Therapeutic drug for disease accompanied by disorders in retinal system cells or retinal tissue

Abstract

To provide a therapeutic drug for a disease accompanied by a disorder in retinal cells or a retinal tissue, wherein the drug includes a retinal tissue being of an allogeneic origin and unlikely to cause rejection.SOLUTION: The present invention relates to a therapeutic drug for a disease accompanied by a disorder in retinal cells or a retinal tissue, the drug including a retinal tissue being of an allogeneic origin and having a three-dimensional structure, wherein an intended patient to be given the therapeutic drug is a patient affected by a disease accompanied by a disorder of retinal cells or a retinal tissue, to be given systemically, for 1 month or more after the administration of the therapeutic drug, no immunosuppressive agent aimed at preventing graft rejection.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a therapeutic agent for a disease associated with damage to retinal cells or tissue. The present invention also relates to a method, a retinal tissue and a kit for treating a disease associated with damage to retinal cells or tissue. [Background technology]

[0002] The intraocular space is considered to be an immune-privileged site where excessive immune responses are unlikely to occur in order to maintain vision, and is recognized as an organ where immune rejection reactions are unlikely to occur. It is believed that the function of the immune-privileged site is achieved by the blood-retinal barrier (BRB), which consists of the inner blood-retinal barrier (inner BRB) made up of retinal capillary endothelial cells and the outer blood-retinal barrier (outer BRB) made up of retinal pigment epithelial (RPE) cells, isolating the retina from the circulating blood, and by cytokines such as TGFβ secreted by RPE cells. On the other hand, when the blood-retinal barrier is damaged by retinal diseases, transplantation procedures, etc., it is believed that the function as an immune-privileged site is weakened and immune rejection reactions are likely to be induced, but the details are not known (Non-Patent Document 1).

[0003] Regarding retinal pigment epithelial cell transplants, it has been demonstrated in recent years that immune responses to retinal pigment epithelial cell grafts can be prevented without the use of immunosuppressants when the major histocompatibility complex antigens (MHC antigens, also known as human leukocyte antigens (HLA)) of the donor and recipient are compatible. However, it has also been reported that when the MHC antigens (HLA) of the donor and recipient are incompatible, an immune response is elicited by the retinal pigment epithelial cell graft, resulting in its elimination, even though the graft was transplanted into an immune-privileged site (Non-Patent Documents 2 and 3).

[0004] In addition, in transplantation of allogeneic retinal pigment epithelial cells with incompatible HLA into humans, immunosuppressants such as tacrolimus (Non-Patent Document 4) or prednisolone (Non-Patent Document 5) are administered systemically by oral administration (Non-Patent Documents 4 and 5). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] J. Wayne Streilein et al., Vision Research, Vol.42(2002),pp.487-495 [Non-Patent Document 2] Sunao Sugita et al., Stem Cell Reports, Vol.7, pp.635-648, October11, 2016 [Non-Patent Document 3] Sunao Sugita et al., Stem Cell Reports, Vol.7, pp.619-634, October11, 2016 [Non-Patent Document 4] Steven D Schwartz et al., LANCET Vol.385,Issue.9967, 7-13 February2015, pp.509-516 [Non-Patent Document 5] Lyndon da Cruz et al., Nature biotechnology, Vol.36, pp.328-337(2018) Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, it is believed that the administration of immunosuppressants is necessary even when transplanting retinal tissue derived from an allogeneic donor into a human. However, the systemic administration of immunosuppressants in particular can have serious side effects, such as increasing the risk of canceration and causing serious infections, and administration to elderly people and cancer patients with weakened immune systems is particularly problematic. In addition, long-term use of immunosuppressants increases the risk of the above-mentioned side effects and significantly reduces the patient's quality of life (QOL).

[0007] Therefore, an object of the present invention is to provide a therapeutic agent for a disease accompanied by damage to retinal cells or retinal tissue, including allogeneic retinal tissue, which is less likely to induce a rejection reaction. [Means for solving the problem]

[0008] The inventors have found that in recipients with MHC (HLA) mismatches with donors, retinal cells in a dispersed state that do not form aggregates induce immune responses, whereas retinal tissues having a three-dimensional structure that forms cell aggregates do not induce immune responses in recipients, but rather suppress immune activation. The present invention is based on this novel finding.

[0009] That is, the present invention relates to the following inventions. [1] A therapeutic agent for a disease accompanied by damage to retinal cells or retinal tissue, comprising allogeneic retinal tissue having a three-dimensional structure, characterized in that the patient to whom the therapeutic agent is administered is a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and is not systemically administered immunosuppressants other than steroidal anti-inflammatory agents and / or calcineurin inhibitors for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent. [2] The therapeutic agent according to [1], wherein the target patient is a patient who has not been systemically administered a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the prevention of transplant rejection after one month or more of administration of the therapeutic agent. [3] A therapeutic drug for a disease accompanied by damage to retinal cells or retinal tissue, comprising allogeneic retinal tissue having a three-dimensional structure, wherein the patient to whom the therapeutic drug is administered is a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and who has not been systemically administered an immunosuppressant for the purpose of preventing transplant rejection for a long period of time (e.g., for one month or more after administration of the therapeutic drug) before, during, and / or after administration of the therapeutic drug. [4] The therapeutic agent according to any one of [1] to [3], wherein the target patient is a patient who is not systemically administered an immunosuppressant for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent. [5] The therapeutic agent according to any one of [1] to [4], wherein the target patient is a patient who has not been locally administered an immunosuppressant other than a steroidal anti-inflammatory agent and a calcineurin inhibitor (preferably a steroidal anti-inflammatory agent) for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent. [6] The therapeutic agent according to any one of [1] to [5], wherein the target patient is a patient to whom one or more immunosuppressants selected from the group consisting of triamcinolone, fluocinolone, and calcineurin inhibitors are locally administered before, during, and / or after administration of the therapeutic agent for the purpose of preventing transplant rejection. [7] The therapeutic agent according to [5], wherein the target patient is a patient who has not been locally administered a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the prevention of transplant rejection after one month or more of administration of the therapeutic agent. [8] The therapeutic agent described in any of [1] to [3], wherein the target patient is a patient who is not administered an immunosuppressant for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent. [9] The therapeutic agent according to any one of [1] to [8], which does not substantially contain retinal pigment epithelial cells.

[10] The therapeutic agent described in any of [1] to [9], wherein TGFβ is present at least on the surface of the retinal tissue.

[11] The therapeutic agent according to any one of [1] to

[10] , wherein the diameter of the retinal tissue in the long axis direction is 0.2 mm or more.

[12] The total number of cells in each retinal tissue sample is 1 × 10 4 The therapeutic agent according to any one of [1] to

[11] , which is a cell or more.

[13] The therapeutic agent according to any one of [1] to

[12] , wherein the surface of the retinal tissue has a continuous epithelial structure in an area ratio of 50% or more.

[14] The therapeutic agent according to any one of [1] to

[13] , wherein 50% or more of the total number of cells contained in each retinal tissue are cells expressing at least one of PAX6, Chx10 and Crx.

[15] The therapeutic agent according to any one of [1] to

[14] , wherein the retinal tissue is derived from pluripotent stem cells. Effect of the Invention

[0010] According to the present invention, it is possible to provide a therapeutic agent for a disease accompanied by damage to retinal cells or tissue, including retinal tissue that is derived from another source but is unlikely to cause a rejection reaction due to its immunosuppressive effect, thereby making it possible to select a method that places a less burden on patients with regard to the concomitant use of immunosuppressants during transplantation, for example, a method that does not require systemic administration of immunosuppressants. [Brief description of the drawings]

[0011] [Figure 1] FIG. 2 shows micrographs of neural retina induced to differentiate from a human ES cell line (khES-1) and a human iPS cell line (TLHD2) in Example 1. [Figure 2A] 1 is a graph showing the results of HLA expression analysis of neural retina induced to differentiate from human ES cells in Example 2. [Figure 2B] 1 is a graph showing the results of HLA expression analysis of neural retina induced to differentiate from human iPS cells in Example 2. [Diagram 3] 13 is a photograph showing the observation results of HLA class I expression in neural retina induced to differentiate from human ES cells in Example 3. [Figure 4] 13 is a photograph showing the observation results of HLA class II expression in the neural retina induced to differentiate from human iPS cells in Example 3. [Figure 5A] 13 is a photograph showing the results of observing the expression of HLA class I and HLA class II after transplantation of neural retina whose differentiation was induced from human ES cells in Example 4. [Figure 5B] 13 is a photograph showing the results of hGFAP staining after transplantation of neural retina whose differentiation was induced from human ES cells in Example 4. [Figure 6] Photographs showing samples for evaluating the ability to suppress the activation of immune cells in Example 5. (1) shows the neural retina itself (whole 3D retina) differentiated from human ES cells, (2) shows a part of the neural retina (1) dissociated (semi dissociate), and (3) shows the neural retina (1) dispersed into single cells (single). [Figure 7A] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human ES cells in Example 5. [Figure 7B] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human ES cells in Example 5. [Figure 8] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human ES cells in Example 5. [Figure 9A] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human iPS cells in Example 5. [Figure 9B] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human iPS cells in Example 5. [Figure 10] FIG. 13 shows the results of an immunogenicity test against immune cells using neural retina induced to differentiate from human iPS cells in Example 5. [Figure 11A]1 shows the results of evaluating the ability of a neural retina induced to differentiate from human iPS cells to suppress activated immune cells in Examples 6 to 7. [Figure 11B] 1 shows the results of evaluating the ability of a neural retina induced to differentiate from human iPS cells to suppress activated immune cells in Examples 6 to 7. [Figure 12] 1 shows the results of evaluating the ability of a neural retina induced to differentiate from human iPS cells to suppress activated immune cells in Examples 6 to 7. [Figure 13] 1 shows the results of evaluating the ability of a neural retina induced to differentiate from human ES cells to suppress activated immune cells in Examples 6 to 7. [Figure 14] 1 shows the results of evaluating the ability of a neural retina induced to differentiate from human ES cells to suppress activated immune cells in Examples 6 to 7. [Figure 15] FIG. 13 shows the results of TGFβ expression analysis in Example 8. [Figure 16] 13 is a photograph showing the expression of TGFβ in the neural retina in Example 9. [Figure 17] FIG. 13 shows the results of measuring the amount of TGFβ2 secreted by ELISA in Example 10. [Figure 18] FIG. 13 shows the results of measuring the amount of TGFβ2 secreted in Example 11. [Figure 19] 1 shows photographs showing the localization of TGFβ2 in Example 11. [Figure 20A] FIG. 13 shows the results of an MLR test using each neural retina 50, 100, 160, and 200 days after the start of floating culture in Example 12. [Figure 20B] FIG. 13 shows the results of an MLR test using each neural retina 50, 100, 160, and 200 days after the start of floating culture in Example 12. [Figure 21] FIG. 13 shows the results of an MLR test using each neural retina 50, 100, 160, and 200 days after the start of floating culture in Example 12. [Figure 22]FIG. 13 shows the results of an immunogenicity test in Example 13. [Diagram 23] FIG. 13 shows the results of an immunogenicity test in Example 13. [Figure 24] FIG. 13 shows the results of microscopic observation of neural retina induced to differentiate from monkey-human iPS cells in Example 14. [Diagram 25] FIG. 15 shows the results of evaluating the immunogenicity of neural retina whose differentiation was induced from monkey iPS cells in Example 15. [Figure 26] 13 is a confocal microscope photograph showing the results of immunostaining of a section transplanted with a neural retina induced to differentiate from monkey iPS cells in Example 16. [Figure 27] 13 is a confocal microscope photograph showing the results of immunostaining of a section transplanted with a neural retina induced to differentiate from monkey iPS cells in Example 16. [Figure 28] FIG. 13 shows the results of evaluating the suppressive ability of activated immune cells in the neural retina whose differentiation was induced from monkey iPS cells in Example 17. [Figure 29] These are micrographs showing the results of immunostaining of neural retina hESC-NR (A-C) induced to differentiate from human ES cells on differentiation day 80, and neural retina hiPSC-NR (D-F) induced to differentiate from human iPS cells. A and D show the results of confirming the expression of CRX, Chx10, and Pax6, B and E show the results of confirming the expression of CRX, Rxr-γ, and Brn3, and C and F show the results of confirming the expression of Islet-1, Prox1, and Brn3. [Diagram 30] FIG. 1 shows the results of flow cytometry analysis of HLA class I, β2-microglobulorin, and HLA class II in hES-NR, hiPSC-NR, and hiPSC-RPE cells cultured for 2 days in the presence or absence of IFN-γ. [Diagram 31] FIG. 13 shows the results of flow cytometry analysis of HLA class I and HLA class II in hESC-NRs cultured for 2 days in the presence or absence of IFN-γ and Crx::Venus-positive fractions of hESC-NRs. [Diagram 32]FIG. 1 shows the results of flow cytometry analysis of β2-Microglobulorin, HLA-E, CD40, CD80, CD86, PD-L1, and PD-L2 in hESC-NR and hiPSC-NR cultured for 2 days in the presence or absence of IFN-γ. [Diagram 33] FIG. 13 shows the results of flow cytometry analysis of CD47 in hESC-NRs and hiPSC-NRs cultured for 2 days in the presence or absence of IFN-γ. [Diagram 34] 13 shows micrographs showing the results of immunostaining of Crx::Venus and HLA class I in hESC-NRs without INF-γ stimulation. [Diagram 35] 13 shows micrographs showing the results of immunostaining of Crx::Venus and HLA class I in hESC-NRs stimulated with INF-γ. [Diagram 36] FIG. 13 shows the results of immunostaining of Crx::Venus and HLA class I in hESC-NRs stimulated with or without INF-γ stimulation over the same period. [Figure 37] Graph showing HLA class I expression levels in neural retina (NR) or single cells (hESC) in the presence or absence of IFN-γ. [Figure 38] This is a graph showing the results of examining the immunosuppressive ability of hESC-NR, using the proliferation of peripheral blood mononuclear cells (PBMCs) activated with CD3 / 28 antibody as an indicator. [Figure 39] These are micrographs showing the results of observing the immunosuppressive ability of hESC-NR, using the proliferation and aggregation of peripheral blood mononuclear cells (PBMCs) activated with CD3 / 28 antibody as indicators. [Diagram 40] FIG. 1 shows the results of examining the dose dependency of the effect of hES-NR (upper panel) / hiPSC-NR (lower panel) in suppressing the activation of CD4-positive T cells and CD8-positive T cells. [Diagram 41] FIG. 13 shows the immunosuppressive pattern of hESC-NR when co-cultured separately using Transwell (registered trademark). [Diagram 42]1 is a graph showing the expression results of TGF-β1, TGF-β2, and TGF-β3 in hiPSC-NR, iPS cells, and iPS-RPE cells. [Diagram 43] 1 is a graph showing the dose-dependence of TGF-β2 expression by hiPSC-NRs. [Diagram 44] 1 is a graph showing the relationship between the number of days of differentiation and the expression level of TGF-β2. [Diagram 45] 13 is a micrograph showing the HLA expression pattern after transplantation in Example 19. [Figure 46] 13 is a micrograph showing the HLA expression pattern after transplantation in Example 19. [Figure 47] 1 is a graph showing the results of examining the immunogenicity of hESC-NR in peripheral blood mononuclear cells (PBMCs) in a state in which HLA-Class I expression was increased through INF-γ treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] [Therapeutic drug] The therapeutic agent of the present specification is a therapeutic agent for a disease accompanied by damage to retinal cells or retinal tissue, and includes retinal tissue of allogeneic origin and having a three-dimensional structure. The retinal tissue may be retinal tissue that is induced to differentiate from allogeneic stem cells and has a three-dimensional structure. The target patient to whom the therapeutic agent is administered may be a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and may be a patient who has not been systemically administered an immunosuppressant for the purpose of preventing transplant rejection for a long period of time (e.g., for one month or more after administration of the therapeutic agent) before, during, and / or after administration of the therapeutic agent. The target patient to whom the therapeutic agent is administered may be a patient who has not been systemically administered an immunosuppressant other than a steroidal anti-inflammatory agent and cyclosporine for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent.

[0013] Examples of diseases associated with damage to retinal cells or retinal tissue include ophthalmic diseases such as macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal disease, retinal detachment, central serous chorioretinopathy, cone dystrophy, cone-rod dystrophy, and macular hole. Diseases associated with damage to retinal cells or retinal tissue also include diseases associated with damage to retinal tissue. Examples of damaged states of retinal tissue include states in which photoreceptor cells are degenerated and dead.

