Composition for inducing functional cirpe cells by means of chemical reprogramming, induction method and use

Abstract

The present invention belongs to the field of biotechnology. Disclosed in the present invention are a composition for inducing functional ciRPE cells by means of chemical reprogramming, an induction method and the use. Provided in the present invention is a two-step chemical reprogramming strategy capable of transdifferentiating fibroblasts into functional RPE (ciRPE) cells via an eye field (EF) / optic vesicle (OV) intermediate state. These ciRPE cells are highly similar in structure and function to native RPE cells, have no tumorigenic risk after transplantation into the retina of RD rats, and can integrate into host tissues.

Description

A composition, induction method and application of chemical reprogramming to induce functional ciRPE cells

[0001] Cross-reference to related applications

[0002] This application claims priority to the following application: Patent application No. 2025103506776, filed on March 24, 2025, entitled “A composition, induction method and application of chemical reprogramming to induce functional ciRPE cells”, the contents of which are incorporated herein by reference in their entirety. Technical Field

[0003] This invention belongs to the field of biotechnology, specifically relating to a composition, induction method and application of chemical reprogramming to induce functional ciRPE cells. Background Technology

[0004] Retinal pigment epithelium (RPE) is crucial for retinal health, playing a key role in maintaining photoreceptor function and the blood-retinal barrier. RPE dysfunction or loss is a key driver in the pathogenesis of destructive retinal degenerative diseases (RD), such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Stargardt's disease, which lead to progressive vision loss and, in later stages, irreversible blindness. Given the vital role of RPE cells in retinal function, repairing or replacing damaged RPE cells is a transformative strategy for treating these debilitating diseases. However, in regenerative medicine, generating fully functional and therapeutically safe RPE cells remains a significant challenge.

[0005] Currently, there are two main methods for preparing RPE cells: isolating RPE cells from natural tissues or differentiating them from pluripotent stem cells. However, both strategies still face certain challenges. RPE cells derived from natural tissues are scarce, and their function rapidly diminishes during culture, limiting their application in large-scale therapy. Conversely, while pluripotent stem cell-derived RPE cells are theoretically an ideal source, they raise tumorigenicity and profound ethical concerns. Another approach involves direct reprogramming using ectopic transcription factors (TFs), which can bypass the pluripotency stage and directly convert fibroblasts into RPE-like cells. Although promising, this method still faces challenges such as the risk of genome integration, low reprogramming efficiency, and high cost, which may limit its wider clinical application. Summary of the Invention

[0006] Identifying appropriate intermediate cell states is crucial for improving the efficiency and stability of retinal reprogramming (RPE). Progenitor cells derived from eye-field (EF) cells or optic vesicle (OV) cells, which naturally differentiate into RPE cells during retinal development, represent ideal intermediate states. These cells possess significant plasticity and differentiation potential, making them the biologically relevant step in guiding somatic cells toward their RPE fate. Using EF (or OV) as an intermediate state not only improves reprogramming efficiency but also minimizes the risk of generating unwanted cell types, ensuring a more stable and reproducible conversion process.

[0007] In view of this, in order to solve the above-mentioned technical problems existing in the prior art, the purpose of the present invention is to provide a composition, induction method and application of chemical reprogramming to induce functional ciRPE cells.

[0008] The above-mentioned objective of this invention is achieved through the following technical solution:

[0009] The first aspect of the present invention provides a method for inducing fibroblasts into functional ciRPE cells through two-step reprogramming using a small chemical molecule composition, comprising: treating fibroblasts with reprogramming medium 1 to induce fibroblasts to be reprogrammed into EF-like or OV-like cells, and then treating EF-like or OV-like cells with reprogramming medium 2 to induce EF-like or OV-like cells to be reprogrammed into ciRPE cells.

[0010] The reprogramming culture medium 1 includes a small chemical molecule composition 1, and the reprogramming culture medium 2 includes a small chemical molecule composition 2.

[0011] When the fibroblasts are mouse-derived fibroblasts, the chemical small molecule composition 1 is specifically a combination of LDN193189, A 83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108 or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof;

[0012] When the fibroblasts are mouse-derived fibroblasts, the chemical small molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof.

[0013] When the fibroblasts are human-derived fibroblasts, the chemical small molecule composition 1 is specifically a combination of CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof;

[0014] When the fibroblasts are human-derived fibroblasts, the chemical small molecule composition 2 is specifically a combination of nicotinamide, Activin A, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof.

[0015] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0016] Furthermore, the reprogramming culture medium 1 and the reprogramming culture medium 2 are respectively composed of a basic culture medium supplemented with the chemical small molecule composition 1 and the chemical small molecule composition 2 for culturing.

[0017] Furthermore, the reprogramming medium 1 uses basal medium 1, specifically an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% vitamin A-free B27, 7.5% BSA, 1% NEAA, 1% P / S, and 10 ng / mL bFGF.

[0018] Furthermore, the reprogramming medium 2 uses basal medium 2, specifically DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055mM 2-mercaptoethanol.

[0019] Furthermore, the method also includes proliferating and culturing ciRPE cells.

[0020] Furthermore, the proliferation culture uses a ciRPE cell proliferation medium with the following components: DMEM / F12 / GlutaMAX as substrate, supplemented with 1% N2, 2% vitamin A-free B27, 1% NEAA, 1% P / S and 0.1mM 2-mercaptoethanol.

[0021] Furthermore, the proliferation medium for ciRPE cells also includes one or more of the following: 10 ng / mL bFGF, 20 ng / mL EGF, 10 μM Y-27632, and 0.5 μM A 83-01.

[0022] Furthermore, the method also includes functional culture of ciRPE cells.

[0023] Furthermore, the functional culture uses a ciRPE cell function maintenance medium with the following components: DMEM / F12 / GlutaMAX plus 1% N2, 2% vitamin A-containing B27, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol.

[0024] Furthermore, when the fibroblasts are mouse-derived fibroblasts, the function maintenance culture is supplemented with 0.2 μM Activin A, 0.5 μM retinoic acid, 1 μM BMP4 and 10 mM nicotinamide.

[0025] Furthermore, when the fibroblasts are human-derived fibroblasts, the function maintenance culture is supplemented with 0.1 μM Activin A, 0.5 μM retinoic acid, and 0.5 μM BMP4.

[0026] Furthermore, when the fibroblasts are mouse-derived fibroblasts, the concentrations of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, and RG108 in the chemical small molecule composition 1 are 0.1 mM, 0.5 mM, 0.2 mM, 0.2 mM, and 10 mM, respectively.

[0027] Furthermore, when the fibroblasts are mouse-derived fibroblasts, the concentration of nicotinamide in the chemical small molecule composition 2 is 10 mM, the concentration of retinoic acid is 1 μM, and the concentration of Activin A is 0.2 mM.

[0028] Furthermore, when the fibroblasts are human-derived fibroblasts, the concentrations of CHIR-99021, LDN193189, Hh-Ag1.5, RG108, BMS-345541, R-268712, BIX-01294, VPA, and SB-431542 in the chemical small molecule composition 1 are 10 μM, 0.2 mM, 0.2 mM, and 10 μM respectively.

[0029] Furthermore, when the fibroblasts are human-derived fibroblasts, the concentration of nicotinamide in the chemical small molecule composition 2 is 10 mM, and the concentration of Activin A is 0.2 mM.

[0030] In some embodiments, LDN193189 is a small molecule compound that acts primarily as a selective inhibitor of the bone morphogenetic protein (BMP) signaling pathway.

[0031] In some implementations, A 83-01 is an effective inhibitor of TGF-βtypeI receptor (ALK5-TD).

[0032] In some implementations, the CKI-7 (dihydrochloride) is an ATP-competitive CKI inhibitor.

[0033] In some embodiments, the Hh-Ag1.5 is a potent Hedgehog (Hh) agonist with an EC50 of 1 nM. The mediated reprogramming breaks the quiescent state of non-damaged liver stem cells, thereby rescuing liver failure.

[0034] In some implementations, CHIR-99021 (Laduviglusib, CT99021) is a GSK-3α and GSK-3β inhibitor.

[0035] In some embodiments, BMS-345541 is a highly selective inhibitor of a catalytic subunit that acts on IKK-2 and IKK-1.

[0036] In some implementations, the RG108 (N-Phthalyl-L-tryptophan) is a DNA methyltransferase inhibitor.

[0037] In some implementations, R-268712 is an orally active and selective ALK-5 inhibitor.

[0038] In some implementations, BIX-01294 is a reversible and highly selective inhibitor of G9a and GLP histone methyltransferases.

[0039] In some implementations, the VPA (NSC-93819, valproic acid, sodium valproate, and sodium divalproate) is an approved medicine primarily used to treat epilepsy and bipolar disorder, and also for the prevention of migraines.

[0040] In some embodiments, SB-431542 is a small molecule inhibitor that blocks internal mediators of TGF-β receptor type I, thereby reducing TGF-β1-mediated proliferation, cytokine and collagen expression.

[0041] In some implementations, the method specifically includes the following steps:

[0042] Phase 1: Chemical transformation of EF cells in MEFs (mouse fibroblasts), specifically the following steps: 1) Four days before chemical transformation, thaw Matrigel overnight at 4°C. The next day, pre-cool the 6-well plate at 4°C for at least 1 hour. Dilute Matrigel 1:40 with DMEM / F12 / GlutaMAX solution, add 1 mL of the diluted Matrigel solution to each well of the 6-well plate, and incubate overnight at 4°C (Note: To ensure optimal performance, Matrigel must be thawed at 4°C and repeated freeze-thaw cycles should be avoided).

[0043] 2) Two days before chemical induction, thaw the cryopreserved primary MEFs in a 37°C water bath and remove the cryoprotectant by centrifugation. Resuspend the cells in MEF medium (DMEM medium containing 10% FBS) at 20 × 10⁶ cells per well. 4 Seed the MEF cells at a density of 1000 cells / well in 6-well plates. Preheat the Matrigel-coated 6-well plates at 37°C for at least 30 minutes before seeding (Note: MEF cell density is crucial for the success of chemical induction. Ensure uniform cell distribution; too few cells will affect proliferation and cell state, while overcrowding may affect induction success rate. Monitor the health and growth of MEF cells regularly after seeding to ensure they adhere well and proliferate as expected. Proper preheating of the matrix-coated plates is essential for optimal cell attachment and performance).

[0044] 3) One day before chemical induction, replace the MEF medium with fresh medium to allow cells to proliferate for one day. Regularly check the growth and status of MEF cells to ensure optimal cell health and density (Note: The growth status of MEF cells is crucial for successful conversion. Long-term storage of MEFs in liquid nitrogen will also reduce conversion efficiency. This study used freshly prepared MEF cells or MEF cells stored in liquid nitrogen for no more than 6 months).

[0045] 4) On the day of chemical induction, prepare the first stage of fresh reprogramming medium 1, prepared in the dark to ensure that all components, especially the chemical reagents, are completely dissolved. After incubating MEF cells in MEF medium overnight, wash the cells twice with 1×PBS and replace the MEF medium with freshly prepared reprogramming medium 1. Replace the reprogramming medium 1 once the next day.

[0046] 5) During the first stage of induction, cells proliferate rapidly within the first 1-4 days, changing their morphology from elongated spindle-shaped to small oval, and cell colonies appear. After one week, the proliferation rate of the colonies slows down. At this point, surrounding non-clonal cells are mechanically removed using the tip of a yellow micropipe to promote continued colony expansion. After about one week, the cells will begin to exhibit a cobblestone-like morphology. Thereafter, the culture medium is changed daily until EF-like cells are formed.

[0047] Phase Two: EF cells generate ciRPE cells, specifically including: In the second phase of induction, cells exhibit the initial RPE cell morphology but still lack maturity and pigmentation. In this phase, pigmentation is induced using reprogrammed medium 2 to promote further maturation of ciRPE cells. The medium is changed daily. After two weeks of treatment, the cells are nearly fully mature and exhibit significant pigmentation. Subsequently, ciRPE cells are passaged and expanded in maintenance medium.

[0048] In some implementations, the specific steps of the dynamic alternating culture of ciRPE cells are as follows: MEF-derived ciRPE cells are cultured in a dynamically alternating medium to balance proliferation and RPE-specific function. After the first passage, during the initial expansion phase (days 0-7), cells are cultured in proliferation medium to maximize cell growth. Subsequently, cells are briefly exposed to maintenance medium for 1-2 days every 3-5 days. This alternating approach effectively supports cell expansion and the preservation of early RPE-like characteristics.

[0049] In some implementations, the method specifically includes the following steps:

[0050] Phase 1: Chemical transformation of HEFs (human fibroblasts) into OV-like cells, specifically including:

[0051] 1) Thaw Matrigel overnight at 4°C. The next day, pre-chill the 6-well plate at 4°C for at least 1 hour. Dilute Matrigel 1:40 with DMEM / F12 / GlutaMAX solution and add the diluted Matrigel solution to each pre-chilled well of the 6-well plate. Then incubate the plate overnight at 4°C.

