CN108753685A - A kind of separation, screening, culture and the Function Identification method of the human aorta vascular wall stem cell of expression c-Kit - Google Patents
A kind of separation, screening, culture and the Function Identification method of the human aorta vascular wall stem cell of expression c-Kit Download PDFInfo
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Abstract
The invention discloses a kind of separation, screening, culture and the Function Identification methods of the human aorta vascular wall stem cell of expression c-Kit.The present invention provides a kind of kits obtaining human aorta vascular wall stem cell, include the substance in conjunction with c-Kit positive cells.In mentioned reagent box, the substance of the combination c-Kit positive cells includes c-Kit antibody；And/or the c-Kit antibody specifics are c-Kit immunomagnetic beads antibody.The present invention is from human aorta tissue separating blood vessel outer membrane, with collagenase digesting, it is filtered by cell sieve and obtains cell suspension, c-Kit positive blood tube wall stem cells are obtained by c-Kit immunomagnetic beads antibody screenings, such stem cell has good in-vitro multiplication and transfer ability, few apoptosis, the not only differentiation potential (can be divided into adipocyte, osteoblast, cartilage cell) with mescenchymal stem cell can also be divided into the tissue-specific differentiation ability of vascular smooth muscle cells with vascular wall stem cell.
Description
Technical field
The invention belongs to stem cell and tissue engineering technique field more particularly to a kind of human aorta blood of expression c-Kit
Separation, screening, culture and the Function Identification method of tube wall stem cell.
Background technology
Adult stem cell is a kind of neoblast being present in adults or human body in differentiated tissue, this thin
Born of the same parents have constantly proliferation and the ability of self-renewing, can break up as the residing particular kind of cell of tissue, to mend in time
The mature cell being damaged in the tissue is filled, histoorgan growth and aging, damage and the dynamic equilibrium repaired are maintained.In recent years,
Numerous studies show that artery in mouse and people and venous blood tube wall outer membrane reside a group adult stem cell, in normal physiological
In the case of, they maintain vessel homeostasis, and the cells of vascular wall of update aging apoptosis is replenished in time.And under pathological state, example
Such as in interior skin lesion injures vascular wall lesion, these stem cells can be by active oxygen, chemotactic factor (CF), cell factor, growth factor, gold
The endothelial cell or smooth of damage is replenished in time by modes such as proliferation, migration, differentiation in a variety of factor activators such as Proteases
Myocyte, participate in injury of blood vessel after repair or vascular remodeling during.Currently, in many researchs, vascular wall stem cell is
It is isolated on mouse aorta, a variety of blood vessels such as grafting vessel, and successfully cultivate in vitro, it is applied to a variety of mouse blood vessels
The research of disease model pathophysiological mechanism.However, deepening continuously with vascular wall stem-cell research in recent years, if energy
Vascular wall stem cell is obtained from the separation of human vas tissue, Pathological Physiology machine that not only can directly to a variety of vascular diseases of human body
In-depth study is made, more establishes normal and a variety of disease vascular wall stem cell banks in clinic in the future, to be applied to group
It knits engineering science and clinical stem-cell therapy provides sufficient resource.
Currently, the research for people vascular wall stem cell, part document simply by immunohistochemical staining method
Prove exist on normal and various disease group human aorta tissue blood vessel slice with the thin of expression stem cell labeling molecule
Born of the same parents, and do not carry out cell separation and in vitro culture.Though separately thering is part document to be reported in normal person's aorta vessel to isolate
Cell express mescenchymal stem cell mark molecule, but do not carry out in separation process the screening of stem cell labeling molecule, lead
Cause the stem cell purity finally actually obtained not high.
C-Kit belongs to one of tyrosine kinase receptor protein family member, as stem cell factor receptor, it can with it is dry thin
Intracellular cytokine is combined, and to start corresponding signal access, regulates and controls the various biologicals function such as proliferation, differentiation of stem cell.c-Kit
It is a kind of vital signs molecule of the identification specific parting of marrow hemopoietic stem cells, the detection of c-Kit can be used for clinically acute white
The classification diagnosis and classification diagnosis of blood disease.
Invention content
It is an object of the present invention to provide a kind of kits obtaining human aorta vascular wall stem cell.
Kit provided by the invention includes the substance in conjunction with c-Kit positive cells.
