Scalable methods for producing retinal pigment epithelium (RPE) cells

Abstract

The present disclosure provides a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) comprising: (a) generating an embryoid from a culture of iPSC wherein the embryoid is in a non-adherent suspension culture state, (b) planking the embryoid in a differentiation induction medium (DIM) on a culture dish coated with a suitable extracellular matrix, wherein the DIM comprises at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors, (c) culturing the neuroectoderm lineage in a differentiation proliferation medium (DPM) for rose knot formation, (d) culturing the rose knot of step (c) in a retinal pigment epithelium maturation medium (RPEMM) to promote retinal progenitor cell formation, and (e) planking the retinal progenitor cells of step (d) in the RPEMM on a petri dish coated with a suitable extracellular matrix to obtain RPE cells.

Description

Technical Field

[0001] The present disclosure relates to the field of cell culture techniques and cell differentiation and methods thereof, and in particular to a cell culture method for generating retinal pigment epithelium (RPE) cells from induced pluripotent stem cells (iPSCs). Background Art

[0002] Macular degeneration is also referred to as age-related macular degeneration (AMD), and it is the leading cause of blindness in people over 60 years old worldwide. Clinically, this disease begins with the distortion of central vision, which is caused by the damage of the macula, and eventually leads to legal blindness. Vision loss has a significant impact on quality of life, and brings huge losses to the economy. In addition, AMD is a complex, progressive neurodegenerative disease, and due to the loss of retinal pigment epithelium (RPE) and photosensitive photoreceptors (retinal pigment epithelium (RPE) supports, protects photosensitive photoreceptors and provides nutrition to them), AMD triggers visual impairment. At present, there is no curative treatment for the most common form (i.e. dry AMD) of the disease. Therefore, there is an urgent need to develop a therapeutic strategy to solve the loss of RPE cells and provide a cure for AMD. Summary of the invention

[0003] In one aspect of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, comprising the following steps: (a) generating embryoid bodies from a culture of iPSC, wherein the embryoid bodies are in a non-adherent suspension culture state, (b) plating the embryoid bodies in a differentiation induction media (DIM) on a culture dish coated with a suitable extracellular matrix, 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 in a differentiation propagation media (DPM) for 11 to 22 days for rosette formation, wherein the DPM does not comprise any inhibitors, (d) culturing the rosettes of step (c) in a retinal pigment epithelium maturation medium (Retinal Pigment Epithelium Maturation Medium) for 11 to 22 days for rosette formation. (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.

[0004] In one aspect of the present disclosure, there is provided a retinal pigment epithelial cell or a population thereof produced by a method as disclosed herein.

[0005] In one aspect of the present disclosure, a pharmaceutical composition is provided, comprising retinal pigment epithelial cells produced by the method as disclosed herein; and a pharmaceutically acceptable carrier.

[0006] In one aspect of the present disclosure, there is provided a method of treating a retinal degenerative disease in a subject, comprising: administering to the subject a composition as disclosed herein.

[0007] These and other features, aspects and advantages of the subject matter will be better understood with reference to the following description. This summary is provided to introduce selected concepts in a simplified form. This summary is not intended to identify key features or essential features of the subject matter claimed, nor is it intended to be used to limit the scope of the subject matter claimed. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] The following drawings constitute part of this specification and are included to further illustrate aspects of the present disclosure. The present disclosure may be better understood by referring to the drawings in conjunction with the detailed description of specific embodiments presented herein.

[0009] Figure 1Depicts the differentiation of RPE, A) embryoid body formation from iPSCs and their specialization to a retinal fate through neuroectoderm induction; B) formation of rosette structures and epithelial clusters, indicating the onset of retinal differentiation; C) retinal progenitor cells show commitment to the fate of RPE cells with the onset of pigmentation; D) RPE cultures with increased pigmentation levels and typical hexagonal morphology; E) RPE culture plates with visible (by naked eye) pigmentation patches; F) mature RPE cell pellets after 70 to 80 days in culture; scale bar is 100 μm, which is consistent with one embodiment of the present disclosure.

[0010] Figure 2 Describe the RPE characterization carried out by immunofluorescence using stage-specific antibodies, A to D) immunostaining of the RPE culture in iPSC source, for checking the marker expression during the progressing day of retinal differentiation.Image shows the expression of following RPE directional differentiation marker (commitment marker): microphthalmia-associated transcription factor (MITF) (A), pigmentation-specific protein tyrosinase-related protein (Tyrosinase related protein, TYRP1) (B) and melanocyte protein (PMEL17) (C), RPE maturation marker (RPE65) (D); Quantification of the RPE differentiation purity carried out by immunophenotyping, flow cytometry shows the late RPE maturation marker of high percentage, such as TYRP1, RPE65 and TYROSINASE-TYR (E), which is consistent with an embodiment of the present disclosure.

[0011] Figure 3 Depicted are gene expression analysis of selected markers using qPCR, expressed as fold change compared to iPSC, in which all positive markers (A) had higher expression, while negative markers (B) showed negligible expression in iPSC-derived RPE; C) ELISA-based quantification of secreted pigment epithelium-derived growth factor (PEDF) from in vitro culture supernatant at two different time points (day 75 and day 120); images and graphs represent a minimum of three independent experiments, which is consistent with one embodiment of the present disclosure.

[0012] Figure 4Depicted are A) phase contrast microscopy images of committed and mature RPE cells at low (10×) and high magnification (20×), showing dense pigmentation; B) photographs of 6-well plates with late-stage RPE cultures and 15 ml tubes (inset) containing RPE cells after centrifugation during the enrichment and expansion process; C) and D) show gene expression profiles of the purity of enriched RPE cells when compared to non-enriched populations, with a representative heat map consisting of important non-RPE gene sets showing downregulation in enriched RPE, and a representative heat map consisting of important RPE-specific gene sets showing upregulation in enriched RPE, which is consistent with an embodiment of the present disclosure.

[0013] Figure 5 Depicted are A) behavioral analysis (functional testing) results of balanced salt solution (BSS, vehicle) and RPE injected RCS rats by measuring optokinetic threshold (OKT); B) quantification of retinal thickness and cone number between the nasal and temporal regions of the retina from animals transplanted with low, medium and high doses of RPE by immunostaining of retinal sections with cone arrestin; C) fundus imaging of saline (BSS) and RPE injected (subretinal) eyes of RCS rats; D) immunostaining (HNM) showed that transplanted RPE cells survived in the subretinal space at P90, with a preserved ONL layer (cone arrestin) and a good-looking retina, indicating that vision was rescued, consistent with one embodiment of the present disclosure. DETAILED DESCRIPTION

[0014] Those skilled in the art will appreciate that, in addition to those specifically described, the present disclosure may be varied and modified. It should be understood that the present disclosure includes all such variations and modifications. The present disclosure also includes all such steps, features, compositions and compounds mentioned or indicated in this specification individually or collectively, and any and all combinations of any one or more of such steps or features.

[0015] definition

[0016] For convenience, certain terms and embodiments used in this specification are explained here before further describing the present disclosure. These definitions should be interpreted and understood by those skilled in the art in light of the remainder of the present disclosure. The terms used herein have meanings that are recognized and known to those skilled in the art, however, for convenience and completeness, specific terms and their meanings are set forth below.

[0017] An unmodified article is used to refer to one or more than one (ie, at least one) of the grammatical object of the article.

[0018] The terms "include" and "comprising" and variations thereof are used in an inclusive open-ended sense, meaning that additional elements may be included. They are not intended to be interpreted as "consisting only of. . . . "

[0019] Throughout this specification, unless the context requires otherwise, the words "comprises / comprising" and variations thereof will be understood to imply the inclusion of stated elements or steps or groups of elements or steps but not the exclusion of any other elements or steps or groups of elements or steps.

[0020] The term "includes / comprising" is used to mean "including / comprising but not limited to." "Includes / comprising" and "including / comprising but not limited to" are used interchangeably.

[0021] The term "induced pluripotent cell (iPSC)" used herein refers to a pluripotent cell derived from an adult somatic cell by ectopic expression of a set of transcription factors. The developmental stages experienced by iPSCs are the formation of neural ectoderm, eye field specialization, and a double-layered optic cup from the optic vesicle.

[0022] As used herein, the term "retinal pigment epithelium (RPE) cells" refers to a single layer of pigmented cells derived from the neuroectoderm of the optic cup and constituting the outermost layer of the retina. The RPE is required to keep the retina alive and enable photoreceptor cells to detect light.

[0023] The term "embryoid body (EB)" used herein refers to a three-dimensional aggregate formed by pluripotent stem cells (pluripotent stem cell, PSC) in suspension, including embryonic stem cells (embryonic stem cell, ESC) and induced pluripotent stem cells (iPSC), which simulate the structure of the developing embryo and have the potential 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 PSC.

[0024] As used herein, the term "rosette" refers to a developmental characteristic of neural progenitor cells in culture in which embryonic stem cells are differentiated; rosettes are radial arrangements of columnar cells that express early neuroectodermal markers such as Pax6 and Sox1 and are capable of differentiating into a variety of region-specific neuronal and glial cell types in response to appropriate developmental cues.

