Stem cell-derived cell cultures, stem cell-derived three-dimensional tissue products, and methods for manufacturing and using the same.

Abstract

To provide methods for generating stem cell-derived retinal pigment epithelial monolayer cultures as well as methods of using the same, and also provide populations of retinal pigment epithelial cells prepared according to these methods.SOLUTION: In addition, three-dimensional tissue products derived from human induced pluripotent stem cells are also provided along with methods of making and using the same.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] Government support This invention was made with government support under grant number R01EY022631, awarded by the National Institutes of Health / National Eye Institute. The government has certain rights in this invention.

[0002] Related applications This application claims priority to U.S. Provisional Application No. 62 / 669,133, filed on 9 May 2018, and U.S. Provisional Application No. 62 / 826,196, filed on 29 March 2019, each of which is incorporated herein by reference in whole.

[0003] Field of Invention This invention generally relates to the field of stem cells. More specifically, this invention provides a method for producing a stem cell-derived retinal pigment epithelium (RPE) monolayer culture from human retinal organoids, three-dimensional tissue products derived from human induced pluripotent stem cells, and methods for manufacturing and using the same. [Background technology]

[0004] Background of the Invention Retinal degenerative diseases are a group of clinical conditions in which dysfunction and death of retinal photoreceptor cells lead to irreversible vision loss, and sometimes complete blindness. Currently, there are no treatments available to prevent many retinal degenerative diseases. Therefore, there remains a need in this field to develop means to study retinal development, cell interactions, and the physiological and disease mechanisms. Furthermore, there is a need to develop additional treatments for these diseases. [Overview of the project] [Problems that the invention aims to solve]

[0005] Summary of the Invention A three-dimensional tissue product derived from human induced pluripotent stem cells (hiPSCs) is provided, comprising functionally mature retinal pigment epithelial (RPE) cells and a portion of three-dimensional neural retina (3DNR), wherein the 3DNR and RPE cells are physically and functionally integrated to form a complex comprising a layer of neural retina and a sublayer of RPE cells. According to various embodiments, in this three-dimensional tissue product, both RPE cells and 3DNR are obtained from human retinal organoids. [Means for solving the problem]

[0006] According to the embodiment, the 3DNR includes i) undifferentiated pseudostratified neuroretinal epithelium; ii) layered neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; and / or iii) highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), wherein the ONL may be rod-enriched, cone-enriched, or any combination thereof.

[0007] In those products, RPE cells may be prepared according to any of the methods described herein, or according to any method known in the art to produce similar RPE tissue. According to various embodiments, RPE cells may be obtained i) from initial plating or subsequent passage; ii) at the early stages of differentiation; and / or iii) at more advanced stages of differentiation, culture time, or a combination thereof.

[0008] Any of the three-dimensional tissue products described herein may include additional biocompatible components that are integrated into the product. In a non-limiting example, the additional biocompatible component may be a natural or synthetic compound (e.g., a hydrogel) in liquid or gel form that provides a suitable biomechanical environment for cell survival and function and / or enables the manipulation of the product. The inclusion of this additional biocompatible component promotes the survival and function of the transplanted cells.

[0009] Furthermore, or to the other, any of the three-dimensional tissue products described herein may further include a biocompatible scaffold, where RPE cells grow on top of the scaffold before integration with 3DNR. In non-limiting examples, such a biocompatible scaffold may include a natural or synthetic scaffold, a scaffold made from a biodegradable material, a scaffold made from a non-biodegradable material, or a combination thereof.

[0010] A three-dimensional tissue product derived from human induced pluripotent stem cells (hiPSCs) is also provided, comprising functionally mature retinal pigment epithelial (RPE) cells and a portion of three-dimensional neural retina (3DNR), wherein the 3DNR and RPE cells are physically and functionally integrated to form a complex comprising a layer of neural retina and a sublayer of RPE cells. According to various embodiments, in this three-dimensional tissue product, both RPE cells and 3DNR are obtained from human retinal organoids.

[0011] According to the embodiment, the 3DNR includes i) undifferentiated pseudostratified neuroretinal epithelium; ii) layered neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; and / or iii) highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), wherein the ONL may be rod-enriched, cone-enriched, or any combination thereof.

[0012] In those products, RPE cells may be prepared according to any of the methods described herein, or according to any method known in the art to produce similar RPE tissue. According to various embodiments, RPE cells may be obtained i) from initial plating or subsequent passage; ii) at the early stages of differentiation; and / or iii) at more advanced stages of differentiation, culture time, or a combination thereof.

[0013] Any of the three-dimensional tissue products described herein may include additional biocompatible components that are integrated into the product. In a non-limiting example, the additional biocompatible component may be a natural or synthetic compound (e.g., a hydrogel) in liquid or gel form that provides a suitable biomechanical environment for cell survival and function and / or enables the manipulation of the product. The inclusion of this additional biocompatible component promotes the survival and function of the transplanted cells.

[0014] Furthermore, or to the other, any of the three-dimensional tissue products described herein may further include a biocompatible scaffold, where RPE cells grow on top of the scaffold before integration with 3DNR. In non-limiting examples, such a biocompatible scaffold may include a natural or synthetic scaffold, a scaffold made from a biodegradable material, a scaffold made from a non-biodegradable material, or a combination thereof.

[0015] According to the embodiment, the three-dimensional tissue product is derived from human induced pluripotent stem cells (hiPSCs) and comprises functionally mature retinal pigment epithelial (RPE) cells, a portion of three-dimensional neuroretina (3DNR), additional biocompatible components, and a biocompatible scaffold, wherein the 3DNR, RPE cells, and additional biocompatible components are physically and functionally integrated to form a complex comprising layers of neuroretina and a sublayer of RPE cells, wherein the 3DNR comprises i) undifferentiated pseudostratified neuroretinal epithelium; ii) iii) Laminated neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; or iii) highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), wherein the ONL may be rod-enriched, cone-enriched, or any combination thereof, wherein the additional biocompatible components comprise natural or synthetic compounds in liquid or gel form that provide a suitable biomechanical environment for cell survival and function, enable the manipulation of the product, or both, and the RPE cells are grown on top of the biocompatible scaffold prior to integration with the 3DNR, and the 3DNR is positioned on top of the RPE cells.

[0016] In those three-dimensional tissue products, both the RPE cells and the 3DNR are obtained from human retinal organoids.

[0017] Similarly, in those three-dimensional tissue products, the RPE cells can be prepared by: a) culturing human retinal organoids in a first medium not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate RPE cells and neural retina (NR); b) isolating RPE cell tissue from the cultured retinal organoids; c) dissociating the isolated RPE tissue into a suspension of single RPE cells; d) plating the single RPE cells in adherent culture; and e) culturing the plated cells in a second medium not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate a monolayer of RPE.

[0018] In any of those three-dimensional tissue products, the RPE cells are obtained from i) an initial plating or subsequent passage; ii) an early stage of differentiation; and / or iii) a more advanced stage of differentiation, culture time, or a combination thereof.

[0019] Suitable biocompatible scaffolds include, but are not limited to: natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, or combinations thereof.

[0020] a) culturing human retinal organoids to generate RPE cells and 3DNR; b) separating the RPE cells and 3DNR; c) seeding a neural retina patch on top of the RPE cells to form a complex; and d) By co-culturing the complex in a suitable medium, a method for producing a three-dimensional tissue product derived from human induced pluripotent stem cells (hiPSCs) is also provided, which comprises functionally matured retinal pigment epithelial (RPE) cells and a neural retina patch obtained from three-dimensional neural retina (3DNR), wherein following co-culturing, the 3DNR and RPE cells are physically and functionally integrated to form a three-dimensional tissue product comprising a neural retina layer and a sublayer of RPE cells.

[0021] According to some embodiments, prior to step c), RPE cells are cultured to generate a monolayer RPE culture.

[0022] According to one non-limiting embodiment, the monolayer RPE culture is generated by: i) dissociating RPE cells into a suspension of single RPE cells; ii) plating the single RPE cells in adherent culture; and iii) culturing the plated cells in a second medium not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways (i.e., agonists and / or antagonists) to generate a monolayer of RPE.

[0023] According to embodiments, the 3DNR can comprise: i) undifferentiated pseudostratified neural retina epithelium; ii) laminated neural retina tissue comprising all retinal layers and their corresponding retinal progenitor cell types; or iii) highly differentiated retinal tissue comprising an outer nuclear layer (ONL) and a bipolar cell layer (BCL), wherein the ONL can be rod-enriched, cone-enriched, or any combination thereof.

[0024] In those products, the RPE cells can be prepared according to any of the methods described herein or according to any methods known in the art for generating similar RPE tissue. According to various embodiments, the RPE cells are obtained from: i) the initial plating or subsequent passages; ii) an early stage of differentiation; or iii) a more advanced stage of differentiation, culture time, or a combination thereof.

[0025] According to any of these methods, RPE cells are dissociated into single RPE cells using enzymatic reactions (e.g., using collagenase, trypsin, dispase, TrypLE, papain, and / or any combination thereof), enzyme-free dissociation solutions, or mechanical means (e.g., mechanical dissociation).

[0026] According to various embodiments, a single RPE cell can be plated at the following densities: approximately 25,000; 50,000; 75,000; 100,000; 125,000; 150,000; 175,000; 200,000; 225,000; 250,000; 275,000; or 300,000 cells / cm². 2 A single RPE cell has approximately 100,000 cells / cm³. 2 It can be plated at a density of [value].

