Compositions and methods for differentiating RPE cells

Abstract

The present disclosure relates to the use of culturing retinal pigment epithelium (RPE) cells. The method comprises: a) providing a PSC population; b) exposing the PSC population to a first culture environment comprising an extracellular matrix protein and a BMP signaling inhibitor; c) culturing the PSC population in the first culture environment for about 0 to 60 hours; and d) obtaining a population of cells that are marked as the retinal lineage.

Description

Cross Reference to Related Applications

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 457,430, filed April 6, 2023, the entirety of which is incorporated by reference herein in its entirety. TECHNICAL FIELD

[0002] The present disclosure relates to cell culture applications, and more specifically to cell differentiation and / or maturation applications. Still more specifically, the present disclosure relates to culturing retinal pigment epithelial (RPE) cells and / or progenitors thereof. BACKGROUND

[0003] The retina is the innermost layer of the eye and is a complex stratified structure of neurons that capture and process light: the light signals are converted into electrical signals that travel through the optic nerve to the visual centers of the brain.

[0004] The retinal pigment epithelium (RPE) is a monolayer of cells located at the outermost layer of the retina. The basal side of the RPE is connected to Bruch’s membrane and the choroid, while the apical side is connected to the outer segments of photoreceptor cells via microvilli structures extending from the RPE cells. RPE cells are a highly phagocytic cell type and are capable of internalizing photoreceptor outer segments (POS) from overlying rod or cone photoreceptor cells. The RPE performs several key functions in vision, such as light absorption, formation of the blood-retinal outer barrier, nutrient and ion transport, retinoid cycling, phagocytosis of spent photoreceptor outer segments, and growth factor secretion (Boulton et al. (2001) Eye 15, 384-389). The RPE has a dark brown color due to its melanin content, which reduces damage to the retina and internal nerves from ultraviolet light.

[0005] RPE structure and function are essential for normal vision, and changes in the RPE can impair function and lead to degenerative retinal diseases such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Stargardt disease (SD). AMD is the leading cause of severe vision loss in adults over 60 years of age and is estimated to affect approximately 196 million people worldwide in 2020. RP causes vision loss in childhood or early adulthood and affects 100,000 people in the United States. SD has a prevalence of 1 in 10,000 live births and is the most common form of inherited juvenile macular degeneration (Yang S et al. (2021) Front Pharmacol. 12:727870). There are currently no therapies for these degenerative disorders. Therefore, understanding the development of RPE in vitro can help elucidate the pathogenesis of related conditions or diseases and can help develop new therapeutic approaches.

[0006] In vitro generated RPE cells can be used to study RPE development, to identify factors that cause damage to RPE, or to identify agents that can be used to promote endogenous RPE cell repair. In addition, in vitro generated RPE cells can be used in cell therapy themselves, which would involve replacing or restoring all or part of a patient's damaged RPE cells.

[0007] Pluripotent stem cells (PSCs), including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), would be ideal starting cell types for in vitro generation of RPE cells. Generation of RPE from PSCs can accelerate research into various eye diseases and can also provide an unlimited source of RPE for retinal cell therapy. Thus, there is a need for a rapid and efficient in vitro protocol to generate RPE from PSCs, and more specifically, a need for a more compliant protocol suitable for cell therapy applications. SUMMARY

[0008] In one aspect of the disclosure, methods of differentiating immature and / or mature RPE, such as from one or more pluripotent stem cells (PSCs), are provided. In one embodiment, the differentiation of immature and / or mature retinal pigment epithelial (RPE) cells proceeds through different stages. Thus, in one aspect of the disclosure, methods of differentiating a population of cells destined for the retinal lineage are provided, the methods comprising providing a population of PSCs and exposing the population of PSCs to a first culture environment comprising an extracellular matrix protein and a bone morphogenetic protein (BMP) signaling inhibitor.

[0009] In one embodiment, the concentration of the BMP signaling inhibitor is about 1000 nM or less, about 750 nM or less, about 600 nM or less, about 500 nM or less, about 250 nM or less, about 100 nM or less, or about 50 nM or less.

[0010] In one embodiment, the BMP signaling inhibitor is comprised in a first cell culture medium. In one embodiment, the first cell culture medium comprises a basal medium.

[0011] In one embodiment, the method can further comprise culturing the population of PSCs in the first culture environment for about 0 to 60 hours to obtain a population of cells destined for the retinal lineage.

[0012] In one embodiment, the first culture environment is free of one or both of: i) a direct wnt signaling antagonist; and ii) an activin signaling agonist.

[0013] In an embodiment, the extracellular matrix protein is coated on the culture surface. In an embodiment, the extracellular matrix protein is vitronectin, laminin, fibronectin, collagen, or a mixture comprising more than one of the foregoing. In an embodiment, the extracellular matrix protein is recombinant.

[0014] In an embodiment, the method of the present disclosure can further comprise exposing the population of cells destined for retinal lineage to a second culture environment and culturing the population of cells for about 0 to 60 hours to obtain a population of ocular zone progenitor cells.

[0015] In an embodiment, the second culture environment comprises an extracellular matrix protein and a second cell culture medium. In an embodiment, the second cell culture medium comprises IGF and a basal medium. In an embodiment, the second cell culture medium comprises an FGF signaling agonist. In an embodiment, the second cell culture medium comprises a basal medium and one or more of IGF and an FGF signaling agonist.

[0016] In an embodiment, the method of the present disclosure can further comprise exposing the population of ocular zone progenitor cells to a third culture environment and culturing the population of ocular zone progenitor cells for about 2 to 5 days to obtain a population of ocular blood vessel progenitor cells. In an embodiment, the ocular blood vessel progenitor cells are bipotent for retinal pigment epithelium or neural retina.

[0017] In an embodiment, the third culture environment comprises an extracellular matrix protein and a third cell culture medium. In an embodiment, the third cell culture medium comprises IGF and a basal medium. In an embodiment, the third cell culture medium comprises an activin signaling agonist. In an embodiment, the third cell culture medium comprises a basal medium and one or more of IGF and an activin signaling agonist.

[0018] In an embodiment, the method of the present disclosure can further comprise exposing the population of ocular blood vessel progenitor cells to a fourth culture environment and culturing the population of ocular blood vessel progenitor cells for about 5 to 10 days to obtain a population of immature retinal pigment epithelial cells.

[0019] In an embodiment, the fourth culture environment comprises an extracellular matrix protein and a fourth cell culture medium. In an embodiment, the fourth cell culture medium comprises a basal medium and one or more of an activin signaling agonist, a wnt signaling agonist, and an FGF signaling antagonist.

[0020] In one embodiment, the methods of the disclosure can further comprise dissociating the population of immature retinal pigment epithelial cells and replating the dissociated cells to enrich for immature retinal pigment epithelial cells. In one embodiment, the population of dissociated immature retinal pigment epithelial cells is not enriched by manual dissection, particle-based enrichment, or ligand-mediated enrichment.

[0021] In one embodiment, about 50% or more of the population of immature retinal pigment epithelial cells express PMEL17.

[0022] In one embodiment, the methods can further comprise maturing the population of immature retinal pigment epithelial cells for about 1 to 5 weeks. In one embodiment, the population of immature retinal pigment epithelial cells is matured upon contact with a maturation medium comprising a basal medium and at least a soluble source of iron. In one embodiment, the maturation medium further comprises one or more of a steroid hormone and a polyamine. In one embodiment, the maturation medium can further comprise progesterone and putrescine.

[0023] In one embodiment, about 80% or more of the mature RPE express RPE65.

[0024] In one embodiment, one or more of the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment is serum-free and / or animal component-free.

[0025] In one embodiment, one or more of the second medium, the third medium, and the fourth medium lacks an exogenously added inhibitor of BMP signaling.

[0026] In one embodiment, one or more of the first medium, the second medium, the third medium, and the fourth medium lacks nicotinamide.

[0027] In one embodiment, the population of PSCs is cultured under non-adherent conditions to produce three-dimensional retinal organoids. In one embodiment, the population of PSCs is seeded into a microwell device.

[0028] In other aspects of the disclosure, methods of differentiating immature or mature RPE do not start from PSCs, but rather start from any of the downstream cell populations described herein using appropriate culture environments and / or media also as described herein.

[0029] In another aspect of the disclosure, systems or kits for differentiating immature or mature RPE from a population of PSCs (or any downstream intermediate) are provided, comprising one or more of the following: a stage-appropriate medium composition (or basal medium and supplements); an extracellular matrix protein; and instructions. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to better understand the various embodiments described herein, and to show clearly how they can be implemented, reference will be made to the accompanying drawings, which show at least one exemplary embodiment and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0031] Figure 1 Stepwise differentiation of immature RPE from pluripotent stem cells is shown. Differentiation of two iPS lines (3A and M001) was performed as described herein and representative images were taken at day 2, 7 and 14 (A). Scale bar represents 200 pm. Flow cytometry analysis of PMEL17 marker expression of day 14 cells in PSC-derived immature RPE cells (B).

[0032] Figure 2 Cell morphology during RPE maturation is shown. Differentiation of two PSC lines (1C and H9) was performed as described herein and representative images were taken at day 18, 21, 28, 42 and 49 (A). Scale bar represents 200 pm.

[0033] Figure 3 Characterization of mature RPE is shown. Flow cytometry results of PMEL17 (91.4% ± 1.2%) marker expression in day 14 differentiated cells and PMEL17 (96.6% ± 0.5%) and RPE65 (93% ± 1.0%) marker expression in day 49 differentiated cells derived from four different PSC lines (A). Data represent mean ± standard error of the mean of 22-25 experiments. Bar graphs quantifying apical and basal secretion of growth factors from mature RPE derived from four different PSC lines. VEGF apical and basal secretion was assessed for day 52 cells (B) and PEDF apical and basal secretion was assessed for day 53 cells (C). Bar graph of barrier function of mature RPE differentiated from four different PSC lines, where transepithelial electrical resistance (TEER) values were measured for day 49 cultures (D). For B) - D), data represent mean of three experiments. Flow cytometry plots quantifying phagocytosis of FITC-conjugated photoreceptor outer segments by mature RPE derived from two PSC lines (H9 and 1C) (E). Representative images of pigmentation of mature RPE derived from four different PSC lines. Day 49 cells were imaged by brightfield microscopy (F). Scale bar represents 50 pm.

[0034] Figure 4The impact of media compositions during differentiation of immature RPE is shown. Differentiation of two PSC lines (H9 and M001) was performed as described herein, but in the presence of cytokines and small molecules at 0.5X, 0.75X, IX, and 1.25X concentrations between days 4 and 14. Representative images were taken of cells at day 14 (A) and day 49 (B). Bar graphs quantifying melanin content in day 49 cells in each media composition are shown in (C). PMEL17 expression was assessed by flow cytometry in day 14 and day 49 cells (D). Data represent the mean of two experiments.

[0035] Figure 5 The impact of different basal media on RPE maturation is shown. Representative brightfield images show pigmentation of 1C-derived RPE cultured in two different supplemented and unsupplemented basal media formulations compared to control basal media (A). Scale bar represents 200 pm. Bar graphs of melanin content following culture in supplemented and unsupplemented media formulations (B). Data represent the mean ± sem of 4 experiments. Representative brightfield images of H9-derived RPE cells cultured in the presence of increasing concentrations of ferric nitrate (10 pM, 25 pM, and 50 pM) or ferric ammonium citrate (10 pM, 25 pM, and 50 pM) compared to unsupplemented media control (C). Scale bar represents 200 pm. Bar graphs showing melanin content in the presence of the indicated concentrations of ferric nitrate or ferric ammonium citrate (D).

[0036] Figure 6 The efficiency of differentiation of hPSCs either single cell passaged or clump passaged is shown. The efficiency of differentiation of cells from 4 different PSC lines was assessed for PMEL17 marker expression by flow cytometry. Data represent the mean ± sem of 8-18 experiments.

