Compositions and methods for differentiating RPE cells

Abstract

This disclosure relates to applications for culturing retinal pigment epithelial (RPE) cells. The method comprises a) providing a population of PSCs; b) exposing the PSC population to a first culture environment containing an inhibitor of extracellular matrix proteins and BMP signaling; c) culturing the PSC population in the first culture environment for approximately 0 to 60 hours; and d) inducing a population of cells whose fate has been determined to the retinal lineage.

Description

Cross-reference of related applications

[0001] This application claims the interests of U.S. Provisional Patent Application No. 63 / 457,430, filed on April 6, 2023, the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] This disclosure relates to cell culture applications, and more specifically to cell differentiation and / or maturation applications. More specifically, this disclosure relates to the culture of retinal pigment epithelial (RPE) cells and / or their progenitor cells. [Background technology]

[0003] The retina is the inner layer of the eye, a complex, layered neuronal structure that captures and processes light, converting light signals into electrical signals that travel through the optic nerve to the visual center of the brain.

[0004] The retinal pigment epithelium (RPE) is a monolayer of cells located in the outermost layer of the retina. The basal side of the RPE connects to the Bruch membrane and choroid, while the apical side connects to the outer segments of photoreceptor cells via microvilli structures extending from the RPE cells. RPE cells are a highly phagocytic cell type and can internalize photoreceptor outer segments (POS) from superior rod or cone photoreceptor cells. The RPE performs several important functions in vision, including light absorption, formation of the outer blood-retinal barrier, nutrient and ion transport, retinoid cycling, phagocytosis of used photoreceptor outer segments, and growth factor secretion (Boulton et al. (2001) Eye 15, 384-389). Due to its melanin content, the RPE is dark brown and reduces UV-induced damage to the retina and internal nerves.

[0005] The structure and function of the retinal pleoplastic arch (RPE) 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's disease (SD). AMD is the leading cause of severe vision loss in adults over 60 years of age, and it was estimated that approximately 196 million people worldwide were affected in 2020. RP causes vision loss in childhood or young adulthood and affects 100,000 people in the United States. SD has a prevalence of 1 in 10,000 births and is the most common form of hereditary juvenile macular degeneration (Yang S et al. (2021) Front Pharmacol. 12:727870). There are currently no treatments for these degenerative diseases. Therefore, understanding the onset of RPE in vitro may help elucidate the pathogenesis of related conditions or diseases and may contribute to the development of new therapeutic approaches.

[0006] RPE cells produced in vitro can be used to study RPE development, identify factors that cause RPE damage, or identify drugs that can be used to promote the repair of endogenous RPE cells. Furthermore, the RPE cells generated in vitro themselves can be used in cell therapies that may involve replacing or repairing 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), may be an ideal starting cell type for in vitro generation of RPE cells. RPE production from PSCs could accelerate research into understanding various eye diseases and provide an unlimited source of RPE for retinal cell therapy. Therefore, there is a need for a rapid and efficient in vitro protocol for generating RPE from PSCs, more specifically, a highly compliant protocol suitable for cell therapy applications. [Overview of the project]

[0008] One aspect of the present disclosure provides a method for differentiating immature RPE and / or mature RPE from, for example, one or more pluripotent stem cells (PSCs). In one embodiment, the differentiation of immature and / or mature retinal pigment epithelium (RPE) involves various stages. Accordingly, one aspect of the present disclosure provides a method for differentiating a cell population destined to the retinal lineage, comprising providing a population of PSCs and exposing the PSC population to a first culture environment containing extracellular matrix proteins and bone morphogenesis protein (BMP) signaling inhibitors.

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

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

[0011] In one embodiment, the method may further include culturing a population of PSCs in a first culture environment for approximately 0 to 60 hours to induce a population of cells whose fate has been determined to become retinal.

[0012] In one embodiment, the first culture environment does not contain either or both of i) a direct WNT signaling antagonist and ii) an activin signaling agonist.

[0013] In one embodiment, the extracellular matrix protein is coated onto the culture surface. In one embodiment, the extracellular matrix protein is vitronectin, laminin, fibronectin, collagen, or a mixture of two or more of the above. In one embodiment, the extracellular matrix protein is recombinant.

[0014] In one embodiment, the method of the present disclosure may further include exposing a cell population determined to be fate-determined into the retinal lineage to a second culture environment, culturing the cell population for about 0 to 60 hours to induce an eye field progenitor cell population.

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

[0016] In one embodiment, the method of the present disclosure may further include exposing the eye field progenitor cell population to a third culture environment, culturing the eye field progenitor cell population for about 2 to 5 days to induce a visual vascular progenitor cell population. In one embodiment, the visual vascular progenitor cells are bipotent with respect to the retinal pigment epithelium or the neural retina.

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

[0018] In one embodiment, the method of the present disclosure may further include exposing the visual vascular progenitor cell population to a fourth culture environment, culturing the visual vascular progenitor cell population for about 5 to 10 days to induce an immature retinal pigment epithelial cell population.

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

[0020] In one embodiment, the method of the present disclosure may further include dissociating an immature retinal pigment epithelial cell population, reseeding the dissociated cells, and concentrating the immature retinal pigment epithelial cells. In one embodiment, the dissociated population of immature retinal pigment epithelial cells is not concentrated by manual dissociation, particle-based enrichment, or ligand-mediated enrichment.

[0021] In one embodiment, at least about 50% of the immature retinal pigment epithelial cell population expresses PMEL17.

[0022] In one embodiment, the method may further include maturing the immature retinal pigment epithelial cell population for about 1 to 5 weeks. In one embodiment, the immature retinal pigment epithelial cell population is matured by contacting a maturation medium that includes a basal medium and at least a soluble iron source. In one embodiment, the maturation medium further includes one or more of a steroid hormone and a polyamine. In one embodiment, the maturation medium may further include progesterone and putrescine.

[0023] In one embodiment, at least about 80% of the matured RPE expresses 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 are serum-free and / or animal component-free.

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

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

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

[0028] In other aspects of this disclosure, the method for differentiating immature or mature RPEs does not begin with PSCs, but rather with any downstream cell population as described herein, using the appropriate culture environment and / or medium described herein.

[0029] In another aspect of the present disclosure, a system or kit is provided for differentiating immature or mature RPEs from a PSC (or any downstream intermediate) population, the system or kit comprising one or more disease-stage-appropriate culture medium compositions (or basal media and supplements), extracellular matrix proteins, and instructions for use.

[0030] To better understand the various embodiments described herein and to more clearly illustrate how these various embodiments can be carried out, the accompanying drawings showing at least one exemplary embodiment are referred to below as examples. The drawings are not intended to limit the scope of the teachings described herein. [Brief explanation of the drawing]

[0031] [Figure 1] This shows the stepwise differentiation of immature RPE cells from pluripotent stem cells. Differentiation of two iPS cells (3A and M001) was carried out as described herein, and representative images were taken on days 2, 7, and 14 (A). The scale bar represents 200 μm. Flow cytometry analysis of cells on day 14 for PMEL17 marker expression among immature RPE cells derived from PSCs (B). [Figure 2]The images show cell morphology during RPE maturation. Differentiation of two PSC lines (1C and H9) was carried out as described herein, and representative images were taken on days 18, 21, 28, 42, and 49. The scale bar represents 200 μm. [Figure 3] This section describes the characteristics of mature RPE. Flow cytometry results (A) of PMEL17 (91.4% ± 1.2%) marker expression between differentiated cells at day 14, and PMEL17 (96.6% ± 0.5%) and RPE65 (93% ± 1.0%) marker expression between differentiated cells at day 49 from four different PSC strains. Data represent the mean ± standard error of 22-25 experiments. Bar graphs quantifying apical and basal secretion of growth factors from mature RPE derived from four different PSC strains. Cells at day 52 were evaluated for apical and basal secretion of VEGF (B), and cells at day 53 were evaluated for apical and basal secretion of PEDF (C). Bar graphs (D) of barrier function of mature RPE differentiated from four different PSC strains with transepithelial electrical resistance (TEER) values ​​measured for cultures at day 49. For B)-D), data represent the mean of three experiments. Flow cytometry plot (E) quantifying phagocytosis of the outer segment of the FITC conjugate photoreceptor by mature RPE from two PSC strains (H9 and 1C). Representative images of pigment deposition in mature RPE from four different PSC strains. Cells at day 49 were imaged by bright-field microscopy (F). Scale bar represents 50 μm. [Figure 4] This study illustrates the effects of culture medium composition during the differentiation of immature RPE cells. Differentiation of two PSC lineages (H9 and M001) was carried out between days 4 and 14, as described herein, in the presence of cytokines and small molecules at concentrations of 0.5x, 0.75x, 1x, and 1.25x. Representative images were taken of cells on day 14 (A) and day 49 (B). A bar graph quantifying the melanin content between cells on day 49 in each culture medium composition is shown in (C). PMEL17 expression was evaluated in cells on day 14 and day 49 by flow cytometry (D). Data represent the average of two experiments. [Figure 5]This shows the effect of different basal media on RPE maturation. Representative bright-field images show pigmentation of 1C-derived RPE cultured in two different supplemented and unsupplemented basal media formulations compared to a control basal medium (A). Scale bars represent 200 μm. Bar graphs (B) show melanin content after culture in supplemented and unsupplemented media formulations. Data represent the mean ± standard error of four experiments. Representative bright-field images (C) of H9-derived RPE cells cultured in the presence of increased concentrations of ferric nitrate (10 μM, 25 μM, and 50 μM) or ferric ammonium citrate (10 μM, 25 μM, and 50 μM) compared to the unsupplemented medium control. Scale bars represent 200 μm. Bar graphs (D) show melanin content in the presence of the indicated concentrations of ferric nitrate or ferric ammonium citrate. [Figure 6] This report describes the differentiation efficiency of either single-cell passaged hPSCs or clamp passaged hPSCs. Cell differentiation efficiency from four different PSC lines was evaluated by flow cytometry for PMEL17 marker expression. Data represent the mean ± standard error of 8–18 experiments. [Figure 7] The efficiency of RPE differentiation using an alternative three-step protocol is demonstrated. Representative bright-field images show the cell morphology of 1C-derived RPE at day 14 and day 49 (A). Flow cytometry analysis of MITF and RPE65 expression between WLS-1C and H9-derived RPE at day 49 (B). Flow cytometry analysis of PMEL17 expression in immature WLS-1C and H9-derived RPE at day 14 using the three-step protocol (C). Flow cytometry analysis of PMEL17 expression in immature WLS-1C and H9-derived RPE cultured in the presence of a combination of wnt signaling surrogate inhibitors and BMP signaling pathway surrogate inhibitors at day 14 (D). [Figure 8] This shows the efficiency of RPE differentiation using an alternative five-step protocol. The box plot summarizes the frequency of immature RPE (day 14) derived from 1C and H9 cells, as assessed by flow cytometry analysis of PMEL17 expression. Data represent the mean ± standard error of 4–12 experiments. [Figure 9]This box plot compares the differentiation efficiency of a 5-step, 3-step, and 4-step RPE differentiation protocol. The data represents the mean ± standard error of 6 to 31 experiments. [Figure 10] The effects of different BMP inhibitors on RPE differentiation efficiency are shown. The percentage of positive immature RPE cells from 3A and H9 cell lines generated on day 14 was analyzed by flow cytometry for PMEL17 marker expression (A). Immature RPE yield per well was calculated for 3A and H9 cell lines on day 14 using a hemocytometer (B). [Figure 11] This image shows the generation and characterization of retinal organoids. Bright-field images show organoid generation over time (A). Immunohistochemical staining (B) characterizes the expression of various protein markers (SNCG, PAX6, VSX2, Prox1, AP2α, OTX2, and CRX) and nuclear markers (DAPI). Each is indicated by an arrow. [Modes for carrying out the invention]

[0032] This disclosure relates to a media composition and kit, and to a method for the stepwise differentiation of retinal pigment epithelial cells (RPEs) and / or their progenitor cells. More specifically, this disclosure relates to a media composition and kit, and to a method for the stepwise differentiation of RPE-like cells and / or their progenitor cells, starting from one or more PSCs.