[0025] As used herein, the term "inhibitor" 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 a variety of inhibitors used as part of the present disclosure. For example, a "WNT inhibitor" inhibits WNT signaling by preventing ligand-receptor interactions or WNT receptor maturation.

[0026] As used herein, the term "subject" refers to both human and veterinary subjects (eg, rats, non-human primates, dogs, cats, horses, rabbits, pigs, mice, and cows).

[0027] The term "pharmaceutically acceptable carrier" as used herein refers to those substances that can be used to implement the methods disclosed herein and to form the compositions disclosed herein. In general, the nature of the carrier will depend on the specific mode of administration adopted. For example, for subretinal delivery carriers, such as balanced salt solution (BSS) or Hank's balanced salt solution (HBSS).

[0028] The term "confluence" 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% confluence indicates that 60 of the 100 growth surfaces are occupied by cells. During cell culture experiments, confluence is used as an indicator of cell growth and expansion.

[0029] 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, the culture medium in contact with the EBs is converted from the growth medium to the DIM by slowly increasing the proportion of the differentiation induction medium (DIM) while reducing the proportion of the growth medium, thereby promoting the development of embryoid bodies. Therefore, in one aspect of the present disclosure, the EBs are developed for 24 hours in a culture medium composition comprising a 3:1 ratio of expansion medium to DIM; then developed for 24 hours in a culture medium composition comprising a 1:1 ratio of expansion medium to DIM; and finally developed for 24 hours in 100% DIM.

[0030] Ratio, concentration, amount and other numerical data can be presented in the form of range in this article.It should be understood that such range form is only used for convenience and brevity, and should be flexibly interpreted to include not only the numerical value clearly recorded as range limit, but also all single numerical values ​​or sub-ranges included in the range, as each numerical value and sub-range are clearly recorded.Unless otherwise defined, all technical terms and scientific terms used in this article have the same meanings as those of ordinary skill in the art to which the present disclosure belongs.Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, preferred methods and materials are now described.All publications mentioned herein are incorporated herein by reference.

[0031] The scope of the present disclosure is not to be limited by the specific embodiments described herein, which are intended for purposes of illustration only. As described herein, functionally equivalent products, compositions, and methods are clearly within the scope of the present disclosure.

[0032] Many ophthalmic diseases (such as age-related macular degeneration) are relevant to the degeneration or deterioration of retina itself or RPE. It is imperative to realize the reservation of photoreceptor rescue and visual function by subretinal transplantation of RPE cells. It is necessary to find the mode of producing RPE cells with a robust and scalable method, such as producing the method of RPE cells from human stem cells, and the RPE cells can be used for treating retinal degenerative diseases and damage.

[0033] According to the present disclosure, there is provided a method for producing RPE by a combination of chemical and manual selection processes, which is used to enrich RPE in the early and late stages of the differentiation process. In principle, pluripotency factors such as FGF2 are suppressed by using dual SMAD and WNT inhibition, so that iPSC is induced into a neural ectoderm fate. This is achieved by using small molecules such as SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-pyridin-2-yl-1H-imidazole-2-yl] benzamide) and LDN193189 (4-(6-(4-(piperazine-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). SB431542 inhibits the activin / TGF-β pathway and LDN193189 acts as a BMP4 / 7 inhibitor, while IWR1 (4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylene-2H-isoindol-2-yl)-N-8-quinolyl-benzamide) silences the canonical WNT signaling pathway.

[0034] In order to promote retinal cell proliferation, the embryoid bodies obtained from iPSC are cultivated in a low serum medium supplemented with N1, so as to form retinal progenitor cells, which are enriched to produce mature RPE.Mature RPE shows as tightly packed hexagonal cells with tight junctions, and brown to black pigmentation, and apical-basal polarity.The method produces pigmented and ciliated RPE, which shows functional characteristics such as polarized secretion of cytokines (PEDF and VEGF).The characterization method for determining its authenticity and purity is also formulated together with the method.The present disclosure also provides a pharmaceutical composition comprising RPE cells.In addition, the present disclosure provides a method for treating retinal degenerative diseases using the pharmaceutical composition.

[0035] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, comprising the following steps: (a) generating embryoid bodies from a culture of iPSC, wherein the embryoid bodies are in a non-adherent suspension culture state, (b) plating the embryoid bodies in a differentiation induction medium (DIM) on a culture dish coated with a suitable extracellular matrix, 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 inhibitor, (c) culturing the neuroectodermal lineage in differentiation proliferation medium (DPM) for 11 to 22 days for rosette formation, wherein the DPM does not contain any inhibitor, (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.

[0036] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the method also includes enriching RPE cells, which includes: (a) enzymatically dissociating RPE cells from an extracellular matrix to obtain a single cell suspension of RPE; (b) plating the single cell suspension of RPE of step (a) on a culture dish coated with a suitable extracellular matrix and culturing in RPEMM for 75 to 100 days.

[0037] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, comprising the following steps: (a) generating embryoid bodies from a culture of iPSC, wherein the embryoid bodies are in a non-adherent suspension culture state, (b) plating the embryoid bodies in a differentiation induction medium (DIM) on a tissue culture dish coated with a suitable extracellular matrix, 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 in a differentiation proliferation medium (DPM) for 11 to 22 days for rosette formation. , wherein the DPM does not contain any inhibitor, (d) the rosettes of step (c) are cultured in retinal pigment epithelium maturation medium (RPEMM) for 23 to 45 days to promote retinal progenitor cell formation, and (e) the retinal progenitor cells of step (d) are plated on a culture dish coated with a suitable extracellular matrix, and cultured in RPEMM for 47 to 75 days to obtain RPE cells; wherein the method also includes enriching RPE cells, which comprises: (a) enzymatically dissociating RPE cells from the extracellular matrix to obtain a single cell suspension of RPE; (b) the single cell suspension of the RPE of step (a) is plated on a culture dish coated with a suitable extracellular matrix, and cultured in RPEMM for 75 to 100 days.

[0038] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the confluence range of the culture of iPSC is 80% to 90%. In another embodiment, wherein the confluence range of the confluent culture of iPSC is 80% to 85%.

[0039] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSCs (induced pluripotent stem cells) is provided, wherein 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 with DIM by: (i) culturing the developing embryoid bodies in a medium composition comprising a 3:1 ratio of expansion medium to DIM for 24 hours, (ii) culturing the developing embryoid bodies obtained in step (i) in a medium composition comprising a 1:1 ratio of expansion medium to DIM for 24 hours, and (iii) culturing the embryoid bodies obtained in step (ii) in DIM for 24 hours, and then plating the embryoid bodies on a tissue culture dish.

[0040] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, 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 culture medium composition comprising a 1:1 ratio of DIM and DPM for 24 to 48 hours; and iii) culturing the neuroectodermal lineage obtained from step (ii) in DPM for 24 hours to promote rosette formation.

[0041] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the retinal progenitor cell formation in step (d) comprises: (i) maintaining rosettes in DPM for 24 hours; (ii) maintaining the rosettes obtained in step (i) in a culture medium composition comprising a 1:1 ratio of DPM and RPEMM 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.

[0042] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, comprising the following steps: (a) generating embryoid bodies from a culture of iPSCs with a confluence range of 80% to 90%, wherein the embryoid bodies are in a non-adherent suspension culture state, (b) plating the embryoid bodies in a differentiation induction medium (DIM) on a culture dish coated with a suitable extracellular matrix, and culturing for 6 to 8 days to obtain a neural ectoderm lineage, wherein the DIM contains at least one WNT pathway inhibitor and at least two SMAD pathway inhibitors, (c) plating the neural ectoderm lineage in a differentiation proliferation medium (DIM) to obtain a neural ectoderm 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 in RPEMM for 47 to 75 days to obtain RPE cells; wherein in step (a), generating embryoid bodies comprises: (a) culturing the confluent iPSCs in growth medium for 24 hours to form embryoid bodies, wherein the growth medium comprises expansion medium and ROCK inhibitor, and (b) gradually contacting the developing embryoid bodies from the growth medium with DIM by: (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 and subsequently plating the embryoid bodies on a culture dish; wherein the rosette formation in step (c) comprises: i) maintaining the neural ectoderm lineage in DIM for 24 hours; ii) plating the neural ectoderm lineage obtained in step (i) The ectodermal lineage is maintained in a culture medium composition comprising a 1:1 ratio of DIM and DPM for 24 to 48 hours; and iii) the neuroectodermal lineage obtained from step (ii) is cultured in DPM for 24 hours to promote rosette formation; and wherein the retinal progenitor cell formation in step (d) comprises: (i) maintaining rosettes in DPM for 24 hours; (ii) maintaining the rosettes obtained in step (i) in a culture medium composition comprising a 1:1 ratio of DPM and RPEMM for 24 hours; and (iii) culturing the rosettes obtained from step (ii) in RPEMM for 24 hours to promote retinal progenitor cell formation.

[0043] In one embodiment of the present disclosure, wherein the growth medium comprises expansion medium and a ROCK inhibitor; wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

[0044] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, 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.

