A moyamoya disease model and a method for constructing the same and an application thereof

Abstract

The application provides a moyamoya disease model and a construction method and application thereof, and belongs to the technical field of moyamoya disease model construction. The method for constructing the moyamoya disease model provided by the application comprises the following steps: extracting CD34+ hematopoietic progenitor cells in peripheral blood of a moyamoya disease patient, generating hiPSCs through reprogramming, and further differentiating the hiPSCs into brain organoids and blood vessel organoids, then fusing the brain organoids and the blood vessel organoids, and culturing to construct a moyamoya disease model of vascularized brain organoids. The moyamoya disease model constructed by using the construction method of the application can simulate and reproduce the phenotypes of increased small branches, reduced large vessels, reduced pericytes, smooth muscle fibrosis deposition and increased abnormal angiogenesis in the clinical core pathological phenotypes of moyamoya disease patients, and is superior to existing animal and cell models to a certain extent.

Description

Technical Field

[0001] This invention belongs to the field of moyamoya disease model construction technology, specifically relating to a moyamoya disease model, its construction method, and its application. Background Technology

[0002] Moyamoya disease is a chronic, progressive cerebrovascular occlusive disease of unknown etiology. It is named for its resemblance to "smoke" in cerebral angiography images. The main pathological features of Moyamoya disease are stenosis or occlusion of the terminal portions of the bilateral internal carotid arteries and the origin of the middle cerebral artery, as well as the formation of an abnormal collateral vascular network. The pathogenesis of Moyamoya disease is complex, and it is currently believed to involve multiple factors, including genetic factors such as gene mutations; and environmental factors such as immune and inflammatory responses, vascular endothelial dysfunction, and oxidative stress.

[0003] Existing models of Moyamoya disease are primarily animal models, mostly constructed using mice and zebrafish. Common construction methods include: surgical models that induce chronic cerebral hypoperfusion by permanently ligating the common carotid or internal carotid artery; immune models that simulate the immune response in Moyamoya disease by inducing vasculitis-like pathology; and genetic models constructed by knocking down or deleting the RNF213 gene. However, all animal and cell models currently fail to simulate the core pathological features of Moyamoya disease, namely, the "smoke-like" abnormal collateral formation or spontaneous vascular stenosis, exhibiting significant limitations. Furthermore, the inability to directly observe the pathological examination of the affected areas in Moyamoya disease patients through biopsy further exacerbates the challenges in clinical and basic research. There is an urgent need to find a new Moyamoya disease model that more closely resembles the symptoms of patients and can completely reproduce the core phenotype of Moyamoya disease. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing a moyamoya disease model, wherein the moyamoya disease model constructed using the method of this invention can completely reproduce the core pathological features of moyamoya disease.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing a moyamoya disease model, comprising the following steps: CD34+ hematopoietic progenitor cells were collected from peripheral blood of patients with Moyamoya disease, reprogrammed to generate hiPSCs, and the hiPSCs were cultured to 70%~80% confluence and differentiation degree not exceeding 10%. hiPSCs were cultured in EB inoculation medium for 45-51 hours to obtain the first-stage embryoids; The first-stage embryoids were cultured for 45-51 hours using EB inoculation medium to obtain the second-stage embryoids. The second-stage embryoids were then cultured for 44-52 hours using ectoderm induction medium to obtain primary brain organoids. The first-stage embryoids were cultured in mesodermal induction medium for 44-52 hours, then in endothelial cell induction medium for 44-52 hours, and then in vascular maturation culture medium for 48-96 hours to obtain primary vascular organoids. The primary brain organoid and the primary blood vessel organoid are fused to obtain a hybrid organoid; The hybrid organoids were co-cultured with neural induction culture medium and vascular maturation culture medium for 68-76 hours to obtain pre-vascularized brain organoids. A Moyamoya disease model was obtained by culturing pre-vascularized brain organoids for more than 28 days using neural maturation culture media and vascular maturation culture media. The EB inoculation medium consists of basal culture medium 1, 2-mercaptoethanol at a final concentration of 0.1 mM, bFGF at a final concentration of 4 ng / mL, and Y-27632 at a final concentration of 50 μM. The basal culture medium 1 consists of the following ingredients by volume fraction: 75% DMEM / F12, 20% KnockOut™ Serum Replacement, 3% fetal bovine serum for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution.

[0006] Preferably, the reprogramming is performed using the Sendai virus reprogramming kit.

[0007] Preferably, the ectoderm induction medium consists of basal culture medium 2 and heparin at a final concentration of 1 μg / mL. The basal culture medium 2 consists of the following ingredients by volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine and 1% MEM non-essential amino acid solution.

[0008] Preferably, the mesodermal induction medium consists of APEL2 and CHIR-99021 at a final concentration of 6 μM; the endothelial cell induction medium consists of APEL2, VEGF165 at a final concentration of 50 ng / mL, BMP4 at a final concentration of 25 ng / mL, and bFGF at a final concentration of 10 ng / mL; and the vascular maturation culture medium consists of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL.

[0009] Preferably, Matrigel is used during the fusion process, and 15-20 μL of Matrigel is required to encapsulate a primary brain organoid and a primary vascular organoid.

[0010] Preferably, the volume ratio of the neural induction culture medium to the vascular maturation culture medium is 1:1. The neural induction culture medium consists of basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, and insulin at a final concentration of 2.5 μg / mL. The basal culture medium 3 consists of the following ingredients by volume fraction: 48% DMEM / F12, 48% Neurobasal medium, 0.5% N-2 supplement, 1% glutamine, 0.5% MEM non-essential amino acid solution, 1% B27 without vitamin A, and 1% penicillin-streptomycin.

[0011] Preferably, the volume ratio of the neural maturation culture medium to the vascular maturation culture medium is (1.5:1) to (1:1.5), and the neural maturation culture medium is composed of the basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, insulin at a final concentration of 2.5 μg / mL, VEGF165 at a final concentration of 20 ng / mL, brain-derived neurotrophic factor at a final concentration of 20 ng / mL, and hydrocortisone at a final concentration of 0.2 µg / mL.

