Method for constructing vascular organoid by using human pluripotent stem cells and application thereof

Abstract

The embodiment of the invention provides a method for constructing a vascular organoid by using human pluripotent stem cells and application of the vascular organoid, and relates to the technical field of medicine. The method for constructing the vascular organoid by using the human pluripotent stem cells comprises the following steps: comprehensively analyzing the quality of hPSCs (human pluripotent stem cells) for differentiating the vascular organoid through morphological observation, RT-qPCR (real-time quantitative polymerase chain reaction) and AP (access point) staining experiments; repeating a differentiation experiment to test the stability of the optimized vascular organoid construction scheme; identifying the vascular organs constructed by the optimization scheme through RT-qPCR (Reverse Transcription-Quantitative Polymerase Chain Reaction) and integral and slice immunofluorescence experiments; the adriamycin is randomly divided into a DMSO (dimethylsulfoxide) control group, a 0.5 mu M group and a 5 mu M group, and the influence of the adriamycin on human vascular organs is analyzed from the transcriptional level through an RT-qPCR experiment. Compared with a traditional vascular organ construction method, the construction method provided by the invention uses fewer reagents and consumables, and the process is more simplified; the vascular organ construction method is feasible and effective, and stable human vascular organs similar to human microvascular cell components and structures are constructed.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to a method for constructing vascular organoids using human pluripotent stem cells and its application. Background Art

[0002] In related technologies, organoids refer to complex 3D multicellular structures similar to organs or original tissues produced by three-dimensional (3D) culture of pluripotent stem cells (including embryonic stem cells and induced pluripotent stem cells), adult stem cells or tumor cells in vitro. Organoids have similar spatial organizations to corresponding organs and can reproduce some of the functions of corresponding organs, thus providing a highly physiologically relevant system, which is a research method at the human tissue level. Researchers have established many organoid culture systems, such as organoids of intestines, livers, lungs, pancreatic islets, kidneys, brains, hearts and various types of tumors; and blood vessel organoids (Blood vessel organoids, BVO) were first reported by Wimmer et al. in 2019. This study showed that under the action of various growth factors and small molecules, hPSCs differentiated into self-organized human vascular organoids composed of endothelial cells and pericytes and containing basement membrane structures;

[0003] In short, in order to form vascular organoids, pluripotent stem cells first spontaneously form spherical aggregates in ultra-low attachment 6-well plates, followed by mesodermal differentiation and vascular lineage induction. When these aggregates after two-stage induction are encapsulated in a mixed matrix of type I collagen and matrigel, they form a vascular network and self-organize into spherical vascular organoids. These vascular organoids are similar to human microvascular structures and can be cultured in single suspension in ultra-low attachment 96-well plates, which will facilitate downstream high-throughput screening;

[0004] In addition, drug-induced vascular toxicity is a common problem that has long troubled drug developers, healthcare professionals, and patients themselves. In preclinical and clinical drug studies, potential vascular toxicity may pose a serious safety threat to patients.

[0005] Doxorubicin (DOX) is an effective antineoplastic drug used to treat various types of cancer. However, its use is limited by the development of cardiotoxicity, which can lead to heart failure;

[0006] However, the above-mentioned scheme for inducing vascular organoids uses a large number of expensive reagents and is complicated to operate. The construction of vascular organoids using this scheme has high requirements for the laboratory. In addition, researchers have been using old schemes to construct vascular organoids. So far, no new schemes for constructing vascular organoids have emerged. So far, studies have shown the effects of doxorubicin on the endothelial system, but there have been no studies on the effects of doxorubicin on human vascular organoids. Currently, research in the field of vascular organoids is still relatively scarce, and there is still much room for improvement in the scheme for constructing human vascular organoids. Summary of the invention

[0007] The present application aims to solve at least one of the technical problems existing in the prior art. To this end, the present application proposes a method for constructing vascular organoids using human pluripotent stem cells and its application, wherein the method for constructing vascular organoids using human pluripotent stem cells has the characteristics of simple operation, low cost and low use requirements.

[0008] In a first aspect, an embodiment of the present invention provides a method for constructing vascular organoids using human pluripotent stem cells, comprising the following steps:

[0009] S1. Comprehensively analyze the quality of hPSCs used for differentiation of vascular organoids through morphological observation, RT-qPCR and AP staining experiments;

[0010] S2. Independently repeat differentiation experiments using ESCs and induced iPSCs to test the stability of the optimized vascular organoid construction scheme;

[0011] S3. The vascular organoids constructed by the optimized scheme were identified by RT-qPCR and whole-body and section immunofluorescence experiments;

[0012] S4. The vascular organoids constructed from three independent differentiation experiments were randomly divided into DMSO control group, 0.5μM group and 5μM group, and the effect of doxorubicin on human vascular organoids was analyzed at the transcriptional level by RT-qPCR experiment;

[0013] S5. The vascular organoids constructed from three independent differentiation experiments were divided into oxidized low-density lipoprotein blank control group, 0.1μM group, 0.2μM group, and 0.3μM group. The deposition of lipids in organoid cells was detected by Oil red staining and Nile red staining. RT-qPCR experiments were used to analyze the effect of oxidized low-density lipoprotein on human vascular organoids at the transcriptional level.

[0014] Advantages of the method for constructing vascular organoids using human pluripotent stem cells according to the embodiment of the present application:

[0015] In order to better popularize the technology of constructing vascular organoids and promote the development of vascular disease research, a lot of optimizations were made to the scheme proposed by Wimmer et al., and a new scheme for constructing human vascular organoids directly in a 96-well U-bottom ultra-low adsorption culture plate was proposed; this scheme eliminates some unnecessary reagents and consumables, and reduces the workload of personnel by modifying the operating procedures; using the optimized scheme, experimenters can spend less time and money in ordinary laboratories and easily construct human vascular organoids.

[0016] Secondly, for the first time, doxorubicin was used to treat vascular organoids to study the effects of doxorubicin on vascular organoids. Low-density lipoprotein was used to treat vascular organoids to study the effects of high fat on vascular organoids. Given the higher physiological relevance of vascular organoids, this construction method not only supplements clinical research data and improves data credibility, but also verifies the ability of vascular organoids constructed with the optimized scheme to correctly respond to injury. In a sense, this also verifies the functionality of vascular organoids.

[0017] Since vascular organoids differentiated from hPSCs are human, have similar structures and functions to human microvessels, and can be cultured in large quantities in vitro, vascular organoids have unique advantages in studying vascular toxicity and modeling certain diseases;

[0018] The optimized protocol does not require dissection, which reduces the workload. It can also flexibly adjust the number of organoids to be cultured according to one's own experimental needs, avoiding the problem of rapid reagent consumption caused by culturing many organoids at a time. In terms of reagent consumption, studies have found that hPSCs can also be well aggregated into spheres in U-bottom ultra-low adsorption 96-well plates when cultured in mTeSR1. Therefore, this protocol uses mTeSR1, which is necessary for culturing hPSCs, to replace the aggregation culture medium in the original protocol. In addition, the hPSCs passaging reagent ReLeSR is used to digest cells to replace the EDTA and Accutase in the original protocol, reducing the purchase of unnecessary experimental reagents;

[0019] 1) Compared with traditional vascular organoid construction methods, the proposed construction method uses fewer reagents and consumables, and the process is simpler;

[0020] 2) The method for constructing vascular organoids is feasible and effective, and can construct stable human vascular organoids in vitro that are similar in composition and structure to human microvascular cells;

[0021] 3) By treating vascular organoids with doxorubicin for 48 hours, it was found that after doxorubicin treatment, the expression levels of the proinflammatory factor IL-1β and the mesenchymal cell marker gene SMA were upregulated in a concentration-dependent manner, the expression of the endothelial cell marker gene CD31 was downregulated, and the pericytes in the vascular organoids transformed into mesenchymal cells; this is information that is difficult to capture in the study of endothelial cells alone; this proves that vascular organoids can be used to study the mechanism of vascular damage and repair by chemotherapeutic drugs.

