Method for producing induced retinal progenitor cells and treatment using such cells

Abstract

The present invention relates to a method for producing induced retinal progenitor cells (induced RPCs) and treatment using said cells.

Description

[Technical Field]

[0001] Related applications This application claims priority under U.S. Provisional Application No. 63 / 505,172, filed on 31 May 2023 pursuant to Section 119 of the U.S. Patent Act, the entirety of which is incorporated herein by reference.

[0002] Technical field The present invention relates to a method for producing induced retinal progenitor cells (induced RPCs) and treatments using such cells. [Background technology]

[0003] Background of the Invention According to the WHO, there are 285 million people worldwide who are visually impaired and 39 million who are blind.[1] Actual studies report that blindness is the most terrifying disease of all human beings.[2] More than 10% of blindness is caused by degeneration of photoreceptor cells, and once the degeneration progresses to an advanced stage, it is considered incurable.[1] Currently, several studies are being conducted that focus on cell replacement therapies for photoreceptor degeneration, but major obstacles have prevented these approaches from being clinically viable. For example, primary retinal progenitor cells (RPCs) derived from the fetal retina suffer from ethical issues and low proliferative capacity, while retinal lineage cells derived from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have relatively low differentiation efficiency and are costly and labor-intensive.

[0004] Based on preclinical studies and clinical trials, RPC has the ability to restore vision in late photoreceptor degeneration. However, the primary availability of RPC has been limited to fetal tissue after miscarriage. [Overview of the Initiative]

[0005] Summary of the Invention In this invention, it was unexpectedly discovered that ocular fibroblasts can be chemically converted into retinal progenitor cells (RPCs) by culturing them in the presence of reprogramming agents, one or a combination of a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-related protein kinase (ROCK) inhibitor, and ascorbic acid. These chemically induced RPCs have been demonstrated to function as primary RPCs and to be effective in rescuing photoreceptor degeneration in cell replacement therapy in animal models.

[0006] In particular, in one aspect, the present invention provides a method for producing induced retinal progenitor cells (induced RPCs), comprising culturing ophthalmic fibroblasts under conditions that enable a portion of them to be reprogrammed into induced RPCs, wherein the conditions include a culture medium comprising a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-related protein kinase (ROCK) inhibitor, and ascorbic acid. The present invention also provides a method for producing induced retinal progenitor cells (induced RPCs), comprising culturing ophthalmic fibroblasts under conditions that enable a portion of them to be reprogrammed into induced RPCs, wherein the conditions include a culture medium comprising a compound selected from the group consisting of a DNMT inhibitor, an HDAC inhibitor, a CDK inhibitor, a cAMP activator, a ROCK inhibitor, ascorbic acid, and any combination thereof.

[0007] In some embodiments, the method of the present invention further includes identifying an induced RPC expressing one or more retinal markers selected from the group consisting of SOX2 (SRY box transcription factor 2), PAX6 (pair box 6), VSX2 (visual system homeobox 2), Neurod1 (neuronal differentiation 1), CRX (corn-rod homeobox protein), and RCVRN (recoverin), and any combination thereof, and isolating the identified induced RPC.

[0008] In some embodiments, induced RPCs express glutamate receptors.

[0009] In one embodiment, ocular fibroblasts are fibroblasts derived from Tenon's capsule.

[0010] In one embodiment, ocular fibroblasts are human fibroblasts derived from Tenon's capsule.

[0011] In some embodiments, DNMT inhibitors, HDAC inhibitors, CDK inhibitors, cAMP activators, ROCK inhibitors, and ascorbic acid are added to the culture medium simultaneously or sequentially.

[0012] In some embodiments, the DNMT inhibitor is RG108, the HDAC inhibitor is VPA, the CDK inhibitor is SU9516, the cAMP activator is forskolin (FSK), and the ROCK inhibitor is Y-27632.

[0013] In some embodiments, RG108 is present in the culture medium at a concentration of 1–100 M, VPA at a concentration of 1–100 mM, SU9516 at a concentration of 1–100 μM, FSK at a concentration of 1–100 μM, Y-27632 at a concentration of 1–100 μM, and ascorbic acid at a concentration of 1–100 μM.

[0014] In some embodiments, RG108 is present in the culture medium at a concentration of 1–50 μM, VPA is present at a concentration of 1–10 mM, SU9516 is present at a concentration of 1–50 μM, FSK is present at a concentration of 1–50 μM, Y-27632 is present at a concentration of 1–50 μM, and ascorbic acid is present at a concentration of 1–50 μM.

[0015] In some embodiments, RG108 is present in the culture medium at a concentration of approximately 20 μM, VPA at a concentration of approximately 3 mM, SU9516 at a concentration of approximately 10 μM, FSK at a concentration of approximately 10 μM, Y-27632 at a concentration of approximately 10 μM, and ascorbic acid at a concentration of approximately 10 μM.

[0016] In some embodiments, the culture medium includes DMEM.

[0017] In some embodiments, the culture medium comprises DMEM / F12 supplemented with N2 and B27 and neuronal basal medium.

[0018] In some embodiments, the method of the present invention is (a) A step of culturing ophthalmic fibroblasts in a culture vessel containing a first culture medium containing a DNMT inhibitor; (b) The step of removing the first culture medium and adding a second culture medium containing a DNMT inhibitor and an HDAC inhibitor; (c) Remove the second culture medium and add a third culture medium containing a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid; (d) The third culture medium is removed and a fourth culture medium containing a cAMP activator, a ROCK inhibitor, and ascorbic acid is added. Includes.

[0019] In a particular embodiment, the method of the present invention is (a) A step of culturing ophthalmic fibroblasts in a culture vessel containing a first culture medium containing a DNMT inhibitor; (b) The step of removing the first culture medium and adding a second culture medium containing a DNMT inhibitor and an HDAC inhibitor; (c) Remove the second culture medium and add a third culture medium containing a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid; (d) The third culture medium is removed, and a fourth culture medium containing a cAMP activator, a ROCK inhibitor, and ascorbic acid is added; (e) Identifying induced RPCs that express one or more retinal markers selected from the group consisting of SOX2, PAX6, VSX2, NEUROD1, CRX, and RCVRN, and any combination thereof; and, (f) Isolating the identified induced RPCs comprising.

[0020] In some embodiments, the cells are cultured in a first culture medium for 1 to 3 days, the cells are cultured in a second culture medium for 1 to 3 days, the cells are cultured in a third culture medium for 1 to 3 days, and / or the cells are cultured in a fourth culture medium for 1 to 3 days.

[0021] In another aspect of the invention, there is provided induced RPCs that highly express SOX2, NESTIN, and protein tyrosine phosphatase receptor type N (PTPRN) as compared to primary RPCs. The invention also provides induced RPCs produced by the methods described herein. The invention further provides a cell population comprising the induced RPCs described herein.

[0022] The invention provides a composition comprising the induced RPCs described herein or a cell population comprising the induced RPCs, and a composition comprising a pharmaceutically acceptable carrier.

[0023] In a further aspect of the invention, there is provided a method of treating a photoreceptor degeneration disease in a subject, the method comprising administering to the subject's eye an effective amount of induced RPCs (retinal progenitor cells) or a cell population comprising the induced RPCs, or a composition described herein. The invention also provides the use of induced RPCs (retinal progenitor cells) or a cell population comprising the induced RPCs, or compositions thereof described herein, for the manufacture of a medicament for the treatment of photoreceptor degeneration diseases.

[0024] In some embodiments, the amount of retinal progenitor cells is effective to rescue color vision and central vision.

[0025] In some embodiments, photoreceptor degenerative diseases are selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy (DR), and Stargardt disease.

[0026] Details of one or more aspects of the present invention are described below. Other features or advantages of the present invention are also evident from some of the aspects detailed below and the appended claims. [Brief explanation of the drawing]

[0027] Brief explanation of the drawing The above summary and the following detailed description of the invention can be better understood by reading them in conjunction with the accompanying drawings. For the purpose of illustrating the present invention, the drawings show the current preferred embodiments. However, it should be understood that the present invention is not limited to the exact configuration or means shown.