[0012] Preferably, the culture temperature is 36.5~37.5℃.

[0013] The present invention also provides a Moyamoya disease model, which is constructed by the above method.

[0014] This invention also provides the application of the above-described method or the above-described moss disease model in the screening of drugs for the prevention and control of moss disease.

[0015] The beneficial effects of this invention are: Traditional animal and cell models currently cannot simulate the core pathological features of Moyamoya disease, namely the "smoke-like" abnormal collateral formation or spontaneous vascular stenosis, exhibiting significant limitations. Compared to traditional models, the Moyamoya disease model constructed using the method of this invention can simulate and reproduce the phenotypes of increased fine branching, reduced large vessels, decreased pericytes, smooth muscle fibrosis deposition, and increased abnormal angiogenesis in the core clinical pathological phenotypes of Moyamoya disease patients, demonstrating superiority over existing animal and cell models to a certain extent. Similarly, this invention achieves complete humanization of the in vitro model, making it more closely resemble the human body, enabling high-throughput preparation and serving as an in vitro drug screening platform, while avoiding the ethical controversies associated with animal models. Attached Figure Description

[0016] Figure 1 The flowchart and bright-field model of vascularized brain organoid construction are shown. A is the flowchart of vascularized brain organoid construction, and B is the bright-field optical microscope image of the brain organoid, vascular organoid and fused vascularized brain organoid. Figure 2Immunofluorescence staining comparison of vascularized brain organoids from healthy controls and patients with moyamoya disease, where star-shaped markers indicate the small branches of the vascular network that the patients could not form; Figure 3 The results of immunofluorescence staining analysis are shown below. A shows the immunofluorescence staining images of CD31 (an endothelial marker of vascularized brain organoids) and TUJ1 (a neural marker); B shows a statistical comparison of the total number of vascular junctions in vascularized brain organoids from healthy controls and patients with Moyamoya disease, based on CD31 staining; C shows a statistical comparison of the total number of major vascular junctions in vascularized brain organoids from healthy controls and patients with Moyamoya disease, based on CD31 staining; D shows a statistical comparison of the total number of major vascular segments in vascularized brain organoids from healthy controls and patients with Moyamoya disease, based on CD31 staining; E shows a statistical comparison of the total branch length of all vascular branches in vascularized brain organoids from healthy controls and patients with Moyamoya disease, based on CD31 staining. T-tests were used, with mean ± SEM, n=9; ns indicates p≥0.05, ** indicates p<0.01. Figure 4 The results of qPCR detection of gene expression levels in vascularized brain organoids from healthy controls and patients with Moyamoya disease were presented. A represents the relative expression level of GFAP gene mRNA in vascularized brain organoids from healthy controls and patients with Moyamoya disease; B represents the relative expression level of MAP2 gene mRNA in vascularized brain organoids from healthy controls and patients with Moyamoya disease; C represents the relative expression level of PDGFRβ gene mRNA in vascularized brain organoids from healthy controls and patients with Moyamoya disease. T-test was used, mean ± SEM, n=3; * indicates p < 0.05, ** indicates p < 0.01. Figure 5 Statistical graph of total NO concentration in vascularized brain organoid culture supernatants from healthy controls and patients with Moyamoya disease, T-test, mean ± SEM, n=6. Detailed Implementation

[0017] This invention provides a method for constructing a moyamoya disease model, comprising the following steps: CD34+ hematopoietic progenitor cells were collected from peripheral blood of patients with Moyamoya disease, reprogrammed to generate hiPSCs, and the hiPSCs were cultured to 70%~80% confluence and differentiation degree not exceeding 10%. hiPSCs were cultured in EB inoculation medium for 45-51 hours to obtain the first-stage embryoids; The first-stage embryoids were cultured for 45-51 hours using EB inoculation medium to obtain the second-stage embryoids. The second-stage embryoids were then cultured for 44-52 hours using ectoderm induction medium to obtain primary brain organoids. The first-stage embryoids were cultured in mesodermal induction medium for 44-52 hours, then in endothelial cell induction medium for 44-52 hours, and then in vascular maturation culture medium for 48-96 hours to obtain primary vascular organoids. The primary brain organoid and the primary blood vessel organoid are fused to obtain a hybrid organoid; The hybrid organoids were co-cultured with neural induction culture medium and vascular maturation culture medium for 68-76 hours to obtain pre-vascularized brain organoids. A Moyamoya disease model was obtained by culturing pre-vascularized brain organoids for more than 28 days using neural maturation culture media and vascular maturation culture media. The EB inoculation medium consists of basal culture medium 1, 2-mercaptoethanol at a final concentration of 0.1 mM, bFGF at a final concentration of 4 ng / mL, and Y-27632 at a final concentration of 50 μM. The basal culture medium 1 consists of the following ingredients by volume fraction: 75% DMEM / F12, 20% KnockOut™ Serum Replacement, 3% fetal bovine serum for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution.

[0018] This invention extracts CD34+ hematopoietic progenitor cells from peripheral blood of patients with Moyamoya disease, reprograms them to generate hiPSCs, and further differentiates them into brain organoids and vascular organoids. Then, the brain organoids and vascular organoids are fused and cultured to construct a vascularized brain organoid Moyamoya disease model. A flowchart of this construction method is shown below. Figure 1 As shown. The method of this invention can construct an in vitro model that fits well with the clinicopathological features of Moyamoya disease. The constructed Moyamoya disease model is superior to all existing animal and cell models. The construction process does not involve manipulation of the embryo, can achieve complete humanization, and avoids ethical issues. The Moyamoya disease model provided by this invention has high throughput and can serve as a good platform for drug screening and mechanism research, providing a new and efficient tool for studying drug targets. Furthermore, since the organoids are derived entirely from the patient's own cells, it lays the foundation for personalized medicine.

