Method for evaluating the quality of neuroretina for transplantation and neuroretina sheet for transplantation

Abstract

To provide a method for evaluating the quality of neural retina for transplantation, and to provide a neural retina sheet for transplantation selected by the method.SOLUTION: The method for evaluating the quality of neural retina for transplantation of the present invention includes: extracting a part or all of a cell aggregate containing a neural retina having an epithelial structure derived from pluripotent stem cells as a quality evaluation sample; detecting the expression of neural retinal cell-related genes and non-neural retinal cell-related genes in the quality evaluation sample; when expression of neural retinal cell-related genes is observed and expression of non-neural retinal cell-related genes is not observed, determining that the following (1) to (3) can be used as neural retina for transplantation: (1) the neural retina (neural retina for transplantation) in the same cell aggregate as the cell aggregate containing the quality evaluation sample, which is a part of the cell aggregate; (2) the neural retina (neural retina for transplantation) in the cell aggregate of the same lot as the cell aggregate containing the quality evaluation sample, which is a part of the cell aggregate; or (3)the neural retina (neural retina for transplantation) in the cell aggregate of the same lot as the cell aggregate of the quality evaluation sample, which is the whole of the cell aggregate, the non-neural retinal cell-related genes including one or more genes selected from the group consisting of cerebrospinal tissue marker genes and ocular-related tissue marker genes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the quality of a neuroretina for transplantation and a neuroretina sheet for transplantation, and more particularly to a method for evaluating the quality of a neuroretina derived from pluripotent stem cells and a neuroretina sheet derived from pluripotent stem cells. [Background technology]

[0002] In living nerve tissue, one or more types of nerve cells form a layered structure. Retinal tissue, one type of nerve tissue, is mainly composed of five types of nerve cells—photoreceptor cells, bipolar cells, horizontal cells, amacrine cells, and ganglion cells—and glial cells, forming a three-dimensional layered structure. While transplantation therapy using nerve tissue has been suggested as an effective treatment for neurological diseases, such as retinal degenerative diseases, it has been difficult to obtain tissue that maintains the layered structure and function that reflects the nerve tissue of living humans, making it difficult to generalize as a treatment method. In recent years, it has become possible to manufacture nerve tissue (e.g., retinal tissue) by differentiating it from pluripotent stem cells (Non-patent documents 1, 2, 3, and 4).

[0003] Retinal tissue derived from pluripotent stem cells has a very complex structure because it contains various types of retinal layer-specific nerve cells and is composed of layers. When using such a complex retinal tissue as a cell therapy for transplantation, it is especially important to strictly control its quality. As a method for evaluating the quality of neuroretina, there are image analysis methods that analyze the presence or absence of continuous epithelial structures in cell aggregates (Patent Document 1). [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] WO2017 / 090741 [Non-patent literature]

[0005] [Non-Patent Document 1] Eiraku M. et al., “Self-organized Formationof Polarized Cortical Tissues From ESCs and Its Active Manipulation byExtrinsic Signals”, Cell Stem Cell, 3(5), 519-32(2008) [Non-Patent Document 2] Eiraku M. et al., “Self-organizing optic-cupmorphogenesis in three-dimensional culture”, Nature,472, 51-56(2011) [Non-Patent Document 3] Nakano T. et al., “Self-formation of OpticCups and Storable Stratified Neural Retina From Human ESCs” Cell Stem Cell, 10(6), 771-775(2012) [Non-Patent Document 4] Kawahara A. et al., “Generation of a ciliarymargin-like stem cell niche from self-organizing human retinal tissue” Nature Communications, 6, 6286(2015) [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, in view of the above circumstances, the present invention aims to provide a method for evaluating the quality of neuroretina for transplantation and a neuroretina sheet for transplantation selected by said method. [Means for solving the problem]

[0007] As a result of diligent research, the inventors have found that in the manufacturing process of cell aggregates containing neuroretina derived from pluripotent stem cells, some cell aggregates may produce not only retina-specific neurons (target cells, neuroretina-related cells) but also cells other than retina-specific neurons (unintended cells, non-neural retina-related cells). Comprehensive analysis of gene expression and other factors in multiple samples revealed that the unintended cells that may be produced are brain and spinal cord tissue and ophthalmoplastic tissue. Further detailed analysis of brain and spinal cord tissue and ophthalmoplastic tissue revealed that the telencephalon (cerebrum), diencephalon (including hypothalamus), midbrain, and spinal cord may be produced as byproducts from the brain and spinal cord tissue, and that the retinal pigment epithelium (RPE), ciliary body, lens, and optic stalk (pedicle and optic nerve tissue) may be produced as byproducts from the ophthalmoplastic tissue.

[0008] Based on these novel findings, the inventors discovered that it is possible to evaluate whether a neuroretina is suitable for transplantation by analyzing the expression of genes related to target cells and genes related to non-target cells, and that neuroretinas for transplantation can be selected accordingly, thus completing the present invention.

[0009] In other words, the present invention relates to the following: [1] A method for evaluating the quality of neuroretina for transplantation, The above method, Extracting a portion or all of a cell aggregate containing a neuroretina with an epithelial structure derived from pluripotent stem cells as a quality evaluation sample, The expression of neuroretinal cell-related genes and non-neuroretinal cell-related genes in the above quality evaluation sample is detected, If the expression of the above neuroretinal cell-related genes is observed, and the expression of the above non-neuroretinal cell-related genes is not observed, (1) The above neuroretina (neuroretinum for transplantation) in the same cell aggregate as the cell aggregate containing the quality evaluation sample which is part of the above, (2) the above-mentioned neural retina (neural retina for transplantation) in cell aggregates of the same lot as the cell aggregates containing the quality evaluation sample which is a part of the above, or (3) the above-mentioned neural retina (neural retina for transplantation) in cell aggregates of the same lot as the cell aggregates of the quality evaluation sample which is all of the above including determining that it can be used as a neural retina for transplantation, wherein the non-neural retina-related gene contains one or more genes selected from the group consisting of a cerebrospinal tissue marker gene and an eye-related tissue marker gene, the method. [2] wherein the above-mentioned cerebrospinal tissue marker gene is one or more genes selected from the group consisting of a telencephalon marker gene, a diencephalon / midbrain marker gene, and a spinal cord marker gene, wherein the above-mentioned eye-related tissue marker gene is 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, the method according to [1] above. [3] wherein the above-mentioned telencephalon marker gene contains one or more genes selected from the group consisting of FoxG1, Emx2, Dlx2, Dlx1, and Dlx5, wherein the above-mentioned diencephalon / midbrain marker gene contains one or more genes selected from the group consisting of OTX1, OTX2, DMBX1, Rx, Nkx2.1, OTP, FGFR2, EFNA5, and GAD1, wherein the above-mentioned spinal cord marker gene contains one or more genes selected from the group consisting of HOXD4, HOXD3, HOXD1, HOXC5, HOXA5, and HOXB2, wherein the above-mentioned Optic Stalk marker gene contains one or more genes selected from the group consisting of GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A, wherein the above-mentioned ciliary body marker gene contains one or more genes selected from the group consisting of Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4, The above lens marker gene contains one or more genes selected from the group consisting of CRYAA and CRYBA1. The method according to [2] above, wherein the retinal pigment epithelium marker gene contains one or more genes selected from the group consisting of MITF, TTR, and BEST1. [4] The method according to any one of [1] to [3] above, wherein the non-neural retinal cell-related gene further contains an undifferentiated pluripotent stem cell marker gene. [5] The method according to [4] above, wherein the undifferentiated pluripotent stem cell marker gene contains one or more genes selected from the group consisting of Oct3 / 4, Nanog, and lin28. [6] The method according to any one of [1] to [5] above, wherein the cell aggregate of the same lot as the cell aggregate of the sample for quality evaluation is a cell aggregate produced under the condition of showing a gene expression profile equivalent to that of the above transplanted neural retina. [6-1] The method according to any one of [1] to [6] above, wherein the transplanted neural retina contains near the center of the epithelial tissue. [6-2] The method according to any one of [1] to [6], [6-1] above, wherein the transplanted neural retina is a continuous epithelial tissue. [7] The above sample for quality evaluation is a part of the cell aggregate. When the expression of the above neural retina cell-related gene is observed and the expression of the above non-neural retina cell-related gene is not observed, the neural retina that was continuous or adjacent to at least a part of the above part in the same cell aggregate as the cell aggregate containing the above part of the quality evaluation sample is determined to be usable as the transplanted neural retina. The method according to any one of [1] to [6] above. [(8)] The method according to [7] above, wherein the transplanted neural retina is contained in the same epithelial tissue as the above sample for quality evaluation. [9] The method according to [8] above, wherein the transplanted neural retina contains near the center of the same epithelial tissue.

[10] The method according to [9] above, wherein the neuroretina for transplantation is continuous epithelial tissue.

[11] The cell aggregate containing the neuroretina comprises a first epithelial tissue containing the neuroretina for transplantation, and a second epithelial tissue having a continuity of tangent slopes on its surface that is different from the continuity of tangent slopes on the surface of the first epithelial tissue, and containing non-neuroretinal cells. The above-mentioned neuroretina for transplantation includes a region on the first epithelial tissue furthest from the second epithelial tissue, The method according to any one of [7] to

[10] above, wherein the above quality evaluation sample is a portion present between the second epithelial tissue and the neuroretina for transplantation.

[12] The method according to

[11] above, wherein the second epithelial tissue is ocular tissue and / or cerebrospinal tissue.

[13] The method according to

[12] above, wherein the eye-related tissues include retinal pigment epithelial cells and ciliary bodies.

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

[13] , wherein the expression of the above neuroretinal cell-related genes and the above non-neuroretinal cell-related genes is performed by quantitative PCR.

[15] The method described in

[14] above, which includes determining that the neuroretina is suitable for transplantation if it meets the following criteria 1 and 2. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of the above neuroretinal cell-related gene and the Ct value of the internal standard gene is 10 or less. Criterion 2: The difference (ΔCt value) between the Ct value of the above non-neuronal retinal cell-related gene and the Ct value of the internal standard gene is 5 or greater.

[16] The method according to

[14] or

[15] above, wherein the quantitative PCR described above is performed by a method comprising the steps (1) to (5) below, thereby simultaneously detecting the expression levels of each neuroretinal cell-related gene and non-neuroretinal cell-related gene in two or more of the above quality evaluation samples. (1) Prepare a channel plate having channels connecting the independent sample wells in the sample well group and the independent primer wells in each of the primer well groups, a solution containing nucleic acids obtained from two or more of the above quality evaluation samples (sample solution), and a solution containing one or more primers specific to one or more of the above neuroretinal cell-related genes or the above non-neuroretinal cell-related genes (primer solution). (2) In the sample well group, add the above sample solution to each quality evaluation sample so that there is 1 sample solution per sample well. (3) Add the primer solution to one or more primer wells in the above group of one or more primer wells so that they become different groups of primer wells. (4) Mixing the nucleic acid and the primer separately via the above-mentioned channel, (5) Perform quantitative PCR using the mixture obtained in (4).

[17] It is a neuroretinal sheet, (1) Derived from pluripotent stem cells, (2) Having a three-dimensional structure, (3) A neuroretinal layer having a multilayer structure including a photoreceptor cell layer and an inner layer, (4) The above photoreceptor layer includes one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, (5) The inner layer contains 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 neuroretinal layer has an apical surface, (7) The inner layer is located inside the photoreceptor layer that is located along the apical surface, (8) The area of ​​the neuroretinal layer is 50% or more of the total surface area of ​​the neuroretinal sheet, (9) The area of ​​the continuous epithelial structure is 80% or more of the total area of ​​the apical surface of the neuroretinal layer, (10) The neuroretinal sheet is characterized in that it shows expression of neuroretinal cell-related genes and does not show expression of non-neuroretinal cell-related genes, and the non-neuroretinal cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eyeball-related tissue marker genes. Neuroretinal sheet.

[18] A neuroretinal sheet as described in

[17] above, having a major axis of 600 μm to 2500 μm.

[19] A neuroretinal sheet as described in

[17] or

[18] above, having a short axis of 200 μm to 1500 μm.

[20] A neuroretinal sheet as described in any of the above

[17] to

[19] , having a height of 100 μm to 1000 μm. [twenty one] The above neuroretinal sheet, (1) It is isolated from cell aggregates including the neuroretina, (2) The cell aggregate includes a region near the center of the continuous epithelial tissue, (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the height is 100 μm to 1000 μm. A neuroretinal sheet as described in any of the above

[17] to

[20] . [twenty two] The above neuroretinal sheet, (1) Isolated from a cell aggregate containing at least a first epithelial tissue and a second epithelial tissue, The above cell aggregate comprises a first epithelial tissue containing human neural retina, and a second epithelial tissue having a continuity of tangent slopes on a surface different from that of the first epithelial tissue, and containing non-neuroretinal cells. (2) Including the region on the first epithelial tissue furthest from the second epithelial tissue, (3) The major axis is 600 μm to 2500 μm, the minor axis is 200 μm to 1500 μm, and the height is 100 μm to 1000 μm. The neuroretinal sheet according to any of

[17] to

[21] above, wherein the second epithelial tissue is selected from the group consisting of ocular-related tissues, cerebrospinal tissues and other tissues different from the neuroretinal tissue of the first epithelial tissue. [twenty three] A neuroretinal sheet according to any of the above

[17] to

[22] , wherein the ratio of Rx-positive cells to the total number of cells in the neuroretinal sheet is 30% to 80%, 40% to 70%, 45% to 60%, or 50% to 60%. [twenty four] A neuroretinal sheet according to any of the above

[17] to

[23] , wherein the proportion of Chx10-positive cells to the total number of cells in the neuroretinal sheet is 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%. [twenty five] A neuroretinal sheet according to any of the above

[17] to

[24] , wherein the proportion of Pax6-positive cells to the total number of cells in the neuroretinal sheet is 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%.

[26] A neuroretinal sheet according to any of the above

[17] to

[25] , wherein the ratio of Crx-positive cells to the total number of cells in the neuroretinal sheet is 10% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%.

[27] A pharmaceutical composition comprising a neuroretinal sheet as described in any of the above

[17] to

[26] .

[28] A method for treating a disease based on neuroretinal cell or neuroretinal disorders or neuroretinal damage, comprising transplanting a neuroretinal sheet described in any of the above

[17] to

[26] to a subject requiring transplantation.

[29] Using any of the methods described in [1] to

[16] above, evaluate cell aggregates containing neuroretina having epithelial structures derived from pluripotent stem cells, and select neuroretina for transplantation that are determined to be suitable for use as neuroretina for transplantation, and Isolating the selected neuroretina for transplantation as described above. A method for manufacturing a neuroretinal sheet according to any of the above

[17] to

[26] , including the above.

[30] To extract quality evaluation samples from cell aggregates containing 2 to 800 cells, each containing a neuroretinal cell with epithelial structures derived from pluripotent stem cells, which are a part of the said cell aggregate. The above-mentioned extracted 2 to 800 quality evaluation samples are evaluated by any of the methods described in [1] to

[16] above, and the neuroretina determined to be suitable for use as a neuroretina for transplantation is selected, and Isolating the selected neuroretina for transplantation as described above. A method for manufacturing a neuroretinal sheet containing [the specified material].

[31] The cell aggregate is a cell aggregate obtained by differentiating pluripotent stem cells, comprising at least a first epithelial tissue and a second epithelial tissue, wherein the first epithelial tissue comprises human neural retina, and the second epithelial tissue has a continuity of tangent slopes on its surface that is different from the continuity of tangent slopes on the surface of the first epithelial tissue, and comprises non-neuroretinal cells. The isolation of the neuroretina for transplantation described above involves isolating the neuroretina for transplantation from the cell aggregate such that it includes the region on the first epithelial tissue that is furthest from the second epithelial tissue. The manufacturing method described in

[29] or

[30] above. [Effects of the Invention]

[0010] According to the present invention, it is possible to provide a method for evaluating the quality of a neuroretina for transplantation, a neuroretina sheet for transplantation selected by said method, and a method for manufacturing said neuroretina sheet for transplantation. [Brief explanation of the drawing]

[0011] [Figure 1] This is a fluorescence microscope image showing the results of immunostaining with Crx and Chx10 on cell aggregates containing the neuroretina for transplantation in Example 1. [Figure 2]This is a fluorescence microscope image showing the results of immunostaining with Rx and Recoverin on cell aggregates containing the neuroretina for transplantation in Example 1. [Figure 3] The results of microarray analysis of RNA extracted from the neuroretina and by-products A, B, C, D, E, and F in Example 2 are shown. [Figure 4] This is a conceptual diagram showing how to fabricate caps and rings from typical cell aggregates. [Figure 5] This is a conceptual diagram illustrating the fabrication of caps and rings from cell aggregates of various shapes. The areas shown in black and gray represent unintended tissues. [Figure 6] The height, major axis, and minor axis of the graft in Example 4 are shown, along with images of a typical graft and a schematic diagram of the graft. [Figure 7] This is a confocal fluorescence microscope image showing the results of immunostaining with Crx and Chx10 on the graft in Example 5. [Figure 8] The results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Example 6 are shown. [Figure 9] The results of quantitative PCR analysis of gene expression in RNA extracted from the cap and ring in Example 7 are shown. [Figure 10] This image shows the results of observing the engraftment after transplantation using a fluorescence microscope, following quantitative PCR analysis of RNA extracted from the ring in Example 8, and subsequent transplantation of the graft (cap) under the rat retina. [Figure 11] This image shows the results of observing the engraftment after transplantation using a fluorescence microscope, following quantitative PCR analysis of RNA extracted from the ring in Example 9, and subsequent transplantation of the graft (cap) under the rat retina. [Figure 12] These images show the ring observed with an inverted microscope in Example 10, and the results of observing the engraftment after transplantation of the ring under the rat retina using a fluorescence microscope. [Figure 13]These are fluorescence microscope images showing the results of immunostaining performed on caps and rings prepared from a single cell aggregate in Example 11. [Figure 14] The results of quantitative PCR analysis of gene expression in RNA extracted from caps and rings prepared from neuroretina and non-neuronal retina (telncephalon tissue, spinal cord tissue, RPE, optic stalk) in Example 12 are shown. [Figure 15] The immunohistochemical images of the stained sections from Example 14, observed using a fluorescence microscope (Keyence), are shown. [Modes for carrying out the invention]

[0012] [Definition] "Stem cells" refer to undifferentiated cells that possess the ability to differentiate and proliferate (especially self-renewal). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation ability. Pluripotent stem cells are those that can be cultured in vitro and possess the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues (pluripotency). Multipotent stem cells are those that have the ability to differentiate into multiple types of tissues and cells, though not all of them. Unipotent stem cells are those that have the ability to differentiate into specific tissues or cells.

[0013] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ cells, tissue-derived 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 created from germ cells (e.g., testes) are also included in the category of pluripotent stem cells.

[0014] Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a culture medium containing bFGF. Methods for producing embryonic stem cells are described, for example, in WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from designated institutions, and can also be purchased commercially. 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 strain Crx::Venus (derived from KhES-1) is available from the RIKEN (National Research and Development Institute).

[0015] "Induced pluripotent stem cells" are somatic cells that have been reprogrammed using known methods to induce pluripotency.

[0016] Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126(4), pp. 663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007 and possess pluripotency and self-renewal ability similar to 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).

[0017] Induced pluripotent stem cells are specifically cells in which pluripotency is induced by reprogramming differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells by expressing one of several combinations of genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, and Esrrb. Preferred reprogramming factor combinations 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).

[0018] In addition to the method of producing induced pluripotent stem cells through direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds, etc. (Science, 2013, 341, pp. 651-654).

