CN106520687A - Method for differentiating induced pluripotent stem cells into mesenchymal stem cells - Google Patents
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Abstract
The invention discloses a method for differentiating induced pluripotent stem cells (iPSC) into mesenchymal stem cells (MSC). The method includes the steps that the induced pluripotent stem cells formed through induction are dissociated into single cells; the single cells are cultivated in a cell plate coated with I type collagen through an iPSC medium and one differential medium including platelet-derived growth factors, and then cultivated in another differential medium to obtain MSC-like cells; and finally, the MSC-like cells are maturated in an MSC medium to obtain the MSC. According to the induction approach, the iPSC is directly differentiated into the MSC, and the method has the beneficial effects of being short in period, high in efficiency, safe, stable and the like.
Description
Technical field
The present invention relates to one kind is by induced multi-potent stem cell（induced pluripotent stem cells）It is divided into
The method of mescenchymal stem cell (mesenchymal stem cells).
Background technology
Mescenchymal stem cell（MSC）From mesoderm, it is a kind of cell with self and multi-lineage potential,
Initially find in marrow, found in the tissue such as umbilical cord, placenta, fat and muscle in succession later.Due to MSC have it is amplifiable
Property, Multidirectional Differentiation and low immunogenicity, also controllable immune response and paracrine function, therefore be considered as to manage in organizational project
The seed cell thought and a kind of approach of clinical cytology replacement therapy.However, the tissue-derived complexity of MSC so as to growth rate, point
Secrete cell factor spectrum and the biological characteristics such as immunoregulation capability has difference, the feasibility extracted in addition and extractible quantity
The problems such as cause the Quality Control to cell more complicated.Therefore unified MSC is extremely important to obtain identical, quality of originating.
Induced multi-potent stem cell（iPSC）It is to be imported by adult cell Jing transcription factors (Oct4, Sox2, Klf4 and c-Myc)
Reprogramming and come with the cell with embryonic stem cell identity function, not only possess self and multi-lineage potential, also
The problems such as overcoming the ethics and immunogenicity of embryonic stem cell presence, becomes research people and comes disease incidence mechanism, histocyte
The important cells source of replacement therapy.Using iPSC as derived cell, which can be expanded in vitro and be induced to differentiate into specific
Histocyte.
At present, the method for being induced to differentiate into MSC from ESC and iPSC is a lot, with embryoid body（Embryoid body, EB) method
Based on, be primarily present Induction Process complicated, time length and it is inefficient the shortcomings of.
The content of the invention
It is an object of the invention to provide a kind of method that induced multi-potent stem cell breaks up mescenchymal stem cell, can solve to pass
The not high shortcoming of system method Induction Process complexity, induction time length, induced efficiency, is to provide unified source, enough, of fine quality
MSC lays the first stone.
The method that the present invention establishes a simplicity, efficiently induction human pluripotent stem cells are divided into mescenchymal stem cell, no
Need Jing embryoid bodies to be formed, iPSC is induced to differentiate into into MSC directly, its scheme is that iPSC is first dissociated into unicellular, subsequently use
Differential medium induces which to be divided into mesenchymal stem cells, and further culture makes which ripe, comprises the following steps：
（1）Prepare iPS single cell suspensions.
IPSC is dissociated into resuspended after individual cells.
Wherein can be using the mTeSR nutrient solution re-suspended cells containing ROCK inhibitor.
（2）Induction differentiation.
Single cell suspension is inoculated on Tissue Culture Plate, is cultivated using differential medium B, then be replaced by differentiation training
Foster base A.
Cell differential medium B under the conditions of 37 DEG C, 5%CO2 cultivates 24 ~ 48h；Subsequently differential medium A continue culture 8 ~
12d。
The Tissue Culture Plate is I-type collagen coated cell culture plate.
（3）Induced maturation.
The MSC-Like cells for being formed are digested using 0.25% pancreatin, MSC nutrient solution re-suspended cells are inoculated in new I types
In the coated cell plates of collagen, cultivated using MSC nutrient solutions.
Add the culture medium of %FBS, GlutaMAX-I and nonessential amino acid based on the MSC nutrient solutions in culture medium.
It is a further object to provide a kind of induced multi-potent stem cell is divided into the differentiation training of mescenchymal stem cell
Foster base.
