Media for tissue transplants

Abstract

A purpose of the present invention is to provide a medium suitable for subretinal transplantation of retinal tissue for treating of retinal degenerative disease such as retinitis pigmentosa, and a transplantation composition that contains retinal tissue and said medium. The medium for transplantation according to the present invention is for subretinal transplantation of retinal tissue, has a viscosity of 5-500 mPa•s when shear speed (1 / s) is 2 at 25°C, and contains hyaluronic acid and a pharmaceutically acceptable aqueous liquid. The transplantation composition according to the present invention contains retinal tissue for transplantation and the medium for transplantation according to the present invention.

Description

[Technical Field]

[0001] The present invention relates to a medium used for transplanting retinal tissue subretinally within an eyeball, and a transplant composition containing retinal tissue and a medium. [Background technology]

[0002] For patients suffering from diseases accompanied by retinal degeneration or damage, such as retinitis pigmentosa or age-related macular degeneration, retinal tissue transplantation is considered a promising treatment, and research and development into this field is currently underway. For example, it is now possible to produce retinal tissue containing photoreceptors by inducing differentiation from pluripotent stem cells (Non-Patent Document 1). Furthermore, as a method for transplanting retinal tissue into the subretinal space within the eye, a case has been reported in which an autologous RPE cell sheet produced from pluripotent stem cells was transplanted into a patient (Non-Patent Document 2). Non-Patent Document 1 reports that RPE tissue was aspirated into the interior of a 20-gauge Surflow needle, the needle was inserted into the subretinal space within the eye, and the RPE tissue was ejected from the tip of the Surflow needle into the subretinal space, resulting in successful engraftment of the RPE cell sheet. However, the medium used for transplanting the RPE cell sheet is not described.

[0003] Furthermore, in transplanting retinal tissue, particularly retinal tissue containing photoreceptors, it is necessary to accurately insert and allow the retinal tissue to take root in the affected area that requires repair. Therefore, there has been a demand for a suitable medium that allows retinal tissue transplantation surgery to be performed without relying on the doctor's technique.

[0004] On the other hand, Patent Document 1 discloses a device for subretinal implant (retinal cell graft) transplantation and describes the use of a hyaluronic acid aqueous solution, but does not describe the specific retinal tissue or hyaluronic acid concentration. Furthermore, Non-Patent Document 3 describes the screening and optimization of media for preserving RPE stem cells before transplantation, and examines the effects of each medium on the survival, attachment, distribution, proliferation, and differentiation ability of RPE stem cells into RPE cells. It also describes the discovery that using a 0.2% hyaluronic acid solution as a storage medium for RPE stem cells promoted good distribution and proliferation of RPE stem cells after 96 hours of storage, as well as cobblestone formation, characteristic of mature RPE cells, and expression of RPE cell markers. However, Non-Patent Document 3 does not describe the effects of using it in actual transplant surgery.

[0005] Furthermore, it has been reported that subretinal injection of hyaluronic acid can cause retinal detachment (see Non-Patent Document 4).

[0006] As described above, a suitable medium for subretinal transplantation of retinal tissue has not yet been established. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Special Publication No. 2005-517468 [Non-patent literature]

[0008] [Non-Patent Document 1] A. Kuwahara et al., “Generation of a ciliary margin-like stem cell niche from self-organizing human retinal tissue” Nature Communications, 6, Article number:6286(2015). [Non-patent document 2] M. Mandai et al., “Autologous InducedStem-Cell-Derived Retinal Cells for Macular Degeneration”, The New England Journal of Medicine, 2017, 376(11), p.1038-1046. [Non-patent document 3] Y. Tian et al., “Screening and optimization of potential injection vehicles for storage of retinalpigment epithelial stem cell before transplantation”, J Tissue Eng Regen Med. 2019;13(1): p76-86. [Non-patent document 4] T. Nakazawa, et al., “Tumor NecrosisFactor-Mediates Photoreceptor Death in a Rodent Model of Retinal Detachment”Investigative Ophthalmology & Visual Science, March 2011, Vol.52, No.3, p1384-1391 Summary of the Invention [Problem to be solved by the invention]

[0009] It is an object of the present invention to provide a vehicle suitable for subretinal transplantation of retinal tissue for the treatment of retinal degenerative diseases such as retinitis pigmentosa (RP), as well as a transplant composition comprising retinal tissue and the vehicle. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors conducted extensive research into transplantation media used for subretinal transplantation of retinal tissue, and as a result discovered a transplantation medium that can be sucked into and ejected from an administration device (transplantation tip) and is useful for transplanting retinal tissue, specifically a neural retinal sheet, into a recipient's subretina while properly maintaining the apical / basal surface orientation, thereby completing the present invention.

[0011] That is, the present invention provides the following. [1] A transplantation medium for subretinal transplantation of retinal tissue, which has a viscosity of 5 to 500 mPa·s at a shear rate of 2 (1 / s) at 25°C and contains hyaluronic acid and a pharmaceutically acceptable aqueous liquid. [2] A transplant medium [1] with a viscosity of 10-100 mPa·s at a shear rate of 2 (1 / s) at 25°C. [3] A transplant medium according to [1] or [2], in which the viscosity at a shear rate of 1000 (1 / s) at 25°C is 100 mPa·s or less, and the difference in viscosity between the viscosity at a shear rate of 1 (1 / s) and the viscosity at a shear rate of 10 (1 / s) is 100 mPa·s or less. [4] A transplant medium with a pH of 7.0 to 7.5, as described in [1] to [3]. [5] The transplant medium according to any one of [1] to [4], wherein the pharmaceutically acceptable aqueous liquid is a balanced salt solution. [6] A transplant medium according to any one of [1] to [5], containing 0.15 w / v% to 1.50 w / v% hyaluronic acid having an average molecular weight of 500,000 to 3,900,000. [7] A transplant medium according to any one of [1] to [6], further comprising chondroitin sulfate. [8] The transplant medium according to [7], which contains 0.3 w / v% to 1.0 w / v% chondroitin sulfate. [9] A transplant medium that does not contain antibacterial agents or preservatives and is any of [1] to [8].

[10] A transplantation composition comprising retinal tissue for transplantation and a transplantation medium according to any one of [1] to [9].

[11] The transplant composition according to

[10] , wherein the retinal tissue for transplantation is a neural retinal sheet for transplantation containing a neural retinal layer.

[12] The transplant composition of

[11] , wherein the neural retinal sheet for transplantation has a front surface and a back surface, the front surface constitutes the apical surface including the neural retinal layer, and the back surface constitutes the basal surface adjacent to the inner layer of the neural retina, the thickness from the front surface to the back surface of the neural retina sheet for transplantation is 100 μm to 1000 μm, the major axis of the neural retinal sheet for transplantation is 600 μm to 2500 μm, and the minor axis of the neural retinal sheet for transplantation is 200 μm to 1500 μm.

[13] The implant composition of

[12] , wherein the surface has a smooth shape with little change in curvature, and the back surface has an irregular shape with large change in curvature.

[14] The transplantation composition according to any one of

[11] to

[13] , comprising 1 to 30 of the neural retinal sheets for transplantation and 5 to 500 μl of the transplantation medium.

[15] The neural retinal sheet for transplantation is (1) derived from pluripotent stem cells; (2) It has a three-dimensional structure. (3) A neural retina layer having a multi-layer structure including a photoreceptor layer and an inner layer, (4) The photoreceptor layer includes one or more cells selected from the group consisting of photoreceptor precursor cells and photoreceptor cells, (5) The inner layer comprises one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells, and bipolar cells; (6) the surface of the neural retinal layer has an apical surface; (7) the inner layer is present inside the photoreceptor layer present along the apical surface, (8) The area of ​​the apical surface of the neural retinal layer is 50% or more of the total area of ​​the surface of the neural retinal sheet; (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neural retina layer; (10) Expression of neural retinal cell-related genes is observed in the neural retinal sheet for transplantation, and expression of non-neural retinal cell-related genes is not observed, and the non-neural retinal cell-related genes include one or more genes selected from the group consisting of cerebrospinal tissue marker genes and eyeball-related tissue marker genes. The composition for transplantation according to any one of

[11] to

[14] , which is a neural retina sheet characterized by:

[16] The neural retinal sheet for transplantation is (1) isolated from a cell aggregate containing the neural retinal layer; (2) comprising a region near the center of the continuous epithelial tissue in the cell aggregate; and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm. A transplant composition according to any one of

[11] to

[15] .

[17] The neural retinal sheet for transplantation is (1) isolated from a cell aggregate containing at least a first epithelial tissue and a second epithelial tissue; the first epithelial tissue comprises a human neural retina, and the second epithelial tissue has a continuity of tangent slope on its surface that is different from the continuity of tangent slope on the surface of the first epithelial tissue, and comprises non-neural retinal cells; (2) including a region on the first epithelial tissue that is furthest from the second epithelial tissue; and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm, The transplant composition according to any one of

[11] to

[16] , wherein the second epithelial tissue is a tissue selected from the group consisting of eye-related tissue, cerebrospinal tissue, and other tissues different from the neural retina of the first epithelial tissue.

[18] A transplant composition according to any one of

[11] to

[17] , wherein the ratio of Rx-positive cells to the total number of cells in the neural retina sheet for transplantation is 30% or more and 80% or less, 40% or more and 70% or less, 45% or more and 60% or less, or 50% or more and 60% or less.

[19] A transplant composition according to any one of

[11] to

[18] , wherein the ratio of Chx10-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% or more and 80% or less, 20% or more and 70% or less, 30% or more and 60% or less, or 40% or more and 50% or less.

[20] A transplant composition according to any one of

[11] to

[19] , wherein the ratio of Pax6-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% or more and 80% or less, 20% or more and 70% or less, 30% or more and 60% or less, or 40% or more and 50% or less. [twenty one] A transplant composition according to any one of

[11] to

[20] , wherein the ratio of Crx-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% to 70% or 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%. [twenty two] A method for treating a disorder of neural retinal cells or the neural retina, or a disease caused by damage to the neural retina, comprising transplanting any one of the transplant compositions

[11] to

[21] into the subretinal space of a subject requiring transplantation. [Effects of the Invention]

[0012] The present invention provides a medium suitable for subretinal transplantation of retinal tissue, and a transplantation composition comprising retinal tissue and the medium, which is useful for retinal tissue transplantation therapy for the treatment of retinal degenerative diseases such as retinitis pigmentosa. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows a method for transplanting a retinal sheet under the retina using a transplantation tip in Example 1. [Figure 2] 1 is a graph showing the results of measuring the correlation between shear rate and viscosity at 30° C. for the media (1) to (10) shown in Table 12 in Example 1. [Figure 3] 1 is a graph showing the results of measuring the correlation between shear rate and viscosity for the media shown in Table 13 at 25° C. in Example 1. [Figure 4] 1 shows fluorescence microscopy images showing the results of immunostaining with Crx and Chx10 on a cell aggregate containing a neural retina for transplantation in Reference Example 1. [Figure 5] 1 shows fluorescence microscopy images showing the results of immunostaining with Rx and Recoverin on a cell aggregate containing a neural retina for transplantation in Reference Example 1. [Figure 6] FIG. 1 is a conceptual diagram of the creation of caps and rings from a typical cell aggregate. [Figure 7] A conceptual diagram of the creation of caps and rings from cell aggregates of various shapes. The black and gray areas represent non-target tissue. [Figure 8] The height, major axis, and minor axis of the graft in Reference Example 3 are shown together with an image of a typical graft and a schematic diagram of the graft. [Figure 9] 10 shows confocal fluorescence microscope images showing the results of immunostaining of a graft with Crx, Chx10, Rx, and Recoverin in Reference Example 4. [Figure 10A]1 shows the results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Reference Example 5. [Figure 10B] 1 shows the results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Reference Example 5. [Figure 11] In Reference Example 6, RNA extracted from the ring was analyzed by quantitative PCR, and then a graft (cap) was transplanted under the retina of a rat. Images showing the results of observing the post-transplant engraftment using a fluorescence microscope. [Figure 12] 10 is a fluorescence microscope image showing the results of immunostaining of a cap and a ring prepared from a single cell aggregate in Reference Example 7. [Figure 13] 10 is a fluorescence microscope image showing the results of immunostaining of a cap prepared from a single cell aggregate in Reference Example 8. DETAILED DESCRIPTION OF THE INVENTION

[0014] 1.Transplantation medium The present invention provides a transplant vehicle for use in implanting retinal tissue grafts under the retina of a mammal.

[0015] The transplant medium of the present invention has a viscosity of 5 to 500 mPa·s, preferably 10 to 100 mPa·s, at a shear rate of 2 (1 / s) at 25°C, and is not limited as long as it contains hyaluronic acid and a pharmaceutically acceptable aqueous liquid.

[0016] The transplant medium of the present invention preferably has a viscosity of 100 mPa·s or less, preferably 30 mPa·s or less, at a shear rate of 1000 (1 / s) at 25°C, and a viscosity change at shear rates of 1 to 10 (1 / s) (i.e., the difference between the viscosity at a shear rate of 1 (1 / s) and the viscosity at a shear rate of 10 (1 / s)) of 100 mPa·s or less, preferably 30 mPa·s or less. Alternatively, the viscosity change rate at shear rates of 1 to 10 (1 / s) (i.e., the percentage obtained by dividing the viscosity difference between the viscosity at a shear rate of 1 (1 / s) and the viscosity at a shear rate of 10 (1 / s) by the viscosity at a shear rate of 1 (1 / s)) is preferably about 10% or less.

[0017] The implantation medium of the present invention preferably further comprises chondroitin sulfate. Hyaluronic acid and chondroitin sulfate function as thickening components of the implantation medium.

[0018] In this specification, hyaluronic acid is a linear polymeric polysaccharide with a molecular weight of tens of thousands to millions, which has a structure in which D-glucuronic acid and DN-acetylglucosamine are alternately linked by β-1,4 and β-1,3 glycosidic bonds. Hyaluronic acid has high water retention capacity and viscosity, and is a substance widely used in pharmaceuticals, cosmetics, etc. In this specification, "hyaluronic acid" refers to a concept that includes both free hyaluronic acid and its salts. Examples of hyaluronic acid salts include alkali metal salts and alkaline earth metal salts of hyaluronic acid, and specifically, commercially available sodium hyaluronate, potassium hyaluronate, etc. can be used.

[0019] The hyaluronic acid used in this specification is not particularly limited as long as it can maintain the viscosity of the transplant medium at 25°C and a shear rate of 2 (1 / s) at 5 to 500 mPa·s, preferably 10 to 100 mPa·s, and hyaluronic acid with an average molecular weight of approximately 500,000 to 3,900,000 can be blended into the transplant medium at a concentration of 0.15 w / v% to 1.50 w / v%, preferably 0.15 w / v% to 0.75 w / v%, and more preferably 0.30 w / v% to 0.50 w / v%.

[0020] Chondroitin sulfate is a mucopolysaccharide having a structure in which sulfate is bound to a sugar chain in which two sugars, D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine (GalNAc), are repeated. In this specification, "chondroitin sulfate" refers to a concept that includes both free chondroitin sulfate and its salts. Examples of chondroitin sulfate salts include alkali metal salts and alkaline earth metal salts of chondroitin sulfate. Specifically, commercially available chondroitin sulfate sodium, chondroitin sulfate potassium, etc. can be used.

[0021] In this specification, the chondroitin sulfate and hyaluronic acid can be blended into the transplant medium at a concentration of 0.3 w / v% to 1.0 w / v%, preferably 0.4 w / v% to 0.7 w / v%, without any particular limitation, as long as the viscosity of the transplant medium at 25°C and a shear rate of 2 (1 / s) can be maintained at 5 to 500 mPa·s, preferably 10 to 100 mPa·s. Chondroitin sulfate having an average molecular weight of approximately 20,000 to 24,000 can be blended into the transplant medium at a concentration of 0.3 w / v% to 1.0 w / v%, preferably 0.4 w / v% to 0.7 w / v%.

[0022] The "pharmaceutically acceptable aqueous liquid" in the medium of the present invention is not particularly limited as long as it is an aqueous solution that can be administered to a living body and has physical properties suitable for transplantation, and may be, for example, an aqueous solution containing a buffer solution, an infusion solution, physiological saline, water for injection, a perfusion solution, etc. A buffer solution is preferred.

[0023] Here, physical properties suitable for transplantation include pH, osmotic pressure, etc. The pH of the transplantation medium is not particularly limited as long as it is in the neutral range, and the transplantation medium of the present invention may be adjusted to a pH of 6.5 to 8.0, preferably about pH 7.0 to 7.5.

[0024] The osmotic pressure of the transplant medium may be hypotonic, isotonic, or hypertonic, but is preferably close to isotonic. The osmotic pressure ratio may be adjusted to 0.7 to 1.3, preferably 0.9 to 1.1.

[0025] Specific examples of "pharmaceutically acceptable aqueous liquids" include aqueous solutions containing balanced salts (i.e., balanced salt solutions). The aqueous solutions containing balanced salts may contain appropriate selected buffering agents, isotonicity agents, pH adjusters, antioxidants, chelating agents, etc., within the range that does not affect the viability of the transplanted retinal tissue.

[0026] Examples of buffers include phosphate buffers, borate buffers, citrate buffers, tartrate buffers, acetate buffers, amino acids, and epsilon-aminocaproic acid.

[0027] Examples of isotonic agents include sugars such as sorbitol, glucose, and mannitol, polyhydric alcohols such as glycerin and propylene glycol, salts such as sodium chloride, and boric acid.

[0028] Chelating agents include sodium edetate and citric acid.

[0029] Examples of pH adjusters include sodium hydroxide, potassium hydroxide, sodium carbonate, sodium bicarbonate, boric acid or its salts (borax), hydrochloric acid, citric acid or its salts (sodium citrate, sodium dihydrogen citrate, etc.), phosphoric acid or its salts (disodium hydrogen phosphate, potassium dihydrogen phosphate, etc.), acetic acid or its salts (sodium acetate, ammonium acetate, etc.), and tartaric acid or its salts (sodium tartrate, etc.).

[0030] Examples of antioxidants include ascorbic acid, glutathione, sodium hydrogen sulfite, dried sodium sulfite, sodium pyrosulfite, and tocopherol.

[0031] The transplantation medium of the present invention can be preferably sterilized by filtration sterilization using a membrane filter or the like.

[0032] Specific examples of "pharmaceutically acceptable aqueous liquids" include aqueous solutions containing one or more components selected from sugars such as glucose, inorganic salts such as calcium chloride, sodium chloride, and magnesium sulfate, inorganic substances such as sodium bicarbonate, sodium acetate, sodium citrate, sodium dihydrogen phosphate, sodium hydrogen phosphate, anhydrous sodium monohydrogen phosphate, and hydrochloric acid, and chelating agents such as edetates (e.g., sodium edetate).

[0033] As hyaluronic acid or its salt, commercially available aqueous solution of hyaluronic acid or its salt can be used.Specifically, OPEGAN (registered trademark) 0.6 ophthalmic viscoelastic agent, which contains 0.6% sodium hyaluronate, sodium chloride, sodium dihydrogen phosphate and sodium hydrogen phosphate as its components, OPEGAN (registered trademark) 1.1 ophthalmic viscoelastic agent, which contains 1.1% sodium hyaluronate, sodium chloride, sodium dihydrogen phosphate and sodium hydrogen phosphate as its components, or Hyaline Mini (registered trademark) ophthalmic solution 0.3%, which contains 0.3% sodium hyaluronate, epsilon-aminocaproic acid, sodium edetate hydrate, potassium chloride, sodium chloride and pH adjuster as its components can be exemplified.