[0019] Furthermore, it is possible to obtain 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, are available from Kyoto University and iPS Academia Japan, Inc. As induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells, established at Kyoto University, are available from Kyoto University.

[0020] In this specification, pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0021] 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).

[0022] 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.

[0023] "Retinal tissue" refers to the tissue in a living retina in which one or more types of retinal cells that make up each retinal layer exist in a certain order, while "neural retina" refers to retinal tissue that includes the inner neural retina layer, which does not include the retinal pigment epithelium layer, as described later.

[0024] "Retinal cells" refers to the cells that make up each retinal layer in a living retina, or their progenitor cells. Retinal cells include, but are not limited to, photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelium (RPE) cells, ciliary body, their progenitor cells (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, etc.), and retinal progenitor cells. Among the retinal cells, the cells that constitute the neuroretinal layer (also called neuroretinal cells or neuroretina-related cells) specifically include photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, and their precursor cells (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, etc.). In other words, neuroretina-related cells do not include retinal pigment epithelial cells or ciliary body cells.

[0025] "Mature retinal cells" refers to cells that may be found in the retinal tissue of an adult human, specifically differentiated cells such as photoreceptor cells (rod and cone cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod and cone cells), Müller glial cells, retinal pigment epithelial (RPE) cells, and ciliary cells. "Immature retinal cells" refers to progenitor cells whose differentiation into mature retinal cells is predetermined (e.g., photoreceptor progenitor cells, bipolar cell progenitor cells, retinal progenitor cells, etc.).

[0026] Photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, Müller glial cell progenitor cells, and retinal pigment epithelial cell progenitor cells are progenitor cells whose differentiation into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cells, respectively, has been determined.

[0027] "Retinal progenitor cells" refer to progenitor cells that can differentiate into any immature retinal cell type, such as photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, Müller glial cells, and retinal pigment epithelial progenitor cells, and ultimately differentiate into any mature retinal cell type, such as photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.

[0028] Photoreceptor cells are cells found in the photoreceptor layer of the retina in living organisms, and their role is to absorb light stimuli and convert them into electrical signals. There are two types of photoreceptor cells: cones, which function in bright light, and rods, which function in dim light (these are called cone photoreceptor cells and rod photoreceptor cells, respectively). Cone photoreceptor cells include S-cone photoreceptor cells that express S-opsin and receive blue light, L-cone photoreceptor cells that express L-opsin and receive red light, and M-cone photoreceptor cells that express M-opsin and receive green light. Photoreceptor cells differentiate and mature from photoreceptor progenitor cells. Whether a cell is a photoreceptor cell or a photoreceptor progenitor cell can be easily confirmed by a person skilled in the art, for example, by the expression of cell markers described later (Crx and Blimp1 expressed in photoreceptor progenitor cells, Recoverin expressed in photoreceptor cells, rhodopsin, S-Opsin, and M / L-Opsin expressed in mature photoreceptor cells, etc.), the formation of outer segment structures, etc. In one embodiment, photoreceptor progenitor cells are Crx-positive cells, and photoreceptor cells are rhodopsin, S-Opsin, and M / L-Opsin-positive cells. In one embodiment, rod photoreceptor cells are NRL and Rhodopsin-positive cells. In one embodiment, S cone photoreceptor cells are S-opsin-positive cells, L cone photoreceptor cells are L-opsin-positive cells, and M cone photoreceptor cells are M-opsin-positive cells.

[0029] The presence of neuroretinal cells can be confirmed by the presence or absence of expression of neuroretinal cell-related genes (hereinafter sometimes referred to as "neuroretinal cell markers" or "neuroretinal markers"). The presence or absence of expression of neuroretinal cell markers, or the proportion of neuroretinal cell marker-positive cells in a cell population or tissue, can be easily confirmed by those skilled in the art. Examples include antibody-based methods, nucleic acid primer-based methods, and sequencing reaction methods. As an antibody-based method, the expression of the neuroretinal cell marker protein can be confirmed, for example, by flow cytometry or immunostaining using commercially available antibodies, by dividing the number of cells positive for a specific neuroretinal cell marker by the total number of cells. As an antibody-based method, the expression of the neuroretinal cell marker RNA can be confirmed, for example, by PCR, semi-quantitative PCR, or quantitative PCR (e.g., real-time PCR). As an sequencing reaction method, the expression of the neuroretinal cell marker RNA can be confirmed, for example, by a nucleic acid sequencer (e.g., next-generation sequencer).

[0030] Examples of neuroretinal cell markers include Rx (also known as Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6, and Chx10 (also known as Vsx2) expressed in neuroretinal progenitor cells, and Crx and Blimp1 expressed in photoreceptor progenitor cells. Other examples include Chx10, which is strongly expressed in bipolar cells; 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; Recoverin, which is expressed in photoreceptor cells and photoreceptor progenitor cells; Rhodopsin, which is expressed in rod cells; Nrl, which is expressed in rod photoreceptor cells and rod photoreceptor progenitor cells; S-opsin and LM-opsin, which are expressed in cone photoreceptor cells; RXR-γ, which is expressed in cone cells, cone photoreceptor progenitor cells, and ganglion cells; TRβ2, OTX2, and OC2, which are expressed in cone photoreceptor cells or their progenitor cells that appear in the early stages of differentiation among cone photoreceptor cells; and Pax6, which is commonly expressed in horizontal cells, amacrine cells, and ganglion cells.

[0031] A "positive cell" refers to a cell that expresses a specific marker on its surface or inside the cell. For example, a "Chx10 positive cell" refers to a cell that expresses the Chx10 protein.

[0032] "Retinal pigment epithelial cells" refer to epithelial cells located outside the neural retina in a living retina. Whether or not cells are retinal pigment epithelial cells can be easily confirmed by those skilled in the art, for example, by the expression of cell markers (RPE65, MITF, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (dark brown), tight junctions between cells, and characteristic polygonal or cobblestone-like cell morphology. Whether or not cells have the function of retinal pigment epithelial cells can be easily confirmed by the secretion capacity of cytokines such as VEGF and PEDF. In one embodiment, retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.

[0033] The term "retinal layer" refers to the various layers that make up the retina, and specifically includes the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane.

[0034] The term "neuroretinal layer" refers to the various layers that make up the neuroretina, specifically the photoreceptor cell layer, outer limiting membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. The "photoreceptor cell layer" refers to the outermost layer of the neuroretina, which contains many photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), photoreceptor progenitor cells, and retinal progenitor cells. The layers other than the photoreceptor cell layer are called inner layers. Which retinal layer each cell belongs to can be confirmed by known methods, such as the presence or absence or degree of expression of cell markers.

[0035] In retinal tissue at a stage where the proportion of photoreceptor cells or photoreceptor progenitor cells is low, the layer containing proliferating neuroretinal progenitor cells is called the "neuroblastic layer," and both an inner neuroblastic layer and an outer neuroblastic layer exist. As is well known to those skilled in the art, this can be determined by the intensity of the color (the outer neuroblastic layer is lighter and the inner neuroblastic layer is darker) under a bright-field microscope.

[0036] The term "ciliary body" includes the developmental and adult "ciliary body," "ciliary margin," and "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, the tissue located at the boundary between the neuroretina and the retinal pigment epithelium in the living retina, and is a region that contains 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 the supply of retinal progenitor cells and differentiated cells to retinal tissue, and in maintaining the structure of retinal tissue. Examples of marker genes for the ciliary body periphery include the Rdh10 gene (positive), the Otx1 gene (positive), and the Zic1 gene (positive). A "ciliary body periphery-like structure" refers to a structure that resembles the ciliary body periphery.

[0037] A "cell aggregate" is defined as any structure formed by the adhesion of multiple cells, without any particular limitations. For example, it refers to a mass formed by the aggregation of cells dispersed in a medium such as a culture medium, or a mass of cells formed through cell division. Cell aggregates also include those that form specific tissues.

[0038] A "sphere-like cell aggregate" refers to a cell aggregate that has a nearly spherical, three-dimensional shape. A nearly spherical, three-dimensional shape is a shape with a three-dimensional structure, and when projected onto a two-dimensional plane, it may appear as a spherical shape that is circular or elliptical, or a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by the overlapping of 2 to 4 circular or elliptical shapes when projected onto a two-dimensional plane). In one embodiment, the core of the aggregate has a vesicular layered structure, and under a bright-field microscope, it is observed that the central part is dark and the outer edge is bright.

[0039] In one embodiment, epithelial tissue undergoes polarization to form an "apical surface" and a "basement membrane." The "basement membrane" refers to the basal layer (basement membrane) produced by epithelial cells, which is 50-100 nm in length and rich in laminin and type IV collagen. The "apical surface" refers to the surface (surface surface) formed on the opposite side of the "basement membrane." In one embodiment, in retinal tissue where the developmental stage has progressed to the point where photoreceptor cells or photoreceptor progenitor cells can be observed, the "apical surface" refers to the surface in contact with the photoreceptor layer (outer granular layer) where photoreceptor cells and photoreceptor progenitor cells are present, after the formation of the outer limiting membrane. Furthermore, such an apical surface can be identified by immunohistochemical staining 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, N-cadherin).

[0040] "Epithelial tissue" is tissue formed when cells completely cover the surface of the body, tubular lumens (such as the digestive tract), and body cavities (such as the pericardial cavity). The cells that make up epithelial tissue are called epithelial cells. Epithelial cells have polarity in the apical-basal direction. Epithelial cells can form strong bonds with each other through adherent junctions and / or tight junctions, creating layers of cells. Epithelial tissue is tissue formed by one to more than ten layers of these cell layers stacked on top of each other. Tissues that can form epithelial tissue include fetal and / or adult retinal tissue, brain and spinal cord tissue, ocular tissue, and nerve tissue. Neuroretina as used herein is also epithelial tissue. "Epithelial structure" refers to structures characteristic of epithelial tissue, such as the apical surface or basement membrane.

[0041] "Continuous epithelial tissue" refers to tissue that has a continuous epithelial structure. A continuous epithelial structure means that the epithelial tissue is continuous. For example, continuous epithelial tissue means that 10 cells to 10 cells are connected tangentially to the epithelial tissue. 7 Cells, preferably 30 to 10 in the tangential direction 7 cells, more preferably 10 2 cells ~10 7 It refers to the state in which cells are arranged in a line.

[0042] For example, the continuous epithelial structure formed in retinal tissue is formed on the surface of the retinal tissue in a manner that is generally parallel and continuous with the photoreceptor layer (outer granular layer), among the layers that make up the neuroretinal layer, where the apical surface is located, and at least the photoreceptor layer (outer granular layer), among the layers that make up the neuroretinal layer. For example, in the case of a cell aggregate containing retinal tissue made from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and 10 or more cells, preferably 30 or more cells, more preferably 100 or more cells, and even more preferably 400 or more photoreceptor cells or photoreceptor progenitor cells are regularly and continuously arranged tangentially to the surface.

[0043] [Method for evaluating the quality of neuroretina for transplantation] One aspect of the present invention is a method for evaluating the quality of a neuroretina for transplantation.

[0044] The method according to the present invention includes: extracting a part or all of a cell aggregate containing a neuroretina having an epithelial structure derived from pluripotent stem cells as a quality evaluation sample; detecting the expression of neuroretinal cell (target cell) related genes and non-neuroretinal cell (non-target cell) related genes in the quality evaluation sample; and determining, if the expression of neuroretinal cell related genes (target cell related genes) is observed and the expression of non-neuroretinal cell related genes (non-target cell related genes) is not observed, that (1) a neuroretina (neuroretina for transplantation) from the same cell aggregate as the cell aggregate containing the part of the quality evaluation sample; (2) a neuroretina (neuroretina for transplantation) from the same lot as the cell aggregate containing the part of the quality evaluation sample; or (3) a neuroretina (neuroretina for transplantation) from the same lot as the cell aggregate of the same lot as the cell aggregate of the entire quality evaluation sample. If the quality evaluation sample shows expression of neuroretinal cell-related genes (target cell-related genes) and does not show expression of non-neurotinic cell-related genes (non-target cell-related genes), the quality evaluation sample is deemed usable as a neuroretina for transplantation. However, since the quality evaluation sample, which is the neuroretina for transplantation, is destroyed for evaluation purposes, it cannot actually be used for transplantation.

[0045] <Cell aggregates including the neuroretina> (Method for producing cell aggregates) The cell aggregates containing neuroretina described herein have an epithelial structure and can be obtained by differentiating pluripotent stem cells. One embodiment describes a method for producing cell aggregates containing neuroretina using differentiation-inducing factors. Examples of differentiation-inducing factors include basement membrane preparations, BMP signaling pathway activators, Wnt signaling pathway inhibitors, and IGF signaling pathway activators. Another embodiment describes a method for producing cell aggregates containing neuroretina by self-organization. Self-organization refers to a mechanism in which a group of cells autonomously creates a complex structure. For example, self-organization can be performed by the SFEB (Serum-free Floating Culture of Embryoid Bodies-like aggregates) method (WO2005 / 12390) or the SFEBq method (WO2009 / 148170).

[0046] Specific differentiation induction methods include, but are not limited to, those disclosed 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, or Nat Commun. 2014 Jun 10;5:4047.

[0047] In one specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A), (B), and (C). (A) A step of culturing pluripotent stem cells in a medium containing undifferentiated maintenance factors in the absence of feeder cells. (B) A step in which cells obtained in step (A) are cultured in suspension to form cell aggregates. (C) A step in which the cell aggregates obtained in step (B) are further cultured in suspension in a culture medium containing a BMP signaling pathway agent. 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 inhibitor, as described later.

[0048] This method is also disclosed in WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732, for example. For more details, please refer to WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732.

[0049] Unless otherwise specified, the culture medium used for preparing cell aggregates including neuroretina may be a basal cell growth medium (also called a basal medium). The basal cell growth medium is not particularly limited as long as it can be used to culture cells, and any commercially available basal medium for cell growth may be used as appropriate. Specifically, examples of media that can be used to culture animal cells include 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 medium, RPMI 1640 medium, Fischer's medium, Leibovitz's L-15 medium, or mixtures thereof. In addition, a medium supplemented with N2 medium may be used.

[0050] TGFβ family signaling pathway inhibitors are substances that inhibit the signaling pathway transmitted by the TGFβ family, i.e., the Smad family. Specifically, these 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.

[0051] Sonic Hedgehog (hereinafter sometimes referred to as "Shh") signaling pathway activators are substances that can enhance signal transduction mediated by Shh. Examples of Shh signaling pathway activators include SHH, partial peptides of SHH, PMA (Purmorphamine), and SAG (Smoothened Agonist).

[0052] The concentrations of TGFβ family signaling pathway inhibitors and Sonic Hedgehog signaling pathway activators should be such that they induce differentiation into retinal cells. For example, SB431542 is typically used at concentrations of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is typically used at concentrations of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is typically used at concentrations of 1 to 2000 nM, preferably 10 to 300 nM. SAG is typically used at concentrations of 1 to 2000 nM, preferably 10 to 700 nM. PMA is typically used at concentrations of 0.002 to 20 μM, preferably 0.02 to 2 μM.

[0053] The undifferentiated maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of undifferentiated maintenance factors commonly used by those skilled in the art include FGF signaling pathway activators, TGFβ family signaling pathway activators, and insulin. Specifically, examples of FGF signaling pathway activators include fibroblast growth factor (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ family signaling pathway activators include TGFβ signaling pathway activators and Nodal / Activin signaling pathway activators. Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2. Examples of Nodal / Activin signaling pathway activators include Nodal, ActivinA, and ActivinB. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the culture medium in the first step preferably contains bFGF as the undifferentiated maintenance factor.

[0054] The concentration of the undifferentiated maintenance factor in the culture medium used in the first step is a concentration capable of maintaining the undifferentiated state of the pluripotent stem cells being cultured, and can be appropriately set by those skilled in the art. For example, specifically, when bFGF is used as the undifferentiated maintenance factor in the absence of feeder cells, its 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.

[0055] Many synthetic culture media containing undifferentiated maintenance factors and suitable for use as feeder-free media for culturing pluripotent stem cells have been developed and are commercially available. An example is Essential 8 medium (manufactured by Life Technologies). Essential 8 medium is DMEM / F12 medium with the following additives: 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 a TGFβ family signaling pathway activator (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), or StemFit (Ajinomoto Co., Inc.). By using these in the first step described above, the present invention can be easily carried out. By using these media, it is possible to culture pluripotent stem cells under feeder-free conditions. The medium used in step (A) is, as an example, a serum-free medium that does not contain any BMP signaling pathway activators, Wnt signaling pathway activators, or Wnt signaling pathway inhibitors.

[0056] In the culture of pluripotent stem cells under feeder-free conditions in step (A), an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold in place of feeder cells. Examples of matrices that can be used as a scaffold 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.

[0057] 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 cultured in the presence of a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator (e.g., 100 nM to 700 nM), but is usually 0.5 to 144 hours. In one embodiment, it 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).

[0058] The culture medium used in step (B) may be a serum-containing medium or a serum-free medium. From the viewpoint of avoiding contamination with chemically undetermined components, a serum-free medium is preferably used. To avoid the complexity of preparation, for example, a serum-free medium to which an appropriate amount of a commercially available serum substitute such as KSR has been added can be used. The amount of KSR added to the serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%.

[0059] In forming aggregates, first, dispersed cells are prepared by dispersing the cells obtained in step (A). The "dispersed cells" obtained by the dispersion operation include, for example, a state in which 70% (preferably 80% or more) or more are single cells and 30% or less (preferably 20% or less) are clusters of 2 to 50 cells. Dispersed cells are in a state in which cell-to-cell adhesion (e.g., surface adhesion) is almost completely eliminated.

[0060] A suspension of dispersed cells is seeded into a culture vessel and spread, and the dispersed cells are cultured in the culture vessel under non-adhesive conditions to aggregate a plurality of cells to form aggregates. As one embodiment, when a fixed 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 statically cultured, the cells rapidly aggregate, and one aggregate is formed in each well (SFEBq method). When culturing cells in suspension using a 96-well plate, a solution prepared to contain about 1×10 3 to about 1×10 5 cells (preferably about 3×10 3 to about 5×10 4 cells, about 4×10 3 to about 2×10 4 cells) per well is added to the well, and the plate is left stationary to form aggregates.

[0061] In one embodiment, the medium used in step (B) contains a sonic hedgehog signaling pathway agonist. That is, as a specific embodiment, a cell aggregate containing a neural retina can be prepared by a method including the following steps (A), (B), and (C): (A) A step of culturing pluripotent stem cells in a medium containing an undifferentiated maintenance factor, which may contain a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist, in the absence of feeder cells. (B) A step of forming cell aggregates by culturing the cells obtained in step (A) in suspension in a medium containing a sonic hedgehog signaling pathway agonist. (C) A step of further culturing the cell aggregates obtained in step (B) in suspension in a medium containing a BMP signaling pathway agonist. In step (B), the sonic hedgehog signaling pathway activators described above can be used at the concentrations described above (e.g., 10 nM to 300 nM). Preferably, the sonic hedgehog signaling pathway activators are included in the culture medium from the start of suspension culture. ROCK inhibitors (e.g., Y-27632) may be added to the culture medium. The culture time is, for example, 12 hours to 6 days. In one example, the culture medium used in step (B) is a medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, Wnt signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0062] BMP signaling pathway activators are substances that can enhance signaling pathways mediated by BMPs. Examples of BMP signaling pathway activators include BMP proteins such as BMP2, BMP4, or BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, or BMP partial peptides. BMP2, BMP4, and BMP7 proteins are available from companies such as R&D Systems, and GDF7 protein is available from companies such as Wako Pure Chemical Industries.