Such as above-mentioned step（1）It is middle that iPSC is dissociated into into dissociation solution used by individual cells for Accutase, the temperature of digestion iPSC
Spend for 37 DEG C, the time is 7min.
Such as above-mentioned step（2）Described in, the differential medium A is to add platelet derived growth factor in basal medium
（PDGF AB）, dexamethasone and 10% hyclone（FBS）Culture medium；The differential medium B be differential medium A and
The mixed liquor of mTeSR1 culture mediums；The Tissue Culture Plate is I-type collagen coated cell culture plate.
As mentioned above in differential medium A PDGF AB final concentration of 0.5 ~ 20ng/ml, dexamethasone it is final concentration of
50~150Mm;Final concentration of 30% ~ 70% (volumn concentration) of mTeSR1 culture mediums in the differential medium B.
Preferably, in above-mentioned differential medium A PDGF AB final concentration of 5ng/ml, dexamethasone it is final concentration of
100nM.Final concentration of the 50% of mTeSR1 in preferably above-mentioned differential medium B.
Such as above-mentioned step（2）In, the basal medium in the differential medium A be DMEM, α-MEM, DMEM/F12, it is excellent
Selection of land basal medium is α-MEM.
Such as above-mentioned step（2）Condition of culture be 37 DEG C culture 24 ~ 48h, preferably 48h, above-mentioned steps（3）Middle culture bar
Part 37 DEG C of cultures 8 ~ 12d, preferably 10d.
Description of the drawings
Fig. 1 is induced to differentiate into the Technology Roadmap of MSC for iPSC.
Fig. 2 is iPSC and MSC aspect graphs.Left figure is iPS colony light field figures, and right figure is to induce MSC under the white light after breaking up
Middle Scale bar=100 μm.
Fig. 3 is induced to differentiate into the relative expression quantity of related gene during MSC for iPSC.
Fig. 4 is the MSC surface markers that Flow cytometry is induced differentiation by iPSC.
Fig. 5 is that iPSC induces the MSC of differentiation growth curve in vitro in incubation, is grown in " S " type.
Fig. 6 be by iPSC induce differentiation MSC skeletonization, into fat and cartilage differentiation poststaining qualification figure.A is induction differentiation
Oil red O stain for lipoblast is identified；B is the alizarin red S dyeing identification of the lipoblast of induction differentiation；C is induction differentiation
Cartilage cell's alcian blue dyeing identification.Wherein multiplication factor be 100 ×.
Fig. 7 be induction differentiation MSC skeletonization, into expression of specific gene detection figure after fat and cartilage differentiation.
Specific embodiment
The invention discloses a kind of method that induced multi-potent stem cell breaks up mescenchymal stem cell, with reference to being embodied as
Example is further described to the present invention, but protection scope of the present invention is not limited thereto, any to be familiar with the art
Technical staff the invention discloses technical scope in, technology according to the present invention scheme and its inventive concept are equal to replace
Change or change, should all be included within the scope of the present invention.
In following embodiments, experimental technique used if no special instructions, is conventional method, used in following embodiments
Material, reagent etc., if no special instructions, commercially obtain.
The embodiment of the present invention is said to the situation of MSC inductions to making one iPSC using differential medium cultural method
It is bright.
（1）The test course schematic diagram of the present invention（Fig. 1）.
