Production method for sheet-like retinal tissue

Abstract

A production method for a cell aggregate containing a retinal tissue having a layer structure, the method comprising performing suspension culturing or adhesive culturing of a dispersed retinal cell population in a medium containing a Wnt signal transduction pathway acting substance, wherein the retinal cell population includes at least one type of cells selected from the group consisting of retinal precursor cells and photoreceptor precursor cells.

Description

Method for producing sheet-shaped retinal tissue

[0001] The present invention relates to a method for producing a sheet-shaped retinal tissue and a sheet-shaped retinal tissue.

[0002] In recent years, it has been reported that when photoreceptor progenitor cells at an appropriate differentiation stage are transplanted into the normal retina of living mice, they functionally survive (Non-Patent Document 1), demonstrating the possibility of transplantation therapy for photoreceptor degenerative diseases such as retinitis pigmentosa.

[0003] Numerous methods for inducing differentiation of pluripotent stem cells into three-dimensional retinal tissue by self-organizing culture have been reported, and it is becoming possible to produce and transplant three-dimensional retinal tissue with a layered structure. For example, a method for obtaining multilayered retinal tissue from pluripotent stem cells (Non-Patent Document 2 and Patent Document 1), a method for forming uniform pluripotent stem cell aggregates in a serum-free medium containing a substance that inhibits the Wnt signaling pathway, and then culturing the resulting aggregates in suspension in the presence of a basement membrane preparation, followed by culturing them in suspension in a serum medium, thereby obtaining multilayered retinal tissue (Non-Patent Document 3 and Patent Document 2), and a method for obtaining retinal tissue by culturing pluripotent stem cell aggregates in suspension in a medium containing a substance that acts on the BMP signaling pathway (Non-Patent Document 4 and Patent Document 3) have been reported. However, these retinal tissues are produced as spherical cell aggregates, and no method for producing sheet-like (flattened) retinal tissue is known.

[0004] On the other hand, it has been reported that Wnt2b has an effect on the formation of retinal epithelial structures in chickens (Non-Patent Document 5), but similar effects have not been reported in organisms other than chickens.

[0005] International Publication No. 2011 / 055855 International Publication No. 2013 / 077425 International Publication No. 2015 / 025967

[0006] Maclaren RE et al., “Retinal Repair by Transplantation of Photoreceptor Precursors”, Nature, 444, 203-207, (2006)Eiraku M. et al., “Self-organizing optic-cup morphogenesis in three-dimensional culture”, Nature, 472, 51-56, (2011)Nakano T. et al., “Self-formation of optic Cups and Storable Stratified Neural Retina From Human ESCs” Cell Stem Cell, 10(6), 771-785, (2012)Kuwahara A. et al., “Generation of a ciliary margin-like stem niche cell from self-organizing human retinal tissue” Nature Communications, 6, 6286, (2015)Nakagawa, S. et al., “Identification of the laminar-inducing factor Wnt-signal from the anterior rim induces correct laminar formation of the neural retina in vitro.” Developmental Biology, 260, 414-425, (2003)

[0007] In view of the above circumstances, the present invention aims to provide a method for regenerating the neuroepithelial structure of retinal tissue from retinal cells, a method for producing sheet-shaped retinal tissue using said method, and sheet-shaped retinal tissue. Secondary objectives of the present invention are to provide a method for purifying retinal progenitor cells, which are more preferable as starting cells, and a method for detaching large sheets of retinal tissue produced by the above method.

[0008] The present inventors conceived the idea of ​​producing sheet-like retinal tissue by re-forming a neuroepithelial structure using dispersed retinal cells as starting cells. However, they found that dissociating retinal cells disrupts the apical-basal polarity of the cells, preventing the re-formation of a neuroepithelial structure.

[0009] In order to address this issue, the present inventors conducted extensive research and found that the above-mentioned problem can be solved by adding a substance acting on the Wnt signaling pathway to dispersed retinal cells. Furthermore, the present inventors conducted extensive research to regenerate a better neuroepithelial structure. As a result, the present inventors further discovered that (1) further adding a ROCK inhibitor, a substance acting on the SHH signaling pathway, and / or a fibroblast growth factor, (2) culturing the cells on a culture plate coated with an extracellular matrix that serves as a scaffold for cell adhesion is effective in regenerating a better neuroepithelial structure, particularly in a sheet form, and (3) improving the purity of retinal progenitor cells used as starting cells can remove impure cells such as retinal pigment epithelial cells (RPE), thereby completing the present invention.

[0010] That is, the present invention relates to the following inventions: [1] A method for producing retinal tissue having an epithelial structure, comprising suspension culture or adhesion culture of a dispersed retinal cell population in a medium containing a substance acting on the Wnt signaling pathway, wherein the retinal cell population comprises one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells. [2] The production method according to [1], wherein the substance acting on the Wnt signaling pathway is one or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b, and Wnt3a. [3] The production method according to [1] or [2], wherein the medium further contains one or more substances selected from the group consisting of a ROCK inhibitor, a substance acting on the SHH signaling pathway, and a substance acting on the FGF signaling pathway. [4] The production method according to [3], wherein the ROCK inhibitor is one or more substances selected from the group consisting of Y-27632, Fasudil (HA1077), and H-1152. [5] The manufacturing method according to [3] or [4], wherein the substance acting on the SHH signaling pathway is one or more substances selected from the group consisting of SAG, PMA, and SHH. [6] The manufacturing method according to any of [3] to [5], wherein the substance acting on the FGF signaling pathway is one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4, and FGF8. [7] The manufacturing method according to any of [1] to [6], wherein the retinal tissue having an epithelial structure is a sheet-like retinal tissue, and the method comprises adhesion culture in a medium containing the substance acting on the Wnt signaling pathway. [8] The manufacturing method according to [7], wherein the adhesion culture is performed using a culture vessel coated with an extracellular matrix and / or a temperature-sensitive polymer. [9] The manufacturing method according to [8], wherein the culture surface of the culture vessel is coated with the temperature-sensitive polymer, and the upper surface of the temperature-sensitive polymer is coated with the extracellular matrix.

[10] The method according to [8] or [9], wherein the extracellular matrix is ​​one or more substances selected from the group consisting of collagen, laminin, fibronectin, matrigel, and vitronectin.

[11] The manufacturing method according to any one of [8] to

[10] , further comprising the step of detaching the sheet-like retinal tissue from the culture vessel by exposing the culture vessel coated with the temperature-sensitive polymer to a temperature at which the properties of the temperature-sensitive polymer change.

[12] The manufacturing method according to any one of [1] to

[11] , further comprising the step of dispersing cell aggregates containing one or more cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells to obtain the dispersed retinal cell population.

[13] The manufacturing method according to

[12] , further comprising the step of differentiating pluripotent stem cells to obtain cell aggregates containing one or more cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells.

[14] The manufacturing method according to any one of [1] to

[13] , further comprising the step of increasing the proportion of retinal progenitor cells in the dispersed retinal cell population before subjecting the dispersed retinal cell population to suspension culture or adhesion culture.

[15] The method according to

[14] , wherein contamination or differentiation of retinal pigment epithelial cells is suppressed.

[16] The method of

[14] or

[15] , wherein the step of increasing the proportion of retinal progenitor cells comprises contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, to obtain a cell population expressing the antigens.

[17] The method of

[16] , wherein the step of increasing the proportion of retinal progenitor cells comprises contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84, to obtain a cell population in which the expression levels of the antigens are below a reference value.

[18] The method according to any one of

[14] to

[17] , wherein the step of increasing the proportion of retinal progenitor cells comprises the following steps: (1) a step of culturing pluripotent stem cells in the presence of one or more selected from the group consisting of a substance acting on the Shh signaling pathway, ATP, and an A2A receptor agonist to produce cell aggregates; (2) a step of inducing the differentiation of the cell aggregates into retinal progenitor cells; and (3) a step of dispersing the cell aggregates and contacting them with a substance that binds to CD39.

[19] The manufacturing method according to any one of [1] to

[18] , wherein the retinal progenitor cells and / or the photoreceptor progenitor cells account for 50% or more of the total number of cells contained in the retinal cell population.

[20] The manufacturing method according to any one of [1] to

[18] , wherein the retinal progenitor cells and / or the photoreceptor progenitor cells account for 80% or more of the total number of cells contained in the retinal cell population.

[21] The manufacturing method according to any one of [1] to

[20] , wherein the dispersed retinal cell population is cultured in a medium containing the substance acting on the Wnt signaling pathway from the start of the suspension culture or adhesion culture.

[22] The manufacturing method according to any one of [1] to

[21] , wherein the orientation of the cells in the epithelial structure is approximately perpendicular to the layer direction.

[23] The manufacturing method according to any one of [1] to

[22] , further comprising a step of cutting out the retinal tissue having an epithelial structure obtained by suspension culture or adhesion culture to a size required for transplantation.

[24] The manufacturing method according to any one of [1] to

[23] , wherein the epithelial structure is a multi-layered structure.

[25] A sheet-like retinal tissue consisting of a retinal cell layer having a multilayer structure, wherein (1) the retinal cell layer having a multilayer structure has polarity of the basal surface and the apical surface, (2) the retinal cell layer having a multilayer structure comprises one or more types of cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells, (3) in each layer of the retinal cell layer, the orientation of the cells is approximately perpendicular to the layer direction, and (4) the diameter of the sheet-like retinal tissue is 8 mm or more.

[26] The sheet-like retinal tissue according to

[25] , wherein the retinal cell layer having a multilayer structure further comprises sheet-like retinal pigment epithelial cells joined to the retinal cell layer, the tangential directions of the surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells are approximately parallel, the apical surface of the retinal cell layer and the apical surface of the sheet-like retinal pigment epithelial cells face each other, and the retinal cell layer and the sheet-like retinal pigment epithelial cells are joined by adhesion factors present between them.

[27] The sheet-shaped retinal tissue according to

[26] , wherein the adhesion factor is an extracellular matrix or a hydrogel.

[28] The sheet-shaped retinal tissue according to

[27] , wherein the adhesion factor is one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.

[29] The sheet-shaped retinal tissue according to

[27] , wherein the adhesion factor is gelatin or fibrin.

[30] A pharmaceutical composition comprising the sheet-shaped retinal tissue according to any of

[25] to

[29] .

[31] A method for treating a disorder of retinal cells or retinal tissue or a disease caused by retinal tissue damage, comprising transplanting the sheet-shaped retinal tissue according to any of

[25] to

[29] into a subject in need of transplantation.

[32] A method for improving the proportion of retinal progenitor cells in a cell population, comprising the step of contacting a cell population containing retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of CD9, CD24, CD29, CD39, CD47, CD49b, CD49c, CD49f, CD57, CD73, CD82, CD90, CD164, CD200, CD340, and CXCR4.

[33] The method described in

[32] , which comprises the step of contacting the cell population containing retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4.

[34] The method described in

[32] or

[33] , which further comprises the step of contacting the cell population containing retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84.

[35] A cell population comprising retinal progenitor cells that are positive for at least one factor selected from the group consisting of CD9, CD39, CD90, and CXCR4 and that are Rx-positive, at 90% or more of the total cell number of the cell population.

[36] The cell population described in

[35] , wherein the retinal progenitor cells are negative for one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84.

[37] The sheet-shaped retinal tissue described in any of

[25] to

[29] , for use in treating a disease caused by a disorder of retinal cells or retinal tissue or damage to retinal tissue.

[38] Use of the sheet-shaped retinal tissue described in any of

[25] to

[29] , in the manufacture of a therapeutic agent for a disease caused by a disorder of retinal cells or retinal tissue or damage to retinal tissue.

[0011] According to the present invention, it is possible to provide a method for regenerating the layer structure of retinal tissue from retinal cells, a method for producing sheet-like retinal tissue using this method, and sheet-like retinal tissue.

[0012] In Example 1, KhES-1-derived aggregates were dispersed into single cells, and this is a bright-field micrograph showing the state of re-formation of the aggregates 1 day after seeding. In Example 1, 1231A3-derived aggregates were dispersed into single cells, and this is a bright-field micrograph showing the state of re-formation of the aggregates 1 day after seeding. In Example 2, KhES-1-derived aggregates were dispersed into single cells, and this is a bright-field micrograph showing the morphology of the aggregates re-formed 1 day after seeding. In Example 2, KhES-1-derived aggregates were dispersed into single cells, and this is a fluorescent micrograph showing the morphology of the aggregates re-formed 7 days after seeding. In Example 2, KhES-1-derived aggregates were dispersed into single cells, and this is a fluorescent micrograph showing the morphology of the aggregates re-formed 14 days after seeding. In Example 2, KhES-1-derived aggregates were dispersed into single cells, and this is a graph showing the results of measuring the area of ​​aggregates re-formed by KhES-1-derived single cells using Image J. (A) shows the area of ​​aggregates on days 1, 7, and 14 after seeding, and (B) shows the ratio (area ratio) of the area of ​​aggregates on days 7 and 14 to the area of ​​aggregates on day 1 after seeding. In Example 2, 1231A3-derived aggregates were dispersed into single cells, and bright-field micrographs showing the morphology of aggregates formed on day 1 after seeding. In Example 3, KhES-1-derived aggregates were dispersed into single cells, and bright-field micrographs showing the morphology of aggregates formed on day 14 after seeding. In Example 3, KhES-1-derived aggregates were dispersed into single cells, and bright-field and fluorescent micrographs showing the morphology of aggregates formed on day 28 after seeding. 1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of the aggregates that have been immunostained (DAPI, Rx::Venus, Chx10) 15 days after seeding, which were obtained by dispersing KhES-1-derived aggregates into single cells in Example 3. 1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of the aggregates that have been immunostained (β-catenin) 15 days after seeding, which were obtained by dispersing KhES-1-derived aggregates into single cells in Example 3.1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of KhES-1-derived aggregates dispersed into single cells and immunostained (Collagen IV, Zo-1) aggregates 15 days after seeding in Example 3. 2 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of KhES-1-derived aggregates dispersed into single cells and immunostained (DAPI, Chx10, Ki67, Pax6) aggregates 28 days after seeding in Example 3. 3 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of KhES-1-derived aggregates dispersed into single cells and immunostained (DAPI, Chx10, Ki67, Pax6) aggregates 28 days after seeding in Example 3. 4 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, Rx::Venus fluorescence aggregates 28 days after seeding in Example 3. 1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of the KhES-1-derived aggregates dispersed into single cells and immunostained (with Collagen IV and Zo-1) on day 28 after seeding in Example 3.

[0034] FIG. 1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of the KhES-1-derived aggregates dispersed into single cells and immunostained (with DAPI, Rx::Venus, Collagen IV and Zo-1) on day 28 after seeding in Example 3.

[0035] FIG. 1 is a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, sections of the KhES-1-derived aggregates dispersed into single cells and immunostained (with DAPI, Chx10, Ki67 and Pax6) on day 28 after seeding in Example 3.

[0033] In Example 3, KhES-1-derived aggregates were dispersed into single cells, and immunostained sections (CRX, RxRg, NRL, Recoverin) of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.

[0034] In Example 3, KhES-1-derived aggregates were dispersed into single cells, and immunostained sections (DAPI, Islet-1, Brn3, Calretinin) of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.

[0035] In Example 4, KhES-1-derived aggregates on days 18, 25, 40, 61, and 75 of differentiation were dispersed into single cells, and bright-field and fluorescent microscope photographs showing the state of the re-formed aggregates 3, 15, and 21 days after seeding were taken.In Example 5, KhES-1-derived aggregates (BMP4+ / BMP4-) on day 40 of differentiation were dispersed into single cells, and bright-field and fluorescent (Rx::Venus) microscope photographs were taken showing the state of the re-formed aggregates on days 3, 15, and 21 after seeding. In Example 6, fluorescent microscope photographs were taken of the morphology of the aggregates formed 1 and 7 days after seeding dispersed retinal system cells cryopreserved in various cryopreservation solutions. In Example 6, graphs were taken showing the viability (A) of dispersed retinal system cells cryopreserved in various cryopreservation solutions after waking them up, and the area (B) of the re-formed aggregates using the waking retinal system cells. In Example 7, photographs were taken of sections immunostained for basement membrane expressed in retinal tissue formed by suspension culture, using a confocal laser scanning fluorescent microscope. In Example 7, immunohistochemical staining images were taken showing the results of examining laminin isoforms expressed in mouse fetal neural retinal tissue.

[0033] Figure 1 shows confocal laser scanning fluorescence microscope photographs confirming the re-formation of sheet-like retinal tissue in adhesion culture from single-cell suspensions of retinal cells under conditions 1 to 3 in Example 8. Figure 2 shows confocal laser scanning fluorescence microscope photographs confirming the re-formation of sheet-like retinal tissue in adhesion culture from single-cell suspensions of retinal cells under conditions 1 to 3 in Example 8. Figure 3 shows bright-field and fluorescence microscope photographs showing the results of confirming the effects of various factors on the re-formation of sheet-like retinal tissue in adhesion culture in Example 9. Figure 4 shows confocal laser scanning fluorescence microscope photographs showing the results of confirming the effects of various factors on the re-formation of sheet-like retinal tissue in adhesion culture in Example 9. Figure 5 shows bright-field, fluorescence, and confocal laser scanning fluorescence microscope photographs showing the results of investigating the concentration and addition period of CHIR99021 on the re-formation of sheet-like retinal tissue in adhesion culture in Example 10. Figure 6 shows confocal laser scanning fluorescence microscope photographs showing the results of investigating the concentration and addition period of CHIR99021 on the re-formation of sheet-like retinal tissue in adhesion culture in Example 10. 11 shows bright-field micrographs, bright-field and fluorescent micrographs, and fluorescent micrographs showing the results of examining the scaffold when seeded onto a Transwell in the regeneration of sheet-like retinal tissue in adhesion culture in Example 11.12. Bright-field and fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. Confocal laser scanning fluorescent micrographs showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. 12 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. 14 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. 15 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesion culture in Example 12. 16 is a fluorescence microscope photograph showing the results of examining the maintenance effects of various factors on retinal cells in Example 13. 17 is a fluorescence microscope photograph showing the results of examining conditions effective for the maintenance culture of retinal progenitor cells in Example 14. 18 is a stereomicroscope photograph showing the results of re-sheeting on collagen in Example 15. 19 is a fluorescence microscope photograph showing a transplant graft prepared from the re-sheeted retinal cell sheet in Example 16. 10 shows fluorescent stereomicroscope and fluorescent microscopic photographs showing the results of observing the fundus of the retina after transplantation in Example 16.17 is a graph showing the results of FACS analysis for sorting an Rx::Venus-positive fraction from a cell population dispersed into single cells in Example 17. (A) is a dot plot, and (B) is a histogram plot. 18 is a stereomicroscope and a fluorescent stereomicroscope photographs of cell populations with and without sorting in Example 17. 19 is a confocal laser scanning fluorescent microscope photographs of cell populations with and without sorting in Example 17. 20 is a confocal laser scanning fluorescent microscope photographs of cell populations with and without sorting in Example 17. 21 is a bright-field microscope and a fluorescent microscope photographs of sorted cell populations in Example 17. 22 is a fluorescent microscope photographs of Rx::Venus-positive cell populations when various proteins are added in Example 18.

[0039] In Example 18, this is a fluorescence microscope photograph of an Rx::Venus-positive cell population when various low molecular weight compounds are added. In Example 18, this is a fluorescence microscope photograph of an Rx::Venus-positive cell population when different concentrations of FGF2, FGF4, and FGF8 are added. In Example 19, this is a bright-field microscope and a fluorescence microscope photograph showing the aggregate reformation and retinal differentiation effect by the addition of sorting and FGF8. In Example 19, this is a bright-field microscope and a fluorescence microscope photograph showing the aggregate reformation and retinal differentiation effect by the addition of sorting and FGF8, as well as a graph showing the results of FACS analysis. In Example 19, this is a bright-field microscope and a fluorescence microscope photograph showing the aggregate reformation and retinal differentiation effect by the addition of FGF8, as well as a graph showing the results of FACS analysis. In Example 20, this is a bright-field microscope and a fluorescence microscope photograph showing the effect of adding FGF8 on sheet re-formation. FIG. 21 is a bright-field microscope and a fluorescent microscope photograph showing the effect of adding FGF8 on re-formation of a sheet in Example 20. FIG. 22 is a bright-field microscope and a fluorescent microscope photograph showing the re-formation of a sheet and the effect of retinal differentiation by adding FGF8 in Example 20, and a graph showing the results of FACS analysis. FIG. 23 is a stereomicroscope and a fluorescent stereomicroscope photograph showing the time course of a re-formed retinal sheet in Example 21. FIG. 24 is a diagram showing markers focused on in surface antigen screening in Example 22.22 is a graph showing the results of cell sorting using various surface antigens in Example 22. 23 is a graph showing the results of cell sorting using various surface antigens in Example 22. 24 is a graph showing the results of cell sorting using various surface antigens in Example 22. 25 is a graph showing the results of cell sorting using various surface antigens in Example 22. 26 is a graph showing the results of cell sorting using various surface antigens in Example 22. 27 is a graph showing the results of cell sorting using various surface antigens in Example 22. 28 is a bright-field microscope and a fluorescence microscope photograph showing the state of differentiation into brain organoid in Example 23. 29 is a confocal laser scanning fluorescence microscope photograph showing the state of differentiation into brain organoid in Example 23. 30 is a graph showing the results of FACS analysis of the expression of CD39, CD73, and CXCR4 in brain organoid in Example 23. 31 is a bright-field microscope and a fluorescence microscope photograph showing the results of differentiation induction by SAG at different concentrations in Example 24. 24 shows the results of FACS analysis of CD39 and CXCR4 expression after differentiation induction with SAG at different concentrations and a numerical graph in Example 24. 25 shows confocal laser scanning fluorescence microscope photographs of immunostained images of tissues induced to differentiate with SAG at different concentrations in Example 24. 26 shows confocal laser scanning fluorescence microscope photographs of immunostained images of tissues induced to differentiate with SAG at different concentrations in Example 24. 27 shows FACS analysis of CD39 and BV421 expression after differentiation induction with SAG at different concentrations and confocal laser scanning fluorescence microscope photographs of immunostained images of tissues induced to differentiate with SAG at different concentrations in Example 24. 28 shows confocal laser scanning fluorescence microscope photographs of immunostained images of tissues induced to differentiate with SAG at different concentrations in Example 24. 29 shows confocal laser scanning fluorescence microscope photographs of immunostained images of tissues induced to differentiate with SAG at different concentrations in Example 24. 1 is a graph showing the results of FACS analysis, illustrating the results of investigating substances capable of enhancing CD39 expression in Example 25. FIG. 2 is a graph showing the numerical results of FACS analysis, illustrating the results of investigating substances capable of enhancing CD39 expression in Example 25.(A) shows the results of CD39 positivity and RX::venus positivity, and (B) shows the results of CXCR4 negativity and RX::venus positivity. In Example 26, bright-field and fluorescent micrographs are shown showing the observation of Islet-1 KO hESC-retina sheets (dd74) prepared with FGF8- and FGF8+, and the process of cutting out grafts for transplantation. In Example 27, FACS dot plots are shown showing the results of screening various surface antigens of NR in comparison with Brain Organoid. In Example 27, FACS dot plots are shown showing the results of screening various surface antigens of NR. In Example 28, FACS dot plots are shown showing the results of changes in CD9 expression over time in hESC-retina. In Example 29, FACS dot plots are shown showing the results of analyzing the expression of CD9 and SSEA-1 in hESC-retina. 30 is a FACS dot plot showing the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Example 30. 31 is a FACS dot plot showing the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Example 30. 32 is a graph showing the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Example 30. 33 is a photograph showing the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Example 30. 34 is a photograph showing the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Example 30. 35 is a FACS diagram showing the results of changes in CD9 and SSEA-1 expression over time in hESC-retina in Example 31. 1 is a schematic diagram showing the process of investigating the composite formation of an RPE sheet and a retinal sheet using gelatin in Example 32. 2 is a stereomicroscope and fluorescence micrographs showing the RPE sheet and re-formed neural retina cultured on the Transwell used for the composite formation using gelatin in Example 32. 3 is a stereomicroscope photograph showing the process of detaching the RPE sheet and re-formed neural retina from the Transwell and adding gelatin in Example 32.32. Stereomicroscope photographs showing the process of adding gelatin to an RPE sheet and a re-sheeted neural retina in Example 32. Stereomicroscope and fluorescent microscope photographs showing the process of combining an RPE sheet and a re-sheeted neural retina in Example 32. Stereomicroscope and fluorescent microscope photographs showing the RPE sheet and a re-sheeted neural retina being lifted with tweezers after combination in Example 32. Stereomicroscope and fluorescent microscope photographs showing the RPE sheet and a re-sheeted neural retina being excised with scissors after combination in Example 32. Stereomicroscope and fluorescent microscope photographs observing the cross-sections of excised composite RPE sheets and re-sheeted neural retina in Example 32. Stereomicroscope and fluorescent microscope photographs observing the suction and ejection of excised composite RPE sheets and re-sheeted neural retina in Example 32. Stereomicroscope and fluorescent microscope photographs observing the RPE sheet and re-sheeted neural retina cultured on the Transwell used for combination using fibron in Example 33.

[0033] Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the process of examining the combination of an RPE sheet and a re-sheeted neural retina by detaching them from a Transwell in Example 33. Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the process of examining the combination of an RPE sheet and a re-sheeted neural retina by adding fibrinogen and thorombin to a recovered RPE sheet and a re-sheeted neural retina in Example 33. Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the process of combining an RPE sheet and a re-sheeted neural retina in Example 33. Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the process of combining an RPE sheet and a re-sheeted neural retina by using tweezers after combining an RPE sheet and a re-sheeted neural retina in Example 33. Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the combined state of an RPE sheet and a re-sheeted neural retina in Example 33. Figure 33 shows stereomicroscopic and fluorescent microscopic photographs showing the process of examining the combination of an RPE sheet by detaching it from a mesh of a Transwell in Example 33.36 is a schematic diagram and photographs showing the results of examining the discharge of unnecessary hydrogel using a cell sifter in Example 34. 37 is a photograph showing the process of preparing and peeling off a planarized sheet on a temperature-sensitive culture dish in Example 35.

