WO2012173207A1 - Procédé d'induction de la différenciation en cellules de la rétine - Google Patents

Procédé d'induction de la différenciation en cellules de la rétine Download PDF

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WO2012173207A1
WO2012173207A1 PCT/JP2012/065285 JP2012065285W WO2012173207A1 WO 2012173207 A1 WO2012173207 A1 WO 2012173207A1 JP 2012065285 W JP2012065285 W JP 2012065285W WO 2012173207 A1 WO2012173207 A1 WO 2012173207A1
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cells
culture
medium
retinal progenitor
retinal
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政代 高橋
理志 岡本
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独立行政法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
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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
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/062—Sensory transducers, e.g. photoreceptors; Sensory neurons, e.g. for hearing, taste, smell, pH, touch, temperature, pain
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • the present invention relates to a method for producing retinal progenitor cells, rod photoreceptor cells, retinal pigment epithelial cells, and the like.
  • Non-Patent Documents 1 and 2 iris tissue
  • Non-Patent Document 3 ciliary body tissue
  • ES embryonic stem cells
  • Non-patent Document 7 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. Showed efficient induction of neural retinal progenitor cells.
  • SFEB serum-free suspension culture
  • 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.
  • 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 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.
  • Non-patent Documents 8 and 9 used another method (inhibition of Wnt signal and Nodal signal by addition of Dkk1 and Lefty-A) that does not rely on retinoic acid to induce cerebral cortex and retinal tissue cells from ES cells ( Non-patent document 10).
  • 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 which is an RAR (retinoic acid receptor) antagonist
  • AGN193109 is a known RAR antagonist that binds to RAR ⁇ , RAR ⁇ , and RAR ⁇ (Patent Document 2, Non-Patent Document 12).
  • 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.
  • 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.
  • a RAR antagonist 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.
  • 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).
  • 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.
  • the present invention relates to the following.
  • 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 retinoic acid receptor antagonist is a compound having at least RAR ⁇ antagonist activity.
  • the pluripotent stem cells are cultured as floating aggregates.
  • 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
  • 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.
  • 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 the living body. A cell.
  • pluripotent stem cells for example, cells derived from warm-blooded animals, preferably mammals, can be used.
  • 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.
  • embryonic stem 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.
  • 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.
  • 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 commercially available products can be purchased.
  • 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 are cells obtained by reprogramming somatic cells (for example, fibroblasts, skin cells, etc.) by introducing reprogramming factors. 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.
  • the maintenance culture of pluripotent stem cells is preferably maintained by serum-free culture using a serum substitute such as Knockout TM Serum Replacement (KSR) or feeder-cell culture.
  • KSR Knockout TM Serum Replacement
  • 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).
  • retinal progenitor cells can be confirmed by a method known per se, for example, expression of a retinal progenitor cell marker.
  • a retinal progenitor cell marker known ones 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.
  • retinal progenitor cells 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.
  • the retinal pigment epithelial progenitor cell marker include Mitf
  • 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.
  • a known method can be used as a method for inducing differentiation into retinal cells, and examples thereof include an adhesion culture method and a suspension culture method.
  • 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) aggregated to form a lump in a suspended state in a culture medium (for example, WO 2008/087917, Nature. 2011 Apr 7: 472 ( 7341): 51-6).
  • 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.
  • a medium widely used for culturing animal cells can be used as a basal medium.
  • 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-Hamhfam12 ' These may be used alone or in combination of two or more, but are not particularly limited thereto.
  • the basal medium can be selected according to the type of cells derived from the target retinal progenitor cells, induction efficiency, etc. .
  • a medium used in the initial stage for 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.
  • a serum-free medium or a medium containing serum is used.
  • 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.
  • 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).
  • the concentration of serum is not limited as long as it is a concentration capable of efficiently inducing differentiation of retinal progenitor cells.
  • concentration of serum is not limited as long as it is a concentration capable of efficiently inducing differentiation of retinal progenitor cells.
