Methods for generating macular, central and peripheral retinal pigment epithelial cells

Abstract

Methods for producing macular, central or peripheral human retinal pigment epithelial (RPE) cells are disclosed. These methods include a) culturing stem cells, such as induced pluripotent stem cells (iPSCs), in a retinal induction medium to initiate differentiation of the cells into RPE progenitor cells; b) culturing the RPE progenitor cells in a retinal differentiation medium to further differentiate the RPE progenitor cells into committed RPE cells; c) culturing the committed RPE cells in a retinal medium to form immature RPE cells; and d) culturing the immature RPE cells in an RPE maturation medium comprising a retinoic acid receptor (RAR) antagonist and / or a canonical Wnt inhibitor, thereby producing macular, central or peripheral human RPE cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Patent Application No. 63 / 194,774, filed May 28, 2021, which is incorporated by reference in its entirety.

[0002] Government support statement This invention was made with Government support under Project No. Z01#: ZIA EY000533-04 by the National Institutes of Health, National Eye Institute. The United States Government has certain rights in this invention.

[0003] Field of the Disclosure It relates to the field of ophthalmology, and specifically to in vitro methods for generating macular, central and peripheral retinal pigmented epithelial (RPE) cells. [Background technology]

[0004] background The retinal pigment epithelium is a single layer of cells located at the back of the eye. On the apical side, RPE cells form contacts with the photoreceptors via their apical processes. On the basal side, they face the capillaries of the choroid. The main function of the retinal pigment epithelium is to exchange metabolic products with the photoreceptors and choroidal vessels, maintaining retinal homeostasis.

[0005] The human central retina contains a cone-enriched region called the macula, which is responsible for acute color vision. Evidence suggests that the human RPE monolayer is a phenotypically and functionally heterogeneous cell population. Phenotypically, macular RPE cells are smaller in size than peripheral RPE cells (Bhatia et al., Molecular Vision, 22, 898-916, 2016) and have petal-like apical processes that primarily support cone photoreceptors. In contrast, peripheral RPE cells have finger-like apical processes that primarily support rod photoreceptors (Pfeffer & Fisher, Journal of Ultrastructure Research, 76(2), 158-172. https: / / doi.org / 10.1016 / S0022-5320(81)80014-71981; Steinberg, Zeitschrift Fur Zellforschung Und Mikroskopische Anatomie, 143(4), 451-463, doi.org / 10.1007 / BF003067651973). Functionally, peripheral RPE cells express higher levels of Na / K ATPase than macular RPE cells (Burke et al., Investigative Ophthalmology & Visual Science, 32(7), 2042-2046, 1991). RPE lysosomal enzyme activity also differs regionally: cathepsin D is more active in macular RPE cells (Boulto et al., The British Journal of Ophthalmology, 78(2), 125-129, 1994; Burke & Twining, The British Journal of Ophthalmology, 78(2), 125-129, 1988; Cabral et al., Investigative Ophthalmology & Visual Science, 31(4), 670-676.1990), whereas acid phosphatase, β-glucuronidase, and N-acetyl-β-glucosaminidase are more active in peripheral RPE cells (Cabral et al., supra, 1990).At least 5% of genes are differentially expressed between macular and peripheral RPE cells (Radeke et al., Experimental Eye Research, 85(3), 366-380, 2007; van Soest et al., Molecular Vision, 13, 1608-1617, 2007). Metabolically, the macular and peripheral human RPE / choroid consume and release different levels of metabolites (Li et al., BioRxiv, 2020.07.10.196295.doi.org / 10.1101 / 2020.07.10.196295, 2020). There remains a need for methods to differentiate different types of RPE cells, such as macular RPE and peripheral RPE. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Bhatia et al.,Molecular Vision,22,898-9162016,2016 [Non-Patent Document 2] Pfeffer & Fisher, Journal of Ultrastructure Research, 76(2), 158-172. https: / / doi.org / 10.1016 / S0022-5320(81)80014-71981 [Non-Patent Document 3] Steinberg, Zeitschrift Fur Zellforschung Und Mikroskopische Anatomie, 143(4), 451-463, doi.org / 10.1007 / BF003067651973 [Non-Patent Document 4] Burke et al.,Investigative Ophthalmology&Visual Science,32(7),2042-2046,1991 [Non-Patent Document 5] Boulto et al., The British Journal of Ophthalmology, 78(2), 125-129, 1994 [Non-Patent Document 6] Burke & Twining, The British Journal of Ophthalmology, 78(2), 125-129, 1988 [Non-Patent Document 7] Cabral et al.,Investigative Ophthalmology&Visual Science,31(4),670-676.1990 [Non-Patent Document 8] Radeke et al.,Experimental Eye Research,85(3),366-380,2007 [Non-Patent Document 9] van Soest et al.,Molecular Vision,13,1608-1617,2007 [Non-Patent Document 10] Li et al.,BioRxiv,2020.07.10.196295.doi.org / 10.1101 / 2020.07.10.196295,2020 Summary of the Invention [Means for solving the problem]

[0007] Summary of the Disclosure Methods for producing macular, central or peripheral RPE cells are disclosed. These methods include: a) culturing pluripotent stem cells, such as induced pluripotent stem cells (iPSCs), in retinal induction medium to initiate the differentiation of cells into RPE progenitor cells; b) culturing RPE progenitor cells in retinal differentiation medium to further differentiate RPE progenitor cells into committed RPE cells; c) culturing committed RPE cells in retinal medium to form immature RPE cells; and d) culturing immature RPE cells in RPE maturation medium containing retinoic acid receptor (RAR) antagonist and / or canonical Wnt inhibitor, thereby producing macular, central or peripheral human RPE cells.

[0008] In some embodiments, the method produces macular human RPE cells, and the RPE maturation medium comprises a RAR antagonist but does not comprise a canonical Wnt inhibitor.

[0009] In a further embodiment, the method produces central human RPE cells and the RPE maturation medium comprises both a RAR antagonist and a canonical Wnt inhibitor.

[0010] In yet other embodiments, the method produces peripheral human RPE cells and the RPE maturation medium comprises a canonical Wnt inhibitor but does not comprise a RAR antagonist.

[0011] The disclosed methods can be used to prepare RPE cells from any mammal, including but not limited to human RPE cells. In some embodiments, the RPE cells produced using the methods disclosed herein can be used to treat retinal degeneration in a subject.

[0012] The above and other features of the present disclosure 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]

[0013] [Figure 1] Macular and peripheral iPSC-RPE differentiation protocols. Scheme of macular and peripheral iPSC-RPE differentiation protocols. AGN 193109 and endo-IWR-1 are added to iPSC-RPE in RPE maturation medium to obtain a macular or peripheral phenotype, respectively. The flow chart shows which drugs are added to the RPE maturation medium and indicates the cell fate states corresponding to each step. [Diagram 2] Human macular and peripheral RPE cells are phenotypically distinct. Human RPE monolayers are phenotypically and functionally heterogeneous cell populations. This figure illustrates two phenotypic differences between macular and peripheral RPE. Above, a scanning electron microscope (SEM) image of ex vivo grown iPSC-RPE cells shows examples of cells enriched with petal-like or finger-like apical processes. Petal-like apical processes are sheet-like projections (with undulations) of the apical RPE cell membrane that encase the cone photoreceptors; these are abundant in the macula. The finger-like apical processes resemble microvilli and selectively support rod photoreceptors, which are abundant in the peripheral retina (Steinberg and Wood, Proceedings of the Royal Society of London-Biological Sciences, 187(1089), 461-478, 1974; Fisher and Steinberg, The Journal of Comparative Neurology, 206(2), 131-145, 1982). Below the figure, two images of RPE cell borders from the macula and peripheral regions show the difference in cell area. The bar graph shows the quantification of cell area between the macula and the periphery (Bhatia et al., Molecular Vision, 22, 898-916, 2016). [Diagram 3] Area morphometric analysis of human RPE cells. Whole human RPE flat mounts were prepared and stained for RPE cell borders to analyze RPE shape metrics (left). A close-up shows an individual RPE cell stained for cell borders with anti-ZO1 antibody. A machine learning algorithm was trained to identify and segment RPE cell borders. The algorithm generates binary images of RPE cells that serve as input for the REShAPE software for cell shape analysis. [Figure 4] Area morphometric analysis of human RPE cells. REShAPE (Retinal Epithelium Shape And Pigment Evaluator) is used to analyze all cells within a successfully segmented field of view, REShAPE provides quantification of more than 25 different shape metrics. The raw data is stored in a spreadsheet to allow statistical analysis. Furthermore, the software creates an image of the segmented cell for each analyzed metric, and all cells are coded according to their raw value. In this image, the segmented RPE cells are coded according to their area. Small cells appear darker (left side of the scale) and larger cells in grey (right side of the scale). The thermal scale is shown as an insert. [Diagram 5] Area morphometric analysis of human RPE cells. Low magnification map of the entire human RPE monolayer. It contains approximately 3-4 million RPE cells, which are coded according to cell area. A thermal scale was used. Dark grey corresponds to RPE cells with small area, and RPE cells with large area are also shown (see scale). The dark spot in the center of the flat mount represents macular RPE cells. RPE cell area grows eccentrically and gradually, except for a peripheral ring of smaller RPE cells. Using cell area as a reference, we identified five significantly different RPE populations, which we named "population 1" (P1) to "population 5" (P5) (see Figure 6). In this figure, we progress from macular to peripheral cells. [Figure 6]Area morphometric analysis of human RPE cells. High magnification images to display single cells for each identified human RPE population (P1-P5). Thermal scale was used. Small and large area RPE cells were identified. Box plots show RPE cell area values ​​for each RPE population. Box limits represent the 1st and 3rd quartiles, center line indicates median, whiskers indicate 5th and 95th percentiles, so range specifies 90% of data. One-way ANOVA table shows multiple comparisons between each RPE population. RPE populations are defined as population median ± 2 standard deviations according to cell size, which encompasses 95% of data. Values ​​are shown in the table on the right. [Figure 7] Comparison of morphometry between human RPE cells and iPSC-RPE. 115 compounds were screened in iPSC-RPE to recapitulate the morphometry of human RPE populations. Compounds are activators and inhibitors of developmental pathways. All compounds were tested by adding to RPE maturation medium for 30 days (as shown in the schematic flow chart). At the end of treatment, cell area was quantified using REShAPE. Results were compared to measurements of macular and peripheral RPE cells to select compounds that recapitulate the dimensions of human RPE. The bottom of the figure shows a schematic of the comparison of cell area between macular and peripheral human RPE (from flat mounts) and iPSC-RPE cells treated with different compounds. [Figure 8]Comparison of morphometry between human RPE cells and iPSC-RPE. Results from compound screening show that the addition of retinoic acid inhibitors, e.g., AGN 193109, or canonical Wnt inhibitors, e.g., endo-IWR-1, to iPSC-RPE recapitulates macular and peripheral cell size in vitro, respectively. Human RPE population P3 was chosen as a reference for peripheral RPE cells. One-way ANOVA and post-hoc tests for multiple comparisons were used for statistical analysis. iPSC-RPE cells treated with DMSO are also shown in the graph as a control for untreated iPSC-RPE. Data are displayed as box plots, with box limits representing the first and third quartiles, center lines indicating the median, and whiskers indicating the fifth and 95th percentiles, so that the range specifies 90% of the data. [Figure 9] Morphometric comparison between human RPE cells and iPSC-derived RPE. Images coded by cell area demonstrate the comparison between human RPE and iPSC-RPE cells. Human RPE population P3 was chosen as a reference for peripheral RPE cells. Addition of retinoic acid inhibitors, e.g. AGN 193109, or canonical Wnt inhibitors, e.g. endo-IWR-1, to iPSC-RPE recapitulates macular and peripheral cell size, respectively, in vitro. The same range of parameters was used for thermal scaling of the images. Thus, areas can be directly compared between images. This data complements the box plot graphs in the previous figure. Scale bar = 100um. [Figure 10] Comparison of apical structure between treated iPSC-derived RPE. Top to bottom view of iPSC-RPE cells shows the structure of the apical process. AGN 193109 enriched cells with undulating, petal-like apical processes characteristic of macular RPE cells. Endo-IWR-1 enriched cells with finger-like apical processes characteristic of peripheral RPE cells. Both types of apical processes can be seen in the DMSO control. [Figure 11]Petaloid (undulating) and finger-like apical processes are defined. The cross sections of the apical processes were measured to precisely define their shape. Petaloid apical processes obtained by treating RPE cells with AGN 193109 have an average transversal length of 1.68 μm (length min 0.5, max 4 μm) and a width of 0.20 μm (width min 0.1, max 1 μm). Petaloid apical processes are also wavy along their length. The width of the undulations ranges from 0.2 to 2 μm. Finger-like apical processes obtained by treating RPE cells with endo-IWR-1 are cylindrical and have the same average transversal length and width of 0.23 μm (min 0.1, max 1 micron). An example of how the measurements of the apical processes were performed is shown for each type of apex treated. [Figure 12] Comparison of cell area. The graph shows a comparison of cell area between macular RPE cells and distal peripheral RPE cells (P1 and P4, respectively) and iPSC-RPE cells treated with different drugs that inhibit the canonical Wnt pathway. The data comes from a screening of compounds that led to the identification of AGN 193109 and endo-IWR-1. iPSC-RPE cells treated with DMSO are also shown in the graph as a control for untreated iPSC-RPE. The graph shows that other canonical Wnt inhibitors can be used to recapitulate the peripheral RPE phenotype. Indeed, all canonical Wnt inhibitors increased iPSC-RPE cell area to the dimensions of peripheral RPE cells (P3). The results showed that canonical Wnt inhibition generates peripheral RPE cells. Data are presented as box plots, with box limits representing the first and third quartiles, center lines indicating medians, and whiskers indicating the fifth and ninety-fifth percentiles, so that the range specifies 90% of the data. One-way ANOVA and post-hoc tests for multiple comparisons were used for statistical analysis. [Figure 13]Different concentrations of AGN 193109 and endo-IWR-1 are tested to recapitulate macular (P1), central (P2) and peripheral (P3) cells. A range of concentrations of AGN 193109 and endo-IWR-1 were tested that can be used to recapitulate macular, central and peripheral RPE populations (labeled P1, P2 and P3, respectively). Population 1 (P1, macular cells) can be recapitulated using 0.1 mM-0.2 mM AGN 193109. Population 3 (P3, peripheral) can be recapitulated using 1 mM-4 mM endo-IWR-1. Using gradients of AGN 193109 and endo-IWR-1, we were able to replicate human RPE population 2 (P2, central)-central RPE cells. In one example, this can be reproduced using 25 nM-50 nM AGN 193109 and 0.1 mM-0.5 mM endo-IWR-1. Box plots for DMSO, P1, P2 and P3 are plotted on the left side of the graph as a reference for cell size comparison. Five concentrations were tested for AGN 193109 and endo-IWR-1. [Figure 14]Different RPE populations are affected by different retinal diseases. Fundus images of patient eyes showing damage in different regions of the RPE in different ocular diseases (A) Choroideremia; (B) Late-onset retinal degeneration (LORD); (C) Undiagnosed retinal degeneration. Different types of retinal degenerative diseases seem to affect different subsets of the RPE population. RPE degeneration in different diseases can be observed in fundus images and their location quantified. Table (D) summarizes which RPE populations are primarily affected by different diseases. Filled squares mean that the entire RPE population is affected. Dotted squares indicate that the population is partially affected. For RPE populations, the numbers in brackets indicate the distance (in millimeters) of each RPE population from the center of the eye. For diseases, the numbers in brackets specify the location of the RPE degeneration expressed as millimeters from the center of the eye. AMD is acute macular degeneration and RD is retinal degeneration in patients with unidentified mutations. The populations with dots or filled squares can be used for treatment of the indicated subjects. P1 is macular, P2 is central, P3 is peripheral RPE, P4 is distal peripheral, and P5 is ora serrata RPE cells. Dots indicate degeneration in later disease stages (also implying partial degeneration in early stages). Squares indicate degeneration in early disease stages. Square areas are transplanted earlier in the disease process than dot areas. Thus, dots and squares provide information regarding the timing when a particular cell population may be used. [Figure 15] Pigmentation and cell morphology. iPSC-RPE pigmentation levels (A, C, E) were examined after treatment with DMSO / control (A, B), endo-IWR-1 (C, D) or AGN 193109 (E, F) to ensure that the compounds were not harmful. Gross cell morphology was also analyzed to detect possible abnormalities (center). Fine subcellular structures were examined for changes using transmission electron microscopy (right). Endo-IWR-1 and AGN 193109 did not alter pigmentation levels or gross or fine cell morphology. [Figure 16]Transepithelial Electrical Resistance (TER). Graph showing the TER (a measure of monolayer tightness) observed for AGN 193109 and endo-IWR-1 treated cells. A cutoff of 400 ohms*cm square was used to exclude cells with low TER. AGN 193109 and endo-IWR-1 treated cells were well above the threshold. Data are displayed as box plots, with box limits representing the first and third quartiles, center line indicating the median, and whiskers indicating the fifth and ninety-fifth percentiles, so the range specifies 90% of the data. [Figure 17] Single-cell RNA sequencing, each dot represents the transcriptome of a single cell. The distance between the dots indicates how different these cells are (closer is more similar). The three sets cluster separately from each other; therefore, their transcriptomes are quite distinct. Shown are AGN 193109-treated cells; endo-IWR-1-treated cells and DMSO-treated cells. [Figure 18]Bulk RNA sequencing. The three heatmaps show the expression levels of specific genes from the literature (Radeke et al., Experimental Eye Research 85(3), 366-380, doi:10.1016 / j.exer.2007.05.006(2007); Whitmore et al., Experimental Eye Research 129, 93-106, doi:10.1016 / J.EXER.2014.11.001(2014); Voigt et al., Experimental Eye Research 184, 234-242, doi:10.1016 / J.EXER.2019.05.001(2019); van Soest et al., Molecular Vision 13, 1608-17, ncbi.nlm.nih.gov / pubmed / 17893662(2007); Li et al. iScience 23(11), 101672, doi:10.1016 / j.isci.2020.101672(2020); Ishibashi et al., Investigative Ophthalmology & Visual Science 45(9), 3291, doi:10.1167 / iovs.04-0168(2004) in the top row, and bulk RNA sequencing in the bottom row. The x-axis is labeled with the gene of interest. Dark grey corresponds to genes more expressed in the macula (reference) or AGN 193109-treated cells (present data, iPSC-RPE). Light grey corresponds to genes more expressed in the periphery (reference) or endo-IWR-1-treated cells (present data, iPSC-RPE). [Figure 19] Data are presented as box plots, with box limits representing the first and third quartiles, center lines indicating the median, and whiskers indicating the fifth and 95th percentiles, so that the range specifies 90% of the data. [Figure 20]Acid phosphatase activity. Graph showing higher acid phosphatase activity in AGN 193109 treated cells (macular iPSC-RPE) than endo-IWR-1. Data are displayed as box plots, with box limits representing the first and third quartiles, center line indicating the median, and whiskers indicating the fifth and ninety-fifth percentiles, so the range specifies 90% of the data. [Figure 21] Metabolic processes in two types of RPE cells. P-RPE have a higher capacity for OXPHOS (lower bar) compared to M-RPE (upper bar), which shows a higher rate of glycolysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS It is disclosed herein that several different types of RPE cells are present in the retina. Methods are disclosed herein for preparing macular, central and peripheral RPE cells from pluripotent stem cells such as iPSCs. These methods include culturing immature RPE cells in RPE maturation medium containing a retinoic acid receptor (RAR) antagonist and / or a canonical Wnt inhibitor to produce macular, central or peripheral human RPE cells. The macular, central and peripheral RPE cells generated by the disclosed methods can be used as cell therapy for treating retinal degeneration, to discover cell type specific drugs, to test drug toxicity to specific cell types, and to perform high throughput drug screening specific to different RPE regions. The macular, central and peripheral RPE cells also provide a model system for studying local RPE effects in different conditions.

[0015] term Unless otherwise stated, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's genes XII, published by Jones&Bartlett Learning, 2017. As used herein, the singular forms "a", "an" and "the" refer to both the singular and the plural, unless the context clearly indicates otherwise. For example, the term "canonical Wnt inhibitor" includes one or more canonical Wnt inhibitors and can be considered equivalent to the phrase "at least one canonical Wnt inhibitor". As used herein, the term "comprises" means "includes". Throughout this application, the term "about" is used to indicate that within 5 percent when a device is specified, or that a value includes the inherent variation of error of the device.

[0016] Furthermore, unless otherwise indicated, any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are understood to be provided for illustrative purposes. Although many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. In order to facilitate review of various embodiments, the following explanations of terms are provided.

[0017] Age-Related Macular Degeneration (AMD): AMD is caused by damage to the macula of the retina. While the onset of AMD can be asymptomatic, AMD gradually worsens over time, generally resulting in blurred or no vision in the center of the visual field in one or both eyes. Difficulty recognizing faces, driving, reading, or performing other activities of daily living may occur, and visual hallucinations may also occur. AMD typically occurs in older adults, such as subjects over the age of about 50. Genetic factors and smoking may play a role. Diagnosis includes a complete vision exam, and severity can range from early, intermediate, and late, which may further include "dry" and "wet" forms.

[0018] Dry AMD develops over time, as macular tissue thins and breaks down. Symptoms may include visual distortion, decreased central vision in one or both eyes, need for brighter light for reading or close tasks, increased difficulty adapting to low light levels, increased blurring of printed words, decreased intensity or brightness of colors, and difficulty recognizing faces. Dry AMD is diagnosed by examining the back of the eye for drusen; testing for defects in the center of vision (e.g., using an Amsler grid to identify whether straight lines in the grid become gradually less visible, broken, or distorted (indicating the presence of dry AMD)); fluorescein or indocyanine green angiograms (to check for abnormal blood vessels or retinal changes); and / or optical coherence tomography (to check for retinal thinning, thickening, or swelling). Currently available treatments include rehabilitation to adjust to the loss of central vision (low vision rehabilitation) and the implantation of telephoto lenses.

