CN110938584B - Method for inducing differentiation of human embryonic stem cells into endothelial cells - Google Patents
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Abstract
The invention relates to the technical field of stem cells, and discloses a method for inducing differentiation of human embryonic stem cells into endothelial cells. The method of the invention prepares human embryonic stem cells into an embryoid body by using a culture medium formed by dispase and the embryoid body; then inducing the embryoid bodies into mesoderm cells by using a mesoderm induction culture medium; finally, the mesodermal cells are directionally induced into endothelial cells by using an endothelial induction culture medium. The invention successfully obtains high-purity endothelial cells from human embryonic stem cells by sequentially inducing the embryoid bodies and the single layer, then obtains the high-quality endothelial cells which have typical shapes of the endothelial cells, normal immunophenotype and high purity and can be continuously subcultured for more than 8 times in vitro by the steps of enriching and amplifying the endothelial cells, and can solve the bottleneck problem of insufficient clinical treatment dosage of the endothelial cells at present.
Description
Technical Field
The invention relates to the technical field of stem cells, in particular to a method for inducing differentiation of human embryonic stem cells into endothelial cells.
Background
In 1988, the first human embryonic stem cell line was established in professor jameson, university of wisconsin, usa, and pioneering the research on human embryonic stem cells. The human embryonic stem cell has strong self-renewal capacity and multidirectional differentiation potential, so that the human embryonic stem cell becomes an attractive seed cell type in the field of functional cell acquisition.
Endothelial cells generally refer to a single layer of flattened epithelium lining the inner surface of the heart, blood vessels, and lymph vessels, which forms the inner wall of the blood vessel. They have the function of phagocytizing foreign bodies, bacteria, necrotic and senescent tissues and also participate in the immune activities of the body. Endothelial cells play an important role in vascular injury repair and revascularization.
At present, endothelial cells can be obtained by primary separation of large vessels, small vessels and micro vessels of arteries or veins, the endothelial cells directly separated from the vessels are generally endothelial cells with better differentiation degree, the in vivo passage frequency and the proliferation capacity are effective, and even the in vitro passage frequency of the umbilical vein/umbilical artery endothelial cells with relatively stronger proliferation capacity is within 10 generations, so that the endothelial cells with clinical dose are difficult to obtain by the culture and amplification way of the primary separated endothelial cells.
In consideration of the strong self-renewal capacity of human embryonic stem cells and the potential of differentiation to cells of various tissue types, the endothelial cells obtained by the induced differentiation of the human embryonic stem cells provide a novel solution with great potential for the endothelial cells, and the important basis is laid for the clinical application of the endothelial cells.
At present, many documents report that endothelial cells are obtained by induced differentiation of human embryonic stem cells, for example, Wu and the like report that the directional induction of human embryonic stem cells to endothelial cells is performed, the research obtains the endothelial cells by sequential induction for 5 days, and establishes an induction method of human embryonic stem cells to the endothelial cells, but the document mainly studies the mechanism and influencing factors of differentiation of human pluripotent stem cells to the endothelial cells, does not carry out deep research on the optimization of an induction system and the preparation of clinical dose endothelial cells, does not form a clear induced differentiation method, has defects on passage times, and cannot effectively solve the bottleneck problem of insufficient clinical treatment dose of the endothelial cells at present.
Disclosure of Invention
In view of the above, the present invention provides a method for inducing differentiation of human embryonic stem cells into endothelial cells, such that the endothelial cells obtained by the method have high purity and differentiation rate, and can be continuously passaged and expanded for more than 8 generations.
In order to achieve the above purpose, the invention provides the following technical scheme:
a method of inducing differentiation of human embryonic stem cells into endothelial cells, comprising:
step 1, preparing a culture medium for forming human embryonic stem cells into an embryoid body by using dispase and the embryoid body;
2, inducing the embryoid bodies into mesoderm cells by using a mesoderm induction medium;
and 3, directionally inducing the mesoderm cells into endothelial cells by using an endothelial induction culture medium.
Aiming at the problem that the induction differentiation of the existing human embryonic stem cells into endothelial cells has less passage times, the invention carries out optimization adjustment from the induction differentiation link to the passage amplification link, so that the induced differentiated endothelial cells can be subjected to passage for more than 8 generations.
