WO2021251419A1 - Procédé de production de cellules capillaires de l'oreille interne, procédé d'évaluation de médicament et composition pour induire la différenciation cellulaire - Google Patents

Procédé de production de cellules capillaires de l'oreille interne, procédé d'évaluation de médicament et composition pour induire la différenciation cellulaire Download PDF

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WO2021251419A1
WO2021251419A1 PCT/JP2021/021863 JP2021021863W WO2021251419A1 WO 2021251419 A1 WO2021251419 A1 WO 2021251419A1 JP 2021021863 W JP2021021863 W JP 2021021863W WO 2021251419 A1 WO2021251419 A1 WO 2021251419A1
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inner ear
cells
medium
wnt
inducer
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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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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • C12N5/06—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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a method for producing inner ear hair cells, a method for evaluating a drug, and a composition for inducing 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.
  • the present invention uses a cell population containing inner ear progenitor cells obtained by inducing differentiation from pluripotent stem cells as a first Wnt / ⁇ catenin pathway inducer and an anti-Flizzled agent. It provides a method for producing inner ear hair cells, which comprises a step of culturing in a contained medium.
  • a cell population containing inner ear progenitor cells induced to differentiate from pluripotent stem cells is used as an inducer for the first Wnt / ⁇ catenin pathway. Since the cells are cultured in a medium containing an anti-frizzled agent, good quality inner ear hair cells can be efficiently obtained.
  • the medium containing the first Wnt / ⁇ -catenin pathway inducer and anti-Frizzled agent was selected from the group consisting of IGF-1, bFGF, and EGF. It is preferable to contain one kind or two or more kinds. According to this, it is possible to more reliably differentiate into good quality inner ear hair cells.
  • the medium containing the first Wnt / ⁇ -catenin pathway inducer and anti-Frizzled agent 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 first 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 method for producing inner ear hair cells includes the following steps (1) and (2).
  • the cell population obtained in step (1) is the first Wnt / ⁇ .
  • Step of Adhesive Culture in Medium Containing Inducer of Catenin Pathway and Anti-Flizzled Agent The suspension culture in the step (1) is preferably carried out in a medium containing an inducer of the second Wnt / ⁇ catenin pathway.
  • the cell population containing the inner ear progenitor cells obtained by inducing differentiation from pluripotent stem cells is subjected to suspension culture in a medium containing an inducer of the Wnt / ⁇ -catenin pathway, so that the quality is good. Differentiation into inner ear hair cells can be made more reliable.
  • the medium containing the second Wnt / ⁇ -catenin pathway inducer is one or 2 selected from the group consisting of IGF-1, bFGF, and EGF. It is preferable to contain seeds or more. According to this, it is possible to more reliably differentiate into good quality inner ear hair cells.
  • the medium containing the second Wnt / ⁇ -catenin pathway inducer 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 second Wnt / ⁇ -catenin pathway inducer is one or more selected from the group consisting of R-spondin1, CHIR99021, and Wnt3a. Is preferable.
  • the cell population containing the inner ear progenitor cells obtained by inducing differentiation from the pluripotent stem cells is retinoic acid and an inducer of the third Wnt / ⁇ catenin pathway. It is preferably obtained by a method including a step of culturing in the contained medium. According to this, it is possible to more reliably induce differentiation of a cell population containing cells expressing PAX2 and / or PAX8, which are markers for the planned inner ear region, from pluripotent stem cells.
  • the third Wnt / ⁇ -catenin pathway inducer may be one or more selected from the group consisting of CHIR99021, BIO, and LiCl. preferable.
  • 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 present invention provides, from the third aspect, a composition for inducing cell differentiation containing a competitor selective for the type of Frizzled receptor.
  • composition for inducing cell differentiation According to the composition for inducing cell differentiation according to the third aspect of the present invention, efficient preparation of inner ear hair cells can be performed more easily by using the composition.
  • the competing agent is preferably a competing agent based on a pseudo-molecule of Frizzled 10.
  • composition for inducing cell differentiation is for inducing hair cells in the inner ear.