[0033] Where used in this disclosure, the term “PSC population” or “PSC” refers to one or more cells that are self-renewing and capable of differentiating into all three germ layers, such as the ectoderm, mesoderm, and endoderm. The conditions and media requirements for culturing PSCs to maintain an undifferentiated state are known. PSC is a broad term that encompasses both embryonic stem cells (“ESCs”) and induced pluripotent stem cells (“iPSCs”). While there may be ethical challenges in sourcing ESCs, several ESC strains are now established in this field. On the other hand, iPSCs can be induced from a wide variety of readily accessible cells, including adult somatic cells, and are therefore associated with fewer ethical constraints. Thus, new iPSC strains emerge regularly. PSCs are important models for studying differentiation mechanisms, modeling diseases, and providing important medical opportunities. PSCs can be obtained from, derived from, or induced from any source species, but in this disclosure, PSCs are preferably human.

[0034] As used in this disclosure, the term “cell population destined to the retinal lineage” refers to a group of cells that have differentiated from one or more progenitor cells (e.g., PSCs) but still retain a variety of potentials (more limited to one or more progenitor cells). A cell population destined to the retinal lineage refers to a population of ectoderm or mesoderm cells capable of further differentiating into RPE cells, i.e., cells destined to retinal pigment epithelial cells, RPE progenitor cells, or cells that differentiate toward the ocular field via a preneurectoderm state. In the context of this disclosure, a cell population destined to the retinal lineage may be biased toward an ectoderm or mesoderm lineage, or may acquire ectoderm or mesoderm potential. One or more subsequent differentiation steps are required to differentiate a cell population destined to the retinal lineage into immature / mature retinal (epithelial) cells. Characteristics of cells destined to the retinal lineage include, but are not limited to, high OCT4, TRA-1-60, and / or loss of NANOG expression. Furthermore, cells destined to become retinal lineage may begin to express relevant markers, such as SIX3, SIX6, LHX2, PAX6, RAX, or NR2E1 levels. In one embodiment, cells destined to become retinal lineage efficiently generate or differentiate into immature and / or mature RPE cells.

[0035] As used in this disclosure, the term “ocular progenitor cell population” refers to a group of cells that have differentiated from one or more cells destined for the retinal lineage but still retain a variety of potentials. The ocular progenitor cell population may be biased toward the ectoderm lineage or may acquire ectoderm potential. The ocular progenitor cell population may also include ocular specialized cells, ocular progenitor cells, preneurial plate-derived cells, or RPE progenitor cells. One or more subsequent differentiation steps are required to differentiate the ocular progenitor cell population into immature / mature RPE. The ocular progenitor cell population may express ocular transcription factors such as PAX6, RAX, SIX3, and LHX2. The cell population destined for the retinal lineage may have some overlap with the ocular progenitor cell population, but the latter may involve the integration or homogenization of relevant marker expressions, such as with respect to SIX3, SIX6, LHX2, PAX6, RAX, or NR2E1 levels. Furthermore, ophthalmic progenitor cells may initiate PMEL17 expression, for example, at relatively low levels (compared to immature RPE cells). In one embodiment, ophthalmic progenitor cells efficiently generate or differentiate immature and / or mature RPE cells.

[0036] As used in this disclosure, the term “optic vascular progenitor cell population” refers to a group of cells differentiated from one or more developmentally early progenitor cells, such as ophthalmic progenitor cells. Optic vascular progenitor cells may have more limited potential than ophthalmic progenitor cells and may develop or differentiate into optic vesicles and optic cups. Optic vesicles may include retinal stem cells (e.g., cells capable of generating all neuroectoderm-derived cells of the eye) and / or transient dipotent progenitor cells (e.g., cells giving rise to retinal pigment epithelium or neuroretina). Optic vascular progenitor cell populations are biased toward the ectoderm lineage and acquire ectoderm potential. Optic vascular progenitor cell populations may include optic vascular specialized cells, optic vascular progenitor cells, cells interacting with surface ectoderm or RPE lineage-determined cells, or RPE progenitor cells. One or more subsequent differentiation steps may be required to differentiate the optic vascular progenitor cell population into immature / mature RPE. The ophthalmic progenitor cell population may partially overlap with the optic vascular progenitor cell population, but the latter may show integration or homogenization of relevant marker expression, such as with respect to MITF or VSX2 levels (compared to the ophthalmic progenitor cell population). Furthermore, optic vascular progenitor cells may express higher levels of PMEL17 and / or PAX6 than ophthalmic progenitor cells. In one embodiment, optic vascular progenitor cells efficiently generate or differentiate immature and / or mature RPE cells.

[0037] Where used in this disclosure, the term “immature retinal pigment epithelial cell population” or “immature RPE” refers to a group of cells differentiated from one or more developmentally early progenitor cells, such as optic vascular progenitor cells. Immature RPEs have a more limited potential compared to optic vascular progenitor cells. An immature RPE population may be biased toward or confined to the RPE lineage. An immature retinal pigment epithelial cell population may include cells destined to become mature RPEs or intermediate RPE progenitor cells. An immature RPE population may be induced or differentiated from RPE progenitor cells. One or more subsequent differentiation / maturation steps may be required to differentiate immature RPEs into mature RPEs. An immature RPE population may have some overlap with the optic vascular progenitor cell population, although the former may encompass the integration or homogenization of relevant marker expressions, such as with respect to PMEL17, MITF, or PAX6 levels. Immature RPEs may be characterized by loss of RAX and / or CHX10 expression, as well as the appearance of one or more of RPE65, ZO1, TYR, and TYRP1 expression. Immature RPEs, particularly those derived from PSCs, may exhibit at least some of the features of mature RPEs, such as polygonal morphology and pigmentation. In one embodiment, immature RPEs efficiently generate or differentiate into mature RPEs.

[0038] Where used herein, the term “mature retinal pigment epithelial cell population” or “mature RPE” refers to a group of cells differentiated from one or more developmentally early progenitor cells, such as immature RPEs. Immature RPE populations can be induced or differentiated from RPE progenitor cells. If derived from one or more PSCs, RPEs may or may not be fully mature, but nevertheless exhibit many of the characteristics of primary mature RPEs, and therefore, where used herein, the term mature RPE may similarly refer to mature RPEs or RPEs that are maturing. Mature RPEs have a more limited potential compared to immature RPEs and may ultimately differentiate. Mature RPE populations may be biased toward or limited to the RPE lineage. Mature RPE populations may have some overlap with immature RPE populations, although the former may encompass the integration or homogenization of relevant marker expressions, such as with respect to RPE65, TYR, TYRP1, BEST1, CRALBP, EZRIN, and ZO-1 levels. Furthermore, mature RPEs, particularly those derived from PSCs, may exhibit at least some or all of the characteristics of mature RPEs, such as polygonal morphology, pigmentation, the ability to phagocytose the outer segments of photoreceptors, polarity, and post-mitotic properties.

[0039] As used in this disclosure, the term “RPE progenitor cells” refers to any of the following: a population of cells destined for the retinal lineage, a population of ocular progenitor cells, and / or a population of optic vascular progenitor cells.

[0040] As used in this disclosure, the term “neuroretinum” refers to a heterogeneous group of cells or structures bounded by RPE and which may include cell types such as retinal progenitor cells, retinal ganglion cells, photoreceptor progenitor cells, rod and cone photoreceptor cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and horizontal cells.

[0041] method One aspect of this disclosure provides a method for differentiating a cell population destined to become a retinal lineage. Another aspect of this disclosure provides a method for differentiating a cell population destined to become a retinal lineage into immature and / or mature RPEs. In one embodiment, the differentiation of immature and / or mature RPEs proceeds through two or more intermediate cell populations, as further described below.

[0042] The methods of the present disclosure may include providing a PSC population and exposing such population to a first culture environment. The PSC population is not particularly limited, as various PSC strains are publicly available and / or commercially available, and techniques for generating PSC strains are now routine. Preferably, the PSC population is undifferentiated or substantially undifferentiated, i.e., the PSC population has the ability to differentiate into all germinal layers. In one embodiment, the PSC population is iPSC or ESC.

[0043] The PSC population may be wild-type or may have mutations or edits at one or more genomic loci. In some embodiments, it may be desirable to model diseases of the retina or other neurological disorders, and therefore, PSCs (present in or edited in reprogrammed cells) with one or more desired mutations may serve as a suitable starting point.

[0044] The PSC population may originate from any animal or mammalian species. In a preferred embodiment, the PSC population may originate from a human, primate, or rodent source. To avoid or limit variability between downstream differentiated cells, such as immature or mature RPEs, it may be desirable for the PSC population to be clonal. The PSC population may originate from a human patient or may be a patient-derived iPSC strain.

[0045] As described above, the method of the present disclosure comprises exposing a PSC population to a first culture environment. The first culture environment is not particularly limited, as it either supports the PSC population or supports the differentiation of the PSC population into a downstream lineage (towards the retinal lineage). In one embodiment, the first culture environment comprises either or both an extracellular matrix protein and a BMP signaling inhibitor. In one embodiment, the first culture environment comprises either or both an extracellular matrix protein and a first culture medium, wherein the BMP signaling inhibitor is contained in the first culture medium.

[0046] Extracellular matrix proteins for culturing / supporting PSCs are known and commercially available. The extracellular matrix proteins used in the first culture environment of this disclosure are not limited to those that support the PSC population and do not inhibit the differentiation of PSCs into desired downstream populations, such as cell populations destined for the retinal lineage.

[0047] Extracellular matrix proteins can be coated onto the culture surface (of the first culture environment). The culture surface can be any surface for culturing PSCs and differentiating them into downstream ectoderm (e.g., neural and / or retinal) lineages. In non-limiting examples, the surface can be a culture flask, cell culture vessel, petri dish, roller bottle, cell culture dish, multiwell plate, or the wall (e.g., bottom wall) of a microcarrier. In one embodiment, the cell culture dish may be untreated and have a hydrophobic surface, or may be treated to have a hydrophilic and electrostatic surface. Furthermore, the surface can be any other type of porous surface supporting the cell population seeded thereon, such as a membrane, filter, or cell culture insert (e.g., Transwell® insert).

[0048] In one embodiment, one or more extracellular matrix (ECM) proteins are coated onto the culture surface before seeding the PSC population. In one embodiment, one or more ECM proteins may be included in the cell culture medium applied to the culture surface. In such embodiments, one or more ECM proteins may be coated onto the surface as the PSC population settles.

