CN104745529B - Leptin is divided into purposes and its application in hematopoietic stem/progenitor in inducing embryo stem cell - Google Patents
Leptin is divided into purposes and its application in hematopoietic stem/progenitor in inducing embryo stem cell Download PDFInfo
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Abstract
The invention discloses leptins to be divided into purposes and its application in hematopoietic stem/progenitor in inducing embryo stem cell, comprising: is divided into the kit of hematopoietic stem/progenitor for inducing embryo stem cell, utilizes the method for kit above-mentioned preparation hematopoietic stem/progenitor and the system of preparation hematopoietic stem/progenitor.Experiment discovery, leptin can significantly improve the efficiency that embryonic stem cell breaks up to hematopoietic stem/progenitor, improve the hematopoietic colonies Forming ability of noble cells, inducing effect is good and stablizes.
Description
Technical field
The present invention relates to bioengineering fields, and in particular, to leptin is divided into Hematopoietic Stem/ancestral in inducing embryo stem cell
Purposes and its application in cell are divided into hematopoietic stem/progenitor more particularly, to leptin in inducing embryo stem cell
It is thin to prepare Hematopoietic Stem/ancestral using kit for purposes, the kit that hematopoietic stem/progenitor is divided into for inducing embryo stem cell
The method of born of the same parents, and the system of preparation hematopoietic stem/progenitor.
Background technique
Hematopoietic stem/progenitor transplanting is for treating Severe Aplastic Anemia, leukaemia, thalassemia, severe
A variety of hematologics such as immunodeficiency symptoms and the maximally efficient therapeutic modality of disease of immune system.Currently, clinically Hematopoietic Stem/ancestral
Cell is mainly derived from peripheral blood and Cord blood after donor's marrow, mobilization.But due to current hematopoietic stem/progenitor donor
Rare numbers and distribution type success rate is extremely low, and the lazy weight of hematopoietic stem/progenitor is led in Cord blood with rebuilding adult artificial blood
Cause a large amount of patients that can not find the hematopoietic stem/progenitor of suitable distribution type, therefore, finding new hematopoietic stem/progenitor source has
Very important economic value and social effect.
Using human embryo stem cell and the appearance for inducing multi-potent stem cell multipotential stem cell technology for representative as biotechnology
Development with regenerative medicine brings huge opportunity.Self-renewal capacity and Multidirectional Differentiation of the multipotential stem cell with height
Potential can directed differentiation be triploblastica and its derivative histocyte broken up under specific inductive condition.Multipotential stem cell institute
These the unique biological characteristics having not only become the good model of research embryonic development rule, and can be to face
Bed substitution or transplantation treatment provide endlessly seed cell.
Currently, foundation of the multipotential stem cell to hematopoietic stem/progenitor differentiation model is derived from people's recognizing for hematopoietic development
Know, it is micro- by co-culturing or adding hematopoietic development in the inducers analogue body such as cell factor and small molecule compound with stroma cell
Environment induces multi-potent stem cell by mesodermal progenitor cell and hemogenic endothelium cell stage, is finally divided into hematopoietic stem/progenitor
And mature blood cell.However, due to people to the understanding of hematopoietic differentiation mechanism there are still compared with big limitation, multipotential stem cell is to making
The induction differentiation efficiency of haemocyte is low, and inducing cell does not have the ability of internal hematopoietic reconstitution still.
Therefore, multipotential stem cell needs further to be improved to the induction differentiated system of hematopoietic cell.
Summary of the invention
The present invention is directed at least solve one of the technical problems existing in the prior art.For this purpose, one object of the present invention
It is to propose a kind of method for improving the induced efficiency that human embryo stem cell breaks up to hematopoietic stem/progenitor using leptin and its answers
With.
It should be noted that the present invention is the following work based on inventor and completes: leptin (Leptin) is a kind of
The hormonal substance secreted by adipose tissue, most important physiological role are that sugar, fat and energy are participated in human body
Metabolism regulation, accelerates body metabolism and energy release, and appetite-suppressing loses weight.Inventors have found that specifically breaking up
Leptin is added in stage culture medium, human embryo stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and is shown
It writes and increases hematopoietic colonies forming quantity, while being also significantly increased to the differentiation capability of mature hematopoietic lineage cell.
Above-mentioned discovery based on inventor as a result, the present invention provides for inducing embryo stem cell be divided into Hematopoietic Stem/
The kit of progenitor cells, device and method.
According to an aspect of the present invention, the present invention provides leptins is divided into Hematopoietic Stem/ancestral in inducing embryo stem cell
Purposes in cell.
Inventor has surprisingly found that leptin can significantly improve the efficiency that embryonic stem cell breaks up to hematopoietic stem/progenitor,
The hematopoietic colonies Forming ability of noble cells is improved, inducing effect is good and stablizes.
According to another aspect of the invention, the present invention provides one kind for inducing embryo stem cell be divided into Hematopoietic Stem/
The kit of progenitor cells.According to an embodiment of the invention, the kit includes: the first culture medium, first culture medium is to add
1640 culture medium of AdvancedRPMI of leptin is added；And second culture medium, second culture medium is to be added to leptin
Stempro34 culture medium.
Inventor has surprisingly found that, is divided into hematopoietic stem/progenitor, colony shape using the kit inducing embryo stem cell
Enhance at ability, and embryonic stem cell is significantly improved to the efficiency that hematopoietic stem/progenitor is broken up, inducing effect is good and stablizes.
To, according to another aspect of the present invention, the present invention provides it is a kind of using kit above-mentioned prepare Hematopoietic Stem/
The method of progenitor cells.This method comprises: pretreated embryonic stem cell is cultivated using first culture medium, so as to
To hemogenic endothelium cell；The hemogenic endothelium cell is cultivated with using second culture medium, so as to obtain Hematopoietic Stem/
Progenitor cells.