Preferably, step 1 is:
culturing human embryonic stem cells until the cell confluency is 80-90%;
removing the culture medium, washing with PBS, digesting with dispase (1mg/ml), sucking dispase and washing with PBS when the cell clone edge is observed to be shiny and curled under an inverted microscope, then uniformly scribing at the bottom of the culture dish in the transverse direction and the longitudinal direction, then blowing and beating the cells until most of the cells are observed to be separated from the bottom of the culture dish under the microscope, and forming cell clusters with relatively uniform sizes;
and standing the cell suspension, sucking and removing a supernatant, then re-suspending the cell clone cluster by using an embryoid body forming culture medium, and transferring the cell clone cluster to a low-adhesion cell culture plate for culturing for 24 hours to obtain an embryoid body.
In a specific embodiment of the present invention, step 1 is:
removing the culture medium, washing with PBS for 2 times, digesting with Dispase (Dispase) for 5-8min, sucking Dispase and washing with PBS for 2 times when the cell clone edge is observed to be shiny and curled under an inverted microscope, then uniformly scribing at the bottom of the culture dish transversely and longitudinally with 1ml Tip head, then blowing and beating the cells for 8-10 times with a Pasteur pipette, and observing that most cells are separated from the bottom of the culture dish under the microscope to form cell clusters with relatively uniform sizes;
the cell suspension was transferred to a 15ml centrifuge tube with a Pasteur pipette, allowed to stand at room temperature for 10min, the supernatant was carefully discarded, and then the cell colony was resuspended in an embryoid body-forming medium and transferred to a low-adhesion cell culture plate (CORNING, #3471) for 24h to obtain embryoid bodies.
Wherein the embryoid body forming culture medium takes DMEM/F-12 as a basic culture medium and contains 20% of KSR,2mM NEAA,2mM L-glutamine and 0.1mM beta-mercaptoethanol; the method comprises the following steps of firstly culturing human embryonic stem cells until the cell confluency is 80-90 percent: culturing human embryonic stem cells in a culture dish coated with glass fibronectin, wherein the culture medium is E8 embryonic stem cell culture medium, and the cell confluency is 80-90%.
Preferably, step 2 is:
collecting the embryoid bodies, standing, sucking and removing the supernatant, then inducing for 96h by using mesoderm induction medium, changing the culture solution for 1 time at 48h to obtain mesoderm cells, wherein the embryoid bodies are performed in a low-adhesion cell culture plate in the whole mesoderm induction process.
In a specific embodiment of the present invention, the step 2 is:
collecting the formed embryoid bodies in a 15ml centrifuge tube, standing for 10min at room temperature, carefully sucking and discarding the supernatant, then using a mesoderm induction medium for 96h, and changing the medium for 1 time at 48h to obtain the mesoderm cells, wherein the embryoid bodies are carried out in a low-adhesion cell culture plate in the whole mesoderm induction process.
Wherein the mesoderm induction medium takes Stemline II serum-free medium (Sigma Aldrich, cat # S0192) as a basic medium and contains 1 XTIS-G (Insulin, Transferrin, Selenium Solution (ITS-G), 100X, GIBCO company, cat # 41400045), 1-100ng/ml BMP4 and 1-50ng/ml bFGF. In a specific embodiment of the invention, the mesoderm induction medium is a Stemline II serum free medium (Sigma Aldrich, cat # S0192) based medium containing 1 XTS-G, 20ng/ml BMP4 and 5ng/ml bFGF.
Preferably, step 3 is:
and collecting and standing the mesoderm cells, sucking and removing supernatant, then re-suspending with an endothelial induction culture medium, inoculating the cells to a cell culture plate, and inducing for 48 hours to obtain the endothelial cells.
In a specific embodiment of the present invention, the step 3 is:
and collecting the mesodermal cells into a 15ml centrifuge tube, standing for 10min at room temperature, carefully sucking and discarding the supernatant, then re-suspending with an endothelial induction culture medium, inoculating the cell culture plate, and inducing for 48h to obtain the endothelial cells.
