A co-differentiated vascularized pancreatic islet organoid and its construction method

Abstract

The present invention belongs to the field of organoid culture technology, and specifically relates to a co-differentiated vascularized pancreatic islet organoid and a method for constructing the same. In view of the problems that the existing pancreatic islet organoid vascularization mostly adopts the co-culture method, which has a long operation process, is difficult, and has randomness and poor uniformity, the present invention provides a method for constructing a co-differentiated vascularized pancreatic islet organoid, which adopts human induced pluripotent stem cells hiPSCs, and sequentially induces and cultures them into directed endoderm cells, primitive intestinal tube cells and posterior foregut cells. In the subsequent induced differentiation, a culture medium containing VEGF, BMP4 and FGF2 is added for culture to obtain co-differentiated vascularized pancreatic islet organoid. For the first time, the present invention directs the differentiation of iPS cells to produce vascularized pancreatic islet organoids containing β, α, δ cells and vascular endothelial cells without adding exogenous endothelial cells. It is more mature than non-vascularized pancreatic islet organoids and has a stronger insulin secretion function, providing new ideas and experimental basis for achieving functional cure of diabetes.

Description

Technical Field

[0001] The present invention belongs to the technical field of organoid culture, and particularly relates to a co-differentiated vascularized pancreatic islet organoid and a construction method thereof. Background Art

[0002] Pancreatic islets are considered to be highly vascularized miniature organs due to their endocrine properties. A rich vascular network is a prerequisite for ensuring oxygen transport and material exchange in the islets. In addition to low heterogeneity, the currently established pancreatic organoid culture system also faces the major challenge of poor vascularization. The degree of vascularization is an important factor affecting the survival and function of pancreatic organoids. Although hPSCs can derive pancreatic organoids with morphology and function similar to native pancreatic islets, their vascularization is obviously insufficient. Due to the lack of blood circulation, nutrients and oxygen have difficulty reaching the core area, and metabolic waste continues to increase. In the later stages of culture and after transplantation into the host, it will cause extensive cell death and premature cell differentiation, which seriously affects the research and application of organoids. Therefore, vascularization is crucial for the development and endocrine function of pancreatic organoids.

[0003] The vascularization of organoids has been a hot topic and a challenge in regenerative medicine. Previous research has focused on enhancing vascularization through the co-culture / co-transplantation of pro-angiogenic support cells with target cells, prevascularization at the transplantation site, the construction of host-integrated microvascular networks using biomaterials or decellularized scaffolds, and the use of microfluidics. Despite significant progress in these strategies, existing vascularization protocols often utilize primary cells such as human umbilical vein endothelial cells (HUVECs), human amniotic epithelial cells (hAECs), and MVFs, which face resource limitations. While engineering approaches such as microfluidics can achieve the construction of vascularized organoids, the size, density, and spatial distribution of their vascular structures differ significantly from those in the in vivo physiological environment. While prevascularization and scaffold engineering offer important pathways for vascularization, they are limited to short-term studies, and long-term functional maintenance and scalable production still lack a stable and reproducible technical system. Therefore, whether it is possible to use hPSCs in vitro to directly construct high-order "vascular-islet organoids" that highly simulate human pancreatic islets, improve the maturity and function of organoids, solve problems such as low vascularization and insufficient oxygen supply after transplantation, improve the success rate of transplantation, and ultimately achieve the goal of constructing organoids with high functional fidelity, has important clinical translational significance for cell transplantation treatment of diabetes.

[0004] Currently, the main strategy in the industry is to induce vascular endothelial cells or vascular organoids through hPSCs and co-culture them with other organoids to achieve vascularization. However, the co-culture method is random and lacks uniformity. Therefore, whether it is possible to develop a co-differentiation strategy in vitro to simultaneously induce the generation of vascular endothelial cells and pancreatic islet cells to achieve the one-step construction of vascularized pancreatic islet organoids is crucial for studying the pathogenesis of diabetes, signaling pathways, target research, and drug screening under the physiological state of real human tissues. It also provides an optimized model and is currently an issue that needs to be solved urgently. Summary of the Invention

[0005] The technical problem to be solved by the present invention is that the existing pancreatic islet organ vascularization mostly adopts the co-culture method, which has long operation procedures, high difficulty, randomness, and poor uniformity.

