CN106350521A - Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron - Google Patents
Preparation method of ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron Download PDFInfo
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Abstract
The invention disclsoes a preparation method of an ALS (Amyotrophic Lateral Sclerosis) patient specific motor neuron. The method comprises the following steps: (1) reprogramming an in-vitro fibroblast carrying ALS related genic mutation from an ALS patient source to obtain induced pluripotent stem cells carrying the ALS related genic mutation (ALS-iPSC); (2) carrying out gene editing on the ALS-iPSC and specifically eliminating the ALS related genic mutation to obtain gene corrected induced pluripotent stem cells (cALS-iPSC); (3) carrying out orient induction and differentiation on the cALS-iPSC and the ALS-iPSC respectively to obtain the ALS patient specific motor neuron carrying the ALS related genic mutation or without the ALS related genic mutation. The specific motor neuron carrying ALS pathogenic gene mutation, obtained by the preparation method, can be used as an effective platform for carrying out efficient and high-throughput individualized drug screening; the ALS patient specific motor neuron without the ALS related genic mutation is hopefully used for treating and preventing ALS. The preparation method of the ALS patient specific motor neuron lays the foundation for ALS disease researches, disease model development, disease pathogenesis researches and disease treatment.
Description
Technical field
The invention belongs to biological technical field, a kind of specifically preparation method of als patient-specific motor neuron.
Background technology
People's amyotrophy lateral schlerosis (amyotrophic lateral sclerosis, abbreviation als) are a kind of nerves
Degenerative disease, major pathologic features are motor neuron degeneration, lead to patient's amyotrophy, paralysis, or even can shadow
Ring respiratory system, jeopardize patients ' lives.Als morbidity is rapid, dead after most patient morbidity 3-5.Als is divided into two types,
Sporadic (sporadic als, sals) and familial (familial als, fals).The two clinical symptoms is similar to, but causes
Anttdisease Mechanism is all unclear, does not also have effective Drug therapy.Sals complex genesis, common because of environmental factorss and inherited genetic factorss
Same-action.Research finds that fals is usually to be caused by certain gene heterozygous mutant, such as sod1, fus, c9orf72, tardbp etc..
Sod1 gene mutation is the earliest discovery Disease-causing gene related to fals, is accounted for by patient als that sod1 gene mutation leads to
The 20% of patient fals.Pathogenesis for sod1 gene mutation have focused largely in model organism (as mice)
OK.Research finds, sod1 gene mutation affects the Redox homeostasis of organism, easily forms hydrophobic condensation product, disturbs egg
White matter degradation pathway, broken cyclase protein matter stable state, affect mitochondrial function, cause apoptosis.Recently also it has been reported that, sod1 makees
Response to oxidative stress for transcription factor regulating cell.Fus is rna associated proteins, participates in the editor of regulation and control mrna, transports and turn over
Translate process.Detailed mechanism research with regard to fus is relatively fewer, the fus cohesion noting abnormalities in the pathologic finding of patient als
Thing.
Although research als contributes to the mechanism it is understood that als in model organism (as mice).But Land use models are given birth to
Thing screens effective medicine and effectively treats making slow progress of scheme with exploring other.So far, only a kind of treat als's
Medicine riluzole is permitted by Food and Drug Administration, but its drug effect is slow.One of reason is species difference, due to people and
The genetic background of model organism is different, and in model organism, the medicine of effectively treatment als of screening clinically can not alleviate als
Symptom, or large side effects.Secondly, the als disease model being produced by overexpression mutant gene can not really reflect patient's
Physiological situation.External source overexpression is mutated the expression of the expression significantly larger than in patient body unit point gene mutation of Disease-causing gene
Level, thus mask some pathogenic key factors it is impossible to really react pathogenesis, the medicine screening on this basis
The clinical symptoms of patient als can not effectively be alleviated.
Content of the invention
It is an object of the invention to provide a kind of preparation of people's amyotrophy lateral schlerosis patient-specific motor neuron
Method.
The preparation method of people's amyotrophy lateral schlerosis patient-specific motor neuron provided by the present invention, can be
Following method a or method b:
Method a: do not carry people's amyotrophy lateral schlerosis patient-specific motor neuron of als associated gene mutation
Preparation method, specifically may include following steps:
(1) the one-tenth fiber to the in vitro people amyotrophy lateral schlerosis patient source carrying als associated gene mutation
Cell is reprogrammed, and obtains carrying the induced multi-potent stem cell of described als associated gene mutation, is designated as als-ipsc；
(2) described als-ipsc is carried out with gene editing, specificity eliminates described als associated gene mutation, obtains gene
The induced multi-potent stem cell corrected, is designated as cals-ipsc；
(3) described cals-ipsc is oriented with induction differentiation, obtains final product the people not carrying described als associated gene mutation
Amyotrophy lateral schlerosis patient-specific motor neuron.
Method b: carry people's amyotrophy lateral schlerosis patient-specific motor neuron of als associated gene mutation
Preparation method, specifically may include following steps:
(1) the one-tenth fiber to the in vitro people amyotrophy lateral schlerosis patient source carrying als associated gene mutation
Cell is reprogrammed, and obtains carrying the induced multi-potent stem cell of described als associated gene mutation, is designated as als-ipsc；
(2) described als-ipsc is oriented with induction differentiation, obtains final product the people's muscle carrying described als associated gene mutation
Amyotrophic lateral sclerosis disease patient-specific motor neuron.
Wherein, in the case that described reprogramming refers to not change gene order, by epigenetic modification (as dna methylates)
To change the process of cell fate.Reprogramming at present refers mainly to two processes: first, the cell reversal of differentiation returns to totipotency
The process of state；Second, be converted into the process of another kind of noble cellss from a kind of noble cellss.
In the step (1) of methods described, described reprogramming be specially by oct3/4 gene, sox2 gene, klf4 gene,
Lmyc gene and lin28 gene import (as electricity turns) described in vitro people's amyotrophy carrying als associated gene mutation jointly
In the fibroblast in lateral schlerosis patient source, and cultivate dry to the induced multi-potent obtaining described als associated gene mutation
Cell.
In the present invention, described " oct3/4 gene, sox2 gene, klf4 gene, l-myc gene and lin28 gene is common
With the fibroblast importing the described in vitro people amyotrophy lateral schlerosis patient source carrying als associated gene mutation
In " particular by the pcxle-hoct3/4-shp53-f plasmid of described oct3/4 gene will be carried, carries described sox2
The pcxle-hsk plasmid of gene and described klf4 gene and carry described l-myc gene and the pcxle- of described lin28 gene
Hul plasmid imports (as electricity turns) described in vitro people's amyotrophy lateral schlerosis disease carrying als associated gene mutation jointly
Realize in the fibroblast of people source.In practical operation, for the ease of the screening of positive colony, can be simultaneously directed
This plasmid of pcxle-egfp plasmid carries and expresses egfp gene.The quality proportioning of four kinds of plasmids can be 1:1:1:1.
In step (1), described culture concretely first in fibroblast culture medium culture change liquid after 1 day, with described
Fibroblast culture medium continues culture and is transferred to containing the mice embryonic crossed with mitomycin inactivation treatment in advance to after the 5th day
Use ipsc culture medium culturing to the 3rd week in fibroblastic raising coating systems, obtain the induction of described als associated gene mutation
Pluripotent stem cell.
Wherein, the composition of described fibroblast culture medium is as follows: by dmem culture medium, (invitrogen, article No. is
11965118) and fbs (hyclone) according to volume ratio for 9:1 ratio mixing after, add penicillin/streptomycin
(invitrogen, article No. is 15070-063), makes the final concentration of 10g/l of described penicillin/streptomycin.
In the present invention, described als associated gene mutation concretely fus g1566a or sod1 a272c.