[0027] In step e) of this method, the second medium may be any medium that promotes the proliferation of RPE cells. In a non-restrictive example, this second medium may contain one or more of the following components: minimal essential medium (MEM) α-modified, N1 supplement, glutamine, penicillin, streptomycin, non-essential amino acids, taurine, hydrocortisone, triiodothyronine, and / or fetal bovine serum. Determining suitable components for the second medium is within the realm of routine knowledge for those skilled in the art.

[0028] In these methods, the second medium may be replaced periodically (e.g., every 1, 2, 3, 4, 5, 6 or more days). Similarly, cells in adherent culture may be passaged periodically. For example, cells may be passaged every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. According to one non-restrictive embodiment, cells are passaged every 10 days to ensure that a complete monolayer develops and that the cells begin to develop an irregular cobblestone shape. According to another embodiment, cells in the resulting monolayer culture retain their RPE differentiation and maturation ability for at least 4 passages (i.e., at least passages 4, 5, 6, 7, 8, 9, 10 or more) without the addition of exogenous factors.

[0029] As shown, human retinal organoids can be prepared by any method known in the art. In a non-restrictive example, human retinal organoids can be prepared by i) culturing hiPSCs to form aggregates; ii) transferring the aggregates to nerve induction medium; iii) seeding the aggregates onto a cell culture substrate coated with extracellular matrix; iv) replacing the nerve induction medium with a chemically defined differentiation medium; v) cleaving the NR domain; vi) culturing in suspension to form three-dimensional retinal organoids; and / or vii) adding animal serum or plasma components and retinoic acid. Routine modifications to this method are within the realm of routine knowledge for those skilled in the art.

[0030] Any method for a three-dimensional tissue product disclosed herein may include the further step of (e) embedding neuroretinal patches, RPE cells, or both neuroretinal patches and RPE cells from 3DNR into an additional biocompatible component integrated into the product. In a non-limiting example, the additional biocompatible component is a natural or synthetic compound in liquid or gel form that provides a suitable biomechanical environment for cell survival and function, enabling the manipulation of the product, or both. The inclusion of this additional biocompatible component promotes the survival and function of the transplanted cells.

[0031] According to any of these methods, the RPE monolayer is grown on top of a biocompatible scaffold before integration with 3DNR. Suitable biocompatible scaffolds include, but are not limited to, natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, or combinations thereof.

[0032] According to any of the methods described herein, 3DNR and RPE cells can be co-cultured at different points in cell maturation. According to some embodiments, 3DNR and RPE cells are co-cultured in a medium that yields a rod-enriched three-dimensional tissue product. According to other embodiments, 3DNR and RPE cells are co-cultured in a medium that yields a cone-enriched three-dimensional tissue product. Those skilled in the art will recognize that any combination of rod-enriched and / or cone-enriched cells can be used in any of the three-dimensional tissue structures described herein.

[0033] According to embodiments, the disclosure provides a method for producing a human induced pluripotent stem cell (hiPSC)-derived three-dimensional tissue product, comprising functionally mature retinal pigment epithelial (RPE) cells, a neuroretinal patch obtained from a three-dimensional neuroretina (3DNR), additional biocompatible components, and a biocompatible scaffold. For example, these methods may include the steps of: a) culturing human retinal organoids to generate RPE cells and 3DNR; b) isolating the RPE cells and 3DNR; c) seeding a neuroretinal patch onto the RPE cells to form a complex; d) co-culturing the complex in a suitable medium; and / or e) embedding the neuroretinal patch from the 3DNR, RPE cells, or both the neuroretinal patch from the 3DNR and RPE cells into an additional biocompatible component integrated into the product, where, following co-culturing, the 3DNR, RPE cells and additional biocompatible component are physically and functionally integrated to form a three-dimensional tissue product including layers of neuroretina and a sublayer of RPE cells, wherein the 3DNR is i) undifferentiated pseudostratified neuroretinal epithelium; ii) iii) a layered neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; and / or iii) a highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), wherein the ONL may be rod-enriched, cone-enriched, or any combination thereof, wherein the additional biocompatible components comprise natural or synthetic compounds in liquid or gel form that provide a suitable biomechanical environment for cell survival and function, enable the manipulation of the product, or both, and the RPE cells are grown on top of the biocompatible scaffold prior to integration with the 3DNR, and the 3DNR is positioned on top of the RPE cells.

[0034] In any of these methods, prior to step c), RPE cells may be cultured to produce an RPE monolayer culture. For example, an RPE monolayer culture may be produced by i) dissociating RPE cells into a suspension of single RPE cells; ii) plating single RPE cells in adherent culture; and / or iii) culturing the plated cells in a second medium that is not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to produce a monolayer of RPE.

[0035] The RPE cells used in these methods are obtained i) from the initial plating or subsequent passage; ii) at the early stages of differentiation; and / or iii) at more advanced stages of differentiation, culture time, or a combination thereof. Furthermore, in any of these methods, the RPE cells are dissociated into single RPE cells using enzymatic reactions, enzyme-free dissociation solutions, or mechanical means (e.g., dissociated RPE tissue is mechanically dissociated).

[0036] According to the embodiment, a single RPE cell population is approximately 25,000 to 300,000 cells / cm³. 2 For example, a density of approximately 100,000 cells / cm³. 2 It is plated at this density.

[0037] In any of these methods, the second medium helps the proliferation of RPE cells.

[0038] Appropriate biocompatible scaffolds include, but are not limited to, natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, or combinations thereof.

[0039] According to the embodiment, 3DNR and RPE cells are co-cultured at different stages of cell maturation; 3DNR and RPE cells are co-cultured in a medium yielding a rod-enriched three-dimensional tissue product; and / or 3DNR and RPE cells are co-cultured in a medium yielding a cone-enriched three-dimensional tissue product.

[0040] Also provided herein is a method for treating retinal diseases, disorders, or conditions by implanting one of the three-dimensional tissue products described herein into the eye of a patient who requires it. In a non-restrictive example, diseases, disorders, or conditions of the retina are selected from the group consisting of retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular diseases, retinal photoreceptor diseases, retinal pigment epithelial diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

[0041] Three-dimensional tissue products described herein for use in the treatment of retinal diseases, disorders, or conditions are also provided. The products are for implantation into the eye of the patient in need. In a non-restrictive example, diseases, disorders, or conditions of the retina are selected from the group consisting of retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular diseases, retinal photoreceptor diseases, retinal pigment epithelial diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

[0042] Furthermore, the present invention provides a method for screening for agents that affect retinal development, function, proliferation, maturation, differentiation, survival, or any combination thereof, by a) contacting any of the three-dimensional tissue products described herein with at least one agent (e.g., a biological agent); and b) determining whether the agent has an effect on retinal development, function, proliferation, maturation, differentiation, survival, or any combination thereof. For example, biological agents may be growth factors, trophic factors, regulatory factors, hormones, antibodies or their antigen-binding fragments, small molecules, and peptides.

[0043] Any of the three-dimensional tissue products described herein may be used to test retinal development. For example, an in vitro method for testing retinal development is provided herein by a) preparing a three-dimensional tissue product; and b) monitoring the cell interactions, function, proliferation, maturation, differentiation, survival, or any combination thereof of the cells within the three-dimensional tissue product. Such monitoring can provide information about normal retinal development (i.e., information about the interaction between the retina and the RPE), and / or information about abnormal retinal development, disease, disorder, or condition (i.e., information about the underlying mechanisms of abnormal retinal development, disease, disorder, or condition).

[0044] Also provided is a method for producing a stem cell-derived retinal pigment epithelium (RPE) monolayer culture by a) culturing human retinal organoids in a first medium unsupplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways (i.e., agonists and / or antagonists) to generate RPE cells and neuroretina (NR); b) isolating RPE cell tissue from the cultured retinal organoids; c) dissociating the isolated RPE tissue into a suspension of single RPE cells (for example, by dissociating into single RPE cells using an enzymatic reaction (e.g., using collagenase, trypsin, dispase, TrypLE, papain, and / or any combination thereof), an enzyme-free dissociation solution, mechanical means, or any combination thereof); d) plating the single RPE cells into adherent culture; and e) culturing the plated cells in a second medium unsupplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate a monolayer of RPE. According to various embodiments, human retinal organoids are three-dimensional retinal organoids derived from human induced pluripotent stem cells (hiPSCs).

[0045] Human retinal organoids can be prepared by any method known in the art. In a non-limiting example, human retinal organoids may be prepared by i) culturing hiPSCs to form aggregates; ii) transferring the aggregates to nerve induction medium; iii) seeding the aggregates onto a cell culture substrate coated with extracellular matrix; iv) replacing the nerve induction medium with a chemically defined differentiation medium; v) cleaving the NR domain; vi) culturing in suspension to form three-dimensional retinal organoids; and / or vii) adding animal serum or plasma components and retinoic acid. Routine modifications to this method are within the realm of common knowledge for those skilled in the art.

[0046] In any of the methods described herein, the resulting RPE cells are found as monolayer polarized RPE tissue associated with retinal organoids or as a disorganized mass of RPE tissue. Those skilled in the art will understand that RPE tissue can be mechanically dissected from retinal organoids and / or dissected RPE tissue can be mechanically dissected.

