CN107083367A - Culture medium and application thereof and the method that mescenchymal stem cell is prepared by urine cell - Google Patents

Culture medium and application thereof and the method that mescenchymal stem cell is prepared by urine cell Download PDF

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CN107083367A
CN107083367A CN201710533052.9A CN201710533052A CN107083367A CN 107083367 A CN107083367 A CN 107083367A CN 201710533052 A CN201710533052 A CN 201710533052A CN 107083367 A CN107083367 A CN 107083367A
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stem cell
cell
mescenchymal stem
culture
embryoid body
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CN107083367B (en
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黄燕飞
车七石
赵澎
刘少辉
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Guangzhou Rainhome Pharm and Tech Co Ltd
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Guangzhou Rainhome Pharm and Tech Co Ltd
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Abstract

The present invention relates to stem cell and gene engineering technology field, more particularly to the preparation method of culture medium and application thereof with mescenchymal stem cell.The culture medium that the present invention is provided can induce urine cell to turn into the method for mescenchymal stem cell, and urine cell derived extensively, is not limited by ethics, and the method provided using the present invention, urine cell sequentially forms IPS cell embryoid body mescenchymal stem cells.All induction can be completed in 20~25 days, and the cell obtained meets the characteristic of mescenchymal stem cell after testing, and quantity is larger, and motility rate is higher.

Description

Culture medium and application thereof and the method that mescenchymal stem cell is prepared by urine cell
Technical field
The present invention relates to stem cell and gene engineering technology field, more particularly to culture medium and application thereof is dry thin with mesenchyma The preparation method of born of the same parents.
Background technology
With the rise of organizational project and clinical transplantation medical science, the research of seed cell is more and more concerned.Mesenchyma is done Cell (MSC, mesenchymal stem cells) be a class be present in Various Tissues (such as marrow, Cord blood and umbilical cord tissue, Placenta tissue, adipose tissue etc.) multipotential stem cell.It has hyperproliferation, the ability of self-renewing, and with Multidirectional Differentiation Potential.The thin of cartilage, bone, skeletal muscle, tendon, fat, corium, nerve and kidney essence can be divided under different inductive conditions Born of the same parents etc..Therefore, mescenchymal stem cell is often used as seed cell.
The clinical research of mescenchymal stem cell is carried out in many countries, and the U.S. have approved 60 remainder clinical tests, with The increasingly mature of mescenchymal stem cell and its correlation technique, China also have approved multinomial clinical test, entered into mesenchyma and done The stage of cell core technical research.At present, clinical test is confirmed, MSCs suppresses available for tissue repair and treatment mesenchyma group Genetic defect disease is knitted, for example：Disease in the blood system, angiocardiopathy, hepatic sclerosis, the nervous system disease, knee joint first quarter moon Plate part cuts off injury repair, autoimmune disease etc..The characteristic of mescenchymal stem cell determines that it has good clinic should Use prospect.
At present, the main source of mescenchymal stem cell is marrow and umbilical cord tissue, and mesenchymal stem cells MSCs is with the age Aging, stem cell population is significantly reduced, Proliferation, Differentiation ability significantly fails, and preparation process is not easy Quality Control, transplants to different Body may cause immune response and larger to human injury.Though umbilical cord mesenchymal stem cells immunity is relatively low but the guarantor of Cord blood Deposit costly.And the quantity of other tissue-derived stem cells is then very rare, separation and culture are also very difficult.Cause This, further the preparation method of research mescenchymal stem cell is still the focus of the current area research.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide culture medium and application thereof and mescenchymal stem cell Preparation method.Urine cell can be reprogrammed into IPS cells by the culture medium that the present invention is provided, then be by ips cell inductions Embryoid body and then directed differentiation are mescenchymal stem cell.
The embryoid body inducing culture that the present invention is provided include basal medium and：
In some embodiments, embryoid body inducing culture is made up of basal medium and following components：
FBS is hyclone, and BMP-4 is bone morphogenic protein -4；VEGF is VEGF, in the present invention, FBS, Glu, beta -mercaptoethanol, BMP-4, VEGF and insulin are added in basal medium, the culture medium can be induced IPS cells are converted to embryoid body.The basal medium is DMEM culture mediums.