[0049] In one embodiment, one or more ECM proteins are added directly to the cell culture medium. In one embodiment, one or more ECM proteins in a desired amount or concentration may be added to a cell culture medium containing a population or suspension of cells. In one embodiment, one or more ECM proteins in a desired amount or concentration may be added to a cell culture medium that will later come into contact with a population or suspension of cells.

[0050] As a non-limiting example, one or more extracellular matrix proteins may be collagen, laminin, decorin, vitronectin, fibronectin, Synthemax®, Synthemax® IIa, Matrigel®, or a mixture of two or more of the above, or may be selected from these. 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.

[0051] In embodiments containing collagen, the collagen may be one or more of type I collagen, type II collagen, type III collagen, or type IV collagen. In embodiments containing laminin, the laminin may 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, a fragment of laminin-511 (e.g., laminin-511-E8), laminin-521, or laminin-213. In embodiments containing elastin, the elastin may be one or more of elastin or tropoelastin. In embodiments containing nidogen (also known as enterin), the nidogen may be one or more of nidogen-1 or nidogen-2. In one embodiment, the extracellular matrix protein may be vivonectin or an isoform thereof. In one embodiment, the extracellular matrix protein may be Matrigel®.

[0052] In one embodiment, the ECM protein may be recombinant and / or of natural origin. In one embodiment, the ECM protein may be genetically engineered (e.g., a fusion protein). In one embodiment, the ECM protein may be a whole protein or a fragment thereof, such as a peptide fragment.

[0053] The methods of this disclosure may include ECM proteins(or ECM proteins) coated on a culture surface or ECM proteins(or ECM proteins) directly added to a cell culture medium. The concentration of ECM proteins(or ECM proteins) is not particularly limited. In one embodiment, the concentration of extracellular matrix proteins may be in the range of about 0.1 μg / mL to 1 mg / mL. In one embodiment, the concentration of one or more (or each) ECM proteins may be in the range of about 1 ng / mL to 1 μg / mL, about 100 ng / mL to 100 μg / mL, about 500 ng / mL to 50 μg / mL, or about 1 μg / mL to 30 μg / mL. In embodiments where the ECM proteins are a combination of two or more pre-mixed ECM proteins such as Matrigel®, they may be diluted in ratios of about 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, or higher.

[0054] As described herein, the first culture environment may include a BMP signaling inhibitor. In non-limiting examples, the BMP signaling inhibitor may be a small molecule, a peptide, or a protein. Examples of small molecule inhibitors of BMP signaling include 4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline dihydrochloride (LDN-193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine (dorsomorphine), 4-[6-(4-propan-2-yloxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), and 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl Examples of BMP signaling protein inhibitors include, but are not limited to, ]-quinoline (DMH2), 3-[(6-amino-5-(3,4,5-trimethoxyphenyl)-3-pyridinyl]phenol (K02288), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]-quinoline (ML-347). Examples of BMP signaling protein inhibitors include, but are not limited to, noggin, chordin, gremlin, crossveinless-2 (CV2), USAG-1 (uterine sensitization-related gene-1), follistatin, and sclerostatin (also known as SOST).

[0055] The concentration of the BMP signaling inhibitor in the first culture environment may affect the efficiency of differentiating the PSC population into immature or mature RPEs via one or more intermediates. In one embodiment, the concentration of the BMP signaling inhibitor in the first culture environment is important for most efficiently influencing the differentiation of the PSC population into immature or mature RPEs via one or more intermediates. In one embodiment, the concentration of the BMP signaling inhibitor is in the range of approximately 0.01 nM to 100 μM, approximately 0.05 nM to 75 μM, approximately 0.1 nM to 50 μM, approximately 0.15 nM to 25 μM, approximately 0.2 nM to 10 μM, and approximately 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 the range of approximately 20 nM to 750 nM. In one embodiment, the concentration of the BMP signaling inhibitor is in the range of approximately 5 ng / ml to 150 ng / ml.

[0056] In a particular embodiment, 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 may not contain a WNT signaling antagonist but may contain an activin signaling agonist. In one embodiment, the first culture environment may not contain an activin signaling agonist but may contain a WNT signaling antagonist. In one embodiment, the first culture environment does not contain either a WNT signaling antagonist or an activin signaling agonist. In one embodiment, the first culture environment may contain both a WNT signaling antagonist and an activin signaling agonist.

[0057] As a non-limiting example, WNT signaling antagonists can be small molecules, cytokines, peptides, or proteins. Examples of small molecule WNT signaling antagonists include N-(6-chloro-1,3-benzothiazole-2-yl)-3-(3,4-dimethoxyphenyl)propenamide (KYO2111), 2-[4-(trifluoromethyl)phenyl]-1,5,7,8-tetrahydrothiopyrano[4,3-d]pyrimidine-4-one (XAV939), 4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl]-N-8-quinolinylbenzamide (IWR-1-endo), and N-(6-methyl-2-benzothiazolyl)-2-[ Examples include, but are not limited to, [3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxyeno[3,2-d]pyrimidine-2-yl]-thio]-aquetamide IWP-4), 2-[[3-(4-fluorophenyl)-3,4,6,7-tetrahydro-4-oxyene[3,2-d]pyrimidine2-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]pyrimidine-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 frizzle-related protein (sFRP), APCDD1, APCDD1L, Draxin, LMBR1L, Notum, SOST / sclerostin, USAG1, and WIF-1.

[0058] As a non-limiting example, activin signaling agonists can be small molecules, cytokines, peptides, or proteins. Examples of activin signaling agonists include, but are not limited to, activin A, 1-(2-cyclopentylidene hydrazide)-heptanediic acid (IDE2), and 1-[2-[(2-carboxyphenyl)methylene]hydrazide]-heptanediic acid (IDE1).

[0059] The PSC population may be exposed to (e.g., cultured) in the first culture environment for approximately 0–120 hours. The duration of exposure to the first culture environment (and the BMP signaling inhibitor), as well as the concentration of the BMP signaling inhibitor, may be important, or absolutely essential, for inducing a population of cells destined to the retinal lineage (or downstream populations such as ocular progenitor cells, optic vascular progenitor cells, and / or immature RPEs). In one embodiment, the PSC population is cultured in the first culture environment for approximately 96 hours or less. In one embodiment, the PSC population is cultured in the first environment for approximately 72 hours or less. In one embodiment, the PSC population is cultured in the first environment for approximately 60 hours or less. In one embodiment, the PSC population is cultured in the first environment for approximately 0–60 hours. In one embodiment, the PSC population is cultured in the first environment for approximately 48 hours, or approximately 48 hours ± 5 hours.

[0060] The first culture medium, which forms part of the first culture environment, may include a basal medium. The basal medium typically contains one or more of the following to support cell growth: amino acids, vitamins, organic and / or inorganic salts, buffers, antioxidants, and energy sources (e.g., carbon). In some embodiments, the basal medium may not contain one or more of the above components and may be supplemented if essential. Numerous commercially available basal media are known, including Dulbecco's Modified Eagle Medium (DMEM), F12, Roswell Park Memorial Institute Medium (RPMI) 1640, Iscove Modified Dulbecco's Medium (IMDM), High DMEM, High DMEM / F-12, Immunocult® brand media, STEMSpan® brand media, and X-VIVO brand media. In one embodiment, the basal medium is an Immunocult® brand media or a STEMSpan® brand media.

[0061] In the first culture environment, after culturing the PSC population under the conditions and duration described herein, a population of cells whose fate has been determined to become the retinal lineage (for example, cells that are no longer PSCs but are on a pathway toward the ectoderm, or are already ectoderm) may be derived.

[0062] The methods of the present disclosure may further include exposing a cell population arising from a first culture environment (e.g., a stage 1 cell population, or a cell population destined to become a retinal lineage) to a second culture environment. Exposure of such cells to the second culture environment may further differentiate them to immature and / or mature RPEs, or along pathways toward them. In one embodiment, the downstream population of cells derived from the population arising from the first culture environment includes a population of ophthalmic progenitor cells.

[0063] The second culture environment may contain the extracellular matrix proteins described above, whether or not they are the same as or different from the first culture environment. In one embodiment, the second culture environment includes a second cell culture medium. In one embodiment, the second culture environment includes both the extracellular matrix (as described above) and the second culture medium.

[0064] The second cell culture medium may also include a basal medium as described above. In one embodiment, the basal medium is an Immunocult® brand medium or a 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 an Immunocult® brand medium, or a STEMSpan® brand medium, or an X-VIVO brand medium.

[0065] The second cell culture medium and its basal medium are appropriately supplemented to differentiate the cell population (fate-determined to the retinal lineage) into immature and / or mature RPE, or toward that direction. In one embodiment, the second cell culture medium contains insulin-like growth factor (e.g., IGF-1 or IGF-2). In one embodiment, the second cell culture medium contains 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 contains basal medium, IGF, and an FGF signaling agonist.

[0066] In one embodiment, the second culture environment and / or the second culture medium contains a BMP signaling inhibitor of the type and concentration described above. In one embodiment, the second culture environment and / or the second culture medium does not contain a BMP signaling inhibitor. In one embodiment, the second culture environment and / or the second culture medium does not contain an exogenously added BMP signaling inhibitor.

[0067] Cell populations exposed to a second culture environment (e.g., those destined to become retinal lineage) can be cultured therein for approximately 0–120 hours. The duration of exposure to the second culture environment (and the presence of BMP signaling inhibitors), as well as the concentration of factors in the second culture medium, may be important, or even absolutely essential, for inducing cell populations destined to become retinal lineage (or downstream populations such as ophthalmic progenitor cells, optic vascular progenitor cells, and / or immature RPEs). The duration of exposure to the second culture environment (and the absence of BMP signaling inhibitors), as well as the absence of BMP signaling inhibitors, may be important, or even absolutely essential, for inducing cell populations destined to become retinal lineage (or downstream populations such as ophthalmic progenitor cells, optic vascular progenitor cells, and / or immature RPEs).

[0068] In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 96 hours or less. In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 72 hours or less. In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 60 hours or less. In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 0 to 60 hours. In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 12 to 60 hours. In one embodiment, the cell population (destined to become the retinal lineage) is cultured in a second culture environment for approximately 48 hours, or approximately 48 hours ± 5 hours.

[0069] After culturing the resulting cell population (destined for the retinal lineage) in a second culture environment under the conditions and for the duration described herein, a progenitor cell population (e.g., an ocular progenitor cell population, or a stage 2 cell population, or a second differentiated cell population) may be induced. Such a population may include all progenitor cells of nerve-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.

[0070] The methods of the present disclosure may further include exposing a cell population arising from a second culture environment (e.g., a stage 2 cell population, or an ophthalmic progenitor cell population) to a third culture environment. Exposure of such cells to the third culture environment may further differentiate them to immature and / or mature RPEs, or along pathways toward them. In one embodiment, the downstream population of cells derived from the population arising from the third culture environment includes an optic vascular progenitor cell population (e.g., a stage 3 cell population, or a third cell population).

[0071] The third culture environment may include extracellular matrix proteins, whether or not they are the same as or different from the first and / or second culture environments, as described above. In one embodiment, the third culture environment includes a third cell culture medium. In one embodiment, the third culture environment includes both the extracellular matrix (as described above) and the third culture medium.