[0034] To prepare a pharmaceutically acceptable aqueous liquid, commercially available intraocular irrigation and irrigation solutions can be used. Specifically, an aqueous solution containing 1.5 mg of glucose, 0.18 mg of calcium chloride hydrate, 0.3 mg of magnesium sulfate hydrate, and 2.1 mg of sodium bicarbonate per mL, as well as additives such as sodium citrate hydrate, sodium acetate hydrate, and hydrochloric acid, commercially available as Opeguard®, or oxyglutathione ocular irrigation solution can be used. Furthermore, to prepare a pharmaceutically acceptable aqueous liquid, Hank's balanced salt solution (HBSS), Eagle's balanced salt solution (EBSS), phosphate-buffered saline (PBS), Dulbecco's phosphate-buffered saline (DPBS), and the like can also be used.

[0035] One embodiment of the transplantation medium of the present invention is a transplantation medium containing only hyaluronic acid as a thickening component, and specifically, for example, it can be prepared by blending OPEGAN (registered trademark) 0.6 ophthalmic viscoelastic agent with an aqueous liquid such as Opeguard at a ratio of 1:1 to 1:3. Another embodiment, for example, it can be prepared by blending Hyalein Mini (registered trademark) eye drops 0.3% with an aqueous liquid such as Opeguard at a ratio of 3:1 to 1:1.

[0036] One embodiment of the transplant medium of the present invention includes a transplant medium containing hyaluronic acid and chondroitin sulfate as viscosity-increasing components. The concentrations of hyaluronic acid and chondroitin sulfate are not particularly limited, as long as the viscosity of the transplant medium at a shear rate of 2 (1 / s) at 25°C can be maintained at 5 to 500 mPa·s, preferably 10 to 100 mPa·s. Another embodiment of the transplant medium of the present invention includes a transplant composition containing 0.15 w / v% to 1.50 w / v% of hyaluronic acid having an average molecular weight of 500,000 to 3,900,000 and 0.3 w / v% to 1.0 w / v% of chondroitin sulfate or a salt thereof having a molecular weight of 20,000 to 24,000.

[0037] One embodiment of the transplantation medium of the present invention is Viscoat® 0.5 ocular viscoelastic agent, which contains sodium hyaluronate, sodium chondroitin sulfate, sodium dihydrogen phosphate, sodium hydrogen phosphate, and an isotonicity agent as ingredients. Specifically, Viscoat® 0.5 ocular viscoelastic agent can be prepared by blending Viscoat® 0.5 ocular viscoelastic agent with an aqueous liquid such as Opeguard at a ratio of 1:3 to 1:7.

[0038] The "pharmaceutically acceptable aqueous liquid" is preferably characterized as being free of antibacterial agents and preservatives.

[0039] 2. Retinal tissue for transplantation (definition) As used herein, the term "tissue" refers to a structure of a cell population in which one or more types of cells with different morphologies and properties are arranged three-dimensionally in a specific pattern.

[0040] As used herein, the term "retinal tissue" refers to a tissue in which one or more types of retinal cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, retinal pigment epithelial cells, their precursor cells, or retinal progenitor cells, that constitute each retinal layer in a living retina, are arranged three-dimensionally, preferably in layers, and may be a cell aggregate or cell sheet, as described below.

[0041] Photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, Müller glial precursor cells, and retinal pigment epithelial precursor cells refer to precursor cells that are committed to differentiating into photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cells, respectively.

[0042] The term "retinal progenitor cell" refers to a precursor cell that can differentiate into any immature retinal cell, such as a photoreceptor precursor cell, horizontal cell precursor cell, bipolar cell precursor cell, amacrine cell precursor cell, retinal ganglion cell precursor cell, Müller glial cell, or retinal pigment epithelial precursor cell, and ultimately can differentiate into any mature retinal cell, such as a photoreceptor cell, rod photoreceptor cell, cone photoreceptor cell, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, or retinal pigment epithelial cell.

[0043] As used herein, the term "retinal layer" refers to a layered cell population in which one or more cells constituting the retina form a single layer or multiple layers in a specific pattern, specifically including the retinal pigment epithelial layer containing retinal pigment epithelial cells and the neural retinal layer containing neural retinal cells. Neural retinal cells are also called retinal layer-specific neurons.

[0044] The neural retinal layers include the outer limiting membrane, photoreceptor layer (external nuclear layer), outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.

[0045] As used herein, "neural retina" refers to retinal tissue that includes the neural retinal layers.

[0046] Neuroretinal cells include photoreceptor cells (including photoreceptor precursor cells and mature photoreceptor cells), bipolar cells, retinal ganglion cells, amacrine cells, horizontal cells, Muller glial cells, and their progenitor cells.

[0047] As used herein, the term "neural retinal progenitor cells" refers to precursor cells of neural retinal cells.

[0048] In living organisms, photoreceptor cells are found in the photoreceptor layer of the retina and are responsible for absorbing light stimuli and converting them into electrical signals. There are two types of photoreceptor cells: cone cells, which function in bright light, and rod cells, which function in the dark. Cone photoreceptors include S-cone photoreceptors, which express S-opsin and perceive blue light; L-cone photoreceptors, which express L-opsin and perceive red light; and M-cone photoreceptors, which express M-opsin and perceive green light. Photoreceptors differentiate and mature from photoreceptor precursor cells. Those skilled in the art can easily confirm whether a cell is a photoreceptor cell or a photoreceptor precursor cell by, for example, the expression of cell markers described below (Crx and Blimp1 expressed in photoreceptor precursor cells, recoverin expressed in photoreceptors, rhodopsin, S-Opsin, and M / L-Opsin expressed in mature photoreceptors, etc.), the formation of an outer segment structure, etc. In one embodiment, photoreceptor precursor cells are Crx-positive cells, and photoreceptors are rhodopsin-, S-Opsin-, and M / L-Opsin-positive cells. In one embodiment, rod photoreceptors are NRL- and rhodopsin-positive cells. In one embodiment, S cone photoreceptors are S-opsin-positive cells, L cone photoreceptors are L-opsin-positive cells, and M cone photoreceptors are M-opsin-positive cells. That is, photoreceptors in this specification are a concept that includes photoreceptor precursor cells and mature photoreceptors.

[0049] It should be noted that neural retinal cells do not include retinal pigment epithelial cells and ciliary body cells, which will be described later.

[0050] An embodiment of the retinal tissue herein is the neural retina, preferably the neural retina comprising a retinal layer-specific nerve cell layer, more preferably the neural retina comprising a photoreceptor cell layer.

[0051] The neural retina preferably contains 10% or more, more preferably 20% or more, of photoreceptor cells or photoreceptor precursor cells.

[0052] The neural retina preferably contains 10% or more neural retinal progenitor cells, more preferably 20% or more. The neural retina also preferably contains 10% or more photoreceptor progenitor cells. In one embodiment, the neural retina contains 3% or more mature photoreceptors.

[0053] An embodiment of the retinal tissue herein includes a retinal tissue containing both a neural retina and a cell layer containing RPE cells, such as the neural retina covered with RPE cells described in WO2019 / 050015.

[0054] An example of a retinal tissue herein is a retinal tissue (composite) in which neural retina and RPE cells are adhered via a polymer hydrogel. Specifically, the example includes a composite comprising a neural retina, retinal pigment epithelial cells, and a hydrogel, wherein the neural retina and the retinal pigment epithelial cells are each derived from human pluripotent stem cells, and the neural retina has formed a neural retinal layer including at least a photoreceptor layer, the photoreceptor layer including at least one cell type selected from the group consisting of photoreceptors, photoreceptor precursor cells, and retinal progenitor cells. The hydrogel is not particularly limited as long as it is a polymeric substance with properties useful for adhering tissues (e.g., glue, adhesive, substance with gelling properties, gelatin, oil, etc.).

[0055] The cells that make up the above-mentioned retinal tissue can be detected or identified using retinal cell markers that are either expressed (positive) or not expressed (negative) in each of them as indicators.

[0056] Retinal cell markers include Rx (also called Rax) and PAX6, which are expressed in retinal progenitor cells, Rx, PAX6, and Chx10 (also called Vsx2), which are expressed in neural retinal progenitor cells, and Crx and Blimp1, which are expressed in photoreceptor progenitor cells. Furthermore, negative markers for retinal progenitor cells or retinal cells include Nkx2.1 and SOX1.

[0057] Markers of retinal layer-specific neurons include Recoverin, which is expressed in photoreceptors (especially mature photoreceptors), Crx and Blimp1, which are expressed in photoreceptors (especially photoreceptor precursors), Rhodopsin, which is expressed in rod cells, Nrl, which is expressed in rod photoreceptors and rod photoreceptor precursors, S-opsin and LM-opsin, which are expressed in cone photoreceptors, cone photoreceptor precursors, and ganglion cells, RXR-γ, which is expressed in cone photoreceptors, cone photoreceptor precursors, and ganglion cells, TRβ2, OTX2, and OC2, which are expressed in cone photoreceptors or their precursors that appear early in the differentiation of cone photoreceptors, Chx10, PKCα, Goα, VSX1, and L7, which are expressed in bipolar cells, TuJ1 and Brn3, which are expressed in retinal ganglion cells, Calretinin and HPC-1, which are expressed in amacrine cells, Calbindin, which is expressed in horizontal cells, and Pax6, which is expressed in horizontal cells, amacrine cells, and ganglion cells.

[0058] Furthermore, the layer in the differentiation stage before the formation of the photoreceptor layer, which contains a large number of neural retinal progenitor cells and has a low ratio of photoreceptor cells or photoreceptor precursor cells, is called the "neuroblastic layer," and it consists of an inner neuroblastic layer and an outer neuroblastic layer. Those skilled in the art can distinguish this by well-known methods, for example, by the color shading (the outer neuroblastic layer is light and the inner neuroblastic layer is dark) under a bright-field microscope.

[0059] Those skilled in the art can easily confirm the presence or absence of retinal cell marker expression, or the proportion of retinal cell marker-positive cells in a cell population or tissue. Examples include antibody-based techniques, nucleic acid primer-based techniques, and sequencing techniques. Antibody-based techniques can confirm the protein expression of retinal cell markers by dividing the number of specific retinal cell marker-positive cells by the total number of cells, using techniques such as flow cytometry or immunostaining with commercially available antibodies. Nucleic acid primer-based techniques can confirm the RNA expression of retinal cell markers by, for example, PCR, semi-quantitative PCR, or quantitative PCR (e.g., real-time PCR). Sequencing techniques can confirm the RNA expression of retinal cell markers by, for example, a nucleic acid sequencer (e.g., a next-generation sequencer).

[0060] "Positive cells" refer to cells that express a specific marker on the cell surface or intracellularly. For example, "Chx10-positive cells" refer to cells that express Chx10 protein.

[0061] "Negative cells" refer to cells that do not have or express a specific marker on the cell surface. For example, "SOX1-negative cells" refer to cells that do not express SOX1 protein. Here, "not expressing" includes cases where the expression level is below the detection limit, or cases where the expression level is 1 / 10 or less, preferably 1 / 50 or less, compared to that of positive cells.

[0062] In another embodiment, for example, "SOX1-negative cells" refer to cells that do not express SOX1 mRNA. Here, "not expressed" includes cases where the expression level is below the detection limit, or cases where the expression level is 1 / 10 or less, preferably 1 / 50 or less, compared to that in positive cells. In particular, when measuring the mRNA level by qPCR, the difference between the target gene (e.g., SOX1) and an internal standard (GAPDH or ACTB) can be evaluated by the ΔCt value, and "not expressed" includes cases where the ΔCt value is 4 or more, more preferably 6 or more, and even more preferably 8 or more.

[0063] "Retinal pigment epithelial cells" refer to epithelial cells present outside the neural retina in a living retina. Those skilled in the art can easily determine whether a cell is a retinal pigment epithelial cell, for example, by the expression of cell markers (RPE65, MITF, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (black-brown color), tight junctions between cells, or the characteristic polygonal or cobblestone-like cell morphology. Whether a cell has the function of a retinal pigment epithelial cell can be easily determined by its ability to secrete cytokines such as VEGF and PEDF. In one embodiment, the retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.

[0064] The term "ciliary body" includes the "ciliary body" during development and in adults, the "ciliary margin," and the "ciliary body." Markers for the "ciliary body" include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The "ciliary marginal zone (CMZ)" is, for example, a tissue present at the boundary between the neural retina and the retinal pigment epithelium in the living retina, and is a region containing retinal tissue stem cells (retinal stem cells). The ciliary margin is also called the ciliary margin or retinal margin, and the ciliary margin, ciliary margin, and retinal margin are equivalent tissues. The ciliary margin is known to play an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, and so on. Marker genes for the ciliary margin include, for example, the Rdh10 gene (positive), the Otx1 gene (positive), and the Zic1 gene (positive). The term "ciliary margin-like structure" refers to a structure similar to the ciliary margin.

[0065] (Structure of retinal tissue) The retinal tissue referred to herein may be in the form of a three-dimensional structure, i.e., a cell aggregate (also referred to as a three-dimensional tissue or organoid), or a cell sheet in which a layered structure is spread two-dimensionally, i.e., a retinal cell sheet. Cell aggregates and cell clusters also include spheres. Here, spheres refer to cell aggregates having a three-dimensional shape close to a sphere. A three-dimensional shape close to a sphere refers to a shape having a three-dimensional structure, such as a spherical shape that is circular or elliptical when projected onto a two-dimensional surface, and a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by two to four overlapping circles or ellipses when projected onto a two-dimensional surface). In one embodiment, the core of the aggregate has a vesicular layered structure and is characterized by a dark center and a bright outer edge when observed under a bright-field microscope.

[0066] In one embodiment, some or all of the cells contained in the cell aggregate or cell sheet are adhered to one another, i.e., some or all of the cells in the aggregate may form cell-cell junctions or cell adhesions, such as adherens junctions.

[0067] The shape and size of the cell aggregate or cell sheet are not particularly limited, and when administered as a graft to a mammal, preferably a monkey or a human, they can be appropriately determined depending on the area of ​​retinal tissue that needs to be repaired in the recipient. That is, the size of the retinal tissue to be transplanted into the recipient is a size that is appropriate for the recipient and can move inside the cell suction part (transplantation needle) of the device used for transplantation.

[0068] A specific example of the retinal tissue contained in the composition for transplantation herein is retinal tissue having a minor axis of 200 to 1500 μm, or 600 to 2500 μm or less, and a thickness of 100 μm to 1000 μm. Retinal tissue of a size suitable for transplantation, i.e., a graft, can be prepared by cutting a piece of retinal tissue of a size suitable for transplantation from a cell aggregate or cell sheet.

[0069] Specifically, a cell sheet of a size suitable for transplantation can be cut out from the cell aggregate to serve as a transplant.

[0070] The retinal tissue herein may have an epithelial structure. The epithelial structure is formed by cells tightly covering the surface of the tissue, and is polarized to have an "apical surface" and a "basal surface." Here, the "basement membrane" refers to a 50-100 nm layer rich in laminin and type IV collagen, and the "apical surface" refers to the surface (superficial layer) formed opposite the "basement membrane." In one aspect, the "apical surface" refers to the surface where the outer limiting membrane is formed and which contacts the photoreceptor layer (external nuclear layer) where photoreceptors and photoreceptor precursors are present in retinal tissue whose differentiation stage has progressed to the point where photoreceptors or photoreceptor precursors are present. Furthermore, such an apical surface can be identified by immunostaining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical PKC (hereinafter abbreviated as "aPKC"), E-cadherin, and N-cadherin).

[0071] The cells that make up the epithelial structure, i.e., epithelial cells, form strong bonds with each other through adherens junctions and / or tight junctions, forming cell layers. These cell layers are stacked in one to several dozen layers to form the epithelial structure.

[0072] As used herein, the epithelial structure containing neural tissue is referred to as neuroepithelium. In particular, the epithelial structure containing the neural retina is referred to as neural retinal epithelium.

[0073] In one embodiment, the neural retina herein may have a polarized layer structure. For example, when the neural retina is in the form of a cell sheet, it may have apical / basal polarity. When the neural retina is a spherical cell aggregate, the cell aggregate may have an epithelial structure, and the epithelial structure may have apical / basal polarity between the surface and the inside of the cell aggregate.

[0074] An example of retinal tissue contained in the transplant composition herein includes retinal tissue having an apical surface and a basal surface, the apical surface comprising a neural retinal layer, which is epithelial tissue formed by intercellular adherens junctions, and the basal surface comprising a basal surface adjacent to the inner layer of the neural retina. Such retinal tissue can be referred to as neural retinal epithelium. Such retinal tissue is preferably a neural retina sheet, which is a sheet-like retinal tissue. The surface has a smooth shape with little change in curvature, while the basal surface has an irregular shape with large changes in curvature. For example, in one embodiment, the change in curvature of the surface of the retinal tissue may resemble the change in curvature of an ellipse (e.g., an ellipse with a minor axis of 1 and a major axis of 1 to 10) (which can also be considered a continuous change in curvature). For example, in one embodiment, the change in curvature of the basal surface of the retinal tissue may resemble the abrupt change in curvature (which can also be considered abrupt change in curvature) that alternates between positive and negative values, like the teeth of a saw.

[0075] The retinal tissue in this specification is preferably a retinal tissue having a continuous epithelial structure. The term "continuous epithelial structure" refers to a state in which the epithelial tissue is continuous, and is also called "continuous epithelium." The term "continuous epithelial tissue" refers to, for example, a state in which 10 cells to 10 cells are connected in a tangential direction to the epithelial tissue. 7 cells, preferably 30 cells to 10 cells in the tangential direction 7 cells, more preferably 10 2 cells ~10 7A continuous epithelial structure does not have a structure in which the apical surface is divided, as seen in rosette-like structures. In one embodiment, the number of cells per area of ​​a cross section of retinal tissue having a continuous epithelial structure is, for example, 100 μm when the number of cell nuclei in a frozen section about 10 μm thick is evaluated. 2 The number of cells per cell is 10 to 900 cells, preferably 30 to 300 cells, more preferably 50 to 250 cells, and even more preferably 75 to 160 cells.

[0076] For example, continuous epithelium formed in retinal tissue has an apical surface characteristic of epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to and continuous with at least the photoreceptor layer (outer nuclear layer) among the layers forming the neural retina. For example, in the case of a cell aggregate containing retinal tissue prepared from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and a continuous neuroepithelium is formed in which 10 or more, preferably 30 or more, more preferably 100 or more, and even more preferably 400 or more photoreceptor cells or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface. The neural retina containing such continuous neuroepithelium is a neural retinal epithelium containing continuous epithelium.

[0077] Whether retinal tissue contains a continuous epithelium can be confirmed by the continuity of the apical surface (i.e., an uninterrupted morphology) of the retinal tissue. Apical surface continuity can be determined, for example, by immunostaining for apical surface markers (e.g., aPKC, E-cadherin, N-cadherin) or markers of apical photoreceptors or photoreceptor precursor cells (e.g., Crx or recoverin) and analyzing the positional relationship between the apical surface and the photoreceptor layer and each retinal layer in the acquired images. Retinal layers other than the apical surface and the photoreceptor layer (outer nuclear layer) can be identified by immunostaining with DAPI staining, PI staining, or Hoechst staining, which stain cell nuclei, or with marker proteins localized in the cell nuclei (e.g., Rx, Chx10, Ki67, Crx, etc.).

[0078] Specifically, the continuity of the apical surface can be identified by the continuous presence of cells that express both markers of cells present on the apical surface, i.e., photoreceptor markers or photoreceptor precursor cell markers and a marker that can stain the cell nuclei.

[0079] As used herein, the retinal tissue preferably includes the neural retina including photoreceptor cells or photoreceptor precursor cells, in other words, the neural retina including a photoreceptor layer.

[0080] As used herein, the retinal tissue preferably includes a neural retina characterized in that the neural retinal layer or photoreceptor layer has a continuous epithelial structure, that is, has a continuous neuroepithelial structure.

[0081] The retinal tissue in this specification may include an "inner layer" containing ganglion cells and amacrine cells inside the photoreceptor layer. The inner layer may be in contact with the basal surface.

