JP2015231365A - Method of inducing inner ear cell - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of inducing an inner ear cell from a pluripotent stem cell.SOLUTION: In a method of inducing an inner ear cell, a process of culturing a pluripotent stem cell under the presence of ROCK Inhibitor; a process of culturing the stem cell under the absence of ROCK Inhibitor; a process of culturing the stem cell on a serum-free medium; a process of culturing the stem cell on a serum-free medium containing a growth factor; and a process of dissociating the stem cell into a single cell are performed in this order, and the cell is induced from the pluripotent stem cell to an inner ear stem cell. The pluripotent stem cell is an embryonic stem cell (ES cell) or an artificial pluripotent stem cell (iPS cell). The method further includes a process of culturing the cell dissociated into the single cell on a culture dish coated by poly-O-fibronectine.

Description

  The present invention relates to a method for guiding inner ear cells.

  The pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) are highly expected to be applied to regenerative medicine or research tools (drug discovery, pathological elucidation).

  However, when used for any purpose, it is necessary to stably supply a large amount of high-quality cells at an appropriate price. Conventionally, when inner ear stem cells are derived from human ES cells, embryoid body formation and accompanying methods are involved. It was necessary to select inner ear stem cells under visual observation, and a long culture period and low efficiency were problems (Non-Patent Document 1-2). Furthermore, although a method for inducing inner ear hair cells from inner ear stem cells has been reported (Non-patent Document 1-2), it is very inefficient and has a quality sufficient to withstand drug toxicity confirmation and evaluation of inner ear hair cell cilia. Hair cells have not been reported, and there have been problems in terms of quality when applied to drug discovery and regenerative medicine.

  In addition, inner ear fiber cells and vascular streak cells, which are cells that constitute the inner ear so far and are thought to be deeply involved in hearing loss due to drug toxicity, have not been induced from pluripotent stem cells, It was a restriction.

  In this way, inner ear cells are difficult to derive from pluripotent stem cells, or even if induced, the quality is poor, and it is extremely difficult to use for regenerative medicine or research tools. In addition, a high-quality method for inducing inner ear cells has been demanded.

  On the other hand, with regard to hearing impairments caused by various causes, and in particular, hearing impairments caused by drugs, it is difficult to measure / compare hearing in laboratory animals, and scientific evaluation is difficult. Development is expected.

Nature 2012 Oct 11; 490 (7419): 278-82 Stem cells Dev. 2014 Mar 10 published online

  It is an object of the present invention to provide a method for inducing inner ear cells.

  One embodiment of the present invention is a method for inducing pluripotent stem cells into inner ear stem cells, the step of culturing the pluripotent stem cells in the presence of ROCK Inhibitor, the step of culturing in the absence of ROCK Inhibitor, This is an induction method in which a step of culturing in a serum-free medium, a step of culturing in a growth factor-containing serum-free medium, and a step of dissociating into single cells are performed in this order. The pluripotent stem cell may be an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell). And further culturing the cells dissociated into single cells in a culture dish coated with poly-O-fibronectine.

  A further embodiment of the present invention is a method for inducing inner ear sensory epithelium (support cells, hair cells) and cochlear ganglion cells from inner ear stem cells, wherein the inner ear stem cells are cultured in suspension in the presence of FGF9 and FGF20. And an adhesion culture method in which the steps of adhesion culture are performed in this order.

  A further embodiment of the present invention is a method for inducing vascular marginal cells from inner ear stem cells, comprising the steps of suspension culture of the inner ear stem cells in the presence of FGF3 and / or FGF10, and adhesion culture. It is a guidance method performed in order.

  A further embodiment of the present invention is a method for inducing Periotic mesenchymal cells from inner ear stem cells, comprising the step of culturing the inner ear stem cells in the presence of bFGF.

  A further embodiment of the present invention is a method for inducing cochlear fibrocytes and vascular streak cells from Periotic mesenchymal cells, the step of culturing the Periotic mesenchymal cells in the presence of bFGF, and culturing in the absence of bFGF. And a step of obtaining cochlear fiber cells and vascular streak cells in this order. The vascular streak cell may be a vascular basal cell.

