CN104388381A - Method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for induced differentiation of inner ear hair cells by virtue of human mesenchymal stem cells. The method comprises the steps of separating bone marrow MSCs (mesenchymal stem cells) from bone marrow of human as a source, and inducing the bone marrow MSCs by combining factor combination with feed layer cells so as to differentiate the mesenchymal stem cells. According the method, the human mesenchymal stem cells can be induced to be differentiated into the inner ear hair cells, and the cells obtained by differentiation express specific protein of the hair cells and have certain hair cell electrophysiological functions.
Description
(1) technical field
The present invention relates to a kind of method utilizing the differentiation-inducing acquisition inner ear hair cells of human marrow mesenchymal stem cell.
(2) background technology
The unrepairable damage of Mammals inner ear hair cells is one of main reason causing hearing loss.It has been generally acknowledged that, human inner ear's auditory hair cell does not have self-regeneration repair ability, and its damage is irreversible.Recent study has pointed out Mammals hair cell under certain inductive condition, realize the possibility regenerated, but still there is very large challenge for the Regeneration and Repair of hair cell in pathological process.Therefore find the cell derived of hair cell regeneration, exploitation damage of hair cell repairs the key that Intervention Strategy is hearing loss treatment.
Stem cell with its all can or versatility feature and become the focus of regeneration and restoration area research.Numerous research has illustrated the feature that embryonic stem cell, adult stem cell and induced multi-potent stem cells are divided into Various Tissues cell, and their application potential in tissue injury reparation and cell therapy.Therefore utilize differentiation of stem cells potential, based on stem cell is differentiation-inducing, inquires into hair cell Regeneration and Repair and there is theoretical basis.Mescenchymal stem cell (Mesenchymal stem cells, MSCs) is the adult stem cell that a class of mesoderma origin has height self-renewal capacity and multi-lineage potential.MSCs is extensively present in the tissues such as marrow, fat, muscle, skin, amniotic fluid and umbilical cord or organ, except breaking up to mesoderm directions such as skeletonization, fat and cartilages, MSCs can also cross over other germinal layer cell types such as being divided into neurocyte, liver cell, myocardial cell, islet cells under certain conditions, namely shows the plasticity-of height.In addition MSCs is easy to be separated, and externally long-term cultivation can increase and keep multi-lineage potential.Therefore MSCs is the focus in stem-cell research field always.Marrow is one of MSCs source of enriching the most, and therefore the induction system of bone marrow MSCs differentiation inner ear hair cells is set up in research, will provide effective cell derived, and have important clinical meaning for hair cell regenerative therapy in the future.
(3) summary of the invention
The object of the invention is to provide a kind of method that inducing bone marrow MSCs breaks up inner ear hair cells, and with people's marrow for source, be separated bone marrow MSCs, the method inducing bone marrow MSCs utilizing combinations of factors to combine with feeder layer cells breaks up inner ear hair cells.
The technical solution used in the present invention is:
The invention provides the method for the differentiation-inducing inner ear hair cells of a kind of human marrow mesenchymal stem cell, described method is: get human marrow mesenchymal stem cell, and inoculation is (preferably with 10
5individual/cm
2density inoculation) to induced liquid I, at 37 DEG C, 5%CO
2in incubator, inducing culture 7 days, then changes induced liquid II, at 37 DEG C, and 5%CO
2incubator continues to cultivate 7-10 days, and 0.25% pancreatin EDTA is seeded on chicken embryo Utriculus mesenchymal cell (i.e. feeder layer cells), adds induced liquid III after digesting, at 37 DEG C, and 5%CO
2incubator continues induction 14-21 days, obtains ripe inner ear hair cells;
Induced liquid I final concentration forms: volumetric concentration 5%FBS, 45-55ng/ml IGF-1 (insulin-like growth factor-i), 20-25ng/ml EGF (epithelical cell growth factor), 10 μMs of RA (all-trans-retinoic acid) and 20-30ng/ml FGF2 (FGF2), and solvent is DMEM/F12 basic culture solution;
Induced liquid II final concentration forms: volumetric concentration 5%FBS, 5-15ng/ml BMP4 (bone morphogenetic protein 4) and 20-30ng/ml FGF2, and solvent is DMEM/F12 basic culture solution;
Induced liquid III final concentration forms: volumetric concentration 5%FBS and 20-30ng/ml FGF2, and solvent is DMEM/F12 basic culture solution;
The pretreated method of described chicken embryo Utriculus mesenchymal cell is: soaked by the DMEM nutrient solution of chicken embryo Utriculus mesenchymal cell (the chicken embryo Utriculus mesenchymal cell of preferred growth to 90% degree of converging) containing final concentration 2 μ g/ml ametycin, at 37 DEG C, 5%CO
2incubator cultivates 3 hours, discards suspension, obtains pretreated chicken embryo Utriculus mesenchymal cell.