[0037] Figure 7 The efficiency of RPE differentiation using an alternative 3-stage protocol is shown. Representative brightfield images show cell morphology of 1C-derived RPE at day 14 and day 49 (A). Flow cytometry analysis of MITF and RPE65 expression in day 49 WLS-1C and H9-derived RPE (B). Flow cytometry analysis of PMEL17 expression in day 14 WLS-1C and H9-derived immature RPE using a 3-stage protocol (C). Flow cytometry analysis of PMEL17 expression in day 14 WLS-1C and H9-derived immature RPE cultured in the presence of a combination of alternative inhibitors of the wnt signaling and BMP signaling pathways (D).

[0038] Figure 8Efficiency of RPE differentiation using an alternative 5-stage protocol is shown. Box plots summarize the frequency of immature RPE (day 14) derived from 1C and H9 cells, assessed by flow cytometry analysis of PMEL17 expression. Data represent mean ± sem of 4-12 experiments.

[0039] Figure 9 Box plots showing the efficiency of differentiation comparing 5-stage, 3-stage and 4-stage RPE differentiation protocols. Data represent mean ± sem of 6-31 experiments.

[0040] Figure 10 Effect of different BMP inhibitors on RPE differentiation efficiency is shown. Percentage of positive immature RPE cells from 3A and H9 cell lines generated at day 14, assessed by flow cytometry analysis for PMEL17 marker expression (A). Immature RPE yield per well for 3A and H9 cell lines was calculated using a hemocytometer at day 14 (B).

[0041] Figure 11 Generation and characterization of retinal organoids is shown. Brightfield images show the generation of organoids over time (A). Immunohistochemical staining was used to characterize the expression of various protein markers (SNCG, PAX6, VSX2, Proxl, AP2a, OTX2 and CRX) and a nuclear marker (DAPI) (B). Each is indicated by an arrow. DETAILED DESCRIPTION

[0042] The present disclosure relates to media compositions and kits, and methods for stepwise differentiation of retinal pigment epithelial cells (RPE) and / or progenitors thereof. More specifically, the present disclosure relates to media compositions and kits, and methods for stepwise differentiation of RPE-like cells and / or progenitors thereof starting from one or more PSCs.

[0043] As used in the present disclosure, the term "PSC population" or "PSCs" refers to one or more cells that are capable of self-renewal and also capable of differentiating into all three germ layers (i.e., ectoderm, mesoderm, and endoderm) and other germ layers. The conditions and media requirements for culturing PSCs to maintain an undifferentiated state are known. PSCs are a broad term that encompasses both capturing embryonic stem cells ("ESCs") and induced pluripotent stem cells ("iPSCs"), among others. While there can be ethical challenges to obtaining ESCs, several ESC lines have been established in the field to date. iPSCs, on the other hand, can be induced from a variety of readily available cells, including adult somatic cells, and thus are associated with fewer ethical constraints. As a result, new iPSC lines are continually emerging. PSCs are important models for studying differentiation mechanisms, modeling diseases, and providing significant medical opportunities. PSCs can be obtained, derived, or induced from any source species, but in the present disclosure, the PSCs are preferably human.

[0044] As used in the present disclosure, the term "a population of cells committed to the retinal lineage" refers to a group of cells that are differentiated from one or more precursors (e.g., PSCs), but still retain various potentials (albeit more limited than the one or more precursors). A population of cells committed to the retinal lineage indicates a population of ectodermal or mesodermal cells that have the ability to further differentiate into RPE cells: cells committed to retinal pigmented epithelial cell fate; RPE precursor cells; or cells that have differentiated via a pre-neural ectodermal state into the eye field. In the context of the present disclosure, a population of cells committed to the retinal lineage can be biased towards the ectodermal lineage or the mesodermal lineage, or can have acquisitive ectodermal potential or mesodermal potential. One or more subsequent differentiation steps are required to differentiate a population of cells committed to the retinal lineage into immature / mature retinal (epithelial) cells. Characteristics of cells committed to the retinal lineage include, but are not limited to, loss of high OCT4, TRA-1-60, and / or NANOG expression. Further, cells committed to the retinal lineage can begin to acquire expression of relevant markers, such as with respect to levels of SIX3, SIX6, LHX2, PAX6, RAX, or NR2E1. In one embodiment, cells committed to the retinal lineage effectively generate or differentiate into immature and / or mature RPE cells.

[0045] As used in the present disclosure, the term "optic zone progenitor cell population" refers to a population of cells that are differentiated from one or more cells destined for the retinal lineage, but that still retain various potentials. The optic zone progenitor cell population can be biased towards the ectoderm lineage or can have acquired ectoderm potential. Further, the optic zone progenitor cell population can comprise optic zone specialized cells, optic zone progenitor cells, pre-neural plate derived cells, or RPE precursor cells. One or more subsequent differentiation steps can be required to differentiate the optic zone progenitor cell population into immature / mature RPE. The optic zone progenitor cell population can express optic zone transcription factors, such as PAX6, RAX, SIX3, and LHX2. While there can be some overlap between the population of cells destined for the retinal lineage and the optic zone progenitor cell population, the latter can encompass consolidation or homogenization of relevant marker expression, such as with respect to levels of SIX3, SIX6, LHX2, PAX6, RAX, or NR2E1. Further, the optic zone progenitor cells can begin to express PMEL17, such as at relatively low levels (as compared to immature RPE). In one embodiment, the optic zone progenitor cells effectively generate or differentiate into immature and / or mature RPE cells.

[0046] As used in the present disclosure, the term "optic vessel progenitor cell population" refers to a population of cells that are differentiated from earlier developmental precursors, such as one or more optic zone progenitor cells. The optic vessel progenitor cells can have more limited potentials relative to the optic zone progenitor cells, and can develop or differentiate into optic vesicles and optic cups. The optic vesicles can comprise retinal stem cells (e.g., cells that are capable of generating all neuroectoderm-derived cells of the eye) and / or transient dual potential progenitor cells (e.g., cells that give rise to retinal pigment epithelium or neural retina). The optic vessel progenitor cell population is biased towards the ectoderm lineage and has acquired ectoderm potential. The optic vessel progenitor cell population can comprise optic vessel specialized cells, optic vessel progenitor cells, cells that interact with surface ectoderm or RPE lineage committed cells, or RPE precursor cells. One or more subsequent differentiation steps can be required to differentiate the optic vessel progenitor cell population into immature / mature RPE. While there can be some overlap between the optic zone progenitor cell population and the optic vessel progenitor cell population, the latter can exhibit consolidation or homogenization of relevant marker expression (relative to the optic zone progenitor cell population), such as with respect to levels of MITF or VSX2. Further, the optic vessel progenitor cells can express higher levels of PMEL17 and / or PAX6 as compared to the optic zone progenitor cells. In one embodiment, the optic vessel progenitor cells effectively generate or differentiate into immature and / or mature RPE cells.

[0047] As used in the present disclosure, the term “immature retinal pigment epithelial cell population” or “immature RPE” refers to a population of cells differentiated from a precursor at an earlier stage of development, such as one or more ocular vascular progenitor cells. Immature RPE has a more limited potential relative to ocular vascular progenitor cells. An immature RPE population can be biased toward or can be limited to the RPE lineage. An immature retinal pigment epithelial cell population can comprise cells destined to become mature RPE or intermediate RPE progenitor stage cells. An immature RPE population can be derived or differentiated from RPE precursor cells. One or more subsequent differentiation / maturation steps can be required to differentiate immature RPE to mature RPE. While an immature RPE population can have some overlap with an ocular vascular progenitor cell population, the former can encompass consolidation or homogenization of relevant marker expression, such as with respect to PMEL17, MITF, or PAX6 levels. Immature RPE can be characterized by loss of RAX and / or CHX10 expression, and appearance of one or more of RPE65, ZOl, TYR, and TYRP1 expression. Immature RPE, and in particular PSC-derived immature RPE, can exhibit at least some characteristics of mature RPE, such as polygonal morphology and pigmentation. In one embodiment, immature RPE effectively generates or differentiates to mature RPE.

[0048] As used in the present disclosure, the term “mature retinal pigment epithelial cell population” or “mature RPE” refers to a population of cells differentiated from a precursor at an earlier stage of development, such as one or more immature RPE. An immature RPE population can be derived or differentiated from RPE precursor cells. When derived from one or more PSCs, RPE can be fully mature or can not be fully mature, but still exhibit many characteristics of primary mature RPE, and thus the term mature RPE can similarly refer to RPE that has matured or is maturing, as used herein. Mature RPE has a more limited potential relative to immature RPE, and can be in a terminally differentiated state. A mature RPE population is biased toward the RPE lineage, and can be limited to the RPE lineage. While a mature RPE population can have some overlap with an immature RPE population, the former can encompass consolidation or homogenization of relevant marker expression, such as with respect to RPE65, TYR, TYRP1, BEST1, CRALBP, EZRIN, and ZO-1 levels. Further, mature RPE, and in particular PSC-derived mature RPE, can exhibit at least some or all characteristics of mature RPE, such as polygonal morphology, pigmentation, ability to phagocytose photoreceptor outer segments, polarity, and post-mitotic.

[0049] As used in the present disclosure, the term “RPE precursor cell” refers to any of a population of cells destined to be of the retinal lineage, a population of ocular field progenitor cells, and / or a population of ocular vascular progenitor cells.

[0050] As used in the present disclosure, the term "neural retina" refers to a heterogeneous group of cells or structures demarcated by the RPE, and which can include cell types including retinal progenitor cells, retinal ganglion cells, photoreceptor progenitor cells, rod and cone photoreceptor cells, bipolar cells, amacrine cells, retinal ganglion cells, Muller glial cells, and horizontal cells Methods In one aspect of the present disclosure, methods of differentiating a population of cells destined for the retinal lineage are provided. In one aspect of the present disclosure, methods of differentiating a population of cells destined for the retinal lineage, as well as other cell populations, into immature and / or mature RPE are provided. In one embodiment, differentiation of immature and / or mature RPE proceeds through a plurality of intermediate cell populations, as further described below.

[0051] Methods of the present disclosure can involve providing a population of PSCs and exposing such a population to a first culture environment. As various PSC lines have been published and / or are commercially available, and techniques for generating PSC lines are now routine, the population of PSCs is not particularly limited. Preferably, the population of PSCs is undifferentiated or substantially undifferentiated, i.e., the population of PSCs has the capacity to differentiate into all germ layers. In one embodiment, the population of PSCs is an iPSC or an ESC.

[0052] The population of PSCs can be wild-type, or can be mutated or edited at one or more genomic loci. In some embodiments, it can be desirable to model a retinal disease or other neurological disease, and thus PSCs carrying one or more mutations of interest (either present in the reprogrammed cells or edited accordingly) would be an appropriate starting point.

[0053] The population of PSCs can be derived from any animal or mammalian species. In preferred embodiments, the population of PSCs is derived from a human, primate, or rodent source. To avoid or limit variability between downstream differentiated cells, such as immature or mature RPE, it can be desirable for the population of PSCs to be derived clonally. The population of PSCs can be derived from a human patient or can be a patient-derived iPSC line.

[0054] As indicated above, methods of the present disclosure include exposing a population of PSCs to a first culture environment. The first culture environment is not particularly limited, so long as it supports the population of PSCs, or supports differentiation of the population of PSCs into a downstream lineage (towards the retinal lineage). In one embodiment, the first culture environment comprises one or both of an extracellular matrix protein and a BMP signaling inhibitor. In one embodiment, the first culture environment comprises one or both of an extracellular matrix protein and a first culture medium, wherein the BMP signaling inhibitor is comprised in the first culture medium.

[0055] Extracellular matrix proteins for culturing / supporting PSCs are known and commercially available. The extracellular matrix protein used in the first culture environment of the present disclosure is not limited, provided that it supports the PSC population and also does not inhibit the differentiation of the PSCs to a downstream population of interest, such as a population of cells destined for the retinal lineage.

[0056] The extracellular matrix protein can be coated on the culture surface (of the first culture environment). The culture surface can be any surface for culturing PSCs and differentiating PSCs to a downstream ectodermal (e.g., neural and / or retinal) lineage. As non-limiting examples, the surface can be the wall (e.g., bottom wall) of a culture flask, cell culture vessel, culture dish, roller bottle, cell culture dish, multi-well plate, or microcarrier. In one embodiment, the cell culture dish can be untreated and have a hydrophobic surface, or can be treated to have a hydrophilic and negatively charged surface. Further, the surface can be a membrane, filter, or any other type of porous surface that supports a population of cells seeded thereon, such as a cell culture insert (e.g., Transwell™ insert, etc.).

