Method to generate induced retinal progenitor cells and treatment using such cells

Abstract

The present invention generally relates to a method for generating induced retinal progenitor cells (induced RPCs) and treatment using such cells.

Description

TITLE OF THE INVENTIONMETHOD TO GENERATE INDUCED RETINAL PROGENITOR CELLS AND TREATMENT USING SUCH CELLSRELATED APPLICATIONS

[0001] This application claims the benefit of U.S. provisional application number 63 / 505,172, filed May 31, 2023 under 35 U.S.C. §119, the entire content of which is incorporated herein by reference.TECHNOLOGY FIELD

[0002] The present invention generally relates to a method for generating induced retinal progenitor cells (induced RPCs) and treatment using such cells.BACKGROUND OF THE INVENTION

[0003] According to WHO, globally there are 285 million people visually impaired, and 39 million blindfl ] . In a real-world survey, blindness is reported to be the most terrifying disease among all human illnesses[2]. There is more than 10% of blindness is caused by photoreceptor degeneration, which considered untreatable once the degeneration progress reaches the end-stage[l]. Currently, there has been some research focusing on cell replacement therapy in photoreceptor degeneration, but major hurdles prevented these approaches from being clinically viable. For example, primary retinal progenitor cells (RPCs) from fetal retina suffer from ethical issues and low proliferative abilities, embry onic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived retinal lineage cells have relatively low efficiency of differentiation and are high-cost and labor-consuming.

[0004] Based on preclinical studies and clinical trials, RPCs are capable to rescue vision in late-stage photoreceptor degeneration. But the availability of primary' RPCs was restricted to post-abortion fetal tissue.SUMMARY OF THE INVENTION

[0005] In the present invention, it is unexpectedly found that eye fibroblasts are chemically converted to retinal progenitor cells (RPCs) by culturing the eye fibroblasts in the presence of reprogramming agents comprising a DNA methyltransferase (DNMT) inhibitor, a histone deacety lase (HD AC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-associated protein kinase (ROCK) inhibitor and ascorbic acid, alone or in combination. It is demonstrated in the examples that the chemically induced RPCsare functional as primary RPCs and effective in cell replacement therapy in animal models to rescue photoreceptor degenerations.

[0006] In particular, in one aspect, the present invention provides a method of generating induced retinal progenitor cells (induced RPCs), comprising culturing eye fibroblasts in a condition which allows a proportion of the eye fibroblasts to reprogramming into induced RPCs, wherein the condition comprises a culture medium which comprises a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HD AC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-associated protein kinase (ROCK) inhibitor and ascorbic acid. The present invention also provides a method of generating induced retinal progenitor cells (induced RPCs), comprising culturing eye fibroblasts in a condition which allows a proportion of the eye fibroblasts to reprogramming into induced RPCs, wherein the condition comprises a culture medium which comprises a compound selected from the group consisting of a DNMT inhibitor, a HD AC 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 comprises identifying induced RPCs that express one or more retinal markers selected from the group consisting of SOX2 SRY-box transcription factor 2 (SOX2), paired box 6 (PAX6), visual system homeobox 2 (VSX2). neuronal differentiation 1 (NEURODI), cone-rod homeobox protein (CRX) and recoverin (RCVRN) and any combination thereof, and isolating the identified induced RPCs.

[0008] In some embodiments, the induced RPCs express a glutamate receptor.

[0009] In some embodiments, the eye fibroblasts are fibroblasts from Tenon’s capsule.

[0010] In some embodiments, the eye fibroblasts are human fibroblasts from Tenon’s capsule.

[0011] In some embodiments, the DNMT inhibitor, the HD AC inhibitor, the CDK inhibitor, the cAMP activator, the ROCK inhibitor, and the ascorbic acid are simultaneously or successively added to the culture medium.

[0012] In some embodiments, the DNMT inhibitor is RG108, the HD AC inhibitor is VP A, 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 in a concentration of 1-100 pM, VPA is present in the culture medium in a concentration of 1-100 mM, SU9516 is present in the culture medium in a concentration of 1-100 pM. FSK is present in the culture medium in a concentration of 1-100 pM, Y-27632 is present in the culture medium in a concentration of 1-100 pM, and the ascorbic acid is present in the culture medium in a concentration of 1-100 pM.

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

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

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

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

[0018] In some embodiments, the method of the present invention comprising the steps of(a) culturing the eye fibroblasts in a culture vessel containing a first medium comprising the DNMT inhibitor;(b) removing the first medium and adding a second medium comprising the DNMT inhibitor and the HD AC inhibitor;(c) removing the second medium and adding a third medium comprising the CDK inhibitor, the cAMP activator, the ROCK inhibitor and the ascorbic acid; and(d) removing the third medium and adding a fourth medium comprising the cAMP activator, the ROCK inhibitor and the ascorbic acid.

[0019] In certain embodiments, the method of the present invention comprises the steps of:(a) culturing eye fibroblasts in a culture vessel containing a first medium comprising a DNMT inhibitor;(b) removing the first medium and adding a second medium comprising a DNMT inhibitor and a HD AC inhibitor;(c) removing the second medium and adding a third medium comprising a CDK inhibitor, a cAMP activator, a ROCK inhibitor and ascorbic acid;(d) removing the third medium and adding a fourth medium comprising a cAMP activator, a ROCK inhibitor and ascorbic acid;(e) identifying induced RPCs that express one or more retinal markers selected from the group consisting of SOX2, PAX6, VSX2, NEURODI, CRX and RCVRN and any combination thereof;and(f) isolating the identified induced RPCs.

[0020] In some embodiments, the cells are cultured in the first medium for a period of one to three days; the cells are cultured in the second medium for a period of one to three days; the cells are cultured in the third medium for a period of one to three days; and / or the cells are cultured in the fourth medium for a period of one to three days.

[0021] In another aspect, the present invention provides induced RPCs having highly expressed SOX2, NESTIN and protein tyrosine phosphatase receptor type N (PTPRN) compared with primary RPC. The present invention also provided induced RPCs produced by a method as described herein. The present invention further provides a cell population comprising the induced RPCs as described herein.

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

[0023] In a further aspect, the present invention provides a method for treating a photoreceptor degenerative disease in a subject in thereof, comprising delivering to the eye of the subject an effective amount of induced RPCs or a cell population comprising the induced RPCs or a composition thereof as described herein. The present invention also provides use of induced RPCs or a cell population comprising the induced RPCs or a composition thereof as described herein for manufacturing a medicament for treating a photoreceptor degenerative disease.

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

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

[0026] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following detailed description of several embodiments, and also from the appending claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presentlypreferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.In the drawings:

[0028] In the drawings:

[0029] Figs. 1 Ato IB show phase-contrast images from Tenon’s capsule fibroblasts (HTFs) to chemically induced retinal progenitor cells (CiRPCs) during and after the reprogramming protocol. Fig. 1 A shows that the cell morphology7was identical to HTFs during the first 3 days of reprogramming. On day 4. all cells underwent a significant morphological change into dome-shaped with bright nuclei, and maintained the morphology after day 6. After subculture, the cells were prone to form clusters and remained viable for up to 2 months. Fig. IB shows that except for significant morphological change from HTFs, all other fibroblast lines we tested (human fetal lung fibroblasts (IMR-90), human newborn foreskin fibroblasts (CRL-2097), human neonatal foreskin fibroblasts (BJ-5ta), and human adult dermal fibroblasts (FB-3652) failed to change into domeshaped with bright nuclei, primary' RPC-like morphology.

[0030] Fig. 2 show comparison of reprogramming protocol on CiRPCs. 5-day protocol of CiRPCs with 6 small molecules.

[0031] Figs. 3 A to 3B show comparison of conversion efficiency on CiRPC protocols. Fig 3 A shows Vsx2-GFP+ cells 5 days after CiRPC protocol. Fig. 3B shows FACS gating strategy of Vsx2- GFP+ cells.

[0032] Figs. 4A to 4B show comparison of gene expression changes. Fig. 4A shows qRT-PCR analysis of CiRPCs after reprogramming from HTFs by six small molecules (6C). Fig. 4B shows WB analysis of VSX2 expression between HTFs. unsorted and FACS-sorted CiRPCs.

[0033] Figs. 5 A to 5B show GO enrichment analysis of HTFs and CiRPCs. Fig. 5 A shows upregulated GOs, related to extracellular matrix component, axon, dendrite, synaptic and postsynaptic membrane, and transport vesicle. Fig. 5B shows downregulated GOs, related cell mitosis and fibrosis.

[0034] Figs. 6Ato 6C shows in vitro calcium imaging on HTFs and unsorted CiRPCs (iRLCs) and sorted iRPCs. Fig. 6A shows that there were no calcium influxes of HTFs upon stimulation with 1 mM glutamate (glutamate stimulation was given from 200 to 400 seconds), while unsorted CiRPCs induced by 6C showed significant calcium influxes upon stimulation with 1 mM glutamate (glutamate stimulation was given from 200 to 400 seconds). Fig. 6B shows that there were no calcium influxes of HTFs upon stimulation with 1 mM glutamate (glutamate stimulation was given from 100 to 400 seconds, indicated by red square), while iRLCs induced by 6C showed significantcalcium influxes upon stimulation with 1 mM glutamate. The calcium influxes response was further enhanced after in FACS-sorted iRPCs induced by 6C. Fig. 6C shows that iRPCs induced by VPA showed significant calcium influxes upon stimulation with 1 mM glutamate (glutamate stimulation was given from 100 to 400 seconds, indicated by red square). CiRPCs = iRPCs = sorted Vsx2 eGFP+ cells induced by 6C; iRLCs = unsorted cells induced by 6C.

[0035] Figs. 7Ato 7B show comparison of therapeutic efficacy in animal models of photoreceptor degenerations. Fig. 7A shows ERG scotopic b-wave of RCS rat received subretinal transplantation of CiRPCs. Fig. 7B shows LDB test of RCS rat received subretinal transplantation of CiRPCs. CiRPCs = iRPCs = sorted Vsx2 eGFP+ cells induced by 6C; iRLCs = unsorted cells induced by 6C.

