Vascular endothelial cell as well as preparation method and application thereof

Abstract

The invention discloses a vascular endothelial cell as well as a preparation method and application thereof. The preparation method comprises the following steps: inoculating induced pluripotent stem cells into a culture medium containing an ROCK signal channel inhibitor for culturing, and inducing the cultured induced pluripotent stem cells by using a culture medium containing a Wnt signal channel activator and a BMP signal channel activator, so that the induced pluripotent stem cells are differentiated into mesoderm cells; inducing the mesoderm cells by using a culture medium containing a vascular endothelial growth factor and a cAMP signaling pathway agonist, so that the mesoderm cells are differentiated into endothelial coarse cells; and performing positive sorting of the endothelial cell marker CD144 on the endothelial coarse cells to obtain purified vascular endothelial cells. The induction path of the method is clear and efficient, the period is short, the vascular endothelial cell population with high purity, complete vascular barrier function and mature phenotype can be obtained, and the cell population can be applied to construction of a vascular network in a gel matrix.

Description

Technical Field

[0001] This invention belongs to the field of cell engineering technology, and relates to a vascular endothelial cell, its preparation method, and its application. Background Technology

[0002] In vitro three-dimensional vascular models are of great value for basic vascular biology research, drug screening, and toxicity assessment. Vascular endothelial cells, as key structural and functional units of the vascular system, play an irreplaceable role in physiological and pathological processes such as maintaining vascular permeability, regulating blood flow, participating in immune responses, and tissue regeneration. Therefore, obtaining high-purity, highly active vascular endothelial cells is a primary prerequisite for conducting vascular biology research, drug screening, and constructing tissue-engineered blood vessels or cell therapy products.

[0003] Currently, the main cell sources for constructing such models include primary mature endothelial cells (such as human umbilical vein endothelial cells) and endothelial cells induced to differentiate into pluripotent stem cells. Although HUVECs are commonly used in in vitro studies, they suffer from problems such as large donor variability and limited expansion capacity, which restricts their standardization and large-scale application.

[0004] To overcome the aforementioned bottlenecks, the directed differentiation of pluripotent stem cells (including embryonic stem cells or induced pluripotent stem cells) into vascular endothelial cells has become a promising alternative strategy. This method has advantages such as abundant cell sources and theoretically unlimited expansion, while avoiding the significant ethical issues of direct cell harvesting, thus providing the possibility of obtaining homogeneous endothelial cell populations that can be used for research and treatment.

[0005] However, existing induction differentiation protocols still have significant shortcomings, limiting their efficiency and application. Mainstream methods often rely on prolonged, multi-stage addition of growth factors or the use of embryoid formation steps, resulting in a lengthy induction cycle, cumbersome procedures, and room for improvement in the purity and functional maturity of the final cell population. In the three-dimensional angiogenesis stage, while existing techniques (such as seeding endothelial cells onto the surface of matrix gel) can induce spontaneous assembly of tubular network structures, these structures often suffer from unstable formation efficiency, random structures, and difficulty in control, affecting experimental reproducibility and the scope of application.

[0006] Therefore, there is an urgent need to develop a new method for inducing vascular endothelial cells that is faster, simpler, more stable, and more controllable, and that can effectively induce them to form well-defined three-dimensional tubular structures in an in vitro environment. This method would have practical value for related basic research and the development of drug development tools. Summary of the Invention

[0007] To address the shortcomings of existing technologies and practical needs, this invention provides vascular endothelial cells, their preparation method, and applications. This improves the efficiency and directionality of induced pluripotent stem cell differentiation into target vascular endothelial cells. The obtained vascular endothelial cells can be used to construct vascular networks in a gel matrix, providing an effective technical platform for basic research in vascular biology, disease model construction, and screening of vasoactive drugs.

[0008] To achieve this objective, the present invention adopts the following technical solution: In a first aspect, the present invention provides a method for preparing vascular endothelial cells, the method comprising: seeding induced pluripotent stem cells in a culture medium containing a ROCK signaling pathway inhibitor for culture; inducing the cultured induced pluripotent stem cells to differentiate into mesodermal cells using a culture medium containing a Wnt signaling pathway activator and a BMP signaling pathway activator; inducing the mesodermal cells to differentiate into crude endothelial cells using a culture medium containing vascular endothelial growth factor and a cAMP signaling pathway agonist; and performing positive sorting of the crude endothelial cells using the endothelial cell marker CD144 to obtain purified vascular endothelial cells.

[0009] The method of this invention has a well-defined, efficient, and short induction pathway, and can obtain a high-purity vascular endothelial cell population with complete vascular barrier function and a mature phenotype. This cell population can be used to construct vascular networks in a gel matrix. A flowchart illustrating the method and application of inducing pluripotent stem cell-directed vascular endothelial cell differentiation is shown below. Figure 1 As shown.

[0010] Preferably, the ROCK signaling pathway inhibitor includes Y-27632.

[0011] Preferably, the Wnt signaling pathway activator includes CHIR-99021.

[0012] Preferably, the BMP signaling pathway activator includes hBMP4.

