Method for accelerated production of layered retinal tissue containing photoreceptor cells

Abstract

The present invention provides a method for producing a layered retinal tissue including photoreceptor cells from pluripotent stem cells or an aggregate including retinal progenitor cells within a short period.

Description

Technical Field

[0001] The present invention relates to a method for promoting the production of a layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells.

Background Art

[0002] One reported method for producing neural tissue, such as retinal tissue, from pluripotent stem cells involves forming homogeneous aggregates of pluripotent stem cells in serum-free medium, culturing them in suspension, and then culturing them again in a differentiation-inducing medium in the presence of appropriate differentiation-inducing factors to induce differentiation from pluripotent stem cells into the desired neural cells. For example, a method to obtain multilayer retinal tissue by forming homogeneous aggregates of pluripotent stem cells in serum-free medium containing a Wnt signaling pathway inhibitor, then suspension culturing these aggregates in the presence of a basement membrane preparation, and then suspension culturing them in serum medium (Patent Document 1); a method to obtain aggregates containing retinal cells or retinal tissue by adhering pluripotent stem cells in a medium containing a TGFβ family signaling pathway inhibitor and / or a Sonic Hedgehog signaling pathway activator, and an undifferentiated maintenance factor, then suspension culturing the obtained cells in a medium containing a Wnt signaling pathway inhibitor, and then suspension culturing the obtained aggregates in a medium containing a BMP signaling pathway activator (Patent Document 2); and a method to obtain aggregates containing retinal tissue by culturing cell aggregates containing retinal tissue in a medium containing a Wnt signaling pathway activator until cells expressing the RPE65 gene appear, and then culturing the resulting cell aggregates that do not express the RPE65 gene in a Wnt Known methods for obtaining cell aggregates containing ciliary body periphery-like structures include: culturing cell aggregates containing retinal tissue in a medium that does not contain signaling pathway activators (Patent Document 3); culturing cell aggregates containing retinal tissue in a medium containing Wnt signaling pathway activators and FGF signaling pathway inhibitors until cells expressing the RPE65 gene appear, and then culturing the resulting cell aggregates that do not express the RPE65 gene in a medium that does not contain Wnt signaling pathway activators to obtain cell aggregates containing ciliary body periphery-like structures (Patent Document 4); and culturing pluripotent stem cells in a medium containing undifferentiated maintenance factors, then suspension culturing the obtained pluripotent stem cells in a medium containing Sonic Hedgehog signaling pathway activators to form cell aggregates, and finally suspension culturing the obtained aggregates in a medium containing BMP signaling pathway activators to obtain aggregates containing retinal cells or retinal tissue (Patent Document 5).Furthermore, although nerve tissue has not yet been obtained, methods for obtaining retinal progenitor cells are known, such as culturing pluripotent stem cells in suspension in serum-free medium to form aggregates of pluripotent stem cells, and then culturing the formed aggregates in suspension in a medium that does not contain Sonic Hedgehog signaling pathway activators but contains BMP signaling pathway activators (Patent Document 6); and culturing pluripotent stem cells in suspension to induce differentiation into retinal precursor cells, culturing the obtained retinal precursor cells in suspension to induce differentiation into retinal ganglion cells, and then culturing the obtained retinal ganglion cells in adherent culture to extend axons to obtain retinal ganglion cells with extended axons (Patent Document 7).

[0003] Furthermore, well-known techniques for producing nerve tissue from pluripotent stem cells include, for example, a differentiation induction method (SEAM method) for obtaining various ocular tissue cells derived from the ectoderm in a concentric pattern (Non-Patent Literature 1), and a differentiation induction method for retinal organoids that starts differentiation induction from two-dimensional culture (Non-Patent Literature 2). Other known facts include: the efficient induction of neural progenitor cells from human ES cells and iPS cells through dual inhibition of BMP signaling and TGFβ family signaling (Non-Patent Literature 3); the induction of differentiation into neuroretinal cells by suspension culture of human pluripotent stem cells in a medium containing BMP4 (Non-Patent Literature 4); the induction of retinal organoids by adherent culture of aggregates of human pluripotent stem cells in a medium containing BMP4 (Non-Patent Literature 5); the promotion of differentiation into rod photoreceptor cells in mammalian retinal cells through the sonic hedgehog signaling pathway (Non-Patent Literature 6); the promotion of differentiation into photoreceptor cells through the promotion of differentiation into nerve cells by Activin (Non-Patent Literature 8); and the promotion of differentiation from optic vesicle-like tissue derived from human pluripotent stem cells into retinal pigment epithelial cells by Activin (Non-Patent Literature 9).

[0004] While these manufacturing methods have made it possible to produce nerve cells or nerve tissue such as retina, the period until the tissue matures is often long, and it remains difficult to obtain layered retinal organoids containing photoreceptor cells from human pluripotent stem cells in a short period of time. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2013 / 077425 [Patent Document 2] International Publication No. 2017 / 183732 [Patent Document 3] International Publication No. 2013 / 183774 [Patent Document 4] International Publication No. 2015 / 087614 [Patent Document 5] International Publication No. 2016 / 063986 [Patent Document 6] International Publication No. 2015 / 025967 [Patent Document 7] International Publication No. 2016 / 021709 [Non-patent literature]

[0006] [Non-Patent Document 1] Nature Protocols, 12, 683-696 (2017) [Non-Patent Document 2] PNAS, 111(23), 8518-8523 (2014) [Non-Patent Document 3] Nat. Biotechnol., 27(3), 275-280 (2009) [Non-Patent Document 4] Nature Communications 6, 6286 (2015) [Non-Patent Document 5] Development. 2019 Jan 1; 146(1): dev171686. [Non-Patent Document 6] J. Neurosci., 17(16), 6277-6288 (1997) [Non-Patent Document 7] Development, 120(8), 2091-2102 (1994) [Non-Patent Document 8] Stem Cell Report, 5(4), 532-545 (2015) [Non-Patent Document 9] Stem Cells., 29(8), 1206-1218 (2011) [Overview of the project] [Problems that the invention aims to solve]

[0007] The problem that this invention aims to solve is to provide a method for producing layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells in a short period of time. [Means for solving the problem]

[0008] The present inventors have conducted extensive studies to solve the above problems and have found that, as an adhesion culture step, pluripotent stem cells are cultured in an adhesion culture medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors, and the obtained cells are cultured in an adhesion culture medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors, and the obtained cells are cultured in an adhesion culture medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators In this process, we obtained aggregates containing retinal progenitor cells by performing adherent culture, and in a subsequent suspension culture step, we cultured the aggregates containing retinal progenitor cells in a culture medium containing an Activin signaling pathway activator, a Sonic Hedgehog signaling pathway activator, and retinoic acid. We then cultured the obtained aggregates in suspension in a culture medium containing a Sonic Hedgehog signaling pathway activator in the absence of the Activin signaling pathway activator and retinoic acid. We discovered that layered retinal tissue containing photoreceptor cells can be produced from pluripotent stem cells in a short period of time, thus completing the present invention. In other words, the present invention relates to the following:

[0009] [1] A method for producing layered retinal tissue containing photoreceptor cells, comprising the following steps (1) to (2); (1) A step of obtaining aggregates containing retinal progenitor cells by suspension culture in a medium containing an Activin signaling pathway agent, a Sonic Hedgehog signaling pathway agent, and retinoic acid, (2) A step of obtaining layered retinal tissue containing photoreceptor cells by culturing aggregates containing retinal ganglion cells and photoreceptor progenitor cells in suspension in a culture medium containing a sonic hedgehog signaling pathway agent in the absence of an Activin signaling pathway agent and retinoic acid. [2] The method according to [1], wherein the layered retinal tissue containing photoreceptor cells includes the outer granular layer, the inner granular layer, and the ganglion cell layer. [3] The method according to [2], wherein photoreceptor cells are included in the outer granular layer. [4] The method according to [3], wherein the photoreceptor cells included in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells. [5] The method according to [4], wherein 50% or more of all the cells included in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells. [6] The method according to any one of [1] to [5], wherein the aggregate containing retinal progenitor cells is an aggregate in which a cell layer co-expressing the retinal progenitor cell markers Rx and Chx10 is formed. [7] The method according to any one of [1] to [6], wherein the Activin signaling pathway agonist is one or more substances selected from the group consisting of Activin A, Activin B, Activin C, and Activin AB. [8] The method according to [7], wherein the Activin signaling pathway agonist is Activin A. [9] The method according to any one of [1] to [8], wherein the Sonic Hedgehog signaling pathway agonist is one or more substances selected from the group consisting of Sonic Hedgehog (Shh), Smoothened Agonist (SAG), and Purmorphamine (PMA).

[10] The method according to [9], wherein the Sonic Hedgehog signaling pathway agonist is SAG.

[11] The method according to any one of [1] to

[10] , wherein in step (1), the aggregate containing retinal progenitor cells is cultured in suspension for 0.5 days to 50 days.

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

[11] , wherein the aggregate containing retinal ganglion cells and photoreceptor cell progenitors is an aggregate in which a cell layer expressing the retinal ganglion cell marker Brn3 in the inner layer and the photoreceptor cell progenitor marker Crx in the outermost layer is formed.

[13] The method according to any one of [1] to

[12] , wherein in step (2), the aggregate containing retinal ganglion cells and photoreceptor cell progenitors is cultured in suspension for 20 days to 320 days.

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

[13] , further including the following step before step (1); (a) In the absence of undifferentiated state-maintaining factors, pluripotent stem cells are adherently cultured in a medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor to obtain neural progenitor cells; (b) Neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway effector in the absence of undifferentiated state-maintaining factors, a TGFβ signaling pathway inhibitor, and a BMP signaling pathway inhibitor to obtain retinal progenitor cells; (c) Retinal progenitor cells are adherently cultured in the absence of undifferentiated state-maintaining factors, a TGFβ signaling pathway inhibitor, a BMP signaling pathway inhibitor, and a BMP signaling pathway effector to obtain aggregates containing retinal progenitor cells.

[15] The method according to

[14] , wherein the pluripotent stem cells are human pluripotent stem cells.

[16] The method according to

[14] or

[15] , wherein the pluripotent stem cells are induced pluripotent stem cells.

[17] The method according to any one of

[14] to

[16] , wherein the TGFβ signaling pathway inhibitor is at least one substance selected from the group consisting of Lefty, SB431542, LY-364947, SB-505124, and A-83-01.

[18] The method according to

[17] , wherein the TGFβ signaling pathway inhibitor is SB431542.

[19] The method according to any one of

[14] to

[18] , wherein the BMP signaling pathway inhibitor is at least one substance selected from the group consisting of LDN193189 and Dorsomorphin.

[20] The method according to

[19] , wherein the BMP signaling pathway inhibitor is LDN193189.

[21] The method according to any one of

[14] to

[20] , wherein in step (a), the pluripotent stem cells are adherently cultured for 1 hour to 50 hours.

[22] The method according to any one of

[14] to

[21] , wherein the BMP signaling pathway effector is at least one substance selected from the group consisting of BMP2, BMP4, BMP7, and GDF7.

[23] The method according to

[22] , wherein the BMP signaling pathway effector is BMP4.

[24] The method according to any one of

[14] to

[23] , wherein neural progenitor cells are adherently cultured for 0.5 to 4 days in step (b).

[25] The method according to any one of

[14] to

[24] , wherein retinal progenitor cells are adherently cultured for 2 to 12 days in step (c).

[26] Isolated, layered retinal tissue containing photoreceptor cells, which is produced by differentiating pluripotent stem cells.