[0036] Fig. 8 shows histology of RCS rat eyes 3 months after subretinal transplantation. The rat eye transplanted with HTFs was found to have significant fibrotic membranes on the retinal surface, with a huge cell mass attached. The cell mass and fibrosis were stained with human nuclei. The rat eyes transplanted with PBS and CiRPCs showed no fibrosis around the area of injection. By IHC, HuNu+ cells were found to integrate into rat retina, at the photoreceptor layer labeled with photoreceptor marker RCVRN.

[0037] Fig. 9 shows high-Content Screening System of HTFs into iRLCs. By applying each chemical compound one at a time and evaluated the consequential alteration in Lsx2::eGFP expression on High-Content Screening System, we found the HTFs were able to be induced into iRLCs with any compound in the 6C protocol, while overall Vsx2 expression pattern was most optimal when all 6 compounds were given. The error bars give the standard deviations. The asterisks mark 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 as shown in Fig. 2.

[0038] Fig. 10 shows comparison of protein expression patterns between HTFs and iRLCs by IF staining. Quantification of marker positive rates among iRLCs. The error bars give the standard deviations.

[0039] Fig. 11 shows OCT images of transplanted cells and rat retinas. A: There was no significant aggregation of HTFs to retinal surface on week one, but the significant fibrotic change of retinal surface was observed on week two (note the curvy retinal surface with cell mass indicated by arrows). B: iRLCs migrated to rat retinal surface one week after transplantation (arrows), and most iRLCs became absent in vitreous without leaving cell debris on week two.

[0040] Figs. 12A to 12B show H&E and immunofluorescent images of rat eyes with transplanted HTFs or iRLCs. Fig. 12A: The rat eye transplanted with HTFs was found to have significant fibrotic membranes on retinal surface, with a huge cell mass (arrow) attached to the epiretinal membrane(triangle). The cell mass and epiretinal membrane were stained with human nuclei in center with rat cells surrounding. There was no integration of transplanted cells with rat retina. Fig. 12B: A few iRLCs with distinct dome shape and bright nucleus were found on retinal surface (triangle) and within retina (arrow). By immunostaining, we found several iRLCs integrated into INL of rat retina with co-expression of photoreceptor marker RCVRN (arrow).

[0041] Fig. 13 shows fundus photography and OCT images of rat eyes right after subretinal transplantation. There was no massive hemorrhage induced by the injection. The eGFP-labeled cells were found at the site of subretinal injection. OCT showed successful subretinal bleb formation in treatment eyes.

[0042] Fig. 14 shows bar charts of ERG b-wave amplitudes. The scotopic responses showed statistically significant improvement in eyes injected with iRPCs at P56, and eyes injected with iRLCs in Pl 12.

[0043] Fig. 15 shows bar charts of LDB tests. The time spent in dark zone was randomly distributed in RCS rats transplanted with PBS at Pl 12 (Additional file 4: Video S2). But RCS rats transplanted with HTFs, iRLCs, and iRPCs (Additional file 5: Video S3), tend to spend more time in dark zone, indicating visual function was rescued 3 months after transplantation.

[0044] Fig. 16 shows H&E and immunofl uores cent images of RCS rat eyes 3 months after subretinal transplantation. The rat eye transplanted with HTFs was found to have significant fibrotic membranes on retinal surface, with a huge cell mass attached. The cell mass and fibrosis were stained with human nuclei. The rat eyes transplanted with iRLCs and iRPCs showed no fibrosis around the area of injection. By immunostaining, HuNu+cells were found to integrate into rat retina w ith co-expression of photoreceptor marker RCVRN.DETAILED DESCRIPTION OF THE INVENTION

[0045] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0046] 1. Definitions

[0047] As used herein, the singular forms “a”, "an", and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a component” includes a plurality of such components and equivalents thereof known to those skilled in the art.

[0048] The term “comprise” or “comprising” is generally used in the sense of include / including w hich means permitting the presence of one or more features, ingredients or components. The term“comprise’’ or “comprising” encompasses the term “consists” or “consisting of.”

[0049] As used herein, the term “retinal progenitor cells (RPCs)” can include progenitor cells that have the ability to differentiate into all ty pes of retinal neurons including rod cells and cone cells, and muller glial cells.

[0050] As used herein, the term “induced retinal progenitor cells (iRPCs) refers to RPC-like cells (i.e. cells having RPC -like features) which are generated (or reprogramed) from other cell types, like fibroblasts.

[0051] As used herein, the term “reprogram” refers to a process that converts cells into different cell types with some different properties or biological functions.

[0052] As used herein, the term “small molecule” refers to organic or inorganic molecules either synthesized or found in nature, generally having a molecular weight less than 10,000 grams per mole, particularly less than 5,000 grams per mole, particularly less than 2,000 grams per mole, and particularly less than 1,000 grams per mole. In some embodiments, a small molecule refers to anon- polymeric, e.g. non-protein or nucleic acid based, chemical molecule.

[0053] As used herein, the term “about” means plus or minus 10% of the numerical value of the number with which it is being used. Therefore, about 1% means in the range of 0.9% to 1.1 %.

[0054] As used herein, the term “express” as used herein refers to the realization of genetic information encoded in a gene to produce a gene product such as an unspliced RNA. an mRNA. a splice variant mRNA, a polypeptide or protein, a post-translationaly modified polypeptide, a splice variant polypeptide and so on.

[0055] As used herein, a DNA methyltransferase (DNMT) inhibitor can refer to an agent that downregulates, decreases or suppresses the amount and / or activity of DNA methyltransferase. Examples of DNMT inhibitors include, but are not limited to, RG108, azatidine, decitabine, thioguanine, zebularine, SGI-110, SGI-1027, lomeguatrib, and procainamide hydrochloride. %

[0056] As used herein, a histone deacetylase (HD AC) inhibitor can refer to an agent that downregulates, decreases or suppresses the amount and / or activity of histone deacetylase to remove acetyl groups from lysine residues on histones. Examples of HD AC inhibitors include, but are not limited to, valproic acid (VPA, 2-propylpentanoic acid), Apicidin, CI 994, FK 228, LMK 235, M 344, MC 1568, MC 1742, MI 192, NCH 51, NSC 3852, PCI 34051, Sodium 4-Phenylbuty rate, Pyroxamide, SAHA. SBHA. Scriptaid, Sodium butyrate, TC-H 106, TCS HDAC6 20b, Trichostatin A, Tubacin, UF 010. Mocetinostat, Pracinostat, and others. %

[0057] As used herein, a cyclin-dependent kinase (CDK) inhibitor can refer to an agent that downregulates. decreases or suppresses the amount and / or activity of cyclin-dependent kinase.Examples of CDK inhibitors as described herein include, but are not limited to, SU9516, PD- 0332991, Roscovitine, SNS-032, Dinaciclib, Flavopiridol, AT7519, Flavopiridol, JNJ-7706621, AZD5438, MK-8776, PHA-793887. BS-181, Palbocichb (PD0332991) Isethionate, A-674563, abemaciclib, BMS-265246, PHA-767491. Milciclib, R547, NU6027. P276-00. MSC2530818, Senexin A, LY2857785, LDC4297, ON123300, Kenpaullone, K03861, THZ1 2HCL AT7519 HC1, Purvalanol A, Ro-3306, XL413, LDC000067, ML167, TG003, Ribociclib, Wogonin, BIO, AZD1080, 1 -Azakenpullone, and others.

[0058] As used herein, a cyclic adenosine monophosphate (cAMP) activator can refer to an agent that increases intracellular levels of cAMP as compared to the background physiological intracellular level when the agent is absent. Examples of cAMP activators include, but are not limited to, forskolin, rolipram, NKH477, PACAP1-27, PACAP1-38 and others.

[0059] As used herein, a Rho-associated protein kinase (ROCK) inhibitor can refer to an agent that downregulates, decreases or suppresses the amount and / or activity of Rho-associated protein kinase. Examples of ROCK inhibitors as described herein include, but are not limited to, Y-27632, AS 1892802, GSK 269962, GSK 429286, H 1152 dihydrochloride, HA 1100 hydrochloride, OXA 06 dihydrochloride, RKI 1447 dihydrochloride, SB 772077B dihydrochloride, etc.

[0060] As used herein, the term "an isolated or purified population of cells" or "isolated or purified cells” refer to a preparation of cells that have been separated from other cellular components or other cells with which the cells are associated. For example, an isolated cell may have been removed from its native environment or group of cells, or may result from propagation of a cell that has been removed from a group of cells. When cells are described as “isolated” or “purified,” it should be understood as not absolutely isolated or purified, but relatively isolated or purified. For example, a preparation comprising isolated cells may comprise the cells in an amount of 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the total cell number in the preparation. In some particular embodiments, a preparation comprising isolated cells may comprise the cells in an amount of 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 100% of the total cell number in the preparation.

[0061] As used here, the term “subject” as used herein includes human and non-human animals such as companion animals (such as dogs, cats and the like), farm animals (such as cows, sheep, pigs, horses and the like), or laboratory animals (such as rats. mice, guinea pigs and the like).

[0062] As used herein, the term “treating” when relating to therapeutically treating refers to the application or administration of a composition including one or more active agents to a subjectafflicted with a disorder, a symptom or conditions of the disorder, or a progression of the disorder, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disorder, the symptoms or conditions of the disorder, the disabilities induced by the disorder, or the progression of the disorder.

[0063] As used herein, the term '‘therapeutically effective amount” used herein refers to the amount of an active ingredient to confer a therapeutic effect in a treated subject. The therapeutically effective amount may change depending on various reasons, such as administration route and frequency, body weight and species of the individual receiving said pharmaceutical, and purpose of administration. As used herein, the term “effective amount” when referring to an application or a process irrelevant to therapeutically treating a disease can refer to the amount of an ingredient or agent to be applied to achieve the intended purpose e.g. the amount of an ingredient or agent to be applied to contact cells e.g. fibroblasts for the purpose of reprogramming.