[0019] Wet AMD follows dry AMD and involves abnormal blood vessel growth and fluid accumulation at the back of the eye, which can cause a bulge in the macula and distort vision loss. In addition to the symptoms of dry AMD, the symptoms of wet AMD can also include clearly defined blurry spots or blind spots in the field of vision, generalized blurring of the entire field of vision, and sudden onset and rapid worsening of symptoms. Currently available treatments include medications aimed at stopping the growth of new blood vessels, such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®) and aflibercept (EYLEA®); photodynamic therapy; photocoagulation; and low vision rehabilitation.

[0020] In some examples, one or more of the cell populations (e.g., macular RPE cells, central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat wet or dry AMD.

[0021] Allele: One of two or more forms of a gene. Diploid organisms, such as humans, contain two copies of each chromosome and therefore have one allele of each.

[0022] The term "homozygous" is defined as containing two identical alleles at a particular locus. The term "heterozygous" refers to containing two different alleles at a particular locus. "Haplotype" refers to a combination of alleles at multiple loci along a single chromosome. A haplotype may be based on a set of single nucleotide polymorphisms (SNPs) on a single chromosome and / or alleles in the major histocompatibility complex. As used herein, the term "haplotype-matched" is defined as a cell (e.g., an iPSC cell) and a subject being treated sharing one or more major histocompatibility locus haplotypes. A subject's haplotype can be easily determined using assays well known in the art. Haplotype-matched iPSC cells can be autologous or allogeneic. Autologous cells grown in tissue culture (e.g., ex vivo) and essentially differentiated into RPE cells are haplotype-matched to the subject. "Substantially the same HLA type" indicates that the donor's HLA type matches the patient's HLA type to the extent that transplant cells obtained by inducing differentiation of iPSCs derived from the donor's somatic cells can engraft when they are transplanted into the patient. "Super donor" refers herein to an individual who is homozygous for certain MHC class I and II genes. These homozygous individuals can serve as super donors, and their cells (including tissues and other materials containing those cells) can be transplanted into individuals who are either homozygous or heterozygous for that haplotype. Super donors can be homozygous for HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DP or HLA-DQ loci / locus alleles, respectively.

[0023] Change: A change in the effective amount of a substance or parameter of interest, such as a polynucleotide, a polypeptide, or a property of a cell. The change in the polypeptide or polynucleotide or activity can affect the physiological properties of a cell, such as cell differentiation, proliferation, or survival. The amount of a substance can be changed by a difference in the amount of the substance produced, a difference in the amount of a substance with a desired function, or a difference in the activation 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 cell differentiation, proliferation, and / or survival.

[0024] Animal: A category that includes living multi-cellular vertebrate organisms, such as 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.

[0025] Antagonist or inhibitor: An agent that blocks or attenuates a biochemical or biological response when it binds to a receptor or a ligand of the receptor. Antagonists mediate their effects through receptor interaction by preventing agonist-induced responses. In one embodiment, Frizzled (Fzd) antagonists bind to Fzd receptors or Fzd ligands (such as Wnt) and reduce or inhibit the Wnt / beta-catenin signaling pathway (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% reduction).

[0026] bear track dystrophy: A condition that forms part of the disorder known as clustered congenital hypertrophy of the retinal pigment epithelium (CHRPE), a distinctive congenital anomaly of the retinal pigment epithelium diagnosed by its characteristic ophthalmoscopic appearance. The disorder is usually without functional consequences in patients with normal visual acuity, color vision, normal visual fields, dark adaptation, electroretinography and electrooculography findings. The main differential diagnosis of CHRPE includes choroidal nevus, choroidal melanoma, chorioretinal scar, subretinal hematoma, pigmented epiretinal membrane, and reactive retinal pigment epithelial hyperplasia.

[0027] 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.).

[0028] The term "cell population" typically refers to a group of cells of a common 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 (e.g., an unfractionated heterogeneous cell population) in which the proportion of a particular cell type, such as macular RPE cells, central RPE cells, or peripheral RPE cells, is greater than the proportion of that cell type in the starting population. A cell population may be enriched in one or more cell types and depleted in one or more cell types.

[0029] Cell adhesion protein: a protein involved in the attachment of cells to other cells or to the extracellular matrix in a process called cell adhesion. Cell adhesion proteins include vitronectin, fibrin, and laminin, but also integrins, which mediate cell-ECM interactions with collagen, fibrinogen, fibronectin, and vitronectin; cadherins, which are homophilic calcium-dependent glycoproteins; and selectins, a family of heterophilic proteins (E-selectin, L-selectin, P-selectin) that depend on fucosylated carbohydrates.

[0030] Chorioderemia: A rare X-linked recessive form of inherited retinal degeneration that affects roughly 1 in 50,000 males. The disease causes gradual loss of vision beginning with night blindness in childhood, followed by loss of peripheral vision, and progressing to central vision loss later in life. The progression continues throughout an individual's life, but both the rate of change and the extent of vision loss can vary among affected individuals, even within the same family. The first symptom many individuals with choroideremia notice is a significant loss of night vision, which begins at a young age. Peripheral vision loss occurs gradually, beginning as a ring of vision loss, continuing to "tunnel vision" in adulthood. Individuals with choroideremia tend to maintain good vision into their 40s, but eventually lose all vision at some point in the age range of 50-70 years. In some examples, one or more of the cell populations (e.g., central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat choroideremia.

[0031] Defined or fully defined: When used in reference to a medium, extracellular matrix or culture condition, it refers to a medium, extracellular matrix or culture condition in which the chemical composition and amount of almost 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. In general, a defined medium includes a basal medium (e.g., Dulbecco's Modified Eagle Medium (DMEM), F12 or Roswell Park Memorial Institute Medium (RPMI) 1640 containing 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.

[0032] Diabetic retinopathy: Diabetic retinopathy is a diabetic complication in which blood vessels in retinal tissue are damaged, and its symptoms can range from asymptomatic or mild, such as at onset, to blindness. In some embodiments, diabetic retinopathy is diagnosed by a comprehensive dilated eye examination to identify, for example, abnormal blood vessels; retinal swelling, blood or fatty deposits; growth of new blood vessels and scar tissue; bleeding of the clear, jelly-like substance that fills the center of the eye (vitreous); retinal detachment; and optic nerve abnormalities. Further diagnostic tests include, for example, vision, glaucoma and cataract tests, as well as fluorescein angiography or optical coherence tomography to determine whether fluid is leaking into retinal tissue. Currently available treatments include photocoagulation, focal laser treatment, panretinal photocoagulation, vitrectomy, and intravitreal administration of drugs such as vascular endothelial growth factor (VEGF) inhibitors. In some examples, one or more of the cell populations (e.g., macular RPE cells, central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat diabetic retinopathy.

[0033] Differentiation: The process by which unspecialized cells become more specialized types, with changes in structural and / or functional properties. Mature cells typically have altered cell structures and tissue-specific proteins. More specifically, in the context of the present method, it refers to the process by which stem cells acquire the cellular characteristics of RPE cells, with characteristics indicating that said RPE cells are macular RPE cells, central RPE cells or peripheral RPE cells.

[0034] As used herein, "undifferentiated" refers to cells that display characteristic markers and morphological features of undifferentiated cells that clearly distinguish them from terminally differentiated cells of embryonic or adult origin.

[0035] Embryo: A mass of cells obtained by one or more divisions of a zygote or activated oocyte with an artificially reprogrammed nucleus, whether or not implanted in a female. A "morula" is a preimplantation embryo 3-4 days after fertilization, when it is a solid mass generally composed of 12-32 cells (blastomeres). A "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, composed of undifferentiated cells, gives rise to what will become a fetus if the blastocyst is implanted in the uterus.

[0036] Embryoid bodies: three-dimensional aggregates of pluripotent stem cells. These cells can differentiate into cells of the endoderm, mesoderm and ectoderm. In contrast to monolayer culture, the spheroid structures formed when pluripotent stem cells aggregate allow non-adherent culture of EBs in suspension, which is useful for bioprocessing approaches. The three-dimensional structure, including the establishment of complex cell adhesion and paracrine signaling within the EB microenvironment, allows differentiation and morphogenesis.

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

[0038] Embryoid bodies (EBs): aggregates of pluripotent stem cells that can differentiate into cells of the endoderm, mesoderm, and ectoderm. Aggregation of pluripotent stem cells forms spheroid structures, allowing non-adherent culture of EBs in suspension.

[0039] 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 the remainder 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 an egg cell with sperm or DNA, nuclear transfer, parthenogenesis, or other means of generating hES cells with homozygosity in the HLA region. Human ES cells can be produced or derived from zygotes, blastomeres, or blastocyst stage mammalian embryos produced by fusion of sperm with an egg cell, nuclear transfer, parthenogenesis, or reprogramming of chromatin and subsequent integration of the reprogrammed chromatin into the plasma membrane to produce embryonic cells. Human embryonic stem cells include, but are not limited to, MAO1, MAO9, ACT-4, No.3, H1, H7, H9, H14 and ACT30 embryonic stem cells. Regardless of their source or the particular method used to produce them, human embryonic stem cells can be identified based on (i) their ability to differentiate into cells of all three germ layers, (ii) their expression of at least Oct-4 and alkaline phosphatase, and (iii) their ability to produce teratomas when transplanted into immunocompromised animals.

[0040] Essentially absent: With respect to a particular component, essentially absent is used herein to mean that none of the particular components are intentionally incorporated into the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the particular component resulting from unintentional contamination of the composition is well below 0.05%, such as less than 0.01%. In some embodiments, the amount of the particular component cannot be detected by standard analytical methods.

[0041] Feeder layer: a coating layer of cells, such as the bottom of a culture dish. Feeder cells can release nutrients into the culture medium and provide a surface to which other cells, such as pluripotent stem cells, can attach.

[0042] Feeder-free or feeder-independent: Cultures supplemented with cytokines and growth factors 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.

[0043] Fibroblast Growth Factor (FGF): Any suitable fibroblast growth factor from any animal, and functional fragments thereof, such as those that bind to a receptor and induce a biological effect associated with receptor activation. Exemplary FGFs 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 a fibroblast growth factor protein, such as FGF-1, FGF-2, FGF-4, FGF-6, FGF-8, FGF-9, or FGF-98, or a biologically active fragment or variant thereof. The FGF may 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 and methods for making many FGFs are known.

[0044] 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 in its entirety. 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 incorporated herein by reference in its entirety. Comparing the 146 residue forms, 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 greater purity) using techniques detailed in U.S. Patent No. 4,956,455.

[0045] FGF inducer comprises an active fragment of FGF. In its simplest form, the active fragment is made by removing N-terminal methionine, using well-known techniques for N-terminal methionine removal, such as treatment with methionine aminopeptidase. The second desired truncation comprises FGF without its leader sequence. Those skilled in the art recognize leader sequence as a series of hydrophobic residues at the N-terminus of a protein that facilitates passage through cell membranes, but is not necessary for activity and is not found on the mature protein. Human and mouse bFGF are commercially available.

[0046] Growth factor: a substance that promotes cell growth, survival and / or differentiation. Growth factors include molecules that function as growth stimulators (mitogens), factors that stimulate cell migration, factors that function as chemotactic factors or inhibit cell migration or invasion of tumor cells, factors that regulate the differentiation function of cells, factors that are involved in apoptosis, or factors that promote cell survival without affecting growth and differentiation. Examples of growth factors are fibroblast growth factors (such as FGF-2), epidermal growth factor (EGF), ciliary neurotrophic factor (CNTF), nerve growth factor (NGF), and activin A.

[0047] Inducer: A molecule that regulates gene expression, such as activating a gene in a cell. Inducers can bind to repressors or activators. Inducers function by disabling repressors.

[0048] Isolated: An "isolated" cell is substantially separated or purified from other cells in an organism or culture. An isolated cell can be, for example, at least 99%, at least 98% pure, at least 95% pure, or at least 90% pure.

[0049] KNOCKOUT™ Serum Replacement: A serum-free formulation optimized to grow and maintain undifferentiated cells, such as stem cells, in culture.

[0050] Late-onset retinal degeneration: a rare autosomal dominant disorder characterized by the presence of thick lipid-rich deposits between the RPE and Bruch's membrane. The disease was first seen in the mid-1990s and is associated with mutations in the C1QTNF5 gene. The C1QTNF5 gene, located on 11q23, encodes a 281 amino acid protein that is highly expressed in the RPE, lens, and ciliary epithelium.

[0051] Individuals with L-ORD often do not exhibit ocular problems until middle age, around 50-60 years of age. Clinically, early symptoms of the disease process include difficulty with light and dark adaptation and the inability to see in dim light or at night. Disease progression then results in loss of central and peripheral vision, choroidal neovascularization and pigmentary retinopathy throughout the retina. Eventually, decreased visual acuity leads to complete vision loss.

[0052] Ophthalmologic examination may be normal initially, even after night vision loss begins. However, as the disease progresses, the appearance of fine yellow-white drusen-like dots in the mid-periphery is the first ophthalmologic sign of L-ORD. These drusen-like "dots" or deposits then form areas of atrophy that extend throughout the retina. Fundus autofluorescence shows widespread, well-defined, corrugated areas of RPE and chorioretinal atrophy, primarily in the mid-periphery and posterior pole of the retina. The macula usually becomes atrophic but may occasionally form a discoid scar. The optic disc also becomes pale.

[0053] In some examples, one or more of the cell populations (e.g., central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat L-ORD.

[0054] Leber congenital amaurosis (LCA): A rare genetic eye disease that appears at birth or early in life (infancy or early childhood) and primarily affects the retina. Presentation can vary because it is associated with multiple genes. However, it is characterized by nystagmus, photophobia, blunted or absent pupillary response, and severe vision loss or blindness. Common modes of inheritance are autosomal recessive and autosomal dominant.

[0055] The pupils normally dilate and constrict in response to the amount of light entering the eye and do not respond normally to light. Instead, they may dilate and constrict more slowly than normal or not respond to light at all. In addition, the clear, front covering of the eye (the cornea) may be cone-shaped and abnormally thin, a condition known as keratoconus. A specific behavior called the Francesetti eye-digital sign is characteristic of LCA. This sign consists of poking, pushing, and rubbing the eye with a fist or finger.

[0056] In some examples, one or more of the cell populations (e.g., macular RPE cells, central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat LCA.

[0057] Mammal: The 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.

[0058] Membrane potential: The electrical potential inside a cell relative to an environment, such as an external bath solution. Those skilled in the art can readily assess the membrane potential of a cell, for example, by using conventional whole-cell techniques. Membrane potential can be assessed using a number of approaches, such as using conventional whole-cell access, or for example, using perforated patch whole-cell and cell-attached configurations.

[0059] Culture medium: A synthetic set of culture conditions that includes nutrients necessary to support the growth (cell proliferation / 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 growth medium includes a minimum essential medium, such as DMEM, supplemented with various nutrients to enhance stem cell proliferation. Additionally, the minimum essential medium may be supplemented with additives, such as horse, calf, or fetal bovine serum.

[0060] Noggin: a protein encoded by the NOG gene. Noggin inhibits TGF-β signaling by binding to TGF-β family ligands and preventing them from binding to their corresponding receptors. Noggin, along with other TGF-β signaling inhibitors (such as chordin and follistatin), plays an important role in neural induction by inhibiting BMP4. Exemplary sequences of Noggin are GENBANK® Accession Nos. NP_005441.1 and NM_005450.4 (January 13, 2013), which are incorporated herein by reference in their entirety.

[0061] Oct-4: A protein also known as POU5-F1 or MGC22487 or OCT3 or OCT4 or OTF3 or OTF4, which 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 portions of Oct-4 that retain the ability to be used to generate iPSCs. Oct-4 protein may have any published sequence for Oct-4 that can be obtained from public sources, such as GENBANK®. Examples of such sequences include, but are not limited to, GENBANK® Accession No. NM_002701.

[0062] Pharmaceutically acceptable carrier: Conventional pharma-ceutically acceptable carriers are useful for carrying out the methods and forming 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 compounds disclosed herein.

[0063] Generally, the nature of the carrier depends on the particular mode of administration used.For example, parenteral formulations usually contain injectable fluids that contain pharma- ceutically and physiologically acceptable fluids as vehicles, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, etc. For solid compositions (e.g., in the form of powder, pill, tablet, or capsule), conventional non-toxic solid carriers can include, for example, pharmaceutical grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, 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, for example, sodium acetate or sorbitan monolaurate.

[0064] Preconfluent: A cell culture in which the entire tissue culture surface is not covered with cells and cell division can occur. The percentage of the culture surface covered by cells can be about 60-80%. Typically, preconfluent refers to a culture in which about 70% of the culture surface is covered by cells.

[0065] Purified: A purified composition does not require absolute purity; rather, it is intended as a relative term. Thus, a purified cell population may be greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% pure, e.g., essentially free of other cell types.

[0066] Retinoic Acid Receptor (RAR) Antagonists: RAR receptors are nuclear receptors that are activated by all-trans and 9-cis retinoic acid. The best-known mechanism of action of these receptors involves their binding to RA response elements (RAREs) in the promoters of retinoid-responsive genes. Retinoid receptors also affect transcription through RARE-independent mechanisms, such as repression of the transcription factor activator protein. There are three subtypes of RAR receptors; pan-RAR antagonists inhibit all subtypes.

[0067] retina light-sensitive layer of tissue that lines the inner surface of the eye.

[0068] Retinal detachment: Retinal detachment is a condition in which the retina pulls away from its normal position, with symptoms including a sudden increase in the number of floating spots (floaters) in vision, flashes of light in one or both eyes, a "curtain" or shadow in the field of vision, and, without immediate treatment, permanent loss of vision.

[0069] In some examples, one or more of the cell populations (e.g., peripheral RPE cells) generated using the methods provided herein are used to treat retinal detachment.

[0070] Retinal Diseases and Disorders: Retinal diseases include diseases in which the function or structure of the retina is damaged or diminished. Includes retinal degenerative diseases in which the structure or function of the retina deteriorates over time. Includes retinal vascular diseases, such as retinal diseases in which the structure or function of the blood vessels of the eye is affected.

[0071] In some examples, one or more of the cell populations (e.g., macular RPE cells, central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat a retinal disease.

[0072] Retinal lineage cell: A cell that can give rise to or differentiate into an RPE cell.

[0073] Retinal Induction Medium (RIM): A growth medium that contains a WNT pathway inhibitor and a BMP pathway inhibitor and can result in differentiation of pluripotent stem cells (PSCs) into retinal lineage cells. RIM also contains a TGFβ pathway inhibitor.

[0074] Retinal Differentiation Medium (RDM): A medium that contains a WNT pathway inhibitor, a BMP pathway inhibitor, and a mitogen-activated protein kinase (MEK) (or FGF) inhibitor to differentiate retinal cells. RDM also contains a TGFβ pathway inhibitor.

[0075] Retinal medium (RM): A growth medium for culturing retinal cells, containing activin A and nicotinamide.

[0076] RPE maturation medium (RPE-MM): A medium for maturation of RPE cells that includes taurine and hydrocortisone. RPE-MM also includes triiodothyronine. RPE-MM may also include PD0325901 or PGE2.

[0077] Retinal Pigment Epithelial (RPE) Cells: RPE cells can be recognized based on pigmentation, epithelial morphology, and apical-basal polarized cells. RPE cells express one or more of Pax6, MITF, RPE65, CRALBP, PEDF, bestrophin, and / or Otx2, both at the mRNA and protein levels. In certain other embodiments, RPE cells express one or more of Pax-6, MitF, and tyrosinase, both at the mRNA and protein levels. RPE cells do not express (at any detectable level) the embryonic stem cell markers Oct-4, nanog, or Rex-1. Specifically, the expression of these genes is approximately 1 / 100-1 / 1000 lower in RPE cells than in ES cells or iPSCs, as assessed by quantitative RT-PCR. Differentiated RPE cells can also be visually recognized by their morphology and initial appearance of pigment. Furthermore, differentiated RPE cells have a high transepithelial resistance / TER and transepithelial potential / TEP across the monolayer (TER>100 ohms*cm 2 ; TEP>2mV), transport fluid and CO2 from the apical to the basal side, and regulate the polarized secretion of cytokines. RPE cells include peripheral, central, and macular RPE cells.

[0078] In humans, the "macular" RPE cells are located approximately 150 μm 2 +33μm 2 The central RPE cells have an area of ​​about 199 μm and a petal-like apical projection that has a length of about 0.5 to about 4 μm, a width of about 0.1 to about 1 μm, and a swell width of about 0.2 to about 2 μm. 2 +40μm2 The "peripheral" RPE cells have an area of ​​approximately 239 μm2 and have a mixture of petal-like and finger-like apical processes. 2 +38μm 2 and a finger-like apical projection having a length of about 0.1 to about 1 μm and a width of about 0.1 to about 1 μm.

[0079] "Mature" RPE cells are referred to herein as RPE cells in which the expression of immature RPE markers, such as Pax6, is downregulated and the expression of mature RPE markers, such as RPE65, is upregulated.

[0080] The "maturation" of RPE cells herein refers to the process of regulating RPE development pathway to generate mature RPE cells.For example, regulating cilia function can lead to RPE maturation.The "retinal lineage cell" herein refers to the cell that can give rise to or differentiate into RPE cells.

[0081] Retinitis pigmentosa (RP): RP is an inherited degenerative eye disease that causes severe vision loss due to progressive degeneration of rod photoreceptor cells in the retina. This form of retinal dystrophy presents with early symptoms independent of age. Early retinal degenerative symptoms of RP are characterized by decreased night vision (night blindness) and loss of mid-peripheral vision. Rod photoreceptor cells are responsible for low light vision, are oriented in the retinal periphery, and are the first retinal projections affected during the nonsyndromic form of the disease. Vision loss progresses relatively rapidly to the far peripheral field and eventually spreads to the central field as tunnel vision increases. Visual acuity and color vision may be impaired due to concomitant abnormalities in the cone photoreceptor cells, which are responsible for color vision, visual acuity, and central vision. Progression of disease symptoms occurs symmetrically, with both eyes experiencing symptoms at a similar rate.

[0082] In some examples, one or more of the cell populations (e.g., macular RPE cells, central RPE cells and / or peripheral RPE cells) generated using the methods provided herein are used to treat RP.