（2）The FLA that MSC is sorted for flow cytometry is as follows：
The anti-human surface recognition molecule CD29 antibody of fluorescein PE marks（SC59829, Santa Cruz）
The anti-human surface recognition molecule CD44 antibody of fluorescein APC marks（170441, Affymetrix eBioscience）
The anti-human surface recognition molecule CD34 antibody of fluorescein FITC marks（110349, Affymetrix eBioscience）
The anti-human surface recognition molecule CD90 antibody of fluorescein PerCP-Cyanine5.5 marks（450909, Affymetrix
eBioscience ）
The isotype control Ab of fluorescein PE marks（Sc2867, Santa Cruz）
The isotype control Ab of fluorescein APC marks（174031, Affymetrix eBioscience）
The isotype control Ab of fluorescein FITC marks（114714, Affymetrix eBioscience）
The isotype control Ab of fluorescein PerCP-Cyanine5.5 marks（454714, Affymetrix eBioscience）
（3）The primer sequence detected for qPCR and RT-PCR is as follows：
Embodiment 1 induces differentiation of the people iPSC to MSC
1. the culture of people iPS cells
In the present embodiment, the people's iPS cells for using（DYR0100, Chinese Academy of Sciences's cell bank）Culture Matrigel (BD,
354277), on, with mTeSRTM1, (05850) STEMCELL TECHNOLOGIES maintain culture, as shown in Figure 2 a.It is concrete to cultivate
Method is comprised the following steps：
1. culture medium and required reagent are taken out from refrigerator, in 37 DEG C of water-baths, preheats 15min or so；
2. cell is taken out, former culture medium is abandoned in suction, with DPBS (no calcium, no magnesium)（Invitrogen,
14190250) cell is washed into one time；
3. 1mg/ml clostridiopetidase A IV are added（It is dissolved in HBSS buffer solutions, 0.22 μm of filtration sterilization）（Invitrogen
17104019）Digested, be placed in 37 DEG C of cell culture incubators and be incubated 5min or so, examine under a microscope, clone edge volume
Rise；
4. inhale and abandon clostridiopetidase A IV, after being washed with DPBS, add appropriate mTeSRTM1 culture mediums；
5. cell clone is scraped from culture dish bottom with cell scraper or sterile glass tube, is transferred to centrifuge tube, gently blow and beat 5
Cell clone is made for ~ 10 times to be changed into the more uniform small cell cluster of size；
6. take out in advance with the coated Tissue Culture Dish of Matrigel, small cell cluster suspension is even added in cell ware, is put
Cultivate in 37 DEG C of cell culture incubators.Fresh culture is changed daily, is passed on 1 time within general 5 ~ 7 days, but cell clone mistake ought occur
It is big or overstocked and then need to be processed in time when there is Spontaneous Differentiation.
People iPS cells break up to the induction of MSC
Differential medium A：In basal medium α-MEM culture mediums（Gibco）Middle addition platelet derived growth factor（PDGF AB）
（PeproTech, AF10014B）And dexamethasone（Sigma, D4902）The culture medium for obtaining.In differential medium A, PDGF AB
Final concentration of 5ng/ml, the final concentration of 100nM of dexamethasone.
Differential medium B：The culture medium that differential medium A and mTeSRTM1 are mixed to get, wherein mTeSRTM1 culture mediums
Final concentration of 50%（Volumn concentration）.
Induction people iPS cells break up to MSC, comprise the following steps：
1. it is dissociated into unicellular：The iPSC of culture is taken, original fluid is abandoned in suction, is washed 1 time using DPBS, adds Accutase
(Invitrogen, A1110501), processes 5 ~ 10min at 37 DEG C, examines under a microscope, iuntercellular between cell rounding, colony
Terminate digestion when gap becomes big, cell is started shedding off, add appropriate α-MEM culture mediums, blow and beat re-suspended cell, 1000rmp centrifugations
5min, abandons supernatant, using differential medium B re-suspended cells；
2. induction differentiation：Take out the pre-coated Tissue Culture Plate of I-type collagen（greiner bio-one）Preheat in room temperature, will
IPS single cell suspensions are added to differential medium B used in Tissue Culture Plate and are cultivated, and are replaced by differential medium A after 48h
Cultured cells, changed fresh culture per 2 ~ 3 days, and culture is until 10 days；
3. airflow classification：The MSC-Like cells for obtaining are digested to using 0.25%Trypsin-EDTA (Gibco) single thin
Born of the same parents, it is resuspended after addition 1mlPBS, density is adjusted after counting to cell, using the streaming antibody (SANTA of anti-CD29 and CD44
CRUZ after) marking cell, using flow cytometer（BD FACS AriaII）First IgG negative control group cell suspensions are flowed
Formula loading, selects negative fluorescence signal region as negative control, collects fluorescence intensity higher than more than 10 times of negative control group
CD29+/CD44+ double positive cells.
4. the amplification of CD29+/CD44+ double positive cells：Cell is inoculated in into new cell plates by 5 × 104/cm2 density
In, add appropriate MSC SFM（Gibco, A1033201）Culture medium is cultivated to cell, as shown in Fig. 2 it is more equal to obtain form
One MSC.