[0063] Examples of media used in step (C) include serum-free media or serum media (preferably serum-free media) to which a BMP signaling pathway activator has been added. Serum-free media and serum media can be prepared as described above. In one example, the media used in step (C) is a media that does not contain one or more (preferably all) selected from the group consisting of Wnt signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators. In another example, the media used in step (C) is a media that does not contain a Sonic Hedgehog signaling pathway activator. In yet another example, the media used in step (C) may contain a Wnt signaling pathway activator.

[0064] The concentration of the BMP signaling pathway activator should be such that it can induce differentiation into retinal cells. For example, in the case of human BMP4 protein, it should be added to the culture medium at a concentration of approximately 0.01 nM to approximately 1 μM, preferably approximately 0.1 nM to approximately 100 nM, more preferably approximately 1 nM to approximately 10 nM, and even more preferably approximately 1.5 nM (55 ng / mL).

[0065] The BMP signaling pathway activator may be added approximately 24 hours after the start of suspension culture in step (A), or it may be added to the culture medium within a few days after the start of suspension culture (for example, within 15 days). Preferably, the BMP signaling pathway activator is added to the culture medium between day 1 and day 15 after the start of suspension culture, more preferably between day 1 and day 9, and most preferably on day 3.

[0066] As a specific embodiment, for example, on days 1 to 9, preferably 1 to 3, after the start of suspension culture in step (B), part or all of the culture medium is replaced with a medium containing BMP4, and the final concentration of BMP4 is adjusted to about 1 to 10 nM. Then, the culture can be performed 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, in order to maintain the same concentration of BMP4, part or all of the culture medium may be replaced with a medium containing BMP4 once or twice. Alternatively, the concentration of BMP4 may be gradually reduced. For example, the concentration of the BMP signaling pathway activator (BMP4) may be maintained from day 2 to 10 after the start of suspension culture in step (B), and then the concentration of the BMP signaling pathway activator (BMP4) may be gradually reduced from day 6 to 20 after the start of suspension culture in step (B).

[0067] The culture conditions in steps (A) to (C) above, such as culture temperature and CO2 concentration, can be set as appropriate. 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%.

[0068] By varying the culture period in step (C) above, it is possible to produce retinal cells at various stages of differentiation as retinal cells contained in cell aggregates. In other words, it is possible to produce retinal cells in cell aggregates containing immature retinal cells (e.g., retinal progenitor cells, photoreceptor progenitor cells) and mature retinal cells (e.g., photoreceptor cells) in various proportions. By extending the culture period in step (C), the proportion of mature retinal cells can be increased.

[0069] Steps (B) and / or (C) described above may also be carried out using the method disclosed in WO2017 / 183732. That is, in steps (B) and / or (C), the cells may be suspended in a medium further containing a Wnt signaling pathway inhibitor to form cell aggregates.

[0070] 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 protein, nucleic acid, small molecule compound, etc. The Wnt-mediated signal is transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and LRP5 / 6 (low-density lipoprotein receptor-related protein 5 / 6). Examples of Wnt signaling pathway inhibitors include substances that directly act 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 protein, etc.), and substances that inhibit physiological activity resulting from signal transduction by Wnt receptors [CKI-7(N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476(4-[4-(2 Examples include, but are not limited to, low molecular weight compounds such as, 3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazole-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-isoindole-2-yl]-N-8-quinolinyl-benzamide), and IWP-2(N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidine-2-yl)thio]acetamide). One or more of these may be included as Wnt signaling pathway inhibitors. CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc., are known Wnt signaling pathway inhibitors and are readily available commercially. IWR1e is preferably used as the Wnt signaling pathway inhibitor.

[0071] The concentration of the Wnt signaling pathway inhibitor in step (B) should be such that it can induce the formation of good cell aggregates. For example, in the case of IWR-1-endo, it should be added to the culture medium at 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 using a Wnt signaling pathway inhibitor other than IWR-1-endo, it is desirable to use it at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo.

[0072] In step (B), it is preferable to add the Wnt signaling pathway inhibitor to the culture medium as early as possible. The Wnt signaling pathway inhibitor is usually added to the culture medium within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, from the start of suspension culture in step (B), and even more preferably at the start of suspension culture in step (B). Specifically, for example, this can be done by adding a basal medium to which the Wnt signaling pathway inhibitor has been added, or by replacing part or all of the basal medium. 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, after being added to the culture medium at the start of suspension culture in step (B), it is allowed to act until the end of step (B) (immediately before the addition of the BMP signaling pathway inhibitor). Even more preferably, as described later, the cells are continuously exposed to the Wnt signaling pathway inhibitor even after the end of step (B) (i.e., during the period of step (C)). In one embodiment, as will be described later, the Wnt signaling pathway inhibitor may be allowed to act continuously even after the completion of step (B) (i.e., during the period of step (C)) until retinal tissue is formed.

[0073] 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).

[0074] The concentration of the Wnt signaling pathway inhibitor in step (C) should be such that it can induce retinal progenitor cells and retinal tissue. For example, in the case of IWR-1-endo, it should be added to the culture medium at 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 using a Wnt signaling pathway inhibitor other than IWR-1-endo, it is desirable to use it at a concentration that shows Wnt signaling pathway inhibitory activity equivalent to that of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the culture medium in step (C) is preferably 50 to 150, more preferably 80 to 120, and even more preferably 90 to 110, when the concentration of the Wnt signaling pathway inhibitor in the culture medium in step (B) is set to 100, and it is more preferable that it is equivalent to the concentration of the Wnt signaling pathway inhibitor in the culture medium in the second step.

[0075] The timing of adding the Wnt signaling pathway inhibitor to the culture medium is not particularly limited as long as aggregate formation including retinal cells or retinal tissue can be achieved, but the earlier the better. Preferably, the Wnt signaling pathway inhibitor is added to the culture medium at the start of step (C). More preferably, after the Wnt signaling pathway inhibitor is added in step (B), it is continuously included in the culture medium in step (C) as well (i.e., from the start of step (B)). Even more preferably, after the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B), it is continuously included in the culture medium in step (C) as well. For example, a BMP signaling agent (e.g., BMP4) may be added to the culture obtained in step (B) (a suspension of aggregates in the culture medium containing the Wnt signaling pathway inhibitor).

[0076] The duration for which the Wnt signaling pathway inhibitor is applied 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), starting from the time of 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 duration for which the Wnt signaling pathway inhibitor is applied is preferably 3 to 15 days (e.g., 5, 6, or 7 days), more preferably 6 to 10 days (e.g., 6 days), starting from the time of 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).

[0077] The cell aggregates obtained by the method described above can also be cultured for approximately 2 to 4 days in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor (step (D)), and then cultured for approximately 30 to 200 days (30 to 150 days, 50 to 120 days, 60 to 90 days) in serum-free medium or serum medium that does not contain the Wnt signaling pathway activator and the FGF signaling pathway inhibitor (step (E)) to produce a neuroretina containing a ciliary body periphery-like structure.

[0078] In one embodiment, a neuroretina containing a ciliary body periphery-like structure can be produced from cell aggregates obtained in steps (A) to (C) at 6 to 30 days and 10 to 20 days (10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th day) after the start of suspension culture in step (B) by steps (D) and (E).

[0079] The Wnt signaling pathway activator is not particularly limited as long as it can enhance Wnt-mediated signal transduction. Specific examples of Wnt signaling pathway activators include GSK3β inhibitors (e.g., 6-Bromoindirubin-3'-oxime (BIO), CHIR99021, Kenpaullone). For example, in the case of CHIR99021, the concentration can range from about 0.1 μM to about 100 μM, preferably from about 1 μM to about 30 μM.

[0080] The FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit FGF-mediated signal transduction. 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.

[0081] In step (D), the culture medium used is, for example, a culture medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, Wnt signaling pathway inhibitors, SHH signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0082] Some or all of the above steps (E) can be performed using a continuous epithelial tissue maintenance medium disclosed in WO2019 / 017492. That is, the continuous epithelial structure of the neuroretina can be maintained by culturing using a continuous epithelial tissue maintenance medium. As an example, a medium prepared by adding B27 supplement (e.g., Thermo Fisher Scientific, 21103049) to Neurobasal medium can be used as a continuous epithelial tissue maintenance medium.

[0083] In step (E) above, it is preferable to gradually replace the culture medium with a medium for maintaining continuous epithelial tissue in order to achieve both differentiation and / or maturation of retinal cells (especially photoreceptor cells) and maintenance of the continuous epithelial structure. For example, the cells can be cultured for the first 10 to 30 days using a basal medium for cell proliferation (e.g., DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine), for the next 10 to 40 days using a mixed medium of basal medium for cell proliferation and continuous epithelial tissue maintenance (a medium prepared by mixing DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine with Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine in a 1:3 ratio), and for the next 20 to 140 days using 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).

[0084] In any part or all of the above step (E), regardless of whether a basal medium for cell proliferation, a medium for maintaining continuous epithelial tissue, or a mixture thereof is used, the medium may further contain a thyroid hormone signaling pathway activator. By culturing in a medium containing a thyroid hormone signaling pathway activator, it becomes possible to produce cell aggregates containing neuroretina in which the proportion of bipolar cells, amacrine cells, ganglion cells, or horizontal cells contained in the neuroretina is low, and the proportion of photoreceptor progenitor cells is increased.

[0085] In this specification, a thyroid hormone signaling pathway agonist is a substance that can enhance signal transduction mediated by thyroid hormones, and is not particularly limited as long as it can enhance the thyroid hormone signaling pathway. Examples of thyroid hormone signaling pathway agonists include triiodothyronine (hereinafter sometimes abbreviated as T3), thyroxine (hereinafter sometimes abbreviated as T4), and thyroid hormone receptor (preferably TRβ receptor) agonists.

[0086] Furthermore, as thyroid hormone receptor agonists well known to those skilled in the art, see International Publication No. 97 / 21993, International Publication No. 2004 / 066929, International Publication No. 2004 / 093799, International Publication No. 2000 / 039077, International Publication No. 2001 / 098256, International Publication No. 2003 / 018515, International Publication No. 2003 / 084915, International Publication No. 2002 / 094319, International Publication No. 2003 / 064369, and Japanese Patent Publication No. 20 Examples of compounds such as diphenylmethane derivatives, diaryl ether derivatives, pyridazine derivatives, pyridine derivatives, or indole derivatives described in Japanese Patent Publication No. 02-053564, Japanese Patent Publication No. 2002-370978, Japanese Patent Publication No. 2000-256190, International Publication Brochure No. 2007 / 132475, International Publication Brochure No. 2007 / 009913, International Publication Brochure No. 2003 / 094845, International Publication Brochure No. 2002 / 051805, or International Publication Brochure No. 2010 / 122980.

[0087] When T3 is used as a thyroid hormone signaling pathway agonist, it can be added to the culture medium in a range of, for example, 0.1 to 1000 nM. Preferably, concentrations that have thyroid hormone signaling-enhancing activity equivalent to T3 concentrations of around 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 used. When T4 is used as a thyroid hormone signaling pathway agonist, it can be added to the culture medium in a range of, for example, 1 nM to 500 μM. Preferably, the range is 50 nM to 50 μM; more preferably 500 nM to 5 μM. When other thyroid hormone receptor agonists are used, any concentration that shows activity comparable to that of T3 or T4 at the above concentrations is acceptable.

[0088] The culture medium used in step (E) may optionally contain L-glutamine, taurine, serum, etc. In one example, the culture medium used in step (E) is a culture medium that does not contain one or more (preferably all) selected from the group consisting of BMP signaling pathway activators, FGF signaling pathway inhibitors, Wnt signaling pathway activators, Wnt signaling pathway inhibitors, SHH signaling pathway activators, TGFβ family signaling pathway inhibitors, and TGFβ family signaling pathway activators.

[0089] In one specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A) to (E): (A) A step of culturing pluripotent stem cells in the absence of feeder cells in a medium that contains undifferentiated maintenance factors and optionally contains TGFβ family signaling pathway inhibitors and / or Sonic Hedgehog signaling pathway activators. (B) A step of forming cell aggregates by suspension culture of the cells obtained in step (A) in a medium which may contain a Wnt signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator. (C) A step in which the cell aggregates obtained in step (B) are further cultured in suspension in a culture medium containing a BMP signaling pathway agent. (D) A step of culturing the cell aggregates obtained in step (C) in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor for a period of 2 to 4 days, and (E) A step in which the cell aggregates obtained in step (D) are cultured for approximately 30 to 200 days in a serum-free medium or serum medium that does not contain Wnt signaling pathway activators and FGF signaling pathway inhibitors, but may contain thyroid hormone signaling pathway activators.

[0090] In one specific embodiment, cell aggregates including the neuroretina can be prepared by a method comprising the following steps (A) to (E): (A) A step of culturing pluripotent stem cells for 12 to 48 hours in a medium containing undifferentiated maintenance factors and TGFβ family signaling pathway inhibitors and / or Sonic Hedgehog signaling pathway activators, in the absence of feeder cells. (B) A step in which cells obtained in step (A) are cultured in suspension for 12 hours to 72 days (24 hours to 48 hours) in a medium containing a Wnt signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator to form cell aggregates. (C) The cell aggregates obtained in step (B) are further cultured in suspension for 8 to 15 days (10 to 13 days) in a medium containing a BMP signaling pathway activator. (D) A step of culturing the cell aggregates obtained in step (C) in serum-free medium or serum medium containing a Wnt signaling pathway activator and / or an FGF signaling pathway inhibitor for 2 to 4 days, and (E) A step of culturing the cell aggregates obtained in step (D) in a serum-free medium or serum medium that does not contain Wnt signaling pathway activators and FGF signaling pathway inhibitors, but may contain thyroid hormone signaling pathway activators, for about 10 to 200 days.

[0091] Step (E) may include culturing the cells in a basal medium for cell proliferation for 10 to 30 days, then culturing them in a mixed medium of the basal medium for cell proliferation and a continuous epithelial tissue maintenance medium containing a thyroid hormone signaling agent for 10 to 40 days, and further culturing them in a continuous epithelial tissue maintenance medium containing a thyroid hormone signaling agent for 20 to 140 days. In one embodiment, step (E) includes culturing for 20 to 60 days (30 to 50 days) in the presence of a thyroid hormone signaling pathway activator. In one embodiment, the incubation period from step (B) to step (E) is 70 to 100 days (80 to 90 days).

[0092] The above-described methods can be used to produce cell aggregates containing neuroretina, but are not limited to these methods. In one embodiment, cell aggregates containing neuroretina can also be obtained as a mixture of cell aggregates. In another embodiment, for example, one cell aggregate can be produced in each well of a 96-well plate, or cell aggregates containing neuroretina can be obtained one at a time. In either case, cell aggregates produced under the same conditions are considered to belong to the same lot. The definition of a cell aggregate belonging to the same lot can be set to any range by a person skilled in the art. For example, cell aggregates contained in the above-described mixture of cell aggregates, or cell aggregates contained in the same cell culture vessel (e.g., a 96-well plate), may be defined as belonging to the same lot. As another example, a range using the same stem cells, culture medium, and other materials prepared simultaneously may be defined as belonging to the same lot. Cell aggregates belonging to the same lot exhibit diversity in cell composition, purity, and morphology. On the other hand, when only a specific tissue (e.g., neuroretina) is evaluated from cell aggregates belonging to the same lot, they usually show equivalent gene expression profiles.

[0093] (Cell aggregates including the neuroretina) Cell aggregates containing neuroretina only need to contain neuroretina, and the structure of the cell aggregate is not relevant. In one embodiment, the cell aggregate containing neuroretina is a sphere-like cell aggregate. In one embodiment, multiple neuroretina may overlap within the cell aggregate containing neuroretina (e.g., see conceptual diagrams (1) and (2) in Figure 5). In one embodiment, the cell aggregate containing neuroretina includes a first epithelial tissue containing the neuroretina for transplantation (target epithelial tissue), and a second epithelial tissue having a continuity of tangent slopes on the surface different from that of the first epithelial tissue and containing non-neuroretinal cells (non-target epithelial tissue). Here, the first epithelial tissue substantially does not contain non-neuroretinal cells (non-target cells) and represents epithelial tissue from which the neuroretina for transplantation can be excised. On the other hand, the second epithelial tissue may contain neuroretina, but is unsuitable for excising the neuroretina for transplantation because it contains non-target cells. In another embodiment, the cell aggregate containing the neuroretina includes only the first epithelial tissue (target epithelial tissue) containing the neuroretina for transplantation, and does not include the non-target epithelial tissue.

[0094] A neuroretina for transplantation is a human neuroretina suitable for human transplantation, preferably consisting solely of the neuroretina. The neuroretina for transplantation includes at least a photoreceptor cell layer, which is formed at least on the outermost part of the cell aggregates, and may also contain photoreceptor cells or photoreceptor progenitor cells on the inner side, or a photoreceptor cell layer may be formed on the inner side as well. The photoreceptor cells are arranged continuously in the tangential direction to the surface of the cell aggregates, i.e., they are adherent to one another, and the continuous arrangement of the photoreceptor cells in the tangential direction to the surface of the cell aggregates forms a photoreceptor cell layer containing the photoreceptor cells. The tangential direction refers to the direction tangential to the surface of the cell aggregates, i.e., the direction in which the photoreceptor cells are arranged in the photoreceptor cell layer, and is parallel or transverse to the neuroretina. Furthermore, the tangential inclination on the surface of epithelial tissue refers to the direction in which cells are arranged when each cell in the epithelial tissue is arranged in a certain direction, and is parallel or transverse to the epithelial tissue (or epithelial sheet).

[0095] The second epithelial tissue included in the cell aggregate is epithelial tissue other than neuroretinal epithelial tissue, i.e., extra-target epithelial tissue. Examples of the second epithelial tissue include ocular tissue and cerebrospinal tissue. Ocular tissue refers to tissue surrounding non-retinal ocular tissue, and includes retinal pigment epithelial cells, ciliary body (e.g., periphery of the ciliary body), and lens. Cerebrospinal tissue refers to the nerve tissue of the brain and spinal cord, and includes the forebrain, telencephalon, cerebrum, diencephalon, hypothalamus, midbrain, hindbrain, cerebellum, and spinal cord. The cells and expressed genes included in the second epithelial tissue are described below.

[0096] Examples of cell aggregates containing primary and secondary epithelial tissues include the conceptual diagrams in Figure 4 and Figures 5 (3) and (5). The conceptual diagram in Figure 4 shows an example of a cell aggregate in which a portion of the neuroretina, which is the primary epithelial tissue, contains ophthalmoplastic tissue (retinal pigment epithelial cells, ciliary body) (black area in Figure 4) as the secondary epithelial tissue. Conceptual diagram (3) in Figure 5 shows an example of a cell aggregate in which multiple neuroretinas overlap (e.g., conceptual diagrams (1) and (2) in Figure 5), and further contains ophthalmoplastic tissue (retinal pigment epithelial cells, ciliary body) (black area in Figure 5 (3)) as the secondary epithelial tissue. Conceptual diagram (5) in Figure 5 shows an example of a cell aggregate in which cerebrospinal tissue (cerebrum, etc.) (gray area in Figure 5 (5)) is present as the secondary epithelial tissue. As shown in conceptual diagram (4) in Figure 5, unintended tissue may be included inside the cell aggregate containing the neuroretina for transplantation. In this case, it does not meet the definition of "having a continuity of tangent slope on a surface different from the continuity of tangent slope on the surface of the first epithelial tissue," and therefore does not qualify as the second epithelial tissue. It is preferable to select the neuroretina for transplantation and the sample for quality evaluation from cell aggregates that do not contain non-target tissue on the inside.

