CN103333920A - Culture medium for establishing pig iPS cell line and culture method thereof - Google Patents
Culture medium for establishing pig iPS cell line and culture method thereof Download PDFInfo
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Abstract
The invention discloses a culture medium for establishing a pig iPS cell line and a culture method thereof. A typical pig iPS cell is obtained in the ninth day through transfection by four transcription factors of OCT4, SOX2, KLF4 and c-MYC and induction culture of the culture medium, and the pig iPS cell can be obtained efficiently. The obtained pig iPS cell clone is flat clone, has a regular edge and is similar to the ES cellular morphology of the human body. According to the pig iPS cell line, the pig iPS cell through subculture keeps the undifferentiated state, shows positive in the AP dyeing displaying result and has a pluripotency mark, and the differentiated cell in vitro expresses NCSTN (entoderm), NESTIN (ectoderm) and DESMIN (mesoblast).
Description
Technical field
The invention belongs to the inducing pluripotent stem cells technical field, relate to a kind of substratum and cultural method thereof of setting up pig iPS clone.
Background technology
Multipotent stem cells (pluripotent stem cells) refers to have the multidirectional differentiation potential that forms humans and animals individual ownership cell type, constantly a class cell of self.Because the multidirectional differentiation potential of multipotent stem cells and the characteristic of self make it that effect and the superiority of its uniqueness be arranged in the research in fields such as cell replacement treatment, gene therapy, developmental biology research, pharmacology and toxicology.At present, the most human known multipotency stem cell is embryonic stem cell (embryonic stem cells, ES cell), generally is that inner cell mass from blastaea is under suitable condition of in vitro culture and the clone of setting up.Yet the acquisition of ES cell, the particularly acquisition of people ES cell have caused many disputes ethically.In addition, the ES cell is applied to the problem that clinical treatment also can face immunological rejection.
(induced pluripotent stem cell is that specific transcription factor is imported somatocyte iPS) to inducing pluripotent stem cells, and making reprogramming of somatic cells is the multipotential stem cell of ES sample.2006, Takahashi and Yamanaka (Takahashi and Yamanaka2006) be first with Oct4, Sox2, and four transcription factors of Klf4 and c-myc import the mouse skin inoblast, and then have obtained to be similar to the iPS cell of ES cell.2007, Takahashi and Yu etc. reported the iPS clone that successfully obtains the people respectively.Research of iPS cell has obtained developing rapidly after this, has obtained a series of breakthroughs, and has set up the special people iPS cell of multiple disease, progressively is applied to the clinical medicine aspect.Up to now, species such as mouse, people, macaque, rat, pig have all been set up iPS clone.The iPS cell has the characteristic similar with the ES cell, and has the ethics avoided dispute, can obtain patient customized a plurality of advantages such as iPS system with respect to the ES cell, and the research of carrying out the iPS cell is significant for the solution of many significant problems of life science.
Pig is a kind of model animal of studying human diseases, and it is highly similar with the people at aspects such as dissect physiology, cardiovascular systems, neural system, endocrine system and skeletal structures.And it and human in the metabolism that particularly heredity determines on the genetics, also have high similarity.This makes pig become the optimal mode animal of human diseases research.
Summary of the invention
The problem that the present invention solves is to provide a kind of substratum and cultural method thereof of setting up pig iPS clone, can efficiently obtain pig iPS clone under no feeder layer cells condition.
The present invention is achieved through the following technical solutions:
A kind of cultural method of setting up pig iPS clone comprises following operation:
1) with Oct4, Sox2, four kinds of transcription factors of Klf4, c-Myc respectively by retroviral vector and viral package carrier transfectional cell, behind transfection 48h, collect the recombinant virus comprised transcription factor respectively;
2) porcine fetus fibroblasts is used trysinization before transfection, be inoculated into then on the porcine fetus fibroblasts substratum, and then add the recombinant virus comprise Oct4, Sox2, Klf4, four kinds of transcription factors of c-Myc and carry out virus infection, in 37 ℃, 5%CO
2Cultivate under the condition, 72h behind the virus infection adds recombinant virus again and carries out the secondary virus infection;
Described porcine fetus fibroblasts substratum is on the DMEM substratum, and the interpolation volumetric concentration is 10% foetal calf serum;
3) 24h behind the secondary virus infection with metainfective porcine fetus fibroblasts trysinization, is inoculated into adherent growth 24h on the porcine fetus fibroblasts substratum, in 37 ℃, 5%CO
2Cultivate under the condition, then the porcine fetus fibroblasts substratum is replaced by pig iPS cell induction substratum, cultivate under the no feeder layer condition;
The described pig iPS of 100ml cell induction substratum is: 85ml DMEM substratum, 15ml10%FBS, 1ml0.1mM NEAA, 1ml2mM glutamine, 1 μ g bFGF, 1 μ g hLIF, the CHIR99021 of 30 μ l10mM, the SB431542 of 20 μ l10mM, 1 μ g hBMP4,185 μ l0.1mM beta-mercaptoethanols, 50IU/mL penicillin and 50 μ g/mL Streptomycin sulphates;
4) treat to occur on the substratum pig iPS cell clone after, choose clone and enlarged culturing.