[0013] Preferably, the vascular endothelial growth factor includes VEGF-165.

[0014] Preferably, the cAMP signaling pathway agonist includes Forskolin.

[0015] Preferably, the final concentration of Y-27632 in the culture medium containing the ROCK signaling pathway inhibitor is 4-12 µM.

[0016] Y-27632, mentioned above, is a small molecule compound and an inhibitor targeting Rho-related protein kinases, which can inhibit cell death of induced pluripotent stem cells during cell dispersion. CHIR-99021 and hBMP4 are both inducible small molecules. CHIR-99021 activates the Wnt / β-catenin signaling pathway by targeting activated GSK-3α / β, rapidly downregulating pluripotency genes while upregulating the expression of key mesodermal markers such as Brachyury and KDR. hBMP4 is human bone morphogenetic protein 4, a key morphogenetic protein in the TGF-β superfamily, which can activate the classical BMP-Smad signaling pathway and synergize with Wnt signaling to finely regulate the specialization of mesodermal subtypes.

[0017] In this invention, CHIR-99021 and hBMP4 together promote the differentiation of induced pluripotent stem cells into lateral plate mesoderm cells.

[0018] Both VEGF-165 and Forskolin are inducible small molecules. VEGF-165 directly drives cells to differentiate into the endothelial lineage by activating its specific receptor VEGFR-2, promoting their proliferation, migration, and lumen formation. Forskolin, a diterpenoid natural product, acts as an adenylate cyclase activator, increasing intracellular cAMP levels. This enhances the expression and functional maturation of endothelial cell markers and upregulates endogenous VEGF signaling, synergistically activating key developmental pathways such as NFATc1 with VEGF.

[0019] In this invention, VEGF-165 and Forskolin form a synergistic effect at the molecular level, promoting the differentiation of lateral plate mesodermal cells into vascular endothelial cells.

[0020] Preferably, the final concentration of CHIR-99021 in the culture medium containing Wnt signaling pathway activator and BMP signaling pathway activator is 8-12 µM (e.g., 8 µM, 10 µM or 12 µM), and the final concentration of hBMP4 in the culture medium containing Wnt signaling pathway activator and BMP signaling pathway activator is 20-30 ng / mL (e.g., 20 ng / mL, 25 ng / mL or 30 ng / mL).

[0021] Preferably, the final concentration of VEGF-165 in the culture medium containing vascular endothelial growth factor and cAMP signaling pathway agonist is 100-300 ng / mL (e.g., 100 ng / mL, 200 ng / mL, or 300 ng / mL), and the final concentration of Forskolin in the culture medium containing vascular endothelial growth factor and cAMP signaling pathway agonist is 1-4 µM (e.g., 1 µM, 2 µM, or 4 µM).

[0022] Preferably, the inoculation density is 40,000-50,000 cells / cm². 2 For example, it could be 40,000 cells / cm³ 2 45,000 cells / cm 2 Or 50,000 cells / cm 2 .

[0023] Preferably, the culture time is 10-20 h, for example, 10 h, 15 h or 20 h.

[0024] Preferably, the induction time for inducing induced pluripotent stem cells after culture is 60-80 h, for example, 60 h, 70 h or 80 h.

[0025] Preferably, the induction time for inducing mesodermal cells is 40-50 h, for example, 40 h, 45 h or 50 h.

[0026] In a second aspect, the present invention provides a vascular endothelial cell, which is induced by the differentiation method described in the first aspect.

[0027] Thirdly, the present invention provides an in vitro construction method for a three-dimensional vascular network, the method comprising the following steps: (1) The vascular endothelial cells described in the second aspect are mixed with fibrinogen and matrix gel to form a cell-gel mixture; (2) The cell-gel mixture is mixed with a solution containing thrombin and aprotinin to obtain a cell-gel-enzyme mixture, which is then coagulated to form a fibrin gel containing vascular endothelial cells. (3) The fibrin gel was cultured using an induction medium containing vascular endothelial growth factor, fibroblast growth factor and neuropeptide Y receptor agonist to induce vascular endothelial cells to self assemble in the gel to form a three-dimensional vascular network.

[0028] Preferably, the final concentration of fibrinogen in the cell-gel mixture is 3-4 mg / mL, for example, 3 mg / mL, 3.5 mg / mL or 4 mg / mL.

[0029] Preferably, the volume percentage of matrix gel in the cell-gel mixture is 5-15%, for example, it can be 5%, 10% or 15%.

[0030] Preferably, the final concentration of thrombin in the cell-gel-enzyme mixture is 5-10 U / mL (e.g., 5 U / mL, 8 U / mL, or 10 U / mL), and the final concentration of aprotinin in the cell-gel-enzyme mixture is 1-3 U / mL (e.g., 1 U / mL, 2 U / mL, or 3 U / mL).

[0031] Preferably, the vascular endothelial growth factor includes VEGF-165.

[0032] Preferably, the fibroblast growth factor includes FGF2.

[0033] Preferably, the neuropeptide Y receptor agonist includes NPY.

