CN104403988B - A kind of inducing mouse embryonic stem cell breaks up the method for inner ear hair cells - Google Patents
A kind of inducing mouse embryonic stem cell breaks up the method for inner ear hair cells Download PDFInfo
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Abstract
The invention discloses the method that a kind of differentiation of inducing mouse embryonic stem cell obtains inner ear hair cells, first with conditioned medium and induction broth inducing embryo stem cell differentiation Hair Cell Progenitors；Then the ripe inner ear hair cells of method induction precursor cell differentiation combined using induction broth with trophocyte；The method of the invention effectively can be divided into inner ear hair cells precursor and ripe inner ear hair cells by inducing embryo stem cell, and differentiation ratio is high, differentiation gained cell expression hair cell specific gene and albumen, and possess certain hair cell electrophysiological function.
Description
(1) technical field
The present invention relates to a kind of differentiation method of inner ear hair cells, more particularly to a kind of inducing mouse embryonic stem cell differentiation
The method for obtaining inner ear hair cells.
(2) background technology
Hearing loss is to have a strong impact on one of worldwide persistent ailment of human life quality, and radical cure method is there is no at present.Lactation
The unrepairable damage of animal inner ear hair cells is considered as to cause one of main reason of hearing loss.Traditional view is recognized
For human inner ear's auditory hair cell is specialization thesocyte, and no longer with Regeneration and Repair ability, its damage is irreversible.Closely
The discovery mammal embryo phase is studied over year or early stage hair cell is possible to regeneration, and promotees hair cell differentiation and development by transfecting
Related gene finds that the dystopy of immature hair cell occurs in adult cochlea, has pointed out mammal hair cell necessarily inducing
Under the conditions of realize the possibility of regeneration, but still suffer from very big challenge for the reparative regeneration of hair cell in pathological process.Therefore seek
The precursor source of hair cell regeneration is looked for, is to be engaged in working as hearing loss treatment to develop damage of hair cell to repair Intervention Strategy
It is anxious.
Stem cell with its all can or versatility and height self duplication ability many advantages, such as and as organizational project
With the focus of cell therapy area research.More and more researchs in recent years illustrate embryonic stem cell and various sources into soma
Great advantage and application potential of the cell in tissue damage reparation and disease treatment research, base is induced to differentiate into stem cell
Plinth, using stem cell correlation technique, the damage and regeneration for inquiring into auditory system injury repair particularly hair cell have been possibly realized.
Simultaneously as hair cell rare numbers, the Study on Molecular Mechanism developed for hair cell is still very limited.Therefore, exploration is built
Vertical effective stem cell differentiation hair cell induction system, can not only provide effective cell derived for hair cell regenerative therapy, and
Research model will be provided to inquire into hair cell differentiation and development correlation molecule and signaling mechanism.Embryonic stem cell have in vitro culture without
The advantages of limit propagation, self-renewing and totipotency, be the emphasis that stem cell breaks up field.The present invention is dry thin using embryo
Born of the same parents, establish the side that a kind of conditioned medium, cell factor and trophocyte are combined effectively induction differentiation inner ear hair cells
Method.
(3) content of the invention
It is an object of the present invention to provide the method that a kind of differentiation of inducing mouse embryonic stem cell obtains inner ear hair cells, the present invention
Method is first with conditioned medium and induction broth inducing embryo stem cell differentiation Hair Cell Progenitors；Then induction is utilized
The ripe interior tragus of method induction precursor cell differentiation that nutrient solution is combined with trophocyte's (i.e. chicken embryo utricle interstitial cell)
Cell, is induced by this method, can obtain a high proportion of Hair Cell Progenitors and ripe inner ear hair cells.