Described fus g1566a refers to that (genome sequence is classified as genbank:ng_012889.2 to fus gene, and cdna sequence is
1566th mutation that there occurs from g to a of gene coded sequence (cds sequence) genbank:nm_004960.3), mutation
Gene afterwards may be simply referred to as fusg1566a.Described sod1 a272c refers to that (genome sequence is classified as genbank:ng_ to sod1 gene
008689.1, cdna sequence is genbank:nm_000454.4) the 272nd of gene coded sequence (cds sequence) there occurs
Mutation from a to c, the gene after mutation may be simply referred to as sod1a272c.
Accordingly, in step (1), the described in vitro people's amyotrophy lateral schlerosis carrying als associated gene mutation
Patient source fibroblast can be skin-derived fibroblast, concretely fus g1566a fibroblast or
Sod1 a272c fibroblast；Described fus g1566a fibroblast specially comes from U.S. coriell cell
The parent cell line of repository is the cell of nd29563；Described sod1 a272c fibroblast is specially
The cell being nd29149 from the parent cell line in U.S. coriell cell repository.
In the step (2) of methods described a, described " specificity eliminates described als associated gene mutation " is by described fus
G1566a corrects as fus g1566g, or described sod1 a272c is corrected as sod1 a272a.
In the present invention, in the step (2) of methods described a, described als-ipsc is carried out with gene editing and is specially utilization
Crispr/cas9 technology carries out gene editing to described als-ipsc.
Accordingly, the step (2) of methods described a is particularly as follows: by grna expression plasmid, cas9 expression plasmid and recombination template
Jointly proceed to described als-ipsc, and cultivate to obtaining described cals-ipsc.
When described als associated gene mutation is described fus g1566a, the knowledge of the grna in described grna expression plasmid
Other regional sequence (i.e. corresponding coded sequence of intervening sequence spacer) is sequence 1 in sequence table；Described recombination template is sequence
Double-strand dna shown in sequence 2 in table；Described grna expression plasmid is specially fus-grna-mcherry；Described fus-grna-
Mcherry be by the dna fragment shown in sequence in sequence table 1 be inserted into grna-mcherry carrier (addgene, article No. be #
78544) recombiant plasmid obtaining after between restriction enzyme site aflii.
When described als associated gene mutation is described sod1 a272c, the knowledge of the grna in described grna expression plasmid
Other regional sequence (i.e. corresponding coded sequence of intervening sequence spacer) is sequence 3 in sequence table；Described recombination template is sequence
Double-strand dna shown in sequence 4 in table.Described grna expression plasmid is specially sod1-grna-mcherry；Described sod1-grna-
Mcherry be by the dna fragment shown in sequence in sequence table 2 be inserted into grna-mcherry carrier (addgene, article No. be #
78544) recombiant plasmid obtaining after between restriction enzyme site aflii.
Described cas9 expression plasmid is cas9-gfp；Described cas9-gfp is purchased from addgene company of the U.S., and article No. is #
59766.
Wherein, the quality proportioning of described grna expression plasmid, described cas9 expression plasmid and described recombination template is 1:2:
2.
In the step (2) of methods described a, described culture is specially and is successfully proceeded to described grna expression plasmid, described
The described als-ipsc of cas9 expression plasmid and described recombination template is transferred to containing being crossed with mitomycin inactivation treatment in advance
Cultivated 2 weeks with cdf12 culture medium (Ju Ti Pei Fang sees below) in the raising coating systems of mouse embryo fibroblasts, obtain described
cals-ipsc.
Described cals-ipsc is carried out with the method for positive identification, and concretely caps identification (is amplified with pcr technology and comprises
The fragment in mutational site, due to the difference of a snp, can be identified with corresponding restricted enzyme).Finally give mutation
Gene fusg1566aCorrect as fusg1566gClone, mutant gene sod1a272cCorrect as sod1a272aClone, as positive gram
Grand.
Fus-f:5 '-gagaaagtggtttcattttgagggctaggtgga-3 '；
Fus-r:5 '-ttgtttgagcctcaccattaaaagggccaaaag-3 '.
Fus identification restricted enzyme: bsaxi.
Sod1-f:5 '-cccatctttcttcccagagcattagtgtgtagacg-3 '；
Sod1-r:5 '-acaaaatgttctgtttaacaagtgagaaacccaatcct-3 '.
Sod1 identification restricted enzyme: apeki.
In the step (3) of methods described a or in the step (2) of methods described b, to described cals-ipsc or described
Als-ipsc is oriented what induction differentiation was specifically carried out according to the method comprising the steps:
(3-1) described cals-ipsc or described als-ipsc is inoculated into containing in advance with mitomycin inactivation treatment mistake
The raising coating systems of mouse embryo fibroblasts in use cdf12 culture medium culturing, obtain treating noble cellss；
(3-2) treat that noble cellss carry out first time differentiation culture to described in mn culture medium 1, after obtaining cultivating for the first time
Cell；
(3-3) in mn culture medium 2, second differentiation culture is carried out to cell after the culture of described first time, obtain second
Cell after culture；
(3-4) after described second being cultivated in mn culture medium 3, cell carries out third time differentiation culture, obtains third time
Cell after culture；
(3-5) in mn culture medium 4, the 4th differentiation culture is carried out to cell after the culture of described third time, obtain the 4th time
Cell after culture；
(3-6) in mn culture medium 5, the 5th differentiation culture is carried out to cell after described 4th time culture, obtain the 5th time
Cell after culture, does not as carry people's amyotrophy lateral schlerosis patient-specific motion of described als associated gene mutation
Neuron or the people's amyotrophy lateral schlerosis patient-specific motor neuron carrying described als associated gene mutation.
Wherein, in step (3-5), when carrying out described 4th differentiation culture, be by described third time cultivate after cell with
Unicellular go to through being cultivated with described mn culture medium 4 in the coated culture plate of matrigel (matrigel).In this step
In, also can be processed with rock inhibitor to improve cell survival rate before and after passing on.
Wherein, the composition of described cdf12 culture medium is as follows: by dmem/f12 culture medium, (invitrogen, article No. is
11320-033) mix according to the volume ratio of 8:2 with knockout serum substitute (invitrogen, article No. is n10828-028)
After conjunction, add non essential amino acid (invitrogen, article No. is 11140-050), glutamax in mixed liquor
(invitrogen, article No. is 35050-061), penicillin/streptomycin (invitrogen, article No. is 15070-063), β-sulfydryl
Ethanol (invitrogen, 21985-023) and people fgf2 (joint protein central) are so that described non-essential amino
Final concentration of 0.1mm, the final concentration of 1mm of described glutamax, the final concentration of 10g/l of described penicillin/streptomycin of acid,
Final concentration of 55 μm of described beta -mercaptoethanol, the final concentration of 10ng/ml of described people fgf2.
The composition of described mn culture medium 1 is as follows: by advanced dmem/f12 culture medium, (invitrogen, article No. is
12634028) and after neurobasal culture medium (invitrogen, article No. be 12348017) mixes according to the volume ratio of 1:1,
Add in mixed liquor n2 (invitrogen, article No. be 17502048), b27 (invitrogen, article No. is 17504044),
Glutamax (invitrogen, article No. be 35050-061), heparin (heparin) (sigma, article No. is h3149),
Chir99021 (cellagentech, article No. be c2447), sb431542 (cell agentech, article No. is c7243),
Compound e (emd chemicals, article No. is 209986-17-4) and dorsomorphin (sigma, article No. is p5499)；
Make final concentration of 1% (volumn concentration) of described n2, final concentration of 2% (volumn concentration) of described b27, described
The final concentration of 2mm of glutamax, the final concentration of 2g/l of described heparin (heparin), described chir99021's is final concentration of
4 μm, final concentration of 3 μm of described sb431542, final concentration of 0.1 μm of described compound e, described dorsomorphin
Final concentration of 1 μm.