[0028] [Figure 1] Figures 1A and 1B show phase-contrast images during and after the reprogramming protocol from capsule of Tenon's fibroblasts (HTF) to chemically induced retinal progenitor cells (CiRPC). Figure 1A shows that the cell morphology was identical to that of HTF during the first three days of reprogramming. By day 4, all cells underwent a significant morphological change to a dome shape with bright nuclei, and maintained this morphology beyond day 6. After subculturing, the cells tended to form clusters and were viable for up to two months. Figure 1B shows that, apart from the significant morphological change from HTF, all other fibroblast cell lines tested by the inventors (human fetal lung fibroblasts (IMR-90), human neonatal foreskin fibroblasts (CRL-2097), human neonatal foreskin fibroblasts (BJ-5ta), and human adult dermal fibroblasts (FB-3652)) did not change to a dome shape with bright nuclei, i.e., a primary RPC-like morphology. [Figure 2] Figure 2 shows a comparison of reprogramming protocols in CiRPC. It presents a 5-day CiRPC protocol using six small molecules. [Figure 3]Figures 3A and 3B show a comparison of conversion efficiencies in the CiRPC protocol. Figure 3A shows Vsx2-GFP+ cells 5 days after the CiRPC protocol. Figure 3B shows the FACS gating strategy of Vsx2-GFP+ cells. [Figure 4] Figures 4A and 4B show a comparison of changes in gene expression. Figure 4A shows qRT-PCR analysis of CiRPCs reprogrammed from HTF by six small molecules (6C). Figure 4B shows WB analysis of VSX2 expression between HTF, unselected CiRPCs, and FACS-selected CiRPCs. [Figure 5] Figures 5A and 5B show GO enrichment analyses of HTF and CiRPC. Figure 5A shows upregulated GO associated with extracellular matrix components, axons, dendrites, synapses and postsynaptic membranes, and transport vesicles. Figure 5B shows downregulated GO associated with cell division and fibrosis. [Figure 6] Figures 6A to 6C show in vitro calcium imaging of HTF, unsorted CiRPCs (iRLCs), and sorted iRPCs. Figure 6A shows no calcium influx in HTF when stimulated with 1 mM glutamate (glutamate stimulation was performed for 200 to 400 seconds). On the other hand, unsorted CiRPCs induced by 6C showed significant calcium influx when stimulated with 1 mM glutamate (glutamate stimulation was performed for 200 to 400 seconds). Figure 6B shows no calcium influx in HTF when stimulated with 1 mM glutamate (glutamate stimulation was performed for 100 to 400 seconds, indicated by the red square). On the other hand, iRLCs induced by 6C showed significant calcium influx when stimulated with 1 mM glutamate. The calcium influx response was further enhanced in FACS sorted iRPCs induced by 6C. Figure 6C shows that VPA-induced iRPCs showed significant calcium influx when stimulated with 1 mM glutamate (glutamate stimulation was performed for 100 to 400 seconds, indicated by the red square). 6C shows iRPCs (selected Vsx2::eGFP+ cells) and iRLCs (unselected cells) induced by 6C. [Figure 7] Figures 7A and 7B show a comparison of therapeutic effects in animal models of photoreceptor degeneration. Figure 7A shows the ERG dark adaptation b wave in RCS rats that received subretinal implantation of CiRPC. Figure 7B shows the LDB test in RCS rats that received subretinal implantation of CiRPC. CiRPC = iRPC = sorted Vsx2::eGFP+ cells; iRLC = unsorted cells, induced by 6C. [Figure 8] Figure 8 shows the ocular histology of RCS rats 3 months after subretinal transplantation. In rats transplanted with HTF, a significant fibrous membrane was observed on the retinal surface, and large cell clumps were found attached. Cell clumps and fibrosis were stained with human nuclei. No fibrosis was observed around the injection site in rats transplanted with PBS and CiRPC. IHC confirmed that HuNu+ cells were integrated into the rat retina and were present in the photoreceptor layer labeled with the photoreceptor marker RCVRN. [Figure 9] Figure 9 shows a high-content screening system for HTF in iRLCs. Each compound was applied one at a time, and the resulting changes in Vsx2::eGFP expression in the high-content screening system were evaluated. It was found that HTF could be induced in iRLCs by any of the compounds in the 6C protocol. On the other hand, the overall Vsx2 expression pattern was optimal when all six compounds were applied. Error ranges indicate standard deviation. Asterisks indicate significant differences between the two cell types by t-test (* for P<0.05, ** for P<0.01, *** for P<0.001). The 5-day reprogramming protocol is shown in Figure 2. [Figure 10] Figure 10 shows a comparison of protein expression patterns between HTF and RLC using IF staining. Quantification of marker positivity in iRLC. Error range indicates standard deviation. [Figure 11]Figure 11 shows OCT images of transplanted cells and rat retina. A: No significant accumulation of HTF on the retinal surface was observed in week 1, but significant fibrotic changes were observed on the retinal surface in week 2 (note the curved retinal surface with cell clumps indicated by arrows). B: iRLCs migrated to the rat retinal surface one week after transplantation (arrows), and most iRLCs disappeared from the vitreous humor by week 2, leaving no cell fragments behind. [Figure 12] Figures 12A and 12B show H&E and immunofluorescence images of rat eyes with transplanted HTF or iRLCs. Figure 12A: In rat eyes transplanted with HTF, a prominent fibrous membrane was observed on the retinal surface, and a large cell mass (arrow) was confirmed to be attached to the epiretinal membrane (triangle). The cell mass and epiretinal membrane had a human nucleus stained in the center, surrounded by rat cells. The transplanted cells were not integrated with the rat retina. Figure 12B: Several iRLCs with a distinct dome shape and bright nucleus were found on the retinal surface (triangle) and within the retina (arrow). Immunostaining revealed that several iRLCs integrated into the inner retina (INL) of the rat retina were co-expressed with the photoreceptor marker RCVRN (arrow). [Figure 13] Figure 13 shows fundus photographs and OCT images of rat eyes immediately after subretinal transplantation. No major hemorrhage was caused by the injection. eGFP-labeled cells were found at the subretinal injection site. OCT showed successful subretinal bulge formation in the treated eye. [Figure 14] Figure 14 shows a bar graph of ERGb wave amplitude. Statistically significant improvements in night vision response were observed in eyes injected with iRPC at P56 and in eyes injected with iRLC at P112. [Figure 15] Figure 15 shows a bar graph of the LDB test. The amount of time spent in the dark was randomly distributed in RCS rats transplanted with PBS at P112 (Addendum File 4: Video S2). However, RCS rats transplanted with HTF, iRLC, and iRPC (Addendum File 5: Video S3) tended to spend more time in the dark, indicating that their visual function recovered 3 months after transplantation. [Figure 16]Figure 16 shows H&E and immunofluorescence images of RCS rat eyes 3 months after subretinal transplantation. In rats transplanted with HTF, a significant fibrous membrane was observed on the retinal surface, and large cell clumps were confirmed to be attached. The cell clumps and fibrosis were stained with human nuclei. No fibrosis was observed around the injection site in the eyes of rats transplanted with iRLC and iRPC. Immunostaining confirmed the integration of HuNu+ cells into the rat retina and their co-expression with the photoreceptor marker RCVRN. [Modes for carrying out the invention]

[0029] Detailed description of the invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0030] 1.Definition In this specification, the singular forms "a," "an," and "the" include the plural form unless the context clearly indicates otherwise. Thus, for example, a reference to "a component" includes the plural form of such component as well as its equivalents, as well as those well known to those skilled in the art.

[0031] The terms "include" or "contain" are generally used to mean "to include / contain" in the sense of allowing the presence of one or more features, components, or elements. The terms "include" or "contain" include the terms "constitute" or "consist of."

[0032] As used herein, the term "retinal progenitor cell (RPC)" may include progenitor cells that have the ability to differentiate into all types of retinal nerve cells, including rod cells, cone cells, and Müller glial cells.

[0033] As used herein, the term "inducible retinal progenitor cell (iRPC)" means an RPC-like cell (i.e., a cell possessing RPC-like characteristics) generated (or reprogrammed) from another cell type, such as a fibroblast.

[0034] As used herein, the term "reprogramming" refers to the process of transforming cells into different cell types that possess different characteristics or biological functions.

[0035] As used herein, the term "small molecule" means a synthesized or naturally occurring organic or inorganic molecule, generally having a molecular weight of less than 10,000 grams per mole, particularly less than 5,000 grams per mole, even more specifically less than 2,000 grams per mole, and especially less than 1,000 grams per mole. In some embodiments, small molecules also mean non-polymeric, i.e., non-protein or nucleic acid-based chemical molecules.

[0036] As used herein, the term "about" means plus or minus 10% of the numerical value used. Therefore, about 1% means a range from 0.9% to 1.1%.

[0037] As used herein, the term "expression" means the realization of genetic information encoded by a gene, and refers to the production of gene products such as unspliceable RNA, mRNA, splice variant mRNA, polypeptide or protein, post-translationally modified polypeptide, or splice variant polypeptide.

[0038] As used herein, DNA methyltransferase (DNMT) inhibitors mean substances that downregulate, reduce, or suppress the amount and / or activity of DNA methyltransferase. Examples of DNMT inhibitors include RG108, azatidine, decitabine, thioguanine, zebralin, SGI-110, SGI-1027, lomeguatrib, and procainamide hydrochloride.

[0039] As used herein, histone deacetylase (HDAC) inhibitors mean substances that downregulate, reduce, or inhibit the amount and / or activity of histone deacetylase in order to remove acetyl groups from lysine residues on histones. Examples of HDAC inhibitors include valproic acid (VPA, 2-propylpentanoic acid), apicidine, CI994, FK228, LMK235, M344, MC1568, MC1742, MI192, NCH51, NSC3852, PCI34051, sodium 4-phenylbutylate, pyroxamide, SAHA, SBHA, scriptide, sodium butyrate, TC-H106, TCSHDAC6 20b, trichostatin A, chuvasin, UF010, mosetinostat, prasinostat, and others.

[0040] As used herein, a cyclin-dependent kinase (CDK) inhibitor means a substance that downregulates, reduces, or inhibits the amount and / or activity of a cyclin-dependent kinase. Examples of CDK inhibitors described herein include SU9516, PD-0332991, roscovitine, SNS-032, dinaciclib, flavopyridol, AT7519, flavopyridol, JNJ-7706621, AZD5438, MK-8776, PHA-793887, BS-181, palbociclib (PD0332991) isethionate, A-674563, abemaciclib, BMS-265246, PHA-767491, milcilib, R547, NU6027, P276-00, MSC2530818, senexin A, LY2857785, LDC4297, ON123300, kempaulon, K03861, and THZ1 Examples include 2HCl, AT7519HCl, pervalanol A, Ro-3306, XL413, LDC000067, ML167, TG003, ribociclib, wogonin, BIO, AZD1080, and 1-azakenepaulon.

[0041] As used herein, the term "cyclic adenosine monophosphate (cAMP) activator" refers to a substance that increases intracellular cAMP levels compared to physiological intracellular levels in the absence of the drug. Examples of cAMP activators include, but are not limited to, forskolin, rolipram, NKH477, PACAP1-27, and PACAP1-38.

[0042] As used herein, Rho-related protein kinase (ROCK) inhibitors mean substances that downregulate, reduce, or suppress the amount and / or activity of Rho-related protein kinases. Examples of ROCK inhibitors described herein include Y-27632, AS1892802, GSK269962, GSK429286, H1152 dihydrochloride, HA1100 hydrochloride, OXA06 dihydrochloride, RKI1447 dihydrochloride, and SB772077B dihydrochloride.

[0043] As used herein, the terms “isolated or purified cell population” or “isolated or purified cells” mean a preparation of cells separated from other cellular components or other cells. For example, isolated cells may be removed from their maternal environment or cell population, or may result from the proliferation of cells removed from a cell population. When cells are expressed as “isolated” or “purified,” it should be understood that they are relatively isolated or purified, not absolutely isolated or purified. For example, a preparation containing isolated cells may contain 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% or more of the total number of cells in the preparation. In certain embodiments, a preparation containing isolated cells may contain 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of the total number of cells in the preparation.

[0044] As used herein, the term "subject" means human and non-human animals, including companion animals (dogs, cats, etc.), farm animals (cattle, sheep, pigs, horses, etc.), or laboratory animals (rats, mice, guinea pigs, etc.).

[0045] In this specification, “to treat” means to apply or administer a composition comprising one or more active ingredients to a subject suffering from a disease, symptoms or conditions of a disease, or the progression of a disease, with the aim of curing, promoting healing, alleviating, reducing, improving, or influencing the disease, symptoms or conditions of a disease, the impairment caused by the disease, or the progression of the disease.

[0046] As used herein, the term "therapeutic effective dose" refers to the amount of an active ingredient necessary to produce a therapeutic effect on a patient. The therapeutically effective dose can vary for various reasons, including the route and frequency of administration, the body weight and species of the individual receiving the drug, and the purpose of administration. As used herein, the term "effective dose" may also refer to the amount of an ingredient or drug applied to achieve a purpose when it refers to an application or process unrelated to the treatment of a disease. For example, the amount of an ingredient or drug applied to bring fibroblasts into contact with the cells.

