WO2021251418A1 - Method for producing inner ear progenitor cells, method for producing inner ear hair cells, method for assessing drug, and composition for inducing differentiation of inner ear cells - Google Patents

Method for producing inner ear progenitor cells, method for producing inner ear hair cells, method for assessing drug, and composition for inducing differentiation of inner ear cells Download PDF

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WO2021251418A1
WO2021251418A1 PCT/JP2021/021862 JP2021021862W WO2021251418A1 WO 2021251418 A1 WO2021251418 A1 WO 2021251418A1 JP 2021021862 W JP2021021862 W JP 2021021862W WO 2021251418 A1 WO2021251418 A1 WO 2021251418A1
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inner ear
cells
medium
cell
wnt
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French (fr)
Japanese (ja)
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翼 佐伯
正人 藤岡
智香 三枝
誠 細谷
栄之 岡野
郁 小川
佐栄子 松▲崎▼
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株式会社オトリンク
学校法人慶應義塾
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing inner ear progenitor cells, a method for producing inner ear hair cells, a method for evaluating a drug, and a composition for inducing inner ear cell differentiation.
  • pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) have been widely used as research tools for regenerative medicine, pathological elucidation, and drug discovery. has been done.
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • hearing impairment caused by various causes it is difficult to measure / compare hearing in experimental animals, and it is expected to develop a method for inducing inner ear cells that contribute to scientific evaluation from pluripotent stem cells. There is.
  • Non-Patent Documents 1 and 2 when inducing inner ear progenitor cells from human ES cells, it is necessary to select germ layer formation and the inner ear progenitor cells under visual observation, and long-term culture is required. Duration and low efficiency were issues. Furthermore, the induction of inner ear progenitor cells to more advanced inner ear hair cells is extremely inefficient, and is of high quality for application to regenerative medicine, pathological elucidation, research tools for drug discovery, etc. There was also a problem in terms of it.
  • Patent Document 1 a method for highly efficiently inducing inner ear progenitor cells from pluripotent stem cells.
  • human iPS cells derived from a patient with Pendred syndrome which is a genetic deafness disease, are established, and Pendred-positive cells are prepared to prepare intracellular aggregates specific to the patient-derived cells.
  • Pendred-positive cells are prepared to prepare intracellular aggregates specific to the patient-derived cells.
  • a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell exfoliation treatment, and then retinoic acid and the first Wnt / ⁇ catenin pathway. It provides a method for producing inner ear progenitor cells, which comprises a step of culturing in a medium containing an inducer.
  • a cell population containing cells expressing PAX2 and / or PAX8, which are markers for the planned inner ear region, which are induced to differentiate from pluripotent stem cells can be obtained. Since cells are separated into individual cells by cell exfoliation treatment and then transferred to the next culture, cells unnecessary for differentiation into inner ear cells can be eliminated to ensure that PAX2 and / or PAX8 expression levels are maintained. can. Then, after the cell exfoliation treatment, the cells are cultured in a medium containing retinoic acid and an inducer of the first Wnt / ⁇ -catenin pathway, so that the expression level of PAX2 and / or PAX8 can be improved. Therefore, this makes it possible to efficiently obtain good quality inner ear progenitor cells.
  • the medium containing the retinoic acid and the inducer of the first Wnt / ⁇ -catenin pathway is one selected from the group consisting of IGF-1, bFGF, and EGF.
  • IGF-1 IGF-1, bFGF, and EGF.
  • the medium containing the retinoic acid and the inducer of the first Wnt / ⁇ -catenin pathway is preferably a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors.
  • the culture in a medium containing the retinoic acid source and the inducer of the first Wnt / ⁇ -catenin pathway is by adhesive culture. According to this, the culture after the cell exfoliation treatment can be efficiently performed. In addition, operations such as medium exchange are easy.
  • the cell detachment treatment includes a step of passing a mesh having a predetermined pore size. According to this, it is possible to more efficiently separate individual cells of a cell population containing cells expressing PAX2 and / or PAX8 which have been induced to differentiate from pluripotent stem cells.
  • the first Wnt / ⁇ -catenin pathway inducer is preferably one or more selected from the group consisting of CHIR99021, BIO, and LiCl. ..
  • a cell population containing PAX2 / PAX8-positive cells induced to differentiate from the pluripotent stem cells was obtained by a method including the following steps (1) to (4). Is preferable.
  • a step of culturing pluripotent stem cells in the absence of a growth factor and in the presence of a ROCK inhibitor (2) A cell population obtained in step (1) in the absence of a growth factor.
  • the cell population obtained in step (3) is cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19 in the absence of BMP4.
  • pluripotent stem cells including cells expressing PAX2 and / or PAX8, which are markers for the planned inner ear region.
  • the inner ear progenitor cells obtained by the above-mentioned production method are subjected to the following steps (i) and (ii) in the state of a cell population containing the inner ear progenitor cells. It provides a method for producing inner ear hair cells, including. (I) Step of suspension-culturing the cell population containing the inner ear progenitor cells (ii) Step of adhesion-culturing the cell population obtained in step (i)
  • the inner ear precursor cells obtained by the method for producing inner ear precursor cells according to the first aspect are used, so that the inner ear has good quality. Hair cells can be obtained efficiently.
  • the suspension culture in the step (i) is carried out in a medium containing an inducer for the second Wnt / ⁇ -catenin pathway. According to this, good quality inner ear hair cells can be obtained more efficiently.
  • the second Wnt / ⁇ -catenin pathway inducer is one or more selected from the group consisting of R-spondin1, CHIR99021, and Wnt3a. Is preferable.
  • the adhesion culture in the step (ii) is carried out in a medium containing an inducer of a third Wnt / ⁇ -catenin pathway and an anti-Frizzled agent. According to this, good quality inner ear hair cells can be obtained more efficiently.
  • the third Wnt / ⁇ -catenin pathway inducer is Wnt3a and / or R-spondin1
  • the anti-Frizzled agent is a competitor with a pseudo-molecule of Frizzled10. Is preferable.
  • the step of treating the inner ear progenitor cells obtained by the above-mentioned production method with a test agent and the step of evaluating the state of the inner ear progenitor cells treated with the test agent provides a method for evaluating a drug, including.
  • the method for evaluating a drug according to the third aspect of the present invention it is possible to effectively and efficiently evaluate a drug that affects inner ear progenitor cells and inner ear differentiation.
  • the step of treating the inner ear hair cells obtained by the above-mentioned production method with the test agent and the state of the inner ear hair cells treated with the test agent are evaluated. It provides a method for evaluating a drug, including a step of performing a drug.
  • the method for evaluating a drug according to the fourth aspect of the present invention it is possible to effectively and efficiently evaluate a drug that affects inner ear hair cells and inner ear differentiation.
  • the present invention provides, from the fifth aspect, a composition for inducing inner ear cell differentiation containing retinoic acid and an inducer of the Wnt / ⁇ -catenin pathway.
  • composition for inducing inner ear cell differentiation according to the fifth aspect of the present invention, it is possible to easily prepare efficient inner ear cells by using the composition.
  • the inducer of the Wnt / ⁇ -catenin pathway is preferably one or more selected from the group consisting of CHIR99021, BIO, and LiCl.
  • composition for inducing inner ear cell differentiation is for inducing inner ear progenitor cells.
  • in the method for producing the inner ear precursor cells or the method for producing the inner ear hair cells "inducing agent of the first Wnt / ⁇ -catenin pathway” and “induction of the second Wnt / ⁇ -catenin pathway”.
  • Agent and “third Wnt / ⁇ -catenin pathway inducer” mean that "Wnt / ⁇ -catenin pathway inducer" is used in different steps, and the material configurations are different from each other. It is not the purpose of distinguishing. Therefore, for example, one or more of the same substances may be used as the inducer of each of the first to third Wnt / ⁇ -catenin pathways, or each of the first to third.
  • an inducer of the Wnt / ⁇ -catenin pathway one or more substances that are completely or partially different may be used.
  • Test Example 1 it is a chart which shows the result of having performed the gene expression analysis by qRT-PCR about the inner ear progenitor cell which was induced to differentiate from the human iPS cell.
  • Test Example 2 it is a chart which shows the result of having compared the transition of the expression level of the inner ear progenitor cell marker in the process of inducing the differentiation from the human iPS cell into the inner ear progenitor cell by qRT-PCR.
  • Test Example 3 the change in the expression level of the inner ear progenitor cell marker by inducing the differentiation of human iPS cells into inner ear progenitor cells and culturing with the addition of three different Wnt / ⁇ -catenin pathway inducers is qRT.
  • -It is a chart showing the result quantified by PCR.
  • Test Example 4 differentiation of inner ear progenitor cells into inner ear hair cells was induced, and the gene expression levels of inner ear progenitor cell markers LGR5 and inner ear hair cell markers ATOH1 and BRN3C were quantified by qRT-PCR. It is a chart.
  • Test Example 5 the change in the expression level of the inner ear hair cell marker due to induction of differentiation from inner ear progenitor cells to inner ear hair cells and suspension culture with the addition of R-spondin1 was quantified by qRT-PCR. It is a chart which shows the result of this.
  • Test Example 6 the results of induction of differentiation of inner ear precursor cells into inner ear hair cells and immunostaining with specific antibodies against the respective marker molecules of inner ear hair cells, sustentacular cells, and spiral ganglion cells are shown. It is a chart.
  • Test Example 7 a chart showing the results of comparing the changes in the expression level of inner ear hair cell markers by adding various antifrizzled agents in the process of inducing differentiation of inner ear progenitor cells into inner ear hair cells by qRT-PCR. Is.
  • the present invention relates to a method for inducing differentiation of pluripotent stem cells into inner ear cells constituting the inner ear organs.
  • the present invention relates to a method for producing an inner ear progenitor cell thus activated so as to promote the induction of differentiation into an inner ear cell in the cell.
  • pluripotent stem cells artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) and the like are known.
  • Pluripotent stem cells may be of human origin or of non-human organisms.
  • iPS cells those prepared by reprogramming from the somatic cells of a healthy person may be used, or those prepared by reprogramming from the somatic cells of a disease carrier may be used.
  • the disease include those related to the inner ear organ, hearing, hearing, and the like, and examples thereof include Pendred syndrome and Usher syndrome.
  • a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell exfoliation treatment and then cultured in a medium containing retinoic acid and an inducer for the first Wnt / ⁇ catenin pathway.
  • the culture can be carried out according to the conventional method.
  • the present invention will be described in detail along with a typical induction method for directing pluripotent stem cells to differentiation into inner ear progenitor cells.
  • the method according to the present invention is not limited to the specific culture conditions and the like described below.
  • adheresive culture means that a target cell or cell population is adhered to the bottom surface of an incubator and cultured
  • suspension culture means a target cell or cell. It means culturing the population without adhering it to the bottom surface of the incubator. In this case, when cells or cell populations adhere to the bottom surface of the incubator during culturing, the cells or cell population adhere to the bottom surface of the incubator through cell-substrate adhesion molecules contained in the extracellular matrix (ECM) or the like. It means an adhered state, and means a state in which cells or cell populations do not float in the culture solution even if the culture solution is shaken lightly.
  • ECM extracellular matrix
  • the fact that cells or cell populations do not adhere to the bottom surface of the incubator during culture means that the cells or cell population adhere to the bottom surface of the incubator through cell-substrate adhesion molecules contained in the extracellular matrix (ECM) or the like. It means a state in which they are not adhered, and even if they are touching the bottom surface, etc., it means a state in which cells or cell populations float in the culture solution when the culture solution is shaken lightly.
  • ECM extracellular matrix
  • the bottom surface of the plastic dish is chemically treated or coated with an adhesive coating (gelatin, polylysine, agar, etc.) to promote adhesion of cells to the substrate. Is preferable.
  • the surface such as the bottom surface of the plastic dish is not treated, or it is coated with an adhesion blocking coating agent (poly (2-hydroxyethyl methacrylate), etc.) to prevent adhesion of cells to the substrate.
  • an adhesion blocking coating agent poly (2-hydroxyethyl methacrylate), etc.
  • the induction of differentiation of pluripotent stem cells into a cell population containing PAX2 / PAX8-positive cells is, for example, the following steps (1) to (4). ) Can be done.
  • First step A step of culturing pluripotent stem cells in the absence of growth factors and in the presence of a ROCK inhibitor (2)
  • Second step Cell population obtained in step (1) In the absence of growth factors and in the absence of ROCK inhibitors (3)
  • Third step Cell populations obtained in step (2) were cultivated in bFGF, FGF3, FGF10, and FGF19.
  • the cell population obtained in step (3) was subjected to the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and was not BMP4. Step of culturing in the presence
  • the medium used in the above-mentioned first step is not particularly limited as long as it is a medium capable of maintaining pluripotent stem cells or cells heading for differentiation from pluripotent stem cells.
  • mTeSR1 serum-free medium that does not require feeder cells for maintaining pluripotent stem cells
  • the culture is carried out in the absence of a growth factor and in the presence of an inhibitor of ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase).
  • the ROCK inhibitor has a cell death inhibitory effect on pluripotent stem cells.
  • the first step is preferably carried out for 1 to 3 days, more preferably for 1 to 2 days.
  • ROCK inhibitor used in the above first step examples include Y-27632 ((R)-(+)-trans-N- (4-Pyridine) -4- (1-aminoethyl) -cyclohexanecarboxamide), Fasudil hydroxylide. , K-115 (ripasudil hydrochloride hydrate), DE-104 and the like are exemplified.
  • the optimum concentration of the ROCK inhibitor may be appropriately determined according to the type of the ROCK inhibitor. For example, in the case of Y-27632, 1 to 100 ⁇ M is preferable, and 10 to 20 ⁇ M is more preferable.
  • the medium used in the above-mentioned second step is not particularly limited as long as it is a medium capable of maintaining pluripotent stem cells or cells heading for differentiation from pluripotent stem cells.
  • mTeSR1 STMCELL Technologies
  • STMCELL Technologies which is a serum-free medium that does not require feeder cells for maintaining pluripotent stem cells
  • the cells are cultured in the absence of a growth factor and in the absence of a ROCK inhibitor.
  • the medium is used as a basal medium used in the subsequent steps (for example, “DMEM / F12” (commodity-free medium).
  • the second step is preferably carried out for a total of 1 to 10 days, more preferably 2 to 8 days, and even more preferably 3 to 6 days.
  • the meaning in the absence of the growth factor and / or the ROCK inhibitor may be that the growth factor and / or the ROCK inhibitor is substantially absent, and the growth factor may be present. And / or the ROCK inhibitor may be included at an ineffective level of concentration.
  • the medium used in the above-mentioned third step is not particularly limited as long as it is a medium capable of maintaining cells heading for differentiation from pluripotent stem cells.
  • DMEM / F12 (trade name “D-MEM / Ham's F-12", Fujifilm Wako Junyaku Co., Ltd., which is a serum-free medium, similar to the medium preferably used in the latter half culture of the second step.
  • Serum-free supplement "B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement “N2” (trade name “Gibco N-2 Supplement” (Thermo Scientific)) Serum-free supplement "Gibco GlutaMAX” (Thermo Fisher Scientific), serum-free supplement "Nonential aminoacid” (Nakalitesk Co., Ltd.), and the like are preferably exemplified.
  • bFGF is preferably used as a growth factor.
  • the concentration range of the growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 in the medium is preferably 10 to 50 ng / mL, and more preferably 10 to 25 ng / mL, respectively.
  • the medium is cultured while being replaced with a fresh medium approximately every day to maintain the cells, thereby further enhancing the effect of the growth factor toward the desired differentiation.
  • the third step is preferably performed for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.
  • the medium used in the above-mentioned fourth step is not particularly limited as long as it is a medium capable of maintaining cells heading for differentiation from pluripotent stem cells.
  • DMEM / F12 (trade name “D-MEM / Ham's F-12", Fujifilm Wako Junyaku Co., Ltd., which is a serum-free medium, similar to the medium preferably used for the culture in the third step.
  • Serum-free supplement “B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement “N2" (trade name “Gibco N-2 Supplement” (Thermo Scientific)
  • Preferred examples include a medium supplemented with the serum supplement "Gibco GlutaMAX” (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid” (Nakalitesk Co., Ltd.).
  • bFGF and FGF3 are used as growth factors.
  • the concentration range of the growth factors bFGF, FGF3, FGF10, and FGF19 in the medium is preferably 10 to 50 ng / mL, more preferably 25 ng / mL, respectively. Further, it is preferable that the medium is cultured while being replaced with a fresh medium approximately every day, for example, to maintain the cells. This further enhances the effect of the growth factors toward the desired differentiation.
  • the fourth step is preferably performed for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.
  • the meaning in the absence of BMP4 may be that BMP4 is substantially non-existent and may be contained at a concentration at a level that has no effect.
  • the series of cultures of the first step to the fourth step described above is an adhesive culture in which the culture is carried out while adhering to the bottom surface of the culture dish or the like. According to this, cells can be efficiently cultured. In addition, it is easy to perform operations such as promoting differentiation induction, suppressing differentiation induction, and exchanging a medium for controlling them. In addition, it is preferable to culture in a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors.
  • PAX2 and PAX8 which are markers for the planned inner ear region
  • PAX2 and PAX8 are markers for the planned inner ear region
  • the expression of PAX2 and PAX8 increases accordingly.
  • the expression of PAX2 and PAX8 tends to be suppressed (Reference 1: Easy M, Ellwanger DC, Kosaric N, Stamper AP, Heller S. “Single-cell analysis delineates a tradition toward the human early”. otic lineage. ”Proc Natl Acad Sci U S A.
  • a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is thus activated so as to promote differentiation induction into inner ear cells by subjecting the cell population to a specific treatment.
  • FIG. 1 illustrates an embodiment of the method for producing inner ear progenitor cells according to the present invention using a flow chart.
  • a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell detachment treatment.
  • the proliferated cells are in a state where the cells adhere to each other. It dissociates into a small number of cell clusters, and individual cells are directly exposed to the medium or incubator and become susceptible to the effects. At this time, if the cells are undifferentiated or the growth activity of the cells is weak, they cannot survive and die.
  • the cell detachment treatment may be any means as long as it can dissociate the adhesion between cells and separate individual cells, and is not particularly limited.
  • trypsin or actase commercial product which is an enzyme preparation for cell detachment. Enzyme treatment with the name "Accutase", Nacalai Tesque Co., Ltd.) and the like can be mentioned. After the enzyme treatment, dissociation can be ensured by pipetting or the like in a liquid medium. Further, the remaining cell mass may be removed by passing through a mesh having a predetermined pore size.
  • a cell strainer provided with a nylon mesh having a predetermined hole diameter in a stepwise range of 1 to 1000 ⁇ m is commercially available, and among such commercially available cell strainers. You may select and use the one having an appropriate hole diameter from the above.
  • the cells or cell populations that have been subjected to the cell detachment treatment are further cultured in a medium containing retinoic acid and an inducer of the first Wnt / ⁇ -catenin pathway. ..
  • serum-free medium "DMEM / F12" (trade name “D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) and the like are preferably exemplified. ..
  • the medium may be supplemented with supplemental nutritional components as appropriate.
  • serum-free supplement "B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific)
  • serum-free supplement "N2” (trade name “Gibco N-2 Supplement” (Thermo Fisher))
  • examples include the supplement “Gibco GlutaMAX” (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid” (Nakalitesk Co., Ltd.).
  • the cells are cultured in a medium containing retinoic acid and an inducer of the first Wnt / ⁇ -catenin pathway.
  • retinoic acid and an inducer of the first Wnt / ⁇ -catenin pathway are contained in a medium in combination and cultured in the medium, the expression level of PAX2 and / or PAX8 is increased. Be done.
  • retinoin acid include all-trans type retinoin acid.
  • CHIR99021 also known as: 6-((2-((4- (2,4-Dichrimidine) -5- (4-Methyl-1H-imidazol-2)) -Yl) pyrimidine-2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride), Wnt3a, R-spondin1 and the like.
  • BIO also known as 6-bro-methylrubin 3'-oxime
  • LiCl lithium chloride
  • Wnt3a Wnt3a
  • R-spondin1 and the like.
  • the lower limit of the concentration of retinoic acid in the medium in this step is preferably 0.1 ⁇ M or more, more preferably 0.3 ⁇ M or more, and further preferably 0.5 ⁇ M or more. More preferred.
  • the upper limit is preferably 5 ⁇ M or less, more preferably 3 ⁇ M or less, and even more preferably 1 ⁇ M or less. Since the above-mentioned serum-free supplement "B27” (trade name “Gibco B-27 Supplement” contains vitamin A as a precursor of retinoic acid, the influence of the vitamin A is eliminated and it is in the medium. To ensure the concentration condition of retinoic acid, use a vitamin A-free serum-free supplement "B27-VA” (trade name "Gibco B-27 Supplement, minus vitamin A", Thermo Fisher Scientific). good.
  • the concentration of the inducer of the first Wnt / ⁇ -catenin pathway in the medium in this step is preferably 0.1 ⁇ M or more as the lower limit value, and 30 mM or less as the upper limit value. Is preferable.
  • the lower limit thereof is preferably 0.1 ⁇ M or more, more preferably 0.5 ⁇ M or more, and more preferably 1 ⁇ M or more. Is even more preferable.
  • the upper limit is preferably 10 ⁇ M or less, more preferably 5 ⁇ M or less, and even more preferably 3 ⁇ M or less.
  • the lower limit thereof is preferably 0.1 ⁇ M or more, more preferably 0.5 ⁇ M or more, and more preferably 1 ⁇ M or more. Is even more preferable.
  • the upper limit is preferably 5 ⁇ M or less, more preferably 3 ⁇ M or less, and even more preferably 1 ⁇ M or less.
  • the inducer of the first Wnt / ⁇ -catenin pathway is LiCl in particular, the lower limit thereof is preferably 1 mM or more, more preferably 3 mM or more, and more preferably 5 mM or more. Even more preferable.
  • the upper limit is preferably 30 mM or less, more preferably 20 mM or less, and even more preferably 10 mM or less.
  • the medium is cultured while being replaced with a fresh medium approximately every 1 to 2 days to maintain the cells. According to this, the effect of the growth factor toward the desired differentiation can be further enhanced. It is preferably performed for 1 to 6 days in total, and more preferably 2 to 5 days. It is even more preferable to carry out for 3 to 4 days.
  • the culture in the medium containing the above-mentioned retinoic acid and the inducer of the first Wnt / ⁇ -catenin pathway is preferably an adhesive culture in which the culture is carried out while adhering to the bottom surface of the incubator or the like. According to this, the culture after the cell exfoliation treatment can be efficiently performed. In addition, operations such as medium exchange are easy. In particular, it is preferable to culture using a culture dish coated with a coating agent. According to this, the cells and cell populations after the cell exfoliation treatment can be more efficiently directed to differentiation.
  • the coating agent one usually used for the purpose of promoting adhesion or differentiation to the incubator may be used, and the coating agent for such a purpose is not particularly limited, but for example, poly-O-fibronectin or the like is preferable. Illustrated. In addition, it is preferable to culture in a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors. Moreover, for the maintenance of stem cell properties, hypoxic conditions (e.g., O 2 4 ⁇ 10%, more preferably 4-6%, even more preferably 4%, CO 2 5%) may but be cultured in , normoxia condition (e.g. O 2 5% to 20% and more preferably 10-20%, even more preferably 20% CO 2 5%) may be cultured in.
  • hypoxic conditions e.g., O 2 4 ⁇ 10%, more preferably 4-6%, even more preferably 4%, CO 2 5%
  • normoxia condition e.g. O 2 5% to 20% and more preferably 10-20
  • Culturing in a medium containing the above-mentioned retinoic acid and an inducer of the first Wnt / ⁇ -catenin pathway has one or more selected from the group consisting of IGF-1, bFGF, and EGF as growth factors. It is preferable to do it below. According to this, it is possible to more reliably differentiate into inner ear progenitor cells. Further, it is even more preferable to have all of the growth factors bFGF, EGF and IGF-1 present.
  • the concentration range of these growth factors in the medium is preferably 10 to 30 ng / mL for each of bFGF and EGF. In IGF-1, it is preferably 10 to 50 ng / mL.
  • the inner ear progenitor cells obtained as described above have improved expression levels of PAX2 and / or PAX8 as compared with those before culturing in the medium containing the above-mentioned retinoic acid and the inducer of the first Wnt / ⁇ -catenin pathway. is doing. For example, when a cell population is collected and mRNA expression is analyzed, the expression level is typically increased 2 to 100 times as compared with that before culturing, and is increased 3 to 50 times. Is more typical, and it is even more typical that the increase is 5 to 20 times.
  • inner ear progenitor cells having improved expression levels of PAX2 and / or PAX8 described above as shown in Examples described later, inner ear cells having further advanced differentiation stages such as inner ear hair cells can be efficiently used. Obtainable.
  • the present invention will be described in more detail along with a typical induction method for directing inner ear progenitor cells to differentiation into inner ear cells.
  • the method according to the present invention is not limited to the specific culture conditions and the like described below.
  • FIG. 2 illustrates an embodiment of the method for producing inner ear hair cells according to the present invention using a flow chart.
  • inner ear progenitor cells having improved expression levels of PAX2 and / or PAX8 are subjected to the following steps (i) and (ii).