Wherein the endothelial induction medium takes Stemline II serum-free medium as a basal medium and contains 1 XTS-G, 1-100ng/ml VEGF, 1-50ng/ml bFGF and 1-200ng/ml SCF. In a specific embodiment of the invention, the endothelial induction medium is a Stemline II serum-free medium as basal medium and contains 1 XTS-G, 40ng/ml VEGF, 5ng/ml bFGF and 50ng/ml SCF.
In addition, the method of the invention also comprises the subculture of endothelial cells:
preparing single cell suspension from induced and differentiated endothelial cells by using TrpLE cells, re-inoculating the single cell suspension to a new cell culture plate, continuously culturing by using a commercial endothelial cell amplification culture medium (lonza EGM-2MV EGM-2CC-3162) until the confluency of the cells reaches about 90%, and carrying out subculture.
In a specific embodiment of the present invention, the subculture method comprises:
digesting the induced and differentiated endothelial cells for 5min by using TrpLE, adding PBS with the same volume, blowing and beating to prepare single cell suspension, collecting the cells, centrifuging (500g for 5min), removing supernatant, re-inoculating the cells to a new cell culture plate, continuously culturing by using a commercial endothelial cell amplification culture medium, and carrying out subculture when the confluency of the cells reaches about 90%.
Compared with the existing induced differentiation, the results show that the invention can still maintain vigorous cell proliferation capacity after being passaged to more than 8 generations, and simultaneously the endothelial cell surface marker is not obviously reduced, while the existing method obviously reduces the passage times if the better cell proliferation capacity is maintained and the endothelial cell surface marker is not obviously reduced.
According to the technical scheme, the invention obtains endothelial cells with high purity and high differentiation rate from human embryonic stem cells by sequentially inducing the embryoid bodies and the single layer, and then obtains the high-quality endothelial cells with typical shapes of the endothelial cells, normal immunophenotype and high purity through the steps of enrichment and amplification of the endothelial cells, wherein the high-quality endothelial cells can be continuously subcultured for more than 8 times in vitro, and the bottleneck problem of insufficient clinical treatment dosage of the endothelial cells at present can be solved.
Drawings
FIG. 1 shows the cell morphology of key nodes in the process of directionally inducing human embryonic stem cells to endothelial cells; wherein D1 indicates the formation of embryoid bodies on day one (fold 40 ×), D5 indicates the directional induction of mesodermal cells to endothelial cells on day 5 (fold 100 ×), and D12 indicates the culture expansion of endothelial cells (p2) on day 12 (fold 100 ×);
FIG. 2 shows flow assay of human embryonic stem cell-derived endothelial cells; wherein, the left graph is the p0 generation flow detection result, and the right graph is the p3 generation flow detection result;
FIG. 3 shows the morphology of endothelial cells derived from P8 generation human embryonic stem cells (invention); wherein, the magnification of the left picture is 40X, and the magnification of the right picture is 100X;
FIG. 4 shows the morphology of endothelial cells derived from p3 generation human embryonic stem cells (prior art literature); wherein the left image is magnified by 40X, and the right image is magnified by 100X.
Detailed Description
The invention discloses a method for inducing differentiation of human embryonic stem cells into endothelial cells, which can be realized by appropriately improving process parameters by the technical personnel in the field by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations of parts, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
The human embryonic stem cells used in the present invention can be obtained through a purchase route, and human embryonic stem cells purchased from WiCell corporation are used in the present invention;
the method for inducing differentiation of human embryonic stem cells into endothelial cells provided by the present invention is further described below.
