CN106701824A - Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells - Google Patents
Method for obtaining spinal motoneurons and functional cells thereof based on human iPS (induced pluripotent stem) cells Download PDFInfo
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Abstract
The invention belongs to the field of biomedicine, and relates to a safe and reliable reprogrammed method for obtaining human iPS (induced pluripotent stem) cells, a method for simply, rapidly and efficiently inducing and differentiating iPS cells into spinal motoneurons, and a method for economically and efficiently obtaining functional spinal motoneurons and application of the method. Human fibroblasts are infected with Sendai virus carrying a reprogramming factor by using vitronectin as the sole growth medium, and are cultured for 3 weeks to obtain human iPS cells without exogenous serum interference; the iPS cells are efficiently induced and differentiated into spinal motoneurons by a combination of multiple small molecule compounds; and further, an astrocyte conditioned culture medium promotes maturation culture and is applied in the formation of neuromuscular junctions. The functional spinal motoneurons obtained by efficient induction can be applied to simulation of disease phenotype and discussion of pathogenesis, and provides a theoretical basis for the screening of effective therapeutic drugs in the future, and even in the future can be applied to the cell transplantation therapy of diseases.
Description
Technical field
The invention belongs to biomedical sector, it is related to the acquisition methods of dynamoneure, in particular, in that including a kind of
The method that safe and reliable reprogramming obtains people's iPS cells, it is a kind of that the simplicity rapidly and efficiently inducing differentiation of people iPS cells is turned into spinal cord
A kind of method of motor neuron, also including acquisition methods of cost-effective feature dynamoneure.
Background technology
Dynamoneure (motorneuron, MN) is sent out in the contractile motion of central nervous system domination skeletal muscle
Bridge Link role is waved, the important vital functions activity such as breathing is controlled, 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.At present studied can by the SF 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, obtain convenience, are not related to apply ethics problem
Etc. advantage, this just provides a kind of new way to set up 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.Being depended on current vitro culture of human iPS cells more
Coating systems are nourished, it has external source；And in terms of dynamoneure differentiation research, although existing phase
To ripe abductive approach, but generally existing step numerous and diverse (four step rule), time-consuming (being up to 7 weeks), differentiation efficiency is low (only
25%) defect such as.Additionally, the functional nerve unit for maintaining culture induction to obtain, often needs to rely on to use expensive neurotrophy
The factor, also considerably increases experimental cost.Therefore, in order to solve existing deficiency, it is quite necessary to explore and set up a kind of whole process not
Rely on and nourish coating systems, the people's iPS cell reprogramming methods without the indefinite material interference of the compositions such as external source serum；Especially explore
A kind of differentiation method of cell differentiations of simplicity rapidly and efficiently inducing people iPS in vitro as dynamoneure is set up, and is built
Found a kind of acquisition methods of cost-effective feature dynamoneure.
In the dynamoneure model for setting up motor neuron disease, 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 studied.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 suppress in, entoderm point
The effect of change, finally realizes that embryoid body breaks up toward neuroderm；Tail veutroization is then mainly logical by activating RA paths and SHH
Road, so as to realize that central nervous system occurs (akrencephalon end to endSpinal cord) and back of the body abdomen (ventricornuRelief angle) the two poles of the earth differentiation；
Maturing is then, by suppressing Notch paths, finally to realize dynamoneure precursor to the spinal motor after mitosis
Neuron is converted：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, essentially consists in its toxic side effects, and be proportionate with its work action concentration.There is phase in existing scheme
Close high workload concentration compound such as DMH1, the especially application of Purmorphamine.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, DIF is that the selection of micromolecular compound is also the key point studied.
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 HSF, 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 with complete medium again, and with 1:5 ratio renewed vaccinations
Cultivated 24 hours in the treated orifice plate of 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 coating in advance
Having carries out amplification cultivation in the orifice plate of vitronectin or matrigel.