[0014] (allogeneic stem cells) As used herein, "allogeneic" refers to a human other than the patient (recipient) to be treated, i.e., a different person (donor). "Allogeneic stem cells" are stem cells derived from a human other than the patient (recipient), i.e., stem cells derived from a different person (donor).

[0015] A "stem cell" is a cell that has both pluripotency (ability to differentiate into multiple types of cells) and self-renewal ability, and can proliferate. Examples of stem cells include pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) artificially produced by introducing reprogramming genes from cells derived from bone marrow, blood, and skin (epidermis, dermis, and subcutaneous tissue), as well as somatic stem cells that exist in bone marrow, fat, hair follicles, brain, nerves, liver, pancreas, kidney, muscle, and other tissues and differentiate into multiple specified types of cells. In this embodiment, the allogeneic stem cells to be induced to differentiate are preferably pluripotent stem cells.

[0016] The term "pluripotent stem cells" is not particularly limited as long as they have pluripotency capable of differentiating into multiple cells present in the living body, preferably all cells other than extraembryonic tissues such as the placenta, and also have proliferation ability. Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, and the like. Examples of pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and spermatogonial stem cells (GS cells) produced from germ cells (e.g., testes) are also included in pluripotent stem cells. Embryonic stem cells were first established in 1981, and have been used to produce knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF (leukemia inhibitory factor). Methods for producing embryonic stem cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from a given institution, 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. Mouse embryonic stem cells EB5 cell line and D3 cell line are available from the National Institute of Physical and Chemical Research and Development and ATCC, respectively.

[0017] Nuclear transfer embryonic stem cells (ntES cells), a type of embryonic stem cell, can be established from a cloned embryo created by transplanting the nucleus of a somatic cell into an egg from which the nucleus has been removed.

[0018] EG cells can be prepared by culturing primordial germ cells in a medium containing mSCF, LIF and bFGF (Cell, 70:841-847, 1992).

[0019] "Artificial pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells by known methods. Specifically, differentiated somatic cells such as fibroblasts or peripheral blood mononuclear cells are reprogrammed by expressing any combination of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc., to induce pluripotency. Preferred combinations of reprogramming factors include (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), or (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466).

[0020] In 2006, Yamanaka et al. established induced pluripotent stem cells in mouse cells (Cell, 2006, 126(4), pp.663-676). In 2007, induced pluripotent stem cells were also established in human fibroblast cells, and they have the same pluripotency and self-renewal ability as embryonic stem cells (Cell, 2007, 131(5), pp.861-872; Science, 2007, 318(5858), pp.1917-1920; Nat. Biotechnol., 2008, 26(1), pp.101-106).

[0021] In addition to the method of producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be produced by a method of inducing induced pluripotent stem cells from somatic cells by adding chemical compounds, etc. (Science, 2013, 341, pp. 651-654).

[0022] Artificial pluripotent stem cells are available as established artificial pluripotent stem cells. For example, human artificial pluripotent stem 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. As established artificial pluripotent stem cells, for example, Ff-I01 cells, Ff-I01s04 cells, QHJ-I01, and Ff-I14 cells established at Kyoto University are available from Kyoto University. In addition, for example, the TLHD2 line established at RIKEN is available from RIKEN.

[0023] Somatic cells used in producing induced pluripotent stem cells include, but are not limited to, tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells (PBMCs), umbilical cord blood mononuclear cells, T cells, etc.), hepatic cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc.

[0024] When producing artificial pluripotent stem cells, in the case of initializing by the expression of several kinds of genes, the means for expressing genes is not particularly limited.The above means include infection method using virus vector (e.g., retrovirus vector, lentivirus vector, Sendai virus vector, adenovirus vector, or adeno-associated virus vector), gene transfer method using plasmid vector (e.g., plasmid vector, or episomal vector) (e.g., calcium phosphate method, lipofection method, retronectin method, or electroporation method), gene transfer method using RNA vector (e.g., calcium phosphate method, lipofection method, or electroporation method), direct protein injection method (e.g., needle method, lipofection method, or electroporation method), etc.

[0025] Artificial pluripotent stem cells can be produced in the presence or absence of feeder cells (feeder-free). When producing artificial pluripotent stem cells in the absence of feeder cells, artificial pluripotent stem cells can be produced in the presence of undifferentiated maintenance factors by a known method. The medium used for producing artificial pluripotent stem cells in the absence of feeder cells is not particularly limited, but a known maintenance medium for embryonic stem cells and / or artificial pluripotent stem cells, or a medium for establishing artificial pluripotent stem cells in a feeder-free manner can be used. Examples of media for establishing artificial pluripotent stem cells in a feeder-free manner include feeder-free media such as Essential 8 medium (E8 medium), Essential 6 medium, TeSR medium, mTeSR medium, mTeSR-E8 medium, Stabilized Essential 8 medium, and StemFit medium. When producing induced pluripotent stem cells, for example, induced pluripotent stem cells can be produced by introducing the four factors Oct3 / 4, Sox2, Klf4, and Myc into somatic cells in a feeder-free manner using a Sendai virus vector.

[0026] The pluripotent stem cells may be human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0027] Allogeneic stem cells may have the same or different HLA genotype as the recipient. There are many HLA loci (HLA-A, B, C, DR, etc.) in the major histocompatibility complex (MHC) region on the short arm of chromosome 6, and each has multiple (several dozen) HLA types (alleles). HLA is a membrane-bound glycoprotein that is involved in self-non-self recognition by presenting antigens to immune cells such as T cells. HLA class 1 and HLA class 2 are mainly involved in self-non-self recognition. HLA class 1 (MHC class 1) is mainly recognized by killer T cells, and HLA class 2 (MHC class 2) is mainly recognized by helper T cells. Antigens presented by HLA class 1 (MHC class 1) are peptide fragments that are formed by decomposing proteins synthesized within cells into small peptide fragments by proteasomes. On the other hand, antigens presented by MHC class 2 are peptide fragments that are formed by decomposing foreign proteins taken up by endocytosis into small peptide fragments by lysosomes.

[0028] (retinal tissue) "Retinal tissue" refers to tissue in which one or more types of retinal cells that make up each retinal layer in a living retina exist according to a certain order, and "neural retina" refers to retinal tissue that includes the inner neural retinal layer, which does not include the retinal pigment epithelium layer, among the retinal layers described below.

[0029] "Retinal cells" refers to cells that constitute each retinal layer in a living retina or their precursor cells. Retinal cells include, but are not limited to, photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelial (RPE) cells, ciliary marginal cells, their precursor cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, etc.), retinal precursor cells, etc. Among the retinal cells, specific examples of cells that make up the neural retinal layer include photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneuron cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, and their precursor cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, etc.).

[0030] The above-mentioned retinal cells can be detected or identified using markers expressed in each cell as indicators. Examples of markers expressed in cells include Rx (also called Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6 and Chx10 expressed in neural retinal progenitor cells, and Crx and Blimp1 expressed in photoreceptor progenitor cells. Other markers 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 precursor cells; Rhodopsin, which is expressed in rod cells; Nrl, which is expressed in rod photoreceptor cells and rod photoreceptor precursor cells; and Rhodopsin, which is expressed in rod photoreceptor cells and rod photoreceptor precursor cells. These include S-opsin and LM-opsin, which are expressed in cone cells, cone photoreceptor precursor cells, and ganglion cells; RXR-γ, which is expressed in cone cells, cone photoreceptor precursor cells, and ganglion cells; TRβ2, OTX2, and OC2, which are expressed in cone photoreceptors or their precursor cells that appear early in the differentiation process of cone photoreceptors; Pax6, which is expressed in horizontal cells, amacrine cells, and ganglion cells; RPE65 and Mitf, which are expressed in retinal pigment epithelial cells; CRALBP, which is expressed in Muller cells; and GS, which is expressed in Muller glial cells.

[0031] "Retinal layer" means each layer that constitutes the retina, and specific examples include the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.

[0032] "Neural retinal layer" refers to each layer constituting the neural retina, specifically, the photoreceptor layer, the outer limiting membrane, the outer nuclear layer, the outer plexiform layer, the inner nuclear layer, the inner plexiform layer, the ganglion cell layer, the nerve fiber layer and the inner limiting membrane. "Photoreceptor layer" refers to a type of retinal layer (neural retinal layer), a retinal layer formed on the outermost side of the neural retina, containing many photoreceptors (rod photoreceptors, cone photoreceptors), photoreceptor precursor cells and retinal precursor cells. It can be confirmed which retinal layer each cell constitutes by a known method, for example, the presence or absence of expression or the degree of expression of a cell marker.

[0033] The retinal tissue in the therapeutic agent of this embodiment has a three-dimensional structure, and therefore, although it is of allogeneic origin (for example, differentiated from human iPS cells, human ES cells, or allogeneic stem cells), there is no need for systemic administration of immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent.

[0034] The retinal tissue in the therapeutic agent of this embodiment has a three-dimensional structure. "Having a three-dimensional structure" means that a certain number or more of retinal cells form an aggregate, the retinal cells in the aggregate form a retinal layer, the retinal cells exist in a layer in the retinal layer, and the retinal tissue has an epithelial structure in which the polarity of the apical surface and the basal surface is maintained at least in a certain range of the tissue. In other words, "the retinal tissue has a three-dimensional structure" means that the retinal tissue is a cell aggregate (also called an organoid) containing retinal cells. Zo-1, β-catenin, atypical PKC, etc. are expressed on the apical surface. Collagen, laminin, etc. are expressed on the basal membrane.

[0035] In one embodiment, the retinal tissue is a spherical cell aggregate. The term "spherical cell aggregate" refers to a cell aggregate having a three-dimensional shape close to a sphere. The three-dimensional shape close to a sphere is a shape having a three-dimensional structure, and includes, for example, a spherical shape that shows a circular or elliptical shape when projected onto a two-dimensional surface, and a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by two to four overlapping circular or elliptical shapes when projected onto a two-dimensional surface (e.g., a cloverleaf shape)). In one embodiment, the spherical cell aggregate has a vesicular structure, and is characterized in that the center is dark and the outer edge is bright under a bright-field microscope.

[0036] The retinal tissue in this specification may not substantially contain retinal pigment epithelial (RPE) cells. The term "retinal pigment epithelial cells" refers to epithelial cells present outside the neural retina in a living retina. Those skilled in the art can easily confirm whether a cell is a retinal pigment epithelial cell by, for example, the expression of cell markers (RPE65, Mitf, CRALBP, MERTK, BEST1, etc.), the presence of melanin granules (black-brown color), tight junctions between cells, and / or characteristic polygonal or paving-stone cellular morphology. Whether a cell has the function of a retinal pigment epithelial cell can be easily confirmed by the secretion ability of cytokines such as VEGF and PEDF. In one embodiment, the retinal pigment epithelial cells are RPE65-positive cells, Mitf-positive cells, or RPE65-positive and Mitf-positive cells. That is, the retinal tissue may not substantially contain RPE65-positive cells, Mitf-positive cells, or RPE65-positive and Mitf-positive cells.

[0037] As used herein, "substantially free of retinal pigment epithelial cells" means that the cells do not stain with the above-mentioned retinal pigment epithelial cell markers (e.g., RPE65 and / or Mitf), or that the cells that stain with such markers account for 5% or less of the total cells (preferably, 4% or less, 3% or less, 2% or less, or 1% or less).

[0038] In another embodiment of the above-mentioned "sphere-shaped cell aggregate", a part of the aggregate may contain retinal pigment epithelial cells and / or a structure for the ciliary body peripheral part. In one embodiment, a part of the continuous interface (constituted by the neural retina) formed by the cell aggregate with respect to the external environment is constituted by retinal pigment epithelial cells, and a structure for the ciliary body peripheral part is present in the boundary region between the neural retina and the retinal pigment epithelial cells. That is, the retinal pigment epithelium and the neural retina are adjacent to the ciliary body peripheral part-like structure along its periphery within the same cell aggregate. As an example, there is a cell aggregate disclosed in WO2013 / 183774 (for example, FIG. 12A in WO2013 / 183774). In the case of retinal tissue containing retinal pigment epithelial cells and a structure for the ciliary body peripheral part, a cell aggregate (retinal tissue) obtained by cutting out a region not containing retinal pigment epithelial cells and a structure for the ciliary body peripheral part by the method described below can be used as a therapeutic agent.

[0039] The ciliary marginal zone-like structure is a structure similar to the ciliary marginal zone. The "ciliary marginal zone (CMZ)" can be, for example, a tissue that exists in the boundary region between the neural retina and the retinal pigment epithelium in a living retina, and can be a region that contains retinal tissue stem cells (retinal stem cells). The ciliary marginal zone is also called the ciliary margin or the retinal margin, and the ciliary marginal zone, the ciliary marginal zone, and the retinal margin are equivalent tissues. It is known that the ciliary marginal zone plays an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, and the like. Marker genes for the ciliary marginal zone can be, for example, the Rdh10 gene (positive), the Otx1 gene (positive), and Zic1 (positive).

[0040] The diameter of the retinal tissue in the long axis direction may be 0.1 mm or more, or 0.2 mm or more (preferably 0.3 mm or more, 0.5 mm or more, 0.8 mm or more, 1.0 mm or more, 1.2 mm or more, 1.4 mm or more, 1.6 mm or more, 1.8 mm or more, or 2.0 mm or more). There is no particular upper limit to the diameter of the retinal tissue in the long axis direction, but it may be, for example, 5.0 mm or less or 10 mm or less. The diameter of the retinal tissue in the long axis direction may be 0.1 mm or more to 10 mm or less, 0.2 mm or more to 10 mm or less, 0.3 mm or more to 10 mm or less, 0.5 mm or more to 10 mm or less, 0.8 mm or more to 10 mm or less, 1.0 mm or more to 10 mm or less, 1.2 mm or more to 5.0 mm or less, 1.4 mm or more to 5.0 mm or less, 1.6 mm or more to 5.0 mm or less, 1.8 mm or more to 5.0 mm or less, or 2.0 mm or more to 5.0 mm or less. In one embodiment of the invention, the long axis diameter of the retinal tissue may be about 1.2, 1.5, or 2 times the short axis diameter. In another embodiment, the long axis diameter and the short axis diameter of the retinal tissue may be approximately equal.

[0041] The diameter of the retinal tissue in the short axis direction may be 0.05 mm or more (preferably 0.1 mm or more, 0.2 mm or more, 0.4 mm or more, 0.6 mm or more, 0.8 mm or more, or 1.0 mm or more). There is no particular upper limit to the diameter of the retinal tissue in the short axis direction, but it may be, for example, 2.5 mm or less or 5 mm or less. The diameter of the retinal tissue in the short axis direction may be 0.05 mm or more and 5 mm or less, 0.1 mm or more and 5 mm or less, 0.2 mm or more and 5 mm or less, 0.4 mm or more and 5 mm or less, 0.6 mm or more and 2.5 mm or less, 0.8 mm or more and 2.5 mm or less, or 1.0 mm or more and 2.5 mm or less.

[0042] Here, the diameter in the long axis direction or the short axis direction of the retinal tissue means, for example, the length of the longest line (diameter in the long axis direction) or the length of the shortest line (diameter in the short axis direction) among the lines connecting any two points on the outer periphery (contour, surface) of the apical side of the retinal tissue when measured based on an image taken using a stereomicroscope. In addition, in an aggregate containing retinal tissue, there are cases where multiple retinal tissues are present overlapping each other (e.g., cloverleaf type). In this case, the diameter in the long axis direction or the diameter in the short axis direction of the retinal tissue means the diameter in the long axis direction or the short axis direction of each retinal tissue in the aggregate, and it is sufficient that the diameter in the long axis direction or the short axis direction of at least one retinal tissue is within the above-mentioned range. In the case of cloverleaf-shaped retinal tissue, specifically, the periphery (contour, surface, apical surface) of the retinal tissue is regarded as a circle or ellipse, and the length of the longest or shortest straight line connecting any two points on the periphery separated by the point where two circles or ellipses overlap in terms of shape (more specifically, when continuous positional information of the periphery of an aggregate containing retinal tissue is hypothetically determined, the point where the continuity of the curve obtained by plotting the positional information on the horizontal axis and the slope of the tangent at that position on the vertical axis is lost) is measured. Note that whether or not multiple retinal tissues are included can be easily determined by a person skilled in the art through observation under a microscope, etc.

