Method for producing retinal tissue

Abstract

The present disclosure includes a method for producing retinal tissue, the method comprising inducing differentiation of pluripotent stem cells to retinal tissue in a region A on a culture substrate, the culture substrate including on the surface thereof the region A, which has cell adhesiveness, and a region B, which is adjacent to at least a portion of the region A and has a lower cell adhesiveness than the region A. The present disclosure also includes a retinal tissue produced by the method, and a composition containing the retinal tissue.

Description

Method for producing retinal tissue

[0001] This application claims priority to Japanese Patent Application No. 2021-120128, the entire contents of which are incorporated herein by reference. The present disclosure includes a method for producing retinal tissue, a retinal tissue produced by the method, and a composition comprising the retinal tissue.

[0002] Pluripotent stem cell culture manipulations are being actively studied for in vitro organ formation for transplantation and drug efficacy evaluation. The serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) method differentiates pluripotent stem cells into central nervous system cells by culturing them in suspension for several days in a medium that does not contain components that inhibit neuronal differentiation, such as serum or transcription factors. This method has also been applied to the formation of retinal tissue from pluripotent stem cells. The SFEBq method mimics the process of ontogeny in vitro by utilizing cellular self-organization, enabling the stable and reproducible generation of three-dimensional tissues. However, because the SFEBq method relies on cellular self-organization, the pattern formation process is inconsistent, making it difficult to control the tissue morphology and size. Furthermore, the thickness of the resulting cell aggregates limits drug penetration into the inner cells, making observation of the inner cells difficult. Furthermore, the SFEBq method is not suitable for automation. Therefore, a more stable supply of retinal tissue and a more automated production method are needed.

[0003] The present disclosure aims to provide a method for producing retinal tissue. The present disclosure also aims to provide retinal tissue produced by the method and compositions containing the retinal tissue.

[0004] In one aspect, the present disclosure relates to a method for producing retinal tissue, comprising inducing differentiation of pluripotent stem cells into retinal tissue in region A on a culture substrate having, on its surface, a region A that has cell adhesive properties and a region B that is adjacent to at least a portion of region A and has lower cell adhesive properties than region A.

[0005] In a further aspect, the present disclosure relates to retinal tissue produced by the method.

[0006] In a further aspect, the present disclosure relates to a composition comprising said retinal tissue.

[0007] The present disclosure provides methods for producing retinal tissue, retinal tissue produced by the methods, and compositions comprising the retinal tissue.

[0008] Figure 1 shows the experimental scheme for retinal differentiation induction. Figure 2 shows gene ontology (GO) analysis of patterned cultured cells before BMP4 addition (Day 8). Figure 3 shows GO analysis of unpatterned cultured cells before BMP4 addition (Day 8). Figure 4 shows the time course of GFP (Rx::venus) expression in cells induced for retinal differentiation by patterned culture (top) and cells induced for retinal differentiation without patterned culture (bottom). Figure 5 shows Chx10 expression (top) and GFP expression (bottom) at Day 18 in patterned cultured cells (Patterning) and unpatterned cultured cells (All). The results are shown for cells with BMP4 addition (BMP+) and cells without BMP4 addition (BMP-), respectively. Figure 6 shows GFP (Rx::venus) and Chx10 expression at Day 18 in cells induced for retinal differentiation by patterned culture. Figure 7 shows the expression of GFP (Rx::venus), Recoverin, and Chx10 on Day 58 in cells induced to differentiate into retinas by patterning culture. Figure 8 shows the expression of GFP (Rx::venus) and FOXG1 on Day 18 in cells induced to differentiate into retinas by patterning culture without BMP4. Figure 9 shows the differentiation induction efficiency of cells induced to differentiate into retinas by patterning culture. Figure 10 shows the expression of GFP on Day 20 in cells induced to differentiate into retinas by patterning culture at various sizes. Figure 11 shows the expression of Crx in tissue on Day 40. Figure 12 shows the induction of retinal differentiation by patterning culture using KthES11 cells. The expression of Crx, Chx10, and Tuj1 on Day 50. Figure 13 shows the induction of retinal differentiation by patterning culture using 201B7 cells (top) and 253G1 cells (bottom). The expression of Crx and Chx10 on Day 34. Figure 14 shows GFP expression on Day 11 with (top) and without (bottom) LDN-193189 addition. Figure 15 shows Chx10 expression (top) and GFP expression (bottom) in patterned and unpatterned cells (Patterning) with and without LDN-193189 addition. Results from Day 18 are shown.Figure 16 shows GFP and Chx10 expression in patterned cultured cells (Patterning) and non-patterned cultured cells (All) with and without LDN-193189. The scale bar represents 1 mm. Results are shown on Day 18. Figure 17 shows the time course of retinal differentiation induction by patterned culture using MPC polymer.

[0009] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0010] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. A range of numerical values ​​includes all values ​​between and at the endpoints. "About" in reference to a range applies to both endpoints of the range. For example, "about 20-30" is intended to include "18%-33%."

[0011] The method for producing retinal tissue of the present disclosure includes inducing differentiation of pluripotent stem cells into retinal tissue in region A on a culture substrate having, on its surface, a region A that has cell adhesive properties and a region B that is adjacent to at least a portion of region A and has lower cell adhesive properties than region A.

[0012] Region A is a region where pluripotent stem cells can be maintained in adherent culture. Region B is a region with lower cell adhesiveness than region A to the extent that, when pluripotent stem cells are seeded in region A, the cells do not spread into region B adjacent to region A during the induction of differentiation into retinal tissue (i.e., the period until differentiation into retinal tissue can be confirmed). Cell adhesiveness is compared using an excess amount (e.g., 5 × 10 5 ~10×10 5 cells / cm 2) pluripotent stem cells are seeded on the cells, cultured at 37°C under 5% CO2 for 24 hours, and then the percentage of the area to which the cells adhere is compared. The cell adhesiveness is usually evaluated after 24 hours of culture, by removing the medium, washing the cells with medium or buffer, and removing unadhered cells.

[0013] For example, the area A is an excess amount (for example, 5 × 10 5 ~10×10 5 cells / cm 2 ) and cultured at 37°C under 5% CO2 for 24 hours, the area may be one in which cells adhere to 70% or more, 80% or more, or 90% or more of the area.

[0014] In one embodiment, region B is a cell-non-adhesive region. A cell-non-adhesive region means that it does not have sufficient cell adhesiveness to maintain pluripotent stem cells in adherent culture. The cell-non-adhesive region may be, for example, a region having an excess amount of cells (e.g., 5×10) relative to its area. 5 ~10×10 5 cells / cm 2 ) and cultured at 37°C under 5% CO2 for 24 hours, the area may be one in which cells adhere to 30% or less, 20% or less, or 10% or less of the area.

[0015] The culture substrate may be made of any material as long as it allows the formation of regions A and B on its surface. The culture substrate may be made of inorganic materials such as metal, glass, and silicone, or organic materials such as plastics (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin, and polytetrafluoroethylene tetrafluoroethylene resin). In one embodiment, the culture substrate is glass or plastic, particularly polystyrene resin.

