WO2022138803A1 - Procédé de forçage et de production pour tissu rétinien stratifié comprenant des cellules photoréceptrices - Google Patents

Procédé de forçage et de production pour tissu rétinien stratifié comprenant des cellules photoréceptrices Download PDF

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WO2022138803A1
WO2022138803A1 PCT/JP2021/047809 JP2021047809W WO2022138803A1 WO 2022138803 A1 WO2022138803 A1 WO 2022138803A1 JP 2021047809 W JP2021047809 W JP 2021047809W WO 2022138803 A1 WO2022138803 A1 WO 2022138803A1
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cells
retinal
photoreceptor
layered
signaling pathway
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Japanese (ja)
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政代 高橋
暁士 大西
登起義 松下
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国立研究開発法人理化学研究所
参天製薬株式会社
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
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  • the present invention relates to a method for forcing and producing layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells.
  • uniform pluripotent stem cell aggregates are formed in a serum-free medium, suspended and cultured, and then used for induction of differentiation.
  • a method for producing neural tissue has been reported by suspending and culturing in a medium in the presence of a differentiation-inducing factor or the like and inducing differentiation from pluripotent stem cells to target nervous system cells (neural cells). ..
  • uniform pluripotent stem cell aggregates are formed in a serum-free medium containing a Wnt signaling pathway inhibitor, which is suspended and cultured in the presence of a basal membrane preparation, and then suspended and cultured in the serum medium.
  • a method for obtaining a multi-layered retinal tissue (Patent Document 1), a medium containing pluripotent stem cells, a TGF ⁇ family signaling pathway inhibitor and / or a sonic hedgehog signaling pathway agonist, and an undifferentiated maintenance factor.
  • the obtained cells are suspended-cultured in a medium containing a Wnt signaling pathway inhibitor, and the obtained aggregates are suspended-cultured in a medium containing a BMP signaling pathway agonist to obtain retinal cells or.
  • Method for obtaining aggregates containing retinal tissue (Patent Document 2), obtained after culturing cell aggregates containing retinal tissue in a medium containing a Wnt signal pathway agonist until cells expressing the RPE65 gene appear.
  • a method for obtaining cell aggregates containing hairy peripheral peripheral-like structures by culturing cell aggregates in which cells expressing the RPE65 gene have not appeared in a medium containing no Wnt signal pathway agonist (Patent Document 3), Cell aggregates containing retinal tissue were cultured in a medium containing a Wnt signal pathway agonist and an FGF signal pathway inhibitor until cells expressing the RPE65 gene appeared, and then the obtained RPE65 was obtained.
  • a method for obtaining a cell aggregate containing a hair-like peripheral periphery-like structure by culturing a cell aggregate in which a cell expressing a gene has not appeared in a medium containing no Wnt signal pathway agonist (Patent Document).
  • the pluripotent stem cells are cultured in a medium containing an undifferentiated maintenance factor, and the obtained pluripotent stem cells are suspended-cultured in a medium containing a sonic hedgehog signal transduction pathway agent to form cell aggregates.
  • a method of obtaining aggregates containing retinal cells or retinal tissues by suspending and culturing the formed aggregates in a medium containing a BMP signal transduction pathway agent (Patent Document 5) is known.
  • pluripotent stem cells are suspended and cultured in a serum-free medium to form aggregates of pluripotent stem cells, and the formed aggregates are transmitted by sonic hedgehog signaling.
  • a method for obtaining retinal precursor cells by suspension culture in a medium containing a BMP signaling pathway agent without a pathway agent (Patent Document 6).
  • Pluripotent stem cells are suspension-cultured and differentiated into retinal precursor cells. Induced, the obtained retinal progenitor cells were suspended and cultured to induce differentiation into retinal ganglion cells, and the obtained retinal ganglion cells were adherently cultured to elongate the axons.
  • a method for obtaining ganglion cells (Patent Document 7) and the like are known.
  • Non-Patent Document 1 a method for inducing differentiation (SEAM method) for obtaining various ocular tissue cells derived from ectoderm concentrically, and two-dimensional culture.
  • SEAM method a method for inducing differentiation of retinal organoids
  • Non-Patent Document 2 a method for inducing differentiation of retinal organoids
  • dual inhibition of BMP signals and TGF ⁇ family signals efficiently induces human ES cells and iPS cells to neural progenitor cells (Non-Patent Document 3), and human pluripotent stem cells can be treated with BMP4.
  • Non-Patent Document 4 Differentiation into neuroretinal cells is induced by suspension culture in a medium containing BMP4 (Non-Patent Document 4), and retinal organoids are produced by adherent culture of aggregates of human pluripotent stem cells in a medium containing BMP4.
  • Non-Patent Document 5 sonic hedgehog signal promotes differentiation into rod photoreceptors in mammalian retinal cells (Non-Patent Document 6), retinoic acid differentiation into photoreceptor cells (Non-Patent Document 7), in the process of inducing the differentiation of pluripotent stem cells into nerve cells, Activin promotes the differentiation into retinal precursor cells (Non-Patent Document 8), human pluripotency It is also known that Activin promotes the induction of differentiation of stem cell-derived ophthalmic tissue into retinal pigment epithelial cells (Non-Patent Document 9).
  • An object to be solved by the present invention is to provide a method for producing a layered retinal tissue containing photoreceptor cells from aggregates containing retinal progenitor cells or pluripotent stem cells in a short period of time.
  • the present inventors used pluripotent stem cells as a TGF ⁇ signaling pathway inhibitor and BMP signaling pathway inhibition in the absence of undifferentiated maintenance factor as an adhesion culture step.
  • Adhesive culture was performed in a medium containing the substance, and the obtained cells were placed in a medium containing a BMP signaling pathway agonist in the absence of an undifferentiated maintenance factor, a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor.
  • the resulting cells are adherently cultured in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists to cause retinal precursors.
  • Cell-containing aggregates are obtained and, as a subsequent suspension culture step, the aggregates containing retinal precursor cells are suspended in a medium containing Activin signaling pathway agonists, Sonic Hedgehog signaling pathway agonists and retinoic acid. It is pluripotent in a short period of time by culturing and suspending the resulting aggregates in a medium containing Sonic Hedgehog signaling pathway agonists in the absence of Activin signaling pathway agonists and retinoic acid.
  • a layered retinal tissue containing photoreceptor cells can be produced from stem cells, and have completed the present invention. That is, the present invention relates to the following.
  • a method for producing a layered retinal tissue containing photoreceptor cells which comprises the following steps (1) and (2); (1) Aggregates containing retinal precursor cells are suspended-cultured in a medium containing Activin signaling pathway agonist, sonic hedgehog signal transduction pathway agonist and retinoic acid, and contain retinal ganglion cells and photoreceptor precursor cells. The process of obtaining aggregates, (2) Aggregates containing retinal ganglion cells and photoreceptor precursor cells were suspended-cultured in a medium containing a sonic hedgehog signal transduction pathway agonist in the absence of Activin signaling pathway agonist and retinoic acid. The process of obtaining layered retinal tissue containing photoreceptor cells.
  • the layered retinal tissue containing photoreceptor cells is a retinal tissue containing an outer nuclear layer, an inner nuclear layer, and a ganglion cell layer.
  • the photoreceptor cells are contained in the outer nuclear layer.
  • the photoreceptors contained in the outer nuclear layer are rod photoreceptors and / or cone photoreceptors.
  • 50% or more of all cells contained in the outer nuclear layer are rod photoreceptors and / or cone photoreceptors.
  • the Activin signaling pathway agent is one or more substances selected from the group consisting of Activin A, Activin B, Activin C, and Activin AB, to any one of [1] to [6]. The method described. [8] The method according to [7], wherein the Activin signaling pathway agent is Activin A.
  • the Sonic hedgehog signaling pathway agonist is one or more substances selected from the group consisting of Sonic Hedgehog (Shh), Smoothened Agonist (SAG), and Purmorphamine (PMA), [1] to [8]. ] The method according to any one of. [10] The method according to [9], wherein the Sonic Hedgehog signaling pathway agonist is SAG. [11] The method according to any one of [1] to [10], wherein the aggregate containing the retinal progenitor cells is suspended-cultured for 0.5 to 50 days in the step (1).
  • [14] The method according to any one of [1] to [13], further comprising the following steps prior to step (1); (a) A step of adhering and culturing pluripotent stem cells in a medium containing a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of an undifferentiated maintenance factor to obtain neural progenitor cells. (b) Neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway agonist in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors, and retinal progenitor cells.
  • Retinal progenitor cells are adherently cultured in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists, and coagulation containing retinal progenitor cells.
  • the pluripotent stem cell is an induced pluripotent stem cell.
  • the TGF ⁇ signaling pathway inhibitor is one or more substances selected from the group consisting of Lefty, SB431542, LY-364947, SB-505124, and A-83-01, [14] to [16].
  • the method according to any one of. [18] The method according to [17], wherein the TGF ⁇ signaling pathway inhibitor is SB431542.
  • the BMP signal transduction pathway inhibitor is one or more substances selected from the group consisting of LDN193189 and Dorsomorphin.
  • the method according to [19], wherein the BMP signaling pathway inhibitor is LDN193189.
  • a method for screening a therapeutic agent for a disorder of layered retinal tissue including photoreceptor cells which comprises the following steps (11) to (13): (11) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the presence of the test substance for a certain period of time under viable culture conditions, the degree of damage to the layered retinal tissue containing the photoreceptor cells is measured.
  • step (12) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the absence of the test substance or in the presence of positive control for a certain period of time under viable culture conditions, the layered retina containing the photoreceptor cells The process of measuring the degree of tissue damage, (13) A step of selecting a test substance in step (11) as a therapeutic agent for a disorder of layered retinal tissue including photoreceptor cells based on the difference in the results measured in step (11) and step (12).
  • a drug efficacy evaluation method including the following steps (21) to (23): (21) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the presence of the test substance for a certain period of time under viable culture conditions, the degree of damage to the layered retinal tissue containing the photoreceptor cells is measured.
  • Toxicity evaluation method including the following steps (31) to (33): (31) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells after culturing the layered retinal tissue containing photoreceptor cells in the presence of a test substance for a certain period of time under viable culture conditions.
  • a layered retinal tissue containing photoreceptor cells from aggregates or pluripotent stem cells containing retinal progenitor cells in a short period of time.
  • FIG. 1 shows the culture schedule of cell line 201B7 in Example 1.