According to an embodiment of the invention, being divided into hematopoietic stem/progenitor using this method inducing embryo stem cell, embryo is dry
Cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and hematopoietic colonies Forming ability enhances, and inducing effect is steady
It is qualitative good.
Further, in accordance with a further aspect of the present invention, the present invention provides it is a kind of prepare hematopoietic stem/progenitor be
System.According to an embodiment of the invention, the system includes: the first culture apparatus, first culture apparatus utilizes the first culture medium
Pretreated embryonic stem cell is cultivated, to obtain hemogenic endothelium cell；With the second culture apparatus, second culture
Device is connected with the first culture apparatus, and second culture apparatus trains the hemogenic endothelium cell using the second culture medium
It supports, to obtain hematopoietic stem/progenitor.
According to an embodiment of the invention, being hematopoietic stem/progenitor using the system induction ES cell differentiation, embryo is dry
Cell is significantly improved to the efficiency that hematopoietic stem/progenitor is broken up, and hematopoietic colonies Forming ability enhances, and inducing effect is good and steady
It is fixed.
Additional aspect and advantage of the invention will be set forth in part in the description, and will partially become from the following description
Obviously, or practice through the invention is recognized.
Detailed description of the invention
Above-mentioned and/or additional aspect of the invention and advantage will become from the description of the embodiment in conjunction with the following figures
Obviously and it is readily appreciated that, in which:
Fig. 1 shows the experimental group that 100ng/ml leptin is added in culture medium according to an embodiment of the invention, embryo
Stem cell is into hematopoietic stem/progenitor induction atomization, the cellular morphology picture of each Fiber differentiation stage microexamination, picture
In, 200 μm of the scale of big figure, 25 μm of the scale of small figure, wherein
Figure A shows that the form picture of preceding human embryo stem cell is broken up in induction,
Figure B shows the form picture of the cell obtained after second stage culture,
Figure C shows the form picture of the cell obtained after phase III culture,
Figure D shows the form picture of the cell obtained after fourth stage culture,
Fig. 2 shows that the embryonic stem cell of experimental group according to an embodiment of the invention is induced to hematopoietic stem/progenitor
Each Fiber differentiation stage in atomization, the expression variation tendency signal of inducing cell hematopoiesis characterizing gene Runx1 and HoxB4
Figure；
Fig. 3 shows that the embryonic stem cell of experimental group according to an embodiment of the invention is induced to hematopoietic stem/progenitor
The expression diagram of the marker protein Brachyury of the specificity for the cell that second stage obtains in atomization；
Fig. 4 shows the embryonic stem cell of experimental group according to an embodiment of the invention and control group to Hematopoietic Stem/ancestral
Cell induces the table of sign face albumen KDR, CD31 and CD144 of the specificity for the cell that the phase III obtains in atomization
It is illustrated up to situation；
Fig. 5 shows the embryonic stem cell of experimental group according to an embodiment of the invention and control group to Hematopoietic Stem/ancestral
Cell induces the expression of specificity marker the surface protein CD34 and CD45 for the cell that fourth stage obtains in atomization
Diagram；
Fig. 6 shows the embryonic stem cell of experimental group according to an embodiment of the invention and control group to Hematopoietic Stem/ancestral
Cell induces the Colony forming situation diagram for the cell that fourth stage obtains in atomization；And
Fig. 7 shows the embryonic stem cell of experimental group according to an embodiment of the invention and control group to Hematopoietic Stem/ancestral
Cell induces the expression feelings of the hematopoiesis idiosyncratic transcription factor RUNX1 albumen for the cell that the 4th cultivation stage obtains in atomization
Condition diagram.
Specific embodiment
The embodiment of the present invention is described below in detail, examples of the embodiments are shown in the accompanying drawings, wherein from beginning to end
Same or similar label indicates same or similar element or element with the same or similar functions.Below with reference to attached
The embodiment of figure description is exemplary, and for explaining only the invention, and is not considered as limiting the invention.
According to an aspect of the present invention, the present invention provides leptins is divided into Hematopoietic Stem/ancestral in inducing embryo stem cell
Purposes in cell.
Wherein, it should be noted that embryonic stem cell mentioned by the application is all from commercialized immortalized cell line,
For example, WA01 and WA09, is all from the U.S. research center WiCell, the embryo for not needing to destroy the mankind is obtained, is not violated
Ethics.
Inventor has surprisingly found that leptin can significantly improve the efficiency that embryonic stem cell breaks up to hematopoietic stem/progenitor,
The hematopoietic colonies Forming ability of noble cells is improved, inducing effect is good and stablizes.
According to another aspect of the invention, the present invention provides one kind for inducing embryo stem cell be divided into Hematopoietic Stem/
The kit of progenitor cells.According to an embodiment of the invention, the kit includes: the first culture medium and the second culture medium.
First culture medium, first culture medium are 1640 culture medium of AdvancedRPMI for being added to leptin.According to this
The specific example of invention, the concentration of the leptin are 10-300ng/ml, it is preferable that 50-150ng/ml.Embryonic stem cell as a result,
The induced efficiency broken up to hematopoietic stem/progenitor significantly improves, and inducing effect stability is good.It is more according to the present invention specifically to show
Example, first culture medium are further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombinant human bone and form egg
White 4 (BMP4), recombination human basic fibroblast growth factor (bFGF) and Recombinant human vascular endothelial growth factor (VEGF), wherein
The concentration of the 1- thioglycerol is 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；The vitamin C phosphoric ester it is dense
Degree is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The concentration of the glutamine is 1mM~3mM, it is preferable that 2mM；
The concentration of the recombination human bone shaping protein 4 is 10ng/ml~30ng/ml, it is preferable that 20ng/ml；The recombination human basic at
The concentration of fibroblast growth factor is 10ng/ml~30ng/ml, it is preferable that 20ng/ml；And the recombinant human endothelial tube is raw
The concentration of the long factor is 40ng/ml~60ng/ml, it is preferable that 50ng/ml.Culture medium is without heterologous group of cow's serum etc. as a result,
Point, and embryonic stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and inducing effect stability is good.