[0083] Retinoic Acid Receptor (RAR) Antagonists: RAR receptors are nuclear receptors that are activated by all-trans and 9-cis retinoic acid. One mechanism of action of these receptors involves their binding to RA response elements (RAREs) in the promoters of retinoid-responsive genes. Retinoid receptors also affect transcription through RARE-independent mechanisms, such as repression of the transcription factor activator protein. There are three subtypes of RAR receptors; pan-RAR antagonists inhibit all subtypes.

[0084] In some examples, the RAR antagonist reduces activity by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.

[0085] Examples of RAR antagonists include AGN 193109 (4-[2-[5,6-dihydro-5,5-dimethyl-8-(4-methylphenyl)-2-naphthalenyl]ethynyl]-benzoic acid), CE 2665, ER 5081, LE 135, LY 2955303, MM 11253, and liarozole dihydrochloride.

[0086] Stem cell: A cell that can differentiate under appropriate conditions into a diverse range of specialized cell types, but can self-renew and maintain an essentially undifferentiated pluripotent state under other appropriate conditions. The term "stem cell" also encompasses pluripotent stem cells, multipotent stem cells, precursor cells, and progenitor cells. Exemplary human stem cells can be derived from hematopoietic or mesenchymal stem cells obtained from bone marrow tissue, embryonic stem cells obtained from embryonic tissue, or embryonic germ cells obtained from fetal reproductive tissue. Exemplary pluripotent stem cells can also be produced from somatic cells by reprogramming them to a pluripotent state through the expression of certain transcription factors associated with pluripotency. These cells are called "induced pluripotent stem cells" or "iPSCs."

[0087] "Embryonic stem (ES) cells" are obtained from earlier stage embryos, such as the inner cell mass of the blastocyst stage, or produced by artificial means (e.g., nuclear transfer), and are undifferentiated pluripotent cells that can give rise to any differentiated cell type, including germ cells (e.g., sperm and eggs), in the embryo or adult.

[0088] "Induced pluripotent stem cells (iPSCs)" are cells 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 certain 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 four reprogramming factors to reprogram somatic cells into pluripotent stem cells.

[0089] The term "pluripotency" refers to the property of cells that differentiate into all other cell types in an organism except extraembryonic or placental cells. Pluripotent stem cells can differentiate into cell types of all three germ layers (e.g., ectodermal, mesodermal and endodermal cell types) even after long-term culture. Pluripotent stem cells are embryonic stem cells derived from the inner cell mass of blastocysts. In other embodiments, pluripotent stem cells are induced pluripotent stem cells obtained by reprogramming somatic cells.

[0090] Tissue replacement implant: a biocompatible structure that contains both matrix and cells produced in vitro that can be used to replace tissue in vivo. The tissue replacement implant can contain macular RPE cells, central RPE cells and / or peripheral RPE cells, and combinations thereof. The tissue replacement implant can also contain rod, cone and / or vascular cells, or combinations thereof.

[0091] Treatment: A therapeutic measure that cures, slows, alleviates symptoms, and / or halts the progression of a diagnosed pathological condition or disorder. In certain 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.

[0092] Undifferentiated: A cell that exhibits characteristic markers and morphological features of undifferentiated cells that distinguish them 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.

[0093] 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, genes of the Wnt family encode cysteine-rich glycoproteins of 38-43 kDa. Wnt proteins have a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence (see, for example, Shimizu et al Cell Growth Differ 8:1349-1358 (1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote stabilization of cytoplasmic beta-catenin, Wnt proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain Wnt proteins is associated with certain cancers.

[0094] 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 / beta-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, which is activated upon receptor binding, is mediated by the cytoplasmic protein Dishevelled (Dvl), which interacts directly with Fzd receptors, resulting in cytoplasmic stabilization and accumulation of β-catenin. In the absence of Wnt signals, beta-catenin localizes to a cytoplasmic destruction complex that contains the tumor suppressor proteins adenomatous polyposis coli (APC) and Axin. These proteins function as critical scaffolds that allow glycogen synthase kinase (GSK)-3beta to bind and phosphorylate beta-catenin, marking it for degradation via the ubiquitin / proteasome pathway. Activation of Dvl results in dissociation of the destruction complex. Accumulated cytoplasmic beta-catenin is then transported into the nucleus where it interacts with DNA-binding proteins of the TCF / LEF family to activate transcription.

[0095] 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 GTPase and ROCK kinase without activating cytoplasmic beta-catenin. ROCK regulates the cytoskeleton to regulate the apical-basal polarity of cells. Due to competition for the same receptors, non-canonical WNT ligands also lead to inhibition of canonical WNT signaling.

[0096] Xeno-free (XF): When used in reference to a medium, extracellular matrix, or culture condition, refers to a medium, extracellular matrix, or culture condition that is essentially free of components derived from a xenogeneic animal. For culturing human cells, any protein of a non-human animal, such as a mouse, is a xenogeneic component. In certain embodiments, a xeno-free matrix is ​​essentially free of components derived from a non-human animal, and thus may exclude mouse feeder cells or MATRIGEL™. MATRIGEL™ is a solubilized basement membrane preparation extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumor enriched in extracellular matrix proteins to include laminin (major component), type IV collagen, heparan sulfate proteoglycan, and entactin / nidogen.

[0097] Therapeutically effective amount: A quantity of a compound or cell, e.g., RPE cell, that, when administered to a subject for the treatment of a disease or condition, is sufficient to effect such treatment or to alleviate the symptoms of the disease or condition. Dysfunction of the retinal pigment epithelium is associated with several vision altering conditions, such as retinal pigment epithelium detachment, dysplasia, atrophy, retinopathy, retinitis pigmentosa, macular dystrophies and degeneration.

[0098] Vascular structure: Vascular structure can include the circulatory system (e.g., the arrangement of blood vessels) or portions thereof, such as the vascular supply to a particular area.

[0099] pluripotent stem cells 1.Embryonic stem cells ES cells are derived from the inner cell mass of blastocysts and have a high in vitro differentiation capacity. ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo and then culturing the inner mass cells on a feeder layer of ungrown cells. The re-plated cells can continue to grow and produce new colonies of ES cells, which can be removed, dissociated, re-plated again, and grown. This process of "passaging" undifferentiated ES cells can be repeated multiple 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 potential to proliferate while maintaining their pluripotency. For example, ES cells are useful in the study of genes that control cells and cell differentiation. The pluripotency of ES cells combined with genetic manipulation and selection can be used for in vivo genetic analysis studies through the creation of transgenic, chimeric, and knockout mice.

[0100] Methods for generating mouse ES cells are known. In one method, preimplantation blastocysts from mouse line 129 are treated with mouse antisera 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 resulting colonies of undifferentiated ES cells are subcultured on the 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 propagated in the absence of a feeder layer by adding the cytokine leukemia inhibitory factor (LIF) to serum-containing culture 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.

[0101] Human ES cells can be produced or derived by previously described methods from zygotes or blastocyst stage mammalian embryos produced by sperm-egg fusion, nuclear transfer, pathogenesis, or reprogramming of chromatin and subsequent integration of the reprogrammed chromatin into the plasma membrane to produce embryonic cells. In one method, human blastocysts are exposed to anti-human serum, and trophectoderm cells are lysed and removed from the inner cell mass which are cultured on a feeder layer of mouse embryonic fibroblasts. Furthermore, clumps of cells 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. In other methods, human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MATRIGEL® or laminin in the presence of a "conditioned" medium containing basic fibroblast growth factor. Human ES cell lines are available. In some embodiments, human ES cells have not required the destruction of human embryos. These include the use of established ES cell lines.

[0102] ES cells can also be derived from other organisms, including rhesus monkeys and marmosets by methods already described, as well as established mouse and human cell lines.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 lineage 129 embryos, and the culture of CGR8 cells can be grown in the presence of LIF without feeder layer.

[0103] 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.

[0104] a. Somatic cell nuclear transfer Pluripotent stem cells can be prepared by the method of somatic cell nuclear transfer. Somatic cell nuclear transfer involves the transfer of a donor nucleus into a spindle-free oocyte. In one method, donor fibroblast nuclei from primate skin fibroblasts are introduced into the cytoplasm of spindle-free mature metaphase II primate oocytes by electrofusion. The fused oocytes are activated by exposure to ionomycin and then incubated until the blastocyst stage. The inner cell mass of the selected blastocysts is then cultured to produce embryonic stem cell lines. The embryonic stem cell lines show normal ES cell morphology, express various ES cell markers, and differentiate into multiple cell types both in vitro and in vivo. The embryo is not destroyed in the production of these ES cells.

[0105] 2. Induced pluripotent stem cells Induction of pluripotency was first achieved in 2006 (Yamanaka et al.) using mouse cells and in 2007 using human cells by reprogramming somatic cells with the introduction of pluripotency-associated transcription factors. 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 grafts may not require lifelong immunosuppressive treatment to prevent graft rejection.

[0106] Any cell can be used as the starting point for iPSCs, except for germ cells. For example, the cell type can be keratinocytes, fibroblasts, hematopoietic cells, mesenchymal cells, liver cells, 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 (U.S. Patent 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 harvested; undifferentiated progenitor cells (including somatic stem cells) and even terminally differentiated mature cells can be used as a source of somatic cells in the methods disclosed herein. In one embodiment, the somatic cells are themselves RPE cells, such as human RPE cells. The RPE cells can be adult or fetal RPE cells. 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.

[0107] Somatic cells and pluripotent stem cells, such as CD34+ cells, can be reprogrammed to produce induced pluripotent stem cells (iPSCs) using methods known to those skilled in the art.Those skilled in the art can easily produce 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 No. WO 2007 / 069666 A1 and US Patent No. 8,268,620 (whole incorporated herein by reference).Generally, nuclear reprogramming factors are used to produce 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.

[0108] Cells are generally treated with nuclear reprogramming agents, which are one or more factors that can induce iPSC from somatic cells, or with nucleic acids that code these agents (including those that are incorporated into vectors). Nuclear reprogramming agents generally include at least Oct3 / 4, Klf4 and Sox2 or nucleic acids that code these molecules. Functional inhibitors of p53, L-myc or nucleic acids that code L-myc, and Lin28 or Lin28b or nucleic acids that code Lin28 or Lin28b can be used as additional nuclear reprogramming agents. Nanog can also be used for nuclear reprogramming.As disclosed in U.S. Patent Application Publication No. 20120196360, exemplary reprogramming factors for the production of iPSCs include: (1) Oct3 / 4, Klf4, Sox2, L-Myc (Sox2 can be replaced with Soxl, Sox3, Soxl5, Soxl7, or Soxl8; Klf4 can be replaced with Klfl, Klf2, or Klf5; (2) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, SV40 large T antigen (SV40LT); (3) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, human papillomavirus (HPV) 16 E6; (4) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E7; (5) Oct3 / 4, Klf4, Sox2, L-Myc, TERT, HPV16 E6, HPV16 E7;(6)Oct3 / 4, Klf4, Sox2, L-Myc, TERT, Bmil;(7)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28;(8)Oct3 / 4 , Klf4, Sox2, L-Myc, Lin28, SV40LT;(9)Oct3 / 4, Klf4, Sox2, L-Myc, Lin28, TERT, SV40LT;(10)Oc t3 / 4, Klf4, Sox2, L-Myc, SV40LT; (11) Oct3 / 4, Esrrb, Sox2, L-Myc (Esrrb can be replaced by Esrrg); (12) Oct3 / 4, Klf4, Sox2; (13) Oct3 / 4, Klf4, Sox2, TERT, SV40LT; (14) Oct3 / 4, Klf4, Sox2, TERT, HP VI 6 E6; (15) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E7; (16) Oct3 / 4, Klf4, Sox2, TERT, HPV16 E6, HPV16 E7; (17) Oct3 / 4, Klf4, Sox2, TERT, Bmil; (18) Oct3 / 4, Klf4, Sox2, Lin28 (19) Oct3 / 4, Klf4, Sox2, Lin28, SV40LT; (20) Oct3 / 4, Klf4, Sox2, Lin28, TERT, SV40LT; (21) Oct3 / 4, Klf4, Sox2, SV40LT; or (22) Oct3 / 4, Esrrb, Sox2 (Esrrb can be replaced by 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, for example, US Patent No. 7,682,828, which is incorporated herein by reference in its entirety. These factors include, but are not limited to, Oct3 / 4, Klf4 and Sox2. In other examples, factors include, but are not limited to, Oct3 / 4, Klf4 and Myc. In some non-limiting examples, Oct3 / 4, Klf4, c-Myc and Sox2 are 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, an integration vector such as a system based on EBV elements can be used (US Patent No. 8,546,140). In further embodiments, reprogramming proteins can be directly introduced into somatic cells by protein transduction. Reprogramming can further include contacting cells with one or more signaling receptors, including glycogen synthase kinase 3 (GSK-3) inhibitors, mitogen-activated protein kinase (MEK) inhibitors, transforming growth factor beta (TGF-β) receptor inhibitors or signaling inhibitors, 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 cell line can be used.

[0109] The mouse and human cDNA sequences of these nuclear reprogramming agents are available by reference to the NCBI accession numbers listed in WO 2007 / 069666, which is incorporated by reference in its entirety. Methods of introducing one or more reprogramming agents, or nucleic acids encoding these reprogramming agents, are known and are disclosed, for example, in U.S. Patent Application No. 2012 / 0196360 and U.S. Patent No. 8,071,369, both of which are incorporated by reference in their entirety.

[0110] Once induced, iPSCs can be cultured in a medium sufficient to maintain pluripotency. iPSCs can be used with various media and techniques developed to culture pluripotent stem cells, more specifically embryonic stem cells, as described in U.S. Patent No. 7,442,548 and U.S. Patent Publication No. 2003 / 0211603. For mouse cells, culture is performed with the addition of leukemia inhibitory factor (LIF) as a differentiation inhibitor to normal medium. For human cells, it is desirable to add basic fibroblast growth factor (bFGF) instead of LIF. Other methods for the culture and maintenance of iPSCs may be used.

[0111] In certain embodiments, undefined conditions can be used. For example, pluripotent cells can be cultured on fibroblast feeder cells or media 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 treated with radiation or antibiotics to terminate cell division, as feeder cells. Alternatively, pluripotent cells can be cultured and maintained in an essentially undifferentiated state using defined feeder-independent culture systems, such as TESR™ medium or E8™ medium.

[0112] In some embodiments, iPSCs can be modified to express exogenous genes, increase expression of endogenous genes, increase gene copy number, correct genetic mutations, or silence expression of mutant genes, etc. In some specific, non-limiting examples, mutations or deletions of endogenous genes are corrected. The genes include, for example, retinoid isomerase (RPE65), bestrophin (BEST) 1, MER tyrosine kinase 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), centrosome protein, It may code for 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, type XVIII, alpha-1 (COL18A1), TTRA serine peptidase (HTRA) 1, ARMS2, tissue inhibitor of metalloproteinases (TIMP) 3, epidermal growth factor (EGF)-containing fibulin-like extracellular matrix protein (EFEMP) 1, microphthalmia-associated transcription factor (MITF), transcription factor EC (TFEC), orthodenticle, Drosophila, 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 performing 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 in its entirety. Any of the methods disclosed therein are useful. This method can include the use of a viral vector, such as an adeno-associated virus vector or a lentivirus vector, that terminates a transgene of interest. This method can include the use of CRISPR / Cas9, TALEN nuclease, zinc finger nuclease, lentivirus-mediated correction, adeno-associated virus-mediated correction, shRNA, siRNA, or F prime editing.

[0113] In some embodiments, iPSCs can be modified to express exogenous nucleic acids, such that they include a nucleic acid sequence encoding a tyrosinase enhancer operably linked to a promoter and a first marker. The tyrosinase gene is disclosed, for example, in GENBANK® Accession No. 22173, available on January 1, 2013. This sequence aligns to positions 5286971-5291691 (in reverse orientation) of chromosome 7 of mouse strain C57BL / 6. A sequence of 4721 base pairs is sufficient for expression in RPE cells (see Murisier et al., Dev. Biol. 303:838-847, 2007, which is incorporated herein by reference in its entirety). This construct is expressed in RPE cells. Other enhancers can be utilized. Other RPE-specific enhancers include D-MITF, DCT, TYRP1, RPE65, VMD2, MERTK, MYRIP and RAB27A. Suitable promoters include any promoter that is expressed in RPE cells, including but not limited to 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 advantageous to transfect cells with the construct. Suitable vectors for stable transfection include but are not limited to retroviral vectors, lentiviral vectors and Sendai virus.

[0114] The plasmid can achieve a regulated high copy number and is compatible for use in mammalian cells, including human cells. In some examples, the plasmid is suitable for maintenance and fermentation in E. coli so that large amounts of DNA can be produced and purified. The plasmid is safe and can be suitable for use in human patients and animals. High copy number plasmids can be relatively easily selected and stably maintained during bacterial fermentation. Elements such as selection markers and other coding sequences can be included in the plasmid. In some embodiments, the plasmid encoding the marker includes: (1) a high copy number origin of replication, (2) a selection marker, such as but not limited to a 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 a tyrosinase promoter. There are numerous plasmid vectors known in the art for introducing nucleic acids encoding proteins, such as those disclosed in U.S. Pat. No. 6,103,470; U.S. Pat. No. 7,598,364; U.S. Pat. No. 7,989,425; U.S. Pat. No. 6,416,998, which are incorporated by reference in their entireties.

[0115] Viral gene delivery systems can be RNA-based 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 lentiviral vectors.

[0116] In some embodiments, cells are transfected with nucleic acid molecules encoding markers. Markers include, but are not limited to, fluorescent proteins (e.g., green fluorescent protein or red fluorescent protein), 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 enzymatically active nucleic acid molecules) or polysaccharides. Included are determinants of any such cellular components detectable by antibodies, lectins, probes, or nucleic acid amplification reactions specific for markers of the cell type of interest. Markers can also be identified by biochemical or enzymatic assays or biological responses that depend on the function of the gene product. Nucleic acid sequences encoding these markers can be operably linked to tyrosinase enhancers. In addition, other genes can be included, such as genes that can affect the differentiation of stem cells to RPE, or the function, physiology, or pathology of RPE. Thus, in some embodiments, nucleic acids encoding one or more of 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 are included.

[0117] A MHC haplotype match The major histocompatibility complex is the primary cause of immune rejection of allogeneic organ transplants. 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 across 4 Mb on chromosome 6. The ability to haplotype HLA genes within this region is clinically important, as this region is associated with autoimmune and infectious diseases, and HLA haplotype compatibility between donor and recipient can affect the clinical outcome of transplantation. HLA corresponding to MHC class I presents peptides from the inside of cells, while HLA corresponding to MHC class II presents antigens from the outside of cells to T lymphocytes. Incompatibility of MHC haplotypes between the graft and the host triggers an immune response against the graft, resulting in its rejection. Thus, subjects can be treated with immunosuppressants to prevent rejection. HLA-matched stem cell lines may overcome the risk of immune rejection.

[0118] Due to the importance of HLA in transplantation, HLA loci are usually typed by serology and PCR to identify favorable donor-recipient pairs. Serological detection of HLA class I and HLA class 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 -B loci. Molecular-based tissue typing can often be more accurate than serological tests. Low-resolution molecular methods such as the SSOP (sequence-specific oligonucleotide probe) method, in which PCR products are tested against a series of oligonucleotide probes, can be used to identify HLA antigens, and currently these methods are the most common methods used for class II-HLA typing. High-resolution techniques such as the SSP (sequence-specific primer) method, which utilizes allele-specific primers for PCR amplification, can identify specific MHC alleles.

[0119] When donor cells are HLA homozygous, i.e., contain identical alleles for each antigen-presenting protein, the MHC compatibility between donor and recipient increases significantly. Most individuals are heterozygous for MHC class I and II genes, but certain individuals are homozygous for these genes. These homozygous individuals can act as super donors, and the grafts generated from their cells can be transplanted into all individuals who are either homozygous or heterozygous for that haplotype. Furthermore, if homozygous donor cells have a haplotype that is frequently found in the population, these cells may have application in transplantation therapy for a large number of individuals.

[0120] Thus, the iPSCs may be expressed as CD34 IgG1-specific IgG1-specific IgG2-specific IgG3-specific IgG4-specific IgG1-specific IgG2-specific IgG1 ... + iPSCs can be produced from cells such as cells. In some cases, the major HLA of the donor (e.g., the three major loci of HLA-A, HLA-B and HLA-DR) is identical to the major HLA of the recipient. In some cases, the somatic cell donor can be a super donor. Thus, iPSCs derived from MHC homozygous super donors can be used to generate RPE cells. Thus, iPSCs derived from super donors can be transplanted into subjects that are either homozygous or heterozygous for their haplotype. For example, iPSCs can be homozygous in two HLA alleles, such as HLA-A and HLA-B. In this way, iPSCs produced from super donors can be used in the methods disclosed herein to generate RPE cells that can potentially "match" to multiple potential recipients.

[0121] B. Episomal vectors In certain embodiments, the reprogramming factors are expressed from an expression cassette contained in one or more exogenous episomal genetic elements (see US Patent Application Publication No. 2010 / 0003757, which is incorporated herein by reference in its entirety). Thus, iPSCs may be essentially free of exogenous genetic elements, such as retroviral or lentiviral vector elements. These iPSCs are prepared by the use of extrachromosomally replicating vectors (i.e., episomal vectors), which are vectors that can replicate episomally to render the iPSCs essentially free of exogenous vectors or viral elements (see US Patent No. 8,546,140, ​​which is incorporated herein by reference in its entirety; 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 or episomally in mammalian cells. These episomal plasmids are essentially free of all these drawbacks associated with integrative vectors (Bode et al., 2001). For example, the lymphotropic herpesvirus systems defined above or the Epstein-Barr Virus (EBV) can replicate extrachromosomally and aid in the delivery of reprogramming genes to somatic cells. Useful EBV elements are OriP and EBNA-1, or their variants or functional equivalents. One advantage of episomal vectors is that exogenous elements are lost over time after being introduced into the cells, resulting in self-sustaining iPSCs that are essentially free of these elements.