  • FIG. 4 shows the culture schedule of cell line 201B7 in Example 2.
  • FIG. 5 shows the expression of the photo
  • FIG. 6 shows the expression of the photoreceptor progenitor cell marker Crx and the retinal ganglion cell marker Brn3 in the aggregate 20 days after the start of differentiation of the cell line M8.
  • FIG. 7 shows the expression of the photoreceptor progenitor cell marker Crx and the retinal ganglion cell marker Brn3 in the aggregate 40 days after the onset of differentiation of the cell line M8.
  • Outer neuroblastic layer outer neuroblastic layer
  • Inner neuroblastic layer inner neuroblastic layer
  • FIG. 8 shows the expression of the pyramidal photoreceptor marker RXR ⁇ and the rod photoreceptor marker NRL in the aggregate 60 days after the start of differentiation of the cell line M8.
  • FIG. 9 shows the expression of pyramidal photoreceptor markers S-opsin, L / M-opsin and rod photoreceptor marker rhodopsin in aggregates 80 days after the onset of differentiation of cell line M8.
  • FIG. 10 shows the culture schedule of cell line 1231A3 in Example 4.
  • FIG. 11 shows microscopic images of aggregates 15 days, 20 days, 40 days, 60 days, 80 days, and 90 days after the start of differentiation induction of cell line 1231A3 in Example 4.
  • the present invention provides a method for producing layered retinal tissue containing photoreceptor cells (hereinafter, production method 1 of the present invention).
  • a tissue is a cell having a structure in which one type of cell having a uniform morphology or property, or a cell layer composed of a plurality of types of cells having a different morphology or property, is arranged in a layered pattern.
  • the layered retinal tissue produced in the production method 1 of the present invention (hereinafter referred to as the retinal tissue of the present invention) is a tissue in which each retinal layer in the living retina or a layer corresponding to each retinal layer is arranged in a layered manner. It means a tissue in which each retinal layer or a layer corresponding to each retinal layer is composed of one type of retinal cells having uniform morphology and properties, or a plurality of types of retinal cells having different morphologies and properties.
  • the retinal layer means each layer constituting the retina, and specifically, the retinal pigment epithelial layer, the photoreceptor layer, the external limiting membrane, the outer nuclear layer, the outer plexiform layer, the inner nuclear layer, and the inner plexiform. Layers, ganglion cell layer, nerve fiber layer and inner limiting membrane can be mentioned.
  • the retinal layer of the present invention may also be a layer corresponding to each of the above-mentioned layers constituting the retina.
  • the term "corresponding" means having the same or substantially the same characteristics as each layer constituting the normally differentiated retina in the living body.
  • the characteristics of each retinal layer constituting the retina include the types of retinal cells contained in each retinal layer and the order of development of each layer in the retina.
  • the retinal layer constituting the retinal tissue of the present invention (hereinafter referred to as the retinal layer of the present invention) is not particularly limited as long as it is the above-mentioned retinal layer, and is composed of each of the above-mentioned one type or two or more types of retinal layers. It may have been done.
  • the retinal tissue of the present invention is preferably a retinal tissue exhibiting the characteristics of the outer nuclear layer, inner nuclear layer, and ganglion cell layer.
  • the retinal tissue of the present invention is preferably a retinal tissue including a layer corresponding to the outer nuclear layer, a layer corresponding to the inner nuclear layer, and a layer corresponding to the ganglion cell layer.
  • retinal cells means cells constituting each retinal layer in a living retina or precursor cells thereof, and are retinal precursor cells, photoreceptor precursor cells, photoreceptor cells (rod photoreceptor cells, pyramidal photoreceptor cells). Includes cells such as horizontal cells, amacrine cells, intervening nerve cells, retinal ganglion cells (ganglion cells), bipolar cells, retinal pigment epithelial cells (RPE), hairline marginal cells, and precursor cells thereof. Not limited to these.
  • the retinal precursor cell is specific to any mature retinal layer such as photoreceptor cells, rod photoreceptors, pyramidal photoreceptors, horizontal cells, bipolar cells, amacrine cells, retinal ganglion cells, and retinal pigment epithelial cells. Precursor cells that can also differentiate into target nerve cells.
  • the photoreceptor cells, horizontal cell precursors, bipolar cell precursor cells, amacrine cell precursor cells, retinal ganglion cell precursor cells, and retinal pigment epithelial precursor cells are photoreceptor cells, horizontal cells, bipolar cells, amacrine cells, and retina, respectively. Precursor cells that have been determined to differentiate into ganglion cells and retinal pigment epithelial cells.
  • the retinal layer-specific nerve cell means a cell constituting the retinal layer and specific to the retinal layer.
  • retinal layer-specific neurons include bipolar cells, retinal ganglion cells, amacrine cells, horizontal cells, photoreceptor cells, retinal pigment epithelial cells, rod photoreceptor cells and pyramidal photoreceptor cells.
  • the retinal cells constituting the retinal layer of the present invention are not particularly limited as long as they are the above-mentioned retinal cells, and may be composed of one or more of the above-mentioned retinal cells.
  • Examples of the retinal system cells constituting the retinal layer of the present invention include retinal pigment epithelial cells (RPE) constituting the retinal pigment epithelial layer and photoreceptor cells constituting the outer granule layer (rod photoreceptor cells, pyramidal photoreceptor cells). , Horizontal cells constituting the inner granule layer, amacrine cells, bipolar cells, retinal ganglion cells (ganglion cells) constituting the ganglion cell layer, and the like. Which cell constitutes each retinal layer can be confirmed by a known method, for example, the presence or absence of expression of a retinal cell marker or the degree thereof.
  • Retinal cell markers include Rx (also called Rax) expressed in neural precursor cells, Rx, PAX6 and Chx10 expressed in retinal precursor cells, and Nkx2 expressed in precursor cells of hypothalamic neurons but not in retinal precursor cells.
  • Rx also called Rax
  • Sox1 which is expressed in the hypothalamic nerve epithelium and not in the retina
  • Crx, Blimp1, etc. which are expressed in precursor cells of photoreceptor cells.
  • Markers for retinal layer-specific neurons include Chx10, PKC ⁇ and L7 expressed in bipolar cells, TuJ1 and Brn3 expressed in retinal ganglion cells, Calretinin expressed in amacrine cells, Calbindin expressed in horizontal cells, and mature photoreceptor cells.
  • Rhodopsin and Recoverin expressed in rod photoreceptors Nrl and Rhodopsin expressed in rod photoreceptors, Rxr-gamma, S-Opsin and M / L-Opsin expressed in pyramidal photoreceptors, RPE65 and Mitf expressed in retinal pigment epithelial cells, Examples thereof include Rdh10 and SSEA1 expressed in hair-like peripheral cells.
  • the retinal tissue of the present invention is characterized by containing photoreceptor cells.
  • the photoreceptor cells contained in the retinal tissue of the present invention are not particularly limited as long as they are contained in one or more types of retinal layers constituting the retinal tissue, but are contained in the outer nuclear layer as described above. Is preferable.
  • the photoreceptor cells contained in the retinal tissue of the present invention are contained in the outer nuclear layer, the photoreceptor cells contained in the outer nuclear layer may be rod photoreceptors and / or cone photoreceptors.
  • the ratio of rod photoreceptors to cone photoreceptors contained in the outer nuclear layer is not particularly limited as long as it is in the range of 100%: 0% to 0%: 100%, and is usually 10%: 90% to 90%.
  • the ratio of rod photoreceptors to pyramidal photoreceptors can be confirmed by a known method, for example, the presence or absence or degree of expression of rod photoreceptors and cone photoreceptors.
  • the proportion of rod photoreceptors and / or pyramidal photoreceptors contained in the outer nuclear layer is not particularly limited, but rod photoreceptors and pyramidal photoreceptors among all cells contained in the outer nuclear layer are not particularly limited.
  • the total number of cells in the cells is usually 50% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more. Of all the cells contained in the outer nuclear layer, the larger the total number of rod photoreceptors and cone photoreceptors, the more mature the retinal tissue of the present invention can be determined.
  • the production method 1 of the present invention includes the following steps. (1) Aggregates containing retinal precursor cells are suspended-cultured in a medium containing Activin signaling pathway agonist, sonic hedgehog signal transduction pathway agonist and retinoic acid, and contain retinal ganglion cells and photoreceptor precursor cells. The process of obtaining aggregates. (2) Aggregates containing retinal ganglion cells and photoreceptor precursor cells were suspended-cultured in a medium containing a sonic hedgehog signal transduction pathway agonist in the absence of Activin signaling pathway agonist and retinoic acid. The process of obtaining layered retinal tissue containing photoreceptor cells.
  • step (1) aggregates containing retinal progenitor cells are suspended-cultured in a medium containing an Activin signaling pathway agonist, a sonic hedgehog signaling pathway agonist, and retinoic acid.
  • the aggregate containing retinal progenitor cells is particularly limited as long as it is an aggregate having a cell layer expressing at least one selected from the group consisting of retinal progenitor cell markers Rx, PAX6 and Chx10.
  • it may be an aggregate containing retinal progenitor cells obtained by adherent culture of pluripotent stem cells, or an aggregate containing retinal progenitor cells obtained by another production method. good.
  • Elizabeth E. Capowski 1 et al., Development. 2019 Jan 1; 146 (1): dev171686, WO 2015/025967 an aggregate containing retinal progenitor cells produced by the method disclosed in the present invention. It may be used in the step (1) of the manufacturing method 1.
  • the aggregate containing the retinal progenitor cells used in the step (1) is uniform.
  • Uniform agglomerates means that the size of each agglomerate is constant when culturing a plurality of agglomerates, and when the size of the agglomerates is evaluated by the length of the maximum diameter, the agglomerates are uniform.
  • Aggregation means that the variance of the maximum diameter length is small. More specifically, 75% or more of the entire population of aggregates is more preferably within the range of an average value of ⁇ 100%, preferably an average value of ⁇ 50%, of the maximum diameter in the population of the aggregates. Means that the average value is within the range of ⁇ 20%.
  • step (1) aggregates containing retinal progenitor cells are suspended and cultured.
  • the term "suspension culture” or “suspension culture method” refers to a method of culturing while maintaining a state in which cells or cell aggregates are suspended and present in a medium, and a method of performing the culture. That is, suspension culture is performed under the condition that cells or cell aggregates are not adhered to the culture equipment or the like, and culture (adhesion culture or adhesion culture method) performed under the condition of adhering to the culture equipment or the like is in the category of suspension culture. Not included in. In this case, cell adhesion means that a strong cell-substratum junction is formed between the cell or cell aggregate and the culture equipment. More specifically, suspension culture refers to culture under conditions that do not form strong cell-substrate bonds between cells or cell aggregates and culture equipment or the like.