[0034] Both FGF2 and NPY are inducible small molecules. FGF2 is a fibroblast growth factor, a key growth factor regulating cell growth and differentiation, and involved in cell proliferation, differentiation, and angiogenesis. Neuropeptide Y (NPY) is a polypeptide neurotransmitter widely distributed in the central and peripheral nervous systems, which regulates angiogenesis through activation of Y2 receptors and precise enzymatic cleavage by DPPIV.

[0035] Preferably, the final concentration of VEGF-165 in the induction medium is 50-200 ng / mL (e.g., 50 ng / mL, 100 ng / mL, or 200 ng / mL), the final concentration of FGF2 in the induction medium is 50-150 ng / mL (e.g., 50 ng / mL, 100 ng / mL, or 150 ng / mL), and the final concentration of NPY in the induction medium is 0-2 µM (e.g., 0 µM, 1 µM, or 2 µM).

[0036] Fourthly, the present invention provides a kit for an in vitro construction method of the three-dimensional vascular network described in the third aspect, the kit comprising: a first culture reagent, a first induction reagent, a second induction reagent, and a purification reagent.

[0037] The first culture reagent includes: a ROCK signaling pathway inhibitor.

[0038] The first inducing agent includes: Wnt signaling pathway activator and / or BMP signaling pathway activator.

[0039] The second inducing agent includes: vascular endothelial growth factor and / or cAMP signaling pathway agonists.

[0040] The purification reagent includes: magnetic bead-conjugated antibodies.

[0041] Preferably, the kit further includes any one or a combination of at least two of the following reagents: (1) Matrix adhesive; (2) Any one or a combination of at least two of the following: vascular endothelial growth factor, fibroblast growth factor, or neuropeptide Y receptor agonist; (3) Any one or a combination of at least two of fibrinogen, thrombin or aprotinin.

[0042] Fifthly, the present invention provides the use of the vascular endothelial cells described in the second aspect in the preparation of products for vascular tissue engineering, disease model construction, or screening of angiogenesis drugs.

[0043] In a sixth aspect, the present invention provides the application of the three-dimensional vascular network constructed by the method described in the third aspect in vascular biology research, construction of angiogenesis-related disease models, or screening of vasoactive drugs.

[0044] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention utilizes an optimized combination of cytokines for precise regulation, enabling the generation of a large number of CD31 and CD144 double-positive, high-purity, and functionally mature vascular endothelial cells from a small number of induced pluripotent stem cells (iPSCs) within a short period of time. This improves the efficiency and directionality of differentiation into target vascular endothelial cells, providing a high-quality cell source for angiogenesis research, disease modeling, and cell therapy. (2) The method of the present invention is designed with three clear stages: pretreatment, mesodermal induction and endothelial induction. The process is standardized and has good reproducibility. The whole method does not involve any animal-derived components, avoids the risk of foreign pathogens and immune rejection, ensures the safety of the differentiation process and the final product, and is more in line with the standards for clinical application. (3) The method of the present invention can obtain a CD144 positive cell population with a purity of more than 90% within about 7 days and form a stable tubular network within 48 hours in three-dimensional culture. The vascular endothelial cells obtained by the method of the present invention can efficiently self-assemble into a three-dimensional vascular network in vitro. The network structure is biomimetic and has rich branches, providing a fully functional in vitro testing platform for vascularized tissue construction, disease model research and drug screening. Attached Figure Description

[0045] Figure 1 A flowchart illustrating the method and application of inducing pluripotent stem cells to differentiate into vascular endothelial cells; Figure 2 Cell morphology diagrams during the process of induced pluripotent stem cell-directed differentiation into vascular endothelial cells; Figure 3 Flow cytometry image of CD144, a marker of vascular endothelial cells; Figure 4 This is a graph showing the detection results of CD31, a single marker of vascular endothelial cells. Figure 5 A morphological diagram of the vascular network constructed to induce vascular endothelial cells. Detailed Implementation

[0046] To further illustrate the technical means and effects of this invention, the following description, in conjunction with embodiments and accompanying drawings, provides a further explanation of the invention. It is understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it.

[0047] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0048] The matrix gel used in the following examples was purchased from Corning (model 354277); Accutase cell separation medium was purchased from STEMCELL Technologies (model #07920); the magnetic bead-coupled anti-CD144 antibody, sorting column, and magnetic rack were all purchased from Miltenyi Biotec (models 130-097-857, 130-042-401, and 130-042-303, respectively); DMEM / F12 complete medium was purchased from Sciencell (model 1001); and mTeSR™ Plus medium was purchased from STEMCELL. Technologies, model number #100-0276; SB216763 purchased from MCE, model number HY-12012; Sb4 purchased from MCE, model number HY-124697; VEGF-B purchased from Sigma-Aldrich, model number SRP3183; prostaglandin E2 purchased from MCE, model number HY-101952.

[0049] The reagent components of N2B27 medium are shown in Table 1, and the reagent components of StemPro-34 SFM complete medium are shown in Table 2.