[0043] The thickness of the epithelial structure of the retinal tissue may be 0.05 mm or more (preferably 0.1 mm or more, 0.12 mm or more, 0.15 mm or more, 0.2 mm or more, 0.25 mm or more, or 0.3 mm or more). There is no particular upper limit to the thickness of the epithelial structure of the retinal tissue, but it may be, for example, 1 mm or less (for example, 0.9 mm or less, 0.8 mm or less, 0.7 mm or less, 0.6 mm or less, or 0.5 mm or less). The thickness of the epithelial structure of the retinal tissue may be, for example, 0.05 mm or more and 1 mm or less, 0.1 mm or more and 0.9 mm or less, 0.12 mm or more and 0.8 mm or less, 0.15 mm or more and 0.7 mm or less, 0.2 mm or more and 0.6 mm or less, 0.25 mm or more and 0.5 mm or less, or 0.3 mm or more and 0.5 mm or less. Here, the thickness of the epithelial structure of the retinal tissue means the thickness from the apical surface of the retinal tissue to the basement membrane.

[0044] The total number of cells in one retinal tissue depends on the size of the retinal tissue. The total number of cells in one retinal tissue is 1 × 10 4 More than cells, 1 x 10 5 Cells or more, 5 x 10 5 Cells or more than 1×10 6 For example, in a retinal tissue having a long axis diameter of 0.5 mm, the total number of cells in the retinal tissue is 1×10 4 cells or more, preferably 1×10 5 Cells or more, 5 x 10 5 Cells or more than 1×10 6 In addition, the outermost cells on the apical surface of the photoreceptor layer (photoreceptor precursor cells, photoreceptor cells, etc.) are continuously arranged at 10 cells or more, 15 cells or more, or 20 cells or more per 0.1 mm.

[0045] In one embodiment, the retinal tissue has a long axis diameter of 0.5 mm or more, an epithelial structure thickness of 0.2 mm or more, and a cell count of 1×10 5 In one embodiment, the retinal tissue may be 1×10 5 cells / mm 3 More than 5×10 5 cells / mm 3 or more, or 1×10 6 cells / mm 3 Contains cells at a density of 100 μg / ml or more.

[0046] The retinal tissue may be a retinal tissue having a continuous epithelial structure. The retinal tissue having a continuous epithelial structure may be, for example, a retinal tissue in which photoreceptors or their precursor cells are continuously present in at least 50% (preferably 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more) of the area ratio of the outermost layer on the surface, i.e., the apical surface, of the retinal tissue. The retinal tissue having a continuous epithelial structure may include, for example, a retinal tissue in which the area of ​​the apical surface present on the surface of the retinal tissue is at least 50% (preferably 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more) of the area of ​​the surface of the retinal tissue.

[0047] In the present specification, the term "continuous epithelial structure" in retinal tissue refers to a structure in which the retinal tissue has an apical surface specific to epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to at least the photoreceptor layer (external nuclear layer) or the neuroblastic layer among the layers forming the neural retinal layer, and is continuous. That is, the continuous epithelial structure maintains the orientation of the apical surface and the basement membrane, and does not have a structure in which the apical surface is divided as observed in rosette-like structures. For example, in the case of a cell aggregate containing retinal tissue prepared from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and 10 or more cells, preferably 30 or more cells, more preferably 100 or more cells, and even more preferably 400 or more cells of photoreceptor cells or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface. The number of continuously arranged photoreceptor cells or photoreceptor precursor cells correlates with the size of the neural retinal tissue contained in the cell aggregate. As used herein, the tangential direction to the epithelial tissue refers to the direction in which the cells that form the apical surface of the epithelial tissue are aligned in a certain direction, and refers to the parallel or lateral direction to the epithelial tissue (or epithelial sheet).

[0048] In one aspect, an apical surface is formed on the surface of the neural retinal tissue, along which photoreceptor cells or photoreceptor precursor cells are arranged in an orderly and continuous manner. In the case of retinal tissue at a stage where the appearance ratio of photoreceptors or photoreceptor precursor cells is low (e.g., retinal tissue at an early stage of development), it is known to those skilled in the art that the layer containing the proliferating neural retinal precursor cells is called the "neuroblastic layer". In addition, in addition to photoreceptors or photoreceptor precursor cells, the surface of the retinal tissue at such a stage may contain neural retinal precursor cells that have polarity and can form an apical surface, or cells that divide and proliferate from neural retinal precursor cells, and / or cells at a stage where neural retinal precursor cells differentiate into cells that constitute the neural retina. For example, by continuing to culture the retinal tissue in such a state under conditions that maintain the "continuous epithelial structure", a retinal tissue in which photoreceptors or photoreceptor precursor cells are regularly and continuously arranged along the apical surface formed on the surface of the neural retinal tissue can be obtained.

[0049] In one embodiment, the area of ​​the apical surface present on the surface of the retinal tissue may be, on average, 30% or more of the area of ​​the surface of the retinal tissue, preferably 50% or more, more preferably 60% or more, more preferably 70% or more, more preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more. The proportion of the area of ​​the apical surface present on the surface of the retinal tissue can be measured by staining with an apical surface marker, as described below.

[0050] In this specification, the term "rosette-like structure" in retinal tissue refers to a structure in which cells are arranged radially or spirally around a central lumen. In retinal tissue in which a rosette-like structure is formed, the apical surface and photoreceptor cells or photoreceptor precursor cells are present along the central portion (lumen), and the apical surface is divided into individual rosette-like structures.

[0051] In the present specification, the term "apical surface" refers to a surface (superficial surface) formed in epithelial tissue on the side opposite to the basement membrane side where a layer (basal membrane) of 50-100 nm produced by epithelial cells is present, which is rich in mucopolysaccharides (positive for PAS staining) and contains a large amount of laminin and type IV collagen. In one aspect, in retinal tissue whose developmental stage has progressed to the point where photoreceptors or photoreceptor precursor cells can be found, the term refers to a surface in contact with the photoreceptor layer (external nuclear layer) where photoreceptors and photoreceptor precursor cells are present, where an outer limiting membrane is formed. In addition, such an apical surface can be identified by immunostaining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical-PKC (hereinafter abbreviated as aPKC), E-cadherin, N-cadherin, Zo-1, beta-catenin, Ezrin). Even in the early stages of development, when photoreceptors or photoreceptor precursor cells have not yet appeared, or when photoreceptors or photoreceptor precursor cells have not yet appeared in sufficient numbers to sufficiently cover the surface of the retinal tissue, the epithelial tissue is polarized and expresses the above-mentioned apical markers on its apical surface.

[0052] Whether or not the retinal tissue has a continuous epithelial structure can be confirmed by the continuity of the apical surface of the retinal tissue (i.e., an undivided morphology). The continuity of the apical surface can be determined, for example, by immunostaining markers of the apical surface (e.g., aPKC, E-cadherin, N-cadherin, Zo-1, beta-catenin, Ezrin) and markers of photoreceptors or photoreceptor precursor cells located on the apical surface side (e.g., Crx or recoverin), and analyzing the positional relationship between the apical surface and the photoreceptor layer and each retinal layer for the acquired images. Retinal layers other than the apical surface and the photoreceptor layer (outer nuclear layer) can be identified by DAPI staining, PI staining, Hoechst staining, which stains cell nuclei, or immunostaining with marker proteins localized in cell nuclei (Rx, Chx10, Ki67, Crx, etc.).

[0053] Whether or not rosette-like structures have formed can be determined by fixing the cell aggregates with 4% paraformaldehyde, preparing frozen sections, and observing the formation of rosette-like structures (e.g., a disrupted apical surface or the intrusion of the apical surface into the cell aggregate) using immunostaining, which is typically performed using antibodies against the apical surface markers aPKC, E-cadherin, and N-cadherin, or DAPI, which specifically stains nuclei.

[0054] In one embodiment, the ratio of retinal progenitor cells, neural retinal progenitor cells, and photoreceptor progenitor cells in the retinal tissue, i.e., the ratio of cells expressing PAX6, Chx10, and / or Crx, may be 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the total number of cells. 50% or more of the total number of cells contained in one retinal tissue may be cells expressing at least one of PAX6, Chx10, and Crx.

[0055] In one embodiment, the ratio of cells expressing PAX6, Chx10 and / or Crx present in the outermost layer (photoreceptor layer or neuroblastic layer) on the apical side of the retinal tissue of the present specification may be 80% or more of the total number of cells present in the outermost layer on the apical side, preferably 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more. In another embodiment, the ratio of photoreceptor precursor cells (Crx positive and rhodopsin, S-Opsin and M / L-Opsin negative cells) and photoreceptors (Recoverin positive cells, or cells positive for any of rhodopsin, S-Opsin and M / L-Opsin) present in the outermost layer (photoreceptor layer or neuroblastic layer) on the apical side of the retinal tissue of the present specification may be 80% or more of the total number of cells present in the outermost layer on the apical side, preferably 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[0056] In one embodiment, the retinal tissue may include photoreceptor cells. "Photoreceptor cells" are present in the photoreceptor layer of the retina and have the role of absorbing light stimuli and converting them into electrical signals. There are two types of photoreceptor cells: cones that function in the light and rods that function in the dark. Photoreceptor cells differentiate and mature from photoreceptor precursor cells. Those skilled in the art can easily confirm whether a cell is a photoreceptor cell or a photoreceptor precursor cell by, for example, the expression of cell markers (Crx and Blimp1 expressed in photoreceptor precursor cells, recoverin expressed in photoreceptor cells, rhodopsin, S-Opsin, M / L-Opsin, etc. expressed in mature photoreceptor cells), the formation of an outer segment structure, etc., as described below.

[0057] In one aspect, the retinal tissue of the present specification may include photoreceptor precursor cells (cells that are Crx positive and rhodopsin, S-Opsin, and M / L-Opsin negative) and photoreceptors (Recoverin positive cells, or cells that are positive for any of rhodopsin, S-Opsin, and M / L-Opsin).

[0058] In one embodiment, the percentage of cells expressing Recoverin in the retinal tissue of the present specification may be 5% or more of the total number of cells, preferably 10% or more, 20% or more, 40% or more, or 50% or more. In one embodiment, the percentage of cells expressing Recoverin in the retinal tissue of the present specification may be 80% or more of the total number of cells present in the photoreceptor layer, preferably 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more.

[0059] The retinal tissue may be characterized by expressing TGF (transforming growth factor) β. TGF β is one of the cytokines that controls cell proliferation and differentiation and maintains homeostasis in the living body. Five subtypes (β1 to β5) of TGF β are known to exist, and three subtypes (β1 to β3) are known in mammals. The expression of these is expected to have an immunosuppressive effect. Of these, the retinal tissue may preferably express TGF β2. TGF β2 is expressed in immune-privileged sites such as the testis. The expression of TGF β2 is expected to have an immunosuppressive effect.

[0060] It is preferable that TGFβ is present on the surface of the retinal tissue, i.e., in cells present on the apical surface and / or in the outermost layer on the apical surface side. The presence of TGFβ on the surface of the retinal tissue refers to a state in which TGFβ is expressed and secreted in cells present on the outermost layer on the apical surface side, and / or TGFβ is present on the apical surface, i.e., TGFβ secreted from the above cells is bound to a matrix (such as N-cadherin) present on the apical surface. More specifically, in a retinal tissue having a continuous epithelial structure and maintaining the directionality of the apical surface and the basement membrane, it is preferable that TGFβ is expressed and secreted in the outermost cells present on the apical surface side (preferably cells present about 1, 2 or 3 cells inside from the outside), and TGFβ is present on the apical surface. Preferably, the proportion of TGFβ present on the apical surface may be 50% or more of the total area of ​​the apical surface in terms of area ratio, and more preferably 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In addition, preferably, TGFβ is expressed in 50% or more of the outermost cells present on the apical surface, and more preferably, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0061] The expression level of TGFβ can be measured by a person skilled in the art using a well-known technique. Specifically, the expression level of TGFβ in the culture medium after culture may be measured by ELISA or the like. In one embodiment, the expression level of TGFβ may be 10 times or more, preferably 50 times or more, 100 times or more, 200 times or more, or 500 times or more, relative to the pluripotent stem cells used as the raw material. In another embodiment, the expression level of TGFβ in one retinal tissue may be 1 pg / mL or more, preferably 2 pg / mL or more, 5 pg / mL or more, or about 10 pg / mL or more, when one retinal tissue is cultured in 1 mL of culture medium for two days. In addition, the expression site of TGFβ can be measured by a person skilled in the art using a well-known technique. Specifically, the retinal tissue may be immunostained using an anti-TGFβ antibody. If necessary, the presence or absence and the proportion of TGFβ on the apical surface can be confirmed by co-staining with the above-mentioned apical surface marker.

[0062] The retinal tissue, which is an active ingredient of the therapeutic agent in this specification, also includes cell aggregates obtained by partially dissociating or partially cutting out retinal tissue induced to differentiate from stem cells. The cell aggregates obtained by partially dissociating or partially cutting out may be, for example, about 1 / 4, 1 / 8, 1 / 16, 1 / 32, 1 / 64, or 1 / 128 of the size (based on the number of cells) of the retinal tissue induced to differentiate from stem cells. The dissociation method may be an enzyme or physical cutting using scissors or a knife.

[0063] That is, in one embodiment, the retinal tissue having a three-dimensional structure that is the active ingredient of the therapeutic agent in this specification has the following characteristics. (1) In the cell aggregate, retinal cells form a retinal layer, and the retinal cells are present in a layered manner in the retinal layer; (2) The diameter of the retinal tissue in the long axis direction is 0.2 mm or more (preferably 0.5 mm or more), the thickness of the epithelial structure of the retinal tissue is 0.2 mm or more, and the total number of cells contained in the retinal tissue is 1 × 10 5 cells or more (or the retinal tissue is 1×10 5 cells / mm 3 containing cells at a density of 100 or more), (3) 50% or more (preferably 60% or more, 70% or more, 80% or more, or 90% or more) of the total number of cells contained in each retinal tissue are cells expressing at least one of PAX6, Chx10, and Crx, and / or 80% or more (preferably 85% or more, 90% or more, or 95% or more) of the total number of cells present in the photoreceptor layer or neuroblastic layer are cells expressing at least one of PAX6, Chx10, and Crx, (4) The retinal tissue has a continuous epithelial structure in an area ratio of at least 50% (preferably 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more) of the surface of the retinal tissue; (5) The retinal tissue expresses TGFβ (preferably TGFβ2), and preferably, TGFβ is present on the surface of the retinal tissue. The retinal tissue may further have one or more of the following characteristics: (A) The retinal tissue is a spherical cell aggregate, preferably having a vesicular structure. (B) Virtually free of retinal pigment epithelial cells. (C) The outermost cells on the apical surface of the retinal tissue are arranged in a continuous pattern with 10 or more cells (preferably 15 or more, or 20 or more cells) per 0.1 mm. (D) The retinal tissue may contain photoreceptor cells, preferably 80% or more of the total number of cells in the photoreceptor layer, or 5% or more of the total number of cells in the retinal tissue. (E) The retinal tissue is derived from pluripotent stem cells.

[0064] In one embodiment, the retinal tissue is induced to differentiate from allogeneic stem cells (e.g., allogeneic pluripotent stem cells). Methods for inducing differentiation 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, Nat Commun. 2014 Jun 10;5:4047, etc. In another embodiment, retinal tissue derived from a living organism can be used. Methods for preparing retinal tissue from a living organism are well known to those skilled in the art. Specifically, retinal tissue can be excised under anesthesia. RPE cells can be excised from the excised retinal tissue.

[0065] A specific embodiment of the method for producing neural retina by differentiation induction includes a method comprising the following steps (A), (B), and (C). (A): Culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway agonist, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells; (B): A step of forming cell aggregates by suspension culture in a serum-free medium; (C): A step of further culturing the cell aggregates obtained in step (B) in a suspension culture medium containing a substance acting on the BMP (bone morphogenetic protein) signaling pathway.

[0066] This method is also disclosed, for example, in WO2016 / 063985 or WO2017 / 183732, and more details can be found in WO2016 / 063985 or WO2017 / 183732.

[0067] The term "TGFβ family signaling pathway inhibitor" refers to a substance that inhibits the TGFβ family signaling pathway, i.e., the signaling pathway transmitted by the Smad family. Specifically, TGFβ signaling pathway inhibitors (e.g., SB431542: 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, LY-364947: 4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline, SB-505124: 2-[4-(1,3-benzodioxol-5-yl)-2-(1, 1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, A-83-01: 3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide, etc.), Nodal / Activin signaling pathway inhibitors (e.g., SB431542, A-83-01, etc.), and BMP signaling pathway inhibitors (e.g., LDN193189: 4-[6-[4-(1-Piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]quinoline dihydrochloride, Dorsomorphin: 6-[4-[2-(1-Piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine, etc.). These substances are commercially available.

