CN108265029A - The screening of the human cardiac ventricle myocyte in human pluripotent stem cell source and preparation method - Google Patents
The screening of the human cardiac ventricle myocyte in human pluripotent stem cell source and preparation method Download PDFInfo
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Abstract
The invention belongs to biomedical researches and application field, it is related to screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source, the present invention carries out human cardiac ventricle myocyte specificity screening purifying and a large amount of preparations after forming cardiac muscle cell from human pluripotent stem cell directed differentiation.The present invention utilizes genome editing technique, build the human pluripotent stem cell cell line that the ending of MYL2 genes carries neomycin or green fluorescent protein screening-gene, and Induction of committed differentiation is cardiac muscle cell, and then purify to obtain human cardiac ventricle myocyte by neomycin or green fluorescent protein screening-gene, the different various engineering human cardiac ventricle muscular tissues of culture forming properties can be combined from any bracket material.The human cardiac ventricle myocyte of the present invention has the phenotype similar to normal human subject ventricular muscle cell and electrophysiologic response, derives from a wealth of sources, and can long-term in vitro culture.Treatment means and the effective drug of screening to be directed to ventricle injury of muscle and disease provide good platform.
Description
Technical field
The invention belongs to biomedical researches and application field, and in particular to pass through human pluripotent stem cell directed differentiation cardiac muscle
Screening and the preparation method of ventricular muscle cell are carried out after cell.
Background technology
It is to endanger human health " number one killer " prior art discloses angiocardiopathy, according to the World Health Organization
(WHO) it counts, the number that angiocardiopathy is died of in the whole world every year is up to 17,100,000, accounts for the 29% of global death toll, and with every
The speed increase incidence and lethality in year 10.42% are in growing trend, Prevention status cause anxiety year by year.Although traditional drug is controlled
The application of the methods for the treatment of, interventional treatment and bypass surgery improves the prognosis of Ischemic Heart Disease, but still can not save
Through dead cardiac muscle cell, reverse remodeling ventricle and subsequent heart failure.Myocardial repair and function weight after myocardial ischemia, infarct
Building has become the global problem in the field, and there is an urgent need for explore new therapy and approach.It is moved by human heart dissection with coronal
The physilogical characteristics of arteries and veins are determined that the position of current most of its myocardial necrosis of myocardial infarction patient is all located at ventricular muscles, because
This, the regenerative therapy of following heart has the ventricular muscle cell of autologous patient very big demand.
For the most of cardiac muscle cell of studies have shown that all in terminal differentiation state, the ability of self-regeneration is extremely limited；
And multipotent stem cells (including hESC (hESCs) and inducing pluripotent stem cells (hiPSCs)) have multidirectional point
Change potential and self-renewal capacity, can infinitely rise in value, can theoretically be divided into the body cell of adult mankind's any one.Cause
Multipotent stem cells directed differentiation cardiovascular system cell is passed through the mechanism such as Myocardial Regeneration and angiogenesis and substitutes infarct by this
Cardiac muscular tissue is increasingly becoming extremely promising cardiac disease treatment new method.
At present, although have it is several about multipotential stem cell toward the report of cardiac muscle cell direction differentiation method, differentiation
Cardiac muscle cell's purity is often inadequate, typically contains a certain proportion of ventricular muscle cell, atrial muscle cell, fibroblast, smoothly
Myocyte, endothelial cell, Sino-atrial Pacemaker Cells, the cell of these Combination, which is implanted into heart infarction animal, causes arrhythmia cordis, leads
Cause animal pattern dead.
Present situation based on the prior art, present inventor intend providing human cardiac ventricle's flesh in human pluripotent stem cell source
The screening of cell and preparation method are not beaten independently high-purity by directed differentiation and drug screening or airflow classification acquisition
Human cardiac ventricle myocyte is spent, will be transplanted available for following self ventricular muscle cell.
Invention content
The purpose of the present invention is defect of the existing technology is overcome to be directed to the limitation in current ventricular muscle cell source, provide
The screening of the human cardiac ventricle myocyte in human pluripotent stem cell source and preparation method.Using genetic engineering means, one group is established
The multipotential stem cell cell of MYL2 gene combination neomycins (Neomycin) and MYL2 gene combining with green fluorescins (eGFP)
Strain, the high-purity human cardiac ventricle myocyte do not beaten independently by directed differentiation and drug screening or airflow classification acquisition,
It is transplanted available for following self ventricular muscle cell.Or conjunctive tissue engineering science, production prepare functional available for transplanting
The artificial mankind's ventricular muscles tissue of engineering.
