Method for increasing cone photoreceptor cells or rod photoreceptor cells by dorsalization signal transmitter or ventralization signal transmitter

Abstract

To provide a retinal tissue rich in cone photoreceptor progenitor cells and / or cone photoreceptor cells, to provide a retinal tissue rich in rod photoreceptor progenitor cells and / or rod photoreceptor cells, and to provide a method for producing the same.SOLUTION: Provided is a method for increasing the proportion of cone photoreceptor progenitor cells and cone photoreceptor cells in photoreceptor progenitor cells and photoreceptor cells contained in retinal tissue, comprising culturing a retinal tissue from the early developmental stage to the stage when the appearance rate of cone photoreceptor progenitor cells is at its maximum i) in a culture medium containing a dorsalization signal transmitter at a concentration to the extent that it suppresses the expression of ventral markers, or a method for increasing the proportion of rod photoreceptor progenitor cells and rod photoreceptor cells in photoreceptor progenitor cells and photoreceptor cells contained in retinal tissue, comprising culturing a retinal tissue from the early developmental stage to the stage when the appearance rate of cone photoreceptor progenitor cells is at its maximum ii) in the presence of a ventralization signal transmitter at a concentration that promotes expression of ventral markers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to retinal tissue rich in cone or rod photoreceptors and a method for producing the same. [Background technology]

[0002] The retinal tissues of mice, rats, humans, and chickens are all said to be regionally determined by dorsalizing and ventralizing signals, but each animal species has its own unique structure. For example, unlike mice and rats, human and chicken retinas have a region in the fundus (or central retina) called the macula, which has a high proportion of cone photoreceptors, but the center of the macula is home to only cone photoreceptors (the rod-free zone). In particular, in the macula of human retinal tissue, the central region has a higher proportion of LM cone photoreceptors, while the proportion of S cone photoreceptors increases toward the periphery. Also, outside the macula, there is a region containing both S cone photoreceptors and rod photoreceptors, with rod photoreceptors clustered at the outer edge of the macula.

[0003] In chicken retinal tissue, it is known that the rod-free zone disappears when the entire dorsalized region of the retinal tissue at the optic vesicle stage of chicken embryos is removed, and that the rod-free zone disappears when ventralizing signals are overexpressed. This suggests that the rod-free zone is formed with the contribution of the dorsalized region (Non-Patent Document 1). On the other hand, several culture methods are known for inducing differentiation of pluripotent stem cells such as ES cells into retinal tissue (Patent Documents 1 to 5). However, it was unknown how the dorsalized region contributes to the formation of the rod-free zone in human or chicken retinal tissue. In particular, no method was known for producing retinal tissue rich in cone photoreceptors (LM cone photoreceptors and / or S cone photoreceptors) or rod photoreceptors. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2009 / 148170 [Patent Document 2] International Publication No. 2011 / 055855 [Patent Document 3] International Publication No. 2013 / 077425 [Patent Document 4] International Publication No. 2013 / 065763 [Patent Document 5] International Publication No. 2015 / 025967 [Non-patent literature]

[0005] [Non-Patent Document 1] J Neurosci. 25(11):2823-2831 (2005) Summary of the Invention [Problem to be solved by the invention]

[0006] The problem to be solved by the present invention is to provide retinal tissue rich in cone or rod photoreceptors and a method for producing the same. [Means for solving the problem]

[0007] The inventors have conducted extensive research to solve the above-mentioned problems and have found that by culturing and maturing retinal tissue at a differentiation stage that contains retinal progenitor cells or neural retinal progenitor cells and in which ganglion cells have not yet appeared, i.e., retinal tissue at the initial developmental stage, in the presence of a dorsalizing signaling substance at a concentration sufficient to suppress the expression of ventral markers, the proportion of cone photoreceptors contained in the photoreceptors can be increased compared to when the tissue is cultured in the absence of the dorsalizing signaling substance. Furthermore, the present inventors discovered that by culturing and maturing retinal tissue in the early stages of development in the presence of a ventralizing signal transduction substance, the proportion of rod photoreceptors among the photoreceptors can be increased compared to when the tissue is cultured in the absence of the ventralizing signal transduction substance, thereby completing the present invention.

[0008] That is, the present invention relates to: [1] A method for increasing the proportion of cone photoreceptor precursors and cone photoreceptors among photoreceptor precursors and photoreceptors contained in retinal tissue, the method comprising the step of culturing retinal tissue from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursors is maximized in a medium containing a dorsalization signaling substance at a concentration sufficient to suppress the expression of ventral markers; [2] The method according to [1] above, wherein the cone photoreceptor progenitor cells and the cone photoreceptor cells are CRX-positive and RXR-γ-positive, or CRX-positive and TRβ2-positive, and NRL-negative cells; [3] The method according to [1] or [2] above, wherein the ventral marker is ALDH1A3 and / or COUP-TF I; [4] The method according to any one of [1] to [3] above, wherein the concentration of the dorsalization signaling substance is such that it does not induce the expression of the most dorsal marker; [5] The method according to any one of [1] to [3] above, wherein the concentration of the dorsalization signaling substance is such that it promotes the expression of a dorsal marker; [6] The method according to any one of [1] to [3] above, wherein the concentration of the dorsalization signaling substance is such that it does not induce the expression of the most dorsal marker but promotes the expression of other dorsal markers; [7] The method according to [5] or [6] above, wherein the dorsal marker is CYP26A1 and / or CYP26C1; [8] The method according to [4] or [6] above, wherein the most dorsal marker is COUP-TF II; [9] The method according to [5] or [6] above, wherein the dorsal marker is ALDH1A1;

[10] The method according to the above item [9], wherein the concentration of the dorsalization signaling substance is such that ALDH1A1 expression is induced to a level of 0.1% to 30% of the level of ALDH1A1 expression promoted by 1.35 nM BMP4;

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

[10] above, wherein the retinal tissue at an early stage of development includes a ciliary margin-like structure;

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

[10] above, wherein the retinal tissue in the early stage of development contains cells that can differentiate into photoreceptors and ganglion cells;

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

[12] above, wherein the retinal tissue at an early stage of development is derived from pluripotent stem cells;

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

[12] above, wherein the retinal tissue at an early stage of development is derived from neuroepithelial cells obtained from adult tissue;

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

[14] above, wherein the retinal tissue at an early stage of development contains PAX6-positive and RX-positive cells;

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

[15] above, wherein the retinal tissue at an early stage of development contains PAX6-positive, RX-positive, and CHX10-positive cells;

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

[16] above, wherein the step of culturing in the presence of a dorsalization signaling substance is continued for 4 to 170 days;

[18] The method according to

[17] above, wherein the step of culturing in the presence of a dorsalizing signal transduction substance is continued until the time when rod photoreceptor precursor cells appear when cultured in the absence of a dorsalizing signal transduction substance;

[19] The method according to any one of the above [1] to

[18] , wherein the dorsalizing signal transduction substance is a substance acting on the BMP signal transduction pathway, a substance acting on the Wnt signal transduction pathway, or a substance inhibiting the SHH signal transduction pathway, capable of inducing a BMP signal equivalent to 0.01 nM to 0.90 nM of BMP4;

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

[19] above, wherein the dorsalization signaling substance is BMP4;

[21] The method according to

[20] above, wherein the concentration of BMP4 is 0.05 nM to 0.45 nM;

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

[19] above, wherein the dorsalization signaling substance is cyclopamine-KAAD;

[23] The method according to

[22] above, wherein the concentration of Cyclopamine-KAAD is 0.01 μM to 5 μM;

[24] The method according to

[23] above, wherein the concentration of Cyclopamine-KAAD is 0.2 μM to 1 μM;

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

[24] above, which is carried out in a medium containing no 9-cis retinoic acid;

[26] Retinal tissue containing photoreceptor progenitors rich in cone photoreceptor progenitors and / or photoreceptors rich in cone photoreceptors, obtained by the method according to any one of [1] to

[25] above;

[27] Retinal tissue containing photoreceptor precursors rich in cone photoreceptor precursors and / or photoreceptors rich in cone photoreceptor precursors, in which the number of cone photoreceptor precursors and cone photoreceptor precursors among the photoreceptor precursors and photoreceptors is at least two times, preferably at least four times, the number of rod photoreceptor precursors and rod photoreceptor precursors, and which has ganglion cells;

[28] The retinal tissue according to

[26] or

[27] above, wherein the number of cone photoreceptor progenitors and cone photoreceptors contained in all photoreceptor progenitors and all photoreceptors is 70% or more, preferably 80% or more;

[29] A retinal tissue that can be matured into the retinal tissue described in

[26] or

[27] above by culturing;

[30] The retinal tissue according to any one of

[26] to

[29] above, wherein 50% or more of the layer structure of the retinal tissue forms a continuous epithelial structure;

[31] The retinal tissue according to

[30] above, wherein the diameter of the retinal tissue in the long axis direction is 0.6 mm or more;

[32] A pharmaceutical composition for transplantation into a retinal tissue in need of transplantation in a patient with a retinal disease, comprising a retinal tissue fragment excised from the retinal tissue according to any one of

[26] to

[31] above;

[33] The pharmaceutical composition according to

[32] above, wherein the retinal tissue requiring transplantation is tissue in a region containing a rod-free zone;

[34] The pharmaceutical composition according to

[33] above, wherein the region containing the rod-free zone has a macular-like structure;

[35] A method for increasing the proportion of rod photoreceptor precursors and rod photoreceptors among photoreceptor precursors and photoreceptors contained in retinal tissue, comprising the step of culturing retinal tissue from an early developmental stage to the stage at which the appearance rate of cone photoreceptor precursors is maximized in the presence of a ventralizing signaling substance at a concentration sufficient to promote the expression of ventral markers for at least one day;

[36] The method according to

[35] above, wherein the rod photoreceptor progenitor cells and the rod photoreceptor cells are NRL-positive and CRX-positive cells;

[37] The method according to

[35] or

[36] above, wherein the ventral marker is ALDH1A3 and / or COUP-TF I;

[38] The method according to any one of

[35] to

[37] above, wherein the retinal tissue at an early stage of development includes a ciliary margin-like structure;

[39] The method according to any one of

[35] to

[38] above, wherein the retinal tissue at an early stage of development contains cells that can differentiate into photoreceptors and ganglion cells;

[40] The method according to any one of

[35] to

[39] above, wherein the retinal tissue at an early stage of development is derived from pluripotent stem cells;

[41] The method according to any one of

[35] to

[39] above, wherein the retinal tissue at an early stage of development is derived from cells including neuroepithelial cells obtained from adult tissue;

[42] The method according to any one of

[35] to

[41] above, wherein the retinal tissue at an early stage of development contains PAX6-positive and RX-positive cells;

[43] The method according to

[42] above, wherein the retinal tissue at an early stage of development contains PAX6-positive, RX-positive, and CHX10-positive cells;

[44] The method according to any one of

[35] to

[43] above, wherein the step of culturing in the presence of a ventralizing signaling substance is continued for 4 days to 170 days;

[45] The method according to

[44] above, wherein the step of culturing in the presence of a ventralizing signaling substance is continued until rod photoreceptor precursor cells appear;

[46] The method according to any one of the above

[35] to

[45] , wherein the ventralizing signal transduction substance is a substance having an SHH signal transduction pathway promoting effect equivalent to 1 nM to 10 μM SAG, or a substance having a BMP signal transduction pathway inhibiting effect equivalent to 0.1 nM to 20 μM LDN193189;

[47] The method according to

[46] above, wherein the ventralization signaling substance is SAG;

[48] ​​The method according to

[47] above, wherein the concentration of SAG is 1 nM to 10 μM;

[49] The method according to

[48] above, wherein the concentration of SAG is 10 nM to 500 nM;

[50] The method according to

[46] above, wherein the ventralization signaling substance is LDN193189;

[51] The method according to

[50] above, wherein the concentration of LDN193189 is 0.1 nM to 20 μM;

[52] The method according to

[51] above, wherein the concentration of LDN193189 is 30 nM to 1 μM;

[53] The method according to any one of

[35] to

[52] above, which is carried out in a medium containing no 9-cis retinoic acid;

[54] Retinal tissue containing photoreceptor progenitors rich in rod photoreceptor progenitors and / or photoreceptors rich in rod photoreceptors, obtained by the method according to any one of

[35] to

[53] above;

[55] Retinal tissue containing rod-rich photoreceptor precursors and / or rod-rich photoreceptors, in which 40% or more, preferably 55% or more, of the number of photoreceptor precursors and photoreceptors are rod photoreceptor precursors and rod photoreceptors, and which has ganglion cells;

[56] Retinal tissue that can be matured into the retinal tissue described in

[55] above by culturing;

[57] The retinal tissue according to any one of

[54] to

[56] above, wherein 50% or more of the layer structure of the retinal tissue forms a continuous epithelial structure;

[58] The retinal tissue according to

[57] above, wherein the diameter of the retinal tissue in the long axis direction is 0.6 mm or more;

[59] A pharmaceutical composition for transplantation into a retinal tissue in need of transplantation in a patient with a retinal disease, comprising a retinal tissue fragment excised from the retinal tissue according to any one of

[54] to

[58] above;

[60] The pharmaceutical composition according to the above-mentioned

[59] , wherein the retinal tissue requiring transplantation is a region including the periphery of the macula and its outer region, which has a high proportion of rod precursors and / or rod photoreceptors;

[61] A method for treating a disease caused by a disorder of retinal cells or retinal tissue, comprising transplanting an effective amount of the retinal tissue according to any one of

[26] to

[31] above or any one of

[54] to

[58] above into a subject in need of transplantation;

[62] Use of any one of the above

[26] to

[31] or any one of the above

[54] to

[58] as a reagent for evaluating toxicity and efficacy of retinal tissue;

[63] A method for producing fully mature retinal tissue that expresses S-opsin, L-opsin, and / or M-opsin, comprising the step of culturing retinal tissue containing cone photoreceptor progenitor cells and cone photoreceptors in a serum-free medium;

[64] The method according to

[63] above, wherein the retinal tissue containing cone photoreceptor progenitor cells and cone photoreceptors is the retinal tissue according to any one of

[26] to

[31] above;

[65] The method according to

[63] or

[64] above, wherein the serum-free medium is a medium containing a dorsalization signaling substance;

[66] The method according to

[65] above, wherein the dorsalization signaling substance is BMP;

[67] The method according to any one of

[63] to

[66] above, wherein the serum-free medium further contains a thyroid hormone signaling substance. [Effects of the Invention]

[0009] According to the present invention, it is possible to produce retinal tissue rich in cone photoreceptors and provide it as retinal tissue for transplantation into the macula of the eye of a patient suffering from age-related macular degeneration or the like, and to produce retinal tissue rich in rod photoreceptors and provide it as retinal tissue for transplantation into the peripheral area of ​​the macula or outside of the eye of a patient suffering from age-related macular degeneration or the like. [Brief explanation of the drawings]

[0010] [Figure 1] 1 shows images of cell aggregates containing retinal tissue prepared from human ES cells taken with a fluorescent stereomicroscope on day 35 (a, b) and day 42 (c, d) after the start of suspension culture. Images a and b are of cell aggregates containing retinal tissue excised with tweezers on day 35 after the start of suspension culture and taken with a fluorescent stereomicroscope, while images c and d are of cells in which CRX::Venus protein fluorescence was observed, i.e., photoreceptor precursor cells, were confirmed to have appeared in the cell aggregates containing retinal tissue on day 42 after the start of suspension culture. [Figure 2] Figure 2 shows images (a–e) of cell aggregates containing retinal tissue generated from human ES cells cultured in suspension for 70 days, taken with a fluorescent stereomicroscope. Images (f–t) of sections of cell aggregates containing retinal tissue harvested from cultures for 75 days were immunostained with anti-CRX, anti-TRβ2, and DAPI. Compared to the untreated control, the addition of dorsalizing signaling agents (+BMP, +Cyclopamine-KAAD) increased the number of CRX::Venus-positive cells, CRX-positive cells, and TRβ2-positive cells, whereas the addition of ventralizing signaling agents (+LDN193189, +SAG) decreased the number of CRX::Venus-positive cells, CRX-positive cells, and TRβ2-positive cells. The CRX::Venus-positive cells, CRX-positive cells, and TRβ2-positive cells that emerge at this differentiation stage are all photoreceptor precursor cells or cone photoreceptor precursor cells. [Figure 3]Figure 3 shows the results of culturing cell aggregates containing retinal tissue from human ES cells for 191–192 days in suspension culture, the differentiation stage at which Müller cells are detected. Sections were prepared from the recovered cell aggregates containing retinal tissue and immunostained using standard methods with anti-GFP (detecting CRX::Venus protein), anti-NRL, anti-RXR-γ, and DAPI. GFP-positive cells were identified, i.e., RXR-γ-positive and NRL-negative cells (cone photoreceptor precursors) among CRX::Venus-positive cells, and NRL-positive cells (rod photoreceptor precursors) among CRX::Venus-positive cells (Figures a–t). Compared to the untreated control, the addition of dorsalizing signaling agents (+BMP4, +Cyclopamine-KAAD) resulted in a higher proportion of cone photoreceptor precursors, whereas the addition of ventralizing signaling agents (+LDN193189, +SAG) resulted in a lower proportion. Conversely, compared to the untreated group (Control), the proportion of rod photoreceptor precursor cells was lower when dorsalizing signaling substances were added (+BMP4, +Cyclopamine-KAAD), and higher when ventralizing signaling substances were added (+LDN193189, +SAG). [Figure 4] Figure 4 shows the results of sectioning the cell aggregates containing retinal tissue recovered from hESC-derived retinal tissue cultured for approximately 70–75 days. Immunostaining was performed with anti-CRX, anti-TRβ2, and DAPI. The percentages of CRX-positive cells and CRX-positive and TRβ2-positive cells relative to DAPI-positive cells were then measured using image analysis software. Compared to the untreated control (control (NUC)), the addition of dorsalizing signaling agents (+BMP4, +Cyclopamine-KAAD) increased the number of CRX-positive cells and CRX-positive and TRβ2-positive cells, whereas the addition of ventralizing signaling agents (+LDN193189, +SAG) decreased the number of CRX-positive cells and CRX-positive and TRβ2-positive cells. At this differentiation stage, CRX-positive cells and CRX-positive and TRβ2-positive cells represent photoreceptor precursor cells and cone photoreceptor precursor cells, respectively. [Figure 5]Figure 5 shows images of cell aggregates containing retinal tissue prepared from human ES cells cultured in suspension for up to 75 days. The collected cell aggregates were then sectioned and immunostained with anti-OC2, anti-OTX2, anti-TRβ2, and DAPI. The retinal tissue was then immunostained in the same manner on retinal tissue approximately 70-75 days after the start of suspension culture. The percentages of OTX2- and OC2-positive cells and the fluorescence signal intensity of OC2-positive cells were measured using image analysis software. These images and graphs show that, compared to the untreated control (Control (NUC)), the addition of dorsalizing signaling agents (+BMP4, +Cyclopamine-KAAD) increased the number of OTX2- and TRβ2-positive cells, whereas the number of OC2-positive cells decreased, with lower expression levels. Conversely, when ventralizing signaling substances were added (+LDN193189, +SAG), both OTX2-positive cells and TRβ2-positive cells decreased, whereas +LDN193189 increased OC2-positive cells. [Figure 6]Figure 6 shows the results of sectioning the cell aggregates containing retinal tissue recovered from hESC-derived retinal tissue-containing cell aggregates cultured for approximately 191–192 days after the initiation of suspension culture. Immunostaining was performed using anti-GFP antibody (detecting CRX::Venus protein), anti-NRL antibody, anti-RXR-γ antibody, and DAPI. The percentage of RXR-γ-positive and NRL-negative cells (cone photoreceptor precursors) among CRX::Venus-positive cells, and the percentage of NRL-positive cells (rod photoreceptor precursors) among CRX::Venus-positive cells, were measured using image analysis software. Compared to the untreated control (control (NUC)), the percentage of cone photoreceptor precursors (Cone) was higher in the dorsalizing signaling pathways (+BMP4, +Cyclopamine-KAAD) and lower in the ventralizing signaling pathways (+LDN193189, +SAG). Conversely, compared to the untreated group (Control (NUC)), the proportion of rod-like cells was lower when dorsalizing signaling substances were added (+BMP4, +Cyclopamine-KAAD), and higher when ventralizing signaling substances were added (+LDN193189, +SAG). [Figure 7-1] Figure 7-1 shows images of cell aggregates containing retinal tissue prepared from human ES cells cultured for approximately 75 days in suspension culture. Sections were prepared from the recovered cell aggregates containing retinal tissue and immunostained using an anti-GFP antibody (detecting CRX::Venus protein), an anti-ALDH1A3 antibody, and DAPI. In the untreated control (Control (NUC)), the number of GFP-positive cells (i.e., CRX::Venus-positive cells) was relatively low in the ALDH1A3-positive region, i.e., the ventralized region of the retina, indicating that differentiation of photoreceptor precursor cells was suppressed. Addition of a dorsalizing signaling agent (+BMP4) resulted in a decrease in the ALDH1A3-positive region and a corresponding increase in CRX::Venus-positive cells. Addition of a ventralizing signaling agent (+LDN193189, +SAG) resulted in an increase in the ALDH1A3-positive region and a corresponding decrease in CRX::Venus-positive cells. [Figure 7-2] Figure 7-2 shows the results of quantitative PCR performed on cell aggregates containing retinal tissue prepared from human ES cells, which were cultured for approximately 40-44 days (d40) or 70-75 days (d70) after the initiation of suspension culture. At approximately 70-75 days (white bars) after the initiation of suspension culture, the addition of a dorsalizing signaling agent (+BMP4) reduced ALDH1A3 gene expression compared to the untreated control (control (NUC)). Consistent with the immunostaining results, the addition of a dorsalizing signaling agent (+LDN193189, +SAG) increased ALDH1A3 gene expression. Furthermore, this ALDH1A3 gene expression was comparable to that observed on approximately 40-44 days after the initiation of suspension culture, suggesting that ALDH1A3 gene expression continues from approximately 40-44 days after the initiation of suspension culture. Furthermore, these findings indicate that the induction of differentiation into photoreceptor precursor cells or photoreceptors is correlated with the suppression of ALDH1A3 expression, and that dorsalizing signaling substances that suppress ALDH1A3 expression or ALDH1A3 gene expression to the extent that they promote the differentiation of CRX::Venus cells. [Figure 8-1] Figure 8-1 shows images of retinal tissue-containing cell aggregates prepared from human ES cells cultured for approximately 40 or 75 days after the start of suspension culture. The collected retinal tissue-containing cell aggregates were sectioned and immunostained using an anti-ALDH1A1 antibody. Approximately 40 days after the start of suspension culture, no ALDH1A1-positive regions were observed in the untreated (Control (NUC)) group or in the groups treated with ventralizing signaling substances (+LDN193189, +SAG). However, in the group treated with dorsalizing signaling substances (+BMP4), ALDH1A1-positive regions were observed approximately 40 days after the start of suspension culture. However, even in the group treated with dorsalizing signaling substances (+BMP4), such clear ALDH1A1-positive regions were no longer observed by approximately 75 days after the start of suspension culture. [Figure 8-2]Figure 8-2 shows quantitative PCR results for cell aggregates containing retinal tissue prepared from human ES cells, which were cultured in suspension culture for approximately 40-44 days (d40) or 70-75 days (d70). At approximately 40-44 days (black bars) after the start of suspension culture, ALDH1A1 gene expression was elevated in the dorsalizing signaling agent (+BMP4) group compared with the untreated group (Control (NUC)) and the ventralizing signaling agent groups (+LDN193189, +SAG), as shown by immunohistochemistry. However, this ALDH1A1 gene expression decreased by approximately 70-75 days after the start of suspension culture, correlating with the immunohistochemistry results, where ALDH1A1 expression was no longer evident. These results suggest that ALDH1A1 expression decreases at the stage when cone differentiation reaches its peak. [Figure 8-3] Figure 8-3 shows quantitative PCR results for cell aggregates containing retinal tissue prepared from human ES cells that were cultured for approximately 70-75 days (d70) after the start of suspension culture. The graph shows that CYP26A1 gene expression increased when a dorsalizing signaling agent (+BMP4) was added compared to when a ventralizing signaling agent (+LDN193189) was added. [Figure 9]Figure 9 shows the results of fluorescence microscopy (upper image), quantitative PCR (graph), and immunostaining (lower image) of cell aggregates containing retinal tissue, which were prepared from human ES cells and cultured for approximately 70-75 days after the start of suspension culture. The results show that the addition of 0.45 nM or 1.35 nM BMP4 reduced the CRX::Venus fluorescence intensity compared to 0.15 nM BMP4, indicating a decrease in CRX::Venus-positive cells (upper image). Furthermore, the addition of a high concentration (1.35 nM) of BMP4 increased the expression of the ALDH1A1 gene, which is expressed in the dorsalized region of the retinal tissue (graph). Furthermore, when 1.35 nM BMP4 was added and cultured, it was found that there were relatively few CRX::Venus-positive cells in areas that stained strongly with anti-ALDH1A1 antibodies, whereas there were relatively many CRX::Venus-positive cells in areas that stained less strongly with anti-ALDH1A1 antibodies (bracket). This indicates that the addition of high concentrations of BMP4 induces ALDH1A1 expression, and that in areas with high ALDH1A1 expression, promotion of photoreceptor differentiation is difficult, and that excessive induction of ALDH1A1 expression actually does not promote the differentiation of photoreceptor precursor cells. [Figure 10]Figure 10 shows the results of immunostaining for S-opsin (S-OPN), LM-opsin (LM-OPN), rhodopsin (RET-P1), cone arrestin (ARR3), and the cone photoreceptor-specific cyclic nucleotide-gated channel (CNGA3), which are expressed with further photoreceptor maturation. Although some S-opsin expression was observed, almost all photoreceptor precursor cells did not express S-opsin (S-OPN), LM-opsin (LM-OPN), rhodopsin (RET-P1), or cone arrestin (ARR3), indicating that final maturation was not observed, unlike in vivo. [Figure 11]Figure 11 shows that cell aggregates containing retinal tissue prepared from human ES cells were cultured for 190 days from the start of culture until they reached a mature stage of differentiation where Muller cells could be detected. After that, they were further cultured for 260 to 338 days from the start of culture in DMEM / F12 medium containing glutamic acid, supplemented with N2 supplement and taurine, but without fetal bovine serum. Sections were prepared from the recovered cell aggregates containing retinal tissue, and the expression of S-opsin (S-OPN), rhodopsin (RET-P1), cone arrestin (ARR3), rod arrestin (SAG), cone photoreceptor-specific cyclic nucleotide-gated channel (CNGA3), rod photoreceptor-specific cyclic nucleotide-gated channel (CNGA1), cone photoreceptor-specific transducin alpha subunit (Gat2), and rod photoreceptor-specific transducin alpha (TFA). This figure shows the results of immunostaining for the retinal phosphodiesterase 6 subunit (Gat1), cone-specific phosphodiesterase 6C (PDE6c), and rod-specific phosphodiesterase 6A (PDE6a). The results showed that these functional molecules, which are expressed during further maturation, are expressed in cone and rod photoreceptors, promoting their final maturation. Furthermore, mature photoreceptors were found throughout the retinal tissue. [Figure 12]Figure 12 shows the results of immunostaining for LM-opsin (LM-OPN), cone arrestin (ARR3), cone photoreceptor-specific cyclic nucleotide-gated channel (CNGA3), cone photoreceptor-specific transducin alpha subunit (Gat2), and cone photoreceptor-specific phosphodiesterase 6C (PDE6c), which are expressed with further photoreceptor maturation. Cell aggregates containing retinal tissue prepared from human ES cells were cultured for 190 days from the start of culture until they reached a mature, differentiated stage of retinal tissue where Müller cells could be detected. Subsequently, BMP4 and T3 were added to DMEM / F12 medium containing glutamate as a component, to which N2 supplement and taurine had been added, but no fetal bovine serum had been added, and the culture was continued for 341 to 344 days from the start of culture. As a result, it was found that these functional molecules, which are expressed as the maturation progresses, are expressed in cone photoreceptors, promoting their final maturation. Furthermore, it was found that the addition of BMP4 and T3 promoted the expression of LM-opsin compared to when BMP4 and T3 were not added. [Figure 13]Figure 13 shows the results of culturing cell aggregates containing retinal tissue prepared from human ES cells for 190 days from the start of culture until they reached a differentiated stage where Müller cells were detected. Then, BMP4 and T3 were added to DMEM / F12 medium containing glutamate as a component, supplemented with N2 supplement and taurine, but without fetal bovine serum, to further mature the retinal tissue. Culture was then initiated for 30 days or more. Quantitative PCR was performed on the recovered cell aggregates containing retinal tissue according to standard methods to examine the expression levels of S-opsin, M-opsin, and L-opsin. The results show that the expression of S-opsin was promoted in the absence of T3, regardless of the presence or absence of BMP4, indicating that further maturation into S-cone photoreceptors, i.e., final maturation, was promoted. On the other hand, the addition of T3 suppressed S-opsin expression, indicating that T3 acts as an inhibitor of final maturation into S-cone photoreceptors. Furthermore, when BMP4 was added in combination with T3, the expression of L- and M-opsin was promoted, and further maturation into L- or M-cone vision, i.e., final maturation, was promoted. DETAILED DESCRIPTION OF THE INVENTION

[0011] 1.Definition As used herein, "stem cells" refer to undifferentiated cells that have the ability to proliferate (particularly the ability to self-renew) and maintain the same differentiation potential even after cell division. Stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells, depending on their differentiation potential. Pluripotent stem cells refer to stem cells that can be cultured in vitro and have the ability (pluripotency) to differentiate into all cell lineages belonging to the three germ layers (ectoderm, mesoderm, and endoderm). Multipotent stem cells refer to stem cells that have the ability to differentiate into multiple types of tissues and cells, although not all types. Unipotent stem cells refer to stem cells that have the ability to differentiate into specific tissues or cells.

[0012] Pluripotent stem cells can be induced from fertilized eggs, cloned embryos, germline stem cells, tissue stem 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). Embryonic stem cells were first established in 1981 and have been used to generate knockout mice since 1989. Human embryonic stem cells were established in 1998 and are now being used in regenerative medicine. ES cells can be produced by culturing inner cell masses on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, and US6,280,718. Embryonic stem cells are available from designated institutions and are also commercially available. For example, human embryonic stem cells KhES-1, KhES-2, and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University. EB5 cells, which are mouse embryonic stem cells, are available from RIKEN, and the D3 strain is available from ATCC. Nuclear transfer ES cells (ntES cells), a type of ES cell, can be established from cloned embryos created by transplanting the nucleus of a somatic cell into an egg from which the cell line has been removed.