[0064] 2. Generation of induced RPCs

[0065] The aim of cell replacement therapy is to replace the lost or damaged photoreceptor cells with healthy ones derived from stem cells or fetal origin. Several preclinical studies in animal models of RP have demonstrated the feasibility and efficacy of cell replacement therapy in restoring visual function. To summarize cunent clinical progress on cell replacement therapy in retinal degenerations. RPE degenerations are successfully investigated in hESCs-RPE or hiPSCs-RPE based clinical trials, but the results were limited too stabilization and minor improvement of patients’ vision since RPEs are non-light-sensitive cells. Photoreceptor degenerations are being studied in fetal tissue-derived hRPC based clinical trials with promising interim results. In this study, we aimed to solve the unmet medical need in photoreceptor degenerations, because the use of fetal tissue-derived hRPC is limited by its availability and ethical problem, and the differentiation of hESC / hiPSC- derived RPC is not only time and labor consuming, but also with low yield efficiency. By chemicalbased direct reprogramming without genome manipulation by exogenous transcription factors, we hypothesized retinal progenitor-like cells could be generated efficiently and safely, with gene expression profile and cellular function similar to those reported in primary hRPCs.

[0066] The present invention relates to a more efficient method to convert eye fibroblasts to induced retinal progenitor cells (induced RPCs).

[0067] The eye fibroblasts used in the method as described herein can be obtained from eye tissues of proper autologous or allogenic donors, such as Tenon’s capsule. The eye tissues are excised from donors and transferred into cell culture flasks containing cell culture medium. After a period of time (e.g. about one week), eye fibroblasts are yield which can be confirmed by themorphologic features of spindle or polygonal shape and expression of fibroblast markers such as SI 00 calcium-binding protein 4 (S100A4). The eye fibroblasts as yielded can be passaged 1 to 15 times. Preferably, the eye fibroblasts for conversion into induced RPCs as used herein are of mammalian origin, most preferably of human origin.

[0068] Culture media suitable for use in the present invention are available in this art which can be further modified as needed. Suitable cell culture media are available commercially, which include, but are not limited to, Dulbecco's Modified Eagle Media (DMEM), Minimal Essential Medium (MEM), Knockout-DMEM (KO-DMEM), Improved Minimum Essential Medium (IMEM), Glasgow Minimal Essential Medium (G-MEM), Basal Medium Eagle (BME) and Ham’s F12 Medium (Fl 2). A common basal medium is a neurobasal medium such as NEUROB AS AL™ which is designed for pre-natal and fetal neuronal cell cultures. Common supplements include N2 supplement, B27 supplement without vitamin A, non-essential amino acid (NEAA), Glutamax supplement, fetal bovine serum (FBS) and bovine serum albumin (BSA). Suitable culture medium as used in the present invention may contain various combinations of medium and supplements. In some embodiments, DMEM / F12 is meant a 1 : 1 mixture of DMEM with Ham’s F12 culture medium.

[0069] In certain embodiments, the culture medium used in the present invention contains a DMEM medium with 5 - 10%FBS.

[0070] In certain embodiments, the culture medium used in the present invention contains a neurobasal medium and a DMEM / F12 medium supplemented with N2 supplement, B27 supplement without vitamin A and non-essential amino acid (NEAA). In particular examples, the culture medium used in the present invention comprises 50% DMEM / F12, 50% Neurobasal medium, lx N2 supplement, lx B27 supplement without vitamin A. and 0. 1 mM NEAA. Comprising lx N2 supplement, lx B27 supplement in the medium means that the final concentration is lx. Specifically, the DMEM / F12 medium can be obtained by mixing the following to obtain a final volume of 1000 ml culture medium:- 480 mL of NEUROBASAL- 480 mL of DMEM / F1210 ml of lOOx N2 supplement;20 mL of 5 Ox B27 supplement; and- 10 mL of lOOx NEAA.

[0071] In general, cells are cultured in a cell culture device such as a cell culture vessel. The cell culture vessel may be a petri dish, a cultivation flask, a roller bottle and a multiwall plate. In particular, the cell culture vessel may be coated with a coating that provides a structural support forthe cells and / or facilitates the cell growth by suppling the cells with metabolites. In some embodiments, the coating may include fibronectin, gelatin, Matrigel™ (BD Bioscience), collagen and / or or laminin.

[0072] According to the present invention, eye fibroblasts are chemically reprogrammed into induced RPCs by using one or more chemical inducers, including a DNMT inhibitor, a HD AC inhibitor, a CDK inhibitor, a cAMP activator, a ROCK inhibitor and ascorbic acid, as described herein, in culture medium. In some embodiments, the chemical inducers are simultaneously or successively added to the culture medium. In some embodiments, the chemical inducers are individually added to the culture medium.

[0073] In some embodiments, eye fibroblasts are chemically reprogrammed into induced RPCs by using six chemical inducers. In particular, when culturing the cells in the method of the present invention, it comprises the steps of (a) culturing in a first medium comprising a DNMT inhibitor; (b) subsequently removing the first medium and culturing in a second medium comprising a DNMT inhibitor and a HDAC inhibitor; (c) subsequently removing the second medium and culturing in a third medium comprising a CDK inhibitor, a cAMP activator and a ROCK inhibitor and ascorbic acid; and (d) subsequently removing the third medium and culturing in a fourth medium comprising a cAMP activator, a ROCK inhibitor and ascorbic acid.

[0074] Table 1 shows certain examples of chemical inducers as used in the present invention.

[0075] In some embodiments, the culture is carried out in a normal condition, for example, 37°C under 1-10% CO2.

[0076] In some embodiments, the culture is carried out for at least 1 day or more (e.g. 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days or 10 days or more). In some embodiments, the culture is carried out for at least 1 day and less than 10 days (e.g. 9 days, 8 days, 7 days, 6 days, 5 days, , 4 days, 3 days, 2 days, 1 day). In some embodiments, the culture is carried out for 1 to 6 days, 1 to 5 days, 1 to 4 days or 1 to 3 days.

[0077] In some embodiments, when culturing the cells in the method of the present invention, a NDMT inhibitor as used herein is RG108. a HD AC inhibitor as used herein is VPA, a CDK inhibitor as used herein is SU9516, a cAMP activator as used herein is forskolin (FSK) and a ROCK inhibitor as used herein is Y-27632.

[0078] In some embodiments, when culturing the cells in the method of the present invention, RG108 is present in the culture medium in a concentration of 1-100 pM, VPA is present in the culture medium in a concentration of 1-100 mM, SU9516 is present in the culture medium in a concentration of 1-100 pM, FSK is present in the culture medium in a concentration of 1-100 pM, Y- 27632 is present in the culture medium in a concentration of 1-100 pM, and the ascorbic acid is present in the culture medium in a concentration of 1-100 pM.

[0079] In some embodiments, when culturing the cells in the method of the present invention, RG108 is present in the culture medium in a concentration of 1 -50 pM, VPA is present in the culture medium in a concentration of 1-10 mM, SU9516 is present in the culture medium in a concentration of 1-50 pM, FSK is present in the culture medium in a concentration of 1-50 pM, Y-27632 is present in the culture medium in a concentration of 1-50 pM, and the ascorbic acid is present in the culture medium in a concentration of 1-50 pM.

[0080] In some embodiments, when culturing the cells in the method of the present invention, RG108 is present in the culture medium in a concentration of about 20 pM, VPA is present in the culture medium in a concentration of about 3 mM, SU9516 is present in the culture medium in aconcentration of about 10 uM. FSK is present in the culture medium in a concentration of about 10 pM, Y-27632 is present in the culture medium in a concentration of about 10 pM, and the ascorbic acid is present in the culture medium in a concentration of about 10 pM.

[0081] In some embodiments, when culturing the cells in the method of the present invention, it comprises step (a) culturing for 1 to 3 days in a first culture medium as described herein; step (b) subsequently removing the first culture medium and culturing for 1 to 3 days in a second culture medium as described herein; step (c) subsequently removing the second culture medium and culturing for 1 to 3 days in a third culture medium as described herein; and step (d) subsequently removing the third culture medium and culturing for 1 to 3 days in a fourth culture medium as described herein.

[0082] In some embodiments, when culturing the cells in the method of the present invention, it comprises step (a) culturing for about 2 days in a first culture medium as described herein; step (b) subsequently removing the first culture medium and culturing for about 1 day in a second culture medium as described herein; step (c) subsequently removing the second culture medium and culturing for about 1 day in a third culture medium as described herein; and step (d) subsequently removing the third culture medium and culturing for about 1 day in a fourth culture medium as described herein.

[0083] In some embodiments, a first culture medium as described herein contains a DMEM medium. In some embodiments, a second culture medium, a third culture medium and a fourth couture medium, as described herein, contains a neurobasal medium and a DMEM / F12 medium, supplemented with N2 supplement and B27 supplement without vitamin A.

[0084] In some embodiments, a first culture medium as described herein contains a DMEM medium with 10% FBS. In some embodiments, a second culture medium, a third culture medium and a fourth couture medium, as described herein, contains a neurobasal medium and a DMEM / F12 medium, supplemented with N2 supplement, B27 supplement without vitamin A and non-essential amino acid (NEAA).

[0085] In some embodiments, a first culture medium as described herein contains a DMEM medium with 10% FBS. In some embodiments, a second culture medium, a third culture medium and a fourth couture medium, as described herein, contains a neurobasal medium with BSA and a DMEM / F12 medium, supplemented with N2 supplement, B27 supplement without vitamin A, non- essential amino acid (NEAA) and Glutamax.

[0086] In some embodiments, when culturing eye fibroblasts in the method of the present invention, it comprises step (a) culturing for about 1 day in a first culture medium (a DMEM mediumwith 10% FBS containing RG108); step (b) subsequently removing the first culture medium and culturing for about 1 day in a second culture medium (a DMEM / F12 medium with N2 supplement, B27 supplement without vitamin A and NEAA containing RG108 and VP A); step (c) subsequently removing the second culture medium and culturing for about 1 day in a third culture medium (a DMEM / F12 medium with N2 supplement, B27 supplement without vitamin A and NEAA containing SU9516, FSK, Y27632 and ascorbic acid); and step (d) subsequently removing the third culture medium and culturing for about 1 day in a fourth culture medium (a DMEM / F12 medium with N2 supplement, B27 supplement without vitamin A and NEAA containing FSK, Y27632 and ascorbic acid).