Further, preferred described induced liquid I final concentration composition: volumetric concentration 5%FBS, 50ng/mlIGF-1,25ng/ml EGF, 10
-6m RA and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
Further, preferred described induced liquid II final concentration composition: volumetric concentration 5%FBS, 10ng/mlBMP4 and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
Further, preferred described induced liquid III final concentration composition: volumetric concentration 5%FBS and 25ng/mlFGF2, solvent is DMEM/F12 basic culture solution.
Further, described feeder layer cells is chicken embryo Utriculus mesenchymal cell, preparation method is mainly through digestion and propagating method separation and Culture Utriculus mesenchymal cell from fertilization 18 days chicken embryos, 3-5 is for cell, after mitomycin C process, as feeder layer cells, preferred described chicken embryo Utriculus mesenchymal cell is prepared as follows: within 18 days, be separated Utriculus chicken embryo from fertilization, 0.25% pancreatin, 37 DEG C of digestion are after 15-20 minute, stop with the DMEM nutrient solution adding volume final concentration 10%FBS, cross 200 eye mesh screens, collecting cell suspension, the centrifugal 6-8 minute of 1000rpm/min, the cell DMEM nutrient solution adding volume final concentration 10%FBS suspends, be inoculated in culturing bottle, put 37 DEG C, 5%CO
2incubator is cultivated, and changes liquid first, discard non-attached cell after 48 hours, adherent continuation is cultured to 90% degree of converging, and by 0.25% pancreatin/EDTA digestion also inoculation culture, is designated as P1 for cell, by Secondary Culture like this, to P3 generation, namely obtain chicken embryo Utriculus mesenchymal cell.
The factor of the present invention all obtains by commercial mode, and described fertilization 18 days chicken embryos cultivate company limited purchased from Hangzhou Tian Yuan.
Compared with prior art, beneficial effect of the present invention is mainly reflected in: the method for the invention effectively can be divided into inner ear hair cells by inducing human mesenchymal stem cells, differentiation gained cell not only expresses hair cell differential protein, and what is more important differentiation and maturation cell possesses certain hair cell electrophysiological function.
(4) accompanying drawing explanation
Fig. 1 is the cellular form microgram (amplifying 100 times) that induced liquid II is induced the 7th day; A figure is the MSCs not starting to induce, and B figure is the cell that induced liquid II is induced the 7th day.
Fig. 2 is the electrophorogram that induced liquid II induces the 7th day cell expressing hair cell marker gene Atoh1; A is that induced liquid II induces cell Atoh1 genetic expression in 7 days to detect gel electrophoresis figure, and band β-actin is as internal reference, and swimming lane 0D represents the MSCs not starting to induce, and swimming lane II-7D represents that induced liquid II induces the cell of the 7th day; B1 is the protein expression of Atoh1 in the MSCs not starting to induce, and B2 is the expression that induced liquid II induces Atoh1 in the cell of the 7th day.
Fig. 3 is the cellular form microgram (amplifying 100 times) of the chicken embryo Utriculus mesenchymal cell in P3 generation.
Fig. 4 is the Immunofluorescence test figure that induced liquid III induces rear cell expressing hair cell specific proteins; A is that Atoh1 and Myosin 7a expresses; B is that Myosin 7a and nucleus C23 express.
Fig. 5 is the Immunofluorescence test figure that induced liquid III induces rear cell expressing hair cell specific proteins; A is that in Espin and nucleus, C23 expresses, and B is that in Myosin 7a and nucleus, C23 expresses, and C is the superposition of A and B two width figure.