[0057] In one embodiment, one or more extracellular matrix (ECM) proteins are coated on the culture surface prior to seeding the PSC population. In one embodiment, the one or more ECM proteins can be included in the cell culture medium applied to the culture surface. In such an embodiment, the one or more ECM proteins can become coated on the surface as the PSC population settles.

[0058] In one embodiment, the one or more ECM proteins are added directly to the cell culture medium. In one embodiment, the desired amount or concentration of the one or more ECM proteins can be added to the cell culture medium containing the cell population or cell suspension. In one embodiment, the desired amount or concentration of the one or more ECM proteins can be added to the cell culture medium that is subsequently contacted with the cell population or cell suspension.

[0059] As non-limiting examples, the one or more extracellular matrix proteins can be or be selected from the group consisting of collagen, laminin, decorin, vitronectin, fibronectin, Synthemax™, Synthemax™ IIa, Matrigel ® or a mixture comprising more than one of the foregoing. In one embodiment, the extracellular matrix protein is one of vitronectin, laminin, fibronectin, or collagen. In one embodiment, the extracellular matrix protein is any combination or mixture of vitronectin, laminin, fibronectin, or collagen.

[0060] In embodiments comprising collagen, the collagen can be one or more of collagen type I, collagen type II, collagen type III, or collagen type IV. In embodiments comprising laminin, the laminin can be one or more of laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-311, laminin-321, laminin-411, laminin-421, laminin-511, fragments of laminin-511 (e.g., laminin 511-E8), laminin-521, or laminin-213. In embodiments comprising elastin, the elastin can be one or more of elastin or tropoelastin. In embodiments comprising nidogen (also known as entactin), the nidogen can be one or more of nidogen-1 or nidogen-2. In one embodiment, the extracellular matrix protein can be vitronectin or isoforms thereof. In one embodiment, the extracellular matrix protein can be Matrigel ® .

[0061] In one embodiment, the ECM protein can be recombinant and / or of natural origin. In one embodiment, the ECM protein can be genetically engineered (e.g., a fusion protein). In one embodiment, the ECM protein can be an intact protein or a fragment thereof, such as a peptide fragment.

[0062] The methods of the present disclosure can involve ECM proteins coated on a culture surface or added directly to the cell culture medium. The concentration of ECM proteins is not particularly limited. In one embodiment, the concentration of extracellular matrix protein can range between about 0.1 pg / mL to 1 mg / mL. In one embodiment, the concentration of one or more (or each) ECM protein ranges between about 1 ng / mL to 1 pg / mL, about 100 ng / mL to 100 pg / mL, about 500 ng / mL to 50 pg / mL, or about 1 pg / mL to 30 pg / mL. In embodiments where the ECM protein is a combination of two or more pre-mixed ECM proteins (such as Matrigel™), it can be diluted by about 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or more.

[0063] As shown herein, the first culture environment can comprise a BMP signaling inhibitor. As non-limiting examples, the BMP signaling inhibitor can be a small molecule, a peptide, or a protein. Examples of small molecule inhibitors of BMP signaling include, but are not limited to: 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline dihydrochloride (LDN-193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine (Dorsomorphin), 4-[6-(4-propan-2-yloxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH2), 3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol (K02288), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (ML-347). Examples of protein inhibitors of BMP signaling include, but are not limited to: Noggin, Chordin, Gremlin, crossveinless-2 (CV2), USAG-1 (uterine sensitization-associated gene-1), follistatin, and sclerostin (also known as SOST).

[0064] The concentration of the BMP signaling inhibitor in the first culture environment can affect the efficiency of the differentiation of the population of PSCs through one or more intermediates to immature or mature RPE. In one embodiment, the concentration of the BMP signaling inhibitor in the first culture environment is critical for most effectively affecting the differentiation of the population of PSCs through one or more intermediates to immature or mature RPE. In one embodiment, the concentration of the BMP signaling inhibitor is in a range of between about 0.01 nM to 100 µM, about 0.05 nM to 75 µM, about 0.1 nM to 50 µM, about 0.15 nM to 25 µM, about 0.2 nM to 10 µM, about 0.25 nM to 5 µM. In one embodiment, the concentration of the BMP signaling inhibitor is less than 25 µM, less than 10 µM, less than 5 µM, less than 2.5 µM, less than 2 µM, less than 1 µM, less than 0.75 µM, less than 0.5 µM, less than 0.25 µM, less than 0.1 µM, less than 0.075 µM, less than 0.05 µM, or less than 0.025 µM. In one embodiment, the concentration of the BMP signaling inhibitor is in a range of between about 20 nM to 750 nM. In one embodiment, the concentration of the BMP signaling inhibitor is in a range of between about 5 ng / ml to 150 ng / ml.

[0065] In certain embodiments, the first culture environment (e.g., the first culture medium) does not contain one or both of: i) a wnt signaling antagonist; and ii) an activin signaling agonist. In one embodiment, the first culture environment can not contain a wnt signaling antagonist, but does contain an activin signaling agonist. In one embodiment, the first culture environment can not contain an activin signaling agonist, but does contain a wnt signaling antagonist. In one embodiment, the first culture environment does not contain a wnt signaling antagonist and an activin signaling agonist. In one embodiment, the first culture environment can contain both a wnt signaling antagonist and an activin signaling agonist.

[0066] By way of non-limiting example, the wnt signaling antagonist can be a small molecule, a cytokine, a peptide, or a protein. Examples of small molecule wnt signaling antagonists include, but are not limited to, N-(6-chloro-l,3-benzothiazol-2-yl)-3-(3,4-dimethoxyphenyl)acrylamide (KYO2111), 2-[4-(trifluoromethyl)phenyl]-l,5,7,8-tetrahydrothiopyrano[4,3-d]pyrimidin-4-one (XAV939), 4-[(3aR,4S,7R,7aS)-l,3,3a,4,7,7a-hexahydro-l,3-dioxo-4,7-methano-2H- isoindol-2-yl]-N-8-quinolinylbenzamide (IWR-1-endo), N-(6-methyl-2- benzothiazolyl)-2-[[3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2- d]pyrimidin-2-yl]thio]-acetamide (IWP-4), 2-[[3-(4-fluorophenyl)-3,4,6,7- tetrahydro-4-oxothieno[3,2-d]pyrimidin-2-yl]thio]-N-(6-methyl-2- benzothiazolyl)-acetamide (IWP-3), and N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP-2). Examples of cytokine or protein-based wnt signaling antagonists include, but are not limited to, Insulin-like growth factor binding protein 4 (IGFBP-4), Dickkopf-related protein 1 (DKK-1), DKK-2, DKK-3, DKK-4, Soggy-1 / DkkL1, secreted Frizzled-related protein (sFRP), APCDD1, APCDD1L, Draxin, LMBR1L, Notum, SOST / sclerostin, USAG1, and WIF-1.

[0067] As non-limiting examples, activin signaling agonists can be small molecules, cytokines, peptides, or proteins. Examples of activin signaling agonists include, but are not limited to, activin A, l-(2-cyclopentylhydrazine)-heptanedioic acid (IDE2), and l-[2-[(2-carboxyphenyl)methylene]hydrazine]-heptanedioic acid (IDE1).

[0068] The PSC population can be exposed to (e.g., cultured in) the first culture environment for between about 0 and 120 hours. Like the concentration of the BMP signaling inhibitor, the duration of exposure to the first culture environment (and BMP signaling inhibitor) can be important or critical to obtaining a population of cells destined for the retinal lineage (or downstream populations, such as ocular field progenitor cells, ocular vascular progenitor cells, and / or immature RPE). In one embodiment, the PSC population is cultured in the first culture environment for about 96 hours or less. In one embodiment, the PSC population is cultured in the first environment for about 72 hours or less. In one embodiment, the PSC population is cultured in the first environment for about 60 hours or less. In one embodiment, the PSC population is cultured in the first environment for about 0 to 60 hours. In one embodiment, the PSC population is cultured in the first environment for about 48 hours, or about 48 hours ± 5 hours.

[0069] The first culture medium forming part of the first culture environment can comprise a basal medium. Basal media generally comprise one or more of the following: amino acids, vitamins, organic and / or inorganic salts, buffers, antioxidants, energy (e.g., carbon) sources, etc. for supporting cell growth. In some embodiments, the basal medium does not comprise one or more of the foregoing components and, if necessary, can be supplemented. Numerous commercially available basal media are known, including Dulbecco’s Modified Eagle Medium (DMEM), F12, Roswell Park Memorial Institute Medium (RPMI) 1640, Iscove’s Modified Dulbecco’s Medium (IMDM), Advanced DMEM, Advanced DMEM / F-12, Immunocult™ brand media, STEMSpan™ brand media, X-VIVO brand media. In one embodiment, the basal medium is an Immunocult™ brand medium or a STEMSpan™ brand medium.

[0070] Following culturing of the PSC population in the first culture environment under the conditions and for the duration as described herein, a population of cells destined for the retinal lineage can be obtained (e.g., cells that are no longer PSCs and are along a path toward ectoderm or are already ectoderm).

[0071] The methods of the present disclosure can also include exposing the population of cells arising from the first culture environment (e.g., the Stage 1 population of cells, or the population of cells destined for the retinal lineage) to a second culture environment. Exposing such cells to a second culture environment can result in their further differentiation into immature and / or mature RPE or further differentiation along the path toward immature and / or mature RPE. In one embodiment, the downstream population of cells derived from the population arising from the first culture environment comprises a population of ocular field progenitor cells.

[0072] The second culture environment can comprise extracellular matrix proteins as described above, whether the same as or different from the first culture environment. In one embodiment, the second culture environment comprises a second cell culture medium. In one embodiment, the second culture environment comprises both an extracellular matrix (as described above) and a second culture medium.

[0073] The second cell culture medium can also comprise a basal medium as described above. In one embodiment, the basal medium is an Immunocult™ brand or STEMSpan™ brand medium. In one embodiment, the basal medium is an X-VIVO brand medium. In one embodiment, the basal medium is an equivalent or substitute for Immunocult™ or STEMSpan™ or X-VIVO brand medium.

[0074] The second cell culture medium, and its basal medium, will be appropriately supplemented, such as to differentiate the population of cells (destined for the retinal lineage) into immature and / or mature RPE or toward immature and / or mature RPE. In one embodiment, the second cell culture medium comprises an insulin-like growth factor (e.g., IGF-1 or IGF-2). In one embodiment, the second cell culture medium comprises an FGF signaling agonist, such as a member of the FGF family. In one embodiment, the FGF signaling agonist is basic fibroblast growth factor (FGFb). In one embodiment, the second cell culture medium comprises a basal medium, an IGF, and an FGF signaling agonist.

[0075] In one embodiment, the second culture environment and / or the second culture medium comprises a BMP signaling inhibitor of the type and concentration described above. In one embodiment, the second culture environment and / or the second culture medium is free of a BMP signaling inhibitor. In one embodiment, the second culture environment and / or the second culture medium is free of an exogenously added BMP signaling inhibitor.

[0076] The population of cells exposed to the second culture environment (e.g., destined to be retinal lineage) can be cultured in the second culture environment for between about 0 and 120 hours. As with the concentration of factors in the second culture medium, the duration of exposure to the second culture environment (and the presence of a BMP signaling inhibitor) can be important or critical to obtaining a population of cells destined to be retinal lineage (or downstream populations, such as ocular field progenitor cells, optic blood vessel progenitor cells, and / or immature RPE). As with the absence of a BMP signaling inhibitor, the duration of exposure to the second culture environment (and the absence of a BMP signaling inhibitor) can be important or critical to obtaining a population of cells destined to be retinal lineage (or downstream populations, such as ocular field progenitor cells, optic blood vessel progenitor cells, and / or immature RPE).

[0077] In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 96 hours or less. In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 72 hours or less. In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 60 hours or less. In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 0 to 60 hours. In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 12 to 60 hours. In one embodiment, the population of cells (destined to be retinal lineage) is cultured in the second culture environment for about 48 hours, or about 48 hours ± 5 hours.

