Method for producing retinal tissue

Abstract

The present disclosure includes: a method for producing a retinal tissue, the method comprising: (1) inoculating pluripotent stem cells to an area A having cell adhesion on a culture substrate at a density of 0.5 * 105 cells / cm < 2 > to 2.5 * 105 cells / cm < 2 >, (2) culturing the pluripotent stem cells inoculated in step (1) in a culture medium containing a ROCK inhibitor for 1-16 hours, and (3) culturing the pluripotent stem cells inoculated in step (1) in a culture medium containing a ROCK inhibitor for 1-16 hours, and (3) culturing the cells obtained after step (2) in a culture medium comprising a BMP signaling activator; a retinal tissue produced by the method; a composition comprising the retinal tissue; and the like.

Description

Technical Field

[0001] This application claims priority to Japanese Patent Application No. 2023-008080, the entire contents of which are incorporated herein by reference.

[0002] This disclosure includes a method for producing retinal tissue, retinal tissue produced by the method, and a composition comprising the retinal tissue. Background Technology

[0003] The culture manipulation of pluripotent stem cells has been actively studied for the in vitro formation of organs for transplantation and efficacy evaluation. The serum-free floating culture of Embryonic Body-like aggregates with quick reaggregation (SFEBq) method induces pluripotent stem cells to differentiate into cells of the central nervous system by suspending them in a medium free of components that inhibit neural differentiation (such as serum and transcription factors) for several days. This method has also been applied to the formation of retinal tissue from pluripotent stem cells. The SFEBq method utilizes cell self-assembly to reproduce the in vitro ontogeny process and can stably produce three-dimensional tissues with good reproducibility. However, because the SFEBq method relies on cell self-assembly, the patterning process can vary and it is difficult to control the morphology and size of the tissue. Furthermore, due to the thickness of the resulting cell clumps, drugs have difficulty penetrating the cells within the clumps, and it is also difficult to observe these internal cells. In addition, the SFEBq method is considered unsuitable for automation. Therefore, a suitable method is needed for the stable supply and automated production of retinal tissue. Summary of the Invention

[0004] The problem the invention aims to solve

[0005] The purpose of this disclosure is to provide a method for producing retinal tissue. A further purpose of this disclosure is to provide retinal tissue produced by this method and a composition comprising the retinal tissue.

[0006] Solution for solving the problem

[0007] In one aspect, this disclosure relates to a method for producing retinal tissue, comprising the following steps:

[0008] (1) Pluripotent stem cells were fed at a rate of 0.5 × 10⁻⁶. 5 cells / cm 2 Up to 2.5×10 5 cells / cm 2The culture medium is inoculated at a density in region A on the surface of the culture medium, wherein the culture medium includes region A and region B on the surface, region A has cell adhesion, and region B is adjacent to at least a portion of region A and has cell adhesion lower than that of region A.

[0009] (2) The pluripotent stem cells seeded in step (1) are cultured in a medium containing ROCK inhibitors for 1 to 16 hours, and

[0010] (3) The cells obtained after step (2) are cultured in a medium containing BMP signal transduction activator.

[0011] In a further aspect, this disclosure relates to retinal tissue produced by the methods of this disclosure.

[0012] In a further aspect, this disclosure relates to compositions comprising the retinal tissue of this disclosure.

[0013] The effects of the invention

[0014] This disclosure provides a method for producing retinal tissue, retinal tissue produced by the method, and a composition comprising the retinal tissue. Attached Figure Description

[0015] Figure 1 The experimental protocol for inducing retinal differentiation is shown.

[0016] Figure 2 Gene ontology (GO) analysis of patterned cultured cells before BMP4 addition (day 8) is shown.

[0017] Figure 3 GO analysis of unpatterned cultured cells before BMP4 addition (day 8) is shown.

[0018] Figure 4 The time-dependent changes in GFP (Rx::venus) expression in cells after induced retinal differentiation are shown with or without patterned culture (top) or without patterned culture (bottom).

[0019] Figure 5 The table shows Chx10 expression (top) and GFP expression (bottom) on day 18 in patterned cultured cells (patterned) and unpatterned cultured cells (all). Results are shown for cells supplemented with BMP4 (BMP+) and cells without BMP4 (BMP-).

[0020] Figure 6 The image shows GFP (Rx::venus) and Chx10 expression in cells on day 18 after retinal differentiation was induced using patterned culture.

[0021] Figure 7 The expression of GFP (Rx::venus), Recoverin, and Chx10 in cells on day 58 after retinal differentiation was induced using patterned culture.

[0022] Figure 8 The patterned culture of cells without BMP4 supplementation shows GFP (Rx::venus) and FOXG1 expression on day 18.

[0023] Figure 9 This shows the differentiation induction efficiency of cells after using patterned culture to induce retinal differentiation.

[0024] Figure 10 The patterned cultured cells of various sizes are shown to express GFP on day 20.

[0025] Figure 11 The Crx expression in the tissue is shown at day 40.

[0026] Figure 12 This demonstrates the induction of retinal differentiation through patterned culture using KthES11 cells. The expression of Crx, Chx10, and Tuj1 at day 50 is shown.

[0027] Figure 13 The image shows the induction of retinal differentiation through patterned culture using 201B7 cells (top) and 253G1 cells (bottom). The expression of Crx and Chx10 at day 34 is shown.

[0028] Figure 14 The image shows GFP expression on day 11 with or without LDN-193189 (top) or without LDN-193189 (bottom).

[0029] Figure 15 The table shows Chx10 expression (top) and GFP expression (bottom) in patterned cultured cells (patterned) and unpatterned cultured cells (all) with or without LDN-193189. Results are shown at day 18.

[0030] Figure 16 The table shows GFP and Chx10 expression in patterned cultured cells (patterned) and unpatterned cultured cells (all) with or without LDN-193189. The scale bar is in 1 mm. Results are shown at day 18.

[0031] Figure 17This illustrates the time-dependent changes in retinal differentiation induced by patterned culture using MPC polymers.

[0032] Figure 18 Human iPS cells (LPF11 line) seeded at various cell densities and cultured via patterned culture are shown. Results are shown at 1 hour, 1 day, 2 days, or 3 days post-seeding.

[0033] Figure 19 The results of immunostaining against Chx10 in human iPS cells (LPF11 strain) after seeding at various cell densities and patterned culture (20 days post-seeding) are shown. BMP(+) indicates patterned culture in the presence of BMP4 and BMP(-) indicates patterned culture in the absence of BMP4.

[0034] Figure 20 The results of altering the action time of Y-27632 in patterned culture of human iPS cells (LPF11 strain) are shown. Bright-field microscopy images and Chx10 immunostaining results are shown 20 days post-inoculation.

[0035] Figure 21 The results of altering the action time of Y-27632 in patterned culture of human iPS cells (DSP-SQ strain) are shown. Immunostaining results of Chx10 are shown 20 days post-inoculation.

[0036] Figure 22 The results of altering BMP4 concentration in patterned culture of human iPS cells (LPF11 strain) are shown. Bright-field microscopy images and Rx immunostaining results are shown 20 days post-inoculation.

[0037] Figure 23 The expression of Chx10 and Rx (retinal progenitor cell marker genes) and Emx2 (non-target cell marker genes) in human iPS cells (LPF11 line) cultured by patterned culture with various BMP4 concentrations is shown.

[0038] Figure 24 The expression of marker genes in cells cultured using the patterned culture method (white) in the reference example and the patterned culture method (black) in the examples are shown respectively. Genes to the left of the dashed lines are neuroretinal marker genes, and genes to the right are non-target cell marker genes. Detailed Implementation

[0039] Unless otherwise defined, the terms used herein are to be understood as such being commonly understood by one of ordinary skill in the art, such as organic chemistry, medicine, pharmacy, molecular biology, and microbiology. Several terms used herein are defined as follows. The definitions herein take precedence over the general understanding.

[0040] When a numerical value is accompanied by the term "approximately," the value is intended to represent any value within the range of -10% to +10% of that value. A range defined by a lower and upper limit encompasses all values ​​from the lower limit to the upper limit, including the values ​​at both endpoints. When a range is accompanied by the term "approximately," the two endpoints are understood to be present simultaneously with the term. For example, "approximately 20–30" is understood as "18–33."

[0041] The method for producing retinal tissue disclosed herein includes inducing pluripotent stem cells to differentiate into retinal tissue on the surface of a culture medium, wherein the culture medium comprises region A and region B on the surface, inducing differentiation in region A, region A having cell adhesion, and region B being adjacent to at least a portion of region A and having cell adhesion lower than that of region A.

[0042] Region A is the area where pluripotent stem cells can be maintained in adherent culture. Region B is the area with lower cell adhesion than region A, thereby preventing cells from extending from region A to the adjacent region B during the induction of retinal tissue differentiation (i.e., until differentiation into retinal tissue is confirmed) when pluripotent stem cells are seeded in region A. This can be achieved by seeding an excess of cells relative to the area (e.g., 5 × 10⁻⁶). 5 ~10×10 5 cells / cm 2 Pluripotent stem cells were used, and cell adhesion was compared by the percentage of cell adhesion area after culturing at 37°C and 5% CO2 for 24 hours. Cell adhesion was typically evaluated after removing the culture medium and washing the cells with culture medium or buffer to remove non-adhering cells after 24 hours of culture.

[0043] Region A can be, for example, the following region, where when the area is excessive relative to its size (e.g., 5 × 10⁻⁶), 5 ~10×10 5 cells / cm 2 When pluripotent stem cells were seeded and cultured at 37°C and 5% CO2 for 24 hours, the cells adhered to more than 70%, 80%, or 90% of the area after 24 hours.

[0044] In some implementations, region B is a region lacking cell adhesion. A region lacking cell adhesion refers to a region that does not possess sufficient cell adhesion to maintain pluripotent stem cells in adherent culture. A region lacking cell adhesion can be, for example, a region where, when an excess of cell adhesion relative to its area (e.g., 5 × 10⁻⁶) is used… 5 ~10×10 5 cells / cm 2When pluripotent stem cells were seeded and cultured at 37°C and 5% CO2 for 24 hours, the cells adhered to less than 30%, 20%, or 10% of the area after 24 hours.