  • Culture is preferably carried out 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.
  • a serum-free medium is preferable from the viewpoint of safety.
  • the effect of improving the efficiency of differentiation induction into retinal progenitor cells by RAR antagonists tends to be impaired.
  • 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.
  • albumin eg, BSA, lipid-rich albumin
  • transferrin fatty acid, insulin, collagen, trace elements, or equivalents and derivatives thereof as appropriate.
  • Such serum replacement can be prepared, for example, by the method described in WO98 / 30679.
  • a commercially available thing can be utilized for a serum substitute. Examples of such commercially available serum substitutes include Knockout TM Serum Replacement (KSR) and N2 and B27 which are serum substitutes for nerve cell culture.
  • KSR Knockout TM Serum Replacement
  • N2 and B27 which are serum substitutes for nerve cell culture.
  • the medium used in Method 1 of the present invention preferably contains KSR.
  • 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. .
  • 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.
  • 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) can be used at, for example, about 0.1 mg / ml to 100 mg / ml, preferably about 1 mg / ml to 20 mg / ml.
  • the lipid for example, the chemically determined concentration of commercially available lipids can be used at, for example, about 0.1 ⁇ to 10 ⁇ , preferably about 0.5 ⁇ to 5 ⁇ .
  • isolation or purification means that an operation to remove factors other than the target component has been performed, and the state existing in nature has been removed.
  • the target component is a protein
  • the purity of the target protein “isolated or purified” 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.
  • RAR antagonist means a compound that suppresses signaling downstream of RAR by inhibiting the binding of all-trans-RA (ATRA) or 9-cis-RA to RAR.
  • RAR includes RAR ⁇ , RAR ⁇ and RAR ⁇ as subtypes.
  • the RAR antagonist used in the present invention has 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 RAR ⁇ , RAR ⁇ , and RAR ⁇ (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
  • At least the RAR ⁇ 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 RAR ⁇ antagonist activity include, for example, compounds having significantly higher RAR ⁇ antagonist activity, compounds having antagonist activity against RAR ⁇ and RAR ⁇ , compounds having antagonist activity against RAR ⁇ and RAR ⁇ , antagonist activity against all of RAR ⁇ , RAR ⁇ and RAR ⁇ (Pan-RAR antagonist), and combinations thereof.
  • a compound having an antagonist activity against two or more RAR subtypes is preferable, and a pan-RAR antagonist is particularly preferably used from the viewpoint of efficiently suppressing signal transduction downstream of RAR.
  • compounds with higher antagonist activity can more efficiently promote differentiation induction from pluripotent stem cells to retinal progenitor cells.
  • RAR antagonists include, for example, 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'-di
  • RAR antagonists include BMS493 (4-[(1E) -2- [5,6-dihydro-5,5-dimethyl-8- (2-phenylethynyl) -2-naphthalenyl] ethenyl] benzoic 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
  • 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.
  • BMS493 is known to have superior antagonist activity against any of RAR ⁇ , ⁇ , and ⁇ , compared to AGN193109, and binding inhibition activity to RAR ⁇ 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.
  • 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 a concentration that can achieve the promotion of differentiation of pluripotent stem cells into retinal progenitor cells.
  • concentration is usually about 0.1 nM to about 100 ⁇ M, preferably about 10 nM to about 10 ⁇ M, more preferably about 100 nM to about 5 ⁇ M.
  • concentration is usually about 0.1 nM to about 10 ⁇ M, preferably about 1 nM to about 1 ⁇ M, 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.
  • the effect of improving the induction of retinal progenitor cells and subsequent differentiated 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.
  • it is preferable that the RAR antagonist is present at the beginning of differentiation induction.
  • 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 differentiation of pluripotent stem cells into retinal progenitor cells can be achieved, but is usually 1 to 3 days.
  • the medium does not substantially contain 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.
  • the medium does not substantially contain B27.
  • 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.
