CN121057811A - Method for producing retina tissue - Google Patents

Abstract

The present invention provides a method for producing a retinal tissue having an epithelial structure, comprising the steps of (1) seeding a pluripotent stem cell onto a culture substrate having a region A having cell adhesion and a region B adjacent to at least a part of the region A having cell adhesion lower than the region A on the surface, (2) culturing the pluripotent stem cell seeded in the step (1) in a medium containing a ROCK inhibitor, (3) culturing the cells obtained after the step (2) in a medium containing a BMP signal transduction pathway-acting substance to obtain a retinal tissue, (4) dispersing the retinal tissue obtained in the step (3) to obtain a dispersed retinal cell population, and (5) subjecting the dispersed retinal cell population obtained in the step (4) to suspension culture or adhesion culture in a medium containing a Wnt signal transduction pathway-acting substance.

Description

Methods for manufacturing retinal tissue

Technical Field

This application claims priority based on Japanese Patent Application No. 2023-054011, which is incorporated herein by reference in its entirety.

This invention relates to a method for manufacturing retinal tissue.

Background Technology

Recent reports indicate that if photoreceptor precursor cells at an appropriate stage of differentiation are transplanted into the normal retina of living mice, they can functionally survive (Non-Patent Literature 1), demonstrating the potential of transplantation therapy for photoreceptor degenerative diseases such as retinitis pigmentosa.

Numerous methods have been reported for inducing three-dimensional retinal tissue by differentiating pluripotent stem cells obtained through autologous tissue culture, gradually enabling the fabrication and transplantation of layered three-dimensional retinal tissue. For example, the following methods have been reported: methods for obtaining multilayered retinal tissue from pluripotent stem cells (Non-Patent Document 2 and Patent Document 1); methods for obtaining multilayered retinal tissue by forming homogeneous aggregates of pluripotent stem cells in a serum-free culture medium containing a substance inhibiting the Wnt signal transduction pathway, suspending the resulting aggregates in the presence of a basement membrane standard, and then suspending them in a serum culture medium (Non-Patent Document 3 and Patent Document 2); and methods for obtaining retinal tissue by suspending aggregates of pluripotent stem cells in a culture medium containing a substance acting on the BMP signal transduction pathway (Non-Patent Document 4 and Patent Document 3), etc. These retinal tissues are fabricated in the form of sphere-shaped cell aggregates. Additionally, a method for obtaining retinal tissue (retinal sheets) from pluripotent stem cells through patterned culture has also been reported (Patent Document 4).

On the other hand, Wnt2b has been reported to be effective in the formation of retinal epithelial structures in chickens (Non-Patent Document 5), but no similar effect has been reported in organisms other than chickens.

Existing technical documents

Patent documents

Patent Document 1: International Publication No. 2011/055855

Patent Document 2: International Publication No. 2013/077425

Patent Document 3: International Publication No. 2015/025967

Patent Document 4: International Publication No. 2023/003025

Non-patent literature

Non-patent literature 1: Maclaren RE et al., “Retinal Repair by Transplantation of Photoreceptor Precursors”, Nature, 444, 203-207, (2006)

Non-patent literature 2: Eiraku M. et al., “Self-organizing optic-cup morphogenesis in three-dimensional culture”, Nature, 472, 51-56, (2011)

Non-patent document 3: Nakano T. et al., "Self-formation of Optic Cups andStorable Stratified Neural Retina From Human ESCs" Cell Stem Cell, 10(6), 771-785, (2012)

Non-patent document 4: Kuwahara A. et al., "Generation of a ciliary margin-likestem cell niche from self-organizing human retinal tissue" Nature Communications, 6, 6286, (2015)

Non-patent document 5: Nakagawa, S. et al., "Identification of the laminar-inducing factor Wnt-signal from the anterior riminduces correct laminar formation of the neural retina in vitro." Developmental Biology, 260, 414-425, (2003)

Summary of the Invention

The problem that the invention aims to solve

Therefore, in view of the above, the object of the present invention is to provide a method for reconstructing the neuroepithelial structure of retinal tissue from retinal cells, and a method for manufacturing retinal tissue using the method.

Methods for solving problems

The inventors discovered that when using dispersed retinal lineage cells as starting cells to reconstruct neuroepithelial structures and manufacture retinal tissue, the neuroepithelial structure can be reconstructed by adding substances acting on the Wnt signaling pathway to the dispersed retinal lineage cells. Furthermore, it was found that this can be achieved by (1) further adding ROCK inhibitors, substances acting on the SHH signaling pathway, and/or fibroblast growth factor; (2) specifically, culturing on culture plates coated with an extracellular matrix that serves as a scaffold for cell adhesion in order to reconstruct a good sheet-like neuroepithelial structure; and (3) increasing the purity of the retinal precursor cells used as starting cells, thereby removing contaminating cells such as retinal pigment epithelial cells (RPE). However, clinical applications require the preparation of large quantities of retinal tissue, necessitating an increase in retinal lineage cells as raw materials for reconstructing neuroepithelial structures. However, the number of cells obtained from a single spherical retinal tissue is limited, and manufacturing multiple spherical retinal tissues and preparing retinal lineage cells from them is cumbersome. The inventors conceived of using retinal lineage cells obtained from retinal sheets as starting cells and found that these cells are suitable for the reconstruction of neuroepithelial structures, thus completing this invention.

That is, the present invention relates to the following inventions.

[1]

A method for manufacturing retinal tissue with an epithelial structure, comprising the following steps:

(1) Pluripotent stem cells are seeded on region A of a culture medium having a region A with cell adhesion and a region B with lower cell adhesion adjacent to at least a portion of region A.

(2) The pluripotent stem cells seeded in step (1) are cultured in a culture medium containing ROCK inhibitors;

(3) The cells obtained after step (2) were cultured in a culture medium containing substances that act on the BMP signal transduction pathway to obtain retinal tissue; and

(4) Disperse the retinal tissue obtained in step (3) to obtain a dispersed population of retinal cells; and

(5) The dispersed retinal cell population obtained in step (4) is cultured in suspension or adhesion in a culture medium containing substances that act on the Wnt signal transduction pathway.

[2]

According to the method described in [1], the aforementioned region A is coated with a cell-adhesive substance.

[3]

According to the method described in [2], the cell adhesion substance is laminin.

[4]

According to any one of [1] to [3], wherein the region A is surrounded by the region B.

[5]

The method according to any one of [1] to [4], wherein the above-mentioned pluripotent stem cells are human iPS cells.

[6]

According to any one of [1] to [5], in step (1) above, the above pluripotent stem cells are seeded at a density of 0.5 × ^10⁵ cells/ ^cm² to 2.5 × ^10⁵ cells/ ^cm² .

[7]

According to any one of [1] to [6], in step (1) above, the above pluripotent stem cells are seeded at a density of 0.5× ^10⁵ cells/ ^cm² to 1.5× ^10⁵ cells/ ^cm² .

[8]

According to any one of [1] to [7], in step (2) above, the culture in the culture medium containing ROCK inhibitor is for 1 hour to 16 hours.

[9]

According to any one of [1] to [8], in step (2) above, the culture in the culture medium containing ROCK inhibitor is for 1 hour to 3 hours.

[10]

According to any one of [1] to [9], in step (2) above, the culture in the culture medium containing ROCK inhibitor is for about 2 hours.

[11]

According to any one of [1] to [10], the ROCK inhibitor is one or more substances selected from the group consisting of Y-27632, fasudil (HA1077) and H-1152.

[12]

According to any one of [1] to [11], in step (3) above, the culture in the culture medium containing the BMP signal transduction pathway active substance is 5 to 10 days.

[13]

According to any one of [1] to [12], the culture medium containing the BMP signal transduction pathway active substance contains 3 nM to 15 nM of BMP4 or a concentration of BMP signal transduction pathway active substance that shows the same activity as the above-mentioned concentration of BMP4.

[14]

According to any one of [1] to [13], the substance acting on the BMP signal transduction pathway is one or more substances selected from the group consisting of BMP2, BMP4, BMP7 and GDF7.

[15]

According to any one of [1] to [14], the substance acting in the BMP signal transduction pathway is BMP4.

[16]

According to any one of [1] to [15], the substance acting on the Wnt signal transduction pathway is one or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b and Wnt3a.

[17]

According to any one of [1] to [16], the culture medium containing the Wnt signal transduction pathway active substance further contains one or more substances selected from the group consisting of ROCK inhibitors, SHH signal transduction pathway active substances and FGF signal transduction pathway active substances.

[18]

According to the manufacturing method described in [17], the ROCK inhibitor in the culture medium containing the Wnt signal transduction pathway active substance is one or more substances selected from the group consisting of Y-27632, fasudil (HA1077) and H-1152.

[19]

According to the manufacturing method described in [17] or [18], wherein the SHH signal transduction pathway active substance in the culture medium containing the Wnt signal transduction pathway active substance is one or more substances selected from the group consisting of SAG, PMA and SHH.

[20]

According to any one of [17] to [19], the FGF signal transduction pathway active substance in the culture medium containing the Wnt signal transduction pathway active substance is one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF8.

[twenty one]

According to any one of [1] to [20], the manufacturing method includes a step of adhesion culture in the culture medium containing the Wnt signal transduction pathway active substance, wherein the retinal tissue with epithelial structure is sheet-like retinal tissue.

[twenty two]

According to the manufacturing method described in [21], the above-mentioned adhesion culture is carried out using a culture vessel coated with an extracellular matrix and/or a temperature-responsive polymer.

[twenty three]

According to the manufacturing method described in [22], the culture surface of the culture container is coated with the temperature-responsive polymer, and the upper surface of the temperature-responsive polymer is coated with the extracellular matrix.

[twenty four]

According to the manufacturing method described in [22] or [23], the extracellular matrix is one or more substances selected from the group consisting of collagen, laminin, fibronectin, matrigol and fibronectin.

[25]

The manufacturing method according to any one of [22] to [24] further includes the step of peeling the sheet-like retinal tissue from the culture container by exposing the culture container coated with the temperature-responsive polymer to a temperature at which the properties of the temperature-responsive polymer change.

[26]

The manufacturing method according to any one of [1] to [25] further includes the following step: before step (5), increasing the proportion of retinal precursor cells contained in the dispersed retinal cell population obtained in step (4).

[27]

According to the method described in [26], the incorporation or differentiation of retinal pigment epithelial cells is inhibited.

[28]

According to the manufacturing method described in [26] or [27], the step of increasing the proportion of the above-mentioned retinal precursor cells includes the following steps: contacting the above-mentioned 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 antigen.

[29]

According to the manufacturing method described in [28], the step of increasing the proportion of the above-mentioned retinal progenitor cells further includes the following step: further contacting the above-mentioned 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 level of the antigen is below a baseline value.

[30]

According to any one of [1] to [29], the manufacturing method wherein retinal precursor cells and/or photoreceptor precursor cells account for more than 50% of the total number of cells contained in the above-mentioned retinal cell group.

[31]

According to any one of [1] to [29], the manufacturing method comprises retinal precursor cells and/or photoreceptor precursor cells accounting for more than 80% of the total number of cells contained in the above-mentioned retinal cell group.

[32]

According to any one of [1] to [31], in the manufacturing method described above, in step (5), the dispersed retinal cell population is cultured in the culture medium containing Wnt signal transduction pathway active substances from the start of the suspension culture or adhesion culture.

[33]

According to any one of [1] to [32], the manufacturing method wherein the culture medium containing the Wnt signal transduction pathway active substance contains 3 μM to 6 μM of CHIR99021 or a concentration of Wnt signal transduction pathway active substance that shows the same activity as the above concentration of CHIR99021.

[34]

According to any one of [1] to [33], in the above-mentioned epithelial structure, the orientation of the cells is approximately perpendicular to the layer direction.

[35]

The manufacturing method according to any one of [1] to [34] further includes the following step: cutting the retinal tissue with epithelial structure obtained by suspension culture or adhesion culture in step (5) above into the size required for transplantation.

[36]

The manufacturing method according to any one of [1] to [35], wherein the epithelial structure is a multilayer structure.

[37]

A method for manufacturing a retinal tissue (a complex of neuroretinal and RPE cells (composite sheet)) comprising: manufacturing a sheet-like retinal tissue by any one of [21] to [36]; and, in the presence of an adhesion factor, attaching the sheet-like retinal tissue to sheet-like or dispersed retinal pigment epithelial cells.

[38]

A method for manufacturing a retinal tissue (a complex of neuroretinal and RPE cells (composite sheet)) comprising: in the presence of an adhesion factor, attaching sheet-like retinal tissue manufactured by any one of [21] to [36] to sheet-like or dispersed retinal pigment epithelial cells.

[39]

According to the manufacturing method described in [37] or [38], the aforementioned adhesion factor is an extracellular matrix or hydrogel.

[40]

The manufacturing method according to any one of [37] to [39], wherein the above-mentioned adhesion factor is one or more substances selected from gelatin, fibroin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan and nestin.

[41]

The manufacturing method according to any one of [37] to [40], wherein the above-mentioned adhesion factor is gelatin or fibroin.

[42]

A retinal tissue, which is manufactured by any one of the methods described in [1] to [41].

[43]

The retinal tissue described in [42] contains a multilayered retinal cell layer.

[44]

According to [43], the retinal tissue contains the above-mentioned multilayered retinal cell layer and sheet-like retinal pigment epithelial cells attached to the above-mentioned retinal cell layer, wherein the tangential directions of the surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells are generally parallel, the apical surfaces of the retinal cell layer and the apical surfaces of the sheet-like retinal pigment epithelial cells are opposite to each other, and the retinal cell layer and the sheet-like retinal pigment epithelial cells are attached by an adhesion factor present between them.

[45]

According to the retinal tissue described in [44], the aforementioned adhesion factor is an extracellular matrix or hydrogel.

[46]

According to the retinal tissue described in [44] or [45], wherein the adhesion factor is one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan and nestin.

[47]

The retinal tissue according to any one of [44] to [46], wherein the adhesion factor is gelatin or fibrin.

[48]

A pharmaceutical composition comprising any one of the following:[42] to [47] retinal tissue.

[49]

A treatment for a disease based on a disorder of retinal cells or retinal tissue or damage to retinal tissue, comprising the step of transplanting retinal tissue as described in any one of [42] to [47] to the recipient of the transplant.

[50]

The retinal tissue according to any one of [42] to [47] is used to treat diseases based on retinal cells or retinal tissue disorders or retinal tissue damage.

[51]

The use of any of the retinal tissues described in [42] to [47] in the manufacture of a therapeutic agent for a disease based on retinal cells or retinal tissue disorders or retinal tissue damage.

Invention Effects

According to the present invention, a method for reconstructing the layered structure of retinal tissue from retinal cells, a method for manufacturing retinal tissue using the method, and retinal tissue can be provided.

Attached Figure Description

Figure 1 is a bright-field microscope photograph showing the reformation state of aggregates from KhES-1 on day 1 after dispersing them into single cells and inoculating them in Reference Example 1-1.

Figure 2 is a bright-field microscope photograph showing the reformation state of aggregates from 1231A3 on day 1 after dispersing them into single cells and inoculating them in Reference Example 1-1.

Figure 3 is a bright-field microscope photograph showing the morphology of aggregates from KhES-1 that were dispersed into single cells and re-formed on day 1 after inoculation, as described in Reference Examples 1-2.

Figure 4 is a fluorescence micrograph showing the morphology of aggregates from KhES-1 that were dispersed into single cells and re-formed on day 7 after inoculation in Reference Examples 1-2.

Figure 5 is a fluorescence micrograph showing the morphology of aggregates from KhES-1 that were dispersed into single cells and reformed on day 14 after inoculation in Reference Examples 1-2.

Figure 6 shows the results obtained by measuring the area of the re-formed aggregates of single cells from KhES-1 in Reference Examples 1-2 using Image J. (A) shows the area of the aggregates on day 1, day 7 and day 14 after inoculation, and (B) shows the ratio of the area of the aggregates on day 7 and day 14 after inoculation to the area of the aggregates on day 1 (area ratio).

Figure 7 is a bright-field microscope photograph showing the morphology of aggregates formed on day 1 after dispersing aggregates from 1231A3 into single cells and inoculating them in Reference Examples 1-2.

Figure 8 is a bright-field microscope photograph showing the morphology of aggregates formed on day 1 after dispersing aggregates from KhES-1 into single cells and inoculating them in Reference Examples 1-3.

Figure 9 is a fluorescence micrograph showing the morphology of aggregates formed on day 14 after dispersing aggregates from KhES-1 into single cells and inoculating them in Reference Examples 1-3.

Figure 10 shows bright-field and fluorescence micrographs of the aggregates formed on day 28 after the aggregates from KhES-1 were dispersed into single cells and inoculated in Reference Examples 1-3.

Figure 11 shows a photograph of the results obtained by observing sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 15 after using confocal laser scanning fluorescence microscopy in Reference Examples 1-3, after immunostaining (DAPI, Rx::Venus, Chx10).

Figure 12 shows a photograph of the results obtained by using confocal laser scanning fluorescence microscopy to observe sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 15 after inoculation, after immunostaining (β-catenin).

Figure 13 is a photograph showing the results obtained by observing the immunostained sections (type IV collagen, Zo-1) of aggregates from KhES-1 dispersed into single cells and inoculated on day 15 after using confocal laser scanning fluorescence microscopy in Reference Examples 1-3.

Figure 14 shows a photograph of the results obtained by using confocal laser scanning fluorescence microscopy to observe sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after inoculation, after immunostaining (DAPI, Chx10, Ki67, Pax6).

Figure 15 shows the results obtained by using confocal laser scanning fluorescence microscopy to observe the Rx::Venus fluorescence of a slice of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after inoculation, as described in Reference Examples 1-3.

Figure 16 shows a photograph of the results obtained by observing the immunostained sections (type IV collagen, Zo-1) of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after using confocal laser scanning fluorescence microscopy in Reference Examples 1-3.

Figure 17 shows photographs of sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after the aggregates were inoculated using confocal laser scanning fluorescence microscopy, as shown in Reference Examples 1-3. The sections were obtained by immunostaining (DAPI, Rx::Venus, type IV collagen, Zo-1).

Figure 18 shows photographs of sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after observation using confocal laser scanning fluorescence microscopy in Reference Examples 1-3, obtained by immunostaining (DAPI, Chx10, Ki67, Pax6).

Figure 19 shows a photograph of the results obtained by observing sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after using confocal laser scanning fluorescence microscopy in Reference Examples 1-3, after immunostaining (CRX, RxRg, NRL, recovery protein).

Figure 20 shows photographs of sections of aggregates from KhES-1 dispersed into single cells and inoculated on day 28 after the aggregates were observed using confocal laser scanning fluorescence microscopy in Reference Examples 1-3, after immunostaining (DAPI, Islet-1, Brn3, calreticulin).

Figure 21 shows bright-field and fluorescence micrographs of aggregates from KhES-1 on days 18, 25, 40, 61, and 75 after differentiation into single cells and then reformed on days 3, 15, and 21 after inoculation.

Figure 22 shows bright-field and fluorescence microscopy (Rx::Venus) images of aggregates (BMP4+/BMP4-) from KhES-1 on day 40 of differentiation in Reference Examples 1-5, which were dispersed into single cells and then re-formed on days 3, 15, and 21 after inoculation.

Figure 23 shows fluorescence micrographs of dispersed retinal cells cryopreserved in various cryopreservation solutions in Examples 1-6, and the morphology of the aggregates formed after 1 day and 7 days.

Figure 24 shows the viability (A) of dispersed retinal cells frozen in various cryopreservation solutions and the area (B) of aggregates re-formed using the revived retinal cells after revival in Examples 1-6.

Figure 25 shows photographs of immunostained sections of retinal tissue formed through suspension culture, obtained using confocal laser scanning fluorescence microscopy to observe the basement membrane expressed in the tissue. (Reference Examples 1-7)

Figure 26 is an immunohistochemical image showing the results obtained from investigating the isoforms of laminin expressed in the neural retina of fetal rats in Reference Examples 1-7.

Figure 27 is a confocal laser scanning fluorescence microscopy image showing the reformation of sheet-like retinal tissue in adhesion culture confirmed by single-cell suspensions of retinal cells under conditions 1 to 3 in Reference Examples 1-8.

Figure 28 is a confocal laser scanning fluorescence microscopy photograph showing the reformation of sheet-like retinal tissue in adhesion culture confirmed by single-cell suspensions of retinal cells under conditions 1 to 3 in Reference Examples 1-8.

Figure 29 shows bright-field and fluorescence micrographs of the results obtained in Reference Examples 1-9, which confirmed the effects of various factors on the reformation of sheet-like retinal tissue in adhesive culture.

Figure 30 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-9 confirming the effects of various factors on the reformation of sheet-like retinal tissue in adhesive culture.

Figure 31 shows bright-field micrographs, fluorescence micrographs, and confocal laser scanning fluorescence micrographs illustrating the results of the study on the concentration and timing of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture in Reference Examples 1-10.

Figure 32 is a confocal laser scanning fluorescence microscopy image showing the results of the study on the concentration and timing of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture in Reference Examples 1-10.

Figure 33 shows bright-field micrographs, bright-field and fluorescence micrographs, and fluorescence micrographs illustrating the results obtained from studying the scaffold seeded on Transwell during the reformation of sheet-like retinal tissue in adhesive culture in Reference Examples 1-11.

Figure 34 shows bright-field and fluorescence micrographs illustrating the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 on the reformation of sheet-like retinal tissue in adhesive culture.

Figure 35 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 36 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 37 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 38 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 39 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 40 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 41 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 42 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 43 is a confocal laser scanning fluorescence microscopy image showing the results obtained in Reference Examples 1-12 confirming the effect of CHIR99021 in the reformation of sheet-like retinal tissue in adhesive culture.

Figure 44 is a fluorescence microscope photograph showing the results of the study on the maintenance effect of various factors on retinal cells in Reference Examples 1-13.

Figure 45 is a fluorescence micrograph showing the results of a study on the conditions for maintaining the culture of retinal precursor cells in Reference Examples 1-14.

Figure 46 is a stereomicrograph showing the results of re-sheetification of collagen in Reference Examples 1-15.

Figure 47 is a fluorescence micrograph showing a graft made from a re-sheetened retinal cell sheet in Reference Examples 1-16.

Figure 48 shows a fluorescence stereomicroscope photograph and a fluorescence microscope photograph illustrating the fundus observation results of the transplanted retina in Reference Examples 1-16.

Figure 49 shows the FACS analysis results of Rx::Venus positive fractions used to sort cell populations dispersed into single cells in Reference Examples 1-17. (A) is a scatter plot, and (B) is a bar chart.

Figure 50 shows stereomicroscope and fluorescence stereomicroscope photographs obtained from observing sorted/unsorted cell populations in Reference Examples 1-17.

Figure 51 shows a confocal laser scanning fluorescence microscope image obtained from observing sorted/unsorted cell populations in Reference Examples 1-17.

Figure 52 shows a confocal laser scanning fluorescence microscope image obtained from observing sorted/unsorted cell populations in Reference Examples 1-17.

Figure 53 shows bright-field micrographs and fluorescence micrographs obtained from observing sorted cell populations in Reference Examples 1-17.

Figure 54 shows a fluorescence microscope image of the Rx::Venus positive cell population observed in Reference Examples 1-18 with various proteins added.

Figure 55 shows a fluorescence microscope image of the Rx::Venus positive cell population observed in Reference Examples 1-18 with the addition of various low molecular weight compounds.

Figure 56 shows fluorescence micrographs of Rx::Venus positive cell populations observed in Reference Examples 1-18 with different concentrations of FGF2, FGF4 and FGF8 added.

Figure 57 shows bright-field and fluorescence micrographs illustrating the effects of sorting and the addition of FGF8 on aggregate reformation and retinal differentiation in Reference Examples 1-19.

Figure 58 shows bright-field micrographs and fluorescence micrographs illustrating the effects of sorting and the addition of FGF8 on aggregate reformation and retinal differentiation in Reference Examples 1-19, as well as a graph showing the results of FACS analysis.

Figure 59 shows bright-field micrographs and fluorescence micrographs illustrating the effects of FGF8 addition on aggregate reformation and retinal differentiation in Reference Examples 1-19, as well as a graph showing the FACS analysis results.

Figure 60 shows bright-field and fluorescence micrographs illustrating the effect of adding FGF8 on re-sheetification in Reference Examples 1-20.

Figure 61 shows bright-field and fluorescence micrographs illustrating the effect of adding FGF8 on re-sheetification in Reference Examples 1-20.

Figure 62 shows bright-field micrographs and fluorescence micrographs illustrating the effects of FGF8 addition on re-sheet formation and retinal differentiation in Reference Examples 1-20, as well as a graph showing the FACS analysis results.

Figure 63 shows stereomicroscopic and fluorescence stereomicroscopic photographs of the temporal changes of the re-sheetped retinal patches in Reference Examples 1-21.

Figure 64 is a diagram showing the biomarkers of interest in the surface antigen screening in Reference Examples 1-22.

Figure 65 is a diagram showing the cell sorting results using various surface antigens in Reference Examples 1-22.

Figure 66 is a diagram showing the cell sorting results using various surface antigens in Reference Examples 1-22.

Figure 67 is a diagram showing the cell sorting results using various surface antigens in Reference Examples 1-22.

Figure 68 is a diagram showing the cell sorting results using various surface antigens in Reference Examples 1-22.

Figure 69 is a diagram showing the cell sorting results using various surface antigens in Reference Examples 1-22.

Figure 70 shows bright-field micrographs and fluorescence micrographs obtained from observing the differentiation of brain organoids in Reference Examples 1-23.

Figure 71 is a confocal laser scanning fluorescence microscopy image obtained from observing the differentiation of brain organoids in Reference Examples 1-23.

Figure 72 shows the FACS analysis results of the expression of CD39, CD73, and CXCR4 in brain organoids in Reference Examples 1-23.

Figure 73 shows bright-field and fluorescence micrographs of the results of differentiation induction using different concentrations of SAG in Reference Examples 1-24.

Figure 74 shows the FACS analysis results and the digitized graphs of CD39 and CXCR4 expression after differentiation induction using different concentrations of SAG in Reference Examples 1-24.

Figure 75 is a confocal laser scanning fluorescence microscopy image obtained from the immunostaining of tissues induced by differentiation using different concentrations of SAG in Reference Examples 1-24.

Figure 76 is a confocal laser scanning fluorescence microscopy image obtained from the immunostaining of tissues induced by differentiation using different concentrations of SAG in Reference Examples 1-24.

Figure 77 shows the FACS analysis results of CD39 and BV421 expression after different concentrations of SAG in Reference Examples 1-24, as well as confocal laser scanning fluorescence microscopy images of the immunostaining of tissues differentiated and induced by different concentrations of SAG.

Figure 78 is a confocal laser scanning fluorescence microscopy image obtained from the immunostaining images of tissues differentiated and induced by different concentrations of SAG in Reference Examples 1-24.

Figure 79 is a confocal laser scanning fluorescence microscopy image obtained from the immunostaining images of tissues differentiated and induced by different concentrations of SAG in Reference Examples 1-24.

Figure 80 is a graph showing the FACS analysis results of the study of substances that enhance CD39 expression in Reference Examples 1-25.

Figure 81 is a digitized FACS analysis of the results of studies showing substances that enhance CD39 expression in Reference Examples 1-25. (A) shows results that are CD39 positive and RX::venus positive, and (B) shows results that are CXCR4 negative and RX::venus positive.

Figure 82 shows bright-field and fluorescence micrographs illustrating the steps of observing Islet-1 KO hESC-retinal patches (dd74) made with FGF8- and FGF8+ and cutting out grafts for transplantation in Reference Examples 1-26.

Figure 83 is a FACS dot plot showing the screening results of various surface antigens of NR in Reference Examples 1-27, compared with brain organoids.

Figure 84 is a FACS dot plot showing the results of screening various surface antigens for NR in Reference Examples 1-27.

Figure 85 is a FACS dot plot showing the time-dependent changes in CD9 expression in hESC-retina in Reference Examples 1-28.

Figure 86 is a FACS dot plot showing the analysis results of CD9 and SSEA-1 expression in hESC-retina in Reference Examples 1-29.

Figure 87 is a FACS dot plot showing the results of purification studies of CD9, CD90, CXCR4 and SSEA-1 in hESC-retina using Reference Examples 1-30.

Figure 88 is a FACS dot plot showing the results of purification studies of CD9, CD90, CXCR4 and SSEA-1 in the hESC-retina used in Reference Examples 1-30.

Figure 89 shows the results of purification studies of CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Examples 1-30.

Figure 90 is a photograph showing the results of purification studies of CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Examples 1-30.

Figure 91 is a photograph showing the results of purification studies of CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Examples 1-30.

Figure 92 is an FACS plot showing the time-dependent changes in the expression of CD9 and SSEA-1 in the hESC-retina in Reference Examples 1-31.

Figure 93 is a schematic diagram illustrating the process of using gelatin in the composite study of RPE film and retinal film in Reference Examples 1-32.

Figure 94 shows stereomicroscopic and fluorescence microscopic photographs of the RPE sheets cultured on Transwell and the re-sheetified neural retina used in the gelatin complexation process in Reference Examples 1-32.

Figure 95 is a stereomicrograph showing the process of peeling the RPE sheet and the re-sheetped neuroretina from the Transwell and adding gelatin in Reference Examples 1-32.

Figure 96 is a stereomicrograph showing the process of adding gelatin to the RPE sheet and the re-sheetened neuroretina in Reference Examples 1-32.

Figure 97 shows stereomicrographs and fluorescence micrographs illustrating the process of combining the RPE sheet with the re-sheetened neuroretina in Reference Examples 1-32.

Figure 98 shows stereomicroscopic and fluorescence microscopic photographs of the RPE sheet after it has been composited with the re-sheetened neuroretina in Reference Examples 1-32, held with tweezers.

Figure 99 shows stereomicroscopic and fluorescence microscopic images of the RPE sheet cut out with scissors after being combined with the re-sheetened neuroretina in Reference Examples 1-32.

Figure 100 shows stereomicroscopic and fluorescence microscopic images of cross-sections of the composite RPE sheet and the re-sheetened neuroretina observed in Reference Examples 1-32.

