observed under a microscope, fluorescence may be measured using a plate reader, and / or cells may be harvested after enzymatic digestion (e.g., trypsin digestion) and analyzed by flow cytometry.

[0050] Non-limiting exemplary assays are as follows: RPE cells generated from pluripotent stem cells (such as, but not limited to, ES cells and iPS cells) as previously described are tested for their phagocytic ability. Cryopreserved RPE cells may be previously frozen and thawed before use. Prior to testing the ability of RPE cells to phagocytose POS labeled with pHrodo® Red dye, which fluoresces upon uptake in the acidic environment of phagosomes, RPE cells are seeded in culture in an appropriate medium, grown to confluence, and maintained in culture. RPE cells are incubated with labeled POS at 37°C to allow phagocytosis, or at 4°C as a negative control. A shift in fluorescence intensity may be detected by flow cytometry for cells incubated at 37°C, indicating phagocytosis of the labeled POS. Statistical integration of the peaks yields the percentage of phagocytosis-positive cells for each lot of RPE cells and incubation temperature.

[0051] As understood by the present disclosure, phagocytosis is detected by incubating RPE cells with labeled POS, which fluoresce in the red spectrum in the acidic phagosomal environment. The percentage of phagocytosis-positive cells, as detected by flow cytometry, is shown for cells incubated at 37°C or 4°C (negative control).

[0052] The obtained RPE cell population can be characterized based on its phagocytosis activity by the method provided herein.The phagocytosis rate can be determined, and thereby RPE cells can be characterized.For example, RPE cells can be characterized as having a phagocytosis rate of photoreceptor outer segments (POS) that is at least 50% higher than the phagocytosis rate of the equivalent number of RPE cells in an isolated adult eyeball, or at least 75, 100, 150 or 200% higher than the phagocytosis rate of the equivalent number of RPE cells in an isolated adult eyeball.Alternatively or additionally, RPE cells can be characterized by a phagocytosis rate of photoreceptor outer segments (POS) that is at least 20% of the total concentration of POS after 24 hours, or at least 25, 30, 25, 40 or 50% of the total concentration of POS after 24 hours.

[0053] Thus, by using the methods described herein, RPE cell populations were achieved that have photoreceptor outer segment (POS) phagocytosis rates that are at least 50% higher, and more preferably at least 75, 100, 150, or 200% higher, than the POS phagocytosis rate for an equivalent number of RPE cells from an isolated adult eye (i.e., a human adult patient aged 25-80 years, more preferably, an adult aged 50-80 years).

[0054] Using the methods described herein, RPE cell populations were achieved that had a phagocytosis rate of photoreceptor outer segments (POS) that was at least 20% of the total concentration of POS after 24 hours, and more preferably at least 25, 30, 25, 40 or 50% of the total concentration of POS after 24 hours.

[0055] Thus, in one aspect, the present disclosure provides a method that includes detecting or measuring fluorescence (test fluorescence or generally considered "test") in RPE cells (or a population of RPE cells) contacted with non-FITC fluorescently labeled, fluorescently labeled photoreceptor outer segments (POS), and comparing the detected or measured fluorescence to a control (control fluorescence or generally considered "control"). The test can be performed at approximately room temperature, or at a temperature of about 15-40°C, or at approximately physiological temperature (e.g., about 37°C). The control can be performed at 4°C. Thus, the control fluorescence can be fluorescence detected or measured following incubation of RPE cells with non-FITC-labeled POS at 4°C. Non-FITC fluorescently labeled POS are POS labeled with a fluorophore other than FITC. Non-FITC fluorescent labels are labels that fluoresce at acidic pH, such as the pH of phagosomes, and especially those of RPE cells, and do not fluoresce or fluoresce only minimally at higher pH, especially at neutral pH (or extracellular environment pH). Non-FITC fluorescent labels are useful for distinguishing between surface labels and internalized labels. An example of such a fluorescent dye molecule is pHrodo® Red dye (Life Technologies, Molecular Probes). A higher degree of phagocytosis in the test is indicated by stronger fluorescence compared to the control.

[0056] Thus, in another aspect, the present disclosure provides a method for manufacturing a semiconductor device comprising: (1) detecting or measuring fluorescence (test fluorescence, or commonly considered a "test") in a first aliquot of RPE cells (or a population of RPE cells) contacted with (and incubated at 37°C with) fluorescently labeled photoreceptor outer segments (POS) labeled with pHrodo® Red dye; and (2) detecting or measuring fluorescence (control fluorescence, or generally considered a "control") in a second aliquot of an adherent cell population of RPE cells (or RPE cell population) contacted with (and incubated at 4°C with) fluorescently labeled POS labeled with pHrodo® Red dye; and (3) optionally, comparing, determining, and / or quantifying test and control fluorescence, wherein test fluorescence greater than control fluorescence is indicative of phagocytic activity of the RPE cell (or cell population); A method is disclosed that includes:

[0057] It should be understood that the methods described herein can also be used to assay phagocytic activity in photoreceptor progenitor (or precursor) cells. In some embodiments, RPE and photoreceptor progenitor cells have phagocytic activity, such as the ability to phagocytose pHrodo® Red photoreceptor outer segments alone, pHrodo® Red E. coli BioParticles, or both, and the methods provided herein assay one or more of these functions.

[0058] In one aspect, the present disclosure provides an assay for determining the ability of a pharmaceutical composition comprising a plurality of retinal pigment epithelial (RPE) cells or photoreceptor precursor cells; and a pharmaceutically acceptable carrier.In one embodiment, the average melanin content of the plurality of RPE cells is less than 8 pg / cell.The RPE cells or photoreceptor precursor cells can be contained in a suspension, gel, colloid, matrix, substrate, scaffold, or graft.

[0059] The pharmaceutically acceptable carrier may comprise a sterile solution having an osmolality of about 290 mOsm / kg to about 320 mOsm / kg, or about 300 mOsm / kg to 310 mOsm / kg, or about 305 mOsm / kg. The pharmaceutically acceptable carrier may comprise a balanced salt solution. The buffered salt solution may comprise, consist of, or consist essentially of, per mL, in water: 7.14 mg sodium chloride, 0.38 mg potassium chloride, 0.154 mg calcium chloride dihydrate, 0.2 mg magnesium chloride hexahydrate, 0.42 mg dibasic sodium phosphate, 2.1 mg sodium bicarbonate, 0.92 mg dextrose, 0.184 mg glutathione disulfide (oxidized glutathione), and hydrochloric acid and / or sodium hydroxide (to adjust the pH to about 7.4).

[0060] The volume of the pharmaceutical composition may be about 100 μL to 1000 μL, or may be at least about 150 μL. The pharmaceutical composition contains about 1,000 to about 1×10 9 of viable RPE cells. The pharmaceutical composition may contain about 333 viable RPE cells / μL to about 2,000 viable RPE cells / μL, about 444 viable RPE cells / μL to about 1766 viable RPE cells / μL, about 333 viable RPE cells / μL, about It may contain 444 viable RPE cells / μL, about 666 viable RPE cells / μL, about 888 viable RPE cells / μL, about 999 viable RPE cells / μL, or about 1,333 viable RPE cells / μL.

