Smoke disease model and construction method and application thereof

Abstract

The invention provides a smoke disease model and a construction method and application thereof, and belongs to the technical field of smoke disease model construction. The method for constructing the smog disease model comprises the following steps: extracting CD34 + hematopoietic progenitor cells in peripheral blood of a smog disease patient, reprogramming to generate hiPSC, further differentiating into brain organs and vascular organs, fusing the brain organs and the vascular organs, and culturing and constructing the vascularized brain organ smog disease model. The smoke disease model constructed by adopting the construction method disclosed by the invention can simulate and reproduce phenotypes such as tiny branch increase, macrovascularization, pericytopenia, smooth muscle fibrosis deposition, abnormal angiogenesis increase and the like in clinical core pathological phenotypes of smoke disease patients, and is superior to existing animal and cell models to a certain extent.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of moyamoya disease model construction, and particularly relates to a moyamoya disease model and a construction method and application thereof. BACKGROUND

[0002] Moyamoya disease is a chronic progressive cerebral vascular occlusive disease with unknown etiology. The disease is named moyamoya disease because it looks like "smoke" in cerebral angiography images. The main pathological features of moyamoya disease are stenosis or occlusion of the bilateral internal carotid artery terminal and the cerebral artery origin, and the formation of abnormal collateral vascular network. The pathogenesis of moyamoya disease is complex, and it is currently believed that its pathogenesis may involve multiple factors, including genetic factors such as gene mutation, environmental factors such as immune and inflammatory response, vascular endothelial dysfunction, and oxidative stress.

[0003] The existing models of moyamoya disease are mainly animal models, most of which are constructed using mice and zebrafish. Common construction methods include: a surgical model that usually induces chronic cerebral hypoperfusion by permanently ligating the common carotid artery or the internal carotid artery; an immune model that simulates the immune response in moyamoya disease by inducing vasculitis-like pathology; and a genetic model constructed by knocking down or knocking out the RNF213 gene. However, all of the current animal models and cell models cannot simulate the core pathological features of moyamoya disease, i.e., "smoke-like" abnormal collateral formation or spontaneous vascular stenosis, which has serious defects. In addition, since the pathological detection of the affected area of moyamoya disease patients cannot be directly observed by biopsy, it further exacerbates the difficulties in clinical and basic research, and there is an urgent need to find a new moyamoya disease model that is closer to moyamoya disease patients and can completely reproduce the core phenotype of moyamoya disease. SUMMARY

[0004] Therefore, the purpose of the present application is to provide a method for constructing a moyamoya disease model, which can completely reproduce the core pathological features of moyamoya disease.

[0005] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions: The present application provides a method for constructing a moyamoya disease model, comprising the following steps: Take CD34+ hematopoietic progenitor cells in the peripheral blood of a moyamoya disease patient, reprogram them to generate hiPSCs, and culture the hiPSCs to a confluence degree of 70% to 80%, with a differentiation degree of no more than 10%; Culture the hiPSCs using EB inoculation medium for 45 to 51 hours to obtain first-stage embryoid bodies; Continue to culture the first-stage embryoid bodies using EB inoculation medium for 45 to 51 hours to obtain second-stage embryoid bodies, and culture the second-stage embryoid bodies using ectoderm induction medium for 44 to 52 hours to obtain primary brain organoids; The first-stage embryoid body is cultured for 44-52 hours using mesoderm induction medium, then cultured for 44-52 hours using endothelial cell induction medium, and then cultured for 48-96 hours using blood vessel maturation culture medium to obtain a primary blood vessel organoid; The primary brain organoid and the primary blood vessel organoid are fused to obtain a mixed organoid; The mixed organoid is cultured for 68-76 hours using neural induction culture medium and blood vessel maturation culture medium to obtain a preliminarily vascularized brain organoid; The preliminarily vascularized brain organoid is cultured for more than 28 days using neural maturation culture medium and blood vessel maturation culture medium to obtain a moyamoya disease model; The EB inoculation medium is composed of basal 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, and the basal medium 1 is composed of the following raw materials in terms of volume fraction: 75% DMEM / F12, 20% KnockOut™ Serum Replacement, 3% fetal bovine serum special for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution.