  • steps (i) and (ii) Step of suspension-culturing the cell population containing the inner ear progenitor cells
  • steps (ii) Step of adhesion-culturing the cell population obtained in step (i)
  • the inner ear precursor cells can be separated into individual cells in the same manner as in the cell detachment treatment described above, suspended in a suitable liquid medium, and then cultured in a non-adherent state. It is preferable to incubate in an incubator for culturing.
  • the incubator for non-adhesive culture specifically, for example, a plastic dish for cell culture can be used.
  • the medium used for the suspension culture is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name “D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) and the like can be used. It is preferably exemplified.
  • the medium may be supplemented with supplemental nutritional components as appropriate.
  • serum-free supplement “B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific)
  • serum-free supplement "N2” (trade name “Gibco N-2 Supplement” (Thermo Fisher)
  • examples include the supplement “Gibco GlutaMAX” (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid” (Nakalitesk Co., Ltd.).
  • bFGF, EGF, IGF-1, heparin and the like are known as growth factors that contribute to the induction of differentiation into inner ear hair cells. Therefore, it is preferable to cultivate one or more of these growth factors in a medium.
  • the concentration range of these growth factors in the medium is preferably 10 to 50 ng / mL, more preferably 10 to 30 ng / mL for bFGF.
  • EGF it is preferably 10 to 50 ng / mL, more preferably 20 to 30 ng / mL.
  • IGF-1 it is preferably 10 to 100 ng / mL, more preferably 20 to 50 ng / mL.
  • heparin it is preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL.
  • This suspension culture is preferably carried out for a total of 2 to 6 days, more preferably 3 to 5 days, and even more preferably 4 days.
  • the spheres (cell clumps) formed by centrifugation or the like are collected so as not to be destroyed, and a fresh medium is replaced or a fresh medium is added, and the suspension culture is further performed. May continue.
  • the additional suspension culture is preferably carried out for 2 to 6 days, more preferably 3 to 5 days, and even more preferably 4 days.
  • the medium to be added is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) is preferably exemplified. Will be done.
  • Serum-free supplement "B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement “N2” (trade name “Gibco N-2 Supplement” (TherciS)) may be used as the medium.
  • Serum-free supplement "Gibco GlutaMAX” (Thermo Fisher Scientific)
  • Serum-free supplement "Nonessential aminoacid” (Nakalitesk Co., Ltd.), etc.
  • bFGF and EGF are used as the growth factors to be contained in the fresh medium to be added.
  • IGF-1, heparin, etc. may be one or more.
  • Preferred concentrations of each are 10 to 30 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, and 10 to 50 ng /. For example, mL.
  • the suspension culture in addition to the above-mentioned growth factors, it is more preferable to carry out the culture in the presence of a second Wnt / ⁇ -catenin pathway inducer as a growth factor.
  • a second Wnt / ⁇ -catenin pathway inducer as a growth factor.
  • the efficiency of inducing differentiation into inner ear hair cells is further improved.
  • the second Wnt / ⁇ -catenin pathway inducer include R-spondin1, CHIR99021 (also known as 6-((2-((4- (2,4-Dichlophoenyl) -5- (4-Methyl-1H-imidazole)).
  • the concentration of the inducer of the second Wnt / ⁇ -catenin pathway in the medium of the suspension culture is preferably 10 ng / mL or more as the lower limit value, and 1000 ng / mL or less as the upper limit value. Is preferable.
  • the second Wnt / ⁇ -catenin pathway inducer is particularly R-spondin1
  • the lower limit thereof is preferably 10 ng / mL or more, more preferably 50 ng / mL or more. Even more preferably, it is 100 ng / mL or more.
  • the upper limit is preferably 1000 ng / mL or less, more preferably 500 ng / mL or less, and even more preferably 200 ng / mL or less.
  • the inducer of the second Wnt / ⁇ -catenin pathway is CHIR99021 in particular, the lower limit thereof is preferably 0.1 ⁇ M or more, more preferably 0.5 ⁇ M or more, and more preferably 1 ⁇ M or more.
  • the upper limit is preferably 10 ⁇ M or less, more preferably 5 ⁇ M or less, and even more preferably 3 ⁇ M or less.
  • the lower limit thereof is preferably 1 ng / mL or more, more preferably 10 ng / mL or more, and more preferably 20 ng / mL. It is even more preferable to use mL or more.
  • the upper limit is preferably 100 ng / mL or less, more preferably 50 ng / mL or less, and even more preferably 20 ng / mL or less.
  • the above-mentioned suspension-cultured cells and cell populations are further adherently cultured.
  • the above-mentioned suspension-cultured cells and cell populations are collected so as not to destroy the spheres (cell clumps) formed by centrifugation or the like, and adhesive culture is performed.
  • the incubator for adhesive culture as described above, it is particularly preferable to culture using a culture dish coated with a coating agent. According to this, cells and cell populations after suspension culture can be more efficiently directed to differentiation.
  • the coating agent one usually used for the purpose of promoting adhesion or differentiation to the incubator may be used, and the coating agent for such a purpose is not particularly limited, but for example, poly-O-fibronectin or the like is preferable. Illustrated. In addition, it is preferable to culture in a serum-free medium.
  • the medium for the adhesive culture the same medium as that used for the suspension culture may be used, or another medium may be used.
  • the above-mentioned serum-free medium "DMEM / F12” (trade name "D-MEM / Ham's F-12", Wako Pure Chemical Industries, Ltd.) is preferably exemplified.
  • Serum-free supplement "B27” (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement” (Thermoscience), as appropriate for the medium.
  • Serum-free supplement “Gibco GlutaMAX” Thermo Fisher Scientific
  • serum-free supplement "Nonessential aminoacid” serum-free supplement "Nonessential aminoacid”
  • bFGF, EGF, IGF-1, heparin and the like As growth factors that contribute to the induction of differentiation into inner ear hair cells, bFGF, EGF, IGF-1, heparin and the like are known. Therefore, one or more of these growth factors are contained in the medium. Cultivation is preferable. In this case, the concentration range of these growth factors in the medium is the same as in the case of the above-mentioned suspension culture.
  • This adhesion culture is preferably carried out for a total of 7 to 21 days, and 10 to 14 days. It is more preferable to do it for a day.
  • a fresh medium may be replaced or a fresh medium may be added to continue the adhesive culture.
  • the culture is preferably carried out for 1 to 4 days, preferably 2 to 3 days, more preferably 2 days from the start of the adhesive culture, and then additional adhesive culture is carried out for 6 to 17 days, preferably 7 to 14 days. It is more preferable, and it is even more preferable to carry out for 8 to 12 days.
  • the medium to be added is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) is preferably exemplified. Will be done.
  • Serum-free supplement "B27” (trade name “Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement “N2” (trade name “Gibco N-2 Supplement” (TherciS)) may be used as the medium.
  • Serum-free supplement "Gibco GlutaMAX” (Thermo Fisher Scientific)
  • Serum-free supplement "Nonessential aminoacid” (Nakalitesk Co., Ltd.), etc.
  • bFGF and EGF are used as the growth factors to be contained in the fresh medium to be added.
  • IGF-1, heparin, etc. may be one or more.
  • Preferred concentrations of each are 10 to 30 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, and 10 to 50 ng /. For example, mL.
  • the adhesive culture in addition to the above-mentioned growth factors, it is more preferable to carry out in the presence of a third Wnt / ⁇ -catenin pathway inducer and an anti-Frizzled agent as growth factors. According to this, as shown in Examples described later, the efficiency of inducing differentiation into inner ear hair cells is further improved.
  • Examples of the inducer of the third Wnt / ⁇ -catenin pathway include Wnt3a, R-spondin1, CHIR99021 (also known as 6- -Imidazole-2-yl) pyrimidin-2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride) and the like.
  • Wnt3a and R-spondin1 are more preferably exemplified, and it is preferable to carry out in the presence of both Wnt3a and R-spondin1.
  • Frizzled receptor As an anti-Frizzled agent, as a competitive agent selective for the type of Frizzled receptor, an extracellular domain fragment of a human Frizzled receptor, a fusion protein composed of a human Fc domain, and the like are known, and thus such Frizzled. Pseudomolecules of the receptor can be used.
  • the pseudo-molecule of Frizzled 10 is preferably exemplified as the anti-Frizzled agent.
  • the anti-Frizzled agent when used together with an inducer of the Wnt / ⁇ -catenin pathway, it is possible to promote the induction of differentiation into cochlear hair cells, which is a form of inner ear hair cells.
  • a pseudo-molecule of Frizzled10 a trade name "Recombinant Human Frizzled-10 Fc Chimera Protein", R & D Systems) and the like are also commercially available, and such a commercially available product may be used.
  • the concentration of the inducer of the third Wnt / ⁇ -catenin pathway in the medium of the adhesive culture is preferably 10 ng / mL or more as the lower limit value, and 1000 ng / mL or less as the upper limit value. Is preferable.
  • the lower limit thereof is preferably 1 ng / mL or more, more preferably 10 ng / mL or more, and more preferably 20 ng / mL. It is even more preferable to use mL or more.
  • the upper limit is preferably 100 ng / mL or less, more preferably 50 ng / mL or less, and even more preferably 20 ng / mL or less.
  • the lower limit thereof is preferably 10 ng / mL or more, more preferably 50 ng / mL or more, and even more preferably 100 ng / mL or more.
  • the upper limit is preferably 1000 ng / mL or less, more preferably 500 ng / mL or less, and even more preferably 200 ng / mL or less.
  • the concentration of the anti-Frizzled agent in the medium of the adhesive culture is preferably 10 ng / mL or more, more preferably 20 ng / mL or more, and more preferably 50 ng / mL or more as the lower limit. Is even more preferable.
  • the upper limit is preferably 200 ng / mL or less, more preferably 100 ng / mL or less, and even more preferably 50 ng / mL or less.
  • the present invention uses the inner ear progenitor cells obtained by the above method to act on any test substance and examine the effect on the inner ear progenitor cells. Thereby, it provides a method for evaluating a drug, which evaluates the test substance.
  • the present invention uses the inner ear hair cells obtained by the above method to act on any test substance and examine the effect on the inner ear hair cells. It provides a method for evaluating a drug for evaluating a test substance.
  • composition for inducing Inner Ear Cell Differentiation provides a composition for inducing inner ear cell differentiation containing retinoic acid and an inducer for the Wnt / ⁇ -catenin pathway.
  • CHIR99021 As an inducer of the Wnt / ⁇ -catenin pathway, CHIR99021 (also known as 6-((2-((4- (2,4-Dichlophoenyl) -5- (4-Methyl-1H-imidazol-2-yl) pyrimidin-) 2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride), Wnt3a, R-spondin1 and the like.
  • BIO also known as 6-bro-methylrubin 3'-oxime
  • LiCl lithium chloride
  • Wnt3a Wnt3a
  • R-spondin1 R-spondin1
  • CHIR99021, BIO, LiCl (lithium chloride) and the like are more preferably exemplified.
  • the content of retinoic acid in the composition for inducing inner ear cell differentiation according to the present invention is not particularly limited, but the lower limit thereof is typically 0.01% by mass or more in the dry content, which is 0. .1% by mass or more is more typical, 1% by mass or more is even more typical, and 5% by mass or more is particularly typical.
  • the upper limit is typically 80% by mass or less, more typically 60% by mass or more, and even more typically 50% by mass or more during the dry content. Yes, it is particularly typical that it is 40% by mass or more.
  • the content of the inducer for the Wnt / ⁇ -catenin pathway in the composition is not particularly limited, but the upper limit thereof is typically 0.01% by mass or more in the dry content.
  • 1% by mass or more is more typical, 1% by mass or more is even more typical, and 5% by mass or more is particularly typical.
  • the upper limit is typically 80% by mass or less, more typically 60% by mass or more, and even more typically 50% by mass or more during the dry content. Yes, it is particularly typical that it is 40% by mass or more.
  • the composition for inducing inner ear cell differentiation according to the present invention is used for inducing differentiation of pluripotent stem cells into inner ear cells.
  • the inner ear according to the present invention is used in the form of a culture premix solution, a liquid medium, or the like for use in a medium containing the above-mentioned retinoic acid and an inducer of the Wnt / ⁇ -catenin pathway.
  • the purpose is to more efficiently carry out a method for producing precursor cells and inner ear hair cells.
  • Growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 are added to serum-free medium (DMEM / F12 + B27 + N2 + GlutaMAX + Nonecential amino acid) at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, 25 ng / mL, and 10 ng / mL, respectively.
  • the medium was replaced with the added medium. After that, the medium was changed every day until Day 7.
  • the medium was replaced with a medium containing growth factors bFGF, FGF3, FGF10, and FGF19 (growth factor concentrations were all 25 ng / mL) in a serum-free medium (DMEM / F12 + B27 + N2 + GlutaMAX + Nonesential amino acid). After that, the medium was changed every day until Day 10.
  • a serum-free medium DMEM / F12 + B27 + N2 + GlutaMAX + Nonesential amino acid
  • the cells were added with actase and incubated at 37 ° C. for 2-3 minutes, stripped from the dish and diluted with PBS.
  • the cells are collected by centrifugation, and the growth factors bFGF, FGF3, FGF10, and FGF19 are added to a serum-free medium (DMEM / F12 + B27 + N2) at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, and 25 ng / mL, respectively.
  • DMEM / F12 + B27 + N2 serum-free medium
  • the remaining cell mass was removed with a nylon mesh (pore diameter 40 ⁇ m) and seeded in wells coated with poly-o-fibronectin. Culturing was carried out under normal oxygen conditions (O 2 20%, CO 2 5%).
  • RNA was extracted using the RNeasy spin column kit (Qiagen), and reverse transcriptase (trade name "Invitrogen SuperScript IV Reverse Transcriptase (Thercimotive) Synthesis (Therci)) from 1 ⁇ g of each sample of total RNA was performed.
  • the expression level of the inner ear precursor cell marker was quantified by qRT-PCR.
  • Figure 3 shows the results. The results are shown as relative values when the quantitative value under the above culture condition 4 is 1.
  • the expression levels of the inner ear progenitor cell markers PAX2 and PAX8 were significantly increased when both CHIR99021 and retinoic acid were added.
  • the addition of CHIR99021 increased the expression of SOX2 and LGR5. This suggests that activation of WNT signal by CHIR99021 and addition of retinoic acid promote the differentiation of inner ear progenitor cells.
  • RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the expression levels of inner ear progenitor cell markers PAX2, PAX8, SOX2 and LGR5 were quantified by qRT-PCR. did.
  • Figure 4 shows the results. The results are shown as relative values when the same quantitative value by qRT-PCR was set to 1 for undifferentiated iPS cells.
  • the expression levels of the inner ear progenitor cell markers PAX2 and LGR5 increased from Day 11 to Day 15.
  • the expression levels of PAX2, PAX8, SOX2, and LGR5 decreased from Day15 to Day20. From this result, it was suggested that the inner ear progenitor cells could be efficiently induced by adding CHIR99021 and retinoic acid from Day 11 to Day 15.
  • Test Example 3 In Test Examples 1 and 2, it was revealed that the expression level of the inner ear progenitor cell marker was increased when CHIR99021, which is an inducer of the Wnt / ⁇ -catenin pathway, was used together with retinoic acid. In this test example, the effects of LiCl and BIO, which are also known as inducers of the Wnt / ⁇ -catenin pathway, on the expression level of inner ear progenitor cell markers were investigated.
  • RNA was extracted, cDNA was synthesized, and the expression level of PAX2, which is a marker for inner ear progenitor cells, was quantified by qRT-PCR in the same manner as in Test Example 1.
  • Figure 5 shows the results. The results are shown as relative values when the quantitative value under the above culture condition 1 (non-addition) is 1.
  • hypoxic conditions O 2 4%, CO 2 5%
  • Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively.
  • medium CHIR99021 and retinoic acid, each concentration were prepared by adding so 3 [mu] M, and 0.5 [mu] M, usually oxygen conditions (O 2 20%, CO 2 5%)
  • Cells were suspended to 8.5 ⁇ 10 4 cells / well and seeded on a low-adhesion 6-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning). At this time, the cells induced with conditions 1 and 2, respectively were suspended in a medium of the following composition, under hypoxic conditions (O 2 4%, CO 2 5%) or normoxic conditions (O 2 20%, It was initiated suspension culture in CO 2 5%).
  • hypoxic conditions O 2 4%, CO 2 5%
  • normoxic conditions O 2 20%, It was initiated suspension culture in CO 2 5%.
  • Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations were 10 ng / mL, 20 ng / ml, and 50 ng / mL, respectively, and further, Y- 27632 was added to a concentration of 10 ⁇ M, Wnt3a was added to a concentration of 20 ng / mL, CHIR99021 was added to a concentration of 3 ⁇ M, and heparin was added to a concentration of 50 ng / mL.
  • DMEM / F12 + B27 + N2 serum-free medium
  • a serum-free medium (DMEM / F12 + B27-VA + N2)
  • the growth factors bFGF, EGF, and IGF-1 were added to the concentrations of 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively.
  • a medium prepared by adding retinoic acid to a concentration of 0.5 ⁇ M and adding retinoic acid to a concentration of 0.5 ⁇ M.
  • the medium was exchanged with a medium prepared by adding growth factors EGF and IGF-1 to Day 28 in serum-free medium (DMEM / F12 + B27 + N2) at concentrations of 25 ng / mL and 10 ng / mL, respectively. .. After that, the medium was changed once every two days.
  • serum-free medium DMEM / F12 + B27 + N2
  • RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the cells were subjected to gene expression analysis by qRT-PCR.
  • CHIR99021 was added so as to have a concentration of 3 ⁇ M, and retinoic acid was added so as to have a concentration of 0.5 ⁇ M to prepare a medium, and the medium was exchanged. Culturing was carried out under normal oxygen conditions (O 2 20%, CO 2 5%).
  • the cells were suspended so as to be 8.5 ⁇ 10 4 cells / well, seeded on a low-adhesion 6-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, using the following conditions 1) or 2) as the medium, the cells were suspended and suspension culture was started.
  • -Condition 1 To the serum-free medium (DMEM / F12 + B27-VA + N2), the growth factors bFGF, EGF, and IGF-1 were added so that the concentrations were 10 ng / mL, 20 ng / ml, and 50 ng / mL, respectively, and further.
  • Y-27632 was added to a concentration of 10 ⁇ M
  • Wnt3a was added to a concentration of 20 ng / mL
  • CHIR99021 was added to a concentration of 3 ⁇ M
  • heparin was added to a concentration of 50 ng / mL.
  • Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) prepared by adding retinoic acid to a concentration of 0.5 ⁇ M.
  • Day24 For the cells of Day24, totalRNA was extracted in the same manner as in Test Example 1, cDNA synthesis was performed, and the cells were subjected to gene expression analysis by qRT-PCR.
  • the expression levels of the inner ear hair cell markers ATOH1, MYO7A, MYO15A, and BRN3C were compared with those in the non-added group.
  • the WNT signal was activated by the addition of R-spondin1, which is an inducer of the Wnt / ⁇ -catenin pathway, and thus the proliferation of inner ear progenitor cells was promoted, or from inner ear progenitor cells to inner ear hair cells. It was considered that the differentiation of the cells was promoted.
  • R-spondin1 was found as a drug for increasing the expression levels of the inner ear hair cell markers ATOH1, MYO7A, MYO15A, and BRN3C. It is possible to evaluate whether the expression level of the inner ear hair cell marker can be increased.
  • the markers and culture conditions used at this time may be set to other markers and culture conditions as appropriate, and can any drug promote the differentiation of inner ear progenitor cells into inner ear hair cells? It is possible to evaluate whether or not.
  • bFGF, EGF, and IGF were placed in serum-free medium (DMEM / F12 + B27-VA + N2) the next day (Day 12) for cells that had been induced to differentiate in the same manner as in Test Example 1 and had undergone cell detachment treatment with Actase on Day 11.
  • -1 was added so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively
  • CHIR99021 was added so that the concentration was 3 ⁇ M
  • retinoic acid was added so that the concentration was 0.5 ⁇ M.
  • the medium was replaced with medium prepared by adding to, were cultured in normoxic conditions (O 2 20%, CO 2 5%).
  • the cells were suspended so as to be 8.5 ⁇ 10 4 cells / well, seeded on a low-adhesion 96-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, as a medium, a serum-free medium (DMEM / F12 + B27-VA + N2) was added with the growth factor EGF so as to have a concentration of 20 ng / mL, and Y-27632 was further added so as to have a concentration of 10 ⁇ M.
  • DMEM / F12 + B27-VA + N2 a serum-free medium
  • Matrigel to a concentration of 1%
  • Wnt3a to a concentration of 20 ng / mL
  • CHIR99021 to a concentration of 10 ⁇ M
  • heparin to a concentration of 50 ng / mL.
  • the cells were suspended and suspended for suspension using a medium prepared by adding retinoic acid to a concentration of 0.5 ⁇ M and adding R-spondin1 to a concentration of 200 ng / mL. Started.
  • mouse anti-MYO7A antibody, mouse anti-BRN3C antibody, rabbit anti-SOX2 antibody, goat anti-SOX2 antibody, goat anti-SOX21 antibody, and rabbit anti-Calbindin antibody 50 times and 100 times, respectively.
  • 100-fold, 100-fold, 1000-fold diluted then labeled with a fluorescent secondary antibody specific for each animal species IgG and observed under a fluorescent microscope.
  • FIG. 8 shows the obtained microscopic observation image (scale bar: upper 50 ⁇ m, lower 20 ⁇ m).
  • hair cell markers MYO7A, BRN3C positive cells, sustentacular cell markers SOX2, SOX21 positive cells, and spiral ganglion cell markers Calbindin positive cells were detected.
  • bFGF, EGF, and IGF were placed in serum-free medium (DMEM / F12 + B27-VA + N2) the next day (Day 12) for cells that had been induced to differentiate in the same manner as in Test Example 1 and had undergone cell detachment treatment with Actase on Day 11.
  • -1 was added so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively
  • CHIR99021 was added so that the concentration was 3 ⁇ M
  • retinoic acid was added so that the concentration was 0.5 ⁇ M.
  • the medium was replaced with medium prepared by adding to, were cultured in normoxic conditions (O 2 20%, CO 2 5%).
  • the cells were suspended so as to be 8.5 ⁇ 10 4 cells / well, seeded on a low-adsorption 6-well plate (trade name “Corning ultra-low adhesive surface (Ultra-Low Attachment) plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, as a medium, growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively.
  • DMEM / F12 + B27-VA + N2 serum-free medium
  • Y-27632 was added so that the concentration was 10 ⁇ M
  • Matrigel was added so that the concentration was 1%
  • Wnt3a was added so that the concentration was 20 ng / mL
  • CHIR99021 was added so that the concentration was 3 ⁇ M.
  • the cells were suspended and suspension culture was started.
  • the medium was exchanged so as to meet the following conditions 1) to 5).
  • Growth factors EGF and IGF-1 were added to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations were 25 ng / mL and 10 ng / mL, respectively, and Wnt3a was further added to a concentration of 10 ng / mL.
  • RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the expression level of hair cell markers under each culture condition was quantified by qRT-PCR.
  • the expression levels of the cochlear hair cell markers MYO7A and MYO15A were enhanced under the conditions of culturing with the addition of Wnt3a, R-spondin1 and FZD10. From this, it was considered that the induction of differentiation into cochlear hair cells is promoted by activating the WNT signal and at the same time regulating the signal mediated by FZD10.
  • retinoic acid powder was dissolved in DMSO to 20 mM. Further, the powder of CHIR99021 was dissolved in DMSO so as to be 10 mM. These were mixed in the same amount to prepare a premix solution for preparing a medium containing retinoic acid and CHIR99021. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.
  • retinoic acid powder was dissolved in DMSO to 20 mM.
  • BIO powder was dissolved in DMSO to 1 mM. These were mixed in the same amount to prepare a premixed solution for preparing a medium containing retinoic acid and BIO. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.
  • retinoic acid powder was dissolved in DMSO to 20 mM. Further, LiCl (lithium chloride) powder was dissolved in MilliQ water so as to have a concentration of 2M. These were mixed in the same amount to prepare a premixed solution for preparing a medium containing retinoic acid and LiCl. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.

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Abstract

Provided is a method that is for producing inner ear progenitor cells and that enables efficient induction of differentiation to inner ear cells.　The method for producing inner ear progenitor cells comprises a step for performing cell detachment treatment on a cell population including PAX2/PAX8 positive cells differentiated through induction from pluripotent stem cells, and culturing said cell population in a medium containing retinoic acid and an inducing agent for a first Wnt/β-catenin pathway.

Description

内耳前駆細胞の製造方法、内耳有毛細胞の製造方法、薬剤の評価方法、及び内耳細胞分化誘導用組成物A method for producing inner ear progenitor cells, a method for producing inner ear hair cells, a method for evaluating a drug, and a composition for inducing inner ear cell differentiation.
　本発明は、内耳前駆細胞の製造方法、内耳有毛細胞の製造方法、薬剤の評価方法、及び内耳細胞分化誘導用組成物に関する。 The present invention relates to a method for producing inner ear progenitor cells, a method for producing inner ear hair cells, a method for evaluating a drug, and a composition for inducing inner ear cell differentiation.
