Composition for inducing functional ciRPE cells through chemical reprogramming, inducing method and application

Abstract

The invention discloses a composition for inducing functional ciRPE cells through chemical reprogramming, an induction method and application. The invention provides a two-step chemical reprogramming strategy, and fibroblasts can be transdifferentiated into functional RPE (ciRPE) cells through an eye domain (EF) / optic bubble (OV) intermediate state. The ciRPE cells are very similar to natural RPE cells in structure and function, have no tumorigenic risk after being transplanted to the retina of an RD rat, can be integrated into host tissues, effectively protect photoreceptor cells and remarkably recover the visual function.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to a composition for inducing functional ciRPE cells by chemical reprogramming, an induction method and applications thereof. Background Art

[0002] Retinal pigment epithelium (RPE) is crucial for retinal health and plays a key role in maintaining photoreceptor function and the blood-retinal barrier. Dysfunction or loss of RPE function is a key driver of the pathogenesis of devastating retinal degenerative diseases (RDs), such as age-related macular degeneration (AMD), retinitis pigmentosa (RP), and Stargardt disease, which lead to progressive vision loss and irreversible blindness in the late stage. Considering the important 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 major challenge.

[0003] Currently, there are mainly two methods for preparing RPE cells: isolating RPE cells from natural tissues or differentiating RPE cells from pluripotent stem cells. However, both of these strategies still pose certain challenges. RPE cells derived from natural tissues are scarce in themselves, and their functions rapidly decline in culture, limiting their application in large-scale treatment. In contrast, pluripotent stem cell-derived RPE cells, although theoretically an ideal source, have tumorigenicity and profound ethical issues. Another method is to directly reprogram 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 genomic integration, low reprogramming efficiency, and high costs, which may limit its wider clinical application. Summary of the Invention

[0004] Determining a suitable intermediate cell state is crucial for improving the efficiency and stability of RPE reprogramming. Progenitor cells derived from eye-field (EF) cells or optic vesicle (OV) cells naturally differentiate into RPE cells during retinal development and are an ideal intermediate state. These cells have significant plasticity and differentiation potential, making them a biologically relevant step in guiding somatic cells towards the RPE fate. Using EF (or OV) as an intermediate state can not only improve the reprogramming efficiency but also minimize the risk of generating unwanted cell types, ensuring a more stable and reproducible conversion process.

[0005] To solve the technical problems existing in the prior art, the present invention provides the following technical solutions.

[0006] The present invention provides a method for reprogramming fibroblasts into functional ciRPE cells by using a chemical small molecule composition in two steps, including: treating fibroblasts with reprogramming medium 1 to induce the reprogramming of fibroblasts into EF-like or OV-like cells, and then treating the EF-like or OV-like cells with reprogramming medium 2 to induce the reprogramming of the EF-like or OV-like cells into ciRPE cells;

[0007] The reprogramming medium 1 includes a chemical small molecule composition 1, and the reprogramming medium 2 includes a chemical small molecule composition 2;

[0008] When the fibroblasts are of murine origin, 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 pharmaceutical products, analogs, isomers, salts, hydrates or precursors equivalent to them;

[0009] When the fibroblasts are of murine origin, the chemical small molecule composition 2 is specifically a combination of nicotinamide, retinoic acid, Activin A or pharmaceutical products, analogs, isomers, salts, hydrates or precursors equivalent to them;

[0010] When the fibroblasts are of human origin, 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 pharmaceutical products, analogs, isomers, salts, hydrates or precursors equivalent to them;

[0011] When the fibroblasts are of human origin, the chemical small molecule composition 2 is specifically a combination of nicotinamide, Activin A or pharmaceutical products, analogs, isomers, salts, hydrates or precursors equivalent to them.

[0012] Furthermore, the composition of the ciRPE cell reprogramming medium is all cultured by adding small molecule compounds to a basal medium.

[0013] Furthermore, the basal medium 1 used for the reprogramming medium 1 is specifically an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% B27 without vitamin A, 7.5% BSA, 1% NEAA, 1% P / S, 10 ng / mL of bFGF.

[0014] Furthermore, the basal medium 2 used in the reprogramming medium 2 is specifically DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055 mM 2-mercaptoethanol.

[0015] Furthermore, the proliferation medium for ciRPE cells is used for the proliferation culture, and the components are as follows: DMEM / F12 / GlutaMAX as the substrate, supplemented with 1% N2, 2% B27 without vitamin A, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol.

[0016] Furthermore, the proliferation medium for ciRPE cells further includes one or more of 10 ng / mL bFGF, 20 ng / mL EGF, 10 μM Y-27632, and 0.5 μM A 83-01.

[0017] Furthermore, the function maintenance medium for ciRPE cells is used for the function culture, and the components are as follows: DMEM / F12 / GlutaMAX plus 1% N2, 2% B27 containing vitamin A, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol.

[0018] Furthermore, when the fibroblasts are of murine origin, the function maintenance culture further adds 0.2 μM Activin A, 0.5 μM retinoic acid, 1 μM BMP4, and 10 mM nicotinamide.

[0019] Furthermore, when the fibroblasts are of human origin, the function maintenance culture further adds 0.1 μM Activin A, 0.5 μM retinoic acid, and 0.5 μM BMP4.

[0020] Furthermore, when the fibroblasts are of murine origin, the concentration of LDN193189 in the chemical small molecule 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.

[0021] Furthermore, when the fibroblasts are of murine origin, 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.

[0022] Further, when the fibroblasts are of human origin, the concentration of CHIR-99021 in the small molecule chemical composition 1 is 10 μM, the concentration of LDN193189 is 0.5 mM, the concentration of Hh-Ag1.5 is 0.5 mM, the concentration of RG108 is 10 μM, the concentration of BMS-345541 is 0.2 mM, the concentration of R-268712 is 10 μM, the concentration of BIX-01294 is 1 μM, the concentration of VPA is 0.2 mM, and the concentration of SB-431542 is 10 μM.

[0023] Further, when the fibroblasts are of human origin, the concentration of nicotinamide in the small molecule chemical composition 2 is 10 mM, and the concentration of Activin A is 0.2 mM.

[0024] In some embodiments, the LDN193189 is a small molecule compound and mainly serves as a selective inhibitor of the bone morphogenetic protein (BMP) signaling pathway.

[0025] In some embodiments, the A 83-01 is an effective inhibitor of TGF-β type I receptor (ALK5-TD).

[0026] In some embodiments, the CKI-7 (dihydrochloride) is an ATP-competitive CKI inhibitor.

[0027] In some embodiments, the Hh-Ag1.5 is an effective Hedgehog (Hh) agonist with an EC50 of 1 nM. The mediated reprogramming breaks the quiescent state of non-injured liver stem cells, thereby rescuing liver failure.

[0028] In some embodiments, the CHIR-99021 (Laduviglusib, CT99021) is a GSK-3α and GSK-3β inhibitor.

[0029] In some embodiments, the BMS-345541 is a highly selective inhibitor of the catalytic subunit, acting on IKK-2 and IKK-1.

[0030] In some embodiments, the RG108 (N-Phthalyl-L-tryptophan) is a DNA methyltransferase inhibitor.

[0031] In some embodiments, the R-268712 is an orally active and selective ALK-5 inhibitor.

[0032] In some embodiments, the BIX-01294 is a reversible and highly selective inhibitor of G9a and GLP histone methyltransferases.

[0033] In some embodiments, the VPA (NSC-93819, valproic acid, sodium valproate, and divalproex sodium) is an approved drug mainly used to treat epilepsy and bipolar disorder and also used to prevent migraines.

[0034] In some embodiments, the SB-431542 is a small molecule inhibitor that can block the intracellular mediators of TGF-β receptor type I, thereby reducing TGF-β1-mediated proliferation, cytokine, and collagen expression.

[0035] In the present invention, the "μM" used represents micromolar concentration. μ is the Greek letter "mu", representing the SI prefix "micro-", i.e., 10⁻ 6 (one millionth), and M represents molar concentration (mol / L), i.e., the amount of substance of the solute in each liter of solution. Therefore, 1 μM = 10⁻ 6 mol / L, that is, there is 1 micromole of solute in each liter of solution.

[0036] In the present invention, mM is used as a special symbol for representing concentration in the SI system. Its full name is millimolar, which is a commonly used method for measuring the concentration of solutions in the fields of chemistry and biology. The definition of this unit is based on the basic unit of amount of substance, the mole (mol), where the prefix "m" represents one thousandth (10 -3 ). Therefore, 1 millimole is equal to 0.001 mole. When applied to a solution system, mM specifically refers to the number of millimoles of solute in each liter of solution, that is, the ratio of the amount of substance of the solute (in millimoles) to the volume of the solution (in liters).

[0037] In some embodiments, the method specifically comprises the following steps:

[0038] The first stage: Chemical transformation of EF cells in MEFs (mouse embryonic fibroblasts), specifically including the steps: 1) Four days before chemical transformation, thaw Matrigel overnight at 4°C. The next day, pre-cool a 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 should not be subjected to repeated freeze-thaw cycles).

[0039] 2) Two days before chemical induction, thaw the cryopreserved primary MEFs in a 37 °C water bath and centrifuge to remove the cryoprotectant. Resuspend the cells in MEF medium (DMEM medium containing 10% FBS) and seed them into a 6-well plate at a density of 20×10 4 cells per well. Before seeding the MEF cells, pre-warm the Matrigel-coated 6-well plate at 37 °C for at least 30 minutes (Note: The density of MEF cells is crucial for the success of chemical induction. Ensure that the cells are evenly distributed; too few cells will affect proliferation and cell state, while overcrowded culture may affect the induction success rate. Regularly monitor the health and growth of MEF cells after seeding to ensure that they adhere well and proliferate as expected. Appropriate pre-warming of the matrix-coated plate is essential for optimal cell attachment and performance).