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

[0007] Preferably, the ectoderm induction medium is composed of basal medium 2 and heparin at a final concentration of 1 μg / mL, and the basal medium 2 is composed of the following raw materials in terms of volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine, and 1% MEM non-essential amino acid solution.

[0008] Preferably, the mesoderm induction medium is composed of APEL2 and CHIR-99021 at a final concentration of 6 μM; the endothelial cell induction medium is composed 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 blood vessel maturation culture medium is composed of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL.

[0009] Preferably, Matrigel is used for the fusion, and 15-20 μL of Matrigel is needed to wrap one primary brain organoid and one primary blood vessel organoid.

[0010] Preferably, the volume ratio of the neural induction culture medium and the vascular maturation culture medium is 1:1, the neural induction culture medium is composed of basal medium 3, 2-mercaptoethanol with a final concentration of 50 μM and insulin with a final concentration of 2.5 μg / mL, and the basal medium 3 is composed of the following raw materials in terms of 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 and the vascular maturation culture medium is (1.5:1)~(1:1.5), the neural maturation culture medium is composed of the basal medium 3, 2-mercaptoethanol with a final concentration of 50 μM, insulin with a final concentration of 2.5 μg / mL, VEGF165 with a final concentration of 20 ng / mL, brain-derived neurotrophic factor with a final concentration of 20 ng / mL and hydrocortisone with a final concentration of 0.2 µg / mL.

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

[0013] The application also provides a moyamoya disease model, which is obtained by the above method.

[0014] The application also provides the application of the above method or the above moyamoya disease model in drug screening for moyamoya disease.

[0015] The application has the following advantages: Traditional animal models and cell models cannot currently simulate the core pathological characteristics of moyamoya disease, i.e. "smoke-like" abnormal collateral formation or spontaneous vascular stenosis, and have serious defects. Compared with traditional models, the moyamoya disease model constructed by 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. Similarly, the application realizes complete humanization of the in vitro model, which is closer to the human body, can realize high-throughput preparation and serve as an in vitro drug screening platform, and can avoid ethical controversy caused by animal models. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 A flow chart for constructing a vascularized brain organoid and a bright field mode image, wherein A is a flow chart for constructing a vascularized brain organoid, and B is a bright field microscope image of a brain organoid, a vascular organoid and a fused vascularized brain organoid; Figure 2Figure 6 is a comparative diagram of immunofluorescence staining of vascularized brain organoids derived from healthy controls and patients with moyamoya disease, wherein the star mark indicates that the patient cannot form fine branches of the vascular network; Figure 3 Figure 7 is an analysis result of immunofluorescence staining, wherein A is an immunofluorescence staining diagram of endothelial marker CD31 and neural marker TUJ1 in a vascularized brain organoid; B is a comparative diagram of total number statistics of vascular junctions of vascularized brain organoids of healthy controls and patients with moyamoya disease based on CD31 staining; C is a comparative diagram of total number statistics of major vascular junctions of vascularized brain organoids of healthy controls and patients with moyamoya disease based on CD31 staining; D is a comparative diagram of total number statistics of major segments of vascularized brain organoids of healthy controls and patients with moyamoya disease based on CD31 staining; E is a comparative diagram of total length statistics of all branches of blood vessels of vascularized brain organoids of healthy controls and patients with moyamoya disease based on CD31 staining, T-test, mean ± SEM, n = 9; ns represents p≥0.05, and ** represents p<0.01; Figure 4 Figure 8 is a result of qPCR detection of expression levels of genes related to vascularized brain organoids of healthy controls and patients with moyamoya disease, wherein A is the relative expression amount of GFAP gene mRNA in vascularized brain organoids derived from healthy controls and patients with moyamoya disease; B is the relative expression amount of MAP2 gene mRNA in vascularized brain organoids derived from healthy controls and patients with moyamoya disease; C is the relative expression amount of PDGFRβ gene mRNA in vascularized brain organoids derived from healthy controls and patients with moyamoya disease; T-test, mean ± SEM, n = 3; * represents p<0.05, and ** represents p<0.01; Figure 5 Figure 9 is a statistical diagram of total NO concentration detection of culture supernatant of vascularized brain organoids derived from healthy controls and patients with moyamoya disease, T-test, mean ± SEM, n = 6. DETAILED DESCRIPTION