[0022] 4) Vascular organoids were treated with low-density lipoprotein for 72 hours, and it was found that after low-density lipoprotein treatment, the expression levels of risk factors for atherosclerosis (PCSK, APOE genes) detected by qPCR were upregulated and concentration-dependent. This proves that the lipid injury model based on vascular organoids can provide an effective means for the study of the mechanism and treatment of atherosclerosis.

[0023] In addition, the method for constructing vascular organoids using human pluripotent stem cells according to the embodiment of the present application also has the following additional technical features:

[0024] In a preferred embodiment of the present invention, the steps of AP staining of hPSCs are as follows:

[0025] 1) Aspirate the medium of hPSCs cultured on the cell culture plate and wash three times with DPBS;

[0026] 2) Add 4% PFA to fix the cells for 20 minutes;

[0027] 3) Aspirate PFA and wash three times with DPBS;

[0028] 4) Prepare BCIP / NBT staining working solution according to the instructions of the AP staining kit;

[0029] 5) Remove the washing solution and add BCIP / NBT staining working solution to fully cover the cell sample;

[0030] 6) Incubate at room temperature in the dark for more than five minutes until the color develops to the expected depth;

[0031] 7) Remove the BCIP / NBT staining solution, wash twice with DPBS to terminate the color development reaction, add new DPBS to cover the cells, observe and take photos for record.

[0032] In a preferred embodiment of the present invention, in the immunofluorescence experiment shown, vascular organoids cultured for 10 to 20 days were selected for fixation, and immunofluorescence experiments were performed on the whole vascular organoids and slices.

[0033] In a preferred embodiment of the present invention, the whole staining steps of vascular organoids are as follows:

[0034] 1) Fixation: Transfer the vascular organoids to a 1.5 mL centrifuge tube, add 1 mL of 4% PFA, fix at room temperature for 2 hours, and remove PFA;

[0035] 2) Add 1 mL of permeabilization blocking solution to each tube and place on a shaker at room temperature for 2 hours;

[0036] 3) Remove the permeabilization blocking solution, add 100 μL of primary antibody diluted with permeabilization blocking solution to fully cover the sample, and shake at 4°C overnight;

[0037] 4) Add 1 mL of PBST to each tube and wash the samples three times on a shaker;

[0038] 5) Add 100 μL of fluorescent secondary antibody diluted with membrane permeation blocking solution to fully cover the sample and incubate at room temperature in the dark;

[0039] 6) Wash twice with PBST;

[0040] 7) Add DAPI dilution and incubate in the dark for 15 minutes;

[0041] 8) Wash twice with PBS.

[0042] In a preferred embodiment of the present invention, in the whole-body staining step of the vascular organoids, the vascular organoids are transferred to a 1.5 mL centrifuge tube, the culture medium is removed, the tubes are washed three times with PBS, 1 mL of 4% PFA is added, the tubes are fixed at room temperature for 2 hours, the PFA is removed, and the tubes are washed three times with PBS.

[0043] In a preferred embodiment of the present invention, in the whole staining step of the vascular organoids, PBST contains a PBS solution with 0.05% Tween20. In the whole staining step of the vascular organoids, step 6) washes with PBST twice, and the operation is protected from light; the time for one PBST wash is 15 minutes.

[0044] In a preferred embodiment of the present invention, the steps of staining the slices of vascular organoids are as follows:

[0045] 1) Fixation: Transfer the vascular organoids to a 1.5 mL centrifuge tube, add 1 mL of 4% PFA, fix at room temperature for 2 hours, and remove PFA;

[0046] 2) Add 1 mL of permeabilization blocking solution to each tube and place on a shaker at room temperature for 2 hours;

[0047] 2) Embedding: Prepare 3% agarose solution with sterile PBS, melt in a microwave oven and place in a 70°C metal bath for preservation; quickly add agarose solution to resuspend the organoids; insert the sample on ice for 10 minutes to fully solidify the agarose; then soak the sample in 70% ethanol overnight;

[0048] 3) Dehydration and transparency: Place the sample in a tissue embedding box, add ethanol of a certain concentration multiple times, and increase the concentration of ethanol added next time;

[0049] 4) Add paraffin wax to the paraffin embedding machine to embed the sample and place it in a freezer to solidify;

[0050] 5) Slice preparation: slice the sample using a microtome to a thickness of 5 μm, spread the slices in a water bath and attach the slices; the prepared white slices can be placed in a slice box and stored at room temperature;

[0051] 6) Dewaxing and rehydration: bake the slides at 60°C for 1 hour, and sequentially perform the following procedures: xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, and PBS for 1 minute;

[0052] 7) Antigen retrieval: Place the sections soaked in citrate buffer in a pressure cooker, heat for 20 minutes, cool to room temperature, and wash twice with PBS, 1 minute each time;

[0053] 8) Draw circles with a tissue pen, add 50 μL of membrane blocking solution, and place in a wet box at room temperature for 2 hours;

[0054] 9) Remove the permeabilization blocking solution, add an appropriate amount of primary antibody diluted with the permeabilization blocking solution to fully cover the sample, and place it in a humidified box at 4°C overnight;

[0055] 10) Place the slices in a histochemical box and place on a shaker, and wash three times with PBST, 15 minutes each time;

[0056] 11) Add an appropriate amount of fluorescent secondary antibody diluted with permeable blocking solution to fully cover the sample, and incubate in a wet box at room temperature in the dark for 2 hours;

[0057] 12) Wash twice with PBST for 15 minutes each time, add DAPI dilution solution, and incubate for 15 minutes in the dark;

[0058] 13) Wash twice with PBS, 15 minutes each time;

[0059] 14) After adding anti-fluorescence quenching sealing agent, seal the slides, dry the slides, and observe and take pictures.

[0060] In a preferred embodiment of the present invention, in the section staining step of the vascular organoids, the vascular organoids are transferred to a 1.5 mL centrifuge tube, the culture medium is removed, the tubes are washed three times with PBS, 1 mL of 4% PFA is added, the tubes are fixed at room temperature for 2 hours, the PFA is removed, and the tubes are washed three times with PBS.

[0061] In a preferred embodiment of the present invention, in the section staining step of the vascular organoid, the proportions and times of addition in step 3 are: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol I for 0.5 hours, anhydrous ethanol II for 0.5 hours, xylene I for 10 minutes, xylene II for 10 minutes, 65°C wax I for 1 hour, and 65°C wax II for 1 hour.

[0062] In a second aspect, an embodiment of the present invention further provides an application of constructing vascular organoids using human pluripotent stem cells, including an application of vascular organoids constructed by any of the above-mentioned methods of constructing vascular organoids using human pluripotent stem cells.

[0063] Additional aspects and advantages of the present application will be given in part in the description below, and in part will become apparent from the description below, or will be learned through the practice of the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0065] Figure 1 It is based on the identification of hPSCs in this application;

[0066] a. Cell colony morphology of H9-ESCs (scale bar: 200 μm);

[0067] b. Cell colony morphology of iPSCs (scale bar: 200 μm);

[0068] c. Expression of stemness genes in iPSCs (**** indicates P < 0.0001);

[0069] Figure 2 It is based on the AP staining results of hPSCs in this application;

[0070] a. Comparison of iPSCs before (left) and after (right) AP staining;

[0071] b. Comparison of H9-ESCs before (left) and after (right) AP staining; (Scale bar: 100 μm);

[0072] Figure 3 is a vascular organoid construction process according to the optimized scheme in this application;

[0073] Figure 4It is a stage bright field image of ES-BVO and iPS-BVO according to the present application (scale bar: 100 μm);

[0074] Figure 5 It is based on the mature BVO bright field image in this application;

[0075] a. Bright field image of ES-BVO on day 20 (scale bar: 200 μm);

[0076] b. Bright field image of iPS-BVO on day 20 (high content imaging, scale bar: 500 μm);

[0077] Figure 6 It is based on the gene expression of iPS-BVO on day 15 in this application (**** indicates P < 0.0001);

[0078] Figure 7 It is based on the overall staining of vascular organoids in this application; CD31+ endothelial cells and CNN1+ parietal cells are shown in red, PDGFRβ+ pericytes are shown in green, and blue fluorescence represents DAPI; scale bars: a, b and c (right) are 200μm, c (left) is 500μm;

[0079] Figure 8 It is based on the section staining of vascular organoids in this application; the figure shows the staining results of iPS-BVO sections on the 15th day of differentiation, in which CD31+ endothelial cells are shown in red, PDGFRβ+ pericytes are shown in green, and blue fluorescence represents DAPI; scale: a, b (left) are 500μm, b (right) is 200μm;

[0080] Fig. 9 are representative bright field images of ES-BVO treated with DOX according to this application (scale bar: 200 μm);

[0081] Fig.10 The results are based on the RT-qPCR results of DOX-treated ES-BVO in this application; the experiment was repeated three times independently, and the significance is: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001, and ns indicates no statistically significant difference;

[0082] Fig.11 are representative bright field images of ES-BVO treated with oxidized LDL according to this application (scale bar: 200 μm);

[0083] Fig.12The results are based on the RT-qPCR results of ES-BVO treated with oxidized low-density lipoprotein in this application; the experiment was repeated three times independently, and the significance is: ** indicates P<0.01, **** indicates P<0.0001, and ns indicates no statistically significant difference. DETAILED DESCRIPTION

[0084] The technical solutions in the embodiments of the present application will be described below in conjunction with the drawings in the embodiments of the present application.