Wherein, as above need to be through vitronectin (1 before porous plate described in method：100 dilutions, the μ g/ml of valid density 10)
Overnight it is coated with 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 iPS cells simplicity rapidly and efficiently inducing
The method of unit, key step is as follows：
(1) the people iPS cell dissociations to 70%-85% degrees of fusion will be cultivated and suspends to be incubated at and contain micromolecular compound
Combine 2 days in the Neuronal induction differential medium of I；
(2) training that suspends is continued in cell being moved into the Neuronal induction differential medium containing micromolecular compound combination II
Support 2 days；
(3) by cell move to containing micromolecular compound combination III Neuronal induction differential medium in sustained suspension
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 described in step (1) is blending constituent, and 1 is contained respectively：1 DMEM/
F12 and Neurobasal culture mediums, 0.5xB27 additives, 0.5xN2 additives, 1x glutamine and 1x nonessential amino acid.
Wherein, 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 of extra addition is (dense in step (1) the Neuronal induction differential medium
Degree) it is as follows：Micromolecular compound combine I+RA (0.1 μM)+SAG (0.5 μM) or micromolecular compound combine I+RA (0.1 μM)+
Purmorphamine(1μM)。
Wherein, the micromolecular compound of extra addition combines III 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)：First by Accutase enzymic digestions, then neuron is seeded to
It is coated with the treated slide of poly-ornithine, laminin and matrigel coating；After cell attachment is complete, addition contains
The Neuronal induction differential medium of micromolecular compound DAPT.
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：The differentiation neuron of the 9th day will be induced to be digested, inoculated 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, adds fresh Neuronal induction differential medium, is collected daily afterwards, and change fresh
Neuronal induction differential medium.
The present invention establishes a kind of whole process and is 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
By with reference to accompanying drawing, to describe the feature situation and its advantage of exemplary embodiments of the present invention, accompanying drawing in detail：
Fig. 1 is to be independent of nourishing coating systems, the people iPS cells reprogramming process without external source serum interference；Wherein A is celestial platform
The HSF of first 2 days of infection of virus, in elongated strip；B is the application on human skin of 1 week after sendai virus infection into fiber
Cell, projection disappears, changes in polygonal；C is the HSF of renewed vaccination after digestion；D is for 3 after virus infection
The primary people iPS cells that week occurs, clone's (shown in white arrow) spherical in shape, cell tight arrangement, refractivity is strong.Scale, A-D
It is 200 microns.
Fig. 2 is the people's iPS cell cultivation process with vitronectin as somatomedin；Wherein A-C is the continuous growth of clone
Dynamic changing process, it is rounded all the time to extend out formula growth；It is identical with the typical embryo's stem cell growth mode in D.Scale, A-D
It is 500 microns.
Fig. 3 is the people's iPS cell cultivation process with matrigel as somatomedin；Wherein A-C is the dynamic of the continuous growth of clone
State change procedure, colony morphology is irregular, growth pattern is not fixed；With in D typical embryo's stem cell growth mode substantially not
Together.Scale, A-D is 500 microns.
Fig. 4 is the 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 marked altogether for SSEA-4 and DAPI；C is marked altogether for TRA-1-60 and DAPI.Scale, A-C is 50 microns.