[0016] The culture substrate may have any shape commonly used for cell culture, and is not particularly limited. The culture substrate may be, for example, a culture vessel such as a petri dish, plate, bottle, chamber, or multi-well plate (e.g., a 6-, 12-, 24-, 48-, 96-, or 384-well plate), a film, or a porous membrane. The culture substrate typically has region A and region B on a surface that is horizontal to the direction of gravity (on the bottom surface in the case of a culture vessel).

[0017] Region A may be an exposed surface of a cell-adhesive culture substrate, or may be a region coated with a cell-adhesive substance to impart cell adhesiveness to the surface of the culture substrate or to enhance the cell adhesiveness of the surface of the culture substrate. Examples of cell-adhesive substances include positively charged polymers such as poly-L-lysine and poly-L-ornithine; laminin; collagens such as type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type VII collagen; tenascin; fibrillin; fibronectin; vitronectin; elastin; entactin; proteoglycans composed of sulfated glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate and a core protein; glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, dermatan sulfate, and hyaluronic acid; Synthemax (registered trademark, a vitronectin derivative), and Matrigel (registered trademark).

[0018] In one embodiment, region A is coated with laminin. Laminin is a heterotrimeric molecule consisting of three subunit chains: an α chain, a β chain, and a γ chain. There are five known types of α chains (α1 to α5), three known types of β chains (β1 to β3), and three known types of γ chains (γ1 to γ3). Each laminin isoform is represented by a number indicating the constituent subunits (e.g., laminin 111 is composed of an α1 chain, a β1 chain, and a γ1 chain). Laminins include laminin-111, laminin-121, laminin-211, laminin-213, laminin-222, laminin-311 (laminin-3A11), laminin-332 (laminin-3A32), laminin-321 (laminin-3A21), laminin-3B32, laminin-411, laminin-421, laminin-423, laminin-521, laminin-522, laminin-523, or fragments thereof. Examples of laminin fragments include E8 fragments, which are fragments of the integrin-binding site, such as laminin-211-E8, laminin-311-E8, laminin-411-E8, and laminin-511-E8. In certain embodiments, the laminin is laminin-511 or a fragment thereof. In further embodiments, the laminin is a laminin-511-E8 fragment. For laminin coating, commercially available products such as iMatrix-511 (Nippi, Inc.) can also be used. iMatrix-511 (Nippi, Inc.) contains laminin 511-E8 fragment.

[0019] Region B may be an exposed region of the culture substrate surface with lower cell adhesiveness than region A, or may be a region in which the surface of the culture substrate is coated with some kind of substance to make it a region with lower cell adhesiveness than region A. In one embodiment, region B is coated with a non-cell-adhesive substance. Examples of non-cell-adhesive substances include MPC (2-methacryloyloxyethyl phosphorylcholine) polymer; celluloses such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymer; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin and derivatives thereof. In one embodiment, the non-cell-adhesive substance is an MPC polymer.

[0020] The shape of region A is not particularly limited, and examples thereof include a circle, an ellipse, and a polygon (e.g., a triangle, a square, a pentagon, a hexagon, an octagon, a decagon, a dodecagon, etc.). In one embodiment, region A is a circle. In this specification, the term "circle" refers to any shape that is recognized in the art as being substantially circular, and is used to mean an approximately circular shape, including a perfect circle. The area of ​​region A can be, for example, 0.01 to 100 cm. 2 , 0.01 to 30 cm 2 , 0.01 to 10 cm 2 , 0.03 to 30 cm 2 , 0.03 to 10 cm 2 , or 0.1 to 10 cm 2 In one embodiment, the area of ​​region A can be, but is not limited to, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm 2 Over 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm 2 For example, the area of ​​region A is 0.5 to 10 cm 2 , 0.7 to 10 cm2 , 1 to 10 cm 2 , 0.5 to 5 cm 2 , 0.7 to 5 cm 2 , or 1 to 5 cm 2 It is possible.

[0021] Region A can be circular or polygonal with an area equivalent thereto, with a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm or more, and 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm or less. In certain embodiments, region A is circular with a diameter of 0.1-10 cm, 0.1-5 cm, 0.1-3 cm, or 0.2-1 cm. In further embodiments, region A is circular with a diameter of 1-10 cm, 1-5 cm, or 1-3 cm.

[0022] At least a portion of region A is adjacent to region B. In one embodiment, the entire outer edge of region A is adjacent to region B (i.e., it is surrounded by region B). The distance between two regions A sandwiching region B is, but is not limited to, about 1 mm or more, for example.

[0023] Regions A and B may be formed on the surface of the culture substrate by any method used for cell patterning, such as soft lithography, photolithography, or 3D printing.

[0024] Regions A and B can be formed by treating a portion of the surface of the culture substrate to differentiate the cell adhesive properties of the treated and untreated regions. For example, a portion of the surface of the culture substrate can be masked and the unmasked region can be coated with a cell adhesive or non-cell adhesive substance. Alternatively, a layer of a cell adhesive or non-cell adhesive substance can be formed on the culture substrate, and a portion of the layer can be treated to change the cell adhesive properties. The treated portion can be an exposed portion of the surface of the culture substrate, or a portion in which the properties of the cell non-adhesive or cell adhesive substance have been changed by the treatment.

[0025] For example, regions A and B can be formed on the culture substrate by creating a sheet with holes of the shape and size of region A using a 3D printer-printed mold, covering the surface of the culture substrate with this sheet, coating the surface not covered with a cell adhesive substance, and then removing the sheet. Alternatively, regions A and B can be formed on the culture substrate by creating a sheet of the shape and size of region A, placing it on the surface of the culture substrate, coating the surface not covered with the sheet with a cell non-adhesive substance, removing the sheet, and coating the surface previously covered with the sheet with a cell adhesive substance. The sheet can be formed from biocompatible materials such as polydimethylsiloxane (PDMS), polyethylene glycol hydrogel, or agarose gel. Coating can be performed, for example, by contacting the culture substrate with a solution of the cell adhesive or non-adhesive substance and allowing it to react at 37°C or room temperature for the required time (e.g., 1 hour or more). The concentration of the cell adhesive or non-adhesive substance can be determined appropriately by those skilled in the art; for example, in the case of laminin, it can be 0.1 to 1 μg / cm. 2 , 0.1 to 0.5 μg / cm 2 , or approximately 0.25 μg / cm 2 It is possible.

[0026] In the present disclosure, the culture substrate may have convex portions (also referred to as pillars) on its surface, and region A may be present on the upper surface of the convex portions. The shape of the convex portions is not particularly limited and may be a cylinder or a prism. In this specification, a cylinder may have a cross-section that is recognized in the art as being substantially circular, and is used to mean a column having a cross-section that is approximately circular, including a perfect circle. In one embodiment, the convex portions are cylindrical. The height of the convex portions is not limited, but may be, for example, 0.1 mm to 10 mm, 0.1 mm to 5 mm, 1 mm to 5 mm, or 3 mm to 5 mm, e.g., 4 mm. The material of the convex portions may be the same as or different from the culture substrate. In addition to the materials exemplified in this specification as materials for the culture substrate, examples of materials for the convex portions include polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), polymethyl glutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), and polyethylene oxide (PEO).