[0122] 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 herpesvirus (KSHV); herpesvirus saimiri (HS) and Marek's disease virus (MDV). Additional sources of episome-based vectors are contemplated, such as yeast ARS, adenovirus, SV40 or BPV.

[0123] Methods for producing RPE cells Disclosed herein are methods for producing macular, central or peripheral RPE cells. In some embodiments, the cells can be produced from pluripotent stem cells such as ESCs or iPSCs.

[0124] RPE cells can be characterized based on their pigmentation, epithelial morphology and apical-basal polarity. Differentiated RPE cells can be visually recognized by their morphology and initial appearance of pigment. In humans, "macular" RPE cells are located approximately 150 μm 2 +33μm 2 The central RPE cells have an area of ​​about 199 μm and a petal-like apical projection that has a length of about 0.5 to about 4 μm, a width of about 0.1 to about 1 μm, and a swell width of about 0.2 to about 2 μm. 2 +40μm 2 The "peripheral" RPE cells have an area of ​​approximately 239 μm2 and have a mixture of petal-like and finger-like apical processes. 2 +33μm 2 and finger-like apical processes having a length of about 0.1 to about 1 μm and a width of about 0.1 to about 1 μm. Methods for producing these types of RPE cells are disclosed.

[0125] Furthermore, all differentiated RPE cells have a high transepithelial resistance / TER and transepithelial potential / TEP across the monolayer (TER>100 ohms*cm 2 ; TEP>2mV), transport fluid and CO2 from apical to basal sides, and regulate polarized secretion of cytokines. RPE cells express several proteins that can serve as markers for detection by use of methodologies such as immunocytochemistry, Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). For example, RPE-specific markers may 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 is approximately 1 / 100-1 / 1000 lower in RPE cells than in ES cells or iPSCs, as assessed by quantitative RT-PCR. RPE cell markers can be detected at the mRNA level, for example, by reverse transcriptase polymerase chain reaction (RT-PCR) using sequence-specific primers with standard amplification methods using publicly available sequence data (GENBANK®), Northern blot analysis, or dot blot hybridization analysis. Expression of tissue-specific markers detected at the protein or mRNA level is considered positive if the level is at least or about 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, or 9-fold, more specifically, more than 10-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold, or higher, than that of control cells (e.g., undifferentiated pluripotent stem cells or other unrelated cell types).

[0126] The exemplary method for producing macular RPE cells, central RPE cells and peripheral RPE cells from iPSCs and ESCs is disclosed below. The description of the inhibitor that can be used in the medium is disclosed at the end of this section. When referring to a class of inhibitor, it should be noted that any specific inhibitor can be used.

[0127] 1. Cultivate pluripotent stem cells to produce RPE progenitor cells The disclosed method includes culturing pluripotent stem cells (PSCs), such as ES cells or iPSCs, in a retinal induction medium to initiate the differentiation of the cells into RPE progenitor cells.PCT Publication No. 2014 / 121077, the entirety of which is incorporated herein by reference, discloses a method in which embryoid bodies (EBs) produced from iPSCs can be utilized in a culture method to produce RPE cells.For example, embryoid bodies are produced from iPSCs by adding a rho-associated coiled-coil kinase (ROCK) inhibitor, and cultured in a first medium containing two WNT pathway inhibitors and a Nodal pathway inhibitor.

[0128] In some embodiments, an essentially single cell suspension of PSCs, such as human iPSCs, can be utilized in the method. In some embodiments, PSCs are cultured to pre-confluence to prevent cell aggregation. In certain aspects, PSCs are dissociated by incubation with cell dissociation enzymes, such as exemplified by TRYPSIN™ or TRYPLE™. PSCs can also be dissociated into an essentially single cell suspension by pipetting. Additionally, blebbistatin (e.g., about 2.5 μM) can be added to the medium to increase PSC survival after dissociation into single cells while the cells are not attached to the culture vessel. Alternatively, a ROCK inhibitor can be used in place of blebbistatin to increase PSC survival after dissociation into single cells.

[0129] To efficiently differentiate RPE cells from single-cell PSCs, accurate counting of input density can increase RPE differentiation efficiency. Therefore, the single-cell suspension of PSCs can be counted before seeding. For example, the single-cell suspension of PSCs can be counted by a hemocytometer or an automated cell counter, such as VICELL® or TC20. The cells can be diluted to a cell density of at least 10,000 cells / mL, such as at least 20,000 cells / mL, at least 50,000 cells / mL, at least 75,000 cells / mL, at least 100,000 cells / mL, at least 150,000 cells / mL, or at least 200,000 cells / mL, such as 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, a single cell suspension of PSCs is diluted to a density of approximately 100,000 cells / mL in a completely defined culture medium, such as ESSENTIAL 8™ (E8™) medium.

[0130] In other embodiments, PSCs, such as iPSCs, are seeded at a cell density of about 1,000 to about 75,000 cells / cm, for example, about 5,000 to about 40,000 cells / cm. In other embodiments, PSCs, such as iPSCs, are seeded at a cell density of at least 1,000 cells / cm, at least 2,000 cells / cm, at least 5,000 cells / cm, at least 10,000 cells / cm, at least 20,000 cells / cm, or at least 50,000 cells / cm, and in a 6-well plate, the cells may be seeded at a cell density of about 50,000 to about 400,000 cells per well. In exemplary methods, cells are seeded at a cell density of about 100,000, about 150,00, about 200,000, about 250,000, about 300,000 or about 350,000 cells per well, for example about 200,00 cells per well.

[0131] Once a single cell suspension of PSCs is obtained at a known cell density, the cells are generally seeded into a suitable culture vessel, such as a tissue culture plate, such as a flask, a 6-well, a 24-well, or a 96-well plate. The culture vessel used to culture the cells can include, but is not limited to, a flask, a tissue culture flask, a dish, a Petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a multi-plate, a multiwell plate, a microslide, a chamber slide, a tube, a tray, a CELLSTACK® chamber, a culture bag, and a roller bottle, as long as the stem cells can be cultured therein. Cells may be cultured in a volume of at least or about 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture. In certain embodiments, the culture vessel may be a bioreactor, which may refer to any ex vivo device or system that supports a biologically active environment such that cells can grow. The bioreactor can have a volume of at least 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 derivable range.

[0132] PSCs, such as iPSCs, are generally cultured on culture plates coated with a matrix comprising one or more cell adhesion proteins to promote cell adhesion while maintaining cell viability. For example, exemplary cell adhesion proteins include extracellular matrix proteins, such as vitronectin, laminin, collagen and / or fibronectin, which may be used to coat culture surfaces as a means of providing a solid support for pluripotent cell growth. In some embodiments, iPSCs are cultured on a matrix comprising at least one cell adhesion protein, such as laminin, vitronectin or fibronectin. Combinations of laminin, vitronectin and / or fibronectin are also useful. In other embodiments, PSCs are grown on culture plates coated with vitronectin or fibronectin. In some embodiments, the cell adhesion protein is a human protein.

[0133] Extracellular matrix can be utilized. The term "extracellular matrix" is art-recognized. Its 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, andurin, epiligrin, and kalinin.

[0134] Extracellular matrix (ECM) proteins can be of natural origin and can be purified from human or animal tissues, or ECM proteins can be genetically engineered recombinant proteins or synthetic in nature. ECM proteins can be in the form of natural or engineered whole proteins or peptide fragments. Examples of ECM proteins that can be useful in matrices for cell culture include laminin, collagen I, collagen IV, 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 xeno-free matrices for culturing human cells, matrix components of human origin can be used and any non-human animal components can be omitted.

[0135] In some aspects, the total protein concentration in the matrix composition can be about 1 ng / mL to about 1 mg / mL. In some embodiments, the total protein concentration in the matrix composition is about 1 μg / mL to about 300 μg / mL. In other embodiments, the total protein concentration in the matrix composition is about 5 μg / mL to about 200 μg / mL.

[0136] a.Culture conditions Cells such as RPE cells or PSCs can be cultured with the nutrients necessary to support the growth of each particular cell population, such as RPE progenitor 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 contain fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidants, pyruvate, buffering agents, and inorganic salts. An exemplary growth medium contains a minimum essential medium, such as Dulbecco's Modified Eagle Medium (DMEM) or ESSENTIAL 8™ (E8™) medium, supplemented with various nutrients, such as non-essential amino acids and vitamins, to enhance stem cell growth. Examples of minimum essential medium include, but are not limited to, Minimum Essential Medium Eagle (MEM) Alpha Medium, Dulbecco's Modified Eagle Medium (DMEM), RPMI-1640 Medium, 199 Medium, and F12 Medium. Additionally, the minimum essential medium may be supplemented with additives, such as horse, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, the growth medium may contain "Knockout serum replacement," referred to herein as a serum-free formulation optimized to grow and maintain 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 its entirety. In some embodiments, PSCs are cultured in a completely defined feeder-free medium.

[0137] Thus, single cell PSCs are generally cultured in fully defined culture medium after seeding. In certain embodiments, about 18-24 hours after seeding, the medium is aspirated and fresh medium, such as E8™ medium, is added to the culture. In certain embodiments, single cell PSCs are cultured in fully defined culture medium for about 1, 2, or 3 days after seeding. In some non-limiting examples, single cell PSCs are cultured in fully defined culture medium for about 2 days before proceeding with the differentiation process.

[0138] In some embodiments, the medium may contain a serum replacement. The serum replacement may include materials that suitably contain albumin (e.g., lipid-rich albumin, albumin substitutes, e.g., recombinant albumin, vegetable starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. The serum replacement may be prepared, for example, by the method disclosed in WO 98 / 30679. Alternatively, for more convenience, any commercially available material may be used. Commercially available materials include KNOCKOUT™ Serum Replacement (KSR), Chemically Defined Concentrated Lipids (Gibco), and GLUTAMAX™ (Gibco). The medium may be serum-free.

[0139] Other culture conditions can be appropriately determined. For example, the culture temperature can be about 30-40°C, for example, at least or about 31, 32, 33, 34, 35, 36, 37, 38, 39°C, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration can be about 1-10%, for example, about 2-5%, or any range derivable therein. The oxygen pressure can be at least, at most, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20%, or any range derivable therein.

[0140] 2. Differentiation Medium and Stepwise Culture a. Retinal induction medium After PSCs such as ES cells or iPSCs are attached to the culture plate, the cells can be cultured in retinal induction medium to start the differentiation process into RPE progenitor cells. The retinal induction medium (RIM) can include a WNT pathway inhibitor and cause the differentiation of PSCs into retinal lineage cells. The RIM further includes a TGFβ pathway inhibitor and a BMP pathway inhibitor. In some embodiments, the RIM includes a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor and insulin growth factor 1 (IGF1).

[0141] The RIM may include DMEM and F12 in a ratio of about 1:1. In an exemplary method, a WNT pathway inhibitor is included in the RIM (such as CKI-7), a BMP pathway inhibitor is included (such as LDN193189), and a TGFβ pathway inhibitor is included (such as SB431542). For example, the RIM includes 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. Additionally, the RIM may include knockout serum replacement, such as about 1% to about 5%, 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 a concentration of about 0.1 ng / mL to about 10 ng / mL, for example about 1 ng / mL. The medium can be aspirated and replaced with fresh RIM daily. The cells are generally cultured in the RIM for about 1 to about 5 days, for example about 1, 2, 3, 4 or 5 days, for example about 2 days, to produce RPE progenitor cells.

[0142] b. Retinal differentiation medium The resulting RPE progenitor cells can then be cultured in retinal differentiation medium (RDM) for further differentiation into committed RPE cells. The RDM can include a WNT pathway inhibitor, a BMP pathway inhibitor, a TGFβ pathway inhibitor, and a MEK inhibitor. The RDM can include a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, a MEK inhibitor, and an IGF1.

[0143] 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 PD032590. 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 compared to the RIM, for example about 9 to about 11 times higher, for example about 10 times higher. In an exemplary method, the RDM comprises about 50 nM to about 200 nM, e.g., about 100 nM, LDN193189, about 1 μM to about 10 μM, e.g., about 5 μM, CKI-7, about 1 μM to about 50 μM, e.g., about 10 μM, SB431542, and about 0.1 μM to about 10 μM, e.g., about 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, or 9 μM, PD0325901.

[0144] Generally, RDM comprises DMEM and F12 in about a 1:1 ratio, knockout serum replacement (e.g., about 1% to about 5%, e.g., 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, e.g., about 10 ng / mL). In certain methods, cells are fed fresh RDM daily after aspirating the medium from the previous day. Generally, cells are cultured in RDM for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days, e.g., about 7 days, to induce committed RPE cells.

[0145] C. Retinal culture medium The resulting committed RPE cells are then further differentiated to form immature RPE cells by culturing in retinal medium (RM). The retinal medium comprises activin A and can further comprise nicotinamide. The RM can comprise about 50 to about 200 ng / mL, e.g., about 100 ng / mL, of activin A, and about 1 mM to about 50 mM, e.g., about 10 mM, of nicotinamide. Alternatively, the RM can comprise other TGF-β pathway activators, e.g., GDF1 and / or WNT pathway activators, e.g., WAY-316606, IQ1, QS11, SB-216763, BIO(6-bromoindirubin-3'-oxime) or 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3-methoxyphenyl)pyrimidine. In some examples, the RM further comprises WNT3a.

[0146] RM may contain about a 1:1 ratio of DMEM and F12, about 1% to about 5%, e.g., about 1.5%, knockout serum replacement, MEM non-essential amino acids (NEAA), sodium pyruvate, N-2 supplement, B-27 supplement, and ascorbic acid. Media may be changed daily in room temperature RM. Cells are generally cultured in RM for about 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 days, e.g., about 10 days, to induce or produce immature RPE cells.

[0147] d.RPE maturation medium For further differentiation of immature RPE cells, the cells are subsequently cultured in RPE maturation medium (RPE-MM) to form macular, central or peripheral RPE cells. RPE maturation medium can include about 100 μg / mL to about 300 μg / mL, for example about 250 μg / mL taurine, about 10 μg / L to about 30 μg / L, for example about 20 μg / L hydrocortisone, and about 0.001 μg / L to about 0.1 μg / L, for example about 0.013 μg / L triiodothyronine. In some examples, RPE-MM includes MEM alpha, N-2 supplement, MEM non-essential amino acids (NEAA), sodium pyruvate, and fetal bovine serum (for example, about 0.5% to about 10%, for example about 1% to about 5%).

[0148] The medium can be changed every other day at room temperature in RPE-MM.RPE-MM can contain RAR antagonist, canonical Wnt inhibitor, or both.Exemplary RAR antagonists include AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253, and liarozole dihydrochloride.In some non-limiting examples, the RAR antagonist is AGN 193109. Exemplary canonical Wnt inhibitors include 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (Endo-1-IWR), calphostin C, cardionogen 1, CCT 031374 hydrobromide, IWP 12, XAV 939, WIKI4, ICG-001, Wnt-C59 (C59), IWR-1-endo, KY02111, LGK-974, IWP-L6, FH535, iCRT 14, IWP 4, JW 67, JW 74, KYA 1797K, NLS-StAx-h, PNU 74654, TAK 715, IWP 2, CKI 7 dihydrochloride, (R)-CR8, D 4476, (R)-DRF053 dihydrochloride, epiblastin A, IC 261, LH 846, PF 4800567 hydrochloride, PF 5006739, PF 670462, SR 3029, AZ 6102, JW 55, MN 64, and TC-E 5001. In some non-limiting examples, the canonical Wnt inhibitor is Endo-1-IWR.

[0149] Immature cells are generally cultured in RPE-MM for about 5 to about 10 days, for example, about 5 days. The cells can then be dissociated, for example, using a cell dissociation enzyme, reseeded, and cultured for an additional period, for example, for another about 5 to about 50 days, about 5 to about 40 days, or about 5 to about 30 days. The cells can be cultured, for example, for about 15 to about 20 days, about 15 to about 25 days, or about 20 to about 25 days, or about 20 to about 30 days, about 30 to about 40 days, about 30 to about 50 days, or about 40 to about 50 days to further differentiate into RPE cells in the presence of a RAR antagonist, a Wnt inhibitor, or both. In specific, non-limiting examples, to produce macular RPE cells, central RPE cells, or peripheral RPE cells, cells are cultured in RPE-MM in the presence of a RAR antagonist, a canonical Wnt inhibitor, or both, for about 15 to about 50 days, e.g., about 20 to about 50 days, about 30 to about 50 days, or about 40 to about 50 days.

[0150] i. Production of macular RPE cells In some embodiments, the method produces macular RPE cells, and RPE-MM comprises RAR antagonist but does not comprise canonical Wnt inhibitor.Macular RPE cells can be human macular RPE cells.The exemplary RAR antagonists that can be present in RPE-MM include one or more of AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253 and liarozole dihydrochloride.

[0151] In some non-limiting examples, the RAR antagonist in the RPE-MM is AGN 193109. The RPE-MM can include about 0.05 to about 0.4 μM AGN 193109, such as about 0.1 to about 0.2 μM AGN 193109. The RPE-MM can include, for example, about 0.05 to about 0.3 μM, about 0.05 to about 0.2 μM, or about 0.05 to about 0.1 μM AGN 193109. RPE-MM can include, for example, about 0.05 to about 0.4 μM, about 0.1 to about 0.2 μM, about 0.15 to about 0.4 μM, about 0.2 to about 0.4 μM, about 0.25 to about 0.4 μM, about 0.3 to about 0.4 μM, or about 0.35 to about 0.4 μM AGN 193109. RPE-MM can include, for example, about 0.1 μM, about 0.15, or about 0.2 μM AGN 193109.

[0152] Immature RPE cells are typically cultured in RPE-MM containing a RAR antagonist but no canonical Wnt inhibitor for about 5 to about 50 days, e.g., about 5 to about 25 days, e.g., about 5 to about 10 days, e.g., about 5 days. The cells can then be dissociated, e.g., with a cell dissociation enzyme, reseeded, and cultured for an additional period, e.g., about 5 to about 50 days, e.g., about 5 to about 40 days, e.g., about 5 to about 30 days, e.g., about 15 to about 20 days, about 15 to about 25 days, or about 20 to about 25 days, about 20 to about 30 days, about 30 to about 40 days, about 40 to about 50 days, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, to further differentiate into central RPE cells in the presence of a RAR antagonist. In a specific, non-limiting example, to produce central RPE cells, immature RPE cells are cultured in RPE-MM containing a RAR antagonist for about 15 to about 25 days.

[0153] In a specific, non-limiting example, the RAR antagonist is AGN 193109.

[0154] ii. Production of central RPE cells In other embodiments, the method produces central RPE cells, and RPE-MM comprises both RAR antagonist and canonical Wnt inhibitor.Central RPE cells can be human central RPE cells.Exemplary RAR antagonists that can be present in RPE-MM include AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253 and liarozole dihydrochloride. Exemplary canonical Wnt inhibitors that may be present in RPE-MM include 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (Endo-1-IWR), calphostin C, cardionogen 1, CCT 031374 hydrobromide, IWP 12, XAV 939, WIKI4, ICG-001, Wnt-C59 (C59), IWR-1-endo, KY02111, LGK-974, IWP-L6, FH535, iCRT 14, IWP 4, JW 67, JW 74, KYA 1797K, NLS-StAx-h, PNU 74654, TAK 715, IWP 2, CKI 7 dihydrochloride, (R)-CR8, D 4476, (R)-DRF053 dihydrochloride, epiblastin A, IC 261, LH 846, PF 4800567 hydrochloride, PF 5006739, PF 670462, SR 3029, AZ 6102, JW 55, MN 64, and TC-E 5001.

[0155] In some non-limiting examples, the RAR antagonist is AGN 193109 and the canonical Wnt inhibitor is ENDO1-1WR. The RPE-MM can include, for example, about 10 nM to about 50 nM AGN 193109 and about 0.025 to about 0.5 μM Endo-1-IWR, for example, about 25 nM to about 50 nM AGN 193109 and about 0.1 μM to about 0.2 μM Endo-1-IWR.

[0156] RPE-MM can include about 10 nM to about 40 nM, about 10 nM to about 30 nM, about 10 nM to about 20 nM, about 20 nM to about 50 nM, about 30 nM to about 50 nM, or about 40 nM to about 50 nM of ANG 193109. RPE-MM can include, for example, about 0.025 to about 0.4 μM, about 0.025 to about 0.3 μM, about 0.025 to about 2 μM, or about 0.025 to about 1 μM of ANG 193109. RPE-MM can contain, for example, about 0.05 to about 0.5 μM, about 0.1 to about 0.5 μM, about 0.15 to about 0.5 μM, about 0.2 to about 0.5 μM, about 0.25 to about 0.5 μM, about 0.3 to about 0.5 μM, about 0.35 to about 0.5 μM, about 0.4 to about 0.5 μM, or about 0.45 to about 0.5 μM Endo-1-IWR.

[0157] The RPE-MM can include about 25 to about 45 nM, about 25 to about 40 nM, about 25 to about 35 nM, or about 25 to about 30 nM AGN 193109. The RPE-MM can include about 0.1 to about 0.125 μM, about 0.1 to about 0.15 μM, or about 0.1 to about 0.175 μM Endo-1-IWR.

[0158] Immature RPE cells are typically cultured in RPE-MM containing a RAR antagonist and a canonical Wnt inhibitor for about 5 to about 50 days, e.g., about 5 to about 25 days, e.g., about 5 to about 10 days, e.g., about 5 days. The cells can then be dissociated, e.g., with a cell dissociation enzyme, reseeded, and cultured for an additional period, e.g., about 5 to about 50 days, e.g., about 5 to about 40 days, e.g., about 5 to about 30 days, e.g., about 15 to about 20 days, about 15 to about 25 days, or about 20 to about 25 days, about 20 to about 30 days, about 30 to about 40 days, about 40 to about 50 days, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, to further differentiate into central RPE cells in the presence of a RAR antagonist and a canonical Wnt inhibitor. In a specific, non-limiting example, to produce central RPE cells, immature RPE cells are cultured in RPE-MM containing a RAR antagonist and a canonical Wnt inhibitor for about 15 to about 25 days.