Fig. 5 is the schematic flow sheet that inducing in vitro human iPS cell differentiations turn into dynamoneure and its application
Fig. 6 is DP group two-step method induced motion neuron differentiation flows and HB9 and ISLET1 immunofluorescence dyeings；Wherein A
It is the embryoid body for suspending the 2nd day, in typical sphere, sharpness of border；B is the neural ball for suspending the 9th day, and volume is significantly increased, thoroughly
Brightness, refractivity increase；C is the dynamoneure of digestion adherent latter 3rd day (induction the 12nd day), with obvious neuron
Projection form, intertexture are reticulated；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 marks dyeing altogether for HB9, ISLET1 and DAPI.Scale, A is 500 microns；B
200 microns are 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
It is the embryoid body for suspending the 2nd day, in typical sphere, sharpness of border；B is the neural ball for suspending the 9th day, and volume is significantly increased, thoroughly
Brightness, refractivity increase；C is the dynamoneure of digestion adherent latter 3rd day (induction the 12nd day), with obvious neuron
Projection form, intertexture are reticulated；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 marks dyeing altogether for HB9, ISLET1 and DAPI.Scale, A is 500 microns；B
200 microns are 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
It is the embryoid body for suspending the 2nd day, in typical sphere, sharpness of border；B is the neural ball for suspending the 9th day, and volume is significantly increased, thoroughly
Brightness, refractivity increase；C is the dynamoneure of digestion adherent latter 3rd day (induction the 12nd day), with obvious neuron
Projection form, intertexture are reticulated；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 marks dyeing altogether for HB9, ISLET1 and DAPI.Scale, A is 500 microns；B
200 microns are 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
It is the embryoid body for suspending the 2nd day, in typical sphere, sharpness of border；B is the neural ball for suspending the 9th day, and volume is significantly increased, thoroughly
Brightness, refractivity increase；C is the dynamoneure of digestion adherent latter 3rd day (induction the 12nd day), with obvious neuron
Projection form, intertexture are reticulated；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 marks dyeing altogether for HB9, ISLET1 and DAPI.Scale, A is 500 microns；B
200 microns are 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
It is 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), with typical garland sample nerve tubular construction (shown in arrow)；C continues the training that suspends for piping and druming after wall
The 8th day neural spherical structure of (induction differentiation the 23rd day) is supported, spherical in shape, sharpness of border, refractivity is strong；D is adherent 3rd day for digestion
The motor neuron of (induction differentiation the 28th day), projection interweaves and reticulates；E is that HB9 and DAPI marks dyeing altogether；F be ISLET1 with
DAPI marks dyeing altogether.Scale, A is 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, tight adherent growth；B-C is dyeed for the common mark of astroglia Specific marker GFAP and DAPI.Scale, A-C is equal
It is 100 microns.
Figure 12 is the immunofluorescence dyeing of ripe motor neuron；Wherein A-D, respectively motor neuron specificity marker
The common mark dyeing of the TUJ1 of thing CHAT, SMI32 and neuronal marker.Scale, A-D is 50 microns.
Figure 13 is the immunofluorescence dyeing of functional maturation neuron；Wherein A-C is respectively functional maturation neuron
The common mark dyeing of Specific marker Synaptophysin and MAP2.Scale, A-C is 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 EAP of record under current-clamp mode；C is electricity
The sodium potassium channel current recorded under pressing tongs pattern.Scale, A is 80 microns.
Figure 15 is under ripe motor neurons innervate, 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：Every time shrink continue 1 second (1 second and 5 seconds be the systole phase, arrow institute
It is shown as the myotubes of contractile motion), shrink interval 4 seconds every time.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 end of wherein BTX (red-label) specific recognition neuromuscular junction
Hardened 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 is that record shrinks
The independence of myotubes provides action potential.
Specific embodiment
One aspect of the present invention provides a kind of whole process and is independent of nourishing coating systems, is done 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) it is independent of nourishing the people iPS cells reprogramming of coating systems：By the HSF 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) be 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), will infect
Fibroblast afterwards is digested.DMEM/F12 is washed 3 times, adds 0.25% pancreas enzyme -EDTA 0.5ml/ holes 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, carries out full dose and changes liquid daily afterwards.After 2 weeks, can obtain reprogram it is thin in the iPS of non-trophoblast system
Born of the same parents.