[0045] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the at least one WNT pathway inhibitor is selected from 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylene-2H-isoindol-2-yl)-N-8-quinolyl-benzamide, 5-(phenylsulfonyl)-N-piperidin-4-yl -2(trifluoromethyl)benzenesulfonamide, 2-(2',3-dimethyl-[2,4'-bipyridyl]-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.

[0046] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSCs (induced pluripotent stem cells) is provided, wherein the at least two SMAD pathway inhibitors are selected from 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)- -yl)pyridin-2-amine, 5-chloro-2-N-[2-methoxy-4-[4-(4-methylpiperazin-1-yl)piperidin-1-yl]phenyl]-4-N-(2-propane-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-propane-2-yloxyphenyl)-4-N-(2-propane-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, or a combination thereof. In another embodiment of the present disclosure, at least two of the 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.

[0047] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the ROCK inhibitor is (1R,4r)-4-((R)-1-aminoethyl)-N-(pyridin-4-yl)cyclohexanecarboxamide.

[0048] In one embodiment of the present disclosure, a method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells) is provided, wherein the enzymatic dissociation of RPE cells in step (a) is performed using an enzyme selected from the group consisting of Accutase, Tryple select, TrypLE, Gentle Cell Dissociation Reagent (GCDR) and Dispase.

[0049] In one embodiment of the present disclosure, there is provided a retinal pigment epithelial cell or a population thereof produced by a method as disclosed herein.

[0050] In one embodiment of the present disclosure, a pharmaceutical composition is provided, which comprises a retinal pigment epithelial cell or a population thereof as disclosed herein; and a pharmaceutically acceptable carrier.

[0051] In one embodiment, the pharmaceutically acceptable carrier is selected from balanced salt solution (BSS) or Hank's balanced salt solution (HBSS).

[0052] In one embodiment of the present disclosure, there is provided a pharmaceutical composition as disclosed herein for use in the treatment of retinal degenerative diseases.

[0053] In one embodiment of the present disclosure, a composition is provided, wherein the retinal degenerative disease is selected from age-related macular degeneration and retinal diseases associated with early and late photoreceptor degeneration.

[0054] In one embodiment of the present disclosure, there is provided a method of treating a renal degenerative disease in a subject, comprising: administering to the subject a pharmaceutical composition as disclosed herein.

[0055] Although the present subject matter has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense.

[0056] After referring to the description of the subject matter, various modifications of the disclosed embodiments and alternative embodiments of the subject matter will become apparent to those skilled in the art. Therefore, it is contemplated that such modifications can be made without departing from the spirit or scope of the subject matter of the invention as defined.

[0057] Example

[0058] The disclosure will now be illustrated by working examples, which are intended to illustrate the work of the disclosure and are not intended to restrictively imply any limitation to the scope of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the disclosure belongs. Although methods and materials similar or equivalent to the methods and materials described herein can be used to implement the disclosed methods and compositions, exemplary methods, devices and materials are described herein. It should be understood that the disclosure is not limited to the described ad hoc methods and experimental conditions, because such methods and conditions are applicable.

[0059] Example 1

[0060] Material

[0061] The iPSC line TC-1133 (RUCDR / NIH) was obtained from Eyestem Biobank and is available at https: / / eyestem.com / collaborations / research-alliance / .

[0062] All important reagents used in this disclosure were purchased as mentioned in the Key Resources Table.

[0063] Procure / thaw iPSCs and maintain them as undifferentiated cultures in mTeSR-based medium (expansion medium). Prepare the medium according to the recipe mentioned in the method.

[0064] iPSC Maintenance and Differentiation Media can be used for up to 2 weeks when stored at 4°C. Therefore, it is not recommended to prepare all of the media at once and it is best to prepare it as needed for specific steps in the procedure.

[0065] All procedures were performed in a BSL-2 certified laboratory biosafety cabinet using standard aseptic techniques. Cultures were grown and maintained at 37°C and 5% CO2 in a humidified incubator.

[0066] Cultures were routinely tested for sterility and karyotyping.

[0067] Standard cell culture practices (such as aseptic handling of cultures, wearing personal protective equipment) and appropriate equipment (such as biosafety cabinets, CO2 incubators, centrifuges, water baths, and microscopes) were used. Cell culture in plates coated with extracellular matrix (ECM) was performed in a humidified incubator at 37°C with 5% CO2 levels. Prior to differentiation, iPSCs were characterized to check for expression of pluripotent markers, chromosomal aberrations by karyotyping, and sterility testing to determine whether the culture was free of contamination (bacteria and mycoplasma).

[0068] Example 2

[0069] General Experimental Preparation

[0070] The reproducibility of RPE differentiation method has been 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 Pre-clinical and ClinicalApplications. Stem cell reports, 5 (4), 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 (PBMC) of Indian patients suffering from inherited retinal diseases carrying different mutations. Stem Cell Research, 45, 101757). Therefore, according to the detailed method as described herein, similar differentiation results are expected in other iPSC lines.

[0071] The entire method is carried out in a sterile environment such as a biological safety cabinet. The method comprises:

[0072] 1. Cell therapy system for iPSC expansion - vitronectin (CTS TM -VTN) preparation.

[0073] Time: 1 hour

[0074] a. Thaw CTS-VTN (0.9 mg / ml) at room temperature (20 to 25° C.) for 5 to 10 minutes and then place on ice.

[0075] CTS-VTN was divided into use-sized aliquots in polypropylene tubes and stored at -60°C to -80°C.

[0076] b. Room temperature storage and / or shaking may cause slight turbidity, which does not affect product performance.

[0077] For coating wells of a 6-well plate, add 60 μL of CTS-VTN to a well containing 6 ml of sterile CTS at room temperature.TM DPBS (1×)(-Ca 2+ / -Mg 2+ ) in a 15 ml tube.

[0078] c. Gently resuspend by pipetting the CTS-VTN solution up and down.

[0079] This yields a working concentration of 9 μg / ml (i.e., 1:100 dilution). The diluted CTS-VTN solution can be stored at 4°C for no more than 7 days. Then, 1 ml of the diluted CTS-VTN solution is added to each well of a 6-well plate. The plate is rotated and / or shaken to ensure uniform coating.

[0080] d. When 1 ml / well is used to coat a 6-well plate (10 cm 2 / well), the final concentration will be 0.9μg / cm 2 .

[0081] e. Incubate the coated plates at 37°C for 1 hour.

[0082] The culture plate can be used immediately or wrapped in laboratory film and stored at 2°C to 8°C for up to one week, ensuring that the wells do not dry out. One hour after coating, a volume of basal medium can be added to the wells to ensure that the wells do not dry out. If part of a well dries out, the well cannot be used. The culture plate must be pre-warmed to room temperature (20 to 25°C) before use.

[0083] f. Aspirate CTS before use TM -VTN solution and discard. TM After VTN, there is no need to rinse the culture plate. Cells can be plated directly onto the CTS-treated TM on VTN-coated plates.

[0084] 2. iPSC culture and maintenance.

[0085] Time: 6 to 10 days

[0086] Before starting the method, prepare the CTS-VTN coated plates. If using CTS-VTN coated plates stored at 4°C, the plates must be equilibrated to room temperature for 1 hour before starting.

[0087] A cryovial containing 1 million cells should be thawed into 1 well of a 6-well plate (10 cm 2 surface area).

[0088] a. Add five ml of cold mTeSR plus medium to a sterile 15 ml tube.

[0089] A 1:5 ratio is recommended for efficient dilution of Cryostor CS10 (1 ml cells to 5 ml mTeSR plus).

[0090] b. Take the cells out of the liquid nitrogen storage tank.

[0091] c. Using a "figure 8" motion, rapidly thaw cells in a 37°C water bath until pea-sized ice balls are observed.

[0092] To avoid cell death, cells should not be completely thawed in a water bath.

[0093] d. Using a 2 ml pipette, slowly add mTeSR plus dropwise to the cells and collect in a 15 ml tube.

[0094] e. Cap the 15 ml tube and gently invert the tube 4 to 5 times to mix the CryoStor CS10 and mTeSR plus.

[0095] f. The tubes were then centrifuged at 200 g for 3 minutes at 25 ± 5 °C.

[0096] g. While the cells are spinning, aspirate CTS-VTN from the plate.

[0097] h. Gently aspirate the supernatant from the cell pellet and resuspend the cells in 2 ml of fresh mTeSR plus containing 10 μM ROCK inhibitor (Y-27632).

[0098] The use of the ROCK inhibitor Y-27632 improved cell survival and cell health. Y-27632 was reconstituted and stored according to the manufacturer's instructions. (https: / / www.tocris.com / products / y-27632-dihydrochloride_1254).

[0099] Due to the known toxicity of DMSO, ensure that DMSO present in the storage medium is completely removed by washing. This should be done in a gentle and rapid manner.

[0100] i Cells were plated on CTS-VTN-coated wells.

[0101] j. Place the culture plate in the incubator; gently rock the plate in a positive direction to evenly distribute the cells.

[0102] k. The next day, replace the medium with 2 ml of fresh mTeSR plus to remove Y-27632.

[0103] For medium changes, always carefully add / remove medium near the well wall to avoid detaching cells.

[0104] l. Colonies are expected to appear within 2 to 3 days.