[27] The tissue according to

[26] , wherein the isolated layered retinal tissue containing photoreceptor cells comprises the outer granular layer, the inner granular layer, and the ganglion cell layer.

[28] The tissue described in

[27] , in which photoreceptor cells are contained in the outer granular layer.

[29] The tissue described in

[28] , wherein the photoreceptor cells contained in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells.

[30] The tissue described in

[29] , wherein more than 50% of all cells in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells. A therapeutic agent for diseases based on disorders of layered retinal tissue containing photoreceptor cells, comprising any one of the following:

[31] ,

[26] , to

[30] .

[32] A method for screening therapeutic agents for disorders of layered retinal tissue containing photoreceptor cells, comprising the following steps (11) to (13): (11) A step of culturing layered retinal tissue containing damaged photoreceptor cells for a certain period of time under viable culture conditions in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (12) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (13) A step of selecting the test substance in step (11) as a therapeutic agent for damage to layered retinal tissue containing photoreceptor cells, based on the difference in the results measured in step (11) and step (12).

[33] A method for evaluating drug efficacy, including the following steps (21) to (23): (21) A step of culturing layered retinal tissue containing damaged photoreceptor cells for a certain period of time under viable culture conditions in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (22) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (23) A step to evaluate the efficacy of the test substance in step (21) based on the difference between the results measured in step (21) and step (22).

[34] Toxicity assessment method including the following steps (31) to (33): (31) A step of culturing layered retinal tissue containing photoreceptor cells in viable culture conditions for a certain period of time in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (32) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (33) A step to evaluate the toxicity of the test substance in step (31) based on the difference between the results measured in steps (31) and (32). [Effects of the Invention]

[0010] According to the present invention, it is possible to produce layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells in a short period of time. [Brief explanation of the drawing]

[0011] [Figure 1] Figure 1 shows the culture schedule for cell line 201B7 in Example 1. [Figure 2] Figure 2 shows the expression of the retinal progenitor cell marker Rx in aggregates of cell line 201B7 5 days after the start of differentiation. Bar = 100 μm [Figure 3]Figure 3 shows the expression of retinal progenitor cell markers Rx and CHX10 in aggregates of cell line 201B7 10 days after differentiation initiation. Bar = 200 μm [Figure 4] Figure 4 shows the culture schedule for cell line 201B7 in Example 2. [Figure 5] Figure 5 shows the expression of the photoreceptor progenitor cell marker Crx and the retinal ganglion cell marker Brn3 in aggregates of cell line 201B7 30 days after the start of differentiation. Bar = 200 μm [Figure 6] Figure 6 shows the expression of the photoreceptor progenitor cell marker Crx and the retinal ganglion cell marker Brn3 in aggregates of cell line M8 20 days after the start of differentiation. Neuroblastic layer: neuroblast layer Bar = 50 μm [Figure 7] Figure 7 shows the expression of the photoreceptor progenitor cell marker Crx and the retinal ganglion cell marker Brn3 in aggregates of cell line M8 40 days after the start of differentiation. Outer neuroblastic layer: outer neuroblastic layer Inner neuroblastic layer: inner neuroblastic layer Ganglion cell tlayer: ganglion cell layer Bar = 50 μm [Figure 8] Figure 8 shows the expression of the cone photoreceptor marker RXRγ and the rod photoreceptor marker NRL in aggregates of cell line M8 60 days after the start of differentiation. (Outer nuclear layer (ONL): outer granular layer) (Inner nuclear layer (INL): inner granular layer) Bar = 50 μm [Figure 9] Figure 9 shows the expression of cone photoreceptor markers S-opsin, L / M-opsin, and rod photoreceptor marker rhodopsin in aggregates of cell line M8 80 days after differentiation initiation. (Outer nuclear layer (ONL): outer granular layer) (Inner nuclear layer (INL): inner granular layer) Bar = 50 μm [Figure 10] Figure 10 shows the culture schedule for cell line 1231A3 in Example 4. [Figure 11]Figure 11 shows microscopic images of aggregates of cell line 1231A3 in Example 4 15, 20, 40, 60, 80, and 90 days after the start of differentiation induction. [Figure 12] Figure 12 shows the expression of rhodopsin and L / M opsin in human retinal organoids 90 days after differentiation, prepared using the differentiation induction method of Example 4. Bar = 100 μm [Modes for carrying out the invention]

[0012] 1. Method for manufacturing layered retinal tissue containing photoreceptor cells The present invention provides a method for producing layered retinal tissue containing photoreceptor cells (hereinafter referred to as "production method 1 of the present invention").

[0013] In this specification, "tissue" refers to a cell population structure having a layered structure in which cell layers composed of one type of cell with uniform morphology and properties, or multiple types of cells with different morphologies and properties, are arranged in a certain pattern. Accordingly, the layered retinal tissue produced in manufacturing method 1 of the present invention (hereinafter, the retinal tissue of the present invention) means tissue in which each retinal layer or layers corresponding to each retinal layer in a living retina are arranged in layers, and each retinal layer or layer corresponding to each retinal layer is composed of one type of retinal cell system with uniform morphology and properties, or multiple types of retinal cell system with different morphologies and properties.

[0014] In this specification, the term "retinal layer" refers to each layer that constitutes the retina, and specifically includes the retinal pigment epithelium layer, photoreceptor layer, outer limiting membrane, outer granular layer, outer plexiform layer, inner granular layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. The retinal layer of the present invention may also be a layer corresponding to any of the above-mentioned layers that constitute the retina. Here, "corresponding" means having the same or substantially the same characteristics as each layer that constitutes a normally differentiated retina in vivo. Characteristics of each retinal layer that constitutes the retina include the types of retinal cells contained in each retinal layer and the developmental order of each layer in the retina. Furthermore, "substantially identical" means that as long as the types of retinal cells contained in each retinal layer are the same, the number of cells does not matter. The retinal layer that constitutes the retinal tissue of the present invention (hereinafter, the retinal layer of the present invention) is not particularly limited as long as it is one of the above-mentioned retinal layers, and may be composed of one or more of the above-mentioned retinal layers. The retinal tissue of the present invention is preferably a retinal tissue that exhibits the characteristics of the outer granular layer, inner granular layer, and ganglion cell layer. Alternatively, the retinal tissue of the present invention is preferably retinal tissue that includes a layer corresponding to the outer granular layer, a layer corresponding to the inner granular layer, and a layer corresponding to the ganglion cell layer.

[0015] In this specification, retinal cells mean cells that constitute each retinal layer in a living retina, or their progenitor cells, and include, but are not limited to, retinal progenitor cells, photoreceptor progenitor cells, photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells), horizontal cells, amacrine cells, interneurons, retinal ganglion cells (ganglion cells), bipolar cells, retinal pigment epithelial cells (RPE), periceral ciliary cells, and their progenitor cells. In this specification, retinal progenitor cells mean progenitor cells that can differentiate into any of the mature retinal layer-specific neurons, such as photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells.

[0016] Photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, retinal ganglion cell progenitor cells, and retinal pigment epithelial progenitor cells are progenitor cells whose differentiation into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells, respectively, has been determined.

[0017] In this specification, retinal layer-specific neurons refer to cells that constitute the retina and are specific to the retina. Examples of retinal layer-specific neurons include bipolar cells, retinal ganglion cells, amacrine cells, horizontal cells, photoreceptor cells, retinal pigment epithelial cells, rod photoreceptor cells, and cone photoreceptor cells.

[0018] The retinal cells constituting the retinal layer of the present invention are not particularly limited as long as they are the above-mentioned retinal cells, and may be composed of one or more of the above-mentioned retinal cells. Examples of retinal cells constituting the retinal layer of the present invention include retinal pigment epithelial cells (RPE) constituting the retinal pigment epithelium layer, photoreceptor cells (rod photoreceptor cells, cone photoreceptor cells) constituting the outer granular layer, horizontal cells, amacrine cells, bipolar cells constituting the inner granular layer, and retinal ganglion cells (ganglion cells) constituting the ganglion cell layer. Which retinal layer each cell constitutes can be confirmed by known methods, such as the presence or absence or degree of expression of retinal cell markers.

[0019] Retinal cell markers include Rx (also called Rax), which is expressed in neural progenitor cells; Rx, PAX6, and Chx10, which are expressed in retinal progenitor cells; Nkx2.1, which is expressed in hypothalamic neuron progenitor cells but not in retinal progenitor cells; Sox1, which is expressed in hypothalamic neuroepithelium but not in the retina; and Crx and Blimp1, which are expressed in photoreceptor progenitor cells. Markers of retinal layer-specific neurons include Chx10, PKCα, and L7 expressed in bipolar cells; TuJ1 and Brn3 expressed in retinal ganglion cells; Calretinin expressed in amacrine cells; Calbindin expressed in horizontal cells; Rhodopsin and Recoverin expressed in mature photoreceptor cells; Nrl and Rhodopsin expressed in rod photoreceptor cells; Rxr-gamma, S-Opsin, and M / L-Opsin expressed in cone photoreceptor cells; RPE65 and Mitf expressed in retinal pigment epithelial cells; and Rdh10 and SSEA1 expressed in peripheral ciliary cells.

[0020] The retinal tissue of the present invention is characterized by containing photoreceptor cells. The photoreceptor cells contained in the retinal tissue of the present invention are not particularly limited as long as they are contained in one or more retinal layers that constitute the retinal tissue, but as described above, it is preferable that they be contained in the outer granular layer. Furthermore, if the photoreceptor cells contained in the retinal tissue of the present invention are contained in the outer granular layer, the photoreceptor cells contained in the outer granular layer may be rod photoreceptor cells and / or cone photoreceptor cells. The ratio of rod photoreceptor cells to cone photoreceptor cells contained in the outer granular layer is not particularly limited as long as it is in the range of 100%:0% to 0%:100%, and is usually 10%:90% to 90%:10%, preferably 30%:70% to 70%:30%, and more preferably 45%:55% to 55%:45%. The ratio of rod photoreceptor cells to cone photoreceptor cells can be confirmed by known methods, for example, by the presence or absence or degree of expression of rod photoreceptor cell markers and cone photoreceptor cell markers.

[0021] The proportion of rod and / or cone photoreceptor cells in the outer granular layer is not particularly limited, but the total number of rod and cone photoreceptor cells among all cells in the outer granular layer is usually 50% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more. The higher the total number of rod and cone photoreceptor cells among all cells in the outer granular layer, the more mature the retinal tissue of the present invention can be considered to be.

[0022] The manufacturing method 1 of the present invention includes the following steps. (1) A step of obtaining aggregates containing retinal progenitor cells by suspension culture of aggregates containing retinal ganglion cells and photoreceptor progenitor cells in a culture medium containing an Activin signaling pathway activator, a Sonic Hedgehog signaling pathway activator, and retinoic acid. (2) A step of obtaining layered retinal tissue containing photoreceptor cells by culturing aggregates containing retinal ganglion cells and photoreceptor progenitor cells in suspension in a culture medium containing a sonic hedgehog signaling pathway agent in the absence of an Activin signaling pathway agent and retinoic acid.

[0023] In step (1), aggregates containing retinal progenitor cells are cultured in suspension in a culture medium containing an Activin signaling pathway activator, a Sonic Hedgehog signaling pathway activator, and retinoic acid.