The detection that 2 people iPS cells of embodiment break up to mesenchymal cell
（1）Expression of the related gene in induction atomization
Trizol（Invitrogen）Method extracting iPS inductions 0,3,6,9,12d cells and the total serum IgE of iPS-MSC-P2,
Using M-MLV the first chain synthesis systems（Invitrogen）Reverse transcription is cDNA and SYBR Premix Ex Taq
（Takara）Carry out the expression of Realtime PCR detection Nanog, Oct4, CD73, CD105 and reference gene actin（Figure
3）.With the prolongation of induction time, the expression of versatility gene Nanog and Oct4 declines, and the expression of CD73 and CD105
Gradually rise, MSC-P2 only expresses a small amount of Nnaog and Oct4, and CD73 and CD105 almost 100% are expressed.
（2）MSC surfaces Marker is identified
The MSC cells that will be obtained（P2）Single cell suspension is digested to, fluorescently-labeled mouse monoclonal anti-human's antibody is separately added into
CD29, CD34, CD44 and CD90 and homotype Negative control mice anti-mouse IgG monoclonal antibody are simultaneously mixed, 4 DEG C of lucifuges incubations
1% bovine serum albumin(BSA) (BSA) being added after 30min, being mixed, 1000rmp centrifugation 5min abandon supernatant, add appropriate 1%BSA, in stream
Carry out detecting corresponding antigens expression on formula cell instrument, shown in Fig. 4, CD29, CD44 and CD90 expression sun of the MSC of acquisition
Property, positive rate is respectively 100%, 98.62% and 99.1%, and CD34 expression is negative.
The measure of the growth curve of the MSC of 3 people's iPS cell differentiations of embodiment
The MSC (P5) for taking people's iPS cell differentiations is digested to single cell suspension, is laid on 24 by the density of 5 × 103/cm2 after counting
Cultivated in the cell of hole, taken 3 hole cell counts respectively after inoculation daily.Fig. 5 understands, is come by people iPS cell differentiations
MSC is grown in " S " type in incubation, and 2 ~ 5d is exponential phase, enters plateau within the 6th day, and surface is by iPS cell differentiations
MSC can carry out self and propagation well in incubation in vitro.
The three of the MSC of 4 people's iPS cell differentiations of embodiment are the identification of differentiation capability
Take the MSC for cultivating to the 5th generation to be come by people iPS cell differentiations, be respectively adopted mescenchymal stem cell skeletonization, into fat and into
Cartilage differentiation culture medium（Biowit）Cultivated, liquid was changed per 2 ~ 3 days.
Treat that culture, to 2 ~ 3 weeks, carries out alizarin red, oil to the Gegenbaur's cell after induction, lipoblast and cartilage cell respectively
Red O and alcian blue dyeing identification, and PPAR- γ, Osteopontin, ALP, Aggrecan and Sox9 is detected with RT-PCR method
The expression of gene.
Shown in Fig. 6, a is the oil red O stain identification for being induced to differentiate into lipoblast；B is the lipoblast of induction differentiation
Alizarin red S dyeing identification；C is cartilage cell's alcian blue dyeing identification of induction differentiation, is the positive.To lipoblast specificity
Gene PPAR- γ, Osteoblast Specific gene Osteopontin and ALP and cartilage cell specific gene Aggrecan
RT-PCR detections with Sox9 are positive expression（Fig. 7）.Show there is skeletonization by the MSC of people's iPS cell differentiations, into fat and soft
Bone differentiation capability.
Claims (9)
1. a kind of method that induced multi-potent stem cell is divided into mescenchymal stem cell, it is characterised in that methods described include for
Induction people's induced multi-potent stem cell is divided into Derived from Mesenchymal Stem Cells culture medium, and the differential medium includes differential medium
A and two kinds of differential medium B.
2. according to claim 1, it is characterised in that wherein described differential medium A is to add blood in basal medium
Platelet derivative factor（PDGF AB）, dexamethasone and 10% hyclone（FBS）Culture medium.
3. according to claim 1, it is characterised in that wherein described differential medium B is mTeSR1 culture mediums and differentiation training
The mixed liquor of foster base A.
4. according to claim 2, the basal medium in wherein described differential medium A is DMEM, α-MEM, DMEM/
F12, it is preferable that basal medium is α-MEM.