[0017] The present application provides a method for constructing a moyamoya disease model, comprising the following steps: CD34+ hematopoietic progenitor cells in peripheral blood of a patient with moyamoya disease are taken, reprogrammed to generate hiPSC, and the hiPSC is cultured to 70%-80% confluence, and the degree of differentiation is not more than 10%; The hiPSC is cultured using EB inoculation medium for 45-51h to obtain a first-stage embryoid body; The first-stage embryoid body is continuously cultured using EB inoculation medium for 45-51h to obtain a second-stage embryoid body, and the second-stage embryoid body is cultured using ectoderm induction medium for 44-52h to obtain a primary brain organoid; The first-stage embryoid body is cultured for 44-52 hours using mesoderm induction medium, then cultured for 44-52 hours using endothelial cell induction medium, and then cultured for 48-96 hours using blood vessel maturation culture medium to obtain a primary blood vessel organoid; The primary blood vessel organoid and the primary brain organoid are fused to obtain a mixed organoid; The mixed organoid is cultured for 68-76 hours using neural induction culture medium and blood vessel maturation culture medium to obtain a preliminarily vascularized brain organoid; The preliminarily vascularized brain organoid is cultured for more than 28 days using neural maturation culture medium and blood vessel maturation culture medium to obtain a moyamoya disease model; The EB inoculation medium is composed of basal medium 1, 2-mercaptoethanol with a final concentration of 0.1 mM, bFGF with a final concentration of 4 ng / mL, and Y-27632 with a final concentration of 50 μM, and the basal medium 1 is composed of the following raw materials in terms of volume fraction: 75% DMEM / F12, 20% KnockOutTM Serum Replacement, 3% fetal bovine serum special for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution.

[0018] The present application extracts CD34+ hematopoietic progenitor cells from the peripheral blood of a moyamoya disease patient, reprograms the hiPSCs, and further differentiates the brain organoids and blood vessel organoids, and then fuses the brain organoids and blood vessel organoids to culture and construct a vascularized brain organoid moyamoya disease model. The flow chart of the construction method of the present application is shown in the figure. Figure 1 The method of the present application can construct an in vitro model that matches the clinical and pathological characteristics of moyamoya disease well, and the moyamoya disease model obtained by the construction method is superior to all existing animal and cell models; the construction process does not involve manipulation of embryos, can achieve complete humanization, and can avoid ethical issues; the moyamoya disease model provided by the present application has the characteristics of high throughput, can be used as a good platform for drug screening and mechanism research, provides a new and efficient tool for studying drug targets, and lays a foundation for personalized medicine because the organoids are completely derived from the patient's own cells.

[0019] The embryoid body (EB) in the present application refers to a three-dimensional cell aggregate formed by human induced pluripotent stem cells (hiPSC) under in vitro culture conditions, which has an inner, middle and outer three germ layer structure, is highly similar to the early embryonic development stage of mammals in morphology, and can provide a microenvironment related to early development to a certain extent.

[0020] The present application is not particularly limited to the method for obtaining CD34+ hematopoietic progenitor cells in the peripheral blood of a patient with moyamoya disease, and a method for obtaining CD34+ hematopoietic progenitor cells in the peripheral blood in the art can be used. The present application is not particularly limited to the method for culturing hiPSCs, and a method for culturing hiPSCs in the art can be used. In the present application, the reprogramming is preferably performed using a Sendai virus reprogramming kit. In the present application, the culture time for obtaining the first-stage embryoid bodies is preferably 47 to 49 h, and the culture time for obtaining the second-stage embryoid bodies is preferably 47 to 49 h. In the present application, the ectoderm induction medium preferably consists of basal medium 2 and heparin at a final concentration of 1 μg / mL, and the basal medium 2 consists of the following raw materials in terms of volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine, and 1% MEM non-essential amino acid solution. The culture time for obtaining the primary brain organoids is preferably 46 to 50 h, and more preferably 47 to 49 h. In the present application, the mesoderm induction medium preferably consists of APEL2 and CHIR-99021 at a final concentration of 6 μM. The culture time for culturing the first-stage embryoid bodies to the mesoderm using the mesoderm induction medium is preferably 46 to 50 h, and more preferably 47 to 49 h. In the present application, 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 culture time for culturing the mesoderm to endothelial cells using the endothelial cell induction medium is preferably 46 to 50 h, and more preferably 47 to 49 h. In the present application, the blood vessel maturation culture medium preferably consists of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL. The culture time for culturing the endothelial cells to the primary blood vessel organoids using the blood vessel maturation culture medium is preferably 50 to 94 h, and more preferably 55 to 90 h.