  • the cell-substrate bond In the aggregate of cells in suspension culture, cells adhere to each other. In the aggregate of cells in the suspension culture, the cell-substrate bond is hardly formed with the culture equipment or the like, or even if it is formed, its contribution is small. In some embodiments, in the aggregate of cells in suspension culture, the endogenous cell-substrate bond is present inside the aggregate, but the cell-substrate bond is hardly formed with the culture equipment or the like. Or, even if it is formed, its contribution is small.
  • Cell-to-cell plane attachment means that cells adhere to each other on the surface. More specifically, when cells are surface-adhered, the ratio of the surface area of one cell to the surface of another cell is, for example, 1% or more, preferably 3% or more, and more preferably 5%. That is all.
  • the surface of the cell can be observed by staining with a membrane-staining reagent (eg DiI) or by immunostaining with a cell adhesion factor (eg E-cadherin or N-cadherin).
  • a membrane-staining reagent eg DiI
  • a cell adhesion factor eg E-cadherin or N-cadherin
  • the incubator used for the suspension culture is not particularly limited as long as the suspension culture is possible, and a person skilled in the art can appropriately determine the incubator.
  • Examples of such an incubator include a flask, a tissue culture flask, a culture dish (dish), a petri dish, a tissue culture dish, a multidish, a microplate, a microwell plate, a micropore, a multiplate, and a multiwell plate.
  • These incubators are preferably cell non-adhesive to allow suspension culture.
  • the surface of the incubator is artificially treated for the purpose of improving adhesion to cells (for example, extracellular matrix of basement membrane preparation, laminin, entactin, collagen, gelatin, etc.). , Or coating treatment with polymers such as polylysine and polyornithin, or surface treatment such as positive charge treatment), etc. can be used.
  • the surface of the incubator was artificially treated for the purpose of reducing the adhesion to cells (for example, superhydrophilic treatment such as MPC polymer, low protein adsorption treatment, etc.). You can use things.
  • Rotational culture may be performed using a spinner flask, a roller bottle, or the like.
  • the culture surface of the incubator may have a flat bottom or may have irregularities.
  • the medium used in the suspension culture can be prepared using the medium normally used for culturing animal cells as the basal medium.
  • the basal medium include BME medium, BGJb medium, CMRL1066 medium, Glassgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, and F-12.
  • Examples include media that can be used for culturing animal cells, such as medium, DMEM / F12 medium, IMDM / F12 medium, ham medium, RPMI1640 medium, Fischer's medium, or a mixed medium thereof.
  • the medium used in the suspension culture may be a serum-containing medium or a serum-free medium, but since the retinal progenitor cells contained in the aggregate have already been determined in the direction of differentiation, the retinal progenitor cells It is preferred to use serum-containing medium to promote further maturation and proliferation.
  • the serum-containing medium means a medium containing unadjusted or unpurified serum.
  • the medium contains fatty acids or lipids, amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, 1-monothioglycerol, pyruvic acid, buffers, inorganic salts and the like. You may.
  • serum-containing mediums include 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1x Antibiotic-Antimycotic (GIBCO), and 0.1 mM Taurine (Sigma-).
  • DMEM / F-12, GlutaMax supplement medium (Thermo Fisher Scientific) containing Aldrich) may be used.
  • the culture may be carried out under xeno-free conditions, preferably.
  • Xenofree means a condition in which a component derived from a species different from that of the cell to be cultured is excluded.
  • the medium used for suspension culture of aggregates containing retinal progenitor cells in step (1) contains Activin signaling pathway agonist, Sonic Hedgehog signaling pathway agonist and retinoic acid.
  • Activin signaling pathway agonist Activin signaling pathway agonist
  • Sonic Hedgehog signaling pathway agonist Activin signaling pathway agonist
  • retinoic acid Activin signaling pathway agonist
  • in order to prepare retinal tissue culturing in a medium containing a differentiation-inducing factor at the time when pluripotent stem cells are differentiated into retinal progenitor cells is not common, and in a medium containing no differentiation-inducing factor.
  • the method of culturing in was the usual method.
  • step (1) aggregates containing retinal progenitor cells are further suspended-cultured in a medium containing an Activin signaling pathway agonist, a sonic hedgehog signaling pathway agonist, and retinoic acid to obtain retinal progenitor cells. It is possible to promote differentiation and further proliferation. In particular, the process of culturing aggregates containing retinal progenitor cells in a medium containing the Activin signaling pathway agonist, the sonic hedgehog signaling pathway agonist, and retinoic acid at the same time was completely unknown.
  • the Activin signal transduction pathway agent is a substance that can enhance the signal mediated by Activin.
  • the Activin signaling pathway agonist is not particularly limited as long as it can induce the differentiation of pluripotent stem cells into retinal precursor cells by enhancing the Activin signaling pathway, but for example, a protein belonging to the Activin family (for example, Activin A, Activin B, Activin C, Activin AB, etc.), Activin receptor agonists, etc.
  • the Activin signal transduction pathway agent may contain one or more of these.
  • the Activin signaling pathway agent is preferably Activin A.
  • the sonic hedgehog signal (hereinafter, may be referred to as Shh) transmission pathway acting substance is a substance capable of enhancing signal transduction mediated by Shh.
  • the Shh signaling pathway agonist is not particularly limited as long as it can promote the differentiation into rod photoreceptors by enhancing the Shh signaling pathway, but is not particularly limited, and is, for example, a protein belonging to the Hedgehog family (eg, for example).
  • Shh especially Sonic Headgehog N-terminus and Ihh
  • Shh receptor agonist PMA (Purmorphamine; 9-cyclohexyl-N- [4- (4-morpholinyl) phenyl] -2- (1-nagtale) Nyloxy) -9H-purine-6-amine) or SAG (Smoothened Agonist; N-methyl-N'-(3-pyridinylbenzyl) -N'-(3-chlorobenzo [b] thiophen-2-carbonyl) ) -1,4-Diaminocyclohexane) and the like.
  • the Shh signal transduction pathway agonist may contain one or more of these.
  • the Shh signal transduction pathway agonist is preferably SAG, Shh N-terminal. Is.
  • retinoic acid acts to promote differentiation into photoreceptor cells.
  • the concentrations of the Activin signaling pathway agonist, the Sonic Hedgehog signaling pathway agonist and the retinoic acid can be appropriately set within the range in which the above effects can be achieved.
  • Activin A is usually used at a concentration of 0.01 to 1000 ng / mL, preferably 0.1 to 750 ng / mL, more preferably 1 to 500 ng / mL (eg, 100 ng / mL).
  • SAG is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM (eg, 100 nM).
  • the Shh N-terminus is usually used at a concentration of 0.1-300 ⁇ g / mL, preferably 1-100 ⁇ g / mL, more preferably 3-60 ⁇ g / mL (eg, 10 ⁇ g / mL).
  • Tretinoin acid is usually used at a concentration of 0.01 to 100 ⁇ M, preferably 0.1 to 10 ⁇ M, more preferably 0.5 to 5 ⁇ M (eg, 1 ⁇ M).
  • the Activin signaling pathway agent can be appropriately used in an amount having an Activin signaling promoting activity equivalent to that of Activin A at the above concentration.
  • the sonic hedgehog signal transduction pathway agent can be appropriately used in an amount having the same Shh signal transduction promoting activity as the SAG and Shh N-terminals at the above concentrations.
  • the aggregate obtained by the step (1) expresses the retinal ganglion cell marker Brn3 in the inner layer and the photoreceptor progenitor cell marker Crx in the outermost layer. It is not particularly limited as long as it is an aggregate in which the expressed cell layer is formed, but it is usually 0.5 to 50 days.
  • the culture time of the aggregate containing the retinal progenitor cells in the step (1) is preferably 1 day or more, 5 days or more, 10 days or more, 15 days or more, or 20 days or more.
  • the culture time of pluripotent stem cells in the step (1) is preferably within 45 days or within 40 days.
  • the range of culture time for aggregates containing retinal progenitor cells in step (1) is preferably 1 to 45 days, 15 to 45 days, or 20 to 40 days (most preferably 30 days). .. That is, in step (1), aggregates containing retinal progenitor cells were suspended and cultured for 0.5 to 50 days (preferably 1 to 45 days, 15 to 45 days, or 20 to 40 days (most preferably 30 days)). After that, step (2) is continued.
  • step (1) aggregates containing retinal progenitor cells are cultivated in a medium containing Activin A (final concentration 100 ng / mL), SAG (final concentration 100 nM) and retinoic acid (final concentration 1 ⁇ M). Suspension culture can be performed for 30 days.
  • Culture conditions such as culture temperature and CO 2 concentration in step (1) can be set as appropriate.
  • the culture temperature is, for example, about 30 ° C to about 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
  • step (2) aggregates containing retinal ganglion cells and photoreceptor precursor cells are placed in a medium containing a sonic hedgehog signaling pathway agonist in the absence of Activin signaling pathway agonists and retinoic acid. Floating culture is performed to obtain layered retinal tissue containing photoreceptor cells.
  • step (2) aggregates containing retinal ganglion cells and photoreceptor progenitor cells are suspended-cultured.
  • the incubator used in the suspension culture method and suspension culture may be the same as that used in step (1).
  • the basal medium used in the step (2) is not particularly limited, and the basal medium described in the step (1) can be appropriately selected.
  • the medium used in the step (2) may be a serum-containing medium or a serum-free medium as in the step (1), but the retinal ganglion cells and photoreceptor precursors contained in the aggregates. Since the cells have already been determined to differentiate, it is preferable to use a serum-containing medium to promote further maturation and proliferation of these cells.
  • DMEM / F-12 containing 0.1 mM Taurine Sigma-Aldrich
  • GlutaMax supplement medium Thermo Fisher Scientific
  • the medium used for suspension culture of aggregates containing retinal ganglion cells and photoreceptor progenitor cells in step (2) is sonic hedgehog signaling pathway action in the absence of Activin signaling pathway agonist and retinoic acid. Contains substances.
  • step (1) in the conventional method, in order to prepare retinal tissue, after the pluripotent stem cells have differentiated into retinal progenitor cells, a method of culturing in a medium containing no differentiation-inducing factor is usually used. there were.