[0045] The culture medium can be made of any material, as long as it can have regions A and B on its surface. For example, the culture medium can be made of inorganic materials such as metals, glass, or silicone, or organic materials such as plastics (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resins, fluoropolymers, polycarbonate resins, polyurethane resins, methylpentene resins, phenolic resins, melamine resins, epoxy resins, vinyl chloride resins, polytetrafluoroethylene resins). In some embodiments, the culture medium is made of glass or plastic, particularly polystyrene resin.

[0046] Culture media can have any shape commonly used for cell culture. Culture media can be, for example, culture containers such as Piper dishes, culture plates, culture flasks, culture chambers, or multi-well plates (e.g., 6-well, 12-well, 24-well, 48-well, 96-well, or 384-well plates), or can be membranes or porous membranes. Culture media typically have regions A and B on a surface in a horizontal direction relative to the direction of gravity (on the bottom surface when the culture media is a culture container). In some embodiments, the culture media has regions A and B on the same plane. In such embodiments, when the culture container has regions A and B on the plane of its bottom surface, the inner wall surface of the culture container is not region B. As described below, when the culture media has a protrusion on its surface and the protrusion has region A on its upper surface, region B is the lateral surface of the protrusion adjacent to the upper surface of the protrusion; however, in this disclosure, region A on the upper surface of the protrusion and region B on the lateral surface of the protrusion are considered to be on the same plane.

[0047] Region A can be the exposed surface area of ​​a cell-adhesive culture medium, or the surface area of ​​a culture medium coated with cell-adhesive substances to generate or enhance surface cell adhesion. Examples of cell-adhesive substances include positively charged polymers such as poly-L-lysine and poly-L-ornithine; laminin; collagen such as type I, type II, type III, type IV, type V, and type VII collagen; tendinin; fibrils; fibronectin; hyalin; elastin; nestin; proteoglycans composed of sulfated glucosamine glycans such as chondroitin sulfate, heparin sulfate, keratin sulfate, and dermatan sulfate, and core proteins; glucosamine glycans such as chondroitin sulfate, heparin sulfate, keratin sulfate, dermatan sulfate, and hyaluronic acid; (vitin derivatives) and

[0048] In some embodiments, region A is coated with laminin. Laminin is a heterotrimeric molecule comprising three subunit chains α, β, and γ. There are five types of α chains α1-α5, three types of β chains β1-β3, and three types of γ chains γ1-γ3, and each laminin isoform is represented by a number indicating its constituent subunits (e.g., laminin 111 is composed of α1, β1, and γ1 chains). Examples of 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 511, laminin 521, laminin 522, laminin 523, or fragments thereof. Examples of laminin fragments include the E8 fragment, which is a fragment of an integrin binding site, such as laminin 211-E8, laminin 311-E8, laminin 411-E8, and laminin 511-E8. In some embodiments, the laminin is laminin 511 or a fragment thereof. In a further embodiment, the laminin is the laminin 511-E8 fragment. For coating with laminin, a commercially available product such as iMatrix-511 (Nippi.Inc.) can be used. iMatrix-511 (Nippi.Inc.) contains the laminin 511-E8 fragment.

[0049] Region B can be an exposed area of ​​the culture medium surface with lower cell adhesion than region A, or an area coated with a substance to make the cell adhesion of this region lower than that of the culture medium surface in region A. In some embodiments, region B is coated with a non-cell-adhesive substance. Examples of non-cell-adhesive substances include MPC (2-methacryloyloxyethyl phosphocholine) polymers; celluloses such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropyl methylcellulose, and sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymers; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginate, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin and its derivatives. In some embodiments, the non-cell-adhesive substance is an MPC polymer.

[0050] Region A can have any shape, and can be circular, elliptical, or polygonal (e.g., triangular, quadrilateral, pentagonal, hexagonal, octagonal, decagonal, or dodecagonal). In some embodiments, region A has a circular shape. As used herein, a circular shape refers to a shape generally considered to be circular in the art, including a perfect circle. The area of ​​region A can be, but is not limited to, 0.01 to 100 cm². 2 0.01~30cm 2 0.01~10cm 2 0.03~30cm 2 0.03~10cm 2 or 0.1-10cm 2 In some implementations, the area of ​​region A is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm². 2 The above and 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1cm 2 Below (upper and lower limits are selected independently). For example, the area of ​​region A can be 0.5–10 cm². 2 0.7~10cm 2 1-10cm 2 0.5-5cm 2 0.7-5cm 2 1-5cm 2 0.5~2cm 2 0.7~2cm 2 0.5~1cm 2 or 0.7-1cm 2 .

[0051] Region A can be a circular shape 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 (the upper and lower limits are selected independently), or a polygonal shape with the same area as a circular shape. In some embodiments, region A has a circular shape with a diameter of 0.1–10 cm, 0.1–5 cm, 0.1–3 cm, or 0.2–1 cm. In a further embodiment, region A has a circular shape with a diameter of 1–10 cm, 1–5 cm, or 1–3 cm.

[0052] At least a portion of region A is adjacent to region B. In some embodiments, the entire outer edge of region A is adjacent to region B (i.e., region A is surrounded by region B). The culture medium may have multiple regions A and / or multiple regions B on its surface. When the culture medium has multiple regions A on its surface, the distance between two regions A sandwiching region B can be, but is not limited to, more than about 1 mm.

[0053] Region A and Region B can be formed on the surface of the culture medium using any of the methods used for cell patterning. For example, Region A and Region B can be formed on the surface of the culture medium using techniques such as soft etching, photolithography, and 3D printing.

[0054] Regions A and B can be formed by treating a portion of the surface of the culture medium to give the treated and untreated regions different cell adhesion properties. Regions A and B can be formed, for example, by coating a portion of the surface of the culture medium with a cell-adhesive or non-cell-adhesive substance. For example, these regions can be formed by masking a portion of the surface of the culture medium and coating the unmasked area with a cell-adhesive or non-cell-adhesive substance. Alternatively, these regions can be formed by providing a layer of cell-adhesive or non-cell-adhesive substance on the culture medium and treating a portion of that layer to alter the cell adhesion properties. The treated portion can be either the exposed surface of the culture medium or the portion where the properties of the non-cell-adhesive or cell-adhesive substance have been altered by the aforementioned treatment.

[0055] For example, regions A and B can be formed on the culture medium by: preparing a sheet with pores of the shape and size of region A using a 3D-printed mold; covering the surface of the culture medium with the sheet; coating the portions of the surface not covered by the sheet with a cell-adhesive substance; and then removing the sheet. Alternatively, regions A and B can be formed on the culture medium by: preparing a sheet with the shape and size of region A; placing the sheet on the surface of the culture medium; coating the portions of the surface not covered by the sheet with a non-cell-adhesive substance; removing the sheet; and coating the portions of the surface covered by the sheet with a cell-adhesive substance. The sheet can be prepared from a biocompatible material such as polydimethylsiloxane (PDMS), polyethylene glycol hydrogel, or agarose gel. Coating can be performed, for example, by contacting the culture medium with a solution of a cell-adhesive substance or a non-cell-adhesive substance and allowing it to react with the solution at 37°C or room temperature for the required time (e.g., more than 1 hour). The concentration of cell-adhesive or non-cell-adhesive substances can be appropriately determined by those skilled in the art, and for example, for laminin, it can be 0.1–1 μg / cm³. 2 0.1~0.5μg / cm 2 or approximately 0.25 μg / cm2 Alternatively, without using the sheets described above, regions A and B can be formed on the culture medium by dropping droplets of a solution of a cell adhesion substance onto the culture medium and allowing the culture medium to react with the solution.

[0056] In this disclosure, the culture medium may have a protrusion (also referred to as a column) on its surface, and the protrusion may have a region A on its upper surface. In such cases, region B is a side surface of the protrusion adjacent to the upper surface of the protrusion. The shape of the protrusion may be, but is not limited to, a cylinder or a prism. As used herein, a cylinder refers to a column whose cross-section is generally considered in the art to be substantially circular (including a perfect circle). In some embodiments, the protrusion is cylindrical. The height of the protrusion may be, but is not limited to, 0.1 mm to 10 mm, 0.1 mm to 5 mm, 1 mm to 5 mm, or 3 mm to 5 mm, for example, 4 mm. The material of the protrusion may be the same as or different from the culture medium. In addition to those exemplified herein as materials for culture media, examples of materials for protrusions include polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), polymethylglutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), vinyl polyvinyl acetate (PEVA), and polyethylene oxide (PEO).

[0057] Pluripotent stem cells are cells possessing pluripotency (the ability to differentiate into all types of cells that constitute an adult) and self-renewal capacity (the ability to maintain pluripotency after cell division). Pluripotent stem cells can be newly established cells or cell lines. 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 are preferably, but not limited to, mammals (e.g., humans, monkeys, mice, rats, hamsters, guinea pigs, dogs, cats, pigs, cattle, goats, horses, sheep, and rabbits). In some embodiments, the pluripotent stem cells are rodent (e.g., mice, rats, hamsters, guinea pigs) or primate (e.g., humans, monkeys), preferably primates. In further embodiments, the pluripotent stem cells are monkey or human pluripotent stem cells; monkey or human ES or iPS cells; or human iPS cells.

[0058] ES cells are pluripotent stem cells derived from early embryos and can be established from the internal cell mass of the blastocyst or from the ectoderm of an early embryo after implantation. ES cells can be cells from 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 and Regenerative Medicine, Kyoto University). In some embodiments, ES cells are ES cells other than those established through any of the methods involving the destruction of a human embryo. In different embodiments, human ES cells are cells established from human embryos within 14 days of fertilization that have not yet undergone development in vivo.

[0059] iPS cells are cells derived from cells other than pluripotent stem cells (e.g., somatic cells) and can be produced by any method, for example, 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 StemCell3(5):568-574(2008), Cell Stem Cell 4(5):381-384(2009), Nature The methods described in 454:646-650 (2008), Cell 136(3):411-419 (2009), Nature Biotechnology 26:1269-1275 (2008), CellStem 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) are as follows. iPS cells can also be cells from 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.).

