Retinal pigment epithelium and photoreceptor bilayer, and its use

Abstract

The present invention provides a bilayer composed of PR / PRP cells and RPE cells for treating a condition of dysfunction of either retinal epithelial cells (RPE) or photoreceptors (PR). [Solution] A tissue replacement graft is provided, comprising photoreceptor progenitor cells (PRP) and / or photoreceptor cells (PR) combined with retinal pigment epithelial cells (RPE) on a biodegradable scaffold.

Description

[Technical Field]

[0001] Priority Claim This application claims priority to U.S. Provisional Patent Application No. 63 / 032,346, filed on 29 May 2020, the entirety of which is incorporated herein by reference.

[0002] Parties to the joint research agreement This invention was made as a result of activities carried out within the scope of a collaborative research agreement that was in effect at the time the invention was made. The parties to the said collaborative research agreement were the U.S. government, represented by the National Eye Institute, an institution of National Institutes of Health and Fujifilm Cellular Dynamics International, Inc., and the U.S. Department of Health and Human Services.

[0003] 1. Field This disclosure, as a whole, relates to the field of stem cell biology. More specifically, this disclosure relates to a composition comprising a bilayer of retinal epithelial cells (RPE) and photoreceptor (PR) cells and / or photoreceptor precursor (PRP) cells. [Background technology]

[0004] 2. Related Technologies Age-related macular degeneration (AMD) is a debilitating condition affecting 11 million people in the United States and 170 million worldwide as of 2016, and is projected to reach 196 million worldwide by 2020 (Pennington and DeAngelis, 2016; Wong et al., 2014). The cause is thought to be dysfunction of the retinal pigment epithelium (RPE), which leads to the death and dysfunction of photoreceptors (Bhutto and Lutty, 2012). Cell therapy using RPE may be effective in treating AMD, myopic macular degeneration, or the rarer forms of hereditary macular degeneration, and several stem cell-based clinical trials to restore visual function are currently underway or planned (Oner, 2018). While AMD is one of the most common causes of blindness, other functional disorders such as retinitis pigmentosa, cone-rod dystrophy, and Leber congenital amaurosis are primarily caused by photoreceptor dysfunction and can be addressed with photoreceptor (PR) transplantation (Barnea-Cramer et al., 2016; Zhou et al., 2015; Zhao et al., 2017).

[0005] Photoreceptors extend the outer segments involved in light sensing. RPE cells support the overall health of photoreceptors by supporting the reuse of detached outer segments and other photoreceptor debris (Strauss, 2005). Therefore, the delivery of both RPE and PR and / or PRP (referred to herein as PR / PRP) as a bilayer culture therapy has potential to treat conditions of dysfunction of either RPE or photoreceptors, and the associated applications are broader compared to the delivery of either cell type alone. Furthermore, the symbiotic relationship between RPE and PR / PRP may make such treatments more effective. Thus, there is an unmet need for bilayer culture therapy composed of PR / PRP cells and RPE cells for the treatment of these diseases. [Overview of the project] [Means for solving the problem]

[0006] In a first embodiment, the disclosure provides a tissue replacement graft comprising photoreceptor progenitor cells (PRP) and / or photoreceptor cells (PR) in combination with retinal pigment epithelial cells (RPE) on a biodegradable scaffold. In certain embodiments, the graft is defined as heterogeneous component-free and feeder-free.

[0007] In some embodiments, the RPE is a mature RPE expressing bethrofin-1 (BEST1) and / or ZO-1. In specific embodiments, this RPE is polarized.

[0008] In certain embodiments, PR / PRP and RPE exist in a bilayer. In specific embodiments, this bilayer PR / PRP is attached to RPE by intercellular contact or attachment to a shared matrix.

[0009] In some embodiments, the biodegradable scaffold comprises poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLLA), polycaprolactone (PCL), poly(glycerol sebacate) (PGS), polypyrrole (PPy), polyvinyl alcohol (PVA), gelatin, collagen, laminin, fibronectin, fibrin, hyaluronic acid, silk, chitosan, and / or polyhydroxyethyl methacrylate (PHEMA). In certain embodiments, the biodegradable scaffold comprises PLGA. In specific embodiments, this PLGA has a DL-lactide / glycolide ratio of approximately 1:1. In some embodiments, this PLGA has an average pore size of less than approximately 1 micron. In certain embodiments, this PLGA has a fiber diameter of approximately 150 to approximately 650 nm.

[0010] In some embodiments, the biodegradable scaffold is coated with an extracellular matrix (ECM) protein. For example, this ECM protein includes vitronectin, laminin, collagen I, collagen IV, or fibronectin. In certain embodiments, this ECM protein includes vitronectin. In some embodiments, the biodegradable scaffold is about 20 to about 30 microns thick, for example, about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 microns.

[0011] In certain embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 2:1 to approximately 30:1, for example, approximately 3:1 to approximately 5:1, approximately 5:1 to approximately 10:1, approximately 10:1 to approximately 15:1, or approximately 15:1 to approximately 30:1. In some embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 1:1 to approximately 5:1, for example, approximately 1:1 to approximately 2:1, approximately 2:1 to approximately 3:1, approximately 3:1 to approximately 4:1, or approximately 4:1 to approximately 5:1.

[0012] In certain embodiments, RPE and / or PR / PRP are derived from pluripotent stem cells (PSCs), such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). In some embodiments, the iPSCs are universal iPSCs, HLA-matched iPSCs, or hypoimmune iPSCs. In specific embodiments, the iPSCs are human iPSCs (hiPSCs). In specific embodiments, the PR / PRP were not derived from organoids.

[0013] In some embodiments, RPE and / or PR / PRP are pre-frozen. In some embodiments, this pre-frozen RPE and / or PR / PRP are thawed and cultured for at least one week. In certain embodiments, the pre-frozen RPE and / or PR / PRP are thawed and cultured for less than one week.

[0014] In a particular configuration, RPE is approximately 100,000 cells / cm². 2 ~Approximately 1,000,000 cells / cm²2 present at a density of, for example, about 200,000 cells / cm 2 , 300,000 cells / cm 2 , 400,000 cells / cm 2 , 500,000 cells / cm 2 , 600,000 cells / cm 2 , 700,000 cells / cm 2 , 800,000 cells / cm 2 , or 900,000 cells / cm 2 present at a density of. In some embodiments, the RPE is present at a density of about 300,000 cells / cm 2 to about 800,000 cells / cm 2 present at a density of, for example, about 400,000 cells / cm 2 , 500,000 cells / cm 2 , 600,000 cells / cm 2 , or 700,000 cells / cm 2 present at a density of. In some embodiments, the PR / PRP is present at a density of about 100,000 cells / cm 2 to about 10,000,000 cells / cm 2 present at a density of, for example, about 200,000 cells / cm 2 , 300,000 cells / cm 2 , 400,000 cells / cm 2 , 500,000 cells / cm 2 , 600,000 cells / cm 2 , 700,000 cells / cm 2 , 800,000 cells / cm 2 , or 900,000 cells / cm 2 present at a density of. In certain embodiments, the PR / PRP is present at a density of about 200,000 cells / cm 2 to about 20,000,000 cells / cm 2 present at a density of, for example, about 300,000 cells / cm 2 to about 5,000,000 cells / cm 2 present at, for example, about 400,000 cells / cm 2500,000 cells / cm² 2 600,000 cells / cm² 2 700,000 cells / cm² 2 800,000 cells / cm² 2 900,000 cells / cm² 2 1,000,000 cells / cm² 2 2,000,000 cells / cm² 2 3,000,000 cells / cm² 2 4,000,000 cells / cm² 2 5,000,000 cells / cm² 2 6,000,000 cells / cm² 2 7,000,000 cells / cm² 2 8,000,000 cells / cm² 2 9,000,000 cells / cm² 2 10,000,000 cells / cm² 2 11,000,000 cells / cm² 2 12,000,000 cells / cm² 2 13,000,000 cells / cm² 2 14,000,000 cells / cm² 2 15,000,000 cells / cm² 2 , or present at a higher density. In certain embodiments, PR / PRP contains approximately 4 million, 5 million, 6 million, or 7 million cells / cm³ 2 They exist at a density of, for example, 6 million cells / cm³. 2 It exists at that density.

[0015] In some embodiments, RPE and / or PR / PRP are derived from the same donor. In certain embodiments, PR / PRP is rod-predisposed. In some embodiments, PR / PRP is cone-predisposed.

[0016] Further embodiments provide a pharmaceutical composition comprising a tissue replacement graft of this embodiment or an embodiment thereof (for example, a tissue replacement graft comprising photoreceptor progenitor cells (PRP) and / or photoreceptor cells (PR) in combination with retinal pigment epithelial cells (RPE) on a biodegradable scaffold). In additional embodiments, the composition further comprises sodium hyaluronate. In some embodiments, the hyaluronate is present at a concentration of less than about 0.5%, for example, at a concentration of 0.4%, 0.3%, or 0.2%. In some embodiments, the composition further comprises sodium bicarbonate, calcium chloride, potassium chloride, monobasic potassium phosphate, magnesium chloride, magnesium sulfate, sodium chloride, and / or dibasic sodium phosphate.

[0017] Another embodiment provides a method for producing a tissue replacement graft according to this embodiment or a similar aspect (for example, a tissue replacement graft comprising photoreceptor progenitor cells (PRP) and / or photoreceptor cells (PR) in combination with retinal pigment epithelial cells (RPE) on a biodegradable scaffold), comprising: (a) seeding RPE on a biodegradable scaffold; (b) culturing the RPE on the biodegradable scaffold in a first tissue culture medium for a period sufficient to generate polarized RPE; (c) seeding PR / PRP on the RPE to form a tissue replacement graft; and (d) culturing the tissue replacement graft in a second tissue culture medium for a period sufficient to allow PR / PRP to adhere to the RPE.

[0018] In some embodiments, this scaffold is secured with a plastic O-ring. In certain embodiments, the polarized RPE expresses bethrofin 1 (BEST1). In certain embodiments, the second tissue culture medium is essentially identical to the first tissue culture medium.

[0019] In certain embodiments, the biodegradable scaffold comprises poly(lactic acid-coglycolic acid) (PLGA), polylactic acid (PLLA), polycaprolactone (PCL), poly(glycerol sebacate) (PGS), polypyrrole (PPy), polyvinyl alcohol (PVA), gelatin, collagen, laminin, fibronectin, fibrin, hyaluronic acid, silk, chitosan, or polyhydroxyethyl methacrylate (PHEMA). In certain embodiments, the biodegradable scaffold comprises PLGA. In some embodiments, this PLGA has a DL-lactide / glycolide ratio of approximately 1:1. In certain embodiments, this PLGA has an average pore size of less than approximately 1 micron. In some embodiments, this PLGA has a fiber diameter of approximately 150 to approximately 650 nm. In certain embodiments, the biodegradable scaffold is coated with an extracellular matrix (ECM) protein, such as vitronectin, laminin, collagen I, collagen IV, or fibronectin. In specific embodiments, this ECM protein includes vitronectin. In certain embodiments, the amount of vitronectin is 0.5 μg / cm³. 2 It is added to the surface at a concentration of, for example, about 1 μg / cm³. 2 5 μg / cm³ 2 , or 10 μg / cm³ 2 Add it to the surface at this concentration.

[0020] In some embodiments, RPE is measured at approximately 100,000 cells / cm². 2 ~Approximately 1,000,000 cells / cm² 2 Seeds are seeded at a density of, for example, approximately 200,000 cells / cm³. 2 300,000 cells / cm² 2 400,000 cells / cm² 2 500,000 cells / cm² 2 600,000 cells / cm² 2 700,000 cells / cm² 2 800,000 cells / cm² 2 , or 900,000 cells / cm² 2 Seeds are seeded at a density of approximately 300,000 cells / cm³. In a particular configuration, RPEs are seeded at a density of approximately 300,000 cells / cm³.2 ~Approximately 800,000 cells / cm² 2 Seeds are seeded at a density of, for example, approximately 400,000 cells / cm³. 2 500,000 cells / cm² 2 600,000 cells / cm² 2 , or 700,000 cells / cm² 2 Seed at a density of approximately 100,000 cells / cm³. In some embodiments, PR / PRP is seeded at a density of approximately 100,000 cells / cm³. 2 ~Approximately 10,000,000 cells / cm² 2 Seeds are sown at a concentration of, for example, approximately 200,000 cells / cm³. 2 300,000 cells / cm² 2 400,000 cells / cm² 2 500,000 cells / cm² 2 600,000 cells / cm² 2 700,000 cells / cm² 2 800,000 cells / cm² 2 , or 900,000 cells / cm² 2 Seedling is performed at this concentration. In a specific configuration, PR / PRP is applied at approximately 3 million cells / cm². 2 ~Approximately 5 million cells / cm² 2 Seeds are sown at a concentration of, for example, approximately 4 million cells / cm². 2 Sow at this concentration.

[0021] In some embodiments, RPE and / or PR / PRP are pre-frozen.

[0022] In certain embodiments, the biodegradable scaffold is contained in a multiwell support equipped with a tissue culture insert. In certain embodiments, a first tissue culture medium is added to the lower compartment of the multiwell siport equipped with the tissue culture insert. In certain embodiments, a second tissue culture medium is added to the upper compartment of the multiwell siport equipped with the tissue culture insert. In some embodiments, the first tissue culture medium contains taurine and hydrocortisone. In certain embodiments, the first tissue culture medium further contains triiodothyronine. In certain embodiments, the first tissue culture medium is a limited medium or a serum-free medium. In some embodiments, the first tissue culture medium contains a serum substitute. In specific embodiments, the first tissue culture medium further contains prostaglandin E2 (PGE2) at a concentration of, for example, 50 μM to 100 μM (e.g., 50 to 75 μM or 75 to 100 μM). In certain embodiments, the first tissue culture medium is RPE-MM medium. In some embodiments, the second tissue culture medium is essentially identical to the first tissue culture medium. In certain embodiments, the second tissue culture medium is different from the first tissue culture medium. In some embodiments, the second tissue culture medium is minimal medium (RMN). In specific embodiments, the first tissue culture medium is added to the lower compartment of the multiwell support, and the second tissue culture medium is added to the upper compartment of the multiwell support. In some embodiments, the pressure from the medium in the lower compartment to the tissue culture insert is higher than the pressure from the medium in the upper compartment.

[0023] In some embodiments, process (b) lasts for at least about two weeks, for example, three or four weeks. In certain embodiments, process (d) lasts for at least about five days, for example, six, seven, or ten days. In some embodiments, process (d) lasts for about one day, for example, two, three, or four days.

[0024] In certain embodiments, PRP is rod-shaped. In some embodiments, PRP is cone-shaped.

[0025] In some embodiments, the first tissue culture medium and the second tissue culture medium are replaced at least once every five days, for example, at least once every four days, once every three days, or once every other day.

[0026] In certain embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 2:1 to approximately 30:1, for example, approximately 3:1 to approximately 5:1, approximately 5:1 to approximately 10:1, approximately 10:1 to approximately 15:1, or approximately 15:1 to approximately 30:1. In some embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 1:1 to approximately 5:1, for example, approximately 1:1 to approximately 2:1, approximately 2:1 to approximately 3:1, approximately 3:1 to approximately 4:1, or approximately 4:1 to approximately 5:1.

[0027] Further embodiments provide tissue replacement grafts of this embodiment or embodiment, manufactured according to the method of this embodiment or embodiment thereof.

[0028] Another embodiment provides a method for producing a PR / PRP-RPE bilayer, comprising: (a) seeding RPE in tissue culture medium into the upper compartment of a multiwell support equipped with a tissue culture insert; (b) seeding PR / PRP in tissue culture medium into the upper compartment of the multiwell support in direct contact with the RPE, wherein the intermediate pressure in the lower compartment is higher than the intermediate pressure in the upper compartment; and (c) culturing for a period of time sufficient to produce a PR / PRP-RPE bilayer.

[0029] In some embodiments, the culture media in the lower and upper compartments of a multi-well support equipped with a tissue culture insert are essentially identical. In certain embodiments, the culture media in the lower and upper compartments of a multi-well support equipped with a tissue culture insert are different.

[0030] In certain embodiments, the RPE is polarized RPE. In certain embodiments, this polarized RPE expresses BEST1. In some embodiments, the RPE is seeded on a biodegradable scaffold. In certain embodiments, this biodegradable scaffold comprises poly(lactic-co-glycolic acid) (PLGA), poly(lactic acid) (PLLA), poly(caprolactone) (PCL), poly(glycerol sebacate) (PGS), polypyrrole (PPy), poly(vinyl alcohol) (PVA), gelatin, collagen, laminin, fibronectin, fibrin, hyaluronic acid, silk, chitosan, and / or poly(hydroxyethyl methacrylate) (PHEMA). In some embodiments, this biodegradable scaffold comprises PLGA. In certain embodiments, this PLGA has a DL-lactide / glycolide ratio of about 1:1. In some embodiments, this PLGA has an average pore size of less than about 1 micron. In certain embodiments, this PLGA has a fiber diameter of about 150 to about 650 nm.

[0031] In some embodiments, the biodegradable scaffold is coated with an extracellular matrix (ECM) protein. In certain embodiments, this ECM protein comprises vitronectin, laminin, collagen I, collagen IV, or fibronectin. In specific embodiments, this ECM protein comprises vitronectin. In some embodiments, this vitronectin is added to the surface at a concentration of about 0.5 μg / cm 2 sup, for example, at a concentration of about 1 μg / cm 2 and added to the surface, for example, at a concentration of about 5 μg / cm 2 or about 10 μg / cm 2 and added to the surface.

[0032] In some embodiments, the RPE is seeded at a density of about 100,000 cells / cm 2 to about 1,000,000 cells / cm 2 , for example, about 200,000 cells / cm 2 , 300,000 cells / cm 2 , 400,000 cells / cm 2 , 500,000 cells / cm 2, seeded at a density of 600,000 cells / cm 2 , seeded at a density of 700,000 cells / cm 2 , seeded at a density of 800,000 cells / cm 2 , or seeded at a density of 900,000 cells / cm 2 . In certain embodiments, RPE is seeded at a density of about 300,000 cells / cm 2 to about 800,000 cells / cm 2 , for example, at a density of about 400,000 cells / cm 2 , 500,000 cells / cm 2 , 600,000 cells / cm 2 , or at a density of 700,000 cells / cm 2 . In certain embodiments, PR / PRP is seeded at a concentration of about 100,000 cells / cm 2 to about 10,000,000 cells / cm 2 , for example, at a concentration of about 200,000 cells / cm 2 , 300,000 cells / cm 2 , 400,000 cells / cm 2 , 500,000 cells / cm 2 , 600,000 cells / cm 2 , 700,000 cells / cm 2 , 800,000 cells / cm 2 , or at a concentration of 900,000 cells / cm 2 . In some embodiments, PR / PRP is seeded at a concentration of about 3 million cells / cm 2 to about 5 million cells / cm 2 , for example, seeded at a concentration of about 4 million cells / cm 2 .