[0085] In order to make the purpose, technical solutions and advantages of the implementation methods of this application clearer, the technical solutions in the implementation methods of this application will be clearly and completely described below in conjunction with the drawings in the implementation methods of this application. Obviously, the described implementation methods are part of the implementation methods of this application, not all of the implementation methods. Based on the implementation methods in this application, all other implementation methods obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0086] The following describes the method and application of constructing vascular organoids using human pluripotent stem cells according to an embodiment of the present application with reference to the accompanying drawings;

[0087] like Figure 1-12 As shown, according to an embodiment of the present application, a method for constructing vascular organoids using human pluripotent stem cells comprises the following steps:

[0088] S1. Comprehensively analyze the quality of hPSCs used for differentiation of vascular organoids through morphological observation, RT-qPCR and AP staining experiments;

[0089] S2. Improve the traditional vascular organoid construction scheme and use ESCs and induced iPSCs to conduct independent repeated differentiation experiments to test the stability of the optimized vascular organoid construction scheme;

[0090] S3. The vascular organoids constructed by the optimized scheme were identified by RT-qPCR and whole-body and section immunofluorescence experiments;

[0091] S4. The vascular organoids constructed from three independent differentiation experiments were randomly divided into DMSO control group, 0.5μM group and 5μM group, and the effect of doxorubicin on human vascular organoids was analyzed at the transcriptional level by RT-qPCR experiment.

[0092] S5. The vascular organoids constructed from three independent differentiation experiments were randomly divided into oxidized low-density lipoprotein blank control group, 0.1μM group, 0.2μM group, and 0.3μM group. The deposition of lipids in organoid cells was detected by Oil red staining and Nile red staining. RT-qPCR experiments were used to analyze the effect of oxidized low-density lipoprotein on human vascular organoids at the transcriptional level.

[0093] Maintenance and culture of hPSCs (ESCs, iPSCs)

[0094] 1. Preparation of mTeSR1 complete medium:

[0095] 1) The day before preparation, thaw the supplement at 4°C overnight; do not thaw in a 37°C water bath.

[0096] 2) Add 100 mL of the additive to 400 mL of basal medium and shake to mix;

[0097] 3) Dispense the mixed complete medium into 50 mL centrifuge tubes and mark the tubes;

[0098] 4) After affixing the sealing film, freeze the centrifuge tube at -20°C and thaw it at room temperature or 4°C overnight before next use.

[0099] 2. Matrigel coated 6-well plate:

[0100] 1) Thaw Matrigel on ice;

[0101] 2) Dilute Matrigel with DMEM / F12 or PBS at a ratio of 1:200 to prepare coating solution;

[0102] 3) Add 1 mL of coating solution to each well of a 6-well cell culture plate, gently shake the plate to evenly cover the bottom of the well with the coating solution, and store it in an incubator at 37°C for at least 15 minutes before use. Store the remaining prepared coating solution in a refrigerator at 4°C.

[0103] 3. Recovery of hPSCs:

[0104] 1) Prepare the Matrigel-coated 6-well plate and required centrifuge tubes in advance, and place the required reagents at room temperature for later use;

[0105] 2) Gently shake the cryovial in a 37°C water bath to quickly thaw the cells;

[0106] 3) Spray the cryotube with 70% ethanol and wipe it dry with absorbent paper;

[0107] 4) Use a 1000 μL pipette tip to gently transfer the cell suspension to a 15 mL centrifuge tube, add 5 mL of mTeSR1 complete medium dropwise, and gently pipette up and down to mix the cell suspension;

[0108] 5) Centrifuge at 300 g for 4-5 minutes at room temperature;

[0109] 6) Remove the supernatant, add 1 mL of mTeSR1 complete medium, and gently pipette up and down to resuspend the cells;

[0110] 7) Take out the coated 6-well plate from the incubator, remove the coating solution, and add 1 mL of mTeSR1 complete medium to each well;

[0111] 8) Add 0.5 mL of cell suspension to each well of a 6-well plate. The volume can be adjusted appropriately according to the cell quantity. Gently shake the plate up and down and left and right to evenly distribute the cells in the plate.

[0112] 9) Place the culture plate in the cell culture incubator and do not touch the plate within 12 hours to avoid disturbing cell adhesion;

[0113] 10) Use mTeSR1 complete medium to replace the medium every day at a volume of 1.5 mL per well of a 6-well plate and monitor cell growth.

[0114] 4. Passaging of hPSCs:

[0115] 1) Prepare the Matrigel-coated 6-well plate and required centrifuge tubes in advance, and place the required reagents at room temperature for later use;

[0116] 2) Aspirate the culture medium, add 1 mL of DPBS to each well of the 6-well plate to wash the cells, and aspirate;

[0117] 3) Add 0.5-1 mL of ReLeSR to each well and aspirate it after about 1 minute to expose the cells to a thin layer of liquid;

[0118] 4) Place the cell culture plate in a cell culture incubator and incubate at 37°C for 4-5 minutes; the incubation time may vary depending on the cell line;

[0119] 5) Take out the cell culture plate and add 1 mL of mTeSR1 complete medium to each well;

[0120] 6) Hold the cell culture plate tightly with one hand and tap the plate on one side with the other hand for about 30 seconds to separate the small cell aggregates formed after digestion from the bottom of the plate;

[0121] 7) Transfer the separated cell aggregates to a 15 mL centrifuge tube and use a 1000 μL pipette tip to gently pipette up and down 1-2 times to break up any larger cell aggregates that may exist; after digestion, the ideal aggregate diameter is about 50-100 μm.

[0122] 8) Adjust the subculture ratio according to the required density and transfer the cell aggregates to a cell culture plate pre-coated with mTeSR1 complete medium;

[0123] 9) Gently shake the well plate up and down and left and right to evenly distribute the cells in the well plate and place it in a cell culture incubator;

[0124] 10) Use mTeSR1 complete medium to replace the medium every day at a volume of 1.5 mL per well of a 6-well plate and monitor cell growth.

[0125] 5. Cryopreservation of hPSCs:

[0126] 1) Prepare the required centrifuge tubes and cryotubes in advance, and place the required reagents at room temperature for later use;

[0127] 2) Aspirate the culture medium, add 1 mL of DPBS to each well of the 6-well plate to wash the cells, and aspirate;

[0128] 3) Add 0.5-1 mL of ReLeSR to each well and aspirate it after about 1 minute to expose the cells to a thin layer of liquid;

[0129] 4) Place the cell culture plate in a cell culture incubator and incubate at 37°C for 4-5 minutes;

[0130] 5) Add 1 mL of mTeSR1 complete medium to each well, hold the cell culture plate tightly with one hand, and tap the culture plate on one side with the other hand for about 30 seconds;

[0131] 6) Collect all separated cell aggregates into a 15 mL centrifuge tube and gently pipette up and down 1-2 times using a 1000 μL pipette tip;

[0132] 7) Centrifuge at 300 g for 5 minutes at room temperature and remove the supernatant;

[0133] 8) Add an appropriate amount of mTeSR1 complete medium containing 10% DMSO to resuspend the cells according to the cell amount and cryopreservation quantity, and add the cell suspension to the marked cryopreservation tubes according to the volume of 1 mL of cell suspension per cryopreservation tube;

[0134] 9) Place the cryovials into a programmed cooling box and transfer them to a -80°C freezer overnight. Then transfer the cells to a liquid nitrogen tank for storage.