[0047] 2. Production of induced RPCs The goal of cell replacement therapy is to replace lost or damaged photoreceptor cells with stem cells or healthy cells derived from the fetus. Several preclinical studies in animal models of retinal pigment epithelium (RP) have demonstrated the feasibility and efficacy of cell replacement therapy for restoring visual function. Summarizing the current clinical progress of cell replacement therapy in retinal degeneration, RPE (retinal pigment epithelium) degeneration has been successfully investigated in clinical trials based on hESC-RPE or hiPSC-RPE, but because RPE cells are not sensitive to light, the results have been limited to stabilization and slight improvement of patients' vision. Photoreceptor degeneration has been studied with promising interim results in clinical trials using fetal tissue-derived hRPCs. In this study, the inventors aimed to address an unmet medical need in photoreceptor degeneration. The use of fetal tissue-derived hRPCs is limited by their availability and ethical issues, and the differentiation of hESC / hiPSC-derived RPCs is not only time-consuming and laborious but also inefficient. We hypothesized that retinal progenitor cell-like cells with gene expression profiles and cellular functions similar to those reported in primary human retinal progenitor cells (hRPCs) could be efficiently and safely produced by chemical direct reprogramming without genome manipulation using exogenous transcription factors.

[0048] This invention relates to a more efficient method for converting ocular fibroblasts into induced retinal progenitor cells (induced RPCs).

[0049] The ocular fibroblasts used in the method of the present invention can be obtained from ocular tissue of a suitable autologous or allogeneic donor, such as Tenon's capsule. The ocular tissue is excised from the donor and transferred to a cell culture flask containing cell culture medium. After a certain period (e.g., about one week), ocular fibroblasts are obtained that can be identified by morphological characteristics such as a spindle shape or polygonal shape, and by the expression of fibroblast markers such as S100 calcium-binding protein 4 (S100A4). The obtained ocular fibroblasts can be passaged 1 to 15 times. Preferably, the ocular fibroblasts for conversion to induced RPC used herein are of mammalian origin, most preferably human origin.

[0050] Culture media suitable for use in the present invention are available in the art and may be further modified as needed. Suitable cell culture media are commercially available and include, but are not limited to, Dulbecco's Modified Eagle Medium (DMEM), Minimum Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Modified Minimum Essential Medium (IMEM), Glasgow Minimum Essential Medium (G-MEM), Basal Medium Eagle (BME), and Ham's F12 Medium (F12). Common basal media include Neurobasal (商標) This is a basal medium for nerve cells, which is designed for fetal and fetal nerve cell culture. Common supplements include N2 supplements, vitamin A-free B27 supplements, non-essential amino acids (NEAAs), glutamax supplements, fetal bovine serum (FBS), and bovine serum albumin (BSA). Suitable culture media used in this invention may include various combinations of culture media and additives. In one embodiment, DMEM / F12 means a 1:1 mixture of DMEM and Ham's F12 culture medium.

[0051] In certain embodiments, the culture medium used in the present invention comprises a DMEM culture medium containing 5-10% FBS.

[0052] In certain embodiments, the culture medium used in the present invention comprises a neuronal cell basal medium and a DMEM / F12 culture medium supplemented with an N2 supplement, a vitamin A-free B27 supplement, and non-essential amino acids (NEAAs). Specifically, the culture medium used in the present invention comprises 50% DMEM / F12, 50% neurobasal culture medium, 1x N2 supplement, 1x vitamin A-free B27 supplement, and 0.1 mM NEAA. The culture medium containing 1x N2 supplement and 1x B27 supplement means that the final concentration is 1x. Specifically, the DMEM / F12 culture medium is obtained by mixing the following components to obtain 1000 ml of culture medium. - 480mL NEUROBASAL - 480 mL of DMEM / F12 -100×N2 Supplement 10ml -50×B27 supplement 20mL; and -100×NEAA 10mL.

[0053] Generally, cells are cultured in cell culture devices such as cell culture vessels. Cell culture vessels may be Petri dishes, culture flasks, roller bottles, and multi-wall plates. In particular, cell culture vessels may be coated with a coating that provides structural support to the cells and / or promotes cell proliferation by supplying metabolites to the cells. In some embodiments, the coating may be fibronectin, gelatin, or Matrigel. (商標) (BD Bioscience), may contain collagen and / or laminin.

[0054] According to the present invention, ocular fibroblasts are chemically reprogrammed into induced RPCs in a culture medium using one or more chemoinducers, including a DNMT inhibitor, an HDAC inhibitor, a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid, as described herein. In some embodiments, the chemoinducers are added to the culture medium simultaneously or sequentially. In some embodiments, the chemoinducers are added to the culture medium individually.

[0055] In some embodiments, ocular fibroblasts are chemically reprogrammed into inducible RPCs using six chemoinducers. In particular, when culturing cells in the method of the present invention, the steps include: (a) culturing in a first medium containing a DNMT inhibitor; (b) then removing the first medium and culturing in a second medium containing a DNMT inhibitor and an HDAC inhibitor; (c) then removing the second medium and culturing in a third medium containing a CDK inhibitor, a cAMP activator and a ROCK inhibitor and ascorbic acid; and (d) then removing the third medium and culturing in a fourth medium containing a cAMP activator, a ROCK inhibitor and ascorbic acid.

[0056] Table 1 shows some examples of chemical inducers used in the present invention. [Table 1-1] [Table 1-2]

[0057] In some embodiments, the culture is carried out under normal conditions, for example, at 37°C under 1-10% CO2.

[0058] In some embodiments, the culture is carried out for at least one day (e.g., two, three, four, five, six, seven, eight, nine, or ten days or more). In some embodiments, the culture is carried out for at least one day but less than ten days (e.g., nine, eight, seven, six, five, four, three, two, or one day). In some embodiments, the culture is carried out for one to six days, one to five days, one to four days, or one to three days.

[0059] In some embodiments, when culturing cells using the method of the present invention, the NDMT inhibitor used herein is RG108, the HDAC inhibitor used herein is VPA, the CDK inhibitor used herein is SU9516, the cAMP activator used herein is forskolin (FSK), and the ROCK inhibitor used herein is Y-27632.

[0060] In some embodiments, when cells are cultured using the method of the present invention, RG108 is present in the culture medium at a concentration of 1-100 μM, VPA is present in the culture medium at a concentration of 1-100 mM, SU9516 is present in the culture medium at a concentration of 1-100 μM, FSK is present in the culture medium at a concentration of 1-100 μM, Y-27632 is present in the culture medium at a concentration of 1-100 μM, and ascorbic acid is present in the culture medium at a concentration of 1-100 μM.

[0061] In some embodiments, when cells are cultured by the method of the present invention, RG108 is present in the culture medium at a concentration of 1-50 μM, VPA is present in the culture medium at a concentration of 1-10 mM, SU9516 is present in the culture medium at a concentration of 1-50 μM, FSK is present in the culture medium at a concentration of 1-50 μM, Y-27632 is present in the culture medium at a concentration of 1-50 μM, and ascorbic acid is present in the culture medium at a concentration of 1-50 μM.

[0062] In some embodiments, when cells are cultured using the method of the present invention, RG108 is present in the culture medium at a concentration of about 20 μM, VPA is present at a concentration of about 3 mM, SU9516 is present at a concentration of about 10 μM, FSK is present at a concentration of about 10 μM, Y-27632 is present at a concentration of about 10 μM, and ascorbic acid is present at a concentration of about 10 μM.

[0063] In some embodiments, when culturing cells by the method of the present invention, the steps include: (a) culturing in a first culture medium as specified herein for 1 to 3 days; (b) then removing the first culture medium and culturing in a second culture medium as specified herein for 1 to 3 days; (c) then removing the second culture medium and culturing in a third culture medium as specified herein for 1 to 3 days; and (d) then removing the third culture medium and culturing in a fourth culture medium as specified herein for 1 to 3 days.

[0064] In some embodiments, when culturing cells by the method of the present invention, the steps include: (a) culturing in a first culture medium as specified herein for about 2 days; (b) then removing the first culture medium and culturing in a second culture medium as specified herein for about 1 day; (c) then removing the second culture medium and culturing in a third culture medium as specified herein for about 1 day; and (d) then removing the third culture medium and culturing in a fourth culture medium as specified herein for about 1 day.

[0065] In some embodiments, the first culture medium described herein comprises DMEM culture medium. In some embodiments, the second, third, and fourth culture media described herein comprise neuronal basal medium and DMEM / F12 culture medium, supplemented with N2 supplement and B27 supplement without vitamin A.

[0066] In some embodiments, the first culture medium described herein comprises a DMEM culture medium containing 10% FBS. In some embodiments, the second, third, and fourth culture media described herein comprise a neuronal basal medium and a DMEM / F12 culture medium supplemented with an N2 supplement, a vitamin A-free B27 supplement, and non-essential amino acids (NEAAs).

[0067] In some embodiments, the first culture medium described herein comprises a DMEM culture medium containing 10% FBS. In some embodiments, the second, third, and fourth culture media described herein comprise a neuronal basal medium containing BSA, and a DMEM / F12 culture medium supplemented with an N2 supplement, a vitamin A-free B27 supplement, non-essential amino acids (NEAAs), and glutamax.

[0068] In some embodiments, when culturing ocular fibroblasts in the method of the present invention, the method comprises the following steps: (a) culturing in a first culture medium (DMEM culture medium supplemented with 10% FBS containing RG108) for about 1 day; (b) then removing the first culture medium and culturing in a second culture medium (DMEM / F12 culture medium containing N2 supplement, B27 supplement without vitamin A, RG108 and VPA containing NEAA) for about 1 day; (c) then removing the second culture medium (d) The third culture medium is removed and the culture is incubated for about 1 day in a third culture medium (DMEM / F12 culture medium containing N2 supplement, B27 supplement without vitamin A, SU9516, FSK, Y27632, and NEAA containing ascorbic acid); and (d) the third culture medium is then removed and the culture is incubated for about 1 day in a fourth culture medium (DMEM / F12 culture medium containing N2 supplement, B27 supplement without vitamin A, FSK, Y27632, and NEAA containing ascorbic acid).

[0069] In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, or more of ophthalmic fibroblasts in culture are reprogrammed into induced RPCs. In specific embodiments, approximately 30%, approximately 35%, or approximately 40% or more of ophthalmic fibroblasts in culture are reprogrammed into induced RPCs. In specific embodiments, 25% to 45% of ophthalmic fibroblasts in culture are reprogrammed into induced retinal progenitor cells (RPCs).