[0068] Sonic hedgehog (hereinafter sometimes referred to as "Shh") signaling pathway agonists are substances that can enhance signaling mediated by Shh. Examples of Shh signaling pathway agonists include SHH, PMA (Purmorphamine: 9-Cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), and SAG (Smoothened Agonist: 3-Chloro-N-[trans-4-(methylamino)cyclohexyl]-N-[[3-(4-pyridinyl)phenyl]methyl]benzo[b]thiophene-2-carboxamide).

[0069] The concentrations of the TGFβ family signaling pathway inhibitor and the Sonic Hedgehog signaling pathway activator may be any concentration capable of inducing differentiation into retinal cells. For example, SB431542 is usually used at a concentration of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is usually used at a concentration of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is usually used at a concentration of 1 to 2000 nM, preferably 10 to 300 nM. SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM. PMA is usually used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM.

[0070] In the culture of pluripotent stem cells under feeder-free conditions in step (A), the above-mentioned feeder-free medium containing an undifferentiated state maintenance factor may be used as the medium.

[0071] In the culture of pluripotent stem cells under feeder-free conditions in step (A), a suitable matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold instead of feeder cells. Examples of matrices that can be used as 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), e.g., Matrigel), gelatin, collagen, heparan sulfate proteoglycan, entactin, vitronectin, and the like.

[0072] The culture time of the pluripotent stem cells in step (A) may be within a range that can achieve the effect of improving the quality of the cell aggregates formed in step (B), and is not particularly limited, but is usually 0.5 to 144 hours. In one embodiment, the culture time of the pluripotent stem cells in step (A) 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).

[0073] Preparation of serum-free medium and formation of cell aggregates can be performed by methods well known to those skilled in the art. Examples of methods for preparing serum-free medium and forming cell aggregates include the SFEB method: Serum-free Floating culture of Embryoid Bodies-like aggregates (Watanabe et al. Nature Neuroscience, volume 8, pages 288-296 (2005)), and the SFEBq method, which is an improved method of the SFEB method (Eiraku et al. Cell Stem Cell, Volume 3, ISSUE 5, P519-532, November 06, 2008). Specifically, the SFEBq method refers to a method in which approximately 12,000 pluripotent stem cells are placed in a non-cell-adhesive culture dish with a diameter of approximately 1 cm per well (e.g., one well of a 96-well plate: approximately 7 mm), and aggregates are quickly formed within 2 to 3 hours.

[0074] In one embodiment, the medium used in step (B) may contain a Sonic Hedgehog signaling pathway active substance. As the Sonic Hedgehog signaling pathway active substance, the above-mentioned substances may be used at the above-mentioned concentrations. The Sonic Hedgehog signaling pathway active substance is preferably contained in the medium from the start of the suspension culture. A ROCK inhibitor (e.g., Y-27632) may be added to the medium. The culture time may be, for example, 12 hours to 6 days.

[0075] The BMP signaling pathway active substance is a substance that can enhance the signaling pathway mediated by BMP. Examples of the BMP signaling pathway active substance include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2 protein, BMP4 protein, and BMP7 protein are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Fujifilm Wako Pure Chemical Corporation.

[0076] The medium used may be, for example, a serum-free medium or a serum medium (preferably a serum-free medium) supplemented with a BMP signaling pathway agent. The serum-free medium and the serum medium may be prepared as described above.

[0077] The concentration of the substance acting on the BMP signaling pathway may be any concentration capable of inducing differentiation into retinal cells. For example, in the case of human BMP4 protein, it is added to the medium at a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL).

[0078] The substance acting on the BMP signaling pathway may be added to the medium at least about 24 hours after the start of the suspension culture in step (A), and may be added to the medium within several days (for example, within 15 days) after the start of the suspension culture. The substance acting on the BMP signaling pathway may be added to the medium preferably between 1st and 15th days, more preferably between 1st and 9th days, and most preferably on the 3rd day after the start of the suspension culture.

[0079] In a specific embodiment, for example, on the 1st to 9th day after the start of the suspension culture in step (B), preferably on the 1st to 3rd day after the start of the suspension culture in step (B), a part or all of the medium is replaced with a medium containing BMP4, the final concentration of BMP4 is adjusted to about 1 to 10 nM, and the cells are cultured 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, a part or all of the medium can be replaced with a medium containing BMP4 once or twice. Alternatively, the concentration of BMP4 can be reduced stepwise.

[0080] The culture conditions such as the culture temperature and CO2 concentration in the above steps (A) to (C) can be appropriately set. The culture temperature is, for example, about 30° C. to about 40° C., and preferably about 37° C. The CO2 concentration is, for example, about 1% to about 10%, and preferably about 5%.

[0081] By varying the culture period in the above step (C), retinal tissue containing retinal cells at various stages of differentiation can be produced. That is, retinal tissue containing immature retinal cells (e.g., retinal progenitor cells, photoreceptor progenitor cells) and mature retinal cells (e.g., photoreceptors) at various ratios can be produced. By extending the culture period in step (C), the ratio of mature retinal cells can be increased. Note that "immature retinal cells" refers to precursor cells that are committed to differentiate into mature retinal cells.

[0082] The above step (B) and / or step (C) may be performed using the method disclosed in WO2017 / 183732. That is, in step (B) and / or step (C), the cells obtained in step (A) may be cultured in suspension in a medium further containing a Wnt signaling pathway inhibitor to form a neural retina.

[0083] The Wnt signaling pathway inhibitor used in step (B) and / or step (C) is not particularly limited as long as it can suppress signaling mediated by Wnt, and may be any of a protein, a nucleic acid, a low molecular weight compound, etc. The signal mediated by Wnt is transmitted via a Wnt receptor that 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 act directly on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNAs, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus proteins, etc.), substances that inhibit physiological activities resulting from signaling by Wnt receptors [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), and IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide) and other low molecular weight compounds, but are not limited thereto. The Wnt signaling pathway inhibitor may contain one or more of these. CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, and the like are known Wnt signaling pathway inhibitors, and are commercially available products and the like. IWR1e is preferably used as the Wnt signaling pathway inhibitor.

[0084] The concentration of the Wnt signaling pathway inhibitor in step (B) may be any concentration capable of inducing the formation of favorable cell aggregates. For example, in the case of IWR-1-endo, it is added to the 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 a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirable to use it at a concentration that exhibits the same Wnt signaling pathway inhibitory activity as the above-mentioned IWR-1-endo concentration.

[0085] In step (B), the timing of adding the Wnt signaling pathway inhibitor to the medium is preferably early. The Wnt signaling pathway inhibitor is added to the medium usually within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, and even more preferably at the start of the suspension culture in step (B). Specifically, for example, a basal medium containing a Wnt signaling pathway inhibitor may be added, or a part or whole of the basal medium may be replaced. The period during which the cells obtained in step (A) are allowed to act on the Wnt signaling pathway inhibitor in step (B) is not particularly limited, but preferably, the cells are allowed to act on the Wnt signaling pathway inhibitor after being added to the medium at the start of the suspension culture in step (B) until the end of step (B) (immediately before the addition of the substance acting on the BMP signaling pathway). More preferably, the cells are continuously exposed to the Wnt signaling pathway inhibitor even after the end of step (B) (i.e., during step (C)), as described below. In one embodiment, as described below, the Wnt signaling pathway inhibitor may be allowed to continue to act even after completion of step (B) (i.e., during the period of step (C)) until retinal tissue is formed.

[0086] In step (C), any of the above-mentioned Wnt signaling pathway inhibitors can be used as the Wnt signaling pathway inhibitor. Preferably, the same type of Wnt signaling pathway inhibitor used in step (B) is used in step (C).

[0087] The concentration of the Wnt signaling pathway inhibitor in step (C) may be any concentration capable of inducing retinal progenitor cells and retinal tissue. For example, IWR-1-endo may be added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirable to use it at a concentration that exhibits the same Wnt signaling pathway inhibitory activity as the above-mentioned IWR-1-endo concentration. The concentration of the Wnt signaling pathway inhibitor in the 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 medium in step (B) is taken as 100, and it is more preferable that it is the same as the concentration of the Wnt signaling pathway inhibitor in the medium in the second step.

[0088] The timing of adding the Wnt signaling pathway inhibitor to the medium is not particularly limited as long as it can achieve the formation of aggregates containing retinal cells or retinal tissue, but the earlier the better. Preferably, the Wnt signaling pathway inhibitor is added to the medium at the start of step (C). More preferably, after the Wnt signaling pathway inhibitor is added in step (B), it continues to be contained in the medium in step (C) (i.e., from the start of step (B)). Even more preferably, after the Wnt signaling pathway inhibitor is added at the start of the suspension culture in step (B), it continues to be contained in the medium in step (C). For example, a BMP signaling agent (e.g., BMP4) may be added to the culture obtained in step (B) (a suspension of aggregates in a medium containing a Wnt signaling pathway inhibitor).

[0089] The period during which the Wnt signaling pathway inhibitor is allowed to act is not particularly limited, but is preferably 2 to 30 days, more preferably 6 to 20 days, 8 to 18 days, 10 to 18 days, or 10 to 17 days (e.g., 10 days) from the start of suspension culture in step (B) when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B). In another embodiment, the period during which the Wnt signaling pathway inhibitor is allowed to act is preferably 3 to 15 days (e.g., 5, 6, or 7 days), more preferably 6 to 10 days (e.g., 6 days) from the start of suspension culture in step (B) when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B).

[0090] A neural retina containing a ciliary marginal region-like structure can also be produced by further subjecting the cell aggregate of retinal cells obtained by the above-mentioned method to the following steps. By producing a neural retina containing a ciliary marginal region-like structure, it is possible to produce a neural retina that is rich in photoreceptor precursor cells and contains a high frequency of continuous epithelial structures. Step D: A step of culturing for about 3 to 6 days in a serum-free medium or serum medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway. Step E: A step of culturing the cell aggregates obtained by step D in a serum-free medium or serum medium that does not contain a substance active in the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway for approximately 30 to 150 days (preferably, 30 to 120 days, or 30 to 100 days).

[0091] In one embodiment, a ciliary marginal zone-like structure can be produced from the cell aggregates containing neural retina obtained in steps (A) to (C), which are on days 6 to 30, 10 to 20 (10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th or 20th) after the start of suspension culture in step (B), by the above steps (D) and (E).

[0092] The substance acting on the Wnt signaling pathway is not particularly limited as long as it can enhance the signal transduction mediated by Wnt. Specific examples of the substance acting on the Wnt signaling pathway include GSK3β inhibitors (e.g., 6-Bromoindirubin-3'-oxime (BIO), CHIR99021, and Kenpaullone). The concentration of the substance acting on the Wnt signaling pathway in the serum-free medium or serum medium in step D is, for example, in the case of CHIR99021, in the range of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM.

[0093] The FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit signal transduction mediated by FGF. Examples of FGF signaling pathway inhibitors include SU-5402, AZD4547, BGJ398, etc. In step D, the concentration of the FGF signaling pathway inhibitor in the serum-free medium or serum medium is, for example, SU-5402, about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, more preferably about 5 μM.

[0094] The above-mentioned methods can be used to produce retinal tissue (neural retina), but are not limited to these.

[0095] The term "transplant rejection" refers to a defensive reaction by an individual that attempts to reject the transplanted tissue or organ due to the immune response of the transplanted patient (recipient).

[0096] Transplant rejection is classified into, for example, acute and chronic phases depending on the time of onset. Acute rejection occurs from the day of transplantation or a few days after transplantation (e.g., 1, 2, or 3 days after transplantation) to about 2 months, while chronic rejection occurs from about 2 months after transplantation. Acute rejection may be further classified into early acute rejection (within about 10 days after transplantation) and late acute rejection (within about 11 days after transplantation to about 2 months after transplantation).

[0097] Specific symptoms include lymphocyte infiltration and immune cell activation, etc. In addition, fundus examination, fluorescein angiography and / or mixed lymphocyte assay can be used as indicators to assess the degree of transplant rejection. Acute rejection (especially in the early acute stage) is likely to induce a complex and strong immune response that is also influenced by the inflammatory response caused by surgical stress, so in kidney transplants, immunosuppressants such as tacrolimus are generally administered systemically. If no signs of rejection are observed, the dosage of the immunosuppressant is then reduced, but the use of the immunosuppressant is usually continued.

[0098] Immunosuppressants are drugs used to suppress or inhibit the activity of the immune system. They are used to prevent and suppress rejection of transplanted organs or tissues, and to control inflammation in autoimmune diseases and inflammatory diseases such as allergies.

[0099] Immunosuppressants can be classified into several categories based on the similarity of their mechanism of action or structure. For example, classification based on the mechanism of action includes steroidal anti-inflammatory drugs (SAIDs), calcineurin inhibitors, mTOR inhibitors, cytotoxic agents, metabolic antagonists, cytotoxic antibiotics, alkylating agents, microtubule synthesis inhibitors, and the like. There is no limitation on the substances used as immunosuppressants, and they may be any of low molecular weight compounds, proteins (including antibodies), and nucleic acids. Among these immunosuppressants, there are drugs that are used as therapeutic agents for other diseases, but have an immunosuppressive effect and are therefore also used as immunosuppressants to suppress rejection due to transplantation. Examples of immunosuppressants administered to organ transplant patients to suppress rejection due to transplantation include calcineurin inhibitors, mTOR inhibitors, metabolic antagonists, and steroidal anti-inflammatory agents.

[0100] Steroidal anti-inflammatory agents are anti-inflammatory agents containing glucocorticoids or their derivatives as active ingredients. Steroidal anti-inflammatory agents bind to the nuclear receptor glucocorticoid receptor (GR / NR3C1) to suppress the expression of inflammatory genes NF-κB and AP-1, and suppress the proliferation signal of lymphocytes (cells of the acquired immune system, particularly cells of the innate immune system: neutrophils, macrophages, etc.). Examples of such agents include, but are not limited to, prednisolone, methylprednisolone, triamcinolone, dexamethasone, betamethasone, and fluorometholone.

[0101] In this specification, steroidal anti-inflammatory agents can be classified based on the strength of their action. Specifically, they are classified into strong steroidal anti-inflammatory agents and mild steroidal anti-inflammatory agents. Examples of strong steroidal anti-inflammatory agents include dexamethasone, betamethasone, prednisolone, and methylprednisolone. Examples of mild steroidal anti-inflammatory agents include fluorometholone, betamethasone dipropionate (betamethasone valerate), triamcinolone, cortisone, and hydrocortisone.

[0102] Steroidal anti-inflammatory drugs used in the field of ophthalmology include betamethasone valerate or fluorometholone, which are used as eye drops, and triamcinolone acetonide, which is used as an intravitreal injection and / or Tenon's injection. As oral drugs, cortisone or hydrocortisone, which have a short duration of action, prednisolone or methylprednisolone sodium succinate, which have a medium duration of action, and dexamethasone or betamethasone, which have a long duration of action, are used. For steroid pulse therapy, a solution for drip infusion of methylprednisolone is generally used. Steroidal anti-inflammatory drugs are also known to be effective in preventing transplant rejection, such as prednisolone. It has been reported that oral steroidal anti-inflammatory drugs or pulse therapy with steroidal anti-inflammatory drugs are used to prevent immune rejection following transplantation of RPE cells, but there have been no reports of the use of steroidal anti-inflammatory drugs alone as eye drops, intravitreal injection, or Tenon's injection.

[0103] Calcineurin inhibitors are drugs that have a calcineurin inhibitory effect, and in particular, are drugs that mainly act by controlling the activation of T cells. Specifically, calcineurin inhibitors bind to intracellular receptors called immunophilins, and then inhibit the activity of the dephosphorylation enzyme calcineurin PP2B, inhibit the production of interleukin 2 (IL-2) and interferon gamma, and inhibit the proliferation of T cells. Examples of calcineurin inhibitors include cyclosporine and tacrolimus.

[0104] In the field of ophthalmology, cyclosporine is used as an eye drop to treat non-infectious uveitis, whereas tacrolimus has no application in the field of ophthalmology. In addition, although it is known that drugs with calcineurin inhibitory activity are effective in preventing transplant rejection, oral calcineurin inhibitors have been reported to be used to prevent immune rejection following transplants of RPE cells, etc. However, the use of calcineurin inhibitor eye drops has not been reported.

[0105] An mTOR inhibitor is a drug that has an mTOR (mammalian target of rapamycin) inhibitory effect. Specifically, an mTOR inhibitor inhibits mTOR activity after binding to immunophilin, and exhibits effects such as stopping the cell cycle of immune cells and suppressing protein translation. It is known that mTOR inhibitors inhibit the activation of T cells and B cells by reducing the production of interleukin 2 (IL-2). Examples of mTOR inhibitors include sirolimus (rapamycin), sirolimus derivatives such as temsirolimus and everolimus.

