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  • C12N5/0602—Vertebrate cells
  • C12N5/0618—Cells of the nervous system
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  • C12N5/0602—Vertebrate cells
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2501/727—Kinases (EC 2.7.)
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/52—Fibronectin; Laminin

Definitions

• the present invention relates to a method for producing retinal tissue.
• Non-Patent Document 1 demonstrating the possibility of transplantation therapy for photoreceptor degenerative diseases such as retinitis pigmentosa.
• Non-Patent Document 2 and Patent Document 1 a method for obtaining multi-layered retinal tissue from pluripotent stem cells
• Patent Document 3 and Patent Document 2 a method for obtaining multi-layered retinal tissue by forming uniform pluripotent stem cell aggregates in a serum-free medium containing a substance that inhibits the Wnt signaling pathway, and then floating-culture the obtained aggregates in the presence of a basement membrane preparation and then floating-culture in a serum medium
• Non-Patent Document 3 and Patent Document 2 a method for obtaining retinal tissue by floating-culture of pluripotent stem cell aggregates in a medium containing a substance that acts on the BMP signaling pathway
• These retinal tissues are produced as sphere-shaped cell aggregates.
• Non-Patent Document 5 it has been reported that Wnt2b has an effect on the formation of retinal epithelial structures in chickens (Non-Patent Document 5), but a similar effect has not been reported in organisms other than chickens.
• the present invention aims to provide a method for regenerating the neuroepithelial structure of retinal tissue from retinal cells and a method for producing retinal tissue by applying this method.
• a neuroepithelial structure when a neuroepithelial structure is reconstituted using dispersed retinal cells as starting cells to produce retinal tissue, a neuroepithelial structure can be reconstituted by adding a substance acting on the Wnt signaling pathway to the dispersed retinal cells.
• a ROCK inhibitor a substance acting on the SHH signaling pathway, and/or a fibroblast growth factor can be further added, (2) in order to reconstitute a good neuroepithelial structure, particularly in a sheet form, by culturing on a culture plate coated with an extracellular matrix that serves as a scaffold for cell adhesion, and (3) by improving the purity of retinal progenitor cells as starting cells, impure cells such as retinal pigment epithelial cells (RPE) can be removed.
• RPE retinal pigment epithelial cells
• retinal tissues need to be prepared, and retinal cells as a raw material for reconstituting a neuroepithelial structure need to be scaled up, but the number of cells that can be obtained from one spherical retinal tissue is limited, and the production of multiple spherical retinal tissues and the preparation of retinal cells from them are complicated.
• the inventors came up with the idea of using retinal cells obtained from a retinal sheet as starting cells, and discovered that these cells are suitable for regenerating neuroepithelial structures, which led to the completion of the present invention.
• a method for producing retinal tissue having an epithelial structure comprising the steps of: (1) Seeding pluripotent stem cells into region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a portion of region A and having lower cell adhesiveness than region A; (2) culturing the pluripotent stem cells seeded in step (1) in a medium containing a ROCK inhibitor; (3) culturing the cells obtained after step (2) in a medium containing a substance acting on the BMP signaling pathway to obtain retinal tissue; (4) dispersing the retinal tissue obtained in step (3) to obtain a dispersed retinal cell population; and (5) subjecting the dispersed retinal cell population obtained in step (4) to suspension or adhesion culture in a medium containing a substance acting on the Wnt signaling pathway.
• the medium containing the substance acting on the Wnt signaling pathway further contains one or more substances selected from the group consisting of a ROCK inhibitor, a substance acting on the SHH signaling pathway, and a substance acting on the FGF signaling pathway.
• the ROCK inhibitor in the medium containing the substance acting on the Wnt signaling pathway is one or more substances selected from the group consisting of Y-27632, Fasudil (HA1077), and H-1152.
• step of increasing the proportion of retinal progenitor cells further comprises a step of contacting the dispersed retinal cell population with a substance that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69 and CD84, to obtain a cell population in which the expression level of the antigen is below a reference value.
• 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 reference value.
• [36] The method according to any one of [1] to [35], wherein the epithelial structure is a multi-layered structure.
• a method for producing retinal tissue (a composite of neural retina and RPE cells (composite sheet)), comprising producing a sheet-like retinal tissue by the method according to any one of [21] to [36], and bonding the sheet-like retinal tissue to a sheet-like or dispersed retinal pigment epithelial cell in the presence of an adhesion factor.
• a method for producing retinal tissue comprising joining a sheet-like retinal tissue produced by the method according to any one of [21] to [36] with sheet-like or dispersed retinal pigment epithelial cells in the presence of an adhesion factor.
• the adhesion factor is an extracellular matrix or a hydrogel.
• the adhesion factor is one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.
• the retinal tissue described in [43] comprises a retinal cell layer having the above-mentioned multilayer structure and a sheet-like retinal pigment epithelial cell joined to the above-mentioned retinal cell layer, wherein the tangential directions of the surfaces of the above-mentioned retinal cell layer and the sheet-like retinal pigment epithelial cell are roughly parallel, the apical surface of the above-mentioned retinal cell layer faces the apical surface of the above-mentioned sheet-like retinal pigment epithelial cell, and the above-mentioned retinal cell layer and the sheet-like retinal pigment epithelial cell are joined to each other by an adhesion factor present between them.
• a pharmaceutical composition comprising the retinal tissue according to any one of [42] to [47].
• a method for treating a disease caused by a disorder of a retinal cell or retinal tissue or damage to the retinal tissue comprising transplanting the retinal tissue according to any one of [42] to [47] into a subject in need of transplantation.
• the retinal tissue according to any one of [42] to [47] for use in treating a disorder of a retinal cell or retinal tissue, or a disease caused by damage to the retinal tissue.
• the present invention makes it possible to provide a method for regenerating the layered structure of retinal tissue from retinal cells, a method for producing retinal tissue using the method, and retinal tissue.
• this is a bright field micrograph showing the state of re-formation of aggregates one day after dispersing KhES-1-derived aggregates into single cells and seeding them.
• 1231A3-derived aggregates were dispersed into single cells, and this is a bright field micrograph showing the state of re-formation of the aggregates one day after seeding.
• this is a bright field micrograph showing the morphology of aggregates that were re-formed one day after dispersing KhES-1-derived aggregates into single cells and seeding them.
• FIG. 13 is a fluorescence micrograph showing the morphology of aggregates that were reformed 7 days after dispersing KhES-1-derived aggregates into single cells and seeding them in Reference Example 1-2.
• FIG. 13 is a fluorescence micrograph showing the morphology of aggregates re-formed 14 days after dispersing KhES-1-derived aggregates into single cells and seeding them in Reference Example 1-2.
• 1 is a graph showing the results of measuring the area of aggregates re-formed by single cells derived from KhES-1 in Reference Example 1-2 using Image J.
• (A) shows the area of the aggregates on days 1, 7, and 14 after seeding
• (B) shows the ratio (area ratio) of the area of the aggregates on days 7 and 14 to the area of the aggregates on day 1 after seeding.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (DAPI, Rx::Venus, Chx10) sections of the aggregates 15 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained ( ⁇ -catenin) sections of the aggregates 15 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (Collagen IV, Zo-1) sections of the aggregates 15 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (DAPI, Chx10, Ki67, Pax6) sections of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and sections of the aggregates 28 days after seeding were observed for Rx::Venus fluorescence using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (Collagen IV, Zo-1) sections of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and the photographs show the results of observing immunostained (DAPI, Rx::Venus, Collagen IV, Zo-1) sections of the aggregates 28 days after seeding using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (DAPI, Chx10, Ki67, Pax6) sections of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (CRX, RxRg, NRL, Recoverin) sections of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates were dispersed into single cells, and immunostained (DAPI, Islet-1, Brn3, Calretinin) sections of the aggregates 28 days after seeding were observed using a confocal laser scanning fluorescence microscope.
• KhES-1-derived aggregates on the 18th, 25th, 40th, 61st, and 75th days of differentiation were dispersed into single cells, and these are bright field and fluorescence microscopy photographs showing the state of the aggregates that were re-formed on the 3rd, 15th, and 21st days after seeding.
• KhES-1-derived aggregates (BMP4+/BMP4-) on the 40th day of differentiation were dispersed into single cells, and these are bright field microscope and fluorescence microscope (Rx::Venus) photographs showing the state of the re-formed aggregates on days 3, 15, and 21 after seeding.
• Reference Example 1-6 these are fluorescence micrographs showing the morphology of aggregates formed 1 day and 7 days after seeding dispersed retinal cells cryopreserved in various cryopreservation solutions. This is a graph showing the viability (A) of dispersed retinal cells cryopreserved in various cryopreservation solutions in Reference Examples 1-6 after waking them up, and the area (B) of aggregates re-formed using the awake retinal cells.
• Reference Example 1-7 a photograph showing the results of observing, using a confocal laser scanning fluorescence microscope, a section immunostained for the basement membrane expressed in the retinal tissue formed by suspension culture.
• 1 shows immunohistochemical staining images showing the results of investigating Laminin isoforms expressed in mouse fetal neural retinal tissue in Reference Example 1-7.
• 13 shows confocal laser scanning fluorescence microscope photographs confirming the re-formation of sheet-like retinal tissue in adhesion culture from a single cell suspension of retinal cells under conditions 1 to 3 in Reference Example 1-8.
• 13 shows confocal laser scanning fluorescence microscope photographs confirming the re-formation of sheet-like retinal tissue in adhesion culture from a single cell suspension of retinal cells under conditions 1 to 3 in Reference Example 1-8.
• 1 shows bright field and fluorescent micrographs showing the results of confirming the effects of various factors on the re-formation of sheet-like retinal tissue in adhesion culture in Reference Example 1-9.
• 13 is a set of confocal laser scanning fluorescence microscope photographs showing the results of confirming the effects of various factors on the re-formation of sheet-like retinal tissue in adhesion culture in Reference Example 1-9.
• 1 shows bright field micrographs, fluorescence micrographs, and confocal laser scanning fluorescence micrographs showing the results of investigating the concentration and addition period of CHIR99021 in the re-formation of sheet-like retinal tissue in adhesion culture in Reference Example 1-10.
• 13 is a set of confocal laser scanning fluorescence microscope photographs showing the results of investigating the concentration and addition period of CHIR99021 in the re-formation of sheet-like retinal tissue in adhesion culture in Reference Example 1-10.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• 13 is a confocal laser scanning fluorescence microscope photograph showing the results of confirming the effect of CHIR99021 in re-forming sheet-like retinal tissue in adhesion culture in Reference Example 1-12.
• Fluorescence microscopy photographs showing the results of investigating the effect of various factors on maintaining retinal cells in Reference Example 1-13.
• Fluorescence microscopy photographs showing the results of an investigation into effective conditions for the maintenance culture of retinal progenitor cells in Reference Example 1-14.
• 1 is a stereomicroscope photograph showing the results of re-forming a sheet on collagen in Reference Example 1-15.
• 1 is a fluorescence micrograph showing a transplant graft prepared from a re-sheeted retinal cell sheet in Reference Example 1-16. Fluorescence stereomicroscope and fluorescence microscopic photographs showing the results of observing the fundus of the retina after transplantation in Reference Example 1-16.
• 1 is a graph showing the results of FACS analysis for sorting an Rx::Venus-positive fraction from a cell population dispersed into single cells in Reference Example 1-17, (A) is a dot plot, and (B) is a histogram plot.
• 1 shows stereomicroscope and fluorescent stereomicroscope photographs of cell populations with and without sorting in Reference Example 1-17.
• 1 shows confocal laser scanning fluorescence micrographs of cell populations with/without sorting in Reference Example 1-17.
• 1 shows confocal laser scanning fluorescence micrographs of cell populations with/without sorting in Reference Example 1-17.
• 1 shows bright field and fluorescent microscopic photographs of a sorted cell population in Reference Example 1-17. Fluorescence micrographs of Rx::Venus-positive cell populations observed when various proteins were added in Reference Example 1-18. Fluorescence micrographs of Rx::Venus-positive cell populations observed when various low molecular weight compounds were added in Reference Example 1-18. Fluorescence micrographs of Rx::Venus-positive cell populations observed when different concentrations of FGF2, FGF4, and FGF8 were added in Reference Example 1-18.
• FIG. 1 shows bright field and fluorescent micrographs showing the effects of aggregate reformation and retinal differentiation by addition of sorting and FGF8 in Reference Example 1-19.
• FIG. 1 shows bright field microscopic and fluorescent microscopic photographs and a graph showing the results of FACS analysis, showing the aggregate reformation and retinal differentiation effects by addition of sorting and FGF8 in Reference Example 1-19.
• 1 shows bright field microscopic and fluorescent microscopic photographs and a graph showing the results of FACS analysis, which indicate the aggregate reformation and retinal differentiation effects by the addition of FGF8 in Reference Example 1-19.
• 1 shows bright field and fluorescence micrographs showing the effect of addition of FGF8 on re-sheet formation in Reference Example 1-20.
• FIG. 1 shows bright field and fluorescence micrographs showing the effect of addition of FGF8 on re-sheet formation in Reference Example 1-20.
• FIG. 13 is a graph showing bright field microscopic and fluorescent microscopic photographs and FACS analysis results showing the effects of sheet re-formation and retinal differentiation by the addition of FGF8 in Reference Example 1-20.
• 1 shows stereomicroscope and fluorescent stereomicroscope photographs showing the time course of changes in the re-sheeted retinal sheet in Reference Example 1-21.
• FIG. 1 shows markers focused on in surface antigen screening in Reference Example 1-22.
• 1 is a graph showing the results of cell sorting using various surface antigens in Reference Example 1-22.
• 1 is a graph showing the results of cell sorting using various surface antigens in Reference Example 1-22.
• FIG. 1 is a graph showing the results of cell sorting using various surface antigens in Reference Example 1-22.
• 1 is a graph showing the results of cell sorting using various surface antigens in Reference Example 1-22.
• 1 is a graph showing the results of cell sorting using various surface antigens in Reference Example 1-22.
• These are bright field and fluorescence microscope photographs of the differentiation state into brain organoid in Reference Example 1-23. This is a confocal laser scanning fluorescence microscope photograph observing the differentiation state into brain organoid in Reference Example 1-23.
• This is a graph showing the results of FACS analysis of the expression of CD39, CD73, and CXCR4 in Brain Organoid in Reference Example 1-23.
• 1 shows bright field and fluorescent micrographs showing the results of differentiation induction by SAG at different concentrations in Reference Example 1-24.
• 1 shows the results of FACS analysis and a graph showing the numerical values for the expression of CD39 and CXCR4 after differentiation induction with different concentrations of SAG in Reference Example 1-24.
• 1 shows confocal laser scanning fluorescence micrographs of immunostained tissues in which differentiation was induced by SAG at different concentrations in Reference Example 1-24.
• 1 shows confocal laser scanning fluorescence micrographs of immunostained tissues in which differentiation was induced by SAG at different concentrations in Reference Example 1-24.
• 1 shows the results of FACS analysis of the expression of CD39 and BV421 after treatment with different concentrations of SAG in Reference Example 1-24, as well as confocal laser scanning fluorescence micrographs showing immunostained images of tissues in which differentiation was induced by different concentrations of SAG.
• 1 shows confocal laser scanning fluorescence micrographs of immunostained tissues in which differentiation was induced by SAG at different concentrations in Reference Example 1-24.
• 1 shows confocal laser scanning fluorescence micrographs of immunostained tissues in which differentiation was induced by SAG at different concentrations in Reference Example 1-24.
• 1 is a graph showing the results of FACS analysis illustrating the results of investigating substances capable of enhancing CD39 expression in Reference Example 1-25.
• FIG. 1 is a graph showing numerical values of FACS analysis results showing the results of investigating substances capable of enhancing CD39 expression in Reference Example 1-25.
• A shows the results of CD39 positivity and RX::venus positivity
• B shows the results of CXCR4 negativity and RX::venus positivity.
• these are bright field and fluorescence microscopic photographs showing the observation of Islet-1 KO hESC-retina sheets (dd74) produced with FGF8- and FGF8+, and the process of cutting out grafts for transplantation.
• This is a FACS dot plot showing the results of screening various surface antigens of NR in comparison with Brain Organoid in Reference Example 1-27.
• FIG. 1 shows FACS dot plots showing the results of screening various surface antigens of NR in Reference Example 1-27.
• FIG. 13 is a FACS dot plot showing the results of time-course changes in CD9 expression in hESC-retina in Reference Example 1-28.
• FIG. 1 shows FACS dot plots illustrating the results of analyzing the expression of CD9 and SSEA-1 in hESC-retina in Reference Example 1-29.
• 1 is a FACS dot plot showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Example 1-30.
• FIG. 1 is a FACS dot plot showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Example 1-30.
• 13 is a graph showing the results of a purification study using CD9, CD90, CXCR4 and SSEA-1 in hESC-retina in Reference Example 1-30.
• 13 shows photographs illustrating the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Reference Example 1-30.
• 13 shows photographs illustrating the results of a purification study using CD9, CD90, CXCR4, and SSEA-1 in hESC-retina in Reference Example 1-30.
• FIG. 13 is a FACS diagram showing the results of time-dependent changes in the expression of CD9 and SSEA-1 in hESC-retina in Reference Example 1-31.
• FIG. 1 is a schematic diagram showing the process of investigating the combination of an RPE sheet and a retina sheet using gelatin in Reference Example 1-32. 1 shows stereomicroscope and fluorescent microscopic photographs of an RPE sheet and a re-sheet of neural retina cultured on the Transwell used for composite formation using gelatin in Reference Example 1-32. 13 is a stereomicroscope photograph showing the process of peeling the RPE sheet and the re-sheeted neural retina from the Transwell and adding gelatin in Reference Example 1-32.
• 13 shows stereomicroscope photographs showing the process of adding gelatin to an RPE sheet and a re-sheet of neural retina in Reference Example 1-32.
• 1 shows stereomicroscope and fluorescent microscope photographs showing the process of combining an RPE sheet and a re-sheeted neural retina in Reference Example 1-32.
• 13 shows stereomicroscope and fluorescent microscope photographs showing that the RPE sheet and the re-sheeted neural retina are combined and then lifted up with tweezers in Reference Example 1-32.
• 13 shows stereomicroscope and fluorescent microscopic photographs showing that an RPE sheet and a re-sheeted neural retina are combined and then cut out with scissors in Reference Example 1-32.
• 1 shows stereomicroscope and fluorescent micrographs of cross sections of the composite RPE sheet and the reconstituted neural retina in Reference Example 1-32.
• 13 shows stereomicroscope and fluorescent microscopic photographs of the suction and ejection of the cut composite RPE sheet and re-sheeted neural retina in Reference Example 1-32.
• 1 shows stereomicroscope and fluorescent microscopic photographs of an RPE sheet cultured on the Transwell used for conjugation with fibron in Reference Example 1-33, and a re-sheet of neural retina.
• 13 shows stereomicroscope and fluorescent microscope photographs showing the process of a combination study in which an RPE sheet and a re-sheet of neural retina are detached from a Transwell in Reference Example 1-33.
• 13 shows stereomicroscope and fluorescent microscopic photographs showing the process of investigating the combination of Fibrinogen and Thorombin added to the recovered RPE sheets and re-formed sheets in Reference Example 1-33.
• 1 shows stereomicroscope and fluorescent microscope photographs showing the process of combining an RPE sheet and a re-sheeted neural retina in Reference Example 1-33.
• 1 shows stereomicroscope and fluorescent microscope photographs showing the process of combining an RPE sheet and a re-sheeted neural retina in Reference Example 1-33.
• 13 shows stereomicroscope and fluorescent microscope photographs showing that the RPE sheet and the re-sheeted neural retina are combined and then lifted up with tweezers in Reference Example 1-33.
• 13 shows stereomicroscope and fluorescent microscope photographs showing the combined state of an RPE sheet and a re-sheeted neural retina in Reference Example 1-33.
• 13 shows stereomicroscope and fluorescent microscopic photographs showing the process of examining the combination of an RPE sheet and a Transwell mesh in Reference Example 1-33.
• FIG. 1 shows a schematic diagram and photographs illustrating the results of a study on the discharge of unnecessary hydrogel using a cell sifter in Reference Example 1-34. Photographs showing the process of preparing a flattened sheet on a temperature-sensitive culture dish and peeling it off in Reference Example 1-35.
• 112 shows human iPS cells (LPF11 strain) seeded at various cell densities and patterned cultured in Reference Example 2-2. The results are shown 1 hour, 1 day, 2 days, and 3 days after seeding.
• 113 shows the results of immunostaining for Chx10 of human iPS cells (LPF11 strain) (20 days after seeding) seeded at various cell densities and patterned in Reference Example 2-2.
• BMP(+) indicates patterning culture in the presence of BMP4, and BMP(-) indicates patterning culture in the absence of BMP4.
• 114 shows the results of varying the duration of exposure to Y-27632 in patterning culture of human iPS cells (LPF11 strain) in Reference Example 2-3. Bright field microscopy images and immunostaining results for Chx10 are shown 20 days after seeding.
• 115 shows the results of varying the duration of Y-27632 exposure in patterning culture of human iPS cells (DSP-SQ strain) in Reference Example 2-3. The results of immunostaining of Chx10 on the 20th day after seeding are shown.
• 116 shows the results of changing the BMP4 concentration in patterning culture of human iPS cells (LPF11 strain) in Reference Example 2-4.
• FIG. 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) patterned and cultured at various BMP4 concentrations in Reference Example 2-4.
• Figure 118 shows the expression of marker genes in cells cultured in the patterning culture method of WO2023/003025 (white) and the patterning culture method of Reference Example 2 (black) in Reference Example 2. The left side of the dashed line is the neural retina marker gene, and the right side is the non-target cell marker gene.
• FIG. 119 shows an experimental outline of Example 1.
• Figure 120 is a bright-field micrograph showing the morphology of aggregates formed in Example 1 on days 3, 13, and 23 after seeding in three-dimensional culture from a retinal progenitor cell sheet on day 20 of patterning culture.
• Figure 121 is a confocal laser microscope photograph showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1) of aggregates formed 23 days after seeding in three-dimensional culture in Example 1, in which a retinal progenitor cell sheet on the 20th day of patterning culture was dispersed into single cells.
• FIG. 122 shows an experimental outline of Example 2.
• Figure 123 is a bright-field micrograph showing the morphology of aggregates formed in Example 2 on days 3, 13, and 23 after seeding in two-dimensional culture from a retinal progenitor cell sheet on day 20 of patterning culture.
• Figure 124 is a confocal laser microscope photograph showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1) of aggregates formed 23 days after seeding in two-dimensional culture in Example 2, in which a retinal progenitor cell sheet on the 20th day of patterning culture was dispersed into single cells.
• FIG. 125 shows an experimental outline of Example 3.
• FIG. 125 shows an experimental outline of Example 3.