[0072] The third cell culture medium may also include a basal medium as described above. In one embodiment, the basal medium is an Immunocult® brand medium or a 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 an Immunocult® brand medium, or a STEMSpan® brand medium, or an X-VIVO brand medium.

[0073] The third cell culture medium and its basal medium are appropriately supplemented to differentiate the stage 2 cell population (e.g., ophthalmic progenitor cell population) into, or toward, immature and / or mature RPE. In one embodiment, the third cell culture medium contains insulin-like growth factor (e.g., IGF-1 or IGF-2). In one embodiment, the third cell culture medium contains an activin signaling agonist as described above. In one embodiment, the third cell culture medium contains basal medium, IGF, and an FGF signaling agonist.

[0074] A cell population (e.g., ophthalmic progenitor cells) exposed to a third culture environment can be cultured in the third culture environment for about 1 to 10 days. In one embodiment, (ophthalmic) progenitor cells are cultured in the third culture environment for about 1 to 7 days. In one embodiment, (ophthalmic) progenitor cells are cultured in the third culture environment for about 1 to 5 days. In one embodiment, (ophthalmic) progenitor cells are cultured in the third culture environment for about 2 to 5 days. In one embodiment, (ophthalmic) progenitor cells are cultured in the third culture environment for about 2 to 4 days. In one embodiment, (ophthalmic) progenitor cells are cultured in the third culture environment for about 3 or about 4 days.

[0075] After culturing the (ocular field) progenitor cell population in a third culture environment under the conditions and for the duration described herein, a progenitor cell population (e.g., optic vascular progenitor cells, a stage 3 cell population, or a third differentiated cell population) may be derived. Such a population may include dipotent cells such as retinal pigment epithelium and / or neuroretina. Such a cell population may further develop or differentiate into optic vesicles and / or optic cups. Such a cell population may be characterized by the expression of lineage-specific markers such as MITF and OTX2.

[0076] The methods of the present disclosure may further include exposing a cell population arising from a third culture environment (e.g., a stage 3 cell population, or a optic vascular progenitor cell population) to a fourth culture environment. Exposure of such cells to the fourth culture environment may further differentiate them into immature and / or mature RPEs, or along pathways toward them. In one embodiment, the downstream population of cells derived from the population arising from the fourth culture environment includes an immature RPE population.

[0077] In one embodiment, the fourth culture environment includes extracellular matrix proteins, whether or not they are the same as or different from the first and / or second culture environments, as described above. In one embodiment, the fourth culture environment includes a fourth cell culture medium. In one embodiment, the fourth culture environment includes both the extracellular matrix (as described above) and the fourth culture medium.

[0078] The fourth cell culture medium may also include a basal medium as described above. In one embodiment, the basal medium is an Immunocult® brand medium or a 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 an Immunocult® brand medium, or a STEMSpan® brand medium, or an X-VIVO brand medium.

[0079] The fourth cell culture medium and its basal medium are appropriately supplemented to differentiate a population of progenitor cells that will develop into or toward immature and / or mature RPEs (optic vessels). In one embodiment, the fourth cell culture medium comprises one or more of the above-described activin signaling agonists, wnt signaling agonists, and FGF signaling antagonists. In one embodiment, the fourth cell culture medium may further comprise a RHO / ROCK pathway inhibitor. Such a RHO / ROCK pathway inhibitor may be any compound or molecule that inhibits rho kinase or the ROCK pathway.

[0080] As a non-limiting example, WNT signaling agonists can be small molecules, cytokines, peptides, or proteins. Examples of small molecule agonists of WNT signaling include 3-[3-[(acetyloxy)imino]-1,3-dihydro-2H-indole-2-ylidene]-6-bromo-1,3-dihydro-2H-indole-2-one (BIO-acetoxime), 9-bromo-7,12-dihydro-indro[3,2-d][1]benzazepine-6(5H)-one (Kempaolone), 3-[[6-(3-aminophenyl)-7H-pyrrolo[2,3-d]pyrimidine-4-yl]oxy]phenol (TWS119), N6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazole-2-yl)-2-pyrimidadinyl]ethyl]-3-nitro-2,6-pyridinediamine (CHIR98014), 3-(2,4-dichloro-dichlorophenyl) Nyl)-4-(1-methyl-1H-indole-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 Examples include, but are not limited to, -imidazole-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitride (CHIR99021) and 6-bromo-3-[(3E)-1,3-dihydro-3-(hydroxyimino)-2H-indole-2-ylidene]-1,3-dihydro-(3Z)-2H-indole-2-one (BIO).

[0081] As a non-limiting example, FGF signaling antagonists can be small molecules, cytokines, peptides, or proteins. Examples of FGF signaling antagonists include, but are not limited to, 2-[(1,2-dihydro-2-oxo-3H-indole-3-ylidene)methyl]-4-methyl-1H-pyrrole-3-propanoic acid (SU5402) and N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidine-7-yl]-N'-(1,1-dimethylethyl)-urea (PD173074).

[0082] A population of optic vascular progenitor cells (e.g., a stage 3 cell population) exposed to the fourth culture environment can be cultured in the fourth culture environment for about 3 to 12 days. In one embodiment, the resulting progenitor cells are cultured in the fourth culture environment for about 3 to 10 days. In one embodiment, the resulting progenitor cells are cultured in the fourth culture environment for about 5 to 10 days. In one embodiment, the resulting progenitor cells are cultured in the fourth culture environment for about 6 to 9 days. In one embodiment, the resulting progenitor cells are cultured in the fourth culture environment for about 6 to 8 days. In one embodiment, the resulting progenitor cells are cultured in the fourth culture environment for about 6, 7, or 8 days.

[0083] The concentrations of cytokines and small molecules present in the third and fourth culture media may be in the range of 2 to 150 ng / ml or 1 to 15 μM. In one embodiment, the concentrations of cytokines in the third and fourth media may be 50 ng / ml, 75 ng / ml, 100 ng / ml, or 125 ng / ml. In one embodiment, the concentration of cytokines in the third media may be 5 ng / ml, 7.5 ng / ml, 10 ng / ml, or 12.5 ng / mL. In one embodiment, the concentration of small molecules in the fourth media may be 5 μM, 7.5 μM, 10 μM, or 12.5 μM. In one embodiment, the concentration of small molecules in the fourth media may be 1.5, 2.25, 3, or 3.75 μM. In one embodiment, the cytokines present in the third media may be activin A or IGF-1. In one embodiment, the cytokine(s) present in the fourth culture medium may be activin A.

[0084] After culturing the optic vessel (progenitor cell) population in a fourth culture environment under the conditions and for the duration described herein, an immature RPE population (e.g., a stage 4 cell population, or a fourth differentiated cell population) may be derived. The immature RPE population may be destined to become RPE cells. In one embodiment, immature RPE includes cells that have begun to differentiate into or toward mature RPE cells, but have not yet acquired all the characteristics of mature RPE cells. In one embodiment, immature RPE may exhibit certain characteristics, such as cell surface marker expression and morphology, similar to mature RPE cells.

[0085] In one embodiment, or in the embodiments described above, the method of the Disclosure may include dissociating an immature RPE population. In one embodiment, the method of the Disclosure includes dissociating an immature RPE population and reseeding the dissociated cells.

[0086] Immature RPEs dissociated from the substrate may be enriched (e.g., by manual dissociation, particle-based enrichment, or ligand-mediated enrichment) before the dissociated cells are reseeded. In one embodiment, the simple act of dissociating an immature RPE population and reseeding the dissociated cells enriches the immature RPEs. Thus, in one embodiment, the dissociated population of immature RPEs is not enriched by anything other than reseeding the bulk population of dissociated immature RPEs (or a portion thereof, depending on the surface area of ​​the culture surface on which they are reseeded). Therefore, the dissociated population of RPEs does not need to be enriched by manual dissociation, particle-based enrichment, or ligand-mediated enrichment. Without being constrained by theory, immature RPEs may be enriched after dissociation and reseeding by contaminated cells with reduced ability to detach from the substrate or reattach to a new substrate, or they may lose out in competition to immature RPEs for the surface area of ​​the reseeded culture surface.

[0087] In one embodiment, the absence of serum in the culture medium (e.g., concentrated / respraised) reduces or limits the establishment of contaminating cell types (such as neuroretinal cells and / or corneal cells) compared to serum-containing conditions in which dissociated RPEs are reseeded.

[0088] In one embodiment, the reseeding medium (e.g., concentrated and / or maturing medium) contains a RHO / ROCK pathway inhibitor, and such a medium helps to (preferentially) promote the survival / adhesion of PMEL17+ immature RPE compared to contaminated cell types. In one embodiment, the RHO / ROCK pathway inhibitor may be added as a supplement to the concentrated / reseeding / maturing medium. In one embodiment, dissociated immature RPE cells are cultured for 2 to 10 days in concentrated / reseeding / maturing medium containing a RHO / ROCK pathway inhibitor.

[0089] The immature RPE differentiated (and optionally enriched) populations described herein may express characteristic markers. In one embodiment, more than 50% of the immature RPE population expresses premelanosome protein (PMEL17). In one embodiment, more than 60% of the immature RPE population expresses PMEL17. In one embodiment, more than 70% of the immature RPE population expresses PMEL17. In one embodiment, more than 80% of the immature RPE population expresses PMEL17. In one embodiment, more than 90% of the immature RPE population expresses PMEL17. In one embodiment, more than 90-95% of the immature RPE population expresses PMEL17.

[0090] In other embodiments, or in the embodiments described above, the method of the Disclosure may include maturing an immature RPE population by contact with a maturing medium. The maturing medium includes at least a soluble iron source. Examples of soluble iron sources include, but are not limited to, ferric nitrate and ammonium ferric citrate. In one embodiment,

[0091] In one embodiment, the maturation medium includes a basal medium as described above. In one embodiment, the basal medium is an Immunocult® brand medium or a 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 an Immunocult® brand medium, a STEMSpan® brand medium, or an X-VIVO brand medium. Other examples of basal mediums are known and may be substituted for the basal mediums described above.

[0092] The maturation medium may further contain one or more steroid hormones and polyamines. In one embodiment, the maturation medium may further contain progesterone and putrescine. In one embodiment, the maturation medium is non-external or non-animal. In one embodiment, the maturation medium does not contain RHO / ROCK pathway inhibitors.

[0093] Immature RPE populations exposed to a soluble iron source, such as through contact with the maturation medium of this disclosure, can be cultured / matured for approximately 1 to 10 weeks, or approximately 1 to 7 weeks, or approximately 1 to 5 weeks.

[0094] After culturing / maturation of immature RPE populations in the presence of a soluble iron source, mature RPEs may initiate or become more homogeneous in expressing markers of mature RPE. In one embodiment, the marker of mature RPE is RPE65. In one embodiment, more than 50% of mature or aged RPEs express RPE65. In one embodiment, more than 60% of mature or aged RPEs express RPE65. In one embodiment, more than 70% of mature or aged RPEs express RPE65. In one embodiment, more than 80% of mature or aged RPEs express RPE65. In one embodiment, more than 90% of mature or aged RPEs express RPE65. In one embodiment, more than 90-95% of mature or aged RPEs express RPE65.