The composition of described mn culture medium 2 is as follows: by advanced dmem/f12 culture medium, (invitrogen, article No. is
12634028) and after neurobasal culture medium (invitrogen, article No. be 12348017) mixes according to the volume ratio of 1:1,
Add in mixed liquor n2 (invitrogen, article No. be 17502048), b27 (invitrogen, article No. is 17504044),
Glutamax (invitrogen, article No. be 35050-061), heparin (heparin) (sigma, article No. is h3149),
Chir99021 (cellagentech, article No. be c2447), sb431542 (cell agentech, article No. is c7243),
Compound e (emd chemicals, article No. be 209986-17-4), dorsomorphin (sigma, article No. is p5499) and
Ra (sigma, article No. is r2625)；Make final concentration of 1% (volumn concentration) of described n2, described b27's is final concentration of
2% (volumn concentration), the final concentration of 2mm of described glutamax, the final concentration of 2g/l of described heparin (heparin),
Final concentration of 4 μm of described chir99021, final concentration of 3 μm of described sb431542, described compound e's is final concentration of
0.1 μm, final concentration of 1 μm of described dorsomorphin, the final concentration of 100nm of described ra.
The composition of described mn culture medium 3 is as follows: by advanced dmem/f12 culture medium, (invitrogen, article No. is
12634028) and after neurobasal culture medium (invitrogen, article No. be 12348017) mixes according to the volume ratio of 1:1,
Add in mixed liquor n2 (invitrogen, article No. be 17502048), b27 (invitrogen, article No. is 17504044),
Glutamax (invitrogen, article No. be 35050-061), heparin (heparin) (sigma, article No. is h3149),
Chir99021 (cellagentech, article No. be c2447), sb431542 (cell agentech, article No. is c7243),
Compound e (emd chemicals, article No. be 209986-17-4), ra (sigma, article No. is r2625) and sag (Article No. is 364590-63-6)；Make final concentration of 1% (volumn concentration) of described n2, described b27
Final concentration of 2% (volumn concentration), the final concentration of 2mm of described glutamax, the end of described heparin (heparin) is dense
Spend for 2g/l, final concentration of 4 μm of described chir99021, final concentration of 3 μm of described sb431542, described compound e
Final concentration of 0.1 μm, the final concentration of 500ng/ml of the final concentration of 100nm of described ra, described sag.
The composition of described mn culture medium 4 is as follows: by advanced dmem/f12 culture medium, (invitrogen, article No. is
12634028) and after neurobasal culture medium (invitrogen, article No. be 12348017) mixes according to the volume ratio of 1:1,
Add in mixed liquor n2 (invitrogen, article No. be 17502048), b27 (invitrogen, article No. is 17504044),
Glutamax (invitrogen, article No. be 35050-061), heparin (heparin) (sigma, article No. is h3149),
Compound e (emd chemicals, article No. be 209986-17-4), ra (sigma, article No. is r2625) and sag (Article No. is 364590-63-6)；Make final concentration of 1% (volumn concentration) of described n2, described b27
Final concentration of 2% (volumn concentration), the final concentration of 2mm of described glutamax, the end of described heparin (heparin) is dense
Spend for 2g/l, final concentration of 0.1 μm of described compound e, the final concentration of 100nm of described ra, the final concentration of described sag
For 500ng/ml.
The composition of described mn culture medium 5 is as follows: by advanced dmem/f12 culture medium, (invitrogen, article No. is
12634028) and after neurobasal culture medium (invitrogen, article No. be 12348017) mixes according to the volume ratio of 1:1,
Add in mixed liquor n2 (invitrogen, article No. be 17502048), b27 (invitrogen, article No. is 17504044),
Glutamax (invitrogen, article No. be 35050-061), heparin (heparin) (sigma, article No. is h3149),
Compound e (emd chemicals, article No. is 209986-17-4), dapt (sigma, article No. is d5942) and fiber connect
Albumen (fibronectin) (gibco, article No. is phe0023)；Make final concentration of 1% (volumn concentration) of described n2,
Final concentration of 2% (volumn concentration) of described b27, the final concentration of 2mm of described glutamax, described heparin
(heparin) final concentration of 0.1 μm of final concentration of 2g/l, described compound e, final concentration of 10 μm of described dapt,
The final concentration of 10 μ g/ml of described fibronectin (fibronectin).
In step (3-1), the concretely 1-2 days time of described culture.In step (3-2), described culture when
Between be 3 days.In step (3-3), the concretely 3 days time of described culture.In step (3-4), the time of described culture
Concretely 1 day.In step (3-5), the concretely 1 day time of described culture.In step (3-6), described culture
Concretely 3 days time.
The present invention is also claimed one kind and is used for preparing people's amyotrophy lateral schlerosis patient-specific motor neuron
Test kit.
The present invention required for protection for preparing people's amyotrophy lateral schlerosis patient-specific motor neuron
Test kit, containing described mn culture medium 1, described mn culture medium 2, described mn culture medium 3, described mn culture medium 4, described mn culture
Base 5.
Cdf12 culture medium as above can certainly be contained.
Fallen within using people's amyotrophy lateral schlerosis patient-specific motor neuron that methods described prepares
Protection scope of the present invention.
Methods described or described test kit or described people's amyotrophy lateral schlerosis patient-specific motor neuron exist
Application in arbitrary as follows falls within protection scope of the present invention:
A () prepares for screening and/or identifying the clinical medicine that can treat and/or prevent people's amyotrophy lateral schlerosis
The product of thing and/or natural organic matter and/or micromolecular compound (predominantly carries people's muscular atrophy of als associated gene mutation
The application of contracting lateral schlerosis patient-specific motor neuron)；
B () prepares for treating and/or preventing the product of people's amyotrophy lateral schlerosis (predominantly not carry als phase
The application of people's amyotrophy lateral schlerosis patient-specific motor neuron of correlation gene mutation)；
C cell model that () prepares people's amyotrophy lateral schlerosis (predominantly carries people's flesh of als associated gene mutation
The application of meat amyotrophic lateral sclerosis disease patient-specific motor neuron).
The als specificity motor neuron of present invention preparation has the advantage that 1) there is self renewal in vitro and divide
The potential of three germinal layers of chemical conversion, thus the cell line accumulated by disease can be directed differentiation to；2) utilize existing gene targeting
The genome that technology can carry out gene mutation for the induction type pluripotent stem cell of patient's autologous is corrected in situ, thus
Gene level removes endogenouss paathogenic factor；3) ipsc of patient's autologous, can be divided into further and be available for autograft
Cell, it is to avoid the puzzlement of distribution type and rejection.
The skin flbroblast that patient als is originated by the present invention using ipsc technology, by nonconformity additive type plasmid
The method that electricity turns makes it reprogram as its specific ipsc, corrects gene using crispr/cas9 gene editing technology targeting
Mutation, obtains the comparison of genetic background consistent (isogenic), and is directed differentiation to motor neuron simultaneously.The present invention obtains
Carry als Disease-causing gene mutation motor neuron can carry out efficiently high-throughout personalised drug as effective platform
Screening, the motor neuron not carrying the mutation of als Disease-causing gene is expected to be used for treatment and the prevention of als.The present invention is als disease
Research, exploitation disease model, the pathogenesis of study of disease simultaneously carry out disease treatment and lay the foundation, in personalized treatment and conversion
There is in medical science huge application prospect.
Brief description
Fig. 1 is the reprogramming of skin flbroblast and the totipotency identification in two plants of patient als sources.Wherein, a is als
The skin flbroblast (fus g1566a fibroblast and sod1 a272c fibroblast) in patient source and reprogramming
The form of the corresponding ipsc obtaining.The gene mutation entrained by cell that b originates for patient als.C does thin for Immunofluorescence test
Born of the same parents label nanog, oct4, sox2.D is to throw ipsc special for patient als into immunodeficient mouse subcutaneous, is divided into tool
There is the teratoma of three germinal layers, wherein, tuj1 (ectoderm marker), foxa2 (endodermal marker thing), α-sma (mesoderm mark
Note thing).