[0013] In the present invention, "induced pluripotent stem cells" (also referred to as iPS cells) refer to cells in which pluripotency has been induced by reprogramming somatic cells using known methods. Specifically, iPS cells include cells in which pluripotency has been induced by reprogramming somatic cells differentiated into fibroblasts or peripheral blood mononuclear cells through the expression of any combination of multiple genes selected from a group of reprogramming genes including Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28, Esrrb, etc. Preferred combinations of reprogramming factors include (1) Oct3 / 4, Sox2, Klf4, and Myc (c-Myc or L-Myc), and (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem Cells, 2013;31:458-466). In 2006, Yamanaka et al. established induced pluripotent stem cells using mouse cells (Cell, 2006, 126(4) pp.663-676). In 2007, induced pluripotent stem cells were also established using human fibroblasts, and they possess the same pluripotency and self-renewal capabilities as embryonic stem cells (Cell, 2007, 131(5) pp.861-872; Science, 2007, 318(5858) pp.1917-1920; Nat. Biotechnol., 2008, 26(1) pp.101-106). Various improvements have been made since then to methods for inducing induced pluripotent stem cells (e.g., mouse iPS cells: Cell. 2006 Aug 25;126(4):663-76, human iPS cells: Cell. 2007 Nov 30;131(5):861-72). In addition to producing induced pluripotent stem cells by direct reprogramming through gene expression, induced pluripotent stem cells can also be induced from somatic cells by adding chemical compounds (Science, 2013, 341 pp. 651-654). It is also possible to obtain established induced pluripotent stem cells. For example, human induced pluripotent cell lines such as 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells, 1231A3 cells, Ff-I01 cells, and QHJI01 cells established at Kyoto University are available from Kyoto University.

[0014] Somatic cells used in producing induced pluripotent stem cells are not particularly limited, but include tissue-derived fibroblasts, blood cells (e.g., peripheral blood mononuclear cells and T cells), hepatocytes, pancreatic cells, intestinal epithelial cells, smooth muscle cells, etc. Fibroblasts include those derived from the dermis.

[0015] When producing induced pluripotent stem cells, if reprogramming is performed by expressing several types of genes, the means for expressing the genes is not particularly limited. Examples of such means include infection methods using viral vectors (e.g., retroviral vectors, lentiviral vectors, Sendai virus vectors, adenoviral vectors, and adeno-associated virus vectors), gene transfer methods using plasmid vectors (e.g., plasmid vectors and episomal vectors) (e.g., calcium phosphate method, lipofection method, retronectin method, and electroporation method), gene transfer methods using RNA vectors (e.g., calcium phosphate method, lipofection method, and electroporation method), and direct protein injection methods. The pluripotent stem cells used in the present invention are preferably ES cells or induced pluripotent stem cells, more preferably induced pluripotent stem cells (iPS cells). The pluripotent stem cells used in the present invention are preferably primate (e.g., human, monkey) pluripotent stem cells, and more preferably human pluripotent stem cells. Accordingly, the pluripotent stem cells used in the present invention are preferably human ES cells or human induced pluripotent stem cells (human iPS cells), and most preferably human induced pluripotent stem cells (human iPS cells).

[0016] Genetically modified pluripotent stem cells can be produced, for example, by using homologous recombination techniques. Examples of genes on chromosomes that can be modified include cell marker genes, histocompatibility antigen genes, and disease-related genes due to retinal cell damage. Target genes on chromosomes can be modified using methods described in "Manipulating the Mouse Embryo, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1994)"; "Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993)"; "Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells," Yodosha (1995); and the like.

[0017] Specifically, for example, genomic DNA containing the target gene to be modified (e.g., a cell marker gene, a gene for a histocompatibility antigen, or a disease-related gene) is isolated, and a target vector for homologous recombination of the target gene is prepared using the isolated genomic DNA. The prepared target vector is introduced into stem cells, and cells in which homologous recombination has occurred between the target gene and the target vector are selected, thereby producing stem cells in which a gene on the chromosome has been modified.

[0018] Methods for isolating genomic DNA containing a target gene include known methods described in Molecular Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press (1989) and Current Protocols in Molecular Biology, John Wiley & Sons (1987-1997). Genomic DNA containing a target gene can also be isolated using a genomic DNA library screening system (manufactured by Genome Systems) or Universal GenomeWalker Kits (manufactured by CLONTECH). Instead of genomic DNA, a polynucleotide encoding a target protein can also be used. The polynucleotide can be obtained by amplifying the corresponding polynucleotide by PCR.

[0019] Construction of a target vector for homologous recombination of a target gene and efficient selection of homologous recombinants can be performed according to the methods described in Gene Targeting, A Practical Approach, IRL Press at Oxford University Press (1993); Biomanual Series 8, Gene Targeting, Generation of Mutant Mice Using ES Cells, Yodosha (1995); etc. Target vectors can be either replacement or insertion types. Selection methods that can be used include positive selection, promoter selection, negative selection, and poly(A) selection. Methods for selecting the desired homologous recombinant from the selected cell lines include Southern hybridization and PCR for genomic DNA.

[0020] In the present invention, "suspension culture" or "suspension culture method" refers to culturing cells or cell aggregates while maintaining a state in which they exist suspended in a culture medium, and to the method of culturing. In other words, suspension culture is performed under conditions that do not allow cells or cell aggregates to adhere to cultureware, etc., and culture performed under conditions that allow cells or cell aggregates to adhere to cultureware, etc. (adhesion culture or adhesion culture method) is not included in the category of suspension culture. In this case, cell adhesion refers to the formation of strong cell-substratum junctions between cells or cell aggregates and the cultureware, etc. More specifically, suspension culture refers to culture under conditions that do not allow strong cell-substratum junctions to form between cells or cell aggregates and the cultureware, etc., and "adhesion culture" refers to culture under conditions that allow strong cell-substratum junctions to form between cells or cell aggregates and the cultureware, etc. In cell aggregates cultured in suspension, cells adhere to each other through plane attachment. In cell aggregates cultured in suspension, cell-substrate bonds are rarely formed between the aggregate and the cultureware, or even if they are formed, their contribution is small. In some embodiments, cell aggregates cultured in suspension have endogenous cell-substrate bonds present within the aggregate, but cell-substrate bonds are rarely formed between the aggregate and the cultureware, or even if they are formed, their contribution is small. From this perspective, one form of "suspension culture" includes a culture method in which cell aggregates are fixed to a thin, needle-like device that serves as a scaffold for the cell aggregate and cultured in the device filled with culture medium. Examples of such culture methods include the use of the bio3D printer "Regenova (registered trademark)" manufactured by Cyfuse Co., Ltd., which was presented at the 136th Annual Meeting of the Pharmaceutical Society of Japan, 29AB-pm009. Surface adhesion between cells refers to surface adhesion between cells. More specifically, surface adhesion between cells refers to the proportion of the surface area of ​​a cell that is adhered to the surface of another cell, for example, 1% or more, preferably 3% or more, and more preferably 5% or more. Cell surfaces can be observed by staining with a membrane-staining reagent (e.g., DiI) or immunostaining for cell adhesion factors (e.g., E-cadherin or N-cadherin).

[0021] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and those skilled in the art can appropriately determine the appropriate vessel. Examples of such culture vessels include flasks, tissue culture flasks, 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, and roller bottles. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels that can be used include those whose surfaces have not been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as laminin, entactin, collagen, and gelatin, or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Non-cell-adhesive culture vessels can be used that have a surface artificially treated to reduce cell adhesion (e.g., ultrahydrophilic treatment with MPC polymer, low protein adsorption treatment, etc.). Rotational culture can also be performed using spinner flasks or roller bottles. The culture surface of the culture vessel can be flat or uneven. On the other hand, culture vessels used for adhesion culture include those whose surfaces have been artificially treated to improve adhesion to cells (for example, coating with extracellular matrices such as basement membrane preparations, laminin, entactin, collagen, gelatin, Matrigel, Synthemax, vitronectin, etc., or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment).

[0022] As used herein, a cell aggregate (cell mass or cell aggregate) is not particularly limited as long as it is a mass formed by adhesion of multiple cells. It may be a cell aggregate formed by the aggregation of cells dispersed in a medium, a cell aggregate derived from a colony formed by cell culture, or a cell aggregate newly formed by budding from another cell aggregate. Cell aggregates also include embryoid bodies, spheres, and spheroids. Preferably, in a cell aggregate, cells are adhered to each other via a surface. In some embodiments, cells may form cell-cell junctions or cell adhesions, such as adherens junctions, in part or all of the cell aggregate. Aggregates also include cell populations derived from the cell aggregate.

[0023] "Uniform aggregates" means that when multiple aggregates are cultured, the size of each aggregate is constant, and when the size of an aggregate is evaluated by the length of its maximum diameter, uniform aggregates mean that the variance of the maximum diameter is small. More specifically, this means that 75% or more of the aggregates in the entire population of aggregates are within ±100% of the average maximum diameter of the population of aggregates, preferably within ±50%, and more preferably within ±20% of the average.

[0024] "Forming uniform aggregates" refers to the formation of uniformly sized cell aggregates by "rapidly aggregating a certain number of dispersed cells" when cells are aggregated to form cell aggregates and cultured in suspension. In other words, if pluripotent stem cells are rapidly aggregated to form pluripotent stem cell aggregates, epithelial-like structures can be reproducibly formed in cells induced to differentiate from the formed aggregates. Specifically, pluripotent stem cells can be rapidly aggregated in serum-free medium to form cell aggregates with epithelial-like structures (SFEBq method (Serum-free Floating Culture of Embryoid Body-like Aggregates with Quick Reaggregation)). An experimental procedure for forming the aggregates is, for example, a small well plate (e.g., a well with a bottom area of ​​0.1 to 2.0 cm2 in terms of a flat bottom). 2 Examples of such methods include confining cells in a small space using a plate (such as a 96-well plate) or micropores, and aggregating cells by centrifuging them for a short period of time in a small centrifuge tube.

[0025] A small plate with wells, such as a 24-well plate (with an area of ​​1.88 cm2 in flat-bottom equivalent) 2 48-well plate (approximately 1.0 cm2 in flat-bottom equivalent area) 2 96-well plate (area equivalent to a flat bottom is 0.35 cm 2Examples of suitable well plates include a 384-well plate (approximately 6-8 mm in inner diameter) and a 96-well plate. A preferred example is a 96-well plate. The shape of the bottom of a small well plate when viewed from above can be polygonal, rectangular, elliptical, or circular, with a perfect circle being preferred. The shape of the bottom of a small well plate when viewed from the side is preferably a structure with a high outer periphery and a low inner recess, such as a U-bottom, V-bottom, or μ-bottom, with a U-bottom or V-bottom being preferred, and a V-bottom being most preferred. Cell culture dishes (e.g., 60-150 mm dishes, culture flasks) with an uneven or recessed bottom (e.g., EZSPHERE (Asahi Technoglass)) may also be used as small well plates. The bottom of a small well plate is preferably a non-cell-adhesive bottom, preferably a bottom coated with the aforementioned non-cell-adhesive material.

[0026] "Dispersion" refers to separating cells or tissues into small cell fragments (2 to 100 cells, preferably 50 cells or less) or single cells by a dispersion treatment such as enzymatic or physical treatment. A certain number of dispersed cells refers to a collection of a certain number of cell fragments or single cells. Methods for dispersing pluripotent stem cells include, for example, mechanical dispersion treatment, cell dispersion liquid treatment, and treatment with the addition of a cell protective agent. These treatments may also be performed in combination. Preferably, cell dispersion liquid treatment is performed first, followed by mechanical dispersion treatment. Methods for mechanical dispersion treatment include pipetting or scraping with a scraper.

[0027] As used herein, the term "tissue" refers to a structure of a cell population in which multiple types of cells with different morphologies and properties are arranged three-dimensionally in a specific pattern. As used herein, "retinal tissue" refers to a tissue in which at least multiple types of retinal cells, such as photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells, retinal pigment epithelial cells, and Muller cells, which constitute each retinal layer in a living retina, and their precursor cells, such as neural retinal progenitor cells or retinal progenitor cells, are arranged three-dimensionally in layers (however, in the case of retinal progenitor cells, other retinal cells may not be included). The retinal layer to which each cell belongs can be determined by known methods, such as the presence or absence of expression of a cell marker or the degree of expression. As used herein, "retinal tissue" includes retinal tissue obtained by inducing differentiation of pluripotent stem cells, or retinal tissue derived from a living body. Specifically, the term "retinal tissue" includes cell aggregates or parts thereof having epithelial tissue containing retinal progenitor cells and / or neural retinal progenitor cells formed on the surface of aggregates formed from pluripotent stem cells, which are obtained by suspension culture of the aggregates under appropriate differentiation-inducing conditions. As used herein, the term "cell aggregate containing retinal tissue" is not particularly limited as long as it is a cell aggregate containing the retinal tissue.

[0028] As used herein, the term "retinal layer" refers to any layer constituting the retina, specifically the retinal pigment epithelium layer and the neural retinal layer, which include the outer limiting membrane, photoreceptor layer (external nuclear layer), outer plexiform layer, inner nuclear layer, inner plexiform layer, ganglion cell layer, nerve fiber layer, and internal limiting membrane. In addition, in retinal tissue at an intermediate stage between "retinal tissue at an early stage of development" (described below) and mature retinal tissue, the neural retinal layer includes a layer containing neural retinal progenitor cells, called the neuroblastic layer in the neural retinal tissue. As used herein, the term "retinal progenitor cells" refers to precursor cells that can differentiate into any of the mature retinal cells that make up retinal tissue, including photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells, retinal pigment epithelial cells, and Muller cells. As used herein, the term "neural retinal progenitor cells" refers to cells that are destined to become the inner layer of the optic cup, and can be a precursor cell that can differentiate into any of the mature cells that make up the neural retinal layer (a retinal layer that contains retinal layer-specific neurons) that does not contain the retinal pigment epithelium.

[0029] Photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, ganglion cell progenitor cells, and retinal pigment epithelial progenitor cells refer to progenitor cells that are committed to differentiating into photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and retinal pigment epithelial cells, respectively. However, because the differentiation stages are continuous, it is difficult to clearly distinguish the boundary at which the differentiation stage transitions, for example, from photoreceptor progenitor cells to photoreceptor cells. Therefore, in this specification, references to photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, retinal pigment epithelial cells, etc. may also include the respective progenitor cells. Conversely, references to photoreceptor progenitor cells, horizontal cell progenitor cells, bipolar cell progenitor cells, amacrine cell progenitor cells, ganglion cell progenitor cells, and retinal pigment epithelial cell progenitor cells may also include the respective differentiated cells, i.e., photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, and retinal pigment epithelial cells, etc.

[0030] As used herein, "retinal layer-specific neurons" refer to neurons that constitute a retinal layer and are specific to that layer. Examples of retinal layer-specific neurons include bipolar cells, ganglion cells, amacrine cells, horizontal cells, and photoreceptors. Examples of photoreceptors include rod photoreceptor cells and cone photoreceptor cells. Examples of cone photoreceptors include S-cone photoreceptors that express S-opsin and receive blue light, L-cone photoreceptors that express L-opsin and receive red light, and M-cone photoreceptors that express M-opsin and receive green light. As used herein, the term "retinal cells" is a concept that encompasses the above-mentioned retinal pigment epithelial cells, Muller cells, photoreceptors, horizontal cells, bipolar cells, amacrine cells, ganglion cells and their precursor cells, retinal progenitor cells, neural retinal progenitor cells, retinal layer-specific neurons and precursor cells of retinal layer-specific neurons, etc.

[0031] The cells that constitute the above-mentioned retinal tissue can be detected or identified using retinal cell markers that are either expressed or not expressed in each cell as indicators. Retinal cell markers include genes and proteins that are predominantly expressed in retinal cells, and examples for each cell type are listed below. Alternatively, genes and proteins that are predominantly expressed in cells other than retinal cells can be used as negative markers. Negative markers for retinal cells, such as retinal progenitor cells, neural retinal progenitor cells, and photoreceptor progenitor cells, include NKX2.1, which is expressed in hypothalamic neuron progenitor cells but not in retinal progenitor cells, and SOX1, which is expressed in the hypothalamic neuroepithelium but not in the retina. Markers for retinal progenitor cells include RX (also called RAX) and PAX6. Markers of neural retinal progenitor cells include RX, PAX6 and CHX10. Markers of retinal layer-specific neurons include CHX10, PKCα, Goα, VSX1, and L7, which are expressed in bipolar cells; TUJ1 and BRN3, which are expressed in ganglion cells; calretinin and HPC-1, which are expressed in amacrine cells; and calbindin and LIM1, which are expressed in horizontal cells. Markers expressed in photoreceptor progenitors and photoreceptors include CRX, recoverin, BLIMP1, and OTX2. Markers expressed in rod photoreceptors and rod photoreceptor progenitors include NRL and rhodopsin. Therefore, for example, rod photoreceptors and rod photoreceptor progenitors can be identified by using the fact that CRX-positive cells are NRL-positive as an indicator. Markers expressed in cone photoreceptors, cone photoreceptor progenitors, and ganglion cells include RXR-γ. Markers expressed in cone photoreceptors and cone photoreceptor progenitors include TRβ2 and TRβ1. For example, cone photoreceptor progenitors can be identified by the coexpression of TRβ2 and CRX, or TRβ1 and CRX as an indicator. Cone photoreceptor progenitors can also be identified by the coexpression of RXR-γ and CRX, but not NRL as an indicator. OC1 (ONECUT1 / HNF6) and OC2 (ONECUT2) are factors required for the differentiation of cone photoreceptor progenitors and are transiently expressed during differentiation. They are also expressed in some ganglion cells, horizontal cells, and some amacrine cells. For example, when cone photoreceptor progenitors or progenitors of cone photoreceptors and horizontal cells that express OC1 and OC2 differentiate into cone photoreceptor progenitors or cone photoreceptors and horizontal cells, the expression of OC1 and OC2 decreases in the cone photoreceptor progenitors or cone photoreceptors, whereas the expression of OC1 and OC2 increases in the horizontal cells. Therefore, the differentiation efficiency of cone photoreceptor progenitors can be determined by measuring the expression level or ratio. Furthermore, retinal tissue at a stage where cone photoreceptors and cone photoreceptor progenitors have been induced but before the appearance of rod photoreceptor progenitors can be confirmed by checking whether CRX-positive cells are NRL-negative and TRβ2-positive, or NRL-negative and RXR-γ-positive. OTX2 is a marker expressed not only in photoreceptor progenitors and photoreceptors but also in bipolar cells. However, if OTX2-positive cells in neural retinal tissue are CHX10-negative and NRL-negative, they can be used as a marker for cone photoreceptor progenitors and cone photoreceptors. On the other hand, among OTX2-positive cells in neural retinal tissue, NRL-positive cells can be identified as rod photoreceptor progenitors and rod photoreceptors. Furthermore, examples of a marker for S-cone photoreceptors include S-opsin, a marker for L-cone photoreceptors including L-opsin, and a marker for M-cone photoreceptors including M-opsin. Markers commonly expressed in horizontal cells, amacrine cells, and ganglion cells include PAX6. Other markers of retinal cells contained in retinal tissue include RPE65, MITF, and PAX6, which are expressed in retinal pigment epithelial cells, and CRABP and CRALBP, which are expressed in Muller cells.

[0032] Dorsal and ventral markers in retinal tissue refer to genes and proteins expressed in tissues corresponding to the dorsal and ventral sides of the retina, respectively. Dorsal markers include markers such as TBX5, TBX3, TBX2, COUP-TF II, CYP26A1, CYP26C1, and ALDH1A1, which are expressed in the dorsalized region of the neural retina. Of these, COUP-TF II can be classified as the "most dorsal marker," and ALDH1A1 is also a factor whose expression level increases with proximity to the region. Ventral markers include markers such as VAX2, COUP-TF I, and ALDH1A3, which are expressed in the ventral region of the neural retina. It was not known that enhancing dorsalization signals in floating cultures of neural retinal tissue in vivo or neural retinal tissue differentiated from pluripotent stem cells increases the proportion of cone photoreceptors that are induced to differentiate. However, the inventors of the present application have found that: 1) It is possible to induce a region positive for the dorsal marker CYP26A1 (i.e., neural retinal tissue with elevated CYP26A1 expression) using dorsalization signaling substances, and to obtain neural retinal tissue with an increased proportion of cone photoreceptor progenitors and cone photoreceptors; and 2) We found that in neural retinal tissue in which COUP-TF II, the most dorsal marker present in the retina, is induced, the proportion of cone photoreceptor progenitors and cone photoreceptors is suppressed. On the other hand, it is known to those skilled in the art that cone photoreceptor precursor cells and cone photoreceptors do not appear in the retinal pigment epithelium, which is located further dorsally than 2). Therefore, by identifying CYP26A1, COUP-TF II, RPE65, or MITF as a marker and appropriately adjusting the strength of the dorsalization signal to an extent that dorsal markers are enhanced but the expression of the most dorsal marker is not enhanced, a neural retina enriched in cone photoreceptor progenitors and cone photoreceptors can be produced.

[0033] As used herein, the term "serum-free medium" refers to a medium that does not contain unconditioned or unpurified serum. As used herein, media containing purified blood-derived components or animal tissue-derived components (e.g., growth factors) are also included in the term serum-free medium as long as they do not contain unconditioned or unpurified serum. As used herein, "serum-free conditions" refers to conditions that do not contain unconditioned or unpurified serum, specifically conditions that use a serum-free medium. Here, the serum-free medium may contain a serum substitute. Examples of serum substitutes include those that appropriately contain albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, 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. Examples of such commercially available serum substitutes include Knockout TM Serum Replacement (manufactured by Life Technologies; hereinafter, also referred to as KSR), Chemically-defined Lipid concentrated (manufactured by Life Technologies), Glutamax TM (manufactured by Life Technologies), B27 (manufactured by Life Technologies), and N2 (manufactured by Life Technologies). Furthermore, the serum-free medium may contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, etc. as appropriate. To avoid the complicated preparation process, a serum-free medium containing an appropriate amount (e.g., about 0.5% to about 30%, preferably about 1% to about 20%) of commercially available KSR (Life Technologies) may be used as the serum-free medium (e.g., a medium containing 10% KSR and 450 μM 1-monothioglycerol in a 1:1 mixture of F-12 medium and IMDM medium). Examples of a medium equivalent to KSR include the medium disclosed in JP-A-2001-508302.

[0034] As used herein, the term "serum medium" refers to a medium containing unconditioned 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, pyruvic acid, buffers, inorganic salts, and the like. Furthermore, serum medium can be used in the process of maintaining retinal cells or retinal tissues produced by the present invention (Cell Stem Cell, 10(6), 771-775 (2012)).

[0035] The serum-free medium or serum-based medium may be supplemented with known growth factors, proteins, growth-promoting additives, chemical substances, etc. Examples of known growth factors and proteins include EGF, FGF, IGF, insulin, etc. Examples of growth-promoting additives include N2 supplement (N2, Invitrogen), B27 supplement (Invitrogen), etc. Examples of growth-promoting chemical substances include retinoids (e.g., retinoic acid or its derivatives), taurine, glutamine, etc. As used herein, "xeno-free" refers to conditions under which components derived from organisms different from the organism species of the cells to be cultured are excluded.

[0036] As used herein, "a medium containing substance X" or "in the presence of substance X" means a medium to which exogenous substance X has been added, a medium containing exogenous substance X, or the presence of exogenous substance X. In other words, if cells or tissues present in the medium express, secrete, or produce substance X endogenously, endogenous substance X is distinguished from exogenous substance X, and a medium that does not contain exogenous substance X is not considered to fall within the category of "a medium containing substance X," even if it contains endogenous substance X. For example, "medium containing a dorsalizing signaling substance" refers to a medium to which an exogenous dorsalizing signaling substance has been added or a medium containing an exogenous dorsalizing signaling substance, and "in the presence of a dorsalizing signaling substance" refers to the presence of an exogenous dorsalizing signaling substance. Also, "medium not containing a BMP signaling pathway inhibitor" refers to a medium to which an exogenous BMP signaling pathway inhibitor has not been added or a medium not containing an exogenous BMP signaling pathway inhibitor.

[0037] As used herein, the term "dorsalizing signaling substance" refers to a signaling substance that promotes differentiation into retinal tissue, which is formed dorsally during development. Examples of dorsalizing signaling substances include substances active in the BMP signaling pathway that transmit dorsalizing signals, substances active in the Wnt signaling pathway that induce BMP expression, and inhibitors of the Sonic Hedgehog (hereinafter sometimes referred to as SHH) signaling pathway that inhibit ventralizing signals. Substances that inhibit ventralizing signals are also referred to as ventralizing signaling inhibitors. The substance acting on the BMP signaling pathway is a substance that can enhance signal transduction mediated by BMP. Specific examples of the substance acting on the BMP signaling pathway include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies (agonist antibodies), and BMP partial peptides. A preferred example of the substance acting on the BMP signaling pathway is BMP4. The Wnt signaling pathway active substance is a substance that can enhance signal transduction mediated by Wnt. Examples of Wnt signaling pathway active substances include proteins belonging to the Wnt family (e.g., Wnt1, Wnt3A, Wnt7A, and Wnt2B), Wnt receptors, Wnt receptor agonists, anti-Wnt receptor antibodies, Wnt partial peptides, β-catenin signaling substances, and GSK3β inhibitors (e.g., 6-Bromoindirubin-3′-oxime (BIO), CHIR99021, and Kenpaullone). The SHH signaling pathway inhibitor is a substance capable of inhibiting signal transduction mediated by SHH. Examples of the SHH signaling pathway inhibitor include an SHH receptor antagonist, a dominant negative SHH molecule, an antibody against a substance acting on the SHH signaling pathway, and a soluble SHH receptor. Specific examples of the SHH signaling pathway inhibitor include GANT58, GANT61, Jervine, SANT-1, veratramine, cyclopamine, and cyclopamine-KAAD (GENES & DEVELOPMENT 16:2743-2748). A preferred example of the SHH signaling pathway inhibitor is cyclopamine-KAAD.

[0038] As used herein, the term "ventralizing signaling substance" refers to a signaling substance that promotes differentiation into retinal tissue, which is formed ventrally during development. Examples of ventralizing signaling substances include SHH signaling pathway agonists that transmit ventralizing signals, and BMP signaling pathway inhibitors that inhibit dorsalizing signals. Substances that inhibit dorsalizing signals are also referred to as dorsalizing signaling inhibitors. The substance acting on the SHH signaling pathway is a substance capable of enhancing signal transduction mediated by SHH. The substance acting on the SHH signaling pathway is not particularly limited as long as it is a substance capable of enhancing signal transduction mediated by SHH, and examples thereof include proteins belonging to the Hedgehog family (e.g., SHH and Ihh), SHH receptors, SHH receptor agonists, Purmorphamine, and SAG (Smoothened Agonist; N-Methyl-N'-(3-pyridinylbenzyl)-N'-(3-chlorobenzo[b]thiophene-2-carbonyl)-1,4-diaminocyclohexane). A preferred example of the substance acting on the SHH signaling pathway is SAG. The SHH signaling enhancing activity of SAG can be determined by methods well known to those skilled in the art, for example, a reporter gene assay focusing on the expression of the GLI1 gene (Oncogene (2007) 26, 5163-5168).

[0039] The BMP signaling pathway inhibitor is a substance capable of inhibiting signal transduction mediated by BMP. Examples of BMP include BMP2, BMP4, BMP7, and GDF7. Examples of BMP signaling pathway inhibitors include substances that directly act on BMP (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (e.g., antisense oligonucleotides, siRNAs, etc.), substances that inhibit the binding of BMP receptors (BMPRs) to BMP (e.g., BMP receptor antagonists, BMP dominant negatives, soluble BMP receptors, etc.), and substances that inhibit physiological activities resulting from signal transduction by BMP receptors. Examples of BMPRs include ALK2 and ALK3. Specific examples of BMP signaling pathway inhibitors include Noggin, Chordin, Dorsomorphin, and LDN193189 (4-[6-(4-piperazin-1-ylphenyl)pyrazolo[1,5-a]pyrimidin-3-yl]quinoline). A preferred example of a BMP signaling pathway inhibitor is LDN193189.

[0040] 2. A method for increasing the proportion of cone photoreceptors among the photoreceptors contained in retinal tissue (a method for producing retinal tissue rich in cone photoreceptors) When retinal tissue at the early developmental stage is cultured in a medium substantially free of dorsalizing signaling substances, increased expression of ventral markers is observed, and the emergence of photoreceptor precursors and / or cone photoreceptor precursors may be suppressed in the ventral marker ALDH1A3-positive region. In contrast, when cultured in a medium supplemented with a dorsalizing signaling substance, the ALDH1A3-positive region is reduced, and the emergence of photoreceptor precursors and / or cone photoreceptor precursors may be promoted. On the other hand, when cultured in a medium supplemented with a highly active or highly concentrated dorsalizing signaling substance, a strong dorsalizing signal induces the most dorsal marker, COUP-TF II, and ALDH1A1, whose expression increases toward the dorsalized region of the neural retina, thereby suppressing differentiation into cone photoreceptor precursors or cone photoreceptors. Therefore, in order to induce differentiation into retinal tissue with a high proportion of cone photoreceptor precursors or cone photoreceptors, it is necessary to appropriately adjust the strength of the dorsalizing signal, i.e., the degree of dorsalization.

[0041] That is, one aspect of the present invention is a method for increasing the proportion of cone photoreceptor precursors and cone photoreceptors among photoreceptor precursors and photoreceptors contained in retinal tissue, comprising the step of culturing retinal tissue from an early developmental stage to a stage where the appearance rate of cone photoreceptor precursors is maximized in a medium containing a dorsalization signaling substance at a concentration sufficient to suppress the expression of ventral markers. The concentration of the dorsalization signaling substance is preferably a concentration sufficient to not induce the expression of the most dorsal markers. Specific examples of the "ventral marker" include VAX2, COUP-TF I, and ALDH1A3.