[0095] The methods of this disclosure may be carried out under conditions free of animal components or free of serum. In one embodiment, the methods of this disclosure are carried out under conditions containing animal components or serum. 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 contain serum. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment contain animal components. In one embodiment, the first culture environment, the second culture environment, the third culture environment, and the fourth culture environment are free of animal components.

[0096] The methods of this disclosure may be carried out under feeder-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-free or do not contain any feeder cells. In one embodiment, the PSC population may be cultured under feeder-free conditions or in the absence of any feeder cells. In one embodiment, the PSC population may be differentiated under feeder-free conditions or in the absence of any feeder cells.

[0097] In one embodiment, one or more of the second culture medium, the third culture medium, and the fourth culture medium lack an exogenously added inhibitor of BMP signaling.

[0098] In one embodiment, one or more of the first culture medium, the second culture medium, the third culture medium, and the fourth culture medium lack nicotinamide. In one embodiment, the second culture medium, the third culture medium, and the fourth culture medium lack nicotinamide. In one embodiment, the third culture medium and / or the fourth culture medium lack nicotinamide.

[0099] In some embodiments, the Disclosure relates to the use of RPE cells obtained by the methods of the Disclosure for the treatment of retinal-related diseases. Such retinal-related diseases may include retinal diseases or disorders or conditions associated with retinal dysfunction, retinal damage, or loss of RPE function. A non-limiting list of conditions that can be treated with RPE cells of the Invention includes age-related macular degeneration (AMD), glaucoma, cataracts, retinitis pigmentosa (RP), Stargardt disease (SD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), Lebers congenital amaurosis, hereditary or acquired macular degeneration, Best's disease, retinal detachment, gynostosis, congenital choroidal atrophy, retinal pigment streaks, high myopia (degenerative myopia), idiopathic neovascular maculopathy, pattern dystrophy, and other dystrophites of RPE, as well as retinal damage resulting from any type of injury.

[0100] Retinal pigment epithelial cells obtained by the method of this disclosure may be used in cell transplantation to generate transplant tissue or to screen for therapeutic compounds or substances that affect the function or metabolism of RPE cells. In one embodiment, RPE cells obtained by the method of this disclosure may be used to treat retinal disorders, diseases, and conditions by transplanting the generated RPE cells into the retina. In one embodiment, RPE cells may be transplanted into the eye of a subject, for example, into a patient with a retinal disorder, disease, or condition. In one embodiment, RPE cells may be transplanted into an animal model of a retinal disorder, disease, or condition.

[0101] In one embodiment, RPE cells obtained by the method of the present disclosure may be cryopreserved as single cells, aggregates, or tissues to generate a cell bank for cell transplantation that may be required in multiple clinical sites. RPE cells may be seeded on a scaffold and cryopreserved to create a tissue bank of transplant-ready tissues. The scaffold may contain any material known in the art and may be maintained during transplantation by parylene or degradable material such as amniotic membrane used in corneal surgery, fibrin hydrogel scaffolds, PLGA, or porcine collagen scaffolds. The culture medium described in the method of the present disclosure may be used as a recovery medium for RPE cells or RPE seeding scaffolds for preparation for transplantation or recovery from transplantation.

[0102] In one embodiment, RPE cells obtained by the method of the present disclosure may be used for toxicity studies such as phototoxicity, toxicity testing, and retinal excitotoxicity, as well as for evaluating the efficacy of therapeutic agents.

[0103] In one embodiment, RPE cells obtained by the method of the present disclosure may be used for large-scale production, large-scale scaling, or automated production of RPE cells, while ensuring high cell purity (e.g., greater than 80%, greater than 90%, or greater than 95%) and functionality for further downstream applications.

[0104] In one embodiment, RPE cells obtained by the method of the present disclosure may be used for gene therapy-related applications focused on treating patients with RPE-related disease mutations, or to optimize gene therapy parameters in vitro before animal or clinical research.

[0105] In one embodiment, RPE cells obtained by the method of this disclosure from patient-derived iPSCs may be used as a diagnostic tool to identify or predict disease-causing mutations.

[0106] In one embodiment, RPE cells obtained by the method of the present disclosure may function as a therapeutic agent for ocular conditions including, but not limited to, retinal diseases or disorders associated with retinal dysfunction, retinal damage, or loss of RPE function. A non-limiting list of conditions that can be treated with RPE cells of the present invention includes age-related macular degeneration (AMD), glaucoma, cataracts, retinitis pigmentosa (RP), Stargardt disease (SD), proliferative vitreoretinopathy (PVR), diabetic retinopathy (DR), Lebers congenital amaurosis, hereditary or acquired macular degeneration, Best disease, retinal detachment, gynostosis, congenital choroidal atrophy, retinal pigment streaks, high myopia (degenerative myopia), idiopathic neovascular maculopathy, pattern dystrophy, and other RPE dystrophy, as well as retinal damage resulting from any type of injury.

[0107] In one embodiment, RPE cells obtained by the method of the present disclosure can be co-cultured with other cell types for various downstream applications. RPE cells can be co-cultured in vitro or assembled with other retinal cell types, including, but not limited to, retinal progenitor cells, photoreceptor progenitor cells or mature photoreceptors (such as rod and cone photoreceptors), the entire retina or developing retina, endothelial cells, fibroblasts or pericytes, and choroid-like tissue. Such co-cultures or assembly can mimic retinal tissue in vivo.

[0108] In one embodiment, the method of the present disclosure may include a conditioned medium obtained after culturing RPE cell progenitor cells, immature RPE, or mature RPE in a first, second, third, fourth, or mature medium as described above. Such a conditioned medium may be further used for the isolation / purification of cellular factors, exosomes, etc., which may be further used for the treatment, prevention, or diagnosis of RPE-related diseases.

[0109] One aspect of this disclosure provides a method for differentiating PSCs to obtain immature and / or mature RPEs. In one embodiment, a method for differentiating PSCs to obtain immature and / or mature RPEs may include: a) culturing a population of PSCs for about 0 to 60 hours in a first culture environment containing extracellular matrix proteins and a BMP signaling inhibitor; b) culturing the cells from step a) for about 0 to 60 hours in a second cell culture environment containing extracellular matrix proteins and one or more of basal medium, IGF, and an FGF signaling agonist; c) culturing the cells from step b) for about 2 to 5 days in a third cell culture environment containing extracellular matrix proteins and one or more of basal medium, IGF, and an activin signaling agonist; and d) culturing the cells from step c) for about 5 to 10 days in a fourth cell culture environment containing extracellular matrix proteins and basal medium and one or more of activin signaling agonists, wnt signaling agonists, and FGF signaling antagonists to induce an immature RPE population.

[0110] In one embodiment, a method for differentiating PSCs to obtain immature and / or mature RPEs may further include dissociating an immature retinal pigment epithelial cell population and reseeding the dissociated cells to enrich the immature retinal pigment epithelial cells. In one embodiment, the dissociated population of immature retinal pigment epithelial cells is not enriched by manual dissociation, particle-based enrichment, or ligand-mediated enrichment.

[0111] In one embodiment, a method for differentiating PSCs to obtain immature and / or mature RPEs may further include maturing an immature retinal pigment epithelial cell population for about 1 to 5 weeks. In one embodiment, the immature retinal pigment epithelial cell population is matured by contact with a maturing medium containing a basal medium and at least a soluble iron source. In one embodiment, the maturing medium further includes one or more of steroid hormones and polyamines.

[0112] In one embodiment, the components of a method for differentiating PSCs to obtain immature and / or mature RPEs may be as described above.

[0113] In another embodiment, the method of the present disclosure may include culturing a population of PSCs under non-adherent conditions to produce three-dimensional retinal organoids. Such a method for producing three-dimensional retinal organoids may further include differentiating the cell population that has been destined to the retinal lineage (essentially as described above). Briefly, the PSC population may be exposed for about 0 to 60 hours to induce the resulting cell population (e.g., destined to the retinal lineage) by exposure to a first culture environment containing extracellular matrix proteins and BMP signaling inhibitors. The resulting cell population (e.g., destined to the retinal lineage) may then be exposed for about 12 to 60 hours to induce a further resulting (ocular) progenitor cell population (essentially as described above). The further resulting (ocular) progenitor cell population may then be exposed for about 2 to 5 days to a third culture environment (essentially as described above).

[0114] Subsequently, cells obtained after exposure to the third culture environment for approximately 2–5 days (essentially as described above) may be exposed to neuroretinal / RPE induction medium for 4–10 days to form aggregates containing different domains of neuroretina and / or RPE. These aggregates containing different domains of neuroretina may be detached / dissociated from the culture surface via a monolayer, for example by cutting / scratching / etching a grid pattern with a pipette tip, releasing a “sheet” or retinal tissue. The resulting retinal tissue may then be cultured in retinal organoid formation medium for approximately 2–6 days under non-adhesion conditions to form retinal organoids containing neuroretina, which can be further matured in retinal organoid maturation medium (under non-adhesion conditions) for 30–200 days.

[0115] Neuroretinal / RPE induction medium may contain (essentially as described above) a basal medium, as well as one or more of progesterone, putrescine, transferrin, and heparin. Neuroretinal / RPE induction medium may further contain members of the bone morphogenesis protein (BMP) family, preferably BMP-4.

[0116] Retinal organoid formation medium may include the basal medium described above, as well as one or more additional supplements such as putrescine, insulin, or transferrin. Retinal organoid maturation medium may include the basal medium described above, as well as one or more additional supplements added at different stages of retinal organoid maturation. Such additional supplements in retinal organoid maturation medium may include serum, retinoic acid pathway activators, retinoic acid receptor activators, retinoic acid agonists, taurine, lipids, or N2 supplement-A.

[0117] The first culture environment, the second culture environment, and / or the third culture environment for generating retinal organoids may contain nicotinamide.

[0118] In one embodiment, a population of PSCs may be seeded in a microwell device. In one embodiment, a population of PSCs may be seeded in an uncoated microwell device to obtain 3D spheroids or aggregates of uniform size of PSCs. Such 3D spheroids may be exposed sequentially to the first, second, and third culture environments as described above. In one embodiment, the 3D spheroids or aggregates of PSCs are seeded on a plate coated with an extracellular matrix protein (e.g., Matrigel, vitronectin, or Synthemax(II)) before exposure to the first, second, and third culture environments.

[0119] Cell populations that develop after exposure to the third culture environment for approximately 2–5 days may then be exposed to neuroretinal / RPE induction medium for 4–10 days (essentially as described above) to form aggregates containing different domains of neuroretina and / or RPE. Once aggregates containing different domains of neuroretina and / or RPE are formed, the grid-like pattern can be separated via a monolayer, for example by the tip of a pipette, and the released "sheets" or retinal tissue may self-organize in a suitable medium (and in the absence of extracellular matrix proteins) to eventually develop into retinal organoids in suspension.

[0120] In one embodiment, the retinal organoid produced by the method of the present disclosure includes retinal progenitor cells or retinal progenitor cells that produce all cell types of neuroretina, as well as cells that differentiate into RPE. The cell types of neuroretina may include photoreceptor progenitor cells, retinal progenitor cells, retinal ganglion cells, retinal neuron photoreceptor progenitor cells, rod and cone photoreceptor cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and horizontal cells.