[0047] According to various embodiments, a single RPE cell can be plated at the following densities: approximately 25,000; 50,000; 75,000; 100,000; 125,000; 150,000; 175,000; 200,000; 225,000; 250,000; 275,000; or 300,000 cells / cm². 2 A single RPE cell has approximately 100,000 cells / cm³. 2 It can be plated at a density of [value].

[0048] In step e) of this method, the second medium may be any medium that promotes the proliferation of RPE cells. In a non-restrictive example, this second medium may contain one or more of the following components: minimal essential medium (MEM) α-modified, N1 supplement, glutamine, penicillin, streptomycin, non-essential amino acids, taurine, hydrocortisone, triiodothyronine, and / or fetal bovine serum. Determining suitable components for the second medium is within the realm of routine knowledge for those skilled in the art.

[0049] In these methods, the second medium may be replaced periodically (e.g., every 1, 2, 3, 4, 5, 6 or more days). Similarly, cells in adherent culture may be passaged periodically. For example, cells may be passaged every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more days. According to one non-restrictive embodiment, cells are passaged every 10 days to ensure that a complete monolayer develops and that the cells begin to develop an irregular cobblestone shape. According to another embodiment, cells in the resulting monolayer culture retain their RPE differentiation and maturation ability for at least 4 passages (i.e., at least passages 4, 5, 6, 7, 8, 9, 10 or more) without the addition of exogenous factors.

[0050] Using any of the methods described herein, RPE cells in a monolayer express the functional, molecular, and / or cellular characteristics of primary RPE cells.

[0051] According to a non-restrictive example, RPE cells in a monolayer may express certain molecules associated with RPE cell differentiation and functional maturation, including (but not limited to) the following: vascular endothelial growth factor (VEGF), melanogenesis-associated transcription factor (MITF), ezrin, retinal pigment epithelium-specific 65kDa protein (RPE65); zo-1 (ZO-1); bestrophin-1 (BEST1); cell retinaldehyde-binding protein (CRALBP); lecithin-retinol acyltransferase (LRAT); tyrosinase (TYR); pigment epithelium-derived factor (PEDF), trirosinase, premelanosome protein (PMEL), claudin 3, receptor tyrosine kinase (MERKT), orthodenticle homeobox 2 (OTX2), and combinations thereof.

[0052] Furthermore (or conversely), RPE cells in a monolayer achieve appropriate polarization with the formation of specialized functional structures of the RPE, including: abundant apical microvilli, adhesive junctions, tight junctions, transepithelial resistance (TER), or any combination thereof.

[0053] A population comprising retinal pigment epithelial (RPE) cells prepared according to any of the methods described herein is also provided. The resulting population may be used in any of the methods described herein.

[0054] This disclosure also provides a method for treating a retinal disease, disorder, or condition, comprising administering an effective amount of RPE cells prepared according to one of the methods described herein to a patient in need. In a non-restrictive example, the diseases, disorders, or conditions of the retina are selected from the group consisting of retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular disorders, retinal photoreceptor diseases, retinal pigment epithelial diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

[0055] This disclosure also provides RPE cells prepared according to any of the methods described herein for use in the treatment of retinal diseases, disorders, or conditions. In a non-restrictive example, the diseases, disorders, or conditions of the retina are selected from the group consisting of retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular disorders, retinal photoreceptor diseases, retinal pigment epithelial diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

[0056] A method is also provided for screening for agents that affect the function, proliferation, maturation, differentiation, or survival of RPE cells, the method comprising: a) contacting a population of RPE cells prepared according to any of the methods described herein with at least one agent; and b) determining whether the agent has an effect on the function, proliferation, maturation, differentiation, or survival of RPE cells. According to various embodiments, the at least one agent is a biological agent (e.g., consisting of growth factors, trophic factors, regulatory factors, hormones, antibodies or their antigen-binding fragments, small molecules, and / or peptides).

[0057] Furthermore, this disclosure also provides an in vitro method for monitoring the role of RPE cells in retinal development by a) preparing an RPE monolayer culture according to any of the methods described herein, and b) monitoring the function, proliferation, maturation, differentiation, survival, or any combination thereof of cells within the RPE monolayer culture during retinal development. For example, in such a method, the monitoring provides information about normal retinal development and / or information about abnormal retinal development, disease, disorder, or condition (e.g., information about the underlying mechanisms of abnormal retinal development, disease, disorder, or condition).

[0058] Any aspect or embodiment described herein may be combined with any other aspect or embodiment disclosed herein, including the outline, drawings, and / or the following specific non-limiting examples / embodiments of the invention.

[0059] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this application belongs. In the specification, singular nouns are also plural nouns unless the context explicitly indicates otherwise.

[0060] Methods and materials similar to or equivalent to those described herein may be used in carrying out and testing the application, but suitable methods and materials are described below. All publications, patent applications, patents, and other references referred to herein are incorporated by reference.

[0061] The references cited herein are not considered prior art to the claimed application. In case of any conflict, the current specification, including definitions, shall prevail. Furthermore, the materials, methods, and examples are illustrative and not intended to limit the scope of the application.

[0062] Other features and advantages of the application will become apparent from the detailed description below, along with examples.

[0063] The patent or application file shall include at least one drawing drawn in color. Copies of the publication of this patent or patent application and the color drawing shall be provided by the Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0064] [Figure 1] Figures 1A-1I show the isolation and induction of primary retinal pigment epithelium (ipRPE) cultures induced from human three-dimensional (3D) retina. In Figure 1A, retinal pigment epithelial cells (RPE) are dissected from the 3D retina. Clusters of RPE cells isolated from the 3D retina are collected (Figures 1B-1E) and dissociated into single cells for culture (Figure 1F, inset). Under these conditions, they develop into an RPE monolayer exhibiting behavior similar to that observed in human primary RPE cultures (Figures 1G-1I).

[0065] [Figure 2] Figures 2A-2J show the characterization of RPE derived from 3D retina. In Figure 2A, RPE is dissected from 3D retina, separated into single cells, and seeded in transwells (Figure 2B), where it forms a monolayer with a characteristic colored cobblestone pattern (Figure 2B) and appropriate ultrastructural differentiation (Figures 2C-2I). Two-passage ipRPE cultures show cobblestone patterns (Figures 2D-2E), expression of functional proteins (Figures 2F-2G), polarization with tight junctions and microvilli formation (Figures 2H-2I), appropriate intracellular localization of Z01 and EZRIN, and transepithelial resistance comparable to that observed in primary human embryonic RPE cultures (Figure 2J).

[0066] [Figure 3]Figures 3A-3D show the characterization of RPEs derived from 3D retinas. ipRPE cultures are suitable for continuous passaging while maintaining their RPE identity. Figures 3A-3B show gene and protein expression in ipRPE cultures obtained from four different passages (P1-P4). Figures 3C-3D show polar release of VEGF-A into ipRPE cultures measured in apical and basal extracellular medium ipRPE monolayers grown on Transwell inserts after different passages.

[0067] [Figure 4] Figures 4A-4F show 3D retinas obtained from hiPSCs. Figure 4A shows that hiPSCs form a 3D retina composed of the neural retina and RPE bundled at the apex. In Figures 4B-4C, the NR shows a characteristic layer containing rod-enriched ONL. Figures 4D-4F show that the photoreceptors have achieved advanced morphological, molecular, and ultrastructural differentiation, including the formation of outer segments (arrowheads) and photoreactions.

[0068] [Figure 5] Figures 5A–5D show stem cell-derived retinal / RPE grafts. Figures 5A–5B are representative optical microscope images of the top view (Figure 5A) and bottom view (Figure 5B), illustrating the physical relationship between the retina and RPE. The transparent appearance of the retina in A reflects its health. Figure 5Cl shows a 3D reconstruction of 20 consecutive image planes (5 μm depth intervals) that allowed for the measurement of the thickness of the retinal / RPE graft. Figure 5D is a 3D rendering of the retinal / RPE graft labeled with Hoechst (RPE) and SYTO Green (retina).

[0069] [Figure 6]Figures 6A–6J show the generation of rod-enriched versus cone-enriched 3DNRs. In Figure 6A, 3DNRs differentiated over 150 days show well-organized ONLs with highly differentiated photoreceptors. During initial differentiation, it is possible to generate rod-enriched (Figure 6B) and cone-enriched 3DNRs (Figures 6C–6D) by fine-tuning the culture medium composition (e.g., retinoic acid (RA) regime). Based on further differentiation, the retinal bipolar precursor generates all bipolar cell types, including: rod bipolar cells (RB: Chx10+PKCα+ / Islet1+; Figures 6E-6F); cone OFF bipolar cells (OFF-CB: Chx10+ / Scgn+ / Islet1- (arrowhead); Figures 6G-6I); and rod ON bipolar cells (ON-CB: Chx10+ / Scgn- / Islet1+ (arrowhead); Figures 6G-6I). Figure 6J shows SV2 expression defining the boundary of the developing outer plexiform layer. [Modes for carrying out the invention]

[0070] Detailed description of the invention Definition: In this disclosure, “comprises,” “comprising,” “containing,” and “having” may have the meanings attributed to them under U.S. patent law, and may also mean “includes,” “including,” etc.; similarly, terms such as “consisting essentially of” or “consist essentially” have the meanings under U.S. patent law, and these terms allow for existence beyond what is described, insofar as they are basic or novel characteristics, and what is described will not be altered by existence beyond what is described, but exclude embodiments of the prior art.

