CN103194423A - Medium and purpose thereof - Google Patents
Medium and purpose thereof Download PDFInfo
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- CN103194423A CN103194423A CN2013100733188A CN201310073318A CN103194423A CN 103194423 A CN103194423 A CN 103194423A CN 2013100733188 A CN2013100733188 A CN 2013100733188A CN 201310073318 A CN201310073318 A CN 201310073318A CN 103194423 A CN103194423 A CN 103194423A
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Abstract
The present invention discloses a group of mediums and kits for in vitro inducing embryonic stem cells to differentiate into endothelial cells, purpose thereof for in vitro inducing embryonic stem cells to differentiate into endothelial cells, and endothelial cells or derivatives thereof. The group of mediums comprise: a first medium, which is an IMDM/F12 medium added with insulin transferrin selenium, BSA, non-essential amino acids, and rhBMP-4; a second medium, which is an IMDM/F12medium added with insulin transferrin selenium, BSA, non-essential amino acids, rhVEGF and rhbFGF; and a third medium, which is an EGM2 medium. The group of medium disclosed by the present invention can quickly and efficiently induce embryonic stem cells to differentiate to endothelial progenitor cells and endothelial cells.
Description
Technical field
The present invention relates to endotheliocyte regeneration techniques field, relate to substratum and uses thereof particularly.More specifically, the present invention relates to be divided into for external evoked embryonic stem cell one group of substratum and the test kit of endotheliocyte, and be divided into the method that the purposes in the endotheliocyte, external evoked embryonic stem cell are divided into endotheliocyte at external evoked embryonic stem cell, and endotheliocyte or derivatives thereof.
Background technology
Cardiovascular systems is at first to occur in the fetal development and the system of performance function.Growth and the differentiation of organ be very important during the generation of blood vessel and grow not only formed for the embryo, also is significant for trauma repair and the reproductive function of adult.Vascular development also plays an important role in a series of pathological phenomenons, as retina propagation, old and feeble relevant degeneration spot, rheumatoid arthritis and the tumour generation etc. of still not having effective treatment means so far.Vascular endothelial cell is mounted lining at the internal surface of whole cardiovascular systems, is simple squamous epithelium, is the boundary cell between vessel wall and the blood, is the morphological basis that forms cardiovascular closed pipe system.Its running balance, fibrinolysis and aggegation at tissue, blood-tissue element exchange, vasoconstriction are regulated and control, normally and in the activation of the vascularization of tumor tissues and the hemocyte under physiology and the pathological conditions and the migration have all been brought into play vital role.People think that vascular endothelial cell is except the barrier as substance transportation between blood and tissue more at present, topmost biological function is to make blood circulation keep flow state, and vascular endothelial cell still can synthesize with the multiple reticular tissue composition of secretion, participate in the metabolism of some materials and with the white corpuscle interaction etc. in addition.The performance of above-mentioned these physiological processs all depends on the regulation and control to the vascular endothelial cell precision, and imbalance then causes the generation of pathological phenomenon.The molecular mechanism that research determines endothelial cell differentiation and phenotype is for the principle of illustrating the blood vessel genesis and development, have important directive significance to angiogenesis treatment and the antineoplaston of infarct or damaged tissue.
HESC (human embryonic stem cells, hESCs) as a kind of multipotential cell that possesses self and infinite multiplication potential, it provides infusive prospect to the possibility of endothelial cell differentiation for the treatment of cardiovascular disorder and limb ischemia.In recent years, studies confirm that constantly that human embryo stem cell hESCs has the ability of the endotheliocyte of the different developmental phases of being divided into.By cultivating, induce the factor that forms embryoid body (EBs) and add short vascular development altogether with the stroma cell of mouse or building endothelium with the strategies such as common cultivation of special two-dimensional medium and grow microenvironment, existing a plurality of research groups break up, have been separated to endotheliocyte from hESCs.Recent result of study shows that the endotheliocyte in hESCs source can form new blood vessel, improves the function of infarcted hearts, is new cell source in the cell replacement treatment.Though obtained these achievements, but be to use the EBs(embryoid body) the relevant inducible factor of interpolation endothelium, cultivate altogether with stroma cell or use matrigel to add the factor and induce differentiation to produce endotheliocyte, general all can be again mode by sorting obtain purer endotheliocyte relatively, enlarged culturing more afterwards.But because ratio and the quantity of the endotheliocyte that these induction methods of use own produce are just less, limited at the multiple of amplification in vitro after the sorting in addition, also be difficult to use in clinical treatment.Efficiently fast inducing embryo stem cell to break up to endothelial progenitor cells and endotheliocyte still be insoluble problem.
Summary of the invention
The present invention is intended to solve at least one of technical problem that exists in the prior art.For this reason, one object of the present invention is to propose a kind of substratum, test kit and method that external evoked embryonic stem cell (ESCs) is divided into endotheliocyte that can be used in.
According to an aspect of the present invention, the invention provides one group of substratum.According to embodiments of the invention, this group substratum comprises: first substratum, this first substratum are the IMDM/F12 substratum that has added pancreas islet plain sheet selenium transferrin, BSA, non-essential amino acid and rhBMP-4; Second substratum, this second substratum are the IMDM/F12 substratum that has added pancreas islet plain sheet selenium transferrin, BSA, non-essential amino acid, rhVEGF and rhbFGF; And the 3rd substratum, the 3rd substratum is the EGM2 substratum.The contriver is surprised to find, utilize above-mentioned substratum of the present invention efficiently to induce ESCs to break up to endothelial progenitor cells and endotheliocyte rapidly, thereby can prepare a large amount of endotheliocytes effectively, and then can be endotheliocyte for ECs(effectively) treatment that is applied to cardiovascular disorder and limb ischemia provides cell base.
