CN106701824B - The method that dynamoneure and its functional cell are obtained based on people iPS cells - Google Patents
The method that dynamoneure and its functional cell are obtained based on people iPS cells Download PDFInfo
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Abstract
The invention belongs to biomedical sector, it is related to a kind of safe and reliable reprogramming and obtains the method for people's iPS cells, a kind of method by the differentiation of iPS cell simplicity rapidly and efficiently inducing as dynamoneure, and a kind of acquisition methods of cost-effective feature dynamoneure and its application.The present invention is using vitronectin as unique somatomedin, and the sendai virus that the factor is reprogrammed by carrying infects human fibroblasts, and culture obtains people's iPS cells without external source serum interference after 3 weeks；Combined afterwards by a variety of micromolecular compounds and induce differentiation into people's iPS cell high-efficients for dynamoneure；Further promote maturation culture through astroglia conditioned medium and applied in the formation of neuromuscular junction.The feature dynamoneure of the invention that efficiently induction obtains, available for the simulation of disease phenotype and the discussion of pathogenesis, the screening for effective medicine from now on provides theoretical foundation；The cellular transplantation therapy of disease is even can be applied in the future.
Description
Technical field
The invention belongs to biomedical sector, it is related to the acquisition methods of dynamoneure, in particular, in that including one kind
The method that safe and reliable reprogramming obtains people's iPS cells, it is a kind of that the differentiation of people's iPS cell simplicity rapidly and efficiently inducing is turned into spinal cord
The method of motor neuron, in addition to a kind of acquisition methods of cost-effective feature dynamoneure.
Background technology
Dynamoneure (motorneuron, MN) is sent out in the contractile motion that central nervous system dominates skeletal muscle
Bridge Link role is waved, the important vital functions activity such as controls breathing, swallow.Also because of that, dynamoneure into
For the motor neuron disease (motorneurondisease, MND) of uncurable disease, such as ALS
(amyotrophiclateralsclerosis, ALS), spinal muscular atrophy (spinalmuscularatrophy, SMA) etc.
Primary infringement target.It is to limit such disease mechanisms research and explore treatment to answer to lack safe and reliable cell model at present
Main bottleneck.Therefore, how simple and effective obtains the spinal motor without the indefinite material interference of the compositions such as exogenous serum
Neuron is the key point for exploring Disease Clinical treatment from now on.Studied at present can by the skin fibroblasts of people,
Embryonic stem cell and people's inductive pluripotent stem cells (human Induced Pluripotent Stem Cells, abbreviation people iPS
Cell) differentiation acquisition dynamoneure, reference can be made to：1.Wang ZB,Zhang X,Li XJ.Recapitulation of
spinal motor neuron-specific disease phenotypes in a human cell model of
spinal muscular atrophy[J].Cell Res,2013,23(3):378-393；2.Son EY,chida JK,
Wainger BJ,et al.Conversion of mouse and human fibroblasts into functional
spinal motor neurons[J].Cell Stem Cell,2011,9(3):205-218；3.Ebert AD,Yu J,Rose
FF Jr,et al.Induced pluripotent stem cells from a spinal muscular atrophy
patient[J].Nature,2009,457(7227):277-280。
Compared with human embryo stem cell, people's iPS cells have abundance, acquisition is convenient, is not related to using ethics problem
Etc. advantage, this just provides a kind of new way to establish the specific cell model of patient's direct sources.Especially for rare disease
For, equivalent to the immortalized cells storehouse for establishing all cell types of patient.Vitro culture of human iPS cells depend on more at present
Coating systems are nourished, it has the problems such as external source serum interference；And in terms of dynamoneure breaks up research, although existing phase
To the abductive approach of maturation, but generally existing step numerous and diverse (four step rule), time-consuming (be up to 7 weeks), differentiation efficiency is low (only has
25%) the defects of.In addition, the functional nerve member for maintaining culture induction to obtain, often needs to rely on using expensive neurotrophy
The factor, also considerably increase experimental cost.Therefore, in order to solve existing deficiency, it is quite necessary to explore establish it is a kind of it is whole not
Rely on and nourish coating systems, people's iPS cell reprogramming methods without the indefinite material interference of the compositions such as external source serum；Especially explore
Establishing a kind of rapidly and efficiently inducing people iPS cell differentiations easy in vitro turns into the differentiation method of dynamoneure, and builds
Found a kind of acquisition methods of cost-effective feature dynamoneure.
In the dynamoneure model of motor neuron disease is established, induction obtains the quantity of aim cell and corresponding
The required time is two big key points, therefore the foundation of effectively method of inducing differentiation is the core of research.Now there are some researches show ridge
Marrow motor neuron Development And Differentiation process relates generally to three phases：Neuralization, tail veutro and maturing.Wherein, neuralization
Mainly by suppressing TGF-β/Actin/Nodal paths, BMP paths and Wnt paths, so as to play in suppression, entoderm divides
The effect of change, finally realize that embryoid body breaks up toward neuroderm；Tail veutroization is then mainly led to by activating RA paths and SHH
Road, so as to realize that (akrencephalon end to end occurs for central nervous systemSpinal cord) with carrying on the back abdomen (ventricornuRelief angle) the two poles of the earth differentiation；
Maturing is then by suppressing Notch paths, finally realizing dynamoneure precursor to the spinal motor after mitosis
Neuron converts：Reference can be made to Dasen JS, Jessell TM.Hox networks and the origins of motor
neuron diversity[J].Curr Top Dev Biol,2009,88:169-200.Correlative study proves, small molecule chemical combination
Thing plays an important role during people's iPS cell Differentiating Into Neurons.But the use of micromolecular compound there is also
Bottleneck effect, its toxic side effects is essentially consisted in, and be proportionate with its work action concentration.Phase in existing scheme be present
Close high workload concentration compound such as DMH1, especially Purmorphamine application.Wherein, the latter is that generally acknowledged toxicity is made at present
With strong, and a kind of effectively narrow compound of working concentration window：Reference can be made to Hu BY, Zhang SC.Differentiation of
spinal motor neuronsfrompluripotenthuman stem cells[J].Nat Protoc,2009,4(9):
1295-1304.Therefore, induced differentiation factor is the key point of selection and the research of micromolecular compound.
The content of the invention
The purpose of the present invention (1) is to provide a kind of method that safe and reliable reprogramming obtains people's iPS cells, key step
For：Minimally invasive acquisition human skin fibroblasts, through the celestial being for carrying the reprogramming factor (hOct3/4, hSox2, hKlf4 and hc-Myc)
After platform virus infection, cultivated 7 days in DMEM complete mediums；It is resuspended again with complete medium, and with 1:5 ratio renewed vaccinations
Cultivated 24 hours in the orifice plate treated to vitronectin coating；E8 stem cell medias are replaced by afterwards, and it is every from next day
Day carries out full dose and changes liquid, until can obtain iPS cells after 2 weeks.Early stage picking iPS cell monoclonal, is seeded to advance coating
Have and amplification cultivation is carried out in the orifice plate of vitronectin or matrigel.