[0159] iii. Generation of peripheral RPE cells In a further embodiment, the method produces peripheral RPE cells, and the RPE maturation medium comprises a canonical Wnt inhibitor but does not comprise a RAR antagonist. The peripheral RPE cells can be human peripheral RPE cells.

[0160] Exemplary canonical Wnt inhibitors that may be present in RRPE MM include 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (Endo-1-IWR), calphostin C, cardionogen 1, CCT 031374 hydrobromide, IWP 12, XAV 939, WIKI4, ICG-001, Wnt-C59 (C59), IWR-1-endo, KY02111, LGK-974, IWP-L6, FH535, iCRT 14, IWP 4, JW 67, JW 74, KYA 1797K, NLS-StAx-h, PNU 74654, TAK 715, IWP 2, CKI 7 dihydrochloride, (R)-CR8, D 4476, (R)-DRF053 dihydrochloride, epiblastin A, IC 261, LH 846, PF 4800567 hydrochloride, PF 5006739, PF 670462, SR 3029, AZ 6102, JW 55, MN 64, and TC-E 5001. In a non-limiting example, the canonical Wnt inhibitor present in RRPE MM is Endo-1-IWR.

[0161] In some non-limiting examples, the RPE-MM contains about 0.5 to about 8 μM Endo-1-IWR, for example, about 1 to about 4 μM Endo-1-IWR. The RPE-MM can contain, for example, about 0.5 to about 7 μM, about 0.5 to about 6 μM, about 0.5 to about 5 μM, about 0.05 to about 4 μM, about 0.05 to about 3 μM, about 0.05 to about 2 μM, or about 0.05 to about 1 μM Endo-1-IWR. The RPE-MM can contain, for example, about 1 to about 2 μM or about 1 to about 3 μM Endo-1-IWR.

[0162] Immature RPE cells are typically cultured in RPE-MM containing a canonical Wnt inhibitor but no RAR antagonist for about 5 to about 50 days, e.g., about 5 to about 25 days, e.g., about 5 to about 10 days, e.g., about 5 days. The cells can then be dissociated, e.g., with a cell dissociation enzyme, reseeded, and cultured for an additional period of time, e.g., about 5 to about 50 days, e.g., about 5 to about 40 days, e.g., about 5 to about 30 days, e.g., about 15 to about 20 days, about 15 to about 25 days, or about 20 to about 25 days, about 20 to about 30 days, about 30 to about 40 days, about 40 to about 50 days, or about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 days, to further differentiate into central RPE cells in the presence of a canonical Wnt inhibitor. In a specific, non-limiting example, to produce central RPE cells, immature RPE cells are cultured in RPE-MM containing a canonical Wnt inhibitor for about 15 to about 25 days.

[0163] 3. Cryopreservation Macular RPE cells, central RPE cells and / or peripheral RPE cells produced by the methods disclosed herein can be cryopreserved, see, e.g., PCT Publication No. 2012 / 149484 A2, which is incorporated herein by reference in its entirety. The cells can 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 thereof. 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 preserve the cells. In further embodiments, the cells are preserved for more than about 6 hours. In further embodiments, the cells are preserved for about 72 hours. In some embodiments, the cells are preserved for 48 hours to about 1 week. In yet other embodiments, the cells are stored for about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In further embodiments, the cells are stored for 1, 2, 3, 4, 5, 67, 8, 9, 10, 11, or 12 months. The cells can also be stored for longer periods of time. The cells can be cryopreserved separately or on a substrate, such as any of the substrates disclosed herein.

[0164] In some embodiments, additional cryoprotectants can be used.For example, cells can be cryopreserved in a cryopreservation solution that contains one or more cryoprotectants, such as DM80, serum albumin, such as human or bovine serum albumin.Cryoprotectants can intercalate into cell membranes and change the properties of cells so that they survive freezing.Central RPE cells, peripheral RPE cells and macular RPE cells can be cryopreserved as isolated populations, or can be mixed in desired ratios before cryopreservation.

[0165] In certain embodiments, the solution comprises 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 comprises 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 comprises 2.5% DMSO. In another specific embodiment, the solution comprises 10% DMSO.

[0166] The cells may be cooled, for example, at about 1° C. / min during cryopreservation. In some embodiments, the cryopreservation temperature is about −80° C. to about −180° C., or about −125° C. to about −140° C. In some embodiments, the cells are cooled to 4° C. before cooling at about 1° C. / min. Cryopreserved cells may be transferred to the vapor phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells reach about −80° C., they are transferred to a liquid nitrogen storage area. Cryopreservation may also be performed using a controlled rate freezer. Cryopreserved cells may be thawed, for example, at a temperature of about 25° C. to about 40° C., for example, at a temperature of about 37° C. The cells may then be matured on the scaffold, as discussed below.

[0167] 4. Maturation of RPE cells on the scaffold The RPE cells generated by the disclosed methods can be cultured in RPE-MM (e.g., containing a canonical Wnt inhibitor, a RAR antagonist, or both) for a sustained period of maturation. In some embodiments, the RPE cells are grown in wells, such as 6-well, 12-well, 24-well, or 10 cm plates (e.g., with a plastic non-permeable or semi-permeable surface). The RPE cells can be maintained in the RPE-MM on the scaffold 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, the RPE cells are cultured in medium on the scaffold for about 2 to 6 weeks, such as about 5 weeks, to obtain a monolayer of mature and functional macular, central, or peripheral RPE cells. This culture results in polarized macular, central, or peripheral RPE cells on the scaffold, which together form a tissue implant.

[0168] A variety of biological or synthetic solid matrix materials (i.e., solid support matrices, biological adhesives or dressings, and biological / medical scaffolds) are suitable for use as scaffolds. The materials can be biodegradable or non-biodegradable. Any implantable surface can be utilized.

[0169] The material is generally physiologically acceptable and suitable for use in in vivo applications.Non-limiting examples of such physiologically acceptable materials include, but are not limited to, biodegradable solid matrix materials, such as cross-linked or non-cross-linked alginate, hydrocolloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) meshes, polyglactin (PGL) meshes, and bioadhesives (e.g., fibrin glue and fibrin gel).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), e.g., having copolymer ratios of about 90:10, 75:25, 50:50, 25:75, 10:90 (PLLA:PLGA) (see Thomson et al., J. Biomed. Mater Res. A 95:1233-42, 2010).

[0170] Suitable polymeric carriers that can include matrices include porous meshes or sponges made of synthetic or natural polymers. A non-limiting example is a polymer hydrogel. Natural polymers that can be used include proteins such as collagen, albumin and fibrin; and polysaccharides such as alginate and hyaluronic acid polymers. Synthetic polymers can be biodegradable. Examples of biodegradable polymers include polymers of hydroxy acids such as polylactic acid (PLA), polyglycolic acid (PGA), and polylactic-glycolic acid (PLGA), polyorthoesters, polyanhydrides, polyphosphazenes, and combinations thereof.

[0171] In some embodiments, the scaffold is a PLGA scaffold. PLGA is a copolymer of polylactic acid (PLA) and polyglycolic acid (PGA). Polylactic acid contains an asymmetric α-carbon, typically described in classical stereochemical terms as D- or L-forms, and sometimes as R- and S-forms, respectively. The enantiomeric forms of the polymer PLA are poly D-lactic acid (PDLA) and poly L-lactic acid (PLLA). PLGA is poly D, L-lactic-co-glycolic acid, with the D-lactic acid and L-lactic acid forms generally in equal ratios. PLGA biodegrades by hydrolysis of its ester bonds. In some embodiments, the PLGA scaffold is cultured for a sufficient time such that the majority of the lactate release from the scaffold occurs in vitro. In some embodiments, more than 50%, 60%, 70%, 80%, 90% or 95% of the lactate release occurs in vitro. The lactate release occurs over time. Thus, in some embodiments, this effect is achieved by maintaining the RPE in RPE-MM on the scaffold 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 in medium on the scaffold for about 2 to 6 weeks, such as about 5 weeks.

[0172] In some embodiments, the PLGA scaffold has 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. In one specific, non-limiting example, the DL-lactide / glycolide ratio is 1:1. In this context, "about" refers to within 5%.

[0173] 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 PLGA scaffold can be about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 microns thick. In this context, about refers to within 5%.

[0174] The scaffold can include nanofibers that cross over one another to form crossover junctions. The scaffold can be treated to fuse the fibers of the scaffold at the junctions of fiber crossovers in the PLGA scaffold to increase mechanical strength. The average pore size is the space between the fibers in the PLGA scaffold.

[0175] In further 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 refers to within 5%.

[0176] 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" refers to within 5%.

[0177] Any of the PLGA scaffold characteristics of thickness, pore size, and fiber diameter described herein can be combined. One skilled in the art can understand that varying the DL-lactide / glycotide ratio will adjust the pore size and fiber diameter. Thus, one skilled in the art can produce PLGA scaffolds of the disclosed thickness, pore size, and fiber diameter. Any of the characteristics can be combined to arrive at a particular combination produced by varying the DL-lactide / glycotide ratio. 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.

[0178] In some embodiments, to increase the mechanical strength of the PLGA scaffold, the scaffold is treated with heat to fuse the fibers of the scaffold at the junctions (fiber intersections) within the PLGA scaffold. This heat treatment also reduces the pore size by fusing the fibers at the junctions, thus allowing cells to form a monolayer on the scaffold. In some non-limiting examples, in some embodiments, the scaffold is placed on a suitable surface, for example, a metal surface such as aluminum foil, such as in the form of an envelope placed in an oven set at the desired temperature for treatment. Suitable temperatures include about 35°C to about 55°C, for example about 40°C to about 50°C, 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, for example about 10 to about 15 minutes, for example about 10, 11, 12, 13, 14 or 15 minutes. In one embodiment, the scaffold is treated at about 45°C for about 10 minutes. The temperature can then be increased relative to the first temperature, for example to about 50°C to about 70°C, for example to about 55°C to about 60°C, for example to about 55°C, 56°C, 57°C, 58°C, 59°C or 60°C. The higher temperature treatment can be for about 45 minutes to about 75 minutes, for example to about 50 minutes to about 70 minutes, or 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°C for about 60 minutes. In one non-limiting example, the scaffold can be treated at about 45°C for about 10 minutes, then at about 60°C for about 60 minutes. After heat treatment, the scaffold can be stored.

[0179] 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 occur after heating the scaffold as disclosed above.

[0180] In other embodiments, the PLGA scaffold is coated with an extracellular matrix, which is a complex mixture of structural and functional biomolecules and / or biopolymers, including but not limited to structural proteins, specialized proteins, proteoglycans, glycosaminoglycans, and growth factors that surround and support cells in mammalian tissues, and is acellular unless otherwise indicated. Extracellular matrices can be prepared by, for example, but not limited to, 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; 6,485,72 Nos. 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 produced from any tissue or from any in vitro source, where the ECM is produced by cultured cells and contains one or more polymeric components of native ECM. ECM preparations can be considered "decellularized" or "acellular," meaning that the cells have been removed from the source tissue or culture.

[0181] 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, the 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, for example, but not limited to, the submucosa, epithelial basement membrane, lamina propria, etc. In one non-limiting embodiment, the ECM is isolated from the bladder.

[0182] The scaffold can include one or more pharmaceutical agents. In some embodiments, the scaffold provides sustained release of one or more pharmaceutical agents. In more embodiments, the pharmaceutical agent is a molecule that inhibits dedifferentiation (or epithelial-mesenchymal transition) of RPE cells, inhibits the formation of drusen deposits under RPE cells, or suppresses reactive oxygen species of RPE cells. In some non-limiting examples, the inhibitor of RPE cell dedifferentiation 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 pharmaceutical agent can be metformin, Nox4 inhibitor, reactive oxygen inhibitor aminocaproic acid, riluzole, or NK-κβ inhibitor. In a specific non-limiting example, the pharmaceutical agent is L,745,870. In another specific non-limiting example, the pharmaceutical agent is metformin. In a further non-limiting example, the pharmaceutical agent is a Nox4 inhibitor (VAS2870, GKT 137831 or GLX7013114). In a further non-limiting example, the pharmaceutical agent is a reactive oxygen species inhibitor (GKT 137831 or GLX7013114 or N-acetylcysteine).

[0183] Before seeding the macular RPE cells, central RPE cells and / or peripheral RPE cells on the scaffold, the scaffold can be sterilized. In some embodiments, gamma irradiation is used to sterilize the scaffold. In other embodiments, an electron beam (ebeam) 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)).

[0184] In some embodiments, macular RPE cells, central RPE cells and / or peripheral RPE cells are seeded onto a scaffold (such as a PLGA scaffold) at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold, e.g., about 150,000 cells per 12 mm diameter, about 200,000 cells per 12 mm diameter, about 250,000 cells per 12 mm diameter, about 300,000 cells per 12 mm diameter, about 350,000 cells per 12 mm diameter, about 400,000 cells per 12 mm diameter, or about 450,000 cells per 12 mm diameter of the scaffold (such as a PLGA scaffold).

[0185] In some embodiments, mature macular, central or peripheral RPE cells develop into a monolayer that behaves as intact macular, central or peripheral RPE tissue by continued culture in RPE-MM (e.g., containing a canonical Wnt inhibitor, a RAR antagonist, or both) on the scaffold. Additional small molecules can be included in the RPE-MM (e.g., containing a canonical Wnt inhibitor, a RAR antagonist, or both). In some embodiments, these small molecules are primary cilia inducers such as prostaglandin E2 (PGE2) or aphidicolin. PGE2 can be added to the RPE-MM at a concentration of about 25 μM to about 250 μM, for example, about 50 μM to about 100 μM.

[0186] In some embodiments, for continued maturation of RPE cells, immature RPE cells can be dissociated in cell dissociation enzymes, such as TRYPLE™, in RPE-MM (e.g., containing a canonical Wnt inhibitor, a RAR antagonist, or both) with a MEK inhibitor, such as PD0325901, and reseeded on scaffolds, such as specialized SNAPWELL™ designs, for at least about 1-2 weeks. Alternatively, the RPE-MM (e.g., containing a canonical Wnt inhibitor, a RAR antagonist, or both) can include a bFGF inhibitor instead of a MEK inhibitor. Suitable methods for culturing RPE cells on degradable scaffolds are taught and described in PCT Publication No. WO 2014 / 121077 and PCT Publication No. WO 2020 / 106622, both of which are incorporated herein by reference in their 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 disclosed above. The SNAPWELL™ plate provides a structure and platform for the biodegradable scaffold. A microporous membrane that creates an apical and basal side supports the scaffold and isolates separate sides of the polarized layer of cells. The ability of the SNAPWELL™ insert to peel off the membrane allows the support ring of the insert to be used as an anchor for the scaffold (see below). The resulting monolayer of macular, central or peripheral RPE cells on the scaffold can then be isolated and used as a tissue replacement implant.

[0187] In yet other embodiments, macular, central or peripheral RPE cells on a scaffold are maintained at a resting potential of about -50 to about -60 mV and a concentration of about 5 to about 10 μl cm -2 h -1has a fluid transport rate of In further embodiments, the macular RPE cells, central RPE cells or peripheral 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 are induced by a vasoconstriction of about 5 to about 10 μl cm -2 h -1 In other embodiments, the RPE cells have a fluid transport rate of 100 Ω*cm 2 Ultra high, for example 200Ω*cm 2 In a further embodiment, the macular RPE cells, central RPE cells or peripheral RPE cells have a transcutaneous resistance of greater than 100 Ω*cm 2 ~500Ω*cm 2 of skin resistance, e.g. 200Ω*cm 2 ~400Ω*cm 2 It has a percutaneous resistance of .

[0188] In one non-limiting example, a) a method is provided for obtaining a PLGA coated with vitronectin, wherein the PLGA scaffold comprises fibers forming a mesh structure, the PLGA scaffold has an upper surface and a lower surface, the PLGA scaffold has a thickness of about 5 to about 30 microns, a DL-lactide / glycolide ratio of about 1:1, an average pore size of less than about 1 micron, and a viscosity of about 150 to about 650 μm. a) obtaining a PLGA scaffold having a fiber diameter of about 100 nm; b) treating the scaffold with heat to fuse the fibers of the scaffold at the junctions of fiber intersections within the PLGA scaffold to increase the mechanical strength of the PLGA scaffold and reduce the pore size; c) seeding macular, central and / or peripheral RPE cells on the PLGA scaffold at about 125,000 to about 500,000 cells per 12 mm diameter of the PLGA scaffold; and d) culturing the macular, central and / or peripheral RPE cells on the PLGA scaffold in vitro in tissue culture medium with medium present on both the top and bottom surfaces of the PLGA scaffold for a time sufficient for i) polarization of the RPE cells and ii) bulk degradation of the PLGA scaffold.

[0189] 5. Useful inhibitors in the differentiation of peripheral, central and macular RPE cells Inhibitors useful in the preparation of RPE cells are disclosed below and can be included in the retinal induction medium, retinal differentiation medium, retinal medium, and / or RPE maturation medium, as described above.

[0190] 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 cysteine-rich glycoproteins of 38–43 kDa. WNT proteins have a hydrophobic signal sequence, a conserved asparagine-linked oligosaccharide consensus sequence (see, for example, Shimizu et al Cell Growth Differ 8:1349-1358(1997)), and 22 conserved cysteine ​​residues. Due to their ability to promote stabilization of cytoplasmic beta-catenin, WNT proteins can act as transcriptional activators and inhibit apoptosis. Overexpression of certain WNT proteins has been shown to be associated with certain cancers.

[0191] WNT inhibitor in this specification generally refers to 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.A particular embodiment of the method relates to the WNT inhibitor in differentiation medium. Examples of suitable WNT inhibitors already known in the art include N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide dihydrochloride (CKI-7), N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]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 1 (DKK1), and secreted frizzled-related protein (SFRP1) 1. In addition, inhibitors of WNT may 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 using RNA-mediated interference (RNAi). Useful exemplary Wnt inhibitors are listed in Table 5.

[0192] BMP pathway inhibitors Bone morphogenetic proteins (BMPs) are multifunctional growth factors that belong to the transforming growth factor beta (TGFβ) superfamily. BMPs are thought to constitute a group of crucial morphogenetic signals that coordinate structure throughout the body. The critical function of BMP signals in physiology is highlighted by the numerous roles for dysregulated BMP signaling in pathological processes.

[0193] BMP pathway inhibitors may include inhibitors of BMP signaling in general, 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-[ These include 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).

[0194] c.TGFβ pathway inhibitors Transforming growth factor beta (TGFβ) is a secreted protein that controls proliferation, cell differentiation, and other functions in 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 in at least three isoforms called TGF-β1, TGF-β2, and TGF-β3. The TGF-β family is part of a superfamily of proteins known as the transforming growth factor beta superfamily, which includes inhibin, activin, anti-Müllerian hormone, bone morphogenetic protein, decapentaplegic, and Vg-1.

[0195] TGFβ pathway inhibitors can generally include any inhibitor of TGFβ signal transduction. 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[l,3]dioxol-5-yl-2-yellow-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB525334), ... 4-(5-Benzol[l,3]dioxol-5-yl-4-pyridin-2-yl-lH-imidazol-2-yl)-benzamide hydrate, 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-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 (D4476), 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).

[0196] d. MEK inhibitors MEK inhibitors are chemicals or drugs that inhibit the mitogen-activated protein kinase enzymes MEK1 or MEK2. MEK inhibitors 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-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).

[0197] e. bFGF inhibitor Basic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF is present in the basement membrane and the subendothelial extracellular matrix of blood vessels. In addition, bFGF is a common component of human ESC culture medium, which is necessary for cells to remain in an undifferentiated state.

[0198] bFGF inhibitor generally refers to bFGF inhibitor. For example, bFGF inhibitor includes N-[2-[4-(diethylamino)butyl]amino-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]-N'-(l,l-dimethylethyl)urea (PD173074), 2-(2-amino-3-methoxyphenyl)-4H-l-benzopyran-4-one (PD 98059), l-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.

[0199] f.RAR antagonist RAR antagonists are useful in the disclosed method. Useful RAR antagonists are listed in table 4. Exemplary RAR antagonists include AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253 and liarozole dihydrochloride.In some non-limiting examples, the RAR antagonist is AGN 193109.

[0200] 6. Kit In some embodiments, the kit may include one or more media and components for producing, for example, macular RPE cells, central RPE cells, and / or peripheral RPE cells. The reagent system may be packaged in either aqueous media or lyophilized form, as appropriate. The container means of the kit will generally include at least one vial, test tube, flask, bottle, syringe, or other container means into which the components may be placed, and preferably appropriately aliquoted. If there is more than one component in the kit, the kit will also generally include a second, third, or other additional container into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The components of the kit may be provided as dry powder(s). When the reagents and / or components are provided as dry powders, the powders can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may be provided in another container means.

[0201] In some examples, the kit includes a retinal induction medium, such as one that includes a WNT pathway inhibitor, a transforming growth factor (TGF)-β pathway inhibitor, a bone morphogenetic protein (BMP) pathway inhibitor, and insulin growth factor 1 (IGF1).

[0202] In some examples, the kit includes a retinal differentiation medium, such as one that includes a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, IGF1, and an additional inhibitor (e.g., a MEK inhibitor or a fibroblast growth factor (FGF) inhibitor).

[0203] In some examples, the kit includes a retinal culture medium, such as one that includes activin A, and may further include nicotinamide.

[0204] In some examples, the kit includes an RPE maturation medium, the RPE maturation medium containing a retinoic acid receptor (RAR) antagonist (e.g., one or more of AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253, and liarozole dihydrochloride) and / or a canonical Wnt inhibitor (e.g., 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (Endo-1-IWR), calphostin C, cardionogen 1, CCT 031374 hydrobromide, IWP 12, XAV 939, WIKI4, ICG-001, Wnt-C59 (C59), IWR-1-endo, KY02111, LGK-974, IWP-L6, FH535, iCRT 14, IWP 4, JW 67, JW 74, KYA 1797K, NLS-StAx-h, PNU 74654, TAK 715, IWP 2, CKI 7 dihydrochloride, (R)-CR8, D 4476, (R)-DRF053 dihydrochloride, epiblastin A, IC 261, LH 846, PF 4800567 hydrochloride, PF 5006739, PF 670462, SR 3029, AZ 6102, JW 55, MN 64, and TC-E 5001 or more). In one example, the RAR antagonist is AGN 193109. In one example, the canonical Wnt inhibitor is Endo-1-IWR. In one example, the RPE maturation medium comprises a RAR antagonist (such as AGN 193109) but does not comprise a canonical Wnt inhibitor. In one example, the RPE maturation medium comprises a canonical Wnt inhibitor (such as Endo1-IWR) but does not comprise a RAR antagonist. In one example, the RPE maturation medium comprises both a canonical Wnt inhibitor (such as Endo1-IWR) and a RAR antagonist (such as AGN 193109).