[0061] The concentration of RPE cells in the pharmaceutical composition may be high enough so that about 30% or less of the RPE cells lose viability within 60 minutes, and optionally, about 10% or less of the RPE cells lose viability within 4 hours. The concentration of RPE cells may be at least about 1,000 cells / μL, at least about 2,000 cells / μL, about 1,000-10,000 cells / μL, or about 2,000-5,000 cells / μL. The pharmaceutical product may contain less than about 25%, 20%, 15%, 10%, 5%, 1%, 0.5%, 0.1%, 0.01%, 0.001%, or 0.0001% of cells that may not be RPE cells.

[0062] The average melanin content of the RPE cells may be less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, less than 5 pg / cell, less than 4 pg / cell, less than 3 pg / cell, less than 2 pg / cell, and at least 0.1 pg / cell, and optionally at least 0.5 pg / cell or 1 pg / cell; 0.1-8 pg / cell, 0.1-7 pg / cell, 0.1-6 pg / cell, 0.1-5 pg / cell, 0.1-4 pg / cell, 0.1-3 pg / cell, 0.1-2 pg / cell, 0.1-1 pg / cell, 1-7 pg / cell, 0.5-6 pg-cell, or 1-5 pg / cell.

[0063] At least 50%, at least 60%, at least 70%, or at least 80% of the cells in the pharmaceutical composition may be bestrophin+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be PAX6+ and / or MITF+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be PAX6+ and / or bestrophin+. At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be ZO-1+. At least 50%, at least 60%, or at least 70% of the cells in the pharmaceutical composition may be PAX6+ and bestrophin+. At least 90%, at least 95%, or at least 99% of the cells in the pharmaceutical composition may be PAX6+.

[0064] In typical embodiments, no more than about 1 cell per million cells, and optionally no more than 2 cells per 9 million cells in the pharmaceutical composition may be positive for both OCT-4 and alkaline phosphatase (AP) expression.

[0065] The needle or injection cannula may contain at least a portion of the RPE cells. The concentration of the RPE cells may be about 444 viable cells / μL to about 1,766 viable cells / μL when loaded into the needle or injection cannula. The concentration of viable RPE cells delivered from the needle or injection cannula may be about 333 viable cells / μL to about 1,333 viable cells / μL. The diameter of the needle or injection cannula may be about 0.3 mm to about 0.9 mm. The diameter of the needle or injection cannula may be about 0.5 mm to about 0.6 mm. The needle or injection cannula may include a tip having a diameter of about 0.09 mm to about 0.15 mm. The cannula may be a MEDONE POLYTIP® cannula 25 / 38g (0.50mm (25g) x 28mm cannula with a 0.12mm (38g) x 5mm tip) or a Synergetics Angled 39g injection cannula.

[0066] The RPE cells may comprise cryopreserved and thawed RPE cells. The RPE cells may be human. The RPE cells, such as human RPE cells, can be produced from any source, including pluripotent cells, such as embryonic stem cells or induced pluripotent stem cells, as well as adult donor tissue or fetal tissue. The pluripotent stem cells may be positive for the expression of one or more markers, including OCT-4, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-81. The pluripotent cells may be human pluripotent cells, which may be cultured in a multilayered population or embryoid body for a sufficient time for pigmented epithelial cells to appear in culture. The sufficient time for pigmented epithelial cells to appear in culture may include at least about 1 week, at least about 2 weeks, at least about 3 weeks, at least about 4 weeks, at least about 5 weeks, at least about 6 weeks, or at least about 7 weeks, or at least about 8 weeks.

[0067] The multilayered population or embryoid bodies may be cultured in a medium that may include DMEM. The medium may include, consist essentially of, or consist of EB-DM. The pigmented epithelial cells may be isolated and cultured to produce an RPE cell population. The isolation may include enzymatically, chemically, or physically separating cells or cell clusters from the culture and selecting pigmented epithelial cells or cell clusters that may contain pigmented epithelial cells. The embryoid bodies may be cultured in suspension and / or as adherent cultures (e.g., suspension followed by adherent culture). The embryoid bodies cultured as adherent cultures may produce one or more outgrowths, including pigmented epithelial cells. The pluripotent stem cells have reduced HLA antigen complexity. Prior to RPE formation, the pluripotent cells may be cultured on a matrix selected from the group consisting of laminin, fibronectin, vitronectin, proteoglycan, entactin, collagen, collagen I, collagen IV, collagen VIII, heparan sulfate, Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells), CellStart, human basement membrane extract, and any combination thereof. The matrix may include Matrigel™ (a soluble preparation derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells).

[0068] The pharmaceutical composition may comprise cells that lack substantial expression of one or more embryonic stem cell markers, which may include OCT-4, NANOG, Rex-1, alkaline phosphatase, Sox2, TDGF-1, SSEA-3, SSEA-4, TRA-1-60, and / or TRA-1-81. The RPE cells may be positive for expression of one or more RPE cell markers, which may include RPE65, CRALBP, PEDF, bestrophin, MITF, Otx2, PAX2, PAX6, ZO-1, and / or tyrosinase.

[0069] The RPE cells are allowed to grow for a sufficient time to reach the average melanin content. The RPE cells may be produced by a method comprising maintaining RPE cells as quiescent cells for a time sufficient to establish bestrophin expression in at least 50% of the RPE cells. The pharmaceutical composition may be substantially free of mouse embryonic feeder cells (MEFs) and human embryonic stem cells (hES).

[0070] The RPE cells may meet at least one of the criteria listed in Table 1 and / or may be manufactured in accordance with Good Manufacturing Practices (GMP). The cryopreserved retinal pigment epithelial (RPE) cells or photoreceptor precursor cells may be provided as a cryopreserved composition.

[0071] The RPE cells may exhibit a photoreceptor outer segment (POS) phagocytosis rate that may be at least 50% faster than the phagocytosis rate of a comparable number of RPE cells derived from an isolated adult human eye, or may be at least 75, 100, 150, or 200% faster than the phagocytosis rate of a comparable number of RPE cells derived from an isolated adult human eye; or may exhibit a POS phagocytosis rate that may be at least 20% of the total concentration of photoreceptor outer segments (POS) after 24 hours, or may be at least 25, 30, 25, 40, or 50% of the total concentration of POS after 24 hours. The photoreceptor progenitor cells may exhibit a photoreceptor outer segment (POS) phagocytosis rate that may be at least 50% faster than the phagocytosis rate of a comparable number of photoreceptor progenitor cells from an isolated adult human eye, or at least 75, 100, 150, or 200% faster than the phagocytosis rate of a comparable number of photoreceptor progenitor cells from an isolated adult human eye; or a photoreceptor outer segment (POS) phagocytosis rate that may be at least 20% of the total concentration of POS after 24 hours, or at least 25, 30, 25, 40, or 50% of the total concentration of POS after 24 hours. The phagocytosis rate and degree of phagocytosis may depend on the incubation time and the maturity of the cells. The binding and uptake rates of POS may vary depending on the maturity and pigmentation of the cells. The percentage of cells capable of phagocytosis may also vary depending on the maturity of the cell culture.