Figure 101 shows stereomicroscopic and fluorescence microscopic photographs of the aspiration/ejection of the cut composite RPE sheet and the re-sheetped neuroretina observed in Reference Examples 1-32.

Figure 102 shows stereomicroscopic and fluorescence microscopic images of RPE sheets cultured on Transwell and re-sheetified neural retina used in the complexation of fibrin in Reference Examples 1-33.

Figure 103 shows stereomicroscopic and fluorescence microscopic images illustrating the process of detaching the RPE sheet and the re-sheetened neuroretina from Transwell in Reference Examples 1-33.

Figure 104 shows stereomicroscopic and fluorescence microscopic images illustrating the process of studying the recovery of RPE sheets and the re-sheetification with added fibrinogen and thrombin in Reference Examples 1-33.

Figure 105 shows stereomicroscopic and fluorescence microscopic images illustrating the process of combining the RPE sheet with the re-sheetened neuroretina in Reference Examples 1-33.

Figure 106 shows stereomicroscopic and fluorescence microscopic images illustrating the process of combining the RPE sheet with the re-sheetened neuroretina in Reference Examples 1-33.

Figure 107 shows stereomicroscopic and fluorescence microscopic images of the RPE sheet after it has been composited with the re-sheetened neuroretina in Reference Examples 1-33, held with tweezers.

Figure 108 shows stereomicroscopic and fluorescence microscopic photographs of the state in which the RPE sheet was combined with the re-sheetened neuroretina in Reference Examples 1-33.

Figure 109 shows stereomicroscopic and fluorescence microscopic images illustrating the composite study process of peeling RPE sheets from the Transwell mesh in Reference Examples 1-33.

Figure 110 is a schematic diagram and photograph illustrating the results of the study using CellShifter to remove unwanted hydrogels in Reference Examples 1-34.

Figure 111 is a photograph showing the process of preparing and peeling planarized slides on temperature-responsive culture dishes in Reference Examples 1-35.

Figure 112 shows human iPS cells (LPF11 line) obtained by seeding at various cell densities and patterning culture in Reference Example 2-2. Results are shown at 1 hour, day 1, day 2, or day 3 after seeding.

Figure 113 shows the Chx10 immunostaining results of human iPS cells (LPF11 line) obtained by seeding at various cell densities and performing patterned culture in Reference Example 2-2 (day 20 post-seeding). BMP(+) indicates patterned culture in the presence of BMP4, and BMP(-) indicates patterned culture in the absence of BMP4.

Figure 114 shows the results obtained in Reference Examples 2-3 by varying the treatment time of Y-27632 in patterned culture of human iPS cells (LPF11 line). Bright-field microscopy images and Chx10 immunostaining results are shown on day 20 post-inoculation.

Figure 115 shows the results obtained in Reference Examples 2-3 by varying the treatment time of Y-27632 in patterned culture of human iPS cells (DSP-SQ strain). The immunostaining results of Chx10 on day 20 post-inoculation are shown.

Figure 116 shows the results obtained in Reference Examples 2-4 by varying the BMP4 concentration in patterned culture of human iPS cells (LPF11 line). Bright-field microscopy images and Rx immunostaining results are shown on day 20 post-inoculation.

Figure 117 shows the expression of Chx10 and Rx (retinal progenitor cell marker genes) and Emx2 (non-target cell marker gene) in human iPS cells (LPF11 line) obtained by patterned culture at various BMP4 concentrations in Reference Examples 2-4.

Figure 118 shows the expression of marker genes in cells cultured using the patterned culture method (white) with WO2023/003025 in Reference Examples 2-5 and the patterned culture method (black) in Reference Example 2, respectively. The left side of the dashed line represents neuroretinal marker genes, and the right side represents non-target cell marker genes.

Figure 119 shows an experimental summary of Example 1.

Figure 120 is a bright-field microscope photograph showing the morphology of aggregates formed on days 3, 13, and 23 after patterned retinal precursor cell sheets cultured for 20 days in Example 1 were dispersed into single cells and inoculated under three-dimensional culture.

Figure 121 is a confocal laser microscope image showing the immunostaining images (Chx10, Pax6, Rx, Crx, ZO-1) of aggregates formed on day 23 after dispersing patterned retinal progenitor cell sheets into single cells and inoculating them under three-dimensional culture in Example 1 on day 20.

Figure 122 shows an experimental summary of Example 2.

Figure 123 is a bright-field microscope photograph showing the morphology of aggregates formed on days 3, 13, and 23 after patterned retinal precursor cell sheets cultured for 20 days were dispersed into single cells and inoculated in two-dimensional culture in Example 2.

Figure 124 is a confocal laser microscope image showing the immunostaining images (Chx10, Pax6, Rx, Crx, ZO-1) of aggregates formed on day 23 after dispersing patterned retinal progenitor cell sheets into single cells and inoculating them in two-dimensional culture in Example 2 on day 20.

Figure 125 shows an experimental summary of Example 3.

Figure 126 shows the results obtained in Example 3 by dispersing patterned retinal precursor cell sheets on day 20 of culture into single cells and purifying CD9-positive cell fractions.

Figure 127 shows a bright-field microscope image (right) illustrating the morphology of aggregates formed on day 23 after inoculation with CD9-positive cells sorted from patterned retinal progenitor cell sheets in Example 3 under three-dimensional culture, and a confocal laser microscope image (left) showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1).

Figure 128 shows the results obtained by measuring the expression levels of Rx (neuroretinal marker) and Emx2 (telobrain dorsal marker) in aggregates formed from CD9-positive cells sorted from retinal progenitor cell sheets in Example 3 using real-time PCR.

Figure 129 shows an experimental summary of Example 4.

Figure 130 shows a bright-field microscope image (right) illustrating the morphology of aggregates formed on day 23 after inoculation in two-dimensional culture using CD9-positive cells sorted from patterned retinal progenitor cell sheets on day 20 of culture in Example 4, and a confocal laser microscope image (left) showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1).

Figure 131 is a bright-field micrograph showing the morphology of aggregates formed on day 23 after inoculation in three-dimensional culture in the presence of Y27632 (final concentration 0 or 10 μM), SAG (final concentration 0, 150, 300 or 600 nM), and CHIR99021 (final concentration 0, 1.5, 3 or 6 μM) in Example 5, after dispersing patterned retinal precursor cell sheets into single cells and inoculating them into single cells.

Figure 132 is a bright-field micrograph showing the morphology of aggregates formed on day 23 after dispersing patterned retinal precursor cell sheets into single cells and inoculating them in three-dimensional culture in the presence of Y27632 (final concentration 10 μM), SAG (final concentration 300 nM), and CHIR99021 (final concentration 0, 1.5, 3, or 6 μM) in Example 5.

Detailed Implementation

[definition]

Stem cells are undifferentiated cells with the ability to differentiate and proliferate (especially self-replicate). Based on their differentiation capacity, stem cells are categorized into subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells. Pluripotent stem cells are those that can be cultured in vitro and possess the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and/or extraembryonic tissues (pluripotency). Multipotent stem cells are those that can differentiate into two or more types of tissues or cells, but not all types. Unipotent stem cells are those that can differentiate into specific tissues or cells.

Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ 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) derived from mesenchymal stem cells (MSCs) and GS cells derived from germ cells (e.g., testes) are also included in the category of pluripotent stem cells.

Human embryonic stem cells were established in 1998 and have begun to be used in regenerative medicine. Embryonic stem cells can be produced by culturing internal cell aggregates on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described, for example, in WO96/22362, WO02/101057, US5843780, US6200806, and US6280718. Embryonic stem cells can be obtained from designated institutions or purchased commercially available products. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 can be obtained from the Institute for Regenerative Medicine, Kyoto University. Human embryonic stem cell lines Crx::Venus and Rx::Venus (both derived from KhES-1) can be obtained from the RIKEN National Research and Development Corporation.

"Induced pluripotent stem cells" are cells that are induced to become pluripotent by reprogramming somatic cells using known methods.

Induced pluripotent stem cells (iPSCs) were established in 2006 by Yamanaka et al. using mouse cells (Cell, 2006, 126(4), pp. 663-676). In 2007, iPSCs were also established from human fibroblasts, and like embryonic stem cells, they possess pluripotency and self-replication capabilities (Cell, 2007, 131(5), pp. 861-872; Science, 2007, 318(5858), pp. 1917-1920; Nat. Biotechnol., 2008, 26(1), pp. 101-106).

Specifically, induced pluripotent stem cells can be categorized as cells that are induced to differentiate into multiple cells by reprogramming differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells through the expression of any combination of genes from a reprogrammed genome, including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sal4, 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).

Induced pluripotent stem cells can be induced from somatic cells, in addition to methods that utilize direct reprogramming based on gene expression, by adding compounds (Science, 2013, 341, pp. 651-654).

Alternatively, pre-lined induced pluripotent stem cells can also be obtained. For example, human induced pluripotent cell lines such as 201B7, 201B7-Ff, 253G1, 253G4, 1201C1, 1205D1, 1210B2, and 1231A3, established by Kyoto University, can be obtained from Kyoto University and iPS Academiya Japan Co., Ltd. Pre-lined induced pluripotent stem cells, such as Ff-I01, Ff-I14, and QHJI01s04, established by Kyoto University, can also be obtained from Kyoto University.

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

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).

Human iPS cells and other pluripotent stem cells can be used for maintenance and expansion culture by methods known to those skilled in the art.

"Nervous tissue" refers to the tissue composed of nerve cells from the brain, midbrain, cerebellum, spinal cord, retina, peripheral nerves, forebrain, hindbrain, telencephalon, diencephalon, etc., during the developmental or adult stages. Nervous tissue sometimes forms layered epithelial structures (neuroepithelium), and the amount of neuroepithelium in cell aggregates can be evaluated by observing it in bright field using an optical microscope.

"Neural cells" refer to cells derived from ectodermal tissues other than epidermal cells. This includes neural progenitor cells, neurons (nerve cells), glial cells, neural stem cells, neuronal progenitor cells, and glial cell progenitor cells. Neural cells also include cells that make up the following retinal tissues: retinal cells, retinal progenitor cells, retinal layer-specific neurons, neuroretinal cells, and retinal pigment epithelial cells. Neural cells can be identified using markers such as Nestin, TuJ1, PSA-NCAM, and N-cadherin.

"Neuron/Neuronal cell" is a functional cell that forms neural circuits and helps in information transduction. It can be identified by the expression of immature nerve cell markers such as TuJ1, Dcx, and HuC/D and/or mature nerve cell markers such as Map2 and NeuN.

Neural precursor cells are a population of precursor cells including neural stem cells, neuronal precursor cells, and glial cell precursor cells, possessing proliferative capacity and the ability to generate neurons and glial cells. Neural precursor cells can be identified using markers such as neural epithelial stem cell proteins, GLAST, Sox2, Sox1, Musashi, and Pax6. Alternatively, cells that are positive for both neural stem cell markers and proliferation markers (Ki67, pH3, MCM) can be identified as neural precursor cells.

"Retinal tissue" refers to one or more types of retinal cells that make up the various retinal layers in an organism's retina, existing in a certain order. "Neural retina" refers to the retinal tissue that contains the inner neuroretinal layer of the retinal layers, excluding the retinal pigment epithelium layer.

"Retinal cells" refer to the cells or precursor cells that make up the various retinal layers in the retina of an organism. Retinal cells include photoreceptor cells (rod cells, cone cells), horizontal cells, amacrine cells, intermediate nerve cells, retinal ganglion cells, bipolar cells (rod bipolar cells, cone bipolar cells), Müller's glial cells, retinal pigment epithelium (RPE) cells, ciliary body, and their precursor cells (such as photoreceptor precursor cells, bipolar cell precursor cells, and retinal pigment epithelium precursor cells), neuroretinal precursor cells, and other retinal precursor cells, but are not limited to these. The retinal cell system, which comprises the cells that make up the neuroretinal layer (also called neuroretinal cells or neuroretina-related cells), specifically includes photoreceptor cells (rod cells, cone cells), horizontal cells, amacrine cells, intermediate nerve cells, retinal ganglion cells (ganglionic cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller's glial cells, and their precursor cells (such as photoreceptor precursor cells, bipolar cell precursor cells, etc.). That is, the neuroretinal cell system does not include retinal pigment epithelial cells and ciliary body cells.

"Mature retinal cells" refer to the cells found in the retinal tissue of adult humans. Specifically, they include differentiated cells such as photoreceptor cells (rod cells, cone cells), horizontal cells, amacrine cells, intermediate nerve cells, retinal ganglion cells (ganglionic cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller's glial cells, retinal pigment epithelium (RPE) cells, and ciliary body cells. "Immature retinal cells" refer to precursor cells that are determined to differentiate into mature retinal cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, retinal precursor cells, etc.).

Precursor cells of photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller's glial cells, and retinal pigment epithelial cells refer to the precursor cells that are known to differentiate into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller's glial cells, and retinal pigment epithelial cells, respectively.

"Retinal precursor cells" refer to precursor cells that can differentiate into any of the following immature retinal cell types: photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, Müller's glial cells, and retinal pigment epithelial cell precursor cells. They are also precursor cells that can ultimately differentiate into any of the following mature retinal cell types: photoreceptor cells, rod cells, cone cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells. "Neural retinal precursor cells" refer to precursor cells that can differentiate into any of the following immature neural retinal cell types: photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, and Müller's glial cells. They are also precursor cells that can ultimately differentiate into any of the following mature neural retinal cell types: photoreceptor cells, rod cells, cone cells, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Neural retinal precursor cells do not have the ability to differentiate into retinal pigment epithelial cells.

Photoreceptor cells are located in the photoreceptor layer of the retina in organisms and have the function of absorbing light stimuli and converting them into electrical signals. Photoreceptor cells include two types: cones, which function in bright light, and rods (or rod bodies, or rods), which function in dark light (referred to as cone cells and rod cells, respectively). Examples of cone cells include S-cones, which express S-opsin and receive blue light; L-cones, which express L-opsin and receive red light; and M-cones, which express M-opsin and receive green light. Photoreceptor cells differentiate and mature from photoreceptor precursor cells. Those skilled in the art can easily confirm whether a cell is a photoreceptor cell or a photoreceptor precursor cell by, for example, through the expression of cell markers (Crx and Blimp1 expressed in photoreceptor precursor cells, Recoverin expressed in photoreceptor cells, rhodopsin, S-opsin, and M/L-opsin expressed in mature photoreceptor cells) and the formation of outer segment structures, as described later. In one embodiment, the photoreceptor precursor cells are Crx-positive cells, and the photoreceptor cells are rhodopsin, S-opsin, and M/L-opsin-positive cells. In another embodiment, the rod cells are NRL and rhodopsin-positive cells. In yet another embodiment, the S-cone cells are S-opsin-positive cells, the L-cone cells are L-opsin-positive cells, and the M-cone cells are M-opsin-positive cells.

The presence of neural retinal cells can be confirmed by the expression of neural retinal cell-related genes (hereinafter sometimes referred to as "neural retinal cell markers" or "neural retinal markers"). Those skilled in the art can readily confirm the expression of neural retinal cell markers or the proportion of neural retinal cell marker-positive cells in a cell population or tissue. Examples include methods using antibodies, methods using nucleic acid primers, and methods using sequencing reactions. As an antibody-based method, flow cytometry (FACS) using commercially available antibodies, immunostaining, etc., can be used to divide the number of specific neural retinal cell marker-positive cells by the total number of cells, thereby confirming the protein expression of the neural retinal cell marker. As a method using nucleic acid primers, PCR, semi-quantitative PCR, and quantitative PCR (e.g., real-time PCR) can be used to confirm the RNA expression of neural retinal cell markers. As a method using sequencing reactions, a nucleic acid sequencer (e.g., a next-generation sequencer) can be used to confirm the RNA expression of neural retinal cell markers.

Examples of neuroretinal cell markers include Rx (also known as Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6 and Chx10 (also known as Vsx2) expressed in neuroretinal progenitor cells, and Crx and Blimp1 expressed in photoreceptor progenitor cells. In addition, examples include Chx10, which is strongly expressed in bipolar cells; PKCα, Goα, VSX1, and L7, which are expressed in bipolar cells; TuJ1 and Brn3, which are expressed in retinal ganglion cells; calreticulin and HPC-1, which are expressed in amacrine cells; calcium-binding proteins, which are expressed in horizontal cells; recovery proteins, which are expressed in photoreceptor cells and photoreceptor precursor cells; rhodopsin, which is expressed in rod cells; Nrl, which is expressed in rod cells and rod cell precursor cells; S-opsin and LM-opsin, which are expressed in cone cells; RXR-γ, which is expressed in cone cells, cone cell precursor cells, and ganglion cells; TRβ2, OTX2, and OC2, which are expressed in cone cells or their precursor cells that appear in the early stages of differentiation; and Pax6, which is co-expressed in horizontal cells, amacrine cells, and ganglion cells. Surface antigens of retinal precursor cells or neural retinal precursor cells identified in the reference examples of this application may also be used as markers for retinal precursor cells or neural retinal precursor cells. Details are as follows.

"Positive cells" are cells that express specific markers on their surface or inside the cell. For example, "Chx10 positive cells" are cells that express the Chx10 protein.

"Retinal pigment epithelial cells" refer to the epithelial cells located on the outer side of the neuroretina in the retina of an organism. Whether a cell is a retinal pigment epithelial cell can be easily confirmed by those skilled in the art, for example, through the expression of cell markers (MITF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (dark brown), tight junctions between cells, and the characteristic polygonal/cobblestone morphology. Whether a cell possesses the functions of a retinal pigment epithelial cell can be easily confirmed by the secretion capacity of cytokines such as VEGF and PEDF, and the phagocytic capacity of the outer segments of the photoreceptor cells. In one embodiment, the retinal pigment epithelial cells are RPE65-positive cells, MITF-positive cells, or RPE65-positive and MITF-positive cells.

"Retinal pigment epithelial cell sheet" refers to a sheet-like structure consisting of one or more cells that are biologically bonded together along at least two dimensions and are composed of single or multiple layers.

The “retinal layer” refers to the various layers that make up the retina. Specifically, these include the retinal pigment epithelium, photoreceptor cell layer, external membrane, external granular layer, external retinal network layer, internal granular layer, internal retinal network layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane.

The term "neuroreretinal layer" refers to the layers that make up the neuroretina. Specifically, these include the photoreceptor cell layer, outer membrane, outer granular layer, outer retinal layer, inner granular layer, inner retinal layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. The "photoreceptor cell layer" is the outermost layer of the neuroretina, containing a large number of cells selected from one or more of the following groups: photoreceptor cells (rod cells, cone cells), photoreceptor precursor cells, and retinal precursor cells. All layers outside the photoreceptor cell layer are called inner layers. The specific retinal layer into which each cell belongs can be determined using known methods, such as the presence or degree of expression of cell markers.

In retinal tissue at stages where photoreceptor cells or photoreceptor precursor cells are present in small proportions, the layer containing proliferating neural retinal precursor cells is called the "neuroblastic layer," which consists of an inner neuroblastic layer and an outer neuroblastic layer. Those skilled in the art can determine this layer using known methods, such as the intensity of color under a bright-field microscope (the outer neuroblastic layer is lighter, and the inner neuroblastic layer is darker).

The ciliary body encompasses both the developmental and adult forms of the ciliary body, the ciliary marginal zone, and the ceric body itself. Markers for the ciliary body include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1, and AQP4. The ciliary marginal zone (CMZ) is the tissue located at the boundary between the neuroretina and the retinal pigment epithelium in the retina, containing retinal stem cells. The ciliary marginal zone is also known as the ciliary margin or retinal margin; the ciliary marginal zone, ciliary margin, and retinal margin are equivalent tissues. The ciliary marginal zone is known to play a crucial role in supplying retinal progenitor cells and differentiating cells to the retinal tissue, and in maintaining retinal tissue structure. Marker genes for the ciliary marginal zone include, for example, Rdh10 (positive), Otx1 (positive), and Zic1 (positive). "Ciliary body marginal zone-like structures" refers to structures similar to the ciliary body marginal zone.

"Brain tissue" refers to the tissue formed by one or more of the cells that constitute the embryonic or adult brain, arranged in a three-dimensional, layered manner, including cortical neural precursor cells, dorsal neural precursor cells, ventral neural precursor cells, brain layer structure-specific neurons (neurons), first-layer neurons, second-layer neurons, third-layer neurons, fourth-layer neurons, fifth-layer neurons, sixth-layer neurons, glial cells (astrocytes and oligodendrocytes), and their precursor cells. The embryonic brain is also called the forebrain or telencephalon. The presence of each cell can be confirmed by known methods, such as the presence or degree of expression of cell markers.

The “brain layers” refer to the various layers that make up the adult brain or the embryonic brain. Specifically, they can be listed as the molecular layer, outer granular layer, outer cone cell layer, inner granular layer, nerve cell layer (inner cone cell layer), polymorphic cell layer, layer one, layer two, layer three, layer four, layer five, layer six, cortical area, intermediate area, subventricular area, and ventricular zone.

As "precursor cells of the brain's neural system," examples include neuronal precursor cells, first-layer neuronal precursor cells, second-layer neuronal precursor cells, third-layer neuronal precursor cells, fourth-layer neuronal precursor cells, fifth-layer neuronal precursor cells, sixth-layer neuronal precursor cells, astrocyte precursor cells, and oligodendrocyte precursor cells. Each cell is a precursor cell for differentiating into first-layer neurons, second-layer neurons, third-layer neurons, fourth-layer neurons, fifth-layer neurons, sixth-layer neurons, astrocytes, and oligodendrocytes.

"Brain neural progenitor cells" include multipotent stem cells (multipotent neural stem cells) that have the ability to differentiate into at least two or more differentiation lineages among first-layer neurons, second-layer neurons, third-layer neurons, fourth-layer neurons, fifth-layer neurons, sixth-layer neurons, astrocytes, and oligodendrocytes.

"Brain layer-specific nerve cells" refer to nerve cells that are specific to the brain layer and constitute the brain layer. Examples of brain layer-specific nerve cells include neurons in the first layer, second layer, third layer, fourth layer, fifth layer, sixth layer, excitatory neurons of the brain, and inhibitory neurons of the brain.

Examples of brain cell markers include FoxG1 (also known as Bf1) expressed in brain cells, Sox2 and neural epithelial stem cell proteins expressed in brain neural progenitor cells, Pax6 and Emx2 expressed in dorsal brain neural progenitor cells, Dlx1, Dlx2 and Nkx2.1 expressed in ventral brain neural progenitor cells, Tbr2, Nex, and Svet1 expressed in neuronal progenitor cells, Tbr1 expressed in the sixth layer of neurons, Ctip2 expressed in the fifth layer of neurons, RORβ expressed in the fourth layer of neurons, Cux1 or Brn2 expressed in the third or second layer of neurons, and Reelin expressed in the first layer of neurons.

The term "cell aggregate" is not specifically defined as any three-dimensional structure formed by the adhesion of two or more cells together. For example, it refers to a mass of cells dispersed in a medium such as a culture medium, or a block of cells formed through cell division. Cell aggregates also include those that form specific tissues.

"Sphere-like cell aggregates" refer to cell aggregates with a near-spherical three-dimensional shape. Examples of near-spherical three-dimensional shapes include: spheres with a three-dimensional structure that appear as circles or ellipses when projected onto a two-dimensional plane, and shapes formed by the fusion of two or more spheres (e.g., shapes formed by the overlapping of 2 to 4 circles or ellipses when projected into a two-dimensional plane). In one embodiment, the core of the aggregate has a vesicular, layered structure, characterized by a dark central portion and a bright outer edge observed under a bright-field microscope.

"Epithelial tissue" refers to tissue formed by cells seamlessly covering the surface of the body, lumens (such as the digestive tract), and body cavities (such as the pericardial cavity). The cells that form epithelial tissue are called epithelial cells. Epithelial cells possess apical-basal polarity. Epithelial cells form strong connections with each other through adhesion junctions and/or tight junctions, creating cell layers. Tissue formed by one to more than ten overlapping cell layers is epithelial tissue. Other tissues that can form epithelial tissue include the retina in embryonic and/or adult stages, brain and spinal cord tissue, eyeball tissue, and nerve tissue. The neuroretina mentioned in this specification is also epithelial tissue. "Epithelial structure" refers to a structure characteristically possessing the polarity of epithelial tissue (e.g., having basal and apical surfaces).

"Continuous epithelial tissue" refers to tissue with a continuous epithelial structure. A continuous epithelial structure indicates a continuous state of epithelial tissue. For example, continuous epithelial tissue means a state in which 10 to ^10⁷ cells are arranged along a tangential direction relative to the epithelial tissue, preferably 30 to ^10⁷ cells, and more preferably ^10² to ^10⁷ cells.

For example, in a continuous epithelial structure formed in retinal tissue, the retinal tissue has an apical surface characteristic of epithelial tissue, which is formed substantially parallel and continuously on the surface of the retinal tissue to at least the photoreceptor cell layer (outer granular layer) among the layers forming the neuroretinal layer. For example, in the case of a cell aggregate containing retinal tissue made from pluripotent stem cells, an apical surface is formed on the surface of the aggregate, and photoreceptor cells or photoreceptor precursor cells are arranged regularly and continuously along a tangential direction relative to the surface, consisting of 10 or more cells, preferably 30 or more cells, more preferably 100 or more cells, and even more preferably 400 or more cells.

In one approach, the epithelial tissue undergoes polarization, forming an "apical surface," a "basal surface," and a "basement membrane." The "basement membrane" refers to the basal layer (basement membrane) produced by epithelial cells, containing abundant laminin and type IV collagen, and having a thickness of 50–100 nm. The "basal surface" refers to the surface (surface layer) formed on the side of the "basement membrane." The "apical surface" refers to the surface (surface layer) formed on the side opposite to the "basement membrane." In another approach, the "apical surface" refers to the surface in retinal tissue that forms an outer membrane and contacts the photoreceptor layer (outer granular layer) containing photoreceptors or photoreceptor precursor cells, at a stage where photoreceptor development has progressed to the point where photoreceptor cells or photoreceptor precursor cells are observed. Furthermore, such an apical surface can be identified using immunostaining methods known to those skilled in the art, using antibodies against markers for the apical surface (e.g., atypical PKC (hereinafter referred to as "aPKC"), tight junction markers (Zo-1), Ezioproteins as ERM proteins, E-cadherin, N-cadherin, etc.).

[Methods for manufacturing cell aggregates]

One aspect of the present invention is a method for manufacturing cell aggregates containing retinal tissue having an epithelial structure (or multilayer structure) from a dispersed retinal cell line population (in this specification, "cell aggregates containing retinal tissue" is sometimes simply referred to as "retinal tissue"). The method includes the step of culturing the dispersed retinal cell line population in suspension or adhesion in a culture medium containing substances acting on the Wnt signal transduction pathway.

As defined above, in one embodiment, the retinal cells contain one or more types of cells selected from the group consisting of retinal precursor cells and photoreceptor precursor cells. Additionally, they may contain photoreceptor cells (rod cells, cone cells), horizontal cells, amacrine cells, retinal ganglion cells (ganglionic cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller's glial cells, etc. The retinal cells are preferably neural retinal cells.

(Differentiation induction method for retinal cell lineage as initiating cells)

The retinal lineage cells derived from pluripotent stem cells used as starting cells in the manufacturing method of the present invention can be obtained by inducing differentiation of pluripotent stem cells. Specifically, the retinal lineage cell population used as starting cells can be obtained by a method including the following steps:

(1) Pluripotent stem cells are seeded on region A of a culture medium having a region A with cell adhesion and a region B with lower cell adhesion adjacent to at least a portion of region A.

(2) The pluripotent stem cells seeded in step (1) are cultured in a culture medium containing ROCK inhibitors;

(3) The cells obtained after step (2) were cultured in a culture medium containing substances that act on the BMP signal transduction pathway to obtain retinal tissue; and

(4) Disperse the retinal tissue obtained in step (3) to obtain a dispersed retinal cell population.

Region A is the region where pluripotent stem cells can be maintained through adhesion culture. Region B is the region with lower cell adhesion compared to Region A, such that when pluripotent stem cells are seeded in Region A, during the induction of differentiation into retinal tissue (i.e., the period prior to confirming differentiation into retinal tissue), the cells cannot extend into Region B adjacent to Region A. Cell adhesion can be compared as follows: Pluripotent stem cells are seeded in excess of their area (e.g., 5 × ^10⁵ to 10 × ^10⁵ cells/ ^cm² ), cultured at 37°C and 5% _CO₂ for 24 hours, and the proportion of area to which cells adhere is compared. The cell adhesion described above is typically evaluated after 24 hours of culture, after removing the culture medium and washing the cells with culture medium or buffer to remove unadhered cells.

Region A can be, for example, an area in which pluripotent stem cells are inoculated in excess of their area (e.g., 5× ^10⁵ to 10× ^10⁵ cells/ ^cm² ), and after being cultured at 37°C and 5% _{CO₂ for} 24 hours, the cells adhere to more than 70%, 80%, or 90% of their area.

In one approach, region B is a cell-nonadhesive region. A cell-nonadhesive region is one that does not possess sufficient cell adhesion to maintain pluripotent stem cells through adhesive culture. A cell-nonadhesive region can be, for example, a region in which pluripotent stem cells, after being seeded in excess of their area (e.g., 5 × ^10⁵ to 10 × ^10⁵ cells/ ^cm² ), adhere to less than 30%, 20%, or 10% of their area after culturing at 37°C and 5% _CO₂ for 24 hours.

Regarding the culture medium, it can be formed from any material, as long as its surface can form regions A and B. Culture mediums can be, for example, inorganic materials such as metals, glass, and silica gel; and organic materials such as plastics (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluoropolymer resin, polycarbonate resin, polyurethane resin, methyl terpene resin, phenolic resin, melamine resin, epoxy resin, vinyl chloride resin, polytetrafluoroethylene resin). In one embodiment, the culture medium is glass or plastic, particularly polystyrene resin.