[0027] Pluripotent stem cells refer to cells that have the pluripotency to differentiate into all cells that constitute an adult organism and the ability to self-renew, maintaining this pluripotency even after cell division. Pluripotent stem cells may be newly established or established cell lines. Examples of pluripotent stem cells include embryonic pluripotent stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). The species of pluripotent stem cells is not particularly limited, but is preferably mammalian (e.g., human, monkey, mouse, rat, hamster, guinea pig, dog, cat, pig, cow, goat, horse, sheep, or rabbit). In certain embodiments, the pluripotent stem cells are from rodents (e.g., mouse, rat, hamster, or guinea pig) or primates (e.g., human or monkey), preferably primates. In further embodiments, the pluripotent stem cells are monkey or human pluripotent stem cells; monkey or human ES cells or iPS cells; or human iPS cells.

[0028] ES cells are pluripotent stem cells derived from early embryos and can be established from the inner cell mass of blastocysts or the epiblast of early embryos after implantation. ES cells may be established cell lines, such as Kh-ES-1 cells, Kh-ES-2 cells, Kh-ES-13 cells, and KthES11 cells (available from the Institute for Virus Research and Frontier Medical Sciences, Kyoto University). In one embodiment, the ES cells are ES cells other than those established by a method involving the destruction of human embryos.

[0029] iPS cells are cells induced from cells other than pluripotent stem cells, such as somatic cells, and may be cells produced by any method, and are described, for example, in WO2007 / 069666, WO2009 / 006930, WO2009 / 006997, WO2009 / 007852, WO2008 / 118820, Cell 126(4): 663-676 (2006), Cell 131(5): 861-872 (2007), Science 318(5858): 1917-1920 (2007), Nature Biotechnology 26(1): 101-106 (2008), Cell Stem Cell 3(5): 568-574 (2008), Cell Stem Cell 4(5): 381-384. (2009), Nature 454: 646-650 (2008), Cell 136(3): 411-419 (2009), Nature Biotechnology 26: 1269-1275 (2008), Cell Stem Cell 3: 475-479 (2008), Nature Cell Biology 11: 197-203 (2009), Cell 133(2): 250-264 (2008), Science, 2013, 341 pp. 651-654, Stem Cells 31: 458-466 (2013)). iPS cells may also be established cell lines, such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells, Ff-I01 cells, Ff-I14 cells, and QHJI01 cells (available from Kyoto University or iPS Academia Japan, Inc.).

[0030] In one embodiment, iPS cells are cells produced by introducing a combination of reprogramming factors selected from Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, and Esrrb into somatic cells such as fibroblasts, skin cells, and blood cells (e.g., peripheral blood mononuclear cells or T cells, and umbilical cord blood-derived cells). Preferred combinations of reprogramming factors include: (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc.

[0031] Pluripotent stem cells are usually seeded onto the culture substrate at a cell number such that the cells occupy 70% or more of region A. The cells may be seeded only in region A, or may be seeded over the entire culture substrate including regions A and B. For example, when region B is a non-cell-adhesive region, the cells can be seeded over the entire culture substrate. For example, pluripotent stem cells can be seeded at a cell density of 5×10 5 ~10×10 5 cells / cm 2 Typically, pluripotent stem cells are collected and dispersed using a cell dispersion solution containing an enzyme (e.g., trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain) and / or a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)), suspended in a desired medium, and the resulting cell suspension is added to a culture substrate. Examples of cell dispersion solutions include TrypLE. TM Select (Thermo Fisher Scientific), TrypLE TM Commercially available products such as Express (Thermo Fisher Scientific) may also be used.

[0032] The differentiation of pluripotent stem cells into retinal tissue is induced by adhesion culture on the culture substrate. After seeding the pluripotent stem cells onto the culture substrate, they may be cultured for a certain period of time in a medium containing a factor for maintaining undifferentiation, and then the differentiation of the pluripotent stem cells into retinal tissue may be initiated. In the present disclosure, the time at which the culture in the medium not containing the factor for maintaining undifferentiation is initiated is defined as the time at which the differentiation induction into retinal tissue is initiated.

[0033] In the present disclosure, inducing differentiation of pluripotent stem cells into retinal tissue may be performed by any method available to those skilled in the art. In one embodiment, inducing differentiation of pluripotent stem cells into retinal tissue comprises culturing the pluripotent stem cells in a medium containing a BMP signaling agonist. In a further embodiment, inducing differentiation of pluripotent stem cells into retinal tissue comprises the following steps: (1) culturing the pluripotent stem cells in a medium containing a BMP signaling inhibitor, and (2) culturing the cells obtained in step (1) in a medium containing a BMP signaling agonist.

[0034] Culturing cells in the presence of a BMP signal inhibitor prior to the addition of a BMP signal agonist can increase the efficiency of differentiation into retinal tissue. That is, in one embodiment, the present disclosure provides a method for producing retinal tissue, comprising steps (1) and (2). In this embodiment, the culture in steps (1) and (2) is preferably adherent culture.

[0035] The medium used in the method of the present disclosure can be prepared using a medium commonly used for culturing animal cells as a basal medium, such as IMDM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, Medium 199 medium, Eagle MEM medium, αMEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof.

[0036] The medium may be a basal medium supplemented with one or more components selected from serum, serum replacements, growth factors, factors for maintaining undifferentiated cells, proteins (e.g., cytokines, insulin), fatty acids, lipids, vitamins, amino acids (e.g., non-essential amino acids, retinoids, glutamine, taurine), antioxidants, 2-mercaptoethanol, 1-thioglycerol, antibiotics, buffers, and inorganic salts, as needed. Serum replacements include, for example, albumins such as bovine serum albumin (BSA), transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 1-thioglycerol, and equivalents and mixtures thereof. The medium may be a KnockOut medium. TM Serum Replacement (Thermo Fisher Scientific), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), GlutaMAX TM Supplement (Thermo Fisher Scientific), Ham's F-12 Nutrient Mix, GlutaMAX TM The medium may contain commercially available serum substitutes such as basal medium supplemented with the above ingredients, for example, Ham's F-12 Nutrient Mix, GlutaMAX, etc. TM Supplement (Thermo Fisher Scientific), DMEM / F-12, GlutaMAX TM Supplement (Thermo Fisher Scientific) or the like may also be used.

[0037] The medium is preferably a serum-free medium. A serum-free medium refers to a medium that does not contain unconditioned or unpurified serum, and includes media containing purified blood-derived components or animal tissue-derived components as long as they do not contain unconditioned or unpurified serum. The serum-free medium may also contain a serum substitute.

[0038] Differentiation induction is preferably performed in the absence of feeder cells (also referred to as "feeder-free conditions"). Differentiation induction is also preferably performed under xeno-free conditions. In the present disclosure, "xeno-free" means that the culture does not contain components derived from a species different from that of the cells to be cultured.

[0039] Factors for maintaining undifferentiated states include FGF signaling agents, TGFβ family signaling agents, and insulin. Examples of FGF signaling agents include FGFs (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ family signaling agents include TGFβ signaling agents and Nodal / Activin signaling agents. Examples of TGFβ signaling agents include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling agents include Nodal, Activin A, and Activin B. In the case of human pluripotent stem cells, the factor for maintaining undifferentiated states preferably includes bFGF. The concentration of the factor for maintaining undifferentiated states may be any concentration that can maintain the undifferentiated state of pluripotent stem cells and can be appropriately determined by those skilled in the art. For example, when bFGF is used to culture human pluripotent stem cells, the concentration of bFGF may be 4 ng to 500 ng / mL, 10 ng to 200 ng / mL, or 30 ng to 150 ng / mL. Examples of media containing factors for maintaining undifferentiated state include StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.), S-medium (DS Pharma Biomedical Co.), and StemPro TM (Thermo Fisher Scientific), mTeSR1 TM (STEMCELL Technologies), mTeSR2 TM(STEMCELL Technologies), TeSR TM -E8 TM (STEMCELL Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14), etc. may also be used. In one embodiment, the medium containing factors for maintaining undifferentiation is StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.).

