CN102206610A - Preparation method of hemopoietic progenitor cells and special medium for the same - Google Patents
Preparation method of hemopoietic progenitor cells and special medium for the same Download PDFInfo
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Abstract
The invention provides a preparation method of hemopoietic progenitor cells and a special medium for the same. The special medium consist of a differentiation medium I-1 or a differentiation medium I-2, a differentiation medium II-1 or a differentiation medium II-2, and a differentiation medium III-1 or a differentiation medium III-2. The differentiation medium I-2 is obtained by adding a human BMP4 and a human Wnt3a into a first phase differentiation medium; the differentiation medium II-2 is obtained by adding a human vascular endothelial growth factor and a human basic fibroblast growth factor into a second phase differentiation medium; the differentiation medium III-1 is a third phase differentiation medium; and the differentiation medium III-2 is obtained by adding an all-trans-RA (retinoic acid) into a third phase differentiation medium. The hemopoietic progenitor produced by the method of the invention is suitable for being used as a substitute of a human primary hemopoietic progenitor.
Description
Technical field
The present invention relates to a kind of preparation method and special culture media thereof of hemopoietic progenitor cell.
Background technology
Human embryo stem cell has self and is divided into all somatic ability (Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S., and Jones, J.M. (1998) .Embryonic Stem Cell Lines Derived from HumanBlastocysts.Science 282,1145-1147).These abilities mean that human embryo stem cell can be used as the research platform of human mesoderm and hemopoietic system growth.And then, stable donorcells can also be provided for the clinical transplantation of human hematopoietic progenitor cells.At present existing a plurality of laboratories have reported that human embryo stem cell for directional is divided into the method (Murry of hematopoietic cell, C.E., and Keller, G. (2008) .Differentiation of Embryonic Stem Cellsto Clinically Relevant Populations:Lessons from Embryonic Development.Cell132,661-680).Though these methods have all produced hematopoietic stem by the human embryo stem cell differentiation, their atomization all is uncertain.This uncertainly mainly show: adopted stroma cell to cultivate differentiated system with blood serum medium altogether, cultivated altogether, or the nutrient solution that contains foetal calf serum is cultivated as the trophocyte; The growth course that does not have hemopoietic system in the complete analogue body.
Hematopoiesis-endothelial precursor cell (hemangioblast): be meant a precursor cell (Xiong who has roughly the same the time to hematopoietic cell and endotheliocyte both direction differentiation capability who in the hematopoietic development process, produces, J.-W. (2008) .Molecular and developmental biology of the hemangioblast.DevelopmentalDynamics 237,1218-1231.).
In view of present research does not all have the growth course of hemopoietic system in the complete analogue body, therefore, the vitro differentiation of human embryo stem cell is still the focus of research.
Summary of the invention
An object of the present invention is to provide a kind of substratum that inducing human embryo stem cell is divided into hemopoietic progenitor cell that is used for.
Inducing human embryo stem cell provided by the invention is divided into the substratum of hemopoietic progenitor cell, and by division culture medium I, division culture medium II and division culture medium III form; Division culture medium I is division culture medium I-1 or division culture medium I-2; Division culture medium II is division culture medium II-1 or division culture medium II-2; Division culture medium III is division culture medium III-1 or division culture medium III-2;
The substratum that former protein-4 (BMP4) obtains takes place for add the bone form in the fs division culture medium in described division culture medium I-1; The final concentration that former protein-4 takes place described bone form among the described division culture medium I-1 is 10-100ng/ml;
The substratum that former protein-4 and Wnt3a obtain takes place for add the bone form in the fs division culture medium in described division culture medium I-2; The final concentration that former protein-4 takes place described bone form among the described division culture medium I-2 is 10-100ng/ml, and the final concentration of described Wnt3a is 50-150ng/ml;
Described division culture medium II-1 is for adding the substratum that vascular endothelial growth factor obtains in the subordinate phase division culture medium; The final concentration of the described vascular endothelial growth factor among the described division culture medium II-1 is 50-150ng/ml;
Described division culture medium II-2 is for adding the substratum that vascular endothelial growth factor and Prostatropin obtain in the subordinate phase division culture medium; The final concentration of the described vascular endothelial growth factor among the described division culture medium II-2 is 50-150ng/ml, and the final concentration of described Prostatropin is 50-150ng/ml;
Described division culture medium III-1 is the phase III division culture medium;
Described division culture medium III-2 is for adding the substratum that all-trans retinoic acid (RA) obtains in the phase III division culture medium; The final concentration of the described all-trans retinoic acid among the described division culture medium III-2 is 1-5 μ M;
Former protein-4 (BMP4) takes place described bone form can former protein-4 take place for people's bone form, also can former protein-4 take place for mouse bone form; Described Wnt3a can be people Wnt3a, also can be mouse Wnt3a; Described vascular endothelial growth factor (VEGF) can be human vascular endothelial growth factor or mouse vascular endothelial growth factor; Described Prostatropin (bFGF) can be rh-bFGF or mouse Prostatropin;
Described fs division culture medium is for adding the substratum that bovine serum albumin (BSA) obtains being used to cultivate on the basic cell culture medium of mammalian cell; The final concentration of the described bovine serum albumin in the described fs division culture medium is 0.1-1mg/ml;
Described subordinate phase division culture medium is for adding the substratum that Regular Insulin-Transferrins,iron complexes-selenium salt (ITS), vitamins C acid and thioglycerin obtain on described fs division culture medium; The final concentration of the described Regular Insulin-Transferrins,iron complexes in the described subordinate phase division culture medium-selenium salt is 0.5-1.5% (volumn concentration), and the final concentration of described vitamins C acid is 0.1-1mmol/l, and the final concentration of described thioglycerin is 0.1-1mmol/l;
Described phase III division culture medium is for to replace to the substratum that B27 additive (B27) obtains with the Regular Insulin-Transferrins,iron complexes in the described subordinate phase division culture medium-selenium salt; The final concentration of the described B27 additive in the described phase III division culture medium is 0.5-1.5% (volumn concentration).
The final concentration that former protein-4 takes place described bone form among described division culture medium I-1 and the I-2 all can be 10,20,40,50,60,80 or 100ng/ml, and the final concentration of the described Wnt3a among the described division culture medium I-2 can be 50,60,80,100,120 or 150ng/ml;
The final concentration of the described vascular endothelial growth factor among described division culture medium II-1 and the II-2 all can be 50,60,80,100,120 or 150ng/ml, and the final concentration of the described vascular endothelial growth factor among the described division culture medium II-2 can be 50,60,80,100,120 or 150ng/m1;
The final concentration of the described all-trans retinoic acid among the described division culture medium III-2 can be 1,1.2,1.5,2,2.5,3,4 or 5 μ M;
The final concentration of the bovine serum albumin in the described fs division culture medium can be 0.1,0.2,0.4,0.5,0.6,0.8 or 1mg/ml;
The final concentration of the described vitamins C acid in described second division culture medium can be 0.1,0.2,0.4,0.5,0.6,0.8 or 1mmol/l, the final concentration of described thioglycerin can be 0.1,0.2,0.4,0.5,0.6,0.8 or 1mmol/l, and the final concentration of described Regular Insulin-Transferrins,iron complexes-selenium salt can be 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.4% or 1.50%;
The final concentration of the described B27 additive in the described phase III division culture medium can be 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.4% or 1.50%.
The final concentration that former protein-4 takes place described bone form among described division culture medium I-1 and the I-2 all is preferably 20-80ng/ml, especially be preferably 50ng/ml, the final concentration of Wnt3a is preferably 80-120ng/ml described in the described division culture medium I-2, especially is preferably 100ng/ml; The final concentration of the described vascular endothelial growth factor among described division culture medium II-1 and the II-2 all is preferably 80-120ng/ml, especially be preferably 100ng/ml, the final concentration of Prostatropin is preferably 80-120ng/ml described in the described division culture medium II-2, especially is preferably 100ng/ml; The concentration of the described all-trans retinoic acid among the described division culture medium III-2 is preferably 1.2-3 μ M, especially is preferably 2 μ M;
The final concentration of bovine serum albumin is preferably 0.4-0.6mg/ml in the described fs division culture medium, especially is preferably 0.5mg/ml; The final concentration of the described vitamins C acid in the described subordinate phase division culture medium is preferably 0.2-0.8mmol/l, especially be preferably 0.5mmol/l, the final concentration of described thioglycerin all is preferably 0.2-0.8mmol/l, especially be preferably 0.45mmol/l, the final concentration of described Regular Insulin-Transferrins,iron complexes-selenium salt is preferably 0.7-1.2%, especially is preferably 1%; The final concentration of the described B27 additive in the described phase III division culture medium is preferably 0.7-1.2%, especially is preferably 1%;
Above-mentioned basic cell culture medium can be selected from following at least a substratum: α-MEM, DMEM, DMEM/F12, RPMI1640, F12 or IMDM, can be IMDM, α-MEM, DMEM, DMEM/F12, F12, RPMI1640 or its combination, be preferably the combination of IMDM and F12.