• Figure 126 shows the results of dispersing a retinal progenitor cell sheet on day 20 of patterning culture into single cells and purifying a CD9-positive cell fraction in Example 3.
• Figure 127 is a bright field micrograph (right) showing the morphology of aggregates formed 23 days after seeding under 3D culture from CD9-positive cells sorted from a retinal progenitor cell sheet on day 20 of patterning culture in Example 3, and a confocal laser microscope photograph (left) showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1).
• Figure 128 shows the results of measuring the expression levels of Rx (neural retina marker) and Emx2 (dorsal telencephalon marker) by real-time PCR for aggregates formed from CD9-positive cells sorted from a retinal progenitor cell sheet in Example 3.
• FIG. 129 shows an experimental outline of Example 4.
• Figure 130 shows a bright field micrograph (right) showing the morphology of aggregates formed 23 days after seeding under two-dimensional culture from CD9-positive cells sorted from a retinal progenitor cell sheet on day 20 of patterning culture in Example 4, and a confocal laser microscope photograph (left) showing immunostained images (Chx10, Pax6, Rx, Crx, ZO-1).
• Figure 131 is a bright-field micrograph showing the morphology of aggregates formed 23 days after seeding in 3D culture in the presence of Y27632 (final concentration 0 or 10 ⁇ M), SAG (final concentration 0, 150, 300, or 600 nM), and CHIR99021 (final concentration 0, 1.5, 3, or 6 ⁇ M) in Example 5, in which a retinal progenitor cell sheet on day 20 of patterning culture was dispersed into single cells.
• Y27632 final concentration 0 or 10 ⁇ M
• SAG final concentration 0, 150, 300, or 600 nM
• CHIR99021 final concentration 0, 1.5, 3, or 6 ⁇ M
• Figure 132 is a bright-field micrograph showing the morphology of aggregates formed 23 days after seeding in 3D culture in the presence of Y27632 (final concentration 10 ⁇ M), SAG (final concentration 300 nM), and CHIR99021 (final concentrations 0, 1.5, 3, or 6 ⁇ M) in Example 5, in which a retinal progenitor cell sheet on day 20 of patterning culture was dispersed into single cells.
• Stem cells refer to undifferentiated cells that have differentiation and proliferation capabilities (particularly self-renewal capabilities). Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation capabilities.
• Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, endoderm) and/or extraembryonic tissues.
• Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, but not all types.
• Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues and cells.
• pluripotent stem cells can be derived from fertilized eggs, cloned embryos, germline stem cells, tissue stem cells, somatic cells, etc.
• pluripotent stem cells include embryonic stem cells (ES cells), EG cells (embryonic germ cells), and induced pluripotent stem cells (iPS cells).
• Muse cells multi-lineage differentiating stress ending cells obtained from mesenchymal stem cells (MSCs) and GS cells created from germ cells (e.g. testes) are also included in pluripotent stem cells.
• Embryonic stem cells Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. Embryonic stem cells can be produced by culturing inner cell aggregates on feeder cells or in a medium containing bFGF. Methods for producing embryonic stem cells are described in, for example, WO96/22362, WO02/101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from a designated institution, and can also be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. Human embryonic stem cells Crx::Venus and Rx::Venus (both derived from KhES-1) are available from the National Institute of Physical and Chemical Research.
• “Induced pluripotent stem cells” are cells whose pluripotency has been induced by reprogramming somatic cells using known methods.
• induced pluripotent stem cells in mouse cells (Cell, 2006, 126(4), pp.663-676).
• induced pluripotent stem cells were also established in human fibroblast cells, and they have the same pluripotency and self-renewal ability as embryonic stem cells (Cell, 2007, 131(5), pp.861-872; Science, 2007, 318(5858), pp.1917-1920; Nat. Biotechnol., 2008, 26(1), pp.101-106).
• induced pluripotent stem cells include cells in which pluripotency is induced by reprogramming somatic cells differentiated into fibroblasts or peripheral blood mononuclear cells through the expression of any combination of multiple genes selected from a group of reprogramming genes including Oct3/4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc.
• Preferred combinations of reprogramming factors include (1) Oct3/4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3/4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466).
• induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341, pp. 651-654).
• induced pluripotent stem cell lines for example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells established at Kyoto University are available from Kyoto University and iPS Academia Japan Inc.
• established induced pluripotent stem cell lines for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells established at Kyoto University are available from Kyoto University.
• the pluripotent stem cells are preferably embryonic stem cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.
• pluripotent stem cells are human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).
• iPS cells human induced pluripotent stem cells
• ES cells human embryonic stem cells
• Pluripotent stem cells such as human iPS cells can be subjected to maintenance culture and expansion culture using methods well known to those skilled in the art.
• Nervous tissue refers to tissue composed of nervous system cells, such as the developing or adult cerebrum, midbrain, cerebellum, spinal cord, retina, peripheral nerves, forebrain, hindbrain, telencephalon, and diencephalon. Nervous tissue may form a layered epithelial structure (neuroepithelium), and the amount of neuroepithelium present in a cell aggregate can be evaluated by bright-field observation using an optical microscope.
• nervous system cells such as the developing or adult cerebrum, midbrain, cerebellum, spinal cord, retina, peripheral nerves, forebrain, hindbrain, telencephalon, and diencephalon.
• Nervous tissue may form a layered epithelial structure (neuroepithelium), and the amount of neuroepithelium present in a cell aggregate can be evaluated by bright-field observation using an optical microscope.
• Neural cells refers to cells of ectoderm-derived tissues other than epidermal cells. In other words, it includes cells such as neural progenitor cells, neurons (nerve cells), glia, neural stem cells, neuronal progenitor cells, and glial progenitor cells. Neural cells also include cells that make up the retinal tissue described below (retinal cells), retinal progenitor cells, retinal layer-specific nerve cells, neural retinal cells, and retinal pigment epithelial cells. Neural cells can be identified using markers such as nestin, TuJ1, PSA-NCAM, and N-cadherin.
• Neuronal cells are functional cells that form neural circuits and contribute to signal transmission, and can be identified using the expression of immature neuronal markers such as TuJ1, Dcx, and HuC/D, and/or mature neuronal markers such as Map2 and NeuN.
• Neural precursor cells are a collection of precursor cells, including neural stem cells, neuronal precursor cells, and glial precursor cells, and have the ability to proliferate and produce neurons and glia. Neural precursor cells can be identified using markers such as Nestin, GLAST, Sox2, Sox1, Musashi, and Pax6. Alternatively, cells that are positive for neural cell markers and proliferation markers (Ki67, pH3, MCM) can also be identified as neural precursor cells.
• Retinal tissue refers to tissue in which one or more types of retinal cells that make up each retinal layer in a living retina exist in a certain order
• neural retina refers to retinal tissue that includes the inner neural retinal layer, which does not include the retinal pigment epithelium layer, among the retinal layers described below.
• Retinal cells refers to cells that make up each retinal layer in a living retina, or their precursor cells.
• Retinal cells include, but are not limited to, photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelial (RPE) cells, ciliary bodies, their precursor cells (e.g. photoreceptor precursor cells, bipolar cell precursor cells, retinal pigment epithelial precursor cells, etc.), neural retinal precursor cells, retinal precursor cells, etc.
• precursor cells e.g. photoreceptor precursor cells, bipolar cell precursor cells, retinal pigment epithelial precursor cells, etc.
• the cells that make up the neural retinal layer specifically include photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneuron cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, and their precursor cells (e.g. photoreceptor precursor cells, bipolar cell precursor cells, etc.).
• neural retinal cells do not include retinal pigment epithelial cells and ciliary body cells.
• “Mature retinal cells” refers to cells that can be contained in the retinal tissue of human adults, specifically, differentiated cells such as photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), Müller glial cells, retinal pigment epithelial (RPE) cells, and ciliary cells.
• “Immature retinal cells” refers to precursor cells that are committed to differentiating into mature retinal cells (e.g., photoreceptor precursor cells, bipolar cell precursor cells, retinal precursor cells, etc.).
• Photoreceptor precursor cells horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, Müller glial precursor cells, and retinal pigment epithelial precursor cells refer to precursor cells that are committed to differentiating into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, Müller glial cells, and retinal pigment epithelial cells, respectively.
• Retinal progenitor cells refer to precursor cells that can differentiate into any immature retinal cell, such as photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, Müller glial cells, and retinal pigment epithelial precursor cells, and ultimately, precursor cells that can differentiate into any mature retinal cell, such as photoreceptors, rod photoreceptors, cone photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.
• Neural retinal progenitor cells refer to precursor cells that can differentiate into any immature neural retinal cell, such as photoreceptor precursor cells, horizontal cell precursor cells, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, and Muller glial cells, and ultimately, can differentiate into any mature neural retinal cell, such as photoreceptors, rod photoreceptors, cone photoreceptors, horizontal cells, bipolar cells, amacrine cells, and retinal ganglion cells. Neural retinal progenitor cells do not have the ability to differentiate into retinal pigment epithelial cells.
• photoreceptor cells exist in the photoreceptor layer of the retina and have the role of absorbing light stimuli and converting them into electrical signals.
• photoreceptor cells There are two types of photoreceptor cells: cones that function in bright places and rods that function in dark places (called cone photoreceptors and rod photoreceptors, respectively).
• cone photoreceptor cells include S cone photoreceptor cells that express S-opsin and receive blue light, L cone photoreceptor cells that express L-opsin and receive red light, and M cone photoreceptor cells that express M-opsin and receive green light.
• Photoreceptor cells differentiate and mature from photoreceptor precursor cells.
• a cell is a photoreceptor cell or a photoreceptor precursor cell by, for example, the expression of cell markers (Crx and Blimp1 expressed in photoreceptor precursor cells, Recoverin expressed in photoreceptor cells, rhodopsin, S-Opsin, and M/L-Opsin expressed in mature photoreceptor cells, etc.) described below, the formation of an outer segment structure, etc.
• the photoreceptor precursor cells are Crx-positive cells
• the photoreceptor cells are rhodopsin, S-Opsin, and M/L-Opsin-positive cells.
• the rod photoreceptor cells are NRL and Rhodopsin-positive cells.
• the S cone photoreceptor cells are S-opsin-positive cells
• the L cone photoreceptor cells are L-opsin-positive cells
• the M cone photoreceptor cells are M-opsin-positive cells.
• neural retinal cell markers neural retinal cell markers
• neural retinal markers neural retinal cell markers
• proportion of neural retinal cell marker-positive cells in a cell population or tissue can be easily confirmed by a person skilled in the art.
• a method using an antibody, a method using a nucleic acid primer, and a method using a sequence reaction can be mentioned.
• the expression of the protein of the neural retinal cell marker can be confirmed by dividing the number of cells positive for a specific neural retinal cell marker by the total number of cells, for example, by flow cytometry (FACS) using a commercially available antibody, immunostaining, etc.
• FACS flow cytometry
• the expression of the RNA of the neural retinal cell marker can be confirmed, for example, by the PCR method, semi-quantitative PCR method, or quantitative PCR method (e.g., real-time PCR method).
• the expression of the RNA of the neural retinal cell marker can be confirmed, for example, by using a nucleic acid sequencer (e.g., next-generation sequencer).
• Neural retinal cell markers include Rx (also called Rax) and PAX6 expressed in retinal progenitor cells, Rx, PAX6 and Chx10 (also called Vsx2) expressed in neural retinal progenitor cells, Crx and Blimp1 expressed in photoreceptor progenitor cells, etc.
• Chx10 which is strongly expressed in bipolar cells, PKC ⁇ , Go ⁇ , VSX1 and L7 expressed in bipolar cells, TuJ1 and Brn3 expressed in retinal ganglion cells, Calretinin and HPC-1 expressed in amacrine cells, Calbindin expressed in horizontal cells, Recoverin expressed in photoreceptor cells and photoreceptor progenitor cells, Rhodopsin expressed in rod cells, Rod photoreceptor cells and Examples of such antigens include Nrl expressed in rod photoreceptor precursor cells, S-opsin and LM-opsin expressed in cone photoreceptor cells, RXR- ⁇ expressed in cone cells, cone photoreceptor precursor cells and ganglion cells, TR ⁇ 2, OTX2 and OC2 expressed in cone photoreceptor cells or their precursor cells that appear in the early differentiation stage among cone photoreceptor cells, and Pax6 expressed in common in horizontal cells, amacrine cells and ganglion cells.
• “Positive cells” refer to cells that express a particular marker on the cell surface or intracellularly.
• Chx10 positive cells refer to cells that express Chx10 protein.
• Retinal pigment epithelial cells refers to epithelial cells present outside the neural retina in a living retina. Whether a cell is a retinal pigment epithelial cell can be easily confirmed by a person skilled in the art, for example, by the expression of cell markers (MITF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.), the presence of melanin granules (black-brown), tight junctions between cells, characteristic polygonal/cobblestone cell morphology, etc.
• cell markers MIRF, Pax6, PMEL17, TYRP1, TRPM1, ALDH1A3, GPNMB, RPE65, CRALBP, MERTK, BEST1, TTR, etc.
• 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 monolayer or multilayer sheet-like structure consisting of single or multiple retinal pigment epithelial cells that are biologically bonded to each other at least in two dimensions.
• Retinal layers refers to the layers that make up the retina, and specifically includes the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.
• Neuronal retinal layer refers to each layer that constitutes the neural retina, and specifically includes the photoreceptor layer, outer limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and inner limiting membrane.
• Photoreceptor layer refers to the retinal layer that is formed on the outermost side of the neural retina and contains a large amount of one or more types of cells selected from the group consisting of photoreceptors (rod photoreceptors, cone photoreceptors), photoreceptor precursor cells, and retinal progenitor cells. Each layer other than the photoreceptor layer is called an inner layer. It can be confirmed which retinal layer each cell constitutes by a known method, such as the presence or absence of expression or the degree of expression of a cell marker.
• the layer containing the proliferating neural retinal precursor cells is called the "neuroblastic layer", and there is an inner neuroblastic layer and an outer neuroblastic layer.
• the neutral layer there is an inner neuroblastic layer and an outer neuroblastic layer.
• Ciliary body includes the developing and adult “ciliary body”, “ciliary margin” and “ciliary body”. Markers of the “ciliary body” include Zic1, MAL, HNF1beta, FoxQ1, CLDN2, CLDN1, GPR177, AQP1 and AQP4.
• the "ciliary marginal zone (CMZ)" can be, for example, a tissue that exists in the boundary region between the neural retina and the retinal pigment epithelium in a living retina, and can be an area that contains retinal tissue stem cells (retinal stem cells).
• the ciliary margin is also called the ciliary margin or retinal margin, and the ciliary margin, ciliary margin and retinal margin are equivalent tissues.
• the ciliary body margin is known to play an important role in supplying retinal progenitor cells and differentiated cells to retinal tissue, maintaining retinal tissue structure, and the like.
• Marker genes for the ciliary body margin include, for example, the Rdh10 gene (positive), the Otx1 gene (positive), and the Zic1 (positive).
• the "ciliary body margin-like structure" refers to a structure similar to the ciliary body margin.
• Cerebral tissue refers to tissue in which one or at least multiple types of cells constituting the fetal or adult cerebrum (e.g., cortical neural precursor cells, dorsal cerebral nervous system precursor cells, ventral cerebral nervous system precursor cells, cerebral layer structure-specific nerve cells (neurons), layer I neurons, layer II neurons, layer III neurons, layer IV neurons, layer V neurons, layer VI neurons, glial cells (astrocytes and oligodendrocytes), their precursor cells, etc.) are arranged three-dimensionally in layers.
• the fetal cerebrum is also called the forebrain or telencephalon.
• the presence of each cell can be confirmed by known methods, such as the presence or absence or the degree of expression of cell markers.
• Cerebral layers refers to each layer that constitutes the adult or fetal cerebrum, specifically including the molecular layer, external granular layer, external pyramidal cell layer, internal granular layer, neuronal cell layer (inner pyramidal cell layer), polymorphic cell layer, first layer, second layer, third layer, fourth layer, fifth layer, sixth layer, cortical zone, intermediate zone, subventricular zone, and ventricular zone.
• Cerebral nervous system precursor cells include neuron precursor cells, layer 1 neuron precursor cells, layer 2 neuron precursor cells, layer 3 neuron precursor cells, layer 4 neuron precursor cells, layer 5 neuron precursor cells, layer 6 neuron precursor cells, astrocyte precursor cells, oligodendrocyte precursor cells, etc.
• Each cell is a precursor cell that is determined to differentiate into layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, astrocytes, and oligodendrocytes.
• “Cerebral nervous system progenitor cells” include multipotent stem cells (multipotent neural stem cells) that have the ability to differentiate (multi-differentiation ability) into at least several of the following lineages: layer I neurons, layer II neurons, layer III neurons, layer IV neurons, layer V neurons, layer VI neurons, astrocytes, and oligodendrocytes.
• Cerebral layer-specific neurons refers to cells that make up the cerebral layer and are specific to the cerebral layer. Examples of cerebral layer-specific neurons include layer 1 neurons, layer 2 neurons, layer 3 neurons, layer 4 neurons, layer 5 neurons, layer 6 neurons, cerebral excitatory neurons, and cerebral inhibitory neurons.
• Cerebral cell markers include FoxG1 (also known as Bf1) expressed in cerebral cells, Sox2 and Nestin expressed in cerebral nervous system progenitor cells, Pax6 and Emx2 expressed in dorsal cerebral nervous system progenitor cells, Dlx1, Dlx2 and Nkx2.1 expressed in ventral cerebral nervous system progenitor cells, Tbr2, Nex and Svet1 expressed in neuronal progenitor cells, Tbr1 expressed in layer 6 neurons, Ctip2 expressed in layer 5 neurons, ROR ⁇ expressed in layer 4 neurons, Cux1 or Brn2 expressed in layer 3 or layer 2 neurons, and Reelin expressed in layer 1 neurons.
• FoxG1 also known as Bf1 expressed in cerebral cells
• Sox2 and Nestin expressed in cerebral nervous system progenitor cells Pax6 and Emx2 expressed in dorsal cerebral nervous system progenitor cells
• Dlx1, Dlx2 and Nkx2.1 expressed in ventral cerebral nervous system progenitor cells Tbr2, Nex and Svet1
• cell aggregate is not particularly limited as long as it is a three-dimensional structure formed by adhesion of multiple cells, and refers to, for example, a mass formed by the gathering of cells dispersed in a medium such as a culture medium, or a mass of cells formed through cell division. Cell aggregates also include those that form a specific tissue.
• Spherical cell aggregate refers to a cell aggregate having a three-dimensional shape close to a sphere.
• a three-dimensional shape close to a sphere is a shape having a three-dimensional structure, and examples of such shapes include a spherical shape that is circular or elliptical when projected onto a two-dimensional surface, and a shape formed by the fusion of multiple spherical shapes (for example, a shape formed by two to four overlapping circular or elliptical shapes when projected onto a two-dimensional surface).
• the core of the aggregate has a vesicular layered structure, and is characterized by being observed under a bright-field microscope with a dark center and a bright outer edge.
• Epithelial tissue refers to tissue formed by cells tightly covering the surface of the body, lumen (such as the digestive tract), and body cavity (such as the pericardial cavity). Cells forming epithelial tissue are called epithelial cells. Epithelial cells have apical-basal polarity. Epithelial cells can form strong bonds between themselves through adherens junctions and/or tight junctions to form cell layers. Epithelial tissue is a tissue formed by stacking one to a dozen or so cell layers. Tissues that can form epithelial tissue include fetal and/or adult retinal tissue, cerebrospinal tissue, ocular tissue, and neural tissue. The neural retina in this specification is also epithelial tissue. "Epithelial structure” refers to a structure that epithelial tissue has characteristically (e.g., having basal and apical polarity).
• Continuous epithelial tissue refers to tissue having a continuous epithelial structure.
• a continuous epithelial structure refers to a state in which epithelial tissue is continuous.
• a continuous epithelial tissue refers to a state in which, for example, 10 cells to 10 cells are lined up in the tangential direction of the epithelial tissue, preferably 30 cells to 10 cells, and more preferably 10 cells to 10 cells are lined up in the tangential direction.
• the continuous epithelial structure formed in retinal tissue has an apical surface characteristic of epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to and continuous with at least the photoreceptor layer (outer nuclear layer) among the layers that form the neural retina.
• the apical surface is formed on the surface of the aggregate, and 10 or more, preferably 30 or more, more preferably 100 or more, and even more preferably 400 or more photoreceptor cells or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface.
• epithelial tissue is polarized to form an "apical surface," a “basal surface,” and a “basement membrane.”
• Basal membrane refers to the layer on the basal side (basement membrane) produced by epithelial cells, contains a lot of laminin and type IV collagen, and has a thickness of 50 to 100 nm.
• Basal surface refers to the surface (superficial surface) formed on the “basement membrane” side.
• “Apical surface” refers to the surface (superficial surface) formed on the opposite side to the “basement membrane.” In one aspect, “apical surface” refers to the surface in contact with the photoreceptor layer (outer nuclear layer) where the outer limiting membrane is formed and photoreceptors and photoreceptor precursor cells are present in retinal tissue at a developmental stage where photoreceptors or photoreceptor precursor cells can be found, in which the outer limiting membrane is formed and the photoreceptor layer (outer nuclear layer) where the photoreceptors and photoreceptor precursor cells are present.
• apical surfaces can be identified by immunostaining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical PKC (hereinafter abbreviated as "aPKC”), tight junction marker (Zo-1), ERM proteins Ezrin, E-cadherin, and N-cadherin).
• aPKC atypical PKC
• Zo-1 tight junction marker
• ERM proteins Ezrin E-cadherin
• E-cadherin E-cadherin
• N-cadherin N-cadherin
• One aspect of the present invention is a method for producing a cell aggregate containing retinal tissue having an epithelial structure (or a multi-layered structure) from a dispersed retinal cell population (herein, a "cell aggregate containing retinal tissue” may be simply referred to as "retinal tissue”), which comprises subjecting a dispersed retinal cell population to suspension or adhesion culture in a medium containing a substance acting on the Wnt signaling pathway.
• the retinal system cells are as defined above.
• the retinal system cells include one or more types of cells selected from the group consisting of retinal progenitor cells and photoreceptor progenitor cells, and may also include other cells such as photoreceptors (rod photoreceptors, cone photoreceptors), horizontal cells, amacrine cells, retinal ganglion cells (ganglion cells), bipolar cells (rod bipolar cells, cone bipolar cells), and Müller glial cells.
• the retinal system cells are preferably neural retinal system cells. .
• the retinal cells derived from pluripotent stem cells as starting cells in the production method of the present invention can be obtained by inducing differentiation of pluripotent stem cells.