[0040] The culturing in the medium containing the undifferentiated state maintenance factor may be, but is not limited to, for example, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 2 to 3 days.

[0041] The medium containing factors for maintaining undifferentiation may further contain a ROCK inhibitor. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. The concentration of the ROCK inhibitor is determined appropriately depending on the type of inhibitor, but may be, for example, 1-100 μM, 5-50 μM, or approximately 10 μM, or a concentration equivalent to the ROCK inhibitory activity of Y-27632 at the aforementioned concentrations. The culture period in a medium further containing a ROCK inhibitor may be part of or the entire culture period in a medium containing factors for maintaining undifferentiation. For example, cells may be cultured for 1-2 days in a medium containing factors for maintaining undifferentiation and a ROCK inhibitor, followed by 1-2 days in a medium containing factors for maintaining undifferentiation but not a ROCK inhibitor.

[0042] BMPs (bone morphogenetic proteins) include, for example, BMP2, BMP4, BMP7, and BMP12 (GDF7). The BMP signal agonist and inhibitor may be an agonist and inhibitor, respectively, of one or more of these BMPs.

[0043] BMP signal inhibitors refer to substances that inhibit BMP-mediated signal transduction, including substances that act on BMP or its receptor, substances that suppress gene expression of BMP or its receptor, and substances that inhibit the binding of BMP to its receptor. Examples of BMP signal inhibitors include LDN-193189 (4-[6-(4-Piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), dorsomorphin (6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), and DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). In one embodiment, the BMP signal inhibitor is LDN-193189. The concentration of the BMP signal inhibitor is determined appropriately depending on the type of inhibitor, but is, for example, 1 to 1000 nM, 10 to 500 nM, 30 to 300 nM, or approximately 100 nM, or a concentration equivalent to the BMP inhibitory activity of LDN-193189 at the above concentrations.

[0044] The culturing in the medium containing the BMP signal inhibitor is, but is not limited to, for example, 1 to 10 days, 2 to 9 days, 3 to 7 days, 4 to 6 days, or about 5 days.

[0045] A BMP signal agonist refers to a substance that activates BMP signaling, and includes substances that act on BMP or its receptor, substances that enhance gene expression of BMP or its receptor, and substances that promote the binding of BMP to its receptor. Examples of BMP signal agonists include BMP2, BMP4, BMP7, BMP12 (GDF7), or fragments thereof, or anti-BMP receptor antibodies. In one embodiment, the BMP signal agonist is BMP4. The concentration of the BMP signal agonist is determined appropriately depending on the type of agonist, but is, for example, 0.01 to 1000 nM, 0.1 to 100 nM, 1 to 10 nM, 1 to 3 nM, or approximately 1.5 nM, or a concentration equivalent to the activity of BMP4 at the aforementioned concentrations.

[0046] Culture in a medium containing a BMP signaling agonist is carried out for a period required for differentiation into retinal tissue. The culture period is not limited to, for example, 1 to 30 days, 2 to 20 days, 3 to 15 days, 4 to 12 days, or 5 to 10 days (e.g., 5, 6, 7, 8, 9, or 10 days). The concentration of the BMP signaling agonist may be kept constant or varied during the culture period. For example, the concentration of the BMP signaling agonist may be gradually reduced by 40 to 60% every 2 to 4 days. For example, after initiating culture in a medium containing a BMP signaling agonist, the concentration of the BMP signaling agonist in the medium may be gradually reduced by replacing a portion (e.g., half) of the medium with medium not containing a BMP signaling agonist every 2, 3, or 4 days.

[0047] In one embodiment, the medium containing a BMP signal inhibitor or a BMP signal agonist is added to a 1:1 mixture of F-12 medium and IMDM medium, and the KnockOut TM The medium is supplemented with Serum Replacement (Thermo Fisher Scientific) (e.g., 0.5% to 30%, 1% to 20%, or 10%), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), BSA, and 1-thioglycerol.

[0048] After culturing in a medium containing a BMP signaling agent, the medium may be replaced with one not containing a BMP signaling agent, and the culture may be continued for a period of time that is not limited to, for example, 1 to 100 days, 10 to 90 days, 20 to 80 days, 30 to 70 days, 40 to 60 days, or about 50 days.

[0049] After culturing in a medium containing a BMP signal agonist, cells may be cultured in a medium containing a Wnt signal inhibitor for part or all of the culture period. A Wnt signal inhibitor refers to a substance that inhibits signal transduction by Wnt, and includes substances that act on Wnt or its receptor, substances that suppress gene expression of Wnt or its receptor, and substances that inhibit the binding of Wnt to its receptor. Examples of Wnt signal inhibitors include CKI-7 (N-(2-Aminoethyl)-5-chloro-8-isoquinolinesulfonamide), D4476 (4-(4-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide), and IWR-1-endo (IWR1e). (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide), and IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]-acetamide). The concentration of the Wnt signal inhibitor is determined appropriately depending on the type of inhibitor, but is, for example, 0.1 to 100 μM, 0.3 to 30 μM, or 1 μM to 10 μM.

[0050] During the culture period, the medium may be replaced as appropriate. For example, part or all of the medium may be replaced every 1 to 4 days. When starting culture in a medium containing a specific component, the entire medium may be replaced with a medium containing that component at a desired concentration, or part of the medium may be replaced so that the component reaches the desired final concentration (for example, half of the medium may be replaced with a medium containing twice the final concentration). Furthermore, as described for BMP signal agonists, the concentration of a specific component may be constant or may be varied during the culture period in a medium containing that component.

[0051] In one embodiment, inducing the differentiation of pluripotent stem cells into retinal tissue comprises: (1) culturing the pluripotent stem cells in a medium containing a BMP signaling inhibitor for 4 to 6 days; and (2) culturing the cells obtained in step (1) in a medium containing a BMP signaling agonist for 5 to 10 days. In a further embodiment, inducing the differentiation of pluripotent stem cells into retinal tissue comprises: (1) culturing the pluripotent stem cells in a medium containing a BMP signaling inhibitor for 4 to 6 days; and (2) culturing the cells obtained in step (1) in a medium containing a BMP signaling agonist for 5 to 10 days; and further comprising: prior to step (1), culturing the pluripotent stem cells for 1 to 2 days in a medium containing an undifferentiated state maintenance factor and a ROCK inhibitor; and culturing the cells obtained in step (2) in a medium not containing a BMP signaling agonist for 1 to 100 days. In a further embodiment, the differentiation of pluripotent stem cells into retinal tissue comprises: (1) culturing the pluripotent stem cells in a medium containing a BMP signal inhibitor for 4 to 6 days; and (2) culturing the cells obtained in step (1) in a medium containing a BMP signal agonist for 5 to 10 days; and further comprising: prior to step (1), culturing the pluripotent stem cells for 1 to 2 days in a medium containing an undifferentiated state maintenance factor and a ROCK inhibitor, and then culturing them for 1 to 2 days in a medium containing an undifferentiated state maintenance factor but not a ROCK inhibitor; and culturing the cells obtained in step (2) in a medium not containing a BMP signal agonist for 1 to 100 days.

