JPWO2012173207A1 - Method for inducing differentiation into retinal cells - Google Patents

Abstract

  The present invention provides a method for producing retinal progenitor cells, comprising culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist, and obtaining retinal progenitor cells from the culture.

Description

  The present invention relates to a method for producing retinal progenitor cells, rod photoreceptor cells, retinal pigment epithelial cells, and the like.

  An increasing number of patients suffer from blindness due to retinal degenerative diseases such as age-related macular degeneration and retinitis pigmentosa. In these diseases, visual cell damage is a direct cause of blindness, so the in vitro production of photoreceptors (or photoreceptor precursors) is in the study of these diseases and the development of treatments for these diseases. Can contribute greatly.

  Many groups, including the present inventors, include iris tissue (Non-Patent Documents 1 and 2), ciliary body tissue (Non-Patent Document 3), or embryonic stem (ES) cells (Non-Patent Documents 4-6). We are trying to create photoreceptor cells. Compared to tissue stem cells, ES cells are superior because they have the ability to proliferate indefinitely and can produce a sufficient number of cells for research and treatment. Recent studies have demonstrated that retinal progenitor cells can be efficiently produced from ES cells in vitro.

  The present inventors used mouse ES cells using serum-free suspension culture (SFEB) system (SFEB / DLFA) of Embryoid body-like aggregates combined with Dkk1, LeftyA, serum and activin treatment (Non-patent Document 7). Showed efficient induction of neural retinal progenitor cells.

  In addition, the present inventors differentiated pluripotent stem cells into retinal progenitor cells by culturing pluripotent stem cells by the SFEB method, and markers such as Rx (Rax) positive from the cultured cells as necessary. Developed a method for producing photoreceptor cells, including sorting retinal progenitor cells based on expression and differentiating retinal progenitor cells into photoreceptor cells by culturing the isolated retinal progenitor cells in a medium containing DAPT or the like. (Patent Document 1). However, in this method, the differentiation efficiency into retinal progenitor cells is not high enough to be sufficiently satisfied, and thus it may be necessary to isolate the differentiated retinal progenitor cells by sorting. In this case, since the number of cells tends to decrease during the sorting operation, it is difficult to prepare a large amount of retinal progenitor cells and photoreceptor cells for the purpose of transplantation or the like.

  It is known that when mouse ES cells are dissociated with trypsin and then reassembled to form a sphere (cell mass), individual cells become embryoid bodies that differentiate into various types of cells. In addition, it is known that neuronal differentiation is promoted when retinoic acid is added during this culture (Non-patent Documents 8 and 9), but almost no cerebral cortex or retinal tissue cells are produced during this culture. . Therefore, Sakurai et al. Used another method (inhibition of Wnt signal and Nodal signal by addition of Dkk1 and Lefty-A) not to rely on retinoic acid to induce cerebral cortex and retinal tissue cells from ES cells ( Non-patent document 10).

  On the other hand, in Non-Patent Document 11, when mouse ES cells are cultured in a medium containing AGN193109, which is an RAR (retinoic acid receptor) antagonist, in an adhered state, neuronal differentiation is inhibited and differentiation into mesoderm is prevented. It is disclosed to be promoted. According to this report, the effect of AGN 193109 is weakened by the addition of Dkk1 and SB, and induces the direction of differentiation rather to the nerve. Further addition of sub-nM retinoic acid rescues neuronal differentiation that was inhibited by AGN 193109. The expression of the neural stem cell marker Sox1 is reduced by the addition of the RAR antagonist, and the peak timing of Sox1 expression is earlier when retinoic acid is added than when it is not added.

  AGN193109 is a known RAR antagonist that binds to RARα, RARβ, and RARγ (Patent Document 2, Non-Patent Document 12).

International Publication No. 2008/087917 Japanese Patent No. 4295357

Haruta, M. et al., Nat. Neurosci., 4, 1163-1164 (2001) Sun, G. et al., Dev. Biol., 289, 243-252 (2006) Tropepe, V. et al., Science, 287, 2032-2036 (2000) Zhao, X., Liu, J. & Ahmad, I., Biochem. Biophys. Res. Commun. 297, 177-184 (2002) Hirano, M. et al., Dev. Dyn., 228, 664-671 (2003) Ikeda, H. et al., Proc. Natl. Acad. Sci. USA, 102, 11331-11336 (2005) Engberg, N. et al., Stem Cells, 28 (9), 1498-1509 (2010) Bain, G. et al., Dev Biol., 168, 342-357 (1995) Bain, G. et al., Biochem Biophys Res Commun., 223, 691-694 (1996) Watanabe, K et al., Nat. Neurosci., 8, 288-296 (2005) Engberg et al., Stem Cells, 28, 1498-1509 (2010) Agarwal, C. et al., J. Biol. Chem., 271 (21), 12209-12212 (1996)

  In view of the above circumstances, an object of the present invention is to provide a method for efficiently producing retinal cells such as retinal progenitor cells, rod photoreceptor cells, and retinal pigment epithelial cells from mammalian pluripotent cells.

As a result of earnest research to achieve the above object, RAR antagonists suppress differentiation into neurons and mesoderm other than the retina at the early stage of differentiation induction of pluripotent stem cells, and from pluripotent stem cells to retinal progenitor cells. It was found to promote the induction of differentiation. When a RAR antagonist is used, since the proportion of retinal progenitor cells contained in the cell population obtained by induction culture is high, cells derived from retinal progenitor cells, particularly visual cells, are isolated without isolating retinal progenitor cells by sorting or the like. Cells could be induced with high efficiency. Furthermore, it has been found that retinal progenitor cells can be produced earlier from pluripotent stem cells when an extracellular matrix is added. In addition, retinal progenitor cells produced in large quantities in the presence of RAR antagonists form a layered structure having a thickness in the process of differentiation induction from pluripotent stem cells. Due to the similar structure, cells derived from retinal progenitor cells could be efficiently produced. In particular, among cells derived from retinal progenitor cells, it was useful for the production of photoreceptor cells and retinal color epithelial cells, particularly for the production of photoreceptor cells with a large number of cells (more preferably rod photoreceptor cells). Moreover, rod photoreceptors were induced with high efficiency by culturing cells containing retinal progenitor cells obtained by the above culture in a medium containing a gamma secretase inhibitor in the absence of a RAR antagonist. Furthermore, when an extracellular matrix was added to the serum-free medium used in the above-described retinal progenitor cell induction culture, it differentiated early into retinal progenitor cells.
Based on the above findings, the present invention has been completed.

That is, the present invention relates to the following.
[1] A method for producing retinal progenitor cells, comprising culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist, and obtaining retinal progenitor cells from the culture.
[2] The method according to [1], wherein the retinoic acid receptor antagonist is a compound having at least RARα antagonist activity.
[3] The method according to [1] or [2], wherein the pluripotent stem cells are cultured as floating aggregates.
[4] The method according to any one of [1] to [3], wherein the medium does not substantially contain retinoic acids.
[5] The method according to any one of [1] to [4], wherein the medium does not substantially contain B27.
[6] The method according to any one of [1] to [5], wherein the medium further contains an extracellular matrix.
[7] The method according to any one of [1] to [6], wherein the medium further contains albumin and / or lipid.
[8] The method according to any one of [1] to [7], wherein the medium further contains a Nodal signal inhibitor and / or a Wnt signal inhibitor.
[9] (a) Obtaining retinal progenitor cells by the method according to any one of [1] to [8], and (b) treating the obtained retinal progenitor cell culture with a retinoic acid receptor antagonist A method for producing retinal cells, comprising culturing in a substantially free medium to obtain cells derived from retinal progenitor cells.
[10] The method according to [9], wherein the medium used for the culture in (a) contains an extracellular matrix, and the cells derived from the retinal progenitor cells in (b) are photoreceptors or retinal pigment epithelial cells.
[11] The medium used for the culture in (b) contains any factor selected from a serum substitute for nerve culture, a gamma secretase inhibitor, and bFGF, and cells derived from retinal progenitor cells are The method according to [9] or [10], which is a cell.
[12] The method according to [10] or [11], wherein the photoreceptor is a rod photoreceptor.
[13] The medium used for culture in (a) and / or (b) contains a Nodal signal inhibitor and / or a Wnt signal inhibitor, and the cells derived from retinal progenitor cells are photoreceptor cells or retinal pigment epithelial cells. The method according to [9], wherein

By using the method of the present invention, retinal progenitor cells can be induced from pluripotent stem cells with high efficiency. Since the ratio of retinal progenitor cells contained in cells obtained by induction culture is high, retinal cells such as photoreceptors are induced with high efficiency from cells after induction culture without isolating retinal progenitor cells by sorting or the like. be able to.
The present invention can greatly facilitate the development of transplantation therapy for retinal diseases based on human pluripotent stem cells.

The time-dependent change of the ratio of a GFP positive cell in the presence or absence of AGN193109 is shown. The number of colonies and the ratio (%) of GFP positive cells in each test example are shown.

  The present invention provides an improved method for producing retinal progenitor cells from pluripotent stem cells and cells derived from retinal progenitor cells such as photoreceptor cells such as rod photoreceptor cells, photoreceptor precursors, and retinal pigment epithelial cells. To do. Details of the present invention will be described below.

(1. Pluripotent stem cells)
A “pluripotent stem cell” is a cell that has self-renewal ability and pluripotency, can be cultured in vitro, and has pluripotency capable of differentiating into all cells constituting a living body. A cell.

  As pluripotent stem cells, for example, cells derived from warm-blooded animals, preferably mammals, can be used. Examples of mammals include primates such as humans and monkeys, rodents such as mice, rats, guinea pigs and hamsters, rabbits, cats, dogs, sheep, pigs, cows, horses and goats.

  Pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells.

  As embryonic stem cells (ES cells) used in the method of the present invention, for example, embryonic stem cells such as mammals established by culturing early embryos before implantation, or by nuclear transfer of somatic cell nuclei. Examples include embryonic stem cells established by culturing the prepared early embryos, and embryonic stem cells obtained by modifying genes on these chromosomes using a known genetic engineering technique.

  Specifically, embryonic stem cells include embryonic stem cells established from internal cell masses or single blastomeres constituting early embryos, EG cells established from primordial germ cells, and early embryos before implantation. Examples thereof include cells isolated from a cell population having multipotency (for example, primitive ectoderm) or cells obtained by culturing the cells. In addition, these embryonic stem cells can respond to the expression of the marker gene by using a known method by knocking the marker gene (for example, a fluorescent protein such as GFP) into the gene encoding the differentiation marker in frame. It may be a cell that can be identified as having reached the differentiation stage.

  Embryonic stem cells can be obtained from a predetermined institution or a commercial product can be purchased. For example, human embryonic stem cells KhES-1, KhES-2 and KhES-3 are available from the Institute of Regenerative Medicine, Kyoto University.

  Fusion ES cells obtained by cell fusion of ES cells and somatic cells are also included in the embryonic stem cells used in the method of the present invention.

  Induced pluripotent stem cells (iPS cells) are cells obtained by reprogramming somatic cells (for example, fibroblasts, skin cells, etc.) by introducing a reprogramming factor. Induced pluripotent stem cells were first discovered by a method of introducing reprogramming factors consisting of Oct3 / 4, Sox2, Klf4 and c-Myc into somatic cells (eg, fibroblasts, skin cells, etc.) (Cell, 126 : p. 663-676, 2006). Since then, many researchers have made various improvements on combinations of reprogramming factors and methods for introducing factors, and various methods for producing induced pluripotent stem cells have been reported. The induced pluripotent stem cells in the present invention include cells produced by such a method.

Pluripotent stem cells can be maintained and cultured by a method known per se. For example, from the viewpoint of clinical application, the maintenance culture of pluripotent stem cells is preferably maintained by serum-free culture using a serum substitute such as Knockout ^™ Serum Replacement (KSR) or feeder-cell culture.

(2. Production of retinal progenitor cells)
The present invention provides a method for producing retinal progenitor cells from pluripotent stem cells (method 1 of the present invention). Method 1 of the present invention comprises culturing pluripotent stem cells in a medium with or without serum and obtaining retinal progenitor cells from the culture. Details will be described below.

  The retinal cells include retinal progenitor cells, progenitor cells derived therefrom (neural retinal progenitor cells, retinal pigment epithelial progenitor cells, photoreceptor precursors, etc.) and mature cells (visual cells, horizontal cells, bipolar cells, amacrine). Cells, ganglion cells, Mueller glial cells, retinal pigment epithelial cells, etc.). The photoreceptor cells include rod photoreceptors and cone photoreceptors. A retinal progenitor cell is a neural retina (visual cells, horizontal cells, bipolar cells, amacrine cells, ganglion cells, Mueller glial cells), retinal pigment epithelial cells, and their progenitor cells (neural retinal progenitor cells, retinal pigment epithelial progenitors) Cell or cell precursor).

  Whether or not the obtained cells are retinal progenitor cells can be confirmed by a method known per se, for example, expression of a retinal progenitor cell marker. As the retinal progenitor cell marker, a known marker can be used, and examples thereof include Rx (Rax) (retinal and anterior neural fold homeobox), Pax6, and the like. Rx (Rax) is one of the most upstream transcription factors in the development of the eye, and has an expression pattern confined to the retina later in development, and plays an important role in the proliferation and differentiation of retinal progenitor cells. Has been suggested.

  As a marker for cells derived from retinal progenitor cells, known ones can be used. For example, Otx2, Crx and the like as retinal photoreceptor markers, Nrl, rhodopsin, recoverin, arrestin and the like as rod photoreceptor markers. Opsin etc. are mentioned as a somatic cell marker. Examples of the retinal pigment epithelial progenitor cell marker include Mitf, and examples of the retinal pigment epithelial cell marker include Pax6, RPE65, MERTK, CRALBP, BEST1, and the like. Other examples include Brn-3b as a ganglion cell marker, CRALBP as a Mueller glial cell marker, Calbindin as a horizontal cell marker, PKC as a bipolar cell marker, and the like.

  As culture conditions for pluripotent stem cells, a known method can be used as a method for inducing differentiation into retinal cells, and examples thereof include methods such as adhesion culture and suspension culture. As an adhesion culture method, for example, a method using an adhesive cell culture device, a method of culturing pluripotent stem cells adhered on feeder cells (for example, WO2001 / 088100) and the like are known. The suspension culture method is a method of culturing a group of pluripotent stem cells (floating aggregates) that aggregate and form a mass in a suspended state in a culture medium (for example, WO 2008/087917, Nature. 2011 Apr 7; 472 ( 7341): 51-6). From the viewpoint of improving the efficiency of differentiation into retinal progenitor cells, preferably, pluripotent stem cells are cultured by a suspension culture method. The floating culture method is preferably used in that it is advantageous in clinical development because it does not require feeder cells for adhering cells, and is easy to handle and scale up a large amount of cells.

  As the medium, a medium widely used for culturing animal cells can be used as a basal medium. Such a medium is not particularly limited as long as differentiation can be induced from a pluripotent stem cell to a target retinal progenitor cell. Commercially available basal media include, for example, Glasgow MEM (GMEM), IMDM (Iscove's Modified Dulbecco's Medium), DMEM, NeuroBasal medium, NeuroBasal-A medium, F12-Hamn, Ka12 These may be used alone or in combination of two or more, but are not particularly limited thereto. When further inducing differentiation of retinal progenitor cells to obtain cells derived from retinal progenitor cells, the basal medium can be selected according to the type of cells derived from the target retinal progenitor cells, induction efficiency, etc. . Among them, a medium used in the initial stage of inducing differentiation from pluripotent stem cells is preferable, specifically, GMEM and a basal medium for nerve cells are preferable, and in particular, GMEM, NeuroBasal medium, NeuroBasal-A medium, and the like are preferable. Used.

  In the method 1 of the present invention, a serum-free medium or a medium containing serum is used. Here, the serum-free medium means a medium that does not contain unconditioned or unpurified serum, and a medium that contains purified blood-derived components or animal tissue-derived components (for example, growth factors) is serum-free. It shall correspond to the culture medium. As the serum, serum derived from any animal, preferably a mammal, can be used. The mammal from which the serum is derived is the same as the mammal from which the pluripotent stem cells are derived (described above).

  When using a medium containing serum, the concentration of serum is not limited as long as it is a concentration capable of efficiently inducing differentiation of retinal progenitor cells. For example, about 0.5 to about 30% (v / v), preferably about It may be from 1.0 to about 20% (v / v), more preferably from about 3 to about 10% (v / v), and most preferably about 5% (v / v).

  The culture is preferably performed in a serum-free medium from the viewpoint of avoiding the risk of contamination of viruses with serum, stabilization of differentiation induction by controlling culture conditions, or improvement of differentiation efficiency into retinal progenitor cells. In particular, in the case of producing transplanted cells, a serum-free medium is preferable from the viewpoint of safety. In the presence of serum, the effect of improving the efficiency of differentiation induction into retinal progenitor cells by RAR antagonists tends to be impaired. In addition, when human cells are used, it is difficult to recognize a significant difference in the efficiency of differentiation induction due to the presence or absence of serum. For this reason, a serum-free medium is preferably used.

The medium may contain, for example, a serum replacement. Serum substitutes contain, for example, isolated or purified albumin (eg, BSA, lipid-rich albumin), transferrin, fatty acid, insulin, collagen, trace elements, or equivalents and derivatives thereof as appropriate. obtain. Such serum replacement can be prepared, for example, by the method described in WO98 / 30679. Moreover, in order to implement the method 1 of this invention more simply, a commercially available thing can be utilized for a serum substitute. Examples of such commercially available serum substitutes include Knockout ^™ Serum Replacement (KSR) and N2 and B27 which are serum substitutes for nerve cell culture. The medium used in Method 1 of the present invention preferably contains KSR.

  In addition, the medium is a component of basal medium or serum substitute or a separately added component, such as isolated or purified albumin (eg, BSA), fatty acid or lipid (preferably chemically determined), Can contain amino acids (eg, non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, reducing agents (2-mercaptoethanol, 1-thioglycerol, etc.), pyruvic acid, buffers, inorganic salts, additives, etc. . For example, 2-mercaptoethanol or 1-thioglycerol used as the reducing agent is not limited as long as it is used at a concentration suitable for the culture of embryonic stem cells, but for example, about 0.05 to about 1.0 mM, preferably about 0.1. It can be used at a concentration of 1 to about 0.5 mM, more preferably about 0.2 mM. The medium used in Method 1 of the present invention preferably contains isolated or purified albumin (eg BSA) and chemically determined lipids. The concentration of albumin (eg, BSA) is, for example, about 0.1 mg / ml to 100 mg / ml, preferably about 1 mg / ml to 20 mg / ml. As the lipid, for example, the chemically determined concentration of commercially available lipid can be used at, for example, about 0.1 × to 10 ×, preferably about 0.5 × to 5 ×.

  In the present specification, the term “isolation or purification” means that an operation for removing factors other than the target component has been performed, and the state existing in nature has been removed. When the target component is a protein, the purity of the target protein “isolated or purified” (percentage of the target protein weight in the total protein weight) is usually 70% or more, preferably 80% or more, more preferably Is 90% or more, most preferably substantially 100%.