Second culture medium, second culture medium are 34 culture medium of Stempro for being added to leptin.It is according to the present invention
Specific example, the concentration of the leptin are 10-300ng/ml, it is preferable that 50-150ng/ml.Embryonic stem cell is to hematopoiesis as a result,
The induced efficiency of ancestral cells differentiation significantly improves, and inducing effect stability is good.Some specific examples according to the present invention, institute
State the second culture medium be further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, SCF, IL-3, FLT-3L and
CHIR99021.Wherein, the concentration of the 1- thioglycerol is 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；The dimension
The concentration of raw element C phosphate is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The concentration of the glutamine be 1mM~
3mM, it is preferable that 2mM；The concentration of the rhMGF is 40ng/ml~60ng/ml, it is preferable that 50ng/ml；Institute
The concentration for stating recombination human interleukin 3 (IL-3) is 40ng/ml~60ng/ml, it is preferable that 50ng/ml；The people FMS sample junket ammonia
The concentration of 3 ligand of acid kinase (FLT-3L) is 40ng/ml~60ng/ml, it is preferable that 50ng/ml；And the CHIR99021
Concentration be 2 μM~4 μM, it is preferable that 3 μM.Culture medium is without the heterologous components such as cow's serum as a result, and embryonic stem cell to
The induced efficiency of hematopoietic stem/progenitor differentiation significantly improves, and inducing effect stability is good.
Inventor has surprisingly found that, is divided into hematopoietic stem/progenitor, colony shape using the kit inducing embryo stem cell
Enhance at ability, and embryonic stem cell is significantly improved to the efficiency that hematopoietic stem/progenitor is broken up, inducing effect is good and stablizes.
According to some embodiments of the present invention, which further comprises: third culture medium and the 4th culture medium,
In, the third culture medium is the Advanced RPMI for being added to 1- thioglycerol, vitamin C phosphoric ester, glutamine
1640 culture mediums, the 4th culture medium are to be added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombination activation element
A (Activin A), 3 μM of BMP4, bFGF and CHIR99021 of 1640 culture medium of Advanced RPMI.Wherein, according to this hair
Bright specific example, the concentration of the 1- thioglycerol are 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；The dimension life
The concentration of plain C phosphate is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The concentration of the glutamine be 1mM~
3mM, it is preferable that 2mM；The concentration of the Activin A is 15ng/ml~35ng/ml, it is preferable that 25ng/ml；The BMP4
Concentration be 10ng/ml~30ng/ml, it is preferable that 25ng/ml；The concentration of the bFGF is 10ng/ml~30ng/ml, preferably
Ground, 25ng/ml；And the concentration of the CHIR99021 is 2 μM -4 μM, it is preferable that 3 μM.Culture medium is not necessarily to cow's serum as a result,
Equal heterologous components, and embryonic stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, inducing effect stability
It is good.
According to another aspect of the present invention, using kit above-mentioned, to prepare Hematopoietic Stem/ancestral thin the present invention provides a kind of
The method of born of the same parents.This method comprises: being cultivated using first culture medium pretreated embryonic stem cell, to be given birth to
Blood endothelial cell；The hemogenic endothelium cell is cultivated with using second culture medium, it is thin to obtain Hematopoietic Stem/ancestral
Born of the same parents.
According to an embodiment of the invention, being divided into hematopoietic stem/progenitor using this method inducing embryo stem cell, embryo is dry
Cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and hematopoietic colonies Forming ability enhances, and inducing effect is steady
It is qualitative good.
According to a particular embodiment of the invention, the pretreatment includes: using the third culture medium to embryonic stem cell
It is cultivated, to obtain losing the embryonic stem cell of part totipotency；Portion is lost to described with using the 4th culture medium
The embryonic stem cell of point totipotency is cultivated, to obtain mesodermal progenitor cell.What is obtained as a result, loses part totipotency
Embryonic stem cell and mesodermal progenitor cell, cell activity is good, and differentiation capability is strong.
Some specific embodiments according to the present invention, using first culture medium to the pretreated embryonic stem cell
Culture 84-108 hours utilizes described the using second culture medium to hemogenic endothelium cell culture 84-108 hours
Three culture mediums were to embryonic stem cell culture 12-26 hours, using the 4th culture medium to the embryo for losing part totipotency
Tire stem cell is cultivated 36-60 hours.The hematopoietic stem/progenitor in a large amount of human embryo stem cell sources is obtained as a result, and height expression is made
The distinctive gene of blood ancestral cells and surface protein, the Forming ability with hematopoietic colonies.
Further, in accordance with a further aspect of the present invention, the present invention provides it is a kind of prepare hematopoietic stem/progenitor be
System.According to an embodiment of the invention, the system includes: the first culture apparatus, first culture apparatus utilizes the first culture medium
Pretreated embryonic stem cell is cultivated, to obtain hemogenic endothelium cell；With the second culture apparatus, second culture
Device is connected with the first culture apparatus, and second culture apparatus trains the hemogenic endothelium cell using the second culture medium
It supports, to obtain hematopoietic stem/progenitor.
According to an embodiment of the invention, dry for hematopoietic stem/progenitor embryo using the system induction ES cell differentiation
Cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and hematopoietic colonies Forming ability enhances, and inducing effect is steady
It is qualitative good.