[0121] kit In another aspect of this disclosure, a culture medium or kit is provided which may include instructions for performing the differentiation methods described herein (e.g., stepwise differentiation of progressively more committed progenitor cells into immature RPEs and / or mature RPEs). The kits described herein may further include, but are not limited to, other materials such as supplements, buffers, diluents, solutions, and accompanying documentation, which include instructions for performing any of the methods described herein.

[0122] In another aspect of this disclosure, a culture medium or kit is provided for carrying out a method intended herein, such as differentiating developmentally early progenitor cells into immature RPEs (e.g., immature RPEs derived from PSCs). In other words, a culture medium or kit is provided for carrying out a method of stepwise differentiation of progenitor cells into immature RPEs and / or more committed mature RPEs.

[0123] Such a kit may include the first, second, third, and fourth culture media as described above. The kit may further include extracellular matrix proteins as described above. In one embodiment, developmentally early progenitor cells may include a population of cells destined for the retinal lineage, an ocular progenitor cell population, and / or an optic vascular progenitor cell population. In one embodiment, developmentally early progenitor cells may be derived from PSCs as described above. In one embodiment, the first culture medium may include a BMP signaling inhibitor as described above. In one embodiment, the first culture medium may not include either or both of i) a direct wnt signaling antagonist and ii) an activin signaling agonist as described above. In one embodiment, the second cell culture medium may include the basal medium as described above, as well as one or more IGFs and an FGF signaling agonist. In one embodiment, the third cell culture medium may include the basal medium as described above, as well as one or more IGFs and an activin signaling agonist. In one embodiment, the fourth cell culture medium comprises a basal medium and one or more of the above-mentioned activin signaling agonists, wnt signaling agonists, and FGF signaling antagonists. The duration of exposure to the first, second, third, and fourth culture media, and the concentrations of the components of the first, second, third, and fourth culture media, may be as described above.

[0124] In another aspect of the present disclosure, a medium or kit is provided for differentiating developmentally early progenitor cells into mature RPEs (e.g., PSC-derived RPEs), which may comprise the first, second, third, fourth, and maturation media as described above. The kit may further comprise extracellular matrix proteins as described above. In one embodiment, the maturation medium comprises the basal medium and at least a soluble iron source as described above. In one embodiment, the maturation medium further comprises one or more steroid hormones and polyamines as described above.

[0125] In another aspect of this disclosure, a medium or kit for enriching immature RPEs (e.g., PSC-derived RPEs) is provided, which may comprise the first, second, third, fourth, and enrichment / resowing culture media as described above. The kit may further comprise extracellular matrix proteins as described above. In one embodiment, dissociating an immature RPE population and reseeding the dissociated cells enriches the immature RPEs. In one embodiment, the dissociated population of RPEs does not need to be enriched by manual dissociation, particle-based enrichment, or ligand-mediated enrichment. In one embodiment, the kit for enriching immature RPEs may comprise instructions for dissociating an immature RPE population and reseeding the dissociated cells to enrich the immature RPEs. In one embodiment, the enrichment / resowing medium may be serum-free. In one embodiment, the enrichment / resowing medium may comprise a RHO / ROCK pathway inhibitor as described above, or a RHO / ROCK pathway inhibitor added as a supplement.

[0126] In another aspect of the present disclosure, a culture medium or kit for producing three-dimensional retinal organoids (e.g., PSC-derived retinal organoids) is provided, which may comprise, as described above, the first, second, and third intermediate, neuroretinal / RPE induction medium, retinal organoid formation medium, and optionally retinal organoid maturation medium. The kit may further comprise extracellular matrix proteins as described above. The kit may further comprise a microwell device as described above.

[0127] The following non-limiting examples illustrate the present disclosure. [Examples]

[0128] Example 1: Maintenance and differentiation of PSCs Human pluripotent stem cells (PSCs) were maintained in mTeSR® branded media such as mTeSR® Plus (STEMCELL Technologies) with either Corning® Matrigel® or Vitronectin (Thermo Fisher Scientific). PSCs were passaged as described by the manufacturers, but generally, the cultures were ready for passage when the majority of the colonies were large, compact, and had dense, multilayered centers (e.g., approximately 70–80% confluent and less than 10% differentiated). PSCs were divided in a 1 / 10 ratio and seeded to initiate RPE differentiation. Various iPSC strains (WLS-1C, SCTi003-A, and STiPS-M001) and ESC strain (H9) were used in downstream differentiation experiments. Whether maintaining or differentiating hPSCs, cells could be seeded as clamps or single cells.

[0129] Example 2: Selected Validation Protocol Although they are at different stages of differentiation, typically immature RPE at day 14 and mature RPE at day 49, the cells can be analyzed by flow cytometry. Briefly, 0.1 × 10⁻⁶ 6Each RPE was aliquoted into each well of a 96-well plate, centrifuged at 500xg for 3 minutes, and the supernatant was removed. The pellet was resuspended in 100 μL of Zombie Violet Fixable Viability Dye (Biolegend) in D-PBS (1 / 1000 stock) and incubated in the dark at room temperature for 10 minutes. 100 μL of D-PBS was added to each well, centrifuged at 500xg for 3 minutes, and the supernatant was removed. The pellet was resuspended in 200 μL of 4% PFA and incubated in the dark at room temperature for 15 minutes. The cells were washed in 200 μL of buffer, the pellet was resuspended in 200 μL of 0.2% saponin, incubated in the dark at room temperature for 15 minutes, centrifuged at 500xg for 3 minutes, and the supernatant was removed. Cells were resuspended in 0.2% saponin with either 100 μL of anti-PMEL17 (Biolegend AF647, Clone HMB-45) or anti-RPE65 (Novus Biologicals Clone: ​​401.8B11.3D9) antibody and incubated in the dark at room temperature for 30–60 minutes. After incubation, cells were washed once or twice with 100 μL or 200 μL of 0.2% saponin. Depending on the primary antibody used, secondary antibody staining may be required. For PMEL17 staining, 200 μL of 0.2% saponin was added to each well, resuspended, and analyzed by fluorescence-activated cell sorting (FACS). For RPE65 staining, 100 μL of secondary donkey anti-mouse antibody (Jackson Immuno, AF647 Fab2) in 0.2% saponin was added to each well and incubated in the dark at room temperature for 30–60 minutes. After incubation, the cells were washed once or twice as described above. The cells were then prepared for analysis by flow cytometry.

[0130] Although at different differentiation stages, the level of RPE pigmentation in cells at mature RPE, typically on day 49, can be analyzed by a melanin content assay. To prepare a standard melanin curve, synthetic melanin (Sigma-Aldrich) was reconstituted in 4.26 mM NaOH, and three concentration ranges of 0, 7.825, 15.65, 31.3, 62.5, 125, 250, and 500 μg / mL were prepared in 96-well tissue culture plates. Total 4 × 10 5 RPE was added to each well of a 96-well u-bottom plate and centrifuged at 500xg for 3 minutes. After removing the supernatant, the cells were resuspended in 100 μL of D-PBS and transferred to a 96-well tissue culture plate. The RPE was centrifuged at 300xg for 3 minutes and the absorbance was measured at 490 nm. Melanin content was calculated by comparison with a synthetic standard.

[0131] Example 3: Retinal pigment epithelium (RPE) differentiation i) Generation of cells whose fate has been determined to become retinal lineage and ocular progenitor cells (Day 0-4) The PSC was maintained as described in Example 1, and the culture on day 0 (approximately 60-80% confluent) was washed twice with 1 ml of sterile 0.5 mM EDTA, after which 1 ml of the first differentiation medium (medium A) was added to each well of a 6-well plate. Such a medium may alternatively be referred to as the first culture medium, and BMP signaling inhibitors (BMPi + ) may include Wnt signaling inhibitors (WNTi - and activin A (A - ) may not contain. Next, the PSC is ground and crushed several times, then approximately 1.2 × 10 5 Approximately 100 μL of clamp cells, each containing one cell, were seeded into one well of a 12-well plate coated with Matrigel® or vitronectin in 1 ml of medium A to initiate differentiation. The plates were shaken to disperse the clamp cells, and the plates were incubated at 5% CO2 and 37°C. The medium was changed on day 1.

[0132] On the second day, Medium A was removed, and 1 ml of a second differentiation medium (Medium B) was added per well. This may alternatively be referred to as the second culture medium and may contain an inhibitor of BMP signaling (BMPi - ), but may not contain an inhibitor of Wnt signaling (WNTi - ) and activin A (A - ). The plate was incubated at 5% CO2 and 37 °C. At this stage, the culture should have clumps showing a rosette-like morphology as shown in Figure 1A.

[0133] ii) Generation of angiogenic precursor cells (days 4 - 7) On the fourth day, the medium was removed, and 1 ml of a third differentiation medium (Medium C) was added per well. Such a medium may alternatively be referred to as the third culture medium and may contain activin A (A + ), but may not contain an inhibitor of BMP signaling (BMPi - ) or an inhibitor of Wnt signaling (WNTi - ). The plate was incubated at 5% CO2 and 37 °C. Preferably, the culture should reach approximately 100% confluence within the next 1 - 2 days.

[0134] On the sixth day, the medium was changed, and the plate was incubated at 5% CO2 and 37 °C for an additional day. An exemplary day 7 culture is shown in Figure 1A.

[0135] iii) Generation of immature retinal pigment epithelial (RPE) cells (days 7 - 14) On the seventh day, the medium was replaced with 1 ml of a fourth differentiation medium (Medium D) per well, and the medium was changed on days 9, 11, and 13. Such a medium may alternatively be referred to as the fourth culture medium and may contain activin A (A + ) and a wnt agonist, but may not contain an inhibitor of BMP signaling (BMPi - ) and / or an inhibitor of Wnt signaling (WNTi - ). The plate was incubated at 5% CO2 and 37 °C. An exemplary uniform monolayer of polygonal immature RPE on day 14 is shown in Figure 1A.

[0136] iv) Passaging and maturation of RPE (day 14, day 49) On day 14, the culture medium was removed, washed twice with 0.5 ml of D-PBS, and contacted with 0.5 ml of trypsin-based enzyme solution. The monolayer was detached using a cell lifter, pulverized several times, and the single-cell suspension containing immature RPE was transferred to a tube and increased to a maximum volume of 10 ml in basal medium containing progesterone, putrescine, and soluble iron ("+3F"). The filtrate was centrifuged at 300xg for 5 minutes through a 70 μm cell strainer, and the pellet was resuspended in 4 ml of basal medium +3F (further containing RHO / ROCK pathway inhibitors). Cell counting was performed by conventional methods, resulting in 1 × 10⁶ cells. 5 cells / cm 2 Cells were seeded on plates coated with Matrigel® / vitronectin (as prepared according to the manufacturer's instructions). Before seeding, cells may be analyzed by flow cytometry as described in Example 2. Maturation may begin when more than approximately 50% of cells are positive for PMEL17. On day 14, cultures with more than 50% PMEL17 expression can be reasonably expected to enrich to more than 80%, and potentially more than 90%, in the enrichment passaging described above. Therefore, the RPE enrichment described herein does not involve manual dissociation, magnetically activated cell sorting (MACS), or separation methods using magnetic particles.

