Method for preparing retinal pigment epithelial cells

Abstract

The present invention provides a method for preparing retinal pigment epithelial cells, comprising (1) a first step of subjecting pluripotent stem cells to suspension culture in a serum-free medium to form aggregates of the pluripotent stem cells, (2) a second step of subjecting the aggregates of the pluripotent stem cells to suspension culture in a serum-free medium to form aggregates of the pluripotent stem cells. (1) a third step in which the aggregate formed in step (1) is subjected to suspension culture in a serum-free medium or a serum-containing medium, each of which does not contain a substance acting on the Sonic hedgehog signal transduction pathway and contains a substance acting on the BMP signal transduction pathway, to obtain an aggregate containing retinal progenitor cells; the aggregate formed in step (2) is subjected to suspension culture in a serum-free medium or a serum-containing medium that does not contain a substance that acts on the Sonic hedgehog signal transduction pathway and a substance that acts on the BMP signal transduction pathway, but contains a substance that acts on the Wnt signal pathway, thereby obtaining an aggregate containing retinal pigment epithelium cells.

Description

[0001] This application is a divisional application of a patent application with international application number PCT / JP2014 / 076471, international filing date October 2, 2014, Chinese application number 201480068651.2, and invention title "Method for Preparing Retinal Pigment Epithelial Cells". Technical Field

[0002] The present invention relates to a method for preparing retinal pigment epithelial cells and the like. Background Art

[0003] There are known reports on the preparation of retinal pigment epithelial cells using pluripotent stem cells (Non-Patent Document 1). A method for obtaining retinal pigment epithelial cells is shown: uniform aggregates of pluripotent stem cells are formed in a serum-free medium containing a substance that inhibits the Wnt signaling pathway, they are subjected to suspension culture in the presence of a basement membrane preparation, then subjected to suspension culture in a serum-containing medium, and then subjected to suspension culture in a serum-free medium or a serum-containing medium containing a substance that acts on the Wnt signaling pathway and does not contain a substance that acts on the Sonic hedgehog signaling pathway (Non-Patent Documents 2 and Patent Document 1).

[0004] [Literature List]

[0005] Patent Documents

[0006] Patent Document 1: WO 2013 / 077425

[0007] Non-Patent Documents

[0008] Non-Patent Document 1: Cell Stem Cell, 5, 396 - 408 (2009)

[0009] Non-Patent Document 2: Cell Stem Cell, 10(6), 771 - 785 (2012) Summary of the Invention

[0011] Problems to be Solved by the Invention [[ID=3)5]]

[0012] There is a desire to develop a method for preparing retinal pigment epithelial cells from pluripotent stem cells.

[0013] Means for Solving the Problems

[0014] The present invention provides a method for preparing retinal pigment epithelial cells or a retinal pigment epithelial cell sheet from pluripotent stem cells and the like.

[0015] That is, the present invention provides:

[0016] [1] A method for preparing retinal pigment epithelial cells, which comprises

[0017] (1) In the first step, pluripotent stem cells are cultured in suspension in a serum-free medium to form aggregates of pluripotent stem cells.

[0018] (2) In the second step, the aggregates formed in step (1) are cultured in suspension in a serum-free medium or a serum-containing medium that each does not contain a substance acting on the Sonic hedgehog signal transduction pathway but contains a substance acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells, and

[0019] (3) In the third step, the aggregates obtained in step (2) are cultured in suspension in a serum-free medium or a serum-containing medium that each does not contain a substance acting on the Sonic hedgehog signal transduction pathway and a substance acting on the BMP signal transduction pathway but contains a substance acting on the Wnt signal pathway, thereby obtaining aggregates containing retinal pigment epithelial cells (sometimes hereinafter referred to as Preparation Method 1 of the present invention);

[0020] [2] A method for preparing a sheet of retinal pigment epithelial cells, which comprises

[0021] (1) In the first step, pluripotent stem cells are cultured in suspension in a serum-free medium to form aggregates of pluripotent stem cells.

[0022] (2) In the second step, the aggregates formed in step (1) are cultured in suspension in a serum-free medium or a serum-containing medium that each does not contain a substance acting on the Sonic hedgehog signal transduction pathway but contains a substance acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells.

[0023] (3) In the third step, the aggregates obtained in step (2) are cultured in suspension in a serum-free medium or a serum-containing medium that each does not contain a substance acting on the Sonic hedgehog signal transduction pathway and a substance acting on the BMP signal transduction pathway but contains a substance acting on the Wnt signal pathway, thereby obtaining aggregates containing retinal pigment epithelial cells, and

[0024] (4) In the fourth step, the aggregates obtained in step (3) are dispersed and the resulting cells are cultured adherently (sometimes hereinafter referred to as Preparation Method 2 of the present invention);

[0025] [3] The preparation method according to [2] above, wherein in the step (4), the adherent culture is carried out in the presence of a serum replacement.

[0026] [4] The preparation method according to [2] or [3] above, wherein in the step (4), the adherent culture is carried out in the presence of a ROCK inhibitor;

[0027] [5] The preparation method according to any one of [2] to [4] above, wherein in the step (4), the adherent culture is carried out in a serum-free medium or a serum-containing medium further comprising one or more substances selected from the group consisting of: substances acting on the Wnt signaling pathway, substances inhibiting the FGF signaling pathway, substances acting on the Activin signaling pathway, and substances acting on the BMP signal transduction pathway;

[0028] [6] The preparation method according to any one of [2] to [5] above, wherein in the step (4), the adherent culture is carried out on a culture vessel material having a surface treated with a culture substrate;

[0029] [7] The preparation method according to [6] above, wherein the culture substrate is a synthetic culture substrate;

[0030] [8] The preparation method according to [6] above, wherein the culture substrate is laminin;

[0031] [9] The preparation method according to any one of [1] to [8] above, wherein the pluripotent stem cell is a primate pluripotent stem cell;

[0032]

[10] The preparation method according to any one of [1] to [9] above, wherein the pluripotent stem cell is a human pluripotent stem cell;

[0033]

[11] The method according to any one of [1] to

[10] above, wherein the steps (1) and (2) are carried out in the presence of a serum replacement;

[0034]

[12] The method according to any one of [1] to

[11] above, wherein the suspension culture is carried out in the absence of a basement membrane preparation;

[0035]

[13] The method according to any one of [1] to

[12] above, wherein the substance acting on the BMP signal transduction pathway is one or more proteins selected from the group consisting of: BMP2, BMP4, BMP7, and GDF7;

[0036]

[14] The method according to any one of [1] to

[13] above, wherein the serum-free medium or serum-containing medium in step (2) each of which does not contain substances acting on the Sonic hedgehog signal transduction pathway and substances acting on the BMP signal transduction pathway but contains substances acting on the Wnt signal pathway further contains substances that inhibit the FGF signal pathway;

[0037]

[15] A reagent for evaluating toxicity or efficacy, which comprises retinal pigment epithelial cells or a retinal pigment epithelial cell sheet prepared by the method according to any one of [1] to

[14] above;

[0038]

[16] A method for evaluating the toxicity or efficacy of a test substance, which includes contacting retinal pigment epithelial cells or a retinal pigment epithelial cell sheet prepared by the method according to any one of [1] to

[14] above with the test substance, and examining the effect of the substance on the cells or the cell sheet;

[0039]

[17] A therapeutic agent for a disease caused by a retinal tissue disorder, which comprises retinal pigment epithelial cells or a retinal pigment epithelial cell sheet prepared by the method according to any one of [1] to

[14] above;

[0040]

[18] A method for treating a disease caused by a retinal tissue disorder, which includes transplanting an effective amount of retinal pigment epithelial cells or a retinal pigment epithelial cell sheet prepared by the method according to any one of [1] to

[14] above into a subject in need of transplantation; and

[0041]

[19] Retinal pigment epithelial cells or a retinal pigment epithelial cell sheet prepared by the method according to any one of [1] to

[14] above, which are used for treating a disease caused by a retinal tissue disorder; etc.

[0042] Advantages of the Invention

[0043] According to the preparation method of the present invention, retinal pigment epithelial cells or a retinal pigment epithelial cell sheet can be prepared very efficiently. In the preparation method of the present invention, since retinal pigment epithelial cells or a retinal pigment epithelial cell sheet can be obtained by suspension-culturing aggregates without adding a basement membrane preparation to the medium (i.e., in the absence of a basement membrane preparation), the risk that the obtained cells or cell sheet are contaminated by components derived from heterologous species is reduced. According to the preparation method of the present invention, retinal pigment epithelial cells or a retinal pigment epithelial cell sheet can be effectively provided for evaluating the toxicity or efficacy of chemical substances, etc., transplantation treatment, etc. Brief Description of the Drawings

[0045] Figure 1Show a bright-field image of aggregates cultured in suspension in a medium supplemented with BMP4 on day 3 of suspension culture, derived from human embryonic stem cells, on day 18 of suspension culture (A), and a bright-field image of aggregates cultured in suspension in a medium supplemented with BMP4 on day 3 of suspension culture, derived from human embryonic stem cells, and cultured in suspension in a medium without BMP4 but containing CHIR99021 from day 18 of suspension culture on day 20 of suspension culture (B).