[0042] The "most dorsal marker" refers to a marker expressed in cells present in the most dorsal region of retinal tissue during development, and can also be considered a marker whose expression is increased by excessive or relatively strong dorsalizing signals. Specific examples of the most dorsal marker in retinal tissue include RPE65, MITF, and COUP-TF II, which are expressed in cells differentiated into retinal pigment epithelium or its precursor cells by excessive dorsalizing signals, and preferably COUP-TF II, which is expressed in neural retinal tissue by relatively strong dorsalizing signals. Here, the "concentration sufficient to suppress the expression of a ventral marker" can be easily determined by those skilled in the art. For example, using techniques such as immunostaining using antibodies against the ventral marker, regions in retinal tissue at any stage from the stage at which photoreceptor precursor cells first appear to the stage at which the rate of appearance of cone photoreceptor precursor cells is maximized, for example, at the stage at which the rate of appearance of cone photoreceptor precursor cells is maximized, where the expression of the ventral marker is observed compared to when the dorsalizing signaling substance is not added, can be analyzed using image analysis software such as ImageJ. The concentration of the dorsalizing signaling substance that increases the region where the expression is suppressed can be determined as the appropriate concentration for increasing the proportion of cone photoreceptors. That is, the concentration of the dorsalizing signaling substance to be added can be determined so that the region in which the expression of the ventral marker is observed in neural retinal tissue containing photoreceptor precursor cells and / or photoreceptors is 50% or less, preferably 20% or less, more preferably 1% or less, and even more preferably 0.01% or less. Alternatively, retinal tissue that has been ventralized by a ventralizing signaling substance, a BMP signaling pathway inhibitor, and in which ventral markers such as ALDH1A3 are highly expressed can be used as an indicator, and the concentration of the dorsalizing signaling substance to be added can be determined so that the gene expression level of ALDH1A3 is 50% or less, preferably 20% or less, and more preferably 5% or less compared to this.

[0043] Furthermore, a "concentration that does not induce the most dorsal marker" can be determined appropriately by one skilled in the art. For example, using techniques such as immunostaining using an antibody against the most dorsal marker, the region in which expression of the most dorsal marker is observed in retinal tissue at the stage where the appearance rate of cone photoreceptor precursor cells is maximized can be analyzed using image analysis software such as ImageJ, and the concentration of the dorsalization signaling substance can be determined appropriately so that the region in which induced expression of the most dorsal marker is not observed (here, "no induced expression" means that the expression level is 1 / 5 or less, preferably 1 / 10 or less, and more preferably 1 / 50 or less of the expression level in the most dorsal neural retinal tissue in the living body) accounts for 50% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more of the neural retinal tissue.

[0044] One aspect of the present invention is a method for increasing the proportion of photoreceptor precursors and cone photoreceptors among photoreceptors contained in retinal tissue, the method comprising the step of culturing retinal tissue from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursors is maximized in a medium containing a dorsalization signaling substance at a concentration sufficient to promote the expression of dorsal markers. Those skilled in the art can easily determine the "concentration sufficient to promote expression of dorsal markers." For example, it can be easily determined by analyzing the expression levels of the proteins or genes (mRNA) of the dorsal markers. Specifically, various concentrations of dorsalization signaling substances are added to the medium, and the concentration at which the dorsal expression level or gene expression level is highest can be determined by immunostaining or quantitative PCR. For example, the concentration of the dorsalization signaling substance may be determined so that it is at a concentration at which the expression of CYP26A1 and / or CYP26C1 is most strongly induced. Specifically, the concentration is such that the expression level of CYP26A1 is at least 1.2-fold, preferably at least 1.5-fold, and more preferably at least 2-fold higher than in retinal tissue ventrally transformed with the above-mentioned BMP signaling pathway inhibitor and in which CYP26A1 expression has been suppressed. However, when determining the concentration of the dorsalization signaling substance based on the expression level of CYP26A1 and / or CYP26C1, it is preferable to determine the concentration using a medium substantially free of retinoic acids, since retinoic acids such as all-trans retinoic acid and 9-cis retinoic acid excessively induce the expression of these genes regardless of the degree of dorsalization, making it difficult to determine the concentration of the dorsalization signaling substance.

[0045] On the other hand, ALDH1A1 is weakly expressed in CYP26A1-positive regions, and its expression level increases continuously with increasing dorsal location. This means that ALDH1A1 is more highly expressed in the COUP-TF II-positive region, the most dorsal region of neural retinal tissue, than in CYP26A1- or CYP26C1-positive regions. Therefore, it is desirable to limit ALDH1A1 expression. Specifically, at the stage where the emergence rate of cone photoreceptor precursor cells reaches its peak, the frequency of ALDH1A1-positive cells is approximately 1% or less. Therefore, it is desirable to adjust the concentration of the dorsalization signaling substance so that ALDH1A1 expression is sufficiently low at that stage. Here, the "concentration at which ALDH1A1 expression is sufficiently low" can be determined by those skilled in the art. For example, retinal tissue at the stage where the incidence of cone photoreceptor precursor cells is at its maximum can be analyzed using a commonly performed immunostaining technique, such as an antibody against ALDH1A1, and image analysis software, such as ImageJ, to determine the ALDH1A1-expressing region, which accounts for 50% or less, preferably 20% or less, more preferably 10% or less, and even more preferably 1% or less of the neural retinal tissue in the retinal tissue. Alternatively, the gene expression level in such retinal tissue can be measured by quantitative PCR or other methods. Specifically, the addition of BMP4 at a relatively high concentration, for example, 0.45 nM to 1.35 nM, induces excessive ALDH1A1 expression. Therefore, the ALDH1A1 expression level can be determined to be 30%, more preferably 15%, and even more preferably 10% or less compared to when differentiation is induced with the addition of 1.35 nM BMP4.

[0046] In one embodiment of the present invention, the "dorsalizing signal transduction substance at a concentration that suppresses the expression of ventral markers and does not induce the expression of the most dorsal marker" refers to a substance that, in retinal tissue at a stage where the appearance rate of cone photoreceptor precursor cells among cells contained in the retinal tissue is at its maximum, results in a ratio of the number of cells expressing OC2 to the total number of cells contained in the neural retinal tissue of about 20% to 70%, preferably about 30% to 70%, more preferably about 50% to 65% compared to when no dorsalizing signal transduction substance is added (for example, when 0.15 nM BMP4 or 500 nM Cyclopamine-KAAD is added, about 60% to 65% of cells express OC2), or about 30% to 60%, preferably about 40% to 50% compared to when the BMP signal transduction pathway inhibitor is added (for example, when 0.15 nM BMP4 or 500 nM Cyclopamine-KAAD is added, about 60% to 65% of cells express OC2). Examples of the dorsalizing signal transduction substance include a concentration at which the expression level of OC2 protein in OC2-positive cells is suppressed to an average of about 20% to 70%, preferably about 30% to 70%, and more preferably about 30% to 40% compared to when no dorsalizing signal transduction substance is added and / or when the BMP signaling pathway inhibitor is added (for example, about 34% to 36% when 0.15 nM BMP4 or 500 nM Cyclopamine-KAAD is added). Here, the proportion of OC2-positive cells in neural retinal tissue can be determined by performing immunostaining or the like using an anti-OC2 antibody, DAPI, or the like, which is routinely performed by those skilled in the art, measuring the number of OC2-positive cells, the number of DAPI-positive cells, or the like, and identifying the proportion of OC2-positive cells in neural retinal tissue. The expression level of OC2 protein can be determined by performing immunostaining or the like using an anti-OC2 antibody, DAPI, or the like, which is routinely performed by those skilled in the art, and analyzing the area stained with the anti-OC2 antibody using image analysis software such as ImageJ, thereby identifying the signal intensity of OC2-positive cells. Alternatively, the proportion of OC2-positive cells in neural retinal tissue and / or the expression level of OC2 protein in OC2-positive cells can be determined by routine flow cytometry analysis using an antibody against OC2 protein. Furthermore, those skilled in the art can identify the "stage at which photoreceptor progenitor cells first appear" by immunostaining with a photoreceptor progenitor marker and / or a cone photoreceptor progenitor marker, nuclear staining with DAPI, or the like. Specifically, for example, retinal tissue undergoing differentiation is fixed with paraformaldehyde or the like, and then frozen sections are prepared. The frozen sections are stained with, for example, a CRX antibody, a TRβ2 antibody, an RXR-γ antibody, or the like, and simultaneously cell nuclei are stained with DAPI or the like, and the stage at which photoreceptor progenitor cells and / or cone photoreceptor progenitor cells first appear in the retinal tissue can be identified.

[0047] Furthermore, the above-mentioned "stage at which the appearance rate of cone photoreceptor progenitor cells reaches a maximum" can be identified by a person skilled in the art using immunostaining with a photoreceptor progenitor marker and / or a cone photoreceptor progenitor marker and nuclear staining with DAPI, or the like. Specifically, for example, retinal tissue undergoing differentiation is collected at regular intervals (e.g., 1-20 days) (e.g., 40, 50, 60, 70, and 80 days after the start of culture), fixed with paraformaldehyde, and then frozen sections are prepared. The frozen sections are stained with, for example, CRX, TRβ2, or RXR-γ antibodies, and cell nuclei are simultaneously stained with DAPI or other antibodies. The proportion of cone photoreceptor progenitor cells (i.e., cells expressing CRX and RXR-γ, or CRX and TRβ2) relative to the total number of cells contained in the neural retina is then determined. The proportion of cone photoreceptor progenitor marker-positive cells relative to the total number of cells, i.e., the appearance rate, is determined at multiple time points. The period with the highest appearance rate of cone photoreceptor progenitor marker-positive cells can be identified as the "stage at which the appearance rate of cone photoreceptor progenitor cells reaches its maximum." Furthermore, BrdU, EdU, or the like, which is incorporated into cells in the cell proliferation phase (here, retinal progenitor cells or neural retinal progenitor cells with the ability to proliferate), can be added to the culture medium for a specific period (e.g., 1 to 7 days), and the proportion of cells that have incorporated BrdU, EdU, or the like that have differentiated into cells expressing the above-mentioned cone photoreceptor progenitor markers can be measured by immunostaining or other methods well known to those skilled in the art. The relationship between this proportion and the differentiation stage (e.g., the time (stage) at which this proportion is highest) can be determined, thereby identifying the "stage at which the appearance rate of cone photoreceptor progenitors reaches its maximum." Specifically, the identification can be performed, for example, by the following procedure: 1) adding BrdU or EdU to a culture medium for culturing retinal tissue at any differentiation stage for one day at any time and culturing the tissue (for example, adding BrdU every other day and culturing for one day, for example, 40 to 41 days, 41 to 42 days, and 42 to 43 days after the start of culture, and repeating this process until 80 days after the start of culture), and measuring the percentage of CRX and RXR-γ positive cells or the percentage of CRX and TRβ2 positive cells among BrdU or EdU positive cells in the retinal tissue recovered immediately thereafter; 2) comparing the measurement results and identifying the retinal tissue in which the proportion of CRX and RXR-γ positive cells or the proportion of CRX and TRβ2 positive cells is highest among BrdU or EdU positive cells; and 3) A process of identifying the period (e.g., 1 day) during which BrdU or EdU is added to the culture medium when culturing retinal tissue that results in the highest increase in the proportion of CRX and RXR-γ positive cells or the highest increase in the proportion of CRX and TRβ2 positive cells among BrdU or EdU positive cells as the "stage at which the appearance rate of cone photoreceptor progenitor cells reaches its maximum." Specifically, the "stage at which the appearance rate of cone photoreceptor precursor cells reaches a maximum" corresponds to 30 to 50 days, preferably 30 to 40 days, after the appearance of cone photoreceptor precursor cells is observed.

[0048] As used herein, the term "early developmental stage" refers to a stage in which retinal progenitor cells have appeared but ganglion cells have not. At this stage, neural retinal progenitor cells may also have appeared. That is, at this stage, RX (RAX)-positive and PAX6-positive cells (and may also be CHX10-positive cells) are included, but TUJ1-positive cells, BRN3-positive cells, and cells positive for at least two of the markers TUJ1, BRN3, and PAX6 are not included. "Retinal tissue at an early developmental stage" includes retinal progenitor cells and / or neural retinal progenitor cells, i.e., cells that can differentiate into photoreceptors and ganglion cells, and is not particularly limited as long as it does not contain ganglion cells, and may also include ciliary marginal structures. Retinal tissue in the early developmental stage corresponds to 22 days (d22) to 33 days (d33) after the start of suspension culture when produced in accordance with raw material production methods 5 to 7 described below, and corresponds to 12 days (d12) to 27 days (d27) after the start of suspension culture when produced in accordance with raw material production methods 1 to 4. "Retinal tissue at an early stage of development" can be identified by confirming the expression status of retinal progenitor cell markers, neural retinal progenitor cell markers, and ganglion cell markers. Retinal tissue in the early stages of development includes tissue that corresponds to the "optic vesicle" or the "earliest stage of the optic cup" retinal tissue that has undergone some differentiation from the optic vesicle and contains neural retinal progenitor cells that are RX-positive, PAX6-positive, and CHX10-positive but does not contain ganglion cells. In the present invention, the step of culturing in a medium containing a dorsalization signaling substance (step (1)) may be carried out at any time between the "early developmental stage" and the "stage at which the appearance rate of cone photoreceptor precursor cells reaches its maximum," and there are no limitations on the time or duration for starting step (1) as long as it is within this period. Step (1) is preferably started within about 40 days from the early developmental stage, more preferably within 20 days, and even more preferably at the early developmental stage.

[0049] The retinal tissue at the early stage of development includes retinal tissue at a differentiation stage that contains retinal progenitor cells or neural retinal progenitor cells induced to differentiate from pluripotent stem cells and that has not yet produced ganglion cells.Furthermore, the retinal tissue at the early stage of development may contain cells that can differentiate into photoreceptors or neurons. The method for producing retinal tissue at the early developmental stage is not particularly limited, and may be either suspension culture or adherent culture, such as those described in WO2013 / 077425 (and US2014 / 341864), WO2015 / 025967 (and US2016 / 251616), WO2016 / 063985 (and US2017 / 313976), WO2016 / 063986 (and US2017 / 313981), and WO2017 / 183732. Examples of such a method include those described in non-patent literature: Proc Natl Acad Sci US A. 111(23): 8518-8523(2014), Nat Commun. 5:4047(2014), Stem Cells.(2017):35(5), 1176-1188, etc. Specifically, examples include aggregates containing retinal progenitor cells or neural retinal progenitor cells, which are obtained by suspension culture of cell aggregates (cell masses) prepared from pluripotent stem cells such as ES cells or iPS cells using the SFEBq method (see Nat Commun. 6:6286 (2015)) in the presence of a differentiation inducer such as BMP4. Furthermore, retinal tissue in the early developmental stage may be cells derived from a cell population containing neuroepithelial cells, and the cell population may be obtained by inducing differentiation from pluripotent stem cells such as ES cells or iPS cells, or by collecting stem cells present in the adult retina and inducing their differentiation.

[0050] Specifically, retinal tissue in the early developmental stage is retinal tissue that contains retinal progenitor cells that are positive for retinal progenitor cell markers (preferably RX positive and PAX6 positive) or neural retinal progenitor cells that are positive for neural retinal progenitor cell markers (preferably CHX10 positive, PAX6 positive and RX positive) in an amount of 30% or more, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 99% or more of the total number of cells contained in the retinal tissue, and in which the proportion of ganglion cells that are positive for ganglion cell markers (preferably BRN3 positive) is 40% or less of the total number of cells, preferably 20% or less, 10% or less, 5% or less, more preferably 1% or less, even more preferably 0.1% or less, and even more preferably 0.01% or less.

[0051] The medium used to culture retinal tissue at an early stage of development in the presence of a dorsalization signaling substance is not particularly limited, as long as it does not contain a substance that inhibits the effect of the dorsalization signaling substance in an amount sufficient to inhibit said effect. Commercially available cell culture media with appropriate additives added as needed can be used. Preferably, the medium is one that can maintain the continuous epithelial structure of the retinal tissue. Specific examples of media that can be used include media supplemented with Wnt2b, Neurobasal medium, and media containing Neurobasal medium, and Neurobasal medium supplemented with Wnt2b is also acceptable. Alternatively, a medium for maintaining continuous epithelial tissue, as described below, can be used.

[0052] As used herein, the continuous epithelial tissue maintenance medium contains at least one of a methyl group donor or a methyl group donor substrate at a concentration capable of inhibiting cell differentiation of retinal progenitor cells or neural retinal progenitor cells, and a neurite outgrowth inhibitor at a concentration capable of inhibiting neurite outgrowth. Preferably, the continuous epithelial tissue maintenance medium contains a methyl group donor or a methyl group donor substrate at a concentration capable of inhibiting cell differentiation of retinal progenitor cells or neural retinal progenitor cells, and a neurite outgrowth inhibitor at a concentration capable of inhibiting neurite outgrowth. In vivo, methyl groups are transferred from methyl donors to proteins containing DNA or histones by methyltransferase. As used herein, a methyl donor refers to a substance (methyl donor) capable of donating a methyl group for transfer to DNA or proteins containing histones. Furthermore, as used herein, a substrate for a methyl donor refers to a substrate required for the biosynthesis of the methyl donor. Specifically, examples of methyl donors include S-adenosylmethionine (also referred to as SAM), and examples of methyl donor substrates include methionine, S-methyl-5'-thioadenosine (sometimes abbreviated as MTA), and homocysteine ​​(sometimes abbreviated as Hcy). In the present invention, methionine is preferably used.

[0053] To determine whether a substance inhibits differentiation of retinal progenitor cells or neural retinal progenitor cells, for example, a substance to be evaluated can be applied to retinal tissue at an early stage of development, containing retinal progenitor cells or neural retinal progenitor cells, and then cultured for a certain period of time. The proportion of retinal progenitor cells or neural retinal progenitor cells in the retinal tissue can be determined, or the proportion of differentiated cells or differentiated cells that have ceased to proliferate can be determined. Specifically, for example, after a certain period of time (e.g., 5 to 50 days) following the appearance of ganglion cells, the rate of decrease in retinal progenitor cells or neural retinal progenitor cells, the rate of increase in differentiated cells, cells that have ceased to proliferate, or cells expressing bHLH transcription factors required for terminal differentiation in the retinal tissue, or the expression level of bHLH transcription factors can be determined by immunostaining, quantitative PCR, or other methods. Retinal progenitor cell or neural retinal progenitor cell markers that can be used include, for example, a combination of RX and PAX6, or a combination of RX, PAX6, and CHX10. Furthermore, examples of markers for differentiated cells contained in retinal tissue, markers for differentiated progenitor cells, or bHLH transcription factor markers required for terminal differentiation include BRN3, CRX, HuNu, Cath5, NeuroM, NGN1, NGN2, ISLET-1 (also known as ISL1), and OLIG2. Examples of markers for cells that have stopped proliferation due to cell differentiation include p53, p27, and p21.

[0054] When methionine is used as the substrate for the methyl group donor, the methionine concentration in the medium for continuous epithelial tissue maintenance is usually greater than 17.24 mg / L (preferably 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, or 30 mg / L or more). The upper limit of the methionine concentration in the medium for continuous epithelial tissue maintenance is not particularly limited as long as continuous epithelial tissue maintenance is achieved, but is usually 100 mg / L or less (preferably 75 mg / L or less). The range of methionine concentration in the medium for maintaining continuous epithelial tissue is, for example, more than 17.24 mg / L and not more than 100 mg / L, preferably 23.62 mg / L to 75 mg / L, 25 mg / L to 75 mg / L, 25.75 mg / L to 75 mg / L, 26 mg / L to 75 mg / L, 26.81 mg / L to 75 mg / L, 27 mg / L to 75 mg / L, or 30 mg / L to 75 mg / L. When a methyl group donor other than methionine or its substrate is used, it is preferably used at a concentration that exhibits the same inhibitory effect on retinal progenitor cell or neural retinal progenitor cell differentiation as the above-mentioned concentrations of methionine.

[0055] As used herein, a neurite outgrowth inhibitor refers to a substance that inhibits the neurite outgrowth of ganglion cells, and specific examples include neurite outgrowth-inhibitory hormones such as glucocorticoids, and neurite outgrowth-inhibitory proteins such as semaphorin, Nogo, Mag, OMgp protein, and chondroitin sulfate proteoglycan. Examples of glucocorticoids include corticosterone, cortisol, and cortisone. The neurite outgrowth inhibitor is preferably a glucocorticoid, more preferably corticosterone.

[0056] The inhibitory effect on neurite outgrowth can be evaluated, for example, by culturing retinal tissue or ganglion cells contained in retinal tissue in the presence of the substance to be evaluated as an adherent culture, and measuring the length of the extended neurites using image analysis software (e.g., Image J).

[0057] When corticosterone is used as the neurite outgrowth inhibitor, the concentration of corticosterone in the continuous epithelial tissue maintenance medium is a concentration that inhibits neurite outgrowth of ganglion cells and other cells contained in retinal tissue, and is typically 0.1 nM or higher (preferably 1 nM or higher, 5 nM or higher, 10 nM or higher, 50 nM or higher, or 100 nM or higher). The upper limit of the corticosterone concentration in the continuous epithelial tissue maintenance medium is not particularly limited as long as continuous epithelial tissue maintenance is achieved, but is typically 10 μM or lower (preferably 5 μM or lower, 1 μM or lower). The range of corticosterone concentration in the continuous epithelial tissue maintenance medium is, for example, 0.1 nM to 10 μM, preferably 1 nM to 5 μM. When a neurite outgrowth inhibitor other than corticosterone is used, it is preferably used at a concentration that exerts the same neurite outgrowth inhibitory effect as corticosterone at the above-mentioned concentrations.

[0058] In one embodiment, the medium for maintaining continuous epithelial tissue contains methionine and corticosterone at the above concentrations. In one embodiment, the medium for maintaining continuous epithelial tissue contains more than 17.24 mg / L (preferably, 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, or 30 mg / L or more) of methionine and 0.1 nM or more (preferably, 1 nM or more, more preferably 5 nM or more, even more preferably 10 nM or more, even more preferably 50 nM or more, and even more preferably 100 nM or more) of corticosterone.

[0059] The medium for maintaining continuous epithelial tissue may further contain an acidic amino acid, an antioxidant and a retinal neuronal cell protective substance, but the concentrations of each are preferably lower.

[0060] As used herein, acidic amino acids specifically include glutamic acid and aspartic acid, each of which includes the L- and D-isomers. As used herein, L-glutamic acid is referred to as L-glutamic acid, L-aspartic acid as L-aspartic acid, D-glutamic acid as D-glutamic acid, and D-aspartic acid as D-aspartic acid. As used herein, when there is no need to distinguish between the D- and L-isomers, they are referred to as "glutamic acid" or "aspartic acid."

[0061] The concentration of L-glutamic acid in the medium for maintaining continuous epithelial tissue is preferably less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and especially preferably 0.1 μM or less). In one embodiment, the concentration of glutamic acid in the medium for maintaining continuous epithelial tissue is preferably less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and especially preferably 0.1 μM or less).

[0062] The concentration of L-aspartic acid in the continuous epithelial tissue maintenance medium is preferably less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and particularly preferably 0.1 μM or less). In one embodiment, the concentration of aspartic acid contained in the continuous epithelial tissue maintenance medium is preferably less than 50 μM (more preferably 10 μM or less, even more preferably 1 μM or less, and even more preferably 0.1 μM or less).

[0063] In a preferred embodiment, the medium for maintaining continuous epithelial tissue contains at least one (preferably both) of more than 17.24 mg / L of methionine (preferably 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, or 30 mg / L or more) and 0.1 nM or more (preferably 1 nM or more, more preferably 5 nM or more, even more preferably 10 nM or more, even more preferably 50 nM or more, and even more preferably 100 nM or more) of corticosterone, and the concentration of L-glutamic acid is less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and particularly preferably 0.1 μM or less). In a further preferred embodiment, the continuous epithelial tissue maintenance medium contains at least one (preferably both) of more than 17.24 mg / L of methionine (preferably 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, or 30 mg / L or more) and 0.1 nM or more (preferably 1 nM or more, 5 nM or more, 10 nM or more, 50 nM or more, or 100 nM or more) of corticosterone, the L-glutamic acid concentration is less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, especially preferably 0.1 μM or less), and the L-aspartic acid concentration is less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, especially preferably 0.1 μM or less).

[0064] As used herein, antioxidants include glutathione, catalase, superoxide dismutase, alpha tocopherol, cysteine, etc. In one embodiment, the concentration of at least one, preferably multiple, and more preferably all antioxidants selected from the group consisting of glutathione, catalase, superoxide dismutase, alpha tocopherol, and cysteine ​​in the medium for continuous epithelial tissue maintenance is within the following range: Glutathione: 100 ng / mL or less (preferably 10 ng / mL or less, more preferably 1 ng / mL or less, even more preferably 0.1 ng / mL or less); Catalase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Superoxide dismutase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Alpha-tocopherol: 50 nM or less (preferably 5 nM or less, more preferably 0.5 nM or less, and even more preferably 0.05 nM or less); and Cysteine: 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less). In one embodiment, the concentration of at least one, preferably multiple, and more preferably all antioxidants selected from the group consisting of glutathione, catalase, superoxide dismutase, and alpha-tocopherol in the medium for maintaining continuous epithelial tissue is a concentration at which no antioxidant effect is observed to the extent that it affects the continuous epithelial structure, and the concentration of cysteine ​​is 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less). When other antioxidants are contained, their concentration is preferably a concentration at which they exert an antioxidant effect equivalent to that of the antioxidant at the above-mentioned concentration or less, or a concentration at which no antioxidant effect is observed. The antioxidant effect can be evaluated, for example, by directly measuring some active oxygen species similar to free radicals in the presence of a spin trap using an electron spin resonance (ESR) device. Antioxidant activity can also be evaluated by various other methods for measuring reactive oxygen species (e.g., measuring the amount of lipid peroxides produced by reactive oxygen species, or the amounts of 8-hydroxydeoxyguanosine and 8-nitroguanosine, which are used as oxidative stress markers). Commercially available assay kits (Cosmo Bio, Dojindo Laboratories, Thermo Fisher Scientific, etc.) can also be used to measure the amount of reactive oxygen species.

[0065] As used herein, examples of retinal neuronal protective substances include progesterone. In one embodiment, the concentration of progesterone in the continuous epithelial tissue maintenance medium is 100 nM or less, preferably 50 nM or less, more preferably 20 nM (or 6.3 μg / mL (20.033708 nM)) or less, even more preferably 10 nM or less, and even more preferably 3 nM or less. In one embodiment, the concentration of progesterone in the continuous epithelial tissue maintenance medium is a concentration at which no protective effect on ganglion cells is observed. When other retinal neuronal protective substances are contained, they are preferably at a concentration below which they exert a retinal neuronal protective effect equivalent to that of progesterone at the above-mentioned concentration, or at a concentration at which no protective effect on retinal neuronal cells is observed. The protective effect on retinal neuronal cells can be confirmed, for example, by identifying the proportion of ganglion cells contained in retinal tissue or the proportion of ganglion cells positive for cleaved caspase 3, known as an apoptosis marker, and determining whether these proportions increase or decrease. If the substance being evaluated exhibits a protective effect on ganglion cells, the proportion of ganglion cells in retinal tissue exposed to the substance for a certain period of time will increase compared to when the substance is not applied, and conversely, the proportion of cleaved caspase 3-positive ganglion cells will decrease. The proportion of ganglion cells can be identified using immunohistochemical staining with antibodies against the above-mentioned ganglion cell markers (e.g., BRN3), DAPI staining, PI staining, Hoechst staining, etc.

[0066] In a preferred embodiment, the medium for maintaining continuous epithelial tissue contains more than 17.24 mg / L (preferably, 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, 30 mg / L or more) of methionine and 0.1 nM or more (preferably, 1 nM or more, more preferably, 5 nM or more, even more preferably, 10 nM or more, even more preferably, 50 nM or more, even more preferably, 100 nM or more). the concentration of L-glutamic acid is less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and especially preferably 0.1 μM or less); and the concentrations of at least one, preferably more than one, and more preferably all, compounds selected from the group consisting of L-aspartic acid, glutathione, catalase, superoxide dismutase, alpha tocopherol, cysteine, and progesterone are within the following ranges: L-aspartic acid: less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, particularly preferably 0.1 μM or less); Glutathione: 100 ng / mL or less (preferably 10 ng / mL or less, more preferably 1 ng / mL or less, even more preferably 0.1 ng / mL or less); Catalase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Superoxide dismutase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Alpha-tocopherol: 50 nM or less (preferably 5 nM or less, more preferably 0.5 nM or less, even more preferably 0.05 nM or less); Cysteine: 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less); and Progesterone: 100 nM or less (preferably 50 nM or less, more preferably 20 nM or less (or 6.3 μg / mL (20.033708 nM) or less), even more preferably 10 nM or less, even more preferably 3 nM or less).

[0067] The continuous epithelial tissue maintenance medium may further contain hypoxanthine, thymidine and vitamin B12, although lower concentrations of each are preferred. In one aspect, the hypoxanthine concentration in the medium for maintaining continuous epithelial tissue is, for example, less than 15 μM (preferably 7.5 μM or less, more preferably 3.75 μM or less, even more preferably 1 μM or less, and even more preferably 0.1 μM or less (e.g., 0 μM)). In one aspect, the thymidine concentration in the medium for maintaining continuous epithelial tissue is less than 1.5 μM (preferably 0.75 μM or less, more preferably 0.375 μM or less, even more preferably 0.1 μM or less, and even more preferably 0.01 μM or less (e.g., 0 μM)). In one embodiment, the vitamin B12 concentration in the medium for maintaining continuous epithelial tissue is less than 0.5 μM (preferably 0.253 μM or less, more preferably 0.129 μM or less, and even more preferably 0.005 μM or less). In a preferred embodiment, the concentrations of two or three compounds selected from the group consisting of hypoxanthine, thymidine and vitamin B12 in the continuous epithelial tissue maintenance medium are within the above-mentioned ranges.

[0068] In a preferred embodiment, the medium for maintaining continuous epithelial tissue contains at least one (preferably both) of methionine at more than 17.24 mg / L (preferably 23.62 mg / L or more, 25 mg / L or more, 25.75 mg / L or more, 26 mg / L or more, 26.81 mg / L or more, 27 mg / L or more, 30 mg / L or more) and corticosterone at 0.1 nM or more (preferably 1 nM or more, more preferably 5 nM or more, even more preferably 10 nM or more, even more preferably 50 nM or more, even more preferably 100 nM or more), and the concentration of L-glutamic acid is less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, particularly preferably 0.1 μM or less). and the concentration of at least one, preferably more than one, more preferably all compounds selected from the group consisting of L-aspartic acid, glutathione, catalase, superoxide dismutase, alpha tocopherol, cysteine, progesterone, hypoxanthine, thymidine, and vitamin B12 is within the following ranges: L-aspartic acid: less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and particularly preferably 0.1 μM or less); Glutathione: 100 ng / mL or less (preferably 10 ng / mL or less, more preferably 1 ng / mL or less, even more preferably 0.1 ng / mL or less); Catalase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Superoxide dismutase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Alpha-tocopherol: 50 nM or less (preferably 5 nM or less, more preferably 0.5 nM or less, even more preferably 0.05 nM or less); Cysteine: 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less); Progesterone: 100 nM or less (preferably 50 nM or less, more preferably 20 nM or less (or 6.3 μg / mL (20.033708 nM) or less), even more preferably 10 nM or less, even more preferably 3 nM or less) Hypoxanthine: less than 15 μM (preferably 7.5 μM or less, more preferably 3.75 μM or less, even more preferably 1 μM or less, even more preferably 0.1 μM or less (e.g., 0 μM)); Thymidine: less than 1.5 μM (preferably 0.75 μM or less, more preferably 0.375 μM or less, even more preferably 0.1 μM or less, even more preferably 0.01 μM or less (e.g., 0 μM)); and Vitamin B12: less than 0.5 μM (preferably 0.253 μM or less, more preferably 0.129 μM or less, and even more preferably 0.005 μM or less).