  • 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 viewpoint of stability of differentiation efficiency and handling.
  • the Wnt signal inhibitor is used at a concentration that can achieve promotion of differentiation of pluripotent stem cells into retinal progenitor cells.
  • 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.
  • the medium used for the culture contains a ROCK (p160-Rho-associated coiled-coil kinase) inhibitor in order to improve / stabilize differentiation efficiency into retinal progenitor cells or suppress cell death.
  • a ROCK inhibitor is a substance that exhibits a very strong cell death inhibitory action upon cell dispersion.
  • Y-27632 ((R)-(+)-trans-N- (4-pyridyl) -4 -(1-aminoethyl) -cyclohexanecarboxamide ⁇ 2HCl ⁇ H 2 O), Fasudil (HA-1077), Thiazovivin and the like are known.
  • the concentration of the ROCK inhibitor is usually about 50 nM to about 10 ⁇ M.
  • activin for example, activin A
  • the concentration of activin used for suspension culture can be a concentration that can produce retinal progenitor cells more efficiently.
  • 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.
  • the culture medium includes 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.
  • 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.
  • retinal progenitor 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.
  • 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).
  • retinal progenitor cells there is also an advantage that the production amount of retinal pigment epithelial cells induced to differentiate thereafter is also improved.
  • fibrous proteins such as collagen and elastin; glucosaminoglycans and proteoglycans such as hyaluronic acid, chondroitin sulfate and heparan sulfate; cell adhesion proteins such as fibronectin, vitronectin and laminin; enteractin, a mixture thereof, etc.
  • 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.
  • 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.
  • 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.
  • 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.
  • a cell adhesive culture vessel for example, a culture vessel coated with an extracellular matrix or the like.
  • culture temperature is not particularly limited, but 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 culture period can be long enough to efficiently produce retinal progenitor cells.
  • the length of time from the start of pluripotent stem cell culture to the production of retinal progenitor cells varies depending on the animal species, differentiation induction method, and the like. 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.
  • the SFEB / DLFA method can be used to obtain a cell population containing 15% of Rx-positive cells from mouse ES cells on the ninth day of culture (Nature Biotechnology 26, 101-106, pages 215, etc.).
  • 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).
  • Rx retinal progenitor cell marker
  • 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).
  • the culture may be performed while monitoring the expression of a retinal progenitor cell marker (eg, Rx) in cells contained in the culture.
  • a retinal progenitor cell marker eg, Rx
  • 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.
  • 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.
  • retinal progenitor cells can be produced with high efficiency.
  • a 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.
  • cells derived from retinal progenitor cells can be efficiently obtained by subsequent differentiation induction.
  • 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.
  • 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.
  • 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. It is suitable for inducing differentiation into a large number of photoreceptor cells (more preferably rod photoreceptor cells).
  • rod photoreceptors can be obtained with high efficiency by subjecting to further culture described in detail below.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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 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. .
  • 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 the RAR antagonist inhibits its RAR antagonist activity (all-trans-RA (ATRA) or 9-cis-RA binding to the RAR, thereby It means that it is low enough not to exhibit the activity of suppressing transmission).
  • the medium used for further cultivation does not contain a RAR antagonist.
  • 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. Of these, neurobasal media such as NeuroBasal medium and NeuroBasal-A medium, particularly NeuroBasal medium are preferably used.
  • a serum-free medium or a medium containing serum may be used.
  • a serum-free medium is preferably used.
  • the serum-free medium used in the culture in step (b) can contain, for example, a serum substitute.
  • 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 the step (b) may contain FGF.
  • FGF include aFGF and bFGF.
  • 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.
  • retinal progenitor cells 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 for adding FGF may be temporary or may be continuously added during the culture period.
  • 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.
  • 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.
  • 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 tethering culture is not limited as long as it can promote differentiation into the target cells derived from retinal progenitor cells. For example, it is 1 to 10 days, preferably 2 to 4 days, more preferably 3 days.