2) the passage amplification cultivation of the people's iPS cells without the indefinite material interference of the compositions such as external source serum：By previous step weight
Program the iPS cells for obtaining and pass through the isolated monoclonal of mechanical picking, contrast is seeded to is coated with vitronectin in advance respectively
(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 dilutions primary antibody (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 antibody 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 showed, by the weight of 3 weeks or so
Programming can obtain whole process and be independent of nourishing coating systems, people iPS cells (Fig. 1) without external source serum interference.And in contrast without taste
Support during people's iPS cell amplification cultivations of coating systems, we have obtained a kind of training with vitronectin as somatomedin at optimization
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 provide it is a kind of from 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 vitronectin (DMEM/F12,1 after dilution:100)
1ml/ holes are overnight coated with 6 orifice plates, 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) to easy, the efficient induction differentiation of dynamoneure：The good people's iPS cells of growth conditions are taken, it is treated
When growing to 4-5 days up to 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 lured with the use of the orientation that the combination of different micromolecular compounds carries out dynamoneure
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) combinations of LDN193189 (0.3 μM)+SB431542 (2 μM)+CHIR99021；
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, 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.Result find, with combine compound LDN193189, SB431542, CHIR99021, RA, SAG and
In the presence of DAPT, induction differentiation can obtain the spinal motor nerve (Fig. 9, table 1) of high-purity (about 95%) on the 12nd day.Relatively
Multi-step, poorly efficient differentiation research (about 25%, 4 weeks, Figure 10, table 1) in conventional dynamoneure, are obviously improved.
And inventor also has found the application of small molecule DMH1 first, dynamoneure mark HB9 and ISL1 can be caused to occur
Segregation phenomenon (table 1).Additionally, the induction differentiation scheme success of easy, quick, the efficient dynamoneure that the present invention is provided
Related high workload concentration compound such as DMH1 is avoided, the especially application of Purmorphamine 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 differentiation neuron of 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
Support base, and addition 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：First passing through low Serum System, to induce into flesh thin
Born of the same parents' differentiation obtains myotubes.To then induce the differentiation neural ball of the 9th day to be digested, be seeded in the form of small clone point
The myotubes changed are co-cultured, while adding micromolecular compound DAPT (10 μM).Per 2-3 days, half amount changed fresh
Culture medium once, until co-culture the 14th day untill.
Detected by immunofluorescence dyeing and patch-clamp, be as a result displayed in and induce differentiation early stage (a 4th week left side of induction differentiation
It is right) it is that can obtain functional ripe motor neuron (Figure 12,13,14), the divergaence time compared to conventional 7 weeks or so, effect
Rate aspect is significantly improved.What is more important, the performance of ripe kinesitherapy nerve meta function is, can be thin with skeletal muscle myotube
Born of the same parents form neuromuscular junction, and play a part of to arrange Skeletal Muscle Contraction.The present invention is further observed by captured in real-time, exempted from
Epidemic disease is dyeed and Patch-clamp techniques, under as a result showing that ripe motor neuron is co-cultured with Skeletal Muscle solencyte, can form nerve
Neuromuscular junction structure simultaneously arranges skeletal muscle rhythm and pace of moving things contraction (Figure 15,16,17).
Based on the above, the present invention has been successfully established the people iPS cells training for being independent of nourishing coating systems, serum-free interference
The system of supporting；People's iPS cells of culture in the system, after the micromolecular compound combination after use in conjunction optimization, can with 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 set up motor neuron disease spy
Idioblas model provides good application prospect.
Embodiment
Material and method
Material source：
Skin histology sample takes 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,
The U.S.)；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 (contain 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 with DMEM 1 time, 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, are coated with 6 holes
Plate, 1ml/ holes, in incubator overnight.Cultivate the 7th day, metainfective fibroblast is digested.DMEM/F12 washings 3
It is secondary, 0.25% pancreas enzyme -EDTA 0.5ml/ holes after preheating are added, digest 1 minute at room temperature, it is then centrifuged for 1200rpm, 1.5 points
Clock.Finally, complete medium is resuspended with 1:5 ratio renewed vaccinations are to vitronectin (DMEM/F12 1:100 dilutions) overnight it is coated with
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
The monoclonal that reprogramming is obtained, contrast is seeded to is coated with vitronectin (DMEM/F12 1 in advance respectively:100 dilutions, 37 DEG C,
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 density long about 70-85%.Under room temperature environment, 0.5mM EDTA digestion iPSCs 1 minute, DMEM/F12 is washed
Wash 1 time, blown and beaten from parietal cell with E8 culture mediums, with 1:In 10 ratio renewed vaccination cells to the treated orifice plate of coating, 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 daily time that Continuous Observation clone occurs under inverted microscope and the metamorphosis of amplification cultivation process.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 shows：
It is calculated as the 0th day (Day 0) with infecting the viral same day.Virus first 2 days (Day-2) of infection, SF patch
Wall grows into elongated fibrous form (Fig. 1 .A).After virus infection 7 days (Day 7), there is significant change, projection in 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 amplification clone with vitronectin as somatomedin, culture visible is closely arranged on the 1st day in many cells
Row growth (Fig. 2 .A), as incubation time extends, clone uniformly gradually increases, 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 with 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 the clone for obtaining 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 the SSEA4 (Fig. 4 .B) and the positive that endonuclear NANOG (Fig. 4 .A), positive signal are located at cell membrane
Signal is located at cytoplasmic TRA-1-60 (Fig. 4 .C).