[0033] In certain embodiments, RPE and / or PR / PRP have been cryopreserved.

[0034] In some embodiments, the first tissue culture medium comprises taurine and hydrocortisone. In additional embodiments, the first tissue culture medium further comprises triiodothyronine. In certain embodiments, the first tissue culture medium is a restricted medium or a serum-free medium. In some embodiments, the first tissue culture medium comprises a serum substitute. In certain embodiments, the first tissue culture medium is RPE-MM medium. In certain embodiments, the second tissue culture medium comprises taurine and hydrocortisone. In some embodiments, the second tissue culture medium further comprises triiodothyronine. In specific embodiments, the second tissue culture medium is a restricted medium or a serum-free medium. In certain embodiments, the second tissue culture medium comprises a serum substitute. In some embodiments, the second tissue culture medium is RPE-MM medium.

[0035] In some embodiments, PR / PRP is rod-shaped. In certain embodiments, PR / PRP is cone-shaped.

[0036] In some embodiments, the first tissue culture medium and the second tissue culture medium are replaced at least once every five days, for example, at least once every four days, once every three days, or once every other day.

[0037] In certain embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 2:1 to approximately 30:1, for example, approximately 3:1 to approximately 5:1, approximately 5:1 to approximately 10:1, approximately 10:1 to approximately 15:1, or approximately 15:1 to approximately 30:1. In some embodiments, the PR / PRP to RPE ratio in the tissue replacement graft is approximately 1:1 to approximately 5:1, for example, approximately 1:1 to approximately 2:1, approximately 2:1 to approximately 3:1, approximately 3:1 to approximately 4:1, or approximately 4:1 to approximately 5:1.

[0038] Further embodiments provide an RPE-PR / PRP bilayer cell composition comprising a distinct bilayer of mature PRP cultured on polarized RPE. In certain embodiments, this polarized RPE is positive for bethrophine and / or ZO-1. In some embodiments, this mature PR / PRP is positive for peripherin-2 and / or neuroretinal leucine zipper (NRL).

[0039] A method for treating an injury or disorder of an eye, comprising implanting an effective amount of retinal epithelial cells (RPE) and a PR / PRP (RPE-PR / PRP) bilayer composition into the eye.

[0040] In some embodiments, the eye disorder is due to RPE dysfunction or photoreceptor dysfunction. In certain embodiments, this eye disorder is age-related macular degeneration, retinitis pigmentosa, cone-rod dystrophy, or Leber congenital amaurosis. In certain embodiments, the RPE-PR / PRP bilayer composition is implanted into the target retina. In some embodiments, the RPE-PR / PRP bilayer composition is implanted onto a scaffold. In certain embodiments, the RPE-PR / PRP bilayer composition comprises a tissue replacement graft of this embodiment or embodiment thereof, or a pharmaceutical composition of this embodiment or embodiment thereof. In some embodiments, this tissue replacement graft is implanted into the subretinal space. In certain embodiments, the tissue replacement graft is implanted using a subretinal injection device. In certain embodiments, the RPE and / or PR / PRP are derived from human induced pluripotent stem cells (hiPSCs). In some embodiments, the RPE and / or PR / PRP are cryopreserved in advance. In certain embodiments, the RPE is a mature RPE, for example, a mature RPE positive for bethrophage and / or ZO1. In some embodiments, the RPE resides on a surface coated with an extracellular matrix (ECM) protein. In certain embodiments, this ECM protein is vitronectin, laminin, collagen I, collagen IV, or fibronectin. In certain embodiments, this ECM protein is vitronectin. In some embodiments, the RPE resides on a copolymer scaffold. In certain embodiments, the copolymer scaffold comprises poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLLA), polycaprolactone (PCL), poly(glycerol sebacate) (PGS), polypyrrole (PPy), polyvinyl alcohol (PVA), gelatin, collagen, laminin, fibronectin, fibrin, hyaluronic acid, silk, chitosan, and / or polyhydroxyethyl methacrylate (PHEMA). In certain embodiments, the PR / PRP was not derived from organoids. In some embodiments, the RPE-PR / PRP bilayer is present in a medium containing taurine and hydrocortisone. In certain embodiments, the medium further comprises triiodothyronine. In certain embodiments, the medium is a limited medium or a serum-free medium.In specific embodiments, this medium contains a serum substitute. In some embodiments, this medium is RPE-MM medium. In certain embodiments, PR / PRP is positive for peripherin-2 and / or neuroretinal leucine zipper (NRL). In some embodiments, the PR / PRP to RPE ratio in the distinct bilayer is 1:1 to 5:1, e.g., approximately 1:1 to 2:1, 2:1 to 3:1, 3:1 to 4:1, or 4:1 to 5:1.

[0041] Further embodiments provide the use of tissue replacement grafts of this embodiment or its embodiment as model retinas.

[0042] Another embodiment provides the use of a tissue replacement graft of this embodiment or an embodiment thereof as a substrate for growing tissue.

[0043] Other purposes, features, and advantages of this disclosure will become apparent from the detailed description below. However, it should be understood that the detailed description and specific examples are given only as examples, illustrating preferred embodiments of the invention, for various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

[0044] The following drawings form part of this specification and are intended to further illustrate certain aspects of the invention. The invention may be better understood by referring to one or more of these drawings in conjunction with the detailed description of the specific embodiments presented herein. [Brief explanation of the drawing]

[0045] [Figure 1A-1E]RPE / PRP co-culture. (Figure 1A) Flow profile of PRP before co-culture. (Figures 1B-1C) RPE / PRP co-culture after 1 day. Phase-contrast microscopy at 2x (Figure 1B) and 10x (Figure 1C) shows the PRP layer on a confluent RPE layer. (Figures 1D-1E) RPE / PRP co-culture after 7 days. Fluorescence microscopy shows (Figure 1D) all nuclei (blue) and (Figure 1E) RPE-specific CRALBP (green), PRP-specific recabinetin (red), and retinal precursor-specific CHX10 (purple). Both RPE and PRP are evident, and PRP (recabinet-positive cells) are attached in subconfluent clusters. Retinal precursors were hardly observed.

[0046] [Figure 2A-2B] Co-staining with ZO-1 and recabinet. (Figure 2A) Focused on PRP, and (Figure 2B) focused on RPE. Two distinct layers are demonstrated by the clear presence of recabinet-positive cells in the PRP focus and the typical hexagonal tight junction morphology highlighted by ZO-1 around the RPE.

[0047] [Figure 3A-3H] Immunocytochemistry of PRP-mediated (RPE / PRP) bilayers (counting after thawing at 3 × 10⁶ and 1 × 10⁷ cells / cm²). Cells at low density were stained for (Figure 3A) ZO-1 (green), (Figure 3B) Recabilin (red), and (Figure 3) CRALBP (green) / Recabilin (red). Cells at high density were stained for (Figure 3D) ZO-1 and (Figure 3E) Recabilin, and (Figure 3F) magnified images of cells stained with ZO-1 show the expected RPE "cobblestone" tight junction morphology. Reconstructed confocal images of the lateral view show multiple layers of RPE / PRP bilayers with (Figure 3G) ZO-1 (green) / Recabilin (red) and (Figure 3H) CRALBP (green) / Recabilin (red), illustrating the lamination of these two cell types.

[0048] [Figures 4A-4F]Quantification of the RPE / PRP ratio 7 days after seeding 4e6 PRP cells onto a confluent monolayer of RPE. RPE is shown as TYRP1 and PRP as AIPL1. (Figure 4A) Flow cytometry plot of iPRP control, (Figure 4B) Flow cytometry plot of iRPE control, (Figure 4C) Flow cytometry plot of RPE cultured in snapwells in monoculture, (Figure 4D) Flow cytometry plot of PRP cultured on polarized RPE for 7 days in coculture. (Figure 4E) Marker quantification shows that both RPE (TYRP1) and PRP (AIPL1) are present in the coculture sample, (Figure 4F) The ratio of the proportion of cells expressing AIPL1 divided by the proportion of cells expressing TYRP1 (a surrogate for the PRP / RPE ratio) is approximately 6 in this sample. Cells were derived from the iPSC lineage HLA-A.

[0049] [Figures 5A-5C] Characterization of RPE / PRP co-cultures on Snapwell scaffolds. (Figure 5A) Flow plots show distinct populations for each cell type, but recabinant expression is higher in co-cultures with RPE-MM (5% FBS). (Figure 5B) Morphology by ICC in RPE-MM (5% FBS) and (Figure 5C) morphology by ICC in RPE-MM (15% KOSR) were similar.

[0050] [Figure 6A-6C] Confocal microscopy images of RPE / PRP bilayers on a PLGA scaffold coated with VTN. (Figure 6A) Bilayers with 5 × 10⁶ cells / cm² and (Figure 6B) Bilayers with 1 × 10⁷ cells / cm², and (C) Side view of a Z-stack section clearly showing distinct RPE and PRP layers.

[0051] [Figures 7A-7D]Improved adhesion when adjusting the amount of culture medium in snapwells. Schematic diagram (A) shows the volume of a snapwell with relatively high pressure at the apical end, and schematic diagram (B) shows the volume of a snapwell with relatively high pressure at the basal end. Immunocytochemistry with ZO-1 (green) and recoverin (red) showed that (C) PRP adhesion decreases as the pressure at the apical end increases, but (D) PRP adhesion improves as the pressure at the basal end increases.

[0052] [Figures 8A-8D] (Figures 8A, 8B) PRP seeded on RPE at low or high vivo concentrations, and at 3 million PRP / cm² or 10 million PRP / cm² (Figures 8A, 8B) respectively. PRP was stained with recabinet (red).

[0053] [Figures 9A-9C] Immunocytochemistry of porcine retina two months after iPSC-RPE / PRP scaffold transplantation. Transplanted human cells were identified with human nuclear antibodies. Transplanted human nuclei colocalize with cones (indicated by ARR3 in Figure 9A) and rods (indicated by NRL in Figure 9B) and rhodopsin in Figure 9C). The photoreceptor layer is in contact with the host neuroretina. This pig is a laser-treated pig model in which RPE and photoreceptors are destroyed.

[0054] [Figure 10] This image shows immunocytochemistry of porcine retina-RPE-choroidal sections two months after iPSC-RPE / PRP scaffold transplantation, demonstrating duplication of human nuclei (Ku80) and RPE (MITF). The transplanted human RPE layer is located on top of the transplanted PRP layer.

[0055] [Figure 11] This image shows the immunocytochemistry of porcine retina-RPE-choroidal sections two months after iPSC-RPE / PRP scaffold transplantation, illustrating the photoreceptors indicated by recabin.

[0056] [Figure 12A-12C]Immunocytochemistry of porcine retina-RPE-choroid sections 2 months after iPSC-RPE / PRP scaffold transplantation (Figure 12A) shows that Müller glia (GFAP) reforms the outer limiting membrane but is arrested in the transplanted RPE layer (Figure 12B). DIC images show the condition and orientation of the sectioned retina, clearly showing colored transplanted and host cells (Figure 12C). No Ki67-positive proliferating cells are observed.

[0057] [Figures 13A-13B] This image shows the immunocytochemistry of porcine retina-RPE-choroidal sections two months after iPSC-RPE / PRP scaffold transplantation (Figure 13A), indicating proximity of the presynaptic marker VGLUT1 to the transplanted photoreceptor, and (Figure 13B) migration of host bipolar cells (PKCα) to the transplanted photoreceptor layer. The possibility of integration is indicated by arrows.

[0058] [Figures 14A-14B] This is immunocytochemistry of porcine retinal-RPE-choroidal sections 2 months after iPSC-RPE / PRP scaffold transplantation, showing (Figure 14A) widespread expression of synaptophysin, a human-specific presynaptic marker, in transplanted cells, and (Figure 14B) co-labeling with ARR3-positive cones. [Modes for carrying out the invention]

[0059] 1. Description of an Exemplary Invention RPE and neuroretina exist in vivo as an ordered series of layered structures. In therapeutic applications, reproduction of the original structural relationships between RPE and PR / PRP may be essential, as proper juxtaposition and correct arrangement of these layers, in particular, can enable function. Furthermore, in vitro reproduction of layered RPE-PR / PRP may also be feasible as a platform for testing drug or cellular disease models. Therefore, in certain embodiments, this disclosure provides a method for culturing human induced pluripotent stem cell (hiPSC)-derived RPE (iRPE) with hiPSC-derived PR cells and / or hiPSC-derived PRP (iPRP) in a “dual therapy” bilayer. In some embodiments, the RPE and / or PR / PRP are derived from PSCs such as embryonic stem cells. From this study, it has been found that the distinct bilayer of RPE-PR / PRP provided herein exhibits layer adhesion, maintenance of expected markers, and layer stratification in several culture formats.

[0060] In certain embodiments, RPE is initially cultured on a surface. This RPE can be cultured to produce polarized RPE that is positive for bethrophine, a late polarization marker. This RPE may be derived from hiPSCs (e.g., by the methods disclosed in PCT / US2016 / 050543 and PCT / US2016 / 050554, which are incorporated herein by reference in whole), or it may be embryonic RPE or fetal RPE. This PR / PRP may also be derived from hiPSCs (e.g., by the methods disclosed in PCT / US2019 / 028557, which are incorporated herein by reference in whole), or it may be derived from ES cells. Next, immature PRP having the ability to mature into rods and cones and / or photoreceptor cells at the end of the PRP stage may be layered on top of this RPE. The PR / PRP may be thawed and seeded directly onto the RPE, or it may be cultured for a certain period of time before being seeded onto the RPE. In some embodiments, factors such as ROCK inhibitors (e.g., Y-27632), laminin-521, peanut agglutinin (PNA), high concentrations of vitronectin (VTN), or PGE2 can be added to this culture system to enhance RPE-PR / PRP adhesion.

[0061] The RPE-PR / PRP bilayer provided herein can be used in a variety of in vivo and in vitro methods. For example, this RPE-PR / PRP bilayer can be used in vivo to treat conditions of the retina or RPE, including, but not limited to, age-related or hereditary macular degeneration and retinitis pigmentosa, or other hereditary extretinal degenerative diseases, or damage causing dysfunction and / or loss of RPE and / or PR / PRP. This RPE-PR / PRP bilayer can also be used in vitro in screening assays to identify potential therapeutic or prophylactic treatment candidates. Similarly, the RPE-PR / PRP bilayer can be used as a substrate for constructing more complex tissues. Further embodiments and advantages of this disclosure are described below.

[0062] I. Definition The term "purified" is intended as a relative term, not to imply absolute purity. Therefore, a purified cell population is more than approximately 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure, or 100% pure, and most preferably essentially free of other cell types.

[0063] As used herein, "a" or "an" may mean one or more. As used in the claims, when used with the word "including," the terms "a" or "an" may mean one or more.

[0064] The use of the term “or” in the claims is used to mean “and / or” unless explicitly indicated to mean only substitutes, or unless the substitutes are mutually exclusive; however, this disclosure supports the definitions of substitutes only and “and / or.” As used herein, “another” may mean at least two or more.

[0065] The term “essentially” should be understood to include only those methods or compositions that do not substantially affect the specified process or materials and the fundamental, novel properties of those methods and compositions.

[0066] As used herein, a composition or culture medium that is "substantially free" of a particular substance or material contains that substance or material in amounts of 30%, 20%, 15%, more preferably 10%, even more preferably 5%, and most preferably 1% or less.

[0067] The terms “substantially” or “approximately,” as used herein, may be applied to modify any quantitative comparison, value, measurement, or other expression that may vary within an acceptable range without altering the fundamental function of the expression in question.

[0068] The term "about" generally refers to a value within the standard deviation of the specified value, as determined using standard analytical techniques for measuring the specified value. This term may also be used to refer to ±5% of the specified value.

[0069] As used herein, “essentially absent” with respect to a characteristic component is used herein to mean that this particular component is not intentionally included in the composition and / or is present only as a contaminant or in trace amounts. Accordingly, the total amount of this particular component due to any unintentional contamination of the composition is well below 0.05%, preferably less than 0.01%. Most preferably, the amount of this particular component in the composition is undetectable by standard analytical methods.

[0070] The term “cell population” is used herein to refer to a group of cells (typically of a common type). A cell population may originate from a common progenitor cell or may include multiple cell types. A “enriched” cell population refers to a cell population derived from a starting cell population (e.g., an unfractionated heterogeneous cell population) in which the proportion of a particular cell type is higher than the proportion of that cell type in the starting population. A cell population may be enriched with one or more cell types and depleted with one or more cell types.

[0071] The term “stem cell” as used herein refers to a cell that, under suitable conditions, can differentiate into a variety of specific cell types, but under other suitable conditions, can self-replicate and remain essentially undifferentiated in a pluripotent state. The term “stem cell” also encompasses pluripotent cells, multipotent cells, precursor cells, and progenitor cells. Exemplary human stem cells can be obtained from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state by expressing certain transcription factors associated with pluripotency; these cells are called “induced pluripotent stem cells” or “iPSCs.”

[0072] The term "pluripotency" refers to the property of cells to differentiate into all other cell types of an organism, except for extraembryonic or placental cells. Pluripotent stem cells can differentiate into all three germ layer cell types (e.g., ectoderm, mesoderm, and endoderm cell types) even after long-term culture. In some embodiments, pluripotent stem cells are embryonic stem cells derived from the inner cell mass of a blastocyst. In other embodiments, pluripotent stem cells are induced pluripotent stem cells obtained by reprogramming somatic cells.

[0073] The term "differentiation" refers to the process by which undifferentiated cells become more differentiated, involving changes in their structural and / or functional properties. Mature cells typically possess altered cellular structures and tissue-specific proteins.

[0074] As used herein, “undifferentiated” refers to cells that exhibit characteristic markers and morphological features that clearly distinguish them from terminally differentiated cells of embryonic or adult origin.