[0105] m. Change the medium daily, and when colonies cover 70% to 80% of the plate, they are ready to be passaged.

[0106] 3. iPSC Passaging

[0107] Time: 1 hour

[0108] a. Prepare CTS-VTN-coated plates as described above and pre-warm mTeSR plus.

[0109] b. Aspirate the medium and wash the wells with (1 ml / well of 6 wells) 1X PBS.

[0110] c. ReLeSR TM Add to culture plate and aspirate ReLeSR within 1 minute TM , so that the colonies are exposed only to a thin film of liquid.

[0111] Table-1: Correlation between culture medium volume and culture plate surface area.

[0112]

[0113] d. Incubate the plate at 37°C for 4 minutes ± 30 seconds.

[0114] e. Gently rinse the plate or well with 1 ml of mTeSR plus to completely detach the cells.

[0115] f. Detach the colonies by holding the plate with one hand and tapping the side of the plate vigorously with the other hand for approximately 30 to 60 seconds.

[0116] g. Use a 5 ml serological pipette to transfer the detached cell aggregates to a 15 ml tube.

[0117] h. The size of the cell aggregates should be suitable for plating (average aggregate size is approximately 50 to 200 μm)

[0118] i. Centrifuge sterile 15 ml or 50 ml serum tubes at 200 g for 3 minutes by keeping the acceleration and deceleration of the centrifuge at maximum 9.

[0119] j. After centrifugation is complete, remove the supernatant, gently tap the pellet, and add the appropriate volume of mTeSR plus with 10 μM Y-27632 to a 15 ml tube or a 50 ml tube depending on the split ratio.

[0120] k. Plate the cell colonies onto CTS-VTN-coated culture dishes.

[0121] 1. Shake the plate in the plus direction to ensure even distribution of colonies in the wells.

[0122] m. Place the plate in a 37°C incubator with 5% CO2. Make sure not to move the plate for 24 hours.

[0123] n. After 24 hours, observe the plate under a microscope to confirm that the colonies have attached to the plate. The next day, replace the medium with 2 ml of fresh mTeSR plus to remove Y-27632.

[0124] o. Change medium daily until plates reach 70% confluence in less than 7 days.

[0125] Any plates that did not reach 70% confluence within 7 days were discarded. Prior to each passaging, supernatants from all plates were combined into sterile 15 ml tubes and stored at -80 °C until tested for sterility.

[0126] 4. Matrigel aliquoting and coating

[0127] g. Aliquot and store Matrigel at -20°C. It is important that any material that comes in contact with Matrigel is ice cold. Matrigel will solidify and adhere to anything above 10°C. Therefore, make sure to avoid multiple freeze / thaw cycles.

[0128] h. Before starting, place chilled serological pipettes, pipette tips, 15 ml tubes, and Matrigel aliquots on ice in a bio-safety cabinet (BSC). Make sure to spray the ice bucket thoroughly with 70% ethanol before placing it in the bio-safety cabinet.

[0129] i. Dilute Matrigel 1:100 in cold DMEM-F12 medium. For example, add 100 μL Matrigel to 10 ml DMEM-F12 medium.

[0130] Be careful to keep your fingertips above the level of the Matrigel, as the heat from your fingertips will cause the Matrigel to solidify. Make sure to frequently replace pipette tips with new, cold ones.

[0131] This 1:100 dilution will make a 1% Matrigel solution ready for coating culture plates.

[0132] j. Using a cold serological pipette, transfer 1% Matrigel solution to the culture plate at 1 ml / well of a 6-well plate.

[0133] k. Vortex and / or shake the plate to ensure even coating.

[0134] 1. The plate was then incubated at 37°C for at least 1 hour.

[0135] Alternatively, if the Matrigel-coated plates are not used on the same day of coating, wrap the plates in parafilm and store in a 4°C refrigerator for up to 1 week, ensuring that the wells do not dry out.

[0136] One hour after coating, a volume of basal medium can be added to the well to ensure that the well does not dry out. If part of a well dries out, the well cannot be used.

[0137] Before use, remove the Matrigel solution. Avoid air drying of Matrigel-coated wells before seeding cells.

[0138] Example 3

[0139] Differentiation of iPSCs into retinal pigment epithelial cells (RPE)

[0140] 1. Formation of embryoid bodies (EBs) by forced iPSC aggregation.

[0141] Time: 0 to 2 days

[0142] a. mTeSR plus (expansion medium: cGMP, commercially purchased stable feeder-free maintenance medium for iPS cells), CTS TM DPBS (1×)(-Ca 2+ / -Mg 2+ )Pre-warm to 37°C.

[0143] One 6-well plate (80% confluence, approximately 1 million cells / well) is sufficient to initiate RPE differentiation through EB formation ( Figure 1 A).

[0144] b. Cells from 6 wells will go into 6 wells of an ultra-low attachment plate (to avoid any form of attachment) for EB formation.

[0145] c. Prepare ultra-low attachment plates by adding 1 ml of mTeSR plus medium to each well.

[0146] d. Aspirate the spent media from the plate containing iPSCs and place the plate in CTS TM DPBS (1×)(-Ca 2+ / -Mg 2+ ) Rinse once (refer to Table-1 for volume).

[0147] e. Aspirate CTS TM DPBS (1×)(-Ca 2+ / -Mg 2+), and add the ReLeSR solution to the plate containing iPSCs. The volume of the ReLeSR solution was adjusted for different plate sizes (refer to the volumes in Table-1).

[0148] f. Add 1 ml / well of ReLeSR TM , and aspirate ReLeSR within 1 minute TM , so that the colonies are exposed to a thin film of liquid.

[0149] g. Incubate the plate at 37°C for 4 minutes ± 30 seconds.

[0150] h. Add an appropriate amount of mTeSR plus medium to each plate (refer to Table-1 for volume) to terminate the dissociation reaction.

[0151] i. Gently pipette the dissociated cells up and down until the colonies are fully dispersed into a single cell suspension.

[0152] j. Make sure to pipette gently to minimize the formation of bubbles.

[0153] k. Transfer the iPSC suspension from each well into a separate 15 ml tube and centrifuge the tube at 200 g for 3 minutes to pellet the cells.

[0154] l. Carefully aspirate the supernatant from the iPSC pellet.

[0155] m. Resuspend the pellet with an appropriate amount (about 6 ml) of mTeSR plus medium.

[0156] n. Add the resuspended cells to each 6-well ultra-low attachment plate containing 6 ml mTeSR plus medium (1 ml / well) and 10 μM ROCK inhibitor Y-27632 (growth medium).

[0157] o. This day is considered as day 0. The cells were cultured in an incubator at 37°C, 5% CO2 for 24 hours to allow them to form EBs. The medium was replaced with 2 ml of fresh mTeSR plus (expansion medium) to remove Y-27632.

[0158] p. Day 1 and Day 2: Gently rotate the EB plate and keep the EB in the center of the dish. Remove 1 ml of medium from the corner of the well and add 1 ml of fresh mTeSR plus medium. ( Figure 1 A) 2. Differentiation induction and plating of embryoid bodies (EBs) to obtain the neural ectodermal lineage

[0159] Time: 3 to 8 days

[0160] a. Prepare differentiation induction medium DIM according to Table-2.

[0161] Table-2: Differentiation Induction Medium (DIM)

[0162]

[0163]

[0164] Growth factors were reconstituted and stored according to the manufacturer's instructions.

[0165] ( https: / / www.sigmaaldrich.com / IN / en / product / sigma / i0161 ;

[0166] https: / / www.tocris.com / products / sb-4315421614 ;

[0167] https: / / www.tocris.com / products / ldn-193189-dihydrochloride_6053 ;

[0168] https: / / www.stemcell.com / products / human-recombinant-igf-i.html) .

[0169] b. The medium of the suspension culture with EBs was gradually switched from mTeSR plus (expansion medium) to differentiation induction medium (DIM).

[0170] c. Day 3: Replace a portion of the mTeSR plus medium with DIM (1:3) (Example: for 1 ml of medium, 750 μl mTeSR plus medium, and 250 μl DIM).

[0171] d. Day 4: Replace a portion of the mTeSR plus medium with DIM (1:1) (Example: for 1 ml of medium, 500 μl mTeSR plus medium and 500 μl DIM).

[0172] e. Day 5: Complete medium change (100%) with DIM.

[0173] f. Day 6: Matrigel coating and EB plating.

[0174] EBs were plated on matrigel (extracellular matrix) coated culture dishes. At this point, EBs were expected to be dense, spherical, with well-defined borders, and to easily aggregate to the center when the plate was rotated.

[0175] i. Gently swirl the EB plate and transfer EBs from 3 wells of the 6-well plate into a 15 ml tube.

[0176] ii. At room temperature (20 to 25°C), inside a biosafety cabinet, allow the EBs to settle to the bottom of the tube (this will take about 3 minutes). Make sure not to centrifuge the EBs. Remove the supernatant (retain 1 ml of medium), add 2 ml of DIM, and then add 500 μl of resuspended medium in a dropwise manner using a 1 ml pipette tip so that the EBs are evenly distributed in the plate.

