CN103361310B - Substratum, test kit and uses thereof - Google Patents
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Abstract
The invention discloses substratum, test kit, the early stage mesodermal progenitor cell of preparation method and utilize test kit of the present invention to prepare the method for hemogenic endothelium cell.Wherein, this substratum comprises: basic medium, and this basic medium is SFDM substratum; PGE2 and BMP4.Utilize this substratum of the present invention efficiently can prepare a large amount of early stage mesodermal progenitor cell fast.
Description
Technical field
The present invention relates to early stage mesodermal progenitor cell and hemogenic endothelium cell regeneration techniques field, relate to substratum, test kit and uses thereof particularly.More specifically, the present invention relates to substratum, test kit, the early stage mesodermal progenitor cell of preparation method and utilize test kit of the present invention to prepare the method for hemogenic endothelium cell.
Background technology
Hemocyte infusion and hematopoietic stem cell transplantation are the common methods of cell therapy, but its application is limited by donor source puzzlement in short supply for a long time, strongly limit the promotion and application of this treatment means.Thus, the new source finding hemopoietic stem cell and mature blood cell becomes one of research direction that regenerative medicine field attracts most attention.
HESC (human embryonic stem cells, or inductive pluripotent stem cells (inducedpluripotent stem cells hESCs), iPSCs) multipotential cell of self and infinite multiplication potential is possessed as a class, there is the potential to hemopoietic stem cell differentiation, for the regeneration of hemopoietic stem cell and mature blood cell and a large amount of preparation provide infusive prospect.But there is the deficiencies such as differentiation-inducing efficiency is low, noble cells function difference in the scheme of inducing hESCs/iPSCs to be divided into hemopoietic stem cell at present, this has coverd with shade to the potential applicability in clinical practice of this technology.HESCs/iPSCs needs to experience early stage mesodermal progenitor cell and hemogenic endothelium cell stage successively to hemopoietic stem cell differentiation, optimize the scheme of hESCs/iPSCs to these two phase cell differentiation, these cells will inevitably be improved further to the differentiation efficiency of hemopoietic stem cell, make noble cells better play function.
But the method that current external evoked hESCs/iPSCs is divided into early stage mesodermal progenitor cell or hemogenic endothelium cell still haves much room for improvement.
Summary of the invention
The present invention is intended at least to solve one of technical problem existed in prior art.For this reason, one object of the present invention is to propose a kind ofly to can be used in external evoked hESCs/iPSCs and be divided into the substratum of early stage mesodermal progenitor cell or hemogenic endothelium cell, test kit and method.
It should be noted that, the present invention completes based on the following discovery of contriver:
Current known person embryonic stem cells into hematopoietic cells atomization simulates each period that hematopoietic cell is grown in vivo substantially, i.e. early stage mesodermal progenitor cell stage, hemogenic endothelium cell stage, and is finally divided into hemopoietic stem cell and ripe hemocyte.Therefore, inducing human embryo stem cell is efficiently divided into early stage mesodermal progenitor cell and hemogenic endothelium cell is improve the key link of human embryo stem cell to hemopoietic stem cell differentiation efficiency.Current research shows that cytokine profiles and transcriptional regulation protein play a significant role to hemopoietic stem cell is differentiation-inducing in early days at human embryo stem cell.But hematopoietic cell differentiation in vivo and maturation are the symphyogenetic results of many factors in Time and place aspect, wherein not only comprise numerous signal path and the regulation and control of transcribing molecule in cell, also relate to interaction and the feedback regulation of the complexity between cell and microenvironment simultaneously.
