Biodegradable tissue replacement implant and its use

Abstract

To provide a tissue replacement implant that is clinically effective for treatment of a variety of retinal diseases and retinal dysfunction, and can be produced using Good Manufacturing Practice (GMP) / clinical-grade manufacturing, and a method of treating a subject.SOLUTION: Tissue replacement implants are provided that include polarized retinal pigment epithelial cells on a poly(lactic-co-glycolic acid) (PLGA) scaffold. The PLGA scaffold is 20-30 microns in thickness, and has a DL-lactide / glycotide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 769,484, filed November 19, 2018, which is incorporated herein by reference.

[0002] Government Support Statement This invention was made with Government support under Project No. ZA1#: ET000542-03 awarded by the National Institutes of Health, National Eye Institute. The Government has certain rights in this invention.

[0003] Field of the Disclosure The present disclosure relates to the field of cell-based therapeutics, and in particular to tissue replacement implants comprising retinal pigment epithelial (RPE) cells, and methods of making and using the tissue replacement implants. [Background technology]

[0004] background Cell-based therapies hold promise for "permanent" replacement of degenerated tissues. The eye is an area of ​​interest due to its accessibility and the urgent need for effective therapies to help the growing elderly population experiencing vision loss (Fischbach, et al., Sci Transl. Med 5, 179ps177 (2013); Song and Bharti, Brain Res 1638, 2-14 (2016)). Previous success using surgical techniques to transplant autologous RPE / choroidal cells obtained from the peripheral part of the eye of the same patient provided the definitive proof of principle required to develop pluripotent stem cell-derived RPE-based cell therapy (Joussen et al., Ophthalmology 114, 551-560 (2007); van Zeeburg, et al., Am J Ophthalmol 153, 120-127.e122 (2012)). In recent preliminary clinical trials, embryonic stem cell (ESC)-based therapies and induced pluripotent stem cell (iPSC)-based therapies have been tested in patients with age-related macular degeneration (AMD), the leading cause of blindness among the elderly (Schwartz et al., Lancet 379, 713-720 (2012); Schwartz et al., Lancet 385, 509-516 (2015); Mandai et al., N Engl J Med 376, 1038-1046 (2017); Bharti et al., Pigment Cell Melanoma Res 24, 21-34 (2011); da Cruz et al., Nat Biotechnol 36, 328-337 (2018); Kashani et al., Sci Transl Med 10, (2018) (Non-patent Document 10)).AMD has two progression stages: "dry" AMD, or geographic atrophy, is caused by the death of the retinal pigment epithelium (RPE), a single layer of pigment-bearing cells located at the back of the eye; "wet" or choroidal neovascular AMD is caused by the proliferation of blood vessels in the choroid that penetrate the RPE and leakage of exudate and blood under the retina (Ambati and Fowler, Neuron 75, 26-39 (2012); Bird, et al., JAMA Ophthalmol 132, 338-345 (2014)). Either abnormality can result in the death of photoreceptor cells, causing severe vision loss, and resulting in blindness.

[0005] Several studies have explored subretinal delivery of stem cell-derived RPE cells in AMD patients: suspensions of ESC-derived RPE cells were tested in phase I clinical trials in patients with geographic atrophy ("dry") stage AMD (Joussen et al., Ophthalmology 114, 551-560 (2007); van Zeeburg, et al., Am J Ophthalmol 153, 120-127.e122 (2012)); autologous iPSC-RPE (iRPE) cells were recently transplanted into one patient with "wet" AMD (Schwartz et al., Lancet 385, 509-516 (2015); Mandai et al., N Engl J Med 376, 1038-1046 (2017)). In addition, ESC-derived RPE on a parenchymal scaffold was tested in four “dry” AMD patients, and the same cells on a polyester scaffold were tested in two “wet” AMD patients (da Cruz et al., Nat Biotechnol 36, 328-337 (2018); Kashani et al., Sci Transl. Med 10 (2018)). Although these studies demonstrated the safety of stem cell-based therapies, they also revealed the need for innovation to develop commercially approved, clinical stem cell therapies. For example, RPE cells in suspension do not self-organize into a confluent polarized monolayer, and they do not provide barrier function at the back of the patient's eye, affecting the long-term viability of the cells (Diniz et al., Investigative Ophthalmology & Visual Science 54, 5087-5096 (2013)). Previous approaches have not provided methods that have been functionally validated in multiple patients (Kamao et al., Stem Cell Reports 2, 205-218 (2014)).Provided herein are tissue replacement implants that are clinically effective for the treatment of various retinal diseases and retinal dysfunctions, and that can be made using Good Manufacturing Practice (GMP) / clinical grade manufacturing methods. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Fischbach, et al., Sci Transl. Med 5, 179ps177 (2013) [Non-Patent Document 2] Song and Bharti, Brain Res 1638, 2-14 (2016) [Non-Patent Document 3] Joussen et al., Ophthalmology 114, 551-560 (2007) [Non-Patent Document 4] van Zeeburg, et al., Am J Ophthalmol 153, 120-127.e122 (2012) [Non-Patent Document 5] Schwartz et al., Lancet 379, 713-720 (2012) [Non-Patent Document 6] Schwartz et al., Lancet 385, 509-516 (2015) [Non-Patent Document 7] Mandai et al., N Engl J Med 376, 1038-1046 (2017) [Non-Patent Document 8] Bharti et al., Pigment Cell Melanoma Res 24, 21-34 (2011) [Non-Patent Document 9] da Cruz et al., Nat Biotechnol 36, 328-337 (2018) [Non-Patent Document 10] Kashani et al., Sci Transl Med 10, (2018) [Non-Patent Document 11] Ambati and Fowler, Neuron 75, 26-39 (2012) [Non-Patent Document 12] Bird, et al., JAMA Ophthalmol 132, 338-345 (2014) [Non-Patent Document 13] Diniz et al., Investigative Ophthalmology & Visual Science 54, 5087-5096 (2013) [Non-Patent Document 14] Kamao et al., Stem Cell Reports 2, 205-218 (2014) Summary of the Invention

[0007] Summary of the Disclosure In some embodiments, a tissue replacement implant is disclosed that includes polarized retinal pigment epithelial cells on a poly(lactic-co-glycolic acid) (PLGA) scaffold, where the PLGA scaffold is 20-30 microns thick, the PLGA scaffold has a DL-lactide / glycolide ratio of about 1:1, an average pore size between adjacent fibers of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm.

[0008] In additional aspects, methods of treating a subject having a retinal degenerative disease, retinal or retinal pigment epithelial dysfunction, retinal deterioration, retinal damage, or retinal pigment epithelial loss are disclosed, comprising locally administering to the eye of the subject a disclosed tissue replacement implant.

[0009] In a further aspect, methods for making tissue replacement implants are disclosed. These methods include: a) obtaining a vitronectin coated PLGA, the PLGA scaffold comprising fibers forming a mesh structure, and the PLGA scaffold having an upper surface and a lower surface, the PLGA scaffold being about 20 to about 30 microns thick, the PLGA scaffold having a DL-lactide / glycolide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm; b) heat treating the PLGA scaffold such that the fibers of the scaffold fuse at the fiber intersections in the PLGA scaffold, increasing the mechanical strength of the PLGA scaffold and decreasing the pore size; c) seeding retinal pigment epithelial cells onto the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold; and d) on a PLGA scaffold in tissue culture medium, with medium present on both the top and bottom surfaces of the PLGA scaffold; i) Retinal pigment epithelial cell polarization, and ii) Bulk degradation of PLGA in the scaffold Culturing the retinal pigment epithelial cells in vitro for a period of time sufficient for the

[0010] [The present invention 1001] 1. A tissue replacement implant comprising polarized retinal pigment epithelial cells on a poly(lactic-co-glycolic acid) (PLGA) scaffold, The tissue replacement implant, wherein the PLGA scaffold is 20 to 30 microns thick, the PLGA scaffold having a DL-lactide / glycolide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm. [The present invention 1002] The tissue replacement implant of the present invention 1001, wherein the PLGA scaffold is coated with vitronectin. [The present invention 1003] The tissue replacement implant of invention 1001 or invention 1002, wherein the polarized retinal pigment epithelial cells are human. [The present invention 1004] The tissue replacement implant of any of claims 1001 to 1003, wherein the polarized retinal pigment epithelial cells are produced from induced pluripotent stem cells or ES cells. [The present invention 1005] The tissue replacement implant of any of claims 1001 to 1004, wherein the polarized retinal pigment epithelial cells are derived from a single subject. [The present invention 1006] The tissue replacement implant of the present invention 1004, wherein the polarized retinal pigment epithelial cells are generated from induced pluripotent stem cells, and the induced pluripotent stem cells are generated from CD34+ cells. [The present invention 1007] The tissue replacement implant of any of claims 1001-1006, wherein the PLGA scaffold has an average pore size of less than 1 micron or greater than 1 micron. [The present invention 1008] A method for treating a subject having a retinal degenerative disease, retinal or retinal pigment epithelial dysfunction, retinal deterioration, damage to the retina, physical injury to the retina, or loss of the retinal pigment epithelium, comprising the step of locally administering to the eye of the subject any of the tissue replacement implants of inventions 1001-1007, thereby treating the subject. [The present invention 1009] The method of the present invention 1008, wherein the retinal degenerative disease is Stargardt's macular dystrophy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, Leber's congenital amaurosis, late-onset retinal degeneration, hereditary macular degeneration or retinal degeneration, Best's disease, Sorsby's fundus dystrophy, retinal detachment, gyrate atrophy, eye trauma, or choroideremia, pattern dystrophy. [The present invention 1010] The method of claim 1008, wherein the damage to the retina or to the retinal pigment epithelium is caused by a laser, inflammation, infection, radiation, neovascularization, or trauma. [The present invention 1011] The method of any of claims 1008 to 1010, wherein the tissue replacement implant is introduced into the subretinal space of the eye, or into the outer retina, into the peripheral retina, into the macula or the area surrounding the macula, or into the choroid. [The present invention 1012] The method according to any one of claims 1008 to 1011, wherein the subject is a human. [The present invention 1013] The method of claim 1012, wherein the subject has age-related macular degeneration. [The present invention 1014] a) obtaining a vitronectin coated PLGA, the PLGA scaffold comprising fibers forming a mesh structure, and the PLGA scaffold having an upper surface and a lower surface, the PLGA scaffold being about 20 to about 30 microns thick, the PLGA scaffold having a DL-lactide / glycolide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm; b) heat treating the PLGA scaffold such that the fibers of the scaffold fuse at the fiber intersections in the PLGA scaffold, increasing the mechanical strength of the PLGA scaffold and decreasing the pore size; c) seeding retinal pigment epithelial cells onto the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold; and d) on a PLGA scaffold in tissue culture medium, with medium present on both the top and bottom surfaces of the PLGA scaffold; i) Retinal pigment epithelial cell polarization, and ii) Bulk degradation of PLGA scaffolds Culturing the retinal pigment epithelial cells in vitro for a period of time sufficient to thereby producing the tissue replacement implant. [The present invention 1015] The method of claim 1014, wherein the retinal pigment epithelial cells are human. [The present invention 1016] The method of any one of claims 1014 to 1015, wherein the retinal pigment epithelial cells are cultured in vitro on the PLGA scaffold until the peak release of lactic acid from the PLGA scaffold has ended. [The present invention 1017] The retinal pigment epithelium is 200 ohms*cm 2 The method according to any one of claims 1014 to 1016, wherein the subject has a trans-epithelial resistance (TER) exceeding that of the subject. [The present invention 1018] Any of the methods of claims 1014 to 1017, wherein step d) comprises culturing retinal pigment epithelial cells on the PLGA scaffold in tissue culture medium for about 3.5 to about 6 weeks. [The present invention 1019] The method of any of claims 1014-1018, wherein the PLGA scaffold is coated with vitronectin or other ECM proteins. [The present invention 1020] The method of any of claims 1014 to 1018, further comprising, prior to step c, a step of generating retinal pigment epithelial cells from the induced pluripotent stem cells or ES cells. [The present invention 1021] The method of claim 1020, wherein induced pluripotent stem cells are generated from the subject's CD34+ cells. [The present invention 1022] The method of any of claims 1014 to 1020, wherein the retinal pigment epithelial cells are derived from a single subject. [The present invention 1023] The method of any of claims 1014 to 1022, wherein the implant has an average pore size of less than 1 micron. [The present invention 1024] The tissue replacement implant of any of claims 1001-1007 or the method of any of claims 1008-1023, wherein the PLGA scaffold is sterilized using an electron beam (e-beam). The above and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief description of the drawings]

[0011] [Figure 1A] Figure 1A-1F: Clinical-grade, 2D, three-step iPSC-RPE differentiation protocol produces pure RPE cells. (A) Workflow illustrating the pipeline for producing and testing clinical-grade autologous iPSC-RPE patches for submission of an Investigational New Drug (IND) application to the FDA for approval to initiate Phase I clinical trials. [Figure 1B] Figure 1A-1F: Clinical-grade, 2D, three-step iPSC-RPE differentiation protocol produces pure RPE cells. (B) Timeline of clinical-grade iRPE differentiation. Clinical-grade iRPE differentiation takes 77 days, is initiated with monolayer iPSCs, and is performed using xeno-free reagents: Neuroectoderm Induction Medium (NEIM); RPE Induction Medium (RPEIM); RPE Commitment Medium (RPECM); RPE Proliferation Medium (RPEGM); RPE Maturation Medium (RPEMM). [Figure 1C] Figure 1A-1F: Clinical-grade, 2D three-step iPSC-RPE differentiation protocol produces pure RPE cells. (C) Sequencing of the coding regions of 223 cancer genes at a depth of 2000x was performed on all nine clinical-grade AMD iPSC clones. Kinship analysis of the sequences confirms identity between the iPSC clones and their respective donors, except for clone B of donor 2, which shows some changes in the sequence. [Figure 1D]Figure 1A-1F: Clinical grade, 2D three-stage iPSC-RPE differentiation protocol produces pure RPE cells. (D, E) Flow cytometry analysis of clinical grade iPSC-RPE from all three AMD patients performed at the RPE progenitor stage, day 17 (D17), RPE committed stage (D27), and immature RPE stage (D42) reveals that PAX6 / MITF double+ cells are abundant at the progenitor stage, which progressively decrease in number as the cells mature toward the RPE lineage (trend line). Consistent with this, MITF+ cells increase in number from the RPE progenitor stage to the immature RPE stage. Furthermore, RPE progenitor marker PMEL17+ cells and pigment-forming enzyme TYRP1+ cells are abundant at the RPE progenitor stage, while mature RPE markers CRABP+ cells and BEST1+ cells begin to arise at the RPE commitment stage and continue to increase towards the immature RPE stage (trend line) (n = 6). [Figure 1E] See legend to Figure 1D. [Figure 1F] Figure 1A-1F: A clinical-grade, 2D, three-step iPSC-RPE differentiation protocol produces pure RPE cells. (F) Progressive increase in RPE-specific gene expression from D5 to D42 of clinical-grade iPSC-RPE differentiation (n = 6). [Figure 2A] Figure 2A-2I: Biodegradable PLGA scaffolds are useful for creating functionally mature AMD iRPE patches. (A) Young's modulus of different PLGA scaffolds is determined to identify the optimal scaffold for implantation (**p < 0.001). [Figure 2B] Figures 2A-2I: Biodegradable PLGA scaffolds are useful for creating functionally mature AMD iRPE patches. (B) SEM confirms the surface topology of fused single-layer PLGA scaffolds. [Figure 2C]Figures 2A-2I: Biodegradable PLGA scaffolds are useful for generating functionally mature AMD iRPE patches. (C) Maturation of iRPE patches confirmed by immunostaining for: mature RPE marker RPE65 (cytoplasmic) and human specific antigen STEM121 (cytoplasmic) in the top panel; RPE pigment-forming protein GPNMB (cytoplasmic) and Bruch's membrane protein collagen IV (basal) in the middle panel; and Bruch's membrane marker collagen VIII (basal) in the bottom panel (n = 3). [Figure 2D] Figures 2A-2I: Biodegradable PLGA scaffolds are useful for generating functionally mature AMD iRPE patches. (D) TEM of iRPE patches on Transwell membranes or on PLGA scaffolds demonstrates similar maturation. Basal invaginations are only present in the case of PLGA scaffolds (inset) (n = 3). [Figure 2E] Figures 2A-2I: Biodegradable PLGA scaffolds are useful for generating functionally mature AMD iRPE patches. (E) ΔcT values ​​of RPE-specific genes are shown for all eight iRPE patches derived from three AMD donors (n = 8). [Figure 2F] Figure 2A-2I: Biodegradable PLGA scaffolds are useful for creating functionally mature AMD iRPE patches. (F) Live TER measurements during the last 3 weeks (D54-D77) of iRPE patch maturation show a progressive increase in patch electrical intactness and maturity. Representative data from three clones (3A, 3D, 4A) are shown (n = 8). [Figure 2G] Figures 2A-2I: Biodegradable PLGA scaffolds are useful for creating functionally mature AMD iRPE patches. (G) The graph shows that the percentage of phagocytosis was similar for most AMD-iRPE patches (n = 8). [Figure 2H]Figures 2A-2I: Biodegradable PLGA scaffolds are useful for generating functionally mature AMD iRPE patches. (H, I) Principal component analysis (PCA) combining data from all assays (morphometry, gene expression, TER, and phagocytosis) shows that most variation is between clones across PC1, except for sample D2B (H). PCA plotted without data for D2B revealed greater donor-to-donor variation compared to between-clone variation (I) (n = 8). [Figure 2I] See legend to Figure 2H. [Figure 3A] Figures 3A-3K: Clinical-grade AMD iRPE patches show improved integration compared to injected RPE cells in rodent models. (A-C) En face infrared images (A), OCT (B), and immunohistochemical analysis of human antigens (STEM121 - intracytoplasmic) (C) demonstrate that 0.5 mm diameter clinical-grade AMD-iRPE patches (light arrows; dark arrows point to rat RPE cells - see inset for higher magnification) were correctly positioned subretinal and fully integrated in the subretinal space of immunodeficient rats 10 weeks after surgery. (n = 20) [Figure 3B] See legend to Figure 3A. [Figure 3C] See legend to Figure 3A. [Figure 3D] Figures 3A-3K: In rodent models, clinical grade AMD iRPE patches show improved integration compared to injected RPE cells. (D) Immunohistochemical analysis for STEM121 - (arrow, see inset for higher magnification) confirms the presence of clinical grade AMD-iRPE cells injected into rat eyes. Rat RPE is not positive for STEM121 (arrow in inset indicates higher magnification). [Figure 3E]Figures 3A-3K: In rodent models, clinical grade AMD iRPE patches show improved integration compared to injected RPE cells. (E) The absence of Ki67 immunostaining demonstrates the lack of proliferation of injected human cells (light arrows, see inset for higher magnification; rat RPE is indicated by dark arrows). [Figure 3F] Figures 3A-3K: In rodent models, clinical grade AMD iRPE patches show improved integration compared to injected RPE cells. (F) STEM121 immunostaining (light arrow) also reveals integration of a small number of human cells (dark arrow; see inset for higher magnification) in rat RPE (n = 10). [Figure 3G] Figure 3A-3K: In rodent models, clinical grade AMD iRPE patches show improved incorporation compared to injected RPE cells. (G,I) Photomontages of RCS rat retinas show that the ONL is rescued (arrows) by transplanted iPSC-RPE patches (~2,500 cells on a 0.5 mm diameter patch) (G) or by iRPE cell suspensions (100,000 cells) (I) compared to the degenerated outer nuclear layer (ONL) in non-transplanted areas (arrows, n = 10). (H,J) Immunofluorescent staining (HuNu is human nuclear antigen - nuclear, human specific anti-PMEL17 - cytoplasmic) of retinas transplanted with iRPE patches (H) or with iRPE cell suspensions (J) with rescue of the ONL (light colored arrows). Note that the dark colored arrows in (H) point to iRPE cells that appear to have migrated out of the scaffold during transplantation. [Figure 3H] See legend to Figure 3G. [Figure 3I] See legend to Figure 3G. [Figure 3J] See legend to Figure 3G. [Figure 3K]Figures 3A-3K: In rodent models, clinical grade AMD iRPE patches show improved integration compared to injected RPE cells. (K) Optokinetic tracking threshold P90. Even using a 40-fold lower dose, iRPE patches perform similarly to cell suspensions at p90, suggesting superior efficacy of the iRPE patch (n = 10, *p < 0.05, **p < 0.001). [Figure 4-1] Figures 4A-4I: Development of an iRPE patch efficacy model by laser-induced RPE ablation in pigs. (A) Illustrated lesion formation in porcine RPE by micropulse laser; inset is fluorescein angiogram showing laser-induced breakdown of the outer blood-retinal barrier. (B, C) OCT reveals RPE detachment 24 hours after 1% duty cycle laser and RPE thinning (arrows) after 48 hours. (D) Heat map of P1 values ​​of visual streak area after 1% or 3% duty cycle laser (laser area is surrounded by dashed line). Scale bar of P1 values ​​is indicated. (E) Average mfERG waveforms from healthy area, 1% duty cycle laser area, and 3% duty cycle laser area. (F-I) Immunostaining for TUNEL (nuclear), RPE65 (cytoplasmic in RPE), and PNA (cytoplasmic in photoreceptors) at 48 hours (F,G) and H&E staining (H,I) reveal a damaged, detached, and apoptotic RPE monolayer (arrows) with some damage to the ONL layer. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 4-4] See description of Figure 4-1. [Figure 5-1]Figures 5A-5N: Clinical grade AMD iRPE patch is effective in a porcine model of laser-induced retinal degeneration. (A-C) OCT comparison of retinas on healthy areas, retinas implanted with empty PLGA scaffolds, or retinas implanted with clinical grade AMD iRPE patches (horizontal lines) shows incorporation of the iRPE patch and healthy retina on the patch compared to the empty scaffold where retinal tubulation can be observed. (D-F) Immunostaining for STEM 121 (intracytoplasmic - RPE, arrow, F) and RPE65 (intracytoplasmic - RPE) demonstrates incorporation of clinical grade AMD iRPE patches in porcine eyes. PNA staining (bright signal in photoreceptor outer segments) shows better preservation of photoreceptors in retinas implanted with iRPE patches (F) compared to retinas implanted with empty scaffolds (E, arrow points to retinal tubulation). (G) Immunostaining for (arrows pointing to cone opsin) demonstrates preservation of cone photoreceptors over the area where the human (STEM121, RPE cytoplasm) iRPE patch was implanted. (H,I) Rhodopsin immunostaining shows photoreceptor outer segments (POS) phagocytosed (arrows pointing to bright signal) by healthy porcine RPE immunostained with RPE65 (intracytoplasmic in RPE) and by human iRPE cells immunostained with STEM121 (intracytoplasmic in RPE). Z-sections show POS localized to the medial side of porcine and human RPE cells. (J-L) Heatmaps of N1P1 mfERG responses show weakened signal in the visual streak area after laser ablation of the RPE (compare J and K) and partial recovery of signal after implantation of a clinical grade iRPE patch (L). (M, N) Mean mfERG waveforms (M) and combined data (N) show significantly improved signal (p < 0.05) in clinical grade iRPE patches on PLGA (top line) compared to blank scaffolds (bottom line) over a 10-week follow-up. n = 7. [Figure 5-2]See description of Figure 5-1. [Figure 5-3] See description of Figure 5-1. [Figure 5-4] See description of Figure 5-1. [Figure 6-1] Figures 6A-6N: iRPE patches show better incorporation and better retinal recovery compared to iRPE cell suspensions in a porcine model of laser-induced retinal degeneration. (A-H) OCT shows improved retinal health in PLGA iRPE patches and Transwell iRPE patches compared to blank PLGA scaffolds and iRPE cell suspensions in pigs with laser-damaged RPE at 2 and 5 weeks after implantation (horizontal line and arrows point to implants). (I-L) Immunostaining for the human antigens STEM121 (an intracytoplasmic signal in RPE) and RPE65 (an intracytoplasmic signal in RPE) demonstrates incorporation of the human iRPE patch in the pig eye (horizontal line and light arrow in J) with an organized outer nuclear layer (PNA white, dark arrow, J) in healthy and iRPE patch-grafted areas compared to minimal PNA staining in empty scaffold areas and cell suspension implants, suggesting recovery of photoreceptors overlying the iRPE patch. n = 3. (M, N) Individual and average mfERG responses show improved retinal health in Transwell iRPE and PLGA iRPE patches compared to empty scaffolds and iRPE cell injections (n ​​= 3; *p < 0.05). [Figure 6-2] See description of Figure 6-1. [Figure 6-3] See description of Figure 6-1. [Figure 6-4] See description of Figure 6-1. [Figure 6-5] See description of Figure 6-1. [Figure 7-1]Figure 7A-7O: (A) Timeline for research grade iPSC-RPE differentiation. Differentiation into mature RPE takes 107 days and is initiated with 3D iPSC aggregates in neuroectoderm induction medium (NEIM). (B, C) Removal of FGF2 from RPE induction medium (RPEIM) doubles the number of GFP-positive RPE cells as measured using a reporter iPSC line expressing GFP under the control of the tyrosinase gene promoter42. (D) Inhibition of MEK with PD0325901 in RPEIM reduced the variability of iPSC differentiation into GFP-positive RPE progenitor cells. (E) Weak SMAD dual inhibition with low amounts of NOGGIN (50 ng / ml) in RPEIM combined with Activin A (100 ng / ml) in RPE commitment medium (RPECM) further increases the number of GFP-positive RPE progenitor cells. (F) As recently shown in 15, Activin A and WNT3a in RPECM do not show any synergistic increase in the number of GFP-positive RPE progenitors (n = 3). (G) Research-grade, 3D iPSC aggregate-based differentiation protocol reproducibly produces RPE from multiple healthy and patient iPSC lines (fibroblast- or blood-derived) (n = 4). (H, I) Flow cytometric analysis of PAX6- and MITF-positive RPE cells at D42 of the research-grade differentiation protocol. (J) Confirmation of epithelial phenotype in cells at day 12 of the clinical-grade differentiation protocol. (K, L) Flow cytometric analysis of OCT4 and TRA1-81 at D42 of the clinical-grade iRPE differentiation protocol confirms the absence of iPSCs. (M-O) Three additional users (data from the first user in Figure 1D) reproduced the clinical-grade iRPE differentiation protocol as shown by flow cytometry analysis of PAX6 and MITF double positive cells at D17. [Figure 7-2] See description of Figure 7-1. [Figure 7-3] See description of Figure 7-1. [Figure 7-4] See description of Figure 7-1. [Figure 7-5] See description of Figure 7-1. [Figure 7-6] See description of Figure 7-1. [Figure 7-7] See description of Figure 7-1. [Figure 7-8] See description of Figure 7-1. [Figure 7-9] See description of Figure 7-1. [Figure 8-1] Figures 8A-8H: (A-H) SEM images of PLGA scaffolds from the side (A-D) and front (E-H) during degradation showing changes in surface topology and overall thinning of the scaffold due to bulk degradation of the PLGA fibers (n = 3). [Figure 8-2] See description of Figure 8-1. [Figure 9-1]Figure 9A-9J: (A,B) SEM demonstrates the presence of comparable apical processes in PLGA and Transwell iRPE patches (n = 3). (C,D) Electrophysiological tracing demonstrates similar electrical properties of PLGA and Transwell iRPE patches (n = 3). (E) Comparison of fold changes in RPE-specific genes between PLGA and Transwell iRPE patches compared to iPSCs showed similar levels of monolayer maturation on the two substrates. Note, however, that separate biological replicates show less variation on PLGA scaffolds compared to Transwell (n = 3). (F,G) Brightfield and ZO-1 immunostained images of iRPE derived from iPSC clone C (D4C) of AMD donor 4. (H) ZO-1 immunostained images are segmented using a convolutional neural network to highlight cell boundaries. Segmented images were used for morphometric analysis (Schaub et al., manuscript in preparation). (I) Morphometric analysis performed with REShAPE software on ZO-1 stained images of iRPE patches from all three AMD donors. 75,000-100,000 cells per patch are imaged, and images are analyzed to determine the hexagonality score (how hexagonal the cell is) of each RPE cell. Data are shown as violin plots with a central dot representing the mean and a horizontal line in each plot representing the 99% data points. RPE patches from different clones are indicated by different dots (n = 8). (J) Graph shows polarized VEGF secretion (basal / apical ratio) for different AMD-iRPE patches (n = 8). [Figure 9-2] See description of Figure 9-1. [Figure 9-3] See description of Figure 9-1. [Figure 9-4] See description of Figure 9-1. [Figure 9-5] See description of Figure 9-1. [Figure 9-6] See description of Figure 9-1. [Figure 9-7] See description of Figure 9-1. [Figure 10A] (A, B) In vitro spike-in studies were performed by seeding mixed cultures of iPSCs and RPE on scaffolds (100% iPSCs; 10% iPSCs + 90% RPE; 1% iPSCs + 99% RPE; and 100% RPE). iPSC markers (OCT4 and TRA 1-81) were assessed by flow cytometry 0, 2, 7, and 14 days after seeding (A). Gene expression analysis of iPSCs (ZFP42, OCT4, NANOG, LIN28A, LEFTY1, DNMT38) and lineage-specific markers (VWF, S100A4, KDR for mesoderm; GATA6, FOXA2, AFP for endoderm; SOX10, MAP2, GFAP for non-RPE ectoderm) assessed 0, 7, and 14 days after seeding of mixed cultures of iPSCs and RPE. Data are shown as a heatmap of relative ΔcT values. All values ​​are normalized to day 0 data (B). This experiment demonstrates that PLGA scaffolds and RPE maturation medium do not support iPSC proliferation or non-RPE lineage proliferation. Also, non-RPE lineage genes are not expressed in iRPE cells. [Figure 10B] See legend to Figure 10A. [Figure 11A] (A) Schematic of implantation of a 0.5 mm diameter iRPE patch in the subretinal space of a rat. The surgery begins with a 1.2 mm sclerotomy, followed by vitreous displacement with hyaluronic acid (HA), retinal detachment with HA injection into the subretinal space, loading of the iRPE patch onto the implantation device, delivery of the patch to the subretinal space, and flattening of the retinal detachment with hyaluronic acid. [Figure 11B] (B) Visualization of an iRPE patch successfully transplanted into the subretinal space of a rat eye. [Figure 11C] (C) Optical coherence tomography images demonstrating successful delivery of an iRPE patch implanted into the subretinal space of a rat eye. [Figure 11D] (D-F) Immunohistochemical analysis for human-specific PMEL17 (arrow, D) confirms the RPE phenotype of injected AMD iRPE cell suspensions in the subretinal space 10 weeks after injection. In contrast, injected human iPSC suspensions (STEM121, E) lead to teratoma formation (F). n = 10 [Figure 11E] See legend to Figure 11D. [Figure 11F] See legend to Figure 11D. [Figure 12A] (A) Heatmap showing the average mfERG electrical waveform response of a porcine eye before laser ablation of the RPE was performed in the visual streak (dotted line), the area of ​​highest electrical activity in the porcine eye. [Figure 12B] (B, C) OCT of the laser at 3% duty cycle 24 and 48 hours after laser shows damage to the RPE and ONL, as well as subretinal edema (arrows). [Figure 12C] See legend to Figure 12B. [Figure 12D] (D) A 3% duty cycle laser area stained with TUNEL (nuclear signal) shows some apoptotic cells in the ONL at 24 hours. The line indicates the RPE monolayer. [Figure 12E] (E) At 48 hours, both the ONL and RPE appeared to be heavily damaged by the 3% duty cycle laser, with some apoptotic cells (nuclear signal) and weak staining for PNA (cytoplasmic signal in photoreceptor outer segments) and RPE65 (cytoplasmic signal in the RPE). [Figure 12F] (F, G) H&E staining after 48 hours with 1% (F) and 3% (G) laser duty cycles. Healthy areas are seen in the left portion at 1% duty cycle (F, arrow). Note the significant destruction of photoreceptor cells in the 3% laser compared to the 1% laser (arrow). [Figure 12G] See legend to Figure 12F. [Figure 13-1] FIG. 13A-13M: (A,B) Images of the implantation tool and the tool tip loaded with an empty scaffold (arrow). (C-F) Schematic of the porcine surgery. After a standard 4-port vitrectomy, a 38G blunt tip cannula is used to detach the retina (C), followed by retinotomy (D), extension of the sclerotomy (E), and delivery of a human RPE patch to the subretinal space (F). (G-I) Intraoperative fundus photography and intraoperative optical coherence tomography (iOCT) performed during surgery demonstrate delivery of the 4 x 2 mm scaffold in its intended location under the retina. (J,K) OCT of porcine eyes 2 weeks (J) and 10 weeks (K) after surgery demonstrate that the empty scaffold degrades and that inflammation is not caused by the by-products of the degrading PLGA in non-immunosuppressed pigs. (L) Post-surgery confirmation that empty scaffolds degrade and that inflammation is not caused by by-products of degrading PLGA in non-immunosuppressed pigs. (M) N1P1 mfERG signal on empty scaffolds shows a significant decrease by week 3, likely caused by the surgical procedure. Signal recovers over time as surgical damage is repaired and the scaffold degrades (2-way ANOVA, p < 0.0001) n = 3. [Figure 13-2] See description of Figure 13-1. [Figure 13-3] See description of Figure 13-1. [Figure 13-4] See description of Figure 13-1. [Figure 13-5] See description of Figure 13-1. [Figure 13-6] See description of Figure 13-1. [Figure 14-1]14A-14I: (A) Human iRPE patch expressing GFP. (B,C) Fundus autofluorescence images showing a subretinal GFP-positive 4 x 2 mm iRPE patch 2 weeks (white arrow) and 10 weeks after transplantation (white arrow indicates iRPE patch and grey arrow indicates laser area). Note that human cells do not migrate out of the patch. (D-F) Images of areas of pig retina transplanted with human iRPE patch stained for photoreceptors (PNA in white, D); and immunostained for human iRPE cells (STEM121, intracytoplasmic, E) and RPE (RPE65, intracytoplasmic, F). (G) Quantification of the number of nuclei in the outer nuclear layer (ONL) of pig retina above areas transplanted with iRPE patch compared to areas transplanted with empty scaffold. Numbers are expressed as a ratio to healthy retina. (H) Immunostaining for human nuclear antigens STEM101 (nucleus) and RPE65 (in the cytoplasm) demonstrates the integration of human iRPE patches in pig eyes. (I) 3D reconstruction of a section immunostained for rhodopsin (photoreceptor outer segments) and STEM121 (in the RPE cytoplasm) (arrows) of a human iRPE patch transplanted into a pig eye shows the photoreceptor outer segments (POS) inside the human RPE cells. [Figure 14-2] See description of Figure 14-1. [Figure 14-3] See description of Figure 14-1. [Figure 14-4] See description of Figure 14-1. [Figure 14-5] See description of Figure 14-1. [Figure 15-1]Figures 15A-15L: (A-I) Fluorescein angiography (A, D, G) confirm micropulse laser-based RPE injury in porcine eyes used for implantation of empty scaffolds (A), Transwell iRPE patches (D), and iRPE cell injections (G). Intraoperative fundus photography (B, E, H) and intraoperative OCT (C, F, I) confirm accurate delivery (white arrows) of the empty scaffolds (B, C), Transwell iRPE patches (E, F), and iRPE cell injections (H, I). (J-L) Images of an area of ​​pig retina where a human iRPE patch was grafted, stained for photoreceptors (PNA, intracytoplasmic in photoreceptor outer segments, J); and immunostained for human iRPE cells (STEM121, intracytoplasmic in RPE, K) and RPE (RPE65, intracytoplasmic in RPE, L). Note that STEM121 labeling for human cells (see underline in K) stops where the pig RPE begins. [Figure 15-2] See description of Figure 15-1. [Figure 15-3] See description of Figure 15-1. [Figure 15-4] See description of Figure 15-1. [Figure 16] CRISPR-mediated gene correction in albinism iPSCs. SEQ ID NO: 1 where X is C or T is shown in the top panel, and SEQ ID NO: 1 where X is C is shown in the bottom panel. [Figure 17] RPE patches derived from patient cells and from CRISPR-corrected iPSCs. [Figure 18] Histology of OCA2 patient RPE cells and CRISR-corrected OCA2 RPE cells. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Sequence Listing The nucleic acid and amino acid sequences listed in the attached sequence listing are shown using standard abbreviations for nucleotide bases and three-letter code for amino acids as specified in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but any reference to the strand shown is understood to include the complementary strand. The sequence listing is submitted as an ASCII text file [Sequence_Listing, November 11, 2019, 709 bytes], which is incorporated herein by reference. In the attached sequence listing: SEQ ID NO:1 is the nucleic acid sequence of a portion of the OCA2 gene.