[0087] In some embodiments, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% or more of eye fibroblasts in the culture are reprogrammed into induced RPCs. In some certain embodiments, about 30% or more, about 35% or more or about 40% or more of eye fibroblasts in the culture are reprogrammed into induced RPCs. In some certain embodiments, 25% to 45% of eye fibroblasts in the culture are reprogrammed into induced RPCs.

[0088] After culturing, the induced RPCs or a cell population comprising the induced RPCs as generated are identified with their features.

[0089] In some embodiments, the induced RPCs as described herein have RPC-like features. Specifically, the induced RPCs have RPC-like morphology, including domed shape of cells with bright nuclei and prone to form clusters. More specifically, the induced RPCs as generated can express typical RPC markers. Examples of typical RPC markers include but are not limited to SOX2 SRY-box transcription factor 2 (SOX2), Paired box 6 (PAX6), visual system homeobox 2 (VSX2). neuronal differentiation 1 (NEURODI), cone-rod homeobox protein (CRX) and recoverin (RCVRN).

[0090] In some embodiments, the induced RPCs as described herein have distinct features from primary RPCs. Specifically, the induced RPCs highly express one or more markers, such as SOX2, NESTIN and protein tyrosine phosphatase receptor type N (PTPRN). compared with primary RPC.

[0091] In some embodiments, the induced RPCs as described herein express a glutamate receptor, which are functional in responding to transmitter stimulation by glutamate.

[0092] In some embodiments, the induced RPCs are unsorted. A population comprising the induced RPCs further include anterior neuroepithelium, eye field, photoreceptor progenitor, retinal ganglion cell, Muller glia, and / or retinal pigment epithelium.

[0093] In some embodiment, to further enrich induced RPCs, the cells can be sorted with one or more RPC markers. Cell sorting can be achieved by various techniques as know n in the art. Examplesof cell sorting techniques include fluorescence-activated cell sorting (FACS), immunoaffinity column separation or immunomagnetic separation (MACS) or any technique which is capable of obtaining enrichment of one certain cell type on the basis of physical characteristics (density) or structural characteristics (in particular specific antigens).

[0094] 5. Applications using induced RPCs

[0095] The induced RPCs as describe herein are effective in rescuing photoreceptor deficiency in eyes, and therefore are useful in therapy, in particular for treating a photoreceptor degenerative disease in a subject in thereof.

[0096] Therapeutic uses of induced RPCs include delivering (or transplanting) induced RPCs to the eye of the subject. In some embodiments, the cells may be injected to the subretinal space of the subject.

[0097] Examples of a photoreceptor degenerative disease include but not limited to retinitis pigmentosa (RP), age-related macular degeneration (AMD), diabetic retinopathy (DR) and Stargardt’s disease.

[0098] Retinitis pigmentosa (RP) is a major cause of visual deficiency in the population below the middle age, impacting more than 2 million individuals worldwide[3]. It is a spectrum of hereditary retinal degenerative diseases that exhibit heterogeneity in genotypes and phenotypes. Out of more than 200 different genotypes in RP. only patients with RPE65 mutation could benefit from gene therapy [4] . Otherwise, once the loss of photoreceptor cells, most patients experience night visual impairment early in the disease progression, to central and color vision loss by age 40[5] .

[0099] Age-related macular degeneration (AMD) is the leading cause of visual impairment in the elderly, affecting nearly 200 million people globally [6] . In the early phase of AMD, the key sign is the presence of drusen in the retinal pigment epithelium (RPE), which is made out of lipids, proteins, and lipofuscin granules)?]. The aggregation of drusen can instigate inflammasome enactment and cause photoreceptor degeneration. Currently, clinically available treatment options include several anti- vascular endothelial growth factors (anti-VEGF) treatments, such as bevacizumab, ranibizumab, and aflibercept. The progression of vision loss related to neovascularization or defective vessels could be slowed by anti-VEGFs. However, once AMD progressed into the late stage with significant photoreceptor degeneration, it remains incurable and the patients become blind.[000100] Diabetic retinopathy (DR) is the most common complication of diabetes mellitus (DM), affecting 93 million people worldwide[8], DR has multifactorial pathogenesis, including microvasculopathy, inflammation, and retinal neurodegeneration. Clinically, the progression of vision loss could be slowed by anti-VEGFs, anti-inflammatory drugs, and laser treatments. However, withdisease progression, late-stage DR with significant photoreceptor degeneration remains incurable. [000101] Stargardt's disease (Stargardt macular dystrophy juvenile macular degeneration, or fundus flavimaculatus), estimated to affect approximately 1 in 8,000 to 10,000 individuals worldwide, is the most common form of inherited juvenile macular degeneration[9]. This hereditary disease is characterized by the deposition of hpofuscin-like substance in RPE, followed by loss of photoreceptor cells inthe macula