Fig. 6 is that induced liquid III induces rear cell hair cell electrophysiological function detection figure; A is that in FM1-43FX dyeing and nucleus, C23 expresses, and B is that in Myosin 7a and nucleus, C23 expression C is the superposition of A and B two width figure.
(5) embodiment
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:
Embodiment 1: the separation of human marrow mesenchymal stem cell
The marrow of Adult Healthy Volunteers, pH value is after 7.4 phosphate buffered saline buffers (PBS, purchased from Sheng Gong biotech firm) wash twice, and with PBS, marrow is diluted to 10
7the cell suspension of individual/ml, add to human lymphocyte parting liquid upper strata (Ficoll-Hypaque, D=1.077 ± 0.002g/ml, biological purchased from raw work) gently, both volume ratios are 3:2; Room temperature (25 DEG C), 2200rpm/min, after centrifugal 25 minutes, draw middle white cellular layer (mononuclearcell layer); Add and add volume final concentration 10% foetal calf serum (FBS, purchased from Gibco company) IMDM basic culture solution (purchased from Corning company), washed cell, then the centrifugal 6-8 minute of 1000rpm/min, remove nutrient solution, with same nutrient solution suspension cell, be inoculated in culturing bottle, put 37 DEG C, 5%CO
2incubator is cultivated, liquid is changed first after 48 hours, discard non-attached cell, adherent continuation is cultured to 90% degree of converging, by 0.25% pancreatin/EDTA (purchased from Corning company) digestion also inoculation culture, be designated as P1 for cell, by Secondary Culture like this, to P3-P5 generation, obtain the fiber-like human marrow mesenchymal stem cell (MSCs) that form is single.
Embodiment 2: the separation of chicken embryo Utriculus mesenchymal cell
Separation Utriculus from fertilization 18 days chicken embryos (cultivating company limited purchased from Hangzhou Tian Yuan), 0.25% pancreatin, 37 DEG C of digestion, after 15-20 minute, stop with the DMEM nutrient solution (purchased from Corning company) adding volume final concentration 10%FBS, cross 200 eye mesh screens, collecting cell suspension, the centrifugal 6-8 minute of 1000rpm/min, the cell DMEM nutrient solution adding volume final concentration 10%FBS suspends, and is inoculated in culturing bottle, put 37 DEG C, 5%CO
2incubator is cultivated, liquid is changed first after 48 hours, discard non-attached cell, adherent continuation is cultured to 90% degree of converging, digest with 0.25% pancreatin/EDTA and be seeded to Tissue Culture Flask and cultivate, be designated as P1 for cell, by Secondary Culture like this, to P3 generation, namely obtain the chicken embryo Utriculus mesenchymal cell that form is single.
Embodiment 3: the induction of human marrow mesenchymal stem cell vitro differentiation inner ear hair cells
The chicken embryo Utriculus mesenchymal cell growing to 90% degree of converging prepared by Example 2, soaks with the DMEM nutrient solution containing final concentration 2 μ g/ml ametycin (available from Sigma), 37 DEG C, 5%CO
2incubator cultivates 3 hours, discards suspension, after embathing, obtains pretreated chicken embryo Utriculus mesenchymal cell with PBS.
The P3-P5 generation human marrow MSCs that Example 1 method obtains, is seeded to induced liquid I, 37 DEG C, 5%CO
2inducing culture 7 days in incubator, wherein often changes liquid in 2-3 days, then changes induced liquid II, 37 DEG C, 5%CO
2incubator continues to cultivate 7-10 days, wherein often within 2-3 days, changes liquid, after 0.25% pancreatin EDTA digests, with 10
5individual/cm
2density be seeded on pretreated chicken embryo Utriculus mesenchymal cell, add induced liquid III, 37 DEG C, 5%CO
2incubator continues induction 14-21 days, collects inducing cell and identifies, obtain ripe inner ear hair cells.
Induced liquid I final concentration forms: volumetric concentration 5%FBS, 50ng/ml IGF-1,25ng/mlEGF, 10
-6m RA and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution (purchased from Corning company).
