Pluripotent stem cell-derived RPE cells, formulations, pharmaceutical compositions, reagents, methods, and kits

Abstract

This invention belongs to the field of regenerative medicine and relates to cell therapy, specifically providing pluripotent stem cell-derived RPE cells, formulations, pharmaceutical compositions, reagents, methods, and kits. Obtaining RPE cells by differentiation of iPSCs is achieved by adding small chemical molecules as inhibitors or activators to E6 medium and includes a neuroectoderm cell induction phase, an RPE progenitor cell induction phase, an RPE cell induction phase, and an RPE cell maturation phase. In this differentiation process, there is no need to manually remove heterogeneous cells with non-RPE-like morphology, and pure RPE cells can be obtained by subculturing.

Description

[Technical Field]

[0001] This invention belongs to the field of regenerative medicine and relates to cell therapy, and more specifically to RPE cells derived from pluripotent stem cells, formulations, pharmaceutical compositions, reagents, methods, and kits. [Background technology]

[0002] The macula is a vital part of the retina located at the posterior pole of the eyeball, and is primarily associated with fine visual functions such as visual acuity and color vision. Lesions in the macula cause progressive damage to the retinal pigment epithelium (RPE), ultimately leading to loss of vision. Age-related macular degeneration (AMD) is a common chronic and progressive macular degeneration disease, with a global incidence of approximately 8.7%, and the related pharmaceutical market is expected to continue growing. Currently, mainstream treatments for AMD are mainly divided into three categories: laser intervention, surgery, and medication.

[0003] (1) Neither laser intervention nor drug therapy can fundamentally cure neovascular AMD. As the number of injections increases, the effect of anti-VEGF drugs gradually weakens, and in some patients, there is a risk of accelerated development of atrophic AMD. Furthermore, laser therapy costs 4,000 to 60,000 yuan per year, and drug therapy costs approximately 20,000 yuan or more per year, and requires long-term administration of medication.

[0004] (2) However, retinal pigment epithelial (RPE) cells are polygonal pigment cells located at the base of the retina, arranged regularly and tightly. Microvilli are distributed on their apical surfaces, surrounding the outer segment (POS) of photoreceptor cells and participating in the phagocytic action of RPE cells. Numerous wrinkles are present at their base to facilitate substance exchange, and they are in contact with the choroid via Bruch's membrane, together forming the blood-retinal barrier. Surgical treatment of RPE cells has been actively researched in recent years, and many RPE cell transplantation clinical trials have been conducted both domestically and internationally. Patient vision improves to some extent within one year after surgery, but long-term use of immunosuppressants is necessary, immune rejection can occur during long-term follow-up, the number of RPE cells at the transplant site decreases, and the patient's vision may decline again.

[0005] iPSC Reprogramming Technology: The advent of induced pluripotent stem cell (iPSC) technology has provided an unlimited cell source that meets the ethical requirements for cell therapy of macular degeneration. Currently, several clinical studies have demonstrated the efficacy and safety of stem cell-derived RPE cells for the treatment of macular degeneration. However, RPE cells derived from autologous iPSCs (hereinafter abbreviated as iPSC-RPE) have long differentiation times (leading to delayed or insufficient treatment timing), high manufacturing costs, and unstable quality. On the other hand, RPE derived from allogeneic iPSCs require long-term administration of immunosuppressants and have factors that prevent permanent engraftment, making rapid clinical introduction difficult.

[0006] CRISPR / Cas9 Technology: With the continued development of gene editing technology, iPSC-RPEs (Remotely Immunized Cells) offer another important advantage: because genome selection and gene editing (immune privilege) can be performed at the iPSC stage, the challenge of immune rejection associated with allogeneic iPSC-RPE cells in clinical use is resolved, and mass production of RPE cells and rigorous quality control before transplantation become possible. [Overview of the Initiative] [Problems that the invention aims to solve]

[0007] Technical challenges and drawbacks regarding the differentiation of iPSCs into RPE cells: To date, numerous RPE cell differentiation methods reported in clinical studies and literature based on in vivo differentiation are based on spontaneous differentiation and have the following problems.

[0008] RPE cell differentiation is labor-intensive, requires highly skilled technicians, and most differentiation methods still require manual sorting of pigment spots to achieve high purity. This manual sorting makes large-scale production of RPE cells extremely difficult, and there are concerns about the risk of tumor formation if undifferentiated cells remaining in the final product are not detected.

[0009] Different stem cell lines exhibit significant differences in differentiation cycles, yields, and quality standards, resulting in poor reproducibility. For example, Reference 1 reports that when a differentiation cycle of 60 days is used and CD140b, CD56, CD104, CD184, and GD2 are used as markers, 6,500 to 13,000 therapeutic doses of RPE cells were obtained from 1 million undifferentiated human iPSCs (Reference 1: Plaza Reyes, A. et al. Identification of cell surface markers and establishment of monolayer differentiation to retinal pigment epithelial cells. Nat Commun 11, 1609 (2020). https: / / doi.org / 10.1038 / s41467-020-15326-5). Reference 2 shows that when the differentiation cycle is set to 42 days and LUM, FN1, MITF, and BEST are used as markers, 16 billion therapeutic RPE cells were obtained in 12 weeks using an automated robot (Reference 2: Regent, F., Morizur, L., Lesueur, L. et al. Automation of human pluripotent stem cell differentiation toward retinal pigment epithelial cells for large-scale productions. Sci Rep 9, 10646 (2019). https: / / doi.org / 10.1038 / s41598-019-47123-6).

[0010] Furthermore, while all differentiation methods described in the literature induce RPE cell differentiation using animal-derived components, in recent years, as our understanding of RPE cell development in vivo and in vitro has deepened, directed differentiation methods have emerged one after another. [Means for solving the problem]

[0011] According to the present application, there is no need to manually remove heterogeneous cells that are not in the form of RPE-like during the differentiation process, and it contains no animal-derived components at all. By subculturing, pure iPSC-RPE cells can be obtained to provide safe and highly reliable RPE cell products for subsequent clinical trials. Provided are pluripotent stem cell-derived RPE cells, formulations, pharmaceutical compositions, reagents, methods, and kits.

[0012] To achieve the above technical objective, the technical solution according to the present application is RPE cells derived from pluripotent stem cells obtained based on the following differentiation stages from pluripotent stem cells: A neuroectoderm cell induction stage in which differentiation induction is performed by any one or more of three signal transduction pathways: inhibition of the TGF-β / nodal / activin signal transduction pathway, inhibition of the BMP signal transduction pathway, and inhibition of the WNT signal transduction pathway; An RPE progenitor cell induction stage in which differentiation induction is performed by activation of the nicotinamide metabolism signal transduction pathway; An RPE cell induction stage in which differentiation induction is performed by any one or more of three signal transduction pathways: activation of the TGF-β / nodal / activin signal transduction pathway, inhibition of the FGF signal transduction pathway, and activation of the WNT signal transduction pathway; An RPE cell maturation stage in which differentiation induction is performed by one or both of two signal transduction pathways: activation of the nicotinamide metabolism signal transduction pathway and activation of the WNT signal transduction pathway.

[0013] As an improved technical solution of the present application, the pluripotent stem cells are those obtained by differentiation of ESC, iPSC or universal iPSC HLA-KO The universal iPSC HLA-KO is an iPSC with immune privilege characteristics, in which HLA expression is specifically reduced by gene editing.

[0014] As an improved technical solution of the present application, the ESC is ESC cells subcultured 4 to 7 times with a subculture ratio of 1:(3-12) each time; the cultured ESC is dissociated into single cells and then cultured in iPSC medium for 1 day, after which the ESC differentiates to obtain RPE cells. The iPSC or the general-purpose iPSC HLA-KO is iPSC cells subcultured 4 to 7 times with a subculture ratio of 1:(3-12) each time; the cultured iPSC is dissociated into single cells and then cultured in iPSC medium for 1 day, after which the iPSC differentiates to obtain RPE cells.

[0015] As an improved technical solution of the present application, the iPSC medium is based on E8, Stem Flex or mTeSR1, and the additive Y27632 is added at a dose of 10 μM.

[0016] As an improved technical solution of the present application, obtaining RPE cells by differentiating iPSC is achieved by adding chemical small molecules as inhibitors or activators to E6 medium, and includes a neuroectodermal cell induction stage, an RPE progenitor cell induction stage, an RPE cell induction stage, and an RPE cell maturation stage.

[0017] As an improved technical solution of the present application, in the neuroectodermal cell induction stage, low molecular weight SB431542 is used at an addition amount of 1-50 μM for inhibiting the TGF-β / Nodal / Activin signaling pathway, low molecular weight LDN193183 is used at an addition amount of 10-100 nM for inhibiting the BMP signaling pathway, low molecular weight IWR-1 is used at an addition amount of 10-100 μM for inhibiting the WNT signaling pathway, or IWR-2 is used at an addition amount of 10-100 μM.

[0018] As an improved technical solution of the present application, in the RPE progenitor cell induction stage, activation of the nicotinamide metabolism signaling pathway is achieved by adding nicotinamide at an addition amount of 1-50 mM.