[0071] Where used herein, the terms “a,” “an,” and “the” shall be understood to be singular or plural unless specifically mentioned or evident from the context.

[0072] Where used herein, the term “or” is understood to be inclusive unless specifically mentioned or evident from the context.

[0073] As used herein, the term “about” refers, unless otherwise specified, to listed values ​​such as quantity, dose, temperature, time, and percentage, within ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1%.

[0074] As used herein, the terms “patient” or “subject” are used interchangeably herein to refer to any mammal, including: humans, livestock, and zoo, sports, and pet animals, such as dogs, horses, cats, and agricultural animals such as cattle, sheep, pigs, and goats. One preferred mammal is humans, including adults, children, and the elderly. Subjects may also be pet animals, including dogs, cats, and horses. Preferred agricultural animals would be pigs, cattle, and goats.

[0075] When used herein, the terms “treat,” “treating,” and “treatment” refer, unless otherwise specified, to the reversal, mitigation, inhibition, or prevention of the process of one or more symptoms of a disease, disorder, or condition, or symptoms or complications of such disease, disorder, or condition, or to the elimination of such symptoms or complications, by administering any of the compositions, pharmaceutical compositions, or dosage forms described herein, in order to prevent the onset of such disease, disorder, or condition, or symptoms or complications of such disease, disorder, or condition. Preferably, treatment is curative or ameliorative.

[0076] As used herein, “preventing” means preventing, improving, suppressing, reducing, or stopping the occurrence or development of a disease, disorder, or condition, whether in whole or in part.

[0077] The terms “therapeutic effective dose” and “effective dose,” as used herein, refer to the amount necessary to administer to a patient, or to the patient’s cells, tissues, or organs, in order to achieve a therapeutic effect, such as improvement or, conversely, a therapeutic effect. An effective dose is sufficient to elicit a biological or medical response in cells, tissues, systems, animals, or humans, as sought by researchers, veterinarians, physicians, or clinicians. Determining an appropriate effective dose or therapeutic effective dose is within the realm of routine knowledge for those skilled in the art.

[0078] The terms “administering,” “administer,” and “administration,” as used herein, refer to any mode of delivery, delivery, introduction, or transport of a therapeutic agent to a subject in need of treatment with such agent. Such modes include, but are not limited to, intraocular, oral, topical, intravenous, intraperitoneal, intramuscular, intradermal, intranasal, and subcutaneous administration.

[0079] The terms “RPE” and “ipRPE,” etc., are used interchangeably herein to refer to retinal pigment epithelial cells cultured according to any of the methods described herein and / or used in the three-dimensional tissue products described herein.

[0080] In this specification, terms such as "human retinal organoid," "3DNR," and "3D neuroretina" are used interchangeably to refer to the retina.

[0081] The terms "retinal / RPE graft" and "3DNR / RPE graft," etc., are used interchangeably herein to refer to any of the three-dimensional tissue products described herein. Retinal development and retinal organoid formation:

[0082] Retinal development occurs within a highly dynamic and complex microenvironment involving highly regulated intercellular interactions via direct contact or diffusive signaling. (See Adler et al., Dev Biol 305:1-12 (2007); Bassett et al., Trends in Neurosciences 35:5650573 (2012)). Previous studies have demonstrated that hiPSCs can be induced to differentiate into retinal precursors that self-organize into three-dimensional retinal cups using a simple procedure. (See US2016 / 033312, which is incorporated herein by reference in its entirety).

[0083] Eye development in the embryonic neural plate begins with the formation of the visual field (EF), a central tissue domain consisting of a subpopulation of anterior neuroepithelial cells that have come to be more specifically identified as retinal precursors. The EF is characterized by the expression of a group of transcription factors including PAX6, RX, LHX2, SIX3, and SIX6, while the surrounding anterior neuroepithelial cells express PAX6 and SOX1. (See Zuber, Curr Top Dev Biol 93:29-60 (2010); Zhang et al., Cell Stem Cell 7:90-100 (2010); Peny et al, Development 125:1967-78 (1998)). In parallel with native events, hiPSC-derived aggregates, after 8 days of differentiation (D8) in chemically defined neuronal differentiation medium, adhered to Matrigel-coated culture dishes and acquired the fate of preneuroepithelium expressing PAX6 and SOX1. Shortly thereafter, LHX2-expressing retinal precursor cells appeared in the central region of the differentiating aggregates. By D12, well-defined EF-like domains expressing appropriate transcription factors could be observed, surrounded by preneuroepithelial-like cells. These preneuroepithelial-like cells typically formed rosettes, a characteristic feature of these cells in culture, which is not observed in their natural context. (See Xia et al., Methods Mol Biol 549:51-58 (2009)).

[0084] In vivo, EF induces the generation of left and right optic vesicles, the retinal precursors of which ultimately form the future neuroretina (NR) and retinal pigment epithelium (RPE). The designation of cell fate to either NR or RPE is critically regulated by two transcription factors, VSX2 and MITF, which are co-expressed in pluripotent precursor cells but subsequently restricted to NR and RPE, respectively. (See Adler et al., Dev Biol 305:1-13 (2007); Nguyen et al., Development 127:3581-3591 (2000); Horsford et al., Development 132:177-187 (2005)). Cells within the EF-like domain in our culture followed the same differentiation sequence. Between D17 and D25 during culture, their NR and RPE domains transition to an optic cup-like structure, with the NR gradually acquiring a horseshoe-shaped dome reminiscent of the inner wall of an optic cup, surrounded by the RPE.

[0085] In these cultures, the retinal precursors in the EF domain underwent efficient and reproducible spontaneous differentiation into NR and RPE, strictly mimicking their in vivo topological configuration in the correct temporal order.

[0086] The optic cup-like shape of the NR domains in culture made them easily identifiable and suitable for collection one by one for mechanical detachment and further culture in suspension. NR domains collected at D21–D28 had a high enrichment of NR precursors and formed 3D retinal cups when cultured in suspension. The retinal cups consisted of thick, transparent NRs continuous with adjacent RPEs, which appeared to be bundled at the tip of the retinal cup and gradually became pigmented. From NR domain collection to D35 (Week 5 or W5), the NRs exhibited molecular and histological features similar to those of actual human embryonic retinas of the same age (see O'Rahilly et al., Developmental Stages in Human Embryos (Carnagie Institution of Washington) (1987)), containing polarized pseudostratified epithelium in which proliferating cells undergo interkinetic nuclear migration and express appropriate transcription factors. Between weeks 5 and 7, NR cells spontaneously began to differentiate and migrated to their corresponding retinal layers, following the characteristic central-to-peripheral wave of neurogenesis. Summary of previous methods used to generate stem cell-derived RPE monolayer cultures:

[0087] Most conventional methods are based on the differentiation of stem cells in an adherent state (see Table 1). Specifically, stem cells are plated and cultured in the presence of growth factors (or without specific growth factors), morphogens, or modulators (i.e., agonists and / or antagonists) to induce RPE differentiation. Such methods generate a mixed population of cells containing islands or patches of RPE cells. Following culture, RPE (pigmentation) patches are manually selected, expanded, and enriched until an RPE monolayer is achieved.

[0088] Another group of conventional methods is based on an initial step consisting of embryoid bodies (suspension aggregates of mixed cell populations) (see Table 2). In such a method, stem cells are first differentiated into embryoid bodies and cultured for a period of time. Next, the embryoid bodies are plated in an attached state, and differentiated cells grow from the embryoid bodies and differentiate into patches of RPE, which are then manually selected, expanded, and enriched until they form an RPE monolayer.

[0089] Ultimately, the third group of methods is based on an initial step consisting of optic vesicle or retinal organoid differentiation (see Table 3). For example, in Meyer et al., Stem Cells 29(8):1206-18 (2011), stem cells are first differentiated into 3D optic vesicle structures and then treated with activin A for RPE cell differentiation. The 3D colored vesicles are then plated, and RPE cells grow from the optic vesicles to form an RPE monolayer. In Wu et al., Oncotarget 7(16):22819-33 (2016), stem cells are first differentiated into neurospheres containing optic vesicles. Following prolonged culture, the optic vesicles develop into RPE pigmented masses or lesions that are excised and plated. RPE cells grow from the lesions to form an RPE monolayer. [Table 1-1] [Table 1-2] [Table 2] [Table 3] hESC: Human embryonic stem cells; hiPSC: Human induced pluripotent stem cells; R: Regular plate; TW: Transwell insert; GN: Gelatin; MEF: Mouse embryonic fibroblasts; MG: Matrigel; VN-PAS: Vitronectin peptide-acrylate surface; PLD: Poly-D-lysine; FN: Fibronectin; LN: Laminin; C-IV: Collagenase-IV; GX: Geltrex; RPE: Retinal pigment epithelium; TER: Transepithelial resistance; PH: Phagophagia; TEM: Transmission electron microscope; ICC: Intracellular calcium concentration; VEGF: Vascular endothelial growth factor; PEDF: Pigment epithelial-derived factor; N / A: Not applicable, unavailable, or no response; X: Completed; *One-step approach: selection or manual picking *PDF only Isolation and characterization of artificial primary retinal pleoplasm (ipRPE) from human retinal organoids:

[0090] In contrast to those conventional methods, in the culture method described herein, RPE cells undergo a spontaneous differentiation process without the need for exogenous factors to promote the fate, differentiation, and / or maturation of RPE cells. A pure RPE monolayer is obtained from the first step of this method without the need for manual selection and / or purification / enrichment steps, and achieves functional maturity by 30 days in culture. In those methods, stem cells are first differentiated into retinal organoids. As the retinal organoids differentiate, they also produce RPE tissue that forms clumps or RPE tissue attached to the retinal organoids. Importantly, exogenous growth factors, morphogens, or modulators (i.e., agonists and / or antagonists) are not used to differentiate the retinal organoids and RPE. Rather, their cultures undergo spontaneous differentiation. RPE clumps are excised from the retinal organoids and dissociated into a single-cell suspension. Single-cell RPE is seeded in a petri dish and cultured until they form a monolayer of RPE. Again, no exogenous factors, such as morphogens or modulators (i.e., agonists and / or antagonists), are added here.