[0013] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS The retina is a layer of specialized nervous tissue that is sensitive to light and is located on the inner surface of the vertebrate eye. Light that reaches the retina after passing through the cornea, lens, and vitreous humor is converted into chemical and electrical events that trigger nerve impulses. The cells responsible for transduction, the process for converting light into these biological processes, are specialized neurons called photoreceptors.

[0014] The retinal pigment epithelium (RPE) is a polarized monolayer of densely packed hexagonal cells in mammalian eyes, which separates the neural retina from the choroid. The cells in the RPE contain pigment granules, and play an important role in the physiology of the retina by forming the blood-retinal barrier and by closely interacting with photoreceptor cells to maintain visual function by absorbing the energy of light focused by the lens on the retina. These cells also transport ions, water, and metabolic end products from the subretinal space to the blood, and take up nutrients from the blood, such as glucose, retinol, and fatty acids, and deliver these nutrients to photoreceptor cells.

[0015] RPE cells are also part of the retinal visual cycle: all-trans-retinal is formed after absorption of a photon and reverts to 11-cis-retinal, but because photoreceptor cells cannot reisomerize all-trans-retinal, retinal is transported to the RPE, where it is reisomerized to 11-cis-retinal and then sent back to the photoreceptor cells.

[0016] Many ophthalmic diseases, such as (age-related) macular degeneration, macular dystrophies such as Stargardt's-like disease, Best's disease (vitelliform macular dystrophy), and adult vitelliform dystrophy, or subtypes of retinitis pigmentosa, are associated with degeneration or deterioration of the retina itself, or of the RPE. It has been demonstrated in animal models that photoreceptor rescue and preservation of visual function can be achieved by subretinal transplantation of RPE cells (Coffey et al. Nat. Neurosci. 2002:5, 53-56; Lin et al. Curr. Eye Res. 1996:15, 1069-1077; Little et al. Invest. Ophthalmol. Vis. Sci. 1996:37, 204-211; Sauve et al. Neuroscience 2002:114, 389-401). In addition, there is a need for methods and implants that can be used to treat physical damage to the retina.

[0017] term Unless otherwise stated, technical terms are used according to normal usage. Definitions of common terms in molecular biology can be found in: Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd, 1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8).

[0018] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided:

[0019] Activin: A member of the transforming growth factor beta (TGF-β) superfamily involved in the regulation of several biological processes, including cell differentiation and proliferation. Activin A is a member of this family and exerts its biological effects through a transmembrane receptor serine / threonine kinase complex and binds to a specific Activin A receptor. It is a dimer composed of two subunits. Activin A is involved in the regulation of stem cell maintenance through SMAD-dependent activation of the transcription of markers of pluripotency such as POU class 5 homeobox 1 (Oct-3 / 4), nanog, nodal, and the regulators of nodal signaling, left-right determinants 1 and 2 (Lefty B and Lefty A). Activin A also stimulates the transcription of several hormones, such as gonadotropin-releasing hormone. An exemplary sequence of Activin A is provided as GENBANK® Accession No. NM_002192.

[0020] Agent: Any protein, nucleic acid molecule (including chemically modified nucleic acids), compound, small molecule, organic compound, inorganic compound, or other molecule of interest. Agents can include therapeutic agents, diagnostic agents, or drugs. A therapeutic agent or drug is one that, alone or together with additional compounds, induces a desired response (e.g., induces a therapeutic or prophylactic effect when administered to a subject).

[0021] Agonist or inducer: An agent that binds to a cell's receptor or to a ligand of such a receptor and causes a response by the cell, often mimicking the action of a naturally occurring substance. In one embodiment, Frizzled (Fzd) agonists bind to Fzd receptors and enhance or potentiate the Wnt / β-catenin signaling pathway.

[0022] Change: A change in the effective amount of a substance of interest or in a parameter of interest, such as, for example, in a polynucleotide, polypeptide, or cell characteristic. A change in a polypeptide or polynucleotide, or enzyme activity, can affect the physiological characteristics of a cell, such as, for example, cell differentiation, proliferation, or aging. The amount of a substance can be changed by a difference in the amount of the substance produced, by a difference in the amount of a substance with a desired function, or by a difference in the activity of the substance. The change can be an increase or decrease. The change can be in vivo or in vitro. In some embodiments, the change is an increase or decrease of at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% in the effective amount (level) of a substance, in the proliferation and / or viability of a cell, or in the activity of a protein, such as an enzyme.

[0023] Animal: A living multi-cellular vertebrate organism, a category that includes, by way of example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term "subject" includes both human and veterinary subjects, such as non-human primates, dogs, cats, horses, rabbits, pigs, mice, rats, and cows.

[0024] Antagonist or inhibitor: An agent that blocks or attenuates a biochemical or biological response when it binds to a receptor or to a ligand of the receptor. Antagonists exert their effect through interaction with the receptor by blocking agonist-induced responses. In one embodiment, Frizzled (Fzd) antagonists bind to Fzd receptors or to Fzd ligands (such as Wnt) and inhibit the Wnt / β-catenin signaling pathway.

[0025] Antibody: A polypeptide ligand that contains at least one light or heavy immunoglobulin chain variable region and that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy and light chains, each of which contains a heavy chain variable (V H ) region and the light chain variable (V L ) region. H Area and V L Both regions are responsible for binding to the antigen recognized by the antibody.

[0026] Antibodies include intact immunoglobulins, as well as antibody variants and portions known in the art, such as Fab fragments, Fab' fragments, F(ab)'2 fragments, single chain Fv proteins ("scFv"), and disulfide stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light chain variable region of an immunoglobulin and the heavy chain variable region of an immunoglobulin are linked by a linker, while in dsFv, the chains are mutated to introduce disulfide bonds that stabilize the association of the chains. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies), heteroconjugate antibodies (e.g., bispecific antibodies, etc.). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd Ed., WH Freeman & Co., New York, 1997.

[0027] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. There are two types of light chains, lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes), which determine the functional activity of the antibody molecule: IgM, IgD, IgG, IgA, and IgE.

[0028] CD34: A protein that is a transmembrane phosphorylated glycoprotein encoded by the CD34 gene in humans and other mammalian species. CD34 was first described on hematopoietic stem cells. Cells expressing CD34 (CD34 + CD34+ cells are normally found in the umbilical cord and bone marrow as hematopoietic cells, or among other cells, in mesenchymal stem cells and endothelial progenitor cells. CD34+ cells can be isolated from blood samples using immunomagnetic or immunofluorescence techniques.

[0029] Cell: A structural and functional unit of an organism that can replicate independently, is surrounded by a membrane, and contains biomolecules and genetic material. As used herein, a cell may be a naturally occurring cell or an artificially modified cell (e.g., a fusion cell, a genetically modified cell, etc.).

[0030] The term "cell population" refers to a group of cells, typically of the same type. A cell population may be derived from a common progenitor cell or may contain more than one cell type. An "enriched" cell population refers to a cell population derived from a starting cell population (such as an unfractionated heterogeneous cell population) that contains a percentage of a particular cell type that is greater than the percentage of that cell type in the starting population. A cell population may be enriched for one or more cell types and depleted for one or more cell types.

[0031] The term "stem cell" refers to a cell that can differentiate under appropriate conditions into various specialized cell types, while under other appropriate conditions it can self-renew and remain in an essentially undifferentiated 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 stem cells or mesenchymal stem cells obtained from bone marrow tissue, from embryonic stem cells obtained from embryonic tissue, or from embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be generated from somatic cells by reprogramming the somatic cells into a pluripotent state by expressing several transcription factors associated with pluripotency; these cells are called "induced pluripotent stem cells" or "iPSCs".

[0032] Differentiation: Refers to the process by which a relatively unspecialized cell (such as an embryonic or other stem cell) acquires specialized structural and / or functional characteristics characteristic of a mature cell. Similarly, "differentiate" refers to this process. Typically, during differentiation, the structure of the cell changes and tissue-specific proteins appear.

[0033] Defined or "fully defined": refers to a medium, extracellular matrix, or culture environment in which the chemical composition and amount of nearly 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. Typically, a defined medium includes a basal medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, which includes amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy sources) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An exemplary fully defined medium is ESSENTIAL 8™ medium.

[0034] Embryoid bodies: three-dimensional aggregates of pluripotent stem cells. These cells can differentiate into endodermal, mesodermal, and ectodermal cells. In contrast to monolayer culture, the spheroid structures formed upon aggregation of pluripotent stem cells allow non-adherent culture of EBs in suspension, which is useful for bioprocessing approaches. The three-dimensional structures include the establishment of complex cell adhesion and paracrine signaling within the EB microenvironment, allowing differentiation and morphogenesis.

[0035] Embryo: A mass of cells resulting from one or more divisions of a zygote, whether or not it implants in a female, or from one or more divisions of an activated oocyte with an artificially reprogrammed nucleus. "Morula" refers to a preimplantation embryo 3-4 days after fertilization, which when it is a solid mass is generally composed of 12-32 cells (blastomeres). "Blastocyst" refers to a preimplantation embryo in placental mammals (about 3 days after fertilization in mice and about 5 days after fertilization in humans) of about 30-150 cells. The blastocyst stage follows the morula stage and can be distinguished by its unique morphology. Blastocysts are generally spheres composed of a layer of cells (trophectoderm), a fluid-filled cavity (blastocoel or blastocyst cavity), and an inner cluster of cells (inner cell mass, ICM). The ICM is composed of undifferentiated cells that give rise to what will become a fetus when the blastocyst implants in the uterus.

[0036] Embryonic stem cell: An embryonic cell derived from the inner cell mass of a blastocyst or morula, optionally serially passaged as a cell line. The term includes cells isolated from one or more blastomeres of an embryo, preferably without destroying other parts of the embryo. The term also includes cells produced by somatic cell nuclear transfer. "Human embryonic stem cell" (hES cell) includes embryonic cells derived from the inner cell mass of a human blastocyst or morula, optionally serially passaged as a cell line. hES cells can be derived from fertilization of eggs with sperm or DNA, nuclear transfer, parthenogenesis, or by means of producing hES cells with homozygosity in the HLA region. Human ES cells may be generated or derived from mammalian embryos at the zygote, blastomere, or blastocyst stage, produced by the fusion of sperm and egg, by nuclear transfer, by parthenogenesis, or by reprogramming of chromatin and subsequent integration of the reprogrammed chromatin into the cell membrane to generate embryonic cells. Human embryonic stem cells include, but are not limited to, embryonic stem cells MAO1, MAO9, ACT-4, No. 3, H1, H7, H9, H14, and ACT30. Human embryonic stem cells, regardless of their source or the particular method used to generate them, may be identified based on the following: (i) the ability to differentiate into cells of all three germ layers; (ii) expression of at least Oct-4 and alkaline phosphatase; and (iii) the ability to generate teratomas when transplanted into immunodeficient animals.

[0037] Expand: A process in which the number or amount of cells in a cell culture increases due to cell division. Similarly, the terms "expansion" or "expanded" also refer to this process. The terms "proliferate", "proliferation", and "expanded" may be used interchangeably with the words "expand", "expand", or "expanded". Typically, during the expansion phase, cells do not differentiate to form mature cells, but divide to form additional cells.

[0038] Expression: The process by which a gene's coded information is converted into active, inactive, or structural parts of a cell, such as the synthesis of a protein. Gene expression can be influenced by external signals. For example, exposing a cell to a hormone can stimulate hormone-induced gene expression. Different types of cells can respond differently to the same signal. Gene expression can also be regulated anywhere along the pathway from DNA to RNA to protein. Regulation can include control in transcription, in translation, in the transport and processing of RNA, in the degradation of intermediate molecules such as mRNA, or by the control of specific protein molecules after they are produced, by activation, inactivation, compartmentalization, or degradation.

[0039] Feeder layer: non-proliferating cells (e.g., irradiated cells) that can be used to support the proliferation of stem cells. Protocols for making feeder layers are known in the art and available on the Internet, such as, for example, at the National Stem Cell Resource website maintained by the American Type Culture Collection (ATCC). "Feeder-free" or "feeder-independent" is used herein to refer to cultures to which cytokines and growth factors (e.g., TGFβ, bFGF, LIF) are added as an alternative to a feeder cell layer. Thus, "feeder-free" or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferative state. 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 allow human stem cells to remain essentially undifferentiated without the need for a "feeder layer" of mouse fibroblasts.

[0040] Fetal: A developing mammal in the pre-natal, embryonic stage. In humans, pre-natal development, the fetal stage, begins at the beginning of the 9th week after fertilization. In humans, the eye and RPE are already formed by the 4th week after conception. The RPE continues to mature over the following weeks.

[0041] Fibroblast growth factor (FGF): Any suitable fibroblast growth factor from any animal, and functional fragments thereof, such as fragments that bind to a receptor and induce biological effects associated with activation of the receptor. A variety of FGFs are known, and include, but are not limited to, FGF-1 (acidic fibroblast growth factor), FGF-2 (basic fibroblast growth factor, bFGF), FGF-3 (int-2), FGF-4 (hst / K-FGF), FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, and FGF-98. "FGF" refers to fibroblast growth factor proteins, such as, for example, FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, FGF-9, or FGF-98, or biologically active fragments thereof or variants thereof. FGFs can be from any animal species. In one embodiment, the FGF is a mammalian FGF, including but not limited to rodent, avian, canine, bovine, porcine, equine, and human.The amino acid sequences of many FGFs and the methods for making them are well known in the art.

[0042] The amino acid sequence of human bFGF and methods for its recombinant expression are disclosed in U.S. Patent No. 5,439,818, which is incorporated herein by reference. The amino acid sequence of bovine bFGF and various methods for its recombinant expression are disclosed in U.S. Patent No. 5,155,214, which is also incorporated herein by reference. When compared in 146-residue form, their amino acid sequences are nearly identical, differing at only two residues. Recombinant bFGF-2, and other FGFs, can be purified to pharmaceutical quality (98% or higher purity) using techniques detailed in U.S. Patent No. 4,956,455.

[0043] FGF inducers include active fragments of FGF. In the simplest form, active fragments are generated by removing N-terminal methionine using well-known techniques for removing N-terminal methionine, such as treatment with methionine aminopeptidase. A second desirable truncation includes FGF without its leader sequence. Those skilled in the art understand leader sequence as a series of hydrophobic residues at the N-terminus of a protein that facilitates the protein's passage through the cell membrane but is not necessary for activity and is not found in the mature protein. Human and mouse bFGF are commercially available.

[0044] Frizzled (Fzd): A family of seven-transmembrane mammalian proteins that have characteristics of G protein-coupled receptors and bind to Wnt family lipoglycoproteins, secreted Frizzled-related proteins (sFRPs), R-spondin, and Norrin proteins, and activate downstream signaling. Frizzled proteins (also called frizzled receptors) contain a cysteine-rich domain (CRD) that binds to its cognate ligand, a carboxy-terminal PDZ (homologous to Psd-95 / disc large / ZO-1) binding domain, and various consensus sites for serine / threonine and tyrosine kinases. Amino acid hydropathy analysis shows that frizzled proteins contain one extracellular amino terminus, three extracellular protein loops, three intracellular protein loops, and one intracellular carboxy terminus.

[0045] Frizzled proteins have important regulatory roles during embryonic development and have also been linked to several diseases in humans and animal models, including various cancers, cardiac hypertrophy, familial exudative vitreoretinopathy, and schizophrenia.