The culture medium can be any shape commonly used for cell culture, without particular limitation. Examples of culture media include dishes, plates, bottles, cavities, multi-well plates (e.g., 6, 12, 24, 48, 96, or 384-well plates), membranes, or porous membranes. The culture medium typically has regions A and B on a surface horizontal relative to the direction of gravity (or, in the case of a culture container, on the bottom surface). In one embodiment, the culture medium has regions A and B on the same plane. In another embodiment, when the culture container has regions A and B on the plane of its bottom surface, the inner wall surface of the culture container is not region B. In this specification, as described later, when the culture medium has a protrusion on its surface and region A is located on the upper surface of that protrusion, the side surface of the protrusion adjacent to the upper surface of the protrusion is considered region B, but it is assumed that region A on the upper surface of the protrusion and region B on the side surface of the protrusion exist on the same plane.

Region A can be the exposed surface area of a cell-adhesive culture medium, or it can be the area where the surface of the culture medium is coated with a cell-adhesive substance to impart cell adhesion or to enhance cell adhesion. Examples of cell-adhesive substances include positively charged polymers such as poly-L-lysine and poly-L-ornithine; laminin; collagens such as type I, type II, type III, type IV, type V, and type VII collagen; tendinin; fibroin; fibronectin; vitronectin; elastin; nestin; proteoglycans composed of sulfated glycosaminoglycans such as chondroitin sulfate, heparin sulfate, keratin sulfate, and dermatan sulfate, along with a core protein; glycosaminoglycans such as chondroitin sulfate, heparin sulfate, keratin sulfate, dermatan sulfate, and hyaluronic acid; Synthemax (registered trademark, vitronectin derivative); Matrigel (registered trademark); etc.

In one approach, region A is coated with laminin. Laminin is a heterotrimeric molecule composed of three subunit chains: α, β, and γ. It is known that there are five types of α chains (α1–α5), three types of β chains (β1–β3), and three types of γ chains (γ1–γ3). Each laminin isoform is represented by a number indicating its structural subunit (e.g., laminin 111 is composed of α1, β1, and γ1 chains). Laminins include laminin 111, laminin 121, laminin 211, laminin 213, laminin 222, laminin 311 (laminin 3A11), laminin 332 (laminin 3A32), laminin 321 (laminin 3A21), laminin 3B32, laminin 411, laminin 421, laminin 423, laminin 511, laminin 521, laminin 522, laminin 523, or fragments thereof. Examples of laminin fragments that serve as integrin binding sites include the E8 fragment, such as laminin 211-E8, laminin 311-E8, laminin 411-E8, laminin 511-E8, etc. In one embodiment, the laminin is laminin 511 or a fragment thereof. In another embodiment, the laminin is the laminin 511-E8 fragment. For coating with laminin, commercially available products such as iMatrix-511 (Nippi.Inc.) can also be used. iMatrix-511 (Nippi.Inc.) contains the laminin 511-E8 fragment.

Region B can be an area where the surface of the culture medium has lower cell adhesion than region A, or it can be an area where the surface of the culture medium is coated with a substance to create a region with lower cell adhesion than region A. In one embodiment, region B is coated with a cell-nonadhesive substance. Examples of cell-nonadhesive substances include MPC (2-methacryloyloxyethylphosphorylcholine) polymers; celluloses such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymers; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginate, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin; and derivatives thereof. In one embodiment, the cell-nonadhesive substance is an MPC polymer.

The shape of region A is not particularly limited, and can be, for example, a circle, an ellipse, and a polygon (e.g., a triangle, quadrilateral, pentagon, hexagon, octagon, decagon, dodecagon, etc.). In one embodiment, region A is a circle. In this specification, "circle" refers to any shape that is substantially circular in the art, and is used to mean a roughly circular shape, including a perfect circle. The area of region A can be, for example, 0.01–100 ^cm² , 0.01–30 ^cm² , 0.01–10 ^cm² , 0.03–30 ^cm² , 0.03–10 ^cm² , or 0.1–10 ^cm² , but is not limited thereto. In one method, the area of region A is greater than or equal to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or ^{1 cm²} , and less than or equal to ¹⁰ , 9, ⁸ , 7, 6, ⁵ , 4, 3, 2, or 1 cm² (the upper and lower limits can be selected independently). For example, the area of region A can be 0.5–10 ^cm² , 0.7–10 ^cm² , 1–10 cm², 0.5–5 ^cm² , 0.7–5 cm², 1–5 ^cm² , 0.5–2 ^cm² , 0.7–2 cm², 0.5–1 ^cm² , or 0.7–1 ^cm² .

Region A can be a circle or a polygon with an area equal to its diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or more than 1 cm, or less than 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm (the upper and lower limits can be selected independently). In one embodiment, region A is a circle with a diameter of 0.1–10 cm, 0.1–5 cm, 0.1–3 cm, or 0.2–1 cm. In another embodiment, region A is a circle with a diameter of 1–10 cm, 1–5 cm, or 1–3 cm.

At least a portion of region A is adjacent to region B. In one embodiment, the entire outer edge of region A is adjacent to region B (i.e., surrounded by region B). The culture medium may have multiple regions A and/or multiple regions B on its surface. When the culture medium contains multiple regions A on its surface, the spacing between two regions A sandwiching region B is not limited, for example, it may be about 1 mm or more.

Region A and Region B can be formed on the surface of the culture medium using any method for cell patterning. Region A and Region B can be formed on the surface of the culture medium, for example, using techniques such as soft lithography, photolithography, and 3D printing.

Regions A and B can be formed by treating a portion of the surface of the culture medium to create differences in cell adhesion between the treated and untreated areas. Regions A and B can be formed, for example, by coating a portion of the surface of the culture medium with a cell-adhesive or cell-non-adhesive substance. For instance, it is possible to cover a portion of the culture medium surface and coat the uncovered area with a cell-adhesive or cell-non-adhesive substance. Alternatively, a layer of cell-adhesive or cell-non-adhesive substance can be formed on the culture medium, and a portion of it can be treated to alter cell adhesion. The treated portion can be the exposed portion of the culture medium surface or the portion where the properties of the cell-non-adhesive or cell-adhesive substance are altered by the aforementioned treatment.

For example, regions A and B can be formed on the culture medium by creating a sheet with holes of the shape and size of region A using a template printed by a 3D printer, covering the surface of the culture medium with this sheet, coating the uncovered surface with a cell-adhesive material, and then removing the sheet. Alternatively, a sheet of the shape and size of region A can be created and placed on the surface of the culture medium, the uncovered surface can be coated with a cell-non-adhesive material, the sheet can be removed, and the covered surface can be coated with a cell-adhesive material, thus forming regions A and B on the culture medium. The sheet can be formed from biocompatible materials such as polydimethylsiloxane (PDMS), polyethylene glycol hydrogel, or agarose gel. Coating can be performed, for example, by contacting the culture medium with a solution of a cell-adhesive or cell-non-adhesive material and reacting at 37°C or room temperature for the required time (e.g., more than 1 hour). The concentrations of cell-adhesive and cell-nonadhesive substances can be appropriately determined by those skilled in the art; for example, in the case of laminin, they can be 0.1–1 μg/ ^cm² , 0.1–0.5 μg/ ^cm² , or about 0.25 μg/ ^cm² . Alternatively, without using sheets as described above, regions A and B can be formed on the culture medium by forming droplets of the cell-adhesive substance solution on the medium and allowing them to react.

In this invention, the culture medium may have a protrusion (also called a pillar) on its surface, and the upper surface of the protrusion may have a region A. In this case, the side of the protrusion adjacent to the upper surface of the protrusion is designated as region B. The shape of the protrusion is not particularly limited and may be cylindrical or prismatic. In this specification, the cross-section of a cylinder is considered substantially circular in the art, and is used to mean a cylinder with a roughly circular cross-section including a perfect circle. In one embodiment, the protrusion is cylindrical. The height of the protrusion is not limited, and may be, for example, 0.1 mm to 10 mm, 0.1 mm to 5 mm, 1 mm to 5 mm, or 3 mm to 5 mm, for example, 4 mm. The material of the protrusion may be the same as or different from that of the culture medium. In addition to the materials used as culture media as illustrated in this specification, other materials that can be used as protrusions include polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), polymethylglutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), vinyl polyethylene acetate (PEVA), and polyethylene oxide (PEO).

Cells can be seeded only in region A, or they can be seeded onto the entire culture medium including regions A and B. For example, if region B is a non-adhesive region, it can be seeded onto the entire culture medium. In one approach, pluripotent stem cells are seeded onto the culture medium with a cell count comprising more than 70% of the cells in region A. In another approach, pluripotent stem cells are selected at concentrations of 0.5× ^10⁵ , 0.6× ^10⁵ , 0.7× ^10⁵ , 0.8× ^10⁵ , 0.9× ^10⁵ , 1.0× ^10⁵ , 1.1× ^10⁵ , 1.2×10⁵, ^{1.3×10⁵ ,} 1.4× ^10⁵ , 1.5× ^10⁵ , 1.6×10⁵, 1.7× ^10⁵ , 1.8× ^10⁵ , 1.9× ^10⁵ , 2.0× ^10⁵ , 2.1× ^10⁵ , 2.2× ^10⁵ , 2.3× ^10⁵ , or ^2.4 × ^10⁵ cells/cm² ^or higher, and at concentrations of 2.5× ^10⁵ , 2.4× ^10⁵ , or 2.3× ^10⁵. 2.2× ^10⁵ , 2.1× ^10⁵ , 2.0×10⁵, 1.9× ^10⁵ , 1.8× ^10⁵ , 1.7× ^10⁵ , 1.6× ^10⁵ , 1.5× ^10⁵ , 1.4× ^10⁵ , 1.3×10⁵, 1.2× ^10⁵ , 1.1× ^10⁵ , 1.0 ^{× 10⁵} , 0.9× ^10⁵ , 0.8× ^10⁵ , 0.7× ^10⁵ or 0.6× ^10⁵ cells/ ^{cm² or} less (upper and lower limits can be selected independently) are inoculated onto the culture medium. For example, pluripotent stem cells can be seeded at concentrations of 0.5 × ^10⁵ –2.5 × ^10⁵ cells/ ^cm² , 1.0 × ^10⁵ –2.5 × ^10⁵ cells/ ^cm² , 1.5 × ^10⁵ –2.5 × ^10⁵ cells/ ^cm² , 0.5 × ^10⁵ –2.0 × ^10⁵ cells/ ^cm² , 1.0 × ^10⁵ –2.0 × ^10⁵ cells/cm², or 0.5 × ^10⁵ –1.5 × ^10⁵ cells/ ^cm² . Typically, pluripotent stem cells are recovered and dispersed using a cell dispersion containing enzymes (e.g., trypsin, collagenase, hyaluronidase, elastase, streptomycin, DNase, papain) and/or a chelating agent (e.g., ethylenediaminetetraacetic acid (EDTA)) and suspended in the desired culture medium. The resulting cell suspension is then added to the culture medium. As a cell dispersion, commercially available products such as TrypLE ^™ Select (Thermo Fisher Scientific) and TrypLE ^™ Express (Thermo Fisher Scientific) can be used.

Differentiation induction of pluripotent stem cells into retinal lineage cells can be performed via adhesion culture on the aforementioned culture medium. After seeding pluripotent stem cells onto the culture medium, and culturing them in a medium containing undifferentiated maintenance factor for a certain period of time, differentiation induction into retinal lineage cells can begin. When documenting the production of retinal lineage cells as the starting cells, the moment when culture begins in a medium without undifferentiated maintenance factor is defined as the moment when differentiation induction into retinal lineage cells begins.

In this invention, the differentiation induction of pluripotent stem cells into retinal cells includes the step of culturing pluripotent stem cells in a culture medium containing substances acting on the BMP signaling pathway. Culture in the culture medium containing substances acting on the BMP signaling pathway can begin at the start of differentiation induction or some time after the start of differentiation induction (e.g., 4–6 days later).

The induction of pluripotent stem cell differentiation into retinal cells may include the following steps: culturing pluripotent stem cells in a medium containing BMP signaling pathway inhibitors before culturing them in a medium containing BMP signaling pathway inhibitors. Culturing cells in the presence of BMP signaling pathway inhibitors before adding BMP signaling pathway inhibitors can improve the differentiation efficiency into retinal tissue.

The culture medium used for inducing differentiation of pluripotent stem cells into retinal cells can be prepared using the medium commonly used for culturing animal cells as the basal medium. Examples of basal media include, for example, IMDM medium, DMEM medium, F-12 medium, DMEM/F12 medium, IMDM/F12 medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, Medium 199 medium, Eagle MEM medium, αMEM medium, ham medium, RPMI 1640 medium, Fischer’s medium, or mixtures thereof.

Culture media can be prepared by adding one or more components selected from serum, serum substitutes, growth factors, undifferentiated maintenance factors, proteins (e.g., cytokines, insulin), fatty acids, lipids, vitamins, amino acids (e.g., non-essential amino acids, retinoids, glutamine, taurine), antioxidants, 2-mercaptoethanol, 1-thioglycerol, antibiotics, buffers, and inorganic salts to a basal medium as needed. Examples of serum substitutes include albumins such as bovine serum albumin (BSA), transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 1-thioglycerol, and their equivalents, as well as mixtures thereof. The culture medium can contain commercially available serum substitutes such as KnockOut ^™ serum substitute (Thermo Fisher Scientific), chemically defined lipid concentrate (Thermo Fisher Scientific), GlutaMAX ^™ supplement (Thermo Fisher Scientific), Ham's F-12 Nutrient Mix, GlutaMAX ^™ supplement (Thermo Fisher Scientific), B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and ITS supplement (Thermo Fisher Scientific). The culture medium can be a commercially available product with the above-mentioned components added to a basal medium, such as Ham's F-12 Nutrient Mix, GlutaMAX ^™ supplement (Thermo Fisher Scientific), DMEM/F-12, and GlutaMAX ^™ supplement (Thermo Fisher Scientific).

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

Differentiation induction is preferably performed in the absence of feeder cells (also known as feeder-free conditions). Differentiation induction is also preferably performed under conditions free of heterologous components. In this invention, "free of heterologous components" means the absence of components from biological species different from those of the cultured cells.

Examples of undifferentiated maintenance factors include substances acting on the FGF signaling pathway, substances acting on the TGFβ family signaling pathway, and insulin. Examples of substances acting on the FGF signaling pathway include FGF (e.g., bFGF, FGF4, FGF8). Examples of substances acting on the FGF signaling pathway and 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. In the case of human pluripotent stem cells, bFGF is preferably included as an undifferentiated maintenance factor. The concentration of the undifferentiated maintenance factor should be such that it can maintain the undifferentiated state of pluripotent stem cells. It can be set appropriately by those skilled in the art. For example, when bFGF is used in the culture of human pluripotent stem cells, the concentration of bFGF can be 4 ng to 500 ng/mL, 10 ng to 200 ng/mL, or 30 ng to 150 ng/mL. As a culture medium containing undifferentiated maintenance factors, one can use, for example, StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.), S-medium (DS Pharma Biomedical Co.), StemPro ^™ (ThermoFisher Scientific), mTeSR1 ^™ (STEMCELL Technologies), mTeSR2 ^™ (STEMCELL Technologies), TeSR ^™ -E8 ^™ (STEMCELL Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9; 105(36): 13409-14). In one embodiment, the culture medium containing undifferentiated maintenance factors is StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.).

There are no restrictions on the culture time in a medium containing undifferentiated maintenance factors, for example, 1–10 days, 1–9 days, 1–8 days, 1–7 days, 1–6 days, 1–5 days, 1–4 days, 1–3 days, or 2–3 days.

Pluripotent stem cells are cultured in a medium containing a ROCK inhibitor before culturing in a medium containing substances acting on the BMP signaling pathway. The ROCK inhibitor can be added to the medium containing undifferentiated maintenance factors. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. The concentration of the ROCK inhibitor can be appropriately determined depending on the type of inhibitor, for example, 1–100 μM, 5–50 μM, or about 10 μM, or a concentration that shows equivalent activity to Y-27632 at the above concentrations. In one method, the culture time in the medium containing ROCK inhibitor is 1 hour to 16 hours, 1 hour to 14 hours, 1 hour to 12 hours, 1 hour to 10 hours, 1 hour to 8 hours, 1 hour to 6 hours, 1 hour to 4 hours, 1 hour to 3 hours, 1 hour to 2 hours, 2 hours to 16 hours, 2 hours to 14 hours, 2 hours to 12 hours, 2 hours to 10 hours, 2 hours to 8 hours, 2 hours to 6 hours, 2 hours to 4 hours, or 2 hours to 3 hours. Preferably, the culture time in the medium containing ROCK inhibitor is 1 hour to 3 hours. The culture time in the medium containing undifferentiated maintenance factor and ROCK inhibitor can be part or all of the culture time in the medium containing undifferentiated maintenance factor. For example, after culturing for 1 hour to 16 hours in a medium containing undifferentiated maintenance factor and ROCK inhibitor, culture can be carried out in a medium containing undifferentiated maintenance factor but without ROCK inhibitor until the culture time in the medium containing undifferentiated maintenance factor reaches the specified culture time.

BMPs (bone morphogenetic proteins) include, for example, BMP2, BMP4, BMP7, and BMP12 (GDF7). BMP signaling pathway agonists and BMP signaling pathway inhibitors can be one or more agonists or inhibitors targeting these BMPs.

BMP signal transduction pathway inhibitors are substances that inhibit BMP-mediated signal transduction, including substances that act on BMP or its receptors, substances that inhibit gene expression of BMP or its receptors, and substances that inhibit the binding of BMP to its receptors. Examples of BMP signal transduction pathway inhibitors include LDN-193189 (4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), Dorsomorphin (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidinyl), and DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline). In one embodiment, the BMP signal transduction pathway inhibitor is LDN-193189. The concentration of BMP signal transduction pathway inhibitors can be appropriately determined according to the type of inhibitor, for example, 1–1000 nM, 10–500 nM, 30–300 nM or about 100 nM, or a concentration that shows equivalent activity to the above concentrations of LDN-193189.

There are no restrictions on the culture time in the medium containing BMP signal inhibitors, for example, 1–10 days, 2–9 days, 3–7 days, 4–6 days or about 5 days.

BMP signaling pathway active substances refer to substances that enhance BMP-mediated signal transduction, including substances acting on BMP or its receptor, substances enhancing gene expression of BMP or its receptor, and substances promoting the binding of BMP to its receptor. Examples of BMP signaling pathway active substances include BMP2, BMP4, BMP7, BMP12 (GDF7), fragments of these, or anti-BMP receptor antibodies. In one embodiment, the BMP signaling pathway active substance is BMP4. The concentration of the BMP signaling pathway active substance can be appropriately determined depending on the type of active substance, for example, 0.01–1000 nM, 0.1–100 nM, 1–10 nM, 1–3 nM, or about 1.5 nM, or a concentration showing equivalent activity to the above-mentioned concentrations of BMP4. In one approach, the concentration of the substance acting on the BMP signaling pathway is 3–15 nM or 3–12 nM, for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nM, or a concentration that shows equivalent activity to BMP4 at the above concentrations.

Culture in a medium containing BMP signaling pathway active substances is performed for the time required for differentiation into retinal cells. The culture time is not limited, but can be, for example, 1–30 days, 2–20 days, 3–15 days, 4–12 days, or 5–10 days (e.g., 5, 6, 7, 8, 9, or 10 days). The concentration of BMP signaling pathway active substances can be fixed or varied during the culture time. For example, the concentration of BMP signaling pathway active substances can be reduced in stages by decreasing it by 40–60% every 2–4 days. For example, after the start of culture in a medium containing BMP signaling pathway active substances, a portion (e.g., half the volume) of the medium can be replaced with a medium without BMP signaling pathway active substances every 2, 3, or 4 days, thereby gradually reducing the concentration of BMP signaling pathway active substances in the medium.

In one embodiment, the culture medium containing BMP signaling pathway inhibitors or BMP signaling pathway activators is a medium in which KnockOut ^™ serum substitute (ThermoFisher Scientific) (e.g., 0.5%–30%, 1%–20%, or 10%), a chemically defined lipid concentrate (ThermoFisher Scientific), BSA, and 1-thioglycerol are added to a 1:1 mixture of F-12 medium and IMDM medium.

After culturing in a medium containing substances that act on the BMP signal transduction pathway, the medium can be replaced with one that does not contain substances that act on the BMP signal transduction pathway, and culturing can continue. There is no limit to the culturing time; for example, it can be 1–100 days, 10–90 days, 20–80 days, 30–70 days, 40–60 days, or about 50 days.

During part or all of the culture time following culturing in a medium containing BMP signaling agents, cells can be cultured in a medium containing Wnt signaling pathway inhibitors. Wnt signaling pathway inhibitors are substances that inhibit Wnt-mediated signal transduction, including substances acting on Wnt or its receptors, substances inhibiting gene expression of Wnt or its receptors, and substances inhibiting the binding of Wnt to its receptors. Examples of Wnt signaling pathway inhibitors include CKI-7 (N-(2-aminoethyl)-5-chloro-8-isoquinoline sulfonamide) and D4476 (4-(4-(2,3-dihydrobenzo[1,4]di)). Examples of inhibitors of the Wnt signaling pathway include (e.g., 1-(6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzamide), IWR-1-endo(IWR1e)(4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methylbridge-2H-isoindol-2-yl]-N-8-quinolinylbenzamide), and IWP-2(N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthiopheno[3,2-d]pyrimidin-2-yl)thio]-acetamide). The concentration of inhibitors in the Wnt signaling pathway is determined appropriately based on the type of inhibitor, for example, 0.1–100 μM, 0.3–30 μM, or 1 μM–10 μM.

During the incubation period, the culture medium can be exchanged appropriately. For example, part or all of the culture medium can be exchanged every 1 to 4 days. When starting in a medium containing a specific component, all the medium can be replaced with a medium containing that component at the desired concentration, or a portion of the medium can be replaced in such a way that the component reaches the desired final concentration (for example, half the volume of medium can be replaced with a medium containing twice the final concentration). Furthermore, as described for substances acting in the BMP signal transduction pathway, the concentration of that component can be fixed or varied during the incubation period in a medium containing that specific component.

Culture conditions, such as culture temperature and _CO2 concentration, can be set appropriately. For example, the culture temperature can be 30℃–40℃ or approximately 37℃. The _CO2 concentration can be, for example, 1%–10% or approximately 5%.

Differentiation into retinal tissue can be confirmed by detecting the expression of neuroretinal cell markers in the cells of the tissue. These neuroretinal cell markers are as described above. Marker expression can be confirmed, for example, between days 10 and 100 of differentiation induction (e.g., days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100) or thereafter. In one approach, marker expression can be confirmed between days 16 and 22 of differentiation induction.

The retinal tissue obtained using the method described in this specification as the starting cells can also be called a retinal sheet, which usually contains retinal progenitor cells and is also referred to as a "retinal progenitor cell sheet" in this specification. In one embodiment, the proportion of Chx10-positive cells or the proportion of Chx10-positive and Rx-positive cells in the retinal tissue (retinal sheet) as the starting cells is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. The proportion of the above-mentioned marker-positive cells can be the proportion at or after days 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 of differentiation induction. In one approach, the proportion of Chx10 and Rx positive cells in retinal tissue is 70% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher at days 16–22 (e.g., days 16, 18, or 20) during differentiation induction. In another approach, the proportion of Chx10 and Rx positive cells in retinal tissue is 90% or higher or 95% or higher at days 16–22 (e.g., days 16, 18, or 20) during differentiation induction.

In one embodiment, the proportion of neuroretinal cells in the retinal tissue (retinal sheet) serving as the initiating cells is 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. This cell proportion can be the proportion observed from day 16 to 100 of differentiation induction (e.g., day 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100) or thereafter. The retinal tissue preferably has a cambium structure.

The thickness of the retinal tissue (retinal sheet) used as the starting cells can be 10, 20, 30, 40, 50, 60, 70, 80, 90 or more than 100 μm, or 500, 400, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210 or less than 220 μm (the upper and lower limits can be selected independently). In one embodiment, the thickness of the retinal tissue is 50 μm to 300 μm. The retinal tissue does not need to be of uniform thickness; when specifying a thickness range for the retinal tissue, it is acceptable as long as the thickness at any location of the retinal tissue falls within that range. In the retinal tissue (retinal sheet) used as the starting cells, the number of cells perpendicular to the culture medium is usually about 30 or less, or the thickness is less than 500 μm.

In one approach, retinal tissue (retinal sheet) serving as the starting cells is obtained through a method comprising the following steps:

(1) Pluripotent stem cells are seeded at a density of 0.5× ^10⁵ cells/ ^cm² to 2.5× ^10⁵ cells/ ^cm² on a culture medium having a region A on the surface with cell adhesion and a region B adjacent to at least a portion of the region A with lower cell adhesion.

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

(3) The cells obtained after step (2) are cultured in a culture medium containing substances that act on the BMP signal transduction pathway to obtain retinal tissue (retinal sheet).

According to this method, retinal tissue with few pores and gaps (areas without cells) and a uniform structure can be created in region A. In one embodiment, the retinal tissue (retinal sheet) used as the starting cell covers more than 95%, 96%, 97%, 98%, or 99% of region A. The method for determining the proportion of region A covered by retinal tissue is not particularly limited; for example, it can be determined by staining the retinal tissue. For example, the presence or absence of expression of the aforementioned neuroretinal cell marker (e.g., Chx10) can be determined by staining (e.g., immunostaining using a fluorescently labeled anti-Chx10 antibody). Then, the area of the neuroretinal cell marker-positive region relative to the area of region A can be calculated using, for example, imaging software (e.g., ImageJ software (NIH)). Furthermore, according to this method, retinal tissue with a low proportion of non-target cells and a high proportion of retinal cells can be efficiently created. Specifically, retinal tissue with high expression levels of genes expressed in neuroretinal cells and low expression levels of genes expressed in non-target cells can be created. Neuroretinal cells and their markers are as described above. As non-target cells, examples include ocular-related cells (e.g., ciliary body, lens, retinal pigment epithelium, etc.) and brain and spinal cord-related cells (teloencephalon, midbrain, spinal cord, etc.), the markers of which are as described above.

In one approach, retinal tissue (retinal sheet) serving as the starting cells is obtained through a method comprising the following steps:

(1) Pluripotent stem cells are seeded at a density of 0.5× ^10⁵ cells/ ^cm² to 2.5× ^10⁵ cells/ ^cm² on a culture medium having a region A on the surface with cell adhesion and a region B adjacent to at least a portion of the region A with lower cell adhesion.

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

(3) The cells obtained after step (2) are cultured in a culture medium containing substances that act on the BMP signal transduction pathway to obtain retinal tissue (retinal slices).

The culture medium in step (2) contains undifferentiated maintenance factor, while the culture medium in step (3) does not contain undifferentiated maintenance factor.

The method further includes the following steps: after step (2) and before step (3), culturing cells in a medium containing undifferentiated maintenance factor and without ROCK inhibitor, and then culturing cells in a medium without undifferentiated maintenance factor and ROCK inhibitor.

In one approach, retinal tissue (retinal sheet) serving as the starting cells is obtained through a method comprising the following steps:

(1) Pluripotent stem cells are seeded at a density of 0.5× ^10⁵ cells/ ^cm² to 2.5× ^10⁵ cells/ ^cm² on a culture medium having a region A on the surface with cell adhesion and a region B adjacent to at least a portion of the region A with lower cell adhesion.

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

(3) The cells obtained after step (2) are cultured in a culture medium containing substances that act on the BMP signal transduction pathway to obtain retinal tissue (retinal sheet).

The culture medium in step (2) contains undifferentiated maintenance factor, while the culture medium in step (3) does not contain undifferentiated maintenance factor.

Step (3) begins 5 to 10 days after vaccination, starting from day 6 to 8 following step (1).

The method further includes the following steps: after step (2) and before step (3), culturing cells in a medium containing undifferentiated maintenance factor and without ROCK inhibitor until day 1 to 3 after inoculation in step (1), and then culturing cells in a medium without undifferentiated maintenance factor and ROCK inhibitor until day 6 to 8 after inoculation in step (1).

The retinal tissue (retinal sheets) obtained in steps (1) to (3) is dispersed as described later. The retinal tissue (retinal sheets) can be dispersed directly without being recovered from the culture medium, or it can be dispersed after recovery from the culture medium. The retinal tissue (retinal sheets) can be recovered from the culture medium using conventional methods. For example, the retinal tissue can be recovered using instruments such as forceps. Alternatively, if the culture medium is coated with a stimulus-responsive polymer (e.g., a temperature-responsive polymer or a photoresponsive polymer), the retinal tissue can be recovered by applying a corresponding stimulus. For example, if a temperature-responsive polymer is used, whose properties reversibly change from cell-adhesive (hydrophobic) to cell-non-adhesive (hydrophilic) at a specified temperature, the retinal tissue can be recovered by maintaining the culture medium at that specified temperature.

(Dispersed retinal cell population)

"Dispersion" refers to the separation of cells or tissues into small cell sheets or cell clumps (2 to 100 cells, preferably 50, 30, 20, 10, or 5 cells; for example, a clump of 2 to 5 cells) or single cells through dispersion treatments such as enzyme treatment or physical treatment. A dispersed cell population refers to a substance composed of a certain number of cell sheets, cell clumps, or single cells. "Dispersed retinal cell population" refers to a dispersed cell population, which can be obtained by dispersing cell clumps of organisms, tissues, or cell aggregates. A dispersed retinal cell population is preferably obtained by dispersing the aforementioned retinal cell aggregates.

In one approach, the dispersion method can be any method capable of dispersing cells in a viable state, such as mechanical dispersion treatment, cell dispersion treatment, and treatment with the addition of a cell protectant. These treatments can also be combined. Preferably, cell dispersion treatment is performed followed by mechanical dispersion treatment.

Methods of mechanical dispersion include blowing or scraping with a scraper.

Cell dispersions used in cell dispersion treatment can include, for example, solutions containing any one of the following enzymes: trypsin, collagenase, hyaluronidase, elastase, streptokinase, DNase, papain, etc., and chelating agents such as EDTA. Commercially available cell dispersions can be used, such as TrypLE Select (manufactured by Life Technologies), TrypLE Express (manufactured by Life Technologies), and nerve cell dispersion (FujiFilm).

Cell death can be inhibited by treating cells with a cell protectant during dispersion. Examples of cell protectants used in this treatment include substances acting on the FGF signaling pathway, heparin, substances acting on the IGF signaling pathway, serum, or serum substitutes. Additionally, to inhibit dispersion-induced cell death (especially in human pluripotent stem cells), inhibitors of Rho-associated coiled-coil containing protein kinase (“ROCK” or “Rho kinase”) or myosin inhibitors can be added during dispersion. Examples of ROCK inhibitors include Y-27632, fasudil (HA1077), and H-1152. Examples of myosin inhibitors include bupirocin. ROCK inhibitors are preferred cell protectants.