　近年、人工多能性幹細胞（ｉＰＳ細胞）や胚性幹細胞（ＥＳ細胞）などの多能性幹細胞は、再生医療あるいは病態解明、創薬のためのリサーチ・ツールなどとして、広汎にその利用が実現されている。一方で、種々の原因により惹起される聴覚障害に関しては、実験動物では聴覚の測定／比較が困難であり、科学的評価に資する内耳細胞を多能性幹細胞から誘導する方法の開発が期待されている。 In recent years, pluripotent stem cells such as induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) have been widely used as research tools for regenerative medicine, pathological elucidation, and drug discovery. Has been done. On the other hand, regarding hearing impairment caused by various causes, it is difficult to measure / compare hearing in experimental animals, and it is expected to develop a method for inducing inner ear cells that contribute to scientific evaluation from pluripotent stem cells. There is.
　しかしながら、例えば、非特許文献１、２にみられるように、ヒトＥＳ細胞から内耳前駆細胞を誘導する場合、胚葉体形成及びそれにともなう目視下における内耳前駆細胞の選択が必要であり、長期の培養期間及び低い効率が問題であった。更に、内耳前駆細胞から更に分化段階の進んだ内耳有毛細胞への誘導は、非常に効率が悪く、再生医療あるいは病態解明、創薬のためのリサーチ・ツールなどに応用するには、品質の面でも問題があった。 However, as seen in Non-Patent Documents 1 and 2, for example, when inducing inner ear progenitor cells from human ES cells, it is necessary to select germ layer formation and the inner ear progenitor cells under visual observation, and long-term culture is required. Duration and low efficiency were issues. Furthermore, the induction of inner ear progenitor cells to more advanced inner ear hair cells is extremely inefficient, and is of high quality for application to regenerative medicine, pathological elucidation, research tools for drug discovery, etc. There was also a problem in terms of it.
　このような課題に対して、本出願人らは、多能性幹細胞から高効率に内耳前駆細胞を誘導する方法を確立した（特許文献１）。また、その誘導方法を応用して、遺伝性難聴疾患であるペンドレッド症候群患者に由来するヒトｉＰＳ細胞を樹立したうえ、ペンドリン陽性細胞を調製し、患者由来細胞に特異的な細胞内凝集体の形成の確認やプロテアソーム阻害負荷に抗することができる薬剤の評価を行っている（特許文献２）。 To address these issues, the applicants have established a method for highly efficiently inducing inner ear progenitor cells from pluripotent stem cells (Patent Document 1). In addition, by applying the induction method, human iPS cells derived from a patient with Pendred syndrome, which is a genetic deafness disease, are established, and Pendred-positive cells are prepared to prepare intracellular aggregates specific to the patient-derived cells. We are confirming the formation and evaluating drugs that can resist the proteasome inhibitory load (Patent Document 2).
特許第６２１８１５２号公報Japanese Patent No. 6218152 国際公開第２０１６／１１７４３１号International Publication No. 2016/117431
　しかしながら、高品質の細胞を安定的に大量かつ適切な価格で供給するには、より効率的な分化誘導方法の開発が必要であった。 However, in order to stably supply high-quality cells in large quantities and at an appropriate price, it was necessary to develop a more efficient method for inducing differentiation.
　よって、本発明の目的は、内耳細胞への効率的な分化誘導を可能にする、内耳前駆細胞の製造方法を提供することにある。また、これにより得られた内耳前駆細胞を用いた内耳有毛細胞の製造方法を提供することにある。また、それら方法により得られた内耳前駆細胞や内耳有毛細胞に対する、薬剤の評価方法を提供することにある。また、内耳細胞への分化誘導を簡便に行うようにするための、内耳細胞分化誘導用組成物を提供することにある。 Therefore, an object of the present invention is to provide a method for producing inner ear progenitor cells, which enables efficient induction of differentiation into inner ear cells. Another object of the present invention is to provide a method for producing inner ear hair cells using the inner ear progenitor cells thus obtained. Another object of the present invention is to provide a method for evaluating a drug for inner ear progenitor cells and inner ear hair cells obtained by these methods. Another object of the present invention is to provide a composition for inducing differentiation of inner ear cells in order to easily induce differentiation into inner ear cells.
　本発明者らは、上記目的を達成するため鋭意検討を重ね、本発明を完成するに至った。 The present inventors have made extensive studies in order to achieve the above object, and have completed the present invention.
　すなわち、本発明は、その第１の観点において、多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団を、細胞剥離処理したうえ、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養する工程を含む、内耳前駆細胞の製造方法を提供するものである。 That is, in the first aspect of the present invention, a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell exfoliation treatment, and then retinoic acid and the first Wnt / β catenin pathway. It provides a method for producing inner ear progenitor cells, which comprises a step of culturing in a medium containing an inducer.
　本発明による、上記第１の観点における内耳前駆細胞の製造方法によれば、多能性幹細胞から分化誘導した、内耳予定領域マーカーであるＰＡＸ２及び／又はＰＡＸ８を発現した細胞を含む細胞集団を、細胞剥離処理により個々の細胞に分離したうえ、次の培養に移行するので、内耳細胞への分化にとって不必要な細胞を淘汰して、ＰＡＸ２及び／又はＰＡＸ８の発現レベルを確実に維持することができる。そして、細胞剥離処理後には、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養するので、ＰＡＸ２及び／又はＰＡＸ８の発現レベルを向上させることができる。よって、これにより、品質の良好な内耳前駆細胞が効率よく得られる。 According to the method for producing inner ear progenitor cells according to the first aspect of the present invention, a cell population containing cells expressing PAX2 and / or PAX8, which are markers for the planned inner ear region, which are induced to differentiate from pluripotent stem cells, can be obtained. Since cells are separated into individual cells by cell exfoliation treatment and then transferred to the next culture, cells unnecessary for differentiation into inner ear cells can be eliminated to ensure that PAX2 and / or PAX8 expression levels are maintained. can. Then, after the cell exfoliation treatment, the cells are cultured in a medium containing retinoic acid and an inducer of the first Wnt / β-catenin pathway, so that the expression level of PAX2 and / or PAX8 can be improved. Therefore, this makes it possible to efficiently obtain good quality inner ear progenitor cells.
　本発明による内耳前駆細胞の製造方法においては、前記レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地は、ＩＧＦ－１、ｂＦＧＦ、及びＥＧＦからなる群から選ばれた１種又は２種以上を含有することが好ましい。これによれば、内耳前駆細胞への分化をより確実にすることができる。 In the method for producing inner ear progenitor cells according to the present invention, the medium containing the retinoic acid and the inducer of the first Wnt / β-catenin pathway is one selected from the group consisting of IGF-1, bFGF, and EGF. Alternatively, it is preferable to contain two or more kinds. According to this, it is possible to more reliably differentiate into inner ear progenitor cells.
　本発明による内耳前駆細胞の製造方法においては、前記レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地は、無血清培地であることが好ましい。これによれば、血清因子に起因して目的外の細胞に分化してしまうリスクを抑えることができる。 In the method for producing inner ear progenitor cells according to the present invention, the medium containing the retinoic acid and the inducer of the first Wnt / β-catenin pathway is preferably a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors.
　本発明による内耳前駆細胞の製造方法においては、前記レチノイン酸源及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地による培養は、接着培養によるものであることが好ましい。これによれば、細胞剥離処理後の培養を効率よく行うことができる。また、培地交換等の操作が容易である。 In the method for producing inner ear progenitor cells according to the present invention, it is preferable that the culture in a medium containing the retinoic acid source and the inducer of the first Wnt / β-catenin pathway is by adhesive culture. According to this, the culture after the cell exfoliation treatment can be efficiently performed. In addition, operations such as medium exchange are easy.
　本発明による内耳前駆細胞の製造方法においては、前記細胞剥離処理は、所定の孔径を有するメッシュを通す工程を含むことが好ましい。これによれば、多能性幹細胞から分化誘導したＰＡＸ２及び／又はＰＡＸ８を発現した細胞を含む細胞集団について、その細胞の個々を分離するのを、より効率よく行うことができる。 In the method for producing inner ear progenitor cells according to the present invention, it is preferable that the cell detachment treatment includes a step of passing a mesh having a predetermined pore size. According to this, it is possible to more efficiently separate individual cells of a cell population containing cells expressing PAX2 and / or PAX8 which have been induced to differentiate from pluripotent stem cells.
　本発明による内耳前駆細胞の製造方法においては、前記第１のＷｎｔ／βカテニン経路の誘導剤は、ＣＨＩＲ９９０２１、ＢＩＯ、及びＬｉＣｌからなる群から選ばれた１種又は２種以上であることが好ましい。 In the method for producing inner ear progenitor cells according to the present invention, the first Wnt / β-catenin pathway inducer is preferably one or more selected from the group consisting of CHIR99021, BIO, and LiCl. ..
　本発明による内耳前駆細胞の製造方法においては、前記多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団が、以下の工程（１）～（４）を含む方法で得られたものであることが好ましい。
　（１）多能性幹細胞を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の存在下で培養する工程
　（２）工程（１）で得られた細胞集団を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の非存在下で培養する工程
　（３）工程（２）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子、及びＢＭＰ４の存在下で培養する工程と、
　（４）工程（３）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子の存在下であって、ＢＭＰ４の非存在下で培養する工程 In the method for producing inner ear progenitor cells according to the present invention, a cell population containing PAX2 / PAX8-positive cells induced to differentiate from the pluripotent stem cells was obtained by a method including the following steps (1) to (4). Is preferable.
(1) A step of culturing pluripotent stem cells in the absence of a growth factor and in the presence of a ROCK inhibitor (2) A cell population obtained in step (1) in the absence of a growth factor. (3) Growth of at least one cell population obtained in step (2) selected from the group consisting of bFGF, FGF3, FGF10, and FGF19. The step of culturing in the presence of factors and BMP4, and
(4) The cell population obtained in step (3) is cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19 in the absence of BMP4. Process to do
　これによれば、多能性幹細胞から、内耳予定領域マーカーであるＰＡＸ２及び／又はＰＡＸ８を発現した細胞を含む細胞集団を、より確実に分化誘導することができる。 According to this, it is possible to more reliably induce differentiation from pluripotent stem cells, including cells expressing PAX2 and / or PAX8, which are markers for the planned inner ear region.
　本発明は、その第２の観点において、上記の製造方法で得られた内耳前駆細胞を、該内耳前駆細胞を含む細胞集団の状態で、以下の工程（ｉ）及び（ｉｉ）に処することを含む、内耳有毛細胞の製造方法を提供するものである。
　（ｉ）前記内耳前駆細胞を含む細胞集団を浮遊培養する工程
　（ｉｉ）工程（ｉ）で得られた細胞集団を接着培養する工程 In the second aspect of the present invention, the inner ear progenitor cells obtained by the above-mentioned production method are subjected to the following steps (i) and (ii) in the state of a cell population containing the inner ear progenitor cells. It provides a method for producing inner ear hair cells, including.
(I) Step of suspension-culturing the cell population containing the inner ear progenitor cells (ii) Step of adhesion-culturing the cell population obtained in step (i)
　本発明による、上記第２の観点における内耳有毛細胞の製造方法によれば、上記第１の観点における内耳前駆細胞の製造方法で得られた内耳前駆細胞を利用するので、品質の良好な内耳有毛細胞を効率的に得ることができる。 According to the method for producing inner ear hair cells according to the second aspect of the present invention, the inner ear precursor cells obtained by the method for producing inner ear precursor cells according to the first aspect are used, so that the inner ear has good quality. Hair cells can be obtained efficiently.
　本発明による内耳有毛細胞の製造方法においては、前記工程（ｉ）における該浮遊培養は、第２のＷｎｔ／βカテニン経路の誘導剤を含有する培地で行うことが好ましい。これによれば、品質の良好な内耳有毛細胞をより効率的に得ることができる。 In the method for producing inner ear hair cells according to the present invention, it is preferable that the suspension culture in the step (i) is carried out in a medium containing an inducer for the second Wnt / β-catenin pathway. According to this, good quality inner ear hair cells can be obtained more efficiently.
　本発明による内耳有毛細胞の製造方法においては、前記第２のＷｎｔ／βカテニン経路の誘導剤は、Ｒ－ｓｐｏｎｄｉｎ１、ＣＨＩＲ９９０２１、及びＷｎｔ３ａからなる群から選ばれた１種又は２種以上であることが好ましい。 In the method for producing inner ear hair cells according to the present invention, the second Wnt / β-catenin pathway inducer is one or more selected from the group consisting of R-spondin1, CHIR99021, and Wnt3a. Is preferable.
　本発明による内耳有毛細胞の製造方法においては、前記工程（ｉｉ）における該接着培養は、第３のＷｎｔ／βカテニン経路の誘導剤及び抗Ｆｒｉｚｚｌｅｄ剤を含有する培地で行うことが好ましい。これによれば、品質の良好な内耳有毛細胞をより効率的に得ることができる。 In the method for producing inner ear hair cells according to the present invention, it is preferable that the adhesion culture in the step (ii) is carried out in a medium containing an inducer of a third Wnt / β-catenin pathway and an anti-Frizzled agent. According to this, good quality inner ear hair cells can be obtained more efficiently.
　本発明による内耳有毛細胞の製造方法においては、前記第３のＷｎｔ／βカテニン経路の誘導剤は、Ｗｎｔ３ａ及び／又はＲ－ｓｐｏｎｄｉｎ１であり、前記抗Ｆｒｉｚｚｌｅｄ剤は、Ｆｒｉｚｚｌｅｄ１０の擬似分子による競合剤であることが好ましい。 In the method for producing inner ear hair cells according to the present invention, the third Wnt / β-catenin pathway inducer is Wnt3a and / or R-spondin1, and the anti-Frizzled agent is a competitor with a pseudo-molecule of Frizzled10. Is preferable.
　本発明は、その第３の観点において、上記の製造方法で得られた内耳前駆細胞を、被検薬剤で処理する工程と、前記被検薬剤で処理した前記内耳前駆細胞の状態を評価する工程とを含む、薬剤の評価方法を提供するものである。 From the third aspect of the present invention, the step of treating the inner ear progenitor cells obtained by the above-mentioned production method with a test agent and the step of evaluating the state of the inner ear progenitor cells treated with the test agent. It provides a method for evaluating a drug, including.
　本発明による、上記第３の観点における薬剤の評価方法によれば、内耳前駆細胞や内耳分化に影響を与える薬剤の評価を有効かつ効率的に行うことができる。 According to the method for evaluating a drug according to the third aspect of the present invention, it is possible to effectively and efficiently evaluate a drug that affects inner ear progenitor cells and inner ear differentiation.
　本発明は、その第４の観点において、上記の製造方法で得られた内耳有毛細胞を、被検薬剤で処理する工程と、前記被検薬剤で処理した前記内耳有毛細胞の状態を評価する工程とを含む、薬剤の評価方法を提供するものである。 From the fourth aspect of the present invention, the step of treating the inner ear hair cells obtained by the above-mentioned production method with the test agent and the state of the inner ear hair cells treated with the test agent are evaluated. It provides a method for evaluating a drug, including a step of performing a drug.
　本発明による、上記第４の観点における薬剤の評価方法によれば、内耳有毛細胞や内耳分化に影響を与える薬剤の評価を有効かつ効率的に行うことができる。 According to the method for evaluating a drug according to the fourth aspect of the present invention, it is possible to effectively and efficiently evaluate a drug that affects inner ear hair cells and inner ear differentiation.
　本発明は、その第５の観点において、レチノイン酸及びＷｎｔ／βカテニン経路の誘導剤を含有する内耳細胞分化誘導用組成物を提供するものである。 The present invention provides, from the fifth aspect, a composition for inducing inner ear cell differentiation containing retinoic acid and an inducer of the Wnt / β-catenin pathway.
　本発明による、上記第５の観点における内耳細胞分化誘導用組成物によれば、これを用いて、効率的な内耳細胞の調製を簡便に行うことができる。 According to the composition for inducing inner ear cell differentiation according to the fifth aspect of the present invention, it is possible to easily prepare efficient inner ear cells by using the composition.
　本発明による内耳細胞分化誘導用組成物においては、前記Ｗｎｔ／βカテニン経路の誘導剤は、ＣＨＩＲ９９０２１、ＢＩＯ、及びＬｉＣｌからなる群から選ばれた１種又は２種以上であることが好ましい。 In the composition for inducing inner ear cell differentiation according to the present invention, the inducer of the Wnt / β-catenin pathway is preferably one or more selected from the group consisting of CHIR99021, BIO, and LiCl.
　本発明による内耳細胞分化誘導用組成物においては、該内耳細胞分化誘導用組成物は、内耳前駆細胞誘導用のものであることが好ましい。 In the composition for inducing inner ear cell differentiation according to the present invention, it is preferable that the composition for inducing inner ear cell differentiation is for inducing inner ear progenitor cells.
　なお、本明細書において、上記内耳前駆細胞の製造方法又は上記内耳有毛細胞の製造方法における、「第１のＷｎｔ／βカテニン経路の誘導剤」、「第２のＷｎｔ／βカテニン経路の誘導剤」、「第３のＷｎｔ／βカテニン経路の誘導剤」とは、それぞれ異なる工程において「Ｗｎｔ／βカテニン経路の誘導剤」を用いるという趣旨であり、物質的構成を相互に別異なものとして区別する趣旨ではない。よって、例えば、第１～第３のそれぞれのＷｎｔ／βカテニン経路の誘導剤として、１種又は２種以上の同一の物質を用いる場合があってよく、あるいは、第１～第３のそれぞれのＷｎｔ／βカテニン経路の誘導剤として、全部又は一部が異なる１種又は２種以上の物質を用いる場合があってもよい。 In the present specification, in the method for producing the inner ear precursor cells or the method for producing the inner ear hair cells, "inducing agent of the first Wnt / β-catenin pathway" and "induction of the second Wnt / β-catenin pathway". "Agent" and "third Wnt / β-catenin pathway inducer" mean that "Wnt / β-catenin pathway inducer" is used in different steps, and the material configurations are different from each other. It is not the purpose of distinguishing. Therefore, for example, one or more of the same substances may be used as the inducer of each of the first to third Wnt / β-catenin pathways, or each of the first to third. As an inducer of the Wnt / β-catenin pathway, one or more substances that are completely or partially different may be used.
本発明による内耳前駆細胞の製造方法の一実施形態を説明するフロー図である。It is a flow figure explaining one Embodiment of the manufacturing method of the inner ear progenitor cell by this invention. 本発明による内耳有毛細胞の製造方法の一実施形態を説明するフロー図である。It is a flow figure explaining one Embodiment of the manufacturing method of the inner ear hair cell by this invention. 試験例１において、ヒトｉＰＳ細胞から分化誘導した内耳前駆細胞について、ｑＲＴ－ＰＣＲによる遺伝子発現解析を行った結果を示す図表である。In Test Example 1, it is a chart which shows the result of having performed the gene expression analysis by qRT-PCR about the inner ear progenitor cell which was induced to differentiate from the human iPS cell. 試験例２において、ヒトｉＰＳ細胞から内耳前駆細胞に分化誘導する過程における内耳前駆細胞マーカーの発現量の推移をｑＲＴ－ＰＣＲで比較した結果を示す図表である。In Test Example 2, it is a chart which shows the result of having compared the transition of the expression level of the inner ear progenitor cell marker in the process of inducing the differentiation from the human iPS cell into the inner ear progenitor cell by qRT-PCR. 試験例３において、ヒトｉＰＳ細胞から内耳前駆細胞への分化誘導を行い、３種類の異なるＷｎｔ／βカテニン経路の誘導剤を添加して培養したことによる内耳前駆細胞マーカーの発現量の変化をｑＲＴ－ＰＣＲで定量した結果を示す図表である。In Test Example 3, the change in the expression level of the inner ear progenitor cell marker by inducing the differentiation of human iPS cells into inner ear progenitor cells and culturing with the addition of three different Wnt / β-catenin pathway inducers is qRT. -It is a chart showing the result quantified by PCR. 試験例４において、内耳前駆細胞から内耳有毛細胞への分化誘導を行い、内耳前駆細胞マーカーのＬＧＲ５及び内耳有毛細胞マーカーのＡＴＯＨ１及びＢＲＮ３Ｃの遺伝子発現量をｑＲＴ－ＰＣＲで定量した結果を示す図表である。In Test Example 4, differentiation of inner ear progenitor cells into inner ear hair cells was induced, and the gene expression levels of inner ear progenitor cell markers LGR5 and inner ear hair cell markers ATOH1 and BRN3C were quantified by qRT-PCR. It is a chart. 試験例５において、内耳前駆細胞から内耳有毛細胞への分化誘導を行い、Ｒ－ｓｐｏｎｄｉｎ１を添加して浮遊培養を行ったことによる内耳有毛細胞マーカーの発現量の変化をｑＲＴ－ＰＣＲで定量した結果を示す図表である。In Test Example 5, the change in the expression level of the inner ear hair cell marker due to induction of differentiation from inner ear progenitor cells to inner ear hair cells and suspension culture with the addition of R-spondin1 was quantified by qRT-PCR. It is a chart which shows the result of this. 試験例６において、内耳前駆細胞から内耳有毛細胞への分化誘導を行い、内耳有毛細胞、支持細胞、及びらせん神経節細胞のそれぞれのマーカー分子に対する特異抗体による免疫染色を行った結果を示す図表である。In Test Example 6, the results of induction of differentiation of inner ear precursor cells into inner ear hair cells and immunostaining with specific antibodies against the respective marker molecules of inner ear hair cells, sustentacular cells, and spiral ganglion cells are shown. It is a chart. 試験例７において、内耳前駆細胞から内耳有毛細胞への分化誘導の過程で各種抗Ｆｒｉｚｚｌｅｄ剤を添加することによる内耳有毛細胞マーカーの発現量の変化をｑＲＴ－ＰＣＲで比較した結果を示す図表である。In Test Example 7, a chart showing the results of comparing the changes in the expression level of inner ear hair cell markers by adding various antifrizzled agents in the process of inducing differentiation of inner ear progenitor cells into inner ear hair cells by qRT-PCR. Is.
　本発明は、多能性幹細胞から内耳器官を構成する内耳細胞を分化誘導する方法に関し、より詳細には、多能性幹細胞から内耳細胞への分化の途中段階にあり、幹細胞性を残す内耳前駆細胞において、内耳細胞への分化誘導が促進されるよう、そのように賦活化した内耳前駆細胞を製造する方法に関する。 The present invention relates to a method for inducing differentiation of pluripotent stem cells into inner ear cells constituting the inner ear organs. The present invention relates to a method for producing an inner ear progenitor cell thus activated so as to promote the induction of differentiation into an inner ear cell in the cell.
　多能性幹細胞としては、人工多能性幹細胞（ｉＰＳ細胞）や胚性幹細胞（ＥＳ細胞）などが知られている。多能性幹細胞は、ヒト由来のものであってもよく、ヒト以外の生物に由来するものであってもよい。また、ｉＰＳ細胞である場合には、健常人の体細胞から初期化して調製されたものを用いてもよく、疾患保有者の体細胞から初期化して調製されたものを用いてもよい。疾患としては、特には、内耳器官、聴覚、聴力等に関するものが挙げられ、例えば、ペンドレッド症候群、アッシャー症候群等が挙げられる。 As pluripotent stem cells, artificial pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) and the like are known. Pluripotent stem cells may be of human origin or of non-human organisms. Further, in the case of iPS cells, those prepared by reprogramming from the somatic cells of a healthy person may be used, or those prepared by reprogramming from the somatic cells of a disease carrier may be used. Examples of the disease include those related to the inner ear organ, hearing, hearing, and the like, and examples thereof include Pendred syndrome and Usher syndrome.
　本発明においては、多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団を、細胞剥離処理したうえ、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養する必要があるが、それ以外は、従来の方法に従って培養を行うことができる。以下、多能性幹細胞を内耳前駆細胞への分化に向かわせるための典型的な誘導方法に沿って、本発明を詳細に説明する。ただし、本発明にかかる方法は、以下に説明する特定の培養条件等に限定されるものではない。 In the present invention, a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell exfoliation treatment and then cultured in a medium containing retinoic acid and an inducer for the first Wnt / β catenin pathway. Other than that, the culture can be carried out according to the conventional method. Hereinafter, the present invention will be described in detail along with a typical induction method for directing pluripotent stem cells to differentiation into inner ear progenitor cells. However, the method according to the present invention is not limited to the specific culture conditions and the like described below.