The technical solution adopted by the present invention is：
The present invention provides a kind of method that inducing mouse embryonic stem cell breaks up inner ear hair cells, and described method is：(1)
Mouse embryo stem cell is seeded to conditioned medium, in 37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days, formation cell mass；Take thin
Born of the same parents group is transferred in induction liquid I, in 37 DEG C, 5%CO2Condition Fiber differentiation 12-14 days, obtains the capillary of ES cell differentiation
Born of the same parents' precursor；The conditioned medium final concentration is constituted：Volumetric concentration 30-50%HepG2 conditioned mediums, 1mM is non-must
Amino acid, 5 μM of beta -mercaptoethanols, 2mM Glus, volumetric concentration 10%FBS (hyclone) are needed, solvent is DMEM/
F12 basic culture solutions；The HepG2 conditioned mediums are prepared as follows：By HepG2 cells with 5 × 104cells/cm2It is close
Degree is inoculated in culture dish, by 1.75 × 105The amount of cells/mL nutrient solutions adds the 10%FBS of final concentration containing volume DMEM trainings
Nutrient solution, is placed in 37 DEG C, 5%CO2Under the conditions of culture collect culture supernatants after 4-5 days, be centrifuged off cell fragment, acquisition upper strata
Clear liquid and lower sediment, discard lower sediment, take supernatant liquor through suction filtration (preferably 0.22 μm filter membrane suction filtration), filtrate is
HepG2 conditioned mediums；Induction liquid I final concentration, which is constituted, is：Volumetric concentration 5%FBS, 5-15ng/ml IGF-1 (insulin
Like growth factor -1), 20-30ng/ml EGF (epithelical cell growth factor), 1 × N2,1 × B27,45-55ng/mL FGF3
(fibroblast growth factor 3), 45-55ng/mL FGF10 (Fibroblast growth factor-10), 45-55ng/mL heparin
Sodium, solvent is DMEM/F12 basic culture solutions；
(2) by chicken embryo utricle interstitial cell (the chicken embryo utricle interstitial cell of preferred growth to 90% degree of converging) with containing
The DMEM nutrient solutions immersion of the μ g/ml mitomycin Cs of final concentration 2, in 37 DEG C, 5%CO2Cultivated 3 hours in incubator, discard culture
Liquid, cell is washed with pH value for 7.4 PBS, as pretreated chicken embryo utricle interstitial cell；
(3) Hair Cell Progenitors for obtaining step (1) carry out digestion process, staticly settle, abandon precipitation, cell is hanged
Liquid is inoculated with (preferably by cell suspension with 105Individual/cm2Density inoculation) on pretreated chicken embryo utricle interstitial cell, plus
Enter to induce liquid II, in 37 DEG C, 5%CO2Continue to induce 3-4 weeks in incubator, obtain the nutrient solution containing inner ear hair cells, collect interior
Tragus cell；The induction liquid II final concentration composition：Volumetric concentration 5%FBS, 20-30ng/ml EGF, 1 × N2,1 × B27,5-
15 μM of RA (ATRA), 5 μM of nitrogen-[nitrogen-(3,5- difluoros phenylacetyl)-L- alanyls]-S- phenylglycine butyl esters
(DAPT), solvent is DMEM/F12 basic culture solutions.
Further, preferably described conditioned medium final concentration composition is：Volumetric concentration 40%HepG2 conditioned mediums, 1mM
Nonessential amino acid, 5 μM of beta -mercaptoethanols, 2mM Glus, volumetric concentration 10%FBS, solvent are DMEM/F12 bases
Nutrient solution.
Further, preferably described induction liquid I final concentration composition is：Volumetric concentration 5%FBS, 10ng/mlIGF-1,25ng/
Ml EGF, 1 × N2,1 × B27,50ng/mL FGF3,50ng/mL FGF10,50ng/mL liquaemins, solvent are DMEM/F12 bases
Plinth nutrient solution.
Further, chicken embryo utricle interstitial cell of the present invention is trophocyte, and preparation method is mainly by disappearing
Change and propagating method is separately cultured utricle interstitial cell for 18 days from fertilization in chicken embryo, 3-5 is for cell, at mitomycin C
After reason, as trophocyte, the specific chicken embryo utricle interstitial cell is prepared as follows：From 18 days chicken embryos of fertilization
Utricle is separated, 0.25% pancreatin after 37 DEG C digest 15-20 minutes, is stopped with volume final concentration 10%FBS+DMEM nutrient solutions,
200 eye mesh screens are crossed, cell suspension is collected, 1000rpm/min is centrifuged 6-8 minutes, cell volume final concentration 10%FBS+DMEM
Nutrient solution suspends, and is inoculated in blake bottle, puts 37 DEG C, 5%CO2Incubator culture, liquid is changed after 48 hours first, is discarded not adherent
Cell, adherent continuation is cultivated to 90% degree of converging, and is digested with 0.25% pancreatin/EDTA and is seeded to Tissue Culture Flask culture, note
It is P1 for cell, by such Secondary Culture, to P3 generations, that is, obtains shape chicken embryo utricle interstitial cell.