[0046] Figure 2 Show (A) a bright-field image of aggregates derived from human embryonic stem cells cultured in suspension in a medium containing BMP4 from day 3 to day 18 of suspension culture on day 27 of suspension culture in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture and then in a medium without BMP4 and CHIR99021, (B) a bright-field image of aggregates cultured in suspension in a medium without BMP4 but containing CHIR99021 from day 18 to day 27 of suspension culture on day 27 of suspension culture, and (C) a bright-field image of aggregates cultured in suspension in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture and in a medium without BMP4 but containing CHIR99021 and SU-5402 from day 21 to day 27 of suspension culture on day 27 of suspension culture.

[0047] Figure 3 Show (A) a bright-field image, (B) a fluorescence immunostaining image of the retinal pigment epithelium marker Mitf, and (C) a fluorescence immunostaining image of the tight junction marker ZO-1 of cells cultured in adhesion in a medium containing CHIR99021 on day 27 from the start of suspension culture, wherein the cells are obtained by culturing aggregates derived from human embryonic stem cells in suspension in a medium containing BMP4 from day 3 to day 18 of suspension culture, in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture, and dissociating the aggregates on day 21 of culture.

[0048] Figure 4 Show (A) an image of a retinal pigment epithelium cell sheet and (B) an enlarged light-field image of cells cultured in adhesion in a medium containing Y27632 on day 40 from the start of suspension culture, wherein the cells are obtained by culturing aggregates derived from human embryonic stem cells in suspension in a medium containing BMP4 from day 3 to day 18 of suspension culture, in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture, dispersing the aggregates on day 21 of suspension culture, and inoculating the resulting cells on a surface coated with Synthemax TMIt is obtained in a treated Boyden chamber.

[0049] Figure 5 An enlarged light field image of a retinal pigment epithelium cell sheet on day 40 from the start of suspension culture is shown. Aggregates derived from human embryonic stem cells are suspension-cultured in a medium containing BMP4 from day 3 to day 18 of suspension culture, and in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture. They are dispersed on day 21 of culture, and the resulting cells are seeded on a Boyden chamber surface treated with Synthemax TM and subjected to adherent culture. The adherent culture is continued until day 40 from the start of suspension culture. On day 24 from the start of suspension culture or day 3 from the start of adherent culture (A), no component is added, or (B) 3 μM CHIR99021, (C) 10 μM SU-5402, (D) 3 μM CHIR99021 and 10 μM SU-5402, (E) 1 nM human BMP4 protein, or (F) 50 ng / ml human activin is added.

[0050] Figure 6 An image of a retinal pigment epithelium sheet on day 26 from the start of suspension culture of cells in adherent culture seeded in a Boyden chamber surface-treated with (A) Matrigel TM or (B) Synthemax TM is shown. The cells are obtained by suspension-culturing aggregates derived from human embryonic stem cells in a medium containing BMP4 from day 3 to day 18 of suspension culture, and in a medium without BMP4 but containing CHIR99021 from day 18 to day 21 of suspension culture, and dispersing them on day 21 of suspension culture.

[0051] Embodiment description

[0052] The following details the ways to implement the present invention.

[0053] "Suspension culture" in the present invention means culturing under conditions that prevent cells or cell aggregates from adhering to the cell culture container material, etc.

[0054] The culture vessel for suspension culture is not particularly limited as long as it can perform "suspension culture", and those skilled in the art can appropriately determine the cell culture vessel. Examples of such culture vessels include flasks, tissue culture flasks, dishes, culture dishes, tissue culture dishes, multidishes, microplates, microwell plates, micropores, multiplates, multiwell plates, chamber slides, schales, test tubes, trays, culture bags, and spinner flasks. Since these culture vessels are used for suspension culture, they are preferably non-cell-adhesive. As a non-cell-adhesive vessel, a vessel whose surface has not been artificially treated to improve cell adhesion (for example, coating treatment using extracellular matrix, etc.) can be used, etc.

[0055] The culture medium generally used in the present invention can be prepared from a culture medium for culturing animal cells as a basal medium. Examples of the basal medium include those media that can be used for culturing animal cells, such as BME medium, BGJb medium, CMRL1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, Ham's medium, RPMI1640 medium, Fischer's medium, and their mixed media.

[0056] The "serum-free medium" in the present invention means a medium that does not contain unconditioned or unpurified serum. In the present invention, a medium containing purified components derived from blood and components derived from animal tissues (for example, growth factors) is considered a serum-free medium unless it contains unconditioned or unpurified serum.

[0057] The serum-free medium can contain serum substitutes. Examples of serum substitutes include those appropriately containing, for example, the following substances: albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiol glycerol, their equivalents, etc. Such serum substitutes can be prepared by the method described in, for example, WO98 / 30679. In addition, serum substitutes can be commercially available products. Examples of such commercially available serum substitutes include Knockout TM SerumReplacement (produced by Invitrogen: sometimes hereinafter referred to as KSR), Chemically defined lipid concentrate (chemically defined lipid concentrate, produced by Gibco), and Glutamax TM (produced by Gibco).

[0058] Serum-free media for suspension culture may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, etc.

[0059] To avoid complex preparation, serum-free media supplemented with an appropriate amount (e.g., about 1 - about 20%) of commercially available KSR can be used as serum-free media (e.g., a medium obtained by adding 10% KSR and 450 μM 1-thioglycerol to a 1:1 mixture of F-12 medium and IMDM medium).

[0060] The "serum-containing medium" in the present invention refers to a medium containing unregulated or unpurified serum. The medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-thioglycerol, pyruvate, buffers, inorganic salts, etc.

[0061] The "basement membrane preparation" in the present invention refers to a preparation containing basement membrane components, which have the function of controlling cell morphology, differentiation, growth, movement, functional expression, etc. similar to epithelial cells when target cells capable of forming a basement membrane are placed thereon and cultured. Here, the "basement membrane components" refer to extracellular matrix molecules existing in the form of a thin film between the epithelial cell layer and the interstitial cell layer in animal tissues, etc. The basement membrane preparation can be prepared, for example, by using a solution (such as an alkaline solution) capable of dissolving cell lipids to remove cells capable of forming a basement membrane (the cells adhere to the support via the basement membrane). Examples of preferred basement membrane preparations include commercially available products as basement membrane products (e.g., Matrigel TM (produced by Beckton Dickinson: sometimes hereinafter referred to as Matrigel)), and extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.).

[0062] Matrigel TM is a basement membrane product extracted from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel TMIts main components are type IV collagen, laminin, heparan sulfate proteoglycan, and nestin. In addition to these, it also contains TGF-β, fibroblast growth factor (FGF), tissue plasminogen activator, and growth factors naturally produced by the EHS tumor. Matrigel TM 's "product with reduced growth factors" has a lower growth factor concentration than ordinary Matrigel TM The standard EGF concentration is <0.5 ng / ml, the standard NGF concentration is <0.2 ng / ml, the standard PDGF concentration is <5 pg / ml, the standard IGF-1 concentration is 5 ng / ml, and the standard TGF-β concentration is 1.7 ng / ml.

[0063] In the present invention, "a medium containing substance X" means a medium supplemented with exogenous substance X or a medium containing exogenous substance X, and "a medium without substance X" means a medium not supplemented with exogenous substance X or a medium not containing exogenous substance X. Here, "exogenous substance X" means substance X that is exogenous to the cells or tissues to be cultured in the medium, and endogenous substance X produced by the cells or tissues is not included therein.

[0064] For example, "a medium containing a substance that acts on the BMP signal transduction pathway" is a medium supplemented with an exogenous substance that acts on the BMP signal transduction pathway or a medium containing an exogenous substance that acts on the BMP signal transduction pathway. "A medium without a substance that acts on the Sonic hedgehog signal transduction pathway" is a medium not supplemented with an exogenous substance that acts on the Sonic hedgehog signal transduction pathway or a medium not containing an exogenous substance that acts on the Sonic hedgehog signal transduction pathway.