[0075] Embryoid bodies (EBs) are aggregates of pluripotent stem cells that can differentiate into endoderm, mesoderm, and ectoderm cells. Spheroid structures arise when pluripotent stem cells aggregate under non-adherent culture conditions, thus forming EBs in suspension.

[0076] "Isolated" cells are actually isolated or purified from other cells in an organism or culture. Isolated cells may be, for example, at least 99%, at least 98%, at least 95%, or at least 90% pure.

[0077] An "embryo" refers to a zygote with an artificially reprogrammed nucleus, or a mass of cells obtained from one or more divisions of an activated oocyte.

[0078] Embryonic stem (ES) cells are undifferentiated pluripotent cells obtained from early stages of the embryo, such as the inner cell mass at the blastocyst stage, or undifferentiated pluripotent cells produced by artificial means (e.g., nuclear transfer), and can give rise to any differentiated cell type of the embryo or adult, such as germ cells (e.g., sperm and eggs).

[0079] Induced pluripotent stem cells (iPSCs) are cells produced by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be produced using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In certain embodiments, factors that may be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct 3 / 4), Sox2, c-Myc, and Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed by expressing at least two, at least three, or four reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0080] An "allele" refers to one of two or more forms of a gene. Diploid organisms such as humans have two copies of each chromosome, and therefore each chromosome contains one allele.

[0081] The term "homozygous" is defined as having two of the same alleles at a particular locus. The term "heterozygous" refers to having two different alleles at a particular locus.

[0082] A "haplotype" refers to a combination of alleles at multiple loci along a single chromosome. Haplotypes can be based on a set of single nucleotide polymorphisms (SNPs) and / or alleles at the major histocompatibility complex on a single chromosome.

[0083] As used herein, the term “haplotype-matched” is defined as a cell (e.g., iPS cells) and the subject being treated sharing one or more major histocompatibility locus haplotypes. The haplotype of the subject can be readily determined using assays known in the art. Haplotype-matched iPS cells may be autologous or allogeneic. Autologous cells grown in tissue culture and differentiated into PRP cells are inherently haplotype-matched to the subject.

[0084] "Substantially identical HLA types" indicates that the donor's human leukocyte antigen (HLA) type and the patient's HLA type match to a degree that the transplanted cells, obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can engraft when transplanted into the patient.

[0085] "Superdonor" as used herein refers to an individual that is homozygous for certain MHC class I and II genes. These homozygous individuals can function as superdonors, and their cells (including tissues or other materials containing these cells) can be transplanted into individuals that are homozygous or heterozygous for this haplotype. Superdonors may be homozygous for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus / locular alleles, respectively.

[0086] In this specification, “feeder-free” or “feeder-independent” refers to cultures in which cytokines and growth factors (e.g., TGFβ, bFGF, LIF) are supplemented as a substitute for the feeder cell layer. Thus, pluripotent cells can be cultured and maintained in an undifferentiated growth state using “feeder-free” or feeder-independent culture systems and media. In some cases, feeder-free cultures utilize animal-based matrices (e.g., MATRIGEL®) or are grown on substrates such as fibronectin, collagen, or vitronectin. These approaches make it possible to maintain human stem cells in an essentially undifferentiated state without requiring a mouse fibroblast “feeder layer.”

[0087] In this specification, the “feeder layer” is defined, for example, as a coating layer of cells on the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can adhere.

[0088] The terms “defined” or “fully defined,” when used in relation to culture media, extracellular matrices, or culture conditions, refer to culture media, extracellular matrices, or culture conditions in which the chemical composition and quantities of virtually all components are known. For example, a defined medium does not contain undefined factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Generally, a defined medium includes a basic medium supplemented with recombinant albumin, chemically distinct lipids, and recombinant insulin (e.g., Dulbecco’s Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Laboratory Medium (RPMI) 1640, which contains amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources). An example of a fully defined medium is Essential 8® medium.

[0089] With regard to culture media, extracellular matrices, or culture systems used with human cells, the term "xeno-free (XF)" refers to a state in which the substances used are not of non-human animal origin.

[0090] "Preconfluent" refers to a cell culture in which approximately 60-80% of the culture's surface is covered by cells. Typically, preconfluent refers to a culture in which approximately 70% of the culture's surface is covered by cells.

[0091] The term "retinal progenitor cells" is also known as "retinal precursor cells" or "RPCs" and includes cells capable of generating all cell types of the retina (e.g., neuroretinal cells (e.g., rods, cones, photoreceptor precursor cells), and cells that can differentiate into RPEs).

[0092] The term "neuroretinal progenitor cells" or "NRP" refers to cells whose ability to differentiate into neuroretinal cells is limited.

[0093] The term "photoreceptor" or "PR" cell refers to a cell that is in the photoreceptor lineage (i.e., maturation) pathway both before and after the upregulation of the expression of either rhodopsin (rod) or one of the three cone opsins (cone), and includes both early and late markers of photoreceptor cells (rod, cone, or both).

[0094] The terms "photoreceptor progenitor cells" or "PRP" refer to cells differentiated from embryonic stem cells or induced pluripotent stem cells that can differentiate into photoreceptor cells expressing the cell marker rhodopsin or one of the three cone opsins. The photoreceptors may be rod photoreceptors and / or cone photoreceptors.

[0095] The "retinal pigment epithelium" refers to the layer of pigment cells between the choroid, which is a layer filled with blood vessels, and the neuroretina.

[0096] The term “Retinal Degeneration-Related Disorders” is intended to refer to any disorder resulting from congenital or postnatal retinal degeneration or retinal abnormalities. Examples of retinal degeneration-related disorders include: retinal dysplasia, retinal degeneration, age-related macular degeneration, Stargardt disease, Best's disease, total choroidal atrophy, hereditary macular degeneration, myopic degeneration, RPE rupture, macular hole, diabetic retinopathy, retinitis pigmentosa, hereditary retinal disease or retinal degeneration, hereditary macular degeneration, cone-form dystrophy, rod-cone dystrophy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, and retinal trauma.

[0097] As used herein, “therapeutically effective amount” means an amount of a compound sufficient to have an effect on a treatment when administered to a subject for the treatment of a disease or condition.

[0098] In this specification, "mature" RPE cells are defined as RPE cells in which the expression of immature RPE markers such as Pax6 is downregulated and the expression of mature RPE markers such as RPE65 is upregulated.

[0099] In this specification, "maturation" of RPE cells refers to the process by which the RPE developmental pathway is regulated to produce mature RPE cells. For example, regulation of ciliary function can lead to RPE maturation.

[0100] In this specification, an "inducer" is defined as a molecule that regulates gene expression, such as the activation of genes within a cell. Inducers may bind to repressors or activators. Inducers function by neutralizing repressors.

[0101] As used herein, the term “clear bilayer of RPE-PR / PRP” refers to a co-culture having layer adhesion, maintenance of expected markers in each cell type, and layer stratification.

[0102] As used herein, the term “engrafted” bilayer refers to the engraftment of transplanted cells into the host retina, as well as the formation of presynaptic and postsynaptic mechanisms such that the transplanted cells and host cells are ready to form synapses.

[0103] As used herein, the term "biodegradable" refers to a substance that provides initial structural support to the delivered cells but degrades over time into products that are not toxic to the transplant host and do not contribute to the pathological condition of the donor site.

[0104] II. Induced pluripotent stem cells Pluripotency induction was first achieved in 2006 using mouse cells (Yamanaka et al., 2006) and in 2007 using human cells (Yu et al., 2007; Takahashi et al., 2007) by reprogramming somatic cells through the introduction of transcription factors associated with pluripotency. Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into any adult cell type.

[0105] Any somatic cell, excluding germ cells, can be used as a starting point for iPSCs. For example, the cell type may be keratinocyte, fibroblast, hematopoietic cell, mesenchymal cell, hepatocyte, or gastric cell. T cells can also be used as a source of somatic cells for reprogramming (U.S. Patent No. 8,741,648). There are no restrictions on the degree of cell differentiation or the age of the animal from which the cells are collected, and even undifferentiated progenitor cells (e.g., somatic stem cells) and finally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is itself a PR / PRP cell or RPE cell (e.g., a human PR / PRP cell or RPE cell). This PR / PRP cell or RPE cell may be an adult PR / PRP cell or RPE cell, or a fetal PR / PRP cell or RPE cell. iPSCs can be proliferated under conditions known to differentiate human ES cells into specific cell types and can express human ES cell markers (e.g., SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81).

[0106] A. HLA of the initiating cell Major histocompatibility complexes (MHCs) are the primary cause of immune rejection in allogeneic organ transplantation. There are three major class I MHC haplotypes (A, B, and C) and three major class II MHC haplotypes (DR, DP, and DQ).

[0107] MHC compatibility between donor and recipient is significantly increased when donor cells are HLA homozygous (i.e., contain identical alleles for each antigen-presenting protein). While most individuals are heterozygous for MHC class I and MHC class II genes, certain individuals are homozygous for these genes. These homozygous individuals can function as superdonors, and grafts generated from these cells can be transplanted into all individuals that are either homozygous or heterozygous for this haplotype. Furthermore, if homozygous donor cells possess a haplotype that is frequently observed in the population, these cells may have applications in transplant therapy for large numbers of individuals.

[0108] Therefore, iPSCs can be produced from somatic cells of the patient to be treated, or from somatic cells of another subject having the same or substantially the same HLA type as the patient. In some cases, the donor's major HLA (e.g., the three major loci of HLA-A, HLA-B, and HLA-DR) is the same as the recipient's major HLA. In some cases, a somatic cell donor may be a superdonor, and therefore, PR / PRP cells can be generated using iPSCs derived from an MHC homozygous superdonor. Thus, iPSCs derived from a superdonor can be transplanted into a subject that is either homozygous or heterozygous for this haplotype. For example, an iPSC may be homozygous for two HLA alleles, such as HLA-A and HLA-B. Therefore, iPSCs produced from a superdonor can be used in the manner disclosed herein to produce PR / PRP cells that are potentially "compatible" with a large number of potential recipients.

[0109] B. Reprogramming Factors Induced pluripotent stem cells (iPSCs) can be produced by reprogramming somatic cells using methods known to those skilled in the art. Those skilled in the art can easily produce induced pluripotent stem cells, see, for example, U.S. Patent Publication Nos. 20090246875, 2010 / 0210014, 20120276636, U.S. Patent Nos. 8,058,065, 8,129,187, 8,278,620, PCT International Publication No. 2007 / 069666A1, and U.S. Patent No. 8,268,620 (these are incorporated herein by reference). Generally, pluripotent stem cells are produced from somatic cells using nuclear reprogramming factors. In some embodiments, at least two, at least three, or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are used. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are used.

[0110] These cells are treated with a nuclear reprogramming agent, which is generally one or more factors capable of inducing iPSCs from somatic cells, or nucleic acids encoding these substances (e.g., in a form incorporated into a vector). Common examples of nuclear reprogramming agents include at least Oct3 / 4, Klf4, and Sox2, or nucleic acids encoding these molecules. Additional nuclear reprogramming agents that may be used include functional inhibitors of p53, L-myc or nucleic acids encoding L-myc, and Lin28 or Lin28b or nucleic acids encoding Lin28 or Lin28b. Nanog may also be used for nuclear reprogramming.As disclosed in U.S. Patent Application Publication No. 20120196360, exemplary reprogramming factors for the manufacture of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 may be replaced with Soxl, Sox3, Soxl5, Soxl7, or Soxl8, and Klf4 may be replaced with Klfl, Klf2, or Klf5); (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7;(5)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oct 3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced with Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HP VI 6 E6;(15)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7;(16)Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7;(17)Oct3 / 4, Klf4, Sox2, TERT, Bmil;(18)Oct3 / 4, Klf4, Sox2, Lin28;(19)Oct3 / 4, Klf4, Sox2, Lin28, SV40LT;(20)Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT;(21)Oct3 / 4, Klf4, Sox2, SV40LT;or(22)Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced with Esrrg).In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are used. In other embodiments, Oct4, Nanog, and Sox2 are used; see, for example, U.S. Patent No. 7,682,828, incorporated herein by reference. Examples of these factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other examples, these factors include, but are not limited to, Oct3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are used. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal 4 are used. Factors such as Nanog, Lin28, Klf4, or c-Myc can enhance reprogramming efficiency and can be expressed from several different expression vectors. For example, embedded vectors such as EBV element-based systems can be used (U.S. Patent No. 8,546,140). In a further embodiment, the reprogrammed protein may be directly introduced into somatic cells by protein transduction. Reprogramming may further involve contacting the cell with one or more signaling receptors, such as glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signaling inhibitors, leukemia suppressor (LIF), p53 inhibitors, NF-kappa B inhibitors, or combinations thereof. The regulators may be small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that substantially any iPS cells or cell lineage may be used.

[0111] The mouse and human cDNA sequences of this nuclear reprogramming material are available by reference to the NCBI accession numbers mentioned in International Publication No. 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming materials or nucleic acids encoding these reprogramming materials are known in the art and are disclosed, for example, in U.S. Patent Application Publication No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated herein by reference.

[0112] Once obtained, iPSCs can be cultured in a medium sufficient to maintain pluripotency. As described in U.S. Patent No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603, iPSCs can be used with various media and techniques developed for culturing pluripotent stem cells (more specifically, embryonic stem cells). In the case of mouse cells, culture is carried out by adding leukemia suppressor (LIF) as a differentiation inhibitor to the usual medium. In the case of human cells, it is preferable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs, as known to those skilled in the art, may be used.

[0113] In certain embodiments, undefined conditions may be used, for example, to maintain stem cells in an undifferentiated state, pluripotent cells may be cultured on fibroblast feeder cells or on a medium exposed to fibroblast feeder cells. In some embodiments, these cells are cultured in the presence of mouse embryonic fibroblasts treated with radiation or antibiotics to terminate cell division, acting as feeder cells. Alternatively, pluripotent cells may be cultured and maintained in an essentially undifferentiated state using defined feeder-independent culture systems (e.g., TESR® medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8® medium (Chen et al., 2011)).

[0114] C. plasmid In some embodiments, iPSCs may be modified to express exogenous nucleic acids, for example, to include an enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker. Suitable promoters include, but are not limited to, any promoter expressed in photoreceptor cells (e.g., rhodopsin kinase promoters). The construct may also include other elements (e.g., a ribosome binding site for translation initiation (internal ribosome binding sequence), and a transcription / translation terminator). Generally, it is advantageous to transfect cells with this construct. Suitable vectors for stable transfection include, but are not limited to, retroviral vectors, lentiviral vectors, and Sendai viruses.

[0115] In some embodiments, the plasmid encoding the marker comprises: (1) a high copy number origin; (2) a selection marker for antibiotic selection by kanamycin (e.g., a neogene, but not limited to these); (3) a transcription termination sequence (e.g., a tyrosinase enhancer); and (4) a multicloning site for the incorporation of various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to a tyrosinase promoter. Numerous plasmid vectors for inducing protein-encoding nucleic acids are known in the art. These include, but are not limited to, the vectors disclosed in U.S. Patent No. 6,103,470; No. 7,598,364; No. 7,989,425; and No. 6,416,998 (these are incorporated herein by reference).

[0116] Viral gene delivery systems may be RNA-based or DNA-based viral vectors. Episome gene delivery systems may be plasmids, Epstein-Barr virus (EBV)-based episome vectors, yeast-based vectors, adenovirus-based vectors, simian virus 40 (SV40)-based episome vectors, bovine papillomavirus (BPV)-based vectors, or lentiviral vectors.

[0117] Markers may include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), enzymes (e.g., horseradish peroxidase, alkaline phosphatase, firefly / sea mushroom luciferase, or nanoluc), or other proteins. Markers may be proteins (e.g., secreted cell surface proteins or cellular proteins; synthesized or taken up by cells); nucleic acids (e.g., mRNA, or enzymatically active nucleic acid molecules), or polysaccharides. These may include antibodies, lectins, probes, or determinants of any such cellular component detectable by nucleic acid amplification reactions specific to the marker of the cell type of interest. Markers may also be identified by biochemical assays or enzymatic assays or biological reactions that depend on the function of the gene product. Nucleic acid sequences encoding these markers may be operably ligated to tyrosinase enhancers. In addition, other genes (e.g., genes that may affect stem cells for PRP differentiation, or photoreceptor function, or for physiological or pathological effects) may be included.

[0118] D. Delivery System The introduction of nucleic acids (e.g., DNA or RNA) into pluripotent stem cells programmed into RPE or PR / PRP as described herein may be performed using any method suitable for nucleic acid delivery for cell transformation, as described herein or known to those skilled in the art. Such methods include, but are not limited to, the following: direct delivery of DNA by means of: ex vivo transfection (Wilson et al., 1989, Nabel et al., 1989), injection (U.S. Patent Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466, and 5,580,859 (each incorporated herein by reference)), for example, by microinjection (Harland and Weintraub, 1985; U.S. Patent No. 5,789,215 (incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253 (incorporated herein by reference); Tur-Kaspa et al., 1986; Potter et al., 1984); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by DEAE-dextran followed by polyethylene glycol (Gopal, 1985); by direct ultrasonic filling (Fechheimer et al., 1987); by liposome-mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al.By means of transfection (Wu and Wu, 1987; Wu and Wu, 1988); by means of transfection (International Publication Nos. 94 / 09699 and 95 / 06128; U.S. Patent Nos. 5,610,042; U.S. Patent Nos. 5,322,783, 5,563,055, 5,550,318, 5,538,877, and 5,538,880 (each incorporated herein by reference)); by means of transfection by means of transfection (Kaeppler et al., 1991); by means of transfection by means of transfection (Kaeppler et al., 1991); by means of transfection (Kaeppler et al., 1998; Wu and Wu, 1987; Wu and Wu, 1988); by means of transfection by means of transfection (Kaeppler et al., 1991);Wu and Wu, 1987; Wu and Wu, 1988); by means of transfection (Kaeppler et al., 1991); by means of transfection (Wu and Wu, 1991; Wu and Wu, 1991; Wu and Wu, 1991); by means of transfection (Kaeppler et al., 1991); by means of transfection (Kaeppler et al., 1991); by means of transfection (Kaeppler et al., 1991; Wu and Wu, 1991; Wu and Wu, 1991; Wu and W al., 1990; U.S. Patent Nos. 5,302,523 and 5,464,765 (each incorporated herein by reference); by Agrobacterium-mediated transfection (U.S. Patent Nos. 5,591,616 and 5,563,055 (each incorporated herein by reference); by desiccation / inhibition-mediated DNA incorporation (Potrykus et al., 1985), and any combination of such methods. The application of such techniques may enable the stable or excessive transformation of organelles, cells, tissues, or organisms.