  • the bridging culture may be either suspension culture or adhesion culture, but is preferably suspension culture.
  • 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.
  • albumin eg, BSA
  • fatty acid e.g., fatty acid
  • lipid preferably chemically determined
  • 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.
  • 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.
  • a method of adding a RAR antagonist to a medium used until retinal progenitor cells are produced from pluripotent stem cells can be mentioned.
  • 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).
  • the medium used in step (b) contains 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).
  • 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.
  • 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).
  • retinoic acid 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.
  • the medium used in step (b) contains a gamma secretase inhibitor.
  • the gamma secretase inhibitor include N- [N- (3,5-difluorophenacetyl) -L-alanyl] -S-phenylglycine tert-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.
  • target cells eg, photoreceptor cells (preferably rod photoreceptor cells)
  • concentrations are for example about 0.1 to about 1000 ⁇ M, preferably about 1 to about 100 ⁇ M, more preferably about 30 to about 50 ⁇ M.
  • 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). .
  • 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.
  • the further culture is at least one selected from the group consisting of IGF-1, taurine and retinoic acid (for example, two, in addition to the above) It may be carried out in a medium containing preferably three factors.
  • 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).
  • FGF FGF
  • 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.
  • the culture is preferably carried out in a medium containing retinoic acid and / or taurine (WO2008 / 987917).
  • further culturing is preferably performed in a medium containing a serum substitute (N2 or the like).
  • a serum substitute N2 or the like
  • the medium used for further cultivation contains a Nodal signal inhibitor and / or a Wnt signal inhibitor (WO2008 / 987917).
  • a Nodal signal inhibitor and / or a 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, photoreceptors (preferably rod photoreceptors)).
  • target cells eg, photoreceptors (preferably rod photoreceptors)
  • the length of such 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 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 simultaneously induced. Depending on the application, it is preferable to produce such two or more cell populations.
  • a differentiation induction method suitable for one type of cell is applied and a case where a plurality of types of retinal cells are simultaneously induced.
  • 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.
  • production of retinal pigment epithelial cells is also observed, both of which are the same as when in vivo. Has been produced.
  • 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.
  • 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.
  • known methods can be used 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 as a method for inducing differentiation of horizontal cells, bipolar cells, amacrine cells, ganglion cells, and Müller glial cells from pluripotent stem cells.
  • pluripotent stem cells are used.
  • 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.
  • retinal diseases such as age-related macular degeneration, retinitis pigmentosa, diabetic retinopathy, and retinal detachment
  • retinal cell damage caused by other causes can be used to replenish the cells.
  • 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 the purity of cells.
  • 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 (e.g. Monoclonal Antibodies: principles and practice, Acad. Press) (1993), Antibody Engineering, A Practical Approach, IRL Press at Oxford University Press (1996), J. Immunol., 141, 2797 (1988)), cell fraction using density difference of sucrose concentration (For example, see the tissue culture technique (third edition), Asakura Shoten (1996)).
  • This problem can be overcome by using inducible pluripotent stem cells derived from somatic cells.
  • 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)).
  • Example 1 Mouse ES cells (Rx-GFP mouse ES cells) that express the GFP gene under the promoter control of the Rx gene (Proc Natl Acad Sci USA 105.11796-11801, 2008) were used.
  • Cell culture The above 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 30000 / well Plates (LIPIDURE-COAT PLATE A-U96: NOF, 51011610) were seeded.
  • G-MEM mouse ES differentiation medium
  • KSR knockout serum replacement
  • 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.
  • 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 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.
  • Example 2 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
  • the medium for suspending cells was 5 mg / ml BSA (Sigma, A4503), 1 ⁇ Chemically defined lipid concentrates (Invitrogen, 11905), 100 unit / ml Penicillin ⁇ 100 ⁇ g / ml StreptomycinP 1-Thioglycerol (Sigma, M6145), Iscove's modified Dulbecco's Medium (Sigma, I3390), 481N, including 1OnM AGN 193109 (Toronto Research Chemicals, A427000) and 48M 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 instead 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%.