[0040] 3) One day before chemical induction, replace the MEF medium with fresh medium to allow the cells to proliferate for 1 day. Regularly check the growth and condition of the MEF cells to ensure that the cells are healthy and the density is in the optimal state (Note: The growth condition of MEF cells is crucial for successful conversion. Long-term storage of MEFs in liquid nitrogen will also reduce the conversion efficiency. In this study, freshly prepared MEF cells or MEF cells stored in liquid nitrogen for no more than 6 months were used).

[0041] 4) On the day of chemical induction, prepare the fresh reprogramming medium 1 for the first stage, prepared in the dark to ensure that all components, especially the chemical reagents, are completely dissolved. After the MEF cells are incubated in MEF medium overnight, wash the cells twice with 1× PBS and replace the MEF medium with the newly prepared reprogramming medium 1. Replace the reprogramming medium 1 once the next day.

[0042] 5) During the induction in the first stage, the cells proliferate rapidly within the first 1 - 4 days, the cell morphology changes from long and slender spindle-shaped to small oval-shaped, and cell clone colonies appear. After one week, the proliferation rate of the clones will slow down. At this point, mechanically remove the surrounding non-clone cells using the tip of a yellow micropipette tip to promote the continued expansion of the clones. After about one week, the cells will start to show a cobblestone-like morphology. Thereafter, update the medium daily until EF-like cells are formed.

[0043] Second stage: EF cells generate ciRPE cells, specifically including: During the induction in the second stage, the cells show the initial RPE cell morphology but still lack maturity and pigmentation. In this stage, reprogramming medium 2 is used to induce pigmentation and promote the further maturation of ciRPE cells. The medium is changed daily. After two weeks of treatment, the cells are nearly fully mature and show obvious pigmentation. Subsequently, the ciRPE cells are passaged and amplified in maintenance medium.

[0044] In some embodiments, the specific steps for the dynamic alternating culture of the ciRPE cells are as follows: To balance proliferation and RPE-specific functions, MEF-derived ciRPE cells are cultured using a dynamic alternating medium. After the first passage, during the initial expansion period (0 - 7 days), the cells are cultured in a proliferation medium to maximize cell growth. Subsequently, every 3 - 5 days, the cells are briefly exposed to a maintenance function medium for 1 - 2 days. This alternating method effectively supports cell expansion and the preservation of early RPE-like characteristics.

[0045] In some embodiments, the method specifically comprises the following steps:

[0046] Phase 1: Chemical conversion of HEFs (human fibroblasts) to OV-like cells, specifically including:

[0047] 1) Matrigel is thawed overnight at 4°C. The next day, a 6-well plate is pre-cooled at 4°C for at least 1 hour. Matrigel is diluted 1:40 with DMEM / F12 / GlutaMAX solution, and the diluted Matrigel solution is added to each pre-cooled well of the 6-well plate. Then the plate is incubated overnight at 4°C.

[0048] 2) The next day, the cryopreserved primary HEFs are thawed in a 37°C water bath, and the cryoprotectant is removed by centrifugation. The cells are resuspended in 15% FBS-DMEM medium and seeded into the 6-well plate at a density of 15×10 4 cells per well. Before seeding the HEF cells, the Matrigel-coated plate is pre-warmed at 37°C for at least 30 minutes (Note: In this study, freshly prepared HEF cells or HEF cells stored in liquid nitrogen for no more than 2 months are used).

[0049] 3) When the density of HEF cells reaches 60 - 70%, chemical induction can be initiated. For the induction in the first phase, fresh reprogramming medium 1 is prepared in the dark to ensure that all components, especially the chemical reagents, are completely dissolved. The HEF cells are washed twice with 1× PBS, and the newly prepared reprogramming medium 1 is replaced, which is replaced every two days.

[0050] 4) In the first phase, the cells proliferate rapidly within the first week, and the morphology changes from long and slender spindle-shaped to small oval-shaped. Around the 12th day, some stable clones with consistent size and morphology will form. At this stage, the surrounding non-clone cells are mechanically removed with the tip of a yellow pipette microtip to promote the continued expansion of the clones. Thereafter, the medium is renewed daily until OV-like cells are formed.

[0051] Phase 2: Generation of hciRPE cells from OV-like cells

[0052] Induce pigmentation with reprogramming medium 2 to promote further maturation of hciRPE cells, and replace reprogramming medium 2 once a day. After treating the cells with the medium for two weeks, the cells showed an RPE-like morphology and had certain pigmentation. Subsequently, passage and expand hciRPE cells in maintenance medium.

[0053] For long-term culture, passage hciRPE cells using mechanical dissociation methods and seed them onto Matrigel-coated plates, maintaining a confluency of 60 - 80%. Update the maintenance medium every 2 - 3 days to ensure optimal cell health. Regularly monitor the RPE-specific characteristics of the cells, including morphology, pigmentation, and expression of key markers.

[0054] The present invention provides a chemical small molecule composition, which is divided into the aforementioned chemical small molecule composition 1 and chemical small molecule composition 2.

[0055] Furthermore, the chemical small molecule composition includes a pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient includes one or more selected from the group consisting of: 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, solubilizing agents, emulsifying agents, pH buffering substances, binders, fillers, lubricants, or other pharmaceutical excipients; or, the pharmaceutical dosage forms that the chemical small molecule composition can be prepared into include: solid dosage forms, including: powders, powders for external use, tablets, pills, capsules, sustained-release agents, controlled-release agents, or other solid dosage forms; liquid dosage forms, including: injections, infusions, suspensions, or other liquid dosage forms; gas dosage forms; or semi-solid dosage forms; or the small molecule composition is a reprogramming preparation or reagent, preferably further including an organic solvent, physiological saline, or other carrier or excipient.

[0056] The general requirements for excipients are that they are stable in nature, have no incompatibility with the main drug, do not produce side effects, do not affect the curative effect, are not easily deformed, cracked, mildewed, infested with insects at room temperature, are harmless to the human body, have no physiological effects, do not produce chemical or physical effects with the main drug, and do not affect the content determination of the main drug, etc. A full discussion of pharmaceutically acceptable carriers or excipients can be found in Remington’s Pharmaceutical Sciences (Mack Pub. Co., N.J. 1991). Its carriers or excipients include but are not limited to: aqueous solutions such as water, saline, phosphate buffer solution, etc.; organic solvents such as DMSO (dimethyl sulfoxide), glycerol, and ethanol; microspheres, liposomes, microemulsions, polymeric surfactants; colloidal drug delivery systems, novel polymeric drug delivery systems, novel drug carriers, and other pharmaceutically carriers; preservatives, antioxidants, flavoring agents, fragrances, solubilizing agents, emulsifiers, pH buffering substances in liquid preparations, binders, fillers, lubricants, and other drug excipients in tablets, etc.

[0057] The present invention provides a reprogramming medium used in the foregoing method, and the reprogramming medium includes reprogramming medium 1 and reprogramming medium 2;

[0058] The reprogramming medium 1 includes the foregoing chemical small molecule composition 1 and basal medium 1, and the basal medium 1 is an equal volume of Neurobasal and DMEM / F12 / GlutaMAX added with 1% N2, 1% B27 without vitamin A, 7.5% BSA, 1% NEAA, 1% P / S, bFGF;

[0059] The reprogramming medium 2 includes the foregoing chemical small molecule composition 2 and basal medium 2, and the basal medium 2 is DMEM / F12 / GlutaMax added with 10% KSR, 1% NEAA, 1% P / S, 0.055 mM 2-mercaptoethanol.

[0060] The present invention provides an application of a chemical small molecule composition in the preparation of a product for chemically reprogramming fibroblasts into functional ciRPE cells, and the chemical small molecule composition includes the foregoing chemical small molecule composition.

[0061] The present invention provides a kit or reagent kit for two-step reprogramming of fibroblasts into ciRPE cells with a chemical small molecule composition, and the kit or reagent kit includes the foregoing chemical small molecule composition or the foregoing reprogramming medium.

[0062] Furthermore, the fibroblasts include fibroblasts of human or non-human mammals.

[0063] Furthermore, the fibroblasts include dermal fibroblasts, hepatic fibroblasts, pulmonary fibroblasts, renal fibroblasts, and pancreatic fibroblasts.