The medium used in Method 1 of the present invention contains a retinoic acid receptor (RAR) antagonist. A RAR antagonist means a compound that suppresses signal transduction downstream of RAR by inhibiting the binding of all-trans-RA (ATRA) or 9-cis-RA to RAR. RAR includes RARα, RARβ and RARγ as subtypes. The RAR antagonist used in the present invention is an activity that inhibits the binding of all-trans-RA (ATRA) or 9-cis-RA to at least one RAR selected from the group consisting of RARα, RARβ and RARγ (antagonist activity). ) And can be used alone or in combination. Antagonist activity and RAR subtype selectivity, for example an IC ₅₀ value and K _i values, a reporter assay using cells (e.g., Chem. Biol. 2009 16 479-489 ) can be compared using known methods, such as.

  In the present invention, at least the RARα antagonist is capable of improving the efficiency of differentiation induction from pluripotent stem cells to retinal progenitor cells, photoreceptor cells, and retinal pigment epithelial cells (hereinafter sometimes referred to as “retinal progenitor cells”). A compound having activity is preferably used. Compounds having at least RARα antagonist activity include, for example, compounds having significantly higher RARα antagonist activity, compounds having antagonist activity against RARα and RARβ, compounds having antagonist activity against RARα and RARγ, antagonist activity against all of RARα, RARβ and RARγ (Pan-RAR antagonist), and combinations thereof. Especially, the compound which has antagonist activity with respect to 2 or more RAR subtype is preferable at the point which can suppress the signal transduction downstream of RAR efficiently, and especially a pan-RAR antagonist is used preferably. Furthermore, among pan-RAR antagonists, compounds with higher antagonist activity can more efficiently promote differentiation induction from pluripotent stem cells to retinal progenitor cells and the like.

  Examples of RAR antagonists include AGN 193109; AGN 194310; ER27191; Ro415253; 3- (4-methoxy-phenylsulfanyl) -3-methylbutyric acid; 6-methoxy-2,2-dimethylthiochroman-4-one, 2,2 -Dimethyl-4-oxo-thiochroman-6-yltrifluoromethanesulfonate; ethyl 4-((2,2-dimethyl-4-oxo-thiochroman-6-yl) ethynyl) benzoate; 4-((2,2- Dimethyl 1-4-trifluoromethanesulfonyloxy-2 (H) -thiochromen-6-yl) ethynyl) ethyl benzoate; thiochromen-6-yl] ethynyl] benzoate (yl); p-[(E) -2- [ 3'4'-dihydro-4,4'-dimethyl-7 '-(heptyloxy -2'H-1-benzothiopyran-6'yl] propenyl] benzoic acid 1'1'-dioxide; 2E, 4E, 6E- [7- (3,5-di-t-butyl-4-n-butoxyphenyl) ) -3-Methyl] -octa2,4,6-trienoic acid; 2E, 4E, 6E- [7- (3,5-di-t-butyl-4-n-propoxyphenyl) -3-methyl]- Octa 2,4,6-trienoic acid; 2E, 4E, 6E- [7- (3,5-di-tert-butyl-4-n-pentoxyphenyl) -3-methyl] -octa 2,4,6 -Trienoic acid; 2E, 4E, 6E- [7- (3,5-di-t-butyl-4-n-hexoxyphenyl) -3-methyl] -octa2,4,6-trienoic acid; 2E, 4E, 6E- [7- (3,5-di-tert-butyl-4-n-heptoxyphenyl) -3-methyl L] -octa2,4,6-trienoic acid; 2E, 4E, 6E- [7- (3,5-di-t-butyl-4-n-octoxyphenyl) -3-methyl] -octa2, 4,6-trienoic acid; (2E, 4E, 6E) -7- [3-tert-butyl-5- (1-phenylvinyl) -phenyl] -3-methyl-octa2,4,6-trienoic acid; 2E, 4E, 6E- [7- (3,5-di-tert-butyl-4 {[4,5-3H2] -n-pentoxy} phenyl) -3-methyl] -octa2,4,6-triene Acid; (2E, 4E)-(1RS, 2RS) -5- [2- (3,5-di-tert.butyl-2-ethoxyphenyl) cyclopropyl] -3-methyl-penta-2,4-diene Acid ethyl ester: (2E, 4E)-(1RS, 2RS) -5- [2- (3,5-di-te t. (Butyl-2-ethoxyphenyl) cyclopropyl] -3-methyl-penta-2,4-dienoic acid; (2E, 4E)-(1RS, 2RS) -5- [2- (3,5-di-tert. (Butyl-2-butoxyphenyl) cyclopropyl] -3-methyl-penta-2,4-dienoic acid; (2E, 4E, 6Z) -7- [3,5-di-tert. Butyl-2-ethoxyphenyl] -3-methyl-2,4,6-octatrienoic acid; (2E, 4E, 6Z) -7- [3,5-di-tert. Butyl-2-butyloxyphenyl] -3-methyl-2,4,6-octatrienoic acid; 4- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalene (Carboxamide) benzoic acid; (2E, 4E) -3-methyl-5-[(1R, 2S) -2- (5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalene- 2-yl) cyclopropyl] penta-2,4-dienoic acid; p-[(E) -2- [3 ′, 4′-dihydro-4 ′, 4′-dimethyl-7 ′-(heptyloxy)- 2′H-1-benzothiopyran-6′-yl] propenyl] benzoic acid; 1 ′, 1′-dioxide, 4- (7,7,10,10-tetramethyl-1-pyridin-3-ylmethyl-4, 5,7,8,9,10-hexahydro-1H-naphtho [ , 3-g] indol-3-yl) -benzoic acid; (2E, 4E, 6Z) -7- [3,5- di-tert.. Butyl-2-methoxyphenyl] -3-methyl-2,4,6-octatrienoic acid; (2E, 4E, 6Z) -7- [3,5-di-tert. Butyl-2-ethoxyphenyl] -3-methyl-2,4,6-octatrienoic acid; (2E, 4E, 6Z) -7- [3,5-di-tert. Butyl-2-hexyloxyphenyl] -3-methyl-2,4,6-octatrienoic acid; (2E, 4E, 6Z) -7- [3,5-di-tert. Butyl-2-octyloxyphenyl] -3-methyl-2,4,6-octatrienoic acid; and (2E, 4E)-(1RS, 2RS) -5- [2- (3,5-di-tert. Butyl-2-butoxyphenyl) cyclopropyl] -3-methyl-penta-2,4-dienoic acid, (2E, 4E, 6Z) -7- (3-n-propoxy-5,6,7,8-tetrahydro -5,5,8,8-tetramethylnaphthalen-2-yl) -3-methylocta-2,4,6-trienoic acid; 4- (5H-2,3 (2,5-dimethyl-2,5- Hexano) -5-n-propyldibenzo [b, e] [1,4] diazepin-11-yl) benzoic acid; 4- (5H-2,3- (2,5-dimethyl-2,5-hexano) -5-methyl-8-nitrodibenzo [b, e] [1,4] dia Zepin-11-yl) benzoic acid; 4-{[4- (4-ethylphenyl) 2,2-dimethyl- (2H) -thiochromen-6-yl] ethynyl} benzoic acid; 4- [4-2 methyl- 1,2-dicarba-croso-dodecaborane-1-yl-phenylcarbamoyl] benzoic acid; 4- [4,5,7,8,9,10-hexahydro-7,7,10,10-tetramethyl -1- (3-pyridylmethyl) anthra [1,2-b] pyrrol-3-yl] benzoic acid; (3-pyridylmethyl)-(5-thiaanthra [2,1-b] pyrrol-3-yl) Benzoic acid; and (3-pyridylmethyl) anthra [2m1-d] pyrazol-3-yl) benzoic acid and the like can be mentioned, but are not limited thereto. Moreover, BMS189453, BMS204493, etc. are known as a RAR antagonist currently applied clinically.

  In a further aspect, RAR antagonists include BMS493 (4-[(1E) -2- [5,6-dihydro-5,5-dimethyl-8- (2-phenylethynyl) -2-naphthalenyl] ethenyl] benzoic acid. Acid), ER50891 (4- [5- [8- (1-methylethyl) -4-phenyl-2-quinolinyl] -1H-pyrrol-2-yl] benzoic acid), LE135 (4-[(7,8 , 9,10-Tetrahydro-5,7,7,10,10-pentamethyl-5H-benzo [e] naphtho [2,3-b] [1,4] diazepine) -13-yl] benzoic acid), MM11253 (6- [2- (5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) -1,3-dithiolan-2-yl] -2-naphthalenecarboxylic acid) Is included.

  In the present invention, from the viewpoint of application to cell transplantation treatment, a clinically usable compound is preferably used. The RAR antagonist used in the present invention is preferably a pan-RAR antagonist, and for example, AGN 193109 and BMS 493 having the following structures are available. It is known that BMS493 is superior to AGN193109 in antagonist activity against any of RARα, β, and γ, and binding inhibition activity against RARα is about twice that of AGN193109 (Chemistry & Biology (2009) 16, 479-489). 481), and the differentiation induction efficiency into retinal progenitor cells is also superior to AGN193109. On the other hand, AGN 193109 is preferable in that it contributes to improvement of differentiation induction efficiency of retinal progenitor cells and the like even when used at a relatively low concentration.

  The concentration of the RAR antagonist is such a concentration that can promote the differentiation of pluripotent stem cells into retinal progenitor cells. Such a concentration is usually about 0.1 nM to about 100 μM, preferably about 10 nM to about 10 μM, more preferably about 100 nM to about 5 μM. For example, for AGN 193109, it is usually about 0.1 nM to about 10 μM, preferably about 1 nM to about 1 μM, more preferably about 10 to about 500 nM.