[0078] Following culture of the resulting population of cells (destined to be retinal lineage) in a second culture environment under conditions and for durations as described herein, a progenitor cell population (e.g., ocular field progenitor cells, or a Stage 2 cell population, or a second differentiated cell population) can be obtained. Such a population can comprise all progenitor cells of neural-derived ocular structures. Exemplary markers of such a population include, but are not limited to, PAX6, RAX, SIX3, SIX6 (Optx2), LHX2, NR2E1, ET, and tll.

[0079] The methods of the present disclosure can further comprise exposing the population of cells resulting from the second culture environment (e.g., a Stage 2 cell population, or an ocular field progenitor cell population) to a third culture environment. Exposure of such cells to a third culture environment can result in their further differentiation into immature and / or mature RPE or further differentiation along the path toward immature and / or mature RPE. In one embodiment, a downstream population of cells derived from the population resulting from the third culture environment comprises a population of optic blood vessel progenitor cells (e.g., a Stage 3 cell population, or a third cell population).

[0080] The third culture environment can comprise extracellular matrix proteins as described above, whether the same as or different from the first culture environment and / or the second culture environment. In an embodiment, the third culture environment comprises a third cell culture medium. In an embodiment, the third culture environment comprises both an extracellular matrix (as described above) and a third culture medium.

[0081] The third cell culture medium can also comprise a basal medium as described above. In an embodiment, the basal medium is an Immunocult™ brand or STEMSpan™ brand medium. In an embodiment, the basal medium is an X-VIVO brand medium. In an embodiment, the basal medium is an equivalent or substitute of Immunocult™ or STEMSpan™ or X-VIVO brand medium.

[0082] The third cell culture medium, and its basal medium, will be appropriately supplemented to differentiate the Stage 2 cell population (e.g., optic zone progenitor cell population) into immature and / or mature RPE or to differentiate toward immature and / or mature RPE. In an embodiment, the third cell culture medium comprises an insulin-like growth factor (e.g., IGF-1 or IGF-2). In an embodiment, the third cell culture medium comprises an activin signaling agonist as described above. In an embodiment, the third cell culture medium comprises a basal medium, IGF, and FGF signaling agonists.

[0083] The cell population (e.g., optic zone progenitor cells) exposed to the third culture environment can be cultured in this third culture environment for between about 1 and 10 days. In an embodiment, the (optic zone) progenitor cells are cultured in the third culture environment for between about 1 and 7 days. In an embodiment, the (optic zone) progenitor cells are cultured in the third culture environment for between about 1 and 5 days. In an embodiment, the (optic zone) progenitor cells are cultured in the third culture environment for between about 2 and 5 days. In an embodiment, the (optic zone) progenitor cells are cultured in the third culture environment for between about 2 and 4 days. In an embodiment, the (optic zone) progenitor cells are cultured in the third culture environment for about 3 days or about 4 days.

[0084] Following culture of the (optic zone) progenitor cell population in the third culture environment under conditions and for durations as described herein, a progenitor cell population (e.g., a retinal vascular progenitor cell, a Stage 3 cell population, or a third differentiated cell population) can be obtained. Such a population can comprise cells that are dual potential cells, such as dual potential cells for retinal pigment epithelium and / or neural retina. Such a cell population can further develop or differentiate into optic vesicles and / or optic cups. Such a cell population can be characterized by expression of lineage-specific markers, such as MITF and OTX2.

[0085] The methods of the present disclosure can also include exposing the population of cells that results from the third culture environment (e.g., a Stage 3 population of cells, or a population of ocular vascular progenitor cells) to a fourth culture environment. Exposing such cells to a fourth culture environment can result in their further differentiation into immature and / or mature RPE or further differentiation along the path toward immature and / or mature RPE. In one embodiment, a downstream population of cells derived from the population that results from the fourth culture environment comprises a population of immature RPE.

[0086] In one embodiment, the fourth culture environment comprises extracellular matrix proteins as described above, whether the same as or different from the first culture environment and / or the second culture environment. In one embodiment, the fourth culture environment comprises a fourth cell culture medium. In one embodiment, the fourth culture environment comprises both an extracellular matrix (as described above) and a fourth culture medium.

[0087] The fourth cell culture medium can also comprise a basal medium as described above. In one embodiment, the basal medium is an Immunocult™ brand or STEMSpan™ brand medium. In one embodiment, the basal medium is an X-VIVO brand medium. In one embodiment, the basal medium is an equivalent or substitute for Immunocult™ or STEMSpan™ or X-VIVO brand medium.

[0088] The fourth cell culture medium, and its basal medium, will be appropriately supplemented to differentiate the resulting population of (ocular vascular) progenitor cells into immature and / or mature RPE or to differentiate toward immature and / or mature RPE. In one embodiment, the fourth cell culture medium comprises one or more of an activin signaling agonist, a wnt signaling agonist, and an FGF signaling antagonist as described above. In one embodiment, the fourth cell culture medium can also comprise a RHO / ROCK pathway inhibitor. Such a RHO / ROCK pathway inhibitor can be any compound or molecule that inhibits the rho kinase or ROCK pathway.

[0089] As non-limiting examples, the wnt signaling agonist can be a small molecule, a cytokine, a peptide, or a protein. Examples of small molecule agonists of wnt signaling include, but are not limited to, 3-[3-[(acetyloxy)imino]-1,3-dihydro-2H-indol-2-ylidene]-6-bromo-1,3-dihydro-2H-indol-2-one (BIO-acetoxime), 9-bromo-7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one (kenpaullone), 3-[[6-(3- aminophenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]oxy]phenol (TWS119), N6-[2-[[4-(2,4- dichlorophenyl)-5-(1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6- pyridinediamine (CHIR98014), 3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H- pyrrole-2,5-dione (SB216763), 2-[2-(4-acetylphenyl)diazenyl]-2-(3,4-dihydro-3,3- dimethyl-1(2H)-isoquinolinylidene)-acetamide (IQ-1), 6-[[2-[[4-(2,4-dichlorophenyl)-5- (5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile (CHIR99021), and 6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indol-2-ylidene]-1,3- dihydro-(3Z)-2H-indol-2-one (BIO).

[0090] As non-limiting examples, the FGF signaling antagonist can be a small molecule, a cytokine, a peptide, or a protein. Examples of FGF signaling antagonists include, but are not limited to, 2-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-4-methyl-1H-pyrrole-3- propionic acid (SU5402) and N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5- dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD173074).

[0091] The population of ocular vascular progenitor cells (e.g., population of stage 3 cells) exposed to the fourth culture environment can be cultured in the fourth culture environment for between about 3 and 12 days. In one embodiment, the generated progenitor cells are cultured in the fourth culture environment for between about 3 and 10 days. In one embodiment, the generated progenitor cells are cultured in the fourth culture environment for between about 5 and 10 days. In one embodiment, the generated progenitor cells are cultured in the fourth culture environment for between about 6 and 9 days. In one embodiment, the generated progenitor cells are cultured in the fourth culture environment for between about 6 and 8 days. In one embodiment, the generated progenitor cells are cultured in the fourth culture environment for about 6 days, 7 days, or 8 days.

[0092] The concentration of the cytokine and small molecule present in the third and fourth culture media can be in the range of 2-150 ng / ml or 1-15 μΜ. In one embodiment, the concentration of the cytokine in the third and fourth culture media can be 50 ng / ml, 75 ng / ml, 100 ng / ml, or 125 ng / ml. In one embodiment, the concentration of the cytokine in the third culture media can be 5 ng / ml, 7.5 ng / ml, 10 ng / ml, or 12.5 ng / mL. In one embodiment, the concentration of the small molecule in the fourth culture media can be 5 μΜ, 7.5 μΜ, 10 μΜ, or 12.5 μΜ. In one embodiment, the concentration of the small molecule in the fourth culture media can be 1.5, 2.25, 3, or 3.75 μΜ. In one embodiment, the cytokine present in the third culture media can be Activin A or IGF-1. In one embodiment, the cytokine present in the fourth culture media can be Activin A.

[0093] Following culture of the population of (ocular vascular) progenitor cells in the fourth culture environment under the conditions and for the duration as described herein, a population of immature RPE (e.g., population of stage 4 cells or fourth differentiated cell population) can be obtained. The population of immature RPE can be destined to become RPE cells. In one embodiment, the immature RPE includes cells that have begun to differentiate into mature RPE cells or differentiate toward mature RPE cells but have not acquired all the characteristics of mature RPE cells. In one embodiment, the immature RPE can exhibit certain characteristics such as cell surface marker expression and morphology similar to mature RPE cells.

[0094] In one aspect, or in the foregoing embodiments, the methods of the present disclosure can include dissociating the population of immature RPE. In one embodiment, the methods of the present disclosure include dissociating the population of immature RPE and replating the dissociated cells.

[0095] Prior to replating the dissociated cells, the immature RPE dissociated from the substrate can be enriched (such as by manual dissection, particle-based enrichment, or ligand-mediated enrichment). In one embodiment, the simple act of dissociating the immature RPE population and replating the dissociated cells enriches the immature RPE. Thus, in one embodiment, the dissociated immature RPE population is not enriched except by virtue of the large population of dissociated immature RPE (or portion thereof, depending on the surface area of the culture surface into which they are replated) being replated. Thus, the dissociated RPE population need not be enriched by manual dissection, particle-based enrichment, or ligand-mediated enrichment. Without being bound by theory, the immature RPE can become enriched upon dissociation and replating due to the reduced ability of contaminating cells to detach from the substrate or reattach to the new substrate, or can be outcompeted by the immature RPE in terms of the surface area of the culture surface into which they are replated.

[0096] In one embodiment, the absence of serum in the (e.g., enrichment / replating) medium reduces or limits the establishment of contaminating cell types (such as neural retinal cells and / or corneal cells) compared to the serum-containing conditions into which the dissociated RPE are replated.

[0097] In one embodiment, the replating medium (e.g., enrichment and / or maturation medium) comprises a RHO / ROCK pathway inhibitor, and such medium facilitates (preferentially) the survival / attachment of PMEL17+ immature RPE compared to contaminating cell types. In one embodiment, the RHO / ROCK pathway inhibitor can be added as a supplement to the enrichment / replating / maturation medium. In one embodiment, the dissociated immature RPE cells are cultured in the enrichment / replating / maturation medium comprising a RHO / ROCK pathway inhibitor for 2 to 10 days.

[0098] The immature RPE population differentiated (and optionally enriched) as described herein can express characteristic markers. In one embodiment, about 50% or more of the immature RPE population expresses premelanosome protein (PMEL17). In one embodiment, 60% or more of the immature RPE population expresses PMEL17. In one embodiment, 70% or more of the immature RPE population expresses PMEL17. In one embodiment, 80% or more of the immature RPE population expresses PMEL17. In one embodiment, 90% or more of the immature RPE population expresses PMEL17. In one embodiment, 90-95% or more of the immature RPE population expresses PMEL17.

[0099] In another aspect, or in the foregoing embodiments, the methods of the present disclosure can comprise maturing the population of immature RPE by contacting with a maturation medium. The maturation medium comprises at least a soluble iron source. Examples of the soluble iron source can include, but are not limited to, ferric nitrate and ferric ammonium citrate. In one embodiment.

[0100] In one embodiment, the maturation medium comprises a basal medium as described above. In one embodiment, the basal medium is an Immunocult™ brand or STEMSpan™ brand medium. In one embodiment, the basal medium is an X-VIVO brand medium. In one embodiment, the basal medium is an equivalent or substitute of Immunocult™ or STEMSpan™ or X-VIVO brand medium. Other examples of basal media are known and can substitute the foregoing basal media.

[0101] The maturation medium can further comprise one or more of a steroid hormone and a polyamine. In one embodiment, the maturation medium can further comprise progesterone and putrescine. In one embodiment, the maturation medium is free of xeno components or free of animal components. In one embodiment, the maturation medium does not comprise a RHO / ROCK pathway inhibitor.