Contriver is found by research, and prostaglandin E2 (prostaglandin, PGE2) plays key player at human embryo stem cell to hemopoietic stem cell differentiation commitment.PGE2 is the important growth of one and regulatory factor that extensively exist in human body.It is the unsaturated fatty acids that a class contains 20 carbon atoms, and its basic framework is five carbocyclic rings and two side chains, is generated in vivo by arachidonic acid (arachidonic acid, AA) through oxidative pathway.When body cell be subject to some machinery, physics or chemical stimulation time, Phospholipase A2 (the phospholipase of cell, PLA2) be activated, make to dissociate with membrane-bound AA, then PGH2(prostaglandin H2 is converted to by cyclooxygenase (COX-1 or COX-2) effect), PGH2 generates rapidly through the effect of cell-specific enzyme has bioactive prostanoid medium.PGE2 take part in immunne response, acute phase reaction as autocrine and the Paracrine factor in normal body, maintains the complete and tension force of blood vessel, nerve growth and the important physiological process such as growth and hematopoiesis adjustment.In addition, further research finds as a kind of important regulatory factor, PGE2 participates in that hemopoietic stem cell is grown, bred, in the process such as to go back to the nest to have laboratory to utilize zebra fish and mice develop model to carry out.
The research work of contriver shows, PGE2 human embryo stem cell to hemopoietic stem cell break up early stage, namely early stage mesodermal progenitor cell and hemogenic endothelium stage play extremely important regulating and controlling effect.Particularly, the means such as activity inhibitor or RNA interference are utilized to block PGE2 speed limit synthetic enzyme---after the effect of cyclooxygenase, human embryo stem cell obviously reduces to the differentiation-inducing efficiency of mesodermal progenitor cell and hemogenic endothelium cell; And in differentiation-inducing process, add PGE2 effectively can strengthen the induced efficiency of human embryo stem cell to early stage mesodermal progenitor cell and hemogenic endothelium cytodifferentiation.
Thus, based on the above-mentioned discovery of contriver, the invention provides and can be used in substratum, test kit and the method that external evoked ES cell differentiation is early stage mesodermal progenitor cell or hemogenic endothelium cell.
According to an aspect of the present invention, the invention provides a kind of substratum.According to embodiments of the invention, this substratum comprises: basic medium, and this basic medium is SFDM substratum; PGE2 and BMP4(and bone morphogenetic protein 4).Contriver is surprised to find, above-mentioned substratum of the present invention is utilized efficiently to induce ESCs to break up to early stage mesodermal progenitor cell rapidly, thus effectively can prepare a large amount of early stage mesodermal progenitor cell, and then effectively for the induction of hemopoietic stem cell and mature blood cell, thus can be applied to the treatment of hematologic disease.
In addition, substratum according to the above embodiment of the present invention can also have following additional technical characteristic:
According to one embodiment of present invention, in the substratum of the invention described above, the concentration of PGE2 is 0.5-10 μM, preferably 1 μM.Thereby, it is possible to effectively inducing human embryo stem cell breaks up to early stage mesodermal progenitor cell.
According to one embodiment of present invention, in the substratum of the invention described above, the concentration of BMP4 is 10-50ng/ml, preferred 25ng/ml.Thus, utilize this substratum, the efficiency that inducing human embryo stem cell is divided into early stage mesodermal progenitor cell can be significantly improved.
According to another aspect of the invention, present invention also offers a kind of test kit.According to embodiments of the invention, this test kit comprises: the first substratum, and this first substratum is foregoing substratum; Second substratum, this second substratum is SFDM substratum, is added with PGE2, VEGF(and vascular endothelial growth factor in this SFDM substratum) and FGF2(and Prostatropin).According to some embodiments of the present invention, utilize test kit of the present invention, can break up to early stage mesodermal progenitor cell and hemogenic endothelium cell by inducing embryo stem cell efficiently, and then effectively for the induction of hemopoietic stem cell and mature blood cell, thus can be applied to the treatment of hematologic disease.
According to one embodiment of present invention, in the second substratum, the concentration of PGE2 is 0.5-25 μM, preferably 1 μM.According to another embodiment of the invention, in the second substratum, the concentration of VEGF is 25-100ng/ml, and the concentration of preferred 50ng/ml, FGF2 is 25-100ng/ml, preferred 50ng/ml.Thus, the second substratum in test kit of the present invention can significantly improve the differentiation-inducing efficiency of early stage mesodermal progenitor cell to hemogenic endothelium cell of external evoked acquisition.