[0205] In some examples, the kit includes one, two, three or four of retinal induction medium, retinal differentiation medium, retinal medium and RPE maturation medium. In some examples, the kit includes RPE maturation medium and one, two or three of retinal induction medium, retinal differentiation medium and retinal medium. In some examples, such kits can further include a matrix, such as one that includes at least one recombinant cell adhesion protein (e.g., laminin, vitronectin or fibronectin). In some examples, such kits can further include a scaffold, such as one that is degradable. In some examples, the scaffold includes PLGA. The kit also typically includes a container for housing the kit component(s) in tightly confined container for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials are held. In some examples, the container is made of glass.

[0206] The kit can also include instructions for use in printed or electronic format, e.g., digital format, etc. The instructions can be for producing the cells and / or for introducing the cells and / or an implant containing the cells into a subject.

[0207] How to use Macular, central and / or peripheral RPE cells can be derived from iPSCs using the disclosed methods and thus can be used to provide "personalized medicine" to patients with ocular diseases. In some embodiments, cells obtained from a patient, such as somatic or CD34+ cells, or umbilical cord cells, can be used to produce iPSCs, which are then used to produce macular, central and / or peripheral RPE cells. In some embodiments, macular, central and / or peripheral RPE cells (or starting iPSCs) can be genetically engineered to correct disease-causing mutations, differentiate into macular, central and / or peripheral RPE, and engineered to form a tissue implant. This tissue replacement implant can be used to replace endogenous degenerated RPE in the same subject. Macular, central and / or peripheral RPE cells can be included in the tissue implant. The type of RPE cells can be selected based on the disease to be treated in the subject. The implant can include a scaffold.

[0208] In some embodiments, iPSCs can be produced from healthy donors or from HLA homozygous "super donor" or "universal" donor pluripotent (iPS) cells and used to prepare tissue implants. RPE cells can be treated in vitro with specific factors such as pigment epithelium-derived factor (PEDF), transforming growth factor (TGF)-beta, and / or retinoic acid to generate an anti-inflammatory and immunosuppressive environment in vivo. These "super donor" iPSCs are commercially available, see Cellular Dynamics International (see, e.g., 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).

[0209] The subject may be a human or veterinary subject. The macular RPE cells, central RPE cells and / or peripheral RPE cells may be derived from a single subject, or several populations of macular RPE cells, central RPE cells and / or peripheral RPE cells, such as different types of RPE, each derived from a different subject, may be used to produce implants for treating subjects with retinal degeneration, etc.

[0210] As shown in Figures 14A-14D, different types of RPE (e.g., P1 macular, P2 central and / or P3 peripheral) produced using the methods provided herein can be used to customize the treatment of different retinal diseases. For example, macular RPE can be used to support the function and activity of cone photoreceptors, while central and peripheral RPE can be used to support the activity of rod photoreceptors. In some instances, macular RPE can support dense iPSC-derived choroidal vasculature when developed adjacent to them, while central and peripheral RPE can support relatively less dense iPSC-derived choroidal vasculature. By combining the correct type of iPSC-derived photoreceptors with the correct type of RPE and iPSC-derived vasculature, different portions of the back of the eye can be generated (e.g., the entire macula or the entire central or the entire peripheral or the entire distal peripheral portion of the eye).

[0211] Different forms of retinal degenerative diseases affect different parts of the eye. For example, choroideremia (CHM; Figure 14A) causes peripheral RPE dysfunction that spreads to central and P4 RPE cells in early disease stages. As the disease progresses, the corresponding photoreceptors and choroidal vasculature also degenerate. Similarly, in late-onset retinal degeneration (L-ORD, Figure 14B), disease-related damage begins in central and peripheral RPE cells, and finally, in later disease stages, photoreceptors and choroidal vasculature also degenerate. In AMD (Figure 14C), damage is initially seen in the macula, P4 and P5 RPE that spreads to photoreceptors and choroidal vasculature in advanced stages of AMD.

[0212] In one example, early stage CHM may be treated using central RPE, intermediate stage may be treated using peripheral and P4 iPSC-RPE, and advanced stage may be treated utilizing central and peripheral RPE with corresponding rod-enriched photoreceptors, with or without choroidal vasculature for P4 iPSC-RPE with cone-enriched photoreceptors.

[0213] In one example, L-ORD is treated using central and peripheral RPE, with or without corresponding photoreceptors and choroidal vasculature, depending on the stage of the disease.

[0214] In one example, macular RPE (or P4 RPE), with or without corresponding photoreceptors and choroidal vasculature, is used to treat AMD.

[0215] In some embodiments, a method of treating a subject in need thereof is provided, comprising producing macular, central or peripheral cells according to the disclosed method, and transplanting the macular, central or peripheral cells into the subject's retina.In some embodiments, the subject has a retinal degenerative disease, retinal dysfunction, retinal decomposition, retinal damage, or loss of retinal pigment epithelium.In some non-limiting examples, the retinal degenerative disease is Stargardt's macular dystrophy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, Leber's congenital amaurosis, acquired macular degeneration, hereditary macular degeneration, late-onset retinal degeneration, Best's disease, retinal detachment, gyrate atrophy, choroideremia, pattern dystrophy.In other non-limiting examples, the retinal damage is caused by laser, inflammatory, infectious, radiation, neovascular or traumatic injury. In further non-limiting examples, central macular and / or peripheral retinal pigment epithelial cells are introduced into the subretinal space, vitreous cavity, inner or outer retina, peripheral retina or choroid of the eye. Methods for producing single and multi-layered retinal implants are disclosed, for example, in PCT Publication No. WO 2018 / 089515, which is incorporated herein by reference.

[0216] Macular RPE and distal peripheral RPE mainly support the function and activity of cone photoreceptors, while central RPE cells and peripheral RPE cells mainly support the activity of rod photoreceptors.Similarly, macular RPE supports dense iPSC-derived choroidal vasculature when it develops adjacent to them, while central and peripheral RPE supports relatively less dense iPSC-derived choroidal vasculature.By combining one or more specific types of iPSC-derived photoreceptors with selected types of RPE as disclosed herein, and optionally with iPSC-derived vasculature, an implant can be designed to treat retinal degeneration in the eye region of interest.

[0217] The cells of the retina that are directly sensitive to light are photoreceptor cells. Photoreceptors are light-sensitive neurons in the outer part of the retina and can be either rods or cones. In the process of phototransduction, photoreceptor cells convert the incoming light energy focused by the lens into an electrical signal that is then sent to the brain via the optic nerve. Vertebrates have two types of photoreceptors: cones and rods. Cones are adapted to detect fine details, central vision and color vision and perform well in bright light. Rods are responsible for peripheral and dim light vision. The neural signals from the rods and cones are processed by other neurons in the retina.

[0218] The retinal pigment epithelium acts as a barrier between the bloodstream and the retina and interacts closely with photoreceptors in maintaining visual function. It is composed of a single layer of hexagonal cells, including macular, central and peripheral RPE cells, densely packed with granules of melanin that absorb the light energy reaching the retina. The functions of these RPE cells include transport of nutrients such as glucose, retinol, and fatty acids from the blood to the photoreceptors; transport of water, metabolic end products and ions from the subretinal space to the blood; light absorption and protection against photooxidation; reisomerization of all-trans-retinol to 11-cis-retinal; phagocytosis of sloughed photoreceptor membranes; and secretion of various essential factors for the structural integrity of the retina.

[0219] Dysfunction, damage and loss of RPE cells are factors in many eye diseases and disorders, including age-related macular degeneration (AMD) and hereditary macular degeneration (such as Best's disease), as well as retinitis pigmentosa. Other diseases are discussed below. Damage to the retina, such as from physical injury, also requires treatment. Exemplary conditions that can be treated are shown in FIG. 14.

[0220] Pharmaceutical compositions of macular, central and / or peripheral RPE cells produced by the methods disclosed herein. These compositions comprise at least about 1×10 3 RPE cells, approximately 1 x 10 4 RPE cells, approximately 1 x 10 5 RPE cells, approximately 1 x 10 6 RPE cells, approximately 1 x 10 7 RPE cells, approximately 1 x 10 8 RPE cells or approximately 1 x 10 9 The composition may comprise RPE cells. In certain embodiments, the composition is a substantially purified preparation (with respect to non-RPE cells) that comprises the differentiated RPE cells produced by the method disclosed herein. The composition can be formulated for delivery to the eye of a subject in need thereof, such as the subretinal space, vitreous space, inner or outer retina, retinal periphery, intrachoroidal or suprachoroidal location of the eye.

[0221] Also provided are compositions comprising a scaffold, such as a polymeric carrier and / or extracellular matrix, and a therapeutically effective amount of macular RPE cells, central RPE cells, and peripheral RPE cells produced by the methods disclosed herein. For example, the cells are provided as a monolayer of cells. The compositions can also include multiple layers and can include photoreceptor and / or vascular cells. Scaffolds, such as physiologically acceptable degradable or non-biodegradable scaffolds suitable for use in in vivo applications, can also be included. For example, physiologically acceptable materials include, but are not limited to, absorbable and / or non-absorbable solid materials, such as small intestinal submucosa (SIS), cross-linked or non-cross-linked alginate, hydrocolloids, foams, collagen gels, collagen sponges, polyglycolic acid (PGA) mesh, fleece, and bioadhesives. The scaffolds can be PLGA scaffolds. These scaffolds can be delivered to the eye of a subject in need thereof.

[0222] The disclosed macular, central and peripheral RPE, and tissue replacement implants comprising macular, central and / or peripheral RPE are useful for treating retinal degenerative diseases, retinal or retinal pigment epithelial dysfunction, retinal decomposition, retinal or retinal pigment epithelial damage, such as damage caused by light, laser, inflammatory, infectious, radiation, neovascularization or traumatic injury. The disclosed macular RPE, central RPE and / or peripheral RPE, and tissue replacement implants comprising these cells are also useful for treating loss of retinal pigment epithelium. The method includes locally administering macular RPE, central RPE and / or peripheral RPE, or tissue-positioned implant to the eye of a subject. The pharmaceutical composition comprising a therapeutically effective amount of RPE cells can be introduced into the subretinal space, vitreous space, inner or outer retina, retinal periphery or choroid of the eye. RPE cells can also be included on the implant.

[0223] 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, Batten's disease, Beart's dystrophy, traumatic eye injury or choroideremia, pattern dystrophy. Further conditions include retinal detachment, pattern dystrophy, and other dystrophies of RPE. In certain non-limiting examples, the subject has age-related macular degeneration. In certain embodiments, a method for treating or preventing a condition characterized by retinal degeneration is provided, comprising administering the disclosed RPE cells or tissue replacement implants comprising these RPE cells to a subject in need thereof. The type of RPE contained in the implant can be selected based on the disorder to be treated.

[0224] These methods can include selecting a subject having one or more of these conditions and administering a tissue replacement implant to treat the condition and / or ameliorate the symptoms of the condition. The implant can include macular RPE, central RPE and / or peripheral RPE, optionally in combination with other retinal cells such as photoreceptor and / or vascular cells.

[0225] Various forms of retinal degenerative diseases affect different parts of the eye. For example, choroideremia (CHM) causes peripheral RPE dysfunction in early disease stages that spreads to central and distal peripheral RPE cells. As the disease progresses, the corresponding photoreceptors and choroidal vasculature also degenerate. The implants used to treat CHM include central RPE cells, and optionally distal peripheral RPE cells, as well as photoreceptors and vascular cells. Similarly, in the disease late-onset retinal degeneration (L-ORD), disease-related damage begins in central and peripheral RPE cells, and eventually in later disease stages, photoreceptors and choroidal vasculature also degenerate. Thus, for early treatment, useful implants include central and / or peripheral RPE cells. In later disease stages, these same implants can be used, or the implants can also include photoreceptors and / or vascular cells. In AMD, damage is initially seen in the macular RPE, distal peripheral RPE, and ora serrata RPE that spreads to photoreceptors and choroidal vasculature in advanced stages of AMD. Thus, for early treatment, a useful implant can include macular RPE, and optionally distal peripheral and ora serrata RPE cells. At later disease stages, these same implants can be used, or the implant can include associated photoreceptor and / or macular and peripheral specific vascular cells.

[0226] These methods can include selecting a subject having one or more of these conditions and administering macular RPE cells, central RPE cells and / or peripheral RPE cells, or a tissue replacement implant comprising RPE cells, to treat the condition and / or ameliorate the symptoms of the condition. The disclosed RPE cells can also be transplanted together with other retinal cells, such as photoreceptor and / or vascular cells (co-transplantation).

[0227] The disclosed method allows for the development of customized cell therapy for different diseases and different stages of those diseases. For example, in early stages of CHM, implants containing central RPE can be used for treatment, and in intermediate stages, implants containing peripheral RPE and distal peripheral RPE can be used for treatment. In advanced stages, implants can include central and peripheral RPE with corresponding rod-rich photoreceptors. Alternatively, implants can be produced for distal peripheral RPE with cone-rich photoreceptors with or without choroidal vasculature. In some embodiments, implants can be produced with central or peripheral RPE with or without corresponding photoreceptors and choroidal vasculature. The selection of cells can be based on the disease stage.

[0228] In other embodiments, implants can be made that contain macular RPE or distal peripheral RPE, with or without corresponding photoreceptors and choroidal vasculature, for the treatment of AMD. These implants can then be used for treatment. In the early stages of AMD, it may be sufficient to replace only macular RPE cells and / or other types of RPE cells (see FIG. 14). Without being bound by theory, transplanted macular RPE cells may mediate the prevention of further loss of RPE cells and / or degeneration of Bruch's membrane. In advanced stages of AMD, patients may experience loss of both RPE cells and photoreceptor cells. Thus, in some embodiments, the method can further include implanting a composition that includes photoreceptor cells. Methods of implanting scaffolds and RPE cells are disclosed, for example, in PCT Publication No. WO2012177968 and PCT Publication No. 2016 / 007852, both of which are incorporated herein by reference.

[0229] In some embodiments, the macular, central and / or peripheral RPE cells in the tissue replacement implant are derived from the subject to be treated, and therefore are autologous.In other embodiments, the macular, central and / or peripheral RPE cells in the tissue replacement implant are produced from an MHC-matched donor or a universal donor.In another embodiment, the macular, central and / or peripheral RPE cells in the tissue replacement implant are allogeneic.

[0230] RPE cells, such as tissue replacement implants, can be introduced into various target sites within the subject's eye. In some embodiments, the tissue replacement implant is introduced, such as by implantation, into the subretinal space of the eye, which is the anatomical location of RPE in mammals (between the photoreceptor outer segments and the choroid). Exemplary methods are disclosed, for example, in PCT Publication No. WO2018 / 089521, which is incorporated herein by reference in its entirety. In some embodiments, the tissue replacement implant is introduced into the outer retina, retinal periphery, macula, or perimacular region, or choroid. Furthermore, depending on the migratory ability and / or positive paracrine action of the cells, introduction into additional ocular compartments, such as the vitreous cavity, the inner or outer retina, the retinal periphery, and the choroid, can be considered.

[0231] The size of the tissue replacement implant to be implanted may generally be determined by comparing a clinical assessment of the size of the area of ​​retinal pathology present in a particular patient with the constraints imposed by the surgical feasibility of delivering an implant of a particular size. For example, in degenerations involving the central retina (e.g., age-related macular degeneration), a circular implant of about 1.0-2.5 mm diameter (e.g., about 1.5 mm diameter) that approximates the anatomical fovea is often appropriate. In some cases, a larger implant may be appropriate, which maximally corresponds to the area of ​​the posterior retina lying between the temporal vascular arcade (histological macula, clinical posterior pole), an ovoid area of ​​about 6.0 mm (vertical) × 7.5 mm (horizontal) centered on the fovea or located in the extrafoveal region. In some instances, it may be appropriate to similarly form a polymer scaffold of smaller dimensions, about 0.5 mm or so, to be placed in the area of ​​circumscribing pathology. Additionally, it may be of interest to custom-make an irregularly shaped implant to fit the patient, e.g., to cover the area of ​​pathology while avoiding areas of residual high vision.

[0232] Tissue replacement implants can be introduced by various techniques known in the art. Methods for performing transplantation 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 transplantation 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 via pars pana vitrectomy surgery followed by delivery of the tissue replacement implant to the subretinal space through a small retinal opening. Alternatively, the tissue replacement implant can be delivered to the subretinal space via a transscleral, transchoroidal, or suprachoroidal approach. Additionally, direct transscleral insertion into the anterior retinal periphery in close proximity to the ciliary body can be performed.

[0233] 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 and / or reduces degeneration of retinal neurons. In still other embodiments, the method may include administering a therapeutically effective amount of an agent to inhibit unwanted angiogenesis, for example, to combat choroidal neovascular (CNV) growth below the fovea in AMD patients. An exemplary therapeutic agent 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 preventing the interaction of VEGF with its receptor. A therapeutically effective amount that suppresses the expression of VEGF by inhibiting pathways that lead to VEGF secretion, such as STAT3, NF-kB, HIF-1α. Other drugs can prevent atrophy of RPE cells by targeting the complement pathway, autophagy, or NF-kB pathway. Treatments available for AMD include drug therapies aimed at stopping the growth of new blood vessels, such as bevacizumab (AVASTIN®), ranibizumab (LUCENTIS®), and aflibercept (EYLEA®); photodynamic therapy; photocoagulation; and low vision rehabilitation.

[0234] 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 can be used, for example, to promote the development or function of neurons, such as photoreceptor cells. 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 transforming 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, -4 / 5, -6, and vitamin E. Such agents may be provided alone or in combination.

[0235] A personalized medicine application is disclosed herein. In some embodiments, a method is disclosed for treating a subject with retinal degenerative disease, retinal or retinal pigment epithelium dysfunction, retinal decomposition, retinal damage, or retinal pigment epithelium loss. The method can include determining which population of RPE cells, such as macular RPE cells, peripheral RPE cells, and / or central RPE cells, is affected for the subject, producing these cells, and then administering these macular RPE cells, peripheral RPE cells, and / or central RPE cells to the subject. In some embodiments, the method includes locally administering the disclosed tissue replacement implant to the eye of the subject. The tissue replacement implant can include macular RPE cells, central RPE cells, and / or peripheral RPE cells depending on the disease process of the subject. The tissue replacement can include photoreceptors such as rods and / or cones and / or vascular cells as required. In a specific non-limiting example, the subject has CHM, AMD, L-ORD, or RD.

[0236] The present disclosure is illustrated by the following non-limiting examples. EXAMPLES

[0237] Example 1 material and method Tissue donor information: 17 eyes from 9 healthy donors were obtained. Eyes were considered for study if there was no history of retinal degeneration. Eyes were enucleated, immediately stored, and transported in ice-cold PBS 1x or RPMI on wet ice. Eyes were received, dissected, and preserved in 4% PFA within 24 hours of donor death.

[0238] Staining and imaging: RPE monolayers were incubated for 1 hour at RT in PBS buffer containing 1% bovine serum albumin (BSA), 0.5% Tween® 20 and 0.5% Triton® X to allow permeabilization and blocking of non-specific sites. Cell borders were then stained with a 1:200 dilution of anti-ZO1 (catalog no. 339100, Thermo Fisher Scientific) and anti-pan-cadherin antibody (catalog no. ab6529, Abcam) overnight at room temperature. Secondary antibodies anti-mouse-647 (catalog no. A-21235, Thermo Fisher Scientific) and anti-rabbit-633 (catalog no. A-21071, Thermo Fisher Scientific) were added at a 1:500 dilution for 1 hour at room temperature in the dark. Both secondary antibody fluorophores were chosen to have similar emission spectra in the far-red wavelengths to increase the signal-to-noise ratio. In fact, due to their physiological activity and the elevated presence of oxygen in the retina, the RPE accumulate many waste products such as lipofuscin, which is highly autofluorescent. The choice of wavelengths also aims to avoid the wide bandwidth of lipofuscin autofluorescence. To further increase the signal-to-noise ratio, phalloidin-iFluor 647 (catalog no. ab176759, Abcam) was added together with the secondary antibody at a dilution of 1:250. Nevertheless, the signal-to-noise ratio was not high enough to be able to distinguish most of the RPE border. After staining, TRUEBLACK® (catalog no. 23007, Biotium), a lipofuscin autofluorescence quencher, was used. TRUEBLACK® was diluted 1:20 in ethanol 70% and applied to the samples with the RPE facing up for up to 2 minutes. All petals of the flat mount were further cut to better flatten the tissue. Finally, the samples were mounted on 50 × 75 mm glass slides (catalog no. 5075, Brain Research Laboratories). A weight was placed on top of the samples overnight to ensure uniform flatness. The RPE monolayer was imaged using a Zeiss Axio Scan.Z1 widefield scanner (Carl Zeiss).A maximum of 250,000-275,000 tiles and 120 µm z-stacks were set up to contain each sample. Stacks were compressed online into single slices such that the final files had a maximum dimension of 10 GB with CZI compression.