[0060] In some implementations, iPS cells are prepared by introducing a combination of reprogramming factors selected from Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sal4, lin28, and Esrrb into somatic cells such as fibroblasts, skin cells, or hematopoietic cells (e.g., peripheral blood mononuclear cells or T cells, or cells derived from umbilical cord blood). Examples of 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.

[0061] Pluripotent stem cells are usually sized at a rate of 0.5 × 10⁻⁶. 5 ~2.5×10 5 cells / cm 2 Cells are seeded onto a culture medium. Cells can be seeded only in region A or across the entire area of ​​the culture medium, which includes regions A and B. For example, when region B is a non-cell-adhesive region, cells can be seeded across the entire area of ​​the culture medium. In some embodiments, pluripotent stem cells are seeded at a rate of 0.5 × 10⁻⁶. 5 0.6×10 5 0.7×10 5 0.8×10 5 0.9×10 5 1.0×10 5 1.1×10 5 1.2×10 5 1.3×10 5 1.4×10 5 1.5×10 5 1.6×10 5 1.7×10 5 1.8×10 5 1.9×10 5 2.0×10 5 2.1×10 5 2.2×10 5 2.3×10 5 Or 2.4×10 5 cells / cm 2 Above and 2.5×10 5 2.4×10 5 2.3×10 5 2.2×10 5 2.1×10 5 2.0×10 5 1.9×10 5 1.8×10 5 1.7×10 5 1.6×10 5 1.5×10 5 1.4×10 5 1.3×10 5 1.2×10 5 1.1×10 5 1.0×10 5 0.9×10 5 0.8×10 5 0.7×10 5 Or 0.6×105 cells / cm 2 The following (upper and lower limits can be selected independently) are seeded onto the culture medium. For example, pluripotent stem cells can be seeded at a rate of 0.5 × 10⁻⁶. 5 ~2.5×10 5 cells / cm 2 1.0×10 5 ~2.5×10 5 cells / cm 2 1.5×10 5 ~2.5×10 5 cells / cm 2 0.5×10 5 ~2.0×10 5 cells / cm 2 1.0×10 5 ~2.0×10 5 Cells / cm, or 0.5×10 5 ~1.5×10 5 cells / cm 2 Inoculation. Typically, pluripotent stem cells are collected and dispersed using a cell dispersion containing enzymes (e.g., trypsin, collagenase, hyaluronidase, elastase, streptokinase, DNase, or papain) and / or a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)), and suspended in the desired culture medium. The resulting cell suspension is then added to the culture medium. As a cell dispersion, for example, a commercially available product such as TrypLE can be used. TM Select (Thermo Fisher Scientific) or TrypLE TM Express (Thermo Fisher Scientific).

[0062] Differentiation induction of pluripotent stem cells into retinal tissue is performed by adherent culture on the aforementioned culture medium. Differentiation induction into retinal tissue can begin after the pluripotent stem cells seeded on the culture medium have been cultured for a certain period in a medium containing undifferentiated maintenance factors. In this disclosure, the time at which culture begins in a medium without undifferentiated maintenance factors is defined as the time at which differentiation induction into retinal tissue begins.

[0063] In this disclosure, the differentiation induction of pluripotent stem cells into retinal tissue includes culturing pluripotent stem cells in a culture medium containing a BMP signaling activator. Cell culture in the BMP signaling activator culture medium can be initiated at the start of differentiation induction or some time after the start of differentiation induction (e.g., 4–6 days later).

[0064] Inducing pluripotent stem cell differentiation into retinal tissue can involve culturing pluripotent stem cells in a medium containing a BMP signaling inhibitor before culturing them in a medium containing a BMP signaling activator. Culturing cells in the presence of a BMP signaling inhibitor before adding the BMP signaling activator can improve the efficiency of differentiation into retinal tissue.

[0065] The culture medium used in the methods of this disclosure can be prepared using a culture medium commonly used for culturing animal cells as a basal medium. For example, the basal medium can be IMDM, DMEM, F-12, DMEM / F12, IMDM / F12, BME, BGJb, CMRL 1066, Glasgow MEM (GMEM), Improved MEM Zinc Option, Medium 199, EagleMEM, αMEM, Ham's medium, RPMI 1640, Fischer's medium, or a mixture thereof.

[0066] The culture medium may contain one or more components selected from serum, serum substitutes, growth factors, undifferentiated maintenance factors, 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, added to the basal medium as needed. Examples of serum substitutes include, for example, albumins such as bovine serum albumin (BSA), transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 1-thioglycerol, and their equivalents, and mixtures thereof. The culture medium may contain commercially available serum substitutes such as KnockOut. TM Serum alternatives (Thermo Fisher Scientific), chemically defined lipid concentrates (Thermo Fisher Scientific), GlutaMAX TM Supplements (Thermo Fisher Scientific), Ham's F-12 Nutritional Blend, GlutaMAX TM Supplements (Thermo Fisher Scientific), B27 supplements (Thermo Fisher Scientific), N2 supplements (Thermo Fisher Scientific), or ITS supplements (Thermo Fisher Scientific). The culture medium can be a commercially available product containing any of the above components in the basal medium, such as Ham's F-12 nutrient mixture, GlutaMAX, etc. TMSupplements (Thermo Fisher Scientific), DMEM / F-12, GlutaMAX TM Supplement (ThermoFisher Scientific).

[0067] The culture medium is preferably serum-free. Serum-free culture medium refers to a culture medium that does not contain unadjusted or unpurified serum. Culture media containing purified blood-derived components or animal tissue-derived components are also included in serum-free culture media, provided they do not contain unadjusted or unpurified serum. Serum-free culture media may contain serum substitutes.

[0068] Differentiation induction is preferably performed in the absence of feeder cells (also described as under feeder-free conditions). Differentiation induction is also preferably performed under xeno-free conditions. In this disclosure, the term xeno-free means free from components derived from a species different from the species of the cells to be cultured.

[0069] Examples of undifferentiated maintenance factors include FGF signaling activators, TGFβ family signaling activators, and insulin. Examples of FGF signaling activators include FGF (e.g., bFGF, FGF4, FGF8). Examples of TGFβ family signaling activators include TGFβ signaling activators and Nodal / Activin signaling activators. Examples of TGFβ signaling activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling activators include Nodal, Activin A, and Activin B. For human pluripotent stem cells, the undifferentiated maintenance factor preferably includes bFGF. The concentration of the undifferentiated maintenance factor can be suitably determined by those skilled in the art, as long as it can maintain the undifferentiated state of the pluripotent stem cells. For example, when using bFGF to culture human pluripotent stem cells, the concentration of bFGF can be 4 ng–500 ng / mL, 10 ng–200 ng / mL, or 30 ng–150 ng / mL. The culture medium containing the undifferentiated maintenance factor can be, for example... AK02N (Ajinomoto Co., Inc.), AK03N (Ajinomoto Co., Inc.), S-medium (DS Pharma Biomedical Co.), StemPro TM (Thermo Fisher Scientific), mTeSR1 TM (STEMCELL Technologies), mTeSR2 TM(STEMCELL Technologies)、TeSR TM -E8 TM (STEMCELL Technologies) or hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep9; 105(36): 13409-14) and other media. In some embodiments, the media containing undifferentiated maintenance factors are AK02N (Ajinomoto Co., Inc.).

[0070] The culture time in a medium containing undifferentiated maintenance factors can be, but is not limited to, 1–10 days, 1–9 days, 1–8 days, 1–7 days, 1–6 days, 1–5 days, 1–4 days, 1–3 days, or 2–3 days.

[0071] Pluripotent stem cells are cultured in a medium containing a ROCK inhibitor before being cultured in a medium containing a BMP signaling activator. The ROCK inhibitor can be added to a medium containing an undifferentiated maintenance factor. Examples of ROCK inhibitors include Y-27632, fasudil (HA1077), and H-1152. The concentration of the ROCK inhibitor is appropriately determined based on the inhibitor to be used and can be 1–100 μM, 5–50 μM, or about 10 μM, or a concentration providing equivalent activity to Y-27632 as described above. In this disclosure, the culture time in the medium containing the ROCK inhibitor is 1–16 hours. In some embodiments, the culture time in the medium containing the ROCK inhibitor is 1–14 hours, 1–12 hours, 1–10 hours, 1–8 hours, 1–6 hours, 1–4 hours, 1–3 hours, 1–2 hours, 2–16 hours, 2–14 hours, 2–12 hours, 2–10 hours, 2–8 hours, 2–6 hours, 2–4 hours, or 2–3 hours. In a preferred embodiment, the culture time in the medium containing the ROCK inhibitor is 1–3 hours. The culture time in the medium containing both undifferentiated maintenance factor and the ROCK inhibitor can be part or all of the culture time in the medium containing the undifferentiated maintenance factor. For example, cells can be cultured in a medium containing both the undifferentiated maintenance factor and the ROCK inhibitor for 1–16 hours, and then in a medium containing the undifferentiated maintenance factor but not the ROCK inhibitor, until the culture time in the medium containing the undifferentiated maintenance factor reaches the predetermined culture time.

[0072] BMPs (bone morphogenetic proteins) include, for example, BMP2, BMP4, BMP7, and BMP12 (GDF7). BMP signaling activators and inhibitors can be one or more of these BMPs, respectively.

[0073] BMP signaling inhibitors are substances that inhibit signal transduction via BMP, and include substances that act on BMP or its receptors, substances that inhibit gene expression of BMP or its receptors, and substances that inhibit the binding between BMP and its receptors. Examples of BMP signaling inhibitors include LDN-1931894-[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]pyrimidinyl), and DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). In some embodiments, the BMP signaling inhibitor is LDN-193189. The concentration of the BMP signaling inhibitor is determined based on the inhibitor to be used, and can be 1–1000 nM, 10–500 nM, 30–300 nM, or about 100 nM, or a concentration that provides equivalent activity to LDN-193189 as described above.

[0074] The culture time in a medium containing BMP signaling inhibitors can be, but is not limited to, 1–10 days, 2–9 days, 3–7 days, 4–6 days, or about 5 days.

