A scalable method for producing retinal pigment epithelial (RPE) cells

Abstract

The present disclosure provides a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), comprising the steps of: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in non-adherent suspension culture; (b) plating the embryoid bodies onto a culture dish coated with a suitable extracellular matrix in a differentiation-inducing medium (DIM), wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; (c) culturing neuroectodermal lineages in a differentiation-proliferation medium (DPM) for rosette formation; (d) culturing the rosettes of step (c) in a retinal pigment epithelium maturation medium (RPEMM) to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) onto a culture dish coated with a suitable extracellular matrix in RPEMM to obtain RPE cells.

Description

[Technical Field]

[0001] FIELD OF THE INVENTION The present disclosure relates to cell culture techniques and cell differentiation and methods thereof, and in particular to cell culture methods for producing retinal pigment epithelial (RPE) cells from induced pluripotent stem cells (iPSCs). [Background technology]

[0002] Background of the Invention Macular degeneration, also known as age-related macular degeneration (AMD), is the leading cause of blindness in people over 60 years of age worldwide. Clinically, the disease begins with distortion of central vision caused by damage to the macula and ultimately leads to legal blindness. Vision loss has a significant impact on quality of life and incurs significant costs to the economy. Furthermore, AMD is a complex, progressive neurodegenerative disorder that causes visual impairment due to loss of the retinal pigment epithelium (RPE) and the light-sensitive photoreceptors that they support, protect, and nourish. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. Therefore, there is an urgent need to develop therapeutic strategies that address RPE cell loss and provide care for AMD. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Baghbaderani, BA et al. (2015).cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Pre-clinical and Clinical Applications. Stem cell reports, 5(4), pp. 647-659 [Non-patent document 2] Konala et al., Derivation of three induced pluripotent stem cell lines under feeder-free culture conditions from peripheral blood mononuclear cells (PBMC) of Indian patients suffering from inherited retinal diseases carrying different mutations. Stem Cell Research, 45, 101757. Summary of the Invention

[0004] SUMMARY OF THE INVENTION In an embodiment of the present disclosure, the steps include: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in non-adherent suspension culture; (b) plating the embryoid bodies onto a suitable extracellular matrix-coated culture dish in differentiation-inducing medium (DIM) and culturing for 6-8 days to obtain a neuroectodermal lineage, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; and (c) culturing the embryoid bodies in differentiation-proliferation medium (DPM) for 11-22 days for rosette formation to obtain a neuroectodermal lineage. wherein DPM does not contain any inhibitors; (d) culturing the rosettes of step (c) in retinal pigment epithelium maturation medium (RPEMM) for 23 to 45 days to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) on a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 47 to 75 days to obtain RPE cells.

[0005] In an aspect of the present disclosure, there is provided a retinal pigment epithelial cell, or a population thereof, produced by the methods disclosed herein.

[0006] In an aspect of the present disclosure, a pharmaceutical composition is provided comprising retinal pigment epithelial cells produced by the methods disclosed herein; and a pharmaceutically acceptable carrier.

[0007] In an aspect of the present disclosure, a method of treating a retinal degenerative disease in a subject is provided, comprising administering to the subject a composition disclosed herein.

[0008] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This Summary is provided to introduce a selection of concepts in a simplified form. It is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

[0009] BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of this specification and are included to further illustrate aspects of the present disclosure. The present disclosure may be better understood by reference to the drawings in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0010] [Figure 1] Illustrates RPE differentiation according to embodiments of the present disclosure, showing: A) embryoid body formation from iPSCs and their specification to a retinal fate through neuroectoderm induction; B) formation of rosette-like structures and epithelial clusters indicating the onset of retinal differentiation; C) retinal progenitor cells showing fate commitment to RPE cells, including the onset of pigmentation; D) RPE culture with increasing pigmentation levels and typical hexagonal morphology; E) RPE culture plate with visible (by eye) patches of pigmentation; F) mature RPE cell pellet after 70-80 days of culture; Scale bar 100 μm. [Figure 2]Illustrated are RPE characterization by immunofluorescence using stage-specific antibodies according to embodiments of the present disclosure. A-D) Immunostaining of iPSC-derived RPE cultures to examine marker expression during progressive days of retinal differentiation. Images show expression of the RPE-restricted marker microphthalmia-associated transcription factor (MITF) (A), the pigmentation-specific proteins tyrosinase-related protein (TYRP1) (B) and melanocyte protein (PMEL17) (C), and the RPE maturation marker RPE65 (D); immunophenotyping and quantification of RPE differentiation purity by flow cytometry show a high percentage of late RPE maturation markers, such as TYRP1, RPE65, and tyrosinase-TYR (E). [Figure 3] Illustrated are gene expression analysis using qPCR for selected markers, expressed as fold change compared to iPSCs, where all positive markers (A) have relatively high expression and negative markers (B) show negligible expression in iPSC-derived RPE; C) ELISA-based quantification of secreted pigment epithelium-derived growth factor (PEDF) from in vitro culture supernatants at two different time points (days 75 and 120); images and graphs are representative of a minimum of three independent experiments, according to embodiments of the present disclosure. [Figure 4] Illustrated are: A) phase contrast images of committed and mature RPE cells at low (10x) and high (20x) magnification showing heavy pigmentation; B) photographs of a 6-well plate containing late-stage RPE cultures and a 15 mL tube containing RPE cells after centrifugation (inset) during the enrichment and scale-up process; C) and D) gene expression profiles showing the purity of RPE cells after enrichment compared to non-enriched populations - representative heat maps with significant non-RPE gene sets showing downregulation in enriched RPE and significant RPE-specific gene sets showing upregulation in enriched RPE, according to embodiments of the present disclosure. [Figure 5]According to embodiments of the present disclosure, A) behavioral analysis (functional testing) results in balanced salt solution (BSS, vehicle) and RPE-injected RCS rats by measuring the optokinetic threshold (OKT); B) quantification of retinal thickness and cone number from low, medium, and high dose RPE transplanted animals between the nasal and temporal regions of the retina was calculated by immunostaining retinal sections with cone arrestin; C) Fundus images of saline solution (BSS) and RPE-injected (subretinal) eyes of RCS rats; D) Immunostaining (HNM) illustrates survival of transplanted RPE cells in the subretinal space at P90, with a preserved ONL layer (cone arrestin) and well-appearing retina, indicating visual recovery. DETAILED DESCRIPTION OF THE INVENTION

[0011] Description of the invention Those skilled in the art will recognize that the present disclosure is subject to variations and modifications other than those specifically described. The present disclosure should be understood to include all such variations and modifications. The present disclosure also includes all such steps, features, compositions, and compounds, individually or collectively, referenced or shown herein, and any and all combinations of any or more of such steps or features.

[0012] definition For convenience, before further description of the present disclosure, certain terms used in the specification and examples will be detailed here. These definitions should be read in light of the remainder of the disclosure and understood by one of ordinary skill in the art. Terms used herein have meanings that are recognized and known to those of ordinary skill in the art; however, for convenience and completeness, specific terms and their meanings are explained below.

[0013] The articles "a", "an" and "the" are used to refer to one or to more than one (ie to at least one) of the grammatical object of the article.

[0014] The terms "comprise" and "comprising" are used in an inclusive and open sense, meaning that additional elements may be included. The term is not intended to be construed as "consisting only of."

[0015] Throughout this specification, unless the context requires otherwise, the word "comprise", as well as variations such as "comprises" and "comprising", will be understood to imply the inclusion of the specified element or step or group of elements or steps, but not the exclusion of any other element or step or group of elements or steps.

[0016] The term "including" is used to mean "including, but not limited to," and "including" and "including but not limited to" are used interchangeably.

[0017] The term "induced pluripotent cells (iPSCs)" as used herein refers to pluripotent cells derived from adult somatic cells upon ectopic expression of a set of transcription factors. The developmental stages that iPSCs go through are neuroectoderm, eye field specification, and formation of a bilayered optic cup from the optic vesicle.

[0018] The term "retinal pigment epithelial (RPE) cells," as used herein, refers to a single layer of pigmented cells derived from the neuroectodermal layer of the optic cup, which constitutes the outermost layer of the retina. The RPE is required for the continued survival of the retina and enables photoreceptor cells to detect light.

[0019] The term "embryoid bodies (EBs)," as used herein, refers to three-dimensional aggregates formed in suspension by pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), that mimic the structure of a developing embryo and have the capacity to develop into cells of all three germ layers—ectoderm, mesoderm, and endoderm. EB differentiation is a common platform for generating specific cell lineages from PSCs.

[0020] The term "rosette," as used herein, refers to a developmental characteristic of neural precursors in culture from differentiating embryonic stem cells; rosettes are radial arrays of columnar cells that express early neuroectodermal markers, such as Pax6 and Sox1, and are capable of differentiating into various region-specific neural and glial cell types in response to appropriate developmental cues.