• the retinal cell population as starting cells is (1) Seeding pluripotent stem cells into region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a portion of region A and having lower cell adhesiveness than region A; (2) culturing the pluripotent stem cells seeded in step (1) in a medium containing a ROCK inhibitor; (3) culturing the cells obtained after step (2) in a medium containing a substance acting on the BMP signaling pathway to obtain retinal tissue; and (4) dispersing the retinal tissue obtained in step (3) to obtain a dispersed retinal cell population.
• Region A is a region where pluripotent stem cells can be maintained in adhesion culture.
• Region B is a region with lower cell adhesiveness than region A to such an extent that, when pluripotent stem cells are seeded in region A, the cells do not spread into region B adjacent to region A during induction of differentiation into retinal tissue (i.e., the period until differentiation into retinal tissue can be confirmed).
• Comparison of cell adhesiveness can be performed by seeding an excess amount (e.g., 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2 ) of pluripotent stem cells relative to the area, culturing at 37° C. and 5% CO 2 for 24 hours, and then comparing the ratio of the area to which the cells adhere.
• the cell adhesiveness is usually evaluated after removing the medium after 24 hours of culture, washing the cells with medium or buffer solution, and removing unadhered cells.
• Region A may be, for example, a region where pluripotent stem cells are seeded in an excess amount relative to the area (e.g., 5 x 10 to 10 x 10 cells/cm), and after culturing for 24 hours at 37°C and 5% CO2 , the cells adhere to 70% or more, 80% or more, or 90% or more of the area.
• region B is a cell non-adhesive region.
• a cell non-adhesive region means a region that does not have sufficient cell adhesiveness to maintain pluripotent stem cells in adherent culture.
• a cell non-adhesive region may be, for example, a region in which cells adhere to 30% or less, 20% or less, or 10% or less of the area after seeding pluripotent stem cells in an excess amount relative to the area (e.g., 5 ⁇ 10 5 to 10 ⁇ 10 5 cells/cm 2 ) and culturing for 24 hours at 37° C. and 5% CO 2.
• the culture substrate may be made of any material as long as it is possible to form regions A and B on its surface.
• the culture substrate may be made of inorganic materials such as metal, glass, and silicone, or organic materials such as plastics (e.g., polystyrene resin, polyethylene resin, polypropylene resin, ABS resin, nylon, acrylic resin, fluororesin, polycarbonate resin, polyurethane resin, methylpentene resin, phenol resin, melamine resin, epoxy resin, polyvinyl chloride resin, polytetrafluoroethylene tetrafluoroethylene resin).
• the culture substrate is glass or plastic, particularly polystyrene resin.
• the culture substrate may have any shape generally used for cell culture, and is not particularly limited.
• the culture substrate may be, for example, a culture vessel such as a petri dish, a plate, a bottle, a chamber, a multi-well plate (e.g., a 6-, 12-, 24-, 48-, 96-, or 384-well plate), a film, or a porous membrane.
• the culture substrate usually has region A and region B on a surface that is horizontal to the direction of gravity (on the bottom surface in the case of a culture vessel). In one embodiment, the culture substrate has region A and region B on the same plane. In one embodiment, when the culture vessel has region A and region B on the plane of the bottom surface, the inner wall surface of the culture vessel is not region B.
• the culture substrate has a convex portion on its surface and has region A on the upper surface of the convex portion
• the side surface of the convex portion adjacent to the upper surface of the convex portion is region B, but region A on the upper surface of the convex portion and region B on the side surface of the convex portion are considered to be in the same plane.
• Area A may be an area where the surface of a cell-adhesive culture substrate is exposed, or it may be an area where the surface of the culture substrate is coated with a cell-adhesive substance in order to impart cell adhesiveness to the surface of the culture substrate or to increase the cell adhesiveness of the surface of the culture substrate.
• cell adhesive substances include positively charged polymers such as poly-L-lysine and poly-L-ornithine; laminin; collagens such as type I collagen, type II collagen, type III collagen, type IV collagen, type V collagen, and type VII collagen; tenascin; fibrillin; fibronectin; vitronectin; elastin; entactin; proteoglycans composed of sulfated glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate, and core proteins; glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, dermatan sulfate, and hyaluronic acid; Synthemax (registered trademark, a vitronectin derivative), Matrigel (registered trademark), and the like.
• region A is coated with laminin.
• Laminin is a heterotrimeric molecule consisting of three subunit chains, an ⁇ chain, a ⁇ chain, and a ⁇ chain. There are five known types of ⁇ chains, ⁇ 1 to ⁇ 5, three known types of ⁇ chains, ⁇ 1 to ⁇ 3, and three known types of ⁇ chains, ⁇ 1 to ⁇ 3. Each laminin isoform is represented by a number indicating the constituent subunits (for example, laminin 111 is composed of an ⁇ 1 chain, a ⁇ 1 chain, and a ⁇ 1 chain).
• 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. Fragments of laminins include E8 fragments, which are fragments of the integrin binding site, such as laminin 211-E8, laminin 311-E8, laminin 411-E8, laminin 511-E8, and the like.
• the laminin is laminin 511 or a fragment thereof. In a further embodiment, the laminin is a laminin 511-E8 fragment.
• commercially available products such as iMatrix-511 (Nippi, Inc.) can be used.
• iMatrix-511 (Nippi, Inc.) contains the laminin 511-E8 fragment.
• Region B may be an exposed region of the culture substrate having a lower cell adhesiveness than region A, or may be a region in which the surface of the culture substrate is coated with some substance to make the region have a lower cell adhesiveness than region A. In one embodiment, region B is coated with a cell non-adhesive substance.
• cell non-adhesive substances examples include MPC (2-methacryloyloxyethyl phosphorylcholine) polymer; celluloses such as methylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose; polyethylene oxide; carboxyvinyl polymer; polyvinylpyrrolidone; polyethylene glycol; polyamides such as polyacrylamide and poly-N-isopropylacrylamide; polysaccharides such as chitin, chitosan, hyaluronic acid, alginic acid, starch, pectin, carrageenan, guar gum, gum arabic, and dextran; albumin and derivatives thereof.
• the cell non-adhesive substance is an MPC polymer.
• region A is not particularly limited, and examples thereof include a circle, an ellipse, and a polygon (e.g., a triangle, a rectangle, a pentagon, a hexagon, an octagon, a decagon, a dodecagon, etc.).
• region A is a circle.
• a circle is used to mean a shape that is recognized in the art as being substantially circular, and is used to mean an approximately circular shape including a perfect circle.
• the area of region A may be, for example, but is not limited to, 0.01 to 100 cm 2 , 0.01 to 30 cm 2 , 0.01 to 10 cm 2 , 0.03 to 30 cm 2 , 0.03 to 10 cm 2 , or 0.1 to 10 cm 2 . In one embodiment, 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 are independently selected).
• 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 .
• Area A may be circular or polygonal with an area equivalent thereto, with a diameter of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 cm or more, and 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cm or less (the upper and lower limits are independently selected).
• area A is circular with a diameter of 0.1-10 cm, 0.1-5 cm, 0.1-3 cm, or 0.2-1 cm. In a further embodiment, area A is circular with a diameter of 1-10 cm, 1-5 cm, or 1-3 cm.
• Area A is adjacent to region B at least in part. In one embodiment, the entire outer edge of region A is adjacent to region B (i.e., surrounded by region B).
• the culture substrate may have multiple regions A and/or multiple regions B on its surface. When the culture substrate includes multiple regions A on its surface, the distance between two regions A that sandwich region B is, but is not limited to, about 1 mm or more, for example.
• Area A and area B may be formed on the surface of the culture substrate by any method used for cell patterning. Area A and area B can be formed on the surface of the culture substrate by 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 substrate to make the cellular adhesiveness of the treated and untreated regions different. Regions A and B can be formed, for example, by coating a portion of the surface of the culture substrate with a cell adhesive or non-cell adhesive substance. For example, it is conceivable to mask a portion of the surface of the culture substrate and coat the unmasked region with a cell adhesive or non-cell adhesive substance. Alternatively, a layer of a cell adhesive or non-cell adhesive substance may be formed on the culture substrate, and a portion of the layer may be treated to change the cellular adhesiveness. The treated portion may be an exposed portion of the surface of the culture substrate, or a portion in which the properties of the cell non-adhesive or cell adhesive substance have been changed by the treatment.
• a sheet having holes of the shape and size of region A can be created using a mold printed with a 3D printer, the surface of the culture substrate can be covered with this sheet, the surface not covered with the sheet can be coated with a cell adhesive substance, and then the sheet can be removed to form region A and region B on the culture substrate.
• a sheet of the shape and size of region A can be created, placed on the surface of the culture substrate, the surface not covered with the sheet can be coated with a cell non-adhesive substance, and then the sheet can be removed and the surface covered with the sheet can be coated with a cell adhesive substance to form region A and region B on the culture substrate.
• the sheet can be formed from a biocompatible material such as polydimethylsiloxane (PDMS), polyethylene glycol hydrogel, or agarose gel.
• the coating can be performed, for example, by contacting the culture substrate with a solution of a cell adhesive substance or a cell non-adhesive substance and reacting it at 37° C. or room temperature for a required time (e.g., 1 hour or more).
• the concentrations of the cell adhesive substance and the cell non-adhesive substance can be appropriately determined by one skilled in the art, but in the case of laminin, for example, the concentrations can be 0.1 to 1 ⁇ g/cm 2 , 0.1 to 0.5 ⁇ g/cm 2 , or about 0.25 ⁇ g/cm 2.
• Regions A and B can also be formed on the culture substrate by forming droplets of a solution of the cell adhesive substance on the culture substrate and allowing them to react, without using the above-mentioned sheet.
• the culture substrate may have a convex portion (also called a pillar) on its surface, and may have region A on the upper surface of the convex portion.
• the side surface of the convex portion adjacent to the upper surface of the convex portion is designated as region B.
• the shape of the convex portion is not particularly limited, and may be a cylinder or a prism.
• a cylinder is used to mean a column whose cross section is substantially circular, including a perfect circle, as long as it has a shape that is recognized in the industry as being substantially circular.
• the convex portion is cylindrical.
• the height of the convex portion 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 convex portion may be the same as or different from the culture substrate.
• Examples of materials for the convex portion include, in addition to those exemplified in this specification as materials for the culture substrate, polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyamide (PA), polymethyl glutarimide (PMGI), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene vinyl acetate (PEVA), polyethylene oxide (PEO), etc.
• PDMS polydimethylsiloxane
• PMMA polymethyl methacrylate
• PET polyethylene terephthalate
• PA polyamide
• PMGI polymethyl glutarimide
• PVA polyvinyl alcohol
• PEG polyethylene glycol
• PEVA polyethylene vinyl acetate
• PEO polyethylene oxide
• the cells may be seeded only in region A, or may be seeded over the entire culture substrate including regions A and B. For example, when region B is a non-cell-adhesive region, the cells may be seeded over the entire culture substrate.
• the pluripotent stem cells are seeded onto the culture substrate at a cell number such that the cells occupy 70% or more of region A.
• the pluripotent stem cells are at or above 0.5x10 , 0.6x10, 0.7x10 , 0.8x10 , 0.9x10 , 1.0x10 , 1.1x10 , 1.2x10, 1.3x10 , 1.4x10 , 1.5x10, 1.6x10, 1.7x10, 1.8x10, 1.9x10 , 2.0x10 , 2.1x10 , 2.2x10, 2.3x10 , or 2.4x10 cells/ cm2 .
• pluripotent stem cells collected and dispersed using a cell dispersion solution containing an enzyme e.g., trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain
• an enzyme e.g., trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain
• a chelating agent e.g., ethylenediaminetetraacetic acid (EDTA)
• EDTA ethylenediaminetetraacetic acid
• the cell dispersion solution for example, commercially available products such as TrypLE TM Select (Thermo Fisher Scientific) and TrypLE TM Express (Thermo Fisher Scientific) may be used.
• the induction of differentiation of pluripotent stem cells into retinal lineage cells is carried out by adhesion culture on the culture substrate. After seeding the pluripotent stem cells on the culture substrate, they may be cultured for a certain period of time in a medium containing factors for maintaining undifferentiation, and then the induction of differentiation of the pluripotent stem cells into retinal lineage cells may be initiated.
• the time when the culture in a medium not containing factors for maintaining undifferentiation is initiated is regarded as the time when the induction of differentiation into retinal lineage cells is initiated.
• inducing differentiation of pluripotent stem cells into retinal lineage cells includes culturing the pluripotent stem cells in a medium containing a substance that acts on the BMP signaling pathway.
• the culture in the medium containing the substance that acts on the BMP signaling pathway may be started at the start of differentiation induction or may be started a certain period of time after the start of differentiation induction (e.g., 4 to 6 days).
• Inducing differentiation of pluripotent stem cells into retinal lineage cells may include culturing the pluripotent stem cells in a medium containing a BMP signaling pathway inhibitor prior to culturing in a medium containing a BMP signaling pathway agonist. Culturing the cells in the presence of a BMP signaling pathway inhibitor prior to adding the BMP signaling pathway agonist can increase the efficiency of differentiation into retinal tissue.
• the medium used to induce differentiation of pluripotent stem cells into retinal cells can be prepared using a medium normally used for culturing animal cells as the basal medium.
• basal media include 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, ⁇ MEM medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof.
• the medium may be a basal medium to which one or more components selected from serum, serum replacements, 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 have been added as necessary.
• serum replacements include albumins such as bovine serum albumin (BSA), transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 1-thioglycerol, and equivalents thereof, as well as mixtures thereof.
• the medium was KnockOut TM Serum Replacement (Thermo Fisher Scientific), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), GlutaMAX TM Supplement (Thermo Fisher Scientific), Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement (Thermo Fisher Scientific), B 27 Supplement (Thermo Fisher).
• the medium may contain commercially available serum substitutes such as basal medium, N2 Supplement (Thermo Fisher Scientific), ITS Supplement (Thermo Fisher Scientific), etc.
• the medium may be a commercially available product in which the above components are added to a basal medium, such as Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement (Thermo Fisher Scientific), DMEM/F-12, GlutaMAX TM supplement (Thermo Fisher Scientific), etc.
• a basal medium such as Ham's F-12 Nutrient Mix, GlutaMAX TM Supplement (Thermo Fisher Scientific), DMEM/F-12, GlutaMAX TM supplement (Thermo Fisher Scientific), etc.
• the medium is preferably a serum-free medium.
• Serum-free medium means a medium that does not contain unconditioned or unpurified serum, and also includes media containing purified blood-derived components or animal tissue-derived components as long as they do not contain unconditioned or unpurified serum.
• the serum-free medium may contain a serum substitute.
• Differentiation induction is preferably performed in the absence of feeder cells (also referred to as under feeder-free conditions). Differentiation induction is also preferably performed under xeno-free conditions.
• xeno-free means not containing components derived from a biological species different from the biological species of the cells to be cultured.
• the undifferentiated state maintaining factor includes FGF signaling pathway active substance, TGF ⁇ family signaling pathway active substance and insulin.
• the FGF signaling pathway active substance includes FGF (e.g., bFGF, FGF4, FGF8).
• the FGF signaling pathway active substance and the TGF ⁇ family signaling pathway active substance include TGF ⁇ signaling pathway active substance and Nodal/Activin signaling pathway active substance.
• the TGF ⁇ signaling pathway active substance includes TGF ⁇ 1 and TGF ⁇ 2.
• the Nodal/Activin signaling pathway active substance includes Nodal, ActivinA and ActivinB.
• the undifferentiated state maintaining factor preferably includes bFGF.
• the concentration of the undifferentiated state maintenance factor may be any concentration capable of maintaining the undifferentiated state of pluripotent stem cells, and can be appropriately set by one skilled in the art.
• concentration of bFGF may be 4 ng to 500 ng/mL, 10 ng to 200 ng/mL, or 30 ng to 150 ng/mL.
• Examples of media containing factors for maintaining undifferentiation include StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03N (Ajinomoto Co., Inc.), S-medium (DS Pharma Biomedical Co.), StemPro TM (Thermo Fisher Scientific), mTeSR1 TM (STEMCELL Technologies), mTeSR2 TM (STEMCELL Technologies), TeSR TM -E8 TM (STEMCELL Technologies), and hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9; 105(36):13409-14) may be used.
• the medium containing an undifferentiated maintenance factor is StemFit (registered trademark) AK02N (Ajinomoto Co., Inc.).
• Culturing in a medium containing an undifferentiated maintenance factor is, but is not limited to, for example, 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 2 to 3 days.
• pluripotent stem cells Prior to culturing in a medium containing a substance acting on the BMP signaling pathway, pluripotent stem cells are cultured in a medium containing a ROCK inhibitor.
• the ROCK inhibitor may be added to a medium containing an undifferentiated maintenance factor.
• Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152.
• the concentration of the ROCK inhibitor is appropriately determined depending on the type of inhibitor, and is, for example, 1 to 100 ⁇ M, 5 to 50 ⁇ M, or about 10 ⁇ M, or a concentration that shows the same activity as Y-27632 at the above concentrations.
• the culture period in the medium containing the 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.
• the culture period in the medium containing the ROCK inhibitor is 1 hour to 3 hours.
• the culture period in the medium containing the undifferentiation maintenance factor and the ROCK inhibitor may be a part or the whole of the culture period in the medium containing the undifferentiation maintenance factor.
• the cells after culturing for 1 hour to 16 hours in the medium containing the undifferentiation maintenance factor and the ROCK inhibitor, the cells may be cultured in a medium containing the undifferentiation maintenance factor but not the ROCK inhibitor until the culture period in the medium containing the undifferentiation maintenance factor reaches a predetermined culture period.
• BMPs (bone morphogenetic proteins) include, for example, BMP2, BMP4, BMP7, and BMP12 (GDF7).
• BMP signaling pathway agonists and BMP signaling pathway inhibitors may be agonists and inhibitors of one or more of these BMPs, respectively.
• BMP signaling pathway inhibitors refers to substances that inhibit signal transduction mediated by BMP, and include substances that act on BMP or its receptor, substances that suppress the gene expression of BMP or its receptor, and substances that inhibit the binding between BMP and its receptor.
• BMP signaling pathway inhibitors include LDN-193189 (4-[6-(4-Piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline), Dorsomorphin (6-[4-[2-(1-Piperizinyl)ethoxy]phenyl]-3-(4-pyridinyl)-pyrazolo[1,5-a]pyrimidine), and DMH1 (4-(6-(4-isopropoxyphenyl)pyrazolo[1,5-a]pyrimidin-3-yl)quinoline).
• the BMP signaling pathway inhibitor is LDN-193189.
• the concentration of the BMP signaling pathway inhibitor is determined appropriately depending on the type of inhibitor, but is, for example, 1 to 1000 nM, 10 to 500 nM, 30 to 300 nM, or about 100 nM, or a concentration that exhibits activity equivalent to that of LDN-193189 at the above concentrations.
• Culture in a medium containing a BMP signal inhibitor is, but is not limited to, for example, 1 to 10 days, 2 to 9 days, 3 to 7 days, 4 to 6 days, or about 5 days.
• a substance acting on the BMP signaling pathway means a substance that enhances signaling mediated by BMP, and includes substances that act on BMP or its receptor, substances that enhance gene expression of BMP or its receptor, and substances that promote the binding of BMP to its receptor.
• substances acting on the BMP signaling pathway include BMP2, BMP4, BMP7, BMP12 (GDF7), or fragments thereof, or anti-BMP receptor antibodies.
• the substance acting on the BMP signaling pathway is BMP4.
• the concentration of the substance acting on the BMP signaling pathway is appropriately determined depending on the type of the substance, and is, for example, 0.01 to 1000 nM, 0.1 to 100 nM, 1 to 10 nM, 1 to 3 nM, or about 1.5 nM, or a concentration that exhibits activity equivalent to that of BMP4 at the aforementioned concentrations.
• the concentration of the BMP signaling pathway agent is 3 to 15 nM or 3 to 12 nM, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 nM, or a concentration that exhibits activity equivalent to that of BMP4 at said concentrations.
• the culture in the medium containing the BMP signaling pathway active substance is carried out for a period required for differentiation into retinal cells.
• the culture period is not limited, but may be, for example, 1 to 30 days, 2 to 20 days, 3 to 15 days, 4 to 12 days, or 5 to 10 days (e.g., 5, 6, 7, 8, 9, or 10 days).
• the concentration of the BMP signaling pathway active substance may be constant or may be changed during the culture period. For example, the concentration of the BMP signaling pathway active substance may be gradually reduced at a rate of 40 to 60% reduction every 2 to 4 days.
• the concentration of the BMP signaling pathway active substance in the medium can be gradually reduced by replacing a part (e.g., half) of the medium with a medium not containing the BMP signaling pathway active substance every 2, 3, or 4 days.
• a medium containing a BMP signaling pathway inhibitor or a BMP signaling pathway activator is a 1:1 mixture of F-12 medium and IMDM medium, to which KnockOut TM Serum Replacement (Thermo Fisher Scientific) (e.g., 0.5% to 30%, 1% to 20%, or 10%), Chemically Defined Lipid Concentrate (Thermo Fisher Scientific), BSA, and 1-thioglycerol are added.
• KnockOut TM Serum Replacement e.g. 0.5% to 30%, 1% to 20%, or 10%
• Chemically Defined Lipid Concentrate Thermo Fisher Scientific
• BSA 2-thioglycerol
• the medium After culturing in a medium containing a substance acting on the BMP signaling pathway, the medium may be replaced with one not containing the substance acting on the BMP signaling pathway, and culturing may be continued.
• This culturing period is not limited, but may be, for example, 1 to 100 days, 10 to 90 days, 20 to 80 days, 30 to 70 days, 40 to 60 days, or about 50 days.
• the cells may be cultured in a medium containing a Wnt signaling pathway inhibitor.
• a Wnt signaling pathway inhibitor means a substance that inhibits signal transduction mediated by Wnt, and includes substances that act on Wnt or its receptor, substances that suppress gene expression of Wnt or its receptor, and substances that inhibit the binding of Wnt to its receptor.
• inhibitors of the Wnt signaling pathway include CKI-7 (N-(2-aminoethyl)-5-chloro-8-isoquinolinesulfonamide), D4476 (4-(4-(2,3-Dihydrobenzo[1,4]dioxin-6-yl)-5-pyridin-2-yl-1H-imidazol-2-yl)benzoamide), and IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3 a,4,7,7a-Hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinylbenzamide), and IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthioeno[3,2-d]pyrimidin-2-yl)thi
• the medium may be replaced as appropriate. For example, part or all of the medium may be replaced every 1 to 4 days.
• the entire medium may be replaced with a medium containing that component at a desired concentration, or part of the medium may be replaced so that the component reaches the desired final concentration (for example, half of the medium may be replaced with a medium containing twice the final concentration).
• the concentration of that component may be constant or may be changed.
• Culture conditions such as culture temperature and CO2 concentration can be set appropriately.
• the culture temperature is, for example, 30° C. to 40° C., or about 37° C.
• the CO2 concentration is, for example, 1% to 10%, or about 5%.