  • Neurobasal Invitrogen, 21103
  • 1 mixed medium of Iscove's modified Dulbecco's Medium Sigma, I3390
  • F12-Ham Sigma, N4888
  • Example 6 Example 3 Example 3 except that GMEM (Sigma, 51492C) was used instead of the 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%.
  • GMEM Sigma, 51492C
  • F12-Ham Sigma, N4888
  • 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
  • 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).
  • NAVEC Sendai virus
  • Cell culture The cells were dissociated with 0.25% trypsin-1 mM EDTA, 5 mg / ml BSA (Sigma, A4503), 1 ⁇ Chemically defined lipid concentrates (Invitrogen, 11905), 100 unit / ml Penicillin-100 ⁇ g / ml Strep0 ), Suspended in NeuroBasal (Invitrogen, 21103) medium containing 450 ⁇ M 1-Thioglycerol (Sigma, M6145), 100 nM AGN 193109 (Toronto Research Chemicals, A427000), and then 30000 cells / well each in a multi-well plate of 96 cells per well.
  • Example 9 In Example 8, the culture was carried out in the same manner as in Example 8 except that NeuroBasal (Invitrogen, 21103) was used instead of Neurobasal-A medium constituting the medium whose whole volume 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%.
  • NeuroBasal Invitrogen, 21103
  • cells expressing rhodopsin and recoverin were obtained by immunostaining using an anti-rhodopsin antibody (Chemicon, CA, mouse-anti-rhodopsin) and an anti-recoverin 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 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 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 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 volume was changed on the 9th day of culture.
  • 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 ⁇ g / ml Strep0mStrep0 ), Suspended in Neurobasal-A medium (Invitrogen, 10888-022) containing 450 nM 1-Thioglycerol (Sigma, M6145), 100 nM AGN 193109 (Toronto Research Chemicals, A427000), and then 30000 cells / well. Well plate (LIPIDURE-COAT PLATE A-U9 6: NOF, 51011610).
  • the whole medium was replaced 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 ⁇ g / ml Streptomycin (Sigma, P0781) and 10ng / ml basic FGF ( The medium was changed to Neurobasal-A medium (Invitrogen, 10888-022) containing Wako, 060-04543), and 1xB27 (Invitrogen, 10889-038), 100 unit / ml Penicillin-100 ⁇ g / ml every 3 days after that.
  • Example 14 In Example 8, on day 9 from 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 then washed twice with PBS (-). Furthermore, PE-conjugated goat anti-rabbit antibody (BD, 732752) was added and left 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.
  • LIPIDURE-COAT PLATE A-U96: NOF, 51011610 low-cell adhesion 96-well multi-well plate
  • 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 each 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.
  • G-MEM mouse ES differentiation medium
  • KSR knockout serum replacement
  • 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.
  • Example 16 instead of 100 nM AGN 193109, 1 ⁇ M AGN 193109, 1 ⁇ M BMS493 (pan-RAR antagonist; Tocris Bioscience), 1 ⁇ M ER50891 (RAR ⁇ selective antagonist; Tocris Bioscience), 1 ⁇ M LE135 (TARcce selective antagonist; RAR ⁇ selective antagonist; Differentiation was induced in the same manner as in Example 16 except that 1 ⁇ M MM11253 (RAR ⁇ 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.
  • a pan-RAR antagonist is preferred in that it can efficiently induce retinal progenitor cells, and the induction efficiency by BMS493 is particularly higher than in Examples 17-18, and a compound having RAR ⁇ antagonist activity is preferred from Examples 19-21. From Examples 16 to 17, it was clarified 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.
