CN108048390B - Method for preparing vascular endothelial cells and special kit thereof - Google Patents

Method for preparing vascular endothelial cells and special kit thereof Download PDF

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CN108048390B
CN108048390B CN201711395640.7A CN201711395640A CN108048390B CN 108048390 B CN108048390 B CN 108048390B CN 201711395640 A CN201711395640 A CN 201711395640A CN 108048390 B CN108048390 B CN 108048390B
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那洁
朱咏林
张凤枝
段福宇
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Abstract

The invention discloses a method for preparing vascular endothelial cells and a special kit thereof. The kit contains a culture solution II and a culture solution I; the culture solution II comprises serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF and a serum-free basic culture solution; culture medium I comprises serum albumin, transferrin, vitamin C, sodium selenite, BMP4, GSK3 inhibitor and a serum-free basal culture medium. Experiments prove that the vascular endothelial cells can be obtained by adopting the preparation method provided by the invention, and the culture solution used by the preparation method not only can enable the human pluripotent stem cells to be quickly and efficiently differentiated into the vascular endothelial cells, but also has determined chemical components and no animal-derived protein or insulin. The preparation method provided by the invention can be used for producing human vascular endothelial cells in a large scale, and has stable quality and high safety. The invention has great application value.

Description

Method for preparing vascular endothelial cells and special kit thereof
Technical Field
The invention relates to the field of biomedicine, in particular to a method for preparing vascular endothelial cells and a special kit thereof.
Background
Human pluripotent stem cells (including human induced pluripotent stem cells and human embryonic stem cells) can be differentiated into functional cells of various tissues and organs, used for researching human development, making disease models, and replacing cells with damaged lesions through cell transplantation, thereby promoting body wound repair and treating diseases. Stem cells and regenerative medicine change the traditional treatment means for diseases such as necrotizing and injuring diseases, and revolutionize the research on the mechanism of the diseases and the clinical treatment.
The vascular endothelial cells are important constitutional cells of human blood vessels, and have important application values in the aspects of injury repair, blood vessel reconstruction, ischemic disease treatment, tissue engineering, drug research and development, disease model establishment and the like. At present, a large amount of vascular endothelial cells are difficult to obtain, the amplification capacity is limited, the price is high, the components of a culture solution are uncertain, and animal-derived components are contained, so that the related clinical application is greatly limited.
Disclosure of Invention
The technical problem to be solved by the invention is how to prepare the vascular endothelial cells.
In order to solve the technical problems, the invention firstly provides a kit for preparing vascular endothelial cells; the kit may contain culture solution II; the culture solution II may include serum albumin, transferrin, vitamin C, sodium selenite, Vascular Endothelial Growth Factor (VEGF), basic fibroblast growth factor (bFGF), and a serum-free basal culture solution.
In the kit, the culture solution II can be specifically composed of serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF and a serum-free basic culture solution.
In the above kit, each 1mL of the culture medium II may comprise 0.1-2.5mg of serum albumin (such as 0.1-0.5mg of serum albumin, 0.5-2.5mg of serum albumin, 0.1mg of serum albumin, 0.5mg of serum albumin or 2.5mg of serum albumin), 1-25 μ g of transferrin (1-5 μ g of transferrin, 5-25 μ g of transferrin, 1 μ g of transferrin, 5 μ g of transferrin or 25 μ g of transferrin), 0.04-1mg of vitamin C (0.04-0.2mg of vitamin C, 0.2-1mg of vitamin C, 0.04mg of vitamin C, 0.2mg of vitamin C or 1mg of vitamin C), 2-50ng of sodium selenite (2-10ng of sodium selenite, 10-50ng of sodium selenite, 2ng of sodium selenite, 10ng of sodium selenite or 50ng of sodium selenite), 10-250ng of VEGF (10-50ng of VEGF), 50-250ng VEGF, 10ng VEGF, 50ng VEGF or 250ng VEGF) and 2-50ng bFGF (2-10ng bFGF, 10-50ng bFGF, 2ng bFGF, 10ng bFGF or 50ng bFGF), wherein the base solution of the culture solution II can be a serum-free base culture solution, and the pH value can be 7.0-7.6 (such as 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).