A further embodiment of the present invention is a method for inducing Pendrin positive cells from Periotic mesenchymal cells, comprising culturing the Periotic mesenchymal cells in the presence of bFGF, and culturing in the presence of NaHCO _{3 in the} absence of bFGF. Then, the process of obtaining Pendrin positive cells is performed in this order. It is a guidance method.

  According to the present invention, a method for inducing inner ear cells can be provided.

(A) In one Example of this invention, it is a phase-contrast micrograph of the obtained inner ear stem cell. (B) In one Example of this invention, it is the microscope picture which showed the result of having dyed the obtained inner ear stem cell with (a) anti-PAX2 antibody, (b) anti-PAX8 antibody, (c) anti-SOX2 antibody. In one embodiment of the present invention (hair cell differentiation induction), inner ear stem cells are cultured in suspension and cultured in the presence of FGF9 and FGF20 to express hair cell markers espin, myosin 7a, and prestin. It is the microscope picture which showed that. (Left figure; triple staining) Stained with Espin, Myosin 7a, Prestin (upper right) Myosin 7a (lower right) Espin antibody. In one example of the present invention (induction of support cell differentiation), a micrograph showing that the inner ear stem cells are cultured in suspension and cultured in the presence of FGF9 and FGF20, and the support cell markers p27kip1 and ISLET1 are expressed. It is. (Left figure; triple staining) p27kip1, ISLET1, Prestin (right figure; double staining) ISLET1, Prestin antibody was stained. In one embodiment of the present invention (induction of differentiation of cochlear ganglion cells), inner ear stem cells are cultured in suspension and cultured in the presence of FGF9 and FGF20, so that GFAP, a marker of cochlear ganglion glial cells, It is the microscope picture which showed that the calbindin and beta III tubulin which are markers express. Triple staining was performed with antibodies of GFAP, calbindin and beta III tubulin. In one embodiment of the present invention (induction of differentiation of Periotic mesenchymal cells), a microscope showing that S100, POU3F4, caldesmon and TBX18, which are markers for Periotic mesenchymal cells, are expressed by culturing inner ear stem cells in the presence of bFGF. It is a photograph. (Upper left) S100, Caldesmon, TBX18 (Upper right) Triple staining was performed with antibodies of cx26, TBX18, and POU3f4. (Bottom) A phase contrast micrograph when cultured for a long time in the presence of bFGF. In one example of the present invention (induction of differentiation of inner ear fiber cells and PENDRIN positive cells), by culturing Periotic mesenchymal cells in the presence of bFGF and then in the absence of bFGF, cochlear fiber cells and cochlear vascular strips are obtained. Expression of basal cell markers carbonic acid dehydrogenase, aquaporin 1, sodium potassium ATPase, vimentin, connexins 26 and 3, Periotic mesenchymal cells are cultured in the presence of bFGF and then cultured in the presence of NaHCO ₃ Is a photomicrograph showing that Pendrin is expressed. (Upper left) CAII and connexin 26 (upper middle) Sodium potassium ATPase and connexin 26 (upper right) Aquaporin 1 and connexin 26 (lower left) Vimentin and connexin 26 were double-stained. (Lower right) Double staining with CAII and Pendrin antibodies. In one Example of this invention, it is the result which showed that inner ear sensory epithelial disorder was induced by gentamicin administration. FIG. 3 is a micrograph showing that inner ear stem cells are induced to differentiate into vascular marginal cells and NaKatpase A1 and NKCC1 are expressed in one example of the present invention.

  Hereinafter, embodiments of the present invention will be described in detail with reference to examples. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

  The inner ear cell induction method from pluripotent stem cells (ES / iPS cells) according to the present invention can be performed along the following flow.

(1) Differentiation induction from pluripotent stem cells to inner ear stem cells In the method for induction from pluripotent stem cells to inner ear stem cells according to the present invention,
1st step: a step of culturing pluripotent stem cells in the presence of a ROCK inhibitor 2nd step: a step of culturing in the absence of a ROCK inhibitor 3rd step: a step of culturing in a serum-free medium 4th step: containing a growth factor Step of culturing in serum-free medium Step 5: The step of dissociating into single cells is performed in this order. It should be noted that steps that are not essential to the present invention may be inserted between the steps.