[0050] Table 1 Table 2 Example 1 This embodiment describes the preparation of vascular endothelial cells and the construction of vascular networks, as detailed below: Step 1: Dilute the matrix gel at a ratio of 1:100 in DMEM / F12 medium, and then add the diluted liquid at a concentration of 200 µL / cm³. 2 The appropriate amount of liquid was added to the well plate. The well plate was then transferred to a cell culture incubator (37°C, 5% CO2) and incubated for 30 min to obtain induced pluripotent stem cell culture plates.

[0051] Step 2: Remove induced pluripotent stem cells (iPSCs) with 80% confluence from the cell culture incubator (37°C, 5% CO2), discard the culture medium in the iPSC culture plate, and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0052] Step 3: After 20 h, discard the supernatant from the centrifugation product of Step 2, resuspend the induced pluripotent stem cells and count them. Discard the culture medium in the induced pluripotent stem cell culture plate prepared in Step 1, and resuspend the cells at 45,000 cells / cm³. 2 Induced pluripotent stem cells were seeded at a density in induced pluripotent stem cell culture plates at 300 µL / cm³ of mTeSR™ Plus complete medium. 2 The volume of liquid added was such that the culture medium contained 10 µM Y-27632.

[0053] Step 4: Discard the culture medium in the cell culture plate from Step 3, and add N2B27 medium containing a final concentration of 8 µM CHIR-99021 and a final concentration of 25 ng / mL hBMP4 at 600 µL / cm³. 2 The appropriate amount of liquid was added to the cell culture plate. The plate was incubated in a cell culture incubator (37°C, 5% CO2) for 72 h. Cell morphology during the induced pluripotent stem cell-directed vascular endothelial cell differentiation process is shown in the figure below. Figure 2 As shown, iPSCs begin to differentiate from the second day and proliferate significantly on the third day. On the sixth day, the cells exhibit a cobblestone morphology similar to endothelial cells.

[0054] Step 5: Discard the culture medium in the cell culture plate from Step 4, and add StemPro-34 SFM complete medium containing a final concentration of 300 ng / mL VEGF-165 and a final concentration of 2 µM Forskolin at a concentration of 500 µL / cm³. 2The liquid volume was added to the cell culture plate and cultured in a cell culture incubator (37°C, 5% CO2) for 48 h.

[0055] Step 6: Discard the culture medium in the induced pluripotent stem cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0056] Discard the supernatant and incubate the cells with PBS at a concentration of 10⁻⁶. 7 Resuspend cells at a density of 1 / mL, add magnetic bead-conjugated anti-CD144 antibody, and incubate at 4°C for 15 min to label the cells.

[0057] Dilute the cell suspension to 10 μL using PBS. 6 Cells / mL, centrifuged at 300 g for 3 min. Subsequently, CD144-positive cells were separated using a sorting column under the magnetic field provided by a magnetic rack. Flow cytometry images of single-labeled CD144, a pre-endothelial marker, are shown below. Figure 3 As shown, the CD144 positivity rate of the induced cells increased significantly on the fifth day of induction, reaching 59.3%.

[0058] After enriching CD144-positive cells, immunofluorescence detection was performed. Cells were proliferated to 80% in culture dishes and fixed with 4% paraformaldehyde. They were permeabilized with 0.1% Triton X-100 and blocked with 2% BSA for 1 h at room temperature. Subsequently, the samples were incubated overnight at 4°C with a specific primary antibody (CD31 monoclonal antibody, purchased from Proteintech, model 66065-2-Ig). After thorough elution with PBS, the samples were incubated with a fluorescently labeled secondary antibody (goat anti-mouse Alexa Fluor® 594, purchased from Abcam, model ab150116) at room temperature in the dark for 1 h. After PBS elution, the cell nuclei were counterstained with Hoechst 33342, and images were observed and acquired under a fluorescence microscope. Hoechst 33342 is a nuclear dye that emits blue fluorescence upon excitation by ultraviolet light. The CD31 fluorescence detection results are shown in the figure below. Figure 4 As shown, the induced endothelial cells universally express the endothelial cell marker CD31.

[0059] Step 7: The cells collected in Step 6 are processed at a rate of 30,000 cells / cm³. 2The cells were seeded at a density suitable for inducing vascular endothelial cells in a culture plate and cultured overnight in a cell incubator to obtain induced vascular endothelial cells. In step 7, the induced vascular endothelial cell culture plate was obtained by the following method: fibronectin was diluted to 25 µg / mL in sterile water, and the diluted liquid was then injected at a concentration of 150 µL / cm³. 2 The required volume of liquid is placed in the well plate. The culture plates for inducing vascular endothelial cells can be sealed with sealing film and stored at 4°C for use within 7 days of preparation.

[0060] Step 8: Place the induced vascular endothelial cells in a cell culture incubator (37°C, 5% CO2) until they expand to 80%. Discard the culture medium in the induced vascular endothelial cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of water. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator (37°C, 5% CO2) for 3 min. Gently pipette the sides and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with warm mTeSR Plus complete medium at 37°C to stop digestion.

[0061] Collect cell suspension using centrifuge tubes and centrifuge at 300 g for 3 min.