Further, preferably described Hair Cell Progenitors carry out digestion process with accutase.
Further, preferably described induction liquid II final concentration composition：Volumetric concentration 5%FBS, 25ng/mlEGF, 1 × N2,1 ×
B27,10 μM of RA, 5 μM of DAPT, solvent are DMEM/F12 basic culture solutions.
Further, the method for the inducing mouse embryonic stem cell differentiation inner ear hair cells recommends to enter as follows
OK：(1) by mouse embryo stem cell ES-D3 be seeded to containing the agar of mass concentration 1% (i.e. agar water dissolving mass concentration is made
1% agar solution, is then down flat plate) culture plate on, add conditioned medium, in 37 DEG C, 5%CO2Under the conditions of cultivate 4-5
My god, liquid is changed daily；Take cell mass to be transferred in culture orifice plate, induction liquid I is added, in 37 DEG C, 5%CO2Condition Fiber differentiation 12-
14 days, liquid is changed daily, obtains the Hair Cell Progenitors of ES cell differentiation；The conditioned medium final concentration is constituted：
Volumetric concentration 40%HepG2 conditioned mediums, 1mM nonessential amino acid, 5 μM of beta -mercaptoethanols, 2mM GlutaMAX, 10%
FBS, solvent is DMEM/F12 basic culture solutions；The HepG2 conditioned mediums are prepared as follows：By HepG2 cells with 5
×104cells/cm2Density is inoculated in culture dish, by 1.75 × 105The amount of cells/mL nutrient solutions adds final concentration containing volume
10%FBS DMEM nutrient solutions, are placed in 37 DEG C, 5%CO2Under the conditions of culture 4-5 days after collect culture supernatants, be centrifuged off carefully
Born of the same parents' fragment, supernatant liquor is through 0.22 μm of filter membrane suction filtration, and filtrate is HepG2 conditioned mediums；The induction liquid I final concentration composition
For：Volumetric concentration 5%FBS, 10ng/ml IGF-1,25ng/ml EGF, 1 × N2,1 × B27,50ng/mL FGF3,50ng/mL
FGF10,50ng/mL liquaemin, solvent are DMEM/F12 basic culture solutions；
(2) chicken embryo utricle interstitial cell μ g/ml mitomycin Cs containing final concentration 2 of 90% degree of converging will be grown to
DMEM nutrient solutions soak, in 37 DEG C, 5%CO2Cultivated 3 hours in incubator, discard nutrient solution, cell pH value is 7.4 PBS
Buffer solution is washed, as pretreated chicken embryo utricle interstitial cell；
(3) Hair Cell Progenitors obtained with accutase digestion steps (1), staticly settle the undispersed cell of removing
Agglomerate, abandons precipitation, induction liquid II on cell suspension inoculation to pretreated chicken embryo utricle interstitial cell, will be added, 37
DEG C, 5%CO2Continue to induce 3-4 weeks in incubator, obtain the nutrient solution containing inner ear hair cells, collect inner ear hair cells；It is described to lure
The final concentration of drain II is constituted：Volumetric concentration 5%FBS, 25ng/ml EGF, 1 × N2,1 × B27,10 μM of RA, 5 μM of DAPT, it is molten
Agent is DMEM/F12 basic culture solutions.
The factor in heretofore described nutrient solution and induction liquid can be obtained by mode purchased in market, the training of DMEM/F12 bases
Nutrient solution is preferably purchased from Corning companies, and 1 × N2 and 1 × B27 are purchased from Invitrogen, and 18 days chicken embryos of the fertilization are purchased from Hangzhoupro
State day source cultivation Co., Ltd.