Band is useful for by the present invention using such as TALEN or CRISPR/CAS9 technologies mediation genetic recombination of gene editing technology
The neomycin of ventricular muscle cell selection markers or green fluorescent protein screening-gene import human pluripotent stem cell (including the mankind
Embryonic stem cell and inducing pluripotent stem cells) in genome after specific MYL2 gene locis, establish for human cardiac ventricle
The multipotential stem cell strain of flesh screening, and then (technology is published by multipotential stem cell directed differentiation cardiac muscle cell technology
Mature technology, specific implementation step are shown in Lian et al., Nature Protocols, 2012；8：162-75), pass through neomycin
Drug screening or green fluorescent protein airflow classification prepare the human cardiac ventricle myocyte that can be used for research and application.
Specifically, the present invention provides the human cardiac ventricle myocyte's after human pluripotent stem cell directed differentiation cardiac muscle cell
Screen purification process, which is characterized in that using genome editing technique, such as the effective base of TALEN, CRISPR/CAS9 or any
Because of recombinant technique, human pluripotent stem cell genome is edited, specific MYL2 genes ending is built and carries neomycin or green glimmering
The human pluripotent stem cell cell strain of photoprotein screening-gene, including step：
A, structure is for target gene MYL2's (albumen of target gene editor is the specific markers of ventricular muscle cell)
TALEN or CRISPR/CAS9 genome editor's plasmids；
B, homologous gene combination neomycin or green fluorescent protein selection markers of the structure with target gene described in step A
Donor plasmid (as shown in Figure 1)；
C, cultivate good undifferentiated human pluripotent stem cell, including hESC with by reprogramming in vitro
Mankind's inducing pluripotent stem cells (people iPS cells) that the body cell of normal human subject individual is obtained；
D, it is TALEN the or CRISPR/CAS9 genome editor plasmids that step A is obtained and MYL2 that step B is obtained is homologous
Genetic donor plasmid common-battery is transferred to human pluripotent stem cell, by genetic recombination by MYL2 homologous gene combination neomycins or green
Fluorescin selection markers import the genome of human pluripotent stem cell；
E, by the screening of gene sequencing and monoclonal succeed homologous recombination with MYL2 combinations neomycin or green
The human pluripotent stem cell of fluorescin selection markers, and establish corresponding cell strain；
F, the human pluripotent stem cell cell marked with MYL2 combinations drug or fluorescent screening that will be obtained described in step E
Strain by existing disclosed human pluripotent stem cell directed differentiation cardiac muscle cell's technology, obtains the mankind without screening purifying
Cardiac muscle cell including human cardiac ventricle myocyte, atrial muscle cell, Sino-atrial Pacemaker Cells and belongs in human heart other
The cell of classification；
G, it by human myocardium's cell after the differentiation without screening purifying in step F, is screened by neomycin drug or green
Color fluorescin selected by flow cytometry apoptosis is purified, and the ratio of mankind's ventricular muscle cell is made to reach more than 95% (such as Fig. 2 institutes
Show)；
H, the human cardiac ventricle myocyte screened by neomycin or green fluorescent protein described in step G is tested
Card, as a result shows its corresponding phenotype (as shown in Figure 3) with normal human subject ventricular muscle cell.
The present invention provides the preparation method of human pluripotent stem cell cell strain screened for human cardiac ventricle myocyte, i.e.,
Using genome editing technique, such as TALEN, CRISPR/CAS9 or any efficient gene recombinant technique, by genetic recombination side
Method editor's human pluripotent stem cell genome, structure human cardiac ventricle flesh-multipotential stem cell cell strain.