Second purpose of the present invention provides the method that a kind of substratum inducing human embryo stem cell that adopts inducing human embryo stem cell to be divided into hemopoietic progenitor cell is divided into hemopoietic progenitor cell, and it may further comprise the steps:
1) be that human embryo stem cell is cultivated in arbitrary described division culture medium I-1 or described division culture medium I-2;
2) will change over to by the cell that step 1) obtains among arbitrary described division culture medium II-1 or the described division culture medium II-2 and cultivate;
3) will be through step 2) cell that obtains changes among arbitrary described division culture medium III-1 or the described division culture medium III-2 and cultivates, and obtains hemopoietic progenitor cell.
Described human embryo stem cell specifically can be the human embryonic stem cell that can obtain from commercial channels shown in the table 1, as table 1.
Table 1. human embryonic stem cell
Described human embryo stem cell is preferably human embryonic stem cell H1 or human embryonic stem cell H9.
Described human embryo stem cell clone H1 is available from U.S. Wicell company, and catalog number is WA01 (H1)-DL-4; Described human embryo stem cell clone H9 is available from U.S. Wicell company, and catalog number is WA09-DL-2.
The culture condition of the described step 1) in the aforesaid method can be 37 ℃ and cultivated 1-2 days, is preferably 1.5 days;
Described step 2) culture condition can be 37 ℃ and cultivated 2-3 days, is preferably 2.5 days;
The culture condition of described step 3) can be 37 ℃ and cultivated 2-4 days, is preferably 3 days.
The following at least a purposes of the hemopoietic progenitor cell that above-mentioned arbitrary method obtains also is the scope that the present invention will protect, and described purposes is: the 1) application of the hemopoietic progenitor cell of arbitrary described method acquisition in the preparation hemocyte;
2) application of hemopoietic progenitor cell in drug toxicity detects of arbitrary described method acquisition, it is adriamycin (Adriamycin) or Tyke rope (Taxol) that described drug toxicity detects the drug toxicity that adopts.
Human vascular endothelial growth factor (VEGF) and the rh-bFGF (bFGF) of experimental results show that of the present invention can induce mesoderm (mesoderm) cell to produce hematopoiesis-endothelial progenitor cells, all-trans retinoic acid (RA) can induce mesoblastema to produce paraxial mesoderm (paraxial mesoderm), in the latter half, all-trans retinoic acid (RA) has suppressed the growth of red corpuscle pedigree (erythroid), has promoted the generation of hemopoietic progenitor cell; The nutrient solution of atomization all is that chemical ingredients is determined in the method provided by the invention, break up by three one-step inducings, simulated the cytocerastic process of embryonic hematopoiesis, and then the hemopoietic progenitor cell and the intravital hemopoietic progenitor cell that produce have bigger similarity, be suitable as the substitute of people's hemopoietic progenitor cell of former generation, carry out fundamental research and various action oriented research, have broad application prospects.
Description of drawings
Fig. 1 is human embryo stem cell is expressed mesoderm gene T under different inducible factor effects a detected result.
Fig. 2 is mesoblastema produces the positive PDGFR α of KDR negative hematopoietic-endothelial precursor cell under different inducible factor effects a detected result.
Fig. 3 produces the detected result of the expression of hematopoiesis-endothelial precursor cell marker gene for mesoblastema under different inducible factor effects.
Hematopoiesis-endothelial precursor cell that Fig. 4 produces for the human embryo stem cell differentiation forms the colony of Blast.
Fig. 5 is mesoblastema forms the Blast colony 2.5 days time the under different inducible factor effects a detected result.
The hemopoietic progenitor cell that Fig. 6 produces for the human embryo stem cell differentiation forms the detected result of various hemocyte colonies.
Fig. 7 forms the detected result of dissimilar hemocyte colonies when inducing the hematopoiesis-endothelial precursor cell of generation to break up 3 days under different inducible factor effects for human embryo stem cell.
Fig. 8 breaks up the detected result that the special sign of hemocyte CD45 is expressed in the back for hematopoiesis-endothelial precursor cell under different inducible factor effects.
Hematopoiesis and endothelium Expression of Related Genes detected result when Fig. 9 additionally breaks up 3 days for hematopoiesis-endothelial precursor cell under different inducible factor effects.
Figure 10 produces the genetic expression detected result of this complete procedure of hemopoietic progenitor cell for the human embryo stem cell differentiation.
Figure 11 is that human embryo stem cell is through 7 days differentiation generation hemopoietic progenitor cell process CD34 and the detected result after the C-Kit enrichment.
Figure 12 is for expressing the detected result of hematopoiesis, endothelium and other specific gene through the hemopoietic progenitor cell after CD34 and the C-Kit enrichment.
Figure 13 is that human embryo stem cell (H1) source hemopoietic progenitor cell and human cord blood source hemopoietic progenitor cell do not have in the methylcellulose gum substratum of erythropoietin at no beta-mercaptoethanol, in the presence of the Adriamycin of specific concentrations or Taxol, the formation detected result of medullary system hemocyte colony.
Figure 14 is that human embryo stem cell (H9) source hemopoietic progenitor cell and human cord blood source hemopoietic progenitor cell do not have in the methylcellulose gum substratum of erythropoietin at no beta-mercaptoethanol, in the presence of the Adriamycin of specific concentrations or Taxol, the formation detected result of medullary system hemocyte colony.
Embodiment
Be example with human embryo stem cell clone H1 and H9 below, illustrate technical scheme of the present invention.
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Human embryo stem cell clone H1 among the following embodiment is available from U.S. Wicell company, and catalog number is WA01 (H1)-DL-4; Human embryo stem cell clone H9 is available from U.S. Wicell company, and catalog number is WA09-DL-2.
The DMEM substratum is available from U.S. Gibco company, and catalog number is 11960; Foetal calf serum (FBS) is from U.S. Gibco company, and catalog number is 16000.
Resuspended liquid: the IMDM nutrient solution contains 3% foetal calf serum.The IMDM nutrient solution is available from U.S. Gibco company, and catalog number is 12440; Foetal calf serum is available from U.S. Gibco company, and catalog number is 16000.
Be untreated 3.5 centimetres of culture dish available from Canadian stemcell technology company, and catalog number is 27150.
Used detection CD45 antibody is PE mark CD45, and available from U.S. company BD, catalog number is 555483.
StemSpan SFEM nutrient solution is available from U.S. stemcell technology company, and catalog number is 09650.
Non-attaching 24 orifice plates are available from U.S. corning company, and catalog number is 3473.
SCF is available from Britain peprotech company, and catalog number is 300-07; Flt-3L is available from Britain peprotech company, and catalog number is 300-19; IL-3 is available from Britain peprotech company, and catalog number is 200-03; IL-6 is available from Britain peprotech company, and catalog number is 200-06.
Phosphoric acid buffer (PBS): take by weighing NaCl 8g, KCl 0.2g, Na
2HPO
40.62g, KH
2PO
40.2g, be dissolved in the 1L distilled water, regulate pH to 7.2, autoclaving.
Pancreatin: available from U.S. gibco company, catalog number is 25200.
The pregnant mouse of ICR is available from dimension tonneau China laboratory animal technology company.
The aseptic ametycin of mitomycin c solution: 2mg is dissolved among the DMEM that 200ml contains 10% foetal calf serum, and making the final concentration of ametycin in solution is 10 μ g/ml, (ametycin is available from German Roche company, and catalog number is 107409).Attention is carried out in sterile environment.
0.1% gelatin solution: take by weighing 0.1g gelatin powder (available from U.S. sigma company, catalog number is G1890), be dissolved in the 100ml distilled water autoclaving.
The substratum that contains serum is the DMEM substratum that contains 10% (volumn concentration) foetal calf serum: the adding foetal calf serum obtains in the DMEM substratum, and the concentration of foetal calf serum in the DMEM substratum is 10% (volumn concentration).
Foetal calf serum and DMEM are all available from U.S. Gibco company, and it is 16000 and 11960 that catalog number is respectively.
Serum substitute is available from U.S. Gibco company, and catalog number is 10828; Glutamine is available from U.S. Gibco company, and catalog number is 35050; The β mercaptoethanol is available from U.S. Gibco company, and catalog number is 21985; Non-essential amino acid is available from U.S. Gibco company, and catalog number is 11140; Human alkaline fibroblast somatomedin (bFGF) is available from Britain peprotech company, and catalog number is AF-100-18B.
1mg/ml collagenase IV: take by weighing 20mg collagenase IV powder, be dissolved among the 20ml DMEM/F12 filtration sterilization.Collagenase IV is available from U.S. Gibco company, and catalog number is 17104.