[0021] The term "inhibitor," as used herein, refers to an agent that blocks or inhibits a biochemical or biological response when bound to a receptor or a ligand of a receptor. WNT inhibitors, SMAD inhibitors, and ROCK inhibitors are various inhibitors that are used as part of this disclosure. For example, a "WNT inhibitor" inhibits WNT signaling by interfering with ligand-receptor interaction or WNT receptor maturation.

[0022] The term "subject," as used herein, refers to both human and veterinary subjects, such as rats, non-human primates, dogs, cats, horses, rabbits, pigs, mice, and cows.

[0023] The term "pharmaceutically acceptable carrier," as used herein, refers to a substance useful for carrying out the methods and forming the compositions disclosed herein. Generally, the nature of the carrier will depend on the particular mode of administration used. For example, for subretinal delivery carriers, balanced salt solution (BSS) or Hank's balanced salt solution (HBSS) are examples.

[0024] The term "confluency," as used herein, refers to the percentage of the growth medium area (surface area of ​​the culture dish) covered by adherent cells. For example, 60% confluency indicates that 60 parts of the growth surface out of 100 parts are occupied by cells. Confluency is used as an indicator of cell growth and expansion during cell culture experiments.

[0025] The term "gradually," as used herein, refers to an action performed slowly or in small increments over a period of time. According to the present disclosure, embryoid body development is promoted by shifting the medium in contact with the EBs from a growth medium to a differentiation-inducing medium (DIM) by slowly increasing the proportion of DIM while decreasing the proportion of growth medium. Thus, in an embodiment of the present disclosure, EBs are developed in a medium composition comprising an expansion medium and DIM in a 3:1 ratio for 24 hours; subsequently, in a medium composition comprising an expansion medium and DIM in a 1:1 ratio for 24 hours; and finally, in 100% DIM for 24 hours.

[0026] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It should be understood that such range format is used merely for convenience and ease and should be flexibly interpreted not only to include the numerical values ​​expressly recited as the limits of the range, but also to include all individual numerical values ​​and subranges subsumed within that range, as if each numerical value and subrange were expressly recited. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference.

[0027] The present disclosure is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of illustration only. Functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure described herein.

[0028] Many ocular diseases, such as age-related macular degeneration, are associated with degeneration or deterioration of the retina itself or the RPE. It is essential to achieve photoreceptor recovery and preserve visual function through subretinal transplantation of RPE cells. There is a need to find a robust and scalable method for generating RPE cells, for example, from human stem cells, that can be used to treat retinal degenerative diseases and injuries.

[0029] According to the present disclosure, a method for generating RPE cells through a combination of chemical and manual selection processes for enrichment of RPE cells at early and late stages of differentiation is provided. In principle, iPSCs are committed to a neuroectodermal fate by suppressing pluripotency factors, such as FGF2, using dual SMAD and WNT inhibition. This is achieved through the use of small molecules, such as SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide) and LDN193189 (4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). SB431542 inhibited the activin / TGF-β pathway, LDN193189 acted as a BMP4 / 7 inhibitor, while IWR1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide) silenced the canonical WNT signaling pathway.

[0030] To promote retinal cell proliferation, embryoid bodies derived from iPSCs are cultured in low-serum medium supplemented with N1, thus forming retinal progenitor cells, which are enriched to produce mature RPE. Mature RPE exhibit tight junctions and densely packed hexagonal cells with brown to black pigmentation and apical-basal polarity. This method yields pigmented and ciliated RPE cells that exhibit functional properties, such as polarized secretion of the cytokines PEDF and VEGF. Characterization methods for determining their authenticity and purity are also provided in conjunction with this method. The present disclosure also provides pharmaceutical compositions comprising RPE cells. Furthermore, the present disclosure provides methods for treating retinal degenerative diseases using the pharmaceutical compositions.

[0031] In an embodiment of the present disclosure, the method comprises the steps of: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in non-adherent suspension culture; (b) plating the embryoid bodies onto a suitable extracellular matrix-coated culture dish in differentiation-inducing medium (DIM) and culturing for 6-8 days to obtain a neuroectodermal lineage, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; and (c) culturing the embryoid bodies in differentiation-proliferation medium (DPM) for 11-22 days for rosette formation to obtain a neuroectodermal lineage. (d) culturing the rosettes of step (c) in retinal pigment epithelium maturation medium (RPEMM) for 23 to 45 days to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) on a culture dish coated with a suitable extracellular matrix and culturing them in RPEMM for 47 to 75 days to obtain RPE cells.

[0032] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), further comprising the steps of: (a) enzymatically dissociating RPE cells from the extracellular matrix to obtain a single-cell suspension of RPE; and (b) plating the RPE single-cell suspension of step (a) onto a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 75 to 100 days to enrich the RPE cells.

[0033] In an embodiment of the present disclosure, the method comprises the steps of: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in non-adherent suspension culture; (b) plating the embryoid bodies onto a tissue culture dish coated with a suitable extracellular matrix in differentiation-inducing medium (DIM) and culturing for 6-8 days to obtain a neuroectodermal lineage, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; (c) culturing the neuroectodermal lineage for rosette formation in differentiation-proliferation medium (DPM) for 11-22 days, wherein the DPM does not comprise any inhibitors; and (d) culturing the rosettes of step (c) in retinal pigment epithelium maturation medium (RPEMM) for 23-45 days. to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) onto a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 47 to 75 days to obtain RPE cells, the method further comprising the steps of: (a) enzymatically dissociating the RPE cells from the extracellular matrix to obtain a single-cell suspension of RPE; and (b) plating the single-cell suspension of RPE of step (a) onto a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 75 to 100 days to enrich the RPE cells.

[0034] In an embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells) is provided, wherein the iPSC culture has a confluency in the range of 80% to 90%. In another embodiment, the confluent iPSC culture has a confluency in the range of 80% to 85%.

[0035] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the step of generating embryoid bodies in step (a) comprises: (a) culturing confluent iPSCs in a growth medium for 24 hours to form embryoid bodies, wherein the growth medium comprises a ROCK inhibitor; and (b) gradually contacting the developing embryoid bodies from the growth medium to the DIM by: (i) culturing the developing embryoid bodies in a medium composition comprising an Expansion Growth Medium and DIM at a ratio of 3:1 for 24 hours, (ii) culturing the developing embryoid bodies obtained in step (i) in a medium composition comprising an Expansion Growth Medium and DIM at a ratio of 1:1 for 24 hours; and (iii) culturing the embryoid bodies obtained in step (ii) in DIM for 24 hours, followed by plating the embryoid bodies onto a tissue culture dish.

[0036] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the rosette formation in step (c) comprises: i) maintaining the neuroectodermal lineage in DIM for 24 hours; ii) maintaining the neuroectodermal lineage obtained in step (i) in a medium composition comprising DIM and DPM in a 1:1 ratio for 24 to 48 hours; and iii) culturing the neuroectodermal lineage obtained from step (ii) in DPM for 24 hours to promote rosette formation.

[0037] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the formation of retinal progenitor cells in step (d) comprises: (i) maintaining the rosettes in DPM for 24 hours; (ii) maintaining the rosettes obtained in step (i) in a medium composition comprising DPM and RPEMM in a 1:1 ratio for 24 hours; and (iii) culturing the rosettes obtained from step (ii) in RPEMM for 24 hours to promote the formation of retinal progenitor cells.

[0038] In an embodiment of the present disclosure, the method comprises the steps of: (a) generating embryoid bodies from a culture of iPSCs having a confluency in the range of 80% to 90%, wherein the embryoid bodies are in a non-adherent suspension culture; (b) plating the embryoid bodies onto a culture dish coated with a suitable extracellular matrix in a differentiation-inducing medium (DIM) and culturing for 6 to 8 days to obtain a neuroectodermal lineage, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; and (c) culturing the embryoid bodies in a differentiation-proliferation medium (DP) for rosette formation. (d) culturing the rosettes of step (c) in retinal pigment epithelium maturation medium (RPEMM) for 23 to 45 days to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) on a culture dish coated with a suitable extracellular matrix and culturing them in RPEMM for 47 to 75 days to obtain RPE cells. 1. A method for obtaining iPSCs (e.g., iPSCs), wherein the step of producing embryoid bodies in step (a) comprises the steps of: (a) culturing confluent iPSCs in a growth medium for 24 hours to form embryoid bodies, the growth medium comprising an expansion medium and a ROCK inhibitor; and (b) (i) culturing the developing embryoid bodies in a medium composition comprising an expansion medium and DIM at a ratio of 3:1 for 24 hours; (ii) culturing the developing embryoid bodies obtained in step (i) in a medium composition comprising an expansion medium and DIM at a ratio of 1:1 for 24 hours. and (iii) culturing the embryoid bodies obtained in step (ii) in DIM for 24 hours, followed by plating the embryoid bodies onto a culture dish, thereby gradually contacting the developing embryoid bodies from the growth medium to DIM; and the rosette formation in step (c) is characterized by: i) maintaining the neuroectodermal lineage in DIM for 24 hours; ii) maintaining the neuroectodermal lineage obtained in step (i) in a medium composition comprising DIM and DPM in a 1:1 ratio for 24 to 48 hours;and iii) culturing the neuroectodermal lineages obtained from step (ii) in DPM for 24 hours to promote rosette formation; wherein the retinal progenitor cell formation in step (d) comprises (i) maintaining the rosettes in DPM for 24 hours; (ii) maintaining the rosettes obtained in step (i) in a medium composition comprising DPM and RPEMM in a 1:1 ratio for 24 hours; and (iii) culturing the rosettes obtained from step (ii) in RPEMM for 24 hours to promote the formation of retinal progenitor cells.