The embryoid body culture medium that the present invention is provided include basal medium and：
In some embodiments, embryoid body culture medium is made up of basal medium and following components：
ITS is insulin transferrins sodium selenite, and EGF is EGF, and the present invention adds in basal medium Plus FBS, ITS, Sodium Pyruvate, EGF and Glu, beta -mercaptoethanol, gained culture medium can be used in the culture of embryoid body. The basal medium is DMEM culture mediums.
The mescenchymal stem cell inducing culture that the present invention is provided include basal medium and：
In some embodiments, mescenchymal stem cell induction is made up of basal medium and following components：
BFGF is basic fibroblast growth factor, and the present invention is in basal medium, addition FBS, insulin, L- paddy Embryoid body can be induced to differentiate into mescenchymal stem cell by glutamine, bFGF and dexamethasone, gained culture medium.The basis training It is DMEM culture mediums to support base.
The mescenchymal stem cell culture medium that the present invention is provided include basal medium and：
In some embodiments, mescenchymal stem cell is made up of basal medium and following components：
HGF be HGF, PDGF be platelet derived growth factor, TGF-β is transforming growth factor-β.The present invention FBS, EGF, HGF, bFGF, PDGF and TGF-β are added into basal medium, gained culture medium can be used in mescenchymal stem cell Culture.The basal medium is DMEM culture mediums.
Present invention also offers a kind of method of induction IPS cells formation embryoid body, this method is to induce to train with embryoid body Support base induction IPS cells；The time of the induction is 3~10 days.
In some embodiments, the time of induction is 7~10 days.Inductive condition is 37 DEG C, saturated humidity, 5%CO2.Induction The density of inoculation is 1 × 104~0.5 × 104cell/mL.The induction is using mass fraction as 0.1%~0.02% agarose Carried out in coated culture plate.In the incubation, liquid is changed daily.
Experiment shows, IPS cells are induced in this way, and 3~4d begins with embryoid body appearance, culture 7~10d embryoid bodies Increment reaches top.
Present invention also offers a kind of method for inducing embryoid body to be divided into mescenchymal stem cell, this method is：By embryoid Body is inoculated in after embryoid body culture medium, 10~14h of culture to fill between mescenchymal stem cell inducing culture induction acquisition in 5~7 days Matter stem cell.
In the present invention, with embryoid body medium culture with progress on the coated culture plate of gelatin.Condition of culture is 37 DEG C, Saturated humidity, 5%CO2.Embryoid body is adherent after 10~14h, changes mescenchymal stem cell and carries out Fiber differentiation.After induction 5~7 days Mescenchymal stem cell is formed.
Expression conditions, the table of NAONG and OCT-4 in mescenchymal stem cell in RT-PCR detection mescenchymal stem cells Hardly expressed up to amount, illustrate that the mescenchymal stem cell of induction loses the expression of multipotency dryness gene, mescenchymal stem cell category It is that expression quantity of the mesoderm gene in mescenchymal stem cell significantly increases in mesoderm MSX-1, meets mescenchymal stem cell Feature.By flow cytometry analysis showed, CD29, CD44, CD73, CD90, CD105 are expressed as sun in mescenchymal stem cell Property, and CD34, CD45 are expressed as feminine gender, meet the expression of results of mescenchymal stem cell surface marker.
Present invention also offers a kind of method that mescenchymal stem cell is prepared by urine cell, this method includes following step Suddenly：
Transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 are imported into urine cell, it is thin that culture obtains IPS Born of the same parents；
IPS cells are induced with embryoid body inducing culture, after 3~10 days, embryoid body are collected with embryoid body medium culture After 10~14h, induced 5~7 days with mescenchymal stem cell inducing culture, obtain mescenchymal stem cell.
In the present invention, obtaining also includes the step of propagation, passage after mescenchymal stem cell, between the propagation, passage are used Mesenchymal stem cell media.
In the present invention, the preparation method of urine cell is：Urine is centrifuged, after precipitation is sterilized, to contain Primocin's REGM medium cultures, obtain urine cell.
In the present invention, the specific preparation method of urine cell is：
Step 1：Collect urine in the container dual anti-containing penicillin/streptomycin, 400g centrifugation 10min, precipitation with containing Have after PBS (adding 5mL penicillin/streptomycins per 95mL PBS) cleaning once of penicillin/streptomycin, be transferred to through 0.1% The coated culture plate of gelatin；
Step 2：REGM culture mediums (adding 3 μ LPrimocin per 2mLREGM culture mediums) culture containing Primocin, training The condition of supporting is 37 DEG C, saturated humidity, 5%CO2；After after cell attachment, culture medium is sucked, is washed with PBS one time, then change at liquid Reason；When urine cell fusion degree reaches 80%, Secondary Culture is carried out.
