Vein vessel organoid based on human pluripotent stem cell induction and construction method thereof

Abstract

The invention relates to a vein vessel organoid based on human pluripotent stem cell induction and a construction method thereof. The construction method comprises the following steps: S1, culturing human pluripotent stem cells in a pore plate by using an mTESR1 culture medium; s2, preparing the human pluripotent stem cells into a single-cell suspension, and forming uniform cell aggregates in an ultra-low adsorption culture plate; step S3, inducing mesoderm cell spheres, collecting cell aggregates, discarding supernatant, adding into a first culture medium which contains a Glutamax additive, L-ascorbic acid, CHIR99021 and BMP4, culturing for 2-3 days, and inducing to form the mesoderm cell spheres; s4, vein vessel organoid differentiation: adding a second culture medium for culture, promoting mesoderm cells to continue to differentiate to the vessel pedigree, and inducing to form a vein vessel network. According to the invention, the directional differentiation of the human pluripotent stem cells to the vein vessel organoid is realized, and the capillary vessel organoid with vein characteristics is successfully constructed.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid construction, and specifically relates to venous organoids induced by human pluripotent stem cells and a construction method thereof. Background Art

[0002] In recent years, the incidence and burden of vascular diseases in my country have shown a sustained upward trend, encompassing categories such as small vessel disease, microcirculatory disorders, tumor-related vascular abnormalities, diabetic vasculopathy, and immune-mediated vascular disease. These diseases have become the leading cause of death in my country, a dire situation that highlights the urgent need to strengthen research on the pathogenesis of vascular diseases and accelerate the development of innovative diagnostic and treatment technologies. As an essential component of the vascular system, malfunctioning veins can cause deep vein thrombosis, chronic venous disease, and venous malformations, leading to pain, ulcers, tissue hypoxia, and multi-organ complications such as pulmonary embolism and skin abnormalities, severely impacting patients' lives and health and increasing the medical burden. However, due to the lack of precise research models, research on their pathological mechanisms and the development of treatments have been slow.

[0003] Vascular organoid technology, a breakthrough in biomedical engineering, has made significant progress in vascular biology research, but critical technical gaps remain in the specific study of the venous system. Current mainstream vascular organoid models suffer from the following limitations: First, the organoid technology pioneered by Wimmer's team (Nature, 2019, Vol. 565, No. 7740, pp. 505-510) can simulate capillary networks but lacks the ability to autonomously differentiate into arteries and veins, requiring in vivo transplantation to form a functional vascular network. Second, the CTGF regulatory mechanism discovered by Romeo's team based on this model (Nature Communications, 2023, Vol. 14, No. 1, p. 5552) provides new insights into diabetic microangiopathy but cannot specifically mimic the unique pathological features of the venous system. Third, while existing biomaterial construction strategies have made progress in the development of vascular grafts, the lack of a natural vascular differentiation process hinders their application to studying venous development and disease mechanisms. These technical limitations severely hinder the mechanistic research and drug development for venous-specific diseases such as deep vein thrombosis and venous malformations.

[0004] Therefore, establishing a vascular organoid model with clear venous characteristics and achieving the specific differentiation and functional reconstruction of venous endothelial cells and pericytes is a technical problem that needs to be urgently solved by technicians in this field. Summary of the Invention

[0005] The first object of the present invention is to provide a method for constructing venous organoids induced by human pluripotent stem cells to address the problems in the prior art.

[0006] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] The method for constructing venous organoids induced by human pluripotent stem cells includes the following steps:

[0008] Step S1, culturing human pluripotent stem cells, culturing human pluripotent stem cells in a well plate using mTESR1 medium;

[0009] Step S2, cell aggregate formation, preparing the human pluripotent stem cells cultured in step S1 into a single cell suspension and forming uniform cell aggregates in an ultra-low attachment culture plate;

[0010] Step S3, inducing mesodermal cell spheroids, collecting cell aggregates, discarding the supernatant, and adding a first culture medium containing Glutamax additive, L-ascorbic acid, CHIR99021, and BMP4, and culturing for 2-3 days to induce the formation of mesodermal cell spheroids;

[0011] Step S4, venous organoid differentiation, venous organoid differentiation, collagen I and matrix gel are mixed in a ratio of 3:1 to prepare a mixed gel, a portion of the mixed gel is taken to solidify to form a lower layer of gel, another portion of the mixed gel is taken to resuspend the mesodermal cell spheres, and then inoculated on the surface of the lower layer of gel to form an upper layer of gel, after the upper layer of gel solidifies, a second culture medium is added, and the cells are placed in an incubator and cultured for 6-8 days to promote the mesodermal cells to continue to differentiate into the vascular lineage and induce the formation of a venous vascular network, wherein the second culture medium contains: Glutamax additive, L-ascorbic acid, VEGFα, bFGF and 10% fetal bovine serum.