• Differentiation into retinal tissue can be confirmed by detecting the expression of neural retinal cell markers in cells in the tissue.
• the neural retinal cell markers are as described above. Expression of the markers is confirmed, for example, on days 10 to 100 (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 later after differentiation induction. In one embodiment, expression of the markers is confirmed on days 16 to 22 after differentiation induction.
• the retinal tissue as starting cells obtained by the method described herein can also be referred to as a retinal sheet, and typically contains retinal progenitor cells, and is also referred to herein as a "retinal progenitor cell sheet.”
• the retinal tissue (retinal sheet) as starting cells has a percentage of Chx10-positive cells or a percentage of Chx10-positive and Rx-positive cells of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.
• the percentage of marker-positive cells may be the percentage on day 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or later after differentiation induction.
• the retinal tissue has a percentage of Chx10 and Rx positive cells on days 16 to 22 (e.g., days 16, 18, or 20) of differentiation induction of 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more. In a further embodiment, the retinal tissue has a percentage of Chx10 and Rx positive cells on days 16 to 22 (e.g., days 16, 18, or 20) of differentiation induction of 90% or more, or 95% or more.
• the retinal tissue (retinal sheet) as starting cells has a proportion of neural retinal cells of 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 cells may be the proportion on days 16 to 100 (e.g., days 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, or 100) of differentiation induction, or thereafter.
• the retinal tissue preferably has cells that form a layer structure.
• the thickness of the retinal tissue (retinal sheet) as starting cells can be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ⁇ m or more, or 500, 400, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, or 220 ⁇ m or less (upper and lower limits are independently selected).
• the retinal tissue is 50 ⁇ m to 300 ⁇ m thick.
• the retinal tissue does not need to be the same thickness throughout, and when specifying a range of thickness of the retinal tissue, it is sufficient that the thickness of any part of the retinal tissue is within that range.
• the retinal tissue (retinal sheet) as starting cells usually has up to about 30 cells in the direction perpendicular to the culture substrate, or a thickness of 500 ⁇ m or less.
• the retinal tissue (retinal sheet) as the starting cells is cultured by the following steps: (1) Seeding pluripotent stem cells at a density of 0.5 ⁇ 10 5 cells/cm 2 to 2.5 ⁇ 10 5 cells/cm 2 into region A on a culture substrate having , on its surface, a region A having cell adhesiveness and a region B adjacent to at least a portion of region A and having lower cell adhesiveness than region A ; (2) culturing the pluripotent stem cells seeded in step (1) in a medium containing a ROCK inhibitor for 1 to 16 hours; and (3) culturing the cells obtained after step (2) in a medium containing a substance that acts on a BMP signaling pathway to obtain a retinal tissue (retinal sheet).
• the retinal tissue (retinal sheet) as the starting cells covers 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more of region A.
• the method for measuring the proportion of the area covered by the retinal tissue in region A is not particularly limited, but can be measured, for example, by staining the retinal tissue.
• the presence or absence of expression of the above-mentioned neuroretinal cell marker e.g., Chx10
• can be stained e.g., immunostaining with fluorescently labeled anti-Chx10 antibody).
• imaging software or the like e.g., ImageJ software (NIH)
• NIR ImageJ software
• retinal tissue with a high expression level of genes expressed in neuroretinal cells and a low expression level of genes expressed in non-target cells are as described above.
• the non-target cells include eye-related cells (e.g., ciliary body, lens, retinal pigment epithelium, etc.) and brain/spinal cord-related cells (telencephalon, midbrain, spinal cord, etc.), and the markers thereof are as described above.
• eye-related cells e.g., ciliary body, lens, retinal pigment epithelium, etc.
• brain/spinal cord-related cells telencephalon, midbrain, spinal cord, etc.
• the retinal tissue (retinal sheet) as the starting cells is obtained by the following method:
• the method includes the following steps: (1) seeding pluripotent stem cells at a density of 0.5 ⁇ 10 5 cells/cm 2 to 2.5 ⁇ 10 5 cells /cm 2 in a region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a part of the region A and having lower cell adhesiveness than the region A; (2) culturing the pluripotent stem cells seeded in step (1) in a medium containing a ROCK inhibitor for 1 to 16 hours; and (3) culturing the cells obtained after step (2) in a medium containing a substance acting on a BMP signaling pathway to obtain a retinal tissue (retinal sheet),
• the medium in step (2) contains a factor for maintaining undifferentiation, and the medium in step (3) does not contain a factor for maintaining undifferentiation;
• the method further comprises, after step (2), culturing the cells in a medium containing
• the retinal tissue (retinal sheet) as the starting cells is obtained by the following method:
• the method includes the following steps: (1) seeding pluripotent stem cells at a density of 0.5 ⁇ 10 5 cells/cm 2 to 2.5 ⁇ 10 5 cells /cm 2 in a region A on a culture substrate having, on its surface, a region A having cell adhesiveness and a region B adjacent to at least a part of the region A and having lower cell adhesiveness than the region A; (2) culturing the pluripotent stem cells seeded in step (1) in a medium containing a ROCK inhibitor for 1 to 16 hours; and (3) culturing the cells obtained after step (2) in a medium containing a BMP signaling pathway agent to obtain a retinal tissue (retinal sheet), wherein: The medium in step (2) contains a factor for maintaining undifferentiation, and the medium in step (3) does not contain a factor for maintaining undifferentiation; Step (3) is carried out for 5 to 10 days from 6 to 8 days after the sowing in step (1);
• the retinal tissue (retinal sheet) obtained by steps (1) to (3) is dispersed as described below.
• the retinal tissue (retinal sheet) may be dispersed without being recovered from the culture substrate, or may be dispersed after recovery from the culture substrate.
• the retinal tissue (retinal sheet) can be recovered from the culture substrate by a conventional method.
• the retinal tissue can be recovered with a tool such as tweezers.
• the culture substrate is coated with a stimuli-responsive polymer (e.g., a temperature-responsive polymer or a light-responsive polymer), the retinal tissue can be recovered by applying a corresponding stimulus.
• a stimuli-responsive polymer e.g., a temperature-responsive polymer or a light-responsive polymer
• the retinal tissue when a temperature-responsive polymer is used, the polymer properties of which reversibly change from cell adhesive (hydrophobic) to cell non-adhesive (hydrophilic) at a certain temperature, the retinal tissue can be recovered by placing the culture substrate at that certain temperature.
• Dispersion refers to separating cells or tissues into small cell fragments or cell clumps (2 to 100 cells, preferably 50 or less, 30 or less, 20 or less, 10 or less, 5 or less; for example, a clump of 2 to 5 cells) or single cells by a dispersion treatment such as an enzyme treatment or a physical treatment.
• a dispersed cell population refers to a collection of a certain number of cell fragments or cell clumps or single cells.
• a "dispersed retinal cell population” refers to a cell population in a dispersed state, and can be obtained by dispersing a cell clump such as a biological tissue or a cell aggregate. The dispersed retinal cell population is preferably obtained by dispersing the above-mentioned clump of retinal cells.
• the dispersion method may be any method capable of dispersing cells alive, such as mechanical dispersion treatment, cell dispersion liquid treatment, and cell protective agent addition treatment. These treatments may be combined. Preferably, cell dispersion liquid treatment is performed first, followed by mechanical dispersion treatment.
• Mechanical dispersion methods include pipetting or scraping with a scraper.
• Cell dispersion liquids used in cell dispersion treatment include solutions containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain, and chelating agents such as ethylenediaminetetraacetic acid.
• enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, papain, and chelating agents such as ethylenediaminetetraacetic acid.
• Commercially available cell dispersion liquids such as TrypLE Select (Life Technologies), TrypLE Express (Life Technologies), and nerve cell dispersion liquid (FujiFilm), can also be used.
• cell death When cells are dispersed, cell death may be suppressed by treating the cells with a cell protective agent.
• cell protective agents used in the cell protective agent treatment include substances acting on the FGF signaling pathway, heparin, substances acting on the IGF signaling pathway, serum, or serum substitutes.
• a Rho-associated coiled-coil containing protein kinase (“ROCK” or "Rho kinase”) inhibitor (ROCK inhibitor) or a myosin inhibitor may be added during dispersion.
• ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152.
• myosin inhibitors include blebbistatin.
• a preferred cytoprotective agent is a ROCK inhibitor.
• the dispersed retinal cell population includes single cells, and for example, 70% or more, preferably 80% or more of the total number of cells in the cell population are single cells, and clumps of 2 to 50 cells account for 30% or less, preferably 20% or less of the total number of cells in the cell population.
• the dispersed retinal cell population there is almost no adhesion between cells (e.g., surface adhesion). It is also preferable that the dispersed retinal cell population is 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 process.
• dispersed cells are in a state in which there is almost no cell-cell bonding (e.g., adherens bonding).
• the retinal tissue may contain unnecessary cells such as retinal pigment epithelial cells.
• the area containing unnecessary cells such as retinal pigment epithelial cells can be separated and the retinal cell population dispersed.
• Retinal pigment epithelial cells can be distinguished by their morphology and pigments, and can be easily excised by a person skilled in the art.
• a step for increasing the purity of the retinal progenitor cells or neural retinal progenitor cells may be carried out as described below, and further, the dispersed retinal cell population may be maintained and expanded.
• the medium is not particularly limited as long as it is a medium in which the retinal cells can survive and proliferate (such as DMEM medium). Since a frozen and thawed retinal cell population can also be used as the starting cells for the production method of the present invention, it is also possible to freeze and store the dispersed retinal cell population. There is no particular restriction on the freezing and storage liquid, and a commercially available freezing and storage liquid can be used.
• a step (purification step) of increasing the proportion (purity) of retinal progenitor cells, preferably the proportion (purity) of neural retinal progenitor cells, may be performed on the dispersed retinal cell population.
• an operation such as cell sorting using a specific marker expressed in retinal progenitor cells and/or neural retinal progenitor cells may be performed.
• Cell sorting can be performed using well-known techniques such as FACS and MACS.
• retinal pigment epithelial progenitor cells and/or neural retinal progenitor cells By performing a purification step of retinal progenitor cells and/or neural retinal progenitor cells, it is possible to reduce the contamination of RPE cells and non-target cells in the manufacturing method described herein.
• the generation of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells is suppressed by the step of increasing the proportion of retinal progenitor cells contained in the dispersed retinal cell population. Whether the generation of retinal pigment epithelial progenitor cells and/or retinal pigment epithelial cells is suppressed may be determined by whether retinal pigment epithelial cells are generated based on the markers, morphology, properties, etc.
• retinal pigment epithelial precursor cells and/or retinal pigment epithelial cells are inhibited if the ratio of retinal pigment epithelial cells to the total number of cells after the above-mentioned culture is inhibited compared to the case where the step of increasing the ratio of retinal progenitor cells is not carried out. If the ratio 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 as markers (positive markers) for retinal progenitor cells.
• markers positive markers
• Rx and Chx10 are well known as markers (positive markers) for retinal progenitor cells.
• these genes are expressed intracellularly, it is necessary to use ingenuity, such as using cells in which the gene is linked to a fluorescent protein by recombinant gene technology, or cells in which the gene has been replaced with a fluorescent protein (e.g., Rx::Venus cells).
• CD9 Genbank ID: NM_001769.4, NM_001330312.2
• CD24 Genbank ID: NM_001291737.1, NM_001291738.1, NM_001291739.1, NM_001359084.
• CD49f (Genbank ID: NM_000210.4, NM_001079818.3, NM_001316306.2, NM_001365529.2, NM_001365530.2), CD57 (Genbank ID: NM_00136 7973.1, NM_018644.3, NM_054025.3), CD73 (Genb ank ID: NM_001204813.1, NM_002526.4), CD82 (Genbank ID: NM_001024844.2, NM_002231.4), CD90 (Genbank ID: NM_001311160.2, NM_00 1311162.2, NM_001372050.1, NM_006288.5), CD16 4 (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,
• Preferred cell surface markers include, for example, CD9, CD39, CD90 and CXCR4.
• a method for improving the proportion of retinal progenitor cells in a cell population which includes a step of contacting the cell population containing retinal progenitor cells with a substance (e.g., an antibody, a peptide, etc.) that binds to one or more antigens selected from the group consisting of CD9, CD39, CD90, and CXCR4, and isolating a positive fraction, may be performed before starting culture in a medium containing the above-mentioned Wnt signaling pathway active substance. This method makes it possible to reduce contamination with RPE cells in the production method described herein.
• negative markers for these cells i.e., markers whose expression is not observed in these cells, have been found, such as 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).
• SSEA1 Genebank ID: NM_002033.3
• CD66b Genebank ID: NM_001816.4
• CD69 Genebank ID: NM_001781.2
• CD84 Genebank ID: NM_001184879.2, NM_001184881.2, NM_001184882.1, NM_001330742.2, NM_003874.4.
• a method for improving the proportion of retinal progenitor cells in a cell population may be performed before starting culture in a medium containing a substance acting on the Wnt signaling pathway, the method including a step 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 with a substance (e.g., an antibody) that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84, and separating a positive fraction of the former marker and a negative fraction of the latter marker.
• a substance e.g., an antibody
• the step of contacting the dispersed retinal cell population with a substance (e.g., an antibody) that binds to one or more antigens selected from the group consisting of SSEA1, CD66b, CD69, and CD84 and separating a negative fraction of these markers is a step of obtaining a cell population in which the expression level of the antigen is equal to or lower than a reference value.
• a reference value can be set arbitrarily by a person skilled in the art.
• a negative fraction can be isolated by treating a cell population with a fluorescently labeled antibody against the antigen that has the same fluorescence intensity as that obtained when the cell population is treated with a fluorescently labeled isotype control antibody.
• the method of increasing the proportion of retinal progenitor cells and/or neural retinal progenitor cells in a cell population using a substance (e.g., an antibody) that binds to the above-mentioned positive markers and negative markers is not limited to being a method as one step of the manufacturing method described in this specification.
• the method of increasing the proportion of retinal progenitor cells and/or neural retinal progenitor cells using a cell surface marker can also be used as one step of the manufacturing method for other retinal tissues, for example.
• the retinal progenitor cells and/or neural retinal progenitor cells may, for example, account for 30% or more of the total number of cells, preferably 50% or more of the total number of cells, more preferably 80% or more of the total number of cells, and even more preferably 90% or more of the total number of cells.
• 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 are included in 50% or more of the total number of cells in the cell population, preferably 80% or more, 85% or more, 90% or more, or 95% or more.
• the cell population is 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.
• negative means that it is below the above-mentioned reference value.
• the method for producing retinal tissue according to the present invention comprises adhesion culture or suspension culture of a dispersed retinal cell population in a medium containing a substance acting on the Wnt signaling pathway. Either culture method makes it possible to re-form an epithelial structure (multi-layered structure) from the dispersed retinal cell population. In order to produce a sheet-like retinal tissue having an epithelial structure (or multi-layered structure), adhesion culture is preferred.
• the medium used for culturing the dispersed retinal cell population is not particularly limited as long as it is a medium in which the retinal cells can survive and grow.
• the medium used for culturing the dispersed retinal cell population is a medium for maintaining continuous epithelial tissue.
• a medium for maintaining continuous epithelial tissue is a medium obtained by compounding Neurobasal medium (e.g., Thermo Fisher Scientific, 21103049) with B27 supplement (e.g., Thermo Fisher Scientific, 12587010).
• the Wnt signaling pathway active substance contained in the culture medium is not particularly limited as long as it is capable of enhancing signaling mediated by Wnt.
• Wnt signaling pathway active substances include GSK3 ⁇ inhibitors (e.g., 6-bromoindirubin-3'-oxime (BIO)), CHIR99021, Kenpaullone, Wnt proteins such as Wnt2b and Wnt3a, and partial peptides thereof, and may be one substance or two or more substances.
• the Wnt signaling pathway active substance is preferably one or more substances, two or more substances, or three or more substances selected from the group consisting of CHIR99021, BIO, Wnt2b, and Wnt3a.
• the Wnt signaling pathway active substance can increase the size of the aggregates and can re-form an epithelial structure (or multi-layer structure) with apical/basement membrane polarity.
• the Wnt signaling pathway active substance can also suppress the formation of rosette-like structures.
• the concentration of the Wnt signaling pathway active substance may be any concentration that can induce the formation of a desired cell aggregate (e.g., re-formation of an epithelial structure (or multi-layer structure)).
• the concentration of the Wnt signaling pathway active substance may be 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M, more preferably 1 ⁇ M to 10 ⁇ M, more preferably 3 ⁇ M to 6 ⁇ M.
• the concentration may be any concentration that exhibits the same level of Wnt signal activation activity as CHIR99021 at the above-mentioned concentration.
• the Wnt signal activation activity can be measured by a person skilled in the art, for example, by a method such as confirming the expression of ⁇ -Catenin.
• the timing of adding the substance acting on the Wnt signaling pathway is not particularly limited, but it is preferable to add it as soon as possible after the start of suspension culture or adhesion culture for re-forming the sheet. In one embodiment, it is preferable to culture in a medium containing a substance acting on the Wnt signaling pathway from the start of culture.
• the number of days of culture in a medium containing a substance acting on the Wnt signaling pathway is not particularly limited as long as the effect of re-forming an epithelial structure (or multi-layered structure) with apical/basement membrane polarity is observed, but is, for example, 1 to 14 days.
• the medium may further contain one or more substances selected from the group consisting of a ROCK inhibitor, a substance acting on the SHH (Sonic Hedgehog) signaling pathway, and a substance acting on the fibroblast growth factor (FGF) signaling pathway.
• a ROCK inhibitor has the effect of promoting the re-aggregation of dispersed retinal progenitor cell populations
• the addition of a substance acting on the SHH signaling pathway has the effect of promoting the proliferation of cell aggregates and enlarging the cell aggregates
• the addition of a substance acting on the FGF signaling pathway e.g., FGF2, FGF8 has the effect of reducing cell damage and suppressing the induction of differentiation into RPE cells.
• the ROCK inhibitor is not particularly limited as long as it can suppress the function of Rho kinase (ROCK), and examples thereof include Y-27632 (see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983(2000); Narumiya et al., Methods Enzymol. 325, 273-284(2000)), Fasudil/HA1077 (see, for example, Uenata et al., Nature 389:990-994(1997)), and the like.
• Y-27632 see, for example, Ishizaki et al., Mol. Pharmacol. 57, 976-983(2000); Narumiya et al., Methods Enzymol. 325, 273-284(2000)
• Fasudil/HA1077 see, for example, Uenata et al., Nature 389:990-994(1997)
• antibodies include those described herein include ribonucleic acid (RIA)-dependent agonists (e.g., ribonucleic acid (ROCK)), H-1152 (e.g., Sasaki et al., Pharmacol. Ther. 93:225-232(2002)), Wf-536 (e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324(2003)) and their derivatives, as well as antisense nucleic acids against ROCK, RNA interference-inducing nucleic acids (e.g., siRNA), dominant negative mutants, and expression vectors thereof.
• RIA ribonucleic acid
• ROCK ribonucleic acid
• H-1152 e.g., Sasaki et al., Pharmacol. Ther. 93:225-232(2002)
• Wf-536 e.g., Nakajima et al., Cancer Chemother Pharmacol. 52(4):319-324(2003)
• ROCK inhibitors other low molecular weight compounds are also known as ROCK inhibitors, and such compounds or their derivatives can also be used in the present invention (see, for example, U.S. Patent Application Publication Nos. 20050209261, 20050192304, 20040014755, 20040002508, 20040002507, 20030125344, 20030087919, and International Publication Nos. 2003/062227, 2003/059913, 2003/062225, 2002/076976, and 2004/039796).
• 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 may be any concentration capable of promoting reaggregation of the dispersed retinal progenitor cell population.
• the concentration may be 0.1 ⁇ M to 1 mM, preferably 1 ⁇ M to 100 ⁇ M, and more preferably 5 ⁇ M to 20 ⁇ M.
• the concentration may be any concentration that exhibits the same degree of ROCK inhibitory activity as the above-mentioned concentration of Y-27632.
• a person skilled in the art can measure the ROCK inhibitory activity, for example, by a method such as expression analysis of phosphorylation of MLC2.
• a substance acting on the SHH (Sonic Hedgehog) signaling pathway is a substance that can enhance signaling mediated by SHH (sometimes written as Shh).
• substances acting on the SHH signaling pathway include proteins belonging to the Hedgehog family (e.g., Shh and Ihh), SHH receptors, Shh receptor agonists, PMA (Purmorphamine; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-naphthalenyloxy)-9H-purin-6-amine), and SAG (Smoothened Agonist; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane).
• the substance acting on the Shh signaling pathway may contain one or more of these.
• the Shh signaling pathway active substance is preferably one or more substances selected from the group consisting of Shh (Genbank accession numbers: NM_000193, NP_000184), SAG, or PMA.
• the concentration of the substance acting on the SHH signaling pathway can be appropriately set by a person skilled in the art depending on the experimental conditions. In one embodiment, the concentration may be within a range in which the effect of enlarging cell aggregates is observed.
• SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM.
• PMA is usually used at a concentration of 0.002 to 20 ⁇ M, preferably 0.02 to 2 ⁇ M.
• SHH is usually used at a concentration of 4 to 500 ng/mL, preferably 10 to 200 ng/mL.
• the concentration may be such that they exhibit the same degree of SHH signal activation as SAG at the above-mentioned concentration.
• the SHH signal activation can be measured by a person skilled in the art, for example, by a method such as expression analysis of downstream signals (SMO and GLI).
• the FGF signaling pathway active substance is not particularly limited as long as it is a substance that can enhance signal transduction mediated by FGF.
• FGF signaling pathway active substances include fibroblast growth factors (e.g., bFGF, FGF4, FGF8, and FGF9).
• the FGF signaling pathway active substance is preferably one or more fibroblast growth factors selected from the group consisting of FGF2, FGF4, and FGF8.
• the concentration of the substance acting on the FGF signaling pathway can be set as appropriate depending on the experimental conditions.
• the concentration may be within a range in which the effect of suppressing differentiation induction into RPE cells is observed.
• the concentration is about 4 to 500 ng/mL, preferably about 10 to 200 ng/mL, and more preferably about 25 to 100 ng/mL.
• the concentration may be such that they exhibit the same degree of FGF signal activation effect as FGF8 at the above-mentioned concentration.
• a person skilled in the art can measure the FGF signal activation effect, for example, by a method such as expression analysis of downstream signals (Akt, MEK).
• the timing of adding the ROCK inhibitor, the substance acting on the SHH (Sonic Hedgehog) signaling pathway, and/or the substance acting on the FGF signaling pathway is not particularly limited, but it is preferable to culture in a medium containing a ROCK inhibitor, a substance acting on the SHH (Sonic Hedgehog) signaling pathway, and/or a substance acting on the FGF signaling pathway from the start of culture.