Compared with prior art, the beneficial effects are mainly as follows：The method of the invention can be induced effectively
ES cell differentiation is inner ear hair cells precursor and ripe inner ear hair cells, and differentiation gained cell expression hair cell is special
Gene and albumen, and possess certain hair cell electrophysiological function.And higher proportion of inner ear is resulted in by this method induction
Hair Cell Progenitors Nuclear maturity inner ear hair cells.
(4) illustrate
Fig. 1 Hair Cell Progenitors marker gene detection figure；ES represents not induce the embryonic stem cell of differentiation in Fig. 1,
Progenitor represents Hair Cell Progenitors obtained by ES cell differentiation, and β-actin are used as internal reference.
Fig. 2 Hair Cell Progenitors protein immunization fluoroscopic examination figures；A1 expresses for Pax2, and A2 expresses for Pax8, and A3 is thin
Karyon (DAPI dyeing), A4 is the superposition of the width figure of A1, A2 and A3 tri-；B1 expresses for Atoh1, and B2 expresses for Pax2, and B3 is cell
Core (DAPI dyeing), B4 is the superposition of the width figure of B1, B2 and B3 tri-；C1 expresses for Atoh1, and C2 expresses for Brn3C, and C3 is nucleus
(DAPI dyeing), C4 is the superposition of the width figure of C1, C2 and C3 tri-.
Fig. 3 maturation hair cell protein immunization fluoroscopic examination figures；A1 is that Espin and nucleus C23 is expressed, and A2 is F-actin
With nucleus C23 expression, A3 is the superposition of A1 and the width figures of A2 two；B1 is that Espin and nucleus C23 is expressed, and B2 is a of Myosin VII
With nucleus C23 expression, B3 is the superposition of B1 and the width figures of B2 two；C1 is FM1-43FX dyeing and nucleus C23 expression, and C2 is
The a of Myosin VII and nucleus C23 expression, C3 is the superposition of C1 and the width figures of C2 two.
(5) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1：Conditioned medium and the method inducing embryo stem cell of combinations of factors differentiation Hair Cell Progenitors
Mouse embryo stem cell ES-D3 (is purchased from Shanghai Inst. of Life Science, CAS biochemistry and cell
Biological study institute) passage is followed by the culture plate for overlaying the agar of mass concentration 1%, add conditioned medium 37 DEG C, 5%
CO2CMC model 4-5 days, changes liquid daily.Then cell mass is transferred in 12 orifice plates, per about 30, hole, changes induction liquid I,
37 DEG C, 5%CO2Condition Fiber differentiation 12-14 days, changes liquid daily, obtains the Hair Cell Progenitors of ES cell differentiation.
Conditioned medium final concentration is constituted：Volumetric concentration 40%HepG2 conditioned mediums, 1mM nonessential amino acid (are purchased from
Gibco companies, numbering：11140-050), 5 μM of beta -mercaptoethanols (being purchased from Gibco companies), 2mM Glus (are purchased from
Gibco companies), volumetric concentration 10%FBS, solvent is DMEM/F12 basic culture solutions (be purchased from Corning companies).
The preparation of HepG2 conditioned mediums：By HepG2 cells (purchased from Shanghai Inst. of Life Science, CAS life
Thing chemistry and Institute of Cell Biology) with 5 × 104cells/cm2Density is inoculated in culture dish, by 1.75 × 105cells/
The DMEM nutrient solutions that the amount of mL nutrient solutions adds the 10%FBS of final concentration containing volume (being purchased from Gibco companies) are (public purchased from Corning
Department), it is placed in 37 DEG C, 5%CO2Under the conditions of culture 4-5 days after collect culture supernatants, be centrifuged off cell fragment, supernatant liquor
Through 0.22 μm of filter membrane suction filtration, filtrate is HepG2 conditioned mediums.
Induce the final concentration of liquid I composition：Volumetric concentration 5%FBS, 10ng/ml IGF-1 (being purchased from Peprotech companies),
25ng/ml EGF (be purchased from Peprotech companies), 1 × N2 (being purchased from Invitrogen), 1 × B27 (being purchased from Invitrogen),
50ng/mL FGF3 (being purchased from R＆D companies), 50ng/mL FGF10 (being purchased from R＆D companies), 50ng/mL liquaemins (are purchased from Sigma
Company), solvent is DMEM/F12 basic culture solutions (being purchased from Corning companies).