[0106] Cytotoxic agents are drugs that have the effect of inhibiting cell division and are also used as anticancer drugs. Cytotoxic agents are also used as immunosuppressants when used in smaller doses than in cancer treatment. Cytotoxic agents can be further classified into metabolic antagonists that interfere with nucleic acid synthesis, cytotoxic antibiotics, alkylating agents, microtubule synthesis inhibitors, etc.

[0107] Examples of metabolic antagonists include purine analogs and their precursors mercaptopurine and azathioprine, folic acid analogs metronexate, mycophenolic acid, a guanosine production inhibitor, and leflunomide, a pyrimidine synthesis inhibitor. Azathioprine and its active metabolite 6-mercaptopurine become 6-MP ribonucleic acid, and mycophenolic acid and mizoribine suppress the activity of inosine-1-phosphate dehydrogenase (IMPDH), which is the main pathway for lymphocyte proliferation, and inhibit lymphocyte proliferation by inhibiting de novo diffusion synthesis. It has also been reported that oral preparations of mycophenolic acid are used to prevent immune rejection following transplantation of RPE cells, etc.

[0108] Cytotoxic antibiotics are antibiotics that have cytotoxic activity, such as dactinomycin, anthracycline, mitomycin C, bleomycin, and mithramycin.

[0109] Alkylating agents are a type of cytotoxic anticancer drug. Examples of alkylating agents include cyclophosphamide.

[0110] Microtubule synthesis inhibitors are drugs that inhibit microtubule synthesis and inhibit cell migration, etc. Examples of microtubule synthesis inhibitors include colchicine and vinblastine.

[0111] Proteins and antibodies include proteins and antibodies that act on cytokines (IL-2, TNFα, etc.) and their receptors, and surface markers of immune cells such as T cells (CD3, CD25, etc.). Antibodies can be classified into polyclonal and monoclonal antibodies. For example, basiliximab is an anti-CD25 antibody, and rituximab is an anti-CD20 antibody. These antibodies suppress the function of the targeted immune cells.

[0112] Immunosuppressants can be classified into oral agents, injections, and topical agents according to their dosage form. Oral agents are pharmaceutical preparations (medicines) that are administered orally. Oral agents include, but are not limited to, various dosage forms such as tablets, capsules, and granules. Injectable agents are liquid agents that are administered directly into the skin or subcutaneous tissue, into blood vessels, or into the eye (sub-Tenon's or intravitreal) using a needle, or are dissolved and made into a liquid when used. Topical agents are a general term for all drugs that are used directly on the human body, excluding oral agents and injections. Topical agents include, but are not limited to, eye drops, eye drop ointments, inhalants such as liquids, aerosols, and dry powders, and transdermal agents such as ointments, creams, poultices, and tapes. Dosage forms of immunosuppressants used in ocular transplants include oral agents, injections (intravenous injections, sub-Tenon injections, intravitreal injections, etc.), and external agents, particularly eye drops.

[0113] "Administering an immunosuppressant systemically" means administering the immunosuppressant in a manner that distributes the immunosuppressant throughout the body, i.e., in a manner in which the active ingredient of the immunosuppressant is present in the bloodstream, specifically, administering the immunosuppressant orally or intravenously, etc.

[0114] "Administering an immunosuppressant locally" means distributing the immunosuppressant locally, such as intraocularly or on the skin, and administering the immunosuppressant in such a manner that the active ingredient of the immunosuppressant is not substantially present in the bloodstream. In this embodiment, "administering an immunosuppressant locally" specifically means administering the immunosuppressant by intraocular injection (sub-Tenon injection, intravitreal injection, etc.) or by eye drops. From the viewpoint of reducing the burden on patients, such as side effects, local administration of the immunosuppressant is preferred, and administration by eye drops is particularly preferred. However, in current transplantation treatments, immunosuppressants are usually administered systemically, and it has not been reported that they can be administered locally for the purpose of preventing transplant rejection.

[0115] Sub-Tenon injection is a method of administration in which a small incision is made in the conjunctiva and the drug is injected into the Tenon's capsule at the back of the eye. It is a much more powerful treatment than eye drops and is expected to have a long-lasting effect on the posterior segment of the eye with a single injection. Intravitreal injection is a method of administering drugs directly into the eye, which reduces the risk of systemic side effects and provides a stronger therapeutic effect against intraocular lesions.

[0116] Administration of immunosuppressants for the purpose of preventing transplant rejection means prophylactic administration from before transplant rejection is observed, i.e. does not include administration of immunosuppressants for the purpose of preventing and / or treating other diseases, or administration of immunosuppressants for the purpose of controlling transplant rejection that has already occurred.

[0117] Usually, the administration of immunosuppressants to prevent transplant rejection begins immediately before or after transplantation and continues indefinitely. During the period of continued immunosuppression after transplantation, the type, dosage form, or dosage of the immunosuppressant may change. Specifically, high doses of immunosuppressants are administered frequently in the early stages of transplantation, but the dosage is increased or decreased as appropriate depending on the symptoms thereafter. After a stable condition is achieved, the dosage may be gradually reduced and maintained at the minimum effective dose. However, systemic administration of immunosuppressants to prevent transplant rejection, including ophthalmic transplants, continues.

[0118] The therapeutic agent of this embodiment is characterized in that it does not induce an immune response that leads to a rejection reaction associated with transplantation, or suppresses the immune response. That is, by using the therapeutic agent of this embodiment for transplantation, it is possible to reduce the burden on the patient caused by administration of an immunosuppressant for the purpose of preventing rejection. Therefore, the therapeutic agent of this embodiment is characterized in that it is possible to locally administer and / or administer for a shorter period of time an immunosuppressant that is intended to prevent rejection reaction associated with transplantation is not administered before or after administration of the therapeutic agent, or an immunosuppressant that has fewer side effects and thus is less of a burden on the patient.

[0119] The shorter the period of use of the immunosuppressant, the better. Considering that the invasion of surgery induces immunity, it is permissible to administer the immunosuppressant before, during, and / or about one week (two weeks, one month, two months) after administration of the therapeutic agent of this embodiment (or a period during which acute rejection may occur). In this case, as described below, local administration of the immunosuppressant is preferred, and the use of a steroidal anti-inflammatory agent is preferred.

[0120] To prevent systemic effects, local administration of immunosuppressants is preferred, and when using the therapeutic agents of the present invention, local administration to the eye is preferred.

[0121] Considering the strength of the immunosuppressive effect, the presence or absence of side effects or the degree of concern about side effects, the frequency of occurrence, and / or the severity, etc., an example of an "immunosuppressant with few side effects" is a steroidal anti-inflammatory agent. That is, when transplantation is performed using the therapeutic agent of this embodiment, systemic or local administration of a steroidal anti-inflammatory agent is permitted. The use of a steroidal anti-inflammatory agent for a certain period of time is also permitted in that it is possible to suppress immune induction induced by invasion due to surgical operation. On the other hand, considering that the therapeutic agent of this embodiment can strongly suppress the activation of T cells and activated T cells, it is possible to suppress the use of calcineurin inhibitors and mTOR inhibitors that mainly target T cells. It is clinically significant that calcineurin inhibitors and mTOR inhibitors, which are most commonly used in current transplant treatments and have high concerns about side effects, are not used, are not administered for a long period of time, are not administered systemically, or the amount of use can be suppressed.

[0122] In one aspect of the therapeutic agent of this embodiment, the subject patient is not systemically administered immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection before, during, and / or after administration of the therapeutic agent described above. The subject patient is preferably not systemically administered immunosuppressants other than steroidal anti-inflammatory agents. In one aspect of the therapeutic agent of this embodiment, preferably, an immunosuppressant is not administered systemically, more preferably, an immunosuppressant is not administered systemically, and an immunosuppressant other than a steroidal anti-inflammatory agent and a calcineurin inhibitor is not administered locally either (i.e., only a steroidal anti-inflammatory agent or a calcineurin inhibitor is administered locally as an immunosuppressant), and more preferably, no immunosuppressant is administered at all.

[0123] Not being administered at the time of administration of a therapeutic agent means not being administered simultaneously with administration of the therapeutic agent of this embodiment, or not being administered immediately before or after administration of the therapeutic agent at a timing considered to be simultaneous administration, for example, not being administered within 24 hours before or after administration of the therapeutic agent or not being administered within 12 hours before administration of the therapeutic agent. Not being administered before administration of a therapeutic agent means not being administered at least one week before the timing considered to be simultaneous administration before administration of the therapeutic agent of this embodiment. Not being administered after administration of a therapeutic agent means not being administered at least within 6 months or one year after the timing considered to be simultaneous administration after administration of the therapeutic agent of the present invention.

[0124] The therapeutic agent of this embodiment is permitted to administer a steroidal anti-inflammatory agent and / or a calcineurin inhibitor systemically or locally (preferably locally) before, during or after administration of the above-mentioned therapeutic agent. In addition, the therapeutic agent of this embodiment may administer an immunosuppressant other than a steroidal anti-inflammatory agent and a calcineurin inhibitor locally before, during or after administration of the above-mentioned therapeutic agent. Administration of the immunosuppressant can be terminated within one year, preferably within six months, more preferably within three months, more preferably within two months, more preferably within one month, more preferably within two weeks, and more preferably within one week after administration of the above-mentioned therapeutic agent.

[0125] The therapeutic agent of the present embodiment may contain an effective amount of retinal tissue. The effective amount of retinal tissue varies depending on the purpose of administration, the administration method, and the condition of the subject (sex, age, weight, medical condition, etc.), but may be, for example, 1.0×10 cell count. 4 10 or more (e.g., 1×10 5 pieces, 1×10 6 1 piece or 1 x 10 7 pcs) may also be used.

[0126] The therapeutic agent of this embodiment may contain a medicament acceptable carrier. As the medicament acceptable carrier, a physiological aqueous solvent (physiological saline, buffer, serum-free medium, etc.) can be used. If necessary, in transplantation medicine, a medicine containing tissue or cells to be transplanted may be mixed with a preservative, stabilizer, reducing agent, isotonicity agent, pH adjuster, etc. that are commonly used.

[0127] The therapeutic agent of the present embodiment can be prepared as a cell suspension by suspending retinal tissue in an appropriate physiological aqueous solvent. If necessary, the therapeutic agent of the present embodiment can be cryopreserved by adding a cryopreservative, thawed at the time of use, washed with a buffer solution, and used in transplantation medicine.

[0128] The retinal tissue contained in the therapeutic agent of this embodiment can be cut into pieces of an appropriate size using, for example, tweezers, a knife, scissors, etc., and a portion of the aggregate can be cut out for transplantation. The shape of the cut out portion is not particularly limited, and can be, for example, a cell sheet.

[0129] The therapeutic agent of this embodiment is administered to a patient by transplantation. The administration to a patient is performed, for example, by a method of subretinal transplantation using an injection needle, or by a method of incising a part of the eyeball and transplanting from the incision site to the damaged or diseased site. When administering the therapeutic agent of this embodiment, immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection are not administered systemically before, during, and / or after administration. When administering the therapeutic agent of this embodiment, steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection may be administered systemically before, during, and / or after administration, and immunosuppressants for the purpose of preventing transplant rejection may be administered locally.

[0130] (patient) The target patient for the therapeutic agent of this embodiment is a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and the therapeutic agent is a therapeutic agent for transplantation into the target patient. The target patient to whom the therapeutic agent is administered is not administered systemically any immunosuppressant other than a steroidal anti-inflammatory agent and a calcineurin inhibitor for the purpose of preventing transplant rejection before, during, and / or after administration of the above-mentioned therapeutic agent. However, the target patient may be administered systemically a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the purpose of preventing transplant rejection before, during, and / or after administration of the above-mentioned therapeutic agent, and may also be administered locally an immunosuppressant for the purpose of preventing transplant rejection.

[0131] Preferably, the subject patient is not systemically administered with a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the prevention of transplant rejection at or after 2 months (or 1 month) after administration of the therapeutic agent. That is, the subject patient may be systemically or locally administered with a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the prevention of transplant rejection before, at the time of, and / or up to 2 months (or 1 month) after administration of the therapeutic agent, but may be locally administered with a steroidal anti-inflammatory agent and / or a calcineurin inhibitor at or after 2 months (or 1 month) after administration of the therapeutic agent.

[0132] The subject patient is preferably not administered systemic immunosuppressants for the prevention of transplant rejection before, during, and / or after administration of the Therapeutic Agent, i.e., the subject patient is not administered systemic immunosuppressants, including steroidal anti-inflammatory agents and / or calcineurin inhibitors, for the prevention of transplant rejection before, during, and / or after administration of the Therapeutic Agent, but may be administered locally.

[0133] The subject patient is preferably not administered locally any immunosuppressant other than steroidal anti-inflammatory agents and calcineurin inhibitors (preferably other than steroidal anti-inflammatory agents) for the purpose of preventing transplant rejection before, during and / or after administration of the therapeutic agent. That is, the subject patient is not administered systemically or locally any immunosuppressant other than steroidal anti-inflammatory agents and calcineurin inhibitors (preferably other than steroidal anti-inflammatory agents) for the purpose of preventing transplant rejection before, during and / or after administration of the therapeutic agent, but may be administered systemically or locally (preferably locally) any steroidal anti-inflammatory agent and / or calcineurin inhibitor. Preferred local administration here includes Tenon's injection or vitreous injection.

[0134] The subject may be administered one or more immunosuppressants selected from the group consisting of triamcinolone, fluocinolone, and calcineurin inhibitors (cyclosporine, tacrolimus, etc.) locally before, during, and / or after administration of the therapeutic agent for the purpose of preventing transplant rejection. Preferred local administrations include Tenon's injection or vitreous injection.

[0135] The subject patient preferably does not receive topical steroidal anti-inflammatory agents and / or calcineurin inhibitors for the prevention of transplant rejection at least 2 months (or 1 month) after administration of the therapeutic agent.

[0136] The subject patient preferably does not receive immunosuppressive drugs prior to, during and / or after administration of the therapeutic agent for the prevention of transplant rejection.

[0137] Diseases based on damage to retinal tissue or retinal cells include, but are not limited to, ophthalmic diseases such as macular degeneration, age-related macular degeneration, retinitis pigmentosa, cataracts, glaucoma, corneal diseases, retinopathy, and macular hole.

[0138] In one embodiment of the present invention, a therapeutic drug is provided for a patient suffering from a disease caused by a disorder of a retinal tissue or a retinal cell, in which the retinal tissue for transplantation (therapeutic drug) is incompatible with the HLA type. A patient in which the retinal tissue for transplantation is incompatible with the HLA type is, for example, a patient in which one, two, three or all of the HLAs selected from the group consisting of HLA-A, B, C and DR are incompatible. Those skilled in the art can determine the degree of compatibility of HLA types by well-known techniques such as serological tests and DNA typing tests.

[0139] As an aspect of the present invention, a therapeutic agent for patients with a damaged blood-retinal barrier is provided. When the blood-retinal barrier is damaged, it is believed that an immune response is likely to occur, and transplant rejection is likely to occur. The therapeutic agent of this embodiment does not induce an immune response, but rather can suppress the immune response. Therefore, the therapeutic agent of this embodiment can be used for patients with a damaged blood-retinal barrier, with reduced use of immunosuppressants. The damage to the blood-retinal barrier also includes damage due to surgery. Whether or not the blood-retinal barrier is damaged can be determined by fundus examination, fluorescein angiography, or optical coherence tomography (OCT) examination. Examples of diseases in which the blood-retinal barrier is damaged include age-related macular degeneration, diabetic retinopathy, Behcet's disease, and sarcoidosis.

[0140] In organ transplantation, there are patients who cannot undergo transplantation, or who, even if transplantation is possible, are forced to undergo long-term hospitalization or outpatient care, and various tests for sufficient follow-up. These patients suffer a particularly large decline in QOL. One of the reasons for this is that patients are subject to contraindications for use of immunosuppressants that require concomitant use, contraindications for concomitant use, and careful administration. Specifically, these include elderly people, pregnant women, women who may be pregnant, breastfeeding women, children (including low birth weight babies, newborns, and infants), patients with infectious diseases, virus carriers such as hepatitis B or C virus, patients with malignant tumors or a history of malignant tumors, patients with renal dysfunction, patients with liver dysfunction, and patients with pancreatic dysfunction. The therapeutic agent of this embodiment does not use an immunosuppressant, or the amount of immunosuppressant used can be reduced. Thus, in one embodiment, a therapeutic agent is provided for one or more patients selected from the group consisting of elderly people, pregnant women, women who may be pregnant, lactating women, children (including low birth weight babies, newborns, and infants), patients with infectious diseases, virus carriers such as hepatitis B or C virus, patients with malignant tumors or a history of malignant tumors, patients with renal dysfunction, patients with liver dysfunction, and patients with pancreatic dysfunction.