Described Oct4, Sox2, four kinds of transcription factors of Klf4, c-Myc are respectively by retroviral vector pMXs and viral package carrier pCL-Eco transfection 293T cell, to express the recombinant virus that comprises Oct4, Sox2, Klf4, c-Myc respectively.
Describedly be seeded in 293T cell on the 100mm culture dish, 5%CO after the transfection by the calcium phosphate method transfection
2, change fresh medium after 37 ℃ of overnight incubation, 48h behind the 293T cell transfecting collects viral supernatant; By volume, four kinds of viral supernatants are joined carry out virus infection in the PEF substratum.
Described behind the secondary virus infection 9d, choose form iPS clone preferably at microscopically, behind IV Collagen Type VI enzyme processing 10～15min, it is inoculated on mouse fetal inoblast (MEF) feeder layer, the substratum of this moment is the DMEM+10% foetal calf serum, the cultivation of going down to posterity.
The described iPS cell cultivated of going down to posterity can keep the state that do not break up, and is positive through the alkaline phosphatase staining display result.
Described iPS cell expressing NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 mark.
Described pig iPS cell inoculation is carried out vitro differentiation inducing culture EB cell, differentiation that can be spontaneous behind the EB cell attachment with the DMEM+10%KSR substratum in the culture dish of low absorption.
The described pig iPS of 100ml cell induction substratum is: 85ml DMEM substratum, 15ml10%FBS, 1ml0.1mM NEAA, 1ml2mM glutamine, 1 μ g bFGF, 1 μ g hLIF, the CHIR99021 of 30 μ l10mM, the SB431542 of 20 μ l10mM, 1 μ g hBMP4,185 μ l0.1mM beta-mercaptoethanols, 50IU/mL penicillin and 50 μ g/mL Streptomycin sulphates.
Compared with prior art, the present invention has following beneficial technical effects:
Substratum and the cultural method thereof of setting up pig iPS clone provided by the invention, a kind of substratum and cultural method of uniqueness are provided, carry out transfection through OCT4, SOX2, KLF4 and four transcription factors of c-MYC, and the inducing culture by substratum, occur typical pig iPS cell in the time of the 9th day and can efficiently obtain pig iPS cell; The pig iPS cell clone that obtains is flat clone, and neat in edge is similar with people's ES cellular form.
The pig iPS clone that the present invention makes up, the pig iPS cell cultivated of going down to posterity can keep the state that do not break up, and is positive through the AP display result that dyes; And has NANOG, SOX2, SSEA-1, SSEA-4, versatility marks such as TRA-1-60 and TRA-1-81; Cell expressing NCSTN(entoderm after external, vitro differentiation), NESTIN(ectoderm) and the DESMIN(mesoderm).
Description of drawings
Fig. 1 is GFP luciferase expression situation behind the retrovirus report carrier pMXs-GFP transfection packing cell 293T, photo under the A. fluorescent microscope; B. photo under the common light microscopic;
Fig. 2 is the GFP luciferase expression situation behind the 48h behind the retroviral infection PEF, photo under the A. fluorescent microscope; B. photo under the common light microscopic;
Fig. 3 is the typical iPS clone that metainfective PEF formed at the 9th day;
Fig. 4 is pig iPS cell (A) and the AP dyeing situation (B) that goes down to posterity and cultivate;
Fig. 5 detects SOX2 in the pig iPS cell, NANOG, SSEA1, SSEA4, the expression of TRA-1-60 and TRA-1-81 for immunofluorescence dyeing;
Fig. 6-1 identifies for vitro differentiation, the differentiation situation (right side) after the embryoid (left side) that pig iPS cell suspension culture forms and embryoid are adherent;
Fig. 6-2 detects the expression of triploblastica marker gene for PCR;
Fig. 7-1 is expelled to the teratoma of the subcutaneous formation in nude mice back for pig iPS;
Fig. 7-2 is teratoma HE dyeing situation.