[0064] Advantages and beneficial effects of the present invention:

[0065] The present invention addresses the complex challenge of generating functional RPE cells by developing an innovative two-stage chemical induction protocol implemented by a single-cell reprogramming compound screening platform (scRCF), which is an advanced platform that combines single-cell transcriptomics-driven computational prediction with a sophisticated drug sequence screening system. This platform systematically identifies the optimal small molecules to induce fibroblasts into an EF-like state and ultimately generate functional chemically induced RPE (ciRPE) cells. These ciRPE cells are highly similar to native RPE cells in terms of morphology, gene expression, and basic functional characteristics. In an RD rat model, transplantation of ciRPE cells can integrate into the host RPE layer, significantly protect photoreceptors, and significantly restore visual function. Multi-omics analysis provides mechanistic insights into this dynamic, showing that the compounds synergistically activate endogenous TFs, such as Ascl1 and Olig2, to reprogram fibroblasts towards the RPE cell phenotype. This study presents a scalable, non-integrating, cost-effective chemical method for generating functional RPE cells, providing a promising innovative strategy for cell replacement therapy for RD diseases. Brief Description of the Drawings

[0066] Figure 1It is the result graph of screening EF reprogramming small molecules by the scRCF system. Among them, A is the workflow of scRCF. The input of scRCF includes scRNA-seq data from the initial and target cell types, as well as three pre-established databases: 1) A small molecule perturbation database that integrates data from GEO and LINCS L1000, and only retains the TFs identified in AnimalTFDB3.0; 2) A small molecule target database and classification information, including information from STITCH, Drug Repurposing Hub, and MedChemExpress; 3) A signaling network database, derived from Reactome and OmniPath. scRCF contains three main steps: 1) Identify differentially expressed transcription factors based on scRNA-seq data, and use signaling network analysis to preliminarily screen candidate signaling proteins; 2) Perform community partitioning on the pre-screened signaling proteins, and perform cumulative scoring according to the distribution of small molecule targets in the community. Classified by signaling pathways, the small molecule with the highest score is selected from each pathway subset as the final output; 3) Further screen candidate small molecules using DRUG-seq2. B is the 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 the differentially expressed TFs (DETFs) identified from scRNA-seq data of MEF and EFs, analyzed using Seurat. DETFs are defined as P<0.05, log2>1. D is to determine candidate small molecules through preliminary screening by scRCF for reprogramming MEF into EF cells. E is a bar graph showing the Z-scores of cells treated with the LAC+1 small molecule combination, based on gene sets related to neuroectoderm and EF. The Z-score represents the relative gene expression change of each drug, and a higher value reflects a stronger impact on the gene set. The bar graph is sorted from the highest to the lowest average Z-score, and the dashed line represents a baseline score of zero. F is a heat map of the gene expression profiles of neuroectoderm and EF-related genes in cells after treatment with the LAC+1 small molecule combination. The color scale represents the log2-transformed expression values, with red indicating high expression, blue indicating low expression, and white indicating medium-level expression.

[0067] Figure 2It is the result graph of establishing a two-stage chemical reprogramming strategy to generate ciRPE cells. Among them, A shows the representative morphological changes of MEF cells exposed to the reprogramming medium (RM) containing 10 small molecules (stage 1) at different time points. Scale bar, 400 μm. B shows the qRT-PCR analysis of the expression of EF-related genes and early RPE development-related genes at the specified time points. C shows the representative morphological images of cells after exposure to the differentiation and maturation medium (DM) containing three compounds (stage 2). DMSO, dimethyl sulfoxide is used as a negative control. M3: NIC, RA, Activin A, scale bar, 200 μm. D shows the qRT-PCR analysis of the expression of RPE-related marker genes at the specified time points. E shows the genetic lineage tracing strategy and chemical reprogramming schematic diagram of MEF-derived ciRPE cells. F shows the morphological and tdTomato fluorescence expression changes of cells at different days during the induction process. Scale bar, 400 μm. G shows the percentage of tdTomato + cells induced by the candidate medium at different days. H shows the percentage of tdTomato + cells treated with the candidate medium (all 13 compounds), and the specified compounds are subtracted from the mixture. Each point represents a biological replicate. "-" indicates the removal of the specified compound. I shows the percentage of tdTomato + cells before and after the optimization of the reprogramming medium at different time points. J shows the schematic diagram of the reprogramming system for inducing MEF into ciRPE cells by compounds, and the representative cell morphological changes at the key time points of reprogramming. MM represents MEF medium, RM represents reprogramming medium, and DM represents differentiation / maturation medium. Scale bar, 200 μm. Figure 2 All representative examples with at least three independent experimental data are given. Data are Mean ± SD; *P<0.05, **P<0.01, ***P<0.001.

[0068] Figure 3It is a characteristic diagram of ciRPE cells. Among them, A shows that ciRPE cells derived from MEF express ZO-1, Pax6, Rpe65, Mitf, Best1, and Cralbp by immunostaining. 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 (upper), while Best1 (red) shows basolateral localization (lower). Scale bar, 10 μm. C is a transmission electron microscopy image of ciRPE cells showing apical microvilli (yellow arrows), melanin granules (red arrows), and tight junctions (black arrows). Scale bar, 1 μm. D is a confocal micrograph showing that the outer segments of photoreceptors (green) are phagocytosed by ciRPE cells. The apical side of ciRPE cells is stained with ZO-1 (purple), and the nucleus is counterstained with DAPI (blue). Scale bar, 50 μm. E shows the apical and basolateral polarized secretion of PEDF and VEGF by ciRPE cells grown on Transwells. F is a morphological image showing the dome structure formed by ciRPE cells derived from MEF during in vitro culture. Red arrows indicate the dome morphology observed under different phase contrast microscopy conditions. Scale bar, 50 μm. G measures the transepithelial electrical resistance (TEER) value of ciRPE cells over 30 days. Figure 3 All representative examples with at least three independent experimental data are given. Data are Mean ± SD.

[0069] Figure 4It is the molecular roadmap of ciRPE chemical reprogramming. Among them, A is a schematic diagram of the multi-omics sequencing analysis strategy for the reprogramming process from MEF to ciRPE cells. B is the principal component analysis (PCA) of RNA-seq and CUT&Tag data (H3K4me3, H3K27ac, and H3K27me3) for samples collected on days 0, 7, 18, and 32 (ciRPE) of reprogramming, with pRPE cells as the control. C is a heatmap of genes differentially expressed in MEF to ciRPE cell reprogramming samples at the specified time points. The numbers on the heatmap represent independent biological replicates. Representative genes (left side of the heatmap) and related gene ontologies (GO; right side of each block of the heatmap) are shown. Red and blue represent upregulated and downregulated genes, respectively. D is the dynamic change of module-specific gene CUT&Tag peaks (H3K4me3, H3K27ac, H3K27me3) in (C). The red line represents the median CUT&Tag peak over time, the blue line represents the median RNA expression level, and the gray background lines represent the peaks at each time point. E is the UMAP analysis of scRNA-seq data for cells collected at the specified time points during the reprogramming of MEF to ciRPE cells. F is the UMAP plot of the identified cell types in samples collected at the specified time points during the reprogramming process. G is a bubble plot of the expression of representative marker genes among different cell types during the reprogramming process. H is the RNA velocity streamline plot drawn using the scVelo method to predict the transformation of cell populations during the reprogramming process. The arrows represent the flow determined by the ratio of unspliced to spliced transcripts, predicting the dynamic change of cell identity. Black arrows represent the 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 is the similarity analysis of gene expression among different cell types during the reprogramming process. pEF cells represent the data used for small molecule prediction above, while pRPE data are from the GSE183572 dataset.

[0070] Figure 5Transcriptional activation control of master regulators of neural and eye development reprograms ciRPE. Among them, A is a volcano plot showing the differential regulatory active transcription factors (TFs) among different cell types during reprogramming, and the data is from SCENIC. B is a bar chart showing the MCC scores of the top 15 key TFs. C is a heat map showing the expression of the top 15 TFs at specified time points during cell reprogramming. D is the normalized analysis of RNA-seq and CUT&Tag sequencing (for histone modifications H3K4me3, H3K27ac, and H3K27me3) of the genomic loci of Ascl1, Olig2, Zic1, Pou3f2, and Lhx2 at specified time points during cell reprogramming. E-F show the evaluation of the 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 as "1", and DMSO was used as the negative control. WT represents wild type; Control KD represents scramble shRNA-mediated knockdown. G is the visualization of the scRCF network of the potential signaling cascades induced by small molecules targeting Ascl1 and Olig2. Orange rectangles represent perturbing compounds, blue diamonds represent signaling protein targets, white ellipses represent intermediate signaling proteins, and green hexagons represent TFs. Figure 5 All representative examples are given with at least three independent experimental data. Data are Mean ± SD; *P<0.05, **P<0.01.

[0071] Figure 6 Chemical reprogramming of HEFs into human ciRPE (hciRPE) cells. Among them, A is a list of candidate small molecules for the preliminary screening of HEF reprogramming into OV cells by scRCF. B is a schematic diagram of the procedure for reprogramming HEFs into hciRPE cells and the 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 is FACS purification of reprogrammed BEST1-EGFP +The proportion of hciRPE cells. D shows the optical microscopy and TEM images of hciRPE cells, revealing melanin granules (red arrows). Scale bar, 1 μm. E shows the qRT-PCR data analysis of the expression of RPE-related genes at specified time points during cell reprogramming. F shows the immunostaining analysis of hciRPE cells derived from BEST1-EGFP-HEFs, indicating positive expression of ZO-1, RPE65, MITF, and BEST1. Scale bar, 20 μm. G shows the PCA analysis of samples of hciRPE cells and control primary hRPE cells at day 0, day 12, day 24, and day 38 (hciRPE) during cell reprogramming. H shows the heatmap analysis of differentially expressed genes in cell samples at specified time points during the reprogramming of HEFs into hciRPE cells. The numbers on the heatmap represent independent biological replicates. Representative genes for each cluster (left side of the heatmap) and related GO (right side of the heatmap) are shown. Red and blue indicate upregulated and downregulated genes, respectively. I shows the apical and basolateral polarized secretion of VEGF and PEDF by hciRPE cells grown on Transwells. J shows the 30-day epithelial resistance (TEER) values of hciRPE cells. Figure 6 All representative examples are given with at least three independent experimental data. Data are Mean ± SD.