In the above kit, each 1mL of the culture medium II may contain 0.1-2.5mg of serum albumin (such as 0.1-0.5mg of serum albumin, 0.5-2.5mg of serum albumin, 0.1mg of serum albumin, 0.5mg of serum albumin or 2.5mg of serum albumin), 1-25 μ g of transferrin (1-5 μ g of transferrin, 5-25 μ g of transferrin, 1 μ g of transferrin, 5 μ g of transferrin or 25 μ g of transferrin), 0.04-1mg of vitamin C (0.04-0.2mg of vitamin C, 0.2-1mg of vitamin C, 0.04mg of vitamin C, 0.2mg of vitamin C or 1mg of vitamin C), 2-50ng of sodium selenite (2-10ng of sodium selenite, 10-50ng of sodium selenite, 2ng of sodium selenite, 10ng of sodium selenite or 50ng of sodium selenite), 10-50ng of VEGF (10-50ng of VEGF), 50-250ng VEGF, 10ng VEGF, 50ng VEGF or 250ng VEGF) and 2-50ng bFGF (2-10ng bFGF, 10-50ng bFGF, 2ng bFGF, 10ng bFGF or 50ng bFGF); the pH can be 7.0-7.6 (e.g., 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).
In the culture solution II, the VEGF may be VEGF-A.
In the culture solution II, the serum albumin can be recombinant human serum albumin, bovine serum albumin, or recombinant human serum albumin of other non-animal sources, such as recombinant human serum albumin of plant source.
In the culture solution II, the transferrin can be human transferrin or other recombinant human transferrin derived from non-animal sources, such as recombinant human transferrin derived from plant sources.
In the culture solution II, the serum-free basic culture solution can be RPMI 1640 culture medium or other basic culture solutions commonly used for cell culture.
In the kit, the culture solution II may be specifically a culture solution II-1, a culture solution II-2 or a culture solution II-3 shown in Table 1.
TABLE 1
Figure BDA0001518399540000021
Any one of the above kits may further comprise a culture solution I; the medium I may comprise serum albumin, transferrin, vitamin C, sodium selenite, bone morphogenic protein 4(BMP4), glycogen synthase kinase 3(GSK3) inhibitors, and a serum-free basal medium.
The culture solution I can be specifically composed of serum albumin, transferrin, vitamin C, sodium selenite, BMP4, GSK3 inhibitor and a serum-free basal culture solution.
In the above kit, each 1mL of the culture medium I may comprise 0.1-2.5mg of serum albumin (such as 0.1-0.5mg of serum albumin, 0.5-2.5mg of serum albumin, 0.1mg of serum albumin, 0.5mg of serum albumin or 2.5mg of serum albumin), 1-25 μ g of transferrin (1-5 μ g of transferrin, 5-25 μ g of transferrin, 1 μ g of transferrin, 5 μ g of transferrin or 25 μ g of transferrin), 0.04-1mg of vitamin C (0.04-0.2mg of vitamin C, 0.2-1mg of vitamin C, 0.04mg of vitamin C, 0.2mg of vitamin C or 1mg of vitamin C), 2-50ng of sodium selenite (2-10ng of sodium selenite, 10-50ng of sodium selenite, 2ng of sodium selenite, 10ng of sodium selenite or 50ng of sodium selenite), 2-50ng of BMP4 (4 ng of BMP), 2-10ng of BMP), 10-50ng BMP4, 2ng BMP4, 10ng BMP4 or 50ng BMP4) and 1-10 μ M GSK3 inhibitor (1-5 μ M GSK3 inhibitor, 5-10 μ M GSK3 inhibitor, 1 μ M GSK3 inhibitor, 5 μ M GSK3 inhibitor or 10 μ M GSK3 inhibitor), wherein the culture solution II base solution can be a serum-free base culture solution with a pH value of 7.0-7.6 (such as 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5 or 7.6).