  • step (2) aggregates containing retinal ganglion cells and photoreceptor precursor cells were further subjected to a medium containing a sonic hedgehog signaling pathway agonist in the absence of Activin signaling pathway agonist and retinoic acid.
  • a medium containing a sonic hedgehog signaling pathway agonist in the absence of Activin signaling pathway agonist and retinoic acid.
  • the transfer method from the step (1) to the step (2) is not particularly limited.
  • a medium containing the medium used in step (1) that is, an Activin signaling pathway agonist, a sonic Hedgehog signaling pathway agonist, and retinoic acid.
  • the medium may be exchanged directly from the medium used in the step (1) to the medium used in the step (2), or the medium used in the step (1) may be exchanged with the basal medium. It may be replaced with the medium used in step (2).
  • step (2) aggregates containing retinal ganglion cells and photoreceptor precursor cells are suspended-cultured in a medium containing a sonic hedgehog signaling pathway agonist in the absence of Activin signaling pathway agonists and retinoic acid. ..
  • absence means that a specific substance is not contained in the medium. Therefore, for example, in the absence of the Activin signal transduction pathway agent, the condition that the Activin signal transduction pathway agent is not added, or the activin signal transduction pathway agent is substantially not contained (for example, Activin A).
  • the condition that the concentration is 0.1 ng / mL or less) can be mentioned.
  • the absence of retinoin acid includes a condition in which A retinoinic acid is not added or a condition in which retinoinic acid is substantially not contained (for example, the concentration of retinoinic acid is 0.1 nM or less).
  • step (2) the Activin signaling pathway agonist and retinoic acid not contained in the medium, and the sonic hedgehog signaling pathway agonist contained in the medium are the same as those described in step (1). It may be a thing.
  • the concentration of the Sonic Hedgehog signaling pathway agent can be appropriately set within the range in which the above effects can be achieved.
  • SAG is typically used at concentrations of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM (eg, 100 nM).
  • the Shh N-terminus is usually used at a concentration of 0.1-300 ⁇ g / mL, preferably 1-100 ⁇ g / mL, more preferably 3-60 ⁇ g / mL (eg, 10 ⁇ g / mL).
  • the sonic hedgehog signaling pathway agonist can be appropriately used in an amount having Shh signaling promoting activity equivalent to that of SAG at the above concentration.
  • the culture time of the aggregate in the step (2) is particularly long as long as the outermost layer of the aggregate obtained in the step (2) can contain pyramidal photoreceptor marker-positive cells or rod photoreceptor marker-positive cells. Not limited, but usually 20-320 days.
  • the culture time of the aggregate containing the retinal ganglion cells and the photoreceptor progenitor cells in the step (2) is preferably 25 days or more, 30 days or more, 35 days or more, or 40 days or more.
  • the culture time of the aggregate containing the retinal ganglion cells and the photoreceptor progenitor cells in the step (2) is preferably within 200 days, within 100 days, within 75 days, or within 60 days.
  • the range of culture times for aggregates containing retinal ganglion cells and photoreceptor progenitor cells in step (2) is preferably 25-200 days, 30-100 days, 35-75 days, or 40-60 days. Days (most preferably 50 days).
  • step (2) aggregates containing retinal ganglion cells and photoreceptor precursor cells are placed in a medium containing SAG (final concentration 100 nM) in the absence of Activin signaling pathway agonist and retinoic acid.
  • a layered retinal tissue containing photoreceptor cells can be obtained by performing suspension culture for 50 days.
  • Culture conditions such as culture temperature and CO 2 concentration in step (2) can be set as appropriate.
  • the culture temperature is, for example, about 30 ° C to about 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
  • the production method 1 of the present invention may further include the following steps before the step (1).
  • (b) Neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway agonist in the absence of undifferentiated maintenance factor, TGF ⁇ signaling pathway inhibitor and BMP signaling pathway inhibitor, and retinal progenitor cells.
  • Retinal progenitor cells are adherently cultured in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists, and coagulation containing retinal progenitor cells.
  • pluripotent stem cells are adherently cultured in a medium containing a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors.
  • a pluripotent stem cell can be cultured in vitro and can differentiate into all three germ layers (outer germ layer, middle germ layer, endoderm) and / or all cell lineages belonging to extraembryonic tissues. Stem cells with ability (pluripotency).
  • Pluripotent stem cells can be derived from fertilized eggs, cloned embryos, reproductive stem cells, tissue stem cells, somatic cells, etc.
  • pluripotent stem cells include embryonic stem cells (ES cells: Embryonic stem cells), EG cells (Embryonic germ cells), and induced pluripotent stem cells (iPS cells: induced pluripotent stem cells).
  • Muse cells Multi-lineage differentiating stress ending cells obtained from mesenchymal stem cells (MSC) and GS cells produced from germ cells (for example, testis) are also included in pluripotent stem cells.
  • Embryonic stem cells were first established in 1981 and have been applied to knockout mouse production since 1989. Human embryonic stem cells were established in 1998 and are being used in regenerative medicine.
  • ES cells can be produced by culturing the inner cell mass on feeder cells or in a medium containing LIF. Methods for producing ES cells are described in, for example, WO96 / 22362, WO02 / 101057, US5,843,780, US6,200,806, US6,280,718 and the like.
  • Embryonic stem cells can be obtained from predetermined institutions or can be purchased commercially. For example, human embryonic stem cells KhES-1, KhES-2 and KhES-3 are available from the Institute for Frontier Medical Sciences, Kyoto University.
  • the human embryonic stem cell Rx :: GFP strain (derived from KhES-1) is available from RIKEN. Both EB5 cells, which are mouse embryonic stem cells, are available from RIKEN, and the D3 strain is available from ATCC.
  • the pluripotent stem cells used in the present invention are preferably ES cells or induced pluripotent stem cells, and more preferably induced pluripotent stem cells.
  • the induced pluripotent stem cells in the present specification are cells in which pluripotency is induced by reprogramming somatic cells by a known method or the like. Specifically, differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells are treated as Oct3 / 4, Sox2, Klf4, Myc (c-Myc, N-Myc, L-Myc), Glis1, Nanog, Sall4, lin28. , Esrrb, etc., and cells that have been reprogrammed to induce pluripotency by expression of any combination of a plurality of genes selected from the reprogramming gene group.
  • differentiated somatic cells such as fibroblasts and peripheral blood mononuclear cells are treated as 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), (2) Oct3 / 4, Sox2, Klf4, Lin28, and L-Myc (Stem). Cells, 2013; 31: 458-466) can be mentioned.
  • Induced pluripotent stem cells were established in mouse cells by Yamanaka et al. In 2006 (Cell, 2006, 126 (4), pp.663-676). Induced pluripotent stem cells were also established in human fibroblasts in 2007, and have pluripotency and self-renewal ability similar to embryonic stem cells (Cell, 2007, 131 (5), pp.861-872; Science. , 2007, 318 (5858), pp.1917-1920; Nat. Biotechnol., 2008, 26 (1), pp.101-106).
  • induced pluripotent stem cells In addition to the method of producing induced pluripotent stem cells by direct reprogramming by gene expression, it is also possible to induce induced pluripotent stem cells from somatic cells by adding a compound or the like (Science, 2013, 341, pp. 651- 654).
  • induced pluripotent stem cells for example, 201B7 cells, 201B7-Ff cells, 253G1 cells, 253G4 cells, 1201C1 cells, 1205D1 cells, 1210B2 cells established at Kyoto University.
  • 1231A3 cells and other human induced pluripotent cell lines are available from Kyoto University and iPS Academia Japan Co., Ltd.
  • the induced pluripotent stem cells for example, Ff-I01 cells, Ff-I14 cells and QHJI01s04 cells established at Kyoto University are available from Kyoto University.
  • the somatic cells used for producing artificial pluripotent stem cells are not particularly limited, but are tissue-derived fibroblasts, blood cell lineage cells (for example, peripheral blood mononuclear cells (PBMC) and T cells), and liver. Examples thereof include cells, pancreatic cells, intestinal epithelial cells, smooth muscle cells and the like.
  • the means for expressing the genes is not particularly limited when the cells are reprogrammed by the expression of several kinds of genes.
  • an infection method using a virus vector for example, a retrovirus vector, a lentivirus vector, an adenovirus vector, an adeno-associated virus vector, a Sendai virus vector which is a cytoplasmic RNA vector), or a plasmid vector which is a non-viral vector.
  • Gene transfer method using for example, plasmid vector, episomal vector
  • plasmid vector, episomal vector for example, calcium phosphate method, lipofection method, retronectin method, electroporation method
  • gene transfer method using RNA vector for example, calcium phosphate method
  • lipofection method electroporation method
  • direct protein injection method for example, a method using a needle, a lipofection method, an electroporation method
  • Induced pluripotent stem cells can be produced in the presence of feeder cells or in the absence of feeder cells (feeder-free).
  • artificial pluripotent stem cells can be produced in the presence of undifferentiated maintenance factor by a known method.
  • the medium used for producing induced pluripotent stem cells in the absence of feeder cells that is, a medium containing an undifferentiated maintenance factor (undifferentiated maintenance medium) is not particularly limited, but known embryonic stem cells and / Alternatively, a medium well known to those skilled in the art can be used as a maintenance medium for induced pluripotent stem cells or a medium for establishing artificial pluripotent stem cells in a feeder-free manner.
  • Essential 8 medium is DMEM / F12 medium with L-ascorbic acid-2-phosphate magnesium (64 mg / l), sodium selenite (14 ⁇ g / 1), insulin (19.4 mg / l), ⁇ 3 ( Includes 543 mg / l), transferrin (10.7 mg / l), bFGF (100 ng / mL), and TGF ⁇ family signaling pathway agonist (TGF ⁇ 1 (2 ng / mL) or Nodal (100 ng / mL)) (Nature Methods, 8, 424-429 (2011)).
  • feeder-free media include, for example, Essential 6 medium (manufactured by Life Technologies), Stabilized Essential 8 medium (manufactured by Life Technologies), and S-medium (manufactured by DS Pharma Biomedical).
  • Essential 6 medium manufactured by Life Technologies
  • Stabilized Essential 8 medium manufactured by Life Technologies
  • S-medium manufactured by DS Pharma Biomedical
  • StemPro manufactured by Life Technologies
  • hESF9 Proc Natl Acad Sci U S A. 2008 Sep 9; 105 (36): 13409-14
  • TeSR medium manufactured by STEMCELL Technologies
  • mTeSR1 manufactured by STEMCELL Technologies
  • Examples include mTeSR2 (manufactured by STEMCELL Technologies) and TeSR-E8 (manufactured by STEMCELL Technologies).