[0012] While adopting the above technical solutions, the present invention may also adopt or combine the following technical solutions:

[0013] As a preferred technical solution of the present invention: the cell confluence of the human pluripotent stem cells cultured in step S1 is 80%-90%.

[0014] As a preferred technical solution of the present invention: in step S2, the human pluripotent stem cells cultured in step S1 were rinsed with PBS, digested with Accutase enzyme and prepared into a single cell suspension, and the cell density was adjusted to 2.0×10 5 -5.0×10 5 The cells were seeded into ultra-low adsorption culture plates and ultra-low adsorption 6-well plates and cultured in mTESR1 medium containing 10 μM Y27632 for 20-24 h to form uniform cell aggregates.

[0015] As a preferred technical solution of the present invention: in step S3, when collecting the cell aggregates, the supernatant is discarded after centrifugation at 500 r / min for 1 min, and the first culture medium is added.

[0016] As a preferred technical solution of the present invention: in step S4, a collagen I solution is first prepared, and then a mixed gel solution is prepared in a ratio of collagen I to matrix gel of 3:1. The lower layer of the mixed gel is first laid in the well plate, and then the well plate is placed in a 37°C incubator for 20-30 minutes to allow it to solidify. After solidification, the cell spheres are resuspended in the mixed gel and spread on top of the lower layer of gel to form an upper layer of gel that wraps the cell spheres. Then, the well plate is placed in a 37°C incubator to allow it to solidify. After the upper layer of gel has also solidified, a second culture medium is added and cultured for 6-8 days to form a venous vascular network.

[0017] As a preferred technical solution of the present invention: the collagen I solution is composed of collagen I stock solution, 10X Ham's F-12 solution and buffer solution, with a composition ratio of 8:1:1. The buffer solution is an ultrapure aqueous solution containing 2.2g NaHCO3, 0.05N NaOH and 200mM HEPES.

[0018] As a preferred technical solution of the present invention: the first culture medium contains: Glutamax additive, 60 μg / mL L-ascorbic acid, 12 μM CHIR99021 and 30 ng / mL BMP4.

[0019] As a preferred technical solution of the present invention: the second culture medium comprises: Glutamax additive, 60 μg / mL L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF and 10% fetal bovine serum.

[0020] The second object of the present invention is to provide a venous organoid derived from the directed differentiation of human pluripotent stem cells to address the problems in the prior art.

[0021] To this end, the above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0022] Venous organoids differentiated from human pluripotent stem cells have a distinct vascular network, including endothelial cells and pericytes. The pericytes cover the endothelial cells, the endothelial cells form an inner lumen, and specifically express venous markers. The vascular diameter is between 15μm and 25μm, and has the characteristics of microvenules in the human body.

[0023] Compared with the existing technology, the venous organoids induced by human pluripotent stem cells and the construction method thereof of the present invention have the following beneficial effects: the present invention achieves efficient directional differentiation of venous organoids through two-stage culture medium induction of mesoderm differentiation and venous blood vessel formation, combined with a three-dimensional culture microenvironment formed by a collagen I / matrigel mixed matrix; the mesodermal pathway is synergistically activated by combining the GSK3 inhibitor CHIR99021 with BMP4 and L-ascorbic acid; endothelial cell proliferation and angiogenesis are promoted by combining VEGFα with bFGF, co-inducing the venous phenotype and forming a network structure, establishing a vascular organoid model with clear venous characteristics, and achieving the specific differentiation and functional reconstruction of venous endothelial cells and pericytes, which has become an urgent need in current vascular biology research and will also provide a revolutionary research platform for precision medicine of venous diseases.