In addition, one group of substratum according to the above embodiment of the present invention can also have following additional technical characterictic:
According to one embodiment of present invention, this IMDM/F12 substratum comprises: 75% interpolation GlutaMAX
TMThe IMDM substratum of-I; And 25% interpolation GlutaMAX
TMThe F12 substratum of-I.Wherein, described " GlutaMAX in this article
TM-I " expression NSC 334200 dipeptides.
According to one embodiment of present invention, comprising 1% pancreas islet plain sheet selenium transferrin (Insulin-Transferrin-Selenium, ITS), 0.05%BSA(is bovine serum albumin), 1% non-essential amino acid (its english abbreviation is " NEAA ") and 25ng/ml rhBMP-4(in this first substratum is recombinant human bone morphogenetic protein 4).Thus, first substratum that utilizes this not contain serum, definite ingredients and contain DIF rhBMP-4, in the suitable concentration of rhBMP-4 and can efficiently induce ESCs to break up to mesendoderm under action time, this be the basis that is obtained endotheliocyte by the ESCs source.
According to one embodiment of present invention, comprise 1% pancreas islet plain sheet selenium transferrin, 0.05%BSA, 1%NEAA(non-essential amino acid in this second substratum), 50ng/ml rhVEGF(is the recombinant human vascular endothelial growth factor) and 50ng/ml rhbFGF(be Prostatropin).Thus, utilize this not contain serum, definite ingredients and contain for endothelium and induce differentiation and enlarged culturing that the rhVEGF of obvious promoter action and second substratum of rhbFGF somatomedin are arranged, can induce the mesendoderm cell in ESCs source to endothelium ancestral/endothelial cell differentiation effectively.
According to one embodiment of present invention, further comprise: the 4th substratum, the 4th substratum are the D-MEM/F-12 substratum that has added Knockout serum substitute, L-glutaminate, beta-mercaptoethanol, non-essential amino acid and rhbFGF; And the 5th substratum, the 5th substratum is the mTeSR substratum.
According to one embodiment of present invention, comprise 20%Knockout serum substitute, 1mM L-glutaminate, 0.1mM beta-mercaptoethanol, 1% non-essential amino acid and 5ng/ml bFGF in the 4th substratum.According to some embodiments of the present invention, this definite ingredients, nutritious, contain and can keep the ESCs(embryonic stem cell) the 4th substratum of the rhbFGF somatomedin of dryness, can and can effectively keep the characteristic of ESCs with the common long-term cultivation ESCs of trophocyte.
According to another aspect of the invention, the present invention also provides a kind of test kit that is divided into endotheliocyte for external evoked embryonic stem cell.According to embodiments of the invention, this test kit comprises foregoing one group of substratum of the present invention.According to some embodiments of the present invention, utilize test kit of the present invention, adopt culture medium culturing embryonic stem cell wherein, can realize efficiently that external evoked embryonic stem cell is divided into endotheliocyte, thereby can prepare a large amount of endotheliocytes effectively, significant for the treatment of cardiovascular disorder and limb ischemia.
According to a further aspect in the invention, the present invention also provides foregoing one group of substratum, and perhaps test kit is divided into purposes in the endotheliocyte at external evoked embryonic stem cell.Thereby, utilize above-mentioned one group of substratum or test kit, can realize effectively that external evoked embryonic stem cell is divided into endotheliocyte, and induce the efficient of differentiation especially high.
In accordance with a further aspect of the present invention, the present invention also provides a kind of external evoked embryonic stem cell to be divided into the method for endotheliocyte.According to embodiments of the invention, this method may further comprise the steps: utilize foregoing one group of substratum, cultivate embryonic stem cell.Thus, can efficiently induce ESCs to break up to endothelial progenitor cells and endotheliocyte rapidly, thereby can prepare a large amount of endotheliocytes effectively, its treatment to cardiovascular disorder and limb ischemia be significant.
According to one embodiment of present invention, be divided in the method for endotheliocyte at external evoked embryonic stem cell of the present invention, embryonic stem cell derives from the mankind.
According to one embodiment of present invention, be divided in the method for endotheliocyte at external evoked embryonic stem cell of the present invention, utilize foregoing one group of substratum, cultivating embryonic stem cell further comprises: utilize this first substratum that this embryonic stem cell is carried out first and induce differentiation culture, in order to obtain the cell of inducing differentiation culture through first; Utilize this second substratum to induce the cell of differentiation culture to carry out second to this through first and induce differentiation culture, in order to obtain the cell of inducing differentiation culture through second; And utilize the 3rd substratum to induce the cell of differentiation culture to carry out the 3rd to this through second and induce differentiation culture, in order to obtain endotheliocyte.The contriver is surprised to find, utilize aforesaid method efficiently to induce ESCs to break up to endothelial progenitor cells and endotheliocyte rapidly, thereby can prepare a large amount of endotheliocytes effectively, and then the treatment that can be effectively be applied to cardiovascular disorder and limb ischemia for ECs provides cell base, and is significant.
Need to prove, when utilizing the external evoked embryonic stem cell of method of the invention described above to be divided into endotheliocyte, second induce the differentiation culture stage to finish after, there have been endothelium ancestral and endotheliocyte to produce, namely induced in the cell of differentiation culture through second to include part endothelium ancestral and endotheliocyte.
According to one embodiment of present invention, be divided in the method for endotheliocyte at external evoked embryonic stem cell of the present invention, carry out this and first induced preferred 2 days differentiation culture 1.5-3 days.Thus, can effectively promote ESCs to break up to mesendoderm.According to another embodiment of the invention, be divided in the method for endotheliocyte at external evoked embryonic stem cell of the present invention, carrying out this, second to induce differentiation culture be 4-8 days, preferred 4.5 days.Thus, can further induce through first and induce the cell of differentiation culture to endothelium ancestral/endothelial cell differentiation.According to still a further embodiment, be divided in the method for endotheliocyte at external evoked embryonic stem cell of the present invention, carrying out the 3rd, to induce differentiation culture be 3-20 days.Thus, can effectively promote to induce the cell of differentiation culture to the differentiation of endothelium ancestral/endotheliocyte through second, and the further differentiation and maturation of endothelium ancestral/endotheliocyte.In addition, according to another embodiment of the invention, induce differentiation culture through the 3rd after, can continue to utilize the 3rd substratum that the endotheliocyte that obtains is carried out enlarged culturing.Thus, can effectively prepare a large amount of endotheliocytes.