[0082] These neural retinas can be obtained by producing cell aggregates containing neural retina using the production method described below.

[0083] (cell aggregates that contain the neural retina) In one embodiment, the cell aggregate containing neural retina is a spherical cell aggregate. In one embodiment, multiple neural retinas may be present overlapping within the cell aggregate containing neural retina (see, for example, conceptual diagrams (1) and (2) in Figure 7). In one embodiment, the cell aggregate containing neural retina includes a first epithelial tissue (target epithelial tissue) containing neural retina for transplantation, and a second epithelial tissue (non-target epithelial tissue) having a surface tangent slope continuity different from that of the first epithelial tissue and containing non-neural retinal cells. Here, the first epithelial tissue is substantially free of non-neural retinal cells (non-target cells) and represents an epithelial tissue from which neural retina for transplantation can be excised. On the other hand, the second epithelial tissue may contain neural retina, but is an epithelial tissue that is unsuitable for excising neural retina for transplantation because it contains non-target cells. In another embodiment, the cell aggregate containing neural retina includes only the first epithelial tissue (target epithelial tissue) containing neural retina for transplantation, and does not include non-target epithelial tissue.

[0084] (Retinal tissue for transplantation) The retinal tissue for transplantation herein refers to a retinal tissue, which is a human retinal tissue suitable for human transplantation, preferably a neural retina, and more preferably consists solely of neural retina.

[0085] Retinal tissue for transplantation can be prepared by cutting out a portion suitable for transplantation from the cell aggregate described above. In one embodiment, retinal tissue for transplantation can be prepared by cutting out the neural retina from a cell aggregate containing the neural retina. When retinal tissue, preferably the neural retina, contains a continuous epithelium, a sheet-like retinal tissue (hereinafter also referred to as a retinal sheet), preferably the neural retina (hereinafter also referred to as a neural retinal sheet), containing the continuous epithelium can be cut out.

[0086] The neural retina for transplantation includes at least a photoreceptor layer, which is formed at least on the outermost surface of the cell aggregate. Furthermore, photoreceptors or photoreceptor precursor cells may also be present on the inner surface, or a photoreceptor layer may also be formed on the inner surface. The photoreceptor cells and the like are present continuously in the tangential direction of the surface of the cell aggregate, i.e., adhered to each other. The continuous presence of the photoreceptor cells and the like in the tangential direction of the surface of the cell aggregate forms a photoreceptor layer containing the photoreceptor cells and the like. The "tangential direction" refers to the tangential direction to the surface of the cell aggregate, i.e., the direction in which the photoreceptor cells and the like in the photoreceptor layer are arranged, and can be parallel or lateral to the neural retina. The inclination of the tangent to the surface of the epithelial tissue refers to the direction in which the cells are arranged when each cell in the epithelial tissue is arranged in a uniform direction, and can be parallel or lateral to the epithelial tissue (or epithelial sheet).

[0087] The cell aggregates used to prepare neural retina for transplantation may contain extraneous tissues other than the neural retina. Examples of extraneous tissues include epithelial tissues other than the neural retina, i.e., second epithelial tissues containing extraneous epithelial tissues. Examples of second epithelial tissues include eye-related tissues and cerebrospinal tissues. Eye-related tissues refer to tissues surrounding non-neural retinal eye tissues, including retinal pigment epithelial cells, ciliary bodies (e.g., peripheral ciliary bodies), lenses, and corneas. Cerebrospinal tissues refer to neural tissues of the brain and spinal cord, including the forebrain, telencephalon, cerebrum, diencephalon, hypothalamus, midbrain, hindbrain, cerebellum, and spinal cord. In one embodiment, the cerebrospinal tissue may include the pituitary gland.

[0088] Examples of cell aggregates containing first and second epithelial tissues include the cell aggregates shown in the conceptual diagrams in FIG. 6 and the conceptual diagrams (3) and (5) in FIG. 7. The conceptual diagram in FIG. 6 shows an example of a cell aggregate in which, in a portion of the neural retina as the first epithelial tissue, eye-related tissues (retinal pigment epithelial cells, ciliary body) (black portion in FIG. 6) are present as the second epithelial tissue. The conceptual diagram (3) in FIG. 7 shows an example of a cell aggregate in which, in a portion of the neural retina as the first epithelial tissue, eye-related tissues (retinal pigment epithelial cells, ciliary body) (black portion in conceptual diagram (3) in FIG. 7) are present as the second epithelial tissue, in addition to multiple overlapping neural retinas (e.g., conceptual diagrams (1) and (2) in FIG. 7). The conceptual diagram (5) in FIG. 7 shows an example of a cell aggregate in which cerebrospinal tissue (such as the cerebrum) (gray portion in conceptual diagram (5) in FIG. 7) is present as the second epithelial tissue. As shown in conceptual diagram (4) in FIG. 7, non-target tissue may be present inside the cell aggregate containing the neural retina for transplantation. In this case, the first epithelial tissue does not meet the definition of "having a continuity of the slope of the tangent on the surface that is different from the continuity of the slope of the tangent on the surface of the first epithelial tissue," and therefore does not qualify as a second epithelial tissue. The neural retina for transplantation and the sample for quality assessment are preferably selected from cell aggregates that do not contain any unintended tissue inside.

[0089] <Extraction process> When a neural retina for transplantation is excised from a cell aggregate, it is desirable to use as an indicator that the excised neural retina contains cells that are positive for markers of photoreceptors or photoreceptor precursor cells.When a neural retina for transplantation is excised from a cell aggregate, it is desirable to use as an indicator that the excised neural retina contains cells that are positive for markers of retinal progenitor cells or neural retinal precursor cells.It is also desirable to use as an indicator that the excised neural retina does not contain cells that are positive for markers of non-target cells.

[0090] In order to obtain a neural retina for transplantation that contains photoreceptor or photoreceptor progenitor marker-positive cells and has a standard level of non-target cell marker-positive cells (or is non-target cell marker-negative cells), it is desirable to evaluate the quality of the neural retina for transplantation in advance.

[0091] For example, the quality of neural retina for transplantation can be evaluated by extracting a portion or all of a cell aggregate containing a neural retina with an epithelial structure derived from pluripotent stem cells as a quality assessment sample (hereinafter referred to as the "extraction step") and detecting markers expressed in the extracted sample using methods well known to those skilled in the art. Extracting a portion of a cell aggregate as a quality assessment sample means selecting a portion of cell aggregates (one or more) or all of the cell aggregates from multiple cell aggregates and isolating (e.g., dissecting) a portion of the selected cell aggregate as an assessment sample using tweezers, scissors, and / or a knife. Extracting all of the cell aggregates as a quality assessment sample means selecting a portion of cell aggregates (one or more) from multiple cell aggregates and separately picking up all of the selected cell aggregates as a quality assessment sample. When selecting one or more cell aggregates from multiple cell aggregates, random extraction is preferred. In this specification, a cell aggregate in which a portion of the cell aggregate is extracted as a quality assessment sample is referred to as a "cell aggregate including a quality assessment sample," and a cell aggregate in which the entire cell aggregate is extracted as a quality assessment sample is referred to as a "cell aggregate of a quality assessment sample."

[0092] In one aspect, the quality assessment sample is a portion of a cell aggregate containing a neural retina with an epithelial structure derived from pluripotent stem cells. Extracting a portion of the cell aggregate as the quality assessment sample has the advantage that the cell aggregate is not completely destroyed, and the neural retina contained in the remaining portion can be used for transplantation. In other words, if the quality assessment sample, which is a portion of the cell aggregate, is determined to be acceptable in the evaluation process described below, the neural retina with an epithelial structure in the cell aggregate containing the quality assessment sample can be used as a neural retina for transplantation and can be used for transplantation.

[0093] A region of the cell aggregate that has a continuous epithelial structure and appears to be divided into two layers, an outer neuroblastic layer and an inner neuroblastic layer, can be determined to be the neural retina. On the other hand, eye-related tissues, particularly retinal pigment epithelial cells, as second epithelial tissues, appear black when observed visually or under a microscope, and therefore can be easily distinguished from the neural retina by those skilled in the art. Furthermore, cerebrospinal tissues as second epithelial tissues can be easily distinguished from the neural retina by those skilled in the art by focusing on morphological characteristics, such as the absence of a continuous epithelial structure on the surface of the cell aggregate, the absence of morphological characteristics specific to the neural retina, and / or a dull color, when observed visually or under a microscope. Therefore, even if the cell aggregate contains the second epithelial tissue, those skilled in the art can isolate a neural retina for transplantation and a sample for quality assessment from the first epithelial tissue containing the neural retina.

[0094] As described above, in one embodiment, a quality assessment sample is set and extracted based on a certain positional relationship with the neural retina for transplantation or a candidate for neural retina for transplantation. That is, the region to be cut out as the quality assessment sample can be determined by setting the neural retina for transplantation or its candidate. Here, in one embodiment, the neural retina for transplantation (also referred to as a graft or cap) and its candidate can be identified by its position in the above-mentioned cell aggregate (e.g., near the center of the epithelial tissue (continuous epithelial tissue), or, if a second epithelial tissue is present, the region on the first epithelial tissue furthest from the second epithelial tissue), as well as its size, as described below in the section (Neural Retinal Sheet for Transplantation). Therefore, a person skilled in the art can set a neural retina having these characteristics as a neural retina for transplantation or its candidate. When extracting a neural retina for transplantation and a quality assessment sample from the same cell aggregate, a person skilled in the art can determine that the quality assessment sample (also referred to as a "ring") is a region that is at least partially continuous with or adjacent to the neural retina for transplantation set as described above, and can be set as narrow as possible within the scope that allows quality assessment. When extracting a portion of one or more cell aggregates from the same lot of cell aggregates as a quality assessment sample, a person skilled in the art can extract the above-mentioned portion of the neural retina for transplantation or its candidate from the cell aggregate. In this case, the size cut out as the quality assessment sample may be the size described below (neural retina sheet for transplantation), or it may be even smaller. Therefore, the quality assessment sample can be set and extracted based on the relationship with the position of the neural retina for transplantation or its candidate and the above size.

[0095] <Detection process> The method for evaluating the quality of neural retina for transplantation according to the present invention comprises detecting the expression of neural retinal cell-associated genes and non-neural retinal cell-associated genes (non-target cell-associated genes) in a quality evaluation sample (detection step). The detection step preferably quantitatively detects the expression levels of the genes. The non-target cell-associated genes include one or more genes selected from the group consisting of cerebrospinal tissue marker genes and ocular tissue marker genes.

[0096] (Neural retinal cell-related genes) A neural retinal cell-associated gene (target cell-associated gene) refers to a gene expressed by neural retinal cells. Preferred neural retinal cell-associated genes are those that are expressed at higher levels in photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, or precursor cells of these cells, neural retinal progenitor cells, etc., compared to non-target cells. Examples of neural retinal cell-associated genes include the above-mentioned neural retinal cell markers, with RAX, Chx10, SIX3, SIX6, RCVRN, CRX, NRL, and NESTIN being preferred. The GenBank IDs of the neural retinal cell markers are listed in Table 1 below. [Table 1]

[0097] The neural retinal cell-associated gene is preferably, but not limited to, the genes listed in Table 1. Other neural retinal cell-associated genes include Rax2, Vsx1, Blimp1, RXRG, S-opsin, M / L-opsin, Rhodopsin, Brn3, and L7.

[0098] (Non-neural retinal cell-related genes) Unintended cells can be identified by detecting unintended cells that are induced as by-products during the process of manufacturing cell aggregates containing neural retina as pharmaceutical raw materials, and genes expressed in cells or tissues from which such unintended cells may be produced as by-products (hereinafter referred to as non-neural retinal cell-related genes or unintended cell-related genes).

[0099] In one embodiment, non-neural retinal cell-associated genes (non-target cell-associated genes) include cerebrospinal tissue marker genes and eye-related tissue marker genes. In another embodiment, non-neural retinal cell-associated genes may include undifferentiated iPS cell marker genes.

[0100] In one embodiment, the cerebrospinal tissue marker gene may be one or more genes selected from the group consisting of a telencephalon marker gene, a diencephalon / mesencephalon marker gene, and a spinal cord marker gene. The diencephalon / mesencephalon marker gene may be one or more genes selected from the group consisting of a diencephalon marker gene, a mesencephalon marker gene, and a hypothalamic marker gene related to the hypothalamus, which is part of the diencephalon.

[0101] In one embodiment, the eye-related tissue marker gene may be one or more genes selected from the group consisting of an optic stalk marker gene, a ciliary body marker gene, a lens marker gene, and a retinal pigment epithelium marker gene.

[0102] Telencephalic marker genes refer to genes expressed in the telencephalon. Telencephalic marker genes may include one or more genes selected from the group consisting of FoxG1 (also known as Bf1), Emx2, Dlx2, Dlx1, and Dlx5. GenBank IDs of telencephalic marker genes are shown in Table 2 below. [Table 2]

[0103] Telencephalic marker genes are preferably, but not limited to, the genes listed in Table 2. Other telencephalic marker genes include Emx1, LHX2, LHX6, LHX7, Gsh2, etc.

[0104] The diencephalon / mesencephalon marker gene refers to a gene expressed in the diencephalon and / or mesencephalon. The diencephalon / mesencephalon marker gene may include one or more genes selected from the group consisting of OTX1, OTX2, and DMBX1. The GenBank IDs of the diencephalon / mesencephalon marker genes are shown in Table 3 below. The diencephalon / mesencephalon marker gene may also include a hypothalamic marker, which will be described later with respect to the hypothalamus, a region of the diencephalon. That is, the diencephalon / mesencephalon marker gene may include one or more genes selected from the group consisting of OTX1, OTX2, OTX2, DMBX1, Rx, Nkx2.1, OTP, FGFR2, EFNA5, and GAD1. [Table 3]

[0105] The hypothalamic marker gene refers to a gene expressed in the hypothalamus. The hypothalamic marker gene may include one or more genes selected from the group consisting of Rx, Nkx2.1, Dmbx1, OTP, gad1, FGFR2, and EFNA5. The GenBank IDs of the hypothalamic marker genes are shown in Table 4 below. [Table 4]

[0106] Spinal cord marker genes refer to genes expressed in the spinal cord. Spinal cord marker genes may include one or more genes selected from the group consisting of HoxB2, HoxA5, HOXC5, HOXD1, HOXD3, and HOXD4. GenBank IDs of spinal cord marker genes are shown in Table 5 below. [Table 5]

[0107] Spinal cord marker genes are preferably, but are not limited to, the genes listed in Table 5. Other spinal cord marker genes include genes that form Hox clusters.

[0108] On the other hand, in one embodiment, when retinoids (e.g., retinoic acid, retinal, retinol, all-trans-retinoic acid, and 11-cis-retinoic acid) are used in the manufacturing process, expression of HOX genes (e.g., HOXC5, HOXA5, and HOXB2) can be observed even when good retinal tissue is produced. HOX gene expression is thought to be regulated by retinoic acid signaling, and HOX gene expression increases to a degree that does not affect the differentiation induction of retinal tissue. This effect of retinoic acid signaling is thought to be due to promoting posteriorization along the anterior-posterior axis. Therefore, when retinoids are used in the manufacturing process (e.g., after the start of retinal differentiation induction), HOX genes (e.g., HOXC5, HOXA5, and HOXB2) can be excluded from the genes to be evaluated for quality, or the quality of the neural retina for transplantation can be determined to be good even if expression of these genes is observed.

[0109] Optic stalk marker genes refer to genes expressed in the optic stalk. Optic stalk marker genes may include one or more genes selected from the group consisting of GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A. GenBank IDs of the optic stalk marker genes are shown in Table 6 below. [Table 6]

[0110] Lens marker genes refer to genes expressed in the lens. Lens marker genes may include one or more genes selected from the group consisting of CRYAA and CRYBA1. GenBank IDs of lens marker genes are shown in Table 7 below. [Table 7]

[0111] The ciliary body marker gene refers to a gene expressed in the ciliary body, the ciliary margin, and / or the ciliary body. The ciliary body marker gene may include one or more genes selected from the group consisting of Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The GenBank IDs of the ciliary body marker genes are shown in Table 8 below. [Table 8]

[0112] The retinal pigment epithelial marker gene refers to a gene expressed in retinal pigment epithelial cells. Examples of the retinal pigment epithelial marker gene include the retinal pigment epithelial markers described above, and may include one or more genes selected from the group consisting of MITF, TTR, and BEST1. The GenBank IDs of the retinal pigment epithelial marker genes are shown in Table 9 below. [Table 9]

[0113] In one embodiment, the non-target cell-associated genes may further include undifferentiated pluripotent stem cell marker genes.

[0114] The undifferentiated pluripotent stem cell marker gene may include one or more genes selected from the group consisting of Oct3 / 4, Nanog, and lin28. Preferably, the undifferentiated pluripotent stem cell marker gene is one or more genes selected from the group consisting of Oct3 / 4, Nanog, and lin28. The GenBank IDs of the undifferentiated pluripotent stem cell marker genes are shown in Table 10 below. [Table 10]

[0115] (Detection method) In one embodiment, the expression of neural retinal cell-related genes and non-neural retinal non-target cell-related genes can be detected by techniques such as, but not limited to, Western blotting, immunostaining, flow cytometry analysis / flow cytometer (FACS (registered trademark, manufactured by BD), etc.), Northern blotting, electrophoresis, PCR (preferably quantitative PCR (qPCR) and / or real-time PCR), gene chip analysis, and next-generation sequencer. Of these, quantitative PCR is useful in terms of quantitation, detection sensitivity, stability of results, and speed. Furthermore, by applying a device used for single-cell quantitative PCR (e.g., Biomark HD (manufactured by Fluidigm), etc.) to conventional quantitative PCR, it is possible to evaluate multiple quality assessment samples in a short period of time.

[0116] In one embodiment, the expression levels of neural retinal cell-associated genes and non-neural retinal cell-associated genes in two or more quality assessment samples may be simultaneously detected by quantitative PCR. The quantitative PCR may be performed, for example, by a method comprising the following steps (1) to (5). Specific methods are well known to those skilled in the art. (1) Preparing a flow path plate having a sample well group consisting of one group of 8 to 800 independent sample wells, a primer well group consisting of one or more groups of 8 to 800 independent primer wells, and flow paths connecting the independent sample wells in the sample well group to the independent primer wells in each primer well group, a solution containing nucleic acids obtained from two or more of the above-mentioned quality assessment samples (sample solution), and a solution containing one or more primers specific to one or more of the above-mentioned neural retinal system cell-related genes or the above-mentioned non-neural retinal system cell-related genes (primer solution); (2) adding the sample solution to each of the quality evaluation samples in a group of sample wells so that one sample solution is added to one sample well; (3) adding the primer solution to one or more primer wells in the one or more primer well groups so that the primer wells are in different primer well groups; (4) separately mixing the primers with the nucleic acid through the flow path; and (5) Perform quantitative PCR using the mixture obtained in (4).

[0117] Flow cytometry analysis using a flow cytometer capable of detecting the proportion of expressing cells is also useful. In recent years, advances in detection speed have been made, and high-throughput flow cytometers (such as FACS (registered trademark)) capable of evaluating a large number of samples are now available. Therefore, the use of a high-throughput flow cytometer is also useful for detecting the expression of neural retinal cell-related genes and non-neural retinal non-target cell-related genes. Commercially available high-throughput flow cytometers (e.g., MACSQuant (registered trademark) Analyzers, manufactured by Miltenyi Biotec) can be used for such high-throughput flow cytometers.

[0118] <Judgment process> Retinal tissue for transplantation, preferably a neural retinal sheet for transplantation, excised from a cell aggregate can be determined to be usable for transplantation, i.e., suitable for transplantation (determination process), if expression of neural retinal cell-related genes is observed and expression of non-neural retinal cell-related genes is not observed.