[0137] The seeded cells were incubated in 5% CO2 at 37°C, with 4 mL of medium changed on days 16 and 18. On day 21 and thereafter, the cells were cultured in basal medium + 3F (e.g., mature medium) with medium changes every 3-4 days until subculturing on day 49. Figure 2 shows the cobblestone-like morphology and the formation of visible intercellular or tight junctions observed on days 18, 21, 28, and 42. On day 49, a uniform monolayer of colored RPE with visible tight junctions should be observed, as shown in Figure 2.

[0138] On day 49, the cells were washed twice with 1 ml of D-PBS, and then 1 ml of trypsin-based enzyme solution was added to each well. The monolayer was detached using a cell lifter, pulverized to produce a single-cell suspension containing mature RPE, transferred to a tube, and increased to a maximum volume of 10 ml in basal medium + 3F. The filtrate was centrifuged at 300xg for 5 minutes through a 70 μm cell strainer, and the pellet was resuspended in 4 ml of basal medium + 3F. Cell counting was performed by conventional methods. 1 × 10⁶ cells were suspended in 4 ml of basal medium + 3F. 5 cells / cm 2 Cells were seeded onto plates coated with Matrigel® / vitronectin (as prepared according to the manufacturer's instructions). Prior to seeding, the cell suspension may be analyzed by flow cytometry, as described in Example 2. At this stage, the majority of cells (e.g., 90–95%) may be positive for PMEL17 and / or RPE65 (Figure 3).

[0139] Example 4: Characterization of mature RPE Four different hPSC strains were essentially differentiated as described in Example 3, and the cells at day 49 were evaluated for maturity through marker expression, growth factor secretion, barrier function, ability to phagocytose the outer segment of photoreceptors, and melanin production.

[0140] Mature RPE cells derived from four different PSC strains were analyzed by flow cytometry on day 49 for PMEL17 and RPE65 expression (essentially as described in Example 2) (Figure 3A). 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 cells on day 49 expressed the maturation marker RPE65.

[0141] Cells that are at different differentiation stages but are typically immature RPEs on day 14 can be evaluated for apical and basal secretion of vascular endothelial growth factor (VEGF) and pigment epithelial-derived factor (PEDF) using commercially available VEGF (Thermo Fisher) and PEDF (Abcam) ELISA kits. Briefly, cells were washed twice with 0.5 ml of D-PBS, and then 0.5 ml of trypsin-based enzyme solution was added to each well. The monolayer was detached using a cell lifter and pulverized to obtain a single cell suspension containing immature RPEs, which was then divided into up to 10 ml of basal medium and RPE maturation medium containing progesterone, putrescine, and soluble iron ("+3F"). This was passed through a 70 μm cell strainer to filter out RPE tissue or contaminated cells or tissues. The filtrate was resuspended in 4 ml of RPE maturation medium further containing a RHO / ROCK pathway inhibitor. Approximately 4 × 10 5 The cells were seeded at the apical end of cell culture inserts (0.4 μm, 12 mm PET, Sterlitech) pre-coated with either Corning® Matrigel® or Vitronectin. Approximately 0.5 ml and 1.5 ml of culture medium were added to 37 cells. 0 During incubation in 1C and 5% CO2, the apical and basal sides of the insert were maintained, respectively. On days 16 and 18, the media from the basal and apical sides were changed. On day 21 and thereafter, the RPE was maintained in mature medium without RHO / ROCK pathway inhibitors, and changed every 3-4 days until days 46-47. On day 49 or 50, 20 μL of acclimatized medium was collected from the apical and basal chambers and analyzed by ELISA essentially according to the manufacturer's instructions.

[0142] PEDF ELISA was performed in a similar manner to the VEGF ELISA protocol, with a few modifications, as described above. On day 49 or 50, 10 μL of conditioned medium was collected from the apical and basal chambers, and the conditioned medium was diluted 100-fold in RPE maturation medium and further diluted in sample dilution buffer to achieve a final dilution of 2000-fold. The samples were then run with minor modifications according to the manufacturer's instructions. The results showed that mature RPE cells exhibited increased basal secretion of VEGF in all cell lines tested (Figure 3B) and increased apical secretion of PEDF in all cell lines tested (Figure 3C).

[0143] Cells that are at different differentiation stages but are typically immature RPEs on day 14 can be evaluated for barrier function by measuring transepithelial electrical 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 of D-PBS, and contacted with 0.5 ml of trypsin-based enzyme solution. The monolayer was detached using a cell lifter, pulverized, and the single-cell suspension containing immature RPEs was resuspended in 10 ml of RPE maturation medium, passed through a 70 μm cell strainer, and further centrifuged. The cell pellet was resuspended in 4 ml of RPE maturation medium containing a RHO / ROCK pathway inhibitor and approximately 4 × 10⁶ 5Cells were seeded at the apical end of cell culture inserts (0.4 μm, 12 mm PET, Sterlitech) pre-coated with Corning® Matrigel®. One blank cell culture insert coated with Corning® Matrigel® was used as a control. Approximately 0.5 ml and 1.5 ml of culture medium were maintained in the apical and basal chambers during incubation at 37°C and 5% CO2, with the medium changed on days 16 and 18. On day 21 and thereafter, RPE was maintained in RPE maturation medium without RHO / ROCK pathway inhibitors, and changed every 3-4 days until days 46-47. After allowing the cells grown in the inserts to stand at room temperature for 10-15 minutes, the TEER was measured. The TEER values ​​of differentiated cells on day 49 were measured using an Epithelial Voltohmmeter EVOM2® equipped with a chopstick electrode (World Precision Instruments) sterilized in 70% ethanol. The electrodes were immersed in the apical and basal chambers, and electrical measurements were recorded for each well. The TEER value was subtracted from the blank control and multiplied by the insertion area coefficient. Mature RPE cells from all four hPSC strains tested measured 200 Ω × cm. 2 The cells showed TEER values ​​exceeding [value] (Figure 3D). These results suggest that the cells on day 49 form tight junctions and sufficient barriers to enable polarization and polar secretion of factors such as PEDF and VEGF. The barrier function could be maintained for a further 4 weeks (data not shown).

[0144] Cells at different differentiation stages but typically mature (at least 77-day) RPEs can be evaluated for their ability to phagocytose the outer segment (POS) of photoreceptors. 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 on a rotating platform at room temperature for 1 hour, protected from light. The unresuspended FITC solids were then centrifuged at over 3000 g, and the pellet was resuspended in 5 mL of DMEM / F12 medium. While rotating, the FITC isomer I solution was rotated approximately 200 × 10⁻⁶ times. 6 The POS was slowly added, and the combination was incubated on a rotating platform at room temperature for 1.5 hours, protected from light. The pellet of labeled POS was resuspended in 1.5 ml of DMEM / F12, pelletized again, washed to remove any non-conjugated FITC isomer I, and repeated. The FITC-conjugated POS was then added to a final volume of 5 mL of DMEM / F12 and 12 × 10⁻¹⁶ 12 × 10⁻¹⁶ POS. 6 Aliquots were prepared for the POS / vials, the vials were centrifuged at over 3000 g, and the pellets were frozen at -80°C. In the RPE POS assay, immature RPE cells on day 14 were matured in tissue culture plates or on 12 mm cell culture inserts until at least day 49. Approximately 1 × 10⁶ cells were matured in 12-well tissue culture plates. 5 cells / cm 2 The seeds were seeded in wells pre-coated with Corning® Matrigel® and incubated at 37°C with 2 mL of maturation medium in 5% CO2, changing every 3-4 days until subculturing on day 49. On day 49, the frozen FITC-POS vials were thawed on ice and subculturized in 10 × 10 units per 2 mL of RPE maturation medium. 6FITC-POS was added. Baseline fluorescence was obtained using untreated cells without POS or cells containing FITC-POS in the presence of 50 μg / mL anti-αvβ5. After 16 hours of incubation, the medium was removed and the wells were rinsed 3-5 times with 2 mL of D-PBS to remove any unbound FITC-POS. 0.5 mL of trypsin-based enzyme solution was added to each well and incubated at 37°C for 10 minutes. The monolayer was detached using a cell lifter, pulverized, and the single cell suspension was resuspended in 10 mL of RPE maturation medium, passed through a 70 μm cell strainer, and further centrifuged. The cell pellet was resuspended in 0.4% trypan blue in D-PBS to quench bound FITC-POS, the volume was adjusted to a maximum of 10 mL in RPE maturation medium, and centrifuged at 300 × g for 5 minutes. The pellet was resuspended in 1 mL of buffer using 1:1000 Hoechst 33342, and then flow cytometry was performed as described in Example 2 to quantify FITC-POS digestion.

[0145] The data show that cells stain positively for FITC only upon the addition of FITC-conjugated POS (Figure 3E). Untreated cells without POS (vehicle) represent the majority of non-phagocytic cells (99.52%) that have not internalized POS. Very low cell populations (0.25% and 0.19% in H9 and 1C-derived RPEs, respectively) showed POS that had adhered to the cells but been released by proteolysis, which we refer to as "unbound POS". Overall, the data suggest that the majority of RPEs from 1C and H9 are capable of phagocytizing POS.

[0146] Mature RPE cells derived from four different PSC lines were imaged on day 49 (P0). Representative microscopic images show significant levels of pigmentation between cells differentiated from each hPSC cell line (Figure 3F).

[0147] Example 5: Effects of culture medium composition on differentiation, purity, and pigmentation of RPE The cells were cultured and differentiated essentially as described in Example 3, except that i) the cytokine and small molecule concentrations in the third and fourth culture media, and ii) the differences in media composition, including different basal media for RPE maturation, were investigated.

[0148] As used in Example 3, the concentrations of cytokines and small molecules present in the third and fourth culture media were varied to evaluate their effects on the differentiation of immature and mature RPEs. The concentrations in the third medium were tested at 0.5x, 0.75x, 1x, and 1.25x, and the concentrations in the fourth medium were matched at 0.5x, 0.75x, 1x, and 1.25x. The results showed similar efficiency in immature RPE generation in both H9 and M001 for all tested conditions (Figure 4A). However, at lower concentrations of cytokines and small molecules (e.g., 0.5x and 0.75x), the level of RPE pigmentation appeared to be reduced (Figure 4B). This was further confirmed by a melanin content assay performed as described in Example 2 (Figure 4C). The results also showed comparable RPE purity between differentiated H9 and M001 for all tested conditions, as assessed by PMEL17 flow cytometry (Figure 4D).

[0149] To test the effects of different basal media during the maturation stage, immature RPEs derived from WLS-1C at day 14 were further cultured in three different basal media: X-VIVO 10 (Lonza) as a control; and two different similar basal media prepared by STEMCELL Technologies (basal media under the StemSpan® and ImmunoCult® brands), essentially as described in Example 3. Compared to X-VIVO-10, cells matured in STEMCELL brand media appeared to show reduced pigmentation. However, supplementation with sources of progesterone, putrescine, and soluble iron (e.g., ferric ammonium citrate or ferric nitrate) restored pigmentation among cells matured in STEMCELL brand media (Figure 5A). These findings were further confirmed by a melanin content assay (Figure 5B). Furthermore, both ferric nitrate and ferric ammonium citrate can induce increased levels of melanin production with increasing concentrations of both iron sources (10 μM, 25 μM, and 50 μM), as observed through bright-field microscopy and melanin content assays (Figures 5C and D). The results indicate that supplementation of the basal medium as described above can result in higher levels of melanin formation than in the control medium.