According to one embodiment of present invention, carry out this first induce differentiation culture before, further comprise: utilize the 5th substratum that this embryonic stem cell is cultivated in advance, digest successively then and the processing of going down to posterity.Thus, can effectively remove the ESCs that part trophocyte and enlarged culturing non-trophoblast are cultivated, for the follow-up differentiation culture of inducing is prepared.Wherein, according to one embodiment of present invention, carry out this pre-cultivation 5-7 days.According to another embodiment of the invention, utilize 1% dispase enzyme to carry out this digestion process.According to still a further embodiment, carry out this processing 2-3 time of going down to posterity according to the ratio of 1:3.Thus, after handling through going down to posterity of non-trophoblast repeatedly, can remove the trophocyte fully, thereby can avoid the trophocyte to follow-up influence of inducing differentiation culture, simultaneously, the processing of going down to posterity repeatedly also can obtain a large amount of ESCs cells, lays a good foundation for further inducing differentiation to obtain a large amount of endothelium ancestral/endotheliocytes.
According to one embodiment of present invention, before this embryonic stem cell is cultivated in advance, further comprise: utilize the 4th substratum that this embryonic stem cell is carried out the trophocyte and cultivate altogether.Thus, can be in the characteristic of long term maintenance ESCs under the vitro conditions.In addition, according to another embodiment of the invention, comprise that further the embryonic stem cell that will cultivate altogether through the trophocyte digests and the processing of going down to posterity.Thus, can realize the further enlarged culturing of the embryonic stem cell of cultivating altogether through the trophocyte, thereby can obtain a large amount of ESCs, in order to prepare for the follow-up differentiation culture of inducing.Wherein, according to one embodiment of present invention, utilize IV Collagen Type VI enzyme to carry out this digestion process, carry out this processing of going down to posterity according to the ratio of 1:3 or 1:5.According to one embodiment of present invention, carry out the 3rd induce differentiation culture before, further comprise: will be somebody's turn to do through second and induce the cell of differentiation culture to digest and the processing of going down to posterity.Thus, can avoid owing to the hyper-proliferative of cell the efficient of inducing differentiation being had a negative impact.Wherein, according to one embodiment of present invention, utilize 0.25% pancreatin to carry out this digestion process.According to another embodiment of the invention, carry out this processing of going down to posterity according to the ratio of 1:2 or 1:3.
According to another aspect of the invention, the present invention also provides a kind of endotheliocyte or derivatives thereof, and it is to obtain by the method that foregoing external evoked embryonic stem cell of the present invention is divided into endotheliocyte.According to embodiments of the invention, endotheliocyte or derivatives thereof of the present invention can be effective to angiogenesis, limb ischemia and antineoplastic treatment of infarct or damaged tissue, and is significant for the development of modern medicine.
Need to prove that one group of substratum of the present invention and utilize the external evoked embryonic stem cell of this group substratum to be divided into the method for endotheliocyte all is that the present inventor just finishes through arduous creative work and a large amount of optimization work.
Additional aspect of the present invention and advantage part in the following description provide, and part will become obviously from the following description, or recognize by practice of the present invention.
Description of drawings
Above-mentioned and/or additional aspect of the present invention and advantage are from obviously and easily understanding becoming the description of embodiment in conjunction with following accompanying drawing, wherein:
Fig. 1 has shown that according to one embodiment of the invention external evoked embryonic stem cell of the present invention is divided into the schematic flow sheet of the general method of endotheliocyte;
Fig. 2 has shown among the embodiment 1 cell of inducing differentiation culture through first, RT-PCR detected result and the cellular form figure of its dryness and mesendoderm correlating markings gene expression dose, wherein,
Fig. 2 A has shown the RT-PCR detected result of dryness and mesoderm correlating markings gene expression dose,
Fig. 2 B has shown cellular form figure;
Fig. 3 has shown among the embodiment 1 cell of inducing differentiation culture through second, the RT-PCR detected result of its endotheliocyte correlating markings gene expression dose, Flow cytometry result, capillary structure formation and phytohemagglutinin are in conjunction with experimental result and cellular form figure, wherein
Fig. 3 A has shown the RT-PCR detected result of endotheliocyte correlating markings gene expression dose,
Fig. 3 B has shown the Flow cytometry result of endotheliocyte correlating markings gene expression dose,
Fig. 3 C has shown cellular form figure,
Fig. 3 D has shown that capillary structure formation and phytohemagglutinin are in conjunction with experimental result;
Fig. 4 has shown among the embodiment 1 endotheliocyte of inducing differentiation culture to obtain in 6 days through the 3rd, the RT-PCR detected result of its endotheliocyte correlating markings gene expression dose, streaming instrument detected result and cellular form figure, wherein,
Fig. 4 A is the RT-PCR detected result that is biased into ripe endotheliocyte correlating markings gene expression dose,
Fig. 4 B is the streaming instrument detected result of endotheliocyte correlating markings gene C D31 and KDR expression level,
Fig. 4 C is cellular form figure.