[0069] In one preferred embodiment, the medium for maintaining continuous epithelial tissue has the following composition: Methionine: greater than 17.24 mg / L (preferably greater than or equal to 23.62 mg / L, greater than or equal to 25 mg / L, greater than or equal to 25.75 mg / L, greater than or equal to 26 mg / L, greater than or equal to 26.81 mg / L, greater than or equal to 27 mg / L, greater than or equal to 30 mg / L); Corticosterone: 0.1 nM or more (preferably 1 nM or more, more preferably 5 nM or more, even more preferably 10 nM or more, even more preferably 50 nM or more, even more preferably 100 nM or more); L-glutamic acid: less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and particularly preferably 0.1 μM or less); L-aspartic acid: less than 50 μM (more preferably 25 μM or less, even more preferably 12.5 μM or less, even more preferably 1 μM or less, and particularly preferably 0.1 μM or less); Hypoxanthine: less than 15 μM (preferably 7.5 μM or less, more preferably 3.75 μM or less, even more preferably 1 μM or less, even more preferably 0.1 μM or less (e.g., 0 μM)); Thymidine: less than 1.5 μM (preferably 0.75 μM or less, more preferably 0.375 μM or less, even more preferably 0.1 μM or less, even more preferably 0.01 μM or less (e.g., 0 μM)); and Vitamin B12: less than 0.5 μM (preferably 0.253 μM or less, more preferably 0.129 μM or less, and even more preferably 0.005 μM or less).

[0070] In such embodiments, the concentration of at least one, preferably more than one, more preferably all compounds selected from the group consisting of glutathione, catalase, superoxide dismutase, alpha tocopherol, L-cysteine, and progesterone is within the following ranges: Glutathione: 100 ng / mL or less (preferably 10 ng / mL or less, more preferably 1 ng / mL or less, even more preferably 0.1 ng / mL or less); Catalase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Superoxide dismutase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Alpha-tocopherol: 50 nM or less (preferably 5 nM or less, more preferably 0.5 nM or less, even more preferably 0.05 nM or less); Cysteine: 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less); and Progesterone: 100 nM or less (preferably 50 nM or less, more preferably 20 nM (6.3 μg / mL) or less, even more preferably 10 nM or less, and even more preferably 3 nM or less).

[0071] The medium for maintaining continuous epithelial tissue can be prepared by appropriately blending commercially available media. Basal media that can be used to prepare a medium for maintaining continuous epithelial tissue include, for example, media that do not contain at least one (e.g., acidic amino acids), preferably two or more (e.g., acidic amino acids and any other substances), and more preferably all, of the aforementioned retinal neuronal protective substances such as acidic amino acids, antioxidants, and progesterone, or media that contain these substances within the aforementioned concentration ranges. In one embodiment of the basal medium, one, preferably two, and more preferably three of hypoxanthine, thymidine, and vitamin B12 are within the aforementioned concentration ranges. In another embodiment of the basal medium, hypoxanthine and thymidine are absent, and the concentration of vitamin B12 is within the aforementioned concentration range.

[0072] A basal medium that can be used to prepare a continuous epithelial tissue maintenance medium is preferably one in which the concentration of at least one of the methyl group donor (e.g., S-adenosylmethionine), methyl group donor substrate (e.g., methionine, MTA, Hcy), and neurite outgrowth inhibitor (e.g., corticosterone) is within the above-mentioned concentration range. A continuous epithelial tissue maintenance medium can be prepared by appropriately supplementing the basal medium with necessary substances so that the concentrations fall within the above-mentioned ranges.

[0073] Basal media that can be used to prepare continuous epithelial tissue maintenance medium can be appropriately selected from commercially available basal media according to the above-mentioned selection criteria, based on the ingredient list published by the manufacturer, etc. Examples of basal media that can be used to prepare continuous epithelial tissue maintenance medium include commercially available Neurobasal medium (including Neurobasal-A medium, phenol red-free Neurobasal medium, etc.), Improved MEM Zinc Option medium, MEM, DMEM, or Leibovitz's L-15, E-MEM, G-MEM, etc. It is also possible to order and purchase media with individually customized ingredients from medium manufacturers, or to use media customized for basal medium that can be used to prepare continuous epithelial tissue maintenance medium as described above.

[0074] A supplementary medium may be appropriately blended to supplement corticosterone. Specific examples of such supplementary medium include B27 supplements. Specific examples of a medium for maintaining continuous epithelial tissue include a medium in which B27 supplements are blended with Neurobasal medium. The medium may also contain L-glutamine, taurine, serum, and the like.

[0075] Neurobasal medium is a known basal medium developed for neuronal cell culture (J. Neurosci. Res., vol. 35, pp. 567-576, 1993). Neurobasal medium, partially modified from the original description, is available commercially from media manufacturers (e.g., Thermo Fisher Scientific, product number 21103049). The composition of Neurobasal medium (product number 21103049) available from Thermo Fisher Scientific is characterized by the absence of acidic amino acids (L-glutamic acid and L-aspartic acid), progesterone, hypoxanthine, and thymidine, and by a higher methionine concentration (30 mg / L), a higher cysteine ​​concentration (0.26 mM), and a lower vitamin B12 concentration (0.005 μM) compared to DMEM / F12. The specific composition is as follows:

[0076] [Table 1-1]

[0077] [Table 1-2]

[0078] B27 supplement is a well-known supplementary medium developed for neuronal cell culture (J. Neurosci. Res., vol. 35, pp. 567-576, 1993). B27 supplement is typically added to basal media such as Neurobasal medium at a volume ratio of approximately 2%. By combining B27 supplement containing corticosterone with Neurobasal medium, it can be used as a medium for maintaining continuous epithelial tissue. For example, a medium for maintaining continuous epithelial tissue can be prepared by adding B27 supplement containing corticosterone to a basal medium containing methionine (e.g., Neurobasal medium) so that the above-mentioned methionine and corticosterone concentrations are achieved.

[0079] B27 supplement (J. Neurosci. Res., vol. 35, pp. 567-576, 1993) can be purchased from a culture medium manufacturer (e.g., Thermo Fisher Scientific, 12587010), and its composition is as follows:

[0080] [Table 2]

[0081] In another aspect, the medium for maintaining continuous epithelial tissue comprises a medium containing a Neurobasal medium containing B27 supplement in a volume ratio of 1:1 or more (preferably 2:1 or more, more preferably 3:1 or more) to a basal medium for cell proliferation (e.g., a DMEM / F12 mixed medium (DMEM:F12=1:1)). The basal cell growth medium is not particularly limited, and commercially available basal media can be used alone or in appropriate mixtures. The basal cell growth medium may contain additives (supplements) as appropriate, and N2 supplements can be given as a specific example of such supplements.

[0082] The concentrations of L-methionine, L-glutamic acid, L-aspartic acid, L-cysteine, hypoxanthine, thymidine, and vitamin B12 in the medium prepared by adding B27 supplement (Thermo Fisher Scientific, 12587010) to the commercially available Neurobasal medium (Thermo Fisher Scientific, 21103049) and the medium prepared by mixing Neurobasal medium with B27 supplement and DMEM / F12 mixed medium (DMEM:F12=1:1) with N2 supplement at a ratio of 3:1, 2:1, or 1:1 are as follows:

[0083] [Table 3]

[0084] In one embodiment, a medium for maintaining continuous epithelial tissue may be used that has a composition of L-methionine, L-glutamic acid, L-aspartic acid, L-cysteine, hypoxanthine, thymidine, and vitamin B12 at concentrations equivalent to those in Table 3. Here, "equivalent concentrations" refers to the concentration of each factor independently falling within a range of ±20% (preferably ±10%, more preferably ±5%, even more preferably ±2.5%, and even more preferably ±1%).

[0085] In one embodiment, a medium having a composition of L-methionine, L-glutamic acid, L-aspartic acid, hypoxanthine, thymidine, and vitamin B12 at concentrations equivalent to those shown in Table 3, and containing at least one, preferably more than one, and more preferably all compounds selected from the group consisting of corticosterone, glutathione, catalase, superoxide dismutase, alpha-tocopherol, L-cysteine, and progesterone at concentrations within the following ranges, may be used as a medium for maintaining continuous epithelial tissue: Corticosterone: 0.1 nM or more (preferably 1 nM or more, more preferably 5 nM or more, even more preferably 10 nM or more, even more preferably 50 nM or more, even more preferably 100 nM or more); Glutathione: 100 ng / mL or less (preferably 10 ng / mL or less, more preferably 1 ng / mL or less, even more preferably 0.1 ng / mL or less); Catalase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Superoxide dismutase: 100 U / mL or less (preferably 10 U / mL or less, more preferably 1 U / mL or less, even more preferably 0.1 U / mL or less); Alpha-tocopherol: 50 nM or less (preferably 5 nM or less, more preferably 0.5 nM or less, even more preferably 0.05 nM or less); Cysteine: 0.26 mM or less (preferably 0.22 mM or less, more preferably 0.18 mM or less, and even more preferably 0.1 mM or less); and Progesterone: 100 nM or less (preferably 50 nM or less, more preferably 20 nM or less (or 6.3 μg / mL (20.033708 nM) or less), even more preferably 10 nM or less, even more preferably 3 nM or less).

[0086] The continuous epithelial tissue maintenance medium may contain L-glutamine, taurine, N2, etc. as appropriate. The concentration of taurine is usually 1 μM to 1000 μM, preferably 10 μM to 500 μM. When N2 is contained, it is more preferable to add a glucocorticoid such as corticosterone at the above-mentioned concentration instead of adding B27.

[0087] The medium for maintaining continuous epithelial tissue may contain one or more additives appropriately selected from among those typically contained in a medium, such as buffers (e.g., HEPES), salts (e.g., inorganic salts such as sodium chloride and sodium bicarbonate) or antioxidants (e.g., 2-mercaptoethanol; in one embodiment, no antioxidants are contained), nutrients such as amino acids (e.g., non-essential amino acids; in one embodiment, no acidic amino acids are contained), fatty acids, sugars, vitamins, lipids, or pyruvic acid, antibiotics (e.g., penicillin, streptomycin), extracellular matrices (e.g., Matrigel, laminin, laminin fragments, laminin 511-E8 fragments), and dyes (e.g., phenol red), to the extent that continuous epithelial tissue can be maintained, but is not limited to these.

[0088] The medium for maintaining continuous epithelial tissue may be either a serum medium or a serum-free medium. A serum medium is preferred. The serum concentration in a serum medium is usually 0.1 to 20% (v / v), preferably 0.1 to 12% (v / v) (e.g., 10% (v / v)). In this specification, changes in the concentration of components contained in the medium due to the addition of serum at a concentration of 0.1 to 20% (v / v) are not taken into consideration.

[0089] The medium may or may not contain retinoids (e.g., retinoic acid or a derivative thereof), and in one embodiment, it may contain 9-cis retinoic acid. A more preferred embodiment is a medium that is substantially free of retinoids, preferably retinoids that are biosynthesized by ALDH1A3 and inhibit the differentiation of cone photoreceptor precursor cells. Specific examples of retinoids that are biosynthesized by ALDH1A3 and inhibit the differentiation of cone photoreceptor precursor cells include all-trans retinoic acid (also known as atRA).

[0090] The term "continuous epithelial structure" in retinal tissue refers to a structure in which the retinal tissue has an apical surface characteristic of epithelial tissue, and the apical surface is formed on the surface of the retinal tissue generally parallel to and continuous with at least the photoreceptor layer (external nuclear layer) or the neuroblastic layer among the layers that make up the neural retina. In other words, a continuous epithelial structure does not have a structure in which the apical surface is divided, as seen in rosette-like structures. For example, in the case of a cell aggregate containing retinal tissue prepared from pluripotent stem cells, the apical surface is formed on the surface of the aggregate, and 10 or more, preferably 30 or more, more preferably 100 or more, and even more preferably 400 or more photoreceptors or photoreceptor precursor cells are regularly and continuously arranged in the tangential direction to the surface. The number of continuously arranged photoreceptors or photoreceptor precursor cells correlates with the size of the neural retinal tissue contained in the cell aggregate. As used herein, the tangential direction to the epithelial tissue refers to the direction in which the cells forming the apical surface of the epithelial tissue are aligned in a certain direction, and refers to the direction parallel or lateral to the epithelial tissue (or epithelial sheet). In one embodiment, an apical surface is formed on the surface of the neural retinal tissue, and photoreceptor cells or photoreceptor precursor cells are arranged in an orderly and continuous manner along the apical surface. Those skilled in the art are aware that in retinal tissue at a stage where the proportion of photoreceptors or photoreceptor precursor cells is low (e.g., retinal tissue at an early developmental stage), the layer containing proliferating neural retinal precursor cells is called the "neuroblastic layer." Furthermore, in addition to photoreceptors or photoreceptor precursor cells, the surface of retinal tissue at this stage may contain, in addition to photoreceptors or photoreceptor precursor cells, polarized neural retinal precursor cells capable of forming an apical surface, cells that divide and proliferate from neural retinal precursor cells, and / or cells at a stage where neural retinal precursor cells are differentiating into cells that constitute the neural retina. By culturing retinal tissue in this state under conditions that maintain a "continuous epithelial structure," retinal tissue can be obtained in which photoreceptors or photoreceptor precursor cells are regularly and continuously arranged along the apical surface that forms on the surface of the neural retinal tissue. In one embodiment, the area of ​​the apical surface present on the surface of the retinal tissue is, on average, 30% or more, preferably 50% or more, more preferably 80% or more, and even more preferably 95% or more of the area of ​​the surface of the retinal tissue. The proportion of the area of ​​the apical surface present on the surface of the retinal tissue can be measured by staining with an apical surface marker, as described below.

[0091] As used herein, the term "rosette-like structure" in retinal tissue refers to a structure in which cells are arranged radially or spirally around a central lumen. In retinal tissue that has formed a rosette-like structure, the apical surface and photoreceptor cells or photoreceptor precursor cells are present along the central portion (lumen), and the apical surface is divided into individual rosette-like structures.

[0092] As used herein, the term "apical surface" refers to the surface (superficial surface) of epithelial tissue that is rich in mucopolysaccharides (positive for PAS staining) and is formed on the side opposite the basement membrane where a 50-100 nm layer (basement membrane) produced by epithelial cells is present, which is rich in laminin and type IV collagen. In one aspect, in retinal tissue whose developmental stage has progressed to the point where photoreceptors or photoreceptor precursor cells can be found, the term refers to the surface where the outer limiting membrane is formed and that contacts the photoreceptor layer (external nuclear layer) where photoreceptors and photoreceptor precursor cells are present. Furthermore, such apical surfaces can be identified by immunostaining methods well known to those skilled in the art using antibodies against apical surface markers (e.g., atypical PKC (hereinafter abbreviated as aPKC), E-cadherin, and N-cadherin). Even in the early stages of development, when photoreceptors or photoreceptor precursor cells have not yet appeared, or when the photoreceptors or photoreceptor precursor cells have not yet appeared in sufficient numbers to fully cover the surface of the retinal tissue, the epithelial tissue remains polarized and expresses the above-mentioned apical markers on its apical surface.

[0093] Whether retinal tissue has a continuous epithelial structure can be confirmed by the continuity of the apical surface (i.e., an uninterrupted morphology) of the retinal tissue. Apical surface continuity can be determined, for example, by immunostaining for apical surface markers (e.g., aPKC, E-cadherin, N-cadherin) or markers of apical photoreceptors or photoreceptor precursor cells (e.g., Crx or recoverin) and analyzing the positional relationship between the apical surface and the photoreceptor layer and each retinal layer in the acquired images. Retinal layers other than the apical surface and the photoreceptor layer (outer nuclear layer) can be identified by immunostaining with DAPI staining, PI staining, or Hoechst staining, which stain cell nuclei, or with marker proteins localized in the cell nuclei (e.g., Rx, Chx10, Ki67, Crx, etc.).

[0094] Whether or not rosette-like structures have formed can be determined by fixing the cell aggregates with 4% paraformaldehyde, preparing frozen sections, and observing the formation of rosette-like structures (e.g., fragmented apical surfaces or intrusion of the apical surfaces into the cell aggregates) using immunostaining, typically performed using antibodies against the apical surface markers aPKC, E-cadherin, and N-cadherin, or DAPI, which specifically stains nuclei.

[0095] One embodiment of the present invention is an aggregate containing retinal tissue with a continuous epithelial structure, i.e., an aggregate containing retinal tissue in which photoreceptors or their progenitor cells are continuously present over at least 50% (preferably 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more) of the surface of the retinal tissue. Preparations containing such aggregates and a medium for maintaining continuous epithelial tissue also fall within the scope of the present invention. One embodiment of the retinal tissue having a continuous epithelial structure of the present invention is an aggregate containing retinal tissue in which the area of ​​the apical surface of the retinal tissue is at least 50% (preferably 60% or more, 70% or more, 80% or more, 85% or more, or 90% or more) of the area of ​​the surface of the retinal tissue. One embodiment of the present invention is an aggregate containing retinal tissue having a continuous epithelial structure, whose diameter in the longitudinal direction is 0.5 mm or more (preferably 0.6 mm or more, 0.8 mm or more, 1.0 mm or more, 1.2 mm or more, 1.4 mm or more, 1.6 mm or more, 1.8 mm or more, or 2.0 mm or more). From the viewpoint of being able to cover a wide area of ​​the retinal tissue of a damaged recipient, a larger retinal tissue to be transplanted is preferable. Generally, retinal tissue exceeding 0.5 mm to 1.0 mm in size is prone to forming a rosette structure during transplantation. However, the present invention can provide an aggregate containing retinal tissue having a continuous epithelial structure, even with retinal tissue exceeding 0.5 mm to 1.0 mm, without forming a rosette structure. The structure of the retinal tissue is as described below in "5. Retinal tissue with a high proportion of cone photoreceptors" or "6. Retinal tissue with a high proportion of rod photoreceptors."

[0096] Here, the long-axis diameter of retinal tissue refers to the length of the longest line connecting any two points on the periphery (contour, surface) of retinal tissue, for example, when measured based on an image captured using a stereomicroscope. Note that, in some aggregates containing retinal tissue, multiple retinal tissues may be present overlapping each other (e.g., cloverleaf-shaped). Whether multiple retinal tissues are present can be easily determined by those skilled in the art. In this case, the long-axis diameter of retinal tissue refers to the long-axis diameter of each retinal tissue in the aggregate, and it is sufficient that the long-axis diameter of at least one retinal tissue is 0.5 mm or greater. Preferably, the long-axis diameter of all retinal tissues in the aggregate is 0.5 mm or greater. More specifically, the long-axis diameter of the retinal tissue refers to the point where two circles or ellipses overlap (more specifically, the point where the continuity of the curve obtained by hypothetically determining continuous positional information on the periphery of an aggregate containing retinal tissue is lost when the positional information is plotted on the horizontal axis and the slope of the tangent at that position on the vertical axis). Furthermore, the aggregate containing retinal tissue may also contain retinal pigment epithelial cells and / or the peripheral ciliary body. In this case, as in the case where multiple retinal tissues are present in the aggregate, the length of the longest straight line connecting any two points on the periphery separated by the contact points between the retinal pigment epithelium and / or the peripheral ciliary body and the retinal tissue is measured.

[0097] The proportion of cells expressing RAX, CHX10 and / or CRX in the retinal tissue is preferably 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0098] In the case of an aggregate containing multiple retinal tissues, it is preferable that the ratio of the number of aggregates containing retinal tissues that meet the above conditions to the total number is at least 50% or more (preferably 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more). Any of the above-mentioned aggregates containing retinal tissue having a continuous epithelial structure can be formed with the apical surface of the surface in contact with the culture medium.

[0099] The continuous epithelial tissue maintenance medium contained in the preparation containing the aggregate and the continuous epithelial tissue maintenance medium is the continuous epithelial tissue maintenance medium defined herein, and may contain, for example, antibiotics, antiseptics, stabilizers, preservatives, etc., as long as it is capable of maintaining continuous epithelial tissue.

[0100] The concentration of the dorsalization signaling substance contained in the medium may be (preferably) a concentration that activates the BMP signaling pathway to a degree that does not inhibit the emergence of cone photoreceptor precursor cells. The concentration of the dorsalization signaling substance that does not inhibit the emergence of cone photoreceptor precursor cells can be appropriately determined based on factors such as the proportion of cone photoreceptor precursor cells that appear in the neural retinal tissue. Specifically, the concentration should be set so that, 30 to 50 days, preferably 30 to 40 days after the initial emergence of photoreceptor precursor cells, an average of 10% or more, preferably 13% or more, and more preferably 16% or more of the total number of cells in the neural retinal tissue become photoreceptor precursor cells. Specifically, when a substance acting on the BMP signaling pathway, particularly BMP4, is used as the dorsalization signaling substance, BMP4 is preferably used at a concentration of 0.01 nM to 0.90 nM, more preferably 0.05 nM to 0.45 nM. The concentration of the dorsalization signaling substance contained in the medium may be a concentration that activates the Wnt signaling pathway to an extent that does not induce the appearance of retinal pigment epithelial cells, specifically, a concentration of 0.01 nM to 0.90 nM, preferably 0.05 nM to 0.45 nM, that activates the Wnt signaling pathway to an extent equivalent to that of BMP4. When the medium is substantially free of exogenous retinoids such as 9-cis retinoic acid, the dorsalization signaling substance contained in the medium is preferably 0.05 nM to 0.15 nM, more preferably 0.1 nM to 0.15 nM BMP4. Alternatively, a substance acting on the Wnt signaling pathway may be applied at a concentration that exerts the same BMP signaling pathway activation effect as BMP4, preferably 0.05 nM to 0.15 nM, more preferably 0.1 nM to 0.15 nM. When the medium contains a retinoid such as 9-cis retinoic acid, the dorsalization signaling substance contained in the medium is preferably 0.15 nM to 0.90 nM, more preferably 0.15 nM to 0.45 nM BMP4. Alternatively, the substance acting on the Wnt signaling pathway may be applied at a concentration that exerts the same BMP signal activation effect as BMP4, preferably 0.15 nM to 0.90 nM, more preferably 0.15 nM to 0.45 nM.

[0101] Furthermore, examples of dorsalizing signal transduction substances include SHH signal transduction pathway inhibitors that inhibit ventralizing signal transduction, specifically cyclopamine-KAAD. The concentration of cyclopamine-KAAD contained in the medium is specifically 0.01 μM to 5 μM, more preferably 0.2 μM to 1 μM. When producing retinal tissue at an early stage of development, a medium containing a substance acting on the Wnt signal transduction pathway may be used, as in Raw Material Production Methods 5 to 7, or a medium not containing a substance acting on the Wnt signal transduction pathway may be used. When a ventralizing signal transduction inhibitor is used as the dorsalizing signal transduction substance, it is more preferable to use retinal tissue at an early stage of development prepared using a medium containing the substance acting on the Wnt signal transduction pathway, as in Raw Material Production Methods 5 to 7. Furthermore, the dorsalization signaling substance may be a combination of the above-mentioned substance acting on the BMP signaling pathway, the above-mentioned substance acting on the Wnt signaling pathway, and / or the above-mentioned SHH signaling pathway inhibitor that inhibits ventralization signaling. When the above-mentioned SHH signaling pathway inhibitor that inhibits ventralization signaling is used as the dorsalization signaling substance, it is preferably used in combination with the above-mentioned substance acting on the BMP signaling pathway and / or the above-mentioned substance acting on the Wnt signaling pathway to prepare retinal tissue containing a higher proportion of cone photoreceptor precursor cells.

[0102] The period of culture in a medium containing a dorsalizing signal transduction substance should be such that the effect of the dorsalizing signal transduction substance continues until the time when rod photoreceptor precursor cells appear when cultured in the absence of the dorsalizing signal transduction substance, and can be set appropriately to typically 4 days or more, preferably 20 days or more, and more preferably 70 days or more. Specifically, the culture can be performed for, for example, 50 to 170 days. More specifically, the culture can be performed for 70 to 100 days, but from the viewpoint of suppressing the appearance of rod photoreceptor precursor cells, a longer period of addition is preferable. Furthermore, cone photoreceptor precursor cells or cone photoreceptors can be matured into L cone photoreceptors, M cone photoreceptors, and S cone photoreceptors. Preferably, cone photoreceptor precursor cells or cone photoreceptors can be further cultured in the presence of a dorsalizing signal transduction substance to obtain neural retinal tissue rich in L cone photoreceptors and M cone photoreceptors, and cultured in the presence or absence of a dorsalizing signal transduction substance can obtain neural retinal tissue rich in S cone photoreceptors. In this case, differentiation into L cone photoreceptors and M cone photoreceptors is more preferably performed in combination with a thyroid hormone signal transduction substance simultaneously with a dorsalizing signal transduction substance, and differentiation into S cone photoreceptors is preferably performed in a medium substantially free of a thyroid hormone signal transduction substance. Further preferably, differentiation is performed in a serum-free medium. Furthermore, to induce selective differentiation into L-cone photoreceptors or M-cone photoreceptors, it is preferable to use a BMP4 signaling substance as the dorsalization signaling substance, which is added to the culture medium at a final concentration of 0.01 nM to 100 nM, preferably 0.05 nM to 10 nM, and more preferably 0.1 nM to 1.5 nM. When T3 is used as the thyroid hormone signaling substance, it may be used at a concentration of 0.01 nM to 100 nM, preferably 0.5 nM to 10 nM, and more preferably 2 nM to 10 nM. When T4 is used as the thyroid hormone signaling substance, it may be used at a concentration of T4 that exhibits the same effect as T3. The period of culture in which the thyroid hormone signaling substance is combined with the dorsalization signaling substance is not particularly limited, but is usually 50 days or more, preferably 70 days or more, more preferably 100 days or more, and even more preferably 150 days or more. Regarding the timing of initiating culture with a dorsalizing signal transduction substance and a thyroid hormone signal transduction substance in combination, the culture is preferably initiated 100 days or more, preferably 150 days or more, after the first appearance of photoreceptor precursor cells, and the culture is continued until usually 250 days, preferably 300 days, more preferably 400 days or more after the first appearance of photoreceptor precursor cells. Furthermore, to mature cone photoreceptor precursor cells or cone photoreceptors into L cone photoreceptors or M cone photoreceptors, preferably they are co-cultured with retinal pigment epithelium, or a conditioned medium (condition medium) used after culturing retinal pigment epithelium is used. Conversely, it is also possible to maintain 90% or more, preferably 99% or more, of the photoreceptor precursor cells or photoreceptors (cone photoreceptor precursor cells or cone photoreceptors, rod photoreceptor precursor cells or rod photoreceptors) in retinal tissue in a state in which they do not differentiate into L cone photoreceptors, M cone photoreceptors, and / or S cone photoreceptors, do not express or produce molecules necessary for light responses such as visual pigments, and do not reach final maturation. Since photoreceptor precursor cells are known to connect to bipolar cells as they mature, this method suppresses the connection between photoreceptor precursor cells and bipolar cells in the transplanted retinal tissue, thereby increasing the connection efficiency between photoreceptor precursor cells in the prepared retinal tissue and the recipient's bipolar cells upon transplantation. Specifically, a state in which they do not reach final maturation can be maintained by culturing them in a medium that does not contain glutamate, more preferably a medium that does not contain glutamate or aspartate, and even more preferably a medium that does not contain neurotransmitters such as glutamate or aspartate. Even more preferably, a medium containing serum is used, specifically a medium containing 5% or more, preferably 10% or more, of serum, and more specifically, the medium for maintaining continuous epithelial tissue described above is used. The serum used here is not particularly limited, but a specific example is fetal bovine serum (FBS). In other words, a method for suppressing the final maturation of photoreceptor progenitor cells, which includes a step of culturing retinal tissue containing photoreceptor progenitor cells or photoreceptors in a medium that does not contain neurotransmitters and contains serum, also falls within the scope of the present invention. Here, "final maturation" refers to a state in which a portion, preferably 15% or more, more preferably 30% or more, and even more preferably 60% or more of the photoreceptor precursor cells or photoreceptors have formed synapses or expressed functional molecules such as visual pigments. That is, neural retinal tissue that has matured to a stage where differentiation has occurred to the point where Muller cells can be observed but has not yet reached final maturation is preferably neural retinal tissue at a stage in which synapse formation has not occurred or in which functional molecules such as visual pigments have not yet been expressed. Whether photoreceptor precursor cells have finally matured can be identified using antibodies against photoreceptor-specific photoreceptors such as S-opsin, L-opsin, and M-opsin, or functional factors required for light stimulus response. That is, cells expressing functional molecules such as S-opsin, L-opsin, M-opsin, Rhodopsin, Cone-arrestin, arrestin, CNGA3, CNGA1, G alpha t2, G alpha t1, PDE6c, PDE6a, and PDE6b can be identified as finally mature photoreceptors. Furthermore, whether photoreceptor precursor cells and bipolar cells have contacted and formed a neural circuit within the retinal tissue can be identified by multiple staining using a combination of antibodies against RIBEYE and CtBP2, which are expressed in the region where photoreceptors and bipolar cells form a neural circuit; mGluR6, which is expressed in bipolar cells in that region; Arrestin and Recoverin, which are expressed in the synaptic endings of photoreceptor cells in that region; cone-specific Arrestin (gene symbol; ARR3); PNA, which is said to be specific to cone photoreceptors; and ELFN1, which is said to be specific to rod photoreceptors. Alternatively, the formation of a neural circuit can be identified by observing the synaptic structure of the photoreceptor synaptic endings and bipolar cells using an electron microscope. On the other hand, by culturing retinal tissue containing photoreceptor progenitor cells or photoreceptors in a serum-free medium, it is possible to obtain fully matured retinal tissue containing L-cone photoreceptors, M-cone photoreceptors, and / or S-cone photoreceptors. In other words, a method for promoting the complete maturation of photoreceptor progenitor cells or photoreceptors, which comprises the step of culturing retinal tissue containing photoreceptor progenitor cells or photoreceptors in a serum-free medium, also falls within the scope of the present invention.

