Method for producing retinal pigment epithelial cells

Abstract

The present invention aims to provide a method for producing RPE cells, RPE cells produced by the method, and reagents suitable for producing RPE cells. The method of the present invention comprises the following steps: introducing the MITF (microphthalmia-associated transcription factor) gene or its expression product, the OTX2 (orthodenticle homeobox 2) gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product into mammalian somatic cells as exogenous factors.

Description

Technical Field

[0001] The present invention relates to a method for producing retinal pigment epithelial cells. More specifically, the present invention relates to a method for producing retinal pigment epithelial cells by direct conversion (direct reprogramming), and reagents for converting somatic cells into retinal pigment epithelial cells.

Background Art

[0002] A treatment method including producing retinal pigment epithelial (RPE) cells from iPS cells and administering them to patients with exudative age-related macular degeneration requires a culture period of about six months and is very costly. It is realistic to use an iPS stock for iPS cells, in which case allogeneic transplantation is performed. There is a need for a method that can shorten the culture period and reduce costs, and can particularly treat patients who require autologous transplantation. A method for producing RPE-like cells by forcibly expressing multiple transcription factors in fibroblasts has been reported (Patent Document 1, Non-Patent Document 1). In the former, at least four transcription factors selected from 10 types of transcription factors, and in the latter, at least four transcription factors selected from 8 types of transcription factors are overexpressed to produce RPE-like cells. However, stable cell lines have not been established. Each combination is different from that of the present invention.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present invention aims to provide a method for producing RPE cells capable of autologous transplantation for patients in whom transplantation of RPE cells is effective, such as patients with exudative age-related macular degeneration, the RPE cells produced by the method, and reagents (for example, vectors) for producing RPE cells suitable for the method.

Means for Solving the Problems

[0006] In view of the above problems, the present inventors conducted intensive studies based on the previous findings. Specifically, the present inventors attempted to produce RPE cells from somatic cells by direct conversion (direct reprogramming) technology. Direct reprogramming can be achieved by transducing a gene encoding a transcription factor that plays an important role in the differentiation stage of target cells, or a gene that promotes reprogramming of somatic cells. The present inventors found that somatic cells can be directly converted into RPE cells by introducing several genes, established the conditions for the conversion, analyzed the phenotypes and functions of the obtained directly converted RPE cells, and completed the present invention. Therefore, the present invention provides the following. (1) As an exogenous factor, the MITF (microphthalmia-associated transcription factor) gene or its expression product, the OTX2 (Orthodenticle homeobox 2) gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product A method for producing retinal pigment epithelial cells, comprising the step of introducing into mammalian somatic cells. (2) The method according to (1) above, further comprising the step of introducing as an exogenous factor the CRX (cone-rod homeobox) gene or its expression product. (3) A method according to (1) or (2) above, comprising three phases: a period (phase 1) of overexpressing the introduced exogenous factor in somatic cells to generate genomic plasticity, a period (phase 2) of converting somatic cell identity into RPE cells, and a period (phase 3) of maturing the converted RPE cells. (4) A method according to (3) above, comprising using a medium containing bFGF and 2-ME in phase 1. (5) A method according to (3) above, comprising using a medium containing ketamine and nicotinamide for a part of the period of phase 2. (6) A method according to (3) above, comprising using a medium containing SB431542 and bFGF in phase 3. (7) A method according to any one of (1) to (6) above, wherein the somatic cell is a human fibroblast. (8) RPE cells produced by a method according to any one of (1) to (7) above. (9) Derived from mammalian somatic cells and, as exogenous factors, the MITF gene or its expression product, the OTX2 gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product RPE cells containing the same. (10) The cells according to (9) above, further comprising the CRX gene or its expression product as an exogenous factor. (11) The cells according to (9) or (10) above, wherein the somatic cell is a human fibroblast. (12) The MITF gene or its expression product, the OTX2 gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product Reagents for directly producing retinal pigment epithelial cells from somatic cells, containing the same. (13) The reagents according to (12) above, further comprising the CRX gene or its expression product. (14) The reagents according to (12) or (13) above, wherein the somatic cell is a human fibroblast.