[0119] "Detection of neuroretinal cell-related gene expression" means that, in a method for detecting gene expression, neuroretinal cell-related gene expression is observed at a level that is substantially detectable by the method (e.g., equal to or greater than the lower limit of detection). "No detection of non-neural retinal cell-related gene expression" means that, in a method for detecting gene expression, the expression of the non-neural retinal cell-related gene cannot be substantially detected by the method (e.g., below the lower limit of detection). "Substantial detectability" means that the gene is detected to a level beyond which it can be said that the gene is substantially functional. Those skilled in the art can appropriately determine this depending on the gene and the detection method. For example, in the case of a quantitative method for detecting gene expression, a range of more than 0% to 10% or more than 0% to 5% based on the lower limit of detection of the gene expression can be determined as not being substantially detectable (i.e., the expression of the gene cannot be detected).

[0120] In one aspect, it is preferable to determine that the tissue can be used as a neural retina for transplantation when the quantitative PCR method satisfies the following criteria 1 and 2. Criterion 1: The difference (ΔCt value) between the threshold cycle (Ct) value of the neural retinal cell-related gene and the Ct value of the internal control gene is 10 or less. Criterion 2: The difference (ΔCt value) between the Ct value of the non-neural retinal cell-related gene and the Ct value of the internal control gene is 5 or more.

[0121] The threshold cycle (Ct) value refers to the cycle number at which a constant amount of amplified product is reached in the exponential region of gene amplification by PCR. Because the Ct value is inversely correlated with the initial amount of gene, it is used to calculate the initial copy number of the gene. In one embodiment, the "2^Ct value (2 raised to the Ct value)" is inversely proportional to the initial amount of gene, and is therefore used to calculate the initial copy number of the gene. Specifically, a sample containing twice the initial amount of gene will have a Ct value one cycle earlier than a sample containing only half the number of copies of the gene before amplification. The constant amount of amplified product may be within the exponential region of gene amplification by PCR, and can be determined by one skilled in the art.

[0122] The internal standard gene refers to a gene whose expression level varies little between samples. As the internal standard gene, any gene known to those skilled in the art can be used appropriately, and examples thereof include 18S ribosomal RNA, β-actin, HPRT, α-tubulin, transferrin receptor, ubiquitin, and GAPDH, with GAPDH being preferred.

[0123] The Ct value is inversely correlated with the initial amount of a gene and therefore depends on the expression level of the gene in the cell. That is, when the concentration of a nucleic acid-containing solution is constant, the Ct value varies depending on the internal standard gene used, and the difference between the Ct value of a specific gene and the Ct value of the internal standard gene (ΔCt value) is affected by the internal standard gene used. Unless otherwise specified, the ΔCt value in this specification is stated based on the value when GAPDH is used as the internal standard gene.

[0124] When an internal standard gene other than GAPDH is used as the internal standard gene, the ΔCt values ​​of the above criteria 1 and 2 can be corrected by comparing the expression levels of GAPDH and the internal standard gene other than GAPDH.

[0125] In one embodiment, when β-actin is used as an internal standard gene, in the production method of the present application, the Ct value of GAPDH is lower than the Ct value of β-actin by about 1, i.e., the absolute amount of GAPDH RNA is about twice the absolute amount of β-actin RNA. Criterion 1: The difference (ΔCt value) between the threshold cycle (Ct) value of the neural retinal cell-related gene and the Ct value of the internal control gene is 9 or less. Criterion 2: The difference (ΔCt value) between the Ct value of the non-neural retinal cell-related gene and the Ct value of the internal standard gene can be 4 or more.

[0126] In one embodiment, when HPRT is used as an internal standard gene, the Ct value of GAPDH is about 7 times lower than that of HPRT in the production method of the present invention. In other words, the absolute amount of GAPDH RNA is 2 times lower than the absolute amount of HPRT RNA. 7 (128 times), Criterion 1: The difference (ΔCt value) between the threshold cycle (Ct) value of the neural retinal cell-related gene and the Ct value of the internal control gene is 3 or less. Criterion 2: The difference (ΔCt value) between the Ct value of the non-neural retinal cell-related gene and the Ct value of the internal standard gene can be -2 or more.

[0127] The neural retinal cell-related gene may be any of the genes described above. There are multiple neural retinal cell-related genes. That is, even when extracted from the same neural retinal cell, the Ct value of the reference value 1 may differ depending on the type of neural retinal cell-related gene. Those skilled in the art can determine a ΔCt value that allows for determining whether a neural retinal cell-related gene is expressed for each gene, based on publicly known information such as the expression site and expression level of the neural retinal cell-related gene.

[0128] For example, for the Chx10 gene, when GAPDH is used as an internal standard, the ΔCt value may be 20 or less, preferably 15 or less, and more preferably 10 or less.

[0129] For example, for the Recoverin gene, when GAPDH is used as an internal standard, the ΔCt value may be 16 or less, preferably 11 or less, and more preferably 6 or less.

[0130] In general, the difference (ΔCt value) between the Ct value of the neuroretinal cell-associated gene and the Ct value of the internal standard gene (e.g., GAPDH) may be, for example, 25 or less, 20 or less, 15 or less, or 10 or less. The difference between the Ct value of the neuroretinal cell-associated gene and the Ct value of the internal standard gene may be, for example, -10 or more, -5 or more, 0 or more, or 5 or more.

[0131] The non-neural retinal cell-associated gene may be any of the genes described above. There are multiple non-neural retinal cell-associated genes. That is, even when extracted from the same non-retinal cell, the Ct values ​​vary depending on the non-neural retinal cell-associated gene. Those skilled in the art can determine a ΔCt value for each gene that allows for determining whether the non-neural retinal cell-associated gene is expressed, based on publicly known information such as the expression site and expression level of the non-neural retinal cell-associated gene. For example, when GAPDH is used as an internal standard for the PAX2 gene, the ΔCt value may be 5 or greater. When GAPDH is used as an internal standard for the HOXB2 gene, the ΔCt value may be 5 or greater. Generally, the difference between the Ct value of the non-neural retinal cell-associated gene and the Ct value of the internal standard gene may be 30 or less, 25 or less, or 20 or less. Furthermore, the difference between the Ct value of the non-neural retinal cell-associated gene and the Ct value of the internal standard gene may be, for example, 0 or greater, 3 or greater, or 5 or greater.

[0132] (Neural retinal sheet for transplantation) The retinal tissue for transplantation herein is preferably a neural retina for transplantation, and more preferably a neural retina transplant sheet containing a neural retinal layer. Examples of the neural retina transplant sheet include a neural retina transplant sheet having a front and back surface, the front surface constituting an apical surface containing the neural retinal layer, and the back surface constituting a basal surface containing basement membrane components, the thickness of the neural retina transplant sheet from the basal surface to the apical surface being 100 μm to 1000 μm, the major axis of the neural retina transplant sheet being 600 μm to 2500 μm, and the minor axis of the neural retina transplant sheet being 200 μm to 1500 μm.

[0133] In one aspect of the present invention, the retinal tissue for transplantation is a neural retinal sheet for transplantation, (1) derived from pluripotent stem cells; (2) It has a three-dimensional structure. (3) A neural retina layer having a multi-layer structure including a photoreceptor layer and an inner layer, (4) The photoreceptor layer includes one or more cells selected from the group consisting of photoreceptor precursor cells and photoreceptor cells; (5) The inner layer comprises one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells, and bipolar cells; (6) the surface of the neural retinal layer has an apical surface; (7) There is an inner layer inside the photoreceptor layer along the apical surface, (8) The area of ​​the apical surface of the neural retinal layer is 50% or more of the total surface area of ​​the neural retinal sheet for transplantation; (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neural retina layer; (10) Expression of neural retinal cell-related genes is observed in the neural retinal sheet for transplantation, and expression of non-neural retinal cell-related genes is not observed, and the non-neural retinal cell-related genes include one or more genes selected from the group consisting of cerebrospinal tissue marker genes and eyeball-related tissue marker genes. The present invention relates to a neural retinal sheet for transplantation.

[0134] The neural retinal sheet for transplantation includes (3) a neural retinal layer having a multi-layer structure including a photoreceptor layer and an inner layer. As described in (6) and (7), the photoreceptor layer is present on the outer side (surface) of the neural retinal sheet for transplantation, but an ectopic photoreceptor layer may also be present in the inner layer.

[0135] The neural retinal sheet for transplantation (5) has an inner layer that contains one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells, and bipolar cells, and may also contain one or more cells selected from the group consisting of ectopic photoreceptor progenitor cells and photoreceptor cells. In one embodiment, there is also provided a neural retinal sheet for transplantation in which the content of ganglion cells, amacrine cells, and horizontal cells is 30% or less of the total number of cells, a neural retinal sheet for transplantation in which the content of ganglion cells, amacrine cells, horizontal cells, and bipolar cells is 30% or less of the total number of cells, and / or a neural retinal sheet for transplantation in which the content of bipolar cells is 10% or less of the total number of cells.

[0136] In the neural retinal sheet for transplantation, (8) the area of ​​the neural retinal layer is 40% or more, preferably 50% or more, more preferably 60% or more of the total area of ​​the surface of the neural retinal sheet for transplantation. In the neural retinal sheet for transplantation, (9) the area of ​​the continuous epithelial structure is 60% or more, preferably 70% or more, more preferably 80% or more of the total area of ​​the apical surface of the neural retinal layer.

[0137] The neural retinal cell-associated genes and non-neural retinal cell-associated genes (cerebrospinal tissue marker genes and eyeball-associated tissue marker genes) are the genes described above.

[0138] (10) Whether neural retinal cell-related gene expression and non-neural retinal cell-related gene expression in a neural retinal sheet for transplantation are detected can be determined by detecting gene expression in a portion of the neural retinal sheet for transplantation. Furthermore, if the neural retinal sheet for transplantation is isolated from a cell aggregate in which neural retinal cell-related gene expression is substantially detected and non-neural retinal cell-related gene expression is substantially not detected in a quality assessment sample using the above-described method for evaluating the quality of the neural retina for transplantation, there is no need to detect gene expression in the neural retinal sheet for transplantation itself. Whether or not a gene is substantially expressed is determined by whether the gene expression is substantially detectable by the method for detecting gene expression, as described above.

[0139] The neural retinal cell-associated gene in the neural retinal sheet for transplantation may be, for example, one or more selected from the group consisting of Rx, Chx10, Pax6, and Crx. The proportion of cells expressing a neural retinal cell-associated gene (positive cells) relative to the total number of cells varies depending on the differentiation stage of the neural retina.

[0140] In one embodiment, the ratio of Rx-positive cells to the total number of cells in the neural retina sheet for transplantation may be 30% or more, 40% or more, 50% or more, or 60% or more. In one embodiment, the ratio of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neural retina sheet for transplantation may be 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more. In one embodiment, the ratio of Crx-positive cells to the total number of cells in the neural retina sheet for transplantation may be 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0141] In one embodiment, the ratio of Rx-positive cells to the total number of cells in the neural retina sheet for transplantation may be 30% to 80%, 40% to 70%, 45% to 60%, or 50% to 60%. In one embodiment, the ratio of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neural retina sheet for transplantation may be 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%. In one embodiment, the ratio of Crx-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%.

[0142] In one aspect, the proportion of Chx10-positive and Pax6-positive cells (neural retinal progenitor cells) relative to the total number of cells in the neural retinal sheet for transplantation may be: (1) 10% to 50% or 10% to 30%; (2) 10% to 25% or 15% to 25%; and (3) 10% to 25% or 10% to 20%.

[0143] In another aspect, the proportion of (1) Chx10-positive and Pax6-positive cells (neural retinal progenitor cells) relative to the total number of cells in the neural retinal sheet for transplantation may be 20% or more and 40% or less, (2) Chx10-positive and Pax6-negative cells (progenitor cells biased toward bipolar cells) may be 5% or more and 20% or less, and (3) Chx10-negative and Pax6-positive cells (ganglion cells and amacrine cells) may be 5% or more and 20% or less, or 5% or more and 15% or less.

[0144] In one aspect, the neural retina sheet for transplantation according to the present invention is a neural retina for transplantation that has been determined to be usable as a neural retina for transplantation by the above-mentioned method for evaluating the quality of a neural retina for transplantation, and may be an isolated sheet-like neural retina for transplantation.

[0145] In one aspect, the neural retinal sheet for transplantation according to the present invention is isolated from a cell aggregate containing a neural retina, and may be a neural retinal sheet for transplantation comprising a region near the center of continuous epithelial tissue in the cell aggregate.

[0146] In one embodiment, the retinal tissue for transplantation is a neural retinal sheet for transplantation, (1) isolated from a cell aggregate containing the neural retinal layer; (2) comprising a region near the center of the continuous epithelial tissue in the cell aggregate; and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm. This is a neural retinal sheet for transplantation.

[0147] In one aspect, the neural retinal sheet for transplantation according to the present invention is isolated from a cell aggregate comprising a neural retina, retinal pigment epithelial cells, and ciliary marginal structures, and may be a neural retinal sheet for transplantation that has a continuity of the tangent slope on its surface that is different from the continuity of the tangent slope on the surface of the neural retinal epithelial structure, and that includes the region on the epithelial structure that is farthest from the part containing the retinal pigment epithelial cells.

[0148] In one aspect, the neural retinal sheet for transplantation according to the present invention is isolated from a cell aggregate containing neural retina and cerebrospinal tissue, and may be a neural retinal sheet for transplantation that has a continuity of tangent slope on the surface that is different from the continuity of tangent slope on the surface of the neural retinal epithelial structure, and that includes the region on the epithelial structure that is farthest from the part containing cerebrospinal tissue.

[0149] In one aspect, the neural retinal sheet for transplantation according to the present invention is isolated from a cell aggregate containing two or more neural retinal epithelia, and may be a neural retinal sheet for transplantation comprising the central portion of a neural retinal epithelium that is morphologically favorable and / or large in size and suitable for isolation.

[0150] In one embodiment, the retinal tissue for transplantation is a neural retinal sheet for transplantation, (1) Isolated from a cell aggregate comprising at least a first epithelial tissue and a second epithelial tissue, wherein the first epithelial tissue comprises a human neural retina, and the second epithelial tissue has a continuity of tangent slope on the surface that is different from the continuity of tangent slope on the surface of the first epithelial tissue, and comprises non-neural retinal cells; (2) including a region on the first epithelial tissue that is furthest from the second epithelial tissue; and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm, The second epithelial tissue is a tissue selected from the group consisting of eye-related tissue, cerebrospinal tissue, and other tissues different from the neural retina of the first epithelial tissue. This is a neural retinal sheet for transplantation.

[0151] In one embodiment, the major axis of the neural retinal sheet for transplantation according to the present invention may be, for example, 300 μm to 3300 μm, preferably 600 μm to 2500 μm, and more preferably 1100 μm to 1700 μm.

[0152] In one embodiment, the minor axis of the neural retina sheet for transplantation according to the present invention may be, for example, 100 μm to 2000 μm, preferably 200 μm to 1500 μm, and more preferably 400 μm to 1100 μm.

[0153] In one embodiment, the height of the neural retina sheet for transplantation according to the present invention may be, for example, 50 μm to 1500 μm, preferably 100 μm to 1000 μm, and more preferably 200 μm to 700 μm.

[0154] In one embodiment, the volume of the neural retinal sheet for transplantation according to the present invention is, for example, 0.001 mm 3 ~4.0mm 3 may be, preferably 0.01 mm 3 ~1.5mm 3 , more preferably 0.07 mm 3 ~0.57mm 3 is.

[0155] The method for measuring the major axis, minor axis, and height of a neural retinal sheet for transplantation is not particularly limited, and can be measured, for example, from images captured under a microscope. For example, a neural retinal sheet for transplantation cut from a cell aggregate can be captured using a stereomicroscope: a front image with the cut surface facing the objective lens, and a lateral image with the cut surface tilted so that it is perpendicular to the objective lens. Measurements can be made from the captured images. Here, the major axis refers to the longest line segment and its length among the line segments connecting two endpoints on the cross section of the sheet in the front image. The minor axis refers to the longest line segment and its length among the line segments connecting two endpoints on the cross section of the sheet in the front image that intersect with the major axis. The height refers to the longest line segment and its length among the line segments that intersect with the cross section of the sheet and have their endpoints at the vertex of the retinal sheet. The volume of the sheet means the volume calculated according to the following formula, assuming that the graft is an ellipsoid cut in half so that the cross section passes through the major axis. Volume = 2 / 3 x pi (π) x (major axis / 2) x (minor axis / 2) x height

[0156] There is no particular limitation on the retinal tissue that can be administered using the vehicle of the present invention.

[0157] In one embodiment, the retinal tissue may be the retinal sheet described in Bryce T. McLelland et al., IOVS, May 2018, Vol. 59, No. 6, p. 2586.

[0158] In one embodiment, a retinal sheet may be produced from retinal tissue produced by the production method described in the following document. Nakano, T. et al. Cell Stem Cell 10, 771-785 (2012). KawaharaA. et al. Nature Communications, 6, p.6286(2015). KuwaharaA, Yamasaki S, et al. Sci Rep. 2019 Dec 12;9(1):18936. Lamba, DA, Gust, J. & Reh, TA Cell Stem Cell 4, 73-79, (2009). Zhu, J., Cifuentes, H., Reynolds, J. & Lamba, DA Cell Stem Cell 20,374-384.e375(2017) Meyer, JS et al. Stem Cells 29, 1206-1218, (2011). Zhong,X. et al. Nat Commun 5, doi:10.1038 / ncomms5047 (2014). Boucherie, C., et al. Stem Cells 31, 408-414, doi:10.1002 / stem.1268 (2013). Gonzalez-Cordero, A. et al. Nat Biotechnol 31, 741-747, (2013). Mellough, CB et al. Stem Cells 33, 2416-2430, (2015). Hallam, D. et al. Stem Cells, doi:10.1002 / stem.2883 (2018). Reichman, S. et al.PNAS 111, 8518-8523, (2014). Gagliardi, G. et al. Stem Cell Reports 11, 665-680, (2018). Tucker, BA, et al. Stem Cells Transl Med 2, 16-24, (2013). Wahlin, KJ et al. Sci Rep 7, 766, (2017). DiStefano, T. et al. Stem Cell Reports 10, 300-313, (2018).

[0159] (Retinal tissue production) Herein, the retinal tissue used for transplantation may be retinal tissue excised from a living organism (e.g., a fetus) or retinal tissue differentiated from autologous or allogeneic pluripotent stem cells (e.g., embryonic stem cells (ES cells) established from an embryo within 14 days of fertilization, or induced pluripotent stem cells (iPS cells)). The retinal tissue may be the neural retina containing photoreceptor cells.

[0160] Methods for preparing retinal tissue from living organisms are well known to those skilled in the art. Specifically, retinal tissue can be excised under anesthesia.

[0161] Examples of retinal tissues induced to differentiate from pluripotent stem cells include retinal tissues obtained by suspension culture of cell aggregates formed from pluripotent stem cells under appropriate differentiation induction conditions.

[0162] The "pluripotent stem cells" used as raw materials here can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Muse cells (multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells generated from germ cells (e.g., testes) are also included in the pluripotent stem cells.

[0163] Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, and US6,280,718. Embryonic stem cells are available from designated institutions and are also commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. The human embryonic stem cell Crx::Venus line (derived from KhES-1) is available from the RIKEN Institute.

[0164] "Induced pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells using known methods.

[0165] In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4), pp. 663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, and they possess the same pluripotency and self-renewal capabilities as embryonic stem cells (Cell, 2007, 131(5), pp. 861-872; Science, 2007, 318(5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26(1), pp. 101-106).

[0166] Specifically, induced pluripotent stem cells include cells in which pluripotency is induced by reprogramming somatic cells differentiated from fibroblasts or peripheral blood mononuclear cells by expressing any combination of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. 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 (Stem Cells, 2013;31:458-466).