[0065] In the present invention, "primate" means a mammal belonging to the order Primates. Examples of primates include prosimians (Strepsirrhini) such as lemurs, lorises, and Tsubai, and anthropoids (Haplorhini) such as monkeys, apes, and humans.

[0066] In the present invention, "stem cell" refers to a cell that can still maintain the same differentiation ability after cell division, and such cells can contribute to tissue regeneration when their tissues are damaged. Here, the stem cell can be an embryonic stem cell (sometimes hereinafter referred to as an ES cell) or a tissue stem cell (also called a stem cell of tissue, tissue-specific stem cell, or somatic stem cell), or an induced pluripotent stem cell (iPS cell: induced pluripotent stem cell). As understood from the fact that tissue cells derived from the above stem cells can regenerate tissues, it is known that stem cells can differentiate into normal cells close to the cells in vivo.

[0067] Stem cells can be obtained from designated institutions or commercially available products can also be purchased. For example, human embryonic stem cells, KhES-1, KhES-2, and KhES-3, can be obtained from the Institute for Frontier Medical Sciences of Kyoto University. EB5 cells can be obtained from RIKEN, and the D3 cell line can be obtained from ATCC, each of which is a mouse embryonic stem cell.

[0068] Stem cells can be cultured and maintained by methods known per se. For example, human stem cells can be maintained by culturing in a medium supplemented with Knockout TM Serum Replacement (Invitrogen). Mouse stem cells can be maintained by adding fetal bovine serum (FCS) and Leukemia Inhibitory Factor (LIF) and culturing without feeder cells.

[0069] In the present invention, "pluripotent stem cells" refer to such stem cells that can be cultured in vitro and have the ability to differentiate into any cell (tissue derived from the three germ layers (ectoderm, mesoderm, endoderm)) (pluripotency) that constitutes a living body (except for the placenta), and embryonic stem cells (ES cells) are included in the pluripotent stem cells. "Pluripotent stem cells" are obtained from fertilized eggs, cloned embryos, regenerative stem cells, and stem cells in tissues. Cells with artificial differentiation pluripotency similar to that of embryonic stem cells (also called induced pluripotent stem cells) after introducing several genes into somatic cells are also included in the pluripotent stem cells. Pluripotent stem cells can be prepared by methods known per se. Examples of the preparation methods include the methods described in Cell, 2007, 131(5) pp. 861-872 and Cell, 2006, 126(4) pp. 663-676.

[0070] In the present invention, "embryonic stem cells (ES cells)" refer to cells having self-renewal ability and multipotency (particularly, "pluripotency"), which are pluripotent stem cells derived from early embryos. Embryonic stem cells were first created in 1981 and have also been used for the production of knockout mice since 1989. In 1998, human embryonic stem cells were created and have also been used in regenerative medicine.

[0071] In the present invention, "induced pluripotent stem cells" refer to cells such as fibroblasts that are induced to become pluripotent by directly reprogramming and differentiating through the expression of several genes such as Oct3 / 4, Sox2, Klf4, and Myc. They were created in mouse cells in 2006 by Yamanaka et al. (Cell. 2006, 126(4), pp. 663-676). In 2007, induced pluripotent stem cells were also established in human fibroblasts, and they have pluripotency similar to that of embryonic stem cells (Cell, 2007, 131(5) pp. 861-872; Science, 2007, 318(5858) pp. 1917-1920; Nat. Biotechnol., 2008, 26(1) pp. 101-106).

[0072] Genetically modified pluripotent stem cells can be prepared, for example, using homologous recombination techniques. Examples of genes on the chromosome to be modified include cell marker genes, histocompatibility antigen genes, genes related to diseases caused by disorders of nervous system cells, etc. The target gene on the chromosome can be modified by the methods described in Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994); Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Bio Manual series 8, gene targeting, Production of mutant mouse using ES cells, YODOSHA Co., LTD. (1995), etc.

[0073] Specifically, for example, genomic genes of the target gene to be modified (such as cell marker genes, histocompatibility antigen genes, disease-related genes, etc.) are isolated, and a target vector for homologous recombination of the target gene is prepared using the isolated genomic genes. The prepared target vector is introduced into stem cells, and cells showing homologous recombination between the target gene and the target vector are selected, whereby stem cells having a modified gene on the chromosome can be prepared.

[0074] As a method for isolating genomic genes of a target gene, known methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989), Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997), etc. can be mentioned. In addition, genomic genes of the target gene can be isolated using a genomic DNA library screening system (manufactured by Genome Systems), Universal GenomeWalker Kits (manufactured by CLONTECH), etc.

[0075] According to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Bio Manual series 8, gene targeting, Production of mutant mouse using ES cells, YODOSHA Co., LTD. (1995), etc., a target vector for homologous recombination of the target gene can be prepared, and homologous recombinants can be effectively selected. The target vector can be of any replacement type and insertion type, and the selection method can be positive selection, promoter selection, negative selection, polyA selection, etc.

[0076] As a method for selecting a target homologous recombinant from the selected cell line, DNA hybridization methods, PCR methods, etc. for genomic DNA can be mentioned.

[0077] The "aggregates" in the present invention refer to cell clumps that are dispersed in the culture medium but aggregate to form aggregates. The "aggregates" in the present invention include aggregates formed from cells dispersed at the start of suspension culture and aggregates that have already formed at the start of suspension culture.

[0078] When cells aggregate to form cell aggregates and the aggregates are subjected to suspension culture, "forming aggregates" means "rapidly aggregating a certain number of dispersed cells" to form homogeneous cell aggregates.

[0079] In the present invention, it is preferable to rapidly aggregate pluripotent stem cells to allow the formation of aggregates of pluripotent stem cells. By forming aggregates of pluripotent stem cells in this manner, an epithelial-like structure with good regenerative ability can be formed in the cells induced to differentiate from the formed aggregates.

[0080] Examples of experimental operations for forming aggregates include methods involving holding cells in a small space using a plate with small holes (96-well plate), micropores, etc., and methods involving aggregating cells by short-time centrifugation using small centrifuge tubes.

[0081] Whether aggregates of pluripotent stem cells have been formed and whether an epithelial-like structure has been formed in the cells forming the aggregates can be determined based on the size and number of cells of the aggregates, macroscopic morphology, microscopic morphology and its uniformity analyzed by tissue staining, expression and its uniformity of differentiation and undifferentiated markers, control and synchrony of expression of differentiation markers, reproducibility of differentiation efficiency between aggregates, etc.

[0082] In the present invention, "tissue" refers to a cell group structure having a configuration in which more than one type of cell different in shape and property is spatially arranged in a specific pattern.

[0083] In the present invention, "retinal tissue" means a retinal tissue in which at least two or more types of cells such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, their progenitor cells or their retinal progenitor cells constituting each retinal layer in a living retina are spatially arranged in multiple layers. For each type of cell, which cell constitutes which retinal layer can be confirmed by known methods, for example, by the presence or expression level of cell markers, etc.

[0084] "Retinal layer" in the present invention means each layer constituting the retina. Specific examples thereof include the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer and inner limiting membrane.

[0085] "Retinal layer-specific nerve cell" in the present invention means a nerve cell that constitutes a retinal layer and is specific to the retinal layer. Examples of retinal layer-specific nerve cells include bipolar cells, ganglion cells, amacrine cells, horizontal cells, photoreceptors, pigment epithelial cells, rod cells and cone cells.

[0086] The "retinal pigment epithelial cells" in the present invention refer to epithelial cells existing on the outer side of the neural retina tissue in the biological retina. Whether a cell is a retinal pigment epithelial cell can be confirmed by those skilled in the art based on, for example, the expression of cell markers (such as RPE65 (pigment epithelial cell), Mitf (pigment epithelial cell), etc.), the presence of melanosomes, the characteristic cell form of polygons, etc.

[0087] The "retinal progenitor cells" in the present invention refer to progenitor cells that can differentiate into any mature retinal cells (photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells).

[0088] Photoreceptor progenitor cells, horizontal progenitor cells, bipolar progenitor cells, amacrine progenitor cells, retinal ganglion progenitor cells, and retinal pigment epithelial progenitor cells are progenitor cells that are determined to differentiate into photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells, respectively.

[0089] Examples of retinal cell markers include Rax and PAX6 expressed in retinal progenitor cells, Nkx2.1 expressed in progenitor cells of hypothalamic neurons but not in retinal progenitor cells, Sox1 expressed in hypothalamic neuroepithelium but not in the retina, Crx expressed in progenitor cells of photoreceptors, etc. Examples of neural cell markers specific to retinal layers include Chx10 and L7 expressed in bipolar cells, Tuj1 and Brn3 expressed in ganglion cells, calretinin expressed in amacrine cells, calbindin expressed in horizontal cells, rhodopsin and recoverin expressed in photoreceptors, RPE65 and Mitf expressed in pigment epithelial cells, Nrl expressed in rod cells, Rxr-γ expressed in cone cells, etc.