[0239] iPSC-RPE Differentiation: The iPSC line used for screening and subsequent phenotypic characterization, termed TJP1-mEGFP, was derived from the widely used parent line WTC-11, in which the tight junction protein 1 gene (TJP1; or tight junction 1, ZO1) was CRISPR-modified such that the N-terminal exon of one allele had a monomeric enhanced green fluorescent protein (mEGFP) sequence insertion. The resulting TJP1 protein was conjugated to mEGFP, allowing live visualization of cell boundaries. Numerous genomic and cellular validations were performed to rule out any obvious adverse effects of tagging. The assays and their results are available on the Allen laboratory website (allencell.org / cell-catalog.html). The iPSC line was differentiated into RPE using the developed protocol (Sharma et al., Science Translational Medicine, 11(475), doi.org / 10.1126 / scitranslmed.aat5580, 2019). RPE differentiation from stem cells can be induced by activation of the TGF and WNT pathways at the neuroectoderm stage (Idelson et al., Cell Stem Cell, 5(4), 396-408, 2009; Lamba et al., Proceedings of the National Academy of Sciences of the United States of America, 103(34), 12769-12774, 2006; Leach et al., Investigative Ophthalmology&Visual Science, 56(2), 1002-1013, 2015; Reh et al., Directing Human Embryonic Stem Cells to a Retinal Fate, doi.org / 10.1007 / 978-1-60761-691-7_9, 2010). We developed a triphasic differentiation protocol that further improves the efficiency and reproducibility of differentiation (Sharma et al., op.cit., 2019).First, dual SMAD inhibition was combined with FGF inhibition to induce the formation of neuroectodermal cells from iPSCs, since dual SMAD inhibition promotes a neuronal fate and FGF pathway activation inhibits differentiation of the eye field into RPE (Bharti et al., PLoS Genetics, 8(7). doi.org / 10.1371 / journal.pgen.1002757, 2012; Chambers et al., Nature Biotechnology, 27(3), 275-280, 2009; Fuhrmann, Current Topics in Developmental Biology, 93, 61-84, 2010; Meyer et al., Proceedings of the National Academy of Sciences of the United States of America, 106(39), 16698-16703, 2009). Second, we activated the TGF-β and WNT pathways to promote commitment of neuroectodermal cells to an RPE fate (see Carr et al., PLoS ONE, 4(12), e81522009; Fuhrmann, Organogenesis, 4(2), 60-67, 2010; Idelson et al., Cell Stem Cell, 5(4), 396-408, 2009). Third, we matured committed RPE by treatment with prostaglandin E2 (PGE2) to actively suppress the canonical WNT pathway by stimulation of the primary cilia (May-Simera et al., Cell Reports, 22(1), 189-205, 2018).

[0240] For differentiation, iPSCs were seeded onto vitronectin (catalog no. A1700, Thermo Fisher Scientific)-coated 6-well plates. After 2 days in Essential 8 medium (E8, Catalog No. A1517001, Thermo Fisher Scientific), cells were cultured in basal differentiation medium (DMEM / F12 (Cat. No. 11330032, Thermo Fisher Scientific), N2 supplement (Cat. No. A1370701, Thermo Fisher Scientific), B27 (Cat. No. 17504044, Thermo Fisher Scientific), KSR (Cat. No. 12618013, Thermo Fisher Scientific) supplemented with 10 nM LDN (Cat. No. 04-0074 Stemgent), 0.5 μM CK1-7 dihydrochloride (Cat. No. C0742, Sigma), 1 μM SB431542 hydrate (Cat. No. S4317, Sigma) and 1 ng / ml IGF-1 (Cat. No. AFL291, R&D Systems). Neuroectodermal cells were treated with neuroectodermal induction medium (NEIM) consisting of 100 nM LDN (catalog number 04-0074 Stemgent), 200 μM ascorbic acid (catalog number A4544, Sigma) for 2 days. Neuroectodermal cells were then cultured in RPE induction medium (RPEIM, basal differentiation medium containing 100 nM LDN (catalog number 04-0074 Stemgent), 5 μM CK1-7 dihydrochloride (catalog number C0742, Sigma), 10 μM SB 431542 hydrate (catalog number S4317, Sigma) and 10 ng / ml IGF-1 (catalog number AFL291, R&D Systems), 1 μM PD 0325901 (catalog number PZ0612, Sigma) for 10 days. Cells were cultured in RPE committing medium (basal differentiation medium containing RPECM, 10 mM nicotinamide (catalog number N0636 Sigma), 150 ng / ml activin A (catalog number 338-AC / CF R&D Systems)) for 10 days.Committed RPE were maintained in RPE growth medium for 5 days and then reseeded to eliminate neurogenesis (RPEGM, MEM+GLUTAMAX™ (Cat. No. 32561037, Thermo Fisher Scientific), 5% FBS (Cat. No. SH30071.03, Hyclone), Taurine (Cat. No. T-0625, Sigma), Thyronine (Cat. No. T-5516, Sigma), Hydrocortisone (Cat. No. H-0396-10, Sigma)). Immature RPE were then cultured in RPEGM for 15 days and then enriched by negative selection using anti-CD24 (Cat. No. 655154, BD Biosciences) and anti-CD56 (Cat. No. 340723, BD Biosciences) antibodies. Finally, immature RPE cells were seeded on vitronectin-coated transwells (Cat. no. 3460, Corning) and cultured in RPE maturation medium (RPEMM (Cat. no. 2296 / 10, Tocris), an RPEGM containing 50 μM PGE2) for 6 weeks to obtain fully mature iPSC-derived RPE. AGN 193109 and endo-IWR-1 were added to the D21-derived RPEGM, respectively, until the mature cells were ready to be used to generate macular (P1) and peripheral (P4) iPSC-RPE cells (committed RPE cells) (Figure 1).

[0241] Screening and imaging of the compound library: 115 drugs targeting various developmental pathways or cytoskeletal stressors were selected to form a new developmental library of compounds. Drugs are activators or inhibitors of various signaling pathways, targeting proteins at different levels of the signaling cascade. For each drug, an initial concentration was selected and the concentration of each drug was adjusted. The assay was performed on committed RPE derived from iPSCs. For all drugs, the concentration was lowered if toxicity was detected and increased if there was no obvious effect on cell morphology compared to the control. A list of compounds tested can be found in Table 1. High content screening was performed in 384-well plates (catalog no. 6057300, Perkin Elmer) using three different concentration ranges (3x, 1x, 0.3x). All drugs were reconstituted in dimethyl sulfoxide (DMSO, catalog no. D2650, Sigma) and 40 μl of each drug was aliquoted in triplicate into 384-well master plates. Serial dilutions with RPEGM were then performed to obtain the final desired working concentrations and the final plates were stored at -80 °C. After 5 days in RPEGM, the arrested RPE was approximately 50 cells / mm 2(500 cells / well) in 384-well plates and allowed to adhere and adapt for 24 h with 10 μM rock inhibitor (catalog no. 1254, Tocris). Each day, one plate of compound at the final concentration was thawed and used to add fresh drug to the cells. This processing procedure allows for a minimum number of freeze / thaw cycles of compound, promoting precision compared to direct addition of very small volumes of drug to the cultured cells, and minimizes the time the cultured cells spend outside the incubator by transferring the drug with a multichannel pipette. Two time points were selected for imaging: 15 and 30 days from cell seeding in 384-well plates. A Yokogawa Cell Voyager (CV7000) high-throughput spinning disk confocal was used with a 20x air immersion objective. Each well was included in 9 fields of view, z-stacks were set to account for the heterogeneity of the monolayer, and images were projected on the fly into single slices. Live imaging was performed for the first time point, and cells fixed in 4% PFA were imaged for the second time point. No staining was necessary as the cells express the TJP1-mEGFP protein throughout their lifespan. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0242] Segmentation using convolutional networks: A convolutional neural network (CNN) algorithm was used to recognize RPE cell boundaries from images of samples labeled by immunofluorescence. The algorithm generates a binary mask of the segmented RPE boundaries; this output was manually corrected to provide a "correct answer" from which the algorithm learns. Once trained, the CNN algorithm was fed with images of fluorescently labeled RPE cells, and the generated binary mask image became the input for the REShAPE software for cell shape analysis (Figure 3).

[0243] Morphometric analysis using REShAPE: The binary segmentations generated by the CNN algorithm were transferred to REShAPE for cell shape analysis. REShAPE (Retinal Epithelium Shape and Pigment Evaluator) is a Fiji plugin for image analysis. For all cells in a successfully segmented field of view, REShAPE provides quantification of more than 25 different shape metrics. The raw data was stored in a spreadsheet to allow statistical analysis. Furthermore, the software creates an image of the segmented cell for each metric analyzed, and all cells are color-coded according to their raw values ​​(Figure 4). The color-coded image displays the location of the analyzed cells. The shape metrics were analyzed by the software to obtain measurements of cell dimensions such as area and perimeter, measurements of elongation such as the length of the major and minor axes, and measurements of cell regularity such as hexagonality score and number of neighbors. The analysis was performed in pixels or in μm, as long as a pixel-to-μm conversion was provided. Data and graph analysis was performed using R software (R Core Team (2018). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https: / / www.R-project.org / ). The R packages "plyr" and "ggplot2" were used for data handling and plotting, respectively (Wickham, 2009, 2011). Statistical analysis was calculated by comparing several treatment groups with the control group using Dunnett's test, a method for post hoc pairwise multiple comparisons. A family-wise confidence level of 95% was used."DescTools" is the R package used to perform the Dunnett's test (Signorell, DescTools: Tools for Descriptive Statistics [R package DescTools version 0.99.31], cran.r-project.org / web / packages / DescTools / index.htm, retrieved October 25, 2019 from 2019). Assumptions of normality were tested using the Shapiro-Wilk test. Because tests of normality are sensitive to sample size, tests were performed on unbiased subsets of data for each group. In addition, quantile-quantile (QQ) plots were drawn to assess normality for the full dataset for each group. Homogeneity of variance was tested by plotting the residuals in a "residuals versus fitted plot". One-way ANOVA was used to assess between-group variance before performing the Dunnett's test. Shapiro-Wilk tests and one-way ANOVA were performed using the R package "dplyr" (taken from Wickham et al., dplyr: A Grammar of Data Manipulation. https: / / cran.r-project.org / package=dplyr, 2019), whereas QQ plots were drawn using the R package "ggpubr" (taken from Kassambara, ggpubr: "ggplot2" Based Publication Ready Plots. cran.r-project.org / package=ggpubr, 2020). Data are presented as box plots, with box limits representing the first and third quartiles, center lines indicating the median, and whiskers indicating the fifth and 95th percentiles, so that the range specifies 90% of the data. For REShAPE morphometric analysis, single-cell measurements were considered as technical replicates, whereas each eye or each well of the drug screen was considered as a biological replicate.

[0244] Quantification of human ocular populations: Several morphometric criteria parameters revealed the presence of distinct RPE populations across human RPE flatmounts (Figure 5). We developed custom-built software to isolate these RPE populations. The software retrieves the xy coordinates of all cells from the spreadsheet generated by REshAPE and reconstructs a color-coded image. RPE populations are cropped directly on the color-coded image, and the information of selected cells is retrieved and saved in a new spreadsheet. As the RPE populations are arranged in rings, we used the circular selection tool to isolate them. Even though the circular areas were cropped according to the gradient of color-coded cells, the variation in measurements for each ring is less than 0.6 mm.

[0245] Example 2 A complete morphometric map of the adult human RPE To analyze the morphometry of adult human RPE cells, 17 cadaveric eyes from 9 non-AMD patients were obtained. Eyes were transported from the eye bank on wet ice and processed within 24 hours of the patient's death. Eyes were injected intravitreally with 1700 mOsm mannitol solution to induce retinal detachment from the RPE, after which the RPE / choroid complex was dissected and mounted as flat tissue. RPE cell borders were immunolabeled with anti-ZO1 and pan-cadherin antibodies to recognize tight and adherens junctions, respectively. As in RPE cells, actin filaments form polygonal rings connecting tight junctions along cell borders, fluorophore-conjugated phalloidin was also used to enhance cell border staining. Whole RPE / choroid flat mounts were imaged and cell shape was analyzed using the custom-built software REShAPE. Color-coded images of the entire epithelium visually display quantification of cell shape for every metric analyzed. As previously reported on a smaller scale rather than across the whole eye (Bhatia et al., op.cit.,2016), clear heterogeneity was observed across the RPE for certain shape metrics. For example, cells were observed in a smaller area corresponding to macular RPE compared to cells located in the periphery of the epithelium (Figures 5-6). RPE cell area increases gradually eccentrically. Interestingly, a ring of small RPE cells was observed in the area contained within 14-17 mm from the center of the eye. The change in cell area between the peripheral RPE inside the ring and the peripheral ring of small RPE is abrupt, and the transition between the ring of small RPE and the peripheral RPE outside the ring is also abrupt. Outside the ring of small RPE, the cell area increases dramatically. However, all RPE cells appear to be of similar regular shape across the entire epithelium, except for the cells at the very periphery, which appear very elongated and irregular. Figure 2 shows the differences between human macular and peripheral RPE cells.

[0246] Example 3 Comparison of macular cells and RPE cells Five different RPE populations were distinguished according to their cell area (Figure 6). Images of flat mounts were divided into concentric rings centered near the center of the eye, approximately halfway between the optic nerve and the macula, and a ring around the macula. From the center to the periphery, the RPE populations were named P1, macular RPE, and P5, the RPE at the very edge of the epithelium. P1 is macular, P2 is central, P3 is peripheral; P4 is distal peripheral; and P5 is ora serrata RPE. Note that even though the circular regions were manually cut according to the gradient of color-coded cells, the average circumferential spread is less than 0.6 mm. Thus, the RPE populations in adult eyes have a very precise location. Moreover, there is a slight asymmetry between the central and peripheral populations of RPE (P2 and P3). The central (P2) population always extends further to the temporal side, while the peripheral (P3) population extends further to the nasal side. As a result, in color-coded images of cell area, the color gradient in the temporal region was less pronounced than in the nasal region, suggesting that RPE cell area tends to remain smaller in the temporal region.

[0247] The mean RPE cell size increases eccentrically from 11.2 ± 0.6 mm to 14.3 ± 0.5 mm from the center of the eye to population P3 (periphery). This RPE population is 238.6 ± 37.8 μm 2 This corresponds to the macular RPE cell area in population P1 (macula, 149.6 ± 33.4 μm 2 ) is nearly 67% larger than P3 (peripheral) (14.6±0.6 mm to 17.2±0.4 mm). At the periphery of P3 (peripheral), RPE cell size is significantly reduced in population P4 (181.8±39.5 μm 2 ). Due to variability in anatomy, this kind of quantification always includes parts of the P3 (peripheral) and P5 populations, which biases measurements towards larger areal dimensions in the P4 population. Thus, as can be seen from the color-coded image (Figure 6), the P4 population may have a very similar average area as the macular cells (P1). Overall, this indicates the presence of macular-type RPE in the periphery.

[0248] In the very periphery of the flat mount, from 17.6 ± 0.4 mm to 21.1 ± 0.5 mm, the mean area of ​​the RPE population P5 was 1.1 times smaller than that of the rest of the eye (336.8 ± 49.6 μm 2 ) is considerably larger and more variable. Visual inspection reveals that in the first few millimeters, the P5 region contains cells with areas comparable to the peripheral population (P3), but the more eccentric cell areas are much larger. Most peripheral RPE cells likely belong to the region of the ora serrata, where the light-sensitive retina transitions into the non-light-sensitive region of the ciliary body.

[0249] Example 4 Comparison of morphometric measurements of iPSC-RPE and adult human RPE We next recapitulated the morphological RPE heterogeneity of P1 (macular) and P3 (peripheral) in vitro. Among the non-macular RPE populations (P2–P5), we chose the peripheral (P3) because it is most likely to support a pure population of rod photoreceptors. Despite the importance of location in focal retinal degeneration, there are no reports in the literature linking stem cell-derived RPE to any specific human RPE population. Generating in vitro RPE populations that recapitulate differential susceptibility to degeneration would provide a better system to study diseases such as AMD. Distal peripheral (P4) cells can be damaged in late AMD and traumatic injury. Serrata (P5) RPE cells can be damaged in traumatic injury.

[0250] Morphometry of fully mature iPSC-derived RPE (iPSC-RPE) generated in ex vivo culture was compared to human RPE populations identified in 17 flat mounts from adult healthy donors. Interestingly, when comparing cell size, the in vitro generated iPSC-RPE cells appear to have a smaller area than any human RPE population. The population with the most similar dimensions is the macular population labeled P1 (iPSC-RPE: 107.0 ± 61.7 μm 2 For P1: 149.6±33.4μm 2 ) (Figure 8).

[0251] Example 5 Comparison of treated iPSC-RPE and adult human RPE morphometry Because REShAPE was shown to be a highly sensitive tool to quantify cell morphology, and because morphometric analysis is a very simple and scalable technique, morphometry was used as a screening assay to identify macular RPE (P1) and peripheral RPE populations (P3) derived from iPSCs. To enrich and separate macular and peripheral RPE, we performed a high-content screening of compounds on iPSC-RPE during differentiation. The 115 selected compounds include activators and inhibitors of different developmental pathways and stress factors of the cytoskeleton. Committed RPE cells were seeded in 384-well plates and treated for 30 days before performing cell shape analysis. To intervene early during RPE differentiation, where developmental pathways still play a major role but do not change RPE fate, cells were treated while committed to an RPE fate. An iPSC-RPE cell line with TJP1 (ZO1) protein conjugated to mEGFP was used. High-content imaging was performed and the fluorescent images were analyzed using REShAPE. Three concentrations separated by 3-fold from each other were evaluated. Data for each shape metric was analyzed and plotted. Cell area was the metric chosen to compare treated iPSC-RPE to human RPE due to the clear distinction between RPE populations (Figure 7).

[0252] All drugs that induced a shift in the cell size distribution in any concentration range were selected and the raw images were checked for non-specific effects caused by toxicity. The remaining drugs are listed in Table 2. [Table 2-1] [Table 2-2]

[0253] The selected drugs were grouped according to the pathway in which they act and their role (activator or inhibitor). For each group, if there were more than one drug, those that induced the strongest shift in cell size were carried forward for characterization of their effect on iPSC-RPE phenotype. Eleven drugs were carried forward for iPSC-RPE phenotype analysis (Table 3). [Table 3]

[0254] Example 6 Phenotypic characterization of selected drug treatments Eleven compounds were selected for further phenotypic characterization based on their initial effects on RPE cell size. The purpose of this characterization was twofold: 1) to analyze the iPSC-RPE phenotype after drug treatment to ensure that the drug did not adversely affect cell health; and 2) to confirm the presence of macular or peripheral type apical processes on the cells. The structure of the apical processes of the RPE has been described as cone or rod specific: petal-like apical processes have been observed to wrap around the outer segments of cones, whereas finger-like structures are apposed to the outer segments of rods (Fisher & Steinberg, J. Comparative Neurology, 206(2), 131-145, 1982; Steinberg & Wood, Proceedings of the Royal Society of London-Biological Sciences, 187(1089), 461-478, 1974). Because the macula is innervated by cones and the periphery by rods, RPE cells in these two distinct regions may be enriched with one of two apical process structures. It was hypothesized that the phenotype of the RPE apical process would serve as an additional discriminator beyond RPE morphometry.

[0255] Different techniques were used to study the morphological features of the RPE. Transepithelial electrical resistance was used to examine tight junction integrity, bright field imaging was useful to determine RPE pigmentation defects, hematoxylin and eosin staining and transmission electron microscopy were useful to examine gross and microscopic morphological changes, respectively, while scanning electron microscopy revealed alterations in the apical process structure. Two compounds, ENDO-1-IWR and AGN 193109, did not disrupt the health of iPSC-RPE and revealed different types of apical process phenotypes.

[0256] Starting from D21, iPSC-RPE were treated with different compounds. On D40, immature RPE were enriched by negative selection according to the differentiation protocol and seeded in transwells for maturation. iPSC-RPE were treated with compounds for another 6 weeks until the cells were fully matured (Figure 1 and Figure 11).

[0257] At this point, transepithelial electrical resistance (TER) was assayed to confirm the quality of differentiation and to examine the effect of drugs on tight junction integrity. 2 A minimum threshold of 0.01 μm is used to determine a good differentiated batch. DMSO-treated iPSC-RPE have a resistance of approximately 1100 Ω cm. 2 The mean TER measurements of 100 Ω cm were obtained, confirming the quality of this differentiation batch. While the other compounds (see Table 3) disrupted the integrity of the epithelial barrier, ENDO-1-IWR and AGN 193109 not only maintained tight junction integrity but also induced an increase in TER resistance (ENDO-1-IWR: approx. 1200 Ω cm). 2 , AGN 193109: Approx. 1600Ωcm 2 ).

[0258] Shape metrics were then analyzed to confirm the effect of the drug on cell area. Compared to DMSO-treated iPSC-RPE, AGN 193109 induced an increase in iPSC-RPE cell area comparable to the dimensions of the macular RPE population (P1) (iPSC-RPE area = 161.8 ± 149.7 µm 2, P1=149.6±33.4μm 2 ), whereas ENDO-1-IWR induced a larger increase in cell size comparable to cells of the peripheral RPE population P3 (iPSC-RPE area = 251.8 ± 218.4 μm 2 , P3=238.6±37.8μm 2 ) The graphs and color-coded images in Figures 8-9 show the comparison between the different groups.

[0259] Brightfield imaging was used to determine RPE pigmentation defects, while hematoxylin and eosin (H&E) staining was useful to examine gross morphological changes in the monolayer. Sections of the transwell membrane were cut and mounted on glass slides for brightfield imaging. Pigmentation levels were preserved after ENDO-1-IWR and AGN 193109 treatment compared to DMSO-treated iPSC-RPE. Another area of ​​the transwell was sectioned and stained with H&E. After drug treatment, the cells were still arranged as a monolayer, no gross morphological changes were detected, and pigment granules were still present on the apical side of the RPE cells.

[0260] Finally, although transmission electron microscopy (TEM) was used to detect fine morphological changes, scanning electron microscopy (SEM) was essential to study the modifications in apical process structure. Although no defects in fine subcellular structures were identified after ENDO-1-IWR and AGN 193109 treatment, the two drugs induced changes in apical process structure. In DMSO-treated iPSC-RPE, we found both types of apical processes, petal-like and finger-like. Notably, upon ENDO-1-IWR treatment, all RPE cells were uniquely enriched with finger-like apical processes, which are associated with RPE interactions with rod outer segments. Meanwhile, AGN 193109 treatment enriched RPE cells with petal-like apical processes (with undulations) that support the cone outer segments. The fact that ENDO-1-IWR increases cell size to the dimensions of the peripheral RPE population P3 and enriches for cells with finger-like apical processes proves that this compound induces a peripheral RPE phenotype. Conversely, AGN 193109 shifts iPSC-RPE cell size to the macular RPE population (P1) and induces the formation of petal-like apical processes (with undulations), providing evidence that this compound induces a macular RPE phenotype (Figures 10 and 11).

