CN107858328A - A kind of neural crest pedigree mesenchymal cell and its method of inducing differentiation from multipotential stem cell - Google Patents
A kind of neural crest pedigree mesenchymal cell and its method of inducing differentiation from multipotential stem cell Download PDFInfo
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- CN107858328A CN107858328A CN201711129757.0A CN201711129757A CN107858328A CN 107858328 A CN107858328 A CN 107858328A CN 201711129757 A CN201711129757 A CN 201711129757A CN 107858328 A CN107858328 A CN 107858328A
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Abstract
The invention discloses a kind of neural crest pedigree mescenchymal stem cell and its method of inducing differentiation from multipotential stem cell, it is that dissociation multipotential stem cell is prepared into cell mass, suspend culture, multipotential stem cell is set to form embryoid body, nutrient solution is replaced by neural crest cell nutrient solution, suspend culture embryoid body, collects idiosome daily and changes nutrient solution；Make embryoid body adherent growth certain time after culture certain time；Collection cell is digested again；The cell that adhere-wall culture is collected, and nutrient solution is replaced by Mesenchymal stem cell nutrient solution, the cell phenotype of mesenchyma is detected after Secondary Culture, is produced.The present invention solves the mescenchymal stem cell heterogeneity of adult origin's mescenchymal stem cell and other non-limiting ways of regeneration multipotential stem cell sources and the shortcomings that mixing, and the neural crest pedigree mescenchymal stem cell of acquisition has stronger immunological regulation and Osteoblast Differentiation ability；The cell mass that standardization induction differentiation program can ensure to obtain between different batches has good uniformity.
Description
Technical field
The invention belongs to biology and medical domain, more particularly it relates to a kind of nerve from multipotential stem cell
Ridge pedigree mescenchymal stem cell and preparation method thereof, including the use of special condition of culture and selection technique.The invention further relates to
Treatment method or its clinical practice using this group of neural crest pedigree mescenchymal stem cell.
Background technology
Mescenchymal stem cell (MSCs) belongs to one kind of adult stem cell, is many tissues such as marrow, navel in adult body
All existing major class pluripotent cell in band blood, fat and peripheral blood etc., remain static when normal, when the tissue at place
Or organ, when sustaining damage, they can quickly enter vegetative state and keep great multiplication potentiality, and be divided into different
Cell type is to repair damaged tissues.A kind of MSCs found at first is mesenchymal stem cells MSCs (Friedenstein A
J,J Embryol Exp Morphol,1966,16(3):381-90), it can be under different inductive conditions to osteocyte, soft
The directions such as osteocyte, adipocyte, muscle cell, nerve cell, hematopoiesis support cell break up, and have typical stem cell special
Point.
It is considered as organizational project earliest because MSCs has the advantages that to be easily isolated amplification and has multi-lineage potential
Preferable seed cell and turn into study hotspot.Being subsequently found MSCs has powerful immunoregulation capability, further promotes
Research related MSCs, becomes and is most hopeful to realize one of star's cell of clinicization.But MSCs is difficult to scale expansion
Increase and heterogeneity is the current two big obstacles for limiting the application of MSCs clinicizations.
In view of the extensive sources of MSCs and heterogeneity, international in order to which experiment related specification MSCs and clinical treatment are studied
Mescenchymal stem cell treats committee ISCT in three standards for proposing identification of M SCs in 2006：(1) cell can be adhered to modeling
Expect culture vessel；(2) cell expresses specific surface antigen, and wherein CD44, CD73, CD90, STRO-1 and CD105/SH2 are positive
Rate>95%, CD11, CD14, CD34 and CD45 positive rates<2%；(3) multi-lineage potential of cell, you can soft to be divided into
Osteocyte, osteocyte and adipocyte etc..