[0090] Preparation method 1 of the present invention is a method for preparing retinal pigment epithelial cells, which comprises the following steps (1), (2), and (3):

[0091] (1) In the first step, pluripotent stem cells are cultured in suspension in a serum-free medium to form aggregates of pluripotent stem cells.

[0092] (2) In the second step, the aggregates formed in step (1) are cultured in suspension in a serum-free medium or a serum-containing medium that does not contain substances acting on the Sonic hedgehog signal transduction pathway but contains substances acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells, and

[0093] (3) In the third step, the aggregates obtained in step (2) are subjected to suspension culture in a serum-free medium or a serum-containing medium that does not contain substances acting on the Sonic hedgehog signaling pathway and substances acting on the BMP signaling pathway but contains substances acting on the Wnt signaling pathway, thereby obtaining aggregates containing retinal pigment epithelial cells.

[0094] Describe step (1) for suspension culture of pluripotent stem cells in a serum-free medium to form aggregates of pluripotent stem cells.

[0095] The serum-free medium used for step (1) is not particularly limited as long as it is as described above. For example, a serum-free medium that does not supplement any substances acting on the BMP signaling pathway and substances that inhibit the Wnt signaling pathway can be used. To avoid a complex preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of serum substitute such as commercially available KSR (for example, a medium obtained by adding 10% KSR, 450 μM 1-mercaptoethanol, and 1 x Chemically Defined Lipid Concentrate to a 1:1 mixture of IMDM and F-12). In the case of human ES cells, for example, the amount of KSR added to the serum-free medium is generally about 1% to about 20%, preferably about 2% to about 20%.

[0096] The culture conditions in step (1), such as the culture temperature and CO2 concentration, can be appropriately determined. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0097] The concentration of pluripotent stem cells in step (1) can be determined as needed to form aggregates of pluripotent stem cells more uniformly and effectively. For example, when suspension culturing human ES cells using a 96-well microplate, a liquid prepared to contain about 1×10 3 to about 5×10 5 cells, preferably about 3×10 3 to about 5×10 4 cells, more preferably about 5×10 3 to about 3×10 4 cells, and most preferably about 1.2×10 4 cells is added to the wells, and the plate is left stationary to form aggregates.

[0098] The time required for suspension culture to form aggregates can be appropriately determined according to the pluripotent stem cells used to allow uniform aggregation of the cells. To form uniform aggregates, ideally, the time should be as short as possible. For example, in the case of human ES cells, aggregates are preferably formed within about 24 hr, more preferably within about 12 hr. The time required for aggregate formation can be appropriately adjusted by controlling the tools for aggregating cells, concentration conditions, etc.

[0099] Whether aggregates of pluripotent stem cells have been formed can be determined based on the size and cell number of the aggregates, macroscopic morphology, microscopic morphology and its uniformity analyzed by tissue staining, expression and its uniformity of differentiation and undifferentiated markers, control and synchrony of differentiation marker expression, reproducibility of differentiation efficiency between aggregates, etc.

[0100] Describe step (2), which includes suspension culture of the aggregates formed in step (1) in a serum-free medium or a serum-containing medium, each of which does not contain substances acting on the Sonic hedgehog signal transduction pathway but contains substances acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells.

[0101] As the medium for step (2), for example, a serum-free medium or a serum-containing medium that is not supplemented with substances acting on the Sonic hedgehog signal transduction pathway but is supplemented with substances acting on the BMP signal transduction pathway is used, and it is unnecessary to add a basement membrane preparation.

[0102] The serum-free medium or serum-containing medium used as such a medium is not particularly limited as long as it is as described above. To avoid a complicated preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of a serum substitute such as commercially available KSR (for example, a medium obtained by adding 10% KSR, 450 μM 1-thioglycerol and 1 x chemically defined lipid concentrate to a 1:1 mixture of IMDM and F-12). In the case of human ES cells, for example, the amount of KSR added to the serum-free medium is generally about 1% to about 20%, preferably about 2% to about 20%.

[0103] As the serum-free medium for step (2), the serum-free medium used in step (1) can be used as it is as long as it does not contain substances acting on the Sonic hedgehog signal transduction pathway, or it can be replaced with a fresh serum-free medium. When directly using the serum-free medium used in step (1) for step (2), it is only necessary to add substances acting on the BMP signal transduction pathway to the medium.

[0104] A substance that acts on the Sonic hedgehog (sometimes hereinafter referred to as Shh) signaling pathway is a substance that can enhance the signal transduction mediated by Shh. Examples of substances that act on the Shh signaling pathway include proteins belonging to the Hedgehog family (e.g., Shh), Shh receptors, Shh receptor agonists, Purmorphamine, SAG, etc.

[0105] A culture medium "free of substances that act on the Sonic hedgehog signaling pathway" also includes a culture medium substantially free of substances that act on the Sonic hedgehog signaling pathway, such as a culture medium in which the concentration of substances that act on the Sonic hedgehog signaling pathway will not have an adverse effect on the selective differentiation into retinal progenitor cells and retinal tissue.

[0106] A culture medium "not supplemented with substances that act on the Sonic hedgehog signaling pathway" also includes a culture medium substantially not supplemented with substances that act on the Sonic hedgehog signaling pathway, such as a culture medium in which the concentration of substances that act on the Sonic hedgehog signaling pathway will not have an adverse effect on the selective differentiation into retinal progenitor cells and retinal tissue.

[0107] A substance that acts on the BMP signaling pathway is a substance that can enhance the signal transduction pathway mediated by BMP. Examples of substances that act on the BMP signaling pathway include BMP proteins such as BMP2, BMP4 or BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, BMP partial peptides, etc. BMP2 protein, BMP4 protein and BMP7 protein can be obtained from, for example, R&D Systems, while GDF7 protein can be obtained from, for example, Wako Pure Chemical Industries, Ltd.

[0108] The concentration of the substance that acts on the BMP signaling pathway only needs to be a concentration that can induce the differentiation of cells forming pluripotent stem cell aggregates into retinal cells. In the case of BMP4, for example, it is added to the culture medium at a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1.5 nM.

[0109] The substance acting on the BMP signal transduction pathway only needs to be added about 24 hours after the start of the suspension culture in step (1), and can be added to the culture medium within several days (for example, within 15 days) since the start of the suspension culture. Preferably, the substance acting on the BMP signal transduction pathway is added to the culture medium from the 1st day to the 15th day, more preferably from the 1st day to the 9th day, and most preferably on the 3rd day since the start of the suspension culture.

[0110] After the substance acting on the BMP signal transduction pathway is added to the culture medium and the induction of differentiation of the cells forming the pluripotent stem cell aggregates into retinal cells starts, it is not necessary to add the substance acting on the BMP signal transduction pathway to the culture medium, and the culture medium can be replaced with a serum-free culture medium or a serum-containing culture medium, each of which does not contain the substance acting on the BMP signal transduction pathway, thereby reducing the cost of the culture medium. It is possible to determine the cells in which the induction of differentiation into retinal cells has started, for example, by detecting the expression of the Rax gene in the cells. It is also possible to confirm the time when the induction of differentiation into retinal cells starts by culturing the aggregates formed from pluripotent stem cells in which a fluorescent reporter protein gene such as GFP has been knocked into the Rax locus in step (1) in the presence of the substance acting on the BMP signal transduction pathway at a concentration required for the induction of differentiation into retinal cells, and detecting the fluorescence emitted by the expressed fluorescent reporter protein. One embodiment of step (2) is a step that includes culturing the aggregates formed in step (1) in a serum-free culture medium or a serum-containing culture medium until cells expressing the Rax gene appear, each of which contains the substance acting on the BMP signal transduction pathway at a concentration required for the induction of differentiation into retinal cells and does not contain the substance acting on the Sonic hedgehog signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells.

[0111] The culture conditions in step (2) such as the culture temperature and CO2 concentration can be appropriately determined. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0112] The aggregates containing retinal progenitor cells that have been obtained can be confirmed, for example, by detecting the presence of cells expressing the retinal progenitor cell markers Rax or PAX6 in the aggregates.

[0113] Describe step (3). Step (3) includes suspension-culturing the aggregates obtained in step (2) in a serum-free medium or a serum-containing medium that does not contain substances acting on the Sonichedgehog signaling pathway and substances acting on the BMP signaling pathway but contains substances acting on the Wnt signaling pathway, thereby obtaining aggregates containing retinal pigment epithelial cells.