Wherein, as above porous plate described in method with before needing through vitronectin (1：100 dilutions, the μ g/ml of valid density 10)
It is coated with overnight in 37 DEG C；Or need to be through matrigel (1：50 dilutions) it is coated with 30 minutes in 37 DEG C.
The purpose of the present invention (2) is to provide one kind turns into spinal motor nerve by people's iPS cell simplicity rapidly and efficiently inducing
The method of member, key step are as follows：
(1) it will cultivate to the people iPS cell dissociations of 70%-85% degrees of fusion and suspension and be incubated at containing micromolecular compound
Combine in I Neuronal induction differential medium 2 days；
(2) cell is moved to combine containing micromolecular compound and continues the training that suspends in II Neuronal induction differential medium
Support 2 days；
(3) cell is moved into sustained suspension in the Neuronal induction differential medium that III is combined containing micromolecular compound
Culture 5 days；
(4) by cell dissociation adhere-wall culture 3 days, micromolecular compound is now only added in Neuronal induction differential medium
DAPT。
Wherein, Neuronal induction differential medium is blending constituent described in step (1), contains 1 respectively：1 DMEM/
F12 and Neurobasal culture mediums, 0.5xB27 additives, 0.5xN2 additives, 1x glutamine and 1x nonessential amino acid.
Wherein, the micromolecular compound combination I (concentration) of step (1) described addition is：DMH1(2μM)+SB431542(2μ
M)+CHIR99021 (3 μM) or LDN193189 (0.3 μM)+SB431542 (2 μM)+CHIR99021 (3 μM).
Wherein, the micromolecular compound combination II additionally added in step (1) the Neuronal induction differential medium is (dense
Degree) it is as follows：Micromolecular compound combination I+RA (0.1 μM)+SAG (0.5 μM) or micromolecular compound combination I+RA (0.1 μM)+
Purmorphamine(1μM)。
Wherein, the micromolecular compound combination III additionally added in the Neuronal induction differential medium described in step (3)
(concentration) is as follows：RA (0.1 μM)+SAG (0.5 μM) or RA (0.1 μM)+Purmorphamine (1 μM).
Wherein, process is specially described in step (4)：It is seeded to first by Accutase enzymic digestions, then by neuron
It is coated with the slide that poly-ornithine, laminin and matrigel coating treat；After cell attachment is complete, addition contains
Micromolecular compound DAPT Neuronal induction differential medium.
The purpose of the present invention (3) is to provide a kind of acquisition methods of cost-effective feature dynamoneure, main
The step is wanted to be：It will induce and break up the neuron of the 9th day and digested, inoculate to being coated with poly-ornithine, layer adhesion in advance
On albumen and matrigel slide, the culture medium based on star spongiocyte conditioned medium, and small molecule is added simultaneously
Compound DAPT (10 μM)；Every 3 days, half amount changed fresh culture once, untill induction differentiation the 31st day.
Wherein, the preparation process of the astroglia conditioned medium is：Using DMEM complete medium culture stars
Shape spongiocyte 3 days, fresh Neuronal induction differential medium is added, is collected daily afterwards, and changed fresh
Neuronal induction differential medium.
The present invention establishes a kind of whole independent of nourishing coating systems, the people iPS cell culture sides without external source serum interference
Method, and under the conditions of certain effect can easy, quick, efficiently and directionally differentiation obtain dynamoneure, further, it would be desirable to provide
The acquisition methods of cost-effective feature dynamoneure, these will carry for the motor neuron disease research of uncurable disease
A kind of cell model of great application prospect is supplied.
Brief description of the drawings
In conjunction with the accompanying drawings, so that the feature situation of exemplary embodiments of the present invention and its advantage, accompanying drawing is described in detail：
Fig. 1 is to reprogram process independent of nourishing coating systems, the people iPS cells without external source serum interference；Wherein A is celestial platform
The human skin fibroblasts of 2 days before virus infection, in elongated strip；B is the application on human skin of 1 week after sendai virus infection into fiber
Cell, projection disappear, changed in polygonal；C is the human skin fibroblasts of renewed vaccination after digestion；D is after virus infection 3
The primary people iPS cells that week occurs, clone spherical in shape is (shown in white arrow), cell tight arrangement, and refractivity is strong.Scale, A-D
It is 200 microns.
Fig. 2 is people's iPS cell cultivation process using vitronectin as somatomedin；Wherein A-C clones continuously grow
Dynamic changing process, it is rounded all the time to extend out formula growth；It is identical with typical embryo's stem cell growth mode in D.Scale, A-D
It is 500 microns.
Fig. 3 is people's iPS cell cultivation process using matrigel as somatomedin；Wherein A-C continuously grows dynamic for clone
State change procedure, colony morphology is irregular, growth pattern is not fixed；It is obvious not with typical embryo's stem cell growth mode in D
Together.Scale, A-D are 500 microns.
Fig. 4 is people's iPS Cells Embryonic stem cells mark immunofluorescence dyeings without external source serum interference；Wherein A is
NANOG and DAPI are marked altogether；B is that SSEA-4 and DAPI is marked altogether；C is that TRA-1-60 and DAPI is marked altogether.Scale, A-C are 50 microns.
Fig. 5 is that inducing in vitro human iPS cell differentiations turn into dynamoneure and its schematic flow sheet of application
Fig. 6 is DP group two-step method induced motion neuron differentiation flows and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
For the embryoid body of the 2nd day of suspending, in typical sphere, sharpness of border；B is the neural ball to suspend the 9th day, and volume significantly increases, thoroughly
Brightness, refractivity increase；C is the dynamoneure for digesting adherent latter 3rd day (induction the 12nd day), has obvious neuron
Projection form, intertexture reticulate；D is that motor neuron specific markers HB9 and DAPI marks dyeing altogether；E is hindbrain and spinal cord god
Through first mark ISLET1 and DAPI immunostainings；F is that HB9, ISLET1 and DAPI mark dyeing altogether.Scale, A are 500 microns；B
It it is 200 microns with C；D-F is 100 microns.
Fig. 7 is LP group two-step method induced motion neuron differentiation flows and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
For the embryoid body of the 2nd day of suspending, in typical sphere, sharpness of border；B is the neural ball to suspend the 9th day, and volume significantly increases, thoroughly
Brightness, refractivity increase；C is the dynamoneure for digesting adherent latter 3rd day (induction the 12nd day), has obvious neuron
Projection form, intertexture reticulate；D is that motor neuron specific markers HB9 and DAPI marks dyeing altogether；E is hindbrain and spinal cord god
Through first mark ISLET1 and DAPI immunostainings；F is that HB9, ISLET1 and DAPI mark dyeing altogether.Scale, A are 500 microns；B
It it is 200 microns with C；D-F is 100 microns.