5%BSA solution: (bovine serum albumin BSA), is dissolved among the 50ml PBS, is stored in 4 ℃ after the filtration sterilization to take by weighing other bovine serum albumin of 2.5g cell cultures level.(bovineserum albumin, BSA) available from U.S. Gibco, catalog number is 15260 to other bovine serum albumin of cell cultures level.
The IMDM substratum is available from U.S. Gibco, and catalog number is 12440.The F12 substratum is available from U.S. Gibco, and catalog number is 11765.
People BMP4 is available from Britain Peprotech company, and catalog number is 120-05; People Wnt3a is available from U.S. R﹠amp; D company, catalog number is 1324-WN.
Sealing saturatingization liquid: take by weighing 0.5g BSA and be dissolved among the 45ml PBS, and add 55 μ l Triton X-100 and 5ml sealing with normal donkey serum, mixing is put in 4 and spends preservation; Normal donkey serum is available from U.S. JacksonImMunoresearch company, and catalog number is 017-000-121.
Test used Paraformaldehyde 96 stationary liquid and purchase sub-Beijing ancient cooking vessel state company, catalog number is DF021.
Experiment antibody is red fluorescence mark goat anti-mouse/people T antibody, available from U.S. R﹠amp; D systems company, catalog number is IC2085P; Control antibodies is a red fluorescence mark goat IgG, available from U.S. R﹠amp; D systems company, catalog number is IC108P.
ITS is available from U.S. Gibco company, and catalog number is 51300; Vitamins C acid (ascorbic acid) is available from U.S. sigma company, and catalog number is A4544; Thioglycerin (monothioglycerol) is available from U.S. sigma company, and catalog number is M-6145.
People VEGF is available from Britain peprotech company, and catalog number is 100-20.
All-trans retinoic acid (RA) is available from U.S. sigma company, and catalog number is R2625.
Serum-free methylcellulose gum substratum is available from Canadian stemcell technology company, and catalog number is SF H4436
B27 (no vitamin A) is available from U.S. Gibco company, and catalog number is 12587.
The phosphoric acid buffer that contains 2% (volumn concentration) foetal calf serum: the adding foetal calf serum obtains in the phosphoric acid buffer liquid culture medium, and the concentration of foetal calf serum in phosphoric acid buffer is 2% (volumn concentration).
But wash-out CD34 magnetic bead antibody, magnetic bead separation agent, separation stop reagent and all take from the multiple sorting test kit of CD34.The multiple sorting test kit of CD34 was available from beautiful day Ni biotech company, and catalog number is 130-056-701.
The MACS separator column is available from beautiful day Ni biotech company of Germany, and catalog number is 130-042-201.
No β mercaptoethanol does not have erythropoietin methylcellulose gum substratum available from Canadian stemcell technology company, and catalog number is H4001.
CD34 magnetic bead antibody is available from beautiful day Ni biotech company of Germany, and catalog number is 130-046-702.
Adriamycin (adriamycin) is available from U.S. Calbiochem, and catalog number is 324380.
Taxol (Tyke rope) is available from Calbiochem, and catalog number is 580555.
I, embryonic stem cell are induced to differentiate into mesoblastema
One, preparation method
1.37 ℃ water-bath preheating division culture medium I-2a.
2. in human embryonic stem cell H1 (people ES clone H1), add division culture medium I-2a, and the clone is transferred in non-attaching six orifice plates, put into 37 ℃ of cultivations of incubator, carry out the mesoderm differentiation;
3.36 after hour, the mesoderm differentiation finishes, and obtains people BMP4 and people Wnt3a combination treatment group cell (BMP4+Wnt3a), is mesoblastema (H1).
Use the same method and adopt fs division culture medium and division culture medium I-1 cultivation to obtain not contain inducible factor cellular control unit (No cytokine) and people BMP4 treatment group cell (BMP4) respectively people ES clone H1.
Above-mentioned division culture medium I-2a is for adding the substratum that people BMP4 and people Wnt3a obtain in the fs division culture medium, the final concentration of people BMP4 and people Wnt3a is respectively 50ng/ml and 100ng/ml among the division culture medium I-2a.
Above-mentioned fs division culture medium is to add 5%BSA solution in the nutrient solution that 50ml is made up of 75% (volumn concentration) IMDM and 25% (volumn concentration) F12, mixing, and the final concentration that makes BSA is the substratum that 0.5mg/ml obtains.
Above-mentioned division culture medium I-1 adds people BMP4 in above-mentioned fs division culture medium, making people BMP4 final concentration is the substratum that 50ng/ml obtains.
Two, differentiated result detects
Detect with the method for born of the same parents' inner cell dyeing, be specially in conjunction with flow cytometry:
With the people BMP4 and the people Wnt3a combination treatment group cell (BMP4+Wnt3a) that obtain among the division culture medium I-2a, do not contain inducible factor cellular control unit (No cytokine) and people BMP4 treatment group cell (BMP4) is used trysinization respectively, the digestion that adds the substratum effect termination pancreatin that contains serum, use 40 μ m cell screen filtrations not to be digested to single celled cell mass, centrifugal collecting cell;
2. eccentric cell, and resuspended with the Paraformaldehyde 96 stationary liquid, and room temperature was placed 20 minutes;
3. eccentric cell, and resuspended with sealing saturatingization liquid, room temperature placement 20 minutes;
4. eccentric cell, and resuspended with sealing saturatingization liquid, with concentration adding red fluorescence mark goat anti-mouse/people T antibody of 1: 50, room temperature placement 1 hour;
5. eccentric cell, and with saturatingization of sealing liquid re-suspended cell;
6. repeating step 4;
7. flow cytometer detects.
The result as shown in Figure 1.Fig. 1 be human embryo stem cell under different inducible factor effects, through 1.5 days the differentiation of inducing, express the detected result of mesoderm gene T.The Isotype group is homotype antibody control group; No cytokine group is not for adding the control group of any inducible factor; BMP4 is a people BMP4 treatment group; BMP4+Wnt3a is people BMP4 and people Wnt3a combination treatment group.Fig. 1 shows: the cell that people BMP4 treatment group (BMP4) produces has 62% to express mesoderm special sign T (Brachyury), and people BMP4 and people Wnt3a combination treatment group (BMP4+Wnt3a) have 94% cell expressing T.Efficiently inducing human embryo stem cell generation mesoblastema of people BMP4 and people Wnt3a combination treatment is described.
3 multiple experiments are all established in the preparation of above cell and detection.Show that to sum up people ES clone H1 produces mesoblastema (H1) expeditiously under people BMP4 and 1.5 days effect of people Wnt3a processing.
In addition, on the basis of division culture medium I-2a, the final concentration preparation that only changes inducible factor obtains division culture medium I-2b and division culture medium I-2c; Described division culture medium I-2b is for adding the substratum that people BMP4 and people Wnt3a obtain in the fs division culture medium, the final concentration of people BMP4 and people Wnt3a is respectively 20ng/ml and 80ng/ml among the described division culture medium I-2b; Described division culture medium I-2c is for adding the substratum that people BMP4 and people Wnt3a obtain in the fs division culture medium, the final concentration of people BMP4 and people Wnt3a is respectively 80g/ml and 120ng/ml among the described division culture medium I-2c; Adopt described division culture medium I-2b and described division culture medium I-2c according to the method described above respectively inducing human embryo stem cell be H1, experimental result shows: people BMP4 and people Wnt3a combination treatment group (BMP4+Wnt3a) have 90% and 92% cell expressing T respectively.
II, mesoblastema are divided into hematopoiesis-endothelial precursor cell
One, preparation
1. the mesoblastema (H1) that obtains in division culture medium I-2a among the step I is taken out, and transfer in the 15ml centrifuge tube; After leaving standstill 5 minutes, the sucking-off nutrient solution, and add division culture medium II-2a, and the clone is transferred in non-attaching six orifice plates, put into incubator, 37 ℃ of cultivations.
2. after cultivating 36 hours, take out cell, the sucking-off substratum, and add fresh division culture medium II-2a again;
3. after cultivating 24 hours, hematopoiesis-endothelial precursor cell differentiation finishes, and obtains people VEGF and people bFGF combination treatment group cell (BW+VF), i.e. hematopoiesis-endothelial precursor cell (H1).
The mesoblastema (H1) that will obtain among the I that uses the same method again adopts subordinate phase division culture medium (the RA final concentration the is 2 μ M) cultivation of subordinate phase division culture medium, division culture medium II-1 and adding RA to obtain not add inducible factor cellular control unit (BW+NC), people VEGF treatment group cell (BW+V) and RA cellular control unit (BW+RA) respectively.
Above-mentioned division culture medium II-2a adds people VEGF and people bFGF in the subordinate phase division culture medium, make the final concentration of people VEGF and people bFGF be 100ng/ml.