[0141] As one aspect of the retinal tissue of the present embodiment, there is provided retinal tissue of allogeneic origin and having a three-dimensional structure, for use in treating a disease accompanied by damage to retinal cells or retinal tissue in a patient who is suffering from a disease accompanied by damage to retinal cells or retinal tissue and who is not systemically administered immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection.

[0142] In addition, as another embodiment of the retinal tissue of the present invention, there is provided a retinal tissue for use in treating a disease accompanied by damage to retinal cells or retinal tissue, which is allogeneically derived and has a three-dimensional structure, and which is administered without systemic administration of immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection before, during, and / or after administration of the retinal tissue.

[0143] [Treatment method] As one aspect of the treatment method of this embodiment, there is provided a treatment method for a disease accompanied by damage to retinal cells or retinal tissue, which comprises administering (transplanting) an effective amount of allogeneic retinal tissue having a three-dimensional structure to a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and is characterized in that the subject patient is not systemically administered with immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors for the purpose of preventing transplant rejection before (pre-transplantation), at the time of (transplantation), and / or after (post-transplantation) the administration of the retinal tissue. The retinal tissue is transplanted into the patient as a preparation, specifically a preparation for transplantation.

[0144] Another aspect of the treatment method of this embodiment provides a method for treating a disease accompanied by damage to retinal cells or retinal tissue, which comprises administering an effective amount of allogeneic retinal tissue having a three-dimensional structure to a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and is characterized in that no immunosuppressants other than steroidal anti-inflammatory agents and calcineurin inhibitors are systemically administered before, during and / or after the administration of the retinal tissue for the purpose of preventing transplant rejection. One aspect of the present invention includes a method of administering the therapeutic agent of the present invention to a patient in whom the therapeutic agent is found to have an inhibitory effect on the immune response in the above-mentioned preliminary test.

[0145] That is, as one aspect, the treatment method of this embodiment includes the steps of inducing differentiation of retinal tissue having a three-dimensional structure from allogeneic stem cells, and administering (transplanting) an effective amount of the retinal tissue to a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue. The target patient and the administration method may be as described above.

[0146] The retinal tissue may be preserved in a medium necessary for maintaining the viability of the retinal tissue during treatment (transplantation). Examples of the "medium necessary for maintaining viability" include culture medium, physiological buffer solution, etc., but are not particularly limited as long as the cell population containing retinal cells such as retinal progenitor cells survives, and a person skilled in the art can select an appropriate medium. An example of the medium is a medium prepared as a basal medium using a medium normally used for culturing animal cells. Examples of the basal medium include media that can be used for culturing animal cells, such as BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, Neurobasal medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof.

[0147] Transplantation is carried out, for example, by subretinal implantation using an injection needle, or by incising a part of the eyeball and transplanting from the incision site to the damaged or diseased site.

[0148] 〔kit〕 In one embodiment, a kit is provided that includes retinal tissue that has been induced to differentiate from allogeneic stem cells and has a three-dimensional structure, and a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and, at the time of or after administration of a therapeutic drug, (1) a notice that an immunosuppressant will not be administered, (2) a notice that an immunosuppressant will not be administered systemically (including a case in which the period during which an immunosuppressant will not be administered systemically for one month or more after administration of the therapeutic drug is limited), (3) a notice that an immunosuppressant will be administered only locally, (4) a notice that only a steroidal anti-inflammatory drug or a calcineurin inhibitor (preferably only a steroidal anti-inflammatory drug) will be administered systemically or locally as an immunosuppressant, or (5) an instruction manual, instruction manual, package insert, or product label that shows a statement substantially equivalent to any of the above (1) to (4). Statements substantially equivalent to any of (1) to (4) include, for example, disclosure of non-clinical or clinical data to the extent that a general physician can understand that administration of (1) to (4) is possible. The above data may be from any animal species, and may not be limited to in vivo data, but may also include data showing immune tolerance or immune suppression effects in vitro. In addition, the kit may also include transplantation equipment (e.g., needles and syringes for intraocular administration), transplantation media, and media required to maintain the viability of retinal tissue.

[0149] The retinal tissue is preferably packaged with instructions, instructions, inserts, or product labels. The instructions, instructions, inserts, or product labels may be printed or may be electronic information posted on the internet, etc. In the case of electronic information, instructions on how to obtain the information are preferably included. EXAMPLES

[0150] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0151] All animals used in the following examples were treated in accordance with the Association for Research in Vision and Ophthalmology statement for the Animals in Ophthalmology and Vision Research. Animal experiments were approved by the RIKEN BDR Ethics Committee and were conducted in accordance with the animal experiment guidelines of the RIKEN Biomechanics Research Center.

[0152] <Example 1: Preparation of neural retina (NR) from human ES / iPS cells using SFEBq method> Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Crx::Venus reporter gene and human iPS cells (TLHD2 line) established at the RIKEN Institute were cultured under feeder-free conditions according to the method described in "Scientific Reports, 4, 3594 (2014)". StemFit medium (product name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0153] The KhES-1 and TLHD2 lines expressed pluripotency markers (Oct3 / 4, Nanog, SSEA-4) in feeder-free medium.

[0154] Specific maintenance culture procedures for human ES and human iPS cells (human ES / iPS cells) were performed as follows. First, human ES / iPS cells (KhES-1 and TLHD2 strains) that had become subconfluent (about 60% of the culture area was covered with cells) were washed with PBS and then dispersed into single cells using TrypLE Select (product name, manufactured by Life Technologies). The human ES cells dispersed into single cells were then seeded onto a plastic culture dish coated with Laminin511-E8 and cultured under feeder-free conditions in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 μM). A 6-well plate (manufactured by Iwaki, for cell culture, culture area 9.4 cm) was used as the plastic culture dish. 2 ), the number of human ES / iPS cells dispersed into single cells was 0.4–1.2 × 10 per well. 4 Human ES cells were used. One day after seeding, the medium was replaced with StemFit medium not containing Y27632. Thereafter, the medium was replaced with StemFit medium not containing Y27632 once every 1 to 2 days. Thereafter, the cells were cultured under feeder-free conditions until one day before subconfluency. The human ES cells one day before subconfluency were cultured under feeder-free conditions for one day in the presence of SB431542 (TGFβ signaling pathway inhibitor, 5 μM) and SAG (Shh signaling pathway agonist, 300 nM) (preconditioning treatment).

[0155] After washing with PBS, human ES / iPS cells were treated with TrypLE Select for cell dispersion and further dispersed into single cells by pipetting. The dispersed human ES cells were then cultured in a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) at 1.2 × 10 cells per well. 4The cells were suspended in 100 μL of serum-free medium and cultured at 27 °C and 5% CO2. The serum-free medium (gfCDM + KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1 × chemically defined lipid concentrate.

[0156] At the start of suspension culture (day 0 after suspension culture), Y27632 (ROCK inhibitor, final concentration 20 μM) and SAG (Shh signaling pathway active substance, 300 nM, 30 nM, 0 nM) were added to the serum-free medium. On the third day after suspension culture was started, 50 μL of medium containing exogenous human recombinant BMP4 at a final concentration of 1.5 nM was added using a medium containing human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Co.) without Y27632 or SAG. From the sixth day after suspension culture was started, half of the medium was replaced once every three days with medium containing no Y27632, SAG, or human recombinant BMP4.

[0157] The aggregates on the 15th to 18th day after the start of the floating culture were transferred to a 90 mm low-adhesion culture dish (manufactured by Sumitomo Bakelite Co., Ltd.) and cultured for 3 to 4 days at 37 ° C. and 5% CO2 in a serum-free medium (medium in which 1% N2 Supplement was added to DMEM / F12 medium) containing a substance acting on the Wnt signaling pathway (CHIR99021, 3 μM) and an FGF signaling pathway inhibitor (SU5402, 5 μM). Thereafter, the aggregates were cultured in a 90 mm low-adhesion culture dish (manufactured by Sumitomo Bakelite Co., Ltd.) in a serum-containing DMEM / F12 medium (hereinafter sometimes referred to as NucT0 medium) without a substance acting on the Wnt signaling pathway and an inhibitor of the FGF signaling pathway. After the 40th day after the start of the floating culture (differentiation induction), the aggregates were cultured in a serum-free medium (mixed medium of NucT0 medium and NucT2 medium, hereinafter sometimes referred to as NucT1 medium) without a substance acting on the Wnt signaling pathway and an inhibitor of the FGF signaling pathway. From day 60 onwards after the start of suspension culture (differentiation induction), the cells were cultured in Neurobasal medium containing T3, a substance that acts on the thyroid hormone signaling pathway (hereinafter sometimes referred to as NucT2 medium), until day 80 to 95 after the start of suspension culture (differentiation induction).

[0158] Observation using a microscope revealed that in a human ES cell line (khES-1) and a human iPS cell line (TLHD2), a neural retina (NR) with a layered structure was formed 80 to 95 days after differentiation induction (Figure 1).

[0159] Figure 29 shows micrographs of the expression of CRX, Chx10, Pax6, RXy-γ, Brn3, Islet-1, and Prox1 in neural retina hES-NR (A-C) and hiPSC-NR (D-F) induced to differentiate from human ES cells and human iPS cells, respectively, on the 80th day after the start of suspension culture prepared by the above method. DAPI was used for nuclear staining. The scale bar in Figure 29 indicates 30 μm.

[0160] Chx10 and Pax6, markers of neural retinal progenitor cells, were expressed in the middle of the cell layer of hES-NR and hiPSC-NR (A, D in Fig. 29). Pax6 and Brn3, a marker of retinal ganglion cells, were expressed on the basement membrane side of the neural retina (A-B, D-E in Fig. 29). At the stage of 80 days after the start of suspension culture, Crx, a marker of photoreceptor precursors, and Rxr-γ, a marker of cone cells, cone photoreceptor precursors, and ganglion cells, were expressed in the apical layer (B, E in Fig. 29). Islet-1 and Prox1 were expressed on both the apical side and the basement membrane side (C, F in Fig. 29). Islet-1 and Prox1 are markers of inner retinal cells at the early differentiation stage.

[0161] Most Crx was located in the apical layer together with Rxr-γ, but not Brn3 (Fig. 29B and E).

[0162] hES-NRs and hiPSC-NRs expressed Islet-1, Prox1, and Brn3 (FIG. 29C, F). The results indicate the presence of retinal ganglion cells, amacrine cells, and horizontal cells.

[0163] <Example 2: HLA expression analysis of neural retina 1> Recombinant IFN-γ (100 ng / mL) (R&D Systems) was added to the culture medium of human ES / iPS cell-derived neural retina 80 to 100 days after the start of floating culture prepared in Example 1, and the cells were cultured for 2 days. A group was also prepared to which recombinant IFN-γ was not added.

[0164] After 2 days, the human ES / iPS cell-derived neural retina was washed with PBS, and a neural cell dispersion medium (WAKO) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were immunostained using anti-HLA class I antibodies (FITC anti-human HLA-A, -B, -C; Sigma-Aldrich, #F5662), anti-HLA class II antibodies (FITC anti-human HLA-DR, -DP, -DQ; DakoCytomation, #F0817 or BD PharMingen, #555558), anti-HLA E antibodies (Biolegend, #342604), anti-CD86 (B7-2) antibodies (eBioscience, #12-0862), and control isotype antibodies (mouse IgG2a, κ isotype control, FITC; BD PharMingen, #555573 or mouse IgG2b, κ isotype control, FITC; eBioscience, #11-4732). These immunostained cells were measured using flow cytometry (BD FACSCanto flow cytometer) and analyzed using FlowJo software. As a result, it was found that under conditions without the addition of recombinant interferon-γ (-), human ES / iPS cell-derived neural retina hardly expressed HLA-class I, and did not express HLA-class II, HLA-E, or CD86. On the other hand, under conditions with the addition of recombinant interferon-γ (+IFN-γ), it was found that HLA-class I was expressed but at a low level, HLA-E was hardly expressed, and HLA-class II and CD86 were not expressed (Figures 2A and 2B).

[0165] These results demonstrated that the neural retina differentiated from human ES / iPS cells has extremely low immunogenicity.

[0166] <Example 3: Analysis of HLA expression in neural retina 2> Recombinant IFN-γ (100 ng / mL) (R&D Systems) was added to the culture medium of the human ES cell-derived neural retina 27, 91, 149, or 239 days after the start of floating culture prepared in Example 1, and the cells were cultured for 2 days. On the other hand, a group was also prepared to which recombinant IFN-γ was not added. The human ES cell-derived neural retina 27, 91, 149, or 239 days after the start of floating culture typically have the following characteristics. 27 days after the start of floating culture: Chx10+ neural retinal progenitor cells are predominant, and Brn3+ / Pax6+ ganglion cells are also present. 91 days after the start of suspension culture: Rxrg+ / recoverin+ cone photoreceptors begin to appear. 149 days after the start of suspension culture: The number of various Crx+ photoreceptor cells on the apical surface increases. 239 days after the start of suspension culture: PKCα+ rod bipolar cells and GS+ / GFAP- Muller glial cells begin to appear.

[0167] After 2 days, the human ES / iPS cell-derived neural retina was washed with PBS and fixed with 4% PFA for 15 minutes at 4°C. After washing with PBS, it was immersed in 30% sucrose solution. Then, it was embedded in a cryomold using OCT compound, and 12 μm sections were prepared with a cryostat. These sections were immunostained with anti-HLA class I antibody (eBioscience, #14-9983) and HLA class II antibody (BD pharmingen, #555557). These immunostained cells were observed under a fluorescent microscope (Keyence Corporation). As a result, it was found that in the neural retina at any differentiation stage, under conditions without the addition of recombinant interferon-γ, the human ES / iPS cell-derived neural retina hardly expressed HLA-class I and did not express HLA-class II. On the other hand, under conditions with the addition of recombinant interferon-γ, it was found that HLA-class I was expressed but at a low level, and HLA-class II was not expressed (Figures 3 and 4).

[0168] <Example 4: Analysis of HLA expression in neural retina 3> The human ES cell-derived neural retina prepared in Example 1 from 80 to 100 days after the start of floating culture was transplanted subretinaly into immunodeficient retinal degeneration rats.

[0169] More than five months after transplantation, the eyes were removed, washed with PBS, and fixed with 4% PFA for 60 minutes at room temperature. After washing with PBS, the eyes were immersed in 30% sucrose solution. After embedding in a cryomold with OCT compound, 12-μm sections were prepared using a cryostat. These sections were immunostained with anti-HLA class I antibody (eBioscience, #14-9983), HLA class II antibody (BD pharmingen, #555557), Stem121 antibody (TaKaRa), and Stem123 (human GFAP) (TaKaRa). These immunostained sections were observed under a fluorescence microscope (Keyence Corporation).

[0170] As a result, it was found that after transplantation, some cells in the human ES cell-derived neural retina expressed HLA-class I, but the majority of photoreceptor cells did not express HLA-class II (Fig. 5A). Furthermore, since cells expressing HLA-class I were also stained for hGFAP, it was found that the cells expressing HLA class I after transplantation were likely to be Muller cells derived from the graft (Fig. 5B).

[0171] <Example 5: Evaluation 1 by immunogenicity test against neural retina immune cells> As samples for evaluating the immunogenicity of neural retina against immune cells, (1) human ES / iPS cell-derived neural retina (sample of neural retina not dispersed into single cells) 80 to 100 days after the start of suspension culture prepared in Example 1, (2) a sample in which the neural retina of (1) was washed with PBS, to which a neural cell dispersion liquid (manufactured by Sumitomo Bakelite Co., Ltd.) was added, incubated at 37°C, and then gently pipetted to prepare a semi-dissociated state, and (3) a sample in which the neural retina of (1) was completely dispersed into single cells were prepared. Blood was collected from healthy individuals who had given informed consent, and the collected PBMCs (peripheral blood mononuclear cells) were mixed and cultured for 2 days with the above (1), (2), and (3) using low-adhesive 24-well plates (manufactured by Sumitomo Bakelite Co., Ltd.). The microscopic observation results of (1), (2), and (3) for human ES-derived neural retina are shown in FIG. 6.