[0013] [Definition] "Stem cells" refer to undifferentiated cells that have the ability to differentiate and proliferate (especially the ability to self-renew). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential. Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues. Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues or cells.

[0014] "Pluripotent stem cells" can be induced from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Muse cells (multi-lineage differentiating stress-enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells prepared from germ cells (e.g., testes) are also included in pluripotent stem cells.

[0015] Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described, for example, in WO 96 / 22362, WO 02 / 101057, US 5,843,780, US 6,200,806, and US 6,280,718. Embryonic stem cells are available from designated institutions and are also commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Human embryonic stem cells Crx::Venus and Rx::Venus (both derived from KhES-1) are available from RIKEN, a national research and development agency.

[0016] "Induced pluripotent stem cells" are cells in which pluripotency has been induced by reprogramming somatic cells using known methods.

[0017] In 2006, Yamanaka et al. established induced pluripotent stem cells from mouse cells (Cell, 2006, 126(4), pp. 663-676). In 2007, induced pluripotent stem cells were also established from human fibroblasts, and they possess the same pluripotency and self-renewal capacity 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).

[0018] Specifically, induced pluripotent stem cells include cells in which pluripotency is induced by reprogramming somatic cells differentiated into fibroblasts or peripheral blood mononuclear cells by expressing any combination of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. Preferred combinations of reprogramming factors include (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013; 31: 458-466).

[0019] In addition to methods for producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds, etc. (Science, 2013, 341, pp. 651-654).

[0020] It is also possible to obtain established induced pluripotent stem cell lines, for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University, which are available from Kyoto University and iPS Academia Japan, Inc. As established induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University are available from Kyoto University.

[0021] In this specification, pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0022] In this specification, pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0023] Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture by methods well known to those skilled in the art.

[0024] "Nervous tissue" refers to tissue composed of nervous system cells, such as the cerebrum, midbrain, cerebellum, spinal cord, retina, peripheral nerves, forebrain, hindbrain, telencephalon, diencephalon, etc., at the developmental or adult stage. Nervous tissue may form a stratified epithelial structure (neuroepithelium), and the abundance of neuroepithelium in cell aggregates can be evaluated by bright-field observation using an optical microscope.

[0025] "Neural cells" refer to cells of ectoderm-derived tissues other than epidermal cells. Specifically, they include cells such as neural progenitor cells, neurons (nerve cells), glia, neural stem cells, neuronal progenitor cells, and glial progenitor cells. Neural cells also include cells that constitute retinal tissue (retinal cells), retinal progenitor cells, retinal layer-specific neurons, neural retinal cells, and retinal pigment epithelial cells, which are described below. Neural cells can be identified using markers such as nestin, TuJ1, PSA-NCAM, and N-cadherin.

[0026] "Neurons (neuron / neuronal cells)" are functional cells that form neural circuits and contribute to signal transmission, and can be identified using the expression of immature neuron markers such as TuJ1, Dcx, and HuC / D, and / or mature neuron markers such as Map2 and NeuN.

[0027] "Neural precursor cells" are a collection of precursor cells including neural stem cells, neuronal precursor cells, and glial precursor cells, and have the ability to proliferate and produce neurons and glia. Neural precursor cells can be identified using markers such as Nestin, GLAST, Sox2, Sox1, Musashi, and Pax6. Alternatively, cells that are positive for neural cell markers and proliferation markers (Ki67, pH3, MCM) can also be identified as neural precursor cells.

[0028] "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 in 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] The term "retinal cells" refers to cells that constitute each retinal layer in a living retina, or their progenitor 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 bodies, their progenitor cells (e.g., photoreceptor progenitors, bipolar cell progenitors, retinal pigment epithelial progenitors, etc.), neural retinal progenitor cells, retinal progenitor cells, etc. Among retinal cells, cells that constitute the neural retinal layer (also referred to as neural retina cells or neural retina-related cells) specifically include 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, and their precursor cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, etc.). In other words, neural retinal cells do not include retinal pigment epithelial cells and ciliary body cells.

[0030] "Mature retinal cells" refers to cells that can be contained in the retinal tissue of an adult human, and specifically refers to differentiated cells such as 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 cells, etc. "Immature retinal cells" refers to precursor cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, retinal precursor cells, etc.) that are committed to differentiating into mature retinal cells.

[0031] Photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, Müller glial precursor cells, and retinal pigment epithelial precursor cells refer to precursor cells that are committed to differentiating into photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cells, respectively.

[0032] The term "retinal progenitor cells" refers to precursor cells that can differentiate into any immature retinal cell, such as a photoreceptor precursor cell, horizontal cell precursor cell, bipolar cell precursor cell, amacrine cell precursor cell, retinal ganglion cell precursor cell, Müller glial cell, or retinal pigment epithelial precursor cell, and ultimately can differentiate into any mature retinal cell, such as a photoreceptor cell, rod photoreceptor cell, cone photoreceptor cell, horizontal cell, bipolar cell, amacrine cell, retinal ganglion cell, or retinal pigment epithelial cell. "Neural retinal progenitor cells" refer to precursor cells that can differentiate into any immature neural retinal cell, such as photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, and Müller glial cells, and ultimately to precursor cells that can differentiate into any mature neural retinal cell, such as photoreceptors, rod photoreceptors, cone photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Neural retinal progenitor cells do not have the ability to differentiate into retinal pigment epithelial cells.

[0033] "Photoreceptor cells" are present in the photoreceptor layer of the retina in living organisms 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 bright light and rods that function in dark light (referred to as cone photoreceptors and rod photoreceptors, respectively). Examples of cone photoreceptors include S-cone photoreceptors that express S-opsin and receive blue light, L-cone photoreceptors that express L-opsin and receive red light, and M-cone photoreceptors that express M-opsin and receive green light. Photoreceptors differentiate and mature from photoreceptor precursor cells. Those skilled in the art can easily confirm whether a cell is a photoreceptor cell or a photoreceptor precursor cell by, for example, the expression of cell markers described below (Crx and Blimp1 expressed in photoreceptor precursor cells, Recoverin expressed in photoreceptors, rhodopsin, S-Opsin, and M / L-Opsin expressed in mature photoreceptors, etc.), the formation of an outer segment structure, etc. In one aspect, the photoreceptor precursor cells are Crx-positive cells, and the photoreceptors are rhodopsin-, S-Opsin-, and M / L-Opsin-positive cells. In one aspect, the rod photoreceptors are NRL- and Rhodopsin-positive cells. In one aspect, the S cone photoreceptors are S-opsin-positive cells, the L cone photoreceptors are L-opsin-positive cells, and the M cone photoreceptors are M-opsin-positive cells.

[0034] The presence of neural retinal cells can be confirmed by the presence or absence of expression of neural retinal cell-associated genes (hereinafter sometimes referred to as "neural retinal cell markers" or "neural retinal markers"). The presence or absence of expression of a neural retinal cell marker, or the proportion of neural retinal cell marker-positive cells in a cell population or tissue, can be easily confirmed by those skilled in the art. Examples include antibody-based techniques, nucleic acid primer-based techniques, and sequencing techniques. Antibody-based techniques can confirm the protein expression of a neural retinal cell marker by, for example, flow cytometry (FACS) or immunostaining using commercially available antibodies, by dividing the number of cells positive for a specific neural retinal cell marker by the total number of cells. Nucleic acid primer-based techniques can confirm the RNA expression of a neural retinal cell marker by, for example, PCR, semi-quantitative PCR, or quantitative PCR (e.g., real-time PCR). Sequencing techniques can confirm the RNA expression of a neural retinal cell marker by, for example, a nucleic acid sequencer (e.g., a next-generation sequencer).

[0035] Examples of neural retinal cell markers include Rx (also referred to as Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6 and Chx10 (also referred to as Vsx2) expressed in neural retinal progenitor cells, Crx and Blimp1 expressed in photoreceptor progenitor cells, etc. In addition, Chx10 strongly expressed in bipolar cells, PKCα, Goα, VSX1 and L7 expressed in bipolar cells, TuJ1 and Brn3 expressed in retinal ganglion cells, calretinin and HPC-1 expressed in amacrine cells, calbindin expressed in horizontal cells, recoverin expressed in photoreceptor cells and photoreceptor progenitor cells, rhodopsin expressed in rod cells, rod photoreceptor cells and Examples of surface antigens include Nrl, which is expressed in rod photoreceptor progenitor cells, S-opsin and LM-opsin, which are expressed in cone photoreceptor cells, cone photoreceptor progenitor cells, and ganglion cells; TRβ2, OTX2, and OC2, which are expressed in cone photoreceptor cells or their progenitor cells that appear in the early differentiation stage of cone photoreceptor cells; and Pax6, which is expressed in common in horizontal cells, amacrine cells, and ganglion cells. The surface antigens of retinal progenitor cells or neural retinal progenitor cells identified in the examples of the present application can also be used as markers of retinal progenitor cells or neural retinal progenitor cells. Details will be described later.

[0036] "Positive cells" refer to cells that express a particular marker on the cell surface or intracellularly. For example, "Chx10-positive cells" refer to cells that express Chx10 protein.

[0037] "Retinal pigment epithelial cells" refer to epithelial cells present outside the neural retina in a living retina. Whether a cell is a retinal pigment epithelial cell can be easily confirmed by those skilled in the art, for example, by the expression of cell markers (MITF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (black-brown), tight junctions between cells, and characteristic polygonal or cobblestone-like cell morphology. Whether a cell has the function of a retinal pigment epithelial cell can be easily confirmed by the ability to secrete cytokines such as VEGF and PEDF, the phagocytic ability of photoreceptor outer segments, etc. In one embodiment, the retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.

[0038] The term "retinal pigment epithelial cell sheet" refers to a monolayer or multilayer sheet-like structure composed of single or multiple retinal pigment epithelial cells that are biologically bonded to one another at least in two dimensions.

[0039] The term "retinal layer" refers to 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.

[0040] "Neural retinal layer" refers to each layer that constitutes the neural retina, and specifically includes the photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. "Photoreceptor layer" refers to the retinal layer that is formed on the outermost side of the neural retina and contains a large number of one or more cells selected from the group consisting of photoreceptors (rod photoreceptors, cone photoreceptors), photoreceptor precursor cells, and retinal progenitor cells. Layers other than the photoreceptor layer are called inner layers. Which retinal layer each cell constitutes can be confirmed by known methods, such as the presence or absence of expression or the degree of expression of a cell marker.

[0041] In retinal tissue at a stage where the proportion of photoreceptors or photoreceptor precursor cells is low, the layer containing proliferating neural retinal precursor cells is called the "neuroblastic layer," and it comprises an inner neuroblastic layer and an outer neuroblastic layer. Those skilled in the art can distinguish between these layers by well-known methods, such as by the color shading (the outer neuroblastic layer is light and the inner neuroblastic layer is dark) under a bright-field microscope.

[0042] The term "ciliary body" includes the "ciliary body" during development and in adults, the "ciliary margin," and the "ciliary body." Markers for the "ciliary body" include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The "ciliary marginal zone (CMZ)" can be, for example, a tissue present in the boundary region between the neural retina and the retinal pigment epithelium in a living retina, and can include a region containing retinal tissue stem cells (retinal stem cells). The ciliary margin is also called the ciliary margin or retinal margin, and the ciliary margin, ciliary margin, and retinal margin are equivalent tissues. The ciliary margin is known to play an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, etc. Marker genes for the ciliary margin include, for example, the Rdh10 gene (positive), the Otx1 gene (positive), and the Zic1 (positive). The "ciliary margin-like structure" refers to a structure similar to the ciliary margin.

[0043] "Cerebral tissue" refers to tissue in which one or at least multiple types of cells constituting the fetal or adult cerebrum (e.g., cortical neural precursor cells, dorsal cerebral nervous system precursor cells, ventral cerebral nervous system precursor cells, cerebral layer structure-specific nerve cells (neurons), layer I neurons, layer II neurons, layer III neurons, layer IV neurons, layer V neurons, layer VI neurons, glial cells (astrocytes and oligodendrocytes), their precursor cells, etc.) are arranged three-dimensionally in layers. The fetal cerebrum is also called the forebrain or telencephalon. The presence of each cell can be confirmed by known methods, such as the presence or absence of expression of cell markers or the level thereof.

[0044] "Cerebral layers" refers to each layer that constitutes the adult or fetal cerebrum, and specifically includes the molecular layer, external granular layer, external pyramidal cell layer, internal granular layer, neuronal cell layer (inner pyramidal cell layer), polymorphonuclear cell layer, layer I, layer II, layer III, layer IV, layer V, layer VI, cortical zone, intermediate zone, subventricular zone, and ventricular zone.

[0045] Examples of "cerebral nervous system progenitor cells" include neuron progenitor cells, layer 1 neuron progenitor cells, layer 2 neuron progenitor cells, layer 3 neuron progenitor cells, layer 4 neuron progenitor cells, layer 5 neuron progenitor cells, layer 6 neuron progenitor cells, astrocyte progenitor cells, oligodendrocyte progenitor cells, etc. Each cell is a progenitor cell that is committed to differentiate into layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes.

[0046] "Cerebral nervous system progenitor cells" include multipotent stem cells (multipotent neural stem cells) that have the ability to differentiate (multi-differentiate) into at least several lineages among layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes.

[0047] "Cerebral layer-specific neurons" refer to cells that constitute the cerebral layers and are specific to the cerebral layers. Examples of cerebral layer-specific neurons include layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, cerebral excitatory neurons, and cerebral inhibitory neurons.

[0048] Cerebral cell markers include FoxG1 (also known as Bf1), which is expressed in cerebral cells; Sox2 and Nestin, which are expressed in cerebral nervous system progenitor cells; Pax6 and Emx2, which are expressed in dorsal cerebral nervous system progenitor cells; Dlx1, Dlx2, and Nkx2.1, which are expressed in ventral cerebral nervous system progenitor cells; Tbr2, Nex, and Svet1, which are expressed in neuronal progenitor cells; Tbr1, which is expressed in layer 6 neurons; Ctip2, which is expressed in layer 5 neurons; RORβ, which is expressed in layer 4 neurons; Cux1 or Brn2, which is expressed in layer 3 or layer 2 neurons; and Reelin, which is expressed in layer 1 neurons.

[0049] The term "cell aggregate" is not particularly limited as long as it is a three-dimensional structure formed by adhesion of multiple cells to each other, and refers to, for example, a mass formed by the aggregation of cells dispersed in a medium such as a culture medium, or a mass of cells formed through cell division. Cell aggregates also include those that form specific tissues.

[0050] "Spherical cell aggregate" refers to a cell aggregate having a three-dimensional shape that is close to a sphere. A three-dimensional shape that is close to a sphere refers to a shape having a three-dimensional structure, such as a spherical shape that appears circular or elliptical when projected onto a two-dimensional surface, and a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by two to four overlapping circular or elliptical shapes when projected onto a two-dimensional surface). In one aspect, the core of the aggregate has a vesicular layered structure and is characterized by being dark in the center and bright in the outer periphery when observed under a bright-field microscope.

[0051] "Epithelial tissue" refers to tissue formed by cells tightly covering the surface of the body, a lumen (such as the digestive tract), a body cavity (such as the pericardial cavity), etc. Cells forming epithelial tissue are called epithelial cells. Epithelial cells have apical-basal polarity. Epithelial cells form strong bonds with each other through adherens junctions and / or tight junctions, and can form cell layers. Epithelial tissue is a tissue formed by one to a dozen or so cell layers stacked together. Tissues that can form epithelial tissue include fetal and / or adult retinal tissue, cerebrospinal tissue, ocular tissue, nervous tissue, etc. The neural retina in this specification is also epithelial tissue. "Epithelial structure" refers to a structure characteristic of epithelial tissue (e.g., having basal and apical polarity).

[0052] "Continuous epithelial tissue" refers to tissue that has a continuous epithelial structure. A continuous epithelial structure refers to a state in which epithelial tissue is continuous. For example, continuous epithelial tissue refers to a state in which 10 cells to 10 cells are connected in a tangential direction to the epithelial tissue. ７ cells, preferably 30 cells to 10 ７ cells, more preferably 10 ２ cells ~10 ７ It refers to the state of cells lined up.

[0053] For example, a continuous epithelial structure formed in retinal tissue has an apical surface characteristic of epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to and continuous with at least the photoreceptor layer (outer nuclear layer) among the layers forming the neural retina. For example, in the case of a cell aggregate containing retinal tissue prepared from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and 10 or more, preferably 30 or more, more preferably 100 or more, and even more preferably 400 or more photoreceptor cells or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface.

[0054] In one aspect, epithelial tissue is polarized to form an "apical surface," a "basal surface," and a "basement membrane." The "basement membrane" refers to the basal layer (basement membrane) produced by epithelial cells, which contains a lot of laminin and type IV collagen and has a thickness of 50 to 100 nm. The "basal surface" refers to the surface (superficial surface) formed on the "basement membrane" side. The "apical surface" refers to the surface (superficial surface) formed on the opposite side of the "basement membrane." In one aspect, the "apical surface" refers to the surface in contact with the photoreceptor layer (external nuclear layer) where the outer limiting membrane is formed and photoreceptors and photoreceptor precursor cells are present in retinal tissue whose developmental stage has progressed to the point where photoreceptors or photoreceptor precursor cells can be observed, in which the outer limiting membrane is formed and the photoreceptor precursor cells are present. Furthermore, such apical surfaces 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"), tight junction marker (Zo-1), ERM proteins Ezrin, E-cadherin, and N-cadherin).

[0055] [Method for producing cell aggregates] One aspect of the present invention is a method for producing cell aggregates comprising retinal tissue having an epithelial structure (or multi-layered structure) from a dispersed retinal cell population (herein, a "cell aggregate comprising retinal tissue" may be simply referred to as "retinal tissue"). The method comprises suspension or adhesion culture of the dispersed retinal cell population in a medium containing a substance acting on the Wnt signaling pathway. Here, the dispersed retinal cell population comprises one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells. Retinal tissue is as defined above. In one aspect, the retinal tissue comprises neural retinal tissue, and may also comprise both neural retinal tissue and retinal pigment epithelial tissue.

[0056] The retinal system cells are as defined above. In one embodiment, the retinal system cells include one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells, and may also include other cells such as photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), and Müller glial cells. The retinal system cells are preferably neural retinal system cells. The retinal system cell population used may be derived from a living tissue or from pluripotent stem cells, and is preferably obtained by inducing differentiation of pluripotent stem cells.

[0057] Those skilled in the art can prepare retinal cells derived from biological tissue from retinal tissue using well-known techniques, such as a method of recovering the cells by detaching them from the RPE after eye removal, or a method of directly extracting the neural retina.

[0058] One aspect of the present invention is a method for producing cell aggregates comprising neural tissue (e.g., cerebral (telencephalic) tissue) with an epithelial structure (or multilayered structure) from a dissociated neural cell population. The method comprises culturing the dissociated neural cell population in an adherent or suspension culture medium containing a substance acting on the Wnt signaling pathway. The neural cells preferably comprise neural progenitor cells (e.g., cerebral neural progenitor cells). The neural cell population used may be derived from a living tissue or from pluripotent stem cells, and is preferably obtained by inducing differentiation of pluripotent stem cells.

[0059] (Method for inducing differentiation of a retinal cell population as starting cells) Retinal cells derived from pluripotent stem cells as starting cells for the production method of the present invention can be obtained by inducing differentiation of the pluripotent stem cells. In one embodiment, retinal cell aggregates can be obtained by inducing differentiation of pluripotent stem cells using a differentiation inducer. Examples of differentiation inducers include basement membrane preparations, substances acting on the BMP signaling pathway, inhibitors of the Wnt signaling pathway, and substances acting on the IGF signaling pathway. In one embodiment, there is a method for producing retinal cell aggregates by self-organization. Self-organization refers to a mechanism by which a population of cells autonomously generates a complex structure. For example, self-organization can be achieved by the SFEB (serum-free floating culture of embryoid bodies-like aggregates) method (WO 2005 / 12390) or the SFEBq method (WO 2009 / 148170).

[0060] Specific differentiation induction methods include those described in WO2011 / 055855, WO2013 / 077425, WO2015 / 025967, WO2016 / 063985, WO2016 / 063986, WO2017 / 183732, PLoS One. 2010 Jan 20; 5(1): e8763, Stem Cells. 2011 Aug; 29(8): 1206-18, Proc Natl Acad Sci USA. 2014 Jun 10; 111(23): 8518-23, or Nat Commun. 2014 Jun 10;5:4047, but is not particularly limited thereto.

[0061] In one specific embodiment, retinal cell aggregates can be prepared by a method comprising the following steps (A), (B), and (C): (A) culturing pluripotent stem cells in a medium containing a factor for maintaining undifferentiated state in the absence of feeder cells, (B) forming cell aggregates by suspension culture of the cells obtained in step (A), and (C) further suspension culture of the cell aggregates obtained in step (B) in a medium containing a substance acting on the BMP signaling pathway.

[0062] Step (A) may further include a substance inhibiting the TGFβ family signaling pathway and / or a substance acting on the sonic hedgehog signaling pathway.

[0063] Furthermore, step (B) may include a sonic hedgehog signaling pathway active substance and / or a Wnt signaling inhibitor, as described below.

[0064] This method is also disclosed in, for example, WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732, and more details can be found in WO2015 / 025967, WO2016 / 063985, and WO2017 / 183732.

[0065] Unless otherwise specified, a basal cell growth medium (also referred to as a basal medium) can be used as the medium used to prepare aggregates of retinal cells. The basal cell growth medium is not particularly limited as long as it allows cells to be cultured, and any basal medium commercially available as a cell growth medium can be used as appropriate. Specific examples include media that can be used to culture animal cells, such as BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, MEM medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Leibovitz's L-15 medium, and mixtures thereof. Alternatively, a medium supplemented with N2 medium, which is a supplementary medium, may be used.

[0066] The 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, and specific examples include TGFβ signaling pathway inhibitors (e.g., SB431542, LY-364947, SB505124, A-83-01, etc.), Nodal / Activin signaling pathway inhibitors (e.g., SB431542, A-83-01, etc.), and BMP signaling pathway inhibitors (e.g., LDN193189, Dorsomorphin, etc.). These substances are commercially available.

[0067] A substance acting on the sonic hedgehog (hereinafter sometimes referred to as "Shh" or "SHH") signaling pathway is a substance that can enhance signal transduction mediated by Shh. Examples of substances acting on the Shh signaling pathway include SHH, partial peptides of SHH, PMA (purmorphamine), SAG (smoothened agonist), etc.

[0068] The concentrations of the TGFβ family signaling pathway inhibitor and the sonic hedgehog signaling pathway agonist may be any concentration capable of inducing differentiation into retinal cells. For example, SB431542 is typically used at a concentration of 0.1 to 200 μM, preferably 2 to 50 μM. A-83-01 is typically used at a concentration of 0.05 to 50 μM, preferably 0.5 to 5 μM. LDN193189 is typically used at a concentration of 1 to 2000 nM, preferably 10 to 300 nM. SAG is typically used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM. PMA is typically used at a concentration of 0.002 to 20 μM, preferably 0.02 to 2 μM.

[0069] The undifferentiated state maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of undifferentiated state maintenance factors commonly used by those skilled in the art include substances acting on the FGF signaling pathway, substances acting on the TGFβ family signaling pathway, insulin, etc. Specific examples of substances acting on the FGF signaling pathway include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Furthermore, examples of substances acting on the TGFβ family signaling pathway include substances acting on the TGFβ signaling pathway and substances acting on the Nodal / Activin signaling pathway. Examples of substances acting on the TGFβ signaling pathway include TGFβ1 and TGFβ2. Examples of substances acting on the Nodal / Activin signaling pathway include Nodal, Activin A, and Activin B. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the medium in step (A) preferably contains bFGF as an undifferentiated state maintenance factor.

[0070] The concentration of the undifferentiation maintenance factor in the medium used in step (A) is a concentration that allows the pluripotent stem cells to be cultured in an undifferentiated state, and can be appropriately determined by those skilled in the art. For example, specifically, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually about 4 to 500 ng / mL, preferably about 10 to 200 ng / mL, and more preferably about 30 to 150 ng / mL.

[0071] Many synthetic media have been developed and are commercially available as feeder-free media that contain factors for maintaining undifferentiated states and can be used to culture pluripotent stem cells, such as Essential 8 Medium (Life Technologies). Essential 8 medium contains the following additives in DMEM / F12 medium: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and a TGFβ family signaling pathway active substance (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Other commercially available feeder-free media include S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), and StemFit (manufactured by Ajinomoto Co.). By using these media in step (A) above, the present invention can be carried out easily. By using these media, it is possible to culture pluripotent stem cells under feeder-free conditions. The medium used in step (A) is, for example, a serum-free medium to which none of a substance acting on the BMP signaling pathway, a substance acting on the Wnt signaling pathway, and a substance inhibiting the Wnt signaling pathway has been added.

[0072] In the feeder-free culture of pluripotent stem cells in step (A), an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold in place of feeder cells. Examples of matrices that can be used as a scaffold include laminin (Nat Biotechnol 28, 611-615, (2010)), laminin fragments (Nat Commun 3, 1236, (2012)), basement membrane preparations (Nat Biotechnol 19, 971-974, (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin.