Embodiment
The present invention is described in further detail below in conjunction with specific embodiment, and the explanation of the invention is not limited.
A kind of method that obtains pig iPS cell, after importing people OCT4, SOX2, KLF4 and c-MYC by retroviral vector pMXs in the PEF respectively, cultivate at inducing culture, can efficiently induce PEF to form pig iPS cell under the condition at no feeder layer, it induces efficient is 0.54%.
Described inducing culture is: DMEM+15%FBS+hLIF(10ng/ml)+bFGF(10ng/ml)+and CHIR99021(5 μ M)+SB431542(2 μ M)+hBMP4(10ng/ml)
Formed its alkaline phosphatase of pig iPS cell (AP) dyeing is positive, express Nanog, Sox2, and SSEA-1, can realize external and the interior triploblastica of body breaks up at versatility marks such as 4.
Concrete, a kind of cultural method of setting up pig iPS clone, operating process is as follows:
1) cultivation of 293T cell and PEF cell (porcine fetus fibroblasts);
The substratum of 293T cell and PEF cell is: DMEM+10% foetal calf serum (FBS, volumetric concentration), cultivate under the normal condition;
2) with four kinds of retroviral plasmids (pMXs-Oct4-GFP, pMXs-Sox2-GFP, pMXs-Klf4-GFP, pMXs-c-Myc-GFP are all available from Addgene) respectively transfection 293T cell to prepare viral liquid; Wherein transfection pMXs-GFP is in order to indicate transfection efficiency.
2.1) preparation of 293T cell
Preceding 24 hours of transfection, with the 293T cell with 0.25% trysinization, 5 the 10cm-wares in shop, counting back, each ware kind 4 * 10
6Individual cell.
2.2) calcium phosphate method transfection 293T cell
Before the transfection each ware is changed the fresh PEF nutrient solution of 7ml.Each ware transfection virus vector and package carrier pCL-Eco(are available from Addgene) each 12 μ g, concrete steps are as follows:
2.2.1) deionized water and the virus vector for the treatment of transfection, package carrier are mixed;
2.2.2) in above-mentioned mixed solution, add 156.25 μ l2M CaCl
2, mixing;
2.2.3) upwards go on foot in the mixed solution and to add 1250 μ l2 * HBS(280mM NaCl, 10mM KCl, 1.5mM Na
2HPO
4, 50mM Heapes, 12mM Glucose), blow and beat mixing repeatedly 30 times;
2.2.4) will go up the step mixed solution and leave standstill 2 minutes in room temperature, dropwise be added drop-wise to uniformly then and prepare to jiggle mixing in the cells transfected;
2.2.5) 5%CO
2, 37 ℃ of overnight incubation (11-14 hour);
2.2.6) carefully change liquid, every ware 8ml fresh medium, 5%CO behind the 11-14 hour
2, 37 ℃ of cultivations.
Behind the transfection 293T cell 24h, under fluorescent microscope, observe the green fluorescence expression, the cell expressing green fluorescence of 80-90% as shown in Figure 1, the transfection efficiency that shows plasmid is 80-90%.