[0019] As an improved technical solution of this application, during the RPE cell induction phase, To activate the TGF-β / nodal / activin signaling pathway, use low molecular weight activin A at an additive dose of 10-200 ng / mL. To inhibit the FGF signaling pathway, use the low molecular weight SU5402 at an additive dose of 1-10 μM. To activate the WNT signaling pathway, use the low-molecular-weight CHIR99021 at an additive dose of 1-10 μM.

[0020] As an improved technical solution of this application, during the maturation phase of RPE cells, Low molecular weight nicotinamide is used to activate the nicotinamide metabolic signaling pathway at an additive dose of 1-50 mA. To activate the WNT signaling pathway, use the low-molecular-weight CHIR99021 at an additive dose of 1-10 μM.

[0021] An improved technical solution of this application is that the passage ratio during the RPE cell induction phase and the RPE cell maturation phase is 1:(3~20).

[0022] Another object of this application is to provide an RPE cell preparation containing the aforementioned pluripotent stem cell-derived RPE cells.

[0023] Another object of this application is to provide an ophthalmic pharmaceutical composition containing the aforementioned pluripotent stem cell-derived RPE cells as an active ingredient.

[0024] Another object of this application is to provide reagents for evaluating the toxicity or efficacy of a test substance, including the aforementioned pluripotent stem cell-derived RPE cells.

[0025] Another object of this application is to provide a method for evaluating the toxicity or efficacy of a test substance, comprising contacting the aforementioned pluripotent stem cell-derived RPE cells with a substance and measuring the effect of the substance on the cells.

[0026] A further object of this application is a first medium comprising a basal medium and an additive A selected from the group consisting of SB431542, LDN193183, IWR-1, IWR-2 and combinations thereof in any molar ratio (where the amount of SB431542 added is 1 to 50 μM, the amount of LDN193183 added is 10 to 100 nM, the amount of IWR-1 added is 10 to 100 μM, and the amount of IWR-2 added is 10 to 100 μM). A basal medium and a second medium containing additive B, which is nicotinamide (however, the amount of nicotinamide added is 1 to 50 mM), A third medium comprising a basal medium and additive C selected from the group consisting of activin A, SU5402, CHIR99021, and combinations thereof in any molar ratio (where the amount of activin A added is 10-200 ng / mL, the amount of SU5402 added is 1-10 μM, and the amount of CHIR99021 added is 1-10 μM), and, The objective is to provide a kit comprising a combination of culture media, including a base medium and a fourth medium containing additive D selected from the group consisting of nicotinamide, CHIR99021, and combinations of the two in any molar ratio (where the amount of nicotinamide added is 1 to 50 mM and the amount of CHIR99021 added is 1 to 10 μM).

[0027] As an improved technical solution of this application, the basal culture medium is E6.

[0028] Further objectives of this application include the following steps: Step 1 involves culturing pluripotent stem cells in the first culture medium for two days to obtain neuroectoderm cells, Step 2 involves culturing the neuroectoderm cells in the second medium for 4 days to obtain RPE progenitor cells, Step 3 involves culturing the RPE progenitor cells in the third medium for 4 days to obtain RPE cells, The objective is to provide a method for obtaining RPE cells using the aforementioned kit, comprising step 4, which involves culturing the RPE cells in the fourth medium for 20 days to obtain mature RPE cells. [Effects of the Invention]

[0029] Conventional prior art methods for obtaining RPE cells by inducing differentiation of ESCs / iPSCs have problems such as unclear cell states, reduced efficiency in obtaining RPE cells, and prolonged differentiation cycles (for example, Chinese patent CN110573610A, titled "Method for Producing Retinal Pigment Epithelial Cells," required a 43-day differentiation cycle). In contrast, this application combines the differentiation process with the in vivo differentiation signaling pathway of RPE cells, thereby clarifying the cell state during the differentiation process, significantly improving differentiation efficiency, shortening the differentiation cycle to just 30 days, and obtaining cells with high purity and high yield.

[0030] In the differentiation process described in this application, by clearly defining the necessary signal transduction pathway activators or inhibitors added to the differentiation process, it becomes possible to fixate the differentiation method and reproduce it efficiently.

[0031] Since the differentiation process described in this application does not use any animal-derived components, the possibility of entry by exogenous viruses is blocked, improving safety in future clinical applications.

[0032] The differentiation process described in this application involves iPSCs and general-purpose iPSCs with immunoprivileged characteristics. HLA-KO It is applicable to both. Both methods show nearly identical differentiation effects to RPE, and the resulting RPE cells have nearly identical functional effects. Through extensive experimental research, the applicants have found that the differentiation method described in this application is universally applicable in the differentiation direction from iPSCs to RPE cells.

[0033] This application aims to reduce immunogenicity at the iPSC stage by combining state-of-the-art induced pluripotent stem cell (iPSC) technology with gene editing technology, and to minimize immune rejection during RPE cell transplantation by differentiating RPE cells according to different differentiation stages and signaling pathways of RPE cells in vivo.

[0034] Thus, the RPE cells obtained in this application are highly safe, stable, donor-independent, and immunogenic general-purpose iPSCs obtained by directional differentiation induced by non-animal-derived small molecule compounds. HLA-KO - As these are RPE cells, treatment and clinical application of retinal degenerative diseases can be realized, and safety issues such as immune rejection associated with intercellular subretinal transplantation of RPE cells in conventional technology can be resolved. [Brief explanation of the drawing]

[0035] [Figure 1A] A flowchart of differentiation induced by small molecules is shown. [Figure 1B] Bright-field images of typical cell morphologies during the process of inducing differentiation of iPSC-RPE or iPSCHLA-KO-RPE at different time points are shown. [Figure 2A] The results of immunofluorescence staining analysis of specific protein expression in differentiated cells iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, and the positive control fRPE (fRPE) are shown. In the figure, iKO-RPE cells refer to RPE cells obtained by differentiation from iPSCHLA-KO (abbreviated as iPSCHLA-KO-RPE), and iRPE cells refer to RPE cells obtained by differentiation from iPSCs (abbreviated as iPSC-RPE). [Figure 2B] The expression of specific proteins in differentiated cells iRPE-1, iRPE-2, and iRPE-3, as analyzed by immunofluorescence staining, is shown. iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 cells are iPSCHLA-KO-RPE cells obtained using three different experimental methods in Example 1, while iRPE-1, iRPE-2, and iRPE-3 cells are iPSC-RPE cells obtained using three different experimental methods in Example 2. In the figure, iKO-RPE cells refer to RPE cells obtained by differentiation from iPSCHLA-KO (abbreviated as iPSCHLA-KO-RPE), and iRPE cells refer to RPE cells obtained by differentiation from iPSC (abbreviated as iPSC-RPE). [Figure 3A]The expression of PAX6 in iPSCs, fRPEs, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3 is shown. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 3B] The expression of MITF in iPSCs, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3 is shown. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE cells refer to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 3C] The expression of RPE65 in RPE cells derived from iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3 is shown. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSC (iPSC-RPE). [Figure 3D] The expression of BEST1 in iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3 is shown. In Figure 3, "normalized to GAPDH" refers to the expression level relative to GAPDH. [Figure 4A]The expression of the specific protein MITF in fRPE, iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 cells, as analyzed by flow cytometry, is shown. A shows the expression of the specific protein MITF in fRPE (98.98%), B shows the expression of the specific protein MITF in iKO-RPE-1 (98.52%), C shows the expression of the specific protein MITF in iKO-RPE-2 (99.41%), and D shows the expression of the specific protein MITF in iKO-RPE-3 (99.26%). In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 4B] The expression of the specific protein MITF in iPSC-RPEs, as analyzed by flow cytometry, is shown. A shows the expression of the specific protein MITF in iRPE-1 (98.56%), B shows the expression of the specific protein MITF in iRPE-2 (97.93%), and C shows the expression of the specific protein MITF in iRPE-3 (99.02%). iRPE-1, iRPE-2, and iRPE-3 are iRPEs obtained using three different experimental methods in Example 2. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 5A]The expression of the specific protein RPE65 in iPSCHLA-KO-RPE cells and positive control fRPE, as analyzed by flow cytometry, is shown. A shows the expression of the specific protein RPE65 in fRPE (99.19%), B shows the expression of the specific protein RPE65 in iKO-RPE-1 (99.02%), C shows the expression of the specific protein RPE65 in iKO-RPE-2 (99.13%), and D shows the expression of the specific protein RPE65 in iKO-RPE-3 (99.59%). iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 were obtained using the three different experimental methods described in Example 1. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 5B] The expression of the specific protein RPE65 in iPSC-RPE cells analyzed by flow cytometry is shown. A shows the expression of the specific protein RPE65 in iRPE-1 (99.54%), B shows the expression of the specific protein RPE65 in iRPE-2 (99.83%), and C shows the expression of the specific protein RPE65 in iRPE-3 (99.87%). iRPE-1, iRPE-2, and iRPE-3 were obtained using three different experimental methods in Example 2. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 6A]The expression of the specific protein PAX6 in iPSCHLA-KO-RPE cells and positive control fRPE, as analyzed by flow cytometry, is shown. A represents PAX6 expression in fRPE (99.10%), B represents PAX6 expression in iKO-RPE-1 (99.23%), C represents PAX6 expression in iKO-RPE-2 (99.37%), and D represents PAX6 expression in iKO-RPE-3 (98.18%). iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 were obtained using three different experimental methods in Example 1. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 6B] The figure shows the expression of PAX6 in iPSC-RPE cells analyzed by flow cytometry. A shows the expression of PAX6 in iRPE-1 (98.23%), B shows the expression of PAX6 in iRPE-2 (97.01%), and C shows the expression of PAX6 in iRPE-3 (98.65%). iRPE-1, iRPE-2, and iRPE-3 were obtained using three different experimental methods in Example 2. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells differentiated from iPSCs (iPSC-RPE). [Figure 7A] This figure shows the detection of phagocytic activity of fRPE, iRPE (iRPE-1, iRPE-2, and iRPE-3), and iKO-RPE (iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3) at 37°C. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 7B]This figure shows the detection of phagocytic activity at 4°C for fRPE, iRPE (iRPE-1, iRPE-2, and iRPE-3), and iKO-RPE (iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3). In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 7C] The negative control groups for fRPE, iRPE (iRPE-1, iRPE-2, and iRPE-3), and iKO-RPE (iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3) are shown. In the figure, iKO-RPE refers to RPE cells obtained by differentiation from iPSCHLA-KO (iPSCHLA-KO-RPE), and iRPE refers to RPE cells obtained by differentiation from iPSCs (iPSC-RPE). [Figure 8A] This diagram shows the effect detection of class I immune privilege for the HLA gene group encoding HLA-A, B, and C (HLA-ABC). [Figure 8B] This diagram shows the effect detection of class I immune privilege for the HLA gene group encoding HLA-DR, DQ, and DP (HLA-DR, DQ, DP). [Modes for carrying out the invention]