In mentioned reagent box, the substance of the combination c-Kit positive cells includes c-Kit antibody；
And/or the c-Kit antibody specifics are c-Kit immunomagnetic beads antibody.
In mentioned reagent box, the kit further includes the erythrocyte cracked liquid for splitting erythrocyte.
The kit further includes dissociating buffer and splitter.
The dissociating buffer is the sterile PBS solution of the EDTA containing 0.5%BSA and 2mM.
Mentioned reagent box further includes the culture medium for cultivating c-Kit positive cells,
The culture medium is grouped as by following group：DMEM-F12+GlutaMAX-1 basal mediums, volumn concentration are
20% FBS, 100U/ml mycillin, 10ng/ml human LIF, 0.1mM beta -mercaptoethanols and 20ng/ml human
BFGF is formed.
Mentioned reagent box further includes recording the module of separation method or comparing to block；
The separation method includes the following steps：
1) from vitro human aorta tissue separating blood vessel outer membrane, then the externa is digested to cell；
2) substance of combination c-Kit positive cell of the cell in above-mentioned kit is passed through positive with c-Kit
C-Kit on cell is combined, and isolates c-Kit positive cells, that is, obtains human aorta vascular wall stem cell.
In step 1) and 2) further include following steps between：Postdigestive cell is filtered through cell sieve, collects filtrate,
The cell precipitation obtained in the filtrate is centrifuged again；Finally remove the red blood cell in the cell precipitation.
C-Kit is being also the model of the invention protected as the application in acquisition human aorta vascular wall stem cell markers
It encloses.
Application of the above-mentioned kit in preparing human aorta vascular wall stem cell products is also protection of the present invention
Range；
Or, application of the above-mentioned kit in obtaining human aorta vascular wall stem cell is also the model that the present invention protects
It encloses.
Or, the substance of above-mentioned combination c-Kit positive cells is in preparing human aorta vascular wall stem cell products
Using being also the scope of protection of the invention；
Or, application of the substance of above-mentioned combination c-Kit positive cells in obtaining human aorta vascular wall stem cell is also
The scope of protection of the invention；
Or, application of the mentioned reagent box in preparing human aorta vascular wall stem cell products is also protection of the present invention
Range；
Or, application of the mentioned reagent box in obtaining human aorta vascular wall stem cell is also the scope of protection of the invention；
Or, application of the above-mentioned culture medium in cultivating c-Kit positive cells is also the scope of protection of the invention.
Second purpose of the invention is to provide a kind of method obtaining human aorta vascular wall stem cell.
Method provided by the invention, includes the following steps：Using c-Kit as aortic blood tube wall stem cell markers, into
The separation of row aorta vascular wall stem cell.
In the above method, the separating step is as follows：
1) from vitro human aorta tissue separating blood vessel outer membrane, then the externa is digested to cell；
2) substance of combination c-Kit positive cell of the cell in above-mentioned kit is passed through positive with c-Kit
C-Kit on cell is combined, and isolates c-Kit positive cells, that is, obtains human aorta vascular wall stem cell.
It is described that externa is digested to cell using collagenase digesting；
The substance of the combination c-Kit positive cells is the antibody of such as c-Kit, specially c-Kit immunomagnetic beads antibody；
Further include following steps between the step 1) and step 2)：The cell is filtered through cell sieve, collects filtrate,
The cell precipitation obtained in the filtrate is centrifuged again；Finally remove the red blood cell in the cell precipitation.
The above method further includes following steps：By the c-Kit positive cells above-mentioned culture c-Kit positive cells training
It supports and is cultivated in base.
3rd purpose of the invention is to provide a kind of culture medium of culture c-Kit positive cells.
Culture medium provided by the invention is the culture medium in above-mentioned kit.
The present invention is obtained thin from human aorta tissue separating blood vessel outer membrane with collagenase digesting by cell sieve filtering
Born of the same parents' suspension obtains c-Kit positive blood tube wall stem cells by c-Kit immunomagnetic beads antibody screenings, such stem cell has good
In-vitro multiplication and transfer ability, few apoptosis, not only the differentiation potential with mescenchymal stem cell (can be divided into fatty thin
Born of the same parents, osteoblast, cartilage cell), the tissue specificity of vascular smooth muscle cells can be also divided into vascular wall stem cell
Differentiation capability；It screens, leads due to a lack of specific stem cell markers in short, the method for the present invention overcomes in previous separation method
It causes the stem cell purity of separation low, grows slow problem, human aorta vascular wall stem cell can be significantly improved by providing one kind
The new isolated culture method of purity and cell activity.