[0097] <Extraction process> The method for evaluating the quality of a neuroretina for transplantation according to the present invention includes extracting (sampling) a portion or all of a cell aggregate containing a neuroretina having an epithelial structure derived from pluripotent stem cells as a quality evaluation sample (hereinafter referred to as the "extraction step"). Extracting a portion of a cell aggregate as a quality evaluation sample means selecting a portion of a cell aggregate (one or more) or all of a cell aggregate from a group of cell aggregates, and isolating a portion of the selected cell aggregate as an evaluation sample using tweezers, scissors and / or a knife, etc. (e.g., dissecting). Extracting all of a cell aggregate as a quality evaluation sample means selecting a portion of a cell aggregate (one or more) from a group of cell aggregates, and separately picking up all of the selected one or more cell aggregates as a quality evaluation sample. When selecting one or more cell aggregates from a group of cell aggregates, it is preferable to extract them randomly. In this specification, when a portion of a cell aggregate is extracted as a quality evaluation sample, the cell aggregate is referred to as "a cell aggregate containing a quality evaluation sample," and when the entire cell aggregate is extracted as a quality evaluation sample, the cell aggregate is referred to as "a cell aggregate of a quality evaluation sample."

[0098] (Extraction of all cell aggregates) In one embodiment, the quality evaluation sample is the entirety of a cell aggregate containing a neuroretina having an epithelial structure derived from pluripotent stem cells. To evaluate the quality of cell aggregates containing neuroretina from the same lot, the quality evaluation sample may be the entirety of one or more cell aggregates from the same lot. Here, a mixture of cell aggregates from the same lot contains, for example, two to 10,000 cell aggregates.

[0099] Specifically, in quality evaluation of cell aggregates contained in the same lot, one or more cell aggregates are selected from the same lot, and all of the selected one or more cell aggregates are extracted as quality evaluation samples. When all of the selected one or more cell aggregates are used as quality evaluation samples, all of the cell aggregates may be extracted together, or all of the cell aggregates may be extracted separately. Cell aggregates used as quality evaluation samples cannot be used for transplantation.

[0100] If, as a result of evaluation using quality evaluation samples, it is determined that all of the cell aggregates used as quality evaluation samples are suitable for use as neuroretina transplants, then it can be determined that the neuroretina in cell aggregates from the same lot as the quality evaluation samples, manufactured under conditions that show a gene expression profile equivalent to that of the neuroretina transplanted from the cell aggregates, is also suitable for use as neuroretina transplants. In this case, the epithelial tissue containing neuroretina from these other cell aggregates can be used for transplantation (also known as same-lot evaluation). Therefore, this extraction method is useful for efficient quality evaluation.

[0101] If, as a result of the evaluation using quality evaluation samples, even one cell aggregate among multiple quality evaluation samples is determined to be unsuitable for use as a neuroretina for transplantation, then the neuroretina from other cell aggregates in the same lot will also be determined to be unsuitable for use as a neuroretina for transplantation. Therefore, this method (same lot evaluation) is usually appropriate when, for example, there are many samples in the same lot and the possibility of contamination with unsuitable samples is low. However, since the entire cell aggregate is extracted as an evaluation sample, it is preferable not to use this method for lots containing cell aggregates that include a second epithelial tissue. In this case, it is more appropriate to extract and evaluate only a portion of the cell aggregate (neuroretina) as described below, rather than the entire cell aggregate. Furthermore, for lots containing cell aggregates that include a second epithelial tissue, the full evaluation described below is preferable to same-lot evaluation.

[0102] (Extraction of a portion of the cell aggregate) In one embodiment, the quality evaluation sample is a portion of a cell aggregate containing a neuroretina with an epithelial structure derived from pluripotent stem cells. By extracting a portion of the cell aggregate as a quality evaluation sample, there is an advantage in that the entire cell aggregate is not destroyed, and the neuroretina contained in the remaining portion can be used for transplantation. That is, if the quality evaluation sample, which is a portion of the cell aggregate, is judged to be acceptable by the judgment process described later, the neuroretina with an epithelial structure in the cell aggregate containing the quality evaluation sample can be used as a neuroretina for transplantation and can be used for transplantation.

[0103] In one embodiment, the extraction method can be used to individually evaluate the quality of cell aggregates containing neuroretina (also called individual evaluation). In this case, there is also the advantage that all cell aggregates containing neuroretina to be evaluated can be selected and all of them can be evaluated for quality (total evaluation). This evaluation method (individual evaluation / total evaluation) can be said to be the most accurate quality evaluation method because it can individually evaluate the quality of neuroretina for transplantation. On the other hand, if the number of samples is large, it requires a great deal of effort and cost. It should be noted that this evaluation method is premised on the existence of a region in the cell aggregate excluding the neuroretina for transplantation that shows a gene expression profile equivalent to that of the neuroretina (quality evaluation sample), and that this quality evaluation sample can be excised. In particular, whether or not this premise is met is a major issue in cell aggregates containing secondary epithelial tissue. The inventors have found for the first time, by evaluating a large number of samples, that cell aggregates containing neuroretina meet this premise and that this evaluation method can be used.

[0104] To take advantage of the above benefits, it is preferable that a portion of the cell aggregate extracted as a quality evaluation sample does not include the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted. It is also preferable that the quality evaluation sample is a portion that exhibits a gene expression profile equivalent to that of the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted. In order for the neuroretina to be transplanted and the quality evaluation sample to exhibit equivalent gene expression profiles, it is preferable that the quality evaluation sample is in close proximity to or continuous with the neuroretina to be transplanted in at least a portion, and is contained in the same epithelial tissue as the neuroretina to be transplanted. Furthermore, it is preferable that the neuroretina to be transplanted includes the central part of the same epithelial tissue as the best portion of the cell aggregate (e.g., containing the neuroretina but not the non-neuroretina, and having a continuous epithelial structure), and is of the size described in the [neuroretina sheet to be transplanted] section below. Therefore, it is preferable that the quality evaluation sample does not include the central part of the same epithelial tissue. Here, the same epithelial tissue means continuous epithelial tissue with continuity in the tangential slope on the surface of the epithelial tissue, and the same epithelial tissue is preferably continuous epithelial tissue. Here, the area near the center of the epithelial tissue (continuous epithelial tissue) is the region on the surface of the same epithelial tissue that is equal in distance from both ends, and can be estimated by a person skilled in the art through microscopic observation. Furthermore, the quality evaluation sample is a portion that is continuous with or adjacent to the region to be used as the neuroretina for transplantation (e.g., a region used for transplantation that includes the area near the center of a single epithelial tissue), and it is preferable that it be the narrowest possible portion within the range in which quality evaluation is possible. For example, the neuroretina for transplantation is a region near the center of the same epithelial tissue, and the quality evaluation sample is a portion of the same epithelial tissue that is continuous with or adjacent to the neuroretina for transplantation in at least a part.

[0105] In one embodiment of individual and / or mass evaluation, if a cell aggregate containing a neuroretina for transplantation includes a first epithelial tissue containing the neuroretina for transplantation, and an off-target epithelial tissue (second epithelial tissue) that has a continuity of tangent slopes on a surface different from the continuity of tangent slopes on the surface of the first epithelial tissue and contains off-target cells, it is preferable that the neuroretina for transplantation includes the region on the first epithelial tissue furthest from the off-target epithelial tissue, in which case the quality evaluation sample may be a portion existing between the off-target epithelial tissue and the neuroretina for transplantation. The region on the first epithelial tissue furthest from the off-target epithelial tissue is, for example, the region including the point on the outer periphery of the first epithelial tissue when a straight line is drawn from the center of the off-target epithelial tissue toward the outer periphery of the first epithelial tissue and the length of the line is the longest. Furthermore, it is preferable that the quality evaluation sample is a portion adjacent to or continuous with the neuroretina for transplantation (e.g., the site used for transplantation including the vicinity of the center of one epithelial tissue), and is as narrow as possible within the range in which quality evaluation is possible. An example of a graft and a quality evaluation sample is shown in the conceptual diagram in Figure 4.

[0106] Alternatively, in order to perform quality evaluation of cell aggregates containing neuroretina from the same lot (same-lot evaluation), instead of using all of the cell aggregates from the same lot, a portion of one or more cell aggregates from the same lot can be extracted as a quality evaluation sample. That is, one or more cell aggregates can be selected from the same lot of cell aggregates, and only a portion cut out from the selected cell aggregates can be used as a quality evaluation sample. This extraction method is particularly useful when it is not possible to use the entire cell aggregate for quality evaluation, such as a cell aggregate containing a second epithelial tissue, and when it is desirable to perform quality evaluation efficiently. In this case, it is preferable to use neuroretina for transplantation as the quality evaluation sample. Neuroretina for transplantation used as a quality evaluation sample here refers to neuroretina in cell aggregates manufactured under conditions that exhibit a gene expression profile equivalent to that of transplantation retina. By using a portion that would have been used as transplantation retina if not extracted as a quality evaluation sample for quality evaluation, it is possible to perform a more accurate quality evaluation of other cell aggregates containing neuroretina for transplantation from the same lot.

[0107] In cell aggregates, areas exhibiting a continuous epithelial structure, where the outer neuroblastic layer and inner neuroblastic layer appear separated into two distinct layers, can be identified as the neuroretina. On the other hand, ocular tissues, particularly retinal pigment epithelial cells, which are a second epithelial tissue, appear black under the naked eye or microscope, and can therefore be easily distinguished from the neuroretina by those skilled in the art. Furthermore, cerebrospinal fluid tissues, which are a second epithelial tissue, can be easily distinguished from the neuroretina by those skilled in the art, focusing on their morphological characteristics, such as the absence of a continuous epithelial structure on the surface of the cell aggregate, the absence of morphological features specific to the neuroretina, and / or their dull color. Therefore, even in cell aggregates containing a second epithelial tissue, those skilled in the art can isolate neuroretina for transplantation and samples for quality evaluation from the first epithelial tissue containing the neuroretina.

[0108] As described above, in one embodiment, a quality evaluation sample is set and extracted based on a certain positional relationship with the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted. That is, by setting the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted, the region to be cut out as a quality evaluation sample can be determined. Here, the neuroretina to be transplanted (also called a graft or cap) and its candidates can be identified in one embodiment by their position within the cell aggregate described above (e.g., near the center of the epithelial tissue (continuous epithelial tissue), and if there is a second epithelial tissue, further, the region on the first epithelial tissue furthest from the second epithelial tissue), and the size described in the [neuroretina sheet to be transplanted] below. Therefore, a person skilled in the art can set a neuroretina having these characteristics as the neuroretina to be transplanted or a candidate for the neuroretina to be transplanted.

[0109] When extracting a neuroretina for transplantation and a quality evaluation sample from the same cell aggregate, a person skilled in the art can define the quality evaluation sample (also called a ring) as a region that is at least partially continuous with or adjacent to the neuroretina for transplantation as defined above, and as the narrowest possible region within which quality evaluation is possible. When extracting a portion of one or more cell aggregates from the same lot of the cell aggregate as a quality evaluation sample, a person skilled in the art can define the quality evaluation sample as the portion of the neuroretina for transplantation or a candidate thereof from the cell aggregate as defined above. In this case, the size of the sample cut out for quality evaluation may be the size described in the [Neuroretina for Transplantation Sheet] section below, or it may be even smaller. Therefore, the quality evaluation sample can be defined and extracted based on its position relative to the neuroretina for transplantation or a candidate thereof, and the size described above.

[0110] <Detection Process> The method for evaluating the quality of a neuroretina for transplantation according to the present invention includes detecting the expression of neuroretinal cell-related genes and non-neurotinic cell-related genes (off-target cell-related genes) in a quality evaluation sample (detection step). It is preferable that the detection step quantitatively detects the expression levels of the genes. The off-target cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eyeball-related tissue marker genes.

[0111] (Neuroretinal cell-related genes) Neuroretinal cell-related genes (target cell-related genes) refer to genes expressed by neuroretinal cells. Preferred neuroretinal cell-related genes include photoreceptor cells (rod and cone cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells, bipolar cells (rod and cone bipolar cells), Müller glial cells, or their progenitor cells, neuroretinal progenitor cells, etc., which are expressed at higher levels compared to non-target cells. Examples of neuroretinal cell-related genes include the neuroretinal cell markers listed above, with RAX, Chx10, SIX3, SIX6, RCVRN, CRX, NRL, and NESTIN being preferred. The GenBank IDs for the neuroretinal cell markers are shown in Table 1 below. [Table 1]

[0112] The neuroretinal cell-related genes are preferably those listed in Table 1, but are not limited to these. Other neuroretinal cell-related genes include Rax2, Vsx1, Blimp1, RXRG, S-opsin, M / L-opsin, Rhodopsin, Brn3, and L7.

[0113] (Non-neuroretinal cell-related genes) There was absolutely no knowledge of how to detect unintended cells induced as byproducts during the process of manufacturing cell aggregates containing neuroretina as pharmaceutical raw materials, or how to detect such unintended cells for quality evaluation of pharmaceuticals. In the process of diligently investigating a method for continuously producing neuroretina of a quality that meets the standards for pharmaceutical raw materials, the inventors discovered non-neuroretinal cell-related genes (unintended cell-related genes) as genes that can be used to identify cells or tissues that may be produced as byproducts, to efficiently and effectively identify them, and to perform quality evaluation of neuroretina.

[0114] In one embodiment, non-neuroretinal cell-related genes (non-target cell-related genes) include brain and spinal cord tissue marker genes and eyeball-related tissue marker genes. In one embodiment, undifferentiated iPS cell marker genes may also be included as non-neuroretinal cell-related genes.

[0115] In one embodiment, the brain and spinal cord tissue marker gene may be one or more genes selected from the group consisting of telencephalon marker genes, diencephalon / midbrain marker genes, and spinal cord marker genes. The diencephalon / midbrain marker gene may be one or more genes selected from the group consisting of diencephalon marker genes, midbrain marker genes, and hypothalamic marker genes related to the hypothalamus, which is part of the diencephalon.

[0116] In one embodiment, the eye-related tissue marker gene may be one or more genes selected from the group consisting of optic stalk marker genes, ciliary body marker genes, lens marker genes, and retinal pigment epithelial marker genes.

[0117] Telencephalon marker genes refer to genes expressed in the telencephalon. Telencephalon marker genes may include one or more genes selected from the group consisting of FoxG1 (also known as Bf1), Emx2, Dlx2, Dlx1, and Dlx5. The GenBank IDs of telencephalon marker genes are shown in Table 2 below. [Table 2]

[0118] The telencephalon marker genes are preferably those listed in Table 2, but are not limited to these. Other telencephalon marker genes include Emx1, LHX2, LHX6, LHX7, and Gsh2.

[0119] Diencephalon / midbrain marker genes refer to genes expressed in the diencephalon and / or midbrain. Diencephalon / midbrain marker genes may include one or more genes selected from the group consisting of OTX1, OTX2, and DMBX1. The GenBank IDs of diencephalon / midbrain marker genes are shown in Table 3 below. Diencephalon / midbrain marker genes may also include hypothalamic markers, which will be described later, with respect to the hypothalamus, a region of the diencephalon. That is, diencephalon / midbrain marker genes 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]

[0120] Hypothalamic marker genes refer to genes expressed in the hypothalamus. Hypothalamic marker genes may include one or more genes selected from the group consisting of Rx, Nkx2.1, Dmbx1, OTP, gad1, FGFR2, and EFNA5. The GenBank IDs for hypothalamic marker genes are shown in Table 4 below. [Table 4]

[0121] 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. The GenBank IDs of spinal cord marker genes are shown in Table 5 below. [Table 5]

[0122] The spinal cord marker genes are preferably those listed in Table 2, but are not limited to these. Other examples of spinal cord marker genes include gene groups that form Hox clusters.

[0123] On the other hand, in one embodiment, when retinoic acid is used in the manufacturing process, the expression of HOX genes (e.g., HOXC5, HOXA5, and HOXB2) may be observed even when good quality retinal tissue is produced. It is thought that the expression of HOX genes is regulated by retinoic acid signaling, and HOX gene expression increases to an extent that does not affect the differentiation induction of retinal tissue. The effect of this retinoic acid signaling is thought to be due to the promotion of posteriorization along the anterior-posterior axis. Therefore, when retinoic acid is used in the manufacturing process (especially when retinoic acid is used after the start of differentiation induction into the retina), HOX genes (e.g., HOXC5, HOXA5, and HOXB2) can be excluded from the genes targeted for quality evaluation, or even if the expression of these genes is observed, the quality of the neuroretina for transplantation can be judged as good quality.

[0124] An Optic Stalk marker gene refers to a gene expressed in the Optic Stalk. An Optic Stalk marker gene may include one or more genes selected from the group consisting of GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A. The GenBank IDs of Optic Stalk marker genes are shown in Table 6 below. [Table 6]

[0125] Lens marker genes refer to genes expressed in the lens of the eye. Lens marker genes may include one or more genes selected from the group consisting of CRYAA and CRYBA1. The GenBank IDs of lens marker genes are shown in Table 7 below. [Table 7]

[0126] Ciliary body marker genes refer to genes expressed in the ciliary body, the periphery of the ciliary body, and / or the ciliary body. Ciliary body marker genes 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 ciliary body marker genes are shown in Table 8 below. [Table 8]

[0127] Retinal pigment epithelial marker genes refer to genes expressed in retinal pigment epithelial cells. Retinal pigment epithelial marker genes include the retinal pigment epithelial markers listed above and may include one or more genes selected from the group consisting of MITF, TTR, and BEST1. The GenBank IDs of retinal pigment epithelial marker genes are shown in Table 9 below. [Table 9]

[0128] In one embodiment, the off-target cell-related gene may further include an undifferentiated pluripotent stem cell marker gene.

[0129] 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]

[0130] (Detection method) In one embodiment, the detection of expression of neuroretinal cell-related genes and non-neuroretinal off-target cell-related genes is not particularly limited, but examples include Western blotting, immunohistochemistry, flow cytometry / flow cytometry (FACS®, BD Inc., etc.), Northern blotting, electrophoresis, PCR (preferably quantitative PCR (qPCR) and / or real-time PCR), gene tip analysis, and next-generation sequencing. Of these, quantitative PCR is useful in terms of quantitative accuracy, detection sensitivity, result stability, and speed. Furthermore, by applying equipment used for single-cell quantitative PCR (e.g., Biomark HD (Fluidigm Inc.), etc.) to normal quantitative PCR, it is possible to evaluate multiple quality assessment samples in a short time. In particular, when performing 100% evaluation, especially when the number of samples used for quality assessment and the number of genes to be evaluated are large, this method enables rapid quality assessment.

[0131] In one embodiment, the expression levels of neuroretinal cell-related genes and non-neuroretinal cell-related genes in two or more quality evaluation samples may be simultaneously detected by quantitative PCR. This quantitative PCR may be performed, for example, by a method including the following steps (1) to (5). (1) Prepare a channel plate having channels connecting the independent sample wells in the sample well group and the independent primer wells in each of the primer well groups, a solution containing nucleic acids obtained from two or more of the above quality evaluation samples (sample solution), and a solution containing one or more primers specific to one or more of the above neuroretinal cell-related genes or the above non-neuroretinal cell-related genes (primer solution). (2) In the sample well group, add the above sample solution to each quality evaluation sample so that there is 1 sample solution per sample well. (3) Add the primer solution to one or more primer wells in the above group of one or more primer wells so that they become different groups of primer wells. (4) Mixing the nucleic acid and the primer separately via the above-mentioned channel, (5) Perform quantitative PCR using the mixture obtained in (4).