[0135] Alkaline phosphatase staining (AP staining) of hPSCs

[0136] 1) Aspirate the medium of hPSCs cultured on the cell culture plate and wash with DPBS three times, 3 minutes each time;

[0137] 2) Add 4% PFA to fix the cells for 20 minutes;

[0138] 3) Aspirate PFA and wash with DPBS three times, 3 minutes each time;

[0139] 4) Prepare BCIP / NBT staining working solution according to the instructions of AP staining kit (Biyuntian, Cat. No.: C3206);

[0140] 5) Remove the last wash solution and add an appropriate amount of BCIP / NBT staining working solution to fully cover the cell sample;

[0141] 6) Incubate at room temperature in the dark for 5-30 minutes or longer until the color develops to the desired depth;

[0142] 7) Remove the BCIP / NBT staining solution, wash twice with DPBS to terminate the color development reaction, add new DPBS to cover the cells, observe and take photos;

[0143] hPSCs-derived vascular organoid construction protocol

[0144] Note that freshly thawed hPSCs must be passaged at least once before they can be used for differentiation or cryopreservation. During operation, the liquid should be gently aspirated or added along the side wall of the culture plate, and the culture medium and other reagents used should be placed in the clean bench to room temperature in advance.

[0145] 1. Preparation of main reagents:

[0146] Table 4: Formula of Mesoderm Induction Medium M1

[0147]

[0148] Table 5: Formulation of Vascular Induction Medium M2

[0149]

[0150]

[0151] Table 6: Formula of type I collagen solution (taking preparation of 2 mL as an example, placed on ice)

[0152]

[0153] Note: After removing PureCol, the remaining additives can be prepared in proportion at one time and stored in a 4℃ refrigerator for later use.

[0154] Table 7: Formulation of Vascular Organoid Maturation Medium M3

[0155]

[0156] 2. Operation process:

[0157] 1) On DAY-2, remove the cell culture medium of hPSCs cultured in 6-well plates, add 1 mL of DPBS to each well to wash the cells for at least 10 seconds, and remove the DPBS;

[0158] 2) Add 1 mL of ReLeSR to each well and incubate in an incubator at 37°C for 5-7 minutes; the digestion time can be appropriately extended to fully digest and dissociate the cells.

[0159] 3) Add 1 mL of mTeSR1 complete medium containing 10 μM Y-27632 to each well and gently pipette the cells to detach them to prepare a single-cell suspension;

[0160] 4) Transfer the single cell suspension to a 15 mL centrifuge tube and centrifuge at 300 g for 3-5 minutes;

[0161] 5) Discard the supernatant, add 1 mL of new mTeSR1 complete medium containing 10 μM Y-27632, and resuspend the cells;

[0162] 6) Cell counting;

[0163] 7) Add an appropriate amount of mTeSR1 complete medium containing 10 μM Y-27632 according to the counting results to dilute the cells to 100,000 cells / mL;

[0164] 8) Add 50 μL of cell suspension to each well of a U-bottom ultra-low attachment 96-well plate, and then add 100 μL of mTeSR1 complete medium containing 10 μM Y-27632 to make each well have 5,000 cells;

[0165] 9) Place the U-bottom ultra-low adsorption 96-well plate in an incubator and culture for 1 day;

[0166] 10) On DAY-1, remove 100 μL of medium from each well and add 100 μL of fresh mTeSR1 complete medium (without Y-27632) to make each well contain 150 μL of medium;

[0167] 11) Place the culture plate in an incubator and continue culturing for 1 day;

[0168] 12) On DAY0, aspirate the culture medium, leaving a small amount of liquid to avoid touching the cell balls at the bottom, and carefully add 200 μL of mesoderm induction medium M1 to each well for mesoderm induction for 3 days;

[0169] 13) On DAY3, aspirate the culture medium, leaving a small amount of liquid to avoid touching the cell balls at the bottom, carefully add 200 μL of vascular induction medium M2 to each well, and culture for 2 days to initiate vascular fate;

[0170] 14) On DAY5, calculate the required volume of type I collagen and matrix gel mixed solution and prepare it according to the corresponding ratio;

[0171] Type I collagen: matrix gel = 3:1, each cell ball requires approximately 60-70 μL of mixed matrix solution.

[0172] 15) Take a new U-bottom ultra-low adsorption 96-well plate, add 30 μL of type I collagen and matrix gel mixed solution to each well, and place it in an incubator for 1 hour to solidify the mixed matrix solution;

[0173] 16) Use a 1000 μL pipette with the tip cut off to transfer the cell aggregates in the original 96-well plate to each well of the new 96-well plate that has been solidified, and try to reduce excess liquid to avoid interfering with subsequent solidification. Add 40 μL of a mixed solution of type I collagen and matrix gel to each well to cover the cell aggregates and place in an incubator for 1 hour to solidify.

[0174] 17) After solidification, add about 180 μL of vascular organoid maturation medium M3 to each well, change the medium every 2 days, and place in an incubator to continue culturing and growing to produce self-organized vascular organoids.

[0175] RT-qPCR experiments

[0176] 1. RNA extraction from vascular organoids:

[0177] 1) Sample processing: Collect 7-10 vascular organoids into a 1.5 mL centrifuge tube, remove excess culture medium, add 1 mL of DPBS and wash repeatedly 2-3 times, then add 1 mL of fresh DPBS, centrifuge at 300 g for 3 minutes, remove the supernatant, and place the sample on ice;

[0178] 2) Preparation: Use ethanol to spray the desktop, gloves, pipettes, etc. Prepare chloroform, isopropanol, and 75% ethanol in advance according to the number of samples. If you have a grinder, clean it with 75% ethanol and ddH2O in turn before use, and wipe it clean with absorbent paper; Chloroform and isopropanol need to be protected from light, and 75% ethanol is prepared with RNAfreeddH2O;

[0179] 3) Add 500 μL of TRIZOL to each tube of the processed vascular organoids for lysis, and choose: a. Vortex for 30 seconds, centrifuge briefly, and keep on ice for 30 seconds, repeat 4-5 times; b. Grind thoroughly on ice with a grinder;

[0180] 4) Lyse on ice for 5 minutes;

[0181] 5) Centrifuge at 12,000 g for 5 min at 4°C;

[0182] 6) Use a pipette to transfer about 480 μL of the supernatant to a new 1.5 mL RNase-free centrifuge tube, add 100 μL of chloroform, vortex for 15-30 seconds, and incubate at room temperature for 3-4 minutes;

[0183] 7) Centrifuge at 12,000 g for 15 min at 4°C;

[0184] 8) After centrifugation, the liquid will be divided into 3 layers. Use a pipette to draw the top layer of liquid (RNA layer, do not draw the middle protein layer) into a new 1.5mL RNase-free centrifuge tube, add an equal volume of isopropanol, vortex to mix, centrifuge briefly, and let stand in a -20℃ refrigerator for 20 minutes;

[0185] 9) Centrifuge at 12,000 g for 10 min at 4°C;

[0186] 10) Discard the supernatant, add 1 mL of 75% ethanol, and vortex for 30 seconds;

[0187] 11) Centrifuge at 4°C, 12000g for 5 minutes. A white precipitate can be seen at the bottom of the tube after centrifugation;

[0188] 12) Remove as much ethanol as possible, open the centrifuge tube lid and let it sit at room temperature for 10 minutes. (You can extend the time to allow the ethanol to evaporate completely);

[0189] 13) Add 20 μL RNase-free H2O to dissolve the white precipitate, mix well, and measure the concentration.