  • StemFit registered trademark
  • a feeder-free medium undifferentiated maintenance medium
  • the pluripotent stem cell used in the present invention is a mammalian pluripotent stem cell, preferably a rodent (eg, mouse, rat) or primate (eg, human, monkey) pluripotent stem cell. More preferably, it is a human induced pluripotent stem cell, and even more preferably, it is a human induced pluripotent stem cell (iPS cell) or a human embryonic stem cell (ES cell).
  • iPS cell human induced pluripotent stem cell
  • ES cell human embryonic stem cell
  • pluripotent stem cells established from somatic cells of transplant recipients can be used to solve this problem.
  • pluripotent stem cells eg, induced pluripotent stem cells
  • the recipient It produces a layered retinal tissue containing immunological autologous photoreceptor cells, which is transplanted into the recipient.
  • pluripotent stem cells eg, induced pluripotent stem cells
  • somatic cells of another person whose recipient and immunity are compatible for example, HLA type and MHC type are compatible
  • Layered retinal tissue containing photoreceptor cells may be produced and transplanted into the recipient.
  • pluripotent stem cells are adherently cultured.
  • the term "adhesive culture” or “adhesive culture method” refers to culturing cells or cell aggregates under conditions of adhering them to a culture device or the like, and a method of performing the culture. That is, adhesive culture is performed under the condition that cells or cell aggregates are adhered to culture equipment or the like, and suspension culture is not included in the category of adhesive culture. More specifically, adhesive culture refers to culture under conditions that allow a strong cell-substrate bond to be formed between cells or cell aggregates and culture equipment or the like.
  • the incubator used for adhesive culture is not particularly limited as long as it is capable of adhesive culture, and a person skilled in the art should appropriately select an incubator according to the scale of culture, culture conditions and culture period. Is possible.
  • Examples of such an incubator include a flask, a tissue culture flask, a culture dish (dish), a tissue culture dish, a multi-dish, a micro plate, a micro-well plate, a multi-plate, a multi-well plate, a chamber slide, and a petri dish.
  • Examples include tubes, trays, culture bags, microcarriers, beads, stack plates, spinner flasks or roller bottles. These incubators are preferably cell adherent in order to enable adherent culture.
  • Examples of the cell-adhesive incubator include an incubator in which the surface of the incubator is artificially treated for the purpose of improving the adhesion to cells, specifically, a surface-processed incubator or an incubator.
  • An incubator whose inside is coated with a coating agent can be mentioned.
  • Examples of the coating agent include laminin [including laminin ⁇ 5 ⁇ 1 ⁇ 1 (hereinafter, laminin 511), laminin ⁇ 1 ⁇ 1 ⁇ 1 (hereinafter, laminin 111) and the like, and laminin fragments (including laminin 511E8, iMatrix-511 (Nippi), etc.)], entactin, collagen.
  • Extracellular matrix such as gelatin, Vitronectin, Synsemax (Corning), Matrigel, etc., or polymers such as polylysine, polyornithin, etc.
  • the surface-treated incubator include a surface-treated incubator such as a positive charge treatment.
  • the medium usually used for culturing animal cells can be prepared as the basal medium in the same manner as in the suspension culture.
  • the basal medium include BME medium, BGJb medium, CMRL1066 medium, Glassgow MEM (GMEM) medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, DMEM medium, and F-12.
  • media that can be used for culturing animal cells such as medium, DMEM / F12 medium, IMDM / F12 medium, ham medium, RPMI1640 medium, Fischer's medium, or a mixed medium thereof.
  • the medium used in the adhesion culture may be a serum-containing medium or a serum-free medium, and may be a differentiation-inducing factor necessary for differentiating pluripotent stem cells into aggregates containing retinal progenitor cells. To limit it, it is preferable to use a serum-free medium.
  • the serum-free medium means a medium containing no unadjusted or unpurified serum.
  • a medium containing purified blood-derived components or animal tissue-derived components is also included in the serum-free medium unless unadjusted or unpurified serum is contained.
  • the serum-free medium may contain a serum substitute.
  • the serum substitute include those appropriately containing albumin, transferrin, fatty acid, collagen precursor, trace element, 2-mercaptoethanol or 3'thiolglycerol, or an equivalent thereof.
  • Such serum substitutes can be prepared, for example, by the method described in WO98 / 30679.
  • Commercially available products may be used as serum substitutes. Examples of such commercially available serum substitutes include Knockout TM Serum Replacement (manufactured by Life Technologies; currently Thermo Fisher: hereinafter, also referred to as KSR), Chemically-defined Lipid concentrated (manufactured by Life Technologies), and Glutamax TM . (Life Technologies), B27 (Life Technologies), N2 supplement (Life Technologies), ITS supplement (Life Technologies).
  • the serum-free medium used in the adhesion culture is appropriately fatty acids or lipids, amino acids (for example, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, 2-mercaptoethanol, pyruvate, buffers, inorganic salts and the like. May be contained.
  • the medium used for adherent culture of pluripotent stem cells in step (a) contains a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors.
  • agglomerates of pluripotent stem cells are prepared by adherent culture of pluripotent stem cells in the presence of undifferentiated maintenance factors, and the aggregates are differentiated and cultured in the absence of undifferentiated maintenance factors. It was a common method to make retinal tissue by means of.
  • pluripotent stem cells are adherently cultured in a medium containing a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor in the absence of undifferentiated maintenance factors. The purpose is to directly differentiate pluripotent stem cells into neural progenitor cells without going through stem cell aggregates.
  • the undifferentiated maintenance factor in the step (a) is not particularly limited as long as it is a substance having an action of suppressing the differentiation of pluripotent stem cells.
  • undifferentiated maintenance factors widely used by those skilled in the art, in the case of primed pluripotent stem cells (for example, human ES cells and human iPS cells), FGF signaling pathway agonists and TGF ⁇ signaling pathways are used. Activators, insulin and the like can be mentioned.
  • the FGF signal transduction pathway agent include fibroblast growth factors (eg, bFGF, FGF4 and FGF8).
  • TGF ⁇ signal transduction pathway agent include TGF ⁇ 1 and TGF ⁇ 2.
  • the absence of the undifferentiated maintenance factor means that the undifferentiated maintenance factor is not contained in the medium. Therefore, in the absence of the undifferentiated maintenance factor, the condition that the undifferentiated maintenance factor is not added or that the undifferentiated maintenance factor is substantially not contained (for example, the concentration of the undifferentiated maintenance factor is 10 pg / mL).
  • concentration of the undifferentiated maintenance factor is 10 pg / mL.
  • the TGF ⁇ signaling pathway is a signal transduction pathway that uses TGF ⁇ as a ligand and is transmitted intracellularly by the Smad family. Therefore, the TGF ⁇ signaling pathway inhibitor represents a substance that inhibits the TGF ⁇ signaling pathway, that is, the signaling pathway transmitted by the Smad family.
  • the TGF ⁇ signal transduction pathway inhibitor is not particularly limited as long as it is a substance that inhibits the signal transduction pathway caused by TGF ⁇ , and may be any of nucleic acid, protein, and low molecular weight organic compound.
  • Such substances include, for example, substances that directly act on TGF ⁇ (for example, proteins, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding TGF ⁇ (for example, antisense oligonucleotides, siRNA, etc.), TGF ⁇ receptors and TGF ⁇ .
  • examples thereof include a substance that inhibits binding and a substance that inhibits physiological activity caused by signal transduction by the TGF ⁇ receptor (for example, an inhibitor of TGF ⁇ receptor, an inhibitor of Smad, etc.).
  • TGF ⁇ signal transduction pathway inhibitors examples include Lefty and the like.
  • TGF ⁇ signal transduction pathway inhibitor a compound well known to those skilled in the art can be used, and specific examples thereof include SB431542, LY-364947, SB-505124, and A-83-01.
  • SB431542 (4- (5-benzol [1,3] dioxol-5-yl-4-pyridin-2-yl-1H-imidazol-2-yl) -benzamide) and A-83-01 (3-( 6-Methyl-2-pyridinyl) -N-phenyl-4- (4-quinolinyl) -1H-pyrazole-1-carbothioamide) is an inhibitor of TGF ⁇ receptor (ALK5) and Activin receptor (ALK4 / 7). It is a compound known as (that is, a TGF ⁇ R inhibitor). As the TGF ⁇ signal transduction pathway inhibitor, one or more of these may be contained.
  • the TGF ⁇ signaling pathway inhibitor is preferably SB431542 or A-83-01.
  • the BMP signal transduction pathway inhibitor is not particularly limited as long as it is a substance that inhibits the signal transduction pathway caused by BMP, and may be any of nucleic acid, protein, and small molecule organic compound.
  • examples of the BMP include BMP2, BMP4, BMP7 and GDF7.
  • Such substances include, for example, substances that directly act on BMP (for example, antibodies, aptamers, etc.), substances that suppress the expression of genes encoding BMP (for example, antisense oligonucleotides, siRNA, etc.), BMP receptors (BMPR) and BMP. Examples thereof include substances that inhibit binding and substances that inhibit physiological activity caused by signal transduction by BMP receptors.
  • BMPR ALK2 or ALK3 can be mentioned.
  • LDN193189 4- [6- (4-piperazine-1-ylphenyl) pyrazolo [1,5-a] pyrimidin-3-yl] quinoline
  • BMPR inhibitor BMPR inhibitor
  • proteins known as BMP signal transduction pathway inhibitors may be used.
  • the BMP signal transduction pathway inhibitor may contain one or more of these.
  • the BMP signaling pathway inhibitor is preferably LDN193189.
  • the concentrations of the TGF ⁇ signal transduction pathway inhibitor and the BMP signal transduction pathway inhibitor can be appropriately set within the range in which the above effects can be achieved.
  • SB431542 is typically used at a concentration of 0.025 to 1000 ⁇ M, preferably 0.1 to 250 ⁇ M, more preferably 0.5 to 50 ⁇ M (eg, 5 ⁇ M).
  • Lefty is usually used at a concentration of 0.1 to 3000 ng / mL, preferably 1 to 1000 ng / mL, more preferably 10 to 300 ng / mL (eg, 100 ng / mL).