[0072] Labeling photoreceptor outer segments with dyes that are non-fluorescent or weakly fluorescent at neutral pH but become more strongly fluorescent upon acidification allows for more sensitive measurement of endocytosed POS in RPE cells or photoreceptor precursor cells in phagocytosis assays. Many quantitative methods used to assess phagocytosis (FACS, fluorescent plate readers) do not distinguish between endocytosed and surface-bound fluorescent particles. Additionally, FITC fluorescence is pH-sensitive and significantly decreases below pH 6, whereas the pH of lysosomes and lysosome-fused phagosomes is below pH 5. Therefore, the fluorescence of FITC-labeled OS may not represent the actual amount of endocytosed OS. According to its manufacturer (Life Technologies, Molecular Probes), the pH-sensitive rhodamine-based pHrodo® Red dye is non-fluorescent at neutral pH and becomes bright red upon acidification. The dye is both fluorescent and pH sensitive and can therefore be used as a specific sensor of phagocytic events, with red fluorescence indicating acidification of the phagosome following phagocytosis.

[0073] The use of dyes that are non-fluorescent or weakly fluorescent at neutral pH but become more strongly fluorescent upon acidification, such as pHrodo® and CypHer5ERed (which also fluoresces maximally in an acidic environment), and the Life Technologies dye-based LysoSensor, to label photoreceptor outer segments offers a significant improvement over prior art methods and reagents. We used this pH-sensitive rhodamine-based pHrodo® Red dye to label bovine OS for specific measurement of internalized particles.

[0074] Comparing FITC-labeled photoreceptor outer segments, pHrodo®-BioParticles, and pHrodo®-labeled photoreceptor outer segments, all showed a significant increase in fluorescence in the phagocytosis assay at 37°C compared to 4°C (Figure 1A–C). However, quantitative analysis revealed that only about half of the RPE cell population at 37°C showed an increase in fluorescence compared to the 4°C control when incubated with FITC-labeled particles in the phagocytosis assay, and that about half of the RPE cell population also showed a significant increase in fluorescence in the 4°C control, likely indicating the presence of particles bound to the cell surface but not internalized. Such particles on the cell surface may represent both specific and nonspecific binding. In addition, FITC-labeled photoreceptor outer segment FACS data are less accurate because some FITC fluorescence can be lost at low pH (Figure 2). That is, once particles are internalized and phagosomes fuse with lysosomes, some FITC fluorescence is lost at the final low pH (4.5-5.5). Therefore, the measured fluorescence includes both the loss of some signal from internalized particles and additional signal from particles nonspecifically bound to the cell surface. Both pHrodo®-labeled BioParticles and pHrodo®-labeled photoreceptor outer segments show no increase in fluorescence at 4°C (Figures 1B and 1C), but do show an increase at 37°C, thus allowing specific measurement of only internalized particles fusing with lysosomes.

[0075] As described herein, pHrodo®-labeled ROS are fluorescent at low pH, so the observed shift in fluorescence is indicative of internalized particles. The use of ROS labeled with FITC or other non-pH-sensitive dyes tends to indicate particles nonspecifically bound to the cell surface, specifically bound but not internalized POS, and / or internalized POS that have not fused with lysosomes. Labeling photoreceptor outer segments with pHrodo® or other pH-sensitive dyes instead of the traditionally used FITC, whose fluorescence is inversely correlated to acidity, or using pHrodo®-labeled POS in combination with other pH-sensitive and / or non-sensitive dyes, improves the accuracy of phagocytosis assays, thereby enabling measurement of phagocytosis of physiologically relevant targets and enabling detailed analysis of phagocytic mechanisms.

[0076] In some embodiments, POS are fragmented prior to use with the cells of interest. POS can be fragmented, for example, by sonication or shearing, or other methods known in the art. It has been found that fragmented POS can result in higher phagocytosis measurements from cells. This can be useful for distinguishing positive activity from control activity.

[0077] Phagocytosis assays can be performed using cells in a single cell suspension or in a monolayer. Thus, cells can be provided as a cell suspension or as a monolayer, including cultured monolayers. This latter embodiment is particularly useful when cells normally grow as a monolayer, allowing for the determination of phagocytic activity of cells as they normally exist. Cells can be incubated with labeled POS as an adherent layer, such as a monolayer, and then enzymatically digested (trypsin digestion) to form a single cell population that can be analyzed, for example, by flow cytometry.

[0078] In some embodiments, cells may be incubated with POS at a temperature of 17° C. or higher, or 18° C. or higher, or 19° C. or higher, or 20° C. or higher. The upper end of the temperature range may be 42° C. or lower, or 41° C. or lower, or 40° C. or lower, or 39° C. or lower, or 38° C. or lower, or 37° C. or lower. Test phagocytic activity may be measured at these temperatures. Cells may be provided as a monolayer, including a cultured monolayer.

[0079] In some embodiments, the test cells or test cell populations are incubated with POS at a temperature ranging from 17-40°C, or 20-40°C, or 25-40°C, or 30-40°C, or 35-40°C, or at a temperature of 37°C. The negative control may correspond to cells incubated with POS at a temperature ranging from about 4-16°C, 5-16°C, 6-16°C, 7-16°C, 8-16°C, 9-16°C, 10-16°C, 11-16°C, or 12-16°C. The negative control may correspond to cells incubated with POS at a temperature ranging from about 4-15°C, 5-15°C, 6-15°C, 7-15°C, 8-15°C, 9-15°C, 10-15°C, 11-15°C, or 12-15°C. The cells may be provided as a monolayer, including a cultured monolayer.

[0080] In some embodiments, the cells are previously cryopreserved, thawed, and briefly cultured to establish a monolayer. Once in monolayer form, the phagocytic activity of the cells can be tested as described herein. In some embodiments, cells are exposed to unlabeled POS for a period of time, and then exposed to fluorescently labeled POS to measure phagocytosis of the latter POS. In this manner, cells may be prepared prior to introduction of labeled POS.

[0081] definition In order that the invention described herein may be fully understood, the following detailed description is set forth. Various aspects of the invention are described in detail and may be further illustrated by the examples provided. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Similar or equivalent methods and materials to those described herein can be used in or testing the present invention, and suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. The following terms and definitions are provided herein.

[0082] As used throughout this description and the claims that follow, the meanings of "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Also, as used in this description, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Throughout this specification, the word "comprise" or variations such as "comprises" or "comprising" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0083] As used herein, "embryonic stem cells" (ES cells) broadly refer to cells derived from the inner cell mass of a blastocyst or morula that have been serially passaged as a cell line. ES cells may be derived from fertilization of an egg cell with sperm, or from DNA, nuclear transfer, parthenogenesis, or by means of generating ES cells homozygous for HLA regions. ES cells may also refer to cells derived from mammalian embryos at the zygote, blastomere, or blastocyst stage resulting from the fusion of a sperm and an egg cell, nuclear transfer, parthenogenesis, or chromatin reprogramming and subsequent integration of the reprogrammed chromatin into a cell membrane for cell production. Regardless of their source or the particular method used to produce them, embryonic stem cells can be identified based on (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunodeficient animals.