Advantages of the Invention

[0007] The current state-of-the-art approach for generating patient-specific retinal pigment epithelium (RPE) is to induce its somatic cells into induced pluripotent stem (iPS) cells. Those skilled in the art must amplify and confirm the quality of those iPS cells and then differentiate those iPS cells into RPE cells, which requires cell culture exceeding 180 days, which is prohibitively expensive for practical medicine. The technology proposed by the present invention rapidly changes somatic donor cells into cells having an RPE-like identity within 2 to 3 weeks and into sufficiently stable initial RPE cells within 40 to 60 days, thereby dramatically reducing the transition period from somatic cells to RPE cells. The present invention utilizes special cell culture conditions and overexpression of important transcription factors to rapidly and efficiently generate RPE cells from easily obtainable patient cells such as skin or blood. The cost of patient-specific cells for medical or research purposes is almost entirely derived from the labor and materials required daily, and the current state-of-the-art production of patient-specific RPE cells is not cost-effective for patients or scientists. The present invention dramatically reduces the cost of patient-specific autologous RPE cells and enables realistic cell products for normal medical and research applications. The present invention is compared with RPE induced by the current state-of-the-art iPS cell technology (Figure 1).

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0009] The present invention will be described below. Unless otherwise indicated, the terms used in this specification have the meanings generally used in the art.

[0010] The present invention relates to a method for producing retinal pigment epithelial (RPE) cells by converting differentiated somatic cells of mammals into RPE cells. "Converting" means changing somatic cells into desired RPE cells. One of the features of the method of the present invention is a method of converting somatic cells into RPE cells, called "direct reprogramming" or "direct conversion", which does not involve a step of completely reprogramming cells typified by the production of iPS cells.

[0011] Induction into RPE cells can theoretically be carried out both in vitro and in vivo.

[0012] RPE cells The "retinal pigment epithelial cells" in the present invention refer to epithelial cells constituting the retinal pigment epithelium and their progenitor cells. Whether a cell is a retinal pigment epithelial cell can be confirmed, for example, by the expression of cell markers (such as RPE65, CRALBP, MERTK, BEST1, etc.), cell morphology (intracellular melanin pigment deposition, polygonal and flat cell morphology, formation of polygonal actin bundles, etc.). The progenitor cells of retinal pigment epithelial cells mean cells that are instructed to be induced to differentiate into retinal cells, and whether a cell is a progenitor cell can be confirmed by the expression of cell markers (such as Mitf, Pax6, Rx, Crx, etc.). These functional evaluations and confirmation operations can be carried out by those skilled in the art by setting appropriate conditions. The RPE cells induced and prepared by the present invention are also called induced RPE cells (iRPE cells).

[0013] Somatic cells In the present invention, the somatic cells targeted for direct programming are not particularly limited as long as RPE cells can be induced and can be derived from mammals. When iRPE cells are transplanted into a living body, somatic cells (autologous cells) derived from the subject to receive the transplantation are preferably used in order to reduce risks such as infection and rejection reactions. However, depending on the purpose, RPE cells prepared in advance from somatic cells of others or other animals instead of autologous cells can be used for transplantation.

[0014] In the present specification, examples of mammals include mice, rats, hamsters, humans, dogs, cats, monkeys, rabbits, cows, horses, pigs, etc., and particularly humans.

[0015] As the somatic cells, somatic cells that can be easily collected from a living body can be used. Examples thereof include fibroblasts, keratinocytes, oral mucosal epithelial cells, nasal mucosal epithelial cells, respiratory mucosal epithelial cells, gastric mucosal epithelial cells, intestinal mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, adipocytes, gingival cells (gingival fibroblasts and gingival epithelial cells), dental pulp cells, periodontal ligament cells, bone marrow cells, bone marrow-derived stromal cells, white blood cells, lymphocytes, conjunctival epithelial cells, and osteoclasts, and preferably fibroblasts, keratinocytes, oral mucosal epithelial cells, gingival cells, white blood cells, and lymphocytes. In the present invention, the above cells collected from a living body are preferably used.

[0016] Gene or its expression product In the method of the present invention, as exogenous factors, the MITF (microphthalmia-associated transcription factor) gene or its expression product, the OTX2 (orthodenticle homeobox 2) gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product are introduced into somatic cells. If desired, the CRX (cone-rod homeobox) gene or its expression product can be introduced as an exogenous factor, and from the viewpoint of colony formation efficiency, such introduction is preferred. As used herein, the "expression product" is, for example, the mRNA or protein of the MITF gene, OTX2 gene, LIN28 gene, L-MYC gene, or CRX gene.

[0017] All of the above genes are highly conserved in vertebrates. In this specification, unless a specific animal name is described, they refer to genes including homologs. As genes, those having a function equivalent to that of the wild-type gene product (even if the gene contains mutations such as polymorphisms) can be further mentioned.

