CN109797132B - Method for promoting directional differentiation of human pluripotent stem cells into endothelial cells - Google Patents
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Abstract
The invention discloses a method for promoting the directional differentiation of human pluripotent stem cells into endothelial cells, which comprises the following steps: on day 0-1, inducing differentiation of human pluripotent stem cells with a cell density of more than 95% by using a CDM3 differentiation medium containing 4-8 μ M of GSK 3I; on day 2, the cells after the further induction culture are further induced to differentiate by using a CDM3 differentiation culture medium added with 40-60 ng/ml bFGF; on days 3-5, further inducing differentiation of the cells subjected to the further induction culture by using a CDM3 differentiation medium added with 40-60 ng/ml VEGF and 20-30 ng/ml BMP 4; digesting the cells subjected to the induction culture in the last step on the 6 th day, and then continuously culturing for 3-4 days by adopting an endothelial cell culture medium. The method for differentiating the endothelial cells is more economic and effective, and the differentiation process is more convenient and faster; serum is not used in the differentiation process, the interference of various factors is eliminated, and the differentiation result is more reliable; and the addition of RA can promote the efficiency of endothelial differentiation, and more endothelial cells can be obtained by one-time differentiation.
Description
Technical Field
The invention relates to a method for promoting directional differentiation of human pluripotent stem cells into endothelial cells, belonging to the technical field of cell culture.
Background
Vascular disease affects the health of millions of people. Severe vascular disease leads to amputation and life threatening stroke. Endothelial cells lining all vascular lumens play a crucial role in regulating vascular permeability, angiogenesis and tissue regeneration. Under physiological conditions, blood vessels can be produced by angiogenesis and vasculogenesis. Angiogenesis refers to the formation of new blood vessels from progenitor cells, while angiogenesis refers to the formation of new blood vessels from existing blood vessels by migration and proliferation of existing vascular cells. However, these processes are impaired in patients with stroke, diabetes or senile dementia. In vascular diseases, the function of blood vessels deteriorates due to abnormal lesion or death of endothelial cells, and recent studies have shown that endothelial cells have the ability to form blood vessels and are very helpful in improving vascular diseases, and thus, a large amount of human-derived endothelial cells are required to be obtained for treating these vascular diseases.
Human pluripotent stem cells, such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hipscs), are capable of self-renewal and differentiation into any cell type in the human body, and are therefore ideal resources for the generation of Endothelial Cells (ECs). Many groups have demonstrated that ECs can be derived from hiPSCs and hESCs. The endothelial cells obtained by the induction can be used for vascular reconstruction of autologous ischemic regions and customization of artificial blood vessels and tissues, and in addition, the in vitro differentiation of the endothelial cells by utilizing pluripotent stem cells also provides a new way for researching and controlling molecular mechanisms of endothelial fate. The mechanism research is helpful for understanding the pathogenesis of the vascular diseases and provides effective measures for treating the vascular diseases. However, prior to the development of clinical applications, it was necessary to establish cost-effective and efficient methods for obtaining ECs from pluripotent stem cells. In addition, there is a need to further explore mechanisms that regulate the differentiation of endothelial cells by pluripotent stem cells.
At present, a plurality of methods for differentiating endothelial progenitor cells exist, but the defects of complex differentiation process, long differentiation period, high cost, unstable differentiation and the like generally exist. First, in the adherent cell differentiation method: in 2015, Gop μ S Rermam et al used a differentiation medium such as StemDiff APEL and ECGM-MV2, and when BMP4, FGF2 and VEGF were continuously added, the proportion of cells that were CD31/CD144 double positive was 80.7%. However, the differentiation medium of StemDiff APEL and ECGM-MV2 used in the method is too expensive, so that the differentiation cost is greatly increased. In 2017, Kit Man Tscan et al used BMP4, FGF2, VEGF under anaerobic conditions for seven consecutive days, which increased the endothelial differentiation efficiency from 12.4% at normoxia to 29.3%. Under the conditions of suspension cell differentiation, 2010, Hongkui Deng et al, using EB differentiation, 27% of CD34 positive progenitors were obtained over the course of seven days using BMP4, VEGF.
Currently, the following drawbacks exist with respect to differentiated endothelial cells: the cost is too high, and the mTeSR culture medium, StemDiff APEL and ECGM-MV2 culture medium have too high price and are not suitable for culturing and differentiating endothelial cells in large quantity; the prior art has low differentiation efficiency and low yield.