[0010] . Gene mutations are most commonly in Abca4 alleles (also known as Abcr)[l l], To date, there are no clinically available treatments to slow this progressive degeneration, patients experience significant vision loss in childhood or adolescence.[000102] In some embodiments, the induced RPCs as describe herein are effective in improving scotopic a-wave and b-wave in eyes. In some embodiments, the induced RPCs as describe herein are effective in rescuing color and central vision.[000103] According to the present invention, induced RPCs as described herein may be used an active ingredient for treating a disease in a subject in need. In some embodiments, a therapeutically effective amount of the active ingredient may be formulated with a pharmaceutically acceptable carrier into a pharmaceutical composition in an appropriate form for the purpose of delivery and absorption. Depending on the mode of administration, the pharmaceutical composition of the present invention preferably comprises about 0.1% by weight to about 100% by weight of the active ingredient, wherein the percentage by weight is calculated based on the weight of the whole composition. The composition can be used directly as an implant or further modified to a suitable form for transplantation.[000104] As used herein, "pharmaceutically acceptable'’ means that the carrier is compatible with the active ingredient in the composition, and preferably can stabilize said active ingredient and is safe to the individual receiving the treatment. Examples of a pharmaceutically acceptable carrier include conventional buffers (phosphoric acid, citric acid, other organic acids, etc.), physiological saline, sterilized water, anti-oxidants (ascorbic acid, etc ), isotonic agents, and preservatives.[000105] In some embodiments, the composition according to the present invention is formulated into a dosage form suitable for injection, where the cells are suspended in a pharmaceutically acceptable carrier e.g. sterilized water or physiological saline or frozen for storage before use. In some embodiments, the composition can further comprise a biodegradable polymer which is useful in stabilizing, supporting and fixing the cell cluster after being locally injected into the defective site. The composition according to the present invention can be formulated as a unit dosage form or incorporated into a multiple dose container. The dosage forms may be a suspension, solution, or emulsion in oil or aqueous medium, or powders, granules, tablets, or capsules. The composition ofthe invention may be delivered through a physiologically acceptable route, typically via injection. [000106] The present invention is further illustrated by the following examples, which are provided for the purpose of demonstration rather than limitation. Those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.[000107] Examples[000108] In this project, we developed a method to directly reprogram human fibroblasts into RPC- like cells by small molecules, aiming to overcome the current bottleneck in translational medicine. In our preliminary experiments, qRT-PCR, immunofluorescence assay, and Western Blot showed the induced cells expressed classical markers of RPCs. In bulk RNA-seq analysis, we compared the transcriptome between human fibroblasts and induced RPC-like cells, induced RPC-like cells demonstrated upregulated expression of transcripts related to extracellular matrix component, axon, dendrite, synaptic and postsynaptic membrane, and transport vesicle formation. By coculture assay, the induced cells were found being able to integrate with host rat retina in vivo. By calcium imaging, intracellular calcium concentrations were found to elevate in the induced cells upon glutamate stimulation, thus confirmed the functionality’ of these cells. Last but not least, we investigated the therapeutic effect in animal model of photoreceptor degeneration. Induced RPC-like cells were found to rescue visual function of transplanted rats without causing significant adverse events.[000109] 1. Materials and methods[000110] 1.1 HTF isolation and primary culture[000111] The leftover samples were obtained anonymously from tissue explants taken during vitrectomy or strabismus surgery' from patients visiting Hualien Tzu Chi Hospital, with the approval of the ethical committees of Hualien Tzu Chi Hospital and Academia Sinica. We thoroughly reviewed the donors’ clinical records and excluded patients with ocular surface diseases and systemic conditions such as infections or diabetes. During the operations, small samples of approximately 0.5 ml of vitreous or 1*1 mm of Tenon’s capsule were excised. The tissues were immediately transferred into cell culture flasks and maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), and 1% penicillin / streptomycin was supplemented during the first week of culture. About one week after the procedure, we observed only cell debris from the vitreous sample, but abundant spindle or polygonal-shaped flat cells were yielded from Tenon’s capsule samples. By their characteristic morphology as well as the expression of fibroblast marker, SI 00 calcium-binding protein A4 (S100A4), we confirmed these cells were human Tenon’s capsulefibroblasts (HTFs). HTFs from passages 3 to 10 were then used for the following experiments.[000112] 1.2 Preparation of Vsx2::eGFP promoter reporter cells[000113] To prepare the Vsx2 eGFP promoter reporter, we digested out the promoter fragment from a commercially available vector. pLKO_AS7w.eGFP.puro (Academia Sinica RNAi core), using restriction enzymes. These promotor sequence of Vsx2 were then cloned into the original vector. Positive clones were then sent for Sanger sequencing for confirmation. The final product was then used for lentivirus preparation. HTFs were transduced with the lentivirus for 3 days.[000114] 1.3 Generation of CiRPCs[000115] All small molecules were diluted in water or DMSO according to the manufacturer’s instructions. Approximately 1,000,000 HTFs (passage <10) were seeded into a 10-cm dish (40X Matri gel-coated). On day 1, the medium was replaced with HTF medium (10% FBS and DMEM) containing RG108 (20 pM). On day 3, fresh CiRPC medium (50% Neural basal with 100X BSA, 50% DMEM / F12 / Glutamax, IX N2, IX B27 without vitamin A, 0.1 mM nonessential amino acids) containing RG108 (20 pM) and VPA (3 mM) were replaced into the dish. The medium was then replaced with CiRPC medium containing SU9516 (10 pM), forskolin (10 pM), Y-27632 (10 pM), and vitamin C (10 pM), on day 4, and CiRPC medium containing forskolin (10 pM), Y-27632 (10 pM), and vitamin C (10 pM), on day 5. On day 6, GFP+cells were collected for further analysis and experiments. On day 6, the cell population was determined to include cells which became domeshaped with bright nuclei, and were defined as induced retinal progenitor cells (iRPCs) or chemically induced retinal progenitor cells (CiRPCs). The unsorted cell population further include cells with stochastic expression of genes related to anterior neuroepithelium, eye field, photoreceptor progenitor, retinal ganglion cell. Muller glia, and retinal pigment epithelium, such unsorted cell population named induced retinal lineage-like cells (iRLCs). iRLCs can be further sorted to provide a cell population enriched with iRPCs. The 5-day reprogramming protocol is as shown in Fig. 2.[000116] 1.4 Fluorescence-activated cell sorting (FACS)[000117] For FACS. CiRPCs were dissociated with TrypLE Express (Gibco) and passed through a 40-pm Nylon cell strainer (Fisher Scientific, Cat# 08-777-1) and suspended in PBS containing 1% bovine serum. Initiating HTFs were used as a negative control. Cells were then sorted in a Beckton- Dickinson FACS Aria IIu Flow cytometer at the core facility7. Sorted cells were collected in CiRPC medium, centrifuged, and processed for RNA extraction and other downstream applications.[000118] 1.5 Quantitative PCR with reverse transcription[000119] RNA was isolated from cells using the RNeasy Micro Kit (Qiagen, Germantown, MD, USA) and was reverse-transcribed (100 ng). RT-PCR analysis was performed with KAPA SYBRFAST qPCR Kits (Kapa Biosystems, Wilmington, MA, USA), and the housekeeping gene, Succinate dehydrogenase complex, subunit A (Sdha), was used for the endogenous reference.[000120] 1.6 Western blotting[000121] Cells were collected by scraping in lysis buffer (1% NP40, 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA, 1 mM Na3VO4) and protease inhibitor cocktail (Sigma, Burlington, MA, USA). Protein quantification was performed with a Bio-Rad protein assay (Bio-Rad, Hercules, CA, United States). Samples were boiled for 15 min at 100 °C, 30 pg protein was loaded on 10% acrylamide gels, and electrophoresis was started at 90 V for 15 min, then turned to 120 V for 1 h. Amersham Protran 0.45 pm nitrocellulose membranes were used for blotting. Blots were blocked for 30 min by shaking in 5% BSA in phosphate-buffered saline with Tween 20 (PBST) (Sigma, Burlington, MA, USA). Primary' antibodies (VSX2, Novus Biologicals; NBP184476) were diluted in 5% BSA in PBST and incubated overnight at 4 °C on the shaker. Secondary' antibodies were diluted in 5% BSA in PBST and cultured for 1 h at room temperature on the shaker. Detection of protein signals was performed with the UVP BioSpectrumAC system (Jena, Thuringia, Germany).[000122] 1.7 Bulk RNA-sequencing[000123] 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 number (RIN) > 8. Stranded TruSeq cDNA libraries with poly dT enrichment were prepared from total RNA from each sample according to the manufacture’s protocol. Libraries for the cDNA samples were sequenced using the Illumina HiSeq sequencing platform yielding 24.8-32 million 150 bp paired end (PE) sequence reads per sample. PE FASTQ files received back from Genomics were analyzed using a customized bioinformatics workflow.[000124] 1.8 Calcium imaging of glutamate responses[000125] Intracellular calcium dynamics, in response to glutamate stimulation, were analyzed for HTFs and CiRPCs. The cells were plated at a low density to allow for high-resolution imaging of individual cells and recording of changes in fura-2 fluorescence, an indicator of intracellular calcium influxes. Cells were rinsed with Ringer’s solution containing (in mM) NaCl 119, KC1 4.16, CaCI2 2.5, MgC12 0.3, MgSO4 0.4, Na2HPO4 0.5, NaH2PO4 0.45, HEPES 20, and glucose 19 (pH 7.4), then incubated in Ringer’s solution containing 0.5 pM fura-2 tetra-acetoxy- methyl ester for 40 minutes at 22°C. Fura-2 was excited by alternating 340- and 380-nm light with the use of a filter changer, under the control of commercial software (InCytIM-2; Intracellular Imaging Corp. Cincinnati, OH) paired with a phase-contrast microscope (Eclipse T5100; Nikon). A new ratio (340 / 380) image was obtained every 0.35 seconds as a measure of Ca2+ concentration. HTFs andCiRPCs were then stimulated with either 1 mM L-glutamate (Sigma-Aldrich), and increases in cytosolic fura fluorescence were analyzed.[000126] 1.9 Subretinal transplantation in RCS rats[000127] Royal College of Surgeons (RCS) rats were provided by the Experimental Animal Center of Tzu Chi University. RCS rats are an established model for retinal degeneration due to retinal pigment epithelium dysfunction. All animal work has been approved by the Institutional Review Board of Hualien Tzu Chi Hospital. The animals, regardless of sex, were raised in a specific pathogen-free room in the Animal Care Center and maintained under a 12-h light / dark cycle. The animals were allocated randomly for experiments. At least four individuals were used for each experiment. All P21 rats were anesthetized through the intramuscular injection of a mixture of ketamine. (40 mg / kg) and xylazine (4 mg / kg) (Sigma), and topical 0.5% Alcaine eye drops (Alcon). Their pupils were dilated using eyedrops containing a mixture of 0.5% tropicamide and 0.5% phenylephrine hydrochloride (Mydrin-P) for post-transplantation examinations with optical coherence tomography (OCT). After sclera exposure, a pilot hole was created tangentially using a 31 -gauge sterile needle, injector needle was guided into a hole, and 5 pL of cell suspension was injected. Subsequently, the needle was retracted slowly, and light pressure was applied for the selfsealing wound. Then, a drop of TobraDex solution (Alcon) was applied to eyeball. After that, rats were returned to the recovery cage and any signs of hemorrhage were observed. Rats were allowed normal food with oral administration of cyclosporine (200 mg / 1 L) (Neoral 100; Novartis).[000128] 1.10 Electroretinogram[000129] Scotopic and photopic full-field electroretinograms (ERG) were recorded for testing retinal function to make comparisons between nontransplanted and transplanted eyes. All rats were adapted in the dark for at least 24 hours and then anesthetized following the abovementioned method. After pupils were dilated, rats were placed in a Ganzfeld ColorDome controlled by an Espion visual electrophysiology system (Diagnosys). ERGs were recorded for each eye using contact lens electrodes placed directly on the comeal surface with 2% Methocel (OmniVision). A reference electrode was placed subdermally in the center of the scalp, a ground electrode was placed in the proximal portion of the tail skin, and the rat was placed on a heating pad. The impedance level was checked periodically for an acceptable green range (i.e., electrodes were properly installed). Dark- adapted (Scotopic) stimuli were set at an intensity of 0.01 cd sec / m2, and 5 trial responses were averaged to create a standard waveform. For the light-adapted (Photopic) readings, rod responses were first saturated by exposing rats for at least 10 min at an intensity of 20 cd sec / m2, and light- adapted stimuli were set at 3.0 cd sec / m2. Ten trial responses were averaged to create a standardwaveform. Finally, the mean value of the b-wave was compared between the control and transplant groups for both scotopic and photopic ERG.[000130] 1.11 Light-dark box test[000131] The visual discrimination light-dark box (LDB) test was conducted in an apparatus that consists of black opaque (100%) acrylic test chambers (30.48 x 15.24 x 30.48 cm (length, width, height)). This chamber was further divided into equal-sized compartments (15.24 x 15.24 x 30.48 cm) by the addition of an insert, to create a dividing wall in the center. Further, to create light and dark zones, one compartment was illuminated with dim ambient light (50 ± 1.5 lx) and the other compartment was kept dark (~ 0. 1 lx). The light and dark compartments were connected by an opening (5 x 5 cm). The position of the rat within the apparatus was recorded using camera. The acrylic chambers were housed separately in sound-attenuating space. Ambient noise within the chambers was 64 dB and testing took place under dim illumination. Rats were maintained in the testing room overnight (about 12 h) in dark conditions in their home cage with free access to food and water. Each rat was allowed to habituate to the testing apparatus (both sides) for 10 min while in the dark. After habituation, one side of the apparatus was illuminated with an ambient light at around 50 lx and the rat was allowed to roam freely between each compartment for 5 min. The time spent in the dark and light compartments was recorded by a camera-based system.[000132] 1.12 Histological examination[000133] The rats were euthanatized through carbon dioxide inhalation; the eyeballs were enucleated and fixed in a 4% paraformaldehyde for 48 hours at 4°C. Then, eyeballs were processed in gradient sucrose concentrations (10%, 20%, and 30%) each for overnight at 4°C. After that, eyeballs were embedded in an OCT compound (Tissue-Tek), cut into 20 pm sections using a cryostat microtome (Leica CM-3050-S) and collected on glass slides. These slides were used for immunostaining and immunofluorescence analysis.[000134] 1.13 Immunofluorescence assay[000135] Cells were fixed with 4% formaldehyde for 15 min at room temperature and washed once with 1 x phosphate-buffered saline (PBS). Cells were permeabilized with 0.3% Triton X-100 for 5 min, washed twice with 1 x PBS, and blocked in 2% bovine serum albumin (BSA) in PBS for 30 min. Then, the cells were incubated with neural stem cell (NSC) marker anti-SOX2 (GeneTex GTX101507, 1:200), anterior neuroepithelium marker anti-OTX2 (R&D MAB1979, 1:50), eye field marker anti-LHX2 (Santa Cruz Biotechnology SC 19344, 1:50), retinal progenitor cell (RPC) marker anti-VSX2 (Novus Biologicals NBP 184476, 1 : 1000), photoreceptor precursor marker anti-RCVRN (Millipore AB5585, 1:1000), retinal ganglion cell (RGC) marker anti-HUD (Invitrogen A21271,1 : 100), Muller glia marker anti-GFAP (Proteintech 16825-1 -AP, 1 :200) , and retinal pigment epithelium (RPE) marker anti-BESTl (Abeam abl4927, 1: 100) in blocking buffer (2% BSA in PBS) overnight at 4 °C. Cells were washed twice in 1 * PBS, followed by incubation in CF555 goat antimouse 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) in blocking buffer for 1 h in the dark at room temperature, DAPI dihydrochloride (MilliporeSigma D9542, 0.5 pg / ml) was used to stain the nuclei. Cells were washed twice in 1 x PBS. The fluorescence intensity’ of each image was analyzed by image analysis software (Image-Pro plus v.4.5; Media Cybernetics. Rockville, MD, USA).