In accordance with a further aspect of the present invention, present invention also offers a kind of method preparing early stage mesodermal progenitor cell.According to embodiments of the invention, the method comprises: utilize foregoing substratum of the present invention, cultivate stem cell, to obtain early stage mesodermal progenitor cell.Wherein, it should be noted that, substratum adopted here is aforesaid first substratum.Contriver is surprised to find, and utilizes the method can prepare a large amount of early stage mesodermal progenitor cell efficiently, and then can be effective to the induction of hemopoietic stem cell and mature blood cell, thus can be applied to the treatment of hematologic disease.
According to one embodiment of present invention, stem cell is embryonic stem cell, multipotential stem cell or inductive pluripotent stem cells in the method, preferred embryo stem cell.
According to a further aspect in the invention, present invention also offers a kind of method utilizing foregoing test kit to prepare hemogenic endothelium cell.According to embodiments of the invention, the method comprises: utilize this first substratum to cultivate embryonic stem cell, to obtain early stage mesodermal progenitor cell; And utilize this second substratum to cultivate, to obtain hemogenic endothelium cell this early stage mesoderm group cell.Contriver is surprised to find, and utilizes the method can prepare a large amount of hemogenic endothelium cells efficiently, and then can be effective to the induction of hemopoietic stem cell and mature blood cell, thus can be applied to the treatment of hematologic disease.
According to one embodiment of present invention, utilize the first substratum to cultivate 24-96 hour to this embryonic stem cell, utilize the second substratum to cultivate 72-96 hour to this early stage mesoblastema.Thereby, it is possible to effectively prepare hemogenic endothelium cell.
It should be noted that substratum of the present invention, test kit and prepare the method for early stage mesodermal progenitor cell and hemogenic endothelium cell are all that the present inventor just completes through arduous creative work and a large amount of Optimization Works.
Additional aspect of the present invention and advantage will part provide in the following description, and part will become obvious from the following description, or be recognized by practice of the present invention.
Accompanying drawing explanation
Above-mentioned and/or additional aspect of the present invention and advantage will become obvious and easy understand from accompanying drawing below combining to the description of embodiment, wherein:
Fig. 1 shows human embryo stem cell in embodiment 1 to mesodermal progenitor cell differential period, cellular form figure when human embryo stem cell is through cultivating 0h, 24h and 48h;
Fig. 2 shows human embryo stem cell in embodiment 1 to mesodermal progenitor cell differential period, the real-time quantitative PCR detected result of each marker gene when human embryo stem cell is through cultivating 0h, 24h and 48h;
Fig. 3 shows human embryo stem cell in embodiment 1 to mesodermal progenitor cell differential period, the identified by immunofluorescence result of marker protein when human embryo stem cell is through cultivating 0h, 24h and 48h;
Fig. 4 shows human embryo stem cell in embodiment 1 to mesodermal progenitor cell differential period, the real-time quantitative PCR detected result of human embryo stem cell mesoderm marker gene Brachyury after different concns PGE2 process; And
Fig. 5 shows PGE2 process inducing human embryo stem cell in embodiment 1 and, after hemogenic endothelium cytodifferentiation, adopts its figuratrix marker protein of flow cytometry to express.
Embodiment
Below in conjunction with embodiment, the solution of the present invention is made an explanation.It will be understood to those of skill in the art that the following examples only for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition in embodiment, according to the technology described by the document in this area or condition or carry out according to product description.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
Embodiment 1
1, the cultivation of human embryonic stem cell
Contriver introduces U.S. Wicell research centre and sets up internationally recognized human embryonic stem cell H1 and H9.Using for reference the human embryo stem cell culture system domestic and international data of literatures basis set up and is applicable to actual working environment and condition, the feeder free system based on the mTeSR substratum comprising Dual culture culture system that the inoblast that utilizes E13.5 days ICR Strains of Mouse embryos to be separated to obtain is feeder layer and utilize Matrigel matrigel and definite ingredients.Culturing cell is differentiated by flow cytometry and immunofluorescence technique the expression detecting the totipotency marker proteins such as SSEA4, Oct4, Nanog, Sox2, and ensure that more than 90% cell is positive cell, alkaline phosphatase (AP) stained positive, in the differentiation-inducing experiment of external triploblastica and body, teratoma is formed after experimental result is the positive, for subsequent use.