To sum up result shows that we successfully reprogram and have obtained the whole people's iPS cells for being independent of nourishing coating systems, and
Establish a kind of optimization culture scheme with 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.
Induction of committed differentiation from the people iPS cells of embodiment 2 to dynamoneure, identification and differentiation efficiency count
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 from wall, collected to 15ml centrifuge tubes
(Guangzhou Jie Te biofiltrations Products Co., Ltd, China), is centrifuged 1000 revs/min, 1 minute.Using 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 adding different micromolecular compound combination DMH1+SB431542+CHIR99021 or LDN193189+SB431542+
CHIR99021, 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 and change liquid, with above-mentioned differential medium.Induction differentiation the 2nd day, full dose changes liquid, training used
Support base and micromolecular compound ibid, it is extra again in addition to add RA+SAG or RA+Purmorphamine various combination compounds,
To promote to form it into neural spherical structure.Induction differentiation the 4th day, full dose changes liquid, and used medium ibid, now only adds RA+SAG
Or the various combination compound of RA+Purmorphamine；The afterwards next day, full dose changed liquid.Induction differentiation the 9th day, collects differentiation
Neural ball, is centrifuged 1000 revs/min, 1 minute, supernatant discarded；Add the Accutase digestive juices of 1ml/ pipes, 37 DEG C of 8 points of water-baths
Clock；Add 8ml DMEM/F12 to terminate digestion, 1000 revs/min, 1 minute, supernatant discarded is centrifuged；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 inoculation in upper strata to used poly-ornithine (0.1mg/ml),
Laminin (40ug/ml) and matrigel (1：50 dilutions) the treated slide of coating 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
Addition micromolecular compound DAPT, to promote the maturation of motor neuron, until culture to 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 from wall with 0.5mM and suspension is incubated at dress
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 the culture dish (Corning companies, the U.S.) for treating in matrigel, 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 adding small molecule Purmorphamine (1 μM) again.When induction is broken up the 25th day,
Accutase digestion suspends and clones and be seeded to again used poly-ornithine, laminin and matrigel coating treated
Adhere-wall culture in slide.
Identification：
The metamorphosis of the daily Continuous Observation clone under inverted microscope.When induction is broken up the 12nd day, terminated,
It is fixed with fresh 4% paraformaldehyde.The 12nd day spinal cord is transported after detecting induction with immunofluorescence dyeing method to above-mentioned cell
The expression of dynamic neuron HB9 and ISLET1 (the motor neuron Specific marker after mitosis).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, as a result one-way analysis of variance is 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) of addition 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, 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), 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
Immunofluorescence dyeing of the born of the same parents in induction differentiation the 12nd day, it is seen that positive particle signal is respectively positioned in nucleus, referring to Fig. 6-9.D-
F.Wherein, Fig. 6 .D-F displays DP group HB9 and ISLET1 immunofluorescence positive cells；Fig. 7 .D-F display 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 is obtained
Dynamoneure rate highest, and there is not the segregation phenomenon (table 1) of motor neuron mark HB9 and ISLET1.