Above-mentioned division culture medium II-1 adds people VEGF in the subordinate phase division culture medium, the final concentration that makes people VEGF is 100ng/ml.
Above-mentioned subordinate phase division culture medium is to add 5%BSA solution, ITS, vitamins C acid (ascorbic acid) and thioglycerin (monothioglycerol) mixing in the nutrient solution of 50ml 75%IMDM and 25%F12, the final concentration that makes BSA is 0.5mg/ml, the final concentration of ITS is 1%, the final concentration of vitamins C acid (ascorbic acid) is 0.5mmol/l, and the final concentration of thioglycerin (monothioglycerol) is 0.45mmol/l.
Two, differentiated result detects
(1) adopt the flow cytometry method to detect hematopoiesis-endothelial precursor cell
Employing flow cytometry method detects people VEGF and the people bFGF combination treatment group cell (BW+VF) that obtains among the division culture medium II-2a, the expression that does not add middle KDR of inducible factor cellular control unit (BW+NC), people VEGF treatment group cell (BW+V) and RA cellular control unit (BW+RA) and PDGFR α (platelet derived growth factor receptor α type) respectively.Wherein, it is APC mark KDR antibody that fluidic cell detects antibody, available from U.S. R﹠amp; D company, catalog number is FAB357A; PE-mark PDGFR Alpha antibodies, available from U.S. company BD, catalog number is 556002;
The result as shown in Figure 2.Fig. 2 under different inducible factor effects, produces the detected result of the positive PDGFR α of KDR negative hematopoietic-endothelial precursor cell for the mesoblastema of human embryo stem cell formation after 1.5 days through people BMP4 and people Wnt3a processing in the time of 2.5 days.BW+NC group is not add the control group of any inducible factor in 2.5 days; BW+VF group is 2.5 days people VEGF and people bFGF combination treatment group; The BW+V group is 2.5 days people VEGF treatment group; BW+RA is 2.5 days RA control groups.Fig. 2 shows: add in the treatment group of people VEGF, 52% cell is the positive PDGFR α of KDR feminine gender; Add in the treatment group of people VEGF and people bFGF, 86% cell is the positive PDGFR α of KDR feminine gender; Add in the control group of RA, 82% cell is the positive KDR feminine gender of PDGFR α; And subordinate phase does not add in the control group of any inducible factor, and 76% cell is the positive PDGFR α of the KDR positive.Because KDR specifically expressing in hematopoiesis-endothelial precursor cell, PDGFR α is specifically expressing in the paraxial mesoderm cell, therefore illustrate that people VEGF and people bFGF have decisive role for inducing mesoblastema to produce hematopoiesis-endothelial precursor cell, and RA is for inducing the paraxial mesoderm cell to have decisive role.
(2) detection of the gene of cell specific expression
Trizol handles respectively and obtains people VEGF and people bFGF combination treatment group cell (BW+VF) among the above-mentioned division culture medium II-2a, do not add inducible factor cellular control unit (BW+NC), people VEGF treatment group cell (BW+V) and RA cellular control unit (BW+RA), extracts the total RNA of sample; Perhaps work as cell quantity and be less than 5 * 10
5The time, in order to obtain high-quality RNA, then use a small amount of RNA to extract test kit (RNAqueousTM-Micro) (available from Ambion company) and extract the total RNA of sample.Each total RNA reverse transcription is obtained cDNA, is that template is carried out the PCR evaluation with these four kinds of cDNA respectively.
Primer sequence is as follows:
The primer that detects gene OCT4 is: Sense:CCAGCCGCAGCCTTTGTGA; Anti:GGTTCAAGGGCTTTATTCCATCT;
The primer that detects gene T is: Sense:AGGTAATTGATCTGGGTGGTG; Anti:GTCCTCGTTGATTGTCATGGTT;
The primer that detects gene KDR is: Sense:CCTCGCCTTTGCCGATCC; Anti:GGATCTTCATGAGGTAGTCAGTC;
The primer that detects gene SCL is: Sense:AGAAAGGGACAATGATAGTAGAGTG; Anti:ATTCGGAACATAGACCAGACC;
The primer that detects gene LMO2 is: Sense:ACATAGCATCCAAGTGGCATAA; Anti:TCTAGTTCGCAAGCGTCAAA;
The primer that detects gene RUNX1 is: Sense:ATGTGGTCCTATTTAAGCCAGCCC; Anti:TCATCTGGCTGAAGACACCAGCTT;
The primer that detects gene GATA2 is: Sense:GCGTCTCCAGCCTCATCTT; Anti:GGAAGAGTCCGCTGCTGTAG;
The primer that detects gene M EOX1 is: Sense:AGAACCGAAGGATGAAGTGG; Anti:AGTCAGGGAAAGATGTGGAGA;
The primer that detects gene PDGFR α is: Sense:CCTCCCACTCCATACCC; Anti:GCAAGGCTCACAGCAATA;
The primer that detects gene GAPDH is: Sense:AATCCCATCACCATCTTCC; Anti:CATCACGCCACAGTTTCC.
The result as shown in Figure 3.Fig. 3 handles the mesoblastema that (adopting division culture medium I-2a) formed after 1.5 days for human embryo stem cell through people BMP4 and people Wnt3a, under different inducible factor effects, and the detected result of the expression of hematopoiesis in the time of 2.5 days-endothelial precursor cell marker gene.BW+NC group is not add the control group of any inducible factor in 2.5 days; BW+VF group is 2.5 days people VEGF and people bFGF combination treatment group; The BW+V group is 2.5 days people VEGF treatment group; BW+RA is 2.5 days RA control groups.Fig. 3 shows: add in the treatment group of people VEGF cell expressing hematopoiesis endothelium specific gene KDR, SCL, LMO2, RUNX1, GATA2 etc.When adopting people VEGF and people bFGF combined action, cell is significantly increased to the expression of hematopoiesis endothelium specific gene.And add in the control group of RA cell high expression level paraxial mesoderm gene M EOX1 and PDGFR α.The control group that subordinate phase does not add any inducible factor has only very low-level hematopoiesis endothelium specific gene to express.Illustrate that people VEGF and people bFGF have decisive role to inducing mesoblastema to produce hematopoiesis-endothelial precursor cell, and RA is for inducing the paraxial mesoderm cell to have decisive role.
(3) adopt the Blast colony to form experiment and detect hematopoiesis-endothelial precursor cell;
Method is:
With the people VEGF and the people bFGF combination treatment group cell (BW+VF) that obtain among the above-mentioned division culture medium II-2a, do not add inducible factor cellular control unit (BW+NC), people VEGF treatment group cell (BW+V) and RA cellular control unit (BW+RA), use trysinization respectively, the digestion that adds the substratum termination pancreatin that contains serum, use 40 μ m cell screen filtrations not to be digested to single celled cell mass, centrifugal collecting cell; The substratum that contains serum is the aforementioned DMEM substratum that contains 10% (volumn concentration) foetal calf serum;
With cell with in the resuspended liquid of the concentration of 20000/100ul;
3. add the serum-free methylcellulose gum substratum that 1ml contains 50ng/ml people BMP4 and 50ng/ml, and abundant mixing;
4. the mixing cell is left standstill 2-5 minute, so that bubble is eliminated;
5. the cell with mixing joins in the 3.5 centimetres of culture dish that are untreated, and puts into incubator and cultivates 7 days for 37 ℃.Keep humidity;
6.7 after it, take out cell, counting;
Result such as Fig. 4, shown in Figure 5.Hematopoiesis-endothelial precursor cell that Fig. 4 produces for the human embryo stem cell differentiation forms the detected result of Blast colony.BW+NC group is not add the control group of any inducible factor in 2.5 days; BW+VF group is 2.5 days people VEGF and people bFGF combination treatment group; The BW+V group is 2.5 days people VEGF treatment group; BW+RA is 2.5 days RA control groups, and Fig. 4 is 200 microns for scale among the blast colony figure of standard.
Fig. 5 under different inducible factor effects, forms the detected result of Blast colony for the mesoblastema of human embryo stem cell formation after 1.5 days through people BMP4 and people Wnt3a processing in the time of 2.5 days.BW+NC group is not add the control group of any inducible factor in 2.5 days; BW+VF group is 2.5 days people VEGF and people bFGF combination treatment group; The BW+V group is 2.5 days people VEGF treatment group; BW+RA is 2.5 days RA control groups.Fig. 5 shows, adds in the treatment group of people VEGF and people bFGF, and cell produces maximum blast colonies; Add in the control group of RA, cell can not produce the blast colony.Illustrate that people VEGF and people bFGF have decisive role for inducing mesoblastema to produce hematopoiesis-endothelial precursor cell.