2, human embryo stem cell is to early stage mesodermal progenitor cell and hemogenic endothelium cell induction differentiation model
With reference to the method for the early stage mesodermal progenitor cell of aforesaid preparation of the present invention and hemogenic endothelium cell, according to following human embryo stem cell to early stage mesodermal progenitor cell and hemogenic endothelium cell induction differentiation model, prepare early stage mesodermal progenitor cell and hemogenic endothelium cell:
2.1 human embryo stem cells are to the differentiation-inducing model of early stage mesodermal progenitor cell:
The human embryo stem cell obtained above is proceeded to feeder free system by MEF feeder layer co-culture system and cultivates 5 generations more than, with Dispase enzymic digestion after its growth traits is stable, digestion is stopped after clone edge is rolled, blow and beat into less clone and resuspended with mTeSR substratum, and be inoculated into incubated overnight in the orifice plate of pre-coated Matrigel matrigel, clone changes to the first substratum when attaching and start normal amplification, namely the SFDM(Serum-Free Differentiation Medium of 25ng/ml BMP4 cytokine and 1 μM of prostaglandin E2 is added with) substratum, in the 37 DEG C of fixed temperature and humidity cell culture incubators containing 5% carbonic acid gas, induction is after 48 hours, obtain early stage mesodermal progenitor cell.
Wherein, the formula of SFDM substratum is as follows:
IMDM/F12 mixed culture medium (wherein IMDM substratum and F12 culture volume are than 3:1)
In addition, MEF feeder layer co-culture system is: carry and being layered in the orifice plate of 0.1% gelatin bag quilt the inoblast that E13.5 days ICR mice embryonics of removing mitogen activation are separated as feeder layer the day before yesterday, wait to spend the night human embryo stem cell to be inoculated in this feeder layer cells and to change and cultivate at 37 DEG C of fixed temperature and humidity cell culture incubators containing 5% carbonic acid gas with special culture media, change liquid to every day this cell, with 1mg/ml collagenase IV human embryo stem cell digested after average 5-7 days and be inoculated in new feeder layer cells with the ratio of going down to posterity of 1:3-1:5.
Wherein, the substratum of raising human embryo stem cell consists of:
Feeder free system is: be inoculated into by human embryo stem cell in the orifice plate of pre-coated Matrigel matrigel and cultivate at 37 DEG C of fixed temperature and humidity cell culture incubators containing 5% carbonic acid gas with mTeSR substratum, change liquid every day and observation of cell state, with Dispase enzyme cell dissociation got off after 5-7 days and be inoculated on new pre-coated orifice plate with the ratio of going down to posterity of 1:3-1:5.
Wherein, Fig. 1 to show in the present embodiment human embryo stem cell to mesodermal progenitor cell differential period, cellular form figure when human embryo stem cell is through cultivating 0h, 24h and 48h.Wherein, as shown in the figure, from top to bottom, figure below is the enlarged view of upper figure.As seen from Figure 1, human embryo stem cell increases rapidly clone in the atomization of mesodermal progenitor cell, and clone's inner cell is loose gradually, and iuntercellular is apart from increasing, and cell volume becomes large, and cellular form is irregular, and nuclear-cytoplasmic ratio obviously reduces.
2.2 human embryo stem cells are to hemogenic endothelium cell induction differentiation model:
On the basis of above-mentioned human embryo stem cell to the differentiation-inducing model of early stage mesoderm, early stage for acquisition mesodermal progenitor cell is changed to the second substratum, namely the SFDM substratum of 50ng/ml VEGF and 50ng/ml FGF2 cytokine and 1 μM of prostaglandin E2 is added with, then cultivate in the 37 DEG C of fixed temperature and humidity cell culture incubators containing 5% carbonic acid gas after 48 hours and change liquid, cultivation is continued 48 hours, to obtain hemogenic endothelium cell after changing liquid.Wherein, the formula of SFDM substratum as previously mentioned.