[0006] The present invention solves the above technical problems by providing a method for constructing co-differentiated vascularized pancreatic islet organoids. The method comprises the following steps:

[0007] Human induced pluripotent stem cells (hiPSCs) were sequentially induced and cultured into committed endoderm cells, primitive intestinal tube cells, and posterior foregut cells. During the differentiation of posterior foregut cells into pancreatic progenitor cells, S4 medium containing vascular endothelial growth factor (VEGF) was added for induction culture for 5-10 days; then S5 medium containing VEGF, bone morphogenetic protein 4 (BMP4), and basic fibroblast growth factor (FGF2) was added for induction culture for 5-10 days to obtain vascularized pancreatic endocrine progenitor cells; then S6 medium containing VEGF was added for induction culture for 5-10 days to finally obtain co-differentiated vascularized islet organoids.

[0008] Wherein, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the VEGF concentration in the S4 culture medium is 10-100 ng / mL.

[0009] Furthermore, the composition of the S4 medium includes: DMEM basal medium, added with a final concentration of 0.5-2% glutamine, 0.5-2% serum-free additive B27, 50-200 U / mL penicillin-streptomycin, 50-200 ng / ml epidermal growth factor EGF, 0.1-0.5 μM benzolactam-derived protein kinase C activator TPPB, 5-20 mM niacinamide, 0.1-1 μM (E)-N-(4-benzyl-1-piperazinyl)-1-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methanimine SANT1, 0.1-1 mM vitamin C, 10-100 ng / mL VEGF and 5-20 uM ROCK inhibitor Y27632.

[0010] In the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the VEGF concentration in the S5 culture medium is 10-200 ng / mL, the BMP4 concentration is 5-100 ng / mL, and the FGF2 concentration is 5-100 ng / mL.

[0011] Furthermore, the composition of the S5 culture medium includes: DMEM basal medium, added with a final concentration of 0.5-2% glutamine, 0.5-2% B27, 50-200 U / mL penicillin-streptomycin, 5-20 μM ALK5 inhibitor II, 0.1-1 μM BMP type I receptor inhibitor LDN193189, 0.1-3 μM 3,3′,5-triiodo-L-thyronine T3, 5-20 μM Isoxazole9, 5-20 μg / mL heparin sodium, 0.5-3 μM γ-secretase inhibitor XXi, 50-200 nM PORCN inhibitor Wnt-C59, 5-20 uM Y27632, 10-200 ng / mL VEGF, 5-100 ng / mL BMP4, 5-100 ng / mL FGF2 and 0.1-1 mM vitamin C.

[0012] Wherein, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the VEGF concentration in the S6 culture medium is 10-100 ng / mL.

[0013] Among them, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the composition of the S6 culture medium includes: DMEM basal culture medium, added with a final concentration of 0.5-2% B27, 50-200 U / mL penicillin-streptomycin, 5-20 μM ALK5 inhibitor II, 0.1-1 μM besentinib, 0.1-3 μM T3, 5-20 μM forskolin, 5-20 μg / mL heparin sodium, 5-20 μM zinc sulfate, 0.5-5 mM N-acetyl-L-cysteine, 10-100 ng / mL VEGF and 0.1-1 mM vitamin C.

[0014] Furthermore, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the specific steps include:

[0015] a. Human induced pluripotent stem cells (hiPSCs) were revived and passaged until the confluence of the hiPSCs reached 90%. The cells were then seeded and induced to differentiate. Culture medium S1 was added for 1-5 days to obtain committed endoderm cells; culture medium S2 was added for 1-5 days to obtain primitive intestinal tube cells; and culture medium S3 was added for 1-5 days to obtain posterior foregut cells.

[0016] b. Digest the posterior foregut stage cells and inoculate them into aggregates. Then inoculate the aggregates and add culture medium S4 for induction culture for 5-10 days to obtain vascularized pancreatic progenitor cells; add culture medium S5 for induction culture for 5-10 days to obtain vascularized pancreatic endocrine progenitor cells; add culture medium S6 for induction culture for 5-10 days to obtain vascularized islet organoids.