[0167] In addition to producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds, etc. (Science, 2013, 341, pp. 651-654).

[0168] It is also possible to obtain established induced pluripotent stem cell lines, for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University, which are available from Kyoto University and iPS Academia Japan, Inc. Examples of established induced pluripotent stem cell lines available from Kyoto University include Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University.

[0169] In this specification, pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0170] In this specification, pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0171] Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture by methods well known to those skilled in the art.

[0172] The method for producing the retinal tissue described in 2 above is not particularly limited, and the tissue can be produced by any method known to those skilled in the art.

[0173] The retinal tissue described in 2 above is preferably a retinal tissue containing a neural retinal layer (neural retina). The neural retinal tissue used for transplantation may be a cell aggregate containing the neural retina or a retinal sheet excised from a portion thereof.

[0174] The cell aggregates containing neural retina herein have an epithelial structure and can be obtained by inducing differentiation of pluripotent stem cells.

[0175] One embodiment includes a method for producing cell aggregates containing neural retina using differentiation-inducing factors. Examples of differentiation-inducing factors include basement membrane preparations, substances acting on the BMP signaling pathway, substances inhibiting the Wnt signaling pathway, and substances acting on the IGF signaling pathway. One embodiment includes a method for producing cell aggregates containing neural retina by self-organization. Self-organization refers to a mechanism by which a group of cells autonomously generates complex structures. For example, self-organization can be achieved by the SFEB (Serum-free Floating Culture of Embryoid Body-like Aggregates) method (WO2005 / 12390) or the SFEBq method (WO2009 / 148170).

[0176] Methods for forming cell aggregates from pluripotent stem cells include, for example, the Serum-free Floating Culture of Embryoid Body-like Aggregates (SFEB) method (WO2005 / 12390) and the SFEBq method (WO2009 / 148170).

[0177] Methods for inducing differentiation of pluripotent stem cells into retinal tissue include those described in WO2011 / 055855, WO2013 / 077425, WO2015 / 025967, WO2016 / 063985, WO2016 / 063986, WO2017 / 183732, PLoS One. 2010 Jan 20;5(1):e8763, Stem Cells. 2011 Aug;29(8):1206-18, Proc Natl Acad Sci USA. 2014 Jun 10;111(23):8518-23, and Nat Commun. 2014 Jun 10;5:4047", WO2012 / 173207, WO2015 / 053375, WO2015 / 053376, WO2015 / 068505, WO2017 / 043605, WO2017 / 183732, WO2019 / 017492, WO2019 / 054514, WO2019 / 054515, "Stem Cell Reports, 2(2), 205-218 (2014)", "Cell Stem Cell, 10(6), 771-785 (2012)", "Nature Communications | 6:6286 (2015)" and the like. However, these methods are not particularly limited.

[0178] Furthermore, examples of methods for producing retinal tissue include the method described in Bryce T. McLelland et al., IOVS, May 2018, Vol. 59, No. 6, p. 2586.

[0179] Furthermore, methods for producing retinal tissue include those described in the following documents. Nakano, T. et al. Cell Stem Cell 10, 771-785 (2012). KawaharaA. et al. Nature Communications, 6, p.6286(2015). KuwaharaA, Yamasaki S, et al. Sci Rep. 2019 Dec 12;9(1):18936. Lamba,D. A., Gust, J. & Reh, T. A. Cell Stem Cell 4, 73-79, (2009). Zhu,J., Cifuentes, H., Reynolds, J. & Lamba, D. A. Cell Stem Cell 20,374-384.e375(2017) Meyer,J. S. et al. Stem Cells 29, 1206-1218, (2011). Zhong,X. et al. Nat Commun 5, doi:10.1038 / ncomms5047 (2014). Boucherie,C., et al. Stem Cells 31, 408-414, doi:10.1002 / stem.1268 (2013). Gonzalez-Cordero,A. et al. Nat Biotechnol 31, 741-747, (2013). Mellough,C. B. et al. Stem Cells 33, 2416-2430, (2015). Hallam,D. et al. Stem Cells, doi:10.1002 / stem.2883 (2018). Reichman,S. et al.PNAS 111, 8518-8523, (2014). Gagliardi,G. et al. Stem Cell Reports 11, 665-680, (2018). Tucker,B. A., et al. Stem Cells Transl Med 2, 16-24, (2013). Wahlin,K. J. et al. Sci Rep 7, 766, (2017). DiStefano,T. et al. Stem Cell Reports 10, 300-313, (2018).

[0180] In one specific embodiment, a cell aggregate containing neural retina can be prepared by a method comprising the following steps (A), (B), (C) and (D). (A) culturing pluripotent stem cells in a medium for pluripotent stem cell culture in the absence of feeder cells; (B) forming cell aggregates by suspension culture of the cells obtained in step (A); (C) further culturing the cell aggregates obtained in step (B) in suspension in a medium containing a substance acting on the BMP signaling pathway; and (D) A step of culturing the cell aggregates obtained in step (C) in suspension and allowing them to mature. Step (A) may further include a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway activator. Furthermore, step (B) may include a sonic hedgehog signaling pathway activator and / or a Wnt signaling pathway inhibitor, as described below.

[0181] This method is also disclosed in, for example, WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732, and for more details, reference can be made to WO2015 / 025967, WO2016 / 063985, WO2017 / 183732, WO2019 / 017492, WO2019 / 054514, WO2019 / 054515, etc.

[0182] The "pluripotent stem cell culture medium" used in step (A) is a medium in which pluripotent stem cells can be cultured under feeder-free conditions, and examples of such a medium include a medium containing a factor for maintaining undifferentiated state.

[0183] As used herein, the factor for maintaining an undifferentiated state is not particularly limited as long as it has the effect of suppressing the differentiation of pluripotent stem cells. Examples of factors commonly used by those skilled in the art for maintaining an undifferentiated state include substances acting on the FGF signaling pathway, substances acting on the TGFβ family signaling pathway, and insulin. Specific examples of substances acting on the FGF signaling pathway include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Furthermore, examples of substances acting on the TGFβ family signaling pathway include substances acting on the TGFβ signaling pathway and substances acting on the Nodal / Activin signaling pathway. Examples of substances acting on the TGFβ signaling pathway include TGFβ1 and TGFβ2. Examples of substances acting on the Nodal / Activin signaling pathway include Nodal, Activin A, and Activin B. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the medium in step (A) preferably contains bFGF as a factor for maintaining an undifferentiated state.

[0184] The concentration of the undifferentiation maintenance factor in the medium used in step (A) is a concentration that allows the cultured pluripotent stem cells to be maintained in an undifferentiated state, and can be appropriately determined by those skilled in the art. For example, specifically, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually about 4 ng to 500 ng / mL, preferably about 10 ng to 200 ng / mL, and more preferably about 30 ng to 150 ng / mL.

[0185] Many synthetic media that can be used for pluripotent stem cell culture under feeder-free conditions have been developed and are commercially available, such as Essential 8 medium (Life Technologies). Essential 8 medium contains the following additives to DMEM / F12 medium: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and TGFβ family signaling pathway agents (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Other commercially available feeder-free media include S-medium (DS Pharma Biomedical), StemPro (Life Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14), mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), TeSR-E8 (STEMCELL Technologies), and StemFit (Ajinomoto Co.). By using these media in step (A), the present invention can be easily implemented. The use of these media enables pluripotent stem cells to be cultured under feeder-free conditions. The medium used in step (A) is, for example, a serum-free medium to which no substance acting on the BMP signaling pathway, no substance acting on the Wnt signaling pathway, or no substance inhibiting the Wnt signaling pathway is added.

[0186] The medium used to prepare cell aggregates containing neural retina, i.e., the medium used in steps (B), (C), and (D) above, can be a basal cell growth medium (also referred to as a basal medium) unless otherwise specified. The basal cell growth medium is not particularly limited as long as it allows cell culture, and any commercially available basal medium for cell growth can be used. Specific examples include media that can be used to culture animal cells, such as BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, MEM medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Leibovitz's L-15 medium, and mixtures thereof. Alternatively, a medium supplemented with N2 medium, a supplementary medium, may be used.

[0187] As used herein, a TGFβ family signaling pathway inhibitor refers to a substance that inhibits the TGFβ family signaling pathway, i.e., the signaling pathway mediated by the Smad family, and specific examples include TGFβ signaling pathway inhibitors (e.g., SB431542, LY-364947, SB505124, A-83-01, etc.), Nodal / Activin signaling pathway inhibitors (e.g., SB431542, A-83-01, etc.), and BMP signaling pathway inhibitors (e.g., LDN193189, Dorsomorphin, etc.). These substances are commercially available.

[0188] As used herein, a sonic hedgehog (hereinafter sometimes referred to as "Shh") signaling pathway agonist refers to a substance that can enhance signal transduction mediated by Shh. Examples of Shh signaling pathway agonists include SHH, partial peptides of SHH (e.g., sonic hedgehog N-terminus (Shh-N), recombinant human sonic hedgehog (C24II) N-terminus (SHH-C24II), and recombinant mouse sonic hedgehog (C25II) N-terminus (SHH-C25II)), hedgehog family proteins other than Shh (e.g., Hh, IHH, DHH, EHH, and TwHH), PMA (purmorphamine), and SAG (smoothened agonist).

[0189] In step (A), the concentrations of the TGFβ family signaling pathway inhibitor and the sonic hedgehog signaling pathway activator may be any concentration capable of inducing differentiation into retinal cells. For example, SB431542 is typically used at a concentration of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is typically used at a concentration of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is typically used at a concentration of 1 to 2000 nM, preferably 10 to 300 nM. SAG is typically used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM. PMA is typically used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM.

[0190] In the feeder-free culture of pluripotent stem cells in step (A), an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold instead of feeder cells. Examples of matrices that can be used as scaffolds include laminin (Nat Biotechnol 28, 611-615, (2010)), laminin fragments (Nat Commun 3, 1236, (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974, (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin.

[0191] The culture time of pluripotent stem cells in step (A) is not particularly limited as long as the effect of improving the quality of the cell aggregates formed in step (B) can be achieved when the cells are cultured in the presence of a TGFβ family signaling pathway inhibitor and / or a Sonic hedgehog signaling pathway agonist (e.g., 100 nM to 700 nM), but is typically 0.5 to 144 hours. In one aspect, the culture time is preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours).

[0192] The medium used in step (B) can be a serum-containing medium or a serum-free medium. A serum-free medium is preferably used to avoid contamination with chemically undefined components. To avoid the complicated preparation process, for example, a serum-free medium supplemented with an appropriate amount of a serum substitute such as commercially available KSR can be used. The amount of KSR added to a serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%.

[0193] When forming aggregates, first, dispersed cells are prepared by dispersing the cells obtained in step (A). The "dispersed cells" obtained by the dispersion procedure are, for example, cells in a state where 70% (preferably 80% or more) or more are single cells and 30% or less (preferably 20% or less) are clumps of 2 to 50 cells. Dispersed cells are cells in a state where there is almost no adhesion (for example, surface adhesion) between cells.

[0194] A suspension of dispersed cells is seeded in a culture vessel, and the dispersed cells are cultured under non-adherent conditions to allow multiple cells to aggregate and form aggregates. In one embodiment, a certain number of dispersed stem cells are placed in each well of a multi-well plate (U-bottom, V-bottom) such as a 96-well plate, and when this is subjected to static culture, the cells rapidly aggregate to form one aggregate in each well (SFEBq method). When cells are cultured in suspension using a 96-well plate, approximately 1 × 10 cells are cultured per well. 3 to approximately 1 x 10 5 Cells (preferably about 3 x 10 3 to about 5 × 10 4 cells, approximately 4 x 10 3 to approximately 2 × 10 4 A solution prepared to form aggregates (cells) is added to the wells, and the plate is left to stand to form aggregates.

[0195] In one embodiment, the medium used in step (B) comprises a sonic hedgehog signaling pathway agent. That is, in one specific embodiment, a cell aggregate containing neural retina can be prepared by a method comprising the following steps (A), (B), and (C): (A) culturing pluripotent stem cells in a medium containing an undifferentiated state maintenance factor, in the absence of feeder cells, and optionally containing a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist; (B) forming cell aggregates by culturing the cells obtained in step (A) in suspension in a medium containing a sonic hedgehog signaling pathway active substance; (C) A step of further culturing the cell aggregates obtained in step (B) in suspension in a medium containing a substance acting on the BMP signaling pathway. The substance acting on the Sonic Hedgehog signaling pathway in step (B) may be any of the above-mentioned substances at the above-mentioned concentrations (e.g., 10 nM to 300 nM). The substance acting on the Sonic Hedgehog signaling pathway is preferably included in the medium from the start of suspension culture. A ROCK inhibitor (e.g., Y-27632) may be added to the medium. The culture time is, for example, 12 hours to 6 days. In one example, the medium used in step (B) is a medium that does not contain one or more (preferably all) selected from the group consisting of a substance acting on the BMP signaling pathway, a substance acting on the Wnt signaling pathway, a TGFβ family signaling pathway inhibitor, and a substance acting on the TGFβ family signaling pathway.

[0196] In one embodiment, the cell aggregates in step (B) are at a differentiation stage where they express pluripotency markers, specifically, where one or more markers selected from Oct3 / 4, Sox2, Klf4, Nanog, Sall4, lin28, Esrrb, and Esrrb can be detected.

[0197] As used herein, a substance acting on the BMP signaling pathway refers to a substance that can enhance the signaling pathway mediated by BMP. Examples of substances acting on the BMP signaling pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2 protein, BMP4 protein, and BMP7 protein are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries, Ltd.

[0198] The medium used in step (C) may be, for example, a serum-free medium or a serum medium (preferably a serum-free medium) supplemented with a substance acting on the BMP signaling pathway. Serum-free medium and serum medium can be prepared as described above. In one example, the medium used in step (C) is a medium to which one or more (preferably all) selected from the group consisting of a substance acting on the Wnt signaling pathway, a substance inhibiting the TGFβ family signaling pathway, and a substance acting on the TGFβ family signaling pathway are not added. In another example, the medium used in step (C) is a medium to which no substance acting on the Sonic Hedgehog signaling pathway is added. In another example, the medium used in step (C) may be a medium to which a substance acting on the Wnt signaling pathway is added.

[0199] The concentration of the substance acting on the BMP signaling pathway may be any concentration that can induce differentiation into retinal cells. For example, in the case of human BMP4 protein, it is added to the medium to a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL).

[0200] The substance acting on the BMP signaling pathway may be added to the medium at least about 24 hours after the initiation of suspension culture in step (A), and may be added within several days (e.g., within 15 days) after the initiation of suspension culture. Preferably, the substance acting on the BMP signaling pathway is added to the medium between days 1 and 15, more preferably between days 1 and 9, and most preferably on day 3 after the initiation of suspension culture.

[0201] In a specific embodiment, for example, on days 1 to 9, preferably days 1 to 3, after the initiation of suspension culture in step (B), the medium is partially or completely replaced with a medium containing BMP4, and the medium is adjusted to a final BMP4 concentration of approximately 1 to 10 nM. Culture can then be continued in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, and more preferably 2 to 5 days. Here, to maintain the same BMP4 concentration, the medium can be partially or completely replaced with a medium containing BMP4 once or twice. Alternatively, the BMP4 concentration can be reduced in stages. For example, the concentration of the substance acting on the BMP signaling pathway (BMP4) can be maintained from days 2 to 10 after the initiation of suspension culture in step (B), and then reduced in stages from days 6 to 20 after the initiation of suspension culture in step (B).

[0202] Culture conditions such as culture temperature and CO2 concentration in steps (A) to (C) can be set appropriately. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0203] By varying the culture period in step (C) above, retinal cells at various stages of differentiation can be produced as retinal cells contained in the cell aggregates. That is, retinal cells in cell aggregates containing various ratios of immature retinal cells (e.g., retinal progenitor cells, photoreceptor progenitor cells) and mature retinal cells (e.g., photoreceptors) can be produced. By extending the culture period in step (C), the proportion of mature retinal cells can be increased.

[0204] The method disclosed in WO2017 / 183732 can also be used for the above step (B) and / or step (C). That is, in step (B) and / or step (C), cell aggregates can be formed by suspension culture in a medium further containing a Wnt signaling pathway inhibitor.

[0205] The Wnt signaling pathway inhibitor used in step (B) and / or step (C) is not particularly limited as long as it can suppress Wnt-mediated signaling, and may be any of a protein, nucleic acid, low-molecular-weight compound, etc. Wnt-mediated signals are transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and low-density lipoprotein receptor-related protein 5 / 6 (LRP5 / 6). Examples of inhibitors of the Wnt signaling pathway include substances that act directly on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus proteins, etc.), substances that inhibit physiological activities resulting from signal transduction by Wnt receptors [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2 ,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-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-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), and small molecule compounds such as 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 Wnt signaling pathway inhibitor may contain one or more of these. CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc. are known Wnt signaling pathway inhibitors, and are appropriately available as commercially available products, etc. IWR1e is preferably used as the Wnt signaling pathway inhibitor.

[0206] The concentration of the Wnt signaling pathway inhibitor in step (B) may be any concentration capable of inducing the formation of favorable cell aggregates. For example, IWR-1-endo is added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR-1-endo.

[0207] In step (B), the timing of adding the Wnt signaling pathway inhibitor to the medium is preferably early. The Wnt signaling pathway inhibitor is added to the medium usually within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, and even more preferably at the initiation of suspension culture in step (B). Specifically, for example, a basal medium supplemented with the Wnt signaling pathway inhibitor may be added, or a partial or complete medium exchange with the basal medium may be performed. The period during which the cells obtained in step (A) are exposed to the Wnt signaling pathway inhibitor in step (B) is not particularly limited, but preferably, the inhibitor is added to the medium at the initiation of suspension culture in step (B) and allowed to act until the end of step (B) (immediately before the addition of the substance acting on the BMP signaling pathway). More preferably, the cells are continuously exposed to the Wnt signaling pathway inhibitor even after the end of step (B) (i.e., during step (C)), as described below. In one embodiment, as described below, the Wnt signaling pathway inhibitor may be allowed to continue acting even after step (B) is completed (i.e., during step (C)), until retinal tissue is formed.

[0208] In step (C), any of the aforementioned Wnt signaling pathway inhibitors can be used as the Wnt signaling pathway inhibitor, but preferably, the same type of Wnt signaling pathway inhibitor used in step (B) is used in step (C).

[0209] The concentration of the Wnt signaling pathway inhibitor in step (C) may be any concentration capable of inducing retinal progenitor cells and retinal tissue. For example, IWR-1-endo is added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the medium in step (C), relative to the concentration of the Wnt signaling pathway inhibitor in the medium in step (B) taken as 100, is preferably 50 to 150, more preferably 80 to 120, and even more preferably 90 to 110. More preferably, it is equivalent to the concentration of the Wnt signaling pathway inhibitor in the medium in the second step.

[0210] The timing of adding the Wnt signaling pathway inhibitor to the medium is not particularly limited as long as it achieves the formation of aggregates containing retinal cells or retinal tissue, but the earlier the better. Preferably, the Wnt signaling pathway inhibitor is added to the medium at the start of step (C). More preferably, after the Wnt signaling pathway inhibitor is added in step (B), it continues to be contained in the medium in step (C) (i.e., from the start of step (B)). Even more preferably, the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B), and it continues to be contained in the medium in step (C). For example, a substance acting on the BMP signaling pathway (e.g., BMP4) may be added to the culture obtained in step (B) (a suspension of aggregates in a medium containing a Wnt signaling pathway inhibitor).