More specifically, the preparation side of the human pluripotent stem cell cell strain for human cardiac ventricle myocyte's screening of the invention
Method, including step：
1) structure of TALEN or CRISPR/CAS9 plasmids and MYL2 homologous gene donor plasmids
It is prominent using PCR, restriction enzyme, ligase, point on the basis of TALEN or CRISPR/CAS9 skeleton plasmids
Becoming equimolecular biological method structure, (plasmid carries puromycin for TALEN the or CRISPR/CAS9 plasmids of MYL2
(puromycin) resistant gene receives the positive cell clone of the plasmid available for screening), while it is homologous to build band MYL2
Genetic donor plasmid；
2) for the Activity determination of the TALEN or CRISPR/CAS9 plasmids of MYL2 genes
With TALEN or CRISPR/CAS9 plasmid transfection 293T cells, the gene of survivaling cell after extraction puromycin screening
Group DNA carries out PCR, and set peak intensity by observing DNA sequencing result is weak, T7E1 mispairing enzyme digesting efficiencies and TA cloning and sequencing ratios
Detection activity；
3) the high TALEN or CRISPR/CAS9 plasmids of activity are hit with MYL2 homologous gene donor plasmid common-batteries transfect into
Enter human pluripotent stem cell
Suitable cell concentration is counted, the TALEN or CRISPR/CAS9 and MYL2 homologous genes for adding in corresponding proportion supply constitution
Grain is selected suitable voltage, pulse strength and pulse number to carry out electricity and is turned；It is selected for different multipotential stem cell cell lines
Voltage, pulse strength and pulse number need to determine optimal values by experiment, voltage range is generally at 1100 millivolt -1400
Millivolt, pulsating sphere are generally 0.5-2 milliamperes, and pulse number is generally 1-3 times；
4) the multipotential stem cell system for the screening of human cardiac ventricle's flesh of screening successful homologous recombination
Select the clone that survives after puromycin screening, extract genomic DNA, using PCR, TA cloning and sequencing and
Southern Blot equimoleculars biological method identifies human cardiac ventricle's flesh-multipotential stem cell system of heterozygosis or homozygosis；
5) the in vitro culture mankind are normal and multipotential stem cell for the screening of human cardiac ventricle's flesh, differentiation cardiac muscle cell
It (can in the market be bought in biological reagent, such as Canada using the inducing pluripotent stem cells culture solution of profession
Stemcell Technology companies), contain at 37 DEG C and human pluripotency stem cell is cultivated in 5% carbon dioxide incubator culture,
After cell covers with, with known method, the accelerating agent (small-molecule drug CHIR99021) and inhibitor of conjunctive use Wnt accesses
(small-molecule drug IWR-1) makes its cells into cardiomyocytes directed differentiation；
6) cardiac muscle cell purifies：
It after differentiation 20 days, is acted on 7 days, and with the DMEM culture solution cultures containing 5%FBS, obtained with the neomycin of 100ug/ml
To the ventricular muscle cell of purity higher (being more than 90%)；Or ventricular muscle cell is gone out by airflow classification；
7) ventricular muscle cell phenotypic evaluation and function, Mechanism Study
Utilize the ventricular muscle cell that verification induction is broken up and purified the methods of immunofluorescence, patch-clamp, multi-electrode microarray
Have the phenotype similar to normal adult's ventricular muscle cell and electrophysiological characteristics, for research dilated cardiomyopathy mechanism and find newly
Therapy target provides cell screening platform.
On the other hand, it the present invention provides the application of the multipotential stem cell screened for human cardiac ventricle's flesh, is done by multipotency
Cell directional differentiation cardiac muscle cell's technology obtains the higher human cardiac ventricle myocyte of purity；
The ventricular muscle cell of acquisition can be used for establishing the external drug evaluation of angiocardiopathy and screening system；It can be used for
The cellular replacement therapy of heart disease；It can also will break up the ventricular muscle cell obtained and culture shape is combined with any bracket material
Into all kinds of external human cardiac ventricle's muscular tissues, for example, the animal hearts matrix of decellularization may be used as timbering material, knot
The human cardiac ventricle myocyte that differentiation obtains is closed, prepares all kinds of external human cardiac ventricle's muscular tissues.
The human cardiac ventricle myocyte of the present invention has the phenotype similar to mankind normal ventricle myocyte and electrophysiologic response,
It derives from a wealth of sources, and can long-term in vitro culture.
Advantages of the present invention has：
The present invention using known activating transcription factor sample effector nuclease (TALEN) or often between palindrome repetitive sequence clump
Collect related protein system (CRISPR/CAS9) genome editing technique, editor's human pluripotent stem cell is (thin including human embryonic stem
Born of the same parents human embryonic stem cells and mankind's inductivity versatile stem cell (human induced
Pluripotent stem cells)) genome, by MYL2 drived Neo or MYL2 drived eGFP import the mankind
Multipotential stem cell genome simultaneously replaces corresponding normal gene, establishes human cardiac ventricle's muscle model, then do by known multipotency
Cell directional breaks up cardiac muscle cell's technology, and then is purified into human cardiac ventricle myocyte.The present invention utilizes TALEN or CRISPR/
The ventricular muscle cell that CAS9 gene recombination technologies obtain, the phenotype for having normal person's ventricular muscle cell similar and electrophysiological characteristics.