[0036] Optimal form for carrying out the invention The overall methodology of the experimental group is described below. (1) Culture of human induced pluripotent stem cells (hiPSCs): iPSCs obtained by knocking out the B2M gene of HLA class I molecules and the CIITA gene, a major regulator of HLA class II. HLA-KOThe cells were seeded in a 1:8 ratio in 12-well plates incubated with laminin and cultured in a 37°C, 5% CO2 incubator, with daily microscopic observation of growth. Fresh E8 medium was changed daily, and subculturing was performed every 3-4 days at a 1:8 subculturing ratio. At the time of subculturing, the cells were treated with 0.25% EDTA by mass / volume for 5-10 minutes and washed once with DPBS buffer. Next, the cells were collected in a 12-well plate incubated with a separate volume of laminin by gentle pipetting no more than 8 times using 1 mL of E8 medium, and culture was continued. On the first day after each subculturing, 10 μM Y-27632 was added to the medium. After repeated culturing for 4 generations, differentiation was performed. The specific differentiation process is shown in Figure 1A.

[0037] (2) General-purpose human induced pluripotent stem cells (iPSCs) HLA-KO ) human retinal pigment epithelial cells (iPSCs) HLA-KO Induction to differentiate into RPE: When the cultured hiPSCs reached 80% confluence (this point is defined as day -1), the cells were degraded, seeded in a 1:10 subculture ratio in a 12-well plate incubated with laminin, and cultured in E8 medium supplemented with 10 μM Y27632. Day 0 was defined as the point after 1 day of treatment.

[0038] On days 0-2, the culture medium was replaced with "neuroectoderm medium" differentiation medium (the first medium in the kit) to induce neuroectoderm cells. However, this medium (neuroectoderm medium) was E6 medium to which 10-100 ng / mL of SB431542, 50-100 ng / mL of LDN193183, and 10-50 mM of IWR-1 were added in any combination.

[0039] When induction into neuroectoderm cells was performed by day 6, the culture medium was changed to "retinal precursor medium" differentiation medium (the second medium in the kit), that is, 1-50 mM nicotinamide was added to E6 medium to induce RPE progenitor cells.

[0040] When the induction to RPE progenitor cells was carried out by the 10th day, the medium was changed to the "progenitor RPE medium", a differentiation medium (the third medium in the kit), to induce RPE progenitor cells. However, this medium (progenitor RPE medium) was a medium obtained by adding activin A at 10 - 200 ng / mL, SU5402 at 1 - 10 μM, and CHIR99021 at 1 - 10 μM to the E6 medium in an arbitrary combination.

[0041] When the induction of RPE progenitor cells was carried out by passage 0 (16th day), the differentiating cells were dissociated with 1×Triple and passaged in a 37°C, 5% CO₂ incubator for 5 - 10 minutes. After passage, a 12 - well plate for culture was pre - incubated with laminin, and the obtained naive - type iPSC HLA-KO - RPE cells were passaged at a ratio of 1:(3 - 20) and cultured in a mature RPE medium (the fourth medium in the kit). This medium (mature RPE medium) was mainly obtained by adding nicotinamide at 1 - 50 mM and CHIR99021 at 1 - 10 μM to the E6 basal medium in an arbitrary combination.

[0042] The culture was continued using the medium (mature RPE medium), and dissociation and passage were performed by dissociating with 1×Triple in a 37°C, 5% CO₂ incubator for 5 - 10 minutes on the 16th day - passage 1 and the 22nd day - passage 2. The more mature iPSC HLA-KO - RPE cells were passaged at a ratio of 1:4 and the culture was continued until the 30th day using the mature RPE medium.

[0043]

Table 1

[0044] Through repeated experiments, the applicant revealed that, in the case of the same signal transduction pathway, adding SB431542 (1-50 μM), LDN193183 (10-100 nM), IWR-1 (10-100 μM), nicotinamide (1-50 mM), activin A (10-200 ng / mL), CHIR99021 (1-10 μM), and SU5402 (1-10 μM) in different doses within their respective specified ranges produced the same or similar effects as those shown in Table 1.

[0045] Modes for carrying out the invention The technical solution of this application will be described clearly and completely below, with reference to specific implementation details. 1.Definition

[0046] iPSC stands for induced pluripotent stem cell. hiPSC stands for human induced pluripotent stem cell. hiPSCs are stem cells obtained by reprogramming human peripheral blood mononuclear cells, possessing both self-renewal ability and the ability to differentiate into three germ layer cells. Three germ layer differentiation refers to differentiation into cells of the ectoderm, mesoderm, and endoderm cell lineages (Source: T / CSCB0005-2021, "Human Induced Pluripotent Stem Cells," population standard published by the Chinese Society for Cell Biology).

[0047] General-purpose iPSC HLA-KOThis refers to iPSCs in which HLA gene class I (e.g., B2M) and / or class II (e.g., CIITA) genes have been knocked out using CRISPR / Cas9 technology. The method of obtaining these cells is similar to the procedure described in the specification of Chinese Patent No. 2022106036092, titled "Method for Producing Hypoimmunogenic iPSC Cells, Hypoimmunogenic iPSC Cells and Composition," but differs in that the iPSCs described herein are not dependent on CD47 editing, or that hPSCs that do not express classical human leukocyte antigen (HLA) class I protein on their surface may be manipulated by knocking out β2 microglobulin (B2M) within the framework of the endogenous B2M locus or by knocking in both alleles of the HLA-G1 gene. [Reference: Shi L, Li W. et al. Generation of hypoimmunogenic human pluripotent stem cells via expression of membrane-bound and secreted β2m-HLA-G fusion proteins. Stem Cells. 2020 Nov;38(11):1423-1437. doi:10.1002 / stem.3269. Epub 2020 Sep 15. PMID: 32930470.]

[0048] RPE cells refer to retinal pigment epithelial cells (RPE cells) located on the outer retina, forming a dense pigment monolayer between the choroid and the neuroretina, and playing a role in supporting, nourishing, and circulating photoreceptor cells. In this application, RPE cells are identified firstly by the positive expression of RPE-specific marker proteins (TYR, RPE65, MITF, PAX6, BEST1), secondly by the presence of melanin granules (brownish-black), and thirdly by characteristic cell morphology such as close intercellular connectivity, typical polygonal shape, and cobblestone-like cell morphology. In this specification, the function of RPE cells is verified by an in vitro phagocytic activity detection test of RPE cells.

[0049] iRPE (iPSC-RPE, induced pluripotent stem cell-derived retinal pigment epithelium) refers to retinal pigment epithelial cells derived from human induced pluripotent stem cells. iRPE cells were seeded on laminin-coated plates and cultured in differentiation medium. Examples of laminins include laminin 521, laminin α4 antibody (laminin α4), laminin α5β1γ1, and laminin α1β1γ1, and viralectin (VTN) may also be used.

[0050] Signaling Pathways: Cell lineage development is typically regulated by multiple signaling pathways that control cell proliferation and differentiation. Each of these pathways is controlled by a set of complex genetic factors, epigenetic factors (e.g., histone modifications), and exogenous signaling factors that can regulate cell fate and behavior during cell development and differentiation. The pathways regulated herein are TGF-β / Nodal / activin signaling, BMP signaling, WNT signaling, nicotine metabolism signaling, and FGF signaling. Pathways not described herein are not regulated by the addition of small molecules.