[0114] The medium used in step (3) is, for example, a serum-free medium or a serum-containing medium that is not supplemented with any of the substances acting on the Sonic hedgehog signaling pathway and the substances acting on the BMP signaling pathway but is supplemented with a substance acting on the Wnt signaling pathway.

[0115] The medium of "not containing substances acting on the Sonic hedgehog signaling pathway and substances acting on the BMP signaling pathway" also includes a medium that is substantially free of substances acting on the Sonic hedgehog signaling pathway and substances acting on the BMP signaling pathway. For example, a medium in which the concentrations of the substances acting on the Sonic hedgehog signaling pathway and the substances acting on the BMP signaling pathway do not have an adverse effect on the selective differentiation into retinal tissue.

[0116] The medium of "not supplemented with any of the substances acting on the Sonic hedgehog signaling pathway and the substances acting on the BMP signaling pathway" also includes a medium that is substantially not supplemented with substances acting on the Sonic hedgehog signaling pathway and substances acting on the BMP signaling pathway. For example, a medium in which the concentrations of the substances acting on the Sonic hedgehog signaling pathway and the substances acting on the BMP signaling pathway do not have an adverse effect on the selective differentiation into retinal tissue.

[0117] Examples of the serum-free medium or the serum-containing medium used as such a medium include the media mentioned above. Preferred is such a serum-free medium, which is a basal medium such as DMEM-F12 medium supplemented with a mixture of neurotrophic factors such as N2 supplement (Invitrogen) and B27 supplement (Invitrogen) without supplementing KSR.

[0118] A substance acting on the Wnt signaling pathway is a substance that can enhance Wnt-mediated signal transduction. Examples of substances acting on the Wnt signaling pathway include proteins belonging to the Wnt family (e.g., Wnt1, Wnt3a, Wnt7a), Wnt receptor agonists, GSK3β inhibitors (e.g., 6-Bromoindirubin-3’-oxime (BIO), CHIR99021, Kenpaullone), etc.

[0119] The concentration of the substance acting on the Wnt signaling pathway can be arbitrary as long as it can induce the differentiation of cells forming pluripotent stem cell aggregates into retinal cells. For example, in the case of CHIR99021, it is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 3 μM.

[0120] When using human ES cells, for example, the substance acting on the Wnt signaling pathway is added not earlier than the 12th day from the start of suspension culture in step (1), preferably on the 18th day.

[0121] In step (3), it is more preferably cultured in a serum-free medium or a serum-containing medium that does not contain substances acting on the Sonic hedgehog signal transduction pathway and substances acting on the BMP signal transduction pathway, but contains substances acting on the Wnt signaling pathway and substances inhibiting the FGF signal pathway.

[0122] A substance inhibiting the FGF signal pathway is a substance that can inhibit FGF-mediated signals. Examples of substances inhibiting the FGF signal pathway include FGF receptors, FGF receptor inhibitors (e.g., SU-5402, AZD4547, BGJ398), MAP kinase cascade inhibitory substances (e.g., MEK inhibitors, MAPK inhibitors, ERK inhibitors), PI3 kinase inhibitors, Akt inhibitors, etc.

[0123] The concentration of the substance inhibiting the FGF signal pathway used in step (3) can be arbitrary as long as it can induce the differentiation of cells forming pluripotent stem cell aggregates into retinal cells. For example, in the case of SU-5402, it is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 10 μM.

[0124] Since the retinal pigment epithelial cells thus generated are present on the surface of the aggregates, they can be confirmed by microscopic observation or the like. It is also possible to obtain highly pure retinal pigment epithelial cells by dispersing the aggregates containing retinal pigment epithelial cells (for example, trypsin / EDTA treatment) and selecting the obtained cells using FACS. It is also possible to physically remove the retinal pigment epithelial cells from the aggregates using forceps or the like and culture the cells. The retinal pigment epithelial cells thus dispersed or removed can be cultured under adhesion conditions. It is also possible to disrupt the aggregates containing retinal pigment epithelial cells by pipetting using a micropipette or the like and further culture the obtained cells under adhesion conditions. In the case of adhesion culture, it is preferable to use a cell adhesion culture container, for example, a culture container treated with an extracellular matrix or the like (for example, poly-D-lysine, laminin, fibronectin). The culture conditions for adhesion culture such as culture temperature, CO2 concentration, and O2 concentration can be determined as appropriate. In this case, the cells can be cultured in the presence of serum, known growth factors, and growth-promoting additives and chemicals. Examples of known growth factors include EGF, FGF, etc. Examples of growth-promoting additives include N2 supplement (Invitrogen), B27 supplement (Invitrogen), etc.

[0125] Preparation method 2 of the present invention is a method for preparing a retinal pigment epithelial cell sheet, which comprises the following steps (1), (2), (3), and (4):

[0126] (1) First step, suspension-culturing pluripotent stem cells in a serum-free medium to form aggregates of pluripotent stem cells,

[0127] (2) Second step, suspension-culturing the aggregates formed in step (1) in a serum-free medium or a serum-containing medium each containing a substance acting on the BMP signal transduction pathway and not containing a substance acting on the Sonic hedgehog signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells,

[0128] (3) Third step, suspension-culturing the aggregates obtained in step (2) in a serum-free medium or a serum-containing medium each containing a substance acting on the Wnt signal pathway and not containing a substance acting on the Sonic hedgehog signal transduction pathway and a substance acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells, and

[0129] (4) Fourth step, dispersing the aggregates obtained in step (3) and performing adhesion culture on the obtained cells.

[0130] Steps (1), (2), and (3) of Preparation Method 2 of the present invention can be carried out similarly to steps (1), (2), and (3) of Preparation Method 1 of the present invention.

[0131] Describe step (4), which is used to disperse the aggregates obtained in step (3) and perform adherent culture on the resulting cells.

[0132] Step (4) is carried out within 60 days, preferably within 30 days, and more preferably within 3 days after the start of step (3).

[0133] The cells derived from aggregates dispersed in step (4) are inoculated into an adherent culture container to a concentration capable of effectively forming a uniform retinal pigment epithelial cell sheet. For example, when adherent culturing cells derived from aggregates using a 65 mm Boyden chamber, a solution containing about 1×10 3 to about 1×10 7 cells, preferably about 3×10 3 to about 5×10 6 cells, more preferably about 5×10 3 to about 1×10 6 cells, and more preferably about 2×10 5 cells / well is added to the wells, and the plate is left stationary to allow the cells derived from aggregates to adhere and culture.

[0134] Examples of the serum-free medium or serum-containing medium for adherent culture in step (4) include the above media. To avoid a complex configuration process, for example, it is preferred to use a serum-free medium supplemented with an appropriate amount of a serum substitute such as commercially available KSR (for example, a medium containing a 1:1 mixture of DMEM / F-12 and Neurobasal, as well as 1 / 2x N2 supplement, 1 / 2x B27 supplement, and 100 μM 2-mercaptoethanol). In the case of cells derived from human ES cells, for example, the concentration of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%.

[0135] In step (4), the cells are preferably cultured in a serum-free medium or serum-containing medium containing a ROCK inhibitor.

[0136] The ROCK inhibitor is a substance capable of inhibiting the signal mediated by Rho kinase. Examples of the ROCK inhibitor include Y-27632 and Fasudil.

[0137] The concentration of the ROCK inhibitor used in step (4) can be arbitrary, as long as it can effectively form a uniform retinal pigment epithelial cell sheet. For example, in the case of Y-27632, it is added at a concentration of about 0.01 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 20 μM.

[0138] In step (4), it is more preferable to culture the cells in a serum-free medium or a serum-containing medium further containing one or more substances selected from the group consisting of substances acting on the Wnt signaling pathway, substances inhibiting the FGF signaling pathway, substances acting on the activin signaling pathway, and substances acting on the BMP signal transduction pathway.

[0139] Examples of substances acting on the Wnt signaling pathway include proteins belonging to the Wnt family (e.g., Wnt1, Wnt3a, Wnt7a), Wnt receptor agonists, GSK3β inhibitors (e.g., 6-Bromoindirubin-3’-oxime (BIO), CHIR99021, Kenpaullone), etc.

[0140] The concentration of the substance acting on the Wnt signaling pathway used in step (4) can be arbitrary, as long as it can effectively form a uniform retinal pigment epithelial cell sheet. For example, in the case of CHIR99021, it is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 3 μM.

[0141] For example, within 18 days starting from step (4), it is preferable to add the substance acting on the Wnt signaling pathway on day 6.