[0052] Culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is, for example, 30°C to 40°C, or about 37°C. The CO2 concentration is, for example, 1% to 10%, or about 5%.

[0053] Differentiation into retinal tissue can be confirmed by detecting the expression of cell markers in cells in the tissue. Marker expression is confirmed, for example, on or after 10 to 100 days of differentiation induction (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days). In one embodiment, marker expression is confirmed on or after 18 to 22 days of differentiation induction.

[0054] The living retina has a layered structure and includes photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells, Muller cells, and their progenitor cells. Herein, the cells that make up the retina are referred to as "retinal cells," and each layer that makes up the retina is referred to as a "retinal layer." Retinal layers in mature retinal tissue 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. Retinal tissue in the developmental stage prior to reaching mature retinal tissue may include a neuroblast layer.

[0055] As used herein, "retinal progenitor cells" refer to precursor cells that can differentiate into one or more types of mature retinal cells selected from photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells, and Muller cells. As used herein, "neural retinal progenitor cells" refer to precursor cells that can differentiate into one or more types of mature retinal cells selected from photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Typically, neural retinal progenitor cells do not differentiate into retinal pigment epithelial cells.

[0056] As used herein, the term "retinal layer-specific neurons" refers to cells that constitute the retinal layer and are specific to the retinal layer. Retinal layer-specific neurons include photoreceptor cells (including rod cells and cone cells), horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells.

[0057] Retinal cell markers include Rx (also called Rax), Pax6, and Chx10, which are expressed in retinal progenitor cells; Nkx2.1, which is expressed in hypothalamic neuron precursor cells but not in retinal progenitor cells; Sox1, which is expressed in the hypothalamic neuroepithelium but not in the retina; and Crx and Blimp1, which are expressed in photoreceptor precursor cells. Retinal layer-specific neuronal markers include Chx10, PKCα, and L7, which are expressed in bipolar cells; Tuj1 and Brn3, which are expressed in retinal ganglion cells; Calretinin, which is expressed in amacrine cells; Calbindin, which is expressed in horizontal cells; Rhodopsin and Recoverin, which are expressed in mature photoreceptors; Nrl and Rhodopsin, which are expressed in rod cells; Rxr-gamma and S-Opsin, which are expressed in cone cells; and RPE65 and Mitf, which are expressed in retinal pigment epithelial cells. Other cell markers used in the examples can also be used.

[0058] In one embodiment, the retinal tissue produced by the method of the present disclosure has a percentage of Chx10-positive cells or a percentage of Chx10-positive and Rx-positive cells of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The percentage of marker-positive cells may be the percentage on days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or later after differentiation induction. In one embodiment, the retinal tissue has a percentage of Chx10- and Rx-positive cells of 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more on days 18 to 22 (e.g., day 20) after differentiation induction. In a further embodiment, the retinal tissue has a percentage of Chx10 and Rx positive cells of 90% or more or 95% or more on days 18 to 22 (eg, day 20) after differentiation induction.

[0059] In certain embodiments, the retinal tissue produced by the method of the present disclosure has a retinal cell percentage of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The cell percentage may be the percentage at or after 20 to 100 days of differentiation induction (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100 days). The cells in the retinal tissue preferably form a layered structure.

[0060] Retinal tissue can be recovered from the culture substrate using conventional methods. For example, retinal tissue can be recovered using tools such as tweezers. Alternatively, if the culture substrate is coated with a stimulus-responsive polymer (e.g., a temperature-responsive polymer or a photoresponsive polymer), retinal tissue can be recovered by applying a corresponding stimulus. For example, if a temperature-responsive polymer is used, the polymer's properties reversibly change from cell-adhesive (hydrophobic) to cell-non-adhesive (hydrophilic) at a predetermined temperature, retinal tissue can be recovered by maintaining the culture substrate at that predetermined temperature.

[0061] The collected retinal tissue can be maintained in a conventional culture vessel, or may be transferred to a suitable storage vessel and frozen for storage.

[0062] The shape and size of the retinal tissue depend on the shape and size of area A. The area of ​​the retinal tissue ranges from 0.01 to 100 cm. 2 , 0.03 to 30 cm 2 , 0.1 to 10 cm 2 In certain embodiments, the area of ​​retinal tissue can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm. 2 Over 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm 2 For example, the area of ​​retinal tissue is between 0.5 and 10 cm 2 , 0.7 to 10 cm 2 , 1 to 10 cm 2 , 0.5 to 5 cm 2 , 0.7 to 5 cm 2 , or 1 to 5 cm 2The retinal tissue may be circular or polygonal with an area equivalent thereto, with a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm or more, and 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm or less. In certain embodiments, the retinal tissue is circular with a diameter of 0.1 to 10 cm, 0.1 to 5 cm, 0.1 to 3 cm, or 0.2 to 1 cm. In further embodiments, the retinal tissue is circular with a diameter of 1 to 10 cm, 1 to 5 cm, or 1 to 3 cm. As used herein, a retinal tissue being circular or polygonal means that it has a shape similar to that of retinal tissue produced by the method of the present disclosure using a culture substrate having an area A of the same shape. As used herein, the area and diameter of retinal tissue refer to the area within the periphery of the retinal tissue and the length of the longest line connecting any two points on the periphery, respectively, measured based on an image captured using a stereomicroscope. The thickness of the retinal tissue can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm or more, and 500, 400, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, or 220 μm or less. In certain embodiments, the retinal tissue is 50 μm to 300 μm thick. The retinal tissue does not need to be uniform in thickness throughout; when specifying a thickness range for the retinal tissue, the thickness of any point in the retinal tissue may be within that range. The retinal tissue obtained by the methods of the present disclosure typically has up to about 30 cells perpendicular to the culture substrate or a thickness of 500 μm or less, and can be referred to as a sheet-like retinal tissue or retinal sheet. Therefore, in one aspect, the present disclosure provides a sheet-like pluripotent stem cell-derived retinal tissue.

[0063] The retinal tissue produced by the method of the present disclosure can be used as a transplant material for treating diseases, particularly diseases involving or caused by damage to retinal tissue or retinal cells. That is, the present disclosure provides a composition for treating diseases involving the retinal tissue of the present disclosure and a method for treating diseases comprising transplanting the retinal tissue of the present disclosure into a subject.

[0064] Diseases involving or caused by damage to retinal tissues or cells include retinal degeneration, retinitis pigmentosa, age-related macular degeneration, organic mercury poisoning, chloroquine retinopathy, glaucoma, diabetic retinopathy, neonatal retinopathy, etc. In one embodiment, the disease is retinitis pigmentosa.

[0065] When used for transplantation, the retinal tissue is preferably derived from pluripotent stem cells that are highly histocompatible with the subject (i.e., recipient) into which the retinal tissue is to be transplanted. In one embodiment, the retinal tissue is a pluripotent stem cell that matches some or all of the histocompatibility antigens (e.g., HLA type) of the subject, or a pluripotent stem cell established from the subject's cells.