Fig. 2 corrects, for targeting, the gene mutation that patient als carries.Wherein, a is gene correction solution schematic diagram；B is caps
Identification eliminates the result of gene mutation；C is that sequencing confirms to eliminate gene mutation.
Fig. 3 is that described ipsc is directed differentiation to motor neuron cell.Wherein, a is to be directed differentiation to move by ipsc
The scheme schematic diagram of neuronal cell；B is fusg1566a- mn, sod1a272c- mn, fusg1566g- mn, sod1a272a- mn motion god
Through first cellular morphology；C is label hb9 and isl1 of Immunofluorescence test motor neuron cell, and neuronal cell
Label map2.
Fig. 4 is to carry difference between the motor neuron of als Disease-causing gene mutation and the motor neuron through gene rectification
The transcriptome analysis result of gene expression.Wherein, a is fusg1566a-mn/fusg1566g- mn group and sod1a272c-mn/
sod1a272aThe upper mediation down-regulated gene analysis of-mn group；B is fusg1566a-mn/fusg1566g- mn group and sod1a272c-mn/
sod1a272aIn-mn group group, the upper down-regulated gene that is in harmonious proportion is analyzed；C is fusg1566a-mn/fusg1566g- mn group and sod1a272c-mn/
sod1a272a- mn organizes two groups of upper mediation down-regulated gene analyses jointly.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Cell culture condition in following embodiments if no special instructions, is 37 DEG C, 5%co2.
Fus g1566a fibroblast (nd29563) in following embodiments, sod1 a272c fibroblast
(nd29149) all buy the coriell cell repository in the U.S..
Pcxle-hoct3/4-shp53-f (#27077) in following embodiments, pcxle-hsk (#27078), pcxle-
Hul (#27080) and pcxle-egfp (#27082) all buys in addgene.Wherein, pcxle-hoct3/4-shp53-f carries
And express oct3/4 gene；Pcxle-hsk carries and expresses sox2 gene and klf4 gene；Pcxle-hul carries and expresses l-
Myc gene and lin28 gene；Pcxle-egfp carries and expresses egfp gene.
The antibody for immunofluorescence in following embodiments is as follows:
Anti-human oct4 antibody (sc-5279), santa cruz biotechnology.
Anti-human sox2 antibody (sc-17320), santa cruz biotechnology.
Anti-human nanog antibody (ab21624), abcam.
Anti-human tuj1 antibody (t2220), sigma.
Anti-human foxa2 antibody (8186), cell signaling technology.
Anti-human sma antibody (a5228), sigma.
Anti-human map2 antibody (4403), sigma.
Anti-human hb9 antibody (81.5c10), developmental studies hybridoma bank
Anti-human isl1 antibody (ab20670), abcam.
Culture medium prescription in following embodiments is as follows:
(1) fibroblast culture medium: (invitrogen, article No. is by 90% (volumn concentration) dmem culture medium
11965118), after and 10% (volumn concentration) fbs (hyclone, article No. is sh30084.03) mixing, add following thing
Matter: 1% (10g/l) penicillin/streptomycin (invitrogen, article No. is 15070-063), the concentration of wherein each material is to become
Final concentration in fibrocyte culture medium.
(2) ipsc culture medium prescription (cdf12 culture medium): by 80% (volume fraction) dmem/f12 culture medium
(invitrogen, article No. is 11320-033) and 20% (volumn concentration) knockout serum substitute
After (invitrogen, article No. is n10828-028) mixing, add following material: 0.1mm non essential amino acid
(invitrogen, article No. be 11140-050), 1mm glutamax (invitrogen, article No. is 35050-061), 1%
(10g/l) penicillin/streptomycin (invitrogen, article No. is 15070-063), 55 μm of beta -mercaptoethanols (invitrogen, goods
Number be 21985-023), 10ng/ml human fgf2 (joint protein central), the concentration of wherein each material be
Final concentration in ipsc culture medium.
(3) mn culture medium 1 formula: by 50% (volumn concentration) advanced dmem/f12 culture medium
(invitrogen, article No. is 12634028) and 50% (volumn concentration) neurobasal culture medium (invitroge, goods
Number it is 12348017) after mixing, add following material: (invitrogen, article No. is 1% (volumn concentration) n2
17502048), 2% (volumn concentration) b27 (invitrogen, article No. is 17504044), 2mm glutamax
(invitrogen, article No. be 35050-061), 0.2% (2g/l) heparin (sigma, article No. is h3149), 4 μm
Chir99021 (cell agentech, article No. be c2447), 3 μm of sb431542 (cellagentech, article No. is c7243),
0.1 μm of compound e (emd chemicals, article No. is 209986-17-4), 1 μm of dorsomorphin (sigma, article No.
For p5499), the concentration of wherein each material is the final concentration in mn culture medium 1.
(4) mn culture medium 2 formula: by 50% (volumn concentration) advanced dmem/f12 culture medium
(invitrogen, article No. is 12634028) and 50% (volumn concentration) neurobasal culture medium (invitroge, goods
Number it is 12348017) after mixing, add following material: (invitrogen, article No. is 1% (volumn concentration) n2
17502048), 2% (volumn concentration) b27 (invitrogen, article No. is 17504044), 2mm glutamax
(invitrogen, article No. be 35050-061), 02% (2g/l) heparin (sigma, article No. is h3149), 4 μm
Chir99021 (cell agentech, article No. be c2447), 3 μm of sb431542 (cellagentech, article No. is c7243),
0.1 μm of compound e (emd chemicals, article No. is 209986-17-4), 1 μm of dorsomorphin (sigma, article No.
For p5499), 100nm retinoic acid (sigma, article No. be r2625), the concentration of wherein each material is in mn culture medium 2
In final concentration.
(5) mn culture medium 3 formula: by 50% (volumn concentration) advanced dmem/f12 culture medium
(invitrogen, article No. is 12634028) and 50% (volumn concentration) neurobasal culture medium (invitroge, goods
Number it is 12348017) after mixing, add following material: (invitrogen, article No. is 1% (volumn concentration) n2
17502048), 2% (volumn concentration) b27 (invitrogen, article No. is 17504044), 2mm glutamax
(invitrogen, article No. be 35050-061), 0.2% (2g/l) heparin (sigma, article No. is h3149), 4 μm
Chir99021 (cellagentech, article No. be c2447), 3 μm of sb431542 (cellagentech, article No. is c7243),
0.1 μm of compound e (emd chemicals, article No. is 209986-17-4), 100nm retinoic acid (sigma,
Article No. is r2625), 500ng/ml sag (calbiochem, article No. be 364590-63-6), the concentration of wherein each material be
Final concentration in mn culture medium 3.
(6) mn culture medium 4 formula: by 50% (volumn concentration) advanced dmem/f12 culture medium
(invitrogen, article No. is 12634028) and 50% (volumn concentration) neurobasal culture medium (invitroge, goods
Number it is 12348017) after mixing, add following material: (invitrogen, article No. is 1% (volumn concentration) n2
17502048), 2% (volumn concentration) b27 (invitrogen, article No. is 17504044), 2mm glutamax
(invitrogen, article No. be 35050-061), 0.2% (2g/l) heparin (sigma, article No. is h3149), 0.1 μm
(sigma, article No. is for compound e (emd chemicals, article No. be 209986-17-4), 100nm retinoic acid
r2625)、500ng/ml sag(Article No. is 364590-63-6), the concentration of wherein each material is in mn training
Final concentration in foster base 4.