Disclosure of Invention
In order to solve the technical problems, the invention provides a method for promoting the directional differentiation of human pluripotent stem cells into endothelial cells, which reduces the cost of the human embryonic stem cell H1 line and the cost of the human pluripotent induced stem cells for inducing the endothelial cells in vitro, and further improves the differentiation efficiency of the endothelial cells by retinoic acid RA, thereby obtaining more endothelial cells in the primary differentiation process.
The first object of the present invention is to provide a method for promoting the directed differentiation of human pluripotent stem cells into endothelial cells, comprising the steps of:
(1) on day 0-1, inducing differentiation of human pluripotent stem cells with a cell density of more than 95% by using a CDM3 differentiation medium supplemented with 4-8 μ M of GSK 3I;
(2) on day 2, further inducing differentiation of the cells after induction culture in the step (1) by using a CDM3 differentiation medium added with 40-60 ng/ml bFGF;
(3) further inducing the differentiation of the cells after the induction culture in the step (2) by using a CDM3 differentiation medium added with 40-60 ng/ml VEGF and 20-30 ng/ml BMP4 on days 3-5;
(4) on the 6 th day, adding cell digestive enzyme to digest the cells induced and cultured in the step (3), and then continuously culturing the cells obtained after digestion for 3-4 days by adopting an endothelial cell culture medium;
the CDM3 culture medium contains RPMI-1640 basic culture medium, double antibiotics, bovine serum albumin and ascorbic acid.
Further, in the step (2), 0.8 to 1.2 μ M Retinoic Acid (RA) is added simultaneously with bFGF.
Further, the human pluripotent stem cell is a human embryonic stem cell line H1 or a human induced pluripotent stem cell (hiPSC).
Further, before the step (1), subculturing the human pluripotent stem cells by using a stem cell culture medium until the cell density reaches more than 95%.
Further, the stem cell culture medium is PSeasy-E8 or mTeSR.
Further, the culturing and the subculturing specifically comprise the following steps: culturing human pluripotent stem cells until the cell density reaches 85-90%, washing cell fragments by using a calcium phosphate free buffer solution (DPBS), adding 0.5 mu M EDTA, digesting for 5-10 min, then blowing the cells out, subculturing the cells into a Matrigel coated culture dish according to the proportion of 1: 6-1: 10 after blowing out, and continuously culturing until the cell density is more than 95%.
Further, in step (1), the GSK3I is CHIR 99021.
Furthermore, the concentration of bovine serum albumin in the CDM3 culture medium is 0.4-0.6 mg/ml, the concentration of ascorbic acid is 0.20-0.25 mg/ml, and the diabesin is penicillin and streptomycin, wherein the concentration of penicillin is 100U/ml, and the concentration of streptomycin is 0.1 mg/ml.
Further, the cell digestive enzyme is pancreatin cell digestive juice or Accutase cell digestive juice.
Further, the endothelial cell culture medium is an ECM culture medium.
Further, in step (4), the cells obtained after digestion were seeded into a Gelatin-coated culture dish and cultured. The culture dish coated by Gelatin is beneficial to the anchorage of auxiliary cells and the growth of endothelial cells.
In the invention, the 0 th day is 0-24 h, the 1 st day is 24-48 h, and the like.
The invention has the beneficial effects that:
the method for differentiating the endothelial cells is more economic and effective, and the differentiation process is more convenient and faster; serum is not used in the differentiation process, the interference of various factors is eliminated, and the differentiation result is more reliable; and the addition of RA can promote the efficiency of endothelial differentiation, and more endothelial cells can be obtained by one-time differentiation.
Drawings
FIG. 1 is a photomicrograph of undifferentiated embryonic stem cells;
FIG. 2 is a flow chart comparing differentiation efficiency at day 6 with the control group with RA addition;
FIG. 3 is a flow chart comparing differentiation efficiency at day 10 with the control group with RA addition;
FIG. 4 is a flow chart of endothelial cells purified by CD144 magnetic beads on the tenth day;
FIG. 5 shows immunofluorescent staining of endothelial cells after purification.
Detailed Description
The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.
Example 1: induction factor for directed differentiation and small molecule compound
Induction factor: CHIR99021, action concentration 6. mu.M; basic fibroblast growth factor (bFGF) with the action concentration of 100 ng/mu l; vascular Endothelial Growth Factor (VEGF) at a concentration of 50 ng/. mu.l; bone morphogenetic protein 4(BMP4) at an action concentration of 25 ng/. mu.l;
small molecule compounds: retinoic Acid (RA), at a concentration of 1 μ M.