[0073] The culture time of pluripotent stem cells in step (A) is not particularly limited as long as the effect of improving the quality of the cell aggregates formed in step (B) can be achieved when the pluripotent stem cells are cultured in the presence of a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist (e.g., 100 nM to 700 nM), but is typically 0.5 to 144 hours. In one aspect, the culture time is preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours).

[0074] The medium used in step (B) may be a serum-containing medium or a serum-free medium. A serum-free medium is preferably used to avoid contamination with chemically undefined components. To avoid the complexity of the preparation, for example, a serum-free medium supplemented with an appropriate amount of a serum substitute such as commercially available KSR may be used. The amount of KSR added to the serum-free medium is usually about 1% to about 30%, preferably about 2% to about 20%.

[0075] When forming aggregates, first, dispersed cells are prepared by dispersing the cells obtained in step (A). The "dispersed cells" obtained by the dispersion procedure are, for example, cells in which 70% (preferably 80% or more) or more are single cells and 30% or less (preferably 20% or less) are clumps of 2 to 50 cells. Dispersed cells are cells in which there is almost no adhesion (e.g., surface adhesion) between cells.

[0076] A suspension of dispersed cells is seeded in a culture vessel, and the dispersed cells are cultured under non-adhesive conditions to allow a plurality of cells to aggregate and form aggregates. In one embodiment, a certain number of dispersed stem cells are placed in each well of a multi-well plate (U-bottom, V-bottom) such as a 96-well plate, and when this is subjected to static culture, the cells rapidly aggregate to form a single aggregate in each well (SFEBq method). When cells are cultured in suspension using a 96-well plate, approximately 1 × 10 cells are cultured per well. ３ to about 1 × 10 ５ Cells (preferably about 3 x 10 ３ From about 5 x 10 ４ cells, approximately 4 x 10 ３ From about 2 x 10 ４ A solution prepared to form aggregates (cells) is added to the wells, and the plate is left to stand to allow the formation of aggregates.

[0077] In one embodiment, the medium used in step (B) contains a substance acting on the Sonic Hedgehog signaling pathway. That is, in one specific embodiment, aggregates of retinal cells can be prepared by a method comprising the following steps (A), (B), and (C): (A) culturing pluripotent stem cells in a medium containing an undifferentiated maintenance factor, in the absence of feeder cells, and optionally containing a TGFβ family signaling pathway inhibitor and / or a substance acting on the Sonic Hedgehog signaling pathway; (B) forming cell aggregates by suspension culturing the cells obtained in step (A) in a medium containing a substance acting on the Sonic Hedgehog signaling pathway; and (C) further suspension culturing the cell aggregates obtained in step (B) in a medium containing a substance acting on the BMP signaling pathway. The substance acting on the Sonic Hedgehog signaling pathway in step (B) can be any of the above-described substances at the above-described concentrations (e.g., 10 nM to 300 nM). The substance acting on the Sonic Hedgehog signaling pathway is preferably included in the medium from the start of suspension culture. A ROCK inhibitor (e.g., Y-27632) may be added to the medium. The culture time is, for example, 12 hours to 6 days. In one example, the medium used in step (B) is a medium to which one or more (preferably all) selected from the group consisting of a substance acting on the BMP signaling pathway, a substance acting on the Wnt signaling pathway, a substance inhibiting the TGFβ family signaling pathway, and a substance acting on the TGFβ family signaling pathway are not added.

[0078] In step (B), culturing in a medium containing a substance acting on the sonic hedgehog signaling pathway increases the expression of CD39, a cell surface marker of retinal progenitor cells or neural retinal progenitor cells (Rx-positive cells), as described below. In one embodiment, CD39 expression is increased by adding a high concentration (100 nM to 1000 nM, e.g., 300 nM) of SAG from the start of suspension culture (Day 0) in step (B). This method allows for the purification of retinal progenitor cells or neural retinal progenitor cells using CD39, thereby improving production efficiency. The tissue containing CD39-positive cells obtained by adding 300 nM SAG is Chx10-positive, Lhx2-positive, and NKX2.1-negative, and is therefore retinal tissue. In particular, ventral (CoupTF1-positive) retinal tissue is induced. In addition, the retinal tissue obtained without adding SAG in step (B) induces dorsal (ALDH1A1-positive) retinal tissue.

[0079] In step (B), the proportion of CD39-positive cells in the induced tissue is also increased by culturing the cells in a medium containing one or more selected from the group consisting of ATP, an ATP analog (e.g., AMP-PNP), and an A2A receptor agonist (adenosine, CGS 21680 (3-[4-[2-[[6-amino-9-[(2R,3R,4S,5S)-5-(ethylcarbamoyl)-3,4-dihydroxy-oxolan-2-yl]purin-2-yl]amino]ethyl]phenyl]propanoicacid), etc.).

[0080] A substance acting on the BMP signaling pathway is a substance that can enhance the signaling pathway mediated by BMP. Examples of substances acting on the BMP signaling pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2 protein, BMP4 protein, and BMP7 protein are available from, for example, R&D Systems, and GDF7 protein is available from, for example, Wako Pure Chemical Industries.

[0081] The medium used in step (C) may be, for example, a serum-free medium or a serum medium (preferably a serum-free medium) supplemented with a substance acting on the BMP signaling pathway. Serum-free medium and serum medium can be prepared as described above. In one example, the medium used in step (C) is a medium to which one or more (preferably all) selected from the group consisting of a substance acting on the Wnt signaling pathway, a substance inhibiting the TGFβ family signaling pathway, and a substance acting on the TGFβ family signaling pathway are not added. In one example, the medium used in step (C) is a medium to which no substance acting on the Sonic Hedgehog signaling pathway is added. In another example, the medium used in step (C) may be a medium to which a substance acting on the Wnt signaling pathway is added.

[0082] 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 to a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL).

[0083] The substance acting on the BMP signaling pathway may be added to the medium at least about 24 hours after the initiation of suspension culture in step (A), and may be added within several days (e.g., within 15 days) after the initiation of suspension culture. Preferably, the substance acting on the BMP signaling pathway is added to the medium between days 1 and 15, more preferably between days 1 and 9, and most preferably on day 3 after the initiation of suspension culture.

[0084] In a specific embodiment, for example, on days 1 to 9, preferably days 1 to 3, after the initiation of suspension culture in step (B), part or all of the medium is replaced with medium containing BMP4, adjusting the final BMP4 concentration to about 1 to 10 nM, and culturing in the presence of BMP4 for, for example, 1 to 12 days, preferably 2 to 9 days, and more preferably 2 to 5 days. Here, to maintain the same BMP4 concentration, part or all of the medium can be replaced with medium containing BMP4 once or twice. Alternatively, the BMP4 concentration can be reduced in stages. For example, the concentration of the substance acting on the BMP signaling pathway (BMP4) may be maintained from days 2 to 10 after the initiation of suspension culture in step (B), and then the concentration of the substance acting on the BMP signaling pathway (BMP4) may be reduced in stages from days 6 to 20 after the initiation of suspension culture in step (B).

[0085] The culture temperature and CO ２ Culture conditions such as concentration can be appropriately set. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. ２ The concentration is, for example, about 1% to about 10%, preferably about 5%.

[0086] By varying the culture period in step (C), retinal cells at various stages of differentiation can be produced as retinal cells contained in the cell aggregate. That is, retinal cells in the cell aggregate 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 proportion of mature retinal cells can be increased.

[0087] The method disclosed in WO 2017 / 183732 can also be used for step (B) and / or step (C). That is, in step (B) and / or step (C), cell aggregates can be formed by suspension culture in a medium further containing a Wnt signaling pathway inhibitor.

[0088] The Wnt signaling pathway inhibitor used in step (B) and / or step (C) is not particularly limited as long as it can suppress signal transduction mediated by Wnt, and may be any of a protein, a nucleic acid, a low molecular weight compound, etc. Wnt-mediated signals are transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and LRP5 / 6 (low-density lipoprotein receptor-related protein 5 / 6). Examples of Wnt signaling pathway inhibitors include substances that act directly on Wnt or Wnt receptors (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus proteins, etc.), and substances that inhibit physiological activities resulting from signal transduction by Wnt receptors [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2 ,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), and 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. The Wnt signaling pathway inhibitor may contain one or more of these. CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, etc. are known Wnt signaling pathway inhibitors, and are appropriately available as commercially available products, etc. IWR1e is preferably used as the Wnt signaling pathway inhibitor.

[0089] The concentration of the Wnt signaling pathway inhibitor in step (B) may be any concentration capable of inducing the formation of favorable cell aggregates. For example, IWR-1-endo is added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR-1-endo.

[0090] 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 suspension culture in step (B). Specifically, for example, a basal medium supplemented with a Wnt signaling pathway inhibitor can be added, or a partial or complete medium exchange with the basal medium can be performed. The period during which the cells obtained in step (A) are exposed to the Wnt signaling pathway inhibitor in step (B) is not particularly limited, but preferably, the cells are exposed to the Wnt signaling pathway inhibitor from the time of the start of 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, as described below, the cells are continuously exposed to the Wnt signaling pathway inhibitor even after the end of step (B) (i.e., during step (C)). In one embodiment, as described below, the Wnt signaling pathway inhibitor may be allowed to continue acting even after step (B) is completed (i.e., during step (C)), until retinal tissue is formed.

[0091] In step (C), any of the aforementioned 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).

[0092] The concentration of the Wnt signaling pathway inhibitor in step (C) may be any concentration capable of inducing retinal progenitor cells and retinal tissue. For example, IWR-1-endo is added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the medium in step (C) is preferably 50 to 150, more preferably 80 to 120, and even more preferably 90 to 110, relative to the concentration of the Wnt signaling pathway inhibitor in the medium in step (B) taken as 100. More preferably, it is equivalent to the concentration of the Wnt signaling pathway inhibitor in the medium in step (B).

[0093] The timing of adding the Wnt signaling pathway inhibitor to the medium is not particularly limited as long as it achieves the formation of aggregates containing retinal cells or retinal tissue, but the earlier the better. Preferably, the Wnt signaling pathway inhibitor is added to the medium at the start of step (C). More preferably, after the Wnt signaling pathway inhibitor is added in step (B), it continues to be contained in the medium in step (C) (i.e., from the start of step (B)). Even more preferably, the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B), and it continues to be contained in the medium in step (C). For example, a BMP signaling inhibitor (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).

[0094] The period during which the Wnt signaling pathway inhibitor is allowed to act is not particularly limited, but is preferably 2 to 30 days, more preferably 6 to 20 days, 8 to 18 days, 10 to 18 days, or 10 to 17 days (e.g., 10 days), counting from the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B). In another aspect, the period during which the Wnt signaling pathway inhibitor is allowed to act is preferably 3 to 15 days (e.g., 5 days, 6 days, or 7 days), more preferably 6 to 10 days (e.g., 6 days), counting from the start of suspension culture in step (B), when the Wnt signaling pathway inhibitor is added at the start of suspension culture in step (B).

[0095] The cell aggregates obtained by the above-mentioned method can be cultured for about 2 to 4 days in a serum-free medium or a serum-based medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway (step (D)), and then cultured for about 30 to 200 days (30 to 150 days, 50 to 120 days, or 60 to 90 days) in a serum-free medium or a serum-based medium that does not contain a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway (step (E)), thereby producing a neural retina containing ciliary marginal zone-like structures.

[0096] In one embodiment, a neural retina containing a ciliary margin-like structure can be produced by the above steps (D) and (E) from cell aggregates obtained in steps (A) to (C), which are on days 6 to 30, or 10 to 20 (10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, or 20th) after the start of suspension culture in step (B).

[0097] The substance acting on the Wnt signaling pathway is not particularly limited as long as it can enhance signal transduction mediated by Wnt. Specific examples of substances acting on the Wnt signaling pathway include GSK3β inhibitors (e.g., 6-bromoindirubin-3'-oxime (BIO), CHIR99021, and Kenpaullone). For example, in the case of CHIR99021, the range of concentration can be about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM.

[0098] The FGF signaling pathway inhibitor is not particularly limited as long as it can inhibit signal transduction mediated by FGF. Examples of FGF signaling pathway inhibitors include SU-5402, AZD4547, and BGJ398. For example, SU-5402 is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 5 μM.

[0099] In one example, the medium used in step (D) is a medium to which one or more (preferably all) selected from the group consisting of a BMP signaling pathway active substance, a Wnt signaling pathway inhibitor, an SHH signaling pathway active substance, a TGFβ family signaling pathway inhibitor, and a TGFβ family signaling pathway active substance are not added.

[0100] Some or all of the steps in step (E) above can be performed using the continuous epithelial tissue maintenance medium disclosed in WO 2019 / 017492. That is, by culturing using a continuous epithelial tissue maintenance medium, the continuous epithelial structure of the neural retina can be maintained. As an example, a medium for maintaining continuous epithelial tissue can be prepared by adding B27 supplement (e.g., Thermo Fisher Scientific, 12587010) to Neurobasal medium (e.g., Thermo Fisher Scientific, 21103049).

[0101] In the culture in the step (E), in order to simultaneously achieve differentiation and / or maturation of retinal cells (particularly photoreceptors) and maintenance of a continuous epithelial structure, it is preferable to gradually change the medium to a continuous epithelial tissue maintenance medium. For example, for the first 10 to 30 days, the medium is a basal cell proliferation medium (e.g., a medium containing 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine in DMEM / F12 medium), and for the next 10 to 40 days, a mixed medium of the basal cell proliferation medium and a continuous epithelial tissue maintenance medium (e.g., a medium containing 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine in DMEM / F12 medium and a mixed medium containing 10% fetal bovine serum, 2% B27 supplement, and 2 mM taurine in Neurobasal medium). For the first 20 to 140 days, the cells can be cultured in a medium for maintaining epithelial tissue (e.g., a medium prepared by mixing a medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine at a ratio of 1:3 with the medium supplemented with 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine), or for the second 20 to 140 days, the cells can be cultured in a medium for maintaining epithelial tissue (e.g., a medium prepared by adding Neurobasal medium to 10% fetal bovine serum, 2% B27 supplement, 2 mM glutamine, and 100 μM taurine).

[0102] In some or all of the steps of step (E), whether a basal cell proliferation medium, a continuous epithelial tissue maintenance medium, or a mixed medium thereof is used, the medium may further contain a substance acting on the thyroid hormone signaling pathway. Culturing in a medium containing a substance acting on the thyroid hormone signaling pathway makes it possible to produce aggregates of retinal cells that have a low proportion of bipolar cells, amacrine cells, ganglion cells, horizontal cells, etc. contained in the neural retina and an increased proportion of photoreceptor precursor cells.

[0103] As used herein, a substance acting on the thyroid hormone signaling pathway refers to a substance that can enhance signal transduction mediated by thyroid hormone, and is not particularly limited as long as it can enhance the thyroid hormone signaling pathway. Examples of substances acting on the thyroid hormone signaling pathway include triiodothyronine (hereinafter sometimes abbreviated as T3), thyroxine (hereinafter sometimes abbreviated as T4), and thyroid hormone receptor (preferably TRβ receptor) agonists.

[0104] Further, examples of thyroid hormone receptor agonists known to those skilled in the art include those described in WO 97 / 21993, WO 2004 / 066929, WO 2004 / 093799, WO 2000 / 039077, WO 2001 / 098256, WO 2003 / 018515, WO 2003 / 084915, WO 2002 / 094319, WO 2003 / 064369, JP 2004-2007066, and the like. Examples of the diphenylmethane derivatives include compounds such as diphenylmethane derivatives, diaryl ether derivatives, pyridazine derivatives, pyridine derivatives, and indole derivatives described in JP-A-2002-053564, JP-A-2002-370978, JP-A-2000-256190, WO 2007 / 132475, WO 2007 / 009913, WO 2003 / 094845, WO 2002 / 051805, and WO 2010 / 122980.

[0105] When T3 is used as the substance acting on the thyroid hormone signaling pathway, it can be added to the medium at a concentration in the range of, for example, 0.1 to 1000 nM. Concentrations that exhibit thyroid hormone signaling enhancing activity equivalent to T3 concentrations of 1 to 500 nM, more preferably 10 to 100 nM, even more preferably 30 to 90 nM, and even more preferably around 60 nM are preferred. When T4 is used as the substance acting on the thyroid hormone signaling pathway, it can be added to the medium at a concentration in the range of, for example, 1 nM to 500 μM. Preferably, the concentration is in the range of 50 nM to 50 μM, more preferably 500 nM to 5 μM. When other thyroid hormone receptor agonists are used, they may be added at any concentration that exhibits activity comparable to the agonistic activity exhibited by T3 or T4 at the aforementioned concentrations.

[0106] The medium used in step (E) may contain, as appropriate, L-glutamine, taurine, serum, etc. In one example, the medium used in step (E) is a medium to which one or more (preferably all) selected from the group consisting of a substance acting on a BMP signaling pathway, a substance inhibiting an FGF signaling pathway, a substance acting on a Wnt signaling pathway, a substance inhibiting a Wnt signaling pathway, a substance acting on an SHH signaling pathway, a substance inhibiting a TGFβ family signaling pathway, and a substance acting on a TGFβ family signaling pathway.

[0107] In one specific embodiment, retinal cell aggregates can be prepared by a method comprising the following steps (A) to (E): (A) culturing pluripotent stem cells in a medium containing an undifferentiated maintenance factor in the absence of feeder cells, and optionally containing a TGFβ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway active substance; (B) forming cell aggregates by suspension culturing the cells obtained in step (A) in a medium which may contain a Wnt signaling pathway inhibitor and / or a sonic hedgehog signaling pathway active substance; (C) further suspension culturing the cell aggregates obtained in step (B) in a medium containing a BMP signaling pathway active substance; (D) culturing the cell aggregates obtained in step (C) in a serum-free medium or serum medium containing a Wnt signaling pathway active substance and / or an FGF signaling pathway inhibitor for a period of about 2 to 4 days; and (E) A step of culturing the cell aggregates obtained in step (D) for approximately 30 to 200 days in a serum-free medium or a serum-free medium that does not contain a substance acting on the Wnt signaling pathway or a substance inhibiting the FGF signaling pathway, and that may contain a substance acting on the thyroid hormone signaling pathway.

[0108] In one specific embodiment, retinal cell aggregates can be prepared by a method comprising the following steps (A') to (E'): (A') culturing pluripotent stem cells for 12 to 48 hours in a medium containing an undifferentiated maintenance factor and containing a substance inhibiting the TGFβ family signaling pathway and / or a substance acting on the sonic hedgehog signaling pathway in the absence of feeder cells; (B') forming cell aggregates by suspension culturing the cells obtained in step (A') in a medium containing a substance inhibiting the Wnt signaling pathway and / or a substance acting on the sonic hedgehog signaling pathway for 12 to 72 days (24 to 48 hours); (C') further culturing the cell aggregates obtained in step (B') in suspension for 8 to 15 days (10 to 13 days) in a medium containing a substance acting on the BMP signaling pathway; (D') a step of culturing the cell aggregates obtained in step (C') for 2 to 4 days in a serum-free medium or a serum medium containing a substance acting on the Wnt signaling pathway and / or a substance inhibiting the FGF signaling pathway; and (E') a step of culturing the cell aggregates obtained in step (D') for approximately 10 to 200 days in a serum-free medium or a serum medium that does not contain a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway, but which may contain a substance acting on the thyroid hormone signaling pathway.

[0109] Here, step (E') may comprise culturing the cells in a basal cell proliferation medium for 10 to 30 days, then culturing the cells in a mixed medium of the basal cell proliferation medium and a continuous epithelial tissue maintenance medium containing a substance that acts on thyroid hormone signaling for 10 to 40 days, and further culturing the cells in a continuous epithelial tissue maintenance medium containing a substance that acts on thyroid hormone signaling for 20 to 140 days.

[0110] In one embodiment, step (E') comprises culturing in the presence of a thyroid hormone signaling pathway agonist for 20 to 60 days (30 to 50 days).

[0111] In one embodiment, the culture period from step (B') to step (E') is 70 to 100 days (80 to 90 days).

[0112] The timing of collecting retinal tissue containing retinal system cells as starting cells is not particularly limited, as long as the retinal tissue contains one or more types of cells selected from the group consisting of retinal progenitor cells, neural retinal progenitor cells, and photoreceptor progenitor cells. A timing when the retinal tissue contains a large number of retinal progenitor cells and / or neural retinal progenitor cells is preferred. In the above-described method, for example, a dispersed retinal system cell population as starting cells may be prepared from retinal tissue 10 to 100 days (preferably 9 to 45 days) after the initiation of suspension culture, as described below.

[0113] (Method for inducing differentiation of a neural cell population as starting cells) Neural cells derived from pluripotent stem cells as starting cells in the production method of the present invention can be induced by inducing differentiation of the pluripotent stem cells. Many methods have been reported for inducing pluripotent stem cell aggregates into neural cells or neural tissue by suspension culture. For example, methods described in WO2005 / 123902, WO2009 / 148170, WO2008 / 035110, WO2011 / 055855, WO2016 / 063985, Cell Stem Cell, 3, 519-32 (2008), Nature, 472, 51-56 (2011), Cell Stem Cell, 10(6), 771-775 (2012), Nature Biotechnology, 27(3), 275-80 (2009), ProcNatl Acad Sci USA, 110(50), 20284-9 (2013), etc. are known, but are not limited to these. By applying such various methods for inducing nervous system cells or nervous tissue to the aggregates obtained in step (B) and culturing the aggregates obtained in step (B) under appropriate neuronal differentiation induction conditions, aggregates containing nervous system cells or nervous tissue can be produced.

[0114] An example of a method for producing nerve cells (e.g., cerebral cells (cerebral tissue)) as starting cells in the production method of the present invention is shown below.

[0115] This method for producing cerebral tissue comprises the following steps (A'') to (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'') culturing the cells obtained in step (A'') in suspension to form cell aggregates; and (C'') culturing the aggregates obtained in step (B'') in suspension in the absence of a differentiation inducer (e.g., a substance agonist of the BMP signaling pathway) or in the presence of a differentiation inducer (e.g., a TGFβ family signaling pathway inhibitor and / or a Wnt signaling pathway inhibitor), to obtain aggregates containing neural tissue (cerebral tissue).

[0116] Steps (A'') and (B'') can be carried out in the same manner as steps (A) and (B) of the above-mentioned method for producing retinal tissue.

[0117] In step (C"), the cells may be cultured in a medium to which a differentiation inducer, for example, a substance that activates or inhibits a specific signal transduction, such as a substance acting on the BMP signaling pathway, is not added. Since the cell aggregates are destined to differentiate into neurons through steps (A") and (B"), they spontaneously differentiate into neurons (e.g., cerebrum) when cultured in a basal medium.

[0118] In step (C"), a TGFβ family signaling pathway inhibitor or a Wnt signaling pathway inhibitor may be added alone to the medium as a differentiation inducer, but it is preferable to use both in combination. These differentiation inducers may be added from step (B") onwards.

[0119] Examples of the TGFβ family signaling pathway inhibitor used in steps (B″) and (C″) include a TGFβ signaling pathway inhibitor, a Nodal / Activin signaling pathway inhibitor, and a BMP signaling pathway inhibitor. These factors are the same as those described above. The TGFβ family signaling pathway inhibitor is preferably SB431542, A-83-01, or LDN193189.

[0120] The Wnt signaling pathway inhibitor used in steps (B'') and (C'') is not particularly limited as long as it can suppress signal transduction mediated by Wnt, and may be any of a protein, a nucleic acid, a low molecular weight compound, etc. Wnt-mediated signals are transmitted via the Wnt receptor, which exists as a heterodimer of Frizzled (Fz) and LRP5 / 6 (low-density lipoprotein receptor-related protein 5 / 6). Examples of Wnt signaling pathway inhibitors include substances that act directly on Wnt or Wnt receptors (anti-Wnt antibodies, anti-Wnt receptor antibodies, etc.), substances that suppress the expression of genes encoding Wnt or Wnt receptors (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptors to Wnt (soluble Wnt receptors, dominant-negative Wnt receptors, Wnt antagonists, Dkk1, Cerberus protein, etc.), and substances that inhibit physiological activities resulting from signal transduction by Wnt receptors [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e), (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), and small molecular weight compounds such as IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide). CKI-7, D4476, IWR-1-endo (IWR1e), IWP-2, and the like are known Wnt signaling pathway inhibitors, and are appropriately available as commercially available products, etc. A preferred example of a Wnt signaling pathway inhibitor is IWR1e.

[0121] The concentration of the Wnt signaling pathway inhibitor may be any concentration capable of inducing differentiation of cells forming pluripotent stem cell aggregates into cerebral cells. For example, IWR-1-endo is added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the concentration of IWR1e described above.

[0122] By such culturing, differentiation of the cells forming the aggregates obtained in step (B") into cerebral nervous system progenitor cells can be induced, and aggregates containing cerebral nervous system progenitor cells can be obtained. The fact that aggregates containing cerebral nervous system progenitor cells have been obtained can be confirmed, for example, by detecting whether the aggregates contain cells expressing cerebral nervous system progenitor cell markers such as FoxG1, Lhx2, PAX6, and Emx2. One embodiment of step (C") includes a step of culturing the aggregates formed in step (B") in suspension in a serum-free or serum-free medium until cells expressing the FoxG1 gene begin to appear. In one aspect, the culturing in step (C") is carried out until 20% or more (preferably 30% or more, 40% or more, 50% or more, or 60% or more) of the cells contained in the aggregates express FoxG1.

[0123] The timing of recovery of neural tissue containing neural cells as starting cells is not particularly limited as long as the neural tissue (cerebral tissue) contains neural progenitor cells or cerebral neural progenitor cells, but is preferably a time when the tissue contains a large number of these cells. In the above-mentioned method, for example, a dispersed neural cell population as starting cells may be prepared from neural tissue (cerebral tissue) 10 to 100 days (preferably 15 to 40 days) after the initiation of suspension culture, as described below.