  • 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 ⁇ g / ml streptomycin (Invitrogen))
  • Primary differentiation induction medium (Reprostem medium (Reprocell), 10 ⁇ M Y-27632 (Wako Pure Chemical Industries), 5 ⁇ M SB431542 (SIGMA), 3 ⁇ M CKI-7 (SIGMA))
  • Second differentiation induction medium (basic medium containing 20% KSR, 10 ⁇ M Y-27632 (Wako Pure Chemicals), 5 ⁇ M SB431542 (SIGMA), 3 ⁇ M CKI-7 (SIGMA))
  • Third differentiation induction medium (basic medium containing 15% KSR, 5 ⁇ M
  • Cell culture The above cells are dissociated into a cell mass composed of 5-10 cells using a dissociation solution, suspended in a medium supplemented with 100 nM AGN 193109 in a primary differentiation induction medium (containing reprostem medium), 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).
  • the cells were fixed with Day 20 in 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 ( ⁇ ).
  • FIG. 2 shows a box plot, and numerical values corresponding to Table 3.
  • Example 23 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.
  • 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 Ra
  • FIG. 2 shows a box plot, and numerical values corresponding to Table 3.
  • 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.

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Abstract

L'invention concerne un procédé de production de cellules précurseurs de la rétine qui comprend la mise en culture de cellules souches pluripotentes dans un milieu contenant un antagoniste d'un récepteur de l'acide rétinoïque et l'obtention de cellules précurseurs de la rétine à partir de la culture.
PCT/JP2012/065285 2011-06-14 2012-06-14 Procédé d'induction de la différenciation en cellules de la rétine WO2012173207A1 (fr)

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WO2015068505A1 (fr) * 2013-11-11 2015-05-14 住友化学株式会社 Procédé de production de cellules épithéliales de pigment rétinien
WO2015107738A1 (fr) * 2014-01-17 2015-07-23 住友化学株式会社 Procédé d'obtention de cellules souches de bord ciliaire
WO2015152330A1 (fr) * 2014-04-02 2015-10-08 国立大学法人東京大学 Système de reconstitution acellulaire utilisant un extrait d'ovocytes de mammifères
WO2016067629A1 (fr) * 2014-10-31 2016-05-06 京都府公立大学法人 Nouveau traitement de la rétine et des nerfs utilisant la laminine
WO2018097253A1 (fr) 2016-11-25 2018-05-31 国立研究開発法人理化学研究所 Population cellulaire pour transplantation, et procédé de fabrication de celle-ci
WO2018164240A1 (fr) 2017-03-08 2018-09-13 大日本住友製薬株式会社 Procédé de production de cellules épithéliales pigmentaires rétiniennes
WO2019050015A1 (fr) 2017-09-08 2019-03-14 国立研究開発法人理化学研究所 Agrégat cellulaire comprenant du tissu rétinien, et méthode de production correspondante
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WO2020218480A1 (fr) 2019-04-26 2020-10-29 国立研究開発法人理化学研究所 Composite comprenant une rétine neuronale, des cellules épithéliales de pigment rétinien et un hydrogel, et son procédé de production
EP3892718A1 (fr) * 2015-09-11 2021-10-13 Propagenix Inc. Prolifération de cellules épithéliales ex vivo
EP3709990A4 (fr) * 2017-11-17 2021-12-01 The Regents of the University of California Manipulation de la voie de signalisation de l'acide rétinoïque
WO2022054924A1 (fr) 2020-09-11 2022-03-17 大日本住友製薬株式会社 Milieu pour tissu destiné à la transplantation
EP4092109A1 (fr) 2015-09-08 2022-11-23 Sumitomo Pharma Co., Ltd. Procede de production de cellules epitheliales de pigment retinien
US11624053B2 (en) 2013-11-27 2023-04-11 Kyoto Prefectural Public University Corporation Application of laminin to corneal endothelial cell culture
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WO2023149565A1 (fr) * 2022-02-07 2023-08-10 国立大学法人大阪大学 Procédé pour induire la différenciation de cellules souches pluripotentes en cellules épithéliales pigmentaires rétiniennes

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