[0070] After culturing, induced RPCs or cell populations containing induced RPCs are identified by their characteristics.

[0071] In some embodiments, the induced RPCs described herein have RPC-like characteristics. Specifically, the induced RPCs have an RPC-like morphology, contain dome-shaped cells with bright nuclei, and readily form clusters. More specifically, the produced induced RPCs can express typical RPC markers. Examples of typical RPC markers include, but are not limited to, SOX2 (SRY box transcription factor 2), PAX6 (pair box 6), VSX2 (visual system homeobox 2), NEUROD1 (neuronal differentiation 1), CRX (corn-rod homeobox protein), and RCVRN (recoverin).

[0072] In some embodiments, the induced RPCs described herein have characteristics that differ from primary RPCs. Specifically, induced RPCs express one or more markers, such as SOX2, NESTIN, and protein tyrosine phosphatase receptor type N (PTPRN), compared to primary RPCs.

[0073] In some embodiments, the induced RPC described herein expresses glutamate receptors that function in response to neurotransmitter stimulation by glutamate.

[0074] In some embodiments, the induced RPCs are unselected. The population containing the induced RPCs further includes preneuroepithelium, ocular regions, photoreceptor progenitor cells, retinal ganglion cells, Müller glia, and / or retinal pigment epithelium.

[0075] In one embodiment, cells can be sorted with one or more RPC markers to further enrich the induced RPC. Cell sorting can be achieved by various techniques known in the art. Examples of cell sorting techniques include fluorescence-activated cell sorting (FACS), immunoaffinity column separation or immunomagnetic separation (MACS), or any technique that can obtain enrichment of specific cell types based on physical properties (density) or structural properties (particularly specific antigens).

[0076] 5. Adaptation using Induction RPC The induced RPC described herein is effective in relieving ocular photoreceptor deficiency and is therefore useful for treatment, particularly in the treatment of photoreceptor degenerative diseases in the subject.

[0077] Therapeutic applications of induced RPCs include supplying (or implanting) induced RPCs into the target eye. In some embodiments, the cells may be injected into the subretinal space of the target eye.

[0078] Examples of photoreceptor degenerative diseases include, but are not limited to, retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy (DR), and Stargardt disease.

[0079] Retinitis pigmentosa (RP) is the leading cause of visual impairment in the under-middle-aged population, affecting more than 2 million people worldwide.[3] It is a spectrum of genetic retinal degenerative diseases, exhibiting heterogeneity in genotype and phenotype. Of the more than 200 different genotypes in RP, only patients with the RPE65 mutation can benefit from gene therapy.[4] Otherwise, as photoreceptor cells are lost, most patients experience impaired night vision early in the progression of the disease, followed by loss of central vision and color vision by age 40.[5]

[0080] Age-related macular degeneration (AMD) is the leading cause of visual impairment in older adults, affecting approximately 200 million people worldwide.[6] An important sign of early-stage AMD is the presence of drusen, composed of lipids, proteins, and lipofuscin granules, within the retinal pigment epithelium (RPE).[7] Accumulation of drusen can trigger inflammasome activation, leading to photoreceptor degeneration. Currently, clinically available treatment options include several anti-vascular endothelial growth factor (anti-VEGF) therapies, such as bevacizumab, ranibizumab, and aflibercept. The progression of vision loss associated with neovascularization or defective vessels can be slowed with anti-VEGF. However, once AMD progresses to the later stages, characterized by significant photoreceptor degeneration, there remains no cure, and patients become blind.

[0081] Diabetic retinopathy (DR) is the most common complication of diabetes mellitus (DM), affecting 93 million people worldwide. Diabetic retinopathy (DR) has a multifactorial etiology, including microvascular disease, inflammation, and retinal neurodegeneration. Clinically, the progression of vision loss can be slowed with anti-VEGF drugs, anti-inflammatory drugs, and laser therapy. However, as the disease progresses, end-stage diabetic retinopathy, with its degeneration of key photoreceptors, remains untreatable.

[0082] Stargardt disease (Stargardt macular dystrophy, juvenile macular degeneration, or retinal maculopathy) is estimated to affect approximately 1 in 8,000 to 10,000 people worldwide and is the most common form of hereditary juvenile macular degeneration.[9] This hereditary disorder is characterized by the deposition of lipofuscin-like material in the retinal pigment epithelium (RPE), followed by the loss of photoreceptor cells in the macula.

[10] Gene mutations are most commonly found in the Abca4 allele (also known as Abcr).

[11] To date, there are no clinically available therapies to slow this progressive degeneration, and patients experience significant vision loss in childhood or adolescence.

[0083] In some embodiments, the induced RPC described herein is effective in improving the a-wave and b-wave night vision of the eye. In some embodiments, the induced RPC described herein is effective in improving color vision and central visual acuity.

[0084] According to the present invention, the induced RPC described herein can be used as an active ingredient to treat target diseases requiring it. In some embodiments, an appropriate amount of the therapeutic active ingredient can be prepared with a pharmaceutically acceptable carrier in a formulation suitable for administration and absorption. The formulations of the present invention preferably contain an active ingredient by weight ratio ranging from about 0.1% to about 100%, depending on the method of administration. This weight ratio is calculated based on the weight of the entire formulation. Its composition can be used directly as an implant or further modified into a form suitable for implantation.

[0085] As used herein, “pharmaceutically acceptable” means that the carrier is compatible with the active ingredient in the composition, preferably stabilizes the active ingredient, and is safe for the individual being treated. Examples of pharmaceutically acceptable carriers include conventional buffers (such as phosphoric acid, citrate, and other organic acids), physiological saline, sterile water, antioxidants (such as ascorbic acid), isotonic agents, and preservatives.

[0086] In some embodiments, the compositions according to the present invention are prepared in a dosage form suitable for injection, and the cells are suspended in a pharmaceutically acceptable carrier, such as sterile water or saline, or frozen for storage before use. In some embodiments, the compositions may further contain a biodegradable polymer, which is useful for stabilizing, supporting, and fixing cell clusters after topical injection into the defect site. The compositions of the present invention may be prepared as a single-dose form or incorporated into a container for multiple doses. The dosage form may be a suspension, a solution, or an emulsion in an oily or aqueous culture medium, or a powder, granules, tablet, or capsule. The compositions of the present invention may be administered via a physiologically acceptable route, usually by injection.

[0087] The present invention is further illustrated by the following embodiments, which are provided for demonstrative purposes rather than limitation. In light of the description herein, those skilled in the art should understand that many modifications are possible in the particular embodiments disclosed, and that similar or comparable results can be obtained without departing from the spirit and scope of the invention. [Examples]

[0088] Examples In this invention, the inventors developed a method to directly reprogram human fibroblasts into RPC-like cells using small molecules, aiming to overcome current bottlenecks in developmental medicine. Preliminary experiments by the inventors, using qRT-PCR, immunofluorescence assays, and Western blotting, showed that the induced cells expressed classical markers of RPC. Bulk RNA-seq analysis compared the transcriptomes of human fibroblasts and induced RPC-like cells, revealing that the induced RPC-like cells showed increased expression of extracellular matrix components, axons, dendrites, synapses and postsynaptic membranes, and transcripts related to transport vesicle formation. Co-culture assays confirmed that the induced cells could integrate with the host rat retina in vivo. Calcium imaging confirmed increased intracellular calcium concentration in the induced cells in response to glutamate stimulation, thus confirming the function of these cells. Finally, the therapeutic effect in an animal model of photoreceptor degeneration was investigated. The induced RPC-like cells were able to restore visual function in transplanted rats without causing serious side effects.

[0089] 1. Materials and Methods 1.1 HTF Isolation and Primary Culture The remaining samples were obtained anonymously from tissue explants taken during vitrectomy or strabismus surgery from patients who visited Hualien Tzu Chi Hospital, and were approved by the ethics committees of Hualien Tzu Chi Hospital and Academia Sinica. The inventors thoroughly reviewed the clinical records of the donors and excluded patients with ocular surface diseases and infections, systemic conditions such as diabetes, etc. During surgery, small samples of approximately 0.5 ml of vitreous humor or 1 x 1 mm of Tenon's capsule were excised. The tissue was immediately transferred to cell culture flasks and maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), with 1% penicillin / streptomycin added during the first week of culture. Approximately one week after surgery, only cell fragments were observed from the vitreous samples, while abundant spindle-shaped or polygonal flat cells were obtained from the Tenon's capsule samples. Their distinctive morphology and expression of the fibroblast marker S100 calcium-binding protein A4 (S100A4) confirmed that these cells were human capsule of Tenon fibroblasts (HTFs). HTFs from passages 3 to 10 were used in subsequent experiments.

[0090] 1.2 Preparation of Vsx2::eGFP promoter reporter cells To prepare the Vsx2::eGFP promoter reporter, promoter fragments were excised from the commercially available vector pLKO_AS7 w.eGFP.puro (Academia Sinica RNAi core) using restriction enzymes. These Vsx2 promoter sequences were cloned into the original vector. Positive clones were confirmed by Sanger sequencing. The final product was used to prepare lentiviruses. HTF was transduced with lentivirus for 3 days.

[0091] 1.3 Preparation of CiRPC All small molecules were diluted in water or DMSO according to the manufacturer's instructions. Approximately 1,000,000 HTF cells (passage <10) were seeded in 10 cm dishes (40 × Matrigel coated). On day 1, the HTF culture medium was replaced with HTF culture medium (10% FBS and DMEM) containing RG108 (20 μM). On day 3, the dishes were replaced with fresh CiRPC culture medium (50% neuronal matrix with 100 × BSA, 50% DMEM / F12 / Glutamax, 1 × N2, 1 × B27 without vitamin A, 0.1 mM non-essential amino acids) containing RG108 (20 μM) and VPA (3 mM). Subsequently, on day 4, the culture medium was replaced with CiRPC culture medium containing SU9516 (10 μM), forskolin (10 μM), Y-27632 (10 μM), and vitamin C (10 μM), and on day 5, it was replaced with CiRPC culture medium containing forskolin (10 μM), Y-27632 (10 μM), and vitamin C (10 μM). On day 6, GFP+ cells were collected for further analysis and experimentation. On day 6, the cell population was confirmed to contain dome-shaped cells with bright nuclei, which were defined as induced retinal progenitor cells (iRPCs) or chemically induced retinal progenitor cells (CiRPCs). Unselected cell populations included cells with stochastic expression of genes related to preneuroepithelium, ocular field, photoreceptor progenitor cells, retinal ganglion cells, Müller glia, and retinal pigment epithelium, and such unselected cell populations are referred to as induced retinal system-like cells (iRLCs). iRLCs can be further classified to provide iRPCs with a richer cell population. A 5-day reprogramming protocol is shown in Figure 2.