[0021] In the present application, Matrigel is preferably used to fuse the primary brain organoids and the primary blood vessel organoids. 15 to 20 μL of Matrigel is preferably used to wrap one primary brain organoid and one primary blood vessel organoid, and more preferably 17 to 19 μL of Matrigel is used. The present application is not particularly limited to the specific source of Matrigel, and a commercially available product in the art can be used.

[0022] In the present application, the volume ratio of the neural induction culture medium and the vascular maturation culture medium is preferably 1:1, the neural induction culture medium preferably consists of basal medium 3, 2-mercaptoethanol at a final concentration of 50 μM, and insulin at a final concentration of 2.5 μg / mL, the basal medium 3 consisting of the following raw materials in terms of 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. In the present application, the time for culturing the mixed organoids into the preliminarily vascularized brain organoids is preferably 70 to 74 h, more preferably 72 to 73 h.

[0023] In the present application, the volume ratio of the neural maturation culture medium and the vascular maturation culture medium is preferably (1.5:1) to (1:1.5), more preferably 1:1, the neural maturation culture medium preferably consisting of the basal 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 the present application, the temperature of the culture is preferably 36.5 to 37.5°C, more preferably 37°C. The present application is not particularly limited to the specific source of each raw material in each of the above culture media, and a conventional commercially available product in the art can be used.

[0024] In the present application, the brain organoid (BOr), also known as brain-like, is formed by in vitro three-dimensional culture of pluripotent stem cells, contains neurons and various glial cells, and can reproduce a miniature model of the mimicked organ part function. The vascular organoid (VOr) is generated by inducing differentiation of hiPSC, composed of various cell types (such as endothelial cells, smooth muscle cells, pericytes and stromal cells), and can simulate the formation, development and function of a miniature model of blood vessels. The vascularized brain organoid (VBOr), also known as vascularized brain, is formed by fusion culture of vascular organoids and brain organoids, completely removes the mouse-derived components in the traditional brain organoid vascularization technology, and realizes in vitro humanization. On the basis of brain organoids, vascularized brain adds the vascular system including vascular endothelial cells to support the neural development of brain organoids, promote the maturation of neurons and glial cells and reduce the apoptosis of cells in the central region of the organoid, and can reproduce the blood-brain barrier structure to a certain extent, thereby more truly simulating the development structure of human brain and providing a more accurate model and platform for moyamoya disease research and drug screening. The moyamoya disease model constructed by the vascularized brain of the present application can provide new possibilities for exploring the pathological mechanism of moyamoya disease and the interaction between neural-vascular networks, as well as the development of new targets for disease treatment.

[0025] The moyamoya disease model provided by the present application has the following advantages compared with other existing models in the art: the alpha-SMA in the smooth muscle tissue of the moyamoya disease model of the present application is increased, fibrosis occurs, spontaneous main vessel stenosis and occlusion occur, and small branches increase, while the above phenomena do not exist in the existing mouse moyamoya disease model in the art.

[0026] The present application also provides a moyamoya disease model, which is obtained by the above method. The present application also provides the application of the above method or the above moyamoya disease model in drug screening for moyamoya disease prevention and treatment.

[0027] The technical solutions provided by the present application will be described in detail below in combination with examples, but they should not be understood as limiting the scope of protection of the present application.

[0028] In the following examples, unless otherwise specified, all are conventional methods.

[0029] In the following examples, unless otherwise specified, all are conventional methods.