[0052] 2) On the second day, thaw the cryopreserved primary HEFs in a 37°C water bath and centrifuge to remove the cryoprotectant. Resuspend the cells in 15% FBS-DMEM medium at 15 × 10⁻⁶ cells per well. 4 The cells were seeded at a density of 100 cells per well in 6-well plates. The Matrigel-coated plates were preheated at 37°C for at least 30 minutes before seeding the HEF cells (Note: Freshly prepared HEF cells or HEF cells stored in liquid nitrogen for no more than 2 months were used in this study).

[0053] 3) When the HEF cell density reaches 60-70%, chemical induction can be initiated. For the first stage of induction, prepare fresh reprogramming medium 1 in the dark, ensuring all components, especially the chemical reagents, are completely dissolved. Wash HEF cells twice with 1×PBS, replacing with freshly prepared reprogramming medium 1 every two days.

[0054] 4) In the first stage, cells proliferate rapidly within the first week, changing their morphology from a slender spindle shape to a small oval shape. Around day 12, some stable clones of uniform size and morphology will form. At this stage, surrounding non-clonal cells are mechanically removed with the tip of a yellow pipette micropipette to promote further clonal expansion. Thereafter, the culture medium is changed daily until OV-like cells are formed.

[0055] Phase 2: OV-like cells generate hciRPE cells

[0056] Pigmentation was induced in reprogrammed medium 2 to promote further maturation of hciRPE cells, with the medium being replaced daily. After two weeks of treatment with the medium, the cells exhibited RPE-like morphology and some pigmentation. Subsequently, hciRPE cells were passaged and expanded in maintenance medium.

[0057] For long-term culture, passage hciRPE cells using mechanical dissociation methods and seed them onto Matrigel-coated plates, maintaining a confluence of 60-80%. The maintenance medium should be refreshed every 2-3 days to ensure optimal cell health. Regularly monitor the cells for RPE-specific characteristics, including morphology, pigmentation, and expression of key markers.

[0058] A second aspect of the present invention provides a small molecule chemical composition comprising small molecule chemical composition 1 and small molecule chemical composition 2, wherein small molecule chemical composition 1 and small molecule chemical composition 2 are selected from any one of the following groups:

[0059] Chemical small molecule composition 1 is specifically a combination of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108 or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof; chemical small molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof.

[0060] Chemical small molecule composition 1 is specifically a combination of CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542 or equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors thereof; chemical small molecule composition 2 is specifically a combination of nicotinamide, Activin A or equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors thereof.

[0061] Furthermore, the chemical small molecule composition includes a pharmaceutically acceptable carrier or excipient.

[0062] Furthermore, the carrier or excipient is selected from one or more of the following groups: water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymeric surfactants; colloidal drug delivery systems or polymeric drug delivery systems; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, pH buffers; binders, fillers, lubricants or other pharmaceutical excipients.

[0063] Furthermore, the pharmaceutical dosage forms that can be prepared from the chemical small molecule composition are selected from one or more of the following groups: solid dosage forms, including: powders, granules, tablets, pills, capsules, sustained-release preparations, controlled-release preparations, or other solid dosage forms; liquid dosage forms, including: injections, infusions, suspensions, or other liquid dosage forms; gaseous dosage forms; semi-solid dosage forms; reprogrammed formulations or reagents.

[0064] Furthermore, the chemical small molecule composition also includes organic solvents, physiological saline, or other carriers or excipients.

[0065] In this invention, the general requirements for excipients are that they are stable, have no incompatibility with the active pharmaceutical ingredient, do not produce side effects, do not affect efficacy, are not easily deformed, cracked, moldy, or infested by insects at room temperature, are harmless to the human body, have no physiological effects, do not produce chemical or physical reactions with the active pharmaceutical ingredient, and do not affect the content determination of the active pharmaceutical ingredient. A thorough discussion of pharmaceutically acceptable carriers or excipients can be found in Remington's Pharmaceutical Sciences (MackPub.Co., NJ1991). These carriers or excipients include, but are not limited to: aqueous solutions such as water, saline, and phosphate buffer; organic solvents such as DMSO (dimethyl sulfoxide), glycerol, and ethanol; microspheres, liposomes, microemulsions, and polymeric surfactants; colloidal drug delivery systems, novel polymeric drug delivery systems, novel drug carriers, and other pharmaceutical carriers; preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, and pH buffers in liquid formulations; and binders, fillers, lubricants, and other pharmaceutical excipients in tablets.

[0066] A third aspect of the present invention provides a reprogrammable culture medium assembly, the reprogrammable culture medium assembly comprising reprogrammable culture medium 1 and reprogrammable culture medium 2;

[0067] The reprogramming culture medium 1 comprises a small chemical molecule composition 1 and a basal culture medium 1. Specifically, the small chemical molecule composition 1 is a combination of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof; or a combination of CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof. The basal culture medium 1 is an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1%... N2, 1% Vitamin A-free B27, 7.5% BSA, 1% NEAA, 1% P / S, bFGF;

[0068] The reprogrammed culture medium 2 comprises a small chemical molecule composition 2 and a basal culture medium 2. The small chemical molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof, or a combination of nicotinamide, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof. The basal culture medium 2 is DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055mM 2-mercaptoethanol.

[0069] Further, in the small molecule chemical composition 1, the concentrations of LDN193189 are 0.1 mM, A83-01 are 0.5 mM, CKI-7 are 5 μM, Hh-Ag1.5 are 0.5 mM, CHIR-99021 are 3 μM, BMS-345541 are 0.2 mM, and RG108 are 10 μM, or the concentrations of CHIR in the small molecule chemical composition 1 are... The concentrations of -99021, LDN193189, Hh-Ag1.5, RG108, BMS-345541, R-268712, BIX-01294, VPA, and SB-431542 were 10 μM.

[0070] Furthermore, the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM, the concentration of retinoic acid is 1 μM, and the concentration of Activin A is 0.2 mM, or the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM and the concentration of Activin A is 0.2 mM.

[0071] A fourth aspect of the present invention provides the use of a small chemical molecule composition in chemically reprogrammed fibroblasts induced to become functional ciRPE cells, wherein the small chemical molecule composition is the same as that described in the second aspect of the present invention.

[0072] Furthermore, the fibroblasts are fibroblasts of humans or non-human mammals.

[0073] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0074] In some embodiments, the chemical small molecule composition is used in stages according to a two-step reprogramming strategy. In the first stage, composition 1 induces fibroblasts to transform into intermediate cells resembling the eye field (EF) / optic vesicle (OV). In the second stage, composition 2 induces the intermediate cells to further differentiate and mature into functional ciRPE cells. The two-stage composition synergistically regulates intracellular signaling pathways and epigenetic modifications to achieve a directional change in cell fate.

[0075] In some embodiments, the chemical small molecule composition is adapted to different components and concentration ratios according to the reprogramming requirements of mouse and human fibroblasts. Mouse fibroblasts are adapted to composition 1 containing LDN193189, A83-01, CKI-7, etc., and composition 2 containing nicotinamide, retinoic acid, and Activin A. Human fibroblasts are adapted to composition 1 containing CHIR-99021, R-268712, BIX-01294, etc., and composition 2 containing nicotinamide and Activin A. Both can efficiently achieve the transdifferentiation of fibroblasts into functional ciRPE cells.

[0076] The fifth aspect of the present invention provides the use of a chemical small molecule composition in the preparation of a product in which chemically reprogrammed fibroblasts are induced to become functional ciRPE cells, wherein the chemical small molecule composition is the chemical small molecule composition described in the second aspect of the present invention.

[0077] Furthermore, the fibroblasts are fibroblasts of humans or non-human mammals.

[0078] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0079] In some implementations, the product is a cell reprogramming reagent, culture medium additive, or special inducing agent. The product can be prepared into corresponding specifications according to the reprogramming requirements of mouse or human fibroblasts. Each small molecule component is encapsulated in a stable form and can be directly mixed with basal culture medium to form reprogramming culture medium 1 and reprogramming culture medium 2, which are suitable for large-scale in vitro induction and preparation of functional ciRPE cells.

[0080] In some implementations, the product can also be formulated into stable formulations, including powders, lyophilized powders, suspensions, etc., in combination with pharmaceutically acceptable carriers and excipients, which facilitates storage, transportation and experimental operations. The small molecule components in the formulation can maintain stable biological activity and can efficiently initiate the two-step chemical reprogramming process of fibroblasts in an in vitro culture system, inducing them to transdifferentiate into functional ciRPE cells.

[0081] A sixth aspect of the present invention provides the application of a reprogramming culture medium combination in chemically reprogrammed fibroblasts induced into functional ciRPE cells, wherein the reprogramming culture medium combination is the reprogramming culture medium combination described in the third aspect of the present invention.

[0082] Furthermore, the fibroblasts are fibroblasts of humans or non-human mammals.

[0083] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0084] In some implementations, the reprogramming culture medium combination is used in stages according to a two-step reprogramming strategy. In the first stage, reprogramming culture medium 1 is used to induce fibroblasts to be reprogrammed into eye domain (EF)-like / optical vesicle (OV)-like intermediate cells. In the second stage, reprogramming culture medium 2 is used to induce the intermediate cells to further differentiate and mature into functional ciRPE cells. The two stages of culture medium work together to achieve a directional conversion of cell fate.

[0085] In some implementations, the reprogramming culture medium combination is adapted to the corresponding basal culture medium and chemical small molecule composition concentration ratio according to the reprogramming requirements of mouse and human fibroblasts. All of them are serum-free and chemically defined culture medium systems, which can stably induce fibroblasts to transdifferentiate through a specific intermediate state into functional ciRPE cells with morphology and function highly similar to natural RPE cells in vitro.

[0086] The seventh aspect of the present invention provides the use of a reprogramming culture medium combination in the preparation of a product in which chemically reprogrammed fibroblasts are induced to become functional ciRPE cells, wherein the reprogramming culture medium combination is the reprogramming culture medium combination described in the third aspect of the present invention.

[0087] Furthermore, the fibroblasts are fibroblasts of humans or non-human mammals.

[0088] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0089] In some implementations, the product is a specialized culture medium kit adapted for reprogramming mouse or human fibroblasts. The kit contains pre-mixed solutions or dry powder formulations of reprogramming culture medium 1 and reprogramming culture medium 2, which can be directly reconstituted or diluted for use. It can meet the full-stage culture needs of fibroblasts from EF-like / OV-like intermediate state to functional ciRPE cells.

[0090] In some implementations, the product can be prepared as a concentrated culture medium, ready-to-use culture medium, or culture medium additive kit for laboratory-scale culture, with each component maintaining stable biological activity and adaptable to conventional cell culture operation systems. The prepared product can achieve efficient and stable two-step chemical reprogramming of fibroblasts and ensure the morphological and functional integrity of the induced ciRPE cells.

[0091] The eighth aspect of the present invention provides a kit or reagent kit for inducing fibroblasts to undergo two-step reprogramming into ciRPE cells using a small chemical molecule composition, said kit or reagent kit comprising the small chemical molecule composition described in the second aspect of the present invention, or the reprogramming culture medium composition described in the third aspect of the present invention.

[0092] Furthermore, the fibroblasts are fibroblasts of humans or non-human mammals.

[0093] Furthermore, the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts, and / or pancreatic fibroblasts.

[0094] In some embodiments, the kit or reagent kit also includes auxiliary reagents required for cell culture, which are selected from one or more of Matrigel, PBS buffer, cell digestion solution, sterile culture consumables, and cell cryopreservation solution, and can meet the needs of the entire process of fibroblast culture and reprogramming induction.

[0095] In some embodiments, the chemical small molecule composition in the kit or reagent is packaged into reprogramming composition 1 and reprogramming composition 2 according to the reprogramming requirements of mouse / human fibroblasts. Each component is prepared at the optimal working concentration and packaged independently, and can be directly added to the corresponding basal culture medium to prepare reprogramming culture medium 1 and reprogramming culture medium 2.

[0096] In some implementations, the kit or reagent kit also includes a matching ciRPE cell proliferation medium, a dry powder or pre-prepared solution of a function maintenance medium, and an RPE-specific marker detection reagent for cell identification, which can enable subsequent culture, expansion and preliminary identification of ciRPE cells.

[0097] In some implementations, the kit or reagent kit comes with detailed instructions for use, which specify the fibroblast seeding density, culture medium replacement cycle, culture conditions for each stage of reprogramming, key points for cell morphology observation, and methods for detecting reprogramming efficiency, and is suitable for large-scale induction and preparation of functional ciRPE cells in the laboratory.

[0098] A ninth aspect of the present invention provides a functional ciRPE cell, wherein the functional ciRPE cell is a functional ciRPE cell prepared by the method described in the first aspect of the present invention.

[0099] In some embodiments, the functional ciRPE cells are obtained by two-step chemical reprogramming induction of fibroblasts from human or non-human mammals, with mouse fibroblasts differentiated from ocular (EF)-like cells in an intermediate state, and human fibroblasts differentiated from optic vesicle (OV)-like cells in an intermediate state.