[0024] The venous organoids constructed by the present invention based on human pluripotent stem cell induction are prepared through complex growth factor temporal induction and three-dimensional microenvironment regulation, realizing the directed differentiation of human pluripotent stem cells into venous organoids, and successfully constructing microvascular organoids with venous characteristics, providing a highly biomimetic venous organoid three-dimensional biological model for vascular disease research, and providing an innovative platform for revealing the pathophysiological mechanisms of the venous system, promoting high-throughput screening of targeted drugs, and developing personalized precision intervention plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the process of constructing venous organoids based on human pluripotent stem cells of the present invention;

[0026] Figure 2 This is a morphological observation diagram of the venous organoids at different differentiation times in Example 1;

[0027] Figure 3 This is the immunofluorescence staining result of the venous organoid in Example 1, where CD31 represents vascular endothelial cells and PDGFRβ represents perivascular cells;

[0028] Figure 4 Figures 1 and 2 show the RT-qPCR results, diameter statistics, and immunofluorescence staining of the venous organoids in Example 1. Figure A shows the RT-qPCR results of the vascular organoids, Figure B shows the diameter statistics of the vascular organoids, and Figure C shows the immunofluorescence staining of the vascular organoids.

[0029] Figure 5 Figures 2 and 3 show the immunofluorescence staining results and branch point statistics of vascular organoids after high sugar and high fat stimulation, respectively. Figure A shows the immunofluorescence staining results of vascular organoids after high sugar and high fat stimulation, and Figure B shows the branch point statistics of vascular organoids after high sugar and high fat stimulation.

[0030] Figure 6 Figures 2 and 3 show the RT-qPCR results of oxidative stress-related genes in vascular organoids after high sugar and high fat stimulation; Figure A shows the RT-qPCR results of oxidative stress-related genes in vascular organoids after high sugar stimulation, and Figure B shows the RT-qPCR results of oxidative stress-related genes in vascular organoids after high fat stimulation;

[0031] Figure 7 This is the bright field morphology of the venous organoid in Comparative Example 1. DETAILED DESCRIPTION

[0032] The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.

[0033] like Figure 1 As shown, the method for constructing venous organoids based on human pluripotent stem cells of the present invention comprises the following steps:

[0034] Step S1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium;

[0035] mTESR1 medium is a feeder-free, chemically defined medium specifically designed for the in vitro culture of human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0036] Step S2: When the confluence of the human pluripotent stem cells cultured in Step 1 reaches 80%-90%, rinse the cells twice with PBS, add Accutase enzyme to digest the cells for 5 minutes, add mTESR1 medium to terminate the digestion, collect the cells into a centrifuge tube, centrifuge at 1000 rpm for 3 minutes, and discard the supernatant. Resuspend the cells in cell aggregation medium to obtain a single-cell suspension. The cell density of the suspension is then adjusted. 2.5×105 single cells are plated into ultra-low attachment six-well plates and cultured in cell aggregation medium for 20-24 hours to obtain cell aggregates. The cell aggregation medium is mTESR1 medium containing 10 μM Y27632 inhibitor.

[0037] Among them, PBS is phosphate buffered saline.

[0038] In the present invention, the ultra-low adsorption plate is combined with the ROCK inhibitor Y27632 to reduce apoptosis and promote the formation of uniform aggregates.

[0039] Step S3: Collect cell aggregates, centrifuge at 500 rpm for 1 min, discard the supernatant, and add the first culture medium, which consists of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021, and 30 ng / mL BMP4, and culture for 2-3 days for mesodermal differentiation.

[0040] Among them, Advanced DMEM / F12 is a high-performance cell culture medium optimized for the in vitro culture of primary cells and stem cells.

[0041] Glutamax is a stable L-glutamine substitute, chemically known as L-alanyl-L-glutamine.

[0042] CHIR99021 is a highly potent and selective inhibitor of glycogen synthase kinase-3 (GSK-3).

[0043] BMP4 is bone morphogenetic protein 4.