[0172] Two days later, PBMCs were collected and washed with PBS, and then immunostained with anti-APC-CD4 (BioLegend, #317416), anti-APC-CD8 (eBioscience, #17-0088-42), anti-APC-CD11b (Miltenyi Biotec, #130-091-241), anti-APC-NKG2A (Miltenyi Biotec, #130-098-809), and anti-PE-Ki-67 (BioLegend, #350504). Mouse IgG1κ isotype control APC (Miltenyi Biotec, #130-113-196), and mouse IgG1)κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as control antibodies. These immunostained cells were measured using a flow cytometer (BD FACSCanto flow cytometer) and analyzed using FlowJo software. Figures 7A, 7B, and 8 show the results of analysis using neural retina induced to differentiate from human ES cells, and Figures 9A, 9B, and 10 show the results of analysis using neural retina induced to differentiate from human iPS cells.

[0173] As a result, when the neural retina was mixed with the single-cell dispersed sample (3), a very weak rejection reaction of CD4+ T cells, CD8+ T cells, CD11b+ monocytes, and microglia was confirmed. On the other hand, in contrast to (3), the neural retina not dispersed into single cells and the semi-dissociated samples (1) and (2) did not induce an immune rejection reaction in CD4+ T cells, CD8+ T cells, CD11b+ monocytes, microglia, and NKG2A+ NK cells.

[0174] Regarding the expression of HLA class I in the human ES cell-derived neural retina, no significant difference was observed between sample (1) (whole retina) and sample (3) (Figure 37). On the other hand, as shown in Figures 7B and 9B, the results of measuring IFN-γ by ELISA showed that sample (1) (whole retina) strongly suppressed the expression of IFN-γ by PBMC in both human ES cell-derived neural retina and human iPS cell-derived neural retina (comparison of sample (1) (whole retina) with sample (2) (semi dissociate), sample (3) (single cell), and control).

[0175] These results indicate that human ES / iPS cell-derived neural retina and the semi-dissociated state are not subject to immune rejection, but rather have an inhibitory effect on immune cells.

[0176] <Example 6: Evaluation of ability to suppress immune cell activation (activated immune cells)> As a sample for evaluating the suppressive ability against activated immune cells by stimulation with the addition of CD3 and CD28 antibodies, human ES / iPS cell-derived neural retina (a sample of neural retina not dispersed into single cells) was prepared 80 to 100 days after the start of suspension culture produced in Example 1. Blood was collected from healthy subjects who gave informed consent, and the collected PBMCs (peripheral blood mononuclear cells) were mixed and cultured for 2 days in a 24-well plate (Sumitomo Bakelite Co., Ltd.).

[0177] Two days later, PBMCs were collected and washed with PBS. Immunostaining was performed using anti-APC-CD4 antibody (BioLegend, #317416), anti-APC-CD8 antibody (eBioscience, #17-0088), anti-APC-CD11b antibody (Miltenyi Biotec, #130-091-241), anti-APC-CD19 antibody, anti-NKG2A antibody, and anti-PE-Ki-67 antibody (BioLegend, #350504). In addition, mouse immunoglobulin 2a (IgG2a) κ isotype control fluorescein isothiocyanate (FITC) (BioLegend, #400208), mouse IgG1 κ isotype control APC, and mouse IgG1 (BioLegend, #400122) κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (FACSCanto flow cytometer, BD) and analyzed using FlowJo software. Figures 11 to 14 show the analysis results when neural retina differentiated from human iPS cells was used.

[0178] As a result, it was found that activation of activated CD4 positive T cells, CD8 positive T cells, CD11b positive monocytes, CD19 positive B cells, and NKG2A positive NK cells was suppressed (Fig. 11A, Fig. 11B, Fig. 12, Fig. 38). This effect was comparable to that of human iPS cell-derived RPE cells (Fig. 11A, Fig. 11B).

[0179] Control PBMCs (PBMCs not co-cultured with hESC-derived neural retina) proliferated and formed large aggregates, whereas PBMCs co-cultured with hESC-derived neural retina formed smaller aggregates, and the degree of aggregation was smaller the closer to the hESC-derived neural retina (Figure 39). The inhibitory effect of hESC-NR was evident in the vicinity of the neural retina induced to differentiate from human ES cells (hES-NR) in the co-culture dish. In addition, the inhibitory effect of immune cells in the activated state of the hES / iPS cell-derived neural retina was dose-dependent (Figure 40).

[0180] These results indicate that the human ES / iPS cell-derived neural retina suppresses the activation of immune cells even when the immune cells are activated, which means that the human iPS cell-derived neural retina has an immunosuppressive ability.

[0181] For the culture assay using Transwell®, a transwell chamber with a 0.3 mm pore size membrane (Corning Costar, Cambridge, MA) was prepared, along with the above-mentioned PBMCs (peripheral blood mononuclear cells) and RPMI 1640 containing recombinant IL2 (Sugita et al., 2015) as a medium.

[0182] In the presence of culture medium, PBMCs were added to the lower well of a transwell plate, and hESC-derived neural retina was added to the upper well. The hESC-derived neural retina was cultured in the upper well for 4-5 days. After 5 days of culture, PBMCs were harvested. The harvested PBMCs were stained with PE-conjugated Ki67 antibody and APC-conjugated CD4, CD8, CD11b, CD19, and NKG2A antibodies, and proliferation was evaluated by flow cytometry.

[0183] The immunosuppressive pattern of hESC-NR was similar in isolated co-cultures using Transwell®, indicating that secreted humoral molecules contribute to the immunosuppression induced by hESC / iPS cell-derived neural retina (Figure 41).

[0184] Compared with hiPS cells (hiPSCs), the human iPS cell-derived neural retina (hiPSC-NR) produced in Example 1 highly expressed TGF-β1, TGF-β2, and TGF-β3 (Figure 42). TGF-β1, TGF-β2, and TGF-β3 are also expressed by human iPS cell-derived retinal pigment epithelial cells (hiPSC-RPE cells). ELISA showed that TGF-β2 in the culture medium supernatant increased in a dose-dependent manner with hiPSC-NR (Figure 43). TGF-β2 was always secreted at different differentiation stages of hESC-NR from differentiation day (DD) 50 to differentiation day (DD) 240. Secretion of TGFβ2 showed the highest value at DD100 and DD160. The secretion levels of TGFβ2 at DD100 and DD160 were similar to those secreted by hiPSC-RPE cells (Figure 44).

[0185] Human iPSC-derived retinal pigment epithelial cells (hiPSC-RPE cells) were prepared as follows. RPE cells were induced by culturing hiPSCs in GMEM medium containing 10uM Y-27632 (Wako), 5uM SB431542 (Sigma), and 3uM CKI-7 (Sigma). When RPE cell-like colonies appeared, they were scraped off and transferred to dishes coated with CELL start (Thermo), and RPE cells were cultured in DMEM / F12 (Thermo) medium containing B27 (Thermo), bFGF (Wako), and SB431542 (SIGMA).

[0186] <Example 7: Evaluation of ability to suppress immune cell activation (TGFβ antibody, receptor inhibitor)> Human ES / iPS cell-derived neural retinas were prepared 80 to 100 days after the start of suspension culture as prepared in Example 1. Blood was collected from healthy individuals who provided informed consent, and the collected PBMCs and neural retinas were mixed and cultured for two days using a 24-well plate (manufactured by Sumitomo Bakelite Co., Ltd.) under the following four conditions: (1) PBMCs only, (2) PBMCs and neural retina mixed, (3) PBMCs and neural retina plus mouse IgG antibody (isotype control) added to the culture medium, (4) PBMCs and neural retina plus anti-TGFβ antibody (manufactured by R&D Systems) added to the culture medium, and (5) PBMCs and neural retina plus TGFβ receptor inhibitor SB431542 added to the culture medium.

[0187] After 2 days, PBMCs were collected and washed with PBS, and then immunostained using anti-human CD4 antibody (BioLegend, catalog #317416) and anti-human CD8 antibody (eBioscience, #17-0088). In addition, mouse IgG1 (BioLegend, #400122) and κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (BD FACSCanto flow cytometer) and analyzed using FlowJo software. As a result, it was found that the inhibition of activation of CD4 positive T cells and CD8 positive T cells was canceled under the conditions where TGFβ antibodies or TGFβ receptor inhibitors (4) and (5) were added to the culture medium, compared to the control (Figures 13 to 14).

[0188] These results indicate that human ES / iPS cell-derived neural retina suppresses the activation of immune cells primarily via TGFβ signaling.

[0189] <Example 8: Expression of TGFβ 1> The neural retina (iPS-3D retina) induced to differentiate from human iPS cells 80 to 100 days after the start of floating culture prepared in Example 1 was washed with PBS, and then a nerve cell dispersion liquid (manufactured by Sumitomo Bakelite Co., Ltd.) was added, and the cells were incubated at 37°C and dispersed into single cells. RNA samples were collected from these cells using a High Pure RNA Isolation Kit (manufactured by Roche). Then, cDNA was synthesized using a Transcriptor First Strand cDNA Synthesis Kit (Roche: 04 897 030 001) with a LightCycler. qPCR of TGFβ1, TGFβ2, and TGFβ3 was performed using a LightCycler 480 Probes Master (Roche: 04 707 494 001) kit. Measurements were performed using a LightCycler.

[0190] As a result, it was found that the expression of TGFβ1 was increased by about 2 to 4 times, TGFβ2 by about 100 times, and TGFβ3 by about 5 to 8 times compared to iPS cells (Figure 15).

[0191] <Example 9: Expression of TGFβ 2> The human ES cell-derived neural retina prepared in Example 1 80 to 100 days after the start of floating culture was washed with PBS, and then fixed with 4% PFA for 15 minutes at 4°C. After washing with PBS, it was immersed in 30% Sucrose solution. Then, it was embedded in a cryomold using OCT compound, and 12 μm sections were prepared with a cryostat. These sections were immunostained using anti-N-cadherin antibody (BD), anti-Ezrin antibody (R&D systems), anti-TGFβ1 antibody (R&D systems), TGFβ2 antibody (R&D systems), and anti-TGFβ3 antibody (R&D systems). These immunostained cells were observed with a confocal microscope (Leica SP8). As a result, it was found that all of TGFβ1, TGFβ2, and TGFβ3 were localized on the apical surface marker N-cadherin and Ezrin positive sides.

[0192] Therefore, it was suggested that TGF-β expressed in the neural retina was localized on the apical side and secreted (FIG. 16).

[0193] <Example 10: Measurement of TGFβ2 secretion amount by ELISA> The human ES cell-derived neural retinas prepared in Example 1 50, 100, 160, and 240 days after the start of suspension culture were washed with PBS, and then 15 neural retinas were placed in a 24-well plate (Sumitomo Bakelite) and cultured in 1 ml of DMEM / F12 (ThermoFisher) for 1 or 2 days.

[0194] After 1 and 2 days, the culture supernatant was collected and the amount of TGFβ2 secreted was measured using a TGFβ2 ELISA kit (R&D systems). The results showed that TGFβ2 was secreted at all stages of differentiation, and that at 100 days, a very large amount of TGFβ2 was secreted after two days of culture (Figure 17).

[0195] <Example 11: Relationship between TGFβ2 expression and ELISA with or without enzyme treatment> The human ES cell-derived neural retina 80-100 days after the start of suspension culture prepared in Example 1 was (1): untreated, (2): washed with PBS, treated with neural cell dispersion liquid (Sumitomo Bakelite), and collected without pipetting, (3): the neural retina of (1) was washed with PBS, to which neural cell dispersion liquid (Sumitomo Bakelite) was added, incubated at 37°C, and then lightly pipetted to prepare a semi-dissociated sample, and (4): the neural retina of (1) was completely dispersed into single cells. The samples were cultured for 2 days in 1 ml of DMEM / F12 (ThermoFisher) using a 24-well plate (Sumitomo Bakelite).

[0196] After 2 days, the culture supernatant was collected and the amount of TGFβ2 secreted was measured using a TGFβ2 ELISA kit (R&D systems). The measurement results are shown in Figure 18(a).

[0197] In addition, RNA samples were collected from cells (1), (2), (3), and (4) using the High Pure RNA Isolation Kit (Roche). Then, cDNA was synthesized using the Transcriptor First Strand cDNA Synthesis Kit (Roche: 04 897 030 001) in a LightCycler. qPCR of TGFβ2 was performed using the LightCycler 480 Probes Master (Roche: 04 707 494 001) kit. The measurement was performed using a LightCycler. The measurement results are shown in Figure 18(b).

[0198] Furthermore, the human ES cell-derived neural retina immediately after the enzyme treatments (1) and (2) was washed with PBS and then fixed with 4% PFA for 15 minutes at 4°C. After washing with PBS, it was immersed in 30% sucrose solution. It was then embedded in a cryomold using OCT compound and cut into 12 μm sections using a cryostat. These sections were immunostained using anti-N-cadherin antibody (BD) and TGFβ2 antibody (R&D systems). These immunostained cells were observed using a confocal microscope (Leica SP8).

[0199] As a result, TGFβ2 secretion was detected by ELISA in the untreated sample (1), but was below the detection limit in samples (2), (3), and (4). On the other hand, qPCR showed that the expression level was highest in sample (1), but was also expressed in samples (2), (3), and (4). In addition, observation with a confocal microscope revealed that TGFβ2 and the apical surface marker N-cadherin were aligned and localized on the surface without enzyme treatment, but the surface was disordered in the enzyme-treated sample (2), and the localization of TGFβ2 was sparse (Figure 19).

[0200] This suggests that the structure of the apical surface (for example, the apical surface being aligned) is important for the neural retina to secrete TGFβ2. On the other hand, it was found that the amount of secretion decreased when the structure of the matrix of the membrane surface was disrupted by enzyme treatment or other methods.

[0201] <Example 12: Differentiation days and MLR> Human ES cell-derived neural retinas were prepared 50, 80, 150, and 200 days after the start of the floating culture produced in Example 1. Blood was collected from healthy individuals who gave informed consent, and the PBMCs and neural retinas were collected and co-cultured in a 24-well plate (Sumitomo Bakelite Co., Ltd.).

[0202] After 2 days, PBMCs were collected and washed with PBS, and then immunostained using anti-human CD4 antibody (BioLegend, #317416), anti-human CD8 antibody (eBioscience, #17-0088), anti-CD19 antibody, and anti-NKG2A antibody. In addition, mouse IgG1 (BioLegend, #400122) and κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (FACSCanto flow cytometer, BD) and analyzed using FlowJo software. As a result, it was found that activation of immune cells was suppressed at all differentiation stages (Figure 20A, Figure 20B, Figure 21).

[0203] <Example 13> To examine the difference between autologous and allogeneic cells in the above MLR assay, blood was drawn from the donor of the iPS cells (TLHD2) and from a healthy individual who had given informed consent. The collected PBMCs and neural retina were made into single cells using a neural cell dispersion solution (Sumitomo Bakelite Co., Ltd.) and were co-cultured for two days in a low-adhesion 24-well plate (Sumitomo Bakelite Co., Ltd.).

[0204] Two days later, PBMCs were collected and washed with PBS. Immunostaining was performed using anti-APC-human CD4 antibody (BioLegend, #317416), anti-human APC-CD8 antibody (eBioscience, #17-0088), anti-APC-human CD11b antibody (Miltenyi Biotec, #130-091-241), anti-CD19 antibody, anti-APC-NKG2A antibody (Miltenyi Biotec, #130-098-809), and anti-human PE-Ki-67 antibody (BioLegend, #350504). In addition, mouse immunoglobulin 2a (IgG2a), κ isotype control fluorescein isothiocyanate (FITC) (BioLegend, #400208), mouse IgG1, κ isotype control APC (BioLegend, #400122), and mouse IgG1 (BioLegend, #400122), κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (FACSCanto flow cytometer, BD) and analyzed using FlowJo software. Figures 22 and 23 show the analysis results when neural retina differentiated from human iPS cells was used.

[0205] As a result, no immune activation was observed when autologous PBMCs were mixed with neural retina. In addition, for allogeneic allografts, very weak rejection reactions were observed in CD4+ T cells, CD8+ T cells, CD11b+ monocytes, and microglia, but no significant differences were observed.

[0206] These results indicate that human iPS cell-derived neural retinas are less susceptible to immune rejection if they are autologous, but do not induce significant rejection reactions even if they are allogeneic.

[0207] <Example 14: Preparation of neural retina (NR) from monkey iPS cells using SFEBq method> Monkey iPS cells (1121A1 line) established at Kyoto University were cultured under on-feeder conditions using MEFs (Millipore PMEF-CFX-C). The undifferentiated medium was a mixture of Primate ES medium (Reprocell) and mTeSR1 (STEMCELL TECHNOLOGIES) in a ratio of 3:1, and supplemented with 10 mg / ml bFGF (Wako) and 10 6 Each of the samples was used after adding 1000-fold dilution of LIF (Millipore) at 1.000 units / ml.