[0103] Here, cells expressing functional molecules such as S-opsin, L-opsin, M-opsin, rhodopsin, cone-arrestin, arrestin, CNGA3, CNGA1, G alpha t2, G alpha t1, PDE6c, PDE6a, or PDE6b can be finally identified as mature photoreceptors. That is, fully matured retinal tissue is retinal tissue that expresses one or more, preferably three or more, of these functional molecules, just like normal retinal tissue in a living organism. When promoting the final maturation of L cone photoreceptors and M cone photoreceptors, the serum-free medium is preferably a medium containing a dorsalizing signal transduction substance. Examples of dorsalizing signal transduction substances include BMP signal transduction pathway promoters, specifically BMPs such as BMP2, BMP4, BMP7, and GDF7. Similarly, when promoting the final maturation of S cone photoreceptors, a medium containing a dorsalizing signal transduction substance can be preferably used. However, when specifically promoting the final maturation of S cone photoreceptors, a medium not containing a dorsalizing signal transduction substance can be preferably used. When promoting the final maturation of L cone photoreceptors and M cone photoreceptors, the serum-free medium preferably contains a thyroid hormone signaling substance in addition to a dorsalization signaling substance. Specific examples of thyroid hormone signaling substances include T3 and T4. On the other hand, since thyroid hormone signaling substances inhibit the final maturation of S cone photoreceptors, when promoting the final maturation of S cone photoreceptors, a medium substantially free of thyroid hormone signaling substances is preferably used. The period for culturing retinal tissue containing photoreceptor precursor cells or photoreceptors in serum-free medium can be appropriately determined by monitoring the degree of maturation. Specifically, retinal tissue at a differentiation stage where Müller cells have been detected can be cultured in serum-free medium for approximately 20 days or more, preferably 30 days or more, more preferably 60 days or more, and even more preferably 100 days or more. To promote the final maturation of L cone photoreceptors and M cone photoreceptors, the tissue can be cultured in serum-free medium containing a dorsalization signaling substance at a concentration equivalent to 0.5 nM to 3 nM, preferably 1 nM to 2 nM, of BMP4, and optionally containing a thyroid hormone signaling substance equivalent to 0 to 10 nM of T3, for approximately 20 days or more, preferably 30 days or more, more preferably 60 days or more, and even more preferably 100 days or more.

[0104] Alternatively, the above-mentioned substances acting on the Wnt signaling pathway can be used as the dorsalization signaling substance. In this case, specifically, the following steps (1) and (2) can be repeated to adjust the intensity of the Wnt signal: (1) adding the substance acting on the Wnt signaling pathway to a medium and culturing for 1 to 5 days, preferably 1 to 3 days; (2) The cells are cultured in a medium that is substantially free of the above-mentioned substance acting on the Wnt signaling pathway for 1 to 15 days, 1 to 10 days, preferably 1 to 7 days or 5 to 10 days. That is, by repeatedly carrying out the above steps (1) and (2), it is possible to induce BMP4 to the same extent as when 0.1 to 0.45 nM of BMP is added.

[0105] 3. Method for increasing the proportion of rod photoreceptors among photoreceptors contained in retinal tissue (method for producing retinal tissue rich in rod photoreceptors) One aspect of the present invention is a method for increasing the proportion of rod photoreceptor precursors and rod photoreceptors among photoreceptor precursors and photoreceptors contained in retinal tissue, the method comprising the step of culturing retinal tissue from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursors is maximized in the presence of a ventralizing signaling substance at a concentration sufficient to suppress the expression of dorsal markers. The medium used for culturing retinal tissue from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursor cells reaches its maximum in the presence of a ventralizing signaling substance is not particularly limited, and can be prepared using a medium commonly used for cell culture, as in the above item 2, so long as it does not contain a substance that inhibits the effect of the ventralizing signaling substance in an amount sufficient to inhibit the effect. Examples of basal media include media that can be used for culturing animal cells, such as Neurobasal medium, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, MEM medium, Eagle MEM medium, αMEM medium, DMEM medium, F-12 medium, DMEM / F12 medium, IMDM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, Leibovitz's L-15 medium, and mixtures thereof. Specific media that can be used include media supplemented with Wnt2b, Neurobasal media, media containing Neurobasal media, etc., and may be Neurobasal media supplemented with Wnt2b.

[0106] The medium may or may not contain a retinoid (e.g., retinoic acid or a derivative thereof), and in one embodiment, it may contain all-trans retinoic acid or 9-cis retinoic acid. A medium that is substantially free of retinoids is also a preferred embodiment.

[0107] The concentration of the ventralizing signal transduction substance contained in the culture medium may be a concentration that activates the SHH signal transduction pathway to an extent that suppresses the appearance of photoreceptor precursor cells during the period when cone photoreceptor precursor cells appear in retinal tissue as a result of development and differentiation, or a concentration that inhibits the BMP signal transduction pathway. Such concentrations can be easily determined by those skilled in the art. Specifically, the concentration of the ventralizing signal transduction substance may be set to an extent that promotes the expression of ALDH1A3. The concentration may be determined using commonly performed techniques such as immunostaining so that the area expressing the ventral markers COUP-TF I and / or ALDH1A3 accounts for 60% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more of the total area in the neural retinal tissue at the stage when the appearance rate of cone photoreceptor precursor cells relative to the cells in the retinal tissue is at its maximum. Specifically, the concentration includes a concentration that exerts the same SHH signaling pathway activation effect as 1 nM to 10 μM, preferably 10 nM to 500 nM SAG, and a concentration that exerts the same BMP signaling pathway inhibitory effect as 0.1 nM to 20 μM, preferably 30 nM to 1 μM LDN193189. Examples of ventralizing signal transduction substances include substances acting on the SHH signaling pathway, preferably 1 nM to 10 μM, more preferably 10 nM to 500 nM SAG. Furthermore, examples of ventralizing signal transduction substances include BMP signal transduction pathway inhibitors that inhibit dorsalizing signal transduction, specifically LDN193189. The concentration of LDN193189 contained in the medium is specifically 0.1 nM to 20 μM, and preferably 30 nM to 1 μM. When a BMP signaling pathway inhibitor such as LDN193189 is used as the ventralizing signal transduction substance, the differentiation efficiency of S cone photoreceptor precursors and / or S cone photoreceptors is improved in addition to rod photoreceptor precursors and rod photoreceptors. That is, another aspect of the present invention is a method for increasing the proportion of S cone photoreceptor precursors and S cone photoreceptors among photoreceptor precursors and photoreceptors contained in retinal tissue, comprising the step of culturing retinal tissue from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursors is maximized in the presence of a BMP signaling pathway inhibitor, specifically, for example, LDN-193189, at a concentration sufficient to suppress the expression of dorsal markers. Here, from the viewpoint of reducing the proportion of cone photoreceptor precursor cells contained, it is more preferable to use a substance that acts on the SHH signaling pathway than a substance that inhibits the BMP4 signaling pathway. Alternatively, one or more of the above-mentioned ventralizing signal transduction substances may be used in appropriate combination as the ventralizing signal transduction substance, for example, a substance acting on the SHH signal transduction pathway and / or a substance inhibiting the BMP signal transduction pathway that inhibits dorsalizing signal transduction may be used in combination. The period of culture in the presence of a ventralizing signal transduction substance should be such that the effect of the ventralizing signal transduction substance continues until the time when rod photoreceptor precursor cells appear when cultured in the absence of the ventralizing signal transduction substance, and can be appropriately set to typically 4 days or more, preferably 20 days or more, and more preferably 70 days or more. Specifically, the culture can be performed for, for example, 50 to 170 days. More specifically, the culture can be performed for 70 to 100 days, but from the viewpoint of promoting the appearance of rod photoreceptor precursor cells, a longer period of addition is preferable.

[0108] 4. Preparation of Early Developmental Retinal Tissue A method for producing retinal tissue at an early stage of development, which is the starting material used in methods 2 and 3 above, from pluripotent stem cells such as human iPS cells will be described. Pluripotent stem cells such as human iPS cells can be obtained or produced by methods well known to those skilled in the art as described above, and can be subjected to maintenance culture and expansion culture. Maintenance culture and expansion culture of pluripotent stem cells can be performed in either suspension culture or adherent culture, but is preferably performed in adherent culture. Maintenance culture and expansion culture of pluripotent stem cells can be performed in the presence or absence of feeder cells (feeder-free), but is preferably performed in the absence of feeder cells.

[0109] Using cultured pluripotent stem cells, retinal tissue at an early stage of development can be produced by methods well known to those skilled in the art. Examples of such methods include those described in WO2013 / 077425 (& US2014 / 341864), WO2015 / 025967 (& US2016 / 251616), WO2016 / 063985, WO2016 / 063986, and WO2017 / 183732. Examples of such methods include those described in non-patent literature: Proc Natl Acad Sci US A. 111(23): 8518-8523(2014), Nat Commun. 5:4047(2014), Stem Cells.(2017):35(5), 1176-1188, etc.

[0110] 4-1. Raw material manufacturing method 1 A preferred embodiment of producing retinal tissue at an early stage of development is a method described in WO2015 / 025967, which comprises the following steps: (1) a first step of forming cell aggregates by culturing pluripotent stem cells in suspension in a serum-free medium; (2) A second step in which the aggregates formed in the first step are cultured in suspension in a serum-free medium or serum-free medium containing a substance acting on the BMP signaling pathway but not a substance acting on the SHH signaling pathway, to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells. The aggregates containing retinal progenitor cells or neural retinal progenitor cells obtained by this method can be used as retinal tissue at an early stage of development, which serves as the starting material for methods 2 and 3 above.

[0111] [Regarding the first step] The first step can be carried out in accordance with the method described in WO2015 / 025967 (& US2014 / 341864), in which pluripotent stem cells are cultured in suspension in a serum-free medium to form cell aggregates. The serum-free medium used in the first step is not particularly limited as long as it is as described above. For example, a serum-free medium containing neither a substance acting on the BMP signaling pathway nor a substance inhibiting the Wnt signaling pathway can be used. To avoid the complicated preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of a commercially available serum substitute such as KSR (e.g., a medium containing a 1:1 mixture of IMDM and F-12 supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x Chemically Defined Lipid Concentrate). As a serum substitute, bovine serum albumin (BSA) can also be added to the serum-free medium at a concentration of 0.1 mg / mL to 20 mg / mL, preferably 4 mg / mL to 6 mg / mL. Furthermore, the amount of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%, for human ES cells or human iPS cells, for example. The culture conditions in the first step, such as culture temperature and CO2 concentration, can be set appropriately. 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%. The concentration of pluripotent stem cells that can be used in the first step can be appropriately set so as to form pluripotent stem cell aggregates more uniformly and efficiently. For example, when human ES cells are cultured in suspension in a 96-well plate, the concentration is about 1 × 10 per well. 3 to approximately 1 × 10 5 cells, preferably about 3 x 10 3 to about 5 × 10 4 cells, more preferably about 5 x 10 3 ~Approx. 3×10 4 cells, even more preferably about 0.9 x 10 4 ~1.2×10 4A solution prepared to form cells is added to the wells, and the plate is left to stand to allow aggregates to form. The suspension culture time required for forming aggregates can be determined appropriately depending on the pluripotent stem cells used, but it is desirable to keep the time as short as possible to form uniform aggregates (e.g., the SFEBq method). The process by which dispersed cells form cell aggregates can be divided into a cell aggregation process and a cell aggregation process in which the aggregated cells form cell aggregates. For example, in the case of human ES cells or human iPS cells, the time from seeding the dispersed cells (i.e., the start of suspension culture) to cell aggregation is preferably within about 24 hours, more preferably within about 12 hours. For example, in the case of human pluripotent stem cells (e.g., human iPS cells), the time from seeding the dispersed cells (i.e., the start of suspension culture) to forming cell aggregates is preferably within about 72 hours, more preferably within about 48 hours. This time until aggregate formation can be adjusted appropriately by adjusting the cell aggregation tool, centrifugation conditions, etc. The formation of cell aggregates can be determined based on the size and cell number of the aggregates, macroscopic morphology, microscopic morphology and its uniformity determined by tissue staining analysis, expression and uniformity of differentiated and undifferentiated markers, control of expression and synchronization of differentiation markers, and reproducibility of differentiation efficiency between aggregates.

[0112] [Regarding the second step] The second step involves suspension-culturing the aggregates formed in the first step in a serum-free medium or serum-free medium containing a substance acting on the BMP signaling pathway but not an SHH signaling pathway active substance, thereby obtaining aggregates containing retinal progenitor cells or neural retinal progenitor cells as retinal tissue at an early stage of development. The medium used in the second step is, for example, a serum-free medium or a serum-based medium to which a substance acting on the BMP signaling pathway is added but no substance acting on the SHH signaling pathway is added, and it is not necessary to add a basement membrane preparation. The serum-free or serum-free medium used in such a medium is not particularly limited, as long as it is as described above. To avoid the complicated preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of a commercially available serum substitute such as KSR (e.g., a medium containing a 1:1 mixture of IMDM and F-12 supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1x Chemically Defined Lipid Concentrate). BSA can also be added to the serum-free medium as a serum substitute at a concentration of 0.1 mg / mL to 20 mg / mL, preferably 4 mg / mL to 6 mg / mL. Furthermore, the amount of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%, for human ES cells, for example.

[0113] As long as the serum-free medium used in the first step does not contain a substance acting on the SHH signaling pathway, the serum-free medium used in the second step can be used as is or can be replaced with a new serum-free medium. When the serum-free medium used in the first step, which does not contain a substance acting on the BMP signaling pathway, is used as is in the second step, a substance acting on the BMP signaling pathway can be added to the medium. A medium that is "free of SHH signaling pathway active substances" includes a medium that is substantially free of SHH signaling pathway active substances, for example, a medium that does not contain SHH signaling pathway active substances at concentrations that adversely affect selective differentiation into retinal progenitor cells and retinal tissue. A medium "not containing an SHH signaling pathway active substance" also includes a medium to which an SHH signaling pathway active substance has not been added substantially, for example, a medium to which an SHH signaling pathway active substance has not been added at a concentration that would adversely affect selective differentiation into retinal progenitor cells and retinal tissue.

[0114] Examples of substances acting on the BMP signaling pathway used in the second step include BMP proteins such as BMP2, BMP4, and BMP7, GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides. BMP2, BMP4, and BMP7 are available from R&D Systems, for example, and GDF7 is available from Wako Pure Chemical Industries, Ltd. A preferred example of a substance acting on the BMP signaling pathway is BMP4. The concentration of the substance acting on the BMP signaling pathway used in the second step may be any concentration capable of inducing differentiation of the cells contained in the aggregates obtained in the first step into retinal cells. For example, BMP4 is added to the medium to a concentration of about 0.01 nM to about 1 μM, preferably about 0.1 nM to about 100 nM, more preferably about 1 nM to about 10 nM, and even more preferably about 1.5 nM (55 ng / mL). When a substance acting on the BMP signaling pathway other than BMP4 is used, it is preferably used at a concentration that exerts the same BMP signaling pathway activation effect as the above-mentioned BMP4 concentration. The substance acting on the BMP signaling pathway may be added to the medium at least about 24 hours after the initiation of suspension culture in the first step, and may be added to the medium within several days (e.g., within 15 days) after the initiation of suspension culture in the first step. Preferably, the substance acting on the BMP signaling pathway is added to the medium on days 1 to 15, more preferably days 1 to 9, even more preferably days 2 to 9, even more preferably days 3 to 8, still more preferably days 3 to 6, and even more preferably day 6 after the initiation of suspension culture. After the substance acting on the BMP signaling pathway is added to the culture medium and the differentiation induction of the cells contained in the aggregate obtained in the first step into retinal cells is initiated, there is no need to further add a substance acting on the BMP signaling pathway to the culture medium, and the culture medium may be replaced with a serum-free medium or serum medium that does not contain a substance acting on the BMP signaling pathway. Alternatively, the concentration of the substance acting on the BMP signaling pathway in the medium may be varied during the second step. For example, the concentration of the substance acting on the BMP signaling pathway may be set within the above range at the start of the second step, and then gradually or stepwise reduced by 40 to 60% every 2 to 4 days. In a specific embodiment, on days 1 to 9, preferably days 2 to 9, more preferably days 3 to 8, and even more preferably days 3 to 6 after the start of suspension culture (i.e., after the start of the first step), part or all of the medium is replaced with a medium containing BMP4, and the final concentration of BMP4 is adjusted to about 1 to 10 nM. The cells can be cultured in the presence of BMP4 for, for example, 1 to 16 days, preferably 2 to 9 days, and more preferably 6 to 9 days. It is also possible to culture for a longer period, specifically 20 days or more, 30 days or more. That is, in the second step, the culture carried out in the presence of a substance acting on the BMP signaling pathway is The process is continued for an appropriate period of time until the aggregates obtained in the first step are induced to differentiate into retinal tissue at an early stage of development; specifically, retinal tissue at an early stage of development can be obtained 6 to 12 days after adding the substance acting on the BMP signaling pathway. In this case, to maintain the same BMP4 concentration, part or all of the medium can be replaced with a medium containing BMP4 once or twice, or, as described above, the BMP4 concentration can be reduced stepwise. In one aspect, after initiating culture in a medium containing a substance acting on the BMP signaling pathway, the concentration of the substance acting on the BMP signaling pathway in the medium can be gradually or stepwise reduced by 40 to 60% every 2 to 4 days by replacing the medium with a serum-free medium or serum medium that does not contain the substance acting on the BMP signaling pathway.

[0115] The induction of differentiation into retinal tissue at an early stage of development can be confirmed, for example, by detecting the expression of a retinal progenitor cell marker or a neural retinal progenitor cell marker in cells in the tissue. The time when differentiation induction into retinal cells has begun can also be confirmed by detecting the expression of a retinal progenitor cell marker or a neural retinal progenitor cell marker in cells in the tissue. The aggregates formed in the first step using pluripotent stem cells in which a fluorescent reporter protein gene such as GFP has been knocked into the RX locus are cultured in suspension in the presence of a substance acting on the BMP signaling pathway at a concentration required for induction of differentiation into retinal cells, and detecting the fluorescence emitted from the expressed fluorescent reporter protein. One embodiment of the second step includes a step of suspension-culturing the aggregates formed in the first step in a serum-free medium or a serum-free medium containing a substance acting on the BMP signaling pathway at a concentration necessary to induce differentiation into retinal cells but not containing a substance acting on the SHH signaling pathway, until cells expressing retinal progenitor cell markers or neural retinal progenitor cell markers (e.g., RX, PAX6, CHX10) begin to appear, thereby obtaining aggregates containing retinal progenitor cells or neural retinal progenitor cells as retinal tissue at an early stage of development.

[0116] When a medium exchange operation is performed in the second step, examples include an operation in which new medium is added without discarding the original medium (medium addition operation), an operation in which about half of the original medium (about 40 to 80% of the volume of the original medium) is discarded and about half of the new medium (40 to 80% of the volume of the original medium) is added (half medium exchange operation), and an operation in which about the entire volume of the original medium (90% or more of the volume of the original medium) is discarded and about the entire volume of new medium (90% or more of the volume of the original medium) is added (full medium exchange operation). When adding a specific component (e.g., BMP4) at a certain point, for example, after calculating the final concentration, approximately half of the original medium may be discarded and approximately half of new medium containing the specific component at a concentration higher than the final concentration (specifically, 1.5 to 3.0 times the final concentration, for example, approximately twice the final concentration) may be added (half medium exchange operation, half medium exchange). If, at a certain point, the concentration of a particular component contained in the original medium is to be maintained, for example, about half of the original medium may be discarded and about half of a new medium containing the particular component at the same concentration as that contained in the original medium may be added. When components contained in the original medium are diluted to reduce their concentrations at a certain point, for example, the medium exchange procedure may be performed multiple times a day, preferably multiple times within an hour (e.g., 2 to 3 times).Furthermore, when components contained in the original medium are diluted to reduce their concentrations at a certain point, the cells or aggregates may be transferred to another culture vessel. The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used. In a preferred embodiment, the concentration of the substance acting on the SHH signaling pathway in the medium used in the second step is 700 nM or less, preferably 300 nM or less, more preferably 10 nM or less, even more preferably 0.1 nM or less, and even more preferably does not contain any substance acting on the SHH signaling pathway, calculated in terms of the SHH signaling-promoting activity of SAG. A medium "free of a substance acting on the SHH signaling pathway" also includes a medium that is substantially free of a substance acting on the SHH signaling pathway, for example, a medium that does not contain a substance acting on the SHH signaling pathway at a concentration that would adversely affect selective differentiation into retinal progenitor cells and retinal tissue. A medium "not supplemented with a substance acting on the SHH signaling pathway" also includes a medium that is substantially free of a substance acting on the SHH signaling pathway, for example, a medium that is not supplemented with a substance acting on the SHH signaling pathway at a concentration that would adversely affect selective differentiation into retinal progenitor cells and retinal tissue.

[0117] The culture conditions in the second step, such as culture temperature and CO2 concentration, can be set appropriately. 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%. By such culturing, differentiation of the cells forming the aggregates obtained in the first step into retinal tissue at an early stage of development can be induced. Whether aggregates containing retinal progenitor cells or neural retinal progenitor cells have been obtained as retinal tissue at an early stage of development can be confirmed, for example, by detecting whether the aggregates contain cells expressing RX or PAX6, which are markers for retinal progenitor cells, or RX, PAX6, or CHX10, which are markers for neural retinal progenitor cells. One embodiment of the second step is to culture the aggregates formed in the first step in suspension in a serum-free medium or serum medium containing a substance acting on the BMP signaling pathway at a concentration necessary for inducing differentiation into retinal cells until cells expressing the RX gene begin to appear, thereby obtaining aggregates containing retinal progenitor cells or neural retinal progenitor cells. In one aspect, the culture in the second step is carried out until 20% or more (preferably 30% or more, 40% or more, 50% or more, or 60% or more) of the cells contained in the aggregates express RX.

[0118] The aggregates obtained by the above method can be used as retinal tissue in the early developmental stage, which serves as a starting material for the production method of the present invention, after being subjected to suspension culture in a serum-free medium or serum-free medium that does not contain any substance acting on the SHH signaling pathway, BMP signaling pathway, or Wnt signaling pathway. The suspension culture period is not particularly limited as long as it is long enough for ganglion cells to appear, but can be 1 to 50 days, preferably 1 to 15 days, and more preferably 1 to 7 days. The medium used in the suspension culture is, for example, a serum-free medium or a serum-free medium to which none of a substance acting on the SHH signaling pathway, a substance acting on the BMP signaling pathway, and a substance acting on the Wnt signaling pathway is added. The medium "containing no SHH signaling pathway active substance, no BMP signaling pathway active substance, and no Wnt signaling pathway active substance" also includes a medium that is substantially free of any of SHH signaling pathway active substances, BMP signaling pathway active substances, and Wnt signaling pathway active substances, for example, a medium that does not contain SHH signaling pathway active substances, BMP signaling pathway active substances, and Wnt signaling pathway active substances at concentrations that adversely affect selective differentiation into retinal tissue. The medium "to which none of an SHH signaling pathway active substance, a BMP signaling pathway active substance, and a Wnt signaling pathway active substance has been added" also includes a medium to which none of an SHH signaling pathway active substance, a BMP signaling pathway active substance, and a Wnt signaling pathway active substance has been added substantially, for example, a medium to which none of an SHH signaling pathway active substance, a BMP signaling pathway active substance, and a Wnt signaling pathway active substance has been added at concentrations that would adversely affect selective differentiation into retinal tissue.

[0119] The serum-free or serum-containing medium used for such a medium is not particularly limited as long as it is as described above. To avoid the complicated preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of a commercially available serum substitute such as KSR (e.g., a medium containing 10% KSR, 450 μM 1-monothioglycerol, and 1x Chemically Defined Lipid Concentrate in a 1:1 mixture of IMDM and F-12). The serum-free medium can also be supplemented with bovine serum albumin (BSA) at 0.1 mg / mL to 20 mg / mL, preferably 4 mg / mL to 6 mg / mL. For human ES cells, the amount of KSR added to the serum-free medium is usually about 1% to about 20%, preferably about 2% to about 20%. To avoid the complicated preparation process for a serum-containing medium, it is more preferable to use a serum-free medium supplemented with an appropriate amount of commercially available serum (e.g., a medium containing serum and N2 supplement in a 1:1 mixture of DMEM and F-12). The amount of serum added to serum-containing media is usually about 1% to about 20%, and preferably about 2% to about 20%, in the case of human ES cells, for example. Taurine and the like may be added to any of the above media. Culture conditions such as culture temperature, CO2 concentration, and O2 concentration can be set appropriately. 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%. The O2 concentration is about 5% or higher, for example, about 20% to about 70%, preferably about 20% to about 60%, more preferably about 20% to about 40%, and particularly preferably about 20%.

[0120] As described above, the retinal tissue obtained by Raw Material Production Method 1 can be identified as being at an early developmental stage, i.e., an early differentiation stage in which retinal progenitor cells or neural retinal progenitor cells are present and ganglion cells have not yet appeared, by measuring the expression status of at least one of retinal progenitor cell markers such as RX and PAX6, neural retinal progenitor cell markers such as CHX10, RX, and PAX6, and ganglion cell markers such as BRN3. That is, it can be confirmed that the retinal tissue is at a differentiation stage in which cells expressing retinal progenitor cell markers and / or neural retinal progenitor cell markers account for 30% or more, preferably 50% or more, more preferably 80% or more, even more preferably 90% or more, and even more preferably 99% or more of all cells contained in the retinal tissue, and cells expressing ganglion cell markers account for 40% or less, preferably 20% or less, 10% or less, 5% or less, 1% or less, even more preferably 0.1% or less, and even more preferably 0.01% or less of all cells contained in the retinal tissue. At this time, the expression of ventral markers and / or the most dorsal markers (e.g., ALDH1A3 and / or ALDH1A1) is not an issue, as long as it is a differentiation stage that can be suppressed or promoted by a substance acting on the dorsalization signaling pathway.

[0121] 4-2. Raw material manufacturing method 2 A preferred embodiment of producing retinal tissue at an early developmental stage includes a method described in WO2016 / 063985 and WO2017 / 183732, which comprises the following steps: (1) a first step of culturing pluripotent stem cells in a medium containing 1) a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway activator, and 2) an undifferentiated state maintenance factor, in the absence of feeder cells; (2) a second step of culturing the cells obtained in the first step in suspension to form cell aggregates; and (3) A third step in which the aggregates obtained in the second step are cultured in suspension in the presence of a substance acting on the BMP signaling pathway to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells. The aggregates containing retinal progenitor cells or neural retinal progenitor cells obtained by this method can be used as the starting material for methods 2 and 3 above as retinal tissue at an early stage of development.

[0122] [Regarding the first step] The first step can be carried out in accordance with the method described in WO2016 / 063985. That is, in the first step, "in the absence of feeder cells" (hereinafter also referred to as "feeder-free") means conditions in which feeder cells are substantially not included (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less). Preferably, the first step is carried out under conditions in which feeder cells are not included. The medium used in the first step is not particularly limited as long as it is a medium (feeder-free medium) that allows pluripotent stem cells to be cultured under feeder-free conditions to maintain the undifferentiated state, but preferably contains factors for maintaining the undifferentiated state in order to enable the culture to maintain the undifferentiated state. The undifferentiated state maintenance factor is not particularly limited as long as it is a substance that has the effect of suppressing the differentiation of pluripotent stem cells. Examples of factors commonly used by those skilled in the art for maintaining undifferentiated states include substances acting on the FGF signaling pathway, substances acting on the TGFβ family signaling pathway, and insulin. Specific examples of substances acting on the FGF signaling pathway include fibroblast growth factors (e.g., bFGF, FGF4, and FGF8). Furthermore, examples of substances acting on the TGFβ family signaling pathway include substances acting on the TGFβ signaling pathway and substances acting on the Nodal / Activin signaling pathway. Examples of substances acting on the TGFβ signaling pathway include TGFβ1 and TGFβ2. Examples of substances acting on the Nodal / Activin signaling pathway include Nodal, Activin A, and Activin B. When culturing human pluripotent stem cells (human ES cells, human iPS cells), the medium in the first step preferably contains bFGF as an undifferentiated state maintenance factor. The factors for maintaining undifferentiation state used in the present invention are not particularly limited as long as they are derived from mammals, but preferably are derived from the same mammalian species as the cells to be cultured. For example, human factors for maintaining undifferentiation state (e.g., bFGF, FGF4, FGF8, EGF, Nodal, Activin A, Activin B, TGFβ1, TGFβ2, etc.) are used to culture human pluripotent stem cells, and isolated factors for maintaining undifferentiation state can be added exogenously (or exogenously). Alternatively, factors for maintaining undifferentiation state may be added in advance to the medium used in the first step. The concentration of the undifferentiation maintenance factor in the medium used in the first step is a concentration that allows the pluripotent stem cells to be cultured in an undifferentiated state, and can be appropriately determined by those skilled in the art. For example, specifically, when bFGF is used as the undifferentiation maintenance factor in the absence of feeder cells, the concentration is usually about 4 ng to 500 ng / mL, preferably about 10 ng to 200 ng / mL, and more preferably about 30 ng to 150 ng / mL. Many synthetic media containing factors for maintaining undifferentiated states and usable as feeder-free media for culturing pluripotent stem cells have been developed and are commercially available, such as Essential 8 medium (Life Technologies). Essential 8 medium is a DMEM / F12 medium supplemented with the following additives: L-ascorbic acid-2-phosphate magnesium (64 mg / L), sodium selenium (14 μg / L), insulin (19.4 mg / L), NaHCO3 (543 mg / L), transferrin (10.7 mg / L), bFGF (100 ng / mL), and TGFβ family signaling pathway agents (TGFβ1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)). Other commercially available feeder-free media include S-medium (manufactured by DS Pharma Biomedical), StemPro (manufactured by Life Technologies), hESF9 (Proc. Natl. Acad. Sci. USA. 2008 Sep 9;105(36):13409-14), mTeSR1 (manufactured by STEMCELL Technologies), mTeSR2 (manufactured by STEMCELL Technologies), TeSR-E8 (manufactured by STEMCELL Technologies), and StemFit (manufactured by Ajinomoto Co., Inc.). By using these media in the first step, the present invention can be carried out easily.