[0084] The term also includes cells isolated from one or more blastomeres of an embryo, preferably without destroying the remainder of the embryo (see, e.g., Chung et al., Cell Stem Cell. 2008 Feb 7;2(2):113-7; U.S. Patent No. 20060206953 (pre-grant publication); U.S. Patent No. 2008 / 0057041 (pre-grant publication), each of which is incorporated by reference in its entirety). The term also includes cells produced by somatic cell nuclear transfer, even if non-embryonic cells are used in the process. ES cells may be obtained from the fertilization of an egg cell with sperm, or from DNA, nuclear transfer, parthenogenesis, or by means of generating ES cells that are homozygous for HLA regions. ES cells are also cells derived from zygote, blastomere, or blastocyst stage mammalian embryos resulting from the fusion of sperm and an egg cell, nuclear transfer, parthenogenesis, or reprogramming of chromatin and incorporation of the reprogrammed chromatin into the cell membrane for subsequent cell production. Human embryonic stem cells of the present disclosure include, but are not limited to, MA01, MA09, ACT-4, No. 3, H1, H7, H9, H14, and ACT30 embryonic stem cells. In certain embodiments, human ES cells used to produce RPE cells are derived and maintained according to GMP standards.

[0085] "Macular degeneration," as used herein, broadly refers to diseases characterized by progressive loss of central vision associated with abnormalities of Bruch's membrane, the neural retina, and the membrane pigment epithelium. Macular degeneration diseases include, but are not limited to, age-related macular degeneration, North Carolina macular dystrophy, Thorsby's fundus dystrophy, Stargardt's disease, pattern dystrophy, Best's disease, Malachialeventinis, Doyne's honeycomb choroidopathy, dominant drusen, and radial drusen.

[0086] "Pluripotent stem cells," as used herein, refer broadly to cells that are capable of long-term or virtually indefinite in vitro proliferation while maintaining their undifferentiated state, exhibit a stable (preferably normal) karyotype, and have the capacity to differentiate into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) under appropriate conditions.

[0087] As used herein, the terms "RPE cells," "differentiated RPE cells," and "ES-derived RPE cells" may be used interchangeably throughout to broadly refer to RPE cells differentiated from pluripotent stem cells, for example, using the methods disclosed herein. The terms are used to generally refer to differentiated RPE cells regardless of the level of cellular maturity and thus may encompass RPE cells of various levels of maturity. RPE cells can be visually recognized by their cobblestone morphology and early pigment appearance. RPE cells can also be molecularly identified based on the substantial lack of expression of embryonic stem cell markers such as Oct-4 and NANOG, and based on the expression of RPE markers such as RPE65, PEDF, CRALBP, and bestrophin. For example, cells may be considered positive for a marker if expected staining patterns are observed, such as PAX6 localized in the nucleus, bestrophin localized to the cell membrane in a polygonal pattern (showing bestrophin staining localized in a distinct line at the periphery of the cell), ZO-1 staining present at tight junctions that outline the cell in a polygonal pattern, and MITF staining detected as restricted to the nucleus.Unless otherwise specified, as used herein, RPE cells refer to RPE cells differentiated in vitro from pluripotent stem cells.

[0088] As used herein, the terms "mature RPE cells" and "mature differentiated RPE cells" may be used interchangeably throughout to broadly refer to the changes that occur following the initial differentiation of RPE cells. Specifically, RPE cells may be recognized in part by the initial appearance of pigment, but after differentiation, mature RPE cells may be recognized by their advanced pigmentation.

[0089] As used herein, "pigment" broadly refers to any level of pigmentation, such as pigmentation that occurs early during differentiation of RPE cells from ES cells. Pigmentation may vary depending on the cell density and maturity of differentiated RPE cells. After terminal differentiation of RPE cells, the pigmentation of RPE cells may be similar to that of average RPE cells. After terminal differentiation of RPE cells, the pigmentation of RPE cells may be more strongly pigmented than average RPE cells. After terminal differentiation of RPE cells, the pigmentation of RPE cells may be less strongly pigmented than average RPE cells.

[0090] "Photoreceptor progenitor" refers to a cell of the neural retina that can be differentiated from embryonic stem cells or derived from pluripotent stem cells and that expresses the marker PAX6 but not the marker CHX10 (i.e., CHX10(-)). These cells transiently express CHX10 at the retinal neural progenitor stage, but CHX10 expression disappears when the cells differentiate to the photoreceptor progenitor stage. Other markers expressed by photoreceptor progenitor cells can include Pax6, Nr2e3, Trβ2, Mash1, RORβ, and NRL. "Photoreceptor" may also refer to a postmitotic cell that can be differentiated from embryonic stem cells or derived from pluripotent stem cells and that expresses the cell markers rhodopsin or any of the three cone opsins, and optionally rod or cone cGMP phosphodiesterase. The photoreceptor cells may also express recoverin, a marker found in photoreceptor cells. The photoreceptor cells may be rod and / or cone photoreceptor cells.

[0091] Cellular Markers: Exemplary cellular markers that may be assessed for expression include the following: PAX6, RX1, SIX3, SIX6, LHX2, TBX3, SOX2, CHX10, nestin, TRβ2, NR2E3, NRL, MASH1, RORβ, recoverin, opsin, rhodopsin, rod and cone cGMP phosphodiesterase, which may be assessed at protein and / or mRNA level (Fischer AJ, Reh TA, Dev Neurosci. 2001;23(4-5):268-76; Baumer et al., Development. 2003 Jul;130(13):2903-15, Swaroop et al., Nat Rev Neurosci. 2010 Aug;11(8):563-76, Agathocleous and Harris, Annu. Rev. Cell Dev. Biol. 2009. 25:45-69, each of which is incorporated herein by reference in its entirety.) The marker identifiers are commonly used in the literature and in the art, particularly in the art relevant to the context in which the gene identifiers are listed herein, which may include literature related to photoreceptors, rods, cones, photoreceptor differentiation, photoreceptor precursor cells, neural differentiation, neural stem cells, pluripotent stem cells, and other areas as indicated by the context. In addition, for example, unless the context dictates otherwise, the markers are typically human. Cell markers can be identified using standard immunocytochemical methods or standard PCR methods, techniques well known to those of skill in the art.

[0092] A "sign" of disease, as used herein, refers broadly to any abnormality detectable upon examination of a patient that is indicative of disease; an objective indicator of disease, as opposed to a symptom, which is a subjective indicator of disease. A "symptom" of disease, as used herein, refers broadly to any morbid phenomenon or deviation from the normal in structure, function, or perception experienced by a patient that is indicative of disease.