Even if the cell in culture complies fully with three standards that ISCT is proposed, the differences of MSCs functionally are also suitable
Substantially, the difference that MSCs shows each side such as multiplication capacity, differentiation capability is directly resulted in.Registered on ClinicalTrials
MSC clinical researches in significant proportion all have to give up the study of because of unstable result and count out, cause this otherness
As a result the main reason for and clinical effectiveness is bad is that MSCs is heterogeneous population body, Different Individual, different tissue sources, even
The MSCs that same tissue extraction arrives is to include the heterogeneous populations of a variety of hypotypes, the MSCs Various Functions of different subtype.
Anokhina is by by the generation of MSCs amplification in vitros 50, comparing MSCs heterogeneity, finding the increase with passage number, MSCs
Heterogeneity declined, Different Individual MSCs expression identical surface marker, illustrate that amplification in vitro can be screened optionally
MSCs subgroups, but MSCs differentiation function then generates larger difference.These results of study show long-term or a large amount of external
The selection pressure that amplification is added in MSCs colonies will greatly influence the composition, differentiation performance and therapeutic efficiency of MSCs populations, lead
Cause different or even opposite results.Even if the change that MSCs Surface Phenotype not can detect, MSCs is in lasting succeeding generations
In can also lose its versatility, or specifically filter out the bad hypotype of performance.Therefore, different subgroup MSCs are distinguished, and then
The MSCs subgroups to be played a crucial role in related application are filtered out, it is at present by MSCs clinical practices to eliminate its heterogeneous influence
One of key technology.
To solve the problems, such as MSCs source and amplification, many researchs are directed to ES and iPS being induced to differentiate into MSCs.According to
Document report, Chunhui Xu in 2004 are earliest by transfecting human telomerase reverse transcriptase
(hTERT) hESCs of the gene into breaking up induces ESC to break up to HEF directions, and obtained cell has the ability of Osteoblast Differentiation.But
It does not carry out MSC detailed identification.Subsequent Tiziano Barberi by co-culturing 40d with mouse OP9cell line,
With the addition of in 20%FBS alpha MEM nutrient solutions induces ESC to break up to MSC, airflow classification CD73+ cells, is considered to be
The report that first induced multi-potent stem cell is broken up to MSC.
Co-culture within 05 year to obtain MSC seminar, the induction for reporting non-co-cultivation in 08 year by OP9 cells：People ESCs
(H1, H7 and H9 cell line) is incubated in the orifice plate for being coated with Matrigel, and in order to induce it to break up to MSC, liquid week is changed in increase
Phase is 3-5d.After 9-10d, 40%-50% cells show goes out into fiber-like form, mechanical removal ESC cells, collagenase digesting
The cell of differentiation is simultaneously accessed in the coated new wares of Matrigel.Fibroblast further increases, and scrapes off for the second time undifferentiated thin
Born of the same parents, pancreatin digest and accessed in the coated ware of gelatin, cultivated in the aMEM for added 10%FBS, and the time of about 4-6 weeks can be with complete
Into induction.
The ES sources MSC that Lian reports a kind of clinical grade in 2007, it digests ESC by pancreatin, with containing bFGF and
PDGF KSR culture mediums are cultivated in the coated culture dish of gelatin, cover with rear pancreatin had digestive transfer culture, after one week with CD105+ with
CD24- sorts to obtain MSC cells.
Existing patent also describes the method that interstitial cell is divided into from iPS, such as (the patent application publication of JieLong Co., Ltd of the U.S.
Number CN101696397A), the country pay off tinkling of pieces of jade (patent application publication CN105754936A) etc. all describe it is a kind of from more competent
Cell differentiation is the method for interstitial cell.
But the cell that the method for these documents or patent report obtains does not control differentiation path, only focuses on solution
MSCs's carrys out source problem, and the heterogeneity for cell does not provide corresponding solution, therefore induces the cell of gained may ratio
The mesenchyma of derived from bone marrow is more heterogeneous；Meanwhile these schemes are using the function of cell as high spot reviews object, and treat effect
The bad product of fruit inherently greatly limits its application.