[0046] There are at least ten mammalian frizzled proteins, and the genes encoding mammalian frizzled proteins are related to the frizzled gene in Drosophila. Human frizzled proteins include: frizzled 1 (Fzd1; GENBANK® Accession No. AB017363), frizzled 2 (Fzd2; GENBANK® Accession No. L37882 / NM_001466), Frizzled 3 (Fzd3; GENBANK® Accession No. AJ272427), Frizzled 4 (Fzd4; GENBANK® Accession No. AB032417), Frizzled 5 (Fzd5; GENBANK® Accession No. U43318), Frizzled 6 (Fzd6; GENBANK® Accession No. AB012911), Frizzled 7 (Fzd7; GENBANK® Accession No. AB010881), Frizzled 8 (Fzd8; GENBANK® Accession No. AB043703), Frizzled 9 (Fzd9; GENBANK® Accession No. U82169 / NM_003508), and Frizzled 10 (Fzd10; GENBANK® Accession No. AB027464). All of the GENBANK® entries available on January 1, 2013 are incorporated herein by reference.

[0047] Growth factor: a substance that promotes cell proliferation, survival, and / or differentiation. Growth factors include: molecules that function as growth stimulants (mitogens), factors that stimulate cell migration, factors that function as chemotactic agents or inhibit cell migration or tumor cell invasion, factors that regulate the differentiation function of cells, factors involved in apoptosis, or factors that promote cell survival without affecting proliferation and differentiation. Examples of growth factors are fibroblast growth factors (such as FGF-2), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), and nerve growth factor (NGF), and activin A.

[0048] Haplotype: A combination of alleles at multiple loci on a chromosome. A haplotype can be based on a set of single nucleotide polymorphisms (SNPs) on a chromosome and / or on alleles in the major histocompatibility complex. The term "haplotype-matched" refers to a common haplotype at one or more major histocompatibility loci between a cell (e.g., iPSC) and a subject to be treated. The haplotype of a subject can be easily determined using assays well known in the art. Haplotype-matched iPSCs can be autologous or allogeneic. Autologous cells grown in tissue culture and differentiated into RPE cells are naturally haplomatched to the subject. "Substantially the same HLA type" refers to the donor's HLA type matching that of the patient to the extent that the transplanted cells, which are obtained by inducing differentiation of iPSCs derived from the donor's somatic cells, can engraft when transplanted into the patient.

[0049] Host cell: A cell in which a vector can propagate and express the DNA of the vector. The cell may be a prokaryotic or eukaryotic cell. The term also includes any progeny of the subject host cell. It is understood that not all progeny may be identical to the parent cell, since there may be mutations that occur during replication. Nevertheless, when the term "host cell" is used, such progeny are also included.

[0050] Isolated: "isolated" biological components, such as nucleic acids, proteins, or organelles, which are substantially separated from or substantially purified from other biological components in the environment in which they naturally occur (e.g., cells), i.e., chromosomal DNA and RNA, extrachromosomal DNA and RNA, proteins, and organelles. "Isolated" nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids and proteins. Similarly, an "isolated cell" is substantially separated from, produced substantially separately from, or substantially purified from other cells of the organism in which it naturally occurs. An isolated cell can be, for example, at least 99%, at least 98%, at least 97%, at least 96%, 95%, at least 94%, at least 93%, at least 92%, or at least 90% pure.

[0051] Mammal: This term includes both human and non-human mammals. Examples of mammals include, but are not limited to, humans, and veterinary and laboratory animals, such as pigs, cows, goats, cats, dogs, rabbits, and mice.

[0052] Marker or label: an agent that can be detected, for example, by ELISA, spectrophotometry, flow cytometry, immunohistochemical analysis, immunofluorescence, microscopy, Northern analysis, or Southern analysis. For example, a marker can be attached to a nucleic acid molecule or protein, thereby allowing the detection of said nucleic acid molecule or protein. Examples of markers include, but are not limited to, radioisotopes, nitroimidazoles, enzyme substrates, cofactors, ligands, chemiluminescent substances, fluorophores, haptens, enzymes, and combinations thereof. Methods for labeling and guidance in selecting appropriate markers for various purposes are discussed, for example, in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989), and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1998).

[0053] In some embodiments, the marker is a fluorophore ("fluorescent label"). A fluorophore is a compound that, upon excitation by exposure to a particular wavelength of light, emits light (i.e., fluoresces), for example at a different wavelength. Fluorophores can be described in terms of their emission profile, or "color." Green fluorophores, such as Cy3, FITC, and Oregon Green, are generally characterized by their fluorescence in the wavelength range of 515-540 λ. Red fluorophores, such as Texas Red, Cy5, and tetramethylrhodamine, are generally characterized by their fluorescence in the wavelength range of 590-690 λ. In other embodiments, the marker is a protein tag recognized by an antibody, such as a histidine (His) tag, a hemagglutinin (HA) tag, or a c-Myc tag.

[0054] Membrane potential: The electrical potential inside a cell relative to its environment, e.g., to the external bath fluid. Those skilled in the art can easily assess the membrane potential of a cell, e.g., by using conventional whole-cell techniques. Membrane potential can be assessed using a number of approaches, e.g., using conventional whole-cell access or, e.g., using the perforated patch whole-cell mode, and the cell-attached mode.

[0055] Culture medium: A comprehensive set of culture environments with nutrients necessary to support the proliferation (cell growth / expansion) and / or differentiation of a particular cell population. In one embodiment, the cells are stem cells, such as iPSCs. In another embodiment, the cells are RPE cells. Culture medium generally includes a carbon source, a nitrogen source, and a buffer to maintain pH. In one embodiment, the proliferation medium includes a minimum essential medium, such as DMEM, supplemented with various nutrients that enhance the proliferation of stem cells. In addition, additives can be added to the minimum essential medium, such as horse serum, calf serum, or fetal bovine serum.

[0056] "Retinal induction medium (RIM)" refers to a growth medium that contains a WNT pathway inhibitor and a BMP pathway inhibitor and can induce differentiation of PSCs into retinal lineage cells. RIM also contains a TGFβ pathway inhibitor.

[0057] "Retinal differentiation medium (RDM)" is a medium that contains a WNT pathway inhibitor, a BMP pathway inhibitor, and a MEK inhibitor and allows differentiation into retinal cells. RDM also contains a TGFβ pathway inhibitor.

[0058] "Retinal medium (RM)" refers to a growth medium for culturing retinal cells, comprising activin A and nicotinamide.

[0059] "RPE maturation medium (RPE-MM)" refers to a medium for maturing RPE cells that includes taurine and hydrocortisone. RPE-MM also includes triiodothyronine. RPE-MM may also include PD0325901 or PGE2.

[0060] Nodal: a secreted protein encoded by the NODAL gene, belonging to the transforming growth factor (TGF-β) superfamily. During embryonic development, left-right (LR) asymmetry of internal organs in vertebrates is established through nodal signaling. Nodal is expressed on the left side of the organism in early development, and is highly conserved among deuterostomes. Exemplary nodal sequences can be found in GENBANK® Accession Nos. NM_018055.4 and NP_060525.3, dated January 6, 2013, which are incorporated herein by reference.

[0061] Nucleic acid: A polymer composed of nucleotide units (ribonucleotides, deoxyribonucleotides, related naturally occurring structural variants thereof, and synthetic non-naturally occurring analogs thereof) linked via phosphodiester bonds. Thus, the term includes nucleotide polymers in which the nucleotides and the bonds between them include synthetic non-naturally occurring analogs, such as, but not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNAs), etc. Such polynucleotides can be synthesized, for example, using an automated DNA synthesizer. When a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), it is understood that this also includes RNA sequences (i.e., A, U, G, C) in which "T" is replaced by "U".

[0062] "Nucleotide" includes, but is not limited to, a monomer containing a base bound to a sugar, such as, for example, a pyrimidine, a purine, or a synthetic analogue thereof, or a base bound to an amino acid as in peptide nucleic acid (PNA). A nucleotide is one monomer in a polynucleotide. A nucleotide sequence refers to the sequence of bases in a polynucleotide.

[0063] Conventional notation is used herein to describe nucleotide sequences: the left end of a single-stranded nucleotide sequence is the 5' end; the leftward direction of a double-stranded nucleotide sequence is referred to as the 5' direction. The 5' to 3' direction in which nucleotides are added to the nascent RNA transcript is referred to as the transcription direction. The DNA strand that has the same sequence as the mRNA is referred to as the "coding strand;" the sequence on the DNA strand that has the same sequence as the mRNA transcribed from the DNA and is located 5' to 5' of the RNA transcript is referred to as the "upstream sequence;" the sequence on the DNA strand that has the same sequence as the RNA and is 3' to 3' of the coding RNA transcript is referred to as the "downstream sequence."

[0064] "cDNA" refers to a DNA that is complementary to or identical to an mRNA, in either single- or double-stranded form.

[0065] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to act as a template in biological processes for the synthesis of other polymers and macromolecules that have either a defined sequence of nucleotides (e.g., rRNA, tRNA, and mRNA) or a defined sequence of amino acid sequences and have biological properties due to them. Thus, a gene codes for a protein when transcription and translation of the mRNA produced by the gene produces a protein in a cell or other biological system. Both the coding strand, which is the nucleotide sequence identical to the mRNA sequence and usually provided in the sequence listing, and the non-coding strand used as a template to transcribe the gene or cDNA, can be said to code for a protein or other product of the gene or cDNA. Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that code for the same amino acid sequence. Protein and RNA encoding nucleotide sequences can include introns. In some examples, the nucleic acid encodes the disclosed antigen.

[0066] Noggin: a protein encoded by the NOG gene. Noggin binds to TGF-β family ligands and inhibits TGF-β signaling by preventing the ligand from binding to its corresponding receptor. Noggin plays an important role in neural induction by inhibiting BMP4 along with other TGF-β signaling inhibitors, such as chordin and follistatin. Exemplary sequences of Noggin are GENBANK® Accession Nos. NP_005441.1 and NM_005450.4, dated January 13, 2013, which are incorporated herein by reference.

[0067] Oct-4: Also known as POU5-F1, or MGC22487, or OCT3, or OCT4, or OTF3, or OTF4, is a protein that is the gene product of the Oct-4 gene. The term includes Oct-4 from any species or source and includes analogs and fragments or parts of Oct-4 that retain their ability to be used for the generation of iPSCs. Oct-4 protein can have any of the publicly known sequences of Oct-4, which can be obtained from public sources, such as GENBANK®. An example of such a sequence includes, but is not limited to, GENBANK® accession number NM_002701.

[0068] Operatively linked: A first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is positioned such that it has a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

[0069] Pharmaceutically acceptable carrier: Pharmaceutically acceptable conventional carriers are useful for carrying out the methods disclosed herein and for forming the compositions disclosed herein. Remington's Pharmaceutical Sciences by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975, describes examples of compositions and formulations suitable for pharmaceutical delivery of the antimicrobial compounds disclosed herein.

[0070] Typically, the nature of the carrier will vary depending on the particular mode of administration being employed. For example, parenteral formulations usually contain as a vehicle an injectable fluid that contains pharma- ceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like. For solid compositions (e.g., powder, pill, tablet, or capsule forms), typical non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to carriers that have no biological properties, the pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, such as sodium acetate or sorbitan monolaurate.

[0071] Pluripotent stem cells: Stem cells that are: (a) It is capable of inducing teratomas when transplanted into immunodeficient (SCID) mice; (b) capable of differentiating into cell types of all three germ layers (e.g., capable of differentiating into ectodermal, mesodermal, and endodermal cell types); and (c) express one or more markers of embryonic stem cells (e.g., express Oct 4, alkaline phosphatase, SSEA-3 surface antigen, SSEA-4 surface antigen, nanog, TRA-1-60, TRA-1-81, SOX2, REX1, etc.) but are incapable of forming embryonic and extraembryonic membranes (are not totipotent).

[0072] Exemplary pluripotent stem cells include embryonic stem cells derived from the inner cell mass (ICM) of a blastocyst stage embryo, as well as embryonic stem cells derived from one or more blastomeres of a cleavage stage or morula stage embryo (optionally without destroying other parts of the embryo). These embryonic stem cells can be generated from embryonic material produced by fertilization or asexual means, including somatic cell nuclear transfer (SCNT), parthenogenesis, and androgenetic reproduction. PSCs alone cannot develop into fetuses or adult animals when transplanted into the uterus, because they lack the ability to generate all extraembryonic tissues (e.g., placenta in vivo, or trophoblast in vitro).

[0073] Pluripotent stem cells also include "induced pluripotent stem cells (iPSCs)" that are generated by reprogramming somatic cells by expressing or inducing the expression of a combination of factors (referred to herein as reprogramming factors). iPSCs can be generated using fetal, postnatal, neonatal, juvenile, or adult somatic cells. In some embodiments, factors that can 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 reprogramming factors, at least three reprogramming factors, or at least four reprogramming factors that reprogram somatic cells into pluripotent stem cells. The properties of iPSCs are similar to those of embryonic stem cells.

[0074] Polypeptide: A polymer in which the monomers are amino acid residues linked together by amide bonds. When the amino acids are alpha amino acids, either the L-optical isomer or the D-optical isomer can be used, with the L-optical isomer predominating in nature. The term polypeptide is specifically intended to encompass naturally occurring proteins as well as recombinantly or synthetically produced proteins.

[0075] As used herein, a substantially purified polypeptide refers to a polypeptide that is substantially free of other proteins, lipids, carbohydrates, and other materials with which it is naturally associated. In one embodiment, the polypeptide is at least 50% free, such as at least 80% free, of other proteins, lipids, carbohydrates, and other materials with which it is naturally associated. In another embodiment, the polypeptide is at least 90% free, of other proteins, lipids, carbohydrates, and other materials with which it is naturally associated. In yet another embodiment, the polypeptide is at least 95% free, of other proteins, lipids, carbohydrates, and other materials with which it is naturally associated.

[0076] Conservative substitution tables of amino acids resulting in functionally similar amino acids are well known to those of skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0077] Non-conservative substitutions of amino acids can result from changes in: (a) the structure of the amino acid backbone in the area of ​​the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of the amino acid side chain. Substitutions that are generally expected to produce the greatest changes in the properties of a protein are those that: (a) a hydrophilic residue replaces (or is substituted by) a hydrophobic residue; (b) proline replacing (or being replaced by) any other residue; (c) a residue having a large side chain, e.g., phenylalanine, replaces (or is replaced by) a residue having no side chain, e.g., glycine; or (d) A residue having a positively charged side chain, such as a lysyl, arginyl, or histadyl residue, replaces (or is replaced by) a negatively charged residue, such as a glutamyl or aspartyl residue.

[0078] The amino acid sequence of the variant can be, for example, 80%, 90%, or even 95% or 98% identical to the original amino acid sequence. Programs and algorithms for determining percentage identity are available on the NCBI website.

[0079] Pre-confluent: A cell culture in which the percentage of the culture surface covered by cells is about 60 to 80%. In one embodiment, pre-confluent refers to a culture in which about 70% of the culture surface is covered by cells.

[0080] Prenatal: existing or occurring before birth. Similarly, "postnatal" is existing or occurring after birth.

[0081] Recombinant: A recombinant nucleic acid or polypeptide molecule is one that has a sequence that is not found in nature or that is made by the artificial combination of two otherwise separate segments of sequence, which can be accomplished by chemical synthesis of the polypeptide or nucleic acid molecule, or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

[0082] Retinal pigment epithelial (RPE) cells: RPE cells can be identified based on pigmentation, epithelial morphology, and apical-basal polarized cells. RPE cells express one or more of the following at both the mRNA and protein levels: Pax6, MITF, RPE65, CRALBP, PEDF, bestrophin, and / or Otx2. In certain other embodiments, RPE cells express one or more of Pax-6, MitF, and tyrosinase at both the mRNA and protein levels. RPE cells do not express any of the embryonic stem cell markers Oct-4, nanog, or Rex-1 (at any detectable level). Specifically, the expression of these genes in RPE cells is approximately 100-1000 times lower than in ES cells or iPSCs, as assessed by quantitative RT-PCR. Differentiated RPE cells can also be visually identified by their cobblestone morphology and initial appearance of pigment. In addition, differentiated RPE cells have a trans epithelial resistance / TER and transepithelial potential / TEP across the monolayer (TER > 100 ohms.cm 2 ; TEP > 2 mV), transport fluid and CO2 from apical to basolateral, and regulate polarized secretion of cytokines.

[0083] As used herein, "mature" RPE cells refer to 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.

[0084] As used herein, the "maturation" of RPE cell refers to the process in which the developmental pathway of RPE is regulated to produce mature RPE cell.For example, the regulation of cilia function can lead to the maturation of RPE.As used herein, the "retinal lineage cell" refers to the cell that can give rise to RPE cell or can differentiate into RPE cell.

[0085] Retinal pigment epithelium: A pigmented layer of hexagonal cells that lies just outside the neurosensory retina and is attached to the choroid below in vivo in mammals. These cells are densely packed with pigment granules and protect the retina from incident light. The retinal pigment epithelium also acts as a limiting transporter, maintaining the retinal environment by providing small molecules, such as amino acids, ascorbic acid, and D-glucose, while being a tight barrier to substances carried by the choroidal blood.

[0086] Secreted frizzled-related proteins (sFRPs): The sFRP family of proteins are glycoproteins of approximately 32-40 kDa that have been identified as antagonists of Wnt signaling (Rattner et al. (1997) Proc. Natl. Acad. Sci. USA 94:2859-63; Melkonyan et al. (1997) Proc. Natl. Acad. Sci. USA 94:13636-41; Finch et al. (1997) Proc. Natl. Acad. Sci. USA 94:6770-5; Uren et al. (2000) J. Biol. Chem. 275:4374-82; Kawano et al. (2003) J. Cell. Sci. 116:2627-34). In mammals, there are five sFRPs. Human sFRPs include sFRP1 (GENBANK® Accession No. AF001900.1), sFRP2 (GENBANK® Accession No. NM_003013.2), sFRP3 (GENBANK® Accession No. U91903.1), sFRP4 (GENBANK® Accession No. NM_003014.3), and sFRP5 (GENBANK® Accession No. NM_003015.3), available as of January 1, 2013.

[0087] sFRPs contain three structural units: an amino-terminal signal peptide, a frizzled-type cysteine-rich domain (CRD), and a carboxy-terminal netrin (NTR) domain. The CRD is approximately 120 amino acids long, contains 10 conserved cysteine ​​residues, and has 30-50% sequence similarity to the CRDs of Fzd receptors. The netrin domain is defined by six cysteine ​​residues and several segments of conserved hydrophobic residues as well as secondary structure. The biological activity of sFRPs is largely attributable to their role as regulators of Wnt function.

[0088] Sonic Hedgehog (SHH): Sonic Hedgehog (SHH) is one of three mammalian homologs of the Drosophila hedgehog signaling molecule and is expressed at high levels in the notochord and floor plate of developing embryos. SHH is known to play an important role in neural tube patterning (Echerlard et al., Cell 75:1417-30, 1993) and in the development of limbs, somites, lungs, and skin. In addition, overexpression of SHH has been found in basal cell carcinoma. An exemplary amino acid sequence of SHH is shown in U.S. Patent No. 6,277,820. An exemplary sequence of human Sonic is shown as GENBANK Accession No. NG_007504.1 (January 1, 2013), which is incorporated herein by reference.

[0089] Subject: An animal or human that is the object of treatment, observation, or experiment.

[0090] Super donor: an individual who is homozygous for certain MHC class I and class II genes. These homozygous individuals can be super donors and their cells, including tissues and other materials that contain their cells, can be transplanted into individuals who are either homozygous or heterozygous for their haplotype. A super donor can be homozygous for one or more alleles of the HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP, or HLA-DQ locus.

[0091] Tissue replacement implant: A biocompatible structure, produced in vitro, containing both matrix and cells, that can be used to replace tissue in vivo.

[0092] Totipotent, or totipotent: The ability of a cell to divide and ultimately produce an entire organism in vivo, including all extraembryonic tissues. In one aspect, the term "totipotent" refers to the ability of a cell to progress through a series of divisions in vitro to a blastocyst. The blastocyst contains an inner cell mass (ICM) and a trophectoderm. Cells found in the ICM give rise to pluripotent stem cells (PSCs) that have the ability to proliferate indefinitely, while, if properly induced, differentiating into all cell types that make up an organism. Trophectoderm cells make the extraembryonic tissues, including the placenta and amnion.

[0093] Treatment: A therapeutic measure that cures a diagnosed condition or disorder, slows the condition or disorder, reduces the symptoms of the condition or disorder, and / or stops the progression of the condition or disorder. In some embodiments, treating a subject with a retinal disorder results in reduced retinal deterioration; an increase in the number of retinal pigment epithelial cells, improved vision, or some combination of effects.

[0094] Tyrosinase: A copper-containing oxidase that catalyzes the production of melanin and other pigments from tyrosine by oxidation. This enzyme is the rate-limiting enzyme that controls the production of melanin. Tyrosinase acts in the hydroxylation of monophenols and in the conversion of o-diphenols to the corresponding o-quinones, which undergo several reactions to eventually produce melanin. In humans, the tyrosinase enzyme is encoded by the TYR gene. Exemplary amino acid and nucleic acid sequences are set forth in GENBANK® Accession Nos. NM_000372.4 (human) and NM_011661.4 (mouse) on January 5, 2013, and are incorporated herein by reference.

[0095] Undifferentiated: Cells that show the markers characteristic of undifferentiated cells and the morphological characteristics of undifferentiated cells, which distinguish undifferentiated cells from differentiated cells of embryonic or adult origin.Thus, in some embodiments, undifferentiated cells do not express lineage-specific markers, including but not limited to RPE markers.

[0096] Vector: A nucleic acid molecule that, when introduced into a host cell, results in a transformed host cell. A vector may contain a nucleic acid sequence that allows the vector to replicate in a host cell, such as an origin of replication. A vector may also contain one or more selectable marker genes and other genetic elements known in the art. A vector may also contain a sequence that codes for an amino acid motif that facilitates the isolation of a desired protein product, such as a sequence that codes for maltose binding protein, c-myc, or GST.

[0097] Wnt: A family of highly conserved secreted signaling molecules that regulate cell-cell interactions and are related to the Drosophila segment polarity gene, wingless. In humans, the Wnt family of genes encode 38-43 kDa cysteine-rich glycoproteins. Wnt proteins have a hydrophobic signal sequence, a conserved asparagine-linked 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 stabilization of β-catenin in the cytoplasm, Wnt proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain Wnt proteins has been shown to be associated with certain cancers.

[0098] The Wnt family includes at least 19 mammalian members. Exemplary Wnt proteins include Wnt-1, Wnt-2, Wnt2b, Wnt-3, Wnt-3a, Wnt-4, Wnt-5a, Wnt5b, Wnt-6, Wnt-7a, Wnt-7b, Wnt-8a, Wnt-8b, Wnt9a, Wnt9b, Wnt10a, Wnt-10b, Wnt-11, and Wnt 16. These secreted ligands activate at least three different signaling pathways. In the canonical (or Wnt / β-catenin) Wnt signaling pathway, Wnt activates a receptor complex consisting of frizzled (Fzd) receptor family members and low-density lipoprotein (LDL) receptor-related protein 5 or 6 (LRP5 / 6). To form receptor complexes that bind Fzd ligands, Fzd receptors interact with LRP5 / 6, a single-pass transmembrane protein with four extracellular EGF-like domains separated by six YWTD amino acid repeats (Johnson et al., 2004, J. Bone Mineral Res. 19:1749). The canonical Wnt signaling pathway, activated upon receptor binding, is mediated by Dishevelled (Dvl), a cytoplasmic protein that directly interacts with Fzd receptors, and leads to the cytoplasmic stabilization and accumulation of β-catenin. In the absence of Wnt signaling, β-catenin is localized in a cytoplasmic degradation complex that contains the tumor suppressor protein adenomatous polyposis coli (APC) protein and axin. These proteins function as critical scaffolds that allow glycogen synthase kinase (GSK) 3β to bind and phosphorylate β-catenin, marking it for degradation by the ubiquitin / proteasome pathway. Activation of Dvl leads to dissociation of the degradation complex. Accumulated cytoplasmic β-catenin is then transported into the nucleus where it interacts with DNA-binding proteins of the TCF / LEF family to activate transcription.

[0099] The non-canonical WNT pathway is regulated by three of these WNT ligands - WNT4, WNT5a, and WNT11. These ligands bind to the WNT receptor Frizzled in the absence of co-receptors (LRP5 / 6). This leads to activation of RHO GTPases and ROCK kinases without activating cytoplasmic β-catenin. ROCK regulates the cytoskeleton to regulate apical-basal polarization of cells. Due to competition for the same receptors, non-canonical WNT ligands also lead to inhibition of canonical WNT signaling.

[0100] Xeno-Free (XF): With respect to a medium, extracellular matrix, or culture environment, refers to a medium, extracellular matrix, or culture environment that is essentially free of components derived from a xenogeneic animal. With respect to the culture of human cells, any protein from a non-human animal, e.g., mouse, can be a xenogeneic component. In certain aspects, a xeno-free matrix can be essentially free of any components derived from a non-human animal, and thus does not include mouse feeder cells or MATRIGEL™. MATRIGEL™ is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor rich in extracellular matrix proteins, that contains laminin (major component), type IV collagen, heparan sulfate proteoglycan, and entactin / nidogen.

[0101] Unless otherwise explained, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The singular terms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. It should be further understood that all base or amino acid sizes, and all given molecular weight or molecular mass values ​​for nucleic acids or polypeptides are approximate and are provided for illustration purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, suitable methods and methods are described below. The term "comprise" means "include." "About" refers to within 5 percent, unless otherwise indicated. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0102] Tissue replacement implants and methods of making In the retina, the cells that are directly sensitive to light are photoreceptors. Photoreceptors are light-sensitive neurons in the outer part of the retina, and can be either rods or cones. In the process of light signaling, photoreceptors convert the energy of the incident light focused by the lens into an electrical signal, which is then sent to the brain via the optic nerve. Vertebrates have two types of photoreceptors, including cones and rods. Cones are adapted to detect high-definition central vision and color vision, and perform well in bright light. Rods are responsible for peripheral vision and vision in dim light. The neural signals from rods and cones are processed by other neurons in the retina.

[0103] The retinal pigment epithelium acts as a barrier between the bloodstream and the retina, and interacts closely with photoreceptor cells in maintaining visual function. It is composed of a single layer of hexagonal cells densely packed with melanin granules, and absorbs the light energy that reaches the retina. The main functions of specialized RPE cells include: transport of nutrients, such as glucose, retinol, and fatty acids, from the blood to photoreceptor cells; transport of water, metabolic end products, and ions from the subretinal space to the blood; light absorption and protection from photooxidation; reisomerization of all-trans-retinal to 11-cis-retinal; phagocytosis of sloughed photoreceptor membranes; and secretion of various factors essential for the structural integrity of the retina.