[0024] In this specification, the aggregates containing retinal progenitor cells are not particularly limited as long as they are aggregates in which a cell layer expressing at least one selected from the group consisting of retinal progenitor cell markers Rx, PAX6, and Chx10 is formed. As described later, these aggregates may be retinal progenitor cells obtained by adherent culture of pluripotent stem cells, or retinal progenitor cells obtained by other production methods. For example, aggregates containing retinal progenitor cells prepared by the method disclosed in Elizabeth E. Capowski1 et al., Development. 2019 Jan 1; 146(1): dev171686, International Publication No. 2015 / 025967 may be used in step (1) of production method 1 of the present invention.

[0025] It is preferable that the aggregates containing retinal progenitor cells used in step (1) are uniform. Uniform aggregates mean that when multiple aggregates are cultured, the size of each aggregate is constant, and when the size of the aggregates is evaluated by the length of the maximum diameter, uniform aggregates mean that the variance of the length of the maximum diameter is small. More specifically, it means that 75% or more of the aggregates in the entire population of aggregates are within the range of the mean value ± 100%, preferably within the range of the mean value ± 50%, and more preferably within the range of the mean value ± 20%.

[0026] In step (1), aggregates containing retinal progenitor cells are cultured in suspension. In this specification, suspension culture or suspension culture method means culturing cells or cell aggregates while maintaining their state of suspension in a culture medium, and the method of performing such culture. That is, suspension culture is performed under conditions in which cells or cell aggregates do not adhere to culture equipment, etc., and culture performed under conditions in which cells adhere to culture equipment, etc. (adherent culture, or adherent culture method) is not included in the category of suspension culture. In this case, cell adhesion means that a strong cell-substrate junction is formed between the cells or cell aggregates and the culture equipment. More specifically, suspension culture means culture under conditions in which a strong cell-substrate junction is not formed between cells or cell aggregates and the culture equipment, etc.

[0027] In cell aggregates during suspension culture, cells adhere to each other across surfaces. In cell aggregates during suspension culture, cell-matrix junctions are hardly formed between cells and culture equipment, or if they are formed, their contribution is small. In some embodiments, in cell aggregates during suspension culture, intrinsic cell-matrix junctions exist within the aggregate, but cell-matrix junctions are hardly formed between cells and culture equipment, or if they are formed, their contribution is small.

[0028] Plane attachment between cells refers to the adhesion of cells across a surface. More specifically, plane attachment between cells means that the proportion of a cell's surface area that is in contact with the surface of another cell is, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. The cell surface can be observed by staining with a membrane staining reagent (e.g., DiI) or by immunostaining for cell adhesion factors (e.g., E-cadherin or N-cadherin).

[0029] The incubator used for suspension culture is not particularly limited as long as it is capable of suspension culture, and can be appropriately determined by those skilled in the art. Examples of such incubators include flasks, tissue culture flasks, culture dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, spinner flasks, Erlenmeyer flasks, or roller bottles. These incubators are preferably cell-non-adherent in order to enable suspension culture. Cell-non-adherent incubators can be those whose surfaces have not been artificially treated to improve cell adhesion (for example, coating with basement membrane preparations, extracellular matrix such as laminin, entactin, collagen, or gelatin, or polymers such as polylysine or polyornithine, or surface treatment such as positive charge treatment). As a cell-non-adherent incubator, the surface of the incubator can be artificially treated to reduce cell adhesion (e.g., superhydrophilic treatment with MPC polymer, low protein adsorption treatment, etc.). Rotation culture may also be performed using spinner flasks or roller bottles. The culture surface of the incubator may be flat-bottomed or have an uneven surface.

[0030] The culture medium used in suspension culture can be prepared using a basal medium that is commonly used for culturing animal cells. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof, which are media that can be used for culturing animal cells. The culture medium used in suspension culture may be serum-containing medium or serum-free medium, but since the retinal progenitor cells contained in the aggregates have already determined their differentiation direction, it is preferable to use a serum-containing medium to promote further maturation and proliferation of the retinal progenitor cells.

[0031] In this specification, serum-containing medium means a medium containing unprocessed or unpurified serum. The medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvate, buffers, inorganic salts, etc.

[0032] To avoid the complexity of preparation, DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich) may be used as the serum-containing medium.

[0033] In one embodiment, the culture may be carried out under preferably xeno-free conditions. Xeno-free means conditions in which components from a different species than the species of the cells to be cultured are excluded.

[0034] In step (1), the culture medium used for suspension culture of aggregates containing retinal progenitor cells contains an Activin signaling pathway activator, a Sonic Hedgehog signaling pathway activator, and retinoic acid. In conventional methods for producing retinal tissue, it was not common to culture pluripotent stem cells in a medium containing differentiation-inducing factors at the stage when they have been differentiated into retinal progenitor cells; it was common to culture them in a medium that did not contain differentiation-inducing factors. However, in step (1), by further suspension culture of aggregates containing retinal progenitor cells in a medium containing an Activin signaling pathway activator, a Sonic Hedgehog signaling pathway activator, and retinoic acid, it is possible to promote the differentiation and further proliferation of retinal progenitor cells. In particular, the process of culturing aggregates containing retinal progenitor cells in a medium containing all three simultaneously—Activin signaling pathway activator, Sonic Hedgehog signaling pathway activator, and retinoic acid—was completely unknown.

[0035] In this specification, an Activin signaling pathway agonist is a substance that can enhance the signal mediated by Activin. The Activin signaling pathway agonist is not particularly limited as long as it can induce differentiation of pluripotent stem cells into retinal progenitor cells by enhancing the Activin signaling pathway. Examples include proteins belonging to the Activin family (e.g., Activin A, Activin B, Activin C, Activin AB, etc.) and Activin receptor agonists. The Activin signaling pathway agonist may consist of one or more of these. The Activin signaling pathway agonist is preferably Activin A.

[0036] In this specification, a sonic hedgehog signaling pathway agent (hereinafter sometimes referred to as Shh) is a substance that can enhance Shh-mediated signal transduction. The Shh signaling pathway activators are not particularly limited as long as they can promote differentiation into rod photoreceptor cells by enhancing the Shh signaling pathway. Examples include proteins belonging to the Hedgehog family (e.g., Shh (especially Sonic Headgehog N-terminus (Shh N-terminus) and Ihh)), Shh receptor agonists, PMA (Purmorphamine; 9-cyclohexyl-N-[4-(4-morpholinyl)phenyl]-2-(1-nagutarenyloxy)-9H-purine-6-amine), or SAG (Smoothened Agonist; N-methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane). One or more of these may be included as Shh signaling pathway activators. The Shh signaling pathway activators are preferably SAG and the Shh N-terminus.

[0037] In this specification, retinoic acid acts to promote differentiation into photoreceptor cells.

[0038] The concentrations of the Activin signaling pathway activator, the Sonic Hedgehog signaling pathway activator, and retinoic acid can be appropriately set within a range that achieves the effects described above. For example, Activin A is typically used at concentrations of 0.01 to 1000 ng / mL, preferably 0.1 to 750 ng / mL, and more preferably 1 to 500 ng / mL (e.g., 100 ng / mL). SAG is typically used at concentrations of 1 to 2000 nM, preferably 10 to 700 nM, and more preferably 30 to 600 nM (e.g., 100 nM). The Shh N-terminus is typically used at concentrations of 0.1 to 300 μg / mL, preferably 1 to 100 μg / mL, and more preferably 3 to 60 μg / mL (e.g., 10 μg / mL). Retinoic acid is typically used at a concentration of 0.01 to 100 μM, preferably 0.1 to 10 μM, and more preferably 0.5 to 5 μM (e.g., 1 μM). In one embodiment, an Activin signaling pathway activator can be used in an amount having Activin signaling activity equivalent to that of Activin A at the aforementioned concentration. In another embodiment, a Sonic Hedgehog signaling pathway activator can be used in an amount having Shh signaling activity equivalent to that of SAG and the N-terminus of Shh at the aforementioned concentration.

[0039] The culture time for aggregates containing retinal progenitor cells in step (1) is not particularly limited as long as the aggregates obtained in step (1) have a cell layer formed in which the inner layer expresses the retinal ganglion cell marker Brn3 and the outermost layer expresses the photoreceptor progenitor cell marker Crx, but is usually 0.5 to 50 days. The culture time for aggregates containing retinal progenitor cells in step (1) is preferably 1 day or more, 5 days or more, 10 days or more, 15 days or more, or 20 days or more. The culture time for pluripotent stem cells in step (1) is preferably within 45 days or within 40 days. In one embodiment, the range of culture time for aggregates containing retinal progenitor cells in step (1) is preferably 1 to 45 days, 15 to 45 days, or 20 to 40 days (most preferably 30 days). Specifically, in step (1), aggregates containing retinal progenitor cells are cultured in suspension for 0.5 to 50 days (preferably 1 to 45 days, 15 to 45 days, or 20 to 40 days (most preferably 30 days)), after which step (2) is carried out.

[0040] In one embodiment, in step (1), aggregates containing retinal progenitor cells can be cultured in suspension for 30 days in a culture medium containing Activin A (final concentration 100 ng / mL), SAG (final concentration 100 nM), and retinoic acid (final concentration 1 μM).

[0041] The culture conditions in step (1), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0042] In step (2), aggregates containing retinal ganglion cells and photoreceptor progenitor cells are cultured in suspension in a medium containing a sonic hedgehog signaling pathway activator in the absence of Activin signaling pathway activator and retinoic acid to obtain layered retinal tissue containing photoreceptor cells.

[0043] In step (2), aggregates containing retinal ganglion cells and photoreceptor progenitor cells are cultured in suspension. Here, the suspension culture method and the culture vessel used for suspension culture may be the same as those used in step (1).

[0044] There are no particular limitations on the basal medium used in step (2), and any of the basal media described in step (1) can be appropriately selected. The medium used in step (2) may be either a serum-containing medium or a serum-free medium, as in step (1). However, since the retinal ganglion cells and photoreceptor progenitor cells contained in the aggregates have already determined their differentiation direction, it is preferable to use a serum-containing medium to promote further maturation and proliferation of these cells. To avoid the complexity of preparation, as in step (1), DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich) may be used as such a serum-containing medium.

[0045] In step (2), the culture medium used for suspension culture of aggregates containing retinal ganglion cells and photoreceptor progenitor cells contains a Sonic Hedgehog signaling pathway agent in the absence of Activin signaling pathway agents and retinoic acid. As described in step (1), in conventional methods for producing retinal tissue, it was common to culture pluripotent stem cells in a medium that did not contain differentiation-inducing factors after they had differentiated into retinal progenitor cells. However, in step (2), by further suspension culture of aggregates containing retinal ganglion cells and photoreceptor progenitor cells in a medium containing a Sonic Hedgehog signaling pathway agent in the absence of Activin signaling pathway agents and retinoic acid, it is possible to promote the differentiation and proliferation of photoreceptor progenitor cells contained in the outermost layer of the aggregates, and as a result, obtain layered retinal tissue containing photoreceptor cells, which is the target product of production method 1 of the present invention.

[0046] The suspension culture in step (2) is not particularly restricted in terms of the method of transition from step (1) to step (2), as long as it is performed after the completion of step (1). For example, the transition from step (1) to step (2) can be performed by exchanging the culture medium used in step (1) (i.e., the medium containing the Activin signaling pathway activator, the Sonic Hedgehog signaling pathway activator, and retinoic acid) to the culture medium used in step (2) (the medium containing the Sonic Hedgehog signaling pathway activator). The culture medium exchange may be performed directly from the culture medium used in step (1) to the culture medium used in step (2), or it may be performed via a culture medium exchange from the culture medium used in step (1) to a basal medium before exchanging to the culture medium used in step (2).