[0019] In this invention, embryoid bodies (EB) refer to three-dimensional cell aggregates that spontaneously form from human induced pluripotent stem cells (hiPSCs) under in vitro culture conditions. These aggregates have endoderm, mesoderm, and ectoderm structures and are morphologically highly similar to the early embryonic development stages of mammals, and can provide a microenvironment related to early development to a certain extent.

[0020] This invention does not specifically limit the method for obtaining CD34+ hematopoietic progenitor cells from peripheral blood of patients with Moyamoya disease; any conventional method for obtaining CD34+ hematopoietic progenitor cells from peripheral blood in the art is acceptable. This invention also does not specifically limit the culture method for hiPSCs; existing conventional culture methods in the art are sufficient. In this invention, the reprogramming is preferably performed using the Sendai virus reprogramming kit. In this invention, the culture time for obtaining the first-stage embryoid is preferably 47-49 hours, and the culture time for obtaining the second-stage embryoid is preferably 47-49 hours. In this invention, the ectoderm induction medium preferably consists of basal culture medium 2 and heparin at a final concentration of 1 μg / mL. The basal culture medium 2 consists of the following ingredients by volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine, and 1% MEM non-essential amino acid solution; the culture time for obtaining primary brain organoids is preferably 46-50 hours, more preferably 47-49 hours. In this invention, the mesodermal induction medium preferably consists of APEL2 and CHIR-99021 at a final concentration of 6 μM. The first-stage embryoids are cultured to the mesoderm using the mesodermal induction medium, and the culture time is preferably 46-50 h, more preferably 47-49 h. In this invention, the endothelial cell induction medium preferably consists of APEL2, VEGF165 at a final concentration of 50 ng / mL, BMP4 at a final concentration of 25 ng / mL, and bFGF at a final concentration of 10 ng / mL. The mesoderm is induced to the endothelial cells using the endothelial cell induction medium, and the culture time is preferably 46-50 h, more preferably 47-49 h. In this invention, the vascular maturation culture medium preferably consists of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL. Endothelial cells are cultured to the primary vascular organoids using the vascular maturation culture medium, and the culture time is preferably 50-94 h, more preferably 55-90 h.

[0021] In this invention, Matrigel is preferably used to fuse primary brain organoids and primary vascular organoids. Encapsulating one primary brain organoid and one primary vascular organoid preferably requires 15-20 μL of Matrigel, more preferably 17-19 μL. This invention does not have a specific limitation on the specific source of Matrigel; commercially available products conventional in the art can be used.

[0022] In this invention, the volume ratio of the neural induction culture medium to the vascular maturation culture medium is preferably 1:1. The neural induction culture medium preferably consists of basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, and insulin at a final concentration of 2.5 μg / mL. The basal culture medium 3 is composed of the following ingredients by volume fraction: 48% DMEM / F12, 48% Neurobasal medium, 0.5% N-2 supplement, 1% glutamine, 0.5% MEM non-essential amino acid solution, 1% B27 (vitamin A-free), and 1% penicillin-streptomycin. In this invention, the time for culturing the mixed organoids to the initial vascularization stage of the brain organoids is preferably 70-74 hours, more preferably 72-73 hours.

[0023] In this invention, the volume ratio of the neural maturation culture medium to the vascular maturation culture medium is preferably (1.5:1) to (1:1.5), more preferably 1:1. The neural maturation culture medium preferably consists of the basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, insulin at a final concentration of 2.5 μg / mL, VEGF165 at a final concentration of 20 ng / mL, brain-derived neurotrophic factor at a final concentration of 20 ng / mL, and hydrocortisone at a final concentration of 0.2 µg / mL. In this invention, the culture temperature is preferably 36.5~37.5℃, more preferably 37℃. This invention does not specifically limit the source of each raw material in the above culture media; conventional commercially available products in the art can be used.

[0024] In this invention, the brain organoid (BOr), also known as a brain-like organism, is a miniature model formed by the self-organization of pluripotent stem cells through in vitro three-dimensional culture. It contains neurons and various types of glial cells and can replicate some functions of the imitated organ. Vascular organoids (VOr) are generated by inducing differentiation of hiPSCs and are composed of various cell types (such as endothelial cells, smooth muscle cells, pericytes, and stromal cells). They can simulate the formation, development, and function of blood vessels. Vascularized brain organoids (VBOr), also known as vascularized brain-like organisms, are organoids formed by fusing and culturing vascular organoids and brain organoids. They completely remove the mouse-derived components from traditional brain organoid vascularization technology, achieving in vitro humanization. Building upon brain organoids, vascularized brain-like models incorporate a vascular system, including vascular endothelial cells, to support neural development, promote neuronal and glial cell maturation, reduce apoptosis in the central region of the organoid, and reproduce the blood-brain barrier structure to a certain extent. This allows for a more realistic simulation of human brain development, providing a more accurate model and platform for Moyamoya disease research and drug screening. This invention utilizes vascularized brain-like models to construct Moyamoya disease models, offering new possibilities for exploring the pathological mechanisms of the disease, the interactions between neural-vascular networks, and the development of novel therapeutic targets.

[0025] Compared with other existing models in the field, the Moyamoya disease model provided by this invention has the following advantages: the smooth muscle tissue of the Moyamoya disease model of this invention shows elevated α-SMA, fibrosis, spontaneous stenosis and occlusion of major blood vessels, and an increase in fine branches, while existing mouse Moyamoya disease models in the field do not exhibit the above phenomena.

[0026] This invention also provides a moyamoya disease model, constructed using the above method. Furthermore, this invention provides the application of the above method or the above moyamoya disease model in the screening of drugs for the control of moyamoya disease.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, the following embodiments are all conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] The sources of some of the raw materials used in the following embodiments are shown in Table 1: Table 1. Sources of some raw materials

[0031] Example 1 A Moyamoya disease model is constructed as follows: I. CD34+ hematopoietic progenitor cell reprogramming and hiPSC culture (1) Peripheral blood was collected from patients with Moyamoya disease. CD34+ progenitor cells were screened by peripheral blood magnetic bead sorting. The kit and magnetic bead were manufactured by Stemcell Technology, catalog number: magnetic bead 18000; kit 05925. CD34+ hematopoietic progenitor cells were obtained from peripheral blood of patients with Moyamoya disease and reprogrammed according to the method shown in the CytoTune-iPS Sendai virus reprogramming kit to generate hiPSCs.