[0104] Dysfunction, damage and loss of RPE cells are the cause of many eye diseases and disorders, including age-related macular degeneration (AMD) and hereditary macular degeneration, such as Best's disease and retinitis pigmentosa. Other diseases are discussed below. Damage to the retina, such as physical damage, also requires treatment. Disclosed herein is a tissue replacement implant that contains RPE cells and can be locally introduced into the eye of people who need treatment. The tissue replacement implant improves retinal function and prevents blindness caused by such abnormalities.

[0105] In some embodiments, disclosed is a tissue replacement implant comprising RPE cells, and the method for making said tissue implant.These tissue replacement implants can be used to treat retinal degenerative disease, retinal or retinal pigment epithelial dysfunction, retinal deterioration, retinal damage, or retinal pigment epithelial loss in subjects.These RPE cells can be made by using the method disclosed in, for example, PCT Publication WO2014 / 121077 and PCT Publication WO2017 / 044483, both of which are incorporated herein by reference.

[0106] In some embodiments, the RPE cells are autologous to the subject that is to be treated with the tissue implant. Autologous cells include cells that are derived from the same subject and are modified, such as modified to correct mutations in genes of interest, to express heterologous proteins, or to silence the expression of mutant genes. In other embodiments, the RPE cells are allogeneic to the subject that is to be treated with the tissue implant. In some non-limiting examples, the RPE cells are MHC-matched to the subject that is to be treated with the tissue implant. The RPE cells can be derived from a single subject, or from multiple subjects, such as 2, 3, 4, or 5 subjects. In further embodiments, the RPE cells are made from pluripotent stem cells. Methods for making tissue implants and tissue replacement implants that include RPE cells are disclosed.

[0107] A. Pluripotent stem cells 1. Embryonic stem cells ES cells originate from the inner cell mass of blastocysts and have a high differentiation potential in vitro. ES cells can be isolated by removing the outer trophectoderm layer of developing embryos and then culturing the cells of the inner cell mass on a feeder layer of non-proliferating cells. The replated cells can continue to proliferate and produce new colonies of ES cells, and the cells can be transferred, dissociated, replated again, and propagated. This process of "passaging" undifferentiated ES cells can be repeated several times to create cell lines containing undifferentiated ES cells (U.S. Patent Nos. 5,843,780; 6,200,806; 7,029,913). ES cells have the ability to proliferate while maintaining their pluripotency. For example, ES cells are useful for research in cells and in genes that control cell differentiation. The pluripotency of ES cells, combined with genetic manipulation and selection, can be used for in vivo genetic analysis studies by the generation of transgenic, chimeric, and knockout mice.

[0108] Methods for generating mouse ES cells are well known. In one method, preimplantation blastocysts from mouse strain 129 are treated with anti-mouse serum to remove the trophectoderm, and the inner cell mass is cultured on a feeder cell layer of chemically inactivated mouse embryonic fibroblasts in medium containing fetal bovine serum. The developing, undifferentiated ES cell colonies are subcultured on a mouse embryonic fibroblast feeder layer in the presence of fetal bovine serum to generate a population of ES cells. In some methods, mouse ES cells can be grown in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing medium (Smith, 2000). In another method, mouse ES cells can be grown in serum-free medium in the presence of bone morphogenetic protein and LIF (Ying et al., 2003).

[0109] Human ES cells can be generated or derived from: zygote or blastocyst stage mammalian embryos generated by sperm and egg fusion, by nuclear transfer, by blastogenesis, or by previously described methods for generating embryonic cells (Thomson and Marshall, 1998; Reubinoff et al., 2000) by chromatin reprogramming and subsequent integration of the reprogrammed chromatin into the cell membrane. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass, which is cultured on a feeder layer of mouse embryonic fibroblasts. Additionally, cell clumps derived from the inner cell mass are chemically or mechanically dissociated and replated, and colonies with undifferentiated morphology are selected by micropipette, dissociated, and replated (U.S. Patent No. 6,833,269). In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al., 2000). In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix, such as on MATRIGEL® or on laminin, in the presence of "conditioned" medium that contains basic fibroblast growth factor (Xu et al., 2001).

[0110] ES cells can also be derived from established mouse and human cell lines, as well as from other organisms, including rhesus monkeys and marmosets, by methods previously described (Thomson, and Marshall, 1998; Thomson et al., 1995; Thomson and Odorico, 2000). For example, established human ES cell lines include MAOI, MA09, ACT-4, HI, H7, H9, H13, H14, and ACT30. As a further example, established mouse ES cell lines include the CGR8 cell line established from the inner cell mass of mouse line 129 embryos, and cultures of CGR8 cells can be grown without a feeder layer in the presence of LIF.

[0111] ES stem cells can be detected by protein markers including: transcription factor Oct4, alkaline phosphatase (AP), stage-specific embryonic antigen SSEA-1, stage-specific embryonic antigen SSEA-3, stage-specific embryonic antigen SSEA-4, transcription factor NANOG, tumor rejection antigen 1-60 (TRA-1-60), tumor rejection antigen 1-81 (TRA-1-81), SOX2, or REX1.

[0112] a. Somatic cell nuclear transfer Pluripotent stem cells can be prepared by the technique of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, a donor fibroblast nucleus derived from a primate skin fibroblast is introduced into the cytoplasm of a spindle-free, mature, metaphase II primate oocyte by electrofusion (Byrne et al., 2007). The fused oocyte is activated by exposure to ionomycin and then incubated until the blastocyst stage. The inner cell mass of the selected blastocyst is then cultured to generate embryonic stem cell lines. The embryonic stem cell lines display normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo.

[0113] 2. Induced pluripotent stem cells Induction of pluripotency was initially achieved by reprogramming somatic cells by introducing transcription factors associated with pluripotency in mouse cells in 2006 (Yamanaka et al. 2006) and in human cells in 2007 (Yu et al. 2007; Takahashi et al. 2007). Pluripotent stem cells can be maintained in an undifferentiated state and can differentiate into almost any cell type. The use of iPSCs avoids most of the ethical and practical issues associated with the large-scale clinical use of ES cells, and patients with iPSC-derived autologous transplants would not require lifelong immunosuppressive treatments to prevent graft rejection.

[0114] Any cell can be used as the starting point for iPSC, except for germ cells. For example, the cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, hepatocytes, or gastric cells. The cell can be a multipotent cell, such as but not limited to hematopoietic stem cells, such as but not limited to CD34+ cells. T cells can also be used as a source of somatic cells for reprogramming (US Pat. No. 8,741,648). There is no limit to the degree of cell differentiation or the age of the animal from which the cells are collected; either undifferentiated progenitor cells (including somatic stem cells) or terminally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cell is an RPE cell itself, such as a human RPE cell. The RPE cell can be an adult RPE cell or a fetal RPE cell. iPSCs can be grown under conditions known to differentiate human ES cells into specific cell types and express human ES cell markers including: SSEA-1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81.

[0115] Somatic cells and pluripotent stem cells, such as CD34+ cells, can be reprogrammed to generate induced pluripotent stem cells (iPSCs) by using methods known to those skilled in the art. Those skilled in the art can easily generate induced pluripotent stem cells, for example, see US Patent Application Publication No. 20090246875, US Patent Application Publication No. 2010 / 0210014; US Patent Application Publication No. 20120276636; US Patent No. 8,058,065; US Patent No. 8,129,187; US Patent No. 8,278,620; PCT Publication WO 2007 / 069666 A1 and US Patent No. 8,268,620. In general, nuclear reprogramming factors are used to generate pluripotent stem cells from somatic cells. In some embodiments, at least three or at least four of Klf4, c-Myc, Oct3 / 4, Sox2, Nanog, and Lin28 are utilized. In other embodiments, Oct3 / 4, Sox2, c-Myc, and Klf4 are utilized.

[0116] Cells are treated with nuclear reprogramming material, which is generally one or more factors that can induce iPSC from somatic cells, or the nucleic acid that codes these materials (including those that are incorporated in vector).Nuclear reprogramming material generally includes at least Oct3 / 4, Klf4, and Sox2, or the nucleic acid that codes these molecules.Function inhibitor of p53, L-myc or the nucleic acid that codes L-myc, and Lin28 or Lin28b or the nucleic acid that codes Lin28 or Lin28b can be used as additional nuclear reprogramming material.Nanog can also be used for nuclear reprogramming. As disclosed in U.S. Patent Application Publication No. 20120196360, exemplary reprogramming factors for generating iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Sox1, Sox3, Sox15, Sox17, or Sox18; Klf4 can be replaced with Klf1, 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) Oct3 / 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 substituted for Esrrg). In one non-limiting example, Oct3 / 4, Klf4, Sox2, and c-Myc are utilized. In other embodiments, Oct4, Nanog, and Sox2 are utilized, see, e.g., U.S. Patent No. 7,682,828, which is incorporated herein by reference. These factors include, but are not limited to, Oct3 / 4, Klf4, and Sox2. In other examples, factors include, but are not limited to, Oct 3 / 4, Klf4, and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc, and Sox2 are utilized. In other non-limiting examples, Oct3 / 4, Klf4, Sox2, and Sal 4 are utilized. Factors such as Nanog, Lin28, Klf4, or c-Myc can increase reprogramming efficiency and can be expressed from several different expression vectors. For example, integrating vectors can be used, such as systems based on EBV elements (US Pat. No. 8,546,140). In a further aspect, reprogramming proteins can be directly introduced into somatic cells by protein transduction. Reprogramming can further include contacting the cells with one or more signaling receptors, including: glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, receptor or signaling inhibitors of transforming growth factor beta (TGF-β), leukemia inhibitory factor (LIF), p53 inhibitors, NF-κB inhibitors, or combinations thereof. These regulators can include small molecules, inhibitory nucleotides, expression cassettes, or protein factors. It is expected that virtually any iPS cell or iPS cell line can be used;

[0117] The mouse and human cDNA sequences of these nuclear reprogramming agents are available by reference to the NCBI accession numbers referenced in WO 2007 / 069666, which is incorporated herein by reference. Methods for introducing one or more reprogramming agents, or nucleic acids encoding these reprogramming agents, 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.

[0118] Once induced, iPSCs can be cultured in a medium that can adequately maintain pluripotency. As described in U.S. Pat. No. 7,442,548 and U.S. Patent Application Publication No. 2003 / 0211603, various media and techniques developed for culturing pluripotent stem cells, more specifically embryonic stem cells, can be used for iPSCs. For mouse cells, leukemia inhibitory factor (LIF) is added to normal culture medium as a differentiation inhibitor. For human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for culturing and maintaining iPSCs that will be known to those skilled in the art can be used.

[0119] In some embodiments, an undefined environment may be used; for example, pluripotent cells may be cultured on fibroblast feeder cells or in a medium exposed to fibroblast feeder cells to maintain stem cells in an undifferentiated state. In some embodiments, cells are cultured in the presence of mouse embryonic fibroblasts, which have been irradiated or antibiotic-treated to stop cell division, as feeder cells. Alternatively, pluripotent cells may be cultured using a defined feeder-independent culture system, such as TESR™ medium (Ludwig et al., 2006a; Ludwig et al., 2006b) or E8™ medium (Chen et al., 2011), and maintained in an essentially undifferentiated state.

[0120] In some embodiments, iPSCs can be modified, such as expressing foreign genes, increasing the expression of endogenous genes, increasing the copy number of genes, correcting gene mutations, or silencing the expression of mutant genes, etc. In some specific, non-limiting examples, mutations or deletions in endogenous genes are corrected.The gene may code for, for example, retinoid isomerohydrolase (RPE65), bestrophin (BEST) 1, MER tyrosine kinase proto-oncogene (MER tyrosine kinase 1), or the like. proto-oncogene (MERTK), RAB escort protein (REP1), cellular retinaldehyde-binding protein (CRALBP), pre-mRNA processing factor (PPRF), complement factor H (CFH), complement component 3a receptor (C3aR)1, complement component 5 receptor (C5aR1), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), complement factor I (CFI), complement factor 2B (C2B), ATP-binding cassette subfamily A member 4 (ABCA4), ATP-binding cassette subfamily A member 1 (ABCA1), membrane-type frizzled-related protein (MFRP), C1q and tumor necrosis factor-related protein 5 (C1qTNF5), spermatogenesis-associated protein 7 (SPATA7), 290 Centrosomal protein of kd (CEP290), myosin VIIA (MYO7A), ciliary neurotrophic factor (CNTF), FMS-related tyrosine kinase 1 (FLT-1), Usher syndrome type I (USH1A), tyrosinase, plasminogen (PLG), collagen XVIII alpha 1 (COL18A1), TTRA serine peptidase 1 (HTRA) 1, ARMS2, tissue inhibitor of metalloproteinases (TIMP) 3, epithelial growth factor (EGF) containing fibulin-like extracellular matrix protein (EFEMP) 1, microphthalmia-associated transcription factor (MITF), transcription factor EC (TFEC), Drosophila orthodenticle homolog 2 (OTX2), zinc finger protein 503 (ZNF503 or NLZ2). In one non-limiting example, the gene is RPE65. In another non-limiting example, the gene is BEST1. In a further non-limiting example, the gene encodes METRK.Methods for carrying out gene editing in iPSCs are disclosed, for example, in Hockenmeyer and Jaenisch, "Induced Pluripotent Stem Cell Meets Genome Editing," Cell Stem Cell 18: 573-586, 2016, which is incorporated herein by reference. Any of the methods disclosed therein can be used. The method can include the use of viral vectors, such as adeno-associated viral vectors or lentiviral vectors, with transgenes of interest at the end. The method can include the use of CRISPR / Cas9, TALEN nucleases, zinc finger nucleases, lentiviral modification, adeno-associated viral modification, shRNA, siRNA, or F prime editing.

[0121] In some embodiments, iPSCs can be modified to express exogenous nucleic acid, such as by including a tyrosinase enhancer operably linked to a promoter and a nucleic acid sequence encoding a first marker. The tyrosinase gene is disclosed, for example, in GENBANK® Accession No. 22173 available on January 1, 2013. This sequence is aligned to positions 5286971-5291691 (reverse orientation) of chromosome 7 of mouse C57BL / 6 strain. A sequence of 4721 base pairs is sufficient for expression in RPE cells, see Murisier et al., Dev. Biol. 303: 838-847, 2007, incorporated herein by reference. This construct is expressed in retinal pigment epithelial cells. Other enhancers are also available. Other RPE-specific enhancers include D-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP, and RAB27A. Suitable promoters include any promoter that is expressed in retinal pigment epithelial cells, including but not limited to the tyrosinase promoter. The construct can also include other elements, such as a ribosome binding site (internal ribosome binding sequence) for translation initiation, and a transcription / translation terminator. In general, it is beneficial to transfect the construct into cells. Suitable vectors for stable transfection include but are not limited to retroviral vectors, lentiviral vectors, and Sendai virus.

[0122] Plasmids are designed with several goals in mind, such as achieving a controlled high copy number and avoiding potential sources of plasmid instability in bacteria, as well as providing a means for plasmid selection that is compatible with use in mammalian cells, including human cells. For use in human cells, there are two requirements for plasmids. First, the plasmid must be suitable for maintenance and culture in E. coli, so that large amounts of DNA can be produced and purified. Second, the plasmid must be safe and suitable for use in human patients and animals. The first requirement requires a high copy number plasmid that can be relatively easily selected and stably maintained during bacterial culture. The second requirement requires consideration of elements, such as selectable markers and other coding sequences. In some embodiments, the plasmid encoding the marker is composed of: (1) a high copy number origin of replication; (2) a selectable marker, such as but not limited to the neo gene for antibiotic selection with kanamycin; (3) a transcription termination sequence including a tyrosinase enhancer; and (4) a multiple cloning site for integrating various nucleic acid cassettes; and (5) a nucleic acid sequence encoding the marker operably linked to the tyrosinase promoter. There are numerous plasmid vectors known in the art for inducing nucleic acids encoding proteins. These include but are not limited to the vectors disclosed in U.S. Patent No. 6,103,470; U.S. Patent No. 7,598,364; U.S. Patent No. 7,989,425; and U.S. Patent No. 6,416,998, which are incorporated herein by reference.

[0123] Viral gene delivery systems can be RNA- or DNA-based viral vectors. Episomal gene delivery systems can be plasmids, Epstein-Barr Virus (EBV)-based episomal vectors, yeast-based vectors, adenovirus-based vectors, Simian Virus 40 (SV40)-based episomal vectors, bovine papilloma virus (BPV)-based vectors, or lentivirus vectors.

[0124] In some embodiments, cells are transfected with a nucleic acid molecule encoding a marker. Markers include, but are not limited to, fluorescent proteins (e.g., green or red fluorescent proteins), enzymes (e.g., horseradish peroxidase, or alkaline phosphatase, or firefly / renilla luciferase, or nanoluc), or other proteins. Markers can be proteins (including secreted proteins, cell surface proteins, or internal proteins; either synthesized or taken up by the cell); nucleic acids (e.g., mRNA, or nucleic acid molecules with enzymatic activity, etc.), or polysaccharides. Any such determinants of cellular components detectable by antibodies, lectins, probes, or nucleic acid amplification reactions that are specific for the markers of the cell type of interest are included. Markers can also be identified by biochemical or enzymatic assays, or biological responses, depending on the function of the gene product. The nucleic acid sequences encoding these markers can be operably linked to the tyrosinase enhancer. In addition, other genes can be included, such as genes that can affect the differentiation of stem cells to RPE, or the function or physiology or pathology of RPE. Thus, in some embodiments, the nucleic acid includes those that code for one or more of the following: MITF, PAX6, TFEC, OTX2, LHX2, VMD2, CFTR, RPE65, MFRP, CTRP5, CFH, C3, C2B, APOE, APOB, mTOR, FOXO, AMPK, SIRT1-6, HTRP1, ABCA4, TIMP3, VEGFA, CFI, TLR3, TLR4, APP, CD46, BACE1, ELOLV4, ADAM 10, CD55, CD59, and ARMS2.

[0125] a. MHC haplotype matching Major histocompatibility complex is the main cause of immune rejection in allogeneic organ transplantation. There are three major class I MHC haplotypes (A, B, and C) and three major MHC class II haplotypes (DR, DP, and DQ). The HLA locus is highly polymorphic and distributed over 4 Mb on chromosome 6. The ability to haplotype HLA genes in this region is clinically important because this region is associated with autoimmune and infectious diseases, and the compatibility of HLA haplotypes between donors and recipients can affect the clinical outcome of transplantation. To T lymphocytes, HLA corresponding to MHC class I presents peptides from the inside of the cell, and HLA corresponding to MHC class II presents antigens from the outside of the cell. Incompatibility of MHC haplotypes between the graft and the host causes an immune response against the graft and leads to its rejection. Therefore, subjects can be treated with immunosuppressants to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.

[0126] Because of the importance of HLA in transplantation, HLA loci are usually typed by serological tests and PCR to identify favorable donor-recipient pairs. Serological detection of HLA class I and II antigens can be achieved using complement-mediated lymphotoxicity tests with purified T or B lymphocytes. This procedure is primarily used to match HLA-A and HLA-B loci. Molecular-based, tissue typing can often be more accurate than serological tests. Low-resolution molecular methods, such as SSOP (sequence-specific oligonucleotide probe) methods, in which PCR products are tested against a series of oligonucleotide probes, can be used to identify HLA antigens, and these methods are currently the most widely used methods for class II-HLA typing. High-resolution techniques, such as SSP (sequence-specific primer) methods, in which allele-specific primers are utilized for PCR amplification, can identify specific MHC alleles.

[0127] When donor cells are HLA homozygous, i.e., contain the same alleles for each of antigen-presenting proteins, the MHC compatibility between donor and recipient increases significantly.Most individuals are heterozygous for MHC class I and class II genes, but some individuals are homozygous for these genes.These homozygous individuals can be super donors, and the grafts made from their cells can be transplanted into all individuals who are either homozygous or heterozygous for their haplotypes.Furthermore, if the cells of homozygous donors have haplotypes that are found frequently in the population, these cells can be applied to transplantation therapy for a large number of individuals.

[0128] Thus, iPSCs may be derived from the cells of the subject to be treated or from the cells of another subject having the same or substantially the same HLA type as that of the patient, e.g., CD34 +The iPSCs can be generated from the donor, such as from the cell. In one example, the major HLA of the donor (e.g., three major loci, HLA-A, HLA-B, and HLA-DR) is the same as the major HLA of the recipient. In some cases, the somatic cell donor can be a super donor; thus, the iPSCs from the MHC homozygous super donor can be used to generate RPE cells. Thus, the iPSCs from the super donor can be transplanted into subjects who are either homozygous or heterozygous for their haplotype. For example, the iPSCs can be homozygous for two HLA alleles, such as HLA-A and HLA-B. Thus, the iPSCs generated from the super donor can be used in the methods disclosed herein to generate RPE cells that can potentially "match" with a large number of potential recipients.

[0129] b. Episomal vectors In some aspects, reprogramming factors are expressed from an expression cassette that contains one or more exogenous episomal genetic elements (see US Patent Publication No. 2010 / 0003757, which is incorporated herein by reference). Thus, iPSCs can be essentially free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared by using extrachromosomal replicating vectors (i.e., episomal vectors), which can replicate episomally to generate iPSCs that are essentially free of exogenous vectors or viral elements (see US Patent No. 8,546,140, ​​which is incorporated herein by reference; Yu et al., 2009). Some DNA viruses, such as adenovirus, Simian Virus 40 (SV40), or bovine papilloma virus (BPV), or Saccharomyces cerevisiae ARS (autonomously replicating sequence)-containing plasmids, replicate extrachromosomally, i.e., episomally, in mammalian cells. These episomal plasmids are essentially free of all of these disadvantages associated with integrative vectors (Bode et al., 2001). For example, those based on lymphotrophic herpes viruses, as defined above, or Epstein-Barr Virus (EBV) can replicate extrachromosomally and serve to deliver reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or variants or functional equivalents thereof. An additional advantage of episomal vectors is that the foreign elements are eventually lost after introduction into the cells, resulting in self-perpetuating iPSCs that are essentially free of these elements.

[0130] 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 during 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 herpes virus (KSHV); Herpesvirus saimiri (HS), and Marek's disease virus (MDV). Additional sources of episomal-based vectors may include, for example, yeast ARS, adenovirus, SV40, or BPV.

[0131] B Retinal pigment epithelial cells RPE cells are utilized in the disclosed tissue implants. In some embodiments, the cells can be generated from stem cells, such as ESCs or iPSCs.

[0132] The retinal pigment epithelium expresses markers such as cellular retinaldehyde-binding protein (CRALBP), RPE65, Best vitelliform macular dystrophy gene (VMD2), and pigment epithelium-derived factor (PEDF). Dysfunction of the retinal pigment epithelium is associated with a number of vision-altering disorders, such as retinal pigment epithelial detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophies, or degeneration.

[0133] Retinal pigment epithelial (RPE) cells can be characterized based on their pigmentation, epithelial morphology, and apical-basal polarity. Differentiated RPE cells can be visually identified by their cobblestone morphology and initial appearance of pigment. In addition, differentiated RPE cells have a transepithelial resistance / TER and transepithelial potential / TEP throughout the monolayer (TER > 200 ohms*cm 2; TEP > 2 mV), transport fluid and CO2 from apical to basolateral, and regulate polarized secretion of cytokines.

[0134] RPE cells express several proteins that can act as markers for detection, for example, by using means such as immunohistochemical analysis, Western blot analysis, flow cytometry, and enzyme-linked immunoassay (ELISA). For example, RPE-specific markers can include: cellular retinaldehyde-binding protein (CRALBP), microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TYRP-1), retinal pigment epithelium-specific 65 kDa protein (RPE65), premelanosome protein (PMEL17), bestrophin 1 (BEST1), and c-mer proto-oncogene tyrosine kinase (MERTK). RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-2. Specifically, expression of these genes in RPE cells is approximately 100-1000-fold lower than in ES cells or iPSCs, as assessed by quantitative RT-PCR.

[0135] RPE cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot analysis, or dot blot hybridization analysis, using sequence-specific primers in standard amplification methods using publicly available sequence data (GENBANK®). The expression of tissue-specific markers detected at the protein or mRNA level is considered positive when the level is at least 2, 3, 4, 5, 6, 7, 8, or 9 times, or about 2, 3, 4, 5, 6, 7, 8, or 9 times, and more specifically, when the level is higher than 10, 20, 30, 40, 50 times, or even higher than the level of control cells, such as undifferentiated pluripotent stem cells or other cell types.

[0136] Exemplary methods for generating RPE from iPSCs and ESCs are disclosed below. Descriptions of inhibitors that can be used in the medium are disclosed at the end of this section. When reference is made to a class of inhibitors, it should be noted that any of the specific inhibitors can be used.

[0137] 1. Induction of RPE cells from PSC embryoid bodies iPSCs, such as, but not limited to, iPSCs generated from CD34+ cells, can be reprogrammed with known reprogramming factors to generate RPE cells. PCT Publication No. 2014 / 121077, the entirety of which is incorporated herein by reference, discloses a method of treating embryoid bodies (EBs) generated from iPSCs in suspension culture with Wnt and nodal antagonists to induce the expression of markers of retinal progenitor cells. This publication discloses a method of deriving RPE cells from iPSCs by a process of differentiating iPSC EBs into a culture highly enriched for RPE cells. For example, embryoid bodies are generated from iPSCs by the addition of a rho-associated coiled-coil kinase (ROCK) inhibitor and cultured in a first medium containing two WNT pathway inhibitors and one nodal pathway inhibitor. Further, to generate differentiating RPE cells, the EBs are plated on tissue culture coated with MATRIGEL® in a second medium, where the second medium does not contain basic fibroblast growth factor (bFGF), contains a nodal pathway inhibitor, contains about 20 ng to about 90 ng noggin, and contains about 1 to about 5% knock out serum replacement. The differentiating RPE cells are cultured in a third medium containing activin and / or WNT3a. The RPE cells are then cultured in RPE medium to generate human RPE cells, where the RPE medium contains about 5% fetal serum, a canonical WNT inhibitor, a non-canonical WNT inhibitor, and an inhibitor of the sonic hedgehog pathway and the FGF pathway.