The content of the invention
Based on this, the defects of in order to overcome above-mentioned prior art, the invention provides a kind of god from multipotential stem cell
Through ridge pedigree mescenchymal stem cell and its abductive approach.
In order to realize foregoing invention purpose, this invention takes following technical scheme：
A kind of method of inducing differentiation of the neural crest pedigree mescenchymal stem cell from multipotential stem cell, including following step
Suddenly：
(1) external adhere-wall culture multipotential stem cell, keeps its undifferentiated state；
(2) after cell amplification is to requirement in (1), cell is prepared into cell mass, suspend culture, makes more competent
Cell forms embryoid body；
(3) nutrient solution is replaced by neural crest cell nutrient solution, suspend culture embryoid body；Daily or 2~4 days change once
Nutrient solution；
(4) suspend after cultivating 4~10 days, adhere-wall culture embryoid body, daily or 2~4 days change a nutrient solution；
(5) after growing 2~10 days, with EDTA or collected by trypsinisation cell；
(6) cell that adhere-wall culture is collected, and nutrient solution is replaced by Mesenchymal stem cell nutrient solution, Secondary Culture 2~6
The cell phenotype of detection mesenchyma, is produced after secondary；
In wherein some embodiments, the cell number in step (1) contained by each embryoid body is 2 × 102~2 ×
106。
In wherein some embodiments, the usable dissociation reagent such as Dispase of step (2) described cell mass,
It is prepared by Accutase or EDTA；The operative temperature for dissociating reagent is 30~37 DEG C.
In wherein some embodiments, step (2) described cell mass can also be used machinery to draw the method scraped and prepare.
In wherein some embodiments, step (3) the neural crest cell nutrient solution includes following components： DMEM/F12:
20-80%, Neuralbasal:20-80%.
In wherein some embodiments, step (3) the neural crest cell nutrient solution also including following components one kind or
It is several：N2:0.1-2%, B27:0.1-2%, L-Glu:0.1-5%, 2-Mercaptoethanol: 0.01mM-1mM、SB
431542:0.01mM-1mM、BIO:0.1-10ng/ml、bFGF:2-50ng/ml、 EGF:2-50ng/ml、Penicillin-
Streptomycin:50-200U/ml。
In wherein some embodiments, step (4) described adhere-wall culture is：Embryoid body is transferred to coating
The surface of the matrigel such as Matrigel or laminin is to promote the adherent of embryoid body.
In wherein some embodiments, step (5) is also including the use of P75 and the cell of HNK1 antibody labelings collection, then lead to
Overflow-type sorts the cell of the double sun of P75 and HNK1.
In wherein some embodiments, step (6) described Mesenchymal stem cell nutrient solution is that with the addition of 5% to 20% blood
Clear DMEM complete culture solutions, or commercialized serum-free complete culture solution.
Present invention also offers the neural crest spectrum from multipotential stem cell obtained by the induction differentiation of above-mentioned method of inducing differentiation
It is mescenchymal stem cell.
A kind of the defects of present invention induces obtained mescenchymal stem cell for prior art, there is provided mesenchyma of optimization
Stem cell subgroup, to strengthen the cytology of cell or treatment function.Present invention simultaneously provides mescenchymal stem cell cell subset
Acquisition methods.Compared with prior art, the invention has the advantages that：
(1) present invention is neural crest cell by first inducing multipotential stem cell, and further induction is dry thin for mesenchyma
Born of the same parents, can solve adult origin's mescenchymal stem cell and the mescenchymal stem cell in other non-limiting ways of regeneration multipotential stem cell sources
Heterogeneity and the shortcomings that mixing, can solve the problems, such as that source for mesenchymal stem cells is limited, the neural crest pedigree mesenchyma of acquisition is done
Cell has stronger immunological regulation and Osteoblast Differentiation ability；
(2) present invention induces differentiation program by standardizing, it is ensured that the cell mass obtained between different batches has good
Uniformity.