In the above kit, each 1mL of the culture medium I may contain 0.1-2.5mg of serum albumin (such as 0.1-0.5mg of serum albumin, 0.5-2.5mg of serum albumin, 0.1mg of serum albumin, 0.5mg of serum albumin or 2.5mg of serum albumin), 1-25 μ g of transferrin (1-5 μ g of transferrin, 5-25 μ g of transferrin, 1 μ g of transferrin, 5 μ g of transferrin or 25 μ g of transferrin), 0.04-1mg of vitamin C (0.04-0.2mg of vitamin C, 0.2-1mg of vitamin C, 0.04mg of vitamin C, 0.2mg of vitamin C or 1mg of vitamin C), 2-50ng of sodium selenite (2-10ng of sodium selenite, 10-50ng of sodium selenite, 2ng of sodium selenite, 10ng of sodium selenite or 50ng of sodium selenite), 2-50ng of BMP4(2-10ng of BMP4), 10-50ng BMP4, 2ng BMP4, 10ng BMP4, or 50ng BMP4) and 1-10 μ M GSK3 inhibitor (1-5 μ M GSK3 inhibitor, 5-10 μ M GSK3 inhibitor, 1 μ M GSK3 inhibitor, 5 μ M GSK3 inhibitor, or 10 μ M GSK3 inhibitor); the pH can be 7.0-7.6 (e.g., 7.0-7.3, 7.3-7.6, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, or 7.6).
In the culture solution I, the serum albumin can be recombinant human serum albumin, bovine serum albumin, or recombinant human serum albumin of other non-animal sources, such as plant sources.
In the culture solution I, the transferrin can be human transferrin or other recombinant human transferrin derived from non-animal sources, such as recombinant human transferrin derived from plant sources.
In the culture solution I, the serum-free basic culture solution can be RPMI 1640 culture medium or a basic culture solution commonly used for cell culture.
In the culture solution I, the GSK3 inhibitor can be a1) or a2) or a3) or a 4): a1) CHIR-99021; a2) b216763; a3) BIO; a4) the TWS 119.
In the kit, the culture solution I can be specifically a culture solution I-1, a culture solution I-2, a culture solution I-3, a culture solution I-4 or a culture solution I-5 shown in Table 2.
TABLE 2
Figure BDA0001518399540000031
Figure BDA0001518399540000041
Any one of the above kits may further comprise a culture solution III-1 and/or a culture solution III-2;
the culture solution III-1 can be TeSR-E8 complete culture solution or other culture solutions for culturing human pluripotent stem cells;
the culture solution III-2 may be a TeSR-E8 complete culture solution containing 2-10. mu.M (e.g., 2-5. mu.M, 5-10. mu.M, 2. mu.M, 5. mu.M or 10. mu.M) of ROCK inhibitor.
Any one of the TeSR-E8 complete culture solutions can be specifically STEM CELL Technologies, Cat # 05940.
The TeSR-E8 complete culture solution (500mL) can be prepared by mixing 474mL of TeSR-E8 basic culture solution, 25mL of 20 × supplement 1 and 1mL of 500 × supplement 2. TeSR-E8 basal medium, 20 Xsupplement 1 and 500 Xsupplement 2 may be components in TeSR-E8 complete medium.
In the culture solution III-2, the Rho-associated protein kinase (ROCK) inhibitor may specifically be Y27632.
The kit of any one of the above aspects may comprise any one of the above-mentioned culture solutions II and/or any one of the above-mentioned culture solutions I and/or any one of the above-mentioned culture solutions III-2 and/or any one of the above-mentioned culture solutions III-1.
In order to solve the technical problems, the invention also provides a method for culturing the vascular endothelial cells.
The method for culturing the vascular endothelial cells provided by the invention can comprise the following steps:
(3) inoculating human pluripotent stem cells into any culture solution I, and culturing to obtain cardiovascular precursor cells;
(4) after the step (3) is finished, inoculating the cardiovascular precursor cells to any one of the culture solution II, and culturing; vascular endothelial cells were obtained.