The dispersed retinal cell population can be characterized by the following states: containing single cells, for example, 70% or more, preferably 80% or more, of the total number of cells in the population are single cells; and having clumps of 2 to 50 cells, representing 30% or less, preferably 20% or less, of the total number of cells in the population. In the dispersed retinal cell population, cell adhesion (e.g., surface adhesion) is essentially absent. Furthermore, the dispersed retinal cell population is more preferably composed of single cells as much as possible, and such a dispersed retinal cell population can be obtained by removing clumps of cells that have not become single cells after the dispersion treatment. The method for removing cell clumps is not particularly limited, and examples include removing clumps through a filter membrane (cell filter). In one embodiment, the dispersed cells can be characterized by the substantial absence of cell-cell connections (e.g., adhesive connections).

When preparing a retinal cell line population as starting cells from retinal tissue, the retinal tissue sometimes contains unwanted cells such as retinal pigment epithelial cells. In this case, the area containing unwanted cells such as retinal pigment epithelial cells can be removed, and then the retinal cell line population can be dispersed. In the case of retinal pigment epithelial cells, they can be identified by morphology and pigmentation, and can be easily removed by those skilled in the art.

Before culturing in the culture medium containing substances involved in the Wnt signal transduction pathway (described later), a step to improve the purity of retinal progenitor cells or neuroretinal progenitor cells (purification step) can be performed as described later, thereby enabling the maintenance and expansion culture of the dispersed retinal cell line population. The culture medium is not particularly limited as long as it is a medium in which retinal cells can survive and proliferate (DMEM medium, etc.). Since freeze-thawed retinal cell line populations can also be used as starting cells in the manufacturing method of the present invention, the dispersed retinal cell line populations can also be cryopreserved. The cryopreservation solution is not particularly limited, and commercially available cryopreservation solutions can be used.

Purification of retinal precursor cells

Before culturing the dispersed retinal cell line population in a culture medium containing substances acting on the Wnt signal transduction pathway, a purification step (increasing the proportion (purity) of retinal precursor cells, preferably the proportion (purity) of neural retinal precursor cells, can be performed on the dispersed retinal cell line population. In the purification step, cell sorting and other operations can be performed using specific markers expressed in retinal precursor cells and/or neural retinal precursor cells. Cell sorting can be performed using known techniques such as FACS and MACS. By performing the purification step of retinal precursor cells and/or neural retinal precursor cells, the contamination of RPE cells and non-target cells in the manufacturing method described in this specification can be reduced. By increasing the proportion of retinal precursor cells in the dispersed retinal cell line population, the generation of retinal pigment epithelial (RPE) precursor cells and/or RPE cells can be inhibited. Whether the generation of RPE precursor cells and/or RPE cells has been inhibited can be determined after culturing the dispersed retinal cell line population using the culture method described later, based on the markers, morphology, and properties of the aforementioned RPE cells. Inhibition of the generation of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells can mean that, compared to the absence of steps to increase the proportion of retinal precursor cells, the proportion of retinal pigment epithelial cells relative to the total number of cells after the above culture is inhibited. If the proportion of retinal pigment epithelial cells is at the level described in paragraph 0151, it can be determined that the generation of retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells is inhibited.

Rx and Chx10 are well-known positive markers for retinal progenitor cells. However, these genes are expressed intracellularly, thus requiring the use of cells that link these genes to fluorescent proteins using gene recombination technology or cells where these genes have been replaced with fluorescent proteins (e.g., Rx::Venus cells). Therefore, positive markers for retinal progenitor cells and/or neural retinal progenitor cells have been explored, and 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), and CD29 (Genbank ID: NM_001291737.1, NM_001291738.1, NM_001291739.1, NM_001359084.1, NM_013230.3), and CD29 (Genbank ID: NM_001291738.1, NM_001291739.1, NM_001359084.1, NM_013230.3) have been identified. ID: NM＿002211.4, NM_033668.2, NM_133376.2), CD39 (GenbankID: NM_001098175 .2, NM_001164178.1, NM_001164179.2, NM_001164181.1, NM_001164182.2, NM_00 1164183.2, NM＿001312654.1, NM_001320916.1, NM_001776.6), CD47(GenbankID : NM＿001777.3, NM_198793.2), CD49b (GenbankID: 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 (GenbankID: NM _001367973.1, NM_018644.3, NM_054025.3), CD73 (GenbankID: NM_001204813.1, NM_002526.4), CD82 (Genbank ID: NM＿001024844.2, NM_002231.4), CD90 (GenbankID: 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_001365852.1, NM_001365853.1, NM_001365854.1, NM_ 001365855.1, NM_005944.7), CD340 (GenbankID: NM＿001005862.2, NM_0012 89936.1, 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) are expressed on the cell surface of these cells. Preferred cell surface markers include, for example, CD9, CD39, CD90, and CXCR4. As an alternative, a method for increasing the proportion of retinal precursor cells in the cell population can be performed before initiating culture in the aforementioned culture medium containing substances acting on the Wnt signal transduction pathway. This method includes the steps of contacting the aforementioned cell population containing retinal precursor cells with a substance (e.g., antibody, peptide, etc.) that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, and then separating the positive fraction. This method reduces the contamination of RPE cells in the manufacturing method described in this specification.

Furthermore, SSEA1 (Genbank ID: NM_002033.3), CD66b (Genbank ID: NM_001816.4), CD69 (Genbank ID: NM_001781.2), and CD84 (Genbank ID: NM_001184879.2, NM_001184881.2, NM_001184882.1, NM_001330742.2, NM_003874.4) were identified as negative markers for these cells, i.e., markers not expressed in these cells. Combining these markers with the aforementioned positive markers can further improve the purity of retinal progenitor cells and/or neural retinal progenitor cells. As one approach, a method for increasing the proportion of retinal progenitor cells in a cell population can be implemented before initiating culturing in a culture medium containing substances that act on the Wnt signal transduction pathway. This method includes the steps of: 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, also contacting it 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 positive fractions of the former markers from negative fractions of the latter markers. 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 negative fractions of these markers is a step to obtain a cell population where the expression level of the antigen is below a baseline value. Here, the baseline value can be arbitrarily set by those skilled in the art. For example, by obtaining a cell population treated with a fluorescently labeled antibody against the aforementioned antigen, which has the same fluorescence intensity as the cell population treated with a fluorescently labeled isotype control antibody, a negative fraction can be obtained.

It should be noted that the method described above for using substances (e.g., antibodies) that bind to positive and negative markers to increase the proportion of retinal precursor cells and/or neuroretinal precursor cells in the cell population is not limited to the method described as a step in the manufacturing process described herein. The method of using cell surface markers to increase the proportion of retinal precursor cells and/or neuroretinal precursor cells may also be used as a step in other methods for manufacturing retinal tissues, for example.

In one embodiment, in the dispersed retinal cell population, retinal progenitor cells and/or neuroretinal progenitor cells may, for example, constitute more than 30% of the total number of cells, preferably more than 50% of the total number of cells, more preferably more than 80% of the total number of cells, and even more preferably more than 90% of the total number of cells. Furthermore, the dispersed retinal cell population may contain, for example, more than 50%, preferably more than 80%, more than 85%, more than 90%, or more than 95% of cells (retinal progenitor cells or neuroretinal 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, relative to the total number of cells in the cell population. Furthermore, the aforementioned cell population is preferably negative for one or more, preferably two or more, three or more, or all of the antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84. The term "negative" here simply means below the aforementioned reference values.

(Reconstructing the lamina structure by culturing dispersed retinal cell populations)

The method for manufacturing retinal tissue according to the present invention includes the step of adhesioculturing or suspension culturing a dispersed retinal cell line population in a culture medium containing substances acting on the Wnt signal transduction pathway. Either culture method allows the dispersed retinal cell line population to reform an epithelial structure (multilayer structure). Adhesioculturing is preferred for manufacturing sheet-like retinal tissue with an epithelial structure (or multilayer structure).

The culture medium used for culturing dispersed retinal cell populations is not particularly limited as long as it allows retinal cells to survive and proliferate. One approach is to use a culture medium for maintaining continuous epithelial tissue as the medium for culturing dispersed retinal cell populations. For example, a culture medium containing Neurobasal medium (e.g., Thermo Fisher Scientific, 21103049) supplemented with B27 (e.g., Thermo Fisher Scientific, 12587010) can be used as a medium for maintaining continuous epithelial tissue.

As a substance that acts on the Wnt signal transduction pathway in the culture medium, there are no particular limitations as long as it can enhance Wnt-mediated signal transduction. Specific examples of substances acting on the Wnt signal transduction pathway include, for instance, GSK3β inhibitors (e.g., 6-indirubin-3'-oxime (BIO), CHIR99021, kamparone, Wnt2b, Wnt3a, and some peptides thereof, which can be one or more substances). Preferably, the substance acting on the Wnt signal transduction pathway is one or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b, and Wnt3a. Substances acting on the Wnt signal transduction pathway can increase the size of aggregates and reform polar epithelial structures (or multilayer structures) with apical/basement membranes during the reaggregation of dispersed retinal cell populations. Substances acting on the Wnt signal transduction pathway can also inhibit the formation of rosette-like structures.

The concentration of the substance acting on the Wnt signaling pathway is sufficient to induce the formation of the desired cell aggregates (e.g., the reformation of epithelial structures (or multilayer structures)). For example, when using CHIR99021, the concentration can be 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 10 μM, and even more preferably 3 μM to 6 μM. When using other substances acting on the Wnt signaling pathway, the concentration is sufficient to exhibit the same level of Wnt signal activation as the aforementioned concentration of CHIR99021. Those skilled in the art can determine Wnt signal activation, for example, by confirming the expression of β-catenin.

There is no particular limitation on the timing of adding the Wnt signaling pathway active ingredient, but it is preferable to add it as early as possible after the start of suspension or adhesion culture for re-sheet formation. Alternatively, it is preferable to culture in a medium containing the Wnt signaling pathway active ingredient from the start of the culture. The number of days of culture in the medium containing the Wnt signaling pathway active ingredient is not particularly limited, as long as the effect of re-formation of polar epithelial structures (or multilayer structures) with apical/basement membranes can be observed, for example, from 1 to 14 days.

In one embodiment, the culture medium may further contain one or more substances selected from the group consisting of ROCK inhibitors, substances acting on the SHH (sound hedgehog) signaling pathway, and substances acting on the fibroblast growth factor (FGF) signaling pathway. For example, by adding ROCK inhibitors, the effect of promoting the reaggregation of dispersed retinal progenitor cell populations can be observed; by adding substances acting on the SHH signaling pathway, the effect of promoting the proliferation of cell aggregates and increasing the size of cell aggregates can be observed; and by adding substances acting on the FGF signaling pathway (e.g., FGF2, FGF8), the effect of reducing cell damage and inhibiting the induction of differentiation into RPE cells can be observed.

ROCK inhibitors are not specifically limited to any substance that can inhibit the function of Rho kinase (ROCK). Examples include Y-27632 (see Ishizaki et al., Mol. Pharmacol. 57, 976-983 (2000); Narumiya et al., Methods Enzymol. 325, 273-284 (2000)), fasudil/HA1077 (see Uenata et al., Nature 389: 990-994 (1997)), H-1152 (see Sasaki et al., Pharmacol. Ther. 93: 225-232 (2002)), and Wf-536 (see Nakajima et al., Cancer Chemother). Pharmacol. 52(4):319-324(2003)) and their derivatives, as well as antisense nucleic acids against ROCK, RNA interference-inducible nucleic acids (e.g. siRNA), dominant-negative mutants and their expression vectors. In addition, other low-molecular-weight compounds are known as ROCK inhibitors, and therefore such compounds or their derivatives may also be used in this invention (e.g., see U.S. Patent Application Publication No. 20050209261, U.S. Patent Application Publication No. 20050192304, U.S. Patent Application Publication No. 20040014755, U.S. Patent Application Publication No. 20040002508, U.S. Patent Application Publication No. 20040002507, U.S. Patent Application Publication No. 20030125344, U.S. Patent Application Publication No. 20030087919, and International Publications Nos. 2003/062227, 2003/059913, 2003/062225, 2002/076976, and 2004/039796). One or more ROCK inhibitors may be used. The ROCK inhibitor preferably contains one or more substances selected from the group consisting of Y-27632, fasudil (HA1077), and H-1152.

The concentration of the ROCK inhibitor can be appropriately set by those skilled in the art based on experimental conditions. As a general rule, any concentration that promotes the reaggregation of dispersed retinal progenitor cell populations is acceptable. For example, when using Y-27632, 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 using a ROCK inhibitor other than Y-27632, any concentration that exhibits the same degree of ROCK inhibitory effect as the aforementioned concentration of Y-27632 is acceptable. Those skilled in the art can determine the ROCK inhibitory effect, for example, through methods such as phosphorylation expression analysis of MLC2.

Substances acting in the SHH (Sound Hedgehog Factor) signal transduction pathway are substances that enhance SHH-mediated signal transduction. Examples of substances acting in the SHH signal transduction pathway include proteins belonging to the hedgehog protein family (e.g., Shh, Ihh), SHH receptors, Shh receptor agonists, PMA (Purmorphamine; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthoxy)-9H-purine-6-amine), or SAG (Smoothened Agonist; N-methyl-N'-(3-pyridylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane), etc. Substances acting in the Shh signal transduction pathway may contain one or more of these. The active substance in the Shh signal transduction pathway is preferably one or more substances selected from the group consisting of Shh (Genbank accession number: NM_000193, NP_000184), SAG, or PMA.

The concentration of substances acting on the SHH signaling pathway can be appropriately set by those skilled in the art based on experimental conditions. As a general rule, any concentration within the range where an increase in cell aggregates can be observed is acceptable. For example, SAG is typically used at concentrations of 1–2000 nM, preferably 10–700 nM. PMA is typically used at concentrations of 0.002–20 μM, preferably 0.02–2 μM. SHH is typically used at concentrations of 4–500 ng/mL, preferably 10–200 ng/mL. When using other substances acting on the SHH signaling pathway, any concentration that demonstrates the same level of SHH signal activation as the aforementioned concentrations of SAG is acceptable. Those skilled in the art can determine SHH signal activation, for example, through methods such as downstream signal (SMO, GLI) expression analysis.

Substances acting on the FGF signal transduction pathway are not particularly limited as long as they can enhance FGF-mediated signal transduction. Specifically, fibroblast growth factors (e.g., bFGF, FGF4, FGF8, and FGF9) can be listed as substances acting on the FGF signal transduction pathway. Preferably, substances acting on the FGF signal transduction pathway are one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4, and FGF8.

The concentration of substances acting on the FGF signaling pathway can be appropriately set by those skilled in the art based on experimental conditions. As a general rule, any concentration within the range where an inhibitory effect on differentiation-inducing into RPE cells can be observed is acceptable. For example, when using FGF2, 4, or 8, a concentration of approximately 4–500 ng/mL is preferred, approximately 10–200 ng/mL is more preferably approximately 25–100 ng/mL. When using other substances acting on the FGF signaling pathway, any concentration that exhibits the same level of FGF signal activation as the aforementioned concentrations of FGF8, etc., is acceptable. Those skilled in the art can determine the FGF signal activation effect, for example, through methods such as downstream signal (Akt, MEK) expression analysis.

There is no particular limitation on the timing of adding ROCK inhibitors, SHH (sound hedgehog factor) signaling pathway active substances, and/or FGF signaling pathway active substances, but it is preferable to culture the cells in a medium containing ROCK inhibitors, SHH (sound hedgehog factor) signaling pathway active substances, and/or FGF signaling pathway active substances from the start of the culture. These substances are preferably added simultaneously to the medium, and more preferably simultaneously with Wnt signaling pathway active substances. Alternatively, dispersed retinal cells can be cultured in a medium containing these substances for 1 to 14 days.

Suspension culture refers to culturing cells in a non-adhesive state to the culture container. There are no particular limitations, and the following culture containers can be used: culture containers that have not been artificially treated to improve cell adhesion (e.g., coating with extracellular matrix, etc.); or culture containers that have been artificially treated to inhibit adhesion (e.g., poly-HEMA, or coated with nonionic surfactants such as Pluronic F-127 or phospholipid-like structures such as Lipidure, a water-soluble polymer with 2-methacryloyloxyethyl phosphocholine as the structural unit).

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

Adhesion culture refers to culturing cells in a state of adhesion to a culture container. There are no particular limitations; culture containers artificially treated to improve cell adhesion can be used. Adhesion culture is preferably performed using culture containers coated with extracellular matrix and/or temperature-responsive polymers. One method for manufacturing retinal tissue includes the step of exposing a culture container coated with a temperature-responsive polymer to a temperature at which the properties of the polymer change, thereby causing sheet-like retinal tissue to detach from the culture container.

Sheet-like retinal tissue, described later, can be produced by adhering to culture containers coated with extracellular matrix.

By culturing in the presence of an extracellular matrix, cells can recognize the basement membrane side, thus easily forming apical surfaces. Furthermore, by oriented the cells in a direction approximately perpendicular to the layer direction, retinal tissue with a better layered structure can be obtained. By using culture containers coated with temperature-responsive polymers, the retinal tissue with an epithelial (or multilayered) structure formed can be easily detached from the culture container simply by temperature changes, without the need for enzyme treatment. Therefore, robust sheet-like retinal tissue can be recovered without weakening intercellular connections due to enzyme treatment. Therefore, it is preferable to use culture containers coated with an extracellular matrix and/or a temperature-responsive polymer, particularly both. Examples include a culture container where the culture surface is coated with the aforementioned temperature-responsive polymer, and the upper surface of the polymer is coated with the aforementioned extracellular matrix. Here, the culture surface refers to the surface in the culture container where cells adhere, and the upper surface of the polymer refers to the side of the polymer coating opposite to the culture surface.

The extracellular matrix refers to the biological macromolecules that constitute the space outside the cell. Examples include cell adhesion proteins such as fibronectin, hyalin, and laminin; fibrous proteins such as collagen and elastin; fragments of these proteins; glycosaminoglycans or proteoglycans such as hyaluronic acid and chondroitin sulfate; and matrix gum. Preferably, the extracellular matrix consists of one or more substances selected from the group consisting of collagen, laminin, fibronectin, matrix gum, hyalin, and fragments of these proteins. Examples of laminin fragments include commercially available products such as iMatrix-511, iMatrix-411, and iMatrix-221.

Matricene is a preparation derived from the basement membrane of Engelbreth Holm Swarn (EHS) mouse sarcoma. Matricene can be prepared using methods disclosed, for example, in US patent No. 4829000, or commercially available products can be purchased. The main components of matricene are laminin, type IV collagen, heparan sulfate proteoglycans, and nestin.

Temperature-responsive polymers are polymers whose properties change with temperature. Specifically, they possess a lower critical solution temperature (LCST) in water, exhibiting a phase transition behavior at a certain temperature: above this temperature, intramolecular or intermolecular hydrophobic bonds strengthen, and polymer chains aggregate; conversely, below this temperature, the polymer chains bind water molecules and become hydrated. Specifically, examples include temperature-responsive polymers that maintain a hydrophobic surface at the culture temperature (approximately 37°C) and become hydrophilic at temperatures below the culture temperature, such as around 20–30°C, allowing for easy peeling of cultured cells. Therefore, cell peeling does not require enzyme treatment, and cells can be recovered in large flakes without damaging intercellular proteins. Poly-N-isopropylacrylamide (PIPAAm) (LCST 32°C) is a preferred example of such a temperature-responsive polymer. Cell culture is typically performed at approximately 37°C. However, considering the potential damage to cells caused by low temperatures, LCST (Liquid Cell Culture Standard) is preferably a temperature-responsive polymer in the range of approximately 20°C to 35°C. Commercially available products include temperature-responsive cell culture equipment for cell sheet recovery with a temperature-responsive polymer immobilized on its surface (Selseed Corporation: UpCell (registered trademark)).

Regarding the cultivation conditions, there are no particular limitations on the cultivation temperature, which is approximately 30–40°C, preferably approximately 37°C, and the cultivation is carried out in an air atmosphere containing _CO2 , with the _CO2 concentration preferably being approximately 2–5%.

The Wnt signal transduction pathway is essential for the formation of layered structures in sheet-like retinal tissue, particularly the formation of the apical/basal plane (polarity of the apex/basal plane).

The concentration of the substance acting on the Wnt signal transduction pathway only needs to be sufficient to induce sheet-like retinal tissue with a layered structure and an apical surface. Regarding the concentration of the substance acting on the Wnt signal transduction pathway, for example, when using CHIR99021, it can be 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 1 μM to 10 μM. When using other substances acting on the Wnt signal transduction pathway, it is sufficient to use a concentration that exhibits the same level of Wnt signal activation as the above-mentioned concentration of CHIR99021. Whether a layered structure has formed can be easily determined by those skilled in the art, for example, through microscopic observation or by measuring the thickness using an OCT device. Whether an apical surface has formed can be confirmed, for example, by staining with anti-Zo-1 antibody, anti-Etz protein antibody, or anti-atypical PKC antibody.

There is no particular limitation on the timing of adding the Wnt signaling pathway active ingredient, but it is preferable to add it as early as possible after the start of adhesion culture. Alternatively, it is preferable to culture in a medium containing the Wnt signaling pathway active ingredient from the start of the culture. The number of days of culture in the medium containing the Wnt signaling pathway active ingredient is not particularly limited, as long as the effect of observing the reformation of a multilayer structure (or multilayer structure) with polarity of the apical/basement membrane can be observed; for example, 1 to 15 days, preferably 1 to 9 days.

It can be continuously cultured in a medium from which substances involved in the Wnt signaling pathway have been removed. Through continuous culture, the layered structure thickens, and retinal cell differentiation occurs. There is no particular limitation on the culture time, as long as it is the time until the seeded, dispersed retinal cell population proliferates and at least forms an epithelial structure (or multilayer structure). Culture can be performed until sheet-like retinal tissue at the target differentiation stage is produced. From the viewpoint of forming an epithelial structure (or multilayer structure), a culture period of at least 7 days is desirable. The culture time can be, for example, from 7 days to less than 60 days, specifically less than 40 days, less than 30 days, less than 20 days, or less than 16 days (e.g., 16 days).

After the formation of the epithelial structure (or multilayer structure), cells can be further cultured to promote proliferation, differentiation, or maturation. The culture medium used for further culture may contain or not contain substances acting on the Wnt signaling pathway, ROCK inhibitors, substances acting on the SHH (sound hedgehog factor) signaling pathway, and/or substances acting on the integrin pathway.

"Multilayer structure" refers to a structure in tissue with basement membrane side and apical side polarity (i.e., epithelial structure) in which two or more layers of cells arranged in the same direction overlap, forming a structure in which the tangential directions of the surfaces of different layers are approximately parallel to each other. This multilayer structure preferably has polarity on the basement and apical surfaces. In one embodiment, the retinal tissue with a multilayer structure can be sheet-like retinal tissue, and the cell aggregates containing the multilayer retinal tissue can be sheet-like cell aggregates. In another embodiment, the cell layers can be a layer of neural retinal anterior cells, a layer of ganglion cells, or a layer of photoreceptor cells.

In one aspect of the invention, in a multilayer structure, the orientation of the cells can be a direction substantially perpendicular to the layer direction. Here, "cell orientation" refers to the directionality of the shape of the nucleus and the orientation of the cell body extending along the basement membrane side and the apical side. Furthermore, a direction substantially perpendicular to the layer direction refers to a direction orthogonal to the direction in which individual cells in the layers of the multilayer structure contact and are arranged (i.e., the tangential direction of the layer surface), meaning a direction perpendicular to the layer or longitudinal.

In one embodiment of the invention, the method may further include the step of cutting retinal tissue (particularly sheet-like retinal tissue) with an epithelial structure (or multilayer structure) obtained through suspension culture or adhesion culture into the size required for transplantation. For example, it can be cut using forceps, a knife, scissors, etc.

On the other hand, the following issue was discovered: during the fabrication of sheet-like retinal tissue using the aforementioned method, RPE cells are manufactured and mixed in at a certain proportion. Although these RPE cells can be visually identified and removed, from the viewpoint of quality and manufacturing efficiency, it is preferable to have no RPE cells from the outset. Therefore, in-depth research was conducted to address this issue, and as a result, the contamination of RPE cells can be significantly reduced by using the method disclosed below.

[Panel retinal tissue]

One aspect of the present invention is a sheet-like retinal (neuroretinal) tissue with an epithelial structure. In one embodiment of this sheet-like retinal tissue, a multilayered retinal cell layer is included, the multilayered structure having polarity of its basal and apical surfaces. This multilayered retinal cell layer contains one or more cell types selected from the group consisting of retinal precursor cells, photoreceptor precursor cells, and photoreceptor cells. In this retinal cell layer, the cells are oriented in a direction substantially perpendicular to the layer direction. Here, "sheet-like" refers to a single-layered or multilayered structure composed of single or multiple cells that are biologically bound together in at least two dimensions.

One of the advantages of this invention is that sheet-like retinal tissue of any size can be manufactured depending on the culture equipment used. That is, it is possible to manufacture sheet-like retinal tissue of sizes that were previously impossible to manufacture, and when the disease involves a large area, treatment can be performed by transplanting a single sheet.

In one embodiment, the major axis (also referred to as diameter) of the sheet-like retinal tissue of 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 another embodiment, the minor axis of the sheet-like retinal tissue of 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 it is limited by the size of the culture dish or the like used in the culture. As one embodiment, the major axis can be less than 10 cm, less than 5 cm, less than 4 cm, less than 3 cm, less than 2 cm, or less than 1 cm. In one embodiment, the height of the sheet-like retinal tissue of the present invention can be, for example, 50 μm to 1500 μm, preferably 200 μm to 700 μm.

There is no particular limitation on the method for determining the major axis, minor axis, and height of sheet-like retinal tissue; for example, they can be determined from images taken under a microscope. For instance, for sheet-like retinal tissue, frontal and transverse images can be taken using a stereomicroscope, with the top surface facing the objective lens, and the transverse image taken at an angle, perpendicular to the cross-section viewed from the objective lens. Here, the major axis refers to the longest line segment connecting the two endpoints of the sheet cross-section in the frontal image and its length. The minor axis refers to the longest line segment orthogonal to the major axis among the line segments connecting the two endpoints of the sheet cross-section in the frontal image and its length. The height refers to the longest line segment orthogonal to the sheet cross-section, with its endpoints being the intersection point with the sheet cross-section and the apex of the retinal patch.

The base surface and the top surface are as defined above. "A multilayer structure having polarity of base and top surfaces" means that a base surface exists on one side of the multilayer structure and a top surface exists on the other side. A basement membrane may be present on the base surface.

"The orientation of cells is approximately perpendicular to the layer direction" means that the long axis of cells in each layer of the retinal cell system is oriented in a direction approximately perpendicular to the layer direction. Approximately perpendicular means that the acute angle formed by the layer direction and the long axis of the cell is approximately 75° (or 80°) or more and less than 90°. 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 in each layer are oriented in a direction approximately perpendicular to the layer direction, then it is determined that "the orientation of cells is approximately perpendicular to the layer direction".

The sheet-like retinal tissue preferably does not contain RPE cells. In one embodiment, the proportion of RPE cells in the sheet-like retinal tissue relative to the total number of cells 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. Regarding the proportion of RPE cells in the sheet-like retinal tissue relative to the total number of cells, the proportion of cells expressing the aforementioned RPE cell markers can be determined, for example, using flow cytometry (FACS). In one embodiment, the area of RPE cells in the total area of the sheet-like 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. RPE cells are black, therefore the proportion of RPE cell area in the total area of the sheet-like retinal tissue can be calculated from the proportion of black area under a microscope. Alternatively, cells expressing RPE cell markers can be detected by PCR or the like. It should be noted that the method for reducing the proportion of RPE cells in the sheet-like retinal (neuroretinal) tissue is as described above.

In one mode of sheet-like retinal tissue composed of multilayered retinal cell layers,

(1) This multilayered retinal cell layer has polarity on its basal and apical surfaces.

(2) The above-mentioned multilayered retinal cell layer contains one or more types of cells selected from the group consisting of retinal precursor cells, photoreceptor precursor cells and photoreceptor cells.

(3) In each of the above-mentioned retinal cell layers, the orientation of the cells is approximately perpendicular to the layer direction, and

(4) The diameter is 8mm or more.

(A complex of sheet-like retinal cell layers (neuroretina) and retinal pigment epithelial cells (complex sheet))

As one aspect of the present invention, the retinal tissue may contain the aforementioned multilayered retinal cell layer and sheet-like retinal pigment epithelial cells attached to the aforementioned retinal cell layer. It may be a composite sheet in which the tangential directions of the surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells are substantially parallel, the apical surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cells are opposite each other, and the retinal cell layer and the sheet-like retinal pigment epithelial cells are attached by an adhesion factor present between them. The sheet-like neuroretina is also called a neuroretinal sheet or NR sheet, and the sheet-like retinal pigment epithelial cells are also called retinal pigment epithelial cell sheets or RPE cell sheets (RPE sheets). That is, one aspect of the present invention also provides a composite sheet of neuroretina and RPE cells. As another aspect of the present invention, a composite sheet in which dispersed retinal pigment epithelial cells are attached to the apical surface of the sheet-like retinal cell layer (sheet-like neuroretina) by an adhesion factor can also be cited.

In the composite of this specification, the tangential directions of the neuroretinal sheet and the retinal pigment epithelial cell sheet are substantially parallel. "Substantially parallel tangential directions" means that the tangential directions of the opposing surfaces of the neuroretinal sheet and the retinal pigment epithelial cell sheet are parallel. Furthermore, in the composite of this invention, the apical surface of the neuroretinal sheet and the apical surface of the retinal pigment epithelial cells are opposite each other. That is, the apical surface of the neuroretinal sheet and the apical surface of the retinal pigment epithelial cells are located close to each other.