[0138] The use of EBs to generate differentiated cell types has several disadvantages. For example, EB generation is an inconsistent and non-reproducible process due to varying efficiencies. The size and shape of EBs generated from iPSC or ES cells are not uniform, and EB generation also requires a rate-limiting centrifugation process. The present disclosure provides a method that allows for the large-scale, EB-independent generation of iPSC- or ES-derived cells required for clinical, research, or therapeutic applications.

[0139] 2. Induction of RPE cells from PSCs, which are essentially single cells In some embodiments, RPE cells are made from essentially single-cell suspensions of pluripotent stem cells (PSCs), such as human iPSCs. In some embodiments, PSCs are cultured to preconfluence to prevent any cell aggregates. In some aspects, PSCs are dissociated by incubation with cell dissociation enzymes, such as those exemplified by TRYPSIN™ or TRYPLE™. PSCs can also be dissociated into essentially single-cell suspensions by pipetting. In addition, blebbistatin (e.g., about 2.5 μM) can be added to the medium to increase the viability of PSCs after dissociation into single cells when the cells are not attached to the culture vessel. Alternatively, a ROCK inhibitor can be used instead of blebbistatin to increase the viability of PSCs after dissociation into single cells.

[0140] To efficiently differentiate RPE cells from single-cell PSCs, accurate counting of the density of input cells can increase the efficiency of RPE differentiation. Thus, single-cell suspensions of PSCs are generally counted before seeding. For example, single-cell suspensions of PSCs are counted by a hemocytometer or by an automated cell counter, such as VICELL® or TC20. Cells can be diluted to a cell density of about 10,000 to about 500,000 cells / mL, about 50,000 to about 200,000 cells / mL, or about 75,000 to about 150,000 cells / mL. In a non-limiting example, single-cell suspensions of PSCs are diluted to a density of about 100,000 cells / mL in a fully defined medium, such as ESSENTIAL 8™ (E8™) medium.

[0141] Once a single cell suspension of PSCs with a known cell density is obtained, the cells are typically seeded into a suitable culture vessel, such as a tissue culture plate, such as a flask, a 6-well plate, a 24-well plate, or a 96-well plate. Culture vessels used to culture the cells include, but are not limited to, flasks, tissue culture flasks, petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, microslides, chamber slides, test tubes, trays, CELLSTACK® chambers, culture bags, and roller bottles, so long as stem cells can be cultured therein. Depending on the needs of the culture, the cells may be cultured in a volume of at least 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 derivable therein, 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 derivable therein. In some embodiments, the culture vessel can be a bioreactor, which can refer to any ex vivo device or system that supports a biologically active environment for cells to grow in. The bioreactor can 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 derivable, or about 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 derivable.

[0142] In one aspect, PSCs, such as iPSCs, are plated at a cell density suitable for efficient differentiation. Typically, the cells are plated at a cell density of about 1,000 to about 75,000 cells / cm2, such as about 5,000 to about 40,000 cells / cm2. In a 6-well plate, the cells can be seeded at a cell density of about 50,000 to about 400,000 cells per well. In an exemplary method, the cells are seeded at a cell density of about 100,000 cells, about 150,000 cells, about 200,000 cells, about 250,000 cells, about 300,000 cells, or about 350,000 cells per well, such as about 200,000 cells per well.

[0143] PSCs, such as iPSCs, are typically cultured on culture plates coated with one or more cell adhesion proteins to promote cell attachment while maintaining cell viability. For example, preferred cell adhesion proteins include extracellular matrices, such as vitronectin, laminin, collagen, and / or fibronectin, which can be used to coat the surface of culture vessels as a means of providing a solid support for the growth of pluripotent cells. The term "extracellular matrix" is understood in the art. The components include one or more of the following proteins: fibronectin, laminin, vitronectin, tenascin, entactin, thrombospondin, elastin, gelatin, collagen, fibrillin, merosin, anchorin, chondronectin, link protein, bone sialoprotein, osteocalcin, osteopontin, epinectin, hyaluronectin, undulin, epiligrin, and kalinin. In an exemplary method, PSCs are grown on vitronectin or fibronectin-coated culture plates. In some embodiments, the cell adhesion protein is a human protein.

[0144] The extracellular matrix (ECM) proteins may be of natural origin and may be purified from human or animal tissue, or alternatively, the ECM proteins may be genetically engineered recombinant proteins or may be naturally synthesized. The ECM proteins may be in the form of full-length proteins or peptide fragments, and may be natural or engineered. Examples of ECM proteins that may be useful as matrices for cell culture include laminin, type I collagen, type IV collagen, fibronectin, and vitronectin. In some embodiments, the matrix composition comprises synthetically produced peptide fragments of fibronectin or recombinant fibronectin. In some embodiments, the matrix composition is xeno-free. For example, in a xeno-free matrix for culturing human cells, matrix components of human origin may be used, in which any non-human animal components may be excluded.

[0145] In some aspects, 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.

[0146] a. Culture conditions Cells, such as RPE cells or PSCs, can be cultured with the nutrients necessary to support the growth of each particular population of cells. Generally, cells are cultured in a growth medium that includes a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also include fatty acids or lipids, amino acids (such as non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, and inorganic salts. Exemplary growth media include minimal essential media, such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients that enhance the growth of stem cells, such as non-essential amino acids and vitamins. Examples of minimal essential media include, but are not limited to, Eagle's Minimum Essential Medium (MEM) alpha medium, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 medium, 199 medium, and F12 medium. In addition, additives, such as horse serum, calf serum, or fetal bovine serum, may be added to the minimal essential medium. Alternatively, the medium can be serum-free. In other cases, the growth medium can include "Knockout serum replacement", which as used herein refers to a serum-free formulation optimized for growing and maintaining undifferentiated cells, such as stem cells, in culture. KNOCKOUT™ serum replacement is disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference. In some embodiments, PSCs are cultured in a fully defined medium and a feeder-free medium.

[0147] Thus, single cell PSCs are generally cultured in fully defined medium after plating. In some aspects, about 18-24 hours after plating, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In some aspects, single cell PSCs are cultured in fully defined medium for about 1, 2, or 3 days after plating. In some non-limiting examples, single cell PSCs are cultured in fully defined medium for about 2 days before proceeding with the differentiation process.

[0148] In some embodiments, the medium may include a serum replacement. The serum replacement may include materials that suitably include: albumin (such as lipid-rich albumin, or albumin substitutes, such as 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 equivalents thereof. The serum replacement may be prepared, for example, by the method disclosed in International Publication No. WO 98 / 30679. Alternatively, any commercially available material may be used for added convenience. Commercially available materials include KNOCKOUT™ Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and GLUTAMAX™ (Gibco). The medium may be serum-free.

[0149] Other culture conditions can be appropriately determined. For example, the culture temperature can be about 30-40 degrees, for example, at least 31, 32, 33, 34, 35, 36, 37, 38, 39 degrees, or about 31, 32, 33, 34, 35, 36, 37, 38, 39 degrees, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37 degrees. The CO2 concentration can be about 1-10%, for example, about 2-5%, or any range derivable therein. The oxygen partial pressure can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein, or up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0150] b. Differentiation medium i. Retinal induction medium After the single cell PSC adheres to the culture plate, the cell is preferably cultured in retinal induction medium to start the differentiation process into retinal lineage cells.The retinal induction medium (RIM) comprises a WNT pathway inhibitor and can induce the differentiation of PSC into retinal lineage cells.The RIM further comprises a TGFβ pathway inhibitor and a BMP pathway inhibitor.

[0151] The RIM may comprise DMEM and F12 at a ratio of about 1:1. In an exemplary method, the RIM comprises a WNT pathway inhibitor, such as CKI-7, a BMP pathway inhibitor, such as LDN193189, and a TGFβ pathway inhibitor, such as SB431542. For example, the RIM comprises about 5 nM to about 50 nM, such as about 10 nM, LDN193189, about 0.1 μM to about 5 μM, such as about 0.5 μM, CKI-7, and about 0.5 μM to about 10 μM, such as about 1 μM, SB431542. In addition, the RIM may include knockout serum replacement, such as about 1% to about 5% knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and insulin growth factor 1 (IGF1). In some embodiments, the IGF1 is animal-free IGF1 (AF-IGF1) and is included in the RIM at about 0.1 ng / mL to about 10 ng / mL, such as about 1 ng / mL. The medium is, for example, aspirated and replaced with fresh RIM every day. To generate retinal lineage cells, the cells are generally cultured in the RIM for about 1 to about 5 days, such as about 1, 2, 3, 4, or 5 days, such as about 2 days.

[0152] ii. Retinal Differentiation Medium The retinal lineage cells can then be cultured in retinal differentiation medium (RDM) for further differentiation. The RDM comprises a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. In one embodiment, the RDM comprises a WNT pathway inhibitor, such as CKI-7, a BMP pathway inhibitor, such as LDN193189, a TGFβ pathway inhibitor, such as SB431542, and a MEK inhibitor, such as PD0325901. Alternatively, the RDM can comprise a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a bFGF inhibitor. In general, the concentrations of the Wnt pathway inhibitor, the BMP pathway inhibitor, and the TGFβ pathway inhibitor are higher in the RDM than in the RIM, such as about 9 to about 11 times higher, such as about 10 times higher. In an exemplary method, the RDM comprises about 50 nM to about 200 nM of LDN193189, such as about 100 nM, about 1 μM to about 10 μM of CKI-7, such as about 5 μM, about 1 μM to about 50 μM of SB431542, such as about 10 μM, and about 0.1 μM to about 10 μM of PD0325901, such as about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM.

[0153] Typically, the RDM comprises about 1:1 ratio of DMEM and F12, knockout serum replacement (e.g., about 1% to about 5%, such as about 1.5%), MEM NEAA, sodium pyruvate, N-2 supplement, B-27 supplement, ascorbic acid, and IGF1 (e.g., about 1 ng / mL to about 50 ng / mL, such as about 10 ng / mL). In certain methods, the cells are fed with fresh RDM every day after aspirating the medium from the previous day. Typically, to induce differentiated retinal cells, the cells are cultured in RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, such as about 7 days.

[0154] iii. Retinal culture medium The differentiated retinal cells can then be further differentiated by culturing the cells in retinal medium (RM). The retinal medium contains activin A and may additionally contain nicotinamide. The RM may contain about 50 to about 200 ng / mL, such as about 100 ng / mL, of activin A and about 1 mM to about 50 mM, such as about 10 mM, of nicotinamide. Alternatively, the RM may contain other TGF-β pathway activators, such as GDF1 and / or WNT pathway activators, such as WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindirubin-3'-oxime), or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. Alternatively, the RM may additionally contain WNT3a.

[0155] The RM may include about 1:1 ratio of DMEM and F12, about 1% to about 5%, such as about 1.5%, of knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. The medium may be replaced daily with room temperature RM. Typically, to induce differentiating RPE cells, cells are cultured in RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days, such as about 10 days.

[0156] The retinal pigment epithelial cells produced by the methods disclosed herein may be cryopreserved, see, e.g., PCT Publication 2012 / 149484 A2, incorporated herein by reference. The cells may be cryopreserved with or without a substrate. In some embodiments, the storage temperature is in the range of 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 therebetween. In some embodiments, lower temperatures are used for storage (e.g., maintenance) of the cryopreserved cells. In some embodiments, liquid nitrogen (or other similar liquid coolant) is used to store the cells. In further embodiments, the cells are stored for more than about 6 hours. In additional embodiments, the cells are stored for about 72 hours. In some embodiments, the cells are stored for 48 hours to about 1 week. In yet other embodiments, cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, cells are stored for 1, 2, 3, 4, 5, 67, 8, 9, 10, 11, or 12 months. Cells can also be stored for longer periods. Cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.

[0157] In some embodiments, additional cryoprotectants may be used. For example, cells may be cryopreserved in a cryopreservation solution containing one or more cryoprotectants, such as DM80, serum albumin, and the like, such as human serum albumin or bovine serum albumin. In some 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 another particular embodiment, the solution comprises 10% DMSO.

[0158] During cryopreservation, the cells may be cooled, for example, at about 1 degree per minute. In some embodiments, the temperature of cryopreservation is about -80 degrees to about -180 degrees, or about -125 degrees to about -140 degrees. In some embodiments, the cells are cooled to 4 degrees before cooling at about 1 degree per minute. The cryopreserved cells may be transferred to liquid nitrogen in vapor phase before thawing for use. In some embodiments, for example, once the cells reach about -80 degrees, they are transferred to a storage area using liquid nitrogen. Cryopreservation may also be performed using a controlled rate freezer. The cryopreserved cells may be thawed, for example, at a temperature of about 25 degrees to about 40 degrees, and typically at a temperature of about 37 degrees. The cells are then matured on the scaffold, as discussed below.

[0159] iv. RPE maturation medium For further differentiation of RPE cells, the cells are preferably cultured in RPE maturation medium (RPE-MM). The RPE maturation medium may contain about 100 μg / mL to about 300 μg / mL of taurine, such as about 250 μg / mL, about 10 μg / L to about 30 μg / L of hydrocortisone, such as about 20 μg / L, and about 0.001 μg / L to about 0.1 μg / L of triiodothyronine, such as about 0.013 μg / L. In addition, the RPE-MM may contain MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), and sodium pyruvate, and fetal bovine serum (e.g., about 0.5% to about 10%, such as about 1% to about 5%). The medium may be replaced with RPE-MM at room temperature every other day. Typically, the cells are cultured in the RPE-MM for about 5 to about 10 days, such as about 5 days. The cells can then be dissociated, such as with a cell dissociation enzyme, reseeded, and cultured for an additional period of time for further differentiation into RPE cells, such as for an additional period of about 5 to about 30 days, such as about 15 to 20 days. In a further embodiment, the RPE-MM does not contain a WNT pathway inhibitor. The RPE cells can be cryopreserved at this stage.

[0160] b. Maturation of RPE cells on scaffolds and on tissue replacement implants The RPE cells can then be cultured in RPE-MM for a continuous period of maturation. In some embodiments, the RPE cells are grown in wells, such as in 6-well plates, 12-well plates, 24-well plates, or 10 cm plates. The RPE cells can be maintained on the scaffold in RPE medium for about 4 to about 10 weeks, such as about 6 to 8 weeks, such as 4, 5, 6, 7, or 8 weeks. In some non-limiting examples, to obtain a mature and functional RPE cell monolayer, the RPE cells are cultured on the scaffold in medium for about 2 to 6 weeks, such as about 5 weeks. This culture produces polarized RPE cells on the scaffold, which together form a tissue implant.

[0161] A variety of biological or synthetic solid matrix materials (i.e., solid support matrices, biological adhesives or coatings, and biological / medical scaffolds) are suitable for use as scaffolds. The matrix materials are generally physiologically acceptable and suitable for use in in vivo applications. Non-limiting examples of such physiologically acceptable matrices include, but are not limited to, biodegradable solid matrix materials, such as cross-linked or non-cross-linked alginates, hydrocolloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, polyglactin (PGL) meshes, and bioadhesives (e.g., fibrin glues and fibrin gels). The polymer can be poly(DL)-lactic-co-glycolic acid (PLGA) (see Lu et al., J. Biomater Sci Polym Ed. 9(11): 1187-205, 1998). In other embodiments, the matrix comprises poly-L-lactic acid (PLLA) and poly(D,L-lactic-co-glycolic acid) (PLGA), such as those having copolymer ratios (PLLA:PLGA) of about 90:10, 75:25, 50:50, 25:75, 10:90, etc. (see Thomson et al., J. Biomed. Mater Res. A 95: 1233-42, 2010).

[0162] Suitable polymeric carriers also include porous meshes or sponges formed from synthetic or natural polymers. A non-limiting example is a polymeric hydrogel. Natural polymers that can be used can include proteins, such as collagen, albumin, and fibrin; and polysaccharides, such as alginates and polymers of hyaluronic acid. Synthetic polymers can be biodegradable. Examples of biodegradable polymers include polymers of hydroxy acids, such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic acid-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof.

[0163] In some embodiments, the scaffold is a PLGA scaffold as disclosed below. PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). Polylactic acid contains an alpha chiral carbon and is typically described using traditional stereochemical terminology as D or L, and sometimes as R and S, respectively. The enantiomeric forms of the polymer PLA are poly D-lactic acid (PDLA) and poly L-lactic acid (PLLA). PLGA is a poly D, L-lactic-co-glycolic acid with approximately equal ratios of D-lactic acid and L-lactic acid. PLGA is biodegradable by hydrolysis of its ester bonds.

[0164] In some embodiments, the PLGA scaffold is cultured for a sufficient time to cause bulk lactate release from the scaffold in vitro. In some embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 90%, or more than 95% lactate release occurs in vitro. Lactate release occurs over time. Thus, in some embodiments, this effect is achieved by maintaining the RPE on the scaffold in RPE medium for about 4 to about 10 weeks, such as about 6 to 8 weeks, such as 4, 5, 6, 7, or 8 weeks. In some non-limiting examples, this effect is achieved by culturing the RPE cells on the scaffold in medium for about 2 to 6 weeks, such as about 5 weeks.

[0165] In some embodiments, the PLGA scaffold can have a DL-lactide / glycolide ratio of about 5:1 to about 1:5, such as about 4:1 to about 1:4, about 3:1 to about 3:3, about 2:1 to 1:2, etc. In one specific, non-limiting example, the DL-lactide / glycolide ratio is 1:1. In this context, "about" means within 5%.

[0166] In further embodiments, the PLGA scaffold is about 10 to about 50 microns thick, such as about 20 to about 40 microns thick, such as about 20 to about 30 microns thick. The thickness of the PLGA scaffold can be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 microns. In this context, about means within 5%.

[0167] The scaffold has nanofibers that cross each other, such that the nanofibers cross and form contacts. In the PLGA scaffold, the scaffold can be treated so that the fibers of the scaffold fuse at the contacts where the fibers cross, increasing mechanical strength. The average pore size is the gap between the fibers in the PLGA scaffold.

[0168] In additional embodiments, the PLGA scaffold has a pore size of less than about 2 microns, such as less than about 1.5 microns, less than about 1.25 microns, or less than about 1 micron. In some embodiments, the PLGA scaffold has a pore size of about 0.5 microns to about 2 microns, about 0.5 to 1 micron, about 1 to about 2 microns. The PLGA scaffold can have a pore size of about 0.5, 0.75, 1, 1.25, 1.5, 1.75, or 2 microns. In this context, about means within 5%.

[0169] In further embodiments, the PLGA scaffold has a fiber diameter of about 100 to about 700 nm, such as about 150 to about 650 nm, such as about 200 to about 600 nm, such as about 300 to about 500 nm. In some embodiments, the PLGA scaffold has a fiber diameter of about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, or 650 nm. In this context, "about" means within 5%.

[0170] It should be noted that any of the PLGA scaffold characteristics can be combined: thickness, pore size, and fiber diameter. One skilled in the art can understand that varying the DL-lactide / glycotide ratio will adjust the pore and fiber diameter. Thus, one skilled in the art can create PLGA scaffolds of the disclosed thickness, pore size, and fiber diameter. Any characteristics created by varying the DL-lactide / glycotide ratio can be combined to produce specific combinations. All of these are understood to be disclosed herein. In a specific, non-limiting example, the PLGA scaffold has a DL-lactide / glycotide ratio of 1:1, an average pore size of less than 1 micron, and a fiber diameter of 150-650 nm.

[0171] In some embodiments, in PLGA scaffolds, the scaffolds are heat treated so that the fibers of the scaffold fuse at the contact points (fiber intersections) to increase the mechanical strength of the PLGA scaffold. This heat treatment also reduces the pore size by fusing the fibers at the contact points, and thus allows cells to form a monolayer on top of the scaffold. In some non-limiting examples, in some embodiments, the scaffold is placed on a suitable surface and placed in an oven set at a desired temperature for treatment, where the surface is, for example, a metal surface, such as aluminum foil, which is, for example, in the shape of an envelope. Suitable temperatures include about 35°C to about 55°C, such as, for example, about 40°C to about 50°C, such as, for example, about 43°C, 44°C, 45°C, 46°C, or 47°C. The scaffold can be heated for about 5 to about 20 minutes, such as for about 10 to about 15 minutes, such as for about 10, 11, 12, 13, 14, or 15 minutes. In one embodiment, the scaffold is treated at about 45 degrees for about 10 minutes. The temperature can then be increased above the first temperature, such as for example, from about 50 degrees to about 70 degrees, such as for example, from about 55 degrees to about 60 degrees, such as for example, about 55 degrees, 56 degrees, 57 degrees, 58 degrees, 59 degrees, or 60 degrees. The higher temperature treatment can be for about 45 minutes to about 75 minutes, such as for example, from about 50 minutes to about 70 minutes, or from about 55 minutes to about 65 minutes. The higher temperature treatment can be applied for about 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 minutes. In some embodiments, the scaffold is treated at about 60 degrees for about 60 minutes. In one non-limiting example, the scaffold can be treated at about 45 degrees for about 10 minutes and then at about 60 degrees for about 60 minutes. After heat treatment, the scaffold can be stored.

[0172] In some embodiments, the PLGA scaffold is coated with vitronectin. In other embodiments, the PLGA scaffold is coated with extracellular matrix or gelatin. This can be done after heating the scaffold as disclosed above.

[0173] In other embodiments, the PLGA scaffold is coated with extracellular matrix. Extracellular matrix is ​​a complex mixture of structural and functional biomolecules and / or biopolymers, and is acellular unless otherwise indicated, including but not limited to structural proteins, specialized proteins, proteoglycans, glycosaminoglycans, and growth factors that surround and support cells in mammalian tissues. Extracellular matrix is ​​disclosed, for example, but not limited to, in the following U.S. Patents: 4,902,508; 4,956,178; 5,281,422; 5,352,463; 5,372,821; 5,554,389; 5,573,784; 5,645,860; 5,771,969; 5,753,267; 5 ,762,966;5,866,414;6,099,567;6,485,723;6,576,265;6,579,538;6,696,270;6,783,776;6,793,939;6,849,273;6,852,339;6,861,074;6,887,495;6,890,562;6,890,563; 6,890,564; and 6,893,666; each of which is incorporated by reference in its entirety. However, ECM can be made from any tissue or any in vitro source in which the ECM is produced from cultured cells and which contains one or more polymeric components (constituents) of native ECM. ECM preparations can be considered to be "decellularized" or "acellular," meaning that cells have been removed from the source tissue or culture.

[0174] In some embodiments, the ECM is isolated from a vertebrate, for example, from a mammalian vertebrate, including but not limited to human, monkey, pig, cow, sheep, etc. The ECM can be derived from any organ or tissue, including but not limited to bladder, intestine, liver, heart, esophagus, spleen, stomach, and dermis. In a specific non-limiting example, the extracellular matrix is ​​isolated from esophageal tissue, bladder, small intestinal submucosa, dermis, umbilical cord, pericardium, cardiac tissue, or skeletal muscle. The ECM can include any part or tissue obtained from an organ, including but not limited to, for example, submucosa, epithelial basement membrane, lamina propria, etc. In one non-limiting embodiment, the ECM is isolated from the bladder.

[0175] The scaffold may include a drug of interest. In some embodiments, the scaffold provides sustained release of one or more drugs. In further embodiments, the drug is a molecule that inhibits dedifferentiation (or epithelial-mesenchymal transition) of RPE cells, or inhibits the formation of drusen deposits under RPE cells, or suppresses reactive oxygen species in RPE cells. In some non-limiting examples, the inhibitor of dedifferentiation of RPE cells can be L,745,870 (3-([4-(4-chlorophenyl)piperazin-1-yl]methyl)-1H-pyrrolo[2,3-b]pyridine), or a dopamine receptor inhibitor. The drug can be metformin, Nox4 inhibitor, reactive oxygen inhibitor aminocaproic acid, riluzole, or an NK-κβ inhibitor. In a specific non-limiting example, the drug is L,745,870. In another specific non-limiting example, the drug is metformin. In further non-limiting examples, the agent is a Nox4 inhibitor (VAS2870, GKT 137831, or GLX7013114). In further non-limiting examples, the agent is a reactive oxygen species inhibitor (GKT 137831, or GLX7013114, or N-acetylcysteine).

[0176] Prior to seeding the retinal pigment epithelial cells onto the scaffold, the scaffold may be sterilized. In some embodiments, gamma irradiation is utilized to sterilize the scaffold. In other embodiments, electron beam (e-beam) is used to sterilize the scaffold. Exemplary methods are disclosed, for example, in Bruyas et al., Tissue Eng. Part A, doi: 10.1089 / ten.TEA.2018.0130 (September 20, 2018), and Proffen et al., J. Orthop. Res. 33(7) 1015-1023 (2015).

[0177] In some embodiments, retinal pigment epithelial cells are seeded onto the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold, such as, for example, about 150,000 cells per 12 mm diameter of the PLGA scaffold, about 200,000 cells per 12 mm diameter of the PLGA scaffold, about 250,000 cells per 12 mm diameter of the PLGA scaffold, about 300,000 cells per 12 mm diameter of the PLGA scaffold, about 350,000 cells per 12 mm diameter of the PLGA scaffold, about 400,000 cells per 12 mm diameter of the PLGA scaffold, or about 450,000 cells per 12 mm diameter of the PLGA scaffold.

[0178] In some embodiments, mature RPE cells develop into a functional RPE cell monolayer, and the monolayer acts as an intact RPE tissue by continued culture in RPE-MM with additional chemicals or small molecules that promote maturation of RPE on the scaffold. In some embodiments, these small molecules are inducers of primary cilia, such as prostaglandin E2 (PGE2) or aphidicolin. PGE2 can be added to the medium at a concentration of about 25 μM to about 250 μM, such as about 50 μM to about 100 μM. Alternatively, the RPE-MM can include a canonical WNT pathway inhibitor. Exemplary canonical WNT pathway inhibitors are N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP2), or 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (endo-IWR1).