[0047] In step (2), aggregates containing retinal ganglion cells and photoreceptor progenitor cells are cultured in suspension in a medium containing a sonic hedgehog signaling pathway activator in the absence of Activin signaling pathway activator and retinoic acid.

[0048] In this specification, "absence" means that a particular substance is not contained in the culture medium. Therefore, for example, "absence of Activin signaling pathway agents" refers to conditions in which Activin signaling pathway agents are not added, or conditions in which Activin signaling pathway agents are substantially absent (for example, a concentration of Activin A of 0.1 ng / mL or less). Similarly, "absence of retinoic acid" refers to conditions in which retinoic acid A is not added, or conditions in which retinoic acid is substantially absent (for example, a concentration of retinoic acid of 0.1 nM or less).

[0049] In step (2), the Activin signaling pathway activator and retinoic acid not contained in the culture medium, and the Sonic Hedgehog signaling pathway activator contained in the culture medium may be the same as those described in step (1).

[0050] The concentration of the Sonic Hedgehog signaling pathway activator can be appropriately set within a range that can achieve the effects described above. For example, SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM, and more preferably 30 to 600 nM (e.g., 100 nM). For example, Shh N-terminus is usually used at a concentration of 0.1 to 300 μg / mL, preferably 1 to 100 μg / mL, and more preferably 3 to 60 μg / mL (e.g., 10 μg / mL). In one embodiment, the Sonic Hedgehog signaling pathway activator can be used in an amount having Shh signaling-promoting activity equivalent to that of SAG at the above concentrations.

[0051] The culture time of the aggregates in step (2) is not particularly limited as long as cone cell marker-positive cells or rod cell marker-positive cells can be observed in the outermost layer of the aggregates obtained in step (2), but is usually 20 to 320 days. The culture time of the aggregates containing retinal ganglion cells and photoreceptor progenitor cells in step (2) is preferably 25 days or more, 30 days or more, 35 days or more, or 40 days or more. The culture time of the aggregates containing retinal ganglion cells and photoreceptor progenitor cells in step (2) is preferably within 200 days, within 100 days, within 75 days, or within 60 days. In one embodiment, the range of culture time for the aggregates containing retinal ganglion cells and photoreceptor progenitor cells in step (2) is preferably 25 to 200 days, 30 to 100 days, 35 to 75 days, or 40 to 60 days (most preferably 50 days).

[0052] In one embodiment, in step (2), aggregates containing retinal ganglion cells and photoreceptor progenitor cells are cultured in suspension for 50 days in a medium containing SAG (final concentration 100 nM) in the absence of Activin signaling pathway activators and retinoic acid, thereby obtaining layered retinal tissue containing photoreceptor cells.

[0053] The culture conditions in step (2), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0054] The aggregate containing retinal progenitor cells cultured in suspension in step (1) can be obtained by adhering pluripotent stem cells as follows. Therefore, the production method 1 of the present invention may further include the following steps prior to step (1). (a) A step of obtaining neural progenitor cells by adhering pluripotent stem cells to a culture medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors. (b) A step of obtaining retinal progenitor cells by adhering neural progenitor cells in a culture medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors. (c) A step of performing adherent culture of retinal progenitor cells in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators to obtain aggregates containing retinal progenitor cells.

[0055] In step (a), pluripotent stem cells are adherently cultured in a culture medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors.

[0056] In this specification, pluripotent stem cells are defined as stem cells that can be cultured in vitro and that possess the ability to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm) and / or extraembryonic tissues (pluripotency).

[0057] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germ cells, tissue-derived stem cells, somatic cells, etc. Examples of pluripotent stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), and induced pluripotent stem cells (iPS cells). Muse cells (Multi-lineage differentiating stress enduring cells) obtained from mesenchymal stem cells (MSCs) and GS cells produced from germ cells (e.g., testes) are also included in the category of pluripotent stem cells. Embryonic stem cells were first established in 1981 and have been applied to the creation of knockout mice since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine. ES cells can be produced by culturing the inner cell mass on feeder cells or in a culture medium containing LIF. Methods for producing ES cells are described, for example, in WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718, etc. Embryonic stem cells can be obtained from designated institutions, or they can be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. The human embryonic stem cell strain Rx::GFP (derived from KhES-1) is available from the RIKEN Institute. Both are mouse embryonic stem cells; EB5 cells are available from the RIKEN Institute, and D3 cells are available from ATCC.

[0058] The pluripotent stem cells used in the present invention are preferably ES cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.

[0059] In this specification, induced pluripotent stem cells are cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, this includes cells in which pluripotency has been induced by reprogramming differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells by expressing one of several combinations of genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, 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).

[0060] Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. in 2006 (Cell, 2006, 126(4), pp.663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007 and possess pluripotency and self-renewal ability similar to embryonic stem cells (Cell, 2007, 131(5), pp.861-872;Science, 2007, 318(5858), pp.1917-1920;Nat. Biotechnol., 2008, 26(1), pp.101-106).

[0061] In addition to the method of producing induced pluripotent stem cells through direct reprogramming via gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding compounds (Science, 2013, 341, pp. 651-654).

[0062] Furthermore, it is possible to obtain induced pluripotent stem cell lines. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, and 1231A3 cells, established at Kyoto University, are available from Kyoto University and iPS Academia Japan, Inc. As induced pluripotent stem cell lines, for example, Ff-I01 cells, Ff-I14 cells, and QHJI01s04 cells, established at Kyoto University, are available from Kyoto University.

[0063] There are no particular limitations on the somatic cells used when producing induced pluripotent stem cells, but examples include tissue-derived fibroblasts, hematopoietic cells (e.g., peripheral blood mononuclear cells (PBMCs) and T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, and smooth muscle cells.

[0064] When reprogramming induced pluripotent stem cells by expressing several types of genes, the means for expressing these genes are not particularly limited. Examples of such means include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors, which are cytoplasmic RNA vectors), gene transfer methods using non-viral vectors such as plasmid vectors (e.g., plasmid vectors, episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, electroporation method), and direct protein injection methods (e.g., needle method, lipofection method, electroporation method).

[0065] Induced pluripotent stem cells (PPTs) can be produced in the presence or absence of feeder cells (feeder-free). When producing PPTs in the presence of feeder cells, known methods can be used to produce PPTs in the presence of undifferentiated maintenance factors. When producing PPTs in the absence of feeder cells, there are no particular limitations on the culture medium used, i.e., the culture medium containing undifferentiated maintenance factors (undifferentiated maintenance medium). However, known maintenance media for embryonic stem cells and / or PPTs, or media well known to those skilled in the art for establishing PPTs in a feeder-free environment, can be used. Many synthetic media have been developed and are commercially available as undifferentiated maintenance media for establishing PPTs in a feeder-free environment, such as Essential 8 (Life Technologies). Essential 8 medium is DMEM / F12 medium containing, as an additive, L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenite (14 μg / l), insulin (19.4 mg / l), NaHCO3 (543 mg / l), transferrin (10.7 mg / l), bFGF (100 ng / mL), and a TGFβ family signaling pathway activator (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)).Other commercially available feeder-free media (undifferentiated maintenance media) include, for example, Essential 6 medium (Life Technologies), Stabilized Essential 8 medium (Life Technologies), S-medium (DS Pharma Biomedical), StemPro (Life Technologies), hESF9 (Proc Natl Acad Sci US A. 2008 Sep 9;105(36):13409-14), TeSR medium (STEMCELL Technologies), mTeSR1 (STEMCELL Technologies), mTeSR2 (STEMCELL Technologies), and TeSR-E8 (STEMCELL Technologies). In addition, StemFit® (Ajinomoto Co., Inc.) is another example of a feeder-free media (undifferentiated maintenance media). In the production of induced pluripotent stem cells, for example, induced pluripotent stem cells can be created by introducing four factors—Oct3 / 4, Sox2, Klf4, and Myc—into somatic cells using a Sendai virus vector in the absence of feeder cells.

[0066] The pluripotent stem cells used in the present invention are mammalian pluripotent stem cells, preferably rodent (e.g., mouse, rat) or primate (e.g., human, monkey) pluripotent stem cells, more preferably human pluripotent stem cells, and even more preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells).

[0067] In transplant medicine, rejection due to differences in histocompatibility antigens is often a problem, but this problem can be overcome by using pluripotent stem cells (e.g., induced pluripotent stem cells) established from the somatic cells of the transplant recipient. That is, in a preferred embodiment, in the manufacturing method 1 of the present invention, pluripotent stem cells (e.g., induced pluripotent stem cells) established from the somatic cells of the recipient are used as pluripotent stem cells to produce layered retinal tissue containing immunological autologous photoreceptor cells from the recipient, which is then transplanted into the recipient.

[0068] Alternatively, layered retinal tissue containing photoreceptor cells may be produced using the manufacturing method 1 of the present invention from pluripotent stem cells (e.g., induced pluripotent stem cells) established from somatic cells of another person whose immune system is compatible with that of the recipient (for example, whose HLA type or MHC type is compatible), and then transplanted into the recipient.

[0069] In step (a), pluripotent stem cells are cultured in an adherent culture. In this specification, adherent culture or adherent culture method means culturing cells or cell aggregates under conditions that cause them to adhere to culture equipment, etc., and the method of performing such culture. That is, adherent culture is performed under conditions that cause cells or cell aggregates to adhere to culture equipment, etc., and suspension culture is not included in the category of adherent culture. More specifically, adherent culture is a culture in which a strong cell-matrix bond is formed between the cells or cell aggregates and the culture equipment, etc. This refers to culturing under conditions that promote the formation of bonds.

[0070] The incubator used for adherent culture is not particularly limited as long as it is capable of adherent culture, and those skilled in the art can appropriately select an incubator according to the culture scale, culture conditions, and culture period. Examples of such incubators include flasks, tissue culture flasks, culture dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, petri dishes, tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks, or roller bottles. These incubators are preferably cell-adherent in order to enable adherent culture. Examples of cell-adherent incubators include incubators whose surfaces have been artificially treated to improve adhesion to cells, specifically, surface-treated incubators or incubators whose interiors are coated with a coating agent. Examples of coating agents include laminins [including laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin α1β1γ1 (hereinafter referred to as laminin 111), etc., and laminin fragments (laminin 511E8, iMatrix-511 (Nippi Corporation), etc.)], extracellular matrix such as entactin, collagen, gelatin, Vitronectin, Synthemax (Corning Corporation), Matrigel, or polymers such as polylysine and polyornithine. Examples of surface-treated culture vessels include culture vessels with surface treatments such as positive charge treatment.

[0071] The culture medium used in adherent culture can be prepared using a basal medium that is commonly used for culturing animal cells, similar to suspension culture. Examples of basal media include BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, or a mixture thereof, which are media that can be used for culturing animal cells. The culture medium used in adherent culture may be serum-containing medium or serum-free medium, but it is preferable to use a serum-free medium in order to limit the differentiation-inducing factors necessary for differentiating pluripotent stem cells into aggregates containing retinal progenitor cells.

[0072] In this specification, serum-free medium means a medium that does not contain unprocessed or unpurified serum. In the present invention, a medium containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) is also included in serum-free medium as long as it does not contain unprocessed or unpurified serum.

[0073] The serum-free medium may contain a serum substitute. Examples of serum substitutes include those appropriately containing albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol or 3'-thiolglycerol, or equivalents thereof. Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679. Commercially available serum substitutes may also be used. An example of such a commercially available serum substitute is Knockout. TM Serum Replacement (manufactured by Life Technologies; now Thermo Fisher; hereafter sometimes referred to as KSR), Chemically-defined Lipid concentrated (manufactured by Life Technologies), Glutamax TMExamples include (manufactured by Life Technologies), B27 (manufactured by Life Technologies), N2 supplement (manufactured by Life Technologies), and ITS supplement (manufactured by Life Technologies).