Description of the drawings
Fig. 1 is the c-Kit that flow cytometry identifies stem cell labeling developed by molecule level and immunofluorescence staining label
Positive cell results figure；
Wherein, A-I is isolated cell from human aorta vascular wall adventitial tissue, is sieved in c-Kit immunomagnetic beads
It is horizontal through flow cytometry identification stem cell labeling developed by molecule before choosing；J is after c-Kit immunomagnetic beads antibody screenings, with exempting from
The cell of the c-Kit positives of epidemic disease fluorescence colour label.
Fig. 2 is the c-Kit positive blood tube wall stem cells of normal donors and Replacement of aorta patient after cultivating 24 hours
Proliferative cell correlation nuclear antigen ki67 expressions.
Fig. 3 is that normal donors and Replacement of aorta patient c-Kit positive blood tube wall stem cells wither after cultivating 72 hours
Die horizontal identification；(A) AnnexinV/PI detection methods, (B) TUNEL decoration methods.
Fig. 4 is to normal donors and Replacement of aorta patient's c-Kit positive bloods tube wall stem cell migration 6-8 hours
Level detection；A is Transwell migration experiments, and (B) is scratch experiment.
Fig. 5 be normal donors and Replacement of aorta patient c-Kit positive blood tube wall stem cells be divided into adipocyte,
The horizontal detection of osteoblast, cartilage cell and smooth muscle cell.
Fig. 6 is that normal donors and Replacement of aorta patient c-Kit positive blood tube wall stem cells are divided into vascular smooth
The horizontal detection of myocyte.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Separation, screening and the culture of embodiment 1, human aorta vascular wall stem cell
One, the separation and screening of human aorta vascular wall stem cell
1, the separation of human aorta vascular wall stem cell
1) separating blood vessel outer membrane
It takes normal organ to contribute donor or receives patient sustainer tissue (patient, donor or the donor of Replacement of aorta
Family members know), area is about 2 × 5cm2, it is immediately placed in the ice-cold sterile tissues of 20ml and preserves (EDTA-Na containing 1mg in liquid2And
The sterile PBS solution of 3%FBS).Vascular tissue is taken out from tissue preserration liquid with aseptic nipper in super-clean bench, with containing 1mg
EDTA-Na2Sterile PBS solution repeatedly rinse vascular tissue, removal residual bloodstain, until rinsing liquid clarification is visible by naked eyes blood
Color.Externa is gently detached with middle film with two aseptic nippers, retains adventitial tissue, obtains externa, and float again
It washes three times, after exhausting liquid as possible, outer membrane is cut into the fractionlet of 2-3mm with sterile scissors.
2) collagenase digesting
Fragment of tissue is put into (the Liberase of clostridiopetidase A containing 0.25mg/ml of 37 DEG C of preheatings of about 4mlTM TL
Research Grade, 5401020001, ROCHE) (0.1mg clostridiopetidase As in serum-free DMEM/F12 culture mediums:1cm2Actively
Arteries and veins tissue) in 37 DEG C of incubators, it is shaken 3-4 hours on shaking table.
When digestion suspension muddiness at chyle sample, when having no apparent tissue block, you can stop digestion.It is blown and beaten repeatedly with pipette
Digestive juice, as possible dispersion tissue's cell mass.The steril cell that aperture is 100 μm and 70 μm is used to be sieved through filter tissue successively again outstanding
Liquid is used in combination full culture medium to rinse each strainer two or three times.It collects filtered fluid to centrifuge 5 minutes with 1000rpm, abandons supernatant, collect thin
Born of the same parents are precipitated.
2, the screening of c-Kit positive humans aortic blood tube wall stem cell
The human aorta vascular wall stem cell obtained to 1 carries out the screening of c-Kit mark molecules, and method is specific as follows：
1), splitting erythrocyte
To avoid splitter blocking caused by abundant residues red blood cell non-specific binding magnetic bead in histocyte suspension
5ml erythrocyte cracked liquids are added before screening c-Kit positive cells with splitter in situation in the cell precipitation first obtained one
Cell is resuspended in (the green skies, C3702) (5 times of erythrocyte cracked liquids that cell concentration is added), is incubated at room temperature 10 minutes, 20ml is added
After PBS mixings, 1000rpm is centrifuged 5 minutes, abandons supernatant, until without apparent red precipitate, the cell for obtaining removal red blood cell is heavy
It forms sediment.