[0092] 1.4 Fluorescence-Activated Cell Selection (FACS) For FACS, CiRPC cells were dispersed in TrypLE Express (Gibco), passed through a 40 μm nylon cell strainer (Fisher Scientific, catalog number 08-777-1), and suspended in PBS containing 1% bovine serum. Initial HTF cells were used as a negative control. Subsequently, cells were fractionated using a Beckton-Dickinson FACS Aria IIu flow cytometer at the core facility. The fractionated cells were collected in CiRPC medium, centrifuged, and subjected to RNA extraction and other downstream processing.

[0093] 1.5 Quantitative PCR with reverse transcription RNA was isolated from cells using the RNeasy Micro Kit (Qiagen, Germantown, MD, USA) and reverse transcribed (100 ng). RT-PCR analysis was performed using the KAPA SYBR FAST qPCR kit (Kapa Biosystems, Wilmington, MA, USA), with succinate dehydrogenase complex subunit A (Sdha), a housekeeping gene, used as the endogenous reference.

[0094] 1.6 Western blot Cells were recovered by scraping using lysis buffer (1% NP40, 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4) and a protease inhibitor cocktail (Sigma, Burlington, MA, USA). Protein quantification was performed using Bio-Rad's protein assay (Bio-Rad, Hercules, CA, United States). The sample was boiled at 100°C for 15 minutes, 30 μg of protein was loaded onto a 10% acrylamide gel, and electrophoresis was started at 90 V for 15 minutes, then switched to 120 V and run for 1 hour. A 0.45 μm nitrocellulose membrane with Amersham Protran was used for blotting. The blot was blocked by shaking in phosphate-buffered saline (PBST) containing 5% BSA (Sigma, Burlington, MA, USA) for 30 minutes. The primary antibody (VSX2, Novus Biologicals; NBP184476) was diluted in 5% BSA in PBST and incubated overnight at 4°C on a shaker. The secondary antibody was diluted in 5% BSA in PBST and cultured on a shaker at room temperature for 1 hour. Protein signals were detected using the UVP BioSpectrumAC system (Jena, Thuringia, Germany).

[0095] 1.7 Bulk RNA Sequencing Total RNA samples were submitted to the Genomics commercial sequencing facility for bioanalyzer quality control analysis and Illumina next-generation sequencing. All submitted samples had an RNA integrity index (RIN) greater than 8. Stranded TruSeq cDNA libraries, enriched with polydT, were prepared using total RNA from each sample according to the manufacturer's protocol. The cDNA sample libraries were sequenced using the Illumina HiSeq sequencing platform, yielding 24.8 to 32 million 150 bp paired-end (PE) sequence reads per sample. The PE FASTQ files returned from Genomics were analyzed using a customized bioinformatics workflow.

[0096] 1.8 Calcium imaging of glutamate response Intracellular calcium dynamics in response to glutamate stimulation were analyzed using HTF and CiRPC. Cells were seeded at low density, allowing for high-resolution imaging of individual cells and recording of changes in Fura-2 fluorescence, an indicator of intracellular calcium influx. Cells were washed with Ringer's solution containing NaCl 119 mM, KCl 4.16 mM, CaCl 22.5 mM, MgCl 20.3 mM, MgSO 40.4 mM, Na2HPO 40.5 mM, NaH2PO 40.45 mM, HEPES 20 mM, and glucose 19 mM (pH 7.4), and then incubated in Ringer's solution containing 0.5 μM Fura-2 tetraacetoxymethyl ester at 22°C for 40 minutes. Fura-2 was controlled by a system combining commercially available software (InCytIM-2; Intracellular Imaging Corp. Cincinnati, OH) and a phase-contrast microscope (Eclipse T5100; Nikon), and was alternately excited with 340 nm and 380 nm light using a filter changer. 2+As an indicator of concentration, a new colorimetric image (340 / 380) was acquired every 0.35 seconds. Subsequently, HTF and CiRPC were stimulated with 1 mM L-glutamate (Sigma-Aldrich), and the increase in cytoplasmic flara fluorescence intensity was analyzed.

[0097] 1.9 Subretinal transplantation in RCS rats Royal College of Surgeons (RCS) rats were provided by the Laboratory Animal Center of Tzu Chi University. RCS rats are an established model of retinal degeneration due to retinal pigment epithelial dysfunction. All animal experiments were approved by the institutional review board of Hualien Tzu Chi Hospital. Regardless of sex, the animals were housed in a specific pathogen-free room at the Animal Care Center and maintained under a 12-hour light-dark cycle. Animals were randomly assigned to experiments. At least four individuals were used in each experiment. All P21 rats were anesthetized by intramuscular injection of a mixture of ketamine (40 mg / kg) and xylazine (4 mg / kg) (Sigma), followed by local administration of 0.5% alkaine eye drops (Alcon). For post-transplant optical coherence tomography (OCT) examination, the pupils were dilated using a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride eye drops (Mydrin-p). After scleral exposure, a pilot hole was created tangentially using a 31-gauge sterile needle, and the injection needle was guided into the hole to inject 5 μL of cell suspension. The needle was then slowly withdrawn, and light pressure was applied to induce self-closing wounds. Subsequently, one drop of Tobladex solution (Alcon) was instilled into the eyeball. The rats were then returned to their recovery cages, and signs of bleeding were observed. The rats were given a normal diet along with oral administration of cyclosporine (200 mg / 1L) (Neoral 100; Novartis).

[0098] 1.10 Electroretinography To examine retinal function and compare the transplanted eye with the untransplanted eye, full-field electroretinograms (ERGs) were recorded in both dark-adapted and light-adapted states. All rats were dark-adapted for at least 24 hours before being anesthetized according to the method described above. After pupil dilation, the rats were placed in a Ganzfeld ColorDome controlled by the Espion Visual Electrophysiology System (Diagnosys). ERGs were recorded for each eye using contact lens electrodes directly attached to the corneal surface with 2% methocell (OmniVision). A reference electrode was placed subcutaneously in the mid-scalp, and a ground electrode was placed proximal to the tail skin. The rats were placed on a warming pad. Impedance levels were checked periodically to maintain an acceptable green indicator (i.e., the electrodes were properly attached). Dark adaptation (Scotopic) stimulation was 0.01 cd sec / m 2 The intensity was set, and the standard waveform was created by averaging the response of 5 trials. In the photopic measurement, first, 20 cd sec / m 2 After irradiating with light at this intensity for more than 10 minutes to saturate the rod reaction, then apply 3.0 cd sec / m². 2 Photopic stimuli were set. Responses from 10 trials were averaged to create a standard waveform. Finally, the mean values ​​of the b-waves in the dark-adapted and light-adapted ERGs were compared between the control group and the transplanted group.

[0099] 1.11 Light-Dark Box Test The Visual Discrimination Light-Dark Box (LDB) test was conducted using an apparatus consisting of a black opaque (100%) acrylic test chamber (30.48 × 15.24 × 30.48 cm (length, width, height)). This chamber was further divided into equal-sized compartments (15.24 × 15.24 × 30.48 cm) by adding an insert to create a partition wall in the center. Furthermore, to create light and dark zones, one compartment was illuminated with dim light (50 ± 1.5 lux), while the other compartment was kept dark (approximately 0.1 lux). The bright and dark compartments were connected by an opening (5 × 5 cm). The position of the rats within the apparatus was recorded using a camera. The acrylic chamber was placed individually in a soundproofed space. The ambient noise level inside the chamber was 64 dB, and the test was conducted under dim light. The rats were housed in a cage in the test room overnight (approximately 12 hours) under dark conditions, with free access to food and water. Each rat was allowed to acclimate to the test apparatus (both sides) in a dark room for 10 minutes. After acclimatization, one side of the apparatus was illuminated with ambient light of approximately 50 lux, and the rats were allowed to move freely between the two compartments for 5 minutes. The time spent in the dark and bright compartments was recorded by a camera-based system.

[0100] 1.12 Histological examination Rats were euthanized by carbon dioxide inhalation. Their eyeballs were removed and fixed with 4% paraformaldehyde at 4°C for 48 hours. Subsequently, the eyeballs were treated overnight at 4°C with gradient sucrose concentrations of 10%, 20%, and 30%. The eyeballs were then embedded in an OCT compound (Tissue-Tek), cut into 20 μm sections using a cryostat microtome (Leica CM-3050-S), and collected on glass slides. These slides were used for immunostaining and immunofluorescence analysis.

[0101] 1.13 Immunofluorescence The cells were fixed with 4% formaldehyde at room temperature for 15 minutes and washed once with 1× phosphate-buffered saline (PBS). The cells were permeabilized with 0.3% Triton X-100 for 5 minutes, washed twice with 1× PBS, and then blocked with 2% bovine serum albumin (BSA) in PBS for 30 minutes. Next, we have the neural stem cell (NSC) marker anti-SOX2 (GeneTex GTX101507, 1:200), the anterior neuroepithelial marker anti-OTX2 (R&D MAB1979, 1:50), the ocular region marker anti-LHX2 (Santa Cruz Biotechnology SC19344, 1:50), the retinal progenitor cell (RPC) marker anti-VSX2 (Novus Biologicals NBP184476, 1:1000), the photoreceptor progenitor cell marker anti-RCVRN (Millipore AB5585, 1:1000), the retinal ganglion cell (RGC) marker anti-HUD (Invitrogen A21271, 1:100), the Müller glial cell marker anti-GFAP (Proteintech 16825-1-AP, 1:200), and the retinal pigment epithelium (RPE) marker anti-BEST1 (Abcam). ab14927 (1:100) was incubated overnight at 4°C in blocking buffer (2% BSA in PBS). After washing the cells twice with 1×PBS, CF555-labeled goat anti-mouse secondary antibody (Life Technologies, Carlsbad, CA, USA), CF555 goat anti-rabbit secondary antibody (Life Technologies, Carlsbad, CA, USA), or CF555 donkey anti-goat secondary antibody (Life Technologies, Carlsbad, CA, USA) were incubated in blocking buffer for 1 hour at room temperature in the dark. DAPI dihydrochloride (MilliporeSigma D9542, 0.5 μg / ml) was used for nuclear staining. The cells were washed twice with 1×PBS. The fluorescence intensity of each image was analyzed using image analysis software (Image-Pro Plus v.4.5; Media Cybernetics, Rockville, MD, USA).