3) preparation of PEF cell
3.1) separation and the cultivation of porcine fetus fibroblasts (PEF)
3.1.1) give the slaughterhouse to slaughter the farrowing sow in 35d pregnant age, fetch the uterus;
3.1.2) under aseptic condition, fetus is taken out from intrauterine, fully clean more than 3 times with the PBS that contains high density green grass or young crops/Streptomycin sulphate (500IU/mL), remove head respectively, internal organ, four limbs are chosen muscle tissue, and it is fully shredded;
3.1.3) add 0.1% IV Collagen Type VI enzyme in each fetal tissue, it is transferred in the 15mL centrifuge tube 37 ℃ of digestion 4-6h;
3.1.4) add isopyknic nutrient solution (DMEM+15%FBS+100IU/mL mycillin), blow and beat mixing;
3.1.5) with the filter yarn filtration of cell/cell mass suspension with 100 order apertures, the centrifugal 5min of 1000rpm;
3.1.6) supernatant discarded, add cell culture fluid re-suspended cell (5 * 10
5Individual/mL);
3.1.7) get 3mL and move in the 100mm culture dish, and add the 7mL nutrient solution, shake up be placed on overnight incubation in the incubator (37 ℃, 5%CO
2, saturated humidity);
3.1.8) inhale second day and to remove nutrient solution, clean 2 times with PBS after, add the 10mL fresh medium and continue to cultivate, change liquid later on every other day, carry out frozen or the cultivation of going down to posterity to it after at the bottom of cell covers with ware;
3.1.9) treat that cell grows to about 80% density, can be to its cultivation of going down to posterity.Original fluid is removed in suction, after PBS cleaning 2 times, adds the cell dissociation buffer of 2～3mL0.25% pancreatin and 0.04%EDTA, hatches digestion 2～3min for 37 ℃;
3.1.10) add isopyknic cell culture fluid termination digestion, blow and beat cell then gently, be transferred in the 15mL centrifuge tube after discrete the suspension;
3.1.11) the centrifugal 5min of 1000rpm, supernatant discarded with cell culture fluid re-suspended cell agglomerate, is re-seeded in the new Tissue Culture Dish, adds nutrient solution and continues to cultivate, at the bottom of cell is paved with ware again.
3.2) the preceding PEF preparation of transfection
Behind the 293T cell transfecting 36 hours, with 0.25% trysinization, the counting back was by 5 * 10 with PEF
5The amount of individual cell/10cm-ware is received in the 10cm-ware.
4) collection virus also infects PEF
4.1) 48h behind the 293T cell transfecting, collect viral supernatant with pasteur pipet, and with in 0.45 μ M membrane filtration and the 15ml centrifuge tube.Adding final concentration is 8 μ g/ml polybrene, mixing.Add 8ml293T substratum (DMEM+10% foetal calf serum) in the 293T culture dish again, continue to cultivate.
48h behind the 293T cell transfecting collects four transcription factors and GFP virus.Infect the PEF cell in the culture dish with retrovirus collection back adding PEF cell.Wherein use GFP virus infection PEF cell separately for the indicator virus efficiency of infection.
4.2) with Oct4, Sox2, four kinds of viral liquid equal-volumes of Klf4 and c-myc add in the PEF culture dish (every kind of virus of 10cm ware adds 5ml); Another ware adds 5ml pMXs-GFP virus, as infecting contrast, Test Virus efficiency of infection.After spending the night, infection renews bright PEF nutrient solution (DMEM+10% foetal calf serum).
As shown in Figure 2, behind the 48h, approximately the PEF cell expressing green fluorescence of 80-90% shows that the efficiency of infection of virus is 80-90% behind the GFP virus infection PEF.
4.3) 72h after the transfection, repeat 4.1) and 4.2) the virus infection operation carry out the secondary virus infection.
5) infect back PEF bed board again
24h behind the virus infection for the second time counts metainfective PEF after with 0.05% trysinization, with 4 * 10
4Individual cell inoculation is in the 10cm-ware.The substratum of this moment is PEF substratum (DMEM+10% foetal calf serum).
6) change pig iPS cell induction substratum
Metainfective PEF cell is adherent growth 24h behind the bed board again, the PEF substratum is replaced by pig iPS cell induction substratum, its composition is: DMEM+15%FBS+hLIF(10ng/ml)+bFGF(10ng/ml)+and CHIR99021(5 μ M)+SB431542(2 μ M)+hBMP4(10ng/ml), change liquid every day.
The preparation of inducing culture (be example with 100ml) is as follows: get 85ml DMEM substratum, add 15ml FBS(Hyclone), 1ml0.1mM NEAA(Gibco), 1ml2mM glutamine (Gibco), 1 μ g bFGF(StemRD), 1 μ g hLIF(Millipore), the CHIR99021(StemRD of 30 μ l10mM), the SB431542(StemRD of 20 μ l10mM), 1 μ g hBMP4(StemRD), 185 μ l0.1mM beta-mercaptoethanols, 50IU/mL penicillin and 50 μ g/mL Streptomycin sulphates.