Embodiment
Make an explanation below in conjunction with the solution of the present invention of embodiment.It will be understood to those of skill in the art that the following examples only are used for explanation the present invention, and should not be considered as limiting scope of the present invention.Unreceipted concrete technology or condition among the embodiment are carried out according to the document in this area described technology or condition or according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
General method:
According to some embodiments of the present invention, with reference to Fig. 1, the method that external evoked embryonic stem cell of the present invention is divided into endotheliocyte generally comprises following steps:
At first, utilizing the 4th substratum that embryonic stem cell is carried out the trophocyte successively cultivates altogether, and utilize the 5th substratum that embryonic stem cell is cultivated in advance, then, utilize this first substratum that this embryonic stem cell is carried out first and induce differentiation culture, in order to obtain the cell of inducing differentiation culture through first.
Then, utilize this second substratum to induce the cell of differentiation culture to carry out second to this through first and induce differentiation culture, in order to obtain the cell of inducing differentiation culture through second.
Then, utilize the 3rd substratum to induce the cell of differentiation culture to carry out the 3rd to this through second and induce differentiation culture, in order to obtain endotheliocyte.
With reference to Fig. 1, adopt external evoked embryonic stem cell of the present invention to be divided into the method for endotheliocyte, cultivate embryonic stem cell according to following steps:
1, the trophocyte cultivates altogether
Adopt the 4th substratum, l cell is cultivated altogether as trophocyte and undifferentiated human embryo stem cell cell (being hESCs, H1 and H9).Wherein, basic composition is of the 4th substratum: 78%D-MEM/F-12 minimum medium (GIBCO, No.12400-016), added 20%Knockout serum substitute (GIBCO, 10828), 1mM L-glutaminate (GIBCO, 35050), and the 0.1mM beta-mercaptoethanol (Invitrogen, 21985-023), 1% non-essential amino acid (NEAA(english abbreviation), Invitrogen, 11140-050), and 5ng/ml Prostatropin (rhbFGF(english abbreviation), Millipore, GF003).Through after the trophocyte cultivates altogether, hESCs is observed, go down to posterity if desired, then with type will be to be gone down to posterity hESCs be digested to little cell mass, afterwards according to cell density according to 1:3 or the 1:5 processing of going down to posterity.
2, the pre-cultivation
Then, cultivate altogether through the trophocyte above-mentioned, the hESCs that growth conditions is good is passaged to the ultrapure layer of the Matrigel(fibronectin of no feeder layer, BD, 354277) on, be that (Stem Cell #05850) cultivates special-purpose mTeSR substratum with the 5th substratum.When treating that hESCs grows to 5-7 days, carry out digestion process according to size and the density of cell, use 1mg/ml dispase enzyme (neutral protease, stem cell, 07923) during digestion.Then, will be through the cell inoculation of digestion process to the orifice plate that is covered with Matrigel, routine leaves standstill cultivation.
To carry out the processing of going down to posterity of 2-3 non-trophoblast through pre-incubated hESCs before inducing differentiation.
Wherein, carry out the above-mentioned processing of going down to posterity, the main instrument that adopts is: 5910 type whizzers (Japanese Kubo field), microscope (OLYMPUS, CKX31), concrete grammar comprises:
A, abandon old substratum, give a baby a bath on the third day after its birth time with 1 * PBS.
B, adding 1mg/ml dispase enzyme (stem cell, 07923) carry out digestion process, and in the microscopically observation of cell, if crimping takes place in the clone, then abandon the dispase enzyme and stop digestion, and with 1 * PBS give a baby a bath on the third day after its birth time (avoid firmly blowing and beating cell, prevent from cloning and lose).
C, (Stem Cell #05850), and softly blows and beats the clone with this substratum, makes embryonic stem cell (ESCs) clone come off from orifice plate to be added into new mTeSR substratum.Wherein, require all clones all to be blown and beaten as far as possible, and will clone the cell mass of blowing and beating into the homogeneous size as far as possible.
D, according to the gained cell density, according to the ratio of 1:2 or 1:3, above-mentioned hESCs clone is inoculated in to contain the mTeSR substratum and handle to be had in the orifice plate of Matrigel, cell is placed 37 ℃ afterwards, 5%CO
2Incubator in standing over night cultivate.
E, after 24 hours, change liquid according to clone's size and handle (namely continuing to cultivate) or the differentiation culture of inducing described below.
3, induce differentiation culture
According to following steps, above-mentioned process cultivated in advance and the hESCs that handles that goes down to posterity carries out the differentiation of inducing of endotheliocyte:
3.1 first induces differentiation culture
At first, treat that above-mentioned process is cultivated in advance and the hESCs that handles of going down to posterity adherent after, select whether to carry out the differentiation of inducing of endotheliocyte according to clone's size.Choose suitable orifice plate, require to contain in the orifice plate sizeable hESCs clone as much as possible (clone who namely contains tens to tens cells), utilize first substratum that it is carried out first and induced differentiation culture 48 hours, in order to obtain the cell of inducing differentiation culture through first.
Wherein, first substratum is to have added 1% pancreas islet plain sheet selenium transferrin (ITS(english abbreviation), GIBCO, 41400-045), the 0.05%BSA(bovine serum albumin, GIBCO, 11021-029), 1% non-essential amino acid (NEAA(english abbreviation), Invitrogen, 11140-050) with 25ng/ml recombinant human bone morphogenetic protein 4(rhBMP-4, RD, IMDM/F12 substratum 314-BP).Wherein, described " IMDM/F12 substratum " comprises in this article: 75% interpolation GlutaMAX
TMThe IMDM substratum of-I (GIBCO, 31980), and 25% interpolation GlutaMAX
TMThe F12 substratum of-I (GIBCO, 37165).
3.2 second induces differentiation culture
With above-mentioned acquisition through first cell of inducing differentiation culture, abandon first substratum and with the PBS washing once, utilize second substratum to carry out second afterwards and induced differentiation culture 4-8 days, in order to obtain the cell of inducing differentiation culture through second.