• These substances are preferably added to the medium at the same time, and more preferably added to the medium at the same time as a substance acting on the Wnt signaling pathway.
• the dispersed retinal cells may be cultured in a medium containing these substances for 1 to 14 days.
• Suspension culture is the cultivation of cells in a non-adherent state to a culture vessel, and can be carried out using, but is not limited to, a culture vessel that has not been artificially treated (e.g., coated with an extracellular matrix, etc.) to improve adhesion to the cells, or a culture vessel that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA), nonionic surface-active polyol (Pluronic F-127, etc.), or a phospholipid-like structure (e.g., a water-soluble polymer (Lipidure) whose constituent unit is 2-methacryloyloxyethyl phosphorylcholine).
• a culture vessel that has not been artificially treated e.g., coated with an extracellular matrix, etc.
• a culture vessel that has been artificially treated to suppress adhesion e.g., coated with polyhydroxyethyl methacrylate (poly
• Floating culture can be performed, for example, by using dispersed retinal cells as starting cells and the SFEB (serum-free floating culture of embryoid bodies-like aggregates) method (WO2005/12390) or the SFEBq method (WO2009/148170).
• SFEB serum-free floating culture of embryoid bodies-like aggregates
• Adhesion culture refers to culturing cells in a state of adhesion to a culture vessel, and can be carried out using, but is not limited to, a culture vessel that has been artificially treated to improve adhesion to the cells. Adhesion culture is preferably carried out using a culture vessel coated with an extracellular matrix and/or a temperature-sensitive polymer.
• One embodiment of the method for producing retinal tissue includes a step of exposing the culture vessel coated with a temperature-sensitive polymer to a temperature at which the properties of the temperature-sensitive polymer change, thereby peeling off the sheet-like retinal tissue from the culture vessel.
• sheet-like retinal tissue By culturing the cells in an adhesion culture vessel coated with an extracellular matrix, sheet-like retinal tissue can be produced, as described below.
• Culturing in the presence of an extracellular matrix allows the cells to recognize the basement membrane side, making it easier for the apical surface to be formed, and the cells are oriented approximately perpendicular to the layer direction, resulting in retinal tissue with a better layer structure.
• the retinal tissue with the formed epithelial structure (or multilayer structure) can be easily peeled off from the culture vessel by temperature change alone, and no enzyme treatment is required. Therefore, strong sheet-like retinal tissue can be recovered without weakening the bonds between cells due to enzyme treatment.
• a culture vessel coated with an extracellular matrix and/or a temperature-sensitive polymer particularly both an extracellular matrix and a temperature-sensitive polymer.
• the culture surface of the culture vessel is coated with the above-mentioned temperature-sensitive polymer, and the upper surface of the polymer is coated with the above-mentioned extracellular matrix.
• the culture surface refers to the surface of the culture vessel to which the cells adhere
• the upper surface of the polymer refers to the surface opposite to the surface in contact with the culture surface of the polymer coating.
• extracellular matrix refers to biopolymers that make up the space outside cells, and includes cell adhesive proteins such as fibronectin, vitronectin, laminin, etc., fibrous proteins such as collagen and elastin, fragments of these proteins, glycosaminoglycans or proteoglycans such as hyaluronic acid and chondroitin sulfate, and matrigel.
• the extracellular matrix is preferably one or more substances selected from the group consisting of collagen, laminin, fibronectin, matrigel, vitronectin, and fragments of these proteins.
• laminin fragments include commercially available products such as iMatrix-511, iMatrix-411, and iMatrix-221.
• Matrigel is a basement membrane preparation derived from the Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared, for example, by the method disclosed in US Patent No. 4,829,000, or it can be purchased commercially. The main components of Matrigel are laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.
• EHS Engelbreth Holm Swarn
• a temperature-sensitive polymer is a polymer whose properties change with temperature. That is, it has a lower critical solution temperature (LCST) in water, and at a certain temperature above which the hydrophobic bonds within or between the molecules become stronger, causing the polymer chains to aggregate, while at a lower temperature the polymer chains bind water molecules and become hydrated, showing a phase transition behavior.
• LCST critical solution temperature
• LCST poly-N-isopropylacrylamide
• LCST poly-N-isopropylacrylamide
• Cell culture is usually carried out at approximately 37°C, and considering the damage that low temperatures can cause to cells, a temperature-sensitive polymer with an LCST in the range of approximately 20°C to 35°C is preferable.
• Temperature-responsive cell culture equipment for cell sheet recovery with a temperature-sensitive polymer fixed to the surface (CellSeed: UpCell (registered trademark)) is also available commercially.
• the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed in an atmosphere of CO2- containing air, and the CO2 concentration is preferably about 2 to 5%.
• Substances acting on the Wnt signaling pathway are required for the formation of layered structures in sheet-like retinal tissue, and in particular for the formation of apical/basal polarity.
• the concentration of the Wnt signaling pathway active substance may be any concentration capable of inducing sheet-like retinal tissue having a layer structure and an apical surface.
• the concentration of the Wnt signaling pathway active substance may be 0.01 ⁇ M to 100 ⁇ M, preferably 0.1 ⁇ M to 10 ⁇ M, and more preferably 1 ⁇ M to 10 ⁇ M.
• the concentration may be such that they exhibit the same degree of Wnt signal activation effect as CHIR99021 at the above-mentioned concentration.
• a layer structure has been formed, for example, by observing with a microscope or measuring the thickness with a device such as OCT. Whether an apical surface has been formed can be confirmed by staining with, for example, an anti-Zo-1 antibody, an anti-Ezrin antibody, or an anti-typical-PKC antibody.
• the timing of adding the substance acting on the Wnt signaling pathway is not particularly limited, but it is preferable to add it as soon as possible after the start of adhesion culture. In one embodiment, it is preferable to culture in a medium containing a substance acting on the Wnt signaling pathway from the start of culture.
• the number of days of culture in a medium containing a substance acting on the Wnt signaling pathway is not particularly limited as long as the effect of reforming a multilayer structure (or multilayer structure) with apical/basement membrane polarity is observed, but is, for example, 1 to 15 days, preferably 1 to 9 days.
• Culture may be continued in a medium that does not contain substances that act on the Wnt signaling pathway. Continuing culture thickens the layer structure and advances differentiation of retinal cells.
• the culture period is not particularly limited, and may be a period during which the seeded dispersed retinal cell population proliferates and at least an epithelial structure (or multi-layered structure) is formed, and may be cultured until a sheet-like retinal tissue at the desired differentiation stage is produced. From the viewpoint of forming an epithelial structure (or multi-layered structure), it is desirable to culture for at least 7 days.
• the culture period may be, for example, 7 to 60 days or less, or may be 40 days or less, 30 days or less, 20 days or less, or 16 days or less (e.g., 16 days).
• the cells may be further cultured to proliferate, differentiate, or mature.
• the medium used for further culture may or may not contain a substance acting on the Wnt signaling pathway, a ROCK inhibitor, a substance acting on the SHH (Sonic Hedgehog) signaling pathway, and/or a substance acting on the integrin transmission pathway.
• multilayer structure refers to a structure formed by stacking two or more cell layers in which cells are aligned in the same direction in tissue that has a polarity between the basement membrane side and the apical side (i.e., has an epithelial structure), and the tangential directions of the surfaces of the different layers are approximately parallel to each other.
• the multilayer structure preferably has a polarity of the basal surface and the apical surface, and in one embodiment, the retinal tissue having a multilayer structure may be a sheet-like retinal tissue, and the cell aggregate containing the retinal tissue having a multilayer structure may be a sheet-like cell aggregate.
• the cell layer may be a neural retinal progenitor cell layer, a ganglion cell layer, or a photoreceptor cell layer.
• the orientation of the cells may be approximately perpendicular to the layer direction.
• cell orientation refers to the direction in which the shape of the nucleus and the orientation of the cell body extend toward the basement membrane side and the apical side.
• approximately perpendicular to the layer direction refers to a direction perpendicular to the direction in which the individual cells are lined up in contact with each other in the layers of the multi-layer structure (i.e., the tangent direction of the surface of the layer), and refers to a perpendicular or longitudinal direction to the layers.
• the method may further include a step of cutting out the retinal tissue (particularly sheet-like retinal tissue) having an epithelial structure (or multi-layer structure) obtained by suspension culture or adhesion culture to a size required for transplantation.
• the tissue can be cut out using tweezers, a knife, scissors, etc.
• One aspect of the present invention is a sheet-like retina (neural retina) tissue having an epithelial structure.
• One aspect of the sheet-like retina tissue is composed of a retinal cell layer having a multi-layer structure, the multi-layer structure having basal and apical polarities, the retinal cell layer having the multi-layer structure containing one or more types of cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells, and the orientation of the cells in the retinal cell layer is approximately perpendicular to the layer direction.
• the term "sheet-like” refers to a single-layer or multi-layer structure composed of a single or multiple cells having biological connections in at least two-dimensional directions.
• One advantage of the present invention is that it is possible to produce sheet-shaped retinal tissue of any size depending on the culture equipment used. In other words, it is possible to produce sheet-shaped retinal tissue of a size that was previously impossible to produce, and in cases where a disease affects a wide area, treatment is possible by transplanting a single sheet.
• the long 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.
• the short 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 long axis and short axis, but the size of the dish or the like used for culture is the limit.
• the long axis may be 10 cm or less, 5 cm or less, 4 cm or less, 3 cm or less, 2 cm or less, or 1 cm or less.
• the height of the sheet-like retinal tissue of the present invention may be, for example, 50 ⁇ m to 1500 ⁇ m, and is preferably 200 ⁇ m to 700 ⁇ m.
• the method for measuring the long diameter, short diameter and height of the sheet-like retinal tissue is not particularly limited, and may be, for example, measured from an image captured under a microscope.
• a front image of the sheet-like retinal tissue captured with the apical surface facing the objective lens and a lateral image captured with the cut surface tilted so that it is perpendicular to the objective lens can be captured with a stereomicroscope, and measurements can be made from the captured images.
• the long diameter means the longest line segment and its length among the line segments connecting two end points on the sheet cross section in the front image.
• the short diameter means the longest line segment and its length among the line segments connecting two end points on the sheet cross section in the front image that are perpendicular to the long diameter.
• the height means the longest line segment and its length among the line segments perpendicular to the sheet cross section and having the intersection with the sheet cross section and the apex of the retinal sheet as their end points.
• the basal and apical surfaces are as defined above. "The multilayer structure has a polarity of basal and apical surfaces” means that the multilayer structure has a basal surface on one side and an apical surface on the other side. A basement membrane may be present on the basal surface.
• the sheet-shaped retinal tissue does not contain RPE cells.
• the ratio of the number of RPE cells to the total number of cells in the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less.
• the ratio of the number of RPE cells to the total number of cells in the sheet-shaped retinal tissue can be measured, for example, by using flow cytometry (FACS) or the like to measure the ratio of cells expressing the above-mentioned RPE cell markers.
• FACS flow cytometry
• the ratio of the area of RPE cells to the total area of the sheet-shaped retinal tissue is 10% or less, preferably 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less. Since RPE cells are black, the ratio of the area of RPE cells to the total area of the sheet-shaped retinal tissue can be calculated from the ratio of the area that shows black under a microscope. In addition, cells expressing RPE cell markers can also be detected by PCR or the like. The method for reducing the proportion of RPE cells in sheet-like retinal (neural retinal) tissue is as described above.
• a sheet-like retinal tissue comprising retinal cell layers having a multilayer structure
• the retinal cell layer having a multilayer structure has basal and apical polarity
• the retinal cell layer having the multilayer structure includes one or more types of cells selected from the group consisting of retinal progenitor cells, photoreceptor progenitor cells, and photoreceptor cells; (3) In each layer of the retinal cell layer, the cells are oriented approximately perpendicular to the layer direction, and (4) The diameter is 8 mm or more.
• the retinal tissue may include a retinal cell layer having the above-mentioned multi-layer structure and a sheet-like retinal pigment epithelial cell bonded to the retinal cell layer, and the retinal cell layer and the sheet-like retinal pigment epithelial cell may be a complex (composite sheet) in which the tangential directions of the respective surfaces of the retinal cell layer and the sheet-like retinal pigment epithelial cell are approximately parallel, the apical surface of the retinal cell layer and the apical surface of the sheet-like retinal pigment epithelial cell face each other, and the retinal cell layer and the sheet-like retinal pigment epithelial cell are bonded to each other by an adhesion factor present between them.
• the sheet-like neural retina is also called a neural retina sheet or NR sheet
• the sheet-like retinal pigment epithelial cell is also called a retinal pigment epithelial cell sheet or RPE cell sheet (RPE sheet).
• RPE sheet retinal pigment epithelial cell sheet
• one embodiment of the present invention also provides a complex (composite sheet) of neural retina and RPE cells.
• Another embodiment of the present invention includes a complex (composite sheet) in which dispersed retinal pigment epithelial cells are bonded to the apical surface side of a sheet-like retinal cell layer (sheet-like neural retina) by an adhesion factor.
• the tangential directions of the neural retina sheet and the retinal pigment epithelial cell sheet are approximately parallel to each other.
• approximately parallel tangential directions means that the tangential directions of the opposing surfaces of the neural retina and the retinal pigment epithelial cell sheet are parallel to each other.
• the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cells face each other. In other words, the apical surface of the neural retina and the apical surface of the retinal pigment epithelial cells are in close proximity to each other.
• a composite of neural retina and RPE cells can be prepared by joining a sheet-like retinal cell layer (neural retina sheet) with an RPE cell sheet or dispersed RPE cells in the presence of an adhesion factor, as described below.
• the sheet-like retinal cell layer can be produced by the above-mentioned method for producing a cell aggregate containing retinal tissue having an epithelial structure (or multi-layer structure).
• the RPE cell sheet can be produced by the method for producing a retinal pigment epithelial cell sheet, which is described below.
• Retinal pigment epithelial (RPE) cells are derived from pluripotent stem cells, and can be obtained by inducing differentiation of pluripotent stem cells.
• Methods for producing retinal pigment epithelial cells include, but are not limited to, those disclosed 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).
• the retinal pigment epithelial cells may be produced as a cell sheet or a spherical cell aggregate.
• the RPE cell sheet can be prepared by cutting open the cell aggregate using tweezers, a knife, scissors, or the like.
• pluripotent stem cells are cultured in the absence of feeder cells under the conditions of 1) treating with a TGF ⁇ family signaling pathway inhibitor and a sonic hedgehog signaling pathway activating substance one day before differentiation induction, and 2) not treating with a sonic hedgehog signaling pathway activating substance at the start of differentiation induction. Then, the above-mentioned steps (B) and (C) are performed. Furthermore, it is preferable to start the above-mentioned step (D) earlier. Specifically, step (D) is started about 9 days (e.g., 7, 8, 9, 10, or 11 days) after the start of the suspension culture in step (B).
• a spherical cell aggregate of RPE cells can be obtained.
• the cell aggregate may be dispersed to prepare a cell suspension, or an RPE cell sheet may be prepared by cutting the cell aggregate open using tweezers, a knife, scissors, or the like.
• RPE cell sheets can also be prepared by culturing the dispersed cell suspension in adherent culture.
• Dispersed RPE cells can also be obtained by dispersing RPE cell sheets or aggregates of RPE cells.
• the retinal pigment epithelial cell sheet may be further cultured until it has a polygonal or pavement-like cell morphology before being brought into contact with the neural retina cell aggregate.
• the medium is not particularly limited, but it can be replaced with a maintenance medium for retinal pigment epithelial cells (hereinafter, sometimes referred to as RPE maintenance medium) and further cultured. This allows for more clear observation of melanin pigmented cell groups and cell groups with polygonal flat morphology adhering to the basement membrane.
• the culture in RPE maintenance medium is not limited as long as colonies that grow while maintaining the properties of retinal pigment epithelial cells are formed, but for example, the culture is performed for 5 days or more (e.g., about 5 to 20 days) with the entire medium replaced at least once every 3 days.
• the maintenance medium for retinal pigment epithelial cells may be, for example, the medium 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., ebruary 2008, Vol. 49, No. 2, and Gamm et al. can be used.
• the long diameter of the retinal pigment epithelial cell sheet may be the same as the long diameter of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the long diameter of the retinal pigment epithelial cell sheet may be in the range of, for example, 3 mm to 50 mm, 5 mm to 30 mm, or 10 mm to 20 mm.
• the minor axis of the retinal pigment epithelial cell sheet may be the same as the minor axis of the sheet-like retinal tissue (neural retinal sheet). In one embodiment, the minor axis of the retinal pigment epithelial cell sheet may be in the range of, for example, 2 mm to 40 mm, 5 mm to 30 mm, or 10 mm to 20 mm.
• the degree of melanin pigmentation in the retinal pigment epithelial cell sheet is not particularly limited. It is preferable that the degree of melanin pigmentation in the retinal pigment epithelial cells contained in the retinal pigment epithelial cell sheet is the same among the cells.
• the average melanin content of the retinal pigment epithelial cell sheet may be less than 20 pg/cell, less than 15 pg/cell, less than 10 pg/cell, less than 8 pg/cell, less than 7 pg/cell, less than 6 pg/cell, less than 5 pg/cell, less than 4 pg/cell, less than 3 pg/cell, less than 2 pg/cell, or less than 1 pg/cell.
• the average melanin content of the retinal pigment epithelial cell sheet may also be 0.1 pg/cell or more, 0.5 pg/cell or more, 1 pg/cell or more, 2 pg/cell or more, or 5 pg/cell or more.
• the melanin content in the retinal pigment epithelial cell sheet can be measured, for example, by dispersing the retinal pigment epithelial cell sheet, followed by extraction of the cells with NaOH or the like, using a spectrophotometer or the like.
• 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 sheet.
• the above-mentioned composite can be produced by joining a neural retina sheet and dispersed RPE cells or an RPE cell sheet.
• a composite sheet in which a neural retina sheet and an RPE cell sheet are joined is preferable.
• the above-mentioned neural retina sheet and RPE cell sheet can be easily removed from the cultureware using tweezers, a knife, scissors, etc. Both sheets that have been removed may be transferred to a new container (such as cultureware), or one may be left in the cultureware and the other transferred to the cultureware. By producing them in cultureware of the same size, the size of the sheet-like tissue to be joined can be made uniform.
• the start date of production may be staggered.
• the above-mentioned neural retinal sheet and retinal pigment epithelial cells are preferably brought into contact in the presence of an adhesion factor.
• the adhesion factor means a substance that has the effect of adhering cells to each other, and is not particularly limited, but examples thereof include the above-mentioned extracellular matrix and artificial hydrogels.
• the adhesion factor does not need to be an isolated single substance, and includes preparations from living organisms or cells, such as Matrigel, interphotoreceptor matrix, and serum.
• Matrigel is a basement membrane preparation derived from Engelbreth Holm Swarn (EHS) mouse sarcoma. Matrigel can be prepared, for example, by the method disclosed in US Patent No. 4829000, and can also be purchased commercially.
• the main components of Matrigel are laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.
• Interphotoreceptor matrix is a general term for the extracellular matrix present between retinal cells such as photoreceptors in the living retina, and includes, for example, hyaluronic acid. Those skilled in the art can collect interphotoreceptor matrix from a living retina by, for example, placing the retina in distilled water and expanding it to separate it, and it can also be purchased commercially.
• the adhesion factor is preferably an extracellular matrix or a hydrogel.
• the extracellular matrix is preferably one or more extracellular matrices selected from the group consisting of hyaluronic acid, fibrin, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin.
• the hydrogel is preferably one or more hydrogels selected from the group consisting of gelatin, fibrin, collagen, pectin, hyaluronic acid, and alginic acid.
• the adhesion factor may be one or more substances selected from gelatin, fibrin, fibronectin, hyaluronic acid, laminin, type IV collagen, heparan sulfate proteoglycan, and entactin, and is particularly preferably gelatin or fibrin.
• Fibrin gel is a gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution.
• the commercially available Bolheal (registered trademark) tissue adhesive can also be used.
• the fibrinogen solution and the thrombin solution can be reacted by contacting or mixing them.
• “Gelatin” is water-insoluble collagen that has been pretreated with, for example, an acid or alkali and then solubilized by thermal hydrolysis. It is also possible to obtain gelatin commercially, such as Gelatin LS-H (Nitta Gelatin Co., Ltd., pigskin alkali-treated gelatin, non-heat-treated gelatin, high jelly strength) and Gelatin LS-W (Nitta Gelatin Co., Ltd., pigskin alkali-treated gelatin, heat-treated gelatin, low jelly strength). Alkali-treated (lime-treated) gelatin (type B gelatin) is preferred, and heat-treated gelatin is also preferred.
• Hydrogels undergo a phase change from gel to sol and from sol to gel when heated or cooled. When cooled, hydrogels lose fluidity and become gel (jelly), and when heated, they become sol (aqueous solution).
• melting point means the temperature at which a sol forms under a constant pressure
• freezing point means the temperature at which a gel forms under a constant pressure. It is preferable that the hydrogel is decomposed in the living body.
• a hydrogel e.g., gelatin having a melting point close to body temperature (25°C to 40°C) is preferable.
• the hydrogel in this specification may have a melting point of 20°C to 40°C (e.g., 20°C to 35°C, 25°C to 35°C, 30°C to 40°C, 35°C to 40°C).
• the melting point of a gel is a measure of the strength of the network
• the melting point of a hydrogel e.g., gelatin
• increasing the solid content by adding sugars tends to raise the melting point and freezing point. In this way, it is possible to vary the melting point and freezing point within a certain range.
• There are no particular limitations on the method for measuring the melting point of a hydrogel and it can be measured, for example, by the method specified in JIS K6503.
• the strength of a hydrogel may be such that the hydrogel does not collapse during the operation for transplantation.
• "Jelly strength” is an index of the strength of a hydrogel.
• the “jelly strength” of a hydrogel means the mechanical strength of an object formed from 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, dyne (s)/ cm2 or g/ cm2 ), and is mainly a measure of the hardness of the gel.
• 1 dyne is defined as the force that, when acting on an object with a mass of 1 gram (g), gives an acceleration of 1 centimeter per second per second (cm/ s2 ) in that direction.
• the jelly strength of gelatin can be measured by the method specified in JIS K6503.
• the jelly strength of a hydrogel (e.g., gelatin) gel may be 50 g or more, 100 g or more, 200 g or more, 500 g or more, 1000 g or more, 1200 g or more, 1300 g or more, 1400 g or more, or 1500 g or more.
• the jelly strength of a hydrogel (gelatin) may be 3000 g or less, 2500 g or less, or 2000 g or less.
• the concentration of the adhesion factor varies depending on the size of the neural retina sheet or retinal pigment epithelial cell sheet and the number of retinal pigment epithelial cells, but a person skilled in the art can easily determine the concentration by checking the adhesion state of the RPE cells.