As described later, a complex (composite sheet) of neuroretinal and RPE cells can be prepared by attaching a sheet-like retinal choroidal cell layer (sheet-like neuroretinal) to a sheet of RPE cells or dispersed RPE cells in the presence of an adhesion factor. The sheet-like retinal choroidal cell layer (sheet-like neuroretinal) can be manufactured using the method described above for manufacturing cell aggregates containing retinal tissue with epithelial structures (or multilayer structures). The RPE cell sheet can be manufactured using the method described later for manufacturing retinal pigment epithelial cell sheets.

(Method for manufacturing retinal pigment epithelial cell sheets)

Retinal pigment epithelial (RPE) cells are derived from pluripotent stem cells, specifically through the induction of differentiation of pluripotent stem cells. Methods for producing RPE cells can be listed in WO2005/070011, WO2006/080952, WO2011/063005, 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), but are not particularly limited thereto. Furthermore, RPE cell sheets can also be prepared by modifying the method described in WO2016/063985. Retinal pigment epithelial cells can be manufactured in the form of cell sheets or spherical cell aggregates. When manufactured in the form of spherical cell aggregates, RPE cell sheets can be prepared, for example, by cutting the cell aggregates with forceps, a knife, scissors, etc.

As an improved method described in WO2016/063985, in the above method, for pluripotent stem cells, under the condition of the absence of feeder cells, 1) treatment with TGFβ family signal transduction pathway inhibitors and Hedgehog signal transduction pathway inhibitors one day before differentiation induction, and 2) culture under conditions without Hedgehog signal transduction pathway inhibitors at the start of differentiation induction. Then, steps (B) and (C) are performed. Furthermore, it is preferable to start step (D) earlier. Specifically, step (D) is started approximately 9 days after the start of suspension culture in step (B) (e.g., 7, 8, 9, 10, or 11 days later). Then, step (E) is performed. This method yields spherical RPE cell aggregates. These cell aggregates can be dispersed to prepare a cell suspension, or the cell aggregates can be cut open with forceps, a knife, scissors, etc., to prepare RPE cell sheets. Alternatively, the dispersed cell suspension can be cultured via adhesion culture to prepare RPE cell sheets. Alternatively, dispersed RPE cells can also be obtained by dispersing RPE cell sheets or aggregates of RPE cells.

Before contacting the cell aggregates of the neuroretina, retinal pigment epithelial (RPE) cell sheets can be further cultured to achieve a polygonal or cobblestone-like cell morphology. The culture medium at this stage is not particularly limited and can be exchanged for RPE maintenance medium (hereinafter sometimes referred to as RPE maintenance medium) for further culture. This allows for clearer observation of melanin-depositing cell populations and polygonal, flattened cell populations adhering to the basement membrane. Culture using RPE maintenance medium is not limited as long as it forms colonies that proliferate while maintaining the properties of RPE cells; for example, it can be cultured for more than 5 days (e.g., approximately 5–20 days) while undergoing complete medium exchange at a frequency of once every 3 days or more. Those skilled in the art can easily determine the culture time while confirming the morphology. The maintenance culture medium for retinal pigment epithelial cells can be, for example, the media described in IOVS, March 2004, Vol.45, No.3, Masatoshi Haruta et al., IOVS, November 2011, Vol.52, No.12, Okamoto et al., Cell Science 122(17), Fumitaka Osakadar et al., February 2008, Vol.49, No.2, Gamm et al.

The long axis of the retinal pigment epithelial cell sheet can be the same as the long axis of the sheet-like retinal tissue (neuroretinal sheet). In one embodiment, the long axis of the retinal pigment epithelial cell sheet can be, for example, in the range of 3 mm to 50 mm, 5 mm to 30 mm, or 10 mm to 20 mm.

The short axis of the retinal pigment epithelial cell sheet can be the same as the short axis of the sheet-like retinal tissue (neuroretinal sheet). In one embodiment, the short axis of the retinal pigment epithelial cell sheet can be, for example, in the range of 2 mm to 40 mm, 5 mm to 30 mm, or 10 mm to 20 mm.

There is no particular limitation on the degree of melanin deposition in the retinal pigment epithelial cell sheet. Preferably, the degree of melanin deposition in the retinal pigment epithelial cells contained in the retinal pigment epithelial cell sheet is equal between cells. Alternatively, the average melanin content of the retinal pigment epithelial cell sheet can 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. Furthermore, the average melanin content of the retinal pigment epithelial cell sheet can 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.

The melanin content in retinal pigment epithelial cell slices can be determined, for example, as follows: After dispersing the retinal pigment epithelial cell slices, a cell extract obtained using NaOH or similar methods is used, and the result is measured using a spectrophotometer. The average melanin content can be calculated by dividing the melanin content by the total number of cells contained in the retinal pigment epithelial cell slice.

The aforementioned composite can be manufactured by conjugating a neuroretinal sheet with dispersed RPE cells or RPE cell sheets. Preferably, it is a composite sheet obtained by conjugating a neuroretinal sheet with an RPE cell sheet. The neuroretinal sheet and RPE cell sheet can be easily removed from the culture apparatus using tweezers, a knife, scissors, etc. The removed sheets can be transferred to a new container (culture apparatus, etc.), or one sheet can be left in the culture apparatus while the other is transferred to the same apparatus. By using culture apparatus of the same size, the size of the conjugated sheet tissues can be made uniform. Furthermore, when using neuroretinal sheets and retinal pigment epithelial cells cultured for different numbers of days in the above manufacturing method as the contacting neuroretinal sheets and retinal pigment epithelial cells, the start dates of manufacturing can be staggered.

Preferably, the aforementioned neuroretinal sheet is brought into contact with retinal pigment epithelial cells in the presence of an adhesion factor. An adhesion factor is a substance that enables cells to adhere to each other; it is not particularly limited and examples include the aforementioned extracellular matrix and artificial hydrogels. The adhesion factor is not necessarily a separated single substance and also includes preparations derived from organisms or cells, such as matrix gel, intercellular matrix, and serum. The matrix gel is a preparation of the basement membrane from Engelbreth Holm Swarn (EHS) mouse sarcoma. The matrix gel can be prepared, for example, by the method disclosed in US Patent No. 4829000, or it can be a commercially available product. The main components of the matrix gel are laminin, type IV collagen, heparan sulfate proteoglycan, and nestin. The intercellular matrix is the general term for the extracellular matrix existing between retinal cells, such as photoreceptors, in the retina of an organism, and includes, for example, hyaluronic acid. The intercellular matrix can be collected from the retina of an organism by a person skilled in the art, for example, by placing the retina in distilled water to swell and separate it, or it can be a commercially available product. As an adhesion factor, the extracellular matrix or hydrogel is preferred. Furthermore, as an extracellular matrix, it is preferably one or more extracellular matrices selected from the group consisting of hyaluronic acid, fibrin, laminin, type IV collagen, heparan sulfate proteoglycan, and nestin. As a hydrogel, it is preferably one or more hydrogels selected from the group consisting of gelatin, fibrin, collagen, pectin, hyaluronic acid, and alginate. Substances that are classified as both extracellular matrices and hydrogels also exist; in this specification, when used as a gel-like adhesion factor, it is treated as a hydrogel. Commercially available extracellular matrices include Corning (registered trademark) matrix basement membrane matrix, iMatrix511, etc. As an adhesion factor, it can be one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and nestin, with gelatin or fibrin being particularly preferred.

Fibrin gel refers to a gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution. Alternatively, commercially available BOLHEAL (registered trademark) tissue adhesion compound can be used. The reaction is achieved by contacting or mixing the fibrinogen solution with or without the thrombin solution.

"Gelatin" refers to a substance that makes water-insoluble collagen soluble through pretreatment with acids or alkalis, or hydrolysis with hot water. Commercially available gelatin is also available, such as gelatin LS-H (Nitta Gelatin Co., Ltd., alkali-treated pigskin gelatin, non-heat-treated gelatin, high gel strength) and gelatin LS-W (Nitta Gelatin Co., Ltd., alkali-treated pigskin gelatin, heat-treated gelatin, low gel strength). Alkali-treated (lime-treated) gelatin (Type B gelatin) is preferred, and heat-treated gelatin is even more preferred.

For hydrogels, the solution changes from a gel phase to a sol phase and from a sol phase to a gel phase through heating or cooling. Hydrogels can gel (gelatinize) by cooling and losing fluidity, and sol (aqueous solution) by heating and gaining fluidity. Here, "melting point" refers to the temperature at which sol-gelation occurs under a certain pressure, and "freezing point" refers to the temperature at which gelation occurs under a certain pressure. Hydrogels that are biodegradable are preferred. As an option, hydrogels (e.g., gelatin) with a melting point near body temperature (25°C to 40°C) are preferred. The melting point of the hydrogels in this specification can be 20°C to 40°C (e.g., 20°C to 35°C, 25°C to 35°C, 30°C to 40°C, 35°C to 40°C). Generally, the melting point of a gel is a measure of network strength; the melting point of the hydrogel (e.g., gelatin) increases with increasing concentration and molecular weight. Furthermore, there is a tendency for the melting point and freezing point to increase when the solid content is increased, for example, by using sugars. Thus, the melting point and freezing point can be varied within a certain range. There are no particular limitations on the method for determining the melting point of hydrogels; for example, it can be determined by the method specified in JIS K6503.

The strength of a hydrogel is simply the degree to which it does not disintegrate during the transplantation process. As an indicator of hydrogel strength, there is "gel strength." The "gel strength" of a hydrogel refers to the mechanical strength of the object forming the gel. It is usually expressed as the force required to deform a gel of a certain shape or the force required to break the gel (unit: g, dynes (s)/ ^cm² , or g/ ^cm² ), primarily a measure of gel hardness. It should be noted that 1 dyne (dyne (s)) is defined as the force required to provide an acceleration of 1 centimeter per second (cm/ ^s² ) along the direction of an object with a mass of 1 gram (g). For example, the gel strength of gelatin can be determined using the method specified in JIS K6503. In this specification, the gel strength of hydrogels (e.g., gelatin) can be 50g or more, 100g or more, 200g or more, 500g or more, 1000g or more, 1200g or more, 1300g or more, 1400g or more, or 1500g or more. In addition, the gel strength of hydrogels (gelatin) can be below 3000g, below 2500g, or below 2000g.

The concentration of adhesion factors (extracellular matrix) varies depending on the size of the neuroretinal sheet or retinal pigment epithelial cell sheet and the number of retinal pigment epithelial cells. Those skilled in the art can easily determine this concentration by confirming the adhesion status of the RPE cells. For example, in the case of matrix gel, it is preferable to add it at a concentration that is 200 to 10,000 times diluted with an existing product (Corning Corporation), and in the case of iMatrix511, it is preferable to add it at a concentration of 0.1 to 5 μg/mL.

As one method for manufacturing the aforementioned complex using an extracellular matrix, a culture for adhering a neuroretinal sheet to retinal pigment epithelial cells or retinal pigment epithelial cell sheets can be performed in a medium containing adhesion factors (extracellular matrix). The medium used is not particularly limited, and examples include culture media used for culturing retinal pigment epithelial cells or neuroretina (e.g., DMEM/F12 medium, Neurobasal medium, mixed media of these, RPE maintenance medium, etc.). Alternatively, the culture for adhesion can be performed in the presence of other components such as growth factors like EGF along with the extracellular matrix. During the culture period for adhering the aforementioned neuroretinal sheet to retinal pigment epithelial cells or retinal pigment epithelial cell sheets, the culture can be continuously performed in the aforementioned medium containing adhesion factors, or the culture can be performed in the aforementioned medium containing adhesion factors for a certain period (e.g., 1 to 10 days) and then replaced with a medium without adhesion factors for continued culture.

Before culturing to allow the neuroretinal sheet to adhere to retinal pigment epithelial cells (RPCs) or RPCs, an adhesion factor can be used to coat the neuroretinal sheet, RPCs, or RPCs. Specifically, the neuroretinal sheet, RPCs, or RPCs can be cultured in the aforementioned medium containing the adhesion factor. Those skilled in the art can appropriately set the culture time, which can be approximately 10 minutes to 5 hours (e.g., 10 minutes to 60 minutes). After culture, the sheet can be washed with a medium such as PBS.

Adhesion factors, hydrogels, or matrix gels are preferably fibrin gels. Fibrin gels are gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution. In this specification, substances that have the property of forming a gel through reaction are called matrix precursors; for example, thrombin and fibrinogen, which form fibrin gels through reaction, are examples of matrix precursors. Fibrinogen solutions can be prepared by dissolving fibrinogen powder or the like in a solution containing an inhibitory peptide capable of dissolving fibrinogen. The concentration is not particularly limited, but is, for example, 40–480 mg/mL, preferably 80 mg/mL–320 mg/mL (e.g., 160 mg/mL). Furthermore, when the activity of coagulation factor VIII contained in 1 mL of normal human plasma is defined as 1 unit, it can be 37.5 units/mL to 225 units/mL (e.g., 75 units/mL). Thrombin solution can be prepared by dissolving thrombin powder or the like in a thrombin lysate containing calcium chloride hydrate. The concentration is not particularly limited, but can be, for example, 125 units/mL to 750 units/mL (e.g., 75 units/mL). By contacting or mixing fibrinogen solution with thrombin solution, the two can react. In this case, it is preferable to use fibrinogen solution and thrombin solution in an activity ratio of 1:1 to 1:9, preferably 1:3 to 1:4.

As one method for manufacturing the aforementioned composite using hydrogels, a method for manufacturing the composite using fibrin gel is shown below. Specifically, a fibrin gel, generated by reacting fibrinogen with thrombin, is used to adhere a neuroretinal sheet to retinal pigment epithelial cells or a sheet of retinal pigment epithelial cells.

In one approach, the fibrinogen and thrombin can react and gel by bringing one tissue (neuroretinal retina or retinal pigment epithelial cells) that has been in contact with the fibrinogen solution and the other tissue (retinal pigment epithelial cells or neuroretina) that has been in contact with the thrombin solution. Here, contact with the solution means that at least the adhesive surface of the tissue (the side facing other tissues when using fibrin gel to adhere to other tissues) comes into contact with the fibrinogen solution or thrombin solution to the extent that the solution adheres to the adhesive surface of the tissue.

More specifically, for example, when adhering a neuroretinal sheet to a retinal pigment epithelial cell sheet, the neuroretinal sheet can be immersed in a fibrinogen solution, and the retinal pigment epithelial cell sheet can be immersed in a thrombin solution (the solutions can be reversed). Preferably, the fibrinogen and thrombin solutions are used in a volume ratio of 3:1 to 1:3. Excess fibrinogen or thrombin adhering to the tissue can be removed before adhesion. This process allows adjustment of the fibrin gel thickness.

To facilitate adhesion between the neuroretinal sheet after contact with fibrinogen solution and the retinal pigment epithelial cell sheet after contact with thrombin solution, the two can be brought into contact. Here, tissue contact means bringing the surface with fibrinogen solution into contact with the surface with thrombin solution, i.e., overlapping. Furthermore, it is preferable to adhere the two sheets such that the tangential directions of their respective surfaces are approximately parallel, and the apical surfaces of the neuroretinal sheet and the retinal pigment epithelial cell sheet face each other.

In one approach, the tissue can be contacted with a fibrinogen solution, followed by the addition of a thrombin solution to the fibrinogen solution to cause the fibrinogen to react with the thrombin. In this case, the tissue is embedded in a fibrin gel.

In one embodiment, the manufacturing method of the present invention may further include the step of cutting out a composite of the desired size for transplantation from the composite. Since two or more tissues are firmly adhered by fibrin gel, the transplant piece can be easily cut to the desired size without detaching the tissue from the composite when cutting it out. Forceps, knives, scissors, etc., can be used for cutting.

[Pharmaceutical compositions, treatment methods, therapeutic agents, and applications]

As one aspect of the present invention, pharmaceutical compositions containing retinal tissue (e.g., sheet-like retinal tissue) are examples. Preferably, the pharmaceutical composition contains, in addition to the retinal tissue of the present invention, a drug-permissible carrier. The pharmaceutical composition can be used to treat diseases based on disorders or damage to the neuroretinal system cells or neuroretina. Examples of diseases based on disorders of the neuroretinal system cells or neuroretina include, for example, retinal degeneration, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal diseases, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone-rod dystrophy. Examples of neuroretinal damage include, for example, the state of photoreceptor cell degeneration and death.

Regarding permissible carriers for drugs, physiological aqueous solvents (physiological saline, buffer solutions, serum-free culture media, etc.) can be used. Depending on the need, the drug composition may include preservatives, stabilizers, reducing agents, isotonic agents, etc., commonly used in transplantation medicine for drugs containing transplanted tissues or cells.

As one aspect of the present invention, a therapeutic agent for diseases based on neuroretinal disorders is provided, comprising retinal tissue (sheet-like retinal tissue) obtained in the present invention. Additionally, as another aspect of the present invention, a method for treating diseases based on neuroretinal cell or neuroretinal disorders or neuroretinal damage, including the step of transplanting the retinal tissue obtained in the present invention to a recipient (e.g., the subretinal region of an eye with an ophthalmic disease), can be listed. The retinal tissue of the present invention can be used as a therapeutic agent for diseases based on neuroretinal disorders, or to replenish the damaged site in a state of neuroretinal damage. By transplanting the sheet-like retinal tissue of the present invention to a patient with a disease based on neuroretinal cell or neuroretinal disorders or a state of neuroretinal damage that requires transplantation, the neuroretinal cells or the damaged neuroretina can be replenished, thereby treating diseases based on neuroretinal cell or neuroretinal disorders or a state of neuroretinal damage. As a transplantation method, methods such as transplanting retinal tissue to the subretinal region of the damaged site by incising the eyeball can be listed. As a transplantation method, methods such as injection using a thin tube or transplantation by forceps can be listed; the thin tube may include an injection needle, etc.

As one aspect of the present invention, retinal tissue (sheet-like retinal tissue) obtained in the present invention is provided for treating diseases based on disorders or damage to retinal cells or retinal tissue. Additionally, as another aspect of the present invention, the use of the retinal tissue obtained in the present invention in the manufacture of a therapeutic agent for diseases based on disorders or damage to retinal cells or retinal tissue is provided.

Example

The present invention is illustrated in detail below with examples, but the present invention is not limited to these examples in any way.

<Refer to Example 1-1: Exploration of Aggregate Reforming Factors>

Human ES cells (KhES-1 strain (Non-Patent Document 3)) and human iPS cells (1231A3 strain, obtained from Kyoto University) that were genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The specific maintenance culture procedures for human ES and human iPS cells (human ES/iPS cells) are as follows. First, human ES/iPS cells that have reached sub-confluence (60% of the culture area is covered by cells) are washed with PBS and then dispersed into single cells using TrypLESelect (trade name, manufactured by Life Technologies). It should be noted that, here, dispersion into single cells means dispersion in a manner that results in single cells, and the dispersed single-cell population may contain not only single cells but also clumps of 2 to 50 cells. Next, the dispersed single-cell human ES cells are seeded into plastic culture dishes coated with laminin 511-E8 and cultured in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM) under feeder-free conditions. Using the aforementioned plastic culture dishes, 6-well plates (manufactured by Iwaki Corporation, for cell culture, culture area 9.4 ^cm² ) were used. The seeding number of human ES/iPS cells dispersed into single cells was 0.4–1.2 × ^10⁴ cells per well. One day after seeding, the medium was exchanged for StemFit medium without Y-27632. Thereafter, the medium was exchanged every 1–2 days with StemFit medium without Y-27632. The cells were then cultured without feeder cells until one day before reaching sub-confluence. These human ES cells, one day before sub-confluence, were then cultured for one day without feeder cells in the presence of SB431542 (a TGFβ signaling pathway inhibitor, 5 μM) and SAG (a Shh signaling pathway activator, 300 nM).

Human ES/iPS cells were washed with PBS, treated with TrypLE Select cell dispersion medium, and then dispersed into single cells by pipetting. The dispersed single-cell human ES cells were then suspended in 100 μL of serum-free medium at a density of 1.2 × ^10⁴ cells per well on a non-cell-adhesive 96-well plate (trade name: PrimeSurface 96-well V plate, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured at 37°C and 5% _CO₂ . The serum-free medium (gfCDM+KSR) was prepared by adding 10% KSR, 450 μM 1-thioglycerol, and 1× chemically defined lipid concentrate to a 1:1 mixture of F-12 and IMDM media.

At the start of suspension culture (at the start of differentiation induction, day 0), 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 described above. On day 3 after the start of suspension culture, 50 μL of medium without Y-27632 and SAG and containing exogenous recombinant human 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 onwards after the start of suspension culture, half-volume exchange was performed every 3 days using medium without Y-27632, SAG, and recombinant human BMP4.

For cell aggregates from KhES-1, the aggregates were further transferred from day 14 to day 18 after the start of suspension culture to 90 mm low-adhesion culture dishes (90Φ (deep) suspension culture dishes, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured for 3–4 days at 37°C and 5% CO2 in serum-free medium (medium prepared by adding 1% N2 supplement to DMEM/F12 medium) containing Wnt signal transduction pathway active ingredient (CHIR99021, 3 μM) and _FGF signal transduction pathway inhibitor (SU5402, 5 μM). Afterwards, they were cultured long-term in 90 mm low-adhesion culture dishes (90Φ (deep) suspension culture dishes, manufactured by Sumitomo Bakelite Co., Ltd.) in serum-free medium (NucTO medium) without Wnt signal transduction pathway active ingredient and FGF signal transduction pathway inhibitor.

Aggregates from day 16 (from 1231A3) or day 27 (from KhES-1) of suspension culture were washed with PBS and added with neural cell dispersion (Wako). After incubation at 37°C for 20–30 minutes, the cells were dispersed into single cells by pipetting. These cells were then suspended in 100 μL of serum-free medium at a density of 2 × ^10⁵ cells per well (from KhES-1) or 5 × ^10⁵ cells per well (from 1231A3) on non-cell-adhesive 96-well plates (Sumitomo Bakelite Co., Ltd.) and cultured at 37°C and 5% _CO₂ . While adding cells, (1) no addition (control), (2) addition of 10 μM or 20 μM (from 1231A3) Y-27632 (Wako), (3) addition of 300 nM SAG (Enzo), (4) addition of 10 μg/mL bFGF (Wako), (5) addition of 1 μM LDN193189 (Stemgent), (6) addition of 3 μM CHIR99021 (Wako), (7) addition of 10 μM SB431542 (Wako), and (8) addition of 3 μM IWR-1 (Wako).

When cultured for approximately 2 weeks under any of the conditions in (1) to (8), the reformation of aggregates was confirmed (see the results of Reference Examples 1-3). With the addition of Y-27632, it was confirmed that aggregates reformed from day 1 to day 2 after suspension culture for aggregate reformation (Fig. 1: KhES-1, Fig. 2: 1231A3). On the other hand, no promoting effect on early aggregate reformation was observed with compounds other than Y-27632 (Fig. 1 and Fig. 2). Therefore, it can be seen that by adding Y-27632 to retinal progenitor cells dispersed into single cells, the reformation of aggregates can be promoted from an early stage.

<Example 2: Exploration of Layer Formation Promoting Factors and Aggregate Proliferation Promoting Factors>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 at days 20 (1231A3) and 27 (KhES-1) after the start of suspension culture were washed with PBS and added with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were then suspended in 100 μL of serum-free medium containing 10 μM Y-27632 (Wako) at a density of 2–5 × ^10⁵ cells per well on non-cell-adhesive 96-well plates (trade name: PrimeSurface 96-well V plate, Sumitomo Bakelite Co., Ltd.) and cultured in suspension at 37°C and 5% _CO₂ . While adding cells, (1) no addition (only 10 μM Y-27632), (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), (8) 3 μM IWR-1 (from KhES-1) or 10 ng/mL EGF (from 1231A3) were added.

On days 1, 7, and 14 after suspension culture for aggregate reformation, the reformation status of aggregates was observed using a bright-field microscope and a fluorescence microscope (Keyence BZ-X810). Results from KhES-1 cells are shown in Figures 3–5. Additionally, the area of the aggregates was measured using Image J. Results from KhES-1 cells are shown in Figure 6 (average value obtained from 12 measurements). Furthermore, results from 1231A3 cells on day 1 after suspension culture for aggregate reformation are shown in Figure 7. Based on Figures 3–7, it was confirmed that the groups supplemented with SAG (300 nM) or CHIR99021 (3 μM) to Y-27632 reformed larger aggregates compared to the group supplemented with Y-27632 alone. This confirmed the effects of SAG and CHIR99021 in promoting aggregate proliferation and reformation.

<An exploration of layer formation promoting factors and aggregate proliferation promoting factors in reference examples 1-3>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 26 after the start of suspension culture were washed with PBS and added with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were then suspended in 100 μL of serum-free medium at a density of 3.0 × ^10⁴ cells per well on non-cell-adhesive 96-well plates (commercial name: PrimeSurface 96-well V plate, Sumitomo Bakelite Co., Ltd.) and cultured in suspension at 37°C and 5% _CO₂ . While adding cells, (1) no addition (control), (2) addition of 10 μM Y-27632 (Wako), (3) addition of 300 nM SAG (Enzo), (4) addition of 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo), (5) addition of 3 μM CHIR99021 (Wako), (6) addition of 10 μM Y-27632 (Wako) and 3 μM CHIR99021 (Wako), (7) addition of 300 nM SAG (Enzo) and 3 μM CHIR99021 (Wako), (8) addition of 10 μM Y-27632 (Wako), 300 nM SAG (Enzo) and 3 μM CHIR99021 (Wako).

The reformation status of aggregates was observed on days 1, 14, and 28 after suspension culture for aggregate reformation (days 1, 14, and 28), and on days 27, 40, and 54 after the start of suspension culture for differentiation induction (days dd27, dd40, and dd54). The results are shown in Figures 8, 9, and 10. Figures 8, 9, and 10 confirm that aggregates reformed upon the addition of CHIR99021. Furthermore, it was confirmed that adding SAG to CHIR99021 resulted in larger aggregates.

Aggregates used for re-aggregation were fixed with 4% PFA on day 15 (equivalent to day 41 of differentiation) and day 28 (equivalent to day 54 of differentiation) after suspension culture, and frozen sections were prepared. For these frozen sections, immunostaining was performed using DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-β-catenin antibody (R&D Systems), anti-type IV collagen 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 Systems), anti-recovery protein antibody (Proteintech), anti-Islet-1 antibody (DSHB), anti-GS antibody (Sigma), anti-Brn3 antibody (Santa Cruz), and anti-calcium reticulum protein antibody (R&D Systems).

These immunostained sections were observed using a bright-field microscope and a fluorescence microscope (Keyence BZ-X810), and a confocal laser scanning fluorescence microscope (Leica SP-8). The results are shown in Figures 11-20. According to Figures 11, 14, 15, and 18, the addition of CHIR99021 confirmed the formation of a layered structure containing Rx::Venus-positive and Chx10-positive neural retinal progenitor cells on the outermost side of the aggregates. Furthermore, according to Figures 12, 16, and 17, the addition of CHIR99021 resulted in the formation of a Zo-1-positive apical surface on the outermost side of the aggregates and a collagen-positive basal surface on the inner side, confirming the formation of epithelial tissue with apical and basal polarity. On the other hand, particularly as shown in Figure 17, in the group without CHIR99021, acellular polarity or a rosette-like structure was observed. Furthermore, based on Figures 19 and 20, it was confirmed that cone precursor cells differentiated into RxR-γ (RxRg) positive and CRX positive, and photoreceptor precursor cells differentiated into Islet-1 positive and Brn3 positive, and calreticulin positive amacrine cells differentiated into retinal ganglion cells.

These results show that by adding CHIR99021 to a single-cell suspension of temporarily dispersed retinal cells, retinal tissue can be reorganized and differentiated into retinal tissue as a polar epithelial tissue with an apical base.

<Refer to Examples 1-4 regarding the differentiation period of aggregate reformation>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 at days 18, 25, 40, 61, and 75 (dd18, dd25, dd40, dd61, dd75) were washed with PBS and supplemented with neural cell dispersion (WAKO). After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were then suspended in 100 μL of serum-free medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako) at 37°C and 5% CO2 in non-cell-adhesive 96-well plates (commercial name: PrimeSurface 96-well V plate, Sumitomo Bakelite Co., Ltd.) at a concentration of 2–5 × ^10⁵ cells per well on non-cell-adhesive ₉₆ -well plates.

On days 3, 15, and 21 (days 3, 15, and 21) of culture used for aggregate reformation, the reformation status of the aggregates was observed using bright-field microscopy and fluorescence microscopy, and the results are shown in Figure 21. According to Figure 21, it was confirmed that aggregates and lamellae were reformed in cell aggregates at any of the differentiation days dd18, dd25, dd40, dd61, and dd75. Furthermore, the properties of Rx::Venus-positive retinal tissue were observed to be maintained (Figure 21).

These results show that adding Y-27632, SAG, and CHIR99021 to retinal tissue at any stage of differentiation can reform aggregates.

<Refer to Examples 1-5 regarding the reformation of brain organoid aggregates>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

On day 3 after the start of suspension culture, 50 μL of medium containing exogenous recombinant human BMP4 (trade name: Recombinant Human BMP-4, manufactured by R&D Systems) at a final concentration of 1.5 nM was used. Additionally, a group without added BMP4 was set up for brain organoid production. From day 6 onwards after the start of suspension culture, half-volume exchange was performed every 3 days using medium without Y-27632, SAG, and recombinant human BMP4.

Aggregates prepared in this manner were washed with PBS on day 40 after the start of suspension culture and then treated with neural cell dispersion (WAKO). After incubation at 37°C, the cells were dispersed into single cells by pipetting. The dispersed cells were then suspended in 100 μL of serum-free medium containing 10 mM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako) at a density of 5 × ^10⁵ cells per well on non-cell-adhesive 96-well plates (trade name: PrimeSurface 96-well V plate, Sumitomo Bakelite Co., Ltd.) and cultured in suspension at 37°C and 5% _CO₂ .

On days 3, 15, and 21 of the culture used for the reformation of aggregates, the reformation status of the aggregates was observed using a bright-field microscope and a fluorescence microscope. The results are shown in Figure 22. According to Figure 22, it was confirmed that aggregates and layered structures were reformed in Rx::Venus-negative telencephalonoids, similar to those in retinal organoids.