[0066] The retinal tissue may be used for treatment in the same shape and size after differentiation induction, or may be cut into appropriate sizes using means such as tweezers while maintaining the layer structure. The composition comprising the retinal tissue of the present disclosure may contain a pharmaceutically acceptable carrier in addition to the retinal tissue. Examples of the pharmaceutically acceptable carrier include physiological aqueous solvents such as physiological saline, buffer, serum-free medium, etc. The composition may further contain additives such as preservatives, stabilizers, reducing agents, and tonicity agents. The composition comprising the retinal tissue of the present disclosure may be cryopreserved, in which case the composition may contain a suitable cryoprotectant.

[0067] Retinal tissue produced by the method of the present disclosure can also be used to evaluate the toxicity or efficacy of drugs on retinal tissue. In certain embodiments, the retinal tissue of the present disclosure is used to evaluate the toxicity or efficacy of therapeutic agents for diseases involving damage to retinal tissue or retinal cells or diseases caused by damage to retinal tissue or retinal cells. In this case, the retinal tissue is preferably produced from pluripotent stem cells established from a subject suffering from the disease.

[0068] The retinal tissue produced by the method of the present disclosure has a structure suitable for transplantation or toxicity and pharmacological evaluation, and is used while maintaining its layered structure. However, in some cases, it can be dispersed (e.g., by conventional means such as proteolytic enzymes or pipetting) to prepare a cell suspension, which can then be administered to a subject; such methods of using retinal tissue are within the scope of the present disclosure.

[0069] Exemplary embodiments of the present disclosure are described below. [1] A method for producing retinal tissue, comprising inducing differentiation of pluripotent stem cells into retinal tissue in region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a portion of region A and having lower cell adhesiveness than region A. [2] The method described in 1 above, wherein region A is coated with a cell adhesive substance. [3] The method described in 2 above, wherein the cell adhesive substance is laminin. [4] The method described in any one of 1 to 3 above, wherein region A is surrounded by region B. [5] The area of ​​region A is 0.03 to 10 cm 2[6] The method according to any one of items 1 to 4 above, wherein region A is a circle having a diameter of 0.1 to 3 cm. [7] The method according to any one of items 1 to 6 above, wherein region B is coated with a cell-non-adhesive substance. [8] The method according to item 7 above, wherein the cell-non-adhesive substance is an MPC polymer. [9] The method according to any one of items 1 to 8 above, wherein inducing the differentiation of pluripotent stem cells into retinal tissue comprises culturing the pluripotent stem cells in a medium containing a BMP signal agonist.

[10] The method according to any one of items 1 to 9 above, wherein inducing the differentiation of pluripotent stem cells into retinal tissue comprises the following steps: (1) culturing the pluripotent stem cells in a medium containing a BMP signal inhibitor, and (2) culturing the cells obtained in step (1) in a medium containing a BMP signal agonist.

[11] The method according to item 9 or 10 above, wherein the cells are cultured in a medium containing a BMP signal agonist for 5 to 10 days.

[12] The method according to any one of items 9 to 11 above, wherein the BMP signal agonist is BMP4.

[13] The method according to any one of 10 to 12 above, wherein the cells are cultured for 4 to 6 days in a medium containing a BMP signal inhibitor.

[14] The method according to any one of 10 to 13 above, wherein the BMP signal inhibitor is LDN-193189.

[15] The method according to any one of 1 to 14 above, wherein the pluripotent stem cells are human iPS cells or ES cells.

[16] The method according to any one of 1 to 15 above, comprising culturing the pluripotent stem cells in a medium containing a factor for maintaining undifferentiation prior to inducing differentiation into retinal tissue.

[17] The method according to 16 above, wherein the medium containing the factor for maintaining undifferentiation further contains a ROCK inhibitor.

[0070]

[18] A method for producing retinal tissue, comprising the following steps: (1) culturing pluripotent stem cells in a medium containing a BMP signaling inhibitor, and (2) culturing the cells obtained in step (1) in a medium containing a BMP signaling agonist.

[19] The method described in claim 18, wherein the cells are cultured in a medium containing a BMP signaling agonist for 5 to 10 days.

[20] The method described in claim 18 or 19, wherein the BMP signaling agonist is BMP4.

[21] The method described in any one of claims 18 to 20, wherein the cells are cultured in a medium containing a BMP signaling inhibitor for 4 to 6 days.

[22] The method described in any one of claims 18 to 21, wherein the BMP signaling inhibitor is LDN-193189.

[23] The method described in any one of claims 18 to 22, wherein the pluripotent stem cells are human iPS cells or ES cells.

[24] The method described in any one of claims 18 to 23, wherein, prior to step (1), the pluripotent stem cells are cultured in a medium containing a factor for maintaining undifferentiation.

[25] The method according to 24, wherein the medium containing the undifferentiated maintenance factor further contains a ROCK inhibitor.

[0071]

[26] A retinal tissue produced by the method according to any one of 1 to 17.

[27] A retinal tissue produced by the method according to any one of 18 to 25.

[0072]

[28] Area between 0.5 and 10 cm 2

[29] A sheet-shaped pluripotent stem cell-derived retinal tissue, which is circular and has a diameter of 1 to 10 cm.

[0073]

[30] A composition comprising the retinal tissue according to any one of claims 26 to 29.

[31] The composition according to claim 30, for use in treating a disease.

[32] The composition according to claim 31, wherein the disease is a disease accompanied by damage to retinal tissue or retinal cells or a disease caused by damage to retinal tissue or retinal cells.

[33] The composition according to claim 31 or 32, wherein the disease is retinitis pigmentosa.

[34] The composition according to claim 30, for use in toxicity evaluation or pharmacological efficacy evaluation.

[0074]

[35] A method for treating a disease accompanied by damage to retinal tissue or retinal cells or a disease caused by damage to retinal tissue or retinal cells, the method comprising transplanting the retinal tissue according to any one of 26 to 29 to a subject in need of said treatment.

[36] The retinal tissue according to any one of 26 to 29 for treating a disease accompanied by damage to retinal tissue or retinal cells or a disease caused by damage to retinal tissue or retinal cells.

[37] Use of the retinal tissue according to any one of 26 to 29 for the manufacture of a medicament for treating a disease accompanied by damage to retinal tissue or retinal cells or a disease caused by damage to retinal tissue or retinal cells.

[0075] The present invention will be further illustrated by the following examples, but the present invention is not limited to these examples in any way.

[0076] I. Patterned Culture I-1. Preparation of PDMS Sheets 1. A pillar-shaped mold with the desired cell adhesion area at its tip was created using a 3D printer (Formlab3). 2. PDMS (polydimethylsiloxane) (SILPOT184 W / C, Dow Corning Toray Co., Ltd.) was mixed with a curing agent (10:1) and vacuum degassed. 3. The mold created using the 3D printer was placed on the surface of a Petri dish, and PDMS was poured into it. After curing at 80°C for 2 hours, the PDMS sheet was removed from the mold and the excess material was trimmed off to obtain the desired PDMS sheet.