Embodiment 2：The separation of chicken embryo utricle interstitial cell
From 18 days chicken embryo (cultivating Co., Ltd purchased from Hangzhou Tian Yuan) middle separation utricles of fertilization, 0.25% pancreatin (is purchased from
Corning companies), after 37 DEG C digest 15-20 minutes, stopped, mistake with DMEM nutrient solutions (addition volume final concentration 10%FBS)
200 eye mesh screens, collect cell suspension, and 1000rpm/min is centrifuged 6-8 minutes, cell DMEM nutrient solutions (addition volume final concentration
10%FBS) suspend, be inoculated in blake bottle, put 37 DEG C, 5%CO2Incubator culture, changes liquid after 48 hours, discards and do not paste first
Parietal cell, adherent continuation is cultivated to 90% degree of converging, and is digested and is inoculated with 0.25% pancreatin/EDTA (being purchased from Corning companies)
To Tissue Culture Flask culture, P1 is designated as cell, by such Secondary Culture, to P3 generations, that is, the single chicken embryo of form is obtained ellipse
Circle capsule interstitial cell.
Embodiment 3：The ripe inner ear hair cells of method induction precursor cell differentiation that combinations of factors is combined with trophocyte
The chicken embryo utricle interstitial cell for growing to 90% degree of converging first prepared by embodiment 2 μ g/ containing final concentration 2
The DMEM nutrient solutions immersion of ml mitomycin Cs (being purchased from Sigma companies), in 37 DEG C, 5%CO2Cultivate 3 hours, abandon in incubator
Nutrient solution is removed, cell is washed with pH value for 7.4 PBS (being purchased from Sheng Gong biotech firms), is removed cleaning solution, is pre- place
Chicken embryo utricle interstitial cell after reason.
With the Hair Cell Progenitors of accutase (being purchased from Gibco companies) digestion induction differentiation of embodiment 1, it is heavy slightly to stand
Form sediment and remove undispersed cell mass, by cell suspension with 105Individual/cm2Density be seeded between pretreated chicken embryo utricle
On cell plastid, induction liquid II is added, in 37 DEG C, 5%CO2Continue to induce 3-4 weeks in incubator, collect inducing cell and reflected
It is fixed, obtain ripe inner ear hair cells.
Induce the final concentration of liquid II composition：Volumetric concentration 5%FBS, 25ng/ml EGF, 1 × N2,1 × B27,10 μM of RA, 5 μ
M DAPT (are purchased from Sigma companies), and solvent is DMEM/F12 basic culture solutions.
Embodiment 4：The identification of noble cells
(1) Hair Cell Progenitors specific gene is detected：The precursor that Example 1 is obtained (is purchased from TRizol
Invetigen total serum IgE) is extracted, extracting method by specification is carried out, then with Reverse Transcriptase kit RNA PCR kit (AMV)
Ver3.0 (being purchased from TaKaRa companies) carries out reverse transcription.The cDNA obtained using reverse transcription as template, respectively primer Pax2,
Pax8, Dlx5, Eya1, Six1, Atoh1 and Brn3c effect under enter performing PCR reaction, detection Pax2, Pax8, Dlx5, Eya1,
Six1, Atoh1 and Brn3c gene expression, β-actin are used as internal reference.PCR programs are：94 DEG C of pre-degeneration 5min, 94 DEG C of denaturation
30s, 50 DEG C~65 DEG C annealing 30s, 72 DEG C of extension 30s, totally 30~40 circulate, 72 DEG C of extension 10min, annealing temperature and circulation
It is several to be selected according to the concrete condition of primer.Pcr amplification product is entered into row agarose gel electrophoresis analysis respectively, while with small
Mouse embryonic stem cell ES-D3 is as control, as a result as shown in Figure 1.