[0074] The serum-free medium used in adherent culture may, as appropriate, contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts, etc.

[0075] To avoid the complexity of preparation, GMEM medium (Thermo Fisher Scientific) containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin, and 450 μM 1-monothioglycerol (Sigma-Aldrich) may be used as such serum-free medium.

[0076] In step (a), the culture medium used for adherent culture of pluripotent stem cells contains a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors. Conventional methods typically involve creating aggregates of pluripotent stem cells by adherent culture of pluripotent stem cells in the presence of undifferentiated maintenance factors, and then creating retinal tissue by differentiating these aggregates in the absence of undifferentiated maintenance factors. However, step (a) aims to directly differentiate pluripotent stem cells into neural progenitor cells without going through aggregates of pluripotent stem cells by adherent culture of pluripotent stem cells in a medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors.

[0077] In step (a), the undifferentiated maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of undifferentiated maintenance factors commonly used by those skilled in the art include FGF signaling pathway activators, TGFβ signaling pathway activators, and insulin in the case of primed pluripotent stem cells (e.g., human ES cells and human iPS cells). Specifically, examples of FGF signaling pathway activators include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Examples of TGFβ signaling pathway activators include TGFβ1 and TGFβ2.

[0078] In step (a), "absence of undifferentiated maintenance factors" means that undifferentiated maintenance factors are not contained in the culture medium. Therefore, "absence of undifferentiated maintenance factors" includes conditions in which undifferentiated maintenance factors are not added, or conditions in which undifferentiated maintenance factors are substantially absent (for example, a concentration of undifferentiated maintenance factors of 10 pg / mL or less).

[0079] In this specification, the TGFβ signaling pathway is a signaling pathway that uses TGFβ as a ligand and is transmitted within cells by the Smad family. Therefore, a TGFβ signaling pathway inhibitor refers to a substance that inhibits the TGFβ signaling pathway, that is, the signaling pathway transmitted by the Smad family.

[0080] The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be a nucleic acid, protein, or small organic compound. Examples of such substances include substances that act directly on TGFβ (e.g., proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGFβ (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of TGFβ receptors to TGFβ, and substances that inhibit the physiological activity caused by signaling by TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). Lefty is an example of a protein known to inhibit the TGFβ signaling pathway. Compounds well known to those skilled in the art can be used as TGFβ signaling pathway inhibitors, specifically SB431542, LY-364947, SB-505124, A-83-01, etc. Here, SB431542 (4-(5-Benzol[1,3]dioxol-5-yl-4-pyridine-2-yl-1H-imidazole-2-yl)-benzamide) and A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide) are compounds known as inhibitors of the TGFβ receptor (ALK5) and Activin receptor (ALK4 / 7) (i.e., TGFβR inhibitors). One or more of these may be included as TGFβ signaling pathway inhibitors. The TGFβ signaling pathway inhibitors are preferably SB431542 or A-83-01.

[0081] The BMP signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by BMP, and may be a nucleic acid, protein, or small organic compound. Here, BMPs include BMP2, BMP4, BMP7, and GDF7. Examples of such substances include substances that act directly on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMP, and substances that inhibit the physiological activity caused by signaling by BMP receptors. BMPRs can be ALK2 or ALK3. Compounds well known to those skilled in the art can be used as BMP signaling pathway inhibitors, specifically LDN193189, Dorsomorphin, etc. Here, LDN193189 (4-[6-(4-piperazine-1-ylphenyl)pyrazolo[1,5-a]pyrimidine-3-yl]quinoline) is a known BMPR (ALK2 / 3) inhibitor (hereinafter referred to as a BMPR inhibitor) and is usually commercially available in the form of its hydrochloride salt. Alternatively, proteins known as BMP signaling pathway inhibitors (such as Chordin and Noggin) may be used. One or more of these may be included as BMP signaling pathway inhibitors. The BMP signaling pathway inhibitor is preferably LDN193189.

[0082] The concentrations of TGFβ signaling pathway inhibitors and BMP signaling pathway inhibitors can be appropriately set within a range that achieves the effects described above. For example, SB431542 is typically used at concentrations of 0.025 to 1000 μM, preferably 0.1 to 250 μM, and more preferably 0.5 to 50 μM (e.g., 5 μM). Lefty is typically used at concentrations of 0.1 to 3000 ng / mL, preferably 1 to 1000 ng / mL, and more preferably 10 to 300 ng / mL (e.g., 100 ng / mL). LY-364947 is typically used at concentrations of 0.025 to 1000 μM, preferably 0.1 to 250 μM, and more preferably 0.5 to 50 μM (e.g., 10 μM). SB505124 is typically used at concentrations of 0.025 to 1000 μM, preferably 0.1 to 250 μM, more preferably 0.5 to 50 μM (e.g., 5 μM). A-83-01 is typically used at concentrations of 0.001 to 300 μM, preferably 0.01 to 30 μM, more preferably 0.1 to 3 μM (e.g., 0.5 μM). LDN193189 is typically used at concentrations of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM (e.g., 100 nM). Dorsomorphin is typically used at concentrations of 0.01 to 1000 μM, preferably 0.1 to 100 μM, more preferably 1 to 10 μM (e.g., 3 μM). In one embodiment, the TGFβ signaling pathway inhibitor can be used in an amount having TGFβ signaling inhibitory activity equivalent to that of SB431542 at the aforementioned concentration. In another embodiment, the BMP signaling pathway inhibitor can be used in an amount having BMP signaling inhibitory activity equivalent to that of LDN193189 at the aforementioned concentration.

[0083] The culture time for pluripotent stem cells in step (a) is not particularly limited as long as the cells obtained in step (a) are cells that express the neural progenitor cell marker Rx, but is usually 1 to 120 hours. The culture time for pluripotent stem cells in step (a) is preferably 5 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more. The culture time for pluripotent stem cells in step (a) is preferably within 100 hours, within 80 hours, within 60 hours, or within 50 hours. In one embodiment, the range of culture time for pluripotent stem cells in step (a) is preferably 5 to 100 hours, 10 to 80 hours, 15 to 60 hours, or 20 to 50 hours (most preferably 24 hours). That is, after pluripotent stem cells are adherently cultured for 1 to 50 hours (preferably 5 to 100 hours, 10 to 80 hours, 15 to 60 hours, or 20 to 50 hours (most preferably 24 hours)) in step (a), step (b) is carried out.

[0084] In one embodiment, in step (a), pluripotent stem cells can be adherently cultured for 24 hours in a culture medium containing SB431542 (final concentration 5 μM) and LDN193189 (final concentration 100 nM).

[0085] The culture conditions in step (a), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0086] In step (b), neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0087] In step (b), the neural progenitor cells are cultured using adherent culture. Here, the adherent culture method and the culture vessel used for adherent culture may be the same as those used in step (a).

[0088] There are no particular limitations on the basal medium used in step (b), and any of the basal media described in step (a) can be appropriately selected. The medium used in step (b) may be either a serum-containing medium or a serum-free medium, as in step (a), but it is preferable to use a serum-free medium in order to limit the differentiation-inducing factors necessary for differentiating neural progenitor cells into retinal progenitor cells. To avoid the complexity of preparation, as in step (a), DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich) may be used as such a serum-free medium.

[0089] In step (b), the culture medium used for adherent culture of neural progenitor cells contains a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors. Conventional methods have generally involved inducing differentiation into retinal progenitor cells by suspension culture of human pluripotent stem cells in a medium containing BMP4, or by adherent culture of aggregates of human pluripotent stem cells in a medium containing BMP4. However, in this step (b), it is possible to promote differentiation into retinal progenitor cells by adherent culture of neural progenitor cells differentiated from pluripotent stem cells in a medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0090] The method of transitioning from step (a) to step (b) is not particularly limited, as long as the adherent culture in step (b) is performed after the completion of step (a). For example, the transition from step (a) to step (b) can be performed by exchanging the culture medium used in step (a) (i.e., the medium containing the TGFβ signaling pathway inhibitor and the BMP signaling pathway inhibitor) to the culture medium used in step (b) (the medium containing the BMP signaling pathway activator). The culture medium exchange may be performed directly from the culture medium used in step (a) to the culture medium used in step (b), or it may be performed via a culture medium exchange from the culture medium used in step (a) to a basal medium before exchanging to the culture medium used in step (b).

[0091] In step (b), neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0092] In step (b), the absence of undifferentiated maintenance factors may be the same conditions as in step (a). Furthermore, the absence of TGFβ signaling pathway inhibitors may be conditions in which no TGFβ signaling pathway inhibitors are added, or conditions in which TGFβ signaling pathway inhibitors are substantially absent (for example, a concentration of TGFβ signaling pathway inhibitors of 10 nM or less). The absence of BMP signaling pathway inhibitors may be conditions in which no BMP signaling pathway inhibitors are added, or conditions in which BMP signaling pathway inhibitors are substantially absent (for example, a concentration of BMP signaling pathway inhibitors of 100 pM or less).

[0093] In step (b), the undifferentiated maintenance factor, TGFβ signaling pathway inhibitor, and BMP signaling pathway inhibitor not contained in the culture medium may be the same as those described in step (a).

[0094] In this specification, a BMP signaling pathway activator is a substance that can enhance signals mediated by BMPs. The BMP signaling pathway activator is not particularly limited as long as it can induce differentiation of neural progenitor cells into retinal progenitor cells by enhancing the BMP signaling pathway, but examples include BMP proteins such as BMP2, BMP4, or BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, or BMP partial peptides. The BMP signaling pathway activator may contain one or more of these. BMP2, BMP4, and BMP7 proteins are available, for example, from R&D Systems, and GDF7 protein is available, for example, from Wako Pure Chemical Industries. The BMP signaling pathway activator is preferably BMP4.

[0095] The concentration of the BMP signaling pathway activator can be appropriately set within a range that can achieve the effects described above. For example, BMP4 is usually used at a concentration of 0.02 to 500 nM, preferably 0.1 to 100 nM, and more preferably 0.3 to 30 nM (e.g., 3 nM). In one embodiment, the BMP signaling pathway activator can be used in an amount having BMP signaling-promoting activity equivalent to that of BMP4 at the above concentration.

[0096] The culture time of neural progenitor cells in step (b) is not particularly limited as long as the cells obtained by step (b) express at least one selected from the group consisting of retinal progenitor cell markers Rx, PAX6, and Chx10, but is usually 0.5 to 6 days. The culture time of neural progenitor cells in step (b) is preferably 1 day or more. The culture time of neural progenitor cells in step (b) is preferably 3 days or less. In one embodiment, the range of culture time of neural progenitor cells in step (b) is preferably 1 to 3 days (most preferably 2 days).

[0097] In one embodiment, in step (b), retinal progenitor cells can be obtained by adhering neural progenitor cells to a culture medium containing BMP4 (final concentration 3 nM) for 2 days in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors.

[0098] The culture conditions in step (b), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0099] In step (c), retinal progenitor cells are cultured in a culture medium in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators.

[0100] In step (c), the retinal progenitor cells are cultured using adherent culture. Here, the adherent culture method and the culture vessel used for adherent culture may be the same as those used in steps (a) and (b).