[0075] BMP signaling activators are substances that activate signal transduction via BMP and include substances that act on BMP or its receptor, substances that enhance gene expression of BMP or its receptor, and substances that promote the binding between BMP and its receptor. Examples of BMP signaling activators include BMP2, BMP4, BMP7, BMP12 (GDF7), fragments thereof, and anti-BMP receptor antibodies. In some embodiments, the BMP signaling activator is BMP4. The concentration of the BMP signaling activator is determined depending on the activator to be used and can be 0.01–1000 nM, 0.1–100 nM, 1–10 nM, 1–3 nM, or about 1.5 nM, or a concentration providing equivalent activity to the concentration of BMP4 described above. In some embodiments, the concentration of the BMP signaling activator is 3–15 nM or 3–12 nM, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nM, or a concentration that provides equivalent activity to BMP4 as described above.

[0076] The time required for cells to differentiate into retinal tissue in a culture medium containing BMP signaling activator. Culture time can be, but is not limited to, 1–30 days, 2–20 days, 3–15 days, 4–12 days, or 5–10 days (e.g., 5, 6, 7, 8, 9, or 10 days). The concentration of BMP signaling activator can be constant or varied during culture. For example, the concentration of BMP signaling activator can be gradually reduced at a rate of 40%–60% every 2–4 days. For example, the concentration of BMP signaling activator in the culture medium can be gradually reduced by replacing a portion (e.g., half) of the culture medium containing BMP signaling activator with a portion of the medium without BMP signaling activator every 2, 3, or 4 days after initiating culture in the medium containing BMP signaling activator.

[0077] In some implementations, the culture medium containing a BMP signaling inhibitor or a BMP signaling activator is derived from a medium containing KnockOut TM Serum substitutes (Thermo Fisher Scientific) (e.g., 0.5%–30%, 1%–20%, or 10%), chemically defined lipid concentrates (Thermo Fisher Scientific), and a 1:1 mixture of BSA and 1-thioglycerol in F-12 medium and IMDM medium are used to prepare the solution.

[0078] After culturing in a medium containing BMP signal transduction activator, the medium can be replaced with a medium without BMP signal transduction activator for continued culturing. The culturing time can be, but is not limited to, 1–100 days, 10–90 days, 20–80 days, 30–70 days, 40–60 days, or approximately 50 days.

[0079] Cells may be cultured in a medium containing a Wnt signaling inhibitor for part or all of the culture time in a medium containing a BMP signaling activator. A Wnt signaling inhibitor is a substance that inhibits signaling via Wnt and includes substances that act on Wnt or its receptor, substances that inhibit gene expression of Wnt or its receptor, and substances that inhibit the binding between Wnt and its receptor. Examples of Wnt signaling inhibitors include CKI-7 (N-(2-aminoethyl)-5-chloro-8-isoquinoline sulfonamide, D4476 (4-(4-(2,3-dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide), IWR-1-endo (IWR1e)(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo- [4,7-bridged methylene-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 signaling inhibitor is determined according to the inhibitor to be used and can be 0.1–100 μM, 0.3–30 μM, or 1 μM–10 μM.

[0080] During cultivation, the culture medium can be changed as needed. For example, a portion or all of the culture medium can be replaced every 1 to 4 days. To initiate cultivation in a medium containing a specific component, the entire medium can be replaced with a medium containing that component at the desired concentration, or a portion of the medium can be replaced to achieve the desired final concentration of that component (e.g., half of the medium can be replaced with a medium at twice the final concentration). As described for BMP signal transduction activators, the concentration of this component can be constant or varied during cultivation in a medium containing the specific component.

[0081] In some embodiments, the method for producing retinal tissue disclosed herein includes:

[0082] (1) Pluripotent stem cells were fed at a rate of 0.5 × 10⁻⁶. 5 cells / cm 2 Up to 2.5×10 5 cells / cm 2 The culture medium is inoculated at a density in region A on the surface of the culture medium, wherein the culture medium includes region A and region B on the surface, region A has cell adhesion, and region B is adjacent to at least a portion of region A and has cell adhesion lower than that of region A.

[0083] (2) The pluripotent stem cells seeded in step (1) are cultured in a medium containing ROCK inhibitors for 1 to 16 hours, and

[0084] (3) The cells obtained after step (2) are cultured in a medium containing BMP signal transduction activator.

[0085] The culture medium in step (2) contains undifferentiated maintenance factors, while the culture medium in step (3) does not contain undifferentiated maintenance factors.

[0086] The method further includes culturing cells in a medium containing undifferentiated maintenance factor but not ROCK inhibitor after step (2) and before step (3), and then culturing cells in a medium containing neither undifferentiated maintenance factor nor ROCK inhibitor.

[0087] In some embodiments, the method for producing retinal tissue disclosed herein includes:

[0088] (1) Pluripotent stem cells were fed at a rate of 0.5 × 10⁻⁶. 5 cells / cm 2 Up to 2.5×10 5 cells / cm 2 The culture medium is inoculated at a density in region A on the surface of the culture medium, wherein the culture medium includes region A and region B on the surface, region A has cell adhesion, and region B is adjacent to at least a portion of region A and has cell adhesion lower than that of region A.

[0089] (2) The pluripotent stem cells seeded in step (1) are cultured in a medium containing ROCK inhibitors for 1 to 16 hours, and

[0090] (3) The cells obtained after step (2) are cultured in a medium containing BMP signal transduction activator.

[0091] The culture medium in step (2) contains undifferentiated maintenance factors, while the culture medium in step (3) does not contain undifferentiated maintenance factors.

[0092] The culture in step (3) begins 6 to 8 days after inoculation in step (1) and lasts for 5 to 10 days.

[0093] The method further includes culturing cells in a medium containing undifferentiated maintenance factor but not ROCK inhibitor until 1 to 3 days after inoculation in step (1), and then culturing cells in a medium containing neither undifferentiated maintenance factor nor ROCK inhibitor until 6 to 8 days after inoculation in step (1).

[0094] Culture conditions, such as culture temperature and CO2 concentration, can be set appropriately. The culture temperature can be, for example, 30°C to 40°C or approximately 37°C. The CO2 concentration can be, for example, 1% to 10% or approximately 5%.

[0095] Differentiation into retinal tissue can be confirmed by detecting the expression of cellular markers in the cells of the tissue. Marker expression can be confirmed, for example, at any of days 10–100 of differentiation induction (e.g., day 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) or thereafter. In some embodiments, marker expression is confirmed at any of days 16–22 of differentiation induction.

[0096] The retinal tissue in organisms has a layered structure and contains photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells, Müller cells, and their progenitor cells. As used herein, the cells that make up the retina are called "retinal cells," and the layers that make up the retina are called "retinal layers." The retinal layers in mature retinal tissue include the retinal pigment epithelium layer, photoreceptor cell layer, outer membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. Retinal tissue in the developmental stages before becoming mature retinal tissue may include a neuroblastocyte layer.

[0097] As used herein, the term "retinal progenitor cell" refers to a progenitor cell that can differentiate into one or more mature retinal cells selected from photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, retinal pigment epithelial cells, and Müller cells. As used herein, the term "neuroreretinal progenitor cell" refers to a progenitor cell that can differentiate into one or more mature retinal cells selected from photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Typically, neural retinal progenitor cells do not differentiate into retinal pigment epithelial cells.

[0098] As used herein, the term "retinal layer-specific neuron" refers to the neuron (neuron) that makes up and is specific to the retinal layer. Examples of retinal layer-specific neurons include photoreceptor cells (including rod and cone cells), horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells.

[0099] Examples of retinal cell markers include Rx (also known as Rax), Pax6, and Chx10 expressed in retinal progenitor cells; Nkx2.1 expressed in hypothalamic neuronal progenitor cells but not in retinal progenitor cells; Sox1 expressed in hypothalamic neuroepithelium but not in the retina; and Crx and Blimp1 expressed in photoreceptor progenitor cells. Examples of markers for retinal layer-specific neurons include Chx10, PKCα, and L7 expressed in bipolar cells; Tuj1 and Brn3 expressed in retinal ganglion cells; calreticulin expressed in amacrine cells; calcium-binding protein expressed in horizontal cells; rhodopsin and recovery protein expressed in mature photoreceptor cells; Nrl and rhodopsin expressed in rod cells; Rxr-γ and S-Opsin expressed in cone cells; and RPE65 and Mitf expressed in retinal pigment epithelial cells. Other cell markers used in the examples may also be used.

[0100] In some embodiments, retinal tissue produced by the methods of this disclosure contains Chx10-positive cells or Chx10-positive and Rx-positive cells in a 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 percentage of marker-positive cells can be a percentage at day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 after differentiation induction. In some embodiments, the retinal tissue contains Chx10-positive and Rx-positive cells at a percentage of 70%, 80%, 85%, 90%, or 95% or more on any of days 16–22 of differentiation induction (e.g., day 16, day 18, or day 20). In further embodiments, the retinal tissue contains Chx10-positive and Rx-positive cells at a percentage of 90% or more or 95% or more on any of days 16–22 of differentiation induction (e.g., day 16, day 18, or day 20).

[0101] In some embodiments, the retinal tissue produced by the methods of this disclosure comprises retinal cells in a 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 percentage of cells can be any of days 16 to 100 of differentiation induction (e.g., day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100) or thereafter. The retinal tissue preferably has a layered cell structure.

[0102] Retinal tissue can be collected from the culture medium using conventional methods. For example, it can be collected using instruments such as forceps. Alternatively, when the culture medium is coated with a stimulus-responsive polymer (e.g., a temperature-responsive polymer or a light-responsive polymer), retinal tissue can be collected by applying the corresponding stimulus. For example, when using a temperature-responsive polymer whose properties reversibly change from cell-adhesive (hydrophobic) to non-cell-adhesive (hydrophilic) at a specific temperature, retinal tissue can be collected by placing the culture medium at that temperature.

[0103] The collected retinal tissue can be maintained in a standard culture container or transferred to a suitable storage container for cryopreservation.