[0119] 1. Viral vector In certain embodiments of this disclosure, viral vectors may be provided. The production of recombinant viral vectors involves replacing non-essential genes with genes or coding sequences of heterologous (or non-natural) proteins. Viral vectors are a type of expression construct that utilizes viral sequences to introduce nucleic acids and, optionally, proteins, into cells. The ability of certain viruses to infect or enter cells via receptor-mediated endocytosis, and their ability to integrate into the host cell genome to stably and efficiently express viral genes, makes them attractive candidates for introducing exogenous nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of viral vectors that may be used to deliver nucleic acids in certain embodiments of this disclosure are described below.

[0120] Retroviruses are considered promising as gene delivery vectors due to their ability to integrate their genes into the host genome, introduce large amounts of foreign genetic material, infect a wide range of species and cell types, and package in specific cell lineages (Miller, 1992).

[0121] To construct retroviral vectors, nucleic acids are inserted into the viral genome in place of a specific viral sequence to produce non-replicating viruses. To produce virians, a packaging cell line is constructed containing the gag, pol, and env genes but lacking the LTR and packaging components (Mann et al., 1983). When a recombinant plasmid containing cDNA is introduced into a specific cell line (e.g., by calcium phosphate precipitation) along with the retroviral LTR and packaging sequence, the packaging sequence can package the RNA transcript of the recombinant plasmid into viral particles, which are then secreted into the culture medium (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The medium containing this recombinant retrovirus is then collected, selectively concentrated, and used for gene transfer. Retroviral vectors can infect a wide range of cell types. However, host cell division is required for integration and stable expression (Paskind et al., 1975).

[0122] Lentiviruses are complex retroviruses that, in addition to the common retroviral genes gag, pol, and env, contain other genes with regulatory or structural functions. Lentiviral vectors are known in the art (see, for example, Naldini et al., 1996; Zufferey et al., 1997; Blomer et al., 1997; U.S. Patent Nos. 6,013,516 and 5,994,136).

[0123] Recombinant lentiviral vectors have the ability to infect non-dividing cells and can be used for gene transfer and nucleic acid sequence expression both in vivo and ex vivo. For example, recombinant lentiviruses with the ability to infect non-dividing cells (suitable host cells are transfected with two or more vectors carrying packaging functions (i.e., gag, pol, and env, as well as rev and tat)) are described in U.S. Patent No. 5,994,136, incorporated herein by reference.

[0124] 2. Episome vectors In certain embodiments of this disclosure, the use of plasmid-based or liposome-based extrachromosomal (i.e., episomal) vectors may also be provided. Examples of such episomal vectors include oriP-based vectors and / or vectors encoding derivatives of EBNA-1. These vectors enable large fragments of DNA to be introduced into cells, maintained extrachromosomally, replicated once per cell cycle, efficiently distributed to daughter cells, and substantially non-inducing immune responses.

[0125] In particular, EBNA-1, the only viral protein required for replication of oriP-based expression vectors, does not induce a cellular immune response because EBNA-1 generates an efficient mechanism to bypass the processing required for the presentation of its antigen to MHC class I molecules (Levitskaya et al., 1997). Furthermore, EBNA-1 can act in trans to enhance the expression of cloned genes, and in some cell lines, the expression of cloned genes can be induced up to 100-fold (Langle-Rouault et al., 1998; Evans et al., 1997). Finally, the production of such oriP-based expression vectors is inexpensive.

[0126] In certain embodiments, the reprogramming factor is expressed from an expression cassette contained within one or more exogenous episomal gene elements (see U.S. Patent Application Publication 2010 / 0003757, incorporated herein by reference). Therefore, the iPSC may not inherently contain exogenous gene elements (e.g., derived from retroviral or lentiviral vector elements). This iPSC is prepared using an extrachromosomal replication vector (i.e., an episomal vector), which is capable of episomal replication to produce an iPSC that is essentially free of exogenous vector or viral elements (see U.S. Patent No. 8,546,140, ​​incorporated herein by reference; see Yu et al., 2009). Many DNA viruses (e.g., adenovirus, monkey vacuolar virus 40 (SV40), or bovine papillomavirus (BPV)) or budding yeast ARS (autonomous replication sequence)-containing plasmids replicate extrachromosomally or episomatically in mammalian cells. This episomal plasmid essentially does not contain all of these drawbacks associated with vector integration (Bode et al., 2001). For example, lymphotropic herpes virus (heRPE virus) based inclusion, as defined above, or Epstein-Barr virus (EBV) can replicate extrachromosomally and facilitate the delivery of reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. A further advantage of episomal vectors is that exogenous elements are lost over time after introduction into cells, resulting in self-persistent iPSCs that essentially do not contain these elements.

[0127] Other extrachromosomal vectors include other lymphotropic herpesvirus-based vectors. Lymphotropic herpesviruses are herpesviruses that replicate in lymphoblasts (e.g., human B lymphoblasts) and become plasmids as part of their natural life cycle. Herpes simplex virus (HSV) is not a "lymphotropic" herpesvirus. Exemplary lymphotropic herpesviruses include, but are not limited to, EBV, Kaposi's sarcoma herpesvirus (KSHV); herpesvirus thymili (HS); and Marek's disease virus (MDV). Similarly, other sources of episome-based vectors (e.g., yeast ARS, adenovirus, SV40, or BPV) are considered.

[0128] Those skilled in the art will have sufficient ability to construct vectors using standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both incorporated herein by reference).

[0129] The vector may also include other components or functionalities that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to target cells. Such other components include, for example: components that affect cell binding or targeting (e.g., components that mediate cell type or tissue-specific binding); components that affect the uptake of vector nucleic acids by cells; components that affect the localization of polynucleotides within cells after uptake (e.g., factors that mediate nuclear localization); and components that affect polynucleotide expression.

[0130] Such components may include markers, for example, detectable markers and / or selection markers that can be used to detect or select cells that have taken up and expressed nucleic acids delivered by a vector. Such components may be provided as innate features of the vector (e.g., by using a particular viral vector with a component or functionality that mediates binding and uptake), or the vector may be modified to provide such functionality. A wide variety of such vectors are known and generally available in the art. If the vector is maintained in a host cell, it may be stably replicated by the cell during mitosis as an autonomous structure, incorporated into the host cell's genome, or maintained in the nucleus or cytoplasm of the host cell.

[0131] 3. Adjustment element The expression cassettes contained in the reprogramming vectors useful in this disclosure preferably include (in a 5' to 3' manner) a eukaryotic transcription promoter operably linked to a protein coding sequence, a splice signal such as an intervening sequence, and a transcription termination / polyadenylation sequence.

[0132] b. Promoter / Enhancer The expression constructs provided herein include a promoter to drive the expression of a programming gene. A promoter generally contains a sequence that functions to position the start site of RNA synthesis. The most well-known example of this is the TATA box, but in some promoters lacking a TATA box (e.g., the promoter for the mammalian terminal deoxynucleotidyltransferase gene and the SV40 late gene), a separate element overlapping the start site itself helps to fix the start site. Further promoter elements regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the start site, but many promoters have been shown to also contain functional elements downstream of the start site. To place the coding sequence "under the control" of the promoter, the 5' end of the transcription start site of the transcription read frame is positioned "downstream" (i.e., 3') of the selected promoter. The "upstream" promoter stimulates the transcription of the DNA and promotes the expression of the encoded RNA.

[0133] The spacing between promoter elements is often flexible, so that promoter function is maintained even when elements are inverted or moved relative to each other. In the tk promoter, the spacing between promoter elements can be widened to 50 bp before activity begins to decline. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription. Promoters may or may not be used with "enhancers," which refer to cis-acting regulatory elements involved in the transcriptional activation of nucleic acid sequences.

[0134] A promoter may be naturally associated with a nucleic acid sequence, such as by isolating a 5' non-coding sequence located upstream of the coding segment and / or exon. Such a promoter may be referred to as “endogenous.” Similarly, an enhancer may be naturally associated with a nucleic acid sequence and located upstream or downstream of it. Alternatively, certain advantages may be obtained by placing the coding nucleic acid segment under the control of a recombinant or heterologous promoter, and this promoter may refer to a promoter that is not normally associated with the nucleic acid sequence in its natural environment. Recombinant or heterologous enhancers also refer to enhancers that are not normally associated with the nucleic acid sequence in its natural environment. Examples of such promoters or enhancers include promoters or enhancers of other genes, and promoters or enhancers isolated from any other virus, or from prokaryotic or eukaryotic cells, and promoters or enhancers that are “not naturally occurring” (i.e., containing different elements of different transcriptional regulatory regions), and / or mutations that alter expression. For example, the most commonly used promoters in recombinant DNA construction include β-lactamase (penicillinase), lactose, and tryptophan (trp) promoter systems. In addition to the synthetic production of promoter and enhancer nucleic acid sequences, sequences may be synthesized using recombinant cloning and / or nucleic acid amplification techniques (e.g., PCR®) in relation to the compositions disclosed herein (see U.S. Patent Nos. 4,683,202 and 5,928,906, respectively, incorporated herein by reference). Furthermore, regulatory sequences that direct the transcription and / or expression of sequences within non-nuclear organelles (e.g., mitochondria, chloroplasts, and similar) may also be utilized.

[0135] Naturally, it will be important to utilize promoters and / or enhancers that effectively direct the expression of a DNA segment in the organelle, cell type, tissue, organ, or organism selected for expression. Those skilled in molecular biology are generally familiar with the use of promoter, enhancer, and cell type combinations for protein expression (see, for example, Sambrook et al. 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inductive, and / or useful under conditions appropriate for directing high levels of expression of the introduced DNA segment (e.g., advantageous in the large-scale production of recombinant proteins and / or peptides). Promoters may be heterogeneous or endogenous.

[0136] In addition, any promoter / enhancer combination (e.g., according to the Eukaryotic Promoter Database EPDB) may also be used to drive expression. The use of T3, T7, or SP6 cytoplasmic expression systems is another possible embodiment. Eukaryotic cells may support cytoplasmic transcription from certain bacterial promoters when the appropriate bacterial polymerase is provided as part of a delivery complex or as an additional gene expression construct.

[0137] Non-limiting examples of promoters include: early or late viral promoters, e.g., early or late SV40 promoter, very early cytomegalovirus (CMV) promoter, early Roussarcoma virus (RSV) promoter; eukaryotic cell promoters, e.g., beta-actin promoter (Ng, 1989; Quitsche et al., 1989), GADPH promoter (Alexander et al., 1988, Ercolani et al., 1988), metallothionein promoter (Karin et al., 1989; Richards et al., 1984); and ligation element promoters, e.g., cyclic AMP reaction element promoter (cre), serum reaction element promoter (sre), phorbol ester promoter (TPA), and reaction element promoter near the minimum TATA box (tre). It is also possible to use a human growth hormone promoter sequence (e.g., the human growth hormone minimal promoter described in Genbank, accession number X05244, nucleotides 283-341) or a mouse mammary tumor promoter (ATCC, available in catalog number ATCC 45007).

[0138] Tissue-specific transgene expression (particularly the expression of reporter genes in hematopoietic cells and hematopoietic cell precursors derived from programming) may be desirable as a method for identifying the hematopoietic cells and precursors from which the transgenes originate. The use of cis-acting regulatory elements is intended to enhance both specificity and activity. For example, hematopoietic cell-specific promoters may be used. Many such hematopoietic cell-specific promoters are known in the art.

[0139] In certain embodiments, the methods of the present disclosure also relate to enhancer sequences (i.e., nucleic acid sequences that increase promoter activity and have the potential to act in cis and regardless of orientation, even at relatively long distances (up to several kilobases away from the target promoter)). However, the function of enhancers is not necessarily limited to such long distances, as they can also function in close proximity to a given promoter.

[0140] The promoter and enhancer sequences of many hematopoietic cells have been identified and may be useful in this method. See, for example, U.S. Patent No. 5,556,954; U.S. Patent Application Publication No. 20020055144; and U.S. Patent Application Publication No. 20090148425.

[0141] c. Initiation signal and linked expression For efficient translation of coding sequences, the expression constructs provided in this disclosure may also utilize specific start signals. These signals may include ATG start codons or adjacent sequences. It may be necessary to provide exogenous translational control signals, such as ATG start codons. Those skilled in the art will readily be able to determine this and provide the necessary signals. It is known that, for reliable translation of the entire insertion, the start codon must be "in-frame" with the reading frame of the desired coding sequence. Exogenous translational control signals and start codons may be either natural or synthetic. The inclusion of appropriate transcriptional enhancer elements may increase expression efficiency.

[0142] In certain embodiments, internal ribosome entry site (IRES) elements are used to construct multiple gene (i.e., polycistronic) messages. IRES elements can bypass the ribosome scanning model of 5' methylation-dependent translation and initiate translation at an internal site (Pelletier and Sonenberg, 1988). IRES elements derived from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), and IRES derived from mammalian messages have also been described (Macejak and Sarnow, 1991). IRES elements can be ligated to heterologous open reading frames. Multiple open reading frames, each separated by the IRES, can be transcribed together to construct a polycistronic message. The IRES element makes each open reading frame available to the ribosome for efficient translation. Multiple genes can be efficiently expressed using a single promoter / enhancer to transcribe a single message (see U.S. Patent Nos. 5,925,565 and 5,935,819, respectively, incorporated herein by reference).

[0143] In addition, certain 2A sequence elements may be used to result in linked or co-expression of programming genes in the constructs provided in this disclosure. For example, genes may be co-expressed using cleavage sequences by linking open reading frames to form a single cistron. Exemplary cleavage sequences are F2A (foot-and-mouth disease virus 2A) or “2A-like” sequences (e.g., Thosea asigna virus 2A; T2A) (Minskaia and Ryan, 2013). In certain embodiments, F2A cleavage peptides are used to link gene expression in multilineage constructs.

[0144] d. Origin of replication To propagate the vector within a host cell, the host cell may contain one or more origin sites (often referred to as "ori"), which may include, for example, a nucleic acid sequence corresponding to the oriP of EBV described above, or a nucleic acid sequence corresponding to a genetically modified oriP having similar or improved programming function, which is a specific nucleic acid sequence from which replication is initiated. Alternatively, an origin of replication of another extrachromosomal replicating virus or autonomous replicating sequence (ARS) described above may be used.

[0145] e. Selection markers and screening markers In certain embodiments, cells containing nucleic acid constructs can be identified in vitro or in vivo by incorporating a marker into the expression vector. Such a marker would confer an identifiable change to the cell, enabling the easy identification of the cell containing the expression vector. Generally, a selection marker confers a property that enables selection. A positive selection marker is one whose presence enables selection, while a negative selection marker is one whose presence prevents selection. An example of a positive selection marker is a drug resistance marker.

[0146] Typically, the inclusion of drug selection markers assists in the cloning and identification of transformants. For example, genes conferring resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeosin, and histidinol are useful selection markers. In addition to markers that confer phenotypes enabling the identification of transformants based on performance of conditions, other types of markers, such as screenable markers like GFP based on colorimetric analysis, are also considered. Alternatively, screenable markers as negative selection markers (e.g., heRPE simplex viral thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT)) may be used. Those skilled in the art will also be familiar with methods of using immunological markers in combination with FACS analysis, in some cases. The marker used is considered irrelevant as long as it can be expressed simultaneously with the nucleic acid encoding the gene product. Further examples of selection markers and screenable markers are known to those skilled in the art.

[0147] III. Differentiation of iPSCs into retinal pigment epithelial cells or photoreceptor / photoreceptor progenitor cells A.RPE differentiation In some embodiments, RPE cells are produced from iPSCs by the methods disclosed herein, for example, by the methods disclosed in PCT / US2016 / 050543 and PCT / US2016 / 050554. Cells in the retina that directly sense light are photoreceptor cells. Photoreceptors are photosensitive nerve cells in the outer part of the retina and can be either rods or cones. In the process of light transmission, photoreceptor cells convert the incident light energy focused by the cornea and lens into electrical signals, which are ultimately sent to the brain by the optic nerve. Vertebrates have two types of photoreceptor cells, including cones and rods. Cones are adapted to detect fine detail, central vision, and color vision and function well in bright light. Rods are involved in peripheral vision and dim vision. Nerve signals from rods and cones are processed by other nerve cells in the retina.

[0148] The retinal pigment epithelium functions as part of the barrier between the bloodstream and the retina, and interacts closely with photoreceptors in maintaining visual function and choroidal blood supply. The retinal pigment epithelium consists of a single layer of hexagonal cells densely packed with melanin granules. The main functions of these specialized RPE cells include: transport of nutrients (e.g., glucose, retinol, and fatty acids) from the blood to photoreceptors; transport of water, metabolic end products, and ions from the subretinal space to the blood; absorption of light and protection from photo-oxidation; re-isomerization of all trans-retinol to 11-cis-retinol; phagocytosis of detached photoreceptor membranes; and secretion of various factors essential for the structural integrity of the retina.

[0149] Mature retinal pigment epithelium expresses markers such as cellular retinal dehydrate-binding protein (CRALBP), RPE65, the best vitiligo macular dystrophy gene (VMD2), and pigment epithelial-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with many visual alteration conditions, such as retinal pigment epithelial detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophy, or age-related macular degeneration.

[0150] Mature retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually recognized by their cobblestone-like morphology and the initial appearance of their pigmentation. In addition, the differentiated RPE cell layer possesses transepithelial resistance / TER throughout the entire monolayer and generates transepithelial potential / TEP (TER > 100 ohms / cm²; TEP > 2 mV), transporting fluid, lactate, and CO2 from the apical to the basal side, and regulating the polarized secretion of cytokines.

[0151] RPE cells express several proteins that can function as markers for detecting identity and maturity status using methodologies such as immunocytochemistry, Western blotting, flow cytometry, and enzyme-linked immunoassay (ELISA). For example, RPE-specific markers may include: cellular retinal dehydride-binding protein (CRALBP), microphthalmia-associated transcription factor (MITF), tyrosinase-associated protein 1 (TYRP-1), retinal pigment epithelium-specific 65kDa protein (RPE65), premelanosome protein (PMEL17), bethroffin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). At the same time, RPE cells do not express the embryonic stem cell markers Oct-4, nanog, or Rex-1 (at any detectable level). Specifically, the expression of these genes is approximately 100 to 1000 times lower in RPE cells compared to ES cells or iPSC cells, as assessed by quantitative RT-PCR.