Described transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 are imported after urine cell, and cell is with REGM With MEF mixed culture medium (volume ratio 1:1) cultivated on the culture plate after being coated with gelatin；Import transcription regulatory factor The 2nd day afterwards, culture medium is replaced by IPS culture medium mTesR, fresh culture is changed daily, make clone continue to breed；Import and turn Record after regulatory factor the 7th day, the Microscopic observation form picking monoclonal close with human embryo stem cell, and be inoculated in and use gelatin bag The culture plate of quilt, culture medium mTesR cultures obtain induced multi-potent stem cell (IPS cells).
IPS cells with 0.03%-0.3% clostridiopetidase As by cell dissociation into after individual cells, induced synthesis embryoid body, then Induced synthesis mescenchymal stem cell again.
The time of the induction of the IPS inductions as embryoid body is 7~10 days.Inductive condition is 37 DEG C, saturated humidity, 5%CO2.The density of challenge inoculation is 1 × 104~0.5 × 104cell/mL.The induction using mass fraction as 0.1%~ Carried out in the coated culture plate of 0.02% agarose.In the incubation, liquid is changed daily.
With embryoid body medium culture with progress on the coated culture plate of gelatin.Condition of culture is 37 DEG C, saturated humidity, 5%CO2.Embryoid body is adherent after 10~14h, changes mescenchymal stem cell and carries out Fiber differentiation.Mesenchyma is done after inducing 5~7 days Cell is formed.
Urine cell is induced to turn into the side of mescenchymal stem cell the invention provides culture medium and using these culture mediums Method, urine cell derived extensively, is not limited by ethics, the method provided using the present invention, and it is thin that urine cell sequentially forms IPS Born of the same parents-embryoid body-mescenchymal stem cell.All induction can be completed in 20~25 days, between the cell obtained meets after testing The characteristic of mesenchymal stem cells, quantity is larger, and motility rate is higher.
Brief description of the drawings
Fig. 1 shows that embodiment 1 prepares the expression conditions of mescenchymal stem cell；
Fig. 2 shows that embodiment 1 prepares the surface markers antigen detection case of mescenchymal stem cell.
Embodiment
The invention provides the preparation method of culture medium and application thereof and mescenchymal stem cell, those skilled in the art can be with Present disclosure is used for reference, technological parameter realization is suitably modified.In particular, all similar replacements and change are to ability It is it will be apparent that they are considered as being included in the present invention for field technique personnel.The method of the present invention and application have been led to Cross preferred embodiment to be described, related personnel substantially can be not departing from present invention, in spirit and scope to this paper's Methods and applications are modified or suitably change is with combining, to realize and apply the technology of the present invention.
The examination material that the present invention is used is all common commercially available product, can all be bought in market.
With reference to embodiment, the present invention is expanded on further：
Embodiment 1
1st, the preparation of culture medium
IPS inducing cultures (configuration culture medium A)：REGM and MEF culture mediums by volume (1:1) ratio mixing.
Embryoid body inducing culture (configuration culture medium B)：450mLDMEM, 50mL FBS, 5mML- glutamine, 0.1mM Beta -mercaptoethanol, 50ng/mL BMP-4,25ng/mLVEGF, 60pM insulin.
Embryoid body culture medium (configuration culture medium C)：400mLDMEM, 100mL FBS, 5ug/ml insulin transferrins are sub- Sodium selenate (ITS), 0.23mM Sodium Pyruvates, 1ng/ml EGFs (EGF), 2mML- glutamine, 0.1mM β-sulfydryl Ethanol.
Mescenchymal stem cell inducing culture (configuration culture medium D)：450mLDMEM basal mediums, 50mLFBS, 60pM Insulin, 2mML- glutamine, 75ug/LbFGF, 3 × 10-8mol/L dexamethasone.
Mescenchymal stem cell culture medium (configuration culture medium E)：450mLDMEM basal mediums, 50mLFBS, 10ng/mL hEGF、10ng/mL bFGF、3ng/mL HGF、5ng/mLPDGF、5ng/mLTGF-β。
2nd, the acquisition of urine cell
1) collection cups each add 2mL penicillin/streptomycins dual anti-.