[0100] In some implementations, the functional ciRPE cells can be stably passaged in vitro, maintaining typical retinal pigment epithelial cell morphology, expression of specific markers, and complete biological function after passage, with normal karyotype and no risk of abnormal proliferation or tumorigenesis.

[0101] In some implementations, the functional ciRPE cells highly express retinal pigment epithelial cell-specific markers such as ZO-1, Pax6, Rpe65, Mitf, and Best1, and the markers exhibit a polarized distribution. They also possess a complete tight junction structure and a stable epithelial resistance (TEER) value.

[0102] In some implementations, the functional ciRPE cells possess the core biological functions of natural retinal pigment epithelial cells, enabling them to phagocytose and clear the outer segment of photoreceptors, and to secrete growth factors such as VEGF and PEDF in a polar manner. Furthermore, after transplantation into animal models of retinal degenerative diseases, they can integrate into the host retinal tissue, effectively protecting photoreceptor cells, inhibiting their apoptosis, and restoring visual function.

[0103] The tenth aspect of the present invention provides the application of the method described in the first aspect of the present invention or the functional ciRPE cells described in the ninth aspect of the present invention in any of the following aspects:

[0104] (1) Use in the preparation of drugs or preparations for treating retinal degenerative diseases;

[0105] (2) Application in the preparation of cell therapy products for repairing or replacing damaged retinal pigment epithelial (RPE) cells;

[0106] (3) Application in cell replacement therapy for retinal degenerative diseases;

[0107] (4) Application in constructing in vitro cell models of retinal degenerative diseases;

[0108] (5) Application in screening candidate drugs for the treatment of retinal degenerative diseases;

[0109] (6) Applications in basic research on the mechanisms of retinal development;

[0110] (7) Application in research on the regulation of retinal pigment epithelial cell function;

[0111] (8) Application in the preparation of biological products that improve or restore visual function;

[0112] (9) Application in the treatment of retinal degenerative diseases;

[0113] (10) Application in cell therapy for repairing or replacing damaged retinal pigment epithelial (RPE) cells;

[0114] (11) Application in improving or restoring visual function.

[0115] Furthermore, the aforementioned retinal degenerative diseases include age-related macular degeneration, retinitis pigmentosa, Stargardt's disease, cone / rod cell dystrophy, Leber congenital amaurosis, myopic macular degeneration, or retinal degenerative changes secondary to diabetic retinopathy and retinal vein occlusion.

[0116] In some embodiments, the drug or formulation is a cell preparation comprising functional ciRPE cells and pharmaceutically acceptable carriers, excipients or diluents, and can be prepared into ophthalmic dosage forms such as injections and suspensions, suitable for subretinal space administration.

[0117] In some implementations, the cell therapy product is a functional ciRPE cell preparation that meets clinical application standards. This product is prepared after in vitro expansion, purification, and quality control, and has the characteristics of being non-tumorigenic, highly active, and able to stably integrate into the host retinal tissue and exert physiological functions.

[0118] In some implementations, the in vitro cell model is an in vitro model constructed by co-culturing functional ciRPE cells with retinal-associated cells or simulating the pathological microenvironment of retinal degenerative diseases, which can be used to study the pathogenesis, pathological process and intercellular interactions of the disease.

[0119] In some embodiments, the biological product uses functional ciRPE cells as the core active ingredient, or contains active factors secreted by functional ciRPE cells that have retinal protective and repair functions, and can be used alone or in combination with other ophthalmic therapeutic drugs.

[0120] The eleventh aspect of the present invention provides a method for treating retinal degenerative diseases, the method comprising administering a therapeutically effective amount of the functional ciRPE cells described in the ninth aspect of the present invention to a subject in need.

[0121] In some implementations, the subject refers to a human or non-human mammal suffering from retinal degenerative diseases. The non-human mammal includes animals such as rodents and primates. The subject exhibits symptoms of visual impairment such as retinal pigment epithelial cell dysfunction / loss, photoreceptor cell damage and apoptosis, accompanied by decreased vision, visual field defects, and abnormal color vision. This includes individuals suffering from retinal degenerative diseases such as age-related macular degeneration, retinitis pigmentosa, and Stargardt's disease.

[0122] In some implementations, the administration refers to transplanting the functional ciRPE cells to the retinal lesion site of the subject through an ophthalmologically acceptable administration method, preferably subretinal space transplantation. It also includes cell injection combined with ophthalmic surgical procedures, sheet transplantation, etc. The administration process must follow aseptic operation principles to ensure that the cells can be successfully integrated into the host retinal pigment epithelium and perform physiological functions.

[0123] In some implementation schemes, the therapeutically effective dose refers to the number of functional ciRPE cells that can effectively improve the symptoms of retinal degenerative diseases in the subject, repair damaged retinal tissue, protect photoreceptor cells from apoptosis, and restore or enhance visual function. This dose can be reasonably adjusted according to the subject's species, age, weight, disease severity, and lesion location. Furthermore, cell administration at this dose will not produce significant toxic side effects on the subject, while simultaneously enabling cell survival, integration, and functional performance in the host retinal tissue.

[0124] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows:

[0125] This invention addresses the complex challenges of generating functional regenerated polymorphic fibroblast (RPE) cells by developing an innovative two-stage chemical induction protocol. This protocol utilizes a single-cell reprogramming compound screening platform (scRCF), an advanced platform that combines single-cell transcriptomics-driven computational prediction with a sophisticated drug sequence screening system. This platform systematically identifies optimal small molecules to induce fibroblasts into an EF-like state, ultimately generating functional chemically induced RPE (ciRPE) cells. These ciRPE cells are highly similar to natural RPE cells in morphology, gene expression, and fundamental functional properties. In a rat model of renal dysplasia (RD), ciRPE cell transplantation integrates into the host RPE layer, significantly protecting photoreceptors and significantly restoring visual function. Mechanistic studies providing insights into this dynamic demonstrate that compounds synergistically activate endogenous TFs, such as Ascl1 and Olig2, directing fibroblast reprogramming towards the RPE cell phenotype. This study introduces a scalable, non-integrative, and cost-effective chemical approach to generate functional RPE cells, offering a promising and innovative strategy for cell replacement therapy targeting RD, with broad application prospects. Attached Figure Description

[0126] Figure 1: Results of screening EF reprogrammed small molecules using the scRCF system, where A represents the scRCF workflow. The inputs to scRCF include scRNA-seq data from the initial and target cell types, and three pre-established databases: 1) a small molecule perturbation database integrating data from GEO and LINCS L1000, retaining only TFs identified in AnimalTFDB3.0; 2) a small molecule target database and classification information, including information from STITCH, Drug Repurposing Hub, and MedChemExpress; and 3) a signaling network database derived from Reactome and OmniPath. scRCF comprises three main steps: 1) Identifying differentially expressed transcription factors based on scRNA-seq data and initially screening candidate signaling proteins using signaling network analysis; 2) Dividing the pre-screened signaling proteins into communities and accumulating scores based on the distribution of small molecule targets within these communities. Small molecules with the highest scores are selected from each pathway subset as the final output, categorized by signaling pathway; and 3) Further screening of candidate small molecules using DRUG-seq2. B is a UMAP visualization of scRNA-seq data from MEF and primary EF (pEF) cells, used to predict small molecules that promote reprogramming between the two cell types. C is a volcano plot showing differentially expressed TFs (DETFs) identified from scRNA-seq data of MEF and EFs, analyzed using Seurat. DETFs were defined as P < 0.05, log2 > 1. D shows candidate small molecules identified through preliminary screening by scRCF for reprogramming MEF into EF cells. E is a bar chart showing the Z-scores of cells treated with the LAC+1 small molecule combination, based on the gene sets of neuroectoderm and EF-related genes. The Z-score represents the relative change in gene expression for each drug; higher values ​​reflect a stronger impact on the gene set. The bars are sorted from highest to lowest average Z-score, with a dashed line indicating a baseline score of zero. F is a heatmap of gene expression profiles of neuroectoderm and EF-related genes in cells after LAC+1 small molecule combination treatment. The color scale represents the expression values ​​after log2 transformation; red indicates high expression, blue indicates low expression, and white indicates intermediate expression.

[0127] Figure 2: Results of generating ciRPE cells using a two-stage chemical reprogramming strategy. A shows representative morphological changes of MEF cells exposed to reprogramming medium (RM) containing 10 small molecules (Phase 1) at different time points. Scale bar: 400 μm. B shows the expression of EF-related genes and early RPE development-related genes at specified time points as shown by qRT-PCR analysis. C shows representative morphological images of cells exposed to differentiation and maturation medium (DM) containing three compounds (Phase 2). DMSO and dimethyl sulfoxide were used as negative controls. M3: NIC, RA, Activin A, scale bar: 200 μm. D shows the expression of RPE-related marker genes at specified time points as shown by qRT-PCR analysis. E is a schematic diagram of the genetic lineage tracing strategy and chemical reprogramming of MEF-derived ciRPE cells. F shows changes in cell morphology and tdTomato fluorescence expression at different days during induction. Scale bar: 400 μm. G shows the percentage of tdTomato+ cells induced by the candidate medium at different days. H represents the percentage of tdTomato+ cells treated with candidate media (all 13 compounds), minus the specified compounds from the mixture. Each point represents one biological replication. "-" indicates the removal of the specified compound. I represents the percentage of tdTomato+ cells before and after reprogramming medium optimization at different time points. J is a schematic diagram of the compound-induced MEF to ciRPE cell reprogramming system, and representative cell morphological changes at key reprogramming time points. MM represents MEF medium, RM represents reprogramming medium, and DM represents differentiation / maturation medium. Scale bar, 200 μm. Figure 2 shows all representative examples of data from at least three independent experiments. Data are Mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001;

[0128] Figure 3: Characteristic images of ciRPE cells. A shows immunostaining of MEF-derived ciRPE cells expressing ZO-1, Pax6, Rpe65, Mitf, Best1, and Cralbp. Scale bar, 50 μm. B is a Z-stack confocal micrograph showing the polarized expression of typical RPE markers in ciRPE cells. ZO-1 (green) shows apical localization (top), while Best1 (red) shows basal lateral localization (bottom). Scale bar, 10 μm. C is a transmission electron microscopy image of ciRPE cells showing apical microvilli (yellow arrow), melanin granules (red arrow), and tight junctions (black arrow). Scale bar, 1 μm. D is a confocal micrograph showing the phagocytosis of the photoreceptor outer segment (green) by ciRPE cells. The apical side of ciRPE cells is stained with ZO-1 (purple), and the nuclei are reverse-stained with DAPI (blue). Scale bar, 50 μm. E shows the apical and basal polarization of ciRPE cells grown on Transwells, secreting PEDF and VEGF. F shows morphological images of the dome structure formed by MEF-derived ciRPE cells during in vitro culture. Red arrows indicate dome morphology observed under different phase-contrast microscopy conditions. Scale bar, 50 μm. G shows the TEER value of ciRPE cells measured at 30 days. Figure 3 provides all representative examples of data from at least three independent experiments. Data are mean ± SD;

[0129] Figure 4: Molecular roadmap of ciRPE chemical reprogramming, where A is a schematic diagram of the multi-omics sequencing analysis strategy for MEF to ciRPE cell reprogramming process. B shows principal component analysis (PCA) of RNA-seq and CUT&Tag data (H3K4me3, H3K27ac, and H3K27me3) of samples collected at reprogramming days 0, 7, 18, and 32 (ciRPE), with pRPE cells as a control. C is a heatmap of differentially expressed genes in MEF to ciRPE cell reprogramming samples at specified time points. The numbers on the heatmap represent independent biological replication. Representative genes (left side of the heatmap) and related gene ontology (GO; right side of the heatmap for each block) are shown. Red and blue represent upregulated and downregulated genes, respectively. D shows the dynamic changes of module-specific gene CUT&Tag peaks (H3K4me3, H3K27ac, H3K27me3) in (C). The red line represents the median peak of CUT & Tag over time, the blue line represents the median RNA expression level, and the gray background line represents the peak at each time point. E shows the UMAP analysis of scRNA-seq data collected at specified time points during MEF reprogramming into ciRPE cells. F shows the UMAP plot of the identified cell types in samples collected at specified time points during the reprogramming process. G shows the bubble chart of representative marker gene expression among different cell types during the reprogramming process. H shows the RNA velocity streamline plot used to predict cell population transitions during the reprogramming process. Arrows indicate the flow rate determined by the proportion of unspliced ​​to spliced ​​transcripts, predicting dynamic changes in cell identity. Black arrows represent RNA velocity flow based on the ratio of unspliced ​​to spliced ​​transcripts, while gray to blue-green arrows are used for visual enhancement to highlight specific trajectories. I shows the similarity analysis of gene expression among different cell types during the reprogramming process. pEF cells represent the data used for small molecule prediction, while pRPE data comes from the GSE183572 dataset;