  • LY-364947 is usually used at a concentration of 0.025 to 1000 ⁇ M, preferably 0.1 to 250 ⁇ M, more preferably 0.5 to 50 ⁇ M (eg, 10 ⁇ M).
  • SB505124 is typically used at a concentration of 0.025 to 1000 ⁇ M, preferably 0.1 to 250 ⁇ M, more preferably 0.5 to 50 ⁇ M (eg, 5 ⁇ M).
  • A-83-01 is usually used at a concentration of 0.001 to 300 ⁇ M, preferably 0.01 to 30 ⁇ M, and more preferably 0.1 to 3 ⁇ M (for example, 0.5 ⁇ M).
  • LDN193189 is usually used at a concentration of 1 to 2000 nM, preferably 10 to 700 nM, more preferably 30 to 600 nM (eg, 100 nM).
  • Dorsomorphin is usually used at a concentration of 0.01 to 1000 ⁇ M, preferably 0.1 to 100 ⁇ M, more preferably 1 to 10 ⁇ M (eg, 3 ⁇ M).
  • the TGF ⁇ signaling pathway inhibitor can be appropriately used in an amount having a TGF ⁇ signaling inhibitory activity equivalent to that of SB431542 at the above concentration.
  • the BMP signal transduction pathway inhibitor can be appropriately used in an amount having a BMP signal transduction inhibitory activity equivalent to that of LDN 193189 at the above concentration.
  • the culture time of pluripotent stem cells in the step (a) is not particularly limited as long as the cells obtained in the step (a) are cells expressing the neural progenitor cell marker Rx, but are usually 1 to 120 hours.
  • the culture time of the pluripotent stem cells in the step (a) is preferably 5 hours or more, 10 hours or more, 15 hours or more, or 20 hours or more.
  • the culture time of the pluripotent stem cells in the step (a) is preferably 100 hours or less, 80 hours or less, 60 hours or less, or 50 hours or less.
  • the range of culture time for pluripotent stem cells in step (a) is preferably 5-100 hours, 10-80 hours, 15-60 hours, or 20-50 hours (most preferably 24 hours). Is.
  • step (a) pluripotent stem cells are adherently cultured for 1 to 50 hours (preferably 5 to 100 hours, 10 to 80 hours, 15 to 60 hours, or 20 to 50 hours (most preferably 24 hours)). After that, step (b) is continued.
  • pluripotent stem cells in step (a), can be adherently cultured in a medium containing SB431542 (final concentration 5 ⁇ M) and LDN193189 (final concentration 100 nM) for 24 hours.
  • Culture conditions such as culture temperature and CO 2 concentration in step (a) can be set as appropriate.
  • the culture temperature is, for example, about 30 ° C to about 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
  • neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway agonist in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors. ..
  • step (b) neural progenitor cells are adherently cultured.
  • the incubator used for the adhesive culture method and the adhesive culture may be the same as that used in the step (a).
  • the basal medium used in the step (b) is not particularly limited, and the basal medium described in the step (a) can be appropriately selected.
  • the medium used in the step (b) may be a serum-containing medium or a serum-free medium as in the step (a), but is necessary for differentiating the neural progenitor cells into retinal progenitor cells. It is preferable to use a serum-free medium in order to limit the differentiation-inducing factors.
  • serum-free medium 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1 x Antibiotic-Antimycotic (1 x Antibiotic-Antimycotic) as in step (a).
  • the medium used for adherent culture of neural progenitor cells in step (b) contains BMP signaling pathway agonists in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors.
  • BMP signaling pathway agonists in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors.
  • neural progenitor cells differentiated from pluripotent stem cells are subjected to BMP signaling pathway action in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors.
  • Adhesion culture in a medium containing a substance can promote differentiation into retinal progenitor cells.
  • the transfer method from the step (a) to the step (b) is not particularly limited.
  • the medium used in step (a) that is, the medium containing the TGF ⁇ signal transduction pathway inhibitor and the BMP signal transduction pathway inhibitor
  • step (b) Can be transferred by exchanging the medium with the medium used in () (medium containing a BMP signal transduction pathway agonist).
  • the medium may be exchanged directly from the medium used in the step (a) to the medium used in the step (b), or the medium used in the step (a) may be exchanged with the basal medium. It may be replaced with the medium used in step (b).
  • neural progenitor cells are adherently cultured in a medium containing a BMP signaling pathway agonist in the absence of an undifferentiated maintenance factor, a TGF ⁇ signaling pathway inhibitor and a BMP signaling pathway inhibitor.
  • the absence of the undifferentiated maintenance factor in the step (b) may be the same conditions as in the step (a). Further, in the absence of the TGF ⁇ signal transduction pathway inhibitor, the condition that the TGF ⁇ signal transduction pathway inhibitor is not added or that the TGF ⁇ signal transduction pathway inhibitor is substantially not contained (for example, TGF ⁇ signal transduction pathway inhibition). The condition that the concentration of the substance is 10 nM or less) can be mentioned.
  • the absence of a BMP signal transduction pathway inhibitor means that the BMP signal transduction pathway inhibitor is not added or that the BMP signal transduction pathway inhibitor is substantially not contained (for example, a BMP signal transduction pathway inhibitor). The condition that the concentration is 100 pM or less) can be mentioned.
  • the undifferentiated maintenance factor, the TGF ⁇ signaling pathway inhibitor and the BMP signaling pathway inhibitor not contained in the medium may be the same as those described in the step (a).
  • a BMP signal transduction pathway agent is a substance that can enhance a signal mediated by BMP.
  • the BMP signaling pathway agonist is not particularly limited as long as it can induce the differentiation of neural precursor cells into retinal precursor cells by enhancing the BMP signaling pathway, but for example, BMP proteins such as BMP2, BMP4 or BMP7, Examples thereof include GDF proteins such as GDF7, anti-BMP receptor antibodies, and BMP partial peptides.
  • the BMP signal transduction pathway agent may contain one or more of these.
  • the BMP2 protein, BMP4 protein and BMP7 protein are available, for example, from R & D Systems, and the GDF7 protein is available, for example, from Wako Pure Chemical Industries.
  • the BMP signaling pathway agent is preferably BMP4.
  • the concentration of the BMP signal transduction pathway agent can be appropriately set within the range in which the above effects can be achieved.
  • BMP4 is usually used at a concentration of 0.02 to 500 nM, preferably 0.1 to 100 nM, more preferably 0.3 to 30 nM (eg, 3 nM).
  • the BMP signaling pathway agent can be appropriately used in an amount having a BMP signaling promoting activity equivalent to that of BMP4 at the above concentration.
  • the culture time of neural progenitor cells in step (b) is such that the cells obtained in step (b) express at least one selected from the group consisting of retinal progenitor cell markers Rx, PAX6, and Chx10. As long as it is not particularly limited, it is usually 0.5 to 6 days.
  • the culture time of neural progenitor cells in step (b) is preferably 1 day or longer.
  • the culture time of neural progenitor cells in step (b) is preferably within 3 days. In one embodiment, the range of culture time of neural progenitor cells in step (b) is preferably 1 to 3 days (most preferably 2 days).
  • neural progenitor cells are placed in a medium containing BMP4 (final concentration 3 nM) in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors.
  • Retinal progenitor cells can be obtained by adhering culture for 2 days.
  • Culture conditions such as culture temperature and CO 2 concentration in step (b) can be set as appropriate.
  • the culture temperature is, for example, about 30 ° C to about 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
  • retinal progenitor cells are adherently cultured in a medium in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists.
  • step (c) retinal progenitor cells are adherently cultured.
  • the incubator used in the adhesive culture method and the adhesive culture may be the same as those used in the steps (a) and (b).
  • the basal medium used in the step (c) is not particularly limited, and the basal medium described in the steps (a) and (b) can be appropriately selected.
  • the medium used in the step (c) may be a serum-containing medium or a serum-free medium as in the step (a), but the retinal progenitor cells are differentiated into aggregates containing the retinal progenitor cells. It is preferable to use a serum-free medium in order to limit the differentiation-inducing factors required for this.
  • serum-free medium 10% Fetal Bovine Serum (BioSera), 1% N2 Supplement (Thermo Fisher Scientific), 1x, as in steps (a) and (b).
  • the medium used for adherent culture of retinal progenitor cells in step (c) does not contain undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists.
  • TGF ⁇ signaling pathway inhibitors when human pluripotent stem cells are differentiated into neuroretinal cells by culturing them in a medium containing BMP4, it is common to continue culturing in the medium containing BMP4 until they are differentiated into neuroretinal cells. It was a method.
  • retinal precursor cells are adherently cultured in a medium containing not only undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors and BMP signaling pathway inhibitors, but also BMP signaling pathway agonists. This makes it possible to promote differentiation into aggregates containing neuroretinal cells.
  • the transfer method from the step (b) to the step (c) is not particularly limited.
  • the medium used in step (b) that is, the medium containing the BMP signal transduction pathway agonist
  • the medium used in step (c) Can be migrated by doing.
  • retinal progenitor cells are adherently cultured in a medium in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists.
  • the absence of the undifferentiated maintenance factor, the TGF ⁇ signaling pathway inhibitor, and the BMP signaling pathway inhibitor in the step (c) may be the same conditions as in the step (b).
  • the absence of a BMP signal transduction pathway agent means that the BMP signal transduction pathway agent is not added, or that the BMP signal transduction pathway agent is substantially not contained (for example, the BMP signal transduction pathway agent).
  • the condition that the concentration is 10 pg / mL or less) can be mentioned.
  • step (c) undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists not contained in the medium are described in steps (a) and (b). It may be the same as the one that was used.
  • the culture time of the retinal progenitor cells in the step (c) is at least one in which the aggregate containing the retinal progenitor cells obtained in the step (c) is selected from the group consisting of the retinal progenitor cell markers Rx, PAX6 and Chx10. It is not particularly limited as long as the cell layer to be expressed is an aggregate formed, but it is usually 2 to 27 days.
  • the culture time of the retinal progenitor cells in the step (c) is preferably 3 days or more, 4 days or more, 5 days or more, or 6 days or more.
  • the culture time of the retinal progenitor cells in step (c) is preferably within 25 days, within 20 days, within 15 days, or within 8 days.
  • the range of culture time of retinal progenitor cells in step (b) is preferably 3 to 11 days, 4 to 10 days, 5 to 9 days, or 6 to 8 days (most preferably 7 days). be.
  • retinal precursor cells are placed in a medium in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists for 7 days.