[0091] A simple and efficient strategy for isolating and culturing RPE cells from human retinal organoids (hRetO) is provided herein. Briefly, hRetO were kept alive as described above (see Zhong et al., Nature Communications 5:4047 (2014) and U.S. Patent Application No. 2016 / 033312, each of which is incorporated herein by reference in whole). Their hiPSC-derived 3D retinal organoids contained functional photoreceptors, were appropriately stacked (with a highly organized outer granular layer containing highly differentiated rods and cones (red, green, and blue)), exhibited medial and lateral segments and photoresponses (see Figure 4), and showed spatial and temporal features that replicated the development of human retina in vivo. Human iPS cells expressed the pluripotency marker OCT4. Nanog, SEEA1, and Nestin were maintained on Matrigel-coated plates.

[0092] On day 0 of differentiation (D0), iPS cell colonies were isolated, mechanically dissociated into small clumps, and cultured in suspension to induce aggregate formation. The aggregates were gradually transferred to nerve induction medium to induce anterior neurite differentiation. On D7, the nerve aggregates were seeded on a Matrigel-coated dish.

[0093] After differentiation begins, at D14, domains expressing neuroretinal precursor markers start to appear, possessing a typical RPE cobblestone structure, and surrounded by non-pigmented cells expressing MITF. These NR domains gradually acquire a horseshoe shape with surrounding MITF-expressing RPE cells.

[0094] On D21, individual horseshoe-shaped NR and RPE domains are mechanically separated and collected, and based on further culture in suspension, they gradually form 3D retinal tissue. By D30, the 3D retinal tissue is fully folded into 3D retinal organoids, and remains similar in characteristics to the actual human embryonic retina of the same age. RPE tissue attached to the 3D retinal organoids can be observed as early as 25-30 days of differentiation (see Figure 1).

[0095] Between D25 and D50, RPEs attached to 3D retinal organoids become polarized, pigment settle, and express some of the following key RPE markers: RPE65 (an isomerohydrolase crucial for the regeneration of visual pigments); EST1 (a calcium-activated anion channel); OTX2 (a transcription factor essential for the development and maintenance of RPEs); and / or EZRIN (a protein localized to the apical process).

[0096] Pigmented RPE was mechanically isolated from 3D retinal organoids (e.g., using a tungsten needle), dissociated into single-cell RPE, and plated onto a Transwell filter (semi-porous polyester membrane) to obtain a polarized RPE monolayer (see Figure 1). Using this technique, pure pigmented RPE tissue was reproducibly isolated from hiPSC-derived 3D retinal organoids after 4 weeks of total differentiation. To increase the yield of isolated RPE tissue, pure pigmented RPE tissue can be isolated at differentiation D50.

[0097] Where used herein, isolated RPE was obtained from 3D retinal organoids as passage 0 (P0). The isolated RPE was used to generate EPE isolation cultures, which would be called artificial primary RPE monolayers (ipRPE). Characterization and development of human artificial primary RPE (ipRPE) monolayer:

[0098] RPE(P0) was isolated from hiPSC-derived 3D retinal organoids and cultured on Transwell (P1) to establish an artificial primary RPE (ipRPE) monolayer. In D1 after plating the ipRPE, pigment deposition was initially lost in most cells. However, as the cells continued to divide, the pigment density increased, indicating de novo synthesis of the pigment (see Figure 1). The newly dividing cells retained their epithelioid morphology.

[0099] As the cultured cells matured, the characteristic polygonal shape and pigment density became more uniform (see Figure 2). Once the P1 ipRPE monolayer was established, subsequent passage and proliferation of RPE cells were performed every 10 days. The ipRPE monolayer was characterized from P1 to P4, and it was found that the ipRPE monolayer retained the RPE phenotype and expressed the major RPE markers by RT-PCR and Western blot in all passages (see Figure 3). The ipRPE has been shown to be suitable for continuous passage while maintaining their RPE differentiation and maturation capabilities up to at least passage 4.

[0100] P1 and P2 were compared to determine the best passage to use (see Figure 3). ipRPE-P1 achieved higher transepithelial resistance (TER) levels compared to P2. Both cell passages were able to release polarized VEGF (see Figure 3). To validate ipRPE-P2, cells were characterized at D50. The ipRPE monolayer expressed MITF and RPE65 (see Figure 2). ZOI expression on the apical side of the cells and BEST1 on the basal side confirmed the well-polarized RPE monolayer (see Figure 2).

[0101] The pigmented cells possessed the structural features of RPE, including numerous apical microvilli, adhesive junctions, and tight junctions (as evidenced by the measurement of transepithelial resistance, which gradually increased as the ipRPE cells matured until they reached a plateau), as observed under a transmission electron microscope (see Figure 2). In summary, these data demonstrated that the ipRPE pigmented monolayer in passage 2 was polarized, functional, and expressed key features of genuine RPE cells. RPE cell monolayer used:

[0102] RPE cells generated according to the methods of this disclosure can be used in a variety of ways. For example, the cells can be used as grafts for stem cell-based regenerative medicine for retinal diseases (see Bharti et al., Invest. Ophthalmolol. Vis Sci 55:1191 -1201 (2014); Trounson et al., Cell Stem Cell 17:11-22 (2015)). Since there are currently no treatments available for retinal diseases, such as the dry form of age-related macular degeneration, there is a large potential market for therapies utilizing RPE cells prepared according to any of the methods described herein.

[0103] Similarly, these cells can also be used as in vitro disease models to elucidate disease mechanisms and develop treatments. On the other hand (or further), such RPE cells can also be used for drug screening to identify agents that affect the function, proliferation, maturation, differentiation, and / or survival of RPE cells. Stem cell-derived retinal / RPE complex:

[0104] A stem cell-based product comprising a three-dimensional tissue product biological unit including integrated 3D retinal tissue and RPE tissue is provided herein. This three-dimensional tissue product is derived from stem cells (e.g., human induced pluripotent stem cells (hiPSCs)) and consists of functionally mature RPE and differentiated neuroretina. By co-culture, stem cell-derived retina and RPE can be combined at different points in cellular maturation.

[0105] The three-dimensional tissue product described herein can be distinguished from other products. For example, Eiraku et al., Nature 472(7341):51-6 (2011) and Nakano et al., Cell Stem Cell 10:771-85 (2012) describe the initial formation of the optic cup, where undifferentiated neuroretina invaginates into the optic cup and is juxtaposed with still undifferentiated RPE tissue. This spatial configuration is merely transient, and the two tissues have not yet achieved differentiation as a combined complex. Similarly, Zhu et al., PLoS One. 2013;8(1):e54552 (2013) describes one experiment involving the co-culture of hESC-derived RPE cells and mouse retinal explants (explants of retinal tissue obtained directly from the mouse eye). Finally, Yanai et al., Tissue Eng Part A. (11-12):1763-71 (2015) uses a co-culture system containing hESC-derived RPE monolayers and retinal explants from humans and rodents (retinal tissue directly obtained from human and rodent eyes).

[0106] In contrast, this technique utilizes hiPSC-derived 3D retinas having functional photoreceptors and functionally mature RPEs generated from hiPSC-derived 3D retinas to produce the stem cell-derived 3D tissue products described herein. The hiPSC-derived 3D retinal tissue containing functional photoreceptors is produced according to the method described in Zhong et al., Nature Communications 5:4047 (2014) and U.S. Patent Application No. 2016 / 033312. These 3D retinas follow the same program and differentiation timing as native human retinas, starting from undifferentiated neuroretinal epithelium and ending in fully laminated retinal tissue. As shown in Figure 4, these hiPSC-derived 3D retinas achieve proper lamination with medial and lateral segments and a highly organized outer granular layer (ONL) containing highly differentiated rods and cones (red, green, and blue) exhibiting photoresponses.

[0107] RPE cells also differentiate in this system, but they do not form a monolayer covering the outer granular layer (see Figure 4A). Importantly, RPE cells can be readily dissected, allowing for independent culture of 3D neural retina (3DNR) and RPE cells (see Figure 2). Thus, a novel methodology for inducing RPE monolayer culture from 3D retina has been established. As described herein, RPE tissue is dissected from 3D retina, dissociated into single cells, and seeded on transwells, where they form a characteristic RPE monolayer exhibiting behavior similar to that observed in primary human RPE cultures. By day 50 of differentiation, the RPE monolayer exhibits differentiation of the normal ultrastructure, including specialized functional structures such as microvilli, tight junctions, and basal folds (see Figures 4B-4C), and appropriate intracellular expression and localization of characteristic genes indicating maturity, as observed in native human RPE cells and primary cultures.