　なお、以下の説明において「接着培養」とは、目的の細胞や細胞集団を培養器の底面等に接着させて培養することを意味し、また、「浮遊培養」とは、目的の細胞や細胞集団を培養器の底面等に接着させずに培養することを意味する。この場合、培養中、細胞や細胞集団が培養器の底面等に接着するとは、細胞や細胞集団が、細胞外マトリクス（ＥＣＭ）などに含まれる細胞－基質接着分子を通じて、培養器の底面等と接着している状態を意味し、培養液を軽く揺らしても細胞や細胞集団が培養液中に浮かんでこない状態をいう。一方、培養中、細胞や細胞集団が培養器の底面等に接着しないとは、細胞や細胞集団が、細胞外マトリクス（ＥＣＭ）などに含まれる細胞－基質接着分子を通じて、培養器の底面等と接着していない状態を意味し、たとえ底面等に触れていても培養液を軽く揺らすと細胞や細胞集団が培養液中に浮かんでくるような状態をいう。接着培養の際は、細胞の基質への接着を促進するために、プラスティックディッシュの底表面を化学処理したり、接着を促進する接着用コーティング剤（ゼラチン、ポリリジン、寒天など）でコートしたりすることが好ましい。浮遊培養の際は、プラスティックディッシュの底面等の表面は処理しないか、細胞の基質への接着を阻止するための接着阻止用コーティング剤（ポリ（２－ヒドロキシエチルメタクリレート）など）でコートしたりすることが好ましい。なお、接着培養であっても、目的の細胞が接着するまでに時間がかかり、ある程度の時間、浮遊状態にある場合があるが、時間がかかってもその細胞が最終的に接着する場合は、接着培養に含めることとする。 In the following description, "adhesive culture" means that a target cell or cell population is adhered to the bottom surface of an incubator and cultured, and "suspension culture" means a target cell or cell. It means culturing the population without adhering it to the bottom surface of the incubator. In this case, when cells or cell populations adhere to the bottom surface of the incubator during culturing, the cells or cell population adhere to the bottom surface of the incubator through cell-substrate adhesion molecules contained in the extracellular matrix (ECM) or the like. It means an adhered state, and means a state in which cells or cell populations do not float in the culture solution even if the culture solution is shaken lightly. On the other hand, the fact that cells or cell populations do not adhere to the bottom surface of the incubator during culture means that the cells or cell population adhere to the bottom surface of the incubator through cell-substrate adhesion molecules contained in the extracellular matrix (ECM) or the like. It means a state in which they are not adhered, and even if they are touching the bottom surface, etc., it means a state in which cells or cell populations float in the culture solution when the culture solution is shaken lightly. During adhesive culture, the bottom surface of the plastic dish is chemically treated or coated with an adhesive coating (gelatin, polylysine, agar, etc.) to promote adhesion of cells to the substrate. Is preferable. During suspension culture, the surface such as the bottom surface of the plastic dish is not treated, or it is coated with an adhesion blocking coating agent (poly (2-hydroxyethyl methacrylate), etc.) to prevent adhesion of cells to the substrate. Is preferable. Even in the case of adhesive culture, it takes time for the target cells to adhere and may be in a suspended state for a certain period of time, but if the cells finally adhere even if it takes a long time, It will be included in the adhesive culture.
　［１］多能性幹細胞から内耳前駆細胞への分化誘導
　本発明において、多能性幹細胞からＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団への分化誘導は、例えば、以下の工程（１）～（４）を経ることにより行うことができる。
　（１）第１工程：多能性幹細胞を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の存在下で培養する工程
　（２）第２工程：工程（１）で得られた細胞集団を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の非存在下で培養する工程
　（３）第３工程：工程（２）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子、及びＢＭＰ４の存在下で培養する工程と、
　（４）第４工程：工程（３）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子の存在下であって、ＢＭＰ４の非存在下で培養する工程 [1] Induction of differentiation of pluripotent stem cells into inner ear progenitor cells In the present invention, the induction of differentiation of pluripotent stem cells into a cell population containing PAX2 / PAX8-positive cells is, for example, the following steps (1) to (4). ) Can be done.
(1) First step: A step of culturing pluripotent stem cells in the absence of growth factors and in the presence of a ROCK inhibitor (2) Second step: Cell population obtained in step (1) In the absence of growth factors and in the absence of ROCK inhibitors (3) Third step: Cell populations obtained in step (2) were cultivated in bFGF, FGF3, FGF10, and FGF19. A step of culturing in the presence of at least one growth factor selected from the group consisting of BMP4 and BMP4.
(4) Fourth step: The cell population obtained in step (3) was subjected to the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19, and was not BMP4. Step of culturing in the presence
　上記の第１工程で用いる培地としては、多能性幹細胞もしくは多能性幹細胞から分化に向かう細胞を維持できる培地であればよく、特に限定されない。例えば、多能性幹細胞維持用のフィーダー細胞不要な無血清培地である「ｍＴｅＳＲ１」（ＳＴＥＭＣＥＬＬ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）などが好ましく例示される。ただし、第１工程においては、成長因子の非存在下であって、ＲＯＣＫ（Ｒｈｏ－ａｓｓｏｃｉａｔｅｄ　ｃｏｉｌｅｄ－ｃｏｉｌ　ｆｏｒｍｉｎｇ　ｋｉｎａｓｅ／Ｒｈｏ結合キナーゼ）の阻害剤の存在下で培養を行う。ＲＯＣＫ阻害剤により、多能性幹細胞に対して細胞死抑制効果がある。第１工程は、１～３日間行うことが好ましく、１～２日間行うことがより好ましい。 The medium used in the above-mentioned first step is not particularly limited as long as it is a medium capable of maintaining pluripotent stem cells or cells heading for differentiation from pluripotent stem cells. For example, "mTeSR1" (STEMCELL Technologies), which is a serum-free medium that does not require feeder cells for maintaining pluripotent stem cells, is preferably exemplified. However, in the first step, the culture is carried out in the absence of a growth factor and in the presence of an inhibitor of ROCK (Rho-associated coiled-coil forming kinase / Rho-binding kinase). The ROCK inhibitor has a cell death inhibitory effect on pluripotent stem cells. The first step is preferably carried out for 1 to 3 days, more preferably for 1 to 2 days.
　上記の第１工程で用いるＲＯＣＫ阻害剤としては、例えば、Ｙ－２７６３２（（Ｒ）－（＋）－ｔｒａｎｓ－Ｎ－（４－Ｐｙｒｉｄｙｌ）－４－（１－ａｍｉｎｏｅｔｈｙｌ）－ｃｙｃｌｏｈｅｘａｎｅｃａｒｂｏｘａｍｉｄｅ）、Ｆａｓｕｄｉｌ　ｈｙｄｒｏｃｈｌｏｒｉｄｅ、Ｋ－１１５（リパスジル塩酸塩水和物）、ＤＥ－１０４などが例示される。ＲＯＣＫ阻害剤の濃度は、ＲＯＣＫ阻害剤の種類に応じて、適宜最適濃度を決定すればよいが、例えばＹ－２７６３２の場合、１～１００μＭが好ましく、１０～２０μＭがより好ましい。 Examples of the ROCK inhibitor used in the above first step include Y-27632 ((R)-(+)-trans-N- (4-Pyridine) -4- (1-aminoethyl) -cyclohexanecarboxamide), Fasudil hydroxylide. , K-115 (ripasudil hydrochloride hydrate), DE-104 and the like are exemplified. The optimum concentration of the ROCK inhibitor may be appropriately determined according to the type of the ROCK inhibitor. For example, in the case of Y-27632, 1 to 100 μM is preferable, and 10 to 20 μM is more preferable.
　上記の第２工程で用いる培地としては、多能性幹細胞もしくは多能性幹細胞から分化に向かう細胞を維持できる培地であればよく、特に限定されない。例えば、第１工程の培養に好ましく使用される培地と同様に、多能性幹細胞維持用のフィーダー細胞不要な無血清培地である「ｍＴｅＳＲ１」（ＳＴＥＭＣＥＬＬ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）などが好ましく例示される。ただし、第２工程では、成長因子の非存在下であって、ＲＯＣＫ阻害剤の非存在下で培養する。また、その際、第２工程の好ましい態様においては、上記ｍＴｅＳＲ１メディウムで１日程度培養した後、培地を、続く工程で使用する基礎培地（例えば、無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）に、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）を補充した培地）などに交換し、１日毎に新鮮培地に交換しつつ培養して、細胞を維持することが好ましい。これによれば、続く工程で成長因子を作用させるために用いる基礎培地に細胞をよくなじませることができる。第２工程は、トータルで１～１０日間行うことが好ましく、２～８日間行うことがより好ましい。３～６日間行うことが更により好ましい。 The medium used in the above-mentioned second step is not particularly limited as long as it is a medium capable of maintaining pluripotent stem cells or cells heading for differentiation from pluripotent stem cells. For example, "mTeSR1" (STEMCELL Technologies), which is a serum-free medium that does not require feeder cells for maintaining pluripotent stem cells, is preferably exemplified as the medium preferably used for culturing in the first step. However, in the second step, the cells are cultured in the absence of a growth factor and in the absence of a ROCK inhibitor. At that time, in a preferred embodiment of the second step, after culturing in the above mTeSR1 medium for about one day, the medium is used as a basal medium used in the subsequent steps (for example, “DMEM / F12” (commodity-free medium). Name "D-MEM / Ham's F-12", Fujifilm Wako Junyaku Co., Ltd.), serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermo Fisher Scientific), serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific), serum-free supplement "Nonesys" It is preferable to replace the cells with a medium) or the like and culture them while replacing them with a fresh medium every day to maintain the cells. The second step is preferably carried out for a total of 1 to 10 days, more preferably 2 to 8 days, and even more preferably 3 to 6 days.
　なお、上記の第１工程及び第２工程において、成長因子及び／又はＲＯＣＫ阻害剤の非存在下の意味は、成長因子及び／又はＲＯＣＫ阻害剤が実質的に非存在であればよく、成長因子及び／又はＲＯＣＫ阻害剤は効果がないレベルの濃度で含まれていてもよい。 In the first and second steps described above, the meaning in the absence of the growth factor and / or the ROCK inhibitor may be that the growth factor and / or the ROCK inhibitor is substantially absent, and the growth factor may be present. And / or the ROCK inhibitor may be included at an ineffective level of concentration.
　上記の第３工程で用いる培地としては、多能性幹細胞から分化に向かう細胞を維持できる培地であればよく、特に限定されない。例えば、第２工程の後半培養で好ましく使用される培地と同様に、無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）に、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）を補充した培地などが好ましく例示される。ただし、第３工程では、成長因子としてｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子、及びＢＭＰ４の存在下に培養する。その際、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９は、それらの全部を存在させることが好ましい。成長因子であるｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、ＦＧＦ１９、及びＢＭＰ４の培地中の濃度範囲としては、それぞれにおいて１０～５０ｎｇ／ｍＬであることが好ましく、１０～２５ｎｇ／ｍＬであることがより好ましい。また、培地は、例えば、およそ１日毎に新鮮なものに交換しつつ培養して、細胞を維持することが好ましい。これによれば、上記成長因子による所望の分化に向かわせる効果を更に高めることができる。第３工程は、トータルで１～６日間行うことが好ましく、２～５日間行うことがより好ましい。３～４日間行うことが更により好ましい。 The medium used in the above-mentioned third step is not particularly limited as long as it is a medium capable of maintaining cells heading for differentiation from pluripotent stem cells. For example, "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Junyaku Co., Ltd., which is a serum-free medium, similar to the medium preferably used in the latter half culture of the second step. Serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermo Scientific)) Serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific), serum-free supplement "Nonential aminoacid" (Nakalitesk Co., Ltd.), and the like are preferably exemplified. However, in the third step, bFGF is preferably used as a growth factor. Incubate in the presence of at least one growth factor selected from the group consisting of FGF3, FGF10, and FGF19, and BMP4, in which bFGF, FGF3, FGF10, and FGF19 may be present in all of them. The concentration range of the growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 in the medium is preferably 10 to 50 ng / mL, and more preferably 10 to 25 ng / mL, respectively. In addition, it is preferable that the medium is cultured while being replaced with a fresh medium approximately every day to maintain the cells, thereby further enhancing the effect of the growth factor toward the desired differentiation. The third step is preferably performed for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.
　上記の第４工程で用いる培地としては、多能性幹細胞から分化に向かう細胞を維持できる培地であればよく、特に限定されない。例えば、第３工程の培養に好ましく使用される培地と同様に、無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）に、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）を補充した培地などが好ましく例示される。ただし、第４工程では、成長因子としてｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子の存在下であって、ＢＭＰ４の非存在下に培養する。その際、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９は、それらの全部を存在させることが好ましい。成長因子であるｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９の培地中の濃度範囲としては、それぞれにおいて１０～５０ｎｇ／ｍＬであることが好ましく、２５ｎｇ／ｍＬであることがより好ましい。また、培地は、例えば、およそ１日毎に新鮮なものに交換しつつ培養して、細胞を維持することが好ましい。これによれば、上記成長因子による所望の分化に向かわせる効果を更に高めることができる。第４工程は、トータルで１～６日間行うことが好ましく、２～５日間行うことがより好ましい。３～４日間行うことが更により好ましい。 The medium used in the above-mentioned fourth step is not particularly limited as long as it is a medium capable of maintaining cells heading for differentiation from pluripotent stem cells. For example, "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Junyaku Co., Ltd., which is a serum-free medium, similar to the medium preferably used for the culture in the third step. ), Serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermo Scientific)) Preferred examples include a medium supplemented with the serum supplement "Gibco GlutaMAX" (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid" (Nakalitesk Co., Ltd.). However, in the fourth step, bFGF and FGF3 are used as growth factors. , FGF10, and FGF19, in the presence of at least one growth factor selected from the group, and in the absence of BMP4, in which bFGF, FGF3, FGF10, and FGF19 are all of them. The concentration range of the growth factors bFGF, FGF3, FGF10, and FGF19 in the medium is preferably 10 to 50 ng / mL, more preferably 25 ng / mL, respectively. Further, it is preferable that the medium is cultured while being replaced with a fresh medium approximately every day, for example, to maintain the cells. This further enhances the effect of the growth factors toward the desired differentiation. The fourth step is preferably performed for a total of 1 to 6 days, more preferably 2 to 5 days, and even more preferably 3 to 4 days.
　なお、第４工程において、ＢＭＰ４の非存在下の意味は、ＢＭＰ４が実質的に非存在であればよく、効果がないレベルの濃度で含まれていてもよい。 In the fourth step, the meaning in the absence of BMP4 may be that BMP4 is substantially non-existent and may be contained at a concentration at a level that has no effect.
　上記した第１工程～第４工程の一連の培養は、培養皿の底面等に付着させつつ培養する、接着培養によることが好ましい。これによれば、細胞を効率よく培養することができる。また、分化誘導を促進したり、逆に分化誘導を抑えたり、それらを調節するための培地交換等の操作が容易である。また、無血清培地で培養することが好ましい。これによれば、血清因子に起因して目的外の細胞に分化してしまうリスクを抑えることができる。 It is preferable that the series of cultures of the first step to the fourth step described above is an adhesive culture in which the culture is carried out while adhering to the bottom surface of the culture dish or the like. According to this, cells can be efficiently cultured. In addition, it is easy to perform operations such as promoting differentiation induction, suppressing differentiation induction, and exchanging a medium for controlling them. In addition, it is preferable to culture in a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors.
　ここで、一般に、多能性幹細胞から内耳細胞への分化を評価するには、内耳予定領域マーカーであるＰＡＸ２やＰＡＸ８の発現を指標にして評価することができる。すなわち、多能性幹細胞は、所定の培養を経ることにより内耳細胞への分化に向かうと、それにともなってＰＡＸ２やＰＡＸ８の発現が上昇する。また、分化度が進むにつれて、ＰＡＸ２やＰＡＸ８の発現は抑制傾向となる（参考文献１：Ealy M, Ellwanger DC, Kosaric N, Stapper AP, Heller S.「Single-cell analysis delineates a trajectory toward the human early otic lineage.」Proc Natl Acad Sci U S A. 2016 Jul 26;113(30):8508-13.;参考文献２：Koehler KR, Nie J, Longworth-Mills E, Liu XP, Lee J, Holt JR, Hashino E.「Generation of inner ear organoids containing functional hair cells from human pluripotent stem cells.」Nat Biotechnol. 2017 Jun;35(6):583-589.）。よって、ＰＡＸ２やＰＡＸ８の発現をタンパク発現レベルやｍＲＮＡ発現レベルで調べることにより、内耳細胞へ分化の程度を評価することができる。したがって、上記に説明した方法を経ることにより、多能性幹細胞を内耳細胞に向けて分化誘導すると、ＰＡＸ２やＰＡＸ８の発現をタンパク発現レベルやｍＲＮＡ発現レベルで調べたとき、少なくともその発現を検知することができるレベルに達した細胞集団となっている。 Here, in general, in order to evaluate the differentiation of pluripotent stem cells into inner ear cells, the expression of PAX2 and PAX8, which are markers for the planned inner ear region, can be used as an index for evaluation. That is, when pluripotent stem cells undergo differentiation into inner ear cells through a predetermined culture, the expression of PAX2 and PAX8 increases accordingly. In addition, as the degree of differentiation progresses, the expression of PAX2 and PAX8 tends to be suppressed (Reference 1: Easy M, Ellwanger DC, Kosaric N, Stamper AP, Heller S. “Single-cell analysis delineates a tradition toward the human early”. otic lineage. ”Proc Natl Acad Sci U S A. 2016 Jul 26; 113 (30): 8508-13 .; Reference 2: Koehler KR, Nie J, Longworth-Mills E, Liu XP, Lee J, Holt JR, Hashino E. "Generation of inner ear organics containing functional hair cells from human pluripotent stem cells." Nat Biotechnol. 2017 Jun; 35 (6): 583-589.). Therefore, by examining the expression of PAX2 and PAX8 at the protein expression level and the mRNA expression level, the degree of differentiation into inner ear cells can be evaluated. Therefore, when pluripotent stem cells are induced to differentiate toward inner ear cells by the method described above, at least the expression of PAX2 and PAX8 is detected at the protein expression level and the mRNA expression level. It is a cell population that has reached a level where it can be used.
　本発明においては、多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団に対して、特定の処理を施すことにより、内耳細胞への分化誘導が促進されるよう、そのように賦活化した内耳前駆細胞を得る。 In the present invention, a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is thus activated so as to promote differentiation induction into inner ear cells by subjecting the cell population to a specific treatment. Obtain the differentiated inner ear progenitor cells.
　具体的に、例えば、図１では、本発明による内耳前駆細胞の製造方法の一実施形態をフロー図により説明している。 Specifically, for example, FIG. 1 illustrates an embodiment of the method for producing inner ear progenitor cells according to the present invention using a flow chart.
　図１に示されるように、本発明においては、多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団について、細胞剥離の処理を施す。多能性幹細胞の培養により内耳細胞へ分化させる過程では、増殖した細胞は細胞同士が接着した状態となるところ、細胞の個々を分離したうえで次の培養に移すと、単細胞化もしくは２～１０個の少数の細胞塊などに解離して、細胞の個々が培地や培養器に直接に暴露して影響を受けやすくなる。このとき、細胞が未分化であったり、細胞の生育活性が弱かったりすると、生き残れずに死滅する。これにより、内耳細胞への分化にとって不必要な細胞が淘汰されて、ＰＡＸ２及び／又はＰＡＸ８の発現レベルを確実に維持することができる。細胞剥離の処理は、細胞同士の接着を解離して、細胞の個々を分離することができる手段であればよく、特に制限はないが、例えば、トリプシンや細胞剥離用酵素製剤であるアクターゼ（商品名「Ａｃｃｕｔａｓｅ」、ナカライテスク株式会社）、などによる酵素処理が挙げられる。酵素処理後には、液体培地中でのピペッティングなどで解離を確実にすることができる。また、所定の孔径を有するメッシュを通すことにより、残存する細胞塊を除去してもよい。そのような目的で用いられるメッシュとしては、孔径１～１０００μｍなどの範囲で段階的に所定の孔径を有するナイロンメッシュを備えたセルストレーナーが市販されているので、そのような市販のセルストレーナーのなかから適宜適当な孔径のものを選択して利用してもよい。 As shown in FIG. 1, in the present invention, a cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell detachment treatment. In the process of differentiating into inner ear cells by culturing pluripotent stem cells, the proliferated cells are in a state where the cells adhere to each other. It dissociates into a small number of cell clusters, and individual cells are directly exposed to the medium or incubator and become susceptible to the effects. At this time, if the cells are undifferentiated or the growth activity of the cells is weak, they cannot survive and die. As a result, cells unnecessary for differentiation into inner ear cells can be culled out, and the expression level of PAX2 and / or PAX8 can be reliably maintained. The cell detachment treatment may be any means as long as it can dissociate the adhesion between cells and separate individual cells, and is not particularly limited. For example, trypsin or actase (commercial product) which is an enzyme preparation for cell detachment. Enzyme treatment with the name "Accutase", Nacalai Tesque Co., Ltd.) and the like can be mentioned. After the enzyme treatment, dissociation can be ensured by pipetting or the like in a liquid medium. Further, the remaining cell mass may be removed by passing through a mesh having a predetermined pore size. As a mesh used for such a purpose, a cell strainer provided with a nylon mesh having a predetermined hole diameter in a stepwise range of 1 to 1000 μm is commercially available, and among such commercially available cell strainers. You may select and use the one having an appropriate hole diameter from the above.
　図１に示されるように、本発明においては、上記細胞剥離の処理を施した細胞や細胞集団を、更に、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養する。 As shown in FIG. 1, in the present invention, the cells or cell populations that have been subjected to the cell detachment treatment are further cultured in a medium containing retinoic acid and an inducer of the first Wnt / β-catenin pathway. ..
　本工程の培養に用いる培地としては、無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）などが好ましく例示される。培地には、適宜、補充栄養成分を補充してもよい。例えば、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）等が挙げられる。 As the medium used for culturing in this step, serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) and the like are preferably exemplified. .. The medium may be supplemented with supplemental nutritional components as appropriate. For example, serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermo Fisher)) Examples include the supplement "Gibco GlutaMAX" (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid" (Nakalitesk Co., Ltd.).
　ただし、本工程では、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養する。後述の実施例で示されるように、レチノイン酸と第１のＷｎｔ／βカテニン経路の誘導剤を併用して培地に含有せしめて、その培地で培養すると、ＰＡＸ２及び／又はＰＡＸ８の発現レベルが高められる。レチノイン酸としては、オールトランス型レチノイン酸等が挙げられる。また、第１のＷｎｔ／βカテニン経路の誘導剤としては、ＣＨＩＲ９９０２１（別名：６－（（２－（（４－（２，４－Ｄｉｃｈｌｏｒｏｐｈｅｎｙｌ）－５－（４－Ｍｅｔｈｙｌ－１Ｈ－ｉｍｉｄａｚｏｌ－２－ｙｌ）ｐｙｒｉｍｉｄｉｎ－２－ｙｌ）ａｍｉｎｏ）ｅｔｈｙｌ）ａｍｉｎｏ）ｎｉｃｏｔｉｎｏｎｉｔｒｉｌｅ）、ＢＩＯ（別名：６－ｂｒｏ－ｍｏｉｎｄｉｒｕｂｉｎ　３’－ｏｘｉｍｅ）、ＬｉＣｌ（塩化リチウム）、Ｗｎｔ３ａ、Ｒ－ｓｐｏｎｄｉｎ１等が挙げられる。これらのうち、ＣＨＩＲ９９０２１、ＢＩＯ、ＬｉＣｌ（塩化リチウム）などがより好ましく例示される。 However, in this step, the cells are cultured in a medium containing retinoic acid and an inducer of the first Wnt / β-catenin pathway. As shown in the examples below, when retinoic acid and an inducer of the first Wnt / β-catenin pathway are contained in a medium in combination and cultured in the medium, the expression level of PAX2 and / or PAX8 is increased. Be done. Examples of retinoin acid include all-trans type retinoin acid. Further, as an inducer of the first Wnt / β-catenin pathway, CHIR99021 (also known as: 6-((2-((4- (2,4-Dichrimidine) -5- (4-Methyl-1H-imidazol-2)) -Yl) pyrimidine-2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride), Wnt3a, R-spondin1 and the like. Of these, CHIR99021, BIO, LiCl (lithium chloride) and the like are more preferably exemplified.
　本工程での培地中でのレチノイン酸の濃度は、その下限値としては、０．１μＭ以上とすることが好ましく、０．３μＭ以上とすることがより好ましく、０．５μＭ以上とすることが更により好ましい。その上限値としては、５μＭ以下であることが好ましく３μＭ以下であることがより好ましく、１μＭ以下であることが更により好ましい。なお、上記した無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」には、レチノイン酸の前駆物質としてビタミンＡが配合されているので、そのビタミンＡの影響を排除して培地中でのレチノイン酸の濃度条件を確実にするには、ビタミンＡ非含有の無血清サプリメント「Ｂ２７－ＶＡ」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ，　ｍｉｎｕｓ　ｖｉｔａｍｉｎ　Ａ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）などを用いるとよい。 The lower limit of the concentration of retinoic acid in the medium in this step is preferably 0.1 μM or more, more preferably 0.3 μM or more, and further preferably 0.5 μM or more. More preferred. The upper limit is preferably 5 μM or less, more preferably 3 μM or less, and even more preferably 1 μM or less. Since the above-mentioned serum-free supplement "B27" (trade name "Gibco B-27 Supplement" contains vitamin A as a precursor of retinoic acid, the influence of the vitamin A is eliminated and it is in the medium. To ensure the concentration condition of retinoic acid, use a vitamin A-free serum-free supplement "B27-VA" (trade name "Gibco B-27 Supplement, minus vitamin A", Thermo Fisher Scientific). good.