[0152] For example, RPE cell markers can be detected at the mRNA level by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis using sequence-specific primers with standard amplification methods using publicly available sequence data (GENBANK®). Expression of tissue-specific markers detected at the protein or mRNA level is considered positive if the level is at least or about 2, 3, 4, 5, 6, 7, 8, or 9 times higher than that of control cells such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0153] Dysfunction, injury, and loss of RPE cells are the cause of many eye diseases and disorders, such as age-related macular degeneration (AMD), hereditary macular degenerations such as Best's disease, Stargardt disease, and total choroidal atrophy, as well as other forms of hereditary retinal diseases, and acquired retinal dysfunction, diseases, and injuries, such as, but not limited to, RPE tearing / laceration. A potential treatment for such diseases is the transplantation of RPE cells into the subretinal space of those who require such treatment. It is hypothesized that this transplantation of RPE cells may delay, stop, or reverse degradation, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining RPE cells directly from human donors and embryos is difficult.

[0154] 2. Induction of RPE cells from PSC embryoid bodies iPSCs reprogrammed using known reprogramming factors can give rise to ophthalmic cells of the nervous system, such as RPE cells (Hirami et al., 2009). PCT Publication No. 2014 / 121077, which is incorporated herein by whole reference, discloses a method for inducing the expression of retinal progenitor cell markers by treating embryoid bodies (EBs) produced from iPSCs with a Wnt antagonist and a Nodal antagonist in a suspension culture. This publication discloses a method by which RPE cells are induced from iPSCs through the process of differentiation of iPSC EBs into a highly enriched culture. For example, embryoid bodies are produced from iPSCs by adding a rho-related coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor. Furthermore, these EB cells are seeded on MATRIGEL®-coated tissue cultures in a second medium that does not contain basic fibroblast growth factor (bFGF) but contains a Nodal pathway inhibitor, approximately 20 ng to 90 ng of Noggin, and approximately 1 to 5% of a knockout serum substitute to form differentiated RPE cells. The differentiated RPE cells are cultured in a third medium containing ACTIVIN and WNT3a. Subsequently, the RPE cells are cultured in RPE medium containing approximately 5% fetal serum, a reference WNT inhibitor, a non-reference WNT inhibitor, and inhibitors of the Sonic Hedgehog pathway and the FGF pathway to produce human RPE cells.

[0155] The use of EBs for the production of differentiated cell types has several drawbacks. For example, the production of EBs from iPSCs is an inconsistent and inreproducible process due to variations in efficiency and changes in the size or shape of the EBs. This disclosure provides a method that enables the large-scale production of iPSC or ES-derived cells required for clinical, research, or therapeutic applications, without relying on EBs.

[0156] 3. Induction of RPE cells from essentially single-cell PSCs In some embodiments, a method is provided for producing RPE cells from a suspension of essentially single-cell pluripotent stem cells (PSCs), such as human iPSCs. In some embodiments, the PSCs are cultured to preconfluence to prevent any cell aggregation. In certain embodiments, the PSCs are dissociated by incubation with a cell dissociation enzyme, such as TRYPSIN or TRYPLE®. The PSCs may also be dissociated into a suspension of essentially single cells by pipetting. In addition, brevistatin (e.g., about 2.5 μM) may be added to the culture medium after dissociation into single cells to increase the viability of the PSCs without causing the cells to adhere to the culture vessel. Alternatively, a ROCK inhibitor may be used instead of brevistatin to increase the viability of the PSCs after dissociation into single cells.

[0157] Once a single-cell suspension of PSCs is obtained, the cells are generally seeded into a suitable culture vessel, such as a tissue culture plate (e.g., a flask, multilayer flask, 6-well plate, 12-well plate, 24-well plate, 96-well plate, or 10 cm plate). The following are possible but not limited to culture vessels used to culture these cells: flasks, tissue culture flasks, petri dishes, petri dishes, tissue culture dishes, multi-petri dishes, microplates, microwell plates, multiplates, multiwell plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as stem cells can be cultured inside. These cells may be cultured in the following volumes, depending on the need for culture: at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range that can be derived from these. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment capable of growing cells. The bioreactor may have a volume of at least 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range that can be derived from these.

[0158] In certain configurations, PSCs such as iPSCs are seeded at a cell density suitable for efficient differentiation. Generally, these cells are seeded at a density of approximately 1,000 to 75,000 cells / cm³. 2 (For example, approximately 5,000 to 40,000 cells / cm²) 2Seed at a cell density of approximately 50,000 to 400,000 cells per well in a 6-well plate. In an exemplary method, these cells are seeded at a cell density of approximately 100,000, 150,000, 200,000, 250,000, 300,000, or 350,000 cells per well (for example, approximately 200,000 cells per well).

[0159] PSCs, such as iPSCs, are generally cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins (e.g., vitronectin, laminin, collagen, and / or fibronectin), and these extracellular matrix proteins can be used to coat the culture surface as a means of providing a solid support for the proliferation of pluripotent cells. The term "extracellular matrix" is recognized in the art. Components of the ECM include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosine, ancholine, chondronectin, binding proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. In an exemplary method, PSCs are grown on a culture plate coated with vitronectin or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0160] Extracellular matrix (ECM) proteins may be of natural origin and purified from human or animal tissue, or they may be genetically engineered recombinant proteins or naturally synthesized. ECM proteins may be whole proteins or in the form of natural or engineered peptide fragments. Examples of ECM proteins that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition includes peptide fragments produced by the synthesis of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is heterogeneous. For example, a heterogeneous-free matrix for culturing human cells may use matrix components of human origin and exclude any non-human animal components.

[0161] In some embodiments, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0162] Cells such as RPE cells or PSCs can be cultured with nutrients necessary to support the growth of each specific population of cells. Generally, cells are cultured in a growth medium containing buffers to maintain pH. This medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, and inorganic salts. Exemplary growth media include minimal essential media (e.g., Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8® (E8®) medium) supplemented with various nutrients (e.g., non-essential amino acids and vitamins) to enhance stem cell growth. Examples of minimal essential media include, but are not limited to, Minimum Essential Medium Eagle (MEM), AlphaMEM, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. In addition, minimal essential media may be supplemented with additives (e.g., horse, calf, or fetal bovine serum). Alternatively, the medium may be serum-free. In other cases, the growth medium may include serum-free formulations, referred herein as “Knockout Serum Substitutes,” which are optimized for growing and maintaining undifferentiated cells (e.g., stem cells) during culture. KNOCKOUT® serum substitutes are disclosed, for example, in U.S. Patent Application Publication No. 2002 / 0076747, incorporated herein by reference. Preferably, PSCs are cultured in a fully defined and feeder-free medium.

[0163] Therefore, single-cell PSCs are generally cultured in a fully defined culture medium after seeding. In certain embodiments, the medium is aspirated and fresh medium (e.g., E8® medium) is added to the culture approximately 18–24 hours after seeding. In certain embodiments, single-cell PSCs are cultured in a fully defined culture medium for approximately 1, 2, or 3 days after seeding. Preferably, single-cell PSCs are cultured in a fully defined culture medium for approximately 2 days before the differentiation process can proceed.

[0164] In some embodiments, the culture medium may or may not contain any serum substitute. Examples of serum substitutes include albumin (e.g., lipid-rich albumin, albumin substitutes (e.g., recombinant albumin), vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 1'-thioglycerol, or substances appropriately containing these equivalents. Serum substitutes may be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679. Alternatively, any commercially available material may be used more conveniently. Examples of commercially available materials include KNOCKOUT® serum substitute (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX® (Gibco).

[0165] Other culture conditions can be appropriately defined. For example, the culture temperature may be about 30–40°C, and may be, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not particularly limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration may be about 1–10% (e.g., about 2–5%), or any range that can be derived within this range. The oxygen partial pressure may be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range that can be derived within this range.

[0166] 4. Differentiation medium Retinal induction medium After single-cell PSCs have attached to a culture plate, these cells are preferably cultured in retinal induction medium to initiate the differentiation process into retinal lineage cells. This retinal induction medium (RIM) contains a WNT pathway inhibitor and can induce differentiation of PSCs into retinal lineage cells. RIM further contains a TGFβ pathway inhibitor and a BMP pathway inhibitor.

[0167] RIM may contain DMEM and F12 in a ratio of approximately 1:1. In an exemplary manner, RIM may contain a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), and a TGFβ pathway inhibitor (e.g., SB431542). For example, RIM may contain approximately 5 nM to approximately 50 nM (e.g., approximately 10 nM) of LDN193189, approximately 0.1 μM to approximately 5 μM (e.g., approximately 0.5 μM) of CKI-7, and approximately 0.5 μM to approximately 10 μM (e.g., approximately 1 μM) of SB431542. In addition, RIM may contain knockout serum substitute (e.g., approximately 1% to approximately 5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). Preferably, IGF1 is animal-free IGF1 (AF-IGF1) and is included in the RIM at a concentration of approximately 0.1 ng / mL to approximately 10 ng / mL (e.g., approximately 1 ng / mL). This medium is aspirated daily and replaced with fresh RIM. Cells are cultured in the RIM for approximately 1 to 5 days (e.g., approximately 1, 2, 3, 4, or 5 days, e.g., approximately 2 days) to produce retinal lineage cells.

[0168] Retinal differentiation medium These retinal cell lineages can then be cultured in retinal differentiation medium (RDM) for further differentiation. RDM contains a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In one embodiment, RDM contains a WNT pathway inhibitor (e.g., CKI-7), a BMP pathway inhibitor (e.g., LDN193189), a TGFβ pathway inhibitor (e.g., SB431542), and a MEK inhibitor (e.g., PD0325901). Alternatively, RDM may contain a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor. Generally, the concentrations of Wnt pathway inhibitors, BMP pathway inhibitors, and TGFβ pathway inhibitors are higher in RDM compared to RIM, for example, about 9 to 11 times higher, or for example, about 10 times higher. In an exemplary method, the RDM includes LDN193189 in approximately 50 nM to approximately 200 nM (e.g., approximately 100 nM), CKI-7 in approximately 1 μM to approximately 10 μM (e.g., approximately 5 μM), SB431542 in approximately 1 μM to approximately 50 μM (e.g., approximately 10 μM), and PD0325901 in approximately 0.1 μM to approximately 10 μM (e.g., approximately 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM).

[0169] Generally, RDM contains DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute (approximately 1% to 5%, e.g., approximately 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., approximately 1 ng / mL to 50 ng / mL, e.g., approximately 10 ng / mL). In certain methods, cells are given fresh RDM daily after aspirating the culture medium from the previous day. Generally, cells are cultured in RDM for approximately 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days (e.g., approximately 7 days) to induce differentiated retinal cells.

[0170] Retinal culture medium Next, these differentiated retinal cells can be further differentiated by culturing them in retinal medium (RM). Retinal medium contains activin A and may further contain nicotinamide. RM may contain about 50 to about 200 ng / mL (e.g., about 100 ng / mL) of activin A and about 1 mM to about 50 mM (e.g., about 10 mM) of nicotinamide. Alternatively, RM may contain other TGF-β pathway activators (e.g., GDF1) and / or WNT pathway activators (e.g., CHIR99021, WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindilbine-3'-oxime), or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine). Alternatively, RM may further contain WNT3a.

[0171] The RM may contain DMEM and F12 in a ratio of approximately 1:1, approximately 1% to approximately 5% (e.g., approximately 1.5%) of knockout serum substitute, MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. This medium may be changed daily with RM at room temperature. Cells are generally cultured in the RM for approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, and 17 days (e.g., approximately 10 days) to induce differentiated RPE cells.

[0172] RPE maturation medium For further differentiation of RPE cells, these cells are preferably cultured in RPE maturation medium (RPE-MM). An example of RPE-MM medium is shown in Table 3. RPE-Maturation medium may contain approximately 100 μg / mL to approximately 300 μg / mL (e.g., approximately 250 μg / mL) of taurine, approximately 10 μg / L to approximately 30 μg / L (e.g., approximately 20 μg / L) of hydrocortisone, and approximately 0.001 μg / L to approximately 0.1 μg / L (e.g., approximately 0.013 μg / L) of triiodothyronine. In addition, RPE-MM may contain MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), and sodium pyruvate, and fetal bovine serum (or KnockOut® serum substitute) (e.g., approximately 0.5% to approximately 10%, e.g., approximately 1% to approximately 5%). This medium may be replaced every other day with RPE-MM at room temperature. These cells are generally cultured in RPE-MM for about 5 to 10 days (e.g., about 5 days). These cells can then be dissociated, for example, with a cell dissociation enzyme, re-seeded, and cultured for a further period (e.g., another 5 to 30 days, e.g., about 15 to 20 days) to further differentiate into RPE cells. In a further embodiment, RPE-MM does not contain a WNT pathway inhibitor. The RPE cells can be cryopreserved at this stage.

[0173] b. Maturation of RPE cells Next, RPE cells can be continuously cultured and matured in RPE-MM. In some embodiments, RPE cells can be grown in wells (e.g., flasks, multilayer flasks, 6-well, 12-well, 24-well, or 10 cm plates). RPE cells can be maintained in RPE medium for about 4 to about 10 weeks (e.g., about 6 to 8 weeks, e.g., 6, 7, or 8 weeks). In an exemplary method for the continuous maturation of RPE cells, these cells can be dissociated with a cell dissociation enzyme such as TRYPLE® and re-seed in RPE-MM on a degradable scaffold assembly such as a special SNAPWELL® design for about 1 to 10 weeks (e.g., 5 weeks). RPE-MM may contain a bFGF inhibitor or a MEK inhibitor. Methods for culturing RPE cells on a degradable scaffold are taught and described in International Publication No. 2014 / 121077, which is incorporated herein by reference in its entirety. In short, the main components of this method are CORNING®, COSTAR®, and SNAPWELL® plates, bioinert O-rings, and a biodegradable scaffold. The SNAPWELL® plate (e.g., with a pore size of 0.4 μm, or smaller or larger pore sizes (e.g., but not limited to 0.3 μm or 0.5 μm)) provides the structure and platform for the biodegradable scaffold. The microporous membranes forming the apical and basal sides not only provide support for the scaffold but are also ideal for isolating different sides of the polarized layers of the cells. The ability of the SNAPWELL® insert to separate the membranes allows the support ring of this insert to be used as an anchor for the scaffold. The resulting differentiated, polarized, and confluent monolayer functional RPE cells can be cryopreserved at this stage (e.g., in heterogeneous component-free CS10 medium).

[0174] In some embodiments, mature RPE cells can be further developed into a monolayer of functional RPE cells that function as intact RPE tissue by continuous culture in RPE-MM with additional chemicals or small molecules that promote RPE maturation. For example, these small molecules are primary ciliary inducers such as prostaglandin E2 (PGE2) or affidicorin. PGE2 may be added to the culture medium at a concentration of about 25 μM to about 250 μM (e.g., about 50 μM to about 100 μM). Alternatively, RPE-MM may contain a canonical WNT pathway inhibitor. Exemplary canonical WNT pathway inhibitors include N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP2) or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindole-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1). Cells can be cultured in this medium for a further period (e.g., about 1 to 5 weeks, e.g., about 2 to 4 weeks) to obtain a monolayer of mature and functional RPE cells. Thus, the method of this disclosure provides mature RPE cells from a single-cell suspension of pluripotent cells that can be replicated on a large scale and consistently for clinical use.

[0175] B. Photoreceptor cells In some embodiments, photoreceptors and / or photoreceptor precursor cells are produced by the methods disclosed herein. Cells in the retina that directly sense light are photoreceptor cells. Photoreceptors are photosensitive nerve cells in the outer part of the retina and can be either rods or cones. In the process of light transmission, photoreceptor cells convert the incident light energy focused by the cornea and lens into electrical signals, which are ultimately sent to the brain by the optic nerve. Vertebrates have two types of photoreceptor cells, including cones and rods. Cones are adapted to detect fine detail, central vision, and color vision and function well in bright light. Rods are involved in peripheral vision and dim vision. Nerve signals from rods and cones are processed by other nerve cells in the retina.

[0176] Photoreceptors can express markers such as OTX2, CRX, PRDM1 (BLIMP1), Neurod1, RCVRN, TUBB3, and L1CAM (CD171). Photoreceptors express several proteins that can function as markers for detection using methodologies such as immunocytochemistry, Western blotting, flow cytometry, and enzyme-linked immunoassays (ELISA). For example, one characteristic photoreceptor marker is RCVRN. Photoreceptors cannot express embryonic stem cell markers OCT-4, NANOG, or REX-1 (at any detectable level). Specifically, the expression of these genes is approximately 100 to 1000 times lower in photoreceptors compared to ES cells or iPSC cells, as assessed by quantitative RT-PCR.

[0177] For example, photoreceptor markers can be detected at the mRNA level by RNA sequencing, such as reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, microarrays, or single-cell RNA sequencing dot blot hybridization analysis using sequence-specific primers with standard amplification methods using publicly available sequence data (GENBANK®). Expression of tissue-specific markers detected at the protein or mRNA level is considered positive if the level is at least or about 2, 3, 4, 5, 6, 7, 8, or 9 times higher than that of control cells such as undifferentiated pluripotent stem cells or other unrelated cell types.

[0178] Dysfunction, damage, and loss of photoreceptor cells are the cause of many eye diseases and disorders, such as age-related macular degeneration (AMD), hereditary macular degenerations such as Best's disease, Stargardt disease, and total choroidal atrophy, retinitis pigmentosa, and other forms of hereditary retinal diseases, as well as acquired retinal dysfunction, disease, and injury. A potential treatment for such diseases is the transplantation of PRP and / or PR into the retina of those who require such treatment. It is hypothesized that supplementation of PRP and / or PR by this transplantation may delay, stop, or reverse degradation, improve retinal function, and prevent blindness resulting from such conditions. However, obtaining PRP and / or PR directly from human donors and embryos is difficult.