  The pluripotent stem cell is not particularly limited as long as it has differentiation pluripotency (multipotency or pluripotency), and examples thereof include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), Muse cells, and the like. Particularly preferred are cells having totipotency.

  The ROCK (Rho-associated coiled-coil forming kinase) inhibitor is not particularly limited, and examples thereof include Y-27632, Fasudil hydrochloride, K-115 (lipasyl hydrochloride hydrate), DE-104 and the like. . As for the concentration of the ROCK inhibitor, an optimal concentration can be easily determined as appropriate, but it is preferably 0.05% to 0.2%, more preferably 0.1%.

  The medium used in the first step and the second step is not particularly limited as long as it can maintain pluripotent stem cells, and mTeSR1 can be exemplified. The first step is preferably performed for 1 to 3 days, and more preferably for 1 to 2 days. The second step is preferably performed for 1 to 3 days, and more preferably for 1 to 2 days.

  In the second step, the meaning in the absence of the ROCK inhibitor may be contained at a concentration at which the ROCK inhibitor is substantially absent as long as the ROCK inhibitor is substantially absent.

  Examples of the serum-free medium used in the third step include DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid. The culture in the third step is preferably performed for 2 to 6 days, more preferably 2 to 4 days, and most preferably 3 days.

  The serum-free medium used in the fourth step can be exemplified by DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid, but the same serum-free medium used in the third step is preferably used. As the growth factor, at least one growth factor in the group consisting of bFGF, FGF3, FGF10, and FGF19 may be added, but it is preferable to add all of them. Preferred concentrations for each are 10-50 ng / ml, 10-50 ng / ml, 10-50 ng / ml, 10-50 ng / ml. In addition, it is preferable to culture in the presence of BMP4 in the first stage of culture and in the absence of BMP4 in the second stage. The concentration of BMP4 added in the previous period is preferably 5 to 50 ng / ml. The meaning of absence of BMP4 in the latter culture is sufficient if BMP4 is substantially absent, and it may be contained at a level at which there is no effect. The first culture is preferably performed for 2 to 6 days, more preferably 2 to 4 days, and most preferably 3 days. Further, the late culture is preferably performed for 2 to 6 days, more preferably 2 to 4 days, and most preferably 3 days. The inner ear stem cells thus obtained have the ability to differentiate into inner ear stem cells.

  As the fifth step, the cell mass obtained in the fourth step is dissociated into a single cell. The method for dissociating into single cells is not particularly limited, and for example, trypsin or actase can be used. After dissociating with an enzyme or physical treatment (such as pipetting), to remove undifferentiated cells and cell clumps, use nylon mesh etc. to select cells dissociated into single cells and remove the remaining cell clumps It is preferable to do this.

In order to culture inner ear stem cells, the culture dish coated with a coating agent is used, and the cells are cultured under hypoxic conditions in order to maintain stem cell ability (sixth step). The coating agent to be used is not particularly limited, but poly-O-fibronectine is most preferable. The stem cells to be cultured may be those obtained in the fourth step or those obtained in the fifth step. Examples of the serum-containing medium used in this step include DMEM and F12, but DMEM / F12 containing N2 and B27 is most preferable in addition to serum (for example, FBS). Further, in the low oxygen condition of this step, the oxygen concentration is preferably 4% to 10%, more preferably 4% to 6%, and most preferably 4%. The growth factor added to the culture may be at least one growth factor selected from the group consisting of bFGF, EGF, and IGF1, but it is preferable to add all of them. Preferred concentrations for each are 10-30 ng / ml, 10-30 ng / ml, 10-50 ng / ml.