The primer (being synthesized by Shanghai JaRa biotech firm) is as follows：
Pax2(5'-CGAATACAAGCGACAGAACC-3',5'-GGGACAGAGCCCTCAGACA-3')；
Pax8(5'-GCAGAAGGCGTTTGTGAC-3',5'-GAGCCCGTTGATAGAGTAGG-3')；
Dlx5(5'-GCCAAGGCTTATGCCGACTA-3',5'-TTCTGGCAGGGCGAGGTAC-3')；
Eya1(5'-GTCCACCAATGCCACTTA-3',5'-AGGTCCCAGATGAACACT-3')；
Six1(5'-CCTCCTCCAACAAGCAGAATC-3',5'-GGAACCCAAGTCCACCAA-3')；
Atoh1(5'-GCTCCCTGAAAACTGAGACAACCAA-3',5'-ATGAAGTGCGTGTATTCTGGGTCCG-
3')；
Brn3c(5'-AGCCTACACTCCGGCTCTGAG-3',5'-TGACTCCACATCGCTGAGACACG-3')；
β-actin (5'-ACACCTTCTACAATGAGCTG-3',
5'-CTGCTTGCTGATCCACATCT-3')。
(2) Hair Cell Progenitors Immunofluorescence test：The precursor that Example 1 is obtained, through 4% paraformaldehyde room
After the fixed 15min of temperature, paraformaldehyde is removed with the PBS rinsings of pH value 7.4, then (Sigma is purchased from 0.05%Triton X-100
Company) (25 DEG C) incubation 10min of room temperature, then close 1h, PBS with 5% lowlenthal serum (biological purchased from raw work) in room temperature
Twice, cell is divided into 3 groups, is separately added into primary antibody, group 1 is that anti-Pax2 and Pax8, group 2 are that anti-Atoh1 and Pax2, group 3 are
Atoh1 and Brn3c, primary antibody is purchased from Santa cruz companies, and 4 DEG C of wet box are incubated overnight；PBS embathes three times, each 10min；
Secondary antibody is sequentially added, group 1 adds Alexa488 and Dylight649 marks, group 2 and adds Alexa488 and Alexa647 marks, group
3 add Alexa488 and Dylight649 marks, and secondary antibody is purchased from Lian Ke biotech firms, and 37 DEG C are incubated 1h, and PBS embathes three times；
DAPI (being purchased from Sigma companies) dye core, laser confocal microscope (Carl Zeiss) observation is taken pictures.As a result as shown in Figure 2.
(3) ripe hair cell differential protein Immunofluorescence test:The inducing cell that Example 3 is obtained, through 4% poly first
Aldehyde room temperature is fixed after 15min, removes paraformaldehyde with the PBS rinsings of pH value 7.4, then (be purchased from 0.05%Triton X-100
Sigma companies) (25 DEG C) incubation 10min of room temperature, then close 1h, PBS with 5% lowlenthal serum (biological purchased from raw work) in room temperature
Cleaning twice, is divided into 3 groups, is separately added into primary antibody, and group 1 is anti-Espin and C23, group 2 be anti-Espin, group 3 be Myosin7a and
C23, primary antibody is purchased from Santa cruz companies, and 4 DEG C of wet box are incubated overnight；PBS embathes three times, each 10min；Sequentially add two
Anti-, group 1 adds Alexa488 and Dylight405 marks, group 2 add Alexa488, group 3 add Dylight649 and
Dylight405 is marked, and secondary antibody is purchased from Lian Ke biotech firms, and 37 DEG C of incubations 1h, PBS embathe three times, laser confocal microscope
(Carl Zeiss) observation is taken pictures.Wherein Espin and C23 detections group secondary antibody, which is incubated, completes and after PBS embathes, and is coupled with TRITC
Phalloidine (be purchased from Invitrogen companies) flag F-actin, then carry out confocal laser scanning microscope and take pictures.
37 DEG C of incubation 1h are directly marked after secondary antibody.As a result as shown in Figure 3.
Hair cell electrophysiological function is detected：Utilize FM1-43FX (being purchased from Invitrogen companies) Coloration experiment detection induction
Cellular electrophysiologicalsensor function, after inducing cell is cleaned with HBSS (being purchased from Sheng Gong biotech firms), adds 5 μM of FM1-43FX room temperatures and incubates
30s is educated, is cleaned immediately with HBSS, then is embathed with HBSS immunofluorescence experiment detection Myosin7a is carried out after 20min, step and anti-
Body is as described in embodiment 4- (3).As a result as shown in Figure 3.