[0051] Neuroectoderm cell induction stage: Cells reach 100% confluence, and their morphology becomes neuroectoderm-like.

[0052] RPE progenitor cell induction phase: Cells are densely packed, cell boundaries are indistinct, and cell morphology is irregularly stacked.

[0053] RPE cell induction phase: Approximately 10% of cells other than RPE cells undergo apoptosis, and non-apoptotic cells emerge as dense, monolayered, stone-like RPE-like cells, i.e., RPE precursor cells.

[0054] RPE cell maturation: RPE progenitor cells gradually form a pebble-like structure, and melanin is uniformly distributed. When functional monolayer RPE cells are formed, the cells exhibit a more regular pebble-like structure, and the melanin becomes more concentrated.

[0055] One important indicator of "functionality" is the in vitro phagocytic activity of RPE cells. Phagocytosis of cells is divided into three stages: binding, internalization, and degradation. In the binding process, the inner microvilli cell membrane of RPE cells binds to the outer segment of fallen photoreceptor cells, is taken up into the cell, and is finally transported to lysosomes by the cytoskeleton and vesicles, where it is degraded.

[0056] TGF-β / Nodal / Activin signaling pathway: This refers to the simultaneous activation or inhibition of the TGF-β signaling pathway, the Nodal signaling pathway, and the activin signaling pathway.

[0057] Nicotinamide metabolic signaling pathway: This pathway depends on low-molecular-weight nicotine [Theoretical basis: Hong S. et al. Nicotinamide N-methyltransferase regulators hepatic nutrient metabolism through Sirt1 protein stabilization. Nat Med. 2015 Aug;21(8):887-94. doi:10.1038 / nm.3882. Epub 2015 Jul 13. PMID:26168293;PMCID:PMC4529375.].

[0058] The culture medium used in the differentiation process described herein is a "serum-free medium." In this specification, the medium used in the iPSC culture stage is E8 medium, and the medium used to obtain RPE cells from iPSC differentiation is E6 medium supplemented with low molecular weight compounds. E6 medium refers to a medium in which two components, FGF2 and TGF-β, are reduced compared to E8 medium.

[0059] Unless otherwise specified, all reagents, materials, and equipment used in this example are commercially available. Furthermore, unless otherwise specified, all experimental methods are conventional methods in this field.

[0060] Activin A belongs to the superfamily of transforming growth factors and plays a role in early embryonic development, vascular smooth muscle proliferation, arteriosclerosis, induction of neural differentiation, proliferation and differentiation of hematopoietic cells, and regulation of pituitary hormone secretion in the endocrine center. In this application, activin A functions to activate the TGF-β / activin signaling pathway and differentiate retinal progenitor cells into RPE progenitor cells.

[0061] In this specification, various chemical substances such as WNT inhibitors, BMP inhibitors, and FGF inhibitors include forms such as free compounds and salts. For example, Y-27632 includes the free form of Y-27632, its hydrochloride salt, and Y-27632 dihydrochloride salt.

[0062] In this specification, "within" means less than or equal to that amount.

[0063] Day 0 represents the 0th day, Day 1 represents the 1st day, and so on.

[0064] The English name for basal culture medium is Differentiation Bassal Medium.

[0065] The English name for retinal progenitor cell culture medium is Retinal Progenitor Medium.

[0066] The English name for the pioneer RPE medium is Immature RPE Medium.

[0067] Human fetal retinal pigment epithelial cells (fRPEs) are used as a positive control for iRPEs. fRPEs are obtained by isolating and culturing them from the eyeballs of aborted fetuses. Specific methods of preparation include, but are not limited to, conventional methods such as those described in Chinese Patent CN201310552005.0, titled "Method for Isolation and Culture of Human Fetal Retinal Pigment Epithelial Cells."

[0068] 2. Design of culture conditions. In all steps of this specification, adherent culture is used for cell culture and differentiation. The culture vessel used for the adherent culture and differentiation of human induced pluripotent stem cells is not particularly limited, as long as it allows cells to adhere to the surface of the culture vessel and enables differentiation of human induced pluripotent stem cells into RPE cells in the culture medium. In this application, a well plate incubated with laminin is used.

[0069] The most important aspect of this application is that by combining low-molecular-weight compounds with basal culture media, high-quality and stable RPE cells can be obtained in a short time under simplified and clearly defined culture media components, and the differentiated RPE cells exhibit better phagocytic activity.

[0070] Therefore, the culture medium used in this application is primarily based on E6 medium, or a medium with equivalent or similar functions to E6 and with clearly defined components. Of course, the technical effects of this application can also be achieved by using a medium with more complex components than E6. However, the first objective of this application is to clarify the differentiation pathway, and the second objective is to perform differentiation using a medium with simplified and clear components in order to ensure subsequent industrial production.

[0071] The culture medium is changed at the end of each stage. Here, changing the culture medium means changing the medium for the neuroectoderm cell induction stage to the medium for RPE progenitor cells, changing the medium for RPE progenitor cells to the medium for the RPE cell induction stage, or changing the medium for the RPE cell induction stage to the medium for the RPE cell maturation stage.

[0072] In this specification, the culture medium is changed daily or every other day during each stage: the neuroectoderm cell induction phase, the RPE progenitor cell phase, the RPE cell induction phase, and the RPE cell maturation phase. Here, changing the culture medium means changing the same basal medium to which the same low molecular weight compound has been added.

[0073] The culture conditions (such as culture temperature and CO2 concentration) should be set appropriately. The culture temperature is not particularly limited, but is 30°C to 40°C, preferably 37°C. The CO2 concentration is approximately 1% to 10%, preferably approximately 5%.

[0074] 3. Selection of iPSCs. The iPSCs used during the neuroectoderm cell induction stage are either standard iPSCs or general-purpose iPSCs with immunogenicity restriction, in which the B2M and CIITA genes have been knocked out. HLA-KO It is preferable that these general-purpose iPSCs HLA-KO These can be produced using the methods known to those skilled in the art. This avoids immune rejection of differentiated RPE cells and improves their versatility. iPSCs can be produced using the methods known to those skilled in the art.

[0075] In this application, compared to using embryonic stem cells (ESCs derived from the commercially available embryonic stem cell line H9), iPSCs or general-purpose iPSCs are particularly effective. HLA-KO During the process of differentiation into RPE cells using general-purpose iPSCs, HLA-KO Many other factors need to be considered, such as the stability of their own pluripotency and whether they can effectively maintain low immunogenicity after differentiation. Therefore, these factors also represent technical difficulties in the differentiation process of this application. In order to reduce the impact of these problems on the stability of obtaining RPE cells from iPSC differentiation and to further improve the differentiation effect of obtaining RPE cells from iPSC differentiation, this application uses iPSC or general-purpose iPSC HLA-KO Specific preprocessing is applied to the iPSC or general-purpose iPSC. HLA-KO The purpose of culturing the iPSCs in iPSC medium for 4-7 passages, preferably 4-5 passages, is to create more stable iPSCs or general-purpose iPSCs. HLA-KO To obtain iPSC or general-purpose iPSC HLA-KO By removing impure cells, iPSCs or general-purpose iPSCs are produced. HLA-KO To ensure that it has a higher purity, and iPSC or general-purpose iPSC HLA-KO The goal is to ensure that the differentiation process is stable and that the differentiated RPE cells have low immunogenicity.

[0076] The passaging ratio for each stage is designed to be 1:(3-12), preferably 1:(3-10), with exemplary ratios being 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, and 1:10. If the passaging ratio falls below 1:3, the cells become overcrowded after subculturing, leading to cell death. If the passaging ratio exceeds 1:12, the cells differentiate into other foreign cells. In actual use, setting the passaging ratio to 1:3-10 minimizes the impact on the performance, differentiation efficiency, and stability of the final RPE cells. In the subsequent examples in this application, verification was performed with a passaging ratio of 1:8.

[0077] In this application, any commercially available stem cell culture medium can be selected for pluripotent stem cells. To clarify the components and ensure a reproducible differentiation process, the culture medium for pluripotent stem cells consists of the medium and the additive Y27632. The dose of Y27632 is the amount necessary to adhere the pluripotent stem cells to the culture wall and can be appropriately determined by a person skilled in the art. For example, the specific dose of Y27632 is set to 10 μM (10 μmol / L). Furthermore, commercially available E8, Stem Flex, or mTeSR1 can be used as the basal medium to maintain the undifferentiated state. These media can also be used for the maintenance and expansion culture of pluripotent stem cells.

[0078] To degrade iPSCs after subculturing, they were primarily treated with EDTA at a mass-to-volume ratio of 0.25% for 5-10 minutes, washed once with DPBS buffer, pipetted with 1 mL of iPSC medium, and then re-seeded in a 12-well plate incubated with laminin, and culture was continued. In this embodiment, EDTA with more clearly defined chemical components is used instead of proteases, mainly to clarify the differentiation pathway at each stage of differentiation. The ultimate goal is to ensure that the components of each operational step in this application are sufficiently clear, to avoid karyotype changes in treated cells due to proteases during degradation, and to ensure that the post-differentiation process has high stability and reproducibility.