[0177] iii. Gently shake the plate to aid even distribution of the aggregates and carefully return the plate to the incubator. Make sure not to disturb the plate for 24 hours.

[0178] AggreWell TM 800 microwell culture plates (Stem Cell Technologies) are used for the EB formation of identical RPE differentiation method.Observe the homogeneity of EB produced using multiwell plates in size.However, do not observe the significant advantage in final RPE number, processing time or the cost produced.Therefore, continue to use ultra-low attachment dish to form EB.

[0179] g. Day 7 to 8:

[0180] i. After 24 hours, observe the plate for EB attachment. Most EBs will be attached and begin to show outgrowth from the edge.

[0181] At this point floating EBs were discarded and the culture medium was replaced.

[0182] ii. Perform complete medium replacement with 100% DIM (refer to Table 1 for volumes).

[0183] 3. Introduce differentiation proliferation medium (DPM) for rosette formation.

[0184] a. Prepare DPM according to the ingredient list provided in Table-3.

[0185] Table-3: Differentiation Proliferation Medium (DPM)

[0186]

[0187]

[0188] N1 supplement was added to the filtered medium.

[0189] At this stage, the culture medium can be stored at 4°C for 2 weeks.

[0190] b. Day 9: Gradually switch the culture medium from DIM to Differentiated Proliferation Medium (DPM). Replace a portion of the DIM medium with DPM (1:1) (Example: for 1 ml of culture medium, 500 μl DIM and 500 μl DPM).

[0191] c. Perform complete medium exchange (100%) with DPM.

[0192] d. Days 11 to 22: The cultures were fed with DPM every other day until day 22 (refer to Table-1 for volumes).

[0193] Around day 20, the formation of rosette-like structures and epithelial clusters was observed, indicating the onset of retinal differentiation ( Figure 1 B).

[0194] 4. Introduction of Retinal Pigment Epithelial Maturation Medium (RPEMM) for Formation of Retinal Progenitor Cells

[0195] a. Prepare RPEMM as shown in Table 4.

[0196] Table-4: Retinal Pigment Epithelial Maturation Medium (RPEMM)

[0197]

[0198]

[0199] Growth factors and supplements were added to the filtered medium. Growth factors were reconstituted and stored according to the manufacturer's instructions.

[0200] ( https: / / www.sigmaaldrich.com / IN / en / product / sigma / t0625;

[0201] https: / / www.sigmaaldrich.com / IN / en / product / sigma / h6909:

[0202] https: / / www.sigmaaldrich.com / IN / en / product / sigma / t5516) .

[0203] The prepared medium can be stored at 4°C for 2 weeks.

[0204] b. Gradually switch the culture medium from DPM to retinal pigment epithelium maturation medium (RPEMM).

[0205] c. Day 23: Replace a portion of the DPM medium with RPEMM (1:1) (Example: for 1 ml of medium, 500 μl DIM and 500 μl RPEMM).

[0206] d. Day 24: On day 24, switch to full RPEMM ( Figure 1 D).

[0207] e. Days 25 to 45: The cultures were fed with RPEMM every other day until day 40 (refer to Table-1 for volumes).

[0208] 5. Day 40 (±5): Enrichment step of RPE by passage

[0209] like Figure 1 As shown in C, the onset of pigmentation is observed in the culture, indicating that the retinal progenitor cells are committed to the fate of RPE cells. Additional visible pigmentation patches are observed in the RPE culture plate ( Figure 1 E) On day 35 to 45 after the onset of pigmentation, RPE cells can be further passaged to enrich for pigmented cells and obtain mature RPE cells.

[0210] a. Matrigel coating: Matrigel-coated plates were prepared as described previously (see step 4 in Example 2).

[0211] b. For 1 x 6 well plate, aliquot 3 ml of Gentle Cell Dissociation Reagent (GCDR) and 3 ml of StemPro Accutase (enzyme for dissociation) into 15 ml tubes and pre-warm in an incubator at 37°C for 30 minutes.

[0212] c. Aspirate the culture medium and use CTS TM DPBS (1×)(-Ca 2+ / -Mg 2+ ) Wash gently once with 1 ml / well.

[0213] d. Incubate cells with a 1:1 ratio of pre-warmed GCDR to Accutase at 37°C for 11 to 12 minutes.

[0214] If necessary, incubate the cells for an additional 2 minutes in step d to ensure complete dissociation into single cells.

[0215] e. Gently pipette to dissociate into single cells or aggregates of 5 to 10 cells and dilute with RPEMM medium.

[0216] f. Collect the cell suspension in a 15 ml tube and centrifuge at 200 g for 3 minutes. Remove the supernatant.

[0217] g. Aspirate the Matrigel (extracellular matrix) just before seeding the cells and add RPEMM medium to each well (refer to Table-1 for volume).

[0218] h. Resuspend the cell pellet in RPEMM and seed the cells at a ratio of 1:2 or 1:3 on Matrigel-coated plates.

[0219] i. Allow cells to attach without disturbing for 24 to 48 hours.

[0220] j. After cells have sufficiently attached, continue with medium changes.

[0221] 6. Day 47 to 75 or later

[0222] a. Cultures were fed with RPEMM every other day until day 75 to 80.

[0223] b. Day 60 (±5): Passage RPE.

[0224] By following the step mentioned in the enrichment step of going down to posterity RPE (with reference to step as described above), can be gone down to posterity again at about the 60th (± 5) day, and by changing culture medium every other day, maintain up to the 90th to 100th day.If it is necessary to produce a large amount of RPE cells for preclinical safety and toxicology research, then this step can be implemented to further expand.

[0225] 7. Day 75 or later: Selection and cryopreservation

[0226] At day 75, RPE cells showed a cuboidal monolayer with hexagonal morphology with visible melanin pigmentation. In addition, at day 120, increased pigmentation accompanied by hexagonal morphology was observed, as shown in Figure 1 As shown in D. Using commercially available cryoprotectants - CS10 Freezing Medium - BioLife Solution, freeze the cultures in 1.8 ml NUNC cryovials.

[0227] a. Treat cells with CTS TM DPBS (1×)(-Ca 2+ / -Mg 2+ )Wash twice.

[0228] b. Add pre-warmed GCDR and Accutase at a 1:1 ratio and incubate at 37°C for 10 to 12 minutes.

[0229] If the enrichment step by passage RPE cells was not performed on day 40 / 45, proceed to step c, otherwise proceed directly to step d.

[0230] c. Use a 20 to 200 μl pipette tip to manually remove visible non-pigmented (non-black) cluster areas (if any) and discard. Use a 1 ml pipette tip to gently rinse out pigmented plaques from the well. Carefully repeat the rinse in the presence of RPEMM until all visible pigmented plaques are successfully detached.

[0231] d. Collect the cells and mix with an equal volume of RPEMM. Centrifuge the cells at 200 g for 2 minutes at room temperature (20 to 25°C). Figure 1 F).

[0232] After counting the cells, determine the number of cryovials needed to freeze approximately 1 million cells per vial in 1 ml of cold freezing medium. Label each cryovial accordingly with the cell line name, cell type, initials and date.

[0233] e. Add Cryostor CS10 freezing medium to the RPE cell pellet, and then gently mix the RPE cell suspension.

[0234] f. Cryopreserved cells (suspension state) in cryovials at 1 million cells / vial.

[0235] g. Place cryovials in Mr. Frosty freezing containers and store at -80°C for 24 hours. After 24 hours, transfer cells to a liquid nitrogen storage tank and update the cryotank binder.

[0236] Figure 4 A depicts phase contrast microscopic images of directed differentiated and mature RPE cells at low (10×) and high magnification (20×), showing dense pigmentation and Figure 4 B depicts a photograph of a 6-well plate with late-stage RPE cultures and a 15 ml tube containing RPE cells after centrifugation (inset) during the enrichment and expansion process.

[0237] Example 4

[0238] Characterization of RPE in vitro

[0239] Protein expression by immunocytochemistry

[0240] Immunocytochemistry of differentiated cultures was performed to observe the expression levels and localization of stage-specific retinal markers and it verified the successful differentiation of iPSCs to RPE ( Figure 2 A to D).

[0241] Time: 4 hours to 2 days

[0242] Table-5: Immunocytochemistry (ICC) reagents

[0243]

[0244]

[0245] Day 1:

[0246] i. Aspirate the culture medium and wash with CTS-DPBS (1×)(-Ca 2+ / -Mg 2+ ) and fixative solution three times and incubate at room temperature (20 to 25°C) for 15 to 20 minutes.

[0247] ii. Remove the fixative and wash with CTS-DPBS (1×)(-Ca 2+ / -Mg 2+ ) Wash gently three times.

[0248] iii. Permeabilization and blocking of cells was performed by adding blocking solution at room temperature (20 to 25°C) for 30 minutes.

[0249] iv. The cells were then washed with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) Wash gently three times.

[0250] v. Add primary antibody diluted in Antibody Diluent to cells and incubate overnight (16 to 20 hours) at 4°C.

[0251] Day 2

[0252] vi. Remove the primary antibody and wash the cells with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) Wash three times.