[0132] A solution containing nucleic acids obtained from two or more quality evaluation samples can be prepared by a reverse transcription reaction using reverse transcriptase and primers on RNA extracted from the quality evaluation samples. RNA extraction and reverse transcription can be carried out as appropriate using methods well known to those skilled in the art. Furthermore, those skilled in the art can prepare primers capable of amplifying the gene to be quantified.

[0133] In one embodiment, the nucleic acid-containing solution (sample solution) may be a solution that has undergone a multiplex-PCR reaction (Pre-Run) using all the primers to be used in a PCR instrument before being added to each well of the sample well group. By amplifying the nucleic acid to a certain extent through Pre-Run, quantitative PCR can be effectively performed. As a condition for Pre-Run, for example, the nucleic acid-containing solution may be subjected to a PCR reaction for about 10 to 15 cycles with all the primers to be used.

[0134] In one embodiment, quantitative PCR may use a flow channel plate having channels connecting the aforementioned different groups of wells. The flow channel plate has channels (e.g., accumulating fluid circuits), multiple wells for adding nucleic acid-containing solutions, and multiple wells for adding primers, and each well may be connected to one or more channels (accumulating fluid circuits). One nucleic acid-containing solution and one primer are added to each well. In one embodiment, the nucleic acid-containing solution and primer added to each well can be flowed into the channels (accumulating fluid circuits) by applying air pressure to the wells using a well-known device (e.g., IFC Controller HX, IFC Controller MX, IFC Controller RX, all manufactured by Fluidigm). The channels (accumulating fluid circuits) may have a structure in which the sample solution and primer solution are mixed in a 1:1 ratio. This allows for the preparation of a sample solution and primer solution mixture as many times as there are combinations of sample solutions and primer solutions (for example, if 96 sample solutions and 96 primer solutions are used, the number of combinations is 96 × 96 = 9216) at once. After preparing the mixture, the expression levels of each gene in the solution containing each nucleic acid can be simultaneously measured by performing a quantitative PCR reaction on a flow channel plate using a quantitative PCR instrument (e.g., Biomark HD).

[0135] Furthermore, flow cytometry analysis using a flow cytometer capable of detecting the proportion of expression cells is also useful. In recent years, improvements in detection speed have led to the availability of high-throughput flow cytometers (such as FACS®) capable of evaluating a large number of samples. Therefore, the use of high-throughput flow cytometers is also useful for detecting the expression of neuroretinal cell-related genes and non-neuroretinal off-target cell-related genes. Such high-throughput flow cytometers can be commercially available (e.g., MACSQuant® Analyzers: manufactured by Miltenyi Biotec).

[0136] <Judgment process> The method according to the present invention includes a determination step of determining whether the epithelial tissue containing the neuroretina (neuroretina for transplantation) in a cell aggregate containing a part of the above-mentioned quality evaluation sample, or the epithelial tissue containing the neuroretina (neuroretina for transplantation) in a cell aggregate of the same lot as the cell aggregate containing the entire above-mentioned quality evaluation sample, is suitable for use as a neuroretina for transplantation, when the expression of neuroretinal cell-related genes is observed and the expression of non-neuroretinal cell-related genes is not observed. Here, being suitable for use as a neuroretina for transplantation means that it is a neuroretina suitable for transplantation, and is also said to be suitable as a neuroretina for transplantation, or to be acceptable as a neuroretina for transplantation.

[0137] The detection of neuroretinal cell-related gene expression means that, in a gene expression detection method, the expression of neuroretinal cell-related gene is detected at a level that is substantially detectable by the method (e.g., above the detection limit). Conversely, the absence of non-neuroretinal cell-related gene expression means that, in a gene expression detection method, the expression of non-neuroretinal cell-related gene cannot be substantially detected by the method (e.g., below the detection limit). Substantial detectability means that the gene is detected to a degree that goes beyond the point at which it cannot be said that the gene is substantially functional. A person skilled in the art can appropriately set this depending on the gene and the detection method. For example, in the case of a quantitative gene expression detection method, ranges of over 0% to 10% and over 0% to 5% can be judged as not being substantially detectable (i.e., the expression of the gene cannot be detected) based on the detection limit of the gene expression.

[0138] In one embodiment, it is preferable to determine that the material is suitable for use as a neuroretina for transplantation if the following criteria 1 and 2 are met in the quantitative PCR method. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 10 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes is 5 or greater.

[0139] The Threshold Cycle (Ct) value refers to the number of cycles at which a constant amount of amplification product is reached in the region where gene amplification by PCR occurs exponentially. Since the Ct value is inversely correlated with the initial amount of gene, it is used to calculate the initial copy number of gene. In one embodiment, the "2^Ct value (2 to the power of Ct)" is inversely proportional to the initial amount of gene and is therefore used to calculate the initial copy number of 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 gene before amplification. A constant amount of amplification product can be set by anyone skilled in the art, as long as it falls within the region where gene amplification by PCR occurs exponentially.

[0140] An internal standard gene refers to a gene whose expression level differs little between samples. Suitable internal standard genes include those well known to those skilled in the art, such as 18S ribosomal RNA, β-actin, HPRT, α-tubulin, transferrin receptor, ubiquitin, and GAPDH, but GAPDH is preferred.

[0141] The Ct value is inversely correlated with the initial amount of the gene and therefore depends on the gene expression level within the cell. That is, when the concentration of the nucleic acid-containing solution is constant, the Ct value will differ 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 values ​​in this specification are based on the values ​​obtained when GAPDH is used as the internal standard gene.

[0142] When using an internal standard gene other than GAPDH as the internal standard gene, the ΔCt values ​​of Criterion 1 and Criterion 2 can be corrected by comparing the expression levels of GAPDH and the internal standard gene other than GAPDH.

[0143] In one embodiment, when β-actin is used as the internal standard gene, in the present invention's method, the Ct value of GAPDH is approximately 1 lower than that of β-actin, meaning that the absolute amount of RNA in GAPDH is about twice the absolute amount of RNA in β-actin. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 9 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes is 4 or greater. It can be done this way.

[0144] In one embodiment, when HPRT is used as the internal standard gene, in the present invention's manufacturing method, the Ct value of GAPDH is approximately 7 lower than that of HPRT, meaning that the absolute amount of RNA in GAPDH is approximately 2^7 (2 to the power of 7, 128 times) greater than that of HPRT. Criterion 1: The difference (ΔCt value) between the Threshold Cycle (Ct) value of a neuroretinal cell-related gene and the Ct value of an internal standard gene is 3 or less. Criterion 2: The difference (ΔCt value) between the Ct value of non-neuronal retinal cell-related genes and the Ct value of internal standard genes is -2 or greater. It can be done this way.

[0145] The neuroretinal cell-related genes can be any of the genes mentioned above. There are multiple genes that can be considered neuroretinal cell-related. That is, even when extracted from the same neuroretinal cell, the Ct value of reference value 1 may differ depending on the type of neuroretinal cell-related gene. A person skilled in the art can determine whether a neuroretinal cell-related gene is being expressed for each gene by setting a ΔCt value that can be determined from publicly known information such as the expression site and expression level of the neuroretinal cell-related gene.

[0146] For example, with respect to the Chx10 gene, if GAPDH is used as the internal standard, the ΔCt value may be 20 or less, preferably 15 or less, and more preferably 10 or less.

[0147] For example, with respect to the Recoverin gene, if GAPDH is used as the internal standard, the ΔCt value may be 16 or less, preferably 11 or less, and more preferably 6 or less.

[0148] Generally, the difference (ΔCt value) between the Ct value of a neuroretinal cell-related gene and the Ct value of an 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 a neuroretinal cell-related gene and the Ct value of an internal standard gene may be, for example, -10 or greater, -5 or greater, 0 or greater, or 5 or greater.

[0149] Non-neuroretinal cell-related genes can be any of the genes mentioned above. There are multiple genes that can be considered non-neuroretinal cell-related. That is, even when extracted from the same non-retinal cell, the Ct value will differ depending on the non-neuroretinal cell-related gene. A person skilled in the art can determine that a neuroretinal cell-related gene is being expressed for each gene based on publicly known information such as the expression site and expression level of the non-neuroretinal cell-related gene. For example, for the PAX2 gene, if GAPDH is used as the internal standard, the ΔCt value may be 5 or higher. For the HOXB2 gene, if GAPDH is used as the internal standard, the ΔCt value may be 5 or higher. Generally, the difference between the Ct value of a non-neuroretinal cell-related gene and the Ct value of the internal standard gene may be 30 or less, 25 or less, or 20 or less. Also, the difference between the Ct value of a non-neuroretinal cell-related gene and the Ct value of the internal standard gene may be, for example, 0 or more, 3 or more, or 5 or more.

[0150] The quality evaluation method described above can be used as a quality control method for pharmaceuticals (neuroretina for transplantation) or as a quality control technique in the manufacturing process of pharmaceuticals (neuroretina for transplantation).

[0151] [Neuroretinal sheet for transplantation] One aspect of the present invention is a neuroretinal sheet for implantation, (1) Derived from pluripotent stem cells, (2) Having a three-dimensional structure, (3) A neuroretinal layer having a multilayer structure including a photoreceptor cell layer and an inner layer, (4) The photoreceptor layer comprises one or more cells selected from the group consisting of photoreceptor progenitor cells and photoreceptor cells, (5) The inner layer contains 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 neuroretinal layer has an apical surface, (7) Inside the photoreceptor layer located along the apical surface, (8) The area of ​​the neuroretinal layer is 50% or more of the total surface area of ​​the neuroretinal sheet for transplantation. (9) The area of ​​continuous epithelial structures is 80% or more of the total area of ​​the apical surface of the neuroretinal layer. (10) Expression of neuroretinal cell-related genes is observed in the neuroretinal sheet for transplantation, and expression of non-neuroretinal cell-related genes is not observed, and the non-neuroretinal cell-related genes include one or more genes selected from the group consisting of brain and spinal cord tissue marker genes and eye-related tissue marker genes. This is a neuroretinal sheet for transplantation characterized by the following features.

[0152] In one embodiment, the neuroretinal sheet for transplantation is a neuroretina for transplantation cut out using the quality evaluation method described above. Therefore, the characteristics of the neuroretinal sheet for transplantation described later also correspond to the characteristics of a neuroretina for transplantation cut out using the quality evaluation method described above.

[0153] The neuroretinal sheet for transplantation includes (3) a neuroretinal layer having a multilayer structure including a photoreceptor cell layer and an inner layer. As described in (6) and (7), the photoreceptor cell layer is located on the outer (surface) side of the neuroretinal sheet for transplantation, but an ectopic photoreceptor cell layer may also be present in the inner layer.

[0154] The neuroretinal sheet for transplantation (5) has an inner layer which contains one or more cells selected from the group consisting of retinal progenitor cells, ganglion cells, amacrine cells and bipolar cells, but may also contain one or more cells selected from the group consisting of ectopic photoreceptor progenitor cells and photoreceptor cells. In one embodiment, there are also provided neuroretinal sheets for transplantation in which the content of ganglion cells, amacrine cells and horizontal cells is 30% or less of the total number of cells, neuroretinal sheets 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 neuroretinal sheets for transplantation in which the content of bipolar cells is 10% or less of the total number of cells.

[0155] (8) The area of ​​the neuroretinal layer of the transplantable neuroretinal sheet is 40% or more, preferably 50% or more, and more preferably 60% or more, of the total surface area of ​​the neuroretinal sheet. (9) The area of ​​the continuous epithelial structure is 60% or more, preferably 70% or more, and more preferably 80% or more, of the total surface area of ​​the apical surface of the neuroretinal layer.

[0156] The genes related to neuroretinal cells and non-neuroretinal cells (brain and spinal cord tissue marker genes and eye-related tissue marker genes) are the genes mentioned above.

[0157] (10) Whether the expression of neuroretinal cell-related genes is observed in the neuroretinal sheet for transplantation and whether the expression of non-neurotinic cell-related genes is not observed can be determined by taking a portion of the neuroretinal sheet for transplantation and detecting the gene expression. Furthermore, if the neuroretinal sheet for transplantation is isolated from a cell aggregate in which the expression of neuroretinal cell-related genes is substantially observed and the expression of non-neurotinic cell-related genes is substantially not observed in the quality evaluation sample according to the neuroretinal sheet for transplantation quality evaluation method described above, then it is unnecessary to detect the gene expression of the neuroretinal sheet for transplantation itself. Whether the gene expression is substantially observed or not is determined, as described above, by whether or not it is at a level that can be substantially detected by the gene expression detection method.

[0158] The neuroretinal cell-related genes in the neuroretinal sheet for transplantation may be one or more selected from the group consisting of, for example, Rx, Chx10, Pax6, and Crx. The proportion of cells expressing neuroretinal cell-related genes (positive cells) relative to the total number of cells varies depending on the differentiation stage of the neuroretina.

[0159] In one embodiment, the proportion of Rx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 30% or more, 40% or more, 50% or more, or 60% or more. In one embodiment, the proportion of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neuroretinal 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 proportion of Crx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 10% or more, 20% or more, 30% or more, 40% or more, or 50% or more.

[0160] In one embodiment, the proportion of Rx-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 30% to 80%, 40% to 70%, 45% to 60%, or 50% to 60%. In one embodiment, the proportion of Chx10-positive cells or Pax6-positive cells to the total number of cells in the neuroretinal sheet for transplantation may be 10% to 80%, 20% to 70%, 30% to 60%, or 40% to 50%. In one embodiment, the proportion of Crx-positive cells to the total number of cells in the neuroretinal sheet for transplantation is 10% to 70%, 10% to 60%, 20% to 60%, 30% to 60%, 40% to 60%, or 50% to 60%.

[0161] In one embodiment, the proportion of cells in the neuroretinal sheet for transplantation may be: (1) Chx10-positive and Pax6-positive cells (neuroretinal progenitor cells) being 10% to 50% or 10% to 30%; (2) Chx10-positive and Pax6-negative cells (bipolar progenitor cells) being 10% to 25% or 15% to 25%; and (3) Chx10-negative and Pax6-positive cells (ganglion cells and amacrine cells) being 10% to 25% or 10% to 20%.

[0162] In another embodiment, the proportion of (1) Chx10-positive and Pax6-positive cells (neuroretinal progenitor cells) relative to the total number of cells in the neuroretinal sheet for transplantation may be 20% to 40%, (2) Chx10-positive and Pax6-negative cells (bipolar progenitor cells) may be 5% to 20%, and (3) Chx10-negative and Pax6-positive cells (ganglion cells and amacrine cells) may be 5% to 20% or 5% to 15%.

[0163] In one aspect, the neuroretinal sheet for transplantation according to the present invention is a neuroretinal sheet for transplantation that has been determined to be usable as a neuroretinal sheet for transplantation by the above-described quality evaluation method for neuroretinal sheet for transplantation, and may be an isolated sheet-like neuroretinal sheet for transplantation.

[0164] In one embodiment, the neuroretinal sheet for transplantation according to the present invention may be isolated from a cell aggregate containing neuroretina and may include a region near the center of the continuous epithelial tissue in the cell aggregate.

[0165] In one embodiment, the neuroretinal sheet for transplantation according to the present invention is isolated from a cell aggregate comprising at least a first epithelial tissue and a second epithelial tissue, wherein the first epithelial tissue comprises human neuroretina, and the second epithelial tissue has a continuity of tangent slopes on a surface different from that of the first epithelial tissue and comprises non-neuroretinal cells, and the neuroretinal sheet for transplantation may be a neuroretinal sheet for transplantation that comprises the region on the first epithelial tissue furthest from the second epithelial tissue. Here, the second epithelial tissue may be a tissue selected from the group consisting of ocular-related tissues, telencephalonal tissues, and tissues different from the neuroretina of the first epithelial tissue.

[0166] In one embodiment, the neuroretinal sheet for transplantation according to the present invention may include a region near the center of the continuous epithelial tissue in the cell aggregate.

[0167] As described above, the neuroretinal sheet for transplantation can be isolated from cell aggregates containing the neuroretina. It can also be obtained by the manufacturing method for the neuroretinal sheet for transplantation described later.

[0168] In one embodiment, the major axis of the neuroretinal sheet for implantation 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.

[0169] In one embodiment, the short diameter of the neuroretinal sheet for implantation 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.

[0170] In one embodiment, the height of the neuroretinal sheet for implantation 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.

[0171] In one embodiment, the volume of the neuroretinal sheet for implantation according to the present invention is, for example, 0.001 mm 3~4.0mm 3 It may be, preferably 0.01 mm 3 ~1.5mm 3 More preferably 0.07 mm 3 ~0.57mm 3 That is the case.

[0172] The method for measuring the major axis, minor axis, and height of a neuroretinal sheet for transplantation is not particularly limited and can be measured, for example, from images taken under a microscope. For example, for a neuroretinal sheet for transplantation excised from a cell aggregate, a frontal image taken with the cut surface facing the objective lens and a lateral image taken with the cut surface tilted so that it is perpendicular to the objective lens can be taken with a stereomicroscope and measured from the images. Here, the major axis refers to the longest line segment and its length among the line segments connecting the two endpoints on the sheet cross-section in the frontal image. The minor axis refers to the longest line segment and its length among the line segments that connect the two endpoints on the sheet cross-section in the frontal image and are perpendicular to the major axis. The height refers to the longest line segment and its length among the line segments perpendicular to the sheet cross-section, with the intersection with the sheet cross-section and the vertex of the retinal sheet as endpoints. The volume of the sheet refers to the volume calculated according to the following formula, assuming that the graft is an ellipsoid obtained by approximating it as a cross-section obtained by halving it so that the cross-section passes through the major axis. Volume = 2 / 3 × Pi (π) × (major axis / 2) × (minor axis / 2) × height

[0173] [Pharmaceutical compositions, treatment methods, therapeutic drugs, and manufacturing methods] One aspect of the present invention is a pharmaceutical composition comprising a neuroretinal sheet for implantation. The pharmaceutical composition comprises the neuroretinal sheet for implantation of the present invention, preferably further comprising a pharmaceutically acceptable carrier. The pharmaceutical composition can be used to treat diseases based on disorders of neuroretinal cells or the neuroretina, or damage to the neuroretina. Examples of diseases based on disorders of neuroretinal cells or the neuroretina 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 neuroretinal damage include conditions in which photoreceptor cells have degenerated and died.

[0174] Acceptable carriers for pharmaceutical use include physiological aqueous solvents (such as physiological saline, buffer solutions, and serum-free culture media). If necessary, the pharmaceutical composition may also contain preservatives, stabilizers, reducing agents, isotonic agents, etc., that are commonly used in pharmaceuticals containing transplanted tissue or cells in transplant medicine.

[0175] One aspect of the present invention provides a therapeutic agent for diseases based on neuroretinal disorders, including a neuroretinal sheet for transplantation obtained in the present invention. Another aspect of the present invention includes a method for treating diseases based on neuroretinal cell disorders or neuroretinal disorders or neuroretinal damage, which involves transplanting the neuroretinal sheet for transplantation obtained in the present invention to a subject requiring transplantation (for example, subretinal area of ​​an eye with an ophthalmic disease). The neuroretinal sheet for transplantation of the present invention can be used as a therapeutic agent for diseases based on neuroretinal disorders, or to replenish the damaged area in a neuroretinal damage state. By transplanting the neuroretinal sheet for transplantation of the present invention to a patient with a neuroretinal cell disorder or neuroretinal disorder requiring transplantation, or to a patient with a neuroretinal damage state, the neuroretinal cell disorder or damaged neuroretinal disorder can be treated by replenishing the neuroretinal cell disorder or the damaged neuroretina. An example of a transplantation method is to transplant the neuroretinal sheet for transplantation subretinal area of ​​the damaged site by making an incision in the eyeball. Methods of transplantation include injecting the transplanted tissue using a thin tube or grasping it with forceps and transplanting it. Examples of thin tubes include hypodermic needles.