[0030] In the following examples, unless otherwise specified, all are conventional methods. Table 1 Sources of some raw materials

[0031] Example 1 A model of moyamoya disease was constructed as follows: I. Reprogramming of CD34+ hematopoietic progenitor cells and culture of hiPSCs (1) Peripheral blood of a patient with moyamoya disease was taken, and CD34+ progenitor cells were screened using peripheral blood magnetic bead sorting. The kit and magnetic pole were produced by stemcell technology, with item number: magnetic pole 18000; kit 05925. CD34+ hematopoietic progenitor cells in the peripheral blood of the patient with moyamoya disease were obtained, and reprogramming was performed according to the method shown in the CytoTune-iPS Sendai virus reprogramming kit to generate hiPSCs.

[0032] (2) Use rhVTN-N vitronectin to coat the hiPSC culture plate Under sterile operating conditions, 0.5 mg / mL of rhVTN-N vitronectin storage solution was diluted according to a ratio of 1:100 to 5 μg / mL as a working solution, which was prepared immediately before use. After adding, gently shake to make the solution evenly cover the entire surface of the cell culture plate, and then place it in a 37°C cell culture incubator for at least 30 min (or use a sealing film to seal and place it at 4°C overnight for coating. The cell culture plate coated at 4°C overnight needs to be warmed at room temperature for 30 min before use). After the coating solution supernatant is aspirated, the cells are inoculated immediately without using DPBS phosphate buffer for surface washing.

[0033] (3) Preparation of mTeSR™Plus cell medium (mTeSR medium) mTeSR™Plus 5x additive was thawed at room temperature or 4°C overnight. Before use, it needs to be fully restored to room temperature and gently mixed to avoid the appearance of flocculent precipitate, and avoid repeated freeze-thawing. Under sterile operating conditions, 100 mL of mTeSR™Plus 5x additive was added to 400 mL of mTeSR™Plus base medium, and mixed thoroughly. The complete medium (mTeSR medium) can be stored at 4°C for 2 weeks, or stored at -20°C for 6 months after being divided into 50 mL centrifuge tubes, and then thawed at 4°C overnight before use, avoiding repeated freeze-thawing. Before use, the required amount of mTeSR medium needs to be warmed to room temperature or heated to 37°C in a metal bath.

[0034] If TeSR™-E8™ cell medium (E8 medium) is used, it is prepared according to the instructions of the reagent supplier, using a similar configuration scheme as mTeSR medium.

[0035] (4) Preparation of hiPSC cryopreservation solution KOSR and DMSO were mixed at a ratio of 9:1 and then were placed in a 4℃ refrigerator for standby use. The iPS cells were cryopreserved at a concentration of 500 μL per tube.

[0036] The hiPSCs obtained after reprogramming were expanded at P5-P10 generations and the cells were cryopreserved for preservation.

[0037] The hiPSCs were cultured to a confluence of 70%-80% (specifically, the cells were cultured for 3-4 days at 37℃ and 5% CO2 after subculture), the differentiation degree was estimated by microscopic observation, and the differentiation degree was ensured to be no more than 10% to perform the following steps.

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

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

[0040] (1) Take the hiPSCs obtained in the above step one, observe under a light microscope whether there are differentiated areas in the hiPSCs, scrape or suck off the differentiated cells with a pipette tip, then suck off the cell medium, and gently wash the cells in the well with 1 mL of DPBS. Suck off the DPBS, add 1 mL of GCDR gentle cell dissociation reagent, and incubate at 37℃ for 8 min; use a 1 mL pipette to slowly and gently blow 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 inoculation medium, and transfer the rinse liquid to the 15 mL centrifuge tube containing the cells, and centrifuge at 800 rpm for 3 min; discard the supernatant, add 1 mL of EB inoculation medium, and gently blow to resuspend the cells, then use a hemocytometer to count the cells; calculate the volume of the cell suspension required to obtain 50,000 cells / mL, and add this volume of cell suspension to the appropriate volume of EB inoculation medium.