[0062] Discard the supernatant of the centrifuged product and use endothelial cell culture medium at 10... 7 Resuspend cells at 1 / mL. Add an equal volume of fibrinogen solution (final concentration 4 mg / mL) to the cell suspension, mix well, and add 10% (v / v) of matrix gel. Mix thrombin and aprotinin in a 1:1 ratio (final concentration of thrombin: 5 U / mL, final concentration of aprotinin: 1 U / mL) to prepare a mixed enzyme. Transfer 1 µL of the mixed enzyme to a new PCR tube. Pipette to form a single-cell suspension. Using a pipette, quickly mix 9 µL of endothelial cell suspension with 1 µL of the mixed solution containing thrombin and aprotinin, and drop the mixture into 48-well plates to form droplets, 10 µL per well.

[0063] The well plates with the added gel were placed in a cell culture incubator (37°C, 5% CO2) for 20 min to allow fibrin gel formation. The plates were then removed, and 300 µL of DMEM / F12 complete endothelial cell culture medium (containing 200 ng / mL VEGF-165, 100 ng / mL FGF2, and 1 µM NPY) was carefully and gently added to each well. Immunofluorescence staining was performed after 48 h. The immunofluorescence staining procedure was as follows: fixation with 200 µL of 4% paraformaldehyde. Permeabilization was performed using 0.1% Triton X-100, followed by blocking with 2% BSA at room temperature for 1 h. Subsequently, the samples were incubated overnight at 4°C with a specific primary antibody (CD31 monoclonal antibody, purchased from Proteintech, model number 66065-2-Ig). After thorough elution with PBS, they were incubated with a fluorescently labeled secondary antibody (goat anti-mouse Alexa Fluor® 488, purchased from Abcam, model number ab150113) at room temperature in the dark for 1 h. After PBS elution, the cell nuclei were counterstained with Hoechst, and the images were observed and acquired under a fluorescence microscope. The morphological diagram of the vascular network constructed by the induced vascular endothelial cells is shown below. Figure 5 As shown, the formation of a vascular network was observed.

[0064] Example 2 This embodiment describes the preparation of vascular endothelial cells and the construction of vascular networks, as detailed below: Step 1: Dilute the matrix gel at a ratio of 1:100 in DMEM / F12 medium, and then add the diluted liquid at a concentration of 200 µL / cm³. 2 The appropriate amount of liquid was added to the well plate. The well plate was then transferred to a cell culture incubator (37°C, 5% CO2) and incubated for 30 min to obtain induced pluripotent stem cell culture plates.

[0065] Step 2: Remove induced pluripotent stem cells (iPSCs) with 80% confluence from the cell culture incubator (37°C, 5% CO2), discard the culture medium in the iPSC culture plate, and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0066] Step 3: After 20 h, discard the supernatant from the centrifugation product of Step 2, resuspend the induced pluripotent stem cells, and count them. Discard the culture medium in the induced pluripotent stem cell culture plate prepared in Step 1, and resuspend the cells at 40,000 cells / cm³. 2 Induced pluripotent stem cells were seeded at a density in induced pluripotent stem cell culture plates at 200 µL / cm³ of mTeSR™ Plus complete medium. 2 The volume of liquid added was such that the culture medium contained 4 µM Y-27632.

[0067] Step 4: Discard the culture medium in the cell culture plate from Step 3, and add N2B27 medium containing a final concentration of 8 µM CHIR-99021 and a final concentration of 20 ng / mL hBMP4 at 700 µL / cm³. 2 The required amount of liquid was added to the cell culture plate. The plate was then incubated in a cell culture incubator (37°C, 5% CO2) for 80 h.

[0068] Step 5: Discard the culture medium in the cell culture plate from Step 4, and add StemPro-34 SFM complete medium containing a final concentration of 300 ng / mL VEGF-165 and a final concentration of 1 µM Forskolin at a concentration of 500 µL / cm³. 2 The liquid volume was added to the cell culture plate and cultured in a cell culture incubator (37°C, 5% CO2) for 50 h.

[0069] Step 6: Discard the culture medium in the induced pluripotent stem cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0070] Discard the supernatant and incubate the cells with PBS at a concentration of 10⁻⁶. 7 Resuspend cells at a density of 1 / mL, add magnetic bead-conjugated anti-CD144 antibody, and incubate at 4°C for 15 min to label the cells.

[0071] Dilute the cell suspension to 10 μL using PBS. 6 Cells / mL, centrifuged at 300 g for 3 min. Subsequently, CD144-positive cells were separated and enriched by using a sorting column in the magnetic field provided by a magnetic rack.

[0072] Step 7: The cells collected in Step 6 are processed at a rate of 30,000 cells / cm³.2 The cells were seeded at a density suitable for inducing vascular endothelial cells in a culture plate and cultured overnight in a cell incubator to obtain induced vascular endothelial cells. In step 7, the induced vascular endothelial cell culture plate was obtained by the following method: fibronectin was diluted to 25 µg / mL in sterile water, and the diluted liquid was then injected at a concentration of 150 µL / cm³. 2 The required volume of liquid is placed in the well plate. The culture plates for inducing vascular endothelial cells can be sealed with sealing film and stored at 4°C for use within 7 days of preparation.