According to a particular embodiment of the invention, which further comprises: pretreatment unit, the pretreatment unit and institute
It states the first culture apparatus to be connected, for pre-processing the embryonic stem cell, the pretreatment unit includes: third culture
Unit, the third culture unit cultivate embryonic stem cell using third culture medium, all-round to obtain losing part
The embryonic stem cell of property；With the 4th culture unit, the 4th culture unit loses part entirely to described using the 4th culture medium
The embryonic stem cell of energy property is cultivated, to obtain mesodermal progenitor cell.It is lost as a result, using what the pretreatment unit obtained
The embryonic stem cell and mesodermal progenitor cell of part totipotency, cell activity is good, and differentiation capability is strong.
Some specific embodiments according to the present invention, using first culture medium to the pretreated embryonic stem cell
Culture 84-108 hours utilizes described the using second culture medium to hemogenic endothelium cell culture 84-108 hours
Three culture mediums were to embryonic stem cell culture 12-26 hours, using the 4th culture medium to the embryo for losing part totipotency
Tire stem cell is cultivated 36-60 hours.The hematopoietic stem/progenitor in a large amount of human embryo stem cell sources is obtained as a result, and height expression is made
The distinctive gene of blood ancestral cells and surface protein, the Forming ability with hematopoietic colonies.
According to some embodiments of the present invention, first culture medium is the AdvancedRPMI 1640 for being added to leptin
Culture medium.Specific example according to the present invention, the concentration of the leptin are 10-300ng/ml, it is preferable that 50-150ng/ml.By
This, embryonic stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and inducing effect stability is good.According to this hair
Bright some specific examples, first culture medium be further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine,
BMP4, bFGF and VEGF, wherein the concentration of the 1- thioglycerol is 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；
The concentration of the vitamin C phosphoric ester is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The concentration of the glutamine is
1mM~3mM, it is preferable that 2mM；The concentration of the recombination human bone shaping protein 4 is 10ng/ml~30ng/ml, it is preferable that
20ng/ml；The concentration of the recombination human basic fibroblast growth factor is 10ng/ml~30ng/ml, it is preferable that 20ng/ml；
And the concentration of the Recombinant human vascular endothelial growth factor is 40ng/ml~60ng/ml, it is preferable that 50ng/ml.It trains as a result,
Base is supported without heterologous components such as cow's serums, and embryonic stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up,
Inducing effect stability is good.
According to some embodiments of the present invention, second culture medium is 34 culture medium of Stempro for being added to leptin.
Specific example according to the present invention, the concentration of the leptin are 10-300ng/ml, it is preferable that 50-150ng/ml.Embryo as a result,
Stem cell significantly improves to the induced efficiency that hematopoietic stem/progenitor is broken up, and inducing effect stability is good.It is more according to the present invention
Specific example, second culture medium are further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, SCF, IL-
3, FLT-3L and CHIR99021.Wherein, the concentration of the 1- thioglycerol is 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；The concentration of the vitamin C phosphoric ester is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The glutamine it is dense
Degree is 1mM~3mM, it is preferable that 2mM；The concentration of the rhMGF is 40ng/ml~60ng/ml, it is preferable that
50ng/ml；The concentration of the recombination human interleukin 3 is 40ng/ml~60ng/ml, it is preferable that 50ng/ml；The people FMS sample
The concentration of 3 ligand of tyrosine kinase is 40ng/ml~60ng/ml, it is preferable that 50ng/ml；And the CHIR99021's is dense
Degree is 2 μM~4 μM, it is preferable that 3 μM.Culture medium is without the heterologous components such as cow's serum as a result, and embryonic stem cell is to hematopoiesis
The induced efficiency of ancestral cells differentiation significantly improves, and inducing effect stability is good.
According to some embodiments of the present invention, the third culture medium is to be added to 1- thioglycerol, vitamin C phosphoric acid
1640 culture medium of Advanced RPMI of ester, glutamine, the 4th culture medium are to be added to 1- thioglycerol, vitamin
C phosphate, glutamine, Activin A, 3 μM of BMP4, bFGF and CHIR99021 of Advanced RPMI 1640 are cultivated
Base.Wherein, the concentration of the 1- thioglycerol is 3 × 10-4M~5 × 10-4M, it is preferable that 4 × 10-4M；The vitamin C phosphorus
The concentration of acid esters is 40 μ of μ g/ml~60 g/ml, it is preferable that 50 μ g/ml；The concentration of the glutamine is 1mM~3mM, preferably
Ground, 2mM；The concentration of the Activin A is 15ng/ml~35ng/ml, it is preferable that 25ng/ml；The concentration of the BMP4 is
10ng/ml~30ng/ml, it is preferable that 25ng/ml；The concentration of the bFGF is 10ng/ml~30ng/ml, it is preferable that 25ng/
ml；And the concentration of the CHIR99021 is 2 μM -4 μM, it is preferable that 3 μM.Culture medium is without heterologous group of cow's serum etc. as a result,
Point, and embryonic stem cell is significantly improved to the induced efficiency that hematopoietic stem/progenitor is broken up, and inducing effect stability is good.
Below with reference to specific embodiment, the present invention will be described, it should be noted that these embodiments are only explanation
Property, and be not considered as limiting the invention.
The solution of the present invention is explained below in conjunction with embodiment.It will be understood to those of skill in the art that following
Embodiment is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.Particular technique or item are not specified in embodiment
Part, it described technology or conditions or is carried out according to the literature in the art according to product description.Agents useful for same or instrument
Production firm person is not specified in device, and being can be with conventional products that are commercially available, such as can purchase from Gibco company.