2) urine is collected, if do not carried out subsequent operation at once, then urine 4 DEG C of refrigerators is stored in and completed follow-up in the same day Operation.
3) every part of urine prepares the hole of six orifice plate 1, and this hole is coated with into more than 20min with 0.1% gelatin, sucked before use in hole Liquid.
4) urine is poured into appropriate number of 50mL centrifuge tubes, centrifuges 400g, 10min.
5) supernatant is sucked, often pipe leaves about 1-5mL, be mixed into a centrifuge tube.
6) PBS (PBS 95mL add 5mL penicillin/streptomycins and mixed) about 10- containing penicillin/streptomycin is added 30mL.Gently mix.
7) 400g, 10min are centrifuged.
8) supernatant is sucked to remaining 0.5-1mL liquid.
9) remaining liquid is added in the hole being coated with, adds 2mLREGM culture mediums and add 3 μ LPrimocin.
10) culture dish is positioned in 37 DEG C of incubators and cultivated, after (7 days) after urine cell attachment, suck culture medium, used PBS is washed one time, then carries out changing liquid processing.
11) when urine cell fusion degree reaches 80%, then Secondary Culture can be carried out.
3rd, the reprogramming of urine cell is ips cells
1) various groups of transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 or other transcription factor will be expressed Amount to urine cell is imported, cell point is cultivated to advance with coated 6 orifice plates of matrigel with nutrient solution A；
2) the 2nd day after transfecting, culture medium is replaced by IPS culture medium mTesR, fresh culture is changed daily；Make clone Continue to breed
3) the 7th day after transfecting, the Microscopic observation form picking monoclonal close with human embryo stem cell, and it is inoculated in use In coated 12 orifice plates of matrigel, culture medium mTesR cultures obtain induced multi-potent stem cell.
4th, Ips cell inductions are embryoid body
1) by with the ips induced multi-potent stem cells 0.03%-0.3% clostridiopetidase As IV that gets ready by cell dissociation into single thin Born of the same parents, collect cell and are washed with PBS three times, and the suspension that 1 × 104-0.5 × 104 are made with nutrient solution B progress suspensions is standby.
2) Tissue Culture Plate 0.1%-0.02% agar glycolyx 0.5-1h.
3) prepared cell suspension in 1) is added with the good culture plate of agar glycolyx, carrying out changing at liquid daily Reason.
4) 3-4d to be cultivated begins with embryoid body appearance, and the increment of culture 7-10d embryoid bodies reaches top, collects and intend Idiosome suspension, is staticly settled, and suspension is carried out with nutrient solution C standby.
5th, embryoid body directed differentiation is mescenchymal stem cell
1) by the embryoid body suspension inoculation prepared into the culture plate being coated with by matrigel.
2) embryoid body 10-14h is cultivated, treats that its adherent addition nutrient solution D carries out Fiber differentiation.
3) filling stem cell between culture 5-7d will form, and change nutrient solution E and continue to cultivate, continue to propagation.
Embodiment 2
1st, the preparation of culture medium
IPS inducing cultures (configuration culture medium A)：REGM and MEF culture mediums by volume (1:1) ratio mixing.
Embryoid body inducing culture (configuration culture medium B)：450mL MEM, 50mL FBS, 1mM- glutamine, 0.05mM Beta -mercaptoethanol, 10ng/mL BMP-4,1ng/mLVEGF, 1pM insulin.
Embryoid body culture medium (configuration culture medium C)：400mLDMEM, 100mL FBS, 1ug/ml insulin transferrins are sub- Sodium selenate (ITS), 0.1mM Sodium Pyruvates, 0.2ng/ml EGFs (EGF), 1mML- glutamine, 0.01mM β-mercapto Base ethanol.
Mescenchymal stem cell inducing culture (configuration culture medium D)：450mLDMEM basal mediums, 50mLFBS, 1pM pancreas Island element, 1mML- glutamine, 50ug/LbFGF, 2 × 10-8mol/L dexamethasone.
Mescenchymal stem cell culture medium (configuration culture medium E)：450mLDMEM basal mediums, 50mLFBS, 1ng/mL hEGF、1ng/mL HGF、1ng/mL bFGF、2ng/mLPDGF、1ng/mLTGF-β。
2nd, the acquisition of urine cell
1) collection cups each add 2mL penicillin/streptomycins dual anti-.