[0044] Step S4: Prepare collagen I solution first, then prepare a mixed gel solution at a ratio of 3:1 collagen I to matrigel. Layer the lower layer of mixed gel onto the plate. Place the plate in a 37°C incubator for 20-30 minutes to allow it to solidify. Once solidified, resuspend the cell spheroids in the mixed gel and layer them onto the lower layer of gel, forming an upper layer of gel that encapsulates the cell spheroids. Place the plate in a 37°C incubator to allow it to solidify. Once the upper layer of gel has solidified, add the second culture medium and culture for 6-8 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax Supplement, 60 μg / mL L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0045] bFGF refers to basic fibroblast growth factor;

[0046] VEGFα refers to vascular endothelial growth factor alpha.

[0047] In the present invention, the basement membrane in vivo is simulated by using collagen I and matrigel in a ratio of 3:1 to enhance the self-assembly ability of the vascular network.

[0048] Specifically, the collagen I solution is composed of collagen I stock solution, 10X Ham's F-12 solution and buffer solution, with a composition ratio of 8:1:1. The buffer solution is an ultrapure aqueous solution containing 2.2g NaHCO3, 0.05N NaOH and 200mM HEPES, wherein HEPES is 4-hydroxyethylpiperazineethanesulfonic acid.

[0049] The reagents and abbreviations involved in the present invention are as follows:

[0050] 10×Ham's F-12 solution refers to a 10-fold concentrated solution of Ham's F-12 medium, containing nutrients such as amino acids, vitamins, and inorganic salts.

[0051] BMP4: bone morphogenetic protein 4;

[0052] bFGF: basic fibroblast growth factor;

[0053] VEGFα: vascular endothelial growth factor alpha.

[0054] According to the present invention, L-ascorbic acid has a beneficial effect on endothelial growth, survival, and maintenance of vascular responsiveness and integrity. It can also promote the formation of new blood vessels in tumors and induce tumor vascular normalization. In the present invention, the addition of L-ascorbic acid achieves the induced differentiation of vascular organoids.

[0055] Example 1

[0056] 1.1 Culture of human pluripotent stem cells

[0057] Step 1: Place the matrix gel in an ice box and place it in a 4°C refrigerator to melt overnight. Pre-cool the pipette tip and EP tube at -20°C.

[0058] Step 2: Dispense the melted Matrigel in step 1 into EP tubes on ice, 500 μL per tube;

[0059] Step 3: Dilute the Matrigel aliquoted from step 2 in 6 mL of 4°C pre-cooled PBS at a ratio of 1:100 and place on ice until ready for use.

[0060] Step 4: Culture cells in a six-well plate. Place 1 mL of diluted Matrigel in each well of the six-well plate. Rotate the plate to evenly distribute the Matrigel. Then, place the plate at 37°C for 40 minutes. Aspirate and discard the supernatant.

[0061] Step 5: Collect the culture supernatant from step 4, rinse the cells with PBS, add 0.5 mL of Accutase digestion enzyme to each well, and digest for 3-5 minutes; use a P1000 pipette tip to blow the digested cells twice to break up the cell colonies into single cells, add culture supernatant to neutralize the digestion enzyme, and centrifuge at 300 × g at room temperature for 5 minutes;

[0062] Step 6: Resuspend the cells obtained in step 5 in mTESR1 medium containing 10 μM Y27632 inhibitor, and then subculture at 37°C. Change the medium 24 hours after subculture.

[0063] 1.2 Human pluripotent stem cells induced to differentiate into venous organoids:

[0064] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium.

[0065] Step 2: When the confluence of human pluripotent stem cells cultured in step 1 reaches 80%-90%, rinse the cells twice with PBS, add digestive enzyme to digest the cells for 5 minutes, add mTESR1 medium to terminate the digestion, collect the cells into a centrifuge tube, centrifuge at 1000r / min for 3 minutes, and discard the supernatant. Resuspend the cells in cell aggregation medium to obtain a single cell suspension, and then adjust the cell density of the suspension to 2.5×10 5 Single cells were seeded into ultra-low attachment six-well plates and cultured for 24 h in mTESR1 medium containing 10 μM Y27632 inhibitor to obtain cell aggregates.

[0066] Step 3: Collect cell aggregates, centrifuge at 500 rpm for 1 min, discard the supernatant, and add the first culture medium, which consists of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021, and 30 ng / mL BMP4, for 3 days for mesodermal differentiation.