　本工程での培地中での第１のＷｎｔ／βカテニン経路の誘導剤の濃度は、その下限値としては、０．１μＭ以上とすることが好ましく、その上限値としては、３０ｍＭ以下であることが好ましい。また、第１のＷｎｔ／βカテニン経路の誘導剤が、特にＣＨＩＲ９９０２１である場合、その下限値としては、０．１μＭ以上とすることが好ましく、０．５μＭ以上とすることがより好ましく、１μＭ以上とすることが更により好ましい。その上限値としては、１０μＭ以下であることが好ましく、５μＭ以下であることがより好ましく、３μＭ以下であることが更により好ましい。また、第１のＷｎｔ／βカテニン経路の誘導剤が、特にＢＩＯである場合、その下限値としては、０．１μＭ以上とすることが好ましく、０．５μＭ以上とすることがより好ましく、１μＭ以上とすることが更により好ましい。その上限値としては、５μＭ以下であることが好ましく、３μＭ以下であることがより好ましく、１μＭ以下であることが更により好ましい。また、第１のＷｎｔ／βカテニン経路の誘導剤が、特にＬｉＣｌである場合、その下限値としては、１ｍＭ以上とすることが好ましく、３ｍＭ以上とすることがより好ましく、５ｍＭ以上とすることが更により好ましい。その上限値としては、３０ｍＭ以下であることが好ましく、２０ｍＭ以下であることがより好ましく、１０ｍＭ以下であることが更により好ましい。 The concentration of the inducer of the first Wnt / β-catenin pathway in the medium in this step is preferably 0.1 μM or more as the lower limit value, and 30 mM or less as the upper limit value. Is preferable. Further, when the inducer of the first Wnt / β-catenin pathway is CHIR99021 in particular, the lower limit thereof is preferably 0.1 μM or more, more preferably 0.5 μM or more, and more preferably 1 μM or more. Is even more preferable. The upper limit is preferably 10 μM or less, more preferably 5 μM or less, and even more preferably 3 μM or less. Further, when the inducer of the first Wnt / β-catenin pathway is BIO in particular, the lower limit thereof is preferably 0.1 μM or more, more preferably 0.5 μM or more, and more preferably 1 μM or more. Is even more preferable. The upper limit is preferably 5 μM or less, more preferably 3 μM or less, and even more preferably 1 μM or less. Further, when the inducer of the first Wnt / β-catenin pathway is LiCl in particular, the lower limit thereof is preferably 1 mM or more, more preferably 3 mM or more, and more preferably 5 mM or more. Even more preferable. The upper limit is preferably 30 mM or less, more preferably 20 mM or less, and even more preferably 10 mM or less.
　本工程においては、培地は、例えば、およそ１日～２日毎に新鮮なものに交換しつつ培養して、細胞を維持することが好ましい。これによれば、上記成長因子による所望の分化に向かわせる効果を更に高めることができる。トータルで１～６日間行うことが好ましく、２～５日間行うことがより好ましい。３～４日間行うことが更により好ましい。 In this step, it is preferable that the medium is cultured while being replaced with a fresh medium approximately every 1 to 2 days to maintain the cells. According to this, the effect of the growth factor toward the desired differentiation can be further enhanced. It is preferably performed for 1 to 6 days in total, and more preferably 2 to 5 days. It is even more preferable to carry out for 3 to 4 days.
　上記したレチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含む培地での培養は、培養器の底面等に接着させつつ培養する、接着培養によることが好ましい。これによれば、細胞剥離処理後の培養を効率よく行うことができる。また、培地交換等の操作が容易である。特に、コーティング剤でコートした培養皿を用いて培養することが好ましい。これによれば、細胞剥離処理後の細胞や細胞集団を、更に効率よく分化に向かわせることができる。コーティング剤は、通常、培養器への接着や分化を促す目的で使用されるものを用いればよく、そのような目的のコーティング剤は、特に限定されないが、例えば、ｐｏｌｙ－Ｏ－ｆｉｂｒｏｎｅｃｔｉｎ等が好ましく例示される。また、無血清培地で培養することが好ましい。これによれば、血清因子に起因して目的外の細胞に分化してしまうリスクを抑えることができる。また、幹細胞性の維持のためには、低酸素条件（例えばＯ２４～１０％、より好ましくは４～６％、更により好ましくは４％、ＣＯ２５％）において培養してもよいが、通常酸素条件（例えばＯ２５～２０％、より好ましくは１０～２０％、更により好ましくは２０％、ＣＯ２５％）において培養してもよい。 The culture in the medium containing the above-mentioned retinoic acid and the inducer of the first Wnt / β-catenin pathway is preferably an adhesive culture in which the culture is carried out while adhering to the bottom surface of the incubator or the like. According to this, the culture after the cell exfoliation treatment can be efficiently performed. In addition, operations such as medium exchange are easy. In particular, it is preferable to culture using a culture dish coated with a coating agent. According to this, the cells and cell populations after the cell exfoliation treatment can be more efficiently directed to differentiation. As the coating agent, one usually used for the purpose of promoting adhesion or differentiation to the incubator may be used, and the coating agent for such a purpose is not particularly limited, but for example, poly-O-fibronectin or the like is preferable. Illustrated. In addition, it is preferable to culture in a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors. Moreover, for the maintenance of stem cell properties, hypoxic conditions (e.g., O 2 4 ~ 10%, more preferably 4-6%, even more preferably 4%, CO 2 5%) may but be cultured in , normoxia condition (e.g. O 2 5% to 20% and more preferably 10-20%, even more preferably 20% CO 2 5%) may be cultured in.
　上記したレチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含む培地での培養は、成長因子としてＩＧＦ－１、ｂＦＧＦ、及びＥＧＦからなる群から選ばれた１種又は２種以上の存在下に行うことが好ましい。これによれば、内耳前駆細胞への分化をより確実にすることができる。また、成長因子であるｂＦＧＦ及びＥＧＦ及びＩＧＦ－１のうち、それらの全部を存在させることが更により好ましい。これらの成長因子の培地中の濃度範囲としては、ｂＦＧＦ及びＥＧＦのそれぞれにおいて１０～３０ｎｇ／ｍＬであることが好ましい。ＩＧＦ－１において１０～５０ｎｇ／ｍＬであることが好ましい。 Culturing in a medium containing the above-mentioned retinoic acid and an inducer of the first Wnt / β-catenin pathway has one or more selected from the group consisting of IGF-1, bFGF, and EGF as growth factors. It is preferable to do it below. According to this, it is possible to more reliably differentiate into inner ear progenitor cells. Further, it is even more preferable to have all of the growth factors bFGF, EGF and IGF-1 present. The concentration range of these growth factors in the medium is preferably 10 to 30 ng / mL for each of bFGF and EGF. In IGF-1, it is preferably 10 to 50 ng / mL.
　以上のようにして得られた内耳前駆細胞は、上記したレチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含む培地での培養前に比べて、ＰＡＸ２及び／又はＰＡＸ８の発現レベルが向上している。例えば、細胞集団を集めてｍＲＮＡ発現解析を行ったとき、その発現レベルは、培養前に比べて２～１００倍に増加していることが典型的であり、３～５０倍に増加していることがより典型的であり、５～２０倍に増加していることが更により典型的である。 The inner ear progenitor cells obtained as described above have improved expression levels of PAX2 and / or PAX8 as compared with those before culturing in the medium containing the above-mentioned retinoic acid and the inducer of the first Wnt / β-catenin pathway. is doing. For example, when a cell population is collected and mRNA expression is analyzed, the expression level is typically increased 2 to 100 times as compared with that before culturing, and is increased 3 to 50 times. Is more typical, and it is even more typical that the increase is 5 to 20 times.
　上記に説明したＰＡＸ２及び／又はＰＡＸ８の発現レベルが向上した内耳前駆細胞によれば、後述する実施例で示されるように、内耳有毛細胞等、更に分化段階の進んだ内耳細胞を効率的に得ることができる。以下、内耳前駆細胞を内耳細胞への分化に向かわせるための典型的な誘導方法に沿って、本発明を更に詳細に説明する。ただし、本発明にかかる方法は、以下に説明する特定の培養条件等に限定されるものではない。 According to the inner ear progenitor cells having improved expression levels of PAX2 and / or PAX8 described above, as shown in Examples described later, inner ear cells having further advanced differentiation stages such as inner ear hair cells can be efficiently used. Obtainable. Hereinafter, the present invention will be described in more detail along with a typical induction method for directing inner ear progenitor cells to differentiation into inner ear cells. However, the method according to the present invention is not limited to the specific culture conditions and the like described below.
　［２］内耳前駆細胞から内耳有毛細胞への分化誘導
　上記［１］で説明した方法により、ＰＡＸ２及び／又はＰＡＸ８の発現レベルを向上させた内耳前駆細胞を得、更に分化誘導を進めて内耳有毛細胞を得ることができる。 [2] Induction of differentiation of inner ear progenitor cells into inner ear hair cells By the method described in [1] above, inner ear progenitor cells with improved expression levels of PAX2 and / or PAX8 were obtained, and differentiation induction was further promoted in the inner ear. Hair cells can be obtained.
　具体的に、例えば、図２では、本発明による内耳有毛細胞の製造方法の一実施形態をフロー図により説明している。 Specifically, for example, FIG. 2 illustrates an embodiment of the method for producing inner ear hair cells according to the present invention using a flow chart.
　図２に示されるように、本発明においては、ＰＡＸ２及び／又はＰＡＸ８の発現レベルを向上させた内耳前駆細胞を、以下の工程（ｉ）及び（ｉｉ）に処する。
　（ｉ）前記内耳前駆細胞を含む細胞集団を浮遊培養する工程
　（ｉｉ）工程（ｉ）で得られた細胞集団を接着培養する工程 As shown in FIG. 2, in the present invention, inner ear progenitor cells having improved expression levels of PAX2 and / or PAX8 are subjected to the following steps (i) and (ii).
(I) Step of suspension-culturing the cell population containing the inner ear progenitor cells (ii) Step of adhesion-culturing the cell population obtained in step (i)
　浮遊培養のためには、内耳前駆細胞は、上記した細胞剥離の処理と同様にして個々の細胞に分離し、適当な液体培地に懸濁させたうえ、非接着状態で培養することができる浮遊培養用の培養器に入れて培養することが好ましい。非接着培養用の培養器としては、具体的には、例えば、細胞培養用プラスティックディッシュなどを用いることができる。 For suspension culture, the inner ear precursor cells can be separated into individual cells in the same manner as in the cell detachment treatment described above, suspended in a suitable liquid medium, and then cultured in a non-adherent state. It is preferable to incubate in an incubator for culturing. As the incubator for non-adhesive culture, specifically, for example, a plastic dish for cell culture can be used.
　浮遊培養に用いる培地は、特に限定されず、上述した無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）などが好ましく例示される。培地には、適宜、補充栄養成分を補充してもよい。例えば、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）等が挙げられる。 The medium used for the suspension culture is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) and the like can be used. It is preferably exemplified. The medium may be supplemented with supplemental nutritional components as appropriate. For example, serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermo Fisher)) Examples include the supplement "Gibco GlutaMAX" (Thermo Fisher Scientific) and the serum-free supplement "Nonential aminoacid" (Nakalitesk Co., Ltd.).
　一般に、内耳有毛細胞への分化誘導に寄与する成長因子としては、ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１、ヘパリンなどが知られている。よって、これら成長因子の１種又は２種以上を培地に含有せしめて培養することが好ましい。この場合、これら成長因子の培地中の濃度範囲としては、ｂＦＧＦでは、１０～５０ｎｇ／ｍＬであることが好ましく、１０～３０ｎｇ／ｍＬであることがより好ましい。ＥＧＦでは、１０～５０ｎｇ／ｍＬであることが好ましく、２０～３０ｎｇ／ｍＬであることがより好ましい。ＩＧＦ－１では、１０～１００ｎｇ／ｍＬであることが好ましく、２０～５０ｎｇ／ｍＬであることがより好ましい。ヘパリンでは、１～１００ｎｇ／ｍＬであることが好ましく、１０～５０ｎｇ／ｍＬであることがより好ましい。この浮遊培養は、トータル２～６日間行うことが好ましく、３～５日間行うことがより好ましく、４日間行うことが更により好ましい。 Generally, bFGF, EGF, IGF-1, heparin and the like are known as growth factors that contribute to the induction of differentiation into inner ear hair cells. Therefore, it is preferable to cultivate one or more of these growth factors in a medium. In this case, the concentration range of these growth factors in the medium is preferably 10 to 50 ng / mL, more preferably 10 to 30 ng / mL for bFGF. For EGF, it is preferably 10 to 50 ng / mL, more preferably 20 to 30 ng / mL. For IGF-1, it is preferably 10 to 100 ng / mL, more preferably 20 to 50 ng / mL. For heparin, it is preferably 1 to 100 ng / mL, more preferably 10 to 50 ng / mL. This suspension culture is preferably carried out for a total of 2 to 6 days, more preferably 3 to 5 days, and even more preferably 4 days.
　また、その浮遊培養においては、遠心分離等により形成されたスフェア（細胞塊）を壊さないように回収して、新鮮な培地を入れ替えたり、新鮮な培地を追加したりして、更にその浮遊培養を続けてもよい。この場合、追加的な浮遊培養を２～６日間行うことが好ましく、３～５日間行うことがより好ましく、４日間行うことが更により好ましい。追加する培地は、特に限定されず、上述した無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）などが好ましく例示される。培地には、適宜、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）等の補充栄養成分を補充してもよい。ただし、細胞や細胞集団に刺激を与えない観点からは、成長因子以外は、最初の浮遊培養で用いた培地と同一のものを用いるのが好ましい。追加する新鮮培地に含有せしめる成長因子としては、上記したもののうちで、ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１、ヘパリン等から選ばれた１種又は２種以上であればよい。それぞれの好ましい濃度は、１０～３０ｎｇ／ｍＬ、１０～３０ｎｇ／ｍＬ、２０～５０ｎｇ／ｍＬ、１０～５０ｎｇ／ｍＬなどである。 Further, in the suspension culture, the spheres (cell clumps) formed by centrifugation or the like are collected so as not to be destroyed, and a fresh medium is replaced or a fresh medium is added, and the suspension culture is further performed. May continue. In this case, the additional suspension culture is preferably carried out for 2 to 6 days, more preferably 3 to 5 days, and even more preferably 4 days. The medium to be added is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) is preferably exemplified. Will be done. Serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (TherciS)) may be used as the medium. ), Serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific), Serum-free supplement "Nonessential aminoacid" (Nakalitesk Co., Ltd.), etc. may be supplemented with supplemental nutritional components, but stimulate cells and cell populations. From the viewpoint of not giving, it is preferable to use the same medium used in the first suspension culture except for the growth factor. Among the above-mentioned growth factors, bFGF and EGF are used as the growth factors to be contained in the fresh medium to be added. , IGF-1, heparin, etc., may be one or more. Preferred concentrations of each are 10 to 30 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, and 10 to 50 ng /. For example, mL.
　浮遊培養においては、上記した成長因子のほか、成長因子として、更に、第２のＷｎｔ／βカテニン経路の誘導剤の存在下で行うことがより好ましい。これによれば、後述する実施例に示されるように、内耳有毛細胞への分化誘導の効率が更に向上する。第２のＷｎｔ／βカテニン経路の誘導剤としては、Ｒ－ｓｐｏｎｄｉｎ１、ＣＨＩＲ９９０２１（別名：６－（（２－（（４－（２，４－Ｄｉｃｈｌｏｒｏｐｈｅｎｙｌ）－５－（４－Ｍｅｔｈｙｌ－１Ｈ－ｉｍｉｄａｚｏｌ－２－ｙｌ）ｐｙｒｉｍｉｄｉｎ－２－ｙｌ）ａｍｉｎｏ）ｅｔｈｙｌ）ａｍｉｎｏ）ｎｉｃｏｔｉｎｏｎｉｔｒｉｌｅ）、ＢＩＯ（別名：６－ｂｒｏ－ｍｏｉｎｄｉｒｕｂｉｎ　３’－ｏｘｉｍｅ）、ＬｉＣｌ（塩化リチウム）、Ｗｎｔ３ａ等が挙げられる。これらのうち、Ｒ－ｓｐｏｎｄｉｎ１、ＣＨＩＲ９９０２１、Ｗｎｔ３ａがより好ましく例示される。浮遊培養の培地中での第２のＷｎｔ／βカテニン経路の誘導剤の濃度は、その下限値としては、１０ｎｇ／ｍＬ以上とすることが好ましく、その上限値としては、１０００ｎｇ／ｍＬ以下であることが好ましい。また、第２のＷｎｔ／βカテニン経路の誘導剤が、特にＲ－ｓｐｏｎｄｉｎ１である場合、その下限値としては、１０ｎｇ／ｍＬ以上とすることが好ましく、５０ｎｇ／ｍＬ以上とすることがより好ましく、１００ｎｇ／ｍＬ以上とすることが更により好ましい。その上限値としては、１０００ｎｇ／ｍＬ以下であることが好ましく、５００ｎｇ／ｍＬ以下であることがより好ましく、２００ｎｇ／ｍＬ以下であることが更により好ましい。また、第２のＷｎｔ／βカテニン経路の誘導剤が、特にＣＨＩＲ９９０２１である場合、その下限値としては、０．１μＭ以上とすることが好ましく、０．５μＭ以上とすることがより好ましく、１μＭ以上とすることが更により好ましい。その上限値としては、１０μＭ以下であることが好ましく、５μＭ以下であることがより好ましく、３μＭ以下であることが更により好ましい。また、第２のＷｎｔ／βカテニン経路の誘導剤が、特にＷｎｔ３ａである場合、その下限値としては、１ｎｇ／ｍＬ以上とすることが好ましく、１０ｎｇ／ｍＬ以上とすることがより好ましく、２０ｎｇ／ｍＬ以上とすることが更により好ましい。その上限値としては、１００ｎｇ／ｍＬ以下であることが好ましく、５０ｎｇ／ｍＬ以下であることがより好ましく、２０ｎｇ／ｍＬ以下であることが更により好ましい。 In the suspension culture, in addition to the above-mentioned growth factors, it is more preferable to carry out the culture in the presence of a second Wnt / β-catenin pathway inducer as a growth factor. According to this, as shown in Examples described later, the efficiency of inducing differentiation into inner ear hair cells is further improved. Examples of the second Wnt / β-catenin pathway inducer include R-spondin1, CHIR99021 (also known as 6-((2-((4- (2,4-Dichlophoenyl) -5- (4-Methyl-1H-imidazole)). -2-yl) pyrimidin-2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride), Wnt3a and the like. Of these, R-spondin1, CHIR99021, and Wnt3a are more preferably exemplified. The concentration of the inducer of the second Wnt / β-catenin pathway in the medium of the suspension culture is preferably 10 ng / mL or more as the lower limit value, and 1000 ng / mL or less as the upper limit value. Is preferable. Further, when the second Wnt / β-catenin pathway inducer is particularly R-spondin1, the lower limit thereof is preferably 10 ng / mL or more, more preferably 50 ng / mL or more. Even more preferably, it is 100 ng / mL or more. The upper limit is preferably 1000 ng / mL or less, more preferably 500 ng / mL or less, and even more preferably 200 ng / mL or less. Further, when the inducer of the second Wnt / β-catenin pathway is CHIR99021 in particular, the lower limit thereof is preferably 0.1 μM or more, more preferably 0.5 μM or more, and more preferably 1 μM or more. Is even more preferable. The upper limit is preferably 10 μM or less, more preferably 5 μM or less, and even more preferably 3 μM or less. Further, when the inducer of the second Wnt / β-catenin pathway is Wnt3a in particular, the lower limit thereof is preferably 1 ng / mL or more, more preferably 10 ng / mL or more, and more preferably 20 ng / mL. It is even more preferable to use mL or more. The upper limit is preferably 100 ng / mL or less, more preferably 50 ng / mL or less, and even more preferably 20 ng / mL or less.
　図２に示されるように、本発明においては、上記浮遊培養した細胞や細胞集団を、更に接着培養する。 As shown in FIG. 2, in the present invention, the above-mentioned suspension-cultured cells and cell populations are further adherently cultured.
　接着培養のためには、上記浮遊培養した細胞や細胞集団を、遠心分離等により形成されたスフェア（細胞塊）を壊さないように回収して、接着培養を行う。接着培養のための培養器としては、上述したように、特に、コーティング剤でコートした培養皿を用いて培養することが好ましい。これによれば、浮遊培養後の細胞や細胞集団を、更に効率よく分化に向かわせることができる。コーティング剤は、通常、培養器への接着や分化を促す目的で使用されるものを用いればよく、そのような目的のコーティング剤は、特に限定されないが、例えば、ｐｏｌｙ－Ｏ－ｆｉｂｒｏｎｅｃｔｉｎ等が好ましく例示される。また、無血清培地で培養することが好ましい。これによれば、血清因子に起因して目的外の細胞に分化してしまうリスクを抑えることができる。接着培養の培地は、浮遊培養で用いた培地と同一のものを用いてもよく、他の培地を用いてもよい。具体的には、例えば、上述した無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）などが好ましく例示される。培地には、適宜、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）等の補充栄養成分を補充してもよい。ただし、上述したように、一般に、内耳有毛細胞への分化誘導に寄与する成長因子としては、ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１、ヘパリンなどが知られている。よって、これら成長因子の１種又は２種以上を培地に含有せしめて培養することが好ましい。この場合、これら成長因子の培地中の濃度範囲としては、上記した浮遊培養の際と同様である。この接着培養は、トータル７～２１日間行うことが好ましく、１０～１４日間行うことがより好ましい。 For adhesive culture, the above-mentioned suspension-cultured cells and cell populations are collected so as not to destroy the spheres (cell clumps) formed by centrifugation or the like, and adhesive culture is performed. As the incubator for adhesive culture, as described above, it is particularly preferable to culture using a culture dish coated with a coating agent. According to this, cells and cell populations after suspension culture can be more efficiently directed to differentiation. As the coating agent, one usually used for the purpose of promoting adhesion or differentiation to the incubator may be used, and the coating agent for such a purpose is not particularly limited, but for example, poly-O-fibronectin or the like is preferable. Illustrated. In addition, it is preferable to culture in a serum-free medium. According to this, it is possible to suppress the risk of differentiating into unintended cells due to serum factors. As the medium for the adhesive culture, the same medium as that used for the suspension culture may be used, or another medium may be used. Specifically, for example, the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Wako Pure Chemical Industries, Ltd.) is preferably exemplified. Serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (Thermoscience), as appropriate for the medium. ), Serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific), serum-free supplement "Nonessential aminoacid" (Nakalitesk Co., Ltd.), etc. may be supplemented. However, as described above, in general, it may be supplemented. As growth factors that contribute to the induction of differentiation into inner ear hair cells, bFGF, EGF, IGF-1, heparin and the like are known. Therefore, one or more of these growth factors are contained in the medium. Cultivation is preferable. In this case, the concentration range of these growth factors in the medium is the same as in the case of the above-mentioned suspension culture. This adhesion culture is preferably carried out for a total of 7 to 21 days, and 10 to 14 days. It is more preferable to do it for a day.
　また、その接着培養においては、新鮮な培地を入れ替えたり、新鮮な培地を追加したりして、更にその接着培養を続けてもよい。この場合、接着培養開始から１～４日間、好ましくは２～３日間、より好ましくは２日間培養を行い、その後、追加的な接着培養を６～１７日間行うことが好ましく、７～１４日間行うことがより好ましく、８～１２日間行うことが更により好ましい。追加する培地は、特に限定されず、上述した無血清培地である「ＤＭＥＭ／Ｆ１２」（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）などが好ましく例示される。培地には、適宜、無血清サプリメント「Ｂ２７」（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎ２」（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）、無血清サプリメント「Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ」（ナカライテスク株式会社）等の補充栄養成分を補充してもよい。ただし、細胞や細胞集団に刺激を与えない観点からは、成長因子以外は、最初の接着培養で用いた培地と同一のものを用いるのが好ましい。追加する新鮮培地に含有せしめる成長因子としては、上記したもののうちで、ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１、ヘパリン等から選ばれた１種又は２種以上であればよい。それぞれの好ましい濃度は、１０～３０ｎｇ／ｍＬ、１０～３０ｎｇ／ｍＬ、２０～５０ｎｇ／ｍＬ、１０～５０ｎｇ／ｍＬなどである。 Further, in the adhesive culture, a fresh medium may be replaced or a fresh medium may be added to continue the adhesive culture. In this case, the culture is preferably carried out for 1 to 4 days, preferably 2 to 3 days, more preferably 2 days from the start of the adhesive culture, and then additional adhesive culture is carried out for 6 to 17 days, preferably 7 to 14 days. It is more preferable, and it is even more preferable to carry out for 8 to 12 days. The medium to be added is not particularly limited, and the above-mentioned serum-free medium "DMEM / F12" (trade name "D-MEM / Ham's F-12", Fujifilm Wako Pure Chemical Industries, Ltd.) is preferably exemplified. Will be done. Serum-free supplement "B27" (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific), serum-free supplement "N2" (trade name "Gibco N-2 Supplement" (TherciS)) may be used as the medium. ), Serum-free supplement "Gibco GlutaMAX" (Thermo Fisher Scientific), Serum-free supplement "Nonessential aminoacid" (Nakalitesk Co., Ltd.), etc. may be supplemented with supplemental nutritional components, but stimulate cells and cell populations. From the viewpoint of not giving, it is preferable to use the same medium used in the first adhesive culture except for the growth factor. Among the above-mentioned growth factors, bFGF and EGF are used as the growth factors to be contained in the fresh medium to be added. , IGF-1, heparin, etc., may be one or more. Preferred concentrations of each are 10 to 30 ng / mL, 10 to 30 ng / mL, 20 to 50 ng / mL, and 10 to 50 ng /. For example, mL.