[0077] I-2. Differentiation Induction Retinal differentiation was induced using the following procedure. Figure 1 shows the experimental scheme. <Day 0: Dish Preparation> Procedure: 1. The prepared PDMS sheet was disinfected with 70% ethanol and placed on a dish (μ-Dish 35mm, High, 81156, Ibidi). 2. The dish lid was removed and the sheet was incubated in a heat block at 60°C for 5-10 minutes. If the sheet was difficult to dry, it was dried in a dryer set at 60°C. 3. 7μL of iMatrix-511 (Nippi Inc.) was added to 500μL of PBS, and 200μL was added to a circle (1cm diameter) on the PDMS sheet. 4. The sheet was incubated for at least 1 hour at 37°C or room temperature (RT).

[0078] <Day 0: Seeding cells onto dishes> Preparation ・TrypLE TM Select (containing 1 mM EDTA) (Thermo Fisher Scientific) was mixed with an equal volume of 0.5 mM EDTA / PBS and diluted with 0.5X TrypLE. TM Select was prepared (final concentration: 0.75 mM EDTA). StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.) (a mixture of the attached solutions A, B, and C, hereafter referred to as AK02N) was added with 10 mM Y-27632 (TOCRIS) at 1 / 1000 the volume of the medium and mixed well (final concentration: 10 μM) (hereafter referred to as AK02N+Y). Procedure: 1. The medium was removed from KhES-1_Rx::Venus cells (KhES-1 cells in which a modified GFP form of Venus was knocked into the Rx locus, a retinal marker) that had reached optimal confluence, and the cells were washed twice with 1 mL of PBS(-). 2. 0.5X TrypLE was added to the cells. TM Select was added at 500 μL / well and incubated at 37°C under 5% CO2. 3. 0.5X TrypLE TM Remove the Select plate, add 2 mL / well of PBS(-), and spray with PBS(-) to detach the cells. 4. Collect the cells in a 15 mL Falcon tube and perform a cell count. 5. Determine the required number of cells (5 x 10 per dish). 5 The resulting solution (5 × 10 cells) was transferred to another tube and centrifuged at 1000 rpm at 20°C for 5 minutes. 6. The supernatant was removed, and the cells were transferred to AK02N+Y at a concentration of 5 × 10 cells. 5 7. The iMatrix coating solution in the PDMS sheet was removed with an aspirator, and the cell suspension (5 × 10 5 8. Using sterilized tweezers, the PDMS sheet was removed from the dish. 9. An additional 300 μL of AK02N+Y was added, and the dish was cultured at 37°C under 5% CO2.

[0079] <Day 1: Medium change with AK02+PS> Procedure 1. Penicillin-Streptomycin (15070-062, Thermo Fisher Scientific) was added to AK02N and mixed well (Y-27632 not included, hereafter referred to as AK02N+PS). 2. The medium was removed using an aspirator, and 500 μL of AK02N+PS was added.

[0080] <Day 2: Initiation of differentiation induction> Procedure 1. Cells were washed three times with 1 mL of gfCDM (10% KSR) (Table 1). 2. The entire medium was replaced with 1 mL of gfCDM (10% KSR). * 20 mL of water for cell culture (W3500, Sigma Aldrich) was added to BSA (5 g) and left for several hours to dissolve.

[0081] <Day 3, Day 6: Addition of BMP inhibitor> 1. The entire medium was replaced with 1 mL of gfCDM (10% KSR) containing LDN-193189 (04-0074-02, ReproCELL Inc.) (hereinafter referred to as LDN) (final concentration: 100 nM LDN).

[0082] <Day 8: BMP addition> 1. Wash three times with 1 mL of gfCDM (10% KSR). 2. Replace the entire medium with 1 mL of BMP4-containing gfCDM (10% KSR) (final concentration: 1.5 nM BMP4). BMP4-containing medium was prepared by diluting 10 μg of BMP4 (R&D Systems) with 275 μL of 0.1% BSA to prepare a 1 μM BMP4 solution, and adding it to gfCDM (10% KSR).

[0083] <Day 11, Day 14: Half-medium exchange> 1. 500 μL of medium was removed and 500 μL of gfCDM (10% KSR) was added (half-exchange). Thereafter, half of the medium was exchanged every 3 days.

[0084] <Day 17: Medium change with DMEM / F-12+N2> 1. 1 mL of DMEM / F-12, GlutaMAX TMThe cells were washed three times with a supplement (10565-042, Thermo Fisher Scientific). 2. 1 mL of DMEM / F-12 + N2 (Table 2) was added. 3. Cultured until day 20.

[0085] <Day 20 and after: Culture in control maturation medium> 1. Wash three times with 1 mL of control maturation medium (Table 3). 2. Add 1 mL of control maturation medium and culture. Thereafter, the medium was changed every 2 to 3 days.

[0086] For cells not to be patterned, the entire surface was coated with iMatrix-511 (Nippi Inc.) (0.25 μg / cm ) without using a PDMS sheet. 2 ) and cultured in the same manner using a microdish (μ-Dish 35 mm, High, 81156, Ibidi).

[0087] II. Effect of Patterned Culture on Retinal Differentiation II-1. Analysis Methods - RNA Sequencing (RNA-seq) 1. Total RNA was prepared from Day 8 samples of patterned and unpatterned cultured cells using the RNeasy Micro kit (QIAGEN). 2. Sequence libraries were prepared using the Next Ultra II Directional RNA Library Prep Kit for Illumine E7760 (NEB), and sequenced using a next-generation sequencer (NextSeq, Illumina). 3. Quality checks of the sequence data were performed using FastQC (v0.11.9), TrimGalore (v0.6.1), cutadapt (v1.18), and Bowtie2 (v2.4.19). Mapping was performed using STAR (v2.7.5a), samtools (v1.1), and SUBREAD (v2.0.1), followed by secondary analysis using R (version 3.6.3) and edgeR (v3.28.1).

[0088] - Gene Ontology (GO) analysis: Metascape was used to perform enrichment analysis of GO Biological processes on the list of differentially expressed genes obtained by the secondary analysis in II-1.

[0089] Single-cell RNA-seq (scRNA-seq) 1. Day 100 retinal tissue was cultured in TrypLE with 150 μg / ml DNase I (Roche). TM Cells were dissociated into single cells using Select (Thermo Fisher Scientific) at 37°C for 5 minutes and then neutralized with 0.04% BSA / PBS. 2. Dissociated cells were pelleted, resuspended in 0.04% BSA / PBS, and passed through a 35 μm filter to obtain a single-cell suspension. 3. A cDNA library from approximately 10,000 single cells was prepared using the Chromium Single Cell 3' Reagent Kit v3 (10x, Genomics). Sequencing was performed on an Illumina NovaSeq sequencer using a paired-end 100-cycle kit (28 + 8 + 91). 4. Mapping was performed using CellRanger (version 3.0.2), and secondary analysis was performed using Seurat (version 3.0). The following markers were used for each cell: Stalk cells: VAX1 / PAX2 Retinal pigment epithelium (RPE): MITF / PMEL Amacrine cells (AC): ONECUT3 / PRDM13 Retinal ganglion cells (RGC): POU4F2 Photoreceptor cells (PR): CRX / NEUROD4 Neural precursor cells (NPC): HES6 / NEUROD1 Retinal progenitors: CHX10 / PAX6

[0090] GFP expression observation GFP-expressing cells were observed using a Keyence BZ-X all-in-one microscope. Phase contrast and GFP fluorescence images were acquired using a 10X lens, and then an image of the entire colony was created using a tiling process.