Fig. 8 is DS group two-step method induced motion neuron differentiation flows and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
For the embryoid body of the 2nd day of suspending, in typical sphere, sharpness of border；B is the neural ball to suspend the 9th day, and volume significantly increases, thoroughly
Brightness, refractivity increase；C is the dynamoneure for digesting adherent latter 3rd day (induction the 12nd day), has obvious neuron
Projection form, intertexture reticulate；D is that motor neuron specific markers HB9 and DAPI marks dyeing altogether；E is hindbrain and spinal cord god
Through first mark ISLET1 and DAPI immunostainings；F is that HB9, ISLET1 and DAPI mark dyeing altogether.Scale, A are 500 microns；B
It it is 200 microns with C；D-F is 100 microns.
Fig. 9 is LS group two-step method induced motion neuron differentiation flows and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
For the embryoid body of the 2nd day of suspending, in typical sphere, sharpness of border；B is the neural ball to suspend the 9th day, and volume significantly increases, thoroughly
Brightness, refractivity increase；C is the dynamoneure for digesting adherent latter 3rd day (induction the 12nd day), has obvious neuron
Projection form, intertexture reticulate；D is that motor neuron specific markers HB9 and DAPI marks dyeing altogether；E is hindbrain and spinal cord god
Through first mark ISLET1 and DAPI immunostainings；F is that HB9, ISLET1 and DAPI mark dyeing altogether.Scale, A are 500 microns；B
It it is 200 microns with C；D-F is 100 microns.
Figure 10 is traditional four-step method induced motion neuron differentiation process and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
For the embryoid body of the 4th day of suspending, spherical in shape, sharpness of border, but translucency is weaker, and dead cell is more；B is embryoid body the adherent rear 8th
My god (induction differentiation the 15th day), there is typical garland sample nerve tubular construction (shown in arrow)；C trains for piping and druming from continuing to suspend after wall
The neural spherical structure of foster 8th day (induction differentiation the 23rd day), spherical in shape, sharpness of border, refractivity are strong；D is adherent 3rd day of digestion
The motor neuron of (induction differentiation the 28th day), projection, which interweaves, to be reticulated；E is that HB9 and DAPI marks dyeing altogether；F be ISLET1 with
DAPI marks dyeing altogether.Scale, A are 500 microns；B-D is 200 microns；F is 100 microns.
Figure 11 is the mark GFAP immunofluorescence dyeings of astroglia；Wherein A is astroglia, in piece
Shape, close adherent growth；The common mark that B-C is astroglia Specific marker GFAP and DAPI dyes.Scale, A-C are equal
For 100 microns.
Figure 12 is the immunofluorescence dyeing of ripe motor neuron；Wherein A-D, respectively motor neuron specificity marker
The TUJ1 of thing CHAT, SMI32 and neuronal marker common mark dyeing.Scale, A-D are 50 microns.
Figure 13 is the immunofluorescence dyeing of functional maturation neuron；Wherein A-C is respectively functional maturation neuron
Specific marker Synaptophysin and MAP2 common mark dyeing.Scale, A-C are 50 microns.
Figure 14 is the functional maturation motor neuron electrophysiological characteristics under patch-clamp whole cell recording pattern；Wherein A is
The ripe motor neuron form that patch-clamp direct-view is recorded；B is the induced action potential recorded under current-clamp mode；C is electricity
The sodium potassium channel current recorded under pressing tongs pattern.Scale, A are 80 microns.
Figure 15 is under ripe motor neurons innervate, and the rhythm and pace of moving things of myotubes shrinks；Ripe motor neuron and myotube are thin
After born of the same parents co-culture 2 weeks, the lasting of myotubes, rule are shunk：Shrink every time continue 1 second (be within 1 second and 5 seconds the systole phase, arrow institute
It is shown as the myotubes of contractile motion), interval is shunk every time 4 seconds.Scale, 50 microns.
Figure 16 is the immunostaining that ripe motor neuron and myotubes form neuromuscular junction；Neuromuscular junction
Multidimensional angled arrangement (shown in " ten " word intersection point).The motion of wherein BTX (red-label) specific recognition neuromuscular junction is whole
Harden structure；MHC (Green Marker) and DAPI (blue markings) common designation have the myotubes (striated muscle) of coenocytism；
Synapsin (grey mark) specific recognition motor neuron end synaptic structure.Scale, 10 microns.
Figure 17 is that the rhythm and pace of moving things under patch-clamp whole cell recording pattern shrinks myotubes electrophysiological characteristics；It shrinks for record
The independence of myotubes provides action potential.
Embodiment
One aspect of the present invention provides a kind of whole process and done independent of nourishing coating systems, without the indefinite material of the compositions such as external source serum
The people's iPS cell culture processes disturbed, comprise the following steps：
1) independent of the people iPS cells reprogramming for nourishing coating systems：By the human skin fibroblasts of original cuiture with
0.15-0.3x 106Cells/well (preferably 0.2x 106Cells/well) density is seeded in 12 orifice plates, cultivates 1-3 days (preferably 2
My god).With infection multiplicity (multiplicity of infection, MOI) for 3-5 (preferably 5), using carry reprogramming because
The sendai virus (hOct3/4, hSox2, hKlf4 and hc-Myc) of son is infected it.After 5-8 days (preferably 7 days), it will infect
Fibroblast afterwards is digested.DMEM/F12 is washed 3 times, the 0.25% pancreas enzyme -EDTA 0.5ml/ holes added after preheating,
It is preferred that digesting 1 minute at room temperature, 1000-1500rpm, 1-3 minutes (preferably 1200rpm, 1.5 minutes) are then centrifuged for.Finally, it is complete
Full culture medium is resuspended with 1:3-1:10 (preferably 1:5) ratio renewed vaccination is to vitronectin (DMEM/F12 1:100 dilutions) overnight
In 6 treated orifice plates of coating, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivate.It is replaced by after 24 hours fresh
Stem cell media E8, carry out full dose daily afterwards and change liquid.After 2 weeks, it is thin that the iPS reprogrammed in non-trophoblast system can be obtained
Born of the same parents.
2) the passage amplification cultivation of people's iPS cells without the indefinite material interference of the compositions such as external source serum：By previous step weight
The iPS cells obtained are programmed by the isolated monoclonal of mechanical picking, contrast is seeded to respectively is coated with vitronectin in advance
(DMEM/F12 1:100 dilutions, 37 DEG C, overnight) or matrigel (DMEM/F121:50 dilution, 37 DEG C, 30 minutes) 6 orifice plates
It is interior, in 37 DEG C, 5%CO2, saturated humidity incubator in amplification cultivation.
The identification of people's iPS cells：
When cell density reaches 70-85%, had digestive transfer culture is carried out to people iPS cells and is seeded at vitronectin coating
On the cover glass managed.2nd day, cell, lower 10 minutes of room temperature environment are fixed with fresh 4% paraformaldehyde.0.01MPBS washings 3
It is secondary, 5 minutes every time.20% donkey serum+0.2%Triton-X-100+0.01M PBS are closed, room temperature 1 hour.With confining liquid 1：
1000 dilution primary antibodies (NANOG, SSEA4, TRA-1-60), 4 DEG C overnight.PBS is washed 3 times, every time 5 minutes.0.01M PBS are with 1:
1000 dilution secondary antibodies and DAPI, are incubated 1 hour at room temperature.PBS is washed 3 times, every time 5 minutes.Finally carry out anti-fluorescence quenching
Mounting.