(2) Differentiation induction from inner ear stem cells to inner ear sensory epithelium (support cells, hair cells), cochlear ganglion cells and vascular marginal cells According to the present invention, inner ear stem cells to inner ear sensory epithelium, cochlear ganglion cells and blood vessels In the induction method to the limbic cell,
First step: Suspension culture of the inner ear stem cells in the presence of a growth factor;
2nd process: The process of adhesion culture is performed in this order. It should be noted that steps that are not essential to the present invention may be inserted before and after the step or between the steps.

  In the first step, inner ear stem cells are cultured in suspension in the presence of a growth factor. Specifically, first, inner ear stem cells are dissociated into single cells as in the fifth step. The dissociated cells are cultured in a floating state. Therefore, a dish for floating culture that can be cultured in a non-adherent state is used as the culture dish. For example, a non-adhesive culture dish such as a plastic culture dish may be used. Here, suspension culture means culturing the target cells or cell mass without adhering to the bottom of the incubator, and adhesion culture means adhering the target cells or cell mass to the bottom of the incubator. It means to culture. Here, during culture, cells and cell clumps adhere to the bottom of the incubator means that the cells and cell clumps adhere to the bottom of the incubator through cell substrate adhesion molecules contained in ECM, A state in which cells and cell masses do not float in the culture solution even if the culture solution is shaken lightly. On the other hand, during culture, cells and cell clusters do not adhere to the bottom of the incubator, which means that the cells and cell clusters do not adhere to the bottom of the incubator through cell-substrate adhesion molecules contained in ECM, Even if it touches the bottom surface, it means a state where cells and cell masses float in the culture solution when the culture solution is shaken lightly. During adhesion culture, the bottom surface of the plastic dish is chemically treated or coated with an adhesive coating agent (gelatin, polylysine, agar, etc.) that promotes adhesion in order to promote cell adhesion to the substrate. It is preferable. During suspension culture, the bottom surface of the plastic dish may not be treated, or may be coated with an adhesion-preventing coating agent (such as poly (2-hydroxyethyl methacrylate)) to prevent cells from adhering to the temperament. preferable. In addition, even in adhesion culture, it takes time until the target cell adheres, and it may be in a floating state for a certain amount of time, but if the cell eventually adheres even if it takes time, Include in adherent culture. The medium used for the suspension culture is not particularly limited, and examples thereof include DMEM and F12. A medium containing external factors such as serum (for example, FBS), N2, B27, and growth factors is preferable, and the medium includes DMEM / Most preferably, F12 is used. When inducing inner ear sensory epithelium (supporting cells, hair cells), cochlear ganglion cells, external factors to be added are among the group consisting of bFGF, EGF, IGF1, Wnt3a, FGF9, FGF20, heparin, TGFβ inhibitor However, it is preferable to include at least one factor (particularly including FGF9 and FGF20), but it is preferable to include a growth factor and heparin, and more preferable to include all of them. The preferred concentrations of bFGF, EGF, IGF1, Wnt3a, FGF9, FGF20, heparin are 10-50, 10-50, 10-100, 10-50, 10-100, 10-100, 1-50 ng / ml, respectively. There is no particular limitation. The induction of vascular cells may include at least one factor (particularly preferably including FGF3 and / or FGF10) from the group consisting of bFGF, EGF, IGF1, heparin, FGF3, and FGF10. It is preferable that heparin is included, and it is more preferable that heparin is included. Preferred concentrations of bFGF, EGF, IGF1, heparin, FGF3, and FGF10 are 10 to 50, 10 to 50, 10 to 100, 1 to 50, 25 to 100, and 25 to 100 ng / ml, respectively, but are not particularly limited. This suspension culture is preferably performed for 3 to 7 days, more preferably 4 to 6 days, and most preferably 5 days.

  Thereafter, the culture medium is added and the suspension culture is further continued. This culture is preferably performed for 3 to 7 days, more preferably 4 to 6 days, and most preferably 5 days. The medium to be added is not particularly limited, and examples thereof include DMEM and F12. A medium containing external factors such as serum (for example, FBS), N2, B27, and growth factors is preferable, and DMEM / F12 is used as the medium. Although it is most preferable to use it, it is preferable to use the same medium as that used in the initial suspension culture except for external factors. As an external factor to be added, at least one growth factor may be included in the group consisting of bFGF, EGF, and IGF1, but it is more preferable to include all of them. Preferred concentrations for each are 10-30 ng / ml, 10-30 ng / ml, 10-50 ng / ml.