[0079] 4. Generation of retinal pigment epithelial (RPE) cells. Figure 1A shows a flowchart of induced differentiation by low-molecular-weight compounds in basal medium E6. Figure 1B shows iPSC-RPE or iPSC at different time points. HLA-KO -Bright-field images showing typical cell morphology during the induction and differentiation process of RPE.

[0080] In this application, RPE cells refer to iPSCs (iPSCs or general-purpose iPSCs). HLA-KO These are cells derived from (abbreviated as ). Obtaining RPE cells from iPSC differentiation is achieved by adding small molecule compounds as inhibitors or activators to E6 medium, and includes the neuroectoderm cell induction phase, RPE progenitor cell induction phase, RPE cell induction phase, and RPE cell maturation phase.

[0081] The main procedure is as follows: (1) Days 1-2 (D0-D2), Neuroectoderm induction phase: Differentiation is induced by inhibiting one or more of the three signaling pathways: the TGF-β / Nodal / activin signaling pathway, the BMP signaling pathway, and the WNT signaling pathway.

[0082] Specifically, the inhibitors used to inhibit the TGF-β / Nodal / activin signaling pathway are limited to small molecule compounds, including but not limited to SB431542. The amount of SB431542 added is preferably 1 to 50 μM. Too little concentration prevents differentiation from stem cells to ectoderm, while too much increases costs. As long as retinal pigment epithelial cells can be obtained using the method of this application, the concentration can be kept constant daily or varied from day to day.

[0083] The inhibitors used to inhibit the BMP signaling pathway are limited to small molecule compounds, including but not limited to LDN193183, and the amount added is 10-100 nM. If the amount is too low, differentiation of stem cells into ectoderm becomes impossible, while if it is too high, the cost increases. As long as retinal pigment epithelial cells can be obtained using the method of this application, the concentration can be kept constant every day or varied from day to day.

[0084] Inhibitors used to inhibit the WNT signaling pathway are limited to small molecule compounds, mainly including but not limited to IWR-1 or IWR-2, with IWR-1 added at concentrations of 10-100 μM and IWR-2 added at concentrations of 10-100 μM. If the added amount is too low, stem cells will differentiate into mesoderm, while if it is too high, the cost will increase. In practical use, as long as retinal pigment epithelial cells can be obtained using the method of this application, the concentration can be kept constant daily or varied from day to day.

[0085] There are no particular restrictions on the number of days during the neuroectoderm cell induction period, but it is usually within 5 days, preferably within 3 days.

[0086] As shown in Figure 1B, the neuroectoderm cell induction phase ends when the cells reach 100% confluence and their morphology becomes neuroectoderm-like. At this point, differentiation induction occurs during the RPE progenitor cell induction phase.

[0087] (2) Day 2 to Day 6: RPE progenitor induction phase: Differentiation is induced by activation of the nicotinamide metabolic signaling pathway.

[0088] Specifically, the substances used to activate the nicotinamide metabolic signaling pathway are limited to low-molecular-weight compounds, including but not limited to nicotinamide. The dose of nicotinamide is 1 to 50 mM. If the dose is too low, the cells will not be able to differentiate into retinal progenitor cells, while if the dose is too high, a large number of cells will die, making the cost too high. As long as retinal pigment epithelial cells can be obtained using the method of this application, the concentration can be kept constant every day or varied from day to day.

[0089] There are no particular restrictions on the number of days during the RPE progenitor cell induction period, but it is usually within 6 days, preferably within 4 days.

[0090] As shown in Figure 1B, the RPE progenitor cell induction phase can be represented by several indicators used to determine the completion of the process during cell morphology or experiments.

[0091] (3) Day 6 to Day 10: RPE cell induction phase (RPE specialization): Differentiation is induced by one or more of the following three signaling pathways: activation of the TGF-β / Nodal / activin signaling pathway, inhibition of the FGF signaling pathway, and activation of the WNT signaling pathway.

[0092] Specifically, inhibitors used to inhibit the FGF signaling pathway in this process are limited to small molecule compounds containing but not limited to SU5402, and the dosage is 1-10 μM. Too little dosage may reduce the purity of differentiated RPE progenitor cells and lead to the emergence of numerous neurons, while too much dosage may cause cell death. To activate the TGF-β / Nodal / activin signaling pathway in this process, the inhibitors used are limited to small molecule compounds containing but not limited to activin A, and the dosage is 10-200 ng / mL. Too little dosage may reduce the purity of differentiated RPE progenitor cells and lead to the emergence of numerous neurons, while too much dosage becomes too costly. To activate the WNT signaling pathway in this process, the inhibitors used are limited to small molecule compounds containing but not limited to CHIR99021, and the dosage is 1-10 μM. If the amount added is too small, the purity of differentiated RPE precursor cells may decrease, potentially leading to the emergence of numerous neurons. Conversely, if the amount added is too large, the cells may develop a fibroblast-like morphology.

[0093] The number of days in the RPE cell induction phase is not particularly limited, but is usually within 10 days, preferably within 6 days. Here, "within" means less than or equal to that number.

[0094] As shown in Figure 1B, the RPE cell induction phase ends when approximately 10% of non-RPE cells undergo apoptosis, and the non-apoptotic cells form dense, monolayered, stone-like RPE-like cells, i.e., RPE precursor cells.

[0095] (4) Day 10 to Day 30 (PRE maturation): Differentiation is induced by either or both of the following: activation of the nicotinamide metabolic signaling pathway and / or activation of the WNT signaling pathway.

[0096] Specifically, the late-stage RPE medium contains E6 medium, a small molecule compound to activate the nicotinamide metabolic signaling pathway, and a small molecule compound to activate the WNT signaling pathway. The small molecule compound to activate the nicotinamide metabolic signaling pathway is nicotinamide, and the amount added is 1 to 50 mM. If the amount is too low, the maturation of RPE cells will be delayed and the time it takes to mature may be affected, while if the amount is too high, the cost will be too high. The small molecule compound to activate the CWNT signaling pathway is HIR99021, and the amount added is 1 to 10 μM. If the amount is too low, the maturation of RPE cells will be delayed and the time it takes to mature may be affected and the proliferation of cells, while if the amount is too high, the cost will be too high.

[0097] There are no particular restrictions on the number of days during the neuroectoderm cell induction period, but it is usually within 15 days, preferably within 1 day.

[0098] As shown in Figure 1B, during the maturation phase of RPE cells, RPE precursor cells gradually form a pebble-like structure, and when melanin is uniformly distributed and functional monolayer RPE cells are formed, the cells exhibit a more regular pebble-like structure and the melanin becomes more concentrated.

[0099] In the differentiation process of this application, there is no need to manually remove heterogeneous cells with non-RPE-like morphology during differentiation, and pure iPSCs-RPE (iPSC-RPE or iPSC) can be obtained by subculturing the RPE cells during their maturation stage.HLA-KO -RPE) can be obtained. The basic components of the differentiation medium at this stage consist only of E6 medium (with reduced bFGF and TGF-β compared to E8 medium) and do not contain any other animal-derived components. Furthermore, according to the control of the signaling pathway of this application, iPS cells (iPS cells or general-purpose iPSCs) can be obtained. HLA-KO RPE cells differentiated from ) were approximately 10 times stronger than the initial inoculated iPS cells. 11 This makes it possible to achieve double the cell yield.

[0100] Compared to the differentiation process in the prior art, the differentiation method disclosed in Reference 1 requires approximately 60 days to obtain RPE cells with uniform pigmentation, and Reference 2 requires 42 days to obtain stable RPE cells, whereas in this application, the cycle for obtaining RPE cells with uniform pigmentation is only about 30 days.

[0101] To obtain a larger number of RPE cells or to ensure the functional stability of RPE cells, the passage ratio during the RPE cell induction and RPE cell maturation phases is preferably 1:(3-20). Examples of ratios include 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, or 1:20.

[0102] In this application, RPE cells differentiated from iPSCs (iPSCs or general-purpose iPSCs) HLA-KO The differentiation efficiency and RPE cell performance of the cells were primarily verified based on flow cytometry, immunofluorescence staining, reverse transcription PCR (RT-PCR), and in vitro phagocytic activity detection tests.

[0103] I. Identical cell line (iPSC) HLA-KO Verification of various differentiation induction methods. The overall method of the experimental group will be described. (1) Culture of human induced pluripotent stem cells (hiPSCs): iPSCs obtained in which the B2M gene of HLA class I molecules and the CIITA gene, a major regulator of HLA class II, were knocked out. HLA-KOThe cells were seeded in a 1:8 ratio in 12-well plates incubated with laminin and cultured in a 37°C, 5% CO2 incubator, with daily microscopic observation of growth. The E8 medium was replaced daily with fresh medium, and subculturing was performed every 3-4 days in a 1:8 ratio. During subculturing, the cells were treated with 0.25% EDTA (mass-to-volume ratio) for 5-10 minutes and washed once with DPBS buffer. Next, the cells were gently pipetted with 1 mL of E8 medium (but not exceeding 8 pipetting cycles) and collected in a 12-well plate incubated with a separate volume of laminin, where culture was continued. On the first day after each subculturing, 10 μM Y-27632 was added to the medium. After repeated culturing for 4 generations, differentiation was performed. The specific differentiation process is shown in Figure 1A.