[0018] For example, the nucleotide sequences of the human (Homo sapiens) MITF gene, OTX2 gene, LIN28 gene, L-MYC (MYCL) gene, CRX gene, and the amino acid sequences of the proteins encoded by these sequences are registered in GenBank provided by the National Center for Biotechnology Information (NCBI). As embodiments, the following accession numbers are exemplified (it should be understood that when multiple revisions are registered, each number refers to the latest revision): Human MITF gene: For example, AB006909.1, Human MITF protein: For example, BAA32288.1, Human OTX2 gene: For example, NM_021728.4, Human OTX2 protein: For example, NP_068374.1, Human LIN28 gene: For example, NM_024674.6, Human LIN28 protein: For example, NP_078950.1, Human L-MYC gene: For example, NM_001033081.3, Human L-MYC protein: For example, NP_001028253.1, Human CRX gene: For example, NM_000554.6, Human CRX protein: For example, NP_000545.1. It is obvious that other sequences can be used as long as they correspond to the same or similar functional genes and / or proteins respectively.

[0019] Introduction The method of the present invention can be carried out according to known reprogramming methods, except that a specific gene is selected. Specifically, a desired gene (hereinafter, also simply referred to as the desired gene) is introduced as an exogenous factor and expressed in target somatic cells. As a method for introducing a gene, for example, a method involving infection with a viral vector such as a retroviral vector, an adenoviral vector, a lentiviral vector, an adeno-associated viral vector, a herpes viral vector, or a Sendai viral vector; and when introducing a gene and its expression product, a non-viral vector such as a cationic liposome or a cationic polymer, or a method involving transfection with a plasmid vector, an episomal vector, or a gene expression product (mRNA, protein) by electroporation can also be used. Alternatively, mRNA can also be introduced. Genome editing techniques such as the CRISPR / Cas9 system (CRISPR target transactivation) can also be utilized.

[0020] A preferred embodiment is a method of introducing an expression cassette designed to control the expression of a desired gene in a period-specific manner in response to an external stimulus. Such an expression cassette is a nucleic acid construct containing at least a promoter capable of inducing gene expression downstream in response to an external stimulus and a desired gene whose expression is controlled by the promoter.

[0021] The promoter is not particularly limited as long as it is a promoter capable of inducing gene expression downstream in response to an external stimulus. As an example, when the external stimulus is the presence of a tetracycline-based antibiotic (tetracycline derivatives such as tetracycline and doxycycline), a promoter capable of inducing gene expression downstream by the binding of a complex of the tetracycline-based antibiotic and a tetracycline transactivator can be mentioned.

[0022] The expression cassette can contain, if necessary, an enhancer, a silencer, a selectable marker gene (for example, a drug resistance gene such as the neomycin resistance gene), an SV40 origin of replication, etc.

[0023] Therefore, external stimuli include culturing in the presence or absence of a drug. For example, the expression of a desired gene is controlled in the presence of doxycycline and using a doxycycline expression induction system (for example, a doxycycline expression induction system).

[0024] The doxycycline expression induction system may be a commercially available product (for example, Takara, Clontech, etc.), or may be produced by the methods described in known literature.

[0025] The method for introducing the above expression cassette into somatic cells is not particularly limited, and known methods can be appropriately selected and used. For example, the above expression cassette is inserted into an appropriate expression vector, and introduced by known transformation methods such as viral infection using a viral vector such as a retroviral vector or an adenoviral vector, lipofection method, liposome method, electroporation method, calcium phosphate method, DEAE dextran method, microinjection method, etc.

[0026] Examples of expression vectors include viral vectors such as lentivirus, retrovirus, herpes virus, adenovirus, adeno-associated virus, Sendai virus, etc., and animal cell expression plasmids. From the perspective of introduction efficiency, lentivirus is preferred.

[0027] Regarding the above external stimulus, a person skilled in the art can appropriately adjust the amount (level) of the stimulus to be added in consideration of the type of the promoter to be used and the like, and in consideration of the addition timing. For example, when the external stimulus is the presence of doxycycline, the preferable addition concentration of doxycycline is 0.1 to 2 μg / ml, more preferably 0.5 to 1 μg / ml. In one embodiment, the addition amount is 1 μg / ml in Phase 1 and Phase 2, then temporarily decreased to 0.5 μg / ml in Phase 3, and then immediately completely removed there.

[0028] External stimuli such as doxycycline may be present in somatic cells into which a desired gene has been introduced for 60 days after the introduction of the desired gene, preferably at least 50 days. As a result, the somatic cells can be induced into RPE cells and mature in Phase 3.