[0130] Figure 5: A volcano plot showing the differentially active transcription factors (TFs) among different cell types during reprogramming, with data from SCENIC. B is a bar chart showing the MCC scores of the top 15 key TFs. C is a heatmap showing the expression of the top 15 TFs at specified time points during cell reprogramming. D is a normalized analysis of RNA-seq and CUT&Tag sequencing (targeting histone modifications H3K4me3, H3K27ac, and H3K27me3) of the Ascl1, Olig2, Zic1, Pou3f2, and Lhx2 genomic loci at specified time points during cell reprogramming. EF assesses relative reprogramming efficiency by detecting the proportion of tdTomato-positive cells on day 32 after knocking down Ascl1 (E) or Olig2 (F) at specified time points under M7+M3 induction. The reprogramming efficiency of M7+M3 induced cells on day 32 was set to "1", with DMSO as the negative control. WT represents wild-type; Control KD represents scramble shRNA-mediated knockdown. G is a visualization of the scRCF network of potential signaling cascades induced by small molecules targeting Ascl1 and Olig2. Orange rectangles represent perturbation compounds, blue rhombuses represent signal protein targets, white ellipses represent intermediate signal proteins, and green hexagons represent TFs. Figure 5 shows all representative examples of data from at least three independent experiments. Data are Mean ± SD; *P < 0.05, **P < 0.01;

[0131] Figure 6: Chemical reprogramming diagram of HEF-induced human ciRPE (hciRPE) cells. A shows the preliminary list of candidate small molecules for HEF reprogramming into OV cells using scRCF. B is a schematic diagram of the HEF reprogramming process into hciRPE cells, and representative morphological changes at specified time points. HM represents HEF medium, RM represents reprogramming medium, and DM represents differentiation / maturation medium. Scale bar: 300 μm. C shows the proportion of FACS-purified reprogrammed BEST1-EGFP+hciRPE cells. D shows optical microscopy and TEM images of hciRPE cells, displaying melanin granules (red arrows). Scale bar: 1 μm. E shows qRT-PCR data analysis of RPE-related gene expression at specified time points during cell reprogramming. F shows immunostaining analysis of BEST1-EGFP-HEFs-derived hciRPE cells, showing positive expression of ZO-1, RPE65, MITF, and BEST1. Scale bar: 20 μm. G represents PCA analysis of hciRPE cells and control primary hC cells at days 0, 12, 24, and 38 of cell reprogramming. H represents a heatmap analysis of differentially expressed genes in cell samples at specified time points during HEF reprogramming of hciRPE cells. Numbers on the heatmap represent independent biological replication. Representative genes (left side of the heatmap) and associated GOs (right side of the heatmap) for each block are shown. Red and blue represent upregulated and downregulated genes, respectively. I represents the apical and basal polarization secretion of VEGF and PEDF in hciRPE cells grown on Transwells. J represents the 30-day TEER values ​​of hciRPE cells. Figure 6 provides all representative examples of data from at least three independent experiments. Data are mean ± SD;

[0132] Figure 7: Results of ciRPE cell transplantation restoring retinal function in RCS rats. A is a schematic diagram of subretinal ciRPE cell transplantation in RCS rats. B shows OCT images of RCS rats 0, 1, 2, and 3 weeks after subretinal ciRPE cell transplantation. Scale bar: 600 μm. C is a representative in vivo imaging photograph showing tumor formation in nude mice after subretinal transplantation of tdTomato-labeled mESCs (via lentivirus) and tdTomato+ciRPE cells. The right-hand statistical graph shows quantitative analysis data, N=10. D shows representative bright-field (top) and immunofluorescence images (bottom) of eye tissue sections 8 weeks after subretinal ciRPE cell transplantation in RCS rats. The dashed box indicates the cell transplantation area. Cell nuclei are reverse-stained with DAPI (blue). Scale bar: 200 μm. E shows immunofluorescence analysis of a whole retinal section 12 weeks after transplantation, revealing tdTomato+ciRPE cell clusters in the transplantation area. The magnified view of the area depicted by the dashed box shows: (i) the non-transplanted area and (ii) the transplanted area. The statistical graph on the right shows the quantitative analysis data. N = 10. Scale bar, 500 μm. FH is an immunostaining image of tdTomato+ciRPE cells co-expressing Mitf(F), Best1(G), and Pax6(H). Scale bar, 50 μm. I is a representative TUNEL-stained micrograph of a frozen section of the retina of RCS rats 12 weeks after tdTomato+ciRPE cell transplantation (left), with the sham transplantation group as a control. The statistical results are shown on the right. N = 10. Scale bar, 50 μm. J is an immunostaining image of tdTomato+ciRPE cells co-expressing with Rhodopsin 12 weeks after subretinal transplantation in RCS rats. Scale bar, 50 μm. K is the fERG response intensity of 0.48 log cd*s / m in the ciRPE transplantation group and the sham transplantation group at 4, 8, 12, and 16 weeks after transplantation. 2 Representative b-wave response data at (darkness 3.0) (left). The right side shows the statistical analysis of b-wave amplitude in the ciRPEs transplant group and the sham transplant group. N=6 for each group. L is a schematic diagram of the quantitative visual-motor response (qOMR) testing device (left). Quantitative evaluation of visual acuity in the ciRPEs transplant group and the sham transplant group at 4, 8, 12, and 16 weeks post-transplantation (right), N=20. Figure 7 shows all representative examples of data from at least three independent experiments. All data are expressed as Mean±SD. *P<0.05, **P<0.01, ***P<0.001;

[0133] Figure 8: Screening diagram of small molecules for EF fate reprogramming using scRCF. A is a visualization of the UMAP scRNA-seq dataset used to predict MEF and EF conversion small molecules for cell type transformation. B is the gene co-expression network of signal proteins (upper layer) and TFs (lower layer). C is the protein-protein interaction network using a random walk algorithm to partition the signal protein community, showing the top 10 largest communities. D is a schematic diagram of the compound screening scheme used for DRUG-seq2 sequencing. LDN193189, A83-01, and CKI-7 (LAC) were used as the base compound combination, and predictive small molecules were added to form the LAC+1 combination. MEF was treated for 14 days before sequencing to assess changes in gene expression. E shows the small molecules targeting MEF conversion to EF selected using scRCF comprehensive screening.

[0134] Figure 9: Schematic diagram of the strategy for establishing a two-stage chemical reprogramming to generate ciRPE cells. A shows a bright-field image of the eye of an embryonic mouse with a Best1-Cre / ROSA26 tdTomato genetic background. Red fluorescence indicates the localization of Best1 in the eye tissue, tracked by tdTomato. Cell nuclei are reverse-stained with DAPI (blue). Scale bar: 750 μm. B shows immunostaining analysis of whole retinal sections of the eye of an embryonic mouse with a Best1-Cre / ROSA26 tdTomato genetic background. Blue indicates DAPI staining, and red fluorescence indicates the localization of Best1 tracked by tdTomato. Scale bar: 500 μm. C shows FACS sorting of E13.5 mouse embryonic MEF cells with a Best1-Cre / ROSA26 tdTomato background. D shows qRT-PCR analysis of RPE-specific gene expression in tdTomato-MEF and pRPE cells, with β-actin as a control. E shows immunostaining analysis revealing negative RPE-specific genes such as Mitf, Cralbp, Best1, and Rpe65 in tdTomato-MEFs. Scale bar, 50 μm. F shows representative morphological images of different fibroblast types (C57BL / 6MEF, 129MEF, C57BL / 6TTF, 129TTF) induced by M7+M3 compound culture medium. Scale bar, 400 μm. G shows flow cytometry analysis of the percentage of Best1-positive cells after induction of different fibroblast types (C57BL / 6MEF, 129MEF, C57BL / 6TTF, 129TTF) using the compound culture medium.

[0135] Figure 10: Characteristic images of ciRPE cells. A shows G-band karyotype analysis indicating normal ciRPE cell karyotype. B shows morphological images of ciRPE and pRPE cells at different passages (P1, P3, and P6). Scale bar: 300 μm. C shows flow cytometry analysis of ethyldeoxyuridine (EdU) incorporation in ciRPE and pRPE cells at different passages (P1, P3, and P6). D shows the distribution of ciRPE and pRPE cells in the cell cycle (G1, S, and G2 phases) (left) and quantitative analysis of cell percentage at each phase (right). E shows confocal microscopy images of ciRPE and pRPE cells phagocytizing latex beads (green). Cell apical side stained with ZO-1 (red). Scale bar: 10 μm.

[0136] Figure 11: Lineage tracing to confirm the results of MEF-induced ciRPE cells. A is a schematic diagram of the genetic lineage tracing strategy. B shows the starting cells, Best1- / tdTomato+MEF cells selected from E13.5 mouse embryonic MEF cells with the Fsp1-Cre / ROSA26 tdTomato genetic background. C shows qRT-PCR analysis of RPE-specific gene expression in Best1- / tdTomato+MEF and pRPE cells. β-actin was used as a control. D shows immunostaining analysis, indicating that Best1, Rpe65, and Cralbp were all negative in Best1- / tdTomato+MEF. Scale bar, 50 μm. E shows bright-field and fluorescence images of induced Best1- / tdTomato+MEF and ciRPE cells. Scale bar, 300 μm. F shows immunostaining analysis, indicating that ZO-1, Rpe65, Mitf, and Best1 were positive in ciRPE cells derived from Best1- / tdTomato+MEF. Scale bar, 20μm;

[0137] Figure 12: Molecular roadmap of ciRPE chemical reprogramming. A shows a heatmap of gene expression specific to 16 fibroblasts and 18 RPE cells at specified time points during reprogramming. Representative genes for each population are listed (right). Red and blue indicate upregulated and downregulated genes, respectively. B shows a hierarchical cluster analysis of cells at specified time points during MEF reprogramming to ciRPE cells, including days 0, 7, 18, 32, and pRPE cells. C is a radar plot showing the average transcriptional activity of genes in the ectoderm, mesoderm, endoderm, extraembryonic development, and stem cell maintenance at specified time points during reprogramming. D shows changes in protein modifications at different time points. “Gain” indicates a peak with a significant increase in signal value between time points (logFC>1), and “Loss” indicates a peak with a significant decrease in signal value (logFC<-1). E shows enrichment curves of histone modifications H3K4me3 (left), H3K27ac (middle), and H3K27me3 (right) for differentially expressed genes at different time points during reprogramming. The x-axis represents the distance (±3kb) from the transcription start site (TSS) to the transcription end site (TES), and the y-axis represents the signal intensity. Each line corresponds to a specific time point, illustrating the dynamic changes in histone modification patterns during reprogramming.

[0138] Figure 13: scRNA-seq analysis results of reprogramming. A shows the UMAP density map of marker gene expression levels in four different cell populations during reprogramming. Expression levels are represented by a color gradient, with darker red indicating higher gene expression. B shows the proportion of cell types at different time points during reprogramming. C shows the GO-term biological process enrichment analysis of different cell types identified from scRNA-seq data. D shows the UMAP density map of proliferation-related gene expression levels in neural progenitor-like intermediate cells during reprogramming. Expression levels are represented by a color gradient, with darker red indicating higher gene expression. E shows the pseudo-time trajectory displaying the proportion of cell types during reprogramming. F shows the pseudo-time for each cell in the Monocle 2 predicted trajectory.

[0139] Figure 14: Analysis results of key TFs during reprogramming, where A is a STRING network diagram of interactions between TFs identified by SCENIC during reprogramming. Nodes represent TFs and are color-coded by cell type: MEF (green), Intermediate cells (light blue), EF-like cells (blue), and ciRPE cells (red). BC assesses gene knockdown and overexpression efficiency in MEFs 72 hours after infection with shAscl1 / oeAscl1 (B) or shGli2 / oeGli2 (C) viruses. Gene expression levels were normalized to wild-type (WT) levels (set to 1), with cells infected with scramble shRNA (scramble) as controls. D shows the cell morphology and proportion of tdTomato+ cells on day 32 after knockdown of Ascl1 or Olig2 at specified time points during M7+M3 induction. WT, wild-type; Control KD, scramble shRNA-mediated knockdown. Scale bar, 200 μm. E represents the relative reprogramming efficiency after overexpression of Ascl1 or Olig2 under specified conditions. The reprogramming efficiency induced by M7 in the first stage is set to "1", and DMSO is the negative control. F is a visualization of the scRCF network of possible signal cascades induced by M7 targeting key TFs. Orange rectangles represent perturbation molecules, blue rhombuses represent signal protein targets, white ellipses represent intermediate signal proteins, and green hexagons represent TFs.

[0140] Figure 15: Results of HEF chemical reprogramming inducing human ciRPE cells. AB is a UMAP visualization of scRNA-seq data from HEF and OV, showing cell types (A) and datasets (B), used to identify small molecules promoting reprogramming between the two cell types. C is a bar chart showing the Z-score of HEF treated with the LCHRB+1 small molecule combination based on the neuroectoderm and EF-related gene set. The Z-score represents the relative gene expression change of each drug on the gene set; a higher value reflects a stronger effect on the gene set. The bars are sorted from highest to lowest by average Z-score, with a dashed line indicating a baseline score of zero. D shows the gene expression of mesoectoderm and EF-related genes after HEF treatment with the LCHRB+1 small molecule combination. The color scale represents the expression value after log2 transformation; red indicates high expression, blue indicates low expression, and white indicates moderate expression. E is a schematic diagram of the BEST1-Pr-EGFP-HEF cell line strategy used to monitor the hciRPE cell reprogramming process. F shows the expression of RPE-specific genes in BEST1-Pr-EGFP-HEF and hRPE cells analyzed by qRT-PCR. β-actin was used as a control. G shows that immunostaining analysis revealed negative results for the RPE-specific genes MITF, CRALBP, BEST1, and RPE65 in BEST1-Pr-EGFP-HEFs. Scale bar, 50 μm. H shows that the G-band karyotype analysis indicated that hciRPE cells had a normal karyotype. I shows the expression of OV and retinal progenitor cell-related marker genes at specified time points during reprogramming by qRT-PCR analysis.