2) urine is collected, if do not carried out subsequent operation at once, then urine 4 DEG C of refrigerators is stored in and completed follow-up in the same day Operation.
3) every part of urine prepares the hole of six orifice plate 1, and this hole is coated with into more than 20min with 0.1% gelatin, sucked before use in hole Liquid.
4) urine is poured into appropriate number of 50mL centrifuge tubes, centrifuges 400g, 10min.
5) supernatant is sucked, often pipe leaves about 1-5mL, be mixed into a centrifuge tube.
6) PBS (PBS 95mL add 5mL penicillin/streptomycins and mixed) about 10- containing penicillin/streptomycin is added 30mL.Gently mix.
7) 400g, 10min are centrifuged.
8) supernatant is sucked to remaining 0.5-1mL liquid.
9) remaining liquid is added in the hole being coated with, adds 2mLREGM culture mediums and add 3 μ LPrimocin.
10) culture dish is positioned in 37 DEG C of incubators and cultivated, after (7 days) after urine cell attachment, suck culture medium, used PBS is washed one time, then carries out changing liquid processing.
11) when urine cell fusion degree reaches 80%, then Secondary Culture can be carried out.
3rd, the reprogramming of urine cell is ips cells
1) various groups of transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 or other transcription factor will be expressed Amount to urine cell is imported, cell point is cultivated to advance with coated 6 orifice plates of matrigel with nutrient solution A；
2) the 2nd day after transfecting, culture medium is replaced by IPS culture medium mTesR, fresh culture is changed daily；Make clone Continue to breed
3) the 7th day after transfecting, the Microscopic observation form picking monoclonal close with human embryo stem cell, and it is inoculated in use In coated 12 orifice plates of matrigel, culture medium mTesR cultures obtain induced multi-potent stem cell.
4th, Ips cell inductions are embryoid body
1) by with the ips induced multi-potent stem cells 0.03%-0.3% clostridiopetidase As IV that gets ready by cell dissociation into single thin Born of the same parents, collect cell and are washed with PBS three times, and carrying out suspension with nutrient solution B is made 1 × 104-0.5×104Suspension it is standby.
2) Tissue Culture Plate 0.1%-0.02% agar glycolyx 0.5-1h.
3) prepared cell suspension in 1) is added with the good culture plate of agar glycolyx, carrying out changing at liquid daily Reason.
4) 3-4d to be cultivated begins with embryoid body appearance, and the increment of culture 7-10d embryoid bodies reaches top, collects and intend Idiosome suspension, is staticly settled, and suspension is carried out with nutrient solution C standby.
5th, embryoid body directed differentiation is mescenchymal stem cell
1) by the embryoid body suspension inoculation prepared into the culture plate being coated with by matrigel.
2) embryoid body 10-14h is cultivated, treats that its adherent addition nutrient solution D carries out Fiber differentiation.
3) filling stem cell between culture 5-7d will form, and change nutrient solution E and continue to cultivate, continue to propagation.
Embodiment 3
1st, the preparation of culture medium
IPS inducing cultures (configuration culture medium A)：REGM and MEF culture mediums by volume (1:1) ratio mixing.
Embryoid body inducing culture (configuration culture medium B)：450mL MEM, 50mL FBS, 10mM- glutamine, 0.5mM Beta -mercaptoethanol, 85ng/mL BMP-4,50ng/mLVEGF, 100pM insulin.
Embryoid body culture medium (configuration culture medium C)：400mLDMEM, 100mL FBS, 10ug/ml insulin transferrins are sub- Sodium selenate (ITS), 0.65mM Sodium Pyruvates, 5ng/ml EGFs (EGF), 5mM Glus, 100mM β-sulfydryl Ethanol.
Mescenchymal stem cell inducing culture (configuration culture medium D)：450mLDMEM basal mediums, 50mLFBS, 100pM Insulin, 10mML- glutamine, 150ug/LbFGF, 5 × 10-8Mol/L dexamethasone.
Mescenchymal stem cell culture medium (configuration culture medium E)：450mLDMEM basal mediums, 50mLFBS, 100ng/mL hEGF、100ng/mL bFGF、50ng/mL HGF、、20ng/mLPDGF、25ng/mLTGF-β。
2nd, the acquisition of urine cell
1) collection cups each add 2mL penicillin/streptomycins dual anti-.
2) urine is collected, if do not carried out subsequent operation at once, then urine 4 DEG C of refrigerators is stored in and completed follow-up in the same day Operation.