[0072] Figure 7 It is a figure showing the results of the restoration of retinal function in RCS rats by transplantation of ciRPE cells. Among them, A is a schematic diagram of subretinal transplantation of ciRPE cells in RCS rats. B are OCT images of RCS rats at 0, 1, 2, and 3 weeks after subretinal transplantation of ciRPE cells. Scale bar: 600 μm. C is a representative photograph of in vivo imaging, showing tdTomato-labeled mESCs (by lentivirus) and tdTomato + The situation of tumor formation in nude mice after subretinal transplantation of ciRPE cells. The statistical chart on the right shows the quantitative analysis data, N = 10. D is a representative bright-field (upper) and immunofluorescence image (lower) of an eye tissue section 8 weeks after subretinal transplantation of tdTomato + ciRPE cells. The dashed box indicates the cell transplantation area. Nuclei are counterstained with DAPI (blue). Scale bar, 200 μm. E shows the immunofluorescence analysis of whole retina sections at 12 weeks after transplantation, showing tdTomato + ciRPE cell clusters in the transplantation area. Magnified views of the areas outlined by the dashed boxes show: (i) the non-transplanted area and (ii) the transplanted area. The statistical chart on the right shows the quantitative analysis data. N = 10. Scale bar, 500 μm. F - H are for tdTomato +Immunostaining images of ciRPE cells co-expressing Mitf (F), Best1 (G), and Pax6 (H). Scale bar, 50 μm. I shows tdTomato with the sham transplantation group as the control + Representative micrographs of TUNEL staining of cryosections of the retina of RCS rats after transplantation of ciRPE cells for 12 weeks (left), and the statistical results are shown on the right. N = 10. Scale bar, 50 μm. J shows tdTomato transplanted subretinally in RCS rats + Immunostaining images of co-expression with Rhodopsin in ciRPE cells after 12 weeks. Scale bar, 50 μm. K shows representative b-wave response data (left) of the ciRPE transplantation group and the sham transplantation group at 4w, 8w, 12w, and 16w after transplantation with a fERG response intensity of 0.48 log cd*s / m² (dark 3.0). The statistical analysis of the b-wave amplitude of the ciRPEs transplantation group and the sham transplantation group is shown on the right. N = 6 per group. L shows a schematic diagram of the quantitative optokinetic response (qOMR) test device (left). Quantitative evaluation of the visual acuity of the ciRPEs transplantation group and the sham transplantation group at 4w, 8w, 12w, and 16w after transplantation (right), N = 20 Figure 7 All representative examples with at least three independent experimental data are presented. All data are expressed as Mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

[0073] Figure 8 It is a diagram of the small molecule system screening for EF fate reprogramming by scRCF. Among them, A is the UMAP visualization of the scRNA-seq datasets of MEF and EF for predicting small molecules for cell type conversion. B is the gene co-expression network of signaling proteins (upper layer) and TFs (lower layer). C is the protein-protein interaction network for community partitioning of signaling proteins using the random walk algorithm, showing the top 10 largest communities. D is a schematic diagram of the compound screening protocol for DRUG-seq2 sequencing. LDN193189, A 83-01, and CKI-7 (LAC) are used as the basic compound combination, and predictive small molecules are added respectively to form the LAC+1 combination. MEF is treated for 14 days before sequencing to evaluate changes in gene expression. E is the small molecule targeting the conversion of MEF to EF comprehensively screened by scRCF

[0074] Figure 9It is a schematic diagram of the strategy for generating ciRPE cells through two-stage chemical reprogramming. Among them, A is a bright-field image of the eyes of embryonic mice with a Best1-Cre / ROSA26 tdTomato genetic background. The red fluorescence indicates the localization of Best1 in eye tissues, traced by tdTomato. The cell nuclei are counterstained with DAPI (blue). Scale bar: 750 μm. B is an immunostaining analysis of whole retinal sections of the eyes of embryonic mice with a Best1-Cre / ROSA26 tdTomato genetic background. Blue indicates DAPI staining, and the red fluorescence indicates the localization of Best1 traced by tdTomato. Scale bar, 500 μm. C is the FACS sorting of E13.5 mouse embryonic MEF cells with a Best1-Cre / ROSA26 tdTomato background. D is the qRT-PCR analysis of tdTomato with β-actin as a control - The expression of RPE-specific genes in MEF and pRPE cells. E is the immunostaining analysis showing tdTomato - The RPE-specific genes such as Mitf, Cralbp, Best1, and Rpe65 are negative in MEFs. Scale bar, 50 μm. F is the representative morphological images of different fibroblast types (C57BL / 6 MEF, 129 MEF, C57BL / 6 TTF, 129 TTF) induced by M7+M3 compound medium. Scale bar, 400 μm. G is the flow cytometry analysis of the percentage of Best1-positive cells after the compound medium induces different fibroblast types (C57BL / 6 MEF, 129 MEF, C57BL / 6 TTF, 129 TTF).

[0075] Figure 10 It is a characteristic diagram of ciRPE cells. Among them, A shows that the G-banding karyotype analysis indicates that ciRPE cells have a normal karyotype. B is the morphological images of ciRPE and pRPE cells at different passages (P1, P3, and P6). Scale bar, 300 μm. C is the flow cytometry analysis of the incorporation of ethynyl deoxyuridine (EdU) in ciRPE and pRPE cells at different passages (P1, P3, and P6). D is the distribution of ciRPE and pRPE cells in the cell cycle (G1, S, and G2 phases) (left) and the quantitative analysis of the percentage of cells in each phase (right). E is the confocal microscopic images of ciRPE and pRPE cells phagocytosing latex beads (green). The apical side of the cells is stained with ZO-1 (red). Scale bar, 10 μm.

[0076] Figure 11It is a result figure of lineage tracing to confirm the induction of ciRPE cells from MEF. Among them, A is a schematic diagram of the genetic lineage tracing strategy. B shows that the starting cells were Best1 − / tdTomato + MEF cells screened from E13.5 mouse embryo MEF with Fsp1-Cre / ROSA26 tdTomato genetic background. C is the qRT-PCR analysis of Best1 - / tdTomato + The expression of RPE-specific genes in MEF and pRPE cells. β-actin was used as a control. D shows that the immunostaining analysis indicates that Best1 - / tdTomato + in MEF was negative for Best1, Rpe65, and Cralbp. Scale bar, 50 μm. E shows the bright-field and fluorescence images of the induced Best1 - / tdTomato + MEF and ciRPE cells. Scale bar, 300 μm. F shows that the immunostaining analysis indicates that in Best1 - / tdTomato + ciRPE cells derived from MEF were positive for ZO-1, Rpe65, Mitf, and Best1. Scale bar, 20 μm.

[0077] Figure 12 It is the molecular roadmap of ciRPE chemical reprogramming. Among them, A is a heatmap of the expression of 16 fibroblast and 18 RPE cell-specific genes at specified time points during the reprogramming process. Representative genes for each population are listed (right). Red and blue indicate upregulated and downregulated genes, respectively. B is the hierarchical clustering analysis of cells at specified time points during the reprogramming of MEF into ciRPE cells, including days 0, 7, 18, 32, and pRPE cells. C is a radar chart showing the average transcriptional activity of genes during ectoderm, mesoderm, endoderm, extra-embryonic development, and stem cell maintenance at specified time points during the reprogramming process. D shows the changes in protein modifications at different time points. "Gain" indicates peaks with a significant increase in signal values between time points (logFC > 1), and "Loss" indicates peaks with a significant decrease in signal values (logFC < -1). E is the enrichment curve of histone modifications H3K4me3 (left), H3K27ac (middle), and H3K27me3 (right) of differentially expressed genes at different time points during the reprogramming process. The x-axis represents the distance from the transcription start site (TSS) to the transcription end site (TES) (±3 kb), 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 the reprogramming process.

[0078] Figure 13 It is the result of scRNA-seq analysis of reprogramming, where A is the UMAP density map of the expression level of marker genes in four different cell populations during the reprogramming process. The expression level is represented by a color gradient, and the darker the red, the higher the gene expression. B is the proportion of cell types at different time points during the reprogramming process. C is the GO-term biological process enrichment analysis of different cell types identified from scRNA-seq data. D is the UMAP density map of the expression level of proliferation-related genes in neural progenitor-like intermediate cells during the reprogramming process. The expression level is represented by a color gradient, and the darker the red, the higher the gene expression. E is a pseudo-time trajectory showing the proportion of cell types during the reprogramming process. F is the pseudo-time of each cell in the trajectory predicted by Monocle 2.

[0079] Figure 14 is the analysis result diagram of key TFs in the reprogramming process, where A is the STRING network diagram of the interactions between TFs identified by SCENIC during the reprogramming process. Nodes represent TFs, color-coded by cell type: MEF (green), Intermediate cells (light blue), EF-like cells (blue), and ciRPE cells (red). BC is the evaluation of 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), and cells infected with scramble shRNA (scramble) were used as controls. D is tdTomato on day 32 after knocking down Ascl1 or Olig2 at designated time points during the M7+M3 induction process. + Cell morphology and proportion of cells. WT, wild type; Control KD, scramble shRNA-mediated knockdown. Scale bar, 200 μm. E, relative reprogramming efficiency after overexpression of Ascl1 or Olig2 under the indicated conditions. The reprogramming efficiency of M7 induction in the first stage was set as “1”, and DMSO was the negative control. F, scRCF network visualization of possible signaling cascades induced by M7 targeting key TFs. Orange rectangles represent perturbing small molecules, blue diamonds represent signaling protein targets, white ovals represent intermediate signaling proteins, and green hexagons represent TFs.