• concentration of the adhesion factor extracellular matrix
• Matrigel it is preferable to add a concentration of 200 to 10,000 times diluted ready-made product (Corning), and in the case of iMatrix511, it is preferable to add it at a concentration of 0.1 to 5 ⁇ g/mL.
• the neural retina sheet and the retinal pigment epithelial cells or the retinal pigment epithelial cell sheet may be cultured in a medium containing an adhesion factor (extracellular matrix) to adhere to each other.
• the medium to be used is not particularly limited, but examples thereof include media used for culturing retinal pigment epithelial cells or neural retina (e.g., DMEM/F12 medium, Neurobasal medium, a mixed medium of these, RPE maintenance medium, etc.).
• the culture for adhesion may be performed in the presence of other components such as growth factors such as EGF together with the extracellular matrix.
• the culture may be continued in the above-mentioned medium containing the adhesion factor, or after a certain period of culture (e.g., 1 to 10 days) in the above-mentioned medium containing the adhesion factor, the medium may be replaced with a medium not containing the adhesion factor, and the culture may be continued.
• a certain period of culture e.g. 1 to 10 days
• the neural retina sheet or the retinal pigment epithelial cell sheet Before culturing to adhere the neural retina sheet to the retinal pigment epithelial cells or retinal pigment epithelial cell sheet, the neural retina sheet or the retinal pigment epithelial cell sheet may be coated with an adhesion factor.
• the neural retina sheet or the retinal pigment epithelial cell or the retinal pigment epithelial cell sheet may be cultured in the above-mentioned medium containing the adhesion factor.
• a person skilled in the art can set the culture time appropriately, but it is sufficient to culture for about 10 minutes to 5 hours (for example, 10 minutes to 60 minutes). After culturing, the sheet may be washed with a medium such as PBS.
• the adhesive factor, hydrogel or matrix gel is preferably fibrin gel.
• Fibrin gel is a gel-like fibrin obtained by reacting a fibrinogen solution with a thrombin solution.
• a substance that has the property of forming a gel by reaction is called a matrix precursor, and for example, thrombin and fibrinogen that react to form a fibrin gel are an example of a matrix precursor.
• the fibrinogen solution can be prepared by dissolving fibrinogen powder or the like in a dissolving solution that contains aprotinin and can dissolve fibrinogen.
• the concentration is not particularly limited, but is, for example, 40 to 480 mg/mL, preferably 80 mg/mL to 320 mg/mL (e.g., 160 mg/mL).
• the concentration may be 37.5 units/mL to 225 units/mL (e.g., 75 units/mL).
• the thrombin solution can be prepared by dissolving thrombin powder or the like in a thrombin dissolving solution containing calcium chloride hydrate, and the concentration is not particularly limited, but is, for example, 125 units/mL to 750 units/mL (e.g., 75 units/mL).
• the fibrinogen solution and the thrombin solution can be reacted by contacting or mixing them, and in this case, the fibrinogen solution and the thrombin solution are preferably used in an activity ratio of 1:1 to 1:9, preferably 1:3 to 1:4.
• a method for producing a composite using a fibrin gel is shown below. Specifically, the neural retina sheet and the retinal pigment epithelial cells or retinal pigment epithelial cell sheet are adhered to each other using a fibrin gel produced by reacting fibrinogen with thrombin.
• one tissue that has been contacted with a fibrinogen solution and the other tissue (retinal pigment epithelial cells or neural retina) that has been contacted with a thrombin solution may be brought into contact with each other to cause a reaction between the fibrinogen and thrombin, resulting in gelation.
• contact with the solution refers to contacting at least the adhesive surface of the tissue (the surface that faces the other tissue when adhering to the other tissue with the fibrin gel) with the fibrinogen solution or thrombin solution to the extent that the solution adheres to the adhesive surface of the tissue.
• the neural retina sheet when adhering a neural retina sheet to a retinal pigment epithelial cell sheet, the neural retina sheet is immersed in a fibrinogen solution, and the retinal pigment epithelial cell sheet is immersed in a thrombin solution (the solutions can be reversed). It is preferable to use a fibrinogen solution and a thrombin solution in a volume ratio of 3:1 to 1:3. Before adhesion, excess fibrinogen or thrombin attached to the tissues may be removed. This operation allows the thickness of the fibrin gel to be adjusted.
• the two may be brought into contact.
• bringing the tissues into contact means bringing the surface with the fibrinogen solution and the surface with the thrombin solution into contact, i.e., overlapping them.
• the tissue may be contacted with a fibrinogen solution, and then a thrombin solution may be added to the fibrinogen solution to react the fibrinogen and thrombin.
• a thrombin solution may be added to the fibrinogen solution to react the fibrinogen and thrombin.
• the tissue is embedded in a fibrin gel.
• the manufacturing method of the present invention may further include a step of cutting out a composite of a size required for transplantation from the composite. Since two or more tissues are firmly adhered by the fibrin gel, when a graft is cut out from the composite, the tissues can be easily cut out to the desired size without peeling off from the composite. When cutting out, tweezers, a knife, scissors, etc. can be used.
• compositions, methods of treatment, therapeutic agents and uses includes a pharmaceutical composition comprising retinal tissue (e.g., sheet-like retinal tissue).
• the pharmaceutical composition preferably further comprises a medicamentously acceptable carrier in addition to the retinal tissue of the present invention.
• the pharmaceutical composition may be used to treat disorders of neural retinal cells or neural retina or diseases caused by damage to the neural retina.
• diseases caused by disorders of neural retinal cells or neural retina include ophthalmic diseases such as retinal degeneration disease, macular degeneration, age-related macular degeneration, retinitis pigmentosa, glaucoma, corneal disease, retinal detachment, central serous chorioretinopathy, cone dystrophy, and cone-rod dystrophy.
• damaged states of the neural retina include states in which photoreceptor cells are degenerated and dead.
• a physiological aqueous solvent such as physiological saline, a buffer solution, or a serum-free medium
• the pharmaceutical composition may contain preservatives, stabilizers, reducing agents, isotonicity agents, etc. that are commonly used in medicines containing tissues or cells to be transplanted in transplantation medicine.
• One aspect of the present invention provides a therapeutic agent for a disease caused by a disorder of the neural retina, comprising the retinal tissue (e.g., sheet-like retinal tissue) obtained by the present invention.
• Another aspect of the present invention includes a method for treating a disease caused by a disorder of neural retinal cells or the neural retina, or a disease caused by damage to the neural retina, comprising transplanting the retinal tissue obtained by the present invention into a subject requiring transplantation (e.g., under the retina of an eye with an ophthalmic disease).
• the retinal tissue of the present invention can be used as a therapeutic agent for a disease caused by a disorder of the neural retina, or to replenish the damaged site of the neural retina in a damaged state of the neural retina.
• the sheet-like retinal tissue of the present invention can be transplanted into a patient having a disease caused by a disorder of the neural retina or a patient having a damaged state of the neural retina, requiring transplantation, to replenish the neural retinal cells or the damaged neural retina, thereby treating the disease caused by a disorder of the neural retina or the damaged state of the neural retina.
• An example of a transplantation method is a method of transplanting retinal tissue under the retina at the damaged site by incising the eyeball, etc. Methods for transplantation include, for example, injecting using a thin tube or pinching with tweezers, and examples of thin tubes include injection needles.
• a retinal tissue obtained by the present invention for use in treating a disease caused by a disorder of retinal cells or retinal tissue or damage to retinal tissue.
• a retinal tissue obtained by the present invention in the manufacture of a therapeutic agent for a disease caused by a disorder of retinal cells or retinal tissue or damage to retinal tissue.
• a specific maintenance culture operation for human ES and human iPS cells was performed as follows. First, human ES/iPS cells that had become subconfluent (about 60% of the culture area was covered with cells) were washed with PBS and then dispersed into single cells using TrypLE Select (trade name, manufactured by Life Technologies).
• dispersion into single cells means dispersion to form single cells, and the cell population dispersed into single cells may contain clumps of 2 to 50 cells in addition to single cells.
• the human ES cells dispersed into single cells were seeded on a plastic culture dish coated with Laminin511-E8, and cultured under feeder-free conditions in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• Y-27632 ROCK inhibitor, 10 ⁇ M
• the number of human ES/iPS cells dispersed into the single cells was 0.4 to 1.2 ⁇ 10 4 cells per well.
• the medium was replaced with StemFit medium not containing Y-27632.
• the medium was replaced with StemFit medium not containing Y-27632 once every 1 to 2 days.
• the medium was cultured under feeder-free conditions until one day before reaching subconfluency.
• the human ES cells one day before reaching subconfluency were cultured under feeder-free conditions for one day in the presence of SB431542 (TGF ⁇ signaling pathway inhibitor, 5 ⁇ M) and SAG (Shh signaling pathway agonist, 300 nM) (Preconditioning treatment).
• human ES/iPS cells were treated with cell dispersion liquid using TrypLE Select, and further dispersed into single cells by pipetting.
• the human ES cells dispersed into single cells were suspended in 100 ⁇ L of serum-free medium so that the number of cells was 1.2 ⁇ 10 4 per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.), and cultured in suspension at 37 ° C. and 5% CO 2.
• the serum-free medium (gfCDM + KSR) used at that time was a serum-free medium prepared by adding 10% KSR, 450 ⁇ M 1-monothioglycerol, and 1 ⁇ chemically defined lipid concentrate to a 1:1 mixture of F-12 medium and IMDM medium.
• Y-27632 (ROCK inhibitor, final concentration 10 ⁇ M or 20 ⁇ M) and SAG (Shh signaling pathway active substance, 300 nM, 30 nM, or 0 nM) were added to the above serum-free medium.
• SAG Shh signaling pathway active substance, 300 nM, 30 nM, or 0 nM
• 50 ⁇ L of medium containing no Y-27632 or SAG but exogenous human recombinant BMP4 product name: Recombinant Human BMP-4, manufactured by R&D Systems
• half of the medium was replaced once every three days with medium containing no Y-27632, SAG, or human recombinant BMP4.
• the aggregates from the 14th to 18th days after the start of the suspension culture were transferred to a 90 mm low-adhesion culture dish (suspension culture dish 90 ⁇ (deep type), manufactured by Sumitomo Bakelite Co., Ltd.) and cultured for 3 to 4 days at 37 ° C. and 5% CO 2 in serum-free medium (DMEM / F12 medium with 1% N2 Supplement added) containing a Wnt signaling pathway active substance (CHIR99021, 3 ⁇ M) and an FGF signaling pathway inhibitor ( SU5402 , 5 ⁇ M).
• serum-free medium DMEM / F12 medium with 1% N2 Supplement added
• the aggregates were cultured for a long period of time in a serum medium (NucT0 medium) that does not contain a Wnt signaling pathway active substance and an FGF signaling pathway inhibitor in a 90 mm low-adhesion culture dish (suspension culture dish 90 ⁇ (deep type), manufactured by Sumitomo Bakelite Co., Ltd.).
• the aggregates on the 16th day (derived from 1231A3) or 27th day (derived from KhES-1) after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37°C for 20-30 minutes, the cells were dispersed into single cells by pipetting.
• Example 2 Search for factors promoting layer structure formation and aggregate growth Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports, 4, 3594 (2014)".
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd. was used as a scaffold instead of feeder cells.
• the aggregates prepared as in Reference Example 1-1 on the 20th day (1231A3) and 27th day (KhES-1) after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37 ° C, they were dispersed into single cells by pipetting.
• the state of aggregate reformation was observed using a bright field microscope and a fluorescent microscope (Keyence BZ-X810) on the 1st, 7th, and 14th days after suspension culture for aggregate reformation.
• the results for KhES-1-derived cells are shown in Figures 3 to 5.
• the area of the aggregates was measured using Image J.
• the results for KhES-1-derived cells are shown in Figure 6 (average value of 12 measurements).
• the results for 1231A3-derived cells, in which the state of aggregate reformation was observed on the 1st day after suspension culture, are shown in Figure 7.
• ⁇ Reference Example 1-3 Search for factors promoting layer structure formation and aggregate growth>
• Human ES cells KhES-1 line, (Non-Patent Document 3)
• Rx::Venus reporter gene Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports, 4, 3594 (2014)”.
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd.
• the aggregates prepared as in Reference Example 1-1 on the 26th day after the start of suspension culture were washed with PBS, and a nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. These cells were suspended in 100 ⁇ L of serum-free medium to 3.0 ⁇ 10 4 cells per well of a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.), and cultured in suspension at 37°C and 5% CO 2 .
• a nerve cell dispersion liquid manufactured by Wako
• the aggregates were fixed with 4% PFA on the 15th day (corresponding to the 41st day of differentiation) and 28th day (corresponding to the 54th day of differentiation) and frozen sections were prepared. These frozen sections were stained with DAPI and anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EXalpha), anti- ⁇ -Catenin antibody (R&D Systems), anti-Collagen IV antibody (Abcam), anti-Zo-1 antibody (Invitrogen), anti-Ki67 antibody (BD), anti-Pax6 antibody (BioLegend), and anti-RxR- ⁇ (RxRg) antibody.
• DAPI and anti-Chx10 antibody trade name: Anti CHX10 Antibody, EXalpha
• anti- ⁇ -Catenin antibody R&D Systems
• Anti-Collagen IV antibody Abcam
• Anti-Zo-1 antibody Invitrogen
• anti-Ki67 antibody BD
• Anti-Pax6 antibody BioLegend
• RxRg anti-RxR- ⁇
• Immunostaining was performed using anti-IL-1 antibody (Spring Bioscience), anti-CRX antibody (Abnova), anti-NRL antibody (R&D Systems), anti-Recoverin antibody (Proteintech), anti-Islet-1 antibody (DSHB), anti-GS antibody (Sigma), anti-Brn3 antibody (Santa Cruz), and anti-Calretinin antibody (R&D Systems).
• Figures 11 to 20 These immunostained sections were observed using a bright field microscope, a fluorescence microscope (Keyence BZ-X810), and a confocal laser scanning fluorescence microscope (Leica SP-8), and the results are shown in Figures 11 to 20.
• Figures 11, 14, 15, and 18 confirm that when CHIR99021 was added, a layer structure containing Rx::Venus-positive and Chx10-positive neural retinal progenitor cells was formed on the outermost side of the aggregate.
• Figures 12, 16, and 17 also confirm that when CHIR99021 was added, a Zo-1-positive apical surface was formed on the outermost side of the aggregate, and a collagen-positive basal surface was formed inside, and epithelial tissue with apical and basal polarity was formed.
• Figure 17 in particular, in the group to which CHIR99021 was not added, it was observed that there was no cell polarity or that a rosette-like structure was shown.
• Figures 19 and 20 confirm that RxR-gamma (RxRg)-positive and CRX-positive cone precursor cells, Recoverin-positive and CRX-positive photoreceptor precursor cells were differentiated, and differentiation of Islet-1-positive and Brn3-positive retinal ganglion cells and Calretinin-positive amacrine cells was also confirmed.
• RxR-gamma (RxRg)-positive and CRX-positive cone precursor cells Recoverin-positive and CRX-positive photoreceptor precursor cells were differentiated, and differentiation of Islet-1-positive and Brn3-positive retinal ganglion cells and Calretinin-positive amacrine cells was also confirmed.
• the aggregates prepared as in Reference Example 1-1 on the 18th, 25th, 40th, 61st, and 75th days after the start of suspension culture (dd18, dd25, dd40, dd61, dd75) were washed with PBS and nerve cell dispersion liquid (manufactured by WAKO) was added. After incubation at 37 ° C, the cells were dispersed into single cells by pipetting.
• Figure 21 shows the results of observing the state of aggregate reformation using a bright field microscope and a fluorescent microscope on the 3rd, 15th, and 21st days (Day 3, Day 15, and Day 21) of culture for the reformation of aggregates.
• Figure 21 confirms that aggregates were reformed and a layered structure was formed in cell aggregates on all differentiation days, dd18, dd25, dd40, dd61, and dd75. It was also observed that the characteristics of Rx::Venus-positive retinal tissue were maintained (Figure 21).
• the aggregates thus prepared 40 days after the start of suspension culture were washed with PBS, and a nerve cell dispersion liquid (manufactured by WAKO) was added. After incubation at 37 ° C, the cells were dispersed into single cells by pipetting.
• the dispersed cells were suspended in 100 ⁇ L of serum-free medium containing 10 mM Y-27632 (Wako), 300 nM SAG (Enzo) and 3 ⁇ M CHIR99021 (Wako) so that the number of cells per well of a non-cell-adhesive 96-well culture plate (trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) was 5 ⁇ 10 5 cells, and the suspension culture was performed at 37 ° C and 5% CO 2 .
• a non-cell-adhesive 96-well culture plate trade name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.
• Figure 22 shows the results of bright field and fluorescence microscopy of the state of aggregate reformation on days 3, 15, and 21 of culture for the reformation of aggregates.
• Figure 22 confirms that aggregates are reformed and a layered structure is formed in Rx::Venus-negative telencephalic organoids as well, just like in retinal organoids.
• the aggregates prepared as in Reference Example 1-1 on the 31st day after the start of suspension culture were washed with PBS and nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting.
• PBS nerve cell dispersion liquid
• the cells were suspended in 100 ⁇ L of serum-free medium containing 10 ⁇ M Y-27632 (Wako), 300 nM SAG (Enzo), and 3 ⁇ M CHIR99021 (Wako) to give a density of 2.0 ⁇ 10 cells per well of a non-cell-adhesive 96-well culture plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.), and cultured in suspension at 37°C and 5% CO2 .
• a non-cell-adhesive 96-well culture plate product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.
• the single cells were suspended at 1.0 x 10 6 cells/mL in the following cryopreservation solutions, Cell Banker 1 (TaKaRa), Stem Cell Banker (TaKaRa), CultureSure (registered trademark) cryopreservation solution (CultureSure (registered trademark) Freezing Medium; Fujifilm Wako Pure Chemical Corporation), STEMdiff (trademark) Neural Progenitor Freezing Medium (Stemcell), StemSure (registered trademark) cryopreservation solution (Fujifilm Wako Pure Chemical Corporation), and Bambanker (Nippon Genetics Co., Ltd.), and were cryopreserved at -80 °C using Bicell as the frozen group.
• cryopreservation solutions Cell Banker 1 (TaKaRa), Stem Cell Banker (TaKaRa), CultureSure (registered trademark) cryopreservation solution (CultureSure (registered trademark) Freezing Medium; Fujifilm Wako Pure Chemical Corporation),
• the aggregates prepared as in Reference Example 1-1 25 to 35 days after the start of suspension culture were fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained using DAPI and anti-Laminin antibody (trade name: Anti Laminin Antibody, Abcam), anti-Fibronectin antibody (R&D Systems), and anti-Collagen IV antibody (Abcam). These immunostained sections were observed using a confocal laser microscope. As a result, it was confirmed that the aggregates expressed at least Laminin, Fibronectin, and Collagen IV, which are components of the basement membrane (Figure 25). In other words, in this self-organizing culture system, it was found that human retinal tissue formed its own basement membrane and expressed laminin, fibronectin, and collagen IV.
• Laminin 511, Laminin 521, Laminin 411, Fibronectin, Collagen IV, etc. may be useful as extracellular matrices for regenerating retinal sheets in adhesive culture.
• the aggregates prepared as in Reference Example 1-1 on the 16th day after the start of suspension culture were washed with PBS and nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting.
• PBS nerve cell dispersion liquid
• Matrigel As an extracellular matrix to promote re-sheet formation, Matrigel (Corning) and Laminin511-E8 (trade name iMatrix511, manufactured by Nippi) were used.
• the above-mentioned dispersed single cells of retinal system cells were suspended and seeded in 300 ⁇ L of serum- free medium containing 10 ⁇ M Y-27632 (Wako), 300 nM SAG (Enzo) and 3 ⁇ M CHIR99021 (Wako) in 24-well Transwell (Corning) under the following three conditions so that the number of cells was 0.5 to 4 ⁇ 10 5 per well.
• Condition 1 Seeding in 24-well Transwells pre-coated with Matrigel (Matrigel (Pre coat))
• Condition 2 Seeded onto 24-well Transwells pre-coated with Laminin511-E8 (iMatrix511 (Pre coat))
• Condition 3 Cells were seeded in a culture medium containing Laminin 511-E8 onto a 24-well Transwell that had not been previously coated (Mix method).
• the aggregates prepared as in Reference Example 1-1 on days 15 to 30 after the start of suspension culture were washed with PBS, and a nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37° C., the aggregates were dispersed into single cells by pipetting.
• the cells were suspended in 300 ⁇ L of serum-free medium containing (1) 10 ⁇ M Y-27632 (Wako), (2) 10 ⁇ M Y-27632 (Wako) and 300 nM SAG (Enzo), (3) 10 ⁇ M Y-27632 (Wako) and 5 ⁇ M CHIR99021 (Wako), or (4) 10 ⁇ M Y-27632 (Wako), 300 nM SAG (Enzo), and 3 ⁇ M CHIR99021 (Wako) at 8 ⁇ 10 5 cells per well in a 12-well Transwell coated with Laminin 511-E8, and cultured as adherent cells at 37°C and 5% CO 2 .
• the medium was replaced with serum-free medium (NucT0) not containing Y-27632, SAG, or CHIR99021 once every 3 to 4 days.
• the aggregates prepared as in Reference Example 1-1 33 days after the start of suspension culture (dd33) were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37 ° C, they were dispersed into single cells by pipetting.
• retinal sheets can be regenerated in single-cell dispersed retinal cells (NR) by adding CHIR99021 at a concentration of at least 1-9 ⁇ M for 3-9 days.
• the aggregates prepared as in Reference Example 1-1 24 days after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37°C, the aggregates were dispersed into single cells by pipetting.
• retinal sheets can be recreated from single-cell dispersed aggregates (NR) using not only iMatrix511 and Matrigel, but also a variety of scaffold proteins.
• the aggregates prepared as in Reference Example 1-1 on the 15th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by 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 ⁇ L of serum-free medium containing 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021 in a 24-well Transwell coated with Laminin511-E8 so that the number of cells per well was 2.0 ⁇ 10 5 , and cultured at 37 ° C, 5% CO 2 for 3 days. After 3 days from seeding, the medium was replaced once every 3 to 4 days with serum-free medium (NucT0) medium not containing Y-27632, SAG, and CHIR99021.
• serum-free medium NucT0
• the aggregates on the 29th day after the start of suspension culture were used to re-form a sheet in the same manner.
• the cell sheet on the 28th day (Day 67) of the reorganization culture was fixed with 4% PFA and frozen sections were prepared. These frozen sections were immunostained using DAPI and anti-Ki67 antibody (R&D Systems), anti-Chx10 antibody (trade name: Anti CHX10 Antibody, EX alpha), anti-Pax6 antibody (BD Pharmingen), anti-Brn3 antibody (Santa Cruz), anti-RxR- ⁇ (RxRg) antibody (Spring Bioscience), and anti-Crx antibody (abnova).