Embodiment 1
One, culture is broken up in the induction of cell
1. the culture of human embryo stem cell
Inventor cultivates MA01 and MA09 system human embryo stem cell (coming from the research center U.S. Wicell).Specifically
MA01 and MA09 system human embryo stem cell is cultivated respectively in culture medium containing mTeSR (hole 2.5ml/) and is coated with matrigel by ground
In six orifice plates, fresh culture medium is replaced daily.After clonal growth to 70-80% convergence degree, with dispersion enzyme treated cell, to
It after clone rolls at edge, inhales and abandons digestive ferment, and sufficiently terminate the effect of digestive ferment with culture medium rinse cell.Clone is blown out
Cellule agglomerate is inoculated into new culture hole in the passage ratio of 1:4 and continues to cultivate, and the passage period is 4-6 days, is obtained
Human embryo stem cell after massive amplification, using the form of microscope (OLYMPUS, CKX31) observation cell, as shown in Figure 1A,
Cell is in clone's shape growth, and cell arrangement is close, and intracellular nucleocytoplasmic ratio is high, the embryonic stem cell can freeze saved in liquid nitrogen it is standby
With.
Break up 2. human embryo stem cell is induced to hematopoietic stem/progenitor
Human embryo stem cell after the amplification that step 1 is obtained carries out induction differentiation, mainly includes that following 4 inductions are broken up
Stage:
First stage: the culture medium for abandoning human embryo stem cell is inhaled, to promote human embryo stem cell to hematopoietic stem/progenitor
Differentiation, after 24 hours, with DF12 culture medium (be purchased from Thermo company, HyClone SH30023.01B) rinse cell, then,
Culture medium 1 is added, handles cell 24 hours, embryonic stem cell is made to lose part totipotency.
Wherein, 1 component of culture medium are as follows:
1640 culture medium of Advanced RPMI
1- thioglycerol 4 × 10-4M
50 μ g/ml of vitamin C phosphoric ester
Glutamine 2mM
Second stage: inhaling the culture medium for cell of abandoning that treated the first stage, replace 2 Fiber differentiation of culture medium 48 hours,
The form of cell after Fiber differentiation is observed using microscope (OLYMPUS, CKX31), as shown in Figure 1B, cell arrangement is gradually dredged
Pine clones center and half raised cell mass occurs, and clone border cell stretches to outside clone, and cell is turned from epithelium shape to interstitial sample
Become, cell nucleocytoplasmic ratio reduces.Wherein, 2 component of culture medium are as follows:
1640 culture medium of Advanced RPMI
1- thioglycerol 4 × 10-4M
50 μ g/ml of vitamin C phosphoric ester
Glutamine 2mM
Activin A 25ng/ml
BMP4 25ng/ml
bFGF 25ng/ml
CHIR99021 3μM
Phase III: inhaling the culture medium for abandoning second stage treated cell, and with 1 rinse of culture medium 1-2 times.Then,
It replaces culture medium 3 to carry out Fiber differentiation 96 hours, during which change the liquid once, observed and induced using microscope (OLYMPUS, CKX31)
The form of cell after culture, as shown in Figure 1 C, interstitial like cell further increases, and cell arrangement is loose, a part occurs and paves the way
The endothelial cell of stone sample form.
Wherein 3 component of culture medium are as follows:
1640 culture medium of Advanced RPMI
1- thioglycerol 4 × 10-4M
50 μ g/ml of vitamin C phosphoric ester
Glutamine 2mM
BMP4 20ng/ml
bFGF 20ng/ml
VEGF 50ng/ml
Leptin 100ng/ml
Fourth stage: inhaling the culture medium for cell of abandoning that treated the phase III, after cell 1 rinse of culture medium 1-2 times,
Change to (4 component of culture medium: 34 culture medium+1- thioglycerol 4 × 10 of Stempro of culture medium 4-450 μ g/ of M+ vitamin C phosphoric ester
Ml+ glutamine 2mM+SCF 50ng/ml+IL-3 50ng/ml+FLT-3L 50ng/ml+CHIR990213 μM+Leptin
Fiber differentiation 100ng/ml) is carried out, is induced 96 hours, hematopoietic stem/progenitor is obtained, it is experimental group that the cell, which is arranged, using aobvious
Micro mirror (OLYMPUS, CKX31) observes the form for the hematopoietic stem/progenitor that Fiber differentiation obtains, as shown in figure iD, adherent thin
Occur a large amount of suspension cells in born of the same parents.
Culture medium 3 and 4 without leptin is set, and the hematopoietic stem/progenitor of acquisition is control group.
Two, the identification and detection of cell
1, cell RNA extraction, reverse transcription and the expression of real-time quantitative PCR testing goal gene
Using the human embryo stem cell of real-time quantitative PCR test experience group into hematopoietic stem/progenitor induction atomization,
The expression of the target gene of embryonic stem cell each induction differential period, specific as follows:
1.1 experimental method
1.1.1 cell is collected
(1) with without Ca2+And Mg2+Phosphate buffer (PBS) rinse cell after, will be lured with Accutase digestive ferment
Guided cell is digested to unicellular.
(2) it is inhaled after being centrifuged cell and abandons supernatant.
1.1.2 RNA is extracted
(1) lysate (RLT, QIAGEN, 74104) is added into the cell of above-mentioned collection, it is thin that high speed shakes sufficiently cracking
Born of the same parents.
(2) kit (RNeasy Mini Kit, QIAGEN, 74104) is used, to thin after cracking the step of by specification
Born of the same parents isolate and purify, and extract total serum IgE in cell.
1.1.3 RNA solution concentration and purity are detected
RNA concentration and purity data pass through ultraviolet specrophotometer (eppendorf, biophotometer plus)
260nm/280nm light absorption value (A260/280) detection obtains, and purity requirement A260/280 ratio is between 1.8-2.0.
1.1.4 reverse transcription
It is accurate to draw the solution for containing 1 μ g total serum IgE, with reverse transcription reagent box (TOYOBO, FSQ-201), the step of by specification
Suddenly it is cDNA by RNA reverse transcription, obtains the cDNA of sample as a result, and save at -20 DEG C, it is spare.
1.1.5 real-time quantitative PCR
Real-time quantitative reaction system is as shown in the table.It is added in PCR pipe after reagent is mixed, and uses real-time PCR
The expression of (BIO-RAD, iQ5) testing goal gene.