[0080] Figure 15Results of chemically reprogramming human ciRPE cells with HEF. Among them, A - B are UMAP visualizations of scRNA - seq data of HEF and OV, showing cell types (A) and datasets (B), used to identify small molecules that promote reprogramming between the two cell types. C is a bar graph showing the Z - score of HEF treated with the LCHRB + 1 small - molecule combination based on neuroectoderm - and EF - related gene sets. The Z - value represents the relative gene expression change of each drug on the gene set, and the higher the value, the stronger the impact on the gene set. The bars are sorted from high to low according to the average Z - value, and the dashed line represents a baseline score of zero. D shows the gene expression of mid - neuroectoderm - and EF - related genes after treating HEF with the LCHRB + 1 small - molecule combination. The color scale represents the log2 - transformed expression values, with red indicating high expression, blue indicating low expression, and white indicating medium - level expression. E is a schematic diagram of the BEST1 - Pr - EGFP - HEF cell line strategy for monitoring the reprogramming process of hciRPE cells. F is a qRT - PCR analysis of the expression of RPE - specific genes in BEST1 - Pr - EGFP - HEF and hRPE cells. β - actin was used as a control. G is an immunostaining analysis showing that the RPE - specific genes MITF, CRALBP, BEST1, and RPE65 are negative in BEST1 - Pr - EGFP - HEFs. Scale bar, 50 μm. H is a G - band karyotype analysis showing that hciRPE cells have a normal karyotype. I is a qRT - PCR analysis showing the expression of OV - and retinal progenitor - related marker genes at specific time points during the reprogramming process.

[0081] Figure 16 Results of transplanting ciRPE cells to restore retinal function in RCS rats. Among them, A is tdTomato + Schematic diagram of the process of transplanting ciRPE cells into the subretinal space of 3 - week - old RCS rats. B is a fundus bright - field image of RCS rats after subretinal injection of tdTomato + ciRPE cells. The red arrow indicates the bulge formed after transplantation. C is a teratoma assay of ciRPE cells. The right bar graph shows the quantitative analysis data. N = 15. D is a representative histological analysis of retinal sections of RCS rats in the non - injection group and the group injected with tdTomato + ciRPE cells (left), and the average outer nuclear layer (ONL) thickness was quantitatively evaluated 12 weeks after injection (right). N = 10. Scale bar, 50 μm. Figure 16 All representative examples presented data from at least three independent experiments. All data are presented as Mean ± SD. **P < 0.01, **P < 0.001. Detailed implementation methods

[0082] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources without special instructions. The experimental methods in the following embodiments are conventional methods without special instructions. The following cells are cultured at 37°C and 5% (volume percentage) CO 2 in an environment.

[0083] Example 1. Screening of Small Molecules for EF Cell Reprogramming by scRCF System

[0084] We established a two-step chemical induction strategy to reprogram fibroblasts into RPE cells. First, fibroblasts were transformed into an intermediate EF-like state, and then differentiated into ciRPE cells. To address the challenge of identifying small molecules for cell reprogramming, we developed scRCF ( Figure 1 A), 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), improving the precision and efficiency of molecule-driven reprogramming.

[0085] Using this platform, we screened and analyzed scRNA-seq data of mouse embryonic fibroblasts (MEF) and EF cells ( Figure 1 B and Figure 8 A). Differential gene expression analysis identified 258 TFs related to cell type conversion ( Figure 1 C). The improved SiPer20 framework calculated the similarity between these TFs and the perturbation spectra from a small molecule database, pre-screening 489 signaling proteins. Further refinement through gene co-expression networks reduced the selection to 279 functionally related signaling proteins ( Figure 8 B). This method revealed the key signaling relationships and transcriptional regulators necessary for cell transformation. To optimize the screening, a random walk algorithm for modular partitioning of protein networks ( Figure 8 C) achieved effect scoring and prioritized small molecules based on pathway relevance. Finally, 41 highly efficient candidate drugs 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 A 83-01, and NF-κB pathway inhibitor BMS-345541, etc. ( Figure 1 D).

[0086] To further identify small molecules for 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 type I receptor inhibitor) and A 83-01 (a TGF-β type I receptor) for their ability to inhibit mesoderm and endoderm differentiation. The addition of CKI-7, an ATP-competitive casein kinase I inhibitor, promoted neuroectoderm formation. We selected LDN193189, A 83-01, and CKI-7 (LAC) as the basal inducing small molecules for generating EF cells and used DRUG-seq2 to identify other synergistic small molecules ( Figure 8 D). After 14 days of treatment with various small molecule combinations, DRUG-seq2 analysis revealed distinct molecular expression patterns and changes in target gene expression in different treatment groups, reflecting specific responses to each condition ( Figure 1 E and Figure 1 F). Key regulatory small molecules promoting cell reprogramming were identified by effect scores of gene sets related to neuroectoderm and early EF development. Among them, Hh-Ag1.5, a Hedgehog signaling antagonist, obtained 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 1 E). Based on gene expression activation profiles and removal of functionally redundant compounds, 7 molecules, namely Hh-Ag1.5, CHIR-99021, Golvatinib, Pirfenidone, BMS-345541, Masitinib, and RG108, were selected from the top-ranked candidate drugs ( Figure 1 F) to form an optimized induction medium. Using this method, we identified 10 small molecules from 4319 candidate molecules capable of reprogramming MEFs into EF cells. In summary, we established a systematic, single-cell transcriptome-driven integrated computational and high-throughput compound screening system. This method identified 10 small molecule candidate induction components that have the potential to drive efficient cell reprogramming ( Figure 8 E).

[0087] Example 2. Establishment of a two-stage chemical reprogramming strategy to generate ciRPE cells

[0088] First, we optimized the serum-free, chemically defined medium containing these 10 small molecules from Example 1 and used it to treat MEFs. In the first stage, we observed the emergence of epithelial-like cell colonies after 6 days of treatment ( Figure 2A). Then, by day 12, cell proliferation began to slow down, but the clone morphology showed clearer boundaries and obvious epithelial characteristics ( Figure 2 A). To enhance the expansion of cell clone colonies, we mechanically removed some of the surrounding cells. As expected, the cell colonies gradually expanded. By day 18, the cells showed tight junctions, and some cells exhibited a high nuclear-cytoplasmic ratio and typical early RPE characteristics, such as cobblestone or hexagonal morphology ( Figure 2 A). Further qPCR analysis showed that EF-related genes (Pax6, Sox2, Six3, and Vsx2) and genes related to early RPE development (Mitf and Best1) were significantly upregulated ( Figure 2 B). These findings indicate that the 10 small molecule induction protocols we identified have the potential to convert MEFs into EF-like cells with early RPE characteristics.

[0089] 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 stage II induction. NIC inhibits neurogenic differentiation and promotes RPE-specific gene expression by regulating epigenetic and metabolic states. RA can promote RPE differentiation and regulate the expression of RPE-specific genes to promote pigmentation. Activin A regulates pigmentation, maintains epithelial characteristics, and promotes the differentiation of EF cells into RPE by activating the TGF-β / SMAD signaling pathway. After two weeks of treatment, the cells showed typical hexagonal RPE morphology and pigmentation ( Figure 2 C). Subsequent qPCR analysis confirmed the significant upregulation of Best1 and mature RPE genes such as Rpe65, Tyr, Lhx2, Pmel, and Otx2 ( Figure 2 D), indicating the successful generation of ciRPE cells. To further verify the effectiveness of reprogramming, we adopted a lineage tracing strategy to monitor the reprogramming process ( Figure 2 E). The Best1 gene is specifically expressed in RPE cells, and its promoter has been shown to effectively drive the expression of reporter genes. We used Best1-Cre / ROSA26tdTomato fluorescent reporter mice, and the results showed that tdTomato was stably and specifically expressed in RPE cells ( Figure 9 A and Figure 9 B). We used fluorescence-activated cell sorting (FACS) to collect Best1-tdTomato negative (tdTom - ) MEFs ( Figure 9 C). These tdTom -MEFs were negative for the expression of RPE marker genes such as Mitf, Cralbp, Best1, and Rpe65 ( Figure 9 D and Figure 9 E), confirming the absence of residual RPE or progenitor cells. Then, we performed two-step chemical induction on tdTom - MEFs (10 small molecules in the first stage and then M3 in the second stage). After the first-stage induction (day 18), low-level tdTomato expression was observed, with a positive rate of 2.51%. After further induction towards RPE cell maturation in the second stage (day 32), the tdTomato positive rate increased to 16.58% ( Figure 2 F and 2G). These results confirmed that the constructed chemical induction system effectively reprogrammed MEFs into RPE cells.

[0090] To minimize the potential toxic effects of small molecules on cells, we conducted a "-1" experiment to further optimize the chemical reprogramming system. The results showed that removing Golvatinib, Pirfenidone, and Masitinib in the first induction stage had the least effect on the tdTomato positive rate ( Figure 2 H), with a positive rate of 19.66% ( Figure 2 I). Therefore, we finally determined the reprogramming induction system, which consisted of seven small molecules from the first stage (M7: LDN193189, A 83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, and RG108) and three compounds from the second stage (M3: NIC, RA, and Activin A) ( Figure 2 J). Finally, we verified the effectiveness of our reprogramming system in MEFs of different batches (n = 5) and different genetic backgrounds (including C57BL / 6 and 129) ( Figure 9 F and Figure 9 G). In addition, this M7 + M3 system successfully promoted the conversion of tail tip fibroblasts (TTFs) from neonatal mice into ciRPE cells ( Figure 9 F and Figure 9 G). In summary, these results indicate that our optimized M7 + M3 system can effectively reprogram MEFs into ciRPE cells.

[0091] Example 3. Characteristics of ciRPE cells

[0092] To further confirm the phenotypic and functional characteristics of ciRPE cells, we first evaluated the expression of RPE markers by immunofluorescence staining. The results showed that ciRPE cells exhibited tight junction structures (ZO-1) and highly expressed RPE markers, including Pax6, Rpe65, Mitf, Best1, and Cralbp ( Figure 3 A). In addition, these cells showed a polarized morphology, with ZO-1 localized to the apical membrane and Best1 localized to the basolateral membrane ( Figure 3 B). This polarized structure is crucial for RPE function, especially the apical microvilli, which mediate the phagocytosis and clearance of shed photoreceptor outer segments, ensuring normal photoreceptor turnover. Subsequently, we analyzed the structural characteristics of ciRPE cells by transmission electron microscopy. These cells showed distinct apical microvilli, pigment granules, and tight junctions ( Figure 3 C), indicating that their morphology is highly similar to that of native RPE cells in vivo.