[0211] The period during which the Wnt signaling pathway inhibitor is allowed to act is not particularly limited, but is preferably 2 to 30 days, more preferably 6 to 20 days, 8 to 18 days, 10 to 18 days, or 10 to 17 days (e.g., 10 days), counting from the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B). In another embodiment, the period during which the Wnt signaling pathway inhibitor is allowed to act is preferably 3 to 15 days (e.g., 5, 6, or 7 days), more preferably 6 to 10 days (e.g., 6 days), counting from the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B).

[0212] The cell aggregates obtained by the above-mentioned method can be cultured for about 2 to 4 days in a serum-free medium or serum medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway (step (D)), and then cultured for about 30 to 200 days (30 to 150 days, 50 to 120 days, or 60 to 90 days) in a serum-free medium or serum medium not containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway (step (E)), thereby producing a neural retina containing ciliary marginal zone-like structures.

[0213] In one embodiment, a neural retina containing a ciliary margin-like structure can be produced from the cell aggregates obtained in steps (A) to (C), which are on days 6 to 30, 10 to 20 (10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th) after the start of suspension culture in step (B), by the above steps (D) and (E).

[0214] The substance acting on the Wnt signaling pathway is not particularly limited as long as it can enhance signal transduction mediated by Wnt. Specific examples of substances acting on the Wnt signaling pathway include GSK3β inhibitors (e.g., 6-Bromoindirubin-3'-oxime (BIO), CHIR99021, and Kenpaullone). For example, in the case of CHIR99021, the range is about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM.

[0215] The FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit signal transduction mediated by FGF. Examples of FGF signaling pathway inhibitors include SU-5402, AZD4547, and BGJ398. For example, SU-5402 is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 5 μM.

[0216] In one example, the medium used in step (D) is a medium to which one or more (preferably all) selected from the group consisting of a BMP signaling pathway active substance, a Wnt signaling pathway inhibitor, an SHH signaling pathway active substance, a TGFβ family signaling pathway inhibitor, and a TGFβ family signaling pathway active substance are not added.

[0217] Some or all of the steps in step (E) above can be performed using the continuous epithelial tissue maintenance medium disclosed in WO2019 / 017492. Specifically, by culturing using a continuous epithelial tissue maintenance medium, the continuous epithelial structure of the neural retina can be maintained. One example of a continuous epithelial tissue maintenance medium is a medium prepared by adding B27 supplement (e.g., Thermo Fisher Scientific, 21103049) to Neurobasal medium (e.g., Thermo Fisher Scientific, 12587010).

[0218] In the culture in step (E) above, it is preferable to gradually change the medium to a medium for maintaining continuous epithelial tissue in order to achieve both differentiation and / or maturation of retinal cells (particularly photoreceptors) and maintenance of a continuous epithelial structure. For example, the cells can be cultured in a basal cell growth medium (e.g., DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine) for the first 10 to 30 days, a mixed medium of the basal cell growth medium and continuous epithelial tissue maintenance medium (a 1:3 mixture of DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine and Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine) for the next 10 to 40 days, and a continuous epithelial tissue maintenance medium (e.g., Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine) for the next 20 to 140 days.

[0219] In some or all of the steps of step (E), whether a basal cell proliferation medium, a continuous epithelial tissue maintenance medium, or a mixed medium thereof is used, the medium may further contain a substance acting on the thyroid hormone signaling pathway. Culturing in a medium containing a substance acting on the thyroid hormone signaling pathway enables the production of cell aggregates containing neural retina with a low proportion of bipolar cells, amacrine cells, ganglion cells, horizontal cells, etc., contained in the neural retina and an increased proportion of photoreceptor precursor cells.

[0220] As used herein, a substance acting on the thyroid hormone signaling pathway refers to a substance that can enhance signal transduction mediated by thyroid hormone, and is not particularly limited as long as it can enhance the thyroid hormone signaling pathway. Examples of substances acting on the thyroid hormone signaling pathway include triiodothyronine (hereinafter sometimes abbreviated as T3), thyroxine (hereinafter sometimes abbreviated as T4), and thyroid hormone receptor (preferably TRβ receptor) agonists.

[0221] Further, examples of thyroid hormone receptor agonists known to those skilled in the art include those described in WO 97 / 21993, WO 2004 / 066929, WO 2004 / 093799, WO 2000 / 039077, WO 2001 / 098256, WO 2003 / 018515, WO 2003 / 084915, WO 2002 / 094319, WO 2003 / 064369, JP 2003-2006692, and the like. Examples of the diphenylmethane derivatives include compounds such as diphenylmethane derivatives, diaryl ether derivatives, pyridazine derivatives, pyridine derivatives, and indole derivatives described in JP-A No. 02-053564, JP-A No. 2002-370978, JP-A No. 2000-256190, WO 2007 / 132475, WO 2007 / 009913, WO 2003 / 094845, WO 2002 / 051805, and WO 2010 / 122980.

[0222] When T3 is used as the substance acting on the thyroid hormone signaling pathway, it can be added to the medium at a concentration in the range of, for example, 0.1 to 1000 nM. Concentrations that exhibit thyroid hormone signaling enhancing activity equivalent to T3 concentrations of 1 to 500 nM, more preferably 10 to 100 nM, even more preferably 30 to 90 nM, and even more preferably around 60 nM are preferred. When T4 is used as the substance acting on the thyroid hormone signaling pathway, it can be added to the medium at a concentration in the range of, for example, 1 nM to 500 μM. Preferably, the concentration is in the range of 50 nM to 50 μM, more preferably 500 nM to 5 μM. When other thyroid hormone receptor agonists are used, they may be added at any concentration that exhibits activity comparable to the agonistic activity of T3 or T4 at the aforementioned concentrations.

[0223] The medium used in step (E) may contain L-glutamine, taurine, serum, etc. In one example, the medium used in step (E) is a medium to which one or more (preferably all) selected from the group consisting of a substance acting on the BMP signaling pathway, a substance inhibiting the FGF signaling pathway, a substance acting on the Wnt signaling pathway, a substance inhibiting the Wnt signaling pathway, a substance acting on the SHH signaling pathway, a substance inhibiting the TGFβ family signaling pathway, and a substance acting on the TGFβ family signaling pathway.

[0224] In one specific embodiment, a cell aggregate containing neural retina can be prepared by a method comprising the following steps (A) to (E): (A) culturing pluripotent stem cells in a medium containing an undifferentiated maintenance factor in the absence of feeder cells, and optionally containing a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist; (B) forming cell aggregates by culturing the cells obtained in step (A) in suspension in a medium that may contain a Wnt signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist; (C) further culturing the cell aggregates obtained in step (B) in suspension in a medium containing a substance acting on the BMP signaling pathway; (D) culturing the cell aggregates obtained in step (C) in a serum-free medium or a serum-containing medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway for a period of about 2 to 4 days; and (E) A process of culturing the cell aggregates obtained in step (D) for approximately 30 to 200 days in a serum-free medium or a serum-free medium that does not contain a substance active in the Wnt signaling pathway or an inhibitor of the FGF signaling pathway, but may contain a substance active in the thyroid hormone signaling pathway.

[0225] In one specific embodiment, a cell aggregate containing neural retina can be prepared by a method comprising the following steps (A) to (E): (A) culturing pluripotent stem cells for 12 to 48 hours in a medium containing an undifferentiated state maintenance factor and containing a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway activator in the absence of feeder cells; (B) forming cell aggregates by suspension culturing the cells obtained in step (A) in a medium containing a Wnt signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist for 12 hours to 72 days (24 hours to 48 hours); (C) a step of further culturing the cell aggregates obtained in step (B) in suspension in a medium containing a substance acting on the BMP signaling pathway for 8 to 15 days (10 to 13 days); (D) culturing the cell aggregates obtained in step (C) for 2 to 4 days in a serum-free medium or a serum-containing medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway; and (E) A process of culturing the cell aggregates obtained in step (D) for approximately 10 to 200 days in a serum-free medium or serum medium that does not contain a substance active in the Wnt signaling pathway or an inhibitor of the FGF signaling pathway, but may contain a substance active in the thyroid hormone signaling pathway.

[0226] Here, step (E) may include culturing the cells in a basal cell growth medium for 10 to 30 days, then culturing them in a mixed medium of the basal cell growth medium and a continuous epithelial tissue maintenance medium containing a substance acting on the thyroid hormone signaling pathway for 10 to 40 days, and then culturing them in a continuous epithelial tissue maintenance medium containing a substance acting on the thyroid hormone signaling pathway for a further 20 to 140 days. In one embodiment, step (E) comprises culturing in the presence of a thyroid hormone signaling pathway agent for 20 to 60 days (30 to 50 days). In one embodiment, the culture period from step (B) to step (E) is 70 to 100 days (80 to 90 days).

[0227] The above-mentioned method can be used to produce cell aggregates containing neural retina, but is not limited thereto. In one embodiment, cell aggregates containing neural retina can be obtained as a mixture of cell aggregates. In another embodiment, for example, one cell aggregate can be produced per well of a 96-well plate, and one cell aggregate containing neural retina can be obtained per well.

[0228] 3. Composition for transplantation The transplantation composition of the present invention comprises the transplantation medium described in 1 above and the retinal tissue for transplantation described in 2 above. The retinal tissue for transplantation is preferably the neural retinal sheet for transplantation described in 2 above.

[0229] The transplantation composition of the present invention contains the retinal tissue for transplantation, i.e., the graft, to be transplanted into a recipient per time, and a transplantation medium in an amount necessary to smoothly aspirate the graft into a transplantation device and expel it under the recipient's retina without damaging the graft.

[0230] Specifically, the transplantation composition of the present invention contains, for example, 1 to 30 grafts and 5 to 500 μl of transplantation composition. Preferably, it contains 1 to 2 grafts and 5 to 50 μl of transplantation composition. In another embodiment, it contains 3 to 4 grafts and 5 to 100 μl of transplantation composition. In another embodiment, it contains 5 to 6 grafts and 10 to 150 μl of transplantation composition. In another embodiment, it contains 7 to 10 grafts and 20 to 200 μl of transplantation composition. In another embodiment, it contains 11 to 30 grafts and 30 to 300 μl of transplantation composition. The graft is preferably a neural retinal sheet for transplantation.

[0231] Specifically, the transplant composition of the present invention contains, for example, 1 to 30 grafts and 5 to 500 μl of the transplant composition, and the number of cells contained in one graft is 10,000 to 1,000,000 cells, preferably 5,000 to 300,000 cells.

[0232] Preferably, the transplantation composition contains one to two grafts (10,000 to 2,000,000 cells, preferably 5,000 to 600,000 cells) and 5 to 50 μl of transplantation composition. In another embodiment, the transplantation composition contains three to four grafts (30,000 to 4,000,000 cells, preferably 15,000 to 1,200,000 cells) and 5 to 100 μl of transplantation composition. In another embodiment, the transplantation composition contains five to six grafts (50,000 to 6,000,000 cells, preferably 25,000 to 1,800,000 cells) and 10 to 150 μl of transplantation composition. In another embodiment, the transplantation composition contains 7 to 10 grafts (70,000 to 10,000,000 cells, preferably 35,000 to 3,000,000 cells) and 20 to 200 μl of transplantation composition. In another embodiment, the transplantation composition contains 11 to 30 grafts (110,000 to 30,000,000 cells, preferably 55,000 to 9,000,000 cells) and 30 to 300 μl of transplantation composition.

[0233] In one aspect of the present invention, a pharmaceutical composition is provided, which comprises the transplantation composition of the present invention, which comprises retinal tissue for transplantation and a transplantation medium.

[0234] The pharmaceutical composition can be used to treat disorders of neural retinal cells or the neural retina, or diseases caused by damage to the neural retina. Examples of diseases caused by disorders of neural retinal cells or the neural retina include ophthalmic diseases such as retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone-rod dystrophy. Examples of damaged states of the neural retina include states in which photoreceptor cells are degenerated and dead.

[0235] In one aspect, the present invention provides a therapeutic agent for a disease caused by damage to the neural retina, comprising the transplant composition of the present invention.

[0236] The transplant composition of the present invention can be prepared by removing the preservation medium from a composition containing a graft and a preservation medium and replacing it with a transplantation medium. The preservation medium is not particularly limited, but an example is the preservation solution described in WO2019 / 017491.

[0237] 4.Transplantation method / treatment method The transplant composition of the present invention can be transplanted by subretinal injection into a mammal (e.g., human, mouse, rat, etc., preferably human) suffering from a retinal disease such as macular degeneration (e.g., atrophic and wet age-related macular degeneration, Stargardt's disease), hereditary retinal disease, retinitis pigmentosa (also known as retinitis pigmentosa), cone dystrophy, rod dystrophy, cone-rod dystrophy, macular hole, giant macular hole, or glaucoma. Examples of transplantation methods include subretinal injection of the transplant composition at the site of damage, such as by incising the eyeball. Examples of transplantation methods include injection using a transplantation device containing an appropriately sized syringe, needle, or resin tip, or by pinching the composition with tweezers.

[0238] When the transplant composition of the present invention is used to treat retinitis pigmentosa, it may be used, for example, to treat patients with corrected visual acuity of less than 0.7, preferably less than 0.2.

[0239] When the transplant composition of the present invention is used to treat retinitis pigmentosa, for example, diseases with visual field constriction, such as diseases in which a central 20-degree visual field remains in a static visual field test (which can be tested by Humphrey visual field test), preferably diseases in which a central 10-degree visual field remains in a static visual field test (which can be tested by Humphrey visual field test), and more preferably diseases in which a central 5-degree visual field remains in a static visual field test (which can be tested by Humphrey visual field test), can be treated by transplanting the transplant composition of the present invention into the area of ​​the retina where visual function has been lost.

[0240] When the transplant composition of the present invention is used for the treatment of retinitis pigmentosa, it may be used for the treatment of patients with an MD value of less than -30 dB in the Humphrey visual field test (10-2), for example.

[0241] One aspect of the present invention is a method for treating neural retinal cell or neural retinal disorder or a disease caused by neural retinal damage, comprising transplanting the transplantation composition of the present invention into a subject requiring transplantation (e.g., subretinal area of ​​an eye suffering from an ophthalmic disease). The transplantation composition of the present invention can be used as a therapeutic agent for a disease caused by neural retinal disorder, or to replenish the damaged area in a damaged neural retina. By transplanting the transplantation composition of the present invention into a patient requiring transplantation who has a neural retinal cell or neural retinal disorder, or a patient with a damaged neural retina, and replenishing the neural retinal cell or damaged neural retina, the disease caused by neural retinal cell or neural retinal disorder, or the damaged neural retina, can be treated. [Example]

[0242] The present invention will be explained in more detail below by showing examples, but the present invention is not limited to these examples in any way.

[0243] Example 1: Viscosity study of candidate implantation media In order to establish a method for transplanting retinal tissue sheets with a major axis of 1 mm or more under the human retina, we investigated the medium for this transplantation.

[0244] First, we performed a preliminary study on the surgical procedure of subretinal retinal sheet transplantation in monkeys. We used a balanced salt solution (BSS) containing neither hyaluronic acid nor chondroitin sulfate as the transplantation medium.

[0245] Using a cataract and vitreous surgery device, a commonly performed vitreous stalk microsurgical detachment was performed, and a posterior vitreous detachment was created as needed. A balanced salt solution was injected under the retina using the liquid injection function of the cataract and vitreous surgery device, creating a localized retinal detachment (bleb). A partial incision was made in the retina at the detached site using vitreous scissors, and a wound for transplantation was created.

[0246] The plastic tip (transplantation tip) was connected to the ejection device, and the balanced salt solution containing the retinal sheet was aspirated. The transplantation tip was inserted through an incision made in the sclera and brought to the transplantation wound made in the retina. The liquid injection function of the cataract and vitreous surgery device was used to inject the balanced salt solution containing the retinal sheet under the retina (see Figure 1).

[0247] As a result, the retinal sheet moves around within the bleb, making it difficult to stably perform subretinal administration.

[0248] Therefore, the composition of the transplant medium was examined using the aqueous hyaluronic acid solutions shown in Table 11 below. [Table 11]

[0249] Specifically, the following media were prepared as potential transplantation media: (1) Viscoat concentrate, (2) 2x dilution of Viscoat (Viscoat:Opeguard = 1:1), (3) 4-fold dilution of Viscoat (Viscoat:Opeguard = 1:3), (4) Hyalein Mini, (5) Provisque, (6) Heeron, (7) Opegan, (8) Opegan diluted 1.5 times (Opegan:Opeguard = 2:1), (9) Opegan diluted twice (Opegan:Opeguard = 1:1). As mentioned above, Opeguard is an aqueous solution (i.e., a balanced salt solution containing sugar) that contains 1.5 mg of glucose, 0.18 mg of calcium chloride hydrate, 0.3 mg of magnesium sulfate hydrate, 2.1 mg of sodium bicarbonate, and the additives sodium citrate hydrate, sodium acetate hydrate, and hydrochloric acid per mL.

[0250] For comparison, (10) balanced salt solution (BSS) was used. The viscosities of the above media (1) to (10) were measured using a viscosity measuring device under the conditions of "30°C, shear rate 1 / s = 2". The results are shown in Table 12. [Table 12]

[0251] Furthermore, for the above media (1) to (9), the correlation between shear rate and viscosity was measured using a viscosity measuring device at 30° C. The results are shown in FIG. [Table 13]

[0252] Each of the media (1) to (10) above was applied to a retinal sheet and then sucked up and expelled using a plastic tip (a transplantation tip manufactured by Bethel; the retinal sheet shown in Figure 8 could pass through the tip and was between 1 cm and 10 cm long). The behavior of the retinal sheet was observed. First, we found that media (1), (5), (6), and (7) had such high viscoelasticity that the retinal sheet quickly tore apart when attempting to move through the media, making repeated sliding impossible. (2) Although the retinal sheet was able to slide in a 2x diluted Viscoat (2900 mPa·s), it was difficult to move in the liquid and was prone to tearing and breaking. The retinal sheet slid well in (3) a 4x diluted Viscoat, (8) a 1.5x diluted Opegan, (9) a 2x diluted Opegan, and (4) Hyalein Mini (37 mPa·s). On the other hand, when a balanced salt solution with lower viscosity (10) was used, it was found to be expelled forcefully, indicating that low viscosity makes transplantation difficult.

[0253] Similar results can also be obtained by using commercially available transplantation chips other than the plastic chips mentioned above.

[0254] In other words, it was found that a medium with adjusted viscosity is important for transplanting retinal sheets, and that good results are obtained when the viscosity at a shear rate of 2 (1 / s) is between 2 mPa·s and 1040 mPa·s.

[0255] Furthermore, as shown in Figure 2, when the "shear rate" on the X-axis was changed, the "viscosity" on the Y-axis also changed, and it was confirmed that the correlation between these factors differed for each administration medium. Therefore, when optimizing the transplant medium, we thought that we could characterize a better transplant medium by evaluating the relationship between "shear rate" and "viscosity."

[0256] The results in Figure 2 and Table 13 show that when the shear rate is low, for example, 10 (1 / s) or less, a small rate of change in viscosity is preferable. Furthermore, it was found that the change in viscosity at shear rates of 1 to 10 (1 / s) (i.e., the difference in viscosity between a shear rate of 1 (1 / s) and a shear rate of 10 (1 / s)) was 500 mPa s or less (314 mPa s in (8) of Table 12).

[0257] It was also found that the viscosity at a shear rate of 1000 (1 / s) was 100 mPa·s or less (68 mPa·s in (8) of Table 12).

[0258] Further investigation was carried out to find the optimum viscosity. The viscosities measured using the aqueous hyaluronic acid solutions shown in Table 14 below at 25°C and a shear rate of 1 / s = 2 are shown in Table 14. Furthermore, the correlation between shear rate and viscosity at 25°C was measured, and the results are shown in Figure 3 and Table 14, respectively. [Table 14]

[0259] Each of the above media was added to a retinal sheet and then sucked up and expelled with a plastic tip (transplant tip, manufactured by Bethel) to observe the behavior of the retinal sheet, i.e., whether it slid smoothly. The results showed that the retinal sheet slid smoothly with all media except for Opeguard and BSS+. In other words, it was found that a viscosity of 5 to 500 mPa·s at a shear rate of 2 (1 / s) at 25°C is appropriate for the transplant medium.