[0141] Figure 16: Results of ciRPE cell transplantation restoring retinal function in RCS rats. A shows a schematic diagram of the process of transplanting tdTomato+ciRPE cells into the subretinal space of 3-week-old RCS rats. B shows bright-field fundus images after subretinal injection of tdTomato+ciRPE cells in RCS rats. Red arrows indicate bulges formed after transplantation. C shows the teratoma assay of ciRPE cells. The right-hand bar chart shows quantitative analysis data. N=15. D shows representative histological analysis of retinal sections from RCS rats in the un-injected group and the subretinal injection group of tdTomato+ciRPE cells (left), and quantitative assessment of mean outer nuclear layer (ONL) thickness at 12 weeks post-injection (right). N=10. Scale bar, 50 μm. Figure 16 provides all representative examples of data from at least three independent experiments. All data are expressed as Mean±SD. **P<0.01, **P<0.001. Detailed Implementation

[0142] The present invention will be further illustrated below with reference to specific embodiments. These specific embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention. The scope of the invention is defined by the claims and their equivalents. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. To facilitate understanding of the invention, the following terms used herein are explained:

[0143] As used herein, the terms “comprising” or “including” mean that any one or more of the stated elements or components are included, without excluding other elements or other components.

[0144] In this paper, the term "functional ciRPE cells" refers to chemically induced retinal pigment epithelial cells obtained by transdifferentiating fibroblasts from the ocular domain (EF) / optic vesicle (OV) intermediate state through a two-step reprogramming strategy using chemical small molecule compositions. These cells are highly similar to natural retinal pigment epithelial (RPE) cells in morphology, gene expression profile, and biological function, possessing typical RPE cell characteristics, including a hexagonal cobblestone morphology, expression of specific markers such as ZO-1, Pax6, Rpe65, Mitf, and Best1, polarized structure and tight junctions, enabling phagocytosis and clearance of the outer segment of photoreceptors, polarized secretion of growth factors such as VEGF and PEDF, formation of epithelial dome structure and maintenance of stable epithelial resistance, and no tumorigenic risk. After transplantation, they can integrate into the host retinal tissue, effectively protecting photoreceptor cells, inhibiting their apoptosis, and restoring damaged visual function. They can play a core role in maintaining retinal homeostasis, ensuring photoreceptor function, and constructing the blood-retinal barrier, just like natural RPE cells.

[0145] As used herein, the term "fibroblast" refers to mesenchymal-derived cells that are widely present in the connective tissues of animal bodies and are involved in the synthesis and secretion of extracellular matrix and tissue repair and remodeling. The fibroblasts described in this invention encompass various types of fibroblasts from human or non-human mammals, specifically including embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, pancreatic fibroblasts, etc., which can be used as starting cells for chemical reprogramming to induce the generation of functional ciRPE cells. Under the two-step chemical reprogramming strategy of this invention, they can be successfully transdifferentiated into functional ciRPE cells from the eye region (EF) / optic vesicle (OV) intermediate state, and fibroblasts from different tissue sources and different strains can all be used as starting cell materials for this reprogramming process.

[0146] In this article, the term "eye domain (EF)-like cell" refers to a cell with characteristics of an eye domain progenitor cell. This cell can naturally differentiate into retinal pigment epithelial cells during retinal development, possesses significant plasticity and potential for differentiation into RPE cell lineages, and is a key murine intermediate cell in the process of fibroblast reprogramming into ciRPE cells. It expresses eye domain-related specific markers such as Pax6, Sox2, and Six3.

[0147] In this article, the term "optical vesicle (OV)-like cell" refers to a cell with characteristics of an optical vesicle progenitor cell. It is an important precursor cell for the formation of retinal pigment epithelial cells during retinal development, a key human intermediate cell in the process of reprogramming fibroblasts into ciRPE cells, and has the potential to differentiate into mature RPE cells. It expresses optical vesicle-related specific markers such as PAX6, SIX3, and VSX2.

[0148] In this article, the term "chemical reprogramming" refers to the process by which intracellular signaling pathways and epigenetic modifications are regulated without the introduction of exogenous transcription factors, and the directional conversion of cell fate is achieved by inducing fibroblasts through specific chemical small molecule compositions, thereby transforming them into functional ciRPE cells through specific intermediate states. This process has no risk of genome integration and is characterized by its ease of operation, high scalability, and high safety.

[0149] As used herein, the term "pharmaceuticalally acceptable carrier or excipient" refers to a carrier or excipient that is safe to use in the pharmaceutical field for preparing pharmaceutical formulations, has no pharmacological activity, is not incompatible with the small molecule chemical compositions of the present invention, does not produce toxic side effects on the body, and does not affect the reprogramming activity of the composition. It may include various types such as aqueous solvents, organic solvents, microspheres, liposomes, preservatives, antioxidants, binders, and fillers.

[0150] As used herein, the term "retinal degenerative disease" refers to a class of diseases characterized by structural damage, functional impairment, or progressive apoptosis of retinal tissue cells such as retinal pigment epithelial cells and photoreceptor cells, which in turn lead to decreased vision, visual field defects, or even blindness. These include, but are not limited to, age-related macular degeneration, retinitis pigmentosa, Stargardt's disease, cone / rod dystrophy, and Leber congenital amaurosis. These are the main therapeutic targets of the functional ciRPE cells of this invention.

[0151] As used herein, the term "equivalent pharmaceutical preparation" refers to a pharmaceutical preparation that has the same or similar target, signaling pathway regulation effect, and cell reprogramming function as the small chemical molecule described in this invention. This includes structural analogs, isomers, pharmaceutically acceptable salts, hydrates, prodrugs, and derivatives and compound preparations with the same pharmacological activity of the small molecule. All of these can replace the original small molecule in the cell reprogramming process of this invention and are included within the scope of protection of this invention.

[0152] As used herein, the term “epithelial resistance (TEER)” refers to an important indicator used to detect the integrity of tight junctions and barrier function of the epithelial cell layer. It can quantitatively reflect the permeability of the epithelial layer formed by the ciRPE cells of this invention to ions. The higher the TEER value, the more complete the tight junctions of the ciRPE cells are, and the better their blood-retinal barrier function mimics that of natural RPE cells.

[0153] As used herein, the term "cell therapy product" refers to a biological product that meets clinical application standards and is prepared by in vitro expansion, purification, and quality control using the functional ciRPE cells described in this invention as the core active ingredient. It can repair or replace damaged retinal pigment epithelial cells and is used for cell replacement therapy of retinal degenerative diseases. It also has the characteristics of being non-tumorigenic, being able to stably integrate into the host retinal tissue, and performing physiological functions.

[0154] As used herein, the term "two-step reprogramming strategy" refers to the phased induction method of inducing fibroblasts into functional ciRPE cells in this invention. In the first stage, fibroblasts are induced to transdifferentiate into EF-like / OV-like intermediate cells through chemical small molecule composition 1. In the second stage, the intermediate cells are further induced to differentiate and mature into functional ciRPE cells through chemical small molecule composition 2. The two stages work together to achieve the directional conversion of cell fate.

[0155] As used herein, the term "polarized secretion" refers to the biological process by which the functional ciRPE cells of this invention mimic the polarity characteristics of natural RPE cells, and directionally secrete different cytokines and growth factors toward the apical and basal sides of the cells, such as secreting PEDF toward the apical side and VEGF toward the basal side. This process is crucial for maintaining the homeostasis of the retinal tissue microenvironment and the function of the blood-retinal barrier.

[0156] As used in this article, the term "μM" refers to micromolar concentration, μ: the Greek letter "mu", representing the SI prefix "micro-", or 10^- ... -6 (Parts per million), M: represents molar concentration (mol / L), that is, the amount of solute in one liter of solution. Therefore, 1 μM = 10 -6 mol / L means that there are 1 micromoles of solute per liter of solution.

[0157] In this article, the term "mM" refers to millimoles per liter (millimolar), a commonly used unit of measurement for solution concentration in chemistry and biology. This unit is defined based on the mole (mol), a fundamental unit of amount of substance, where the prefix "m" represents one-thousandth (10⁻⁶). -3 Therefore, 1 millimole is equal to 0.001 mole. When applied to solution systems, mM specifically refers to the number of millimoles of solute per liter of solution, that is, the ratio of the amount of solute (in millimoles) to the volume of the solution (in liters).

[0158] The reagents and raw materials used in this invention are readily available to those skilled in the art and, unless otherwise specified, can be obtained commercially. Experimental methods not specifying particular conditions in this invention are generally performed under conventional conditions or according to the manufacturer's recommendations. The invention will be further described in detail below with reference to specific embodiments. The examples given are only for illustrating the invention and not for limiting its scope. Unless otherwise specified, the experimental methods in the following examples are conventional methods. The cells described below were cultured at 37°C in a 5% (v / v) CO2 environment unless otherwise indicated.

[0159] Example 1: Screening of small molecules for reprogramming EF cells using the scRCF system

[0160] This embodiment establishes a two-step chemical induction strategy to reprogram fibroblasts into RPE cells. First, fibroblasts are transformed into an intermediate EF-like state and then differentiate into ciRPE cells. To address the challenge of identifying small molecules for cell reprogramming, we developed scRCF (Figure 1A), which combines single-cell transcriptomics with gene co-expression networks to identify key TFs and target proteins, facilitating the rational selection of compounds. To refine their identification, we incorporated cell-level sequencing (DRUG-seq2) to improve the accuracy and efficiency of molecularly driven reprogramming.

[0161] Using this platform, we screened and analyzed scRNA-seq data from mouse embryonic fibroblasts (MEF) and EF cells (Fig. 1B and Fig. 8A). Differential gene expression analysis identified 258 TFs associated with cell type transition (Fig. 1C). An improved SiPer20 framework was used to calculate the similarity between these TFs and perturbation spectra from a small molecule database, pre-screening 489 signaling proteins. Further refinement using a gene co-expression network reduced the selection to 279 functionally relevant signaling proteins (Fig. 8B). This approach revealed key signaling relationships and transcriptional regulators essential for cell transition. To optimize the screening, a random walk algorithm for modular partitioning of protein networks (Fig. 8C) was used to implement performance scoring and prioritize small molecules based on pathway relevance. Ultimately, 41 highly effective drug candidates were identified, including Wnt pathway regulators CKI-7 and CHIR-99021, RTK pathway inhibitors Orantinib and Rebastinib, epigenetic regulators Trichostatin-A and RG108, TGF-β / Smad inhibitor A83-01, and NF-κB pathway inhibitor BMS-345541, etc. (Figure 1D).

[0162] To further identify small molecules targeting EF reprogramming, we systematically evaluated 41 candidate compounds using DRUG-seq2. Considering the neuroectodermal origin of EF, we prioritized molecules such as LDN193189 (a BMP-I receptor inhibitor) and A83-01 (a TGF-β-I receptor) for their ability to inhibit mesoderm and endoderm differentiation. The addition of CKI-7 (an ATP-competitive casein kinase I inhibitor) promoted neuroectodermal formation. We selected LDN193189, A83-01, and CKI-7 (LAC) as the basic inducing small molecules for EF cell generation and used DRUG-seq2 to identify other synergistic small molecules (Fig. 8D). After 14 days of treatment with various small molecule combinations, DRUG-seq2 analysis revealed different molecular expression patterns and changes in target gene expression across different treatment groups, reflecting specific responses to each condition (Fig. 1E and Fig. 1F). Key regulatory small molecules promoting cell reprogramming were identified by effect scoring of gene sets related to neuroectodermal and early EF development. Hh-Ag1.5, a Hedgehog signaling antagonist, received the highest score. Other high-scoring compounds included GSK-3 inhibitors (CHIR-99021, 1-Azakenpaullone, Kenpaullone) and NF-κB pathway inhibitors (BMS-345541, WHI-P154) (Figure 1E). Based on gene expression activation profiles and removal of functionally redundant compounds, seven molecules—Hh-Ag1.5, CHIR-99021, Golvatinib, Pirfenidone, BMS-345541, Masitinib, and RG108—were selected from the top-ranked candidates (Figure 1F) to form an optimized induction medium. Using this method, we identified 10 small molecules from 4319 candidate molecules capable of reprogramming MEF cells into EF cells. In summary, we established a systematic, single-cell transcriptome-driven compound screening system integrating computation and high throughput. This method identified 10 small molecules as candidate inducible components that have the potential to drive effective cell reprogramming (Figure 8E).