[0123] The culture of pluripotent stem cells in the first step may be carried out under either suspension culture or adherent culture conditions, but is preferably carried out by adherent culture. The culture vessel used for adherent culture is not particularly limited as long as it is capable of "adherent culture," but a cell-adhesive culture vessel is preferred. Examples of cell-adhesive culture vessels include those whose surfaces have been artificially treated to improve cell adhesion, specifically those whose interiors are coated with the aforementioned coating agent. Examples of coating agents include laminin [including laminin α5β1γ1 (hereinafter referred to as laminin 511), laminin α1β1γ1 (hereinafter referred to as laminin 111), and laminin fragments (such as laminin 511E8)], entactin, collagen, gelatin, vitronectin, Synthemax (Corning), and extracellular matrices such as Matrigel, as well as polymers such as polylysine and polyornithine. Alternatively, culture vessels with surface treatments such as positive charging can also be used. Laminin is preferred, and laminin 511E-8 is more preferred. Laminin 511E-8 can be purchased commercially (e.g., iMatrix-511, Nippi). The medium used in the first step contains a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway activator. A TGFβ family signaling pathway inhibitor refers to a substance that inhibits the TGFβ family signaling pathway, i.e., the signaling pathway transmitted by the Smad family, and specific examples include TGFβ signaling pathway inhibitors, Nodal / Activin signaling pathway inhibitors, and BMP signaling pathway inhibitors. The TGFβ signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by TGFβ, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. 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 physiological activities caused by signal transduction by TGFβ receptors (e.g., TGFβ receptor inhibitors, Smad inhibitors, etc.). Proteins known to inhibit the TGFβ signaling pathway include Lefty. Compounds well known to those skilled in the art can be used as TGFβ signaling pathway inhibitors, and specific examples include SB431542 (4[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), LY-364947 (4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]-quinoline), SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine), A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide), and the like. The Nodal / Activin signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by Nodal or Activin, and may be any of nucleic acids, proteins, and low-molecular-weight organic compounds. Examples of such substances include substances that act directly on Nodal or Activin (e.g., antibodies, aptamers, etc.), substances that suppress the expression of genes encoding Nodal or Activin (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Nodal / Activin receptors to Nodal / Activin, and substances that inhibit physiological activities resulting from signal transduction via Nodal / Activin receptors. Compounds well known to those skilled in the art can be used as Nodal / Activin signaling pathway inhibitors, including, for example, SB431542 and A-83-01. Proteins known as Nodal / Activin signaling pathway inhibitors (e.g., Lefty and Cerberus) may also be used. The Nodal / Activin signaling pathway inhibitor is preferably SB431542, A-83-01, or Lefty. The BMP signaling pathway inhibitor is not particularly limited as long as it inhibits the signaling pathway caused by BMP, and the substances mentioned above can be used. Compounds well known to those skilled in the art can be used as the BMP signaling pathway inhibitor, and specific examples include LDN193189, Dorsomorphin, etc. Proteins known as BMP signaling pathway inhibitors (Chordin, Noggin, etc.) may also be used. The BMP signaling pathway inhibitor is preferably LDN193189. The TGFβ family signaling pathway inhibitor is preferably Lefty, SB431542, A-83-01, or LDN193189. Multiple types of TGFβ family signaling pathway inhibitors with different sites of action may be used in combination. Combinations are expected to enhance the effect of improving aggregate quality. Examples include a combination of a TGFβ signaling pathway inhibitor and a BMP signaling pathway inhibitor, a combination of a TGFβ signaling pathway inhibitor and a Nodal / Activin signaling pathway inhibitor, and a combination of a BMP signaling pathway inhibitor and a Nodal / Activin signaling pathway inhibitor. Preferably, a TGFβ signaling pathway inhibitor is used in combination with a BMP signaling pathway inhibitor. A specific preferred combination is the combination of SB431542 and LDN193189. The substance acting on the SHH signaling pathway may be any of those described above. The substance acting on the SHH signaling pathway is preferably SHH protein (Genbank accession numbers: NM_000193, NP_000184), SAG, or PMA. A TGFβ family signaling pathway inhibitor and a substance acting on the SHH signaling pathway may be used in combination. Specific combinations include, for example, a combination of a TGFβ family signaling pathway inhibitor selected from the group consisting of Lefty, SB431542, A-83-01, and LDN193189 with a substance acting on the SHH signaling pathway selected from the group consisting of SHH protein, SAG, and PMA. When a TGFβ family signaling pathway inhibitor and a substance acting on the SHH signaling pathway are used in combination, cells may be cultured in a medium containing both a TGFβ family signaling pathway inhibitor and a substance acting on the SHH signaling pathway, or cells may be treated with either a TGFβ family signaling pathway inhibitor or a substance acting on the SHH signaling pathway, and then subsequently treated with either or both. The concentrations of the TGFβ family signaling pathway inhibitor and the SHH signaling pathway activator can be appropriately set within a range that achieves the above-mentioned effects. For example, SB431542 is typically used at a concentration of 0.1 μM to 200 μM, preferably 2 μM to 50 μM. A-83-01 is typically used at a concentration of 0.05 μM to 50 μM, preferably 0.5 μM to 5 μM. LDN193189 is typically used at a concentration of 1 nM to 2000 nM, preferably 10 nM to 300 nM. Lefty is typically used at a concentration of 5 ng / mL to 200 ng / mL, preferably 10 ng / mL to 50 ng / mL. SHH protein is typically used at a concentration of 20 ng / mL to 1000 ng / mL, preferably 50 ng / mL to 300 ng / mL. SAG is typically used at a concentration of 1 nM to 2000 nM, preferably 10 nM to 700 nM, and more preferably 30 to 600 nM. PMA is typically used at a concentration of 0.002 to 20 μM, preferably 0.02 μM to 2 μM. In one embodiment, a TGFβ family signaling pathway inhibitor can be used appropriately in an amount that has the same inhibitory activity on the TGFβ family signaling pathway as SB431542 at the aforementioned concentrations. In another embodiment, a substance acting on the SHH signaling pathway can be used appropriately in a concentration that has the same SHH signaling pathway activation effect as SAG at the aforementioned concentrations. The medium used in the first step may be a serum-containing medium or a serum-free medium, but is preferably a serum-free medium from the viewpoint of avoiding contamination with chemically undefined components. The medium used in the first step may be a medium whose components are chemically defined, from the viewpoint of avoiding contamination with chemically undefined components. The culture of pluripotent stem cells in the first step may be carried out under either suspension culture or adherent culture conditions, but is preferably carried out by adherent culture.

[0124] In the first step of culturing pluripotent stem cells under feeder-free conditions, an appropriate matrix may be used as a scaffold to provide the pluripotent stem cells with a scaffold instead of feeder cells. The pluripotent stem cells are cultured in an adherent manner in a cell container whose surface is coated with the scaffold matrix. Examples of matrices that can be used as scaffolds include laminin (Nat. Biotechnol. 28, 611-615 (2010)), laminin fragments (Nat. Commun. 3, 1236 (2012)), basement membrane preparations (Nat. Biotechnol. 19, 971-974 (2001)), gelatin, collagen, heparan sulfate proteoglycan, entactin, and vitronectin. Preferably, in the first step of culturing pluripotent stem cells under feeder-free conditions, the pluripotent stem cells are cultured in an adherent manner in a cell container whose surface is coated with isolated laminin 511 or the E8 fragment of laminin 511 (more preferably the E8 fragment of laminin 511).

[0125] The culture time of the pluripotent stem cells in the first step is not particularly limited as long as it is within a range that can achieve the effect of improving the quality of the aggregates formed in the second step, but is usually 0.5 to 144 hours. The culture time of the pluripotent stem cells in the first step is preferably 1 hour or more, 2 hours or more, 6 hours or more, 12 hours or more, 18 hours or more, or 24 hours or more. The culture time of the pluripotent stem cells in the first step is preferably 96 hours or less, or 72 hours or less. In one aspect, the culture time of the pluripotent stem cells in the first step is preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours). That is, the first step is started 0.5 to 144 hours (preferably 18 to 28 hours) before the start of the second step, and the second step is carried out immediately after the first step is completed. In a further aspect, the culture time range of the pluripotent stem cells in the first step is preferably 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours. When cells are treated with either a substance inhibiting the TGFβ family signaling pathway or a substance acting on the SHH signaling pathway, and then subsequently treated with the other substance, the respective treatment times can be set within the above-mentioned culture time ranges. The culture conditions in the first step, such as culture temperature and CO2 concentration, can be set appropriately. 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%.

[0126] In a preferred embodiment, the cells obtained in the first step are cells in which pluripotent-like properties are maintained, and pluripotent-like properties are maintained throughout the first step. Pluripotent-like properties refer to a state in which at least some of the traits common to pluripotent stem cells, including pluripotency, are maintained that are specific to pluripotent stem cells. Strict pluripotency is not required for pluripotent-like properties. Specifically, "pluripotent-like properties" include a state in which all or some of the markers that indicate the pluripotent state are expressed. Examples of markers for pluripotent-like properties include Oct3 / 4 positivity and alkaline phosphatase positivity. In one embodiment, cells in which pluripotent-like properties are maintained are Oct3 / 4 positivity. Even if the expression level of Nanog is lower than that of ES cells or iPS cells, the cells are still considered to be "cells exhibiting pluripotent-like properties." In one embodiment, the cells obtained by the first step are stem cells capable of differentiating into at least retinal tissue, retinal cells, retinal progenitor cells, and retinal layer-specific nerve cells.

[0127] In a preferred embodiment, human pluripotent stem cells (e.g., iPS cells) are cultured in an adherent manner in the absence of feeder cells in a serum-free medium containing a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway agonist, and bFGF. The adherent culture is preferably carried out in a cell container whose surface is coated with laminin-511 or the E8 fragment of laminin-511. The TGFβ family signaling pathway inhibitor is preferably a TGFβ signaling pathway inhibitor (e.g., SB431542, A-83-01, Lefty), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189, Chordin, Noggin), or a combination thereof (e.g., SB431542 and LDN193189). The TGFβ family signaling pathway inhibitor is more preferably Lefty, SB431542, A-83-01, or LDN193189, or a combination thereof (e.g., SB431542 and LDN193189). The substance acting on the SHH signaling pathway is preferably an SHH protein, SAG, or Purmorphamine (PMA), more preferably SAG. A TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189) may be used in combination with a substance acting on the SHH signaling pathway (e.g., an SHH protein, SAG, or PMA). The culture time is 0.5 to 144 hours (preferably 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours (e.g., 18 to 28 hours)). For example, human pluripotent stem cells (e.g., human iPS cells) are maintained and cultured in a serum-free medium containing bFGF in the absence of feeder cells. The maintenance culture is preferably performed by adhesion culture. The adhesion culture is preferably performed in a cell container whose surface is coated with vitronectin, laminin 511, or the E8 fragment of laminin 511. Then, a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway agonist is added to the culture, and the culture is continued. The TGFβ family signaling pathway inhibitor is preferably a TGFβ signaling pathway inhibitor (e.g., SB431542, A-83-01, Lefty), a Nodal / Activin signaling pathway inhibitor (e.g., SB431542, A-83-01, Lefty), a BMP signaling pathway inhibitor (e.g., LDN193189), or a combination thereof (e.g., SB431542 and LDN193189). The TGFβ family signaling pathway inhibitor is more preferably Lefty, SB431542, A-83-01, or LDN193189, or a combination thereof (e.g., SB431542 and LDN193189). The SHH signaling pathway active substance is preferably SHH protein, SAG, or PMA. A TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189) may be used in combination with a substance active on the SHH signaling pathway (e.g., SHH protein, SAG, PMA). After addition, the culture is continued for 0.5 to 144 hours (preferably 18 to 144 hours, 24 to 144 hours, 24 to 96 hours, or 24 to 72 hours (e.g., 18 to 28 hours)).

[0128] [Regarding the second step] The second step, in which the cells obtained in the first step are cultured in suspension in a medium to form cell aggregates, will now be described. The medium used in the second step can be serum-containing or serum-free. To avoid contamination with chemically undefined components, serum-free medium is preferred in the present invention. For example, serum-free medium containing neither a substance acting on the BMP signaling pathway nor a substance inhibiting the Wnt signaling pathway can be used. To avoid the complexity of the preparation process, it is preferable to use a serum-free medium supplemented with an appropriate amount of a commercially available serum substitute such as KSR (e.g., a 1:1 mixture of IMDM and F-12 supplemented with 10% KSR, 450 μM 1-monothioglycerol, and 1× Chemically Defined Lipid Concentrate, or a medium containing GMEM supplemented with 5% to 20% KSR, NEAA, pyruvic acid, and 2-mercaptoethanol). For example, in the case of human pluripotent stem cells, the amount of KSR added to the serum-free medium is typically about 1% to about 30%, preferably about 2% to about 20%. When forming aggregates, first, dispersed cells are prepared by dispersing the cells obtained in the first step. The "dispersed cells" obtained by the dispersion step are, for example, those in which 70% or more are single cells and 30% or less are clumps of 2 to 50 cells. Dispersed cells are preferably those in which 80% or more are single cells and 20% or less are clumps of 2 to 50 cells. Dispersed cells are those in which there is almost no adhesion between cells (for example, surface adhesion). The dispersion procedure of the cells obtained in the first step may include the above-mentioned mechanical dispersion treatment, cell dispersion treatment, and cell protective agent treatment. These treatments may also be performed in combination. Preferably, the cell dispersion treatment is performed simultaneously with the cell protective agent treatment, followed by the mechanical dispersion treatment. Examples of cytoprotective agents used in the cytoprotective agent treatment include substances acting on the FGF signaling pathway (e.g., fibroblast growth factors such as bFGF, FGF4, and FGF8), heparin, substances acting on the IGF signaling pathway (e.g., insulin), serum, or serum substitutes. Furthermore, a Rho-associated coiled-coil kinase (ROCK) inhibitor or a myosin inhibitor may be added as a cytoprotective agent to suppress cell death of pluripotent stem cells (particularly human pluripotent stem cells) induced by dissociation. To suppress cell death and protect pluripotent stem cells (particularly human pluripotent stem cells) induced by dissociation, a ROCK inhibitor or a myosin inhibitor may be added from the start of the second-step culture. Examples of ROCK inhibitors include Y-27632, Fasudil (HA1077), and H-1152. Examples of myosin inhibitors include Blebbistatin. Examples of cell dispersion solutions used in cell dispersion treatment include solutions containing enzymes such as trypsin, collagenase, hyaluronidase, elastase, pronase, DNase, or papain, or chelating agents such as ethylenediaminetetraacetic acid. Commercially available cell dispersion solutions, such as TrypLE Select (Life Technologies) or TrypLE Express (Life Technologies), can also be used. Methods for mechanical dispersion include pipetting and scraping with a scraper. The dispersed cells are suspended in the above medium. The dispersed cell suspension is then seeded in the above-mentioned culture vessel, and the dispersed cells are cultured under non-adhesive conditions to aggregate multiple cells. In this case, the dispersed cells may be seeded in a relatively large culture vessel, such as a 10 cm dish, to simultaneously form multiple cell aggregates in a single culture vessel. However, this results in variations in the size of each aggregate. Therefore, for example, if a certain number of dispersed stem cells are placed in each well of a multi-well plate (U-bottom, V-bottom), such as a 96-well plate, and the plate is subjected to static culture, the cells rapidly aggregate to form a single aggregate in each well. By collecting these aggregates from multiple wells, a uniform population of aggregates can be obtained (e.g., the SFEBq method). The cell concentration in the second step can be appropriately set so as to form cell aggregates more uniformly and efficiently. For example, when human cells (e.g., cells obtained from human iPS cells in the first step) are cultured in suspension in a 96-well plate, the cell concentration is about 1 x 10 per well. 3 From approximately 1 x 10 5 cells, preferably about 3 x 10 3 From about 5 x 10 4 cells, more preferably about 4 x 10 3 From about 2 x 10 4 cells, more preferably about 4 x 10 3 to approximately 1.6 x 10 4 cells, even more preferably about 8 x 10 3 to approximately 1.2 x 10 4 A solution prepared to form cells is added to the wells, and the plate is left to stand to allow aggregates to form. The culture conditions in the second step, such as culture temperature and CO2 concentration, can be set appropriately. 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%.

[0129] When a medium exchange operation is performed in the second step, examples include an operation in which new medium is added without discarding the original medium (medium addition operation), an operation in which about half of the original medium (about 30 to 90% of the volume of the original medium, for example, about 40 to 60%) is discarded and about half of the new medium (about 30 to 90% of the volume of the original medium, for example, about 40 to 60%) is added (half medium exchange operation), and an operation in which about the entire volume of the original medium (90% or more of the volume of the original medium) is discarded and about the entire volume of new medium (90% or more of the volume of the original medium) is added (full medium exchange operation). The tools used for the medium replacement operation are not particularly limited, and examples include pipettors, micropipettes, multichannel micropipettes, repeating dispensers, etc. For example, when a 96-well plate is used as the culture vessel, a multichannel micropipette may be used. The suspension culture time required to form cell aggregates can be determined appropriately depending on the cells used so as to uniformly aggregate the cells, but it is desirable to keep the time as short as possible to form uniform cell aggregates. The process by which dispersed cells form cell aggregates can be divided into a cell aggregation process and a cell aggregate formation process in which the aggregated cells form cell aggregates. For example, in the case of human cells (e.g., stem cells obtained from human iPS cells in the first process) the time from seeding the dispersed cells (i.e., at the start of suspension culture) to cell aggregation is preferably within about 24 hours, more preferably within about 12 hours. For example, in the case of human pluripotent stem cells (e.g., human iPS cells), the time from seeding the dispersed cells (i.e., at the start of suspension culture) to cell aggregate formation is preferably within about 72 hours, more preferably within about 48 hours. This time until aggregate formation can be adjusted appropriately by adjusting the cell aggregation tool, centrifugation conditions, etc. The formation of cell aggregates and their uniformity can be determined based on the size and cell number of the aggregates, macroscopic morphology, microscopic morphology and uniformity determined by tissue staining analysis, expression and uniformity of differentiated and undifferentiated markers, control of expression of differentiation markers and their synchronization, and reproducibility of differentiation efficiency between aggregates. After the formation of aggregates, the culture of the aggregates may be continued as is. The time for suspension culture in the second step may usually be continued until the substance acting on the BMP signaling pathway is added, and specifically, the time may usually be 12 hours to 6 days, and preferably 12 hours to 3 days.

[0130] Examples of the medium used in the second step include a medium containing an SHH signaling pathway agonist (see WO2016 / 063985), a medium containing a Wnt signaling pathway inhibitor, or a medium containing a Wnt signaling pathway inhibitor and an SHH signaling pathway agonist (see WO2017 / 183732). In the first step, pluripotent stem cells are treated with a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway agonist. In the second step, the cells obtained in the first step are subjected to suspension culture in a medium (preferably serum-free medium) containing an SHH signaling pathway agonist and / or a Wnt signaling pathway inhibitor to form aggregates. This further improves the quality of the aggregates and enhances their ability to differentiate into retinal tissue. Using these high-quality aggregates, aggregates containing retinal progenitor cells or neural retinal progenitor cells can be induced with high efficiency.

[0131] The substances acting on the SHH signaling pathway can be those described above. Preferably, the substance acting on the SHH signaling pathway is SHH protein, SAG, or PMA. The concentration of the substance acting on the SHH signaling pathway in the medium can be appropriately set within a range that achieves the above-mentioned effects. SAG is usually used at a concentration of 1 nM to 2000 nM, preferably 10 nM to 700 nM, and more preferably 30 nM to 600 nM. PMA is usually used at a concentration of 0.002 μM to 20 μM, preferably 0.02 μM to 2 μM. SHH protein is usually used at a concentration of 20 ng / mL to 1000 ng / mL, preferably 50 ng / mL to 300 ng / mL. When a substance acting on the SHH signaling pathway other than SHH protein, SAG, or PMA is used, it is desirably used at a concentration that exerts the same SHH signaling pathway activation effect as the above-mentioned concentration of SAG. The concentration of the substance acting on the SHH signaling pathway in the medium may be varied during the second step. For example, the concentration of the substance acting on the SHH signaling pathway may be set within the above range at the start of the second step, and then gradually or stepwise reduced by 40 to 60% every 2 to 4 days. The timing of adding the substance acting on the SHH signaling pathway to the medium is not particularly limited as long as the above-mentioned effects can be achieved, but the earlier the addition, the greater the effect. The substance acting on the SHH signaling pathway is added to the medium usually within 6 days, preferably within 3 days, more preferably within 1 day, and even more preferably at the time of the start of the second step.

[0132] The Wnt signaling pathway inhibitor is not particularly limited as long as it can inhibit signaling mediated by Wnt, and examples thereof include substances that act directly on Wnt or Wnt receptor (anti-Wnt neutralizing antibodies, anti-Wnt receptor neutralizing antibodies, etc.), substances that inhibit the expression of genes encoding Wnt or Wnt receptor (e.g., antisense oligonucleotides, siRNA, etc.), substances that inhibit the binding of Wnt receptor and Wnt (soluble Wnt receptor, dominant-negative Wnt receptor, Wnt antagonist, Dkk1, Cerberus protein, etc.), substances that inhibit physiological activity caused by signaling via Wnt receptor [CKI-7 (N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide), D4476 (4-[4-(2,3-dihydro-1,4-benzodioxin-6-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide), IWR-1-endo (IWR1e) (4-[(3aR,4S,7R,7aS)-1,3,3a,4,7,7a-hexahydro-1,3-dioxo-4,7-methano-2H-isoindol-2-yl]-N-8-quinolinyl-benzamide), and small molecular weight compounds such as IWP-2 (N-(6-methyl-2-benzothiazolyl)-2-[(3,4,6,7-tetrahydro-4-oxo-3-phenylthieno[3,2-d]pyrimidin-2-yl)thio]acetamide). IWR1e is preferably used as the Wnt signaling pathway inhibitor. The concentration of the Wnt signaling pathway inhibitor in the medium can be appropriately set within a range that achieves the above-mentioned effects. IWR1e is usually added to the medium to a concentration of about 0.1 μM to about 100 μM, preferably about 0.3 μM to about 30 μM, more preferably about 1 μM to about 10 μM, and even more preferably about 3 μM. When a Wnt signaling pathway inhibitor other than IWR-1-endo is used, it is desirably used at a concentration that exhibits Wnt signaling pathway inhibitory activity equivalent to the above-mentioned concentration of IWR-1-endo. The concentration of the Wnt signaling pathway inhibitor in the medium may be varied during the second step. For example, the concentration of the Wnt signaling pathway inhibitor may be set within the above range at the start of the second step, and then gradually or stepwise reduced by 40 to 60% every 2 to 4 days. The timing of adding the Wnt signaling pathway inhibitor to the medium is not particularly limited as long as the above-mentioned effects can be achieved, but the earlier the addition, the greater the effect. The Wnt signaling pathway inhibitor is usually added to the medium within 6 days, preferably within 3 days, more preferably within 1 day, more preferably within 12 hours, and even more preferably at the start of suspension culture in the second step. Specifically, for example, basal medium supplemented with the Wnt signaling pathway inhibitor can be added, or a partial or complete medium exchange with the basal medium can be performed. The period during which the cells obtained in the first step are exposed to the Wnt signaling pathway inhibitor in the second step is not particularly limited as long as the above-mentioned effects can be achieved, but preferably, the cells are added to the medium at the start of suspension culture in the second step and allowed to be exposed to the Wnt signaling pathway inhibitor until the end of the second step. Furthermore, the cells can be continuously exposed to the Wnt signaling pathway inhibitor even after the end of the second step (i.e., during the third step). In one embodiment, the Wnt signaling pathway inhibitor can be allowed to act on the cells continuously after the end of the second step (i.e., during the third step) until neuroepithelial tissue and / or neural tissue are formed.

[0133] In a preferred embodiment, the human cells obtained in the first step (e.g., cells obtained from human iPS cells in the first step) are subjected to suspension culture in a serum-free medium containing a substance acting on the SHH signaling pathway (e.g., SAG, PMA, SHH protein) and / or a substance inhibiting the Wnt signaling pathway (e.g., IWR1e) to form aggregates. The substance acting on the SHH signaling pathway is preferably contained in the medium from the start of suspension culture. A ROCK inhibitor (e.g., Y-27632) may also be added to the medium. The culture time is 12 hours to 6 days, preferably 12 hours to 3 days. The formed aggregates are preferably uniform aggregates.

[0134] For example, human cells obtained in the first step (e.g., cells obtained from human iPS cells in the first step) are collected, dispersed into single cells or a state close to single cells, and subjected to suspension culture in a serum-free medium containing an agent acting on the SHH signaling pathway (e.g., SAG, PMA) and / or an agent inhibiting the Wnt signaling pathway (e.g., IWR1e). The serum-free medium may also contain a ROCK inhibitor (e.g., Y-27632). A suspension of human stem cells (e.g., stem cells derived from human iPS cells) is seeded in the above-mentioned culture vessel, and the dispersed cells are cultured under non-adhesive conditions to form aggregates. The culture time is 12 hours to 6 days (preferably 12 hours to 3 days). The aggregates formed are preferably uniform. In this manner, by carrying out the second step, aggregates of the cells obtained in the first step or cells derived therefrom are formed. The aggregates obtained in the second step have higher quality than those obtained without treatment with a TGFβ family signaling pathway inhibitor and / or an SHH signaling pathway agonist in the first step. Specifically, a population of aggregates can be obtained that is round, has a smooth surface, is dense inside, and is enriched in aggregates that do not lose their shape. In one embodiment, when aggregates (e.g., 100 or more) are randomly selected on day 6 from the start of the second step, the proportion of aggregates that do not form cysts is, for example, 70% or more, preferably 80% or more. The aggregates obtained in the second step have the ability to differentiate into retinal tissue. In a preferred embodiment, in the first step, pluripotent stem cells are treated with a TGFβ signaling pathway inhibitor, and in the second step, the cells obtained in the first step are suspension cultured in a medium containing an SHH signaling pathway agonist (e.g., SAG, PMA, SHH protein) and / or a Wnt signaling pathway inhibitor (e.g., IWR1e). Preferably, SB431542 or A-83-01 can be used as the TGFβ signaling pathway inhibitor. In a preferred embodiment, in the first step, pluripotent stem cells are treated with a BMP signaling pathway inhibitor, and in the second step, the cells obtained in the first step are suspension cultured in a medium that does not contain an SHH signaling pathway agonist (e.g., SAG, PMA, SHH protein). Preferably, LDN193189 can be used as the BMP signaling pathway inhibitor. In a preferred embodiment, in the first step, pluripotent stem cells (e.g., human pluripotent stem cells) are treated with a TGFβ family signaling pathway inhibitor (e.g., a TGFβ signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189), or a combination thereof (e.g., SB431542 and LDN193189), etc. ); or a combination of a TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189) and a substance active in the SHH signaling pathway (e.g., SHH protein, SAG, PMA), and in the second step, the cells obtained in the first step are subjected to suspension culture in a medium containing a substance active in the SHH signaling pathway (e.g., SAG, PMA, SHH protein). In another aspect, in the first step, pluripotent stem cells (e.g., human pluripotent stem cells) are treated with a TGFβ family signaling pathway inhibitor (e.g., a TGFβ signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189), or a combination thereof (e.g., SB431542 and LDN193189)); The cells are treated with a substance active in the HH signaling pathway (e.g., SHH protein, SAG, PMA); or a combination of a TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189) and a substance active in the SHH signaling pathway (e.g., SHH protein, SAG, PMA), and in the second step, suspension culture of the cells obtained in the first step is carried out in a medium that does not contain a substance active in the SHH signaling pathway (e.g., SAG, PMA, SHH protein). In either embodiment, the medium in the second step preferably contains a ROCK inhibitor (eg, Y-27632).

[0135] [Regarding the third step] The aggregates formed in the second step are subjected to suspension culture in the presence of a substance acting on the BMP signaling pathway to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells. This step can be carried out in accordance with the second step in the above-mentioned raw material production method 1. In one embodiment, when the concentration of the substance acting on the SHH signaling pathway added to the medium in the second step is relatively low (e.g., 700 nM or less for SAG, or a concentration that activates the SHH signaling pathway equivalent to or less than that of SAG at the aforementioned concentration for other substances acting on the SHH signaling pathway), it is not necessary to change the medium, and a substance acting on the BMP signaling pathway (e.g., BMP4) can be added to the medium used in the second step. On the other hand, when the concentration of the substance acting on the SHH signaling pathway is relatively high (e.g., more than 700 nM for SAG or 1000 nM or more, or a concentration that activates the SHH signaling pathway equivalent to that of SAG at the aforementioned concentration for other substances acting on the SHH signaling pathway), it is desirable to change the medium to fresh one containing a substance acting on the BMP signaling pathway (e.g., BMP4) to suppress the effects of any remaining substance acting on the SHH signaling pathway at the time of addition of the substance acting on the BMP signaling pathway. In a preferred embodiment, the concentration of the substance acting on the SHH signaling pathway in the medium used in the third step is 700 nM or less, preferably 300 nM or less, more preferably 10 nM or less, even more preferably 0.1 nM or less, and even more preferably does not contain any substance acting on the SHH signaling pathway, in terms of the SHH signaling promoting activity of SAG. A medium "free of a substance acting on the SHH signaling pathway" refers to a medium that is substantially free of a substance acting on the SHH signaling pathway, for example, a medium that contains a substance acting on the SHH signaling pathway at a concentration that adversely affects selective differentiation into retinal progenitor cells and retinal tissue. A medium "not supplemented with an SHH signaling pathway active substance" also includes a medium to which an SHH signaling pathway active substance has not been added substantially, for example, a medium to which an SHH signaling pathway active substance has not been added at a concentration that would adversely affect selective differentiation into retinal progenitor cells and retinal tissue. In a preferred embodiment for producing retinal tissue at an early stage of development, in the first step, human pluripotent stem cells (e.g., human iPS cells) are cultured in an adherent manner in a serum-free medium containing a TGFβ signaling pathway inhibitor (e.g., SB431542, A-83-01) and bFGF in the absence of feeder cells; in the second step, the cells are cultured in suspension in a serum-free medium containing an SHH signaling pathway active substance (e.g., SAG, PMA, SHH protein); and in the third step, the aggregates are cultured in suspension in a serum-free medium containing a BMP signaling pathway active substance (e.g., BMP4). Furthermore, in a preferred embodiment for producing retinal tissue at an early stage of development, in the first step, human pluripotent stem cells (e.g., human iPS cells) are cultured in an adherent manner in a serum-free medium containing a BMP signaling pathway inhibitor (e.g., LDN193189) and bFGF in the absence of feeder cells; in the second step, the cells are cultured in suspension in a serum-free medium containing or not containing a substance acting on the SHH signaling pathway (e.g., SAG, PMA); and in the third step, the aggregates are cultured in suspension in a serum-free medium containing a substance acting on the BMP signaling pathway (e.g., BMP4). In a preferred embodiment for producing retinal tissue at an early stage of development, in the first step, human pluripotent stem cells (e.g., human iPS cells) are cultured in an adhesion manner in a serum-free medium containing a substance acting on the SHH signaling pathway (e.g., SAG, PMA) and bFGF in the absence of feeder cells, preferably for 1 to 6 days, more preferably 2 to 4 days; in the second step, the cells are cultured in suspension in a serum-free medium containing a substance acting on the SHH signaling pathway (e.g., SAG, PMA); and in the third step, the aggregates are cultured in suspension in a serum-free medium containing a substance acting on the BMP signaling pathway (e.g., BMP4). In a preferred embodiment of the method for producing retinal tissue at an early stage of development, in the first step, human pluripotent stem cells (e.g., human iPS cells) are cultured in the absence of feeder cells with a TGFβ family signaling pathway inhibitor (e.g., a TGFβ signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a Nodal / Activin signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01), a BMP signaling pathway inhibitor (e.g., LDN193189), or a combination thereof (e.g., SB431542 and LDN193189)); an SHH signaling pathway agonist (e.g., SHH protein, SAG, PMA); or a TGFβ family signaling pathway inhibitor (e.g., Lefty, SB431542, A-83-01, LDN193189). and a substance acting on the SHH signaling pathway (e.g., SHH protein, SAG, PMA); and bFGF. In a second step, the cells obtained in the first step are cultured in suspension in a serum-free medium containing a substance acting on the SHH signaling pathway (e.g., SAG, PMA, SHH protein) to form cell aggregates. In a third step, the aggregates are cultured in suspension in a serum-free medium containing a substance acting on the BMP signaling pathway (e.g., BMP4) to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells.