Wherein, second substratum is for having added 1% pancreas islet plain sheet selenium transferrin (GIBCO, 41400-045), 0.05%BSA(GIBCO, 11021-029), 1% non-essential amino acid (Invitrogen, 11140-050), 50ng/ml recombinant human vascular endothelial growth factor (rhVEGF(english abbreviation), PEPROTECH, 100-20) and 50ng/ml Prostatropin (rhbFGF(english abbreviation), Millipore, IMDM/F12 substratum GF003).
Carry out second and induce differentiation culture after 4.5 days, induce the cell of differentiation culture to grow near more than 90% of orifice plate density through second.
3.3 the 3rd induces differentiation culture
Before differentiation culture is induced in beginning the 3rd, utilize 0.25% pancreatin, will induce the cell of differentiation culture to carry out digestion process through second, and according to the processing of going down to posterity of the ratio of 1:2 or 1:3.
Wherein, digest and the processing of going down to posterity, the main instrument that adopts is: 5910 type whizzers (Japanese Kubo field), microscope (OLYMPUS, CKX31), concrete grammar comprises:
A, abandon old substratum, give a baby a bath on the third day after its birth time with 1 * PBS.
B, adding 0.25% pancreatin (AMRESCO, 0458), use PBS to be configured, filtration sterilization afterwards, preserve or-20 ℃ of preservations for 4 ℃) carry out digestion process, and in the microscopically observation of cell, big if the intercellular substance becomes, take place to break away between cell and the orifice plate that then to add the growth medium that contains 10% serum (be IMDM substratum+10%FBS), stop digestion.
C, collecting cell, under the 1000rpm condition centrifugal 5 minutes.Abandon supernatant afterwards and use the resuspended washed cell of PBS, in order to remove to stop the serum that uses in the digestion, recentrifuge 5 minutes under the 1000rpm condition then.
D, abandon supernatant, (Lonza, cc-4176) re-suspended cell, and according to cell density, inoculating according to the ratio of 1:2 or 1:3 place 37 ℃, 5%CO then with the EGM2 substratum
2Incubator in standing over night cultivate.
E, according to the cell growing state, select to change liquid (namely continuing to cultivate) or the processing of going down to posterity.
Then, utilize the 3rd substratum, induce differentiation culture and the cell handled of going down to posterity carries out the 3rd and induced differentiation culture 3-20 days with above-mentioned through second, in order to obtain endotheliocyte.Wherein, the 3rd substratum be the EGM2 substratum (Lonza, cc-4176).Carry out the 3rd when inducing differentiation culture 3-6 days, consider whether to go down to posterity processing according to cell density.
Wherein, digest and the processing of going down to posterity, the main instrument that adopts is: 5910 type whizzers (Japanese Kubo field), microscope (OLYMPUS, CKX31), concrete grammar comprises:
A, abandon old substratum, give a baby a bath on the third day after its birth time with 1 * PBS.
B, adding 0.25% pancreatin (AMRESCO, 0458) use PBS to be configured, filtration sterilization is afterwards preserved or-20 ℃ of preservations for 4 ℃) carry out digestion process, and in the microscopically observation of cell, if it is big that the intercellular substance becomes, disengaging takes place between cell and the orifice plate then stop digestion.
C, collecting cell, under the 1000rpm condition centrifugal 5 minutes.Abandon supernatant afterwards and use the PBS re-suspended cell, under 1000rpm centrifugal 5 minutes then.
D, abandon supernatant, (Lonza, cc-4176) re-suspended cell, and according to cell density, inoculating according to the ratio of 1:2 or 1:3 place 37 ℃, 5%CO then with the EGM2 substratum
2Incubator in standing over night cultivate.
In addition, when cell growth state is good, enlarged culturing can further be carried out, in order to obtain more endotheliocyte in the 3rd substratum.
Respectively to obtain among the embodiment 1 through first cell of inducing differentiation culture, through second cell of inducing differentiation culture, and through the 3rd endotheliocyte of inducing differentiation culture to obtain in 6 days, carry out flow cytometry and RT-PCR and detect, and the morphological change of observing cell in the three phases in microscopically.
One, concrete detection method:
1, Flow cytometry
Use instrument: 5910 type whizzers (Japanese Kubo field), DH-II rotation mixed instrument (Ningbo Xin Zhi company), the streaming instrument (BD, FACSAria).
Method:
1.1 peptic cell:
(1) abandon old substratum, it is inferior to give a baby a bath on the third day after its birth with 1 * PBS.
(2) add 0.25% pancreatin cell to be detected is carried out digestion process, and in the microscopically observation of cell, big if the intercellular substance becomes, take place between cell and the orifice plate to break away from then to be added into new termination substratum, stop digestion.
(3) collecting cell under the 1000rpm condition centrifugal 5 minutes, is abandoned supernatant afterwards and is used the PBS re-suspended cell.
1.2 traget antibody:
According to the antibody specification sheets, the cell of the process digestion process of above-mentioned acquisition placed centrifuge tube and with 4-7 * 10
5Cell is resuspended among the 100 μ l PBS, adds corresponding antibody afterwards, namely anti-people CD31-APC (eBioscience, 17-0319-42) and anti-people CD309-PE(vascular endothelial growth factor receptor 2, VEGFR2/KDR; R﹠amp; D systems, 560494), abundant mixing then, and centrifuge tube placed on the rotation mixed instrument carry out hatching and mark of streaming antibody, the time is 40 minutes.
1.3 wash antibody:
After mark is finished, use the resuspended above-mentioned cell through antibody labeling of 1.4ml PBS, and under 1000rpm centrifugal 5 minutes.Abandon supernatant afterwards, and use 1.5ml PBS re-suspended cell again, under 1000rpm centrifugal 5 minutes then, repeat this step (namely abandon supernatant, centrifugal after resuspended) afterwards.
1.4 resuspended:
Abandon supernatant, utilize the cell of 300-500 μ l PBS or the above-mentioned processing of the resuspended process of 2% Paraformaldehyde 96.