[0124] (Dispersed Retinal or Nervous System Cell Population) "Dispersion" refers to separating cells or tissues into small cell fragments or cell clumps (2 to 100 cells, preferably 50 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer cells; for example, a cluster of 2 to 5 cells) or single cells by a dispersion treatment such as enzymatic or physical treatment. A dispersed cell population refers to a collection of a certain number of cell fragments, cell clusters, or single cells. A "dispersed retinal cell population" refers to a cell population in a dispersed state, and can be obtained by dispersing a cell cluster such as a biological tissue or a cell aggregate. A dispersed retinal cell population is preferably obtained by dispersing the above-mentioned retinal cell aggregates.

[0125] In one embodiment, the dispersion method may be any method that can disperse cells while keeping them alive, such as mechanical dispersion treatment, cell dispersion treatment, or cell protective agent addition treatment. These treatments may also be performed in combination. Preferably, cell dispersion treatment is performed first, followed by mechanical dispersion treatment.

[0126] Methods for mechanical dispersion include pipetting and scraping with a scraper.

[0127] Examples of cell dispersion solutions used in cell dispersion treatment include solutions containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, and papain, and chelating agents such as ethylenediaminetetraacetic acid. Commercially available cell dispersion solutions, such as TrypLE Select (Life Technologies) and TrypLE Express (Life Technologies), and nerve cell dispersion solution (FujiFilm), can also be used.

[0128] When cells are dispersed, cell death may be suppressed by treating them with a cytoprotective agent. Examples of cytoprotective agents used in the cytoprotective agent treatment include substances acting on the FGF signaling pathway, heparin, substances acting on the IGF signaling pathway, serum, or serum substitutes. Furthermore, in order to suppress cell death induced by dispersion (particularly cell death of human pluripotent stem cells), a Rho-associated coiled-coil containing protein kinase ("ROCK" or "Rho kinase") inhibitor (ROCK inhibitor) or a myosin inhibitor may be added during dispersion. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. Examples of myosin inhibitors include blebbistatin. Preferred cytoprotective agents include ROCK inhibitors.

[0129] Dispersed retinal cell populations (or neural cell populations) contain single cells. For example, 70% or more, preferably 80% or more of the total cell population are single cells, and clumps of 2 to 50 cells account for 30% or less, preferably 20% or less of the total cell population. In dispersed retinal cell populations (or neural cell populations), cell-to-cell adhesion (e.g., surface adhesion) is almost eliminated. Dispersed retinal cell populations (or neural cell populations) are preferably composed of as many single cells as possible. Such dispersed retinal cell populations (or neural cell populations) can be obtained by removing clumps of cells that have not formed into single cells after dispersion treatment. The method for removing clumps of cells is not particularly limited, and examples include removal of clumps using a membrane filter (cell strainer). In one aspect, dispersed cells are those in a state in which cell-to-cell junctions (e.g., adherens junctions) are almost eliminated.

[0130] When preparing a retinal cell population as starting cells from retinal tissue, the retinal tissue may contain unnecessary cells such as retinal pigment epithelial cells. Examples include cases where retinal tissue is collected from a living organism, or where steps (D) and (E) of the above-described method for producing retinal tissue from pluripotent stem cells are performed. In such cases, the retinal cell population can be dispersed after separating the area containing unnecessary cells such as retinal pigment epithelial cells. Retinal pigment epithelial cells can be distinguished by their morphology and pigment, and can be easily excised by a person skilled in the art.

[0131] Before starting the culture in a medium containing a substance acting on the Wnt signaling pathway, as described below, a step (purification step) for increasing the purity of retinal progenitor cells or neural retinal progenitor cells may be carried out, and further, maintenance and expansion culture of the dispersed retinal cell population (or neural cell population) may be carried out. The medium is not particularly limited as long as it is a medium (e.g., DMEM medium) in which retinal cells (or neural cells) can survive and proliferate. Since a frozen-thawed retinal cell population can also be used as the starting cells for the production method of the present invention, it is also possible to freeze and preserve the dispersed retinal cell population. There is no particular restriction on the cryopreservation solution, and commercially available cryopreservation solutions can be used.

[0132] [Purification of Retinal Progenitor Cells] Before starting to culture the dispersed retinal cell population described above in a medium containing a substance acting on the Wnt signaling pathway, a purification step may be performed to increase the proportion (purity) of retinal progenitor cells, preferably the proportion (purity) of neural retinal progenitor cells, in the dispersed retinal cell population. The purification step may involve cell sorting using specific markers expressed in retinal progenitor cells and / or neural retinal progenitor cells. Cell sorting can be performed using well-known techniques such as FACS and MACS. By performing a purification step of retinal progenitor cells and / or neural retinal progenitor cells, it is possible to reduce the contamination of RPE cells and / or non-target cells in the production method described herein. The step of increasing the proportion of retinal progenitor cells in the dispersed retinal cell population suppresses the generation of retinal pigment epithelial progenitor cells and / or retinal pigment epithelial cells. Whether or not the generation of retinal pigment epithelial progenitor cells and / or retinal pigment epithelial cells has been inhibited can be determined by culturing the dispersed retinal cell population using the culture method described below and then determining whether or not retinal pigment epithelial cells have been generated based on the markers, morphology, properties, etc. of the retinal pigment epithelial cells described above. The inhibition of the generation of retinal pigment epithelial progenitor cells and / or retinal pigment epithelial cells means that the proportion of retinal pigment epithelial cells to the total number of cells after the culture is inhibited compared to when the step of increasing the proportion of retinal progenitor cells is not performed. It can be determined that the generation of retinal pigment epithelial progenitor cells and / or retinal pigment epithelial cells has been inhibited if the proportion of retinal pigment epithelial cells is at the level described in paragraph 0175.

[0133] Well-known markers (positive markers) for retinal progenitor cells include Rx and Chx10. However, because these genes are expressed intracellularly, it is necessary to use ingenuity, such as using cells in which the gene is linked to a fluorescent protein by genetic engineering, or cells in which the gene has been replaced with a fluorescent protein (e.g., Rx::Venus cells). Therefore, we searched for positive markers for retinal progenitor cells and / or neural retinal progenitor cells, and found CD9 (Genbank ID: NM_001769.4, NM_001330312.2), CD24 (Genbank ID: NM_001291737.1, NM_001291738.1, NM_001291739.1, NM_001359084.1, NM_013230.3), CD29 (Genbank ID: NM_002211.4, NM_033668.2, NM_133376.2), and CD39 (Genbank ID: NM_002211.4, NM_033668.2, NM_133376.2). ID: NM_001098175.2, NM_001164178.1, NM_001164179.2, NM_001164181.1, NM_00116418 2.2, NM_001164183.2, NM_001312654.1, NM_001320916.1, NM_001776.6), CD47 (Genbank ID: NM_001777.3, NM_198793.2), CD49b (Genbank ID: NM_002203.4), CD49c (Genbank ID: NM_002204.4), CD49f (Genbank ID: NM_000210.4, NM_001079818.3, NM_001316306.2, NM_001365529.2, NM_001365530.2), CD57 (Genbank ID: NM_001367973.1, NM_018644.3, NM_054025.3), CD73 (Genbank ID: NM_001204813.1, NM_002526.4), CD82 (Genbank ID: NM_001024844.2, NM_002231.4), CD90 (Genbank ID: NM_001311160.2, NM_001311162.2, NM_001372050.1, NM_006288.5), CD164 (Genbank ID: NM_001142401.2, NM_001142402.2,NM_001142403.3, NM_001142404.2, NM_001346500.2, NM_006016.6), CD200 (Genbank ID: NM_001004196.3, NM_001318826.1, NM_001318828.1, NM_001318830.1, NM_001365851.2, NM_0 01365852.1, NM_001365853.1, NM_001365854.1, NM_001365855.1, NM_005944.7), CD340 (Genbank These cells were found to express CD9, CD39, CD90, and CXCR4 (Genbank IDs: NM_001005862.2, NM_001289936.1, NM_001289937.1, NM_001289938.1, NM_004448.3) and CXCR4 (Genbank IDs: NM_001008540.2, NM_001348056.2, NM_001348059.2, NM_001348060.2, NM_003467.3) on the cell surface. Preferred cell surface markers include, for example, CD9, CD39, CD90, and CXCR4. In one embodiment, a method for increasing the proportion of retinal progenitor cells in a cell population may be performed before starting culture in a medium containing the above-mentioned substance acting on the Wnt signaling pathway, the method comprising the step of contacting the cell population containing retinal progenitor cells with a substance (e.g., an antibody, a peptide, etc.) that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, and separating and collecting a positive fraction. This method makes it possible to reduce contamination with RPE cells in the production method described herein.

[0134] Furthermore, negative markers for these cells, i.e., markers whose expression is not observed in these cells, have been found, including SSEA1 (Genbank ID: NM_002033.3), CD66b (Genbank ID: NM_001816.4), CD69 (Genbank ID: NM_001781.2), and CD84 (Genbank IDs: NM_001184879.2, NM_001184881.2, NM_001184882.1, NM_001330742.2, NM_003874.4). By combining these with the above-mentioned positive markers, it is possible to further increase the purity of retinal progenitor cells and / or neural retinal progenitor cells. In one embodiment, a method for increasing the proportion of retinal progenitor cells in a cell population may be performed before initiating culture in a medium containing a substance acting on the Wnt signaling pathway. The method includes contacting the cell population containing retinal progenitor cells with a substance (e.g., an antibody) that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, preferably with a substance (e.g., an antibody) that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84, and separating a fraction positive for the former marker and a fraction negative for the latter marker. The step of contacting the dispersed retinal cell population with a substance (e.g., an antibody) that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84 and separating a fraction negative for these markers involves obtaining a cell population in which the expression levels of the antigens are below a reference value. Here, the reference value can be determined arbitrarily by one skilled in the art. For example, a negative fraction can be isolated by treating a cell population with a fluorescently labeled antibody against the antigen, the fluorescent intensity of which is equivalent to that of a fluorescently labeled isotype control antibody.

[0135] It should be noted that the method for increasing the proportion of retinal progenitor cells and / or neural retinal progenitor cells in a cell population using a substance (e.g., an antibody) that binds to the above-mentioned positive marker and negative marker is not limited to a method as a step in the manufacturing method described herein. The method for increasing the proportion of retinal progenitor cells and / or neural retinal progenitor cells using a cell surface marker can also be used as a step in the manufacturing method of other retinal tissues, for example.

[0136] In one aspect, in the dispersed retinal cell population, retinal progenitor cells and / or neural retinal progenitor cells may account for, for example, 30% or more of the total cell number, preferably 50% or more of the total cell number, more preferably 80% or more of the total cell number, and even more preferably 90% or more of the total cell number. Furthermore, for example, in the dispersed retinal cell population, cells (retinal progenitor cells or neural retinal progenitor cells) that are positive for at least one marker selected from the group consisting of CD9, CD39, CD90, and CXCR4 and positive for Rx and / or Chx10 account for 50% or more of the total cell number of the cell population, preferably 80% or more, 85% or more, 90% or more, or 95% or more. Furthermore, the cell population is preferably negative for one or more, preferably two or more, three or more, or all antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84. "Negative" here means that the value is below the above-mentioned standard value.

[0137] In one embodiment, the step of increasing the proportion of retinal progenitor cells (purification step) comprises the following steps: (1) culturing pluripotent stem cells in the presence of one or more selected from the group consisting of a substance acting on the Shh signaling pathway, ATP, and an A2A receptor agonist to produce cell aggregates, (2) inducing the differentiation of the cell aggregates into retinal progenitor cells, and (3) dispersing the cell aggregates and contacting them with a substance (e.g., an antibody) that binds to CD39. Details of steps (1) and (2) are as described in the "Method for inducing differentiation of a retinal cell population as starting cells."

[0138] (Re-formation of a layered structure by culturing a dispersed retinal cell population or a neural cell population) The method for producing retinal tissue according to the present invention comprises adhesion culture or suspension culture of a dispersed retinal cell population in a medium containing a substance acting on the Wnt signaling pathway. Either culture method can be used to re-form an epithelial structure (multi-layered structure) from the dispersed retinal cell population. Adhesion culture is preferred for producing sheet-like retinal tissue having an epithelial structure (or multi-layered structure). The production method according to the present invention can also be carried out using a dispersed neural cell population as the starting cells. The following description will be given for the case where a dispersed retinal cell population is used as the starting cells, but unless otherwise specified, the same applies when a dispersed neural cell population is used as the starting cells.

[0139] The medium used for culturing the dispersed retinal cell population is not particularly limited as long as it allows the retinal cells to survive and proliferate. In one aspect, the medium used for culturing the dispersed retinal cell population is a medium for maintaining continuous epithelial tissue. One example of a medium for maintaining continuous epithelial tissue is a medium obtained by adding B27 supplement (e.g., Thermo Fisher Scientific, 12587010) to Neurobasal medium (e.g., Thermo Fisher Scientific, 21103049).

[0140] The substance acting on the Wnt signaling pathway contained in the medium is not particularly limited, as long as it can enhance signaling 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, Kenpaullone, Wnt proteins such as Wnt2b and Wnt3a, and partial peptides thereof, and the substance may be one or more substances. The substance acting on the Wnt signaling pathway is preferably one or more substances, two or more substances, or three or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b, and Wnt3a. In the process of re-aggregating dispersed retinal cell populations, the substance acting on the Wnt signaling pathway can increase the size of the aggregates and can reform an epithelial structure (or multi-layered structure) with apical / basement membrane polarity. The substance acting on the Wnt signaling pathway can also suppress the formation of rosette-like structures.

[0141] The concentration of the substance acting on the Wnt signaling pathway may be any concentration that can induce the formation of a desired cell aggregate (for example, the re-formation of an epithelial structure (or multi-layered structure)). When CHIR99021 is used, for example, the concentration of the substance acting on the Wnt signaling pathway may be 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, and more preferably 1 μM to 10 μM. When other substances acting on the Wnt signaling pathway are used, the concentration may be such that they exhibit the same level of Wnt signal activation activity as CHIR99021 at the above-mentioned concentrations. Those skilled in the art can measure the Wnt signal activation activity, for example, by methods such as confirming the expression of β-catenin.

[0142] The timing of adding the substance acting on the Wnt signaling pathway is not particularly limited, but it is preferable to add it as soon as possible after the initiation of suspension culture or adhesion culture for re-forming the sheet. In one embodiment, it is preferable to culture in a medium containing a substance acting on the Wnt signaling pathway from the initiation of culture. The number of days of culture in a medium containing a substance acting on the Wnt signaling pathway is not particularly limited as long as the effect of re-forming an epithelial structure (or multi-layered structure) with apical / basement membrane polarity is observed, but is, for example, 1 to 14 days.

[0143] In one aspect, the medium may further contain one or more substances selected from the group consisting of a ROCK inhibitor, a substance acting on the SHH (sonic hedgehog) signaling pathway, and a substance acting on the fibroblast growth factor (FGF) signaling pathway. For example, the addition of a ROCK inhibitor has been shown to have the effect of promoting the re-aggregation of dispersed retinal progenitor cell populations, the addition of a substance acting on the SHH signaling pathway has been shown to have the effect of promoting the proliferation of cell aggregates and increasing the size of the cell aggregates, and the addition of a substance acting on the FGF signaling pathway (e.g., FGF2, FGF8) has the effect of reducing cell damage and suppressing the induction of differentiation into RPE cells.

[0144] The ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), and examples thereof include Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983(2000); Narumiya et al., Methods Enzymol. 325, 273-284(2000)), Fasudil / HA1077 (see, for example, Uenata et al., Nature 389:990-994(1997)), H-1152 (see, for example, Sasaki et al., Pharmacol. Ther. 93:225-232(2002)), and Wf-536 (see, for example, Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324(2003)) and derivatives thereof, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant-negative mutants, and expression vectors thereof. In addition, other low molecular weight compounds are also known as ROCK inhibitors, and such compounds or derivatives thereof can also be used in the present invention (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003 / 062227, 2003 / 059913, 2003 / 062225, 2002 / 076976, and 2004 / 039796). One or more ROCK inhibitors can be used. The ROCK inhibitor preferably comprises one or more substances selected from the group consisting of Y-27632, Fasudil (HA1077), and H-1152.

[0145] The concentration of the ROCK inhibitor can be appropriately determined by one skilled in the art depending on the experimental conditions. In one embodiment, any concentration can be used as long as it is capable of promoting the reaggregation of a dispersed retinal progenitor cell population. For example, when Y-27632 is used, the concentration can be 0.1 μM to 1 mM, preferably 1 μM to 100 μM, and more preferably 5 μM to 20 μM. When a ROCK inhibitor other than Y-27632 is used, any concentration can be used as long as it exhibits the same level of ROCK inhibitory activity as that of Y-27632 at the above-mentioned concentration. The ROCK inhibitory activity can be measured by one skilled in the art, for example, by a method such as expression analysis of MLC2 phosphorylation.

[0146] A substance acting on the SHH (Sonic Hedgehog) signaling pathway is a substance that can enhance signal transduction mediated by SHH (sometimes referred to as Shh). Examples of substances acting on the SHH signaling pathway include proteins belonging to the Hedgehog family (e.g., Shh and Ihh), SHH receptors, Shh receptor agonists, PMA (Purmorphamine; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), and SAG (Smoothened Agonist; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane). The substance acting on the Shh signaling pathway may contain one or more of these. The substance acting on the Shh signaling pathway is preferably one or more substances selected from the group consisting of Shh (Genbank accession numbers: NM_000193, NP_000184), SAG, or PMA.

[0147] The concentration of the substance acting on the SHH signaling pathway can be appropriately determined by one skilled in the art depending on the experimental conditions. In one embodiment, the concentration may be within a range in which the effect of increasing the size of cell aggregates is observed. For example, 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. SHH is usually used at a concentration of 4 to 500 ng / mL, preferably 10 to 200 ng / mL. When other substances acting on the SHH signaling pathway are used, they may be used at a concentration that exhibits the same level of SHH signal activation activity as SAG at the above-mentioned concentrations. The SHH signal activation activity can be measured by one skilled in the art, for example, by methods such as expression analysis of downstream signals (SMO or GLI).

[0148] The substance acting on the FGF signaling pathway is not particularly limited as long as it is a substance that can enhance signal transduction mediated by FGF. Specific examples of substances acting on the FGF signaling pathway include fibroblast growth factors (e.g., bFGF, FGF4, FGF8, and FGF9). The substance acting on the FGF signaling pathway is preferably one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4, and FGF8.

[0149] The concentration of the substance acting on the FGF signaling pathway can be appropriately determined by one skilled in the art depending on the experimental conditions. In one embodiment, the concentration may be within a range in which the effect of suppressing differentiation induction into RPE cells is observed. For example, when FGF2, 4, or 8 is used, the concentration is approximately 4 to 500 ng / mL, preferably approximately 10 to 200 ng / mL, and more preferably approximately 25 to 100 ng / mL. When other substances acting on the FGF signaling pathway are used, the concentration may be such that they exhibit the same level of FGF signal activation activity as FGF8 or the like at the above-mentioned concentrations. The FGF signal activation activity can be measured by one skilled in the art, for example, by methods such as expression analysis of downstream signals (Akt, MEK).

[0150] The timing of adding the ROCK inhibitor, the substance acting on the SHH (Sonic Hedgehog) signaling pathway, and / or the substance acting on the FGF signaling pathway is not particularly limited, but it is preferable to culture in a medium containing the ROCK inhibitor, the substance acting on the SHH (Sonic Hedgehog) signaling pathway, and / or the substance acting on the FGF signaling pathway from the start of culture. These substances are preferably added to the medium simultaneously, and more preferably added to the medium simultaneously with the substance acting on the Wnt signaling pathway. In one embodiment, dispersed retinal cells may be cultured in a medium containing these substances for 1 to 14 days.

[0151] Suspension culture is culture of cells in a non-adherent state to a culture vessel, and is not particularly limited thereto. Suspension culture can be carried out using a culture vessel that has not been artificially treated (for example, coated with an extracellular matrix or the like) for the purpose of improving adhesion to the cells, or a culture vessel that has been artificially treated to suppress adhesion (for example, coated with polyhydroxyethyl methacrylate (poly-HEMA), a nonionic surface-active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (for example, a water-soluble polymer (Lipidure) having 2-methacryloyloxyethyl phosphorylcholine as a constituent unit).

[0152] Floating culture can be performed, for example, using dispersed retinal cells as starting cells and the SFEB (Serum-free Floating Culture of Embryoid Bodies-like Aggregates) method (WO2005 / 12390) or the SFEBq method (WO2009 / 148170).

[0153] Adhesion culture refers to culturing cells in a state of adhesion to a culture vessel, and can be performed using, but is not limited to, a culture vessel that has been artificially treated to improve adhesion to the cells. Adhesion culture is preferably performed using a culture vessel coated with an extracellular matrix and / or a temperature-sensitive polymer. In one embodiment, a method for producing retinal tissue includes a step of exposing a culture vessel coated with a temperature-sensitive polymer to a temperature at which the properties of the temperature-sensitive polymer change, thereby peeling off a sheet-like retinal tissue from the culture vessel.

[0154] By carrying out adhesion culture on a culture vessel coated with an extracellular matrix, sheet-like retinal tissue can be produced as described below.

[0155] Culturing in the presence of an extracellular matrix allows cells to recognize the basement membrane side, facilitating the formation of an apical surface. Furthermore, cells orient roughly perpendicular to the layer direction, resulting in retinal tissue with a better layered structure. By using a culture vessel coated with a temperature-sensitive polymer, retinal tissue with a formed epithelial structure (or multilayered structure) can be easily detached from the culture vessel by temperature change alone, eliminating the need for enzymatic treatment. Therefore, strong sheet-like retinal tissue can be recovered without weakening intercellular bonds due to enzymatic treatment. Therefore, it is preferable to use a culture vessel coated with an extracellular matrix and / or a temperature-sensitive polymer, particularly both an extracellular matrix and a temperature-sensitive polymer. For example, the culture surface of the culture vessel may be coated with the temperature-sensitive polymer, and the top surface of the polymer may be coated with the extracellular matrix. Here, the culture surface refers to the surface of the culture vessel to which cells adhere, and the top surface of the polymer refers to the surface opposite the surface that contacts the polymer-coated culture surface.

[0156] The extracellular matrix refers to a biopolymer that constitutes the space outside cells, and examples thereof include cell adhesive proteins such as fibronectin, vitronectin, and laminin, fibrous proteins such as collagen and elastin, fragments of these proteins, glycosaminoglycans or proteoglycans such as hyaluronic acid and chondroitin sulfate, and Matrigel. The extracellular matrix is ​​preferably one or more substances selected from the group consisting of collagen, laminin, fibronectin, Matrigel, vitronectin, and fragments of these proteins. Examples of laminin fragments include commercially available products such as iMatrix-511, iMatrix-411, and iMatrix-221.

[0157] Matrigel is a basement membrane preparation derived from the Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared, for example, by the method disclosed in U.S. Patent No. 4,829,000, or purchased commercially. The main components of Matrigel are laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.

[0158] A temperature-sensitive polymer is a polymer whose properties change with temperature. Specifically, it exhibits a phase transition behavior in which, at temperatures above a certain threshold, intramolecular or intermolecular hydrophobic bonds strengthen, causing polymer chains to aggregate. Conversely, at lower temperatures, the polymer chains bind water molecules and become hydrated. Specifically, a temperature-sensitive polymer may be used that maintains its hydrophobic state at the incubation temperature (approximately 37°C) and becomes hydrophilic at temperatures lower than the incubation temperature, e.g., around 20-30°C, making it easier for cultured cells to detach. Therefore, cell detachment does not require enzymatic treatment, and cells can be recovered as a large sheet without disrupting the proteins between cells. A preferred example of such a temperature-sensitive polymer is poly-N-isopropylacrylamide (PIPAAm) (LCST: 32°C). Cell culture is usually carried out under conditions of approximately 37°C, and taking into consideration damage to cells due to low temperatures, a temperature-sensitive polymer with an LCST in the range of approximately 20°C to 35°C is preferred. Commercially available products include temperature-responsive cell cultureware for cell sheet recovery (CellSeed Inc.: UpCell (registered trademark)) with a temperature-sensitive polymer fixed to the surface.

[0159] Regarding the culture conditions, the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and CO ２ The culture was carried out under an atmosphere of CO ２ The concentration is preferably about 2-5%.

[0160] The formation of layer structures in sheet-like retinal tissue, and in particular the formation of apical / basal polarity, requires Wnt signaling pathway agents.

[0161] The concentration of the substance acting on the Wnt signaling pathway may be any concentration capable of inducing sheet-like retinal tissue having a layered structure and an apical surface. For example, when CHIR99021 is used, the concentration of the substance acting on the Wnt signaling pathway may be 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, and more preferably 1 μM to 10 μM. When other substances acting on the Wnt signaling pathway are used, the concentration may be such that they exhibit the same level of Wnt signal activation activity as CHIR99021 at the aforementioned concentrations. Those skilled in the art can easily determine whether a layered structure has been formed, for example, by observing with a microscope or measuring the thickness using a device such as OCT. Whether an apical surface has been formed can be confirmed by staining with, for example, an anti-Zo-1 antibody, an anti-Ezrin antibody, or an anti-typical-PKC antibody.

[0162] The timing of adding the substance acting on the Wnt signaling pathway is not particularly limited, but it is preferable to add it as soon as possible after the initiation of adherent culture. In one embodiment, it is preferable to culture in a medium containing a substance acting on the Wnt signaling pathway from the initiation of culture. The number of days of culture in a medium containing a substance acting on the Wnt signaling pathway is not particularly limited as long as the effect of reforming a multilayer structure (or multilayer structure) with apical / basement membrane polarity is observed, but is, for example, 1 to 15 days, preferably 1 to 9 days.