[0041] (2) Add 100 μL of cell suspension from step (1) to each well of an ultra-low attachment 96-well cell culture plate (5000 cells / well); place the 96-well plate in an incubator at 37°C for continued culture, and incubate for 48 h (2 days) with no disturbance to obtain the first-stage embryoid bodies; (3) Gently add 100 μL of EB seeding medium to each well of the 96-well plate containing the first-stage embryoid bodies, and place the plate in an incubator at 37°C for continued culture for 48 h. Observe the second-stage embryoid bodies under an optical microscope. The second-stage embryoid bodies have a diameter of 400-600 μm and have a dense center and a smooth and transparent edge.

[0042] (4) Add 500 μL of ectoderm induction medium to each well of an ultra-low attachment 24-well cell culture plate, and pre-equilibrate the plate in an incubator at 37°C. Transfer 1-2 second-stage embryoid bodies to each well of the 24-well plate: use a 200 μL wide-bore pipette tip to aspirate about 50 μL of liquid from one well of the 96-well plate of step (3) to obtain a second-stage embryoid body; carefully expel the liquid back into the well to remove most of the medium and retain the second-stage embryoid body in the pipette tip; transfer the second-stage embryoid body to one well of the 24-well plate containing the ectoderm induction medium. Shake the plate 3 times before placing it in the incubator to ensure that the second-stage embryoid bodies are evenly distributed in the well and that second-stage embryoid bodies in contact with each other are more likely to fuse. If a large number of second-stage embryoid bodies are observed to merge, transfer only one second-stage embryoid body per well.

[0043] Place the plate in an incubator at 37°C for 48 h. Observe that the second-stage embryoid bodies will grow with smooth edges and form optically translucent edges to obtain the primary brain organoids.

[0044] III. Culture of Primary Vascular Organoids 100 mL of mesoderm induction medium is prepared: 100 mL of APEL2 and CHIR-99021 at a final concentration of 6 μM.

[0045] 100 mL of endothelial cell induction medium is prepared: 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] 100 mL of vascular maturation culture medium is prepared: 100 mL of Endothelial Cell Growth Medium MV 2 and VEGF165 at a final concentration of 50 ng / mL.

[0047] (1) Induction medium for mesoderm was heated to 37°C, 100 μL of the pre-heated medium was added to each well of a new ultra-low attachment 96-well plate, and the first-stage pseudo-embryos obtained in step two of step (2) were transferred to the 96-well plate containing the mesoderm induction medium by using a 1000 μL wide-bore pipette tip, and the culture was continued for 48 h to obtain mesoderm.

[0048] (2) A new ultra-low attachment 96-well plate was prepared, 100 μL of endothelial cell induction medium was added to each well, and the mesoderm obtained in step (1) was transferred to the 96-well plate containing the endothelial cell induction medium by using a 1000 μL wide-bore pipette tip, and the culture was continued for 48 h to obtain endothelial cells.

[0049] (3) The endothelial cell induction medium in the ultra-low attachment 96-well plate was gently aspirated, and care was taken not to disturb the organoids, and then 100 μL of vascular maturation medium was added to each well, and the culture was continued for 48 h to obtain primary vascular organoids.

[0050] Four, mixed organoid culture (1) Matrigel was thawed in advance in a 4°C refrigerator, and during the experiment, Matrigel was operated on ice at all times to prevent solidification, and all experimental supplies in contact with Matrigel, such as pipette tips, were pre-cooled in a -20°C refrigerator for at least 30 min before use.

[0051] (2) Sterile silicone-coated plates containing primary brain organoids were placed in an empty, sterile 10 cm dish for use; using a 1000 μL wide-bore pipette tip, 25-50 μL of medium was aspirated 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 embedding surface of the silicone-coated plate, and this step was repeated until about 12-16 "1+1" organoid groups were collected on the embedding surface of the silicone-coated plate.

[0052] (3) The medium was aspirated, taking care not to aspirate the organoids, and then the organoid balls were placed in close contact; 18 μL of Matrigel (consistent with the same batch) was added to each organoid ball using a 200 μL pre-cooled pipette tip; a new 200 μL pre-cooled pipette tip was used to place the organoid balls as close to the center of the droplet as possible and to ensure their arrangement order; the silicone-coated plate was placed in a 37°C incubator for 45 min to allow the Matrigel to solidify, and mixed organoids were obtained.