• dd70 3D Retina (cell aggregates) that had not been flattened (re-sheeted) were also stained (FIGS. 35 to 37). Furthermore, after the cell sheet on Day 57 was fixed with 4% PFA, whole-body immunostaining was performed without preparing sections.
• DAPI and anti-Chx10 antibody product name: Anti CHX10 Antibody, EX alpha), anti-Sox2 antibody (BD Pharmingen), anti-Ki67 antibody (BD), anti-Pax6 antibody (BioLegend), anti-Brn3 antibody (Santa Cruz) , anti-TUJ1 antibody (Millipore), anti-Islet-1 antibody (R&D Systems), anti-Crx antibody (abnova), anti-Recoverin antibody (Proteintech), anti-Zo-1 antibody (Invitrogen), anti-Collagen IV antibody (Abc Immunostaining was performed using am Co., Ltd.).
• the dd112 cell sheet was immunostained in the sheet state without cutting.
• anti-Ribeye antibody CtBP2, BD
• anti-Recoverin antibody Proteintech
• anti-Pax6 antibody BioLegend
• anti-Chx10 antibody trade name: Anti CHX10 Antibody, SantaCruz
• ⁇ Reference Example 1-13 Maintenance culture of retinal cells using Rx::Venus strain> Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports 4, 3594 (2014)".
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd.
• the aggregates prepared as in Reference Example 1-1 on day 31 after the start of suspension culture were washed with PBS, and a nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37° C., the aggregates were dispersed into single cells by pipetting. After dispersion, 10 ⁇ L of Laminin511-E8-coated 12-well Transwells were seeded with 2.0 ⁇ 10 cells per well in 500 ⁇ L of serum-free DMEM/F12 containing 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021, and N2 was added to the medium.
• a nerve cell dispersion liquid manufactured by Wako
• the medium contained (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 ⁇ g/mL GDNF, (10) 20 ⁇ g/mL BDNF, (11) 2 ⁇ M Pyrintegrin, and (12) 1 ⁇ M BMP4 were added, and the cells were cultured for 3 days at 37° C. and 5% CO 2. From day 3 after seeding, the medium was replaced once every 3 to 4 days with serum-free medium (DMEM/F12 supplemented with N2) that did not contain Y-27632, SAG, or CHIR99021.
• serum-free medium DMEM/F12 supplemented with N2
• the aggregates (NR) prepared in Reference Example 1-1 on the 27th day after the start of suspension culture were washed with PBS, and a nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 500 ⁇ L of the following medium containing 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021 to a density of 8.0 ⁇ 10 5 cells/well and seeded on a 12-well Transwell coated with 10 ⁇ L of Laminin511-E8.
• NeuroCult NS-A (STEMCELL Technologies), (2) STEMdiff Neural Progneitor (STEMCELL Technologies), (3) StemPro NSC SFM (Thermo Fisher), and (4) RHB-A (TaKaRa) were cultured for one month with 20 ng/mL FGF2 and 20 ng/mL EGF.
• NucT0 medium was used as a control. After 3 days from seeding, the medium was replaced once every 3 to 4 days with a medium that did not contain Y-27632, SAG, and CHIR99021.
• a collagen gel was prepared using beMatrix low endotoxin collagen solution (Nitta Gelatin Co., Ltd.). Specifically, collagen AT, 5xDME, and reconstitution buffer were mixed in a ratio of 7:2:1, coated on the mesh of a Transwell, and incubated at 37°C in a 5% CO2 incubator for 30 minutes. After incubation, medium was added to the inside and outside of the insert.
• the aggregates (NR) prepared in Reference Example 1-1 on the 26th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were seeded on the collagen gel of a 12-well Transwell at 8.0 x 10 5 cells/well. For the first three days, the cells were cultured in the presence of 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021. After 3 days from seeding, the medium was replaced once every 3 to 4 days with serum-free medium (NucT0 medium) that does not contain Y-27632, SAG, and CHIR99021.
• serum-free medium NucT0 medium
• the 26th day aggregates (NR) on the collagen gel prepared as in Reference Example 1-15 were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting. After dispersion, the cells were seeded on the collagen gel of a 12-well Transwell at 8.0 x 10 5 cells/well. For the first 3 days, the cells were cultured in the presence of 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021. After 3 days from seeding, the medium was replaced once every 3 to 4 days with serum-free medium (NucT0 medium) that did not contain Y-27632, SAG, and CHIR99021.
• serum-free medium NucT0 medium
• the retinal cell sheets on days 63 and 70 of differentiation after 37 and 44 days of culture were treated with Collagenase (Roche) for 30 minutes at 37°C and detached.
• the detached retinal sheets were cut into strips using tweezers and scissors to prepare grafts for transplantation with a length of approximately 1.8 cm (Figure 47).
• the prepared grafts were transplanted under the retina of immunodeficient retinal-deficient rats (SD Foxn) using a glass Pasteur pipette.
• the sorted cells were seeded on 12-well Transwells (Corning) coated with Laminin511-E8 at 1.6-8.0 x 10 5 cells per well. A sheet without sotting was also prepared as a control.
• the cells were cultured in the presence of 10 ⁇ M Y-27632, 300 nM SAG, and 3 ⁇ M CHIR99021.
• the medium was replaced with serum-free medium (NucT0 medium) that did not contain Y-27632, SAG, or CHIR99021 once every 3 to 4 days.
• the cells were observed using a fluorescent stereomicroscope on the 26th day (FIG. 50).
• the cell sheet purified with Rx::Venus was fixed with 4% PFA, and immunostaining was performed on the sheet without preparing sections. Immunostaining was performed using DAPI and anti-Recoverin antibody (Proteintech). As a control, a retinal sheet that had not been sorted was also stained ( Figures 51, 52, and 53). After that, frozen sections were prepared and immunostaining was performed using DAPI and anti-Ki67 antibody (BD), anti-Chx10 antibody (trade name: Anti CHX10 Antibody, Santa Cruz), and anti-CRX antibody (TaKaRa) ( Figure 53).
• ⁇ Reference Example 1-18 Search for factors that maintain the properties of Retina when re-formed into a sheet>
• Human ES cells KerES-1 line, (Non-Patent Document 3)
• Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports 4, 3594 (2014).
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd. was used as a scaffold instead of feeder cells.
• the aggregates (NR) prepared in Reference Example 1-1 on the 25th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37 ° C., the cells were dispersed into single cells by pipetting, and sorting was performed using Cell sorter ARIAII (BD) with Rx::Venus as an index.
• the cells after sorting were seeded on a 96-well glass bottom plate (Greiner) coated with Laminin511-E8 using Easy iMatrix, with 5 ⁇ 10 4 cells per well, and 100 ⁇ L of NucT0 was added with Y-27632, CHIR99021, and SAG. At the same time as seeding, the proteins shown in Table 1 or the low molecular weight compounds shown in Table 2 were added.
• the aggregates (NR) prepared in Reference Example 1-1 on the 24th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and sorting was performed using Cell sorter ARIAII (BD) with Rx::Venus as an index. The cells after sorting were seeded at 2.0 to 5.0 x 10 4 cells per well on low-adhesion Vplate. As controls, a group without FGF8 (FGF8-) and a group without sorting (no sorting) were also placed.
• FGF8- group without FGF8
• no sorting were also placed.
• the medium was changed once every 3-4 days. FGF8 was also added when changing the medium.
• Rx::Venus-negative cell clusters or RPE cells were observed in the group without sorting and FGF8- ( Figure 57).
• FGF8+ FGF8 addition
• RPE cells were partially observed.
• Rx::Venus-positive aggregates were formed, and RPE cells or Rx::Venus-negative clusters were not observed.
• the aggregates on the 54th day of culture (30 days after reseeding) for the purpose of re-formation of the aggregates were washed with PBS and dispersed into single cells using a neuronal cell dispersion liquid (Wako), and then fixed with formaldehyde using Fixation buffer (BD). After washing with Perm/Wash buffer (BD), the cells were stained with anti-CHX10-Alexa Fluo 647 Conjugate antibody (Santacruz), anti-Sox2-BV421 antibody (Biolegend), anti-Ki67-Alexa Fluo 647 Conjugate antibody (BD), and anti-CRX-Alexa Fluo 647 Conjugate antibody (Santacruz).
• the aggregates (NR) prepared in Reference Example 1-1 on the 24th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37 ° C., the cells were dispersed into single cells by pipetting, and sorting was performed using Cell sorter ARIAII (BD) with Rx::Venus as an index. The cells after sorting were suspended and seeded in 24-well Transwell (Corning) at 2.0 ⁇ 10 5 cells per well in NucT0 medium containing Y-27632, CHIR99021, SAG, and FGF8. As a control, a group without FGF8 (FGF8-) was also placed.
• FGF8- nerve cell dispersion liquid
• the medium was changed once every 3-4 days. FGF8 was also added when changing the medium.
• the sheets on day 63 of differentiation, 40 days after reseeding, were observed under a fluorescent stereomicroscope ( Figures 60 and 61).
• the aggregates on day 64 of differentiation, 41 days after reseeding were washed with PBS and dispersed into single cells using a nerve cell dispersion liquid (Wako), and then fixed with formaldehyde using Fixation buffer (BD).
• Wako nerve cell dispersion liquid
• BD Fixation buffer
• the aggregates prepared as in Reference Example 1-1 on the 27th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (manufactured by Wako) was added. After incubation at 37 ° C, they were dispersed into single cells by pipetting. After dispersion, the cells were suspended in 200 ⁇ L of serum-free medium containing 10 ⁇ M Y-27632, 300 nM SAG, 3 ⁇ M CHIR99021, and 100 ng / mL FGF8 so that there were 2.0 ⁇ 10 5 cells per well in a 24-well Transwell coated with Laminin 511-E8, and cultured at 37 ° C, 5% CO 2 for 3 days. After 3 days from seeding, the medium was replaced once every 3 to 4 days with serum-free medium (NucT0) medium not containing Y-27632, SAG, CHIR99021, and FGF8.
• serum-free medium NucT0
• the above cells were used to initiate suspension culture for differentiation induction as described in Reference Example 1-1.
• 300 nM SAG was added.
• the aggregates (NR) on the 19th and 26th days after the start of suspension culture were washed with PBS, and neural cell dispersion liquid (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and surface antigen screening was performed using a surface antigen screening kit, MACS (registered trademark) Marker Screen, human (Miltenyi) (fluorescent dye conjugate antibody group with Alexa 647). Human ES cells (hESC) were stained as a control. A total of 371 markers were used for screening ( Figures 64-69).
• FACS analysis was performed using MACS Quant10 (Miltenyi) and FlowJo. Gating was performed on the populations shown by FSC and SSC in Figure 65. First, it was confirmed that staining and analysis were performed accurately using E-Cadherin, SSEA-4, and SSEA-5, which are expressed in hESCs and lose their expression upon differentiation ( Figure 66). The results of the subsequent analysis are summarized in Figure 64.
• hESC high or there is expression in human ES cells
• hESC: low means that the expression level is low or there is no expression in human ES cells
• NR: low means that the expression level is low or there is no expression in neural retinal cells.
• 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).
• CD39, CD73, and CD184 were analyzed by FACS analysis using mouse IgG1-APC-Conjugate antibody (Miltenyi), anti-CD39-APC-Conjugate antibody (Miltenyi), anti-CD73-APC-Conjugate antibody (Miltenyi), and anti-CD184 (CXCR4)-APC-Conjugate antibody (Miltenyi) at dd18, dd25, dd53, and dd81 (d18, d25, d53, and d81 in Figure 68) after the start of suspension culture ( Figure 68).
• the morphology of aggregates created 26 days after the start of suspension culture by adding (1) 0 nM, (2) 30 nM, or (3) 300 nM SAG at the start of differentiation induction was observed under a microscope. As a result, it was confirmed that the aggregates were larger and the layer structure was more clearly visible when 0 nM and 30 nM SAG were added compared to when 300 nM SAG was added at the start of suspension culture (dd0) ( Figure 73).
• the above aggregates were dispersed into single cells using a nerve cell dispersion liquid, and FACS analysis was performed on the expression of CD39 and CXCR4 using anti-CD39-APC conjugate antibody (Miltenyi) and anti-CXCR4-APC conjugate antibody (Miltenyi).
• the proportion of expressing cell population was measured using a FACSCantoII (BD Bioscience), and analysis was performed using FlowJo.
• ⁇ Reference Example 1-25 Examination of enhancement of CD39 expression> Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports 4, 3594 (2014)".
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd. was used as a scaffold instead of feeder cells.
• CD39 expression can be enhanced by adding ATP and an A2A receptor agonist.
• BD Cell sorter ARIAII
• the medium was changed once every 3-4 days. FGF8 was also added when changing the medium.
• Crx::Venus cell populations were observed to be sparse in the group where FGF8 was not added, whereas they were uniformly present in the group where FGF8 was added ( Figure 82).
• the sheets thus produced were collected using collagenase on the 88th day after the start of suspension culture, and a long graft for transplantation was then excised using microscissors (Figure 82), which was then transplanted under the retina of immunodeficient retinal dysfunction rats (SD Foxn).
• retinal sheets produced from cells sorted by CD39 can be transplanted.
• the aggregates (NR) were prepared 24-26 days after the start of suspension culture by adding 30 nM SAG at the start of suspension culture (dd0). They were dispersed into single cells using a nerve cell dispersion liquid (Wako) and stained using a surface antigen screening kit, MACS (registered trademark) Marker Screen, human (Miltenyi), to perform surface antigen screening (Alexa 647 fluorescent dye conjugate antibody group).
• a brain organoid prepared without adding BMP4 on the third day after the start of differentiation induction was stained.
• FACS analysis was performed using MACS Quant10 (Miltenyi) and analysis was performed using FlowJo. Searching for surface antigens that were not expressed (or were expressed) in Brain Organoids and were expressed (or not expressed) in the Rx::Venus positive group on days 24-26 after the start of suspension culture, it was shown that CD9, CD15, CD49c, CD66b, CD69, CD82, CD164, EpCAM, and ErbB2 (CD340) could be distinguished from Brain Organoids ( Figure 83).
• CD9, CD24, CD49c, CD90, CXCR4, and EpCAM may be useful markers for distinguishing Rx::Venus positive and negative cells ( Figure 84).
• hESCs and aggregates (NR) of dd4, dd11, dd18, dd25, dd32, and dd46 which were produced by adding 30 nM SAG at the start of suspension culture (dd0, when differentiation induction began), were dispersed into single cells in a neural cell dispersion medium (Wako), stained with anti-CD9 antibody (BioLegend) conjugated with APC, and after washing the antibody, FACS analysis was performed. FACS analysis was performed using a MACS Quant10 (Miltenyi) and analysis was performed using FlowJo.
• NR 25th day aggregates prepared by adding 30nM SAG at the start of suspension culture (at the start of differentiation induction, dd0) were dispersed into single cells in a neural cell dispersion medium (Wako), stained with anti-CD9 antibody conjugated with APC (BioLegend), and anti-SSEA1 antibody conjugated with BV421 (BioLegend), measured using a FACSCantoII (BD Bioscience), and analyzed using FlowJo.
• the percentage of Rx-positive cells in the entire aggregate was approximately 87%, but when CD9-positive cells were gated, it was found that the percentage of Rx-positive cells increased to 94%. Furthermore, when CD9-positive and SSEA1-negative cells were gated, it was found that the percentage of Rx-positive cells increased to approximately 96% ( Figure 86).
• retinal cells can be further purified.
• ⁇ Reference Example 1-30 Study of the combination of CD9, CD90, CXCR4, and SSEA1> Human ES cells (KhES-1 line, (Non-Patent Document 3)) genetically modified to have the Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports 4, 3594 (2014)".
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd. was used as a scaffold instead of feeder cells.
• NR neural cell dispersion medium
• the cells were stained with (5) anti-CD9-APC-Conjugate antibody (BioLegend) and anti-CD90-BV421-Conjugate antibody (BioLegend), and (6) anti-CD90-APC-Conjugate antibody (BD) and anti-SSEA1-BV421-Conjugate antibody (BioLegend). After staining, sorting was performed using ARIA II (BD) with the frame shown in FIG. 87 as an index. The cells collected by sorting were subjected to FACS analysis using MACS Quant10 (Miltenyi) to confirm that the target cell fraction was purified (FIG. 87).
• retinal sheets sorted for CD9 and CD9 and SSEA1 negative or CD90 positive fractions were better than those sorted for Rx::Venus positive, as they were able to remove the RPE fraction.
• hESCs and aggregates (NR) of dd4, dd11, dd18, dd25, dd32, and dd46 which were produced by adding 30 nM SAG at the start of suspension culture (dd0, when differentiation induction began), were dispersed into single cells using neural cell dispersion medium (Wako), stained with anti-CD9-APC-Conjugate antibody (BioLegend) and anti-SSEA1-BV421-Conjugate antibody (BioLegend), and subjected to FACS analysis. Measurements were performed using a FACSCantoII (BD Bioscience), and analysis was performed using FlowJo.
• CD9 and SSEA1 were sorted as indicators, and 10 ⁇ M Y-27632, 3 ⁇ M CHIR99021, 300 nM SAG, and 100 ng/mL FGF8 were added.
• the retinal sheets prepared on the 12-well Transwells were then collected using a scalpel or scissors on the 48th and 62nd days after the start of suspension culture, and long grafts for transplantation were then cut out using microscissors and transplanted under the retina of immunodeficient retinal insufficient rats (SD Foxn) (not shown).
• retinal sheets produced using cells sorted with CD9 and SSEA1 are transplantable.
• the aggregates (NR) prepared in Reference Example 1-1 on the 18th to 30th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37 ° C., the cells were dispersed into single cells by pipetting, and 4.0 to 8.0 ⁇ 10 5 cells were seeded per well in a 12-well Transwell. At the time of seeding, Y-27632, SAG, and CHIR99021 were added to the NucT0 medium. Thereafter, the medium was replaced once every 3 to 4 days with NucT0 medium not containing Y-27632, SAG, and CHIR99021, and the culture was continued for more than one month.
• PBS nerve cell dispersion liquid
• the RPE aggregates which were differentiated at the same time after 80 days from the start of the floating culture prepared in Reference Example 1-1, were seeded on a dish coated with Laminin511-E8 in a medium containing DMEM (SIGMA) and F12 Ham (SIGMA) medium supplemented with B27 (Gibco), L Glutamin, 10 ng/mL FGF2, and SB431542, and cultured.
• SIGMA DMEM
• F12 Ham F12 Ham
• B27 Gibco
• L Glutamin 10 ng/mL FGF2, and SB431542
• 1.0 x 10 6 cells of RPE were seeded on a 12-well TransWell coated with Laminin511-E8, and the medium was changed once every 3 to 4 days, and the culture was continued for more than one month.
• BeMatrix LS-W gelatin (Nitta Gelatin) was used to adhere the RPE sheet and sheet-like retinal tissue cultured as described above. Specifically, each sheet was retrieved from the Transwell using tweezers and scissors, and the sheet-like retinal tissue and RPE sheet were blended with 10% (w/v) gelatin, then blended with 20% gelatin, and finally 30% gelatin was added to adhere the sheet-like retinal tissue and RPE sheet. After adhesion, the sheets were rapidly cooled to 4°C and incubated for 20 minutes to solidify, and then retrieved to produce a composite sheet in which the sheet-like retinal tissue and RPE sheet were adhered ( Figures 93 to 98).
• the retinal tissue-RPE composite sheet thus prepared was cut out using tweezers and scissors. First, the composite sheet was cut in half, and then cut into strips along the cut surface (Figure 99). As a result, both sheets were cut out in a state of being adhered to each other via gelatin. When the cross section of the cut composite sheet was observed, RPE with black pigment was confirmed on one side, and Rx::Venus-positive sheet-like retinal tissue was confirmed on the other side, with gelatin packed between them ( Figure 100). To check whether the cut composite sheet would come off during the aspiration and discharge operations of the transplantation process, a 1 mL tip with its tip cut off was used to perform the aspiration and discharge operations. As a result, the composite sheet could be aspirated and discharged many times without separation (Figure 101).
• a transplantable retinal tissue-RPE composite sheet can be produced by using a sheet-like retinal tissue, an RPE sheet, and gelatin.
• the aggregates (NR) prepared in Reference Example 1-1 on the 18th to 30th day after the start of suspension culture were washed with PBS, and nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and 4.0 to 8.0 x 10 5 cells were seeded per well in a 12-well Transwell. At the time of seeding, Y-27632, SAG, and CHIR99021 were added to the NucT0 medium. Thereafter, the medium was replaced once every 3 to 4 days with NucT0 medium not containing Y-27632, SAG, and CHIR99021, and the culture was continued for more than one month (Figure 102).
• the RPE aggregates which were differentiated at the same time after 80 days from the start of the floating culture prepared in Reference Example 1-1, were seeded on a dish coated with iMatrix511 in a medium containing DMEM (SIGMA) and F12 Ham (SIGMA) medium supplemented with B27 (Gibco), L Glutamin, 10 ng/mL FGF2, and SB431542, and cultured. After the RPE adhered and grew and spread, only the RPE was scraped off using a tip and seeded on another dish coated with iMatrix511 for purification. After expansion culture, 1.0 x 10 6 cells of RPE were seeded on a 12-well TransWell coated with iMatrix511, and the medium was replaced once every 3 to 4 days, and the culture was continued for more than one month (FIG. 102).
• the retinal tissue sheet cultured as described above was attached to the RPE sheet using Bolheal Tissue Adhesive (Teijin Pharma). Specifically, each sheet was retrieved from the Transwell using tweezers and scissors, 100 ⁇ L of Fibrinogen was added to the retinal tissue sheet and 100 ⁇ L of Thorombin was added to the RPE sheet, and after blending, they were removed (Figs. 103-104). Again, 100 ⁇ L of Fibrinogen was added to the retinal tissue sheet and 100 ⁇ L of Thorombin was added to the RPE sheet (Fig. 104).
• the RPE sheet was placed on a culture dish with the apical side facing up and the mesh side facing the culture dish, and the retinal tissue sheet was placed on top so that the apical side faced the RPE side (Fig. 105). After adhesion, the sheet was incubated at room temperature for 5 minutes to solidify, and then retrieved to produce an attached sheet (Figs. 106-107).
• the retinal tissue-RPE composite sheet thus prepared was turned over using tweezers, placed so that the sheet-like retinal tissue was on the bottom and the retinal pigment epithelium sheet was on the top, and observed ( Figure 108). The Transwell mesh that had been attached was also removed ( Figure 109).
• a retinal tissue-RPE composite sheet can be produced by using a sheet-like retinal tissue, an RPE sheet, and fibrin.
• ⁇ Reference Example 1-35 Preparation of a planarized sheet on a temperature-sensitive culture dish and examination of detachment and transplantation>
• Human ES cells KerE-1 line, (Non-Patent Document 3)
• Rx::Venus reporter gene were cultured under feeder-free conditions according to the method described in "Scientific Reports 4, 3594 (2014).