[0032] (2) Use rhVTN-N vitrin to coat hiPSC culture plates Under aseptic conditions, dilute 0.5 mg / mL rhVTN-N polinecin stock solution to 5 μg / mL at a ratio of 1:100 using DPBS phosphate buffer. Prepare fresh before use. After adding the solution, gently shake to ensure even coverage of the entire cell culture plate surface. Then, place the plate in a 37°C cell culture incubator for at least 30 min (or seal with sealing film and coat overnight at 4°C; cell culture plates coated overnight at 4°C require 30 min at room temperature before use). After aspirating the coating supernatant, immediately seed cells without rinsing the surface with DPBS phosphate buffer.

[0033] (3) Prepare mTeSR™Plus cell medium (mTeSR medium) Thaw the mTeSR™Plus 5× additive overnight at room temperature or 4°C. Before use, allow it to fully return to room temperature and mix gently to avoid flocculation and precipitation. Avoid repeated freeze-thaw cycles. Under aseptic conditions, add 100 mL of mTeSR™Plus 5× additive to 400 mL of mTeSR™Plus base medium and mix thoroughly. The complete medium (mTeSR medium) can be stored at 4°C for 2 weeks, or aliquoted into 50 mL centrifuge tubes and stored at -20°C for 6 months. Thaw overnight at 4°C before use, avoiding repeated freeze-thaw cycles. Before use, bring the required amount of mTeSR medium to room temperature or heat it to 37°C in a metal bath.

[0034] If using TeSR™-E8™ cell media (E8 media), follow the reagent manufacturer's instructions and use a preparation method similar to mTeSR media.

[0035] (4) Prepare hiPSC cryopreservation solution After thoroughly mixing KOSR and DMSO at a ratio of 9:1, pre-cool at 4°C for later use. Store iPSCs in 500 μL of cryopreservation solution per tube.

[0036] The reprogrammed hiPSCs were propagated at P5-P10 passages and then cryopreserved for preservation.

[0037] Culture hiPSCs to 70%~80% confluence (specifically, use mTeSR plus to culture for 3~4 days after subculture at 37℃ and 5% CO2), estimate the degree of differentiation by microscopic observation, and ensure that the degree of differentiation does not exceed 10%, then proceed with the following steps.

[0038] II. Primary Brain Organoid Culture Prepare 100 mL of EB inoculation medium: Mix 75 mL of DMEM / F12, 20 mL of KnockOut™ Serum Replacement, 3 mL of fetal bovine serum for embryonic stem cells, 1 mL of glutamine, and 1 mL of MEM non-essential amino acid solution to obtain basal medium 1; add 2-mercaptoethanol, bFGF, and Y-27632 to basal medium 1 to make the final concentration of 2-mercaptoethanol 0.1 mM, the final concentration of bFGF 4 ng / mL, and the final concentration of Y-27632 50 μM to obtain EB inoculation medium.

[0039] Prepare 100 mL of ectoderm induction medium: Mix 97 mL of DMEM / F12, 1 mL of N-2 supplement, 1 mL of glutamine and 1 mL of MEM non-essential amino acid solution to obtain basal medium 2; add heparin to basal medium 2 to make the final concentration of heparin 1 μg / mL to obtain ectoderm induction medium.

[0040] (1) Take the hiPSCs obtained in step one above, observe under a light microscope whether there are differentiated regions in the hiPSCs, scrape or aspirate with a pipette tip to remove differentiated cells, then aspirate the cell medium and gently wash the cells in the wells with 1 mL of DPBS. Aspirate the DPBS, add 1 mL of GCDR mild cell dissociation solution, and incubate at 37°C for 8 min; use a 1 mL pipette to slowly and gently pipette the cells 3 times to resuspend the cells, and transfer the cell suspension to a sterile 15 mL centrifuge tube; then rinse the well plate with 1 mL of EB seeding medium, and then transfer the rinsing solution to a 15 mL centrifuge tube containing cells, and centrifuge at 800 rpm for 3 min; discard the supernatant, add 1 mL of EB seeding medium, gently pipette to resuspend the cells, and then use a hemocytometer to count the cells; calculate the cell suspension volume required to obtain 50,000 cells / mL, and add this volume of cell suspension to an appropriate volume of EB seeding medium.

[0041] (2) Add 100 μL of cell suspension from step (1) to each well of an ultra-low adhesion 96-well cell culture plate (5000 cells / well); place the 96-well plate in a 37°C incubator and continue culturing for 48 h (2 days) to obtain the first stage embryoid body; (3) Add 100 μL of EB inoculation medium to each well of the 96-well plate of the first-stage embryoid and incubate at 37°C for 48 h. Observe the formation of the second-stage embryoid under an optical microscope. The embryoid has a diameter of 400-600 μm and has a dense center and smooth, translucent edges.

[0042] (4) Add 500 μL of ectoderm induction medium to each well of an ultra-low adhesion 24-well cell culture plate and equilibrate it in a 37°C incubator beforehand. Transfer 1-2 second-stage embryoids to each well of the 24-well plate: using a 200 μL wide-mouth pipette tip, aspirate approximately 50 μL of liquid from one well of the 96-well plate from step (3) to obtain second-stage embryoids; carefully drain the liquid back into the well to remove most of the medium, leaving the second-stage embryoids in the pipette tip; transfer the second-stage embryoids to one well of the 24-well plate containing ectoderm induction medium. Before placing it in the incubator, shake the plate back and forth 3 times to ensure that the second-stage embryoids are evenly distributed in the wells. Second-stage embryoids in contact with each other are more likely to fuse. If a large number of second-stage embryoids are observed to merge, only one second-stage embryoid should be transferred per well.