[0108] hiPSC-derived 3D retinal and RPE tissues can be combined to form a functionally coupled complex consisting of a neuroretinal layer and a sublayer of RPE cells. Both the retinal and RPE layers replicate the cellular and topological configuration observed in a normal human retina. This 3D tissue product is the first stem cell-derived system to replicate the physical and functional interactions between neuroretina and RPE that occur in native retina. Importantly, this product solves the current problem of the lack of a stem cell-derived system capable of replicating the physical and functional interactions between neuroretina and RPE.

[0109] hiPSC-derived 3D retinal tissue is produced as described in Zhong et al., Nature Communications 5:4047 (2014) and U.S. Patent Application No. 2016 / 033312. Neuroretinal patches (3DNRs) are prepared from 3D retina. Specifically, hiDSC-derived 3D retina is opened (e.g., using a tungsten needle or any other method known in the art) to expose the inside of the 3D retinal cup, flatten it as a retinal flat mount, and obtain a retinal explant. Next, these retinal explants (or patches) (e.g., approximately 1.5 mm × 1.5 mm) are seeded onto ipRPE (passage 2) and co-cultured for various periods (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or more). Under these conditions, 3DNR adheres to the RPE monolayer, and a 3DNR / RPE complex is formed (see Figure 5). The incorporation of additional biocompatible components into the system (e.g., natural or synthetic compounds in liquid or gel form that provide a suitable biomechanical environment for cell survival and function (e.g., hydrogels), and / or biocompatible scaffolds (e.g., natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, and / or any combination thereof)) further provides an improved biomechanical environment that allows for longer culture periods and manipulation during transplantation. This inclusion of additional biocompatible components promotes the survival and function of transplanted cells.

[0110] Accordingly, the three-dimensional tissue products derived from human induced pluripotent stem cells (hiPSCs) described herein include functionally mature RPE cells and a portion of 3DNR. According to embodiments, such three-dimensional tissue products may also include additional biocompatible components (i.e., natural or synthetic compounds in liquid or gel form (e.g., hydrogels) that provide a suitable biomechanical environment for cell survival and function to promote cell survival and function of the transplanted cells and / or to enable the manipulation of the product), and / or biocompatible scaffolds (i.e., natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, and / or any combination thereof) where the 3DNR, RPE cells and additional biocompatible components are physically and functionally integrated to form a complex including layers of neuroretina and a sub-layer of RPE cells. The RPE cells may be grown on top of the biocompatible scaffold prior to integration with the 3DNR, resulting in the 3DNR being positioned on top of the RPE cells. For example, the 3DNR and RPE may be embedded in the additional biocompatible components.

[0111] Those skilled in the art will recognize that 3DNR may be i) undifferentiated pseudostratified neuroretinal epithelium; ii) layered neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; and / or iii) highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), where the ONL may be rod-enriched, cone-enriched, or any combination thereof, and / or RPE cells may be obtained i) from initial plating or subsequent passages; ii) in the early stages of differentiation; and / or iii) at more advanced stages of differentiation, culture time, or a combination thereof. Any combination of these 3DNR and RPE may be used in any of the three-dimensional tissue products described herein.

[0112] RPE cells may be prepared using any of the methods described herein. In a non-limiting example, RPE cells may be prepared by a) culturing human retinal organoids in a first medium unsupplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate RPE cells and neuroretina (NR); b) isolating RPE cell tissue from the cultured retinal organoids; c) dissociating the isolated RPE tissue into a suspension of single RPE cells; d) plating the single RPE cells into adherent culture; and e) culturing the plated cells in a second medium unsupplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate a monolayer of RPE.

[0113] Using the methods described herein, it is possible to generate both rod-enriched and cone-enriched hiPSC-derived retinal tissue (see Figures 6A-6J). For example, rod-enriched 3DNR can be reproducibly generated using the retinoic acid (RA) regime described in #Zhong et al., Nature Communications 5:4047 (2014) (see also U.S. Patent Application No. 2016 / 033312). Similarly, modifications of the RA regime enable the generation of cone-enriched hiPSC-derived retinal tissue. Furthermore, bipolar cells in 3DNR have the ability to differentiate into all major bipolar cell subtypes, including rod, cone-ON, and cone-OFF bipolar cells. In addition, rods and cones have the ability to establish synaptic connections with bipolar cells.

[0114] Those skilled in the art will recognize that the diversity of co-culture approaches described herein allows for the combination of 3DNR and RPE at different photoreceptor maturation times to produce different three-dimensional tissue products. Method for manufacturing 3D tissue products:

[0115] A method for producing a three-dimensional tissue product derived from human induced pluripotent stem cells (hiPSCs) is also provided, comprising: a) culturing human retinal organoids to generate RPE cells (i.e., obtained from i) initial plating or subsequent passages; ii) the early stages of differentiation; and / or iii) more advanced stages of differentiation, culture time, or a combination thereof) and 3DNR; b) isolating the RPE cells and 3DNR; c) seeding a neuroretinal patch onto the RPE cells to form a complex; d) co-culturing the complex in a suitable medium; and / or e) embedding the neuroretinal patch from the 3DNR, RPE cells, or both the neuroretinal patch from the 3DNR and RPE cells into an additional biocompatible component integrated into the product, thereby providing functionally mature retinal pigment epithelium (RPE) cells, and a neuroretinal patch obtained from a three-dimensional neuroretina (3DNR), an additional biocompatible component, and a biocompatible scaffold, here Following co-culture, 3DNR, RPE cells, and additional biocompatible components are physically and functionally integrated to form a three-dimensional tissue product comprising layers of neuroretina and a sublayer of RPE cells, wherein the 3DNR comprises i) undifferentiated pseudostratified neuroretinal epithelium; ii) layered neuroretinal tissue comprising all retinal layers and their corresponding retinal precursor cell types; and / or iii) highly differentiated retinal tissue comprising the outer granular layer (ONL) and bipolar cell layer (BCL), where the ONL may be rod-enriched, cone-enriched, or any combination thereof; the additional biocompatible components comprise natural or synthetic compounds in liquid or gel form that provide a suitable biomechanical environment for cell survival and function, enabling the manipulation of the product, or both; and the RPE cells are grown on top of the biocompatible scaffold prior to integration with the 3DNR, with the 3DNR positioned on top of the RPE cells.

[0116] According to the embodiment, prior to step c), the RPE cells are cultured to produce an RPE monolayer culture (e.g., i) the RPE cells are dissociated into a suspension of single RPE cells; ii) Plate a single RPE cell in adherent culture (e.g., approximately 25,000 to 300,000 cells / cm²). 2 (That is, approximately 100,000 cells / cm²)2 ) in); and / or iii) by culturing the plated cells in a second medium that is not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways in order to produce a monolayer of RPE). The RPE cells are dissociated into single RPE cells using an enzymatic reaction, an enzyme-free dissociation solution, or mechanical means (i.e., mechanical dissociation). Use of 3D tissue products:

[0117] Any of the three-dimensional tissue products described herein can be used in a variety of ways. For example, it may be used as a transplant for stem cell-based regenerative therapy for retinal diseases, disorders, or conditions. In a non-limiting example, these tissue products may be used to treat AMD and / or retinal dystrophy, such as retinitis pigmentosa (RP). (See Bharti et al., Invest. Ophthalmolol. Vis Sci 55:1191 -1201 (2014); Trounson et al., Cell Stem Cell 17:11-22 (2015)).

[0118] Similarly, it can also be used as an in vitro system for studying normal mechanisms, including retinal development, the retina, and RPE, and / or as a disease model for elucidating physiological and / or disease mechanisms and developing treatments. Furthermore (or on the other hand), three-dimensional tissue products can also be used as in vitro models for drug discovery. For example, it can be used to screen for drugs that affect retinal development, function, proliferation, maturation, differentiation, and / or survival. These products can also be used to study the toxicology of current treatments. Composition:

[0119] A population of cells (e.g., RPE cells cultured according to the method described herein) that can be injected with one or more pharmaceutically or veterinarily acceptable carriers, diluents, excipients, or vehicles is provided herein.

[0120] The terms “medically acceptable” and “veterinary acceptable” refer to materials, compositions, or vehicles that are medically or veterinarily acceptable, such as liquid or solid fillers, excipients, solvents, or encapsulating materials. Each component must be “medically acceptable” or “veterinary acceptable” in the sense that it can be compatible with other components of a pharmaceutical formulation. It must be suitable for use in contact with human and animal tissues or organs without excessive toxicity, irritation, allergic reactions, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio. (Remington: The Science and Practice of Pharmacy, 21st Edition; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 5th Edition; Rowe et al., Eds., The Pharmaceutical Press and the American Pharmaceutical Association: 2005; and Handbook of Pharmaceutical Additives, 3rd Edition; Ash and Ash Eds., Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, Gibson Ed., CRC Press LLC: Boca Raton, FL, 2004).

[0121] The pharmaceutical compositions of this disclosure are formulated to be compatible with their intended route of administration (i.e., intraocular, subretinal, parenteral, intravenous, intra-arterial, intradermal, subcutaneous, oral, inhalation, transdermal, topical, transmucosal, intraperitoneal or intrapleural, and / or rectal administration).