[0179] In some embodiments, a method is provided for producing photoreceptors from essentially single-cell suspensions of PSCs, such as human iPSCs. In some embodiments, the PSCs are cultured to preconfluence. In certain embodiments, the PSCs are dissociated by incubation with a cell dissociation solution or enzyme such as Versene, Trypsin, ACCUTASE®, or TRYPLE®. The PSCs may also be dissociated into essentially single-cell suspensions by pipetting.

[0180] In addition, to enhance the viability of PSCs after dissociation into single cells without causing the cells to adhere to the culture vessel, brevistatin (e.g., approximately 2.5 μM) may be added to the culture medium. Alternatively, a ROCK inhibitor may be used instead of brevistatin to enhance the viability of PSCs after dissociation into single cells.

[0181] Once a single-cell suspension of PSCs is obtained, the cells are generally seeded into a suitable culture vessel, such as a tissue culture plate (e.g., a flask, multilayer flask, 6-well, 12-well, 24-well, 96-well, or 10 cm plate). Culture vessels that can be used to culture these cells include, but are not limited to, flasks, tissue culture flasks, petri dishes, petri dishes, multi-petri dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, as long as stem cells can be cultured inside. These cells may be cultured in the following volumes, depending on the need for culture: at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range that can be derived from these. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment capable of growing cells. The bioreactor may have a volume of at least 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range that can be derived from these.

[0182] In certain configurations, PSCs such as iPSCs are seeded at a cell density suitable for efficient differentiation. Generally, these cells are seeded at a density of approximately 1,000 to 75,000 cells / cm³. 2 (For example, approximately 5,000 to 40,000 cells / cm²) 2Seed the cells at a cell density of approximately 50,000 to 400,000 cells per well in a 6-well plate. In an exemplary method, the cells are seeded at a cell density of approximately 100,000, 150,000, 200,000, 250,000, 300,000, or 350,000 cells per well (for example, approximately 50,000 cells per well).

[0183] PSCs, such as iPSCs, are generally cultured on culture plates coated with one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrix proteins (e.g., vitronectin, laminin, collagen, and / or fibronectin), and these extracellular matrix proteins can be used to coat the culture surface as a means of providing a solid support for the proliferation of pluripotent cells. The term "extracellular matrix (ECM)" is recognized in the art. Components of the ECM may include, but are not limited to, one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosine, ancholine, chondronectin, binding proteins, bone sialoproteins, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. Other ECM components may include synthetic peptides for adhesion (e.g., RGD motif or IKVAV motif), synthetic hydrogels (e.g., PEG, PLGA, etc.), or natural hydrogels (e.g., alginates). In an exemplary method, PSCs are grown on a culture plate coated with vitronectin. In some embodiments, the cell adhesion protein is a human protein.

[0184] Extracellular matrix proteins may be of natural origin and purified from human or animal tissue, or ECM proteins may be genetically engineered recombinant proteins or naturally synthesized. ECM proteins may be whole proteins or in the form of natural or engineered peptide fragments. Examples of ECM proteins that may be useful in the matrix for cell culture include laminin, collagen I, collagen IV, fibronectin, and vitronectin. In some embodiments, the matrix composition is heterogeneous. For example, a heterogeneous matrix for culturing human cells may use matrix components of human origin and exclude any non-human animal components.

[0185] In some embodiments, the total protein concentration in the matrix composition may be about 1 ng / mL to about 1 mg / mL. In some preferred embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In more preferred embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0186] Cells such as photoreceptors or PSCs can be cultured with nutrients necessary to support the proliferation of each specific population of cells. Generally, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. This medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. Exemplary growth media include minimal essential media (e.g., Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8® (E8®) medium) supplemented with various nutrients (e.g., non-essential amino acids and vitamins) to enhance stem cell proliferation. Examples of minimal essential media include, but are not limited to, Minimum Essential Medium Eagle® Alpha, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640, 199, and F12. In addition, minimal essential media may be supplemented with additives (e.g., horse, calf, or fetal bovine serum). Alternatively, the medium may be serum-free. In other cases, the growth medium may include serum-free formulations, referred herein as “Knockout Serum Substitutes,” which are optimized for growing and maintaining undifferentiated cells (e.g., stem cells) during culture. KNOCKOUT® serum substitutes are disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, incorporated herein by reference. Preferably, PSCs are cultured in a fully defined and feeder-free medium.

[0187] Therefore, single-cell PSCs are generally cultured in a fully defined culture medium after seeding. In certain embodiments, the medium is aspirated and fresh medium (e.g., E8® medium) is added to the culture approximately 18–24 hours after seeding. In certain embodiments, single-cell PSCs are cultured in a fully defined culture medium for approximately 1, 2, or 3 days after seeding. Preferably, single-cell PSCs are cultured in a fully defined culture medium for approximately 2 days before the differentiation process can proceed.

[0188] In some embodiments, the culture medium may or may not contain any serum substitute. Examples of serum substitutes include albumin (e.g., lipid-rich albumin, albumin substitutes (e.g., recombinant albumin), vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or substances appropriately containing these equivalents. Serum substitutes may be prepared, for example, by the methods disclosed in International Publication No. 98 / 30679. Alternatively, any commercially available material may be used more conveniently. Examples of commercially available materials include KNOCKOUT® serum substitute (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX® (Gibco).

[0189] Other culture conditions can be appropriately defined. For example, the culture temperature may be about 30–40°C, and may be, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not particularly limited thereto. In one embodiment, cells are cultured at 37°C. The CO2 concentration may be about 1–10% (e.g., about 2–5%), or any range that can be derived within this range. The oxygen partial pressure may be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range that can be derived within this range.

[0190] 2. Differentiation medium Retinal maturation culture medium Differentiated retinal cells cultured in the above RDM can be further differentiated and expanded by culturing these cells in retinal maturation medium (RM) to produce RPC. RM may contain nicotinamide. RM may contain about 1 mM to about 50 mM (e.g., about 10 mM) of nicotinamide. RM may further contain ascorbic acid (e.g., 50 to 500 μm, particularly about 100 to 300 μm, e.g., about 200 μm). Preferably, RM does not contain activin A or is essentially activin A-containing. Exemplary RM media are shown in Table 1. RM (e.g., RM2) may further contain γ-secretase inhibitors (e.g., DAPT, basic FGF) and / or TGFβ pathway inhibitors (e.g., SB431542).

[0191] The RM may contain DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute in approximately 1% to 5% (e.g., approximately 1.5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. This medium may be replaced daily with RM at room temperature. These cells are generally cultured in the RM for approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days (e.g., approximately 10 days) to induce expanded RPCs.

[0192] PRP maturation medium (PM) PRP can be matured in PRP maturation medium (PM). Exemplary PM media are shown in Table 1. PM media contain ascorbic acid, nicotinamide, and a γ-secretase inhibitor (e.g., DAPT (e.g., about 1 μM to about 10 μM (e.g., about 5 μM))). PM (e.g., PM2) may also contain a CDK inhibitor, e.g., a CDK4 / 6 inhibitor, e.g., PD0332991 (e.g., about 1 μM to about 50 μM, e.g., about 10 μM of PD0332991).

[0193] PM medium may contain DMEM and F12 in a ratio of approximately 1:1, knockout serum substitute in approximately 1% to 5% (e.g., approximately 1.5%), MEM non-essential amino acids (NEAAs), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. This medium may be replaced daily with PM medium at room temperature. These cells are generally cultured in RM medium for approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days (e.g., approximately 10 days) to induce mature photoreceptors. [Table 1] JPEG2026120524000002.jpg255166JPEG2026120524000003.jpg255165JPEG2026120524000004.jpg255164JPEG2026120524 000005.jpg255165JPEG2026120524000006.jpg255167JPEG2026120524000007.jpg255167JPEG2026120524000008.jpg84170

[0194] In addition, photoreceptors can be increased and their purity maintained by adding brevistatin (e.g., approximately 2.5 μM) to the culture medium to promote aggregate formation. Alternatively, a ROCK inhibitor can be used instead of brevistatin, such as TRYPLE®, to increase photoreceptor viability after dissociation into single cells.

[0195] C. Cell cryopreservation RPE, photoreceptor cells, or PRP prepared by the methods disclosed herein may be cryopreserved, see, for example, PCT International Publication No. 2012 / 149484A2, incorporated herein by reference. These cells may be cryopreserved with or without a substrate. In some embodiments, the storage temperature ranges from about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and overlapping ranges thereof. In some embodiments, lower temperatures are used for preserving (e.g., maintaining) the cryopreserved cells. In some embodiments, liquid nitrogen (or other similar coolant) is used to preserve the cells. In further embodiments, these cells are preserved for more than about 6 hours. In additional embodiments, these cells are preserved for about 72 hours. In some embodiments, these cells are preserved for 48 hours to about 1 week. In yet another embodiment, these cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In yet another embodiment, these cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. These cells can be stored for even longer periods of time. These cells can be cryopreserved separately or on a substrate such as one of the substrates disclosed herein.

[0196] In some embodiments, additional cryoprotectants may be used. For example, these cells may be cryopreserved in a cryopreservation solution containing one or more cryoprotectants (e.g., DM80, serum albumin (e.g., human serum albumin or bovine serum albumin)). In certain embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In certain embodiments, the solution contains 2.5% DMSO. In yet another particular embodiment, the solution contains 10% DMSO.

[0197] Cells can be cooled, for example, at about 1°C / min during cryopreservation. In some embodiments, the cryopreservation temperature is about -80°C to about -180°C or about -125°C to about -140°C. In some embodiments, cells are cooled to 4°C and then cooled at about 1°C / min. Cryopreserved cells can be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, when the cells reach about -80°C, they are transferred to the liquid nitrogen storage area. Cryopreservation can also be performed using a freezer with a controlled rate. Cryopreserved cells can be thawed, for example, at a temperature of about 25°C to about 40°C, typically at about 37°C.

[0198] D. Inhibitors WNT pathway inhibitors WNTs are a highly conserved family of secretory signaling molecules that regulate intercellular interactions and are associated with the Drosophila segment polarity gene wingless. In humans, the WNT family of genes encodes 38-43 kDa cysteine-rich glycoproteins. WNT proteins possess a hydrophobic signaling sequence, a conserved asparagine-binding oligosaccharide consensus sequence (see, e.g., Shimizu et al. Cell Growth Differ 8:1349-1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote cytoplasmic beta-catenin stabilization, WNT proteins may act as transcriptional activators to inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain cancers.

[0199] In this specification, WNT inhibitors (also referred to as WNT pathway inhibitors) generally refer to WNT inhibitors. Therefore, a WNT inhibitor refers to any inhibitor of a member of the WNT family protein (e.g., Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11, and Wnt16). Certain embodiments of this method relate to WNT inhibitors in differentiation media. Examples of suitable WNT inhibitors known in the art include: N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidine-2-yl)thio]acetamide (IWP4), 2-phenoxybenzoic acid-[( [5-methyl-2-furanyl)methylene]hydrazide (PNU74654), 2,4-diaminoquinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidine-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK715), Dickkopf-related protein 1 (DKK1), and secreted frizzled-related protein (SFRP1) 1. In addition, WNT inhibitors may include antibodies against WNT, dominant-negative variants of WNT, and siRNAs and antisense nucleic acids that suppress WNT expression. WNT inhibition can also be achieved using RNA-mediated interference (RNAi).

[0200] BMP pathway inhibitors Bone morphogenetic proteins (BMPs) are multifunctional growth factors belonging to the transforming growth factor beta (TGFβ) superfamily. BMPs are considered to constitute a crucial group of morphogenetic signaling pathways, organizing structures throughout the body. The important physiological functions of BMP signaling are highlighted by their numerous roles in dysregulation of BMP signaling in pathological processes.

[0201] BMP pathway inhibitors (also referred to herein as BMP inhibitors) may generally include inhibitors of BMP signaling, or may include inhibitors specific to BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Examples of BMP inhibitors include: 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidine-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (dolsomorphine), 4-[6-(4 -(1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline (ML347).

[0202] TGFβ pathway inhibitors Transforming growth factor beta (TGFβ) is a secreted protein that regulates proliferation, cell differentiation, and other functions in most cells. TGFβ is a type of cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. There are at least three isoforms of TGFβ, called TGFβ1, TGFβ2, and TGFβ3. The TGFβ family is part of a superfamily of proteins known as the transforming growth factor beta superfamily (e.g., inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplesic, and Vg-1).

[0203] TGFβ pathway inhibitors (also referred to herein as TGFβ inhibitors) generally include any inhibitor of TGFβ signaling. For example, TGFβ inhibitors include 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazole-4-yl]quinoxaline (SB525334), and 2-(5-benzo[1,3]dioxol-5-yl-2-ieri-butyl-3H -Imidazole-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-Benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]-benzamide hydrate, left-right determinant (Lefty), 3-(6-methyl-2-pyridinyl) Dinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazole-2-yl]benzamide (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide These are Mido (GW788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]quinoline (LY364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R268712), or 2-(3-(6-methylpyridinyl-2-yl)-1H-pyrazole-4-yl)-1,5-naphthiridine (RepSox).

[0204] MEK inhibitors MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2. These chemicals and drugs can be used to affect the MAPK / ERK pathway. Examples of MEK inhibitors include: N-[(2R)-2,3-dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901), N-[3-[3-cyclopropyl-5-(2-fluoro-4-iodoanilino)-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidine-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)- 7-Fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (MEK162), N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-(2,3-dihydroxypropyl)cyclopropane-1-sulfonamide (RDEA119), and 6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide (AZD6244).

[0205] Gamma-secretase inhibitors Gamma-secretase is a multi-subunit protease complex (which is itself an endogenous membrane protein) that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. This type of protease is known as an endometrial protease. The most well-known substrate of gamma-secretase is amyloid precursor protein, a large endogenous membrane protein. When this endogenous membrane protein is cleaved by both gamma-secretase and beta-secretase, it produces a short amino acid peptide called amyloid-beta. The abnormally folded fibril form of this amino acid peptide is a major component of amyloid plaques found in the brains of Alzheimer's disease patients.

[0206] The term "gamma-secretase inhibitor" as used herein generally refers to a γ-secretase inhibitor. Examples of γ-secretase inhibitors include, but are not limited to, the following: N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), 5-chloro-N-[(1S)-3,3,3-trifluoro-1-(hydroxymethyl)-2-(trifluoromethyl)propyl]-2-thiophenesulfonamide (Vegas Estate), MDL-28170, 3,5-bis(4-) Trophenoxy)benzoic acid (compound W), 7-amino-4-chloro-3-methoxy-1H-2-benzopyran (JLK6), (5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)-hydroxy-(2R)-benzylhexanoyl)-L-leucine-L-phenylalaninamide (L-685,485), (R)-2-fluoro-α-methyl[1,1'-biphenyl]-4-acetic acid ((R)-flurbiprofen; flurizan), N-[ (1S)-2-[[(7S)-6,7-dihydro-5-methyl-6-oxo-5H-dibenzo[b,d]azepine-7-yl]amino]-1-methyl-2-oxoethyl]-3,5-difluorobenzeneacetamide (dibenzazepine; DBZ), N-[cis-4-[(4-chlorophenyl)sulfonyl]-4-(2,5-difluorophenyl)cyclohexyl]-1,1,1-trifluoromethanesulfonamide (MRK560), (2S)-2-[[(2S )-6,8-difluoro-1,2,3,4-tetrahydro-2-naphthalenyl]amino]-N-[1-[2-[(2,2-dimethylpropyl)amino]-1,1-dimethylethyl]-1H-imidazole-4-yl]pentanamide hydrobromide (PF3084014 hydrobromide), and 2-[(1R)-1-[[(4-chlorophenyl)sulfonyl](2,5-difluorophenyl)amino]ethyl-5-fluorobenzenebutanoic acid (BMS299897).

[0207] Cyclin-dependent kinase inhibitors Cyclin-dependent kinases (CDKs) are a family of serine kinases first discovered for their role in the control of the cell cycle. CDKs are also involved in the control of neuronal transcription, mRNA processing, and differentiation. In many human cancers, CDKs are either overactive or CDK inhibitor proteins are not functioning. A CDK inhibitor can be a CDK1 inhibitor, a CDK2 inhibitor, a CDK3 inhibitor, a CDK4 inhibitor, a CDK5 inhibitor, a CDK6 inhibitor, a CDK7 inhibitor, a CDK8 inhibitor, and / or a CDK9 inhibitor. In certain embodiments, the CDK inhibitor is a CDK4 / 6 inhibitor.

[0208] Examples of CDK inhibitors include, but are not limited to: palbociclib (PD-0332991) HCl, roscovitine (seliciclib, CYC202), SNS-032 (BMS-387032), dinaciclib (SCH727965), flavopiridol (alvocidib), MSC2530818, JNJ-7706621, AZD5438, MK-8776 (SCH 900776), PHA-793887, BS-181 HCl, A-674563, abemaciclib (LY2835219), BMS-265246, PHA-767491, or milciclib (PHA-848125).

[0209] bFGF inhibitor Basic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the vascular basement membrane and the subendothelial extracellular matrix. In addition, bFGF is a common component of human ESC culture media necessary for cells to maintain an undifferentiated state.

[0210] The bFGF inhibitor refers to the entire bFGF inhibitor in this specification. For example, the following are included as bFGF inhibitors, but not limited thereto: N-[2-[[4-(diethylamino)butyl]amino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one (PD98059), 1-tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]urea (PD161570), 6-(2,6-dichlorophenyl)-2-[[4-[2-(diethylamino)ethoxy]phenyl]amino]-8-methyl-pyrido[2,3-d]pyrimidin-7(8H)-one dihydrochloride hydrate (PD166285), N-[2-amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)-urea (PD166866), and MK-2206.