Induced liquid II final concentration forms: volumetric concentration 5%FBS, 10ng/ml BMP4 and 25ng/mlFGF2, nutrient solution (DMEM/F12) based on solvent;
Induced liquid III final concentration forms: volumetric concentration 5%FBS and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
IGF-1, EGF and FGF2 purchased from Peprotech company, BMP4 purchased from RD company, RA available from Sigma.
Embodiment 4: the qualification of noble cells
(1) morphological observation: induce the cell of the 7th day to utilize microscope observing cell form without the MSCs induced and induced liquid II, and Taking Pictures recording, the results are shown in Figure shown in 1, picture amplifies 100 times.
(2) hair cell specific gene detects: the cell TRizol (purchased from Invitrogen company) after induced liquid II induction extracts total serum IgE, extracting method by specification carries out, and then uses Reverse Transcriptase kit RNA PCR kit (AMV) Ver3.0 (purchased from TaKaRa company) to carry out reverse transcription.The cDNA obtained with reverse transcription is template, under the effect of primer Atoh1 (primer β-actin is as internal reference), carry out PCR reaction, detects Atoh1 expression conditions, and using β-actin as internal reference.PCR program is: 94 DEG C of denaturation 5min, 94 DEG C of sex change 30s, 50 DEG C ~ 65 DEG C annealing 30s, and 72 DEG C extend 30s, totally 30 ~ 40 circulations, and 72 DEG C extend 10min, and annealing temperature and cycle number are selected according to the particular case of primer.PCR primer is carried out agarose gel electrophoresis analysis, to the results are shown in Figure in 2 shown in A.
The primer is as follows: Atoh1 (5'-ATCTACATCAACGCCTTGTCCGAGC-3', 5'-CGAAAGTCGAGAAGTGCAGAGCGTC-3'); β-actin (5'-ACACCTTCTACAATGAGCTG-3', 5'-CTGCTTGCTGATCCACATCT-3'), is synthesized by Shanghai JaRa biotech firm.
(3) hair cell differential protein Immunofluorescence test: induced liquid II and induced liquid III induce after cell after the fixing 15min of 4% paraformaldehyde room temperature (25 DEG C), with PBS (pH value is 7.4) rinsing removing paraformaldehyde, use 0.05%Triton X-100 (available from Sigma) soaking at room temperature process 10 minutes again, then 5% lowlenthal serum (biological purchased from raw work) is used to close 1h in room temperature, PBS cleaning twice, cell is divided into 4 groups, add primary antibodie respectively, group 1 is anti-Atoh1, group 2 is anti-Myosin7a and anti-C23, group 3 is anti-Espin, group 4 is anti-Myosin7a and anti-C23, primary antibodie is all purchased from Santa cruz company, 4 DEG C of wet box overnight incubation, PBS embathes three times, each 10min, add FITC mark two successively to resist, group 1 is Alexa 488, group 2 resists for Alexa 647 and Dylight 405 marks two, group 3 is Alexa 488, group 4 is Dylight 649 and Dylight405 mark two anti-(two is anti-all purchased from Lian Ke biotech firm), hatch 1h for 37 DEG C, PBS embathes three times, wherein anti-Atoh1 primary antibodie group cell DAPI (available from Sigma) contaminates core, and laser confocal microscope (Carl Zeiss) is observed and taken pictures.The results are shown in Figure B1 and B2 in 2 (all amplifying 200 times), Fig. 4 and Fig. 5 (all amplifying 630 times).
(4) hair cell electrophysiological function detects: utilize FM1-43FX (purchased from Invitrogen company) Coloration experiment to detect noble cells electrophysiological function, after induced liquid III induces rear cell HBSS (purchased from Sheng Gong biotech firm) to clean, add 5 μMs of FM1-43FX incubated at room 30s, clean with HBSS immediately, after embathing 20min with HBSS again, carry out immunofluorescence experiment and detect Myosin7a expression, step and antibody are with hair cell differential protein Immunofluorescence test.The results are shown in Figure shown in 6, amplify 630 times.
Result shows, induction gained noble cells can express Hair Cell Progenitors (namely induced liquid II induces gained cell) specific gene and albumen, ripe hair cell specific proteins, what is more important differentiation and maturation cell has certain hair cell electrophysiological function, shows that this induction method can effective inducing bone mesenchymal differentiation of stem cells inner ear hair cells.