Repeated experiments is all established in the preparation of above cell and detection 3 times.To sum up show after going down to posterity people ES clone H1 people BMP4 and people Wnt3a handle produced mesoblastema (H1) in 1.5 days after, add people VEGF and people bFGF again and handled 2.5 days, cell high-efficient produces hematopoiesis-endothelial precursor cell (H1).
In addition, on the basis of division culture medium II-2a, the final concentration preparation that only changes inducible factor obtains division culture medium II-2b and division culture medium II-2c; Described division culture medium II-2b adds people VEGF and people bFGF in the subordinate phase division culture medium, make the final concentration of people VEGF and people bFGF be 80ng/ml; Described division culture medium II-2c adds people VEGF and people bFGF in the subordinate phase division culture medium, make the final concentration of people VEGF and people bFGF be 120ng/ml; The mesoblastema (H1) that adopts described division culture medium II-2b and described division culture medium II-2c to induce respectively according to the method described above to obtain through division culture medium I-2a obtains hematopoiesis-endothelial precursor cell (H1), and experimental result shows: people VEGF and people bFGF combination treatment group (VEGF+bFGF) all can produce the positive PDGFR α of KDR negative hematopoietic-endothelial precursor cell of 85%.
III, hematopoiesis-endothelial precursor cell are divided into hemopoietic progenitor cell
One, preparation
1. hematopoiesis-the endothelial precursor cell (H1) that obtains at division culture medium II-2a among the above-mentioned steps II is taken out, and sucking-off upper strata substratum;
2. add division culture medium III-2a, put into 37 ℃ of cultivations of incubator;
3. every 24 hours, take out cell, the sucking-off substratum, and add identical fresh culture again;
4. after cultivating 3 days, the hemopoietic progenitor cell differentiation is finished, and obtains RA treatment group cell (BW+VF+RA), i.e. hemopoietic progenitor cell (H1).
Use the same method and to adopt phase III division culture medium (being division culture medium III-1) cultivation to obtain not add inducible factor cellular control unit (BW+VF+NC) by hematopoiesis-endothelial precursor cell (H1) that division culture medium II-2a obtains.
Above-mentioned division culture medium III-2a is for to add all-trans retinoic acid (RA) in the phase III division culture medium, the final concentration that makes RA is 2 μ M.
Above-mentioned phase III division culture medium is to add 5%BSA solution, B27 (no vitamin A), vitamins C acid (ascorbic acid) and thioglycerin mixing in the nutrient solution of 50ml 75%IMDM and 25%F12, the final concentration that makes BSA is 0.5mg/ml, the final concentration of B27 (no vitamin A) is 1%, the final concentration of vitamins C acid (ascorbic acid) is 0.5mmol/l, and the final concentration of thioglycerin is 0.45mmol/l.
Two, detect
(1) adopts the hemocyte colony to form experiment and detect hemopoietic progenitor cell
Form method that experiment detects according to above-mentioned hemocyte colony and detect hematopoiesis-endothelial precursor cell (H1) that not adding of obtaining among the above-mentioned division culture medium III-2a obtain by division culture medium II-2a in inducible factor control group (BW+VF+NC), RA treatment group (BW+VF+RA) and the II step (BW+VF).
Result such as Fig. 6, shown in Figure 7.Fig. 6 for human embryo stem cell through people BMP4 and people Wnt3a combination treatment, VEGF and bFGF combination treatment after forming hematopoiesis-endothelial precursor cell in 4 days, pass through all-trans retinoic acid (RA) again and handle the detected result that the hemopoietic progenitor cell that produces after 3 days forms various hemocyte colonies.CFU-E is the red corpuscle colony; BFU-E is a Blast red corpuscle colony; CFU-G is a granular leukocyte colony; CFU-M is a macrophage colony; CFU-GM is granulocyte and macrophage colony; CFU-GEMM is red corpuscle, megalokaryocyte, granulocyte and macrophage colony.Fig. 6 is the hemocyte colony of standard, and scale is 200 microns among the figure.
Fig. 7 induces the hematopoiesis-endothelial precursor cell of generation for human embryo stem cell, under different inducible factor effects, forms the detected result of dissimilar hemocyte colonies when breaking up 3 days.The BW+VF group is directly done the control group that colony forms experiment for hematopoiesis-endothelial precursor cell; When the BW+VF+NC group is broken up 3 days for hematopoiesis-endothelial precursor cell under the condition that does not add any inducible factor, form the control group of hemocyte colony; The BW+VF+RA group forms the treatment group of hemocyte colony for the differentiation under the condition that adds RA of hematopoiesis-endothelial precursor cell in the time of 3 days.Fig. 7 shows, adds in the treatment group of all-trans retinoic acid (RA), and cell produces maximum medullary systems and mixes the hemocyte colony; Do not add in the control group of all-trans retinoic acid, the hemocyte colony overwhelming majority that cell produces is the red corpuscle colony; The control group of hematopoiesis-endothelial precursor cell only produces a small amount of red assembly and falls.In the process of hematopoietic development, red is that the generation of hemocyte is an independent process (early than the generation of hemopoietic progenitor cell), and therefore, producing a large amount of red assemblys independent red of being likely of indication that fall when not adding all-trans retinoic acid is that hemocyte is grown destiny.And behind the adding all-trans retinoic acid, red assembly falls and reduces to some extent, and the generation of medullary system and mixing hemocyte colony significantly increases, and illustrates that (RA) has vital role for inducing hematopoiesis-endothelial precursor cell to produce hemopoietic progenitor cell.More than each colony form experiment and all establish 4 repetitions.Among Fig. 7, red assembly falls and is made up of above-mentioned red corpuscle colony and Blast red corpuscle colony, and the medullary system colony is by above-mentioned granular leukocyte colony, the macrophage colony granulocyte, form with macrophage colony, mixing the hemocyte colony is above-mentioned red corpuscle, megalokaryocyte, granulocyte and macrophage colony.
(2) adopt the method that continues differentiation generation CD45 positive cell to detect
1. hematopoiesis-endothelial precursor cell (H1) that not adding in inducible factor control group (BW+VF+NC), RA treatment group (BW+VF+RA) and the Step II of obtaining among the above-mentioned division culture medium III-2a obtained by division culture medium II-2a (BW+VF), use trysinization respectively, the digestion that adds the substratum effect termination pancreatin that contains serum, use 40 μ m cell screen filtrations not to be digested to single celled cell mass, centrifugal collecting cell;
2. with cell centrifugation, and resuspended with StemSpan SFEM nutrient solution;
3. with the concentration of 40000 cell/ml cell is resuspended in and contains 10% foetal calf serum, 50ng/ml SCF, 50ng/ml Flt-3L is in the StemSpan nutrient solution of 10ng/mlIL-3 and 10ng/mlIL6;
4. re-suspended cell is joined in non-attaching 24 orifice plates;
5. put into incubator and cultivated 7 days, nutrient solution of replacing in per two days;
6.7 after it, adopt above-mentioned flow cytometry method to detect the expression of CD45.
The result as shown in Figure 8.Fig. 8 induces the hematopoiesis-endothelial precursor cell of generation for human embryo stem cell, in differentiation under the different inducible factor effects after 3 days, again through other 7 days induce differentiation after, express the detected result of the special sign of hemocyte CD45.BW+VF group for hematopoiesis-endothelial precursor cell directly through other 7 days control group of inducing after the differentiation; BW+VF+NC group continues differentiation three days for hematopoiesis-endothelial precursor cell under the condition that does not add any inducible factor, pass through other 7 days control group of inducing after the differentiation again; BW+VF+RA group continues differentiation after three days for hematopoiesis-endothelial precursor cell after adding RA, again through other 7 days treatment group of inducing after the differentiation.
Fig. 8 shows: add in the treatment group of all-trans retinoic acid (RA), cell can produce 47% CD45 positive blood cell through differentiation in extra 7 days; The control group that does not add the control group of all-trans retinoic acid and hematopoiesis-endothelial precursor cell produces 20% and 10%CD45 positive blood cell respectively.Because CD45 is the special sign of hematopoietic cell, illustrate that therefore all-trans retinoic acid (RA) has vital role for inducing hematopoiesis-endothelial precursor cell to produce hemopoietic progenitor cell.Each experiment repeats 4 times.
(3) adopt gene expression method to detect hemopoietic progenitor cell
1, detects the expression of the specific gene of the hemopoietic progenitor cell (H1) that is divided into
According to the method for said gene detection of expression detect obtain among the above-mentioned division culture medium III-2a do not add inducible factor control group (BW+VF+NC) and RA treatment group (BW+VF+RA).