[0104] The shape and size of the retinal tissue depend on the shape and size of region A. The area of ​​the retinal tissue can range from 0.01 to 100 cm². 2 0.03~30cm 2 or 0.1-10cm 2 In some implementations, the area of ​​the retinal tissue is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm. 2 The above and 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1cm 2 Below (upper and lower limits are selected independently). For example, the area of ​​retinal tissue can be 0.5–10 cm². 2 0.7~10cm 2 1-10cm 2 0.5-5cm 2 0.7-5cm 2 1-5cm 2 0.5~2cm 2 0.7~2cm 2 0.5~1cm 2 or 0.7-1cm 2Retinal tissue can have a circular shape with a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 cm or more and 10, 9, 8, 7, 6, 5, 4, 3, 2 or less (the upper and lower limits are selected independently) or a polygonal shape with the same area as a circular shape. In some embodiments, the retinal tissue has a circular shape with a diameter of 0.1–10 cm, 0.1–5 cm, 0.1–3 cm or 0.2–1 cm. In further embodiments, the retinal tissue has a circular shape with a diameter of 1–10 cm, 1–5 cm or 1–3 cm. As used herein, retinal tissue having a circular or polygonal shape refers to retinal tissue having a shape similar to that of retinal tissue produced by the method of this disclosure using a culture medium having a circular or polygonal region A. As used herein, the area and diameter of retinal tissue refer to the area within the periphery of the retinal tissue as measured based on images taken using a stereomicroscope, and the length of the longest straight line connecting any two points in the periphery, respectively. The thickness of the retinal tissue can be greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 μm and less than 500, 400, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, or 220 μm (the upper and lower limits are selected independently). In some embodiments, the thickness of the retinal tissue is from 50 μm to 300 μm. The retinal tissue does not need to have the same thickness throughout the tissue. When the retinal tissue is defined by a thickness range, the retinal tissue can have a thickness within that range at any given location. The retinal tissue obtained by the methods of this disclosure typically contains about 30 or fewer cells in a direction perpendicular to the culture medium, or has a thickness of less than 500 μm, and is referred to herein as sheet-like retinal tissue or retinal sheet. Therefore, in one aspect, this disclosure provides sheet-like retinal tissue derived from pluripotent stem cells.

[0105] The method of this disclosure can produce uniform retinal tissue in region A that is virtually free of pores or voids (i.e., areas without cells). In some embodiments, the retinal tissue produced by the method of this disclosure covers more than 95%, more than 96%, more than 97%, more than 98%, and more than 99% of region A.

[0106] The percentage of area A covered by retinal tissue can be measured by any method, and for example, by staining the retinal tissue. For instance, it can be measured by staining for the expression of the aforementioned retinal cell markers (e.g., Chx10) (e.g., by immunostaining with a fluorescently labeled anti-Chx10 antibody). Then, using imaging software such as ImageJ software (NIH), the ratio of the area positive for retinal cell markers to the area of ​​region A can be calculated.

[0107] The method disclosed herein can also efficiently produce retinal tissue with a low percentage of non-target cells and a high percentage of retinal cells. Specifically, the method disclosed herein can produce retinal tissue with high expression of genes expressed in retinal cells and low expression of genes expressed in non-target cells. Retinal cells and their markers are as described above. Non-target cells include eye-related cells (e.g., ciliary body, lens, retinal pigment epithelium) and brain-spinal cord-related cells (e.g., telencephalon, midbrain, spinal cord), and these markers include AQP1 (ciliary body), EMX2 (dorsal telencephalon), FOXG1 (telencephalon), DLX2 (ventral neural tissue, ventral telencephalon), and HOXB2 (spinal cord). These markers can be measured by any method appropriately selected by those skilled in the art, such as real-time PCR.

[0108] Retinal tissue produced by the methods of this disclosure can be used as a transplant material to treat diseases, particularly those related to or caused by conditions of the retinal tissue or retinal cells. Therefore, this disclosure provides compositions comprising the retinal tissue of this disclosure for treating diseases and methods of treating diseases including transplanting the retinal tissue of this disclosure into a subject.

[0109] Examples of diseases associated with or caused by conditions of the retinal tissue or retinal cells include retinal degeneration, retinitis pigmentosa, age-related macular degeneration, organic mercury poisoning, chloroquine retinopathy, glaucoma, diabetic retinopathy, and neonatal retinopathy. In some embodiments, the disease is retinitis pigmentosa.

[0110] When used for transplantation, the retinal tissue is preferably derived from pluripotent stem cells that are highly histocompatible with the subject (i.e., the recipient) to whom the retinal tissue is to be transplanted. In some embodiments, the retinal tissue is derived from pluripotent stem cells that have some or all of the subject's histocompatibility antigens (e.g., HLA type) or from pluripotent stem cells derived from the subject's cells.

[0111] Retinal tissue can be used for treatment in its shape and size after differentiation induction, or it can be cut to an appropriate size using forceps or other means to maintain its layered structure. In addition to retinal tissue, compositions containing the retinal tissue of this disclosure may also contain a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a physiological aqueous solvent such as physiological saline, buffer solution, or serum-free culture medium. The composition may further contain additives such as preservatives, stabilizers, reducing agents, and isotonic agents. Compositions containing the retinal tissue of this disclosure can be cryopreserved, and in this case, the composition may contain a suitable cryoprotectant.

[0112] Retinal tissue produced by the methods of this disclosure can also be used to evaluate the toxicity or efficacy of pharmaceutical agents on retinal tissue. In some embodiments, the retinal tissue of this disclosure is used to evaluate the toxicity or efficacy of therapeutic agents for diseases associated with or caused by conditions of retinal tissue or retinal cells. In this case, retinal tissue is preferably produced from pluripotent stem cells derived from a subject suffering from the disease.

[0113] The retinal tissue produced by the methods of this disclosure has a structure suitable for transplantation or evaluation of the toxicity or efficacy of a drug, and is used in a state that maintains its layered structure. However, in some cases, the retinal tissue 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. This method of using retinal tissue is also within the scope of this disclosure.

[0114] The following illustrates exemplary embodiments of this disclosure.

[0115] 1. A method for producing retinal tissue, comprising the following steps:

[0116] (1) Pluripotent stem cells were fed at a rate of 0.5 × 10⁻⁶. 5 cells / cm 2 Up to 2.5×10 5 cells / cm 2 The culture medium is inoculated at a density in region A on the surface of the culture medium, wherein the culture medium includes region A and region B on the surface, region A has cell adhesion, and region B is adjacent to at least a portion of region A and has cell adhesion lower than that of region A.

[0117] (2) The pluripotent stem cells seeded in step (1) are cultured in a medium containing ROCK inhibitors for 1 to 16 hours, and

[0118] (3) The cells obtained after step (2) are cultured in a medium containing BMP signal transduction activator.

[0119] 2. According to the method of Project 1, wherein the BMP signal transduction activator is contained in the culture medium at a concentration of 3 nM to 15 nM or at a concentration providing equivalent activity to BMP4 at a concentration of 3 nM to 15 nM.

[0120] 3. The method according to Project 1 or 2, wherein the BMP signal transduction activator is selected from BMP2, BMP4, BMP7 and GDF7.

[0121] 4. The method according to any one of items 1 to 3, wherein the BMP signal transduction activator is BMP4.

[0122] 5. According to the method of any one of items 1 to 4, wherein the cells are cultured in a medium containing BMP signal transduction activator for 5 to 10 days.

[0123] 6. According to the method of any one of items 1 to 5, wherein pluripotent stem cells are used at 0.5 × 10 5 cells / cm 2 Up to 1.5×10 5 cells / cm 2 Density of vaccination.

[0124] 7. According to the method of any one of items 1 to 6, wherein the cells are cultured in a medium containing a ROCK inhibitor for 1 to 3 hours.

[0125] 8. Following the method in Project 7, the cells were cultured in a medium containing a ROCK inhibitor for approximately 2 hours.

[0126] 9. According to any one of items 1 to 8, wherein region A is coated with a cell-adhesive substance.

[0127] 10. According to the method in Project 9, the cell adhesion substance is laminin.

[0128] 11. Using any of the methods in items 1 to 10, where region A is surrounded by region B.

[0129] 12. The method of any one of items 1 to 11, wherein the pluripotent stem cells are human iPS cells.

[0130] 13. The method according to any one of items 1 to 12, wherein the retinal tissue is sheet-like retinal tissue.

[0131] 14. According to the method of any one of items 1 to 13, the retinal tissue covers more than 95% of the area A.

[0132] 15. A retinal tissue produced by means of any one of items 1 to 14.

[0133] 16. A composition comprising retinal tissue according to item 15.

[0134] 17. The composition according to item 16, wherein the composition is used to treat a disease.

[0135] 18. The composition according to item 17, wherein the disease is a disease related to or caused by a condition of the retinal tissue or retinal cells.

[0136] 19. The composition according to item 17 or 18, wherein the disease is retinitis pigmentosa.

[0137] 20. The composition according to item 19, wherein the composition is used to evaluate the toxicity or efficacy of a pharmaceutical agent.

[0138] 21. A method for treating a disease associated with or caused by a condition of retinal tissue or retinal cells, comprising transplanting retinal tissue according to item 15 into a subject in need.

[0139] 22. The retinal tissue according to item 15, used to treat diseases related to or caused by conditions of the retinal tissue or retinal cells.

[0140] 23. The use of retinal tissue according to item 15 for the manufacture of a medicament for the treatment of diseases related to or caused by conditions of the retinal tissue or retinal cells.

[0141] The present invention will be further illustrated by the following embodiments, but should not be limited to these embodiments in any way.

[0142] Example

[0143] [Reference Example]

[0144] I. Patterned Culture

[0145] I-1. Preparation of PDMS Sheets

[0146] 1. Prepare a columnar mold with a shape on top designed to be used as a cell adhesion region using a 3D printer (Formlab 3).

[0147] 2. Mix PDMS (polydimethylsiloxane) (SILPOT 184W / C, Dow Corning Toray Co. Ltd.) with a curing agent (10:1) and degas under vacuum.

[0148] 3. After placing the mold prepared by the 3D printer on the surface of the Petri dish, PDMS is poured into the dish and cured at 80°C for 2 hours. Then, the prepared PDMS sheet is removed from the mold and the unwanted parts of the sheet are cut off to obtain the desired PDMS sheet.

[0149] I-2. Differentiation Induction

[0150] Differentiation into the retina is induced through the following process. Figure 1 The experimental protocol is shown.