[0163] The culture may be continued in a medium from which substances acting on the Wnt signaling pathway, etc., have been removed. By continuing the culture, the layer structure thickens and the differentiation of retinal cells progresses. The culture period is not particularly limited, and may be a period until the seeded, dispersed retinal cell population proliferates and at least an epithelial structure (or multi-layered structure) is formed, or until a sheet-like retinal tissue at the desired differentiation stage is produced. From the viewpoint of forming an epithelial structure (or multi-layered structure), it is desirable to culture for at least 7 days. The culture period may be, for example, from 7 days to 60 days or less, or may be 40 days or less, 30 days or less, 20 days or less, or 16 days or less (e.g., 16 days).

[0164] Even after an epithelial structure (or a multi-layered structure) is formed, the culture may be continued for further cell proliferation, differentiation, or maturation. The medium used for further culture may or may not contain a substance acting on the Wnt signaling pathway, a ROCK inhibitor, a substance acting on the SHH (sonic hedgehog) signaling pathway, and / or a substance acting on the integrin signaling pathway.

[0165] The term "multilayer structure" refers to a structure formed by stacking two or more cell layers in which cells are aligned in the same direction in tissue having a basement membrane side and an apical side polarity (i.e., an epithelial structure), and the tangential directions of the surfaces of the different layers are roughly parallel to each other. The multilayer structure preferably has a basal surface and an apical surface polarity, and the basement membrane and in one embodiment, the retinal tissue having a multilayer structure may be a sheet-like retinal tissue, and the cell aggregate containing the retinal tissue having a multilayer structure may be a sheet-like cell aggregate. In another embodiment, the cell layer may be a neural retinal progenitor cell layer, a ganglion cell layer, or a photoreceptor cell layer.

[0166] In one embodiment of the present invention, the orientation of cells in a multilayer structure may be approximately perpendicular to the layer direction. Here, "cell orientation" refers to the direction in which the shape of the nucleus and the orientation of the cell body extend toward the basement membrane and the apical end. Furthermore, "approximately perpendicular to the layer direction" refers to a direction perpendicular to the direction in which individual cells are lined up in contact with each other in a layer of the multilayer structure (i.e., the tangent direction of the surface of the layer), and refers to a direction perpendicular or longitudinal to the layer.

[0167] In one aspect of the present invention, the method may further include a step of cutting out the retinal tissue (particularly sheet-like retinal tissue) having an epithelial structure (or multilayer structure) obtained by suspension culture or adherent culture to a size required for transplantation. For example, the tissue can be cut out using tweezers, a knife, scissors, or the like.

[0168] On the other hand, when producing sheet-shaped retinal tissue using the above-mentioned method, a problem was found in that a certain percentage of RPE cells were produced and mixed in. These RPE cells can be visually confirmed and removed, but from the standpoint of quality and production efficiency, it is preferable that RPE cells are not included from the beginning. Therefore, as a result of intensive research to solve this problem, it has been found that by using the method disclosed below, it is possible to significantly reduce the contamination of RPE cells.

[0169] [Sheet-like retinal tissue] One embodiment of the present invention is a sheet-like retinal (neural retinal) tissue having an epithelial structure. One embodiment of the sheet-like retinal tissue is composed of a multilayered retinal cell layer, the multilayered structure having basal and apical polarity, the multilayered retinal cell layer containing one or more cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells, and the orientation of the cells in the retinal cell layer is approximately perpendicular to the layer direction. Here, the term "sheet-like" refers to a single-layer or multilayer structure composed of a single cell or multiple cells that are biologically connected in at least two dimensions.

[0170] One advantage of the present invention is that it allows the production of sheet-shaped retinal tissue of any size depending on the culture substrate used. That is, it is possible to produce sheet-shaped retinal tissue of a size that was previously impossible to produce, and in cases where disease affects a wide area, it becomes possible to treat the disease by transplanting a single sheet.

[0171] In one aspect, the major axis (also referred to as diameter) of the sheet-shaped retinal tissue according to the present invention is, for example, 2 mm or more, 4 mm or more, 5 mm or more, 7.5 mm or more, or 10 mm or more. In one aspect, the minor axis of the sheet-shaped retinal tissue according to the present invention is, for example, 2 mm or more, 3 mm or more, 4 mm or more, or 5 mm or more. In principle, there is no upper limit to the major and minor axes, but the size of the dish or the like used for culture is the limit. In one aspect, the major axis may be 10 cm or less, 5 cm or less, 4 cm or less, 3 cm or less, 2 cm or less, or 1 cm or less. In one aspect, the height of the sheet-shaped retinal tissue according to the present invention may be, for example, 50 μm to 1500 μm, and preferably 200 μm to 700 μm.

[0172] The method for measuring the major axis, minor axis, and height of sheet-like retinal tissue is not particularly limited, and can be measured, for example, from images captured under a microscope. For example, a front image of the sheet-like retinal tissue is taken with the apical surface facing the objective lens, and a lateral image is taken with the cut surface tilted so that it is perpendicular to the objective lens, using a stereomicroscope, and measurements can be made from the captured images. Here, the major axis refers to the longest line segment and its length among the line segments connecting two end points on the sheet cross section in the front image. The minor axis refers to the longest line segment and its length among the line segments connecting two end points on the sheet cross section in the front image that are perpendicular to the major axis. The height refers to the longest line segment and its length among the line segments perpendicular to the sheet cross section, whose end points are the intersection with the sheet cross section and the apex of the retinal sheet.

[0173] The basal and apical surfaces are as defined above. "The multilayer structure has a polarity of basal and apical surfaces" means that the multilayer structure has a basal surface on one side and an apical surface on the other side. A basement membrane may be present on the basal surface.

[0174] "The orientation of the cells is approximately perpendicular to the layer direction" means that the major axis of the cells present in each layer of the retinal cell layer is approximately perpendicular to the layer direction. The "approximately perpendicular" direction means that the acute angle between the layer direction and the major axis of the cells is approximately 75° (or 80°) or more and 90° or less. In this specification, if approximately 50% or more, preferably 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the cells present in each layer are approximately perpendicular to the layer direction, it is determined that "the orientation of the cells is approximately perpendicular to the layer direction."

[0175] It is preferable that the sheet-shaped retinal tissue does not contain RPE cells. In one embodiment, the ratio of the number of RPE cells to the total number of cells in the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. The ratio of the number of RPE cells to the total number of cells in the sheet-shaped retinal tissue can be measured, for example, by measuring the percentage of cells expressing the above-mentioned RPE cell markers using flow cytometry (FACS) or the like. In one embodiment, the percentage of the area occupied by RPE cells in the total area of ​​the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Since RPE cells are black, the percentage of the area occupied by RPE cells in the total area of ​​the sheet-shaped retinal tissue can be calculated from the percentage of the area that appears black under a microscope. Cells expressing RPE cell markers can also be detected by PCR or the like. The method for reducing the proportion of RPE cells in a sheet-like retinal (neural retinal) tissue is as described above.

[0176] In one embodiment, the sheet-like retinal tissue consisting of a retinal cell layer having a multilayer structure has the following characteristics: (1) the retinal cell layer having a multilayer structure has polarity at the basal surface and the apical surface; (2) the retinal cell layer having a multilayer structure contains one or more types of cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells; (3) in each layer of the retinal cell layer, the orientation of the cells is approximately perpendicular to the layer direction; and (4) the diameter is 8 mm or more.

[0177] (Sheet-like Retinal Cell Layer (Neural Retina)-Retinal Pigment Epithelial Cell Composite (Composite Sheet)) In one aspect of the present invention, the retinal cell layer having the multilayer structure may further include a sheet-like retinal pigment epithelial cell bonded to the retinal cell layer, and the retinal cell layer and the sheet-like retinal pigment epithelial cell are tangentially parallel to each other, the apical surface of the retinal cell layer faces the apical surface of the sheet-like retinal pigment epithelial cell, and the retinal cell layer and the sheet-like retinal pigment epithelial cell are bonded to each other via adhesion factors present between them (composite sheet). A sheet-like neural retina is also referred to as a neural retina sheet or an NR sheet, and a sheet-like retinal pigment epithelial cell is also referred to as a retinal pigment epithelial cell sheet or an RPE cell sheet (RPE sheet). In other words, one aspect of the present invention also provides a composite (composite sheet) of neural retina and RPE cells. Another embodiment of the present invention is a composite (composite sheet) in which dispersed retinal pigment epithelial cells are attached to the apical surface side of a sheet-like retinal cell layer (neural retina sheet) via an adhesion factor.

[0178] In the composite of the present specification, the tangential directions of the neural retina sheet and the retinal pigment epithelial cell sheet are approximately parallel to each other. "Approximately parallel tangential directions" means that the tangential directions of the opposing surfaces of the neural retina and the retinal pigment epithelial cell sheet are parallel to each other. Furthermore, in the composite of the present invention, the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cells face each other. In other words, the apical surfaces of the neural retina and the retinal pigment epithelial cells are in close proximity to each other.

[0179] A composite of neural retina and RPE cells (composite sheet) can be prepared by joining a sheet-like retinal cell layer (neural retina sheet) with an RPE cell sheet or dispersed RPE cells in the presence of an adhesion factor, as described below. The sheet-like retinal cell layer (neural retina sheet) can be produced by the above-mentioned method for producing a cell aggregate containing retinal tissue having an epithelial structure (or multilayer structure). The RPE cell sheet can be produced by the method for producing a retinal pigment epithelial cell sheet described below.

[0180] (Method for producing a retinal pigment epithelial cell sheet) Retinal pigment epithelial (RPE) cells are derived from pluripotent stem cells and can be obtained by inducing differentiation of pluripotent stem cells. Methods for producing retinal pigment epithelial cells are described in WO2005 / 070011, WO2006 / 080952, WO201 １／０６ 3005, WO2012 / 173207, WO2015 / 053375, WO2015 / 053376, WO2015 / 068505, WO2017 / 043605, Stem Cell Reports, 2(2), 205-218 (2014), and Cell Stem Cell, 10(6), 771-785 (2012). Furthermore, a retinal pigment epithelial (RPE) cell sheet can also be prepared by modifying the method described in WO2016 / 063985. Retinal pigment epithelial cells may be produced as a cell sheet or spherical cell aggregates. When produced as spherical cell aggregates, RPE cell sheets can be prepared by cutting open the cell aggregates using, for example, tweezers, a knife, scissors, or the like.

[0181] An improved version of the method described in WO 2016 / 063985 involves culturing pluripotent stem cells in the absence of feeder cells by 1) treating them with a TGFβ family signaling pathway inhibitor and a sonic hedgehog signaling pathway agonist one day before differentiation induction, and 2) culturing them without treatment with a sonic hedgehog signaling pathway agonist at the start of differentiation induction. The above-described steps (B) and (C) are then performed. Furthermore, it is preferable to initiate step (D) earlier. Specifically, step (D) is initiated approximately 9 days (e.g., 7, 8, 9, 10, or 11 days) after the start of suspension culture in step (B). Then, the above-described step (E) is performed. This method yields spherical cell aggregates of RPE cells. The cell aggregates can be dispersed to form a cell suspension, or an RPE cell sheet can be prepared by cutting the cell aggregates open using tweezers, a knife, scissors, or the like. An RPE cell sheet can also be prepared by culturing the dispersed cell suspension in an adherent culture. Dispersed RPE cells can also be obtained by dispersing an RPE cell sheet or an aggregate of RPE cells.

[0182] Before contacting the retinal pigment epithelial cell sheet with neural retinal cell aggregates, it may be further cultured until it acquires a polygonal or cobblestone-like cell morphology. In this case, the medium is not particularly limited, but it can also be replaced with a retinal pigment epithelial cell maintenance medium (hereinafter sometimes referred to as RPE maintenance medium) and further cultured. This allows for more clearly observed melanin-pigmented cell groups and cell groups with polygonal and flattened morphologies adhering to the basement membrane. The culture in RPE maintenance medium is not limited as long as it forms colonies that proliferate while maintaining the properties of retinal pigment epithelial cells. For example, the culture is performed for 5 days or more (e.g., approximately 5 to 20 days) with a complete medium change at least once every 3 days. Those skilled in the art can easily determine the culture period while confirming the morphology. Retinal pigment epithelial cell maintenance medium can be prepared, for example, as described in IOVS, March 2004, Vol. 45, No. 10001. 3, Masatoshi Haruta et al., IOVS, November 2011, Vol. 52, No. 12, Okamoto et al., Cell Science 122 (17), Fumitaka Osakadar et al., bureau 2008, Vol. 49, No. 2, and Gamm et al. can be used.

[0183] The major axis of the retinal pigment epithelial cell sheet may be the same as the major axis of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the major axis of the retinal pigment epithelial cell sheet may be in the range of, for example, 3 mm to 50 mm, 5 mm to 30 mm, or 10 mm to 20 mm.

[0184] The minor axis of the retinal pigment epithelial cell sheet may be the same as the minor axis of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the minor axis of the retinal pigment epithelial cell sheet may be in the range of, for example, 2 mm to 40 mm, 5 mm to 30 mm, or 10 mm to 20 mm.

[0185] The degree of melanin pigmentation in the retinal pigment epithelial cell sheet is not particularly limited. It is preferable that the degree of melanin pigmentation in the retinal pigment epithelial cells contained in the retinal pigment epithelial cell sheet is similar among the cells. In one embodiment, the average melanin content of the retinal pigment epithelial cell sheet may be less than 20 pg / cell, less than 15 pg / cell, less than 10 pg / cell, less than 8 pg / cell, less than 7 pg / cell, less than 6 pg / cell, less than 5 pg / cell, less than 4 pg / cell, less than 3 pg / cell, less than 2 pg / cell, or less than 1 pg / cell. The average melanin content of the retinal pigment epithelial cell sheet may also be 0.1 pg / cell or more, 0.5 pg / cell or more, 1 pg / cell or more, 2 pg / cell or more, or 5 pg / cell or more.

[0186] The melanin content in a retinal pigment epithelial cell sheet can be measured, for example, by dispersing the retinal pigment epithelial cell sheet and then extracting the cell extract with NaOH or the like using a spectrophotometer, etc. The average melanin content can be determined by dividing the melanin content by the total number of cells contained in the retinal pigment epithelial cell sheet.

[0187] The above-mentioned composite can be produced by joining a neural retina sheet with dispersed RPE cells or an RPE cell sheet. A composite sheet formed by joining a neural retina sheet with an RPE cell sheet is preferred. The above-mentioned neural retina sheet and RPE cell sheet can be easily removed from the culture vessel using tweezers, a knife, scissors, or the like. The removed sheets can be transferred to a new container (e.g., culture vessel), or one sheet can be left in the culture vessel and the other transferred to the culture vessel. By producing the sheets using culture vessels of the same size, the size of the sheet-like tissues to be joined can be made uniform. Furthermore, when using neural retina sheets and retinal pigment epithelial cells that have been cultured for different days in the above-mentioned production method, the start dates of production can be staggered.

[0188] The neural retinal sheet and retinal pigment epithelial cells are preferably contacted in the presence of an adhesion factor. The adhesion factor refers to a substance that has the ability to adhere cells to each other, and is not particularly limited, but examples include the above-mentioned extracellular matrix and artificial hydrogels. The adhesion factor does not need to be an isolated single substance, and also includes preparations from living organisms or cells, such as Matrigel, interphotoreceptor matrix, and serum. Matrigel is a basement membrane preparation derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared, for example, by the method disclosed in US Patent No. 4,829,000, or commercially available. The main components of Matrigel are laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Interphotoreceptor matrix is ​​a general term for the extracellular matrix present between retinal cells such as photoreceptors in the living retina, and includes, for example, hyaluronic acid. Those skilled in the art can extract interphotoreceptor matrix from living retinas by, for example, placing the retina in distilled water, swelling it, and separating it. It is also commercially available. The adhesion factor is preferably an extracellular matrix or a hydrogel. Furthermore, the extracellular matrix is ​​preferably one or more extracellular matrices selected from the group consisting of hyaluronic acid, fibrin, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. The hydrogel is preferably one or more hydrogels selected from the group consisting of gelatin, fibrin, collagen, pectin, hyaluronic acid, and alginic acid. Although there are substances classified as both extracellular matrices and hydrogels, in this specification, when used as a gel-like adhesion factor, it is considered to be a hydrogel. Examples of commercially available extracellular matrices include Corning® Matrigel basement membrane matrix and iMatrix 511. The adhesion factor may be one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin, and is particularly preferably gelatin or fibrin.

[0189] Fibrin gel is a fibrin gel obtained by reacting a fibrinogen solution with a thrombin solution. The commercially available Bolheal (registered trademark) tissue adhesive can also be used. The fibrinogen solution and the thrombin solution can be reacted by contacting or mixing them.

[0190] "Gelatin" refers to water-insoluble collagen that has been solubilized by, for example, pretreating with an acid or alkali and then thermally hydrolyzing it. Commercially available gelatin is also available, and examples include Gelatin LS-H (Nitta Gelatin Co., Ltd., pigskin alkali-treated gelatin, non-heat-treated gelatin, high jelly strength) and Gelatin LS-W (Nitta Gelatin Co., Ltd., pigskin alkali-treated gelatin, heat-treated gelatin, low jelly strength). Alkali-treated (lime-treated) gelatin (type B gelatin) is preferred, and heat-treated gelatin is also preferred.

[0191] Hydrogels undergo a phase change from gel to sol and from sol to gel upon heating or cooling. Hydrogels become gels (jelly) when cooled, losing their fluidity, and sols (aqueous solutions) when heated, regaining their fluidity. Here, "melting point" refers to the temperature at which a hydrogel becomes a sol under constant pressure, and "freezing point" refers to the temperature at which a hydrogel becomes a gel under constant pressure. It is preferable for hydrogels to be biodegradable. In one embodiment, hydrogels (e.g., gelatin) have a melting point near body temperature (25°C to 40°C). The hydrogels described herein may have a melting point of 20°C to 40°C (e.g., 20°C to 35°C, 25°C to 35°C, 30°C to 40°C, or 35°C to 40°C). Generally, the melting point of a gel is a measure of the network strength, and the melting point of a hydrogel (e.g., gelatin) increases with increasing concentration and molecular weight of the hydrogel. Furthermore, for example, increasing the solid content by adding sugars tends to increase the melting point and freezing point. In this way, it is possible to vary the melting point and freezing point within a certain range. The method for measuring the melting point of the hydrogel is not particularly limited, and can be measured, for example, by the method specified in JIS K6503.

[0192] The strength of a hydrogel is sufficient as long as it does not collapse during the procedure for transplantation. One indicator of the strength of a hydrogel is "jelly strength." The "jelly strength" of a hydrogel refers to the mechanical strength of an object formed from the gel. It is usually the force required to deform a gel of a certain shape or the force required to break the gel (unit: g, dyne (s) / cm ２ or g / cm ２ ) and is mainly a measure of the hardness of gels. Note that 1 dyne (dyne (s)) is 1 centimeter per second per second (cm / s) in the direction acting on an object with a mass of 1 gram (g). ２ ) acceleration. For example, the jelly strength of gelatin can be measured by the method specified in JIS K6503. In this specification, for example, the jelly strength of a hydrogel (e.g., gelatin) gel may be 50 g or more, 100 g or more, 200 g or more, 500 g or more, 1000 g or more, 1200 g or more, 1300 g or more, 1400 g or more, or 1500 g or more. In addition, the jelly strength of a hydrogel (e.g., gelatin) may be 3000 g or less, 2500 g or less, or 2000 g or less.

[0193] The concentration of the adhesion factor (extracellular matrix) varies depending on the size of the neural retina sheet or retinal pigment epithelial cell sheet and the number of retinal pigment epithelial cells, but can be easily determined by a person skilled in the art by confirming the state of adhesion of RPE cells. For example, it is preferable to add Matrigel at a concentration obtained by diluting a ready-made product (Corning) 200 to 10,000 times, or iMatrix 511 at a concentration of 0.1 to 5 μg / mL.

[0194] In one embodiment of the method for producing the composite using an extracellular matrix, a neural retinal sheet and retinal pigment epithelial cells or retinal pigment epithelial cell sheet may be cultured in a medium containing an adhesion factor (extracellular matrix) to adhere to each other. The medium used is not particularly limited, but examples include media used for culturing retinal pigment epithelial cells or neural retina (e.g., DMEM / F12 medium, Neurobasal medium, a mixture of these, RPE maintenance medium, etc.). Alternatively, the culture for adhesion may be performed in the presence of other components, such as growth factors such as EGF, along with the extracellular matrix. During the culture period for adhering the neural retinal sheet and retinal pigment epithelial cells or retinal pigment epithelial cell sheet described above, the cells may be cultured continuously in the medium containing the adhesion factor, or after a certain period of culture (e.g., 1 to 10 days) in the medium containing the adhesion factor, the medium may be replaced with a medium not containing the adhesion factor, and the culture may be continued.

[0195] Before culturing the neural retinal sheet and the retinal pigment epithelial cells or retinal pigment epithelial cell sheet to adhere to each other, the neural retinal sheet or the retinal pigment epithelial cell sheet may be coated with an adhesion factor. Specifically, the neural retinal sheet or the retinal pigment epithelial cells or retinal pigment epithelial cell sheet may be cultured in the above-mentioned medium containing the adhesion factor. Those skilled in the art can appropriately determine the culture time, but it is sufficient to culture for approximately 10 minutes to 5 hours (e.g., 10 minutes to 60 minutes). After culturing, the sheet may be washed with a medium such as PBS.

[0196] The adhesion factor, hydrogel, or matrix gel is preferably fibrin gel. Fibrin gel is a fibrin gel obtained by reacting a fibrinogen solution with a thrombin solution. In this specification, a substance capable of forming a gel upon reaction is referred to as a matrix precursor. For example, thrombin and fibrinogen, which react to form a fibrin gel, are examples of matrix precursors. The fibrinogen solution can be prepared by dissolving fibrinogen powder or the like in a fibrinogen-dissolving solution containing aprotinin. The concentration of the fibrinogen solution is not particularly limited, but may be, for example, 40 to 480 mg / mL, preferably 80 to 320 mg / mL (e.g., 160 mg / mL). Furthermore, when the blood coagulation factor VIII activity contained in 1 mL of normal human plasma is defined as 1 unit, the fibrinogen solution may have a concentration of 37.5 units / mL to 225 units / mL (e.g., 75 units / mL). The thrombin solution can be prepared by dissolving thrombin powder or the like in a thrombin solution containing calcium chloride hydrate, and the concentration is not particularly limited, but is, for example, 125 units / mL to 750 units / mL (e.g., 75 units / mL). The fibrinogen solution and the thrombin solution can be reacted by contacting or mixing them, and in this case, the fibrinogen solution and the thrombin solution are preferably used in an activity ratio of 1:1 to 1:9, preferably 1:3 to 1:4.

[0197] As one embodiment of the method for producing the above-mentioned composite using a hydrogel, a method for producing a composite using a fibrin gel is described below. Specifically, a neural retina sheet and retinal pigment epithelial cells or a retinal pigment epithelial cell sheet are adhered to each other using a fibrin gel produced by reacting fibrinogen with thrombin.

[0198] In one embodiment, one of the two tissues (neural retina or retinal pigment epithelial cells) contacted with a fibrinogen solution and the other tissue (retinal pigment epithelial cells or neural retina) contacted with a thrombin solution may be brought into contact with each other to cause a reaction between fibrinogen and thrombin and form a gel. Here, "contact with the solution" refers to contacting at least the adhesive surface of the tissue (the surface facing the other tissue when adhering to the other tissue with the fibrin gel) with the fibrinogen solution or thrombin solution to such an extent that the solution adheres to the adhesive surface of the tissue.

[0199] More specifically, for example, when adhering a neural retinal sheet to a retinal pigment epithelial cell sheet, the neural retinal sheet is immersed in a fibrinogen solution, and the retinal pigment epithelial cell sheet is immersed in a thrombin solution (the solutions can be reversed). The fibrinogen solution and the thrombin solution are preferably used in a volume ratio of 3:1 to 1:3. Before adhering, excess fibrinogen or thrombin adhering to the tissues may be removed. This procedure allows the thickness of the fibrin gel to be adjusted.

[0200] To adhere a neural retinal sheet contacted with a fibrinogen solution and a retinal pigment epithelial cell sheet contacted with a thrombin solution, the two may be brought into contact. Here, "contacting the tissues" refers to bringing the surface with the fibrinogen solution into contact with the surface with the thrombin solution, i.e., overlapping them. Furthermore, it is preferable to adhere the retinal pigment epithelial cell sheet and the neural retina so that the tangent directions of their respective surfaces are roughly parallel and the apical surfaces of the neural retina and the retinal pigment epithelial cell sheet face each other.

[0201] In one embodiment, the tissue may be contacted with a fibrinogen solution, and then a thrombin solution may be added to the fibrinogen solution to react the fibrinogen and thrombin, in which case the tissue is embedded in a fibrin gel.

[0202] In one embodiment, the manufacturing method of the present invention may further include a step of cutting out a composite of a size required for transplantation from the composite. Since two or more tissues are firmly adhered by the fibrin gel, when a graft is cut out from the composite, the tissues can be easily cut out to the desired size without peeling off from the composite. When cutting out, tweezers, a knife, scissors, etc. can be used.

[0203] Pharmaceutical Compositions, Treatment Methods, Therapeutic Agents, and Uses One embodiment of the present invention is a pharmaceutical composition comprising a sheet-shaped retinal tissue. The pharmaceutical composition preferably further comprises a pharmaceutically acceptable carrier in addition to the sheet-shaped retinal tissue of the present invention. The pharmaceutical composition can be used to treat disorders of neural retinal system cells or the neural retina, or diseases caused by damage to the neural retina. Examples of diseases caused by damage to neural retinal system cells or the neural retina include ophthalmic diseases such as retinal degenerative diseases, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone-rod dystrophy. Examples of damaged states of the neural retina include states in which photoreceptor cells are degenerated and dead.