[0163] Example 2: Establishing a two-stage chemical reprogramming strategy to generate ciRPE cells

[0164] First, this example optimized the serum-free, chemically defined culture medium containing these 10 small molecules from Example 1 and used it to treat MEFs. In Phase I, we observed epithelial-like cell colonies after 6 days of treatment (Fig. 2A). Then, by day 12, cell proliferation began to slow, but clonal morphology showed more defined boundaries and distinct epithelial features (Fig. 2A). To enhance the expansion of cell clonal colonies, we mechanically removed some surrounding cells. As expected, the cell colonies gradually expanded. By day 18, the cells exhibited tight junctions, with some cells displaying high nucleocytoplasmic ratios and typical early RPE features, such as cobblestone or hexagonal morphology (Fig. 2A). Further qPCR analysis showed that EF-related genes (Pax6, Sox2, Six3, and Vsx2) and early RPE development-related genes (Mitf and Best1) were significantly upregulated (Fig. 2B). These findings suggest that our identified 10 small molecule induction protocol has the potential to transform MEFs into EF-like cells with early RPE features.

[0165] To promote the further differentiation and maturation of EF-like cells into RPE cells, we introduced nicotinamide (NIC), retinoic acid (RA), and Activin A (hereinafter referred to as M3) during the second-stage induction process. NIC inhibited neurogenic differentiation and promoted the expression of RPE-specific genes by regulating epigenetic and metabolic states. RA promoted RPE differentiation and regulated the expression of RPE-specific genes to promote pigmentation. Activin A promoted the differentiation of EF cells into RPE cells by activating the TGF-β / SMAD signaling pathway, regulating pigmentation to maintain epithelial properties. After two weeks of treatment, the cells exhibited typical hexagonal RPE morphology and pigmentation (Figure 2C). Subsequent qPCR analysis confirmed the significant upregulation of Best1 and mature RPE genes such as Rpe65, Tyr, Lhx2, Pmel, and Otx2 (Figure 2D), indicating successful generation of ciRPE cells. To further verify the effectiveness of reprogramming, we used a lineage tracing strategy to monitor the reprogramming process (Figure 2E). The Best1 gene is specifically expressed in RPE cells, and its promoter has been shown to effectively drive reporter gene expression. Using Best1-Cre / ROSA26tdTomato fluorescent reporter mice, we observed stable and specific tdTomato expression in RPE cells (Figs. 9A and 9B). We collected Best1-tdTomato-negative (tdTom-)MEFs using fluorescence activated cell sorting (FACS) (Fig. 9C). These tdTom-MEFs were negative for RPE marker genes such as Mitf, Cralbp, Best1, and Rpe65 (Figs. 9D and 9E), confirming the absence of residual RPE or progenitor cells. We then performed a two-step chemical induction on the tdTom-MEFs (stage I with 10 small molecules, followed by stage II with M3). Low levels of tdTomato expression were observed after the first induction stage (day 18), with a positive rate of 2.51%. Following further induction into RPE cell maturation in stage II (day 32), the tdTomato positivity rate increased to 16.58% (Figures 2F and 2G). These results confirm that the constructed chemical induction system effectively reprogrammed MEF cells into RPE cells.

[0166] To minimize the potential toxicity of small molecules to cells, we performed a "-1" experiment to further optimize the chemical reprogramming system. The results showed that the removal of Golvatinib, Pirfenidone, and Masitinib in the first induction phase had the least impact on the tdTomato positivity rate (Fig. 2H), with a positivity rate of 19.66% (Fig. 2I). Therefore, we finally determined the reprogramming induction system to consist of seven small molecules from the first phase (M7: LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, and RG108) and three compounds from the ⅠⅠ phase (M3: NIC, RA, and Activin A) (Fig. 2J). Finally, we validated the effectiveness of our reprogramming system in MEFs from different batches (n=5) and different genetic backgrounds (including C57BL / 6 and 129) (Fig. 9F and Fig. 9G). Furthermore, this M7+M3 system successfully promoted the transformation of tail tip fibroblasts (TTFs) of newborn mice into ciRPE cells (Fig. 9F and Fig. 9G). In summary, these results demonstrate that our optimized M7+M3 system can effectively reprogram MEFs into ciRPE cells.

[0167] Example 3: Characteristics of ciRPE cells

[0168] To further confirm the phenotypic and functional characteristics of ciRPE cells, we first assessed the expression of RPE markers using immunofluorescence staining. The results showed that ciRPE cells exhibited a tight junction structure (ZO-1) and highly expressed RPE markers, including Pax6, Rpe65, Mitf, Best1, and Cralbp (Fig. 3A). Furthermore, these cells exhibited a polarized morphology, with ZO-1 localized to the apical membrane and Best1 localized to the basement membrane (Fig. 3B). This polarized structure is crucial for RPE function, especially the apical microvilli, which mediates the phagocytosis and clearance of shed photoreceptor segments, ensuring normal photoreceptor switching. Subsequently, we analyzed the structural features of ciRPE cells using transmission electron microscopy. These cells exhibited prominent apical microvilli, pigment granules, and tight junctions (Fig. 3C), indicating a high degree of morphological similarity to native RPE cells in vivo.

[0169] To assess the phagocytic capacity of ciRPE cells, fluorescently labeled porcine photoreceptor posterior segments (POS) and latex beads were introduced into the ciRPE cell culture medium. After incubation, laser scanning confocal microscopy revealed the presence of phagocytosed POS and latex beads within the ciRPE cells (Fig. 3D and Fig. 10E), confirming their phagocytic function. Furthermore, RPE cells are known to exhibit polarized secretion of growth factors, a process crucial for maintaining homeostasis between the retina and choroid. We further analyzed the polarized secretion of growth factors in ciRPE cells, finding that these cells primarily secrete VEGF from the basal side and PEDF from the apical side (Fig. 3E). In addition, ciRPE cells formed dome-shaped structures during in vitro culture (Fig. 3F), indicating that their epithelial layer can effectively transport fluid while maintaining tight junction integrity. Measurements of epithelial resistance (TEER) further confirmed the integrity of the tight junctions and barrier function of ciRPE cells, showing that TEER steadily increased to approximately 80 Ω × cm⁻¹ within the first three weeks. 2 The cells then stabilized (Figure 3G). Overall, these data indicate that ciRPE cells exhibit key functional characteristics of native RPE cells.

[0170] To further validate the proliferative potential of ciRPE cells, we purified them and passaged them continuously in expansion medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The results showed that ciRPE cells could be passaged for at least 20 generations while maintaining stable RPE morphology and normal karyotype (Fig. 10A and Fig. 10B). In contrast, primary RPE (pRPE) cells gradually lost pigmentation and hexagonal morphology by generation 6, and their growth rate significantly decreased (Fig. 10B and Fig. 10C), consistent with previous studies. In different passage batches, the proliferative capacity of ciRPE cells was significantly higher than that of pRPE cells, and the EdU incorporation rate consistently exceeded that of pRPE cells (Fig. 10C). Flow cytometry analysis showed that the cell cycle distribution of ciRPE cells (P3) was similar to that of pRPE cells, with 70%, 17.4%, and 6.87% of the cells in the G0 / G1, S, and G2 / M phases, respectively (Fig. 10D). In summary, our results indicate that ciRPE cells are very similar to native RPE cells in morphology and function, exhibiting typical polarization characteristics, phagocytic capacity, and polarized secretion of growth factors. Furthermore, they possess strong proliferative capacity and stability.

[0171] Finally, we validated the process of MEFs reprogramming into ciRPE cells using a pedigree tracing strategy with Fsp1-Cre and ROSA26-tdTomato mice. MEFs were isolated from E13.5 transgenic mice with an Fsp1-Cre / ROSA26 tdTomato genetic background, and tdTomato+ / Best1- cells were obtained by FACS sorting (Fig. 11A and Fig. 11B). These cells initially did not express RPE markers (Fig. 11C and Fig. 11D). After chemical reprogramming, ciRPE cells began to express RPE-specific genes and co-expressed tdTomato (Fig. 11E and Fig. 11F), further confirming that these cells originated from the original MEFs.

[0172] Example 4: Molecular route diagram of ciRPE chemical reprogramming

[0173] To enhance the understanding of the complex process of MEF reprogramming into ciRPE cells, a comprehensive multi-omics analysis was performed in this embodiment (Figure 4A). RNA sequencing (RNA-seq) was performed on cells at days 0, 7, 18, and 32 of the reprogramming process. Principal component analysis (PCA) of the RNA-seq results revealed significant transcriptional changes throughout the dynamic process, indicating a gradual transition from fibroblasts to ciRPE cells. Notably, the final ciRPE cells showed greater similarity to pRPE cells at the transcriptional level compared to earlier stages (Figure 4B). Temporal fuzzy clustering analysis of differentially expressed genes revealed four distinct gene clusters corresponding to each stage of reprogramming: fibroblasts (day 0), intermediate state (day 7), EF-like cells (day 18), and mature ciRPE cells (day 32) (Figure 4C).

[0174] In the initial stages of the transition from fibroblasts to the intermediate state, downregulated genes such as Fn1, Itga11, and Runx2 were associated with decreased fibroblast function and proliferative activity. In contrast, upregulated genes, including Sox2, Ascl1, Gli2, Gli1, and Gbx2, were associated with neuroectodermal processes (Fig. 4C). These results suggest that early chemical induction promotes the transition to neuronal fate by simultaneously inhibiting fibroblast characteristics through activation of neuroectodermal genes and signaling pathways. Furthermore, in the intermediate stage, transient activation of genes such as Tfap2a, Rorb, and Etv5 was associated with epithelial cell proliferation and inflammation, reflecting enhanced intercellular interactions and adaptation to environmental signals (Fig. 4C). By day 18, the upregulated genes were primarily those related to eye development, such as Pax6, ID4, and Best1. As induction progressed towards mature RPE, early EF-related genes showed a trend of downregulation. By day 32, genes related to RPE function (such as Rpe65, Lhx2, Tyr, Cralbp, Otx2) and genes related to pigment cell differentiation (such as Pmel, Mlana, Slc24a5) were significantly upregulated (Figure 4C), confirming the transformation of cells into mature pigment ciRPE cells.

[0175] To confirm the successful conversion of fibroblasts into ciRPE cells, we analyzed 16 fibroblast-specific genes and 18 RPE-specific genes. During the reprogramming process, fibroblast markers were significantly downregulated, while RPE markers were upregulated, indicating that ciRPE cells acquired RPE characteristics (Fig. 12A). Ultimately, the gene expression profiles of ciRPE cells and pRPE cells were highly matched, further confirming that these cells possess RPE cell characteristics (Figs. 4B, 4C, 12A, and 12B). Notably, the reprogramming process did not involve the iPSC stage; instead, transcriptional activation was primarily concentrated on ectoderm-related genes (Fig. 12C). This suggests that the combination of compounds we identified specifically guides fibroblast differentiation into the neuroectoderm and eye development pathways, highlighting the effectiveness of our screening strategy.

[0176] Epigenetic remodeling plays a crucial role in cell fate reprogramming. We used CUT&Tag sequencing to track the dynamic changes in histone modifications of H3K4me3, H3K27ac, and H3K27me3 near gene transcription start sites. PCA analysis revealed significant changes in chromatin state during ciRPE cell reprogramming, indicating that ciRPE cells are very similar to pRPE cells in histone modification patterns (Fig. 4B). H3K4me3 gradually increased from MEFs to ciRPE cells, peaking at days 18 and 32, and was associated with RPE gene activation. The loss of H3K4me3 decreased over time, indicating its role in establishing new gene expression patterns. Changes in H3K27ac varied at different stages, decreasing from day 7 to day 18, indicating gene repression of EF differentiation. The dynamic changes in H3K27me3 reflected gene silencing: significant loss from MEFs to day 7 of induction promoted differentiation, while the increase from day 7 to day 18 established new silencing. In the ciRPE stage, H3K27me3 levels stabilized, maintaining the mature RPE phenotype (Fig. 12D). Further analysis revealed that the CUT & Tag signals of H3K4me3 and H3K27ac correlated with RNA expression levels, highlighting their role in gene activation (Fig. 4D). Although H3K27me3 generally shows a negative correlation with RNA-seq data, there was some deviation in the fibroblast stage, possibly due to the dominance of activation markers. From day 7 to day 18, the increase in H3K27me3 was associated with a decrease in RNA expression, and the stable trend in the ciRPE stage reflected a balance between gene silencing and activation (Fig. 4D and Fig. 12E). These findings highlight the complex role of dynamic histone modifications in cell type transitions at different stages and underscore the crucial role of epigenetic regulation in the reprogramming of MEFs to RPE. By regulating gene activation and silencing, cells were successfully reprogrammed into the RPE lineage.