[0102] 1.14 Intravitreous transplantation in healthy Wistar rats This study used adult male Wistar rats weighing 150-180g to evaluate the safety of HTF and iRLC in co-culture with healthy rat retinas. The rats were purchased from a BioLASCO breeding colony in Taipei, Taiwan. All animal experiments were approved by the Animal Care and Experiment Committee of Buddhist Tzu Chi General Hospital. The rats were given an HTF suspension (5 μL, 1 × 10⁶). 5 Cells / μL) or iRLC suspension (5μL, 1×10⁶) 5 A single intravitreal injection of cells / μL was administered. In both groups, the right eye was designated as the treated eye, and the left eye as the control eye (untreated). Two weeks after intravitreal injection, fundus photography, optical coherence tomography (OCT), and immunohistochemistry (IHC) analysis were performed on HTF-transplanted rats and iRLC-treated rats to evaluate retinal safety and histological changes.

[0103] 1.15 Optical coherence tomography OCT was performed using a contact lens integrated into a Micron IV retinal microscope (Phoenix Research Laboratories). All rats were anesthetized using the method described above, their pupils were dilated, and the corneal surface was protected with 2% methocel (OmniVision). The rats were then placed on a horizontal platform so that light could pass perpendicularly through the cornea, and the fundus was observed using the Micron IV microscope camera. Retinal OCT images were scanned horizontally to observe the transplanted cells, and at least two OCT images were acquired.

[0104] 1.16 Statistical analysis All data are presented as mean ± standard error (SEM). Statistical significance was determined using Student's t-test and one-way analysis of variance (ANOVA) with GraphPad Prism software (GraphPad Software). P-values ​​are indicated in the figures.

[0105] 2.Results 2.1 Primary cultured ophthalmic fibroblasts derived from Tenon's capsule Primary cultures of ocular fibroblasts from Tenon's capsule were performed. This is a procedure that can be performed in a short time of 15 minutes in a clinic without damaging intraocular structures. Characteristic morphology and expression of the fibroblast marker S100 calcium-binding protein A4 (S100A4) were confirmed (data not presented), and these cells were defined as human Tenon's capsule fibroblasts (HTF). Subsequently, HTFs from passages 3 to 10 were used in the following experiments.

[0106] 2.2 Cell Reprogramming Next, we used HTF as the source of cell reprogramming and tested different chemical cocktail compositions. Using a set of six small molecule compounds (6C), we were able to directly reprogram human fibroblasts into RPCs in 5 days. We confirmed stable Vsx2 expression using a Vsx2-GFP reporter system and named these cells chemi-inducible RPCs (CiRPCs). To optimize the chemical composition in the protocol, we sequentially removed each compound and evaluated the changes in cell morphology and Vsx2 expression patterns. We confirmed that the Vsx2 expression pattern was best overall when all six compounds were added. The induced cells were dome-shaped with bright nuclei, readily formed clusters, expressed the cell markers SOX2, PAX6, VSX2, and RCVRN, and were shown to be stably maintained for more than 2 months after subculturing (Figure 1A).

[0107] The inventors also tested other fibroblasts, including human fetal lung fibroblasts (IMR-90), human neonatal foreskin fibroblasts (CRL-2097), human neonatal foreskin fibroblasts (BJ-5ta), and human adult dermal fibroblasts (FB-3652) (Figure 1B). However, the 6C protocol was found to have a specific ability to reprogram HTF to CiRPC regardless of the origin of the HTF from different patients. Interestingly, other fibroblasts after 6C treatment either did not induce structural changes or showed good survival.

[0108] On the other hand, our protocol required only 5 days to reprogram human adult ocular fibroblasts to CiRPC using 6C (Figure 2). Our approach resulted in 42.8% Vsx2-GFP+ cells being identified after HTF induced by the CiRPC protocol (Figure 3B).

[0109] The inventors further used each chemical in the 6C protocol individually, removing the remaining five substances from the reprogramming process. Vsx2::eGFP expression analysis using a high-content screening system confirmed successful reprogramming to iRLCs even with single-component administration, but the highest efficiency was obtained when all six substances were administered (Figure 9). The inventors concluded that, regardless of which compound in the 6C protocol is applied, HTF can be induced to iRLCs through the cells' inherent reprogramming ability.

[0110] 2.3 Transcriptional Expression Characteristics To investigate changes in the gene expression of retinal cell markers, increased expression of multiple retinal markers, including photoreceptor-specific genes, was observed in CiRPCs converted from HTF. Increased VSX2 expression was also confirmed by qRT-PCR (Figure 4A) and Western blot (WB) analysis (Figure 4B). This protocol demonstrated that, compared to human fibroblasts, qRT-PCR analysis showed that VSX2, an RPC marker, was upregulated more than 1000-fold in CiRPCs, and that photoreceptor-related genes were also upregulated. Western blot analysis detected VSX2 expression in both unselected and selected CiRPCs, but not in HTF.

[0111] The inventors performed a series of immunofluorescence (IF) stainings to confirm the corresponding protein expression patterns. The selected markers were those with elevated mRNA expression. In contrast to HTF, iRLCs (unselected CiRPCs) on day 6 showed elevated expression of SOX2, OTX2, LHX2, VSX2, RCVRN, HUD, GFAP, and BEST1 upon IF staining, with positive rates ranging from 28.28% (BEST1) to 75.01% (SOX2) (Figure 10). Taken together, these results indicate that iRLCs showed elevated expression not only of RPC-related genes but also of genes associated with anterior neuroepithelium, ocular region, photoreceptor progenitor cells, RGCs, Müller glia, and RPE. This suggests that iRLCs may be a mixed cell population exhibiting stochastic expression of retinal system-related genes, and that they are not primary cells of anterior neuroepithelium, ocular region photoreceptor progenitor cells, RGCs, Müller glia, and RPE, which typically express only specific cell type-specific marker genes during development.

[0112] 2.4 Genome-wide transcriptome profiles The inventors also conducted gene ontology (GO) enrichment analyses to explore the biological functions of transcripts whose expression was elevated / degraded between HTF and CiRPC. GOs whose expression was upregulated were associated with extracellular matrix components, axons, dendrites, synapses and postsynaptic membranes, and transport vesicle formation. Downregulated GOs corresponded to cell division and fibrillation. These significant changes in GO enrichment analyses suggest a major shift in biological function from fibroblasts to neurons after reprogramming from HTF to CiRPC (Figures 5A and 5B).

[0113] 2.5 Functional analysis of induced cells 2.5.1 Efficacy in In Vitro Most of the inner retinal neurons have glutamate receptors, but studies have shown that neither RPCs nor retinal neurons show a response with calcium influx to transmission stimulation by glutamate. High-resolution imaging of CiRPCs and recording of the fluorescence change of Fluo-2, an indicator of intracellular calcium influx, showed that both unsorted and sorted CiRPCs induced by 6C showed significant calcium influx in response to 1 mM glutamate stimulation, while HTFs did not respond to glutamate stimulation (Figures 6A and 6B). These results suggest that unsorted and sorted CiRPCs induced by 6C exhibit in vitro functional characteristics similar to those of primary RPCs. Furthermore, VPA-induced iRPCs showed significant calcium influx (Figure 6C).

[0114] 2. 5.2 Efficacy in vivo In the method of the present inventors, CiRPCs derived from HTFs were transplanted into the subretinal space of RCS rats on the 21st day (P21). In the eyes transplanted with CiRPCs, improvement of the scotopic b-wave was observed at P28 and P56. In the light-dark box test (LDB), RCS rats administered with CiRPCs had a significantly longer residence time in the dark space, suggesting a partial recovery of visual function based on the instinctive tendency of rats to avoid bright spaces (Figures 7A and 7B).

[0115] In H&E staining in histological studies, significant fibrosis was observed around the area injected with HTF, but not in the control groups injected with CiRPCs or PBS. No cell infiltration into the extraocular space was observed. Furthermore, in immunohistochemistry (IHC), HuNu + cells labeled as RCVRN-positive were observed in the photoreceptor layer, indicating that human cells survived and integrated in the rat photoreceptor layer 3 months after subretinal transplantation (Figure 8).

[0116] 2.5.3 In vivo co-culture assay To more deeply understand the safety of induced cells against the normal retina, HTFs or unsorted iRLCs induced by 6C were injected into the vitreous cavity of Wistar rats at 1×10 5In vivo co-culture assays were performed, in which individual cells were transplanted and the migration of transplanted cells was tracked by optical coherence tomography (OCT) once a week. One week after transplantation, significant aggregation of transplanted cells on the retinal surface was observed in the iRLC group, but not in the HTF group. Two weeks after transplantation, most of the accumulated iRLCs disappeared within the vitreous humor, leaving no cell residue. In contrast, the HTF group showed marked fibrotic changes on the retinal surface (Figure 11). Subsequently, rat eyeballs were harvested and cryopreserved for H&E staining and immunofluorescence staining. H&E staining revealed a prominent fibrotic membrane on the retinal surface of the HTF-transplanted group, with fibrotic cell aggregates adhering to the epiretinal membrane (Figure 12A). Immunostaining against human nuclei confirmed that the epiretinal membrane and cell aggregates were composed of HTF cells, surrounded by rat retinal cells (Figure 12A). In contrast, the morphology of the vitreous humor and retina was normal in rat eyeballs transplanted with iRLCs. A small number of iRLCs with a characteristic dome shape and a distinct nucleus were identified within the vitreous humor and retina. Immunostaining revealed that multiple iRLCs were integrated into the inner nucleus layer (INL) of the rat retina. The iRLCs within the INL also co-expressed the photoreceptor marker RCVRN (Figure 12B).