[0204] As a pharmaceutically acceptable carrier, a physiological aqueous solvent (such as physiological saline, a buffer solution, or a serum-free medium) can be used. If necessary, the pharmaceutical composition may contain preservatives, stabilizers, reducing agents, isotonicity agents, and the like that are commonly used in medicines containing tissues or cells to be transplanted in transplantation medicine.

[0205] One aspect of the present invention provides a therapeutic agent for a disease caused by neural retinal damage, comprising a sheet-like retinal tissue obtained by the present invention. Another aspect of the present invention includes a method for treating a neuroretinal cell or neuroretinal disorder or a neuroretinal damage-related disease, comprising transplanting the sheet-like retinal tissue obtained by the present invention into a subject requiring transplantation (e.g., subretinal area of ​​an eye suffering from an ophthalmic disorder). The sheet-like retinal tissue of the present invention can be used as a therapeutic agent for a neuroretinal disorder-related disease or to replenish the damaged area of ​​the neuroretina in a damaged state of the neuroretina. By transplanting the sheet-like retinal tissue of the present invention into a patient requiring transplantation who has a neuroretinal cell or neuroretinal disorder-related disease or a patient with a damaged neuroretina, the neuroretinal cell or the damaged neuroretina can be replenished, thereby treating the neuroretinal cell or neuroretinal disorder-related disease or the neuroretinal damage state. Examples of transplantation methods include a method in which a sheet-like retinal tissue is transplanted subretinally at the damaged area via incision in the eyeball. Examples of the transplantation method include injection using a thin tube and transplantation by pinching with tweezers, and examples of the thin tube include an injection needle.

[0206] One aspect of the present invention provides a sheet-shaped retinal tissue obtained by the present invention for use in treating disorders of retinal cells or retinal tissue or diseases caused by retinal tissue damage. Another aspect of the present invention provides use of a sheet-shaped retinal tissue obtained by the present invention in the manufacture of a therapeutic agent for disorders of retinal cells or retinal tissue or diseases caused by retinal tissue damage.

[0207] 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.

[0208] Example 1: Search for Aggregate-Reforming Factors Human ES cells (KhES-1 strain (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene and human iPS cells (1231A3 strain, obtained from Kyoto University) 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0209] The specific maintenance culture procedure for human ES and human iPS cells (human ES / iPS cells) was performed as follows. First, human ES / iPS cells 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 (trade name, manufactured by Life Technologies). Note that dispersion into single cells here means dispersing the cells to form single cells, and the cell population dispersed into single cells may contain clumps of 2 to 50 cells in addition to single cells. The dispersed human ES cells were then seeded onto a plastic culture dish coated with Laminin 511-E8 and cultured under feeder-free conditions in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM). The plastic culture dish used was a 6-well plate (manufactured by Iwaki Co., Ltd., for cell culture, culture area 9.4 cm). ２ ), the number of human ES / iPS cells dispersed into single cells seeded per well was 0.4 to 1.2 × 10 ４ Human ES cells were used. One day after seeding, the medium was changed to StemFit medium without Y-27632. Thereafter, the medium was changed once every 1-2 days with StemFit medium without Y-27632. The cells were then cultured under feeder-free conditions until one day before reaching subconfluence. The human ES cells one day before reaching subconfluence were cultured under feeder-free conditions for one day in the presence of SB431542 (a TGFβ signaling pathway inhibitor, 5 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM) (preconditioning treatment).

[0210] The human ES / iPS cells were washed with PBS, then treated with TrypLE Select to form a cell dispersion solution, and further dispersed into single cells by pipetting. The dispersed human ES cells were then plated at 1.2 × 10 per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ４ The cells were suspended in 100 μL of serum-free medium and incubated at 37°C, 5% CO ２ 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.

[0211] At the initiation of suspension culture (day 0, at the initiation of differentiation induction), Y-27632 (a ROCK inhibitor, final concentration 10 μM or 20 μM) and SAG (a substance acting on the Shh signaling pathway, 300 nM, 30 nM, or 0 nM) were added to the serum-free medium. On day 3 after the initiation of suspension culture, 50 μL of medium containing exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Systems) at a final concentration of 1.5 nM was used. From day 6 after the initiation of suspension culture, half of the medium was replaced every 3 days with medium containing no Y-27632, SAG, or human recombinant BMP4.

[0212] For KhES-1-derived cell aggregates, the aggregates from 14 to 18 days after the start of suspension culture were transferred to a 90 mm low-adhesion culture dish (90Φ (deep) dish for suspension culture, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured in a serum-free medium (DMEM / F12 medium supplemented with 1% N2 Supplement) containing a substance acting on the Wnt signaling pathway (CHIR99021, 3 μM) and a substance inhibiting the FGF signaling pathway (SU5402, 5 μM) at 37°C and 5% CO. ２The cells were cultured for 3 to 4 days in a 90 mm low-adhesion culture dish (suspension culture dish 90Φ (deep type), manufactured by Sumitomo Bakelite Co., Ltd.) and then cultured for a long period in a serum-free medium (NucT0 medium) that did not contain a substance acting on the Wnt signaling pathway or a substance inhibiting the FGF signaling pathway.

[0213] Aggregates on day 16 (1231A3-derived) or day 27 (KhES-1-derived) after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C for 20-30 minutes, the cells were dispersed into single cells by pipetting. These cells were plated at 2 x 10 per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, Sumitomo Bakelite Co., Ltd.). ５ cells (KhES-1 derived) or 5 × 10 ５ The cells (derived from 1231A3) were suspended in 100 μL of serum-free medium and incubated at 37°C, 5% CO ２ The cells were cultured in suspension at 100°C for 1 hour. Simultaneously with the addition of the cells, (1) no addition (control), (2) 10 μM or 20 μM (derived from 1231A3) Y-27632 (Wako), (3) 300 nM SAG (Enzo), (4) 10 μg / mL bFGF (Wako), (5) 1 μM LDN193189 (Stemgent), (6) 3 μM CHIR99021 (Wako), (7) 10 μM SB431542 (Wako), or (8) 3 μM IWR-1 (Wako) were added.

[0214] Under all of the conditions (1) to (8), it was confirmed that aggregates reformed after approximately two weeks of culture (see the results of Example 3). However, when Y-27632 was added, aggregates were confirmed to reform from 1 to 2 days (Day 1 to Day 2) after suspension culture for aggregate reformation (Figure 1: KhES-1, Figure 2: 1231A3). On the other hand, compounds other than Y-27632 did not exhibit any effect of promoting early aggregate reformation (Figures 1 and 2). Therefore, it was found that the addition of Y-27632 to retinal progenitor cells dispersed into single cells promoted early aggregate reformation.

[0215] Example 2: Search for factors promoting layer structure formation and aggregate growth Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0216] The aggregates prepared as in Example 1 on the 20th day (1231A3) and 27th day (KhES-1) after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were plated at 2-5 x 10 per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, Sumitomo Bakelite Co., Ltd.). ５ The cells were suspended in 100 μL of serum-free medium containing 10 μM Y-27632 (Wako Co., Ltd.) and incubated at 37°C in 5% CO ２ Simultaneously with the addition of the cells, (1) no addition (10 μM Y-27632 only), (2) 30 nM SAG (Enzo), (3) 300 nM SAG (Enzo), (4) 10 ng / mL bFGF (Wako), (5) 1 μM LDN193189 (Stemgent), (6) 3 μM CHIR99021 (Wako), (7) 10 μM SB431542 (Wako), or (8) 3 μM IWR-1 (derived from KhES-1) or 10 ng / mL EGF (derived from 1231A3) was added.

[0217] The state of aggregate reformation was observed using a bright-field microscope and a fluorescence microscope (Keyence BZ-X810) on days 1, 7, and 14 after suspension culture for aggregate reformation. The results for KhES-1-derived cells are shown in Figures 3 to 5. The area of ​​the aggregates was measured using Image J. The results for KhES-1-derived cells are shown in Figure 6 (average value of 12 measurements). The results for 1231A3-derived cells, in which the state of aggregate reformation was observed on day 1 after suspension culture, are shown in Figure 7. Figures 3 to 7 confirm that the groups to which SAG (300 nM) or CHIR99021 (3 μM) was added in addition to Y-27632 reformed larger aggregates than the group to which only Y-27632 was added. Therefore, the effects of SAG and CHIR99021 in promoting the growth and reformation of aggregates were confirmed.

[0218] Example 3: Search for factors promoting layer structure formation and aggregate growth Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0219] The aggregates prepared as in Example 1 on day 26 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (manufactured by Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were plated at 3.0 × 10 per well of a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ４ The cells were suspended in 100 μL of serum-free medium and incubated at 37°C, 5% CO ２was cultured in suspension. Simultaneously with the addition of cells, (1) no addition (control), (2) 10 μM Y-27632 (Wako), (3) 300 nM SAG (Enzo), (4) 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo), (5) 3 μM CHIR99021 (Wako), (6) 10 μM Y-27632 (Wako) and 3 μM CHIR99021 (Wako), (7) 300 nM SAG (Enzo) and 3 μM CHIR99021 (Wako), (8) 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako) was added.

[0220] The state of aggregate reformation was observed on days 1, 14, and 28 (Day 1, Day 14, and Day 28) after suspension culture for aggregate reformation, i.e., on days 27, 40, and 54 (dd27, dd40, and dd54) after the initiation of suspension culture for differentiation induction, and the results are shown in Figures 8, 9, and 10. Figures 8, 9, and 10 confirmed that aggregates were reformed when CHIR99021 was added. It was also confirmed that aggregates were larger when SAG was added in addition to CHIR99021.

[0221] After suspension culture for the re-formation of aggregates, the aggregates were fixed with 4% PFA on day 15 (corresponding to day 41 of differentiation) and day 28 (corresponding to day 54 of differentiation) and cryosections were prepared. These cryosections were stained with DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-β-Catenin antibody (R&D Systems), anti-Collagen IV antibody (Abcam), anti-Zo-1 antibody (Invitrogen), anti-Ki67 antibody (BD), anti-Pax6 antibody (BioLegend), anti-RxR-γ (RxRg) antibody (Spring Bioscience), anti-CRX antibody (Abnova), anti-NRL antibody (R&D Immunostaining was performed using anti-Recoverin antibody (Proteintech), anti-Islet-1 antibody (DSHB), anti-GS antibody (Sigma), anti-Brn3 antibody (Santa Cruz), and anti-Calretinin antibody (R&D Systems).

[0222] These immunostained sections were observed using a bright-field microscope, a fluorescence microscope (Keyence BZ-X810), and a confocal laser scanning fluorescence microscope (Leica SP-8), and the results are shown in Figures 11 to 20. Figures 11, 14, 15, and 18 confirm that when CHIR99021 was added, a layered structure containing Rx::Venus-positive, Chx10-positive neural retinal progenitor cells was formed at the outermost part of the aggregates. Figures 12, 16, and 17 also confirm that when CHIR99021 was added, a Zo-1-positive apical surface was formed at the outermost part of the aggregates, and a collagen-positive basal surface was formed internally, forming epithelial tissue with apical and basal polarity. On the other hand, as shown in Figure 17 in particular, in the group to which CHIR99021 was not added, a lack of cell polarity or a rosette-like structure was observed. Furthermore, Figures 19 and 20 confirmed the differentiation of RxR-γ (RxRg)-positive and CRX-positive cone progenitor cells, and Recoverin-positive and CRX-positive photoreceptor progenitor cells, and also confirmed the differentiation of Islet-1-positive and Brn3-positive retinal ganglion cells and Calretinin-positive amacrine cells.

[0223] These results demonstrate that addition of CHIR99021 to a single cell suspension of dispersed retinal cells enables reorganization and differentiation of retinal tissue as epithelial tissue with apical-basal polarity.

[0224] Example 4: Differentiation Period for Aggregate Reformation Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0225] Aggregates prepared as in Example 1 on days 18, 25, 40, 61, and 75 (dd18, dd25, dd40, dd61, and dd75) after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (manufactured by WAKO) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were plated at 2-5 x 10 cells per well of a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ５ The cells were suspended in 100 μL of serum-free medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako), and incubated at 37°C, 5% CO ２ was cultured in suspension.

[0226] The state of aggregate reformation was observed using a bright-field microscope and a fluorescent microscope on days 3, 15, and 21 (Day 3, Day 15, and Day 21) of culture for aggregate reformation. The results are shown in Figure 21. Figure 21 confirmed that aggregates reformed and a layered structure was formed in cell aggregates at all differentiation days (dd18, dd25, dd40, dd61, and dd75). It was also observed that the characteristics of Rx::Venus-positive retinal tissue were maintained (Figure 21).

[0227] These results demonstrated that aggregates can be re-formed in retinal tissue at any differentiation stage by adding Y-27632, SAG, and CHIR99021.

[0228] Example 5: Reaggregation of Cerebral Organoids Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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.

[0229] On the third day after the start of suspension culture, 50 μL of medium containing exogenous human recombinant BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Systems) at a final concentration of 1.5 nM was used. In addition, a group without BMP4 was prepared to prepare cerebral organoids. From the sixth day after the start of suspension culture, half of the medium was replaced every three days with medium containing no Y-27632, SAG, or human recombinant BMP4.

[0230] The aggregates thus prepared on day 40 of suspension culture were washed with PBS, and a neuronal cell dispersion solution (manufactured by WAKO) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. The dispersed cells were plated at 5 x 10 per well of a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ５ The cells were suspended in 100 μL of serum-free medium containing 10 mM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako), and incubated at 37°C in 5% CO ２ was cultured in suspension.

[0231] The results of the state of aggregate reformation observed under bright field and fluorescence microscopes on days 3, 15, and 21 of culture for aggregate reformation are shown in Figure 22. Figure 22 confirmed that aggregates were reformed and a layered structure was formed in the Rx::Venus-negative telencephalic organoids as in the retinal organoids.

[0232] These results demonstrate that aggregates can be re-formed not only in retinal organoids but also in neuroepithelial tissues such as telencephalic organoids.

[0233] Example 6: Cryopreservation Study Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0234] The aggregates prepared as in Example 1 on day 31 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (manufactured by Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting.

[0235] After dispersion, in the non-frozen group, 2.0 × 10 cells were cultured per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ５ The cells were suspended in 100 μL of serum-free medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako), and incubated at 37°C, 5% CO ２ was cultured in suspension.

[0236] After dispersion, the single cells were frozen at a density of 1.0 × 10 using PBS (Gibco) as a negative control and the following cryopreservation solutions: Cell Banker 1 (TaKaRa), Stem Cell Banker (TaKaRa), CultureSure® cryopreservation solution (CultureSure® Freezing Medium; Fujifilm Wako Pure Chemical Corporation), STEMdiff™ Neural Progenitor Freezing Medium (Stemcell), StemSure® cryopreservation solution (Fujifilm Wako Pure Chemical Corporation), and Bambanker (Nippon Genetics Co., Ltd.). ６ The cells were suspended at 1000 cells / mL and stored frozen at -80°C using bicelles.

[0237] After one day of cryopreservation, the cells were awakened and the viability was confirmed. Immediately after awakening, the viability decreased dramatically in the PBS group, but was high in all cryopreservation media. The viability was above 90% in the cryopreservation media other than STEMdiff (Cell Banker 1, Stem Cell Banker, CultureSure, StemSure, and Bambanker), comparable to that of unfrozen cells (Figure 24(A)).

[0238] These cells were cultured at 2 × 10 cells per well in a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.). ５ The cells were suspended in 100 μL of NucT0 medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako), and incubated at 37°C, 5% CO ２ was cultured in suspension.

[0239] One day after seeding, it was confirmed that aggregates had reformed in all cryopreservation media (Cell Banker 1, Stem Cell Banker, CultureSure, STEMdiff, StemSure, and Bambanker) comparable to unfrozen cells (Figures 23 and 24(B)). In particular, with Cell Banker 1, the small Rx::Venus-positive cell aggregates present around the aggregates observed in the other cryopreservation media were not observed, and the observed image was similar to that of unfrozen cells (Figure 23).

[0240] After 7 days of culture for the re-formation of aggregates, it was found that aggregates were re-formed in all of the cryopreservation media in a manner comparable to that of non-frozen samples.

[0241] These results indicate that even if a single-cell suspension of retinal cells obtained by dispersion is cryopreserved, it is possible to re-form aggregates by culturing the cells after waking them up and adding Y-27632, SAG, and CHIR99021.

[0242] Example 7: Regarding scaffold proteins expressed in retinal aggregates. The studies in Examples 1 to 6 demonstrated that retinal cells were dispersed, pluripotent stem cells were differentiated into retina to obtain cell aggregates containing retinal cells, the cell aggregates were dispersed to obtain single-cell suspensions, and the single-cell suspensions were cultured in suspension with the addition of Y-27632 (Wako), SAG (Enzo), and CHIR99021 (Wako) at the time of seeding. This demonstrated that layered, polarized retinal tissue could be reorganized. Next, the authors envisioned performing adhesion culture to study the preparation of large sheet-like retinal tissue. They then decided to investigate the extracellular matrix for adhesion culture.

[0243] Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0244] Aggregates prepared as in Example 1, 25 to 35 days after the initiation of suspension culture, were fixed in 4% PFA and frozen sections were prepared. These frozen sections were immunostained using DAPI and anti-laminin antibody (trade name: Anti-Laminin Antibody, Abcam), anti-fibronectin antibody (R&D Systems), and anti-collagen IV antibody (Abcam). These immunostained sections were observed using a confocal laser microscope. The results confirmed that the aggregates expressed at least laminin, fibronectin, and collagen IV, which are components of the basement membrane (Figure 25). In other words, in this self-organizing culture system, human retinal tissue formed its own basement membrane and expressed laminin, fibronectin, and collagen IV.

[0245] We investigated the laminin isoforms expressed in mouse fetal neural retina tissues using information from a database of mouse fetal tissue staining specialized for basement membranes (MOUSE BASEMENT MEMBRANE BODYMAP; http: / / dbarchive.biosciencedbc.jp / archive / matrixome / bm / home.html). The results revealed that the laminin isoforms are laminin α 1, 4, and 5, laminin β 1 and 2, and laminin γ 1 (Figure 26).

[0246] These results suggest that the combination of Laminin α, Laminin β, and Laminin γ, with Laminin 511, 521, 411, 421, 111, and 121 (particularly 511, 521, and 411) being useful as an extracellular matrix suitable for neural retinal tissue.

[0247] That is, it was suggested that laminin 511, laminin 521, laminin 411, fibronectin, collagen IV, etc. may be useful as extracellular matrices for regenerating retinal sheets in adhesive culture.

[0248] Example 8: Examination of extracellular matrices seeded onto Transwells In the studies of Examples 1 to 6, a method was investigated for re-forming aggregates containing retinal tissue with a layered structure from a single-cell suspension of retinal cells in suspension culture. In Example 7, candidate extracellular matrices for use in adhesion culture were investigated. By combining these, a method for re-forming sheet-like retinal tissue (re-sheet formation) from a single-cell suspension of retinal cells in adhesion culture was investigated.

[0249] Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0250] The aggregates prepared as in Example 1 on day 16 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (manufactured by Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting.

[0251] Matrigel (Corning) and Laminin 511-E8 (trade name iMatrix 511, manufactured by Nippi) were used as extracellular matrices to promote re-sheet formation. The dispersed single cells were plated at 0.5 to 4 × 10 cells per well in a 24-well Transwell (Corning) under the following three conditions: ５To obtain cells, the cells were suspended and seeded in 300 μL of serum-free medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako). From day 3 after seeding, the medium was replaced with serum-free medium (gfCDM) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days. Condition 1: Seeded onto 24-well Transwells pre-coated with Matrigel (Matrigel (Pre-coat)). Condition 2: Seeded onto 24-well Transwells pre-coated with Laminin511-E8 (iMatrix511 (Pre-coat)). Condition 3: Cells were seeded onto 24-well Transwells that had not been pre-coated using culture medium supplemented with Laminin511-E8 (Mix method).

[0252] After 14 days of culture for the re-formation of aggregates, sheet-like cell aggregates (cell sheets) were formed under all conditions. The sheet-like cell aggregates were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained using DAPI and anti-Chx10 antibody (ExAlpha), anti-Zo-1 antibody (trade name: Anti Zo-1 Antibody, Invitrogen), and anti-Collagen IV antibody (Abcam). These immunostained sections were observed using a confocal laser scanning fluorescence microscope. As a result, the presence of Chx10-positive neural retinal progenitor cells was confirmed in all of the Matrigel (pre-coat), iMatrix511 (pre-coat), and Mix methods (Figure 27). Furthermore, under the conditions of Matrigel (pre-coat) and iMatrix511 (pre-coat), Zo-1, a marker of the apical surface (tight junctions of epithelial tissue are formed on the apical surface), was observed on the upper side (the side not in contact with the well) of the sheet-like cell aggregates, while Collagen IV, a marker of the basal surface, was observed on the lower side (the side in contact with the well) of the sheet-like cell aggregates (Figure 28). It was confirmed that a sheet of Rx::Venus-positive retinal progenitor cells with apical and basal polarity was re-formed. On the other hand, it was confirmed that no polarity was formed when the Mix method, in which extracellular matrix was simultaneously administered, was used.

[0253] These results demonstrate that sheet-like retinal tissue with apical-basal polarity can be recreated (re-sheet formation) by pre-coating single-cell dispersed retinal cells (NR) with Laminin511-E8 or Matrigel as a scaffold.

[0254] Example 9: Seeding onto Transwells and Re-creating Sheets: Confirmation of the Effect of CHIR99021 Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0255] The aggregates prepared as in Example 1 on days 15 to 30 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, 8 x 10 cells were added per well to a 12-well transwell coated with Laminin 511-E8. ５ The cells were suspended in 300 μL of serum-free medium containing (1) 10 μM Y-27632 (Wako), (2) 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo), (3) 10 μM Y-27632 (Wako) and 5 μM CHIR99021 (Wako), or (4) 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako), and incubated at 37°C and 5% CO ２ Three days after seeding, the medium was replaced with serum-free medium (NucT0) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days.

[0256] Observation of the cell sheets obtained after 14 days of culture for the re-formation of aggregates revealed that cells survived under all conditions (Figure 29). These cell sheets were then fixed in 4% PFA and cryosectioned. These cryosections were immunostained using DAPI, anti-Zo-1 antibody (trade name: Anti Zo-1 Antibody, Invitrogen), and anti-Collagen IV antibody (Abcam). These immunostained sections were observed using a confocal laser scanning fluorescence microscope. The results confirmed that, when CHIR99021 was added, sheets of Rx::Venus-positive retinal progenitor cells with apical and basal polarity were re-formed. On the other hand, when CHIR99021 was not added, no apical surface was formed, confirming the lack of polarity (Figure 30).

[0257] These results demonstrated that addition of CHIR99021 to single-cell dispersed retinal cells (NR) allowed the re-creation of retinal sheets with apical-basal polarity.

[0258] Example 10: Seeding onto Transwells and Re-creating Sheets: Examination of CHIR99021 Concentration and Addition Period Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0259] The aggregates prepared as in Example 1 on day 33 (dd33) after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were placed in a 24-well transwell coated with Laminin 511-E8 at 2-4 x 10 cells per well. ５The cells were seeded and 300 μL of serum-free medium containing 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo) plus 1 μM, 3 μM, or 9 μM CHIR99021 (Wako) was added. The cells were then incubated at 37°C in 5% CO for 3 days (days 0 to 3), 6 days (days 0 to 6), or 9 days (days 0 to 9). ２ After 3 days from seeding, the medium was changed once every 3 to 4 days.

[0260] After 14 days of culturing (Day 14, dd47) for the re-formation of aggregates, the resulting cell sheets were observed to have survived, except for the groups treated with CHIR99021 at 9 μM for 6 and 9 days (Figure 31). These cell sheets were then fixed in 4% PFA and cryosectioned. These cryosections were immunostained with DAPI. Addition of CHIR99021 at 1 μM to 9 μM for 3 to 9 days confirmed the re-formation of thick sheets of Rx::Venus-positive retinal progenitor cells (Figure 32).

[0261] These results demonstrated that retinal sheets could be regenerated from dispersed single-cell retinal cells (NR) by adding CHIR99021 at a concentration of at least 1 to 9 μM for 3 to 9 days.

[0262] Example 11: Examination of the scaffold used when seeding onto Transwells Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0263] The aggregates prepared as in Example 1 on day 24 of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting. After dispersion, (1) non-coated (Non Coat), (2) 2 μL of iMatrix 511 (trade name, Nippi), (3) 2 μL of iMatrix 411 (trade name, Nippi), (4) 2 μL of iMatrix 211 (trade name, Nippi), (5) 2 μL of Vitronectin (VTN-N) Recombinant Human Protein (Thermo), and (6) 2 μL of Cell Start (Thermo), (7) 5 μL of Laminin 111, 5 μL of Laminin 121, 5 μL of Laminin 211, 5 μL of Laminin 221, 5 μL of Laminin 411, 5 μL of Laminin 421, 5 μL of Laminin 511, 5 μL of Laminin 521 (BioLamina), (8) 5 μL of Laminin 332, (9) 2 μL of Matrigel (Corning) coated 24-well Transwells at 2.0 × 10 per well. ５ The cells were seeded and 200 μL of serum-free medium containing 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo), as well as 3 μM CHIR99021 (Wako) and 100 μg / mL FGF8 (Wako) was added, and the cells were incubated at 37°C, 5% CO for 3 days. ２ After 3 days from seeding, the medium was changed once every 3 to 4 days.

[0264] When the cell sheets obtained 22 days after seeding were observed, good neural retina sheets were obtained on all scaffolds except Cell Start (FIG. 33).