In addition, being 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 shows that present inventor establishes a kind of safe, reliable, efficient dynamoneure and lures
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：
C57BL/6 Strains of Mouse after taking-up is raw in 1 day, after 75% alcohol sufficiently sterilised, quickly takes 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), with 0.25% pancreas enzyme -EDTA in digestion 10 minutes under 37 DEG C of water-baths.DMEM complete mediums (contain 20%FBS)
Terminate digestion, gently blow and beat for several times, be made single cell suspension.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 is removing other cellular layers and the cell fragment on astroglia
Deng, renew fresh DMEM complete mediums (containing 20%FBS), continue to cultivate 3-5 days.When its stand density about 80-90%,
Carry out had digestive transfer culture.DMEM culture mediums are washed 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, is 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 that will cultivate the 3rd generation after expanding is thin
Born of the same parents are with 5x 104Cell/cm2Density is seeded to and 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 are washed 3 times, and are added
Enter fresh Neuronal induction differential medium (comprising 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 lures
Lead differential medium.
Induction the 9th day neural ball of (Day 9) of differentiation 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 addition micromolecular compound DAPT (10 μM) simultaneously.Every 3 days,
Half amount changes fresh culture once, untill induction differentiation the 31st day.
Dynamoneure is co-cultured with Skeletal Muscle solencyte：
After by the induction differentiation of short duration digestion of neural ball of (Day 9) in the 9th day, 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 adding micromolecular compound DAPT (10 μM).Per 2-3 days, half amount was more
Fresh culture is changed once, untill co-culturing the 14th day.It is simultaneously right as feminine gender with independent inoculation myotubes in culture dish
According to using identical cultivating system.
Identification：
The daily metamorphosis of star spongiocyte after Continuous Observation Secondary Culture under inverted microscope, induction differentiation come
After the dynamoneure growing state and dynamoneure in source form neuromuscular junction with myotubes, flesh is arranged
The contraction situation of pipe.Above-mentioned cell detects marker expression situation using immunofluorescence dyeing.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 ripe dynamoneure and the skeletal muscle for shrinking
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 closely (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 be located at Synaptophys in and the positive signal of cell membrane and be 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 Whole-cell recording induction differentiation the 31st day.Culture there is into fortune
The slide of dynamic neuron is as the outer liquid (Mm of 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 (with 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 shows：Under current-clamp mode, generation can be induced after injecting step galvanism
Continuously, the action potential of bunchiness, and with the increase of Injection Current, the frequency of action potential granting is consequently increased (figure
14.B)；Under voltage-clamp mode, the sodium channel electricity for producing quick activation-inactivation type can be induced after step voltage stimulates through injecting
Stream (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 arranges the record of Skeletal Muscle solencyte：Dynamoneure and Skeletal Muscle
After solencyte is co-cultured 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 giving full cell to the myotubes for shrinking
Patch-clamp techniques.System is recorded with neuron, and parameter uses gap-free (not giving any exogenous stimulation).Result shows can be remembered
The autonomous continuous action potential (Figure 17) provided is recorded, the formation in the presence of neural joint design is further demonstrate.
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 without the interference of the compositions such as external source serum indefinite material
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, for the screening for effectively treating drug candidate from now on provides theoretical foundation；Even can
It is applied to cellular transplantation therapy from now on.
The above has been described many specific embodiments of the invention, but the present invention should not be construed as limited to
These specific embodiments.It should be understood that unless otherwise indicated, any or all of example provided here or typical
Term does 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 implementation method for being not limited to specifically describe, and only limited by the scope of claim.
Claims (10)
1. a kind of method that safe and reliable reprogramming obtains people's iPS cells, it is characterised in that mainly comprise the following steps：Minimally invasive acquisition fell
Skin fibroblast, after the sendai virus infection of factor hOct3/4, hSox2, hKlf4 and hc-Myc is reprogrammed through carrying, in
Cultivated 7 days in DMEM complete mediums；It is resuspended with complete medium again, and with 1:5 ratio renewed vaccinations to vitronectin is coated with
Culture 24 hours in treated orifice plate；E8 stem cell medias are replaced by afterwards, and carrying out full dose changes liquid daily from next day,
Until iPS cells can be obtained after 2 weeks.