Example 2: culture and passaging of embryonic stem cells
Culturing a human embryonic stem cell H1 line on a Matrigel-coated culture dish by using a PSeasy-E8 culture medium, sucking the culture medium when the confluence degree of the cells reaches 80% or more, and adding DPBS for washing once; adding EDTA, and incubating in a stable incubator at 37 deg.C for 3-5 min; and (3) sucking out the digestive juice, immediately adding a fresh PSeasy-E8 culture medium, blowing the bottom of the culture dish by using a pipette fan to make cell colonies attached to the bottom of the dish fall off, and gently and slowly sucking and uniformly mixing. The cells were then transferred to a new Matrigel-coated cell culture plate at a ratio of 1:8 or 1:10, subcultured using Pseasy-E8 medium, 10. mu.M ROCK-I (Y27632) was added to the medium 24h before the passage, and then the medium was changed daily until the next passage.
The result of photographing an undifferentiated human embryonic stem cell line H1 under an inverted fluorescence microscope is shown in FIG. 1, and the human embryonic stem cell line H1 is grown in a Martrigel-coated culture dish, cultured in PSeasy-E8 medium, the cells are closely arranged and grow in colony shape, and the cell clone and the periphery have obvious boundaries.
Example 3: in vitro directed differentiation of endothelial cells
And (5) continuously culturing by using PSeasy-E8 culture medium until the density is 95% -100%, and replacing the differentiation culture medium for induction. The differentiation Medium is CDM3 (containing RPMI Medium 1640 basic Medium, double antibody, bovine serum albumin and ascorbic acid).
Retinoic acid treated group (RA group): two days before differentiation (day 0 to day 1), designated as day 0 from the time of induction, 6. mu.M of CHIR99021 was added. On day 2, medium CDM3 was changed and 50ng/ml bFGF and 1. mu.M RA were added. From day 3 to day 5, CDM3 was replaced with 50ng/ml VEGF and 25ng/ml BMP 4. On day 6, the cells were digested with 0.1% trypsin or Accutase, and then inoculated into a Gelatin-coated 60mm dish for further culture for 3-4 days, with the medium being replaced with ECM.
Control group (DMSO group): the culture process was the same as that of the above-described group RA except that the medium CDM3 added on day 2 was supplemented with 50ng/ml bFGF and DMSO in the same volume as that of RA in the group RA.
The induced endothelial cells were subjected to flow cytometric assay on day 6 and day 10, as follows:
1. digesting with 0.1% pancreatin, placing in a 37 ℃ constant temperature incubator for 3-5 minutes, adding an ECM culture medium to stop digestion, blowing cells to be single by using a pipette gun, and adding a cell suspension into a 1.5ml centrifuge tube;
2. centrifuging at 300g for 5 minutes;
3. add 80. mu.l PBS to resuspend the cells (3 x 10)5Individual cells), 20. mu.l of CD34-APC or CD31-FITC or CD31-APC or CD144-PE antibody was added, incubated at 4 ℃ for 30 minutes in the dark, 400. mu.l of PBS was added to resuspend the cells, and the cells were tested on the machine.
As shown in FIGS. 2 and 3, CD34 in FIG. 2 is a marker molecule for endothelial progenitor cells, and the positive rate (29.6%) for CD34 in the RA-treated group was 10.9% higher than that (16.0%) in the DMSO group at day 6. In fig. 3, CD144 and CD31 are both marker molecules for endothelial cells, and a 23.9% increase in the double positive rate (84.6%) for CD144 and CD31 in the RA-treated group over the double positive rate (60.7%) in the DMSO group was detected at day 10.
Example 4: purification of endothelial cells
1. On day 10, trypsinize for 3-5 min with 0.1% pancreatin, resuspend the cells with 10mL Buffer (PBS + 0.5% BSA +2mM EDTA);
2. passing the cell suspension through a 30 micron filter screen to remove cell clumps;
3.300 g, 10 minutes, centrifugate;
4.80 u L Buffer heavy suspension cells, adding 20ml CD144 magnetic beads placed in 4 degrees, incubated for 15 minutes;
washing the magnetic beads with 5.1-2 mL of Buffer for 5 minutes at 300g, and centrifuging;
6. add 300. mu.l Buffer to resuspend the cells, pass the cells through an LS filtration column (Miltenyi Biotec),5ml Buffer over the column three times;
7. adding 5ml Buffer, separating a magnet by an LS filter column to flow out cells marked by CD144 magnetic beads, and centrifuging for 5 minutes at 300 g;
8. the cells were resuspended by adding ECM medium and allowed to continue growing in the fibrinectin coated dish.