  • TGF ⁇ signaling pathway inhibitors TGF ⁇ signaling pathway inhibitors
  • BMP signaling pathway inhibitors BMP signaling pathway agonists
  • Culture conditions such as culture temperature and CO 2 concentration in step (c) can be set as appropriate.
  • the culture temperature is, for example, about 30 ° C to about 40 ° C, preferably about 37 ° C.
  • the CO 2 concentration is, for example, about 1% to about 10%, preferably about 5%.
  • the present invention provides a therapeutic agent (therapeutic agent of the present invention) for a disease caused by a disorder of the retinal tissue, including a layered retinal tissue containing photoreceptor cells.
  • the therapeutic agent of the present invention contains an effective amount of layered retinal tissue containing photoreceptor cells and a pharmaceutically acceptable carrier.
  • the layered retinal tissue containing the included photoreceptor cells may be produced by the production method 1 of the present invention.
  • a physiological aqueous solvent physiological saline solution, buffer solution, serum-free medium, etc.
  • a medicine containing tissues and cells to be transplanted may be mixed with a preservative, a stabilizer, a reducing agent, an tonicity agent, etc., which are usually used.
  • the therapeutic agent of the present invention can be produced as a suspension by suspending the layered retinal tissue containing photoreceptor cells in an appropriate physiological aqueous solvent. If necessary, a cryopreservative may be added, cryopreserved, thawed at the time of use, washed with a buffer solution, and used for transplantation medicine.
  • a layered retinal tissue containing photoreceptor cells can be used as a therapeutic agent for a disease caused by a disorder of the retinal tissue or in order to replenish the damaged site in the damaged state of the retinal tissue.
  • Visualization by transplanting a layered retinal tissue containing photoreceptor cells into a patient with a disorder-based disorder of retinal tissue or a damaged state of retinal tissue that requires transplantation and replenishing the damaged retinal tissue itself. It can treat diseases caused by disorders of layered retinal tissue containing cells, or damaged states of retinal tissue.
  • Diseases based on damage to layered retinal tissue, including photoreceptor cells include, for example, ophthalmic diseases such as age-related macular degeneration, retinitis pigmentosa, pyramidal dystrophy, macular edema, retinal detachment, cancer-related retinopathy, and retinal veins. Examples include obstruction and retinal pigment epithelial detachment.
  • the layered retinal tissue containing photoreceptor cells produced by the production method 1 of the present invention is a layered retinal tissue containing photoreceptor cells. Since it is useful as a disease research material and a drug discovery material in screening of therapeutic agents for diseases based on retinal tissue disorders and drug efficacy evaluation, it can be used as a screening reagent or a drug efficacy evaluation reagent for a test substance.
  • iPS cells are prepared from a human patient with a disease caused by a disorder of layered retinal tissue including photoreceptor cells, particularly a disease caused by a hereditary disorder, and the iPS cells are used for vision according to the production method 1 of the present invention.
  • the retinal tissue can reproduce in vitro damage to the layered retinal tissue containing photoreceptor cells that causes the disease the patient is suffering from. Therefore, the present invention comprises contacting a test substance with a layered retinal tissue containing photoreceptor cells produced by the production method 1 of the present invention and testing the effect of the substance on the retinal tissue.
  • layered retinal tissue containing photoreceptor cells having a specific disorder is cultured in the presence or absence of a test substance (negative control). Then, the degree of damage in the layered retinal tissue containing the photoreceptor cells treated with the test substance is compared with the negative control.
  • a test substance with a reduced degree of the disorder can be selected as a candidate substance for a therapeutic agent for the disease based on the disorder.
  • a test substance that further enhances the physiological activity (for example, survival promotion or maturation) of a layered retinal tissue containing photoreceptor cells can be searched for as a drug candidate substance.
  • a layered retina containing photoreceptor cells produced by preparing artificial pluripotent stem cells from somatic cells having a gene mutation exhibiting a specific disorder such as retinal disease and inducing differentiation of the cells by the production method 1 of the present invention.
  • a test substance can be added to a tissue, and a candidate for a test substance effective as a therapeutic agent or a preventive agent for the disorder can be searched for by using whether or not the disorder is exhibited as an index.
  • the present invention provides a method for screening a therapeutic agent for a disorder of layered retinal tissue including photoreceptor cells, which comprises the following steps (11) to (13). (11) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the presence of the test substance for a certain period of time under viable culture conditions, the degree of damage to the layered retinal tissue containing the photoreceptor cells is measured.
  • step (12) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the absence of the test substance or in the presence of positive control for a certain period of time under viable culture conditions, the layered retina containing the photoreceptor cells The process of measuring the degree of tissue damage, (13) A step of selecting a test substance in step (11) as a therapeutic agent for a disorder of layered retinal tissue including photoreceptor cells based on the difference in the results measured in step (11) and step (12).
  • the present invention provides a drug efficacy evaluation method including the following steps (21) to (23). (21) After culturing the layered retinal tissue containing the impaired photoreceptor cells in the presence of the test substance for a certain period of time under viable culture conditions, the degree of damage to the layered retinal tissue containing the photoreceptor cells is measured.
  • the layered retinal tissue containing photoreceptor cells is not particularly limited as long as it is a layered retinal tissue containing damaged photoreceptor cells, and may be produced by the production method 1 of the present invention.
  • the absence of the test substance includes adding only the medium and the solvent in which the test substance is dissolved instead of the test substance.
  • positive control means a known compound having a therapeutic effect on damage to layered retinal tissue including photoreceptor cells.
  • a method for measuring the degree of damage a method for measuring the number of surviving cells, for example, a method for measuring the amount of intracellular ATP, a method for measuring the number of living cells by cell staining (for example, cell nucleus staining) and morphological observation, etc. Can be mentioned.
  • step (23) as a method of selecting a test substance as a therapeutic agent for a disorder of layered retinal tissue including photoreceptor cells or a method of evaluating the medicinal effect of the test substance, for example, the measured value and step of step (21).
  • the test substance can be selected as a therapeutic agent for layered retinal tissue damage, or the test substance. Can be judged to have medicinal properties.
  • the measured value of step (21) and the measured value of positive control in step (22) are compared, and when the degree of cell damage in step (21) is reduced to the same or higher level, the test substance is applied to the layered retina. It can be selected as a therapeutic agent for tissue disorders, or it can be determined that the test substance has a medicinal effect.
  • Toxicity evaluation method In toxicity evaluation, layered retinal tissue containing photoreceptor cells is cultured in the presence or absence of a test substance (negative control). Then, the degree of toxicity in the layered retinal tissue containing the photoreceptor cells treated with the test substance is compared with the negative control. As a result, the test substance showing toxicity as compared with the negative control can be determined as a substance having toxicity to the layered retinal tissue including photoreceptor cells.
  • the present invention provides a toxicity evaluation method including the following steps (31) to (33).
  • (31) A step of measuring the degree of damage to the layered retinal tissue containing photoreceptor cells after culturing the layered retinal tissue containing photoreceptor cells in the presence of a test substance for a certain period of time under viable culture conditions.
  • (32) Damage to the layered retinal tissue containing photoreceptor cells after culturing the layered retinal tissue containing photoreceptor cells under viable culture conditions for a certain period of time in the absence of a test substance or in the presence of positive control.
  • the process of measuring the degree of (33) A step of evaluating the toxicity of the test substance in the step (31) based on the difference in the results measured in the step (31) and the step (32).
  • the layered retinal tissue containing photoreceptor cells may be produced by the production method 1 of the present invention.
  • the absence of the test substance includes adding only the medium and the solvent in which the test substance is dissolved instead of the test substance.
  • the positive control means a known compound having toxicity.
  • a method for measuring the degree of cell damage a method for measuring the number of surviving cells, for example, a method for measuring the amount of intracellular ATP, or a method for measuring the number of living cells by cell staining (for example, cell nucleus staining) and morphological observation. The method and the like can be mentioned.
  • the measured value of the step (31) is compared with the measured value of the negative control in the step (32), and the cell damage in the step (31) is compared. It can be determined that the test substance is toxic when the degree of is large.
  • the measured value of step (31) and the measured value of positive control in step (32) are compared, and it can be determined that the test substance is toxic when the degree of cell damage in step (31) is equal to or higher than that. ..
  • the present invention also comprises a method for producing aggregates containing suspension-cultured retinal progenitor cells in step (1) of the production method of the present invention (hereinafter, production method 2 of the present invention). I will provide a.
  • the production method 2 of the present invention includes the following steps.
  • Retinal progenitor cells are adherently cultured in the absence of undifferentiated maintenance factors, TGF ⁇ signaling pathway inhibitors, BMP signaling pathway inhibitors and BMP signaling pathway agonists, and coagulation containing retinal progenitor cells. The process of obtaining an aggregate.
  • Example 1 Human iPS cell line 201B7 (Ci) seeded on a 6-well plate coated with iMatrix-511 (Nippi) at a cell density of 5,000 cells per well was cultured in StemFit AK02N medium (Ajinomoto) for 8 days, and then the medium was used.
  • Differentiation Medium (10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin and 450 ⁇ M 1-monothioglycerol (Sigma-Aldrich) ) was replaced with a GMEM medium (Thermo Fisher Scientific)), and differentiation induction was started.
  • GEBCO Non-essential Amino Acid
  • GMEM medium Thermo Fisher Scientific
  • SB431542 final concentration 5 ⁇ M, Fujifilm Wako Pure Chemical Industries, Ltd.
  • LDN193189 hydorochrolide final concentration 100 nM, Sigma-Aldrich
  • BMP4 final concentration 1.5 nM, R & D systems
  • the secondary antibody reaction was carried out at room temperature for 2 hours. After the completion of the secondary antibody reaction, washing was performed using PBS (-), and fluorescence microscopy was performed using a laser cofocal microscope LSM700 (Zeiss). Differentiation started 5 In the aggregate after a day, the formation of a dense cell region consisting of cells expressing the neural precursor cell marker Rx was observed (Fig. 2).
  • Samples 10 days after the start of differentiation were blocked with a blocking solution (5% deactivated horse serum (GIBCO), PBS (-) containing 0.05% Triton-X100) at room temperature for 1 hour, and then subjected to anti-Rx antibody and anti-Rx antibody and anti-Rx antibody.
  • the primary antibody reaction was carried out at 4 ° C., day and night, using CHX10 sheep polyclonal IgG antibody (1: 1000 dilution, X1180P / Exalpha Biologicals).