(7) mn culture medium 5 formula: by 50% (volumn concentration) advanced dmem/f12 culture medium
(invitrogen, article No. is 12634028) and 50% (volumn concentration) neurobasal culture medium (invitroge, goods
Number it is 12348017) after mixing, add following material: (invitrogen, article No. is 1% (volumn concentration) n2
17502048), 2% (volumn concentration) b27 (invitrogen, article No. is 17504044), 2mm glutamax
(invitrogen, article No. be 35050-061), 0.2% (2g/l) heparin (sigma, article No. is h3149), 0.1 μm
Compound e (emd chemicals, article No. is 209986-17-4), 10 μm of dapt (sigma, article No. is d5942), 10 μ
G/ml fibronectin (gibco, article No. be phe0023), the concentration of wherein each material is that the end in mn culture medium 5 is dense
Degree.
Cell culture processes in following embodiments are as follows:
(1) fibroblastic culture
With the culture of described fibroblast culture medium, change liquid every other day, pass a second generation within every 3-4 days.By 1:3 or 1:4 when passing on
Pass on.
(2) culture of ipsc
(2-1) raise coating systems: ipsc is seeded to complete in advance through mitomycin (U.S.'s sigma Products,
Article No.: m0503) inactivation treatment mouse embryo fibroblasts (abbreviation mef, U.S.'s invitrogen Products, article No.:
S1520-100, in culture plate), with mef co-cultivation, using described cdf12 culture medium culturing, pass a second generation within every 5-7 days.
(2-2) coating systems are no raised: ipsc is seeded to and uses extracellular matrix (growth factor reduced- in advance
Matrigel, U.S.'s bd biosciences product, article No.: 354277) in coated culture plate, (beautiful using mtesr culture medium
State's stemcell technologies product, article No.: #05850) culture, pass a second generation within every 5-7 days.
(3) culture of motor neuron
The motor neuron precursor of Induction of committed differentiation is seeded to be used in the coated culture plate of matrigel in advance,
Cultivated with mn culture medium 5.
Embodiment 1, the induced multi-potent stem cell (ipsc) in acquisition als patient source
First, the acquisition of the induced multi-potent stem cell in als patient source
1st, cell culture
Respectively two plants are carried the skin flbroblast in the patient als source of different genes mutation: fus g1566a becomes
Fibrocyte (nd29563) and sod1 a272c fibroblast (nd29149) are enlarged in fibroblast culture medium
Culture.Treat that proper density is arrived in cell culture, digestion, count, two kinds of fibroblasts respectively take 1.5 × 106Individual.
2nd, electricity turns liquid preparation
By 100 μ l opti-mem (life technology product), 1.5 μ g pcxle-hoct3/4-shp53-f, 1.5
μ g pcxle-hsk, 1.5 μ g pcxle-hul and 1.5 μ g pcxle-egfp mix, and obtain electricity and turn liquid, are placed in 1.5ml ep pipe
In.
3rd, with step 2 obtain electricity turn liquid respectively to step 1 obtain fus g1566a fibroblast (nd29563) and
Sod1 a272c fibroblast (nd29149) carry out respectively resuspended, resuspended after transfer in electric revolving cup, and put into electroporation
In (lonza 4d nucleofector), carry out electricity and turn, electricity turns procedure Selection en150, and (concrete steps are with reference to lonza 4d
Nucleofector description), respectively obtain the cell suspension (1.5 × 10 after electricity turns6Cell/100 μ l).
4th, the cell suspension after turning electricity respectively is added to the hole of six orifice plates containing fibroblast culture medium of preheating
In, rock uniformly, put back to culture in incubator.Change within second day liquid, observe electric transfer efficient.
5th, persistently cultivated to the 5th day with fibroblast culture medium, digestive inoculation is to completing in advance through mitomycin
In the culture plate of the mef of inactivation treatment, persistently cultivated using ipsc culture medium.Cultivate to the 3rd week, respectively obtain similar to embryo
The little clone (ipsc) of tire stem cell, the ipsc that fus g1566a fibroblast (nd29563) induction is obtained is named as
fusg1566aIpsc (the referred to as fus of mutationg1566a- ipsc), sod1 a272c fibroblast (nd29149) induction is obtained
Ipsc be named as sod1a272cIpsc (the referred to as sod1 of mutationa272c- ipsc), different fibroblast inductions obtain
, as shown in a in Fig. 1, gene mutation result is as shown in b in Fig. 1 for the form of ipsc.Respectively little clone is chosen to the warp completed in advance
Amplification culture in the culture plate of the mef crossing mitomycin inactivation treatment.
2nd, the identification of the induced multi-potent stem cell in als patient source
Shown to triploblastica Forming ability (teratoma experiment) by detecting stem cell labeling thing and vivo detection in vitro,
Described ipsc has the dryness similar with embryonic stem cell.Specific as follows:
1st, Immunofluorescence test stem cell labeling thing nanog, the expression of oct4, sox2
Respectively by two kinds of the step one gained ipsc (fus carrying gene mutationg1566a- ipsc and sod1a272c-ipsc)
It is inoculated into and completes in advance on the coverslip of the mef of mitomycin inactivation treatment, after it is adherent, first use 4% paraformaldehyde
After fixing 30 minutes, then with phosphate buffer (pbs) rinse 3 times, then with containing 0.4% (volumn concentration) tritonx-
Penetrating 30 minutes of 100 pbs, pbs washes 3 times.Then close 1 hour with the donkey serum of 10% (volumn concentration).One resists
According to proper proportion (anti-human Mus source oct4 antibody, sc-5279,1:100；Anti-human sheep source sox2 antibody, sc-17320,1:100；
Anti-human rabbit source nanog antibody, ab21624,1:250) dilute after 4 DEG C of overnight incubation.Second day, then washed after 3 times with pbs, plus
Enter proper proportion dilution two are anti-little with nucleus dyestuff hoechst33342 (invitrogen, article No.: h3569) incubation 1
When.After pbs washes 3 times, with mountant (vector company, article No. h-1000) mounting.Finally enter under laser confocal microscope
Row is observed, and takes pictures.
Testing result is as shown in c in Fig. 1: it can be seen that two kinds the obtained ipsc carrying gene mutation
(fusg1566a- ipsc and sod1a272c- ipsc) all express oct4, the label of these three stem cell of nanog, sox2, illustrate two
The ipsc that kind carries gene mutation all maintains good dryness.
2nd, teratoma experiment
Respectively by two kinds of the step one gained ipsc (fus carrying gene mutationg1566a- ipsc and sod1a272c-ipsc)
It is inoculated into and complete in advance in the culture plate of the mef of mitomycin inactivation treatment.Treat that ipsc covers with, digestion counts, and with containing
The mixed liquor of 20% (volumn concentration) matrigel and 80% (volumn concentration) cdf12 culture medium to ipsc (3 ×
106Individual) carry out resuspended, obtain cell suspension；And it is subcutaneous that the cell suspension of acquisition is squeezed into immunodeficient mouse.After about 8 weeks,
The teratoma having Semen Glyciness size grows up to.After de- neck puts to death mice, teratoma is taken out, fixes one week in 4% paraformaldehyde,
After making it fully fixing, bubble is dehydrated in 30% (30g/100ml) sucrose solution and sinks to the bottom to teratoma, shows fully to be dehydrated.
Then, embedded using compound o.c.t embedding medium.Freezing microtome is cut into slices, thickness is 12 μm, tissue
After section is dried, you can carry out the expression of three germinal layer labels of Immunofluorescence test.Immunofluorescence operating procedure is with above-mentioned step
Rapid 1.Wherein, an anti-inclusion: anti-human rabbit source tuj1 antibody (sigma company, article No. t2220,1:500)；Anti-human rabbit source foxa2 resists
Body (cell signaling technology company, article No. 8186,1:200)；Anti-human Mus source-sma antibody (sigma company,
Article No. a5228,1:200).