[0017] In the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the seeding density in step a is 1-1.5×10 5 cells / cm 2 .

[0018] Among them, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the composition of the culture medium S1 described in step a includes: fetal bovine serum with a final concentration of 0.2%, 100 U / mL penicillin-streptomycin, 1% glutamine, 0.02% insulin-transferrin-selenium-aminoethanol, 100 ng / mL activin A and 50 ng / mL Wnt3A added to RPIM1640 basal culture medium.

[0019] In the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the composition of the culture medium S2 described in step a includes: DMEM / F12 basal medium, added with a final concentration of 2% fetal bovine serum, 100 U / mL penicillin-streptomycin, 0.1% ITS-X and 50 ng / mL keratinocyte growth factor KGF.

[0020] Among them, in the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the composition of the culture medium S3 described in step a includes: DMEM basal medium, added with a final concentration of 1% serum-free additive, 100 U / mL penicillin-streptomycin, 2 μM retinoic acid, 0.1 μM BMP type I receptor inhibitor, 0.25 μM (E)-N-(4-benzyl-1-piperazinyl)-1-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methanamine and 100 nM PORCN inhibitor.

[0021] In the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the cell seeding density at the posterior foregut stage described in step b is 800-1000 cells / each aggregate.

[0022] In the above-mentioned method for constructing co-differentiated vascularized pancreatic islet organoids, the aggregate seeding density in step b is 3000-5000 cells / cm 2 .

[0023] The present invention also provides a vascularized pancreatic islet organoid constructed by the above method.

[0024] The beneficial effects of the present invention are:

[0025] The present invention adopts a one-step method to gradually induce hiPSC into vascularized pancreatic islet organoids by optimizing the combination of induction factors and culture conditions. There is no need to add exogenous endothelial cells, and the co-differentiation of vascular endothelial cells and pancreatic islet cells is achieved, forming an endogenous vascular network. The method of the present invention not only simplifies the operation process and improves experimental efficiency, but also enhances the physiological relevance of the model, and obtains pancreatic islet organoids with rich vascular networks, high maturity, and complete structure, which reproduces the key structural and functional characteristics of human pancreatic islet organs. The culture system established by the method of the present invention supports the long-term continuous passage culture of pancreatic islet organoids, providing a basis for large-scale production and clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Shown is a schematic diagram of the construction of vascularized pancreatic islet organoids.

[0027] Figure 2 Shown are schematic diagrams of the different stages of vascularized islet organoid induction.

[0028] Figure 3 Shown are the Q-PCR analyses of the expression of islet- and vascular-related genes in pancreatic islet organoids before and after vascularization.

[0029] Figure 4 Shown is the expression of INS, GCG, and SST in vascularized pancreatic islet organoids identified by immunofluorescence.

[0030] Figure 5 Shown is the expression of NKX6.1, INS, and CD31 identified by immunofluorescence in vascularized pancreatic islet organoids.

[0031] Figure 6 Shown is the proportion of vascular endothelial cells in vascularized pancreatic islet organoids identified by flow cytometry.

[0032] Figure 7 Shown is the distribution of hormone particles in vascularized pancreatic islet organoids identified by transmission electron microscopy.

[0033] Figure 8 Shown are vascularized pancreatic islet organoids exhibiting enhanced insulin secretion.

[0034] Figure 9 Shown are random non-fasting blood glucose measurements after islet organoids / vascularized islet organoids were transplanted into NOD-SCID hyperglycemic mice. DETAILED DESCRIPTION

[0035] The present invention provides a method for constructing vascularized pancreatic islet organoids by co-differentiating vascular endothelial cells and pancreatic islet cells. Human induced pluripotent stem cells (hiPSCs) are used to screen and combine specific growth factors to achieve co-differentiation of vascular endothelial cells and pancreatic islet cells, forming an endogenous vascular network, which is a very significant improvement.

[0036] We added growth factors that play a key role in vascular induction and maintenance to the culture system of posterior foregut pancreatic progenitor cell lineage specification, including vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF, also known as FGF2), and bone morphogenetic protein 4 (BMP4), and screened and adjusted the concentration and duration of growth factors.