Brief description of the drawings
Fig. 1 is the pluripotency marker's immunofluorescence photograph for the iPS cells that embodiment 1 is cultivated；
Fig. 2 is suspended in embodiment 1 culture and the embryoid body of adhere-wall culture；
Fig. 3 is the sorting figure of neural crest cell in embodiment 1 and the cellular morphology figure after sorting；
Fig. 4 be embodiment 1 in neural crest cell passed on 4 times in mesenchyma nutrient solution after cellular morphology figure and cell table
Face marks flow cytometer showed figure；
Fig. 5 be obtained in test example 1 of the present invention neural crest pedigree mescenchymal stem cell skeletonization, into fat into cartilage differentiation
Figure；
Fig. 6 is the suppression that neural crest pedigree mescenchymal stem cell is secreted to T cell inflammatory factor TNFalpha in test example 2 of the present invention
Making flow cytometer showed figure.
Embodiment
In order to facilitate the understanding of the purposes, features and advantages of the present invention, below in conjunction with the accompanying drawings to the present invention
Embodiment be described in detail.Many details are elaborated in the following description in order to fully understand this hair
It is bright.But the invention can be embodied in many other ways as described herein, those skilled in the art can be not
Similar improvement is done in the case of running counter to intension of the present invention, therefore the present invention is not limited to the specific embodiments disclosed below.Below
Involved technology in embodiment, it is cell biology well known to those skilled in the art, bioid unless stated otherwise
The routine techniques of the every field such as, molecular biology.
The method of inducing differentiation of neural crest pedigree mescenchymal stem cell of the embodiment 1 from multipotential stem cell
The method of inducing differentiation of the neural crest pedigree mescenchymal stem cell from multipotential stem cell of the present embodiment, including with
Lower step：
First, external adhere-wall culture multipotential stem cell, keeps its undifferentiated state；
Multipotential stem cell, including embryonic stem cell and induced multi-potent stem cell can be conveniently by commercialization or this experiments
Room structure obtains.The further culture that the present embodiment focuses on to be set forth on the basis of the multipotential stem cell system of stable foundation is grasped with differentiation
Make.
Multipotential stem cell can on a large scale expand under conditions of without feeder layer and keep undifferentiated state.Nutrient solution can be selected
The mTeSR series of products of STEMCELL companies, E8 etc., with above-mentioned several nutrient solutions be adapted, it is necessary to be wrapped in nutrient solution matter
By matrigel, such as Matrigel or laminin.The multipotential stem cell for ensureing high quality is the key for carrying out next one-step inducing.
Based on mTeSR1 and Matrigel cultivating systems, specific culture operation is as follows：
1st, the coating of culture dish：Melt Matrigel, DMEM/F12 dilutions Matrigel to 1 on ice:20 to 1:200, add
It is standby to enter room temperature coating more than 1h in orifice plate.
2nd, cryopreservation tube is taken out from liquid nitrogen, the quick-thawing cell line in 37 C water baths, is transferred to added with 5ml
In the 15ml centrifuge tubes of mTeSR1 complete culture solutions, 300g centrifugations 5min collects cell.
3rd, coated Matrigel is sucked, cell is resuspended with 2ml mTeSR1, it is good into coating to be equably inoculated with cell
In orifice plate, 37 degrees Celsius, 5%CO are put into2Adhere-wall culture is stood with the incubator of 95% humidity.
4th, nutrient solution is changed daily, thoroughly sucks old nutrient solution, is added fresh preheated nutrient solution, is continued to train
Support, until size of the cell clone length to suitable passage.
5th, before passage, cell is washed twice using PBS, add 0.5mM EDTA be incubated in 37 degrees Celsius 1 to
10min, Microscopic observation, cell dissociation is set to suck EDTA into small cell mass, beat cell so that PBS is gently soft, ensure cell
Maintain small lumps；
6th, transfer cell mass enters in 15ml centrifuge tubes, and 300g centrifugations 5min collects cell.