In the above method, the method may further include the step (5): after the step (4) is completed, the vascular endothelial cells are inoculated into any one of the culture solutions II and cultured.
In the above method, the method may further include the steps of: performing steps (1) and (2) before performing step (3);
the step (1) may be: inoculating human pluripotent stem cells to any one of the culture solutions III-2, and culturing for 1 day +/-0.5 days;
the step (2) may be: culturing the human pluripotent stem cells subjected to the step (1) in any one of the culture solutions III-1 for 1 day. + -. 0.5 days.
In the step (1), the concentration of the human pluripotent stem cells in the culture system may be 2.0X 104One per square centimeter (cm)2)－5.0×104One per square centimeter (cm)2) (e.g., 2.0X 10)4Per cm2－2.5×104Per cm2、2.5×104Per cm2－5.0×104Per cm2、2.0×104Per cm2、5.0×104Per cm2Or 2.5X 104Per cm2)。
In the step (4), the seeding density for "seeding the cells subjected to the step (3) into any of the culture solutions II" may be 0.25X 105Per cm2-1.0×105Per cm2(e.g., 0.25X 10)5Per cm2-5×104Per cm2、5×104Per cm2-1.0×105Per cm2、0.25×105Per cm2、5×104Per cm2Or 1.0X 105Per cm2)。
In the step (3), the culturing time may be 3 days ± 0.5 day.
In the step (4), the cultivation time may be 3 to 10 days.
In the step (5), the cultivation time may be 3 to 30 days.
In any of the above methods, the culture parameters may be: 36-38 deg.C (such as 36 deg.C, 37 deg.C or 38 deg.C).
Any of the above described cultures may employ CO2The incubator is used for carrying out the culture. The CO is2The parameters of the incubator setup may be: 5% CO2。
In any of the above methods, the human pluripotent stem cells may be single cells after digestion with digestive juices. The digestive juice can be specifically Accutase. The digestive juice Accutase can be specifically a product of Merk Millipore company, and the catalog number is SF 006.
Any one of the human pluripotent stem cells described above may be b1) or b2) or b3) or b4) or b5) or b 6):
b1) a human embryonic stem cell line;
b2) a human induced pluripotent stem cell line;
b3) human embryonic stem cell line H1;
b4) human embryonic stem cell line H7;
b5) human embryonic stem cell line H9;
b6) the human induced pluripotent stem cell line is CD 34-iPSC.
It should be noted that, if the method provided by the present invention is used to prepare human vascular endothelial cells, the serum albumin in the culture solution I and the culture solution II may be recombinant human serum albumin, and the transferrin may be human transferrin. If the method provided by the invention is adopted to prepare non-human vascular endothelial cells, the serum albumin in the culture solution I and the culture solution II can be bovine serum albumin.
The human embryonic stem cell line may be specifically purchased commercially. The human embryonic stem cell line H1, the human embryonic stem cell line H7 and the human embryonic stem cell line H9 can be products of the U.S. WiCell cell bank.
Experiments prove that the vascular endothelial cells can be obtained by adopting the preparation method provided by the invention, and the culture solution used by the preparation method not only can enable the human pluripotent stem cells to be quickly and efficiently differentiated into the vascular endothelial cells, but also has determined chemical components and no animal-derived protein or insulin. The preparation method provided by the invention can be used for producing human vascular endothelial cells in a large scale, has stable quality and high safety, and provides a large amount of cell sources for tissue engineering, drug research and development and cell therapy. The invention has great application value.
Drawings
FIG. 1 is a clone morphology chart of undifferentiated human pluripotent stem cells.
FIG. 2 is the morphological changes at different time points during the differentiation of human induced pluripotent stem cells (CD34-iPSC) into vascular endothelial cells.
FIG. 3 shows the morphological changes at different time points during the differentiation of human embryonic stem cells (H1) into vascular endothelial cells.
FIG. 4 shows the immunofluorescence assay of cardiovascular precursor cells in step two of example 2.
FIG. 5 shows the results of the experiment for measuring the ratio of vascular endothelial cells in step two of example 2.