[0102] The population of immature RPE exposed to the soluble iron source, such as by contacting with the maturation medium of the present disclosure, can be cultured / maturation for about 1 to 10 weeks, or about 1 to 7 weeks, or about 1 to 5 weeks.

[0103] Upon culturing / maturation of the population of immature RPE in the presence of the soluble iron source, the matured / already matured / maturing RPE can begin to express or become more uniform in the expression of markers of mature RPE. In one embodiment, the marker of mature RPE is RPE65. In one embodiment, 50% or more of the matured or already matured or maturing RPE express RPE65. In one embodiment, 60% or more of the matured or already matured or maturing RPE express RPE65. In one embodiment, 70% or more of the matured or already matured or maturing RPE express RPE65. In one embodiment, 80% or more of the matured or already matured or maturing RPE express RPE65. In one embodiment, 90% or more of the matured or already matured or maturing RPE express RPE65. In one embodiment, 90-95% or more of the matured or already matured or maturing RPE express RPE65.

[0104] The methods of the present disclosure can be performed under animal component-free or serum-free conditions. In one embodiment, the methods of the present disclosure are performed under animal component-containing or serum-containing conditions. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are serum-free. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are serum-containing. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are animal component-containing. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are animal component-free.

[0105] The methods of the present disclosure can be performed under feeder cell-free conditions or in the absence of feeder cells. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are feeder cell-free or do not contain any feeder cells. In one embodiment, the population of PSCs can be cultured under feeder cell-free conditions or in the absence of any feeder cells. In one embodiment, the population of PSCs can be differentiated under feeder cell-free conditions or in the absence of any feeder cells.

[0106] In one embodiment, one or more of the second medium, the third medium, and the fourth medium lack an exogenously added inhibitor of BMP signaling.

[0107] In one embodiment, one or more of the first medium, the second medium, the third medium, and the fourth medium lack nicotinamide. In one embodiment, the second medium, the third medium, and the fourth medium lack nicotinamide. In one embodiment, the third medium and / or the fourth medium lack nicotinamide.

[0108] In some aspects, the present disclosure relates to the use of RPE cells obtained by the methods of the present disclosure for the treatment of retinal-related diseases. Such retinal-related diseases can include retinal diseases or disorders or conditions associated with retinal dysfunction, retinal injury, or loss of RPE function. A non-limiting list of conditions that can be treated with the RPE cells of the present disclosure includes age-related macular degeneration (AMD), glaucoma, cataracts, retinitis pigmentosa (RP), Stargardt disease (SD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), Leber congenital amaurosis, inherited or acquired macular degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, angiod streaks, high myopia (degenerative myopia), idiopathic neovascular maculopathy, pattern dystrophy, and other RPE dystrophies and retinal injury due to any kind of trauma.

[0109] Retinal pigment epithelial cells obtained by the methods of the present disclosure can be used for cell transplantation, to generate a transplanted tissue, or to screen for therapeutic compounds or agents that affect the function or metabolism of RPE cells. In one embodiment, RPE cells obtained by the methods of the present disclosure can be used to implant the generated RPE cells into the retina to treat retinal disorders, diseases, and conditions. In one embodiment, the RPE cells can be implanted into the eye of a subject, e.g., a patient having a retinal disorder, disease, and condition. In one embodiment, the RPE cells can be implanted into an animal model of a retinal disorder, disease, and condition.

[0110] In one embodiment, RPE cells derived by the methods of the present disclosure can be cryopreserved as single cells, aggregates, or tissues to generate a cell bank for cell transplantation that can be needed for multiple clinical sites. RPE cells can be seeded onto scaffolds and cryopreserved to prepare a tissue bank of ready-to-implant tissues. The scaffolds can comprise any material known in the art and can be maintained during implantation, such as amniotic membrane, paralyene, or degradable materials such as fibrin hydrogel scaffolds, PLGA, or porcine-derived collagen scaffolds used in corneal surgery. The media described in the methods of the present disclosure can be used as recovery media for RPE cells or RPE-seeded scaffolds to prepare for implantation or recover from implantation.

[0111] In one embodiment, RPE cells obtained by the methods of the present disclosure can be used for toxicity studies, such as phototoxicity, toxicity testing, and retinal excitotoxicity, and for evaluating the efficacy of therapeutic drugs.

[0112] In one embodiment, RPE cells obtained by the methods of the present disclosure can be used for large-scale manufacturing, large-scale expansion, or automated production of RPE cells while ensuring high cell purity (e.g., >80%, or >90%, or >95%) and functionality for further downstream applications.

[0113] In one embodiment, RPE cells obtained by the methods of the present disclosure can be used for gene therapy-related applications that focus on treating patients with RPE-related disease mutations, or for optimizing gene therapy parameters in vitro prior to animal or clinical studies.

[0114] In one embodiment, RPE cells obtained from patient-derived iPSCs by the methods of the present disclosure can be used as a diagnostic tool to identify or predict disease-causing mutations.

[0115] In one embodiment, the RPE cells obtained by the methods of the present disclosure can serve as a therapeutic agent for ocular conditions, including but not limited to retinal diseases or disorders associated with retinal dysfunction, retinal injury, or loss of RPE function. A non-limiting list of conditions that can be treated with the RPE cells of the present application include age-related macular degeneration (AMD), glaucoma, cataracts, retinitis pigmentosa (RP), Stargardt disease (SD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), Leber congenital amaurosis, inherited or acquired macular degeneration, Best disease, retinal detachment, gyrate atrophy, choroideremia, angiod streaks, high myopia (degenerative myopia), idiopathic neovascular maculopathy, pattern dystrophy, and other RPE dystrophies and retinal injury due to any kind of trauma.

[0116] In one embodiment, the RPE cells obtained by the methods of the present disclosure can be co-cultured with other cell types for various downstream applications. The RPE cells can be co-cultured or assembled in vitro with other retinal cell types, such as but not limited to retinal progenitor cells, photoreceptor progenitor cells or mature photoreceptors (such as rod and cone photoreceptors), whole retinas or developing retinas, choroid-like tissue composed of endothelial cells, fibroblasts or pericytes. Such co-cultures or assemblies can mimic in vivo retinal tissue.

[0117] In one embodiment, the methods of the present disclosure can include the conditioned media obtained after culturing RPE cell precursors, immature RPE, or mature RPE in the first media, second media, third media, fourth media, or maturation media as described above. Such conditioned media can be further used to isolate / purify cytokines, exosomes, etc., which can be further used to treat, prevent, or diagnose RPE-related diseases.

[0118] In one aspect of the disclosure, a method for differentiating PSCs to obtain immature and / or mature RPE is provided. In one embodiment, the method for differentiating PSCs to obtain immature and / or mature RPE can comprise: a) culturing a population of PSCs in a first culture environment comprising an extracellular matrix protein and a BMP signaling inhibitor for about 0 to 60 hours; b) culturing the cells of step a) in a second culture environment comprising an extracellular matrix protein and a second cell culture medium comprising a basal medium and one or more of an IGF and FGF signaling agonist for about 0 to 60 hours; c) culturing the cells of step b) in a third culture environment comprising an extracellular matrix protein and a third cell culture medium comprising a basal medium and one or more of an IGF and activin signaling agonist for about 2 to 5 days; and d) culturing the cells of step c) in a fourth culture environment comprising an extracellular matrix protein and a fourth cell culture medium comprising a basal medium and one or more of an activin signaling agonist, a wnt signaling agonist, and a FGF signaling antagonist for about 5 to 10 days to obtain a population of immature RPE.

[0119] In one embodiment, the method for differentiating PSCs to obtain immature and / or mature RPE can further comprise dissociating the population of immature retinal pigment epithelial cells and replating the dissociated cells to enrich for immature retinal pigment epithelial cells. In one embodiment, the population of dissociated immature retinal pigment epithelial cells is not enriched by manual dissection, particle-based enrichment, or ligand-mediated enrichment.

[0120] In one embodiment, the method for differentiating PSCs to obtain immature and / or mature RPE can further comprise maturing the population of immature retinal pigment epithelial cells for about 1 to 5 weeks. In one embodiment, the population of immature retinal pigment epithelial cells is matured upon contact with a maturation medium comprising a basal medium and at least a soluble source of iron. In one embodiment, the maturation medium further comprises one or more of a steroid hormone and a polyamine.

[0121] In one embodiment, the components of the method for differentiating PSCs to obtain immature and / or mature RPE can be as described above.

[0122] In another aspect, the methods of the present disclosure can include culturing a population of PSCs under non-adherent conditions to produce three-dimensional retinal organoids. Such methods for producing three-dimensional retinal organoids can also include differentiating a population of cells destined to the retinal lineage (substantially as described above). Briefly, a population of PSCs can be exposed to a first culture environment comprising extracellular matrix proteins and a BMP signaling inhibitor for about 0 to 60 hours to yield a produced population of cells (e.g., destined to the retinal lineage). Next, the produced population of cells (e.g., destined to the retinal lineage) can be exposed to a second culture environment (substantially as described above) for about 12 to 60 hours to yield a further produced (ocular field) progenitor population. Next, the further produced (ocular field) progenitor population can be exposed to a third culture environment (substantially as described above) for about 2 to 5 days.

[0123] Cells obtained after having been exposed to the third culture environment (substantially as described above) for about 2 to 5 days can then be subsequently exposed to a neural retina / RPE induction medium for 4 to 10 days to form aggregates comprising distinct domains of neural retina and / or RPE. The aggregates comprising distinct domains of neural retina can be released by peeling / detaching from the culture surface through a single layer of engraved / scraped / etched grid-like pattern (such as with a pipette tip or the like) to release “flakes” or retinal tissue. The resulting retinal tissue can then be cultured under non-adherent conditions in a retinal organoid formation medium for about 2 to 6 days to form retinal organoids comprising neural retina, which can be further matured in a retinal organoid maturation medium (under non-adherent conditions) for 30 to 200 days.

[0124] The neural retina / RPE induction medium can comprise a basal medium (substantially as described above), and one or more of progesterone, putrescine, transferrin, and heparin. The neural retina / RPE induction medium can further comprise a member of the bone morphogenetic protein (BMP) family, preferably BMP-4.

[0125] The retinal organoid formation medium can comprise a basal medium as described above and one or more additional supplements such as putrescine, insulin, or transferrin. The retinal organoid maturation medium can comprise a basal medium as described above and one or more additional supplements added at different stages of retinal organoid maturation. Such additional supplements in the retinal organoid maturation medium can include serum, a retinoic acid pathway activator, a retinoic acid receptor activator, a retinoic acid agonist, taurine, a lipid, or N2 Supplement-A.

[0126] The first culture environment, the second culture environment, and / or the third culture environment for generating retinal organoids can comprise nicotinamide. The second culture environment and / or the third culture environment for generating retinal organoids can comprise nicotinamide In one embodiment, a population of PSCs can be seeded into a microwell device. In one embodiment, a population of PSCs can be seeded into an uncoated microwell device to obtain 3D spheroids or uniform-sized aggregates of PSCs. Such 3D spheroids can be sequentially exposed to the first culture environment, the second culture environment, and the third culture environment, as described above. In one embodiment, the 3D spheroids or aggregates of PSCs are seeded onto a plate coated with an extracellular matrix protein (e.g., Matrigel, vitronectin, or Synthemax (II)) prior to exposure to the first culture environment, the second culture environment, and the third culture environment.

[0127] The population of cells produced after exposure to the third culture environment for about 2 to 5 days can then be exposed to a neural retina / RPE induction medium (essentially as described above) for 4 to 10 days to form aggregates comprising distinct domains of neural retina and / or RPE. Once the aggregates comprising distinct domains of neural retina and / or RPE are formed, the monolayer can be engraved with a grid-like pattern by a pipette tip or the like, and the released“flakes” or retinal tissue can self-organize into structures in an appropriate medium as described above (and in the absence of extracellular matrix proteins), which eventually develop into retinal organoids in suspension.

[0128] In one embodiment, the retinal organoids generated by the methods of the present disclosure include retinal precursor cells or retinal progenitor cells that generate all cell types of the neural retina, as well as cells that differentiate into RPE. Cell types of the neural retina can include precursors of photoreceptors, retinal progenitor cells, retinal ganglion cells, retinal neuronal photoreceptor progenitor cells, rod and cone photoreceptor cells, bipolar cells, amacrine cells, retinal ganglion cells, Muller glial cells, and horizontal cells.