3) every part of urine prepares the hole of six orifice plate 1, and this hole is coated with into more than 20min with 0.1% gelatin, sucked before use in hole Liquid.
4) urine is poured into appropriate number of 50mL centrifuge tubes, centrifuges 400g, 10min.
5) supernatant is sucked, often pipe leaves about 1-5mL, be mixed into a centrifuge tube.
6) PBS (PBS 95mL add 5mL penicillin/streptomycins and mixed) about 10- containing penicillin/streptomycin is added 30mL.Gently mix.
7) 400g, 10min are centrifuged.
8) supernatant is sucked to remaining 0.5-1mL liquid.
9) remaining liquid is added in the hole being coated with, adds 2mLREGM culture mediums and add 3 μ LPrimocin.
10) culture dish is positioned in 37 DEG C of incubators and cultivated, after (7 days) after urine cell attachment, suck culture medium, used PBS is washed one time, then carries out changing liquid processing.
11) when urine cell fusion degree reaches 80%, then Secondary Culture can be carried out.
3rd, the reprogramming of urine cell is ips cells
1) various groups of transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 or other transcription factor will be expressed Amount to urine cell is imported, cell point is cultivated to advance with coated 6 orifice plates of matrigel with nutrient solution A；
2) the 2nd day after transfecting, culture medium is replaced by IPS culture medium mTesR, fresh culture is changed daily；Make clone Continue to breed
3) the 7th day after transfecting, the Microscopic observation form picking monoclonal close with human embryo stem cell, and it is inoculated in use In coated 12 orifice plates of matrigel, culture medium mTesR cultures obtain induced multi-potent stem cell.
4th, Ips cell inductions are embryoid body
1) by with the ips induced multi-potent stem cells 0.03%-0.3% clostridiopetidase As IV that gets ready by cell dissociation into single thin Born of the same parents, collect cell and are washed with PBS three times, and the suspension that 1 × 104-0.5 × 104 are made with nutrient solution B progress suspensions is standby.
2) Tissue Culture Plate 0.1%-0.02% agar glycolyx 0.5-1h.
3) prepared cell suspension in 1) is added with the good culture plate of agar glycolyx, carrying out changing at liquid daily Reason.
4) 3-4d to be cultivated begins with embryoid body appearance, and the increment of culture 7-10d embryoid bodies reaches top, collects and intend Idiosome suspension, is staticly settled, and suspension is carried out with nutrient solution C standby.
5th, embryoid body directed differentiation is mescenchymal stem cell
1) by the embryoid body suspension inoculation prepared into the culture plate being coated with by matrigel.
2) embryoid body 10-14h is cultivated, treats that its adherent addition nutrient solution D carries out Fiber differentiation.
3) filling stem cell between culture 5-7d will form, and change nutrient solution E and continue to cultivate, continue to propagation.
Embodiment 4
Mescenchymal stem cell made from embodiment 1~3 is detected：
1st, RT-PCR detects the situation of change of each gene in mescenchymal stem cell, and primer is：
NANOG:F5'-TGAACCTCAGCTACCCCAG-3',
R 5'-TGGTGGTAGCAACACTAAAG-3'
OCT-4：F5'-CCTCACTTCACTGCATGTA-3'
R 5'-CACTTTTTCTTTTCCCCTAGCT-3'
MSX-1：F5'-CGACAGCCCCGTGCCAGAG-3'
R 5'-GGCGGACCCATCTTCTCCAG-3'
GAPDH:F5'-ATCCCATCATCACCATCTTCC-3'
R 5'-GAGTCCTTCCACGATACCA-3'
Experimental procedure
1. by the mescenchymal stem cell got ready when its degrees of fusion reaches 80%, 0.25% pancreatin digestion mescenchymal stem cell And collect cell.
2. the total serum IgE of mescenchymal stem cell is extracted using Trizol methods, quality is carried out to it with ultramicrospectrophotometer Detection and concentration mensuration；
3. RNA is reversed into by cDNA using Reverse Transcriptase kit；
4. 10 μ L amplification system is configured, according to 95 DEG C of reactions 10min, 95 DEG C of reactions 15s, 75 DEG C of reaction 1min, circulation 40 times.