FIG. 6 shows the results of immunofluorescence staining identification of vascular endothelial cells in step two of example 2.
FIG. 7 is the experimental results of 4 in vitro differentiated vascular endothelial cells forming a vascular-like network on Matrigel in step two of example 2.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
RPMI 1640 medium and recombinant human Vitronectin (Vitronectin) were both products of ThermoFisher corporation. VEGF-A is a product of SinoBiological corporation under the catalog number 11066-HNAB. CHIR-99021 is a product of Tocris Biosciences, catalog No. 4423. Y27632 is a targetMol product, catalog number T1725. Vitamin C (catalog number A8960) and human transferrin (catalog number T1147) are both products of Sigma Aldrich. Sodium selenite is available from Sigma Aldrich under catalog number S5261. bFGF is a product of R & D Biosystems, catalog number 233-FB. The PBS buffer was Wincent, Catalogue No. 311-010-CL. Fetal bovine serum is a product of Hyclone company under the catalog number SH 30088.03. Paraformaldehyde is a product of chemical reagents of the national drug group. The digestion solution Accutase is a product of Merk Millipore company, and the catalog number is SF 006. DMEM culture solution (catalog No. 11960044) and 0.25% Trypsin (catalog No. 25200056) are products of Gibco corporation. TeSR-E8 complete medium is STEMCELL, Catalogue No. 05940. BMP4 is a product of R & D Biosystems, catalog number 314-BP. The recombinant human serum albumin is a product of Wuhanhe grass Yuan Biotech company. The anti-human MESP1 antibody was a product of Avia Systems Biology, catalog number ARP39374_ P050. DAPI is a product of ThermoFisher, Inc. under product catalog number D1306. FITC-labeled anti-human CD31 antibody is available from BD Biosciences under catalog number 557508. The anti-human CD31 antibody is available from Abcam under the catalog number ab 76533. Matrigel is a product of BD Biosciences, catalog number 356231.
Human embryonic stem cell line H1, human embryonic stem cell line H7 and human embryonic stem cell line H9 are all products of the U.S. WiCell cell Bank. Hereinafter, the human embryonic stem cell line H1 is abbreviated as H1 cell, the human embryonic stem cell line H7 is abbreviated as H7 cell, and the human embryonic stem cell line H9 is abbreviated as H9 cell.
The human induced pluripotent stem cell line CD34-iPSC is obtained by reprogramming human cord blood hematopoietic stem cells. Hereinafter, the human induced pluripotent stem cell line CD34-iPSC is abbreviated as CD34-iPSC cell.
4% paraformaldehyde: 4% (4g/100mL) paraformaldehyde in PBS buffer.
PBST buffer: PBS buffer containing 0.1% (v/v) Tween-20.
In the following examples, the pore size of the filter was 0.22. mu.m.
Example 1 preparation of culture solution
Preparation of stock solution
And (3) dissolving the recombinant human serum albumin by using sterile PBS buffer solution, and then filtering by using a filter membrane to obtain a recombinant human serum albumin stock solution with the concentration of 50 mg/mL.
Dissolving human transferrin by sterile PBS buffer solution, and then filtering by a filter membrane to obtain a human transferrin stock solution with the concentration of 1 mg/mL.
Vitamin C is taken, dissolved by cell culture grade water and then filtered by a filter membrane to obtain a vitamin C stock solution with the concentration of 50 mg/mL.
Sodium selenite is taken, dissolved by cell culture grade water and then filtered by a filter membrane to obtain a sodium selenite stock solution with the concentration of 100 mug/mL.
CHIR-99021 was taken and dissolved in DMSO to give a 20mM stock solution of CHIR-99021.
Secondly, preparation of the culture solution I
Mixing RPMI 1640 culture medium, recombinant human serum albumin stock solution, human transferrin stock solution, vitamin C stock solution, sodium selenite stock solution, BMP4 and CHIR-99021 stock solution to obtain culture solution I-1, culture solution I-2, culture solution I-3, culture solution I-4 or culture solution I-5. The pH of each culture broth and the concentrations of each component are shown in Table 2.