[0067] Step 4: Prepare collagen I solution first, and then prepare mixed gel solution in a ratio of 3:1 between collagen I and matrix gel. First, spread the lower layer of mixed gel in the well plate, and then place the well plate in a 37°C incubator for 20-30 minutes to solidify it. After solidification, resuspend the cell spheres in the mixed gel and spread them on top of the lower layer of gel to form an upper layer of gel that wraps the cell spheres. After the upper layer of gel also solidifies, add the second culture medium and culture for 7 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0068] In this example, an inverted microscope was used to observe the morphology of vascular organoids at various stages of development. Figure 2 As shown, hiPSCs gradually formed embryoid bodies, relatively compact mesodermal cell spheres, and finally formed a clear venous vascular network.

[0069] In this example, vascular organoids were identified by immunofluorescence staining. Figure 3As shown, the vascular organoids contain endothelial cells and pericytes, and can spontaneously assemble to form a vascular network. At the same time, pericytes can be seen covering the outside of the vascular endothelial cells, and there is a vascular lumen between the endothelial cells, indicating that the vascular organoids of the present invention are relatively similar to the blood vessels in the body.

[0070] In this example, vascular organoids were identified by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and the vascular diameter was counted. Figure 4 As shown, the vascular organoids highly expressed the venous markers NRP2, EphB4, and COUP-TFII, while lowly expressed the arterial marker SOX17 and the arteriolar marker DLL4. The average diameter of the vessels in the vascular organoids was approximately 21 μm, similar to that of venules in the human body. Further immunofluorescence staining revealed that the vascular organoids highly expressed the venule marker EphB4, indicating that the vascular organoids constructed in this example are human venous organoids.

[0071] Example 2

[0072] High glucose stimulates vascular organoids:

[0073] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium.

[0074] Step 2: When the confluence of human pluripotent stem cells cultured in step 1 reaches 80%-90%, rinse the cells twice with PBS, add digestive enzyme to digest the cells for 5 minutes, add mTESR1 medium to terminate the digestion, collect the cells into a centrifuge tube, centrifuge at 1000r / min for 3 minutes, and discard the supernatant. Resuspend the cells in cell aggregation medium to obtain a single cell suspension, and then adjust the cell density of the suspension to 2.5×10 5 Single cells were seeded into ultra-low attachment plates and cultured for 24 h in mTESR1 medium containing 10 μM Y27632 inhibitor to obtain cell aggregates.

[0075] Step 3: Collect cell aggregates, centrifuge at 500 rpm for 1 min, discard the supernatant, and add the first culture medium, which consists of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021, and 30 ng / mL BMP4, for 3 days for mesodermal differentiation.

[0076] Step 4: Prepare collagen I solution first, then prepare a mixed gel solution in a ratio of 3:1 between collagen I and matrigel. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay them on top of the lower gel to form an upper gel that wraps the cell spheres. After the upper gel solidifies, add the second culture medium composed of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum and culture for 6 days to form a venous network.

[0077] Step 5: Replace the secondary medium with high-glucose medium 3 for 5 days, changing the medium every 24 hours. Simultaneously, culture the control group with control medium 5 for another 5 days, changing the medium every 24 hours. High-glucose medium 3 consists of Advanced DMEM / F12, 1× Glutamax supplement, 100 ng / mL VEGFα, 100 ng / mL bFGF, 10% fetal bovine serum, and 25 mM D(+) glucose. Control medium 5 consists of Advanced DMEM / F12, 1× Glutamax supplement, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0078] After step 5 high glucose stimulation treatment, the following tests were performed:

[0079] Immunofluorescence staining: Immunofluorescence staining of vascular organoids after high glucose stimulation;

[0080] Vascular branch point statistics: Statistical analysis of the branch point counts of CD31+ vascular networks;

[0081] RT-qPCR: RT-qPCR experiments were performed on vascular organoids after high glucose stimulation.

[0082] In this example, vascular organoids were identified by immunofluorescence staining. Figure 5 As shown, the number of vascular branch points decreased significantly after high glucose stimulation, indicating that the high glucose environment inhibited angiogenesis in vascular organoids.

[0083] In this example, vascular organoids were identified by RT-qPCR. Figure 6 As shown in A, the expression levels of oxidative stress-related genes in vascular organoids increased significantly after high glucose stimulation, indicating that vascular organoids can respond to oxidative stress in a high glucose environment.