[0101] There are no particular limitations on the basal medium used in step (c), and the basal media described in steps (a) and (b) can be appropriately selected. The medium used in step (c) may be a serum-containing medium or a serum-free medium, as in step (a), but it is preferable to use a serum-free medium in order to limit the differentiation-inducing factors necessary for differentiating retinal progenitor cells into aggregates containing retinal progenitor cells. To avoid the complexity of preparation, as such a serum-free medium, DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich) may be used, as in steps (a) and (b).

[0102] The culture medium used for adherent culture of retinal progenitor cells in step (c) does not contain undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, or BMP signaling pathway activators. In conventional methods, when differentiating human pluripotent stem cells into neuroretinal cells by culturing them in a medium containing BMP4, it was common practice to continue culturing them in a BMP4-containing medium until differentiation into neuroretinal cells occurred. However, in this step (c), by adherent culture of retinal progenitor cells in a medium that does not contain not only undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors, but also BMP signaling pathway activators, it is possible to promote differentiation into aggregates containing neuroretinal cells.

[0103] The method of transitioning from step (b) to step (c) is not particularly restricted, as long as the adherent culture in step (c) is performed after the completion of step (b). For example, the transition from step (b) to step (c) can be carried out by exchanging the culture medium used in step (b) (i.e., the medium containing BMP signaling pathway activators) to the culture medium used in step (c).

[0104] In step (c), retinal progenitor cells are adherently cultured in culture medium in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators.

[0105] In step (c), the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors may be the same conditions as in step (b). The absence of BMP signaling pathway activators may be conditions in which BMP signaling pathway activators are not added, or conditions in which BMP signaling pathway activators are substantially absent (for example, a concentration of BMP signaling pathway activators of 10 pg / mL or less).

[0106] In step (c), the undifferentiated maintenance factor, TGFβ signaling pathway inhibitor, BMP signaling pathway inhibitor, and BMP signaling pathway activator not contained in the culture medium may be the same as those described in steps (a) and (b).

[0107] The culture time for retinal progenitor cells in step (c) is not particularly limited as long as the aggregate containing retinal progenitor cells obtained in step (c) is an aggregate in which a cell layer expressing at least one selected from the group consisting of retinal progenitor cell markers Rx, PAX6, and Chx10 is formed, but is usually 2 to 27 days. The culture time for retinal progenitor cells in step (c) is preferably 3 days or more, 4 days or more, 5 days or more, or 6 days or more. The culture time for retinal progenitor cells in step (c) is preferably within 25 days, within 20 days, within 15 days, or within 8 days. In one embodiment, the range of culture time for retinal progenitor cells in step (b) is preferably 3 to 11 days, 4 to 10 days, 5 to 9 days, or 6 to 8 days (most preferably 7 days).

[0108] In one embodiment, in step (c), retinal progenitor cells can be cultured in a culture medium for 7 days in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators to obtain aggregates containing retinal progenitor cells.

[0109] The culture conditions in step (c), such as culture temperature and CO2 concentration, can be set as appropriate. The culture temperature is, for example, about 30°C to about 40°C, preferably about 37°C. The CO2 concentration is, for example, about 1% to about 10%, preferably about 5%.

[0110] 2. Therapeutic agents for diseases based on damage to layered retinal tissue, including photoreceptor cells. Layered retinal tissue containing photoreceptor cells is useful in transplant medicine for diseases based on damage to said retinal tissue. Therefore, the present invention provides a therapeutic agent for diseases based on damage to said retinal tissue (the therapeutic agent of the present invention), which includes layered retinal tissue containing photoreceptor cells.

[0111] The therapeutic agent of the present invention comprises an effective amount of layered retinal tissue containing photoreceptor cells and a pharmaceutically acceptable carrier. In the therapeutic agent of the present invention, the layered retinal tissue containing photoreceptor cells may be produced by the production method 1 of the present invention.

[0112] Acceptable carriers for pharmaceuticals include physiological aqueous solvents (such as physiological saline, buffer solutions, and serum-free culture media). If necessary, preservatives, stabilizers, reducing agents, isotonic agents, etc., commonly used in transplant medicine, may be added to the pharmaceutical product containing the tissue or cells to be transplanted.

[0113] The therapeutic agent of the present invention can be prepared as a suspension by suspending layered retinal tissue containing photoreceptor cells in a suitable physiological aqueous solvent. If necessary, a cryopreservative may be added, the suspension may be frozen for storage, thawed before use, washed with a buffer solution, and used in transplant medicine.

[0114] Layered retinal tissue containing photoreceptor cells can be used as a treatment for diseases based on damage to the retinal tissue, or to replenish the damaged area in a state of retinal tissue damage. In patients with diseases or retinal tissue damage requiring transplantation, layered retinal tissue containing photoreceptor cells can be transplanted to replenish the damaged retinal tissue itself, thereby treating diseases or retinal tissue damage caused by layered retinal tissue containing photoreceptor cells. Examples of diseases based on layered retinal tissue containing photoreceptor cells include ophthalmic diseases such as age-related macular degeneration, retinitis pigmentosa, cone dystrophy, macular edema, retinal detachment, cancer-associated retinopathy, retinal vein occlusion, and retinal pigment epithelial detachment.

[0115] 3. A screening method or efficacy evaluation method for therapeutic drugs for diseases based on damage to layered retinal tissue including photoreceptor cells. The layered retinal tissue containing photoreceptor cells, produced by the manufacturing method 1 of the present invention, is useful as a disease research material and drug discovery material for screening therapeutic agents for diseases based on damage to layered retinal tissue containing photoreceptor cells, and for evaluating drug efficacy. Therefore, it can be used as a screening reagent or a drug efficacy evaluation reagent for test substances. For example, iPS cells can be generated from human patients with diseases based on damage to layered retinal tissue containing photoreceptor cells, particularly diseases based on hereditary disorders, and layered retinal tissue containing photoreceptor cells can be produced using the manufacturing method 1 of the present invention with these iPS cells. This retinal tissue can reproduce in vitro the damage to layered retinal tissue containing photoreceptor cells that causes the disease suffered by the patient. Therefore, the present invention provides a method for screening therapeutic agents for diseases based on damage to layered retinal tissue containing photoreceptor cells, which includes contacting a test substance with the layered retinal tissue containing photoreceptor cells produced by the manufacturing method 1 of the present invention and testing the effect of the substance on the retinal tissue.

[0116] For example, layered retinal tissue containing photoreceptor cells, exhibiting a specific disorder (e.g., a hereditary disorder), is cultured in the presence or absence (negative control) of the test substance. The degree of disorder in the layered retinal tissue containing photoreceptor cells treated with the test substance is then compared to that of the negative control. As a result, the test substance that reduces the degree of disorder can be selected as a candidate substance for a therapeutic drug for the disease based on that disorder. For example, a test substance that further improves the physiological activity (e.g., survival promotion or maturation) of layered retinal tissue containing photoreceptor cells can be searched for as a candidate drug. Alternatively, induced pluripotent stem cells can be prepared from somatic cells with gene mutations exhibiting a specific disorder such as retinal disease, and the test substance can be added to layered retinal tissue containing photoreceptor cells produced by differentiating these cells using the production method 1 of the present invention. The presence or absence of the disorder can then be used as an indicator to search for a candidate test substance that is effective as a therapeutic or preventive drug for the disorder.

[0117] For example, the present invention provides a method for screening therapeutic agents for disorders of layered retinal tissue containing photoreceptor cells, comprising the following steps (11) to (13). (11) A step of culturing layered retinal tissue containing damaged photoreceptor cells for a certain period of time under viable culture conditions in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (12) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (13) A step of selecting the test substance in step (11) as a therapeutic agent for damage to layered retinal tissue containing photoreceptor cells, based on the difference in the results measured in step (11) and step (12).

[0118] Furthermore, for example, the present invention provides a method for evaluating drug efficacy, comprising the following steps (21) to (23). (21) A step of culturing layered retinal tissue containing damaged photoreceptor cells for a certain period of time under viable culture conditions in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (22) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (23) A step to evaluate the efficacy of the test substance in step (21) based on the difference between the results measured in step (21) and step (22).

[0119] Here, the layered retinal tissue containing photoreceptor cells is not particularly limited as long as it contains damaged photoreceptor cells, and may be manufactured by the manufacturing method 1 of the present invention. Furthermore, "absence of the test substance" includes adding only the culture medium and the solvent in which the test substance is dissolved instead of the test substance. Furthermore, "positive control" refers to a known compound that has a therapeutic effect on damage to layered retinal tissue containing photoreceptor cells. Methods for measuring the degree of damage include methods for measuring the number of viable cells, such as methods for measuring the amount of intracellular ATP, or methods for measuring the number of viable cells by cell staining (e.g., nuclear staining) and morphological observation.

[0120] In step (23), a method for selecting the test substance as a therapeutic agent for damage to layered retinal tissue including photoreceptor cells, or a method for evaluating the efficacy of the test substance, is to compare the measured value in step (21) with the measured value of the negative control in step (22), and if the degree of cell damage in step (21) is improved, the test substance can be selected as a therapeutic agent for damage to layered retinal tissue, or it can be determined that the test substance has efficacy. Alternatively, the measured value in step (21) can be compared with the measured value of the positive control in step (22), and if the degree of cell damage in step (21) is reduced to the same or greater extent, the test substance can be selected as a therapeutic agent for damage to layered retinal tissue, or it can be determined that the test substance has efficacy.

[0121] 4. Toxicity Assessment Methods In toxicity assessments, layered retinal tissue containing photoreceptor cells is cultured in the presence or absence (negative control) of the test substance. The degree of toxicity in the layered retinal tissue containing photoreceptor cells treated with the test substance is then compared to that of the negative control. As a result, the test substance that shows toxicity compared to the negative control can be determined to be a substance that is toxic to layered retinal tissue containing photoreceptor cells.

[0122] For example, the present invention provides a toxicity assessment method comprising the following steps (31) to (33). (31) A step of culturing layered retinal tissue containing photoreceptor cells in viable culture conditions for a certain period of time in the presence of a test substance, and then measuring the degree of damage to the layered retinal tissue containing photoreceptor cells. (32) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (33) A step to evaluate the toxicity of the test substance in step (31) based on the difference between the results measured in steps (31) and (32).

[0123] Here, the layered retinal tissue containing photoreceptor cells may be produced by the manufacturing method 1 of the present invention. Furthermore, "absence of the test substance" includes adding only the culture medium and the solvent in which the test substance is dissolved instead of the test substance. Also, "positive control" refers to a known toxic compound. Methods for measuring the degree of cell damage include methods for measuring the number of viable cells, such as measuring the amount of intracellular ATP, or methods for measuring the number of viable cells by cell staining (e.g., nuclear staining) and morphological observation.

[0124] In step (33), a method for evaluating the toxicity of the test substance is, for example, to compare the measured value in step (31) with the measured value of the negative control in step (32), and if the degree of cell damage in step (31) is greater, it can be determined that the test substance is toxic. Alternatively, to compare the measured value in step (31) with the measured value of the positive control in step (32), it can be determined that the test substance is toxic if the degree of cell damage in step (31) is equal to or greater than that of the positive control.

[0125] 5. Method for producing aggregates containing retinal progenitor cells The present invention also provides a method for producing aggregates containing retinal progenitor cells cultured in suspension in step (1) of the production method of the present invention (hereinafter referred to as production method 2 of the present invention).