The primer that detects gene GYPA is: Sense:ATGATACGCACAAACGG; Anti:CAATAACACCAGCCATC;
The primer that detects gene GATA1. is: Sense:TTAGCCACCTCATGCCTTTCCCT; Anti:CCAGAGACTTGGGTTGTCCAGAAT;
The primer that detects gene KDR is: Sense:CCTCGCCTTTGCCGATCC; Ant:GGATCTTCATGAGGTAGTCAGTC;
The primer that detects gene SCL is: Sense:AGAAAGGGACAATGATAGTAGAGTG; Anti:ATTCGGAACATAGACCAGACC;
The primer that detects gene LMO2 is: Sense:ACATAGCATCCAAGTGGCATAA; Anti:TCTAGTTCGCAAGCGTCAAA;
The primer that detects gene RUNX1 is: Sense:ATGTGGTCCTATTTAAGCCAGCCC; Anti:TCATCTGGCTGAAGACACCAGCTT;
The primer that detects gene GATA2 is: Sense:GCGTCTCCAGCCTCATCTT; Anti:GGAAGAGTCCGCTGCTGTAG;
The primer that detects gene M EOX1 is: Sense:AGAACCGAAGGATGAAGTGG; Anti:AGTCAGGGAAAGATGTGGAGA;
The primer that detects gene HOXB4 is: Sense:GCACGGTAAACCCCAATTA; Anti:GGCAACTTGTGGTCTTTTTT;
The primer that detects gene TIE2 is: Sense:CTGGACCTGTGAGACGC; Ant:CCACCTGGTATGCTGTTT;
The primer that detects gene C myb is: Sense:TGCTACCTCAGACACCCTC; Anti:TGCTCCTCCATCTTTCCA;
The primer that detects gene VE-CAD is: Sense:TGGAGAAGTGCCATCAGTCAACAG; Anti:TCTACAATCCCTTGCAGTGTGAG;
The primer that detects gene GAPDH is: Sense:AATCCCATCACCATCTTCC; Anti:CATCACGCCACAGTTTCC.
The result as shown in Figure 9.Fig. 9 induces the hematopoiesis-endothelial precursor cell of generation for human embryo stem cell, extra differentiation hematopoiesis and endothelium Expression of Related Genes detected result in the time of 3 days under different inducible factor effects.The BW+VF+NC group is broken up 3 days control group for hematopoiesis-endothelial precursor cell under the condition that does not add any inducible factor; The BW+VF+RA group is hematopoiesis-endothelial precursor cell differentiation treatment group in the time of 3 days under the condition that adds RA.
Fig. 9 shows: add in the test group of all-trans retinoic acid (RA), the control group that the expression of red corpuscle marker gene GYPA, SCL, GATA1, LMO2 does not add all-trans retinoic acid (RA) relatively significantly lowers; And the hemopoietic progenitor cell marker gene adds in the treatment group of VEGF, and the expression of cell expressing hematopoiesis endothelium specific gene RUNX1, GATA2, Cmyb, HOXB4 etc. is risen to some extent.Illustrate that all-trans retinoic acid (RA) has vital role for inducing hematopoiesis-endothelial precursor cell to produce hemopoietic progenitor cell.
3 multiple experiments are all established in the preparation of above cell and detection.Show that to sum up all-trans retinoic acid (RA) has vital role for inducing hematopoiesis-endothelial precursor cell to produce hemopoietic progenitor cell.
In addition, on the basis of division culture medium III-2a, the final concentration preparation that only changes inducible factor obtains division culture medium III-2b and division culture medium III-2c; Described division culture medium III-2b is for to add all-trans retinoic acid in the phase III division culture medium, the final concentration that makes described all-trans retinoic acid is 1.2 μ M; Described division culture medium III-2c is for to add all-trans retinoic acid in the phase III division culture medium, the final concentration that makes described all-trans retinoic acid is 3 μ M; Hematopoiesis-the endothelial precursor cell (H1) that adopts described division culture medium III-2b and described division culture medium III-2c to induce respectively according to the method described above to obtain through division culture medium II-2a obtains hemopoietic progenitor cell (H1), and experimental result shows: RA treatment group (BW+VF+RA) cell all produces 45% CD45 positive blood cell through differentiation in extra 7 days.
2, detect the expression of specific gene between different differentiation phases in hemopoietic progenitor cell (H1) preparation process
Method detection according to the said gene detection of expression is differentiated to form in the process of hemopoietic progenitor cell (H1) specific gene of different time sections cell expressing at people ES clone H1.
The primer that wherein detects gene OCT4, T, KDR, SCL, LMO2, RUNX1, GATA2, Cmyb, HOXB4, CD34 and GAPDH is with the above-mentioned primer that provides.
The primer that detects gene RALDH2 is: Sense:AGAAGAAGCGTGGAGCG; Anti:GGGCATTTAAGGCATTGTA;
The primer that detects gene C D45 is: Sense:CATTTGGCTTTGCCTTTCTG; Anti:TTCTCTTTCAAAGGTGCTTGC.
The result as shown in figure 10.Figure 10 for human embryo stem cell through people BMP4 and people Wnt3a handle formed mesoblastema in 1.5 days after, behind remarkable VEGF and people bFGF processing formation in 2.5 days hematopoiesis-endothelial precursor cell, pass through all-trans retinoic acid (RA) again and handle the genetic expression detected result that produces this complete procedure of hemopoietic progenitor cell after 3 days again.D represents fate.Figure 10 shows: human embryo stem cell at first was divided into mesoblastema through about 1.5 days, expressed specific gene T; Be divided into hematopoiesis-endothelial precursor cell through about 2.5 days again subsequently, in this process, express specific gene KDR, SCL, LMO2, RUNX1, GATA2, Cmyb, HOXB4, CD34 etc., and no longer express mesoderm gene T; Be divided into hemopoietic progenitor cell again through about 3 days, rise to some extent in this stage hemopoietic progenitor cell Expression of Related Genes, and the expression of the special marker gene CD45 of hematopoiesis occurs.
3 multiple experiments are all established in the preparation of above cell and detection.To sum up show, human embryo stem cell be divided into hemopoietic progenitor cell process simulation the main process of hematopoietic cell fetal development.
The drug toxicity of embodiment 2, hemopoietic progenitor cell detects
The enriching and purifying of I, hemopoietic progenitor cell
One, method
1. division culture medium III-2a is cultivated the hemopoietic progenitor cell (H1) that obtains and add trysinization, the digestion that adds the substratum termination pancreatin that contains serum again, use 40 μ m cell screen filtrations not to be digested to single celled cell mass, centrifugal collecting cell; The substratum that contains serum is the aforementioned DMEM substratum that contains 10% (volumn concentration) foetal calf serum.
2. cell is resuspended in the phosphoric acid buffer that contains 2% (volumn concentration) foetal calf serum, but cell uses wash-out CD34 magnetic bead antibody 4 ℃ of refrigerator labeled cells 20 minutes;
3. cell centrifugation is collected, and uses the phosphoric acid buffer re-suspended cell that contains 2% foetal calf serum;
4. cell is added in the mounted MACS separator column, cell can be dirty under action of gravity;
5. treat that cell is all dirty, add the phosphoric acid buffer that 0.5ml contains 2% foetal calf serum, and wait for that liquid is dirty
6. repeating step 5;
7. take off the MACS separator column, be placed on the 15ml centrifuge tube, and add the phosphoric acid buffer that 1ml contains 2% foetal calf serum;
8. release nutrient solution and cell with the piston of MACS separator column;
9. the cell that is obtained adds the magnetic bead separation agent, and 4 ℃ of refrigerators were placed 10 minutes;
10. add to separate and stop reagent;
11. cell centrifugation is collected, and uses the phosphoric acid buffer re-suspended cell that contains 2% foetal calf serum;
12. cell is resuspended in the phosphoric acid buffer that contains 2% foetal calf serum, and cell uses C-Kit magnetic bead antibody (beautiful day Ni biotech company of Germany, catalog number is 130-091-332) 4 ℃ of refrigerator labeled cells 15 minutes;
13. cell centrifugation is collected, and uses the phosphoric acid buffer re-suspended cell that contains 2% foetal calf serum;
14. cell is added in the mounted MACS separator column, and cell can be dirty under action of gravity;
15. treat that cell is all dirty, add the phosphoric acid buffer that 0.5ml contains 2% foetal calf serum, and wait for that liquid is dirty;
16. repeating step 5;
17. take off the MACS separator column, be placed on the 15ml centrifuge tube, and add the phosphoric acid buffer that 1ml contains 2% foetal calf serum;
18. the piston with the MACS separator column is released nutrient solution and cell.
Two, the detection of enrichment of cell
The above-mentioned hemopoietic progenitor cell (H1) that obtains enrichment CD34 and C-Kit is detected the situation of its expression CD34, C-Kit and other marker gene with the method for flow cytometry.