[0151] Day 0: Preparation of the Petri Dish

[0152] process

[0153] 1. Sterilize the PDMS sheet prepared therefrom with 70% ethanol and place it on a culture dish (μ-culture dish 35mm, tall type, 81156, ibidi).

[0154] 2. After removing the lid from the petri dish, incubate the petri dish in a heating block at 60°C for 5 to 10 minutes. If the petri dish is difficult to dry, dry it in a desiccator set at 60°C.

[0155] 3. Add 7 μL of iMatrix-511 (Nippi.Inc.) to 500 μL of PBS, and add 200 μL of the resulting solution into the circle (1 cm in diameter) of the PDMS sheet.

[0156] 4. Incubate the petri dish at 37°C or at room temperature (RT) for at least 1 hour.

[0157] <Day 0: Cells seeded into culture dishes>

[0158] Prepare

[0159] TrypLE TM Select (containing 1 mM EDTA) (Thermo Fisher Scientific) was mixed with an equal volume of 0.5 mM EDTA / PBS to prepare 0.5× TrypLE. TM Select (final concentration is 0.75 mM EDTA).

[0160] Will AK02N (Ajinomoto Co., Inc.) (a mixture of solutions A, B and C of this product, hereinafter referred to as AK02N) is thoroughly mixed with 10 mM Y-27632 (TOCRIS) added to the culture medium at a volume of 1 / 1000 (final concentration of 10 μM) (hereinafter referred to as AK02N+Y).

[0161] process

[0162] 1. KhES-1_Rx::Venus cells (KhES-1 cells obtained by knocking in a GFP variant called Venus into the locus of the retinal marker Rx) were collected at optimal confluence by removing the culture medium and washing twice with 1 mL PBS(-).

[0163] 2. Add 0.5×TrypLE to each well at a concentration of 500 μL / well. TM Select was incubated at 37°C and 5% CO2.

[0164] 3. In the case of 0.5×TrypLE TM After the Select was removed, PBS(-) was added to the wells at 2 mL / well and the cells were peeled off by spraying PBS(-).

[0165] 4. Collect the cells into 15 mL Falcon tubes and count the number of cells.

[0166] 5. Add the required number of cells (5 × 10⁶ cells per culture dish) 5 The cells were transferred to another tube and centrifuged at 1000 rpm for 5 minutes at 20°C.

[0167] 6. After removing the supernatant, the cells were resuspended with AK02N+Y to provide 5 × 10⁻⁶ cells / mL. 5 Cell suspension of 200 μL LAK02N+Y per cell.

[0168] 7. Remove the iMatrix coating solution from the PDMS sheet using a pipette and remove the cell suspension (5×10⁻⁶). 5 Add 200 μL of AK02N+Y per cell to the circle and incubate at 37°C for 30 minutes.

[0169] 8. Use sterile forceps to remove the PDMS sheet from the culture dish.

[0170] 9. Incubate the cells with 300 μL of AK02 N+Y added to the culture dish at 37°C and 5% CO2.

[0171] Day 1: Replace the culture medium with AK02+PS

[0172] process

[0173] 1. Add penicillin-streptomycin (15070-062, Thermo Fisher Scientific) to AK02N and mix thoroughly (the resulting culture medium does not contain Y-27632, and is referred to as AK02N+PS below).

[0174] 2. Remove the culture medium with a pipette and add 500 μL of AK02N+PS to the petri dish.

[0175] Day 2: Differentiation Induction Begins

[0176] process

[0177] 1. Wash the cells three times with 1 mL gfCDM (10% KSR) (Table 1).

[0178] 2. Replace all the culture medium with 1 mL gfCDM (10% KSR).

[0179] Table 1

[0180]

[0181] Add BSA (5g) to 20mL of cell culture water (W3500, Sigma Aldrich) and let stand for several hours to dissolve.

[0182] <Day 3, Day 6: Add BMP inhibitor>

[0183] 1. Replace all culture medium with 1 mL gfCDM (10% KSR) containing LDN-193189 (04-0074-02, ReproCELL Inc.) (hereinafter referred to as LDN) (final concentration 100 nM LDN).

[0184] Day 8: Add BMP

[0185] 1. Wash the cells three times with 1 mL gfCDM (10% KSR).

[0186] 2. Replace all the culture medium with 1 mL of gfCDM (10% KSR) containing BMP4 (final concentration 1.5 nM BMP4). Prepare the BMP4-containing culture medium by adding 1 μM BMP4 solution (prepared by diluting 10 μg BMP4 (R&D Systems) with 275 μL of 0.1% BSA) to gfCDM (10% KSR).

[0187] <Day 11, Day 14: Replace half the culture medium>

[0188] 1. After removing 500 μL of culture medium, add 500 μL of gfCDM (10% KSR) to the petri dish (replace half the volume). Then, replace half the volume of culture medium every 3 days.

[0189] <Day 17: Replace the culture medium with DMEM / F-12+N2>

[0190] 1. Use 1 mL of DMEM / F-12 and GlutaMAX to treat the cells. TM The supplement (10565-042, Thermo Fisher Scientific) was rinsed three times.

[0191] 2. Add 1 mL of DMEM / F-12+N2 (Table 2) to the culture dish.

[0192] Table 2

[0193]

[0194] 3. Culture the cells until day 20.

[0195] <Day 20: Cultured in control maturation medium>

[0196] 1. Wash the cells three times with 1 mL of control maturation medium (Table 3).

[0197] 2. Culture the cells in 1 mL of control maturation medium added to the culture dish.

[0198] Table 3

[0199]

[0200] Then, change the culture medium every 2 to 3 days.

[0201] To prepare unpatterned cultured cells (non-patterned cultured cells), cells were cultured in micro-culture dishes (μ-culture dishes, 35 mm, tall, 81156, ibidi) according to the same procedure (without PDMS sheets), with their entire surface coated with iMatrix-511 (Nippi. Inc.) (0.25 μg / cm³). 2 ).

[0202] II. The Effects of Patterned Culture on Retinal Differentiation

[0203] II-1. Analytical Methods

[0204] -RNA sequencing (RNA-seq)

[0205] 1. Total RNA was prepared from day 8 samples of patterned or unpatterned cultured cells using the RNeasy Micro Kit (QIAGEN).

[0206] 2. Sequence libraries were constructed using the Next Ultra II Directional RNA Library Prep Kit for illumine E7760 (NEB), and sequences were determined using a next-generation sequencer (NextSeq, Illumina).

[0207] 3. After checking the quality of the sequence data using FastQC (v0.11.9), TrimGalore (0.6.1), cutadapt (1.18), and Bowtie2 (v2.4.19), mapping was performed on STAR (2.7.5a), samtools (1.1), and SUBREAD (v2.0.1), followed by secondary analysis using R (version 3.6.3) and edgeR (v3.28.1).

[0208] Gene Ontology (GO) Analysis

[0209] For the list of differentially expressed genes obtained through the secondary analysis of II-1, enrichment analysis of GO biological processes was performed using Metascape.

[0210] Single-cell RNA sequencing (scRNA-seq)

[0211] 1. By using TrypLE containing 150 μg / ml DNase I (Roche) at 37°C. TM Select (ThermoFisher Scientific) for 5 minutes to dissociate day 100 retinal tissue into single cells and neutralize with 0.04% BSA / PBS.

[0212] 2. Pellet the dissociated cells, resuspend them in 0.04% BSA / PBS, and pass them through a 35 μm filter to provide a single-cell suspension.

[0213] 3. A cDNA library was constructed from approximately 10,000 single cells using the Chromium Single Cell 3' Kit v3 (10x, Genomics). Sequences were determined using a paired-end 100-cycle kit (28+8+91) and an Illumina NovaSeq sequencer.

[0214] 4. Mapping is performed using CellRanger (version 3.0.2) and secondary analysis is performed using Seurrat (version 3.0).

[0215] The following markers are used for various cell types:

[0216] Stalk cells (stalk): VAX1 / PAX2

[0217] Retinal pigment epithelial cells (RPE): MITF / PMEL

[0218] Amacrine adenoids (AC): ONECUT3 / PRDM13

[0219] Retinal ganglion cells (RGCs): POU4F2

[0220] Photoreceptor cells (PR): CRX / NEUROD4

[0221] Neural progenitor cells (NPCs): HES6 / NEUROD1

[0222] Retinal progenitor cells: CHX10 / PAX6

[0223] Observation of GFP expression

[0224] Cells expressing GFP were observed using the Keyence BZ-X all-in-one microscope. After obtaining phase difference images and GFP fluorescence images with a 10× lens, complete images of the colonies were generated through tiling.

[0225] Immunostaining

[0226] The expression of Chx10, recovery protein, FOXG1, Crx, Tuj1, Zo-1 and Pax6 was evaluated by immunostaining with primary antibodies specific to each marker and secondary antibodies suitable for that primary antibody.

[0227] II-2. Induction of retinal differentiation in patterned culture

[0228] Gene expression in patterned cultured cells was compared with that in unpatterned cultured cells (seeded across the entire surface of a culture dish). RNA-seq and GO analyses were performed on cells before the addition of BMP4 (day 8). In patterned cultured cells, unlike in unpatterned cultured cells, neural differentiation was enhanced before the addition of BMP4, which induces retinal differentiation. Figure 2 In unpatterned cultured cells, BMP and Hippo signaling are enhanced. Figure 3 ).

[0229] Retinal differentiation following BMP4 addition was evaluated by observing the expression of GFP (venus) over time under the promoter of Rx (a retinal marker). Earlier GFP expression was observed in patterned cultured cells, indicating superior differentiation induction efficiency compared to unpatterned cultured cells. Figure 4In addition, on day 18, the expression of Chx10 (a marker of bipolar cells and their progenitor cells) was evaluated. For both patterned and unpatterned cultured cells, expression was evaluated with and without the addition of BMP4. Chx10 expression was assessed by quantitative PCR (using HPRT1 as an endogenous control) and immunostaining. Figure 5 As shown, Chx10 expression was significantly increased in patterned cultured cells compared to unpatterned cultured cells. Figure 6 The image shows cells on day 18 after retinal differentiation via patterned culture. Rx and Chx10 are expressed in almost the entire surface area, indicating that over 95% of the cells express these markers. On day 58, recovery protein (a marker of retinal photoreceptor cells) is expressed in cells after retinal differentiation via patterned culture. Figure 7 In cells after patterned culture without BMP4 addition, FOXG1 (a marker of the telencephalon) was expressed on day 18. Figure 8 ).