[0091] Immunostaining: The expression of Chx10, Recoverin, FOXG1, Crx, Tuj1, Zo-1, and Pax6 was evaluated by immunostaining using primary antibodies specific for each marker and secondary antibodies appropriate for the primary antibodies.

[0092] II-2. Induction of retinal differentiation in patterned cultures. Gene expression was compared between patterned and unpatterned cultured cells (confluent seeding). RNA-seq and GO analysis were performed using cells before BMP4 addition (Day 8). Patterned cultured cells, unlike unpatterned cultured cells, showed enhanced neural differentiation even before the addition of BMP4, which induces retinal differentiation (Figure 2). Unpatterned cultured cells showed enhanced BMP and Hippo signaling (Figure 3).

[0093] Retinal differentiation after BMP4 addition was assessed by monitoring the expression of GFP (venus) under the promoter of the retinal marker Rx over time. GFP expression was observed early in patterned cultured cells, indicating a higher differentiation induction efficiency compared to unpatterned cultured cells (Figure 4). Furthermore, the expression of Chx10, a marker for bipolar cells and their progenitor cells, was assessed at day 18. Expression was assessed in both patterned and unpatterned cultured cells with and without BMP4. Chx10 expression was assessed by quantitative PCR (HPRT1 was used as an endogenous control) and immunohistochemistry. As shown in Figure 5, Chx10 expression was significantly increased in patterned cultured cells compared to unpatterned cultured cells. Photographs of cells induced to differentiate by patterned culture at day 18 are shown in Figure 6. Expression of Rx and Chx10 was confirmed almost entirely, and it was estimated that more than 95% of the cells expressed these markers. At day 58, expression of the retinal photoreceptor marker Recoverin was observed in cells induced to differentiate into the retina by patterning culture (Fig. 7). At day 18, expression of the telencephalic marker FOXG1 was observed in cells cultured by patterning without BMP4 (Fig. 8).

[0094] II-2. Differentiation efficiency. We performed scRNA-seq on cells induced to differentiate into retinal cells in patterned cultures on Day 100. scRNA-seq showed that over 97% of the cells had differentiated into retinal cells (Figure 9, left). GFP expression in the cells was assessed by FACS on Day 74, and over 90% were GFP-positive (Figure 9, right).

[0095] PDMS sheets with circular holes of 2 mm, 5 mm, 7 mm, and 1 cm diameter were prepared and retinal differentiation was similarly induced. GFP expression was assessed on day 20, and differentiation induction was confirmed for all sizes (Figure 10).

[0096] II-4. Polarity of retinal tissue The polarity of the differentiation-induced tissue was evaluated. The expression of Crx, a photoreceptor marker, was evaluated by immunostaining in the retinal tissue on day 40. Crx expression was observed on the apical surface, indicating that the differentiation-induced tissue had retinal polarity (Figure 11).

[0097] II-5. Differentiation induction from other ES or iPS cell lines KthES11 cells were used to induce retinal differentiation by patterning culture in the same manner as above, and the expression of Crx, Chx10, and the neuronal marker Tuj1 was evaluated by immunostaining on Day 50. Expression of these markers was confirmed throughout the tissue, demonstrating that KthES11 cells, like KhES-1_Rx::Venus cells, can be induced to differentiate into the retina (Figure 12).

[0098] Similarly, retinal differentiation was induced using iPS cell lines 201B7 and 253G1 cells, and the expression of Crx and Chx10 was assessed at day 34. Crx and Chx10 expression was confirmed throughout the tissue, demonstrating that these iPS cells also induce retinal differentiation (Figure 13). Furthermore, the expression of Zo-1 and Chx10 was assessed by immunostaining at day 41 in tissue induced from 201B7 cells. Zo-1 is a tight junction marker. Zo-1 and Chx10 expression was observed in layers, indicating that the differentiated tissue had the layered structure of the retina (data not shown).

[0099] The effect of the BMP signal inhibitor LDN-193189 on differentiation induction was evaluated. GFP expression in cells without LDN-193189 from Day 3 to Day 8 of the patterning culture was compared with that in cells with LDN-193189 on Day 11. Cells with LDN-193189 showed uniform GFP expression compared with cells without LDN-193189 (Figure 14).

[0100] Furthermore, GFP and Chx10 expression was assessed on day 18 in both patterned and unpatterned cells with and without LDN-193189. Chx10 expression was assessed by quantitative PCR (HPRT1 was used as an endogenous control) and immunostaining. Even without patterned culture, LDN-193189 increased GFP and Chx10 expression, demonstrating that the cells were more likely to develop into retina (Figures 15 and 16).

[0101] IV. Patterned Culture Using Hydrophilic Polymers. Patterned culture was performed by coating MPC (2-methacryloyloxyethyl phosphorylcholine) polymer to form cell-nonadhesive regions. A 1 cm circular PDMS sheet was attached to a dish (μ-Dish 35 mm, High, 81156, Ibidi) and coated with an ethanol solution containing 0.5 vol% MPC polymer. The PDMS sheet was then removed. 7 μL iMatrix-511 (Nippi Inc.) was added to 500 μL PBS and added to the dish. This resulted in iMatrix not adhering to the MPC polymer-precoated area, and only the 1 cm circular area protected by the PDMS sheet was coated with iMatrix. Retinal differentiation induction was performed using this dish in the same manner as above, except that Iwp2 (REPROCELL) (2 μM) was added from Day 20.

[0102] Even when a cell-non-adhesive region was formed by coating with MPC polymer, efficient and uniform GFP expression was observed (FIG. 17).

Claims

1. A method for producing a retinal tissue, comprising inducing differentiation of pluripotent stem cells into a retinal tissue in a region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a part of the region A and having lower cell adhesiveness than the region A.

2. The method according to claim 1, wherein the region A is coated with a cell adhesive substance.

3. The method according to claim 2, wherein the cell adhesive substance is laminin.

4. The method according to claim 1, wherein the region A is surrounded by the region B.

5. The method according to claim 1, wherein the region B is coated with a cell non-adhesive substance.

6. The method according to claim 5, wherein the cell non-adhesive substance is an MPC polymer.

7. The method according to claim 1, wherein the induction of differentiation of pluripotent stem cells into a retinal tissue includes the following steps: (1) culturing pluripotent stem cells in a medium containing a BMP signal inhibitor, and (2) culturing the cells obtained in step (1) in a medium containing a BMP signal agonist.

8. The method according to claim 7, wherein the BMP signal agonist is BMP4.

9. The method according to claim 7, wherein the BMP signal inhibitor is LDN-193189.

10. A method for producing a retinal tissue, the method including the following steps: (1) culturing pluripotent stem cells in a medium containing a BMP signal inhibitor, and (2) culturing the cells obtained in step (1) in a medium containing a BMP signal agonist.

11. A retinal tissue produced by the method according to claim 1.

12. A retinal tissue produced by the method according to claim 10.

13. A sheet-shaped retinal tissue derived from pluripotent stem cells, which is circular with a diameter of 1 to 10 cm.

14. A composition comprising the retinal tissue according to any one of claims 11 to 13.

15. The composition according to claim 14, for use in the treatment of a disease.

16. The composition according to claim 15, wherein the disease is retinitis pigmentosa.

17. The composition according to claim 14, for use in toxicity evaluation or drug efficacy evaluation.