TABLE 2
Figure BDA0001518399540000071
Thirdly, preparation of culture solution II
Mixing RPMI 1640 culture medium, recombinant human serum albumin stock solution, human transferrin stock solution, vitamin C stock solution, sodium selenite stock solution, VEGF-A and bFGF to obtain culture solution II-1, culture solution II-2 or culture solution II-3. The pH of each culture broth and the concentrations of each component are shown in Table 1.
TABLE 1
Figure BDA0001518399540000081
Fourthly, preparation of culture solution III-1 and culture solution III-2
Culture III-1 was prepared by mixing 474mL of TeSR-E8 basal medium, 25mL of 20 Xsupplement 1 and 1mL of 500 Xsupplement 2.
Culture solution III-2: mixing the culture solution III-1 and Y27632 to obtain a culture solution III-2; in the culture solution III-2, the concentration of Y27632 was 5. mu.M.
Example 2 obtaining of vascular endothelial cells and detection thereof
The human pluripotent stem cell is H1 cell, H7 cell, H9 cell or CD34-iPSC cell.
The culture solution II is culture solution II-1, culture solution II-2 or culture solution II-3.
The culture solution I is culture solution I-1, culture solution I-2, culture solution I-3, culture solution I-4 or culture solution I-5.
First, obtaining vascular endothelial cells
1. Human pluripotent stem cells grown to 70% -80% confluence were collected, the supernatant was discarded, and then washed 2 times with PBS buffer (in order to remove dead cells and residual metabolic wastes from the cells).
The morphology of human pluripotent stem cells is shown in figure 1 (H1 cells at the top left, H7 cells at the top right, H9 cells at the bottom left, and CD34-iPSC cells at the bottom right).
2. After the step 1 is finished, adding a proper amount of digestive juice Accutase, and standing for 3-5 minutes at 37 ℃; then adding a proper amount of DMEM culture solution to terminate digestion, centrifuging, removing supernatant, adding culture solution III-2 for resuspension, and obtaining the suspension of the human pluripotent stem cells.
3. The plates were coated with recombinant human Vitronectin and incubated at 37 ℃ for 2 hours.
4. And (3) after the step 3 is completed, adding the suspension of the human pluripotent stem cells obtained in the step 2 and the culture solution III-2 into the culture dish to obtain a culture system. The concentration of human pluripotent stem cells in the culture system was 2.0X 104Per cm2－5.0×104Per cm2。
5. Placing the culture system obtained in the step 4 at 37 ℃ and 5% CO2Culturing in an incubator for 1 day.
6. After completion of step 5, the supernatant was discarded, followed by addition of the culture medium III-1, and the mixture was left at 37 ℃ with 5% CO2Culturing in an incubator for 1 day.
7. After completion of step 6, the supernatant was discarded, and then culture medium I was added thereto, and the mixture was left at 37 ℃ with 5% CO2The cells were cultured in an incubator for 3 days (liquid change every day during the culture period) to obtain cells B.
8. And 7, removing the supernatant, adding a proper amount of 0.25% Trypsin for digestion to a single cell state, adding a DMEM culture solution containing 10% (v/v) fetal calf serum for terminating digestion, centrifuging, removing the supernatant, and adding a culture solution II for resuspension to obtain a suspension of the cells B.
9. Another fresh culture dish was coated with recombinant human Vitronectin and incubated at 37 ℃ for 2 hours.
10. And (4) after the step 9 is finished, taking the culture dish, and adding the suspension of the cells B and the culture solution II obtained in the step 8 to obtain a culture system. The concentration of cell B in the culture system was 5X 104Per cm2。
11. The culture system was incubated at 37 ℃ with 5% CO2The cells were cultured in an incubator for 5 days (liquid change every day during the culture period) to obtain cells C.
During the culture, morphological changes of human pluripotent stem cells were observed (day 0 is the time of addition of culture solution I). The morphological changes of the partially human pluripotent stem cells are shown in FIG. 2(CD34-iPSC cells) and FIG. 3(H1 cells). The results show that the morphology of human pluripotent stem cells gradually changes to that of vascular endothelial cells.