Reaction condition:
1.1.6 data are acquired, and data are analyzed and processed.
1.2 experimental result
By the analysis processing to data, the expression of target gene by using described above as shown in Fig. 2, lured
Culture medium is led, the expression of the distinctive marker gene Runx and HoxB4 of hematopoietic stem/progenitor is shown as induction time extends
It writes and improves.
2. Flow cytometry Nuclear extract
It is specific as follows using the Nuclear extract for the cell that Flow cytometry experimental group second stage obtains:
2.1 experimental methods:
2.1.1 cell is collected
(1) with without Ca2+And Mg2+Phosphate buffer (PBS) rinse inducing cell after, with Accutase digestive ferment
Inducing cell is digested to unicellular.
(2) it is inhaled after being centrifuged cell and abandons supernatant, and washing cell is resuspended with the PBS containing 2% cow's serum.
(3) cell suspension is removed into cell mass by 70-100 μm of cell screen clothes, is inhaled after being centrifuged again and abandons supernatant.
2.1.2 labelled antibody
According to antibody specification, the cell of above-mentioned collection is placed in centrifuge tube and by 1 × 106Cell is resuspended in 500 μ l
Fixative in, room temperature be protected from light incubation 10 minutes.Then, it is washed cell 2 times with PBS, with film cleaning solution is worn, to wash cell primary,
Cell is resuspended with the film cleaning solution of wearing of 100 μ l, isotype control Ab or Brachyury-PE antibody (10 are added into cell suspension
μ l/test) it mixes afterwards, it is placed in 4 DEG C and is protected from light incubation 45 minutes.
2.1.3 antibody is washed
Cell after label is resuspended in 400 μ l PBS with wearing after film cleaning solution washs 2-3 times.
2.1.4 Flow cytometry
It is according to product description and operating guidance, the resuspension cell of above-mentioned acquisition is intracellular using flow cytomery
The expression of albumen.
2.2. experimental result
The expression of the intracellular albumen is detected using flow cytometer (BD, FACSAria), as shown in figure 3, by
After the induction differentiation of first stage, most cells express the distinctive nuclear factor Brachyury of mesoderm, illustrate the training
Feeding base can efficiently break up inducing human embryo stem cell to mesoblastema.
3, Flow cytometry cell surface membrane protein
Detect the expression of the surface membrane protein of the cell of phase III and fourth stage acquisition respectively using flow cytometry
Situation, specific as follows:
3.1 experimental methods:
The cell of experimental group and control group that Processing Example 1 obtains is distinguished according to the following steps, it is specific as follows
3.1.1 collecting cell:
(1) with without Ca2+And Mg2+Phosphate buffer (PBS) rinse cell after, will be lured with Accutase digestive ferment
Guided cell is digested to unicellular.
(2) it is inhaled after being centrifuged cell and abandons supernatant, and washing cell is resuspended with the PBS containing 2% cow's serum.
(3) cell suspension is removed into cell mass by 70-100 μm of cell screen clothes, is inhaled after being centrifuged again and abandon supernatant, is used
Cell is resuspended in the PBS solution of 100 μ l.
3.1.2 labelled antibody
According to antibody specification, the cell of above-mentioned collection is placed in centrifuge tube and by 2 × 105-1×106Cell is resuspended in
In 100 μ l PBS, corresponding antibody is added later, and isotype control Ab or detection is added into the cell suspension of each detection sample
Antibody, wherein the detection antibody of phase III cell be KDR, CD31 and VE-CAD antibody (KDR antibody BD company, 560494；
CD31 antibody eBioscience, 17-0319-42；CD144 antibody BD company, 561566), the inspection for the cell that fourth stage obtains
Survey antibody is CD34 and CD45 (CD34 antibody eBioscience9012-0349；CD45 antibody BD company, 555485), antibody makes
It is 5 μ l/test with dosage, then mixes, is placed in 4 DEG C and is protected from light incubation 45 minutes.
3.1.3 antibody is washed
After the completion of label, the above-mentioned cell marked by antibody is resuspended using 1.4ml PBS, and be centrifuged 5 under 1000rpm
Minute.Supernatant is abandoned later, is repeated the above steps 3 times (be resuspended and be centrifuged).
3.1.3 being resuspended
Cell is resuspended in 400 μ lPBS.
3.1.4 Flow cytometry
According to product description and operating guidance, the room 7-AAD (1 μ l/test) will be added in the resuspension cell of above-mentioned acquisition
Temperature is protected from light incubation after five minutes, carries out the detection of flow cytometry.
3.2 experimental result
With flow cytometer (BD, FACSAria) detection cell surface protein expression, as shown in Figures 4 and 5, to point
Change after adding leptin in second stage induced medium, the expression of hemogenic endothelium cell surface marker albumen KDR, CD3 and CD144
Ratio significantly improves.After induction differentiation phase III addition leptin, hematopoietic stem/progenitor surface marker PROTEIN C D34 and CD45
Expression efficiency significantly improve.Illustrate that the induction of hemogenic endothelium cell and hematopoietic stem/progenitor can be significantly improved by adding leptin
Differentiation efficiency.
4, hematopoietic colonies form detection
It is counted by cell and cell colony, the Colony forming situation for the hematopoietic stem/progenitor that detection fourth stage obtains.
4.1 experimental method
4.1.1 cell is collected
(1) with without Ca2+And Mg2+Phosphate buffer (PBS) rinse hematopoietic stem/progenitor after, disappeared with Accutase
Inducing cell is digested to unicellular by change enzyme.
(2) it is inhaled after being centrifuged cell and abandons supernatant, and washing cell is resuspended with the PBS containing 2% cow's serum.
(3) cell suspension is removed into cell mass by 70-100 μm of cell screen clothes, is inhaled after being centrifuged again and abandons supernatant, 500 μ
Cell is resuspended in 34 culture medium of Stempro of l.