[0093] "Therapy," "therapeutic," "treating," "treat," or "treatment," as used herein, broadly refer to treating a disease, arresting or reducing the progression of a disease or its clinical symptoms, and / or alleviating a disease, resulting in the alleviation of a disease or its clinical symptoms. Therapy includes preventing, suppressing, treating, curing, curing, reducing, alleviating a disease, and / or providing relief from a disease, signs, and / or symptoms. Treatment includes alleviating signs and / or symptoms in patients with ongoing disease signs and / or symptoms (e.g., blindness, retinal deterioration). Treatment also encompasses "prevention" and "suppression." Prevention includes inhibiting a disease from occurring subsequent to treatment of a disease in a patient or reducing the incidence or severity of a disease in a patient. The term "alleviated," for therapeutic purposes, broadly refers to a clinically significant reduction in signs and / or symptoms. Treatment includes treating recurrences or recurrent signs and / or symptoms (e.g., retinal degeneration, vision loss). Treatment includes, but is not limited to, eliminating the appearance of signs and / or symptoms at any time, as well as reducing and eliminating existing signs and / or symptoms. Treatment includes treatment of chronic disease ("maintenance") and treatment of acute disease. For example, treatment includes treating or inhibiting recurrences or the reappearance of signs and / or symptoms (e.g., blindness, retinal degeneration).

[0094] The RPE or photoreceptor progenitor cells in the preparation may have a photoreceptor outer segment (POS) phagocytosis rate that is at least 50% higher, and more preferably at least 75, 100, 150, or even 200% higher, than the phagocytosis rate of photoreceptor outer segments (POS) for an equivalent number of RPE cells from an isolated adult eye (i.e., a human adult patient aged 25-80 years, more preferably an adult aged 50-80 years). The photoreceptor progenitor cells in the preparation may have a photoreceptor outer segment (POS) phagocytosis rate that is at least 50% higher, and more preferably at least 75, 100, 150, or even 200% higher, than the phagocytosis rate of photoreceptor outer segments (POS) for an equivalent number of photoreceptor progenitor cells from an isolated adult eye (i.e., a human adult patient aged 25-80 years, more preferably an adult aged 50-80 years).

[0095] The RPE or photoreceptor progenitor cells in the preparation may exhibit a photoreceptor outer segment (POS) phagocytosis rate of at least 20% of the total concentration of POS after 24 hours, and more preferably at least 25, 30, 25, 40, or even 50% of the total concentration of POS after 24 hours. POS phagocytosis can be measured, as an illustrative and non-limiting example, with non-FITC-labeled POS as described herein using the protocol described in Bergmann et al., FASEB Journal, March 2004, vol. 18, pp. 562-564.

[0096] The RPE or photoreceptor progenitor cell population may comprise differentiated RPE cells of various levels of maturity, or may be substantially pure with respect to differentiated RPE cells of a particular level of maturity. The RPE cells may be a substantially purified preparation comprising RPE cells of various levels of maturity / pigmentation.

[0097] Cryopreserved preparation of RPE cells RPE cells or photoreceptor precursor cells can be preserved by any suitable method known in the art (e.g., deep freezing), and can be frozen at any temperature suitable for preserving cells.Before using these cells, they can be tested in the assay of the present invention to determine the phagocytic activity and / or capacity of the cells. RPE cells or photoreceptor progenitor cells that demonstrate competence in the phagocytosis assay of the present invention can be used to treat retinal degenerative diseases associated with retinal detachment, retinal dysplasia, angioid streaks, myopic macular degeneration or retinal atrophy, or a number of vision-altering diseases that result in photoreceptor damage and blindness, such as congenital choroideremia, diabetic retinopathy, macular degeneration (e.g., age-related macular degeneration), retinitis pigmentosa, and Stargardt's disease (fundus flava).

[0098] RPE or photoreceptor precursor cells provided herein can be human RPE or photoreceptor precursor cells.However, it should be noted that human cells can be used in animal models or animal patients as well as in human patients.For example, human cells can be tested in mouse, rat, cat, dog or non-human primate models of retinal degeneration.In addition, human cells can also be used therapeutically to treat animals in need, such as in veterinary medicine.

[0099] Screening assays The present disclosure provides methods for identifying agents that modulate the phagocytic activity of RPE cells or photoreceptor progenitor cells.

[0100] example Having now generally described the invention, it will be more readily understood by reference to the following examples, which are included merely to illustrate certain aspects and embodiments of the invention and are not intended to limit the invention.

[0101] RPE cells were derived from human embryonic stem cells and isolated from differentiated cultures of pigmented clusters at passages 2–5, as previously described (Klimanskaya et al., 2004). Cells were cultured in EGM-2 medium (Lonza) until confluence was reached, and then cultured in RPE maintenance medium (Klimanskaya et al., 2004). Cells used in experiments had established a differentiated RPE phenotype characterized by hexagonal morphology, varying levels of brown pigmentation, cuboidal cell appearance, polarized tissue, and tight junctions. Alternatively, cells were used after reaching confluence but before full maturation. Bovine rod outer segments (catalog # 98740) from InVision Bioresources. FITC isomer (catalog # F1906) from Life Technologies. pHrodo® Red Phagocytosis Particle Labeling Kit (catalog # A10026) from Life Technologies.

[0102] Labeling of POS with FITC One vial of 10 mg of FITC isomer I was resuspended at 2 mg / mL in 0.1 M sodium carbonate buffer (pH 9.5). After centrifugation at 3000 g for 10 minutes, the undiluted particles were removed and the supernatant was used to label bovine ROS. Twenty-five bovine eyeballs containing ROS were thawed and resuspended in 5 mL of wash buffer (20 mM phosphate buffer containing 10% sucrose, pH 7.2, and 5 mM taurine). 1.5 mL of the 2 mg / mL FITC supernatant was added to the resuspended ROS and incubated for 1 hour at room temperature with rocking in the dark. After incubation, the ROS-FITC fraction was spun down at 3000 g and resuspended in 10 mL of wash buffer. This wash step was repeated twice. After washing, the cells were resuspended in 10 mL of 2.5% sucrose-containing DMEM (Gibco # 11960) and spun down again at 3000 g for 10 min. Finally, the cells were resuspended in 10 mL of 2.5% sucrose-containing DMEM and the ROS-FITC particles were counted using a hemocytometer to obtain a 1 × 10 6000 ROS-FITC particle count in 2.5% sucrose-containing DMEM. 8 The concentration was adjusted to 1000 particles / mL and the particles were frozen at -80°C.

[0103] pHrodo® POS Labeling Twenty-five bovine eyeballs, each containing ROS, were resuspended in 4.165 mL of 0.1 M sodium bicarbonate buffer from the pHrodo® Red phagocytosis Particle Labeling kit. The ROS were aliquoted into four 750 μL aliquots into microcentrifuge tubes. The tubes were centrifuged at 10,000 RPM for 1 minute and then resuspended in 750 μL of 0.1 M sodium bicarbonate buffer. pHrodo® dye was resuspended in DMSO to a final concentration of 10 mM. pHrodo® dye was added to the ROS in sodium bicarbonate buffer to a final concentration of 0.5 mM and incubated in the dark for 45 minutes. 500 μL of "Component C" (from the kit) was added and centrifuged at 10,000 RPM for 1 minute. The supernatant was aspirated and resuspended in 1 mL of 100% methanol. The tubes were vortexed for 30 seconds and centrifuged at 10,000 RPM for 1 minute. The methanol was aspirated and the particles were resuspended in 1 mL of "Component C" (wash buffer from the kit) and centrifuged again at 10,000 RPM for 1 minute. This wash step was repeated twice in total. All particles were resuspended in a total volume of 20 mL of "Buffer B" (from the kit). ROS-pHrodo® was spun down at 3,000 RPM for 10 minutes, resuspended in DMEM with 2.5% sucrose, and diluted to 1 x 10 8 The concentration was adjusted to 1000 particles / mL and the particles were frozen at -80°C.