Embodiment 2, the early stage mesodermal progenitor cell of differentiation-inducing acquisition to be identified
Collect the early stage mesodermal progenitor cell of inducing acquisition in embodiment 1, and it is identified.
One, authentication method
1, RT-Real Time PCR identifies
The early stage mesodermal progenitor cell (i.e. cell to be measured) obtained embodiment 1 respectively carries out the qualification of cell sign gene (i.e. goal gene) expression level, the goal gene that wherein the present embodiment is chosen comprises totipotency marker gene Nanog, Oct4, early stage mesodermal progenitor cell marker gene Brachyury, Mixl-1, EOMES, PDGFR α, WNT3, CDX2, CXCR4 and GSC, concrete steps are as follows:
First, with TRIZOL (invitrogen, 15596-018) reagent, cell total rna to be measured is extracted according to the method provided in product description.
Then, utilize PCR instrument (eppendorf, Mastercycler) that the mRNA in cell total rna to be measured is carried out reverse transcription.Wherein, reverse transcription system be:
The program of reverse transcription is: mix first three and plant reagent 70 DEG C insulation after 10 minutes rapidly at chilling more than 2 minutes on ice; Then add all the other reagent, 42 DEG C insulation 1 hour after 70 DEG C insulation 15 minutes after cooled on ice.
Thus, the cDNA of cell to be measured is obtained.
Then, utilize real-time PCR (Bio-Rad company, opticon2), according to the process specifications of product, the cDNA of the cell to be measured of above-mentioned acquisition is carried out goal gene amplification.Wherein, Real Time PCR reaction system is:
Real Time PCR program is:
Circulation 1:(1X)
Step 1:95.0 ° of C, 3 minutes.
Circulation 2:(45X)
Step 1:95.0 ° of C, 10 seconds;
Step 2:60.0 ° of C, 30 seconds;
Step 3:72.0 ° of C, 30 seconds;
Collect data;
Step 4:80.0 ° of C, 10 seconds;
Collect data and carry out real-time analysis.
Circulation 3:(81X)
Step 1:55.0 ° of C-95.0 ° of C, 10 seconds;
After the 2nd circulation, each circulation increase by 0.5 degree Celsius,
Collect melting curve data and analyze.
Circulation 4:(1X)
Step 1:4.0 ° of C, keeps.
The primers designed of above-mentioned each goal gene is as follows:
Wherein, each reaction is all independent in triplicate to eliminate systematic error.
Then, image data, utilizes Bio-Rad iQ5 software to process each data, and calculates the expression level of each correlating markings gene using people GAPDH as reference gene.
2, immunofluorescence dyeing (Immunofluorescence)
Cell to be measured is fixed 20 minutes with 4% paraformaldehyde room temperature, then with containing PBS solution (PBST) the room temperature rupture of membranes of 0.2%TritonX-100 after 10 minutes, 30 minutes are closed by the PBS solution room temperature containing 10% lowlenthal serum, again according to the ratio of 1:150, by mouse anti human Oct4(mouse anti human Oct4), goat-anti people Brachyury(goat anti humanBrachyury) dilute with confining liquid, after 4 DEG C of incubated cells spend the night, add sheep anti mouse FITC(goat anti mouseFITC), rabbit anti-goat TRITC(rabbit anti goat TRITC) two anti-Incubating Solution incubated at room 1 hour, then with 4', 6-diamidino-2-phenylindone lining transfect cell core is after 5 minutes, fluorescence is observed under fluorescence inverted microscope (Olympus).Wherein, between each step above, all wash three times with PBS, each 5 minutes.