[0260] Based on the results in Figure 3, the correlation between shear rate and viscosity of the transplant medium at 25°C was examined.

[0261] As a result, the physical properties of the medium are as follows: When the shear rate (1 / s) is 2, the viscosity (mPa·s) ranges from 6.9 to 434.0. When the shear rate (1 / s) is 10, the viscosity (mPa·s) ranges from 6.9 to 410.0. When the shear rate (1 / s) is 100, the viscosity (mPa·s) ranges from 6.8 to 236.0. When the shear rate (1 / s) is 1000, the viscosity (mPa·s) ranges from 5.9 to 78.8. When the shear rate (1 / s) is 10,000, the viscosity (mPa·s) ranges from 4.3 to 20.6. It was found that good results were obtained. This result was consistent with the results in Figure 2. In other words, when the shear rate is small, for example, at or below 10 (1 / s), a small rate of change in viscosity is preferable. For example, it was found that the rate of change in viscosity at shear rates of 1 to 10 (1 / s) (i.e., the percentage when the difference in viscosity between a shear rate of 1 (1 / s) and a shear rate of 10 (1 / s) is divided by the viscosity at a shear rate of 1 (1 / s)) is approximately 10% or less, and the change in viscosity is 30 mPa·s or less (26 mPa·s at a BCT:OGD = 1:3 in Table 14).

[0262] It was also found that the viscosity at a shear rate of 1000 (1 / s) was less than 100 mPa·s.

[0263] Example 2: Study of the viscosity of the transplant composition in subretinal administration to rats The viscosity of the transplant composition was investigated during subretinal administration in rats. The transplantation method for rats was as described by Shirai et al. Proc Natl Acad Sci USA 2016, 113:E81-90.

[0264] Based on the results of Example 1, - Dilute Viscoat twice (Viscoat:Opeguard = 1:1) - Dilute Viscoat 4 times (Viscoat:Opeguard = 1:3) Transplantation was performed under two conditions. The results showed that stable subretinal transplantation was possible when Viscoat was diluted 4 times. In fact, transplantation was performed on 20 nude rats under these conditions, and transplantation was successful in 18 of them. On the other hand, when Viscoat was diluted 2 times, transplantation itself was possible, but it was found to be a little difficult due to its high viscosity.

[0265] Furthermore, pathological analysis of rat eyeballs transplanted with a 4-fold dilution of Viscoat after 6 months revealed no findings of particular safety concern and demonstrated that the retinal sheet took well. It was demonstrated that a 4-fold dilution of Viscoat does not inhibit the take of the retinal sheet.

[0266] In other words, it was demonstrated that there are no concerns about safety or retinal sheet survival even when the viscoelastic substance (hyaluronic acid, etc.) contained in 4-fold diluted Viscoat is administered subretinally at this concentration and volume. In other words, it can be considered that there are no concerns about safety or survival when the concentration and amount of Viscoat is 4-fold diluted or less.

[0267] From the above results, it was found that the 4-fold diluted Viscoat medium is safe and functional, and is also suitable for transplantation techniques.

[0268] Example 3: Study of the viscosity of the transplant composition in subretinal administration to monkeys The viscosity of the transplant composition for subretinal administration in cynomolgus monkeys was investigated. The transplantation was performed according to the method described in Example 1. Furthermore, an experienced surgeon, i.e., a clinical surgeon with over 10 years of experience in ophthalmic surgery, evaluated the ease of use of the transplantation procedure.

[0269] Based on the results of Example 1, the following administration vehicles were used. Viscoat 2x dilution (Viscoat:Opeguard = 1:1) Viscosity (30°C, shear rate 1 / s = 2): 2900 mPa·s Viscoat 4-fold dilution (Viscoat:Opeguard = 1:3) Viscosity (25°C, shear rate 1 / s = 2): 434 mPa·s Viscoat diluted 6 times (Viscoat:Opeguard = 1:5) Viscosity (25°C, shear rate 1 / s = 2): 56 mPa·s Viscoat diluted 7 times (Viscoat:Opeguard = 1:6) Viscosity (25°C, shear rate 1 / s = 2): 32.3 mPa·s Hyalein Mini Viscosity (30°C, shear rate 1 / s=2): 37 mPa·s

[0270] As in Example 2, the 2x diluted Viscoat solution was evaluated as having a high viscosity and being difficult to use in the transplantation method described in Example 1. That is, in the process of aspirating the retinal sheet and discharging it at the appropriate position, the slideability during transplantation was not good, and it was evaluated that a slightly better slideability would make transplantation easier. Furthermore, the 4x diluted Viscoat solution was evaluated as being better than the 2x diluted Viscoat solution, but similarly, its slideability needed improvement.

[0271] On the other hand, transplantation into monkeys was performed using Hyalein Mini, Viscoat 6-fold dilution, and Viscoat 7-fold dilution, and it was found that transplantation could be performed without any problems even with the evaluator's technique. In particular, Viscoat 6-fold dilution was transplanted into two monkeys and it was confirmed that it could be transplanted successfully.

[0272] Specifically, it was found that the retinal sheet could be slowly administered under the monkey's retina, which slowed its movement within the bleb and made transplantation easier.

[0273] Example 4: Study of the viscosity of the transplant composition in subretinal administration using isolated pig eyes The transplant composition was examined for subretinal administration using excised pig eyes. Surgery was performed as described in Example 1. Viscoat diluted 7 times (Viscoat:Opeguard = 1:6, viscosity (30°C, shear rate 1 / s = 2): 32.3 mPa s) was used as the transplant composition.

[0274] First, Example 1 showed that, for example, when using a dilution medium for Viscoat, the dilution ratio was approximately 4 to 14, but a dilution ratio of 5 to 11 was preferable. That is, in terms of viscosity (25°C, shear rate 1 / s = 2), a ratio of 7 mPa·s to 434 mPa·s, preferably 11 to 200 mPa·s, gave good results, and it was found that a 7-fold dilution of Viscoat could be used for subretinal transplantation into pig eyes without any problems.

[0275] After extensive investigations, we have found that Viscoat is a preferable viscoelastic material. We believe that this is related not only to the viscosity but also to the physical properties of Viscoat (stickiness, stringiness). As a physicochemical method for analyzing this physical property, we have considered that the correlation between shear rate and viscosity influences the behavior of the retinal sheet within the bleb. Specifically, as discussed in Example 2, when the shear rate (1 / S) at 25°C is 2, the appropriate viscosity is 5 to 500 mPa·s. Furthermore, the change in viscosity at shear rates of 1 to 10 is 500 or less, preferably 30 or less, and more preferably 2 or less. At a shear rate of 1,000, the viscosity is 100 or less, more preferably 30 or less.

[0276] In this invention, we investigated a completely new form of cell and tissue transplantation medium called a retinal sheet. There was no established technique for stably transplanting three-dimensional tissues, such as retinal sheets, into the human subretina. This invention clarified the physical properties of a transplantation medium suitable for retinal sheets.

[0277] <Reference Example 1: Production of retinal sheet containing neural retina> Human iPS cells (DSP-SQ line, established by Sumitomo Dainippon Pharma Co., Ltd.) were cultured in a feeder-free environment according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (manufactured by Nippi Co., Ltd.) was used as the feeder-free scaffold.

[0278] To induce differentiation, human iPS cells (DSP-SQ strain) were cultured in StemFit medium in a feeder-free manner until they reached subconfluency (approximately 30% of the culture area was covered with cells) for 2 days. These human iPS cells were then cultured in the presence of SAG (300 nM) for 2 days in a feeder-free manner (preconditioning treatment).

[0279] The preconditioned human iPS cells were treated with TrypLE Select (Life Technologies) to prepare a cell dispersion solution, and then dispersed into single cells by pipetting. The dispersed human iPS cells were then placed in a non-adhesive 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite Co., Ltd.) at 1.3 × 10 cells per well. 4 The cells were suspended in 100 μl of serum-free medium and cultured at 37°C and 5% CO2. The serum-free medium (gfCDM+KSR) was a 1:1 mixture of F-12 and IMDM supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× chemically defined lipid concentrate. At the initiation of culture (day 0), Y-27632 (final concentration 20 μM) and SAG (final concentration 10 nM) were added to the serum-free medium. On day 2, 50 μl of fresh serum-free medium was added to the medium containing human recombinant BMP4 (R&D) but without Y-27632 or SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0280] Four days later (six days after the start of suspension culture), the medium was replaced with the serum-free medium described above without Y-27632, SAG, or human recombinant BMP4. The medium replacement procedure involved discarding 60 μl of the medium in the culture vessel and adding 90 μl of fresh serum-free medium described above, bringing the total volume to 180 μl. Thereafter, half of the medium was replaced every 2–4 days with the serum-free medium described above without Y-27632, SAG, or human recombinant BMP4. The half-medium replacement procedure involved discarding half the volume of the medium in the culture vessel (i.e., 90 μl) and adding 90 μl of fresh serum-free medium described above, bringing the total volume to 180 μl.

[0281] The cell masses obtained in this manner on the 13th day after the start of suspension culture were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM) and SU5402 (5 μM) for 3 days, i.e., until the 16th day after the start of suspension culture.

[0282] The resulting cell aggregates on the 16th day after the start of suspension culture were cultured under 5% CO2 conditions using the serum medium shown in [1], [2], and [3] below until the 75th day after the start of suspension culture. [1] From day 16 to day 40 after the start of suspension culture: DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine (hereinafter referred to as medium A). [2] From day 40 to day 60 after the start of suspension culture: A medium was mixed with Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2mM glutamine, 60nM T3, and 100μM taurine (hereinafter referred to as B medium) in a 1:3 ratio. [3] After 60 days of suspension culture: Medium B.

[0283] The cell masses were observed under an inverted microscope on the 75th day after the start of suspension culture to confirm their morphology, and it was found that a neuroepithelial structure had formed.

[0284] Cell clusters 75 days after the start of suspension culture were fixed with 4% paraformaldehyde and cryosectioned. The cryosections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a neural retina marker, and Crx (anti-Crx antibody, Takara, rabbit), a photoreceptor progenitor cell marker (Figure 4). Another cryosection was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), a neural retina marker, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), a photoreceptor cell marker (Figure 5). Cell nuclei were stained with DAPI.

[0285] These stained sections were observed using a fluorescence microscope (Keyence) to obtain immunostained images. Photographs of the produced cells observed under a fluorescence microscope are shown in Figures 4 and 5. In Figures 4 and 5, the upper images were taken with a low-magnification lens, and the lower images were taken with a high-magnification lens.

[0286] The DAPI-stained images in Figures 4 and 5 reveal that densely packed neural tissue forms on the surface of the cell cluster, forming a continuous epithelial structure. Analysis of the image in Figure 4 reveals that a Crx-positive layer (photoreceptor layer) approximately 2–5 cells thick is formed on the surface of the cell cluster, and a Chx10-positive layer approximately 5–20 cells thick is formed inside the Crx-positive layer. Further inside this, a layer containing sparsely distributed Crx-positive cells is formed (Figure 4). The surface of this cell cluster is morphologically determined to be the apical surface. Furthermore, analysis of the image in Figure 5 reveals that a Recoverin-positive layer (photoreceptor layer) and an Rx-positive layer are also formed in the neural tissue. These results suggest that the neural tissue forms a photoreceptor layer containing Crx-positive and Recoverin-positive cells on the surface, a retinal progenitor cell layer containing Chx10-positive cells is formed inside the photoreceptor layer, and a cell layer is also formed inside the retinal progenitor cells. In other words, this method allows the production of neural retina containing a photoreceptor layer and a retinal progenitor cell layer from human iPS cells, and it has been found that this neural retina has a continuous epithelial structure.

[0287] <Reference Example 2: Preparation and evaluation of grafts> The three-dimensional retina created from human iPS cells consists of a neural retina with a neuroepithelial structure that has a continuous cell composition and distribution. This neural retina with a neuroepithelial structure has a layered structure consisting of a photoreceptor layer and an inner layer, and has a characteristic appearance and morphology (Figure 4).

[0288] Each human 3D retina is approximately 1–2 mm in size and has a unique shape. Due to the characteristics of the self-organizing culture production method, the neural retina used for transplantation is the primary product. However, non-neural retinal tissues (e.g., RPE, ciliary body) and cerebrospinal tissues (e.g., telencephalon, spinal cord) are also produced as by-products. Therefore, the central portion of the neural retina, excluding the non-neural retina, was excised to obtain a retinal fragment (graft, cap) (Figures 6 and 7). Specifically, the peripheral portion of the retinal fragment (cap) was used as a quality assessment sample (ring), and analysis (preferably quantitative PCR) was performed to determine whether only caps corresponding to rings that met the criteria were used as neural retina for transplantation.

[0289] 6 and 7 are conceptual diagrams of typical cell aggregates. The area where the neuroepithelial structure (preferably a continuous epithelial structure) specific to the neural retina, in which the photoreceptor layer and the inner layer appear to be separated into two layers, is observed is designated as the graft (cap), the area around the cap where the same neuroepithelial structure (preferably a continuous epithelial structure) as the cap is observed is designated as the quality evaluation sample (ring), and the area other than the cap and ring is called the root.

[0290] The procedure for isolating the cap and ring from neuroepithelial structures contained in a single cell aggregate is as follows.

[0291] <Reference Example 3: Shape of graft (cap)> Grafts (caps) were prepared using the following method (Figure 8). First, bright-field images (phase-contrast images) of cell aggregates prepared from human iPS cells (DSP-SQ strain) on day 99 of suspension culture, according to the method described in Reference Example 1, were taken using an inverted microscope (Olympus). After confirming the presence of neural retina on the cell aggregates, the cell aggregates were transferred under a stereomicroscope, and neural retina of various sizes were excised as grafts using the following method. We also investigated the effect of graft size on the transplantation procedure using a transplantation device.

[0292] (Excision of retinal sheets from neural retina) Bright-field (phase-contrast) images of the cell aggregates were observed under an inverted microscope (Nikon, ECLIPSE Ti). Particular attention was paid to the morphology of individual cells and the state of cell adhesion. Cell aggregates with a continuous epithelial structure and a two-layer structure consisting of an outer neuroblastic layer (including the photoreceptor layer and neural retinal progenitor cell layer) and an inner neuroblastic layer were determined to be neural retina. Tissues lacking a continuous epithelial structure, or areas with a continuous epithelial structure but in which the outer and inner neuroblastic layers were indistinguishable and appeared as a single layer, were classified as by-products. Subsequently, while observing the aggregates under a stereomicroscope, neural retina sections were excised from the cell aggregates using fine-tipped tweezers and scissors to prepare tissue sections.

[0293] (Impact on transplantation of excised retinal sheets) The excised grafts were photographed using a stereomicroscope: frontal images with the cut surface facing the objective lens, and lateral images with the cut surface tilted so that it was perpendicular to the objective lens. The major and minor axes, and height of the grafts were then measured from the photographed images. The major axis was defined as the longest line segment connecting two endpoints on the cross section of the retinal sheet in the frontal image, and its length. The minor axis was defined as the longest line segment connecting two endpoints on the cross section of the retinal sheet in the frontal image, and its length. The height was defined as the longest line segment connecting two endpoints on the cross section of the retinal sheet, and its length, and its length. Furthermore, the volume of the graft was calculated by approximating the graft as an ellipsoid divided in half so that the cross section passes through the major axis, according to the following formula: Volume = 2 / 3 x pi x (major axis / 2) x (minor axis / 2) x height

[0294] The results showed that the placement of the graft into the implantation device, the stability of the graft in the implantation device, and the expulsion of the graft from the implantation device were affected by the size of the graft. It was also suggested that the minor axis was a particularly useful parameter. The major axis, minor axis, height, and volume were calculated for 11 grafts that were successfully implanted using the implantation device. The average, maximum, and minimum values ​​for each parameter were calculated and are summarized in Table 15. From these results, it was found that the grafts (caps) should have at least a major axis of 0.8 to 1.7 mm, a minor axis of 0.4 to 1.1 mm, a height of 0.2 to 0.7 mm, and an apparent volume of 0.07 to 0.57 mm. 3 It was found to be to an extent. [Table 15]

[0295] <Reference Example 4: Cellular composition of the graft (cap)> Grafts (caps) were prepared as follows (numbers: 18001MF, d89, H5). First, grafts (caps) were isolated from cell aggregates prepared from human iPS cells (DSP-SQ strain) on day 89 of suspension culture according to the method described in Reference Example 1, using the methods described in Reference Examples 2 and 3.

[0296] The explants were fixed in 4% paraformaldehyde and cryosectioned. The cryosections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a neural retinal marker, and Crx (anti-Crx antibody, Takara, rabbit), a photoreceptor progenitor cell marker (Figure 9). Another cryosection was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), a neural retinal marker, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), a photoreceptor cell marker (Figure 9). Nuclei were stained with DAPI. These stained sections were observed using a confocal laser scanning microscope (Olympus) to obtain immunostained images.

[0297] The staining images revealed that densely packed neural tissue formed on the surface of the graft (cap) (left side of the image), and that this neural tissue formed a neuroepithelial structure (particularly a continuous epithelial structure) (Figure 9). Furthermore, a Crx-positive layer (photoreceptor layer, Figure 9) approximately 2–10 cells thick was formed on the surface of the cell cluster in this neural tissue. A Chx10-positive layer approximately 5–20 cells thick was formed inside the Crx-positive layer, and a layer containing Crx-positive cells was further formed inside that (Figure 9). The surface of this graft (cap) was morphologically determined to be the parietal surface. Furthermore, a Recoverin-positive layer (photoreceptor layer, Figure 9, arrow) and an Rx-positive layer were also formed in this neural tissue. These results indicate that this neural tissue formed a photoreceptor layer containing Crx-positive and Recoverin-positive cells on the surface, a retinal progenitor cell layer containing Chx10-positive cells was formed inside the photoreceptor layer, and a cell layer was also formed inside the retinal progenitor cells. In other words, the graft (cap) was able to produce a neural retina containing a photoreceptor layer and a retinal progenitor cell layer, and it was found that this neural retina has a continuous epithelial structure.

[0298] <Reference Example 5: Verification of equivalence between cap and ring> The gene expression of the cap and ring was compared using the following method. First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) on day 99 of suspension culture using the method described in Reference Example 1, designated as Lot 1. Furthermore, cell aggregates were prepared from human iPS cells (DSP-SQ strain) on day 82 of suspension culture using the method described in Reference Example 1, designated as Lot 2. For these two lots, the neural retina (the main product) and by-products were identified using a microscope as described in Reference Example 3, and the neural retinal cap and by-product cap were isolated, respectively. The rings were excised and isolated under a stereomicroscope using fine-tipped tweezers and scissors, similar to the explants. Total RNA was extracted from the caps and rings isolated from the neural retina and by-products using a spin column (RNeasy Micro kit, QIAGEN) using the method described in the kit's manual.

[0299] The total RNA concentration was measured using a Nanodrop meter (Thermo Scientific), and then reverse-transcribed to cDNA using reverse transcriptase and a primer (Reverse Transcription Master Mix Kit, Fluidigm). The cDNA was subjected to a multiplex PCR reaction (pre-run) using all probes used in the validation using a PCR device (Veriti 96-well thermal cycler, Applied Biosystems). The pre-run reaction mixture was then injected into a multi-well plate (96.96 Dynamic Array IFC, Fluidigm) with a flow channel using an IFC controller HX (Fluidigm). The expression levels of marker genes for neural retina and non-neural retinal by-products were measured by real-time PCR using a multi-analyte real-time PCR system (Biomark HD, Fluidigm). The PCR probes used in the validation are listed in Table 16.