[0190] 2. Reversal:

[0191] In this study, the NanoDrop ND-2000 instrument was used to analyze the concentration and quality of the obtained RNA. Then, according to the reverse transcription system and process provided by the GoScript reverse transcription kit (Promega, A5000), 1 μg of the total RNA was reversed into cDNA, and the cDNA was diluted 20 times using ddH2O. The obtained samples were labeled and stored in a -20°C refrigerator.

[0192] 3. qPCR experiment:

[0193] The primers in this study were synthesized by Qingke Biotechnology Co., Ltd. GAPDH was selected as the internal reference gene. The primer sequences used are shown in Table 8. 2xSYBR Green qPCR mastermix (Bimake, B21202) was used for real-time fluorescence quantitative PCR experiments, and the reaction system is shown in Table 9. Then we used a Real-time PCR instrument (ThermoFisher) to observe the melting curves of all primers and export the results in Excel tables. The 2-ΔΔCT method was used to calculate the relative expression levels of mRNA or genes in each group. According to the data processing results, Graphpadprism8 was used to draw the result chart.

[0194] Table 8: Primer sequences

[0195]

[0196]

[0197] Table 9: qPCR reaction system

[0198]

[0199]

[0200] Immunofluorescence (IF)

[0201] In order to detect the protein expression of different cell markers in vascular organoids and observe the structure of vascular organoids, this protocol selected vascular organoids cultured for 15 days for fixation, and performed immunofluorescence experiments on the whole vascular organoids and sections; the primary antibodies used are shown in Table 10, and primary antibodies from different species are used for combined staining, and then fluorescently labeled secondary antibodies are used to bind to the primary antibodies that have been bound to the antigen to achieve the purpose of detecting the antigen. In addition, unlike traditional immunofluorescence experiments, this protocol uses permeabilization and blocking at the same time. The preparation of permeabilization and blocking solutions is shown in Table 11.

[0202] Table 10: List of primary antibodies

[0203]

[0204] Table 11: Permeabilization blocking solution formula (taking 50 mL as an example)

[0205]

[0206] Note: Filter through a 0.2 μm filter and store in a 4°C refrigerator.

[0207] 1. Overall staining protocol for vascular organoids:

[0208] 1) Fixation: Use a 1000 μL pipette with the tip cut off to transfer the vascular organoids to a 1.5 mL centrifuge tube (be careful not to damage the organoid structure), remove the culture medium, wash three times with PBS, add 1 mL of 4% PFA, fix at room temperature for 2 hours, remove PFA, and wash three times with PBS, each time for about 10 minutes;

[0209] 2) Add 1 mL of permeabilization blocking solution to each tube and place on a shaker at room temperature for 2 hours;

[0210] 3) Remove the permeabilization blocking solution, add 100 μL of primary antibody diluted with permeabilization blocking solution to fully cover the sample, and shake at 4°C overnight;

[0211] 4) Add 1 mL of PBST to each tube and wash the sample on a shaker for 3 times, 15 minutes each time; wherein PBST contains 0.05% Tween20 in PBS solution.

[0212] 5) Add 100 μL of fluorescent secondary antibody diluted with membrane blocking solution to fully cover the sample and incubate at room temperature in the dark for 2 hours;

[0213] 6) Wash twice with PBST, protecting from light during the operation, each time for 15 minutes;

[0214] 7) Add DAPI dilution and incubate in the dark for 15 minutes;

[0215] 8) Wash twice with PBS, 15 minutes each time;

[0216] 9) Sealing: Use a 1000 μL pipette with the tip cut off to transfer the vascular organoids to a glass slide, paint a circle around it with nail polish to properly raise the cover glass, add anti-fluorescence quenching sealing medium (Yisheng Bio, Catalog No.: 36307ES25), and then cover with a cover glass to seal;

[0217] 10)Confocal observation and photography.

[0218] 2. Staining protocol for vascular organoid sections:

[0219] 1) Fixation: Same as the whole-mount staining protocol described above;

[0220] 2) Embedding: Prepare 3% agarose solution with sterile PBS, melt it in a microwave oven and keep it warm in a 70°C metal bath; briefly preheat the sample and pipette tip, quickly add the agarose solution to resuspend the organoids, try to ensure that the organoids are suspended in the middle of the agarose and avoid introducing bubbles; insert the sample on ice for 10 minutes to fully solidify the agarose; then soak the sample in 70% ethanol overnight;

[0221] 3) Dehydration and transparency: Place the sample in a tissue embedding box and sequentially enter the following procedures: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol I for 0.5 hour, anhydrous ethanol II for 0.5 hour, xylene I for 10 minutes, xylene II for 10 minutes, 65°C wax I for 1 hour, 65°C wax II for 1 hour;

[0222] 4) Add paraffin wax to the paraffin embedding machine to embed the sample and place it in a freezer to solidify;

[0223] 5) Slice preparation: slice the sample using a microtome to a thickness of 5 μm, spread the slices in a water bath and attach the slices; the prepared white slices can be placed in a slice box and stored at room temperature;

[0224] 6) Dewaxing and rehydration: bake the slides at 60°C for 1 hour, and sequentially perform the following procedures: xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, and PBS for 1 minute;

[0225] 7) Antigen retrieval: Place the sections soaked in citrate buffer in a pressure cooker at a temperature above 95°C or close to boiling point for 20 minutes, cool to room temperature, and wash twice with PBS for 1 minute each time;

[0226] 8) Draw circles with a tissue pen, add 50 μL of membrane blocking solution, and place in a wet box at room temperature for 2 hours;

[0227] 9) Remove the permeabilization blocking solution, add an appropriate amount of primary antibody diluted with the permeabilization blocking solution to fully cover the sample, and place it in a humidified box at 4°C overnight;

[0228] 10) Place the slices in a histochemical box and place on a shaker, and wash three times with PBST, 15 minutes each time;

[0229] 11) Add an appropriate amount of fluorescent secondary antibody diluted with permeable blocking solution to fully cover the sample, and incubate in a wet box at room temperature in the dark for 2 hours;

[0230] 12) Wash twice with PBST for 15 minutes each time, add DAPI dilution solution, and incubate for 15 minutes in the dark;

[0231] 13) Wash twice with PBS, 15 minutes each time;

[0232] 14) After adding anti-fluorescence quenching sealing agent, seal and dry the slides, and use Confocal to observe and take pictures.

[0233] In view of the potential differences between organoids constructed from different batches, organoids constructed from the same batch were randomly grouped in each experiment. All data were from three independent differentiation experiments. Graphpadprism8 was used to perform homogeneity of variance test and one-way ANOVA, and the results were plotted. P<0.05 was considered statistically significant.

[0234] Morphological and functional analysis of hPSCs (ESCs, iPSCs)

[0235] Obviously, the key to successfully constructing vascular organoids differentiated from hPSCs in vitro lies in the quality of the hPSCs used before differentiation. Therefore, before the construction of vascular organoids, we first conducted a simple analysis of the hPSCs used in terms of morphology and function to ensure that subsequent experiments can be carried out smoothly. In order to ensure the high quality of each generation of cells, maintaining and expanding ESCs and induced iPSCs requires a suitable culture system and skilled operation techniques. The ESCs and iPSCs in this protocol are cultured in mTeSR1 complete medium produced by Stemcell, and the cells are digested using the passaging reagent ReLeSR. In addition, this protocol uses DMEM / F12 culture medium to dilute the growth factor-reduced matrix gel (Matrigel) to prepare the coating solution. After being added to the cell culture plate and placed in the cell culture incubator for a period of time, Matrigel will solidify at the bottom of the culture plate wells, providing an attachment surface for ESCs and iPSCs to promote cell adhesion and growth. Considering that ESCs and iPSCs are very sensitive to external stimuli and are very likely to undergo spontaneous differentiation during the culture process, all reagents used in cell operations in this protocol were placed in a clean bench in advance to restore to room temperature, and when changing the liquid, the liquid was gently added or removed along the side wall of the culture well to reduce stimulation to the cells.