Testing result is as shown in d in Fig. 1: visible by two kinds of ipsc (fus carrying gene mutationg1566a- ipsc and
sod1a272c- ipsc) teratoma that formed all expresses tuj1, the label of these three germinal layers of foxa2, sma, illustrates that two kinds carry
The ipsc of gene mutation all maintains good dryness.
Embodiment 2, targeting correct the gene mutation that two kinds of als-ipsc carry
Two kinds of ipsc (fus embodiment 1 being obtained using crispr/cas9 gene editing technologyg1566a- ipsc and
sod1a272c- ipsc) gene mutation that carries carries out targeting removing, finally gives the ipsc eliminating gene mutation.Concrete steps are such as
Under:
1st, cell culture
Two kinds of ipsc that described embodiment 1 obtains are cultivated in the coated culture plate of matrigel (matrigel)
(fusg1566a- ipsc and sod1a272c- ipsc), culture medium is mtesr.Treat that Clonal density reaches 70%-80%, tryple disappears
Change, count, collect 5 × 106Individual cell.
2nd, electricity turns liquid preparation
The recombination template that correct 8 μ g cas9-gfp plasmids, 8 μ g corresponding gene and the grna- of 4 μ g corresponding gene rectification
Mcherry plasmid mixes, and is settled to 100 μ l with opti-mem (life technology), obtains electricity and turn liquid, be placed in 1.5ml
In ep pipe.
When the associated gene mutation needing to correct is fus g1566a, the grna-mcherry plasmid that corresponding gene is corrected
For fus-grna-mcherry；The recombination template that corresponding gene is corrected is double-strand dna shown in sequence 2 in sequence table.Described
The construction method of fus-grna-mcherry is as follows: first with phusion high-fidelity pcr master mix
With gc buffer (neb, article No. be m0532) by the fus grna oligo of synthesis (fus-grna-mcherry-f:5 '-
gaggccgtattaattagcc-3’；Fus-grna-mcherry-r:5 '-ggctaattaatacggcctcc-3 ') annealing extension
Become double-strand dna, then utilize gibson assemly master mix (neb, article No. be e2611) by described fus grna
Double-strand dna through homologous recombination connect entrance with restricted enzyme aflii enzyme action grna-mcherry carrier after purification
(addgene, article No. is #78544), thus be configured to described fus-grna-mcherry carrier.Described fus-grna-
The structure of mcherry is described as follows: by the dna fragment shown in sequence in sequence table 1, (the identification region sequence of grna, that is, be spaced
The corresponding coded sequence of sequence spacer) be inserted between the restriction enzyme site aflii of grna-mcherry plasmid after the restructuring that obtains
Plasmid.
When the associated gene mutation needing to correct is sod1 a272c, the grna-mcherry plasmid that corresponding gene is corrected
For sod1-grna-mcherry；The recombination template that corresponding gene is corrected is double-strand dna shown in sequence 4 in sequence table.Described
The construction method of sod1-grna-mcherry is as follows: first with phusion high-fidelity pcr master mix
With gc buffer (neb, article No. be m0532) by the sod1 grna oligo of synthesis (sod1-grna-mcherry-f:
5’-atgtgactgctgacaaaga-3’；Sod1-grna-mcherry r:5 '-ctttggcagcagtcacatc-3 ') annealing
Extend into double-strand dna, then will be described using gibson assemly master mix (neb, article No. is e2611)
Sod1grna double-strand dna through homologous recombination connect entrance with restricted enzyme aflii enzyme action grna-mcherry after purification
Carrier (addgene, article No. is #78544), thus be configured to described sod1-grna-mcherry carrier.Described sod1-
The structure of grna-mcherry is described as follows: by shown in sequence in sequence table 3 dna fragment (the identification region sequence of grna, that is,
The corresponding coded sequence of intervening sequence spacer) be inserted between the restriction enzyme site aflii of grna-mcherry plasmid after obtain
Recombiant plasmid.
Wherein, described cas9-gfp plasmid (article No.: #63592) and grna-mcherry empty carrier (article No.: #62340) are equal
For addgene Products.
3rd, electricity turns
Turn, with the electricity obtaining in step 2, the corresponding cell collected in the resuspended step 1 of liquid, be then transferred into electric revolving cup
In, and put in electroporation (lonza 4d nucleofector), carry out electricity and turn, electricity turns procedure Selection cm113 (concrete steps
With reference to lonza 4d nucleofector description), respectively obtain the cell suspension (5 × 10 after electricity turns6Cell/100 μ l).
4th, the cell suspension after turning electricity respectively is added in the coated culture plate of matrigel, with mtesr culture, puts back to
In incubator.After electricity turns 24 hours, observe electric transfer efficient.With fluorescence microscope green fluorescence and red fluorescence, double positives
Ratio is more than 1%, you can carry out next step experiment.
5th, after electricity turns 48 hours, cell is collected in tryple digestion, uses selected by flow cytometry apoptosis double positive cells.To sort
To cell be placed to and complete in advance in the hole of six orifice plates of the mef of mitomycin inactivation treatment, with the training of cdf12 culture medium
Support, put back in incubator.
6th, persistently use cdf12 culture medium culturing about 14 days, you can see by the unicellular little clone being formed, use caps method
Identification (amplifies, with pcr technology, the fragment comprising mutational site, due to the difference of a snp, can use restricted accordingly interior
Enzyme cutting is identified).
Fus-f:5 '-gagaaagtggtttcattttgagggctaggtgga-3 '；
Fus-r:5 '-ttgtttgagcctcaccattaaaagggccaaaag-3 '.
Fus identification restricted enzyme: bsaxi.
In the presence of associated gene mutation fus g1566a, through the pcr amplification of fus-f/fus-r, contain in products therefrom
The recognition sequence of restrictive restriction endonuclease bsaxi, thus after enzyme action is carried out to pcr product using bsaxi, size can be produced about
For 790bp, 3 bands of 540bp and 250bp.And when associated gene mutation fus g1566a does not exist, through fus-f/
The pcr amplification of fus-r, does not contain the recognition sequence of restricted enzyme bsaxi in products therefrom, thus when using bsaxi pair
After pcr product carries out enzyme action, 2 bands that size is about 540bp and 250bp bp can be produced.
Sod1-f:5 '-cccatctttcttcccagagcattagtgtgtagacg-3 '；
Sod1-r:5 '-acaaaatgttctgtttaacaagtgagaaacccaatcct-3 '.
Sod1 identification restricted enzyme: apeki.
In the presence of associated gene mutation sod1 a272c, through the pcr amplification of sod1-f/sod1-r, in products therefrom
Recognition sequence containing restricted enzyme apeki, thus after enzyme action is carried out to pcr product using apeki, size can be produced
It is about 600bp, 3 bands of 400bp and 200bp bp.And when associated gene mutation sod1a272c does not exist, pass through
The pcr amplification of sod1-f/sod1-r, does not contain the recognition sequence of restricted enzyme apeki in products therefrom, thus when employing
Apeki is carried out after enzyme action to pcr product, can produce 1 band that size is about 600bp.
Identified, finally successfully obtain mutant gene fusg1566aCorrect as fusg1566gClone, mutant gene
sod1a272cCorrect as sod1a272aClone.Successfully obtain two kinds of ipsc correcting through gene, be briefly referred to as
fusg1566g- ipsc and sod1a272a-ipsc.
Specific gene correction solution and qualification result are as shown in a, b and c in Fig. 2.
Embodiment 3, carry als Disease-causing gene mutation motor neuron and through gene correct motor neuron acquisition
And identification
First, the motor neuron of als Disease-causing gene mutation and the acquisition of the motor neuron corrected through gene are carried
By obtain in embodiment 1 two kinds of ipsc (fus carrying gene mutationg1566a- ipsc and sod1a272c- ipsc) and
Ipsc (the fus that two kinds obtaining in embodiment 2 are corrected through geneg1566g- ipsc and sod1a272a- ipsc) orient respectively simultaneously
It is induced to differentiate into motor neuron, respectively obtain fusg1566a- mn, sod1a272c- mn, fusg1566g- mn, sod1a272a-mn.