[0265] These results demonstrated that retinal sheets could be reconstructed from dispersed single-cell aggregates (NR) not only in iMatrix511 and Matrigel but also in various scaffold proteins.

[0266] Example 12: Seeding onto Transwells, Re-Sheet Formation, and Confirmation of Retinal Differentiation Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Crx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0267] The aggregates prepared as in Example 1 on day 15 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting. After dispersion, the aggregates were placed in a 24-well transwell coated with Laminin 511-E8 at a concentration of 2.0 × 10 per well. ５ The cells were suspended in 200 μL of serum-free medium containing 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021, and incubated at 37°C, 5% CO ２ After 3 days from seeding, the medium was replaced with serum-free medium (NucT0) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days.

[0268] When the cell sheet obtained 44 days after seeding (Day 44, dd59) was observed, it was found that Crx::Venus-positive photoreceptor cells had differentiated ( FIG. 34 ).

[0269] Furthermore, aggregates on day 29 after the start of suspension culture were similarly re-formed into sheets. Cell sheets on day 28 (Day 67) of reorganized culture were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained using DAPI and anti-Ki67 antibody (R&D Systems), anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-Pax6 antibody (BD Pharmingen), anti-Brn3 antibody (Santa Cruz), anti-RxR-γ (RxRg) antibody (Spring Bioscience), and anti-Crx antibody (abnova). As a control, dd70 3D Retina (cell aggregates) that had not been flattened (re-formed into sheets) were also stained (Figures 35 to 37). Furthermore, after fixing the cell sheet on Day 57 with 4% PFA, whole-body immunostaining was performed without preparing sections. DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-Sox2 antibody (BD Pharmingen), anti-Ki67 antibody (BD), anti-Pax6 antibody (BioLegend), anti-Brn3 antibody (Santa Cruz), anti-TUJ1 antibody (Millipore), anti-Islet-1 antibody (R&D Immunostaining was performed using anti-Crx antibody (Abnova), anti-Recoverin antibody (Proteintech), anti-Zo-1 antibody (Invitrogen), and anti-Collagen IV antibody (Abcam).

[0270] As a result, Chx10-positive, Pax6-positive and Ki67-positive retinal progenitor cells, Brn3-positive retinal ganglion cells, RxRg-positive, Crx:: Venus-positive and Crx-positive cone cell progenitor cells were confirmed to be differentiated comparable to Control 3D Retina. In addition, when the positional relationship with the Transwell was observed using transmitted light, a retinal sheet was formed on the Transwell, and Pax6-strongly positive and Chx10-negative retinal ganglion cells localized on the basal side were confirmed to be localized on the Transwell side (Figure 38). Furthermore, when the entire specimen was observed using a confocal microscope without preparing slices, it was confirmed that the retinal cells had apical and basal polarity and differentiated according to the polarity (Figures 39, 40, 41).

[0271] After further long-term culture, dd112 cell sheets were immunostained in the sheet state without sectioning. When stained with anti-Ribeyre antibody (CtBP2, BD), anti-Recoverin antibody (Proteintech), anti-Pax6 antibody (BioLegend), and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, SantaCruz), expression of Crx::Venus and Recoverin was observed in the same cells, confirming the expression of Ribeye (Figure 42). Therefore, it was confirmed that photoreceptors differentiated from the re-sheeted retina matured to the extent that they expressed synaptic proteins. Furthermore, it was confirmed that a sheet was formed with Crx::Venus localized on the apical side and Chx10 and Pax6 localized on the medial side (Figure 43).

[0272] These results confirmed the differentiation of retinal cells in the retinal sheet reorganized on the Transwell, and it was observed that polarity was maintained.

[0273] Example 13: Maintenance and culture of retinal cells using the Rx::Venus line Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0274] The aggregates prepared as in Example 1 on day 31 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting. After dispersion, 2.0 × 10 cells per well were added to a 12-well transwell coated with 10 μL of Laminin 511-E8. ５ To 500 μL of serum-free DMEM / F12 containing 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021, and supplemented with N2, were added (1) Control, (2) 20 ng / mL FGF2, (3) 20 ng / mL EGF, (4) 100 nM SAG, (5) 1 unit LIF, (6) 10 ng / mL IGF-1, (7) 100 ng / mL PDGF-AA, (8) 100 ng / mL PDGF-AB, (9) 10 μg / mL GDNF, (10) 20 μg / mL BDNF, (11) 2 μM Pyrintegrin, and (12) 1 μM BMP4, and the cells were cultured at 37°C and 5% CO. ２ After 3 days from seeding, the medium was replaced with serum-free medium (DMEM / F12 supplemented with N2) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days.

[0275] After 34 days, the cell sheets were fixed with 4% PFA and the nuclei were stained with DAPI. The area of ​​Rx::Venus-positive cells was observed to assess the state of retinal cell maintenance. Observation using a fluorescence microscope (Keyence BZ-X810) confirmed that Rx::Venus-positive cells were widely maintained when 20 ng / mL EGF and FGF2 were continuously added ( Figure 44 ).

[0276] Therefore, it was found that adding EGF or FGF during retinal reorganization is effective in maintaining Rx::Venus-positive cells.

[0277] Example 14 Maintenance and culture of retinal progenitor cells during planar culture Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Crx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0278] The aggregates (NR) prepared in Example 1 on day 27 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, 8.0 × 10 cells were added to a 12-well transwell coated with 10 μL of Laminin 511-E8. ５The cells were suspended in 500 μL of the following medium containing 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021 and seeded at 10 cells / well. To examine the medium for maintaining retinal progenitor cells, 20 ng / mL FGF2 and 20 ng / mL EGF were added to (1) NeuroCult NS-A (STEMCELL Technologies), (2) STEMdiff Neural Progneitor (STEMCELL Technologies), (3) StemPro NSC SFM (Thermo Fisher), and (4) RHB-A (TaKaRa) medium and cultured for one month. NucT0 medium was used as a control. From day 3 after seeding, the medium was replaced with a medium not containing Y-27632, SAG, and CHIR99021 every 3 to 4 days.

[0279] After 16 days of culture for the re-formation of aggregates, we examined conditions under which differentiation was not occurring using the photoreceptor progenitor marker Crx::Venus as an indicator (conditions under which Crx::Venus did not glow when observed under a fluorescent microscope). We found that NeuroCult NS-A and RHB-A media had the effect of delaying differentiation to some extent (Figure 45).

[0280] Therefore, it was found that adding NeuroCult NS-A and RHB-A media with EGF and FGF during re-sheet formation has the effect of maintaining retinal progenitor cells and delaying their differentiation.

[0281] Example 15: Culture on Collagen Gel Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0282] Collagen gel was prepared using beMatrix low-endotoxin collagen solution (Nitta Gelatin Co., Ltd.). Specifically, collagen AT, 5x DME, and reconstitution buffer were mixed in a ratio of 7:2:1, coated on a Transwell mesh, and incubated at 37°C and 5% CO. ２ After incubation in an incubator for 30 minutes, medium was added to the inside and outside of the insert.

[0283] The aggregates (NR) prepared in Example 1 on day 26 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, 8.0 × 10 cells were placed on the collagen gel of a 12-well Transwell. ５ Cells were seeded at 1 cell / well. For the first 3 days, they were cultured in the presence of 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021. From day 3 onwards, the medium was replaced with serum-free medium (NucT0 medium) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days.

[0284] After 33 days of culture for the re-formation of aggregates, the state of re-sheet formation was observed under a stereomicroscope using Rx::Venus as an indicator, and it was found that Rx::Venus-positive cells were present on the front of the collagen gel in the Transwell (Figure 46).

[0285] Therefore, it was found that the addition of Y-27632, SAG, and CHIR99021 also allowed the collagen gel to be re-formed into a sheet.

[0286] Example 16: Transplantation Study Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Crx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0287] The aggregates (NR) on the collagen gel prepared as in Example 15 on day 26 were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting. After dispersion, 8.0 × 10 cells were placed on the collagen gel in a 12-well transwell. ５ Cells were seeded at 1 cell / well. For the first 3 days, they were cultured in the presence of 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021. From day 3 onwards, the medium was replaced with serum-free medium (NucT0 medium) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days.

[0288] For the re-formation of aggregates, retinal cell sheets on days 63 and 70 of differentiation after 37 and 44 days of culture were treated with collagenase (Roche) for 30 minutes at 37°C and detached. The detached retinal sheets were cut into strips with a length of approximately 1.8 cm using tweezers and scissors to prepare transplantation grafts (Figure 47).

[0289] The prepared grafts were implanted subretinaly into immunodeficient retinal deficient rats (SD Foxn) using a glass Pasteur pipette.

[0290] The rat eyes were removed one year after transplantation and fixed in 4% PFA. Observation using a fluorescent stereomicroscope and a fluorescent microscope (Keyence BZ-X810) confirmed that Crx::Venus-positive grafts had taken root ( Figure 48 ).

[0291] Therefore, it was found that the regenerated retinal sheet could survive after transplantation.

[0292] Example 17: Sorting study Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0293] The aggregates prepared in Example 1 on days 14 to 25 after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After gating the target cell population by FSC and SSC using a Cell Sorter ARIA II (BD), the Rx::Venus-positive fraction was collected ( Figure 49 ).

[0294] The sorted cells were placed in a volume of 1.6-8.0 x 10 per well. ５ The cells were seeded onto 12-well Transwells (Corning) coated with Laminin 511-E8. An unsoated sheet was also prepared as a control. For the first three days, the cells were cultured in the presence of 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021. From day 3 onwards, the medium was replaced with serum-free medium (NucT0 medium) containing no Y-27632, SAG, or CHIR99021 every 3 to 4 days. On day 26 after seeding, the cells were observed using a fluorescent stereomicroscope (Figure 50).

[0295] Furthermore, cell sheets purified with Rx::Venus by sorting on day 82 of differentiation were fixed in 4% PFA and then immunostained as sheets without sectioning. Immunostaining was performed using DAPI and anti-Recoverin antibody (Proteintech). A control retinal sheet without sorting was also stained (Figures 51, 52, and 53). Subsequently, frozen sections were prepared and immunostained using DAPI and anti-Ki67 antibody (BD), anti-Chx10 antibody (trade name: Anti CHX10 Antibody, Santa Cruz), and anti-CRX antibody (TaKaRa) (Figure 53).

[0296] As a result, when the Rx::Venus-positive fraction was seeded without sorting, clumps of RPE cells were observed in some areas. However, because these clumps could be visually confirmed, it was possible to remove the RPE cells. On the other hand, when the Rx::Venus-positive fraction was sorted, it was observed that an Rx::Venus-positive retinal sheet was produced. Furthermore, it was confirmed that the majority of RPE cells were removed (Figures 49 and 50). However, even after sorting, RPE cells were sometimes mixed in. It is unclear whether this is because the RPE cells could not be completely removed by sorting, or whether they differentiated after sorting. However, it was confirmed that the mixed RPE cells were not mixed with the neural retinal tissue (Figure 52). Because these clumps could be visually confirmed, it was possible to remove the RPE cells.

[0297] Furthermore, in the retinal sheets that had undergone sorting, it was confirmed that recoverin-positive photoreceptor cells had differentiated, comparable to the control retinal sheets that had not been sorted (Figures 51 and 53). Z-stack analysis and immunohistological analysis of sections confirmed the re-formation of the layer structure of the retinal tissue, and the presence of Chx10-positive and Ki67-positive retinal progenitor cells and Crx-positive photoreceptor progenitor cells (Figure 53).

[0298] Therefore, by gating the target cell population using FSC and SSC and then sorting using Rx::Venus as an indicator, it was possible to remove most of the RPE cells, and it was observed that retinal cells differentiated without any problems when the cells were re-formed into a sheet.

[0299] Example 18: Searching for factors that maintain Retina properties upon re-creation into cell sheets Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0300] The aggregates (NR) prepared in Example 1 on day 25 after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and sorting was performed using a Cell Sorter ARIA II (BD) with Rx::Venus as an index. The sorted cells were then plated at 5 x 10 cells per well onto a Laminin 511-E8-coated 96-well glass-bottom plate (Greiner) using Easy iMatrix. ４ The cells were seeded in 100 μL of NucT0 supplemented with Y-27632, CHIR99021, and SAG. At the same time as seeding, the proteins shown in Table 1 or the low molecular weight compounds shown in Table 2 were added.

[0301] Three days after seeding, the medium was similarly changed to maintain the concentrations of the low-molecular-weight compounds or proteins listed in Tables 1 and 2. Then, on day 7, the medium was changed with NucT0 medium containing no low-molecular-weight compounds or proteins listed in Tables 1 and 2. Ten days after seeding, the cells were fixed with 4% PFA, and the cells were evaluated after sorting using Rx::Venus as an indicator under a fluorescence microscope (Keyence BZ-X810). As a result, sheets containing FGF2, FGF4, and FGF8, among the proteins, showed higher Rx::Venus expression intensity compared to the control PBS (Figure 54). Furthermore, sheets containing IWR1 endo, among the low-molecular-weight compounds, showed higher Rx::Venus expression intensity (Figure 55). Furthermore, concentration dependency was confirmed, with the highest Rx::Venus expression intensity observed when 100 μg / mL of FGF8 was added (Figure 56).

[0302] Therefore, it was found that the Rx::Venus-positive cell population can be maintained by activating the FGF signal immediately after cell seeding.

[0303] Example 19: Confirmation of Aggregate Reformation and Retinal Differentiation by Addition of FGF8 Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0304] The aggregates (NR) prepared in Example 1 on day 24 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and sorting was performed using a Cell Sorter ARIA II (BD) with Rx::Venus as an index. After sorting, the cells were plated onto a low-adhesion Vplate at 2.0 to 5.0 x 10 cells per well. ４As controls, a group to which FGF8 was not added (FGF8-) and a group to which sorting was not performed (no sorting) were also included.

[0305] The medium was changed every 3 to 4 days. FGF8 was also added during the medium change. Microscopic observation 10 days after reseeding revealed Rx::Venus-negative cell clusters or RPE cells in the FGF8- group without sorting (Figure 57). Meanwhile, a decrease in these cell clusters was observed in the FGF8+ group. In the FGF8- group with sorting, RPE cells were partially observed. Since RPE cells were removed by sorting, this suggests that a subset of Rx::Venus-positive cells differentiated into RPE cells. Meanwhile, in the FGF8+ group with sorting, Rx::Venus-positive aggregates were formed, and no RPE cells or Rx::Venus-negative clusters were observed.

[0306] These results demonstrate that the addition of FGF8 can reduce the differentiation of RPE cells or Rx::Venus-negative cells.

[0307] Furthermore, for the re-formation of aggregates, the aggregates on day 54 of culture (day 30 after reseeding) were washed with PBS and dispersed into single cells using a neuronal cell dispersion medium (Wako). Then, the cells were fixed with formaldehyde using Fixation buffer (BD). After washing with Perm / Wash buffer (BD), the cells were stained with anti-CHX10-Alexa Fluo 647 conjugate antibody (Santacruz), anti-Sox2-BV421 antibody (Biolegend), anti-Ki67-Alexa Fluo 647 conjugate antibody (BD), and anti-CRX-Alexa Fluo 647 conjugate antibody (Santacruz). After washing the antibody with Perm / Wash buffer (BD), measurements were performed using a FACSCanto II (BD Bioscience) and analysis was performed using FlowJo. As a control, non-reorganized neural retinal tissues with the same differentiation days were also placed.

[0308] As a result, in the group without sorting and without addition of FGF8, a large number of Rx::Venus-negative cell populations were observed, and the Rx::Venus-positive rate tended to be low. On the other hand, even in the group without sorting and with addition of FGF8, the Rx::Venus-positive rate tended to be high (Figure 58).

[0309] Furthermore, when sorting was performed, the Rx::Venus positivity rate tended to be high even in the group without FGF8 addition (Figure 58).On the other hand, in the group with sorting and addition of FGF8, the proportion of Chx10-positive and Sox2-positive neural retinal progenitor cells tended to be high, and the proportion of Crx-positive photoreceptor progenitor cells tended to be low (Figure 59).

[0310] These results demonstrate that the addition of FGF8, in addition to sorting, reduces differentiation of RPE and Rx::Venus-negative cells, and enables the production of uniform and favorable aggregates.

[0311] Example 20: Confirmation of the effect of FGF8 upon re-creation of a sheet Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold in place of feeder cells.

[0312] The aggregates (NR) prepared in Example 1 on day 24 after the start of suspension culture were washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and sorting was performed using a Cell Sorter ARIA II (BD) with Rx::Venus as an index. The sorted cells were then placed in NucT0 medium containing Y-27632, CHIR99021, SAG, and FGF8 in a 24-well Transwell (Corning) at 2.0 x 10 cells per well. ５The cells were suspended and seeded. As a control, a group to which FGF8 was not added (FGF8-) was also included.

[0313] The medium was changed every 3 to 4 days. FGF8 was also added during the medium change. Sheets 63 days after differentiation and 40 days after reseeding were observed under a fluorescent stereomicroscope (Figures 60 and 61). For FACS analysis, aggregates 64 days after differentiation and 41 days after reseeding were washed with PBS and dispersed into single cells using a neuronal cell dispersion solution (Wako), followed by formaldehyde fixation using a fixation buffer (BD). The cells were then fixed using Fixation buffer (BD) and washed with Perm / Wash buffer (BD), followed by staining with anti-CHX10-Alexa Fluo 647 conjugate antibody (Santacruz), anti-Sox2-BV421 conjugate antibody (Biolegend), anti-Ki67 antibody (BD), and anti-CRX-Alexa Fluo 647 conjugate antibody (Santacruz). After washing the antibodies with Perm / Wash buffer (BD), measurements were performed using a FACSCanto II (BD Bioscience) and analysis was performed using FlowJo. As a control, non-reorganized neural retinal tissue with the same differentiation days was also placed (Figure 62).

[0314] As a result, after sorting, in sheets to which FGF8 had not been added, a large number of Rx::Venus-negative cell populations were observed, and Rx::Venus-positive cells tended to be concentrated in clumps around the periphery of the well. On the other hand, in sheets to which FGF8 had been added after sorting, Rx::Venus-positive cells were uniformly present throughout the well, demonstrating the creation of good sheets (Figures 60 and 61). Furthermore, black RPE (Figure 61, arrow) was observed in sheets to which FGF8 had not been added, whereas it was hardly observed in sheets to which FGF8 had been added.

[0315] Furthermore, FACS analysis showed that the Rx::Venus positivity rate was reduced to 60% in sheets without FGF8 addition, but was maintained at over 95% in the FGF8-added group, a positivity rate comparable to that of neural retinal tissue (Figure 62). Furthermore, the proportions of Chx10-positive and Sox2-positive neural retinal progenitor cells and Crx-positive photoreceptor progenitor cells were comparable to those in neural retinal tissue. The proportion of Rx::Venus-positive and Ki67-positive proliferative retinal cells tended to be higher in the sheets compared to neural retinal tissue (Figure 62).

[0316] These results demonstrate that adding FGF8 after sorting when re-forming sheets reduces differentiation of RPE and Rx::Venus-negative cells, enabling the production of uniform, high-quality sheets.

[0317] Example 21: Confirmation of changes over time after seeding onto Transwells and re-forming into sheets Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0318] The aggregates prepared as in Example 1 on day 27 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting. After dispersion, the aggregates were placed in a 24-well transwell coated with Laminin 511-E8 at 2.0 x 10 per well. ５ The cells were suspended in 200 μL of serum-free medium containing 10 μM Y-27632, 300 nM SAG, 3 μM CHIR99021, and 100 ng / mL FGF8, and incubated at 37°C in 5% CO ２ After 3 days from seeding, the medium was replaced with serum-free medium (NucT0) containing no Y-27632, SAG, CHIR99021, or FGF8 every 3 to 4 days.

[0319] The cell sheets obtained after 3, 6, 9, 12, 22, and 40 days (dd30, dd33, dd36, dd39, dd49, and dd67) of culturing for the re-formation of aggregates were fixed with 4% PFA, and the sheet formation was observed using a fluorescent stereomicroscope and a fluorescent microscope with Rx::Venus as an indicator. It was observed that Rx::Venus-positive retinal cells had proliferated, and a thick sheet was being formed (Figure 63). In particular, a thick cell sheet completely covered with Rx::Venus-positive cells was observed from 22 to 40 days.

[0320] From these results, it was observed that reorganized retinal sheets that could be used for transplantation, assays, etc. could be obtained after 22 to 40 days.

[0321] Example 22: RPC surface antigen screening - comparison of hES cells and NR - Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0322] The cells were cultured in suspension for differentiation induction as described in Example 1. 300 nM SAG was added at the start of differentiation induction. The aggregates (NR) on days 19 and 26 after the start of suspension culture were washed with PBS, and neural cell suspension (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and surface antigen screening was performed using the MACS® Marker Screen, human (Miltenyi) surface antigen screening kit (Alexa 647 fluorescent dye-conjugated antibody group). Human embryonic stem cells (hESCs) were stained as a control. A total of 371 markers were used for screening (Figures 64-69).

[0323] FACS analysis was performed using a MACS Quant10 (Miltenyi) and FlowJo. Analysis was performed by gating on the populations indicated by FSC and SSC shown in Figure 65. First, we confirmed that staining and analysis were performed accurately using E-cadherin, SSEA-4, and SSEA-5, which are expressed in hESCs and lose expression upon differentiation (Figure 66). The results of the subsequent analysis are summarized in Figure 64. Note that "hESC: High" indicates high or present expression in human ES cells, "hESC: Low" indicates low or absent expression in human ES cells, "NR: High" indicates high expression in neural retinal cells, and "NR: Low" indicates low or absent expression in neural retinal cells.

[0324] A search for surface antigens that are not expressed (or are expressed) in hESCs but are expressed (or are not expressed) in the Rx::Venus-positive population on days 19 or 26 after the start of suspension culture suggested that CD39, CD73, CXCR4, and further CD29, CD49b, CD49c, CD49f, CD57, CD82, CD90, and CD200 may be markers that can distinguish Rx::Venus-positive cells from other cells (Rx::Venus-negative cells, hESCs) ( Figure 67 ).

[0325] Furthermore, the expression of CD39, CD73, and CD184 (CXCR4) was analyzed by FACS using mouse IgG1-APC-Conjugate antibody (Miltenyi), anti-CD39-APC-Conjugate antibody (Miltenyi), anti-CD73-APC-Conjugate antibody (Miltenyi), and anti-CD184 (CXCR4)-APC-Conjugate antibody (Miltenyi) on days dd18, dd25, dd53, and dd81 after the initiation of suspension culture (d18, d25, d53, and d81 in Figure 68) (Figure 68).

[0326] As a result, it was found that the proportion of cells expressing CD39 and CD73 was highest in dd25, while the proportion of cells expressing CD184 at low levels was high in dd18 and dd25.

[0327] Therefore, when using surface antigens CD39, CD73, and CD184 to purify neural retinal progenitor cells, it was found that the best time to do so was after 18 days and around 25 days after the start of suspension culture (Figure 68).

[0328] Expression Analysis of CD39, CD73, and CXCR4 in Example 23 Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0329] Brain organoids were prepared by adding 300 nM SAG at the start of suspension culture (at the start of differentiation induction), without adding BMP4 on day 3 after the start of suspension culture. A control group was added with BMP4. The brain organoids prepared in this manner were observed under a fluorescence microscope 26 days after the start of suspension culture (Figure 70). As a result, Rx::Venus-positive retinal tissue was observed to have differentiated in the BMP4-added group, while Rx::Venus-negative neuroepithelial cells were observed to have differentiated in the BMP4-unadded group. Furthermore, these aggregates were fixed in 4% PFA, sectioned, stained with DAPI and anti-FoxG1 antibody (TaKaRa), and observed under a fluorescence microscope (Keyence BZ-X810). As a result, FoxG1 was negative in the group to which BMP4 was added, while FoxG1 was positive in the group to which BMP4 was not added, confirming that brain organoid had differentiated (Figure 71).

[0330] The Brain Organoid prepared in this way on day 25 after the start of suspension culture was washed with PBS, and a neuronal cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting and stained with anti-CD39-APC conjugate antibody (Miltenyi), anti-CD73-APC conjugate antibody (Miltenyi), and anti-CXCR4-APC conjugate antibody (Miltenyi). FACS measurement was performed using a FACSCanto II (BD Bioscience) to measure the proportion of expressing cell populations, and analysis was performed using FlowJo.

[0331] As a result, it was observed that CD39 and CD73 were not expressed in the brain organoid, whereas CXCR4 was expressed in most cells of the brain organoid (FIG. 72).

[0332] Therefore, it was confirmed that by using CD39-positive, CD73-positive, and CXCR4-negative cell populations as indicators, it is possible to separate and isolate Rx::Venus-positive cell populations, distinguishing them from Brain Organoid.

[0333] Example 24: CD39 Expression Analysis Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0334] The morphology of aggregates (NR) prepared by adding (1) 0 nM, (2) 30 nM, or (3) 300 nM SAG at the start of differentiation induction was observed under a microscope on day 26 after the start of suspension culture. The results showed that the aggregates formed when 0 nM or 30 nM SAG was added were larger and the layered structure was more clearly visible than when 300 nM SAG was added at the start of suspension culture (dd0) (Figure 73).

[0335] Furthermore, the aggregates were dispersed into single cells using a neuronal cell dispersion medium, and the expression of CD39 and CXCR4 was analyzed by FACS using anti-CD39-APC conjugate antibody (Miltenyi) and anti-CXCR4-APC conjugate antibody (Miltenyi). The proportion of expressing cells was measured using a FACSCanto II (BD Bioscience), and analysis was performed using FlowJo. These aggregates were fixed with 4% PFA, sections were prepared, and then stained with anti-ALDH1A1 antibody (R&D Systems), anti-CoupTF1 antibody (PPMX), anti-LHX2 antibody (Millipore), anti-Chx10 antibody (ExAlpha), anti-Pax2 antibody (Covance), and anti-NKX2.1 antibody (Leica).