[0150] Example 6: High differentiation efficiency of clamp and single-cell passaged hPSCs PSCs derived from four cell lines, 1C, M001, 3A, and H9, were collected at 25,000 / cm³. 2 The cells were seeded as single cells or as clamp cells (seeding ratio of 1 / 10) and differentiated under the conditions described in Example 3. Differentiation was evaluated by flow cytometry analysis of PMEL17 marker expression (Figure 6). Both conditions resulted in significant levels of PMEL17 expression, and a slightly higher efficiency (approximately 94%) was observed in single-cell passaged cells compared to cells differentiated from clamp passaged PSCs.

[0151] Example 7: Three-step RPE differentiation protocol As an alternative to the four-step differentiation protocol described in Example 3, RPE differentiation was performed in three steps. In the first step, hPSCs were differentiated in a medium containing a BMP pathway inhibitor (Noggin) and a wnt pathway inhibitor (DKK1) from day 0 to 4. In the second step, cells were cultured in a medium containing activin-A from day 4 to 8. In the third step, cells were cultured in a medium containing activin-A, an FGFR-1 inhibitor, and a wnt agonist from day 8 to 14.

[0152] Differentiated cells were analyzed for RPE65 and MITF expression using microscopy and flow cytometry. Immature (day 14) and mature (day 49) RPE cells exhibited a polygonal morphology (Figure 7A). Furthermore, flow cytometry analysis of day 49 cells quantified the expression of MITF (optic vesicle and RPE marker) and RPE65 (RPE maturation marker) in RPE cells derived from WLS-1C and H9. The results showed that RPE cells derived from 1C had 99.28% RPE65-positive cells and 81.92% MITF-positive cells, while RPE cells derived from H9 had 98.36% RPE65-positive cells and 75.51% MITF-positive cells (Figure 7B). The results suggest that high differentiation and RPE maturation efficiency was achieved with the three-step differentiation protocol. However, when evaluating PMEL17 expression between WLS-1C-derived cells and H9-derived cells on day 14, greater variability was observed in the three-step differentiation protocol compared to the four-step protocol of Example 3 (Figure 7C).

[0153] Interestingly, the combined use of wnt and BMP signaling pathway inhibitors in the culture medium from days 0 to 4 resulted in higher cell death on day 8 (data not shown). When the BMP pathway inhibitor (Noggin) and wnt pathway inhibitor (DKK1) were replaced with the alternative BMP pathway inhibitor LDN-193189 and the alternative wnt pathway inhibitor IWP2, the RPE differentiation efficiency, as assessed by PMEL17 marker expression, was found to be lower in H9-derived RPE. Flow cytometry analysis of PMEL17 expression showed that H9-derived cells yielded 61.99% PMEL17-positive cells compared to 91.04% PMEL17-positive cells in 1C-derived RPE (Figure 7D).

[0154] Example 8: 5-step RPE differentiation protocol As an alternative to the four-step and three-step differentiation protocols described in Examples 3 and 7, respectively, RPE differentiation was performed in five steps. In the first step, hPSCs were differentiated in a medium containing 50 ng / ml of Noggin, DKK-1, and IGF-1 on days 0-2. In the second step, cells were cultured in a medium containing 10 ng / ml of Noggin, DKK-1, IGF-1, and FGFb on days 2-4. In the third step, cells were cultured in a medium containing activin A, DKK-1, and IGF-1 on days 4-6. In the fourth step, cells were cultured in a medium containing activin A and an FGFR-1 inhibitor on days 6-8. In the fifth step, cells were cultured in a medium containing activin A, an FGFR-1 inhibitor, and a wnt signaling agonist on days 8-14. The effect on RPE differentiation was evaluated by removing or replacing individual culture medium components with substitute factors, and by flow cytometry assessment 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 five-step differentiation protocol described above; (ii) removal of DKK1; (iii) removal of FGFb; (iv) removal of IGF-1; and (v) replacement of Noggin with LDN-193189. The results (Figure 8) showed that the removal of DKK1 or IGF-1 did not adversely affect RPE differentiation, as assessed by flow cytometry of the percentage of immature RPEs at day 14. The results suggest that RPE differentiation can be achieved in the absence of DKK1.

[0155] Example 9: Comparison of differentiation efficiency of three differentiation protocols The efficiencies of the five-step differentiation protocol from Example 8, the three-step differentiation protocol from Example 7, and the four-step differentiation protocol from Example 3 were compared. Flow cytometry analysis of PMEL17 expression in immature RPE at day 14 is summarized in the box plot in Figure 9. Each protocol showed an average efficiency of over 80%, but the four-step differentiation protocol showed the highest efficiency and was the most robust (Figure 9).

[0156] Example 10: Effect of BMP inhibition on RPE differentiation efficiency Cells were cultured and differentiated essentially as described in Example 3, but the first medium was modified to include different types and concentrations of the following BMP signaling inhibitors: (i) no BMP inhibition, (ii) Noggin (12.5 ng / ml, 25 ng / ml, and 100 ng / ml), (iii) Dorsomorphin (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).

[0157] The positivity rate of immature RPE cells at day 14 from 3A and H9 cell lines was analyzed for PMEL17 expression by flow cytometry (Figure 10A). In addition, the yield of immature RPE per well was calculated using a hemocytometer (Figure 10B). The results generally showed improved RPE differentiation at lower concentrations for all BMP inhibitors tested (Figure 10A). Specifically, 0.625 μM or 1.25 μM dolsomorphine and 25 nM or 100 nM LDN-193189 produced the most consistent RPE differentiation efficiency (e.g., over 80%) among the hPSC cell lines tested. The absence of any inhibitor of BMP signaling was observed to result in a high percentage of PMEL17+ immature RPE at day 14, and this absence seemed to correlate with, for example, increased corneal contamination during maturation (data not shown).

[0158] Example 11: Generation and Characteristics of Retinal Organoids PSCs, maintained essentially as described in Example 1, were formed into aggregates in 24-well Aggrewell® 800 (STEMCELL Technologies) plates of mTeSR® 1 or mTeSR® Plus according to the manufacturer's protocol. The aggregates were harvested from the AggreWell® plates, seeded onto Matrigel® coated plates, and cultured for up to day 6, essentially as described in Example 3. Subsequently, the cells were cultured for 3 days in neuroretinal / RPE induction medium containing BMP4, followed by 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 neuroretina and / or RPE. The aggregates containing different domains of neuroretina were then dissociated by cutting through a monolayer into a grid-like pattern using a pipette tip or the like, releasing sheets of retinal tissue. Next, the retinal-like tissue was cultured in retinal organoid formation medium for 4 days, from day 14 to day 18, under non-adherent conditions, to form retinal organoids including the neural retina. Then, the retinal organoids were matured in retinal organoid maturation medium from day 18 to day 150.

[0159] Figure 11A shows the generation of retinal organoids at different developmental stages in the protocol described above, including homogeneous PSC aggregates formed on day 0 in AggreWell® 800, differentiated aggregates adhered to a plate on day 1, cultures on day 14 containing different domains of neuroretina and RPE, retinal structures on day 18 with a bright boundary of the neuroretinal phase, retinal organoids on day 27, and maturation of retinal organoids on days 50, 96, and 123.

[0160] Figure 11B shows immunohistochemical staining to characterize retinal organoid cells at various 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) were stained with SNCG antibody and correctly localized within the retinal organoids. Observation of RGCs generated cellular processes in the neuroblast layer stained with VSX2 antibody (i). The generated retinal organoids contained amacrine cells (AP2α) and horizontal cells (Prox1), further showing that the amacrine cells were more organized toward the RGC layer (ii). The generated retinal organoids were also shown to involve differentiation into retinal progenitor cells (RPCs) and peripherally organized photoreceptor progenitor cells (PRPs) by staining with OTX2 and CRX antibodies (iii). Staining at day 96 with OTX2 and CRX antibodies further showed that photoreceptor cells increased in density along the periphery of the retinal organoids and settled in the developing outer nucleus layer (iv). Staining data from day 96 also showed that the generated retinal organoids contained amacrine cells (AP2α) and horizontal cells (Prox1), with the amacrine cells being more organized toward the RGC layer (v). RGC nerve fiber-like bundles were also observed on day 96 (Prox1 and AP2α) (vi). Similar tissue in retinal organoids containing amacrine cells and horizontal cells was also observed on day 123 (vii).

Claims

1. A method for differentiating a group of cells whose fate has been determined to become the retinal lineage, wherein the method is a) To provide a population of pluripotent stem cells (PSCs), b) Exposing the PSC population to a first culture environment containing an inhibitor of extracellular matrix proteins and bone morphogenesis protein (BMP) signaling, c) Culturing the PSC population in the first culture environment for approximately 0 to 60 hours, d) The method comprising inducing the cell population whose fate has been determined to become the retinal lineage.

2. The method according to claim 1, wherein the first culture environment does not contain i) an antagonist of direct wnt signaling, or ii) an agonist of activin signaling, or both.

3. The method according to claim 1 or 2, wherein the extracellular matrix protein is coated onto a culture surface, and the extracellular matrix protein is a mixture comprising vitronectin, laminin, fibronectin, collagen, or two or more of the above.

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

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

6. Furthermore, e) Exposing the cell population whose fate has been determined to become the retinal lineage to a second culture environment, f) Culturing the aforementioned cell population for approximately 0 to 60 hours, g) The method according to any one of claims 1 to 5, further comprising inducing a population of ocular progenitor cells.

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

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

9. h) Exposing the aforementioned ophthalmic progenitor cell population to a third culture environment, i) Culturing the aforementioned ophthalmic progenitor cell population for approximately 2 to 5 days, j) The method according to any one of claims 6 to 8, further comprising inducing a population of optic vascular progenitor cells.

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

11. The method according to claim 10, wherein the third cell culture medium comprises a basal medium, and one or more IGFs and an activin signaling agonist.

12. The method according to any one of claims 9 to 11, wherein the optic vascular progenitor cells are dipotent with respect to retinal pigment epithelium or neuroretina.

13. k) Exposing the optic vascular progenitor cell population to a fourth culture environment, l) Culturing the aforementioned optic vascular progenitor cell population for approximately 5 to 10 days, The method according to any one of claims 9 to 12, further comprising m) inducing a population of immature retinal pigment epithelial cells.

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

15. The method according to claim 14, wherein 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.

16. The method according to any one of claims 13 to 15, further comprising dissociating the immature retinal pigment epithelial cell population and reseeding the dissociated cells to concentrate the immature retinal pigment epithelial cells.

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

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

19. The method according to 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 according to claim 19, wherein the immature retinal pigment epithelial cell population is matured by contact with a maturation medium containing a basal medium and at least a soluble iron source.

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

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

23. The method according to 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 animal component-free.

24. The method according to 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 lack an exogenously added inhibitor of BMP signaling.

25. The method according to 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 lack nicotinamide.

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

27. The method according to claim 26, wherein the PSC population is seeded into a microwell device.

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