[0236] The study found that the best passaging effect was achieved when the digested ESCs and iPSCs formed cell aggregates with a diameter of about 50-100 μm. In terms of morphology, the ESCs and iPSCs we cultured formed compact multicellular colonies at the bottom of the culture plate wells with clear boundaries ( Figure 1 a. Figure 1 b). The cells were tightly packed, and no irregular cell morphology areas or other cell types were observed, indicating that the cells cultured in this protocol were morphologically healthy and in line with expectations. Subsequent RT-qPCR experiments confirmed that the mRNA levels of OCT4 and SOX2 in iPSCs were significantly increased compared with those in induced differentiated vascular organoids (BVO) ( Figure 1 c, P<0.0001). OCT4 and SOX2 are two stemness genes that are often used to identify hPSCs, which indicates that the hPSCs in this protocol have stemness. In addition, alkaline phosphatase staining experiments (AP staining) of iPSCs and ESCs were also performed. Although alkaline phosphatase is not a specific marker for hPSCs and is expressed in many types of cells, hPSCs strongly express alkaline phosphatase and are therefore often used to identify iPSCs that have been successfully reprogrammed. After AP staining, the iPSCs and ESCs in this protocol appear dark purple-blue, as shown in Figure 2. Figure 2 This is consistent with our expectations. We noticed that spontaneous differentiation areas appeared around the iPSCs colonies, such as Figure 2As shown in a, the coloring is significantly lighter than that of normal cells, which is because the spontaneously differentiated cells have lost their identity as iPSCs.

[0237] In summary, the hPSCs used in this protocol are normal in morphology and function and can be used for subsequent BVO differentiation experiments.

[0238] Optimization of traditional vascular organoid construction protocols

[0239] The construction scheme of vascular organoids was first reported by Wimmer et al., and subsequent studies on vascular organoids have also used this scheme. Based on the established two-dimensional (2D) induction scheme and three-dimensional (3D) co-culture assay, they developed a scheme to differentiate multiple vascular cell types from common mesodermal precursor cells without the need to independently induce and purify multiple homogenous vascular cell populations. The scheme first digests iPSCs into single cells, then inoculates a certain number of iPSCs in a low-adhesion 6-well cell culture plate. These stem cells will spontaneously aggregate into many small spheres, and then under the activation of the Wnt pathway and the action of BMP-4 (bone morphogenetic protein 4), the cells enter the mesodermal lineage. By adding VEGF-A (vascular endothelial growth factor A) and Forskolin (adenylate cyclase activator), both parietal cells and endothelial cells can be differentiated from mesodermal precursor cells. After the induction of vascular fate, these cell spheroids did not undergo much morphological change. In order to induce vascular sprouting, they were embedded in a double-layer extracellular matrix (ECM) composed of a mixture of type I collagen and Matrigel. With the support of ECM, these spheroids will self-organize into vascular organoids containing endothelial cells and pericytes and containing basement membrane structures.

[0240] In the study, it was found that there were many areas that could be optimized in this original vascular organoid construction scheme. A major improvement made in this scheme is to directly inoculate the same number of hPSCs in each well of a U-bottom ultra-low attachment 96-well plate to obtain a single existing stem cell sphere for differentiation ( Figure 3). Considering that the starting point of inducing differentiation is to first obtain embryoid bodies (EBs), which are essentially stem cell spheres composed of hPSCs, this protocol attempts to use a U-bottom ultra-low adsorption 96-well plate to complete the process of hPSCs aggregation into spheres, and successfully obtains EBs that can be used for subsequent differentiation. This approach will bring many conveniences: 1. We do not need to purchase ultra-low adsorption 6-well plates and tissue culture treatment 12-well plates required by the original protocol, reducing the use of funds. 2. Since our cell spheres are grown individually in each well of the U-bottom ultra-low adsorption 96-well plate, this avoids the problem of easy fusion between multiple cell spheres. In the original protocol, since the cell spheres are suspended in the low-adsorption 6-well plate, adjacent cell spheres are easy to fuse, so resuspension work is often required. The optimized protocol does not require this operation, which is more time-saving and labor-saving. Another improvement made in this protocol is to embed the cell spheres directly in the U-bottom ultra-low adsorption 96-well plate. The original scheme was to embed the cell spheroids in a mixed gel in a 12-well plate, which inevitably used a large amount of type I collagen and matrix gel at one time, which are very expensive reagents. In order to obtain vascular organoids that can be stored individually in a U-bottom ultra-low adsorption 96-well plate, it is necessary to peel each vascular organoid from the gel under a dissecting microscope, which is an extremely time-consuming and labor-intensive task and will increase the risk of contamination. The optimized scheme does not require dissection, which reduces the workload, and can flexibly adjust the number of organoids required for culture according to its own experimental needs, avoiding the problem of excessive reagent consumption caused by culturing many organoids at a time. In terms of reagent consumption, studies have found that hPSCs can also be well aggregated into spheres in a U-bottom ultra-low adsorption 96-well plate under the culture of mTeSR1. Therefore, this scheme uses mTeSR1, which is necessary for the culture of hPSCs, to replace the aggregation medium in the original scheme. In addition, the hPSCs subculture reagent ReLeSR is used to digest cells to replace the EDTA and Accutase in the original scheme, reducing the purchase of unnecessary experimental reagents (Table 12).

[0241] In summary, compared with the traditional vascular organoid construction scheme, our optimized scheme is more flexible, simple and economical.

[0242] Table 12: Examples of non-essential reagents, consumables, and instruments

[0243]

[0244] Characterization of vascular organoids constructed using the optimized protocol

[0245] In order to ensure the stability of the optimized vascular organoid construction scheme, two types of hPSCs, H9-ESCs and iPSCs, were used to perform vascular organoid differentiation experiments, and a large number of repetitions were performed. The experiments showed that our differentiation scheme can stably use hPSCs to obtain human vascular organoids.

[0246] First, this protocol records the bright field images of the process from hPSCs differentiation to vascular organoids. It was found that when the digested hPSCs were inoculated into a U-bottom ultra-low attachment 96-well plate and cultured in mTeSR1 complete medium, the hPSCs could spontaneously aggregate into embryoid bodies (EBs) at the bottom of the culture plate without centrifugation ( Figure 4 ). The size of the initial differentiation EB will affect subsequent experiments. If the EB is too small, it is difficult to find it with the naked eye, which is not conducive to the subsequent replacement of the medium and embedding of the gel. If the EB is too large, the cells in the center of the cell spheroid will be more prone to hypoxia and necrosis, which will affect the subsequent differentiation efficiency. Our experiments found that when the number of hPSCs seeded in each well was about 5,000, the experimental effect was ideal. Figure 4 As shown, after several experiments, we found that ESCs seemed to be more susceptible to apoptosis than iPSCs, so that more cell fragments were formed around the cell spheroids. In the early stage of induction, the size changes of the cell spheroids formed by iPSCs were slightly larger. About 2 days after the cell spheroids were embedded in a mixed matrix composed of type I collagen and matrigel, that is, around DAY7, it can be clearly observed that burr-like structures were generated around the cell spheroids, which is a signal of vascular sprouting. On DAY13, the vascular network structure can be clearly observed. It is worth noting that at this time, the vascular organoids (iPS-BVO) differentiated from iPSCs have begun to form clear boundaries, which is consistent with the results of the original protocol reported by Wimmer et al. Compared with the vascular organoids (ES-BVO) differentiated from ESCs, the diameter of mature iPS-BVO is larger. Therefore, in order to capture the full picture, this protocol uses high-content imaging of mature iPS-BVO ( Figure 5 ).

[0247] Secondly, this protocol used RT-qPCR experiments to detect the expression of various cell marker genes in iPS-BVO on day 15 ( Figure 6). Among them, OCT4 is a stemness gene. Once entering the induction stage, iPSCs will lose their stemness identity. Therefore, compared with iPSCs, the expression of OCT4 in iPS-BVO was significantly downregulated (P<0.0001). The expression of endothelial cell marker genes CD31, vWF and VE-Cadherin was significantly upregulated (P<0.0001), suggesting the presence of endothelial cells in iPS-BVO. The expression of typical pericyte marker genes SMA, NG2, RGS-5, and PDGFRβ was significantly upregulated (P<0.0001), suggesting the presence of pericytes in iPS-BVO. Among them, SMA is a mural cell marker gene, that is, it can be expressed by both pericytes and vascular smooth muscle cells, and we observed that the expression of vascular smooth muscle cell maturation marker genes MYH11 and SMMHC was upregulated (P<0.0001), suggesting that iPS-BVO may also contain vascular smooth muscle cells.