Ipsc is directed differentiation to the scheme schematic diagram of motor neuron cell as shown in a in Fig. 3.Concrete operations are as follows:
1st, the little clone of four kinds of ipsc obtaining embodiment 1 and embodiment 2 is inoculated into and completes through mitomycin in advance
In the culture plate of the mef of inactivation treatment, with cdf12 culture medium culturing about 1-2 days, reach 20% about to its density, obtain
Cell to be broken up.
2nd, cell to be broken up is continued to cultivate with mn culture medium 1, change liquid daily, persistently cultivate 3 days, obtain 1 culture
Cell afterwards.
3rd, the mn culture medium 2 of the cell after cultivating 1 time continues to cultivate, and changes liquid daily, persistently cultivates 3 days, obtains 2 trainings
Cell after supporting.
4th, the mn culture medium 3 of the cell after cultivating 2 times continues to cultivate, and changes liquid daily, persistently cultivates 1 day, obtains 3 trainings
Cell after supporting.
5th, the cell after cultivating 3 times uses mn inducing culture 4 with unicellular going in the coated culture plate of matrigel
Cultivated, persistently cultivated 1 day, obtained the cell after 4 cultures.In this step, also rock can be used before and after passing on
Inhibitor is processed to improve cell survival rate.
6th, the cell after cultivating 4 times is cultivated with mn culture medium 5, persistently cultivates 3 days it may be observed that significantly taking out
Silk, obtains the preferable motor neuron cell of form.
fusg1566a- mn, sod1a272c- mn, fusg1566g- mn, sod1a272aIn-mn motor neuron cell form such as Fig. 3
Shown in b.
2nd, the motor neuron of als Disease-causing gene mutation and the identification of the motor neuron corrected through gene are carried
The fus being obtained using Immunofluorescence test technology for detection step oneg1566a- mn, sod1a272c- mn, fusg1566g- mn,
sod1a272aThe expression of motor neuron label hb9 and isl1 of-mn, and the table of neuronal cell label map2
Reach situation.Specific experiment step is with reference to step 21 in embodiment 1.
One is anti-and dilution ratio is as follows: anti-human hb9 antibody (dshb company, article No. 81.5c10,1:50), and anti-human isl1 resists
Body (abcam, ab20670,1:200), anti-human map2 antibody (sigma company, article No. t2220,1:500).
Testing result is as shown in c in Fig. 3: the ipsc of the visible ipsc carrying gene mutation and removing gene mutation can
Directed differentiation is mn cell, and the mn cell obtaining all expresses hb9, isl1, map2 label.
Poor between embodiment 4, the motor neuron carrying the mutation of als Disease-causing gene and the motor neuron through gene rectification
The transcriptome analysis of heterologous gene expression
Obtain in collection embodiment 3 respectively carries the motor neuron of als Disease-causing gene mutation and through gene rectification
Motor neuron, i.e. fusg1566a-mn/fusg1566g- mn and sod1a272c-mn/sod1a272a-mn.Transcribed after extracting rna
Group sequencing analysis (transcriptome analysis are completed by Beijing Nuo Hezhi source Science and Technology Co., Ltd.).In analysis due to gene mutation
fusg1566aThe differential gene leading to and gene mutation sod1a272cOn the basis of the differential gene leading to, compare the two further altogether
Same difference.
Experimental result is as shown in a, b, c in Fig. 4: fusg1566a-mn/fusg1566g- mn group, down-regulated gene 13, raises base
Because of 33；sod1a272c-mn/sod1a272a- mn group, down-regulated gene 575, up-regulated gene 364.Then further by two groups
Relatively find two groups of common down-regulated genes 6, common up-regulated gene 1.
Claims (10)
1. a kind of preparation method of people's amyotrophy lateral schlerosis patient-specific motor neuron, for following method a or side
Method b:
Method a: do not carry the system of people's amyotrophy lateral schlerosis patient-specific motor neuron of als associated gene mutation
Preparation Method, comprises the steps:
(1) fibroblast to the in vitro people amyotrophy lateral schlerosis patient source carrying als associated gene mutation
Reprogrammed, obtain carrying the induced multi-potent stem cell of described als associated gene mutation, be designated as als-ipsc；
(2) described als-ipsc is carried out with gene editing, specificity eliminates described als associated gene mutation, obtain gene and correct
Induced multi-potent stem cell, be designated as cals-ipsc；
(3) described cals-ipsc is oriented with induction differentiation, obtains final product the people's muscle not carrying described als associated gene mutation
Amyotrophic lateral sclerosis disease patient-specific motor neuron；
Method b: carry the preparation of people's amyotrophy lateral schlerosis patient-specific motor neuron of als associated gene mutation
Method, comprises the steps:
(1) fibroblast to the in vitro people amyotrophy lateral schlerosis patient source carrying als associated gene mutation
Reprogrammed, obtain carrying the induced multi-potent stem cell of described als associated gene mutation, be designated as als-ipsc；
(2) described als-ipsc is oriented with induction differentiation, obtains final product the people's amyotrophy carrying described als associated gene mutation
Lateral schlerosis patient-specific motor neuron.
2. method according to claim 1 it is characterised in that: in step (1), described reprogramming be by oct3/4 gene,
Sox2 gene, klf4 gene, lmyc gene and lin28 gene be common to import the described in vitro als associated gene mutation that carries
In the fibroblast in people amyotrophy lateral schlerosis patient source, and cultivate to obtaining described als associated gene mutation
Induced multi-potent stem cell.
3. method according to claim 1 and 2 it is characterised in that: described als associated gene mutation be fusg1566a or
sod1a272c；
In the step (2) of methods described a, described " specificity eliminates described als associated gene mutation " is by described fus
G1566a corrects as fus g1566g, or described sod1a272c is corrected as sod1a272a.
4. method according to claim 3 it is characterised in that: in methods described a, described step (2) is: by grna table
Reach plasmid, cas9 expression plasmid and recombination template and jointly proceed to described als-ipsc, and cultivate to obtaining described cals-ipsc；
When described als associated gene mutation is fus g1566a, the identification region sequence of the grna in described grna expression plasmid
It is classified as sequence 1 in sequence table；Described recombination template is double-strand dna shown in sequence 2 in sequence table；
When described als associated gene mutation is sod1 a272c, the identification region sequence of the grna in described grna expression plasmid
It is classified as sequence 3 in sequence table；Described recombination template is double-strand dna shown in sequence 4 in sequence table.
5. according to described method arbitrary in claim 1-4 it is characterised in that: in step (3) or the described side of methods described a
In the step (2) of method b, described cals-ipsc or described als-ipsc is oriented with induction differentiation is to walk according to inclusion is following
Rapid method is carried out:
(3-1) by described cals-ipsc or described als-ipsc be inoculated into containing in advance with mitomycin inactivation treatment cross little
Use cdf12 culture medium culturing in the raising coating systems of rat embryo fibroblast cell, obtain treating noble cellss；
(3-2) treat that noble cellss carry out first time differentiation culture to described in mn culture medium 1, thin after obtaining cultivating for the first time
Born of the same parents；
(3-3) in mn culture medium 2, second differentiation culture is carried out to cell after the culture of described first time, obtain second culture
Cell afterwards；
(3-4) after described second being cultivated in mn culture medium 3, cell carries out third time differentiation culture, obtains third time and cultivates
Cell afterwards；
(3-5) in mn culture medium 4, the 4th differentiation culture is carried out to cell after the culture of described third time, obtain the 4th culture
Cell afterwards；
(3-6) in mn culture medium 5, the 5th differentiation culture is carried out to cell after described 4th time culture, obtain the 5th culture
Cell afterwards, does not as carry people's amyotrophy lateral schlerosis patient-specific nervus motoriuies of described als associated gene mutation
Unit or the people's amyotrophy lateral schlerosis patient-specific motor neuron carrying described als associated gene mutation.