[0104] RPE cells were incubated with either pHrodo®-conjugated BioParticles or bovine outer segments labeled with either FITC or pHrodo® for 2-24 hours at 37°C. Cells were then washed, harvested with trypsin / dissociation buffer (1:1), centrifuged, and analyzed by flow cytometry. As a negative control, cells were incubated for the same time at 4°C.

[0105] In addition, interpretation of FITC-labeled ROS FACS data is less accurate because some of the FITC fluorescence can be lost at low pH (Figure 2A and B). That is, once particles are internalized and phagosomes fuse with lysosomes, some of the FITC fluorescence is lost at the final low pH (4.5–5.5). Thus, the measured fluorescence includes both a partial signal loss from internalized particles and additional signal from particles nonspecifically bound to the cell surface.

[0106] pHrodo® is a pH-sensitive fluorescent dye, and both pHrodo®-labeled BioParticles and ROS show no increase in fluorescence at 4°C (Figures 1B and 1C), allowing for the specific measurement of only internalized particles fusing with lysosomes. Labeling of ROS with pHrodo® can be used instead of or complementary to FITC-labeled ROS to improve the accuracy of phagocytosis assays and dissect the complex mechanisms of phagocytosis.

[0107] pHrodo® E. coli Labeling with Fluorescent Bioparticles Phagocytosis was assessed by a FACS-based assay using pHrodo® E. coli fluorescent bioparticles (Invitrogen), which fluoresce when incorporated into the reduced pH environment of intracellular phagosomes. Bioparticles were prepared according to the manufacturer's instructions. Confluent RPE cells were incubated with 50–200 μL of bioparticles per well of a 4-well plate in CO2-independent medium (Invitrogen) for 16–20 hours at 37°C. A negative control plate was incubated at 4°C. Cells were observed under a microscope, harvested with trypsin, and analyzed by FACS, counting 10,000 events on a C6 flow cytometer.

[0108] [Table 1-1] [Table 1-2]

[0109] Phagocytosis of pHrodo® Red-labeled ROS by RPE cells hESC-derived RPE and ARPE-19 cells were cultured in RPE growth medium (RPE-GM) consisting of Endothelial Cell Growth Medium (Lonza, cat# CC-3162, CC-3156).

[0110] For the phagocytosis assay, RPE cells were plated at 5x10 in a 96-well plate (Becton-Dickinson). 5 cells / cm 2 Cells were seeded at a density of 1000 kJ / ml and maintained in a humidified incubator at 37°C and 5% CO2. For optimal assay measurements, RPE cells were cultured in RPE-GM for 3–5 days prior to assessment of phagocytic activity. If cells were cultured for longer than 5 days, RPE-GM was switched to RPE Maintenance Medium (RPE-MM), consisting of DMEM supplemented with 10% fetal bovine serum, GlutaMax, and Normocin. The medium was changed every 2–3 days to ensure adequate nutrients were provided. The phagocytic activity of RPE cells was determined using pH-sensitive rhodamine-based pHrodo® Red-labeled rod outer segments (ROS) (InVision Bioresources, cat. # 98740). Labeling of ROS with the pHrodo® Red Microscale Labeling Kit (Thermo Fisher Scientific, cat. # P35363) was previously described. Confluent RPE cells in each well were seeded with 0.1 mL of DMEM medium containing 10% FBS and Normocin, along with 0.1 mL of labeled ROS reconstituted in DMEM supplemented with 10% FBS and Normocin.

[0111] To assess the optimal phagocytic capacity of RPE cells, 1.5x10 cells were cultured in a 96-well plate. 6 , 3x10 6 , 3.75x10 6 , 5x10 6 , 6x10 6 , 7.5x10 6 , 10x10 6 and 13.5x10 6 Different ROS concentrations (ROS / well) were tested. To reduce ROS aggregation and increase phagocytosis efficiency, a direct pulsed sonication step was introduced during rod outer segment reconstitution. RPE cells and ROS were incubated for 20–28 h in a 5% CO2 and 95% air atmosphere at 37°C for test samples and 12–15°C for negative controls. The following day, after incubating ROS with the RPE cell monolayer for 20–28 h, the culture medium was aspirated and each well was washed three times with 0.2 mL of Ca / Mg-free PBS (Gibco / Invitrogen #14190-250). 0.2 mL of 0.25% Trypsin / EDTA (Sigma, cat. # T4049) supplemented with Cell Dissociation Buffer (Gibco / Invitrogen, cat. # 13151) was added to each well at a 1:1 ratio and incubated at room temperature until each cell suspension became visible (10–20 min). The cell suspension for each sample was transferred to an appropriately labeled round-bottom polystyrene tube containing 2 mL of DMEM supplemented with 10% FBS to neutralize the reaction and centrifuged at 160 g for 5 min. The supernatant was decanted, leaving approximately 0.2–0.25 mL of liquid. The sample tubes were vortexed, and ROS uptake by RPE cells was assessed using a BD Accuri C6 Flow Cytometer as previously described.

[0112] To further increase phagocytic capacity, naive RPE cells in monolayers can be made "competent" by exposing them to unlabeled ROS for a defined period of time, with or without a recovery step, before performing phagocytosis of pHrodo® Red-labeled ROS following the procedure described above.

[0113] References Schwartz SD, Hubschman JP, Heilwell G, Franco-Cardenas V, Pan CK, Ostrick RM, Mickunas E, Gay R, Klimanskaya I, Lanza R. Embryonic stem cell trials for macular degeneration: a preliminary report. Lancet. 2012 Feb 25;379(9817):713-20. doi:10.1016 / S0140-6736(12)60028-2. Epub 2012 Jan 24. PubMed PMID: 22281388. Klimanskaya I. Retinal pigment epithelium. Methods Enzymol. 2006;418:169-94. PubMed PMID: 17141036. Lund RD, Wang S, Klimanskaya I, Holmes T, Ramos-Kelsey R, Lu B, Girman S, Bischoff N, Sauve Y embryo, Lanza R. Humannic stem cell-derived cells rescue visual function in dystrophic RCS rats. Cloning Stem Cells. 2006 Fall;8(3):189-99. PubMed PMID: 17009895. Klimanskaya I, Hipp J, Rezai KA, West M, Atala A, Lanza R. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells. 2004;6(3):217-45. PubMed PMID:15671670. Mao Y, Finnemann SC. Analysis of photoreceptor outer segment phagocytosis by RPE cells in culture. Methods Mol Biol. 2013;935:285-95. doi:10.1007 / 978-1-62703-080-9_20. PubMed PMID: 23150376; PubMed Central PMCID:PMC3590840.