It can be used for：
1) seeking leads to the early molecule access and its pathogenesis of human myocardium's disease；
2) seek novel, the effective therapy target of human myocardium's disease；
3) the external drug evaluation of human inheritance's property cardiomyopathy and screening caused by being mutated for different Disease-causing genes are established
System；
4) the transplanting regenerative therapy of heart degenerative disease；
5) reconstruction in vitro human cardiac ventricle muscular tissue.
The human cardiac ventricle myocyte of the present invention is the early molecule access of cardiomyopathy caused by seeking a variety of causes, further
Pathogenesis is studied, seeks effective, novel treatment means and the effective corresponding treatment drug of screening provides good tool
And platform.
Description of the drawings
Fig. 1, MYL2 homologous gene donor plasmid are built, wherein, at the nearly terminator end of 7 exons of MYL2, introduce one section
Neomycin medicine sieves gene or eGFP genes are inserted among the homology arm of left and right.
Fig. 2, design of primers scheme, wherein, in the primer of the nearly terminator end design left and right homology arm of 7 exons of MYL2.
Fig. 3, TALEN plasmid cleavage MYL2 DNA Efficiency testings, which show DNA shearings typically occur in double dyeing
One in body, due to that can generate the increase or deletion of nucleotide in genome repair process, PCR sequencings can have been found
Sequence set peak occurs, and the target DNA in a chromosome is prompted to be sheared, T7EI detects the DNA situations that can test mispairing, and T7EI can
Mismatched dna is cut, occurs two band after electrophoresis.
Fig. 4, genomic DNA PCR detect the clone for being correctly inserted into target gene.
Fig. 5, flow cytometer detection is for ratio of the multipotential stem cell Induction of committed differentiation that human cardiac ventricle's flesh screens for cardiac muscle cell
Example and the ratio by ventricular muscle cell after neomycin medicine sieve.
Fig. 6 filters out ventricular muscle cell by airflow classification.
Fig. 7, action potential of the ventricular muscles with mankind normal ventricle myocyte after purification.
Specific embodiment
The present invention using known activating transcription factor sample effector nuclease (TALEN) or often between palindrome repetitive sequence clump
Collect related protein system (CRISPR/CAS9) genome editing technique, editor's human pluripotent stem cell is (thin including human embryonic stem
Born of the same parents (human embryonic stem cells) and mankind's inductivity versatile stem cell (human induced
Pluripotent stem cells)) genome, according to MYL2 genetic traits design site-specific TALEN or CRISPR/
CAS9 plasmids mediate genetic recombination by genome editing technique, will lead to target gene, import human pluripotent stem cell base
Because of group and corresponding normal gene is replaced, establishes the multipotential stem cell strain for the screening of human cardiac ventricle's flesh.Again by known
Multipotential stem cell directed differentiation cardiac muscle cell's technology, and then purify and prepare human cardiac ventricle myocyte.
The present invention is realized by following technical proposals and step：
A, structure builds TALEN or CRISPR/CAS9 plasmids for MYL2 genetic traits, and detects it and target gene is cut
Cut activity；
B, donor plasmid of the structure with MYL2 homologous genes and selection markers；
C, the inducing pluripotent stem cells of good undifferentiated hESC or normal human subject individual are cultivated；
D, the high TALEN or CRISPR/CAS9 plasmids of activity are hit into infection human pluripotent stem cell with donor plasmid common-battery
(including embryonic stem cell and inductivity versatile stem cell) is done riddled basins importing mankind's multipotency by genetic recombination
Cellular genome；
E, grope suitably to screen drug (such as puromycin Puromycin) concentration screening go out heterozygosis or homozygosis for people
The multipotential stem cell system of class ventricular muscles screening；
F, it is cardiac muscle cell by the multipotential stem cell directed differentiation for the screening of human cardiac ventricle's flesh filtered out；
G, the cardiac muscle cell after differentiation sieve by neomycin medicine or airflow classification purifies, reach its ratio
To more than 95%, verify phenotype and carry out the research of function and mechanism.