[0117] 2.5.4 In vivo functional analysis in animal models of photoreceptor degeneration To evaluate the in vivo functional characteristics of induced cells, the inventors investigated the therapeutic effect using Royal College of Surgeons (RCS) rats. RCS rats are an animal model of human retinitis pigmentosa, and RPE cells in RCS rats are unable to phagocytose detached photoreceptor epithelial cells, leading to the subsequent death of the photoreceptor cells. This has been widely used in previous studies on hRPC. The inventors used 21-day-old (P21) RCS rats and established four groups for comparison: PBS group, HTF control group, iRLC group (6C induction), and iRPC group (6C induction). After successful subretinal injection in P21 rats, OCT examination confirmed that the retinal structure of 3-week-old rats was preserved. Post-transplant OCT confirmed that the cells were normally injected into the subretinal space and that the retina at the injection site was undamaged (Figure 13). In ERG measurements of dark adaptation response across the four groups, statistically significant improvements were observed in the eyes injected with iRPC at P56 and in the eyes injected with iRLC at P112 (Figure 14). In the LDB test, while RCS rats showed severe visual degeneration at P112, the HTF, iRLC, and iRPC transplantation groups tended to show prolonged stay in dark areas, suggesting visual function recovery through cell replacement therapy (Figure 15). Finally, histological studies revealed significant fibrosis around the areas injected with HTF, but not with iRLC or iRPC. Furthermore, in IHC, RCVRN was found in the photoreceptor layer in all groups except the PBS group. + HuNu labeled as + Cells were observed, demonstrating that human cells survived and integrated in the rat photoreceptor layer 3 months after subretinal transplantation (Figure 16). Overall, in eyes transplanted with iRLC or iRPC, layer-by-layer retinal morphology was maintained compared to eyes administered with PBS or HTF, suggesting the preservation of photoreceptors through neurotrophic mechanisms of induced cells (Figure 16).

[0118] 3. Summary In our study, CiRPCs were obtained 5 days after induction, and the reprogramming efficiency, as defined by the reporter system, was 42.8%. Furthermore, CiRPCs induced using our chemical protocol exhibited a dome-shaped morphology with bright nuclei and showed a tendency to cluster. These cells expressed various retinal cell markers, including SOX2, NESTIN, VSX2, and ??, and maintained stability for more than 2 months after passage. To investigate the biological functions of transcripts with increased and decreased expression between parental cells and CiRPCs, Gene Ontology (GO) enrichment analysis was performed. Upregulated GO was associated with extracellular matrix components, axons, dendrites, synapses and postsynaptic membranes, and transport vesicle formation. On the other hand, downregulated GO was associated with cell division and fibrosis. These significant changes indicate that CiRPCs dramatically transitioned from fibroblasts to neurons after reprogramming.

[0119] High-resolution imaging analysis of CiRPCs and recording of Fura-2 fluorescence changes confirmed that CiRPCs exhibit significant calcium influx in the presence of glutamate. The behavior of CiRPCs mimics the majority of inner retinal neurons that possess glutamate receptors. In animal models, dark adaptation b-wave improved in eyes implanted with CiRPCs in P28 and P56. Furthermore, in light-dark box (LDB) tests, rats injected with CiRPCs spent significantly longer in dark areas, suggesting partial visual function recovery due to rats' natural tendency to avoid bright spaces. Immunohistochemical examination showed that CiRPCs were viable and integrated into the rat photoreceptor layer three months after subretinal implantation.

[0120] 4. Discussion 4.1 Progress in cell replacement therapy for photoreceptor degeneration 4.1.1 Replacement of RPE or photoreceptor Retinitis pigmentosa (RP), diabetic retinopathy (DR), age-related macular degeneration (AMD), and Stargardt disease differ in their etiology and prevalence, but all of these retinal diseases cause photoreceptor apoptosis in their late stages. Further classification of the apoptotic cell types involved in these diseases reveals that in the heterogeneous phenotypes of RP and DR, most patients suffer primarily from the loss of photoreceptor cells. AMD and Stargardt disease cause RPE dysfunction and degeneration. When RPE cells are unable to maintain phagocytosis of the photoreceptor outer segment and lose the integrity of the blood-retinal barrier, the disease progresses, and after reaching a high stage, the loss of photoreceptor cells continues. In preclinical and clinical trials, transplantation of RPE cells from various sources in AMD patients has been thoroughly investigated [12-14]. Because RPE cells are not photosensitive, patients' vision after transplantation is usually only stabilized or slightly improved, and long-term efficacy has not been established [15-17]. On the other hand, photoreceptor replacement therapy is the ultimate solution for rescuing and restoring degenerative visual function in patients with retinitis pigmentosa (RP), diabetic retinopathy (DR), age-related macular degeneration (AMD), and late-stage Stargardt disease. It targets photosensitive cells in the retina, including rod and cone cells, and is expected that the transplanted cells will generate an electrical response to light stimulation. However, the number of suitable cell sources is limited, and consistent clinical implementation remains a challenge.

[0121] 4.1.2 Sources of photoreceptor substitution The present invention's approach to replacing deficient photoreceptors can be classified into three methods, using either RPCs (photoreceptor progenitor cells), young post-mitotic photoreceptors (photoreceptor progenitor cells), or three-dimensional retinal tissue. Preclinical studies and clinical trials, particularly those involving RPCs, have shown promising results. Currently, there are 19 published preclinical studies on human RPC therapy for photoreceptor degeneration [18-36]. These preclinical studies have used RPCs derived from fetal retina at 11-20 weeks of gestation, differentiated from pluripotent stem cells (embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells)), or differentiated from pluripotent stem cells (mesenchymal stem cells (MSCs)). Regarding clinical trials, ReNeuron and jCyte have completed Phase II clinical trials using fetal retina-derived RPCs in patients with retinitis pigmentosa (clinicalTrials.gov: NCT02464436; NCT03073733). The transplanted cells were effective in restoring patients' vision for at least one year in both groups [37, 38]. However, ReNeuron discontinued further trials due to concerns about surgical complications. Meanwhile, jCyte announced that visual function recovery depends on the central visual field at baseline and is aiming to move to a definitive trial in the United States.

[0122] Regarding young postmittal photoreceptors (photoreceptor progenitor cells), current evidence is limited to preclinical data only. Previous studies using mice have shown that both immature and mature photoreceptors can be integrated into the retinas of wild-type or diseased mice, but transplantation of mature photoreceptors has shown a significantly higher failure rate due to poor viability during isolation and separation protocols

[39] . Thus, researchers have concluded that the ideal cell type for photoreceptor transplantation is young postmittal photoreceptors (photoreceptor progenitor cells)

[40] . However, human photoreceptor progenitor cells have only been identified in the second trimester, and access to primary human photoreceptor progenitor cells has been limited due to legal and ethical concerns. Instead, human ES cell / iPS cell-derived photoreceptor progenitor cells have been considered in most studies, but the overall efficiency of differentiation protocols to generate photoreceptor progenitor cells has been relatively low, taking more than 100 days and less than 20% of the cells

[41] . Despite the aforementioned challenges, similar results have been shown to those obtained using hRPCs, suggesting that ES cell / iPS cell-derived photoreceptor progenitor cells integrate into the host retina, transfer cytoplasmic material, differentiate into more mature photoreceptors, and restore some degree of visual function [42-44]. In addition to differentiated photoreceptor progenitor cells derived from ES cell / iPS cells, Mahato et al. at the North Texas Eye Institute reported that mouse / human fibroblasts could be converted into rod photoreceptor progenitor cells by administering five small molecule compounds in addition to differentiated photoreceptor progenitor cells derived from ES cell / iPS cells

[45] . In their study, when photoreceptor progenitor cells (CiPCs) chemically induced from mouse fibroblasts were transplanted into the subretinal space of rod photoreceptor-degenerated mice, some recovery of visual function was observed. On the other hand, the therapeutic effect of human CiPCs in animal models at the time of publication had not been verified.

[0123] Regarding three-dimensional retinal tissue, in early 2009, Li et al. reported that transplantation of human fetal (12-24 week) neuroretinal and retinal pigment epithelium (RPE) sheets into photo-induced retinal degeneration miniature pigs resulted in functional improvement in 15 out of 25 eyes, with no graft rejection occurring during a 12-month follow-up period

[46] . With advances in organoid culture systems, researchers have begun to focus on transplanting ES cell / iPS cell-derived 3D retinal sheets. Several studies have shown that mouse and human ES cell / iPS cell-derived 3D retinal sheets can survive for up to 6 months after transplantation into retinal degeneration animal models and can differentiate into mature retinal cells including photoreceptors, bipolar cells, and ganglion cells. However, ESC / iPSC-derived 3D retinal grafts were found to be unable to maintain proper layering structure after long-term follow-up, and the grafts degenerated, forming many disordered rosettes within the host retina [47-49]. The long-term survival and structural maintenance of ESC / iPSC-derived 3D retinal tissue need to be improved in the future.

[0124] 4.2 Direct reprogramming by compounds Both the reprogramming and differentiation processes have raised potential safety concerns in the clinical application of iPS cells. Exogenous gene induction during reprogramming can lead to genomic instability and mutations

[50] . Furthermore, the retention of undifferentiated cells after differentiation protocols increases the risk of undesirable tumor formation after transplantation

[51] . In recent years, significant progress has been made in the field of direct lineage reprogramming using only chemicals

[52] . Cell fate reversal is achieved by regulating the activity of cell signaling pathways and histone / DNA modifying enzymes without the use of transgenes. Small molecule compounds enhance the efficiency of transcription factor-based direct reprogramming and can sometimes substitute for the effects of transcription factors and cytokines, which is clearly useful in cost-effectively preparing large quantities of cells in a defined manner [53, 54]. Notable advantages of the compounds include high storage capacity, high purity, long half-life, non-immunogenicity, and efficacy at low concentrations.

[0125] 4.3 Chemoinducible photoreceptor progenitor cells (CiPCs) Mahato et al. at the North Texas Eye Institute published their method in the journal Nature

[45] . They reported that using an Nrl-GFP reporter system, mouse / human fibroblasts could be converted into rod photoreceptor progenitor cells by administering five small molecules. These chemoinducible photoreceptor progenitor cells (CiPCs) showed partial recovery of visual function after transplantation into the subretinal space of rod photoreceptor-degenerated mice.

[0126] In our project, we developed six small molecule compounds for directly reprogramming human fibroblasts into RPCs using the Vsx2-eGFP reporter system. These chemically induced RPCs (iRPCs) showed partial recovery of visual function after being implanted in the subretinal space of photoreceptor-degenerated rats.

[0127] Firstly, to compare the time required for reprogramming protocols, the protocol by Mahato et al. required 10 days to convert human adult dermal fibroblasts (HADF) into CiPCs. Our protocol, on the other hand, required only 5 days to reprogram adult human ocular fibroblasts (HTF) into iRPCs.

[0128] Secondly, comparing the conversion efficiencies of the reprogramming protocols, Nrl-GFP+ cells treated with the CiPC protocol had a conversion rate of 24.9%. On the other hand, Vsx2-eGFP+ cells induced with our proprietary iRPC protocol had a conversion rate of 42.8%.

[0129] Thirdly, when comparing the changes in retinal marker gene expression, CiPC reprogrammed from HADF showed increased expression of photoreceptor-specific genes. On the other hand, iRPC converted from HTF showed increased expression of multiple retinal marker genes, including photoreceptor-specific genes.