[0039] In an embodiment of the disclosure, the growth medium comprises an expansion growth medium and a ROCK inhibitor; the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

[0040] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein at least one suitable extracellular matrix is ​​selected from Matrigel, laminin, vitronectin, fibronectin, collagen, poly-L-lysine, poly-L-ornithine, or a combination thereof.

[0041] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the at least one WNT pathway inhibitor is selected from the group consisting of 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide, 5-(phenylsulfonyl)-N-piperidin-4-yl-2(trifluoromethyl)benzenesulfonamide, 2-(trifluoromethyl ... -(2',3-dimethyl-[2,4'-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide, 2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide, 8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one, or a combination thereof.

[0042] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the at least two SMAD pathway inhibitors are 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine, 5-chloro-2-N-[2-methyl- The method further comprises the step of: providing a compound selected from the group consisting of 5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, ... or a combination thereof. In another embodiment of the present disclosure, the at least two SMAD pathway inhibitors are 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide and 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline.

[0043] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

[0044] In an embodiment of the present disclosure, there is provided a method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), wherein the enzymatic dissociation of the RPE cells in step (a) is carried out using an enzyme selected from the group consisting of Accutase, Tryple select, TrypLE, Gentle Cell Dissociation Reagent (GCDR), and Dispase.

[0045] In an embodiment of the present disclosure, there is provided a retinal pigment epithelial cell, or a population thereof, produced by the methods disclosed herein.

[0046] In an embodiment of the present disclosure, a pharmaceutical composition is provided comprising the retinal pigment epithelial cells or populations thereof disclosed herein; and a pharmaceutically acceptable carrier.

[0047] In embodiments, the pharmaceutically acceptable carrier is selected from balanced salt solution (BSS) or Hank's balanced salt solution (HBSS).

[0048] In an embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein for use in treating a retinal degenerative disease.

[0049] In an embodiment of the present disclosure, a composition is provided wherein the retinal degenerative disease is selected from the group consisting of age-related macular degeneration and retinal diseases associated with early and late photoreceptor degeneration.

[0050] In an embodiment of the present disclosure, there is provided a method of treating a retinal degenerative disease in a subject, comprising administering to the subject a pharmaceutical composition disclosed herein.

[0051] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense.

[0052] Various modifications of the disclosed embodiments, as well as alternative embodiments of the subject matter, will be apparent to those skilled in the art upon reference to the description of the subject matter, and it is therefore contemplated that such modifications can be made without departing from the spirit or scope of the inventive subject matter as defined. [Example]

[0053] Example The present disclosure is now illustrated using practical examples, which are intended to illustrate the practice of the present disclosure and are not intended to be construed as limiting or implying any limitations on the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein. It should be understood that the present disclosure is not limited to the specific methods and experimental conditions described, as such methods and conditions can be applied.

[0054] Example 1 material The iPSC line TC-1133 (RUCDR / NIH) was obtained from the Eyestem biobank and can be found at https: / / eyestem.com / collaborations / research-alliance / .

[0055] All key reagents used in this disclosure were sourced as mentioned in the Primary Sources Table.

[0056] iPSCs were procured / thawed and maintained as undifferentiated cultures in mTeSR-based medium (expansion medium). The culture medium was prepared according to the recipe mentioned in the methods.

[0057] iPSC maintenance and differentiation medium can be used for up to 2 weeks when stored at 4°C. Therefore, it is not wise to prepare all of the medium at once, but rather when required for specific steps in the procedure.

[0058] All procedures were performed in a BSL-2 certified laboratory biosafety cabinet using standard aseptic techniques. Cultures were grown and maintained in a humidified incubator at 37°C with 5% CO2.

[0059] Cultures were routinely tested for sterility and karyotyping.

[0060] Standard cell culture practices, such as aseptic handling of cultures and wearing personal protective equipment, were used, along with appropriate equipment, such as a biosafety cabinet, CO2 incubator, centrifuge, water bath, and microscope. Cells were cultured in extracellular matrix (ECM)-coated plates in a humidified incubator at 37°C with a 5% CO2 level. Prior to differentiation, iPSCs were characterized for expression of pluripotency markers, chromosomal abnormalities by karyotype analysis, and sterility testing to determine whether the cultures were free of contamination (bacteria and mycoplasma).

[0061] Example 2 General experimental preparation The reproducibility of the RPE differentiation method was tested in multiple iPSC lines, including TC-1133 (RUCDR / NIH; Baghbaderani, BA et al. (2015). cGMP-Manufactured Human Induced Pluripotent Stem Cells Are Available for Preclinical and Clinical Applications. Stem Cell Reports, 5(4), pp. 647-659), ERPLi001-A, ERPLi002-A, and ERPLi003-A (Konala et al., Derivation of three induced pluripotent stem cell lines under feeder-free culture conditions from peripheral blood mononuclear cells (PBMCs) of Indian patients suffering from inherited retinal diseases carrying different mutations. Stem Cell Research, 45, pp. 1017-57). Therefore, similar differentiation results were expected for other iPSC lines following the detailed method described here.

[0062] The entire method was performed in a sterile environment, e.g., a biosafety cabinet. The method comprises the following steps: 1. Preparation of Cell Therapy System-Vitronectin (CTS™-VTN) for iPSC Expansion Timing: 1 hour a. CTS-VTN (0.9 mg / mL) was thawed at room temperature (20-25°C) for 5-10 minutes and then placed on ice. CTS-VTN was aliquoted into working size aliquots in polypropylene tubes and stored at -60°C to -80°C. b. Room temperature storage and / or shaking could result in the appearance of light turbidity which did not affect product performance. To coat the wells of a 6-well plate, add 60 µL of CTS-VTN to 6 mL of sterile CTS™ DPBS (1x) (-Ca) at room temperature. 2+ / -Mg 2+ ) was added to a 15 mL tube containing c. The CTS-VTN solution was gently resuspended by pipetting up and down. This resulted in a working concentration of 9 μg / mL (i.e., a 1:100 dilution). The diluted CTS-VTN solution can be stored at 4°C for up to 7 days. 1 mL of the diluted CTS-VTN solution was then added to each well of a 6-well plate. The plate was swirled and / or rocked to ensure uniform coating. d. 1 mL / well in a 6-well plate (10 cm 2 / well), the final concentration is 0.9 μg / cm 2 It would be. e. The coated plates were incubated at 37°C for 1 hour.

[0063] The culture plates can be used now or stored wrapped in laboratory film at 2-8°C for up to one week, ensuring that the wells do not dry out. A volume of basal medium can be added to the wells one hour after coating to ensure that the wells do not dry out. If any part of the well is dry, the well cannot be used. Prior to use, the culture plates must be pre-warmed to room temperature (20-25°C). f. Immediately prior to use, the CTS™-VTN solution was aspirated and discarded. There is no need to rinse the culture plate after removal of the CTS™-VTN. Cells can be seeded directly onto the CTS™-VTN-coated culture plate. 2. iPSC Culture and Maintenance Timing: 6-10 days

[0064] Before starting the method, CTS-VTN coated plates were prepared. If using CTS-VTN coated plates stored at 4°C, the plates should be allowed to equilibrate to room temperature for 1 hour prior to starting.

[0065] One cryovial containing 1 million cells was placed in a 6-well plate (10 cm 2 surface area) must be thawed into one well. a. 5 mL of chilled mTeSR plus medium was added to a sterile 15 mL tube. To effectively dilute the Cryostor CS10, a 1:5 ratio is recommended (1 mL of cells and 5 mL of mTeSR plus). b. Cells were removed from a liquid nitrogen storage tank. c. Cells were rapidly thawed in a 37°C water bath using a "Figure 8" motion until pea-sized ice was visible. To avoid cell death, cells should not be completely thawed in the water bath. d. Using a 2 mL pipette, mTeSR plus was slowly added dropwise to the cells and collected in a 15 mL tube. e. The 15 mL tube was capped and gently inverted 4-5 times to mix the CryoStor CS10 and mTeSR plus. f. The tubes were then centrifuged at 200 g for 3 minutes at 25±5°C. g. CTS-VTN was aspirated from the plate while the cells were centrifuged. h. The supernatant was gently aspirated from the cell pellet and the cells were resuspended in 2 mL of fresh mTeSR plus containing 10 μM ROCK inhibitor (Y-27632).

[0066] The use of the ROCK inhibitor Y-27632 increased cell viability and cell health. Y-27632 was reconstituted and stored as per the manufacturer's instructions (https: / / www.tocris.com / products / y-27632-dihydrochloride_1254).