As a result show, induction gained noble cells can express Hair Cell Progenitors specific gene, precursor and into
Ripe hair cell specific proteins, and with certain hair cell electrophysiological function, show that the inventive method can effectively induce embryo
Tire stem cell breaks up inner ear hair cells.
Finally, in addition it is also necessary to it is noted that listed above is only several specific embodiments of the invention.Obviously, this hair
It is bright to be not limited to above example, there can also be many deformations.One of ordinary skill in the art can be from present disclosure
All deformations for directly exporting or associating, are considered as protection scope of the present invention.
Claims (8)
1. a kind of inducing mouse embryonic stem cell breaks up the method for inner ear hair cells, it is characterised in that described method is：(1) will
Mouse embryo stem cell is seeded to conditioned medium, in 37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days, formation cell mass；Take cell
Group is transferred in induction liquid I, in 37 DEG C, 5%CO2Condition Fiber differentiation 12-14 days, obtains the hair cell of ES cell differentiation
Precursor；The conditioned medium final concentration is constituted：Volumetric concentration 30-50%HepG2 conditioned mediums, 1mM are nonessential
Amino acid, 5 μM of beta -mercaptoethanols, 2mM Glus, volumetric concentration 10%FBS, solvent are DMEM/F12 basic culture solutions；
The HepG2 conditioned mediums are prepared as follows：By HepG2 cells with 5 × 104cells/cm2Density is inoculated in culture dish
In, by 1.75 × 105The amounts of cells/mL nutrient solutions adds the DMEM nutrient solutions of the 10%FBS containing volumetric concentration, be placed in 37 DEG C,
5%CO2Under the conditions of culture 4-5 days after collect culture supernatants, centrifugation, take supernatant liquor through suction filtration, filtrate is HepG2 conditions
Nutrient solution；Induction liquid I final concentration, which is constituted, is：Volumetric concentration 5%FBS, 5-15ng/ml IGF-1,20-30ng/ml EGF,
1 × N2,1 × B27,45-55ng/mL FGF3,45-55ng/mL FGF10,45-55ng/mL liquaemins, solvent is DMEM/F12
Basic culture solution；
(2) chicken embryo utricle interstitial cell is soaked with the DMEM nutrient solutions of the μ g/ml mitomycin Cs containing final concentration 2, at 37 DEG C,
5%CO2Cultivated 3 hours in incubator, discard nutrient solution, cell is washed for 7.4 PBS with pH value, as pre-processed
Chicken embryo utricle interstitial cell afterwards；
(3) Hair Cell Progenitors for obtaining step (1) carry out digestion process, staticly settle, abandon precipitation, cell suspension is connect
Plant to pretreated chicken embryo utricle interstitial cell, induction liquid II is added, in 37 DEG C, 5%CO2Continue to induce in incubator
3-4 weeks, obtain the inner ear hair cells of differentiation and maturation；The induction liquid II final concentration composition：Volumetric concentration 5%FBS, 20-30ng/
Ml EGF, 1 × N2,1 × B27,5-15 μM of RA, 5 μM of DAPT, solvent are DMEM/F12 basic culture solutions.
2. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that the bar
Part nutrient solution final concentration is constituted：Volumetric concentration 40%HepG2 conditioned mediums, 1mM nonessential amino acid, 5 μM of β-sulfydryl second
Alcohol, 2mM Glus, volumetric concentration 10%FBS, solvent are DMEM/F12 basic culture solutions.
3. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that described to lure
The final concentration of drain I is constituted：Volumetric concentration 5%FBS, 10ng/ml IGF-1,25ng/mlEGF, 1 × N2,1 × B27,50ng/
ML FGF3,50ng/mL FGF10,50ng/mL liquaemins, solvent are DMEM/F12 basic culture solutions.
4. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that the chicken
Embryo utricle interstitial cell is prepared as follows：Utricle, 0.25% pancreatin, 37 DEG C of digestion are separated within 18 days in chicken embryo from fertilization
After 15-20 minutes, stopped with volume final concentration 10%FBS+DMEM nutrient solutions, cross 200 eye mesh screens, collect cell suspension,
1000rpm/min is centrifuged 6-8 minutes, and cell is suspended with volume final concentration 10%FBS+DMEM nutrient solutions, is inoculated in blake bottle,
Put 37 DEG C, 5%CO2Incubator culture, liquid is changed after 48 hours first, discards non-attached cell, and adherent continuation, which is cultivated to 90%, to converge
It is right, digested and be seeded in Tissue Culture Flask with 0.25% pancreatin/EDTA and cultivated, P1 is designated as cell, by so passing on
Culture, to P3 generations, that is, obtains chicken embryo utricle interstitial cell.
5. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that the hair
Cell-progenitor cells carry out digestion process with accutase.
6. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that described to lure
The final concentration of drain II is constituted：Volumetric concentration 5%FBS, 25ng/ml EGF, 1 × N2,1 × B27,10 μM of RA, 5 μM of DAPT, it is molten
Agent is DMEM/F12 basic culture solutions.
7. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that step (3)
The cell suspension is with 105Individual/cm2Density be seeded on pretreated chicken embryo utricle interstitial cell.
8. inducing mouse embryonic stem cell as claimed in claim 1 breaks up the method for inner ear hair cells, it is characterised in that described
Method is carried out as follows：(1) mouse embryo stem cell ES-D3 is seeded on the culture plate of the agar containing mass concentration 1%,
Conditioned medium is added, in 37 DEG C, 5%CO2Under the conditions of cultivate 4-5 days, liquid is changed daily；Cell mass is taken to be transferred to culture orifice plate
It is interior, induction liquid I is added, in 37 DEG C, 5%CO2Condition Fiber differentiation 12-14 days, changes liquid daily, obtains ES cell differentiation
Hair Cell Progenitors；The conditioned medium final concentration is constituted：Volumetric concentration 40%HepG2 conditioned mediums, 1mM is non-must
Amino acid, 5 μM of beta -mercaptoethanols, 2mM Glus, volumetric concentration 10%FBS are needed, solvent is the culture of DMEM/F12 bases
Liquid；The HepG2 conditioned mediums are prepared as follows：By HepG2 cells with 5 × 104cells/cm2Density is inoculated in training
Support in ware, by 1.75 × 105The amount of cells/mL nutrient solutions adds the 10%FBS of final concentration containing volume DMEM nutrient solutions, is placed in
37 DEG C, 5%CO2Under the conditions of culture 4-5 days after collect culture supernatants, be centrifuged off cell fragment, supernatant liquor is through 0.22 μm
Filter membrane suction filtration, filtrate is HepG2 conditioned mediums；Induction liquid I final concentration, which is constituted, is：Volumetric concentration 5%FBS, 10ng/
Ml IGF-1,25ng/ml EGF, 1 × N2,1 × B27,50ng/mLFGF3,50ng/mL FGF10,50ng/mL liquaemin, it is molten
Agent is DMEM/F12 basic culture solutions；
(2) the chicken embryo utricle interstitial cell DMEM of the μ g/ml mitomycin Cs containing final concentration 2 of 90% degree of converging will be grown to
Nutrient solution soaks, in 37 DEG C, 5%CO2Cultivated 3 hours in incubator, discard nutrient solution, cell pH value buffers for 7.4 PBS
Liquid is washed, as pretreated chicken embryo utricle interstitial cell；
(3) Hair Cell Progenitors obtained with accutase digestion steps (1), staticly settle, discard precipitation, by cell suspension
With 105Individual/cm2Density be seeded on pretreated chicken embryo utricle interstitial cell, add induction liquid II, in 37 DEG C, 5%
CO2Continue to induce 3-4 weeks in incubator, obtain the nutrient solution containing inner ear hair cells, collect inner ear hair cells；The induction liquid II
Final concentration is constituted：Volumetric concentration 5%FBS, 25ng/ml EGF, 1 × N2,1 × B27,10 μM of RA, 5 μM of DAPT, solvent is
DMEM/F12 basic culture solutions.
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