[0114] Finnemann SC, Bonilha VL, Marmorstein AD, Rodriguez-Boulan E. Phagocytosis of rod outer segments by retinal pigment epithelial cells requires alpha(v)beta5 integrin for binding but not for internalization. Proc Natl Acad Sci U S A. 1997 Nov 25;94(24):12932-7. PubMed PMID: 9371778; PubMed Central PMCID: PMC24241. Miksa M, Komura H, Wu R, Shah KG, Wang P. A novel method to determine the engulfment of apoptotic cells by macrophages using pHrodo succinimidyl ester. J Immunol Methods. 2009 Mar 15;342(1-2):71-7. doi: 10.1016 / j.jim.2008.11.019. Epub 2009 Jan 9. PubMed PMID: 19135446; PubMed Central PMCID: PMC2675277. Lu B, Malcuit C, Wang S, et al. Long-term safety and function of RPE from human embryonic stem cells in preclinical models of macular degeneration. Stem Cells 2009; 21, 2125-2135. Sparrow JR, Hicks D, Hamel CP. The retinal pigment epithelium in health and disease. Curr Mol Med 2010; 10, 802-823. Strauss O. The retinal pigment epithelium in visual function. Physiol Rev 2005; 85, 845-881.

Claims

1. 1. A method for assessing phagocytic activity, comprising: Incubating the cells with photoreceptor outer segments (POS) for a time and temperature sufficient for the cells to phagocytose the POS, where POS fluoresces more strongly at acidic pH than at higher pH; and Detecting the fluorescence intensity of the cells after incubation, where an increase in fluorescence compared to the control indicates phagocytosis of the POS by the cells; The method comprising:

2. 10. The method of claim 1, wherein the cells are incubated with the POS at a temperature ranging from about room temperature to about 37°C, or from about room temperature to about 40°C.

3. 10. The method of claim 1, wherein the cells are incubated with the POS at about room temperature, about physiological temperature, or about 37°C.

4. The method of any one of claims 1 to 3, wherein the cells are incubated with the POS for about 16 to 20 hours.

5. The method of any one of claims 1 to 4, wherein the cells are provided as a cell culture.

6. The method of any one of claims 1 to 5, wherein the cells are a confluent cell culture.

7. The method of any one of claims 1 to 6, wherein the cells are RPE cells.

8. The method of any one of claims 1 to 7, wherein the cells are human RPE cells.

9. The method according to any one of claims 1 to 6, wherein the cells are photoreceptor precursor cells.

10. The method according to any one of claims 1 to 6, wherein the cells are human photoreceptor precursor cells.

11. The method according to any one of claims 1 to 10, wherein the POS is labeled with pHrodo® Red dye.

12. The method of any one of claims 1 to 11, wherein the control is cells incubated with POS at below room temperature.

13. The method of any one of claims 1 to 12, wherein the control is cells incubated with POS at 4°C.

14. The method of any one of claims 1 to 13, wherein the fluorescence is detected by flow cytometry.

15. The method of any one of claims 1 to 14, wherein the fluorescence is detected using a plate reader.

16. 1. A method for assessing phagocytic activity, comprising: providing photoreceptor outer segments (POS) labeled with a fluorescent label, which have a fluorescent signal that is changed when the POS is phagocytosed into a low pH compartment within the cell relative to the fluorescent signal when the POS is present outside the cell; Incubating the test cells with the labeled POS under conditions that allow phagocytosis of the labeled POS; and Detecting any altered fluorescence in the test cells after incubation with the labeled POS, and quantifying the phagocytic activity of the test cells therefrom. The method comprising:

17. 17. The method of claim 16, wherein the altered fluorescent signal is an increase in fluorescent signal intensity relative to when the fluorescent label is present extracellularly.

18. 18. The method of claim 16 or 17, wherein the altered fluorescent signal is detectable by flow cytometry.

19. 19. The method of any one of claims 16 to 18, wherein the altered fluorescent signal distinguishes between labeled POS that has been internalized by phagocytosis and labeled POS that is bound to the surface of the test cell but has not been internalized.

20. 19. The method of any one of claims 16 to 18, wherein the altered fluorescent signal detected in the test cells is compared to a control cell population incubated with labeled POS to quantify the phagocytic activity of the test cells.

21. 21. The method of claim 20, wherein the control cell population is incubated with the labeled POS at below room temperature.

22. The method of any one of claims 16 to 21, wherein the test cells comprise retinal pigment epithelial (RPE) cells.

23. The method of any one of claims 16 to 21, wherein the test cells comprise photoreceptor precursor cells.

24. 24. The method of claim 22 or 23, wherein the test cell is a human cell.

25. The method of any one of claims 16 to 24, wherein the test cells are produced by in vitro differentiation of pluripotent stem cells.

26. The method of any one of claims 16 to 24, wherein the test cells are cryopreserved and thawed before use.

27. The method of any one of claims 16 to 24, wherein the fluorescent label is pHrodo® Red.

28. 25. The method of any one of claims 16 to 24, wherein the POS is labeled with pHrodo® Red and pHrodo® Red E. coli BioParticles.

29. 1. A labeled photoreceptor outer segment (POS) preparation for assessing phagocytic activity of a test cell population, comprising: The POS is labeled with a fluorescent label, and when the fluorescent label is taken up into a low pH compartment within a cell by phagocytosis, the fluorescent signal has changed relative to the fluorescent signal when the fluorescent label is present outside the cell. Said preparation.

30. 30. The preparation of claim 29, wherein the altered fluorescent signal is an increase in fluorescent signal intensity relative to when present outside the cell.

31. 31. The preparation of claim 29 or 30, wherein the altered fluorescent signal is detectable by flow cytometry.

32. The preparation of any one of claims 29 to 31, wherein the fluorescent label is pHrodo® Red.

33. 32. The preparation according to any one of claims 29 to 31, wherein the POS is labeled with pHrodo® Red and pHrodo® Red E. coli BioParticles.

34. 1. A method for measuring phagocytic activity in a cell population, comprising: measuring test fluorescence in a test cell population contacted with non-FITC fluorescently labeled photoreceptor outer segments (POS); and comparing the measured test fluorescence with the control fluorescence; Including, Here, non-FITC fluorescently labeled POS emits fluorescence at acidic pH, but emits no or minimal fluorescence at higher pH. The method.

35. 35. The method of claim 34, wherein the test cell population is contacted with the non-FITC fluorescently labeled POS at a temperature ranging from about room temperature to about physiological temperature, or from about room temperature to about 40°C.

36. 35. The method of claim 34, wherein the control fluorescence is the fluorescence of a cell population contacted with non-FITC fluorescently labeled POS at below room temperature.

37. 35. The method of claim 34, wherein the test cell population is contacted with the non-FITC fluorescently labeled POS at a temperature of about 15-40°C or at physiological temperature.

38. The method of any one of claims 34 to 37, wherein the control fluorescence is the fluorescence of a cell population contacted with non-FITC fluorescently labeled POS at 4°C.