[0067] Due to its known toxicity, washing ensured complete removal of DMSO present in the storage medium. The procedure was performed in a gentle and rapid manner. i. Cells were plated onto CTS-VTN coated wells. j. Place the culture plate in the incubator; gently rock the plate in a positive direction for uniform distribution of the cells. k. The next day, to remove the Y-27632, replace the medium with 2 mL of fresh mTeSR plus.

[0068] For medium changes, medium was always added / removed carefully near the wall of the well to avoid cell detachment. l. colonies were expected to appear within 2-3 days. The medium was changed daily, and the colonies were ready to be passaged when they covered 70% to 80% of the plate. 3. iPSC Passaging Timing: 1 hour a. CTS-VTN coated plates were prepared as previously described and pre-warmed with mTeSR plus. b. The medium was aspirated and the wells were washed with 1x PBS (1 mL / well of 6 wells). c. ReLeSR™ was added to the culture plate and the ReLeSR™ was aspirated within 1 minute, thereby exposing the colonies to only a thin layer of liquid.

[0069] [Table 1]

[0070] d. The plate was incubated at 37°C for 4 minutes ± 30 seconds. e. For complete cell detachment, the plate or well was gently rinsed with 1 mL of mTeSR plus. f. Holding the plate in one hand, firmly tap the side of the plate with the other hand for approximately 30-60 seconds to detach the colonies. g. The detached cell aggregates were transferred to a 15 mL tube using a 5 mL serological pipette. h. Cell aggregates should be of an appropriate size for plating (average aggregate size of approximately 50-200 µm). i. Sterile 15 mL or 50 mL serum tubes were centrifuged at 200 g for 3 minutes by maintaining acceleration and deceleration at a maximum of 9 on the centrifuge. j. Upon completion of centrifugation, the supernatant was removed, the pellet was gently tapped, and the appropriate volume of mTeSR plus was added along with 10 μM Y-27632 depending on the split ratio into 15 mL or 50 mL tubes. k. Cell colonies were plated onto CTS-VTN coated dishes. l. The plate was rocked in a positive direction to ensure even distribution of colonies in the wells. m. The plates were placed in a 37°C incubator with 5% CO2. Ensure that the plates did not move for 24 hours. After 24 hours, the plates were observed under a microscope to confirm that colonies had adhered to the plates. The next day, to remove the Y-27632, the medium was replaced with 2 mL of fresh mTeSR plus. o. Medium was changed daily until plates reached 70% confluency in less than 7 days.

[0071] Any plates that did not reach 70% confluence within 7 days were discarded. Before each passage, the supernatants from all plates were pooled into a single sterile 15 mL tube and stored at -80°C until tested for sterility. 4. Matrigel aliquoting and coating g. Matrigel was aliquoted and stored at -20°C. It was important to keep any materials that would come into contact with Matrigel on ice. Matrigel will solidify and adhere to any item that is above 10°C. Therefore, multiple freeze / thaw cycles were carefully avoided. h. Before beginning, frozen serological pipettes, pipette tips, 15 mL tubes, and aliquots of Matrigel were placed on ice in a biosafety cabinet (BSC). Ensure that the ice bucket was thoroughly sprayed with 70% ethanol before placing in the BSC. i. Matrigel was diluted 1:100 in cold DMEM-F12 medium. For example, 100 μL Matrigel was added to 10 mL DMEM-F12 medium.

[0072] Care was taken to keep the fingertip above the level of the Matrigel, as the heat from the fingertip solidified the Matrigel. Frequent changes to fresh, chilled pipette tips were ensured. This 1:100 dilution will result in a 1% Matrigel solution ready to use for coating culture plates. j. Using a chilled serological pipette, transfer 1% Matrigel solution to the culture plate at 1 mL / well of a 6-well plate. k. The plate was swirled and / or rocked to ensure an even coating. l. The plates were then incubated at 37°C for at least 1 hour. m. Alternatively, if the Matrigel-coated plates were not used on the same day as coating, the plates were wrapped in parafilm and stored in a 4° C. refrigerator for up to 1 week, ensuring that the wells did not dry out.

[0073] A volume of basal medium can be added to the wells one hour after coating to ensure that the wells do not dry out. If any part of the well is dry, the well cannot be used.

[0074] Prior to use, the Matrigel solution was removed, avoiding allowing the Matrigel-coated wells to air dry before seeding the cells.

[0075] Example 3 Differentiation of iPSCs into retinal pigment epithelial (RPE) cells 1. Embryoid body (EB) formation by forced aggregation of iPSCs Timing: Days 0-2 a. mTeSR plus (Expansion Medium: cGMP, stabilized feeder-free maintenance medium for iPS cells commercially sourced), CTS™ DPBS (1x) (-Ca 2+ / -Mg 2+ ) was pre-warmed to 37°C. One 6-well plate (approximately 1 million cells per well at 80% confluency) was sufficient to initiate RPE differentiation through EB formation (Fig. 1A). b. Cells from 6 wells will be placed into 6 wells of an ultra-low attachment plate (to avoid any form of adhesion) for the formation of EBs. c. Ultra-low attachment plates were prepared by adding 1 mL of mTeSR plus medium to each well. d. Aspirate spent medium from the plate containing iPSCs and replace with CTS™ DPBS (1x) (-Ca 2+ / -Mg 2+ ) (see Table-1 for volumes) was used to rinse the plate once. e.CTS(TM)DPBS(1×)(-Ca 2+ / -Mg 2+ ) was aspirated and ReLeSR solution was added to the plates containing iPSCs. The volume of ReLeSR solution was adjusted for different plate sizes (see Table 1 for volumes). f. 1 mL / well of ReLeSR™ was added and the ReLeSR™ was aspirated within 1 minute, thereby exposing the colonies to a thin layer of liquid. g. The plate was incubated at 37°C for 4 minutes ± 30 seconds. h. To stop the dissociation reaction, an appropriate amount of mTeSR plus medium was added to each plate (for volumes, see Table-1). i. The dissociated cells were gently pipetted up and down sufficiently to disperse the colonies into a single cell suspension. j. Ensure gentle pipetting to minimize bubble formation. k. The iPSC suspension was transferred from each well to a separate 15 mL tube, and the tubes were centrifuged at 200 g for 3 minutes to pellet the cells. l. The supernatant was carefully aspirated from the iPSC pellet. m. An appropriate amount (approximately 6 mL) of mTeSR plus medium was used to resuspend the pellet. n. The resuspended cells were added to each 6-well ultra-low attachment plate containing 6 mL of mTeSR plus medium (1 mL / well) containing 10 μM ROCK inhibitor Y-27632 (growth medium). This day was considered day 0. The cells were incubated in a 37°C, 5% CO2 incubator for 24 hours to allow them to form EBs. To remove the Y-27632, the medium was replaced with 2 mL of fresh mTeSR plus (expansion medium). Days 1 and 2: Gently swirl the EB plate to move the EBs to the center of the dish. 1 mL of medium was removed from the corner of the well, and 1 mL of fresh mTeSR plus medium was added (Figure 1A). 2. Differentiation and plating of embryoid bodies (EBs) to obtain neuroectodermal lineages Timing: Days 3-8 a. Differentiation induction medium DIM was prepared as shown in Table 2.

[0076] [Table 2]

[0077] Growth factors were added according to the manufacturer's instructions ( https: / / www.sigmaaldrich.com / IN / en / product / sigma / i0161 ; https: / / www.tocris.com / products / sb-431542_1614; https: / / www.tocris.com / products / ldn-193189-dihydrochloride_6053; https: / / www.stemcell.com / products / human-recombinant-igf-i.html) The solution was reconstituted and stored as described above. b. The medium of suspension cultures containing EBs was gradually shifted from mTeSR plus (expansion medium) to differentiation-inducing medium (DIM). c. Day 3: One part of mTeSR plus medium was replaced with DIM (3:1) (eg, for 1 mL of medium, 750 μL mTeSR plus medium and 250 μL DIM). d. Day 4: One part of mTeSR plus medium was replaced with DIM (1:1) (e.g., for 1 mL of medium, 500 μL mTeSR plus medium and 500 μL DIM). e. Day 5: A complete medium change (100%) was performed with DIM. f. Day 6: Matrigel was coated and EBs were plated.

[0078] EBs were plated onto Matrigel (extracellular matrix) coated culture dishes, at which point they were expected to be dense, spherical, with clear borders, and would easily converge to the center when the plate was swirled. i. The EB plate was gently swirled to transfer EBs from 3 wells of the 6-well plate into a 15 mL tube. ii. Allow the EBs to settle to the bottom of the tube (this will take approximately 3 minutes) at room temperature (20-25°C) inside a biosafety cabinet. Be sure not to centrifuge the EBs. Remove the supernatant (retaining 1 mL of medium) and add 2 mL of DIM, followed by 500 µL of resuspended medium in a dropwise manner using a 1 mL pipette tip, thereby distributing the EBs evenly across the plate. iii. The culture plate was gently shaken to aid in even distribution of the aggregates and the plate was carefully placed back into the incubator, ensuring that the plate was not rocked for 24 hours.