  The RAR antagonist may be added once, or may be added continuously or intermittently over a plurality of times. Some RAR antagonists are known to have a half-life in the medium of half a day or less, and multiple additions are often effective to sustain the effects of the RAR antagonist. In the present invention, the effect of improving the induction of retinal progenitor cells and subsequent differentiated cells (visual cells and retinal pigment epithelial cells) can be achieved even by a single addition, but the effect can be achieved by adding two or more times. May be further improved. In addition, it is preferable that the RAR antagonist is present at the beginning of differentiation induction. For example, a medium containing the RAR antagonist can be used as a medium at the start of differentiation induction. The interval between additions in the case of adding a plurality of times is not particularly limited as long as it is possible to achieve differentiation promotion of pluripotent stem cells into retinal progenitor cells, but is usually 1 to 3 days.

  In order to ensure or enhance the effect of the RAR antagonist, the medium is preferably substantially free of retinoic acids. Retinoic acids mean retinoic acid, retinoids that are derivatives thereof, and precursors thereof. “Substantially free of retinoic acids” means that retinoic acids are not contained or the concentration of retinoic acids is induced in neurons other than the neural retina (Sox1-positive cells and differentiated cells thereof) in the presence of an RAR antagonist. It means that the concentration is not effective. The concentration of retinoic acids in a medium substantially free of retinoic acids is usually less than 1 nM, preferably less than 10 pM. Most preferably, the medium does not contain any retinoic acids.

  In order to ensure or enhance the effect of the RAR antagonist, it is preferable that the medium does not substantially contain B27. This is because B27, which is usually marketed, contains a certain amount of retinoic acids and exhibits a nerve-inducing effect. “Substantially free of B27” means that it does not contain B27 (including products from which retinoic acids have been removed) or that the content of B27 is an amount that does not exert a nerve-inducing effect. . Most preferably, the medium does not contain any B27.

  In order to improve / stabilize the differentiation efficiency into retinal progenitor cells, the medium used for culture is any inhibitor selected from the group consisting of a Nodal signal inhibitor and a Wnt signal inhibitor, preferably both inhibitors. It may be included (see WO2008 / 088791). By combining the Nodal signal inhibitor and the Wnt signal inhibitor, an excellent effect of improving differentiation efficiency can be expected.

  The Nodal signal inhibitor is not particularly limited as long as it can suppress signal transduction mediated by Nodal. Examples of the Nodal signal inhibitor include Lefty-A, Lefty-B, Lefty-1, Lefty-2, soluble Nodal receptor, Nodal antibody, Nodal receptor inhibitor, and SB-431542. The Nodal signal inhibitor is preferably Lefty-A, SB-431542, more preferably SB-431542 (4- [4- (1,3-benzodioxol- 5-yl) -5- (2-pyridinyl) -1H-imidazol-2-yl] -benzamide or a hydrate thereof. The Nodal signal inhibitor is used at a concentration such that promotion of differentiation of pluripotent stem cells into retinal progenitor cells can be achieved.

  The Wnt signal inhibitor is not particularly limited as long as it can suppress signal transduction mediated by Wnt. Examples of the Wnt signal inhibitor include Dkk1, Cerberus protein, Wnt receptor inhibitor, soluble Wnt receptor, Wnt antibody, casein kinase inhibitor, dominant negative Wnt protein, CKI-7 (N- (2-amino). Ethyl) -5-chloro-isoquinoline-8-sulfonamide), D4476 (4- {4- (2,3-dihydrobenzo [1,4] dioxin-6-yl) -5-pyridin-2-yl-1H -Imidazol-2-yl} benzamide). The Wnt signal inhibitor is preferably Dkk1, CKI-7, more preferably CKI-7, from the viewpoints of stability of differentiation efficiency and handleability. The Wnt signal inhibitor is used at a concentration that can achieve promotion of differentiation of pluripotent stem cells into retinal progenitor cells.

  In one embodiment, suspension culture of pluripotent stem cells is performed in the absence of a Nodal signal inhibitor and / or a Wnt signal inhibitor. Since differentiation into retinal progenitor cells is strongly promoted by the RAR antagonist, differentiation into retinal progenitor cells is induced with high efficiency without adding a Nodal signal inhibitor or a Wnt signal inhibitor.

In one embodiment, in order to improve / stabilize differentiation efficiency into retinal progenitor cells, or to suppress cell death, the culture medium used for culture contains a ROCK (p160-Rho-associated coiled-coil kinase) inhibitor. You may go out. A ROCK inhibitor is a substance that exhibits a very strong cell death inhibitory action upon cell dispersion. For example, Y-27632 ((R)-(+)-trans-N- (4-pyridyl) -4 _{- (1-aminoethyl) -cyclohexanecarboxamide ·} 2HCl · H 2 O), Fasudil (HA-1077), etc. Thiazovivin are known. The concentration of the ROCK inhibitor is usually about 50 nM to about 10 μM.

  For the purpose of promoting differentiation into retinal progenitor cells, activin (for example, activin A) may be added to the medium used for suspension culture (see WO2008 / 088791). The concentration of activin used for suspension culture can be a concentration that can produce retinal progenitor cells more efficiently.

  In one embodiment, the medium used in suspension culture contains a combination of a Nodal signal inhibitor (preferably Lefty-A or SB-431542) and a Wnt signal inhibitor (preferably Dkk1 or CKI-7) in addition to the RAR antagonist. Including.

  In one embodiment, the medium includes an extracellular matrix. In the method 1 of the present invention, when pluripotent stem cells are cultured in the presence of an extracellular matrix, the production time of retinal progenitor cells tends to be accelerated, and the production period of retinal progenitor cells can be shortened. Therefore, it is advantageous in that the culture period for obtaining cells derived from retinal progenitor cells obtained by further differentiation induction can be shortened. In addition, by using an extracellular matrix in combination, a thick layer structure containing a large number of retinal progenitor cells is formed in the process of induction of the effects of an RAR antagonist described later, that is, differentiation induction from pluripotent stem cells. Since it has morphological characteristics similar to the induction of differentiation of retinal cells in vivo, the effect of inducing retinal progenitor cells can be further amplified. Thus, since the environment in which retinal progenitor cells are produced by the method 1 of the present invention using a medium containing an extracellular matrix is suitable as a place for inducing differentiation of the neural retina, it is found in the neural retina in vivo. It is particularly useful for the production of cells, particularly photoreceptors with a large number of cells (more preferably rod photoreceptors). In addition, as a result of producing a large amount of retinal progenitor cells, there is also an advantage that the production amount of retinal pigment epithelial cells induced to differentiate thereafter is also improved.

  As an extracellular matrix, fibrous proteins such as collagen and elastin; glucosaminoglycans and proteoglycans such as hyaluronic acid, chondroitin sulfate and heparan sulfate; cell adhesive proteins such as fibronectin, vitronectin and laminin; enteractin, a mixture thereof, etc. However, it is not limited to these. Especially, as an extracellular matrix, the component which comprises a basement membrane is preferable, for example, laminin, collagen IV, heparan sulfate proteoglycan, entactin, or nidogen 1 can be used individually or in combination of 2 or more types. Particularly preferred are laminin and entactin. Commercial products containing these extracellular matrix and other components can also be used. For example, Matrigel (registered trademark) (Becton, Dickinson and Company) is laminin, collagen IV, heparan sulfate proteoglycan, entactin and nidogen 1 Is commercially available as a suitable extracellular matrix composition.

  The extracellular matrix is added to the medium at a concentration that has the effect of accelerating the production time of retinal progenitor cells.

  The extracellular matrix may be already added to the medium from the beginning of the culture of pluripotent stem cells, but may be added to the medium several days after the start of suspension culture (for example, within 10 days of culture). The extracellular matrix is preferably contained in the medium at a time within 5 days of culture, more preferably from the beginning of suspension culture.

  When pluripotent stem cells are cultured in suspension, the incubator used in suspension culture is not particularly limited as long as the cells can be cultured in suspension, but the incubator is preferably non-cell-adherent. As the non-cell-adhesive incubator, those in which the surface of the incubator has not been artificially treated (for example, coating treatment with an extracellular matrix or the like) for the purpose of improving adhesion to cells can be used.

  When pluripotent stem cells are subjected to adhesion culture, it is preferable to use a cell adhesion culture vessel, for example, a culture vessel coated with an extracellular matrix or the like.

Other culture conditions such as culture temperature and CO ₂ concentration in the culture can be set as appropriate. The culture temperature is not particularly limited and is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is, for example, about 1 to about 10%, preferably about 5%.

  The duration of the culture can be long enough to efficiently produce retinal progenitor cells. The length of time from the start of culture of pluripotent stem cells to the production of retinal progenitor cells varies depending on the animal species, differentiation induction method, etc. In the case of mouse cells, for example, 6 to 20 days, preferably 9 to About 16 days, in the case of human cells, for example, 20 days to 40 days, preferably about 25 days to 35 days. The production of retinal progenitor cells can be confirmed using the appearance of Rx positive cells as an index. For example, the SFEB / DLFA method can be used to obtain a cell population containing 15% of Rx-positive cells from mouse ES cells on the 9th day of culture (such as page 215 of Nature Biotechnology 26, 101-106, 2008).

  After completion of the culture, retinal progenitor cells may be isolated from the culture. This isolation can be performed by a method known per se (cell sorter or the like) using an antibody against the above-mentioned retinal progenitor cell marker (Rx or the like). On the other hand, if a sufficient amount of retinal progenitor cells is produced, differentiation can be continuously induced into photoreceptor cells and retinal pigment epithelial cells without isolating retinal progenitor cells from the culture. Here, a sufficient amount of retinal progenitor cells means a cell population in which Rx positive cells in the culture are, for example, 10% or more, preferably 20% or more.