[0126] The manufacturing method 2 of the present invention includes the following steps. (a) A step of obtaining neural progenitor cells by adhering pluripotent stem cells to a culture medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors. (b) A step of obtaining retinal progenitor cells by adhering neural progenitor cells in a culture medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors. (c) A step of performing adherent culture of retinal progenitor cells in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators to obtain aggregates containing retinal progenitor cells.

[0127] In the manufacturing method 2 of the present invention, the cells, undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, BMP signaling pathway activators, culture conditions (culture medium, culture period, culture temperature, etc.) used in each step (a) to (c) may all be the same as those used in the manufacturing method 1 of the present invention.

[0128] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0129] Example 1 Human iPS cell line 201B7 (Ci), seeded at a cell density of 5,000 cells per well in a 6-well plate coated with iMatrix-511 (Nippi), was cultured for 8 days in StemFit AK02N medium (Ajinomoto). The culture medium was then replaced with differentiation medium (GMEM medium (Thermo Fisher Scientific) containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin, and 450 μM 1-monothioglycerol (Sigma-Aldrich)) to initiate differentiation. From the start of differentiation induction to 1 day after differentiation induction, the TGF-beta signaling inhibitor SB431542 (final concentration 5 μM, Fujifilm Wako Pure Chemical Industries) and the BMP signaling inhibitor LDN193189 hydrochrolide (final concentration 100 nM, Sigma-Aldrich) were added to the differentiation medium. From 1 day after differentiation induction to 3 days after differentiation induction, BMP4 (final concentration 1.5 nM, R&D Systems) was added to the differentiation medium. From 3 days after differentiation induction to 10 days after differentiation induction, the cells were cultured in differentiation medium alone. The culture schedule is shown in Figure 1. Five or ten days after differentiation induction, aggregates containing retinal progenitor cells, which could be observed under a light microscope as well-defined, high-density cell aggregates, were confirmed to have formed in the central region of the iPS cell colony, and the cells were fixed using 4% PFA (paraformaldehyde) / PBS.

[0130] Samples 5 days after the start of differentiation were blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). A primary antibody reaction was then performed at 4°C overnight using anti-Rx rabbit polyclonal IgG antibody (1:1000 dilution, M228 / Takara). After the primary antibody reaction, the samples were washed with PBS(-), and a secondary antibody reaction was performed at room temperature for 2 hours using Alexa546-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific) and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction, the samples were washed with PBS(-), and fluorescence microscopy was performed using a laser confocal microscope LSM700 (Zeiss). In aggregates 5 days after the start of differentiation, the formation of regions with high cell density consisting of cells expressing the neural progenitor cell marker Rx was observed (Figure 2).

[0131] Samples 10 days after the start of differentiation were blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Primary antibody reactions were then carried out at 4°C overnight using anti-Rx antibody and anti-CHX10 sheep polyclonal IgG antibody (1:1000 dilution, X1180P / Exalpha Biologicals). After the primary antibody reaction, the samples were washed with PBS(-) and secondary antibody reactions were carried out at room temperature for 2 hours using Alexa488-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-goat IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was complete, the samples were washed with PBS(-) and fluorescence microscopy was performed using an inverted fluorescence microscope DMi8 (Leica). In well-defined aggregates 10 days after the start of differentiation, a cell layer co-expressing the retinal progenitor cell markers Rx and CHX10 was observed at the outer edge (Figure 3).

[0132] Example 2 Human iPS cell line 201B7, seeded at a cell density of 5,000 cells per well in 6-well plates coated with iMatrix-511 (Nippi), was cultured for 8 days in StemFit AK02N medium (Ajinomoto). The culture medium was then replaced with differentiation medium (GMEM medium (Thermo Fisher Scientific) containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin, and 450 μM 1-monothioglycerol (Sigma-Aldrich)) to initiate differentiation. From the start of differentiation induction to 1 day after differentiation induction, the TGF-beta signaling inhibitor SB431542 (final concentration 5 μM, Fujifilm Wako Pure Chemical Industries) and the BMP signaling inhibitor LDN193189 hydrochrolide (final concentration 100 nM, Sigma-Aldrich) were added to the differentiation medium. From 1 day after differentiation induction to 3 days after differentiation induction, BMP4 (final concentration 1.5 nM, R&D Systems) was added to the differentiation medium.

[0133] From 3 days after the start of differentiation to 10 days after the start of differentiation, the cells were cultured in differentiation medium only. On 10 days after the start of differentiation, aggregates containing retinal progenitor cells, which could be seen under a light microscope as well-defined and dense cell aggregates, were confirmed to have formed in the central part of the iPS cell colony, and these aggregates were detached using a scraper. Unwanted cells were removed from the detached aggregates using a 40 μm cell strainer, and suspension culture was started using maturation medium (DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich)). From 10 days after the start of differentiation induction to 30 days after differentiation induction, cells were cultured in mature medium with 100 ng / mL Activin A (R&D systems, final concentration 100 ng / mL), sonic hedgehog signal activator SAG (Alexis Biochemicals, final concentration 100 nM), and retinoic acid (Fujifilm Wako Pure Chemical Industries, final concentration 1 μM). The culture schedule is shown in Figure 4. Aggregates were collected 30 days after differentiation induction, fixed with a 3% PFA, 7.5% sucrose / PBS solution, and embedded using OCT Compound (Sakura FineTech). After embedding, the frozen blocks were sectioned using a cryomicrotome to prepare 10 μm frozen sections. The prepared frozen sections were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS(-), and then blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Subsequently, primary antibody reactions were carried out at 4°C overnight using anti-Crx rabbit polyclonal IgG antibody (1:1000 dilution, M231 / Takara) and anti-Brn3 goat polyclonal IgG antibody (1:1000 dilution, sc-6026 / Santa Cruz).After the primary antibody reaction, the cells were washed with PBS(-), and then a secondary antibody reaction was performed at room temperature for 2 hours using Alexa488-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-goat IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was complete, the cells were washed with PBS(-), and then mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific). After air drying, fluorescence microscopy was performed using an inverted fluorescence microscope DMi8 (Leica). Retinal ganglion cell marker-positive cells were observed in the inner layer of the aggregates 30 days after differentiation induction, and photoreceptor cell progenitor cell marker-positive cells were scattered (Figure 5).

[0134] Example 3 Human iPS cell line M8 (iPS cells established by the inventors from healthy peripheral blood mononuclear cells according to the "Method for Establishing iPS Cells from Peripheral Blood Using Episomal Vectors" by the Kyoto University iPS Cell Research Institute) was seeded at a cell density of 4,500 cells per well in a 6-well plate coated with iMatrix-511 (Nippi) and cultured in StemFit AK02N medium (Ajinomoto) for 8 days. After that, the medium was replaced with differentiation medium (GMEM medium (Thermo Fisher Scientific) containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin, and 450 μM 1-monothioglycerol (Sigma-Aldrich)) to initiate differentiation induction. From the start of differentiation induction to 1 day after differentiation induction, the TGF-beta signaling inhibitor SB431542 (final concentration 5 μM, Fujifilm Wako Pure Chemical Industries) and the BMP signaling inhibitor LDN193189 hydrochrolide (final concentration 100 nM, Sigma-Aldrich) were added to the differentiation medium. From 1 day after differentiation induction to 3 days after differentiation induction, BMP4 (final concentration 3 nM, R&D Systems) was added to the differentiation medium.

[0135] From 3 days after the start of differentiation to 10 days after the start of differentiation, the cells were cultured in differentiation medium only. On 10 days after the start of differentiation, aggregates containing retinal progenitor cells, which could be seen under a light microscope as well-defined and dense cell aggregates, were confirmed to have formed in the central part of the iPS cell colony, and these aggregates were detached using a scraper. Unwanted cells were removed from the detached aggregates using a 40 μm cell strainer, and suspension culture was started using maturation medium (DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich)). From 10 days after the start of differentiation induction to 40 days after differentiation induction, cells were cultured in mature medium with 100 ng / mL Activin A (R&D Systems, final concentration 100 ng / mL), sonic hedgehog signal activator SAG (Alexis Biochemicals, final concentration 100 nM), and retinoic acid (Fujifilm Wako Pure Chemical Industries, final concentration 1 μM). From 40 days after differentiation induction to 80 days after differentiation induction, cells were cultured in mature medium containing sonic hedgehog signal activator SAG (final concentration 100 nM). Unless there was a change in the medium additives, the medium was changed every two or three days.

[0136] At 20, 40, 60, and 80 days after the start of differentiation induction, aggregates (retinal organoids) were fixed with a 3% PFA, 7.5% sucrose / PBS solution and embedded using OCT Compound (Sakura FineTech). After embedding, the frozen blocks were sectioned using a cryomicrotome to prepare 10 μm frozen sections.

[0137] Frozen sections prepared from samples 20 and 40 days after the start of differentiation induction were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS(-), and then blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Subsequently, primary antibody reactions were carried out at 4°C overnight using anti-Crx rabbit polyclonal IgG antibody (1:1000 dilution, M231 / Takara) and anti-Brn3 goat polyclonal IgG antibody (1:1000 dilution, sc-6026 / Santa Cruz). After the primary antibody reaction, the cells were washed with PBS(-), and then a secondary antibody reaction was performed at room temperature for 2 hours using Alexa488-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-goat IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was completed, the cells were washed with PBS(-), and then mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific). After air drying, fluorescence microscopy was performed using a laser confocal microscope LSM700 (Zeiss). In aggregates 20 days after the start of differentiation induction, retinal ganglion cell marker-positive cells were observed in the inner layer of the aggregates, and a small number of photoreceptor cell progenitor cell marker-positive cells were observed in the outer layer (Figure 6). Furthermore, in aggregates 40 days after the start of differentiation induction, retinal ganglion cell marker-positive cells were observed in the inner layer of the aggregates, and photoreceptor cell progenitor marker-positive cells were observed in the outer layer. In addition, layer formation of photoreceptor cell progenitor marker-positive cells was observed in the outermost layer (Figure 7).

[0138] Frozen sections prepared from samples 60 days after differentiation induction were blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Subsequently, primary antibody reactions were carried out at 4°C overnight using anti-RxRγ rabbit polyclonal IgG antibody (1:200 dilution, sc-555 / Santa Cruz) and anti-NRL goat polyclonal IgG antibody (1:200 dilution, AF2945 / R&D systems). After the primary antibody reaction, the sections were washed with PBS(-), and then secondary antibody reactions were carried out at room temperature for 2 hours using Alexa488-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-goat IgG donkey polyclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific) and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was complete, the cells were washed with PBS(-) and then mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific). After air drying, fluorescence microscopy was performed using a laser confocal microscope LSM700 (Zeiss). At 60 days after the start of differentiation induction, RXRγ-positive cells or NRL-positive cells were observed in the outermost layer of the aggregates (Figure 8). No cells co-positive for RXRγ and NRL were observed.

[0139] Frozen sections prepared from samples 80 days after the start of differentiation induction were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS(-), and then blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Subsequently, primary antibody reactions were carried out at 4°C overnight using anti-rhodopsin mouse monoclonal IgG antibody (1:1000 dilution, Clone RET-P1 / Sigma-Aldrich), anti-L / M opsin rabbit polyclonal IgG antibody (1:1000 dilution, AB5405 / Sigma-Aldrich), and anti-S-opsin goat polyclonal IgG antibody (1:500 dilution, sc-14363 / Santa Cruz). After the primary antibody reaction, the samples were washed with PBS(-), and then a secondary antibody reaction was carried out at room temperature for 2 hours using Alexa488-labeled anti-mouse IgG donapoliclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-rabbit IgG donapoliclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa647-labeled anti-goat IgG donapoliclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was complete, the samples were washed with PBS(-), and then mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific). After air drying, fluorescence microscopy was performed using a laser confocal microscope LSM700 (Zeiss). Eighty days after the start of differentiation induction, the outermost layer of the aggregates showed cone photoreceptor cell marker S-opsin-positive cells, L / M-opsin-positive cells, or rod photoreceptor cell marker rhodopsin-positive cells (Figure 9).