[0211] IV. RPE-photoreceptor cell / photoreceptor precursor bilayer culture In certain embodiments, the RPE can be cultured on any suitable culture surface (particularly a culture surface acceptable for transplantation, such as a scaffold under GMP-compliant conditions). In certain embodiments, the RPE is cultured on a surface (such as a multi-well plate (e.g., 6-well, 12-well, 24-well, 48-well, or 96-well)) coated with ECM (e.g., vitronectin) on a Transwell support (e.g., Snapwell insert) or on a scaffold coated with a polymer (e.g., poly(lactic-co-glycolic acid) (PLGA)). Alternatively, the RPE can be cultured on collagen or laminin. In certain embodiments, the culture surface has a high concentration (e.g., 1 μg / cm 2 sup, particularly, 2 μg / cm 2 , 3 μg / cm 2 , 4 μg / cm 2 , 5 μg / cm 2 , 6 μg / cm 2 , 7 μg / cm 2 , 8 μg / cm2 9 μg / cm³ 2 , 10 μg / cm³ 2 RPE is coated with , or higher than, vitronectin. RPE can be cultured in a medium containing taurine, hydrocortisone, and taurine, for example, in the RPE-MM medium described herein. In certain embodiments, this medium may be serum-free or serum-restricted and may contain a knockout serum substitute. RPE is cultured at 100,000 cells / cm². 2 ~500,000 cells / cm² 2 They can be cultured at densities such as 150,000 cells / cm³. 2 200,000 cells / cm² 2 250,000 cells / cm² 2 300,000 cells / cm² 2 , or 350,000 cells / cm² 2 They can be cultured at a density of approximately 300,000 cells / cm³. 2 It can be cultured at this density.

[0212] In some embodiments, RPEs may be cultured to produce polarized RPEs that are positive for bethrofin and / or ZO1. These polarized RPEs may be positive for PRE65 and / or ezrin. These RPEs may originate from hiPSCs and may be mature (e.g., RPEs positive for PMEL17, TYRP1, and CRALBP). Mature RPEs (e.g., RPEs at day 42 by the above method) may be cultured directly or thawed if they have been previously cryopreserved. The RPEs may then be cultured for a period sufficient to polarize them (e.g., 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, or longer). Specifically, the RPEs may be cultured for 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days to polarize them.

[0213] The photoreceptors layered on the RPE may be immature photoreceptors destined to become either cones or rods. Specifically, photoreceptors can be obtained by the hybrid differentiation method described herein. The photoreceptors may be seeded directly onto the RPE from culture or cryopreservation, or they may be re-seed and cultured before seeding onto the RPE. The photoreceptors may be seeded onto the RPE and then cultured for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 days, or more, to allow adhesion and the formation of a distinct bilayer. The photoreceptors may be seeded at a density of 500,000 cells / cm². 2 1 million cells / cm 2 2 million cells / cm 2 3 million cells / cm 2 4 million cells / cm 2 5 million cells / cm 2 6 million cells / cm 2 7 million cells / cm 2 8 million cells / cm 2 9 million cells / cm 2 10 million cells / cm² 2 or they can be seeded at a higher density. Specifically, photoreceptors can be seeded at a density of approximately 3 million cells / cm³. 2 They can be seeded at a density of . In certain embodiments, the photoreceptors are derived from hiPSCs and not from organoid selection. The seeded photoreceptors may be essentially single cells that do not contain aggregates. RPEs may be cultured in a medium containing taurine, hydrocortisone, and taurine, for example, in the RPE-MM medium described herein. In certain embodiments, this medium may be serum-free or serum-restricted and may contain a knockout serum substitute.

[0214] In some embodiments, before the addition of the photoreceptor, the RPE culture may be treated with a ROCK inhibitor, an ECM protein, and / or prostaglandin E2 (PGE2) to enhance, for example, the adhesion of the photoreceptor to the RPE. The ROCK inhibitor may be Y-27632. The ECM protein may be laminin, collagen I, collagen IV, fibronectin, or vivoronectin, and in particular may be laminin-521. The ROCK inhibitor, ECM protein, or PGE2 may be added for at least 10 minutes before the addition of the photoreceptor, for example, 30 minutes, 60 minutes, or 90 minutes.

[0215] The ratio of photoreceptors to RPEs in this distinct bilayer may be 1:1, 1:2, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, 25:1, or 30:1.

[0216] V. Use of bilayer PR / PRP:RPE co-cultures PR / PRP:RPE bilayer cultures can be used for transplantation in cell rescue therapy or total tissue replacement therapy. Certain embodiments provide the use of PR / PRP:RPE bilayer cultures to enhance the maintenance and repair of ocular tissue for any necessary condition such as retinal degeneration or significant injury. Retinal degeneration may be associated with age-related macular degeneration (AMD), hereditary macular degeneration, Stargardt macular dystrophy, Best's disease, total choroidal atrophy, hereditary retinal degeneration (e.g., retinitis pigmentosa, cone / rod and rod / cone dystrophy), diabetic retinopathy, retinal vascular disease, damage caused by retinopathy of prematurity (ROP), viral infections of the eye, and other retinal / eye diseases or injuries / traumas.

[0217] In another aspect, the Disclosure provides a method for treating an individual in need, comprising implanting a composition comprising a PR / PRP:RPE bilayer into the individual. The composition may be administered into the eye, such as the subretinal space. Such individuals may have hereditary macular degeneration or retinal degeneration, such as retinitis pigmentosa, cone / rod or rod / cone dystrophy, Stargardt disease, Best's disease, total choroidal atrophy, retinal dysplasia, retinal degeneration, diabetic retinopathy, congenital retinal dystrophy, Leber congenital amaurosis, retinal detachment, damage caused by retinopathy of prematurity (ROP), or other retinal trauma or injury.

[0218] To determine the suitability of a cell composition for therapeutic administration, the cells may be first tested in a suitable animal model such as a rodent or a pig. In one embodiment, a PR / PRP:RPE bilayer culture is evaluated for its ability to survive in vivo and maintain its phenotype. This composition is transplanted into an immunodeficient animal (e.g., a nude mouse or rat, or an animal that has been immunodeficient by chemical or radiation). After the proliferation period, tissue is collected and evaluated to determine whether pluripotent stem cell-derived cells are still present.

[0219] Human PR / PRP:RPE bilayer cultures, or pharmaceutical compositions containing such bilayers, as described herein may be used in the manufacture of pharmaceuticals to treat specific patient conditions. The photoreceptors or RPEs may be cryopreserved in advance. In certain embodiments, the photoreceptors or RPEs of this disclosure are derived from iPSCs and can therefore be used to provide “personalized medicine” to patients with eye diseases. In some embodiments, somatic cells obtained from a patient may be genetically engineered to correct disease-causing mutations, differentiate into PR / PRP or RPE, and manipulated to form PR / PRP:RPE bilayer cultures. This tissue may be used to replace endogenous degenerated PR / PRP and RPE from the same patient. Alternatively, iPSCs generated from a healthy donor or an HLA homozygous “superdonor” may be used.

[0220] The introduction of PR / PRP:RPE bilayer cultures obtained using the methods disclosed herein may treat or prevent a variety of ocular conditions. These conditions include retinal dysfunction or deterioration, retinal damage, and / or diseases or disorders of the retina generally associated with the loss of retinal pigment epithelium and / or photoreceptors. Conditions that may be treated include, but are not limited to, the following: degenerative diseases of the retina, e.g., Stargardt macular dystrophy, retinitis pigmentosa, rod / cone and cone / rod dystrophy, macular degeneration (e.g., age-related macular degeneration, myopic macular degeneration, or other acquired or hereditary macular degenerations), retinal damage caused by retinopathy of prematurity (ROP), and diabetic retinopathy. Further conditions include: Leber congenital amaurosis, hereditary or acquired macular degeneration or retinal degeneration, Best's disease, retinal detachment, gynostosis, total choroidal atrophy, pattern dystrophy, other dystrophys of photoreceptor cells, and retinal damage resulting from damage caused by any one of the following: light, laser, inflammation, infection, radiation, neovascularization, or trauma. In certain embodiments, a method is provided for treating or preventing a condition characterized by retinal degeneration, comprising administering an effective amount of a composition containing the PR / PRP:RPE bilayer culture to a subject of interest. This method may include selecting a subject having one or more of these conditions, and administering a therapeutically effective amount of the PR / PRP:RPE bilayer culture sufficient to treat and / or alleviate the symptoms of the condition. The PR / PRP:RPE bilayer culture can be implanted in various forms. For example, PR / PRP:RPE bilayer cultures can be introduced into target sites attached to substrates such as matrix, extracellular matrix, or biodegradable polymers.

[0221] Advantageously, the pharmaceuticals of this disclosure can be used to compensate for the lack or reduction of RPE and / or PR / PRP cell function. Examples of retinal dysfunctions that can be treated by the retinal cell populations and methods of the present invention include, but are not limited to, photoreceptor degeneration (e.g., retinitis pigmentosa, cone dystrophy, cone-rod and / or rod-cone dystrophy, and those occurring in genetic and age-related or myopic macular degeneration); retinal detachment and trauma; photodamage caused by lasers or sunlight; macular holes; macular edema; night blindness and color vision deficiency; ischemic retinopathy such as that caused by diabetes or vascular occlusion; retinopathy / retinal damage resulting from premature birth; infectious conditions, e.g., CMV, retinitis, and toxoplasmosis; inflammatory conditions, e.g., uveitis; tumors, e.g., retinoblastoma and intraocular melanoma.

[0222] In one embodiment, the cells may treat or alleviate symptoms of hereditary retinal degeneration (e.g., retinitis pigmentosa, cone / rod or rod / cone dystrophy, Leber congenital amaurosis, or retinal injury / trauma / damage) in patients requiring treatment. In another embodiment, the cells may treat or alleviate symptoms of acquired or hereditary macular degeneration (e.g., age-related macular degeneration (wet or cyclic), Stargardt disease, Best's disease, myopic macular degeneration, or similar) in patients requiring treatment. For all of these treatments, the cells may be autologous or allogeneic to the patient. In further embodiments, the cells of this disclosure may be administered in combination with other treatments.

[0223] In some embodiments, the PR / PRP:RPE bilayer culture can be used as an autologous graft for subjects suitable for regenerative medicine. This PR / PRP:RPE bilayer culture can be transplanted in combination with other retinal cells. The transplantation of the PR / PRP:RPE bilayer culture produced by the method of this disclosure can be carried out by various techniques known in the art. According to one embodiment, transplantation is carried out by a surgical approach to the ciliary body squamous region and subsequent delivery of cells through a small retinal opening into the subretinal space, or by direct injection. This PR / PRP:RPE bilayer culture can be introduced into a target site attached to a matrix such as an extracellular matrix or provided on a substrate such as a biodegradable polymer.

[0224] This PR / PRP:RPE bilayer culture can be used to generate neurosensory retinal structures. These structures can be used for drug screening, as disease models, or as pharmaceuticals or in pharmaceutical products. In the latter case, the pharmaceutical product may be an RPE-photoreceptor graft, which can be placed on a biocompatible solid support or matrix (preferably a biocompatible matrix or support) that can be implanted like a "patch".

[0225] To further illustrate, the biocompatible support for cells may be a biodegradable synthetic (e.g., polyester) film support of RPE. The biodegradable polyester may be any biodegradable polyester suitable for use as a substrate or scaffold to support the proliferation and differentiation of retinal precursor cells. The polyester should be able to form a thin film (preferably a film with a fine pattern on its surface) and should be biodegradable if used for tissue or cell transplantation. Suitable biodegradable polyesters for use in the present invention include: polylactic acid (PLA), polylactides, polyhydroxyalkanoates, both homopolymers and copolymers, e.g., polyhydroxybutyrate (PHB), polyhydroxybutyrate co-hydroxyvalate (PHBV), polyhydroxybutyrate co-hydroxyhexanoate (PHBHx), polyhydroxybutyrate co-hydroxyoctonoate (PHBO), and polyhydroxybutyrate co-hydroxyoctadecanoate (PHBOd), polycaprolactone (PCL), polyesteramide (PEA), aliphatic copolyesters, e.g., polybutylene succinate (PBS) and polybutylene succinate / adipate (PBSA), and aromatic copolyesters. Both high and low molecular weight polyesters, substituted and unsubstituted polyesters, blocks, branched or random, and mixtures and blends of polyesters may be used. Preferably, the biodegradable polyester is polycaprolactone (PCL).

[0226] Pharmaceutical compositions of PR / PRP:RPE bilayer cultures prepared by the methods disclosed herein are also provided. These compositions contain at least about 1 × 10⁻⁶ 3 A single cell, approximately 1 x 10⁻⁶ 4 A single cell, approximately 1 x 10⁻⁶ 5 A single cell, approximately 1 x 10⁻⁶ 6 A single cell, approximately 1 x 10⁻⁶ 7 A single cell, approximately 1 x 10⁻⁶ 8 A single cell, or approximately 1 × 10⁻⁶ cells. 9It may contain individual cells. In certain embodiments, the composition is a substantially purified preparation (with respect to non-PR / PRP:RPE cells) containing differentiated PR / PRP:RPE cells produced by the method disclosed herein. Compositions also provided that contain a scaffold (e.g., PR / PRP as a polymer carrier and / or extracellular matrix) and an effective amount of photoreceptor RPE cells produced by the method disclosed herein. Matrix materials are generally physiologically acceptable and suitable for use in in vivo applications. Examples of physiologically acceptable materials include, but are not limited to, the following: absorbable and / or non-absorbable solid matrix materials, e.g., intestinal submucosa (SIS), cross-linked or uncross-linked alginates, hydrophilic colloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, fleece, and bioadhesives.

[0227] Suitable polymer carriers include synthetic or natural polymers, and porous meshes or sponges formed from polymer solutions. For example, the matrix may be a polymer mesh or sponge, or a polymeric hydrogel. Natural polymers that can be used include proteins, such as collagen, albumin, and fibrin; and polysaccharides, such as alginates and hyaluronic acid polymers. Synthetic polymers include both biodegradable and non-biodegradable polymers. For example, biodegradable polymers include hydroxy acid polymers, such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PGLA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof. Non-biodegradable polymers include polyacrylates, polymethacrylates, ethylene vinyl acetates, and polyvinyl alcohols.

[0228] A polymer capable of forming malleable, ionically or covalently crosslinked hydrogels may be used. A hydrogel is a substance formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open lattice structure that traps water molecules and forms a gel. Examples of materials that can be used to form hydrogels include ionically crosslinked polysaccharides (e.g., alginates, polyphosphazenes, and polyacrylates) or block copolymers (e.g., PLURON1CS® or TETRON1CS®, or polyethylene oxide-polypropylene glycol block copolymers crosslinked by temperature or H, respectively). Other materials include proteins such as fibrin, polymers such as polyvinylpyrrolidone, hyaluronic acid, and collagen.

[0229] The pharmaceutical composition may be optionally packaged in a suitable container with written instructions for a desired purpose (e.g., reconstitution of photoreceptor function to improve disease or abnormality of retinal tissue). In some embodiments, photoreceptors produced by the method of this disclosure may be used to replace degenerated photoreceptor cells of the target required.

[0230] VI. Kit In some embodiments, for example, a kit is provided that can include one or more media and components for manufacturing a PR / PRP:RPE bilayer culture. Such a formulation can include a cocktail of retinal differentiation factors and / or trophic factors in a form suitable for combination with photoreceptor precursors or photoreceptor cells. This reagent system can be packaged, if desired, in the form of a lyophilized product in an aqueous medium. The container means of this kit generally includes at least one vial, test tube, flask, bottle, syringe, or other container means into which the components can be placed and preferably appropriately dispensed. If the kit contains multiple components, the kit generally also includes a second, third, or other additional container into which additional components can be separately placed. However, various combinations of components may be included in one vial. The components of this kit can be provided as dry powders. If the reagent and / or components are provided as dry powders, the powder can be reconstituted by the addition of a suitable solvent. It is also contemplated that the solvent can be provided in a separate container means. The kit also typically includes means for containing the kit components in a secure enclosure for commercial sale. Such containers can include injection-molded plastic containers or blow-molded plastic containers that hold the desired vials. The kit can also include instructions for use in electronic form, such as in printed or digital form (e.g., for bilayer culture therapy).

Examples

[0231] VII. Examples The following examples are included to demonstrate preferred embodiments of the present invention. Those skilled in the art should recognize that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the implementation of the present invention and, therefore, can be considered to constitute a preferred model for the implementation of the present invention. However, those skilled in the art should recognize that, in light of this disclosure, many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtain similar or comparable results.

[0232] Example 1 - Generation of bilayer cultures PRP seeding on polarized RPE: iPSC-derived RPE was grown according to the method described herein using CD56-negative selection (see PCT / US2016 / 050554), and iPSC-derived PRP was grown according to the hybrid ophthalmic vesicle PRP differentiation method (see PCT / US2019 / 028557). The RPE was thawed on day 42 and seeded at 100,000 or 300,000 cells / cm². 2 The PRP was polarized in a 48-well plate until day 68. At this point, the PRP on day 75 was removed from the laminin-521 coated container and resuspended in RPE-MM at 6 million cells / mL. The PRP was then resuspended in unmodified RPE-MM at 3 million cells / cm³. 2 Seeds were seeded on RPE using 0.5 mL of culture medium.

[0233] One day later, phase-contrast microscopy revealed that PRP had adhered to the RPE layer under all conditions. Representative input PRP flow plots and micrographs after adhesion are shown in Figure 1. PRP adhered to RPE at both RPE densities with and without 30 minutes of lamin-521 incubation prior to PRP seeding, and with both PRP-input cells. Furthermore, RPE and PRP appeared to maintain separate layers (Figure 2). The adhesion of PRP to RPE in RPE-MM, and the clear presence of both cell types after 7 days of co-culture, suggest that this form is a potentially robust basis for bilayer culture therapy.

[0234] Thawing and seeding of PRP on polarized RPE: To minimize the complex timing issues of directly using PRP and simultaneously culturing and maturing RPE and PRP, the PRP was thawed directly onto the polarized RPE layer. In addition, since the PRP layer was subconfluent, PRP seeding was tested at two different densities: 3 million cells / cm². 2 and 10 million cells / cm 2RPE was thawed on day 42 and polarized until day 68. On day 75, PRP was successfully thawed on the RPE layer in RPE-MM without any brevistatin or ROCK inhibitors. After 7 days of culture, the cells were fixed and showed bilayer morphology by immunohistochemistry (Figure 3). In addition, flow cytometry data also showed both cell types as separate populations, with most RPE being mature bethrophine 1 + The phenotype was expressed (Figure 4).

[0235] This experiment demonstrated several advances for bilayer culture products. First, PRP can be thawed directly onto RPE, enabling a modular process that allows the PRP product to be stored and used as needed. The RPE and PRP form separate layers, suggesting that this format could be used both as a treatment in which this cell bilayer is implanted in the retina and as a simple in vitro model. Finally, high concentrations (1 × 10⁻⁶) 7 cells / cm 2 By sowing PRP in this manner, a nearly confluent PRP layer can be obtained. This experiment demonstrates the conditions under which a confluent PRP layer can be achieved.