[0053] Five, primary vascularized brain organoid culture 100 mL of neural induction culture medium was prepared by mixing 48 mL of DMEM / F12, 48 mL of Neurobasal medium, 0.5 mL of N-2 supplement, 1 mL of glutamine, 0.5 mL of MEM non-essential amino acid solution, 1 mL of B27 without vitamin A, and 1 mL of penicillin-streptomycin.

[0054] The neural induction culture medium and the vascular maturation culture medium in step three were mixed at a volume ratio of 1:1 to obtain a mixed solution containing the neural induction culture medium and the vascular maturation culture medium. The silica gel plate in step four was taken out, and a sterile forceps was used to grab the surface of the silica gel plate containing the Matrigel droplets from the corner; the silica gel plate was suspended above one hole of an ultra-low adhesion 6-hole cell culture plate, 1 mL of a pipette was used to suck out the mixed solution containing the neural induction culture medium and the vascular maturation culture medium, and then the Matrigel droplets were gently washed and dropped from the surface layer of the silica gel plate into the hole, 3 mL was used for each hole, and the process was repeated until all the Matrigel droplets entered the hole; the preliminary vascularized brain organoids were obtained by culturing in a 37°C incubator for 72 h.

[0055] Six, culturing the preliminary vascularized brain organoids using the neural maturation culture medium and the vascular maturation culture medium 100 mL of neural maturation culture medium was prepared by mixing 48 mL of DMEM / F12, 48 mL of Neurobasal medium, 0.5 mL of N-2 supplement, 1 mL of glutamine, 0.5 mL of MEM non-essential amino acid solution, 1 mL of B27 without vitamin A, and 1 mL of penicillin-streptomycin.

[0056] The neural maturation culture medium and the vascular maturation culture medium in step three were mixed at a volume ratio of 1:1 to obtain a mixed solution containing the neural maturation culture medium and the vascular maturation culture medium. The mixed solution containing the neural induction culture medium and the vascular maturation culture medium in step five was aspirated, and the culture medium was replaced with the mixed solution containing the neural maturation culture medium and the vascular maturation culture medium, and the culture was placed in a 37°C incubator, and the medium was replaced every 3 days, and the vascularized brain organoids were obtained after 28 days of culture, and the vascularized brain organoids were a moyamoya disease model.

[0057] Sustainable replacement medium for long-term culture, every 3-4 days, 6-well plate per hole 3 mL can be replaced.

[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 a healthy person, and the rest are the same as Example 1. The construction obtains a vascularized brain organoid of a healthy control.

[0059] Test Example 1 The vascularized brain organoids of the patient with moyamoya disease obtained in Example 1 and the vascularized brain organoids of the healthy control obtained in Comparative Example 1 were subjected to frozen section and tissue immunofluorescence staining (the following part of the reagent purchase source: O.C.T tissue embedding agent was purchased from Sakura Japan, the model number is 4583; primary antibody diluent was purchased from Beijing Solaybao Technology Co., Ltd., the model number is A1810; antibody diluent was purchased from Beijing Solaybao Technology Co., Ltd., the model number is A1800; anti-fluorescence quenching mounting agent was purchased from Beijing Solaybao Technology Co., Ltd., the model number is S2100; anti-PDGFRβ-antibody was purchased from Proteintech, the model number is 13449-1-AP; anti-CD31-antibody was purchased from Proteintech, the model number is 66065-2-Ig; anti-β-3Tubulin antibody was purchased from Proteintech, the model number is 10068-1-AP; donkey anti-mouse IgG (Alexa Fluor® 488) secondary antibody was purchased from Abeam USA, the model number is ab150105; donkey anti-mouse IgG (Alexa Fluor® 594) secondary antibody was purchased from Abeam USA, the model number is ab150108; donkey anti-rabbit IgG (Alexa Fluor® 488) secondary antibody was purchased from Abeam USA, the model number is ab150073; donkey anti-rabbit IgG (Alexa Fluor® 594) secondary antibody was purchased from Abeam USA, the model number is ab150076; immunohistochemical pen was purchased from Abeam USA, the model number is ab2601) 1. Preparation of experimental reagents 1) Preparation of permeation solution containing 0.25% Triton X-100 Take 49.875 mL of PBS phosphate buffer into a 50 mL centrifuge tube, use a 200 μL wide-bore pipette to slowly take 125 μL of Triton X-100 stock solution, add it to the above PBS, mix well to prepare 50 mL of cell permeation solution containing 0.25% Triton X-100, and store at room temperature.