  The culture obtained by the method 1 of the present invention contains retinal progenitor cells at a high frequency (content). The cells obtained by the method 1 of the present invention have a high frequency, for example, 5% or more, preferably 10% or more, more preferably 20% or more, still more preferably 30% or more, and still more preferably 40% or more (colony). Frequency) and Rx positive (marker of retinal progenitor cells).

  In the method 1 of the present invention, the culture may be performed while monitoring the expression of a retinal progenitor cell marker (eg, Rx) in cells contained in the culture. For example, some cells are isolated from the culture, and the ratio of retinal progenitor cell marker positive cells in the cells is measured by a flow cytometer, immunohistological staining, or the like. Then, when the ratio of retinal progenitor cell marker positive cells is sufficiently high (for example, 5% or more, preferably 10% or more, more preferably 20% or more, still more preferably 30% or more, still more preferably 40% or more ), The culture containing the retinal progenitor cells can be obtained with high frequency (content) by collecting the culture.

  According to the method 1 of the present invention, retinal progenitor cells can be produced with high efficiency. When pluripotent stem cells were cultured in the presence of a RAR antagonist, expression of Sox1 and goosecoid was suppressed compared to when cultured in the absence. Therefore, without being bound by theory, the RAR antagonist suppresses the differentiation into nerve cells and promotes the differentiation into retinal progenitor cells in the process of inducing differentiation of pluripotent stem cells. It is considered that a cell population included in the concentration was obtained. Furthermore, cells derived from retinal progenitor cells can be efficiently obtained by subsequent differentiation induction. Thus, since the method 1 of the present invention can improve the yield of the target cells by a simple method of adding a RAR antagonist to a known differentiation induction method, it can be easily combined with a known method, Useful.

  According to the method 1 of the present invention, retinal progenitor cells form a layer structure in the process of being induced to differentiate from pluripotent stem cells. Since such a layer structure exhibits morphological characteristics similar to the development process of retinal cells in a living body, the method 1 of the present invention promotes the formation of a “field” suitable for the induction of retinal progenitor cells. It is speculated that there is also a contribution to the increase in production of retinal progenitor cells from a specific aspect. When an extracellular matrix is used in combination, the production of retinal progenitor cells is further improved, and retinal progenitor cells can form a thicker layer structure. Therefore, retinal progenitor cells are produced earlier and shorten the differentiation induction period. The effect to do. It is preferable to use an extracellular matrix in combination with KSR because the effect of shortening the time to induce proliferation and differentiation of retinal progenitor cells is further promoted. This layer structure formed by retinal progenitor cells also shows morphological characteristics similar to the development process of the neural retina in the living body. Therefore, the number of cells in the neural retina in the living body, especially the number of cells in the neural retina. It is suitable for inducing differentiation into a large number of photoreceptor cells (more preferably rod photoreceptor cells). For example, rod photoreceptors can be obtained with high efficiency by subjecting to further culture described in detail below. Moreover, a photoreceptor cell and its precursor cell can be obtained by culturing the retinal progenitor cell obtained by the method 1 of the present invention under a known photoreceptor cell or a precursor differentiation condition thereof. Since the retinal progenitor cells are contained at a high frequency (content) in the culture obtained by the method 1 of the present invention, the photoreceptor cells and their cells are isolated from the culture without isolating the retinal progenitor cells by sorting or the like. Progenitor cells can be induced with high efficiency. In addition, it is known that a cell population of retinal pigment epithelial cells is simultaneously formed in the layer structure formed by retinal progenitor cells by a method using an extracellular matrix (Nature. 2011 Apr 7; 472 (7341) : 51-6), this method is also useful as a method for efficiently producing retinal pigment epithelial cells.

(3. Production of retinal cells)
The present invention
(A) obtaining retinal progenitor cells by the method 1 of the present invention, and (b) culturing a culture containing the obtained retinal progenitor cells in a medium substantially free of a retinoic acid receptor antagonist. The present invention provides a method for producing retinal cells, comprising obtaining cells derived from retinal progenitor cells (Method 2 of the present invention). Retinal cells can be produced by culturing the retinal progenitor cells obtained by the method 1 of the present invention under known conditions suitable for further differentiation into target cells. Examples of the retinal cell include those described in the above item 2. The method 2 of the present invention can be carried out by combining the method 1 of the present invention and a known method for inducing differentiation from retinal progenitor cells to predetermined retinal cells.

  The retinal progenitor cells used in step (b) may be isolated. In the present specification, the term “isolated” means that an operation for increasing the purity (ratio) of a target cell as compared with the case where the cell is not treated is performed. The purity of the isolated cells (the ratio of the desired cells to the total cells) is, for example, 60% or more, preferably 70% or more, more preferably 80% or more, and most preferably 90% or more (for example, 100%). is there.

  Isolation of retinal progenitor cells can be performed by a method known per se (cell sorter or the like) using an antibody against the above-mentioned retinal progenitor cell marker.

  The culture obtained by the method 1 of the present invention contains retinal progenitor cells at a high frequency (content). Therefore, in one embodiment, the cell population containing the retinal progenitor cells obtained by the above method 1 can be directly subjected to the step (b) of the method 2 of the present invention without isolating the retinal progenitor cells by sorting or the like. .

  In step (b), the culture containing retinal progenitor cells is further cultured in a medium substantially free of RAR antagonist. “Substantially free” means that the concentration of a RAR antagonist inhibits its RAR antagonist activity (all-trans-RA (ATRA) or 9-cis-RA binding to RAR, thereby causing a signal downstream of RAR). It means that it is low enough not to exhibit the activity of suppressing transmission). In a preferred embodiment, the medium used for further cultivation does not contain a RAR antagonist.

  As the basal medium of the medium used in the culture in the step (b), the same basal medium as exemplified in the method 1 of the present invention can be used, and it is the same as the medium used in the method 1 of the present invention. It may be different. Among them, a medium for nerve cells such as NeuroBasal medium and NeuroBasal-A medium, particularly NeuroBasal medium is preferably used.

  In the culture in the step (b), either a serum-free medium or a medium containing serum may be used. For the same reason as described in the above item 2, a serum-free medium is preferably used.

  The serum-free medium used in the culture of step (b) can contain, for example, a serum substitute. As such a serum substitute, those exemplified in the above item 2 can be used. Serum substitutes can also be selected according to the type of target cells to be differentiated. Among them, a serum substitute for nerve culture, preferably B27 can be used. By using a serum substitute for nerve cell culture, differentiation induction into photoreceptor cells and retinal pigment epithelial cells can be promoted.

  The medium used in the culture in step (b) may contain FGF. Examples of FGF include aFGF and bFGF. Preferably bFGF is used. The concentration of FGF is not limited as long as it can promote cell growth, but such concentration is, for example, about 0.1 to about 1000 ng / ml, preferably about 1 to about 500 ng / ml, more preferably about 5 to about 50 ng / ml. ml. The addition of FGF after the production of retinal progenitor cells is observed is advantageous in that the overall cell number of a cell population containing a high proportion of retinal progenitor cells can be increased. Furthermore, as a result, retinal progenitor cells can be efficiently proliferated. When FGF is added before retinal progenitor cells are produced, the entire cell population with a low content of retinal progenitor cells is proliferated. As a result, unnecessary cells other than retinal progenitor cells are proliferated, and the yield of retinal progenitor cells is improved. It tends to be difficult.

  The period during which FGF is added may be temporary or may continue to be added during the culture period. In the present invention, the retinal progenitor cells obtained in step (a) are once cultured in a medium containing FGF (hereinafter, the culture in the medium containing FGF is referred to as continuation culture), and obtained by ligation culture. When the obtained cells are further cultured in a medium not containing the above-mentioned RAR antagonist, it is advantageous in that the yield of the target cells derived from retinal progenitor cells can be improved. At this time, a medium having a composition suitable for the production of the target cells can be used. For example, by adding a gamma secretase inhibitor to the medium, photoreceptor cells, particularly rod photoreceptors can be efficiently produced. .

  The period of connection culture is not limited as long as it can promote differentiation into retinal progenitor cells into target cells, but is, for example, 1 to 10 days, preferably 2 to 4 days, and more preferably 3 days.

  The linkage culture may be either suspension culture or adhesion culture, but is preferably suspension culture.

  The other culture conditions for the continuous culture are the same as those for the further culture in step (b) above or below.

  In addition, the medium used in the culture in the step (b) contains additives such as albumin (eg, BSA), fatty acid or lipid (preferably chemically determined) as in the method 1 of the present invention. it can.

  In the culture in the step (b), a culture containing retinal progenitor cells is suspended or cultured under adhesion conditions. The culture conditions for suspension culture and adhesion culture are the same as those of Method 1 of the present invention except for the components of the medium.

(3-1. Production of photoreceptor cells (rod photoreceptor cells or cone photoreceptor cells), retinal pigment epithelial cells, and precursor cells thereof)
In one embodiment of the method 2 of the present invention, the retinal progenitor cells obtained by the method 1 of the present invention are cultured in a medium substantially free of a RAR antagonist to produce photoreceptors (rods). Photoreceptor cells or cone photoreceptor cells), retinal pigment epithelial cells or their precursor cells, in particular obtaining rod photoreceptor cells, photoreceptor cells (rod photoreceptor cells or cone photoreceptor cells), retinal pigment epithelial cells or these A method for producing progenitor cells is provided. Known methods for inducing differentiation of pluripotent stem cells and retinal progenitor cells into photoreceptor cells, rod photoreceptor cells, cone photoreceptor cells, retinal pigment epithelial cells, and these progenitor cells include, for example, WO2001-088100, WO2008 / 087917, Nature. 2011 Apr 7; 472 (7341): 51-6 and the like are known. The method of this embodiment can be carried out by combining the above-described method 1 of the present invention with these known methods. For example, in the known method, a method of adding a RAR antagonist to a medium used until retinal progenitor cells are produced from pluripotent stem cells can be mentioned. In particular, in the method 1 of the present invention, retinal progenitor cells derived from pluripotent stem cells in the presence of an extracellular matrix have the ability to differentiate early into photoreceptor cells (preferably rod photoreceptor cells). This is advantageous for the production of photoreceptor cells (preferably rod photoreceptor cells).