These results show that aggregates can be reformed not only in retinal organoids but also in the neuroepithelial tissues of telencephalomyeloids and other similar organisms.

<Reference Examples 1-6: Cryopreservation Studies>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 31 of the start of suspension culture were washed with PBS and added with nerve cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting.

After dispersion, for the non-frozen group, 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) at 2.0 × ^10⁵ cells per well on non-cell-adhesive 96-well culture plates (trade name: PrimeSurface 96-well V plate, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured at 37°C and 5% _CO₂ .

After dispersion, for the frozen group and the negative control, the single cells were suspended in PBS (Gibco) and the following cryopreservation solutions at a rate of 1.0 × 10⁶ cells/mL: CellBanker 1 (TaKaRa), Stem Cell Banker (TaKaRa), CultureSure cryopreservation solution (Fujifilm and Koko Pure Chemical Industries, Ltd.), STEMdiff NeuralProgenitor cryopreservation solution (Stemcell), StemSure cryopreservation solution (Fujifilm and Koko Pure Chemical Industries, Ltd.), and ^Bambanker (Genetics, Inc., Japan). The cells were then frozen and stored at -80°C using bicel.

After thawing for 1 day, the viable cell rate was confirmed. Regarding the viable cell rate immediately after thawing, the PBS group showed a sharp decline, but the viable cell rate was high in all cryopreservation solutions. The viable cell rate in cryopreservation solutions other than STEMdiff (Cell Banker 1, StemCellBanker, CultureSure, StemSure, Bambanker) was no less than that of the non-frozen group (Figure 24(A)).

These cells were suspended at 2 × ^10⁵ cells per well in 100 μL of NucT₀ medium containing 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), and 3 μM CHIR99021 (Wako) in a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V plate, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured at 37 °C and 5% _CO₂ .

One day after inoculation, aggregates were confirmed to reform in all cryopreservation solutions (Cell Banker 1, Stem Cell Banker, CultureSure, STEMdiff, StemSure, Bambanker) with comparable performance to non-frozen conditions (Figs. 23 and 24(B)). In particular, Cell Banker 1 did not show the small Rx::Venus-positive cell aggregates observed at the periphery of the aggregates in other cryopreservation solutions, yielding images close to those observed in non-frozen conditions (Fig. 23).

It can be seen that, after 7 days of culture for the reformation of aggregates, the reformation of aggregates in any cryopreserved solution was no less effective than that in the non-cryopreserved solution.

These results show that even if the single-cell suspension of retinal cells obtained by temporary dispersion is cryopreserved, it can re-form aggregates after thawing by adding Y-27632, SAG and CHIR99021 and culturing.

<Refer to Examples 1-7 regarding scaffold proteins expressed in retinal aggregates>

As demonstrated in Examples 1-1 to 6, dispersing retinal cells and differentiating pluripotent stem cells into retina to obtain cell aggregates containing retinal cells, then dispersing these cell aggregates to obtain single-cell suspensions, and adding Y-27632 (Wako), SAG (Enzo), and CHIR99021 (Wako) to these single-cell suspensions followed by suspension culture, allows for the reorganization of retinal tissue with a layered structure and polarity. Furthermore, for the purpose of creating large sheet-like retinal tissue, adhesion culture was considered. The extracellular matrix used for adhesion culture was also investigated.

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1, after 25–35 days of suspension culture, were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained with DAPI and anti-laminin antibodies (trade name: Anti-laminin Antibody, Abcam), anti-fibronectin antibodies (R&D Systems), and anti-type IV collagen antibodies (Abcam). These immunostained sections were observed using a confocal laser microscope. The results confirmed that the aggregates expressed at least laminin, fibronectin, and type IV collagen, which are components of the basement membrane (Figure 25). That is, it can be seen that in this self-organized culture system, human retinal tissue spontaneously forms a basement membrane and expresses laminin, fibronectin, and type IV collagen.

Using information from a well-known database of staining of mouse embryonic tissues specialized as the basement membrane (MOUSE BASEMENTMEMBRANE BODYMAP; http://dbarchive.biosciencedbc.jp/archive/matrixome/bm/home.html), we investigated the isoforms of laminin expressed in fetal mouse neuroretinal tissue. The results showed that laminin α has 1, 4, and 5 isoforms; laminin β has 1 and 2 isoforms; and laminin γ has 1 isoform (Figure 26).

This result suggests that, as an extracellular matrix suitable for neural retinal tissue, the combination of laminin α, laminin β, and laminin γ, particularly laminin 511, 521, 411, 421, 111, and 121 (especially 511, 521, and 411) may be useful for laminin α, laminin β, and laminin γ.

This suggests that laminin 511, laminin 521, laminin 411, fibronectin, type IV collagen, etc., may be useful as extracellular matrix for the reformation of sheet-like retinal sheets through adhesion culture.

<Refer to Examples 1-8 for extracellular matrix seeding studies on Transwell>

In the studies of Reference Examples 1-1 to 6, a method for reconstituted aggregates containing layered retinal tissue from single-cell suspensions of retinal cells via suspension culture was investigated. Candidates for the extracellular matrix used in adhesion culture were investigated in Reference Examples 1-7. These were combined to investigate a method for reconstituted sheet-like retinal tissue from single-cell suspensions of retinal cells via adhesion culture (re-sheetification).

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 16 after the start of suspension culture were washed with PBS and nerve cell dispersion (Wako Corporation) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting.

As an extracellular matrix to promote re-sheet formation, matrix gel (Corning) and laminin 511-E8 (trade name iMatrix511, manufactured by Nippi) were used. Single cells of the dispersed retinal cell lineage were suspended at a rate of 0.5–4 × ^10⁵ cells per well in 300 μL of serum-free medium containing 10 μM Y-27632 (Wako), 300 nm SAG (Enzo), and 3 μM CHIR99021 (Wako) and seeded in 24-well Transwell (Corning). Three days after seeding, the medium was exchanged every 3–4 days with serum-free medium (gfCDM) without Y-27632, SAG, and CHIR99021.

Condition 1: Inoculated into 24-well Transwells pre-coated with matrix adhesive (matrix (pre-coated)).

Condition 2: Seedling in 24-well Transwell (iMatrix511 (pre-coated)) pre-coated with laminin 511-E8

Condition 3: In uncoated 24-well Transwells, cells were seeded using culture medium supplemented with laminin 511-E8 (Mix method).

After 14 days of culture for recombination, sheet-like cell aggregates (cell sheets) formed under any conditions. These cell aggregates were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained with DAPI and anti-Chx10 antibody (ExAlpha), anti-Zo-1 antibody (trade name: AntiZo-1 Antibody, Invitrogen), and anti-type IV collagen antibody (Abcam). These immunostained sections were observed using confocal laser scanning fluorescence microscopy. The results confirmed the presence of Chx10-positive neural retinal progenitor cells under matrix gel (pre-coated), iMatrix511 (pre-coated), and Mix methods (Figure 27). Furthermore, under the conditions of matrix gel (pre-coated) and iMatrix511 (pre-coated), Zo-1, a marker of the apical face (where tight junctions of epithelial tissue form), was confirmed on the upper side (the side not in contact with the pores) of the sheet-like cell aggregates, and type IV collagen, a marker of the basal face, was confirmed on the lower side (the side in contact with the pores) of the sheet-like cell aggregates (Fig. 28). Sheets of Rx::Venus-positive retinal progenitor cells with apical and basal polarity were confirmed to have reformed. On the other hand, it was confirmed that no polarity was formed when mixed with extracellular matrix.

These results show that for retinal lineage cells (NR) dispersed into single cells, sheet-like retinal tissue with apical-basal polarity can be recreated (re-sheetification) by using pre-coating with laminin 511-E8 or matrix gel as a scaffold.

<Confirmation of the effects of inoculation and re-flaking of CHIR99021 on Transwell, as referenced in Examples 1-9>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1, after 15–30 days of suspension culture, were washed with PBS and a neural cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 12-well Transwells coated with laminin 511-E8 at a density of 8 × ^10⁵ cells per well in (1) 300 μL of serum-free medium containing 10 μM MY-27632 (Wako), (2) 300 μL of serum-free medium containing 10 μM MY-27632 (Wako) and 300 nM MSAG (Enzo), (3) 300 μL of serum-free medium containing 10 μM MY-27632 (Wako) and 5 μM CHIR99021 (Wako), and (4) 300 μL of serum-free medium containing 10 μM MY-27632 (Wako), 300 nM MSAG (Enzo), and 3 μM CHIR99021 (Wako), and cultured at 37°C and 5% _CO₂ . Three days after inoculation, the culture medium was exchanged every 3 to 4 days using serum-free medium (NucTO) without Y-27632, SAG and CHIR99021.

Cell sheets obtained after 14 days of culture for re-aggregation were observed, and cell viability was observed under all conditions (Fig. 29). Therefore, these cell sheets were fixed with 4% PFA to prepare frozen sections. These frozen sections were immunostained with DAPI and anti-Zo-1 antibody (trade name: Anti Zo-1 Antibody, manufactured by Invitrogen), and anti-type IV collagen antibody (Abcam). These immunostained sections were observed using confocal laser scanning fluorescence microscopy. The results confirmed that, with the addition of CHIR99021, sheets of Rx::Venus-positive retinal progenitor cells with apical and basal polarity were re-formed. On the other hand, it was confirmed that without the addition of CHIR99021, no apical face was formed, and no polarity was established (Fig. 30).

These results show that, for retinal lineage cells (NR) dispersed into single cells, the addition of CHIR99021 can recreate retinal sheets with apical-basal polarity.

<Refer to Examples 1-10: Studies on the concentration and timing of inoculation and re-flaking of CHIR99021 on Transwell>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 and cultured for 33 days (dd33) were washed with PBS and added with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were seeded at 2–4 × ^10⁵ cells per well in 24-well Transwells coated with laminin 511-E8. 300 μL of serum-free medium containing 10 μM MY-27632 (Wako) and 300 nM SAG (Enzo), along with 1 μM, 3 μM, or 9 μM CHIR99021 (Wako), was added. The cells were then cultured at 37°C and 5% _CO₂ for 3 days (days 0–3), 6 days (days 0–6), or 9 days (days 0–9). After 3 days of seeding, the medium was changed every 3–4 days.

After 14 days of culture for the reformation of aggregates (day 14, dd47), the resulting cell sheets were observed to be viable, except for the groups supplemented with 9 μM CHIR99021 for 6 and 9 days (Fig. 31). Therefore, these cell sheets were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained with DAPI. It was confirmed that thicker sheets of Rx::Venus-positive retinal progenitor cells were reformed by supplementing with 1 μM to 9 μM CHIR99021 for 3 to 9 days (Fig. 32).

These results show that for retinal cells (NR) dispersed into single cells, retinal sheets can be re-formed by adding CHIR99021 at least 1–9 μM for 3–9 days.

<Refer to Example 1-11: Study of scaffolds during transwell implantation>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1, after 24 days of suspension culture, were washed with PBS and added with neuronal cell dispersion (Wako). After incubation at 37°C, they were dispersed into single cells by pipetting. After dispersion, the cells were coated in (1) uncoated, (2) with 2 μL of iMatrix511 (trade name, Nippon Pharmaceutical), (3) with 2 μL of iMatrix411 (trade name, Nippon Pharmaceutical), (4) with 2 μL of iMatrix211 (trade name, Nippon Pharmaceutical), (5) with 2 μL of vitrin (VTN-N) recombinant human protein (Thermo Fisher Scientific), and (6) with 2 μL of Cellular Dispersant. (7) Coated with 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, and 5 μL of laminin 521 (BioLamina); (8) Coated with 5 μL of laminin 332; (9) Coated with 2 μL of Corning gel in 24-well Transwells, seeded at 2.0 × ^10⁵ cells per well, and supplemented with 10 μM Y-27632 (Wako) and 300 nM SAG (Enzo), as well as 3 μM CHIR99021 (Wako), 100 μg/mL. 200 μL of serum-free FGF8 (Wako Biotechnology) culture medium was used for adhesion culture at 37°C and 5% _CO2 for 3 days. After 3 days of inoculation, the culture medium was changed every 3-4 days.

The cell sheets obtained 22 days after inoculation were observed. The results showed that good neuroretinal sheets were obtained in all scaffolds except Cell Start (Figure 33).

These results show that, for aggregates dispersed into single cells (NR), not only iMatrix511 and matrix gel, but also various scaffold proteins, can be used to recreate retinal sheets.

<Refer to Examples 1-12 for confirmation of inoculation, repatization, and retinal differentiation on Transwell>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with the Crx::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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 15 after the start of suspension culture were washed with PBS and neuronal cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 200 μL of serum-free medium containing 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021 at a rate of 2.0 × ^10⁵ cells per well in 24-well Transwells coated with laminin 511-E8, and cultured for 3 days at 37°C and 5% _CO₂ . After 3 days of inoculation, the medium was exchanged every 3–4 days with serum-free medium (NucTO) without Y-27632, SAG, and CHIR99021.

The cell slices obtained 44 days after inoculation (day 44, dd59) were observed to show differentiation of Crx::Venus-positive photoreceptor cells (Figure 34).

Then, aggregates from day 29 after the start of suspension culture were similarly re-sheeted. Cell sheets from day 28 (day 67) of reorganized culture were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained with 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, 3D Retina (cell aggregates) of dd70 that were not planarized (re-sheeted) were also stained (Figures 35–37).

In addition, cell slides from day 57 were fixed with 4% PFA and then subjected to whole-cell immunostaining instead of sectioning. Immunostaining was performed using DAPI and the following antibodies: anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-Sox2 antibody (BDPharmingen), anti-Ki67 antibody (BD), anti-Pax6 antibody (BioLegend), anti-Brn3 antibody (SantaCruz), anti-TUJ1 antibody (Millipore), anti-Islet-1 antibody (R&D Systems), anti-Crx antibody (abnova), anti-recovery protein antibody (Proteintech), anti-Zo-1 antibody (Invitrogen), and anti-type IV collagen antibody (Abcam).

As a result, Chx10-positive, Pax6-positive, and Ki67-positive retinal progenitor cells, Brn3-positive retinal ganglion cells, and RxRg-positive, Crx::Venus-positive, and Crx-positive cone cell progenitor cells differentiated as well as the control 3D Retina cells. Furthermore, observation of their positional relationship with the Transwell under transmitted light confirmed the formation of retinal patches on the Transwell, with strongly Pax6-positive and Chx10-negative retinal ganglion cells locally present on the basal side (Fig. 38). Subsequently, observation of the entire cell structure using confocal microscopy without preparing sections confirmed the presence of apical and basal polarities, and the differentiation of various retinal cells according to these polarities (Figs. 39, 40, and 41).

Further long-term culture was conducted. For dd112 cell slices, immunostaining was performed on the slices rather than preparing sections. Staining was performed using anti-Ribeye antibody (CtBP2, BD Biosciences), anti-recovery protein antibody (Proteintech), anti-Pax6 antibody (BioLegend Biosciences), and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, Santa Cruz Biosciences). The results showed expression of Crx::Venus and recovery protein in the same cells, confirming Ribeye expression (Figure 42). This confirmed that photoreceptor cells differentiated from the re-sheet-like retina matured to the point of expressing synaptic proteins. Furthermore, it was confirmed that slices were formed with Crx::Venus locally present on the apical side, and Chx10 and Pax6 locally present on the medial side (Figure 43).

Based on these results, differentiation of retinal cells was confirmed in retinal slices reorganized on Transwell, and polarity was observed to be maintained.

<Refer to Example 1-13 for maintenance culture of retinal cells using Rx::Venus strain>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 31 of the start of suspension culture were washed with PBS and added with nerve cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, in 12-well Transwells coated with 10 μL of laminin 511-E8, at a density of 2.0 × ^10⁵ cells per well, the following were added to a medium containing 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021 in 500 μL of serum-free DMEM/F12 with added N2: (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, (12) 1 μM BMP4 was cultured at 37°C and 5% _CO2 for 3 days. After 3 days of inoculation, the culture medium was changed every 3-4 days using serum-free medium (N2 added to DMEM/F12) without Y-27632, SAG and CHIR99021.

After 34 days, cell slides were fixed with 4% PFA and stained with DAPI for nuclear staining. The maintenance status of retinal cells was investigated by observing the area of Rx::Venus positive cells. Observation using a fluorescence microscope (Keyence BZ-X810) confirmed that Rx::Venus positive cells were widely maintained with continuous addition of 20 ng/mL EGF and FGF2 (Figure 44).

Therefore, it can be concluded that adding EGF and FGF during retinal reorganization is effective in maintaining Rx::Venus positive cells.

<Refer to Example 1-14 for maintenance culture of retinal precursor cells during planar culture>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with the Crx::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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NRs) prepared in Reference Example 1-1 on day 27 after the start of suspension culture were washed with PBS and neural cell dispersion (Wako Pharmaceuticals) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were seeded in 12-well Transwells coated with 10 μL of laminin 511-E8 at a rate of 8.0 × ^10⁵ cells/well in less than 500 μL of medium containing 10 μM MY-27632, 300 nM SAG, and 3 μM CHIR99021. To investigate the culture media for maintaining retinal progenitor cells, 20 ng/mL LFGF2 and 20 ng/mL LEGF were added to (1) NeuroCult NS-A (STEMCELL Technologies), (2) STEMdiff Neural Progneitor (STEMCELL Technologies), (3) StemPro NSC SFM (Thermo Fisher Scientific), and (4) RHB-A (TaKaRa Scientific) media and cultured for 1 month. NucT0 medium was used as a control. Three days after inoculation, the culture medium was exchanged every 3–4 days with medium free of Y-27632, SAG, and CHIR99021.

After 16 days of culture for the re-formation of aggregates, the conditions for non-differentiation were investigated using the visual cell precursor cell marker Crx::Venus as an indicator (the condition under which Crx::Venus does not emit light when observed with a fluorescence microscope). The results confirmed that NeuroCultNS-A and RHB-A media had a certain degree of effect in delaying differentiation (Figure 45).

Therefore, it can be seen that if NeuroCult NS-A and RHB-A culture media and EGF and FGF are added during the re-sheetification process, it can maintain retinal progenitor cells and delay their differentiation.

<Refer to Example 1-15: Culturing on collagen gel>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Collagen gels were prepared using beMatrix low-endotoxin collagen solution (Nitta Gelatin Co., Ltd.). Specifically, collagen AT was mixed with 5xDME and reconstruction buffer at a ratio of 7:2:1, coated onto a Transwell mesh, and incubated at 37°C with 5% _CO2 for 30 minutes. After incubation, culture medium was added to both the inside and outside of the intercalation.

The aggregates (NRs) prepared in Reference Example 1-1 on day 26 of suspension culture were washed with PBS and a neural cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were seeded at 8.0 × ^10⁵ cells/well on 12-well Transwell collagen gels. For the first 3 days, the cells were cultured in the presence of 10 μM MY-27632, 300 nM SAG, and 3 μM CHIR99021. After 3 days of seeding, the culture medium was exchanged every 3–4 days with serum-free medium (NucT0 medium) without MY-27632, SAG, and CHIR99021.

After 33 days of culture for the reformation of aggregates, the re-sheetification was observed using a stereomicroscope with Rx::Venus as the indicator. The results confirmed the presence of Rx::Venus positive cells on the front of the collagen gel in Transwell (Figure 46).

Therefore, it can be seen that collagen gel can also be re-sheetified by adding Y-27632, SAG and CHIR99021.

<Reference Example 1-16 Transplantation Research>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with the Crx::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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates (NRs) on day 26 prepared as in Reference Examples 1-15 were washed with PBS and neural cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, cells were seeded at 8.0 × ^10⁵ cells/well on 12-well Transwell collagen gels. For the first 3 days, cells were cultured in the presence of 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021. After 3 days of seeding, the culture medium was exchanged every 3–4 days with serum-free medium (NucT0 medium) without Y-27632, SAG, and CHIR99021.

For retinal cell slices cultured for 37 and 44 days and differentiated for 63 and 70 days for recombination, collagenase (Roche) was used to treat the slices at 37°C for 30 minutes for dissection. The dissected retinal slices were then cut into thin strips with forceps and scissors to prepare grafts approximately 1.8 cm in length for transplantation (Figure 47).

The prepared grafts were implanted under the retina of immunodeficient retinal-deficient rats (SDFoxn) using glass Pasteur pipettes.

Eyes of rats one year after transplantation were removed and fixed with 4% PFA. Observation with a fluorescence stereomicroscope and a fluorescence microscope (Keyence BZ-X810) confirmed that the Crx::Venus-positive grafts had survived (Figure 48).

This shows that the re-sheet-like retinal patches can survive after transplantation.

<Reference Example 1-17 Sorting Study>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared in Reference Example 1-1 on days 14–25 after the start of suspension culture were washed with PBS and added with nerve cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting. The target cell population was then gated using an ARIAII cell sorter (BD) via FSC and SSC, and Rx::Venus positive fractions were collected (Figure 49).

The separated cells were seeded at a rate of 1.6–8.0 × ^10⁵ cells per well in 12-well Transwell (Corning) plates coated with laminin 511-E8. Unsorted slides were also prepared as a control. For the first 3 days, cells were cultured in the presence of 10 μM Y-27632, 300 nM SAG, and 3 μM CHIR99021. After 3 days post-seeding, the culture medium was exchanged every 3–4 days with serum-free medium (NucTO medium) without Y-27632, SAG, and CHIR99021. Cells were observed using a fluorescence stereomicroscope on day 26 post-seeding (Figure 50).

In addition, cell slides purified by Rx::Venus sorting on day 82 of differentiation were fixed with 4% PFA and then immunostained as slides without being sectioned. Immunostaining was performed using DAPI and anti-recovery protein antibody (Proteintech). As a control, unsorted retinal slides were also stained (Figs. 51, 52, and 53). Subsequently, frozen sections were prepared and immunostained using DAPI and anti-Ki67 antibody (BD Biosciences), anti-Chx10 antibody (trade name: Anti-CHX10 Antibody, Santa Cruz), and anti-CRX antibody (TaKaRa) (Fig. 53).

As a result, if Rx::Venus-positive fractions are inoculated directly without sorting, clumps of RPE cells are observed in some areas. However, these RPE cells can be removed for visual confirmation. On the other hand, when Rx::Venus-positive fractions are sorted, it is observed that Rx::Venus-positive retinal sheets can be prepared. This confirms that most RPE cells can be removed (Figs. 49 and 50). However, even with sorting, RPE cells are sometimes mixed in; it is unclear whether this is due to the inability to completely remove RPE cells through sorting or differentiation after sorting. However, it is confirmed that the mixed RPE cells are not mixed with neuroretinal tissue (Fig. 52). Since these can be confirmed visually, the RPE cells can be removed.

Furthermore, it was confirmed that sorted retinal slices differentiated into recovery protein-positive photoreceptor cells at a rate comparable to that of unsorted retinal slices used as controls (Fig. 51, Fig. 53). Based on Z-Stack analysis and immunohistochemical analysis of the slices, it was confirmed that the retinal tissue layer structure had reformed and that Chx10-positive and Ki67-positive retinal progenitor cells and Crx-positive photoreceptor progenitor cells were present (Fig. 53).

Therefore, it was observed that by gate the target cell population with FSC and SSC and then sorting with Rx::Venus as an indicator, most RPE cells could be removed, and retinal cells could be differentiated without problems during re-sheetification.

<Refer to Example 1-18: Exploration of factors maintaining retinal properties during re-patterning>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NR) prepared in Reference Example 1-1 on day 25 after the start of suspension culture were washed with PBS and neural cell dispersion (Wako Corporation) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting and sorted using an ARIAII cell sorter (BD Corporation) with Rx::Venus as the indicator. For the sorted cells, in 96-well glass plates (Grania Corporation) coated with laminin 511-E8 using Easy iMatrix, Y-27632, CHIR99021, and SAG were added to 100 μL of NucT0, and the cells were seeded at a rate of 5 × ^10⁴ cells per well. Simultaneously with seeding, the proteins shown in Table 1 or the low molecular weight compounds shown in Table 2 were added.

[Table 1]

[Table 2]

Three days after inoculation, the culture medium was exchanged to maintain the concentrations of the low molecular weight compounds or proteins described in Tables 1 and 2. Then, on day 7, the culture medium was exchanged again using NucT0 medium without the low molecular weight compounds or proteins described in Tables 1 and 2. Ten days after inoculation, cells were fixed with 4% PFA and evaluated using a fluorescence microscope (Keyence BZ-X810) with Rx::Venus as an indicator. The results showed that, for proteins, slides supplemented with FGF2, FGF4, and FGF8 exhibited higher Rx::Venus expression intensity compared to the control PBS (Figure 54). Furthermore, for low molecular weight compounds, slides supplemented with IWR1 endo showed higher Rx::Venus expression intensity (Figure 55). Concentration-dependent effects were further confirmed; the highest Rx::Venus expression intensity was observed with the addition of 100 μg/mL FGF8 (Figure 56).

Therefore, it can be concluded that by activating FGF signaling immediately after cell seeding, the Rx::Venus-positive cell population can be maintained.

<Confirmation of aggregate reformation and retinal differentiation induced by FGF8 addition in Example 1-19>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NR) prepared in Reference Example 1-1 on day 24 after the start of suspension culture were washed with PBS and neural cell dispersion (Wako Pharmaceuticals) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting and sorted using an ARIAII cell sorter (BD Pharmaceuticals) with Rx::Venus as the index. Sorted cells were seeded at 2.0–5.0 × ^10⁴ cells per well on low-adhesion V plates. As controls, a group without FGF8 (FGF8-) and a group without sorting were also included.

The culture medium was changed every 3–4 days. It should be noted that FGF8 was also added during the culture medium exchange. Microscopic observation was performed on day 10 after re-inoculation. In the unsorted, FGF8- group, Rx::Venus-negative cell masses or RPE cells were observed (Fig. 57). On the other hand, these cell masses were reduced in the FGF8-added group (FGF8+). In the sorted, FGF8- group, RPE cells were observed locally. Since RPE cells were removed through sorting, this suggests that a subset of Rx::Venus-positive cells differentiated into RPE cells. On the other hand, in the sorted, FGF8+ group, Rx::Venus-positive aggregates formed, and no RPE cells or Rx::Venus-negative masses were observed.

These results indicate that adding FGF8 can reduce the differentiation of RPE cells or Rx::Venus negative cells.

Subsequently, aggregates cultured on day 54 (day 30 after re-inoculation) for re-aggregation were washed with PBS, dispersed into single cells using neural cell dispersion medium (Wako), and fixed with formaldehyde using fixation buffer (BD). After washing with Perm/washing buffer (BD), staining was performed using 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 with Perm/washing buffer (BD), assays were performed using FACSCantoII (BD Bioscience), and analysis was performed using FlowJo. It should be noted that, as a control, neural retinal tissue with the same differentiation days but without reorganization was also included.

The results showed that the unsorted group, which did not have FGF8 added, had a larger population of Rx::Venus negative cells, tending to have a low Rx::Venus positivity rate. On the other hand, the group that did not have FGF8 added, although unsorted, tended to have a high Rx::Venus positivity rate (Figure 58).

In addition, the group that did not have FGF8 added during sorting also tended to have a high Rx::Venus positivity rate (Fig. 58). On the other hand, the group that was sorted and had FGF8 added tended to have a high proportion of Chx10 and Sox2 positive neural retinal precursor cells and a low proportion of Crx positive photoreceptor precursor cells (Fig. 59).

These results show that by not only sorting but also adding FGF8, the differentiation of RPE and Rx::Venus negative cells can be reduced, resulting in uniform and well-formed aggregates.

<Confirmation of the effect of FGF8 during re-flakeification as shown in Example 1-20>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NR) prepared in Reference Example 1-1 on day 24 after the start of suspension culture were washed with PBS and neural cell dispersion (Wako Pharmaceuticals) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting and sorted using an ARIAII cell sorter (BD Pharmaceuticals) with Rx::Venus as the index. The sorted cells were then seeded in 24-well Transwell (Corning Pharmaceuticals) at a suspension density of 2.0 × ^10⁵ cells per well in NucT0 medium containing Y-27632, CHIR99021, SAG, and FGF8. As a control, a group without FGF8 (FGF8-) was also included.

Culture medium was exchanged every 3–4 days. It should be noted that FGF8 was also added during culture medium exchange. The slides were observed using a fluorescence stereomicroscope after 63 days of differentiation and 40 days after reseeding (Figures 60 and 61). Additionally, for FACS analysis, aggregates after 64 days of differentiation and 41 days after reseeding were washed with PBS, dispersed into single cells using neural cell dispersion (Wako), and fixed with formaldehyde using fixation buffer (BD). After fixation with fixation buffer (BD), and washing with Perm/washing buffer (BD), staining was performed 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 antibody with Perm/washing buffer (BD Biosciences), the assay was performed using FACSCantoII (BD Biosciences) and analyzed using FlowJo. It should be noted that, as a control, neural retinal tissue with the same number of days of differentiation and no reorganization was also included (Figure 62).

As a result, in the case of slides without FGF8 after sorting, a large number of Rx::Venus negative cells were observed, with a tendency for Rx::Venus positive cells to concentrate at the periphery of the wells and exist on the block. On the other hand, in the case of slides with FGF8 after sorting, Rx::Venus positive cells were observed to be evenly distributed throughout the wells, resulting in well-prepared slides (Figs. 60 and 61). In addition, black RPE was observed in the case of slides without FGF8 (Fig. 61, arrow), while it was almost not observed in the case of slides with FGF8.

Furthermore, in FACS analysis, the Rx::Venus positivity rate decreased to 60% in the slides without FGF8, while it remained above 95% in the FGF8-added group, indicating that it maintained a positivity rate comparable to that of neural retinal tissue (Figure 62). Moreover, the proportions of Chx10-positive and Sox2-positive neural retinal progenitor cells, and Crx-positive photoreceptor progenitor cells, were also comparable to those in neural retinal tissue. There was a tendency for a higher proportion of proliferative retinal cells with both Rx::Venus positivity and Ki67 positivity compared to neural retinal tissue (Figure 62).

These results show that adding FGF8 after sorting during re-slicing can reduce the differentiation of RPE and Rx::Venus negative cells and obtain uniform and good slices.