2), c-Kit antibody (CD117MicroBead Kit, human, Miltenyi Biotec, 130-091-332) is tied
Close cell
To cell precipitation (the about 1x10 of the above-mentioned removal red blood cell 1) obtained7A cell) in be added precooling 300 μ l
Dissociating buffer (is made of) volumn concentration 0.5%BSA, 2mM EDTA and sterile PBS solution, and cell counting board counts,
Cell quantity is less than 108(it is denoted as the total cell number before screening) when a cell, while 100 μ lFcR confining liquids are added
(being provided in CD117MicroBead kit) and 100 μ l c-Kit immunomagnetic beads coupled antibodies (CD117MicroBead kit),
It mixes well, is incubated 15 minutes in 4 DEG C of refrigerators, cell suspension after being incubated.
3), magnetic bead adsorbs
Cell suspension after the above-mentioned incubation 2) obtained is taken out, is added 2ml dissociating buffers, 300g centrifuges 10 minutes, completely
Supernatant is exhausted, is discarded.
It is centrifuged simultaneously in cell, splitter is fixed on MACS separators, 500 μ l dissociating buffers, rinse column is added
Son avoids being mixed into bubble, blocks pillar.
500 μ l dissociating buffers are added in cell precipitation after centrifugation, liquid-transfering gun blows and beats mixing, slowly by cell suspension
Splitter (MS Columns, Miltenyi Biotec, 130-042-201) is added.At this point, by c-Kit immunomagnetic beads antibody marks
The positive cell of note is attracted to using magnetism in splitter, and not by the negative cells of c-Kit labels then with liquid outflow point
From column.
After cell suspension drop are most, 500 μ l dissociating buffers are added again and rinse the negative cells remained in splitter,
It is repeated twice, obtained positive cell is adsorbed in splitter.
4), c-Kit positive cells detach
Splitter after the above-mentioned adherent cell 3) obtained is gently removed into MACS separators, it is sterile to be placed in a new 15ml
On centrifuge tube, 1ml dissociating buffers are added in splitter, mating piston is used in combination to squeeze into splitter inner cell suspension rapidly
In the centrifuge tube of lower section, collects people's vascular wall stem cell that c-Kit positive cell suspensions are the c-Kit positives and (derive from normal
Organ donation donor or receive Replacement of aorta patient aortic tissue).
The c-Kit positive cells that cell counting board counting collection arrives, by the way that (c-Kit is immune with the total cell number before screening
Magnetic bead coupled antibody combines preceding cell number) it compares, calculate the percentage of c-Kit positive cells.
The percentage of people's vascular wall stem cell (contributing donor from normal organ) is about 10%；
The percentage of people's vascular wall stem cell (from the patient sustainer tissue for receiving Replacement of aorta) is about
5%-12%.
Two, the culture of human aorta vascular wall stem cell
The human aorta vascular wall stem cell that above-mentioned one obtains is cultivated as follows：
3ml complete mediums are added in the people vascular wall stem cell obtained to above-mentioned one, is added and is covered with after cell mixing
The culture dish of a diameter of 35mm of 0.04%B type ox-hide collagens, is put into 37 DEG C, 5%CO2In incubator.After 24 hours, observation is thin
Born of the same parents, 90% or more cell is adherent, and cell is in fusiformis or spindle.The culture medium more renewed, the not adherent cell of removal.3 days
It is primary to replace culture medium, after 3-7 days, when cell is grown to 90%, with 0.05% trypsase EDTA digestion, you can press 1:3 ratios
It is passed on.
Above-mentioned complete medium is grouped as by following group：DMEM-F12+GlutaMAX-1 basal mediums (GibcoTM,
10565018), 20% (volumn concentration) FBS, 100U/ml mycillins, 10ng/ml human LIF, 0.1mM β-sulfydryls
Ethyl alcohol and 20ng/ml human bFGF compositions.