[0336] As a result, CD39 expression and low CXCR4 expression were observed in aggregates added with 300 nM SAG at the start of suspension culture (dd0) (Figure 74). Furthermore, addition of SAG at dd0 decreased the expression of the dorsal marker ALDH1A1 and increased the expression of the ventral marker CoupTF1 (Figures 75 and 76). Furthermore, it was found that the expression level of Rx in cell aggregates was uniform without SAG stimulation (Figure 77). However, expression of LHX2, Chx10, and Pax2 was confirmed even in areas with low Rx expression in aggregates added with 300 nM, and NKX2.1 was negative, confirming that these are neural retinal tissue (Figures 78 and 79).

[0337] Therefore, it was found that adding 300 nM of SAG at the start of differentiation induction makes it possible to obtain a population of CD39-positive, CXCR4-negative neural retinal progenitor cells.

[0338] Example 25: Examination of CD39 Expression Enhancement Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0339] At the start of differentiation induction (dd0), 300 nM SAG was added to the aggregates (NR) to prepare them. On the 17th day after the start of suspension culture, 10 neural retinal tissues were transferred to a 60 mm dish for low-attachment suspension culture (Sumitomo Bakelite Co., Ltd.) and cultured in 4 mL of NucT0 medium containing 1 mM and 0.2 mM adenosine (A2A receptor agonist, Sigma), 1 mM, 0.2 mM, and 0.04 mM AMP-PNP (Tocris), 5 mM, 1 mM, 0.2 mM, and 0.04 mM ATP disodium salt (Tocris), and 1 μM, 0.2 μM, and 0.04 μM CGS 21680 (adenosine A2A receptor agonist). Agonist (Tocris) was added and the cells were cultured until day 25 after the initiation of suspension culture. Changes in CD39 and CXCR4 expression were stained using mouse IgG1-APC conjugate antibody (Miltenyi), anti-CD39-BV421 conjugate antibody (BD Bioscience), and anti-CXCR4-APC conjugate antibody (Miltenyi), and measured using a FACSCanto II (BD Bioscience) and analyzed using FlowJo. A DMSO-added group was used as a control.

[0340] As a result, it was found that ATP and A2A receptor agonists have the effect of increasing CD39 expression and decreasing CXCR4 expression (FIGS. 80 and 81).

[0341] Therefore, it was found that the expression of CD39 can be enhanced by adding ATP and an A2A receptor agonist.

[0342] Example 26: Transplantation of Islet-1 KO hESC-retina Sheets Prepared by Sorting with CD39 and Adding FGF8 Human ES cells (KhES-1 strain, (Non-Patent Document 3)) that had been genetically modified to carry the Rx::Venus reporter gene and in which the Islet-1 gene had been knocked out were cultured under feeder-free conditions according to the methods described in Scientific Reports 4, 3594 (2014) and WO 2018 / 097253. StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0343] Aggregates differentiated with 300 nM SAG at the start of suspension culture (dd0, at the start of differentiation induction) were dispersed into single cells. Aggregates (NR) on day 25 of suspension culture were stained with anti-CD39-APC conjugate antibody (Miltenyi) at 37°C for 15 minutes, and then sorted using a Cell Sorter ARIA II (BD) with CD39-APC as an indicator. The sorted cells were plated at 8.0 x 10 per well on a 12-well transwell coated with collagen gel. ５ Cells were seeded. At the time of seeding, Y-27632 (Wako), SAG (Enzo), CHIR99021 (Wako), and 50 ng / mL FGF8 (Wako) were added. A control group (FGF8-) was also included, to which FGF8 was not added.

[0344] The medium was changed every 3 to 4 days. FGF8 was also added at the time of medium change. Microscopic observation on day 73 after the start of suspension culture revealed that Crx::Venus cell populations were observed sparsely in the group without FGF8 addition, whereas they were uniformly present in the group with FGF8 addition (Figure 82).

[0345] The sheet thus prepared was recovered using collagenase on day 88 after the start of suspension culture, and a long graft for transplantation was excised using microscissors ( Figure 82 ), which was then transplanted under the retina of an immunodeficient retinal dysfunction rat (SD Foxn).

[0346] Therefore, it was confirmed that retinal sheets produced from cells sorted by CD39 are transplantable.

[0347] Example 27: RPC Surface Antigen Screening - Comparison of Brain Organoid and NR - Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0348] The aggregates (NR) were prepared 24-26 days after the start of suspension culture by adding 30 nM SAG at the start of suspension culture (dd0). The aggregates were dispersed into single cells using a neural cell dispersion solution (Wako Co., Ltd.) and stained using the MACS (registered trademark) Marker Screen, human (Miltenyi Co., Ltd.) surface antigen screening kit (Alexa 647 fluorescent dye conjugate antibody group). As a control, a brain organoid prepared without adding BMP4 on the third day after the start of differentiation induction was stained.

[0349] FACS analysis was performed using a MACS Quant10 (Miltenyi) and FlowJo. Surface antigens expressed (or not expressed) by the Rx::Venus-positive population on days 24-26 after the start of suspension culture, but not expressed (or not expressed) by the Brain Organoid, were examined. CD9, CD15, CD49c, CD66b, CD69, CD82, CD164, EpCAM, and ErbB2 (CD340) were found to be distinguishable from the Brain Organoid (Figure 83).

[0350] Furthermore, it was shown that CD9, CD24, CD49c, CD90, CXCR4, and EpCAM may be useful markers for distinguishing Rx::Venus-positive cells from Rx::Venus-negative cells (Figure 84).

[0351] Example 28: Confirmation of changes over time in CD9 expression Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0352] hESCs and aggregates of dd4, dd11, dd18, dd25, dd32, and dd46 (NR) prepared by adding 30 nM SAG at the start of suspension culture (dd0), were dispersed into single cells in a neuronal cell dispersion medium (Wako), stained with an APC-conjugated anti-CD9 antibody (BioLegend), washed, and then subjected to FACS analysis. FACS analysis was performed using a MACS Quant10 (Miltenyi) and FlowJo.

[0353] As a result, it was found that although CD9 is expressed in hESCs, its expression drops off at day 4, when differentiation begins, and then begins to be expressed around day 11 (d11 in Figure 85), with the rate of positive cells increasing from dd18 (d18 in Figure 85) to dd25 (d25 in Figure 85) and around dd32 (d32 in Figure 85). It was confirmed that the best time to purify is after dd11, and more preferably between dd18 and dd32 (Figure 85).

[0354] Example 29: Examination of Removal of Undesired Cells Using SSEA1 as an Indicator Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0355] 25-day-old aggregates (NR) prepared by adding 30 nM SAG at the start of suspension culture (dd0, the start of differentiation induction) were dispersed into single cells in a neuronal cell dispersion medium (Wako). The single cells were then added to an APC-conjugated anti-CD9 antibody (BioLegend) and stained with a BV421-conjugated anti-SSEA1 antibody (BioLegend). Measurements were performed using a FACSCanto II (BD Bioscience) and analysis was performed using FlowJo.

[0356] As a result, the percentage of Rx-positive cells in the aggregate as a whole was approximately 87%, but when CD9-positive cells were gated, the percentage of Rx-positive cells was found to increase to 94%.Furthermore, when CD9-positive and SSEA1-negative cells were gated, the percentage of Rx-positive cells was found to increase to approximately 96% (Figure 86).

[0357] Therefore, it was found that the combination of CD9 and SSEA1 can further purify retinal cells.

[0358] Example 30: Examination of Combinations of CD9, CD90, CXCR4, and SSEA1 Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0359] Two hundred 25-day-old aggregates (NR) prepared by adding 30 nM SAG at the start of suspension culture (dd0, the start of differentiation induction) were dispersed into single cells in a neuronal cell dispersion medium (Wako). The cells were then divided into seven equal parts and stained for surface antigens at 4°C for 1 hour under the following seven conditions: (1) unstained (purified using Rx::Venus as an indicator), (2) anti-CD9-APC-Conjugate antibody (BioLegend), (3) anti-CD90-APC-Conjugate antibody (BD), (4) anti-CD184 (CXCR4)-APC-Conjugate antibody (Miltenyi), (5) anti-CD9-APC-Conjugate antibody (BioLegend) and anti-SSEA1-BV4. The cells were stained using (6) anti-CD9-APC-Conjugate antibody (BioLegend) and anti-CD90-BV421-Conjugate antibody (BioLegend), and (7) anti-CD90-APC-Conjugate antibody (BD) and anti-SSEA1-BV421-Conjugate antibody (BioLegend). After staining, sorting was performed using an ARIA II (BD) with the frame shown in Figure 87 as an index. The cells recovered by sorting were subjected to FACS analysis using a MACS Quant10 (Miltenyi) to confirm that the desired cell fraction had been purified (Figure 87). When the proportion of Rx::Venus-positive cells in the cell population after purification was measured, compared to the state before sorting, (1) the purity was highest in the sample sorted using the Rx::Venus-positive fraction, but the sample purified using the CD9-positive and SSEA-1-negative fraction had a high Rx::Venus-positive rate, comparable to that of samples purified using Rx::Venus (Figures 88 and 89).

[0360] These sorted cells were plated onto Laminin511-E8 coated 24-well transwells at 2.0 × 10 cells per well. ５ Cells were seeded. At the time of seeding, 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), 3 μM CHIR99021 (Wako), and 100 ng / mL FGF8 (Wako) were added. After seeding, the medium was replaced once every 3 to 4 days.

[0361] Bright-field and fluorescent observations were performed using a fluorescence microscope on days 1 and 12 after the start of re-sheet culture. Cells were observed to have grown on the Transwell mesh for all purified cells on day 1 after seeding (Figure 90). Furthermore, observations after 12 days showed that, while some black RPE were observed in the Rx::Venus-positive purified sample, no black RPE were observed in the CD9-purified sheets of CD9-positive, CD9-positive / SSEA1-negative (CD9 / SSEA1) population, or CD9-positive / CD90-positive (CD9 / CD90) population (Figure 91).

[0362] Therefore, it was confirmed that retinal sheets sorted by CD9 and CD9 and SSEA1 negative or CD90 positive fractions were better than those sorted by Rx::Venus positivity, as they were able to remove the RPE fraction.

[0363] Example 31: Confirmation of changes over time in CD9 and SSEA1 expression Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in Scientific Reports 4, 3594 (2014). StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Inc.) was used as the feeder-free medium, and Laminin 511-E8 (trade name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0364] hESCs and aggregates of dd4, dd11, dd18, dd25, dd32, and dd46 (NR) prepared by adding 30 nM SAG at the start of suspension culture (dd0 at the start of differentiation induction) were dispersed into single cells in a neuronal cell dispersion medium (Wako), stained with anti-CD9-APC-Conjugate antibody (BioLegend) and anti-SSEA1-BV421-Conjugate antibody (BioLegend), and subjected to FACS analysis. Measurements were performed using a FACSCanto II (BD Bioscience), and analysis was performed using FlowJo.

[0365] As a result, hESCs were CD9 positive and SSEA1 negative, but by day 4 after the start of differentiation, they became CD9 negative and SSEA1 positive (Figure 92). Furthermore, a CD9 positive and SSEA1 negative population began to appear from day 11 of differentiation, and the proportion of the target population increased on days 18 and 25. It was also determined that expression continued on days 32 and 46.

[0366] Therefore, it was confirmed that the best time to purify is after dd11 (d11 in FIG. 92), and that the best time to purify is from dd18 (d18 in FIG. 92) to dd32 (d32 in FIG. 92) (FIG. 92).

[0367] In this way, CD9 and SSEA1 were sorted as indicators, and 10 μM Y-27632, 3 μM CHIR99021, 300 nM SAG, and 100 ng / mL FGF8 were added to prepare retinal sheets on 12-well Transwell plates. On days 48 and 62 after the start of suspension culture, the sheets were collected using a scalpel or scissors, and long grafts for transplantation were excised using microscissors and transplanted into the subretinal space of immunodeficient retinal dysfunction rats (SD Foxn) (not shown).

[0368] Therefore, it was confirmed that retinal sheets produced using cells sorted by CD9 and SSEA1 are transplantable.

[0369] Example 32: Study of RPE-retinal tissue composite using gelatin Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0370] The aggregates (NR) prepared in Example 1 on day 18 to 30 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and the cells were placed in a 12-well Transwell at 4.0 to 8.0 × 10 per well. ５ Cells were seeded. At the time of seeding, Y-27632, SAG, and CHIR99021 were added to NucT0 medium. Thereafter, the medium was replaced with NucT0 medium without Y-27632, SAG, or CHIR99021 every 3 to 4 days, and the cells were cultured for at least one month.

[0371] The differentiated aggregates of RPE prepared in Example 1 on or after 80 days from the start of suspension culture were seeded and cultured on a Laminin 511-E8-coated dish in a medium consisting of DMEM (SIGMA) and F12 Ham (SIGMA) medium supplemented with B27 (Gibco), L-Glutamin, 10 ng / mL FGF2, and SB431542. After the RPE adhered and proliferated, the RPE alone was scraped off using a 1 mL tip and seeded on another Laminin 511-E8-coated dish for purification. After expansion, the RPE were cultured at 1.0 x 10 ６ RPE cells were seeded, and the medium was changed every 3 to 4 days, and the cells were cultured for more than one month.

[0372] The RPE sheet cultured as described above was attached to the retinal tissue sheet using BeMatrix LS-W gelatin (Nitta Gelatin Co., Ltd.). Specifically, each sheet was retrieved from the transwell using tweezers and scissors. The retinal tissue sheet and RPE sheet were then blended with 10% (w / v) gelatin, followed by 20% gelatin, and finally 30% gelatin was added to bond the retinal tissue sheet and RPE sheet together. After bonding, the sheets were rapidly cooled to 4°C and incubated for 20 minutes to solidify. The sheets were then retrieved to prepare composite sheets of retinal tissue and RPE sheet (Figures 93 to 98).

[0373] The retinal tissue-RPE composite sheet prepared in this manner was excised using tweezers and scissors. First, the composite sheet was cut in half and then cut into strips along the cut surface (Figure 99). As a result, the two sheets were excised while still adhered via gelatin. Observation of the cross-section of the excised composite sheet revealed RPE with black pigment on one side and Rx::Venus-positive sheet-like retinal tissue on the other side, with gelatin packed between them (Figure 100). To ensure that the excised composite sheet would not peel off during the aspiration and discharge procedures of the transplantation process, a 1 mL tip with its tip cut off was used for aspiration and discharge. As a result, the composite sheet could be aspirated and discharged multiple times without separation (Figure 101).

[0374] Therefore, it was found that a transplantable retinal tissue-RPE composite sheet can be prepared by using a sheet-like retinal tissue, an RPE sheet, and gelatin.

[0375] Example 33: Study of RPE-retinal tissue composite formation using fibrin Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0376] The aggregates (NR) prepared in Example 1 on day 18 to 30 after the start of suspension culture were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and the cells were placed in a 12-well Transwell at 4.0 to 8.0 × 10 per well. ５Cells were seeded. At the time of seeding, Y-27632, SAG, and CHIR99021 were added to NucT0 medium. Thereafter, the medium was replaced with NucT0 medium without Y-27632, SAG, or CHIR99021 every 3 to 4 days, and the cells were cultured for at least one month (Figure 102).

[0377] The differentiated aggregates of RPE prepared in Example 1 at 80 days or more after the start of suspension culture were seeded and cultured on iMatrix511-coated dishes in a medium consisting of DMEM (SIGMA) and F12 Ham (SIGMA) medium supplemented with B27 (Gibco), L-Glutamin, 10 ng / mL FGF2, and SB431542. After the RPE adhered and proliferated, the RPE was scraped off using a tip and seeded on another iMatrix511-coated dish for purification. After expansion, the aggregates were cultured on a 12-well TransWell plate coated with iMatrix511 at 1.0 x 10 ６ RPE cells were seeded, and the medium was changed every 3-4 days, and the cells were cultured for more than one month (FIG. 102).

[0378] The retinal tissue sheets cultured as described above were bonded to the RPE sheets using Bolheal Tissue Adhesion Kit (Teijin Pharma). Specifically, each sheet was retrieved from the Transwell using tweezers and scissors. 100 μL of fibrinogen was added to the retinal tissue sheet, and 100 μL of thorombin was added to the RPE sheet. After blending, the sheets were removed (Figures 103-104). Again, 100 μL of fibrinogen was added to the retinal tissue sheet, and 100 μL of thorombin was added to the RPE sheet (Figure 104). The RPE sheet was placed on a culture dish, apical side up, with the mesh side facing the culture dish. The retinal tissue sheet was then placed on top, with the apical side facing the RPE side (Figure 105). After bonding, the sheets were incubated at room temperature for 5 minutes to solidify, and then retrieved to produce the bonded sheets (Figures 106-107). The retinal tissue-RPE composite sheet thus prepared was turned over using tweezers, with the retinal tissue sheet facing downward and the retinal pigment epithelial sheet facing upward, and then observed (Figure 108).The Transwell mesh that had been attached was then removed (Figure 109).

[0379] Therefore, it was found that a retinal tissue-RPE composite sheet can be prepared by using a sheet-like retinal tissue, an RPE sheet, and fibrin.

[0380] Example 34: Examination of removal of unnecessary gelatin between two sheets using a cell sifter It was found that a large amount of gelatin was contained between the two sheets, making them thick, in the composite sheet examined in Example 32. Therefore, a composite device was examined that had a path for the gelatin to escape so that the unnecessary gelatin between the two sheets could be pushed out and removed during the composite process.

[0381] On a glass slide, a silicone sheet was used to create two, four, or five embankment-shaped channels, and two cell sifters (Cell Seed) with gelatin added between them were used to push from above, and it was observed whether the gelatin flowed out.

[0382] As a result, it was found that the device with five flow channels and a silicone sheet placed on top like a bank produced the most excess gelatin (Figure 110).

[0383] Example 35: Preparation of a planarized sheet on a temperature-sensitive culture dish, followed by detachment and transplantation studies Human ES cells (KhES-1 strain, (Non-Patent Document 3)) genetically modified to carry the Rx::Venus reporter gene 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 Laminin 511-E8 (product name, manufactured by Nippi Co., Ltd.) was used as a scaffold instead of feeder cells.

[0384] The aggregates (NR) prepared in Example 1 on days 18 to 30 were washed with PBS, and a nerve cell dispersion solution (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and then plated at 8.0 × 10 cells per well onto a 24-well temperature-sensitive culture dish (Cell Seed) coated with Laminin 511-E8 and with seven stages of detachment. ５ Cells were seeded at 1000 cells / well. The medium was changed every 3-4 days. After 30 days from seeding, when a thick sheet had formed, the plate was cooled to room temperature, incubated for 2 hours, and observed. As a result, it was observed that retinal sheets could be detached by bringing them to room temperature in several wells (2b, 2c, 3, and 4) of the seven temperature-sensitive culture dishes with different detachment levels (Figure 111).

[0385] Therefore, it was found that retinal sheets can be recovered by culturing them on a temperature-sensitive culture dish.

[0386] In this way, Rx::Venus was sorted as an indicator, and 10 μM Y-27632, 3 μM CHIR99021, 300 nM SAG, and 100 ng / mL FGF8 were added. The retinal sheets produced on the temperature-sensitive culture dishes were incubated at 4°C for 1 hour on day 62 after the start of suspension culture and then recovered. After that, a long graft for transplantation was excised using microscissors and transplanted into the subretinal space of immunodeficient retinal dysfunction rats (SD Foxn) (not shown).

[0387] Therefore, it was confirmed that the retinal sheet cultured on the temperature-sensitive culture dish can be transplanted.

Claims

1. A method for producing retinal tissue having an epithelial structure, comprising: The method comprises culturing the dispersed retinal cell population in suspension or adherent culture in a medium containing a Wnt signaling pathway active substance; A manufacturing method in which the retinal cell population comprises one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells.

2. The method of claim 1, wherein the substance acting on the Wnt signaling pathway is one or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b, and Wnt3a.

3. The method of claim 1 or 2, wherein the medium further comprises one or more substances selected from the group consisting of a ROCK inhibitor, a substance acting on the SHH signaling pathway, and a substance acting on the FGF signaling pathway.

4. The method according to claim 3, wherein the ROCK inhibitor is one or more substances selected from the group consisting of Y-27632, Fasudil (HA1077), and H-1152.

5. The method according to claim 3, wherein the substance acting on the SHH signaling pathway is one or more substances selected from the group consisting of SAG, PMA, and SHH.

6. The method according to claim 3 , wherein the substance acting on the FGF signaling pathway is one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4, and FGF8.

7. The method of claim 1 or 2, wherein the retinal tissue having an epithelial structure is a sheet-like retinal tissue, and the method comprises adhesion culture in a medium containing the substance acting on the Wnt signaling pathway.

8. The method according to claim 7 , wherein the adherent culture is carried out using a culture vessel coated with an extracellular matrix and / or a temperature-sensitive polymer.

9. The method according to claim 8 , wherein the culture surface of the culture vessel is coated with the temperature-sensitive polymer, and the upper surface of the temperature-sensitive polymer is coated with the extracellular matrix.

10. The method according to claim 8, wherein the extracellular matrix is ​​one or more substances selected from the group consisting of collagen, laminin, fibronectin, matrigel, and vitronectin.

11. The manufacturing method according to claim 8, further comprising the step of peeling off the sheet-like retinal tissue from the culture vessel by exposing the culture vessel coated with the temperature-sensitive polymer to a temperature at which the properties of the temperature-sensitive polymer change.

12. The manufacturing method according to claim 1 or 2, comprising a step of dispersing cell aggregates containing one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells to obtain the dispersed retinal cell population.

13. The production method according to claim 12, comprising a step of differentiating pluripotent stem cells to obtain cell aggregates containing one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells.

14. The method of claim 1 or 2, further comprising a step of increasing the proportion of retinal progenitor cells contained in the dispersed retinal cell population before the dispersed retinal cell population is cultured in suspension or adhesion.

15. The method of claim 14, wherein contamination or differentiation of retinal pigment epithelial cells is suppressed.

16. The manufacturing method described in claim 14, wherein the step of increasing the proportion of retinal progenitor cells comprises a step of contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, to obtain a cell population that expresses the antigens.

17. The manufacturing method of claim 16, wherein the step of increasing the proportion of retinal progenitor cells further comprises a step of contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84, thereby obtaining a cell population in which the expression level of the antigen is below a reference value.

18. The method of claim 14, wherein the step of increasing the proportion of retinal progenitor cells comprises the steps of: (1) A step of culturing pluripotent stem cells in the presence of one or more selected from the group consisting of a substance acting on the Shh signaling pathway, ATP, and an A2A receptor agonist to produce cell aggregates; (2) inducing differentiation of the cell aggregates into retinal progenitor cells; (3) Dispersing the cell aggregates and contacting them with a substance that binds to CD39.

19. The method of claim 1 or 2, wherein the retinal progenitor cells and / or the photoreceptor progenitor cells account for 50% or more of the total number of cells contained in the retinal cell population.

20. The method of claim 1 or 2, wherein the retinal progenitor cells and / or the photoreceptor progenitor cells account for 80% or more of the total number of cells contained in the retinal cell population.

21. The method of claim 1 or 2, wherein the dispersed retinal cell population is cultured in a medium containing the substance acting on the Wnt signaling pathway from the start of the suspension culture or adhesion culture.

22. The method according to claim 1 or 2, wherein the cells in the epithelial structure are oriented approximately perpendicular to the layer direction.

23. The method for producing retinal tissue according to claim 1 or 2, further comprising a step of cutting out the retinal tissue having an epithelial structure obtained by suspension culture or adhesion culture into a size required for transplantation.

24. The method of claim 1 or 2, wherein the epithelial structure is a multi-layered structure.

25. A sheet-like retinal tissue consisting of retinal cell layers having a multilayer structure, (1) The retinal cell layer having the multilayer structure has basal and apical polarity, (2) The retinal cell layer having the multilayer structure contains one or more types of cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells; (3) In each layer of the retinal cell layer, the orientation of the cells is approximately perpendicular to the layer direction, and (4) A sheet-like retinal tissue having a diameter of 8 mm or more.

26. 26. The sheet-like retinal tissue of claim 25, wherein the retinal cell layer having a multilayer structure further comprises sheet-like retinal pigment epithelial cells joined to the retinal cell layer, the tangential directions of the surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells are approximately parallel, the apical surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells face each other, and the retinal cell layer and the sheet-like retinal pigment epithelial cells are joined by adhesion factors present between them.

27. The sheet-shaped retinal tissue according to claim 26 , wherein the adhesion factor is an extracellular matrix or a hydrogel.

28. The sheet-shaped retinal tissue according to claim 27, wherein the adhesion factor is one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.

29. The sheet-shaped retinal tissue according to claim 27, wherein the adhesion factor is gelatin or fibrin.

30. A pharmaceutical composition comprising the sheet-shaped retinal tissue of claim 25 or 26.

31. A method for improving the proportion of retinal progenitor cells in a cell population, comprising the step of contacting a cell population containing retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of CD9, CD24, CD29, CD39, CD47, CD49b, CD49c, CD49f, CD57, CD73, CD82, CD90, CD164, CD200, CD340, and CXCR4.

32. 32. The method of claim 31, comprising contacting the cell population comprising the retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4.

33. The method of claim 31 or 32, further comprising contacting the cell population comprising the retinal progenitor cells with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84.

34. A cell population comprising retinal progenitor cells that are positive for at least one factor selected from the group consisting of CD9, CD39, CD90, and CXCR4 and are Rx-positive, accounting for 90% or more of the total number of cells in the cell population.

35. The cell population of claim 34, wherein the retinal progenitor cells are negative for one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84.