[0073] Step 8: Place the induced vascular endothelial cells in a cell culture incubator (37°C, 5% CO2) until they expand to 80%. Discard the culture medium in the induced vascular endothelial cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of water. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator (37°C, 5% CO2) for 3 min. Gently pipette the sides and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with warm mTeSR Plus complete medium at 37°C to stop digestion.

[0074] Collect cell suspension using centrifuge tubes and centrifuge at 300 g for 3 min.

[0075] Discard the supernatant of the centrifuged product and use endothelial cell culture medium at 10... 7 Resuspend cells at 1 / mL. Add an equal volume of fibrinogen solution (final concentration 4 mg / mL) to the cell suspension, mix well, and add 5% (v / v) of matrix gel. Mix thrombin and aprotinin at a 1:1 ratio (thrombin final concentration 5 U / mL, aprotinin final concentration 1 U / mL) to prepare a mixed enzyme. Transfer 1 µL of the mixed enzyme to a new PCR tube. Pipette to form a single-cell suspension. Using a pipette, quickly mix 9 µL of endothelial cell suspension with 1 µL of the mixed solution containing thrombin and aprotinin, and drop the mixture into 48-well plates to form droplets, 10 µL per well.

[0076] The well plates with the added gel were placed in a cell culture incubator (37°C, 5% CO2) for 20 min, where fibrin gel formed. The plates were then removed, and 300 µL of DMEM / F12 complete endothelial cell culture medium (containing 50 ng / mL VEGF-165 and 50 ng / mL FGF2) was carefully and gently added to each well. Vascular network formation was observed after 24 h.

[0077] Example 3 Step 1: Dilute the matrix gel at a ratio of 1:100 in DMEM / F12 medium, and then add the diluted liquid at a concentration of 300 µL / cm³. 2 The appropriate amount of liquid was added to the well plate. The well plate was then transferred to a cell culture incubator (37°C, 5% CO2) and incubated for 30 min to obtain induced pluripotent stem cell culture plates.

[0078] Step 2: Remove induced pluripotent stem cells (iPSCs) with 70% confluence from the cell culture incubator (37°C, 5% CO2), discard the culture medium in the iPSC culture plate, and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0079] Step 3: After 20 h, discard the supernatant from the centrifugation product of Step 2, resuspend the induced pluripotent stem cells and count them. Discard the culture medium in the induced pluripotent stem cell culture plate prepared in Step 1, and resuspend the cells at 50,000 cells / cm³. 2 Induced pluripotent stem cells were seeded at a density in induced pluripotent stem cell culture plates at 400 µL / cm³ of mTeSR™ Plus complete medium. 2 The volume of liquid added was such that the culture medium contained 12 µM Y-27632.

[0080] Step 4: Discard the culture medium in the cell culture plate from Step 3, and add N2B27 medium containing a final concentration of 12 µM CHIR-99021 and a final concentration of 30 ng / mL hBMP4 at 500 µL / cm³. 2 The required amount of liquid was added to the cell culture plate. The plate was then incubated in a cell culture incubator (37°C, 5% CO2) for 60 h.

[0081] Step 5: Discard the culture medium in the cell culture plate from Step 4, and add StemPro-34 SFM complete medium containing a final concentration of 100 ng / mL VEGF-165 and a final concentration of 4 µM Forskolin at 700 µL / cm³. 2 The liquid volume was added to the cell culture plate and cultured in a cell culture incubator (37°C, 5% CO2) for 48 h.

[0082] Step 6: Discard the culture medium in the induced pluripotent stem cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of PBS.2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator for 6 min. Gently tap the sidewalls and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with 37°C warm mTeSR Plus complete medium to stop digestion. Collect the cells in centrifuge tubes and centrifuge at 300 g for 3 min.

[0083] Discard the supernatant and incubate the cells with PBS at a concentration of 10⁻⁶. 7 Resuspend cells at a density of 1 / mL, add magnetic bead-conjugated anti-CD144 antibody, and incubate at 4°C for 15 min to label the cells.

[0084] Dilute the cell suspension to 10 μL using PBS. 6 Cells / mL, centrifuged at 300 g for 3 min. Subsequently, CD144-positive cells were separated and enriched by using a sorting column in the magnetic field provided by a magnetic rack.

[0085] Step 7: The cells collected in Step 6 are processed at a rate of 20,000 cells / cm³. 2 The cells were seeded at a density suitable for inducing vascular endothelial cells in a culture plate and cultured overnight in a cell incubator to obtain induced vascular endothelial cells. In step 7, the induced vascular endothelial cell culture plate was obtained by the following method: fibronectin was diluted to 25 µg / mL in sterile water, and the diluted liquid was then injected at a concentration of 150 µL / cm³. 2 The required volume of liquid is placed in the well plate. The culture plates for inducing vascular endothelial cells can be sealed with sealing film and stored at 4°C for use within 7 days of preparation.