Detecting antibody is FITC mark CD34 (available from U.S. company BD, catalog number is 348053), APC mark C-Kit (available from U.S. company BD, catalog number is 340529), and APC mark TIE2 is (available from U.S. R﹠amp; Dsystems company, catalog number is FAB3131A), PE mark VE-Cad is (available from U.S. R﹠amp; D systems company, catalog number is FAB9381P), APC mark GYPA is (available from U.S. company BD, catalog number is 551336), PE mark CD41 is (available from U.S. company BD, catalog number is 555467), APC mark CD73 (available from U.S. company BD, catalog number is 550257), PE mark CD45 is (available from U.S. company BD, catalog number is 555483), APC mark CD34 (available from U.S. company BD, catalog number is 555824), PE mark CD43 is (available from U.S. company BD, catalog number is 560199), APC mark CD90 is (available from U.S. R﹠amp; D systems company, catalog number is FAB2067A), PE mark CD143 is (available from U.S. R﹠amp; D systems company, catalog number is FAB929P), PE mark CD105 (available from U.S. company BD, catalog number is FAB10971P) and PE mark CXCR4 (available from U.S. company BD, catalog number is 557145).
Result such as Figure 11 and shown in Figure 12.
The enrichment result as shown in figure 11, wherein (A) for utilizing CD34 and C-Kit as surface marker, the detected result that the cell that differentiation produced after 7 days to human embryo stem cell carries out sorting; (B) for utilizing the detected result of 4 groups of cell expressing hematopoiesis specific genes that produce after CD34 and the C-Kit sorting hemopoietic progenitor cell; (C) utilize 4 groups of cells that produce after CD34 and the C-Kit sorting hemopoietic progenitor cell to produce the detected result of hemocyte colony; (D) utilize 4 groups of cells that produce after CD34 and the C-Kit sorting hemopoietic progenitor cell to induce differentiation through other 7 days after, express the detected result of the special sign of hemocyte CD45.1 is not sorting; 2 is the CD34 feminine gender; 3 is the CD34 positive; 4 is the CD34 and the C-Kit positive, find out CD34 and the positive hemopoietic progenitor cell of C-Kit that shows after the enrichment from B, high expression level CD34 and C-Kit, and other hematopoietic cell specific gene LMO2, SCL, GATA2, RUNX1, Cmyb, HOXB4 etc. do not express paraxial mesoderm specific gene MEOX1.
The potential of enrichment of cell hematopoietic differentiation is found out from C to show the hemopoietic progenitor cell of utilizing after CD34 and the C-Kit enrichment as shown in figure 11, can be produced high efficiency hemocyte colony; Find out after breaking up from D, can produce the hemocyte of expressing the special sign of hemocyte CD45 more than 95%, be higher than the positive and unsorted cell of CD34 through other 7 days.
The situation that enrichment of cell hematopoiesis and other pedigree specific gene are expressed as shown in figure 12.Show the hemopoietic progenitor cell of utilizing after CD34 and the C-Kit enrichment, high expression level hematopoiesis genes involved CD31, KDR, TIE2, CD90, CD43 etc. express red-macronucleus specific gene GYPA and CD41 on a small quantity.In addition, utilize the hemopoietic progenitor cell after CD34 and the C-Kit enrichment, also express signs such as CD73, CD143, CD105, CXCR4 to some extent.
3 multiple experiments are all established in the preparation of above cell and detection.To sum up show, utilize CD34 and C-Kit can effectively produce the human embryo stem cell source hemopoietic progenitor cell (H1) of enrichment CD34 and C-Kit.
The hemopoietic progenitor cell of II, use enriching and purifying is carried out drug toxicity and is detected
One, preparation method
1. human cord blood source hemopoietic progenitor cell preparation method:
(1) gets one bag of human cord blood from obstetrics and gynecology hospital;
(2) lymphocyte separation medium of the preheating amount branch with the 20ml/50ml centrifuge tube is filled in several 50ml centrifuge tubes;
(3) the 20ml Cord blood is slowly added have in the 50ml of the 20ml lymphocyte separation medium centrifuge tube;
(4) with centrifuge tube centrifugal 30 minutes with 2000rpm;
(5) centrifugal good cell is taken out, this moment, liquid was two-layer about being divided into, and the middle level is a lymphocyte;
(6) lymphocyte is taken out from centrifugal good cell middle level;
(7) cell is resuspended in the phosphoric acid buffer that contains 3% (volumn concentration) foetal calf serum, cell uses CD34 magnetic bead antibody 4 ℃ of refrigerator labeled cells 20 minutes;
(8) cell centrifugation is collected, and uses the phosphoric acid buffer re-suspended cell that contains 2% (volumn concentration) foetal calf serum;
(9) cell is added in the mounted MACS separator column, cell can be dirty under action of gravity;
(10) treat that cell is all dirty, add the phosphoric acid buffer that 0.5ml contains 2% foetal calf serum, and wait for that liquid is dirty;
(11) repeating step is (10) 1 times;
(12) take off the MACS separator column, be placed on the 15ml centrifuge tube, and add the phosphoric acid buffer that 1ml contains 2% foetal calf serum;
(13) release nutrient solution and cell with the piston of MACS separator column;
(14) separation of human cord blood source hemopoietic progenitor cell finishes.
Two, drug toxicity detects
(1) the hemocyte colony forms experiment
1. the human embryo stem cell source hemopoietic progenitor cell (H1) of enrichment CD34 that obtains among the step I with present embodiment and C-Kit is resuspended in the resuspended liquid with the concentration of 5000 cell/100ul.
2. adding 1ml does not have the β mercaptoethanol is not had erythropoietin methylcellulose gum substratum, and abundant mixing.
3. add 1.1ul concentration respectively and be 0.2,1,5,20,100, the medicine Adriamycin of 500ug/ml, make that its final concentration in system is respectively 0.2,1,5,20,100,500ng/ml.Add 1.1ul dimethyl sulfoxide (DMSO) (DMSO) in dimethyl sulfoxide (DMSO) (DMSO) control group.Thorough mixing is even.
4. the mixing cell is left standstill 2-5 minute, so that bubble is eliminated.
5. the cell with mixing joins in the 3.5 centimetres of culture dish that are untreated, and puts into incubator and cultivates 14 days for 37 ℃.Keep humidity.
6.14 after it, the human embryo stem cell source hemopoietic progenitor cell after obtaining handling takes out cell, counting.
Count after adopting same way to handle the human cord blood source hemopoietic progenitor cell of above-mentioned acquisition.
Use the same method and adopt drug toxicity Taxo1 to handle the back counting respectively the enrichment CD34 of above-mentioned acquisition and human embryo stem cell source hemopoietic progenitor cell (H1) and the human cord blood source hemopoietic progenitor cell of C-Kit.
The result as shown in figure 13.Numerical value among Figure 13 is the relative value that relative dimethyl sulfoxide (DMSO) (DMSO) is deposited medullary system colony number.Figure 13 shows that drug toxicity Adriamycin and Taxol to human embryo stem cell (H1) source hemopoietic progenitor cell and human cord blood source hemopoietic progenitor cell, all have similar concentration dependent toxicity.
3 multiple experiments are all established in the preparation of above cell and detection.Show that to sum up the hemopoietic progenitor cell of utilizing the human embryo stem cell source that is obtained by the present invention can be used as the cell sample that drug toxicity detects.
I, embryonic stem cell are induced to differentiate into mesoblastema
One, preparation method
Preparation method's inducing human embryo stem cell (H9) in division culture medium I-2a is divided into mesoblastema (H9) described in the employing embodiment 1-I.
Two, differentiated result detects
Adopt the above-mentioned mesoblastema that in division culture medium I-2a, obtains of method (H9) of flow cytometry.Experimental result shows: in people BMP4 and people Wnt3a combination treatment group (BMP4+Wnt3a) (adopting division culture medium I-2a) 93% cytodifferentiation being arranged is the positive mesoblastema of T.
II, mesoblastema are divided into hematopoiesis-endothelial precursor cell
One, preparation
Adopt preparation method described in the embodiment 1-II in division culture medium II-2a, to induce above-mentioned mesoblastema (H9) to produce hematopoiesis-endothelial progenitor cells (H9).
Two, differentiated result detects
Adopt the mesoblastema (H9) of the above-mentioned acquisition of method of flow cytometry.Experimental result shows: in people VEGF and people bFGF combination treatment group (VEGF+bFGF) (adopting division culture medium II-2a), it is the positive PDGFR α of KDR negative hematopoietic-endothelial progenitor cells that 87% cytodifferentiation is arranged.
III, hematopoiesis-endothelial precursor cell are divided into hemopoietic progenitor cell
One, preparation
Adopt preparation method described in the embodiment 1-III in division culture medium III-2a, to induce hematopoiesis-endothelial precursor cell (H9) to produce hemopoietic progenitor cell (H9).
Two, detect
The method that adopts flow cytometry and hemocyte colony to form detects the above-mentioned mesoblastema that obtains (H9) in division culture medium III-2a.Experimental result shows: under the processing (adopting division culture medium III-2a) of vitamin A acid, the hemopoietic progenitor cell that human embryo stem cell (H9) induces differentiation to produce can produce a large amount of medullary systems and mix the hemocyte colony, can also produce 42% CD45 positive blood cell.