[000136] 1.14 Intravitreal transplantation in healthy Wistar rat[000137] In this study, we used adult male Wistar rats weighing 150-180 g to evaluate the safety’ of HTFs and iRLCs, when co-cultured with healthy rats' retina. The rats were purchased from the breeding colony of BioLASCO Co.. Taipei. Taiwan. All animal experiments were approved by the Institutional Animal Care and Use Committee of Buddhist Tzu Chi General Hospital. The rats received one intravitreal injection of HTFs suspension (5 pL, 1 x 105cells / pL) or iRLCs suspension (5 pL, 1 x io5cells / pL). In both groups, the right eyes served as the treatment eyes, whereas the left eyes served as controls and untreated. Two weeks after intravitreal injection, the HTFs-transplanted rat and the iRLCs-treated rat were analyzed by using fundus photography, optical coherence tomography (OCT), and immunohistochemistry’ (IHC) analysis to evaluate the safety7and histological changes in the retina.[000138] 1.15 Optical coherence tomography[000139] OCT was performed with a contact lens assembled in a Micron IV retinal microscope (Phoenix Research Laboratories). All rats were anesthetized following the aforementioned method, pupils were dilated, and the corneal surface was protected using a 2% Methocel (OmniVision). Then, the rats were placed on a horizontal platform to allow the penetration of light vertical to the cornea, and the ocular fundus was monitored using the camera of the Micron IV microscope. The retinal OCT image was set horizontally, scanning was performed to observe the transplanted cells, and at least 2 OCT images were captured.[000140] 1.16 Statistical analysis[000141] All data are presented as mean ± s.e.m. Statistical significance was determined using Student’s Ltest and one-way ANOVA using GraphPad Prism Software (GraphPad Software); P values are indicated in the figures.[000142] 2. Results[000143] 2.1 Primary culture ocular fibroblast from Tenon’s capsule[000144] We performed primary culture of ocular fibroblast from Tenon’s capsule. It is a 15-minute short procedure in the clinic, without damage to the intraocular structure. By confirming characteristic morphology as well as the expression of fibroblast marker, SI 00 calcium-binding protein A4 (S100A4) (data not shown), we defined these cells being human Tenon’s capsule fibroblasts (HTFs). HTFs from passages 3 to 10 were then used for the following experiments.[000145] 2.2 Cell reprograming[000146] We then used the HTFs as the source of cell reprogramming, to test different compositions of the chemical cocktail. With a set of six small molecules (6C), we were capable of directly reprogramming human fibroblasts into RFCs in 5 days. With Fsx2-GFP reporter system, we confirmed the stable expression of Vsx2 and named these cells chemically induced RPCs (CiRPCs). To optimize the composition of chemical compounds in the protocol, we removed each chemical one at a time and evaluated the consequential alteration in cell morphology and Vsx2 expression pattern. We found the overall Vsx2 expression pattern was most optimal when all 6 compounds were given. The induced cells became dome-shaped with bright nuclei, prone to form clusters, expressed cells markers SOX2, PAX6, VSX2, and RCVRN, and were able to be stably maintained for more than two months after subculture (Fig. 1A).[000147] We also tested other fibroblasts, including human fetal lung fibroblasts (IMR-90), human newborn foreskin fibroblasts (CRL-2097), human neonatal foreskin fibroblasts (BJ-5ta), and human adult dermal fibroblasts (FB-3652) (Fig. IB) However, we found the 6C protocol w as specifically capable to reprogram HTFs into CiRPCs, regardless of HTFs derived from different patients. Interestingly, other fibroblasts after treating with 6C, do not induce conformation change or do not survive well.[000148] Whereas in our protocol, it took only 5 days to reprogram human adult ocular fibroblasts into CiRPCs with 6C (Fig. 2). In our approach, there were 42.8% Fsx2-GFP cells after HTFs induced with the CiRPC protocol (Fig. 3B).[000149] We further used each chemical compound of 6C protocol one at a time, and removed remaining 5 chemical compounds in the reprogramming process. Vsx2 '. eGFP expression on High- Content Screening System showed successful reprogramming of iRLCs when one compound was given at a time, while all 6 compounds given resulted in highest efficiency. (Fig. 9). We concluded w hen applying any chemical compound in the 6C protocol, w ith intrinsic cell reprogramming competence, HTFs were able to be induced into iRLCs.[000150] 2.3 Transcriptional expression characteristics[000151] To investigate the gene expression change of retinal cell markers, CiRPCs after transdifferentiated from HTF, showed increased expression of multiple retinal lineage markers, including photoreceptor-specific genes. The elevation of VSX2 expression was also confirmed by qRT-PCR (Fig. 4A) and Western Blot (WB) analysis (Fig. 4B). In our protocol, qRT-PCR analysis showed comparing to human fibroblasts, CiRPCs had over 1000 folds of upregulation in RPC marker Vsx2, and upregulation in photoreceptor related genes. Western Blot analysis showed that of VSX2 expression was detected in unsorted and sorted CiRPCs but not found in HTFs.[000152] We also performed a series of immunofluorescence (IF) staining to validate if there are correspondent protein expression patterns. The selected markers were those with upregulated mRNA expression. In contrary to HTFs, iRLCs (unsorted CiRPCs) on day 6 showed upregulation of SOX2, OTX2, LHX2, VSX2, RCVRN, HUD, GFAP, BEST1 by IF staining, with positive rate ranging from 28.28% (BEST1) to 75.01% (SOX2) (Fig. 10). Taking the results together, we found the iRLCs had upregulation of genes not only related to RPC, but also genes related to anterior neuroepithelium, eye field, photoreceptor progenitor, RGC, Muller glia, and RPE. This implies iRLCs may be a mixed population of cells with stochastic expression of genes related to retinal lineage, rather than primary cells in anterior neuroepithelium, eye field, photoreceptor progenitor, RGC, Muller glia, and RPE only express designated cell-type-specific marker genes in normal developmental process[000153] 2.4 Genome-wide transcriptome profile[000154] We also performed Gene Ontology (GO) enrichment analysis, to explore the biological function of upregulated / downregulated expression of transcripts between HTFs and CiRPCs. The upregulated GOs were related to the extracellular matrix component, axon, dendrite, synaptic and postsynaptic membrane, and transport vesicle formation. The downregulated GOs were corresponding to cell mitosis and fibrosis. These profound changes in GO enrichment analysis implied after reprogramming from HTFs into CiRPCs, the biological functions shifted profoundly from fibroblasts into neuronal cells (Figs. 5A and 5B).[000155] 2.5 Investigation of function of induced cells[000156] 2.5.1 In vitro potency[000157] While most inner retinal neurons have glutamate receptors, studies have shown in response to transmitter stimulation by glutamate, either RPCs or retinal neurons exhibit a response with calcium influxes. By high-resolution imaging of CiRPCs and recording the changes in fura-2 fluorescence, an indicator of intracellular calcium influxes, we found that unsorted and sorted CiRPCs induced by 6C stimulated with 1 mM of glutamate responded with significant calcium influxes, while HTFs showed no response upon glutamate stimulation (Figs. 6A and 6B). The resultsindicate that unsorted and sorted CiRPCs inducted by 6C show similar in vitro functional characteristics to primary RPCs. In addition, iRPCs induced by VP A showed significant calcium influxes (Fig. 6C).[000158] 2.5.2 In vivo potency[000159] In our approach, HTFs-derived CiRPCs were transplanted into the subretinal space of RCS rats on day 21 (P21). There was an improvement in scotopic b-wave in the eyes in which CiRPCs had been transplanted at P28 and P56. In the light-dark box test (LDB), RCS rats injected with CiRPCs were found to spend significantly more time in the dark space, indicating partial visual function restoration because of the raf s innate tendency to avoid lit spaces (Figs. 7A and 7B).[000160] The H&E stain in the histology study showed significant fibrosis around the area injected with HTFs, but not CiRPCs or the control group injected with PBS. There was no cell migration to extraocular space. Furthermore, IHC showed HuNu+cells in the photoreceptor layer labeled as RCVRN+, except for the PBS group, indicating survival and integration of human cells in layer of rat photoreceptors, 3 months after subretinal transplantation (Fig. 8).[000161] 2.5.3 In vivo coculture assay[000162] To better understand the safety of induced cells on healthy retina, we performed in vivo coculture assay by transplanting l*105of HTFs or unsorted iRLCs induced by 6C into the vitreous cavity of Wistar rats and traced the migration of transplanted cells by optical coherence tomography (OCT) weekly. One week after transplantation, we observed significant aggregation of transplanted cells to the retinal surface in the iRLCs group but not the HTFs group. Two weeks after transplantation, most of the aggregated iRLCs became absent in vitreous and left no cell debris. On the contrary, significant fibrotic change of retinal surface was observed in the HTFs group (Fig. 11). The rat eyes were then collected for cryofixation with H&E and immunofluorescence staining. On H&E staining, the rat eye transplanted with HTFs was found to have significant fibrotic membranes on retinal surface, with a fibrotic cell mass attached to the epiretinal membrane (Fig. 12A). By immunostaining of human nuclei, the epiretinal membrane and cell mass were confirmed to be composed of HTFs, surrounded by rat retinal cells (Fig. 12A). On the contrary, the rat eye transplanted with iRLCs had normal vitreous and retina morphology. A few iRLCs were identified in the vitreous and retina with their distinct dome shape and bright nuclei. By immunostaining, we found several iRLCs integrated into the inner nuclear layer (INL) of rat retina. The iRLCs in INL also co-expressed photoreceptor marker RCVRN (Fig. 12B).[000163] 2.5.4 In vivo functional analysis in animal model of photoreceptor degeneration[000164] To evaluate the in vivo functional property of induced cells, we studied therapeuticefficacy in the Royal College of Surgeons (RCS) rats. RCS rat is an animal model of human retinitis pigmentosa, RPE cells of RCS rats fail to phagocytose shed photoreceptor epithelial cells and subsequently photoreceptor cells die, it is widely utilized in previous studies on hRPCs. We used RCS rats at 21 days old and design four groups for comparison, including PBS, HTF control, iRLCs (induced by 6C), iRPCs (induced by 6C). After successful subretinal injections on P21, OCT examinations demonstrated the intact structure of the retinas on 3-week-old rats. The post-transplant OCT revealed that the cells were successfully injected into the subretinal space, and retinas remained intact at the injection sites (Fig. 13). The ERGs measured in four groups for scotopic responses showed statistically significant improvement in eyes injected with iRPCs at P56. and eyes injected with iRLCs in Pl 12 (Fig. 14). LDB tests revealed when RCS rats had severely degenerated vision at Pl 12, the rats transplanted with HTFs, iRLCs, and iRPCs tend to spend more time in dark zone, indicating rescue of visual function by cell replacement therapy (Fig. 15). Lastly, histology study showed significant fibrosis around the area injected with HTFs. but not iRLCs nor iRPCs.Furthermore, IHC showed HuNu+cells in the photoreceptor layer labeled as RCVRN+, except for the PBS group, indicating survival and integration of human cells in layer of rat photoreceptors, 3 months after subretinal transplantation (Fig. 16). Overall, the layer-by-layer retinal morphology was preserved in eyes transplanted with iRLCs or iRPCs, compared to eyes treated with PBS or HTFs, indicating photoreceptor preservation by neurotrophic mechanism of induced cells (Fig. 16).[000165] 3. Summary[000166] In our studies, CiRPCs were obtained after 5 days of induction, and reprogramming efficiency is 42.8% defined by the reporter system. Furthermore, the CiRPCs induced using our chemical protocol exhibited a dome-shaped morphology with bright nuclei and a tendency to form clusters. These cells expressed various retinal cell markers, including SOX2, NESTIN, VSX2, A, and remained stable for over two months after subculture. To explore the biological functions of the upregulated and downregulated transcripts between parental cells and CiRPCs, we conducted a Gene Ontology (GO) enrichment analysis. The upregulated GOs were associated with extracellular matrix components, axons, dendrites, synaptic and postsynaptic membranes, and transport vesicle formation. On the other hand, the downregulated GOs were linked to cell mitosis and fibrosis. These significant changes indicate that CiRPCs have shifted dramatically from fibroblasts to neuronal cells after reprogramming.[000167] The high-resolution imaging analysis of CiRPCs and recording of the changes in fura-2 fluorescence showed that CiRPCs demonstrated significant calcium influxes in the presence of glutamate. The behaviors of CiRPCs mimic most inner retinal neurons with glutamate receptors. Inanimal models, eyes that received CiRPCs transplants at P28 and P56 improved scotopic b-wave. Additionally, in the light-dark box test (LDB), the CiRPCs-injected rats spent considerably more time in the dark area, indicating partial restoration of visual function due to the rat's natural inclination to avoid bright spaces. The immunohistochemical assay showed CiRPCs survived and integrated into a layer of rat photoreceptors three months after subretinal transplantation.[000168] 4. Discussion[000169] 4.1 Progress on cell replacement therapy in photoreceptor degeneration[000170] 4.1.1 RPE or photoreceptor replacement[000171] While RP, DR, AMD, and Stargardt's disease have different pathogenesis and various demographics, these retinal disorders all cause apoptosis of photoreceptors at their end-stage. To further categorize the apoptotic cell types involved in these diseases, for heterogeneous phenotypes in RP and DR, most patients suffer from loss of photoreceptors cells primarily. AMD and Stargardt's disease cause RPE dysfunction and degeneration. When RPE cells fail to maintain phagocytosis of photoreceptor outer segments and lost blood-retinal barrier integrity, there will be subsequent photoreceptors loss after disease progressed into advanced stage. In both preclinical studies and clinical trials, the transplantation of RPE cells from variable sources has been thoroughly investigated in patients with AMD [12- 14], Since RPE cells are not light-sensitive, there were usually only stabilization and minor improvement of patients’ vision after transplantation, and long-term efficacy remains to be established[15-17]. Alternatively, photoreceptor replacement therapy is the ultimate solution to rescue and reverse patients’ degenerative vision in late stage of RP, DR, AMD, and Stargardt's disease. Targeting the light-sensitive cells in retina, including rods and cones, the transplanted cells are anticipated to generate electrical responses upon light stimulus. However, there is limited number of suitable cell sources, it remains challenging for consistent clinical implementation.[000172] 4.1.2 Source of photoreceptor replacement[000173] Current approaches toward replacement of defective photoreceptors could be classified into three arms, either using RPCs, young post-mitotic photoreceptors (photoreceptor precursors) or 3D retinal tissue. The results of preclinical studies and clinical trials on RPCs have been especially promising. Currently, there are 19 preclinical studies published in literature regarding human RPC treatment in photoreceptor degeneration [18-36], In these preclinical studies, PRCs are either derived from 11-20 weeks gestational age fetal retina, differentiated from pluripotent stem cells (embryonic stem cells, ESCs; induced pluripotent stem cells, iPSCs), or differentiated from multipotent stem cells (mesenchymal stem cells, MSCs). Regarding clinical trials, ReNeuron and jCyte, using RPCsderived from fetal retina, have completed phase 2 clinical trials in patients with RP (clinicalTrials.gov: NCT02464436; NCT03073733). The efficacy of transplanted cells rescued patients’ vision for at least one year in both groups [37, 38], However, ReNeuron halted its further trial, since surgical complications caused concern. On the other hand, jCyte announced the restoration of visual function depended on the patients’ central visual field at baseline, aims to move forward into a US pivotal trial.[000174] Regarding young post-mitotic photoreceptors (photoreceptor precursors), current evidence is limited to preclinical data only. Earlier studies on mice revealed that both immature and mature photoreceptors were able to integrate into wild type or diseased mice retina, but significantly higher failure rates were found in mature photoreceptors transplantation, due to their poor survival ability' during the dissociation and isolation protocol