[0086] Step 8: Place the induced vascular endothelial cells in a cell culture incubator (37°C, 5% CO2) until they expand to 80%. Discard the culture medium in the induced vascular endothelial cell culture plate and wash the cells once with PBS. Aspirate the PBS and rinse with 100 µL / cm³ of water. 2 Add the required volume of Accutase cell separation medium and incubate in a cell culture incubator (37°C, 5% CO2) for 3 min. Gently pipette the sides and bottom of the cell culture plate to completely detach the cells. Dilute the Accutase cell separation medium 1:1 with warm mTeSR Plus complete medium at 37°C to stop digestion.

[0087] Collect cell suspension using centrifuge tubes and centrifuge at 300 g for 3 min.

[0088] Discard the supernatant of the centrifuged product and use endothelial cell culture medium at 10... 7Resuspend cells at 1 / mL. Add an equal volume of fibrinogen solution (final concentration 3 mg / mL) to the cell suspension, mix well, and add 10% (v / v) of matrix gel. Mix thrombin and aprotinin in a 1:1 ratio (thrombin final concentration: 10 U / mL, aprotinin final concentration: 3 U / mL) to prepare a mixed enzyme. Transfer 1 µL of the mixed enzyme to a new PCR tube. Pipette to form a single-cell suspension. Using a pipette, quickly mix 9 µL of endothelial cell suspension with 1 µL of the mixed solution containing thrombin and aprotinin, and drop the mixture into 48-well plates to form droplets, 10 µL per well.

[0089] The well plates with the added gel were placed in a cell culture incubator (37°C, 5% CO2) for 20 min, where fibrin gel formed. The plates were then removed, and 300 µL of DMEM / F12 complete endothelial cell culture medium (containing 200 ng / mL VEGF-165, 150 ng / mL FGF2, and 2 µM NPY) was carefully and gently added to each well. Vascular network formation was observed after 48 h.

[0090] Example 4 The only difference between this embodiment and Example 1 is that the N2B27 culture medium in step 4 does not contain CHIR-99021.

[0091] Result: No vascular endothelial cells were obtained.

[0092] Example 5 The only difference between this embodiment and Example 1 is that the N2B27 culture medium in step 4 does not contain hBMP4.

[0093] Result: No vascular endothelial cells were obtained.

[0094] Example 6 The only difference between this embodiment and Example 1 is that the StemPro-34 SFM complete culture medium in step 5 does not contain VEGF-165.

[0095] Result: No vascular endothelial cells were obtained.

[0096] Example 7 The only difference between this embodiment and Example 1 is that the StemPro-34 SFM complete culture medium in step 5 does not contain Forskolin.

[0097] Results: A small number of vascular endothelial cells could be obtained, and the yield of CD144-positive cells decreased after magnetic bead sorting.

[0098] Example 8 The only difference between this embodiment and Example 1 is that in step 4, CHIR-99021 in the N2B27 culture medium is replaced with SB216763.

[0099] Results: Vascular endothelial cells were obtained, but the yield of CD144-positive cells decreased after magnetic bead sorting.

[0100] Example 9 The only difference between this embodiment and Example 1 is that in step 4, hBMP4 in the N2B27 culture medium is replaced with Sb4.

[0101] Results: Vascular endothelial cells could be obtained, but the yield of CD144-positive cells decreased after magnetic bead sorting.

[0102] Example 10 The only difference between this embodiment and Example 1 is that in step 5, VEGF-165 in the StemPro-34 SFM complete culture medium is replaced with VEGF-B.

[0103] Result: No vascular endothelial cells were obtained.

[0104] Example 11 The only difference between this embodiment and Example 1 is that in step 5, Forskolin in the StemPro-34 SFM complete culture medium is replaced with prostaglandin E2.

[0105] Results: Vascular endothelial cells could be obtained, but the yield of CD144-positive cells decreased after magnetic bead sorting.

[0106] Comparative Example 1 The only difference between this comparative example and Example 1 is that the N2B27 culture medium in step 4 does not contain CHIR-99021 and hBMP4.

[0107] Result: No vascular endothelial cells were obtained.

[0108] Comparative Example 2 The only difference between this comparative example and Example 1 is that the StemPro-34 SFM complete culture medium in step 5 does not contain VEGF-165 and Forskolin.

[0109] Result: No vascular endothelial cells were formed.

[0110] In summary, this invention is the first to develop a method for the directed induction of vascular endothelial cell (viEC) differentiation and vascular network formation using induced pluripotent stem cells (iPSCs). The method involves a three-stage directed induction differentiation of iPSCs to obtain vascular endothelial cells, followed by culturing these endothelial cells under specific three-dimensional culture conditions to assemble them into tubular structures. This invention can induce a high-purity cell population with complete vascular barrier function and a mature phenotype. This cell population can be used to construct vascular networks in a gel matrix, providing an effective technical platform for basic research in vascular biology, disease model construction, and screening of vasoactive drugs.