[0179] In some embodiments, for continued maturation of RPE cells, the cells can be dissociated using a cell dissociating enzyme, such as TRYPLE™, and reseeded on a scaffold, such as a specialized SNAPWELL™ design, in RPE-MM containing a MEK inhibitor, such as PD0325901, for at least about 1-2 weeks. Alternatively, the RPE-MM can contain a bFGF inhibitor rather than a MEK inhibitor. A suitable method for culturing RPE cells on a degradable scaffold is taught and described in PCT Publication WO 2014 / 121077, which is incorporated herein by reference in its entirety. Briefly, the main components of this method are a CORNING® COSTAR® SNAPWELL™ plate, a bioinert O-ring, and a biodegradable scaffold, such as any of the PLGA scaffolds described above. The SNAPWELL™ plate provides a structure and platform for the biodegradable scaffold. The microporous membrane that creates the apical and basal sides supports the scaffold and separates the polarized layer of cells. The ability of the SNAPWELL™ insert to have a removable membrane allows the support ring of the insert to be used as an anchor for the scaffold (see below). The resulting differentiated, polarized, and confluent functional RPE cell monolayer on the scaffold can then be isolated and used as a tissue replacement implant.

[0180] In yet another embodiment, the RPE cells on the scaffold are maintained at a resting potential of about -50 to about -60 mV and at about 5 to about 10 μl cm -2 h -1In additional embodiments, the RPE cells express MITF, PAX6, LHX2, TFEC, CDH1, CDH3, CLDN10, CLDN16, CLDN19, BEST1, TIMP3, TRPM1, TRPM3, TTR, VEGFA, CSPG5, DCT, TYRP1, TYR, SILV, SIL1, MLANA, RAB27A, OCA2, GPR143, GPNMB, MYO6, MYRIP, RPE65, RBP1, RBP4, RDH5, RDH11, RLBP1, MERTK, ALDH1A3, FBLN1, SLC16A1, KCNV2, KCNJ13, and CFTR, express miR204 and miR211, have a resting potential of about -50 to about -60 mV, and have a fluid transport rate of about 5 to about 10 μl cm -2 h -1 In other embodiments, the RPE cells have a fluid transport rate of 150 Ω*cm 2 Above 200 Ω*cm 2 In a further embodiment, the RPE cells have a transepithelial resistance of greater than 200 Ω*cm. 2 ~500 Ω*cm 2 For example, the transepithelial resistance of 200 Ω*cm 2 ~400 Ω*cm 2 and other transepithelial resistance.

[0181] In one non-limiting example, a method for making a tissue replacement implant includes: a) obtaining a vitronectin coated PLGA, the PLGA scaffold comprising fibers forming a mesh structure, and the PLGA scaffold having an upper surface and a lower surface, the PLGA scaffold being about 20 to about 30 microns thick, the PLGA scaffold having a DL-lactide / glycolide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm; b) heat treating the PLGA scaffold such that the fibers of the scaffold fuse at the fiber intersections in the PLGA scaffold, increasing the mechanical strength of the PLGA scaffold and decreasing the pore size; c) seeding retinal pigment epithelial cells onto the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold; and d) on a PLGA scaffold in tissue culture medium, with medium present on both the top and bottom surfaces of the PLGA scaffold; i) Retinal pigment epithelial cell polarization, and ii) Bulk degradation of PLGA scaffolds Culturing the retinal pigment epithelial cells in vitro for a period of time sufficient for the

[0182] Use of inhibitors in the preparation of tissue replacement implants Disclosed below are inhibitors useful in the preparation of RPE cells and the disclosed tissue implants.

[0183] 1. WNT pathway inhibitors WNTs are a family of highly conserved secreted signaling molecules that regulate cell-cell interactions and are related to the Drosophila segment polarity gene, wingless. In humans, the WNT family of genes encode 38-43 kDa cysteine-rich glycoproteins. WNT proteins have a hydrophobic signal sequence, a conserved asparagine-linked 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 stabilization of β-catenin in the cytoplasm, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain cancers.

[0184] In this specification, WNT inhibitor refers to general WNT inhibitor.Therefore, WNT inhibitor refers to any inhibitor of WNT family protein member, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt4, Wnt5A, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt9A, Wnt10a, Wnt11 and Wnt16.Some embodiment of the method relates to WNT inhibitor in differentiation medium. Examples of suitable WNT inhibitors are already known in the art and 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]pyrimidin-2-yl)thio]-acetamide (IWP2), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-3-(2-methoxyphenyl)-4-oxothieno[3,2-d]pyrimidin-2-yl)thio]-acetamide (IWP4), 2-phenoxybenzoic acid-[(5-methyl-2-furanyl)methylene]hydrazide (PNU 74654). 2,4-diamino-quinazoline, quercetin, 3,5,7,8-tetrahydro-2-[4-(trifluoromethyl)phenyl]-4H-thiopyrano[4,3-d]pyrimidin-4-one (XAV939), 2,5-dichloro-N-(2-methyl-4-nitrophenyl)benzenesulfonamide (FH 535), N-[4-[2-ethyl-4-(3-methylphenyl)-5-thiazolyl]-2-pyridinyl]benzamide (TAK 715), Dickkopf-related protein one (DKK1), and secreted frizzled-related protein one (SFRP1). In addition, inhibitors of WNT can include antibodies against WNT, dominant negative variants of WNT, and siRNA and antisense nucleic acids that suppress the expression of WNT. Inhibition of WNT can also be achieved by using RNA interference (RNAi).

[0185] 2. BMP Pathway Inhibitors Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor β (TGFβ) superfamily. BMPs constitute a group of important morphogenetic signals that are thought to regulate architecture throughout the body. The important physiological functions of BMP signals are highlighted by the existence of numerous pathological processes that involve dysregulation of BMP signaling.

[0186] BMP pathway inhibitors may include inhibitors of general BMP signaling or inhibitors specific for BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, or BMP15. Exemplary BMP inhibitors include: 4-(6-(4-(piperazin-1-yl)phenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline hydrochloride (LDN193189), 6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine dihydrochloride (Dorsomorphin), 4-[6-[4- (1-methylethoxy)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline (DMH1), 4-[6-[4-[2-(4-morpholinyl)ethoxy]phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (DMH-2), and 5-[6-(4-methoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline (ML 347).

[0187] 3. TGFβ Pathway Inhibitors Transforming growth factor beta (TGFβ) is a secreted protein that controls the proliferation, differentiation, and other functions of most cells. It is a type of cytokine that plays a role in immunity, cancer, bronchial asthma, pulmonary fibrosis, heart disease, diabetes, and multiple sclerosis. TGF-β exists as at least three isoforms called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a protein superfamily known as the transforming growth factor beta superfamily, which includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1.

[0188] TGFβ pathway inhibitors may include any of the common TGFβ signaling inhibitors. For example, TGFβ pathway inhibitors include: 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542), 6-[2-(1,1-dimethylethyl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-4-yl]quinoxaline (SB525334), 2-(5-benzo[1,3]dioxol-5-yl-2-ieri-butyl-3H-imidazol-4-yl)-6-methyl Pyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[1,3]dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl)-benzamide hydrate, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide hydrate, left-right determinant (lefty), 3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide (A 83-01), 4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (D 4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline (LY 364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazol-4-yl]phenyl]-1H-pyrazole-1-ethanol (R 268712), or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (RepSox).

[0189] 4. MEK Inhibitors MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2. They can be used to affect the MAPK / ERK pathway. For example, 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]pyrimidin-1-yl]phenyl]acetamide (GSK1120212), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120213), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120214), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120215), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120216), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120217), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120218), 6-(4-bromo-2-fluoroanilino)-7-fluoropyrimidin-1-yl]phenyl]acetamide (GSK1120219), 6-(4-bromo-2-fluoroanilino)-7-fluoropy 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).

[0190] 5. bFGF Inhibitors Basic fibroblast growth factor (also known as bFGF, FGF2, or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the basement membrane and in the subendothelial extracellular matrix of blood vessels. In addition, bFGF is a common component of human ESC culture medium and is required to keep cells in an undifferentiated state.

[0191] In this specification, bFGF inhibitor refers to a general bFGF inhibitor. For example, bFGF inhibitors include, but are not limited to, 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 (PD 98059), 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.

[0192] Uses of tissue replacement implants and methods of treatment A tissue replacement implant is provided that comprises a biodegradable scaffold and an effective amount of RPE cells. The tissue replacement implants described herein and pharmaceutical compositions comprising these implants can be used to manufacture medicaments for treating disorders in patients in need thereof.

[0193] In some embodiments, the disclosed RPE cells are derived from iPSCs and can therefore be used to provide "personalized medicine" to patients with eye diseases. In some embodiments, cells obtained from a patient, such as somatic or CD34+ cells, or umbilical cord cells, can be used to generate iPSCs, which can then be used to generate RPE cells. In some embodiments, the RPE cells (or starting iPSCs) can be genetically engineered to correct disease-causing mutations, differentiate into RPE, and engineered to form a tissue implant. This tissue replacement implant can be used to replace the endogenous degenerated RPE of the same subject.

[0194] Alternatively, iPSCs can be generated from iPS cells of healthy donors, or from iPS cells of HLA homozygous "super donors" or "universal" donors, and used to prepare tissue implants. To create an anti-inflammatory and immunosuppressive environment in vivo, RPE cells can be treated in vitro with several factors, such as pigment epithelium-derived factor (PEDF), transforming growth factor (TGF)-β, and / or retinoic acid. These "superdonor" iPSCs are commercially available, see Cellular Dynamics International (see, e.g., internet: globenewswire.com / news-release / 2015 / 02 / 09 / 704392 / 10119161 / en / Cellular-Dynamics-Manufactures-cGMP-HLA-Superdonor-Stem-Cell-Lines-to-Enable-Cell-Therapy-With-Genetic-Matching, February 15, 2015).

[0195] The subject may be a human subject or a veterinary subject. The RPE cells may be derived from a single subject, or several populations of RPE cells, for example 1, 2, 3, 4, or 5 types of RPE, each derived from a different subject, may be used in the implant.

[0196] A variety of eye disorders can be treated or prevented by the introduction of tissue implants. The disorders generally include retinal diseases or disorders associated with retinal dysfunction or deterioration, retinal damage, and / or loss of retinal pigment epithelium. The disclosed tissue replacement implants are useful for treating retinal degenerative diseases, retinal or retinal pigment epithelial dysfunction, retinal deterioration, damage to the retina or to the retinal pigment epithelium, for example, damage caused by light, laser, inflammation, infection, radiation, neovascularization, or trauma. The disclosed tissue replacement implants are also useful for treating loss of retinal pigment epithelium. The method includes locally administering the tissue replacement implant to the subject's eye.

[0197] In some embodiments, the retinal degenerative disease is Stargardt's macular dystrophy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, Leber's congenital amaurosis, late-onset retinal degeneration, hereditary macular degeneration or acquired retinal degeneration, Best's disease, Sorsby's fundus dystrophy, retinal detachment, gyrate atrophy, eye trauma, or choroideremia, pattern dystrophy.Additional abnormalities include retinal detachment, pattern dystrophy, and other dystrophy of RPE.In certain non-limiting examples, the subject has age-related macular degeneration.In some embodiments, a method for treating or preventing abnormalities characterized by retinal degeneration is provided, the method comprises administering tissue replacement implant to a subject in need thereof.

[0198] These methods may include selecting a subject with one or more of these abnormalities, and administering tissue replacement implant to treat the abnormality and / or improve the symptoms of the abnormality.Tissue replacement implant can also be transplanted with other retinal cells, such as photoreceptor cells (co-transplantation).In some embodiments, the RPE cells in tissue replacement implant are derived from the subject to be treated and are therefore autologous.In other embodiments, the RPE cells in tissue replacement implant are made from MHC-matched donors or universal donors.In another embodiment, the RPE cells in tissue replacement implant are allogeneic.

[0199] The tissue replacement implant can be introduced into various target sites in the eye of a subject. In some embodiments, the tissue replacement implant is introduced into the subretinal space of the eye, for example by implantation, where the subretinal space is the anatomical location of the RPE in mammals (between the photoreceptor outer segment and the choroid). Exemplary methods are disclosed, for example, in PCT Publication WO2018 / 089521, which is incorporated herein by reference. In some embodiments, the tissue replacement implant is introduced into the outer retina, the retinal periphery, the macula or the area around the macula, or into the choroid. In addition, depending on the migration ability and / or positive paracrine effect of the cells, introduction into additional compartments of the eye can be considered, such as the vitreal space, the inner or outer retina, the retinal periphery, and into the choroid.

[0200] The size of the tissue replacement implant to be implanted may generally be determined by comparing a clinical assessment of the size of the diseased area of ​​the retina present in a particular patient with the constraints imposed by the feasibility of surgery to deliver an implant of a particular size. For example, in degenerations affecting the central part of the retina (e.g., age-related macular degeneration), a circular implant with a diameter of about 1.0-2.5 mm (e.g., about 1.5 mm in diameter), approximating the size of the anatomical fovea, will often be appropriate. In some cases, a larger implant may be appropriate, corresponding to an oval area of ​​approximately 6.0 mm (vertical) x 7.5 mm (horizontal) at most, in the area of ​​the posterior retina (histological macula, clinical posterior pole) extending between the temporal vascular arcades, located in the center of the fovea or in the extrafoveal region. In some instances, it may be appropriate to create a polymer scaffold with smaller dimensions, as small as about 0.5 mm, for placement in the area of ​​the focal disease. Additionally, it may be of interest to create custom implants of irregular shapes for the patient, for example, to cover diseased areas while avoiding areas of residual high vision.

[0201] Tissue replacement implants can be introduced by various techniques known in the art. Methods for performing transplants are disclosed, for example, in U.S. Patent No. 5,962,027, U.S. Patent No. 6,045,791, and U.S. Patent No. 5,941,250; Biochem Biophys Res Commun Feb. 24, 2000; 268(3): 842-6; and Opthalmic Surg February 1991; 22(2): 102-8). Methods for performing corneal transplants are described, for example, in U.S. Patent No. 5,755,785; Curr Opin Opthalmol August 1992; 3 (4): 473-81; Ophthalmic Surg Lasers April 1998; 29 (4): 305-8; and Opthalmology April 2000; 107 (4): 719-24. In some embodiments, implantation is performed by pars plana vitrectomy followed by delivery of the tissue replacement implant into the subretinal space via a small incision in the retina. Alternatively, the tissue replacement implant can be delivered into the subretinal space via a transscleral or transchoroidal approach. In addition, direct transscleral insertion into the anterior retinal periphery adjacent to the ciliary body can be performed.

[0202] The tissue replacement implant can also be transplanted together with other cells (co-transplantation), such as photoreceptor cells.

[0203] In some embodiments, the method includes administering an immunosuppressant that reduces the immune response, for example, by downregulating the response of inflammatory cells or by inducing apoptosis of inflammatory cells. In other embodiments, the method includes administering a therapeutically effective amount of a neuroprotective agent that promotes survival of retinal neurons and / or reduces degeneration of retinal neurons. In yet other embodiments, the method may include administering a therapeutically effective amount of an agent that inhibits unwanted angiogenesis, for example, preventing the growth of choroidal neovascularization (CNV) under the fovea in AMD patients. Exemplary therapeutic agents can reduce the activity of vascular endothelial growth factor (VEGF), for example, by binding to the receptor site of the active form of VEGF and interfering with the interaction of VEGF with its receptor. Therapeutically effective amounts of agents that suppress the expression of VEGF by inhibiting pathways that lead to the secretion of VEGF, such as STAT3, NF-κB, HIF-1α, etc. Other agents can prevent the atrophy of RPE cells by targeting the complement pathway, autophagy, or NF-kB pathway.

[0204] In further embodiments, the method includes administering to the subject a therapeutically effective amount of ciliary neurotrophic factor (CNTF), brain-derived neurotrophic factor (BDNF), or pigment epithelium-derived factor (PEDF), which may be used, for example, to promote the development or function of neurons, such as photoreceptors. Other exemplary, non-limiting embodiments include administering to the subject a therapeutically effective amount of thrombospondin 1, an anti-inflammatory cytokine (e.g., interleukin (IL)-lra, IL-6, Fas ligand, or tumor growth factor (TGF)-β, a neurotrophic / neuroprotective growth factor, such as, but not limited to, glial cell line-derived growth factor, brain-derived neurotrophic factor, nerve growth factor, neurotrophin 3, neurotrophin 4 / 5, neurotrophin 6, and vitamin E. Such agents may be provided alone or in combination.

[0205] The present disclosure is illustrated by the following non-limiting examples. EXAMPLES

[0206] Good Manufacturing Practice (GMP) / clinical-grade manufacturing methods were used to create AMD patient-specific iPSC-derived RPE (iRPE) patches using biodegradable scaffolds. These patches were tested in two different animal models. Autologous iRPE patches avoid immune rejection by the patient's cells, and these iRPE cells do not display any AMD cellular phenotype, allowing increased viability and integration of the autologous iRPE patch in the patient's eye. This approach of implanting the patch at the border of dry AMD lesions rescues photoreceptors in the transition zone, an area where the RPE is atrophied but photoreceptors still survive (Bird, et al., JAMA Ophthalmol 132, 338-345 (2014)). Furthermore, these patches can be created using major histocompatibility (MHC)-matched RPE cells or using heterologous RPE cells. These patches may contain RPE cells from a single subject or from multiple subjects.

[0207] In the study disclosed herein, the use of CD34+ cells isolated from peripheral blood of AMD patients allowed the development of clinical grade iPSC banks without oncogene mutations. These iPSC banks were used to develop a more efficient and reproducible clinical grade RPE differentiation process compared to research grade differentiation. Evidence is provided that iRPE patches derived from three AMD patients do not display any cellular phenotype of the disease and mature and function to the same extent. In addition, seeding of iRPE cells onto biodegradable substrates was demonstrated to significantly improve the incorporation of RPE monolayers compared to transplanted cell suspensions in immunocompromised rats, in rats with RPE dysfunction associated with retinal degeneration, and in a pig model of laser-induced RPE damage in which clinical doses of iRPE patches were tested. These experiments provide a complete workflow to demonstrate the efficacy of these tissue implants and to perform preclinical trials in support of autologous iPSC-based Phase I clinical trials for macular degeneration and other indications (Figure 1A).

[0208] Example 1 material and method Research- and clinical-grade iPSC derivation, maintenance, and RPE differentiation: Reporter iPSC lines expressing GFP under the control of the tyrosinase enhancer and constitutive RFP were previously published (Maruotti et al., Proc Natl Acad Sci USA 112, 10950-10955 (2015)) and were used to optimize the research-grade differentiation protocol. iPSCs were cultured in MEFs for 4 days before being used for differentiation. To form cell aggregates, iPSCs were treated with collagenase for 20 minutes. After the collagenase was aspirated, NEIM (DMEM / F12, KOSR, supplemented with N2, B27, LDN-193189 10 uM, SB431452 10 nM, CKI-7 hydrochloride 0.5 uM, and IGF-1 1 ng / ml) was added to the wells (1 ml / well) and a cell scraper was used to scrape off the colonies. The aggregates were then cut into 10 cm pieces. 2The cells were grown in NEIM medium in low-attachment Corning dishes for 48 hours. After 48 hours, floating cell aggregates were collected and seeded onto MATRIGEL®-coated plates in RPEIM (DMEM / F12, KOSR, supplemented with N2, B27, LDN-193189 100 uM, SB431452 100 nM, CKI-7 hydrochloride 5 uM, and IGF-1 10 ng / ml, PD0325901 1 uM) and cultured for 3 weeks. After 3 weeks of culture in RPEIM, the cells were transferred to RPECM (DMEM / F12, KOSR, supplemented with N2, B27, nicotinamide 10 mM, and activin A 100 ng / ml) and cultured for another 3 weeks with regular medium changes. Pigmented patches of immature RPE cells were collected by differential trypsinization and seeded in Transwell or T-25 flasks in RPEGM (MEM, Sigma; 1% N2 supplement, ThermoFisher; 1% glutamine, ThermoFisher; 1% non-essential amino acids, ThermoFisher; 125 mg taurine (Sigma) / 500 ml; 10 μg hydrocortisone (Sigma) / 500 ml; 0.0065 μg triiodothyronine (Sigma) / 500 ml; 5% FBS, Sigma) (Maminishkis et al., C Invest Ophthalmol Vis Sci 47, 3612-3624 (2006)). Table 1 provides a list of all the reagents used in the fabrication process. Flow cytometry was performed to confirm GFP expression in differentiating cells at various time points.

[0209] Table 1. Detailed list of clinical grade reagents used for iPSC production and differentiation into RPE. TIFF2025004058000001.tif126167TIFF2025004058000002.tif232167TIFF2025004058000003.tif38167Certificates of Analysis for all reagents have been verified for all of these reagents due to our clinical grade manufacturing process.

[0210] Feeder-free iPSC clones were derived from CD34+ PBMCs using a clinical-grade protocol as previously published (Mack, et al., PLoS One 6, e27956 (2011)). Minibanks of up to eight clones were verified for pluripotency by flow cytometry, sterility (WuXiApp Tech, Marietta, GA), normal G-banded karyotype (Cell Line Genetics, Madison, WI), STR identity (Univ. Madison Clinics, Madison, WI), loss of plasmids (Cellular Dynamics Inc., Madison, WI), and cancer gene sequencing (Q2 Solutions, Morrisville, NC). iPSC cells were then seeded on vitronectin (A14701S, ThermoFisher, Carlsbad, CA)-coated surfaces in E8 medium (A1517001, ThermoFisher, Carlsbad, CA). After 2 days, cells were transferred to RPEIM and cultured for 10 days, and then transferred to RPECM and cultured for another 10 days. On day 22, cells were switched to RPEGM, and on day 27, cells were trypsinized and reseeded on RPEGM. On day 42, cells were reseeded onto scaffolds in RPEMM (MEM, Sigma; 1% N2 supplement, ThermoFisher; 1% glutamine, ThermoFisher; 1% non-essential amino acids, ThermoFisher; 125 mg taurine (Sigma) / 500 ml; 10 ug hydrocortisone (Sigma) / 500 ml; 0.0065 ug triiodothyronine (Sigma) / 500 ml; 5% FBS, Sigma; 50 uM PGE2, R&D Biosystems).

[0211] Real-time PCR: Total RNA was isolated using NucleoSpin RNA (#740955, Machery-Nagel) as per protocol. RNA was quantified using an ND-1000 spectrophotometer (Nanodrop Technologies). cDNA synthesis and custom-made 24 RPE gene array plates were purchased from Bio-rad. SYBR Green-based QPCR was performed on a ViiA 7 real-time PCR system (Thermo Fisher Scientific) according to the manufacturer's protocol. Each sample was tested in at least three biological replicates, and data were analyzed in R-based software.

[0212] Transepithelial resistance: Electrical intactness was measured using an EVOM2 and EndOhm chamber (World Precision Instruments), and each clone had its resistance measured in three biological replicates.

[0213] Methodology for hexagonality measurements: Cells were fixed in 4% paraformaldehyde and stained with Anti-Zonula Occludens-1 (ZO1) conjugated with AlexaFluor 594. Entire Transwells were mounted, and 2 mm x 4 mm x 60 micrometer sections of each well were imaged at 20x magnification using a Zeiss Axio Scan-1. Maximum intensity projections (MIPs) were then applied to the Z-stacks, and cells in the MIPs were analyzed for their boundaries using a convolutional neural network. Once cell boundaries were identified, binary "masks" were created to measure morphological properties. How close the RPE is to an ideal convex regular hexagon was measured using a novel metric known as "hexagonality". Briefly, hexagonality was assessed by taking the average of two ratios 10 times (Equation 3). The two ratios are the Hexagon Side Ratio (HSR), defined as Equation 1, and the Hexagon Area Ratio (HAR), defined as Equation 2. TIFF2025004058000004.tif51128In the above, P Cell is the periphery of the cell, and P Hull is the perimeter of the convex hull that encloses the cell, and A Cell is the area of ​​the cell, and A Hull is the area of ​​the convex hull.

[0214] Phagocytosis of photoreceptor outer segments: The phagocytic capacity of iRPE cells was measured using a published protocol with minor modifications (55). Bovine photoreceptor outer segments (POS) (InVision Bio) were labeled with pH-rodo dye (ThermoFisher) according to the manufacturer's protocol. Mature iRPE cells after 5 weeks of culture on Transwell or PLGA scaffolds were loaded with POS at a concentration of 5 cells / RPE cell for 4 hours at 37°C. Cells were washed three times with DPBS and incubated in 0.25% trypsin for 20 minutes. Trypsinized cells were collected with a 1 ml pipette and suspended in a 15 ml tube containing 10 ml of RPE MM. The 15 ml tube with cells was centrifuged at 400 g for 5 minutes, the cell pellet was washed three times in 10 ml DPBS, resuspended in 10 ml DPBS, and the cell suspension was passed through a 0.44 um cell strainer.

[0215] Cell samples were run through a MACS Miltenyi flow cytometer. Live cells were selected as DAPI negative. A laser channel with excitation at 586 nm and emission at 515 nm was used to determine the fluorescence from the captured pH-rodo-labeled POS. Flow cytometry data was analyzed using flowjo software, and the median fluorescence for the channel Y1 positive population was calculated for spiked and unspiked samples. The ratio of spiked to unspiked samples was calculated and plotted on a graph.

[0216] Lactate measurements: PLGA scaffolds were incubated under the same conditions used to culture iPSC-RPE patches. The medium was changed every other day, and the culture medium was collected in 15 ml tubes. The medium from three consecutive medium changes was combined into one tube. The same procedure was performed for all six technical replicates. The collected medium was promptly frozen and stored at -80 degrees. Lactate measurements were performed. Each of the technical replicate tubes, from three consecutive medium collections, contained 12 ml of medium and was lyophilized to reduce the total volume. The lyophilized material was resuspended in 1 ml of 1x D-PBS. Lactate measurements were performed using a Lactate Gen. 2 instrument with a measurement range of 0.2–15.5 mmol / L (1.8–140 mg / dL).

[0217] Statistical Analysis: All statistical analyses were performed using R software with the Dunn's test package applicable. Data were first assessed for normality by determining the skewness, kurtosis, and qq plots of the data. All data for gene expression, TER, morphometry, and phagocytosis were found to have skewness or kurtosis values ​​outside the range of -1 to 1 and showed significant deviance in the qq plots and were therefore treated as non-normal. Therefore, Dunn's test was used to perform multiple pairwise comparisons after Kruskal-Wallis test was performed for probabilistic superiority among k groups. Bonferroni-Dunn correction was used for all pairwise comparisons and an adjusted alpha of 0.05 was used for significance. For principal component analysis, data from phagocytosis, TER, and gene expression profiles across all days and all clones were scaled from 0 to 1 with all data pooled for each measurement. PCA was performed and clustering was demonstrated based on k nearest neighbors.