[0230] II-2. Differentiation efficiency

[0231] On day 100, scRNA-seq was performed on cells after retinal differentiation using patterned culture. scRNA-seq showed that over 97% of the cells differentiated into retinal cells. Figure 9 (Left). GFP expression in cells on day 74 was evaluated by FACS, and over 90% of cells were GFP-positive. Figure 9 ,right).

[0232] II-3. Size of Patterned Cultures

[0233] PDMS sheets with circular pores of 2 mm, 5 mm, 7 mm, or 1 cm in diameter were prepared, and retinal differentiation was induced using the same procedure. GFP expression on day 20 was evaluated, and differentiation induction was confirmed at any pore size. Figure 10 ).

[0234] II-4. Polarity of retinal tissue

[0235] The polarity of the differentiated tissue was evaluated. Crx (a marker of photoreceptor cells) expression in the retinal tissue at day 40 was evaluated by immunostaining. Crx expression was observed at the apical side of the tissue, indicating that the differentiated tissue possessed retinal polarity. Figure 11 ).

[0236] II-5. Differentiation induction from other ES or iPS cell lines

[0237] Retinal differentiation was induced in KthES11 cells via patterned culture using the same procedure, and the expression of Crx, Chx10, and the neuronal marker Tuj1 was evaluated by immunostaining on day 50. These markers were expressed throughout the tissue, indicating that retinal differentiation was induced in KthES11 cells, as it is in KhES-1_Rx:Venus cells. Figure 12 ).

[0238] Similarly, retinal differentiation was induced using iPS cell lines 201B7 and 253G1, and Crx and Chx10 expression was evaluated on day 34. Crx and Chx10 were expressed throughout the tissue, indicating that retinal differentiation was also induced in these iPS cells. Figure 13 Furthermore, the expression of Zo-1 and Chx10 in tissues derived from 201B7 cells at day 41 was evaluated by immunostaining. Zo-1 is a marker of tight junctions. Layered expression of Zo-1 and Chx10 was observed, indicating that the differentiated tissues possess the layered structure of the retina (data not shown).

[0239] III. BMP signaling inhibitors

[0240] The effect of LDN-193189 (a BMP signaling inhibitor) on differentiation induction was evaluated. GFP expression on day 11 was compared between cells prepared according to the patterned culture described above (which included the addition of LDN-193189 from day 3 to day 8) and cells prepared according to the same procedure but without the addition of LDN-193189. Cells with the addition of LDN-193189 showed more uniform GFP expression compared to cells without LDN-193189. Figure 14 ).

[0241] Furthermore, the expression of GFP and Chx10 on day 18 was evaluated in patterned and unpatterned cultured cells, with or without the addition of LDN-193189. Chx10 expression was assessed by quantitative PCR (using HPRT1 as an endogenous control) and immunostaining. Even without patterned culture, the addition of LDN-193189 increased GFP and Chx10 expression, indicating a promotion of retinal (retinal) induction. Figure 15 and Figure 16 ).

[0242] IV. Patterned culture using hydrophilic polymers

[0243] Patterned cultures were performed using non-cell-adhesive regions prepared by coating with MPC (2-methacryloyloxyethylphosphocholine) polymer. Circular PDMS sheets with a diameter of 1 cm were placed on culture dishes (μ-culture dish 35 mm, tall type, 81156, ibidi). After coating the culture dish with a solution containing 0.5% (v / v) MPC polymer in ethanol, the PDMS sheets were removed from the culture dish. A solution prepared by adding 7 μL of iMatrix-511 (Nippi.Inc.) to 500 μL of PBS was added to the culture dish. Following these procedures, iMatrix did not adhere to the regions pre-coated with MPC polymer, and only the 1 cm circular regions protected by the PDMS sheets were coated with iMatrix. Retinal differentiation was induced on this culture dish according to the same procedure, except that Iwp2 (REPROCELL) (2 μM) was added starting from day 20.

[0244] Even when non-cell-adhesive regions were prepared using MPC polymer coating, efficient and uniform GFP expression was observed. Figure 17 ).

[0245] [Example]

[0246] <Example 1: Conditions for research on retinal tissue production via patterned culture>

[0247] Focusing on patterned culture, we compared the conditions used to prepare retinal tissue from undifferentiated human iPS cells.

[0248] Human iPS cells (LPF11 and DSP-SQ strains established at Sumitomo Pharma Co., Ltd.) were established using commercially available Sendai virus vectors (four factors: Oct3 / 4, Sox2, KLF4, and c-Myc, IDPharma Cytotune kit), based on the methods described in Thermo Fisher Scientific's published protocol (iPS2.0 Sendai Reprogramming Kit, Publication No. MAN0009378, Revision 1.0) and Kyoto University's published protocol (Establishment and maintenance culture of feeder-free human iPS cells, CiRA_Ff-iPSC_protocol_JP_v140310, http: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html), using StemFit medium (AK03, Ajinomoto Co., Inc.) and laminin 511-E8 (Nippi. Inc.).

[0249] Human iPS cells (LPF11 and DSP-SQ strains) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, Ajinomoto Co., Inc.) was used as the feeder-free medium and laminin 511-E8 (Nippi., Inc.) was used as the feeder-free scaffold.

[0250] Specifically, for maintenance culture, subconfluent human iPS cells (LPF11 and DSP-SQ strains) were first washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then seeded onto plastic culture dishes coated with laminin 511-E8 and cultured in StemFit medium without a feeder layer in the presence of Y-27632 (a ROCK inhibitor, 10 μM). When using 6-well plates (Iwaki, for cell culture, 9.4 cm²), the culture was... 2 When used as a plastic culture dish, the number of human iPS cells seeded as single cells was 1.0 × 10⁻⁶. 4 One day after inoculation, the culture medium was replaced with StemFit medium without Y-27632. Thereafter, the culture medium was replaced with StemFit medium without Y-27632 every 1 to 2 days. Cells were then cultured until 6 days after inoculation.

[0251] For patterning, droplets (1 cm in diameter) of a solution containing 0.785 μL of laminin 511-E8 (Nippi. Inc.) in 100 μL PBS (laminin 511-E8 concentration 3.925 ng / μl) were added to 12-well plates (Iwaki, cell culture, culture area 3.8 cm²). 2 The center of the wells was filled with laminin 511-E8, and then incubated at 37°C or room temperature for 1 to 3 hours to prepare patterned 12-well plates for culture (laminin 511-E8: 0.5 μg / cm). 2 ).

[0252] The sub-confluent human iPS cells (LPF11 strain) were then washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then dispersed from 0.5 × 10⁻⁶ cells. 5 Up to 5.0×10 5 cells / cm 2Cells at various densities were seeded into one well of a patterned culture plate and cultured in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM) without a feeder layer. Figure 18 ).

[0253] The culture time using Y-27632 ranged from 2 to 32 hours, after which the medium was replaced with 2 mL of StemFit medium (ROCK inhibitor, 0 μM) without Y-27632. Figure 19 ).

[0254] Two days after iPS cells were seeded into patterned culture plates, the medium was replaced with 2 mL gfCDM (10% KSR) (Table 1) (hereinafter also referred to as serum-free medium (gfCDM+KSR)). Five days later, the medium was replaced with gfCDM+KSR containing recombinant human BMP4 (R&D). Various final concentrations of exogenous recombinant human BMP4 (R&D) from 0 to 12 nM were compared. Figure 20 and Figure 21 ).

[0255] Subsequently, every 2 to 3 days, half of the culture medium was replaced with serum-free medium (gfCDM+KSR) that did not contain recombinant human BMP4. When replacing half of the culture medium, the half-volume culture medium (i.e., 1 ml) in the culture vessel was discarded, and 1 mL of new serum-free medium (gfCDM+KSR) was added to bring the total volume of the culture medium to 2 ml.

[0256] After 17 days of patterned culture, the cells were cultured in 5% CO2 using control maturation medium (Table 3).

[0257] On day 20 of patterned culture, cells were fixed with 4% paraformaldehyde, immunostained for retinal progenitor markers Chx10 (anti-Chx10 antibody, Exalpha) and Rx (anti-Rx antibody, Takara), and observed using a fluorescence microscope (Keyence). Detailed results of the optimized differentiation method are shown in Examples 2 through 4.

[0258] <Example 2: Study on Seeding Density of Human iPS Cells in Patterned Culture>

[0259] Human iPS cells (LPF11 line) were stored at various cell densities (cells / cm²). 2 Inoculate onto patterned culture plates to investigate optimal conditions.

[0260] First, a patterned 12-well plate for culture was provided, prepared by the method described in Example 1. Sub-merging human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then cultured at 0.5 × 10⁻⁶ cells / well. 5 1.0×10 5 2.5×10 5 Or 5.0×10 5 cells / cm 2 The culture medium was seeded at a density of 10 μM into one well of a patterned culture plate and cultured in StemFit medium in the presence of Y-27632 (a ROCK inhibitor, 10 μM) under conditions without a feeder layer. After two hours, the medium was replaced with 2 mL of StemFit medium without Y-27632.

[0261] Bright-field microscopy observations of human iPS cells at 1 hour, 1 day, 2 days, or 3 days after seeding them into patterned culture plates. Figure 18 As shown in the image. Figure 18 Analysis of the images in the image shows that at 5.0×10 5 cells / cm 2 At a seeding density of [insert seeding density here], human iPS cells adhered unevenly to the cell-adhesive region, while at 0.5 × 10 [insert seeding density here], [insert seeding density here]. 5 ~2.5×10 5 cells / cm 2 At the seeding density, cells adhere uniformly to the cell-adhesive region.