[0136] 4-3. Raw material manufacturing method 3 A preferred embodiment of the method for producing retinal tissue at an early stage of development includes a method described in WO2016 / 063986, which comprises the following steps: (1) a first step of culturing pluripotent stem cells in a medium containing factors for maintaining undifferentiated state in the absence of feeder cells; (2) a second step of culturing the pluripotent stem cells obtained in the first step in suspension in the presence of an agent acting on the SHH signaling pathway to form cell aggregates; and (3) A third step in which the aggregates obtained in the second step are cultured in suspension in the presence of a substance acting on the BMP signaling pathway to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells.

[0137] [Regarding the first step] The first step can be carried out according to the method described in WO2016 / 063986. That is, in the first step, human pluripotent stem cells, preferably human induced pluripotent stem cells (iPS cells) or human embryonic stem cells (ES cells), are cultured in a medium containing a factor for maintaining undifferentiated state in the absence of feeder cells. In the first step, "feeder-free" refers to conditions in which feeder cells are substantially absent (for example, the ratio of the number of feeder cells to the total number of cells is 3% or less). Preferably, the first step is carried out under conditions in which feeder cells are not present. The medium used in the first step is not particularly limited as long as it is a medium that allows pluripotent stem cells to be cultured under feeder-free conditions and maintain the undifferentiated state (feeder-free medium), but preferably contains factors for maintaining the undifferentiated state so that the culture can maintain the undifferentiated state. For example, a medium containing factors for maintaining the undifferentiated state but not containing an inhibitor of the TGFβ family signaling pathway or a substance acting on the SHH signaling pathway can be used. Examples of undifferentiated maintenance factors and feeder-free media include those described in the raw material production method 2 above. The culture time for pluripotent stem cells in the first step is not particularly limited as long as it is within a range that can achieve the effect of improving the quality of the aggregates formed in the second step, but is usually 0.5 to 144 hours, preferably 2 to 96 hours, more preferably 6 to 48 hours, even more preferably 12 to 48 hours, and even more preferably 18 to 28 hours (e.g., 24 hours). That is, the first step is started 0.5 to 144 hours (preferably 18 to 28 hours) before the start of the second step, and the second step is carried out immediately after the first step is completed. In the first step, the medium may be replaced as needed, specifically every 1 to 2 days, for example, with a medium that does not contain a cell protective agent or a cell death inhibitor such as a ROCK inhibitor. The culture conditions in the first step, such as culture temperature and CO2 concentration, can be set appropriately. 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%. In a preferred embodiment, human pluripotent stem cells (e.g., human iPS cells) are cultured in an adherent manner in a serum-free medium containing bFGF in the absence of feeder cells. The adherent culture is preferably carried out in a cell container whose surface is coated with laminin-511, the E8 fragment of laminin-511, or vitronectin. The adherent culture is preferably carried out using Essential 8, TeSR medium, mTeSR medium, mTeSR-E8 medium, or StemFit medium as the feeder-free medium, more preferably Essential 8 or StemFit medium.

[0138] [Regarding the second step] The second step, in which the pluripotent stem cells obtained in the first step are cultured in suspension in the presence of a substance acting on the SHH signaling pathway to form pluripotent stem cell aggregates, may be carried out in accordance with the method described in the second step of the above-mentioned raw material production method 2.

[0139] [Regarding the third step] The third step can be carried out in accordance with the second step in the raw material production method 1 or the third step in the raw material production method 2.

[0140] 4-4. Raw material manufacturing method 4 A preferred embodiment of producing retinal tissue at an early stage of development includes a method described in WO2013 / 077425, which comprises the following steps: (1) a first step of forming pluripotent stem cell aggregates by suspension culture of pluripotent stem cells in a serum-free medium containing a Wnt signaling pathway inhibitor; (2) A second step in which the aggregates formed in the first step are cultured in suspension in a serum-free medium containing a basement membrane preparation to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells. Raw material production method 4 can be carried out in accordance with the description in WO2013 / 077425 (& US2014 / 341864).

[0141] [Regarding the first step] Examples of Wnt signaling pathway inhibitors include those mentioned above. The concentration of the Wnt signaling pathway inhibitor used here may be any concentration that allows pluripotent stem cell aggregates to be formed, for example, in the case of a typical Wnt signaling pathway inhibitor such as IWR1e, the concentration is 0.1 μM to 100 μM, preferably 1 μM to 10 μM, and more preferably about 3 μM. The Wnt signaling pathway inhibitor may be added to the serum-free medium before the start of suspension culture, or may be added to the serum-free medium within a few days (e.g., within 5 days) after the start of suspension culture. Preferably, the Wnt signaling pathway inhibitor is added to the serum-free medium within 5 days, more preferably within 3 days, and most preferably simultaneously with the start of suspension culture. Furthermore, suspension culture is continued with the addition of the Wnt signaling pathway inhibitor until day 18, more preferably until day 12, after the start of suspension culture. Culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature is not particularly limited, but 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%. Furthermore, those skilled in the art can appropriately set the concentration of pluripotent stem cells so as to form pluripotent stem cell aggregates more uniformly and efficiently. The concentration of pluripotent stem cells during aggregate formation is not particularly limited as long as it is a concentration that allows the formation of uniform stem cell aggregates. For example, when human ES cells are cultured in suspension on a 96-well plate, the concentration of pluripotent stem cells per well is about 1 × 10 3 ~Approx. 5×10 4 cells, preferably about 3 x 10 3 ~Approx. 3×10 4 cells, more preferably about 5 x 10 3 ~Approx. 2×10 4 cells, most preferably 9 x 10 3 A solution prepared so as to be in front of or behind the cells is added, and the plate is left to stand to form aggregates. The suspension culture time required for aggregate formation can be determined appropriately depending on the pluripotent stem cells used, as long as the cells can be rapidly aggregated; however, it is desirable to keep the time as short as possible to form uniform aggregates (e.g., the SFEBq method). For example, in the case of human ES cells or human iPS cells, it is desirable to form aggregates preferably within 24 hours, more preferably within 12 hours. Those skilled in the art can adjust the time required for aggregate formation as appropriate by adjusting the cell aggregation tool, centrifugation conditions, etc. Those skilled in the art can determine whether pluripotent stem cell aggregates have been formed based on the size and cell number of the aggregates, their macroscopic morphology, the microscopic morphology and its uniformity determined by tissue staining analysis, the expression and uniformity of differentiated and undifferentiated markers, the control and synchronization of expression of differentiation markers, and the reproducibility of differentiation efficiency between aggregates.

[0142] [Regarding the second step] The second step involves suspension culture of the aggregates formed in the first step in a serum-free medium containing a basement membrane preparation to obtain aggregates containing retinal progenitor cells or neural retinal progenitor cells. A "basement membrane preparation" refers to a preparation containing basement membrane components that, when desired cells capable of forming a basement membrane are seeded and cultured, regulate epithelial cell-like cell morphology, differentiation, proliferation, motility, and functional expression. Here, "basement membrane components" refers to thin membrane-like extracellular matrix molecules present between the epithelial cell layer and the interstitial cell layer in animal tissues. Basement membrane preparations can be prepared, for example, by removing cells capable of forming a basement membrane that are attached to a support via a basement membrane using a solution capable of dissolving lipids from the cells or an alkaline solution. Preferred basement membrane preparations include commercially available products containing basement membrane components (e.g., Matrigel (hereinafter sometimes referred to as Matrigel)) and preparations containing extracellular matrix molecules known as basement membrane components (e.g., laminin, type IV collagen, heparan sulfate proteoglycan, entactin, etc.). Matrigel is a basement membrane preparation derived from the Engelbreth Holm Swarn (EHS) murine sarcoma. Its main components are type IV collagen, laminin, heparan sulfate proteoglycan, and entactin, but it also contains TGF-β, fibroblast growth factor (FGF), tissue plasminogen activator, and growth factors naturally produced by EHS tumors. The "growth factor reduced" Matrigel formulation contains lower concentrations of growth factors than standard Matrigel, typically <0.5 ng / mL EGF, <0.2 ng / mL NGF, <5 pg / mL PDGF, 5 ng / mL IGF-1, and 1.7 ng / mL TGF-β. For raw material manufacturing method 4, the "growth factor reduced" formulation is preferred. The concentration of the basement membrane preparation added to the serum-free medium for suspension culture in the second step is not particularly limited as long as the epithelial structure of neural tissue (e.g., retinal tissue) is stably maintained, but when Martigel is used, for example, the concentration can be preferably 1 / 20 to 1 / 200 of the volume of the culture medium, more preferably about 1 / 100 of the volume. The basement membrane preparation may be added to the medium already at the start of culture of pluripotent stem cell aggregates, but is preferably added to the serum-free medium within 5 days, more preferably within 2 days, after the start of suspension culture. The serum-free medium used in the second step can be the same as that used in the first step, or can be replaced with a new serum-free medium. When the serum-free medium used in the first step is used in this step as is, the "basement membrane preparation" can be added to the medium. The serum-free medium used for suspension culture in the first and second steps is not particularly limited as long as it is as described above. However, from the viewpoint of avoiding the cumbersome preparation process, it is preferable to use a commercially available serum-free medium (GMEM or DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid mix, 1 mM sodium pyruvate) supplemented with an appropriate amount of KSR. The amount of KSR added to the serum-free medium is not particularly limited; for example, in the case of human ES cells, it is usually 1 to 20%, preferably 2 to 20%. The culture conditions in the second step, such as the culture temperature and CO2 concentration, can be set appropriately. The culture temperature is not particularly limited, but 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%.

[0143] The aggregates obtained by the second step can be used as retinal tissue at an early stage of development. However, in order to increase the content of retinal progenitor cells or neural retinal progenitor cells, the aggregates can be cultured in suspension in a serum-free medium containing a basement membrane preparation, and then the following third step can be carried out, and the resulting aggregates can be used as retinal tissue at an early stage of development: (3) A third step of culturing the aggregates cultured in the second step in suspension in a serum-containing medium. The serum medium used in the third step may be the serum-free medium used for culture in the second step to which serum has been directly added, or may be replaced with a new serum medium. The serum to be added to the medium in the third step may be, for example, mammalian serum such as bovine serum, calf serum, fetal bovine serum, horse serum, foal serum, fetal horse serum, rabbit serum, baby rabbit serum, fetal rabbit serum, or human serum. Serum is added after the 7th day, more preferably after the 9th day, and most preferably after the 12th day of suspension culture (i.e., the first step). The serum is added at a concentration of 1 to 30%, preferably 3 to 20%, and more preferably about 10%. The serum-containing medium used in the third step is not particularly limited as long as it is as described above, but it is preferable to use the serum-free medium (GMEM or DMEM, 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acid mix, 1 mM sodium pyruvate) to which serum has been added. Furthermore, such serum medium may be used by adding an appropriate amount of a commercially available serum substitute such as KSR. In the third step, the production efficiency of retinal tissue at the early developmental stage can be increased by adding an SHH signaling pathway active substance in addition to serum. The substance acting on the SHH signaling pathway is not particularly limited as long as it is capable of enhancing signaling mediated by SHH, and includes those mentioned above. The concentration of the substance acting on the SHH signaling pathway used in this step is 0.1 nM to 10 μM, preferably 10 nM to 1 μM, and more preferably about 100 nM, in the case of a conventional substance acting on the SHH signaling pathway, such as SAG. The aggregates thus obtained can also be used as retinal tissue at an early stage of development.

[0144] Furthermore, in a preferred embodiment of producing retinal tissue at an early stage of development, after the third step, the following fourth step can be carried out, and the resulting optic-cup-like structure can be used as retinal tissue at an early stage of development: (4) A fourth step of culturing the aggregates cultured in the third step in suspension in a serum-free medium or serum-containing medium containing a substance acting on the SHH signaling pathway and a substance acting on the Wnt signaling pathway. Here, the substance acting on the SHH signaling pathway is not particularly limited as long as it is capable of enhancing signaling mediated by SHH, and includes those mentioned above. The concentration of the substance acting on the SHH signaling pathway used here, for example, in the case of a conventional substance acting on the SHH signaling pathway such as SAG, is added at a concentration of 0.1 nM to 10 μM, preferably 10 nM to 1 μM, and more preferably about 100 nM. The substance acting on the Wnt signaling pathway is not particularly limited as long as it is capable of enhancing signaling mediated by Wnt, and examples thereof include those mentioned above. The concentration of the substance acting on the Wnt signaling pathway used here, for example, in the case of a typical substance acting on the Wnt signaling pathway such as CHIR99021, is added at a concentration of 0.1 μM to 100 μM, preferably 1 μM to 30 μM, and more preferably about 3 μM. The substance acting on the SHH signaling pathway and the substance acting on the Wnt signaling pathway are added within 12 to 25 days after the start of suspension culture (start of the first step), preferably within 15 to 18 days. In this case, it is preferable to use a medium that does not contain the Wnt signaling pathway inhibitor added in the aggregate formation step. After 18 days from the start of suspension culture, optic-cup-like structures are formed in the form of protrusions from the aggregates. The optic-cup-like structures produced by the fourth step can also be used as retinal tissue in the early developmental stage, which serves as the starting material for methods 2 and 3 above.

[0145] The aggregates obtained in the fourth step can be cultured in suspension for 1 to 20 days in a serum-free medium or serum-free medium that does not contain either a substance acting on the SHH signaling pathway or a substance acting on the Wnt signaling pathway, and then used as retinal tissue in the early developmental stage as the starting material for the methods used in steps 2 and 3 above. This method for producing a raw material may also simultaneously form neural tissue other than retinal tissue, which may express dorsalizing signaling substances such as substances acting on the Wnt signaling pathway. Therefore, preferably, to eliminate the effects of excessive dorsalizing signaling substances such as substances acting on the Wnt signaling pathway, the optic cup-like structures present on the surface of the aggregates can be physically excised from the aggregates using tweezers, scissors, a syringe needle, a razor, or similar tools.

[0146] 4-5. Raw material manufacturing method 5 The retinal tissue at an early stage of development may contain ciliary marginal structures, and the retinal tissue at an early stage of development containing ciliary marginal structures can be produced by the methods described in WO2015 / 087614 (& US2016 / 376554). Specifically, cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% to 100% (for example, in Raw Material Production Methods 1 to 4, cell aggregates corresponding to about 9 to 60 days, preferably 9 to 40 days, more preferably about 15 to 20 days, for example, 18 days, after the initiation of suspension culture) can be cultured in a serum-free or serum-based medium containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway, for a period until cells expressing the RPE65 gene appear. Alternatively, aggregates containing ciliary marginal zone-like structures can be obtained by culturing the resulting "cell aggregates in which cells expressing the RPE65 gene have not appeared" in a serum-free or serum-based medium not containing a substance acting on the Wnt signaling pathway. These aggregates can also be used as the starting material for the retinal tissue in the early developmental stage used in the methods 2 and 3 above. Specifically, aggregates containing ciliary marginal structures, prepared by the following method, for example, are also included in retinal tissue at an early stage of development: (1) A method for producing an aggregate containing ciliary margin-like structures, comprising the steps of culturing a cell aggregate containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less, in a serum-free medium or serum medium containing a substance that acts on the Wnt signaling pathway and a substance that inhibits the FGF signaling pathway, for a period until cells that express the RPE65 gene appear, and then culturing the resulting "cell aggregate in which cells that express the RPE65 gene do not appear" in a serum-free medium or serum medium that does not contain a substance that acts on the Wnt signaling pathway.

[0147] "Cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less" can be obtained by the methods described in the above-mentioned raw material production methods 1 to 4. That is, the cell aggregates themselves are aggregates containing retinal tissue in an early stage of development. For example, in the second step of raw material production method 1 or the third step of raw material production method 2 or 3, retinal tissue in an early stage of development can be obtained by culturing for 6 to 15 days in the presence of a substance acting on the BMP signaling pathway, such as BMP4, thereby obtaining "cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less." Furthermore, the above-mentioned "step of culturing in a serum-free or serum-containing medium containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway, for a period limited to the time until cells expressing the RPE65 gene appear" is preferably initiated by continuously culturing in a serum-free or serum-containing medium containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway (for example, for 30 days or more) until 50% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more of the cells contained in the retinal tissue are able to express the RPE65 gene, i.e., until the above percentage of cells contained in the retinal tissue are able to differentiate into retinal pigment epithelium. Specifically, the culture is initiated within 40 days, preferably 30 days, and more preferably 20 days after the start of suspension culture. The cell aggregates obtained in this manner can be used in this process as "cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less." First, "cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% to 100%" are cultured in a serum-free or serum-free medium containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway, for a period until cells expressing the RPE65 gene appear, according to the method described in WO2015 / 087614. Here, suspension culture is a preferred example of the culture method. Examples of serum-free media include those prepared by adding N2 or KSR to a basal medium, more specifically, those prepared by adding N2 supplement (N2, Invitrogen) to DMEM / F-12 medium. Examples of serum-free media include those prepared by adding fetal bovine serum to a basal medium. Culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature can be, for example, in the range of about 30°C to about 40°C. A preferred example is about 37°C. The CO2 concentration can be, for example, in the range of about 1% to about 10%. A preferred example is about 5%. When the cell aggregates are cultured in a serum-free medium or a serum-containing medium, the Wnt signaling pathway active substance contained in the medium is not particularly limited as long as it is capable of enhancing signaling mediated by Wnt, and examples thereof include those listed above. The concentration of a substance acting on the Wnt signaling pathway contained in a serum-free medium or serum medium may range, for example, from about 0.1 μM to about 100 μM in the case of a typical substance acting on the Wnt signaling pathway, such as CHIR99021. A preferred range is, for example, from about 1 μM to about 30 μM. A more preferred concentration is, for example, about 3 μM. When the "cell aggregates containing retinal tissue" are cultured in a serum-free medium or a serum-based medium, the FGF signaling pathway inhibitor contained in the medium is not particularly limited, as long as it can inhibit FGF-mediated signal transduction. Examples of FGF signaling pathway inhibitors include FGF receptors, FGF receptor inhibitors (e.g., SU-5402, AZD4547, BGJ398), MAP kinase cascade inhibitors (e.g., MEK inhibitors, MAPK inhibitors, ERK inhibitors), PI3 kinase inhibitors, and Akt inhibitors. The concentration of the FGF signaling pathway inhibitor contained in the serum-free medium or serum medium may be any concentration that can induce differentiation of cells forming pluripotent stem cell aggregates into retinal cells. For example, in the case of SU-5402, it is added at a concentration of about 0.1 μM to about 100 μM, preferably about 1 μM to about 30 μM, and more preferably about 5 μM. As used herein, "culturing only during the period until cells expressing the RPE65 gene appear" means culturing only during all or part of the period until cells expressing the RPE65 gene appear. In other words, the "cell aggregates containing retinal tissue" present in the culture system may be cultured only during all or part of the period (any period) during which the cells do not substantially express the RPE65 gene, and by employing such culture, cell aggregates in which cells expressing the RPE65 gene do not appear can be obtained. "Cell aggregates in which cells expressing the RPE65 gene are not present" include "cell aggregates in which no cells expressing the RPE65 gene are present at all" and "cell aggregates in which cells expressing the RPE65 gene are not substantially present." "Cell aggregates in which cells expressing the RPE65 gene are not substantially present" include cell aggregates in which the proportion of RPE65-positive cells in the retinal tissue contained in the cell aggregate is approximately 1% or less. To set such a specific period, the presence or absence of RPE65 gene expression in the "cellular aggregate containing retinal tissue" can be measured using conventional genetic engineering or biochemical techniques. Specifically, for example, frozen sections of the "cellular aggregate containing retinal tissue" can be immunostained with an antibody against the RPE65 protein to determine the presence or absence of RPE65 gene expression. The "period until cells expressing the RPE65 gene appear" can be, for example, the period until the proportion of CHX10-positive cells in the retinal tissue decreases to within the range of 30% to 0% compared to the time when the cell aggregates were cultured in a serum-free medium or serum medium containing a substance acting on the Wnt signaling pathway and a substance inhibiting the FGF signaling pathway. The "cell aggregates in which cells expressing the RPE65 gene do not appear" can be, for example, cell aggregates in which the proportion of CHX10-positive cells in the retinal tissue is within the range of 30% to 0%. The number of days in the "period until cells expressing the RPE65 gene appear" varies depending on the type of substance acting on the Wnt signaling pathway and the substance inhibiting the FGF signaling pathway, the type of serum-free or serum-based medium, other culture conditions, etc., but can be, for example, within 14 days. More specifically, when a serum-free medium (e.g., a serum-free medium in which N2 is added to a basal medium) is used, the period is preferably, for example, within 10 days, more preferably, for example, within 2 to 6 days, and even more specifically, for example, within 3 to 5 days. When a serum-based medium (e.g., a serum-based medium in which fetal bovine serum is added to a basal medium) is used, the period is preferably, for example, within 12 days, more preferably, for example, within 6 to 9 days. The aggregates thus obtained can be used as retinal tissue at an early stage of development, which serves as the starting material for the methods used in 2 and 3 above. Next, the "cell aggregates in which cells expressing the RPE65 gene have not appeared" obtained by culturing as described above may be further cultured in a serum-free medium or serum medium that does not contain a substance acting on the Wnt signaling pathway for 1 to 50 days (corresponding to "from the early developmental stage to the stage at which the appearance rate of cone photoreceptor precursor cells reaches its maximum"), preferably for 1 to 15 days (corresponding to within about 5 days after ganglion cells begin to appear), and more preferably for 1 to 7 days (corresponding to the stage at which ganglion cells begin to appear), and then used as retinal tissue in the early developmental stage that serves as the starting material for methods 2 and 3 above; for details of this culture method, see WO2015 / 087614 (e.g., paragraphs

[0076] to

[0079] ).

[0148] 4-6. Raw material manufacturing method 6 Retinal tissue at an early stage of development containing ciliary marginal structures that can be used as a starting material for the production method of the present invention can also be produced by the method described in WO2013 / 183774 (&US2015 / 132787). Specifically, cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less, are cultured in a serum-free medium or serum medium containing a substance acting on the Wnt signaling pathway, for a period until cells expressing the RPE65 gene appear; or aggregates containing ciliary margin-like structures obtained by further culturing the resulting "cell aggregates in which cells expressing the RPE65 gene have not appeared" in a serum-free medium or serum medium not containing a substance acting on the Wnt signaling pathway, are also retinal tissue in an early stage of development.

[0149] The "cell aggregates containing retinal tissue, in which the proportion of CHX10-positive cells in the retinal tissue is 20% or more and 100% or less" or "Wnt signaling pathway active substances" used as raw materials here include the same as those used in raw material production method 5 above. A preferred culture method is, for example, suspension culture, and a preferred medium is, for example, serum-free medium. Culture conditions such as culture temperature and CO2 concentration can be set appropriately. The culture temperature can be, for example, in the range of about 30°C to about 40°C, and preferably, for example, about 37°C. The CO2 concentration can be, for example, in the range of about 1% to about 10%, and preferably, for example, about 5%. The substance acting on the Wnt signaling pathway to be contained in the medium is not particularly limited as long as it is capable of enhancing signaling mediated by Wnt, and examples thereof include those listed above. Furthermore, the concentration of a substance acting on the Wnt signaling pathway contained in a serum-free medium or serum medium can be, for example, in the range of about 0.1 μM to 100 μM in the case of a typical substance acting on the Wnt signaling pathway, such as CHIR99021. Preferably, the concentration can be, for example, in the range of about 1 μM to 30 μM. More preferably, the concentration can be, for example, about 3 μM. The cell aggregates are cultured in the same manner as in Production Method 5, except that they do not need to contain an FGF signaling pathway inhibitor, and are "cultured only until cells expressing the RPE65 gene appear." A preferred "period until cells expressing the RPE65 gene appear" is, for example, the period during which the proportion of CHX10-positive cells in the retinal tissue is within the range of 50% to 1%. In this case, the resulting "cell aggregate in which cells expressing the RPE65 gene have not appeared" will be a cell aggregate in which the proportion of CHX10-positive cells in the retinal tissue is within the range of 50% to 1%. The number of days in the "period until cells expressing the RPE65 gene appear" varies depending on the type of agent acting on the Wnt signaling pathway, the type of serum-free or serum-containing medium, other culture conditions, etc., but can be, for example, within 14 days. More specifically, when a serum-free medium (e.g., a serum-free medium in which N2 is added to a basal medium) is used, the period is preferably, for example, within 10 days, more preferably, for example, within 2 to 6 days, and even more specifically, for example, within 3 to 5 days. When a serum-containing medium (e.g., a serum-containing medium in which fetal bovine serum is added to a basal medium) is used, the period is preferably, for example, within 12 days, more preferably, for example, within 6 to 9 days. The aggregates thus obtained can be used as retinal tissue at an early stage of development, which serves as the starting material for the methods used in 2 and 3 above. Subsequently, the "cell aggregates in which cells expressing the RPE65 gene have not appeared" obtained by culturing as described above may be used as retinal tissue at an early stage of development as is, or may be further cultured in a serum-free medium or serum medium not containing a substance acting on the Wnt signaling pathway for 1 to 50 days, preferably 1 to 15 days, and more preferably 1 to 7 days, before being used as aggregates containing retinal tissue at an early stage of development; for details of such culturing methods, see WO2015 / 087614 (e.g., paragraphs

[0076] to

[0079] ).

[0150] 4-7. Raw material manufacturing method 7 The retinal tissue at an early stage of development may contain ciliary marginal structures, and the retinal tissue at an early stage of development containing ciliary marginal structures can be produced by the method described in WO2015 / 107738 (and U.S. Patent Application No. 15 / 112,187). Specifically, for example, retinosphers prepared by a method comprising the steps of: (1) A step of culturing cells obtained from cell aggregates containing ciliary marginal zone-like structures induced to differentiate from pluripotent stem cells in a suspension culture to obtain retinospheric cells.

[0151] That is, "cell aggregates containing ciliary marginal zone-like structures induced to differentiate from pluripotent stem cells" can be produced according to the above-mentioned raw material production method 5 or 6, and the cells obtained from these can be dispersed and cultured in suspension to obtain retinosphers. Examples of such cells include cells obtained by dispersing the above-mentioned "cell aggregates containing ciliary marginal zone-like structures differentiated from pluripotent stem cells," cells obtained by dispersing ciliary marginal zone-like structures separated from the cell aggregates, and cells obtained by dispersing cells sorted from the cell aggregates. When such cells are cultured in suspension at low density in the presence of growth factors and the like, spherical cell aggregates derived from a single cell or a small number of cells (approximately 2 to 10 cells), i.e., retinospheric cells, are formed. For methods of producing retinospheric cells, see WO2015 / 107738 (and U.S. Patent Application No. 15 / 112,187). Specifically, the dispersed cells can be cultured in suspension in a serum-free or serum-based medium supplemented with neuronal culture additives and growth factors. The medium preferably includes a serum-free or serum-based medium containing one or more substances selected from the group consisting of substances acting on the FGF signaling pathway and substances acting on the EGF signaling pathway. Substances acting on the FGF signaling pathway include FGF proteins such as FGF1, bFGF, FGF4, FGF7, FGF8, and FGF9, and FGF signaling aids such as heparin. Substances acting on the EGF signaling pathway include EGF and TGF-alpha. The retinosphers produced as described above contain retinal progenitor cells or neural retinal progenitor cells, just like retinal tissue, and can therefore be used as retinal tissue at an early stage of development as a starting material for the production method of the present invention. Furthermore, retinosphers cultured in suspension in a serum-free or serum-based medium containing a substance acting on the BMP signaling pathway (e.g., BMP4) after step (1) above can also be used as retinal tissue at an early stage of development as a starting material for the production method of the present invention. The retinosphers obtained as described above may then be further cultured in a serum-free or serum-based medium not containing a substance acting on the Wnt signaling pathway for 1 to 50 days, preferably 1 to 15 days, and more preferably 1 to 7 days, and the resulting cell aggregates may be used as retinal tissue at an early stage of development.