Cellular morphology observation and immunofluorescent staining result under inverted microscope were shown, by the weight of 3 weeks or so
Programming can obtain whole independent of nourishing coating systems, the people iPS cells (Fig. 1) without external source serum interference.And in contrast without taste
During the people's iPS cell amplification cultivations for supporting coating systems, we, which optimize, has obtained a kind of training using vitronectin as somatomedin
Support scheme (Fig. 2), it is important that it has the same characteristic features (Fig. 4) of embryonic stem cell.
Another aspect of the present invention provides a kind of letter from the people iPS cells without external source serum interference to dynamoneure
Just rapidly and efficiently inducing differentiation method (Fig. 5), comprises the following steps：
1) culture of people's iPS cells of non-trophoblast system：Add dilution after vitronectin (DMEM/F12,1:100)
1ml/ holes are coated with 6 orifice plates overnight, the culture medium based on E8, people's iPS cells are then inoculated with, in 37 DEG C, 5%CO2, saturation it is wet
Amplification is cultivated in the incubator of degree.
2) easy, the efficient induction to dynamoneure is broken up：People's iPS cells that growth conditions are good are taken, treat it
When growing to 4-5 days and reaching 70%-85% degrees of fusion (preferably 80% degrees of fusion), digest from wall and be suspended in using 0.5mM EDTA
Neuronal induction differential medium.And the orientation that dynamoneure is carried out with the use of the combination of different micromolecular compounds lures
Lead differentiation.Wherein micromolecular compound combination addition situation is as follows：
Induction differentiation the 0th day to the 2nd day, is separately added into DMH1 (2 μM)+SB431542 (2 μM)+CHIR99021 (3 μM) group
Close or (3 μM) of LDN193189 (0.3 μM)+SB431542 (2 μM)+CHIR99021 is combined；
Induction differentiation the 2nd day to the 4th day, RA is additionally separately added on the basis of the micromolecular compound described in 0-2 days again
(0.5 μM) combination of (1 μM) combination of (0.1 μM)+Purmorphamine or RA (0.1 μM)+SAG；
Induction differentiation the 4th day to the 9th day, it is separately added into (1 μM) combination of RA (0.1 μM)+Purmorphamine or RA (0.1
μM) (0.5 μM) combination of+SAG；
Induction differentiation the 9th day to the 12nd day, only adds DAPT (10 μM).
The present invention have chosen corresponding each path representative activator or inhibitor, and use in conjunction carries out spinal motor
The induction differentiation of neuron.As a result find, with combine compound LDN193189, SB431542, CHIR99021, RA, SAG and
In the presence of DAPT, the 12nd day spinal motor nerve (Fig. 9, table 1) that can obtain high-purity (about 95%) of induction differentiation.Relatively
Multi-step in conventional dynamoneure, poorly efficient differentiation research (about 25%, 4 weeks, Figure 10, table 1), are obviously improved.
And inventor also has found small molecule DMH1 application first, and dynamoneure mark HB9 and ISL1 can be caused to occur
Segregation phenomenon (table 1).In addition, the induction differentiation scheme success of easy, quick, efficient dynamoneure provided by the invention
Related high workload concentration compound such as DMH1 is avoided, especially Purmorphamine application, effectively reduces the secondary work of its poison
With.
The further object of the present invention is to provide dynamoneure and promotees maturation method and its functional study application, function
Property dynamoneure obtain mainly comprise the following steps：The neuron broken up the 9th day will be induced to be digested, inoculated to advance
It is coated with poly-ornithine, laminin and matrigel slide, is trained based on star spongiocyte conditioned medium
Base is supported, and adds micromolecular compound DAPT (10 μM) simultaneously；Every 3 days, half amount changed fresh culture once, until induction point
Untill changing the 31st day.Wherein, the preparation process of the astroglia conditioned medium is：Trained using DMEM complete mediums
Support astroglia 3 days, add fresh Neuronal induction differential medium, be collected daily afterwards, and more renew
Fresh Neuronal induction differential medium.
Further, obtaining the proprietary characteristic main flow of motor neuron is：It is thin into flesh to first pass through low Serum System induction
Born of the same parents break up to obtain myotubes.It will then induce and break up the neural ball of the 9th day and digested, be seeded in the form of small clone point
The myotubes changed are co-cultured, while add micromolecular compound DAPT (10 μM).Per 2-3 days, half amount was changed fresh
Culture medium once, until co-culture the 14th day untill.
Detected by immunofluorescence dyeing and patch-clamp, be as a result shown in and induce differentiation early stage (a 4th week left side of induction differentiation
It is right) it can obtain functional ripe motor neuron (Figure 12,13,14), the divergaence time compared to conventional 7 weeks or so, effect
Significantly improved in terms of rate.What is more important, the performance of ripe kinesitherapy nerve meta function be, can be thin with skeletal muscle myotube
Born of the same parents form neuromuscular junction, and play a part of dominating Skeletal Muscle Contraction.The present invention is further observed by captured in real-time, exempted from
Epidemic disease dyes and Patch-clamp techniques, under as a result showing that ripe motor neuron co-cultures with Skeletal Muscle solencyte, can form nerve
Neuromuscular junction structure simultaneously dominates skeletal muscle rhythm and pace of moving things contraction (Figure 15,16,17).
Based on the above, the present invention has been successfully established independent of the people iPS cells training for nourishing coating systems, serum-free disturbs
The system of supporting；People's iPS cells of culture in the system, after the micromolecular compound combination after use in conjunction optimization, can it is easy,
Quickly, efficiently induction obtains a large amount of, dynamoneure of high-purity；Also, carried in astroglia conditioned medium
Functional ripe motor neuron further can be quickly divided into the microenvironment of confession.This will be to establish motor neuron disease spy
Idioblas model provides good application prospect.
Embodiment
Material and method
Material source：
Skin histology sample is derived from The First Affiliated Hospital, Fujian Medical University Neurology spinal muscular atrophy patient.It is newborn
C57BL/6 rats (after birth in 24 hours) are provided by Chinese Academy of Sciences's Shanghai nerve.