　接着培養においては、上記した成長因子のほか、成長因子として、更に、第３のＷｎｔ／βカテニン経路の誘導剤及び抗Ｆｒｉｚｚｌｅｄ剤の存在下で行うことがより好ましい。これによれば、後述する実施例に示されるように、内耳有毛細胞への分化誘導の効率が更に向上する。第３のＷｎｔ／βカテニン経路の誘導剤としては、Ｗｎｔ３ａ、Ｒ－ｓｐｏｎｄｉｎ１、ＣＨＩＲ９９０２１（別名：６－（（２－（（４－（２，４－Ｄｉｃｈｌｏｒｏｐｈｅｎｙｌ）－５－（４－Ｍｅｔｈｙｌ－１Ｈ－ｉｍｉｄａｚｏｌ－２－ｙｌ）ｐｙｒｉｍｉｄｉｎ－２－ｙｌ）ａｍｉｎｏ）ｅｔｈｙｌ）ａｍｉｎｏ）ｎｉｃｏｔｉｎｏｎｉｔｒｉｌｅ）、ＢＩＯ（別名：６－ｂｒｏ－ｍｏｉｎｄｉｒｕｂｉｎ　３’－ｏｘｉｍｅ）、ＬｉＣｌ（塩化リチウム）等が挙げられる。これらのうち、Ｗｎｔ３ａ、Ｒ－ｓｐｏｎｄｉｎ１がより好ましく例示され、Ｗｎｔ３ａ及びＲ－ｓｐｏｎｄｉｎ１の両方の存在下に行うことが好ましい。また、抗Ｆｒｉｚｚｌｅｄ剤としては、Ｆｒｉｚｚｌｅｄ受容体の種類に選択的な競合剤として、ヒトＦｒｉｚｚｌｅｄ受容体の細胞外ドメイン断片及びヒトＦｃドメインからなる融合タンパク質などが知られているので、そのようなＦｒｉｚｚｌｅｄ受容体の擬似分子を用いることができる。 In the adhesive culture, in addition to the above-mentioned growth factors, it is more preferable to carry out in the presence of a third Wnt / β-catenin pathway inducer and an anti-Frizzled agent as growth factors. According to this, as shown in Examples described later, the efficiency of inducing differentiation into inner ear hair cells is further improved. Examples of the inducer of the third Wnt / β-catenin pathway include Wnt3a, R-spondin1, CHIR99021 (also known as 6- -Imidazole-2-yl) pyrimidin-2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride) and the like. Of these, Wnt3a and R-spondin1 are more preferably exemplified, and it is preferable to carry out in the presence of both Wnt3a and R-spondin1. Further, as an anti-Frizzled agent, as a competitive agent selective for the type of Frizzled receptor, an extracellular domain fragment of a human Frizzled receptor, a fusion protein composed of a human Fc domain, and the like are known, and thus such Frizzled. Pseudomolecules of the receptor can be used.
　本発明の限定されない任意の態様においては、抗Ｆｒｉｚｚｌｅｄ剤として、Ｆｒｉｚｚｌｅｄ１０の擬似分子が好ましく例示される。これによれば、後述する実施例に示されるように、Ｗｎｔ／βカテニン経路の誘導剤とともに使用することで、内耳有毛細胞の一形態である蝸牛有毛細胞への分化誘導を促すことができる。例えば、Ｆｒｉｚｚｌｅｄ１０の擬似分子として、商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　Ｆｒｉｚｚｌｅｄ－１０　Ｆｃ　Ｃｈｉｍｅｒａ　Ｐｒｏｔｅｉｎ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）なども市販されているので、そのような市販品を用いてもよい。 In any non-limiting aspect of the present invention, the pseudo-molecule of Frizzled 10 is preferably exemplified as the anti-Frizzled agent. According to this, as shown in Examples described later, when used together with an inducer of the Wnt / β-catenin pathway, it is possible to promote the induction of differentiation into cochlear hair cells, which is a form of inner ear hair cells. can. For example, as a pseudo-molecule of Frizzled10, a trade name "Recombinant Human Frizzled-10 Fc Chimera Protein", R & D Systems) and the like are also commercially available, and such a commercially available product may be used.
　接着培養の培地中での第３のＷｎｔ／βカテニン経路の誘導剤の濃度は、その下限値としては、１０ｎｇ／ｍＬ以上とすることが好ましく、その上限値としては、１０００ｎｇ／ｍＬ以下であることが好ましい。また、第３のＷｎｔ／βカテニン経路の誘導剤が、特にＷｎｔ３ａである場合、その下限値としては、１ｎｇ／ｍＬ以上とすることが好ましく、１０ｎｇ／ｍＬ以上とすることがより好ましく、２０ｎｇ／ｍＬ以上とすることが更により好ましい。その上限値としては、１００ｎｇ／ｍＬ以下であることが好ましく、５０ｎｇ／ｍＬ以下であることがより好ましく、２０ｎｇ／ｍＬ以下であることが更により好ましい。また、特にＲ－ｓｐｏｎｄｉｎ１である場合、その下限値としては、１０ｎｇ／ｍＬ以上とすることが好ましく、５０ｎｇ／ｍＬ以上とすることがより好ましく、１００ｎｇ／ｍＬ以上とすることが更により好ましい。その上限値としては、１０００ｎｇ／ｍＬ以下であることが好ましく、５００ｎｇ／ｍＬ以下であることがより好ましく、２００ｎｇ／ｍＬ以下であることが更により好ましい。 The concentration of the inducer of the third Wnt / β-catenin pathway in the medium of the adhesive culture is preferably 10 ng / mL or more as the lower limit value, and 1000 ng / mL or less as the upper limit value. Is preferable. Further, when the inducer of the third Wnt / β-catenin pathway is Wnt3a in particular, the lower limit thereof is preferably 1 ng / mL or more, more preferably 10 ng / mL or more, and more preferably 20 ng / mL. It is even more preferable to use mL or more. The upper limit is preferably 100 ng / mL or less, more preferably 50 ng / mL or less, and even more preferably 20 ng / mL or less. Further, particularly in the case of R-spondin1, the lower limit thereof is preferably 10 ng / mL or more, more preferably 50 ng / mL or more, and even more preferably 100 ng / mL or more. The upper limit is preferably 1000 ng / mL or less, more preferably 500 ng / mL or less, and even more preferably 200 ng / mL or less.
　一方、上記接着培養の培地中での抗Ｆｒｉｚｚｌｅｄ剤の濃度は、下限値としては、１０ｎｇ／ｍＬ以上とすることが好ましく、２０ｎｇ／ｍＬ以上とすることがより好ましく、５０ｎｇ／ｍＬ以上とすることが更により好ましい。その上限値としては、２００ｎｇ／ｍＬ以下であることが好ましく、１００ｎｇ／ｍＬ以下であることがより好ましく、５０ｎｇ／ｍＬ以下であることが更により好ましい。 On the other hand, the concentration of the anti-Frizzled agent in the medium of the adhesive culture is preferably 10 ng / mL or more, more preferably 20 ng / mL or more, and more preferably 50 ng / mL or more as the lower limit. Is even more preferable. The upper limit is preferably 200 ng / mL or less, more preferably 100 ng / mL or less, and even more preferably 50 ng / mL or less.
　［３］薬剤の評価方法
　本発明の別の観点では、本発明は、上記した方法によって得られた内耳前駆細胞を用いて、任意の被験物質を作用させ、その内耳前駆細胞への影響を調べることにより、その被験物質を評価する、薬剤の評価方法を提供するものである。 [3] Drug Evaluation Method From another aspect of the present invention, the present invention uses the inner ear progenitor cells obtained by the above method to act on any test substance and examine the effect on the inner ear progenitor cells. Thereby, it provides a method for evaluating a drug, which evaluates the test substance.
　本発明の更に別の観点では、本発明は、上記した方法によって得られた内耳有毛細胞を用いて、任意の被験物質を作用させ、その内耳有毛細胞への影響を調べることにより、その被験物質を評価する、薬剤の評価方法を提供するものである。 In yet another aspect of the present invention, the present invention uses the inner ear hair cells obtained by the above method to act on any test substance and examine the effect on the inner ear hair cells. It provides a method for evaluating a drug for evaluating a test substance.
　［４］内耳細胞分化誘導用組成物
　本発明の別の観点では、本発明は、レチノイン酸及びＷｎｔ／βカテニン経路の誘導剤を含有する内耳細胞分化誘導用組成物を提供する。Ｗｎｔ／βカテニン経路の誘導剤としては、ＣＨＩＲ９９０２１（別名：６－（（２－（（４－（２，４－Ｄｉｃｈｌｏｒｏｐｈｅｎｙｌ）－５－（４－Ｍｅｔｈｙｌ－１Ｈ－ｉｍｉｄａｚｏｌ－２－ｙｌ）ｐｙｒｉｍｉｄｉｎ－２－ｙｌ）ａｍｉｎｏ）ｅｔｈｙｌ）ａｍｉｎｏ）ｎｉｃｏｔｉｎｏｎｉｔｒｉｌｅ）、ＢＩＯ（別名：６－ｂｒｏ－ｍｏｉｎｄｉｒｕｂｉｎ　３’－ｏｘｉｍｅ）、ＬｉＣｌ（塩化リチウム）、Ｗｎｔ３ａ、Ｒ－ｓｐｏｎｄｉｎ１等が挙げられる。これらのうち、ＣＨＩＲ９９０２１、ＢＩＯ、ＬｉＣｌ（塩化リチウム）などがより好ましく例示される。 [4] Composition for Inducing Inner Ear Cell Differentiation From another aspect of the present invention, the present invention provides a composition for inducing inner ear cell differentiation containing retinoic acid and an inducer for the Wnt / β-catenin pathway. As an inducer of the Wnt / β-catenin pathway, CHIR99021 (also known as 6-((2-((4- (2,4-Dichlophoenyl) -5- (4-Methyl-1H-imidazol-2-yl) pyrimidin-) 2-yl) amino) ethyl) amino) nicotinonirile), BIO (also known as 6-bro-methylrubin 3'-oxime), LiCl (lithium chloride), Wnt3a, R-spondin1 and the like. Of these, CHIR99021, BIO, LiCl (lithium chloride) and the like are more preferably exemplified.
　本発明にかかる内耳細胞分化誘導用組成物中のレチノイン酸の含有量は、特に限定されないが、その下限値として、乾燥分中に０．０１質量％以上であることが典型的であり、０．１質量％以上であることがより典型的であり、１質量％以上であることが更により典型的であり、５質量％以上であることが特に典型的である。その上限値としては、乾燥分中に８０質量％以下であることが典型的であり、６０質量％以上であることがより典型的であり、５０質量％以上であることが更により典型的であり、４０質量％以上であることが特に典型的である。また、組成物中のＷｎｔ／βカテニン経路の誘導剤の含有量は、特に限定されないが、その上限値として、乾燥分中に０．０１質量％以上であることが典型的であり、０．１質量％以上であることがより典型的であり、１質量％以上であることが更により典型的であり、５質量％以上であることが特に典型的である。その上限値としては、乾燥分中に８０質量％以下であることが典型的であり、６０質量％以上であることがより典型的であり、５０質量％以上であることが更により典型的であり、４０質量％以上であることが特に典型的である。 The content of retinoic acid in the composition for inducing inner ear cell differentiation according to the present invention is not particularly limited, but the lower limit thereof is typically 0.01% by mass or more in the dry content, which is 0. .1% by mass or more is more typical, 1% by mass or more is even more typical, and 5% by mass or more is particularly typical. The upper limit is typically 80% by mass or less, more typically 60% by mass or more, and even more typically 50% by mass or more during the dry content. Yes, it is particularly typical that it is 40% by mass or more. The content of the inducer for the Wnt / β-catenin pathway in the composition is not particularly limited, but the upper limit thereof is typically 0.01% by mass or more in the dry content. 1% by mass or more is more typical, 1% by mass or more is even more typical, and 5% by mass or more is particularly typical. The upper limit is typically 80% by mass or less, more typically 60% by mass or more, and even more typically 50% by mass or more during the dry content. Yes, it is particularly typical that it is 40% by mass or more.
　本発明にかかる内耳細胞分化誘導用組成物は、多能性幹細胞から内耳細胞への分化誘導のために用いられる。具体的には、上記したレチノイン酸及びＷｎｔ／βカテニン経路の誘導剤を含有する培地での培養に使用するための、培養プレミックス液、液体培地等の形態で使用して、本発明による内耳前駆細胞や内耳有毛細胞の製造方法を、より効率的に実施するためのものである。 The composition for inducing inner ear cell differentiation according to the present invention is used for inducing differentiation of pluripotent stem cells into inner ear cells. Specifically, the inner ear according to the present invention is used in the form of a culture premix solution, a liquid medium, or the like for use in a medium containing the above-mentioned retinoic acid and an inducer of the Wnt / β-catenin pathway. The purpose is to more efficiently carry out a method for producing precursor cells and inner ear hair cells.
　以下に実施例を挙げて本発明を更に具体的に説明する。ただし、これらの実施例は本発明の範囲を限定するものではない。 The present invention will be described in more detail with reference to examples below. However, these examples do not limit the scope of the present invention.
　（試薬）
（１）Ｍａｔｒｉｇｅｌ：コーティング剤（商品名「Ｃｏｒｎｉｎｇ　Ｍａｔｒｉｇｅｌ基底膜マトリックス」、Ｃｏｒｎｉｎｇ社）
（２）アクターゼ：細胞剥離用酵素製剤（商品名「Ａｃｃｕｔａｓｅ」、ナカライテスク株式会社）
（３）Ｙ－２７６３２：ＲＯＣＫ阻害剤（Ｒｈｏ－ａｓｓｏｃｉａｔｅｄ　ｃｏｉｌｅｄ－ｃｏｉｌ　ｆｏｒｍｉｎｇ　ｋｉｎａｓｅ／Ｒｈｏ結合キナーゼの特異的阻害剤）（商品名「Ｙ－２７６３２」、富士フイルム和光純薬株式会社）
（４）ｍＴｅＳＲ１：ｉＰＳ細胞用維持培地（商品名「ｍＴｅＳＲ１」、ＳＴＥＭＣＥＬＬ　Ｔｅｃｈｎｏｌｏｇｉｅｓ社）
（５）ＤＭＥＭ／Ｆ１２：無血清培地（商品名「Ｄ－ＭＥＭ／Ｈａｍ’ｓ　Ｆ－１２」、富士フイルム和光純薬株式会社）
（６）Ｂ２７：無血清サプリメント（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）
（７）Ｂ２７－ＶＡ：無血清サプリメント（商品名「Ｇｉｂｃｏ　Ｂ－２７　Ｓｕｐｐｌｅｍｅｎｔ，　ｍｉｎｕｓ　ｖｉｔａｍｉｎ　Ａ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）
（８）Ｎ２：無血清サプリメント（商品名「Ｇｉｂｃｏ　Ｎ－２　Ｓｕｐｐｌｅｍｅｎｔ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）
（９）ＧｌｕｔａＭＡＸ：無血清サプリメント（商品名「Ｇｉｂｃｏ　ＧｌｕｔａＭＡＸ」、Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）
（１０）Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ：非必須アミノ酸サプリメント（商品名「ＭＥＭ　非必須アミノ酸溶液」、ナカライテスク株式会社）
（１１）ｐｏｌｙ－ｏ－ｆｉｂｒｏｎｅｃｔｉｎ：コーティング剤（商品名「Ｐｏｌｙ－Ｌ－ｏｒｎｉｔｈｉｎｅ」、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）、（商品名「Ｆｉｂｒｏｎｅｃｔｉｎ」、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）
（１２）ｂＦＧＦ（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＦＧＦ－ｂａｓｉｃ」、Ｐｅｐｒｏｔｅｃｈ社）
（１３）ＦＧＦ３（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＦＧＦ－３　ｐｒｏｔｅｉｎ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（１４）ＦＧＦ１０（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＦＧＦ－１０　ｐｒｏｔｅｉｎ」、Ｐｅｐｒｏｔｅｃｈ社）
（１５）ＦＧＦ１９（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＦＧＦ－１９　ｐｒｏｔｅｉｎ」、Ｐｅｐｒｏｔｅｃｈ社）
（１６）ＢＭＰ４（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＢＭＰ－４　ｐｒｏｔｅｉｎ」、Ｐｅｐｒｏｔｅｃｈ社）
（１７）ＩＧＦ－１（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　ＩＧＦ－１　Ｐｒｏｔｅｉｎ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（１８）ＣＨＩＲ９９０２１（商品名「ＣＨＩＲ９９０２１」、Ｆｏｃｕｓｂｉｏｍｏｌｅｃｕｌｅｓ社）
（１９）Ｗｎｔ３ａ（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　Ｗｎｔ３ａ　ｐｒｏｔｅｉｎ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（２０）ＢＩＯ（商品名「ＢＩＯ」、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）
（２１）レチノイン酸（商品名「ｒｅｔｉｎｏｉｃ　ａｃｉｄ」、Ｓｉｇｍａ－Ａｌｄｒｉｃｈ社）
（２２）ヘパリン（商品名「Ｈｅｐａｒｉｎ　Ｓｏｄｉｕｍ　Ｓａｌｔ」、ナカライテスク株式会社）
（２３）Ｆｒｉｚｚｌｅｄ７の疑似分子（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　Ｆｒｉｚｚｌｅｄ－７　Ｆｃ　Ｃｈｉｍｅｒａ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（２４）Ｆｒｉｚｚｌｅｄ８の疑似分子（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　Ｆｒｉｚｚｌｅｄ－８　Ｆｃ　Ｃｈｉｍｅｒａ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（２５）Ｆｒｉｚｚｌｅｄ１０の擬似分子（商品名「Ｒｅｃｏｍｂｉｎａｎｔ　Ｈｕｍａｎ　Ｆｒｉｚｚｌｅｄ－１０　Ｆｃ　Ｃｈｉｍｅｒａ　Ｐｒｏｔｅｉｎ」、Ｒ＆Ｄ　Ｓｙｓｔｅｍｓ社）
（２６）ｑＲＴ－ＰＣＲに用いたプライマーの配列を表１に示す。 (reagent)
(1) Matrix: Coating agent (trade name "Corning Matrix basement membrane matrix", Corning)
(2) Actase: Enzyme preparation for cell exfoliation (trade name "Accutase", Nacalai Tesque Co., Ltd.)
(3) Y-27632: ROCK inhibitor (Rho-associated coiled-coil forming kinase / specific inhibitor of Rho-binding kinase) (trade name "Y-27632", Wako Pure Chemical Industries, Ltd.)
(4) mTeSR1: Maintenance medium for iPS cells (trade name "mTeSR1", STEMCELL Technologies)
(5) DMEM / F12: Serum-free medium (trade name "D-MEM / Ham's F-12", Wako Pure Chemical Industries, Ltd.)
(6) B27: Serum-free supplement (trade name "Gibco B-27 Supplement", Thermo Fisher Scientific)
(7) B27-VA: Serum-free supplement (trade name "Gibco B-27 Supplement, minus vitamin A", Thermo Fisher Scientific)
(8) N2: Serum-free supplement (trade name "Gibco N-2 Supplement", Thermo Fisher Scientific)
(9) GlutaMAX: Serum-free supplement (trade name "Gibco GlutaMAX", Thermo Fisher Scientific)
(10) Nonesential aminoacid: Non-essential amino acid supplement (trade name "MEM non-essential amino acid solution", Nacalai Tesque Co., Ltd.)
(11) poly-o-fibronectin: coating agent (trade name "Poly-L-ornithine", Sigma-Aldrich), (trade name "Fibronectin", Sigma-Aldrich)
(12) bFGF (trade name "Recombinant Human FGF-basic", Proprotech)
(13) FGF3 (trade name "Recombinant Human FGF-3 protein", R & D Systems)
(14) FGF10 (trade name "Recombinant Human FGF-10 protein", Proprotech)
(15) FGF19 (trade name "Recombinant Human FGF-19 protein", Proprotech)
(16) BMP4 (trade name "Recombinant Human BMP-4 protein", Proprotech)
(17) IGF-1 (trade name "Recombinant Human IGF-1 Protein", R & D Systems)
(18) CHIR99021 (trade name "CHIR99021", Focusbiomolecules)
(19) Wnt3a (trade name "Recombinant Human Wnt3a protein", R & D Systems)
(20) BIO (trade name "BIO", Sigma-Aldrich)
(21) Retinoic acid (trade name "retinoic acid", Sigma-Aldrich)
(22) Heparin (trade name "Heparin Sodium Salt", Nacalai Tesque Co., Ltd.)
(23) Pseudo-molecule of Frizzled7 (trade name "Recombinant Human Frizzled-7 FcChimera", R & D Systems)
(24) Pseudo-molecule of Frizzled8 (trade name "Recombinant Human Frizzled-8 FcChimera", R & D Systems)
(25) Pseudo-molecule of Frizzled10 (trade name "Recombinant Human Frizzled-10 FcChimera Protein", R & D Systems)
(26) The sequences of the primers used for qRT-PCR are shown in Table 1.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　［試験例１］
　本試験例では、ヒトｉＰＳ細胞から内耳前駆細胞を分化誘導する過程におけるＰＡＸ２、ＰＡＸ８、ＳＯＸ２、ＬＧＲ５の発現量をｑＲＴ－ＰＣＲで定量した。 [Test Example 1]
In this test example, the expression levels of PAX2, PAX8, SOX2, and LGR5 in the process of inducing differentiation of inner ear progenitor cells from human iPS cells were quantified by qRT-PCR.
　［分化誘導方法］
（Ｄａｙ０）
１）　６ウェルプレートをＭａｔｒｉｇｅｌでコーティングした。
２）　コンフルエントなｆｅｅｄｅｒ－ｆｒｅｅヒトｉＰＳ細胞にアクターゼを添加して３７℃で２～３分インキュベートし、ディッシュから剥離した。
３）　ＰＢＳで希釈後遠心し、細胞を回収した。
４）　上澄みをすてＲＯＣＫ阻害剤（Ｙ－２７６３２）（１０μＭ）を加えたｍＴｅＳＲ１メディウムに細胞を懸濁した。
５）　ナイロンメッシュ（孔径４０μｍ）を通し、血球計算盤で細胞数をカウントした。
６）　前記１）でＭａｔｒｉｇｅｌコーティングしたウェルにＹ－２７６３２を添加したｍＴｅＳＲ１メディウムを加えた。
７）　１ウェルあたり２．０×１０４ｃｅｌｌｓ／ｃｍ２となるように細胞懸濁液を播種した。 [Differentiation induction method]
(Day 0)
1) A 6-well plate was coated with Matrigel.
2) Actase was added to a confluent feeder-free human iPS cell, and the mixture was incubated at 37 ° C. for 2 to 3 minutes and exfoliated from the dish.
3) After diluting with PBS, the cells were collected by centrifugation.
4) The supernatant was removed and the cells were suspended in mTeSR1 medium supplemented with a ROCK inhibitor (Y-27632) (10 μM).
5) The number of cells was counted by a blood cell calculator through a nylon mesh (pore diameter 40 μm).
6) The mTeSR1 medium to which Y-27632 was added was added to the Well that was coated with Matrigel in 1) above.
7) The cell suspension was seeded at 2.0 × 10 4 cells / cm 2 per well.
（Ｄａｙ１）
　ＲＯＣＫ阻害剤を含まないｍＴｅＳＲ１メディウムに培地交換した。 (Day 1)
The medium was replaced with mTeSR1 medium containing no ROCK inhibitor.
（Ｄａｙ２）
　無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２＋ＧｌｕｔａＭＡＸ＋Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ）に培地交換した。以後、Ｄａｙ４まで、毎日培地交換した。 (Day2)
The medium was replaced with serum-free medium (DMEM / F12 + B27 + N2 + GlutaMAX + Nonesential amino acid). After that, the medium was changed every day until Day 4.
（Ｄａｙ５）
　無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２＋ＧｌｕｔａＭＡＸ＋Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ）に、成長因子ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、ＦＧＦ１９、ＢＭＰ４を、それぞれ濃度が２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、１０ｎｇ／ｍＬとなるように添加した培地に培地交換した。以後、Ｄａｙ７まで、毎日培地交換した。 (Day 5)
Growth factors bFGF, FGF3, FGF10, FGF19, and BMP4 are added to serum-free medium (DMEM / F12 + B27 + N2 + GlutaMAX + Nonecential amino acid) at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, 25 ng / mL, and 10 ng / mL, respectively. The medium was replaced with the added medium. After that, the medium was changed every day until Day 7.
（Ｄａｙ８）
　無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２＋ＧｌｕｔａＭＡＸ＋Ｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏａｃｉｄ）に、成長因子ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、ＦＧＦ１９を添加した培地（成長因子の濃度は全て２５ｎｇ／ｍＬ）に培地交換した。以後Ｄａｙ１０まで、毎日培地交換した。 (Day 8)
The medium was replaced with a medium containing growth factors bFGF, FGF3, FGF10, and FGF19 (growth factor concentrations were all 25 ng / mL) in a serum-free medium (DMEM / F12 + B27 + N2 + GlutaMAX + Nonesential amino acid). After that, the medium was changed every day until Day 10.