1.5 Flow cytometry
According to product description and operational guidance, the re-suspended cell of above-mentioned acquisition is carried out the detection of flow cytometry.
2, RT-PCR(real-time quantitative PCR) detect
2.1RNA extract
Use instrument: whizzer (sigma company, 3-18k).
Method:
(1) collection-attached cell of cell: in centrifuge tube, utilize collagenase or pancreatin with cell to be detected digest centrifugal after, PBS washes twice, carry out again abandoning supernatant after centrifugal, utilize 1ml TRIZOL (Invitrogen then, 15596018) lysing cell carries out the extraction of RNA afterwards according to subsequent step.
(2) sample (namely passing through the cell of cracking) was left standstill under room temperature 5-15 minute, the nucleic acid-protein mixture is separated fully.
(3) every pipe adds 0.2ml chloroform (Chemical Reagent Co., Ltd., Sinopharm Group, 10006892) cover lid, acutely fully shakes up 15s, leaves standstill under room temperature 10 minutes then.
(4) in 4 ℃, under the 12000g condition centrifugal 15 minutes, RNA was dissolved in the water layer the inside on upper strata, and volume is approximately 50%.
(5) the colourless aqueous phase layer in the upper strata in the above-mentioned centrifuge tube softly is transferred in the new 1.5ml centrifuge tube.
(6) add 0.5ml Virahol (Chemical Reagent Co., Ltd., Sinopharm Group, 80109292) in the new centrifuge tube, soft mixing afterwards, room temperature is placed 10min.
(7) in 4 ℃, under the 12000g condition centrifugal 15 minutes, abandon supernatant, then the RNA precipitation that the bottom can adularescent.
(8) abandon supernatant, add 75% the alcohol of being prepared by DEPC water (water of no RNA enzyme) (Chemical Reagent Co., Ltd., Sinopharm Group, 10009292) in the centrifuge tube, resuspended RNA, afterwards in 4 ℃, under the 12000g centrifugal 15 minutes.
(9) abandon clean supernatant, leave standstill 5-10min, treat that alcohol volatilization and bottom RNA just add 10-30 μ l DEPC water (water of no RNA enzyme) during bleach, carry out concentration determination afterwards.
(10) will extract the RNA that obtains and preserve down in-70 ℃, standby.
2.2 reverse transcription
Use the reverse transcription system of TaKaRa, according to operation instructions the RNA that 0.8 μ g said extracted obtains is carried out the reverse transcription of 20 μ l systems.
Use instrument: (Eppendorff company, mascercycler), (Hangzhou is difficult to understand contains the small desk whizzer PCR instrument, K6)
Concrete grammar:
(1) according to following proportioning, with the abundant mixing of following each component:
Masterplate RNA 500-800ng(adds corresponding volume according to actual RNA concentration)
The water of no RNA enzyme (RNase Free dH
2O (DEPC water)) making the liquid final volume is 12.0 μ l.
(2) said mixture is carried out centrifugal, place 70 ℃ of following 10min then, chilling 2min the more centrifugal several seconds, concentrates at the bottom of the centrifuge tube pipe liquid on ice, in order to obtain treated masterplate RNA.
(3) according to following proportioning, preparation reverse transcription reaction system in centrifuge tube:
(4) according to following program, in the PCR instrument, carry out reverse transcription reaction:
42 ℃, 60min; 70 ℃, 15min; Be cooled to 4 ℃.
Thus, obtain the cDNA of sample, and preserve down in-20 ℃, standby.
2.3RT-PCR
Get the sample cDNA of the above-mentioned acquisition of 1 μ l, dispose the RT-PCR reaction system according to following proportioning:
Then, the RT-PCR reaction system that configuration is obtained is carried out the RT-PCR reaction according to following program: 94 ° of C, 5 minutes; Carry out following circulation 40 times afterwards: (94 ° of C, 30 seconds; Annealing temperature, 30 seconds; 72 ° of C, 30 seconds); Then, 72 ° of C, 5 minutes; Be cooled to 4 ° of C afterwards.
Then, image data utilizes BIO-Rad software that each data is handled, and calculates each correlating markings expression of gene level with GAPDH as internal control gene.
3, capillary structure formation and phytohemagglutinin are in conjunction with experiment
With 48 orifice plates, shop, every hole 15-20 μ l Marigel (ultrapure layer fibronectin, BD, 354234), and static placement 30 minutes under 37 ° of C solidify Marigel and form very thin one deck.Then, according to the digestion process method in " 3.2 second induce differentiation culture " stage among the embodiment 1, to inducing the cell of differentiation culture to carry out digestion process through second, be seeded to then in above-mentioned 48 orifice plates, utilize the EGM2 substratum, place 37 ℃, 5%CO
2Standing over night is cultivated in the incubator, and (OLYMPUS company CKX31) observes capillary structure down and forms in microscope after 48 hours.
By above-mentioned observation, treat that cell is after Matrigel forms capillary structure, abandon old substratum, cell is placed in 4% Paraformaldehyde 96 for three times with 1 * PBS washing fixes 10 minutes, use 1 * PBS washing three times afterwards, and keep 100 μ lPBS in orifice plate, then to wherein adding 2.4 μ g phytohemagglutinin (Vector companies, v0323-2mg), abandon the PBS that contains phytohemagglutinin after 30 minutes, use 1 * PBS washed cell 3 times, add and use the DAPI of 1 * PBS dilution to dye nuclear, dye after 2 minutes, use 1 * PBS washing three times, add again 100 μ l1 * PBS and in fluorescent microscope (OLYMPUS company, IX70) down the making plant lectin in conjunction with situation.
4, morphocytology observation
In microscopically (Nikon company, TE-2000-V), to among the embodiment 1 through first cell of inducing differentiation culture, induce the cell of differentiation culture through second, and through the 3rd endotheliocyte of inducing differentiation culture to obtain in 6 days, carry out cellular form observation.