Second, detecting
1. Detection of cell B
(1) The cells B obtained in step one, step 7, were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then the 4% paraformaldehyde was aspirated and washed 3 times with PBS buffer (in order to remove residual paraformaldehyde).
(2) After the step (1) is finished, adding PBS buffer solution containing 0.25 percent (v/v) of Triton X-100, and standing for 20 minutes at room temperature; then 5% (v/v) BSA in PBS buffer was added to block for 1h at room temperature.
(3) After completion of step (2), anti-human MESP1 antibody was added (volume ratio 1: 100), incubated at room temperature for 2h, and then washed 3 times with PBST buffer.
(4) After completion of step (3), a secondary antibody (product of Abcam Co.) was added (volume ratio 1: 500), incubated at room temperature for 1h, and then washed 3 times with PBST buffer.
(5) After completion of step (4), DAPI was added (volume ratio 1: 1000), incubated at room temperature for 20 minutes, and then washed 3 times with PBST buffer. The staining of the cells was observed under a fluorescent microscope.
The staining of cell B under fluorescence microscopy is shown in FIG. 4 (left panel for MESP1 expression and right panel for nuclear staining). The results indicate that cell B is a cardiovascular precursor cell.
The result shows that the culture solution I can efficiently induce and differentiate the human pluripotent stem cells into the cardiovascular precursor cells.
2. Detection of cell C-
(1) Taking the cell C obtained in the step one (11), firstly adding a proper amount of 0.25% Trypsin to digest the cell to a single cell state, then centrifuging, removing supernatant, and adding PBS buffer solution containing 5% (v/v) fetal calf serum to obtain cell C suspension.
(2) Taking the suspension (containing 1X 10 cells) of the cells C obtained in the step (1)5Cell C), FITC-labeled anti-human CD31 antibody was added and incubated for 20 minutes at room temperature in the dark (during which time mixing was done every 5 minutes); then washed 2 times with PBS buffer containing 5% (v/v) fetal bovine serum (in order to remove unbound antibody).
(3) After completion of step (2), 500. mu.L of PBS buffer containing 5% (v/v) fetal bovine serum was added for resuspension and then detected by flow cytometry.
The results of cell C detection using CD34-iPSC cells are shown in FIG. 5. The results show that more than 50% of the cells C cultured in the culture solution II express the protein CD31 specifically expressed by endothelial cells on the surface.
3. Detection of cell C
(1) The cells C obtained in step one, 11, were fixed with 4% paraformaldehyde at room temperature for 10 minutes, and then the 4% paraformaldehyde was aspirated and washed 3 times with PBS buffer (in order to remove the residual paraformaldehyde).
(2) After completion of step (1), blocking was performed by adding 5% (v/v) BSA in PBS buffer for 1 hour.
(3) After completion of step (2), anti-human CD31 antibody was added (volume ratio 1: 200), incubated at room temperature for 2 hours, and then washed 3 times with PBST buffer.
(4) After completion of step (3), a secondary antibody (product of Abcam Co.) was added (volume ratio 1: 500), incubated at room temperature for 1h, and then washed 3 times with PBST buffer.
(5) After completion of step (4), DAPI was added (volume ratio 1: 1000), incubated at room temperature for 20 minutes, and then washed 3 times with PBST buffer. The staining of the cells was observed under a fluorescent microscope.
The results of cell C detection using H1 cells are shown in FIG. 6. The results showed that cell C cultured in culture medium II expressed protein CD31 on its surface, indicating that cell C is a vascular endothelial cell.
4. Detection of cell C
(1) The plates were coated with Matrigel and incubated at 37 ℃ for 2 hours.
(2) After the completion of step (1), cells C obtained in step one (11) were plated on Matrigel, and then the formation of the angioid network was observed every 12 hours and recorded.
The results of the experiment using cell C obtained from H1 cells are shown in fig. 7 (left panel is a diagram showing the formation of the angioid network, and right panel is a partial enlarged view of the angioid network). The results indicate that cell C has the ability to form a vascular-like network.