[0208] The specific maintenance culture procedure for monkey iPS cells was as follows. First, monkey iPS cells that had become subconfluent (about 60% of the culture area was covered with cells) were physically detached using a 1 ml tip, and the monkey iPS cells recovered in a colony state were seeded on a plastic culture dish seeded with MEFs, and cultured under on-feeder conditions in a Primate ES and mTeSR1 mixed medium in the presence of bFGF and LIF. As the plastic culture dish, a 60 mm dish or a 100 mm dish (manufactured by Iwaki, for cell culture) was used. After seeding, the medium was changed every day. The cells were then cultured until they became subconfluent.

[0209] After washing with PBS, the monkey iPS cells were detached using CTK (Reprocell) and collected in the form of colonies in a 15 ml tube. After leaving the cells to stand and confirming that the colonies had fallen to the bottom, the supernatant was removed using an aspirator. After washing with PBS, Tryple Select was added and incubated at 37°C for approximately 5 minutes. After dispersing into single cells by pipetting, the monkey iPS cells dispersed into single cells were placed in a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) at 1.2 × 10 per well. 4The cells were suspended in 100 μL of serum-free medium and cultured at 27 °C and 5% CO2. The serum-free medium (gfCDM + KSR) used was a 1:1 mixture of F-12 medium and IMDM medium, supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1 × chemically defined lipid concentrate.

[0210] At the start of suspension culture (day 0 after the start of suspension culture), Y27632 (ROCK inhibitor, final concentration 10-20 μM) and SAG (Shh signaling pathway active substance, 300 nM or 30 nM) were added to the serum-free medium. On the third day after the start of suspension culture, 50 μL of medium containing exogenous human recombinant BMP4 at a final concentration of 1.5 nM was added using a medium containing human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Co.) without Y27632 or SAG. From the sixth day after the start of suspension culture, half of the medium was replaced once every three days with medium containing no Y27632, SAG, or human recombinant BMP4.

[0211] The aggregates on the 15th day after the start of the floating culture were transferred to a 90 mm low-adhesion culture dish (manufactured by Sumitomo Bakelite Co., Ltd.) and cultured for 3 to 4 days at 37 ° C. and 5% CO2 in a serum-free medium (DMEM / F12 medium with 1% N2 Supplement) containing a substance acting on the Wnt signaling pathway (CHIR99021, 3 μM) and an FGF signaling pathway inhibitor (SU5402, 5 μM). Thereafter, the aggregates were cultured in a serum medium (NucT0 medium) not containing a substance acting on the Wnt signaling pathway and an inhibitor of the FGF signaling pathway in a 90 mm low-adhesion culture dish (manufactured by Sumitomo Bakelite Co., Ltd.). After the 40th day after the start of the floating culture (differentiation induction), the aggregates were cultured in a serum medium (NucT1 medium) not containing a substance acting on the Wnt signaling pathway and an inhibitor of the FGF signaling pathway. After the 60th day after the start of the floating culture (differentiation induction), the aggregates were cultured for a long period in NucT2 medium.

[0212] Observation using a microscope revealed that a neural retina (NR) with a layered structure was formed in the monkey iPS cell line (Figure 24).

[0213] The monkey iPS cell-derived neural retina was washed with PBS and fixed with 4% PFA for 15 minutes at 4°C. After washing with PBS, it was immersed in 30% Sucrose solution. After embedding in a cryomold with OCT compound, 12 μm sections were prepared with a cryostat. These sections were immunostained with anti-Recoverin antibody (Millipore), Pax6 antibody (BD pharmingen), CRX antibody (TaKaRa), Islet-1 (DSHB), and BRN3 (Santa Cruz). These immunostained cells were observed under a fluorescent microscope (Keyence Corporation). As a result, it was confirmed that the monkey iPS cell-derived neural retina underwent retinal differentiation similar to that of humans (Figure 24).

[0214] <Example 15> Recombinant IFN-γ (100 ng / mL) (R&D Systems) was added to the culture medium of monkey iPS cell-derived neural retina 45 to 55 days after the start of suspension culture prepared in Example 14, and the cells were cultured for 2 days. On the other hand, a group was also prepared to which recombinant IFN-γ was not added.

[0215] After 2 days, the monkey iPS cell-derived neural retina was washed with PBS, and then a neural cell dispersion solution (Sumitomo Bakelite Co., Ltd.) was added, and the cells were incubated at 37°C and dispersed into single cells. These cells were immunostained using anti-HLA class I antibodies (FITC anti-human HLA-A, -B, -C; Sigma-Aldrich, #F 5662), anti-HLA class II antibodies (FITC anti-human HLA-DR, -DP, -DQ; DakoCytomation, #F0817 or BD PharMingen, #555558), and control isotype antibodies (mouse IgG2a, κ isotype control, FITC; BD PharMingen, #555573 or mouse IgG2b, κ isotype control, FITC; eBioscience, #11-4732). These immunostained cells were measured using flow cytometry (BD FACSCanto flow cytometer) and analyzed using FlowJo software. As a result, it was found that in the condition where recombinant interferon-γ was not added, the neural retina differentiated from monkey iPS cells had almost no expression of MHC-classI (HLA-classI) and no expression of MHC-classII (HLA-classII). On the other hand, in the condition where recombinant interferon-γ was added, it was found that there was expression of MHC-classI (HLA-classI) but it was low, and there was no expression of MHC-classII (HLA-classII) (Figure 25).

[0216] These results demonstrated that the neural retina differentiated from monkey iPS cells has very low immunogenicity, similar to human ES / iPS cells.

[0217] <Example 16: MHC matched and unmatched monkey-monkey transplantation> The neural retina derived from monkey iPS cells prepared in Example 14 on days 45 to 55 after the start of suspension culture was transplanted into cynomolgus monkeys whose MHC type matched with the monkey iPS cells in which a photoreceptor degeneration model had been prepared by laser treatment, and into cynomolgus monkeys whose MHC type did not match. After transplantation, no immunosuppressants were administered.

[0218] Six months later, the eyes transplanted with monkey iPS cell-derived neural retina were washed with PBS and fixed with 4% PFA for 60 minutes at 4°C. After washing with PBS, the eyes were immersed in 30% sucrose solution. After embedding in cryomolds with OCT compound, 12 μm sections were prepared with a cryostat. These sections were immunostained with anti-Rhodopsin antibody (Sigma), anti-Recoverin antibody (Millipore), anti-PKCα antibody (R&D systems), and Cone arrestin antibody (R&D systems). These immunostained tissues were observed with a confocal microscope (Leica SP8). As a result, it was confirmed that rod and cone photoreceptors were engrafted and that Rhodopsin-positive rod photoreceptors were mature in both MHC-matched and unmatched monkeys.

[0219] Thus, it was demonstrated that photoreceptor cells survived and partially matured in both MHC-matched and non-matched monkeys, even without administration of immunosuppressants (FIGS. 26 and 27).

[0220] Example 17: Evaluation of the inhibitory ability of activated immune cells in the neural retina of living monkeys A neural retina (a sample of neural retina not dispersed into single cells) from a living monkey aged 8 years and 6 months was prepared as a sample for evaluating the suppressive ability against activated monkey-derived immune cells by stimulation with anti-CD3 and anti-CD28 antibodies. Blood was collected from an allogeneic monkey, and PBMC (peripheral blood mononuclear cells) were mixed and cultured for 2 days in a 24-well plate (Sumitomo Bakelite Co., Ltd.).

[0221] Two days later, PBMCs were collected and washed with PBS. Immunostaining was performed using anti-APC-CD4 antibody (BioLegend, #317416), anti-APC-CD8 antibody (eBioscience, #17-0088), anti-APC-CD11b antibody (Miltenyi Biotec, #130-091-241), anti-APC-CD19 antibody, anti-NKG2A antibody, and anti-PE-Ki-67 antibody (BioLegend, #350504). In addition, mouse immunoglobulin 2a (IgG2a) κ isotype control fluorescein isothiocyanate (FITC) (BioLegend, #400208), mouse IgG1 κ isotype control APC, and mouse IgG1 (BioLegend, #400122) κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (FACSCanto flow cytometer, BD) and analyzed using FlowJo software. Figure 28 shows the analysis results when using live monkey neural retina.

[0222] As a result, it was found that monkey-derived neural retina suppressed the activation state of activated CD4+ T cells, CD8+ T cells, CD11b+ monocytes and microglia, and NKG2A+ NK cells (Figure 28).

[0223] These results indicate that the neural retina derived from living monkeys suppresses the activation of immune cells even when the immune cells are activated, which means that the neural retina derived from living monkeys has an immunosuppressive ability.

[0224] <Example 18: Expression of HLA class I and II in human ES / iPSC-derived neural retina by IFN-γ stimulation> Recombinant IFN-γ (100 ng / mL) (R&D Systems) was added to the culture medium of human ES / iPS cell-derived neural retina 80 to 100 days after the start of floating culture prepared in Example 1, and the cells were cultured for 2 days. A group was also prepared to which recombinant IFN-γ was not added.

[0225] After 2 days, the human ES / iPS cell-derived neural retina was washed with PBS and added with neural cell dispersion solution (WAKO). After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were immunostained with anti-HLA class I antibodies (APC anti-human HLA-A, -B, -C; Bio Legend, #311410), anti-HLA class II antibodies (APC anti-human HLA-DR, -DP, -DQ; Bio Legend, #361714), anti-β2-microglobulin antibodies (APC Bio Legend, #316312), and control isotype antibodies (mouse IgG2a, κ isotype control, APC; Bio Legend, #400220). The immunostained cells were measured using flow cytometry (FACSCanto flow cytometer BD) and analyzed using FlowJo software.

[0226] To evaluate the antigen-presenting ability of human ES / iPS cell-derived neural retina (hES-NR / hiPSC-NR), the expression of immunogenicity-related molecules, such as human leukocyte antigen (HLA) class I and II, was examined by flow cytometry (Figures 30 and 31).

[0227] hES-NR / hiPSC-NR expressed lower levels of HLA class I and β2-microglobulin (β2-MGat) compared with RPE cells, but did not express HLA class II. Stimulation with the inflammatory cytokine interferon-gamma (IFN-γ) increased the expression of HLA class I.

[0228] The expression level of HLA class I in hiPSC-NR was lower than that in hiPSC-RPE (Figures 30 and 31), indicating that hESC-NR is less immunogenic than RPE cells.

[0229] INF-γ also increased the expression of β2-microglobulin (β2-MG), a protein that constitutes MHC class I molecules, HLA-E, and PD-L1 in both hESC-NRs and hiPSC-NRs. On the other hand, the expression of costimulatory factors CD40, 80, and 86 was not detected regardless of the presence or absence of INF-γ. Furthermore, CD47, a known immunosuppressive surface antigen, was expressed regardless of the presence or absence of INF-γ. The level of HLA class I expression was lower in Crx::venus+ photoreceptor precursors than in other populations (Figure 32, Figure 33).

[0230] Immunohistochemical analysis confirmed the upregulation of HLA class I in hESC-NRs after INF-γ stimulation, whereas it was barely detectable in Crx::Venus+ photoreceptor precursors without INF-γ stimulation (Figures 34-36).

[0231] Example 19: HLA expression levels and distribution of hESC-NR after transplantation Using a retinal degeneration model of Rho mutant nude rats (lymphocyte-depleted nude rats), we investigated HLA expression by transplanted human ES cell-derived neural retina (hES-NR).

[0232] Rho mutant nude rats (SD-Foxn1 Tg (S334ter) 3LavRrrc nude rats, Japan SLC Co., Ltd.) were prepared. Human ES / iPS cell-derived neural retinas 80 to 100 days after the start of suspension culture prepared in Example 1 were prepared as neural retinas for producing grafts.

[0233] A continuous epithelial portion of the neural retina for graft preparation was cut and used as a graft. Rats were anesthetized with ketamine hydrochloride (40-80 mg / kg) and xylazine (5-10 mg / kg) or inhalation of 3-5% isoflurane (Hung-ya et al., 2018EbioMedicine). The pupils were dilated with MydrinP (0.5% phenylephrine + 0.5% tropicamide; Santen Pharmaceutical Co., Ltd., Osaka, Japan). The graft was inserted into the subretinal space of SD-Foxn1Tg (from S334) 3LavRrc nude rats using a glass pipette.

[0234] HLA class I expression was not evident on the first day after graft insertion. However, 5 months after graft insertion, HLA class I-expressing cells were localized between the photoreceptor rosettes and the host RPE. Furthermore, these HLA class I-expressing cells were colocalized with human-specific glial fibrillary acidic protein (GFAP), and activated Müller glia derived from the graft showed HLA-Class I (Figure 46). Thus, after transplantation, HLA-Class I was expressed exclusively in activated Müller glia, and not in other photoreceptor cells, etc.

[0235] No expression of HLA class II was observed at any time point or in any area observed (Figure 45C).

[0236] <Example 20: Activation of NR immune cells with increased HLA-Class I expression by INF-g treatment> Recombinant IFN-γ (100 ng / mL) (R&D Systems) was added to the culture medium of human ES cell-derived neural retina 80 to 100 days after the start of floating culture prepared in Example 1, and the cells were cultured for 2 days. On the other hand, a group was also prepared in which recombinant IFN-γ was not added. Blood was collected from healthy subjects who gave informed consent, and the PBMCs and neural retina were collected and co-cultured in a 24-well plate (Sumitomo Bakelite Co., Ltd.).

[0237] After 5 days, PBMCs were collected and washed with PBS, and then immunostained using anti-human CD4 antibody (BioLegend, #317416), anti-human CD8 antibody (eBioscience, #17-0088), anti-CD19 antibody, and anti-NKG2A antibody. In addition, mouse IgG1 (BioLegend, #400122) and κ isotype control phycoerythrin (PE) (BioLegend, #400112) were used as isotype antibodies. These immunostained cells were measured using flow cytometry (FACSCanto flow cytometer BD) and analyzed using FlowJo software. As a result, it was found that hESC-NRs with INF-g treatment and increased HLA-Class I expression did not activate immune cells (Figure 47).

Claims

1. A therapeutic agent for a disease accompanied by damage to retinal cells or retinal tissue, The present invention relates to a retinal tissue that is derived from another source and has a three-dimensional structure. A therapeutic drug, wherein the target patient to whom the therapeutic drug is administered is a patient suffering from a disease accompanied by damage to retinal cells or retinal tissue, and the patient is not systemically administered an immunosuppressant for the purpose of preventing transplant rejection for one month or more after administration of the therapeutic drug.

2. The therapeutic agent according to claim 1 , wherein the target patient is a patient who is not systemically administered an immunosuppressant for the purpose of preventing transplant rejection before, during and / or after administration of the therapeutic agent.

3. 3. The therapeutic agent according to claim 1 or 2, wherein the target patient is a patient who has not been locally administered an immunosuppressant other than a steroidal anti-inflammatory agent and a calcineurin inhibitor for the purpose of preventing transplant rejection before, during and / or after administration of the therapeutic agent.

4. The therapeutic agent according to any one of claims 1 to 3, wherein the target patient is a patient to whom one or more immunosuppressants selected from the group consisting of triamcinolone, fluocinolone, and calcineurin inhibitors are locally administered before, during, and / or after administration of the therapeutic agent for the purpose of preventing transplant rejection.

5. The therapeutic agent according to claim 3, wherein the target patient is a patient who has not been locally administered a steroidal anti-inflammatory agent and / or a calcineurin inhibitor for the purpose of preventing transplant rejection after one month or more of administration of the therapeutic agent.

6. The therapeutic agent according to claim 1 , wherein the target patient is a patient who is not administered an immunosuppressant for the purpose of preventing transplant rejection before, during and / or after administration of the therapeutic agent.

7. The therapeutic agent according to any one of claims 1 to 6, which is substantially free of retinal pigment epithelial cells.

8. The therapeutic agent according to any one of claims 1 to 7, wherein TGFβ is present at least on the surface of the retinal tissue.

9. The therapeutic agent according to any one of claims 1 to 8, wherein the diameter of the retinal tissue in the long axis direction is 0.2 mm or more.

10. The total number of cells contained in one retinal tissue is 1 × 10 ４ The therapeutic agent according to any one of claims 1 to 9, which is a cell or more.

11. The therapeutic agent according to any one of claims 1 to 10, wherein the surface of the retinal tissue has a continuous epithelial structure in an area ratio of 50% or more.

12. The therapeutic agent according to any one of claims 1 to 11, wherein 50% or more of the total number of cells contained in one retinal tissue are cells expressing at least one of PAX6, Chx10, and Crx.

13. The therapeutic agent according to any one of claims 1 to 12, wherein the retinal tissue is derived from pluripotent stem cells.

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