[0093] To evaluate the phagocytic ability of ciRPE cells, we introduced fluorescently labeled porcine photoreceptor outer segments (POS) and latex beads into the ciRPE cell culture medium. After a period of incubation, laser scanning confocal microscopy showed the presence of phagocytosed POS and latex beads within ciRPE cells ( Figure 3 D and Figure 10 E), confirming their phagocytic function. In addition, RPE cells are known to exhibit polarized secretion of growth factors, a process that is crucial for maintaining homeostasis between the retina and the choroid. We further analyzed the polarized secretion of growth factors in ciRPE cells and found that these cells mainly secreted VEGF from the basolateral side and PEDF from the apical membrane side ( Figure 3 E). In addition, ciRPE cells formed dome-shaped structures during in vitro culture ( Figure 3 F), indicating that their epithelial layer was able to effectively transport fluid while maintaining the integrity of tight junctions. Measurement of transepithelial electrical resistance (TEER) further demonstrated the integrity and barrier function of ciRPE cell tight junctions, with the results showing that TEER steadily increased to approximately 80 Ω×cm² within the first three weeks and then stabilized ( Figure 3 G). Overall, these data indicate that ciRPE cells exhibit the key functional characteristics of native RPE cells.

[0094] To further verify the proliferative potential of ciRPE cells, we purified them and performed serial passage culture in an expansion medium containing basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF). The results showed that ciRPE cells could be passaged at least 20 times while maintaining a stable RPE morphology and normal karyotype ( Figure 10 A and Figure 10B). In contrast, primary RPE (pRPE) cells gradually lost pigmentation and hexagonal morphology at passage 6, and the growth rate decreased significantly ( Figure 10 Band Figure 10 C), which is consistent with previous studies. In different passage batches, the proliferation ability of ciRPE cells was significantly higher than that of pRPE cells, and the EdU incorporation rate always exceeded that of pRPE cells ( Figure 10 C). 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 G0 / G1 phase, S phase, and G2 / M phase, respectively ( Figure 10 D). In summary, our results indicate that ciRPE cells are very similar to native RPE cells in terms of morphology and function, showing typical polarization characteristics, phagocytic ability, and polarized secretion of growth factors. In addition, they also have strong proliferation ability and stability.

[0095] Finally, we used the lineage tracing strategy of Fsp1-Cre and ROSA26-tdTomato mice to verify the reprogramming process of MEFs into ciRPE cells. MEFs were isolated from E13.5 transgenic mice with the Fsp1-Cre / ROSA26 tdTomato genetic background, and tdTomato + / Best1 - cells were obtained by FACS sorting ( Figure 11 A and Figure 11 B). These cells initially did not express RPE markers ( Figure 11 C and Figure 11 D). After chemical reprogramming, ciRPE cells began to express RPE-specific genes and co-express tdTomato ( Figure 11 E and Figure 11 F), further confirming that these cells originated from the original MEFs.

[0096] Example 4. Molecular Roadmap of Chemical Reprogramming of ciRPE

[0097] To enhance the analysis of the complex process of reprogramming MEFs into ciRPE cells, we performed comprehensive multi-omics analysis ( Figure 4 A). Cells were subjected to RNA sequencing (RNA-seq) on day 0, day 7, day 18, and day 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 the early stages ( Figure 4B). Time-series fuzzy clustering analysis of differentially expressed genes revealed four distinct gene clusters corresponding to different stages of reprogramming: fibroblasts (day 0), intermediate state (day 7), EF-like cells (day 18), and mature ciRPE cells (day 32) ( Figure 4 C).

[0098] At the initial stage of the transition from fibroblasts to the intermediate state, downregulated genes such as Fn1, Itga11, and Runx2 were associated with reduced fibroblast function and proliferative activity. In contrast, upregulated genes including Sox2, Ascl1, Gli2, Gli1, and Gbx2 were related to neuroectodermal processes ( Figure 4 C). These results suggest that early chemical induction promotes the transition towards a neuronal fate by simultaneously inhibiting fibroblast characteristics through the activation of neuroectodermal genes and signaling pathways. Additionally, during the intermediate stage, transient activation of genes such as Tfap2a, Rorb, and Etv5 was associated with epithelial cell proliferation and inflammation, reflecting enhanced cell-cell interactions and adaptation to environmental signals ( Figure 4 C). By day 18, the upregulated genes were mainly those related to eye development, such as Pax6, ID4, and Best1. As the 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 4 C), confirming the transformation of cells into mature pigmented ciRPE cells.

[0099] To confirm the successful conversion of fibroblasts into ciRPE cells, we analyzed 16 fibroblast-specific genes and 18 RPE-specific genes. During reprogramming, fibroblast markers were significantly downregulated while RPE markers were upregulated, indicating that ciRPE cells acquired RPE characteristics( Figure 12 A). Finally, the gene expression profile of ciRPE cells highly matched that of pRPE cells, further confirming the RPE cell characteristics of these cells ( Figure 4 B, Figure 4 C, Figure 12 A and Figure 12 B). Notably, the reprogramming process did not involve the iPSC stage; instead, transcriptional activation was mainly concentrated on genes related to the ectoderm ( Figure 12 C). This indicates that the compound combination we identified specifically guides fibroblasts to differentiate along the neuroectodermal and eye development pathways, highlighting the effectiveness of our screening strategy.

[0100] Epigenetic remodeling plays a crucial role in cell fate reprogramming. We used CUT&Tag sequencing to track the dynamic changes of histone modifications H3K4me3, H3K27ac, and H3K27me3 near the gene transcription start sites. PCA analysis showed significant changes in chromatin states during the reprogramming of ciRPE cells, indicating that ciRPE cells are very similar to pRPE cells in histone modification patterns ( Figure 4 B). H3K4me3 gradually increased from MEFs to ciRPE cells, reaching peaks on days 18 and 32, which is related to the activation of RPE genes. The loss of H3K4me3 decreased over time, indicating its role in establishing new gene expression patterns. The changes in H3K27ac were different at different stages, decreasing from day 7 to day 18, indicating that genes inhibited EF differentiation. The dynamic changes in H3K27me3 reflected gene silencing: the significant loss from MEFs to day 7 of induction promoted differentiation, while the increase from day 7 to day 18 established new silencing. By the ciRPE stage, the level of H3K27me3 tended to be stable, maintaining the mature RPE phenotype ( Figure 12 D). Further analysis found that the CUT&Tag signals of H3K4me3 and H3K27ac were correlated with RNA expression levels, emphasizing their role in gene activation ( Figure 4 D). Although H3K27me3 was generally negatively correlated with RNA-seq data, there was a certain deviation at the fibroblast stage, probably due to the dominant activation markers. From day 7 to day 18, the increase in H3K27me3 was correlated with the decrease in RNA expression, and the stable trend at the ciRPE stage reflected the balance between gene silencing and activation ( Figure 4 D and Figure 12 E). These findings emphasized the complex role of dynamic histone modifications in cell type transitions at different stages, highlighting the key 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.

[0101] To accurately describe the reprogramming trajectory, we performed scRNA-seq at three key time points during the reprogramming of MEFs to ciRPE cells. Single-cell transcriptome data of 9679, 13227, and 11282 cells were obtained from samples collected on days 7, 18, and 32, respectively. ( Figure 4 E). Cluster analysis of specific marker genes showed that on day 7 of reprogramming, the cell population consisted of MEF cells, neural progenitor cell-like intermediate cells, and a small fraction of EF-like cells. By day 18, the proportion of intermediate cells decreased, and the proportion of EF-like cells increased. On day 32, the EF-like cells decreased, and a large number of ciRPE cells appeared ( Figure 4 F,Figure 4 G, Figure 13 A, and Figure 13 B). To further validate the reprogramming trajectory of ciRPE cells, we performed single-cell pseudotime trajectory analysis. The analysis revealed a progression from MEFs to neural progenitor-like intermediate cells, followed by differentiation into EF-like cells and finally into ciRPE cells ( Figure 4 H). Unsupervised clustering highlighted the dynamic molecular events during this process, including the fibroblast-to-neural progenitor transition, the progression through eye developmental stages, and the establishment of RPE-specific functions (pigmentation and retinoid metabolism) ( Figure 13 C). Interestingly, among the two identified neural progenitor-like subpopulations, the differentiation trajectory of MEFs mainly corresponded to the subpopulation expressing neurogenic and proliferative markers (such as Mki67, Cdk1, Top2a), while the other subpopulation lacked these markers ( Figure 13 D). This indicates that proliferative neural progenitors are crucial for subsequent differentiation into EF-like cells. RNA velocity analysis similarly reflected the progression of reprogramming towards ciRPE cells, which was consistent with the trajectory observed in pseudotime analysis ( Figure 13 E and Figure 13 F). We further performed a similarity analysis between the reprogrammed cell population and publicly available single-cell datasets. The similarity scores of the induced cells to RPE cells and EF-like cells were 0.80 and 0.64, respectively ( Figure 4 I), indicating that the reprogrammed cells possess 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 intermediate states and EF-like cell stages. This series of processes emphasizes the precise control of lineage-specific gene expression and epigenetic remodeling.