[0142] Examples of substances inhibiting the FGF signaling pathway include FGF receptors, FGF receptor inhibitors (e.g., SU-5402, AZD4547, BGJ398), MAP kinase cascade inhibitors (e.g., MEK inhibitors, MAPK inhibitors, ERK inhibitors), PI3 kinase inhibitors, and Akt inhibitors.

[0143] The concentration of the substance inhibiting the FGF signaling pathway used in step (4) can be arbitrary, as long as it can effectively form a uniform retinal pigment epithelial cell sheet. For example, in the case of SU-5402, it is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 10 μM.

[0144] For example, within 18 days starting from step (4), it is preferable to add the substance inhibiting the FGF signaling pathway on day 6.

[0145] A substance acting on the activin signaling pathway is a substance that can enhance the signal mediated by activin. Examples of substances acting on the activin signaling pathway include proteins belonging to the activin family (e.g., activin A, activin B, activin C, activin AB, etc.), activin receptors, and activin receptor agonists.

[0146] The concentration of the substance acting on the activin signaling pathway used in step (4) can be arbitrary, as long as it can effectively form a uniform retinal pigment epithelial cell sheet. For example, in the case of recombinant human / mouse / rat activin A (R&D Systems, Inc. #338-AC), it is added at a concentration of about 1 ng / ml to about 10 μg / ml, preferably about 10 ng / ml to about 1 μg / ml, and more preferably about 100 ng / ml.

[0147] For example, within 18 days starting from step (4), preferably on day 6, the substance acting on the activin signaling pathway is added.

[0148] Examples of substances acting on the BMP signal transduction pathway include BMP proteins such as BMP2, BMP4, or BMP7 and GDF proteins such as GDF7, anti-BMP receptor antibodies, BMP partial peptides, etc.

[0149] The concentration of the substance acting on the BMP signal transduction pathway used in step (4) can be arbitrary, as long as it can effectively form a uniform retinal pigment epithelial cell sheet. For example, in the case of BMP4, it is added at a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, and more preferably about 1.5 nM.

[0150] For example, within 18 days starting from step (4), preferably on day 6, the substance acting on the BMP signal transduction pathway is added.

[0151] In step (4), for example, the cells are cultured in a serum-free medium or a serum-containing medium further containing a substance selected from the group consisting of a substance acting on the Wnt signaling pathway, a substance inhibiting the FGF signaling pathway, a substance acting on the activin signaling pathway, and a substance acting on the BMP signal transduction pathway, or in a serum-free medium or a serum-containing medium further containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway.

[0152] In step (4), preferably, adhesion culture is performed on a culture container material treated with a culture substrate surface.

[0153] As a culture matrix for processing the culture vessel material in step (4), a cell culture matrix that allows adhesion of cells derived from aggregates and formation of a retinal pigment epithelial cell sheet can be mentioned. To avoid complicated preparation and prevent contamination with components derived from heterologous species, it is preferable to use a synthetic culture matrix, a basement membrane component derived from humans, a recombinant human basement membrane protein, etc. Specific examples of such a culture matrix include a synthetic culture matrix such as Synthemax TM (Corning incorporated), etc., a basement membrane component derived from humans such as Cellstart TM (Invitrogen), human extracellular matrix (BD), etc., and recombinant human laminins such as human recombinant laminin 111 (Biolamina), human recombinant laminin 511 (Biolamina), human recombinant laminin 521 (Biolamina), human recombinant laminin 411 (Biolamina), human recombinant laminin 421 (Biolamina), human recombinant laminin 332 (Biolamina), and human recombinant laminin 211 (Biolamina). As used herein, laminin 111 is a laminin composed of an α1 chain, a β1 chain, and a γ1 chain, laminin 511 is a laminin composed of an α5 chain, a β1 chain, and a γ1 chain, laminin 521 is a laminin composed of an α5 chain, a β2 chain, and a γ1 chain, laminin 411 is a laminin composed of an α4 chain, a β1 chain, and a γ1 chain, laminin 421 is a laminin composed of an α4 chain, a β2 chain, and a γ1 chain, laminin 332 is a laminin composed of an α3 chain, a β3 chain, and a γ2 chain, and laminin 211 is a laminin composed of an α2 chain, a β1 chain, and a γ1 chain. In addition, a portion called the E8 fragment, which is composed of the C-terminal regions of each of the α, β, and γ chains of laminin, is also known to exhibit binding activity to integrins expressed by cells (J. Biol. Chem., 284, 7820-7831 (2009)). Therefore, it is expected to be used similarly to the above-mentioned adhesins.

[0154] The retinal pigment epithelial cells or retinal pigment epithelial cell sheets produced by Preparation Method 1 or 2 of the present invention have characteristics extremely similar to those of in vivo retinal pigment epithelial cells. Therefore, they can also be used for screening therapeutic drugs for diseases caused by disorders of retinal cells, or as transplantation materials for cell therapy, materials for studying diseases, or drug discovery materials for therapeutic drugs for cell damage caused by other etiologies. In addition, they can be used for studying toxicities such as phototoxicity, toxicities of chemical substances, etc., and toxicity tests in pharmacodynamic evaluation.

[0155] Examples of diseases caused by disorders of retinal cells include organic mercury poisoning, chloroquine retinopathy, retinitis pigmentosa, age-related macular degeneration, glaucoma, diabetic retinopathy, neonatal retinopathy, etc.

[0156] The retinal pigment epithelial cells or retinal pigment epithelial cell sheets prepared by Preparation Method 1 or 2 of the present invention can be used as retinal pigment epithelial cells for transplantation, and the cells are used to supplement damaged cells or the diseased tissue itself in a state of cell damage (for example, for transplantation surgery, etc.). Examples

[0157] The present invention will be described in more detail with reference to the following examples, which are not considered to be restrictive.

[0158] Example 1: Preparation of retinal pigment epithelial cells using human ES cells

[0159] Human ES cells (derived from KhES-1) were cultured according to the methods described in "Ueno, M. et al. PNAS 2006, 103(25), 9554-9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681-686". As the culture medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the single-dispersed ES cells were seeded at 1.2×10 per well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) 4Cells were suspended in 100 μl of serum-free medium to allow rapid formation of aggregates and cultured at 37°C in 5% CO2. As the serum-free medium used for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, and 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM, and suspension culture was continued. Half of the medium in the wells was exchanged every 3 days with the above medium without adding substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement) and cultured until the 21st day from the start of suspension culture, and microscopic observation was performed.

[0160] On the 18th day from the start of suspension culture, the formation of neuroepithelium was confirmed ( Figure 1 A). On the 21st day from the start of suspension culture, it was confirmed that the cells differentiated into retinal pigment epithelial cells with thin epithelium ( Figure 1 B).

[0161] Example 2: Preparation of retinal pigment epithelial cells using human ES cells

[0162] Human ES cells (derived from KhES-1) were cultured according to the methods described in "Ueno, M. et al. PNAS 2006, 103(25), 9554-9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681-686". As the medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, and 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the single-dispersed ES cells were seeded at 1.2×10 4Cells were suspended in 100 μl of serum-free medium to allow rapid formation of aggregates, and cultured at 37°C, 5% CO2. As the serum-free medium used for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM, and suspension culture was continued. Every 3 days, half of the medium in the wells was exchanged with the above-mentioned medium without adding substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement), and suspension culture was continued until the 21st day from the start of suspension culture. As a control, the aggregates were cultured similarly even in the absence of CHIR99021. A part of the aggregates cultured in suspension in the aforementioned medium containing CHIR99021 was subjected to suspension culture under the same medium conditions until the 27th day from the start of suspension culture, and another part was subjected to suspension culture from the 21st day to the 27th day from the start of suspension culture at 37°C, 5% CO2 in a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 (a substance acting on the Wnt signal pathway) and 10 μM SU-5402 (a substance inhibiting the FGF signal pathway) and supplemented with 1x N2 supplement).

[0163] When the aggregates were cultured in the absence of a substance acting on the Wnt signal pathway from the 21st day of culture, the retinal pigment epithelium with a thin epithelium reverted to the neuroepithelium with a thick epithelium( Figure 2 A), while in the aggregates that were continuously cultured in the presence of a substance acting on the Wnt signal pathway, dark-colored retinal pigment epithelial cells appeared on the surface of almost all aggregates( Figure 2 B). The aggregates cultured in the presence of a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway showed an increase in the differentiation induction efficiency, and formed aggregates with retinal pigment epithelial cells on the surface of almost all aggregates( Figure 2 C).