[0043] After being cultured in a 37°C incubator for 48 hours, it was observed that the second-stage embryoids maintained smooth edges and formed optically translucent edges, thus obtaining the primary brain organoids.

[0044] III. Primary Vascular Organoid Culture Preparation of 100 mL mesodermal induction medium: consisting of 100 mL APEL2 and CHIR-99021 with a final concentration of 6 μM.

[0045] 100 mL of endothelial cell induction medium was prepared by consisting of 100 mL of APEL2, VEGF165 at a final concentration of 50 ng / mL, BMP4 at a final concentration of 25 ng / mL, and bFGF at a final concentration of 10 ng / mL.

[0046] Preparation of 100mL vascular maturation culture medium: consisting of 100mL Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50ng / mL.

[0047] (1) Heat the mesodermal induction medium to 37°C. Add 100 μL of preheated mesodermal induction medium to each well of a new ultra-low adhesion 96-well plate. Transfer the first-stage embryoid obtained in step (2) of step two to the 96-well plate containing the mesodermal induction medium through a 1000 μL wide-mouth pipette tip. Continue culturing for 48 h to obtain the mesodermal layer.

[0048] (2) Prepare a new ultra-low adhesion 96-well plate, add 100 μL of endothelial cell induction medium to each well, transfer the mesoderm obtained in step (1) to the 96-well plate containing endothelial cell induction medium through a 1000 μL wide-mouth pipette tip, and continue to culture for 48 h to obtain endothelial cells.

[0049] (3) Gently aspirate the endothelial cell induction medium from the ultra-low adhesion 96-well plate, taking care not to disturb the organoids as much as possible. Then, add 100 μL of vascular maturation culture medium to each well and continue culturing for 48 h to obtain primary vascular organoids.

[0050] IV. Mixed Organoid Culture (1) Thaw Matrigel in advance in a 4°C freezer. During the experiment, Matrigel must be kept on ice to prevent solidification. All experimental supplies that come into contact with Matrigel, such as pipette tips, must be pre-cooled by refrigerating at -20°C for at least 30 minutes before use.

[0051] (2) Place the sterile primary brain organoid coated silicone plate into an empty, sterile 10cm dish for later use; using a 1000μL wide-mouth pipette tip, aspirate 25~50μL of medium from each well of the 24-well plate to transfer one primary vascular organoid and one primary brain organoid to the same well on the silicone plate embedding surface. Repeat this step until about 12~16 “1+1” organoid groups are collected on the silicone plate embedding surface.

[0052] (3) Remove the medium, being careful to avoid removing the organoids, and then make the organoid spheres in close contact; using a 200 μL pre-cooled pipette tip, add 18 μL of Matrigel to each organoid sphere (keeping it consistent with the same batch); using a new 200 μL pre-cooled pipette tip, place the organoid spheres as close to the center of the droplet as possible, and ensure their arrangement order; place the silica gel plate in an incubator at 37°C for 45 min to allow the Matrigel to solidify, thus obtaining mixed organoids.

[0053] V. Preliminary vascularized brain organoid culture Preparation of 100 mL neural induction culture medium: Mix 48 mL DMEM / F12, 48 mL Neurobasal medium, 0.5 mL N-2 supplement, 1 mL glutamine, 0.5 mL MEM non-essential amino acid solution, 1 mL B27 vitamin A-free solution, and 1 mL penicillin-streptomycin to obtain basal medium 3; add 2-mercaptoethanol to basal medium 3 to a final concentration of 50 μM and insulin to a final concentration of 2.5 μg / mL to obtain neural induction culture medium.

[0054] Mix the neural induction culture medium and the vascular maturation culture medium from step three at a volume ratio of 1:1 to obtain a mixture containing both media. Remove the silica gel plate from step four, and use sterile forceps to grasp the surface of the plate containing Matrigel droplets from a corner. Suspend the silica gel plate above one well of a 6-well ultra-low adhesion cell culture plate. Use a 1 mL pipette to aspirate the mixture containing the neural induction culture medium and the vascular maturation culture medium. Then, gently rinse the surface of the silica gel plate with 3 mL of Matrigel droplets, dripping them into the well. Repeat until all Matrigel droplets have entered the well. Incubate at 37°C for 72 hours to obtain preliminarily vascularized brain organoids.

[0055] VI. Using neural maturation culture media and vascular maturation culture media to culture preliminarily vascularized brain organoids Preparation of 100 mL neural maturation culture medium: Mix 48 mL DMEM / F12, 48 mL Neurobasal medium, 0.5 mL N-2 supplement, 1 mL glutamine, 0.5 mL MEM non-essential amino acid solution, 1 mL B27 (vitamin A-free), and 1 mL penicillin-streptomycin to obtain basal medium 3; add 2-mercaptoethanol (final concentration 50 μM), insulin (final concentration 2.5 μg / mL), VEGF165 (final concentration 20 ng / mL), brain-derived neurotrophic factor (final concentration 20 ng / mL), and hydrocortisone (final concentration 0.2 µg / mL) to basal medium 3 to obtain neural maturation culture medium.

[0056] The neural maturation culture medium and the vascular maturation culture medium from step three were mixed at a volume ratio of 1:1 to obtain a mixture containing both neural and vascular maturation culture media. The mixture containing both neural induction and vascular maturation culture media from the culture plate in step five was discarded, and the culture medium was replaced with the mixture containing both neural and vascular maturation culture media. The plate was then incubated at 37°C, with the medium changed every 3 days. After 28 days of culture, vascularized brain organoids were obtained, which served as a model of Moyamoya disease.

[0057] Long-term culture can be carried out by continuously changing the medium. Change the medium every 3 to 4 days, and change 3 mL per well of a 6-well plate.

[0058] Comparative Example 1 The difference from Example 1 is that the CD34+ hematopoietic progenitor cells in step one are taken from the peripheral blood of healthy individuals; all other aspects are the same as in Example 1. Vascularized brain organoids were constructed to obtain healthy controls.