5. according to claim 2, in the differential medium A PDGF AB final concentration of 0.5 ~ 20ng/ml, ground plug rice
Final concentration of 50 ~ the 150mM of pine, it is preferable that the final concentration of 5ng/ml of PDGF AB, the end of dexamethasone in differential medium A
Concentration is 100nM.
6. according to claim 3, in the differential medium B mTeSR1 culture mediums final concentration of 30% ~ 70% (volume hundred
Divide content), it is preferable that final concentration of the 50% of wherein mTeSR1.
7. a kind of method that induced multi-potent stem cell is divided into mescenchymal stem cell, it is characterised in that will using claim 1-6
Described in asking for culture medium induced multi-potent stem cell from induced multi-potent stem cell to Derived from Mesenchymal Stem Cells to mesenchyma
Stem cell breaks up, and the method comprises the steps:
（1）IPSC is dissociated into into single disseminated cell；
（2）Make step（1）The cell of middle acquisition is cultivated in proceeding to differential medium B；
（3）Cultivate in proceeding to differential medium A again；
（4）Finally cultivated using mescenchymal stem cell culture medium, obtained ripe mescenchymal stem cell.
8. according to claim 7, described induced multi-potent stem cell kind behaviour, monkey, rat, mouse, ox, rabbit, pig etc..
9. according to claim 7, the step（1）It is middle iPSC is dissociated into into dissociation solution used by individual cells to be
Accutase。
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| WO2019144606A1 (en) * | 2018-01-26 | 2019-08-01 | 皓昇莱生物制药有限公司 | Screening and differentiating method from hpscs to mscs |
| CN110628712A (en) * | 2019-09-27 | 2019-12-31 | 南京市妇幼保健院 | Preparation method and application of therapeutic interstage mesenchymal stem cells based on induced pluripotent stem cells |
| CN112048470A (en) * | 2020-09-17 | 2020-12-08 | 深圳丹伦基因科技有限公司 | Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells |
| EP3828263A1 (en) | 2019-11-27 | 2021-06-02 | Shanghai East Hospital (East Hospital Affiliated to Tongji University) | Method and kit for preparing clinical-grade mesenchymal stem cells derived from human induced pluripotent stem cells |
| CN114438037A (en) * | 2022-01-25 | 2022-05-06 | 深圳市乐土生物医药有限公司 | Method for preparing inductive mesenchymal stem cells |
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019144606A1 (en) * | 2018-01-26 | 2019-08-01 | 皓昇莱生物制药有限公司 | Screening and differentiating method from hpscs to mscs |
| CN114787340A (en) * | 2019-07-26 | 2022-07-22 | 布瑞克斯奥根株式会社 | Mesenchymal stem cell precursor cell for inducing pluripotent stem cell source and preparation method thereof |
| CN110628712A (en) * | 2019-09-27 | 2019-12-31 | 南京市妇幼保健院 | Preparation method and application of therapeutic interstage mesenchymal stem cells based on induced pluripotent stem cells |
| CN110628712B (en) * | 2019-09-27 | 2023-11-24 | 南京市妇幼保健院 | Preparation method and application of therapeutic interstage mesenchymal stem cells based on induced pluripotent stem cells |
| EP3828263A1 (en) | 2019-11-27 | 2021-06-02 | Shanghai East Hospital (East Hospital Affiliated to Tongji University) | Method and kit for preparing clinical-grade mesenchymal stem cells derived from human induced pluripotent stem cells |
| CN112048470A (en) * | 2020-09-17 | 2020-12-08 | 深圳丹伦基因科技有限公司 | Method for preparing clinical-grade mesenchymal stem cell preparation by utilizing human induced pluripotent stem cells |
| CN112048470B (en) * | 2020-09-17 | 2023-05-12 | 深圳丹伦基因科技有限公司 | Method for preparing clinical grade mesenchymal stem cell preparation by using human induced pluripotent stem cells |
| CN114438037A (en) * | 2022-01-25 | 2022-05-06 | 深圳市乐土生物医药有限公司 | Method for preparing inductive mesenchymal stem cells |
| CN117683712A (en) * | 2024-02-02 | 2024-03-12 | 深圳市北科生物科技有限公司 | Three-dimensional induction method for obtaining mesenchymal stem cells by differentiation of pluripotent stem cells |
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