[0122] Pharmaceutical compositions for injectable use include sterile aqueous solutions (if water-soluble) or dispersions, and sterile powders for the immediate preparation of sterile injectable solutions or cell dispersions. In all cases, the composition must be sterile and fluid enough to allow for easy injection. It must be stable under manufacturing and storage conditions and protected from contamination by microorganisms, such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity can be maintained by the use of coatings such as lecithin, in the case of dispersions by maintaining the required particle size, and by the use of surfactants. Prevention of microbial action can be achieved by various antimicrobial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. According to some embodiments, it would be desirable to include isotonic agents, such as sugar, polyhydric alcohols, such as mannitol, sorbitol, and sodium chloride, in the composition. Long-term absorption of injectable compositions can be achieved by including absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0123] Sterile injectable solutions can be prepared by incorporating the required amount of the active compound into a suitable solvent containing one or a combination of the components listed above, as needed, followed by filtration sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterilization vehicle containing a basic dispersion medium and other components required from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and lyophilization to obtain the powder of the active ingredient and any desired components from a previously sterile filtered solution.

[0124] According to one embodiment, the active compound is prepared using a carrier that will protect the compound from rapid elimination from the body, such as in controlled-release formulations including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may be used. Methods for preparing such formulations will be apparent to those skilled in the art. The materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (containing liposomes that target infected cells with monoclonal antibodies against viral antigens) may also be used as pharmaceutically acceptable carriers. They may be prepared according to methods known to those skilled in the art, as described in U.S. Patent No. 4,522,811.

[0125] As used herein, a dosage unit form refers to a physically distinct unit suitable as a single dose for the subject being treated; each unit contains a predetermined amount of the active compound calculated to produce the desired therapeutic effect in relation to the required pharmaceutical carrier. The specifications of the dosage unit forms in this disclosure are determined and directly depend on the inherent properties of the active compound, the specific therapeutic effect to be achieved, and the technically inherent limitations of formulating such active compound for the treatment of an individual. Kits, pharmaceuticals, and manufactured products:

[0126] RPE cells cultured according to the methods of this disclosure, and / or the three-dimensional tissue products of this disclosure, may be used alone or in combination with one or more other therapeutic agents in the manufacture of pharmaceuticals.

[0127] Kits are also provided (optionally, with instructions for use) for treating retinal diseases, disorders, or conditions, for testing the role of RPE cells in retinal development; for screening for drugs that affect retinal development, function, proliferation, maturation, differentiation, and / or survival; and / or for testing retinal development.

[0128] A manufactured product including a container and instructions for use containing any of the cell or three-dimensional tissue products described herein is also provided.

[0129] Any of the compositions described herein may be contained in a container, pack, or dispenser, along with instructions for administration. Treatment method:

[0130] Any of the compositions described herein may be used to treat retinal diseases, disorders, or conditions in mammals.

[0131] It will be understood that the therapeutic entities described herein may be administered with appropriate carriers, excipients, and other agents incorporated into the formulation to provide improved mobility, delivery, tolerance, etc. Numerous suitable formulations can be found in formulation collections known to all medicinal chemists: Remington's Pharmaceutical Sciences (15th ed, Mack Publishing Company, Easton, PA (1975)), particularly Chapter 87 by Blaug and Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids (cationic or anionic) containing vesicles (such as lipofectin®), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, emulsion carbowaxes (polyethylene glycol of various molecular weights), semi-solid gels, and semi-solid mixtures containing carbowaxes. Any of the aforementioned mixtures may be suitable for the treatment and therapy described herein, provided that the active ingredients in the formulation are not activated by the formulation, the formulation is physiologically compatible, and it is resistant to the route of administration.See also: Baldrick P. “Pharmaceutical excipient development: the need for preclinical guidance.” Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. “Lyophilization and development of solid protein pharmaceuticals.” Int. J. Pharm. 203(1-2):1-60 (2000), Charman WN “Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts.” J Pharm Sci.89(8):967-78 (2000), Powell et al. “Compendium of excipients for parenteral formulations” PDA J Pharm Sci Technol. 52:238-311 (1998), and citations in those sources for additional information related to formulations, excipients, and carriers well known to medicinal chemists.

[0132] The following examples are presented to those skilled in the art to provide a complete disclosure and description of how the compounds, compositions, articles, devices, and / or methods described and claimed herein are prepared and evaluated, and are intended purely to illustrate and not to limit the scope that the inventors consider to be their invention. While efforts have been made to ensure accuracy with respect to numerical values ​​(quantities, temperatures, etc.), some errors and deviations should be taken into consideration here. Unless otherwise specified, parts are parts by weight, temperatures are in degrees Celsius or ambient temperature, and pressures are atmospheric pressure or close to it. Many variations and combinations of reaction conditions exist that can be used to optimize the purity and yield of the products obtained from the described processes, e.g., component concentrations, desired solvents, solvent mixtures, temperatures, pressures, and other reaction ranges and conditions. Optimizing such process conditions requires only reasonable and routine experimentation. [Examples]

[0133] Example 1: Induction of retinal pigment epithelium from human stem cell-derived retinal organoids: Method for producing a product : Step 1. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs.

[0134] Three-dimensional retinal tissue is prepared according to the methods described in Zhong et al., Nature Communications 5:4047 (2014) and U.S. Patent Application No. US2016 / 0333312 (each of which is incorporated herein by reference). In such hiPSC-derived 3D retinal organoids, RPE cells are found as clusters at the apex of the retinal organoid.

[0135] Step 2. Isolation of RPE cells from human 3D retina and establishment of induced primary RPE (ipRPE) culture (Figure 1). 1. Dissect the RPE tissue from the 3D retinal cup and / or floating RPE tissue aggregates, collect them in the center of a Petri dish, and aspirate the culture medium. 2. Rinse twice with PBS (approximately 5 ml). 3. After aspirating the final washing solution, add DMEM medium containing 0.25% collagenase IV (or other dissociation reagent) and let it stand for 4 hours (weigh, mix, and warm for 15 minutes, and filter out the collagenase before use). 4. After 4 hours in incubation (37°C, 5% CO2), the RPE tissue is broken down into small pieces by vigorous pipetting. Centrifuge at 5.25°C at 800 rpm for 5 minutes. 6. Aspirate the culture medium, resuspend the mass with Accumax (or other dissociation reagent), and incubate in an incubator for 20-30 minutes. 7. After the allotted time, gently pipette the mass until it dissociates into single cells, and then filter the solution using a 40 μm nylon mesh. 8. Seed the cells at approximately 100,000 cells per 1 cm 2 and plate them, and they need to be grown in RPE medium (Table 4). For example, 12 mm Matrigel (or other coating solution) coated transwell plating can be used. 9. Change the medium daily (rinse the first two times with PBS beforehand to clean up debris and cell death).

Table 4

[0136] Generate hiPSC-derived 3D retinal tissue as described in Zhong et al., Nature Communications 5:4047 (2014) and U.S. Patent Application Publication No. US2016 / 0333312. Prepare neural retinal patches (3DNR) from the 3D retina using any method known in the art. For example, open the hiPSC-derived 3D retina (e.g., using a tungsten needle or any other method known in the art) to expose the inside of the 3D retinal cup, flatten it as a retinal flat mount, and obtain retinal explants. On the other hand, retinal explants can also be obtained directly from the 3D retina using a laser.

[0137] Next, seed those retinal explants (or patches) onto ipRPE (passage 2) and co-culture for different times. Under those conditions, the 3DNR adheres to the RPE isolation and forms a 3DNR / RPE complex. (See Figure 5). Incorporation into a system of additional biocompatible components (e.g., natural or synthetic compounds in liquid or gel form that provide an appropriate biomechanical environment for cell survival and function, such as hydrogels), and / or biocompatible scaffolds (e.g., natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, and / or combinations thereof) further provides an improved biomechanical environment that allows for longer culture periods and handling during transplantation. Inclusion of this additional biocompatible component promotes the survival and function of transplanted cells.