[0164] Example 3: Preparation of retinal pigment epithelial cells using human ES cells

[0165] Human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006, 103(25), 9554-9559” and “Watanabe, K. et al. Nat Biotech 2007, 25, 681-686”. As the culture medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the single-dispersed ES cells were suspended at a density of 1.2×10 4 cells per well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) in 100 μl of serum-free medium to allow rapid formation of aggregates, and cultured at 37°C, 5% CO2. As the serum-free medium for this purpose, a serum-free medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM, and suspension culture was continued. Every 3 days, half of the medium in the wells was exchanged with the above medium without supplementation of substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement), and suspension culture was continued until the 21st day from the start of suspension culture. On the 21st day from the start of suspension culture, the aggregates were disrupted by aspirating and ejecting the solution with a micropipette, and the resulting cells were cultured adherently in a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 (a substance acting on the Wnt signal pathway) and supplemented with 1x N2 supplement) at 37°C, 5% CO2.

[0166] By the 27th day from the start of suspension culture, retinal pigment epithelial cells with a characteristic polygonal cell form were observed ( Figure 3 A). Protein expression was confirmed by cell staining methods. As a result, the cells were positive for the transcription factor Mitf (which is a retinal pigment epithelial cell marker) ( Figure 3B) and positive for ZO-1 (which is a tight junction marker) Figure 3 C), thereby confirming that the obtained cells are retinal pigment epithelial cells.

[0167] Example 4: Preparation of retinal pigment epithelial cell sheets using human ES cells

[0168] Human ES cells (derived from KhES-1) were cultured according to the methods described in "Ueno, M. et al. PNAS 2006, 103(25), 9554 - 9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681 - 686". As the medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the single-dispersed ES cells were seeded at 1.2×10 per well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) 4Cells were suspended in 100 μl of serum-free medium to allow rapid formation of aggregates, and cultured at 37°C, 5% CO2. As the serum-free medium used for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-mercaptoethanol, 1x chemically defined lipid concentrate, and 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM, and suspension culture was continued. Every 3 days, half of the medium in the wells was exchanged with the above medium without adding substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement) and cultured until the 21st day from the start of suspension culture. On the 21st day from the start of suspension culture, the aggregates were dispersed in the cell dispersion solution Accutase (ICT), and debris was removed using a 40 μm cell strainer. The resulting cells were suspended in a serum-free medium (200 μl) (which is a 1:1 mixture of DMEM / F-12 medium and Neurobasal medium supplemented with 10% KSR, 1 / 2 N2 supplement, 1 / 2 B27 supplement, and 20 μM Y27632), and 5 seeded at 2x10 TM cells / well in a 65 mm Boyden chamber (Transwell, Corning Incorporated) coated with Synthemax

[0169] After that, a medium (1 ml) with the same composition was added to the lower well, and adhesion culture was carried out at 37°C, 5% CO2. The medium was exchanged every 3 days, and the level of epithelial formation was observed under a microscope on the 40th day from the start of suspension culture. Figure 4 By the 40th day from the start of suspension culture, a uniform retinal pigment epithelial cell sheet Figure 4 A) with characteristic polygonal cell form (

[0170] Example 5: Preparation of retinal pigment epithelial cell sheet using human ES cells

[0171] Human ES cells (derived from KhES-1) were cultured according to the methods described in “Ueno, M. et al. PNAS 2006, 103(25), 9554 - 9559” and “Watanabe, K. et al. Nat Biotech 2007, 25, 681 - 686”. As the culture medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the above-cultured ES cells were suspended at 1.2×10 4 cells per well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) in 100 μl of serum-free medium to allow rapid formation of aggregates and cultured at 37°C, 5% CO2. As the serum-free medium for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a concentration of 1.5 nM, and suspension culture was continued. Every 3 days, half of the medium in the wells was exchanged with the above medium without adding substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement) and cultured until the 21st day from the start of suspension culture. On the 21st day from the start of suspension culture, the aggregates were dispersed in the cell dispersion solution Accutase (ICT), and debris was removed using a 40 μm cell strainer. The resulting cells were suspended in serum-free medium (200 μl) (which is a 1:1 mixture of DMEM / F-12 medium and Neurobasal medium supplemented with 10% KSR, 1 / 2 N2 supplement, 1 / 2 B27 supplement, 20 μM Y27632) and seeded at 2x10 5 cells / well on Synthemax TMIn a 65 mm Boyden chamber (Transwell, Corning Incorporated) after coating treatment, a culture medium (1 ml) with the same composition was also added to the lower well, and adhesion culture was carried out at 37°C and 5% CO2. On the 24th day from the start of suspension culture, that is, on the 3rd day from the start of adhesion culture, 3 μM CHIR99021 was added, 10 μM SU-5402 was added, 3 μM CHIR99021 and 10 μM SU-5402 were added simultaneously, 1 nM recombinant human BMP4 protein was added, or 50 ng / ml recombinant human activin was added, and adhesion culture was continued. Similar culture was carried out under the condition of not adding the aforementioned substances. The culture medium was exchanged every 3 days, and the level of epithelial formation was observed under a microscope on the 40th day from the start of suspension culture.

[0172] Compared with the control without addition ( Figure 5 A), when 3 μM CHIR99021 was added on the 24th day from the start of suspension culture ( Figure 5 B), the cells differentiated into darker retinal pigment epithelial cells. It was observed that by adding 3 μM CHIR99021 and 10 μM SU-5402 simultaneously ( Figure 5 D), the pigment epithelium was darker in color than by adding 3 μM CHIR99021 alone ( Figure 5 B). Compared with the control without addition ( Figure 5 A), when 1 nM recombinant human BMP4 protein was added ( Figure 5 E), or 50 ng / ml recombinant human activin was added ( Figure 5 F), a more uniform epithelium was obtained.

[0173] Example 6: Preparation of retinal pigment epithelial cell sheets using human ES cells

[0174] Growth Factor Reduced Matrigel TM (diluted 30-fold), or Synthemax TM (Corning Incorporated) (diluted 40-fold) was added to the wells of a 65 mm Boyden chamber (Transwell, Corning Incorporated) at 100 μl each. The chamber added with Matrigel was incubated at 4°C for 24 hr, and the chamber added with Synthemax TM was incubated at room temperature for 2 hr, thereby performing the coating treatment of the culture vessel material. On the 21st day from the start of suspension culture, a single cell suspension was prepared from the aggregates by the method described in Example 4, and the suspended cells were at 2x105 Cells were seeded at 1.2×10⁴ cells / well into serum-free medium (200 μl), which was a 1:1 mixture of DMEM / F-12 medium supplemented with 10% KSR, 1 / 2 N2 supplement, 1 / 2 B27 supplement, 20 μM Y27632 and Neurobasal medium. Medium (1 ml) with the same composition was also added to the lower wells, and adherent culture was performed at 37°C and 5% CO₂. Cell adhesion and epithelial formation were observed on the 5th day after seeding into the Boyden chamber.

[0175] In the culture vessel material coated with Synthemax TM the cells derived from aggregates cultured Figure 6 adhered in the same manner as in the culture vessel material coated with Matrigel (which is a basement membrane preparation) Figure 6 A), and a retinal pigment epithelium-like form in which the cells adhered tightly to each other was observed.

[0176] Example 7: Preparation Example of Retinal Pigment Epithelial Cells Using Induced Pluripotent Stem Cells (iPS Cells)

[0177] Human iPS cell line 201B7 (available from the RIKEN BioResource center or iPS Academia Japan Inc.) was cultured according to the methods described in "Ueno, M. et al. PNAS 2006, 103(25), 9554 - 9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681 - 686". As the medium, a medium obtained by adding 20% KSR (Knockout TM Serum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF to DMEM / F12 medium (Sigma) was used. The cultured iPS cells were dispersed into single cells using TrypLE Express (Invitrogen), and seeded at 1.2×10⁴ cells per well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) 4Cells were suspended in 100 μl of serum-free medium to allow rapid formation of aggregates and cultured in suspension at 37°C and 5% CO2. As the serum-free medium used for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-thioglycerol, 1x chemically defined lipid concentrate, and 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM, and suspension culture was continued. Every 3 days, half of the medium in the wells was exchanged with the above medium without adding any substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement) and cultured until the 21st day from the start of suspension culture. On the 21st day from the start of suspension culture, the aggregates were disrupted by injecting and ejecting the solution with a micropipette, and the resulting cells were cultured in adhesion at 37°C and 5% CO2 in a serum-free medium (DMEM / F-12 medium containing 3 μM CHIR99021 (a substance acting on the Wnt signal pathway) and supplemented with 1x N2 supplement). On the 27th day from the start of suspension culture, the cell form was observed under a microscope, and the expression of retinal pigment epithelium marker genes (Mitf, ZO-1) was confirmed by cell staining method.

[0178] In this way, retinal pigment epithelial cells were prepared from human iPS cells.