Two, detected result
By above-mentioned concrete detection method, the contriver has carried out RT-PCR detection, streaming detection and morphological observation to each stages of cell of differentiation culture of inducing among the embodiment 1, the results are shown in as Fig. 2-4.
Wherein, Fig. 2 has shown among the embodiment 1 cell of inducing differentiation culture through first, RT-PCR detected result and the cellular form figure of its dryness and mesendoderm correlating markings gene expression dose.Wherein, Fig. 2 A has shown the RT-PCR detected result of dryness and mesendoderm correlating markings gene expression dose, and Fig. 2 B has shown cellular form figure.Shown in Fig. 2 A, with the i.e. blank cell shown in the figure of undifferentiated hESCs() compare, finish the cell that the fs induces differentiation (being aforementioned through first cell of inducing differentiation culture), the expression level of its dryness genes involved Oct4, Sox2 obviously reduces, and the expression level of mesendoderm correlating markings gene Brachurary, PDGF1 α and MixL1 all is significantly improved, and the dryness of this explanation cell slowly weakens and begun induces differentiation to mesendoderm.Wherein, in Fig. 2 A, shown detected result is for being the relative result of internal control gene with GAPDH, and * represents p＜0.05, shows that difference has statistical significance and significant difference, and * * represents p＜0.01, shows that difference is extremely remarkable.Shown in Fig. 2 B, " hESCs " represents human embryo stem cell, " fs-2D " expression is induced 2 days cell of differentiation culture through first, wherein last figure is the aspect graph of human embryo stem cell, figure below is for through first aspect graph of inducing 2 days cell of differentiation culture, and contrast is compared with undifferentiated hESCs as can be known, finish the cell that the fs induces differentiation and no longer shown as fine and close clone's sample growth as hESCs, and the cell nuclear-cytoplasmic ratio also decreases.These results show that all cell has begun to break up.
Fig. 3 has shown among the embodiment 1 cell of inducing differentiation culture through second, and the RT-PCR detected result of its endotheliocyte correlating markings gene expression dose, Flow cytometry result, cellular form figure and capillary structure form experiment and phytohemagglutinin in conjunction with result of experiment.Wherein, Fig. 3 A has shown the RT-PCR detected result of endotheliocyte correlating markings gene expression dose, Fig. 3 B has shown the Flow cytometry result of endotheliocyte relevant surfaces sign expression level, Fig. 3 C has shown cellular form figure, and Fig. 3 D has shown that capillary structure formation and phytohemagglutinin are in conjunction with experimental result.As shown in Figure 3A, induce the cell of differentiation to compare with finishing the fs, finish subordinate phase and induce the cell of differentiation (being aforementioned through second cell of inducing differentiation culture), the expression level of its endotheliocyte genes involved CD31, CD34, KDR, VE-cadherin and vWF all is significantly improved.Wherein, in Fig. 3 A, shown detected result is for being the relative result of internal control gene with GAPDH, and * represents p＜0.05, shows that difference has statistical significance and significant difference, and * * represents p＜0.01, shows that difference is extremely remarkable.Shown in Fig. 3 B, the detection of streaming level shows, after subordinate phase induced differentiation to finish, no matter be H9 cell or H1 cell, the expression level of its endotheliocyte relevant surfaces sign CD31 and KDR all had tangible up-regulated expression (and CD31 and KDR not have expression substantially among the hESCs).Shown in Fig. 3 C, " subordinate phase-4.5 day " expression is induced 4.5 days cell of differentiation culture through second, with this figure and foregoing through first induce differentiation culture cell aspect graph contrast as can be known, with respect to through first cell of inducing differentiation culture, finishing subordinate phase induces the nuclear-cytoplasmic ratio of the cell of differentiation obviously to reduce, and tangible change has also taken place in its cellular form.Show that with inducing the cell of differentiation culture to compare through first, this cell has carried out further differentiation, some has broken up endothelium ancestral/endotheliocyte stage.Shown in Fig. 3 D, wherein figure (1) has shown that the formation experiment of process capillary structure and phytohemagglutinin are in conjunction with the cell after testing, picture under its light field state induces the cell after the differentiation to form capillary structure at Matrigel through subordinate phase as can be seen from the figure; Figure (2) shown this cell under fluorescent microscope observed phytohemagglutinin in conjunction with situation; Figure (3) has shown this cell nuclear dyeing situation after DAPI dyeing; Figure (4) is (1), (2) that will scheme D, the picture after (3) figure stack.By Fig. 3 D as can be known, induce the cell of differentiation culture not only can on Matrigel, can form capillary structure through second, and can be in conjunction with phytohemagglutinin.These results further show induce the differentiation gained cell tentatively have the function of endothelium ancestral/endotheliocyte.
Fig. 4 has shown among the embodiment 1 through the 3rd endotheliocyte of inducing differentiation culture to obtain in 6 days, the RT-PCR detected result of its endotheliocyte correlating markings gene expression dose, Flow cytometry result and cellular form figure.Wherein, Fig. 4 A has shown the RT-PCR detected result that is biased into ripe endotheliocyte correlating markings gene expression dose, and Fig. 4 B has shown the streaming instrument detected result of endotheliocyte correlating markings gene C D31 and KDR expression level, and Fig. 4 C has shown cellular form figure.Shown in Fig. 4 A, induce the cell of differentiation to compare with finishing subordinate phase, finish the cell that the phase III induces differentiation (being aforementioned through the 3rd endotheliocyte of inducing differentiation culture to obtain in 6 days), the expression level of its endotheliocyte genes involved vWF, ang2 and tie2 all is significantly improved.Wherein, in Fig. 4 A, shown detected result is for being the relative result of internal control gene with GAPDH, and * * represents p＜0.01, shows that difference is extremely remarkable.Shown in Fig. 4 B, the detection of streaming level shows, after phase III induced differentiation to finish, the expression of Cheng Shu endotheliocyte relevant surfaces sign CD31 had tangible rise relatively, and the expression level of early stage relatively endotheliocyte genes involved KDR has the downward modulation expression of certain level.Shown in Fig. 4 C, " phase III-6D " expression is through the 3rd endotheliocyte of inducing differentiation culture to obtain in 6 days, with this figure and foregoing through second induce differentiation culture cell aspect graph contrast as can be known, with respect to finishing second cell of inducing differentiation culture, induce the cell (being endotheliocyte) of differentiation to carry out further inducing differentiation again through the phase III.