[0138] References 1. Klimanskaya I, Hipp J, Rezai KA, West M, Atala A, Lanza R. Derivation and comparative assessment of retinal pigment epithelium from human embryonic stem cells using transcriptomics. Cloning Stem Cells. 2004;6(3):217-45. 2. Buchholz DE, Hikita ST, Rowland TJ, Friedrich AM, Hinman CR, Johnson LV, et al. Derivation of functional retinal pigmented epithelium from induced pluripotent stem cells. Stem Cells. 2009;27(10):2427-34. 3. Zahabi A, Shahbazi E, Ahmadieh H, Hassani SN, Totonchi M, Taei A, et al. A new efficient protocol for directed differentiation of retinal pigmented epithelial cells from normal and retinal disease induced pluripotent stem cells. Stem Cells Dev. 2012;21(12):2262-72. 4. Buchholz DE, Pennington BO, Croze RH, Hinman CR, Coffey PJ, Clegg DO. Rapid and efficient directed differentiation of human pluripotent stem cells into retinal pigmented epithelium. Stem Cells Transl Med. 2013;2(5):384-93. 5. Maruotti J, Wahlin K, Gorrell D, Bhutto I, Lutty G, Zack DJ. A simple and scalable process for the differentiation of retinal pigment epithelium from human pluripotent stem cells. Stem Cells Transl Med. 2013;2(5):341-54. 6. Singh R, Phillips MJ, Kuai D, Meyer J, Martin JM, Smith MA, et al. Functional analysis of serially expanded human iPS cell-derived RPE cultures. Invest Ophthalmol Vis Sci. 2013;54(10):6767-78. 7. Ferrer M, Corneo B, Davis J, Wan Q, Miyagishima KJ, King R, et al. A multiplex high-throughput gene expression assay to simultaneously detect disease and functional markers in induced pluripotent stem cell-derived retinal pigment epithelium. Stem Cells Transl Med. 2014;3(8):911-22. 8. Reichman S, Terray A, Slembrouck A, Nanteau C, Orieux G, Habeler W, et al. From confluent human iPS cells to self-forming neural retina and retinal pigmented epithelium. P Natl Acad Sci USA. 2014;111(23):8518-23. 9. Croze RH, Buchholz DE, Radeke MJ, Thi WJ, Hu Q, Coffey PJ, et al. ROCK Inhibition Extends Passage of Pluripotent Stem Cell-Derived Retinal Pigmented Epithelium. Stem Cells Transl Med. 2014;3(9):1066-78. 10. Leach LL, Buchholz DE, Nadar VP, Lowenstein SE, Clegg DO. Canonical / beta-catenin Wnt pathway activation improves retinal pigmented epithelium derivation from human embryonic stem cells. Invest Ophthalmol Vis Sci. 2015;56(2):1002-13. 11. Maruotti J, Sripathi SR, Bharti K, Fuller J, Wahlin KJ, Ranganathan V, et al. Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells. P Natl Acad Sci USA. 2015;112(35):10950-5. 12. Lidgerwood GE, Lim SY, Crombie DE, Ali R, Gill KP, Hernandez D, et al. Defined Medium Conditions for the Induction and Expansion of Human Pluripotent Stem Cell-Derived Retinal Pigment Epithelium. Stem Cell Rev. 2016;12(2):179-88. 13. Osakada F, Ikeda H, Mandai M, Wataya T, Watanabe K, Yoshimura N, et al. Toward the generation of rod and cone photoreceptors from mouse, monkey and human embryonic stem cells. Nat Biotechnol. 2008;26(2):215-24. 14. Vugler A, Carr AJ, Lawrence J, Chen LL, Burrell K, Wright A, et al. Elucidating the phenomenon of HESC-derived RPE: anatomy of cell genesis, expansion and retinal transplantation. Exp Neurol. 2008;214(2):347-61. 15. Idelson M, Alper R, Obolensky A, Ben-Shushan E, Hemo I, Yachimovich-Cohen N, et al. Directed Differentiation of Human Embryonic Stem Cells into Functional Retinal Pigment Epithelium Cells. Cell Stem Cell. 2009;5(4):396-408. 16. Vaajasaari H, Ilmarinen T, Juuti-Uusitalo K, Rajala K, Onnela N, Narkilahti S, et al. Toward the defined and xeno-free differentiation of functional human pluripotent stem cell-derived retinal pigment epithelial cells. Mol Vis. 2011;17:558-75. 17. Zhu D, Deng X, Spee C, Sonoda S, Hsieh CL, Barron E, et al. Polarized secretion of PEDF from human embryonic stem cell-derived RPE promotes retinal progenitor cell survival. Invest Ophthalmol Vis Sci. 2011;52(3):1573-85. 18. Plaza-Reyes A, Petrus-Reurer S, Antonsson L, Stenfelt S, Bartuma H, Panula S, et al. Xeno-Free and Defined Human Embryonic Stem Cell-Derived Retinal Pigment Epithelial Cells Functionally Integrate in a Large-Eyed Preclinical Model. Stem Cell Reports. 2016;6(1):9-17. 19. Meyer JS, Shearer RL, Capowski EE, Wright LS, Wallace KA, McMillan EL, et al. Modeling early retinal development with human embryonic and induced pluripotent stem cells. Proc Natl Acad Sci US A. 2009;106(39):16698-703. 20. Zhong X, Gutierrez C, Xue T, Hampton C, Vergara MN, Cao LH, et al. Generation of three-dimensional retinal tissue with functional photoreceptors from human iPSCs. Nat Commun. 2014;5:4047. 21. Wu W, Zeng Y, Li Z, Li Q, Xu H, Yin ZQ. Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures - a new donor for cell therapy. Oncotarget. 2016;7(16):22819-33.

[0139] equivalent Details of one or more embodiments of the present invention are described in the appendix above. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described herein.

[0140] The foregoing description is provided for illustrative purposes only and is not intended to limit the invention to the exact form disclosed, but rather to the claims appended herein.

Claims

1. A three-dimensional tissue product derived from human induced pluripotent stem cells (hiPSCs) that responds to light after transplantation into a subject, The three-dimensional tissue product comprises functionally mature retinal pigment epithelial (RPE) cells, a portion of three-dimensional neuroretina (3DNR), additional biocompatible components including a hydrogel, and a biocompatible scaffold, wherein the 3DNR, RPE cells, and additional biocompatible components are physically and functionally integrated to form a complex. The aforementioned 3DNR, i) Undifferentiated pseudostratified neuroretinal epithelium; ii) Layered neuroretinal tissue including all retinal layers and their corresponding retinal precursor cell types; or iii) A highly differentiated retinal tissue comprising the outer granular layer (ONL) and the bipolar cell layer (BCL), wherein the ONL may be rod-enriched, cone-enriched, or any combination thereof, The RPE cells are located on top of the biocompatible scaffold, and the 3DNRs are located on top of the RPE cells. A three-dimensional tissue product in which both the RPE cells and 3DNRs are obtained from human retinal organoids.

2. A method for preparing RPE cells to prepare the three-dimensional tissue product described in Claim 1, wherein the method comprises the following steps: a) A step of culturing human retinal organoids in a first medium that is not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate RPE cells and neuroretina (NR); b) A step of isolating RPE cell tissue from the cultured retinal organoid; c) The step of dissociating the isolated RPE cell tissue into a suspension of single RPE cells; d) The step of plating a single RPE cell into an adherent culture; and e) A step of culturing the plated cells in a second medium that is not supplemented with exogenous growth factors, morphogens, or modulators of their signaling pathways to generate a monolayer of RPE; Methods that include...

3. The aforementioned RPE cells, i) From the first plating or subsequent subculturing; ii) In the early stages of differentiation; or iii) The method according to claim 2, obtained by a more advanced stage of differentiation, a longer culture time, or a combination thereof.

4. The three-dimensional tissue product according to claim 1, wherein the biocompatible scaffold is selected from the group consisting of natural or synthetic scaffolds, scaffolds made from biodegradable materials, scaffolds made from non-biodegradable materials, or combinations thereof.

5. A three-dimensional tissue product according to any one of claims 1 or 4 for treating a disease, disorder, or condition of the retina, wherein the treatment comprises implanting the three-dimensional tissue product according to any one of claims 1 or 4 into the eye of a patient requiring the treatment.

6. The aforementioned retinal diseases, disorders, or conditions include retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular disease, retinal photoreceptor disease, and retinal pigmentary atrophy. A three-dimensional tissue product according to claim 5, selected from the group consisting of skin diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

7. A screening method for drugs that affect the development, function, proliferation, maturation, differentiation, survival, or any combination thereof of the retina, a) Contacting the three-dimensional tissue product according to any one of claims 1 or 4 with at least one drug; and b) A method comprising determining whether the drug has an effect on the development, function, proliferation, maturation, differentiation, survival, or any combination thereof of the retina.

8. The method according to claim 7, wherein the at least one agent is a biological agent, and the biological agent is selected from the group consisting of growth factors, trophic factors, regulatory factors, hormones, antibodies or antigen-binding fragments thereof, small molecules, and peptides.

9. An in vitro method for testing retinal development, a) Prepare a three-dimensional tissue product according to any one of claims 1 or 4; and b) An in vitro method comprising monitoring cell interactions, function, proliferation, maturation, differentiation, survival, or any combination thereof of cells within the three-dimensional tissue product.

10. By monitoring in step b): i) Normal retinal development; ii) Interaction between the retina and RPE; iii) Abnormal development, disease, disorder, or condition of the retina; or iv) The underlying mechanisms of abnormal development, disease, disorder, or condition of the retina; An in vitro method according to claim 9, wherein information relating to is obtained.

11. A three-dimensional tissue product according to any one of claims 1 or 4, for use in the treatment of retinal diseases, disorders or conditions, for transplantation into the eyes of patients requiring transplantation.

12. The aforementioned retinal diseases, disorders, or conditions include retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), Stargardt disease, Usher syndrome, choroidemia, rod-cone or cone-rod dystrophy, ciliopathy, mitochondrial disorders, progressive retinal atrophy, degenerative retinal diseases, age-related macular degeneration (AMD), wet AMD, dry AMD, geographic atrophy, familial or acquired macular disease, retinal photoreceptor disease, and retinal pigment epithelium. A three-dimensional tissue product according to claim 11, selected from the group consisting of sexually transmitted diseases, diabetic retinopathy, cystic macular edema, uveitis, retinal detachment, traumatic retinal injury, iatrogenic retinal injury, macular hole, macular telangiectasia, ganglion cell disease, optic nerve cell disease, glaucoma, optic neuropathy, ischemic retinal disease, retinopathy of prematurity, retinal vascular occlusion, familial aortic aneurysm, retinal vascular disease, ophthalmic vascular disease, vascular disease, and ischemic optic neuropathy.

Images