[0059] Experimental Example 1 Vascularized brain organoids from patients with Moyamoya disease obtained in Example 1 and vascularized brain organoids from healthy controls obtained in Comparative Example 1 were subjected to frozen sections and tissue immunofluorescence staining. (The following reagents were purchased from: OCT tissue embedding medium from Sakura Japan, catalog number 4583; primary antibody diluent from Beijing Solarbio Science & Technology Co., Ltd., catalog number A1810; antibody diluent from Beijing Solarbio Science & Technology Co., Ltd., catalog number A1800; anti-fluorescence attenuation mounting medium from Beijing Solarbio Science & Technology Co., Ltd., catalog number S2100; anti-PDGFRβ-antibody from Proteintech, catalog number 13449-1-AP; anti-CD31-antibody from Proteintech, catalog number 66065-2-Ig; anti-β-3Tubulin antibody from Proteintech, catalog number 10068-1-AP; donkey anti-mouse IgG (Alexa Fluor® 488) secondary antibody from Abcam USA, catalog number ab150105; donkey anti-mouse IgG...) The secondary antibody (AlexaFluor® 594) was purchased from Abcam USA, catalog number ab150108; the donkey anti-rabbit IgG (AlexaFluor® 488) secondary antibody was purchased from Abcam USA, catalog number ab150073; the donkey anti-rabbit IgG (AlexaFluor® 594) secondary antibody was purchased from Abcam USA, catalog number ab150076; the immunohistochemistry pen was purchased from Abcam USA, catalog number ab2601. 1. Preparation of experimental reagents 1) Prepare a permeation solution containing 0.25% Triton X-100 Measure 49.875 mL of PBS phosphate buffer into a 50 mL centrifuge tube. Using a 200 μL wide-mouth pipette tip, slowly aspirate 125 μL of Triton X-100 stock solution and add it to the PBS. Mix thoroughly to prepare 50 mL of cell permeation buffer containing 0.25% Triton X-100. Store at room temperature.

[0060] 2) Prepare a blocking solution containing 5% donkey serum and 1% BSA. Take 8.5 mL of 0.1% PBS-T solution into a 15 mL centrifuge tube, add 500 μL of donkey serum stock solution for blocking and 1 mL of 10% bovine serum albumin (BSA) (dissolved in ddH2O), mix thoroughly to prepare 10 mL of cell blocking solution containing 5% donkey serum and 1% BSA, store at 4°C, and bring to room temperature before use.

[0061] 3) Prepare 15% sucrose solution and 30% sucrose solution for tissue dehydration. Weigh 15g of sucrose using an electronic balance, dissolve it in 80mL of PBS phosphate buffer into a reagent bottle, stir thoroughly until the sucrose dissolves, and then bring the volume to 100mL in a graduated cylinder to obtain a 15% sucrose solution. Transfer the solution to a reagent bottle and store at 4℃. Prepare a 30% sucrose solution in the same way and store at 4℃.

[0062] 4) Prepare a 0.5% gelatin solution Weigh 1g of gelatin granules using an electronic balance, then heat and stir continuously until fully dissolved in 200mL ddH2O to prepare a 0.5% gelatin solution. After dispensing, store at -20℃ for later use. Before use, thaw thoroughly and allow to return to room temperature.

[0063] 2. Tissue pretreatment (dehydration, fixation and embedding) 1) Gently remove the vascularized brain organoids into a 15mL centrifuge tube using a serum pipette, carefully aspirate any residual medium, wash three times with an appropriate amount of PBS solution, add 2mL of 4% tissue cell fixative that has been brought to room temperature, and fix overnight at 4°C. 2) Discard the fixative, wash three times with an appropriate amount of PBS solution, add at least 10 mL of pre-cooled 15% sucrose solution to the centrifuge tube, place it on a 4°C rotating shaker, and pre-dehydrate overnight; 3) The next day, when the organoids settle to the bottom of the 15% sucrose solution, the 15% sucrose solution is removed and replaced with at least 10 mL of pre-cooled 30% sucrose solution. The organoids are then placed in a 4°C rotating shaker for dehydration overnight. After dehydration, the organoids will settle to the bottom of the 30% sucrose solution. 4) Before tissue embedding, use aluminum foil to fold the tissue embedding box to an appropriate size for later use; 5) Discard the 30% sucrose solution, wash once with PBS solution, then dilute the 0.4% trypan blue staining solution 50 times with PBS solution and stain the tissue for 5 minutes to allow the tissue to be stained and visualize the subsequent sectioning process. 6) Discard the trypan blue staining solution, wash once with PBS solution, and gently transfer the brain-like tissue to absorb excess water on absorbent paper to prevent adhesion. 7) Inject the tissue embedding agent OCT into the embedding cassette, then carefully transfer the brain-like tissue to the OCT center, avoiding the formation of air bubbles, and then immediately place it in dry ice for quick freezing. After freezing, transfer the tissue embedding block to a -20°C freezer for storage. 8) When slicing tissue, remove the foil from the embedding cassette at -20℃ in the cryostat and fix the tissue embedding block to the sample holder using OCT. After low-temperature curing, slice the tissue on the cryostat with a slice thickness of 30μm. Quickly mount the continuous tissue slices onto glass slides pretreated with gelatin, bake the slides at 37℃ for 10 min, and then store them in a -20℃ freezer.