[0129] Kits In another aspect of the present disclosure, a medium or kit is provided, which can comprise instructions for performing a differentiation method as described herein (e.g., stepwise differentiation of progressively more committed precursors into immature RPE and / or mature RPE). The kits described herein can also include other materials such as, but not limited to, supplements, buffers, diluents, solutions, and package inserts with instructions for performing any of the methods described herein.

[0130] In another aspect of the disclosure, there is provided a medium or a kit for implementing a method as contemplated herein, such as differentiating a more early developmental precursor into immature RPE (e.g., PSC-derived immature RPE). In other words, there is provided a medium and a kit for implementing a method of progressively differentiating a progressively more committed precursor into immature RPE and / or mature RPE.

[0131] Such a kit can include a first medium, a second medium, a third medium, and a fourth medium as described above. The kit can also include an extracellular matrix protein as described above. In an embodiment, the more early developmental precursor can include a population of cells destined for the retinal lineage, a population of ocular field progenitor cells, and / or a population of ocular vascular progenitor cells. In an embodiment, the more early developmental precursor can be derived from a PSC as described above. In an embodiment, the first medium can comprise a BMP signaling inhibitor as described above. In an embodiment, the first medium is free of one or both of: i) a direct wnt signaling antagonist; and ii) an activin signaling agonist. In an embodiment, the second cell culture medium comprises a basal medium, and one or more of IGF and FGF signaling agonists, as described above. In an embodiment, the third cell culture medium comprises a basal medium, and one or more of IGF and activin signaling agonists, as described above. In an embodiment, the fourth cell culture medium comprises a basal medium, and one or more of activin signaling agonist, wnt signaling agonist, and FGF signaling antagonist, as described above. The duration of exposure to the first medium, the second medium, the third medium, and the fourth medium, and the concentration of the components of the first medium, the second medium, the third medium, and the fourth medium can be as described above.

[0132] In another aspect of the disclosure, there is provided a medium or a kit for differentiating a more early developmental precursor into mature RPE (e.g., PSC-derived RPE), which can comprise a first medium, a second medium, a third medium, a fourth medium, and a maturation medium as described above. The kit can also include an extracellular matrix protein as described above. In an embodiment, the maturation medium comprises a basal medium and at least a soluble iron source, as described above. In an embodiment, the maturation medium further comprises one or more of a steroid hormone and a polyamine, as described above.

[0133] In another aspect of the disclosure, provided are media or kits for enriching immature RPE (e.g., PSC-derived RPE), which can include the first media, second media, third media, fourth media, and enrichment / replating media as described above. The kits can also include extracellular matrix proteins as described above. In one embodiment, dissociating a population of immature RPE and replating the dissociated cells enriches for immature RPE. In one embodiment, the population of RPE after dissociation does not need to be enriched by manual dissection, particle-based enrichment, or ligand-mediated enrichment. In one embodiment, the kits for enriching immature RPE can include instructions for dissociating a population of immature RPE and replating the dissociated cells to enrich for immature RPE. In one embodiment, the enrichment / replating media can be serum-free. In one embodiment, the enrichment / replating media includes a RHO / ROCK pathway inhibitor or includes a RHO / ROCK pathway inhibitor added as a supplement, as described above.

[0134] In another aspect of the disclosure, provided are media or kits for generating three-dimensional retinal organoids (e.g., PSC-derived retinal organoids), which can include the first media, second media, and third media, neural retina / RPE induction media, retinal organoid formation media, and optionally retinal organoid maturation media, all as described above. The kits can also include extracellular matrix proteins as described above. The kits can also include microwell devices as described above.

[0135] The following non-limiting examples illustrate the disclosure.

[0136] Examples Example 1 : Maintenance and differentiation of PSCs Human pluripotent stem cells (PSCs) were maintained in mTeSR™ brand media, such as mTeSR™ Plus (STEMCELL Technologies) in Corning® Matrigel® in a 37°C, 5% CO2 incubator. ® Matrigel ®PSCs were passaged as described by the manufacturer, but generally when most colonies were large, compact, and had a dense multilayered center (e.g., approximately 70-80% confluent, and exhibited <10% differentiation), the culture was ready for passaging. PSCs were split at a 1 / 10 ratio for seeding to initiate RPE differentiation. In this work, various iPSC lines (WLS-1C, SCTi003-A, and STiPS-M001) and ESC line (H9) were used for downstream differentiation experiments. Whether maintaining hPSCs or differentiating hPSCs, cells can be seeded as a mass or as single cells.

[0137] Example 2: Selected validation protocols At different stages of differentiation, but generally at day 14 for immature RPE and day 49 for mature RPE, cells can be analyzed by flow cytometry. Briefly, 0.1 x 10 6 RPE aliquots were centrifuged at 500 x g for 3 minutes and the supernatant was removed. The pellets were resuspended in 100 pL Zombie Violet Fixable Viability dye (Biolegend) in D-PBS (1 / 1000 of the original volume) and incubated at room temperature in the dark for 10 minutes. 100 pL D-PBS was added to each well, centrifuged at 500 x g for 3 minutes, and the supernatant was removed. The pellets were resuspended in 200 pL of 4% PFA and incubated at room temperature in the dark for 15 minutes. Cells were washed in 200 pL of buffer and the pellets were resuspended in 200 pL of 0.2% saponin, incubated at room temperature in the dark for 15 minutes, centrifuged at 500 x g for 3 minutes, and the supernatant was removed. Cells were resuspended in 100 pL of anti-PMEL17 (Biolegend AF647, clone HMB-45) or anti-RPE65 (Novus Biologicals clone: 401.8B11.3D9) antibody in 0.2% saponin and incubated at room temperature in the dark for 30-60 minutes. After incubation, one or two washes with 100 pL or 200 pL of 0.2% saponin were performed. Depending on the primary antibody used, secondary antibody staining can be required. For PMEL17 staining, 200 pL of 0.2% saponin was added to each well, resuspended, and analyzed by fluorescence-activated cell sorting (FACS). In the case of RPE65 staining, 100 pL of secondary donkey anti-mouse antibody (Jackson Immuno, AF647 Fab2) in 0.2% saponin was added to each well and incubated at room temperature in the dark for 30-60 minutes. After incubation, one or two washes were performed as described above. Cells were ready for analysis by flow cytometry.

[0138] The pigmentation level of RPE cells at different differentiation stages, typically maturing on day 49, can be analyzed by melanin content assay. To prepare a melanin standard curve, synthetic melanin (Sigma-Aldrich) was reconstituted in 4.26 mM NaOH to prepare triplet concentrations of 0, 7.825, 15.65, 31.3, 62.5, 125, 250, and 500 µg / mL in 96-well tissue culture plates. A total of 4 x 10⁻⁶ μg / mL was used. 5 One dose of RPE was added to each well of a 96-well U-plate and centrifuged at 500 x g for 3 min. Once the supernatant was removed, the cells were resuspended in 100 µL of D-PBS and transferred to a 96-well tissue culture plate. The RPE was centrifuged at 300 x g for 3 min, and the absorbance was measured at 490 nm. Melanin content was calculated relative to synthetic standards.

[0139] Example 3: Retinal pigment epithelial (RPE) differentiation i) Generation of cells destined for retinal lineage and ocular field progenitors (days 0-4) Maintain PSC as described in Example 1, and wash the Day 0 culture (approximately 60-80% confluence) twice with 1 ml of sterile 0.5 mM EDTA, then add 1 ml of first differentiation medium (medium A) to each well of a 6-well plate. This medium can be alternatively referred to as first medium and may contain a BMP signaling inhibitor (BMPi). + And it does not contain Wnt signaling inhibitors (WNTi) - And it does not contain activin A (A - Then scrape the PSC and grind it several times, then it will contain approximately 1.2 x 10 5 Approximately 100 μL of cells per molecule were seeded into 1 ml of Matrigel medium A. ® Alternatively, place the contents of each well in a 12-well plate coated with quinone to initiate differentiation. Shake the plate to distribute the clumps and incubate at 5% CO2 and 37°C. Change the medium on day 1.

[0140] On day 2, remove medium A and add 1 ml of secondary differentiation medium (medium B) to each well. This can be alternatively referred to as the secondary medium and may not contain BMP signaling inhibitors (BMPi). - ), does not contain Wnt signaling inhibitors (WNTi) - And it does not contain activin A (A - The plates were incubated at 5% CO2 and 37°C. At this stage, the culture should exhibit a rosette-like clump structure, such as... Figure 1 As shown in Figure A.

[0141] ii) Generation of ocular vascular progenitors (days 4-7) On day 4, remove the culture medium and add 1 ml of third differentiation medium (medium C) to each well. This medium can be alternatively referred to as third medium and may contain activin A (A... + And it does not contain BMP signaling inhibitors (BMPi). - ) or Wnt signaling inhibitors (WNTi - Incubate the plate at 5% CO2 and 37°C. Preferably, the culture should reach approximately 100% confluence within the next 1-2 days.

[0142] On day 6, change the culture medium and incubate the plate for another day at 5% CO2 and 37°C. An example culture on day 7 is shown below. Figure 1 As shown in Figure A.

[0143] iii) Generation of immature retinal pigment epithelial (RPE) cells (days 7-14) On day 7, replace the medium with 1 ml of fourth differentiation medium (medium D) per well, and replace the medium on days 9, 11, and 13. This medium can be alternatively referred to as fourth medium and may contain activator A (A... + ) and Wnt agonists, but not BMP signaling inhibitors (BMPi) - ) and / or Wnt signaling inhibitors (WNTi - The plates were incubated at 5% CO2 and 37°C. An exemplary homogeneous monolayer of polygonal immature RPE on day 14 is shown. Figure 1 As shown in Figure A.

[0144] iv) RPE passaging and maturation (days 14, 49) On day 14, the culture medium was removed, the cells were washed twice with 0.5 ml D-PBS, and contacted with 0.5 ml of trypsin-based enzymatic solution. The monolayer was peeled off using a cell scraper, ground several times, and the single-cell suspension containing immature RPE was transferred to a tube containing progesterone, putrescine, and soluble iron ("+3F") to bring the volume to 10 ml. The filtrate, passed through a 70 μm cell filter, was centrifuged at 300 x g for 5 min, and the pellet was resuspended in 4 ml of basal medium +3F (which also contains RHO / ROCK pathway inhibitors). Cell counting was performed using standard methods, and 1 x 102 cells were counted. 5 cells / cm 2 Cells were seeded into a substrate coated with Matrigel. ® / Pelin plates (prepared according to the manufacturer's instructions). Cells can be analyzed by flow cytometry prior to inoculation, as described in Example 2. Maturation can be initiated when approximately 50% or more of the cells are positive for PMEL17. It is reasonable to expect that cultures with >50% PMEL17 expression at day 14 will be enriched to >80% in the aforementioned enrichment passages, and potentially >90%. Therefore, the RPE enrichment described herein does not involve manual dissection, magnetic activated cell sorting (MACS), or separation methods using magnetic particles.

[0145] The inoculated cells were incubated at 5% CO2 and 37°C, with 4 mL of culture medium replaced on days 16 and 18. From day 21 onwards, the cells were cultured in basal medium + 3F (e.g., maturation medium), with the medium replaced every 3–4 days until passage on day 49. Cobblestone-like morphology and visible intercellular or tight junctions were observed on days 18, 21, 28, and 42. Figure 2 As shown. On day 49, a uniform monolayer of pigmented RPE with visible tight junctions should be observed, as shown. Figure 2 As shown.

[0146] On day 49, cells were washed twice with 1 ml D-PBS, and then 1 ml of trypsin-based enzymatic solution was added to each well. The monolayer was peeled off using a cell scraper, milled to produce a single-cell suspension containing mature RPE, and transferred to tubes in basal medium +3F to bring the volume to 10 ml. The filtrate, passed through a 70 μm cell filter, was centrifuged at 300 x g for 5 min, and the pellet was resuspended in 4 ml basal medium +3F. Cell counting was performed using standard methods. 1 x 10n cells resuspended in 4 ml basal medium +3F were counted. 5 cells / cm 2 Cells were seeded into a substrate coated with Matrigel. ® / The cells were placed on a plate of telogen effluvium (prepared according to the manufacturer's instructions). Prior to inoculation, the cell suspension could be analyzed by flow cytometry as described in Example 2. At this stage, most (e.g., 90-95%) of the cells were likely positive for PMEL17 and / or RPE65. Figure 3 ).