  • Example 2 Human iPS cell line 201B7 strain seeded with iMatrix-511 (Nippi) -coated 6-well plate at a cell density of 5,000 cells per well was cultured in StemFit AK02N medium (Ajinomoto) for 8 days, and then the medium was used as a differentiation medium (Ajinomoto).
  • SB431542 final concentration 5 ⁇ M, Fujifilm Wako Pure Chemical Industries, Ltd.
  • LDN193189 hydorochrolide final concentration 100 nM, Sigma-Aldrich
  • BMP4 final concentration 1.5 nM, R & D systems
  • the prepared frozen sections were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS (-), and then blocked solution (5% deactivated horse serum (GIBCO), 0.05. Blocking treatment was performed at room temperature for 1 hour using PBS (-) containing% Triton-X100. After that, a primary antibody reaction was carried out at 4 ° C., day and night, using an anti-Crx rabbit polyclonal IgG antibody (1: 1000 diluted, M231 / Takara) and an anti-Brn3 goat polyclonal IgG antibody (1: 1000 diluted, sc-6026 / Santa Cruz). ..
  • fluorescence microscopy was performed using an inverted fluorescence microscope DMi8 (Leica). Retinal ganglion cell marker-positive cells were observed in the inner layer of the aggregate 30 days after the induction of differentiation, and photoreceptor progenitor cell marker-positive cells were scatteredly observed (FIG. 5).
  • Example 3 Human iPS cell line M8 strain seeded with iMatrix-511 (Nippi) -coated 6-well plate at a cell density of 4,500 cells per well (Kyoto University iPS Cell Research Institute "iPS from peripheral blood using episomal vector" According to the “cell establishment method", the inventors cultured iPS cells established from healthy human peripheral blood mononuclear cells in StemFit AK02N medium (Ajinomoto) for 8 days, and then used the medium for differentiation medium (10% KnockOut Serum Replacement (Thermo Fisher)).
  • GMEM medium (Thermo Fisher Scientific) containing 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin and 450 ⁇ M 1-monothioglycerol (Sigma-Aldrich). was replaced with, and differentiation induction was started.
  • GEBCO Non-essential Amino Acid
  • SB431542 final concentration 5 ⁇ M, Fujifilm Wako Pure Chemical Industries, Ltd.
  • LDN193189 hydorochrolide final concentration 100 nM, Sigma-Aldrich
  • BMP4 final concentration 3 nM, R & D systems
  • Aggregates were fixed with 3% PFA, 7.5% sucrose / PBS solution 20 days, 40 days, 60 days, and 80 days after the start of differentiation induction, and embedded with O.C.T.Compound (Sakura Finetech). did. After embedding, the frozen block was sliced using a cryomicrotome to prepare 10 ⁇ m frozen sections.
  • Frozen sections prepared from samples 20 and 40 days after the start of differentiation induction were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS (-), and then a blocking solution. Blocking treatment was performed at room temperature for 1 hour using (5% deactivated horse serum (GIBCO), PBS (-) containing 0.05% Triton-X100). After that, a primary antibody reaction was carried out at 4 ° C., day and night, using an anti-Crx rabbit polyclonal IgG antibody (1: 1000 diluted, M231 / Takara) and an anti-Brn3 goat polyclonal IgG antibody (1: 1000 diluted, sc-6026 / Santa Cruz). ..
  • Frozen sections prepared from the sample 60 days after induction of differentiation were blocked with a blocking solution (5% deactivated horse serum (GIBCO), PBS containing 0.05% Triton-X100 (-)) at room temperature for 1 hour. After that, the primary antibody reaction was carried out at 4 ° C. overnight using anti-RxR ⁇ rabbit polyclonal IgG antibody (1: 200 dilution, sc-555 / Santa Cruz) and anti-NRL goat polyclonal IgG antibody (1: 200 dilution, AF2945 / R & D systems). rice field.
  • a blocking solution 5% deactivated horse serum (GIBCO), PBS containing 0.05% Triton-X100 (-)
  • Frozen sections prepared from the sample 80 days after the start of differentiation induction were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS (-), and then blocked solution (5%). Blocking treatment was performed at room temperature for 1 hour using deactivated horse serum (GIBCO) and PBS (-) containing 0.05% Triton-X100. Then anti-rhodopsin mouse monoclonal IgG antibody (1: 1000 diluted, Clone RET-P1 / Sigma-Aldrich), anti-L / M opsin rabbit polyclonal IgG antibody (1: 1000 diluted, AB5405 / Sigma-Aldrich) and anti-S-opsin goat.
  • a primary antibody reaction was carried out at 4 ° C. for 24 hours a day using a polyclonal IgG antibody (1: 500 diluted, sc-14363 / Santa Cruz). After the primary antibody reaction, wash with PBS (-), then Alexa488-labeled anti-mouse IgG donkey polyclonal IgG antibody (1: 1000 diluted, Thermo Fisher Scientific), Alexa546-labeled anti-rabbit IgG donkey polyclonal IgG antibody (1: 1000 diluted).
  • Thermo Fisher Scientific Alexa647-labeled anti-goat IgG donkey polyclonal IgG antibody (1: 1000 diluted, Thermo Fisher Scientific) and DAPI (final concentration 1 ⁇ g / mL, Thermo Fisher Scientific) at room temperature for 2 hours. gone.
  • the cells were washed with PBS (-) and then encapsulated with a PermaFluor aqueous encapsulant (Thermo Fisher Scientific). After air-drying, fluorescence microscopy was performed using a laser confocal microscope LSM700 (Zeiss).
  • pyramidal photoreceptor cell marker S-opsin-positive cells, L / M-opsin-positive cells, or rod photoreceptor marker rhodopsin-positive cells were found in the outermost layer of the aggregate (Fig. 9).
  • Example 4 Human iPS cell line 1231A3 (CiRA) seeded on a 6-well plate coated with iMatrix-511 (Nippi) at a cell density of 5,000 cells per well was cultured in StemFit AK02N medium (Ajinomoto) for 10 days, and then the medium was used.
  • CiRA Human iPS cell line 1231A3
  • Nippi iMatrix-511
  • Differentiation Medium (10% KnockOut Serum Replacement (Thermo Fisher Scientific), 0.1 mM Non-essential Amino Acid (GIBCO), 1 mM Sodium Pyruvate, 100 U / mL Penicilin, 100 mg / mL streptomycin and 450 ⁇ M 1-monothioglycerol (Sigma-Aldrich) ) was replaced with a GMEM medium (Thermo Fisher Scientific)), and differentiation induction was started.
  • GEBCO Non-essential Amino Acid
  • GMEM medium Thermo Fisher Scientific
  • SB431542 final concentration 5 ⁇ M, Fujifilm Wako Pure Chemical Industries, Ltd.
  • LDN193189 hydorochrolide final concentration 100 nM, Sigma-Aldrich
  • BMP4 final concentration 3 nM, R & D systems
  • the layer corresponding to the outer neuroblastoma layer observed at the time of development of the living body corresponds to the outer nuclear layer in the living body, and the layer corresponding to the inner nuclear layer is the outer plexiform layer. It was thought that it showed the appearance of being separated by a layer corresponding to.
  • granular structures and microvilli-like structures, which are thought to be inner segments of photoreceptor cells, have begun to be confirmed on the surface of the aggregates.
  • the retinal organoid was fixed with 3% paraformaldehyde / 7.5% sucrose solution and embedded with O.C.T.Compound (Sakura Finetech).
  • the frozen block was sliced using a cryomicrotome to prepare 10 ⁇ m frozen sections.
  • the prepared frozen sections were activated by heat treatment in 10 mM sodium citrate buffer (pH 6.0), washed with PBS (-), and then blocked solution (5% deactivated horse serum (GIBCO), 0.05. Blocking treatment was performed at room temperature for 1 hour using PBS (-) containing% Triton-X100.
  • Thermo Fisher Scientific and DAPI (final concentration 1 ⁇ g / mL, Thermo Fisher Scientific) were used to carry out a secondary antibody reaction at room temperature for 2 hours. After completion of the secondary antibody reaction, the cells were washed with PBS (-) and then encapsulated with a PermaFluor aqueous encapsulant (Thermo Fisher Scientific). After air-drying, fluorescence microscopy was performed using an inverted fluorescence microscope DMi8 (Leica). A large number of rhodopsin-positive cells and L / M opsin-positive cells were found in the outermost layer of the retinal organoid 90 days after the start of differentiation induction (Fig. 12).

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Abstract

La présente invention concerne un procédé de production d'un tissu rétinien stratifié comprenant des cellules photoréceptrices à partir de cellules souches pluripotentes ou d'un agrégat comprenant des cellules progénitrices rétiniennes dans une courte période.
PCT/JP2021/047809 2020-12-24 2021-12-23 Procédé de forçage et de production pour tissu rétinien stratifié comprenant des cellules photoréceptrices WO2022138803A1 (fr)

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WO2024085251A1 (fr) * 2022-10-21 2024-04-25 住友ファーマ株式会社 Procédé d'évaluation de la qualité d'un implant rétinien

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WO2017043604A1 (fr) * 2015-09-08 2017-03-16 大日本住友製薬株式会社 Procédé de production d'un tissu rétinien
JP2017528163A (ja) * 2014-08-27 2017-09-28 ソウル ナショナル ユニバーシティ アールアンドディービー ファンデーション 幹細胞を網膜神経節細胞に分化させる方法
WO2019017492A1 (fr) * 2017-07-20 2019-01-24 国立研究開発法人理化学研究所 Procédé de maturation de tissu rétinien contenant un épithélium continu
WO2019054514A1 (fr) * 2017-09-14 2019-03-21 国立研究開発法人理化学研究所 Procédé de production de tissus rétiniens
WO2020184720A1 (fr) * 2019-03-13 2020-09-17 大日本住友製薬株式会社 Procédé d'évaluation de la qualité d'une rétine neurale de transplantation, et feuille de rétine neurale de transplantation

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JP2016537967A (ja) * 2013-10-09 2016-12-08 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア 哺乳動物の網膜幹細胞の生産方法及び適用
JP2017528163A (ja) * 2014-08-27 2017-09-28 ソウル ナショナル ユニバーシティ アールアンドディービー ファンデーション 幹細胞を網膜神経節細胞に分化させる方法
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WO2024085251A1 (fr) * 2022-10-21 2024-04-25 住友ファーマ株式会社 Procédé d'évaluation de la qualité d'un implant rétinien

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