[0037] The results showed that the vascularized pancreatic islet organoids obtained after the addition of VEGF, BMP4, and FGF2 growth factors were more mature. Specifically, the organoids showed significant upregulation of endocrine genes related to islet cells, as well as mesothelial and endothelial cell marker genes. The organoids also had a similar endothelial cell composition ratio to human islets, a significant increase in hormone granules within the cells, and glucose-stimulated insulin secretion, closer to that of human islets. When transplanted into the renal capsule of NOD-SCID mice, preliminary observations showed that the vascularized pancreatic islet organoids could restore normal blood sugar levels in STZ-induced mice in the short term.

[0038] The inventors of the present invention co-cultured vascular organoids at different stages of pancreatic islet induced differentiation in a combined manner. Based on the results of co-culture, they developed the concentration and action time of adding vascular network regulatory factors at different stages, and finally obtained the co-differentiated pancreatic islet organoid culture method of the present invention.

[0039] The specific embodiments of the present invention will be further explained below through examples, but it is not intended to limit the scope of protection of the present invention to the scope described in the examples.

[0040] All instruments and reagents used in the examples are common commercially available products.

[0041] Example 1 Construction of vascularized pancreatic islet organoids using the method of the present invention

[0042] The specific steps are as follows:

[0043] (1) HiPSCs were expanded and cultured. When the confluence reached more than 90%, the hiPSCs were digested into single cells using Accutase.

[0044] (2) In a Matrigel-coated cell plate, 1-1.5×10 5 cells / cm 2 Inoculate at a density of

[0045] (3) 24 hours after inoculation, observe that the cell density is above 90% and then start inducing differentiation;

[0046] (4) In the order of directional endoderm, primitive gut, and posterior foregut, different growth factors and small molecule inhibitors or activators are added to prepare culture medium S1-S3 for inducing differentiation. The induced cells are replaced with culture medium every day. On the 10th day of induction, cells of the posterior foregut stage can be obtained (e.g. Figure 1 shown);

[0047] (5) Then, add 500 μL of anti-adhesion buffer to the 24-well AggreWell™400 plate and centrifuge the plate at 1300 g for 5 min.

[0048] (6) Aspirate the anti-adhesion rinse solution from the wells and rinse each well with warm basal medium DMEM (use 2 mL of preheated basal medium DMEM for rinsing);

[0049] (7) Add 1 mL of pre-warmed pancreatic progenitor cell S4 complete medium to the wells for later use;

[0050] (8) After digesting the adherent cultured posterior foregut stage cells into single cells, they were seeded into a 24-well plate containing AggreWell™400 at a density of 800–1000 cells per aggregate, with a volume of 2 mL per well;

[0051] (9) Centrifuge the AggreWell™ 400-well plate containing cells at 300 g for 5 min, and then place the plate in a CO2 incubator for 48 h to allow for the formation of uniformly sized aggregates.

[0052] (10) After the aggregates are formed, they are seeded into a low-adhesion 6-well plate at a density of 3000-5000 aggregates per well and placed on a shaker at 95-105 rpm for subsequent differentiation into vascularized pancreatic progenitor cells, vascularized pancreatic endocrine precursor cells, and vascularized islet cells. The stage-specific differentiation medium S4-S6 is prepared and the medium is changed every day. The induction is continued for 15 days to obtain vascularized islet organoids (such as Figure 2 shown).

[0053] The culture medium used in this embodiment is composed as follows:

[0054] The composition of culture medium S1 includes: RPIM1640 basal medium, with the addition of 0.2% fetal bovine serum, 100 U / mL penicillin-streptomycin, 1% glutamine, 0.02% ITS-X, 100 ng / mL Activin A, and 50 ng / mL Wnt3A at a final concentration.

[0055] The composition of culture medium S2 includes: DMEM / F12 basal medium, with the addition of 2% fetal bovine serum, 100 U / mL penicillin-streptomycin, 0.1% ITS-X and 50 ng / mL KGF at a final concentration.

[0056] The composition of culture medium S3 includes: DMEM basal medium, with the addition of 1% B27, 100 U / mL penicillin-streptomycin, 2 μM retinoic acid, 0.1 μM LDN193189, 0.25 μM Sant1 and 100 nM Wnt-C59 to the final concentration.