[0064] 3. Immunofluorescence staining 1) Vascularized brain organoid tissue sections were gently washed twice in room temperature PBS solution to remove as much gelatin and OCT embedding agent as possible from the patches; 2) After the slides have been allowed to air dry slightly at room temperature, circle the patch area with an immunohistochemical pen. First, add permeabilization buffer containing 0.25% Triton X-100 to the tissue site, place it in a humidified chamber, and incubate at room temperature for 30 minutes to permeabilize the tissue. Remove the permeabilization buffer, and block the tissue in a humidified chamber at room temperature for 30 minutes with blocking buffer containing 5% donkey serum and 1% BSA. After blocking, remove the blocking buffer; no washing is required. 3) Using primary antibody dilution buffer, dilute the primary antibody to the corresponding antibody working solution according to the IHC dilution ratio. After uniformly adding it to the tissue area, incubate it overnight at 4°C in a humidified chamber. 4) The next day, remove the primary antibody working solution and wash three times with PBS solution; prepare the fluorescent secondary antibody working solution using secondary antibody dilution buffer, add it evenly to the tissue area, and incubate in a humidified chamber at room temperature for 2 hours. 5) Remove the secondary antibody working solution, add an appropriate amount of DAPI staining solution, and incubate at room temperature in the dark for 5 minutes to label the cell nuclei; 6) Remove DAPI, wash three times with PBS solution, air dry at room temperature in the dark, then add an appropriate amount of anti-fluorescence decay mounting medium, cover with a coverslip, try to avoid air bubbles, and seal the edges of the coverslip with nail polish or resin. 7) Observe immediately under a fluorescence microscope or a confocal microscope, or place the mounted slide in a refrigerator at 4°C and store it in the dark for no more than 7 days to prevent fluorescence quenching.

[0065] 4. Calculation and Analysis Scheme Angiogenesis Analyzer in ImageJ software was used for vascular-related calculations and analyses.

[0066] 5. Results Immunofluorescence staining results as follows Figure 2As shown, the star-shaped markers indicate the small branches of the vascular network that the patient cannot form, indicating that the vascularized brain organoid constructed in Example 1 of this invention reproduces the pathological features of the "smoke-like" abnormal collateral formation in the clinical presentation of Moyamoya disease.

[0067] The results of immunofluorescence staining analysis are as follows: Figure 3 As shown, compared with healthy controls, patients with moyamoya disease showed a significant decrease in the number of vascularized brain organoid junctions, the total number of major junctions, and the total number of major segments, indicating a reduced ability to form vascular networks and an increase in fine branches (due to...). Figure 3 As can be seen from E, the total branch length is the same, but Figure 3 The reduction in major segments in the D diagram indicates an increase in small branches, while the reduction in large vessel segments closely matches the clinicopathological features.

[0068] Experimental Example 2 RNA extraction and qPCR detection of vascularized brain organoids obtained in Example 1 and Comparative Example 1 (Some reagents were purchased from: TRIzol™ reagent from Invitrogen, USA, catalog number 15596018; DEPC-water from Invitrogen, USA, catalog number AM9915G; RNaseZap™ RNase scavenger from Invitrogen, USA, catalog number AM9780; Transcriptor High Fidelity cDNA synthesis kit from Roche, Switzerland, catalog number 5091284001) 1. Total RNA was extracted from cells using the TRIzol method. 1) Wash tissue samples with PBS, transfer them to RNase-free 1.5mL centrifuge tubes, add 500µL of TRIzol reagent, sonicate to break them up, add TRIzol reagent to make up to 1mL, and let stand at room temperature for 5min. 2) Pre-cool the centrifuge, add 1 / 5 volume (200 μL) of TRIzol in chloroform to the above 1.5 mL EP tube, add 200 μL of chloroform (half the cell count), shake vigorously for 15 seconds (do not vortex), let stand at room temperature for 3 minutes, and centrifuge at 12000 rpm and 4℃ for 15 minutes. 3) After centrifugation, remove the tube and let it stand. Carefully transfer the upper aqueous phase to a clean 1.5 mL EP tube, add an equal volume of isopropanol, mix well, and let it stand at room temperature for 10 min. 4) Centrifuge at 4℃ and 12000 rpm for 10 min, then discard the supernatant; 5) Add 1 mL of 80% ethanol solution (prepared by adding DEPC water to anhydrous ethanol) to the EP tube and invert the tube to wash the RNA; 6) Centrifuge at 7500 rpm and 4℃ for 5 min, carefully discard the supernatant and keep the flaky precipitate; 7) Place in a clean, well-ventilated area at room temperature for 3 minutes to allow residual ethanol to evaporate; 8) Add an appropriate amount of DEPC water to dissolve the RNA (10-30 μL of tissue). 9) After analyzing the total RNA bands by agarose gel electrophoresis and measuring the total RNA concentration by spectrophotometer, immediately reverse transcribe it into cDNA according to the following steps and store it at -20℃. 10) The remaining total RNA was aliquoted and stored at -80°C.

[0069] 2. Reverse transcription of RNA into cDNA The reaction systems are shown in Tables 2 and 3.

[0070] Table 2 Reaction System 1

[0071] Table 3 Reaction System 2

[0072] 1) Prepare reaction system 1, set up the PCR instrument, and react at 65℃ for 10 min in the PCR instrument, then place it on ice for 2 min.

[0073] 2) Prepare reaction system 2, mix gently without vortexing, then add it to the tube in step 1), and mix gently again; 3) In the PCR instrument, react at 25℃ for 10 min, then at 55℃ for 30 min, and then at 85℃ for 5 min to inactivate the enzyme; 4) The reversed cDNA needs to be stored at -20℃ for later use.

[0074] 3. qPCR detection of related gene expression The forward and reverse primers of commercially available kits were used to detect GFAP, MAP2, PDGFRβ, and GAPDH.

[0075] The qPCR reaction system is shown in Table 4: Table 4 qPCR reaction system

[0076] The qPCR reaction program was as follows: pre-denaturation at 95℃ for 10 min; amplification cycle: denaturation at 95℃ for 10 s, annealing at 60℃ for 15 s, extension at 72℃ for 30 s, for 40 cycles; melting curve: 95℃ for 10 s, then slowly cooling to 65℃.

[0077] GAPDH was used as an internal control, and the Ct values ​​of each gene were measured when it was amplified to the baseline level. The relative gene expression level was calculated by the ΔΔCt method.

[0078] The results are as follows Figure 4 As shown, compared with healthy controls, patients with Moyamoya disease showed decreased PDGFRβ expression, indicating a reduction in pericyte numbers and possible vascular dysfunction; decreased MAP2 expression suggested a reduction in the number of mature neurons or delayed development; and increased GFAP expression suggested an increase in the number of astrocytes, consistent with the clinicopathological phenotype.