39. 1. A method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of a cell population incubated with fluorescently labeled photoreceptor outer segments (POS) labeled with pHrodo® Red dye at a temperature ranging from about room temperature to about physiological temperature, or from about room temperature to about 40°C; and (2) measuring control fluorescence in a second aliquot of the cell population incubated at below room temperature with fluorescently labeled POS labeled with pHrodo® Red dye; wherein test fluorescence greater than control fluorescence indicates phagocytic activity of the cell population; The method.

40. 40. The method of claim 39, wherein the cell population is an RPE cell population.

41. 40. The method of claim 39, wherein the cell population is a photoreceptor progenitor cell population.

42. 1. A method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of a cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at a temperature ranging from about room temperature to about physiological temperature, or from about room temperature to about 40° C.; (2) measuring control fluorescence in a second aliquot of the cell population incubated below room temperature with photoreceptor outer segments (POS) fluorescently labeled with pHrodo® Red dye alone or with pHrodo® Red E. coli BioParticles; wherein test fluorescence greater than control fluorescence indicates phagocytic activity of the cell population; The method.

43. 43. The method of claim 42, wherein the cell population is an RPE cell population.

44. 43. The method of claim 42, wherein the cell population is a human RPE cell population.

45. The method of claim 42, wherein the cell population is a photoreceptor progenitor cell population.

46. The method of claim 42, wherein the cell population is a human photoreceptor progenitor cell population.

47. 10. The method of any one of the preceding claims, wherein the cell, cell population, test cell or test cell population is enzymatically digested prior to use.

48. 10. The method of any one of the preceding claims, wherein the cell, cell population, test cell or test cell population is provided as an adherent cell population.

49. 10. The method according to any one of the preceding claims, wherein the POS is a fragmented POS.

50. 10. The method according to any one of the preceding claims, wherein the POS is an ultrasonically treated POS.

51. 1. A method for assessing the phagocytic activity of a cell population, comprising: incubating the adherent cell population with photoreceptor outer segments (POS) for a time and at a temperature sufficient to cause cells in the cell population to phagocytose POS, where POS fluoresces more strongly at acidic pH than at higher pH; and detecting the fluorescence intensity of the cell population after incubation, wherein an increase in fluorescence compared to a control indicates phagocytosis of the POS by the cells; Optionally, the cell population is provided as a monolayer, further optionally as a confluent monolayer. The method.

52. 52. The method of claim 51, wherein the cell population is incubated with the POS at a temperature ranging from about 17 to 40°C, or from about 25 to 40°C, or from about 34 to 40°C, or at a temperature of about 37°C.

53. 53. The method of claim 51 or 52, wherein the cells are RPE cells or photoreceptor progenitor cells.

54. 54. The method of any one of claims 51 to 53, wherein the cell is a human cell.

55. 55. The method of any one of claims 51 to 54, wherein the POS is labeled with pHrodo® Red dye.

56. 56. The method of any one of claims 51 to 55, wherein the control is a cell population incubated with POS at a temperature of about 12-15°C.

57. 57. The method of any one of claims 51 to 56, wherein the fluorescence is detected using a plate reader.

58. 1. A method for assessing phagocytic activity, comprising: providing photoreceptor outer segments (POS) labeled with a fluorescent label, which have a fluorescent signal that is changed when the POS is phagocytosed into a low pH compartment within the cell relative to the fluorescent signal when the POS is present outside the cell; Incubating the adherent test cells with the labeled POS at a temperature ranging from about 17 to 40°C under conditions that allow phagocytosis of the labeled POS; and Detecting any altered fluorescence in the adherent test cells after incubation with the labeled POS, and quantifying the phagocytic activity of the adherent test cells therefrom. The method comprising:

59. 59. The method of claim 58, wherein the adherent test cells are incubated with the labeled POS at a temperature in the range of about 25-40°C, or about 34-40°C, or at a temperature of about 37°C.

60. 60. The method of claim 58 or 59, wherein the altered fluorescent signal detected in the adherent test cells is compared to a control cell population incubated with the labeled POS at a temperature in the range of about 12-15°C to quantify the phagocytic activity of the test cells.

61. 61. The method of any one of claims 58 to 60, wherein the adherent test cells comprise retinal pigment epithelial (RPE) cells or photoreceptor progenitor cells.

62. 62. The method of any one of claims 58 to 61, wherein the adhesion test cells are human cells.

63. 63. The method of any one of claims 58 to 62, wherein the adherent test cells are produced by in vitro differentiation of pluripotent stem cells.

64. 64. The method of any one of claims 58 to 63, wherein the fluorescent label is pHrodo® Red.

65. 65. The method of any one of claims 58 to 64, wherein the POS is pHrodo® Red and pHrodo® Red E. coli BioParticles.

66. 1. A method for measuring phagocytic activity in an adherent cell population, comprising: measuring test fluorescence in an adherent test cell population contacted with non-FITC fluorescently labeled photoreceptor outer segments (POS); and comparing the measured test fluorescence with the control fluorescence; Including, Here, non-FITC fluorescently labeled POS emits fluorescence at acidic pH, but emits no or minimal fluorescence at higher pH. The method.

67. 67. The method of claim 66, wherein the test cell population is contacted with the non-FITC fluorescently labeled POS at a temperature ranging from about 17 to 40°C, or from about 25 to 40°C, or from about 34 to 40°C, or at a temperature of about 37°C.

68. 68. The method of claim 66 or 67, wherein the control fluorescence is the fluorescence of an adherent cell population contacted with non-FITC fluorescently labeled POS at a temperature of about 12-15°C.

69. 1. A method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of an adherent cell population incubated with fluorescently labeled photoreceptor outer segments (POS) labeled with pHrodo® Red dye at a temperature ranging from about 17-40°C, or about 25-40°C, or 34-40°C, or at a temperature of 37°C; and (2) measuring control fluorescence in a second aliquot of the adherent cell population incubated with fluorescently labeled POS labeled with pHrodo® Red dye at a temperature ranging from about 12 to 15°C; wherein test fluorescence greater than control fluorescence indicates phagocytic activity of the cell population; The method.

70. 1. A method for measuring phagocytic activity, comprising: (1) measuring test fluorescence in a first aliquot of an adherent cell population incubated with pHrodo® Red dye-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at a temperature ranging from about 17-40°C, or about 25-40°C, or about 34-40°C, or at a temperature of about 37°C; and (2) measuring control fluorescence in a second aliquot of the adherent cell population incubated with pHrodo® Red-labeled photoreceptor outer segments (POS) alone or with pHrodo® Red E. coli BioParticles at a temperature ranging from about 12-15°C; wherein test fluorescence greater than control fluorescence indicates phagocytic activity of the cell population; The method.

71. 71. The method of any one of claims 51 to 70, wherein the cell, cell population, test cell, or test cell population is enzymatically digested prior to use.

72. The method of any one of claims 51 to 71, wherein the POS is fragmented POS.

73. 73. The method according to any one of claims 51 to 72, wherein the POS is an ultrasonically treated POS.

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