Example 1: induced differentiation of endothelial cells
1. Maintenance culture of human embryonic stem cells
The embryonic stem cells of the invention are cultured in a culture dish coated by Vitronectin (Vitronectin), the culture medium is E8 embryonic stem cell culture medium, and the culture system can avoid the introduction of animal-derived components contained in the traditional coating matrix Matrigel and the traditional human embryonic stem cell culture medium mTeSR;
2. formation of embryoid bodies
Culturing human embryonic stem cells in the culture system for 4-5 days, and performing embryoid body formation experiment when the cell confluency reaches 80-90% (see figure 1); the method comprises the following specific steps:
removing the E8 culture medium, washing with PBS for 2 times, digesting with Dispase (Dispase) for 5-8min, sucking Dispase when the cell clone edge is observed to be shiny and curled under an inverted microscope, washing with PBS for 2 times, then uniformly scribing at the bottom of the culture dish transversely and longitudinally with 1ml Tip, then blowing and beating the cell for 8-10 times with a pasteur pipette, observing that most of the cell is separated from the bottom of the culture dish under the microscope, and forming a cell mass with relatively uniform size;
the cell suspension was transferred to a 15ml centrifuge tube with a Pasteur pipette, allowed to stand at room temperature for 10min, the supernatant carefully discarded, and the cell colony was resuspended in embryoid body-forming medium (DMEM/F-12, 20% KSR,2mM NEAA,2mM L-glutamine,0.1mM beta-mercaptoethanol) and transferred to a low-adhesion cell culture plate (CORNING, #3471) for 24 h.
3. Endothelial cell induction
The endothelial cell induction is divided into 2 steps, namely the directional induction of human embryonic stem cells to mesoderm cells and the directional induction of mesoderm cells to endothelial cells, and comprises the following steps:
1) directional induction of human embryonic stem cells into mesodermal cells
Collecting the formed embryoid bodies to a 15ml centrifuge tube, standing for 10min at room temperature, carefully sucking and discarding the supernatant, then using a mesoderm induction medium (Stemline II +1 × ITS-G + cytokine group A (20ng/ml BMP4+5ng/ml bFGF)), wherein the induction time is 96h, changing the liquid for 1 time in 48h, and carrying out the embryoid bodies in a low-adhesion cell culture plate in the whole mesoderm induction process;
2) induction of mesodermal cells into endothelial cells
Embryoid bodies induced by mesoderm orientation for 96h were collected in 15ml centrifuge tubes, left to stand at room temperature for 10min, the supernatant was carefully discarded, resuspended in endothelial induction medium (Stemline II +1 XTS-G + cytokine combination B (40ng/ml VEGF, 5ng/ml bFGF and 50ng/ml SCF)), seeded onto cell culture plates and induced for 48h, and the results of representative flow assays are shown in D5 in FIG. 1.
4. Enrichment and expansion of endothelial cells
Preparing single cell suspension from the induced cell product by using TrpLE cells, re-inoculating the single cell suspension to a new cell culture plate, continuously culturing by using an endothelial cell amplification culture medium (lonza EGM-2MV EGM-2CC-3162), carrying out subculture (shown as D12 in figure 1) when the confluency of the cells reaches about 90 percent, carrying out endothelial cell morphology and surface marker identification when the cells are cultured to p2-p3, and obtaining a representative flow detection result shown as figure 2.
In FIG. 2, the left graph shows the p0 generation and the right graph shows the p3 generation, and the differentiation efficiency (about 70%) of endothelial cells of p0 generation is obviously lower than that (about 95%) of endothelial cells of p3 generation.
The endothelial cells obtained by the induced differentiation method of the invention can be continuously passaged to more than 8 generations, and the cells can still maintain vigorous proliferation capacity and typical morphological characteristics of the endothelial cells (see figure 3).
Example 2: detection of the number of passages of endothelial cells (comparative experiment)
3 independent experiments were carried out (Human embryonic stem Cells are the same as in example 1) according to the method for inducing differentiation of Human embryonic stem Cells into Endothelial Cells reported in the references (Defining Minimum Essential Factors to derivative high Pure cell from iPS/ES Cells in an Animal substrate-Free System; wu et al) mentioned in the background of the invention as follows:
1) inducing the iPS/ES to mesoderm cells, wherein the induction culture medium is a basic culture medium (12g/L DMEM/F-12, 3.56g/L HEPES, 1.742g/L of sodium pyruvate, 14ug/L sodium selenate, 10.7mg/L of recombinant human transferrin, 19.4mg/L of recombinant human insulin, 64mg/L Vc) + mesoderm inducer combination (10mM Y-27632,250ng/cm2 of VTN,3mM CHIR99021, and 2ng/ml Activin A), and the induction time is 48 h;
2) mesodermal cells induced to endothelial cells: collecting mesodermal cells obtained by induction, preparing single cell suspension, then re-performing endothelial cell induction, wherein the induction differentiation culture medium is a basal culture medium (the same as above) and an endothelial cell inducer combination (2mg/ml PVA, 10ng/ml of hVEGF-A), and the induction time is 72 h.