[0039] . Thus, researchers concluded the ideal cell type for photoreceptor transplantation would be young post-mitotic photoreceptors (photoreceptor precursors)

[0040] . However, human photoreceptor precursors were found only during the second trimester of pregnancy, the legal and ethical concerns restrained the availability of primary human photoreceptor precursors. Human ESC / iPSC-derived photoreceptor precursors were investigated in most studies instead, but the overall efficiencies of differentiation protocols in generating photoreceptor precursors were relatively low, with less than 20% of cells and require more than 100 days[41 J . Despite the aforementioned hurdles, studies have shown similar results to those conducted on hRPCs, suggesting that ESC / iPSC-derived photoreceptor precursors were able to integrate into host retina, transfer cytoplasmic material, differentiate into more mature photoreceptors, and restore some degree of visual function at a certain extent[42-44].In addition to ESC / iPSC-differentiated photoreceptor precursors, Mahato et al. from North Texas Eye Research Institute reported that with administration of a set of five small molecules, mouse / human fibroblasts could be transdifferentiated into rod photoreceptor precursors

[0045] , In their study, the chemically induced photoreceptor precursors (CiPCs) from mouse fibroblasts showed partial restoration of visual function after transplantation to the subretinal space of rod photoreceptor degeneration mice, human CiPCs were not tested for its therapeutic efficacy in animal model of their publication.[000175] As for 3D retinal tissue, early in 2009 Li et al. reported by transplanting human fetal (12- 24 week) neuro-retina and RPE sheets into mini-pigs with light-induced retinal degeneration, there was functional improvement in 15 out of 25 eyes. And there was no graft rejection over 12 months of follow up

[0046] . With the advancement in organoid culture systems, researchers started to focus on transplantation of ESC / iPSC-derived 3D retinal sheet. Several studies showed after transplantation into animal models with retinal degeneration, both mouse and human ESC / iPSC-derived 3D retinalsheets were able to survive for up to 6 months, and differentiate into mature retinal lineage cells including photoreceptors, bipolar and ganglion cells. However, the ESC / iPSC-derived 3D retinal grafts were also found unable to maintain a properly layered structure after long-term follow up, the grafts developed lots of disorganized rosettes in host retina[47-49]. The long-term survival and structure maintenance of ESC / iPSC-derived 3D retinal tissue remain to be improved in the future. [000176] 4.2 Chemical compound-based direct reprogramming[000177] Both the reprogramming and differentiation processes raised potential safety concerns for the clinical use of iPSCs. The exogenous gene induction during reprogramming might cause genomic instability and mutations

[0050] . Besides, the presence of residual undifferentiated cells after differentiation protocol increases the risk of undesirable tumorigenesis after transplantation

[0051] . Over the years, noteworthy progress has been made in the field of direct lineage reprogramming, by means of chemical compounds alone

[0052] , The cell fate conversions are accomplished by regulating cellular signaling pathways and activity of histone / DNA modifying enzymes, without the use of transgenes. Small molecules can promote the efficiency of transcription factor-based direct reprogramming and sometimes replace the effects of transcription factors and cytokines, which is obviously helpful in preparing a large number of cells in a defined and cost-effective manner[53, 54], The unique advantage of chemical compounds is that they are preserved, highly purified, having a long half-life, are non-immunogenic, and effective at a low concentration.[000178] 4.3 Chemically induced photoreceptor precursors (CiPCs)[000179] Mahato et al. from North Texas Eye Research Institute published their approach in scientific journal Nature