[0084] Example 3

[0085] High-fat stimulation of vascular organoids:

[0086] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium.

[0087] Step 2: When the confluence of human pluripotent stem cells cultured in step 1 reaches 80%-90%, rinse the cells twice with PBS, add digestive enzyme to digest the cells for 5 minutes, add mTESR1 medium to terminate the digestion, collect the cells into a centrifuge tube, centrifuge at 1000r / min for 3 minutes, and discard the supernatant. Resuspend the cells in cell aggregation medium to obtain a single cell suspension, and then adjust the cell density of the suspension to 2.5×10 5 Single cells were seeded into ultra-low attachment plates and cultured for 24 h in mTESR1 medium containing 10 μM Y27632 inhibitor to obtain cell aggregates.

[0088] Step 3: Collect cell aggregates, centrifuge at 500 rpm for 1 min, discard the supernatant, and add the first culture medium, which consists of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 12 μM CHIR99021, and 30 ng / mL BMP4, for 3 days for mesodermal differentiation.

[0089] Step 4: Prepare collagen I solution first, then prepare a mixed gel solution in a ratio of 3:1 between collagen I and matrigel. First, lay the lower layer of mixed gel in the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to solidify. After solidification, resuspend the cell spheres in the mixed gel and lay them on top of the lower gel to form an upper gel that wraps the cell spheres. After the upper gel solidifies, add the second culture medium composed of Advanced DMEM / F12, 1× Glutamax supplement, 60 μg / ml L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum and culture for 6 days to form a venous network.

[0090] Step 5: Replace the secondary medium with high-lipid medium 4 for 5 days, changing the medium every 24 hours. Simultaneously, replace the control medium with control medium 5 for another 5 days, changing the medium every 24 hours. High-lipid medium 4 consists of Advanced DMEM / F12, 1× Glutamax supplement, 100 ng / mL VEGFα, 100 ng / mL bFGF, 10% fetal bovine serum, and 0.5 mM palmitic acid. Control medium 5 consists of Advanced DMEM / F12, 1× Glutamax supplement, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0091] After step 5 high-fat stimulation, the following tests were performed:

[0092] Immunofluorescence staining: Immunofluorescence staining of vascular organoids after high-fat stimulation;

[0093] Blood vessel branch point count: CD31 + The number of branch points in the vascular network was statistically analyzed;

[0094] RT-qPCR: RT-qPCR experiments were performed on vascular organoids after high-fat stimulation.

[0095] In this example, vascular organoids were identified by immunofluorescence staining. Figure 5 As shown, the number of vascular branch points was significantly reduced after high-fat stimulation, indicating that the high-fat environment inhibited angiogenesis in vascular organoids.

[0096] In this example, vascular organoids were identified by RT-qPCR. Figure 6 As shown in B, the expression levels of oxidative stress-related genes in vascular organoids increased significantly after high-fat stimulation, indicating that vascular organoids can respond to oxidative stress in a high-fat environment.

[0097] Comparative Example 1

[0098] This comparative example provides a method for preparing vascular organoids, which differs from the method in Example 2 in that L-ascorbic acid is not added to the first culture medium and the second culture medium. The specific operation is as follows:

[0099] Step 1: Culture human pluripotent stem cells in a six-well plate using mTESR1 medium.

[0100] Step 2: When the confluence of the human pluripotent stem cells cultured in step 1 reaches 80%-90%, rinse the cells twice with PBS, add digestive enzymes to digest the cells for 5 minutes, add mTESR1 medium to terminate the digestion, collect the cells into a centrifuge tube, centrifuge at 1000r / min for 3 minutes, and discard the supernatant. Resuspend the cells in cell aggregation medium to obtain a single cell suspension, then adjust the cell density of the suspension, and seed 2.5×105 single cells into ultra-low attachment six-well plates. Culture them in cell aggregation medium for 24 hours to obtain cell aggregates. The cell aggregation medium is mTESR1 medium containing 10 μM Y27632 inhibitor;

[0101] Step 3: Collect cell aggregates, centrifuge at 500 rpm for 1 minute, discard the supernatant, and add the first culture medium, which consists of Advanced DMEM / F12, 1× Glutamax supplement, 12 μM CHIR99021, and 30 ng / mL BMP4, for 3 days for mesodermal differentiation.