[0104] (2) General-purpose human induced pluripotent stem cells (iPSCs) HLA-KO ) from human retinal pigment epithelial cells (iPSCs) HLA-KO Differentiation induction to RPE: Day 1 was defined as the point when the cultured hiPSCs reached 80% confluence. At this point, the cells were degraded and seeded in a 12-well plate incubated with laminin at a subculturing ratio of 1:10. For culturing, the medium was changed to E8 medium and 10 μM Y27632 was added. Day 0 was defined as the point one day after the start of treatment.

[0105] On days 0 and 2, the culture medium was replaced with "neuroectoderm medium" differentiation medium (the first medium in the kit). This medium (neuroectoderm medium) is prepared by adding 10-100 ng / mL of SB431542, 50-100 ng / mL of LDN193183, and 10-50 mM of IWR-1 in any combination to E6 medium, and is used to induce neuroectoderm cells.

[0106] When neuroectoderm cells were induced up to day 6, the culture medium was changed to "retinal precursor medium" differentiation medium (the second medium in the kit), that is, 1-50 mM nicotinamide was added to E6 medium to induce RPE progenitor cells.

[0107] When the induction of RPE progenitor cells progressed to day 10, the culture medium was changed to "Immature RPE Medium" differentiation medium (the third medium in the kit), and RPE progenitor cell induction was performed. This medium (Immature RPE Medium) was prepared by adding 10-200 ng / mL of activin A, 1-10 μM of SU5402, and 1-10 μM of CHIR99021 to E6 medium in any combination.

[0108] When the induction of RPE progenitor cells progressed to passage 0 (day 16), differentiating cells were degraded with 1×3 ions and passaged for 5-10 minutes in a 37°C, 5% CO2 incubator. The 12-well plates of the passaged cultures were pre-incubated with laminin, and the resulting naive iPSCs were obtained. HLA-KO - RPE cells were subculturised in a ratio of 1:(3-20) and cultured in mature RPE medium (the fourth medium in the kit). This medium (Mature RPE Medium) mainly consists of E6 basal medium, 1-50 mM nicotinamide, and 1-10 μM CHIR99021 added in any combination.

[0109] The culture was continued using culture medium (mature RPE medium). Between day 16 and subculturing 1, and between day 22 and subculturing 2, degradation was induced using 1×Triple, and subculturing was carried out for 5-10 minutes in a 37°C, 5% CO2 incubator. The resulting more mature iPSCs were obtained. HLA-KO -RPE cells were subculturised in a 1:4 ratio and cultured in mature RPE medium for up to 30 days.

[0110] [Table 2]

[0111] Through repeated experiments, the applicant revealed that, in the case of the same signal transduction pathway, adding SB431542 (1-50 μM), LDN193183 (10-100 nM), IWR-1 (10-100 μM), nicotinamide (1-50 mM), activin A (10-200 ng / mL), CHIR99021 (1-10 μM), and SU5402 (1-10 μM) in different doses within their respective specified ranges produced the same or similar effects as those shown in Table 1.

[0112] II. Verification of various differentiation induction methods for the same cell line (conventional iPSC). The procedure was the same as in Example 1, except that conventional iPSC was used as the cell line.

[0113] [Table 3]

[0114] Through repeated experiments, the applicant revealed that, in the case of the same signal transduction pathway, adding SB431542 (1-50 μM), LDN193183 (10-100 nM), IWR-1 (10-100 μM), nicotinamide (1-50 mM), activin A (10-200 ng / mL), CHIR99021 (1-10 μM), and SU5402 (1-10 μM) in different doses within their respective specified ranges produced the same or similar effects as those shown in Table 2.

[0115] III. Detection by immunofluorescence staining. Cells from each experimental group, the positive control group, and the negative control group were cultured until confluence, the supernatant was discarded, and the cells were washed once with DPBS. 4% paraformaldehyde by volume was added to each group, and the cells were incubated at room temperature for 15 minutes. They were then washed three times with DPBS for 2 minutes each, permeabilized with 0.2% Triton-X100 (w / v) by mass-volume for 10 minutes, and then blocked at room temperature for 1 hour with a DPBS solution containing 1% bovine serum albumin (BSA) by mass-volume. Next, after removing BSA, the following antibodies were directly added as primary antibodies without washing: mouse monoclonal MITF antibody (diluted at a ratio of 1:100 between the antibody and the diluent), mouse monoclonal OCT2 antibody (diluted at a ratio of 1:300 between the antibody and the diluent), rabbit monoclonal TYROSINASE antibody (diluted at a ratio of 1:100 between the antibody and the diluent), rabbit monoclonal RPE65 antibody (diluted at a ratio of 1:100 between the antibody and the diluent), and rabbit monoclonal ZO-1 antibody (diluted at a ratio of 1:500 between the antibody and the diluent). The mixture was incubated overnight at 4°C. Next, after washing three times with PBS for 5 minutes each, the following secondary antibodies were added: 546-labeled goat anti-rabbit IgG (diluted at a ratio of antibody to diluent of 1:1000), 546-labeled goat anti-mouse IgG (diluted at a ratio of antibody to diluent of 1:1000), FITC-labeled goat anti-rabbit IgG (diluted at a ratio of antibody to diluent of 1:1000), and FITC-labeled goat anti-mouse IgG (diluted at a ratio of antibody to diluent of 1:1000). The mixture was incubated at room temperature for 2 hours. Next, after washing three times with DPBS for 5 minutes each, DAPI staining solution was added, and the mixture was incubated in the dark at room temperature for 15 minutes. Finally, the mixture was washed three times with DPBS solution for 5 minutes each, and images were taken by direct observation under a fluorescence microscope.

[0116] Immunofluorescence staining analysis of differentiated target cells iRPE or iKO-RPE revealed that both subcultured iRPE and iKO-RPE cells were capable of positively expressing the RPE cell-specific marker proteins MITF, ZO-1, TYROSINASE (TYR), BEST1, and RPE65 (see Figure 2). Figure 2A shows the results of immunofluorescence staining analysis of specific protein expression in differentiated iKO-RPE cells and positive control fRPE (fRPE). Figure 2B shows the results of immunofluorescence staining analysis of specific protein expression in differentiated iRPE cells.

[0117] IV. Detection by reverse transcription qPCR. Total RNA was extracted from each cell group using an RNA extraction kit (Vazyme, China), and the OD value was measured. To ensure purity, the RNA OD260 / OD280 value for each cell group was set to 1.8-2.1. Next, RNA was reverse transcribed to cDNA using a reverse transcription kit (Vazyme, China), referring to the reaction system and procedure in the product manual. The resulting cDNA was used for RT-PCR. The primer sequences are shown in Table 3 below. For the reaction system, please refer to the manual for the SYBR kit (Bio, USA). After centrifugation, the product was pre-denatured at 95°C for 5 minutes and reacted for 40 cycles (30 seconds at 94°C, 30 seconds at 59°C, and 30 seconds at 72°C). 2 μL of the reaction product was pipetteed, 3 μL of SYBRGREEN MIX was added and mixed well, and detection was performed.

[0118] [Table 4]

[0119] RPE65, MITF, PAX6, and BEST1 were used to detect RPE expression levels, and GAPDH was used as an internal reference gene to correct and standardize the expression of target genes.

[0120] RT-PCR results showed that differentiated RPE cells could positively express RPE-specific marker genes RPE-65, MITF, PAX6, and BEST1. These RPE-specific marker genes were positively expressed in hRPE cells of the positive control group, but negatively expressed in hiPSCs of the negative control group (see Figure 3). Figure 3 shows the results of reverse transcription PCR analysis of specific gene expression in iRPE or iKO-RPE cells and the positive control fRPE. Figure 3A shows the expression of PAX6 in iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3. Figure 3B shows the expression of MITF in iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3. Figure 3C shows the expression of RPE65 in RPE cells derived from iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3. Figure 3D shows the expression of BEST1 in iPSC, fRPE, iKO-RPE-1, iKO-RPE-2, iKO-RPE-3, iRPE-1, iRPE-2, and iRPE-3. In the figures, "normalized to GAPDH" represents the expression level relative to GAPDH.

[0121] V. Detection by flow cytometry. After the cell culture reached confluence, the cells were degraded, and the cell suspension, which had been degraded to single cells, was washed once with DPBS. 4% paraformaldehyde (v / v) was added by volume and incubated at room temperature for 10 minutes. After washing once with DPBS, the cells were centrifuged, and the supernatant was discarded. Next, the cells were permeabilized with 0.2% Triton-X100 (w / v) by mass / volume for 10 minutes. After washing once with DPBS, the cells were centrifuged, the supernatant was discarded, and a DPBS solution containing 1% bovine serum albumin (BSA) by mass / volume was added, followed by blocking at room temperature for 30 minutes. After removing the BSA by centrifugation, PAX6 was incubated with APC-labeled PAX6 antibody at room temperature for 0.5 hours. Next, the cells were washed once with PBS, the supernatant was discarded, and DPBS was added before injection into a flow cytometer for analysis. For MITF and RPE65, after removing BSA, mouse monoclonal MITF antibody (diluted at a ratio of 1:100 antibody to diluent) and rabbit monoclonal RPE65 antibody (diluted at a ratio of 1:100 antibody to diluent) were added as primary antibodies without washing, and incubated overnight at 4°C. Next, after washing once with PBS, FITC-labeled goat anti-mouse IgG (diluted at a ratio of 1:1000 antibody to diluent) and PE-labeled goat anti-rabbit IgG (diluted at a ratio of 1:1000 antibody to diluent) were added as secondary antibodies, and incubated at room temperature for 1 hour. Next, after washing once with PBS and discarding the supernatant, DPBS was added and the mixture was injected into a flow cytometer for analysis.