The drug toxicity of embodiment 4, hemopoietic progenitor cell (H9) detects
The enriching and purifying of I, hemopoietic progenitor cell (H9)
One, method
Adopt the described method of embodiment 2-I to prepare the hemopoietic progenitor cell (H9) of enrichment CD34 and C-Kit.
Two, the detection of enrichment of cell
Adopt the method detection enrichment CD34 of flow cytometry and the hemopoietic progenitor cell (H9) of C-Kit.After through differentiation in other 7 days, the hemopoietic progenitor cell of enrichment CD34 and C-Kit (H9) can produce the hemocyte of expressing the special sign of hemocyte CD45 more than 96%, is higher than the positive and unsorted cell of CD34.
The hemopoietic progenitor cell (H9) of II, use enriching and purifying is carried out drug toxicity and is detected
Detection method among the employing embodiment 2 detects the hemopoietic progenitor cell (H9) of the enrichment CD34 and the C-Kit of above-mentioned acquisition.The result as shown in figure 14.Numerical value among Figure 14 is the relative value that relative dimethyl sulfoxide (DMSO) (DMSO) is deposited medullary system colony number.Figure 14 shows, drug toxicity Adriamycin and Taxol to human embryo stem cell source hemopoietic progenitor cell (H9) and human cord blood source hemopoietic progenitor cell, all have similar concentration dependent toxicity.
Claims (10)
1. inducing human embryo stem cell is divided into the substratum of hemopoietic progenitor cell, and by division culture medium I, division culture medium II and division culture medium III form; Division culture medium I is division culture medium I-1 or division culture medium I-2; Division culture medium II is division culture medium II-1 or division culture medium II-2; Division culture medium III is division culture medium III-1 or division culture medium III-2;
The substratum that former protein-4 obtains takes place for add the bone form in the fs division culture medium in described division culture medium I-1; Former protein-4 final concentration takes place described bone form among the described division culture medium I-1 is 10-100ng/ml;
The substratum that former protein-4 and Wnt3a obtain takes place for add the bone form in the fs division culture medium in described division culture medium I-2; The final concentration that former protein-4 takes place described bone form among the described division culture medium I-2 is 10-100ng/ml, and the final concentration of described Wnt3a is 50-150ng/ml;
Described division culture medium II-1 is for adding the substratum that vascular endothelial growth factor obtains in the subordinate phase division culture medium; The final concentration of the described vascular endothelial growth factor among the described division culture medium II-1 is 50-150ng/ml;
Described division culture medium II-2 is for adding the substratum that vascular endothelial growth factor and Prostatropin obtain in the subordinate phase division culture medium; The final concentration of the described vascular endothelial growth factor among the described division culture medium II-2 is 50-150ng/ml, and the final concentration of described Prostatropin is 50-150ng/ml;
Described division culture medium III-1 is the phase III division culture medium; Described division culture medium III-2 is for adding the substratum that all-trans retinoic acid obtains in the phase III division culture medium, the final concentration of the described all-trans retinoic acid among the described division culture medium III-2 is 1-5 μ M;
Described fs division culture medium is for adding the substratum that bovine serum albumin obtains being used to cultivate on the basic cell culture medium of mammalian cell; The final concentration of the described bovine serum albumin in the described fs division culture medium is 0.1-1mg/ml;
Described subordinate phase division culture medium is for adding the substratum that Regular Insulin-Transferrins,iron complexes-selenium salt, vitamins C acid and thioglycerin obtain on described fs division culture medium; The final concentration of the described Regular Insulin-Transferrins,iron complexes in the described subordinate phase division culture medium-selenium salt is 0.5-1.5% (volumn concentration), and the final concentration of described vitamins C acid is 0.1-1mmol/l, and the final concentration of described thioglycerin is 0.1-1mmol/l;
Described phase III division culture medium is for to replace to the substratum that B27 obtains with the Regular Insulin-Transferrins,iron complexes in the described subordinate phase division culture medium-selenium salt, and the final concentration of the described B27 in the described phase III division culture medium is 0.5-1.5% (volumn concentration).
2. substratum according to claim 1 is characterized in that:
The final concentration that former protein-4 takes place described bone form among described division culture medium I-1 and the I-2 is 10,20,40,50,60,80 or 100ng/ml, and the final concentration of the described Wnt3a among the described division culture medium I-2 is 50,60,80,100,120 or 150ng/ml;
The final concentration of the described vascular endothelial growth factor among described division culture medium II-1 and the II-2 is 50,60,80,100,120 or 150ng/ml, and the final concentration of the described Prostatropin among the described division culture medium II-2 is 50,60,80,100,120 or 150ng/ml;
The final concentration of the described all-trans retinoic acid among the described division culture medium III-2 is 1,1.2,1.5,2,2.5,3,4 or 5 μ M;
The final concentration of the bovine serum albumin in the described fs division culture medium is 0.1,0.2,0.4,0.5,0.6,0.8 or 1mg/ml;
The final concentration of the described vitamins C acid in described second division culture medium is 0.1,0.2,0.4,0.5,0.6,0.8 or 1mmol/l, the final concentration of described thioglycerin is 0.1,0.2,0.4,0.5,0.6,0.8 or 1mmol/l, and the final concentration of described Regular Insulin-Transferrins,iron complexes-selenium salt is 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.4% or 1.5%;
The final concentration of described B27 in the described phase III division culture medium is 0.5%, 0.7%, 0.9%, 1%, 1.2%, 1.4% or 1.5%.
3. substratum according to claim 1 is characterized in that:
The final concentration that former protein-4 takes place described bone form among described division culture medium I-1 and the I-2 is 20-80ng/ml, and the final concentration of Wnt3a is 80-120ng/ml described in the described division culture medium I-2; The final concentration of the described vascular endothelial growth factor among described division culture medium II-1 and the II-2 is 80-120ng/ml, and the final concentration of Prostatropin is 80-120ng/ml described in the described division culture medium II-2; The final concentration of the described all-trans retinoic acid among the described division culture medium III-2 is 1.2-3 μ M;
The final concentration of bovine serum albumin is 0.4-0.6mg/ml in the described fs division culture medium; The final concentration of the described vitamins C acid in the described subordinate phase division culture medium is 0.2-0.8mmol/l, and the final concentration of described thioglycerin is 0.2-0.8mmol/l, and the final concentration of described Regular Insulin-Transferrins,iron complexes-selenium salt is 0.7-1.2%; The final concentration of described B27 in the described phase III division culture medium is 0.7-1.2%.
4. according to claim 1,2 or 3 described, it is characterized in that: described basic cell culture medium is selected from following 6 kinds of substratum at least a: α-MEM, DMEM, DMEM/F12, RPMI1640, F12 and IMDM.
5. the substratum inducing human embryo stem cell that adopts arbitrary inducing human embryo stem cell among the claim 1-4 to be divided into hemopoietic progenitor cell is divided into the method for hemopoietic progenitor cell, and it may further comprise the steps:
1) be that human embryo stem cell is cultivated among arbitrary described division culture medium I-1 or the described division culture medium I-2 in claim 1-4;
2) will change over to by the cell that step 1) obtains among the claim 1-4 among arbitrary described division culture medium II-1 or the described division culture medium II-2 and cultivate;
3) will be through step 2) cell that obtains changes among the claim 1-4 among arbitrary described division culture medium III-1 or the described division culture medium III-2 and cultivates, and obtains hemopoietic progenitor cell.
6. method according to claim 5 is characterized in that: described human embryo stem cell is the clone that commercial sources obtains.
7. according to claim 5 or 6 described methods, it is characterized in that: described human embryo stem cell is following any clone: BG01, BG02, BG03, BG04, SA01, SA02, SA03, ES01, ES02, ES03, ES04, ES05, ES06, TE03, TE32, TE33, TE04, TE06, TE62, TE07, TE72, UC01, UC06, WA01, WA07, WA09, WA13 and WA14; The described numbering that is numbered NIH.
8. according to arbitrary described method in the claim 5 to 7, it is characterized in that:
The culture condition of described step 1) is 37 ℃ to be cultivated 1-2 days, was preferably 1.5 days;
Described step 2) culture condition is 37 ℃ to be cultivated 2-3 days, was preferably 2.5 days;
The culture condition of described step 3) is 37 ℃ to be cultivated 2-4 days, was preferably 3 days.
9. the application of the hemopoietic progenitor cell that arbitrary described method obtains in the claim 5 to 8 in the preparation hemocyte.
10. the application of hemopoietic progenitor cell in drug toxicity detects that arbitrary described method obtains in the claim 5 to 8, described drug toxicity detects the drug toxicity that adopts and is preferably adriamycin or Tyke rope.
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