With reference to specific embodiment, the present invention is further explained.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.Unless otherwise described, implementation of the invention will use molecular biology, microbiology,
The routine techniques such as biochemistry, cell biology, these are known to those skilled in the art.These technologies are in following documents
In have complete description：For example, Sambrook《Molecular Cloning:A Laboratory guide》Second edition (1989)；《DNA clone》I and II volumes
(D.N.Glover edits 1985)；《Oligonucleotide synthesis》(M.J.Gait is edited, 1984)；《Protein purification》((Richard
R.Burgess it can carry out) or according to the specification that reagent manufacturer is provided.
Embodiment 1
1) TALEN or CRISPR/CAS9 plasmids and the structure with corresponding homologous gene and selection markers donor plasmid
On the basis of skeleton plasmid, structure is directed to TALEN the or CRISPR/CAS9 plasmids of MYL2 genetic traits and carries
Drive the homologous gene donor plasmid of MYL2 promoters；(shown in such as Fig. 1, Fig. 2 legend)；
2) for different target spot TALEN or the Activity determination of CRISPR/CAS9 plasmids
With TALEN or CRISPR/CAS9 plasmid transfection 293T cells, the base of survivaling cell after extraction Puromycin screenings
Because group DNA carries out PCR, weak peak intensity, T7E1 mispairing enzyme digesting efficiencies and TA cloning and sequencings ratio detection activity are covered (such as by observation
Shown in Fig. 3)；
3) the high TALEN or CRISPR/CAS9 plasmids of activity are turned into human pluripotent stem cell with donor plasmid common-battery
Human pluripotency's stem cell medium, 37 DEG C contain 5% carbon dioxide incubator culture, after cell covers with,
Accutase digests, and counts suitable cell concentration, adds in the TALEN or CRISPR/CAS9 and donor plasmid of corresponding proportion, selects
Suitable voltage, pulse strength and pulse number carry out electricity and turn；
4) the multipotential stem cell system for the screening of human cardiac ventricle's flesh of heterozygosis or homozygosis is screened
Select the clone that survives after Puromycin screenings, extract genomic DNA, using PCR, TA cloning and sequencing and
Southern Blot equimoleculars biological method identifies the multipotential stem cell strain for the screening of human cardiac ventricle's flesh of heterozygosis or homozygosis
(as shown in Figure 4)；
5) the in vitro culture mankind are normal and for the multipotential stem cell that human cardiac ventricle's flesh screens, and break up cardiac muscle cell
It (is bought using inducing pluripotent stem cells culture solution in Stemcell Technolgy companies), 37 DEG C contain 5% 2
Carbonoxide incubator culture, after cell covers with, the accelerating agent and inhibitor of conjunctive use Wnt accesses orient its cells into cardiomyocytes
Differentiation (the directed differentiation technology is known mature technology)；The accelerating agent (can be in for micromolecular compound CHIR99021
Selleck companies buy), inhibitor is micromolecular compound IWR-1 (can be bought in Selleck companies)；
6) cardiac muscle cell purifies
After differentiation 20 days, acted on one week with the neomycin of 100ug/ml, gained cell can do fluidic cell with MYL2 genes
It analyzes and identifies and obtains that purity is higher to be contained containing high-purity MYL2 positives ventricular muscle cell (as shown in Figure 5) or airflow classification
The ventricular muscle cell (as shown in Figure 6) of green fluorescent protein；
7) ventricular muscle cell phenotypic evaluation and function, Mechanism Study
Have using the cardiac muscle cell of verification induction differentiation the methods of immunofluorescence, patch-clamp with normal adult or after purification
The similar phenotype of obtained ventricular muscle cell and electrophysiological characteristics (as shown in Figure 7) are pathogenesis and the discovery of study of disease
New therapy target.
The human cardiac ventricle myocyte as made from the above method of the present invention has the table similar to mankind normal ventricle myocyte
Type and electrophysiologic response, not only derived from a wealth of sources but also can long-term in vitro culture, it is rare and not to solve human pathological cardiac muscular tissue source
The drawbacks of easy long-term in vitro culture, and available for the early stage pathogenesis of research cardiomyopathy.
Embodiment 2
The human cardiac ventricle myocyte obtained by the above method, using multi-electrode microarray means detect its contractile function and
Electrophysiological characteristics；Further, different small-molecule chemical drugs or Chinese herbal and crude drugs preparations are added in the myocardial cells culture environment, are led to
Excessive its contractile function of electrode microarray system detectio and electrophysiological function variation, test small-molecule chemical drug or in
Herbal medicinal product may screen novel, effective medicine to the function effect of human heart tissue.