Finally, it is also to be noted that what enumerate above is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be had.All distortion that those of ordinary skill in the art can directly derive from content disclosed by the invention or associate, all should think protection scope of the present invention.
Claims (6)
1. a method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell, is characterized in that described method is: get human marrow mesenchymal stem cell, be seeded to induced liquid I, at 37 DEG C, and 5%CO
2in incubator, inducing culture 7 days, then changes induced liquid II, at 37 DEG C, and 5%CO
2incubator continues to cultivate 7-10 days, and 0.25% pancreatin EDTA is seeded on pretreated chicken embryo Utriculus mesenchymal cell, adds induced liquid III after digesting, at 37 DEG C, and 5%CO
2incubator continues induction 14-21 days, obtains ripe inner ear hair cells;
Induced liquid I final concentration forms: volumetric concentration 5%FBS, 45-55ng/ml IGF-1,20-30ng/mlEGF, 10 μMs of RA and 20-30ng/ml FGF2, and solvent is DMEM/F12 basic culture solution;
Induced liquid II final concentration forms: volumetric concentration 5%FBS, 5-15ng/ml BMP4 and 20-30ng/mlFGF2, and solvent is DMEM/F12 basic culture solution;
Induced liquid III final concentration forms: volumetric concentration 5%FBS and 20-30ng/ml FGF2, and solvent is DMEM/F12 basic culture solution;
Described chicken embryo Utriculus cell pretreatment method is: soaked by the DMEM nutrient solution of chicken embryo Utriculus mesenchymal cell containing final concentration 2 μ g/ml ametycin, at 37 DEG C, and 5%CO
2incubator cultivates 3 hours, discards suspension, obtains pretreated chicken embryo Utriculus mesenchymal cell.
2. the method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell as claimed in claim 1, is characterized in that described induced liquid I final concentration forms: volumetric concentration 5%FBS, 50ng/ml IGF-1,25ng/ml EGF, 10
-6m RA and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
3. the method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell as claimed in claim 1, it is characterized in that described induced liquid II final concentration forms: volumetric concentration 5%FBS, 10ng/ml BMP4 and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
4. the method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell as claimed in claim 1, is characterized in that described induced liquid III final concentration forms: volumetric concentration 5%FBS and 25ng/ml FGF2, solvent is DMEM/F12 basic culture solution.
5. the method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell as claimed in claim 1, it is characterized in that described chicken embryo Utriculus mesenchymal cell is prepared as follows: within 18 days, chicken embryo, be separated Utriculus from fertilization, 0.25% pancreatin, 37 DEG C of digestion are after 15-20 minute, stop with the DMEM nutrient solution adding volume final concentration 10%FBS, cross 200 eye mesh screens, collecting cell suspension, the centrifugal 6-8 minute of 1000rpm/min, the cell DMEM nutrient solution adding volume final concentration 10%FBS suspends, be inoculated in culturing bottle, put 37 DEG C, 5%CO
2incubator is cultivated, and changes liquid first, discard non-attached cell after 48 hours, adherent continuation is cultured to 90% degree of converging, and digests and is seeded in Tissue Culture Flask and cultivate, be designated as P1 for cell with 0.25% pancreatin/EDTA, by Secondary Culture like this, to P3 generation, namely obtain chicken embryo Utriculus mesenchymal cell.
6. the method for the differentiation-inducing inner ear hair cells of human marrow mesenchymal stem cell as claimed in claim 1, is characterized in that described induced liquid II cultivates postdigestive cell with 10
5individual/cm
2density be seeded on pretreated chicken embryo Utriculus mesenchymal cell.
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| WO2017146035A1 (en) * | 2016-02-22 | 2017-08-31 | 学校法人順天堂 | Method for producing inner ear cells |
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| CN107384852A (en) * | 2017-07-27 | 2017-11-24 | 山东兴瑞生物科技有限公司 | A kind of sertoli cell is divided into method and the application of inner ear hair cells |
| CN112852715A (en) * | 2021-03-08 | 2021-05-28 | 浙江大学 | Method for directionally differentiating induced pluripotent stem cells into inner ear hair cell-like cells |
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