<Confirmation of time-related changes after inoculation and re-flaking on Transwell, as referenced in Example 1-21>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates prepared as in Reference Example 1-1 on day 27 after the start of suspension culture were washed with PBS and neuronal cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, 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 in 24-well Transwells coated with laminin 511-E8 at a rate of 2.0 × ^10⁵ cells per well, and cultured for 3 days at 37°C and 5% _CO₂ . Three days after inoculation, the medium was exchanged every 3–4 days with serum-free medium (NucTO) without Y-27632, SAG, CHIR99021, and FGF8.

After culturing for 3, 6, 9, 12, 22, and 40 days (dd30, dd33, dd36, dd39, dd49, dd67) to reform aggregates, the resulting cell sheets were fixed with 4% PFA and observed using a fluorescence stereomicroscope and a fluorescence microscope with Rx::Venus as an indicator. The results showed that Rx::Venus-positive retinal cells proliferated and formed thick sheets (Figure 63). Particularly from 22 to 40 days, thick cell sheets completely covered with Rx::Venus-positive cells were confirmed.

Based on these results, it was observed that reorganized retinal slices were obtained after 22 to 40 days, which could be used for transplantation, analysis, etc.

<Refer to Example 1-22: RPC surface antigen screening – Comparison of hES cells and NR cells >>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Using the cells described above, suspension culture for differentiation induction was initiated as described in Example 1-1. Note that 300 nM SAG was added at the start of differentiation induction. Aggregates (NRs) on days 19 and 26 after the start of suspension culture were washed with PBS and treated with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting and screened for surface antigens using the MACS (registered trademark) Marker Screen, human (Miltenyi) surface antigen screening kit (Alexa 647, fluorescent dye conjugated antibody group). As a control, human ES cells (hESCs) were stained. Note that a total of 371 markers were used in the screening (Figures 64-69).

FACS analysis was performed using a MACS Quant10 (Miltenyi) and analyzed using FlowJo. Gating and analysis were performed using the groups represented by FSC and SSC as shown in Figure 65. First, E-cadherin, SSEA-4, and SSEA-5, expressed in hESCs but whose expression disappears upon differentiation, were used to confirm correct staining and analysis (Figure 66). The subsequent analytical results are summarized in Figure 64. It should be noted that "hESC: High" indicates high or present expression in human ES cells, "hESC: Low" indicates low or no expression in human ES cells, "NR: High" indicates high expression in neural retinal cells, and "NR: Low" indicates low or no expression in neural retinal cells.

The surface antigens expressed (or not expressed) in hESCs and in Rx::Venus positive cells at day 19 or day 26 after the start of suspension culture were explored. The results suggest 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).

Furthermore, FACS analysis was performed on the expression of CD39, CD73, and CD184 (CXCR4) in 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) after the start of suspension culture (Figure 68).

The result was that the highest proportion of cells showed the highest expression of CD39 and CD73 at dd25, while the highest proportion of cells showed low expression of CD184 at dd18 and dd25.

Therefore, when using surface antigens CD39, CD73, and CD184 to purify neural retinal precursor cells, the optimal time is after day 18 to around day 25 after the start of suspension culture (Figure 68).

<Expression analysis of CD39, CD73, and CXCR4 in Example 1-23>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Brain organoids were created by adding 300 nM SAG at the start of suspension culture (at the start of differentiation induction) and not adding BMP4 on day 3 after the start of suspension culture. A control group with added BMP4 was set up. Brain organoids created in this way were observed under a fluorescence microscope on day 26 after the start of suspension culture (Fig. 70). The results showed that Rx::Venus-positive retinal tissue was observed in the BMP4-added group, while Rx::Venus-negative neuroepithelial cells were observed in the group without added BMP4. These aggregates were then fixed with 4% PFA, sectioned, stained with DAPI and anti-FoxG1 antibody (TaKaRa), and observed under a fluorescence microscope (Keyence BZ-X810). The results confirmed FoxG1 negativity in the BMP4-added group and FoxG1 positivity in the group without added BMP4, confirming the differentiation of brain organoids (Fig. 71).

Brain organoids prepared in this manner were washed with PBS on day 25 after the start of suspension culture and then added with neural cell dispersion (Wako). 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). The proportion of expressing cell populations was determined using FACS Canto II (BD Bioscience) and analyzed using FlowJo.

As a result, CD39 and CD73 were not expressed in brain organoids, while CXCR4 was expressed in most cells of brain organoids (Fig. 72).

This confirms that Rx::Venus-positive cell populations can be isolated by using CD39-positive, CD73-positive, and CXCR4-negative cell populations as indicators to differentiate them from brain organoids.

<Example 1-24 CD39 Expression Analysis>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

At the start of differentiation induction, the morphology of aggregates (NR) prepared by adding SAG (1) 0 nM, (2) 30 nM, and (3) 300 nM was observed under a microscope on day 26 after the start of suspension culture. The results showed that the aggregates were larger and the layered structure was clearer when SAG 0 nM and SAG 30 nM were added compared with the case of adding SAG 300 nM at the start of suspension culture (dd0) (Fig. 73).

Furthermore, the aggregates were dispersed into single cells using a neural 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 cell populations was determined using FACSCantoII (BD Bioscience), and analysis was performed using FlowJo. Additionally, these aggregates were fixed with 4% PFA, sectioned, and 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).

The results showed that CD39 expression and low CXCR4 expression were observed in aggregates with 300 nM SAG added at the start of suspension culture (dd0) (Fig. 74). Furthermore, the addition of SAG to dd0 resulted in decreased expression of the dorsal marker ALDH1A1 and increased expression of the ventral marker CoupTF1 (Figs. 75 and 76). Additionally, it was determined that Rx expression was uniform in cell aggregates without SAG stimulation (Fig. 77). However, for LHX2, Chx10, and Pax2, expression was confirmed even in regions of low Rx expression in aggregates with 300 nM added, while NKX2.1 was negative, thus confirming them as neuroretinal tissue (Figs. 78 and 79).

Therefore, it can be seen that by adding 300 nM of SAG at the beginning of differentiation induction, a population of CD39-positive and CXCR4-negative neural retinal progenitor cells can be obtained.

<Reference Example 1-25: Study on Enhanced CD39 Expression>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

For aggregates (NR) prepared by adding 300 nM SAG at the start of differentiation induction (dd0), 10 neural retinal tissue samples were transferred to a 60 mm dish (Sumitomo Bakelite Co., Ltd.) for low-adhesion suspension culture on day 17 after the start of suspension culture. 4 mL of NucT0 medium was supplemented with 1 mM and 0.2 mM adenosine (A2A receptor agonist, Sigma-Aldrich), 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 ATP disodium salts. CGS21680 (adenosine A2A receptor agonist, Tocris) was cultured for 25 days after the start of suspension culture. Changes in CD39 and CXCR4 expression were analyzed using mouse IgG1-APC conjugate antibody (Miltenyi), anti-CD39-BV421 conjugate antibody (BD Bioscience), and anti-CXCR4-APC conjugate antibody (Miltenyi) staining, followed by FACSCantoII (BD Bioscience) assays and FlowJo analysis. Note that a DMSO supplementation group was included as a control.

The results showed that ATP and A2A receptor agonists enhanced CD39 expression and reduced CXCR4 expression (Figures 80 and 81).

Therefore, it can be concluded that CD39 expression can be enhanced by adding ATP and A2A receptor agonists.

<Refer to Example 1-26: Islet-1 KO hESC - Retinal Lens Transplantation Created Using CD39 Sorting and FGF8 Addition>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) that were genetically modified with the Rx::Venus reporter gene and had the Islet-1 gene knocked out were cultured under feeder-free conditions according to the methods described in Scientific Reports 4,3594 (2014) and WO2018/097253. StemFit medium (trade name: AK03N, manufactured by Ajinomoto Co., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

For aggregates obtained from differentiation using SAG 300 nM at the start of suspension culture (at the beginning of differentiation induction, dd0), the aggregates (NR) on day 25 of suspension culture were dispersed into single cells. After staining with anti-CD39-APC conjugated antibody (Miltenyi) at 37°C for 15 minutes, cells were sorted using an ARIAII cell sorter (BD) with CD39-APC as the indicator. The sorted cells were seeded at a rate of 8.0 × ^10⁵ cells per well in 12-well Transwell cells coated with collagen gel. Y-27632 (Wako), SAG (Enzo), CHIR99021 (Wako), and 50 ng/mL FGF8 (Wako) were added at seed. Additionally, a control group (FGF8-) without FGF8 was also included.

The culture medium was changed every 3 to 4 days. It should be noted that FGF8 was also added during the culture medium exchange. Microscopic observation was performed on day 73 after the start of suspension culture. The results showed that Crx::Venus cell populations were sparsely observed in the group without FGF8, while Crx::Venus cell populations were uniformly observed in the group with FGF8 (Figure 82).

For slides prepared in this way, the slides were recovered using collagenase on day 88 after the start of suspension culture. The grafts were then cut into strips using micro-scissors (Fig. 82) and transplanted under the retina of immunodeficient retinal defect rats (SD Foxn).

This confirms that retinal slices made from cells sorted with CD39 can be transplanted.

<Refer to Example 1-27: RPC Surface Antigen Screening – Comparison of Brain Organoids and NR>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

For aggregates (NRs) prepared by adding 30 nM SAG at the start of suspension culture (at the beginning of differentiation induction, dd0) on days 24–26 after the start of culture, single cells were dispersed using neural cell dispersion medium (Wako Pharmaceuticals) and stained using the MACS (registered trademark) Marker Screen, human (Miltenyi Pharmaceuticals) surface antigen screening kit for surface antigen selection (Alexa 647, fluorescent dye conjugated antibody group). As a control, brain organoids prepared without the addition of BMP4 on day 3 after the start of differentiation induction were stained.

FACS analysis was performed using a MACS Quant10 (Miltenyi) and analyzed using FlowJo. The study explored surface antigens expressed (or not expressed) in Rx::Venus positive groups during suspension culture (days 24–26) that were either non-expressed (or expressed). Results showed that CD9, CD15, CD49c, CD66b, CD69, CD82, CD164, EpCAM, and ErbB2 (CD340) might be distinguishable from brain organoids (Figure 83).

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

<Confirmation of the time-related changes in the expression of CD9 in Example 1-28>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates (NRs) of dd4, dd11, dd18, dd25, dd32, and dd46, prepared by adding 30 nM SAG at the start of hESC and suspension culture (at the start of differentiation induction, dd0), were dispersed into single cells using neural cell dispersion medium (Wako Pharmaceuticals), stained with an anti-CD9 antibody conjugated with APC (BioLegend Pharmaceuticals), and after antibody washing, were analyzed using FACS. The FACS analysis was performed using a MACS Quant10 (Miltenyi Pharmaceuticals) and analyzed using FlowJo.

The results showed that CD9 was expressed in hESCs, but expression was lost on day 4 of differentiation, and began to reappear around day 11 (d11 in Figure 85). The percentage of positive cells increased around dd18 (d18 in Figure 85) to dd25 (d25 in Figure 85) and dd32 (d32 in Figure 85). It was determined that the optimal time for purification was after dd11, and more preferably after dd18 to dd32 (Figure 85).

<Refer to Example 1-29: Study on Removal of Non-Target Cells Using SSEA1 as an Indicator>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

For aggregates (NR) prepared by adding 30 nM SAG at the start of suspension culture (at the start of differentiation induction, dd0) on day 25, they were dispersed into single cells using neural cell dispersion (Wako Corporation), stained with anti-CD9 antibody conjugated with APC (BioLegend Corporation), and then stained with anti-SSEA1 antibody conjugated with BV421 (BioLegend Corporation). The staining was performed using FACSCantoII (BD Biosciences) and analyzed using FlowJo.

The results showed that approximately 87% of the aggregates were Rx-positive, but this percentage increased to 94% when gating CD9-positive cells. It was also found that gating CD9-positive and SSEA1-negative cells increased the Rx-positive percentage to approximately 96% (Figure 86).

Therefore, it can be concluded that retinal cells can be further purified by combining SSEA1 with CD9.

<Reference Example 1-30: Study of the combination of CD9, CD90, CXCR4, and SSEA1>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

For 200 day 25 aggregates (NR) prepared by adding 30 nM SAG at the start of suspension culture (at the start of differentiation induction, dd0), the cells were dispersed into single cells using neural cell dispersion (Wako Corporation), divided into 7 equal parts, and stained with surface antigen at 4°C for 1 hour under the following 7 conditions. (1) No staining (purification using Rx::Venus as an indicator), (2) Staining with anti-CD9-APC conjugate antibody (BioLegend), (3) Staining with anti-CD90-APC conjugate antibody (BD), (4) Staining with anti-CD184(CXCR4)-APC conjugate antibody (Miltenyi), (5) Staining with anti-CD9-APC conjugate antibody (BioLegend) and anti-SSEA1-BV421 conjugate antibody (BioLegend), (6) Staining with anti-CD9-APC conjugate antibody (BioLegend) and anti-CD90-BV421 conjugate antibody (BioLegend), and (7) Staining with anti-CD90-APC conjugate antibody (BD) and anti-SSEA1-BV421 conjugate antibody (BioLegend). After staining, the cells were sorted using ARIA II (BD) with the boxes shown in Figure 87 as indicators. The cells recovered through sorting were analyzed by FACS using MACSQuant10 (Miltenyi) to confirm that they could be purified using the target cell fraction (Figure 87). The proportion of Rx::Venus positive cells in the purified cell population was measured. Compared with the samples before sorting, the samples obtained by sorting using the Rx::Venus positive fraction (1) had the highest purity. However, the samples purified using the CD9 positive and SSEA-1 negative fraction were no less effective than the samples purified using the Rx::Venus fraction, with a high Rx::Venus positivity rate (Figures 88 and 89).

The sorted cells were seeded at 2.0 × ^10⁵ cells per well in 24-well Transwell culture media coated with laminin 511-E8. At seeding, 10 μM Y-27632 (Wako), 300 nM SAG (Enzo), 3 μM CHIR99021 (Wako), and 100 ng/mL FGF8 (Wako) were added. The culture medium was changed every 3–4 days post-seeding.

On days 1 and 12 after the start of re-sheet culture, bright-field and fluorescence microscopy observations were performed. The results showed that on day 1 post-inoculation, all purified cells survived on the Transwell mesh (Fig. 90). Furthermore, on day 12, some black RPEs were observed in samples purified using Rx::Venus positive purification. In contrast, no black RPEs were observed in CD9-purified sheets of CD9-positive, CD9-positive/SSEA1-negative (CD9/SSEA1), and CD9-positive/CD90-positive (CD9/CD90) groups (Fig. 91).

This confirms that retinal slices sorted using CD9 and CD9+ SSEA1 negative or CD90 positive grades are better at removing RPE grades than those sorted using Rx::Venus positive grades.

<Confirmation of the time-dependent changes in the expression of CD9 and SSEA1 in Reference Example 1-31>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

Aggregates (NRs) of dd4, dd11, dd18, dd25, dd32, and dd46, prepared by adding 30 nM SAG at the start of hESC and suspension culture (at the start of differentiation induction, dd0), were dispersed into single cells using neural cell dispersion medium (Wako Biosciences). Staining with anti-CD9-APC conjugate antibody (BioLegend) and anti-SSEA1-BV421 conjugate antibody (BioLegend) was performed, followed by FACS analysis. Measurements were taken using FACSCantoII (BD Biosciences), and analysis was performed using FlowJo.

The results showed that hESCs were CD9 positive and SSEA1 negative, but became CD9 negative and SSEA1 positive by day 4 after differentiation began (Figure 92). Furthermore, it was found that 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. Continuous expression was also detected on days 32 and 46.

Therefore, it is confirmed that the preferred time for purification is after dd11 (d11 in Figure 92), especially after dd18 (d18 in Figure 92) to dd32 (d32 in Figure 92) (Figure 92).

Retinal slices prepared in 12-well transwells using CD9 and SSEA1 as indicators and supplemented with 10 μM Y-27632, 3 μM CHIR99021, 300 nM SAG, and 100 ng/mL FGF8 were retrieved with a scalpel and scissors on days 48 and 62 after the start of suspension culture. The grafts were then cut into strips with micro-scissors and transplanted under the retina of immunodeficient retinal defect rats (SD Foxn) (not illustrated).

This confirms that retinal slices made from cells sorted using CD9 and SSEA1 can be transplanted.

<Refer to Example 1-32: Study on RPE-retinal tissue composite using gelatin>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NRs) prepared in Reference Example 1-1, collected on days 18–30 after the start of suspension culture, were washed with PBS and supplemented with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting and seeded into 12-well Transwells at a rate of 4.0–8.0 × ^10⁵ cells per well. Y-27632, SAG, and CHIR99021 were added to the NucT0 medium at seeding. Subsequently, the medium was changed every 3–4 days with NucT0 medium without Y-27632, SAG, and CHIR99021, and cultured for at least one month.

For aggregates obtained by simultaneous differentiation of RPEs prepared 80 days after the start of suspension culture as described in Example 1-1, they were seeded into laminin 511-E8 coated culture dishes and cultured in a medium supplemented with DMEM (SIGMA) and F12 Ham (SIGMA) media, along with B27 (Gibco), L-glutamine, 10 ng/mL FGF2, and SB431542. After adhesion, the RPEs proliferated and gradually spread out; therefore, only RPEs were peeled off using a 1 mL pipette tip and seeded into other laminin 511-E8 coated culture dishes for purification. Following expansion culture, 1.0 × ^10⁶ cells of RPE were seeded into 12-well transwells coated with laminin 511-E8, with medium exchange every 3–4 days, and cultured for more than one month.

The adhesion of the RPE sheet obtained from the above culture to the sheet-like retinal tissue was performed using BeMatrix LS-W gelatin (Nitta Gelatin Co., Ltd.). Specifically, each sheet was retrieved from Transwell using forceps and scissors. The sheet-like retinal tissue and RPE sheet were fused with 10% (w/v) gelatin, followed by 20% gelatin, and finally 30% gelatin was added to adhere the sheet-like retinal tissue to the RPE sheet. After adhesion, the sheet was rapidly cooled to 4°C and incubated for 20 minutes to solidify it before being retrieved, thus creating a composite sheet of sheet-like retinal tissue and RPE sheet (Figures 93-98).

The prepared retinal tissue-RPE composite sheet was cut using forceps and scissors. First, the composite sheet was divided in half, and then cut into strips along the cut surface (Fig. 99). As a result, it was possible to cut the two sheets together with gelatin attached. Observing the cross-section of the cut composite sheet, RPE with black pigment was confirmed on one side, and sheet-like retinal tissue positive for Rx::Venus was confirmed on the opposite side, filled with gelatin (Fig. 100). The cut composite sheet did not detach during transplantation operations, i.e., aspiration and ejection, or during aspiration and ejection using a pipette tip with 1 mL of the tip cut off. As a result, the composite sheet did not separate and could be repeatedly aspirated and ejected (Fig. 101).

Therefore, it can be seen that a transplantable retinal tissue-RPE composite sheet can be made by using sheet-like retinal tissue, RPE sheet, and gelatin.

<Refer to Example 1-33: Study on RPE-retinal tissue complexation using fibrin>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NRs) prepared in Reference Example 1-1, collected on days 18–30 after the start of suspension culture, were washed with PBS and a neural cell dispersion (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting and seeded into 12-well Transwells at a rate of 4.0–8.0 × ^10⁵ cells per well. Y-27632, SAG, and CHIR99021 were added to the NucT0 medium at seeding. Subsequently, the medium was changed every 3–4 days with NucT0 medium without Y-27632, SAG, and CHIR99021, and cultured for more than one month (Figure 102).

For aggregates obtained by simultaneous differentiation of RPEs prepared 80 days after the start of suspension culture as described in Reference Example 1-1, they were seeded into iMatrix511-coated culture dishes and cultured in a medium prepared by adding B27 (Gibco), L-glutamine, 10 ng/mL FGF2, and SB431542 to DMEM (SIGMA) and F12 Ham (SIGMA) media. After adhesion, the RPEs proliferated and gradually spread; therefore, only the RPEs were peeled off with a pipette tip and seeded into other iMatrix511-coated culture dishes for purification. After subsequent expansion culture, 1.0 × ^10⁶ cells of RPE were seeded into 12-well Transwells coated with iMatrix511, with medium exchange performed every 3–4 days, and cultured for more than one month (Figure 102).

The adhesion of sheet-like retinal tissue obtained from the above culture to RPE sheets was performed using BOLHEAL tissue adhesion kit (Teijin Farma). Specifically, each sheet was retrieved from Transwell using forceps and scissors. 100 μL of fibrinogen was added to the sheet-like retinal tissue, and 100 μL of thrombin was added to the RPE sheet. After fusion, the tissue was removed (Figs. 103-104). 100 μL of fibrinogen was added to the sheet-like retinal tissue again, and 100 μL of thrombin was added to the RPE sheet (Fig. 104). On a culture dish, the mesh side of the RPE sheet was placed on the side of the dish with the tip side facing upwards, and the sheet-like retinal tissue was placed from above with the tip side facing the RPE side (Fig. 105). After adhesion, the tissue was incubated at room temperature for 5 minutes to allow it to adhere firmly, and then retrieved, thus creating the adhered sheet (Figs. 106-107). The prepared retinal tissue-RPE composite sheet was flipped over with forceps, positioned so that the sheet of retinal tissue faced downwards and the retinal pigment epithelium sheet faced upwards, and then observed (Fig. 108). Additionally, the adhered Transwell mesh was peeled off (Fig. 109).

Therefore, it can be seen that retinal tissue-RPE composite sheets can be made by using sheet-like retinal tissue, RPE sheets, and fibrin.

<Refer to Example 1-34: Study on the use of CellShifter to remove unwanted gelatin between two sheets>

In the composite sheets studied in Reference Examples 1-32, the layers between the two sheets contain a relatively large amount of gelatin, making them thicker. Therefore, in order to extrude and remove the unwanted gelatin between the two sheets during the composite process, a composite apparatus with channels for gelatin escape was studied.

Two, four, and five channels were made on a glass slide using silicon wafers, forming a dam shape. Two CellShifter (CellSeed) sheets with gelatin added in the middle were then extruded from above, and the gelatin was observed to see if it flowed out.

As a result, it was found that in devices with five flow paths and silicon wafers arranged in a dam-like manner, excess gelatin was most likely to flow out (Figure 110).

<Refer to Example 1-35: Study on the fabrication, dissection, and transplantation of planarized slides on temperature-responsive culture dishes>

Human ES cells (KhES-1 strain, (Non-Patent Literature 3)) genetically modified with 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., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (trade name, manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold instead of feeder cells.

The aggregates (NRs) prepared in Reference Example 1-1, aged 18–30 days, were washed with PBS and supplemented with neural cell dispersion (Wako). After incubation at 37°C, the cells were dispersed into single cells by pipetting and seeded at 8.0 × ^10⁵ cells/well in 24-well temperature-responsive culture dishes (Cellseed) with seven grades of peelability at different peelability levels. The culture medium was changed every 3–4 days. After 30 days from seeding, thicker sheets formed. The culture dishes were then cooled to room temperature and incubated for 2 hours for observation. The results showed that retinal sheets could be peeled off at room temperature in wells 2b, 2c, 3, 4, and several other wells across the seven grades of peelability (Figure 111).

Therefore, it can be concluded that retinal slices can be recovered by culturing them in temperature-responsive petri dishes.

For retinal patches prepared by sorting using Rx::Venus as an indicator and adding 10 μM Y-27632, 3 μM CHIR99021, 300 nM SAG and 100 ng/mL FGF8 in temperature-responsive culture dishes, after incubation at 4°C for 1 hour on day 62 after the start of suspension culture and recovery, the grafts were cut into strips using microscissors and transplanted under the retina of immunodeficient retinal defect rats (SD Foxn) (not illustrated).

This confirms that retinal patches cultured on temperature-responsive culture dishes can be transplanted.

<Refer to Example 2-1: Conditions for the Study of Retinal Tissue Preparation Method Based on Patterned Culture>

As a method for manufacturing cell aggregates containing retinal precursor cells (adhesive cell aggregates), a differentiation method based on patterned culture is known (WO2023/003025). Focusing on patterned culture, a comparative study was conducted on methods for preparing retinal tissue from undifferentiated human iPS cells.

Human iPS cells (LPF11 and DSP-SQ strains, established by Sumitomo Pharmaceutical Co., Ltd.) were established using commercially available Sendai virus vectors (containing four factors: Oct3/4, Sox2, KLF4, and c-Myc, and the CytoTune kit manufactured by IDPharma). The method was based on the publicly available protocol from Thermo Fisher Scientific (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and the publicly available protocol from Kyoto University (Establishment/Maintenance Culture of Human iPS Cells Without Feeder Cells, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/protocol.html) using StemFit medium (AK03, manufactured by Ajinomoto Co., Ltd.) and laminin 511-E8 (manufactured by Nippon Pharmaceutical Co., Ltd.).

Human iPS cells (LPF11 and DSP-SQ strains) were cultured under feeder-free conditions according to the method described in Scientific Reports, 4,3594 (2014). StemFit medium (AK03N, manufactured by Ajinomoto Co., Ltd.) was used as the feeder-free culture medium, and laminin 511-E8 (manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the feeder-free scaffold.

For specific maintenance culture procedures, firstly, sub-confluent human iPS cells (LPF11 and DSP-SQ strains) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). Then, these dispersed single-cell human iPS cells were seeded into plastic culture dishes coated with laminin 511-E8 and cultured without feeder in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM). Using 6-well plates (Iwaki Corporation, for cell culture, 9.4 ^cm² ), the seeding density of the dispersed single-cell human iPS cells was set at 1.0 × ^10⁴ . One day after seeding, the medium was exchanged for StemFit medium without Y-27632. Thereafter, the medium was exchanged every 1-2 days using StemFit medium without Y-27632. The cells were then cultured for 6 days after seeding.

As a patterning step, a droplet (1 cm in diameter) of a solution containing 0.785 μl of laminin 511-E8 (manufactured by Iwaki Corporation, for cell culture, culture area 3.8 ^cm² ) in PBS was first prepared in the center of each well of a 12-well plate (manufactured by Iwaki Corporation, for cell culture, culture area 3.8 cm²). The droplet was then incubated at 37°C or room temperature for 1 hour to 3 hours to prepare a patterned 12-well plate for culture (laminin 511-E8: 0.5 μg/ ^cm² ).

Then, the semi-confluent human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single cells were then seeded at various cell densities ranging from 0.5 × ^10⁵ to 5.0 × ^10⁵ cells/ ^cm² in a well of a patterned culture plate and cultured without feeder in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM) (Figure 112).

Afterwards, Y-27632 was allowed to act for various durations from 2 to 32 hours, and then replaced with 2 ml of StemFit medium (ROCK inhibitor, 0 μM) without Y-27632 (Figure 114).

Two days after seeding iPS cells into patterned culture plates, the culture medium was replaced with 2 ml of serum-free medium (gfCDM+KSR). Five days later, the medium was replaced with gfCDM+KSR containing recombinant human BMP4 (manufactured by R&D Corporation). Multiple concentrations of exogenous recombinant human BMP4 from 0 to 12 nM were compared (Figures 116 and 117).

Subsequently, half of the culture medium was exchanged every 2–3 days using serum-free medium (gfCDM+KSR) without recombinant human BMP4. As a half-volume culture medium exchange operation, half the volume of culture medium in the incubator, i.e., 1 ml, was discarded, and 1 ml of new serum-free culture medium (gfCDM+KSR) was added, so that the total culture medium volume was 2 ml.

For cells after patterned culture day 17, they were cultured in 5% _CO2 using control maturation medium (DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement and 100 μM taurine).

On day 20 of patterned culture, cells were fixed with 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha) and Rx (anti-Rx antibody, Takara), which serve as markers for retinal progenitor cells. The cells were then observed using a fluorescence microscope (Keyence). Detailed results of the optimization of the differentiation method are shown in References 2-2 to 4.

<Refer to Example 2-2: Seeding Density Study of Human iPS Cells in Patterned Culture>

Human iPS cells (LPF11 strain) were seeded at various cell densities (cells/ ^cm² ) on patterned culture plates to study the optimal conditions.

First, a patterned 12-well plate for culture was prepared according to the method described in Reference Example 2-1. Subconfluent human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded into one well of the patterned culture plate at densities of ^0.5 × ^10⁵ , 1.0 × ^10⁵ , 2.5 × 10⁵, or 5.0 × ^10⁵ cells/ ^cm² and cultured without feeder in StemFit medium in the presence of Y-27632 (a ROCK inhibitor, 10 μM). After 2 hours, the medium was exchanged for 2 ml of StemFit medium without Y-27632.

Human iPS cells were observed at 1 hour, day 1, day 2, or day 3 after being seeded into patterned culture plates using a bright-field microscope. The results are shown in Figure 112. Analysis of the images in Figure 112 shows that at a seeding density of 5.0 × ^10⁵ cells/ ^cm² , human iPS cells adhered unevenly to the cell-adhesive regions. On the other hand, at seeding densities of 0.5 × ^10⁵ to 2.5 × ^10⁵ cells/ ^cm² , adhesion was complete.

Subsequently, patterned culture was performed according to the method described in Reference Example 2-1. Cells on day 20 after inoculation were fixed with 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha) as a marker of retinal progenitor cells. These immunostained cells were observed using a fluorescence microscope (Keyence), and the results are shown in Figure 113. Analysis of the images in Figure 113 shows that under inoculation conditions of 1.0 × ^10⁵ and 2.5 × ^10⁵ cells/ ^cm² , retinal tissue with uniformly distributed Chx10-positive cells could be produced. On the other hand, under inoculation conditions of 5.0 × ^10⁵ cells/ ^cm² , pores and gaps formed by the detachment of Chx10-positive cells appeared on the surface of the cell sheet, and the formation efficiency of uniformly differentiated retinal tissue was not good.

These results indicate that, as a method for preparing retinal tissue using patterned culture, the preferred seeding concentration of undifferentiated human iPS cells is 1.0 × ^10⁵ to 2.5 × ^10⁵ cells/ ^cm² .

<Refer to Example 2-3: Study on the duration of action of ROCK inhibitors during cell seeding in patterned culture>

The study determined the optimal Y-27632 exposure time when human iPS cells (LPF11 and DSP-SQ strains) were seeded into patterned culture plates.