The purified endothelial cells were detected by the same flow cytometry method as in example 3, and the results are shown in fig. 4, wherein the double positive rate of flow detection of CD144 and CD31 was 98.3% after the endothelial cells induced on day 10 were purified by CD144 magnetic beads.
Immunofluorescence staining using CD144 antibody resulted in irregular linear cell boundaries and regular elliptical cell nuclei stained with Hoechst, as shown in fig. 5.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (8)
1. A method for promoting the directional differentiation of human pluripotent stem cells into endothelial cells, which comprises the following steps:
(1) on day 0-1, inducing differentiation of human pluripotent stem cells with cell density of more than 95% by using CDM3 differentiation medium added with 4-8 μ M GSK 3I;
(2) on day 2, further inducing differentiation of the cells after induction culture in the step (1) by using a CDM3 differentiation medium added with 40-60 ng/ml bFGF;
(3) further inducing the differentiation of the cells after the induction culture in the step (2) by using a CDM3 differentiation medium added with 40-60 ng/ml VEGF and 20-30 ng/ml BMP4 on days 3-5;
(4) on the 6 th day, adding cell digestive enzyme to digest the cells induced and cultured in the step (3), and then continuously culturing the cells obtained after digestion for 3-4 days by adopting an endothelial cell culture medium;
the CDM3 differentiation culture medium contains RPMI-1640 basic culture medium, double antibiotics, bovine serum albumin and ascorbic acid;
in the step (2), 0.8-1.2 mu M retinoic acid is added while adding bFGF;
the concentration of bovine serum albumin in the CDM3 differentiation medium is 0.4-0.6 mg/ml, the concentration of ascorbic acid is 0.20-0.25 mg/ml, and the double antibiotics are penicillin and streptomycin, wherein the concentration of penicillin is 100U/ml, and the concentration of streptomycin is 0.1 mg/ml.
2. The method of claim 1, wherein the human pluripotent stem cell is a human embryonic stem cell line H1 or a human induced pluripotent stem cell.
3. The method of claim 1, further comprising, prior to step (1), subculturing the human pluripotent stem cells with a stem cell culture medium to a cell density of 95% or more.
4. The method of claim 3, wherein the stem cell culture medium is PSeasy-E8 or mTeSR.
5. The method according to claim 3, wherein the subculture comprises the following steps: culturing human pluripotent stem cells until the cell density reaches 85-90%, washing cell fragments by using a calcium-free phosphate buffer solution, adding 0.5 mu M EDTA, digesting for 5-10 min, then blowing out the cells, subculturing the cells into a Matrigel-coated culture dish according to the proportion of 1: 6-1: 10 after blowing out, and continuously culturing until the cell density is more than 95%.
6. The method of claim 1, wherein in step (1), the GSK3I is CHIR 99021.
7. The method of claim 1, wherein the cellular digestive enzyme is pancreatin cellular digestive fluid or Accutase cellular digestive fluid.
8. The method of claim 1, wherein said endothelial cell culture medium is an ECM medium.
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| EP3875579A1 (en) * | 2020-03-02 | 2021-09-08 | Urgo Recherche Innovation Et Developpement | Method for obtaining endothelial cells from pluripotent stem cells |
| CN111440760B (en) * | 2020-03-09 | 2022-09-23 | 浙江大学 | Method for efficiently differentiating human pluripotent stem cells to obtain endothelial progenitor cells |
| CN112725261B (en) * | 2021-01-19 | 2022-09-02 | 上海爱萨尔生物科技有限公司 | Culture solution for differentiation culture of endothelial cells by pluripotent stem cells and differentiation method |
| CN112980770B (en) * | 2021-03-03 | 2022-08-02 | 华中科技大学同济医学院附属协和医院 | Method for inducing directional endothelial differentiation of human pluripotent stem cells |
| CN113897331B (en) * | 2021-09-29 | 2023-01-17 | 苏州大学 | Differentiation method for inducing human artery endothelial cells |
| CN116574672B (en) * | 2023-07-11 | 2023-10-20 | 北京北启生物医药有限公司 | Culture medium and method for inducing differentiation of chemically induced pluripotent stem cells into hematogenic endothelial cells |
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