[0262] Subsequently, patterned culture was performed as described in Example 1, and on day 20 after inoculation, the cells were fixed with 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha) (a marker of retinal progenitor cells). These immunostained cells were observed under a fluorescence microscope (Keyence), and the results were... Figure 19 As shown in the image. Figure 19 Analysis of the images shows that at 1.0 × 10 5 and 2.5×10 5 cells / cm 2 Under the inoculation conditions, retinal tissue in which Chx10-positive cells were uniformly present was produced. On the other hand, 5.0 × 10 5 cells / cm 2 The inoculation conditions resulted in pores and gaps on the cell sheet surface lacking Chx10 positive cells, indicating a low efficiency in the formation of uniformly differentiated retinal tissue.

[0263] These results indicate that the optimal cell density for inoculating undifferentiated human iPS cells is 1.0 × 10⁻⁶. 5 ~2.5×10 5 cells / cm 2 .

[0264] <Example 3: Study on the duration of action of ROCK inhibitor during cell seeding in patterned culture>

[0265] The optimal incubation time of Y-27632 was investigated when human iPS cells (LPF11 and DSP-SQ strains) were seeded onto patterned culture plates.

[0266] First, a patterned 12-well plate for culture was provided, prepared by the method described in Example 1. Sub-merging human iPS cells (LPF11 and DSP-SQ strains) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then cultured at 2.0 × 10⁻⁶ cells / well. 5 cells / cm 2 Human iPS cells were seeded at a density in one well of a patterned culture plate and cultured in the presence of Y-27632 (a ROCK inhibitor, 10 μM), in StemFit medium, and under feeder-free conditions. Then, after 2, 4, 8, 16, or 32 hours of culture, the medium was replaced with 2 mL of StemFit medium without Y-27632. Under all conditions, human iPS cells survived and could continue to be cultured, indicating that the removal of Y-27632 within a short period is not a problem as long as human iPS cells adhere to the cell-adhesive region in a near-confluent state.

[0267] Then, patterned culture was performed as described in Example 1, and on day 20 after inoculation, the cells were fixed with 4% paraformaldehyde and bright-field images were obtained using a fluorescence microscope (Keyence). Results using human iPS cells (LPF11 line) were... Figure 20The results showed that the longer the Y-27632 treatment time, the more pores were generated on the surface of the cell sheet, resulting in lower efficiency in the formation of uniformly differentiated retinal tissue. Immunostaining with Chx10 (anti-Chx10 antibody, Exalpha) (a marker of retinal progenitor cells) and observation using fluorescence microscopy (Keyence) showed the absence of retinal progenitor cells in the pores on the sheet surface. The percentage of Chx10-positive areas on the sheet surface was calculated using ImageJ software (NIH), and was above 97% for sheets treated with Y-27632 for less than 16 hours (16 hours: 97%, 8 hours: 99%, 4 hours: 98%, 2 hours: 99%), compared to 91% for sheets treated with Y-27632 for 32 hours. Furthermore, counting the number of Chx10-negative pores at each treatment time of Y-27632 showed that treatment time of Y-27632 for less than 16 hours could inhibit the formation of a large number of pores on the sheet surface. Similar to human iPS cells (DSP-SQ strain), shortening the treatment time of Y-27632 resulted in fewer Chx10-negative areas on the sheet surface. Figure 21 ).

[0268] These results indicate that for the preparation of retinal tissue via patterned culture, the optimal duration of action of the ROCK inhibitor during seeding of undifferentiated human iPS cells is 1 to 16 hours.

[0269] <Example 4: Study on the concentration of BMP activator added for retinal differentiation via patterned culture>

[0270] Next, we investigated the optimal concentration of BMP4 to be added to human iPS cells during patterned culture.

[0271] First, a patterned 12-well plate for culture was provided, prepared by the method described in Example 1. Submerged human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then cultured at 2.0 × 10⁻⁶ cells / well. 5 cells / cm 2 The culture medium was seeded at a density into one well of a patterned culture plate and cultured in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM) under conditions without a feeder layer. After two hours, the medium was replaced with 2 mL of StemFit medium without Y-27632, and patterned culture was performed as described in Example 1.

[0272] On day 7 of patterned culture, the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous recombinant human BMP4 (R&D) at a final concentration of 0, 1.5, 3, 6, or 12 nM. Thereafter, half of the medium was replaced with serum-free medium (gfCDM+KSR) without recombinant human BMP4 every 2 to 3 days. For half-volume replacement, half the medium in the culture vessel (i.e., 1 ml) was discarded, and 1 mL of fresh serum-free medium (gfCDM+KSR) was added to bring the total medium volume to 2 ml.

[0273] On day 20 of patterned culture, cells were fixed with 4% paraformaldehyde and immunostained with the retinal progenitor marker Rx (anti-Rx antibody, Takara). These immunostained cells were observed under a fluorescence microscope (Keyence), and the results were... Figure 22 As shown in the image. Figure 22 Analysis of the images showed that the expression of the retinal progenitor cell marker Rx was stronger in retinal tissue induced with BMP4 concentrations of 3–12 nM compared to retinal tissue induced with 1.5 nM BMP4 concentration.

[0274] Total RNA was then extracted from retinal tissue using a centrifuge column (QIAGEN, RNeasy Micro kit) according to the method described in the kit. The concentration of total RNA was measured using a measuring device (Nanodrop, Thermo Scientific), and then reverse transcribed into cDNA using reverse transcriptase and primers (Reverse Transcription Master Mix kit, Fluidigm). Multiplex PCR reactions (pre-run) were performed using the cDNA and all probes used for validation using a PCR system (Veriti 96-well thermal cycler, AppliedBiosystems). The pre-run reaction solution was then injected into flow-through wells (96.96 Dynamic Array IFC, Fluidigm) using an IFC Controller HX (Fluidigm), and the expression levels of marker genes targeting the neuroretina and byproducts outside the neuroretina were determined by real-time PCR using a multi-sample real-time PCR system (BiomarkHD, Fluidigm).

[0275] The analysis results of the relative mRNA expression levels of each gene are as follows: Figure 23The results are shown in the figure. Specifically, the ΔCt value was calculated from the difference between the Ct value of the target gene and the Ct value of GAPDH (the gene used as an internal standard), and the ratio of the expression levels of each gene was calculated from the difference in ΔCt between samples (ΔΔCt). The results showed that, similar to the results of immunostaining for Rx expression, retinal tissue induced to differentiate into retinal cells at concentrations of 3–12 nM BMP4 had higher expression of the retinal progenitor marker genes Chx10 and Rx compared to retinal tissue induced to differentiate into retinal cells at a concentration of 1.5 nM BMP4.

[0276] The study also found that retinal tissues induced to differentiate into retina at BMP4 concentrations of 3–12 nM had low and suppressed Emx2 (a marker gene for non-target cells) expression.

[0277] These results indicate that the optimal final concentration of BMP4 for inducing retinal differentiation is 3–15 nM for preparing retinal tissue via patterned culture.

[0278] <Example 5: Patterned Culture Using Optimized Conditions>

[0279] The quality of retinal tissue differentiated by the method of the reference example and by the method optimized for various conditions according to the studies in Examples 2 to 4 (optimized method) was compared by measuring the expression levels of marker genes for neuroretinal and byproducts other than neuroretinal using real-time PCR.

[0280] First, a patterned 12-well plate for culture was provided, prepared by the method described in Example 1. Sub-merging human iPS cells (LPF11 line) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then cultured at a density of 5.0 × 10⁻⁶ cells using conventional methods. 5 cells / cm 2 The optimization method uses 2.0 × 10. 5 cells / cm 2 The culture medium was seeded at a density into one well of a patterned culture plate and cultured in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM) under feeder-free conditions. Then, for the reference method, the medium was replaced with 2 mL of StemFit medium without Y-27632 after 24 hours and for the optimized method, the patterned culture was continued as described in Example 1.

[0281] On day 7 of patterned culture, the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous recombinant human BMP4 (R&D) (final concentration of 1.5 nM for the reference method and 6 nM for the optimized method). Thereafter, half of the medium was replaced with serum-free medium (gfCDM+KSR) without recombinant human BMP4 every 2 to 3 days. For each half-volume replacement, half the medium in the culture vessel (i.e., 1 ml) was discarded, and 1 mL of fresh serum-free medium (gfCDM+KSR) was added to bring the total medium volume to 2 ml.

[0282] On day 20 of patterned culture, the expression levels of marker genes for the neuroretina and for byproducts other than the neuroretina (Table 4) in the retinal tissue were measured as described in Example 4. The analysis results of the relative mRNA expression levels of each gene are presented in... Figure 24 The results show that, compared with retinal tissue differentiated by the method described in the reference example, retinal tissue differentiated by the optimized method (the optimized method) exhibited higher expression levels of marker genes targeting neural progenitor cells and retinal progenitor cells. Furthermore, the expression of various non-target cell marker genes was found to be suppressed in retinal tissue differentiated by the optimized method.

[0283] [Table 4]

[0284]

Claims

1. A method for producing retinal tissue, comprising the following steps: (1) Pluripotent stem cells were fed at a rate of 0.5 × 10⁻⁶. 5 cells / cm 2 Up to 2.5×10 5 cells / cm 2 The culture medium is seeded at a density in region A on the surface of a culture medium, wherein the culture medium on the surface includes region A and region B, region A has cell adhesion, and region B is adjacent to at least a portion of region A and has lower cell adhesion than region A. (2) The pluripotent stem cells seeded in step (1) are cultured in a medium containing ROCK inhibitors for 1 to 16 hours, and (3) The cells obtained after step (2) are cultured in a medium containing BMP signal transduction activator.

2. The method according to claim 1, wherein the concentration of the BMP signal transduction activator is 3 nM to 15 nM or provides an activity equivalent to that of BMP4 at a concentration of 3 nM to 15 nM.

3. The method according to claim 1 or 2, wherein the BMP signal transduction activator is selected from BMP2, BMP4, BMP7 and GDF7.

4. The method according to any one of claims 1 to 3, wherein region A is coated with a cell-adhesive substance.

5. The method according to claim 4, wherein the cell adhesion substance is laminin.

6. The method according to any one of claims 1 to 5, wherein region A is surrounded by region B.

7. The method according to any one of claims 1 to 6, wherein the retinal tissue is sheet-like retinal tissue.

8. The method according to any one of claims 1 to 7, wherein the retinal tissue covers more than 95% of the region A.

9. A retinal tissue produced by the method according to any one of claims 1 to 8.

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