  Next, as a second step, the formed spheres are collected without breaking them, and adhesion culture is performed. As the medium, the same medium as that used in the subsequent suspension culture may be used. After 1 hour to 2 days, preferably 1 hour to 36 hours, and most preferably the next day, the medium is replaced with fresh medium containing external factors such as serum (eg FBS), N2, B27, and growth factors. To do. At this time, it is preferable to use the same medium as that used in the initial suspension culture except for external factors. When inducing inner ear sensory epithelium (support cells, hair cells), cochlear ganglion cells, the external factor to be added is preferably T3 and / or IGF1, and the preferred concentrations thereof are 10 to 100, 1 ~ 50 ng / ml. For the induction of vascular marginal cells, it is preferable to use EGF or IGF1 as an external factor to be added, and the preferred concentrations are 10 to 50 and 10 to 50 ng / ml, respectively. This culture is preferably performed for 3 days or more, more preferably 5 days or more. During this time, only the medium exchange may be performed.

(3) Induction of differentiation from inner ear stem cells via periotic mesenchymal cells into cochlear fibrocytes, vascular streak cells, and PENDRIN positive cells In the method for induction from inner ear stem cells to Periotic mesenchymal cells according to the present invention, Culturing stem cells in a bFGF-containing medium. It should be noted that steps that are not essential to the present invention may be inserted into this method.

  The medium used here is not particularly limited, and examples thereof include DMEM and F12. A medium containing serum (for example, FBS) and bFGF is preferable, and DMEM is most preferably used as the medium. A preferred concentration of bFGF is 1-50 ng / ml. This culture is preferably carried out for 7 days or more, more preferably for 10 days or more, most preferably for 14 days or more, and after cultivation, Periotic mesenchymal cells having a fibrous cellular structure are obtained.

  If culture is further continued with a new medium in which bFGF is removed from the medium, cochlear fiber cells and vascular streak cells (particularly vascular basal cells) can be obtained. This culture is preferably performed for 7 days or longer, more preferably for 10 days or longer, and most preferably for 14 days or longer.

Further, PENDRIN-positive cells can be obtained by adding NaHCO ₃ to a new medium from which bFGF has been removed and culturing. The preferred concentration of NaHCO ₃ is 0.3% to 1%. This culture is preferably performed for 7 days or longer, more preferably for 10 days or longer, and most preferably for 14 days or longer.

<Example 1>
In this example, inner ear stem cells were induced to differentiate from iPS cell cells.

[Differentiation induction method]
Day 0
1) One well (6 well plate) was coated with Matri Gel.
2) Confluent Feeder-Free human iPS cells were treated with actinase and detached from the dish.
3) After dilution with PBS, the cells were collected by centrifugation.
4) The supernatant was drained, and the cells were suspended in mTeSR1 supplemented with ROCK inhibitor (Y27632) (10 μmol / L).
5) Only cells dissociated into single cells with a nylon mesh were obtained, and the number of cells was counted with a hemocytometer.
6) mTeSR1 containing Y27632 was added to the Matri Gel-coated well in 1) above.
7) Dissociated cells were added at 2.0 × 10 ⁴ / cm ² per well.

Day 1
The medium was changed to mTeSR1 containing no ROCK inhibitor.

Day 2
The medium was changed to a serum-free medium (DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid). Thereafter, the medium was changed every day until Day 4.

Day 5
Serum-free medium (DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid + bFGF, FGF3, FGF10, FGF19 + BMP4 (25ng / ml, 25ng / ml, 25ng / ml, 25ng / ml, 10ng / ml)) The medium was changed. Thereafter, the medium was changed every day until Day 7.

Day 8
The medium was changed to a serum-free medium (DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid + bFGF, FGF3, FGF10, FGF19 (the growth factor concentrations were all 25 ng / ml)). Thereafter, the medium was changed every day until DAY10.