7th, cell is resuspended with mTeSR1, with 1：6 or other suitable ratio uniforms cell is inoculated with into the good hole of coating
In plate, 37 degrees Celsius, 5%CO are put into2Adhere-wall culture is stood with the incubator of 95% humidity.
8 persistently pass on to obtain the induction that enough cell concentrations carry out next step.
Pluripotency marker's immunofluorescence photograph of the iPS cells of culture is as shown in Figure 1.
2nd, the preparation and culture of embryoid body
After cell amplification is to requirement in (one), dissociated cell is prepared into cell mass, and suspend culture, makes more competent
Cell forms embryoid body, wherein, the cell number contained by each embryoid body is 2 × 102~2 × 106；
1st, cell is washed twice using PBS, the EDTA for adding 0.5mM is incubated 1 to 10min in 37 degrees Celsius, Jing Xiaguan
Examine, cell dissociation is sucked EDTA into small cell mass, is beaten cell so that PBS is gently soft, ensure that cell maintains small lumps.
2nd, transfer cell mass enters in 15ml centrifuge tubes, and 50g centrifugations 1min collects cell mass.
3rd, with neural crest cell nutrient solution (49%DMEM/F12,49%Neuralbasal, 0.5%N2,0.5%B27,
1%L-Glu, 0.1mM 2-Mercaptoethanol, 0.1mM SB 431542,2ng/ml BIO, 5ng/ml bFGF, 5ng/
Ml EGF) cell mass is resuspended, with 1：1 to 1：Cell is inoculated with low absorption orifice plate or culture dish 3 ratio uniform.It is put into
37 degrees Celsius, 5%CO2Cultivated with standing to suspend in the incubator of 95% humidity.
4th, suspend culture second day, and cell mass will spontaneously form embryoid body, and embryoid body is transferred into 15ml using suction pipe
In centrifuge tube, 50g centrifugations 1min collects embryoid body.
5th, with neural crest cell nutrient solution be resuspended embryoid body, be inoculated with it is low absorption orifice plate or culture dish in 37 degrees Celsius, 5%
CO2Cultivated with standing to suspend in the incubator of 95% humidity.
6th, the culture embryoid body 5d that suspends is stood, during which daily or 2 to 3d changes liquid once, repeat step 4~5 when changing liquid.
7th, during embryoid body suspension culture 6d, embryoid body is transferred in 15ml centrifuge tubes using suction pipe, 50g centrifugations
1min collects embryoid body, and embryoid body is resuspended with neural crest cell nutrient solution, is inoculated with into the culture dish for being coated with Matrigel
In, in 37 degrees Celsius, 5%CO2With quiescent culture in the incubator of 95% humidity, make embryoid body adherent.
8th, the embryoid body of adhere-wall culture is daily or 2~3d changes liquid once.
3rd, nutrient solution is replaced by neural crest cell nutrient solution, culture dish is no longer coated with matrigel, or is replaced by ultralow suction
Attached culture dish, suspend culture embryoid body, collects idiosome daily and changes nutrient solution；
1st, after embryoid body adhere-wall culture 5d, wash cell twice using PBS, 0.05% pancreatin is added, in 37 degrees Celsius
0.5 to 3min is incubated, Microscopic observation, is added when cell starts to shrink at and stops to disappear with the neural crest cell nutrient solution of pancreatin equivalent
Change, beat cell so that PBS is gently soft, transfer cell enters in 15ml centrifuge tubes, and 250g centrifugations 5min collects cell.
2nd, cell is resuspended with 0.1 to the 1ml PBS containing 1%BSA, be divided into two parts, it is a to be added in right amount according to antibody specification
P75 and HNK1 antibody, another adds IgG antibody, and 30min is incubated at 4 degrees Celsius.
3rd, the cell that PBS washings have been incubated antibody is added, supernatant is abandoned in 250g centrifugations 5min.