  In one embodiment, the medium used in step (b) includes a Nodal signal inhibitor and / or a Wnt signal inhibitor (WO2008 / 987917). These inhibitors are preferably used when producing retinal pigment cells or photoreceptors in step (b).

  When producing retinal pigment cells in the step (b), the culture containing the retinal progenitor cells obtained in the step (a) is contained in a medium substantially free of a retinoic acid receptor antagonist under retinal pigment cell-inducing conditions. Further culturing with, induces differentiation into retinal pigment cells.

  When producing photoreceptor cells (especially rod photoreceptor cells) in the step (b), the culture containing the retinal progenitor cells obtained in the step (a) is treated with retinoic acid under conditions for inducing photoreceptor cells (particularly rod photoreceptor cells). By further culturing in a medium substantially not containing a receptor antagonist, differentiation from retinal progenitor cells to photoreceptor cells (particularly rod photoreceptor cells) is induced.

  In one embodiment, the medium used in step (b) includes a gamma secretase inhibitor. Examples of the gamma secretase inhibitor include N- [N- (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine t-butyl ester (DAPT).

  The concentration of the gamma secretase inhibitor in the medium used in the step (b) is a concentration that can promote differentiation from retinal progenitor cells to target cells (eg, photoreceptor cells (preferably rod photoreceptor cells)). possible. When using DAPT as a gamma secretase inhibitor, such concentrations are for example about 0.1 to about 1000 μM, preferably about 1 to about 100 μM, more preferably about 30 to about 50 μM.

  The gamma secretase inhibitor may already be added to the medium at the start of further culture, or may be added to the culture several days after the start of further culture (for example, within 10 days of culture). . Preferably, the gamma secretase inhibitor is added to the medium at a time within 5 days from the start of further cultivation, more preferably within 3 days.

  Among the photoreceptors, in order to promote differentiation into rod photoreceptors, in addition to the above, the further culture is at least one selected from the group consisting of IGF-1, taurine and retinoic acid (for example, two, It may be carried out in a medium containing preferably three factors.

  In one embodiment, the step (b) comprises at least one selected from the group consisting of FGF (aFGF, bFGF, etc.), shh signal promoter (shh, etc.), retinoic acid and taurine (eg, two, preferably three). , More preferably 4 and most preferably 5). This embodiment is advantageous for the production of photoreceptor cells or precursors thereof. In particular, in the production of primate photoreceptors or precursors thereof, the culture is preferably carried out in a medium containing retinoic acid and / or taurine (WO2008 / 987917). By using a plurality of these factors in combination, a further remarkable effect can be expected.

  In order to promote differentiation into photoreceptor cells or their precursors, it is preferable that further culture is performed in a medium containing a serum substitute (N2 or the like).

  In one embodiment, the medium used for further cultivation includes a Nodal signal inhibitor and / or a Wnt signal inhibitor (WO2008 / 987917). Examples of the Nodal signal inhibitor and / or Wnt signal inhibitor include those described above. This embodiment is advantageous for producing photoreceptor cells or retinal pigment epithelial cells.

  The differentiation induction period may be of a length that allows efficient production of target cells (eg, photoreceptor cells (preferably rod photoreceptor cells)). The length of this period is calculated from the start of induction of differentiation of pluripotent stem cells, for example, about 20 days or more, preferably about 30 to about 50 days for mouse cells, and for example, 60 days or more for human cells. , Preferably from about 90 to about 150 days.

  The method for producing cells derived from retinal progenitor cells includes a case where a differentiation induction method suitable for one type of cell is applied and a case where a plurality of types of retinal cells are induced simultaneously. Depending on the application, it is preferable to produce such two or more cell populations. For example, Nature. 2011 Apr 7; 472 (7341): 51-6 reports a method that allows three-dimensional culture of photoreceptor cells. In this method, conditions suitable for inducing differentiation of photoreceptor cells, such as using a gamma secretase inhibitor, are used. At the same time, production of retinal pigment epithelial cells is also observed, both of which are the same as when in vivo. Has been produced. Since the retinal pigment epithelial cells and the photoreceptor cells are a combination that is expected to improve the therapeutic effect when transplanted at the same time, the above method is also useful as a method for simultaneously obtaining these two types of cells. The present invention improves the production of retinal progenitor cells by combining with the above method, and as a result, simultaneously produces a plurality of cells derived from retinal progenitor cells such as photoreceptor cells and retinal pigment epithelial cells. Can do.

(3-2. Production of horizontal cells, bipolar cells, amacrine cells, ganglion cells, Mueller glial cells, and progenitor cells thereof)
In another embodiment of the method 2, the retinal progenitor cells obtained by the method 1 of the present invention are cultured in a medium not containing an RAR antagonist to obtain horizontal cells, bipolar cells, amacrine cells, nerves. The present invention provides a method for obtaining node cells, Mueller glial cells, or progenitor cells thereof. As a method for inducing differentiation of horizontal cells, bipolar cells, amacrine cells, ganglion cells, and Müller glial cells from pluripotent stem cells, known methods can be used. For example, in the known methods, pluripotent stem cells are used. And a method of adding a RAR antagonist to the medium used until retinal progenitor cells are produced.

(4. Cell culture and use as medicine)
The present invention also provides a cell culture obtained by the method of the present invention. The cell culture of the present invention can be, for example, a suspension aggregate of pluripotent stem cells, a cell obtained by dispersing the suspension aggregate, a cell obtained by culturing a dispersion-treated cell, and the like. The present invention also provides a homogeneous cell isolated and purified to such an extent that it can be administered to a subject from such a cell culture, for example, retinal progenitor cells, rod photoreceptors, neural retinal progenitor cells, retinal pigment epithelial progenitor cells Providing photoreceptor precursors and cone photoreceptors.

  Cells obtained by the method of the present invention can be used in the treatment of retinal diseases such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinal detachment, or in retinal cell damage caused by other causes. It can be used to replenish the cells.

  Further, when the cells obtained by the method of the present invention are used as a therapeutic agent for retinal diseases, it is preferable to transplant the cells after increasing the purity of the cells.

  Any known cell separation and purification method can be used as a method for increasing cell purity. For example, a method using a flow cytometer (for example, Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory). (1988), Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press (1993), Int. Immunol., 10, 275 (1998)), panning methods (eg Monoclonal Antibodies: principles and practice, Third Edition, Acad. Press (1993), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press (1996), J. Immunol., 141, 2797 (1988)), cell fractionation using density difference of sucrose concentration (For example, see the tissue culture technique (third edition), Asakura Shoten (1996)).

  In transplantation medicine, rejection due to differences in histocompatibility antigens is often a problem, but embryonic stem cells obtained by nuclear transfer of the nuclei of somatic cells of patients, or embryonic stem cells obtained by modifying genes on chromosomes, patients This problem can be overcome by using inducible pluripotent stem cells derived from somatic cells.

  In addition, by inducing differentiation using embryonic stem cells obtained by nuclear transplantation of somatic cell nuclei, retinal progenitor cells of individuals who have provided pluripotent cells, and cells further differentiated from the retinal progenitor cells (for example, photoreceptor cells) ) Can be obtained. Such an individual cell is useful not only as a transplantation medicine itself but also as a diagnostic material for determining whether an existing drug is effective for the individual.

  Retinal progenitor cells induced to differentiate from pluripotent stem cells and rod photoreceptors further differentiated from the retinal progenitor cells can be transplanted to a disease site of a patient by a method known per se (for example, Arch Ophthalmol. 122, 1159 -1165 (2004)).

  All references cited in this specification, including publications, patent documents, etc., are cited by reference as if they were individually incorporated by reference specifically and in their entirety. To the same extent, it is incorporated herein by reference.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

[Example 1]
(cell)
Mouse ES cells (Rx-GFP mouse ES cells) (Proc Natl Acad Sci US A. 105: 11796-11801, 2008) expressing the GFP gene under the promoter control of the Rx gene were used.

(Cell culture)
The cells were dissociated with 0.25% trypsin-1 mM EDTA, and mouse ES differentiation medium (G-MEM, 5% knockout serum replacement (KSR; GIBCO), 0) containing 100 ng / ml Dkk1, 500 ng / ml Lefty-A. Suspending in a medium supplemented with 100 nM AGN 193109 (Toronto Research Chemicals, A427000) in 1 mM non-essential amino acids, 1 mM pyruvate and 0.1 mM 2-mercaptoethanol, followed by low cell adhesion 96-well multiwell at 30,000 / well Plates (LIPIDURE-COAT PLATE A-U96: NOF, 51011610) were seeded. The suspension culture was carried out according to the culture method of Osakada et al. (Nat. Biotechnology, 26: 215-24, 2008) except that AGN193109 was contained until the fifth day, and FACSCanto II after 7, 9 and 11 days from the start of the culture. When analyzed by (BD), the ratio of GFP positive cells was 3.6%, 8.4%, and 8.3%, respectively.