First, a patterned 12-well plate for culture was prepared according to the method described in Reference Example 2-1. Human iPS cells (LPF11 and DSP-SQ strains) that had reached near-confluence were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed single-cell human iPS cells were then seeded at a density of 2.0 × ^10⁵ cells/ ^cm² in one well of the patterned culture plate and cultured without feeder in StemFit medium in the presence of Y-27632 (a ROCK inhibitor, 10 μM). After 2, 4, 8, 16, or 32 hours, the medium was replaced with 2 ml of StemFit medium without Y-27632. Under all conditions, the human iPS cells survived and continued to be cultured, indicating that if human iPS cells adhere to cell-adhesive regions in a near-confluenced state, the removal of Y-27632 within a short period is not a problem.

Subsequently, patterned culture was performed according to the method described in Reference Example 2-1. Cells on day 20 after inoculation were fixed with 4% paraformaldehyde, and bright-field images were captured using a fluorescence microscope (Keyence). The results using human iPS cells (LPF11 strain) are shown in Figure 114. These results indicate that the longer the Y-27632 treatment time, the more pores are generated on the cell sheet surface, and the efficiency of uniformly differentiated retinal tissue formation is not good. Immunostaining with Chx10 (anti-Chx10 antibody, Exalpha), a marker of retinal progenitor cells, and observation using a fluorescence microscope (Keyence) revealed the absence of retinal progenitor cells in the pore areas on the sheet surface. The proportion of Chx10-positive areas on the sheet surface was calculated using ImageJ software (NIH). The results showed that 91% of the sheets treated with Y-27632 for 32 hours were positive, while the proportion was above 97% for treatment times of less than 16 hours (16 hours: 97%, 8 hours: 99%, 4 hours: 98%, 2 hours: 99%). Furthermore, the number of wells with Chx10 negativity at various treatment times for Y-27632 was counted. The results showed that by limiting the treatment time of Y-27632 to within 16 hours, most wells formed on the slide surface could be suppressed. It was also found that when using human iPS cells (DSP-SQ strain), shortening the treatment time of Y-27632 reduced the Chx10 negativity area on the slide surface (Figure 115).

These results indicate that, as a method for preparing retinal tissue using patterned culture, the optimal duration of action of the ROCK inhibitor when seeding undifferentiated human iPS cells is 1–16 hours.

<Refer to Example 2-4: Study on the concentration of BMP-acting drug in patterned retinal differentiation>

Then, the optimal concentration of BMP4 acting on human iPS cells during patterned culture was investigated.

First, a patterned culture plate for 12-well culture was prepared according to the method described in Reference Example 2-1. Human iPS cells (LPF11 strain) that had reached sub-confluence were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded at a density of 2.0 × ^10⁵ cells/ ^cm² in one well of the patterned culture plate and cultured without feeder in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 μM). After 2 hours, the medium was replaced with 2 ml of StemFit medium without Y-27632, and patterned culture was performed according to the method described in Reference Example 2-1.

On day 7 of patterned culture, the culture medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous recombinant human BMP4 (manufactured by R&D Corporation) adjusted to final concentrations of 0, 1.5, 3, 6, or 12 nM. Subsequently, half the culture medium was replaced every 2–3 days with serum-free medium (gfCDM+KSR) without recombinant human BMP4. For this half-volume replacement, half the volume of culture medium in the incubator (1 ml) was discarded, and 1 ml of fresh serum-free medium (gfCDM+KSR) was added, bringing the total culture medium volume to 2 ml.

Cells cultured for 20 days were fixed with 4% paraformaldehyde and immunostained with Rx (anti-Rx antibody, Takara) as a marker of retinal progenitor cells. These immunostained cells were observed using a fluorescence microscope (Keyence), and the results are shown in Figure 116. Analysis of the images in Figure 116 showed that retinal tissue induced by BMP4 concentrations of 3–12 nM, compared to a concentration of 1.5 nM BMP4, strongly expressed Rx as a marker of retinal progenitor cells.

Then, total RNA was extracted from retinal tissue using a rotating column (QIAGEN, RNeasy Micro kit) according to the kit instructions. After determining the concentration of total RNA using a Nanodrop (Nanodrop, Thermo Scientific), it was reverse transcribed into cDNA using reverse transcriptase and primers (Reverse Transcription Master Mix Kit, Fluidigm). Using the cDNA and all probes for validation, a multiplex PCR reaction (Pre-Run) was performed using a PCR apparatus (Veriti 96wellthermal cycler, Applied Biosystems). The Pre-Run reaction solution was then injected into multiple wells with flow paths (96.96 Dynamic Array IFC, Fluidigm) using an IFC Controller HX (Fluidigm). The expression levels of marker genes for neuroretinal and extraretinal byproducts were determined by real-time PCR using a multi-sample real-time PCR system (BiomarkHD, Fluidigm).

The results of the analysis of the relative mRNA expression levels of each gene are shown in Figure 117. Specifically, the ΔCt value was calculated by the difference between the Ct value of the target gene and the Ct value of the gene GAPDH used as an internal standard, and the expression ratio of each gene was calculated by the difference in ΔCt between samples (ΔΔCt). The results showed that, similar to Rx expression based on immunostaining, the expression levels of Chx10 and Rx, as marker genes for retinal progenitor cells, were higher in retinal tissue induced by BMP4 concentrations of 3–12 nM compared to a BMP4 concentration of 1.5 nM. Furthermore, the expression of Emx2, a marker gene for non-target cells, was lower in retinal tissue induced by BMP4 concentrations of 3–12 nM.

These results indicate that, as a method for preparing retinal tissue using patterned culture, the final concentration of BMP4 for inducing retinal differentiation is preferably 3–15 nM.

<Refer to Example 2-5 for patterned culture under various optimized conditions>

The quality of retinal tissue differentiated using the method of WO2023/003025 and retinal tissue differentiated using the method optimized for various conditions in the studies of Reference Examples 2-2 to 4 (optimized method) was compared by measuring the expression levels of marker genes of neuroretina and byproducts other than neuroretina using real-time PCR.

First, a patterned culture plate for 12-well culture was prepared according to the method described in Reference Example 2-1. Subconfluent human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). The dispersed human iPS cells were then seeded into one well of the patterned culture plate at a density of 5.0 × ^10⁵ cells/ ^cm² using conventional methods, and at a density of 2.0 × ^10⁵ cells/ ^cm² using optimized methods. The cells were cultured without feeders in StemFit medium in the presence of Y-27632 (a ROCK inhibitor, 10 μM). After 24 hours under the WO2023/003025 method and 2 hours under the optimized method, the medium was replaced with 2 ml of StemFit medium without Y-27632, and patterned culture was continued according to the method described in Reference Example 2-1.

On day 7 of patterned culture, the culture medium was exchanged. Under the WO2023/003025 method, the exchange was performed with serum-free medium (gfCDM+KSR) with a final concentration of exogenous recombinant human BMP4 (manufactured by R&D) adjusted to 1.5 nM. Under the optimized method, the exchange was performed with serum-free medium (gfCDM+KSR) with a final concentration of exogenous recombinant human BMP4 (manufactured by R&D) adjusted to 6 nM. Subsequently, half the culture medium was exchanged every 2–3 days using serum-free medium (gfCDM+KSR) without recombinant human BMP4. For each half-volume exchange, half the culture medium volume (1 ml) in the incubator was discarded, and 1 ml of fresh serum-free medium (gfCDM+KSR) was added, bringing the total culture medium volume to 2 ml.

On day 20 of patterned culture, the expression levels of marker genes for neuroretinal and non-neuroretinal byproducts in the retinal tissue were determined using the methods described in Examples 2-4 (Table 3). The results of the analysis of the relative mRNA expression levels of each gene are shown in Figure 118. The results showed that, compared with retinal tissue differentiated using the method of WO2023/003025, retinal tissue differentiated using the optimized method (optimized method) exhibited higher expression levels of marker genes for neural progenitor cells and retinal progenitor cells. Furthermore, it was found that the expression of various non-target cell marker genes was suppressed to a lower degree in retinal tissue differentiated using the optimized method.

[Table 3]

<Example 1: Manufacturing Reorganized Organoids Using Retinal Precursor Cell Sheets>

As described in Reference Example 1 of this application, cell aggregates containing retinal precursor cells prepared by the SFEBq method can be reorganized and re-sheetified as starting material. Furthermore, as described in WO2023/003025 and Reference Example 2 of this application, a differentiation method based on patterned culture can be used as a method for manufacturing cell aggregates (adhesive cell aggregates) containing retinal precursor cells. Retinal tissue containing retinal precursor cells prepared as cell aggregates by patterned culture (hereinafter also referred to as retinal sheets or retinal precursor cell sheets) shares similarities in cell composition with cell aggregates containing retinal precursor cells prepared by the SFEBq method, but the cell culture states differ. Therefore, the possibility of reorganization and re-sheetification was investigated using patterned retinal precursor cell sheets as raw material.

First, we investigated whether reorganized organoids could be created by dispersing retinal progenitor cell sheets obtained through patterned culture and inducing reorganization in three-dimensional culture (Figure 119).

Human iPS cells (LPF11 strain, established by Sumitomo Pharmaceutical Co., Ltd.) were established using commercially available Sendai virus vectors (containing four factors: Oct3/4, Sox2, KLF4, and c-Myc, and the CytoTune kit manufactured by IDPharma). The method was based on the publicly available protocol from Thermo Fisher Scientific (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and the publicly available protocol from Kyoto University (Establishment/Maintenance Culture of Human iPS Cells Without Feeder Cells, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/protocol.html) using StemFit medium (AK03, manufactured by Ajinomoto Co., Ltd.) and laminin 511-E8 (manufactured by Nippon Pharmaceutical Co., Ltd.).

The human iPS cells (LPF11 line) were cultured without feeders according to the method described in Scientific Reports, 4, 3594 (2014). StemFit medium (AK03N, manufactured by Ajinomoto Co., Ltd.) was used as the culture medium without feeders, and laminin 511-E8 (manufactured by Nippon Pharmaceutical Co., Ltd.) was used as the scaffold without feeders.

As a specific maintenance culture procedure, firstly, the semi-confluent human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). These dispersed single-cell human iPS cells were then seeded into plastic culture dishes coated with laminin 511-E8 and cultured without feeder in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 μM). Using 6-well plates (Iwaki Corporation, for cell culture, culture area 9.4 ^cm² ), the seeding density of the dispersed single-cell human iPS cells was set at 1.0 × ^10⁴ . One day after seeding, the medium was exchanged for StemFit medium without Y27632. Subsequently, the medium was exchanged every 1-2 days with StemFit medium (ROCK inhibitor, 0 μM) without Y27632, and cultured until day 6 post-seeding.

As a patterning step, a droplet (1 cm in diameter) of a solution containing 0.785 μl of laminin 511-E8 (manufactured by Iwaki Corporation, for cell culture, culture area 3.8 ^cm² ) in PBS was first prepared in the center of each well of a 12-well plate (manufactured by Iwaki Corporation, for cell culture, culture area 3.8 cm²). The droplet was then incubated at 37°C or room temperature for 1 hour to 3 hours to prepare a patterned 12-well plate for culture (laminin 511-E8 0.5 μg/ ^cm² ).

Next, the semi-confluenced human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). These dispersed single cells were then seeded at a density of 1.0 × ^10⁵ cells/ ^cm² in one well of a patterned culture plate and cultured without feeder in StemFit medium with Y27632 (ROCK inhibitor, 10 μM). After incubation with Y27632 for 2 hours, the medium was replaced with 2 ml of StemFit medium (ROCK inhibitor, 0 μM) without Y27632.

Two days after seeding iPS cells in patterned culture plates, the culture medium was replaced with 2 ml of serum-free medium (gfCDM+KSR). Five days later, the culture medium was replaced with gfCDM+KSR containing 6 nM of recombinant human BMP4 (manufactured by R&D Corporation).

Subsequently, half of the culture medium was exchanged every 2–3 days using gfCDM+KSR without human recombinant BMP4. As a half-volume culture medium exchange operation, half the volume of culture medium in the incubator, i.e., 1 ml, was discarded, and 1 ml of new gfCDM+KSR was added, so that the total culture medium volume was 2 ml.

After patterning culture for 17 days, retinal precursor cell sheets were prepared by culturing in a control mature medium (DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement and 100 μM taurine) under 5% _CO2 conditions.

Then, patterned retinal precursor cell sheets cultured for 20 days were dispersed into single cells using neural cell dispersion (WAKO Corporation) and suspended in control mature medium at a density of 1.2 × ^10⁴ cells per well in non-cell-adhesive 96-well plates (trade name: PrimeSurface 96-well V-shaped plate, Sumitomo Bakelite Corporation) for three-dimensional culture. At cell seeding, Y27632 (final concentration 10 μM), SAG (final concentration 300 nM), and CHIR99021 (final concentration 3 μM) were added to promote reorganization.

Subsequently, half the volume of culture medium was used to prepare the control mature medium every 3–4 days, and aggregates were observed on days 3, 13, and 23 from the start of suspension culture using a bright-field microscope. The results confirmed the gradual increase in size of the cambium structure on the surface of the reorganized organoids over time (Fig. 120). The reorganized organoids from day 23 of suspension culture were fixed in 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), Rx (anti-Rx antibody, Takara), Crx (anti-Crx antibody, Takara), and ZO-1 (anti-ZO-1 antibody, Invitrogen), which serve as markers of retinal progenitor cells, as well as markers of apical cell polarity. These immunostained cells were observed using a confocal laser microscope (LSM880, ZEISS) (Figure 121). The results showed that a layer of Chx10, Pax6, and Rx-positive retinal progenitor cells, approximately 5–10 cells thick, was formed on the surface of the reorganized organoid, interspersed with Crx-positive photoreceptor progenitor cells. Furthermore, the outer surface was ZO-1 positive, indicating that this reorganized organoid is a polar retinal tissue with its apical surface formed on the outer side.

These results confirm that reorganized retinal organoids can be created using retinal progenitor cell sheets obtained through patterned culture as starting material.

<Example 2: Fabrication of Reticularized Slices Using Retinal Precursor Cell Sheets>

The study investigated whether reorganized sheets could be produced by dispersing patterned retinal progenitor cell sheets and inducing reorganization in two-dimensional culture (Figure 122).

First, patterned retinal precursor cell sheets cultured for 20 days using the method described in Example 1 were dispersed into single cells using neural cell dispersion (WAKO). These cells were then seeded at a rate of 0.5 × ^10⁵ cells per well in a 24-well Transwell (Corning) culture medium pre-coated with laminin 511-E8 (Nippon Chemi-Consumer) for two-dimensional culture. At the time of seeding, Y27632 (final concentration 10 μM), SAG (final concentration 300 nM), and CHIR99021 (final concentration 3 μM) were simultaneously added to promote reorganization.

Subsequently, half the culture medium was exchanged with control mature medium every 3–4 days, and the reorganized slides inoculated on Transwell at days 3, 13, and 23 were observed using a bright-field microscope. The results confirmed that the cells aggregated into sheets on day 3, becoming denser over time (Figure 123). The reorganized slides inoculated on day 23 of Transwell were fixed with 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), Rx (anti-Rx antibody, Takara), Crx (anti-Crx antibody, Takara), and ZO-1 (anti-ZO-1 antibody, Invitrogen), which are markers of retinal progenitor cells, as well as ZO-1 (anti-ZO-1 antibody, Invitrogen), which is a marker of apical cell polarity. These immunostained cells were observed using a confocal laser microscope (LSM880, ZEISS) (Figure 124). The results showed that, similar to the reorganized organoids, the reorganized sheets were formed from Chx10, Pax6, and Rx-positive retinal progenitor cells, interspersed with Crx-positive photoreceptor progenitor cells. Furthermore, the upper side of the sheet (the side not in contact with the pore membrane) was ZO-1 positive, indicating that the reorganized sheet was retinal tissue with a polar apical surface.

These results confirm that reorganized retinal sheets can be produced using retinal precursor cell sheets obtained through patterned culture as starting material.

<Example 3: Manufacturing Reorganized Organoids Using Retinal Precursor Cells Sorted from Retinal Precursor Cell Sheets>

We investigated whether it is possible to create reorganized organoids with fewer non-target cells by sorting CD9-positive retinal progenitor cells from patterned cultured retinal progenitor cell sheets and inducing reorganization in three-dimensional culture (Figure 125).

First, patterned retinal progenitor cell sheets cultured for 20 days using the method described in Example 1 were dispersed into single cells using neural cell dispersion medium (WAKO). The surface antigens were stained with APC-conjugated CD9 antibody (BioLegend) at 4°C for 1 hour, and then sorted using a MACS Quant Tyto cell sorter (Miltenyi). Cells before and after sorting were analyzed by flow cytometry using a MACS Quant Analyzer 10 (Miltenyi) to confirm the purification of CD9-positive cell fractions (Figure 126).

The purified cells were suspended in control maturation medium at a density of 1.2 × ^10⁴ cells per well in non-cell-adhesive 96-well plates (trade name: PrimeSurface 96-well V-shaped plate, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured in three dimensions. At cell seeding, Y27632 (final concentration 10 μM), SAG (final concentration 300 nM), and CHIR99021 (final concentration 3 μM) were added simultaneously to promote reorganization.

Subsequently, the culture medium was exchanged at half the volume using the control mature medium every 3–4 days. Aggregates on day 23 of suspension culture were observed using a bright-field microscope, confirming the formation of reorganized organoids with an epithelial layer on the surface (Fig. 127). These reorganized organoids were then fixed in 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), Rx (anti-Rx antibody, Takara), Crx (anti-Crx antibody, Takara), and ZO-1 (anti-ZO-1 antibody, Invitrogen), which serve as markers for retinal progenitor cells, as well as ZO-1, a marker for apical cell polarity. These immunostained cells were observed using a confocal laser microscope (LSM880, ZEISS) (Fig. 127). As a result, similar to the reorganized organoids prepared under the conditions of Example 1, the reorganized organoids prepared using CD9-positive cells sorted from retinal progenitor cell sheets also formed a Chx10, Pax6, and Rx-positive retinal progenitor cell layer approximately 5–10 cells thick, with Crx-positive photoreceptor progenitor cells scattered throughout. Furthermore, the outer surface was ZO-1 positive, indicating that the apical surface was formed on the outer side.

Then, to confirm that non-target cells had been removed from the reorganized organoids made from CD9-positive cells sorted from retinal progenitor cell slices, the expression levels of the Rx gene, a neuroretinal marker, and Emx2, a dorsal telencephalon marker, were determined by real-time PCR.

First, total RNA was extracted from reorganized organoids made from CD9-positive cells (CD9-sorted) or unsorted cells (unsorted) using a rotating column (QIAGEN, RNeasy Microkit) according to the kit instructions. After determining the total RNA concentration using a Nanodrop (Nanodrop, Thermo Scientific), it was reverse transcribed into cDNA using reverse transcriptase and primers (Reverse Transcription Master Mix Kit, Fluidigm). Using the cDNA and all probes for validation, a multiplex PCR reaction (Pre-Run) was performed using a PCR apparatus (Veriti 96well thermal cycler, Applied Biosystems). Subsequently, the Pre-Run reaction solution was injected into multiple wells with flow paths (96.96 Dynamic Array IFC, Fluidigm) using an IFCController HX (Fluidigm), and the expression levels of the Rx and Emx2 genes were determined by real-time PCR using a multi-sample real-time PCR system (Biomark HD, Fluidigm).

The results of the analysis of the relative mRNA expression levels of the two genes are shown in Figure 128. Specifically, the ΔCt value was calculated by the difference between the Ct value of the target gene and the Ct value of the gene GAPDH used as an internal standard, and the expression ratio of each gene was calculated by the difference in ΔCt between samples (ΔΔCt). The results showed that, compared with unsorted, in the case of CD9-sorted reorganized organoids, the expression level of Rx, a marker of retinal progenitor cells, was higher, while the expression level of Emx2, a marker gene of non-target cells, was lower. These results confirm that reorganized organoids with fewer non-target cells can be produced by using CD9-positive cells sorted from retinal progenitor cell sheets. It should be noted that, in addition to using CD9 as a surface antigen, studies were also conducted using CD90 as a surface antigen, confirming its expression in retinal progenitor cell sheets and its ability to be sorted.

These results confirm that reorganized retinal organoids can be produced using retinal progenitor cells sorted from retinal progenitor cell sheets prepared through patterned culture as starting material.

<Example 4: Manufacturing Reticularized Slices Using Reticular Precursor Cells Sorted from Reticular Precursor Cell Sheets>

The study investigated whether it was possible to produce reorganized sheets with fewer non-target cells by sorting CD9-positive retinal progenitor cells from patterned cultured retinal progenitor cell sheets and inducing reorganization under two-dimensional culture (Fig. 129).

First, patterned retinal progenitor cell sheets cultured for 20 days using the method described in Example 1 were dispersed into single cells using neural cell dispersion medium (WAKO Corporation). Surface antigens were stained with APC-conjugated CD9 antibody (BioLegend Corporation) at 4°C for 1 hour, and sorted using a MACS Quant Tyto cell sorter (Miltenyi Corporation). The recovered cells were seeded at a rate of 0.5 × 10⁵ cells per well in 24-well Transwell (Corning Corporation) pre-coated with laminin 511-E8 (Nippon Pharmaceutical Group). At seeding, Y27632 (final concentration 10 μM), SAG (final concentration 300 nM), and CHIR99021 (final concentration 3 μM) were added simultaneously to promote reorganization.

Subsequently, the culture medium was exchanged at half the volume with the control mature medium every 3–4 days for maintenance culture. Cell sheets inoculated on day 23 after Transwell were observed using a bright-field microscope, confirming that the cells had reorganized into sheet-like structures (Fig. 130). The reorganized sheet was fixed with 4% paraformaldehyde and immunostained with Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), Rx (anti-Rx antibody, Takara), Crx (anti-Crx antibody, Takara), and ZO-1 (anti-ZO-1 antibody, Invitrogen), which are markers of retinal progenitor cells, as well as ZO-1, a marker of apical cell polarity. The staining was then observed using a confocal laser microscope (LSM880, ZEISS) (Fig. 130).

As a result, similar to the reorganized slide prepared under the conditions of Example 2, the reorganized slide prepared using CD9-positive cells sorted from retinal progenitor cell sheets was also formed from Chx10, Pax6, and Rx-positive retinal progenitor cells, with Crx-positive photoreceptor progenitor cells scattered throughout. Furthermore, the upper side of the slide (the side not in contact with the pore membrane) was ZO-1 positive, indicating that this reorganized organoid is a polar retinal tissue with its apical surface formed on the outer side.

These results confirm that reorganized retinal sheets can be produced using retinal progenitor cells sorted from retinal progenitor cell sheets prepared through patterned culture as starting material.

<Example 5: Study on the optimal concentration of CHIR required for reorganization>

By dispersing retinal progenitor cell sheets prepared using patterned culture, the optimal concentrations of ROCK inhibitors, Wnt signaling pathway agents, and Shh signaling pathway agents, which are crucial for reorganization, will be investigated.

First, patterned retinal precursor cell sheets cultured for 20 days using the method described in Example 1 were dispersed into single cells using neural cell dispersion (WAKO Corporation). These cells were then cultured in non-cell-adhesive 96-well plates (trade name: PrimeSurface 96-well V-shaped plate, Sumitomo Bakelite Corporation) suspended in control maturation medium at a density of 1.2 × ^10⁴ cells per well. At cell seeding, Y27632 (final concentration 0 or 10 μM), SAG (final concentration 0, 150, 300, or 600 nM), and CHIR99021 (final concentration 0, 1.5, 3, or 6 μM) were simultaneously added to promote reorganization.

Subsequently, half the amount of culture medium was exchanged with the control mature medium every 3–4 days, and the aggregates on day 23 of suspension culture were observed using a bright-field microscope (Fig. 131). The results showed that epithelium formed on the surface of the reorganized organoids at concentrations above 3 μM CHIR99021, and the layered structure had a better morphology at a concentration of 6 μM (Fig. 132).

As demonstrated above, although patterned culture-derived retinal precursor cell sheets adhere to the dish and possess properties different from suspended cell aggregates, they can serve as starting materials for reorganization and resheetization, and the optimal concentration of reorganization factors added at this stage has been identified. Patterned culture-derived retinal precursor cell sheets can be mass-produced in various shapes and sizes, thus potentially serving as useful starting materials for reorganization and resheetization; this study confirms the feasibility of this approach.

Claims (31)

1. A method of manufacturing retinal tissue having an epithelial structure, comprising the steps of:

(1) Seeding pluripotent stem cells on a culture substrate having a region a having cell adhesion and a region B having cell adhesion lower than the region a adjacent to at least a part of the region a on a surface;

(2) Culturing the pluripotent stem cells inoculated in the step (1) in a culture medium containing a ROCK inhibitor;

(3) Culturing the cells obtained after the step (2) in a culture medium containing BMP signal transduction pathway active material to obtain retinal tissue;

(4) Dispersing the retinal tissue obtained in the step (3) to obtain a dispersed retinal cell population, and

(5) Subjecting the dispersed retinal cell population obtained in the step (4) to suspension culture or adhesion culture in a medium containing a Wnt signaling pathway-acting substance.

2. The method of claim 1, wherein the region a is coated with a cell-adhesive substance.

3. The method of claim 2, wherein the cell-adhesive substance is laminin.

4. A method according to any one of claims 1 to 3, wherein the area a is surrounded by the area B.

5. The method according to any one of claims 1 to 4, wherein in the step (1), the pluripotent stem cells are seeded at a density of 0.5 x 10 ⁵ cells/cm ²～2.5×10⁵ cells/cm ².

6. The method according to any one of claims 1 to 5, wherein in the step (2), the culture in the medium containing the ROCK inhibitor is 1 to 16 hours.

7. The method of any one of claims 1-6, wherein the ROCK inhibitor is one or more selected from the group consisting of Y-27632, fasudil (HA 1077), and H-1152.

8. The method according to any one of claims 1 to 7, wherein the culture medium containing the BMP signaling pathway working substance contains BMP4 in a concentration of 3nm to 15nm or BMP signaling pathway working substance in a concentration showing equivalent activity to BMP4 in the concentration.

9. The method according to any one of claims 1 to 8, wherein the BMP signal transduction pathway working material is one or more selected from the group consisting of BMP2, BMP4, BMP7 and GDF 7.

10. The production method according to any one of claims 1 to 9, wherein the Wnt signaling pathway-acting substance is one or more selected from the group consisting of CHIR99021, BIO, wnt2b and Wnt3 a.

11. The method according to any one of claims 1 to 10, wherein the medium containing the Wnt signaling pathway-acting substance further contains one or more substances selected from the group consisting of a ROCK inhibitor, an SHH signaling pathway-acting substance, and an FGF signaling pathway-acting substance.

12. The method according to claim 11, wherein the ROCK inhibitor in the medium containing the Wnt signaling pathway-acting substance is one or more selected from the group consisting of Y-27632, fasudil (HA 1077) and H-1152.

13. The production method according to claim 11 or 12, wherein the SHH signaling pathway-acting substance in the Wnt signaling pathway-acting substance-containing medium is one or more selected from the group consisting of SAG, PMA and SHH.

14. The production method according to any one of claims 11 to 13, wherein the FGF signaling pathway-acting substance in the Wnt signaling pathway-acting substance-containing medium is one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4 and FGF 8.

15. The method according to any one of claims 1 to 14, wherein the step (5) comprises a step of performing adhesion culture in the medium containing the Wnt signaling pathway-acting substance, and the retinal tissue having the epithelial structure is a sheet-like retinal tissue.

16. The method according to claim 15, wherein the adhesion culture is performed using a culture vessel coated with an extracellular matrix and/or a temperature-responsive polymer.

17. The production method according to claim 16, wherein a culture surface of the culture vessel is coated with the temperature-responsive polymer, and an upper surface of the temperature-responsive polymer is coated with the extracellular matrix.

18. The production method according to claim 16 or 17, wherein the extracellular matrix is one or more selected from the group consisting of collagen, laminin, fibronectin, matrigel, and vitronectin.

19. The method according to any one of claims 16 to 18, further comprising the step of peeling the sheet-like retinal tissue from the culture container coated with the temperature-responsive polymer by exposing the culture container to a temperature at which a property of the temperature-responsive polymer changes.

20. The method according to any one of claims 1 to 19, further comprising the step of increasing the proportion of retinal precursor cells contained in the dispersed retinal cell population obtained in the step (4) before the step (5).

21. The method of claim 20, wherein the mixing or differentiation of retinal pigment epithelial cells is inhibited.

22. The method of claim 20 or 21, wherein the step of increasing the proportion of retinal precursor cells comprises the step of contacting the dispersed population of retinal lineage cells with a substance that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90 and CXCR4, resulting in a population of cells expressing the antigen.

23. The method according to claim 22, wherein the step of increasing the proportion of the retinal precursor cells comprises the step of further contacting the dispersed retinal cell population with a substance binding to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84 to obtain a cell population having an expression level of the antigen of at most a reference value.

24. The method according to any one of claims 1 to 23, wherein in step (5), the dispersed retinal cell population is cultured in the medium containing the Wnt signaling pathway-acting substance from the start of the suspension culture or the adhesion culture.

25. The method according to any one of claims 1 to 24, wherein the medium containing the Wnt signaling pathway-acting substance contains CHIR99021 at a concentration of 3 μm to 6 μm or a concentration of Wnt signaling pathway-acting substance showing the same activity as CHIR99021 at the concentration.

26. The method according to any one of claims 1 to 25, wherein in the epithelial structure, the cells are oriented in a direction substantially perpendicular to the layer direction.

27. The method according to any one of claims 1 to 26, further comprising the step of cutting the retinal tissue having an epithelial structure obtained by the suspension culture or the adhesion culture in the step (5) into a size required for transplantation.

28. The production method according to any one of claims 1 to 27, wherein the epithelial structure is a multilayer structure.

29. A method for producing retinal tissue, comprising joining the sheet-like retinal tissue produced by the method according to any one of claims 15 to 28 with sheet-like or dispersed retinal pigment epithelial cells in the presence of an adhesion factor.

30. The method of claim 29, wherein the adhesion factor is an extracellular matrix or a hydrogel.

31. The production method according to claim 29 or 30, wherein the adhesion factor is one or more selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.