Day 11
The medium was replaced with a fresh serum-free medium (DMEM / F12 + B27 + N2 + GlutaMax + Nonessential aminoacid + bFGF, FGF3, FGF10, FGF19).

Day 12
Centrifuge the cells, collect the cells by centrifugation, and suspend with DMEM / F12 + N2 + B27 medium + bFGF, FGF3, FGF10, FGF19 (25 ng / ml, 25 ng / ml, 25 ng / ml, 25 ng / ml) It became cloudy. Cells dissociated into single cells with a nylon mesh were collected and seeded in wells coated with poly-O-fibronectine (please tell us the number of cells). Culturing was performed under hypoxic conditions (O ₂ 4%, CO ₂ 5%). Thereafter, every 3 days, the medium was changed to DMEM / F12 + N2 + B27 medium + bFGF, EGF, IGF1 (20 ng / ml, 20 ng / ml, 50 ng / ml) and passaged about every 6 days. .

  The thus obtained inner ear stem cells were proliferated and the confluent dish was photographed with a phase contrast microscope (FIG. 1A).

[Antibody staining]
The obtained cells were subjected to fluorescent antibody staining using anti-PAX2 antibody, anti-PAX8 antibody, and anti-SOX2 antibody, which are markers for inner ear stem cells. In the fluorescent antibody staining, the cells fixed on the slide were subjected to antigen activation, and then rabbit anti-PAX2 antibody, mouse anti-PAX8 antibody, and goat anti-SOX2 antibody were added (50 times, 100 times, respectively). 100-fold dilution). Then, it labeled using the fluorescent secondary antibody specific for each animal species IgG, and observed with the fluorescence microscope. As a positive control, nuclear staining was performed with Hoechst. As shown in FIG. 1B, about 80% of cells express a marker of inner ear stem cells, which indicates that the inner ear stem cell differentiation inducing method of the present invention is extremely efficient.

<Example 2>
In this example, hair cells, support cells, and cochlear ganglion cells were induced to differentiate from inner ear stem cells.

[Differentiation induction method]
The inner ear stem cells prepared in Example 1 were treated with actase to detach the cells from the dish, and centrifuged to collect the cells. DMEM / F12 + N2 + B27 medium + bFGF, EGF, IGF1, Wnt3a, FGF9, FGF20, Heparin, (+ TGFβ inhibitor) (concentration of each factor is 25ng / ml, 25ng / ml, 50ng / ml, 20ng / ml , 50ng / ml, 50ng / ml , 10ng / ml) was added and suspension cultured on low suction plate (Corning ultra low adhesive surface (ultra-low Attachment) plate) (about 20,000 cells / well (96 well). after one day On the 5th day after suspension culture, the medium DMEM / F12 + N2 + B27 + bFGF, EGF, IGF1 (the concentration of each factor was 25ng / ml, 25ng / ml, 50ng) / ml) was added in an equal amount.

  On the 10th day after suspension culture, the spheres were collected so as not to break, transferred to a poly-O-fibronectine-coated plate, and cultured for adhesion (about 5-10 spheres). On the next day, the medium was changed to DMEM / F12 + N2 + B27 medium + T3, IGF1 (the concentration of each factor was 60 ng / ml, 10 ng / ml), and then the medium was changed once every three days. Hair cells, support cells, and cochlear ganglion cells were obtained after 5 days of adhesion culture.

[Antibody staining]
In the fluorescent antibody staining, the cells fixed on the slide were subjected to antigen activation, and then rabbit anti-myosin VIIa antibody, mouse anti-espin antibody, and goat anti-prestin antibody were added (200 times and 100 times, respectively). 50-fold dilution). Then, it labeled using the fluorescent secondary antibody specific for each animal species IgG, and observed with the fluorescence microscope.

  The obtained cells were stained with fluorescent antibodies using Escher, Myosin 7a, and Prestin, which are hair cell markers, using the respective antibodies (FIG. 2). Further, fluorescent antibody staining was performed on each of the antibodies for islet1, a marker for nerve cells, p27 / Kip1, a marker for hair cells, and prestin, a marker for hair cells (FIG. 3). Further, GFAP, which is a marker for cochlear ganglia, and calbindin and beta III tubulin, which are markers for mature neurons, were stained with fluorescent antibodies using the respective antibodies (FIG. 4).