（Ｄａｙ１１）
　細胞にアクターゼを添加して３７℃で２～３分インキュベートし、ディッシュから剥離し、ＰＢＳで希釈した。遠心して細胞を回収し、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、ＦＧＦ１９を、それぞれ濃度が２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬ、２５ｎｇ／ｍＬとなるように添加した培地で懸濁した。ナイロンメッシュ（孔径４０μｍ）で残存する細胞塊を除去して、ｐｏｌｙ－ｏ－ｆｉｂｒｏｎｅｃｔｉｎでコーティングしたウェルに播種した。培養は、通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で行った。 (Day 11)
The cells were added with actase and incubated at 37 ° C. for 2-3 minutes, stripped from the dish and diluted with PBS. The cells are collected by centrifugation, and the growth factors bFGF, FGF3, FGF10, and FGF19 are added to a serum-free medium (DMEM / F12 + B27 + N2) at concentrations of 25 ng / mL, 25 ng / mL, 25 ng / mL, and 25 ng / mL, respectively. Suspended in the added medium. The remaining cell mass was removed with a nylon mesh (pore diameter 40 μm) and seeded in wells coated with poly-o-fibronectin. Culturing was carried out under normal oxygen conditions (O 2 20%, CO 2 5%).
（Ｄａｙ１２）
　翌日、下記条件１）～４）のそれぞれの培地条件となるよう、培地交換した。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加して調製した培地
・条件２）上記条件１）の培地に、更に、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地
・条件３）上記条件１）の培地に、更に、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加して調製した培地
・条件４）上記条件１）の培地に、更に、レチノイン酸を濃度が０．５μＭとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加して調製した培地 (Day 12)
The next day, the medium was exchanged so as to meet the following conditions 1) to 4).
-Condition 1) Prepared by adding growth factors bFGF, EGF, and IGF-1 to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. Medium / Condition 2) Medium prepared by adding retinoic acid to the medium of the above condition 1) so as to have a concentration of 0.5 μM ・ Condition 3) Further to the medium of the above condition 1), CHIR99021 was further concentrated. Medium prepared by adding serum to 3 μM ・ Condition 4) Add retinoic acid to the medium of condition 1) above to a concentration of 0.5 μM, and add CHIR99021 to a concentration of 3 μM. Medium prepared by adding
　以後Ｄａｙ１５まで、毎日培地交換した。 After that, the medium was changed every day until Day15.
（Ｄａｙ１５）
　Ｄａｙ１５の細胞について、ＲＮｅａｓｙスピンカラムキット（Ｑｉａｇｅｎ社）を用いてｔｏｔａｌ　ＲＮＡを抽出し、各サンプル１μｇのｔｏｔａｌ　ＲＮＡから逆転写酵素（商品名「Ｉｎｖｉｔｒｏｇｅｎ　ＳｕｐｅｒＳｃｒｉｐｔ　ＩＶ　Ｒｅｖｅｒｓｅ　Ｔｒａｎｓｃｒｉｐｔａｓｅ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ社）によりｃＤＮＡ合成を行い、内耳前駆細胞マーカーの発現量をｑＲＴ－ＰＣＲにより定量した。 (Day15)
For cells of Day15, total RNA was extracted using the RNeasy spin column kit (Qiagen), and reverse transcriptase (trade name "Invitrogen SuperScript IV Reverse Transcriptase (Thercimotive) Synthesis (Therci)) from 1 μg of each sample of total RNA was performed. The expression level of the inner ear precursor cell marker was quantified by qRT-PCR.
　図３に結果を示す。なお、結果は、上記培養条件４のときの定量値を１としたときの相対値で示した。 Figure 3 shows the results. The results are shown as relative values when the quantitative value under the above culture condition 4 is 1.
　その結果、図３に示されるように、内耳前駆細胞マーカーであるＰＡＸ２、ＰＡＸ８の発現レベルはＣＨＩＲ９９０２１とレチノイン酸の両方を添加した際に顕著に増加していた。また、ＣＨＩＲ９９０２１の添加によって、ＳＯＸ２及びＬＧＲ５の発現上昇が認められた。このことから、ＣＨＩＲ９９０２１によるＷＮＴシグナルの活性化、およびレチノイン酸の添加によって内耳前駆細胞の分化が促進されることが示唆された。 As a result, as shown in FIG. 3, the expression levels of the inner ear progenitor cell markers PAX2 and PAX8 were significantly increased when both CHIR99021 and retinoic acid were added. In addition, the addition of CHIR99021 increased the expression of SOX2 and LGR5. This suggests that activation of WNT signal by CHIR99021 and addition of retinoic acid promote the differentiation of inner ear progenitor cells.
　［試験例２］
　本試験例では、ヒトｉＰＳ細胞から内耳前駆細胞を分化誘導する過程におけるＰＡＸ２、ＰＡＸ８、ＳＯＸ２、ＬＧＲ５の発現量の推移をｑＲＴ－ＰＣＲで定量した。 [Test Example 2]
In this test example, changes in the expression levels of PAX2, PAX8, SOX2, and LGR5 in the process of inducing differentiation of inner ear progenitor cells from human iPS cells were quantified by qRT-PCR.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）、培地交換を行った。その培地としては、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、ＣＨＩＲ９９０２１及びレチノイン酸を、それぞれの濃度が３μＭ、０．５μＭとなるように添加して調製した培地を使用した。以後Ｄａｙ１８まで、毎日培地交換を行った。 [Differentiation induction method]
Differentiation was induced in the same manner as in Test Example 1, and the cells subjected to cell exfoliation treatment with actase on Day 11 were subjected to medium exchange on the next day (Day 12). As the medium, growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and further. , CHIR99021 and retinoic acid were added so that their concentrations were 3 μM and 0.5 μM, respectively, and a medium prepared was used. After that, the medium was changed every day until Day 18.
（Ｄａｙ１１、１５、２０）
　Ｄａｙ１１、１５、２０の細胞について、それぞれ試験例１と同様にして、ｔｏｔａｌ　ＲＮＡを抽出、ｃＤＮＡを合成し、ｑＲＴ－ＰＣＲにより内耳前駆細胞マーカーであるＰＡＸ２、ＰＡＸ８、ＳＯＸ２、ＬＧＲ５の発現量を定量した。 ( Day 11, 15, 20)
For cells of Day 11, 15 and 20, total RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the expression levels of inner ear progenitor cell markers PAX2, PAX8, SOX2 and LGR5 were quantified by qRT-PCR. did.
　図４に結果を示す。なお、結果は、未分化ｉＰＳ細胞について同様のｑＲＴ－ＰＣＲによる定量値を１としたときの相対値で示した。 Figure 4 shows the results. The results are shown as relative values when the same quantitative value by qRT-PCR was set to 1 for undifferentiated iPS cells.
　その結果、図４に示されるように、内耳前駆細胞マーカーであるＰＡＸ２とＬＧＲ５の発現レベルはＤａｙ１１からＤａｙ１５にかけて上昇した。一方、Ｄａｙ１５からＤａｙ２０にかけてはＰＡＸ２、ＰＡＸ８、ＳＯＸ２、ＬＧＲ５の発現レベルはいずれも減少した。この結果から、Ｄａｙ１１からＤａｙ１５にかけてＣＨＩＲ９９０２１およびレチノイン酸を添加することで、内耳前駆細胞を効率よく誘導できることが示唆された。 As a result, as shown in FIG. 4, the expression levels of the inner ear progenitor cell markers PAX2 and LGR5 increased from Day 11 to Day 15. On the other hand, the expression levels of PAX2, PAX8, SOX2, and LGR5 decreased from Day15 to Day20. From this result, it was suggested that the inner ear progenitor cells could be efficiently induced by adding CHIR99021 and retinoic acid from Day 11 to Day 15.
　［試験例３］
　試験例１、２では、Ｗｎｔ／βカテニン経路の誘導剤であるＣＨＩＲ９９０２１をレチノイン酸とともに使用すると、内耳前駆細胞マーカーの発現量が高められることが明らかとなった。本試験例では、他にＷｎｔ／βカテニン経路の誘導剤として知られているＬｉＣｌ及びＢＩＯについて、内耳前駆細胞マーカーの発現量に与える影響を調べた。 [Test Example 3]
In Test Examples 1 and 2, it was revealed that the expression level of the inner ear progenitor cell marker was increased when CHIR99021, which is an inducer of the Wnt / β-catenin pathway, was used together with retinoic acid. In this test example, the effects of LiCl and BIO, which are also known as inducers of the Wnt / β-catenin pathway, on the expression level of inner ear progenitor cell markers were investigated.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）、培地交換を行った。その培地としては、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦを、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加し、更に培地に以下の７つの異なる条件でＷｎｔ／βカテニン経路の誘導剤を添加したものを調製して使用した。
・条件１）非添加
・条件２）ＣＨＩＲ９９０２１　３μＭ
・条件３）ＬｉＣｌ　１ｍＭ
・条件４）ＬｉＣｌ　１０ｍＭ
・条件５）ＢＩＯ　０．１μＭ
・条件６）ＢＩＯ　０．５μＭ
・条件７）ＢＩＯ　１μＭ [Differentiation induction method]
Differentiation was induced in the same manner as in Test Example 1, and the cells subjected to cell exfoliation treatment with actase on Day 11 were subjected to medium exchange on the next day (Day 12). As the medium, growth factors bFGF and EGF were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL and 20 ng / mL, respectively, and the concentration of retinoic acid was 0.5 μM. The medium was further added with an inducer for the Wnt / β-catenin pathway under the following seven different conditions, and the medium was prepared and used.
-Condition 1) No addition-Condition 2) CHIR99021 3 μM
・ Condition 3) LiCl 1 mM
・ Condition 4) LiCl 10 mM
・ Condition 5) BIO 0.1 μM
・ Condition 6) BIO 0.5 μM
・ Condition 7) BIO 1 μM
　以後Ｄａｙ１６まで、毎日培地交換を行った。 After that, the medium was changed every day until Day 16.
（Ｄａｙ１６）
　Ｄａｙ１６の細胞について、試験例１と同様にして、ｔｏｔａｌＲＮＡを抽出、ｃＤＮＡを合成し、ｑＲＴ－ＰＣＲにより内耳前駆細胞マーカーであるＰＡＸ２の発現量を定量した。 (Day16)
For Day16 cells, total RNA was extracted, cDNA was synthesized, and the expression level of PAX2, which is a marker for inner ear progenitor cells, was quantified by qRT-PCR in the same manner as in Test Example 1.
　図５に結果を示す。なお、結果は、上記培養条件１（非添加）のときの定量値を１としたときの相対値で示した。 Figure 5 shows the results. The results are shown as relative values when the quantitative value under the above culture condition 1 (non-addition) is 1.
　その結果、図５に示されるように、ＬｉＣｌおよびＢＩＯ添加群は非添加群と比較してＰＡＸ２の発現が上昇した。この結果から、Ｗｎｔ／βカテニン経路の誘導剤は、レチノイン酸とともに使用することにより、内耳前駆細胞の誘導を促進することが示唆された。 As a result, as shown in FIG. 5, the expression of PAX2 was increased in the LiCl and BIO-added groups as compared with the non-added group. This result suggests that the Wnt / β-catenin pathway inducer promotes the induction of inner ear progenitor cells when used in combination with retinoic acid.
　［試験例４］
　本試験例では、内耳前駆細胞から内耳有毛細胞への分化誘導を行い、内耳前駆細胞マーカーのＬＧＲ５及び内耳有毛細胞マーカーのＡＴＯＨ１およびＢＲＮ３Ｃの遺伝子発現量をｑＲＴ－ＰＣＲで定量した。 [Test Example 4]
In this test example, differentiation of inner ear progenitor cells into inner ear hair cells was induced, and the gene expression levels of the inner ear progenitor cell markers LGR5 and the inner ear hair cell markers ATOH1 and BRN3C were quantified by qRT-PCR.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）から、以下の２種類の異なる培養条件で培養を行った。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加して調製した培地、低酸素条件下（Ｏ２４％、ＣＯ２５％）
・条件２）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、ＣＨＩＲ９９０２１及びレチノイン酸を、それぞれの濃度が３μＭ、０．５μＭとなるように添加して調製した培地、通常酸素条件下（Ｏ２２０％、ＣＯ２５％） [Differentiation induction method]
Differentiation was induced in the same manner as in Test Example 1, and cells subjected to cell desquamation treatment with actase on Day 11 were cultured from the next day (Day 12) under the following two different culture conditions.
Condition 1) A medium prepared by adding growth factors bFGF, EGF, and IGF-1 to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations are 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. hypoxic conditions (O 2 4%, CO 2 5%)
-Condition 2) Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. medium CHIR99021 and retinoic acid, each concentration were prepared by adding so 3 [mu] M, and 0.5 [mu] M, usually oxygen conditions (O 2 20%, CO 2 5%)
　以後Ｄａｙ１５まで、毎日培地交換を行った。 After that, the medium was changed every day until Day15.
（Ｄａｙ１５）
　細胞をアクターゼで剥離し、等量のＰＢＳを加え、ナイロンメッシュ（孔径４０μｍ）を通し、血球計算盤で細胞数をカウントした。 (Day15)
The cells were detached with actase, an equal amount of PBS was added, the cells were passed through a nylon mesh (pore diameter 40 μm), and the number of cells was counted by a hemocytometer.
　８．５×１０４ｃｅｌｌｓ／ｗｅｌｌになるように細胞を懸濁し、低接着６ウェルプレート（商品名「Ｃｏｒｎｉｎｇ超低接着表面（Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ）プレート」、Ｃｏｒｎｉｎｇ社）に播種した。このとき、条件１と条件２で誘導した細胞は、それぞれ以下の組成の培地に懸濁し、低酸素条件下（Ｏ２４％、ＣＯ２５％）又は通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で浮遊培養を開始した。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍｌ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加して調製した培地
・条件２）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地 Cells were suspended to 8.5 × 10 4 cells / well and seeded on a low-adhesion 6-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning). At this time, the cells induced with conditions 1 and 2, respectively were suspended in a medium of the following composition, under hypoxic conditions (O 2 4%, CO 2 5%) or normoxic conditions (O 2 20%, It was initiated suspension culture in CO 2 5%).
-Condition 1) Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations were 10 ng / mL, 20 ng / ml, and 50 ng / mL, respectively, and further, Y- 27632 was added to a concentration of 10 μM, Wnt3a was added to a concentration of 20 ng / mL, CHIR99021 was added to a concentration of 3 μM, and heparin was added to a concentration of 50 ng / mL. 2) In a serum-free medium (DMEM / F12 + B27-VA + N2), the growth factors bFGF, EGF, and IGF-1 were added to the concentrations of 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. Add Y-27632 to a concentration of 10 μM, Wnt3a to a concentration of 20 ng / mL, CHIR99021 to a concentration of 3 μM, and heparin to a concentration of 50 ng. A medium prepared by adding retinoic acid to a concentration of 0.5 μM and adding retinoic acid to a concentration of 0.5 μM.
（Ｄａｙ１９、２３）
　条件１と条件２でそれぞれ下記の組成の培地を用いてＤａｙ１９およびＤａｙ２３に全量培地交換を行った。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加した培地
・条件２）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地 (Day 19, 23)
Under conditions 1 and 2, the total amount of medium was exchanged for Day 19 and Day 23 using the medium having the following composition, respectively.
-Condition 1) Add growth factors bFGF, EGF, and IGF-1 to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations are 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and further add Wnt3a. Addition to a concentration of 20 ng / mL, CHIR99021 to a concentration of 3 μM, and heparin to a concentration of 50 ng / mL Medium / Condition 2) Serum-free medium (DMEM / F12 + B27- To VA + N2), growth factors bFGF, EGF, and IGF-1 were added so as to have concentrations of 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and Wnt3a was further added so as to have a concentration of 20 ng / mL. Then, CHIR99021 was added to a concentration of 3 μM, heparin was added to a concentration of 50 ng / mL, and retinoic acid was added to a concentration of 0.5 μM.
（Ｄａｙ２７）
　Ｄａｙ２７に、ｐｏｌｙ－ｏ－ｆｉｂｒｏｎｅｃｔｉｎでコーティングした１２ウェルプレートにスフェア（細胞塊）を接着させた。 (Day27)
A sphere (cell mass) was adhered to Day 27 on a 12-well plate coated with poly-o-fibronectin.
（Ｄａｙ２８）
　Ｄａｙ２８に条件１、２のいずれも無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が２５ｎｇ／ｍＬ、１０ｎｇ／ｍＬ添加して調製した培地で培地交換を行った。以後２日に一度培地交換を行った。 (Day 28)
The medium was exchanged with a medium prepared by adding growth factors EGF and IGF-1 to Day 28 in serum-free medium (DMEM / F12 + B27 + N2) at concentrations of 25 ng / mL and 10 ng / mL, respectively. .. After that, the medium was changed once every two days.
（Ｄａｙ３４）
　Ｄａｙ３４の細胞について、試験例１と同様にして、ｔｏｔａｌＲＮＡを抽出し、ｃＤＮＡ合成を行って、ｑＲＴ－ＰＣＲによる遺伝子発現解析に供した。 (Day34)
For the cells of Day 34, total RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the cells were subjected to gene expression analysis by qRT-PCR.
　その結果、図６に示されるように、内耳前駆細胞をＣＨＩＲ９９０２１及びレチノイン酸を添加した培地で培養した試験群では、非添加群と比べて、内耳前駆細胞マーカーのＬＧＲ５の発現は減少し、一方で初期の内耳有毛細胞マーカーであるＡＴＯＨ１やＢＲＮ３Ｃの発現は亢進していた。このことから、ＣＨＩＲ９９０２１及びレチノイン酸の添加により内耳前駆細胞の誘導効率が向上した結果、内耳有毛細胞への誘導効率についても向上したものと考えられた。 As a result, as shown in FIG. 6, in the test group in which the inner ear progenitor cells were cultured in the medium supplemented with CHIR99021 and retinoic acid, the expression of the inner ear progenitor cell marker LGR5 was decreased as compared with the non-added group. In the early stage, the expression of ATOH1 and BRN3C, which are markers of inner ear hair cells, was enhanced. From this, it is considered that the addition of CHIR99021 and retinoic acid improved the induction efficiency of inner ear progenitor cells, and as a result, the induction efficiency to inner ear hair cells was also improved.
　［試験例５］
　本試験例では、内耳前駆細胞から内耳有毛細胞への分化誘導を行い、Ｒ－ｓｐｏｎｄｉｎ１を添加して浮遊培養を行ったことによる内耳有毛細胞マーカーの発現量の変化をｑＲＴ－ＰＣＲで定量した。 [Test Example 5]
In this test example, the change in the expression level of the inner ear hair cell marker due to induction of differentiation from inner ear progenitor cells to inner ear hair cells and suspension culture with the addition of R-spondin1 was quantified by qRT-PCR. did.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）、培地交換を行った。その培地としては、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加したものを調製し、培地交換を行った。培養は、通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で行った。 [Differentiation induction method]
Differentiation was induced in the same manner as in Test Example 1, and the cells subjected to cell exfoliation treatment with actase on Day 11 were subjected to medium exchange on the next day (Day 12). As the medium, growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and further. , CHIR99021 was added so as to have a concentration of 3 μM, and retinoic acid was added so as to have a concentration of 0.5 μM to prepare a medium, and the medium was exchanged. Culturing was carried out under normal oxygen conditions (O 2 20%, CO 2 5%).
　以後Ｄａｙ１８まで、毎日培地交換を行った。 After that, the medium was changed every day until Day 18.
（Ｄａｙ１８）
　細胞をアクターゼで剥離し、等量のＰＢＳを加え、ナイロンメッシュ（孔径４０μｍ）を通し、血球計算盤で細胞数をカウントした。 (Day18)
The cells were detached with actase, an equal amount of PBS was added, the cells were passed through a nylon mesh (pore diameter 40 μm), and the number of cells was counted by a hemocytometer.
　８．５×１０４ｃｅｌｌｓ／ｗｅｌｌになるように細胞を懸濁し、低接着６ウェルプレート（商品名「Ｃｏｒｎｉｎｇ超低接着表面（Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ）プレート」、Ｃｏｒｎｉｎｇ社）に播種し、低酸素条件下（Ｏ２４％、ＣＯ２５％）で浮遊培養を開始した。このとき、培地として、下記条件１）又は条件２）を使用して、細胞を懸濁して浮遊培養を開始した。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍｌ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地
・条件２）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍｌ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加し、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加して調製した培地 The cells were suspended so as to be 8.5 × 10 4 cells / well, seeded on a low-adhesion 6-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, using the following conditions 1) or 2) as the medium, the cells were suspended and suspension culture was started.
-Condition 1) To the serum-free medium (DMEM / F12 + B27-VA + N2), the growth factors bFGF, EGF, and IGF-1 were added so that the concentrations were 10 ng / mL, 20 ng / ml, and 50 ng / mL, respectively, and further. Y-27632 was added to a concentration of 10 μM, Wnt3a was added to a concentration of 20 ng / mL, CHIR99021 was added to a concentration of 3 μM, and heparin was added to a concentration of 50 ng / mL. 2) Growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) prepared by adding retinoic acid to a concentration of 0.5 μM. Add to a concentration of 10 ng / mL, 20 ng / ml, 50 ng / mL, further add Y-27632 to a concentration of 10 μM, and add Wnt3a to a concentration of 20 ng / mL. CHIR99021 was added to a concentration of 3 μM, heparin was added to a concentration of 50 ng / mL, retinoic acid was added to a concentration of 0.5 μM, and R-spondin1 was added to a concentration of 200 ng / mL. Medium prepared by adding so as to
（Ｄａｙ２２）
　Ｄａｙ２２に条件１）及び条件２）ともにＲＯＣＫ阻害剤であるＹ－２７６３２を添加しないこと以外は、同じ組成の培地で培地交換を行った。 (Day22)
The medium was exchanged with a medium having the same composition except that Y-27632, which is a ROCK inhibitor, was not added to Day 22 in both conditions 1) and 2).
（Ｄａｙ２４）
　Ｄａｙ２４の細胞について、試験例１と同様にしてｔｏｔａｌＲＮＡを抽出し、ｃＤＮＡ合成を行って、ｑＲＴ－ＰＣＲによる遺伝子発現解析に供した。 (Day24)
For the cells of Day24, totalRNA was extracted in the same manner as in Test Example 1, cDNA synthesis was performed, and the cells were subjected to gene expression analysis by qRT-PCR.
　その結果、図７に示されるように、浮遊培養時の培地にＲ－ｓｐｏｎｄｉｎ１を添加した試験群は、非添加群と比べて、内耳有毛細胞マーカーのＡＴＯＨ１、ＭＹＯ７Ａ、ＭＹＯ１５Ａ、ＢＲＮ３Ｃの発現量が上昇していた。このことから、Ｗｎｔ／βカテニン経路の誘導剤であるＲ－ｓｐｏｎｄｉｎ１の添加によりＷＮＴシグナルが活性化されたことで、内耳前駆細胞の増殖が促進されたか、あるいは内耳前駆細胞から内耳有毛細胞への分化が促進されたものと考えられた。 As a result, as shown in FIG. 7, in the test group in which R-spondin1 was added to the medium during suspension culture, the expression levels of the inner ear hair cell markers ATOH1, MYO7A, MYO15A, and BRN3C were compared with those in the non-added group. Was rising. From this, the WNT signal was activated by the addition of R-spondin1, which is an inducer of the Wnt / β-catenin pathway, and thus the proliferation of inner ear progenitor cells was promoted, or from inner ear progenitor cells to inner ear hair cells. It was considered that the differentiation of the cells was promoted.
　なお、この試験例では、内耳有毛細胞マーカーのＡＴＯＨ１、ＭＹＯ７Ａ、ＭＹＯ１５Ａ、ＢＲＮ３Ｃの発現量を上昇させる薬剤として、Ｒ－ｓｐｏｎｄｉｎ１が見出されたが、他の薬剤についても、同様にして、これらの内耳有毛細胞マーカーの発現量を上昇させることができるかどうかについて評価することが可能である。また、このときに用いるマーカーや培養条件は、適宜に他のマーカーや培養条件を設定してもよく、任意の薬剤について、内耳前駆細胞から内耳有毛細胞への分化を促進させることができるかどうかを評価することが可能である。 In this test example, R-spondin1 was found as a drug for increasing the expression levels of the inner ear hair cell markers ATOH1, MYO7A, MYO15A, and BRN3C. It is possible to evaluate whether the expression level of the inner ear hair cell marker can be increased. In addition, the markers and culture conditions used at this time may be set to other markers and culture conditions as appropriate, and can any drug promote the differentiation of inner ear progenitor cells into inner ear hair cells? It is possible to evaluate whether or not.
　［試験例６］
　ＣＨＩＲ９９０２１及びレチノイン酸で処理した内耳前駆細胞から、支持細胞及びらせん神経節細胞をともなう内耳有毛細胞を分化誘導させた。 [Test Example 6]
Inner ear hair cells with sustentacular cells and spiral ganglion cells were induced to differentiate from inner ear progenitor cells treated with CHIR99021 and retinoic acid.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地に培地交換し、通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で培養を行った。 [Differentiation induction method]
Growth factors bFGF, EGF, and IGF were placed in serum-free medium (DMEM / F12 + B27-VA + N2) the next day (Day 12) for cells that had been induced to differentiate in the same manner as in Test Example 1 and had undergone cell detachment treatment with Actase on Day 11. -1 was added so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and CHIR99021 was added so that the concentration was 3 μM, and retinoic acid was added so that the concentration was 0.5 μM. the medium was replaced with medium prepared by adding to, were cultured in normoxic conditions (O 2 20%, CO 2 5%).