[0176] One aspect of the present invention is a method for producing a neuroretinal sheet for transplantation obtained in the present invention. In one embodiment, the method for producing a neuroretinal sheet for transplantation includes evaluating a cell aggregate containing a neuroretina having an epithelial structure derived from pluripotent stem cells using the above-described method for evaluating neuroretina for transplantation, determining that the neuroretina is suitable for use as a neuroretina for transplantation, and isolating the determined neuroretina for transplantation.

[0177] In another embodiment, the method for producing a neuroretinal sheet for transplantation includes extracting quality evaluation samples from a cell aggregate containing 2 to 800 neuroretinas having epithelial structures derived from pluripotent stem cells, selecting neuroretinas that are determined to be suitable for use as neuroretinas for transplantation, and isolating the selected neuroretinas for transplantation.

[0178] Preferably, the cell aggregate is a cell aggregate obtained by differentiating pluripotent stem cells and includes at least a first epithelial tissue and a second epithelial tissue. Preferably, the first epithelial tissue includes human neuroretina, and the second epithelial tissue has a continuity of tangent slopes on its surface that is different from the continuity of tangent slopes on the surface of the first epithelial tissue, and includes non-neuroretinal cells. Preferably, the isolation of the neuroretina for transplantation is performed by isolating the neuroretina for transplantation from the cell aggregate such that the neuroretina for transplantation includes the region on the first epithelial tissue that is furthest from the second epithelial tissue. Isolation is performed by cutting using the method described above.

[0179] The quality evaluation method disclosed in this invention, which uses cell aggregates containing epithelial tissue as raw materials, with a portion of the epithelial tissue used as a transplantation sample and another portion of the epithelial tissue used as a quality evaluation sample, is particularly effective when the morphology and structure of each cell aggregate differ. It can also be applied to cell aggregates containing various types of epithelial tissue. [Examples]

[0180] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0181] <Example 1: Production of cell aggregates containing neuroretina> Human iPS cells (DSP-SQ strain, established by Dainippon Sumitomo Pharma Co., Ltd.) were established using commercially available Sendai virus vectors (four factors: Oct3 / 4, Sox2, KLF4, and c-Myc, IDPharma's SiteTune Kit) based on the methods described in Thermo Fisher Scientific's public protocol (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and Kyoto University's public protocol (Establishment and maintenance culture of human iPS cells in a feeder-free environment, CiRA_Ff-iPSC_protocol_JP_v140310, http: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html), using StemFit medium (AK03; Ajinomoto Co., Ltd.) and Laminin511-E8 (Nippi Corporation).

[0182] The human iPS cells (DSP-SQ strain) were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, Ajinomoto Co., Ltd.) was used as the feeder-free medium, and Laminin511-E8 (Nippi Corporation) was used as the feeder-free scaffold.

[0183] The specific maintenance culture procedure involved first washing subconfident human iPS cells (DSP-SQ strain) with PBS and then dispersing them into single cells using TrypLE Select (Life Technologies). Subsequently, these single-cell human iPS cells were seeded onto plastic culture dishes coated with Laminin511-E8 and cultured feeder-free in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 μM). The plastic culture dishes used were 6-well plates (Iwaki Corporation, for cell culture, culture area 9.4 cm²). 2 When using this method, the number of seeded human iPS cells dispersed into the single cell is 1.0 × 10⁶. 4The medium was changed to Y27632-free StemFit medium one day after sowing. Thereafter, the medium was changed to Y27632-free StemFit medium once every 1-2 days. The culture was then continued until 5 days after sowing.

[0184] For differentiation induction, human iPS cells (DSP-SQ strain) were cultured feeder-free in StemFit medium until two days before subconfretion (approximately 30% of the culture area was covered by cells). These human iPS cells, two days before subconfretion, were then cultured feeder-free for two days in the presence of SAG (300 nM) (preconditioning).

[0185] Preconditioned human iPS cells were treated with a cell dispersion using TrypLE Select (Life Technologies), and then dispersed into single cells by pipetting. These single-cell dispersed human iPS cells were then placed in a non-cell-adherent 96-well culture plate (PrimeSurface 96V bottom plate, Sumitomo Bakelite) at a rate of 1.3 × 10⁶ per well. 4 Cells were suspended in 100 μl of serum-free medium and cultured in suspension at 37°C and 5% CO2. The serum-free medium used was a 1:1 mixture of F-12 medium and IMDM medium to which 10% KSR, 450 μM 1-monothioglycerol, and 1 × Chemically defined lipid concentrate were added. At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (final concentration 20 μM) and SAG (final concentration 10 nM) were added to the serum-free medium. On day 2 after the start of suspension culture, 50 μl of the above serum-free medium was added to a medium containing human recombinant BMP4 (manufactured by R&D Co., Ltd.) but without Y27632 and SAG, so that the final concentration of exogenous human recombinant BMP4 was 1.5 nM (55 ng / ml).

[0186] Four days later (six days after the start of suspension culture), the culture medium was changed with the serum-free medium described above, which does not contain Y27632, SAG, or human recombinant BMP4. The medium change procedure involved discarding 60 μl of the culture medium in the incubator and adding 90 μl of the new serum-free medium described above, so that the total volume of medium was 180 μl. Subsequently, every 2 to 4 days, a half volume of the culture medium was changed with the serum-free medium described above, which does not contain Y27632, SAG, or human recombinant BMP4. The half volume of the culture medium was discarded by half the volume, i.e., 90 μl, of the culture medium in the incubator and added 90 μl of the new serum-free medium described above, so that the total volume of medium was 180 μl.

[0187] The cell aggregates obtained in this manner, 13 days after the start of suspension culture, were cultured for 3 days, or until 16 days after the start of suspension culture, in serum-free medium containing CHIR99021 (3 μM) and SU5402 (5 μM) (DMEM / F12 medium supplemented with 1% N2 supplement).

[0188] The resulting cell aggregates, obtained 16 days after the start of suspension culture, were cultured under 5% CO2 conditions until 75 days after the start of suspension culture, using the serum media described in [1], [2], and [3] below for the culture medium described below. [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: Medium A and medium B, which is Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, 60 nM T3 and 100 μM taurine, are mixed in a 1:3 ratio. [3] 60 days after the start of suspension culture: Medium B.

[0189] Cell aggregates 75 days after the start of suspension culture were observed using an inverted microscope to confirm their morphology. It was found that neuroepithelial structures had formed at this time.

[0190] Cell aggregates 75 days after the start of suspension culture were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor cell precursor cell markers (Figure 1). Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers (Figure 2). Cell nuclei were stained with DAPI.

[0191] These stained sections were observed using a fluorescence microscope (Keyence), and immunohistochemical images were obtained. Photographs of the produced cells observed under a fluorescence microscope are shown in Figures 1 and 2. In Figures 1 and 2, the upper row shows images taken with a low-magnification lens, and the lower row shows images taken with a high-magnification lens.

[0192] From the DAPI staining images of FIGS. 1 and 2, it was found that on the surface of the cell mass, a nerve tissue densely packed with cells was formed, and this nerve tissue formed a continuous epithelial structure. As a result of analyzing the image of FIG. 1, in this nerve tissue, a Crx-positive layer (photoreceptor cell layer) about 2 to 5 cells thick was formed on the surface of the cell mass, and a Chx10-positive layer about 5 to 20 cells thick was formed inside the Crx-positive layer. Furthermore, it was found that a layer in which Crx-positive cells were sparsely present was formed inside that (FIG. 1). The surface of this cell mass was morphologically found to be the apical surface. Furthermore, as a result of analyzing the image of FIG. 2, it was found that a Recoverin-positive layer (photoreceptor cell layer) and an Rx-positive layer were also formed in this nerve tissue. From these results, it was found that in this nerve tissue, a photoreceptor cell layer containing Crx-positive cells and Recoverin-positive cells was formed on the surface, a retinal progenitor cell layer containing Chx10-positive cells was formed inside the photoreceptor cell layer, and a cell layer was also formed inside the retinal progenitor cells. That is, by this production method, it was found that a neural retina containing a photoreceptor cell layer and a retinal progenitor cell layer could be produced from human iPS cells, and this neural retina had a continuous epithelial structure.

[0193] <Example 2 Identification of unwanted cells (non-neural retina cells) and search for marker genes> Human iPS cells (QHJI-01-s04 strain, obtained from the Institute for Integrated Cell-Material Sciences, Kyoto University) were differentiated into the retina under various culture conditions. Specifically, human iPS cells maintained by the method described in Example 1 were cultured feeder-free until 2 days before sub-confluence (about 30% of the culture area was covered with cells) or 1 day before sub-confluence (about 50% of the culture area was covered with cells). The human iPS cells 2 days before sub-confluence were cultured feeder-free for 2 days in the presence of SAG (300 nM), or the human iPS cells 1 day before sub-confluence were cultured feeder-free for 1 day in the presence of SAG (300 nM) and LDN (LDN193189, 100 nM) (Precondition treatment).

[0194] The preconditioned human iPS cells were cultured in suspension by the method described in Example 1. For the serum-free medium (gfCDM+KSR), a serum-free medium obtained by adding 10% or 5% KSR, 450 μM 1-monothioglycerol, and 1×Chemically defined lipid concentrate to a 1:1 mixture of F-12 medium and IMDM medium was used. At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (final concentration 20 μM) and IWR-1e (final concentration 3 μM), or Y27632 (final concentration 20 μM), IWR-1e (final concentration 3 μM), and SAG (30 nM) were added to the serum-free medium. On the 3rd day after the start of suspension culture, a new 50 μl of the above serum-free medium was added such that the final concentration of exogenous human recombinant BMP4 became 1.5 nM (55 ng / ml) and the final concentration of IWR-1e became 3 μM in a medium containing human recombinant BMP4 (manufactured by R&D) and IWR-1e but not containing Y27632 and SAG.

[0195] Three days later (6 days after the start of suspension culture), the medium was changed to the above serum-free medium containing IWR-1e but not containing Y27632, SAG, and human recombinant BMP4. As the medium change operation, 60 μl of the medium in the incubator was discarded, 90 μl of the new above serum-free medium was added, and the total medium volume was 180 μl. At 10 to 12 days after the start of suspension culture, a 67% medium change operation was performed twice using the above serum-free medium not containing IWR1-e, Y27632, SAG, and human recombinant BMP4 so that the concentration of exogenous IWR-1e was about 10% compared to that before the medium change. Thereafter, a half-volume medium change was performed once every 2 to 4 days using the above serum-free medium not containing IWR1-e, Y27632, and human recombinant BMP4. As the half-volume medium change operation, 90 μl, which is half of the volume of the medium in the incubator, was discarded, 90 μl of the new above serum-free medium was added, and the total medium volume was 180 μl.

[0196] The cell aggregates obtained in this manner, 19-20 days after the start of suspension culture, were cultured for 3 days, or 22-23 days after the start of suspension culture, in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing CHIR99021 (3 μM), a Wnt signaling pathway activator, and SU5402 (5 μM), an FGF signaling pathway inhibitor.

[0197] Subsequently, the cultures were incubated under 5% CO2 conditions for 89 to 97 days after the start of suspension culture using the serum media described in [1], [2], and [3] in Example 1, or DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, 0.5 μM retinoic acid, and 100 μM taurine (hereinafter referred to as Retina medium).

[0198] Cell aggregates obtained in this manner, 89 to 97 days after the start of suspension culture, were observed using an inverted microscope (Nikon ECLIPSE Ti) to obtain bright-field images (phase-contrast images). Particular attention was paid to the morphology of each individual cell and the characteristics of cell adhesion. In the cell aggregates, areas with a continuous epithelial structure, where the outer neuroblastic layer (including the photoreceptor cell layer and the neuroretinal progenitor cell layer) and the inner neuroblastic layer appeared to be separated into two layers, were identified as the neuroretina. Tissues where a continuous epithelial structure was not observed, or where a continuous epithelial structure was observed but the outer neuroblastic layer and inner neuroblastic layer could not be distinguished and appeared as a single layer, were designated as by-products (A, B, C, D, E, and F). Subsequently, while observing with a stereomicroscope, the neuroretina or by-products were dissected from the cell aggregates under stereomicroscope conditions using tapered tweezers and scissors to prepare tissue fragments. The excised tissue samples totaled 33 samples: "Neural Retinal #1-9", "By-product A, #10-16", "By-product B, #17-20", "By-product C, #21, #22", "By-product D, #23, #24", "By-product E, #25-29", and "By-product F, #30-33".

[0199] Subsequently, total RNA was extracted from the tissue samples using a spin column (QIAGEN, RNeasy Micro kit) according to the kit's instructions, and then analyzed using a microarray (Affymetrix, Human Genome U133 Plus2.0) (Figure 3). Figure 3 is a heatmap display, where gray corresponds to high gene expression and black corresponds to low gene expression (lighter colors correspond to higher gene expression).

[0200] As a result, it was found that the expression levels of the following markers were generally high in the nine tissue samples labeled "neuroretinum #1-9": cone cell progenitor marker RXRG, rod cell progenitor marker NRL, photoreceptor marker Recoverin (also known as RCVRN), photoreceptor progenitor marker Crx, neuroretinal marker Rax2, and photoreceptor progenitor marker Blimp1 (also known as PRDM1). In other words, it was confirmed that "neuroretinum #1-9" is retinal tissue containing neuroretinal cells.

[0201] Furthermore, the expression of HOX genes (HOXC5, HOXA5, and HOXB2) was observed in "Neuroretina #1-3". Retinoic acid was added to "Neuroretina #1-3" during the manufacturing process, and it is thought that the expression of HOX genes is controlled by retinoic acid. However, as mentioned above, "Neuroretina #1-3" is a good quality retinal tissue containing neuroretinal cells. Therefore, when retinoic acid is added during the manufacturing process, the expression of HOX genes (HOXC5, HOXA5, and HOXB2) is acceptable.

[0202] We analyzed "By-product A, #10-16". As a result, we found that By-product A generally showed low expression levels of neuroretinal cell markers. On the other hand, when we searched for marker genes that were highly expressed in By-product A, we found that GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A were expressed. After carefully examining the literature information on these markers (Baumer N, et al. Development. 2003 Jul;130(13):2903-15, Pfeffer PL, et al. Development. 1998 Aug;125(16):3063-74), we found that By-product A is an optic stalk. In other words, it was found that when neuroretina is created from pluripotent stem cells in vitro, an optic stalk may be produced as a by-product. Furthermore, GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, and SEMA5A were found to be useful marker genes for distinguishing these optic stalks.

[0203] We analyzed "By-product B, #17-20". As a result, we found that by-product B generally showed low expression levels of neuroretinal cell markers. On the other hand, when we searched for marker genes that were highly expressed in by-product B, we found that Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, CRYAA, and CRYBA1 were expressed. After carefully reviewing the literature information on these markers, we found that by-product B is the ciliary body, lens, and peripheral ciliary body (hereinafter referred to as ciliary body and lens). In other words, it was found that the ciliary body and lens may be produced as a by-product when a neuroretina is created from pluripotent stem cells in vitro. Furthermore, it was found that Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, CRYAA, and CRYBA1 are useful as marker genes for distinguishing this ciliary body and lens.

[0204] We analyzed "By-products C, #21, and #22." The results showed that by-product C generally exhibited low expression levels of neuroretinal cell markers. On the other hand, a search for marker genes highly expressed in by-product C revealed the presence of MITF, TTR, and BEST1. A thorough review of the literature on these markers revealed that by-product C is retinal pigment epithelium (RPE). This suggests that RPE may be a by-product when neuroretina is created from pluripotent stem cells in vitro. Furthermore, MITF, TTR, and BEST1 were found to be useful marker genes for distinguishing this RPE.

[0205] We analyzed "By-products D, #23, and #24." The results showed that by-product D generally exhibited low expression levels of neuroretinal cell markers. On the other hand, a search for marker genes highly expressed in by-product D revealed the presence of HOXD4, HOXD3, HOXD1, HOXC5, HOXA5, and HOXB2. A thorough review of the literature regarding these markers revealed that by-product D is spinal cord tissue. In other words, it was found that spinal cord tissue may be a by-product when neuroretina is created from pluripotent stem cells in vitro. Furthermore, HOXD4, HOXD3, HOXD1, HOXC5, HOXA5, and HOXB2 were found to be useful marker genes for distinguishing this spinal cord tissue.

[0206] We analyzed "Byproduct E, #25-29". As a result, we found that byproduct E generally showed low expression levels of neuroretinal cell markers. On the other hand, when we searched for marker genes that were highly expressed in byproduct E, we found that Nkx2.1, OTP, FGFR2, EFNA5, and GAD1 were expressed. After carefully reviewing the literature information on these markers, we found that byproduct E is the diencephalon, midbrain, and hypothalamus (hereinafter referred to as diencephalon and midbrain). In other words, it was found that the diencephalon and midbrain may be produced as byproducts when a neuroretina is created from pluripotent stem cells in vitro. Furthermore, it was found that Nkx2.1, OTP, FGFR2, EFNA5, and GAD1 are useful as marker genes for distinguishing this diencephalon and midbrain.

[0207] We analyzed "Byproduct F, #30-33". As a result, we found that byproduct F generally showed low expression levels of neuroretinal cell markers. On the other hand, when we searched for marker genes that were highly expressed in byproduct F, we found that DLX2, DLX1, DLX5, FOXG1, EMX2, GPR177(Wls), and AQP4 were expressed. After carefully reviewing the literature information on these markers, we concluded that byproduct F is the telencephalon based on the expression of DLX2, DLX1, DLX5, FOXG1, and EMX2. We also found that GPR177 and AQP4 are useful as byproduct markers. In other words, we found that the telencephalon may be a byproduct when a neuroretina is created from pluripotent stem cells in vitro. Furthermore, we found that DLX2, DLX1, DLX5, FOXG1, and EMX2 are useful as marker genes for distinguishing this telencephalon.

[0208] The results above indicate that in the byproducts, the expression levels of marker genes for neuroretinal cells were low, while the expression levels of genes known to be expressed in the tissues of the Optic Stalk, ciliary body / lens, RPE, spinal cord, diencephalon / midbrain, and telencephalon were high. Therefore, it was found that the Optic Stalk, ciliary body / lens, RPE, spinal cord, diencephalon / midbrain, and telencephalon can be byproducts during the differentiation process into the three-dimensional retina. Furthermore, it was found that the genes listed in Table 11 are markers that can detect each tissue.

[0209] [Table 11]

[0210] <Example 3: Development of a method for evaluating grafts> The three-dimensional retina created from human iPS cells consists of a neuroretina with a neuroepithelial structure that exhibits continuity in the composition and distribution of cells. This neuroepithelial structure has a layered structure composed of a photoreceptor cell layer and an inner layer, and possesses a characteristic appearance and morphology (Figure 1).