  As shown in FIGS. 2 to 3, it was confirmed that hair cell markers, myosin 7a, espin, prestin-positive cells, and supporting cell markers p27kip1, ISLET1-expressing cells were induced. In addition, as shown in FIG. 4, cells expressing GFAP expressed by calbindin positive cells and associated glia, which are markers of cochlear neurons, were obtained at the same time. Thus, in this method, hair cells, supporting cells, and cochlear ganglion cells, which are main cells constituting the inner ear sensory epithelial cells, are induced in a three-dimensional manner with the same arrangement as in vivo.

<Example 3>
In this example, differentiation induction of Periotic mesenchymal cells, cochlear fiber cells and vascular streak cells, and PENDRIN positive cells was performed.

  The inner ear stem cells obtained in Example 1 were replaced with POMC medium (DMEM 500 ml (D5796), 1 M HEPES 5 ml, FBS 30 ml, bFGF (10 ng / ml) 2.0 ml) and cultured under normal oxygen. Periotic mesenchymal cells were observed from about day 10 of culture. When the culture was continued thereafter, a fibrous cellular structure was formed after 2 weeks. (Fig. 5)

When the medium of the cells in the form of fibrous cells was changed to FBS (10%) + DMEM medium and cultured for about 2 weeks, cochlear fibrocytes and vascular streak cells (especially vascular basal cells) were obtained. . (Fig. 6)
Moreover, PENDRIN positive cells were obtained by using a medium in which NaHCO ₃ (0.375%) was added to FBS (10%) + DMEM medium. (Fig. 6)

[Antibody staining]
For fluorescent antibody staining, cells immobilized on a slide were subjected to antigen activation, and then rabbit anti-S100 antibody, POU3F4 antibody, mouse anti-caldesmon antibody, and goat anti-TBX18 antibody were added (each 3 times, (100, 100, 50 times dilution). Then, it labeled using the fluorescent secondary antibody specific for each animal species IgG, and observed with the fluorescence microscope.

  As shown in FIG. 5, it was confirmed that cells in which the expression of S100, POU3F4, caldesmon, and TBX18, which are markers for Periotic mesenchymal cells, were induced were induced.

  After cell induction by each induction method, antigen-stimulating operation was performed on the cells fixed on the slide, and then rabbit anti-carbonic acid dehydrogenase II antibody, anti-aquaporin 1 antibody, anti-sodium potassium ATPase Antibody and vimentin antibody, mouse anti-connexin 26, connexin 30 antibody, goat anti-PENDRIN antibody were added (all diluted 100 times). Then, it labeled using the fluorescent secondary antibody specific for each animal species IgG, and observed with the fluorescence microscope.

As shown in FIG. 6, since carbonic acid dehydrogenase, aquaporin 1, sodium potassium ATPase, vimentin, and connexins 26 and 30 expressed in cochlear fiber cells and cochlear vascular basal cells are expressed, these cells Was confirmed to be induced. Moreover, since PENDRIN was expressed, it was confirmed that PENDRIN positive cells were induced.

<Example 4>
In this example, it is shown that inner ear sensory epithelial cells induced to differentiate by the method of the present invention are damaged by administration of gentamicin, which is known for inner ear toxicity.

  A gentamicin injection diluted 80-fold was administered for 10 days to inner ear sensory epithelial cells induced by the method of the present invention.

  The administration of this drug significantly reduced the number of cells compared to the control group, indicating that cytotoxicity caused by gentamicin administration occurred in the same manner as in the living body.

<Example 5>
A blood vessel line consists of a marginal cell, an intermediate cell, and a basal cell, and these three cell types are known as a blood vessel cell. In this example, vascular marginal cells were induced to differentiate from inner ear stem cells.