[0261] Overall, these results indicate that using cell shape as a screening tool not only separates populations of larger cells from those of smaller cells, but also selects for morphological features.Without wishing to be bound by theory, the data indicate that developmental pathways are targeted during RPE differentiation.

[0262] In summary, 17 RPE / choroid flatmounts from nine non-AMD donor eyes were dissected, stained for RPE cell boundaries, imaged, and segmented on average 3–4 million cells per flatmount. For the first time, we generated a complete morphometric map of the entire human RPE for each human eye used in this study. Cell area was chosen among other morphometric criteria to highlight RPE cell heterogeneity across the eye. A gradient of RPE cells was identified with progressively larger regions starting with small RPE cells in the macula and moving eccentrically towards the periphery. A peripheral ring of small RPE cells was detected around a radius of 14–17 mm from the center of the eye. Five unique regions of RPE cells were identified.

[0263] The finding of a peripheral ring of smaller RPE cells indicates the presence of macular-type RPE cells in the distal periphery. RPE population P4 may correspond to the same region where the peripheral edge of cones has been described. For example, there is an abrupt transition from the large RPE cells of population P3 (periphery) to the small cells of population P4 (distal periphery) and again to the large RPE cells of population P5 (serrata). Furthermore, RPE population P4 is approximately 1-2 mm wide, encompassing a region approximately 14-17 mm away from the center of the eye and anterior to the transition zone of the serrata (the same region where the loose band of cones was described). Small RPE cells appear to reside in cone-dominated regions such as the macula and peripheral edge, while large RPE cells occur in rod-dominated regions.

[0264] The disclosed method achieves the recapitulation of regional RPE heterogeneity in vitro. To achieve this, the cell size of iPSC-RPE generated in culture was compared to the average cell area of ​​each human RPE population. The comparison showed that fully mature RPE grown in culture had a smaller cell area than any other human RPE population identified in cadaveric eye samples. The closest human RPE population in terms of size was macular RPE (P1). Although cells were seeded in different plate formats and concentrations were adjusted accordingly, the average cell area did not change substantially, so it is unlikely that the seeding concentration induces a smaller cell size.

[0265] To identify such signals that determine RPE cell size, iPSC-RPE cells were manipulated during the differentiation stage when they are just committed to the RPE fate. This manipulation was performed using 115 activators and inhibitors of different developmental pathways and regulators of the actin cytoskeleton. RPE morphometry was analyzed for all drugs and compared as a reference to a human macular RPE population (P1) and a peripheral RPE population P3.

[0266] The peripheral population P3 was chosen as a reference for the periphery because population P5 contains RPE cells of the ora serrata that may not have “true” RPE characteristics, and population P4 (distal periphery) resembles macular RPE in cell size.

[0267] Compounds were grouped by pathway and their role in activating or inhibiting it, and the compounds that showed the greatest shift in cell size for each group were tested for phenotypic characterization. Two compounds, AGN 193109 and ENDO-1-IWR, were found to be able to recapitulate the cell size of the macular (P1) and peripheral (P3) populations, respectively, enriching RPE cells with petal-like and finger-like apical processes, respectively. Both compounds had no effect on the healthy RPE phenotype, as verified by TER, bright field, H&E and TEM.

[0268] AGN 193109 is a high affinity pan-retinoic acid receptor (RAR) antagonist. Retinoic acid is a form of vitamin A that is not available in the diet, but is synthesized from retinol. Its levels are therefore regulated by its synthesis and degradation (Duester, Molecular and Cellular Biology, 11(3), 1638-164; Napoli, J. Biol. Chem., 261(29), 13592-13597j 1986). Retinoic acid can regulate many important biological processes such as embryogenesis, immunity, promoting or inhibiting cell proliferation and differentiation (Lotan, Biochimica et Biophysica Acta (BBA)-Reviews on Cancer, 605(1), 33-91, 1980). The RPE plays a central role in ocular retinoid metabolism: the RPE is a reservoir for retinyl esters, the storage form of vitamin A, and isomerization and oxidation of all-trans retinyl esters to form 11-cis-retinalaldehyde occurs in RPE cells (Rando, Biochemistry, 30(3), 595-60, 1991). Furthermore, the RPE is one of the tissues that contains the highest concentrations of retinoids in the body, highlighting the importance of RPE cells in vitamin A metabolism (Berman et al., Investigative Ophthalmology, 13(9), 675-687, 1974). Cultured bovine RPE cells have been shown to metabolize retinoic acid via the activity of cytochrome P-450 monooxygenase. The RPE converts retinoic acid to a more polar metabolite, 4-oxo-retinoic acid, which can be rapidly released from the cell (Doyle et al., Investigative Ophthalmology & Visual Science, 36(3), 708-717, 1995). This result indicates that the RPE may be important in the inactivation of this biologically potent retinoid in the retina.

[0269] Localized degradation of retinoic acid from the RPE and retina during early eye development may indicate the location of future macular RPE and fovea. Without wishing to be bound by theory, AGN 193109 may mimic retinoic acid depletion by antagonizing RARs in RPE cells. To test this hypothesis, basal levels of retinoic acid signaling can be examined and cytochrome expression and activity in control and AGN 193109-treated RPE cells can be confirmed.

[0270] Other commercially available retinoic acid inhibitors can be used to generate macular iPSC-RPE (P1). These are listed in Table 4. [Table 4]

[0271] ENDO-1-IWR is a canonical Wnt pathway inhibitor. Wnt is an evolutionarily conserved pathway that regulates key aspects of cell fate determination, cell migration, cell polarity, neural patterning and organogenesis during embryonic development. The Wnt pathway can be subdivided into two main branches downstream of the frizzled receptor: the canonical Wnt pathway, which is dependent on β-catenin, and the non-canonical Wnt pathway. The latter is the planar cell polarity (PCP) pathway and the Wnt / Ca pathway. 2+It can be further subdivided into pathways. Wnt molecules are secreted glycoproteins that bind to the frizzled receptor family. Wnt signaling is initiated by the binding of the frizzled receptor to a co-receptor, e.g., low density lipoprotein-related protein 5 / 6 (LRP5 / 6), in the case of canonical Wnt signaling. The signal is then transduced to the cytoplasmic protein Dishevelled (Dsh), and at this level the pathways branch (Komiya&Habas,Organogenesis,4(2),68-75,2008). A hallmark of the canonical Wnt pathway is the accumulation and translocation in the nucleus of the adherens junction-associated protein β-catenin, which activates the transcription of target genes through binding to DNA-binding transcription factors such as LEF / TCF (Clevers,Cell 127(3),469-480.2006; Reya&Clevers,Nature,434(7035),843-850,2005). In the absence of Wnt signaling, β-catenin is phosphorylated by the β-catenin destruction complex and targeted for degradation by the proteasome. Planar cell polarity has been shown to regulate the organization and orientation of epithelial cells (Mlodzik, Trends in Genetics: TIG, 18(11), 564-571, 2002). This pathway appears to be independent of transcription but functions through direct regulation of the actin cytoskeleton to achieve structural organization. The second branch of the noncanonical pathway is the Wnt / Ca pathway. 2+ This is independent of β-catenin-induced transcription and is a G protein-mediated pathway for intracellular Ca release from the ER. 2+ It is characterized by release of phospholipids (Kohn & Moon, Cell Calcium, 38(3-4), 439-446, 2005; Slusarski & Pelegri, Developmental Biology, 307(1), 1-13, 2007).

[0272] ENDO-1-IWR, a compound used to characterize the RPE phenotype, acts by stabilizing the Axin protein in the β-catenin destruction complex. Stabilization of the destruction complex promotes β-catenin degradation and thus canonical Wnt pathway inhibition.

[0273] The Wnt pathway plays a role in the development of the RPE. First, Wnt pathway inhibition along with BMP inhibition allows the generation of the eye field from the anterior neuroepithelium. Dkk-1 and Noggin endogenous expression (Wnt / β-catenin and BMP inhibitors, respectively) were upregulated in human embryonic stem cells during eye field specification (Meyer et al., Proceedings of the National Academy of Sciences of the United States of America, 106(39), 16698-16703, 2009). Second, during RPE specification, canonical Wnt is activated and β-catenin translocates to the nucleus and directly binds to enhancer sites of the RPE-specific genes Mitf and Otx2, inducing their expression. Loss of β-catenin converts the RPE into a multilayered tissue in which Mitf and Otx2 are downregulated, resulting in transdifferentiation of the RPE into the retina (Westenskow et al., Development, 136(15), 2505-2510, 2009).

[0274] Based on the data disclosed herein, the canonical Wnt pathway further plays a role in the RPE at later stages of development, from the RPE-committed stage to the mature RPE stage. During this stage, canonical Wnt inhibition with ENDO-1-IWR increases RPE cell size to match the human peripheral RPE population P3, enriching these cells with finger-like apical processes to support rod-dominated regions of the retina. A cell size gradient can be achieved by the combination of retinoic acid and Wnt inhibition.

[0275] Other Wnt inhibitors used in the screen showed similar trends in increasing iPSC-RPE cell area, further supporting the role of Wnt inhibition in generating peripheral RPE cells (Figure 12). This evidence indicates that other commercially available canonical Wnt inhibitors can be used to generate peripheral iPSC-RPE (P3). Table 5 provides an exemplary list of these compounds. [Table 5-1] [Table 5-2]

[0276] Canonical Wnt inhibition during the RPE maturation phase (D40–D75, the total number of days in culture) can promote RPE maturation (May-Simera et al., Cell Reports 22(1), 189-205, 2018). Treatment can begin 15 days after the start of culture in maturation medium. In contrast to the data presented here, Wnt inhibition is initiated during cell commitment to the RPE fate and is maintained until the end of RPE maturation (D25–D75). During RPE commitment, developmental pathways still play a major role and may affect the final RPE phenotype without altering the RPE fate.

[0277] Therefore, human RPE populations of macular (P1) and peripheral (P3) RPE cells were reproduced in vitro. Exemplary, non-limiting concentrations used are listed in Table 6. [Table 6] [Table 7]

[0278] This work provides insight into the development of the macula and RPE, improving current iPSC-based disease models of retinal degeneration and can be used to develop cell therapy products or target one or more regions of the retina (Figure 14).

[0279] Example 7 Further characterization of cells treated with AGN 193109 or endo-IWR-1 To demonstrate that DMSO (control, carrier), endo-IWR-1 and AGN 193109 were not harmful, we examined iPSC-RPE pigmentation levels after treatment with DMSO (Figure 15A), endo-IWR-1 (Figure 15C) or AGN 193109 (Figure 15E). AGN 193109 was used at 0.2 μM and endo-IWR-1 was used at 4 uM. Both drugs were dissolved in DMSO, so that the final concentration of DMSO was 0.1% or less. The final concentration of the DMSO control was 0.1%. Compounds were added to RPE maturation medium until cells matured. To detect possible abnormalities, gross cell morphology was also analyzed (Figure 15B, Figure 15E, Figure 15F, left images). Fine subcellular structures were examined for changes using transmission electron microscopy (TEM, Figure 15B, Figure 15E, Figure 15F, right images). These agents did not alter pigmentation levels or affect gross or microscopic cell morphology.

[0280] Transepithelial electrical resistance (TER) is a measure of the compactness of the monolayer. The denser the monolayer, the healthier the cells in it. 400 Ohms*cm 2 A cutoff of 0.01 was used to exclude cells with low TER. As shown in Figure 16, both AGN 193109- and endo-IWR-1-treated cells were well above this threshold. Data are displayed as box plots, with box limits representing the first and third quartiles, center lines indicating the median, and whiskers indicating the fifth and ninety-fifth percentiles. Ranges designate 90% of the data.

[0281] Single-cell RNA sequencing was performed to confirm that the compounds altered the cellular transcriptome. As shown in Figure 17, macular and mid-peripheral iPSC-RPE are functionally distinct from DMSO and each other.

[0282] Bulk RNA sequencing was performed and expression levels of specific genes were compared to data available from the literature (Figure 18). The data demonstrate an incomplete but consistent agreement of genes more expressed in macular RPE; a set of genes with opposite expression; and a near perfect agreement of genes more expressed in peripheral RPE cells.

[0283] iPSC-RPE cells were provided with purified bovine outer segments (mainly rod photoreceptors) for phagocytosis. The outer segments were labeled with a dye that fluoresces when the outer segments are phagocytosed. RPE cells were incubated with the outer segments for 4 hours and then analyzed by flow cytometry to measure outer segment ingestion, or returned to the incubator for 20 hours and then flow cytometry was performed to measure outer segment digestion. As shown in Figure 19, endo-iwr-1 treated cells ingested more outer segments (peripheral cells), indicating a possible affinity for rod outer segments, which are normally predominant in the periphery of the retina. One-way ANOVA showed no differences.

[0284] Acid phosphatase has higher expression in the macula (Boulton et al., The British Journal of Ophthalmology 78(2), 125-9, doi:10.1136 / bjo.78.2.125(1994)). This enzyme plays an important role in the digestion of photoreceptor outer segments. Acid phosphatase activity was determined in cells treated with DMSO, AGN 193109 or endo-IWR-1. For these studies, RPE cells were lysed in distilled water to extract the enzyme. The lysate was then incubated with p-nitrophenyl phosphate, a phosphatase substrate that turns yellow when dephosphorylated by acid phosphatase. The color of the solution was measured with a spectrophotometer to estimate the enzyme activity. As shown in Figure 20, AGN 193109-treated cells (macular iPSC-RPE) had higher acid phosphatase activity than DMSO- or endo-IWR-1-treated cells.

[0285] Peripheral (P)-RPE has higher mitochondrial oxidative phosphorylation (OXPHOS) compared to macular (M)-RPE. Metabolic processes in two types of RPE cells (M-RPE and P-RPE) were measured using Seahorse technology. The Seahorse system measures live cell oxygen consumption rate and extracellular acidification rate, which are indicators of mitochondrial respiration and glycolysis, respectively. As shown in Figure 21, under two different carbon sources (proline and succinate), P-RPE had a higher ability to undergo OXPHOS compared to M-RPE, which showed a higher glycolysis rate.

[0286] In view of the many possible embodiments to which the principles of the present invention may be applied, it is to be recognized that the illustrated embodiments are merely examples of the invention and should not be considered limitations on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

Claim 1 A method for producing macular, central or peripheral human retinal pigment epithelial (RPE) cells, comprising: a) culturing pluripotent stem cells in a retinal induction medium to initiate differentiation of said cells into RPE progenitor cells; b) culturing said RPE progenitor cells in a retinal differentiation medium to further differentiate said RPE progenitor cells into committed RPE cells; c) culturing said committed RPE cells in a retinal medium to form immature RPE cells; and d) culturing said immature RPE cells in an RPE maturation medium containing a retinoic acid receptor (RAR) antagonist and / or a canonical Wnt inhibitor, thereby producing human RPE cells; wherein said human RPE cells are macular human RPE cells, central human RPE cells or peripheral human RPE cells. Claim 2 The method according to claim 1, wherein said stem cells are induced pluripotent stem cells (iPSCs). Claim 3 The method according to claim 1 or claim 2, wherein said RAR antagonist is AGN 193109, CE 2665, ER 5081, LE 135, LY 2955303, MM 11253, or liarozole dihydrochloride. Claim 4 The method according to any one of claims 1 to 3, wherein said RAR antagonist is AGN 193109. Claim 5 The method according to any one of claims 1 to 4, wherein the canonical Wnt inhibitor is 4-(1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl)-N-8-quinolinyl-benzamide (Endo-1-IWR), calphostin C, cardiogen 1, CCT 031374 hydrobromide, IWP 12, XAV 939, WIKI4, ICG-001, Wnt-C59 (C59), IWR-1-endo, KY02111, LGK-974, IWP-L6, FH535, iCRT 14, IWP 4, JW 67, JW 74, KYA 1797K, NLS-StAx-h, PNU 74654, TAK 715, IWP 2, CKI 7 dihydrochloride, (R)-CR8, D 4476, (R)-DRF053 dihydrochloride, epibrastatin A, IC 261, LH 846, PF 4800567 hydrochloride, PF 5006739, PF 670462, SR 3029, AZ 6102, JW 55, MN 64, or TC-E 5001.

6. The method according to claim 5, wherein the canonical Wnt inhibitor is Endo-1-IWR.

7. The method according to any one of claims 1 to 6, wherein the method produces macular human RPE cells, and the RPE maturation medium contains the RAR antagonist but does not contain the canonical Wnt inhibitor.

8. The method according to claim 7, wherein the RAR antagonist is AGN 193109.

9. The method according to claim 8, wherein the RPE maturation medium contains 0.05 to 0.4 μM of AGN 193109.

10. The method according to claim 8, wherein the RPE maturation medium contains about 0.1 to about 0.2 μM of AGN 193109.

11. The method according to any one of claims 1 to 6, wherein the method produces central human RPE cells, and the RPE maturation medium contains both the RAR antagonist and the canonical Wnt inhibitor.

12. The method according to claim 11, wherein the RAR antagonist is AGN 193109 and the canonical Wnt inhibitor is Endo1-IWR.

13. The method according to claim 12, wherein the RPE maturation medium contains about 10 nM to about 50 nM of AGN 193109 and about 0.025 to about 0.5 μM of Endo-1-IWR.

14. The method according to claim 13, wherein the RPE maturation medium comprises AGN 193109 at about 25 nM to about 50 nM and Endo-1-IWR at about 0.1 μM to about 0.2 μM.

15. The method according to any one of claims 1 to 6, wherein the method produces peripheral human RPE cells, and the RPE maturation medium comprises the canonical Wnt inhibitor but does not comprise the RAR antagonist.

16. The method according to claim 15, wherein the canonical Wnt inhibitor is Endo-1-IWR.

17. The method according to claim 16, wherein the RPE maturation medium comprises Endo-1-IWR at about 0.5 to about 8 μM.

18. The method according to claim 17, wherein the RPE maturation medium comprises Endo-1-IWR at about 1 to about 4 μM.

19. The method according to any one of claims 1 to 18, wherein the iPSC in step a) is cultured on a matrix.

20. The method according to claim 19, wherein the matrix comprises at least one recombinant cell adhesion protein.

21. The method according to claim 20, wherein the at least one cell adhesion protein is laminin, vitronectin, or fibronectin.

22. The method according to any one of claims 1 to 21, wherein the retinal induction medium comprises a WNT pathway inhibitor, a transforming growth factor (TGF)-β pathway inhibitor, a bone morphogenetic protein (BMP) pathway inhibitor, and insulin growth factor 1 (IGF1).

23. The method according to any one of claims 1 to 22, wherein the retinal differentiation medium comprises a WNT pathway inhibitor, a TGFβ pathway inhibitor, a BMP pathway inhibitor, IGF1, and an additional inhibitor, and the additional inhibitor is a MEK inhibitor or a fibroblast growth factor (FGF) inhibitor.

24. The method according to any one of claims 1 to 22, further comprising the step of dissociating the RPE cells and the step of re-seeding the RPE cells on a scaffold in the RPE maturation medium.

25. The method according to claim 24, wherein the scaffold is degradable.

26. The method according to claim 24, wherein the scaffold is non-degradable.

27. The method according to claim 25 or 26, wherein the RPE cells are present within one or more eccentric circles on the scaffold.

28. The method according to any one of claims 24 to 27, wherein the RPE maturation medium contains at least one primary cilium inducer.

29. The method according to claim 28, wherein the at least one primary cilium inducer is prostaglandin E2 (PGE2) or afidicolin.

30. The method according to any one of claims 1 to 29, further comprising the step of cryopreserving the RPE cells.

31. The TGFβ pathway inhibitor is 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[l,3]dioxol-5-yl-2-yl-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride hydrate (SB-505124), 4-(5-benzo[l,3]dioxol-5-yl-4-pyridin-2-yl-lH-imidazol-2-yl)-benzamide hydrate, 4-[4-(l,3-benzodioxol-5-yl)-5-(2-pyridinyl)-lH-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 (D4476), 4-[4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide (GW 788388), 4-[3-(2-pyridinyl)-1H-pyrazole-4-yl]-quinoline (LY 364847), 4-[2-fluoro-5-[3-(6-methyl-2-pyridinyl)-1H-pyrazole-4-yl]phenyl]-1H-pyrazole-1-ethanol (R 268712) or 2-(3-(6-methylpyridin-2-yl)-1H-pyrazole-4-yl)-1,5-naphthyridine (RepSox), the method according to any one of claims 22 to 30.

32. The method according to any one of claims 22 to 31, wherein the BMP pathway inhibitor is LDN193189.

33. A composition for use in a method of treating a subject in need of treatment, the composition comprising macular cells, central cells or peripheral cells produced according to the method of any one of claims 1 to 32, the method comprising a step of transplanting the macular cells, central cells or peripheral cells into the retina of the subject A composition.

34. The composition according to claim 33, wherein the subject has a retinal degenerative disease, retinal dysfunction, retinal degeneration, retinal damage, or loss of retinal pigment epithelium.

35. The composition according to claim 34, wherein the retinal degenerative disease is Stargardt macular dystrophy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, Leber congenital amaurosis, acquired macular degeneration, hereditary macular degeneration, Best disease, late-onset retinal degeneration, Bietti dystrophy, retinal detachment, gyral atrophy, coloboma, or pattern dystrophy.

36. The composition according to claim 34, wherein the retinal damage is caused by laser, inflammatory, infectious, radiation, neovascular or traumatic injury.

37. The composition according to any one of claims 33 to 36, wherein the RPE cells are introduced through a position in the subretinal space, vitreous cavity, inner or outer retina, retinal periphery, within the choroid, or suprachoroid of the eye.

38. i) A tissue implant comprising retinal pigment epithelial cells produced by the method of any one of claims 1 to 32, or ii) A pharmaceutical composition comprising the composition of any one of claims 33 to 37.

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