As shown in Figure 3, typical pig iPS cell appearred the 9th day the time.This clone is flat clone, and neat in edge is similar with people's ES cellular form.
7) Ke Long picking and enlarged culturing
7.1) infect back 9d, choose form iPS clone preferably at microscopically, will clone cutting back sucking-off with glass needle, put into 4 orifice plates of completing the MEF feeder layer.
7.2) with the cultivation of going down to posterity of the clone in 4 orifice plates, amplification.
Behind IV Collagen Type VI enzyme treated cell 10-15min, wash one time with PBS.With yellow rifle head the clone is spread to fritter, with pasteur pipet the clone is blown down then.Clone's renewed vaccination of blowing down in the new ware of the MEF feeder layer of completing, is obtained pig iPS clone.
8) evaluation of pig iPS cell
8.1) alkaline phosphatase (AP) dyeing
Will carry out pig iPS cell that going down to posterity of AP dyeing cultivate with the fixing 15min of 4% Paraformaldehyde 96 room temperature, wash twice with PBS, add AP colour developing liquid (among the firm red 0.1M Trish-HCl that is dissolved into 5mL pH8.9 of 0.002g naphthyl alcohol salt and 0.005g), the room temperature lucifuge leaves standstill 15min, wash twice with PBS, add an amount of PBS, take a picture.
As shown in Figure 4, the pig iPS cell cultivated of going down to posterity can keep the state that do not break up, and is positive through the AP display result that dyes.
8.2) identified by immunofluorescence
The cell that will identify is washed twice with PBS then with the fixing 15min of 4% Paraformaldehyde 96 room temperature.Afterwards, in 0.1% Triton X-100, hatch 10min, hatched the back and given a baby a bath on the third day after its birth time with PBS.Seal 30min with 1%BSA then, sealing adds later with BSA dilutes good primary antibodie, spends the night.Second day, with the primary antibodie sucking-off, give a baby a bath on the third day after its birth time with PBS, add then dilute with BSA good two anti-, incubated at room 1 hour.Afterwards, dye nuclear 1min with 10 μ g/ml Hochest33342, wash twice back with PBS and under fluorescent microscope, observe.
Carry out NANOG, SOX2, SSEA-1, SSEA-4, the immunofluorescence dyeing of versatility marks such as TRA-1-60 and TRA-1-81 according to the method described above.As shown in Figure 5, the above-mentioned versatility mark of pig iPS cell expressing that obtains of the present invention.
8.3) vitro differentiation
Pig iPS cell inoculation is cultivated with DMEM+10%KSR substratum (division culture medium) in the culture dish of low absorption, formed embryoid (EB).Changed liquid in per two days, EB was inoculated in the 8th day in the culture dish of gelatin bag quilt and cultivates.When 5 days and 10 days, collecting cell extracts RNA, the marker gene (ectoderm Nestin, mesoderm Desmin, entoderm NCSTN) of three germinal layers is carried out PCR identify.
Used primer sequence is as follows:
NESTIN：S:5’-TAGAGCCCGTGTTGGAAGAT
A:5’-CATCACTTCCACTGTGGTGC
DESMIN：S:5’-CCTCAACTTCCGAGAAACAAGC
A:5’-TCACTGACGACCTCCCCATC
NCSTN：S:5’-CAGCAAAGAACTGGAGTTCATCACTCT
A:5’-AGGAAAAGCTGGGGTCCTCTTCAG
Shown in Fig. 6-1, the pig iPS cell that the present invention obtains is cultivated with division culture medium in the culture dish of low absorption can form the good EB of form, can realize spontaneous differentiation after EB is adherent.
The differentiation different number of days cell utilize RT-PCR to detect tridermic marker gene, shown in Fig. 6-2, the result shows the cell expressing NCSTN(entoderm after the differentiation), the NESTIN(ectoderm) and the DESMIN(mesoderm).
8.4) the interior differentiation of body
With the back subcutaneous location (〉 2 * 10 of pig iPS injection cell to nude mice
6Individual cell), teratoma appearance in about about 25 days was excised teratoma about 40 days from the nude mice back, carried out HE dyeing, observed the triploblastica structure.
Fig. 7-1 is nude mice back, 40d left and right sides teratoma, and Fig. 7-2 is teratoma tissue slice HE dyeing situation.Can find, tangible triploblastica tissue, as: muscle tissue (mesoderm), fatty tissue (mesoderm), neuro epithelium (ectoderm) and glandular tube (entoderm).