3, cellular form observation
Under inverted microscope (Nikon TE2000-U), to observe in embodiment 1 human embryo stem cell respectively to mesodermal progenitor cell differential period, cellular form when human embryo stem cell is cultivated 0h, 24h and 48h.
Two, qualification result
By above-mentioned authentication method, contriver has carried out RT-PCR detection, immunological identification and cellular form to the early stage mesodermal progenitor cell in embodiment 1 and has detected, and the results are shown in Figure 2 and Fig. 3.
Wherein, Fig. 2 to show in embodiment 1 human embryo stem cell to mesodermal progenitor cell differential period, the RT-PCR detected result of each marker gene when human embryo stem cell is through cultivating 0h, 24h and 48h.As shown in Figure 2, in differentiation-inducing process, the expression level of human embryo stem cell dryness gene Oct4, Nanog extends with divergaence time and reduces, and the expression level of mesoderm marker gene Brachyury, Mixl-1, PDGFR α, WNT3, CDX2, CXCR4, GSC, EOMES raises, show that human embryo stem cell loses versatility and to mesodermal progenitor cell specialization under the induction of the BMP4 factor.
Fig. 3 shows in embodiment 1 that human embryo stem cell is to mesodermal progenitor cell differential period, and human embryo stem cell is through the identified by immunofluorescence result of induction sign albumen.As shown in Figure 3, carry out along with differentiation-inducing, the expression of versatility marker protein Oct4 declines gradually, and mesoblastic marker protein Brachyury staining positive cells ratio raises gradually.
Embodiment 3 different concns PGE2 is on the impact of human embryo stem cell to mesodermal progenitor cell and hemogenic endothelium cytodifferentiation
Collect early stage mesodermal progenitor cell and the hemogenic endothelium cell (i.e. cell to be measured) of in embodiment 1, inducing acquisition, and following experiment carried out to it:
1, RT-Real Time PCR identifies
Experimental technique is with embodiment 2.
2, Flow cytometry
By 1 × 10
6to be measured cells trypsinised be unicellular, to be resuspended in after washing one time with cold PBS in 100 μ l PBS and to add and carry the mixing of fluorescently-labeled corresponding antibodies, after lucifuge 4 DEG C hatches 45 minutes, the cold PBS of cell is washed twice to remove non-binding antibody, then be resuspended in 400 μ l PBS after cell 7-AAD being marked process, and machine testing is gone up in fluidic cell instrument (BD, FACSCalibur).Wherein, with 7 Amino Actinomycin D(7-AAD) negative cells group draw a circle to approve analyze door cell respective channel fluorescence is analyzed.
The results are shown in Figure 4 and Fig. 5.
Wherein, Fig. 4 shows in embodiment 1 that human embryo stem cell is to mesodermal progenitor cell differential period, and human embryo stem cell induces the detected result of 48 hours mesoderm marker gene Brachyury through different concns PGE2.This result shows that the expression amount of Brachyury is more and more higher along with the concentration of PGE2 raises within the scope of finite concentration.
Fig. 5 shows PGE2 process inducing human embryo stem cell in embodiment 1 and, after hemogenic endothelium cytodifferentiation, adopts its figuratrix marker protein of flow cytometry to express.As shown in Figure 5, PGE2 promotes that human embryo stem cell is to hemogenic endothelium cytodifferentiation, improves KDR
+the per-cent of cell.The mesodermal progenitor cell that PGE2 promotes human embryo stem cell to break up to obtain further to hemogenic endothelium cytodifferentiation, and promotes the expression of hemogenic endothelium cell sign albumen KDR.The positive correlation of this facilitation effect tool concentration dependant.
In the description of this specification sheets, specific features, structure, material or feature that the description of reference term " embodiment ", " some embodiments ", " example ", " concrete example " or " some examples " etc. means to describe in conjunction with this embodiment or example are contained at least one embodiment of the present invention or example.In this manual, identical embodiment or example are not necessarily referred to the schematic representation of above-mentioned term.And the specific features of description, structure, material or feature can combine in an appropriate manner in any one or more embodiment or example.