[0253] vii. Add the secondary antibody diluted in Antibody Diluent to the cells and then incubate at room temperature (20 to 25° C.) in the dark for 60 minutes.

[0254] viii. Remove the secondary antibody solution and wash the cells with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) Wash three times.

[0255] ix. The cells were counterstained with DAPI at a concentration of 1 μg / ml and incubated in the dark for 10 minutes at room temperature (20 to 25°C).

[0256] x. Remove the DAPI solution and wash the cells with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ )Wash once.

[0257] xi. Add CTS-DPBS (1×)(-Ca 2+ / -Mg 2+) (300 μl for 1 well of a 4-well plate), and the cells were observed under a fluorescence microscope.

[0258] Prior to staining, the fixed cells from step (ii) can be incubated at 4°C in CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) for 1 week.

[0259] observe

[0260] iPSC-derived RPE cultures were immunostained to examine marker expression during the progression of retinal differentiation. The RPE directed differentiation marker MITF ( Figure 2 A), pigmentation-specific proteins (such as tyrosinase-related protein TYRP1 ( Figure 2 B) and melanocyte protein PMEL17 ( Figure 2 C)), and RPE65 ( Figure 2 D) shows higher expression in RPE cells obtained by the method of the present disclosure.

[0261] Immunophenotyping by flow cytometry

[0262] Flow cytometric analysis of RPE cells helped to quantify protein expression levels, thus reaffirming successful differentiation of iPSCs into RPE ( Figure 2 E).

[0263] Time: 3 hours

[0264] Table-6: FACS buffer

[0265] Reagents Final concentration Volume (for 10ml) PBS 1× 10ml FBS 2％ 200 μl Triton-X-100 (10%) 0.25％ 250μl

[0266] Enzymatic dissociation of RPE into single cells

[0267] i. Aspirate the medium and wash the cells with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ )washing.

[0268] ii. Enzymatically harvest cells (GCDR to Accutase at a 1:1 ratio for 10 to 12 minutes; (refer to steps a to d under Section 7, Step-by-Step Method Details) and gently pipette to prepare a single-cell suspension.

[0269] iii. Centrifuge the cells at 200 g for 3 minutes and discard the supernatant.

[0270] iv. By incubating in CTS-DPBS (1×)(-Ca 2+ / -Mg2+ ) to wash the pellet and centrifuge at 200 g for 3 min.

[0271] v. Fix the cells by adding 1 ml 4% PFA for 10 minutes at room temperature (20 to 25°C).

[0272] vi. Cells were washed with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) and centrifuged at 200 g for 3 min, and then the CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ).

[0273] vii. Add 1 ml of ice-cold methanol to the pellet and incubate at 4°C for 20 minutes.

[0274] viii. The cells were washed with FACS buffer (refer to Table-6) and centrifuged at 200 g for 3 minutes.

[0275] ix. Stain the cells with the optimized concentration of primary antibody in 100 μl FACS buffer and incubate at room temperature (20 to 25° C.) for 30 minutes.

[0276] x. Repeat the wash with FACS buffer and centrifuge at 200 g for 3 minutes.

[0277] xi. Add 100 μl of secondary antibody in FACS buffer at optimized concentration to the pellet and incubate in the dark for 30 minutes.

[0278] xii. Vortex the vial every 10 minutes to ensure mixing.

[0279] xiii. Cells were washed with CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) and resuspended in FACS buffer.

[0280] xiv. Transfer cells to flow tubes for flow cytometry analysis.

[0281] The fixed cells from step (f) can be incubated at 4°C in CTS-DPBS (1×) (-Ca 2+ / -Mg 2+ ) for 48 to 72 hours, followed by permeabilization and blocking.

[0282] Permeabilization, blocking, and immunolabeling were performed simultaneously to avoid cell loss during washing steps.

[0283] observe

[0284] from Figure 2 As can be clearly seen in E, the RPE obtained by iPSC differentiation was of high purity, as shown by the high percentages of late RPE maturation markers such as TYRP1, RPE65, and TYROSINASE in the flow cytometry results.

[0285] Gene expression analysis by quantitative real-time PCR

[0286] Gene expression profiling helped to understand the molecular characteristics of these de novo generated RPE cells compared with undifferentiated iPSCs.

[0287] Time: 4 to 5 hours

[0288] Purification of total RNA

[0289] Precipitation preparation:

[0290] i. Collect the cells in a sterile 1.5 ml tube (refer to selection and cryopreservation steps a to d in Example 3) and centrifuge at 200 g for one minute to pellet the cells.

[0291] ii. Carefully remove the supernatant and snap freeze the pellet using liquid nitrogen. Store the snap frozen cells at -80°C until RNA isolation.

[0292] RNA Isolation:

[0293] iii. For RNA isolation, the RNeasy Mini kit (Qiagen) was used and all steps were performed at room temperature (20 to 25°C).

[0294] iv. Add 500 μl of RLT buffer to the cell pellet and add an equal volume of 70% ethanol. Add the mixture to a spin column and spin at 8000 g for 1 minute.

[0295] v. The column was washed once with buffer RW1 and twice with buffer RPE. All wash steps were performed at 8000 g for 1 minute.

[0296] vi. For the final elution, add 20 μl of RNase-free water directly to the spin column, incubate for 1 minute, and spin at 8000 g for 1 minute to elute the RNA.

[0297] vii. RNA was immediately kept on ice and quantified using nanodrop.

[0298] cDNA Synthesis:

[0299] viii. cDNA transcription was performed using the Verso cDNA synthesis kit.

[0300] All reaction set-up steps were performed on ice.

[0301] ix. The recommended template RNA concentration range for the Verso cDNA Kit method is 1 pg to 1 μg. The cDNA synthesis reaction mixture was performed on ice using the following volumes.

[0302] Table-7: cDNA synthesis reaction

[0303] Components volume 5× cDNA synthesis buffer 4μl dNTP Mix 2μl OligodT 1μl RT enhancer 1μl Verso Enzyme Blend 1μl RNA 1 μg Nuclease-free water Make up to 20 μl

[0304] x. Incubate the RNA at 42°C for 45 minutes for cDNA synthesis to occur, and at 95°C for 2 minutes for enzyme inactivation. The synthesized cDNA can be used for qPCR analysis.

[0305] The RT enhancer from the kit used degrades dsDNA during RNA transcription and is inactivated in a second step at 95° C. This eliminates the need for DNase treatment.

[0306] observe

[0307] Gene expression profiles show the purity of enriched RPE cells when compared to unenriched RPE cell populations. Representative heat maps consisting of important non-RPE gene sets (epithelial to mesenchymal transition genes and cranial neural crest-like genes) show downregulation in enriched RPE cells ( Figure 4 C) and representative heatmap consisting of important RPE-specific gene sets (melanogenesis and late RPE) showing upregulation in enriched RPE cells ( Figure 4 D).

[0308] RT-qPCR

[0309] The expression of various genes was analyzed by RT-qPCR.

[0310] RT-qPCR was performed using a QuantStudio3 real-time PCR instrument and 2 -ddCt RT-qPCR experiments are preferably performed under minimal light.

[0311] xi. qPCR using SYBR green: Prepare a 20 μl reaction mixture containing 10 μl SYBR green mix (2×), 1 μl each of forward and reverse primers (stock 10 μM), 2 μl cDNA (50 ng) and 6 μl water.

[0312] xii. The running parameters of the qPCR reaction are as follows.

[0313] Table 8: qPCR Cycling Parameters - SYBR green

[0314]

[0315] xiii. qPCR using TaqMan probe: Prepare a 20 μl reaction mixture containing 10 μl TaqMan master mix (2×), 1 μl gene-specific probe (stock 20×), 2 μl cDNA (50 ng) and 7 μl water.

[0316] xiv. The running parameters of the qPCR reaction are as follows

[0317] Table 9: qPCR Cycling Parameters - TaqMan Reagents

[0318]

[0319] 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).

[0320] observe

[0321] Gene expression analysis of selected markers of RPE Figure 3 The gene expression analysis of the selected markers using qPCR is presented as the fold change compared to iPSC. In the iPSC-derived RPE of the present disclosure, all positive markers (e.g., PMEL17, MITF, RPE 65, TYR, TYRP1 ( Figure 3 A)) showed higher expression, while negative markers (such as OCT4, alpha-fetoprotein (AFP), heart and neural crest derivatives expressed 2 (HAND2), TH ( Figure 3 B)) showed negligible expression.

[0322] Protein was detected by enzyme-linked immunosorbent assay (ELISA). Measurement of exocytosis

[0323] The secretion of pigment epithelium-derived growth factor (PEDF) is an important criterion for assessing the polarity of RPE generated in vitro. Quantification of PEDF was performed enzymatically using a commercially available kit (Human Serpin F1 / PEDF Duoset ELISA Kit).

[0324] Time: 4 to 5 hours

[0325] i. Coat the ELISA plate with 100 μl of capture antibody and incubate overnight at room temperature (20 to 25°C).

[0326] ii. Wash the coated wells 3 times with wash buffer. All steps were performed at room temperature (20 to 25°C).

[0327] iii. Add 300 μl of reagent diluent as blocking solution and incubate the plate for 1 hour. Repeat the washing procedure 3 times with washing buffer.