[0102] Example 5. Transcriptional activation of master regulators of neural and eye development controls ciRPE reprogramming

[0103] To identify the key TFs driving the reprogramming of MEFs into ciRPE cells, we combined single-cell data with SCENIC analysis to systematically investigate the regulatory factors expressed in each cell type and their dynamic changes during reprogramming. Setting logFC > 1.0 as the threshold, we identified 125 TFs in four cell types, highlighting their distinct roles at different reprogramming stages ( Figure 5 A). Using the STRING database, we constructed an interaction network of these TFs, revealing their cooperative roles during reprogramming ( Figure 14A). Network analysis using maximum clique centrality (MCC) identified 15 key TFs, including MEF-specific factors (Dlx2 and Dlx1), transient activation factors on day 7 (Sox11, Foxa2, and Pax2), factors with continuously high expression from day 7 to day 18 (Sox2, Sox9, Olig2, Zic1, Ascl1, Atoh1, Pou3f2, and Gbx2), and ciRPE-related factors (Lhx2 and Otx2) ( Figure 5 B and 5C). Notably, most of these key TFs are related to neuroectodermal development, indicating that the fate towards neuroectoderm is a key transition stage in the reprogramming of MEFs into EF-like cells and ciRPE cells.

[0104] To further explore the regulatory mechanisms of these TFs, we integrated RNA-seq and CUT&Tag-seq data to evaluate the activities of the top 5 TFs ( Figure 5 D). On day 0, neuroectodermal TFs, including Ascl1, Olig2, Zic1, and Pou3f2, showed weak activation marks (H3K4me3 and H3K27ac) and were mainly inhibited by H3K27me3, which is consistent with their inhibition in the stable fibroblast state. By day 7, when the cells transitioned to a neuroprogenitor-like intermediate state, these TFs showed increased activation marks and RNA expression, indicating the activation of the neuroectodermal pathway. This coincided with the inhibition of fibroblast characteristics and the onset of neurogenesis. By day 18, the activities of Ascl1 and Olig2 decreased, while Zic1 and Pou3f2 remained highly activated. On day 32, at the ciRPE stage, Lhx2 showed strong activation and significant RNA upregulation, while early neuroectodermal TFs were silenced ( Figure 5 D). Notably, although H3K27me3 played a key inhibitory role in the early stage (0 - 7 days), its role weakened in the later stage, suggesting that other inhibitory mechanisms (such as DNA methylation or other histone marks) may be regulating TF activities at this stage. These findings highlight the crucial role of dynamic epigenetic modifications and TF expression in guiding cell fate transitions during the reprogramming of MEFs into ciRPE. Overall, neurodevelopment-related TFs are the "switches" for early differentiation into EF, while RPE-related TFs dominate the later stage and drive RPE maturation.

[0105] To determine the effects of Ascl1 and Olig2 on the reprogramming of fibroblasts into EF-like cells, we investigated the impact of separately knocking down these genes with shRNA on the efficiency of ciRPE induction. As expected, knockdown of Ascl1 or Olig2 significantly reduced the reprogramming efficiency, confirming their positive regulatory role in reprogramming.Figure 5 E, Figure 5 F, Figure 14 B- Figure 14 D). Importantly, knocking down these genes at early stages had the most significant impact on reprogramming efficiency (a 9.1-fold, 16.7-fold, and 3.6-fold reduction when transfecting shAscl1 on days 0, 7, and 18 respectively, Figure 5 E; a 12.5-fold, 8.3-fold, and 2.8-fold reduction when transducing shOlig2 on days 0, 7, and 18 respectively, Figure 5 F), 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 M7+M3-mediated ciRPE reprogramming efficiency ( Figure 14 E), verifying the direct involvement of Ascl1 and Olig2 in ciRPE reprogramming.

[0106] These findings highlight the importance of the transition to the neuroectodermal and EF-like cell states for the successful reprogramming of MEFs into ciRPE cells, verifying the effectiveness of our first-phase small molecule compound combination in coordinating these transitions. To gain a deeper understanding of the mechanism by which these small molecules regulate key TFs, we constructed a complex regulatory network using scRCF ( Figure 5 G and Figure 14 E). Analysis showed that the activation of key TFs was the result of the combined action of these small molecules, which regulated TFs through their respective target proteins, coordinated the activation of multiple signaling pathways including Wnt, TGF-β, and Hedgehog, and jointly promoted the reprogramming process and guided the precise transition of cell fate towards ciRPE reprogramming.

[0107] Example 6. Generation of human ciRPE cells from HEFs by chemical reprogramming

[0108] In our efforts to reprogram human ciRPE (hciRPE) cells, we initially attempted to apply the mouse induction system to reprogram human embryonic fibroblasts (HEF). However, the results were not satisfactory, probably due to the higher complexity of human cell reprogramming and more stringent signal requirements compared to the mouse system. Therefore, it was necessary to adjust the small molecule combination to improve reprogramming efficiency and success rate. During the selection process of compounds, we focused on the "OV" stage of early human retinal development, which is a key intermediate state guiding human RPE reprogramming. By integrating single-cell data from human embryos (Carnegie stage 12 and 16) and 30-day retinal organoids ( Figure 15 A and Figure 15 B), we strategically selected the top 15 key TFs identified during the mouse ciRPE reprogramming process (Figure 5 B) Incorporate scRCF. This method can more accurately identify small molecules, thus guiding the reprogramming of hciRPE cells. Finally, we identified 42 candidate small molecules ( Figure 6 A). Notably, LDN193189, CHIR-99021, Hh-Ag1.5, RG108, and BMS-345541 (LCHRB) found in the mouse RPE reprogramming system are present in the basic compound list. Then, we used DRUG-Seq2 (LCHRB+1) to screen other compounds and ranked the top 10 through a comprehensive scoring system ( Figure 15 C and Figure 15 D), and finally selected 15 small molecule combinations. We hypothesized that these combinations would promote the transformation of HEFs into OV-like cells.

[0109] To verify our hypothesis, we constructed a BEST1 Pr-EGFP-HEFs tracking cell line to monitor the reprogramming process ( Figure 15 E). Initially, these cells did not express RPE markers such as MITF, CRALBP, BEST1, and RPE65, nor did they express EGFP ( Figure 15 F and Figure 15 G). After successful reprogramming, the Best1 promoter specifically drives EGFP expression. We added the selected 15 compounds to the reprogramming medium to initiate the reprogramming of HEFs. In the early stage of induction, HEFs showed rapid proliferation; however, by day 12, only a small number of cell clones appeared. To support the growth of clones, we removed the surrounding cells, but their proliferation was still slow. By day 24, only a few cells were positive for BEST1-EGFP. After continued induction, there were no obvious changes in the cells, and on the contrary, the cell number decreased. We then introduced three compounds M3 (NIC, RA, and Activin A) to promote the differentiation and maturation of RPE, but the proportion of BEST1-EGFP positive cells was still very low.

[0110] Since the efficiency of this induction system is not ideal and excessive compounds have potential cytotoxicity, it is necessary to further optimize the induction system. Starting from the initial 15 small molecules and 3 differentiation and 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 and maturation stage, 6.74% of BEST1-EGFP positive cells could be obtained by day 38 ( Figure 6B and Figure 6 C), while other combinations produced a lower proportion of positive cells.

[0111] We further characterized the molecular and biological features of hciRPE cells. After purification and expansion, the induced hciRPE cells exhibited a typical hexagonal morphology and prominent melanin granules ( Figure 6 D), while maintaining a normal karyotype ( Figure 15 H). The results of qPCR analysis and immunofluorescence staining together confirmed that EGFP + hciRPE cells specifically expressed key RPE markers such as MITF, CRALBP, BEST1, and RPE65, ( Figure 6 E and Figure 6 F). Transcriptomic analysis showed that the expression profile of hciRPE cells was very similar to that of primary human RPE (hRPE) cells used as a positive control ( Figure 6 G and Figure 6 H). In hciRPE cells and the intermediate reprogramming 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 occurred at the intermediate state stage, indicating that the neuroectodermal transcriptional program might be activated and might transition towards the RPE lineage. By day 24, markers associated with OV and retinal progenitor cells (PAX6, SIX3, VSX2, MITF, and BEST1) were activated ( Figure 15 I), indicating successful conversion into OV-like cells, which is a key intermediate state in the transition towards the RPE lineage. After continued differentiation, these cells ultimately showed similar expression to hRPE cells ( Figure 6 G and Figure 6 H). Further analysis showed that hciRPE cells secreted growth factors such as VEGF and PEDF ( Figure 6 I), and TEER analysis confirmed the presence of tight junctions and barrier function ( Figure 6 J). These results indicate that our optimized two-stage chemical reprogramming system can effectively convert HEFs into functional hciRPE cells, providing a scalable and non-integrating method for studying RPE biology and developing cell-based therapeutic approaches.

[0112] Example 7. Transplantation of ciRPE cells restores retinal function in RCS rats

[0113] Leveraging the functional and safety advantages of ciRPE cells, we conducted in vivo transplantation studies to evaluate their potential for treating RD diseases. Royal College of Surgeons (RCS) rats in the UK develop retinal degeneration due to impaired phagocytic ability of RPE cells for photoreceptor outer segments caused by Mertk gene mutations, making them a classic model for studying RD diseases. We transplanted FACS-purified tdTomato + ciRPE cells into the subretinal space of 3-week-old RCS rats ( Figure 7 A, Figure 16 A and Figure 16 B). The contralateral non-transplanted eye, PBS-transplanted eye, and mESCs-transplanted eye were used as control groups. Postoperative optical coherence tomography (OCT) imaging showed a distinct bulge at the transplantation site, which decreased significantly after one week and completely regressed after three weeks, indicating successful transplantation and good tissue adaptability ( Figure 7 B). To evaluate the long-term safety of transplantation, we conducted a 4-month follow-up assessment. During this period, no signs of tumor formation were observed in the nude mice transplanted with ciRPE. In contrast, visible intraocular tumors developed in 13 out of 15 nude mice that received tdTomato-labeled mESCs ( Figure 7 C). This result is consistent with the experimental findings of subcutaneous teratomas, further confirming that ciRPE cell transplantation has no risk of tumorigenicity and thus supporting its safety ( Figure 16 C).