[0177] To accurately characterize the reprogramming trajectory, we performed scRNA-seq at three key time points during the reprogramming of MEFs into ciRPE cells. Single-cell transcriptome data were obtained from samples collected on days 7, 18, and 32, respectively (Fig. 4E). Specific marker gene clustering analysis revealed that on day 7 of reprogramming, the cell population consisted of MEF cells, neural progenitor-like intermediate cells, and a small subset of EF-like cells. By day 18, the proportion of intermediate cells decreased, while the proportion of EF-like cells increased. On day 32, the number of EF-like cells decreased, and a large number of ciRPE cells appeared (Fig. 4F, Fig. 4G, Fig. 13A, and Fig. 13B). To further validate the reprogramming trajectory of ciRPE cells, we performed single-cell pseudo-timeline analysis. The analysis revealed the progression from MEFs to neural progenitor-like intermediate cells, followed by differentiation into EF-like cells, and finally differentiation into ciRPE cells (Fig. 4H). Unsupervised clustering highlighted the dynamic molecular events in this process, including the transition from fibroblasts to neural progenitors, the progression of eye development stages, and the establishment of RPE-specific functions (pigmentation and retinoid metabolism) (Fig. 13C). Interestingly, in two identified neural progenitor-like subpopulations, the differentiation trajectory of MEFs primarily aligned with the subpopulation expressing neurogenic and proliferative markers (e.g., Mki67, Cdk1, Top2a), while the other subpopulation lacked these markers (Fig. 13D). This suggests that proliferating neural progenitors are crucial for subsequent differentiation into EF-like cells. RNA rate analysis also reflected the progress of reprogramming into ciRPE cells, consistent with the trajectory observed in pseudo-time analysis (Fig. 13E and Fig. 13F). We further performed similarity analysis on the reprogrammed cell population and publicly available single-cell datasets. The similarity score between induced cells and RPE cells was 0.80, and the similarity score with EF-like cells was 0.64 (Fig. 4I), indicating that the reprogrammed cells possessed both EF-like and RPE cell characteristics. In summary, our two-step chemical reprogramming system effectively guides MEFs to generate functional ciRPE cells through neural progenitor-like intermediates and EF-like cell stages. This series of processes emphasizes precise control over lineage-specific gene expression and epigenetic remodeling.

[0178] Example 5: Transcriptional activation control of master regulators of neural and eye development, ciRPE reprogramming

[0179] To identify the key functional groups (TFs) driving MEF reprogramming into ciRPE cells, we combined single-cell data with SCENIC analysis to systematically investigate the expressed regulatory factors in each cell type and their dynamic changes during the reprogramming process. Using logFC > 1.0 as a threshold, we identified 125 TFs across four cell types, highlighting their distinct roles at different reprogramming stages (Figure 5A). Using the STRING database, we constructed an interaction network of these TFs, revealing their synergistic effects during reprogramming (Figure 14A). Network analysis using maximum cluster centrality (MCC) identified 15 key functional groups (TFs), including MEF-specific factors (Dlx2 and Dlx1), day 7 transient activators (Sox11, Foxa2, and Pax2), factors persistently highly expressed from day 7 to day 18 (Sox2, Sox9, Olig2, Zic1, Ascl1, Atoh1, Pou3f2, and Gbx2), and ciRPE-related factors (Lhx2 and Otx2) (Figures 5B and 5C). Notably, most of these key TFs are associated with neuroectodermal development, suggesting that the fate of neuroectodermal development is a crucial transitional stage for MEFs to reprogram into EF-like cells and ciRPE cells.

[0180] To further explore the regulatory mechanisms of these TFs, we integrated RNA-seq and CUT&Tag-seq data to assess the activity of the top 5 TFs (Fig. 5D). On day 0, neuroectodermal TFs, including Ascl1, Olig2, Zic1, and Pou3f2, showed weak activation markers (H3K4me3 and H3K27ac), primarily suppressed by H3K27me3, consistent with their suppression in the stable fibroblast state. By day 7, as cells transitioned to a neural progenitor-like intermediate state, these TFs showed increased expression of activation markers and RNA, indicating activation of the neuroectodermal pathway. This aligns with the fibroblast-like suppression and the initiation of neurogenesis. By day 18, the activity of Ascl1 and Olig2 decreased, while Zic1 and Pou3f2 remained highly activated. On day 32, during the ciRPE phase, Lhx2 showed robust activation and significant RNA upregulation, while the earlier neuroectodermal TFs were silenced (Fig. 5D). Notably, while H3K27me3 played a crucial inhibitory role in the early stages (0-7 days), its effect diminished in later stages, suggesting that other inhibitory mechanisms (such as DNA methylation or other histone markers) may be regulating TF activity during this phase. These findings highlight the critical role of dynamic epigenetic modifications and TF expression in guiding cell fate transitions during MEF reprogramming to ciRPE. In summary, neurodevelopment-related TFs act as a "switch" for early differentiation into EF, while RPE-related TFs dominate the later stages, driving RPE maturation.

[0181] To determine the effects of Ascl1 and Olig2 on fibroblast to EF-like cell reprogramming, we investigated the effect of knocking down these genes separately with shRNA on the efficiency of ciRPE induction. As expected, knockdown of either Ascl1 or Olig2 significantly reduced reprogramming efficiency, confirming their positive regulatory role in reprogramming (Fig. 5E, Fig. 5F, Fig. 14B–14D). Importantly, knockdown of these genes had the most significant effect on reprogramming efficiency at the early stages (9.1-fold, 16.7-fold, and 3.6-fold reductions at days 0, 7, and 18 when transfected with shAscl1, respectively, Fig. 5E; and 12.5-fold, 8.3-fold, and 2.8-fold reductions at days 0, 7, and 18 when transfected with shOlig2, respectively, Fig. 5F), highlighting their crucial role in initiating the neuroectodermal transition required for subsequent ciRPE differentiation. On the other hand, overexpression of Ascl1 or Olig2 enhanced the efficiency of M7+M3-mediated ciRPE reprogramming (Figure 14E), verifying that Ascl1 and Olig2 directly participate in ciRPE reprogramming.

[0182] These findings highlight the importance of the transition to neuroectodermal and EF-like cell states for the successful reprogramming of MEFs into ciRPE cells, validating the effectiveness of our first-stage small molecule compound ensemble in coordinating these transitions. To further understand the mechanisms by which these small molecules regulate key TFs, we constructed a complex regulatory network using scRCF (Figures 5G and 14E). Analysis revealed that the activation of key TFs is a result of the combined action of these small molecules, which regulate TFs through their respective target proteins, coordinating the activation of multiple signaling pathways, including Wnt, TGF-β, and Hedgehog, to jointly drive the reprogramming process and guide the precise transition of cell fate to ciRPE reprogramming.

[0183] Example 6: Generating human ciRPE cells from HEFs via chemical reprogramming

[0184] In our research on reprogramming human ciRPE (hciRPE) cells, we initially attempted to reprogram human embryonic fibroblasts (HEF) using a mouse induction system. However, the results were not ideal, likely due to the higher complexity and more stringent signal requirements of human cell reprogramming compared to the mouse system. Therefore, it was necessary to tailor the small molecule combination to improve reprogramming efficiency and success rate. In the compound selection process, we focused on the “OV” stage of early human retinal development, a crucial intermediate state guiding human RPE reprogramming. By integrating single-cell data from human embryos (Carnegie stage 12 and 16) and retinal organoids at day 30 (Fig. 15A and Fig. 15B), we strategically incorporated the top 15 key TFs identified during mouse ciRPE reprogramming (Fig. 5B) into the scRCF. This approach enabled more accurate identification of small molecules to guide hciRPE cell reprogramming. Ultimately, we identified 42 candidate small molecules (Fig. 6A). Notably, LDN193189, CHIR-99021, Hh-Ag1.5, RG108, and BMS-345541 (LCHRB), identified in the mouse RPE reprogramming system, were present in the basal compound list. We then screened other compounds using DRUG-Seq2 (LCHRB+1), ranking the top 10 using a comprehensive scoring system (Figures 15C and 15D), ultimately selecting 15 small molecule combinations. We hypothesize that these combinations will promote the transformation of HEFs into OV-like cells.

[0185] To test our hypothesis, we constructed a BEST1 Pr-EGFP-HEFs tracking cell line to monitor the reprogramming process (Fig. 15E). Initially, these cells did not express RPE markers such as MITF, CRALBP, BEST1, and RPE65, nor did they express EGFP (Figs. 15F and 15G). After successful reprogramming, the Best1 promoter specifically drove EGFP expression. We added 15 selected compounds to the reprogramming medium to initiate HEFs reprogramming. In the early stages of induction, HEFs exhibited rapid proliferation; however, by day 12, only a small number of cell clones appeared. To support clonal growth, we removed surrounding cells, but their proliferation remained slow. By day 24, only a few cells were BEST1-EGFP positive. Continued induction did not show significant changes in cells; instead, the cell number decreased. We subsequently introduced three compounds, M3 (NIC, RA, and Activin A), to promote RPE differentiation and maturation, but the proportion of BEST1-EGFP positive cells remained low.

[0186] Due to the unsatisfactory efficiency of this induction system and the potential cytotoxicity of excessive compounds, further optimization of the induction system is necessary. Starting with an initial 15 small molecules and 3 differentiation-maturation compounds, we explored various combinations and culture conditions. We found that using 9 small molecules (M9: CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, VPA, SB-431542) in the initial stage and 2 compounds (M2: NIC and Activin A) in the differentiation-maturation stage resulted in 6.74% BEST1-EGFP positive cells by day 38 (Figures 6B and 6C), while other combinations produced lower proportions of positive cells.

[0187] We further characterized the molecular and biological features of hciRPE cells. After purification and amplification, the induced hciRPE cells exhibited a typical hexagonal morphology and prominent melanin granules (Fig. 6D), while maintaining a normal karyotype (Fig. 15H). qPCR analysis and immunofluorescence staining results jointly confirmed that EGFP+ hciRPE cells specifically expressed key RPE markers such as MITF, CRALBP, BEST1, and RPE65 (Fig. 6E and Fig. 6F). Transcriptomic analysis showed that the expression profile of hciRPE cells was very similar to that of primary human RPE (hRPE) cells, which served as a positive control (Fig. 6G and Fig. 6H). In hciRPE cells and in the intermediate reprogrammed state, fibroblast-specific genes (such as FN1 and RUNX2) were downregulated, while RPE-specific genes (such as RPE65, TYR, LHX2, and CRALBP) were upregulated. Notably, the upregulation of TFs such as RORB, PAX3, DES, and TFAP2A during the intermediate stage indicates that the neuroectodermal transcriptional program may be activated, potentially leading to a transition to the RPE lineage. By day 24, markers associated with OV and retinal progenitor cells (PAX6, SIX3, VSX2, MITF, and BEST1) were activated (Fig. 15I), indicating successful conversion to OV-like cells, a crucial intermediate state for the RPE lineage transition. Upon further differentiation, these cells eventually exhibit similar expression to hRPE cells (Figs. 6G and 6H). Further analysis revealed that hciRPE cells secrete growth factors such as VEGF and PEDF (Fig. 6I), and TEER analysis confirmed the presence of tight junctions and barrier functions (Fig. 6J). These results demonstrate that our optimized two-stage chemical reprogramming system can effectively convert HEFs into functional hciRPE cells, providing a scalable, non-integrative approach for studying RPE biology and developing cell-based therapeutics.

[0188] Example 7: ciRPE cell transplantation restores retinal function in RCS rats

[0189] Leveraging the functional and safety advantages of ciRPE cells, we conducted an in vivo transplantation study to evaluate their potential for treating retinopathy of predisposition (RD). The Royal College of Surgeons (RCS) rats, with their retinal degeneration caused by a mutation in the Mertk gene leading to impaired phagocytosis of the outer segment of photoreceptors by RPE cells, are a classic model for RD research. We transplanted FACS-purified tdTomato+ciRPE cells into the subretinal space of 3-week-old RCS rats (Figs. 7A, 16A, and 16B). The contralateral untransplanted eye, the PBS-transplanted eye, and the mESC-transplanted eye served as controls. Postoperative optical coherence tomography (OCT) imaging showed a significant bulge at the transplantation site, which significantly decreased after one week and completely disappeared after three weeks, indicating successful transplantation and good tissue adaptation (Fig. 7B). To assess the long-term safety of the transplantation, we conducted a 4-month follow-up evaluation. During this period, no signs of tumor formation were observed in the ciRPE-transplanted nude mice. In contrast, 13 out of 15 nude mice receiving tdTomato-labeled mESCs developed visible intraocular tumors (Fig. 7C). This result is consistent with the findings of subcutaneous teratoma experiments, further confirming that ciRPE cell transplantation has no tumorigenic risk, thus supporting its safety (Figure 16C).