[0328] iv. Add 100 μl of sample (on the day of harvesting cell supernatant) and also add standards diluted in reagent diluent to the coated wells and incubate the plate for 2 hours. Carefully wash the plate three times with wash buffer.

[0329] v. Add 100 μl of detection antibody diluted in Reagent Diluent and incubate the plate for 2 minutes.

[0330] vi. Carefully wash the plate three times with wash buffer.

[0331] vii. Add 100 μl of Streptavidin-HRP to each well. Cover the plate and incubate in the dark for 20 minutes.

[0332] viii. Carefully wash the plate three times with wash buffer.

[0333] ix. Add 100 μl of substrate solution and incubate for 20 minutes. Make sure to avoid exposing the plate to direct light.

[0334] x. Add 50 μl of Stop Solution to each well and mix the plate gently.

[0335] xi. The optical density of each well at 450 nm and 540 nm was immediately determined using a Varioskan LUX microplate reader for background subtraction.

[0336] xii. Analyze the concentration of PEDF from the samples by plotting the standard values ​​into a graph.

[0337] observe

[0338] ELISA-based quantification of secreted PEDF Figure 3 Shown in C. ELISA-based quantification of secreted pigment epithelium-derived growth factor (PEDF) from in vitro culture supernatants at two different time points (day 75 and day 120) demonstrated the increased polarity of RPE obtained by the methods of the present disclosure.

[0339] Example 5

[0340] Preclinical safety and efficacy studies in animal models

[0341] 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) enabling preclinical data.

[0342] a. Animal maintenance

[0343] Time: 2 months

[0344] i. A colony of pigmented RCS (RCS-p+ / Lav) rats (NIH Rat Resource and Research Center, #315) was maintained.

[0345] ii. Animals were fed a standard laboratory chow and maintained on a 12-hour dark / light cycle.

[0346] iii. The animals were weaned on postnatal day (PD) 21 and orally administered cyclosporin A (210 mg / L; Gengraf, North Chicago, IL) in the drinking water. The animals continued to receive cyclosporin A until sacrifice.

[0347] e. Cell preparation

[0348] It is extremely important that the cells are prepared according to published protocols under sterile conditions prior to injection for optimal performance of the cells after transplantation into an animal.

[0349] Time: 1 hour

[0350] a. Remove cells from liquid nitrogen storage and thaw in a 37°C water bath for 2 min.

[0351] b. Add 1 ml of pre-warmed DMEM / F12 medium dropwise to the vial.

[0352] c. Transfer cells to a 15 ml tube containing 5 ml pre-warmed DMEM / F12 medium.

[0353] d. Rinse the cryovial with 1 ml of DMEM / F12 medium and add to the 15 ml tube.

[0354] e. Centrifuge the cells at 150 g for 5 minutes at room temperature (20 to 25°C).

[0355] f. Aspirate the supernatant and resuspend the cells in 1 ml of pre-warmed DMEM / F12 medium.

[0356] g. Carefully pipette up and down to resuspend cells.

[0357] h. Remove 10 μl of the cell suspension and add to 10 μl of 0.08% Trypan blue solution.

[0358] i. After pipetting up and down, load the cell suspension onto a hemacytometer (10 μl per side).

[0359] j. Count the viable cells.

[0360] k. Centrifuge the cell suspension again at 150 g for 5 minutes.

[0361] 1. Resuspend the cells in an appropriate volume to achieve the desired concentration for injection.

[0362] f. Subretinal injection

[0363] Subretinal injections of cryopreserved RPE frozen at days 75 to 80 were tested, with the assumption that iPS-RPE at this stage would be suitable for transplantation studies. Transplantation of cells at earlier stages of differentiation is possible, but the behavior of progenitor cells in vivo is not well known.

[0364] Duration: 1 day

[0365] a. One hour before injection, dilate the pupil with 1% tropicamide and 2.5% phenylephrine.

[0366] b. Anesthetize the eye with topical 0.5% proparacaine hydrochloride.

[0367] c. Animals were sedated with intraperitoneal ketamine / xylazine (100 / 10 mg / kg).

[0368] d. The corner of the eyelid is incised to allow visualization of the posterior retina. A hemostatic agent is applied to reduce blood accumulation.

[0369] e. Using the tip of a 27G needle, make a small scleral / choroidal incision (approximately 1 mm) in the dorsotemporal region 2 mm posterior to the limbus.

[0370] f. A small lateral corneal puncture was performed using a 30G needle to limit the increase in intraocular pressure and reduce the outflow of injected cells.

[0371] g. Two microliters of cell suspension containing total cell dose or control medium were delivered to the subretinal space using a fine glass pipette (75 to 150 μm inner diameter) 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) micropipette.

[0372] h. Following delivery, the retina was observed under a microscope using a glass coverslip and subretinal blebs were scored based on size and injection-related issues (eg, hemorrhage, bubbles, cells in the vitreous).

[0373] i. 0.5% erythromycin ointment was placed on the eyes and the animals were allowed to recover from anesthesia.

[0374] j. On the day of surgery, dexamethasone (1.0 mg / kg) was administered intraperitoneally. After cell transplantation, animals were administered dexamethasone every other day for 2 weeks to minimize potential inflammatory responses.

[0375] Make sure the injection should occur between PD 22 to PD 25. The animals need to be placed on cyclosporine A one day prior to the injection.

[0376] g. Optokinetic Tracking (OKT)

[0377] Optomotor thresholds should be assessed using a virtual optomotor system (VOS; Cerebral Mechanics), which consists of four computer monitors arranged in a square with the displays facing inward. On the monitors, a virtual cylinder displays a sine wave grating that rotates either clockwise or counterclockwise, allowing independent assessment of both the left and right eyes; the left eye responds to clockwise movement, and the right eye responds to counterclockwise movement.

[0378] Duration: 1 day

[0379] a. Place the animal on a pedestal centered over four computer monitors. Allow the animal to acclimate to the pedestal for 5 minutes.

[0380] b. Using Cerebral Mechanics software, set up a simple staircase test for both eyes, keeping the contrast set to 100%.

[0381] c. The program will start at a low spatial frequency, the cylinder will rotate, and if the grating is resolved, the animal will respond by tracking the grating with a reflexive head and neck movement. If the animal responds, click "yes", if the animal does not respond, click "no". Based on this input, the spatial frequency of the grating will then be gradually increased until the animal no longer tracks the stimulus, resulting in a maximum spatial frequency threshold.

[0382] d. Once the examination is complete, remove the animals from the pedestals and return them to their home cages.

[0383] The OKT of RCS rats is close to normal within PD 60. To determine the functional rescue caused by cell transplantation, this examination is best performed no earlier than PD 90.

[0384] observe

[0385] Behavioral analysis (functional test) of RCS rats injected with balanced salt solution (BSS, vehicle) and RPE was measured by optomotor threshold (OKT). From birth to 4 months of age, the grating visual acuity of RCS rats decreased rapidly from 0.8 to 0.3 c / d. Within 70 days after injection, the visual acuity of the cell-treated eyes remained consistent in all doses (low, medium, and high) ( Figure 5 A).

[0386] e. Fundus photography

[0387] Fundus cameras or retinal cameras are designed to view the inner area of ​​the eye, primarily the retina. Fundus images are obtained at any time point after injection to verify successful engraftment of cells and length of cell survival.

[0388] Duration: 1 day

[0389] a. Dilate the pupil with 1% tropicamide and 2.5% phenylephrine.

[0390] b. Anesthetize the eye with topical 0.5% proparacaine hydrochloride.

[0391] c. Animals were sedated with intraperitoneal ketamine / xylazine (100 / 10 mg / kg).

[0392] d. Lubricate the eye with 2.5% Goniovisc Hypromellose.

[0393] e. White light was used to capture bright field images of the retina using a Micron IV (Phoenix-Micron, Inc.) imaging system.

[0394] f. Use the optic nerve head as a guide when imaging, but then position the animal so that the peripheral / temporal region of the retina where the injection will occur is in view.

[0395] g. 0.5% erythromycin ointment was placed on the eyes and the animals were allowed to recover from anesthesia.

[0396] observe

[0397] Fundus imaging of saline (BSS) and RPE injected (subretinal) eyes of RCS rats showing patches of pigmented (black) RPE cells in the cell injected group ( Figure 5 C).

[0398] f. Tissue Preparation

[0399] Tissue samples are appropriately harvested and prepared for histological and immunohistochemical studies. Sample preparation includes processes such as fixation, dehydration, embedding, and sectioning.

[0400] Duration: 5 days

[0401] a. Following IACUC guidelines, remove the eye as soon as possible after euthanasia.

[0402] b. Place the enucleated eye in cold 4% paraformaldehyde (PFA) while attempting to maintain a 10 to 20× fixative to tissue ratio.

[0403] c. After fixation for 10 to 60 minutes, the anterior chamber of the eye including the lens is removed and the remaining eye cup is replaced in 4% PFA. The samples are placed in PFA on ice.

[0404] d. Place the eye at 2 to 8°C for 24 to 48 hours.

[0405] e. After 24 to 48 hours, remove the PFA solution and add an equal volume of cold 10% sucrose solution in 1× PBS.