[0114] Histological and immunostaining analyses at 4 weeks after transplantation showed the presence of tdTomato + transplanted cell clusters in the subretinal space ( Figure 7 D). By 12 weeks, these cell tissues had transformed into an orderly monolayer morphology, indicating their successful integration into the host RPE ( Figure 7 E). In addition, the transplanted cells expressed mature RPE markers such as Mitf, Cralbp, Pax6, and Rpe65 ( Figure 7 F, Figure 7 G, Figure 7 H). Typically, RCS rats exhibit severe retinal dysfunction at 2 to 3 months of age, characterized by significant apoptosis of photoreceptors and thinning of the outer nuclear layer (ONL). At 12 weeks after transplantation, the ONL in the ciRPE transplantation group was significantly thicker than that in the non-transplanted group and the Sham group ( Figure 7 E and Figure 16 D), indicating that ciRPE transplantation protected photoreceptor cells and slowed their degeneration, thus maintaining and improving the structure and function of the retina. TUNEL staining further showed that the number of apoptotic cells in the ONL of the ciRPE group was significantly reduced compared to the Sham group ( Figure 7I), highlighting the protective effect of ciRPE cells against photoreceptor apoptosis. In RCS rats, the loss of phagocytic function of RPE cells due to Mertk mutation affects the clearance of photoreceptor outer segments. To evaluate whether ciRPE cells can restore this function in vivo, we monitored their uptake of Rhodopsin, the major component of photoreceptor outer segments. Co-localization of tdTomato and Rhodopsin was observed in the subretinal space of rats 12 weeks after transplantation of ciRPE cells ( Figure 7 J), indicating that the transplanted cells were successfully integrated and restored the phagocytic function of RPE cells.

[0115] To comprehensively evaluate the effect of ciRPE cell transplantation on retinal function, we performed dark-adapted flash electroretinogram (fERG) and behavioral tests. fERG data collected 4 - 16 weeks after 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 after transplantation ( Figure 7 K). This indicates that ciRPE cells support photoreceptor survival and functional recovery. The continuous increase in b-wave amplitude reflects the short-term and long-term effects of cell integration. To evaluate whether these electrophysiological improvements translate into enhanced visual performance, we performed optomotor response (OMR) tests. The ciRPE group showed superior visual performance compared to the control group, such as faster responses to moving stripe stimuli and higher sensitivity to changes in spatial frequency and direction of motion, which could be quantified by increased OMR scores ( Figure 7 L). These findings confirm the long-term survival, safety, and therapeutic efficacy of ciRPE cells in vivo, their ability to successfully integrate into host tissue, reconstruct functional RPE structures, and restore visual function, highlighting their potential as a potential cell source for treating RD diseases.

Claims

1. A method for inducing fibroblasts into functional ciRPE cells by two-step reprogramming using a chemical small molecule composition, comprising: Treat fibroblasts with reprogramming medium 1 to induce reprogramming of fibroblasts into EF-like or OV-like cells, and then treat EF-like or OV-like cells with reprogramming medium 2 to induce reprogramming of EF-like or OV-like cells into ciRPE cells; The reprogramming medium 1 includes a chemical small molecule composition 1, and the reprogramming medium 2 includes a chemical small molecule composition 2; When the fibroblasts are of mouse origin, the chemical small molecule composition 1 is specifically LDN193189, A 83-01, CKI-7, Hh-Ag1.5, CHIR-99021, BMS-345541, RG108 or a combination of pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors equivalent thereto; When the fibroblasts are of mouse origin, the chemical small molecule composition 2 is specifically nicotinamide, retinoic acid, Activin A, or a combination of pharmaceutical preparations, analogs, isomers, salts, hydrates, or precursors equivalent thereto; When the fibroblasts are of human origin, the chemical small molecule composition 1 is specifically CHIR-99021, Hh-Ag1.5, LDN193189, RG108, BMS-345541, R-268712, BIX-01294, Valproic acid (VPA), SB-431542 or a combination of pharmaceutical preparations, analogs, isomers, salts, hydrates or precursors equivalent thereto; When the fibroblasts are of human origin, the chemical small molecule composition 2 is specifically nicotinamide, Activin A, or a combination of their equivalent pharmaceutical preparations, analogs, isomers, salts, hydrates, or precursors.

2. The method according to claim 1, wherein the composition of the ciRPE cell reprogramming medium is to use a basic medium supplemented with a small molecule compound for culture; Preferably, the reprogramming medium 1 uses a basal medium 1, specifically equal volumes of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% B27 without vitamin A, 7.5% BSA, 1% NEAA, 1% P / S, and 10 ng / mL bFGF; Preferably, the reprogramming medium 2 uses a basic medium 2, specifically DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055 mM 2-mercaptoethanol.

3. The method according to claim 1, wherein the proliferation culture uses a proliferation medium for ciRPE cells, the components of which are as follows: DMEM / F12 / GlutaMAX as a substrate, supplemented with 1% N2, 2% B27 without vitamin A, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol; Preferably, the proliferation medium of the ciRPE cells further comprises one or more of 10 ng / mL bFGF, 20 ng / mL EGF, 10 μM Y-27632, and 0.5 μM A 83-01; Preferably, the functional culture uses a function maintenance medium for ciRPE cells, the components of which are as follows: DMEM / F12 / GlutaMAX plus 1% N2, 2% B27 containing vitamin A, 1% NEAA, 1% P / S, and 0.1 mM 2-mercaptoethanol; Preferably, when the fibroblasts are of mouse origin, the function-maintaining culture is supplemented with 0.2 μM Activin A, 0.5 μM retinoic acid, 1 μM BMP4 and 10 mM nicotinamide; Preferably, when the fibroblasts are of human origin, the function-maintaining culture is supplemented with 0.1 μM Activin A, 0.5 μM retinoic acid, and 0.5 μM BMP4.

4. The method according to claim 1, when the fibroblasts are of mouse origin, the concentration of LDN193189 in the chemical small molecule composition 1 is 0.1 mM, the concentration of A 83-01 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; Preferably, when the fibroblasts are of mouse origin, 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; Preferably, when the fibroblasts are of human origin, the concentration of CHIR-99021 in the chemical small molecule composition 1 is 10 μM, the concentration of LDN193189 is 0.5 mM, the concentration of Hh-Ag1.5 is 0.5 mM, the concentration of RG108 is 10 μM, the concentration of BMS-345541 is 0.2 mM, the concentration of R-268712 is 10 μM, the concentration of BIX-01294 is 1 μM, the concentration of VPA is 0.2 mM, and the concentration of SB-431542 is 10 μM; Preferably, when the fibroblasts are of human origin, the concentration of nicotinamide in the chemical small molecule composition 2 is 10 mM, and the concentration of Activin A is 0.2 mM.

5. A chemical small molecule composition, wherein the chemical small molecule composition is divided into the chemical small molecule composition 1 and the chemical small molecule composition 2 according to any one of claims 1 to 4.

6. The chemical small molecule composition as claimed in claim 5, wherein the chemical small molecule composition comprises a pharmaceutically acceptable carrier or excipient; preferably, the carrier or excipient comprises one or more selected from the following groups: water, saline, phosphate buffer or other aqueous solvents; DMSO, glycerol and ethanol or other organic solvents; microspheres, liposomes, microemulsions or polymer surfactants; colloidal drug delivery systems or polymer drug delivery systems; preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, pH buffer substances; Adhesives, fillers, lubricants or other pharmaceutical excipients; or, the pharmaceutical dosage forms that can be prepared by the chemical small molecule composition include: solid dosage forms, including: powders, powders, tablets, pills, capsules, sustained-release agents, controlled-release agents, or other solid dosage forms; liquid dosage forms, including: injections, infusions, suspensions, or other liquid dosage forms; gaseous dosage forms; or semi-solid dosage forms; or the small molecule composition is a reprogramming preparation or reagent, preferably also including an organic solvent, saline, or other carriers or excipients.

7. A reprogramming medium for use in the method according to any one of claims 1 to 4, the reprogramming medium comprising reprogramming medium 1 and reprogramming medium 2; The reprogramming medium 1 comprises the chemical small molecule composition 1 according to claim 5 or 6 and a basic medium 1, wherein the basic medium 1 is an equal volume of Neurobasal and DMEM / F12 / GlutaMAX supplemented with 1% N2, 1% B27 without vitamin A, 7.5% BSA, 1% NEAA, 1% P / S, and bFGF; The reprogramming medium 2 comprises the chemical small molecule composition 2 described in claim 5 or 6 and a basic medium 2, wherein the basic medium 2 is DMEM / F12 / GlutaMax supplemented with 10% KSR, 1% NEAA, 1% P / S, and 0.055 mM 2-mercaptoethanol.

8. Use of a chemical small molecule composition in preparing a product for chemically reprogramming fibroblasts into functional ciRPE cells, the chemical small molecule composition comprising the chemical small molecule composition according to claim 5 or 6.

9. A medicine box or test kit for inducing two-step reprogramming of fibroblasts into ciRPE cells using a chemical small molecule composition, the medicine box or test kit comprising the chemical small molecule composition according to claim 5 or 6, or the reprogramming medium according to claim 7.

10. The kit or reagent box of claim 9, wherein the fibroblasts comprise fibroblasts of human or non-human mammals; Preferably, the fibroblasts include skin fibroblasts, liver fibroblasts, lung fibroblasts, kidney fibroblasts, and pancreatic fibroblasts.

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