[0130] Fourthly, to compare the therapeutic effects in photoreceptor degeneration animal models, mouse CiPCs were implanted into the subretinal space of rd1 mice at 31 days postnatal (P31). Electroretinography (ERG) analysis showed improvement in dark adaptation a-wave in eyes implanted at P45, but the improvement decreased from P59 onward. In our approach, human iRPCs were implanted into the subretinal space of RCS rats at 21 days postnatal (P21). Improvement in dark adaptation b-wave was observed in eyes implanted with either unselected iRLCs or FACS-selected iRPCs. In the light-dark box test (LDB), RCS rats injected with either unselected iRLCs or FACS-selected iRPCs spent significantly longer in the dark room, suggesting partial recovery of visual function, given the rats' innate tendency to avoid lit spaces.

[0131] 5. Conclusion Our research has shown that induced retinal cells can be generated from human ocular fibroblasts using defined small molecule compounds and culture media without the addition of exogenous genetic material. Experimental results showed that iRLCs possess gene expression profiles of multiple retinal cell types. In in vitro functional assays, both unselected iRLCs and FACS-selected iRPCs showed calcium influx upon glutamate stimulation, mimicking the electrophysiological function of primary retinal cells. In co-culture with retinal tissue fragments and healthy rat retinas, iRLCs showed a tendency to migrate to and integrate into endoretinal cells. In photoreceptor transplantation animal models, unselected iRLCs or FACS-selected iRPCs restored ERG and visual function in transplanted rats without causing serious adverse events.

[0132] The inventors expect that their novel approach will overcome current challenges in clinical applications of other cell types, such as ethical issues with fetal RPCs and ES cells, the low proliferative capacity of fetal RPCs, concerns about genetic abnormalities in iPS cells, and the low differentiation efficiency of ES cells and iPS cells, and will provide a new platform for research into cell replacement therapy in retinal degeneration.

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Claims

1. A method for producing induced retinal progenitor cells (induced RPCs), comprising culturing ocular fibroblasts under conditions that enable the reprogramming of a portion of them into induced RPCs, wherein the conditions include a culture medium containing compounds selected from the group consisting of DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors, cyclin-dependent kinase (CDK) inhibitors, cyclic adenosine monophosphate (cAMP) activators, Rho-related protein kinase (ROCK) inhibitors, ascorbic acid, and any combination thereof.

2. A method for producing induced retinal progenitor cells (induced RPCs), comprising culturing ocular fibroblasts under conditions that enable the reprogramming of a portion of them into induced RPCs, wherein the conditions include a culture medium comprising a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HDAC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-related protein kinase (ROCK) inhibitor, and ascorbic acid.

3. The method according to claim 1 or 2, further comprising identifying an induced RPC expressing one or more retinal markers selected from the group consisting of SRY-box transcription factor 2 (SOX2), pairbox 6 (PAX6), visual system homeobox 2 (VSX2), neuron differentiation 1 (NEUROD1), cone-rod homeobox protein (CRX), and recoverin (RCVRN), and any combination thereof, and isolating the identified induced RPC.

4. The method according to any one of claims 1 to 3, wherein the induced RPC expresses a glutamate receptor.

5. The method according to any one of claims 1 to 4, wherein the ocular fibroblasts are fibroblasts derived from Tenon's capsule.

6. The method according to claim 5, wherein the ocular fibroblasts are human fibroblasts derived from Tenon's capsule.

7. The method according to any one of claims 1 to 6, wherein a DNMT inhibitor, an HDAC inhibitor, a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid are added simultaneously or sequentially to the culture medium.

8. The method according to any one of claims 1 to 7, wherein the DNMT inhibitor is RG108, the HDAC inhibitor is VPA, the CDK inhibitor is SU9516, the cAMP activator is forskolin (FSK), and the ROCK inhibitor is Y-27632.

9. The method according to claim 8, wherein RG108 is present at a concentration of 1-100 μM, VPA is present at a concentration of 1-100 mM, SU9516 is present at a concentration of 1-100 μM, FSK is present at a concentration of 1-100 μM, Y-27632 is present at a concentration of 1-100 μM, and ascorbic acid is present at a concentration of 1-100 μM.

10. The method according to claim 9, wherein RG108 is present at a concentration of 1-50 μM, VPA is present at a concentration of 1-10 mM, SU9516 is present at a concentration of 1-50 μM, FSK is present at a concentration of 1-50 μM, Y-27632 is present at a concentration of 1-50 μM, and ascorbic acid is present at a concentration of 1-50 μM.

11. The method according to claim 9, wherein RG108 is present at a concentration of approximately 20 μM, VPA is present at a concentration of approximately 3 mM, SU9516 is present at a concentration of approximately 10 μM, FSK is present at a concentration of approximately 10 μM, Y-27632 is present at a concentration of approximately 10 μM, and ascorbic acid is present at a concentration of approximately 10 μM.

12. (a) A step of culturing ophthalmic fibroblasts in a culture vessel containing a first culture medium containing a DNMT inhibitor; (b) The step of removing the first culture medium and adding a second culture medium containing a DNMT inhibitor and an HDAC inhibitor; (c) The step of removing the second culture medium and adding a third culture medium containing a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid; (d) The third culture medium is removed and a fourth culture medium containing a cAMP activator, a ROCK inhibitor, and ascorbic acid is added. The method according to any one of claims 1 to 11, including the method described in any one of claims 1 to 11.

13. In step (a), the cells are cultured in the first culture medium for 1 to 3 days. In step (b), the cells are cultured in the second culture medium for 1 to 3 days. In step (c), the cells are cultured in a third culture medium for 1 to 3 days. In step (d), the cells are cultured in the fourth culture medium for 1 to 3 days. The method according to claim 12.

14. The method according to claim 12 or 13, wherein the first culture medium comprises DMEM.

15. The method according to claim 12 or 13, wherein the second culture medium, the third culture medium, and the fourth culture medium each contain DMEM / F12 supplemented with N2 and B27 and a neuronal cell basal medium.

16. A method for producing induced retinal progenitor cells (induced RPCs), (a) A step of culturing ophthalmic fibroblasts in a culture vessel containing a first culture medium containing a DNMT inhibitor; (b) The step of removing the first culture medium and adding a second culture medium containing a DNMT inhibitor and an HDAC inhibitor; (c) The step of removing the second culture medium and adding a third culture medium containing a CDK inhibitor, a cAMP activator, a ROCK inhibitor, and ascorbic acid; (d) The third culture medium is removed and a fourth culture medium containing a cAMP activator, a ROCK inhibitor, and ascorbic acid is added; (e) A step of identifying an induced RPC that expresses one or more retinal markers selected from the group consisting of SOX2, PAX6, VSX2, Neurod1, CRX, and RCVRN, and any combination thereof; and, (f) Steps to isolate the identified induced RPC The production method, including the above.

17. The method according to claim 16, wherein the induced RPC expresses a glutamate receptor.

18. The method according to claim 16 or 17, wherein the ocular fibroblasts are fibroblasts derived from Tenon's capsule.

19. The method according to claim 18, wherein the ocular fibroblasts are human fibroblasts derived from Tenon's capsule.

20. The method according to any one of claims 16 to 19, wherein the DNMT inhibitor is RG108, the HDAC inhibitor is VPA, the CDK inhibitor is SU9516, the cAMP activator is forskolin (FSK), the ROCK inhibitor is Y-27632, and the antioxidant is ascorbic acid.

21. The method according to claim 20, wherein RG108 is present at a concentration of 1-100 μM, VPA is present at a concentration of 1-100 mM, SU9516 is present at a concentration of 1-100 μM, FSK is present at a concentration of 1-100 μM, Y-27632 is present at a concentration of 1-100 μM, and ascorbic acid is present at a concentration of 1-100 μM.

22. The method according to claim 21, wherein RG108 is present at a concentration of 1-50 μM, VPA is present at a concentration of 1-10 mM, SU9516 is present at a concentration of 1-50 μM, FSK is present at a concentration of 1-50 μM, Y-27632 is present at a concentration of 1-50 μM, and ascorbic acid is present at a concentration of 1-50 μM.

23. The method according to claim 21, wherein RG108 is present at a concentration of approximately 20 μM, VPA is present at a concentration of approximately 3 mM, SU9516 is present at a concentration of approximately 10 μM, FSK is present at a concentration of approximately 10 μM, Y-27632 is present at a concentration of approximately 10 μM, and ascorbic acid is present at a concentration of approximately 10 μM.

24. The method according to any one of claims 16 to 23, wherein the first culture medium comprises DMEM.

25. The method according to any one of claims 16 to 24, wherein the second culture medium, the third culture medium, and the fourth culture medium each contain DMEM / F12 supplemented with N2 and B27 and a neuronal cell basal medium.

26. In step (a), the cells are cultured in the first culture medium for 1 to 3 days. In step (b), the cells are cultured in the second culture medium for 1 to 3 days. In step (c), the cells are cultured in a third culture medium for 1 to 3 days, and In step (d), the cells are cultured in the fourth culture medium for 1 to 3 days. The method according to any one of claims 16 to 25.

27. Induced retinal progenitor cells (inducible RPCs) produced by any of the methods of claims 1 to 26, or a cell population containing the said induced RPCs.

28. Induced retinal progenitor cells (inducible RPCs) or a cell population containing such induced RPCs that express high levels of SOX2, NESTIN, and protein tyrosine phosphatase receptor type N (PTPRN) compared to primary retinal progenitor cells.

29. A composition comprising an induced RPC according to claim 27 or 28 or a cell population containing the induced RPC, and a pharmaceutically acceptable carrier.

30. A method for treating a photoreceptor degenerative disease, comprising administering to a target eye an effective amount of the induced RPC described in claim 27 or 28, or a cell population containing the induced RPC, or the composition described in claim 29.

31. The method according to claim 30, wherein the amount of induced RPC is effective in saving the subject's color vision and central visual acuity.

32. The method according to claim 30 or 31, wherein the photoreceptor degenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy (DR), and Stargardt disease.

33. Use of the induced RPC or cell population containing the induced RPC described in claim 27 or 28, or the composition described in claim 29, for the purpose of manufacturing a pharmacopoeia for treating photoreceptor degenerative diseases in subjects requiring it.

34. The use according to claim 33, wherein the induced RPC is effective in saving the subject's color vision and central visual acuity.

35. The use according to claim 33 or 34, wherein the photoreceptor degenerative disease is selected from the group consisting of retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy (DR), and Stargardt disease.

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