4th, cell is resuspended in PBS containing 1%BSA, and the cell marked IgG is used as positive control, airflow classification P75 and
The double positive cells of HNK1, so as to isolate neural crest cell.
The culture that suspends is as shown in Figure 2 with the embryoid body of adhere-wall culture.The sorting figure of neural crest cell and the cell after sorting
Form is as shown in Figure 3.
4th, induction differentiation of the neural crest cell to mescenchymal stem cell
1st, neural crest cell nutrient solution resuspension neural crest cell, with 5~10 × 104/cm2Density be inoculated in coating
In the orifice plate of matrigel, in 37 degrees Celsius, 5%CO2Adhere-wall culture is stood with the incubator of 95% humidity.
2nd, second day, it was Mesenchymal stem cell nutrient solution (such as LDMEM+10% tires ox blood to change neural crest cell nutrient solution
Clearly), in 37 degrees Celsius, 5%CO2Liquid is changed with quiescent culture, every 2~3d in the incubator of 95% humidity.
3rd, after cell growth to 80~90% fusions, with pancreatin had digestive transfer culture, it is inoculated with into common tissue culture treated mistake
Tissue Culture Dish, stand adhere-wall culture；Take the antibody such as a part of cell detection CD44, CD73, CD90, CD34, CD45 simultaneously
Expression.
4th, repeat step 3, until expression CD44, CD73, CD90 cell reach more than 95%；Express CD34, CD45
Cell is less than 5%, produces neural crest pedigree mescenchymal stem cell.
Neural crest cell passed on 4 times in mesenchyma nutrient solution after cellular morphology and cell surface marker flow cytometer showed such as
Shown in Fig. 4.
The skeletonization of the neural crest pedigree mescenchymal stem cell of test example 1 breaks up into fat into chondrocyte induction
1st, Osteoblast Differentiation：After neural crest pedigree growth of mesenchymal stem cells to 80~90% fusions, nutrient solution is changed into
Self-bone grafting differentiation nutrient solution (the μ g/ml vitamin Cs of L-DMEM+10%FBS+10mM β-glycerophosphate+ 50 and 100nM
Dexamethasone).Liquid is changed per 3d once, Alizarin red staining analysis is carried out after Fiber differentiation 14d, as shown in figure 5, dyeing is visible bright
Aobvious calcium tubercle.
2nd, break up into fat：After neural crest pedigree growth of mesenchymal stem cells to 80~90% fusions, nutrient solution is changed into
Fat induction differentiation nutrient solution (H-DMEM+10%FBS+100nM Indo+0.5mM IBMX and 1mM dexamethasone).Changed per 3d
Liquid once, carries out oil red O stain analysis after Fiber differentiation 21d, as shown in figure 5, the visible obvious fat drips of dyeing.
3rd, into cartilage differentiation：Neural crest pedigree mescenchymal stem cell is collected in digestion, with 2.5 × 105/ 15ml centrifuge tubes it is thin
Born of the same parents' amount is tapped into 15ml centrifuge tubes, 200 × g centrifugation 5min, is abandoned supernatant, is added 1ml cartilage differentiation induction broths (H-DMEM
+ 10ng/ml rhTGF-BETAs (recombinant human transforming growth factor- β 3,
TGF-β 3), 100nM dexamethasone, 50 μ g/ml vitamin Cs, 1mM Sodium Pyruvates, 40 μ g/ml proline and ITS+premix).
Liquid is changed per 3d once, the staining analysis of A Li Xinlan is carried out after Fiber differentiation 21d, as shown in figure 5, the visible obvious osamine of dyeing
Glycan colours.
The inhibitory action detection that the neural crest pedigree mescenchymal stem cell of test example 2 is secreted to T cell inflammatory factor TNFalpha
Comprise the following steps：
1st, MSCs is resuspended in MSCs nutrient solutions, with 4 × 104/cm2Density be inoculated with orifice plate.