6. method according to claim 5 it is characterised in that:
The composition of described cdf12 culture medium is as follows: by dmem/f12 culture medium and knockout serum substitute according to 8:2 body
After the mixing of long-pending ratio, add non essential amino acid, glutamax, penicillin/streptomycin, beta -mercaptoethanol and people in mixed liquor
Fgf2 is so that the final concentration of 0.1mm of described non essential amino acid, the final concentration of 1mm of described glutamax, described penicillium sp
The final concentration of 10g/l of element/streptomycin, final concentration of 55 μm of described beta -mercaptoethanol, described people fgf2's is final concentration of
10ng/ml；And/or
The composition of described mn culture medium 1 is as follows: by advanced dmem/f12 culture medium and neurobasal culture medium according to 1:
1 volume ratio mixing after, in mixed liquor add n2, b27, glutamax, heparin, chir99021, sb431542,
Compound e and dorsomorphin；Make final concentration of 1% volumn concentration of described n2, the final concentration of described b27
For 2% volumn concentration, the final concentration of 2mm of described glutamax, the final concentration of 2g/l of described heparin, described
Final concentration of 4 μm of chir99021, final concentration of 3 μm of described sb431542, final concentration of 0.1 μ of described compound e
Final concentration of 1 μm of m, described dorsomorphin；And/or
The composition of described mn culture medium 2 is as follows: by advanced dmem/f12 culture medium and neurobasal culture medium according to 1:
1 volume ratio mixing after, in mixed liquor add n2, b27, glutamax, heparin, chir99021, sb431542,
Compound e, dorsomorphin and ra；Make final concentration of 1% volumn concentration of described n2, the end of described b27 is dense
Spend for 2% volumn concentration, the final concentration of 2mm of described glutamax, the final concentration of 2g/l of described heparin, described
Final concentration of 4 μm of chir99021, final concentration of 3 μm of described sb431542, final concentration of 0.1 μ of described compound e
Final concentration of 1 μm of m, described dorsomorphin, the final concentration of 100nm of described ra；And/or
The composition of described mn culture medium 3 is as follows: by advanced dmem/f12 culture medium and neurobasal culture medium according to 1:
1 volume ratio mixing after, in mixed liquor add n2, b27, glutamax, heparin, chir99021, sb431542,
Compound e, ra and sag；Make final concentration of 1% volumn concentration of described n2, final concentration of 2% body of described b27
Long-pending percentage composition, the final concentration of 2mm of described glutamax, the final concentration of 2g/l of described heparin, the end of described chir99021
Concentration is 4 μm, final concentration of 3 μm of described sb431542, final concentration of 0.1 μm of described compound e, the end of described ra
Concentration is 100nm, the final concentration of 500ng/ml of described sag；And/or
The composition of described mn culture medium 4 is as follows: by advanced dmem/f12 culture medium and neurobasal culture medium according to 1:
After 1 volume ratio mixing, add n2, b27, glutamax, heparin, compound e, ra and sag in mixed liquor；Make institute
State final concentration of 1% volumn concentration of n2, final concentration of 2% volumn concentration of described b27, described glutamax's
Final concentration of 0.1 μm of final concentration of 2mm, the final concentration of 2g/l of described heparin, described compound e, the end of described ra is dense
Spend for 100nm, the final concentration of 500ng/ml of described sag；And/or
The composition of described mn culture medium 5 is as follows: by advanced dmem/f12 culture medium and neurobasal culture medium according to 1:
After 1 volume ratio mixing, n2, b27, glutamax, heparin, compound e, dapt and fiber is added to connect in mixed liquor
Albumen；Make final concentration of 1% volumn concentration of described n2, final concentration of 2% volumn concentration of described b27, institute
State the final concentration of 2mm of glutamax, final concentration of 0.1 μm of the final concentration of 2g/l of described heparin, described compound e,
Final concentration of 10 μm of described dapt, the final concentration of 10 μ g/ml of described fibronectin.
7. the method according to claim 5 or 6 it is characterised in that:
In step (3-1), the time of described culture is 1-2 days；And/or
In step (3-2), the time of described culture is 3 days；And/or
In step (3-3), the time of described culture is 3 days；And/or
In step (3-4), the time of described culture is 1 day；And/or
In step (3-5), the time of described culture is 1 day；And/or
In step (3-6), the time of described culture is 3 days.
8. a kind of test kit for preparing people's amyotrophy lateral schlerosis patient-specific motor neuron, wants containing having the right
Ask mn culture medium described in 61, described mn culture medium 2, described mn culture medium 3, described mn culture medium 4, described mn culture medium 5.
9. transported using people's amyotrophy lateral schlerosis patient-specific that methods described arbitrary in claim 1-7 prepares
Dynamic neuron.
10. arbitrary described method or the test kit described in claim 8 or the people described in claim 9 in claim 1-7
Application in arbitrary as follows for the amyotrophy lateral schlerosis patient-specific motor neuron:
A () prepares for screening and/or identifying the clinical medicine that can treat and/or prevent people's amyotrophy lateral schlerosis
And/or the product of natural organic matter and/or micromolecular compound；
B () prepares the product for treating and/or preventing people's amyotrophy lateral schlerosis；
C () prepares the cell model of people's amyotrophy lateral schlerosis.
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| WO2018170794A1 (en) * | 2017-03-22 | 2018-09-27 | Tsinghua University | Mutant fus model for als |
| CN110241138A (en) * | 2019-03-27 | 2019-09-17 | 温州医科大学 | A kind of preparation method of human retinoblastoma model |
| CN110511992A (en) * | 2019-08-27 | 2019-11-29 | 深圳市宝安区妇幼保健院 | A kind of TARDBP mutated gene, detection primer and kit |
| CN113528437A (en) * | 2021-07-07 | 2021-10-22 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
| CN113969264A (en) * | 2021-11-29 | 2022-01-25 | 中南大学 | COG5 gene knockout human embryonic stem cell line, construction method and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018170794A1 (en) * | 2017-03-22 | 2018-09-27 | Tsinghua University | Mutant fus model for als |
| CN110770336A (en) * | 2017-03-22 | 2020-02-07 | 清华大学 | Mutant FUS model for ALS |
| CN110770336B (en) * | 2017-03-22 | 2023-09-12 | 神济昌华(北京)生物科技有限公司 | Mutant FUS model for ALS |
| CN108103027A (en) * | 2018-02-02 | 2018-06-01 | 中国医学科学院血液病医院（血液学研究所） | The method that the reprogramming of high efficiency haemocyte realizes gene editing simultaneously |
| CN110241138A (en) * | 2019-03-27 | 2019-09-17 | 温州医科大学 | A kind of preparation method of human retinoblastoma model |
| CN110511992A (en) * | 2019-08-27 | 2019-11-29 | 深圳市宝安区妇幼保健院 | A kind of TARDBP mutated gene, detection primer and kit |
| CN110511992B (en) * | 2019-08-27 | 2023-06-06 | 深圳市宝安区妇幼保健院 | TARDBP mutant gene, detection primer and kit |
| CN113528437A (en) * | 2021-07-07 | 2021-10-22 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
| CN113528437B (en) * | 2021-07-07 | 2023-08-25 | 中国医学科学院血液病医院(中国医学科学院血液学研究所) | Kit for enhancing gene editing efficiency and application thereof |
| CN113969264A (en) * | 2021-11-29 | 2022-01-25 | 中南大学 | COG5 gene knockout human embryonic stem cell line, construction method and application |
| CN113969264B (en) * | 2021-11-29 | 2023-12-08 | 中南大学 | COG5 gene knockout human embryonic stem cell line, construction method and application |
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