4.1.2 cell and cell colony count
The cell density that cell is resuspended is counted, 500 μ l semisolid culturemediums are added into 24 orifice plate of low adsorption
(Stemcell company, MethoCult 04435), the cell of equivalent is added in culture medium, is sufficiently stirred and mixes cell.
Culture plate is placed in 37 DEG C of incubators of 5% carbon dioxide, to the hematopoietic stem/progenitor collection of formation after continuing culture 7-10 days
Drop into capable counting.
4.2 experimental result
Control group and the hematopoietic stem/progenitor colony sum of experimental group are as shown in fig. 6, the colony sum of experimental group is significantly high
In control group, it is 3 times or so of control group, illustrates that leptin can significantly improve Cells Hematopoietic Colony forming ability.
5、Western Blot
5.1 experimental method
5.1.1 cell is grouped: carrying out induction differentiation training to cell according to the method for " one, the induction of cell break up culture "
It supports, experimental group and control group is set, wherein control group is the culture medium 3 and 4 without leptin, the hematopoietic stem/progenitor of acquisition,
Experimental group is divided into 3 groups, (1) group: leptin is the culture medium 3 and 4 of 20ng/ml, the hematopoietic stem/progenitor of acquisition；(2)
Group: leptin is the culture medium 3 and 4 of 100ng/ml, the hematopoietic stem/progenitor of acquisition；(3) group: leptin 200ng/
The culture medium 3 and 4 of ml, the hematopoietic stem/progenitor of acquisition；
5.1.2 cell is collected
(1) with without Ca2+And Mg2+Phosphate buffer (PBS) rinse hematopoietic stem/progenitor after, disappeared with Accutase
Inducing cell is digested to unicellular by change enzyme.
(2) it is inhaled after being centrifuged cell and abandons supernatant.
5.1.3 total protein content is detected
(1) the RIPA lysate (100 μ l/ pipe) for containing 1% protease inhibitors is added, after 4 DEG C of rotations are incubated for 1-2 hours
130000rpm low-temperature centrifugation 20 minutes.
(2) after being centrifuged, supernatant is drawn into new EP pipe, detects the content of total protein in each sample.
5.1.4SDS-Page electrophoresis
Each group equal protein is drawn respectively, is mixed with 5X Load Buffer, boiling water bath 5 minutes, with 12% acrylamide-
Page gel electrophoresis protein isolate.
5.1.5 transferring film
It will be on protein delivery in glue to 0.45 μm of pvdf membrane using semidry method.
5.1.6 immune response
(1) with 5% skim milk close membrane 1 hour.
(2) by the film after closing, 4 DEG C of rabbit-anti people RUNX1 antibody (being prepared with 5% skim milk) is added and is incubated overnight, uses
PBST buffer washs film 3 times, every time 10 minutes.
(3) above-mentioned film is placed in the goat antirabbit two corresponding anti-solution of cascaded H RP and (is prepared with 5% skim milk) incubation at room temperature
It 1 hour, is washed film 3 times, every time 10 minutes with PBST buffer.
5.1.7 chemiluminescence is developed, fixing
It is incubated for film 10 seconds with ECL solution, with the luminous situation of specific band on photographic film detection film in darkroom.
5.2 experimental result
Film is taken pictures, as shown in fig. 7, object tape is analyzed with gel images processing system, as seen from the figure, to induction
The expression that leptin can significantly improve the transcription factor protein RUNX1 of hematopoietic differentiation key is added in culture medium, and to leptin
There are a certain amount to imitate relationship for concentration, and addition 100ng/ml leptin is best to the activation effect of RUNX1 albumen.
In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specifically show
The description of example " or " some examples " etc. means specific features, structure, material or spy described in conjunction with this embodiment or example
Point is included at least one embodiment or example of the invention.In the present specification, schematic expression of the above terms are not
Centainly refer to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be any
One or more embodiment or examples in can be combined in any suitable manner.
Although an embodiment of the present invention has been shown and described, it will be understood by those skilled in the art that: not
A variety of change, modification, replacement and modification can be carried out to these embodiments in the case where being detached from the principle of the present invention and objective, this
The range of invention is defined by the claims and their equivalents.
Claims (7)
1. a kind of kit for being divided into hematopoietic stem/progenitor for inducing embryo stem cell characterized by comprising
First culture medium, first culture medium are 1640 culture medium of AdvancedRPMI for being added to leptin；And
Second culture medium, second culture medium are 34 culture medium of Stempro for being added to leptin,
Third culture medium, the third culture medium are to be added to 1- thioglycerol, vitamin C phosphoric ester, glutamine
1640 culture medium of Advanced RPMI,
4th culture medium, the 4th culture medium are to be added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombinate and swash
Cerebroysin A, recombination human bone shaping protein 4, recombination human basic fibroblast growth factor and CHIR99021 Advanced RPMI
1640 culture mediums,
Wherein, the concentration of the leptin in first culture medium, in the second culture medium is 10-300ng/ml,
First culture medium is further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombinant human bone and is formed
Albumen 4, recombination human basic fibroblast growth factor and Recombinant human vascular endothelial growth factor,
In first culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The concentration of the vitamin C phosphoric ester is
50μg/ml；The concentration of the glutamine is 2mM；The concentration of the recombination human bone shaping protein 4 is 20ng/ml；The recombination
The concentration of human alkaline fibroblast growth factor is 20ng/ml；And the concentration of the Recombinant human vascular endothelial growth factor is
50ng/ml；
Second culture medium is further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombination human stem cell
The factor, recombination human interleukin 3, people's FMS-like tyrosine kinase 3 ligand and CHIR99021；
In second culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The concentration of the vitamin C phosphoric ester is
50μg/ml；The concentration of the glutamine is 2mM；The concentration of the rhMGF is 50ng/ml；The recombination
The concentration of human interleukin-13 is 50ng/ml；The concentration of people's FMS-like tyrosine kinase 3 ligand is 50ng/ml；And it is described
The concentration of CHIR99021 is 3 μM；
In the third culture medium and the 4th culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The vitamin C phosphorus
The concentration of acid esters is 50 μ g/ml；The concentration of the glutamine is 2mM；The concentration of the recombination activation element A is 25ng/ml；Institute
The concentration for stating recombination human bone shaping protein 4 is 25ng/ml；The concentration of the recombination human basic fibroblast growth factor is 25ng/
ml；And the concentration of the CHIR99021 is 3 μM,
Wherein, the embryonic stem cell is all from commercialized immortalized cell line.