[0300] [Table 16]

[0301] The results are shown in heat maps in Figures 10A and 10B. Gene expression levels were assessed using the delta Ct value, calculated from the difference between the Ct value of the gene of interest and that of GAPDH, the internal standard. A lower delta Ct value indicates higher gene expression, whereas a higher delta Ct value indicates lower gene expression. Gray indicates higher gene expression, while black indicates lower gene expression (lighter colors indicate higher gene expression). Gene expression in each cap and ring was examined. Both Lot 1 and Lot 2 showed expression of neural retinal marker genes in caps and rings isolated from the neural retina. Conversely, gene expression in caps and rings isolated from the by-products showed lower expression of neural retinal marker genes and higher expression of by-product marker genes, in contrast to the neural retina. Furthermore, comparison of gene expression in caps and rings isolated from the same cell aggregates revealed that the expression levels of neural retinal marker genes and by-product marker genes were comparable across all caps and rings isolated from the neural retina and by-products.

[0302] These results demonstrate that if the ring is neural retina, then the cap is also neural retina, and that gene expression is equivalent between the cap and ring.

[0303] <Reference Example 6: Results of graft transplantation> After analyzing gene expression in the rings using the method described in Reference Example 5, the corresponding caps (neural retinal sheets) were used as the retinal sheets described in Example 1. The retinal sheets were immersed in a 4-fold diluted Viscoat solution (Viscoat:Opeguard = 1:3) as a transplantation medium to prepare a transplantation composition. This was transplanted into nude rats with retinal degeneration, and the post-transplant engraftment survival was evaluated.

[0304] First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) according to the method described in Reference Example 1. Subsequently, caps and rings were isolated from cell aggregates formed 75 days or later after the start of suspension culture, according to the method described in Reference Example 3. The isolated caps were stored in a commercially available preservative solution until genetic analysis of the rings was performed. Gene expression analysis of the isolated rings was performed by real-time PCR using Biomark HD (Fluidigm) according to the method described in Reference Example 5. From the results of the gene expression analysis, rings expressing neural retinal marker genes but not by-product marker genes were selected, and the caps corresponding to these rings were selected as grafts (retinal sheets to be used for transplantation). The grafts were washed with buffer solution (Thermo Fisher Scientific) and then immersed in the medium described in Example 1 to prepare a transplantation composition. This was transplanted under the retina of a nude rat with retinal degeneration (photoreceptor degeneration model, SD-Foxn1 Tg(S334ter)3LavRrrc nude rat) using an injector described in a known publication (Shirai et al., PNAS 113, E81-E90).

[0305] Ocular tissues aged 230–240 days after the start of suspension culture were fixed with paraformaldehyde (PFA) and sucrose-substituted medium. The fixed ocular tissues were then cryosectioned using a cryostat. These cryosections were immunostained for human nuclei (anti-HuNu antibody, Millipore, mouse, or anti-HNA antibody), the photoreceptor marker Recoverin (anti-Recoverin antibody, Proteintech, rabbit), and the bipolar cell marker PKCα (anti-PKCα antibody, R&D Systems, goat).

[0306] Table 17 summarizes the results of graft quality assessment and transplantation results based on gene expression analysis of the ring. The ΔCt value was calculated using the method described in Reference Example 5. The ring gene expression analysis was considered to have passed the quality assessment test (ring-PCR test) if the ΔCt value of Recoverin, a neural retinal marker gene, was 10 or less, and the ΔCt values ​​of the by-product marker genes FOXG1, HOXB2, ZIC1, and OCT3 / 4 were each 5 or more. Regarding transplantation results, if human nucleus-positive and Recoverin-positive photoreceptors were detected under the retina, the engraftment was evaluated as good. Furthermore, if the transplant site did not significantly exceed the appropriate engraftment size and thicken, it was determined that no hypertrophy was detected.

[0307] Representative images of engraftment are shown in Figure 11. The 14 eyes that passed the pre-transplant quality assessment test were evaluated for post-transplant engraftment. Recoverin-positive photoreceptors were detected in all 14 eyes, demonstrating good engraftment. These cells were HuNu-positive, indicating that the Recoverin-positive photoreceptors originated from the transplanted cap. Furthermore, no hypertrophy was detected in any of the 14 eyes.

[0308] Based on the above results, gene expression analysis of the ring was performed to examine the expression levels of marker genes for the neural retina and its by-products before transplantation, and it was possible to select grafts that would successfully engraft under the retina, i.e., grafts in which photoreceptors would engraft and not undergo hypertrophy.

[0309] We also demonstrated the safety of subretinal administration of the transplantation medium "Viscoat 4-fold diluted (Viscoat:Opeguard = 1:3)." That is, Viscoat 4-fold diluted contains hyaluronic acid and chondroitin sulfate, and we were able to demonstrate the safety of subretinal administration of a medium containing 0.75 w / v% hyaluronic acid and 1.00 w / v% chondroitin sulfate.

[0310] [Table 17]

[0311] <Reference Example 7: Expression of markers in caps and rings> In Reference Example 5, it was demonstrated by PCR that the cellular composition of the cap and the ring was equivalent. Therefore, we next investigated by immunostaining whether the cellular composition and tissue structure of the cap and the ring were equivalent.

[0312] First, human iPS cells (DSP-SQ strain) were differentiated into retinas using the method described in Reference Example 1. Then, neural retinal caps and rings were isolated from cell aggregates 120 days after the start of suspension culture using the method described in Reference Example 5. The caps and rings were then washed, fixed with 4% paraformaldehyde (PFA), and sucrose-substituted. The fixed caps and rings were then frozen and sectioned using a cryostat. These frozen sections were immunostained for DAPI, which stains nuclei; Crx, a marker for photoreceptor progenitors (anti-Crx antibody, Takara, rabbit); Chx10, a marker for neural retina (anti-Chx10 antibody, Exalpha, sheep); NRL, a marker for rod photoreceptor progenitors (anti-NRL antibody, Bio-Techne, goat); FOXG1, a marker for telencephalon (anti-FOXG1 antibody, Takara Bio, rabbit); PAX2, a marker for optic stalk (anti-PAX2 antibody, Thermo Fisher Scientific, rabbit); and NANOG, a marker for undifferentiated pluripotent stem cells (anti-NANOG antibody, Merck, mouse).

[0313] The results of immunostaining are shown in Figure 12. Caps and rings excised from the same cell aggregates were shown in the upper panel and the lower panel, respectively. These results demonstrate that Crx-positive photoreceptor precursors, Chx10-positive neural retina, and NRL-positive rod photoreceptor precursors are expressed in continuous layers in both the cap and ring. Furthermore, no FOXG1-positive telencephalon, PAX2-positive optic stalk, or NANOG-positive pluripotent stem cells were detected in either the cap or ring. Furthermore, comparison of the stained images for Crx, Chx10, and NRL confirmed that these neural retinal markers were distributed in approximately the same manner in the cap and ring.

[0314] <Reference Example 8 Ratio of photoreceptor progenitor cells and neural retinal progenitor cells constituting neural retinal sheet for transplantation> Among the constituent cells of neural retinal sheets prepared from cell aggregates differentiated from pluripotent stem cells, the proportion of photoreceptor precursor cells and neural retinal precursor cells was analyzed and quantified using immunohistochemistry (IHC), a type of immunostaining method.

[0315] Human iPS cells (DSP-SQ strain) were differentiated into retinas using the method described in Reference Example 1. Subsequently, caps and rings were isolated from cell aggregates on days 84, 92, and 93 after the start of suspension culture using the method described in Reference Example 3. Gene expression analysis of the isolated rings was performed using the method described in Reference Example 6. Rings expressing neural retinal marker genes but not expressing by-product marker genes were selected using the method described in Reference Example 6, and the caps corresponding to these rings were used as neural retinal sheets for transplantation. In this manner, one neural retinal sheet for transplantation was prepared from a cell aggregate on day 84 after the start of suspension culture, two neural retinal sheets for transplantation from a cell aggregate on day 92 after the start of suspension culture, and one neural retinal sheet for transplantation from a cell aggregate on day 93 after the start of suspension culture. A total of four neural retinal sheets for transplantation were thus prepared.

[0316] The resulting neural retinal sheets for transplantation were cultured in medium B for 7 days for analysis. The cultured neural retinal sheets for transplantation were fixed with 4% paraformaldehyde and cryosectioned. The cryosections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), a neural retinal progenitor cell marker, and Crx (anti-Crx antibody, Takara, rabbit), a photoreceptor progenitor cell marker. Another cryosection was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), a neural retinal marker, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), a photoreceptor cell marker. Cell nuclei were stained with DAPI. These stained sections were observed under a fluorescence microscope (Keyence) to obtain immunostained images. An example (D3) is shown in Figure 13.

[0317] The immunostained images were analyzed using ImageJ (version 1.52a, NIH), and the number of DAPI-positive cells, the number of DAPI-positive and Chx10-positive cells, and the number of DAPI-positive and Crx-positive cells were analyzed for each of the four neural retinal sheets for transplantation. Similarly, the immunostained images were analyzed to determine the number of DAPI-positive cells and the number of DAPI-positive and Rx-positive cells. From these values, the percentages of Chx10-positive cells, Crx-positive cells, and Rx-positive cells were calculated. The results are shown in Table 18. [Table 18]

[0318] From these results, it was found that the percentage of Chx10-positive cells in the neural retina sheets for transplantation excised from the cell aggregates was approximately 23-45%, the percentage of Crx-positive cells was approximately 30-56%, and the percentage of Rx-positive cells was approximately 40-54%.

[0319] In other words, it was suggested that the neural retinal sheet for transplantation contained approximately 34% (23-45%) Chx10-positive neural retinal progenitor cells, approximately 40% (30-56%) Crx-positive photoreceptor progenitor cells, and approximately 47% (40-54%) Rx-positive cells.

[0320] <Reference Example 9: Proportion of photoreceptor progenitor cells and neural retinal progenitor cells constituting neural retinal sheet for transplantation> The composition of the constituent cells of neural retinal sheets for transplantation, which were prepared from cell aggregates differentiated from various pluripotent stem cells, was investigated using flow cytometry (also known as FACS), a type of immunostaining method.

[0321] Human iPS cells (QHJI01s04 strain) were differentiated into retinas using the method described in Reference Example 1. Subsequently, caps and rings were isolated from cell aggregates 88 days after the start of suspension culture using the method described in Reference Example 3. The caps were used as neural retinal sheets for transplantation. The neural retinal sheets for transplantation were cryopreserved at 17°C for 2 days. Five of the resulting neural retinal sheets for transplantation were combined into one sample, washed with PBS, and enzymatically treated with a neuronal cell dispersion solution (manufactured by WAKO, containing papain) at 37°C for approximately 30 minutes. The cells were dispersed into single cells by pipetting to obtain a single-cell suspension. The resulting single-cell suspension was fixed using a fixative (BD, CytoFix) to obtain a sample for FACS. The sample for FACS was blocked and permeabilized (perforated the cell membrane) using a serum-containing Perm / Wash solution (manufactured by BD). Immunostaining was performed using the following fluorescently labeled antibodies: anti-Chx10 antibody (Santa Cruz), anti-Pax6 antibody (BD), and anti-Crx antibody (Santa Cruz), followed by flow cytometry analysis using an analyzer (BD).

[0322] As a result, the Chx10-positive and Pax6-positive fraction (neural retinal progenitor cell fraction) was 11.5%, the Chx10-positive and Pax6-negative fraction (bipolar cell-biased progenitor cell fraction) was 23.4%, the Chx10-negative and Pax6-positive fraction (ganglion cell and amacrine cell fraction) was 10.7%, and the Crx-positive cell fraction (photoreceptor progenitor cell fraction) was 17.4%.

[0323] Furthermore, human iPS cells (DSP-SQ strain) were differentiated into retinas using the method described in Reference Example 1. Subsequently, 11 cell aggregates were prepared 88 days after the start of suspension culture, and 11 caps and 11 rings were isolated from each cell aggregate using the method described in Reference Example 3. The 11 caps were combined into one cap sample. Similarly, the 11 rings were combined into one ring sample. The cap sample and ring sample were each washed with PBS and then subjected to an enzyme treatment using a neuronal cell dispersion solution (manufactured by WAKO, containing papain) at 37°C for approximately 30 minutes to obtain single-cell suspensions of the caps and rings. The resulting single-cell suspensions of the caps and rings were fixed using a fixative (manufactured by BD, CytoFix) to obtain samples for FACS. The FACS samples were blocked and perforated using serum-containing Perm / Wash solution (BD) and then immunostained with the following fluorescently labeled antibodies: anti-Chx10 antibody (Santa Cruz), anti-Crx antibody (Santa Cruz), and anti-SSEA-4 antibody. An isotype control was used as a negative control for immunostaining. Analysis was performed by flow cytometry using an analyzer (BD). The percentages of Chx10-positive cells, Crx-positive cells, and SSEA-4-positive cells were calculated as the difference from the respective isotype controls. The results are shown in Table 19. [Table 19]

[0324] In the cap sample, 29.4% of cells were positive for the neural retinal progenitor cell marker Chx10, 15.8% for the photoreceptor progenitor cell marker Crx, and less than 1% for the pluripotent stem cell marker SSEA-4 (unintended cells).In the ring sample, 28.1% of cells were positive for the neural retinal progenitor cell marker Chx10, 21.7% for the photoreceptor progenitor cell marker Crx, and less than 1% for the pluripotent stem cell marker SSEA-4 (unintended cells).

[0325] These results demonstrated that the cap and ring samples were neural retina containing Chx10- and Crx-positive cells and were substantially free of undifferentiated iPS cells. Furthermore, the proportions of Chx10- and Crx-positive cells in the cap sample were comparable to those in the ring sample. Therefore, if the ring sample is neural retina, then the cap is also neural retina.

[0326] Furthermore, when these caps or rings (preferably caps) are used as neural retinal sheets for transplantation, it was found that the Chx10-positive fraction (neural retinal progenitor cell fraction) contained in this neural retinal sheet for transplantation is approximately 30% (approximately 20-40%), and the Crx-positive cell fraction (photoreceptor progenitor cell fraction) is approximately 17% (approximately 10-30%). [Industrial Applicability]

[0327] The vehicle of the present invention and a transplantation composition comprising retinal tissue and the vehicle are extremely useful for subretinal transplantation of retinal tissue for the treatment of retinal degenerative diseases such as retinitis pigmentosa (RP).

Claims

1. A transplantation medium for subretinal transplantation of retinal tissue, which has a viscosity of 5 to 500 mPa·s at a shear rate of 2 (1 / s) at 25°C and contains hyaluronic acid and a pharmaceutically acceptable aqueous liquid.

2. 2. The transplant medium according to claim 1, wherein the viscosity at a shear rate of 2 (1 / s) at 25°C is 10 to 100 mPa·s.

3. 3. The transplant medium according to claim 1, wherein the viscosity at a shear rate of 1000 (1 / s) at 25°C is 100 mPa·s or less, and the difference in viscosity between the viscosity at a shear rate of 1 (1 / s) and the viscosity at a shear rate of 10 (1 / s) is 100 mPa·s or less.

4. The implantation medium according to any one of claims 1 to 3, having a pH of 7.0 to 7.

5.

5. The transplantation vehicle according to any one of claims 1 to 4, wherein the pharmaceutically acceptable aqueous liquid is a balanced salt solution.

6. 6. The transplant medium according to claim 1, comprising 0.15 w / v % to 1.50 w / v % of hyaluronic acid having an average molecular weight of 500,000 to 3,900,000.

7. The implantation medium according to any one of claims 1 to 6, further comprising chondroitin sulfate.

8. 8. The transplant medium of claim 7, comprising 0.3 w / v % to 1.0 w / v % chondroitin sulfate.

9. The implantation medium according to any one of claims 1 to 8, which is free of antimicrobial agents and preservatives.

10. A transplantation composition comprising a retinal tissue for transplantation and the transplantation medium according to any one of claims 1 to 9.

11. The transplant composition according to claim 10 , wherein the retinal tissue for transplantation is a neural retinal sheet for transplantation comprising a neural retinal layer.

12. The transplant composition according to claim 11, wherein the neural retinal sheet for transplantation has a front surface and a back surface, the front surface constituting an apical surface including a neural retinal layer, and the back surface constituting a basal surface including a basement membrane component, the thickness of the neural retinal sheet for transplantation from the basal surface to the apical surface being 100 μm to 1000 μm, the major axis of the neural retinal sheet for transplantation being 600 μm to 2500 μm, and the minor axis of the neural retinal sheet for transplantation being 200 μm to 1500 μm.

13. The implant composition of claim 12, wherein the front surface has a smooth shape with few changes in curvature and the back surface has an irregular shape with many changes in curvature.

14. The transplantation composition according to any one of claims 11 to 13, comprising 1 to 30 neural retinal sheets for transplantation and 5 to 500 µl of the transplantation medium.

15. The neural retinal sheet for transplantation is (1) derived from pluripotent stem cells; (2) has a three-dimensional structure; (3) A neural retina layer having a multi-layer structure including a photoreceptor layer and an inner layer, (4) The photoreceptor layer includes one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, (5) The inner layer comprises one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells, and bipolar cells; (6) The surface of the neural retinal layer has an apical surface; (7) The inner layer is present inside the photoreceptor layer present along the apical surface, (8) The area of ​​the apical surface of the neural retinal layer is 50% or more of the total area of ​​the surface of the neural retinal sheet; (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neural retina layer; (10) Expression of neural retinal cell-related genes is observed in the neural retinal sheet for transplantation, and expression of non-neural retinal cell-related genes is not observed, and the non-neural retinal cell-related genes include one or more genes selected from the group consisting of cerebrospinal tissue marker genes and eyeball-related tissue marker genes. The transplant composition according to any one of claims 11 to 14, which is a neural retina sheet characterized by:

16. The neural retinal sheet for transplantation is (1) isolated from a cell aggregate containing a neural retinal layer; (2) comprising a region near the center of the continuous epithelial tissue in the cell aggregate; and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm. The transplant composition according to any one of claims 11 to 15.

17. The neural retinal sheet for transplantation is (1) Isolated from a cell aggregate containing at least a first epithelial tissue and a second epithelial tissue; the first epithelial tissue comprises a human neural retina, and the second epithelial tissue has a continuity of tangent slope on its surface that is different from the continuity of tangent slope on the surface of the first epithelial tissue, and comprises non-neural retinal cells; (2) The region on the first epithelial tissue that is furthest from the second epithelial tissue, and (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the thickness is 100 μm to 1000 μm; The transplant composition according to any one of claims 11 to 16, wherein the second epithelial tissue is a tissue selected from the group consisting of eye-related tissue, cerebrospinal tissue, and other tissues different from the neural retina of the first epithelial tissue.

18. The transplant composition according to any one of claims 11 to 17, wherein the ratio of Rx-positive cells to the total number of cells in the neural retina sheet for transplantation is 30% or more and 80% or less, 40% or more and 70% or less, 45% or more and 60% or less, or 50% or more and 60% or less.

19. The transplant composition according to any one of claims 11 to 18, wherein the ratio of Chx10-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% or more and 80% or less, 20% or more and 70% or less, 30% or more and 60% or less, or 40% or more and 50% or less.

20. The transplant composition according to any one of claims 11 to 19, wherein the ratio of Pax6-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% or more and 80% or less, 20% or more and 70% or less, 30% or more and 60% or less, or 40% or more and 50% or less.

21. The transplant composition according to any one of claims 11 to 20, wherein the ratio of Crx-positive cells to the total number of cells in the neural retina sheet for transplantation is 10% to 70% or 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%.

22. A transplant composition described in any one of claims 10 to 21, for subretinal transplantation into a subject requiring transplantation.

23. A transplant composition described in any one of claims 10 to 22 for the treatment of disorders of neural retinal cells or the neural retina, or diseases based on damage to the neural retina.

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