[0211] Human 3D retinas are approximately 1-2 mm in size, and each has a different shape. Furthermore, due to the characteristics of the manufacturing method using self-organizing culture, it was found that while the neuroretina used for transplantation is the main product, non-neuronal retinal tissues such as ophthalmoplastic tissue (RPE, ciliary body, etc.) and cerebrospinal tissue (telencephalon, spinal cord, etc.) are also produced as by-products (Example 2). Therefore, we decided to obtain retinal fragments (grafts, caps) by cutting out the central part of the neuroretina that does not contain non-neuronal retina (Figures 4 and 5). While it is desirable to perform quality evaluation regarding the composition and purity of the retinal fragments by testing all of them, it is not possible to destructively test all of the retinal fragments. Therefore, we decided to use the peripheral part of the retinal fragment (cap) as a quality evaluation sample (ring). Then, we decided to analyze all of the rings (preferably by quantitative PCR), and considered using only the caps corresponding to rings that met the criteria as neuroretina for transplantation.

[0212] Note that FIGS. 4 and 5 are conceptual diagrams of typical cell aggregates. A transplant (cap) is a site where a neuroepithelial structure (preferably a continuous epithelial structure) peculiar to the neural retina, where the photoreceptor cell layer and the inner layer are visibly divided into two layers, is observed. A site where a neuroepithelial structure (preferably a continuous epithelial structure) similar to that of the cap is observed at the peripheral site of the cap is used as a quality evaluation sample (ring), and sites other than the cap and the ring are called roots.

[0213] Hereinafter, the usefulness of a method for isolating the cap and the ring from the neuroepithelial structure contained in one cell aggregate was examined.

[0214] <Example 4 Shape of Transplant (Cap)> The transplant (cap) was prepared by the following method (FIG. 6). First, a bright-field image (phase-contrast image) of a cell aggregate on the 99th day after the start of suspension culture prepared from human iPS cells (DSP-SQ strain) according to the method described in Example 1 was taken with an inverted microscope (manufactured by Olympus). After confirming that there was a neural retina on the cell aggregate, the cell aggregate was transferred under a stereomicroscope, and neural retinas of various sizes were excised as transplants using the method described in Example 2. In addition, the influence of the size of the transplant on the transplantation operation using the transplantation device was examined.

[0215] For the excised graft, a frontal image was taken with the cut surface facing the objective lens, and a lateral image was taken with the cut surface tilted so that it was perpendicular to the objective lens, using a stereomicroscope. Subsequently, the major axis, minor axis, and height of the graft were measured from the acquired images. For measurement, the major axis was defined as the longest line segment connecting two endpoints on the retinal sheet cross-section in the frontal image and its length. The minor axis was defined as the longest line segment perpendicular to the major axis connecting two endpoints on the retinal sheet cross-section in the frontal image and its length. The height was defined as the longest line segment perpendicular to the retinal sheet cross-section in the lateral image, with the intersection with the retinal sheet cross-section and the surface of the retinal sheet as endpoints, and its length. Furthermore, the volume of the graft was approximated as an ellipsoid obtained by halving the graft so that its cross-section passes through the major axis, and was calculated according to the following formula. Volume = 2 / 3 × Pi (π) × (major axis / 2) × (minor axis / 2) × height

[0216] The results showed that the placement of grafts in the implantation device, the stability of grafts within the device, and the ejection of grafts from the device were all affected by the size of the graft. Furthermore, the short diameter was suggested to be a particularly useful parameter. For 11 grafts that underwent successful implantation using the implantation device, the long diameter, short diameter, height, and volume were calculated. The average, maximum, and minimum values ​​for each parameter were calculated and summarized in Table 12. From these results, it was found that grafts (caps) should have at least a long diameter of 0.8–1.7 mm, a short diameter of 0.4–1.1 mm, a height of 0.2–0.7 mm, and an apparent volume of 0.07–0.57 mm. 3 It was found to be to that extent. [Table 12]

[0217] <Example 5: Cell composition of the graft (cap)> Grafts (caps) were prepared by the following method (number: 18001MF, d89, H5). First, grafts (caps) were isolated from cell aggregates 89 days after the start of suspension culture, prepared from human iPS cells (DSP-SQ strain) according to the method described in Example 1, using the methods described in Examples 2 and 3.

[0218] The grafts were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor progenitor cell markers (Figure 7). Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers (Figure 7). Cell nuclei were stained with DAPI. These stained sections were observed using a confocal laser microscope (Olympus), and immunostained images were obtained.

[0219] Staining revealed that densely packed nerve tissue was formed on the surface of the graft (cap) (left side in the figure), and that this nerve tissue formed a neuroepithelial structure (particularly a continuous epithelial structure) (Figure 7). Furthermore, it was found that a Crx-positive layer (photoreceptor layer, Figure 7) with a thickness of about 2 to 10 cells was formed on the surface of the cell mass in this nerve tissue, a Chx10-positive layer with a thickness of about 5 to 20 cells was formed inside the Crx-positive layer, and further inside that, a layer containing Crx-positive cells was formed (Figure 7). Morphologically, the surface of this graft (cap) was found to be the apical surface. In addition, it was found that a Recoverin-positive layer (photoreceptor layer, arrow in Figure 7) and an Rx-positive layer were formed in this nerve tissue. From these results, it was found that a photoreceptor layer containing Crx-positive and Recoverin-positive cells was formed on the surface of this nerve tissue, 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, it was found that the graft (cap) could be used to create a neuroretina containing a photoreceptor cell layer and a retinal progenitor cell layer, and that this neuroretina had a continuous epithelial structure.

[0220] <Example 6: Verification of the equivalence of the cap and ring> To compare gene expression in the caps and rings, the following method was used for verification. First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) 99 days after the start of suspension culture according to the method described in Example 1, and designated as Lot 1. Furthermore, cell aggregates from human iPS cells (DSP-SQ strain) 82 days after the start of suspension culture were prepared from human iPS cells (DSP-SQ strain) according to the method described in Example 1, and designated as Lot 2. For these two lots, the main product, the neuroretina, and the by-products were identified using a microscope according to the methods described in Examples 2 and 3, and the caps of the neuroretina and the by-products were isolated, respectively. The rings were isolated by cutting them out under a stereomicroscope using tapered tweezers and scissors, similar to the grafts. Total RNA was extracted from the caps and rings isolated from the neuroretina and by-products using the method described in Example 2. After measuring the total RNA concentration with a measuring instrument (Nanodrop, Thermo Scientific), it was reverse transcribed into cDNA using reverse transcriptase and primer (Reverse Transcription Master Mix Kit, Fluidigm). cDNA was subjected to multiplex-PCR (Pre-Run) using a PCR instrument (Veriti 96-well thermal cycler, Applied Biosystems) with all the probes used for validation. The pre-run reaction mixture was then injected into a multi-well with a flow channel (96.96 Dynamic Array IFC, Fluidigm) using an IFC controller HX (Fluidigm), and the expression levels of marker genes for neuroretina and non-neurotinic byproducts were measured by real-time PCR using a multi-sample real-time PCR system (Biomark HD, Fluidigm). The PCR probes used for validation are shown in Table 13.

[0221] [Table 13]

[0222] The results are shown in Figure 8 as a heatmap. Gene expression levels were evaluated using the ΔCt value, which is calculated from the difference between the Ct value of the target gene and the Ct value of GAPDH, a gene used as an internal standard. A lower ΔCt value indicates higher gene expression, and a higher ΔCt value indicates lower gene expression. Gray indicates high gene expression, and black indicates low gene expression (lighter colors indicate higher gene expression). When gene expression was examined in each cap and ring, the neuroretinal marker gene group was expressed in the caps and rings isolated from the neuroretina in both Lot 1 and Lot 2. On the other hand, when gene expression was examined in the caps and rings isolated from the byproduct, in both lots, contrary to the neuroretina, the expression level of the neuroretinal marker gene group was low, and the expression level of the byproduct marker gene group was high. From this, it was confirmed that the marker gene group found in Example 2 could detect neuroretina and byproduct separately. Furthermore, when comparing gene expression in caps and rings isolated from the same cell aggregate, it was found that the expression levels of neuroretinal marker genes and by-product marker genes were equivalent in caps and rings isolated from any neuroretina or by-product.

[0223] These results demonstrate that if the ring is a neuroretina, then the cap is also a neuroretina. Furthermore, it was demonstrated that gene expression is equivalent in the cap and the ring.

[0224] <Example 7: Verification of the equivalence of the cap and ring in various pluripotent stem cell lines> To investigate whether gene expression in the cap and ring is equivalent in cell aggregates differentiated from various pluripotent stem cells, the following was examined.

[0225] First, Crx::Venus knock-in human ES cells (derived from KhES-1; Nakano, T. et al. Cell Stem Cell 2012, 10(6), 771-785; obtained from Kyoto University, established and used at RIKEN CDB), human iPS cells (QHJI-01-s04 strain), and human iPS cells (DSP-SQ strain) were differentiated into retina using the method described in Example 1. Subsequently, from cell aggregates that had been cultured in suspension for 70 days or more, the neuroretina and by-product caps and rings were excised using the method described in Example 6. Then, total RNA was extracted using the method described in Example 6. After measuring the total RNA concentration with a measuring instrument (Nanodrop, Thermo Scientific), it was reverse transcribed into cDNA using reverse transcriptase and primer (Reverse Transcription Master Mix Kit, Fluidigm). cDNA was subjected to multiplex-PCR (Pre-Run) using a PCR instrument (Veriti 96-well thermal cycler, Applied Biosystems) with all the probes used for validation. The pre-run reaction mixture was then injected into a multi-well with a flow channel (96.96 Dynamic Array IFC, Fluidigm) using an IFC controller HX (Fluidigm), and the expression levels of marker genes for neuroretina and non-neurotinic byproducts were measured by real-time PCR using a multi-sample real-time PCR system (Biomark HD, Fluidigm). The PCR probes shown in Table 3 of Example 6 were used for validation.

[0226] The results are shown in the heatmap in Figure 9. The heatmap was prepared according to the method described in Example 6. When gene expression was examined in the caps and rings, in all cell lines, the caps and rings isolated from the neuroretina showed high expression levels of neuroretinal marker genes and low expression levels of by-product marker genes. On the other hand, in the caps and rings isolated from the by-products, the expression levels of by-product marker genes were high and the expression levels of neuroretinal marker genes were low. Furthermore, when comparing the gene expression of caps and rings isolated from the same cell aggregate, it was found that the neuroretinal and by-product marker genes were expressed to a similar degree in caps and rings isolated from all cell lines.

[0227] These results demonstrate that, in any cell aggregate derived from a pluripotent stem cell line, if the ring is neuroretinal, then the cap is also neuroretinal. Furthermore, it was demonstrated that gene expression is equivalent in the cap and the ring. In other words, even when a cap the size shown in Table 2 is excised, or when a ring is excised from the remaining portion of the cell aggregate, it is possible to excise a ring that has equivalent gene expression to the cap.

[0228] <Example 8: Results of graft transplantation> Examples 6 and 7 showed that the gene expression of the cap and ring were equivalent. Furthermore, it was confirmed that if the ring is a neuroretina, then the cap is also a neuroretina. Therefore, we devised a method to confirm that the ring is a neuroretina by analyzing its gene expression before transplantation, and then transplant the cap corresponding to the ring. To demonstrate the usefulness of this method, we analyzed the gene expression of the ring, then transplanted the corresponding cap into retinal degenerative nude rats, and evaluated the engraftment after transplantation.

[0229] First, cell aggregates were prepared from human iPS cells (DSP-SQ strain) according to the method described in Example 1. Then, caps and rings were isolated from the cell aggregates 75 days or more after the start of suspension culture, according to the method described in Example 6. The isolated caps were stored in a commercially available preservation solution while genetic analysis of the rings was performed. Gene expression analysis of the isolated rings was performed using real-time PCR with Biomark HD (Fluidigm) according to the method described in Example 6. From the results of the gene expression analysis, rings that expressed the neuroretinal marker gene and did not express the byproduct marker gene were selected, and the caps corresponding to these rings were selected as grafts. The grafts were washed with buffer (Thermo Fisher Scientific) and then transplanted under the retina of retinal degenerative nude rats (photoreceptor degeneration model, SD-Foxn1 Tg(S334ter)3LavRrrc nude rat) using an injector described in the publicly available literature (Shirai et al. PNAS 113, E81-E90).

[0230] Eye tissue from mice aged 230-240 days after the start of suspension culture was fixed with paraformaldehyde (PFA) and sucrose. Frozen sections were prepared from the fixed eye tissue using a cryostat. These frozen sections were immunostained for human nuclei (anti-HuNu antibody, Millipore, mouse or anti-HNA antibody), Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers, and PKCα (anti-PKCα antibody, R&D Systems, goat), one of the bipolar cell markers.

[0231] Table 14 summarizes the results of graft quality evaluation based on gene expression analysis of the ring and the transplantation results. The method for calculating the ΔCt value was the same as the method described in Example 6. For gene expression analysis of the ring, the quality evaluation test (ring-PCR test) was considered successful if the ΔCt value of Recoverin, one of the neuroretinal marker genes, 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. For transplantation results, the engraftment was evaluated as good if human nucleus-positive and Recoverin-positive photoreceptor cells were detected under the retina. Furthermore, if the transplantation site did not become significantly thicker than the appropriate engraftment size, it was determined that no hypertrophy was detected.

[0232] A representative engraftment image is shown in Figure 10. The post-transplant engraftment image was evaluated for 14 eyes that passed the pre-transplant quality assessment test. In all 14 eyes, Recoverin-positive photoreceptor cells were detected, indicating good engraftment. Since these cells were HuNu-positive, it was determined that the Recoverin-positive photoreceptor cells originated from the transplanted cap. Furthermore, no hypertrophy was detected in any of the 14 eyes.

[0233] Based on these results, it was demonstrated that by examining the expression levels of neuroretinal and by-product marker genes in the ring before transplantation, it is possible to select grafts that will successfully engraft beneath the retina, i.e., those in which photoreceptor cells will engraft and hypertrophy will not occur.

[0234] [Table 14]

[0235] <Example 9: Graft transplantation results> In Example 8, Biomark HD (Fluidigm) was used to analyze the gene expression of the ring. Biomark HD is a machine originally designed for single-cell analysis, and its use in Example 8 is not typical. Therefore, we investigated whether it was possible to select grafts that would engraft well under the retina by analyzing the gene expression of the ring using a commonly used real-time PCR device, similar to the method used with Biomark HD.

[0236] Human iPS cells (DSP-L strain, established by Dainippon Sumitomo Pharma) were established using commercially available Sendai virus vectors (four factors: Oct3 / 4, Sox2, KLF4, and c-Myc, IDPharma's SiteTune Kit) based on the methods described in Thermo Fisher Scientific's public protocol (iPS 2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and Kyoto University's public protocol (Establishment and maintenance culture of human iPS cells in a feeder-free environment, CiRA_Ff-iPSC_protocol_JP_v140310, http: / / www.cira.kyoto-u.ac.jp / j / research / protocol.html), using StemFit medium (AK03; Ajinomoto Co., Inc.) and Laminin511-E8 (Nippi Co., Ltd.).

[0237] First, cell aggregates were prepared from human iPS cells (DSP-L strain) according to the method described in Example 1. Then, caps and rings were isolated from the cell aggregates 86 days after the start of suspension culture using the method described in Example 6. The isolated caps were stored in a commercially available preservation solution while the gene analysis of the rings was performed. Gene expression analysis of the isolated rings was performed as follows.

[0238] First, total RNA was extracted using the method described in Example 2. After measuring the concentration of total RNA with a measuring instrument (Nanodrop, Thermo Scientific), it was reverse transcribed into cDNA using reverse transcriptase (QuantiTect Revese Transcription Kit (QIAGEN)) and primer (RT primer mix, QIAGEN). Subsequently, the expression levels of marker genes for neuroretina and non-retinal byproducts were measured by real-time PCR using a StepOnePlus real-time PCR system (Applied Biosystems) with a real-time PCR enzyme (TaqMan Fast Advanced Master Mix, Applied Biosystems). The results are shown in Figure 11. A sample of cDNA from undifferentiated iPS cells and cDNA from 3D retina was used as a positive control sample. Calibration curve samples were prepared by serially diluting the positive control sample, and a calibration curve was drawn. Gene expression was normalized using gapdh as an internal standard.

[0239] PCR analysis revealed that Sample1 and Sample2 showed high expression levels of the neuroretinal markers Chx10, Rx, Crx, and Recoverin, and low expression levels of the by-product marker genes Oct3 / 4, FoxG1, Aqp1, Nkx2.1, Dlx6, CDH6, Emx2, Zic1, HoxA5, Pax2, and Pax8. Rings expressing the neuroretinal marker genes but not the by-product marker genes were selected, and caps corresponding to the rings were transplanted subretinally into retinal degenerate nude rats according to the method described in Example 8.

[0240] Eye tissue from mice aged 230-240 days after the start of suspension culture was fixed with paraformaldehyde (PFA) and sucrose. Frozen sections were prepared from the fixed eye tissue using a cryostat. These frozen sections were immunostained for human nuclei (anti-HuNu antibody, Millipore, mouse), Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers, and SCGN (anti-SCGN antibody, BioVendor, sheep), one of the bipolar cell markers.

[0241] Figure 11 shows the engraftment in rat eyes to which the cap corresponding to the aforementioned ring was transplanted. Evaluation of the engraftment after transplantation revealed Recoverin-positive photoreceptor cells, indicating good engraftment. Since these cells were HuNu-positive, the Recoverin-positive photoreceptor cells originated from the transplanted cap. Furthermore, no hypertrophy was observed.

[0242] The results above show that even when analyzing gene expression in the ring using a commonly used real-time PCR system, examining the expression levels of neuroretinal and by-product marker genes before transplantation allows for the selection of grafts that will successfully engraft beneath the retina. Therefore, it was found that multiple genes can be analyzed using a multi-sample real-time PCR system.

[0243] <Example 10: Results of ring transplantation>. In Examples 8 and 9, it was found that when the caps prepared by the above method were implanted under the retina of retinal degenerated nude rats, photoreceptor cells engrafted. To verify the equivalence of the rings and caps, we investigated implanting the rings under the retina in the same manner.

[0244] Crx::Venus knock-in human ES cells (derived from KhES-1; Nakano, T. et al. Cell Stem Cell 2012, 10(6), 771-785; obtained from Kyoto University, established and used at RIKEN CDB) were differentiated into retinal cells using the method described in Example 1. Subsequently, from the cell aggregates 74 days after the start of suspension culture, the neuroretina and the by-product caps and rings were excised using the method described in Example 6 (Figure 12). The rings were then transplanted subretinically into retinal degenerative nude rats (SD-Foxn1 Tg(S334ter)3LavRrrc nude rat), a model of photoreceptor degeneration, using a syringe as described in the publicly available literature (Shirai et al. PNAS 113, E81-E90).

[0245] Retinal degenerated nude rats were raised for one year after transplantation. Subsequently, the eye tissue to which the ring was transplanted was excised, fixed with paraformaldehyde (PFA), and replaced with sucrose. Frozen sections were prepared from the fixed eye tissue using a cryostat. These frozen sections were immunostained for Stem121 (anti-Stem121 antibody, Cellartis, mouse), a human cytoplasmic marker, or Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers.

[0246] Figure 12 shows bright-field images taken immediately after excising the ring used for transplantation, and immunostaining results of sections prepared from eye tissue excised from rats grown for one year after ring transplantation. When the ring shown in Figure 12 (left) was transplanted under the retina of a retinal degenerative nude rat, the immunostaining results shown in Figure 12 (right) were obtained. The immunostaining results showed that Stem121-positive human cells engrafted. It was also found that Recoverin-positive photoreceptor cells engrafted.

[0247] These results indicate that, like the cap, photoreceptor cells can engraft when transplanted into the ring. Furthermore, these results suggest that similar transplantation results can be obtained regardless of whether a cap or ring is transplanted into the neuroretina.