The results show that the culture solution I and the culture solution II can be used for efficiently inducing and differentiating the human pluripotent stem cells into the vascular endothelial cells.

Claims (11)

1. A kit for preparing vascular endothelial cells; the kit contains a culture solution II;
the culture solution II consists of serum albumin, transferrin, vitamin C, sodium selenite, VEGF, bFGF and a serum-free basic culture solution; the serum-free basal culture solution is RPMI 1640 culture medium.
2. The kit of claim 1, wherein: 0.1-2.5mg of serum albumin, 1-25 mug of transferrin, 0.04-1mg of vitamin C, 2-50ng of sodium selenite, 10-250ng of VEGF and 2-50ng of bFGF in every 1mL of the culture solution II, wherein a solvent of the culture solution II is a serum-free basic culture solution, and the pH value is 7.0-7.6.
3. The kit of claim 1 or 2, wherein: the kit also contains a culture solution I;
the culture solution I consists of serum albumin, transferrin, vitamin C, sodium selenite, BMP4, a GSK3 inhibitor and a serum-free basal culture solution; the serum-free basal culture solution is RPMI 1640 culture medium.
4. The kit of claim 3, wherein: every 1mL of the culture solution I consists of 0.1-2.5mg of serum albumin, 1-25 mug of transferrin, 0.04-1mg of vitamin C, 2-50ng of sodium selenite, 2-50ng of BMP4 and 1-10 mug M of GSK3 inhibitor, the solvent of the culture solution I is a serum-free basic culture solution, and the pH value is 7.0-7.6.
5. The kit of claim 1 or 2, wherein: the kit also contains a culture solution III-1 and/or a culture solution III-2;
the culture solution III-1 is a TeSR-E8 complete culture solution;
the culture solution III-2 is a TeSR-E8 complete culture solution containing 2-10 mu M ROCK inhibitor.
6. A method of culturing vascular endothelial cells, comprising the steps of:
(3) inoculating the human pluripotent stem cells into the culture solution I of claim 3 or 4, and culturing to obtain cardiovascular precursor cells;
(4) after the step (3) is completed, inoculating the cardiovascular precursor cells into the culture solution II in the claim 1 or 2, and culturing; vascular endothelial cells were obtained.
7. The method of claim 6, wherein: the method further comprises step (5): after completion of step (4), the vascular endothelial cells are seeded into the culture solution II according to claim 1 or 2 and cultured.
8. The method of claim 7, wherein: the method further comprises the steps of: performing steps (1) and (2) before performing step (3);
the step (1) is as follows: inoculating the human pluripotent stem cells into the culture solution III-2 of claim 5, and culturing for 1d +/-0.5 d;
the step (2) is as follows: the human pluripotent stem cells subjected to the step (1) are cultured in the culture solution III-1 according to claim 5 for 1 d. + -. 0.5 d.
9. The method of any of claims 6 to 8, wherein: the culture parameters are as follows: 36-38 ℃.
10. The method of any of claims 6 to 8, wherein: the human pluripotent stem cell is a human embryonic stem cell line or a human induced pluripotent stem cell line.
11. The method of claim 10, wherein: the human embryonic stem cell line is a human embryonic stem cell line H1 or a human embryonic stem cell line H7 or a human embryonic stem cell line H9;
the human induced pluripotent stem cell line is a human induced pluripotent stem cell line CD 34-iPSC.
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Publication number Priority date Publication date Assignee Title
CN101314766A (en) * 2007-05-31 2008-12-03 中国医学科学院基础医学研究所 Method for isolated culture of human fat mesenchyma stem cell and special culture medium thereof

Family Cites Families (6)

* Cited by examiner, † Cited by third party
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen;Brewer, G.J.等;《 Brain Res》;19890807;第494卷(第1期);第65–74页 *
人胚胎干细胞定向分化为血管内皮细胞的初步研究;王彦等;《组织工程与重建外科杂志》;20100630;第6卷(第02期);第66-69页 *

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