Reagent：
DMEM culture mediums, DMEM-F12 culture mediums, Neurobasal culture mediums, E8 culture mediums, hyclone (FBS),
0.25% pancreas enzyme -EDTA, vitronectin, N2 additives, B27 additives, Accutase digestive juices (Thermo Fisher
Scientific companies, the U.S.)；Laminin (Sigma companies, the U.S.)；Matrigel (Corning companies, the U.S.)；Second two
Amine tetraacethyl (Beijing Sai Bei bio tech ltd, China)；SB431542、CHIR99021、LDN193189(Stemgent
Company, the U.S.)；Poly-ornithine, vitamin A acid (RA) (Sigma companies, the U.S.)；DMH1、Purmorphamine、DAPT
(Abcam companies, Britain)；SAG (Millipore companies, Germany)；Sendai virus iPS cells reprogramming kit (Thermo
Fisher Scientific companies, the U.S.)；PBS pieces (Medicago companies, Sweden)；Goat anti human NANOG (R＆D companies, it is beautiful
State)；Mouse anti human TRA-1-60, Goat anti human CHAT, mouse anti human Synaptophysin (Millipore companies, Germany)；
Mouse anti human SMI32, rabbit against human T UJ1, (Covance companies, the U.S.) mouse anti human SSEA4, mouse anti human ISLET1 (DSHB,
The U.S.)；Goat anti human HB9, rabbit-anti people MAP2 (SANTA CRUZ companies, the U.S.)；Alexa594 donkey anti goat igg,
Alexa594 donkey anti-mouse IgG, Alexa488 donkey anti-mouse IgG, Alexa488 donkey anti-rabbits
IgG、AlexaCy5 donkey anti-rabbits IgG (Thermo Fisher Scientific companies, the U.S.)；DAPI (Sigma, it is beautiful
State)；Anti- fluorescence quenching (SouthernBiotech, the U.S.).
Reprogramming, culture and the identification of the people's iPS cells of embodiment 1
Reprogramming and culture：DMEM complete mediums (containing 10%FBS, 100IU/ml penicillin, 100IU/ml streptomysins)
The skin histology block cell of original cuiture patient's biopsy.After it is covered with, washed 1 time with DMEM, the digestion of 0.25% pancreas enzyme -EDTA
Passage, with 0.2x 106Cells/well density is seeded in 12 orifice plates, is placed in 37 DEG C, 5%CO2, saturated humidity incubator in train
Support.After 2 days, with infection multiplicity (multiplicityof infection, MOI) for 5, the celestial platform of the reprogramming factor will be carried
Viral (hOct3/4, hSox2, hKlf4 and hc-Myc) is infected it.Above-mentioned fresh culture, 1ml/ are changed after 24 hours
Hole；The afterwards next day, carries out full dose and changes liquid.Cultivate the 6th day, with DMEM/F12 with 1:100 dilution proportion vitronectins, it is coated with 6 holes
Plate, 1ml/ holes, in overnight in incubator.Cultivate the 7th day, metainfective fibroblast is digested.DMEM/F12 washings 3
0.25% pancreas enzyme -EDTA 0.5ml/ holes that are secondary, adding after preheating, digest 1 minute, are then centrifuged for 1200rpm at room temperature, 1.5 points
Clock.Finally, complete medium is resuspended with 1:5 ratio renewed vaccinations are to vitronectin (DMEM/F12 1:100 dilutions) it is coated with overnight
In 6 treated orifice plates, in 37 DEG C, 5%CO2, saturated humidity incubator in cultivate.After 24 hours, E8 stem cells are replaced by
Culture medium；Carry out full dose daily afterwards and change liquid.After cultivating 2 weeks, it is seen that people iPS cell clones occur.Now, Mechanical Method picking is given
Obtained monoclonal is reprogrammed, contrast is seeded to respectively is coated with vitronectin (DMEM/F12 1 in advance:100 dilutions, 37 DEG C,
Stay overnight) or matrigel (DMEM/F12 1:50 dilution, 37 DEG C, 30 minutes) 6 orifice plates in culture amplification.After 5-6 days, cell life
Secondary Culture is carried out during long density about 70-85%.Under room temperature environment, 0.5mM EDTA digestion iPSCs 1 minute, DMEM/F12 is washed
Wash 1 time, blown and beaten with E8 culture mediums from parietal cell, with 1:10 ratio renewed vaccination cells are to being coated with treated orifice plate, in 37
DEG C, 5%CO2, saturated humidity incubator in continue amplification cultivation.According to cell density, passage 1 time per 5-6 days, daily
Carry out changing liquid.
Identification：
The time and the metamorphosis of amplification cultivation process that Continuous Observation clone occurs under inverted microscope daily.Culture
After expanding for the 5th generation, the detection of expression of embryonic stem cell marker is carried out by immunofluorescence technique, specific practice is as follows：Treat thin
When intracellular growth density reaches 70-85%, had digestive transfer culture is carried out to it and is seeded on the treated cover glass of vitronectin coating.2nd
My god, fix cell, lower 10 minutes of room temperature environment with fresh 4% paraformaldehyde.0.01M PBS are washed 3 times, every time 5 minutes.20%
Donkey serum+0.2%Triton-X-100+0.01M PBS are closed, room temperature 1 hour.With confining liquid 1：1000 dilution primary antibodies
(NANOG, SSEA4, TRA-1-60), 4 DEG C overnight.PBS is washed 3 times, every time 5 minutes.0.01M PBS are with 1:1000 dilution secondary antibodies
With DAPI, it is incubated 1 hour at room temperature.PBS is washed 3 times, every time 5 minutes.Finally carry out anti-fluorescence quenching mounting.
Observation result is shown：
It was calculated as the 0th day (Day 0) with the virus infection same day.Before virus infection 2 days (Day-2), skin fibroblasts patch
Wall grows into elongated fibrous form (Fig. 1 .A).After virus infection 7 days (Day 7), significant change, projection occur for cellular morphology
Disappear, the increase of cell space volume, spherical in shape or polygonal, refractivity enhancing (Fig. 1 .B).Again the cell after inoculation is passed on
(Day 8), its form (Fig. 1 .C) still based on spherical.After virus infection 21 days (Day 21), there is obvious cell clone (figure
1.D, shown in arrow).Passage using vitronectin as somatomedin expands clone, and culture visible is closely arranged on the 1st day in many cells
Row growth (Fig. 2 .A), as incubation time extends, clone's uniformly gradually increase, refractivity enhancing, sharpness of border (Fig. 2 .B, C),
And there is same modality outward appearance and Reproduction methods (Fig. 2 .D) with embryonic stem cell.And the passage using matrigel as somatomedin is expanded
Increase clone, the metamorphosis and Reproduction methods in incubation greatly differ from each other (Fig. 3 .A-D) with embryonic stem cell.It is further right
The former expands obtained clone and carries out immunofluorescence dyeing identification, as a result shows that it can stablize expression embryonic stem cell correlating markings
Thing：Positive signal is located at endonuclear NANOG (Fig. 4 .A), positive signal is located at the SSEA4 (Fig. 4 .B) and the positive of cell membrane
Signal is located at cytoplasmic TRA-1-60 (Fig. 4 .C).
To sum up result is shown, we successfully reprogram to have obtained whole people's iPS cells independent of nourishing coating systems, and
Establish a kind of optimization culture scheme using vitronectin as somatomedin.Importantly, this whole process is without external source serum interference
People's iPS cells possess the same characteristic features of embryonic stem cell, alternative the latter is used for the research of disease mechanisms and treatment from now on.