[0057] The composition of culture medium S4 includes: DMEM basal medium, with the addition of a final concentration of 1% glutamine, 1% B27, 100 U / mL penicillin-streptomycin, 100 ng / ml EGF, 0.2 μM TPPB, 10 mM niacinamide, 0.25 μM Sant1, 0.25 mM vitamin C, 10 ng / mL VEGF and 10 uM Y27632.

[0058] The composition of culture medium S5 includes: DMEM basal medium, with the addition of a final concentration of 1% glutamine, 1% B27, 10 μM ALK5 inhibitor II, 0.3 μM LDN193189, 1 μM T3, 10 μM ISX9, 10 μg / mL heparin sodium, 1uM Xxi, 100 nM Wnt-C59, 10 μM Y27632, 10 ng / mL VEGF, 10 ng / mL BMP4, 10 ng / mL FGF2, and 0.25 mM vitamin C.

[0059] The composition of culture medium S6 includes: DMEM basal medium, with the addition of B27 at a final concentration of 1%, 100 U / mL penicillin-streptomycin, 10 μM ALK5 inhibitor II, 0.5 μM R428, 1 μM T3, 10 μM forskolin, 10 μg / mL heparin sodium, 10 μM zinc sulfate, 2 mM N-Acetyl-ʟ-cysteine, 10 ng / mL VEGF, and 0.25 mM vitamin C.

[0060] Example 2 Identification of Vascularized Pancreatic Islet Organoids

[0061] The islet organoid construction method of Example 1 was adopted, but VEGF, BMP4 and FGF2 growth factors were not added to the S4-S6 culture medium. The other culture conditions were the same, and the islet organoids obtained were cultured as a control.

[0062] To identify the vascularized pancreatic islet organoids obtained in Example 1, we first extracted RNA and performed real-time fluorescence quantitative PCR. The results showed that the endocrine genes related to pancreatic islet cells, mesothelial cells and endothelial cell marker genes were significantly upregulated (such as Figure 3 As shown in Figure 2, it shows that the vascularized pancreatic islet organoids constructed by the method of the present invention have the genetic characteristics of pancreatic islets and blood vessels. In addition, the vascularized pancreatic islet organoids were observed to have more uniform and more INS, GCG and SST expressions (as shown in Figure 2). Figure 4 ), indicating that vascularization promotes the increase of endocrine cells in pancreatic islet organoids and has a higher expression of vascular endothelial cell marker CD31 (as shown in Figure 5 By flow cytometry, we also found that in vascularized islet organoids, there were about 20% vascular endothelial cells, while in islet organoids, there were only 3-5% endothelial cells (as shown in Figure 6 Furthermore, transmission electron microscopy revealed that the number of hormone secretion granules of various cell types in vascularized pancreatic islet organoids was significantly increased compared to that in pancreatic islet organoids (e.g. Figure 7 shown).

[0063] Example 3 Glucose-stimulated insulin secretion assay in vascularized pancreatic islet organoids

[0064] To verify the functionality of the constructed vascularized pancreatic islet organoids, we first performed a glucose-stimulated insulin secretion test in vitro. The specific steps are as follows:

[0065] (1) Prepare Krebs-Ringer-HEPES (KRH) solution by accurately weighing 23.83 mg HEPES, 5.3676 mg KCl, 2.4648 mg MgSO4·7H2O, 163.632 mg NaCl, 3.3604 mg NaHCO3, 1.1998 mg NaH2PO4, and 7.644 mg CaCl2 powder into 18 mL ultrapure water. Add 0.1 N NaOH to adjust the pH to 7.4. Then, dilute to 20 mL with ultrapure water and filter sterilize using a 0.22 μm filter.

[0066] (2) Prepare Krebs-Ringer-HEPES (KRH) / BSA solution by weighing 20 mg of BSA and dissolving it in 10 mL of Krebs-Ringer-HEPES (KRH) solution to a final concentration of 0.2% w / v. Place on ice for no more than 24 hours.