[0079] AggreWell™ 800 microwell culture plates (Stem Cell Technologies) were used for EB formation using the same RPE differentiation method. Uniformity in size of EBs generated using multiwell plates was observed. However, no significant advantages were observed in terms of final RPE number, processing time, or cost. Therefore, ultra-low attachment dishes were continued to be used for EB formation. g.7~8th day: i. After 24 hours, the plates were observed for EB attachment. The majority of EBs will have attached and will begin to show outgrowth from the outer edge.

[0080] Floating EBs were discarded at this point while the medium was changed. ii. A complete medium change was performed with 100% DIM (see Table-1 for volumes). 3. Introduction of Differentiation Proliferation Medium (DPM) for Rosette Formation a. DPM was prepared as per the ingredient list provided in Table-3.

[0081] [Table 3]

[0082] N1 supplement was added to the filtered medium.

[0083] At this stage, the medium could be stored at 4°C for 2 weeks. b. Day 9: The medium was gradually shifted from DIM to Differentiation and Proliferation Medium (DPM). One part of the DIM medium was replaced with DPM (1:1) (e.g., for 1 mL of medium, 500 μL DIM and 500 μL DPM). c. A complete medium change (100%) was performed using DPM. d. Days 11-22: Every other day until day 22, cultures were fed with DPM (for volumes, see Table-1).

[0084] Around day 20, rosette-like structures and epithelial cluster formation were observed, indicating the onset of retinal differentiation (FIG. 1B). 4. Introduction of Retinal Pigment Epithelium Maturation Medium (RPEMM) for the Formation of Retinal Progenitor Cells a.RPEMM was prepared as shown in Table-4.

[0085] [Table 4]

[0086] Growth factors and additives were added to the filtered medium. Growth factors were added according to the manufacturer's instructions ( https: / / www.sigmaaldrich.com / IN / en / product / sigma / t0625 ; https: / / www.sigmaaldrich.com / IN / en / product / sigma / h6909; https: / / www.sigmaaldrich.com / IN / en / product / sigma / t5516) The solution was reconstituted and stored as described above.

[0087] The prepared medium could be stored at 4°C for 2 weeks. b. The medium was gradually switched from DPM to retinal pigment epithelium maturation medium (RPEMM). c. Day 23: One part of DPM medium was replaced with RPEMM (1:1) (e.g., for 1 mL of medium, 500 μL DIM and 500 μL RPEMM). d. Day 24: On day 24, the mice were switched to complete RPEMM (Figure 1D). e. Days 25-45: Every other day until day 40, cultures were fed with RPEMM (for volumes, see Table-1).

[0088] Day 5.40 (+5): RPE enrichment process by subculturing As shown in Figure 1C, the onset of pigmentation was observed in the cultures, indicating the fate commitment of retinal progenitor cells to RPE cells. Additional visible patches of pigmentation were observed in the RPE culture plates (Figure 1E). RPE cells could be further passaged between days 35 and 45 after the onset of pigmentation to enrich for pigmented cells, resulting in mature RPE cells. a. Matrigel coating: Matrigel coated plates were prepared as previously specified (see step 4 in Example 2). b. For 1 x 6-well plate, 3 mL of gentle cell dissociation reagent (GCDR) and 3 mL of StemPro Accutase (dissociation enzyme) were aliquoted into a 15 mL tube and pre-warmed in an incubator at 37°C for 30 minutes. c. Aspirate the culture medium and replace with CTS™ DPBS (1x) (-Ca 2+ / -Mg 2+ ) and washed gently once with 1 mL / well. d. Cells were incubated with pre-warmed GCDR and Accutase in a 1:1 ratio for 11-12 minutes at 37°C.

[0089] If necessary, cells were incubated for an additional 2 min in step d to ensure complete dissociation into single cells. e. Gently pipette to dissociate into single cells or aggregates of 5-10 cells and dilute with RPEMM medium. f. The cell suspension was collected in a 15 mL tube and centrifuged at 200 g for 3 minutes. The supernatant was removed. g. Matrigel (extracellular matrix) was aspirated immediately before seeding the cells and RPEMM medium was added into each well (see Table-1 for volumes). h. The cell pellet was resuspended in RPEMM and the cells were seeded 1:2 or 1:3 into matrigel-coated plates. i. Cells were allowed to adhere without shaking for 24 to 48 hours. j. After proper attachment of the cells, medium exchange was resumed. 6. Days 47 to 75 or later a. Cultures were fed with RPEMM every other day until days 75-80. b. Day 60 (+5): Passage of RPE.

[0090] By following the steps mentioned in the RPE subculture enrichment process (see steps above), RPE cells can be passaged again around day 60(+5) and maintained up to day 90-100 by changing the medium every other day. This process could be carried out on a larger scale if a larger number of RPE cells need to be generated for preclinical safety and toxicology studies.

[0091] 7. Day 75 or later: Selection and cryopreservation RPE cells exhibited a cubic monolayer with hexagonal morphology at day 75, accompanied by visible deposition of melanin pigment. Furthermore, increased pigmentation with hexagonal morphology was observed at day 120, as shown in Figure 1D. Cultures were frozen using commercially available cryoprotectant CryoStor® CS10 freezing medium-BioLife solution in 1.8 mL NUNC cryotubes. a.CTS(trademark)DPBS(1×)(-Ca 2+ / -Mg 2+ ) and washed the cells twice. b. Pre-warmed GCDR and Accutase were added in a 1:1 ratio and incubated at 37°C for 10-12 minutes. If the enrichment step by passaging RPE cells was not performed on day 40 / 45, then step c followed, otherwise step d followed directly. c. Visually unpigmented cluster areas (non-black), if any, were manually removed and discarded using a 20-200 μL pipette tip. Using a 1 mL pipette tip, the pigmented patches were gently blown off from the wells. The blow-off was carefully repeated in the presence of RPEMM until all visibly pigmented patches were successfully detached. d. The cells were collected and mixed with an equal volume of RPEMM. The cells were centrifuged at 200 g for 2 minutes at room temperature (20-25°C) (Figure 1F).

[0092] After counting the cells, the number of cryovials required to freeze approximately 1 million cells per vial in 1 mL of chilled freezing medium was determined, and each cryovial was labeled with the cell line name, cell type, initials, and date. e. Cryostor CS10 freezing medium was added to the RPE cell pellet, followed by gentle mixing of the RPE cell suspension. f. Cells (in suspension) were cryopreserved in cryovials at 1 million cells per vial. g. The cryovials were placed in a Mr. Frosty freezing container and stored at -80° C. for 24 hours. After 24 hours, the cells were transferred to a liquid nitrogen storage tank and the cryotank binder was renewed.

[0093] Figure 4A depicts phase contrast microscopy images of committed and mature RPE cells at low (10x) and high (20x) magnification showing heavy pigmentation, and Figure 4B depicts photographs of a 6-well plate containing late-stage RPE cultures during the enrichment and scale-up process and a 15 mL tube containing RPE cells after centrifugation (inset).

[0094] Example 4 Characterization of the RPE in vitro Protein expression by immunocytochemistry Immunocytochemistry was performed to visualize the expression levels and localization of stage-specific retinal markers in the differentiation cultures, demonstrating successful differentiation of iPSCs into RPE (Figures 2A-D). Timing: 4 hours to 2 days

[0095] [Table 5]

[0096] Day 1 i. Aspirate the culture medium and replace with CTS-DPBS (1x) containing fixative solution (-Ca 2+ / -Mg 2+ The plate was gently washed three times with PBS and incubated at room temperature (20-25°C) for 15-20 minutes. ii. Remove the fixative solution and resuspend the CTS in 1x CTS-DPBS (-Ca) 2+ / -Mg 2+ ) and gently washed three times with iii. Cells were permeabilized and blocked by adding a blocking solution at room temperature (20 to 25°C) for 30 minutes. iv. The cells were then resuspended in CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and gently washed three times with v. Primary antibody diluted in antibody diluent was added to the cells and incubated overnight (16-20 hours) at 4°C. Day 2 vi. Remove the primary antibody and resuspend the cells in CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and washed three times. vii. Secondary antibody diluted in antibody diluent was added to the cells, followed by incubation in the dark at room temperature (20-25°C) for 60 minutes. viii. Remove the secondary antibody solution and resuspend the cells in CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and washed three times. ix. The cells were counterstained with 1 μg / mL DAPI and incubated in the dark at room temperature (20-25°C) for 10 minutes. Remove the DAPI solution and resuspend the cells in CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and washed once. xi. CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) (300 μL per well of a 4-well plate) was added, and the cells were visualized under a fluorescent microscope.

[0097] Fixed cells from step (ii) were incubated in CTS-DPBS (1x) (-Ca) for 1 week at 4 °C before proceeding to staining. 2+ / -Mg 2+ ) can be stored in

[0098] observation Immunostaining of iPSC-derived RPE cultures was performed to examine marker expression during progression of retinal differentiation. The RPE-restricted marker MITF (Figure 2A); pigmentation-specific proteins, such as the tyrosinase-related protein TYRP1 (Figure 2B) and the melanocyte protein PMEL17 (Figure 2C); and the RPE maturation marker RPE65 (Figure 2D) showed relatively high expression in RPE cells obtained by the disclosed method.