[0147] Example 4: Characterization of mature RPE Basically, as described in Example 3, four different hPSC lines were differentiated, and the maturity of cells on day 49 was assessed by marker expression, growth factor secretion, barrier function, ability to phagocytose photoreceptor segments, and melanin production.

[0148] On day 49 (essentially as described in Example 2), the expression of PMEL17 and RPE65 in mature RPE cells derived from four different PSC lineages was analyzed by flow cytometry. Figure 3 A). Based on PMEL17 marker expression, differentiation efficiencies of 91.4% and 96.6% were observed on day 14 and day 49 (P0), respectively, and 93% of day 49 cells expressed the maturation marker RPE65.

[0149] The apical and basal secretion of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEDF) in immature RPE cells at different differentiation stages, typically day 14, can be assessed using commercially available VEGF (Thermo Fisher) and PEDF (Abcam) ELISA kits. Briefly, cells are washed twice with 0.5 ml D-PBS, followed by the addition of 0.5 ml trypsin-based enzymatic solution to each well. The monolayer is peeled off using a cell scraper, ground, and a single-cell suspension containing immature RPE cells is brought to 10 ml in RPE maturation medium containing basal medium plus progesterone, putrescine, and soluble iron ("+3F"). This medium is then passed through a 70 μm cell filter to remove RPE tissue or contaminating cells or tissue. The filtrate is resuspended in 4 ml of RPE maturation medium also containing RHO / ROCK pathway inhibitors. Approximately 4 x 10⁻⁶ cells are then added to each well. 5 Cells were seeded with Corning ® Matrigel ® Alternatively, use the tip side of a telogen-coated cell culture insert (0.4 µm, 12 mm PET, Sterlitech). During incubation at 37 °C and 5% CO2, maintain approximately 0.5 ml and 1.5 ml of medium, respectively, on the tip and basal sides of the insert. Replace the medium from the basal and tip sides on days 16 and 18. From day 21 onwards, maintain the RPE in mature medium free of RHO / ROCK pathway inhibitors, replacing it every 3–4 days until day 46–47. On day 49 or 50, collect 20 µL of conditioned medium from the tip and basal chambers and analyze by ELISA, substantially according to the manufacturer's instructions.

[0150] The PEDF ELISA was performed in a similar manner to the VEGF ELISA protocol described above, but with some modifications. On day 49 or 50, 10 µL of conditioned medium was collected from both the top and basal chambers and diluted 100-fold in RPE maturation medium, followed by further dilution in sample dilution buffer to achieve a final dilution of 2000-fold. Subsequently, samples were run according to the manufacturer's instructions with slight modifications. Results showed that mature RPE exhibited increased basal secretion of VEGF for all tested cell lines. Figure 3 B), and for all tested cell lines, PEDF showed increased apical secretion ( Figure 3 C).

[0151] Barrier function of cells at different differentiation stages, typically day 14, immature RPEs, can be assessed by measuring transepithelial resistance (TEER). Briefly, as described in Example 3, hPSCs from four different cell lines were differentiated into immature RPEs (day 14), washed twice with 0.5 ml D-PBS, and contacted with 0.5 ml of trypsin-based enzymatic solution. The monolayer was peeled using a cell scraper, milled, and the single-cell suspension containing the immature RPEs was resuspended in 10 ml of RPE maturation medium, then passed through a 70 μm cell filter and further centrifuged. The cell pellet was resuspended in 4 ml of RPE maturation medium containing RHO / ROCK pathway inhibitors, and approximately 4 x 10⁻⁶ cells were centrifuged. 5 Cells were seeded with Corning ® Matrigel ® The top side of the pre-coated cell culture insert (0.4µm, 12mm PET, Sterlitech). A Corning-coated insert is placed on the top side. ® Matrigel ®Blank cell culture inserts were used as controls. During incubation at 37°C and 5% CO2, approximately 0.5 ml and 1.5 ml of culture medium were maintained in the apical and basal chambers, respectively, with the medium being changed on days 16 and 18. From day 21 onwards, RPE was maintained in RPE maturation medium without RHO / ROCK pathway inhibitors, with changes every 3–4 days until day 46–47. Cells grown on the inserts were placed at room temperature for 10–15 minutes before TEER measurement. TEER values ​​of differentiated cells on day 49 were measured using an EVOM2™ epithelial voltammometer equipped with a chopstick electrode sterilized in 70% ethanol (World Precision Instrument). The electrode was immersed in the apical and basal chambers, and the electrophysiological measurements were recorded for each well. The TEER values ​​were subtracted from the blank control and multiplied by the insert area factor. Mature RPE cells from all four hPSC lines tested showed greater than 200 Ω × cm⁻¹. 2 TEER value ( Figure 3 (D). These results indicate that by day 49, cells form tight junctions and a sufficient barrier to allow polarization and polarization secretion of factors such as PEDF and VEGF. Barrier function can be maintained for another 4 weeks (data not shown).

[0152] This study assessed the ability of cells at different differentiation stages, but typically mature (at least day 77) of the RPE, to phagocytose photoreceptor peripheral segments (POS). POS obtained from bovine eyes (InVision BioResources) was conjugated with fluorescein-5-isothiocyanate (FITC). Briefly, 10 mg of FITC isomer I was resuspended in 0.1 M sodium bicarbonate buffer and incubated for 1 hour at room temperature on a rotating platform in the dark. Subsequently, the unresuspended FITC solid was... > Centrifuge at 3000g and resuspend the precipitate in 5 mL of DMEM / F12 medium. Slowly add FITC isomer I solution to approximately 200 × 10⁻⁶. 6 Each POS was rotated, and the combination was incubated on a rotating platform at room temperature in the dark for 1.5 hours. The precipitate of the labeled POS was resuspended in 1.5 mL of DMEM / F12 and precipitated again to elute any unconjugated FITC isomer I, and this process was repeated. The FITC-conjugated POS was resuspended to a final volume of 5 mL of DMEM / F12 and 12 x 10⁻⁶ ppm was prepared. 6 Divide the sample into equal portions per POS / vial, and then use the vials... >Centrifuge at 3000g, then freeze the precipitate at -80°C. For RPE POS assays, allow immature RPEs on day 14 to mature in tissue culture plates or on 12 mm cell culture inserts until at least day 49. In 12-well tissue culture plates, each well (using Corning) ® Matrigel ® Pre-coated (vaccination) approximately 1×10 5 cells / cm 2 The culture medium was incubated at 37°C and 5% CO2, with 2 mL of maturation medium replaced every 3-4 days until subculture on day 49. On day 49, the frozen FITC-POS vials were thawed on ice, and 10 × 10⁻⁶ mg / L RPE maturation medium was added to every 2 mL of the medium. 6 FITC-POS was obtained using untreated cells without added POS or cells containing FITC-POS in the presence of 50 µg / mL anti-αvβ5. After incubation for 16 hours, the medium was removed, and the wells were washed 3–5 times with 2 mL D-PBS to remove any unbound FITC-POS. 0.5 mL of trypsin-based enzymatic solution was added to each well and incubated at 37 °C for 10 min. The monolayer was peeled off using a cell scraper, ground, and the single-cell suspension was resuspended in 10 mL RPE maturation medium, then passed through a 70 μm cell filter and centrifuged further. The cell pellet was resuspended in 0.4% trypan blue in D-PBS to quench bound FITC-POS, and the volume was brought up to 10 mL in RPE maturation medium and centrifuged at 300 × g for 5 min. The pellet was resuspended in 1 mL of buffer containing 1:1000 Hoechst 33342 before performing flow cytometry to quantify FITC-POS digestion as described in Example 2.

[0153] Data show that cells only show positive staining for FITC when FITC-conjugated POS is added. Figure 3 E). Untreated cells without added POS (mediator) showed that the majority (99.52%) of non-phagocytic cells did not internalize POS. A very low cell population (0.25% and 0.19% in H9 and 1C-derived RPEs, respectively) exhibited POS attached to the cells but released through proteolytic hydrolysis, termed "unbound POS". Overall, the data suggest that most 1C and H9-derived RPEs were capable of phagocytizing POS.

[0154] On day 49 (P0), mature RPE cells derived from four different PSC lineages were imaged. Representative microscopic images show distinct levels of pigmentation in cells differentiated from each hPSC lineage. Figure 3 F).

[0155] Example 5: Effect of media composition on RPE differentiation, purity and pigmentation Cells were cultured and differentiated essentially as described in Example 3, but differences in culture medium compositions were explored, including i) the concentrations of cytokines and small molecules in the third and fourth culture media, and ii) different basal culture media for RPE maturation.

[0156] The concentrations of each of the cytokines and small molecules present in the third and fourth culture media as used in Example 3 were varied to assess their effect on the differentiation of immature and mature RPEs. Concentrations in the third culture medium were tested at 0.5X, 0.75X, 1X, and 1.25X, and the concentrations in the fourth culture medium were matched at 0.5X, 0.75X, 1X, and 1.25X. Results showed that the efficiency of immature RPE formation was similar in both H9 and M001 for all tested conditions. Figure 4 A). However, at lower concentrations of cytokines and small molecules (e.g., 0.5X and 0.75X), RPE pigmentation levels appeared to decrease ( Figure 4 B), as further confirmed by the melanin content determination performed as described in Example 2. Figure 4 C). The results also showed that, for all test conditions, the RPE purity assessed by PMEL17 flow cytometry was comparable between differentiated H9 and M001 cells. Figure 4 D).

[0157] To test the effects of different basal media during the maturation phase, WLS-1C-derived immature RPEs were further cultured for day 14 in three different basal media, essentially as described in Example 3: X-VIVO 10 (Lonza) as a control; and two different similar basal media (StemSpan™ and ImmunoCult™ brand basal media) manufactured by STEMCELL Technologies. Compared to X-VIVO-10, cells maturing in the STEMCELL brand medium appeared to exhibit reduced pigmentation. However, pigmentation was restored in cells maturing in the STEMCELL brand medium upon supplementation with progesterone, putrescine, and a soluble iron source (e.g., ferric ammonium citrate or ferric nitrate). Figure 5 A). Melanin content determination further confirmed these findings. Figure 5 B). Furthermore, for both ferric nitrate and ferric ammonium citrate, increasing the concentration of both iron sources (10 µM, 25 µM, and 50 µM) induced an increase in melanin production levels, as observed by bright-field microscopy and melanin content assays. Figure 5(C and D). The results showed that, compared with the control medium, supplementing with the basal medium described above resulted in higher levels of melanin formation.

[0158] Example 6: High differentiation efficiency of hPSCs in clumps and single cell passaging PSCs derived from four cell lines, 1C, M001, 3A, and H9, were used as a control at 25,000 / cm². 2 Seeded single cells or as clumps (1 / 10 seeding ratio) were passaged and differentiated under the conditions described in Example 3. Differentiation was assessed by flow cytometry analysis of PMEL17 marker expression. Figure 6 Both conditions resulted in significant levels of PMEL17 expression, with a slightly higher efficiency (approximately 94%) observed in single-cell passaged cells compared to cells differentiated from PSCs passaged from clumps.

[0159] Example 7: 3-stage RPE differentiation protocol As an alternative to the four-stage differentiation protocol described in Example 3, RPE differentiation is performed in three stages. In the first stage, from day 0 to day 4, hPSCs are differentiated in a medium containing a BMP pathway inhibitor (headin) and a Wnt pathway inhibitor (DKK1). In the second stage, from day 4 to day 8, cells are cultured in a medium containing activin-A. In the third stage, from day 8 to day 14, cells are cultured in a medium containing activin-A, an FGFR-1 inhibitor, and a Wnt agonist.