Although illustrate and describe embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple change, amendment, replacement and modification to these embodiments when not departing from principle of the present invention and aim, scope of the present invention is by claim and equivalents thereof.
Claims (14)
1. a substratum, is characterized in that, comprises:
Basic medium, described basic medium is SFDM substratum;
PGE2; And
BMP4，
Wherein, the formula of described SFDM substratum is as follows:
IMDM/F12 mixed culture medium;
Glutamine 1%;
Insulin-Transferrin-Sodium Selenite 1%;
Non-essential amino acid 1%;
Bovine serum albumin 0.05%;
Penicillin 100U/ml;
Streptomycin sulphate 100 μ g/ml,
In wherein said IMDM/F12 mixed culture medium, IMDM substratum and F12 culture volume are than being 3:1.
2. substratum according to claim 1, is characterized in that, the concentration of described PGE2 is 0.5-10 μM.
3. substratum according to claim 2, is characterized in that, the concentration of described PGE2 is 1 μM.
4. substratum according to claim 1, is characterized in that, the concentration of described BMP4 is 10-50ng/ml.
5. substratum according to claim 4, is characterized in that, the concentration of described BMP4 is 25ng/ml.
6. a test kit, is characterized in that, comprises:
First substratum, described first substratum is the substratum described in any one of Claims 1 to 5;
Second substratum, described second substratum obtains by adding PGE2, VEGF and FGF2 in SFDM substratum,
Wherein, the formula of described SFDM substratum is as follows:
IMDM/F12 mixed culture medium;
Glutamine 1%;
Insulin-Transferrin-Sodium Selenite 1%;
Non-essential amino acid 1%;
Bovine serum albumin 0.05%;
Penicillin 100U/ml;
Streptomycin sulphate 100 μ g/ml,
In wherein said IMDM/F12 mixed culture medium, IMDM substratum and F12 culture volume are than being 3:1.
7. test kit according to claim 6, is characterized in that, in the second substratum, the concentration of described PGE2 is 0.5-25 μM.
8. test kit according to claim 7, is characterized in that, in the second substratum, the concentration of described PGE2 is 1 μM.
9. test kit according to claim 6, is characterized in that, in the second substratum, the concentration of described VEGF is 25-100ng/ml, and the concentration of described FGF2 is 25-100ng/ml.
10. test kit according to claim 9, is characterized in that, in the second substratum, the concentration of described VEGF is 50ng/ml.
11. test kits according to claim 9, is characterized in that, in the second substratum, the concentration of described FGF2 is 50ng/ml.
12. 1 kinds of methods preparing early stage mesodermal progenitor cell, is characterized in that, comprising:
Utilize the substratum described in any one of claim 1 ~ 5, cultivate stem cell, to obtain early stage mesodermal progenitor cell, wherein, described stem cell is multipotential stem cell, or from the embryonic stem cell of human embryonic stem cell H1 or H9.
13. 1 kinds of methods utilizing the test kit described in any one of claim 6 ~ 11 to prepare hemogenic endothelium cell, is characterized in that, comprising:
Described first substratum is utilized to cultivate embryonic stem cell, to obtain early stage mesodermal progenitor cell; And
Described second substratum is utilized to cultivate described early stage mesoderm group cell, to obtain hemogenic endothelium cell,
Wherein, described embryonic stem cell is from human embryonic stem cell H1 or H9.
14. methods according to claim 13, is characterized in that, utilize described first substratum to cultivate 24-96 hour to described embryonic stem cell, utilize described second substratum to cultivate 72-96 hour to described early stage mesoblastema.
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| JP7534070B2 (en) * | 2015-11-04 | 2024-08-14 | フェイト セラピューティクス，インコーポレイテッド | Methods and compositions for inducing hematopoietic cell differentiation - Patents.com |
| CN105886457A (en) * | 2016-05-16 | 2016-08-24 | 中国人民解放军军事医学科学院野战输血研究所 | Method for inducing stem cell differentiation to form epiblast progenitor cell |
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