[0248] In summary, our optimized vascular organoid construction scheme is stable and can construct vascular organoids in vitro with similar cell components and structures to human microvascular cells.

[0249] Effects of DOX on vascular organoids

[0250] This protocol selected ES-BVO on the 20th day of differentiation for subsequent DOX treatment experiments. In view of the potential differences between ES-BVO constructed in different batches, this protocol conducted three independent differentiation experiments. In each experiment, ES-BVO constructed in the same batch were selected and randomly divided into DMSO group, 0.5μM group and 5μM group, 10 in each group, and treated with DOX for 48 hours. Because DMSO used to dissolve DOX is cytotoxic and may affect the experimental results, this protocol uses DMSO as the control group, and prepares the corresponding DOX concentration gradient mother solution in advance to ensure that the same volume of DMSO is added to the culture medium. It was found that after 48 hours of DOX treatment, the morphology of ES-BVO did not seem to change significantly ( Fig. 9 ). RT-qPCR results showed that under the action of DOX, compared with the DMSO control group, the expression levels of the pro-inflammatory factor IL-1β and the mesenchymal cell marker gene SMA in ES-BVO were upregulated in a concentration-dependent manner ( Fig.10). The expression of endothelial cell marker gene CD31 was also downregulated under the action of DOX. This suggests that DOX may cause inflammatory response and endothelial-mesenchymal transition (EndMT) in ES-BVO. In previous studies on endothelial cells, it was found that IL-1β can trigger endothelial-mesenchymal transition, causing endothelial cells to lose endothelial characteristics and transdifferentiate into mesenchymal cells (SMA+), which may also be the mechanism by which DOX causes EndMT in ES-BVO. It is worth noting that DOX treatment also caused the downregulation of the expression of pericyte marker gene PDGFRβ in ES-BVO. Previous studies have shown that pericytes are the source of mesenchymal stem cell precursors in multiple organs in the body to respond to damage. Our results suggest that pericytes in ES-BVO may also transdifferentiate into mesenchymal cells under the action of DOX, which is information that is difficult to capture in simple endothelial cell studies, and also shows the advantages of vascular organoids for the study of certain diseases.

[0251] Effects of oxidized low-density lipoprotein on vascular organoids

[0252] This protocol selected ES-BVO on the 20th day of differentiation for subsequent oxidized low-density lipoprotein treatment experiments. In view of the potential differences between ES-BVO constructed in different batches, we conducted three independent differentiation experiments. In each experiment, ES-BVO constructed in the same batch were randomly divided into a blank group, a 0.1μM group, a 0.2μM group, and a 0.3μM group, with 10 cells in each group, and treated with oxidized low-density lipoprotein for 72 hours. We found that after 72 hours of treatment with oxidized low-density lipoprotein, the morphology of ES-BVO did not seem to change significantly ( Fig.11 ). RT-qPCR results showed that under the action of oxidized low-density lipoprotein, the expression levels of PCSK and APOE genes, risk factors for atherosclerosis, were upregulated in ES-BVO compared with the blank control group ( Fig.10 ), suggesting that the treatment of oxidized low-density lipoprotein may cause changes in lipid levels in organoids, confirming that a high-fat environment is prone to induce atherosclerosis. The expression levels of scavenger receptor SR-A4, inflammatory factors such as IL-1, IL-6, TNF-α, MCP-1, and CRP are also downregulated under the action of oxidized low-density lipoprotein. This suggests that oxidized low-density lipoprotein may cause an inflammatory response in ES-BVO. Previous studies have found that lipid deposition caused by inflammatory response may be an important cause of the occurrence and development of atherosclerosis.

[0253] Specifically, this construction method mainly uses hPSCs as seed cells for differentiation experiments, and uses these cells to construct human vascular organoids through staged induced differentiation. hPSCs include two types of cells, namely ESCs and induced iPSCs, and ESCs and iPSCs each contain different cell lines. The embryonic stem cells we used were H9-ESCs, which came from the organoid platform of the School of Life Sciences of Fudan University, and iPSCs were purchased from Shanghai Jihe Biotechnology Co., Ltd. (Cat. No.: JH-H1681).

[0254] Main reagents, consumables and instruments

[0255] Table 1: Main reagents

[0256]

[0257]

[0258] NOTE: This table contains only the reagents required to construct vascular organoids.

[0259] Table 2: Main consumables

[0260]

[0261] NOTE: This table only contains the consumables required to construct vascular organoids.

[0262] Table 3: Main instruments

[0263]

[0264]

[0265] NOTE: This table contains only the instruments required to construct vascular organoids.

[0266] In a second aspect, an embodiment of the present invention further provides an application of constructing vascular organoids using human pluripotent stem cells, including an application of vascular organoids constructed by any of the above-mentioned methods of constructing vascular organoids using human pluripotent stem cells.

[0267] Among them, hPSCs are human pluripotent stem cells, ESCs are embryonic stem cells, iPSCs are multipotent stem cells, H9-ESCs are H9 embryonic stem cells; PDGFRβ is platelet-derived growth factor receptor β;

[0268] BVO differentiation experiment is a differentiation experiment of specific cells or tissues, the purpose of which is to study the process and mechanism of cell or tissue differentiation in a specific direction under specific conditions;

[0269] Stem cells are a type of cells with the potential for self-renewal and multidirectional differentiation;

[0270] mTeSR1 complete medium is a medium designed specifically for the culture of human pluripotent stem cells (such as embryonic stem cells and induced pluripotent stem cells);

[0271] Matrigel coating is a commonly used technique in cell culture and experiments;

[0272] DMEM / F-12 is a widely used basal medium for the growth of many different mammalian cells, including MDCK, glial cells, fibroblasts, human endothelial cells, and rat fibroblasts;

[0273] DPBS is a phosphate-buffered saline solution used to remove impurities and residual substances; maintain the osmotic pressure and pH balance of cells;

[0274] ReLeSR is a reagent used in cell culture to promote a specific state or function of cells, or play a key role in a certain step of cell processing;

[0275] DMSO (dimethyl sulfoxide) is a sulfur-containing organic compound with excellent solvent properties: it can dissolve many organic and inorganic compounds;

[0276] PFA (paraformaldehyde) is a commonly used chemical reagent in biological and medical research;

[0277] BCIP / NBT is a chromogenic substrate combination commonly used in biochemistry and molecular biology experiments;

[0278] BCIP (5-bromo-4-chloro-3-indolylphosphate) and NBT (nitro blue tetrazolium chloride) are often used together;

[0279] DAY-0" may mean the day the experiment starts, DAY-1" may mean the first day after the experiment starts, DAY-2" may mean the second day after the experiment starts, and so on;

[0280] MY-27632 is a compound commonly used in cell biology research;

[0281] TRIZOL lysis is a commonly used sample processing method in molecular biology experiments; TRIZOL reagent can effectively lyse cells and inhibit the activity of RNase to extract high-quality RNA, DNA or protein;

[0282] RNasefree refers to a state or condition without ribonuclease (RNase) contamination. Dissolving with RNasefree H2O can prevent RNase from degrading possible RNA components.

[0283] NanoDropND-2000 is a micro-volume spectrophotometer commonly used in biochemistry and molecular biology research;

[0284] The GoScript reverse transcription kit (Promega, A5000) is a tool commonly used in molecular biology experiments;

[0285] ddH2O is double distilled water, which is high-purity water obtained by distillation twice;

[0286] 2xSYBRGreenqPCRmastermix (Bimake, B21202) is a premix for real-time fluorescence quantitative PCR (qPCR) experiments;

[0287] ThermoFisher is a scientific instrument manufacturer. Its Real-time PCR instruments (real-time fluorescence quantitative PCR instruments) are widely used in molecular biology, medicine, biotechnology and other fields.

[0288] ThermoFisher is a scientific instrument manufacturer. Its Real-time PCR instruments (real-time fluorescence quantitative PCR instruments) are widely used in molecular biology, medicine, biotechnology and other fields.