[0406] f. Place the eye at 2 to 8°C for 24 to 48 hours.

[0407] g. Remove the 10% sucrose solution and replace it with an equal volume of cold 20% sucrose solution.

[0408] h. Place the eye at 2 to 8°C for 24 hours.

[0409] i. Remove 20% sucrose solution and replace with an equal volume of cold 30% sucrose solution.

[0410] j. Place the eye at 2 to 8°C for 24 hours.

[0411] k. Remove the eye cup from the 30% sucrose solution and gently dry it on a piece of filter paper.

[0412] l. Place the eye cup cut side down on the filter paper to allow the vitreous to drain from the eye cup. Repeat this step until the filter paper remains dry.

[0413] m. Place the eye cup in the labeled cryomol and fill with OCT medium.

[0414] n. Orient the eye cup toward the front of the freezing mold and orient the injection site at approximately 2 o'clock for the OS (oculus sinister left) eye and 10 o'clock for the OD (oculus dextrus right) eye.

[0415] o. Freeze the cryomold containing the eye in a liquid nitrogen bath. Make sure the liquid nitrogen does not directly touch the OCT medium, but only touches the walls of the cryomold ( Figure 3 C), because direct contact between liquid nitrogen and OCT medium generates bubbles, destroying the integrity of the sample.

[0416] p. Once the media is no longer shiny, remove it from the liquid nitrogen bath.

[0417] q. Wrap the frozen mold tightly in aluminum foil to try to limit exposure of the module to air.

[0418] r. Place the wrapped module in a -20°C freezer.

[0419] g. Histology / Immunohistochemistry

[0420] Hematoxylin and eosin (H&E) and immunohistochemical staining are valuable tools for detecting histopathological changes and specific antigens in tissues.

[0421] Duration: 2 to 4 weeks

[0422] a. Eye blocks were cut at 12 μm.

[0423] b. Approximately 40 slides were obtained from each eye, each slide containing 4 sections.

[0424] c. Sections were collected in series of 5 slides to provide a representative section every 60 μm on each slide across the entire eye cup.

[0425] d. The first slide in each series was stained with cresyl violet or hematoxylin and eosin and examined for evidence of photoreceptor rescue and retinal damage / toxicity.

[0426] b. For immunohistochemistry, allow slides to dry for at least 30 minutes after removing them from the refrigerator.

[0427] c. Block the slides with 4% horse serum, 1% bovine serum albumin (BSA), 0.5% triton x in phosphate buffered saline (PBS) for 45 minutes.

[0428] d. Add primary antibody (anti-cone arrestin, generously provided by W. Clay Smith, PhD, University of Florida) to blocking buffer and incubate overnight (at least 15 hours) at 4°C.

[0429] e. The next day, wash the slides 3 x 5 min with 1 x PBS.

[0430] f. Add secondary antibody (1:300) in blocking buffer and incubate in the dark for 45 min at room temperature (20 to 25 °C).

[0431] g. Wash the slides 3 x 5 min with 1 x PBS.

[0432] h. Incubate the slides with DAPI for 10 minutes in the dark.

[0433] i. Wash slides 2 x 5 min with 1 x PBS.

[0434] j. Place a coverslip on the slide with 100 μl of Fluoromount G.

[0435] observe

[0436] Quantification of retinal thickness and cone number between the nasal and temporal regions of the retina from animals transplanted with low, medium, and high doses of RPE was calculated by immunostaining retinal sections with cone arrestin. The data showed significant ONL preservation in the temporal region compared to the nasal region ( Figure 5 B).

[0437] h. Quantification of photoreceptors

[0438] The outer nuclear layer (ONL) thickness, as a major indicator of photoreceptor rescue, was measured with the help of immunohistochemical staining.

[0439] Time: 1 to 2 weeks

[0440] a. A scanning laser confocal microscope (Leica SP5 with LAS AF software; Leica) was used to image the stained slides. The laser intensity setting (gain) was kept constant for each emission wavelength.

[0441] b. Z-stack images were acquired at ×10 and ×20 magnifications at 1024 × 1024 resolution and 1 μm step size.

[0442] c. When each color channel z-stack is flattened, the color channels remain independent. Save each color channel image and save the color merged image as a TIFF file.

[0443] d. ONL cell bodies within ONL rows in both temporal (injected) and nasal (non-injected) regions were counted to obtain retinal thickness-nuclear values.

[0444] e. Cone arrestin positive cells were counted in both temporal (injected) and nasal (non-injected) regions to obtain a cone value for each image.

[0445] f. Three observers were blinded to dose and age group to generate counts.

[0446] g. Using the data collected in steps 4, 5, and 6, average ONL counts and photoreceptor counts for each group, and present data from both the temporal (injected) and nasal (non-injected) regions.

[0447] observe

[0448] Immunostaining of retinal tissue sections (HNM) showed that transplanted RPE cells survived in the subretinal space at P90 (passage 90) with preserved ONL layer (cone arrestin) and good-looking retina, indicating rescue of vision.

[0449] In summary, the disclosed methods provide an efficient protocol to obtain enriched mature RPE cells with high purity and significant therapeutic potential.

[0450] Advantages of the present disclosure

[0451] The present disclosure provides a method for obtaining RPE cells from iPSCs, which exhibits the following advantages.

[0452] (a) This method produces mature, pigmented and polarized RPE that exhibits functional characteristics such as secretion of cytokines - PEDF and VEGF - from both the apical and basal compartments.

[0453] (b) De novo generated RPE cells are mature, ciliated, and mimic the morphological, ultrastructural, and molecular features of native RPE; they are also suitable for in vivo cell transplantation studies.

[0454] (c) The method comprises an enrichment step which renders the method amenable to large-scale GMP manufacture of pure RPE populations.

[0455] (d) RPE cells generated following this method are suitable for cell replacement therapy for macular degeneration.

[0456] (e) RPE cells generated following this method may benefit through their neuroprotective effects against other retinal diseases associated with early and late photoreceptor degeneration.

[0457] (f) Due to their similarity to native RPE tissue in terms of structure, molecular features, and function, they are suitable for modeling diseases associated with retinal degeneration.

Claims

1. A method for obtaining RPE (retinal pigment epithelium) cells from iPSC (induced pluripotent stem cells), comprising the following steps: (a) generating embryoid bodies from a culture of iPSCs, wherein the embryoid bodies are in a non-adherent suspension culture state, (b) plating the embryoid bodies in differentiation induction medium (DIM) on a culture dish coated with a suitable extracellular matrix 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 in differentiation proliferation medium (DPM) for 11 to 22 days for rosette formation, wherein the DPM does not contain any inhibitors, (d) culturing the rosettes of step (c) in retinal pigment epithelial maturation medium (RPEMM) for 23 to 45 days to promote the formation of retinal progenitor cells, and (e) The retinal progenitor cells of step (d) are plated on a culture dish coated with a suitable extracellular matrix and cultured in RPEMM for 47 to 75 days to obtain RPE cells.

2. according to the method described in claim 1, wherein said method also comprises the RPE cell described in enrichment, and it comprises: a) enzymatically dissociating the RPE cells from the extracellular matrix to obtain a single cell suspension of RPE; as well as b) The single cell suspension of RPE from step (a) is plated on a culture dish coated with a suitable extracellular matrix and cultured in RPEMM for 75 to 100 days. 3 . The method of claim 1 , wherein the confluence of the culture of iPSCs ranges from 80% to 90%.

4. The method according to claim 1, wherein generating the embryoid body in step (a) comprises: (a) culturing the iPSCs in a growth medium for 24 hours to form embryoid bodies, wherein the growth medium comprises an expansion medium and a ROCK inhibitor, and (b) gradually contacting the developing embryoid bodies from the growth medium with the DIM by: (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, and subsequently plating the embryoid bodies on a culture dish.

5. The method of claim 1, 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 culture medium composition comprising a 1:1 ratio of DIM to DPM for 24 to 48 hours; as well as (iii) The neuroectodermal lineage obtained from step (ii) was cultured in DPM for 24 hours to promote rosette formation.

6. The method of claim 1, wherein the forming of retinal progenitor cells in step (d) comprises: (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) The rosettes obtained from step (j) were cultured in RPEMM for 24 hours to promote the formation of retinal progenitor cells.

7. The method of claim 1 or claim 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. 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-methylene-2H-isoindol-2-yl)-N-8-quinolyl-benzamide, 5-(phenylsulfonyl)-N-piperidin-4-yl-2(trifluoromethyl)benzenesulfonamide, 2-(2',3-dimethyl-[2,4'-bipyridyl]-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 method of claim 1, wherein the at least two SMAD pathway inhibitors are selected from 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)piperidin-1-yl]phenyl]-4-N-(2-propane-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-propane-2-yloxyphenyl)-4-N-(2-propane-2-ylsulfonylphenyl)pyrimidine-2,4-diamine, or a combination thereof.

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

11. The method according to claim 2, 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.

12. Retinal pigment epithelial cells or a population thereof produced by the method according to 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. The pharmaceutical composition according to claim 13 , for use in treating retinal degenerative diseases.

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

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

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