[0122] hiPSCs-RPE positively expressed specific marker proteins of RPE cells. The positive cell rates for RPE-65, MITF, and PAX6 exceeded 97%, indicating that hiPSCs-RPE exhibited high purity consistent with fRPE. Detection of residual stem cells in differentiated RPE cells by qPCR and flow cytometry revealed no residual stem cells.

[0123] Specifically, Figure 4A shows the results of flow cytometry analysis of the expression of the specific protein MITF in cells fRPE, iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3. Here, (a) is the expression of the specific protein MITF in fRPE (98.98%), (b) is the expression of the specific protein MITF in iKO-RPE-1 (98.52%), (c) is the expression of the specific protein MITF in iKO-RPE-2 (99.41%), and (d) is the expression of the specific protein MITF in iKO-RPE-3 (99.26%). iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 were obtained using the three different experimental methods described in Example 1. Figure 4B shows the results of flow cytometry analysis of the expression of the specific protein MITF in iPSC-RPE cells. Here, (a) shows the expression of the specific protein MITF in iRPE-1 (98.56%), (b) shows the expression of the specific protein MITF in iRPE-2 (97.93%), and (c) shows the expression of the specific protein MITF in iRPE-3 (99.02%). iRPE-1, iRPE-2, and iRPE-3 were obtained using the three different experimental methods described in Example 2.

[0124] Figure 5A shows iPSC HLA-KOThe following shows the results of flow cytometry analysis of the expression of the specific protein RPE65 in RPE cells and positive control fRPE. (a) shows the expression of the specific protein RPE65 in fRPE (99.19%), (b) shows the expression of the specific protein RPE65 in iKO-RPE-1 (99.02%), (c) shows the expression of the specific protein RPE65 in iKO-RPE-2 (99.13%), and (d) shows the expression of the specific protein RPE65 in iKO-RPE-3 (99.59%). iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 were obtained using the three different experimental methods described in Example 1. Figure 5B shows the results of flow cytometry analysis of the expression of the specific protein RPE65 in iPSC-RPE cells. (a) shows the expression of the specific protein RPE65 in iRPE-1 (99.54%), (b) shows the expression of the specific protein RPE65 in iRPE-2 (99.83%), and (c) shows the expression of the specific protein RPE65 in iRPE-3 (99.87%). iRPE-1, iRPE-2, and iRPE-3 were obtained using the three different experimental methods described in Example 2.

[0125] Figure 6A shows iPSC HLA-KOThe results of flow cytometry analysis of PAX6 expression in -RPE cells and positive control fRPE are shown. (a) shows the expression of the specific protein PAX6 in fRPE (99.10%), (b) shows the expression of the specific protein PAX6 in iKO-RPE-1 (99.23%), (c) shows the expression of the specific protein PAX6 in iKO-RPE-2 (99.37%), and (d) shows the expression of the specific protein PAX6 in iKO-RPE-3 (98.18%). iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3 were obtained using the three different experimental methods described in Example 1. Figure 6B shows the results of flow cytometry analysis of PAX6 expression in iPSC-RPE cells. (a) shows the expression of the specific protein PAX6 in iRPE-1 (98.23%), (b) shows the expression of the specific protein PAX6 in iRPE-2 (97.01%), and (c) shows the expression of the specific protein PAX6 in iRPE-3 (98.65%). The iRPE-1, iRPE-2, and iRPE-3 cells were obtained using the three different experimental methods described in Example 2.

[0126] VI. Detection of in vitro phagocytic activity The cells from each experimental group were placed in a 12-well plate in a 1x10⁶ arrangement. 6Cells were seeded in individual wells and cultured at 37°C for 2 days. After 2 days, fluorescent particles were added and incubated at 4°C / 37°C for 8 hours. Trypan blue was added to quench the fluorescence. Next, the cells were washed three times with DPBS, degraded with TrpLE 1×, resuspended in DPBS, and injected into a flow cytometer for analysis [Methodological basis: Parinot C, Rieu Q, Chatagnon J, Finnemann SC, Nandrot EF. Large-scale purification of porcine or bovine photoreceptor outer segments for phagocytosis assays on retinal pigment epithelial cells. J Vis Exp. 2014 Dec 12;(94):52100.doi:10.3791 / 52100.PMID:25548986;PMCID:PMC4396958.].

[0127] Another important function of RPE cells is phagocytosis of the outer segments detached from photoreceptor cells. To detect the phagocytic activity of differentiated RPE cells, fluorescent particles were used for follow-up after cell culture. The results showed that the uptake and intracellular entry of fluorescent particles by iPSCs-RPE cells and fRPE cells could be more intuitively confirmed by flow cytometry. Each experiment was repeated three times. Figure 7A shows the phagocytic activity detection graph for fRPE, iRPE (iRPE-1, iRPE-2, and iRPE-3), and iKO-RPE (iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3) at 37°C. Figure 7B shows the phagocytic activity detection graph for fRPE, iRPE (iRPE-1, iRPE-2, and iRPE-3), and iKO-RPE (iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3) at 4°C. Figure 7C shows the negative control groups for fRPE, iRPE-1, iRPE-2, iRPE-3, iKO-RPE-1, iKO-RPE-2, and iKO-RPE-3. In other words, no fluorescent particles were added during the incubation process. iKO-RPE in the figure represents iPSC HLA-KORPE cells (iPSCs) obtained by differentiation from these cells. HLA-KO -RPE refers to RPE cells obtained by differentiating from iPSCs (iPSC-RPE), while iRPE refers to RPE cells obtained by differentiating from iPSCs.

[0128] VII. Detection of iKO-RPE immune function in vitro IFN-γ, whose abnormal expression is associated with many autoinflammatory and autoimmune diseases, is important to the immune system, as reflected in its immunostimulatory and immunomodulatory functions. IFN-γ is primarily secreted by NK cells and NKT cells, playing a role in innate immunity, and is secreted by CD4+TH1 and CD8 cytotoxic T cells in antigen-specific immunity. IFN-γ functions as a disease marker in various pathological conditions, including infectious diseases, autoimmune diseases, transplant rejection, and allergic reactions. In this application, in an in vivo inflammatory environment simulated by the applicant under IFN-γ stimulation, iKO-RPE cells differentiated after knocking out the B2M and CIITA genes showed MHC inactivation of HLA-class I HLA genes such as HLA-A, B, and C, and HLA-class II HLA genes such as HLA-DR, DQ, and DP, thus eliminating rejection in the body. [Methodological basis: Petrus-Reurer S, Winblad N, Kumar P, Gorchs L, Chrobok M, Wagner AK, Bartuma H, Lardner E, Aronsson M, Plaza Reyes A, Andre H, Alici E, Kaipe H, Kvanta A, Lanner F. Generation of Retinal Pigment Epithelial Cells Derived from Human Embryonic Stem Cells Lacking Human Leukocyte Antigen Class I and II. Stem Cell Reports. 2020 Apr] 14;14(4):648-662.doi:10.1016 / j.stemcr.2020.02.006.Epub 2020 Mar 19.PMID:32197113;PMCID:PMC7160308.].

[0129] Specifically, the cells from each experimental group were placed in a 12-well plate at a rate of 1 × 10⁶ 5 Cells were seeded in individual / wells and cultured at 37°C for 2 days and 5 days under IFN-γ stimulation or without IFN-γ stimulation, respectively. After 2 days, HLA class I proteins (HLA-A / B / C) were detected by flow cytometry. After 5 days, HLA class II proteins (HLA-DP / DQ / DR) were detected by flow cytometry. Flow cytometry visually confirmed that iKO-RPE cells differentiated after knockout of the B2M and CIITA genes under IFN-γ stimulation showed HLA class I MHC inactivation (see Figure 8A) and HLA class II MHC inactivation (see Figure 8B).

[0130] An RPE cell preparation is provided, which includes the RPE cells derived from pluripotent stem cells as described above.

[0131] Another object of this application is to provide an ophthalmic pharmaceutical composition containing the above-mentioned pluripotent stem cell-derived RPE cells as an active ingredient.

[0132] Another object of this application is to provide reagents for evaluating the toxicity or efficacy of a test substance, including the pluripotent stem cell-derived RPE cells described above.

[0133] Another object of this application is to provide a method for evaluating the toxicity or efficacy of a test substance, comprising contacting the aforementioned pluripotent stem cell-derived RPE cells with a substance and measuring the effect of the substance on the cells.