　以後Ｄａｙ１８まで、毎日培地交換を行った。 After that, the medium was changed every day until Day 18.
（Ｄａｙ１８）
　細胞をアクターゼで剥離し、等量のＰＢＳを加え、ナイロンメッシュ（孔径４０μｍ）を通し、血球計算盤で細胞数をカウントした。 (Day18)
The cells were detached with actase, an equal amount of PBS was added, the cells were passed through a nylon mesh (pore diameter 40 μm), and the number of cells was counted by a hemocytometer.
　８．５×１０４ｃｅｌｌｓ／ｗｅｌｌになるように細胞を懸濁し、低接着９６ウェルプレート（商品名「Ｃｏｒｎｉｎｇ超低接着表面（Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ）プレート」、Ｃｏｒｎｉｎｇ社）に播種し、低酸素条件下（Ｏ２４％、ＣＯ２５％）で浮遊培養を開始した。このとき、培地としては、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ＥＧＦを濃度が２０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｍａｔｒｉｇｅｌを濃度が１％となるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が１０μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加し、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加して調製した培地を使用して、細胞を懸濁して浮遊培養を開始した。 The cells were suspended so as to be 8.5 × 10 4 cells / well, seeded on a low-adhesion 96-well plate (trade name “Corning Ultra-Low Attachment Plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, as a medium, a serum-free medium (DMEM / F12 + B27-VA + N2) was added with the growth factor EGF so as to have a concentration of 20 ng / mL, and Y-27632 was further added so as to have a concentration of 10 μM. , Matrigel to a concentration of 1%, Wnt3a to a concentration of 20 ng / mL, CHIR99021 to a concentration of 10 μM, and heparin to a concentration of 50 ng / mL. The cells were suspended and suspended for suspension using a medium prepared by adding retinoic acid to a concentration of 0.5 μM and adding R-spondin1 to a concentration of 200 ng / mL. Started.
（Ｄａｙ２３）
　浮遊培養後５日目（Ｄａｙ２３）に、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ＥＧＦを濃度が２０ｎｇ／ｍＬとなるように添加して調製した培地で全量培地交換を行い、通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で浮遊培養を行った。その後は、３日に１度の頻度で培地交換した。 (Day23)
On the 5th day (Day 23) after the suspension culture, the whole medium was exchanged with a medium prepared by adding growth factor EGF to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentration was 20 ng / mL, and the medium was exchanged under normal oxygen conditions. They were cultured in suspension under (O 2 20%, CO 2 5%). After that, the medium was changed once every 3 days.
（Ｄａｙ４２）
　Ｄａｙ４２にスフェア（細胞塊）を４％パラフォルムアルデヒドで固定し、凍結切片を作成した。 (Day 42)
A sphere (cell mass) was fixed on Day 42 with 4% paraformaldehyde to prepare a frozen section.
　免疫染色は、抗原賦活化操作を行ったのち、マウス抗ＭＹＯ７Ａ抗体、マウス抗ＢＲＮ３Ｃ抗体、ウサギ抗ＳＯＸ２抗体、ヤギ抗ＳＯＸ２抗体、ヤギ抗ＳＯＸ２１抗体、ウサギ抗Ｃａｌｂｉｎｄｉｎ抗体（それぞれ、５０倍、１００倍、１００倍、１００倍、１０００倍希釈）により処理し、その後、それぞれの動物種ＩｇＧに特異的な蛍光２次抗体を用いて標識し、蛍光顕微鏡で観察した。図８には、得らえた顕微鏡観察像を示す（ｓｃａｌｅ　ｂａｒ：上段５０μｍ、下段２０μｍ）。 For immunostaining, after performing an antigen activation operation, mouse anti-MYO7A antibody, mouse anti-BRN3C antibody, rabbit anti-SOX2 antibody, goat anti-SOX2 antibody, goat anti-SOX21 antibody, and rabbit anti-Calbindin antibody (50 times and 100 times, respectively). , 100-fold, 100-fold, 1000-fold diluted), then labeled with a fluorescent secondary antibody specific for each animal species IgG and observed under a fluorescent microscope. FIG. 8 shows the obtained microscopic observation image (scale bar: upper 50 μm, lower 20 μm).
　その結果、図８に示されるように、有毛細胞マーカーであるＭＹＯ７Ａ、ＢＲＮ３Ｃ陽性細胞、支持細胞マーカーであるＳＯＸ２、ＳＯＸ２１陽性細胞、らせん神経節細胞のマーカーであるＣａｌｂｉｎｄｉｎ陽性細胞が検出された。 As a result, as shown in FIG. 8, hair cell markers MYO7A, BRN3C positive cells, sustentacular cell markers SOX2, SOX21 positive cells, and spiral ganglion cell markers Calbindin positive cells were detected.
　このことから、本誘導法を用いて培養することで、多能性幹細胞から、支持細胞及びらせん神経節細胞をともなう内耳有毛細胞を分化誘導できることが明らかとなった。 From this, it was clarified that by culturing using this induction method, differentiation of inner ear hair cells with sustentacular cells and spiral ganglion cells can be induced from pluripotent stem cells.
　［試験例７］
　内耳前駆細胞から内耳有毛細胞を分化誘導する過程で、培地に各種抗Ｆｒｉｚｚｌｅｄ剤を添加することによる効果を検討した。 [Test Example 7]
In the process of inducing differentiation of inner ear hair cells from inner ear progenitor cells, the effect of adding various anti-Frizzled agents to the medium was investigated.
　［分化誘導方法］
　試験例１と同様の方法で分化誘導を行い、Ｄａｙ１１にアクターゼによる細胞剥離処理を行った細胞について、翌日（Ｄａｙ１２）、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加して調製した培地に培地交換し、通常酸素条件下（Ｏ２２０％、ＣＯ２５％）で培養を行った。 [Differentiation induction method]
Growth factors bFGF, EGF, and IGF were placed in serum-free medium (DMEM / F12 + B27-VA + N2) the next day (Day 12) for cells that had been induced to differentiate in the same manner as in Test Example 1 and had undergone cell detachment treatment with Actase on Day 11. -1 was added so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively, and CHIR99021 was added so that the concentration was 3 μM, and retinoic acid was added so that the concentration was 0.5 μM. the medium was replaced with medium prepared by adding to, were cultured in normoxic conditions (O 2 20%, CO 2 5%).
　以後Ｄａｙ１８まで、毎日培地交換を行った。 After that, the medium was changed every day until Day 18.
（Ｄａｙ１８）
　細胞をアクターゼで剥離し、等量のＰＢＳを加え、ナイロンメッシュ（孔径４０μｍ）を通し、血球計算盤で細胞数をカウントした。 (Day18)
The cells were detached with actase, an equal amount of PBS was added, the cells were passed through a nylon mesh (pore diameter 40 μm), and the number of cells was counted by a hemocytometer.
　８．５×１０４ｃｅｌｌｓ／ｗｅｌｌになるように細胞を懸濁し、低吸着６ウェルプレート（商品名「Ｃｏｒｎｉｎｇ超低接着表面（Ｕｌｔｒａ－Ｌｏｗ　Ａｔｔａｃｈｍｅｎｔ）プレート」、Ｃｏｒｎｉｎｇ社）に播種し、低酸素条件下（Ｏ２４％、ＣＯ２５％）で浮遊培養を開始した。このとき、培地としては、無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７－ＶＡ＋Ｎ２）に、成長因子ｂＦＧＦ、ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が１０ｎｇ／ｍＬ、２０ｎｇ／ｍＬ、５０ｎｇ／ｍＬとなるように添加し、更に、Ｙ－２７６３２を濃度が１０μＭとなるように添加し、Ｍａｔｒｉｇｅｌを濃度が１％となるように添加し、Ｗｎｔ３ａを濃度が２０ｎｇ／ｍＬとなるように添加し、ＣＨＩＲ９９０２１を濃度が３μＭとなるように添加し、ヘパリンを濃度が５０ｎｇ／ｍＬとなるように添加し、レチノイン酸を濃度が０．５μＭとなるように添加し、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加して調製した培地を使用して、細胞を懸濁して浮遊培養を開始した。 The cells were suspended so as to be 8.5 × 10 4 cells / well, seeded on a low-adsorption 6-well plate (trade name “Corning ultra-low adhesive surface (Ultra-Low Attachment) plate”, Corning), and hypoxic. It was initiated suspension culture under conditions (O 2 4%, CO 2 5%). At this time, as a medium, growth factors bFGF, EGF, and IGF-1 were added to a serum-free medium (DMEM / F12 + B27-VA + N2) so that the concentrations were 10 ng / mL, 20 ng / mL, and 50 ng / mL, respectively. Further, Y-27632 was added so that the concentration was 10 μM, Matrigel was added so that the concentration was 1%, Wnt3a was added so that the concentration was 20 ng / mL, and CHIR99021 was added so that the concentration was 3 μM. Add heparin to a concentration of 50 ng / mL, add retinoic acid to a concentration of 0.5 μM, and add R-spondin1 to a concentration of 200 ng / mL. Using the prepared medium, the cells were suspended and suspension culture was started.
（Ｄａｙ２２）
　浮遊培養後４日目（Ｄａｙ２２）にＲＯＣＫ阻害剤であるＹ－２７６３２を添加しないこと以外は、同じ組成の培地で全量培地交換を行った。 (Day22)
On the 4th day (Day 22) after the suspension culture, the whole medium was replaced with a medium having the same composition except that the ROCK inhibitor Y-27632 was not added.
（Ｄａｙ２４）
　浮遊培養６日目（Ｄａｙ２４）にスフェア（細胞塊）を回収し、ｐｏｌｙ－ｏ－ｆｉｂｒｏｎｅｃｔｉｎでコーティングしたプレートに接着させた。 (Day24)
On the 6th day of suspension culture (Day 24), spheres (cell clumps) were collected and adhered to a plate coated with poly-o-fibronectin.
　翌日、下記条件１）～５）のそれぞれの培地条件となるよう、培地交換した。
・条件１）無血清培地（ＤＭＥＭ／Ｆ１２＋Ｂ２７＋Ｎ２）に、成長因子ＥＧＦ、ＩＧＦ－１を、それぞれ濃度が２５ｎｇ／ｍＬ、１０ｎｇ／ｍＬとなるように添加し、更に、Ｗｎｔ３ａを濃度が１０ｎｇ／ｍＬとなるように添加して調製した培地
・条件２）上記条件１）の培地に、更に、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加して調製した培地
・条件３）上記条件１）の培地に、更に、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加し、Ｆｒｉｚｚｌｅｄ７の擬似分子（以下「ＦＺＤ７」という場合がある。）を濃度が１００ｎｇ／ｍＬとなるように添加して調製した培地
・条件４）上記条件１）の培地に、更に、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加し、Ｆｒｉｚｚｌｅｄ８の擬似分子（以下「ＦＺＤ８」という場合がある。）を濃度が１００ｎｇ／ｍＬとなるように添加して調製した培地
・条件５）上記条件１）の培地に、更に、Ｒ－ｓｐｏｎｄｉｎ１を濃度が２００ｎｇ／ｍＬとなるように添加し、Ｆｒｉｚｚｌｅｄ１０の擬似分子（以下「ＦＺＤ１０」という場合がある。）を濃度が１００ｎｇ／ｍＬとなるように添加して調製した培地 The next day, the medium was exchanged so as to meet the following conditions 1) to 5).
-Condition 1) Growth factors EGF and IGF-1 were added to a serum-free medium (DMEM / F12 + B27 + N2) so that the concentrations were 25 ng / mL and 10 ng / mL, respectively, and Wnt3a was further added to a concentration of 10 ng / mL. Medium prepared by adding so as to condition 2) Medium prepared by adding R-spondin1 to the medium of the above condition 1) so as to have a concentration of 200 ng / mL 3) The above condition 1) R-spondin1 was further added to the medium so as to have a concentration of 200 ng / mL, and a pseudo-molecule of Frizzled 7 (hereinafter, may be referred to as “FZD7”) was added so as to have a concentration of 100 ng / mL. Prepared medium-Condition 4) R-spondin1 was further added to the medium of the above condition 1) so as to have a concentration of 200 ng / mL, and a pseudo-molecule of Frizzled 8 (hereinafter, may be referred to as "FZD8") was concentrated. Medium prepared by adding so that A medium prepared by adding "FZD10") so as to have a concentration of 100 ng / mL.
（Ｄａｙ３０）
　接着培養６日後（Ｄａｙ２４）の細胞について、試験例１と同様にして、ｔｏｔａｌ　ＲＮＡを抽出、ｃＤＮＡ合成を行い、ｑＲＴ－ＰＣＲにより各培養条件における有毛細胞マーカーの発現量を定量した。 (Day30)
For cells 6 days after adherent culture (Day 24), total RNA was extracted and cDNA was synthesized in the same manner as in Test Example 1, and the expression level of hair cell markers under each culture condition was quantified by qRT-PCR.
　その結果、図９に示されるように、Ｗｎｔ３ａ及びＲ－ｓｐｏｎｄｉｎ１及びＦＺＤ１０を添加して培養した条件においては、蝸牛有毛細胞マーカーであるＭＹＯ７ＡやＭＹＯ１５Ａの発現量が亢進していた。このことから、ＷＮＴシグナルを活性化し、同時にＦＺＤ１０を介したシグナルを調節することで、蝸牛有毛細胞への分化誘導が促進されるものと考えられた。 As a result, as shown in FIG. 9, the expression levels of the cochlear hair cell markers MYO7A and MYO15A were enhanced under the conditions of culturing with the addition of Wnt3a, R-spondin1 and FZD10. From this, it was considered that the induction of differentiation into cochlear hair cells is promoted by activating the WNT signal and at the same time regulating the signal mediated by FZD10.
　（処方例１）
　レチノイン酸の粉末をＤＭＳＯで２０ｍＭとなるように溶解した。また、ＣＨＩＲ９９０２１の粉末をＤＭＳＯで１０ｍＭとなるように溶解した。これらを同量混合し、レチノイン酸及びＣＨＩＲ９９０２１を含有する培地を調製するためのプレミックス液の形態とした。このプレミックス液は、４℃の冷蔵庫で２週間～１ヵ月程度保管可能であった。 (Prescription example 1)
The retinoic acid powder was dissolved in DMSO to 20 mM. Further, the powder of CHIR99021 was dissolved in DMSO so as to be 10 mM. These were mixed in the same amount to prepare a premix solution for preparing a medium containing retinoic acid and CHIR99021. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.
　（処方例２）
　レチノイン酸の粉末をＤＭＳＯで２０ｍＭとなるように溶解した。また、ＢＩＯの粉末をＤＭＳＯで１ｍＭとなるように溶解した。これらを同量混合し、レチノイン酸及びＢＩＯを含有する培地を調製するためのプレミックス液の形態とした。このプレミックス液は、４℃の冷蔵庫で２週間～１ヵ月程度保管可能であった。 (Prescription example 2)
The retinoic acid powder was dissolved in DMSO to 20 mM. In addition, BIO powder was dissolved in DMSO to 1 mM. These were mixed in the same amount to prepare a premixed solution for preparing a medium containing retinoic acid and BIO. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.
　（処方例３）
　レチノイン酸の粉末をＤＭＳＯで２０ｍＭとなるように溶解した。また、ＬｉＣｌ（塩化リチウム）の粉末をＭｉｌｌｉＱ水で２Ｍとなるように溶解した。これらを同量混合し、レチノイン酸及びＬｉＣｌを含有する培地を調製するためのプレミックス液の形態とした。このプレミックス液は、４℃の冷蔵庫で２週間～１ヵ月程度保管可能であった。 (Prescription example 3)
The retinoic acid powder was dissolved in DMSO to 20 mM. Further, LiCl (lithium chloride) powder was dissolved in MilliQ water so as to have a concentration of 2M. These were mixed in the same amount to prepare a premixed solution for preparing a medium containing retinoic acid and LiCl. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.

Claims (17)

  1. 　多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団を、細胞剥離処理したうえ、レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地で培養する工程を含む、内耳前駆細胞の製造方法。 A cell population containing PAX2 / PAX8-positive cells induced to differentiate from pluripotent stem cells is subjected to cell exfoliation treatment and then cultured in a medium containing retinoic acid and an inducer of a first Wnt / β catenin pathway. Method for producing inner ear progenitor cells.
  2. 　前記レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地は、ＩＧＦ－１、ｂＦＧＦ、及びＥＧＦからなる群から選ばれた１種又は２種以上を含有する、請求項１記載の内耳前駆細胞の製造方法。 10. The medium according to claim 1, wherein the medium containing the retinoic acid and the inducer of the first Wnt / β-catenin pathway contains one or more selected from the group consisting of IGF-1, bFGF, and EGF. Method for producing inner ear progenitor cells.
  3. 　前記レチノイン酸及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地は、無血清培地である、請求項１又は２記載の内耳前駆細胞の製造方法。 The method for producing inner ear progenitor cells according to claim 1 or 2, wherein the medium containing the retinoic acid and the inducer of the first Wnt / β-catenin pathway is a serum-free medium.
  4. 　前記レチノイン酸源及び第１のＷｎｔ／βカテニン経路の誘導剤を含有する培地による培養は、接着培養によるものである、請求項１～３のいずれか１項に記載の内耳前駆細胞の製造方法。 The method for producing inner ear progenitor cells according to any one of claims 1 to 3, wherein the culture in the medium containing the retinoic acid source and the inducer of the first Wnt / β-catenin pathway is by adhesive culture. ..
  5. 　前記細胞剥離処理は、所定の孔径を有するメッシュを通す工程を含む、請求項１～４のいずれか１項に記載の内耳前駆細胞の製造方法。 The method for producing inner ear progenitor cells according to any one of claims 1 to 4, wherein the cell exfoliation treatment includes a step of passing a mesh having a predetermined pore size.
  6. 　前記第１のＷｎｔ／βカテニン経路の誘導剤は、ＣＨＩＲ９９０２１、ＢＩＯ、及びＬｉＣｌからなる群から選ばれた１種又は２種以上である、請求項１～５のいずれか１項に記載の内耳前駆細胞の製造方法。 The inner ear according to any one of claims 1 to 5, wherein the first Wnt / β-catenin pathway inducer is one or more selected from the group consisting of CHIR99021, BIO, and LiCl. Method for producing progenitor cells.
  7. 　陽性NKX6.1陰性細胞が、次の２つの工程を含む方法で多能性幹細胞より製造された細胞である、請求項１から５のいずれか１項に記載の方法：
    （１）多能性幹細胞を、アクチビンを含む培地で培養する工程、および
    （２）工程（１）で得られた細胞を、KGFを含む培地で培養する工程。前記多能性幹細胞から分化誘導したＰＡＸ２／ＰＡＸ８陽性細胞を含む細胞集団が、以下の工程（１）～（４）を含む方法で得られたものである、請求項１～６のいずれか１項に記載の内耳前駆細胞の製造方法。
    　（１）多能性幹細胞を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の存在下で培養する工程
    　（２）工程（１）で得られた細胞集団を、成長因子の非存在下であって、ＲＯＣＫ阻害剤の非存在下で培養する工程
    　（３）工程（２）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子、及びＢＭＰ４の存在下で培養する工程と、
    　（４）工程（３）で得られた細胞集団を、ｂＦＧＦ、ＦＧＦ３、ＦＧＦ１０、及びＦＧＦ１９からなる群から選ばれた少なくとも１種の成長因子の存在下であって、ＢＭＰ４の非存在下で培養する工程     The method according to any one of claims 1 to 5, wherein the positive NKX6.1 negative cell is a cell produced from a pluripotent stem cell by a method including the following two steps:
    (1) A step of culturing pluripotent stem cells in a medium containing activin, and (2) a step of culturing the cells obtained in step (1) in a medium containing KGF. Any one of claims 1 to 6, wherein the cell population containing the PAX2 / PAX8-positive cells induced to differentiate from the pluripotent stem cells was obtained by a method including the following steps (1) to (4). The method for producing inner ear progenitor cells according to the section.
    (1) A step of culturing pluripotent stem cells in the absence of a growth factor and in the presence of a ROCK inhibitor (2) A cell population obtained in step (1) in the absence of a growth factor. (3) Growth of at least one cell population obtained in step (2) selected from the group consisting of bFGF, FGF3, FGF10, and FGF19. The step of culturing in the presence of factors and BMP4, and
    (4) The cell population obtained in step (3) is cultured in the presence of at least one growth factor selected from the group consisting of bFGF, FGF3, FGF10, and FGF19 in the absence of BMP4. Process to do
  8. 　請求項１～７のいずれか１項に記載の製造方法で得られた内耳前駆細胞を、該内耳前駆細胞を含む細胞集団の状態で、以下の工程（ｉ）及び（ｉｉ）に処することを含む、内耳有毛細胞の製造方法。
    　（ｉ）前記内耳前駆細胞を含む細胞集団を浮遊培養する工程
    　（ｉｉ）工程（ｉ）で得られた細胞集団を接着培養する工程     The inner ear progenitor cells obtained by the production method according to any one of claims 1 to 7 are subjected to the following steps (i) and (ii) in the state of a cell population containing the inner ear progenitor cells. Methods for producing inner ear hair cells, including.
    (I) Step of suspension-culturing the cell population containing the inner ear progenitor cells (ii) Step of adhesion-culturing the cell population obtained in step (i)
  9. 　前記工程（ｉ）における該浮遊培養は、第２のＷｎｔ／βカテニン経路の誘導剤を含有する培地で行う、請求項８記載の内耳有毛細胞の製造方法。 The method for producing inner ear hair cells according to claim 8, wherein the suspension culture in the step (i) is carried out in a medium containing an inducer for the second Wnt / β-catenin pathway.
  10. 　前記第２のＷｎｔ／βカテニン経路の誘導剤は、Ｒ－ｓｐｏｎｄｉｎ１、ＣＨＩＲ９９０２１、及びＷｎｔ３ａからなる群から選ばれた１種又は２種以上である、請求項９記載の内耳有毛細胞の製造方法。 The method for producing inner ear hair cells according to claim 9, wherein the second Wnt / β-catenin pathway inducer is one or more selected from the group consisting of R-spondin1, CHIR99021, and Wnt3a. ..
  11. 　前記工程（ｉｉ）における該接着培養は、第３のＷｎｔ／βカテニン経路の誘導剤及び抗Ｆｒｉｚｚｌｅｄ剤を含有する培地で行う、請求項８～１０のいずれか１項に記載の内耳有毛細胞の製造方法。 The inner ear hair cell according to any one of claims 8 to 10, wherein the adhesive culture in the step (ii) is carried out in a medium containing an inducer of a third Wnt / β-catenin pathway and an anti-Friszled agent. Manufacturing method.
  12. 　前記第３のＷｎｔ／βカテニン経路の誘導剤は、Ｗｎｔ３ａ及び／又はＲ－ｓｐｏｎｄｉｎ１であり、前記抗Ｆｒｉｚｚｌｅｄ剤は、Ｆｒｉｚｚｌｅｄ１０の擬似分子による競合剤である、請求項１１記載の内耳有毛細胞の製造方法。 The inner ear hair cell according to claim 11, wherein the third Wnt / β-catenin pathway inducer is Wnt3a and / or R-spondin1, and the anti-Frizzled agent is a competitor by a pseudo-molecule of Frizzled10. Production method.
  13. 　請求項１～７のいずれか１項に記載の製造方法で得られた内耳前駆細胞を、被検薬剤で処理する工程と、前記被検薬剤で処理した前記内耳前駆細胞の状態を評価する工程とを含む、薬剤の評価方法。 A step of treating the inner ear progenitor cells obtained by the production method according to any one of claims 1 to 7 with a test agent, and a step of evaluating the state of the inner ear progenitor cells treated with the test agent. Drug evaluation methods, including.
  14. 　請求項８～１２のいずれか１項に記載の製造方法で得られた内耳有毛細胞を、被検薬剤で処理する工程と、前記被検薬剤で処理した前記内耳有毛細胞の状態を評価する工程とを含む、薬剤の評価方法。 The step of treating the inner ear hair cells obtained by the production method according to any one of claims 8 to 12 with a test agent and the state of the inner ear hair cells treated with the test agent are evaluated. A method for evaluating a drug, including the steps to be performed.
  15. 　レチノイン酸及びＷｎｔ／βカテニン経路の誘導剤を含有する内耳細胞分化誘導用組成物。 A composition for inducing inner ear cell differentiation containing retinoic acid and an inducer of the Wnt / β-catenin pathway.
  16. 　前記Ｗｎｔ／βカテニン経路の誘導剤は、ＣＨＩＲ９９０２１、ＢＩＯ、及びＬｉＣｌからなる群から選ばれた１種又は２種以上である、請求項１５記載の内耳細胞分化誘導用組成物。 The composition for inducing inner ear cell differentiation according to claim 15, wherein the Wnt / β-catenin pathway inducer is one or more selected from the group consisting of CHIR99021, BIO, and LiCl.
  17. 　前記内耳細胞分化誘導用組成物は、内耳前駆細胞誘導用のものである、請求項１５又は１６記載の内耳細胞分化誘導用組成物。
     
          The composition for inducing inner ear cell differentiation according to claim 15 or 16, wherein the composition for inducing inner ear cell differentiation is for inducing inner ear progenitor cells.
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