[0099] Immunophenotyping by flow cytometry Flow cytometry analysis of RPE cells helped to quantify the levels of protein expression and provided reconfirmation of successful differentiation of iPSCs into RPE (Figure 2E). Timing: 3 hours

[0100] [Table 6]

[0101] Enzymatic dissociation of RPE into single cells i. Aspirate the culture medium and replace with CTS-DPBS (1x) (-Ca2+ / -Mg 2+ ) and washed the cells. ii. Cells were enzymatically harvested (GCDR and Accutase at a 1:1 ratio, 10-12 minutes); (see section 7, steps a-d under stepwise method details) by gentle pipetting to create a single cell suspension. iii. The cells were centrifuged at 200 g for 3 minutes and the supernatant was discarded. iv. CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ The pellet was washed by resuspending in PBS and centrifuging at 200 g for 3 minutes. v. Cells were fixed by adding 1 mL of 4% PFA for 10 min at room temperature (20–25°C). vi. CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and centrifuged at 200 g for 3 min, followed by resuspending the cells in CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) was completely removed. vii. 1 mL of ice-cold methanol was added to the pellet and incubated at 4°C for 20 minutes. viii. Cells were washed with FACS buffer (see Table-6) and centrifuged at 200 g for 3 minutes. ix. Stain the cells with primary antibodies at optimized concentrations in 100 μL FACS buffer and incubate for 30 min at room temperature (20–25 °C). x. Repeated washing with FACS buffer and centrifuged at 200 g for 3 min. xi. Secondary antibody in 100 μL FACS buffer was added to the pellet at an optimized concentration and incubated in the dark for 30 minutes. xii. The vial was vortexed every 10 minutes to ensure mixing. xiii. CTS-DPBS (1x) (-Ca 2+ / -Mg 2+ ) and resuspended in FACS buffer. xiv. Cells were transferred to flow tubes for flow cytometry analysis.

[0102] Fixed cells from step (f) were incubated in CTS-DPBS (1x) (-Ca) for 48-72 h at 4 °C before proceeding to permeabilization and blocking. 2+ / -Mg 2+ ) can be stored in

[0103] To avoid cell loss during washing steps, permeabilization, blocking, and immunolabeling were performed simultaneously.

[0104] observation From Figure 2E, it is clear that the RPE obtained by differentiation of iPSCs was of high purity, as indicated by the high percentage of late RPE maturation markers, such as TYRP1, RPE65, and tyrosinase, in the flow cytometry results.

[0105] Gene expression analysis by quantitative real-time PCR Gene expression profiling helps understand the molecular characteristics of these de novo generated RPE cells compared to undifferentiated iPSCs. Timing: 4-5 hours Total RNA purification Pellet preparation: i. Cells were collected into a sterile 1.5 mL tube (see selection and cryopreservation steps a-d in Example 3) and centrifuged at 200 g for 1 minute to pellet the cells. ii. The supernatant was carefully removed and the pellet was flash-frozen using liquid nitrogen. The flash-frozen cells were stored at -80°C until RNA isolation. RNA isolation: iii. For RNA isolation, an RNeasy Mini kit (Qiagen) was used, and all steps were carried out at room temperature (20-25°C). iv. 500 μL RLT buffer was added to the cell pellet and an equal volume of 70% ethanol was added. The mixture was added to a spin column and subjected to centrifugation at 8000 g for 1 minute. v. The column was washed once with buffer RW1 and twice with buffer RPE. All washing steps were performed at 8000 g for 1 minute. vi. For the final elution, 20 μL of RNase-free water was added directly to the spin column, incubated for 1 minute, and centrifuged at 8000 g for 1 minute to elute the RNA. vii.RNA was immediately kept on ice and quantified using nanodrop. cDNA synthesis: viii. cDNA transcription was performed using the Verso cDNA synthesis kit. All reaction setup steps were carried out on ice. ix. The Verso cDNA kit method recommends a template RNA concentration ranging from 1 pg to 1 μg. The cDNA synthesis reaction mix was performed on ice using the volumes mentioned below.

[0106] [Table 7]

[0107] The x.RNA was incubated at 42°C for 45 minutes to allow cDNA synthesis to occur and at 95°C for 2 minutes for enzyme inactivation. The synthesized cDNA can be used for qPCR analysis.

[0108] The RT enhancer used from the kit degrades dsDNA during transcription of RNA and is inactivated in a second step at 95° C. This eliminates the need for DNase treatment.

[0109] observation Gene expression profiles demonstrated the purity of enriched RPE cells compared to unenriched populations of RPE cells. A representative heat map comprised of key non-RPE gene sets (epithelial-mesenchymal transition genes and cranial neural crest-like) showed downregulation in enriched RPE cells (Figure 4C), while a representative heat map comprised of key RPE-specific gene sets (melanogenesis and late RPE) showed upregulation in enriched RPE cells (Figure 4D).

[0110] RT-qPCR Analysis of the expression of various genes by RT-qPCR. RT-qPCR was performed using a QuantStudio3 real-time PCR instrument. -ddCt The results were analyzed using the RT-qPCR method. RT-qPCR experiments were preferably performed with minimal light. xi. qPCR using SYBR green: Prepare a 20 μL reaction mixture containing 10 μL of SYBR green mix (2×), 1 μL each of forward and reverse primers (stock 10 μM), 2 μL of cDNA (50 ng), and 6 μL of water. xii. The running parameters for the qPCR reactions were as follows:

[0111] [Table 8]

[0112] xiii. qPCR using TaqMan probes: A 20 μL reaction mixture was prepared containing 10 μL of TaqMan master mix (2×), 1 μL of gene-specific probe (stock 20×), 2 μL of cDNA (50 ng), and 7 μL of water. xiv. The running parameters for the qPCR reactions were as follows:

[0113] [Table 9]

[0114] xv. Quantification of gene expression: Here, the expression of the test gene was normalized to the expression level of β-actin / GAPDH as a housekeeping gene. The fold change was calculated by normalizing the expression of the sample (RPE) to the control (iPSC).

[0115] observation Gene expression analysis results for selected markers of RPE are depicted in Figure 3. Gene expression analysis using qPCR for selected markers was expressed as fold change compared to iPSCs. All positive markers, such as PMEL17, MITF, RPE65, TYR, and TYRP1 (Figure 3A), showed relatively high expression, while negative markers, such as OCT4, alpha-fetoprotein (AFP), heart and neural crest derivatives expressed 2 (HAND2), and TH (Figure 3B), showed negligible expression in the iPSC-derived RPE of the present disclosure.

[0116] Measurement of extracellular secretion of proteins by enzyme-linked immunosorbent assay (ELISA) Secretion of pigment epithelium-derived growth factor (PEDF) is an important criterion for assessing the polarity of in vitro generated RPE. PEDF was quantified enzymatically using a commercially available kit (Human Serpin F1 / PEDF Duoset ELISA Kit). Timing: 4-5 hours i. ELISA plates were coated with 100 μL of capture antibody and incubated overnight at room temperature (20-25°C). ii) The coated wells were washed three times with washing buffer. All steps were carried out at room temperature (20-25°C). iii. 300 μL of reagent diluent was added as blocking solution and the plate was incubated for 1 hour. The washing procedure with wash buffer was repeated three times. iv. 100 μL of sample (on the day the cell supernatant was collected) and standards diluted in reagent diluent were added to the coated wells, and the plate was incubated for 2 hours. The plate was carefully washed three times with wash buffer. v. 100 μL of detection antibody was diluted in reagent diluent and the plate was incubated for 2 minutes. vi. The plate was carefully washed three times with wash buffer. vii. 100 μL of streptavidin-HRP was added to each well. The plate was covered and incubated in the dark for 20 minutes. viii. The plate was carefully washed three times with wash buffer. ix. 100 μL of substrate solution was added and incubated for 20 minutes, making sure to avoid exposing the plate to direct sunlight. 50 μL of stop solution was added to each well and the plate was mixed gently. xi. The optical density of each well was immediately determined using a Varioskan LUX microplate reader at 450 nm and at 540 nm for background subtraction. xii. The concentration of PEDF from the samples was analyzed by plotting the standard values ​​as a graph.

[0117] observation ELISA-based quantification of secreted PEDF is depicted in Figure 3C. ELISA-based quantification of secreted pigment epithelium-derived growth factor (PEDF) from in vitro culture supernatants at two different time points (days 75 and 120) demonstrated an increase in the polarization of RPE obtained by the disclosed method.

[0118] Example 5 Preclinical safety and efficacy studies in animal models Subretinal transplantation of frozen-thawed RPE cells in an animal model (rat) was performed to evaluate the efficacy and safety of RPE cells in vivo, thereby providing important investigational new drug (IND) validating preclinical data.