The people iPS cells of embodiment 2 count to the Induction of committed differentiation of dynamoneure, identification and differentiation efficiency
Take and be grown in 6 orifice plates (Corning companies, the U.S.) people's iPS cells in good condition, treat that it grows to 5-6 days
During up to 70%-85% degrees of fusion, digested 1 minute with 0.5mM EDTA room temperatures, coordinate piping and druming to be collected from wall to 15ml centrifuge tubes
(Guangzhou Jie Te biofiltrations Products Co., Ltd, China), centrifuges 1000 revs/min, 1 minute.Utilize 10ml nerve-inducings point
Change culture medium and (include 1:1DMEM/F12:Neurobasal culture mediums, 0.5x N2 additives, 0.5x B27 additives, L- paddy ammonia
Acid amides, nonessential amino acid) be resuspended in the low absorption culture dishes of 10cm (Guangzhou Jie Te biofiltrations Products Co., Ltd, in
State), while add different micromolecular compound combination DMH1+SB431542+CHIR99021 or LDN193189+SB431542+
CHIR99021, it is placed in 37 DEG C, 5%CO2, saturated humidity incubator in, with promote form it into embryoid body structure, be now calculated as
Induction differentiation the 0th day.The next day carry out full dose change liquid, with above-mentioned differential medium.Induction differentiation the 2nd day, full dose changes liquid, training used
Support base and micromolecular compound be same as above, additionally add RA+SAG or RA+Purmorphamine various combination compounds again in addition,
To promote to form it into neural spherical structure.Induction differentiation the 4th day, full dose changes liquid, and used medium is same as above, and now only adds RA+SAG
Or RA+Purmorphamine various combination compound；The afterwards next day, full dose changed liquid.Induction differentiation the 9th day, collects differentiation
Neural ball, 1000 revs/min, 1 minute are centrifuged, supernatant discarding；The Accutase digestive juices of 1ml/ pipes are added, 37 DEG C of water-baths 8 divide
Clock；Add 8ml DMEM/F12 and terminate digestion, centrifuge 1000 revs/min, 1 minute, supernatant discarding；Add above-mentioned point of 1ml/ pipes
Change culture medium, blow and beat 3-4 times, stand 3 minutes；Draw the thin s born of the same parents' suspension in upper strata be seeded to poly-ornithine (0.1mg/ml),
Laminin (40ug/ml) and matrigel (1：50 dilutions) the treated slide of coating is in 24 orifice plates, 40 μ L/ slides；Put
In 37 DEG C, 5%CO2, saturated humidity incubator in, it is adherent to promote.Culture medium is supplied after 2 hours, 0.5ml/ holes are another extra
Micromolecular compound DAPT is added, to promote the maturation of motor neuron, until culture untill induction differentiation the 12nd day.
Specifically it is grouped as follows：
(1)DMH1(2μM)+SB431542(2μM)+CHIR99021(3μM)+RA(0.1μM)+Purmorphamine(1μ
M)+DAPT (10 μM), abbreviation DP groups；
(2)LDN193189(0.3μM)+SB431542(2μM)+CHIR99021(3μM)+RA(0.1μM)+
Purmorphamine (1 μM)+DAPT (10 μM), abbreviation LP groups；
(3)DMH1(2μM)+SB431542(2μM)+CHIR99021(3μM)+RA(0.1μM)+SAG(0.5μM)+DAPT
(10 μM), abbreviation DS groups；
(4)LDN193189(0.3μM)+SB431542(2μM)+CHIR99021(3μM)+RA(0.1μM)+SAG(0.5μM)
+ DAPT (10 μM), abbreviation LS groups；
In addition, the induction differentiation step of the rapid dynamoneure of traditional four-step approximately as：
When people iPS cell growths to 70%-85% degrees of fusion, digested with 0.5mM and be incubated at dress from wall and suspension
There is the culture dish of nerve-inducing differential medium (comprising DMEM/F12 culture mediums, 1x N2 additives, nonessential amino acid) (wide
Zhou Jiete biofiltrations Products Co., Ltd, China) in.The 0th day (Day 0) is now calculated as, daily half amount changes liquid afterwards.Induction
When breaking up the 7th day, promoted adhere-wall culture and be coated with matrigel in treated culture dish (Corning companies, the U.S.), the next day
Half amount changes liquid.And since the 10th day add small molecule RA (0.1 μM).When induction is broken up the 15th day, directly blow and beat from wall gram
It is grand, and the culture that suspends again, while additionally small molecule Purmorphamine (1 μM) is added again.When induction is broken up the 25th day,
Accutase, which digests to suspend, clones and is seeded to again what is treated with poly-ornithine, laminin and matrigel coating
Adhere-wall culture in slide.
Identification：
The metamorphosis that Continuous Observation is cloned under inverted microscope daily.When induction is broken up the 12nd day, terminated,
It is fixed with fresh 4% paraformaldehyde.Above-mentioned cell is transported with the 12nd day spinal cord after the detection induction of immunofluorescence dyeing method
Dynamic neuron HB9 and ISLET1 (the motor neuron Specific marker after mitosis) expression.Primary antibody is Goat anti human
HB9 antibody (1:And mouse anti human ISLET1 antibody (1 50):100).3 random fields/examination every time is counted by double, double-blind study
Test, independent repeated trials 3 times.Motor neuron differentiation efficiency=HB9 and ISL1 sun that target positive cell number/DAPI is marked altogether
Property cell number.Using SPSS16.0 statistical softwares, one-way analysis of variance, as a result represented with average ± standard error.
As a result：
People iPS cells carry out spinal cord fortune in the micromolecular compound (DP groups, LP groups, DS groups and LS groups) for adding various combination
In the induction atomization of dynamic neuron：Typical EB balls (Fig. 6-9.A) can be formed within 2nd day；Sustained suspension culture was to the 9th day
When, it can form the neural ball (Fig. 6-9.B) that volume is big, light-proofness is strong, bright degree is big；(induction differentiation the 12nd 3rd day after adherent
My god), it can form typical motor neuron form, it will be apparent that projection and be woven into network-like structure (Fig. 6-9.C).Each group is thin
Born of the same parents break up the immunofluorescence dyeing of the 12nd day in induction, it is seen that positive particle signal is respectively positioned in nucleus, referring to Fig. 6-9.D-
F.Wherein, Fig. 6 .D-F show DP group HB9 and ISLET1 immunofluorescence positive cells；Fig. 7 .D-F show LP groups HB9 and ISLET1
Immunofluorescence positive cell；Fig. 8 .D-F show DS group HB9 and ISLET1 immunofluorescence positive cells；Fig. 9 .D-F show LS groups
HB9 and ISLET1 immunofluorescences positive cell (specifically referring to table 1).Obviously, the small molecule combinatorial induction scheme of LS groups obtains
Dynamoneure rate highest, and there is not motor neuron mark HB9 and ISLET1 segregation phenomenon (table 1).