[Differentiation induction method]
The inner ear stem cells prepared in Example 1 were treated with actase to detach the cells from the dish, and centrifuged to collect the cells. DMEM / F12 + N2 + B27 medium + bFGF, EGF, IGF1, FGF3, FGF10, Heparin (concentration of each factor is 20ng / ml, 20ng / ml, 50ng / ml, 100ng / ml, 100ng / ml, 10ng / ml) and suspended culture on a low adsorption plate (Corning Ultra-Low Attachment plate) (approx. 20000 cells / well (96-well plate). After 1 day, formation of spheres (cell mass) was observed. On the third day after suspension culture, medium DMEM / F12 + N2 + B27 + bFGF, EGF, IGF1 (the concentrations of each factor were 25 ng / ml, 25 ng / ml, 50 ng / ml, respectively) were added in equal amounts.

  On the 6th day after suspension culture, the spheres were collected without breaking them, transferred to a poly-O-fibronectine-coated plate, and cultured for adhesion (about 5-10 spheres). On the next day, the medium was changed to DMEM / F12 + N2 + B27 medium + EGF, IGF1 (concentration of each factor was 20 ng / ml, 50 ng / ml), and then the medium was changed once every three days. From 5 days after adhesion culture, vascular marginal cells were obtained.

[Antibody staining]
The obtained cells were subjected to fluorescent antibody staining using NaKatpase A1 and NKCC1, which are markers of vascular marginal cells, using respective antibodies. As a control, the expression of cochlear fibrocytes was examined using an antibody of CRYM (μ crystallin) that is not expressed in vascular marginal cells.

  Fluorescent antibody staining was performed on the cells fixed on the slide after antigen activation, and then added with mouse anti-CRYM antibody, rabbit anti-NaKatpaseA1 antibody, and goat anti-NKCC1 antibody (respectively 300 times, 300 times, 100-fold dilution). Then, it labeled using the fluorescent secondary antibody specific for each animal species IgG, and observed with the fluorescence microscope.

  As shown in FIG. 8, expression of NaKatpaseA1 and NKCC1 was detected, and expression of CRYM was not observed. Thus, it was confirmed that the vascular cells were induced by the treatment of this example.

  The obtained inner ear cells can be used for research tools for inner ear cells such as regenerative medicine or a drug-induced inner ear sensory epithelial disorder evaluation model.

Claims (9)

A method for inducing pluripotent stem cells into inner ear stem cells,
Culturing the pluripotent stem cells in the presence of ROCK Inhibitor;
Culturing in the absence of ROCK Inhibitor;
Culturing in a serum-free medium;
Culturing in a serum-free medium containing a growth factor;
A method of induction in which the step of dissociating into single cells is performed in this order.   The induction method according to claim 1, wherein the pluripotent stem cell is an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell). Culturing the cells dissociated into single cells in a culture dish coated with poly-O-fibronectine;
A guidance method further comprising: A method of inducing inner ear sensory epithelium (support cells, hair cells) and cochlear ganglion cells (neurons, glial cells) from inner ear stem cells,
A step of suspension culture of the inner ear stem cells in the presence of FGF9 and FGF20;
An adhesion culture step;
Are guided in this order. A method for inducing vascular marginal cells from inner ear stem cells,
The suspension culture of the inner ear stem cells in the presence of FGF3 and / or FGF10;
An adhesion culture step;
Are guided in this order. A method for inducing Periotic mesenchymal cells from inner ear stem cells,
An induction method comprising a step of culturing the inner ear stem cells in the presence of bFGF. A method of inducing cochlear fiber cells and vascular streak cells from Periotic mesenchymal cells,
Culturing the Periotic mesenchymal cells in the presence of bFGF;
culturing in the absence of bFGF to obtain cochlear fiber cells and vascular streak cells;
In this order, the guidance method.   The induction method according to claim 7, wherein the vascular cells are vascular basal cells. A method of inducing Pendrin positive cells from Periotic mesenchymal cells,
Culturing the Periotic mesenchymal cells in the presence of bFGF;
culturing in the presence of NaHCO _{3 in the} absence of bFGF to obtain Pendrin positive cells;
In this order. Guidance method.