Embodiment 3
The cardiac toxic of drug be hinder drug development key factor, the medicine heart based on transgenic cell and animal
Toxicity screening, often there are some false negatives and false positive as a result, leading to unnecessary death, and the cardiac toxic of drug
Effect is more easy to be happened at the patient of basal cardiac lesion；Series of human ventricular muscle cell is built by the above method, is contained
The ion channel for having mankind normal ventricle myocyte surface all can reflect complicated interaction between ion channel, more smart
It really predicts the cardiac toxic of drug, becomes the platform of good drug toxicity screening.
Embodiment 4
The high-purity human cardiac ventricle myocyte obtained using the above method, by the quality inspection flow according to national regulation
Meet clinical application standard, can be directly used for preparing drug injection preparation, be injected in cardiac's diseased region or surrounding,
It carries out it and treats the clinical research of heart degenerative disease, final exploitation is human cardiac ventricle's flesh product available for transplanting.
Embodiment 5
The human cardiac ventricle myocyte obtained using the above method, conjunctive tissue engineering technology can be weighed further in vitro
Human cardiac ventricle's muscular tissue is built, from tissue level further investigation cardiomyopathy to ventricular muscles development and the influence of function and the base of cardiomyopathy
This pathologic process, for example, the organizational project ventricle muscular tissue prepared by natural heart matrix and a variety of human cells；The tissue work
After journey ventricle muscular tissue is converted by clinic, available for 1) vitro detection various kinds of drug to the toxic effect of human heart tissue；
2) treat or substitute the downright bad ventricle muscular tissue after myocardial infarction；This engineered human cardiac ventricle's muscular tissue is by decellularization
Natural heart matrix as stent, be inoculated with and be made of the ventricular muscle cell ingredient in human pluripotent stem cell differentiation source；Structure
Engineered ventricle muscular tissue using the heart of the rat of decellularization or pig for nature heart matrix as be engineered ventricle
Muscular tissue matrix after the matrix framework of decellularization, is made the heart matrix segment of different sizes and shape, will be used for the mankind
The multipotential stem cell directed differentiation of ventricular muscles screening for cardiovascular system cell and people umbilical cord mesenchymal stem cells and rat or
Pig heart matrix combines culture, and functional engineering human heart tissue is made；This engineered human cardiac ventricle's flesh group
It knits with the cellular component similar to normal human subject cardiac muscular tissue, extracellular matrix and biological function, external medicine can be carried out
The test and screening of object, the internal transplanting research of preliminary preclinical phase toy and treatment observation of curative effect, it is final to realize people
The individualized treatment of class angiocardiopathy provides significant auxiliary；
Above-mentioned technical proposal is realized by following technical proposals and step：
A. cellularised rat or pig heart are removed, obtains nature heart matrix；
B. the different base material of shape, size is made in the rat of decellularization or pig heart matrix on demand；
C. the multipotential stem cell screened for human cardiac ventricle's flesh is obtained according to the present invention, directed differentiation is the cardiac muscle of the mankind
Cell
D. the cardiac muscle cell in the induced multi-potent stem cell source after differentiation is purified, make its ratio reach 95% with
On, seed cell 1 is made；
E, umbilical cord mesenchymal stem cells are extracted, seed cell 2 is made in vitro culture；
F, it is inoculated on the rat or pig heart matrix of decellularization after mixing seed cell 1 and seed cell 2, in vitro
Dimensional culture builds engineered ventricle muscular tissue；
The separation and culture of umbilical cord mesenchymal stem cells：The umbilical cord of newborn fetus is taken, overburden removing shreds, IV Collagen Type VIs
37 DEG C of digestion of enzyme；FBS terminates digestion, and supernatant is abandoned in centrifugation, and cell is resuspended in PBS, filtering, and supernatant is abandoned in the centrifugation of gained filtrate,
Cell is resuspended in DMEM culture solutions, plants in culture dish 37 DEG C, is cultivated in 5%CO2 insulating boxs, changes within 2-3 days a culture solution, until
It is passed on during cell fusion to 60%-75% density；
Size, variform rat used by ventricular muscle cell after purification and mescenchymal stem cell are inoculated into
Or in pig decellularization heart matrix, through external dimensional culture, the engineered cardiac muscular tissue of different shape and size is built,
Using the natural heart matrix of decellularization as cardiac muscular tissue's matrix, there is good biocompatibility, mechanical property and biology
Degradability, obtained engineered ventricle muscular tissue have various cell components, extracellular matrix and the life of normal heart
Object activity；Engineered human cardiac ventricle's muscular tissue obtained has and normal human subject cardiac muscular tissue similar cellular ingredient and electricity
Physiological function had both kept certain mechanical strength, basic mechanical performance, and has had rational porosity, good cytocompatibility
Property, biological degradability will not cause inflammatory reaction, be a kind of living tissue with biological activity that can be used for transplanting, and
And it because of the effect without pacemaker cells such as sinoatrial nodes, is implanted into organism, ectopic beat will not be caused；It can be further used for
Curative effect sight is studied and is treated in the test and internal transplant of screening and preliminary preclinical phase toy for carrying out external drug
It examines, finally to realizing that the individualized treatment of human cardiovascular disease provides significant auxiliary.