[0067] (3) Prepare 1 M glucose solution by weighing 1.8 g of glucose into 10 mL of Krebs-Ringer-HEPES (KRH) solution to a concentration of 1 M. This solution can be stored at -20°C for 6 months.

[0068] (4) Prepare 2 mM glucose solution by diluting 1 M glucose into Krebs-Ringer-HEPES (KRH) / BSA solution to a final concentration of 2 mM. Store on ice for no more than 24 hours and preheat to 37°C before use.

[0069] (5) Prepare 20 mM glucose solution by diluting 1 M glucose into Krebs-Ringer-HEPES (KRH) / BSA solution to a final concentration of 20 mM. Store on ice for no more than 24 hours and preheat to 37°C before use.

[0070] (6) Rinse vascularized islet organoids, non-vascularized islet organoids, and primary human islets three times with Krebs-Ringer-HEPES (KRH) / BSA buffer;

[0071] (7) Cover the sample with Krebs-Ringer-HEPES (KRH) / BSA buffer and place in an incubator for 30 minutes to equilibrate;

[0072] (8) Remove the buffer, cover the sample with buffer containing 2.8 mM glucose, and incubate in an incubator for 1 hour;

[0073] (9) After collecting the supernatant, transfer the sample to a buffer containing 20 mM glucose, incubate in an incubator for 1 hour, and collect the supernatant again;

[0074] (10) Place the sample in an acid / ethanol solution at -20°C overnight;

[0075] (11) Use ELISA kit to test the collected samples.

[0076] The results showed that vascularized pancreatic islet organoids could respond to different glucose stimuli and had glucose-stimulated insulin secretion function that was closer to that of primary human pancreatic islets (e.g. Figure 8 As shown in Figure 3 ), vascularized islet organoids exhibited a more mature phenotype and enhanced function in improving hyperglycemia.

[0077] Example 4: Kidney capsule transplantation of vascularized pancreatic islet organoids

[0078] Furthermore, we used STZ to establish a type 1 diabetes model in NOD-SCID mice and transplanted vascularized pancreatic islet organoids into the renal capsule. The specific procedures are as follows:

[0079] (1) Collect the suspended islet organoids and vascularized islet organoids and wash them three times with saline;

[0080] (2) Adjust the number of transplanted vascularized islet organoids to 4,000 per mouse, resuspend them in 30 μL of saline, and place them on ice until ready for transplant.

[0081] (3) Place the NOD / SCID mice in an anesthesia machine for anesthesia;

[0082] (4) Place the mouse on the operating table with its back facing up after gas anesthesia, and wipe and disinfect its back with a 75% alcohol cotton ball;

[0083] (5) Cut open the subcutaneous tissue of the mouse and squeeze it to fully expose its kidney;

[0084] (6) Use an insulin needle to aspirate the organoid suspension, gently lift the renal capsule so that the needle tip can move between the renal parenchyma and the renal capsule, and slowly inject 25-30 μL of cell suspension;

[0085] (7) Slowly push the cell-transplanted kidney back to its original position, suture the mouse endothelium and outer skin, and wipe the suture with iodine solution;

[0086] (8) Return the mice to their cages and continue feeding.

[0087] After the experiment, we preliminarily observed that vascularized pancreatic islet organoids could restore STZ model mice to normal blood sugar in a short period of time (e.g. Figure 9 shown).

[0088] The above experimental results collectively demonstrate that the present invention can construct functional vascularized pancreatic islet organoids through a co-differentiation strategy. The vascularized pancreatic islet organoids directly differentiated from iPSCs have a more mature phenotype and enhanced function. The present invention lays a good foundation for subsequent applications, has good in vivo safety, and has good prospects for clinical translation.