[0236] RPE / PRP co-culture in snapwell format: RPE / PRP co-cultures were seeded in snapwell format in RPE-MMs, and in RPE-MMs containing 15% knockout serum substitute (KOSR) instead of 5% FBS. On day 42, RPE was cultured at 300,000 cells / cm². 2 The seeds were sown and allowed to polarize until day 68. PRP was 1 × 10 7 cells / cm 2 The cells were thawed on RPE. Some samples were seeded with RPE-MM containing 15% KOSR instead of RPE-MM. After 7 days of culture, the cells were analyzed by flow cytometry or immunocytochemistry (Figure 5). This experiment demonstrated the feasibility of RPE / PRP co-culture on Snapwell inserts and in both FBS-containing RPE-MM and KOSR-containing RPE-MM.

[0237] RPE / PRP co-culture on PLGA scaffolds: Next, this co-culture was performed on a vitronectin (VTN) coated PLGA scaffold assembled in a snapwell culture system. On day 42, RPE was performed at a rate of 300,000 cells / cm². 2 The seeds were sown and allowed to polarize until day 68. The PRP was thawed at the following two densities: 5 × 10 6 cells / cm 2 and 1 × 10 7 cells / cm 2 After 7 days of culture, the cells were analyzed by immunocytochemistry and confocal microscopy (Figure 6). This experiment confirmed the feasibility of culturing RPE / PRP bilayers on a VTN-coated PLGA scaffold, and demonstrated relatively low (5 × 10⁻¹⁴)⁻¹⁴ 6 cells / cm 2 ) and relatively high (1 × 10 7 cells / cm 2 The PRP morphology after seeding at the given cell density was observed, and this relatively high density resulted in the achievement of a nearly confluent layer of PRP.

[0238] The ability to culture RPE and PRP as a bilayer demonstrates the feasibility of this culture format for cell-based therapies. This “dual therapy” can be relevant to the treatment of a wider range of conditions compared to RPE alone or PRP alone, because both cell types are present and organized in a separate, appropriately layered structure in which polarized RPE is covered by PRP. Furthermore, PRP can be thawed directly onto RPE, thus allowing these two cell types to be prepared and cryopreserved independently. This feature of dual therapy makes it possible to utilize different PRP subtypes (e.g., rod-type or conical-type) in a modular system.

[0239] Example 2 - Optimization of PRP adhesion 10 μg / cm³ of RPE before thawing on a 48-well plate 2 Depending on the viralectin concentration, 0.5 μg / cm³ 2Compared to the above-mentioned vivo concentration, PRP adhesion to RPE was improved (Figure 13). This experiment also demonstrated the effect of PRP seeding density, with PRP being 0.5 μg / cm³. 2 With the Vitronectin, 10 million PRPs / cm 2 Compared to 3 million PRP / cm² 2 When sown, it adhered well.

[0240] Adjustment of Snapwell Culture Volume: The amount of culture medium in the snapwell culture was adjusted so that the pressure from the medium was lower at the top (apex) or bottom (base) of the snapwell. Increasing the volume at the base of the snapwell, and thereby increasing the pressure at this side, had two effects on the culture. First, the cell bilayer was more easily separated from this snapwell. Second, PRP appeared to adhere more strongly to RPE, as demonstrated by complete PRP coating after punching the sample with a biopsy punch (Figure 8).

[0241] PRP density titration: Density titration of PRP on RPE in RM1 in a 48-well plate showed an increase in PRP coverage area with increasing seeding density, but approximately 5 million PRP / cm² 2 Seeding of PRP exceeding a certain density resulted in detachment (Figure 7). This trend suggests that an alternative method to improve RPE / PRP adhesion is to reduce the PRP density. A high PRP:RPE ratio is preferable (ideally 30:1), but lower PRP densities may also allow adhesion.

[0242] In conclusion, several modifications to the culture medium or culture system appeared to qualitatively improve RPE / PRP cell adhesion. Increasing the concentration of vitronectin beneath the RPE layer also improved PRP adhesion. Increasing the amount of medium above the bottom of the snapwell also improved PRP adhesion. Finally, reducing the density of PRP may improve PRP adhesion (3 million PRP cells / cm³). 2 At densities below a certain level, PRPs cluster in colonies rather than networks.

[0243] Example 3 - In vivo implantation of RPE-PRP bilayer We developed a bilayer ophthalmic cell therapy consisting of RPE cells and PRP cells. This bilayer therapy, PLGA-RPE-PRP, was transplanted into pig eyes, and the grafts were analyzed by immunocytochemistry.

[0244] Prior to surgery, a bilayer of RPE and PRP was prepared on a PLGA scaffold. Briefly, RPE (day 42) was thawed and 3 × 10⁻¹⁴⁻¹ 5 cells / cm 2 RPE was seeded on a PLGA scaffold. RPE was cultured for 26 days (until day 68 of differentiation) to polarize it, including PGE2 addition from day 54 to day 68. PRP (day 75 or 77) was added to the RPE at day 68 in 4 × 10⁶ 6 cells / cm 2 The cells were seeded as single cells on RPE. This cell bilayer was cultured for 7 days, with the culture medium (RPE-MM) being changed every 1-2 days.

[0245] Two days prior to surgery, the retinas of some pigs were irradiated with a 532 / 577 micropulse laser using a very short duty cycle (1%–3%) and a threshold energy that limited the energy emitted outside the retina (resulting in lesions that were barely visible). The laser energy was sensed by the host's RPE and damaged photoreceptors. The pigs were also treated with immunosuppressants, which were started 5–8 days after laser treatment and continued until euthanasia.

[0246] On the day of surgery, the pigs were anesthetized, intubated, paralyzed, and prepared for subretinal transplantation. Double-layered samples for double therapy were prepared for surgery. A 0.42% Healon-GV solution was prepared and homogenized by aspirating with an 18G needle approximately 30-50 times. Several droplets of this solution were dropped onto a cutting mat. A Snapwell insert was removed from the 6-well plate, washed with HBSS+, and placed on a droplet of Healon-GV solution. The entire bottom of the Snapwell insert was punched out using an 8mm biopsy punch. The plastic bottom of this Snapwell insert was mechanically removed from the PLGA-RPE-PRP layer using tweezers or a spatula. This PLGA-RPE-PRP was punched out again using a 2x4mm oval biopsy punch and transferred to a droplet of 0.42% Healon-GV. At this point, the PLGA-RPE-PRP grafts, punched out to 2 x 4 mm, were loaded into a subretinal injection device (Figure 9A).

[0247] This subretinal injection device was primed with a hyaluronic acid solution such as Healon-GV. A syringe was used to generate pressure for aspirating the liquid into the device, and the syringe also controlled the loading of the sample into the anterior part of the device. The sample was loaded into the device while the surgeon performed a ciliary body vitrectomy and prepared two 2.4 mm working ports in preparation for subretinal transplantation.

[0248] PLGA-RPE-PRP grafts were delivered into the subretinal space using a subretinal injection device. Intraoperative optical coherence tomography (iOCT) imaging confirmed sample delivery into the subretinal space. After confirming successful sample delivery, fluid / air / gas exchange was performed. The surgeon sutured the working port and removed other sutureless surgical ports in the eye. Postoperatively, OCT imaging was performed every two weeks to monitor the pig's retina.

[0249] After sufficient time had passed for the grafts to engraft in the host retina, the eyes were analyzed by histological examination. Histological examination was performed by cryotome sectioning of the eyes. Briefly, the incised eyes were embedded in an optimal cutting temperature (OCT) compound, frozen, and sectioned perpendicular to the retina using a cryotome. The sectioned samples were mounted on Superfrost Plus slides. The slides were numbered sequentially, so the slide numbers correspond to the medial / lateral regions of the retina.

[0250] Immunocytochemistry of sectioned pig retinas revealed both rod and cone markers (Figure 9). Human RPE was clearly adjacent to the photoreceptor layer indicated by MITF (Figure 10). The presence of photoreceptors was also indicated by recabin (Figure 11). GFAP-expressing cells in the transplanted cell region were human nucleo-negative and host Müller glial cells. Host Müller glial cells attempted to reform the outer limiting membrane but were stopped at the transplanted RPE layer (Figure 12). The potential integration of transplanted cells with the host retina was indicated by the proximity of the presynaptic marker VGLUT1 to the transplanted photoreceptor indicated by PKCα and the proximity of host bipolar cells (Figure 13). The presynaptic marker synaptophysin was also evident in the transplanted cells and appeared to co-localize with the cone marker arrestin-3 (Figure 14).

[0251] RPE / PRP bilayers were successfully implanted into the subretinal space of pig retinas. Postoperatively, the implanted PRP matured into both rod and cone photoreceptors, and the implanted RPE and PRP appeared to have engrafted in the host retina and were ready for integration. Evidence of integration ability is supported by the presence of presynaptic markers adjacent to the implanted photoreceptors and the migration of host bipolar cells to the implanted photoreceptor layer.

[0252] All methods disclosed and claimed herein can be assembled and performed without excessive experimentation in consideration of this disclosure. While the compositions and methods of the present invention are described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications can be made to these methods, and to the steps or order of steps of the methods described herein, without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that the agents described herein can be replaced with certain chemically and physiologically related agents, and that identical or similar results can be achieved. All such similar substitutions and modifications that are apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims. References The following references are incorporated herein by reference insofar as they provide exemplary procedures or other details that supplement those described herein. Alexander et al., Proc. Nat. Acad. Sci. USA, 85:5092-5096, 1988. Barnea-Cramer et al.Sci Rep.6:29784,2016. Bhutto and Lutty.Mol Aspects Med.33(4):295-317,2012. Ercolani et al., J. Biol. Chem., 263:15335-15341, 1988. Hirami et al., Neurosci. Lett., 48:126-131, 2009. Hirami et al., Neurosci. Lett., 48:126-131, 2009. International Publication No. PCT / US2016 / 050543 International Publication No. PCT / US2016 / 050554 International Publication No. PCT / US2019 / 028557 PCT Publication No. WO 98 / 30679. Karin et al., Cell, 36:371-379, 1989. Ludwig et al., Nat. Biotechnol., 24:185-187, 2006b. Ludwig et al., Nat. Methods, 3:637-646, 2006a. Macejak and Sarnow, Nature, 353:90-94, 1991. Ng, Nuc. Acid Res., 17:601-615, 1989. Oner, Turk J Ophthalmol. 48(1):33-8, 2018. PCT International Publication No. WO 2007 / 069666 PCT International Publication No. WO 2014 / 121077. Pelletier and Sonenberg, Nature, 334(6180):320-325, 1988. Pennington and DeAngelis, Eye Vis (Lond). 3:3, 2016. Richards et al., Cell, 37:263-272, 1984. Sambrook and Russel, Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Lab. Press, 2001. Shimizu et al, Cell Growth Differ 8:1349-1358, 1997. Strauss et al., Physiological Reviews, 85:845-881, 2005. Strauss, Physiol Rev. 85(3):845-81, 2005. Takahashi et al., Cell, 126, 663-676, 2006. Takahashi et al., Cell, 131, 861-872, 2007. Thomson and Marshall,Curr.Top.Dev.Biol.,38:133-165,1998. Thomson and Odorico,Trends Biotechnol.,18(2):53-57,2000. Thomson et al.Proc.Natl.Acad.Scie.USA,92:7844-7848,1995. U.S. Patent Application Publication No. 2002 / 0055144 U.S. Patent Application Publication No. 2002 / 0076747 Specification U.S. Patent Application Publication No. 2009 / 0148425 U.S. Patent Application Publication No. 2009 / 0246875 U.S. Patent Application Publication No. 2010 / 0210014 U.S. Patent Application Publication No. 2012 / 0196360 U.S. Patent Application Publication No. 2012 / 0276636 U.S. Patent No. 4,683,202 U.S. Patent No. 5,556,954 U.S. Patent No. 5,843,780 U.S. Patent No. 5,925,565 U.S. Patent No. 5,928,906 U.S. Patent No. 5,935,819 U.S. Patent No. 6,103,470 U.S. Patent No. 6,200,806 U.S. Patent No. 6,416,998 U.S. Patent No. 6,833,269 U.S. Patent No. 7,029,913 U.S. Patent No. 7,442,548 U.S. Patent No. 7,598,364 U.S. Patent No. 7,682,828 U.S. Patent No. 7,989,425 U.S. Patent No. 8,058,065 U.S. Patent No. 8,071,369 U.S. Patent No. 8,129,187 U.S. Patent No. 8,268,620. U.S. Patent No. 8,278,620 U.S. Patent No. 8,546,140 U.S. Patent No. 8,741,648 U.S. Patent and Trademark Publication No. 2003 / 0211603 U.S. Patent and Trademark Publication No. 2010 / 0003757 Wong et al. The Lancet Global Health. 2(2):e106-e1, 2014. Yu et al.,Science,318:1917-1920,2007. Zhao et al. Development.144(8):1368-81,2017. Zhou et al. Development.142(19):3294-306,2015.

Claims

1. A tissue replacement graft comprising photoreceptor progenitor cells (PRP) and / or photoreceptor cells (PR) combined with retinal pigment epithelial cells (RPE) on a biodegradable scaffold, wherein the RPE and PR and / or PRP (RPE-PR / PRP) form a bilayer.

2. The tissue replacement graft according to claim 1, wherein the graft is defined as heterogeneous component-free and feeder-free; and / or the RPE is a mature RPE expressing bethrophine-1 (BEST1) and / or ZO-1.

3. The tissue replacement graft according to claim 1, wherein PR and / or PRP are attached to RPE in a bilayer by intercellular contact or attachment to a shared matrix.

4. The tissue replacement graft according to claim 1, wherein the biodegradable scaffold comprises poly(lactic acid-co-glycolic acid) (PLGA), polylactic acid (PLLA), polycaprolactone (PCL), poly(glycerol sebacate) (PGS), polypyrrole (PPy), polyvinyl alcohol (PVA), gelatin, collagen, laminin, fibronectin, fibrin, hyaluronic acid, silk, chitosan, and / or polyhydroxyethyl methacrylate (PHEMA).

5. i) The biodegradable scaffold includes PLGA, and the PLGA has a DL-lactide / glycolide ratio of approximately 1:

1. ii) The biodegradable scaffold includes PLGA having an average pore size of less than 1 micron, or iii) The biodegradable scaffold includes PLGA having a fiber diameter of 150 to 650 nm, The tissue replacement graft according to claim 1.

6. i) The biodegradable scaffold is coated with ECM protein, ii) The biodegradable scaffold is coated with vitronectin, laminin, collagen I, collagen IV, or fibronectin. iii) The biodegradable scaffold contains approximately 0.5 μg / cm³ ２ It is coated with vitronectin added to the surface at a concentration exceeding that, or iv) The biodegradable scaffold contains approximately 10 μg / cm³ ２ It is coated with vitronectin added to the surface at the following concentration. The tissue replacement graft according to claim 1.

7. The biodegradable scaffold has a thickness of 20 to 30 microns, as described in any one of claims 1 to 6.

8. The tissue replacement graft according to any one of claims 1 to 7, wherein the PR and / or PRP and RPE are present in a ratio of 2:1 to 30:1 or 1:1 to 5:

1.

9. i) RPE and / or PR and / or PRP are derived from pluripotent stem cells (PSCs). ii) RPE and / or PR and / or PRP are derived from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs), or iii) RPE and / or PR and / or PRP are derived from human, universal, HLA-matched, or hypoimmune iPSCs. A tissue replacement graft according to any one of claims 1 to 8.

10. i) The RPE and / or PR and / or PRP have been frozen and stored in advance, ii) The RPE and / or PR and / or PRP have been previously frozen and thawed, and cultured for at least one week or less than one week. A tissue replacement graft according to any one of claims 1 to 9.

11. The RPE has a density of 100,000 cells / cm². ２ ~1,000,000 cells / cm² ２ They exist at a density of 300,000 cells / cm³; the RPE is 300,000 cells / cm³. ２ ~800,000 cells / cm² ２ They exist at a density of 3 million cells / cm²; and / or the PR and / or PRP are 3 million cells / cm². ２ ~7 million cells / cm² ２ A tissue replacement graft according to any one of claims 1 to 10, which is present at a density of [value].

12. A pharmaceutical composition comprising a tissue replacement graft according to any one of claims 1 to 11.

13. i) further containing sodium hyaluronate, ii) Further containing sodium hyaluronate present at a concentration of less than approximately 0.5%, The pharmaceutical composition according to claim 12.

14. The pharmaceutical composition according to claim 12, further comprising sodium bicarbonate, calcium chloride, potassium chloride, monobasic potassium phosphate, magnesium chloride, magnesium sulfate, sodium chloride, and / or dibasic sodium phosphate.

15. A method for producing a tissue replacement graft according to any one of claims 1 to 11, (a) Sowing RPE on a biodegradable scaffold; (b) Culturing the RPE on the biodegradable scaffold in a first tissue culture medium for a period of time sufficient to generate polarized RPE; (c) Seeding PR and / or PRP on the RPE to form tissue replacement grafts; and (d) Culturing the tissue replacement graft in a second tissue culture medium for a period of time sufficient to allow PR and / or PRP to adhere to the RPE. A method that includes this.

16. i) The biodegradable scaffold is coated with extracellular matrix (ECM) proteins. ii) The biodegradable scaffold is coated with vitronectin, laminin, collagen I, collagen IV, or fibronectin. iii) The biodegradable scaffold is coated with vitronectin. iv) The aforementioned biodegradable scaffold has about 0.5 μg / cm ２ is coated with vitronectin added to the surface at a concentration greater than, or v) The biodegradable scaffold contains approximately 10 μg / cm³ ２ It is coated with vitronectin added to the surface at the following concentration. The method according to claim 15.

17. i) The RPE is divided into 100,000 cells / cm². ２ ~1,000,000 cells / cm² ２ Seed at a density of 300,000 cells / cm², and / or the RPE. ２ ~800,000 cells / cm² ２ Sow at this density, or ii) The PR and / or PRP contains 3 million cells / cm² ２ ~7 million cells / cm² ２ Sow at this density. The method according to claim 15.

18. A tissue replacement graft according to any one of claims 1 to 11, manufactured according to the method described in any one of claims 15 to 17.

19. RPE-PR and / or PRP bilayer cell composition comprising a bilayer of mature PRP cultured on polarized RPE.

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