As a result find, in mescenchymal stem cell made from embodiment 1~3, the gene NAONG of induced multi-potent stem cell expression Hardly expressed with expression quantity of the OCT-4 in mescenchymal stem cell, illustrate that the mescenchymal stem cell of induction loses ability more The expression of property gene, it is expression quantity of the mesoderm gene in mescenchymal stem cell that mescenchymal stem cell, which belongs to mesoderm MSX-1, Significantly increase, meet the feature of mescenchymal stem cell.Wherein, the testing result of mescenchymal stem cell embodiment 1 is made as schemed 1.The testing result of stem cell is made similarly in embodiment 2~3.
2nd, flow cytometry surface antigen is detected
Experimental procedure
1. the mescenchymal stem cell each embodiment got ready fills when its degrees of fusion reaches 80% between the digestion of 0.25% pancreatin Matter stem cell simultaneously collects cell, and it is 2 × 10 to make its cell number5Individual/every sample；
2. cell born of the same parents are resuspended in 1mL DPBS and washed 2 times, 200g, centrifuge 5min；
Often pipe sample adds the fixed 2h of alcohol resuspension of the precoolings of 1mL 70%；
3. centrifugation discards fixer, adds 200 μ L mouse source CD29, CD44, CD73, CD90, CD105, CD34, CD45 primary antibodies (dilution factor is 1 to antibody:100) 30min is incubated under normal temperature；
4. after 1mLPBS washings, (dilution factor is 1 with FITC marks secondary antibody respectively:200) normal temperature lucifuge is incubated 1h；
5. after PBS is washed twice, discard, adding appropriate PBS according to cell concentration is resuspended cell, is examined using flow cytometer Survey.
By flow cytometry analysis showed, in mescenchymal stem cell made from embodiment 1~3 CD29, CD44, CD73, CD90, CD105 are expressed as the positive, and CD34, CD45 are expressed as feminine gender, meet mescenchymal stem cell surface marker Expression of results.Fig. 2 shows the testing result for embodiment 1 being made mescenchymal stem cell surface markers antigen.Embodiment 2~3 is made The testing result of stem cell is similarly.
3rd, cell viability is detected
The motility rate that cell is made to each embodiment using mtt assay is detected, is as a result shown, embodiment 1~3 is filled between being made The vigor of matter stem cell is followed successively by 95%, 62%, 55%, and wherein the vigor of stem cell is significantly higher than implementation made from embodiment 1 (the p of example 2~3<0.05)
It the above is only the preferred embodiment of the present invention, it is noted that come for those skilled in the art Say, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should be regarded as Protection scope of the present invention.

Claims (10)

1. embryoid body inducing culture, it is characterised in that including basal medium and：
2. embryoid body culture medium, it is characterised in that including basal medium and：
3. mescenchymal stem cell inducing culture, it is characterised in that including basal medium and：
4. mescenchymal stem cell culture medium, it is characterised in that including basal medium and：
5. the culture medium according to any one of Claims 1 to 4, it is characterised in that the basal medium is cultivated for DMEM Base.
6. a kind of method of induction IPS cells formation embryoid body, it is characterised in that induced with the embryoid body described in claim 1 Culture medium induces IPS cells, and the time of the induction is 3~10 days.
7. a kind of method for inducing embryoid body to be divided into mescenchymal stem cell, it is characterised in that embryoid body is inoculated in into right will Lured after asking the embryoid body culture medium described in 2,10~14h of culture with the mescenchymal stem cell inducing culture described in claim 3 Lead 5~7 days and obtain mescenchymal stem cell.
8. a kind of method that mescenchymal stem cell is prepared by urine cell, it is characterised in that comprise the following steps：
Transcription regulatory factor OCT4, SOX2, NANOG, KLF4 and LIN28 are imported into urine cell, culture obtains IPS cells；
IPS cells are induced with the embryoid body inducing culture described in claim 1, after 3~10 days, collecting embryoid body will with right Ask after 10~14h of embryoid body medium culture described in 2, lured with the mescenchymal stem cell inducing culture described in claim 3 Lead 5~7 days, obtain mescenchymal stem cell.
9. method according to claim 8, it is characterised in that after the acquisition mescenchymal stem cell, in addition to propagation, biography For the step of, the propagation, passage are using the mescenchymal stem cell culture medium described in claim 4.
10. method according to claim 8, it is characterised in that the preparation method of the urine cell is：Centrifuge urine, After precipitation is sterilized, with the REGM medium cultures containing Primocin, urine cell is obtained.
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