[0218] Cancer gene coding variant analysis: Coding and exonic regions across 223 cancer genes were analyzed 2 Each iPSC clone and associated PBMC donor were sequenced at high depth by QMS Solutions (Morissville, NC). Variants labeled as potentially pathogenic were identified using QMS. 2 The variants were detected using Tute Genomics in the variant call files (VCF) provided by . In addition, three different somatic callers (Mutect version 1.15, SomaticSniper version 1.0.5.0; and Strelka version 1.0.15) were used to compare each iPSC clone with matched PBMCs. This parallel analysis found no novel mutations in either exons or splice sites.

[0219] Animals: Castrated Yucatan miniature pigs (RRID: NSRRC_0012) and Yorkshire pigs (age: 5-11 months; weight: 30-40 Kg), Crl: NIH-Foxn1 rnu Immunodeficient rats and Royal College of Surgeon rats were used. All animals were fed a diet appropriate for their weight.

[0220] Surgical delivery of cells in rodents: A. Suspension: RCS rats (RRID: RGD_1358258) or adult immunodeficient rats, Crl:NIH-Foxn1, were cultured on postnatal days (P) 21-28. rnuNude rats (RRID # RGD_2312499) were anesthetized with 2,2,2-tribromoethanol (intraperitoneally; 230 mg / kg; Sigma) and the eyes received topical anesthesia with 0.5% proparacaine HCl. Pupils were dilated with 1% tropicamide and 2.5% phenylephrine HCl, and the eyes were slightly pronated. Using the tips of needles of increasing gauge, small scleral / choroidal incisions (approximately 1 mm) were made 2 mm posterior to the limbus, in the dorsal temporal region. To limit the rise in intraocular pressure and to reduce the outflow of cells after injection, a small puncture in the outer cornea was made with a 30-gauge needle. Using a fine glass pipette (inner diameter 75–150 μm) inserted into the subretinal space, 2 microliters of suspension containing the total cell dose (100,000 cells) was delivered into the subretinal space of one eye. The conjunctiva was then replaced over the scleral incision. All animals included in the study received cell injections with a minimum of 90% pre- and post-injection cell viability. To minimize potential inflammatory responses, all RCS rats received daily intraperitoneal injections of dexamethasone (1.6 mg / Kg) for 2 weeks after cell implantation.

[0221] B. iRPE patch: Placement of the iRPE patch followed a similar procedure to that of the suspension injection, with the following exceptions: A subretinal bleb was created with 2-3 μl of balanced salt solution (BSS+) and the scleral incision was enlarged with an 18 ga needle to accommodate insertion of a 1 mm circular implant. A 1 mm punch of RPE scaffold was extracted from the culture plate using a sterile trephine. To protect and stabilize the scaffold during implantation, the 1 mm implant was held at its distal end with an ILM dissection forceps. The forceps were gently inserted into the subretinal space with the RPE facing the photoreceptors.

[0222] Optokinetic tracking: Optokinetic tracking (OKT) thresholds were measured using a virtual optomotor system (VOS; Cerebral Mechanics, Lethbridge, AB, Canada), which allows for independent assessment of both the left and right eye. Thresholds were assessed as P90 using methods described elsewhere (39, 56). Thresholds were assessed by a primary operator and confirmed by a second operator.

[0223] Immunosuppression: The tetracycline antibiotics doxycline and minocycline were used orally at a dose of 5 mg / Kg twice daily. An intramuscular loading dose of methylprednisolone was used at a dose of 5 mg / Kg followed by a similar oral dose of prednisone once daily. Rapamycin was used orally at a loading dose of 2 mg followed by a daily dose of 1 mg. Tacrolimus was used at an oral dose of 0.5 mg / day.

[0224] Laser Damage Model: An IQ 532 micropulse laser (Iridex, USA) fitted with a TXCELL™ scanning laser delivery device is used to selectively damage the RPE using a Volk HR Centralis contact lens (Volk Optical Inc. Mentor, OH) with a 74° field of view, 1 / 08x magnification, and 0.93x laser spot magnification. A micropulse power (1000-1600 mW) sufficient to lightly blanche the lasered area and an exposure time of 330 ms is used. A 7 x 7 confluent grid was placed over a 49 mm swath using a 1% duty cycle of micropulses (0.100 ms pulse "on" and 9.900 ms pulse "off") and a 200 micron spot size. 2 was created to create a lesion.

[0225] Implantation of iRPE patches in pigs: Sterilization of the surgical area with povidone-iodine, temporal canthotomy, retraction of the superior rectus muscle, and retraction of the nictitating membrane were performed to enlarge the surgical exposure area. A nasal corneal periclinotomy was made to expose the sclera, and four surgical ports (injection, chandelier lighting, and two working ports) were created 3.5 mm from the limbus using a 25G valved trocar cannula (Alcon surgical, Fort Worth USA). After vitrectomy and posterior vitreous detachment, a localized retinal detachment (RD) was made in the visual streak (laser area) using a 25G / 38G cannula (MedOne Surgical Inc. Sarasota, FL), and a retinotomy with scissors was made at the base of the RD. A scleral incision (2.3–2.5 mm) was made in the area of ​​the nasal port to pass the implantation instruments. The tip of the instrument was introduced into the subretinal space through the retinal incision, and the iRPE scaffold was released in the subretinal space with the aid of the viscous fluid injection device of the vitrectomy system (Alcon surgical, Fort Worth, USA). Fluid-air exchange was performed while maintaining intraocular pressure, and the scleral incision was closed with ophthalmic wound clamps to flatten the detached area, as confirmed by intraoperative OCT. The scleral incision was closed with nylon 8-0.

[0226] Optical coherence tomography and fluorescein angiography: After the animals were properly anesthetized, a jet electrode was attached to the eye along with an appropriate amount of GenTeal Tears (Alcon Fort Worth TX, NDC 0078-429-47). OCT was performed using a Spectralis (Heidelberg Engineering) with a 55 degree lens. The region of interest (ROI) was placed at the center, and both a single averaged B-scan across the ROI and a volumetric OCT scan covering the entire ROI were performed. The follow-up function of the instrument was used to allow OCT scans at the same location on the retina for each time point tested. The Spectralis was also used to obtain fluorescein angiograms by intravenous injection of sodium fluorescein (SF, Akorn Inc, Lake Forest, IL). Early (first minute) and late (15 minutes) fundus angiograms are recorded.

[0227] Multifocal electroretinogram (mfERG): mfERG was recorded using the RETImap system (Roland Consult, Brandenburg Germany). Briefly, a bipolar contact lens (The GoldLens Corneal Electrode, Doran Instruments, Inc; Littleton, MA) was placed on the eye. A ground electrode, a Genuine Grass Platinum subdermal needle electrode (F-E2-12, Natus Manufacturing Limited, Galaway Ireland), was placed under the skin on the animal's chin. A minimum of three cycles were used for each recording, and all three overlapping retinal areas were measured. To account for slight differences in optics, the ROI was shifted slightly around the periphery of the field of view for each recording. A low bandpass of 10 hz and a high bandpass of 300 hz were used. The seven components of the mfERG, as determined by the MATLAB program, were as follows: N1 (first main trough) amplitude and P1 (subsequent peak) amplitude (nV / deg2 ), N1P1 (difference in amplitude between N1 and P1), scalar product, AUC, and N1 width and P1 width (time in milliseconds at half maximum amplitude of peak). The seven mfERG components are normalized by subtracting the implant laser signal with the healthy area signal and dividing by the pre-laser signal. Linear mixed models (LME) and analysis of variance were performed to determine if groups were statistically different. The equation function used in the fitlme function was: data ~ week + group * component + (1 | pig name).

[0228] Immunostaining and histological analysis: Tissues were prepared for immunohistochemical analysis by placing the eyes in 4% PFA for up to 4 hours after enucleation. The surgical area was sectioned and placed in 10% sucrose / PBS overnight, then 20% sucrose / PBS for 24 hours. The specimens were then placed in a 2:1 OCT:20% sucrose solution and flash frozen in cryostat molds and stored in a -80 freezer until cryostat sectioning was possible. Cryostat sectioning was performed at 10 μm sections, with tissue sections cut every 50 μm. Immunohistochemical analysis was generally performed as follows: 5% native goat serum (NGS) (Thermo Fisher Scientific, Grand Island NY; #31873) blocking solution for 2 hours, followed by incubation with primary antibodies in 1% NHS overnight at room temperature.Primary antibodies included: RPE65 (1:300, Abcam, Cambridge, MA; ab78036, and custom antibodies from M. Redmond lab / NEI, 1:400), biotinylated peanut agglutinin (PNA) (1:300, Vector Laboratories, Burlingame CA; B-1075), STEM121 (1:300, Takara Clontech Mountain View CA; Y40410), STEM101 (1:300, Takara Clontech, Mountain View, CA), rhodopsin (1:10,000, Encor Biotechnology Inc. Gainesville FL, MCA-B630); red / green opsin (1:300, Millipore Burlingotn MA; AB5405); blue opsin (1:300, Millipore Burlingotn MA; AB5407); iPSC- OCT4 (#653704, Biolegend, RRID: AB_2562018), TRA 1-81 (#560124, BD Biosciences); and SSEA-4 (#560126, BD Biosciences); RPE progenitor cells - MITF (#X2397M, Exalpha), PAX6 (#PRB-278P, Covance); committed RPE - PMEL17 (#HMB45, Dako), TYRP1 (#NBP2-32901, Novus Biologicals), and mature RPE - BEST1 (#NB300-164, Novus Biologicals). Ezrin (#E8897, SigmaAldrich), type IV collagen (#ab6311, Abcam), ALDH1A3 (#ab80176, Santa Cruz Biotechnology). Following overnight incubation, tissue samples were washed three times in 1% NGS solution and incubated with appropriate primary antibodies at a 1:300 dilution in 1% NGS solution, followed by secondary antibodies conjugated to the fluorescent markers Alexa 488, Alexa 555, and / or Alexa 633 (Thermo Fisher Scientific, Grand Island NY).Slides were then imaged on either a Zeiss 800 confocal microscope or a Zeiss Axio Scan Z1 slide scanner. The number of nuclei (DAPI) in the implant area (either empty scaffold or iRPE patch) was normalized to the corresponding healthy area on the same section. For counting, the same distance across the retina (approximately 400 μm) was used for the implant area and the corresponding healthy area. (p < 0.05 in unpaired t-test; N = 4 for empty scaffold, N = 5 for iRPE patch).

[0229] Example 2 A clinical-grade, three-step protocol efficiently and reproducibly generates AMD-iRPE cells iPSCs derived from AMD patient dermal fibroblasts under clinical grade conditions generated chromosome copy number changes, which appeared to occur during the reprogramming process (Mandai et al., N Engl J Med 376, 1038-1046 (2017)). However, dermal fibroblasts are not considered an ideal starting point when deriving iPSCs from older patients (Kang et al., Cell Stem Cell 18, 625-636 (2016)). Thus, a manufacturing workflow for autologous iPSC-derived RPE patches from patient CD34+ peripheral blood cells was developed. A pipeline (Figure 1A, 1B) for the generation, functional validation, and in vivo testing of clinical grade, AMD patient-specific iPSC-RPE patches was developed. Due to their progenitor and proliferative properties, CD34+ cells isolated from patient peripheral blood may provide a good starting point for iPSC generation. Using a clinical-grade episomal reprogramming protocol (Mack, et al., PLoS One 6, e27956 (2011)), multiple iPSC clones were generated from CD34+ cells and dermal fibroblasts from three patients with advanced "dry" AMD (ages 85, 89, and 87 years). CD34+ cell-derived iPSCs were more genomically stable than dermal fibroblast-derived iPSCs (Mandai et al., N Engl J Med 376, 1038-1046 (2017)). Passage 10 banks were successfully generated from three iPSC clones / three patients (derived from CD34+ cells) (Table 2).

[0230] Table 2: Validation of the working bank of GMP grade AMD iPSCs. The iPSC working bank at passage 10 was validated for: sterility (free of bacteria, fungi, and mycoplasma); normal G-banded karyotype, expression of pluripotency markers (positive for SSEA4, TRA1-60, TRA1-81, and OCT4); percent of cells that had lost the reprogramming plasmid; and identity of the iPSCs to patient material. TIFF2025004058000005.tif74160¶ UD = undetectable. Sterility was tested at WuXi AppTec (Mariette, GA); G-banded karyotyping and STR analysis were performed at Cell Line Genetics (Madison, WI); plasmid loss was detected using the Fluidigm single-cell qPCR assay at Cellular Dynamics International, Inc. (Madison, WI).

[0231] These banks were further verified for: sterility; expression of SSEA4, TRA1-60, TRA1-81, and OCT4 >85%; normal G-banded karyotype; loss of reprogramming plasmid; matched STR identity; and matched cancer gene sequences to patients (Table 2). To determine whether CD34+ cell-derived iPSCs acquired any sequence alterations during clinical-grade reprogramming and expansion, the coding regions of all 223 cancer genes were sequenced across all nine iPSC clones. With the exception of clone B from donor 2 (D2B), eight iPSC clones were matched to their respective donor's PBMCs (Figure 1C). However, none of the sequence alterations in iPSC clone D2B were associated with a known cancerous phenotype (Q 2 Solutions, Morrisville, NC) and, unlike Merkle et al., no mutations were observed in the coding region of the tumor suppressor gene p53 (Mandai et al., N Engl J Med 376, 1038-1046 (2017); Merkle et al., Nature 545, 229-233 (2017)). Thus, CD34+ cells appear to generate iPSCs with minimal mutations during reprogramming and can be used for autologous iPSC-based therapies. Using the above criteria, three validated iPSC clones per donor were selected for the fabrication of iRPE patches.

[0232] RPE differentiation can be induced from stem cell-derived neuroectoderm by activation of the TGF pathway or the canonical WNT pathway (Idelson et al., Cell Stem Cell 5, 396-408 (2009).; Leach, et al., Invest Ophthalmol Vis Sci 56, 1002-1013 (2015); Lamba, et al., Proc Natl Acad Sci USA 103, 12769-12774 (2006); Reh, et al., Methods Mol Biol 636, 139-153 (2010)). To further improve differentiation efficiency and reproducibility, as well as to make the iPSC-RPE production method clinically compatible, a three-step differentiation protocol was optimized (Figure 7). The protocol included the following three outcomes: (1) Dual inhibition of SMADs promotes neuronal fates and activation of the FGF pathway inhibits the RPE phenotype (Fuhrmann, Curr Top Dev Biol 93, 61-84 (2010); Bharti et al., PLoS Genet 8, e1002757 (2012); Chambers et al., Nat Biotechnol 27, 275-280 (2009); Meyer et al., Proc Natl Acad Sci USA 106, 16698-16703 (2009)), so based on these observations, appropriate levels of dual inhibition of SMADs and FGF inhibition were combined to drive iPSCs towards RPE-primed neuroectodermal cells, which increased differentiation efficiency from 24% to 81% (Figures 7B-7E); (2) Activation of the TGF or WNT pathway induced a committed RPE fate in these RPE-primed neuroectoderm with much greater efficiency and reproducibility compared to previously published protocols (see Idelson et al., Cell Stem Cell 5, 396-408 (2009); Fuhrmann, Curr Top Dev Biol 93, 61-84 (2010); Carr et al., PLoS One 4, e8152 (2009)) (Figures 7F, 7G); and (3) To actively suppress the canonical WNT pathway, committed RPE was matured by inducing primary cilia in cells by PGE2 treatment. Overall, this three-step differentiation protocol improved the efficiency, reproducibility of iPSC-RPE differentiation, and produced mature RPE cells.

[0233] To test the reproducibility of the clinical-grade differentiation protocol across multiple iPSC clones and multiple patients, it was performed on iPSCs derived from all three AMD patients (two iPSC clones per patient). Differentiation in two different starting conditions was compared: 3D cell aggregates versus 2D monolayer. The advantage of the 2D monolayer protocol is that it improves the efficiency of differentiation and makes it user-independent (Figure 1B, Figure 7A, 7M). Even though the 3D and 2D protocols started with the same number of cells, the 2D protocol induced an epithelial phenotype in the cells more quickly. By day 12 (D12), the cells displayed an epithelial morphology, which prompted a switch to RPE commitment medium (RPECM). By D17, in four of the six clones, over 80% of the cells co-expressed PAX6 / MITF and a small percentage expressed MITF alone, demonstrating that the neuroectodermal cells were at the RPE-primed stage (Figure 1D). As expected, by D27, the number of PAX6 / MITF double-positive RPE progenitors had decreased to 30-40% and the number of MITF-only positive cells had increased dramatically (20-60% across all six clones), suggesting a shift to a committed RPE population. By D42, over 80% of the cells across all six iPSC clones expressed MITF, with a concomitant decline in the PAX6 / MITF double-positive population, demonstrating a shift to an immature RPE phenotype (Figure 1D) (Idelson et al., Cell Stem Cell 5, 396-408 (2009)). In contrast, in the 3D protocol, most cells remained PAX6 positive until D42, suggesting an extended progenitor stage of the cells (Figure 7H, 7I). The results on PAX6 and MITF expressing cells were further supported by analysis of downstream and known targets of these genes (Idelson et al., Cell Stem Cell 5, 396-408 (2009); Fuhrmann, Curr Top Dev Biol 93, 61-84 (2010)).At D17, the majority of cells across all six iPSC clones expressed the RPE progenitor markers PMEL17 (40-85%) and TYRP1 (20-60% across all six iPSC clones), and only a small number of cells expressed the RPE maturation markers CRALBP and BEST1 (Figure 1E). As cells continued to mature, expression of PMEL17 and TYRP1 stabilized at D27 and increased to >99% by D42 after cell enrichment in all six iPSC clones, verifying the purity of the cells (Figure 1E). In comparison, CRALBP- and BEST1-positive cells continued to increase over time, reaching >95% for CRALBP and >70% for BEST1 across all six iPSC clones (Figure 1E). The resulting RPE cells had no detectable iPSCs (no OCT4+ cells and no TRA1-81+ cells; Figure 7K, 7L). Expression analysis of genes related to RPE pigmentation (GPNMB and TYR), visual cycle (ALDH1A3, TRPM1, RPE65), and RPE maturation (RPE65 and BEST1) (Strunnikova et al., Human Molecular Genetics 19, 2468-2486 (2010)) demonstrated that all six AMD-iPSC clones differentiated with similar efficiency and similar progressively acquired maturity, demonstrating the reproducibility of the clinical-grade differentiation process (Figure 1F). Furthermore, the reproducibility of the manufacturing process was confirmed across four different users (Figures 7M-7O). Overall, compared to the 3D research-grade protocol, the 2D protocol was at least 40% faster and more efficient in iPSC-RPE differentiation.

[0234] Example 3 Biodegradable scaffolds aid in functional maturation of clinical-grade AMD-iRPE cells into monolayer tissue It was hypothesized that the biodegradable scaffolds could provide a suitable material for RPE cells to secrete extracellular matrix (ECM) to form a polarized monolayer. As the scaffolds degrade, the ECM and cells could organize native-like RPE tissue, which could enhance the likelihood of long-term integration of the iRPE patch in the patient's eye. The scaffolds used in the clinical grade process were fabricated using poly(lactic-co-glycolic acid) / PLGA (50:50 lactic acid / glycolic acid, IV midpoint 1.0 dl / g) with an average fiber diameter of 350 nm, previously shown to be optimal for RPE growth (Liu, et al., Biomaterials 35, 2837-2850 (2014); Stanzel et al., Stem Cell Reports 2, 64-77 (2014)). A single-layer, heat-fused nanofiber scaffold was selected for iRPE patch fabrication due to its high Young's modulus, which correlates with ease of implantation (Figures 2A, 2B). As expected for a biodegradable scaffold, the scaffold was fully degraded in 80-90 days (SEM confirmed the following scaffold thicknesses: 10 μm at D49; 5 μm at D56; 2-4 μm at D63; and fully degraded at D80-90; Figures 8A-8H; Figure 3C). iRPE from all three patients matured and polarized on the scaffold, as evidenced by high expression of RPE65 and GPNMB, and basal distribution of ECM proteins collagen IV and collagen VIII (representative donor 3 clone C (D3C) is shown in Figure 2C). This suggests that the iRPE cells synthesized an equivalent of Bruch's membrane de novo, supporting our hypothesis that the 3D structure of the PLGA scaffold induces the secretion of ECM proteins by the iRPE cells.

[0235] Previously, maturity and functionality of primary RPE and iRPE monolayers were verified on semi-permeable Transwell (polyester) membranes (May-Simera et al., Cell Reports 22, 189-205 (2018); Maminishkis et al., C Invest Ophthalmol Vis Sci 47, 3612-3624 (2006)). To determine whether clinical grade iRPE patches from AMD patients performed similarly on PLGA scaffolds and Transwell, structural, molecular, and functional comparisons were made between D3C iRPE patches on the two surfaces. TEM and SEM demonstrated the presence of dense apical processes with apically located melanosomes and tight junctions between adjacent cells; basal invaginations were only detected on PLGA scaffolds (inset in Figure 2D; Figures 9A, 9B). Consistent with the similar structure between the two types of patches, both monolayers showed similar electrical properties (Figures 8C, 8D) as well as similar expression of key RPE-specific genes, OCA2, GPNMB, TYRP1, TRPM1, ALDH1A3, RPE65, and BEST1, with lower variability across samples on PLGA scaffolds compared to polyester films (Figure 9E). Taken together, these results demonstrate that iRPE matures similarly on polyester films and PLGA scaffolds, while native-like features are present on the PLGA scaffolds, indicating the superiority of the PLGA scaffolds.

[0236] Donor genetic characteristics are the largest source of variation in cell types derived from iPSCs (Miyagishima et al., Stem Cells Transl Med 5, 1562-1574 (2016); Kajiwara et al., Proceedings of the National Academy of Sciences of the United States of America 109, 12538-12543 (2012)). To determine whether such variation exists in clinical-grade iRPE patches derived from different patients, and to develop criteria to functionally validate iRPE patches prior to transplantation in patients, iRPE patches were generated from all eight iPSC clones (Figure 1C). A performance metric for "hexagonality" was designed to measure how close the iRPE patch is to an ideal convex regular hexagonal pattern (see Example 1). Quantitative morphological assessment of the iRPE patches, performed on images obtained using staining for tight junctions (ZO-1, Figure 9F, 9G), revealed similar hexagonality across all eight iRPE patches (hexagonality score 8.1 ± 0.1; out of 10; Figure 9H), suggesting that the iRPE patches derived from all three patients were of a similar epithelial phenotype. Gene expression analysis of RPE patches derived from all eight iPSC clones demonstrated similar expression of RPE markers and suggested similar maturation of the RPE monolayer across the iRPE of the different patients (Figure 2E). This conclusion was further supported by TER measurements during the final 3 weeks of maturation of iRPE patches derived from D3A, D3D, and D4A clones. All three iRPE patches showed progressively increasing TER (250-1000 ohm.cm 2), suggesting a progressive maturation of the iRPE patch (Fig. 2F). iRPE patches derived from six of the eight iPSC clones phagocytosed POS and secreted VEGF in a polarized manner (Fig. 2G; Fig. 9I). The variation between the different samples could be due to: (1) genetic differences between donors; (2) pluripotent state in the different clones; or (3) technical variation in the different assays. To determine the main sources of variation, a principal component analysis (PCA) was performed on the data obtained from all these assays (Fig. 2H). Interestingly, iRPE patches derived from the D2B iPSC clone, which differs in sequence from its donor, also showed significant variation in functional output (Figs. 1C, 2H). Thus, sequence variation appears to be the cause of variation in functional output from this sample. PCA performed excluding D2B data revealed that iRPE patch functional outputs clustered by donor (Figure 2I), suggesting that patient genetic characteristics are likely the primary source of variation in iRPE patch function. Overall, this validation exercise validates the notion that a combination of sequencing molecular and functional readouts can be used to validate transplantable iRPE patches.

[0237] The purity of differentiated cells in the final product is one of the goals in the development of stem cell therapy. In vitro spike tests were performed to confirm that iPSCs could not survive in the culture environment used for the maturation of iRPE patches. RPE cells mixed with 100%, 10%, 1%, or 0% iPSCs were seeded on PLGA scaffolds and cultured for 35 days. Flow cytometry confirmed that more than 90% of iPSCs died within 2 days of culture, and that no iPSCs could be detected on PLGA scaffolds after D14 (Figure 10A). Gene expression analysis also confirmed the absence of iPSCs and non-RPE cells in all cultures, and the absence of non-RPE lineage markers in RPE cells (except for the analysis of 100% iPSCs, as expected; Figure 10B). In conclusion, the in vitro spiking experiments demonstrated that iPSCs do not survive in the RPE maturation environment and that iRPE patches contained below detectable levels of iPSCs and non-RPE cells, if any at all.

[0238] Example 4 In preclinical studies in rodents, clinical-grade AMD iRPE patches are safely incorporated into the eye and demonstrate improved efficacy over cell suspensions To test the long-term integration and safety profile of the AMD-iRPE patch, clinical grade patches with a diameter of 0.5 mm (approximately 2,500 cells) were applied to immunodeficient (Crl: NIH-Foxn1 rnu) into the subretinal space of rat eyes (Fig. 11A, 11B). Ten weeks after surgery, infrared fundus imaging and optical coherence tomography (OCT) demonstrated successful integration of the patch into the host subretina (Fig. 3A,B, horizontal line) and complete reattachment of the retina over the transplant area (arrow in Fig. 3B). Histological analysis confirmed the OCT data, with the AMD-iRPE patch fully integrated onto the rat Bruch's membrane (STEM121, human cells; arrow in Fig. 3C). In contrast, injected iRPE cell suspensions were poorly integrated into the rat RPE, consistent with previous observations that suspension cells do not form a continuous monolayer in the posterior segment of the eye (Diniz et al., Investigative Ophthalmology & Visual Science 54, 5087-5096 (2013)). (Figure 3D, STEM121; Figure 3F, STEM121; Figure 11D, PMEL17, arrows). AMD-iRPE cells in suspension and as patches were negative for the proliferation marker Ki67 and no tumors / teratomas were found, whereas teratomas were observed when pure iPSCs were injected into the subretinal space (Figures 3E, 11E, 11F; Table 3). In rats, no signs of systemic toxicity of implantation were found, and all animals maintained their food intake and body weight over the 10-week period (Table 3), suggesting the safety of human iRPE cells as an implantable patch. Data showing successful incorporation of iRPE patches suggested that the patches may be more effective compared to cell suspensions.