Three, the functional verification of human aorta vascular wall stem cell
1, human aorta vascular wall stem cell separation purity is identified
It reflects to the 1 different kinds of molecules marker for obtaining cell after collagenase digesting from above-mentioned one using flow cytometry
It is fixed：It being protected from light incubated cell on ice 1 hour with different molecular antibody and its corresponding IgG respectively, 1500rpm is centrifuged after five minutes,
PBS is washed one time, and PBS is resuspended, and immediately passes through each antibody expression of flow cytomery；Including stem cell labeling molecule
CD29, CD73, CD105, CD44, CD90 and c-Kit, endothelial cell marker molecule CD31, leukocyte marker molecule CD45, macrophage
Cell marker molecules CD11b.
As a result as shown in Figure 1A-I, it can be seen that the 1 of the present invention above-mentioned one obtains cell precipitation and expresses a large amount of stem cell marks
It scores son, about 10% is c-Kit positive cells, hardly expresses endothelium, leucocyte, macrophage marker molecule, explanation passes through
The stem cell purity that the separation method is extracted is high, is seldom mixed into other tissues and blood cell.
Immunofluorescence staining detects above-mentioned one 2 obtained c-Kit positive cell suspension cells, and cell climbing sheet is with 4%
Paraformaldehyde fixes 10 minutes, and donkey serum room temperature is closed 60 minutes, and rabbit-anti c-Kit primary antibodies, 4 DEG C of overnight incubations is added to add donkey anti-rabbit
Secondary antibody is incubated at room temperature 1 hour, and PBS is washed three times, and DAPI is dyed 5 minutes, and mounting, fluorescence microscope is as a result, result such as Fig. 1 J
It is shown, it is seen that 90% or more cell expresses c-Kit albumen, illustrates the c-Kit stem cells obtained by this extraction and screening technique
Purity is very high.
2, human aorta vascular wall stem cells hyperplasia ability is identified
To identify the proliferative capacity of c-Kit positive human aortic blood tube wall stem cells, people's vascular wall from above-mentioned two is dry thin
Kind is in 4 hole glass plates after born of the same parents pass on forth generation cell dissociation, by nucleus related antigen ki67 immunofluorescence dyeings, thin
After born of the same parents cultivate 24 hours, 4% formalin fixes 5 minutes, and donkey serum room temperature is closed 1 hour, 4 DEG C of overnight incubations of ki67 antibody,
PBS is washed three times, is incubated at room temperature 1 hour of corresponding secondary antibody, and PBS is washed three times, and DAPI is dyed 5 minutes, mounting, and fluorescence microscope is seen
Examine result.
The results are shown in Figure 2, dry thin from the normal people's vascular wall for contributing donor and Replacement of aorta patient's separation and Extraction
Born of the same parents (c-Kit is positive) all have apparent ability of cell proliferation.
3, human aorta vascular wall stem cell level of apoptosis is identified
For identify c-Kit positive human aortic blood tube wall stem cells level of apoptosis, cell in vitro according to two method
It after culture 72 hours, is dyed by Annexin V/PI apoptosis kits and TUNEL, is utilized respectively flow cytometry and fluorescence is aobvious
Micro mirror detects apoptotic cell quantity (Annexin V/PI kits, the green skies, C1063；TUNEL kits, the green skies,
C1088) (above method is operated according to corresponding reagent box specification).
The results are shown in Figure 3, dry thin from the normal people's vascular wall for contributing donor and Replacement of aorta patient's separation and Extraction
Born of the same parents (c-Kit is positive) minute quantity cell under condition of culture provided by the invention is in apoptosis and necrosis is horizontal, only less than 4%
Apoptosis or death, illustrate that extraction through the invention and the obtained c-Kit Stem Cell Activities of screening technique are also very high.
4, human aorta vascular wall stem cell migration ability is identified
To identify the transfer ability of c-Kit positive human aortic blood tube wall stem cells, migrated respectively by Transwell real
It tests and scratch experiment, counts and migrated to the cells Transwell bottom surface and cut with the cell of inner region after 6-8 hours.
The results are shown in Figure 4, dry thin from the normal people's vascular wall for contributing donor and Replacement of aorta patient's separation and Extraction
Born of the same parents (c-Kit is positive) all have stronger cell migration ability.
5, positive human aortic blood tube wall stem cell differentiation capability is identified
For identify c-Kit positive human aortic blood tube wall stem cells differentiation capability, using routine at fat, skeletonization, at
Stimulation differentiation is carried out in cultured chondrocytes liquid, and (Analytical Chemical Experiment uses Human Mesenchymal Stem Cell
Functional Identification Kit, R&D SC006), (method and step operates to specifications).