[Example 2]
In Example 1, when DKK1 and Lefty-A were not added, the ratios of GFP positive cells 7 days, 9 days and 11 days after the start of the culture were 3.7%, 8.4% and 8.9%, respectively. It was almost the same as the ratio of GFP positive cells in Example 1.
From these results, it was suggested that when a RAR antagonist was added, retinal progenitor cells could be efficiently induced even in the absence of a Wnt signal inhibitor and a Nodal signal inhibitor.

[Comparative Example 1]
In Example 2, when AGN 193109 was not added, the ratios of GFP-positive cells 7 days, 9 days, and 11 days after the start of culture were 0.9%, 1.1%, and 0.8%, respectively.

Example 3
(Cell culture)
In Example 2, the medium in which the cells are suspended is 5 mg / ml BSA (Sigma, A4503), 1 × chemically defined lipid concentrates (Invitrogen, 11905), 100 unit / ml Penicillin−100 μg / ml Streptomycin0P 1-Thioglycerol (Sigma, M6145), Iscove's Modified Dulbecco's Medium (Sigma, I3390), 481G to N-48A, mixed with 100 nM AGN193109 (Toronto Research Chemicals, A427000) Cell seeding, 37 ° C, After culturing for 9 days in a 5% CO ₂ incubator, analysis by FACSCanto II (BD) revealed that the ratio of GFP positive cells was 22.5%.

Example 4
In Example 3, instead of 1: 1 mixed medium of Iscove's modified Dulbecco's Medium (Sigma, I3390) and F12-Ham (Sigma, N4888), NeuroBasal (Invitrogen, 21103) and F12-HamN48 (Sigma, 48103) ) Except that the 1: 1 mixed medium was used, the cells were cultured for 9 days in the same manner as in Example 3, and then analyzed by FACSCanto II (BD). The ratio of GFP-positive cells was 12.9. %Met.

Example 5
Example 3 Example 3 except that NeuroBasal (Invitrogen, 21103) was used in place of 1: 1 mixed medium of Iscove's modified Dulbecco's Medium (Sigma, I3390) and F12-Ham (Sigma, N4888) in Example 3. After culturing for 9 days by the same method as 3 and analyzed by FACSCanto II (BD), the ratio of GFP positive cells was 35.1%.

Example 6
Example 3 Example 3 except that GMEM (Sigma, 51492C) was used instead of 1: 1 mixed medium of Iscove's modified Dulbecco's Medium (Sigma, I3390) and F12-Ham (Sigma, N4888). After culturing for 9 days in the same manner as 3 and analyzed by FACSCanto II (BD), the ratio of GFP positive cells was 10.0%.

Example 7
In Example 5, culture was performed in the same manner as in Example 5 using a medium to which AGN 193109 was added and a medium to which AGN 193109 was not added, and analysis was performed by FACSCanto II (BD). Below, the ratio of the GFP positive cell for every culture | cultivation day is shown. The graph is shown in FIG. As a result, by adding the RAR antagonist, the retinal progenitor cells were induced earlier, and the ratio of retinal progenitor cells tended to increase.

Example 8
(cell)
In accordance with the method described in Proceedings of the Japan Academy. Series B, Physical and biological sciences 85: 348-362, 2009, Nrl that expresses GFP gene under the control of Nrl gene promoter using Sendai virus (DNAVEC) -Mouse iPS cells established by the method of introducing Oct3 / 4, Sox2, Klf4, c-Myc genes into fibroblasts derived from GFP transgenic mice (Cell Transplantation 16: 493-503, 2007) (Nrl-GFP mouse iPS Cells).

(Cell culture)
The cells were dissociated with 0.25% trypsin-1 mM EDTA, and 5 mg / ml BSA (Sigma, A4503), 1 × chemically defined lipid concentrates (Invitrogen, 11905), 100 unit / ml Penicillin-100 μg / ml Strep0mStrep0 ), Suspended in NeuroBasal (Invitrogen, 21103) medium containing 450 μM 1-Thioglycerol (Sigma, M6145), 100 nM AGN 193109 (Toronto Research Chemicals, A427000), and then 30000 cells / well each in low-well plate of 96 cells per well. -COAT PLATE A-U96: NOF, 5101 1610). Suspension culture was performed according to the culture method of Osakada et al. (Nat. Biotechnology, 26: 215-24, 2008) except that AGN193109 was included until the fifth day. On day 9 of culture, the whole medium was replaced with the same medium, and 1xB27 (Invitrogen, 10889-038), 100 unit / ml Penicillin-100 ug / ml Streptomycin (Sigma, P0781) and 10 uM DAPT (Wako, 041-30983) Culturing was performed using Neurobasal-A medium (Invitrogen, 10888-022). Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 31.7%.
Moreover, when the culture was observed under a microscope from the 13th day to the 15th day from the start of the culture, production of brown to black colored cells was observed. From this, it was confirmed that not only rod photoreceptor cells but also retinal pigment epithelial cells were produced in this production method.

Example 9
In Example 8, the culture was performed in the same manner as in Example 8 except that NeuroBasal (Invitrogen, 21103) was used instead of Neurobasal-A medium constituting the medium whose medium was changed on the 9th day of culture. It was. Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 30.4%.

  30 days after the start of culture, cells expressing rhodopsin and recoverin were obtained by immunostaining using anti-rhodopsin antibody (Chemicon, CA, mouse anti-rhodopsin) and anti-recovery antibody (Chemicon, CA, rabbit anti-recoverin). It can be detected.

Example 10
In Example 8, a 1: 1 mixed medium of NeuroBasal (Invitrogen, 21103) and F12-Ham (Sigma, N4888) was used in place of the Neurobasal-A medium that constitutes the medium whose whole volume was changed on the ninth day of culture. The culture was performed in the same manner as in Example 8 except for the above. Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 14.8%.

Example 11
In Example 8, a 1: 1 mixed medium of DMEM-HG (Sigma, D5796) and F12-Ham (Sigma, N4888) was used instead of the Neurobasal-A medium constituting the medium whose whole medium was changed on the ninth day of culture. The culture was performed in the same manner as in Example 8 except that was used. Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 9.6%.

Example 12
In Example 8, the culture was carried out in the same manner as in Example 8 except that OPTI-MEM was used instead of Neurobasal-A medium constituting the medium whose whole medium was replaced on the ninth day of culture. Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 10.7%.

Example 13
Nrl-GFP mouse iPS cells in Example 8 were used. The cells were dissociated with 0.25% trypsin-1 mM EDTA, and 5 mg / ml BSA (Sigma, A4503), 1 × Chemically defined lipid concentrates (Invitrogen, 11905), 100 unit / ml Penicillin-100 μg / ml Strep0mStrep0 ), Suspended in Neurobasal-A medium (Invitrogen, 10888-022) containing 450 μM 1-Thioglycerol (Sigma, M6145), 100 nM AGN 193109 (Toronto Research Chemicals, A427000), then 30,000 cells / well in low numbers per well. Well plate (LIPIDURE-COAT PLATE A-U9 6: NOF, 51011610).

  The whole medium was changed with the same medium on the 9th day of culture, and on the 13th day, 1 × B27 (Invitrogen, 10889-038), 100 unit / ml Penicillin-100 μg / ml Streptomycin (Sigma, P0781) and 10 ng / ml basic FGF ( Neurobasal-A medium (Invitrogen, 10888-022) containing Wako, 060-04543) was changed, and 1 × B27 (Invitrogen, 10889-038), 100 unit / ml Penicillin-100 μg / ml every 3 days after 3 days. Neurobasal-A medium containing Streptomycin (Sigma, P0781) and 10 μM DAPT (Wako, 041-30983) ( nvitrogen, it was subjected to the total amount of medium exchange in 10888-022). Analysis by FACSCanto II (BD) on the 30th day from the start of the culture revealed that the ratio of GFP positive cells was 45.9%. From this, it was shown that the yield of Nrl-positive cells is improved by culturing in the presence of bFGF for a certain period after the generation of retinal progenitor cells.

Example 14
In Example 8, 9 days after the start of culture, a part of the cells was collected, dissociated with 0.25% trypsin-1 mM EDTA, fixed with ice-cold methanol for 10 minutes, and then rabbit anti-Rx antibody (abcam , ab23340) and left on ice for 1 hour, and washed twice with PBS (-). In addition, PE-conjugated goat anti-rabbit antibody (BD, 732752) was added and allowed to stand on ice for 1 hour, washed 3 times with PBS (-), and then analyzed for GFP-positive cells with FACSCanto II (BD). Thus, the ratio of retinal progenitor cells can be determined using Rx positive cells as an index.

Example 15
Rx-GFP mouse ES cells similar to those used in Example 1 were dissociated with 0.25% trypsin-1 mM EDTA, and mouse ES differentiation medium (G-MEM, 5% knockout serum replacement (KSR; GIBCO), After suspending in a medium supplemented with 100 mM AGN 193109 (Toronto Research Chemicals, A427000) in 0.1 mM non-essential amino acids, 1 mM pyruvate and 0.1 mM 2-mercaptoethanol, or 3000 / Each cell was seeded in a low-cell adhesion 96-well multi-well plate (LIPIDURE-COAT PLATE A-U96: NOF, 51011610), and 1 day after the start of culture, 2% (v / v) Matrigel was added. , Nature. 2011 Apr 7; 472 (7341): 51-6. It was subjected to suspension culture in accordance with the method. As a result, at the time after 5 days from the start of culture, the cells were kept alive according to the method described in the section of Live imaging of optic-cup formation in Nature. 2011 Apr 7; 472 (7341): 51-6. When observed below, a very small number of GFP positive cells were observed in the cell population cultured in the medium not added with AGN 193109, whereas the cell population cultured in the medium added with AGN 193109 had a very large number of GFP positive cells. Cells were observed. From this, it was shown that the production time of Rx positive cells was accelerated by the addition of AGN193109.