Since the above documents do not describe the method of passaging, the passaging is carried out by a conventional passaging method as follows: when the confluency of cells reaches about 90%, removing the culture medium by aspiration, washing with PBS, digesting with TrypLE (pancreatic enzyme substitute, GIBCO, cat # 12604013) for 5min, observing most cells under microscope to brighten and round, stopping digestion with PBS of equal volume, collecting cells, centrifuging (300g, 5min), discarding supernatant, resuspending with endothelial cell amplification medium (lonza EGM-2MV EGM-2CC-3162), counting, and resuspending at 2 × 104/cm2The density of the cells was inoculated onto a new plate and the culture was continued (37 ℃ C., 5% CO)2). Observed every day, liquid is changed every other day, and the cells are passaged for 1 time in 4-5 days according to the growth and proliferation conditions of the cells.
The result shows that the induced differentiation efficiency (68.2 +/-5.1%) of the existing literature has no significant difference with the invention, but the obtained cells have poor expansion and passage capacity, and the cell morphology becomes large after passage 2-3 times, loses the proliferation capacity and can not be continuously subcultured (see figure 4).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (3)
1. A method of inducing differentiation of human embryonic stem cells into endothelial cells, comprising:
step 1, culturing human embryonic stem cells in a culture dish coated with glass fibronectin, wherein the culture medium is an E8 embryonic stem cell culture medium until the cell confluency is 80-90%; removing the culture medium, washing with PBS, digesting with dispase, sucking the dispase when the clone edge of the cell is observed to be shiny and curled under an inverted microscope, washing with PBS, uniformly scribing at the bottom of the culture dish transversely and longitudinally, blowing and beating the cell until most of the cell is observed to be separated from the bottom of the culture dish under the microscope, and forming cell clusters with relatively uniform size;
standing the cell suspension, sucking and removing a supernatant, then re-suspending a cell clone cluster by using an embryoid body forming culture medium, and transferring the cell clone cluster to a low-adhesion cell culture plate for culturing for 24 hours to obtain an embryoid body; the embryoid body forming culture medium takes DMEM/F-12 as a basic culture medium and contains 20% of KSR,2mM NEAA,2mM L-glutamine and 0.1mM beta-mercaptoethanol;
2, inducing the embryoid bodies into mesoderm cells by using a mesoderm induction medium; the mesoderm induction culture medium takes Stemline II serum-free culture medium as a basic culture medium and contains 1 × ITS-G, 1-100ng/ml of BMP4 and 1-50ng/ml of bFGF;
step 3, directionally inducing the mesoderm cells into endothelial cells by using an endothelial induction culture medium; the endothelial induction culture medium takes Stemline II serum-free culture medium as a basal culture medium and contains 1 XTS-G, 1-100ng/ml VEGF, 1-50ng/ml bFGF and 1-200ng/ml SCF;
and 4, preparing a single cell suspension from the induced and differentiated endothelial cells by using TrpLE cells, re-inoculating the single cell suspension to a new cell culture plate, continuously culturing by using a commercially available endothelial cell amplification culture medium, and carrying out subculture when the confluency of the cells reaches about 90%.
2. The method of claim 1, wherein step 2 is:
collecting the embryoid bodies, standing, sucking and removing the supernatant, then inducing for 96h by using mesoderm induction medium, changing the culture solution for 1 time at 48h to obtain mesoderm cells, wherein the embryoid bodies are performed in a low-adhesion cell culture plate in the whole mesoderm induction process.
3. The method of claim 1, wherein step 3 is:
and collecting and standing the mesoderm cells, sucking and removing supernatant, then re-suspending with an endothelial induction culture medium, inoculating the cells to a cell culture plate, and inducing for 48 hours to obtain the endothelial cells.
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