[0140] Example 4 Human iPS cell line 1231A3 (CiRA), seeded at a cell density of 5,000 cells per well in 6-well plates coated with iMatrix-511 (Nippi), was cultured for 10 days in StemFit AK02N medium (Ajinomoto), and then differentiation induction was initiated by replacing the medium with differentiation medium (GMEM medium (Thermo Fisher Scientific) containing 10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin, and 450 μM 1-monothioglycerol (Sigma-Aldrich)). From the start of differentiation induction to 1 day after differentiation induction, the TGF-beta signaling inhibitor SB431542 (final concentration 5 μM, Fujifilm Wako Pure Chemical Industries) and the BMP signaling inhibitor LDN193189 hydrochrolide (final concentration 100 nM, Sigma-Aldrich) were added to the differentiation medium. From 1 day after differentiation induction to 3 days after differentiation induction, BMP4 (final concentration 3 nM, R&D Systems) was added to the differentiation medium.

[0141] From 3 days after the start of differentiation to 10 days after the start of differentiation, the cells were cultured in differentiation medium only. On 10 days after the start of differentiation, aggregates containing retinal progenitor cells, which could be seen under a light microscope as well-defined and dense cell aggregates, were confirmed to have formed in the central part of the iPS cell colony, and these aggregates were detached using a scraper. Unwanted cells were removed from the detached aggregates using a 40 μm cell strainer, and suspension culture was started using maturation medium (DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-Aldrich)). From 10 days after the start of differentiation induction to 40 days after differentiation induction, cells were cultured in mature medium with 100 ng / mL Activin A (R&D systems, final concentration 100 ng / mL), sonic hedgehog signal activator SAG (Alexis Biochemicals, final concentration 100 nM), and retinoic acid (Fujifilm Wako Pure Chemical Industries, final concentration 1 μM). From 40 days after differentiation induction to 90 days after differentiation induction, cells were cultured in mature medium containing sonic hedgehog signal activator SAG (final concentration 100 nM). Unless there was a change in the medium additives, the medium was changed every two or three days. The culture schedule is shown in Figure 10. Microscopic images of aggregates were also examined at 15, 20, 40, 60, 80, and 90 days after the start of differentiation induction (Figure 11). Fifteen and twenty days after induction, the aggregates consisted of a high-luminosity outer layer containing a high density of cells and a low-luminosity inner layer that appeared reticular. Forty days after induction, the aggregates had decreased overall luminosity, consisted of a single layer, and appeared hollow. Sixty days after induction, the aggregates had increased overall luminosity again, and the inner boundaries were clearly defined. Eighty or ninety days after induction, the high-luminosity outermost layer of the aggregates appeared to be further divided into two layers.Considering this in conjunction with the examination of tissue sections, it was thought that this indicated a structure in which the layer corresponding to the exoneuroblast layer observed during biological development is separated from the layer corresponding to the outer granular layer in living organisms, and the layer corresponding to the inner granular layer is separated from the layer corresponding to the outer plexiform layer. Furthermore, granular structures, thought to be intracellular segments of photoreceptor cells, and microvilli-like structures began to be observed on the surface of the aggregates.

[0142] Ninety days after differentiation induction, retinal organoids were fixed with a 3% paraformaldehyde / 7.5% sucrose solution and embedded using OCT Compound (Sakura FineTech). After embedding, the frozen block was sectioned using a cryomicrotome to prepare 10 μm frozen sections. The prepared frozen sections were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS(-), and then blocked at room temperature for 1 hour using a blocking solution (PBS(-) containing 5% inactivated horse serum (GIBCO) and 0.05% Triton-X100). Subsequently, primary antibody reactions were carried out at 4°C for 24 hours using anti-rhodopsin mouse monoclonal IgG antibody (1:1000 dilution, Clone RET-P1 / Sigma-Aldrich) and anti-L / M opsin rabbit polyclonal IgG antibody (1:1000 dilution, AB5405 / Sigma-Aldrich). After the primary antibody reaction, the samples were washed with PBS(-), and then secondary antibody reactions were carried out at room temperature for 2 hours using Alexa488-labeled anti-mouse IgG donapoliclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), Alexa546-labeled anti-rabbit IgG donapoliclonal IgG antibody (1:1000 dilution, Thermo Fisher Scientific), and DAPI (final concentration 1 μg / mL, Thermo Fisher Scientific). After the secondary antibody reaction was complete, the samples were washed with PBS(-), and then mounted using PermaFluor aqueous mounting medium (Thermo Fisher Scientific). After air drying, fluorescence microscopy was performed using an inverted fluorescence microscope DMi8 (Leica). Numerous rhodopsin-positive and L / M opsin-positive cells were observed in the outermost layer of retinal organoids 90 days after the start of differentiation induction (Figure 12). [Industrial applicability]

[0143] According to the invention, it is possible to produce layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells in a short period of time. This application is based on Japanese Patent Application No. 2020-215415 (filing date: December 24, 2020), the contents of which are fully incorporated herein by reference.

Claims

1. A method for producing layered retinal tissue containing photoreceptor cells, comprising the following steps (1) to (2); (1) A step of obtaining aggregates containing retinal progenitor cells by suspension culture in a medium containing an Activin signaling pathway agent, a Sonic Hedgehog signaling pathway agent, and retinoic acid, (2) A step of obtaining layered retinal tissue containing photoreceptor cells by culturing aggregates containing retinal ganglion cells and photoreceptor progenitor cells in suspension in a culture medium containing a sonic hedgehog signaling pathway agent in the absence of an Activin signaling pathway agent and retinoic acid.

2. The method according to claim 1, wherein the layered retinal tissue containing photoreceptor cells is retinal tissue comprising an outer granular layer, an inner granular layer, and a ganglion cell layer.

3. The method according to claim 2, wherein the photoreceptor cells are contained in the outer granular layer.

4. The method according to claim 3, wherein the photoreceptor cells contained in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells.

5. The method according to claim 4, wherein 50% or more of all cells contained in the outer granular layer are rod photoreceptor cells and / or cone photoreceptor cells.

6. The method according to any one of claims 1 to 5, wherein the aggregate containing retinal progenitor cells is an aggregate in which a cell layer co-expressing the retinal progenitor cell markers Rx and Chx10 is formed.

7. The method according to any one of claims 1 to 6, wherein the Activin signaling pathway agent is one or more substances selected from the group consisting of Activin A, Activin B, Activin C, and Activin AB.

8. The method according to claim 7, wherein the Activin signaling pathway agent is Activin A.

9. The method according to any one of claims 1 to 8, wherein the sonic hedgehog signaling pathway activator is one or more substances selected from the group consisting of Sonic Hedgehog (Shh), Smoothened Agonist (SAG), and Purmorphamine (PMA).

10. The method according to claim 9, wherein the sonic hedgehog signaling pathway activator is SAG.

11. The method according to any one of claims 1 to 10, wherein in step (1), aggregates containing retinal progenitor cells are cultured in suspension for 0.5 to 50 days.

12. The method according to any one of claims 1 to 11, wherein the aggregate containing retinal ganglion cells and photoreceptor progenitor cells is an aggregate in which a cell layer expressing the retinal ganglion cell marker Brn3 is formed in the inner layer and the photoreceptor progenitor cell marker Crx is formed in the outermost layer.

13. The method according to any one of claims 1 to 12, wherein in step (2), aggregates containing retinal ganglion cells and photoreceptor progenitor cells are cultured in suspension for 20 to 320 days.

14. The method according to any one of claims 1 to 13, further comprising the following steps prior to step (1); (a) In the absence of undifferentiated maintenance factors, pluripotent stem cells are cultured in a culture medium containing a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor to obtain neural progenitor cells. (b) A step to obtain retinal progenitor cells by adhering neural progenitor cells in a culture medium containing a BMP signaling pathway activator in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, and BMP signaling pathway inhibitors. (c) A step of performing adherent culture of retinal progenitor cells in the absence of undifferentiated maintenance factors, TGFβ signaling pathway inhibitors, BMP signaling pathway inhibitors, and BMP signaling pathway activators to obtain aggregates containing retinal progenitor cells.

15. The method according to claim 14, wherein the pluripotent stem cells are human pluripotent stem cells.

16. The method according to claim 14 or 15, wherein the pluripotent stem cells are induced pluripotent stem cells.

17. The method according to any one of claims 14 to 16, wherein the TGFβ signaling pathway inhibitor is one or more substances selected from the group consisting of Lefty, SB431542, LY-364947, SB-505124, and A-83-01.

18. The method according to claim 17, wherein the TGFβ signaling pathway inhibitor is SB431542.

19. The method according to any one of claims 14 to 18, wherein the BMP signaling pathway inhibitor is one or more substances selected from the group consisting of LDN193189 and Dorsomorphin.

20. The method according to claim 19, wherein the BMP signaling pathway inhibitor is LDN193189.

21. The method according to any one of claims 14 to 20, wherein pluripotent stem cells are adherently cultured for 1 to 50 hours in step (a).

22. The method according to any one of claims 14 to 21, wherein the BMP signaling pathway activator is one or more substances selected from the group consisting of BMP2, BMP4, BMP7, and GDF7.

23. The method according to claim 22, wherein the BMP signaling pathway agent is BMP4.

24. The method according to any one of claims 14 to 23, wherein in step (b), neural progenitor cells are adherently cultured for 0.5 to 6 days.

25. The method according to any one of claims 14 to 24, wherein in step (c), retinal progenitor cells are adherently cultured for 2 to 27 days.

26. A screening method for therapeutic drugs for damage to layered retinal tissue containing photoreceptor cells, including the following steps (11) to (13): (11) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, which was produced by the method of any one of claims 1 to 25, after culturing it for a certain period of time in the presence of a test substance under viable culture conditions, (12) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells, which was produced by the method of any one of claims 1 to 25, after culturing it for a certain period of time under viable culture conditions in the absence of a test substance or in the presence of a positive control, (13) A step of selecting the test substance in step (11) as a therapeutic agent for damage to layered retinal tissue including photoreceptor cells, based on the difference in the results measured in steps (11) and (12).

27. A method for evaluating drug efficacy, including the following steps (21) to (23): (21) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells, which was produced by the method of any one of claims 1 to 25, after culturing it for a certain period of time in the presence of a test substance under viable culture conditions, (22) A step of measuring the degree of damage to the layered retinal tissue containing damaged photoreceptor cells, which was produced by the method of any one of claims 1 to 25, after culturing it for a certain period of time under viable culture conditions in the absence of a test substance or in the presence of a positive control, (23) A step to evaluate the efficacy of the test substance in step (21) based on the difference between the results measured in steps (21) and (22).

28. Toxicity assessment method including the following steps (31) to (33): (31) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells, which was produced by the method of any one of claims 1 to 25, after culturing it for a certain period of time in the presence of a test substance under viable culture conditions, (32) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells produced by the method of any one of claims 1 to 25, after culturing the layered retinal tissue containing photoreceptor cells for a certain period of time under viable culture conditions in the absence of the test substance or in the presence of a positive control, (33) A step to evaluate the toxicity of the test substance in step (31) based on the difference between the results measured in steps (31) and (32).

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