[0045] , They reported that with administration of a set of five small molecules, with Nrl-GFP reporter system, mouse / human fibroblasts could be transdifferentiated into rod photoreceptor precursors. These chemically induced photoreceptor precursors (CiPCs) show ed partial restoration of visual function after transplantation to the subretinal space of rod photoreceptor degeneration mice.[000180] In our project, we developed a set of six small molecules to directly reprogram human fibroblasts into RPCs with Esx2-eGFP reporter system. These chemically induced RPCs (iRPCs) also showed partial restoration of visual function after transplantation to the subretinal space of photoreceptor degeneration rat.[000181] Firstly, to compare the time required in reprogramming protocol, the protocol by Mahato et al. took 10 days to transdifferentiate human adult dermal fibroblasts (HADFs) into CiPCs. Whereas our protocol took only 5 days to reprogram human adult ocular fibroblasts (HTFs) into iRPCs. [000182] Secondly, to compare the conversion efficiency of reprogramming protocol, there w as24.9% Nrl-GFP cells after HADFs were treated with the CiPC protocol. In our approach, there was 42.8% Esx2-eGFP+cells after HTFs were induced with our proprietary' iRPC protocol.[000183] Thirdly, to compare the gene expression change of retinal lineage markers, CiPCs after reprogramming from HADF, showed increased expression of photoreceptor-specific genes. While iRPCs after trans differentiated from HTF, showed increased expression of multiple retinal lineage markers, including photoreceptor-specific genes.[000184] Fourthly, to compare the therapeutic efficacy in animal models of photoreceptor degenerations, mouse CiPCs were transplanted into the subretinal space of rdl mice on day 31 (P31). Electroretinogram (ERG) analysis demonstrated improvement of the scotopic a-wave in ey es which CiPCs had been transplanted at P45, but improvement diminished at and after P59. In our approach, human iRPCs were transplanted into the subretinal space of RCS rat on day 21 (P21). There was improvement on scotopic b-wave in eyes which unsorted iRLCs or FACS-sorted iRPCs had been transplanted. In light-dark box test (LDB), RCS rats injected with unsorted iRLCs or FACS-sorted iRPCs were found to spend significantly more time in the dark space, indicating partial visual function restoration because of rat’s innate tendency to avoid lit spaces.[000185] 5. Conclusions[000186] Our study shows that induced retinal lineage cells can be generated from human eye fibroblasts by defined small molecules and culture medium, free of exogenous genetic materials. Our experiment results showed the iRLCs have gene expression profile of multiple retinal lineage cell ty pes, By in vitro functional assay, both unsorted iRLCs or FACS-sorted iRPCs show ed calcium influx upon stimulation of glutamate, mimicking electrophysiology function of primary retinal cells. Upon co-culture with retinal explants and health rat retina, the iRLCs showed tendency to migrate and integrate with inner retinal cells. 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Claims

CLAIMSWhat is claimed is:

1. A method of generating induced retinal progenitor cells (induced RPCs), comprising culturing eye fibroblasts in a condition which allows a proportion of the eye fibroblasts to reprogramming into induced RPCs. wherein the condition comprises a culture medium which comprises a compound selected from the group consisting of a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HD AC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-associated protein kinase (ROCK) inhibitor, ascorbic acid and any combination thereof.

2. A method of generating induced retinal progenitor cells (induced RPCs), comprising culturing eye fibroblasts in a condition which allows a proportion of the eye fibroblasts to reprogramming into induced RPCs, wherein the condition comprises a culture medium which comprises a DNA methyltransferase (DNMT) inhibitor, a histone deacetylase (HD AC) inhibitor, a cyclin-dependent kinase (CDK) inhibitor, a cyclic adenosine monophosphate (cAMP) activator, a Rho-associated protein kinase (ROCK) inhibitor and ascorbic acid.

3. The method of claim 1 or 2, further comprising identifying induced RPCs that express one or more retinal markers selected from the group consisting of SRY-box transcription factor 2 (SOX2), paired box 6 (PAX6), visual system homeobox 2 (VSX2), neuronal differentiation 1 (NEURODI), cone-rod homeobox protein (CRX) and recoverin (RCVRN), and any combination thereof, and isolating the identified induced RPCs.

4. The method of any of claims 1 to 3, wherein the induced RPCs express a glutamate receptor.

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

6. The method of claim 5, wherein the eye fibroblasts are human fibroblasts from Tenon’s capsule.

7. The method of any of claims 1 to 6, wherein the DNMT inhibitor, the HD AC inhibitor, theCDK inhibitor, the cAMP activator, the ROCK inhibitor, and the ascorbic acid are simultaneously or successively added to the culture medium.

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

9. The method of claim 8, wherein RG108 is present in a concentration of 1-100 pM, VPA is present in a concentration of 1-100 mM. SU9516 is present in a concentration of 1-100 pM, FSK is present in a concentration of 1-100 pM, Y-27632 is present in a concentration of 1-100 pM, and the ascorbic acid is present in a concentration of 1-100 pM.

10. The method of claim 9. wherein RG108 is present in a concentration of 1-50 pM, VPA is present in a concentration of 1-10 mM, SU9516 is present in a concentration of 1-50 pM, FSK is present in a concentration of 1-50 pM, Y-27632 is present in a concentration of 1-50 pM, and the ascorbic acid is present in a concentration of 1-50 pM.

11. The method of claim 9. wherein RG108 is present in a concentration of about 20 pM. VPA is present in a concentration of about 3 mM, SU9516 is present in a concentration of about 10 pM, FSK is present in a concentration of about 10 pM, Y-27632 is present in a concentration of about 10 pM, and the ascorbic acid is present in a concentration of about 10 pM.

12. The method of any of claims 1 to 11, comprising the steps of(a) culturing the eye fibroblasts in a culture vessel containing a first medium comprising the DNMT inhibitor(b) removing the first medium and adding a second medium comprising the DNMT inhibitor and the HD AC inhibitor;(c) removing the second medium and adding a third medium comprising the CDK inhibitor, the cAMP activator, the ROCK inhibitor and the ascorbic acid; and(d) removing the third medium and adding a fourth medium comprising the cAMP activator, the ROCK inhibitor and the ascorbic acid.

13. The method of claim 12, whereinin step (a), the cells are cultured in the first medium for one to three days; in step (b), the cells are cultured in the second medium for one to three days; in step (c), the cells are cultured in the third medium for one to three days; and in step (d), the cells are cultured in the fourth medium for one to three days.

14. The method of claim 12 or 13, wherein the first medium comprises DMEM.

15. The method of claim 12 or 13, wherein the second medium, the third medium and the fourth medium comprise DMEM / F 12 and Neural Basal Medium, supplemented with N2 and B27.

16. A method of generating induced retinal progenitor cells (induced RPCs), comprising the steps of:(a) culturing eye fibroblasts in a culture vessel containing a first medium comprising a DNMT inhibitor;(b) removing the first medium and adding a second medium comprising a DNMT inhibitor and a HD AC inhibitor(c) removing the second medium and adding a third medium comprising a CDK inhibitor, a cAMP activator, a ROCK inhibitor and ascorbic acid;(d) removing the third medium and adding a fourth medium comprising a cAMP activator, a ROCK inhibitor and ascorbic acid;(e) identifying induced RPCs that express one or more retinal markers selected from the group consisting of SOX2, PAX6, VSX2, NEURODI. CRX and RCVRN and any combination thereof; and(f) isolating the identified induced RPCs.

17. The method of claim 16, wherein the induced RPCs express a glutamate receptor.

18. The method of claim 16 or 17, wherein the eye fibroblasts are fibroblasts from Tenon’s capsule.

19. The method of claim 18, wherein the eye fibroblasts are human fibroblasts from Tenon’s capsule.

20. The method of any of claims 16 to 19, wherein the DNMT inhibitor is RG108, the HD ACinhibitor 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 of claim 20, wherein RG108 is present in a concentration of 1-100 pM. VPA is present in a concentration of 1-100 rnM, SU9516 is present in a concentration of 1-100 pM, FSK is present in a concentration of 1-100 pM, Y-27632 is present in a concentration of 1-100 pM, and ascorbic acid is present in a concentration of 1-100 pM.

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

23. The method of claim 21, wherein RG108 is present in a concentration of about 20 pM, VPA is present in a concentration of about 3 mM, SU9516 is present in a concentration of about 10 pM, FSK is present in a concentration of about 10 pM, Y-27632 is present in a concentration of about 10 pM, and ascorbic acid is present in a concentration of about 10 pM.

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

25. The method of any of claims 16 to 24, wherein the second medium, the third medium and the fourth medium comprise DMEM / F12 and Neural Basal Medium, supplemented with N2 and B27.

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

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

28. Induced retinal progenitor cells (induced RPCs) having highly expressed SOX2, NESTIN andprotein tyrosine phosphatase receptor type N (PTPRN) compared with primary' RPC, or a cell population comprising the induced RPCs.

29. A composition comprising the induced RPCs of claim 27 or 28 or a cell population comprising the induced RPCs and a pharmaceutically acceptable carrier.

30. A method for treating a photoreceptor degenerative disease in a subject in thereof, comprising delivering to the eye of the subject an effective amount of induced RPCs of claim 27 or 28 or a cell population comprising the induced RPCs or a composition of claim 29.

31. The method of claim 30, wherein the amount of the induced RPCs is effective in rescuing color and central vision of the subject.

32. The method of 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’s disease.

33. Use of induced RPCs of claim 27 or 28 or a cell population comprising the induced RPCs or a composition of claim 29 for manufacturing a medicament for treating a photoreceptor degenerative disease in a subject in need thereof.

34. Use of claim 33, wherein the induced RPCs are effective in rescuing color and central vision of the subject.

35. Use of 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’s disease.

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