[0239] Table 3. Summary of preclinical rat and pig studies conducted to demonstrate the safety and efficacy of clinical-grade AMD iPSC-RPE patches. TIFF2025004058000006.tif101170* In the safety study, the body weight and food consumption of the immunodeficient rats were monitored weekly. Neither the patch nor the cell suspension had any obvious effect on the body weight and food consumption of these animals.

[0240] To compare their efficacy, iRPE patches and iRPE cell suspensions were implanted into a previously established Royal College of Surgeons (RCS) rat model (35-38) between postnatal (p) days 21 and 28 using vehicle control (BSS+ or empty scaffold), a 0.5 mm diameter iRPE patch (~2,500 cells), or 100,000 iRPE cells in suspension. Both the iRPE patch and cell suspension rescued overlying photoreceptors; note the increased thickness of the outer nuclear layer (ONL) of photoreceptors in the transplanted areas compared to non-transplanted areas (arrows point to human cells, human nuclear antigen / HuNu; human specific PMEL17; Figures 3G-3J). The apparent difference in ONL thickness between iRPE patches and iRPE cell suspensions implanted into rat retinas is likely due to the relatively more invasive surgical technique used to deliver the patch compared to injection of cells in suspension. Measurements of optokinetics (OKN) (Douglas et al., Visual Neuroscience 22, 677-684 (2005)) demonstrated that at day p90, animals implanted with iRPE patches and iRPE cell suspensions showed similar recovery compared to vehicle control animals (Figure 3K; Table 2). Overall, these rat experiments show that clinical grade AMD iRPE patches were able to fully integrate into the subretinal space of rodent eyes, in contrast to cell suspensions that showed limited integration. The cell dose in the patch was 1 / 40th of the cells in suspension (2,500 cells on a 0.5 mm patch compared to 100,000 cells in an iRPE cell suspension). Despite the 40-fold difference, OKN showed similar recovery for the iRPE patch and iRPE cell suspension. Thus, the AMD iRPE patch was more effective compared to the cell suspension.

[0241] Example 5 A human clinical dose of AMD iRPE patch was incorporated into eyes of a porcine model of laser-induced RPE damage and rescued the degenerating retina. Unlike what is observed in AMD patients, in RCS rats, the RPE monolayer is dysfunctional but still present. To test the AMD iRPE patch in an animal model with atrophied RPE, utilizing a full human clinical dose of 4 x 2 mm iRPE patch, laser-induced RPE ablation was optimized in pigs. The property of melanin to efficiently absorb 532 nm wavelength was exploited, and a micropulse laser was used to selectively damage the pig RPE (Sivaprasad, et al., Surv Ophthalmol 55, 516-530 (2010)) (Figure 4A). The damage of the RPE was targeted to the pig's visual streak, which contains the highest density of cone photoreceptors (Figure 12A). OCT analysis of RPE / retinal damage caused by 1% or 3% duty cycle (DC) laser with 330 ms exposure time revealed RPE detachment at 24 hours and RPE thinning at 48 hours with 1% DC (Figure 4B, 4C), while 3% DC additionally caused subretinal exudate accumulation at 24 hours and significant damage to the RPE / photoreceptor outer segment interface by 48 hours (Figure 12B, 12C). Pre-laser mfERG confirmed that the electrical response was similar across the visual streak in the retina, while post-laser mfERG confirmed that both 1% and 3% DC laser-treated areas showed similar reductions in mfERG signal (Figure 4D, 4E, dotted lines). The OCT and mfERG results were corroborated at the cellular level by TUNEL staining combined with immunostaining for RPE65 and PNA, which revealed apoptotic RPE and photoreceptor cells in both 1% and 3% DC laser-treated eyes, with the 3% DC laser causing more apoptosis in photoreceptor cells compared to the 1% DC laser (Figures 4F, 4G, arrows; Figures 12D, 12E).H&E staining further demonstrated that both laser powers caused thermal damage to the RPE, but 1% DC caused less damage to photoreceptor outer segments at both 24 and 48 h (Figures 4H, 4I; 12F, 12G, arrows). Taken together, the OCT, mfERG, and histological data suggest that 1% DC micropulse laser is preferred over 3% for causing specific damage to the porcine RPE while preserving retinal electrical responses.

[0242] A special implantation tool was designed to deliver the 4 x 2 mm patch, with an S-shaped cannula that fits the curve of the human (or pig) eye and allows easy delivery of the iRPE patch while maintaining its orientation (Figures 13A, 13B, arrows). The procedure included a four-port vitrectomy, posterior vitreous and retinal detachment, a 2.5 mm retinotomy, an extension of the sclerotomy, and subretinal delivery of the iRPE patch loaded into the tool (Figures 13C-13F, arrows). Intraoperative optical coherence tomography (iOCT) confirmed the accurate delivery of the patch subretinal (Figures 13G-13I, arrows). To determine whether the degradation products of PLGA (lactic acid and glycolic acid) cause any inflammation in the eye, intact PLGA-scaffolds without cells were first tested in the subretinal space of non-immunosuppressed and non-laser-damaged pigs. OCT of the porcine eye 2 weeks after surgery demonstrated that the empty scaffold could be delivered with minimal damage to the retina (Figure 13J, arrow). Ten weeks after surgery, there was no sign of inflammation, but the photoreceptor outer segment layer was thinned and showed retinal tubulation, suggesting damage to the photoreceptors - likely because the empty scaffold prevented nutrient supply from the host RPE, the visual cycle between the RPE and the photoreceptors, and phagocytosis of the photoreceptor outer segments (Figure 13K). Concurrent with scaffold degradation (5 weeks after surgery), the multifocal ERG signal N1P1 over the area of ​​the implant recovered to 80%, suggesting that the damage caused by the empty scaffold was minimal (Figure 13M). These in vivo results are consistent with the lactate release profile of the degrading PLGA scaffold. During in vitro culture, 83% of the total lactate was released from the scaffold. This included approximately 70% of the lactic acid released during the bulk degradation phase of our PLGA scaffolds, which began at week 3 and ended at the end of week 5 while the scaffolds were still in culture (Table 4).Furthermore, the maximum amount of lactate released from the scaffolds during the bulk degradation phase (0.0074±0.0014 mmol / L / scaffold / day) was 310-fold lower than the systemic concentration of lactate in the blood (2.3 mmol / L) (Wacharasint, et al., Shock 38, 4-10 (2012)) and 513-fold lower than the lactate concentration at the apical surface of the RPE in the eye (Adler and Southwick, Ophthalmic Res 24, 243-252 (1992)). Overall, this data demonstrated that the PLGA scaffolds were not inflammatory in the subretinal space of the porcine eye and that the newly developed implantation device safely delivered the patch.

[0243] Table 4: Lactic acid released from PLGA scaffolds during degradation. Days 1-35 are the in vitro stage of the scaffolds, and days 35 and after are the in vivo stage after implantation. The days during the bulk degradation phase of the PLGA scaffolds (days 19-36) are highlighted in gray. Note that bulk degradation of the scaffolds occurs in vitro. TIFF2025004058000007.tif65160

[0244] Fundus photography of pig eyes implanted with GFP-expressing iRPE patches confirmed survival of human cells over 10 weeks (Figures 14A-14C, arrows), which was achieved by suppressing systemic and resident innate immune responses with prednisone, doxycycline, and minocycline, and adaptive immune responses with tacrolimus and sirolimus (Santa-Cecilia et al., Neurotox Res 29, 447-459 (2016); Scholz et al., J Neuroinflammation 12, 209 (2015); Swijnenburg et al., Proc Natl Acad Sci USA 105, 12991-12996 (2008); Xian and Huang, Stem Cell Res Ther 6, 161 (2015)). As the biodegradable PLGA scaffold degraded, a human clinical dose of AMD patient-derived iRPE patch could be incorporated and activated on the pig's Bruch's membrane. A 4 x 2 mm patch was implanted in the pig's eye over an area with laser-induced RPE ablation. As the PLGA scaffold degraded over 10 weeks, OCT demonstrated that the clinical grade iRPE patch was incorporated into the pig's eye and that the retina on the iRPE patch maintained both the inner and outer layers of the retina, compared to animals implanted with a blank PLGA scaffold, in which retinal tube formation was evident (Figures 5A-5C; arrows in 5B and 5E). Immunostaining confirmed the integration of AMD-iRPE patches in laser-injured porcine eyes and the mature phenotype of the transplanted cells, as verified by robust RPE65 immunostaining in iRPE cells (STEM121; RPE65, Figures 5D-5F, Figures 14D-14F). PNA staining confirmed improved organization of photoreceptor outer segments on retinas transplanted with iRPE patches compared to retinas transplanted with empty scaffolds (white PNA, Figures 5D-5F).

[0245] To quantify the difference in photoreceptor rescue between empty scaffold and iRPE patch implants, the number of photoreceptor nuclei in the ONL over both implant areas was counted and the results were compared to adjacent healthy retina. Analysis showed that the number of nuclei in the ONL over the empty scaffold was 42% of the adjacent healthy area, whereas the number of nuclei over the iRPE patch area was 73% of the healthy area (p < 0.05, T test; FIG. 14G). To exclude the possibility that STEM121 labeling was not caused by human iRPE cells phagocytosing the porcine RPE, immunostaining was performed for a nuclear-specific human antigen (STEM101) (FIG. 14H). This data shows specific nuclear labeling only in the RPE area, providing additional evidence of the integration of the human iRPE patch in the porcine eye.

[0246] Laser damage and subsequent implantation of an iRPE patch was performed in the visual streak area of ​​the pig eye to determine whether human RPE cells could preserve pig cone photoreceptors. Immunostaining for specific cone opsins (S, L, and M) confirmed the preservation of pig cone photoreceptors over the area of ​​the iRPE patch (Figure 5G, arrows). To test the functional integration of the human iRPE patch in the pig eye, it was tested whether human RPE cells could phagocytose pig photoreceptor outer segments (POS). Rhodopsin staining of healthy pig retinas and of retinas implanted with clinical grade human iRPE patches revealed phagocytosed POS inside the human RPE cells (arrows in Figures 5H, 5I), similar to what is observed for native pig RPE. The preservation of cone photoreceptors and the functional integration of human RPE inside the pig eye prompted us to test the recovery of electrical responses in laser-damaged pig retinas over the area of ​​the iRPE patch. Heat maps of mfERG responses showed improved signal over the laser-damaged visual streak area in pigs implanted with iRPE patches compared to those implanted with empty scaffolds (Figure 5J-5L). To address issues related to regional variability in the location of laser damage and the extent of laser damage on the effect of the iRPE patch, a linear mixed effects (LME) analysis of all mfERG components was used. LME analysis of all mfERG components (N1, N1 width, P1, P1 width, N1P1, area under the curve (AUC), and scalar product) revealed significant differences between the iRPE patch and empty patch over 10 weeks (p < 0.05) (Figure 5M). This observation was further supported in a linear regression analysis of the area under the curve, which also showed significant differences between the two groups (Figure 5N, difference in y-intercepts, p < 0.05). In summary, these results demonstrated that clinical-grade AMD-iRPE patches were incorporated into porcine retinas and rescued porcine photoreceptors following laser injury of the porcine RPE, and that iRPE patches derived from different patients showed similar efficacy responses.

[0247] To perform comparative analysis of the PLGA iRPE patch versus the Transwell iRPE patch (non-degradable Transwell membrane) and the iRPE cell suspension versus the empty scaffold, all four implants were tested in pigs with laser-ablated RPE (Figures 15A-15I). Two weeks after surgery, OCT confirmed that all four implants were delivered correctly with minimal signs of inflammation (Figures 6A-6D). Five weeks after surgery, the empty scaffold and the iRPE suspension showed disruption of the outer nuclear layer and outer limiting membrane of the retina, and structures reminiscent of outer retinal tubulogenesis were often observed in the degenerating retina (47) (arrows in Figures 6E, 6H). In contrast, OCT showed that retinas overlying iRPE patches (both PLGA and Transwell) did not show any such retinal tubulation, and both the outer nuclear layer and the outer limiting membrane appeared intact (Fig. 6F, G). OCT analysis was consistent across all three pigs evaluated for each treatment. Immunohistochemical analysis of PLGA iRPE patches demonstrated integration and post-transplant maintenance of the mature phenotype of human RPE cells in the posterior segment of the pig eye (compare 6I-6K; white, STEM121, human specific; grey, RPE65), and maintenance of photoreceptors on human iRPE patches but not on empty scaffold or cell suspension implants (PNA, photoreceptors; Fig. 6I-6L; Fig. 8 SD-I; Table 2). In contrast, the ONL on empty scaffolds and on iRPE suspensions show tube formation and degeneration as observed in OCT (arrows in FIG. 6I). Furthermore, unlike iRPE patches, iRPE cells in suspension lose expression of the RPE maturation marker RPE65 (arrows in FIG. 6L). Without being bound by theory, this is likely because trypsinized RPE cells are unable to maintain a stable epithelial phenotype unless reorganized as a confluent monolayer (Radeke et al., Genome Med 7, 58 (2015)).Consistent with the OCT and immunostaining data, mfERG demonstrated greater recovery of mfERG individual waveforms and integrated data over 5 weeks in the laser-irradiated areas in both PLGA iRPE and Transwell iRPE patches compared to blank PLGA scaffolds or iRPE cell suspensions (Figures 6M, 6N). These results demonstrate that monolayer iRPE patches are superior to cell suspensions in rescuing retinal degeneration in laser-injured porcine eyes.

[0248] Successful autologous cell therapy requires efficient and reproducible manufacturing processes that produce safe and effective products. The disclosed clinical grade process provides reproducibility of the manufacturing process and ensures safety and efficacy of clinical products (Schwartz et al., Lancet 385, 509-516 (2015); Mandai et al., N Engl J Med 376, 1038-1046 (2017); Kamao et al., Stem Cell Reports 2, 205-218 (2014)). The manufacturing process was developed using CD34+ cells from three patients with advanced stage AMD (geographic atrophy) (Mack, et al., PLoS One 6, e27956 (2011); Badenes et al., PLoS One 11, e0155296 (2016); Badenes et al., PLoS One 11, e0151264 (2016)). Passage 10 working banks of clinical grade iPSCs were generated from up to three banks per patient and validated for key iPSC quality attributes (Table 2).

[0249] In addition to their pluripotency and correct G-banded karyotype, there were two safety features: loss of reprogramming plasmid and sequencing of oncogenes. All nine clones from three patients lost their reprogramming plasmid by passage 10. Without being bound by theory, this is likely due to the use of low copy number, EBNA origin-based plasmids (Carr et al., PLoS One 4, e8152 (2009)). None of the iPSC clones acquired any oncogene mutations during reprogramming or expansion, and no sequence changes were observed in eight of the nine iPSC clones (Kwon et al., Proc Natl Acad Sci USA 114, 1964-1969 (2017)) (Figure 1C).

[0250] One iPSC clone, D2B, that showed some sequence and copy number changes also showed variation in the functional output of the iRPE patch (Figure 2H). Without being bound by theory, it is possible that these sequence changes are the reason for the variation in functional output at the iRPE patch level. A combination of sequencing of the cancer genes of iPSCs and functional analysis of test articles can be performed to identify transplantable derivatives from iPSCs.

[0251] Clinical-grade differentiation of iPSCs into mature and polarized RPE patches on biodegradable PLGA-based scaffolds (Figs. 1, 2) was demonstrated. Differentiation of iPSCs into implantable RPE patches takes approximately 10 weeks (Kamao et al., Stem Cell Reports 2, 205-218 (2014)). The process is user-independent (Figs. 7M-7O) and scalable to multiple iPSC clones (Figs. 1, 2). PCA suggested that patient genetics were likely the largest single contributor to any variability. iRPE patches from different patients show different functional responses, but the patches can be functionally validated to generate implantable products.

[0252] Compared to ESC-RPE suspensions (Schwartz et al., Lancet 379, 713-720 (2012); Schwartz et al., Lancet 385, 509-516 (2015)), iRPE patches contained a monolayer of fully polarized cells, both integrated into Bruch's membrane in immunodeficient rats and in pigs after laser-induced RPE injury (Figures 3C, 3H, 3J, 5D-5I, 6I-6L). This outcome likely reflects the coordination of continuous PLGA scaffold degradation and ECM production by iRPE, which promotes integration into the host's Bruch's membrane. The results show that iRPE cells on PLGA scaffolds produce Bruch's membrane proteins, collagen types IV and VIII (Figure 2C). Furthermore, unlike cell suspensions, which appear unable to perform most of the functions of the RPE, polarized RPE monolayers on PLGA iRPE patches and Transwell iRPE patches performed many of the functions of the RPE (Figures 2, 9). Combined, these properties of the iRPE patch suggest a mechanism of action for the improved efficacy observed with the patch approach. Ablation of the RPE in a laser wound pig model resembled the loss of RPE in eyes with advanced AMD, resulting in geographic atrophy (Bird, et al., JAMA Ophthalmol 132, 338-345 (2014)). Thus, in AMD patients, the incorporation of transplanted iRPE patches can employ "young" iRPE cells that secrete metalloproteases that can modify "aged" Bruch's membrane (Greene, et al., Journal of Ocular Pharmacology and Therapeutics: The Official Journal of the Association for Ocular Pharmacology and Therapeutics 33, 132-140 (2017)).In comparison to PLGA iRPE patches and Transwell iRPE patches, RPE cell suspensions showed only incidental integration, as previously suggested (Weisz et al., Retina (Philadelphia, Pa.) 19, 540-545 (1999)) (Figures 3C, 3F).

[0253] Although the RCS rat model and the porcine model of laser-induced RPE damage do not fully recapitulate the pathophysiology of AMD, these models provide important insights into the viability, integration, and potential efficacy of iRPE cells derived from AMD patients (Figures 3-6). Restoration of retinal function using RPE suspensions and iRPE patches differed between rodents and pigs. A similar degree of rescue of visual function was observed in both cell suspensions (100,000 cells) and iPSC-RPE patches (2,500 cells). Cells in suspension do not form an intact polarized monolayer in the posterior part of the rat eye, but instead act as chemical bioreactors, isotopically secreting neurotrophic factors. Unlike cell suspensions, intact RPE patches are polarized cell monolayers that integrate into the interior of the rat eye and simultaneously meet the needs of the overlying photoreceptors while maintaining the integrity of its interface with the choroid. In pigs where the same number of cells were implanted as a 4 x 2 mm patch versus a suspension of 100,000 cells, a significantly greater degree of protection of photoreceptors was observed in the patch. Furthermore, clinical-grade iRPE patches derived from different AMD patients performed similarly (Figure 5), suggesting that the manufacturing process is reproducible.

[0254] Thus, provided herein is a more stable clinical grade manufacturing process and a RPE patch on a biodegradable scaffold for better integration and functionality.

[0255] Example 6 CRISPR-mediated gene correction in albinism iPSCs Patient iPSCs were obtained that contained a heterozygous c.593C>T (p.P198L) in the OCA2 gene, which results in the loss of activity of the gene. This mutation was corrected using CRISPR / Cas9 technology. Guide RNAs were designed near the exact genomic location of the c.593C>T mutation in the OCA2 gene and obtained from a commercial source. A donor plasmid containing a wild-type sequence at this location was also obtained commercially. The donor plasmid also contained a puramycin selection cassette and a cDNA encoding green fluorescent protein (GFP). The guide RNA, wild-type donor plasmid, and the plasmid encoding the CAS9 protein were all transfected into iPSCs using Lipofectamine reagent (ThermoFisher). Puramycin-resistant iPSC colonies were selected and expanded. Selected colonies were further purified by flow cytometry for GFP. Correction of the mutation was confirmed by sequencing. The corrected iPSCs were then differentiated into RPE patches using the methods disclosed above.

[0256] In Figure 16, the top panel shows the sequence of the region surrounding the mutated area (SEQ ID NO: 1 where X is C or T). Note that there are two peaks, one cytosine and one thymidine, indicating that the patient is heterozygous at this position. The bottom panel shows the gene after correction, where there is only one peak of a cytosine nucleotide (SEQ ID NO: 1 where X is C).

[0257] As shown in Figure 17, when RPE cells from a patient's iPSC line differentiate into RPE patches, these cells remain pigment-free because they lack the activity of the OCA2 protein product. In contrast, when CRISPR-corrected iPSCs differentiate into RPE patches, they have pigment. Thus, the RPE patch technology works well with genetically engineered cells and can be used to treat patients with single-gene diseases. Figure 18 also shows the RPE of an OCA2 patient and the RPE corrected by CRISPR.

[0258] It will be apparent that the exact details of the methods or compositions described may be changed or modified without departing from the spirit of the described invention, and it is the inventors' intent to encompass all such modifications and variations within the scope and spirit of the appended claims.

[0259] Sequence information SEQUENCE LISTING <110> THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY, DEPARTMENT OF HEALTH AND HUMAN SERVICES <120> BIODEGRADABLE TISSUE REPLACEMENT IMPLANT AND ITS USE <150> US 62 / 769,484 <151> 2018-11-19 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 13 <212> DNA <213> Homo sapiens <400> 1 cagaaayggg att 13 <210> 2 <211> 13 <212> DNA <213> Homo sapiens <400> 2 cagaaacggg that 13

Claims

1. 1. A tissue replacement implant comprising polarized retinal pigment epithelial cells on a poly(lactic-co-glycolic acid) (PLGA) scaffold, comprising: The tissue replacement implant, wherein the PLGA scaffold is 20-30 microns thick, the PLGA scaffold having a DL-lactide / glycotide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm.

2. 10. The tissue replacement implant of claim 1, wherein the PLGA scaffold is coated with vitronectin.

3. 3. The tissue replacement implant of claim 1 or claim 2, wherein the polarized retinal pigment epithelial cells are human.

4. The tissue replacement implant of any one of claims 1 to 3, wherein the polarized retinal pigment epithelial cells are generated from induced pluripotent stem cells or ES cells.

5. The tissue replacement implant of any one of claims 1 to 4, wherein the polarized retinal pigment epithelial cells are derived from a single subject.

6. 5. The tissue replacement implant of claim 4, wherein the polarized retinal pigment epithelial cells are generated from induced pluripotent stem cells, and the induced pluripotent stem cells are generated from CD34+ cells.

7. 7. The tissue replacement implant of any one of claims 1 to 6, wherein the PLGA scaffold has an average pore size of less than 1 micron or greater than 1 micron.

8. 13. A method of treating a subject having a retinal degenerative disease, retinal or retinal pigment epithelial dysfunction, retinal deterioration, damage to the retina, physical injury to the retina, or loss of the retinal pigment epithelium, comprising the step of locally administering to an eye of the subject a tissue replacement implant according to any one of claims 1 to 7, thereby treating the subject.

9. 9. The method of claim 8, wherein the retinal degenerative disease is Stargardt's macular dystrophy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, Leber's congenital amaurosis, late-onset retinal degeneration, hereditary macular or retinal degeneration, Best's disease, Sorsby's fundus dystrophy, retinal detachment, gyrate atrophy, eye trauma, or choroideremia, pattern dystrophy.

10. 9. The method of claim 8, wherein the damage to the retina or to the retinal pigment epithelium is caused by a laser, inflammation, infection, radiation, neovascularization, or trauma.

11. 11. The method of any one of claims 8 to 10, wherein the tissue replacement implant is introduced into the subretinal space of the eye, or into the outer retina, into the peripheral retina, into the macula or perimacular region, or into the choroid.

12. The method according to any one of claims 8 to 11, wherein the subject is a human.

13. 13. The method of claim 12, wherein the subject has age-related macular degeneration.

14. a) obtaining a vitronectin coated PLGA, the PLGA scaffold comprising fibers forming a mesh structure and having an upper surface and a lower surface, the PLGA scaffold being about 20 to about 30 microns thick, the PLGA scaffold having a DL-lactide / glycotide ratio of about 1:1, an average pore size of less than about 1 micron, and a fiber diameter of about 150 to about 650 nm; b) heat treating the PLGA scaffold such that the fibers of the scaffold fuse at the fiber intersections in the PLGA scaffold, increasing the mechanical strength of the PLGA scaffold and decreasing the pore size; c) seeding retinal pigment epithelial cells onto the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold; and d) on a PLGA scaffold in tissue culture medium, with medium present on both the top and bottom surfaces of the PLGA scaffold; i) Retinal pigment epithelial cell polarization, and ii) Bulk degradation of PLGA scaffolds Culturing the retinal pigment epithelial cells in vitro for a period of time sufficient to 2. A method of making the tissue replacement implant of claim 1, comprising:

15. The method of claim 14, wherein the retinal pigment epithelial cells are human.

16. The method according to claim 14 or 15, wherein the retinal pigment epithelial cells are cultured in vitro on the PLGA scaffold until the peak release of lactic acid from the PLGA scaffold is over.

17. The retinal pigment epithelium is 200 ohms*cm 2 The method of any one of claims 14 to 16, wherein the cell has a Trans-Epithelial Resistance (TER) greater than 100%.

18. The method of any one of claims 14 to 17, wherein step d) comprises culturing retinal pigment epithelial cells on the PLGA scaffold in tissue culture medium for about 3.5 to about 6 weeks.

19. The method of any one of claims 14 to 18, wherein the PLGA scaffold is coated with vitronectin or other ECM proteins.

20. The method according to any one of claims 14 to 18, further comprising, prior to step c, generating retinal pigment epithelial cells from induced pluripotent stem cells or ES cells.

21. 21. The method of claim 20, wherein the induced pluripotent stem cells are generated from the subject's CD34+ cells.

22. The method of any one of claims 14 to 20, wherein the retinal pigment epithelial cells are derived from a single subject.

23. The method of any one of claims 14 to 22, wherein the implant has an average pore size of less than 1 micron.

24. The tissue replacement implant of any one of claims 1 to 7 or the method of any one of claims 8 to 23, wherein the PLGA scaffold is sterilized using electron beam (e-beam).

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