[0079] Experimental Example 3 Nitric oxide content detection experiment (using the total nitric oxide detection kit, catalog number S0023, manufactured by Beyotime Biotechnology) 1. Preparation of experimental reagents 1) Preparation of standard products Dilute the 1M NaNO2 standard to 2, 5, 10, 20, 40, 60, and 80 μM using the solution used in preparing or diluting the sample (in this experiment, vascularized brain organoid maintenance culture medium (a mixture of neural maturation culture medium and vascular maturation culture medium)) and keep it on ice for later use.

[0080] 2) Preparation of NADPH solution Add 1 ml of Milli-Q grade ultrapure water to 5 mg NADPH, mix thoroughly by inverting, and then bring the volume up to 3 mL to prepare a 2 mM NADPH working solution. Take out the portion used in this experiment, and aliquot the remaining NADPH solution and store it in a freezer at -80°C.

[0081] 2. Nitric oxide detection 1) Collect 400 μL of cell supernatant from each sample and keep it on ice for later use; 2) Remove FAD, Nitrate Reductase, and LDH and place them on ice; at the same time, bring GriessReagent I and Griess Reagent II to room temperature before use; all reactions must be carried out in the dark. 3) Add 60 μL of sample, 5 μL of NADPH working solution, 10 μL of FAD and 5 μL of Nitrate Reductase to the sample well in sequence, mix well and incubate at 37°C for 30 min, taking care to avoid air bubbles; replace the 60 μL of sample with 60 μL of culture medium and standard for blank control and standard. 4) After incubation, add 10 μL LDH Buffer and 10 μL LDH in sequence, mix gently, and incubate at 37°C for 30 min. 5) After incubation, add 50 μL of Griess Reagent I and 50 μL of Griess Reagent II in sequence, mix gently, incubate at room temperature for 10 min, and then measure A540.

[0082] The results are as follows Figure 5 As shown, compared with healthy controls, the amount of NO secretion in the supernatant of vascularized brain organoid cultures from patients with Moyamoya disease was significantly reduced, indicating abnormal vascular function, endothelial damage, or abnormal angiogenesis.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a Moyamoya disease model, characterized in that, Includes the following steps: CD34+ hematopoietic progenitor cells were collected from peripheral blood of patients with Moyamoya disease, reprogrammed to generate hiPSCs, and the hiPSCs were cultured to 70%~80% confluence and differentiation degree not exceeding 10%. hiPSCs were cultured in EB inoculation medium for 45-51 hours to obtain the first-stage embryoids; The first-stage embryoids were cultured for 45-51 hours using EB inoculation medium to obtain the second-stage embryoids. The second-stage embryoids were then cultured for 44-52 hours using ectoderm induction medium to obtain primary brain organoids. The first-stage embryoids were cultured in mesodermal induction medium for 44-52 hours, then in endothelial cell induction medium for 44-52 hours, and then in vascular maturation culture medium for 48-96 hours to obtain primary vascular organoids. The primary brain organoid and the primary blood vessel organoid are fused to obtain a hybrid organoid; The hybrid organoids were co-cultured with neural induction culture medium and vascular maturation culture medium for 68-76 hours to obtain pre-vascularized brain organoids. A Moyamoya disease model was obtained by culturing pre-vascularized brain organoids for more than 28 days using neural maturation culture media and vascular maturation culture media. The EB inoculation medium consists of basal culture medium 1, 2-mercaptoethanol at a final concentration of 0.1 mM, bFGF at a final concentration of 4 ng / mL, and Y-27632 at a final concentration of 50 μM. The basal culture medium 1 consists of the following ingredients by volume fraction: 75% DMEM / F12, 20% KnockOut™ Serum Replacement, 3% fetal bovine serum for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution. The ectoderm induction medium consists of basal culture medium 2 and heparin at a final concentration of 1 μg / mL. The basal culture medium 2 consists of the following ingredients by volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine and 1% MEM non-essential amino acid solution. The volume ratio of the neural induction culture medium to the vascular maturation culture medium is 1:

1. The neural induction culture medium consists of basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, and insulin at a final concentration of 2.5 μg / mL. The basal culture medium 3 consists of the following ingredients by volume fraction: 48% DMEM / F12, 48% Neurobasal medium, 0.5% N-2 supplement, 1% glutamine, 0.5% MEM non-essential amino acid solution, 1% B27 without vitamin A, and 1% penicillin-streptomycin. The volume ratio of the neural maturation culture medium to the vascular maturation culture medium is (1.5:1) to (1:1.5). The neural maturation culture medium is composed of the basal culture medium 3, 2-mercaptoethanol at a final concentration of 50 μM, insulin at a final concentration of 2.5 μg / mL, VEGF165 at a final concentration of 20 ng / mL, brain-derived neurotrophic factor at a final concentration of 20 ng / mL, and hydrocortisone at a final concentration of 0.2 µg / mL. The fusion process uses Matrigel; 15-20 μL of Matrigel is required to encapsulate a primary brain organoid and a primary vascular organoid. The mesodermal induction medium consists of APEL2 and CHIR-99021 at a final concentration of 6 μM; the endothelial cell induction medium consists of APEL2, VEGF165 at a final concentration of 50 ng / mL, BMP4 at a final concentration of 25 ng / mL, and bFGF at a final concentration of 10 ng / mL; the vascular maturation culture medium consists of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL.

2. The method according to claim 1, characterized in that, The reprogramming was performed using the Sendai virus reprogramming kit.

3. The method according to claim 1, characterized in that, The culture temperature is 36.5~37.5℃.

4. A model of Moyamoya disease, characterized in that, It is obtained by the method described in any one of claims 1 to 3.

5. The application of the method according to any one of claims 1 to 3 or the moss disease model according to claim 4 in the screening of drugs for the prevention and treatment of moss disease.

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