The results are shown in Figure 5 for immunofluorescence dyeing, and after breaking up 14 days, adipocyte mark molecule expression increases.21
After it, chondroblast and osteoblast marker developed by molecule level increase.
In addition, according to Fig. 6 the experimental results showed that, in specific SMC differentiation culture solution (DMEM-F12+
1%FBS, 10ng/ml TGF-β 1 are added in GlutaMAX-1 culture mediums) moderate stimulation break up 7 days after, analyzed by qPCR, smoothly
Myocyte mark molecule Calponin mRNA level in-sites are significantly raised before relatively breaking up, and immunofluorescence dyeing is further discovered that
Calponin protein expression levels are also significantly raised.
Particular embodiments described above has carried out further in detail the purpose of the present invention, technical solution and advantageous effect
Describe in detail it is bright, for the ordinary skill in the art, it is possible to understand that the case where not departing from the principle and spirit of the invention
Under can these embodiments be carried out with a variety of change, modification, replacement and modification, the scope of the present invention by appended claims and its
Equivalent limits.
Claims (10)
1. a kind of kit obtaining human aorta vascular wall stem cell includes the substance in conjunction with c-Kit positive cells.
2. kit according to claim 1, it is characterised in that：The substance of the combination c-Kit positive cells includes c-
Kit antibody；
And/or the c-Kit antibody specifics are c-Kit immunomagnetic beads antibody.
3. kit according to claim 1 or 2, it is characterised in that：The kit further includes culture c-Kit positive thin
The culture medium of born of the same parents,
The culture medium is grouped as by following group：DMEM-F12+GlutaMAX-1 basal mediums, volumn concentration 20%
FBS, 100U/ml mycillin, 10ng/mlhuman LIF, 0.1mM beta -mercaptoethanols and 20ng/ml human bFGF groups
At.
4. according to any kit in claim 1-3, it is characterised in that：The kit further includes red for cracking
The erythrocyte cracked liquid of cell.
5. kit according to any one of claims 1-4, it is characterised in that：The kit further includes recording separation side
The module of method compares card；
The separation method includes the following steps：
1) from vitro human aorta tissue separating blood vessel outer membrane, then the externa is digested to cell；
2) substance of the combination c-Kit positive cells in any kit in the cell claim 1-5 is led to
It crosses and is combined with the c-Kit on c-Kit positive cells, isolate c-Kit positive cells, that is, it is dry thin to obtain human aorta vascular wall
Born of the same parents.
6.c-Kit is as the application in obtaining human aorta vascular wall stem cell markers；
Or, the substance of the combination c-Kit positive cells in claim 1-5 in any kit is preparing people master
Application in arterial blood tube wall stem cell products；
Or, the substance of the combination c-Kit positive cells in claim 1-5 in any kit is obtaining human aorta
Application in vascular wall stem cell；
Or, any kit answering in preparing human aorta vascular wall stem cell products in claim 1-5
With；
Or, application of any kit in obtaining human aorta vascular wall stem cell in claim 1-5；
Or, application of the culture medium in claim 1-5 in any kit in cultivating c-Kit positive cells.
7. a kind of method obtaining human aorta vascular wall stem cell, includes the following steps：Using c-Kit as aortic blood tube wall
Stem cell markers carry out the separation of aortic blood tube wall stem cell.
8. according to the method described in claim 8, it is characterized in that：The separating step is as follows：
1) from vitro human aorta tissue separating blood vessel outer membrane, then the externa is digested to cell；
2) substance of the combination c-Kit positive cells in any kit in the cell claim 1-5 is led to
It crosses and is combined with the c-Kit on c-Kit positive cells, isolate c-Kit positive cells, that is, it is dry thin to obtain human aorta vascular wall
Born of the same parents.
9. according to the method described in claim 8, it is characterized in that：
The method further includes following steps：The c-Kit positive cells is positive in the culture c-Kit of claim 4
It is cultivated in the culture medium of cell.
10. a kind of culture medium of culture c-Kit positive cells, is the culture in claim 1-5 in any kit
Base.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030054973A1 (en) * | 2001-06-06 | 2003-03-20 | Piero Anversa | Methods and compositions for the repair and/or regeneration of damaged myocardium |
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