[0102] Step 4: Prepare collagen I solution first, then prepare mixed gel solution in a ratio of 3:1 between collagen I and matrix gel. First, spread the lower layer of mixed gel on the well plate, then place the well plate in a 37°C incubator for 20-30 minutes to solidify it. After solidification, resuspend the cell spheres in the mixed gel and spread them on top of the lower layer of gel to form an upper layer of gel that wraps the cell spheres. After the upper layer of gel also solidifies, add the second culture medium and culture for 7 days to form a venous network. The second culture medium consists of Advanced DMEM / F12, 1× Glutamax supplement, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

[0103] In this comparative example, an inverted microscope was used to observe the morphology of vascular organoids. Figure 7 As shown, in this comparative example, without the addition of L-ascorbic acid, vascular organoids were unable to form a vascular branching network, demonstrating that L-ascorbic acid plays an important role in angiogenesis.

[0104] Experimental Example 1

[0105] Immunofluorescence staining to identify vascular organoids:

[0106] Step 1: Use a P1000 pipette to aspirate the culture medium from the well plate and fix the sample in 4% tissue cell fixative for 20 minutes at room temperature. Discard the 4% tissue cell fixative and then add PBS.

[0107] Step 2: Under a microscope, use ultrafine scissors and fine tweezers to cut the gel obtained in step 1 into small pieces, avoiding cutting the vascular network. Use tweezers to transfer the gel fragments to a fresh 2 mL EP tube, add blocking buffer, and permeabilize the vascular network at room temperature for 2 h. For the vascular network, add 2 mL of blocking buffer to each tube and rock tube rack (placed vertically on an orbital shaker) for proper permeabilization.

[0108] Step 3: Remove the blocking buffer and add 50-100 μL of primary antibody diluted in blocking buffer. Incubate the vascular network upright on a shaker at 4°C overnight.

[0109] Step 4: The next day, wash the samples three times in PBS-T solution for 10-15 minutes. For vascular networks, place the tube rack vertically on an orbital shaker for washing.

[0110] Step 5: Add secondary antibody to the samples from step 4 in blocking buffer and incubate at room temperature for 2 hours; wash the samples twice in PBS-T solution for 10-15 minutes; then add DAPI solution (1:1000 in PBS) and incubate for 10-15 minutes, and finally wash the samples in PBS for 10-15 minutes.

[0111] Step 6: Transfer the sample to a glass slide, cover it with a coverslip, and take a photo.

[0112] The immunofluorescence staining markers used in the above method are as follows: CD31 represents vascular endothelial cells, PDGFRβ represents perivascular cells, and EphB4 represents venous endothelial cells.

[0113] Experimental Example 2

[0114] RT-qPCR:

[0115] Step 1: Transfer the cultured vascular organoids to a 15 ml centrifuge tube, add fresh PBS until the tube is filled, and then centrifuge for 5 minutes at 1000 rpm. Discard the supernatant.

[0116] Step 2: Add 1 ml of Trizol and pipette repeatedly to fully lyse the sample;

[0117] Step 3: Add 200 μl of chloroform, shake for 15 seconds, let stand at room temperature for 5 minutes, and centrifuge at 4°C for 15 minutes at 12,000 g.

[0118] Step 4: Transfer the supernatant to a new 1.5 ml centrifuge tube without enzyme, add an equal volume of isopropanol, mix by inversion, let stand at room temperature for 10 min, centrifuge at 4°C for 10 min at 12,000 g, and discard the supernatant.

[0119] Step 5: Slowly add 1 ml of 75% ethanol solution along the wall of the centrifuge tube, invert and wash, centrifuge at 4°C for 5 min at 12,000 g, and discard the supernatant;

[0120] Step 6: After drying at room temperature for 5 minutes, add an appropriate amount of enzyme-free water to fully dissolve the RNA;

[0121] Step 7: Add RNA solution and reverse transcriptase in proportion to prepare a reverse transcription reaction mixture. Incubate in a 37°C water bath for 15 minutes, then in an 85°C water bath for 5 seconds to obtain the sample cDNA solution.