Claims (8)
1. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source, which is characterized in that utilize gene
Group editing technique, edits human pluripotent stem cell genome, builds specific MYL2 genes ending and carries neomycin or green glimmering
The human pluripotent stem cell cell strain of photoprotein screening-gene, including step：
A, structure is for TALEN or CRISPR/CAS9 genome editor's plasmids of target gene MYL2；
B, the donor of homologous gene combination neomycin or green fluorescent protein selection markers of the structure with target gene described in step A
Plasmid；
C, the human pluripotent stem cell of good undifferentiated separate sources is cultivated, including the hESC of preparation
With the mankind's inducing pluripotent stem cells people's iPS cells obtained by reprogramming the body cell of normal human subject individual in vitro；
D, the MYL2 homologous genes that the TALEN or CRISPR/CAS9 genome editor plasmids for obtaining step A are obtained with step B
Donor plasmid common-battery is transferred to human pluripotent stem cell, by genetic recombination by MYL2 homologous gene combination neomycins or green fluorescence
Protein screening label imports the genome of human pluripotent stem cell；
E, by the screening of gene sequencing and monoclonal succeed homologous recombination with MYL2 combinations neomycin or green fluorescence
The human pluripotent stem cell of protein screening label, and establish corresponding cell strain；
F, the human pluripotent stem cell cell strain marked with MYL2 combinations drug or fluorescent screening that will be obtained described in step E,
By human pluripotent stem cell directed differentiation cardiac muscle cell's technology, human myocardium's cell without screening purifying is obtained；
G, it is glimmering by neomycin drug screening or green by human myocardium's cell after the differentiation without screening purifying in step F
Photoprotein selected by flow cytometry apoptosis is purified；
H, the human cardiac ventricle myocyte screened by neomycin or green fluorescent protein described in step G is verified, obtained
Corresponding phenotype with normal human subject ventricular muscle cell.
2. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source as described in claim 1,
It is characterized in that, the human pluripotent stem cell cell strain of acquisition is to be screened with MYL2 gene combination neomycins or green fluorescent protein
The human pluripotent stem cell cell strain of label.
3. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source as described in claim 1,
It is characterized in that, the genome editing technique is selected from TALEN, CRISPR/CAS9 or any efficient gene recombinant technique.
4. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source as described in claim 1,
It is characterized in that, in the step A, the albumen that target gene MYL2 is edited is the specific markers of ventricular muscle cell.
5. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source as described in claim 1,
Be characterized in that, in the step F, obtain without screening purifying human myocardium's cell, including human cardiac ventricle myocyte,
Atrial muscle cell, Sino-atrial Pacemaker Cells and the cell for belonging to other classifications human heart Nei.
6. screening and the preparation method of the human cardiac ventricle myocyte in human pluripotent stem cell source as described in claim 1,
It is characterized in that, in the step F, the ratio for making mankind's ventricular muscle cell after purification reaches more than 95%.
7. screening described in claim 1 and preparation method, wherein the human pluripotent stem cell cell strain is being used to prepare
Purposes in human cardiomyopathy's heart of patient viable transplantation product.
8. screening described in claim 1 and preparation method, wherein the human pluripotent stem cell cell strain is being used to prepare
Purposes in engineered in vitro human cardiac ventricle's muscular tissue, wherein, by break up with screening purifying obtain human cardiac ventricle myocyte with
Timbering material combines, and culture forms engineered in vitro human cardiac ventricle's muscular tissue.
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