[0160] The expression of RPE65 and MITF in differentiated cells was analyzed by microscopy and flow cytometry. Immature (day 14) and mature (day 49) RPE cells exhibited a polygonal morphology. Figure 7 A). Further, flow cytometry analysis of cells on day 49 quantified the expression of MITF (a marker of optic vesicles and RPE) and RPE65 (a marker of RPE maturation) in WLS-1C and H9-derived RPE cells. The results showed that 1C-derived RPE cells had 99.28% RPE65-positive cells and 81.92% MITF-positive cells, while H9-derived RPE cells had 98.36% RPE65-positive cells and 75.51% MITF-positive cells. Figure 7 B). The results showed that the 3-stage differentiation protocol achieved high differentiation and RPE maturation efficiency. However, when assessing PMEL17 expression between WLS-1C-derived cells and H9-derived cells on day 14, greater variability was observed in the 3-stage differentiation protocol compared to the 4-stage protocol in Example 3. Figure 7 C).

[0161] Interestingly, the combination of Wnt signaling inhibitors and BMP signaling pathway inhibitors in the culture medium from days 0-4 appeared to lead to higher cell death on day 8 (data not shown). When the BMP pathway inhibitors (headin) and Wnt pathway inhibitors (DKK1) were replaced with the BMP pathway inhibitor LDN-193189 and the Wnt pathway inhibitor IWP2, RPE differentiation efficiency, as assessed by PMEL17 marker expression, was found to be lower in H9-derived RPEs. Flow cytometry analysis of PMEL17 expression showed that H9-derived cells produced 61.99% PMEL17-positive cells compared to 91.04% PMEL17-positive cells in 1C-derived RPEs. Figure 7 D).

[0162] Example 8: 5-stage RPE differentiation protocol As an alternative to the 4-stage and 3-stage differentiation protocols described in Examples 3 and 7, respectively, RPE differentiation was performed in 5 stages. In the first stage, from day 0 to 2, hPSCs were differentiated in a medium containing 50 ng / ml headprotein, DKK-1, and IGF-1. In the second stage, from day 2 to 4, cells were cultured in a medium containing 10 ng / ml headprotein, DKK1, IGF-1, and FGFb. In the third stage, from day 4 to 6, cells were cultured in a medium containing activin A, DKK1, and IGF-1. In the fourth stage, from day 6 to 8, cells were cultured in a medium containing activin A and an FGFR-1 inhibitor. In the fifth stage, from day 8 to 14, cells were cultured in a medium containing activin A, an FGFR-1 inhibitor, and a Wnt signaling agonist. Individual culture medium components were removed or replaced with alternative factors, and their impact on RPE differentiation was assessed by flow cytometry of the percentage of immature RPEs derived from 1C and H9 at day 14. The following conditions were tested: (i) a control with all culture medium components of the above 5-stage differentiation protocol; (ii) removal of DKK1; (iii) removal of FGFb; (iv) removal of IGF-1; and (v) replacement of head protein with LDN-193189. Results ( Figure 8 The results showed that, as assessed by flow cytometry of the percentage of immature RPEs on day 14%, removal of DKK1 or IGF-1 had no negative impact on RPE differentiation. The results indicate that RPE differentiation can be achieved in the absence of DKK1.

[0163] Example 9: Comparison of differentiation efficiency of 3 differentiation protocols The efficiencies of the 5-stage differentiation protocol in Example 8, the 3-stage differentiation protocol in Example 7, and the 4-stage differentiation protocol in Example 3 were compared. Flow cytometry analysis of PMEL17 expression in immature RPEs on day 14 is summarized in […].Figure 9 In the box plots, while each scheme showed an average of about 80% or higher, the 4-stage differentiation scheme demonstrated the highest efficiency and was the most robust. Figure 9 ).

[0164] Example 10: Effect of BMP inhibition on RPE differentiation efficiency Cells were cultured and differentiated essentially as described in Example 3, but the first culture medium was modified to contain different types and concentrations of BMP signaling inhibitors: (i) no BMP inhibition; (ii) head protein (12.5 ng / ml, 25 ng / ml and 100 ng / ml); (iii) doxorphine (0.625 µM, 1.25 µM, 2.5 µM and 5 µM); and (iv) LDN-193189 (25 nM, 100 nM, 200 nM and 500 nM).

[0165] The percentage of positive immature RPE cells on day 14 from 3A and H9 cell lines was analyzed by flow cytometry to target PMEL17 expression. Figure 10 A). In addition, a hemocyte counter was used to calculate the yield of immature RPE per well ( Figure 10 B). For all tested BMP inhibitors, results typically showed improved RPE differentiation at lower concentrations ( Figure 10 A). Specifically, 0.625 µM or 1.25 µM doxomorphine and 25 nM or 100 nM LDN-193189 generated the most consistent RPE differentiation efficiency (e.g., >80%) among the tested hPSC cell lines. Although the absence of any BMP signaling inhibitors was observed to produce a high percentage of PMEL17+ immature RPEs on day 14, this absence appeared to be associated with increased corneal contamination, such as during maturation (data not shown).

[0166] Example 11 : Generation and characterization of retinal organoids According to the manufacturer's protocol, PSCs maintained substantially as described in Example 1 were formed into aggregates in Aggrewell™ 800 (STEMCELL Technologies) 24-well plates in mTeSR™1 or mTeSR™ Plus. The aggregates were recovered from the Aggrewell™ plates and plated in Matrigel™-coated plates, and cultured substantially as described in Example 3 until day 6. Subsequently, the cells were cultured for 3 days in a neuroretinal / RPE induction medium containing BMP4, and then for 5 days in a medium lacking BMP4 and containing one or more of progesterone, putrescine, transferrin, and heparin to form aggregates containing different domains of the neuroretina and / or RPE. Next, the aggregates containing different domains of the neuroretina were dissociated by using a pipette tip or similar means to penetrate a monolayer carved with a grid pattern and release sheets of retinal tissue. The retinal-like tissue was then cultured for 4 days from day 14 to day 18 in retinal organoid formation medium under non-adherent conditions to form retinal organoids containing the neuroretina. Next, from day 18 to day 150, retinal organoids were matured in retinal organoid maturation culture medium.

[0167] Figure 11 A illustrates the generation of retinal organoids at different developmental stages during the above protocol, including: homogeneous day 0 PSC aggregates formed in AggreWell™ 800; day 1 differentiated aggregates adhered to the plate; day 14 cultures containing different domains of the neuroretina and RPE; day 18 retinal structures with phase-bright boundaries of the neuroretina; day 27 retinal organoids; and maturation of day 50, 96, and 123 retinal organoids.

[0168] Figure 11B shows immunohistochemical staining used to characterize the cells of retinal organoids at different stages. Expected markers of retinal organoids were observed at specific stages of the protocol. Staining data at day 60 showed that retinal ganglion cells (RGCs) stained with SNCG antibody were correctly located within the retinal organoids. RGCs were observed projecting cells into a neuroblast layer stained with VSX2 antibody (i). The resulting retinal organoids were further shown to contain amacrine cells (AP2α) and horizontal cells (Prox1), with the amacrine cells more oriented towards the RGC layer tissue (ii). Staining with OTX2 and CRX antibodies also showed that the resulting retinal organoids contained differentiated retinal progenitor cells (RPCs) and photoreceptor progenitor cells (PRPs) of the peripheral tissue (iii). Staining with OTX2 and CRX antibodies at day 96 further showed an increase in photoreceptor density along the periphery of the retinal organoids and filling the developing outer nuclear layer (iv). Staining data from day 96 also indicated that the generated retinal organoids contained amacrine cells (AP2α) and horizontal cells (Prox1), with the amacrine cells more oriented towards the RGC layer tissue (v). RGC nerve fiber-like tracts (Prox1 and AP2α) were also observed on day 96 (vi). Similar tissue containing both amacrine cells and horizontal cells was observed on day 123 (vii).

Claims

1. A method for differentiating a population of cells destined to form the retinal lineage, the method comprising: a) Provide a population of pluripotent stem cells (PSCs); b) Expose the PSC population to a first culture environment containing inhibitors of extracellular matrix proteins and bone morphogenetic protein (BMP) signaling; c) Incubate the PSC population in the first culture environment for approximately 0 to 60 hours; and d) Obtain the cell population that is destined to be of the retinal lineage.

2. The method of claim 1, wherein the first culture environment does not contain one or both of the following: i) a direct Wnt signaling antagonist; and ii) an activin signaling agonist.

3. The method of claim 1 or 2, wherein the extracellular matrix protein is coated on the culture surface, and the extracellular matrix protein is fibronectin, laminin, fibronectin, collagen, or a mixture of more than one of the foregoing substances.

4. The method of any one of claims 1 to 3, wherein the concentration of the BMP signaling inhibitor is about 100 nM or less.

5. The method of any one of claims 1 to 4, wherein the BMP signaling inhibitor is contained in the first cell culture medium.

6. The method according to any one of claims 1 to 5, further comprising: e) Expose the cell population, which is destined to be of the retinal lineage, to a second culture environment; f) Culture the cell population for approximately 0 to 60 hours; as well as g) Obtain the progenitor cell population of the eye region.

7. The method of claim 6, wherein the second culture environment comprises the extracellular matrix protein and the second cell culture medium.

8. The method of claim 7, wherein the second cell culture medium comprises a basal culture medium and one or more of insulin-like growth factor (IGF) and fibroblast growth factor (FGF) signaling agonists.

9. The method of any one of claims 6 to 8, further comprising: h) Expose the ocular progenitor cell population to a third culture environment; i) Culture the ocular progenitor cell population for approximately 2 to 5 days; as well as j) Obtain the population of visual vessel progenitor cells.

10. The method of claim 9, wherein the third culture environment comprises the extracellular matrix protein and the third cell culture medium.

11. The method of claim 10, wherein the third cell culture medium comprises a basal culture medium and one or more of IGF and activin signaling agonists.

12. The method of any one of claims 9 to 11, wherein the optic angiogenesis progenitor cells are bifunctional for either the retinal pigment epithelium or the neuroretina.

13. The method of any one of claims 9 to 12, further comprising: k) Expose the optic angiogenesis progenitor cell population to a fourth culture environment; l) Culture the aforementioned optic angiogenesis progenitor cell population for approximately 5 to 10 days; as well as m) Immature retinal pigment epithelial cell populations were obtained.

14. The method of claim 13, wherein the third culture environment comprises the extracellular matrix protein and the fourth cell culture medium.

15. The method of claim 14, wherein the fourth cell culture medium comprises a basal culture medium and one or more of an activin signaling agonist, a Wnt signaling agonist, and an FGF signaling antagonist.

16. The method of any one of claims 13 to 15, further comprising dissociating the immature retinal pigment epithelial cell population and re-coating the dissociated cells to enrich the immature retinal pigment epithelial cells.

17. The method of claim 16, wherein the dissociated population of immature retinal pigment epithelial cells is not enriched by manual dissection, particle-based enrichment, or ligand-mediated enrichment.

18. The method of any one of claims 13 to 17, wherein about 50% or more of the immature retinal pigment epithelial cell population expresses PMEL17.

19. The method of any one of claims 13 to 18, further comprising maturing the immature retinal pigment epithelial cell population for about 1 to 5 weeks.

20. The method of claim 19, wherein the immature retinal pigment epithelial cell population matures upon contact with a maturation medium comprising a basal medium and at least a soluble iron source.

21. The method of claim 20, wherein the maturation culture medium further comprises one or more of steroid hormones and polyamines.

22. The method of any one of claims 19 to 21, wherein about 80% or more of the mature retinal pigment epithelial (RPE) cells express RPE65.

23. The method of any one of claims 1 to 22, wherein one or more of the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are serum-free and / or free of animal components.

24. The method of any one of claims 6 to 23, wherein one or more of the second culture medium, the third culture medium, and the fourth culture medium are deficient in exogenously added BMP signaling inhibitors.

25. The method of any one of claims 1 to 24, wherein one or more of the first culture medium, the second culture medium, the third culture medium, and the fourth culture medium are deficient in nicotinamide.

26. The method of claim 1, wherein the PSC population is cultured under non-adherent conditions to generate three-dimensional retinal organoids.

27. The method of claim 26, wherein the PSC cluster is seeded into the microporous device.

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