[0119] a. Animal maintenance Timing: 2 months i. Raise a colony of pigmented RCS (RCS-p+ / Lav) rats (NIH Rat Resource and Research Center, #315). ii. Animals were fed a standard laboratory diet and maintained on a 12 hour dark / light cycle. iii. Animals were weaned on postnatal day (PD) 21 and administered oral cyclosporine A (210 mg / L; Gengraf, North Chicago, IL) in drinking water. Animals were maintained on cyclosporine A until sacrifice.

[0120] e.Cell preparation Preparing the cells prior to injection under sterile conditions and following the disclosed protocol was crucial for optimal performance of the cells after implantation into the animal.

[0121] Timing: 1 hour a. Cells were removed from liquid nitrogen storage and thawed in a 37°C water bath for 2 minutes. b. 1 mL of pre-warmed DMEM / F12 medium was added dropwise to the vial. c. The cells were transferred to a 15 mL tube containing 5 mL of pre-warmed DMEM / F12 medium. d. 1 mL of DMEM / F12 medium was used to rinse the cryotube and added to the 15 mL tube. e. The cells were centrifuged at 150 g for 5 minutes at room temperature (20-25°C). f. The supernatant was aspirated and the cells were resuspended in 1 mL of pre-warmed DMEM / F12 medium. g. Carefully pipette up and down to resuspend the cells. h. 10 μL of the cell suspension was removed and added to 10 μL of 0.08% trypan blue solution. i. After pipetting up and down, the cell suspension was loaded onto a hemocytometer (10 μL on each side). j. Viable cells were counted. k. The cell suspension was centrifuged again at 150 g for 5 minutes. l. Cells were resuspended in the appropriate volume to achieve the desired concentration for injection.

[0122] f. Subretinal injection Cryopreserved RPE frozen at days 75-80 were tested for subretinal injection, with the hypothesis that iPS-RPE at this stage would be suitable for application in transplantation studies. Although transplantation of cells at earlier stages of differentiation has been possible, the behavior of progenitor cells in vivo is unknown.

[0123] Timing: 1 day a. One hour before injection, pupils were dilated with 1% tropicamide and 2.5% phenylephrine. b. The eyes were anesthetized with topical 0.5% proparacaine HCl. c. Animals were sedated with intraperitoneal ketamine / xylazine (100 / 10 mg / kg). d. An incision was made at the corner of the eyelid to allow visualization of the posterior retina. A hemostat was used to reduce blood pooling. e. A small scleral / choroidal incision (approximately 1 mm) 2 mm posterior to the limbus was made in the dorsotemporal region using a 27 gauge needle tip. f. A small lateral corneal puncture was made with a 30-G needle to limit the increase in intraocular pressure and reduce cell spillage after injection. g. Two microliters of cell suspension containing the total cell dose or control medium was delivered into the subretinal space using a fine glass pipette (inner diameter, 75-150 μm) inserted into the subretinal space. The glass pipette was connected to a 10 μL Hamilton syringe (Hamilton, Reno, NV) with a small-bore (400 μL total volume) microtube. h. After delivery, the retina was observed under a microscope using a cover slip and subretinal blebs were scored based on size and any problems associated with the injection, such as bleeding, air bubbles, cells in the vitreous. i. 0.5% erythromycin ointment was placed on the eyes and the animals were allowed to recover from anesthesia. j. On the day of surgery, dexamethasone (1.0 mg / kg) was administered intraperitoneally. To minimize a possible inflammatory response, animals received dexamethasone every other day for two weeks after cell transplantation.

[0124] Ensure that injections are performed between PD 22 and PD 25. Animals should be placed on cyclosporine A one day prior to injection.

[0125] g. Optokinetic Tracking (OKT) Optokinetic thresholds were assessed using a virtual optokinetic system (VOS; Cerebral Mechanics, Inc.), consisting of four computer monitors arranged in a square with the displays facing inward. On the monitors, a virtual cylinder displayed a sinusoidal lattice pattern, which rotated either clockwise or counterclockwise, allowing independent assessment of both the left and right eyes; the left eye responded to clockwise movements, and the right eye responded to counterclockwise movements.

[0126] Timing: 1 day a. The animal was placed on a platform in the center of four computer monitors. The animal was allowed to habituate to the platform for 5 minutes. b. Using Cerebral Mechanics software, a simple staircase test was set up for both eyes with contrast set at 100%. c. The program started with a low spatial frequency; when the cylinder rotated and the grid shifted, the animal would respond with a reflexive head and neck movement to track the grid. If the animal responded, a "yes" was clicked; if the animal did not respond, a "no" was clicked. Based on this input, the spatial frequency of the grid was then gradually increased until the animal no longer tracked the stimulus, resulting in the maximum spatial frequency threshold. d. Once testing was completed, the animals were removed from the platform and returned to their home cages.

[0127] RCS rats have near-normal OKT through PD 60. To determine functional recovery resulting from cell transplantation, this test was most optimal from PD 90 onwards.

[0128] observation Behavioral analysis (functional testing) in balanced salt solution (BSS, vehicle)- and RPE-injected RCS rats was measured by optokinetic threshold (OKT). Stripe visual acuity in RCS rats rapidly declined from birth to 4 months of age, ranging from 0.8 to 0.3 c / d. Visual acuity in cell-treated eyes across all doses (low, medium, and high) remained constant for 70 days after injection (Figure 5A).

[0129] e. Fundus photography A fundus or retinal camera was designed to observe the inner region of the eye, i.e., the retina. Fundus images were taken at various time points after injection to verify successful engraftment of cells and the length of cell survival.

[0130] Timing: 1 day a. Mydriasis was achieved with 1% tropicamide and 2.5% phenylephrine. b. The eyes were anesthetized with topical 0.5% proparacaine HCl. c. Animals were sedated with intraperitoneal ketamine / xylazine (100 / 10 mg / kg). d. The eye was lubricated with 2.5% Goniovisc hypromellose. e. A Micron IV (Phoenix-Micron, Inc.) imaging system was used to capture bright-field images of the retina using white light. f. During imaging, the optic nerve head was used as a guide, and the animal was positioned so that the peripheral / temporal region of the retina where the injection was to occur was in view. g. 0.5% erythromycin ointment was placed on the eyes and the animals were allowed to recover from anesthesia.

[0131] observation Fundus imaging of saline solution (BSS) and RPE-injected (subretinal) eyes of RCS rats showed patches of pigmented (black) RPE cells in the cell-injected group (Figure 5C).

[0132] f. Tissue preparation Tissue samples were appropriately collected and prepared for histology and immunohistochemistry studies. Sample preparation included processes such as fixation, dehydration, embedding, and sectioning.

[0133] Timing: 5 days a. After euthanasia according to IACUC guidelines, eyes were enucleated as quickly as possible. b. Enucleated eyes were placed in cold 4% paraformaldehyde (PFA), trying to maintain a fixative to tissue ratio of 10-20x. After 10-60 minutes of fixation, the anterior chamber of the eye, including the lens, was removed and the remaining eye cup was placed back into 4% PFA. The samples were placed on ice in PFA. d. The eyes were placed at 2-8°C for 24-48 hours. e. After 24–48 h, the PFA solution was removed and an equal volume of cold 10% sucrose solution in 1× PBS was added. f. The eyes were placed at 2-8°C for 24 hours. g. The 10% sucrose solution was removed and replaced with an equal volume of cold 20% sucrose solution. h. The eyes were placed at 2-8°C for 24 hours. i. The 20% sucrose solution was removed and replaced with an equal volume of cold 30% sucrose solution. j. The eyes were placed at 2-8°C for 24 hours. k. The eyecup was removed from the 30% sucrose solution and lightly dried on a small piece of filter paper. l. The eyecup was placed cut side down on the filter paper and the vitreous humor was allowed to drain from the eyecup. This process was repeated until the filter paper remained dry. m. The eyecup was placed in a labeled cryomold and filled with OCT medium. n. The eye cup was oriented toward the anterior side of the cryomold, with the injection site oriented at approximately 2 o'clock for the OS (left eye, left) eye and 10 o'clock for the OD (right eye, right) eye. The cryomold containing the eye was frozen in a liquid nitrogen bath. It was ensured that the liquid nitrogen did not directly contact the OCT medium but only the walls of the cryomold (Figure 3C), as direct contact of liquid nitrogen with the OCT medium would create bubbles and compromise sample integrity. p. Once the medium was no longer shiny, it was removed from the liquid nitrogen bath. q. The cryomolds were wrapped tightly in aluminum foil to try to limit the block's exposure to air. r. The wrapped block was placed in a -20°C freezer.

[0134] g.Histology / Immunohistochemistry Hematoxylin and eosin (H&E) and immunohistochemical staining are valuable tools for detecting histopathological changes and specific antigens in tissues.