[0179] Example 8: Preparation of Retinal Pigment Epithelial Cell Sheets Using Induced Pluripotent Stem Cells (iPS Cells) Example

[0180] The human iPS cell line 201B7 (available from the RIKEN BioResource center or iPS Academia Japan Inc.) was cultured according to the methods described in "Ueno, M. et al. PNAS 2006, 103(25), 9554-9559" and "Watanabe, K. et al. Nat Biotech 2007, 25, 681-686". As the medium, a medium obtained by adding 20% KSR (Knockout TMSerum Replacement; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, 5 ng / ml bFGF were added to the medium obtained by adding to DMEM / F12 medium (Sigma). The cultured iPS cells were dispersed into single cells using TrypLE Express (Invitrogen), and 1.2×10 4 cells were suspended in 100 μl of serum-free medium in each well of a non-cell-adhesive 96-well culture plate (sumilon spherical plate, SUMITOMO BAKELITE Co., Ltd.) to allow rapid formation of aggregates, and suspension culture was carried out at 37°C and 5% CO2. As the serum-free medium used for this, a serum-free medium obtained by adding 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, 20 μM Y27632 to a 1:1 mixture of F-12 medium and IMDM medium was used. On the 3rd day from the start of suspension culture, BMP4 was added at a final concentration of 1.5 nM and suspension culture was continued. Every 3 days, half of the medium in the well was exchanged with the above medium without adding substances acting on the BMP signal transduction pathway. On the 18th day from the start of suspension culture, the aggregates were transferred to a serum-free medium (which is DMEM / F-12 medium containing 3 μM CHIR99021 and supplemented with 1x N2 supplement) and cultured until the 21st day from the start of suspension culture. On the 21st day from the start of suspension culture, the aggregates were dispersed in the cell dispersion solution Accutase (ICT), and debris was removed using a 40 μm cell strainer. The resulting cells were suspended in serum-free medium (200 μl) (which is a 1:1 mixture of DMEM / F-12 medium and Neurobasal medium supplemented with 10% KSR, 1 / 2 N2 supplement, 1 / 2 B27 supplement, 20 μM Y27632) and seeded at 2x10 5 cells / well on Synthemax TMIn a 65 mm Boyden chamber (Transwell, Corning Incorporated) after coating treatment, a culture medium (1 ml) having the same composition was also added to the lower well, and adhesion culture was performed at 37°C and 5% CO2. Any one of 3 μM CHIR99021, 10 μM SU-5402, 1 nM recombinant human BMP4 protein, and 50 ng / ml recombinant human activin, or a combination thereof, can be added on the 24th day from the start of suspension culture, that is, on the 3rd day from the start of adhesion culture. The culture medium was exchanged every 3 days, and the level of epithelial formation was observed under a microscope on the 40th day from the start of suspension culture.

[0181] In this way, retinal pigment epithelial cell sheets are prepared from human iPS cells.

[0182] This application is based on Japanese Patent Application No. 2013-232795 (filing date: November 11, 2013), the content of which is incorporated herein in its entirety.

[0183] Industrial Applicability

[0184] According to the production method of the present invention, retinal pigment epithelial cells or retinal pigment epithelial cell sheets can be produced with high efficiency.

Claims

1. A method for preparing retinal pigment epithelial cells, comprising (1) In the first step, culturing pluripotent stem cells in suspension in a serum-free medium to form aggregates of pluripotent stem cells, (2) In the second step, culturing the aggregates formed in step (1) in a serum-free medium or a serum-containing medium each lacking a substance acting on the Sonic hedgehog signal transduction pathway and containing a substance acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells expressing Rax or PAX6, and (3) In the third step, culturing the aggregates obtained in step (2) in a serum-free medium or a serum-containing medium each (a) lacking a substance acting on the Sonic hedgehog signal transduction pathway and a substance acting on the BMP signal transduction pathway and (b) containing a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway, thereby obtaining aggregates containing retinal pigment epithelial cells; wherein the suspension culture is carried out in the absence of a basement membrane preparation.

2. A method for preparing a retinal pigment epithelial cell sheet, comprising (1) In the first step, culturing pluripotent stem cells in suspension in a serum-free medium to form aggregates of pluripotent stem cells, (2) In the second step, culturing the aggregates formed in step (1) in a serum-free medium or a serum-containing medium each lacking a substance acting on the Sonic hedgehog signal transduction pathway and containing a substance acting on the BMP signal transduction pathway, thereby obtaining aggregates containing retinal progenitor cells expressing Rax or PAX6, (3) In the third step, culturing the aggregates obtained in step (2) in a serum-free medium or a serum-containing medium each (a) lacking a substance acting on the Sonic hedgehog signal transduction pathway and a substance acting on the BMP signal transduction pathway and (b) containing a substance acting on the Wnt signal pathway and a substance inhibiting the FGF signal pathway, thereby obtaining aggregates containing retinal pigment epithelial cells, and (4) In the fourth step, dispersing the aggregates obtained in step (3) and performing adherent culture on the resulting cells; wherein the suspension culture is carried out in the absence of a basement membrane preparation.

3. The preparation method according to claim 2, wherein in the step (4), the adherent culture is carried out in the presence of a serum substitute.

4. The preparation method according to claim 2 or 3, wherein in the step (4), the adherent culture is carried out in the presence of a ROCK inhibitor.

5. The preparation method according to claim 2 or 3, wherein in the step (4), the adherent culture is carried out in a serum-free medium or a serum-containing medium further comprising one or more substances selected from the group consisting of: a substance acting on the Wnt signal pathway, a substance inhibiting the FGF signal pathway, a substance acting on the activin signal pathway, and a substance acting on the BMP signal transduction pathway.

6. The production method according to claim 2 or 3, wherein in the step (4), the adhesion culture is carried out on a culture vessel material having a surface treated with a culture substrate.

7. The production method according to claim 6, wherein the culture substrate is a synthetic culture substrate.

8. The production method according to claim 6, wherein the culture substrate is laminin.

9. The production method according to any one of claims 1, 2, and 3, wherein the pluripotent stem cells are primate pluripotent stem cells.

10. The production method according to any one of claims 1, 2, and 3, wherein the pluripotent stem cells are human pluripotent stem cells.

11. The method according to any one of claims 1, 2, and 3, wherein the steps (1) and (2) are carried out in the presence of a serum replacement.

12. The method according to any one of claims 1, 2, and 3, wherein the substance acting on the BMP signal transduction pathway is one or more proteins selected from the group consisting of BMP2, BMP4, BMP7, and GDF7.

13. A method for evaluating the toxicity or efficacy of a test substance, comprising: (1) preparing retinal pigment epithelial cells or a retinal pigment epithelial cell sheet by the method according to any one of claims 1 to 12, (2) contacting the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet with the test substance, and (3) examining the effect of the substance on the cells or the cell sheet.

14. A method for preparing a therapeutic agent for a disease caused by a disorder of the retinal tissue, comprising: (1) preparing retinal pigment epithelial cells or a retinal pigment epithelial cell sheet by the method according to any one of claims 1 to 12, (2) preparing a transplantation material for treating a disease caused by a disorder of the retinal tissue, which contains the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet.

15. The method according to claim 1 or 2, wherein the substance acting on the Wnt signal pathway is selected from the group consisting of proteins belonging to the Wnt family, Wnt receptor agonists, and GSK3β inhibitors.

16. The method according to claim 1 or 2, wherein the substance acting on the Wnt signal pathway is selected from the group consisting of Wnt1, Wnt3a, Wnt7a, 6-bromoindirubin-3'-oxime (BIO), CHIR99021, and Kenpaullone.

17. The method according to claim 1 or 2, wherein the substance inhibiting the FGF signal pathway is selected from the group consisting of FGF receptors and FGF receptor inhibitors.

18. The method according to claim 1 or 2, wherein the substance inhibiting the FGF signal pathway is selected from the group consisting of SU-5402, AZD4547, and BGJ398.

19. The method according to claim 12, wherein the substance acting on the BMP signal transduction pathway in step (2) is added to the culture medium between the 1st day and the 9th day from the start of the suspension culture in step (1).

20. The method according to claim 19, wherein the substance acting on the BMP signal transduction pathway in step (2) is added to the culture medium on the 3rd day starting from the beginning of the suspension culture in step (1), and half of the culture medium is exchanged with the culture medium without supplementing the substance acting on the BMP signal transduction pathway every 3 days.

21. The method according to claim 20, wherein step (3) is carried out on the 18th day starting from the beginning of the suspension culture.

22. The method according to claim 1 or 2, wherein step (2) where the substance acting on the BMP signal transduction pathway is located is the step of culturing until cells expressing the Rax gene appear.

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