Example 16
Rx-GFP mouse ES cells similar to those used in Example 1 were dissociated with 0.25% trypsin-1 mM EDTA, and mouse ES differentiation medium (G-MEM, 5% knockout serum replacement (KSR; GIBCO), After suspending in a medium supplemented with 100 mM AGN 193109 (Toronto Research Chemicals, A427000) in 0.1 mM nonamino acids, 1 mM pyruvate, and 0.1 mM 2-mercaptoethanol, 3000 cells / well with low cell adhesion 96-well multiwell A well plate (LIPIDURE-COAT PLATE A-U96: NOF, 51011610) was inoculated, and 1 day after the start of culture, a final concentration of 2% (v / v) matrigel was added. Analysis was performed by FACSCanto II (BD) 6 days after the start of the culture. As a result, the ratio of GFP positive cells in the medium supplemented with 100 nM AGN 193109 was 8.1%. The results are shown in Table 2. In the method of this example using matrigel, it was possible to induce differentiation of many retinal progenitor cells at an earlier stage than in Example 2.

[Comparative Example 2]
In Example 16, differentiation induction was performed in the same manner as in Example 16 except that AGN193109 was not added, and analysis was performed by FACSCanto II (BD) 6 days after the start of culture. The ratio of GFP positive cells was Was 0.9%. The results are shown in Table 2.

[Examples 17 to 21]
In Example 16, instead of 100 nM AGN193109, 1 μM AGN193109, 1 μM BMS493 (pan-RAR antagonist; Tocris Bioscience), 1 μM ER50891 (RARα selective antagonist; Tocris Bioscience), 1 μM LE135 (TARcce selective antagonist; RARβ selective antagonist; Differentiation was induced in the same manner as in Example 16 except that 1 μM MM11253 (RARγ selective antagonist; Tocris Bioscience) was used, and analysis was performed with FACSCanto II (BD) 6 days after the start of culture. The ratio of each GFP positive cell is shown in Table 2. As a result, the effect of adding RAR antagonists to induce retinal progenitor cells was observed. A pan-RAR antagonist is preferable in that retinal progenitor cells can be efficiently induced, and the induction efficiency by BMS493 is particularly higher than that of Examples 17-18, and the compound having RARα antagonist activity is more preferable than Examples 19-21. From Examples 16 to 17, it was revealed that AGN 193109 had a difference of 0.5% in GFP positive cells even when the concentration used was 1/10, and that sufficient induction activity was obtained at a low concentration.

[Example 22] Human cells (medium)
Basic medium (GMEM medium (Invitrogen), 10%, 15%, 20% KSR (Invitrogen), 0.1 mM MEM non-essential amino acid solution (INVITROGEN), 1 mM sodium pyruvate (SIGMA), 0.1M 2-mercaptoethanol (Wako Pure Chemicals), 100 U / ml penicillin—100 μg / ml streptomycin (Invitrogen))
Primary differentiation induction medium (Reprostem medium (Reprocell), 10 μM Y-27632 (Wako Pure Chemicals), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))
Second differentiation induction medium (basic medium containing 20% KSR, 10 μM Y-27632 (Wako Pure Chemicals), 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))
Third differentiation induction medium (basic medium containing 15% KSR, 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))
Fourth differentiation induction medium (basic medium containing 10% KSR, 5 μM SB431542 (SIGMA), 3 μM CKI-7 (SIGMA))
Dissociation solution (PBS (Invitrogen), 0.25% trypsin (SIGMA), 1 mg / ml collagenase IV (Invitrogen), 20% KSR (Invitrogen), 1 mM calcium chloride (Wako Pure Chemical Industries))
(cell)
Human iPS cells (253G1) described in Nature Biotechnology 26, 101-106, 2008 were used.
(Cell culture)
The above cells are dissociated into a cell mass composed of 5-10 cells using a dissociation solution, suspended in a primary differentiation induction medium (containing reprostem medium) in a medium supplemented with 100 nM AGN 193109, and then treated with 2 × 10 ⁴ cells by MPC. The non-adhesive 6 cm culture dish (Nunc) was inoculated, and the culture was started under the conditions of 37 ° C. and 5% CO ₂ (Day 0). Subsequently, half of the medium was replaced with Day 2 and Day 3 with the second differentiation induction medium (containing 20% KSR), half of the medium was replaced with Day 3, 7 and 9 with the third differentiation induction medium (containing 15% KSR), Day 11 13, half of the medium was replaced with a fourth differentiation induction medium (containing 10% KSR). Cells were collected on Day 15 and 17, seeded on a laminin / fibronectin-coated chamber slide using a fourth differentiation induction medium (containing 10% KSR), and cultured until the cells adhered to the culture dish. The cell mass seen in Day 9 was larger than Comparative Example 3, and the number of colonies was also large.
The cells were fixed with Day 20 on which the cells adhered to the culture dish with 4% paraformaldehyde (Wako Pure Chemical Industries), treated with 0.3% Triton X-100 (Nacalai Tesque), and then rabbit anti-human Rax antibody (abcam, ab 23340). ) Was added and allowed to stand at 4 ° C. overnight and washed twice with PBS (−). Further, Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (Invitrogen, A11034) and DAPI (Invitrogen, D1306) were added, left to stand at room temperature for 1 hour, washed with PBS (−) three times, and then fluorescence microscope (Axioplan2 , ZEISS) and the number of colonies was counted. The average number of colonies of Rax positive cells was 19.3 / well (SD = 10.5, n = 18), and the ratio of Rax positive cell colonies was The average was 36.1% (SD = 11.3, n = 18). FIG. 2 shows a box plot, and numerical values corresponding to Table 3.

Example 23
In Example 22, differentiation induction was performed using the same method as in Example 22 except that a secondary differentiation-inducing medium supplemented with 100 nM AGN 193109 was used during Day 1 medium exchange. The cell mass that appeared on Day 9 was larger than that of Example 22, and the number of colonies was also larger than that of Example 22.
Cells were collected in Day 20 in the same manner as in Example 22, and after the antibody staining, visual observation was performed with a fluorescence microscope (Axioplan 2, ZEISS), and the number of colonies was counted. The number of colonies of Rax positive cells was 27.7 / average. The ratio of wells (SD = 12.9, n = 18) and Rax positive cell colonies was an average of 41.7% (SD = 14.4, n = 18). FIG. 2 shows a box plot, and numerical values corresponding to Table 3. Compared with Example 22 in which AGN 193109 was added only once, in this Example in which AGN 193109 was added twice, the number of colonies and the ratio of Rax positive cells were improved.

[Comparative Example 3]
In Example 22, differentiation induction was performed using the same method as in Example 22 except that the primary differentiation-inducing medium was used without adding AGN193109. The cell mass that appeared in Day 9 was smaller than that in Example 22, and the number of colonies was also smaller than that in Example 22. Cells were collected in Day 20, and visually observed with a fluorescence microscope (Axioplan 2, ZEISS) after antibody staining, and the number of colonies was counted. The average number of colonies of Rax positive cells was 10.3 / well (SD = 7.3). n = 18), the ratio of Rax positive cell colonies averaged 25.6% (SD = 13.5, n = 18). FIG. 2 shows a box plot, and numerical values corresponding to Table 3.

By using the method of the present invention, retinal progenitor cells can be induced from pluripotent stem cells with high efficiency. Since the ratio of retinal progenitor cells contained in cells obtained by induction culture is high, photoreceptor cells and the like can be induced with high efficiency from cells after induction culture without isolating retinal progenitor cells by sorting or the like. .
The present invention can greatly facilitate the development of transplantation therapy for retinal diseases based on human pluripotent stem cells.

  This application is based on a patent application No. 2011-132712 filed in Japan (filing date: June 14, 2011), the contents of which are incorporated in full herein.

Claims (13)

  A method for producing retinal progenitor cells, comprising culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist, and obtaining retinal progenitor cells from the culture.   The method of claim 1, wherein the retinoic acid receptor antagonist is a compound having at least RARα antagonist activity.   The method according to claim 1 or 2, wherein the pluripotent stem cells are cultured as floating aggregates.   The method according to any one of claims 1 to 3, wherein the medium is substantially free of retinoic acids.   The method according to any one of claims 1 to 4, wherein the medium is substantially free of B27.   The method according to any one of claims 1 to 5, wherein the medium further comprises an extracellular matrix.   The method according to any one of claims 1 to 6, wherein the medium further contains albumin and / or lipid.   The method according to any one of claims 1 to 7, wherein the medium further contains a Nodal signal inhibitor and / or a Wnt signal inhibitor. (A) obtaining a retinal progenitor cell by the method according to any one of claims 1 to 8, and (b) obtaining a culture containing the obtained retinal progenitor cell substantially from a retinoic acid receptor antagonist. A method for producing retinal cells, comprising culturing in a medium not containing retinal progenitor cells.   The method according to claim 9, wherein the medium used for culture in (a) contains an extracellular matrix, and the cells derived from retinal progenitor cells in (b) are photoreceptors or retinal pigment epithelial cells.   The medium used for culture in (b) contains any factor selected from a serum substitute for nerve culture, a gamma secretase inhibitor, and bFGF, and the cells derived from retinal progenitor cells are photoreceptor cells. The method according to claim 9 or 10.   The method according to claim 10 or 11, wherein the photoreceptor cell is a rod photoreceptor cell.   The medium used for the culture in (a) and / or (b) contains a Nodal signal inhibitor and / or a Wnt signal inhibitor, and the cells derived from retinal progenitor cells are photoreceptors or retinal pigment epithelial cells. The method of claim 9.