[0152] 5. Retinal tissue with a high proportion of cone photoreceptors The present invention provides retinal tissue containing photoreceptor precursors rich in cone photoreceptor precursors and / or photoreceptors rich in cone photoreceptors (hereinafter referred to as retinal tissue with a high proportion of cone photoreceptors). That is, the present invention provides retinal tissue containing photoreceptor precursors rich in cone photoreceptor precursors and / or photoreceptors rich in cone photoreceptors, in which the number of cone photoreceptor precursors and cone photoreceptors among the photoreceptor precursors and photoreceptors is at least two times, preferably at least four times, and more preferably at least five times, the number of rod photoreceptor precursors and rod photoreceptors. Specifically, the retinal tissue contains ganglion cells in which the number of CRX-positive, TRβ2-positive, and NRL-negative cells or the number of CRX-positive, RXR-γ-positive, and NRL-negative cells is at least two times, preferably at least four times, and more preferably at least five times the number of CRX-positive and NRL-positive cells. The retinal tissue may have undergone differentiation induction to the extent that it includes retinal tissue at a differentiation stage where Muller cells have appeared. That is, the retinal tissue obtained by the manufacturing method described in 2. above includes 1) retinal tissue that has differentiated to the extent that Muller cells can be observed in the retinal tissue, and of the total number of cells contained in the neural retinal tissue, 25% or more, preferably 30% or more, are cone photoreceptor precursors and cone photoreceptors, and 12% or less, preferably 10% or less, more preferably 7% or less are rod photoreceptor precursors and rod photoreceptors, or 2) retinal tissue that can become the retinal tissue of 1) by further maturing in vitro or in vivo after transplantation. Furthermore, in the above-mentioned 1), the proportion of cone photoreceptor progenitors and cone photoreceptors among all photoreceptor progenitors and all photoreceptors in the retinal tissue is preferably 70% or more, more preferably 80% or more. Here, Müller cells can be identified by detecting well-known markers, such as CRABP-positive cells and / or CRALBP-positive cells. Furthermore, neural retinal tissue containing photoreceptor progenitors differentiated to the extent that Müller cells are recognized, obtained by the production method described in the above 2, is characterized in that the proportion of PAX6-positive and CHX10-negative cells (ganglion cells, horizontal cells, and / or amacrine cells) is reduced by 10% or more, preferably 20% or more, compared to when a dorsalizing signaling substance is not applied. In one embodiment of the present invention, the retinal tissue obtained by the manufacturing method described in 2. above is neural retinal tissue at a stage where the occurrence rate of cone photoreceptor precursor cells is at its maximum, and an average of 10% or more, preferably 13% or more, more preferably 16% or more, and even more preferably 17% or more of the total number of cells are photoreceptor precursor cells or photoreceptors. Furthermore, the retinal tissue obtained by the manufacturing method described in 2. above has a higher proportion of cone photoreceptor progenitor cells that can differentiate into L cone photoreceptors and / or M cone photoreceptors than when cultured in the absence of a dorsalizing signal transduction substance or in the presence of a ventralizing signal transduction substance. Furthermore, in the production method described in 2 above, retinal tissue obtained by culturing in the presence of a dorsalization signaling substance at a concentration sufficient to suppress ALDH1A3 expression has an average bipolar cell ratio of 15% or more of the total number of cells contained in the neural retinal tissue (approximately 1.5 to 2 times that in the absence of the substance), resulting in retinal tissue similar to the macula. "Culturing in the presence of a dorsalization signaling substance at a concentration sufficient to suppress ALDH1A3 expression" refers, for example, to a process in which, after initiation of suspension culture, cells are cultured in a medium containing 0.1 nM to 0.15 nM BMP4 for 50 to 130 days.

[0153] Furthermore, retinal tissue obtained by the manufacturing method described in 2 above, which is at an intermediate stage leading up to "retinal tissue at a differentiation stage where Müller cells have appeared," is also retinal tissue with a high proportion of cone photoreceptor precursor cells or cone photoreceptors, or with a high proportion of cone photoreceptors due to maturation, and is therefore encompassed in the "retinal tissue with a high proportion of cone photoreceptors" of the present invention. Specifically, the present invention is directed to retinal tissue at a stage where the appearance rate of cone photoreceptor progenitor cells is at its maximum, and the retinal tissue has at least one, preferably three or more, more preferably five or more, and even more preferably all of the following characteristics (i) to (xi): (i) the continuous epithelial rate is 50% or more, preferably 80% or more, and more preferably 95% or more; (ii) an average of 10% or more, preferably 13% or more, more preferably 16% or more, and even more preferably 17% or more of the total number of cells in the neural retinal tissue are photoreceptor precursor cells or photoreceptors; (iii) the ratio of cone photoreceptor progenitor cells and cone photoreceptors to the total number of cells in the neural retina tissue is preferably 8% or more, and even more preferably 10% or more; (iv) the area in which expression of ventral markers (such as ALDH1A3 and COUP-TF-I) is observed in the neural retinal tissue is 50% or less, preferably 20% or less, more preferably 1% or less, and even more preferably 0.01% or less; (v) the gene expression level of ALDH1A3 in the entire retinal tissue is 50% or less, preferably 20% or less, and more preferably 5% or less, compared to retinal tissue that has been ventralized by a BMP signaling pathway inhibitor and in which ventral markers such as ALDH1A3 are highly expressed; (vi) the region in the neural retina where expression of the most dorsal marker is not induced (i.e., the region in which expression of the most dorsal marker is not observed) is 50% or more, preferably 80% or more, more preferably 90% or more, and even more preferably 99% or more; (vii) the expression level of CYP26A1 is 1.2 times or more, preferably 1.5 times or more, and more preferably 2 times or more, compared to that of retinal tissue ventralized by the above-mentioned BMP signaling pathway inhibitor and in which CYP26A1 expression has been suppressed; (viii) the ratio of the number of cells expressing OC2 to the total number of cells contained in the neural retinal tissue is about 20% to 70%, preferably about 30% to 70%, and more preferably about 50% to 65%, compared to the ratio in the absence of the dorsalizing signaling substance; (ix) the ratio of the number of cells expressing OC2 to the total number of cells contained in the neural retinal tissue is suppressed to about 30% to 60%, preferably about 40% to 50%, compared to the ratio in the case where the BMP signaling pathway inhibitor is added; (x) the ratio of the number of cells expressing OC2 to the total number of cells in the neural retinal tissue is 25% or less, preferably 17% or less; and (xi) The expression level of OC2 protein in OC2-positive cells in neural retinal tissue is on average about 20% to 70%, preferably about 30% to 70%, and more preferably about 30% to 40%, compared to when no dorsalizing signaling substance is added or when the BMP signaling pathway inhibitor is added. In the above, the "continuous epithelial ratio" means the ratio of the area having a continuous epithelial structure to the surface area of ​​the retinal tissue.

[0154] 6. Retinal tissue with a high proportion of rod photoreceptors The present invention provides retinal tissue containing photoreceptor precursors rich in rod photoreceptor precursors and / or photoreceptors rich in rod photoreceptors (hereinafter referred to as retinal tissue with a high proportion of rod photoreceptors). Specifically, the present invention provides retinal tissue in which the number of rod photoreceptor precursors and rod photoreceptor cells is 40% or more, preferably 55% or more, relative to the number of photoreceptor precursors and photoreceptor cells, and the tissue also contains ganglion cells. In other words, in the retinal tissue, the number of rod photoreceptor precursors and rod photoreceptor cells is 0.6 times or more, preferably 1.3 times or more, relative to the number of cone photoreceptor precursors and cone photoreceptor cells. Specifically, the number of CRX-positive and NRL-positive cells is 0.6 times or more, preferably 1.3 times or more, relative to the number of CRX-positive, TRβ2-positive and NRL-negative cells or the number of CRX-positive, RXR-γ-positive and NRL-negative cells. In one embodiment of the retinal tissue, the differentiation induction of the retinal tissue may have progressed to the extent that Muller cells have appeared. That is, the retinal tissue obtained by the production method described in 3 above is 1) Retinal tissue that has differentiated to the extent that Muller cells can be observed in the retinal tissue, and of the total number of cells contained in the neural retinal tissue, preferably 13% or more, preferably 15% or more, more preferably 17% or more are rod photoreceptor precursor cells and rod photoreceptors, and 25% or less, preferably 21% or less, more preferably 15% or less are cone photoreceptor precursor cells and cone photoreceptors, or 2) Retinal tissue that can become the retinal tissue of 1) by further maturing in vitro or in vivo after transplantation. In addition, in the above 1), the proportion of rod photoreceptor precursor cells among all photoreceptor precursor cells in the retinal tissue is preferably 40% or more, more preferably 55% or more. Muller cells can be identified by detecting well-known markers, such as CRABP-positive cells and / or CRALBP-positive cells.

[0155] That is, the retinal tissue having a high proportion of rod photoreceptors according to the present invention is: 1) Retinal tissue that has differentiated to the extent that Muller cells can be observed in the retinal tissue, in which 25% or less, preferably 21% or less, and more preferably 15% or less of the total number of cells contained in the neural retinal tissue are cone photoreceptor precursor cells, and 13% or more, preferably 15% or more, and more preferably 17% or more are rod photoreceptor precursor cells, or 2) Retinal tissue that can become the retinal tissue of 1) above by further maturing in vitro or in vivo after transplantation. Examples include: Furthermore, the proportion of rod photoreceptor precursor cells among all photoreceptor precursor cells in the retinal tissue of 1) above is preferably 40% or more, more preferably 55% or more. Furthermore, retinal tissue obtained by the manufacturing method described in 3. above, which is at an intermediate stage leading up to "retinal tissue at a differentiation stage where Muller cells have appeared," is also retinal tissue that has a high proportion of rod photoreceptors or that can become high in proportion of rod photoreceptors, and is therefore encompassed in the "retinal tissue with a high proportion of rod photoreceptors" of the present invention. Specifically, the retinal tissue is at a stage where the appearance rate of cone photoreceptor precursor cells is at its maximum, and has at least one, preferably three or more, and more preferably all of the following characteristics (i) to (v): (i) the rate of continuous epithelium is 50% or more, preferably 80% or more, and more preferably 95% or more; (ii) photoreceptor precursor cells and photoreceptors account for an average of 10% or less, preferably 7% or less, and more preferably 6% or less of the total number of cells in the retinal tissue; (iii) the ratio of cone photoreceptor progenitor cells and cone photoreceptors to the total number of cells in the retinal tissue is 6% or less, preferably 4% or less; (iv) the number of cells in which expression of a ventral marker (such as ALDH1A3 or COUP-TF-I) is observed in the retinal tissue is 50% or more, preferably 70% or more, more preferably 90% or more, and even more preferably 99% or more of the total number of cells; and (v) The gene expression level of ALDH1A3 is at least 2-fold, preferably at least 5-fold, and more preferably at least 20-fold higher than that of retinal tissue that has been dorsalized by a substance acting on the BMP signaling pathway, which is a dorsalization signaling substance, and in which ventral markers such as ALDH1A3 have been suppressed.

[0156] 7. Pharmaceutical Compositions The present invention provides a pharmaceutical composition comprising an effective amount of retinal tissue with a high proportion of cone or rod photoreceptors, which is produced by the production method of the present invention. The pharmaceutical composition comprises an effective amount of retinal tissue produced by the production method of the present invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include physiological aqueous solvents (e.g., physiological saline, buffer solutions, serum-free media, etc.) If necessary, commonly used preservatives, stabilizers, reducing agents, isotonicity agents, etc. may be added to pharmaceuticals containing tissues or cells to be transplanted in transplantation medicine. The pharmaceutical composition of the present invention can be prepared as a suspension by suspending the retinal tissue prepared by the production method of the present invention in an appropriate physiological aqueous solvent. If necessary, a cryopreservative may be added, followed by cryopreservation, and the tissue may be thawed at the time of use, washed with a buffer solution, and then used in transplantation therapy. The retinal tissue obtained by the production method of the present invention can take various forms suitable for medical applications, such as a sheet, column, block, or plug, and is preferably in the form of a sheet from the viewpoints of excellent therapeutic effect, ease of use, etc. The retinal tissue obtained by the production method of the present invention can be cut into pieces of an appropriate size using a tool such as tweezers to prepare a retinal tissue fragment for administration. Alternatively, the retinal tissue fragment cut into a sheet form can be used as a sheet preparation. When preparing a sheet preparation, an appropriate sheet or mesh-like sheet made of a biocompatible polymer, monomer, or gel for the purpose of stretching the retinal tissue of the present invention may be used. That is, a pharmaceutical composition containing a retinal tissue slice excised from the retinal tissue of the present invention also falls within the scope of the present invention. The retinal tissue obtained by the production method of the present invention can be dispersed in a cell dispersion solution containing a protease such as papain to prepare a retinal cell suspension for administration. Alternatively, cells desired as an active ingredient can be isolated from the cells contained in the cell suspension using specific antibodies, aptamers, purification peptides, etc., against antigenic proteins expressed by the target cells, and used to prepare a pharmaceutical composition. That is, the present invention also includes a pharmaceutical composition comprising a cell suspension prepared by dispersing and / or purifying the retinal tissue of the present invention.

[0157] 8. Therapeutic Drugs and Treatment Methods Retinal tissue produced by the production method of the present invention is useful in transplantation therapy for diseases caused by (or resulting from) damage to the retinal tissue and the retinal cells contained therein. Therefore, the present invention provides a therapeutic agent for diseases caused by retinal tissue damage, which contains retinal tissue produced by the production method of the present invention as an active ingredient, and a treatment method comprising administering the therapeutic agent to a patient. The retinal tissue produced by the production method of the present invention can be used as a therapeutic agent for diseases caused by retinal tissue damage, or to replenish retinal tissue at the site of retinal tissue damage in patients who require transplantation. By transplanting retinal tissue produced by the production method of the present invention into patients with diseases caused by retinal tissue damage or in a damaged state of retinal tissue, the disease caused by retinal tissue damage or the damaged state of retinal tissue can be treated. Examples of diseases caused by retinal tissue damage include retinal degeneration, retinitis pigmentosa, age-related macular degeneration, organic mercury poisoning, chloroquine retinopathy, glaucoma, diabetic retinopathy, neonatal retinopathy, and retinal photodamage such as ultraviolet light-induced or blue light-induced retinal damage.

[0158] Among the retinal tissues of the present invention, retinal tissues containing photoreceptor progenitors enriched in cone photoreceptor progenitors are useful as pharmaceutical compositions for transplantation into regions of a patient's eye that are rich in cone photoreceptors. A specific example of a region rich in cone photoreceptors is a region containing a rod-free zone. A region containing a rod-free zone is a region having a macular-like structure, preferably the macular region. That is, the retinal tissues of the present invention containing photoreceptors that are rich in cone photoreceptor progenitors are useful as pharmaceutical compositions for transplantation into a patient's macula, preferably a more central region of the macula (e.g., fovea). Diseases requiring transplantation into the macular region include conditions such as age-related macular degeneration, in which visual acuity in bright light (i.e., daytime visual acuity) is reduced, visual field constriction in bright light, and total blindness, and the retinal tissues can be used to improve or treat these conditions.

[0159] On the other hand, among the retinal tissues of the present invention, retinal tissues containing photoreceptor progenitors enriched in rod photoreceptor progenitors are useful as pharmaceutical compositions for transplantation into regions of the patient's eye where rod photoreceptors are abundant, such as the periphery of the macula, or into the outer periphery of said region. Specific transplantation sites include the periphery of the macula and its outer regions. That is, the retinal tissues of the present invention containing cells enriched in rod photoreceptor progenitors are useful as pharmaceutical compositions for transplantation into the periphery of the macula and its outer regions of a patient. Diseases requiring transplantation into the periphery of the macula and its outer regions include conditions such as age-related macular degeneration, in which vision is reduced in the dark, and night blindness, and the retinal tissues of the present invention can be used to improve or treat these conditions. Even in cases of age-related macular degeneration, in which early symptoms such as reduced vision in the dark or night blindness are not observed, the retinal tissues of the present invention can be used to treat or prevent retinal tissue degeneration when symptoms such as a ring scotoma or a geographic scotoma are present. Furthermore, by transplanting retinal tissue containing photoreceptor progenitor cells that are rich in rod photoreceptor progenitor cells to the periphery of the macula or outside it, it is possible to suppress (i.e., prevent) the infiltration of degenerated regions into the macula.

[0160] In transplantation medicine, rejection due to differences in histocompatibility antigens is often a problem, but this problem can be overcome by using pluripotent stem cells established from the somatic cells of the transplant recipient (e.g., induced pluripotent stem cells). That is, in a preferred embodiment, in the method of the present invention, pluripotent stem cells established from the somatic cells of the recipient (e.g., induced pluripotent stem cells) are used as pluripotent stem cells, thereby producing neural tissue or nervous system cells that are immunologically autologous to the recipient, and these are transplanted into the recipient (autologous transplantation). Alternatively, allogeneic retinal tissue or cells may be produced from pluripotent stem cells (e.g., induced pluripotent stem cells) established from somatic cells of another person who is immunocompatible with the recipient (e.g., partially or fully compatible with the HLA type or MHC type), and then transplanted into the recipient (allogeneic transplantation).

[0161] 9. Toxicity and efficacy evaluation methods Retinal tissue produced by the production method of the present invention is useful as a disease research material or drug discovery material for screening therapeutic agents for diseases caused by retinal tissue damage or for toxicity evaluation, and can therefore be used as a reagent for evaluating the toxicity and pharmacological efficacy of test substances. For example, iPS cells are established from a human patient with a disease caused by retinal tissue damage, particularly a disease caused by a hereditary disorder, and these iPS cells are used to produce the retinal tissue of the present invention by the method of the present invention. The retinal tissue can reproduce in vitro the retinal tissue damage that causes the disease in the patient. Therefore, the present invention provides a method for evaluating the toxicity and pharmacological efficacy of a test substance, which comprises contacting the test substance with retinal tissue produced by the production method of the present invention and assaying the effect of the substance on the tissue. For example, retinal tissue with a specific disorder (e.g., a genetic disorder) produced by the production method of the present invention is cultured in the presence or absence (negative control) of a test substance. The degree of damage in the retinal tissue treated with the test substance is then compared with that of the negative control. As a result, a test substance that reduces the degree of damage can be selected as a candidate substance for a therapeutic agent for a disease caused by the disorder. For example, a test substance that further improves the physiological activity (e.g., survival promotion, functional improvement, or maturation) of retinal tissue produced by the production method of the present invention can be identified as a candidate drug. Alternatively, induced pluripotent stem cells are prepared from somatic cells with a genetic mutation that exhibits a specific disorder, such as a disease caused by retinal tissue damage, and the test substance is added to retinal progenitor cells or retinal layer-specific neurons produced by inducing differentiation of the cells using the production method of the present invention. Whether or not the disorder is exhibited can be used as an indicator to identify candidate test substances effective as therapeutic or preventive agents for the disorder. In the toxicity evaluation, retinal tissue produced by the production method of the present invention is cultured in the presence or absence (negative control) of a test substance. The degree of toxicity in the retinal tissue treated with the test substance is then compared with that of the negative control. As a result, a test substance that exhibits toxicity compared with the negative control can be determined to be a substance toxic to retinal tissue. That is, the present invention encompasses a toxicity evaluation method comprising the following steps: (Step 1) a step of culturing retinal tissue produced by the production method of the present invention under viable culture conditions in the presence of a test substance for a certain period of time, and then measuring the degree of cell damage; (Step 2) culturing the retinal tissue produced by the production method of the present invention under viable culture conditions for a certain period of time in the absence of a test substance or in the presence of a positive control, and then measuring the degree of cell damage; (Step 3) A step of evaluating the toxicity of the test substance in Step 1 based on the difference between the results measured in (Step 1) and (Step 2). Here, "in the absence of a test substance" includes adding only a culture medium or a solvent dissolving the test substance instead of the test substance. Furthermore, "positive control" refers to a known toxic compound. Methods for measuring the degree of cell damage include counting the number of viable cells, such as measuring the amount of intracellular ATP, or counting the number of viable cells by cell staining (e.g., cell nucleus staining or cytotoxicity markers) and morphological observation. In (Step 3), the toxicity of the test substance can be evaluated, for example, by comparing the measured value in (Step 1) with the measured value of a negative control in (Step 2), and determining that the test substance is toxic if the degree of cell damage in (Step 1) is significant. Alternatively, by comparing the measured value in (Step 1) with the measured value of a positive control in (Step 2), and determining that the degree of cell damage in (Step 1) is equal to or greater than that, the test substance can be determined to be toxic. The obtained retinal tissue may be used as a reagent for evaluating toxicity and efficacy as is. Highly purified retinal progenitor cells (cone photoreceptor progenitors and rod photoreceptor progenitors) can also be obtained by dissociating the retinal tissue (e.g., with trypsin / EDTA or papain) and sorting the resulting cells using FACS or MACS. Furthermore, photoreceptor progenitor cells (S cone photoreceptor progenitors, L cone photoreceptor progenitors, M cone photoreceptor progenitors, or rod photoreceptor progenitors) contained in the neural retinal tissue may be differentiated into photoreceptors expressing visual pigments (S cone photoreceptors, L cone photoreceptors, M cone photoreceptors, or rod photoreceptors) through final maturation and used as a reagent for evaluating toxicity and efficacy. [Example]

[0162] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples in any way. Example 1 (Example of production of cell aggregates containing retinal tissue using human ES cells and method of excising retinal tissue) CRX::Venus knock-in human ES cells (KhES-1 derived; Nakano, T. et al. Cell Stem Cell 2012, 10(6), 771-785) were cultured according to the methods described in Ueno, M. et al. PNAS 2006, 103(25), 9554-9559 and Watanabe, K. et al. Nat Biotech 2007, 25, 681-686. The culture medium for human ES cells was DMEM / F12 medium (Sigma) supplemented with 20% Knockout™ Serum Replacement (KSR; Invitrogen), 0.1 mM 2-mercaptoethanol, 2 mM L-glutamine, 1x non-essential amino acids, and 7.5 ng / mL bFGF. Cell aggregates containing retinal tissue were prepared by a modified method described in "Kuwahara et al. Nat Commun 2015, 19(6), 6286-." Specifically, the cultured ES cells were dispersed into single cells using TrypLE Express (Invitrogen), and the dispersed single cells were then plated into a non-adhesive 96-well plate (Sumilon Spheroid Plate, Sumitomo Bakelite Co., Ltd.) at 9 x 10 per well. 3The cells were suspended in 100 μL of serum-free medium, allowed to rapidly form aggregates, and then cultured at 37°C in 5% CO2. The serum-free medium used was a 1:1 mixture of F-12 and IMDM medium supplemented with 10% KSR, 450 μM 1-monothioglycerol, 1x chemically defined lipid concentrate, 5 mg / mL BSA, and 20 μM Y27632. Six days after the initiation of suspension culture, BMP4 was added to a final concentration of 1.5 nM, and suspension culture was continued. Half of the culture medium in the wells was replaced every 3 or 4 days with the above medium without BMP signaling pathway agonists. Cell aggregates containing retinal tissue on day 18 of suspension culture were cultured in serum-free medium (DMEM / F12 medium supplemented with 1% N2 supplement) containing 3 μM CHIR99021 and 5 μM SU5402 for 4 days, i.e., until day 22 of suspension culture. After that, the cell aggregates containing retinal tissue were cultured in suspension until they were used for analysis. During this period, the serum-free medium described below in [1] to [3] was used, and the cells were cultured under 5% CO2 conditions. [1] From day 22 to day 38 after the start of suspension culture; DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine. [2] From day 38 to day 60 after the start of suspension culture: DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% N2 supplement, and 100 μM taurine, and Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, and 100 μM taurine, mixed at a 1:3 ratio. [3] After 60 days of suspension culture: Neurobasal medium supplemented with 10% fetal bovine serum, 2% B27 supplement, and 100 μM taurine. Furthermore, retinal tissue can be excised from cell aggregates that are mostly retinal tissue by visually confirming the non-retinal tissue portions of the cell aggregates and then appropriately cutting them out with tweezers. An example of retinal tissue excised from a cell aggregate containing retinal tissue on day 35 after the initiation of suspension culture is shown in Figure 1a and b. Furthermore, when the cell aggregates containing retinal tissue cultured according to the above culture method were observed under a fluorescence microscope (Biorevo BZ-9000, Keyence), green fluorescence emitted by the knocked-in CRX::Venus was observed in almost all retinal tissue by day 42 after the initiation of suspension culture (Figure 1c and d).

[0163] Example 2 Aggregates containing retinal tissue enriched in cone or rod photoreceptor progenitors were prepared by the method described in Example 1, except that a dorsalizing signaling agent (BMP4 or Cyclopamine-KAAD) or a ventralizing signaling agent (LDN193189 or SAG) was added as follows: [1] Control group (Fig. 2a, f, k, p): Following the method described in Example 1, the cells were cultured without adding anything until the 70th or 75th day after the start of suspension culture. [2]+BMP group (Fig. 2b, g, l, q): 0.15 nM BMP4 was added from day 22 after the start of suspension culture until the end of suspension culture, and the cells were cultured until day 70 or 75 after the start of suspension culture. [3]+Cyclopamine-KAAD group (Fig. 2c, h, m, r): 500 nM Cyclopamine-KAAD was added from day 22 after the start of suspension culture until the end of suspension culture, and the cells were cultured until day 70 or 75 after the start of suspension culture. [4]+LDN193189 group (Fig. 2d, i, n, s): 100 nM LDN193189 was added from day 22 after the start of suspension culture until the end of suspension culture, and the cells were cultured until day 70 or 75 after the start of suspension culture. [5]+SAG group (Fig. 2e, j, o, t): 100 nM SAG was added from day 22 after the start of suspension culture until the end of suspension culture, and the cells were cultured until day 70 or 75 after the start of suspension culture. Cell aggregates containing retinal tissue cultured under these conditions were observed under a fluorescence microscope (Biorevo BZ-9000, Keyence). After fixation with 4% paraformaldehyde, frozen sections were prepared and immunostained for CRX and TRβ2 (TRb2), or stained for cell nuclei using DAPI. The green fluorescence of the knock-in CRX::Venus was observed in the +BMP4 group, f...

Claims

1. Human retinal tissue containing photoreceptor precursor cells rich in cone photoreceptor precursor cells and / or photoreceptor cells rich in cone photoreceptor precursor cells, in which the number of cone photoreceptor precursor cells and cone photoreceptor cells among the photoreceptor precursor cells and photoreceptor cells is more than twice the number of rod photoreceptor precursor cells and rod photoreceptor cells, and which has ganglion cells.

2. Human retinal tissue as described in claim 1, wherein the number of cone photoreceptor progenitor cells and cone photoreceptor cells contained in all photoreceptor progenitor cells and all photoreceptor cells is 70% or more.

3. Human retinal tissue described in claim 1 or 2, wherein the cone photoreceptor progenitor cells and cone photoreceptors are CRX-positive and RXR-γ-positive, or CRX-positive and TRβ2-positive, and NRL-negative cells.

4. Human retinal tissue described in any one of claims 1 to 3, comprising a CYP26A1 and / or CYP26C1 positive area.

5. Human retinal tissue in which the expression level of CYP26A1 is at least 1.2 times higher than that of ventral retinal tissue in which CYP26A1 expression is suppressed.

6. Human retinal tissue that can be matured into the retinal tissue described in any one of claims 1 to 5 by culturing.

7. Human retinal tissue described in any one of claims 1 to 5, wherein 50% or more of the layer structure of the retinal tissue forms a continuous epithelial structure.

8. Human retinal tissue as described in claim 7, wherein the diameter of the retinal tissue in the long axis direction is 0.6 mm or more.

9. The following steps: A step of culturing human retinal tissue from the early developmental stage when PAX6-positive and RX-positive cells are detected to the stage when the incidence of CRX-positive and RXR-γ-positive or CRX-positive and TRβ2-positive cells in the retinal tissue reaches a maximum in a medium containing a dorsalizing signal transduction substance selected from a substance acting on the BMP signal transduction pathway, a substance acting on the Wnt signal transduction pathway, and a substance inhibiting the sonic hedgehog signal transduction pathway at a concentration sufficient to suppress the expression of ventral markers. The human retinal tissue according to any one of claims 1 to 8, obtained by a production method comprising the steps of:

10. Human retinal tissue as described in claim 9, wherein the stage at which the incidence of CRX-positive and RXR-γ-positive, or CRX-positive and TRβ2-positive cells in the retinal tissue reaches its maximum is 30 to 50 days after the early developmental stage.

11. Human retinal tissue described in claim 9 or 10, wherein the concentration of the dorsalization signaling substance is at a level that does not induce expression of the most dorsal marker, but promotes expression of other dorsal markers.

12. Human retinal tissue as described in claim 11, wherein the ventral marker is ALDH1A3 and / or COUP-TF I, the dorsal marker is CYP26A1, CYP26C1 and / or ALDH1A1, and the most dorsal marker is COUP-TF II.

13. Human retinal tissue as described in claim 12, wherein the concentration of the dorsalizing signaling substance is such that ALDH1A1 expression is induced to be 0.1% to 30% of the expression level of ALDH1A1 promoted by 1.35 nM BMP4.

14. Human retinal tissue described in any one of claims 1 to 13, wherein the retinal tissue in the early developmental stage is derived from pluripotent stem cells.

15. Human retinal tissue described in any one of claims 9 to 14, characterized in that the retinal tissue in the early developmental stage contains PAX6-positive, RX-positive and CHX10-positive cells.

16. Human retinal tissue described in any one of claims 9 to 15, wherein the process of culturing in the presence of a dorsalization signaling substance continues for 4 days to 170 days.

17. Human retinal tissue as described in claim 16, wherein the process of culturing in the presence of a dorsalizing signaling substance is continued until the time when rod photoreceptor precursor cells appear when cultured in the absence of the dorsalizing signaling substance.

18. Human retinal tissue described in any one of claims 9 to 17, wherein the dorsalizing signaling substance is a BMP signaling pathway active substance or an SHH signaling pathway inhibitor capable of inducing a BMP signal equivalent to 0.01 nM to 0.90 nM BMP4.

19. Human retinal tissue as described in claim 18, wherein the BMP signaling pathway active substance is BMP4.

20. Human retinal tissue described in claim 18, wherein the SHH signaling pathway inhibitor is Cyclopamine-KAAD.

21. A pharmaceutical composition for transplantation into retinal tissue requiring transplantation in a patient with retinal disease, comprising a retinal tissue fragment excised from human retinal tissue described in any one of claims 1 to 20.

22. The pharmaceutical composition described in claim 21, wherein the retinal tissue requiring transplantation is tissue in an area including a rod-free zone.

23. The pharmaceutical composition described in claim 22, wherein the area including the rod-free zone has a macular-like structure.

24. A therapeutic agent for a disease caused by damage to retinal cells or retinal tissue, for transplantation into a subject in need of transplantation, comprising an effective amount of retinal tissue described in any one of claims 1 to 20.

25. A method for producing mature retinal tissue expressing S-opsin, L-opsin and / or M-opsin, comprising the step of culturing human retinal tissue described in any one of claims 1 to 20 in a serum-free medium.

26. The method described in claim 25, wherein the serum-free medium is a medium containing a substance acting on the BMP signaling pathway.

27. ​​The method described in claim 25 or 26, wherein the serum-free culture medium further contains a thyroid hormone signaling substance.

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