  • the first Wnt / ⁇ -catenin pathway inducer means that "Wnt / ⁇ -catenin pathway inducer" is used in different steps, and does not mean that the material constituents are distinguished from each other. 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. As 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 organ, and more specifically, has been improved so as to promote differentiation induction from pluripotent stem cells to inner ear hair cells. , Concerning a method for producing inner ear hair cells.
  • 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.
  • the present invention it is necessary to culture the inner ear progenitor cells obtained by inducing differentiation from pluripotent stem cells in a medium containing an inducer of the first Wnt / ⁇ -catenin pathway and an anti-Frizzled agent. Other than that, 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 hair 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.
  • inducing differentiation of pluripotent stem cells into inner ear progenitor cells is, for example, the following steps (1) to (4). It can be done by passing.
  • 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: Every M, Ellwanger DC, Kosaric N, Stopper AP, Heller S. "Single-cell analysis direct lineage technique”. optic lineage. ”Proc Natl Acad Scii US A.
  • FIG. 1 illustrates an embodiment of the method for producing inner ear hair cells according to the present invention using a flow chart.
  • a cell population containing inner ear progenitor cells obtained by inducing differentiation from pluripotent stem cells is subjected to a specific treatment to promote differentiation induction into inner ear hair cells.
  • the inner ear progenitor cells obtained by inducing differentiation from pluripotent stem cells are subjected to a medium containing an inducer of the first Wnt / ⁇ -catenin pathway and an anti-Frizzled agent. Incubate.
  • 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.).
  • 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.
  • the culture is preferably carried out in an environment in which EGF is at least present as a growth factor. According to this, it is possible to more reliably differentiate into inner ear hair cells.
  • the culturing method suspension culturing may be performed, adhesive culturing may be performed, or both culturing methods may be continuously used in combination with each other.
  • the culture period is preferably 15 to 30 days in total, more preferably 18 to 27 days, and even more preferably 21 to 24 days.
  • Examples of the first Wnt / ⁇ -catenin pathway inducer include Wnt3a, 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) 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 first Wnt / ⁇ -catenin pathway in the medium is preferably 10 ng / mL or more as the lower limit value, and preferably 1000 ng / mL or less as the upper limit value. ..
  • 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 is preferably 10 ng / mL or more, more preferably 20 ng / mL or more, and even more preferably 50 ng / mL or more as the lower limit. ..
  • 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.
  • FIG. 2 illustrates another embodiment of the method for producing inner ear hair cells according to the present invention by means of a flow chart.
  • the culture in the medium containing the above-mentioned first Wnt / ⁇ -catenin pathway inducer and anti-Frizzled agent is carried out by the following steps (i) and (ii).
  • the cell population obtained in step (i) is the first Wnt / ⁇ .
  • Step of adhesion culture in a medium containing an inducer of the catenin pathway and the anti-Flizzled agent is carried out by the following steps (i) and (ii).
  • 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.).
  • 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 suspension-cultured cells and cell populations are further adherently cultured in a medium containing an inducer of the first Wnt / ⁇ -catenin pathway and an anti-Frizzled agent.
  • 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”
  • other supplemental nutritional components may be supplemented.
  • 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 preferred concentration range of the first Wnt / ⁇ -catenin pathway inducer and the anti-Frizzled agent in the culture medium of the adhesive culture is the same as the preferred concentration range in the medium described above.
  • inner ear hair cells are induced to differentiate from inner ear progenitor cells obtained from pluripotent stem cells, but in any aspect thereof, the cells are not limited.
  • the inner ear progenitor cells to be used may be activated after being further subjected to the following treatment after the series of cultures of the first step to the fourth step described above.
  • Fifth step The cell population obtained in step (4) is subjected to cell exfoliation treatment and then cultured in a medium containing retinoic acid and a third Wnt / ⁇ -catenin pathway inducer.