[0190] Histological and immunostaining analyses at 4 weeks post-transplantation revealed clusters of tdTomato+ transplanted cells in the subretinal space (Fig. 7D). By 12 weeks, these cells had evolved into an orderly monolayer, indicating successful integration into the host RPE (Fig. 7E). Furthermore, the transplanted cells expressed mature RPE markers such as Mitf, Cralbp, Pax6, and Rpe65 (Fig. 7F, Fig. 7G, Fig. 7H). Typically, RCS rats exhibit severe retinal dysfunction at 2-3 months of age, characterized by significant photoreceptor apoptosis and thinning of the outer nuclear layer (ONL). At 12 weeks post-transplantation, the ONL in the ciRPE transplantation group was significantly thicker than that in the non-transplantation group and the Sham group (Fig. 7E and Fig. 16D), indicating that ciRPE transplantation protected photoreceptor cells and slowed their degeneration, thereby maintaining and improving retinal structure and function. TUNEL staining further revealed a significant reduction in apoptotic cells in the ONL of the ciRPE group compared to the Sham group (Fig. 7I), highlighting the protective effect of ciRPE cells against photoreceptor apoptosis. In RCS rats, the Mertk mutation leads to loss of phagocytic function in RPE cells, affecting the clearance of the outer segment of the photoreceptor. To assess whether ciRPE cells could restore this function in vivo, we monitored their uptake of rhodopsin, a major component of the outer segment of the photoreceptor. Co-localization of tdTomato and Rhodopsin was observed in the subretinal space of rats 12 weeks after ciRPE cell transplantation (Figure 7J), indicating successful integration of the transplanted cells and restoration of the phagocytic function of the RPE cells.

[0191] To comprehensively evaluate the impact of ciRPE cell transplantation on retinal function, we performed dark-adapted flash electroretinography (fERG) and behavioral tests. fERG data collected 4–16 weeks post-transplantation showed that the b-wave amplitude in the ciRPE group was significantly higher than that in the control group, especially in the first few weeks post-transplantation (Fig. 7K). This indicates that ciRPE cells support photoreceptor survival and functional recovery. The sustained increase in b-wave amplitude reflects both short-term and long-term effects of cell integration. To assess whether these electrophysiological improvements translated into improved visual performance, we performed an optical motion response (OMR) test. The ciRPE group exhibited superior visual performance compared to the control group, such as faster responses to moving stripe stimuli and greater sensitivity to changes in spatial frequency and direction of motion, which could be quantified by increased OMR scores (Fig. 7L). These findings confirm the long-term survival, safety, and therapeutic efficacy of ciRPE cells in vivo, their ability to successfully integrate into host tissues, reconstruct functional RPE structures, and restore visual function, highlighting their potential as a cell source for treating RD.

Claims

1. A method for inducing fibroblasts into functional ciRPE cells through a two-step reprogramming process using a small chemical molecule composition, comprising: Fibroblasts were treated with reprogramming medium 1 to induce them to be reprogrammed into EF-like or OV-like cells. Then, EF-like or OV-like cells were treated with reprogramming medium 2 to induce them to be reprogrammed into ciRPE cells. The reprogramming culture medium 1 includes a small chemical molecule composition 1, and the reprogramming culture medium 2 includes a small chemical molecule composition 2. When the fibroblasts are mouse-derived fibroblasts, the chemical small molecule composition 1 is specifically a combination of LDN193189, A 83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108 or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof; When the fibroblasts are mouse-derived fibroblasts, the chemical small molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof. When the fibroblasts are human-derived fibroblasts, the chemical small molecule composition 1 is specifically a combination of CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof; When the fibroblasts are human-derived fibroblasts, the chemical small molecule composition 2 is specifically a combination of nicotinamide, Activin A, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof.

2. The method according to claim 1, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

3. The method as described in claim 1, wherein the reprogramming culture medium 1 and the reprogramming culture medium 2 are respectively composed of a basic culture medium to which the chemical small molecule composition 1 and the chemical small molecule composition 2 are added for culturing.

4. The method of claim 3, wherein the reprogramming medium 1 uses basal medium 1, specifically an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% vitamin A-free B27, 7.5% BSA, 1% NEAA, 1% P / S, and 10 ng / mL bFGF.

5. The method of claim 3, wherein the reprogramming medium 2 uses basal medium 2, specifically DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055 mM 2-mercaptoethanol.

6. The method of claim 1, further comprising proliferating and culturing ciRPE cells.

7. The method of claim 6, wherein the proliferation culture uses a ciRPE cell proliferation medium with the following components: DMEM / F12 / GlutaMAX as substrate, supplemented with 1% N2, 2% vitamin A-free B27, 1% NEAA, 1% P / S and 0.1 mM 2-mercaptoethanol.

8. The method of claim 7, wherein the proliferation culture medium for ciRPE cells further comprises one or more of 10 ng / mL bFGF, 20 ng / mL EGF, 10 μM Y-27632, and 0.5 μM A83-01.

9. The method of claim 1, further comprising functional culture of ciRPE cells.

10. The method of claim 9, wherein the functional culture uses a ciRPE cell function maintenance medium with the following components: DMEM / F12 / GlutaMAX plus 1% N2, 2% vitamin A-containing B27, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol.

11. The method of claim 10, wherein when the fibroblasts are mouse-derived fibroblasts, the function maintenance culture is further supplemented with 0.2 μM Activin A, 0.5 μM retinoic acid, 1 μM BMP4 and 10 mM nicotinamide.

12. The method of claim 10, wherein when the fibroblasts are human-derived fibroblasts, the function maintenance culture is further supplemented with 0.1 μM Activin A, 0.5 μM retinoic acid, and 0.5 μM BMP4.

13. The method of claim 1, wherein when the fibroblasts are mouse-derived fibroblasts, the concentrations of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, and RG108 in the chemical small molecule composition 1 are 0.1 mM, 0.5 mM, 0.2 mM, 0.2 mM, and 10 mM, respectively.

14. The method of claim 1, wherein when the fibroblasts are mouse-derived fibroblasts, the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM, the concentration of retinoic acid is 1 μM, and the concentration of Activin A is 0.2 mM.

15. The method of claim 1, wherein when the fibroblasts are human-derived fibroblasts, the concentrations of CHIR-99021, LDN193189, Hh-Ag1.5, RG108, BMS-345541, R-268712, BIX-01294, VPA, and SB-431542 in the chemical small molecule composition 1 are 10 μM, 0.2 mM, 0.2 mM, and 10 μM, respectively.

16. The method of claim 1, wherein when the fibroblasts are human-derived fibroblasts, the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM and the concentration of Activin A is 0.2 mM.

17. A small molecule chemical composition comprising small molecule chemical composition 1 and small molecule chemical composition 2, wherein small molecule chemical composition 1 and small molecule chemical composition 2 are selected from any one of the following groups: Chemical small molecule composition 1 is specifically a combination of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108 or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof; chemical small molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof. Chemical small molecule composition 1 is specifically a combination of CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542 or equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors thereof; chemical small molecule composition 2 is specifically a combination of nicotinamide, Activin A or equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors thereof.

18. The small molecule chemical composition of claim 17, wherein the small molecule chemical composition comprises a pharmaceutically acceptable carrier or excipient.

19. The small molecule chemical composition of claim 18, wherein the carrier or excipient is selected from one or more of the following: Water, saline, phosphate buffer, or other aqueous solvents; DMSO, glycerol, ethanol, or other organic solvents; Microspheres, liposomes, microemulsions, or high molecular weight surfactants; Colloidal drug delivery systems or polymeric drug delivery systems; Preservatives, antioxidants, flavoring agents, fragrances, solubilizers, emulsifiers, pH buffers; Adhesives, fillers, lubricants or other pharmaceutical excipients.

20. The small molecule chemical composition of claim 17, wherein the pharmaceutical dosage form prepared from the small molecule chemical composition is selected from one or more of the following: Solid dosage forms, including: Powders, granules, tablets, pills, capsules, sustained-release preparations, controlled-release preparations, or other solid dosage forms; Liquid dosage forms, including: injections, infusions, suspensions, or other liquid dosage forms; Gaseous dosage form; Semi-solid dosage form; Reprogramming preparations or reagents.

21. The small molecule chemical composition of claim 17, wherein the small molecule chemical composition further comprises an organic solvent, physiological saline, or other carrier or excipient.

22. A reprogramming culture medium assembly, the reprogramming culture medium assembly comprising reprogramming culture medium 1 and reprogramming culture medium 2; The reprogramming culture medium 1 comprises a small chemical molecule composition 1 and a basal culture medium 1. Specifically, the small chemical molecule composition 1 is a combination of LDN193189, A83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108, or equivalent pharmaceutical products, analogs, isomers, salts, hydrates, or precursors thereof, or CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic. acid (VPA), SB-431542 or a combination of equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors thereof; the basal culture medium 1 is an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% vitamin A-free B27, 7.5% BSA, 1% NEAA, 1% P / S, and bFGF; The reprogrammed culture medium 2 comprises a small chemical molecule composition 2 and a basal culture medium 2. The small chemical molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof, or a combination of nicotinamide, Activin A or equivalent pharmaceutical products, analogs, isomers, salts, hydrates or precursors thereof. The basal culture medium 2 is DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055 mM 2-mercaptoethanol.

23. The reprogrammed culture medium composition of claim 22, wherein the concentration of LDN193189 in the small molecule chemical composition 1 is 0.1 mM, the concentration of A83-01 is 0.5 mM, the concentration of CKI-7 is 5 μM, the concentration of Hh-Ag1.5 is 0.5 mM, the concentration of CHIR-99021 is 3 μM, the concentration of BMS-345541 is 0.2 mM, and the concentration of RG108 is 10 μM, or the small molecule chemical composition 1 is... In composition 1, the concentrations of CHIR-99021 (10 μM), LDN193189 (0.5 mM), Hh-Ag1.5 (0.5 mM), RG108 (10 μM), BMS-345541 (0.2 mM), R-268712 (10 μM), BIX-01294 (1 μM), VPA (0.2 mM), and SB-431542 (10 μM) are all present.

24. The reprogrammed culture medium composition of claim 22, wherein the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM, the concentration of retinoic acid is 1 μM, and the concentration of Activin A is 0.2 mM, or the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM and the concentration of Activin A is 0.2 mM.

25. The application of a small chemical molecule composition in chemically reprogrammed fibroblasts induced into functional ciRPE cells, wherein the small chemical molecule composition is any one of claims 17-21.

26. The application as described in claim 25, wherein the fibroblasts are human or non-human mammalian fibroblasts.

27. The application as described in claim 26, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

28. The use of a small chemical molecule composition in the preparation of a product in which chemically reprogrammed fibroblasts are induced to become functional ciRPE cells, wherein the small chemical molecule composition is any one of claims 17-21.

29. The application as described in claim 28, wherein the fibroblasts are human or non-human mammalian fibroblasts.

30. The application as described in claim 29, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

31. The application of a reprogramming culture medium combination in chemically reprogrammed fibroblasts into functional ciRPE cells, wherein the reprogramming culture medium combination is any one of claims 22-24.

32. The application as described in claim 31, wherein the fibroblasts are human or non-human mammalian fibroblasts.

33. The application as described in claim 32, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

34. The use of a reprogramming culture medium combination in the preparation of a product in which chemically reprogrammed fibroblasts are induced to become functional ciRPE cells, wherein the reprogramming culture medium combination is any one of claims 22-24.

35. The application as described in claim 34, wherein the fibroblasts are human or non-human mammalian fibroblasts.

36. The application as described in claim 35, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

37. A kit or reagent kit for inducing two-step reprogramming of fibroblasts into ciRPE cells using a small chemical molecule composition, said kit or reagent kit comprising the small chemical molecule composition of any one of claims 17-21, or the reprogramming culture medium composition of any one of claims 22-24.

38. The kit or reagent kit of claim 37, wherein the fibroblasts are human or non-human mammalian fibroblasts.

39. The kit or reagent kit of claim 38, wherein the fibroblasts are embryonic fibroblasts, skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, intestinal fibroblasts, bladder fibroblasts and / or pancreatic fibroblasts.

40. A functional ciRPE cell, wherein the functional ciRPE cell is a functional ciRPE cell prepared by the method of any one of claims 1-16.

41. The use of the method of any one of claims 1-16 or the functional ciRPE cells of claim 40 in any of the following aspects: (1) Use in the preparation of drugs or preparations for treating retinal degenerative diseases; (2) Application in the preparation of cell therapy products for repairing or replacing damaged retinal pigment epithelial (RPE) cells; (3) Application in cell replacement therapy for retinal degenerative diseases; (4) Application in constructing in vitro cell models of retinal degenerative diseases; (5) Application in screening candidate drugs for the treatment of retinal degenerative diseases; (6) Applications in basic research on the mechanisms of retinal development; (7) Application in research on the regulation of retinal pigment epithelial cell function; (8) Application in the preparation of biological products that improve or restore visual function; (9) Application in the treatment of retinal degenerative diseases; (10) Application in cell therapy for repairing or replacing damaged retinal pigment epithelial (RPE) cells; (11) Application in improving or restoring visual function.

42. The application as described in claim 41, wherein the retinal degenerative disease is age-related macular degeneration, retinitis pigmentosa, Stargardt's disease, cone / rod cell dystrophy, Leber congenital amaurosis, myopic macular degeneration, or retinal degenerative changes secondary to diabetic retinopathy and retinal vein occlusion.

43. A method for treating retinal degenerative diseases, the method comprising administering a therapeutically effective amount of the functional ciRPE cells of claim 40 to a subject in need.

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