Claims

1. A method for constructing co-differentiated vascularized pancreatic islet organoids, characterized in that: The following steps are involved: Human induced pluripotent stem cells (hiPSCs) were cultured in medium S1 to produce committed endoderm cells, in medium S2 to produce primitive gut cells, and in medium S3 to produce posterior foregut cells. During the differentiation of posterior foregut cells into pancreatic progenitor cells, they were induced in medium S4 containing vascular endothelial growth factor (VEGF) for 5-10 days. Furthermore, they were induced in medium S5 containing VEGF, bone morphogenetic protein 4 (BMP4), and basic fibroblast growth factor (FGF2) for 5-10 days to obtain vascularized pancreatic endocrine progenitor cells. Then, S6 medium containing VEGF was added to induce culture for 5-10 days to finally obtain co-differentiated vascularized pancreatic islet organoids; The composition of the S4 culture medium includes: DMEM basal medium, added with a final concentration of 0.5-2% glutamine, 0.5-2% serum-free additive B27, 50-200 U / mL penicillin-streptomycin, 50-200 ng / mL epidermal growth factor EGF, 0.1-0.5 μM benzolactam-derived protein kinase C activator TPPB, 5-20 mM nicotinamide, 0.1-1 μM (E)-N-(4-benzyl-1-piperazinyl)-1-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methanimine SANT1, 0.1-1 mM vitamin C, and 10-100 ng / mL VEGF and 5-20uM ROCK inhibitor Y27632; the composition of the S5 culture medium includes: DMEM basal culture medium, added with a final concentration of 0.5-2% glutamine, 0.5-2% B27, 50-200U / mL penicillin-streptomycin, 5-20μM ALK5 inhibitor II, 0.1-1μM BMP Type I receptor inhibitor LDN193189, 0.1-3 μM 3,3′,5-triiodo-L-thyronine T3, 5-20 μM Isoxazole9, 5-20 μg / mL heparin sodium, 0.5-3 μM γ-secretase inhibitor XXi, 50-200 nM PORCN inhibitor Wnt-C59, 5-20 μM Y27632, 10-200 ng / mL VEGF, 5-100 ng / mL BMP4, 5-100 ng / mL FGF2 and 0.1-1 mM vitamin C; the composition of the S6 culture medium includes: adding B27 at a final concentration of 0.5-2%, 50-200 U / mL penicillin-streptomycin, 5-20 μM ALK5 inhibitor II, 0.1-1 μM besentinib, 0.1-3 μM T3, 5-20 μM forskolin, 5-20 μg / mL heparin sodium, 5-20 μM zinc sulfate, 0.5-5 mM N-acetyl-L-cysteine, 10-100 ng / mL VEGF and 0.1-1 mM vitamin C; the composition of the culture medium S1 includes: adding RPIM1640 basal medium with a final concentration of 0.2% fetal bovine serum, 100 U / mL penicillin-streptomycin, 1% glutamine, 0.02% ITS-X, 100 ng / mL Activin A and 50 ng / mL Wnt3A; the composition of the culture medium S2 includes: adding DMEM / F12 basal medium with a final concentration of 2% fetal bovine serum, 100 U / mL penicillin-streptomycin, 0.1% ITS-X and 50 ng / mL KGF; the composition of the culture medium S3 includes: DMEM basal medium, added with a final concentration of 1% serum-free supplement, 100 U / mL penicillin-streptomycin, 2 μM retinoic acid, 0.1 μM BMP type I receptor inhibitor LDN193189, 0.25 μM (E)-N-(4-benzyl-1-piperazinyl)-1-(3,5-dimethyl-1-phenyl-1H-pyrazol-4-yl)methanimine, and 100 nM PORCN inhibitor Wnt-C59.

2. The method for constructing co-differentiated vascularized pancreatic islet organoids according to claim 1, characterized in that: The specific steps include: a. Human induced pluripotent stem cells (hiPSCs) were revived and passaged until the confluence of the hiPSCs reached 90%. The cells were then seeded and induced to differentiate. Culture medium S1 was added for 1-5 days to obtain committed endoderm cells; culture medium S2 was added for 1-5 days to obtain primitive intestinal tube cells; and culture medium S3 was added for 1-5 days to obtain posterior foregut cells. b. Digest the posterior foregut stage cells and inoculate them into aggregates. Then inoculate the aggregates and add culture medium S4 for induction culture for 5-10 days to obtain vascularized pancreatic progenitor cells; add culture medium S5 for induction culture for 5-10 days to obtain vascularized pancreatic endocrine progenitor cells; add culture medium S6 for induction culture for 5-10 days to obtain vascularized islet organoids.

3. The vascularized pancreatic islet organoid constructed by the method according to any one of claims 1 or 2.

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