[0135] Timing: 2-4 weeks a. Eye blocks were sectioned at 12 μm. b. Approximately 40 slides containing 4 sections per slide were obtained from each eye. c. Sections were collected in a series of 5 slides to provide representative sections every 60 μm on each slide throughout the eyecup. d. The first slide of each series was stained with cresyl violet or hematoxylin and eosin and examined for evidence of retinal damage / toxicity and photoreceptor recovery. b. For immunohistochemistry, slides were allowed to dry for at least 30 minutes after removal from the freezer. c. Slides were blocked with 4% horse serum, 1% bovine serum albumin (BSA), 0.5% TritonX in phosphate-buffered saline (PBS) for 45 minutes. d. Primary antibody (anti-cone arrestin, generously provided by W. Clay Smith, PhD, University of Florida) was added in blocking buffer and incubated overnight (minimum 15 hours) at 4°C. e. The next day, the slides were washed 3 x 5 min with 1 x PBS. f. Secondary antibody (1:300) was added in blocking buffer and incubated at room temperature (20-25°C) in the dark for 45 minutes. g. The slides were washed 3 x 5 min with 1 x PBS. h. Slides were incubated with DAPI in the dark for 10 minutes. i. Slides were washed 2 x 5 min with 1 x PBS. j. A coverslip was placed on the slide with 100 μL of Fluoromount G.

[0136] observation Quantification of retinal thickness and cone number between the nasal and temporal regions of the retina from low-, medium-, and high-dose RPE transplant animals was calculated by immunostaining retinal sections with cone arrestin. The data showed significant preservation of the ONL in the temporal region compared with the nasal region (Figure 5B).

[0137] h. Photoreceptor quantification The outer nuclear layer thickness (ONL) was measured with the aid of immunohistochemical staining as a primary indicator of photoreceptor recovery.

[0138] Timing: 1-2 weeks a. Stained slides were imaged using a confocal laser scanning microscope (Leica SP5 with LAS AF software; Leica). Laser intensity settings (gain) were kept constant for each emission wavelength. bZ stack images were collected at ×10 and ×20 magnification with a 1 μm step size at 1024 × 1024 resolution. c. Color channels were kept separate as each color channel z-stack was flattened. Each color channel image was saved, and the color merged image was saved as a TIFF file. d. ONL cell bodies were counted within columns of the ONL in both the temporal (injected) and nasal (non-injected) regions to obtain retinal thickness-nuclear values. e. Cone arrestin positive cells were counted in both the temporal (injected) and nasal (non-injected) regions to obtain a cone value per image. f. Counts were performed by three observers blinded to dose and age group. g. Mean ONL counts and photoreceptor counts per group using data collected in steps 4, 5, and 6 and current data from both temporal (injected) and nasal (non-injected) regions.

[0139] observation Immunostaining of retinal tissue sections (HNM) showed survival of transplanted RPE cells in the subretinal space at P90 (passage 90), with a preserved ONL layer (cone arrestin) and a well-appearing retina, indicating visual recovery.

[0140] Overall, the disclosed method provides an efficient protocol for obtaining enriched mature RPE cells of high purity and significant therapeutic potential.

[0141] Advantages of the present disclosure The present disclosure provides a method for obtaining RPE cells from iPSCs that exhibits the following advantages: (a) The method produced mature, pigmented, and polarized RPE that exhibited functional properties, such as secretion of the cytokines PEDF and VEGF from the apical and basal chambers. (b) De novo generated RPE cells are mature, ciliated, and mimic the morphology, ultrastructural, and molecular characteristics of native RPE; they are also suitable for in vivo cell transplantation studies. (c) The method includes an enrichment step, thereby making the method useful for large-scale GMP production of pure RPE populations. (d) RPE cells produced according to this method are suitable for cell replacement therapy for macular degeneration. (e) RPE cells produced according to the present methods may provide benefit through their neuroprotective effects against other retinal diseases associated with early and late photoreceptor degeneration. (f) Its similarity to native RPE tissue in terms of structure, molecular characteristics, and function makes it suitable for modeling diseases associated with retinal degeneration.

Claims

1. A method for obtaining RPE (retinal pigment epithelial) cells from iPSCs (induced pluripotent stem cells), comprising: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in non-adherent suspension culture; (b) plating the embryoid bodies onto a culture dish coated with a suitable extracellular matrix in a differentiation-inducing medium (DIM) and culturing for 6 to 8 days to obtain a neuroectodermal lineage, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors; (c) culturing the neuroectodermal lineage for rosette formation in differentiation and proliferation medium (DPM) for 11 to 22 days, wherein the DPM does not contain any inhibitors; (d) culturing the rosettes of step (c) in retinal pigment epithelium maturation medium (RPEMM) for 23 to 45 days to promote retinal progenitor cell formation; and (e) plating the retinal progenitor cells of step (d) onto a culture dish coated with a suitable extracellular matrix and culturing them in RPEMM for 47 to 75 days to obtain RPE cells; A method comprising:

2. The method comprises: a) enzymatically dissociating the RPE cells from the extracellular matrix to obtain a single cell suspension of RPE; and b) plating the single cell suspension of RPE from step (a) onto a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 75-100 days; a step of concentrating the RPE cells, The method of claim 1 further comprising:

3. 10. The method of claim 1, wherein the culture of iPSCs has a confluency in the range of 80% to 90%.

4. The step of preparing the embryoid bodies in step (a) comprises: (a) culturing the iPSCs in a growth medium for 24 hours to form embryoid bodies, the growth medium comprising an expansion medium and a ROCK inhibitor; and (b) gradually contacting the developing embryoid bodies from the growth medium to the DIM by (i) culturing the developing embryoid bodies in a medium composition comprising the expansion medium and DIM in a ratio of 3:1 for 24 hours, (ii) culturing the developing embryoid bodies obtained in step (i) in a medium composition comprising the expansion medium and DIM in a ratio of 1:1 for 24 hours, and (iii) culturing the embryoid bodies obtained in step (ii) in DIM for 24 hours, followed by plating the embryoid bodies onto a culture dish.

2. The method of claim 1, comprising:

5. The rosette formation in step (c) (i) maintaining the neuroectodermal lineage in DIM for 24 hours; (ii) maintaining the neuroectodermal lineage obtained in step (i) in a medium composition comprising DIM and DPM in a 1:1 ratio for 24 to 48 hours; and (iii) culturing the neuroectodermal lineage obtained from step (ii) in DPM for 24 hours to promote rosette formation.

2. The method of claim 1, comprising:

6. The formation of retinal progenitor cells in step (d) (i) maintaining the rosettes in DPM for 24 hours; (j) maintaining the rosettes obtained in step (i) in a medium composition comprising DPM and RPEMM in a 1:1 ratio for 24 hours; and (k) culturing the rosettes obtained from step (j) in RPEMM for 24 hours to promote the formation of retinal progenitor cells.

2. The method of claim 1, comprising:

7. 3. The method of claim 1 or 2, wherein the at least one suitable extracellular matrix is ​​selected from matrigel, laminin, vitronectin, fibronectin, collagen, poly-L-lysine, poly-L-ornithine, or a combination thereof.

8. 2. The method of claim 1, wherein the at least one WNT pathway inhibitor is selected from 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide, 5-(phenylsulfonyl)-N-piperidin-4-yl-2(trifluoromethyl)benzenesulfonamide, 2-(2',3-dimethyl-[2,4'-bipyridin]-5-yl)-N-(5-(pyrazin-2-yl)pyridin-2-yl)acetamide, 2-(4-(2-methylpyridin-4-yl)phenyl)-N-(4-(pyridin-3-yl)phenyl)acetamide, 8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one, or a combination thereof.

9. The at least two SMAD pathway inhibitors are selected from the group consisting of 4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazol-2-yl]benzamide, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine, 5-chloro-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperazin-1-yl]piperazin-2-yl]benzamide, 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)pyridin-2-amine, and 5-chloro-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperazin-1-yl]piperazin-2-yl]pyrazole.

10. The method of claim 1, wherein the compound is selected from the group consisting of 5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, 9-ethyl-6,6-dimethyl-8-(4-morpholin-4-ylpiperidin-1-yl)-11-oxo-5H-benzo[b]carbazole-3-carbonitrile, 5-chloro-2-N-(5-methyl-4-piperidin-4-yl-2-propan-2-yloxyphenyl)-4-N-(2-propan-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, or a combination thereof.

10. 5. The method of claim 4, wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

11. 3. The method of claim 2, wherein the enzymatic dissociation of RPE cells in step (a) is carried out using an enzyme selected from the group consisting of Accutase, Tryple select, TrypLE, Gentle Cell Dissociation Reagent (GCDR), and Dispase.

12. A retinal pigment epithelial cell or a population thereof produced by the method of claim 1.

13. A pharmaceutical composition comprising the retinal pigment epithelial cells or a population thereof according to claim 12 and a pharmaceutically acceptable carrier.

14. 14. The pharmacetical composition of claim 13 for use in the treatment of a retinal degenerative disease.

15. 15. The pharmaceutical composition of claim 14, wherein the retinal degenerative disease is selected from the group consisting of age-related macular degeneration and retinal diseases associated with early and late photoreceptor degeneration.

16. 1. A method of treating a retinal degenerative disease in a subject, comprising: Administering the pharmaceutical composition of claim 13 to the subject. A method comprising:

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