• StemFit medium product name: AK03N, manufactured by Ajinomoto Co., Inc.
• Laminin511-E8 product name, manufactured by Nippi Co., Ltd. was used as a scaffold instead of feeder cells.
• the aggregates (NR) prepared in Reference Example 1-1 on Day 18-30 were washed with PBS, and a nerve cell dispersion liquid (Wako) was added. After incubation at 37°C, the cells were dispersed into single cells by pipetting, and the cells were seeded at 8.0 x 10 5 cells/well on a 24-well Laminin511-E8-coated temperature-sensitive culture dish (Cell Seed) with seven stages of different detachment degrees. The medium was replaced once every 3-4 days, and after 30 days after seeding, the sheet was formed with a thickness, and then the temperature was lowered to room temperature, incubated for 2 hours, and observed. As a result, it was observed that the retinal sheet could be detached by bringing it to room temperature in multiple wells 2b, 2c, 3, and 4 of the temperature-sensitive culture dishes with seven stages of detachment degrees (Figure 111).
• retinal sheets can be harvested by culturing them in a temperature-sensitive culture dish.
• Rx::Venus was sorted as an indicator, and 10 ⁇ M Y-27632, 3 ⁇ M CHIR99021, 300 nM SAG, and 100 ng/mL FGF8 were added.
• the retinal sheet on the temperature-sensitive culture dish was then incubated at 4°C for 1 hour on the 62nd day after the start of the floating culture, and then harvested.
• a long graft for transplantation was then cut out using microscissors and transplanted under the retina of immunodeficient retinal insufficient rats (SD Foxn) (not shown).
• Human iPS cells (LPF11 and DSP-SQ strains, established by Sumitomo Pharma Co., Ltd.) were reprogrammed using commercially available Sendai virus vectors (Oct3/4, Sox2, KLF4, and c-Myc, four factors, IDPharma Cytotune Kit) according to the published protocol of Thermo Fisher Scientific (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009).
• CiRA_Ff-iPSC_protocol_JP_v140310 the published protocol of Kyoto University (Establishment and maintenance of human iPS cells in a feeder-free environment, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/protocol.html), using StemFit medium (AK03, Ajinomoto Co., Inc.) and Laminin511-E8 (Nippi Co., Ltd.).
• the human iPS cells (LPF11 and DSP-SQ strains) were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014).
• StemFit medium (AK03N, Ajinomoto Co., Inc.) was used as the feeder-free medium
• Laminin511-E8 (Nippon Pharmaceuticals, Inc.) was used as the feeder-free scaffold.
• human iPS cells LPF11 strain and DSP-SQ strain
• TrypLE Select TrypLE Select
• the human iPS cells dispersed into the single cells were seeded on a plastic culture dish coated with Laminin511-E8, and cultured feeder-free in StemFit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• a 6-well plate Iwaki, for cell culture, culture area 9.4 cm 2
• the seeded cell number of the human iPS cells dispersed into the single cells was 1.0 ⁇ 10 4.
• the medium was replaced with StemFit medium not containing Y-27632. Thereafter, the medium was replaced with StemFit medium not containing Y-27632 once every 1 to 2 days, and the cells were then cultured for 6 days after seeding.
• the patterning procedure involved first creating a droplet ( 1 cm in diameter) of a solution (Laminin 511-E8 concentration 3.925 ng/ ⁇ l) made by adding 0.785 ⁇ l of Laminin 511-E8 (Nippon) to 100 ⁇ l of PBS in the center of the well of a 12-well plate (Iwaki, for cell culture, culture area 3.8 cm 2 ), and then incubating the plate at 37°C or room temperature for 1 to 3 hours to prepare a 12-well plate for patterning culture (Laminin 511-E8: 0.5 ⁇ g/cm 2 ).
• the 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 a culture plate for patterning culture at various cell densities of 0.5 ⁇ 10 5 to 5.0 ⁇ 10 5 cells/cm 2 , and cultured in a feeder-free manner in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M) ( FIG. 112 ).
• ROCK inhibitor 10 ⁇ M
• Y-27632 was then applied for various periods ranging from 2 to 32 hours, after which the medium was replaced with 2 ml of StemFit medium (ROCK inhibitor, 0 ⁇ M) that did not contain Y-27632 ( Figure 114).
• DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine
• ⁇ Reference Example 2-2 Examination of seeding density of human iPS cells in patterning culture> Human iPS cells (LPF11 strain) were seeded onto a patterning culture plate at various cell densities (cells/cm 2 ) to examine the optimal conditions.
• the seeding cell concentration of undifferentiated human iPS cells is preferably 1.0 ⁇ 10 5 to 2.5 ⁇ 10 5 cells/cm 2 in the method for producing retinal tissue by the patterning culture method.
• ⁇ Reference Example 2-3 Examination of the duration of action of ROCK inhibitor when cells are seeded in patterning culture> The optimal Y-27632 exposure time when seeding human iPS cells (LPF11 and DSP-SQ strains) onto a patterning culture plate was investigated.
• a 12-well plate for patterning culture prepared by the method described in Reference Example 2-1 was prepared, and subconfluent human iPS cells (LPF11 strain and DSP-SQ strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of a culture plate for patterning culture at a density of 2.0 ⁇ 10 5 cells/cm 2 and cultured feeder-free in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). After that, 2, 4, 8, 16, or 32 hours, the medium was replaced with 2 ml of StemFit medium not containing Y-27632.
• Chx10 anti-Chx10 antibody, Exalpha
• a retinal progenitor cell marker a retinal progenitor cell marker
• observation was performed with a fluorescence microscope (Keyence), revealing that no retinal progenitor cells were present in the holes on the surface of the sheet.
• the percentage of Chx10-positive areas on the sheet surface was calculated using ImageJ software (NIH). The percentage was 91% for sheets exposed to Y-27632 for 32 hours, whereas it was 97% or more for exposure times of 16 hours or less (16 hours: 97%, 8 hours: 99%, 4 hours: 98%, 2 hours: 99%).
• Chx10-negative holes was counted for each exposure time of Y-27632, and it was found that the large number of holes formed on the sheet surface could be suppressed by limiting the exposure time of Y-27632 to 16 hours or less.
• human iPS cells DSP-SQ strain
• a 12-well plate for patterning culture prepared by the method described in Reference Example 2-1 was prepared, and human iPS cells (LPF11 strain) that had become subconfluent were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies). Thereafter, the human iPS cells dispersed into single cells were seeded in one well of a culture plate for patterning culture at a density of 2.0 ⁇ 10 5 cells/cm 2 , and cultured feeder-free in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M). After 2 hours, the medium was replaced with 2 ml of StemFit medium not containing Y-27632, and patterning culture was performed by the method described in Reference Example 2-1.
• human iPS cells LPF11 strain
• TrypLE Select TrypLE Select
• the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous human recombinant BMP4 (R&D) at a final concentration of 0, 1.5, 3, 6, or 12 nM. Thereafter, half of the medium was replaced once every 2-3 days with serum-free medium (gfCDM+KSR) not containing human recombinant BMP4. For half-medium replacement, half the volume of the medium in the culture vessel, i.e. 1 ml, was discarded and 1 ml of new serum-free medium (gfCDM+KSR) was added, making the total medium volume 2 ml.
• serum-free medium gfCDM+KSR
• retinal progenitor cell marker Rx (anti-Rx antibody, Takara). These immunostained cells were observed under a fluorescence microscope (Keyence), and the results are shown in Figure 116. Analysis of the images in Figure 116 revealed that the retinal progenitor cell marker Rx was more strongly expressed in retinal tissue in which retinal differentiation was induced at BMP4 concentrations of 3 to 12 nM compared to 1.5 nM BMP4.
• concentration of total RNA was measured using a measuring device (Nanodrop, Thermo Scientific), and then reverse transcribed into cDNA using reverse transcriptase and primers (Reverse Transcription Master Mix Kit, Fluidigm).
• a multiplex-PCR reaction was performed using the cDNA and all probes used for verification using a PCR device (Veriti 96well thermal cycler, Applied Biosystems).
• the pre-run reaction solution was then injected into a multi-well with flow channels (96.96 Dynamic Array IFC, Fluidigm) using an IFC Controller HX (Fluidigm), and the expression levels of marker genes for neural retina and by-products other than neural retina were measured by real-time PCR using a multi-analyte real-time PCR system (Biomark HD, Fluidigm).
• the final concentration of BMP4, which induces retinal differentiation is preferably 3 to 15 nM when producing retinal tissue using the patterning culture method.
• a 12-well plate for patterning culture prepared by the method described in Reference Example 2-1 was prepared, and subconfluent human iPS cells (LPF11 strain) were washed with PBS and then dispersed into single cells using TrypLE Select (Life Technologies, Inc.).
• the human iPS cells dispersed into single cells were seeded into one well of the culture plate for patterning culture at a density of 5.0 ⁇ 10 5 cells/cm 2 in the conventional method and 2.0 ⁇ 10 5 cells/cm 2 in the optimized method, and cultured feeder-free in Stemfit medium in the presence of Y-27632 (ROCK inhibitor, 10 ⁇ M).
• the medium was replaced with serum-free medium (gfCDM+KSR) containing exogenous human recombinant BMP4 (R&D) at a final concentration of 1.5 nM in the method of WO2023/003025 and 6 nM in the optimized method. Thereafter, half of the medium was replaced with serum-free medium (gfCDM+KSR) not containing human recombinant BMP4 once every 2-3 days. For the half-medium replacement operation, half the volume of the medium in the culture vessel, i.e. 1 ml, was discarded and 1 ml of new serum-free medium (gfCDM+KSR) was added, making the total medium volume 2 ml.
• serum-free medium gfCDM+KSR
• the expression levels of marker genes (Table 3) for neural retina and by-products other than neural retina in the retinal tissue were measured by the method described in Reference Example 2-4.
• the analysis results of the relative mRNA expression levels of each gene are shown in FIG. 118.
• the expression levels of neural progenitor cell and retinal progenitor cell marker genes were higher in the retinal tissue differentiated by the method in which various conditions were optimized (optimization method) than in the retinal tissue differentiated by the method of WO2023/003025. It was also found that the expression of various non-target cell marker genes was suppressed to a low level in the retinal tissue differentiated by the optimization method.
• Example 1 Production of reorganized organoids using retinal progenitor cell sheets
• the cell aggregates containing retinal progenitor cells prepared by the SFEBq method can be used as a starting material to reorganize and re-form into a sheet.
• a differentiation method by patterning culture can be used as a method for producing a cell aggregate (adhesive cell aggregate) containing retinal progenitor cells.
• Retinal tissue containing retinal progenitor cells which is a cell aggregate prepared by patterning culture (also referred to as a retinal sheet or retinal progenitor cell sheet in this specification), has a commonality in cell composition with cell aggregates containing retinal progenitor cells prepared by the SFEBq method, but the cell culture state is different. Therefore, it was examined whether the retinal progenitor cell sheet prepared by patterning can be used as a starting material to reorganize and re-form into a sheet.
• Human iPS cells (LPF11 line, established by Sumitomo Pharma Co., Ltd.) were regenerated using commercially available Sendai virus vectors (four factors: Oct3/4, Sox2, KLF4, and c-Myc, Cytotune Kit manufactured by IDPharma) according to the published protocol of Thermo Fisher Scientific (iPS2.0 Sendai Reprogramming Kit, Publication Number MAN0009378, Revision 1.0) and the published protocol from Kyoto University (Establishment and maintenance of human iPS cells in a feeder-free environment, CiRA_Ff-iPSC_protocol_JP_v140310, http://www.cira.kyoto-u.ac.jp/j/research/protocol.html), using StemFit medium (AK03, Ajinomoto Co.) and Laminin511-E8 (Nippi Co.).
• Sendai virus vectors four factors: Oct3/4, Sox2, KLF4, and c-Myc, Cytotune
• the human iPS cells were cultured in a feeder-free manner according to the method described in Scientific Reports, 4, 3594 (2014).
• StemFit medium (AK03N, Ajinomoto Co., Inc.) was used as the feeder-free medium
• Laminin511-E8 (Nippon Pharmaceuticals, Inc.) was used as the feeder-free scaffold.
• human iPS cells LPF11 strain
• TrypLE Select Life Technologies
• the human iPS cells dispersed into the single cells were then seeded on a plastic culture dish coated with Laminin511-E8 and cultured feeder-free in StemFit medium in the presence of Y27632 (ROCK inhibitor, 10 ⁇ M).
• a 6-well plate Iwaki, for cell culture, culture area 9.4 cm 2
• the seeded cell number of the human iPS cells dispersed into the single cells was 1.0 ⁇ 10 4.
• the medium was replaced with StemFit medium not containing Y27632. Thereafter, the medium was replaced with StemFit medium (ROCK inhibitor, 0 ⁇ M) not containing Y27632 once every 1 to 2 days, and the cells were cultured until the 6th day after seeding.
• StemFit medium (ROCK inhibitor, 0 ⁇ M) not containing Y27632
• the patterning procedure involved first creating a droplet ( 1 cm in diameter) of a solution (Laminin 511-E8 concentration 3.925 ng/ ⁇ l) made by adding 0.785 ⁇ l of Laminin 511-E8 (Nippi) to 100 ⁇ l of PBS in the center of the well of a 12-well plate (Iwaki, for cell culture, culture area 3.8 cm 2 ), and then incubating the plate at 37°C or room temperature for 1 to 3 hours to prepare a 12-well plate for patterning culture (Laminin 511-E8 0.5 ⁇ g/cm 2 ).
• the subconfluent human iPS cells (LPF11 strain) were washed with PBS and dispersed into single cells using TrypLE Select (Life Technologies).
• the human iPS cells dispersed into single cells were then seeded into one well of a culture plate for patterning culture at a cell density of 1.0 ⁇ 10 5 cells/cm 2 and cultured feeder-free in Stemfit medium in the presence of Y27632 (ROCK inhibitor, 10 ⁇ M). After that, Y27632 was allowed to act for 2 hours, and the medium was replaced with 2 ml of StemFit medium (ROCK inhibitor, 0 ⁇ M) that did not contain Y27632.
• the medium was replaced with 2 ml of serum-free medium (gfCDM + KSR). Five days later, the medium was replaced with gfCDM + KSR containing 6 nM human recombinant BMP4 (R&D).
• half of the medium was replaced with gfCDM+KSR not containing human recombinant BMP4 once every 2-3 days.
• half the volume of the medium in the culture vessel i.e. 1 ml, was discarded and 1 ml of new gfCDM+KSR was added, making the total medium volume 2 ml.
• retinal progenitor cell sheets were produced by culturing the cells under 5% CO2 conditions using a control maturation medium (DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine).
• DMEM/F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 ⁇ M taurine.
• the retinal progenitor cell sheet on the 20th day of patterning culture was dispersed into single cells using a nerve cell dispersion liquid (manufactured by WAKO Co., Ltd.), and the cells were suspended in a control maturation medium so that 1.2 x 104 cells were placed per well in a non-cell-adhesive 96-well plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) and cultured three-dimensionally.
• Y27632 final concentration 10 ⁇ M
• SAG final concentration 300 nM
• CHIR99021 final concentration 3 ⁇ M
• the reorganized organoids on the 23rd day of suspension culture were fixed with 4% paraformaldehyde and immunostained with retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), and Rx (anti-Rx antibody, Takara), photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara), and apical cell polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen). These immunostained cells were observed using a confocal laser microscope (LSM880, ZEISS) ( Figure 121).
• Example 2 Production of a reorganized sheet using a retinal progenitor cell sheet
• a reorganized sheet could be produced by dispersing the retinal progenitor cell sheet obtained by patterning culture and inducing reorganization under two-dimensional culture ( FIG. 122 ).
• the retinal progenitor cell sheet on the 20th day of patterning culture prepared by the method described in Example 1 was dispersed into single cells using a nerve cell dispersion liquid (manufactured by WAKO), and the cells were seeded in a control maturation medium at 0.5 x 105 cells per well on a 24-well Transwell (Corning) previously coated with Laminin511-E8 (manufactured by Nippi) and cultured two-dimensionally.
• Y27632 final concentration 10 ⁇ M
• SAG final concentration 300 nM
• CHIR99021 final concentration 3 ⁇ M
• the reorganized sheets on the 23rd day after seeding in the Transwells were fixed with 4% paraformaldehyde and immunostained for the retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), and Rx (anti-Rx antibody, Takara), the photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara), and the apical cell polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen).
• Chx10 anti-Chx10 antibody, Exalpha
• Pax6 anti-Pax6 antibody, BD Biosciences
• Rx anti-Rx antibody, Takara
• the photoreceptor progenitor cell marker Crx anti-Crx antibody, Takara
• the apical cell polarity marker ZO-1 anti-ZO-1 antibody, Invitrogen
• the reorganized sheet like the reorganized organoid, was formed of Chx10-, Pax6-, and Rx-positive retinal progenitor cells, and Crx-positive photoreceptor progenitor cells were scattered throughout. Furthermore, since the upper side of the sheet (the side not in contact with the well membrane) was ZO-1 positive, it was found that this reorganized sheet had polarity and was a retinal tissue with the apical surface formed on the upper side.
• Example 3 Production of reorganized organoids using retinal progenitor cells sorted from retinal progenitor cell sheets
• the retinal progenitor cell sheet on the 20th day of patterning culture prepared by the method described in Example 1, was dispersed into single cells using a neural cell dispersion liquid (WAKO), and the surface antigen was stained with APC-Conjugate CD9 antibody (BioLegend) at 4°C for 1 hour, and sorting was performed using a MACS Quant Tyto Cell Sorter (Miltenyi).
• the cells before and after sorting were analyzed by flow cytometry using a MACS Quant Analyzer 10 Flow Cytometer (Miltenyi), and it was confirmed that the CD9-positive cell fraction had been purified ( Figure 126).
• the purified cells were suspended in control maturation medium and cultured three-dimensionally in a non-cell-adhesive 96-well plate (product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.) at 1.2 x 104 cells per well.
• a non-cell-adhesive 96-well plate product name: PrimeSurface 96-well V-bottom plate, manufactured by Sumitomo Bakelite Co., Ltd.
• Y27632 final concentration 10 ⁇ M
• SAG final concentration 300 nM
• CHIR99021 final concentration 3 ⁇ M
• the reorganized organoids were fixed with 4% paraformaldehyde and immunostained for retinal progenitor cell markers Chx10 (anti-Chx10 antibody, Exalpha), Pax6 (anti-Pax6 antibody, BD Biosciences), Rx (anti-Rx antibody, Takara), photoreceptor progenitor cell marker Crx (anti-Crx antibody, Takara), and apical cell polarity marker ZO-1 (anti-ZO-1 antibody, Invitrogen).
• Chx10 anti-Chx10 antibody, Exalpha
• Pax6 anti-Pax6 antibody, BD Biosciences
• Rx anti-Rx antibody, Takara
• photoreceptor progenitor cell marker Crx anti-Crx antibody, Takara
• apical cell polarity marker ZO-1 anti-ZO-1 antibody, Invitrogen
• the reorganized organoids produced from CD9-positive cells sorted from the retinal progenitor cell sheet also had a Chx10, Pax6, and Rx-positive retinal progenitor cell layer with a thickness of about 5 to 10 cells, in which Crx-positive photoreceptor precursor cells were scattered. Furthermore, since the outer surface was ZO-1 positive, it was found that the apical surface was formed on the outside.
• the expression levels of the neural retina marker Rx gene and the dorsal telencephalon marker Emx2 were measured by real-time PCR.
• the total RNA concentration was measured using a measuring device (Nanodrop, Thermo Scientific), and then reverse transcribed to cDNA using reverse transcriptase and primers (Reverse Transcription Master Mix Kit, Fluidigm).
• a multiplex-PCR reaction pre-run was performed using a PCR device (Veriti 96well thermal cycler, Applied Biosystems).
• the pre-run reaction solution was injected into a multi-well with a channel (96.96 Dynamic Array IFC, Fluidigm) using an IFC controller HX (Fluidigm), and the expression levels of the Rx gene and Emx2 gene were measured by real-time PCR using a multi-analyte real-time PCR system (Biomark HD, Fluidigm).
• Example 4 Production of a reorganized sheet using retinal progenitor cells sorted from a retinal progenitor cell sheet
• the retinal progenitor cell sheet on the 20th day of patterning culture prepared by the method described in Example 1 was dispersed into single cells using a neural cell dispersion liquid (WAKO), and the surface antigen was stained with APC-Conjugate CD9 antibody (BioLegend) at 4°C for 1 hour, and sorted using a MACS Quant Tyto Cell Sorter (Miltenyi).
• the collected cells were seeded at 0.5 x 105 cells per well into 24-well Transwells (Corning) that had been previously coated with Laminin 511-E8 (Nippi). When cells were seeded, Y27632 (final concentration 10 ⁇ M), SAG (final concentration 300 nM), and CHIR99021 (final concentration 3 ⁇ M) were added simultaneously to promote reorganization.
• the reorganized sheet made from CD9-positive cells sorted from the retinal progenitor cell sheet was formed from Chx10-, Pax6-, and Rx-positive retinal progenitor cells, with Crx-positive photoreceptor precursor cells scattered throughout. Furthermore, since the upper side of the sheet (the side not in contact with the well membrane) was ZO-1 positive, it was found that this reorganized organoid had polarity and was retinal tissue with the apical surface formed on the outside.
• Example 5 Examination of the optimal concentration of CHIR required for reorganization We investigated the optimal concentrations of a ROCK inhibitor, a substance acting on the Wnt signaling pathway, and a substance acting on the Shh signaling pathway, which are important when dispersing and reorganizing retinal progenitor cell sheets prepared by the patterning culture method.
• the retinal progenitor cell sheet on the 20th day of patterning culture prepared by the method described in Example 1 was dispersed into single cells using a nerve cell dispersion liquid (manufactured by WAKO Co., Ltd.), and 1.2 x 104 cells per well were suspended and cultured in a control maturation medium on a non-cell-adhesive 96-well plate (product name: PrimeSurface 96-well V-bottom plate , manufactured by Sumitomo Bakelite Co., Ltd.).
• Y27632 final concentration 0 or 10 ⁇ M
• SAG final concentration 0, 150, 300, or 600 nM
• CHIR99021 final concentration 0, 1.5, 3, or 6 ⁇ M
• retinal progenitor cell sheets obtained by patterning culture adhere to the dish and have different properties from floating cell aggregates, they have been demonstrated to be able to be reorganized and remade into sheets as a starting material, and the optimal concentration of the reorganization factors added during this process was clarified.
• Retinal progenitor cell sheets produced by patterning culture can be mass-produced in a variety of shapes and sizes, making them a useful starting material for reorganization and remaking into sheets, and the feasibility of this has now been demonstrated.

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