  • 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, and examples thereof include enzymatic treatment with trypsin, actase, and the like. 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 population treated with the above-mentioned cell exfoliation are further cultured in a medium containing retinoic acid and an inducer of the third Wnt / ⁇ -catenin pathway.
  • a medium containing retinoic acid and an inducer of the third 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 third Wnt / ⁇ -catenin pathway.
  • retinoic acid and a third Wnt / ⁇ -catenin pathway inducer are contained in a medium in combination and cultured in the medium, the expression levels of PAX2 and / or PAX8 are increased. Be done.
  • retinoin acid include all-trans type retinoin acid.
  • CHIR99021 also known as 6-((2-((4- (2,4-Dichlophoreneyl) -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 Wnt3a, R-spondin1 and the like.
  • CHIR99021, BIO, LiCl (lithium chloride) and the like are more preferably exemplified.
  • 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 third 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 third 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 third 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 third 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.
  • IGF-1 it is preferably 10 to 50 ng / mL.
  • the inner ear progenitor cells obtained as described above have improved PAX2 and / or PAX8 expression levels as compared with those before culturing in a medium containing the above-mentioned retinoic acid and a third Wnt / ⁇ -catenin pathway inducer. 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.
  • any test substance is allowed to act on the inner ear hair cells obtained by the above method, and the effect on the inner ear hair cells. It provides a method for evaluating a drug, which evaluates the test substance by examining.
  • the present invention provides a composition for inducing cell differentiation containing a competitor selective for the type of Frizzled receptor.
  • a competitor selective for the type of Frizzled receptor a fusion protein consisting of an extracellular domain fragment of a human Frizzled receptor and a human Fc domain is known, and thus a pseudo-molecule of such Frizzled receptor is used. be able to.
  • a pseudo molecule of Frizzled 10 is preferably exemplified as a competitive agent selective for the type of Frizzled receptor.
  • 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 content of the competing agent selective for the type of Frizzled receptor in the composition for inducing cell differentiation according to the present invention is not particularly limited, but the lower limit thereof is typically 0.5 ng / mL or more. It is more typical that it is 1 ng / mL or more, it is even more typical that it is 10 ng / mL or more, and it is particularly typical that it is 50 ng / mL or more.
  • the upper limit is typically 10 ⁇ g / mL or less, more typically 5 ⁇ g / mL or less, and even more typically 1 ⁇ g / mL or less. It is particularly typical that it is 5 ⁇ g / mL or less.
  • the composition for inducing cell differentiation according to the present invention is preferably used for inducing differentiation of pluripotent stem cells into inner ear hair cells.
  • the method for producing inner ear hair cells according to the present invention which is used in the form of a culture premix solution, a liquid medium, or the like for use in culturing in a medium containing the above-mentioned antifrizzled agent. It can be carried out more easily.
  • 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.
  • Frizzled 10 pseudomolecule (trade name "Recombinant Human Frizzled-10 Fc Chimera Protein", R & D Systems) is dissolved in PBS to 100 ⁇ g / mL to prepare a medium containing an anti-Frizzled agent. It was in the form of a premix solution. This premix solution could be stored in a refrigerator at 4 ° C. for about 2 weeks to 1 month.

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Abstract

L'invention concerne un procédé de production de cellules capillaires d'oreille interne présentant une efficacité d'induction de différenciation améliorée.　Ce procédé de production de cellules capillaires d'oreille interne comprend l'étape de culture, dans un milieu contenant un premier inducteur de la voie Wnt/β-caténine et d'un agent anti-FZD, une population cellulaire comprenant des cellules précurseurs d'oreille interne obtenues par induction de cellules souches pluripotentes à différencier.
PCT/JP2021/021863 2020-06-09 2021-06-09 Procédé de production de cellules capillaires de l'oreille interne, procédé d'évaluation de médicament et composition pour induire la différenciation cellulaire WO2021251419A1 (fr)

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JP2015231365A (ja) * 2014-05-13 2015-12-24 学校法人慶應義塾 内耳細胞誘導方法
WO2016117431A1 (fr) * 2015-01-19 2016-07-28 学校法人慶應義塾 Agent thérapeutique pour perte d'audition neurosensorielle
WO2017132530A1 (fr) * 2016-01-29 2017-08-03 Massachusetts Eye And Ear Infirmary Expansion et différenciation des cellules de support de l'oreille interne, et méthodes d'utilisation de ces dernières

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