[0248] <Example 11: Verification of the equivalence of the cap and ring>. In Examples 6 and 7, the equivalent gene expression of the cap and ring at the RNA level was demonstrated using PCR. Furthermore, it was shown that analyzing the ring's gene expression using PCR could verify whether the cap was a neuroretinal byproduct. Next, we investigated whether analyzing the ring's gene expression using immunohistochemistry could verify whether the cap was a neuroretinal byproduct.

[0249] First, human iPS cells (DSP-SQ strain) were differentiated into retinal cells using the method described in Example 1. Subsequently, the neuroretinal caps and rings were isolated from cell aggregates 120 days after the start of suspension culture using the method described in Example 6. After washing the caps and rings, they were fixed with 4% paraformaldehyde (PFA fixation) and sucrose replacement. Frozen sections were prepared from the fixed caps and rings using a cryostat. These frozen sections were immunostained for DAPI, which stains the nucleus; Crx (anti-Crx antibody, Takara, rabbit), one of the markers for photoreceptor progenitor cells; Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the markers for neuroretinal cells; NRL (anti-NRL antibody, Bio-Techne, goat), one of the markers for rod photoreceptor progenitor cells; FOXG1 (anti-FOXG1 antibody, Takara Bio, rabbit), one of the telencephalon markers; PAX2 (anti-PAX2 antibody, Thermo Fisher Scientific, rabbit), one of the Optic Stalk markers; and NANOG (anti-NANOG antibody, Merck, mouse), one of the markers for undifferentiated pluripotent stem cells.

[0250] The results of immunohistochemical staining are shown in Figure 13. For caps and rings excised from the same cell aggregate, the immunohistochemical staining results for the rings are shown in the upper panel, and the results for the caps are shown in the lower panel. From these results, it was found that in both the caps and rings, Crx-positive photoreceptor progenitor cells, Chx10-positive neuroretina, and NRL-positive rod photoreceptor progenitor cells were expressed in a continuous, layered manner. Furthermore, in neither the caps nor the rings were telencephalon (FOXG1-positive), optic stalk (PAX2-positive), or pluripotent stem cells (NANOG-positive) detected. In addition, a comparison of the staining patterns for Crx, Chx10, and NRL revealed that these neuroretinal markers showed almost identical distributions in the caps and rings.

[0251] The results above demonstrate that the cap and ring are equivalent not only through gene expression analysis but also through immunohistochemistry. Furthermore, these results demonstrate that analyzing the gene expression of the ring using immunohistochemistry can verify whether the cap is a neuroretinal byproduct.

[0252] <Example 12: Verification of the equivalence of the cap and ring in the non-neuronal retina> In Examples 6 and 7, it was found that gene expression was equivalent in the caps and rings of the neuroretina and its by-products. Therefore, we decided to conduct a detailed analysis to determine whether gene expression was equivalent in caps and rings isolated from tissues other than the retina.

[0253] First, human iPS cells (QHJI-01-s04 strain) and human iPS cells (DSP-SQ strain) were differentiated into retina using the method described in Example 1. Subsequently, cell aggregates that were 80 days or older after the start of suspension culture were examined under a microscope, and the presence of epithelial structures was observed. The neuroretina and the caps and rings of the by-products were excised from the cell aggregates that were 80 days or older after the start of suspension culture using the method described in Example 6. Then, RNA was extracted using the method described in Example 6, and the expression levels of marker genes in the neuroretina and by-products other than retina were measured by real-time PCR using Biomark HD (Fluidigm).

[0254] The results are shown in Figure 14 as a heatmap. The heatmap was prepared according to the method described in Example 6. When gene expression was examined in the caps and rings, it was confirmed that neuroretinal marker genes were expressed in the caps and rings excised from the neuroretina, while marker genes from the byproducts were not expressed. On the other hand, when gene expression was examined in the caps and rings excised from the byproducts, caps and rings that highly expressed FOXG1, one of the telencephalon markers, caps and rings that highly expressed HOXB2 and HOXA5, spinal cord markers, caps and rings that highly expressed MITF, one of the RPE markers, and caps and rings that highly expressed PAX2, one of the Optic Stalk markers were found. Therefore, these byproducts were classified into telencephalon tissue, spinal cord tissue, RPE, and Stalk based on the highly expressed marker genes. When gene expression was examined in the caps and rings isolated from these byproducts, the caps and rings showed equivalent gene expression in all byproducts.

[0255] The results above indicate that gene expression is equivalent not only in the neuroretina but also in caps and rings isolated from telencephalon tissue, spinal cord tissue, RPE, and Optic Stalk. Furthermore, these results suggest that examining gene expression in rings isolated from non-neuronal retina such as telencephalon tissue, spinal cord tissue, RPE, and Optic Stalk can determine whether the caps are from the same tissue.

[0256] <Example 13: Percentage of photoreceptor progenitor cells and neuroretinal progenitor cells constituting the neuroretinal sheet for transplantation> The proportion of photoreceptor progenitor cells and neuroretinal progenitor cells among the constituent cells of a neuroretinal sheet for transplantation, which was prepared from cell aggregates differentiated from pluripotent stem cells, was analyzed and quantified using immunohistochemistry (IHC), a type of immunostaining method.

[0257] Human iPS cells (DSP-SQ strain) were differentiated into retina using the method described in 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 Example 6. Gene expression analysis of the isolated rings was performed using the method described in Example 8. Using the method described in Example 8, rings expressing neuroretinal marker genes and not expressing by-product marker genes were selected, and the caps corresponding to these rings were used as neuroretinal sheets for transplantation. In this way, one neuroretinal sheet for transplantation was prepared from the cell aggregate on day 84 after the start of suspension culture, two neuroretinal sheets for transplantation were prepared from the cell aggregate on day 92 after the start of suspension culture, and one neuroretinal sheet for transplantation was prepared from the cell aggregate on day 93 after the start of suspension culture. In other words, a total of four neuroretinal sheets for transplantation were prepared.

[0258] The obtained neuroretinal sheets for transplantation were cultured in medium B for 7 days for analysis. The cultured neuroretinal sheets were fixed with 4% paraformaldehyde, and frozen sections were prepared. The frozen sections were immunostained for Chx10 (anti-Chx10 antibody, Exalpha, sheep), one of the neuroretinal progenitor cell markers, and Crx (anti-Crx antibody, Takara, rabbit), one of the photoreceptor cell progenitor cell markers. Another frozen section was immunostained for Rx (anti-Rx antibody, Takara, guinea pig), one of the neuroretinal markers, and Recoverin (anti-Recoverin antibody, Proteintech, rabbit), one of the photoreceptor cell markers. Cell nuclei were stained with DAPI. These stained sections were observed using a fluorescence microscope (Keyence), and immunostained images were obtained. One example (D3) is shown in Figure 15.

[0259] Immunostaining images were analyzed using ImageJ (version 1.52a, NIH) to determine the number of DAPI-positive cells, DAPI-positive and Chx10-positive cells, and DAPI-positive and Crx-positive cells for each of the four neuroretinal sheets for transplantation. Similarly, immunostaining images were analyzed to determine the number of DAPI-positive cells and DAPI-positive and Rx-positive cells. From these values, the percentage of Chx10-positive cells, Crx-positive cells, and Rx-positive cells were calculated. The results are shown in Table 15. [Table 15]

[0260] From these results, it was found that the proportion of Chx10-positive cells in the neuroretinal sheets for transplantation was approximately 23-45%, the proportion of Crx-positive cells was approximately 30-56%, and the proportion of Rx-positive cells was approximately 40-54%.

[0261] In other words, it was suggested that the neuroretinal sheets for transplantation contained approximately 34% (23-45%) Chx10-positive neuroretinal progenitor cells, approximately 40% (30-56%) Crx-positive photoreceptor progenitor cells, and approximately 47% (40-54%) Rx-positive cells.

[0262] <Example 14: Percentage of photoreceptor progenitor cells and neuroretinal progenitor cells constituting the neuroretinal sheet for transplantation> The composition of the constituent cells of a neuroretinal sheet for transplantation, prepared from cell aggregates differentiated from various pluripotent stem cells, was investigated using flow cytometry (also known as FACS), a type of immunohistochemical staining method.

[0263] Human iPS cells (QHJI-01-s04 strain) were differentiated into retinal cells using the method described in Example 1. Subsequently, caps and rings were isolated from cell aggregates 88 days after the start of suspension culture using the method described in Example 6. The caps were then used as the neuroretinal sheets for transplantation. The neuroretinal sheets for transplantation were stored at 17°C for 2 days. Five of the obtained neuroretinal sheets were bundled together to form one sample, washed with PBS, and enzymatically treated with a neuronal cell dispersion (WAKO, containing papain) at 37°C for approximately 30 minutes. The cells were then dispersed into single cells by pipetting to obtain a single-cell suspension. The obtained single-cell suspension was fixed using a fixative (BD, CytoFix) to obtain a sample for FACS. The FACS sample was blocked and permeabilized (cell membrane puncture) using a serum-containing Perm / Wash solution (BD). Immunostaining was then performed using the following fluorescently labeled antibodies: anti-Chx10 antibody (Santa Cruz), anti-Pax6 antibody (BD), and anti-Crx antibody (Santa Cruz). The samples were then analyzed by flow cytometry using an analyzer (BD).

[0264] The results showed that the Chx10-positive and Pax6-positive fraction (neuroretinal progenitor cell fraction) accounted for 11.5%, the Chx10-positive and Pax6-negative fraction (progenitor cell fraction biased towards bipolar cells) accounted for 23.4%, the Chx10-negative and Pax6-positive fraction (ganglion cells and amacrine cells) accounted for 10.7%, and the Crx-positive cell fraction (photoreceptor cell progenitor cell fraction) accounted for 17.4%.

[0265] Furthermore, human iPS cells (DSP-SQ strain) were differentiated into retinal cells using the method described in Example 1. Subsequently, 11 cell aggregates were prepared 88 days after the start of suspension culture, and 11 caps and rings were isolated from each cell aggregate using the method described in Example 6. The 11 caps were combined to form one cap sample. Similarly, the 11 rings were combined to form one ring sample. The cap samples and ring samples were washed with PBS and enzymatically treated with a nerve cell dispersion (WAKO, containing papain) at 37°C for approximately 30 minutes to obtain single-cell suspensions of the caps and rings, respectively. The obtained single-cell suspensions of the caps and rings were fixed using a fixative (BD, CytoFix) to obtain samples for FACS. FACS samples were blocked and drilled using Perm / Wash solution (BD) containing serum, and 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. The samples were then analyzed 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 compared to the respective isotype controls. The results are shown in Table 16. [Table 16]

[0266] In the cap samples, 29.4% of cells were positive for Chx10, a neuroretinal progenitor cell marker; 21.7% were positive for Crx, a photoreceptor progenitor cell marker; and less than 1% were positive for SSEA-4, a pluripotent stem cell marker (untargeted cells). In the ring samples, 28.1% were positive for Chx10, a neuroretinal progenitor cell marker; 15.8% were positive for Crx, a photoreceptor progenitor cell marker; and less than 1% were positive for SSEA-4, a pluripotent stem cell marker (untargeted cells).

[0267] These results first revealed that the cap and ring samples were neuroretina containing Chx10-positive and Crx-positive cells, and substantially free of undifferentiated iPS cells. Furthermore, it was demonstrated that the proportion of Chx10-positive and Crx-positive cells in the cap sample was equivalent to that in the ring sample. Finally, if the ring sample was neuroretina, then the cap sample was also demonstrated to be neuroretina.

[0268] Furthermore, when these caps or rings (preferably caps) are used as a neuroretinal sheet for transplantation, it was found that approximately 30% (20-40%) of the cells in this neuroretinal sheet are Chx10-positive (neuroretinal progenitor cell fraction), and approximately 17% (10-30%) are Crx-positive cell fraction (photoreceptor progenitor cell fraction).

[0269] While the present invention has demonstrated the usefulness of quantitative PCR as a method for evaluating multiple samples and multiple genes as a quality assessment technique, it has now been found that quality assessment can also be performed using flow cytometry analysis, one of the immunohistochemical staining methods.

[0270] From these results, it was found that in cell aggregates containing epithelial tissue with an epithelial structure (preferably neuroepithelium, more preferably neuroretina), the quality of the cap can be ensured by isolating the cap and ring from a single epithelial structure and examining the gene expression of the ring. It was found that both quantitative PCR and immunohistochemistry methods are useful for examining gene expression. Furthermore, it was found that this quality inspection methodology can be applied whether the epithelial tissue with an epithelial structure is neuroretina or non-neuroretina. [Industrial applicability]

[0271] According to the present invention, it is possible to provide a method for evaluating the quality of a neuroretina for transplantation and a neuroretina sheet for transplantation selected by this method.

Claims

1. A method for evaluating the quality of neuroretina for transplantation, The aforementioned method, Extracting a portion or all of a cell aggregate containing an epithelial structure of the neuroretina, derived from pluripotent stem cells, as a quality evaluation sample, The expression of neuroretinal cell-related genes and non-neuroretinal cell-related genes in the aforementioned quality evaluation sample is detected, If the expression of the neuroretinal cell-related gene is observed, and the expression of the non-neuroretinal cell-related gene is not observed, (1) The neuroretina (neuroretinum for transplantation) in the same cell aggregate as the cell aggregate containing the sample for quality evaluation which is a part of the above (2) The neuroretina (neuroretinum for transplantation) in a cell aggregate of the same lot as the cell aggregate containing the quality evaluation sample which is a part of the above, or (3) The neuroretina (neuroretinum for transplantation) from the cell aggregates of the quality evaluation sample which is the whole of the above, and from the cell aggregates of the same lot as the above, This includes determining that it is suitable for use as a neuroretina for transplantation, The aforementioned non-neuronal retinal cell-related genes include telencephalon marker genes, spinal cord marker genes, ophthalmoplegic tissue marker genes, and undifferentiated pluripotent stem cell marker genes. The neuroretinal cell-related genes include one or more genes selected from the group consisting of Rax, Chx10, SIX3, SIX6, RCVRN, CRX, NRL, NESTIN, RXRG, NRL, and Blimp1. The telencephalon marker gene comprises one or more genes selected from the group consisting of FoxG1, Emx2, Dlx2, Dlx1, and Dlx5. The aforementioned spinal cord marker gene includes one or more genes selected from the group consisting of HOXD4, HOXD3, HOXD1, HOXC5, HOXA5, and HOXB2. The eye-related tissue marker gene comprises one or more genes selected from the group consisting of GREM1, GPR17, ACVR1C, CDH6, Pax2, Pax8, GAD2, SEMA5A, Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, AQP4, CRYAA, CRYBA1, MITF, TTR, and BEST1. The aforementioned undifferentiated pluripotent stem cell marker gene includes one or more genes selected from the group consisting of Oct3 / 4, Nanog, and Lin28. A method comprising the cell aggregate wherein the neuroretina for transplantation is a region near the center of the continuous epithelial tissue containing the neuroretina, the quality evaluation sample which is a part of the continuous epithelial tissue is a ring-shaped portion continuous with the neuroretina for transplantation, the neuroretina for transplantation has a major axis of 600 μm to 2500 μm, a minor axis of 200 μm to 1500 μm and a height of 100 μm to 1000 μm, and the quality evaluation sample has a height of 100 μm to 1000 μm.

2. The aforementioned non-neuronal retinal cell-related genes further include diencephalic / midbrain marker genes, The method according to claim 1, wherein the diencephalon / midbrain marker gene comprises one or more genes selected from the group consisting of OTX1, OTX2, DMBX1, Nkx2.1, OTP, FGFR2, EFNA5, and GAD1.

3. The method according to claim 1 or 2, wherein, when the expression of the neuroretinal cell-related genes is observed and the expression of the non-neuroretinal cell-related genes is not observed, it is determined that the neuroretina that was continuous with or adjacent to at least a portion of the cell aggregate containing the quality evaluation sample, which is a portion of the cell aggregate, is suitable for use as a neuroretina for transplantation.

4. The method according to claim 3, wherein the neuroretina for transplantation is contained in the same epithelial tissue as the quality evaluation sample.

5. The cell aggregate containing the neuroretina comprises a first epithelial tissue containing the neuroretina for transplantation, and a second epithelial tissue having a continuity of tangent slopes on its surface that is different from the continuity of tangent slopes on the surface of the first epithelial tissue, and containing non-neuroretinal cells. The transplantable neuroretina includes a region on the first epithelial tissue furthest from the second epithelial tissue, The method according to claim 3 or 4, wherein the quality evaluation sample is a portion present between the second epithelial tissue and the neuroretina for transplantation.

6. The method according to claim 5, wherein the second epithelial tissue is ocular tissue and / or cerebrospinal tissue.

7. The method according to claim 6, wherein the eye-related tissue includes retinal pigment epithelial cells and ciliary body.

8. The method according to any one of claims 1 to 7, wherein the detection of the expression of the neuroretinal cell-related gene and the non-neuroretinal cell-related gene is performed by quantitative PCR.

9. The method according to claim 8, which includes determining that the neuroretina is suitable for transplantation if it satisfies the following criteria 1 and 2. Criterion 1: The difference (ΔCt value) between the Threhold Cycle (Ct) value of the neuroretinal cell-related gene and the Ct value of the internal standard gene is 10 or less. Criterion 2: The difference (ΔCt value) between the Ct value of the non-neuronal retinal cell-related gene and the Ct value of the internal standard gene is 5 or greater.

10. The method according to claim 8 or 9, wherein the quantitative PCR is performed by a method comprising the following steps (1) to (5), thereby simultaneously detecting the expression levels of each neuroretinal cell-related gene and non-neuroretinal cell-related gene in two or more quality evaluation samples. (1) Prepare a sample well group consisting of 8 to 800 independent sample wells in one group, a primer well group consisting of 8 to 800 independent primer wells in one or more groups, and a channel plate having channels connecting the independent sample wells in the sample well group and the independent primer wells in each primer well group, a solution containing nucleic acids obtained from two or more of the quality evaluation samples (sample solution), and a solution containing one or more primers specific to one or more of the neuroretinal cell-related genes or the non-neuroretinal cell-related genes (primer solution). (2) In the sample well group, add the sample solution so that there is one sample solution per sample well for each quality evaluation sample. (3) Adding the primer solution to one or more primer wells in the group of one or more primer wells so that they form different groups of primer wells. (4) Mixing the primer separately with the nucleic acid via the channel. (5) Perform quantitative PCR using the mixture obtained in (4).

11. To extract quality evaluation samples from cell aggregates containing 2 to 800 cells, each containing a neuroretinal cell with an epithelial structure derived from pluripotent stem cells, which are a part of the said cell aggregate. The extracted 2 to 800 quality evaluation samples are evaluated by the method described in any one of claims 1 to 10, and the neuroretina determined to be suitable for use as a neuroretina for transplantation is selected, and To isolate the selected neuroretina for transplantation. A method for manufacturing a neuroretinal sheet containing [the specified material].

12. The cell aggregate is a cell aggregate obtained by differentiating pluripotent stem cells, comprising at least a first epithelial tissue and a second epithelial tissue, wherein the first epithelial tissue comprises human neuroretina, and the second epithelial tissue has a continuity of tangent slopes on its surface that is different from the continuity of tangent slopes on the surface of the first epithelial tissue, and comprises non-neuroretinal cells. The isolation of the neuroretina for transplantation involves isolating the neuroretina from the cell aggregate such that it includes the region on the first epithelial tissue that is furthest from the second epithelial tissue. The manufacturing method according to claim 11.

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