In addition, sent out in comparing with classical traditional abductive approach (four step rule, suspension-adherent-suspension-adherent, Figure 10)
Existing, no matter the abductive approach of the present application still all has in simplicity (two-step method, suspension-adherent) in terms of differentiation efficiency
There is obvious advantage (table 1).
To sum up result is shown, present inventor establishes a kind of safe, reliable, efficient dynamoneure and lured
Lead differentiation scheme.And easy, the efficient acquisition of high-purity dynamoneure, the application of disease phenotype simulation is beneficial to,
Cellular transplantation therapy even from now on.
Application after the induced myeloid motor neuron fast-ripenin of embodiment 3
Primary star spongiocyte culture：
Take out it is raw after C57BL/6 Strains of Mouse in 1 day, after 75% alcohol sufficiently sterilised, quickly take brain.It is micro- dissecting
After the fibre compositions such as meninx and blood vessel are divested under mirror, move it in 15ml centrifuge tubes that (the clean special biofiltration product in Guangzhou is limited
Company, China), digested 10 minutes under 37 DEG C of water-baths with 0.25% pancreas enzyme -EDTA.DMEM complete mediums (contain 20%FBS)
Digestion is terminated, gently blows and beats for several times, single cell suspension is made.5 points of kinds are staticly settled, supernatant is drawn and is inoculated into advance matrigel
(1:100 dilutions) it is coated with treated T75 blake bottles (Corning), it is placed in 37 DEG C, 5%CO2, saturated humidity incubator
Middle culture.After 24 hours, firmly shaken cultivation bottle with remove be located at astroglia on other cellular layers and cell fragment
Deng, renew fresh DMEM complete mediums (containing 20%FBS), continue culture 3-5 days.When its stand density is about 80-90%,
Carry out had digestive transfer culture.DMEM culture mediums wash 1 time, add the pancreas enzyme -EDTAs of 5ml 0.25% in 3 points of incubation in 37 DEG C of incubators
Clock.Complete medium terminates digestion, and gently blows and beats for several times from parietal cell.Cell suspension is moved in 15ml centrifuge tubes, centrifuged
1500rpm, 2 minutes.Supernatant is abandoned in suction, and DMEM complete mediums (containing 20%FBS) are resuspended, with 1：4 ratios are seeded to new 10cm
In culture dish (Corning), 37 DEG C, 5%CO are placed in2, saturated humidity incubator in cultivate.
Star spongiocyte conditioned medium culture dynamoneure：
The culture medium based on DMEM complete mediums (containing 20%FBS), the star-like colloid in the 3rd generation is thin after culture is expanded
Born of the same parents are with 5x 104Cell/cm2Density, which is seeded to, is coated with matrigel (1:100 dilution) 10cm culture dishes (Corning) in, put
In 37 DEG C, 5%CO2, saturated humidity incubator in.3rd day, original culture medium was abandoned in suction, and DMEM culture mediums wash 3 times, and add
Enter fresh Neuronal induction differential medium and (include 1:1DMEM/F12:Neurobasal culture mediums, 0.5x N2 additives,
0.5x B27 additives, Glu, nonessential amino acid).It is collected daily afterwards, and changes fresh neuron and lure
Lead differential medium.
The neural ball for inducing differentiation the 9th day (Day 9) is digested, with 0.1x 104Cell/cm2Density is seeded to pre-
First it is coated with poly-ornithine (0.1mg/ml), laminin (40ug/ml) and matrigel (1：50 dilution) slide on, with
Culture medium based on star spongiocyte conditioned medium, and micromolecular compound DAPT (10 μM) is added simultaneously.Every 3 days,
Half amount changes fresh culture once, untill induction differentiation the 31st day.
Dynamoneure co-cultures with Skeletal Muscle solencyte：
By after the of short duration digestion of neural ball for inducing differentiation the 9th day (Day 9), being seeded to culture in advance with small cloned version thereof has
In copolymerization Jiao's special culture dish of myotubes (Thermo Fisher Scientific companies, the U.S.), culture medium is replaced by
Neuronal induction differential medium (includes 1:1DMEM/F12:Neurobasal culture mediums, 0.5x N2 additives, 0.5x B27
Additive, Glu, nonessential amino acid), while add micromolecular compound DAPT (10 μM).Per 2-3 days, half amount was more
Fresh culture is changed once, untill co-culturing the 14th day.Simultaneously so that individually inoculation myotubes are right as feminine gender in culture dish
According to using identical cultivating system.
Identification：
The metamorphosis of star spongiocyte, induction differentiation come after Continuous Observation Secondary Culture under inverted microscope daily
After the dynamoneure growing state and dynamoneure in source form neuromuscular junction with myotubes, flesh is dominated
The contraction situation of pipe.Above-mentioned cell utilizes immunofluorescence dyeing detection marker expression situation.Primary antibody is rabbit-anti people's GFAP antibody
(1:500), Goat anti human CHAT (1:300), mouse anti human SMI32 (1:500), rabbit against human T UJ1 (1:10000), mouse anti human
Synaptophys in(1:2000), rabbit-anti people MAP2 (1:10000)、BTX(1：500), the anti-human MHC (1 of mouse：And rabbit 2000)
Anti-human Synaps in (1:2000).Meanwhile using patch clamp technique to the dynamoneure of maturation and the skeletal muscle shunk
Myotubes carry out electrophysiological recording.
As a result：
1. immunofluorescence dyeing：It can be seen that the star spongiocyte growth of passage amplification cultivation is adherent close (Figure 11 .A), and
Expression positive signal is located at cytoplasmic Specific marker GFAP (Figure 11 .B-D).With various, the cheap star of composition
The neurotrophic factor culture dynamoneure of type spongiocyte conditioned medium replacement expensive traditional (Day after about 2 weeks
22), it starts to express ripe motor neuron Specific marker CHAT, SMI32 and TUJ1, and its positive signal is respectively positioned on cell
Matter (Figure 12 .A-D).After continuing adherent about 3 weeks (Day 31), start expressive function motor neuron Specific marker：Sun
Property signal is located at the Synaptophys in of cell membrane and positive signal is located at cytoplasmic TUJ1 (Figure 13 .A-C).