2nd, CD3+T cells are resuspended with 1640+10%FBS nutrient solutions.
3rd, with 1：T cell vaccination is entered to have cultivated in MSCs hole by 5 ratio, in 37 degrees Celsius, 5% CO2It is wet with 95%
Quiescent culture 3d in the incubator of degree.
4th, after 3d, stimulant processing 6h is added.
5th, after 6h, the T cell for shifting suspension enters 15ml centrifuge tubes, and 500g centrifugations 10min collects cell.
6th, cell is resuspended with 100 μ l PBS, the PFA room temperatures for adding 100 μ l 4% fix cell 20min.
7th, add saponin(e after the cell that PBS washings fix to penetrate, while add TNF α antibody at room temperature and be incubated 15min.
8th, the antibody that PBS washings have marked, cell is finally resuspended with PBS and carries out flow cytometer detection.
As a result as shown in fig. 6, result shows that neural crest pedigree mescenchymal stem cell carefully has compared with the mesenchyma of derived from bone marrow
Stronger inflammatory factor inhibitory action.
Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality
Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, the scope that this specification is recorded all is considered to be.
Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously
Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art
Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
- A kind of 1. method of inducing differentiation of the neural crest pedigree mescenchymal stem cell from multipotential stem cell, it is characterised in that bag Include following steps：(1) external adhere-wall culture multipotential stem cell, keeps its undifferentiated state；(2) after cell amplification is to requirement in (1), cell is prepared into cell mass, suspended culture cell agglomerate, made more Can stem cell formation embryoid body；(3) nutrient solution is replaced by neural crest cell nutrient solution, suspend culture embryoid body；Daily or replacing in 2~4 days is once cultivated Liquid；(4) suspend after cultivating 4~10 days, adhere-wall culture embryoid body, daily or 2~4 days change a nutrient solution；(5) adhere-wall culture is after 2~10 days, with EDTA or collected by trypsinisation cell；(6) cell that adhere-wall culture is collected, and nutrient solution is replaced by Mesenchymal stem cell nutrient solution, after Secondary Culture 2~6 times The cell phenotype of mesenchyma is detected, is produced.
- 2. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that the cell number in step (2) contained by each embryoid body is 2 × 102~2 × 106。
- 3. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that step (2) cell mass is prepared using dissociation reagent；It is described dissociation reagent be Dispase, Accutase or EDTA；The operative temperature of the dissociation reagent is 30~37 DEG C.
- 4. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that step (2) described cell mass is to draw the method scraped using machinery to be prepared.
- 5. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that step (3) the neural crest cell nutrient solution includes following components：DMEM/F12:20-80%, Neuralbasal:20-80%.
- 6. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that step (3) the neural crest cell nutrient solution also includes the one or more of following components：N2:0.1- 2%th, B27:0.1-2%, L-Glu:0.1-5%, 2-Mercaptoethanol:0.01mM-1mM、SB 431542:0.01mM- 1mM、BIO:0.1-10ng/ml、bFGF:2-50ng/ml、EGF:2-50ng/ml、Penicillin-Streptomycin:50- 200U/ml。
- 7. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that adhere-wall culture is described in step (4)：Embryoid body is transferred to and has been coated with Matrigel or layer adhesion egg The surface of white matrigel, promote the adherent of embryoid body.
- 8. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that the cell that step (5) is also collected including the use of P75 and HNK1 antibody labelings, then pass through airflow classification P75 With the cell of the double sun of HNK1.
- 9. the induction differentiation side of the neural crest pedigree mescenchymal stem cell according to claim 1 from multipotential stem cell Method, it is characterised in that step (6) described Mesenchymal stem cell nutrient solution is that the DMEM that with the addition of 5% to 20% serum is trained completely Nutrient solution or commercialized serum-free complete culture solution.
- 10. the god from multipotential stem cell as obtained by the method for inducing differentiation induction differentiation described in any one of claim 1~9 Through ridge pedigree mescenchymal stem cell.
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