[0029] Culture In the method of the present invention, differentiated somatic cells of a mammal can be cultured in a medium after the introduction of a gene. For example, it is a preferable embodiment that RPE is induced (prepared) in vitro.

[0030] The culture can be carried out in a container suitable for storing cells and the medium. In the case of adherent culture, a cell adhesion culture container, for example, a culture container after being coated with an extracellular matrix or the like (for example, poly-D-lysine, laminin, fibronectin, iMatrix511 (product name)) is preferably used. Culture conditions for adherent culture such as the culture temperature, CO 2 concentration, and O 2 concentration can be appropriately determined. In this case, the cells can be cultured in the presence of serum, known growth factors, and additives and chemicals that promote growth. Examples of known growth factors include EGF, FGF, etc. Examples of additives that promote growth include N2 supplement (Invitrogen), B27 supplement (Invitrogen), etc.

[0031] The culturing period is not particularly limited as long as the effects of the present invention are not impaired. For example, it can be set to about 30 days, or 40 days, or 50 days, or 60 days, or 70 days as needed. If necessary, the medium can be replaced, and it is preferable to replace it with a medium having components appropriately adjusted according to the stage (see "medium" below).

[0032] Medium The medium used in the method of the present invention is not particularly limited. Ordinary liquid media such as DMEM (Dulbecco's Modified Eagle Medium), EMEM (Eagle's Minimum Essential Medium), αMEM (Alpha Modified Minimum Essential Medium), Ham's F-12 (Nutrient Mixture F-12 Ham) can be used. If necessary, serum components (fetal bovine serum (FBS), human serum (HS)), serum replacements (SR), antibiotics such as streptomycin and penicillin, non-essential amino acids (NEAA), and similar components can be added.

[0033] Considering the high production efficiency of RPE cells by the method of the present invention, it is preferable to divide the production process of RPE cells into three stages (Phase 1, Phase 2, Phase 3) and use a medium suitable for each phase (see Figure 3). These media may be referred to as Phase 1 medium, Phase 2 medium, and Phase 3 medium for convenience. The Phase 1 medium is a medium used during the overexpression period of the desired gene introduced into somatic cells, and is typically used for 8 to 10 days after gene introduction. From the viewpoint of maintaining initial cell growth, the medium preferably contains bFGF and 2-ME. The preferable addition concentration of bFGF is 1 to 100 ng / ml, more preferably about 10 ng / ml. The preferable addition concentration of 2-ME is 10 to 100 μM, more preferably about 55 μM. The Phase 2 medium is a medium used during the conversion period from cells into which a desired gene has been introduced after Phase 1 to RPE cells, and is typically used for 34 to 38 days after Phase 1. The medium preferably contains ketomine and nicotinamide so as to be able to eliminate unwanted cells. The preferred addition concentration of ketomine is 10 to 100 nM, more preferably 40 to 80 nM. The preferred addition concentration of nicotinamide is 1 to 50 mM, more preferably 5 to 10 mM. The Phase 3 medium is a medium used during the maturation period of the converted RPE cells after Phase 2, and is typically used for 7 to 12 days after Phase 2. From the perspective of common RPE cell maturation materials, the medium preferably contains SB431542 and bFGF. The preferred addition concentration of SB431542 is 0.5 to 1 μM, more preferably about 0.5 μM. The preferred addition concentration of bFGF is 1 to 100 ng / ml, more preferably about 10 ng / ml.

[0034] Manufacture (induction) Thus, RPE cells are produced from somatic cells. Whether the cells are retinal pigment epithelial cells can be confirmed by those skilled in the art based on, for example, the expression of cell markers (such as RPE65, Mitf, etc.), the presence of melanin granules, the characteristic cell morphology of polygons, etc.

[0035] Induced RPE cells (iRPE cells) contain exogenous MITF gene, OTX2 gene, LIN28 gene, and L-MYC gene (preferably also the CRX gene). As used herein, the term "exogenous" mainly means a gene or its expression product introduced by the above-described introduction means and different from the natural state.

[0036] iRPE cells can be obtained as a mixture with cells other than iRPE cells (for example, the original somatic cells). In this case, the iRPE cells can be separated from the cells other than iRPE cells as needed. The separation means is not particularly limited. For example, they can be separated using a cell sorter or magnetic beads.