2. the patch-clamp electrophysiological recording of feature dynamoneure：Under room temperature condition (22 DEG C -24 DEG C), carry out
The functional maturation dynamoneure electrophysiological characteristics of the 31st day is broken up in Whole-cell recording induction.Culture there is into fortune
The slide of dynamic neuron is as liquid (Mm outside electrode：NaCl 135,KCl 3,CaCl2 2,MgCl2 1,Glucose1 1,
Sucrose 10 and HEPES10, NaOH adjust pH value to 7.4, osmotic pressure 310mOsm/L) in.Liquid (mM in irrigation recording electrode：
KCl 140,NaCl 9,MgCl21, EGTA 0.2, ATP-Mg 2, GTP-Na 0.25 and HEPES10, KOH adjust pH value to
7.2, osmotic pressure 290mOsm/L), electrode resistance 3-5M Ω.(the 40X under inverted microscope (Nikon companies, Japan) direct-view
Mirror), high resistance seals, rupture of membranes and record (Figure 14 .A) are carried out to record cell.Using current-clamp mode recording parameters：-200pA→
+ 250pA (using 50pA as step), each stimulus intervals 1ms, stimulus duration：Duration A 200ms, duration B
200ms, duration C 100ms；Using voltage-clamp mode recording parameters：Clamping voltages -70mV, -70mV →+30mV (with
10mV is step), each stimulus intervals 1ms, stimulus duration：Duration A 100ms, duration B 400ms,
duration C 100ms.Data by Axon Multiclamp 700B amplifiers, Digidata1440A data acquisition boards and
The softwares of Clampex 10.2 (Molecular Devices, the U.S.) acquisition and recording.Signal sampling frequencies 10kHz, low pass filtered wave frequency
Rate 1kHz.Electric capacity and series resistance compensation value 30%.
Patch-clamp techniques final result is shown：Under current-clamp mode, generation can be induced after injection step electric current stimulates
Continuously, the action potential of bunchiness, and as the increase of Injection Current, the frequency of action potential granting are consequently increased (figure
14.B)；Under voltage-clamp mode, the sodium channel electricity for producing quick activation-inactivation type can be induced after injection step voltage stimulates
Flow (inward electric current) and potassium channel current (outward current) (Figure 14 .C).Thus infer, the functional maturation spinal cord that early stage obtains
Motor neuron possesses excitability speciality.
3. ripe dynamoneure dominates the record of Skeletal Muscle solencyte：Dynamoneure and Skeletal Muscle
After solencyte co-cultures 2 weeks, captured in real-time (20X mirrors) recorded branch of the Skeletal Muscle solencyte in ripe dynamoneure
(picture 15) is shunk with tight knot rule is issued；And the myotubes that any contraction is had no in the culture dish of myotubes are cultivated merely.
Meanwhile the visible neuromuscular junction of immunostaining forms (picture 16).In addition, also give full cell to the myotubes of contraction
Patch-clamp techniques.System records with neuron, and parameter uses gap-free (not giving any exogenous stimulation).As a result display can be remembered
The action potential (Figure 17) independently continuously provided is recorded, further demonstrate the formation of neural joint design be present.
It these results suggest that, the ripe motor neuron efficiently obtained in a short time according to the inventive method tentatively possesses
Function specific to body dynamoneure.
By above content it has been confirmed that the invention provides a kind of indefinite material interference of the compositions such as no external source serum
People iPS cell culture protocols and a kind of easy, fast and efficiently dynamoneure induction differentiation scheme is established, and
The identified functional characteristic met in body dynamoneure of the ripe motor neuron of acquisition.Such cell will can be used for disease
The simulation of sick phenotype and the discussion of pathogenesis, the screening for effectively treatment drug candidate from now on provide theoretical foundation；Even can
Applied to cellular transplantation therapy from now on.
Many embodiments of the invention have been described in the above, but the present invention should not be construed as limited to
These embodiments.It should be understood that unless otherwise indicated, any or all of example provided here or typical
Term is not limit the scope of the invention all merely for the sake of the purpose of the present invention is better described.It should be understood, however, that
On the premise of without departing from the spirit and scope of the present invention, many different modifications can be made.Therefore, it should be clear that, this hair
The bright embodiment for being not limited to specifically describe, and only limited by the scope of claim.
Claims (1)
1. the method for feature dynamoneure is obtained based on people iPS cell high-efficients, it is characterised in that this method mainly walks
Suddenly it is：
First, reprogramming obtains people's iPS cells
Minimally invasive acquisition human skin fibroblasts, through the celestial being for carrying reprogramming factor hOct3/4, hSox2, hKlf4 and hc-Myc
After platform virus infection, cultivated 7 days in DMEM complete mediums；It is resuspended again with complete medium, and with 1:5 ratio renewed vaccinations
Cultivated 24 hours in the orifice plate treated to vitronectin coating；E8 stem cell medias are replaced by afterwards, and it is every from next day
Day carries out full dose and changes liquid, until can obtain iPS cells after 2 weeks；Picking iPS cell monoclonals, it is seeded to and is coated with glass in advance
The induction differentiation of motor neuron after amplification cultivation is even carried out in the orifice plate of albumen or matrigel and is used for；
2nd, the induction differentiation of motor neuron
(1) it will cultivate to the people iPS cell dissociations of 70%-85% degrees of fusion and suspension and be incubated at and combined containing micromolecular compound
2 days in I Neuronal induction differential medium；
(2) cell is moved to combine containing micromolecular compound and continues the culture 2 that suspends in II Neuronal induction differential medium
My god；
(3) cell is moved into sustained suspension culture 5 in the Neuronal induction differential medium that III is combined containing micromolecular compound
My god；
(4) cell dissociation adhere-wall culture can be obtained into motor neuron in 3 days, only added in Neuronal induction differential medium around here
Enter micromolecular compound DAPT；
The Neuronal induction differential medium of the step (1) is blending constituent, contains 1 respectively：1 DMEM/F12 and
Neurobasal culture mediums, 0.5x B27 additives, 0.5x N2 additives, 1x glutamine and 1x nonessential amino acid；
The micromolecular compound combination I that the step (1) is added is formed and concentration is：DMH1 2μM+SB431542 2μM+
3 μM of CHIR9902 or 0.3 μM of LDN193189,2 μM of+SB431542,3 μM of+CHIR99021；
The micromolecular compound combination II compositions and concentration additionally added in the Neuronal induction differential medium of the step (2)
It is as follows：0.5 μM of+SAG of micromolecular compound 0.1 μM of I+RA of combination；
Additionally added in the Neuronal induction differential medium of the step (3) micromolecular compound combination III composition and it is dense
Degree is as follows：RA 0.1μM+SAG 0.5μM；
First by Accutase enzymic digestions in the step (4), then neuron is seeded to and is coated with poly-ornithine, layer glues
On the slide that even albumen and matrigel coating treat；After cell attachment is complete, the god containing micromolecular compound DAPT is added
Through first inductive differentiation medium；
3rd, the cultivation of functional exercise neuron
The neuron that the culture that suspends in step 2 (3) terminates is digested, inoculated to being coated with poly-ornithine, layer in advance
On Fibronectin and matrigel slide, the culture medium based on star spongiocyte conditioned medium, and small point is added simultaneously
Sub- 10 μM of compound DAPT；Every 3 days, half amount changed fresh culture once, untill induction differentiation the 31st day；
The preparation process of the astroglia conditioned medium is：Using DMEM complete medium culture astroglias
3 days, fresh Neuronal induction differential medium is added, is collected daily afterwards, and changes fresh Neuronal induction
Differential medium.
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