Claims (8)
1. a cultural method of setting up pig iPS clone is characterized in that, comprises following operation:
1) with Oct4, Sox2, four kinds of transcription factors of Klf4, c-Myc respectively by retroviral vector and viral package carrier transfectional cell, behind transfection 48h, collect the recombinant virus comprised transcription factor respectively;
2) porcine fetus fibroblasts is used trysinization before transfection, be inoculated into then on the porcine fetus fibroblasts substratum, and then add the recombinant virus comprise Oct4, Sox2, Klf4, four kinds of transcription factors of c-Myc and carry out virus infection, in 37 ℃, 5%CO
2Cultivate under the condition, 72h behind the virus infection adds recombinant virus again and carries out the secondary virus infection;
Described porcine fetus fibroblasts substratum is on the DMEM substratum, and the interpolation volumetric concentration is 10% foetal calf serum;
3) 24h behind the secondary virus infection with metainfective porcine fetus fibroblasts trysinization, is inoculated into adherent growth 24h on the porcine fetus fibroblasts substratum, in 37 ℃, 5%CO
2Cultivate under the condition, then the porcine fetus fibroblasts substratum is replaced by pig iPS cell induction substratum, cultivate under the no feeder layer condition;
The described pig iPS of 100ml cell induction substratum is: 85ml DMEM substratum, 15ml10%FBS, 1ml0.1mM NEAA, 1ml2mM glutamine, 1 μ g bFGF, 1 μ g hLIF, the CHIR99021 of 30 μ l10mM, the SB431542 of 20 μ l10mM, 1 μ g hBMP4,185 μ l0.1mM beta-mercaptoethanols, 50IU/mL penicillin and 50 μ g/mL Streptomycin sulphates;
4) treat to occur on the substratum pig iPS cell clone after, choose clone and enlarged culturing.
2. the cultural method of setting up pig iPS clone as claimed in claim 1, it is characterized in that, described Oct4, Sox2, four kinds of transcription factors of Klf4, c-Myc are respectively by retroviral vector pMXs and viral package carrier pCL-Eco transfection 293T cell, to express the recombinant virus that comprises Oct4, Sox2, Klf4, c-Myc respectively.
3. the cultural method of setting up pig iPS clone as claimed in claim 2 is characterized in that, is seeded in 293T cell on the 100mm culture dish, 5%CO after the transfection by the calcium phosphate method transfection
2, change fresh medium after 37 ℃ of overnight incubation, 48h behind the 293T cell transfecting collects viral supernatant; By volume, four kinds of viral supernatants are joined carry out virus infection in the PEF substratum.
4. the cultural method of setting up pig iPS clone as claimed in claim 1, it is characterized in that, 9d behind the secondary virus infection, choose form iPS clone preferably at microscopically, behind IV Collagen Type VI enzyme processing 10～15min, it is inoculated on mouse fetal inoblast (MEF) feeder layer, and the substratum of this moment is the DMEM+10% foetal calf serum, the cultivation of going down to posterity.
5. as claim 1 or the 4 described cultural methods of setting up pig iPS clone, it is characterized in that the iPS cell cultivated of going down to posterity can keep the state that do not break up, and is positive through the alkaline phosphatase staining display result.
6. as claim 1 or the 4 described cultural methods of setting up pig iPS clone, it is characterized in that described iPS cell expressing NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60 and TRA-1-81 mark.
7. as claim 1 or the 4 described cultural methods of setting up pig iPS clone, it is characterized in that, pig iPS cell inoculation is carried out vitro differentiation inducing culture EB cell, differentiation that can be spontaneous behind the EB cell attachment with the DMEM+10%KSR substratum in the culture dish of low absorption.
8. substratum of setting up pig iPS clone, it is characterized in that, the described pig iPS of 100ml cell induction substratum is: 85ml DMEM substratum, 15ml10%FBS, 1ml0.1mM NEAA, 1ml2mM glutamine, 1 μ g bFGF, 1 μ g hLIF, the CHIR99021 of 30 μ l10mM, the SB431542 of 20 μ l10mM, 1 μ g hBMP4,185 μ l0.1mM beta-mercaptoethanols, 50IU/mL penicillin and 50 μ g/mL Streptomycin sulphates.
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