[0037] The iRPE cells produced by the present invention have characteristics extremely similar to those of retinal pigment epithelial cells in the body. Therefore, they can also be used for screening therapeutic agents for diseases caused by retinal cell disorders, or as transplantation materials for cell therapy, materials for disease research, or drug discovery materials for therapeutic agents for cell damage caused by other etiologies. In addition, they can be used in toxicity studies such as phototoxicity, and toxicity tests in toxicity and efficacy evaluations of chemicals and the like. Examples of diseases caused by retinal cell disorders include organic mercury poisoning, chloroquine retinopathy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, retinopathy of prematurity, and the like. The iRPE cells produced by the present invention can be used as retinal pigment epithelial cells for transplantation, which are used to supplement damaged cells or damaged tissues themselves in a cell-damaged state (for example, used in transplantation surgery) and the like.

[0038] Reagent As described above, RPE cells can be induced by introducing, as exogenous factors, the MITF gene or its expression product, the OTX2 gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product, and preferably, in addition to these, the CRX gene or its expression product into somatic cells. Therefore, the present invention further provides a reagent for producing RPE cells from somatic cells, which contains the MITF gene or its expression product, the OTX2 gene or its expression product, the LIN28 gene or its expression product, and the L-MYC gene or its expression product, and preferably, in addition to these, the CRX gene or its expression product. Here, the somatic cells are preferably fibroblasts. Specifically, the reagent includes a vector incorporated with the above genes in a form that can introduce the above genes into somatic cells. The above genes may be incorporated into different vectors respectively, or two or more genes may be simultaneously incorporated into one vector. The types of vectors that can be used are as described above. The present invention will be described in detail below by referring to examples, but should not be construed in a limiting sense. The reagents and materials used are commercially available unless otherwise specifically limited.

Examples

[0039] Example 1; Induction of RPE cells (Phase 1) Human somatic cells (fibroblasts; ATCC Cat#CRL2522) were cultured and grown in a standard medium and method until reprogramming was desired. For the preparation of reprogramming, the fibroblasts were transduced with a lentiviral dox-inducible conditional gene expression system and dox-inducible lentivirus for MITF, OTX2, LIN28, L-MYC, and CRX. To initiate reprogramming, 100 - 150×10 3 cells / well were seeded on 6-well (6W) plate wells coated with iMatrix511. The next day, reprogramming medium phase 1 (phase 1 medium) was added, and human exogenous transcription factors (MITF, OTX2, LIN28, L-MYC, and CRX) were overexpressed daily using the lentiviral dox-inducible system. The overexpression of reprogramming factors was maintained until it was gradually removed at the early stage of phase 3 when iRPE became stable (Figure 7). The culture using phase 1 medium was maintained for 8 - 10 days.

[0040] (Phase 2) Next, phase 2 medium was added every other day (every two days). Approximately on the 24th - 32nd day, each 6W plate well of reprogramming was reseeded in large quantities into one or more wells (typically 2 wells) with phase 2 medium on a 6W plate coated with iMatrix511 at 400 - 600×10 3 cells / well. Alternatively, a 12-well (12W) plate was seeded at 200 - 300×10 3It can be used in cells / wells. Alternatively, iRPE colonies can be isolated into individual iMatrix511-coated 96-well (96W) plate wells, but bulk passage is recommended. After reseeding, reprogramming was continued in phase 2 medium for an additional ~1 week. Then, optional treatment with 40 - 80 nM ketamine and 5 - 10 mM nicotinamide can be added for 6 - 12 days to improve RPE cell quality (Figure 8), or culturing using phase 2 medium can be continued on alternate days for the same period. Culturing using phase 2 medium was continued for an additional 2 days (one medium change) after removal of ketamine and nicotinamide.

[0041] (Phase 3) Phase 3 medium was added on alternate days, causing RPE-like maturation and increasing polarization and pigmentation. Overexpression of exogenous MITF, OTX2, LIN28, L-MYC, and CRX gradually decreased over 7 - 12 days until they were no longer added. During the process of disappearance of exogenous reprogramming and subsequent culturing, the quality of iRPE and unwanted cells became clear (Figure 7). Culturing using phase 3 medium was maintained, and it was considered that pigmented cells and / or pigmented cells with continuous BEST1::EGFP reporter expression were stably induced into RPE (iRPE). These reprogrammed and differentiated iRPE cells can then be purified, passaged, examined, and transplanted using standard RPE culture methods. Reprogramming of human foreskin fibroblasts into iRPE cells was observed over time. The expression of RPE-related genes RPE65 and BEST1, and ZO-1 that constitutes tight junctions was examined. The results are shown in Figure 5. RPE65: Mouse-anti RPE65, Millipore cat#MAB5428 BEST1: Live Best1::EGFP Transgenic Reporter ZO-1: Rabbit anti-ZO-1, Invitrogen cat#61-7300 Reprogramming medium composition Phase 1 Medium:

[0042] [Table 1]

[0043] Phase 2 Medium:

[0044] [Table 2]

[0045] Phase 3 Medium:

[0046] [Table 3]

[0047] Example 2; Induction of RPE cells (CRX) Exogenous factors with CRX and exogenous factors without CRX introduced in the same manner as in Example 1 were compared. The number of colony formations (per well) in the 6-well plate of iRPE colonies formed on day 9 was counted at the end of Phase 1 of reprogramming gene overexpression. The results are shown in the following table.

[0048] [Table 4]

[0049] The size distribution of each colony was examined on day 12 after gene introduction. The results are shown in Figure 6. The diameter of the colony was used as an index. Day 12 after gene introduction is the initial stage of Phase 2. RPE cells were induced from fibroblasts even in the absence of CRX, but the induction was more effective in the presence of CRX in both its number and size. The addition of CRX was optional, but CRX induced far more colonies in number and far more proliferative colonies as measured by diameter.

[0050] Example 3; Induction of RPE cells (ketamine / nicotinamide) The effects of treatment with ketamine and nicotinamide were examined at the late stage of Phase 2. RPE cells were induced in the same manner as in Example 1 except that ketamine and nicotinamide were added. The results are shown in Fig. 8. Stronger dye deposition was confirmed when both compounds were added. From these results, it was found that treatment with ketamine and nicotinamide at a certain stage of the process of inducing RPE cells from somatic cells is effective.

[0051] Example 4; Transplantation of the iRPE cells of the present invention into the eyes of rats The stable cultured human iRPE cells prepared in Example 1 were transplanted into the eyes of immunodeficient albino rats. The expression of the BEST1 live reporter was examined 4.5 months after transplantation (Fig. 9). Some large areas of the transplanted human iRPE cells expressed the BEST1::EGFP live mature reporter gene transduction. Similarly, tissue sections were prepared 4.5 months after transplantation and stained with hematoxylin and eosin (Fig. 10). Some human iRPE cells became pigment cells, and the pigment cells grew, matured, and connected to the neural retina (such as integration into the normal RPE layer and sheet formation). BEST1: Live BEST1::EGFP transgenic reporter Hematoxylin and eosin staining; Standard Practice

Industrial Applicability

[0052] The present invention dramatically reduces the cost of patient-specific autologous RPE cells and enables a realistic cell product for normal medical and research applications. This application is based on Japanese Patent Application No. 2020-033848 (filing date: February 28, 2020), the content of which is incorporated herein in its entirety.

Claims

**Claim 1** As an exogenous factor, the MITF (Microphthalmia - associated transcription factor) gene or its expression product, the OTX2 (Orthodenticle homeobox 2) gene or its expression product, the LIN28 gene or its expression product, and the L - MYC gene or its expression product are introduced into mammalian somatic cells and expressed in the target somatic cells, a method for producing retinal pigment epithelial cells (excluding methods performed in the human body). **Claim 2** The method according to claim 1, further comprising the step of introducing as an exogenous factor the CRX (Cone - rod homeobox) gene or its expression product. **Claim 3** The method according to claim 1 or 2, comprising three stages: a period (phase 1) of overexpressing the introduced exogenous factor in somatic cells to generate genomic plasticity, a period (phase 2) of converting somatic cell identity to RPE cells, and a period (phase 3) of maturing the converted RPE cells. **Claim 4** The method according to claim 3, comprising using a medium containing bFGF and 2 - ME in phase 1. **Claim 5** The method according to claim 3, comprising using a medium containing ketamine and nicotinamide for a part of the period of phase 2. **Claim 6** The method according to claim 3, comprising using a medium containing SB431542 and bFGF in phase 3. **Claim 7** The method according to any one of claims 1 to 6, wherein the somatic cell is a human fibroblast. **Claim 8** The MITF gene or its expression product, the OTX2 gene or its expression product, the LIN28 gene or its expression product, and the L - MYC gene or its expression product A reagent for directly producing retinal pigment epithelial cells from somatic cells. **Claim 9** The reagent according to claim 8, further comprising the CRX gene or its expression product. **Claim 10** The reagent according to claim 8 or 9, wherein the somatic cell is a human fibroblast.

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