[0289] GraphPadPrism8 is a powerful and widely used software for scientific research and data analysis;

[0290] PBS (Phosphate Buffered Saline) is a buffer solution commonly used in biological and medical experiments;

[0291] DAPI (4',6-diamidino-2-phenylindole) is a fluorescent dye commonly used in biological and cell biology research;

[0292] Confocal usually refers to confocal microscopy; confocal microscopy is a microscopy technique with high resolution and high definition;

[0293] Wnt is a class of secreted glycoproteins that play a key role in various physiological processes such as development, cell proliferation, differentiation and cell fate determination.

[0294] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of changes or substitutions within the technical scope disclosed in the present application, which should be included in the protection scope of the present application. Therefore, the protection scope of the present application should be based on the protection scope of the claims.

Claims

1. A method for constructing vascular organoids using human pluripotent stem cells, characterized in that: The steps include: S1. Comprehensively analyze the quality of hPSCs used for differentiation of vascular organoids through morphological observation, RT-qPCR and AP staining experiments; S2. Independently repeat differentiation experiments using ESCs and induced iPSCs to test the stability of the optimized vascular organoid construction scheme; S3. The vascular organoids constructed by the optimized scheme were identified by RT-qPCR and whole-body and section immunofluorescence experiments; S4. The vascular organoids constructed from three independent differentiation experiments were randomly divided into DMSO control group, 0.5μM group and 5μM group, and the effect of doxorubicin on human vascular organoids was analyzed at the transcriptional level by RT-qPCR experiment; S5. The vascular organoids constructed from three independent differentiation experiments were divided into oxidized low-density lipoprotein blank control group, 0.1μM group, 0.2μM group, and 0.3μM group. The deposition of lipids in organoid cells was detected by Oil red staining and Nile red staining. RT-qPCR experiments were used to analyze the effect of oxidized low-density lipoprotein on human vascular organoids at the transcriptional level.

2. A method for constructing vascular organoids using human pluripotent stem cells according to claim 1, characterized in that: The steps of AP staining of hPSCs are as follows; 1) Aspirate the medium of hPSCs cultured on the cell culture plate and wash three times with DPBS; 2) Add 4% PFA to fix the cells for 20 minutes; 3) Aspirate PFA and wash three times with DPBS; 4) Prepare BCIP / NBT staining working solution according to the instructions of the AP staining kit; 5) Remove the washing solution and add BCIP / NBT staining working solution to fully cover the cell sample; 6) Incubate at room temperature in the dark for more than five minutes until the color develops to the expected depth; 7) Remove the BCIP / NBT staining solution, wash twice with DPBS to terminate the color development reaction, add new DPBS to cover the cells, observe and take photos for record.

3. The method for constructing vascular organoids using human pluripotent stem cells according to claim 1, characterized in that: In the immunofluorescence experiment shown, vascular organoids cultured for 10 to 20 days were fixed, and immunofluorescence experiments were performed on the whole vascular organoids and sections.

4. A method for constructing vascular organoids using human pluripotent stem cells according to claim 3, characterized in that: The steps for whole-mount staining of vascular organoids are as follows: 1) Fixation: Transfer the vascular organoids to a 1.5 mL centrifuge tube, add 1 mL of 4% PFA, fix at room temperature for 2 hours, and remove PFA; 2) Add 1 mL of permeabilization blocking solution to each tube and place on a shaker at room temperature for 2 hours; 3) Remove the permeabilization blocking solution, add 100 μL of primary antibody diluted with permeabilization blocking solution to fully cover the sample, and shake at 4°C overnight; 4) Add 1 mL of PBST to each tube and wash the samples three times on a shaker; 5) Add 100 μL of fluorescent secondary antibody diluted with membrane permeation blocking solution to fully cover the sample and incubate at room temperature in the dark; 6) Wash twice with PBST; 7) Add DAPI dilution and incubate in the dark for 15 minutes; 8) Wash twice with PBS.

5. A method for constructing vascular organoids using human pluripotent stem cells according to claim 4, characterized in that: In the whole-body staining step of the vascular organoids, the vascular organoids were transferred to a 1.5 mL centrifuge tube, the culture medium was removed, the tubes were washed 3 times with PBS, 1 mL of 4% PFA was added, the tubes were fixed at room temperature for 2 hours, the PFA was removed, and the tubes were washed 3 times with PBS.

6. A method for constructing vascular organoids using human pluripotent stem cells according to claim 5, characterized in that: In the whole staining step of the vascular organoids, PBST contains a PBS solution with 0.05% Tween20. In the whole staining step of the vascular organoids, step 6) washes with PBST twice, and the operation is protected from light; the time for one PBST wash is 15 minutes.

7. The method for constructing vascular organoids using human pluripotent stem cells according to claim 3, characterized in that: The steps for staining sections of vascular organoids are as follows: 1) Fixation: Transfer the vascular organoids to a 1.5 mL centrifuge tube, add 1 mL of 4% PFA, fix at room temperature for 2 hours, and remove PFA; 2) Add 1 mL of permeabilization blocking solution to each tube and place on a shaker at room temperature for 2 hours; 2) Embedding: Prepare 3% agarose solution with sterile PBS, melt in a microwave oven and place in a 70°C metal bath for preservation; quickly add agarose solution to resuspend the organoids; insert the sample on ice for 10 minutes to fully solidify the agarose; then soak the sample in 70% ethanol overnight; 3) Dehydration and transparency: Place the sample in a tissue embedding box, add ethanol of a certain concentration multiple times, and increase the concentration of ethanol added next time; 4) Add paraffin wax to the paraffin embedding machine to embed the sample and place it in a freezer to solidify; 5) Slice preparation: slice the sample using a microtome to a thickness of 5 μm, spread the slices in a water bath and attach the slices; the prepared white slices can be placed in a slice box and stored at room temperature; 6) Dewaxing and rehydration: bake the slides at 60°C for 1 hour, and sequentially perform the following procedures: xylene I for 10 minutes, xylene II for 10 minutes, anhydrous ethanol I for 3 minutes, anhydrous ethanol II for 3 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, and PBS for 1 minute; 7) Antigen retrieval: Place the sections soaked in citrate buffer in a pressure cooker, heat for 20 minutes, cool to room temperature, and wash twice with PBS, 1 minute each time; 8) Draw circles with a tissue pen, add 50 μL of membrane blocking solution, and place in a wet box at room temperature for 2 hours; 9) Remove the permeabilization blocking solution, add an appropriate amount of primary antibody diluted with the permeabilization blocking solution to fully cover the sample, and place it in a humidified box at 4°C overnight; 10) Place the slices in a histochemical box and place on a shaker, and wash three times with PBST, 15 minutes each time; 11) Add an appropriate amount of fluorescent secondary antibody diluted with permeable blocking solution to fully cover the sample, and incubate in a wet box at room temperature in the dark for 2 hours; 12) Wash twice with PBST for 15 minutes each time, add DAPI dilution solution, and incubate for 15 minutes in the dark; 13) Wash twice with PBS, 15 minutes each time; 14) After adding anti-fluorescence quenching mounting medium, seal the slides, dry the slides, and observe and take pictures.

8. The method for constructing vascular organoids using human pluripotent stem cells according to claim 7, characterized in that: In the section staining step of the vascular organoids, the vascular organoids were transferred to a 1.5 mL centrifuge tube, the culture medium was removed, the tubes were washed 3 times with PBS, 1 mL of 4% PFA was added, the tubes were fixed at room temperature for 2 hours, the PFA was removed, and the tubes were washed 3 times with PBS.

9. The method for constructing vascular organoids using human pluripotent stem cells according to claim 7, characterized in that: In the section staining step of the vascular organoid, the proportions and times of addition in step 3 are: 75% ethanol for 1 hour, 85% ethanol for 1 hour, 90% ethanol for 1 hour, 95% ethanol for 1 hour, anhydrous ethanol I for 0.5 hours, anhydrous ethanol II for 0.5 hours, xylene I for 10 minutes, xylene II for 10 minutes, 65°C wax I for 1 hour, and 65°C wax II for 1 hour.

10. Use of vascular organoids constructed by the method for constructing vascular organoids using human pluripotent stem cells according to any one of claims 1 to 9.

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