[0111] The applicant declares that the detailed method of the present invention is illustrated by the above embodiments, but the present invention is not limited to the above detailed method, that is, it does not mean that the present invention must rely on the above detailed method to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for preparing vascular endothelial cells, characterized in that, The preparation method includes: seeding induced pluripotent stem cells (iPSCs) in a culture medium containing a ROCK signaling pathway inhibitor and culturing them; inducing the cultured iPSCs to differentiate into mesodermal cells using a culture medium containing a Wnt signaling pathway activator and a BMP signaling pathway activator; inducing the mesodermal cells to differentiate into coarse endothelial cells using a culture medium containing vascular endothelial growth factor (VEGF) and a cAMP signaling pathway agonist; and performing positive sorting of the coarse endothelial cells using the endothelial cell marker CD144 to obtain purified vascular endothelial cells.

2. The method for preparing vascular endothelial cells according to claim 1, characterized in that, The ROCK signaling pathway inhibitor includes Y-27632; Preferably, the Wnt signaling pathway activator includes CHIR-99021; Preferably, the BMP signaling pathway activator includes hBMP4; Preferably, the vascular endothelial growth factor includes VEGF-165; Preferably, the cAMP signaling pathway agonist includes Forskolin; Preferably, the final concentration of Y-27632 in the culture medium containing the ROCK signaling pathway inhibitor is 4-12 µM; Preferably, the final concentration of CHIR-99021 in the culture medium containing Wnt signaling pathway activator and BMP signaling pathway activator is 8-12 µM, and the final concentration of hBMP4 in the culture medium containing Wnt signaling pathway activator and BMP signaling pathway activator is 20-30 ng / mL. Preferably, the final concentration of VEGF-165 in the culture medium containing vascular endothelial growth factor and cAMP signaling pathway agonist is 100-300 ng / mL, and the final concentration of Forskolin in the culture medium containing vascular endothelial growth factor and cAMP signaling pathway agonist is 1-4 µM.

3. The method for preparing vascular endothelial cells according to claim 1 or 2, characterized in that, The inoculation density is 40,000-50,000 cells / cm³. 2 ; Preferably, the culture time is 10-20 h; Preferably, the induction time for inducing induced pluripotent stem cells after culture is 60-80 h; Preferably, the induction time for the induced mesodermal cells is 40-50 h.

4. A type of vascular endothelial cell, characterized in that, The vascular endothelial cells are induced by the differentiation method described in any one of claims 1-3.

5. A method for in vitro construction of a three-dimensional vascular network, characterized in that, The in vitro construction method of the three-dimensional vascular network includes the following steps: (1) The vascular endothelial cells of claim 4 are mixed with fibrinogen and matrix gel to form a cell-gel mixture; (2) The cell-gel mixture is mixed with a solution containing thrombin and aprotinin to obtain a cell-gel-enzyme mixture, which is then coagulated to form a fibrin gel containing vascular endothelial cells. (3) The fibrin gel was cultured using an induction medium containing vascular endothelial growth factor, fibroblast growth factor and neuropeptide Y receptor agonist to induce vascular endothelial cells to self assemble in the gel to form a three-dimensional vascular network.

6. The method for in vitro construction of a three-dimensional vascular network according to claim 5, characterized in that, The final concentration of fibrinogen in the cell-gel mixture is 3-4 mg / mL; Preferably, the volume percentage of matrix gel in the cell-gel mixture is 5-15%; Preferably, the final concentration of thrombin in the cell-gel-enzyme mixture is 5-10 U / mL, and the final concentration of aprotinin in the cell-gel-enzyme mixture is 1-3 U / mL. Preferably, the vascular endothelial growth factor includes VEGF-165; Preferably, the fibroblast growth factor includes FGF2; Preferably, the neuropeptide Y receptor agonist comprises NPY; Preferably, the final concentration of VEGF-165 in the induction medium is 50-200 ng / mL, the final concentration of FGF2 in the induction medium is 50-150 ng / mL, and the final concentration of NPY in the induction medium is 0-2 µM.

7. A kit for the in vitro construction method of the three-dimensional vascular network of claim 5 or 6, characterized in that, The kit includes: a first culture reagent, a first induction reagent, a second induction reagent, and a purification reagent; The first culture reagent includes: a ROCK signaling pathway inhibitor; The first inducing agent includes: a Wnt signaling pathway activator and / or a BMP signaling pathway activator; The second inducing agent includes: vascular endothelial growth factor and / or cAMP signaling pathway agonists; The purification reagent includes: magnetic bead-conjugated antibodies.

8. The kit for in vitro construction of three-dimensional vascular networks according to claim 7, characterized in that, The kit also includes any one or a combination of at least two of the following reagents: (1) Matrix adhesive; (2) Any one or a combination of at least two of the following: vascular endothelial growth factor, fibroblast growth factor, or neuropeptide Y receptor agonist; (3) Any one or a combination of at least two of fibrinogen, thrombin or aprotinin.

9. The use of the vascular endothelial cells of claim 4 in the preparation of products for vascular tissue engineering, disease model construction, or screening of angiogenesis drugs.

10. The application of the three-dimensional vascular network constructed by the method of claim 5 or 6 in vascular biology research, construction of angiogenesis-related disease models, or screening of vasoactive drugs.

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