[0122] Step 8: Using the obtained cDNA as a template, add the primers of the target gene, and use a real-time quantitative fluorescence kit and a real-time fluorescence quantitative PCR instrument to detect the expression level of the target gene.

[0123] The above-mentioned specific implementation methods are used to illustrate the present invention and are only preferred embodiments of the present invention, rather than limiting the present invention. Any modifications, equivalent substitutions, improvements, etc. made to the present invention within the spirit of the present invention and the scope of protection of the claims shall fall within the scope of protection of the present invention.

Claims

1. A method for constructing venous organoids induced by human pluripotent stem cells, characterized in that: The following steps are involved: Step S1, culturing human pluripotent stem cells in a well plate using mTESR1 medium; Step S2, cell aggregate formation, preparing the human pluripotent stem cells cultured in step S1 into a single cell suspension and forming uniform cell aggregates in an ultra-low attachment culture plate; Step S3, inducing mesodermal cell spheroids, collecting cell aggregates, discarding the supernatant, and adding a first culture medium containing Glutamax additive, L-ascorbic acid, CHIR99021, and BMP4, and culturing for 2-3 days to induce the formation of mesodermal cell spheroids; Step S4, venous organoid differentiation, collagen I and matrix gel are mixed in a ratio of 3:1 to prepare a mixed gel, a portion of the mixed gel is solidified to form a lower layer of gel, another portion of the mixed gel is taken to resuspend the mesodermal cell spheres, and then inoculated on the surface of the lower layer of gel to form an upper layer of gel. After the upper layer of gel solidifies, a second culture medium is added and placed in an incubator for culture for 6-8 days to promote the continued differentiation of the mesodermal cells into the vascular lineage and induce the formation of a venous vascular network. The second culture medium contains: Glutamax additive, L-ascorbic acid, VEGFα, bFGF and 10% fetal bovine serum.

2. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: The cell confluence of the human pluripotent stem cells cultured in step S1 is 80%-90%.

3. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: In step S2, the human pluripotent stem cells cultured in step S1 were rinsed with PBS, digested with Accutase enzyme, and prepared into a single cell suspension. The cell density was adjusted to 2.0×10 5 -5.0×10 5 The cells were seeded into ultra-low attachment culture plates and ultra-low attachment 6-well plates, and cultured in mTESR1 medium containing 10 μM Y27632 for 20-24 hours to form uniform cell aggregates.

4. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: In step S3, when collecting the cell aggregates, the supernatant was discarded after centrifugation at 500 r / min for 1 min, and the first culture medium was added.

5. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: In step S4, collagen I solution is first prepared, and then a mixed gel solution is prepared in a ratio of 3:1 between collagen I and matrix gel. The lower layer of mixed gel is first spread on the well plate, and then the well plate is placed in a 37°C incubator for 20-30 minutes to solidify it. After solidification, the cell spheres are resuspended in the mixed gel and spread on top of the lower layer of gel to form an upper layer of gel that wraps the cell spheres. Then, the well plate is placed in a 37°C incubator to solidify it. After the upper layer of gel has also solidified, the second culture medium is added and cultured for 6-8 days to form a venous network.

6. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: Collagen I solution is composed of collagen I stock solution, 10× Ham's F-12 solution and buffer solution, with a composition ratio of 8:1:

1. The buffer solution contains 2.2g NaHCO 3、 Ultrapure solution of 0.05N NaOH and 200mM HEPES in water.

7. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: The first culture medium contained Glutamax supplement, 60 μg / mL L-ascorbic acid, 12 μM CHIR99021, and 30 ng / mL BMP4.

8. The method for constructing venous organoids derived from human pluripotent stem cells according to claim 1, wherein: The second culture medium contains Glutamax supplement, 60 μg / mL L-ascorbic acid, 100 ng / mL VEGFα, 100 ng / mL bFGF, and 10% fetal bovine serum.

9. Human pluripotent stem cell-derived venous organoids, characterized by: The venous organoids are prepared by the method for constructing venous organoids induced by human pluripotent stem cells as described in any one of claims 1 to 8. The venous organoids have a clear vascular network, vascular endothelial cells and pericytes, the pericytes cover the endothelial cells, the endothelial cells form an inner lumen, and specifically express venous markers. The vascular diameter is between 15μm and 25μm, and has the characteristics of microvenules in the human body.

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