In the description of this specification sheets, concrete feature, structure, material or characteristics that the description of reference term " embodiment ", " some embodiment ", " example ", " concrete example " or " some examples " etc. means in conjunction with this embodiment or example description are contained at least one embodiment of the present invention or the example.In this manual, the schematic statement to above-mentioned term not necessarily refers to identical embodiment or example.And concrete feature, structure, material or the characteristics of description can be with the suitable manner combination in any one or more embodiment or example.
Although illustrated and described embodiments of the invention, those having ordinary skill in the art will appreciate that: can carry out multiple variation, modification, replacement and modification to these embodiment under the situation that does not break away from principle of the present invention and aim, scope of the present invention is limited by claim and equivalent thereof.
Claims (10)
1. one group of substratum is characterized in that, comprising:
First substratum, described first substratum are the IMDM/F12 substratum that has added pancreas islet plain sheet selenium transferrin, BSA, non-essential amino acid and rhBMP-4;
Second substratum, described second substratum are the IMDM/F12 substratum that has added pancreas islet plain sheet selenium transferrin, BSA, non-essential amino acid, rhVEGF and rhbFGF; And
The 3rd substratum, described the 3rd substratum is the EGM2 substratum.
2. one group of substratum according to claim 1 is characterized in that, described IMDM/F12 substratum comprises:
75% interpolation GlutaMAX
TMThe IMDM substratum of-I; And
25% interpolation GlutaMAX
TMThe F12 substratum of-I.
3. one group of substratum according to claim 1 is characterized in that, comprises 1% pancreas islet plain sheet selenium transferrin, 0.05%BSA, 1% non-essential amino acid and 25ng/ml rhBMP-4 in described first substratum.
4. one group of substratum according to claim 1 is characterized in that, comprises 1% pancreas islet plain sheet selenium transferrin, 0.05%BSA, 1% non-essential amino acid, 50ng/ml rhVEGF and 50ng/ml rhbFGF in described second substratum.
5. one group of substratum according to claim 1 is characterized in that, further comprises:
The 4th substratum, described the 4th substratum are the D-MEM/F-12 substratum that has added Knockout serum substitute, L-glutaminate, beta-mercaptoethanol, non-essential amino acid and rhbFGF; And
The 5th substratum, described the 5th substratum is the mTeSR substratum,
Wherein, comprise 20%Knockout serum substitute, 1mM L-glutaminate, 0.1mM beta-mercaptoethanol, 1% non-essential amino acid and 5ng/ml rhbFGF in described the 4th substratum.
6. one kind is used for the test kit that external evoked embryonic stem cell is divided into endotheliocyte, it is characterized in that, comprises each described one group of substratum of claim 1-5.
7. each described one group of substratum of claim 1-5, perhaps the described test kit of claim 6 is divided into purposes in the endotheliocyte at external evoked embryonic stem cell.
8. an external evoked embryonic stem cell is divided into the method for endotheliocyte, it is characterized in that, may further comprise the steps:
Utilize each described one group of substratum of claim 1-5, cultivate embryonic stem cell,
Randomly, described embryonic stem cell derives from the mankind.
9. method according to claim 8 is characterized in that, utilizes each described one group of substratum of claim 1-5, cultivates embryonic stem cell and further comprises:
Utilize described first substratum that described embryonic stem cell is carried out first and induce differentiation culture, in order to obtain the cell of inducing differentiation culture through first;
Utilize described second substratum to induce the cell of differentiation culture to carry out second through first to induce differentiation culture to described, in order to obtain the cell of inducing differentiation culture through second; And
Utilize described the 3rd substratum to induce the cell of differentiation culture to carry out the 3rd through second to induce differentiation culture to described, in order to obtain endotheliocyte, wherein
Carry out described first and induced differentiation culture 1.5-3 days, preferred 2 days,
Carry out described second and induced differentiation culture 4-8 days, preferred 4.5 days,
Carry out the described the 3rd and induced differentiation culture 3-20 days,
Carry out described first induce differentiation culture before, further comprise:
Utilize described the 5th substratum that described embryonic stem cell is cultivated in advance, digest successively then and the processing of going down to posterity, wherein, carry out described pre-cultivation 5-7 days, utilize 1% dispase enzyme to carry out described digestion process, carry out described going down to posterity according to the ratio of 1:3 and handle 2-3 time
Before described embryonic stem cell is cultivated in advance, further comprise: utilize described the 4th substratum that described embryonic stem cell is carried out the trophocyte and cultivate altogether, and comprise that further the embryonic stem cell that will cultivate altogether through the trophocyte digests and the processing of going down to posterity, wherein utilize IV Collagen Type VI enzyme to carry out described digestion process, carry out the described processing of going down to posterity according to the ratio of 1:3 or 1:5
Carry out the described the 3rd induce differentiation culture before, further comprise: induce the cell of differentiation culture to digest and the processing of going down to posterity described through second, wherein utilize 0.25% pancreatin to carry out described digestion process, carry out the described processing of going down to posterity according to the ratio of 1:2 or 1:3.
10. endotheliocyte or derivatives thereof, it is to obtain by the method that claim 8 or 9 described external evoked embryonic stem cells are divided into endotheliocyte.
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