2. the method that the reprogramming as described in claim 1 obtains people's iPS cells, it is characterised in that early stage picking iPS cell list
Clone, being seeded to be coated with advance carries out amplification cultivation in the orifice plate of vitronectin or matrigel.
3. it is a kind of by iPS cells simplicity rapidly and efficiently inducing method of the differentiation as dynamoneure, it is characterised in that should
Method is mainly comprised the following steps：
(1) the people iPS cell dissociations to 70%-85% degrees of fusion will be cultivated and suspends to be incubated at and contain micromolecular compound combination
2 days in the Neuronal induction differential medium of I.
(2) culture 2 that suspends is continued in cell being moved into the Neuronal induction differential medium containing micromolecular compound combination II
My god.
(3) by cell move to containing micromolecular compound combination III Neuronal induction differential medium in sustained suspension culture 5
My god.
(4) by cell dissociation adhere-wall culture 3 days, micromolecular compound is only added in Neuronal induction differential medium around here
DAPT。
4. a kind of as described in claim 3 by the simplicity rapidly and efficiently inducing differentiation of iPS cells as dynamoneure
Method, it is characterised in that the Neuronal induction differential medium of the step (1) is blending constituent, and 1 is contained respectively：1 DMEM/
F12 and Neurobasal culture mediums, 0.5x B27 additives, 0.5x N2 additives, 1x glutamine and 1x non-essential aminos
Acid.
5. a kind of as described in claim 3 by the simplicity rapidly and efficiently inducing differentiation of iPS cells as dynamoneure
Method, it is characterised in that the micromolecular compound that the step (1) is added combines I (concentration) and is：DMH1(2μM)+
SB431542 (2 μM)+CHIR99021 (3 μM) or LDN193189 (0.3 μM)+SB431542 (2 μM)+CHIR99021 (3 μM).
6. a kind of as described in claim 3 by the simplicity rapidly and efficiently inducing differentiation of iPS cells as dynamoneure
Method, it is characterised in that the micromolecular compound combination of extra addition in the Neuronal induction differential medium of the step (2)
II (concentration) is as follows：Micromolecular compound combines I+RA (0.1 μM)+SAG (0.5 μM) or micromolecular compound combination I+RA (0.1
μM)+Purmorphamine(1μM)。
7. a kind of as described in claim 3 by the simplicity rapidly and efficiently inducing differentiation of iPS cells as dynamoneure
Method, it is characterised in that the micromolecular compound combination of extra addition in the Neuronal induction differential medium of the step (3)
III (concentration) is as follows：RA (0.1 μM)+SAG (0.5 μM) or RA (0.1 μM)+Purmorphamine (1 μM).
8. a kind of method that iPS cell simple and effectives are induced differentiation into dynamoneure as described in claim 3, its
It is characterised by, first by Accutase enzymic digestions in the step (4), then neuron is seeded to is coated with poly bird ammonia
On the treated slide of acid, laminin and matrigel coating；After cell attachment is complete, addition contains micromolecular compound
The Neuronal induction differential medium of DAPT.
9. a kind of acquisition methods of cost-effective feature dynamoneure, it is characterised in that mainly comprise the following steps：To lure
Lead the differentiation neuron of the 9th day to be digested, inoculate to being coated with poly-ornithine, laminin and matrigel glass in advance
On piece, the culture medium based on star spongiocyte conditioned medium, and addition micromolecular compound DAPT (10 μM) simultaneously；
Every 3 days, half amount changed fresh culture once, untill induction differentiation the 31st day.
10. acquisition methods of a kind of cost-effective feature dynamoneure as described in claim 9, its feature exists
It is in the preparation process of the astroglia conditioned medium：Using DMEM complete medium cultures astroglia 3
My god, fresh Neuronal induction differential medium is added, it is collected daily afterwards, and change fresh Neuronal induction point
Change culture medium.
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