2. kit according to claim 1, which is characterized in that thin in first culture medium, in the second culture medium
The concentration of element is 50-150ng/ml.
3. a kind of method using the preparation hematopoietic stem/progenitor of kit described in as claimed in claim 1 or 22, which is characterized in that packet
It includes:
Pretreated embryonic stem cell is cultivated using first culture medium, to obtain hemogenic endothelium cell；With
The hemogenic endothelium cell is cultivated using second culture medium, to obtain hematopoietic stem/progenitor,
Wherein, described pre-process includes:
Embryonic stem cell is cultivated using the third culture medium, so that the embryo for obtaining losing part totipotency is dry thin
Born of the same parents；With
The embryonic stem cell for losing part totipotency is cultivated using the 4th culture medium, to obtain mesoderm
Progenitor cells,
Wherein, the embryonic stem cell is all from commercialized immortalized cell line.
4. according to the method described in claim 3, it is characterized in that, using first culture medium to the pretreated embryo
Stem cell is cultivated 84~108 hours,
Using second culture medium to the hemogenic endothelium cell culture 84~108 hours,
Using the third culture medium to embryonic stem cell culture 12~26 hours,
Using the 4th culture medium to the embryonic stem cell culture for losing part totipotency 36~60 hours.
5. a kind of system for preparing hematopoietic stem/progenitor characterized by comprising
Pretreatment unit, for the pretreatment unit for pre-processing to embryonic stem cell, the pretreatment unit includes: the
Three culture units, the third culture unit cultivates embryonic stem cell using third culture medium, to obtain losing portion
Divide the embryonic stem cell of totipotency；With the 4th culture unit, the 4th culture unit is lost using the 4th culture medium to described
The embryonic stem cell of part totipotency is cultivated, to obtain mesodermal progenitor cell；
First culture apparatus, first culture apparatus are connected with the pretreatment unit, and first culture apparatus utilizes the
One culture medium cultivates pretreated embryonic stem cell, to obtain hemogenic endothelium cell；With
Second culture apparatus, second culture apparatus are connected with the first culture apparatus, and second culture apparatus utilizes second
Culture medium cultivates the hemogenic endothelium cell, to obtain hematopoietic stem/progenitor,
Wherein,
First culture medium is 1640 culture medium of AdvancedRPMI for being added to leptin；
Second culture medium is 34 culture medium of Stempro for being added to leptin；
The third culture medium is the Advanced RPMI for being added to 1- thioglycerol, vitamin C phosphoric ester, glutamine
1640 culture mediums；
4th culture medium is to be added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombination activation element A, recombination
1640 culture medium of Advanced RPMI of human BMP-4, recombination human basic fibroblast growth factor and CHIR99021,
In first culture medium, in the second culture medium, the concentration of the leptin is 10ng/ml~300ng/ml,
First culture medium is further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombinant human bone and is formed
Albumen 4, recombination human basic fibroblast growth factor and Recombinant human vascular endothelial growth factor,
In first culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The concentration of the vitamin C phosphoric ester is
50μg/ml；The concentration of the glutamine is 2mM；The concentration of the recombination human bone shaping protein 4 is 20ng/ml；The recombination
The concentration of human alkaline fibroblast growth factor is 20ng/ml；And the concentration of the Recombinant human vascular endothelial growth factor is
50ng/ml,
Second culture medium is further added to 1- thioglycerol, vitamin C phosphoric ester, glutamine, recombination human stem cell
The factor, recombination human interleukin 3, people's FMS-like tyrosine kinase 3 ligand and CHIR99021,
In second culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The concentration of the vitamin C phosphoric ester is
50μg/ml；The concentration of the glutamine is 2mM；The concentration of the rhMGF is 50ng/ml；The recombination
The concentration of human interleukin-13 is 50ng/ml；The concentration of people's FMS-like tyrosine kinase 3 ligand is 50ng/ml；And it is described
The concentration of CHIR99021 is 3 μM,
In the third culture medium and the 4th culture medium, the concentration of the 1- thioglycerol is 4 × 10-4M；The vitamin
The concentration of C phosphate is 50 μ g/ml；The concentration of the glutamine is 2mM；The concentration of the recombination activation element A is 25ng/
ml；The concentration of the recombination human bone shaping protein 4 is 25ng/ml；The concentration of the recombination human basic fibroblast growth factor is
25ng/ml；And the concentration of the CHIR99021 is 3 μM,
Wherein, the embryonic stem cell is all from commercialized immortalized cell line.
6. system according to claim 5, which is characterized in that trained using first culture medium to the embryonic stem cell
It supports 84~108 hours,
Using second culture medium to the hemogenic endothelium cell culture 84~108 hours,
Using the third culture medium to the embryonic stem cell culture 12~26 hours,
Using the 4th culture medium to the embryonic stem cell culture for losing part totipotency 36~60 hours.
7. system according to claim 6, which is characterized in that described thin in first culture medium, in the second culture medium
The concentration of element is 50ng/ml~150ng/ml.
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| CN102206610A (en) * | 2010-03-26 | 2011-10-05 | 北京大学 | Preparation method of hemopoietic progenitor cells and special medium for the same |
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