[0134] The technical solutions in Examples 1 and 2 of this application can also be achieved by kit.

[0135] This kit includes a combination of culture media, comprising medium 1, medium 2, medium 3, and medium 4.

[0136] The first medium comprises a basal medium and additive A selected from the group consisting of SB431542, LDN193183, IWR-1, IWR-2, and combinations thereof in any molar ratio. However, the amount of SB431542 added is 1 to 50 μM, the amount of LDN193183 added is 10 to 100 nM, the amount of IWR-1 added is 10 to 100 μM, and the amount of IWR-2 added is 10 to 100 μM. The second medium comprises a basal medium and additive B, which is nicotinamide. However, the amount of nicotinamide added is 1 to 50 mM.

[0137] The third medium comprises a basal medium and additive C selected from the group consisting of activin A, SU5402, CHIR99021, and combinations thereof in any molar ratio. However, the amount of activin A added is 10 to 200 ng / mL, the amount of SU5402 added is 1 to 10 μM, and the amount of CHIR99021 added is 1 to 10 μM.

[0138] The fourth medium comprises a basal medium and additive D selected from the group consisting of nicotinamide, CHIR99021, and combinations thereof in any molar ratio. However, the amount of nicotinamide added is 1 to 50 mM, and the amount of CHIR99021 added is 1 to 10 μM.

[0139] As an improved technical solution of this application, the basal culture medium is E6.

[0140] Using the aforementioned kit to achieve the solutions in Examples 1 and 2 of this application includes the following steps: Pluripotent stem cells are cultured in the first medium for 2 days, and neuroectoderm cells are subjected to step 1; Step 2: The neuroectoderm cells are cultured in the second medium for 4 days to obtain RPE progenitor cells; Step 3: Culturing the RPE progenitor cells in the third medium for 4 days to obtain RPE cells; Step 4 involves culturing the RPE cells in the fourth medium for 20 days to obtain mature RPE cells. [Industrial applicability]

[0141] In this application, by combining the differentiation process with the differentiation signaling pathway within RPE cells, the cellular state during differentiation is clarified, differentiation efficiency is greatly improved, the differentiation cycle is shortened to just 30 days, and cells can be obtained with high purity and high yield.

[0142] This application aims to fixate on the differentiation method and efficiently reproduce it by clearly defining the activators or inhibitors of the necessary signaling pathways that are added during the differentiation process.

[0143] Since the differentiation process described in this application does not use any animal-derived components, the possibility of entry by exogenous viruses is blocked, improving safety in future clinical applications.

Claims

1. From pluripotent stem cells, the following differentiation stages occur: Neuroectoderm cell induction phase in which differentiation is induced by one or more of the following three signaling pathways: inhibition of the TGF-β / nodal / activin signaling pathway, inhibition of the BMP signaling pathway, and inhibition of the WNT signaling pathway; RPE progenitor cell induction phase, in which differentiation is induced by activation of the nicotinamide metabolic signaling pathway; RPE cell induction phase, in which differentiation is induced by one or more of the following three signaling pathways: activation of the TGF-β / nodal / activin signaling pathway, inhibition of the FGF signaling pathway, and activation of the WNT signaling pathway; and RPE cells derived from pluripotent stem cells, characterized by being obtained based on the maturation phase of RPE cells, which undergo differentiation induction by one or both of two signaling pathways: activation of the nicotinamide metabolic signaling pathway and activation of the WNT signaling pathway.

2. The aforementioned pluripotent stem cells are ESCs, iPSCs, or general-purpose iPSCs. ＨＬＡ－ＫＯ It was obtained through differentiation, The aforementioned general-purpose iPSC ＨＬＡ－ＫＯ The pluripotent stem cell-derived RPE cell according to claim 1, characterized in that it is an iPSC having immune privilege characteristics, in which HLA expression is specifically reduced by gene editing.

3. The aforementioned ESCs are ESC cells that have been passaged 4 to 7 times with a passage ratio of 1:(3 to 12) each time; the cultured ESCs are degraded into single cells, then cultured in iPSC medium for 1 day, after which the ESCs differentiate to obtain RPE cells; The iPSC or general-purpose iPSC ＨＬＡ－ＫＯ The pluripotent stem cell-derived RPE cells according to claim 2, characterized in that the iPSC cells are passaged 4 to 7 times with a passage ratio of 1:(3 to 12) each time; the cultured iPSCs are degraded into single cells, then cultured in iPSC medium for 1 day, after which the iPSCs differentiate to obtain RPE cells.

4. The iPSC medium is characterized in that it is prepared by using E8, Stem Flex, or mTeSR1 as the base medium and adding the additive Y27632 at an amount of 10 μM, as described in claim 1, for RPE cells derived from pluripotent stem cells.

5. The RPE cells obtained by differentiation of iPSCs are achieved by adding small chemical molecules as inhibitors or activators to E6 medium, and the pluripotent stem cell-derived RPE cells according to claim 1 are characterized by comprising a neuroectoderm cell induction phase, an RPE progenitor cell induction phase, an RPE cell induction phase, and an RPE cell maturation phase.

6. During the neuroectoderm cell induction period, To inhibit the TGF-β / nodal / activin signaling pathway, low molecular weight SB431542 was used at an additive dose of 1 to 50 μM. To inhibit the BMP signaling pathway, low molecular weight LDN193183 is used at an additive dose of 10 to 100 nM. The pluripotent stem cell-derived RPE cells according to claim 1, characterized in that low molecular weight IWR-1 is used in an amount of 10 to 100 μM, or IWR-2 is used in an amount of 10 to 100 μM, for inhibiting the WNT signaling pathway.

7. The pluripotent stem cell-derived RPE cells according to claim 1, characterized in that, during the RPE progenitor cell induction phase, activation of the nicotinamide metabolic signaling pathway is achieved by adding nicotinamide in an amount of 1 to 50 mM.

8. During the RPE cell induction phase, To activate the TGF-β / nodal / activin signaling pathway, low molecular weight activin A is used at an additive dose of 10 to 200 ng / mL. To inhibit the FGF signaling pathway, low molecular weight SU5402 is used at an additive amount of 1 to 10 μM. The pluripotent stem cell-derived RPE cells according to claim 1, characterized in that low molecular weight CHIR99021 is used in an additive amount of 1 to 10 μM to activate the WNT signaling pathway.

9. During the RPE cell maturation phase, To activate the aforementioned nicotinamide metabolic signaling pathway, low molecular weight nicotinamide is used in an additive amount of 1 to 50 mA. The pluripotent stem cell-derived RPE cells according to claim 1, characterized in that low molecular weight CHIR99021 is used in an additive amount of 1 to 10 μM to activate the WNT signaling pathway.

10. The pluripotent stem cell-derived RPE cell according to claim 1, characterized in that the passage ratio during the RPE cell induction phase and the RPE cell maturation phase is 1:(3-20).

11. An RPE cell preparation characterized by comprising RPE cells derived from pluripotent stem cells as described in any one of claims 1 to 10.

12. An ophthalmic pharmaceutical composition comprising RPE cells derived from pluripotent stem cells as described in any one of claims 1 to 10 as an active ingredient.

13. A reagent for evaluating the toxicity or efficacy of a test substance, comprising RPE cells derived from pluripotent stem cells as described in any one of claims 1 to 10.

14. A method for evaluating the toxicity or efficacy of a test substance, comprising contacting RPE cells derived from pluripotent stem cells as described in any one of claims 1 to 10 with the substance and measuring the effect of the substance on the cells.

15. A first medium comprising a basal medium and additive A selected from the group consisting of SB431542, LDN193183, IWR-1, IWR-2, and combinations thereof in any molar ratio (where the amount of SB431542 added is 1 to 50 μM, the amount of LDN193183 added is 10 to 100 nM, the amount of IWR-1 added is 10 to 100 μM, and the amount of IWR-2 added is 10 to 100 μM), A basal medium and a second medium containing additive B, which is nicotinamide (where the amount of nicotinamide added is 1 to 50 mM), A third medium comprising a basal medium and an additive C selected from the group consisting of activin A, SU5402, CHIR99021, and combinations thereof in any molar ratio (where the amount of activin A added is 10 to 200 ng / mL, the amount of SU5402 added is 1 to 10 μM, and the amount of CHIR99021 added is 1 to 10 μM), and, A kit characterized by comprising a combination of culture media, including a base medium and a fourth medium containing additive D selected from the group consisting of nicotinamide, CHIR99021, and combinations of the two in any molar ratio (where the amount of nicotinamide added is 1 to 50 mM and the amount of CHIR99021 added is 1 to 10 μM).

16. The kit according to claim 15, characterized in that the basal culture medium is E6.

17. A method for obtaining RPE cells using a kit according to any one of claims 15 to 16, Step 1 involves culturing pluripotent stem cells in the first culture medium for two days to obtain neuroectoderm cells, Step 2 involves culturing the neuroectoderm cells in the second culture medium for 4 days to obtain RPE progenitor cells. Step 3 involves culturing the RPE progenitor cells in the third medium for 4 days to obtain RPE cells. A method characterized by comprising step 4, culturing the RPE cells in the fourth medium for 20 days to obtain mature RPE cells.

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