[0060] 2) Preparation of blocking solution containing 5% donkey serum, 1% BSA Take 8.5 mL of 0.1% PBS-T solution into a 15 mL centrifuge tube, add 500 μL of blocking horse serum stock solution and 1 mL of 10% bovine serum albumin BSA (ddH2O dissolved) to it, mix well after preparation of 10 mL of 5% horse serum, 1% BSA cell blocking solution, 4°C storage, restore to room temperature before use.

[0061] 3) Preparation of 15% sucrose solution and 30% sucrose solution for tissue dehydration Take 15 g of sucrose on the electronic balance, dissolve it in 80 mL of PBS phosphate buffer into the reagent bottle, stir well until the sucrose is dissolved, then measure 100 mL in the graduated cylinder, which is 15% sucrose solution, transfer to the reagent bottle and store at 4°C; similarly, prepare 30% sucrose solution and store at 4°C.

[0062] 4) Preparation of 0.5% gelatin solution Weigh 1 g of gelatin particles on the electronic balance, continuously heat and stir to dissolve them in 200 mL of ddH2O, which is 0.5% gelatin solution, aliquot and store at -20°C for later use, thaw completely and restore to room temperature before use.

[0063] 2. Tissue pretreatment (dehydration, fixation and embedding) 1) Use a serum pipette to gently remove the vascularized brain organoids into a 15 mL centrifuge tube, carefully aspirate the remaining medium, wash 3 times with PBS solution, then add 2 mL of 4% tissue cell fixation solution that has been restored to room temperature, and fix overnight at 4°C; 2) Aspirate the fixative, wash 3 times with PBS solution, then add at least 10 mL of pre-cooled 15% sucrose solution to the centrifuge tube, and place it on a 4°C rotary shaker for pre-dehydration overnight; 3) The next day, when the organoids settle at the bottom of the 15% sucrose solution, aspirate the 15% sucrose solution and replace it with at least 10 mL of pre-cooled 30% sucrose solution, and place it on a 4°C rotary shaker for dehydration overnight. The dehydrated organoids will settle at the bottom of the 30% sucrose solution; 4) Before tissue embedding, use tin foil to stack the tissue embedding box to the appropriate size for later use; 5) Aspirate 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 visualize the subsequent sectioning process; 6) Aspirate the trypan blue staining solution, wash once with PBS solution, then gently transfer the brain tissue to the absorbent paper to remove excess water and prevent adhesion; 7) Fill the embedding box with Tissue Embedding Medium O.C.T. and then carefully transfer the brain-like tissue to the center of the O.C.T. avoiding air bubbles, and then immediately place it in dry ice for quick freezing. Transfer the frozen tissue block to -20 °C refrigerator for storage; 8) When sectioning the tissue, remove the tin foil of the embedding box in the cryostat at -20 °C and use O.C.T. to fix the tissue block to the sample holder. After low-temperature solidification, section the tissue on the cryostat with a thickness of 30 pm. Quickly mount the tissue sections on the gelatin-preprocessed glass slides, and after baking the slides at 37 °C for 10 min, store them in the -20 °C refrigerator.

[0064] 3. Immunofluorescence staining 1) Wash the vascularized brain-like organoid tissue sections in PBS solution at room temperature for 2 times to remove as much gelatin and O.C.T. embedding agent as possible. 2) After the sections are slightly air-dried at room temperature, use an immunohistochemical pen to circle the area of the mounted slide. First, add the permeabilization solution containing 0.25% Triton X-100 to the tissue site, and place it in a wet box for incubation at room temperature for 30 min for tissue permeabilization. Remove the permeabilization solution, and use the blocking solution containing 5% donkey serum and 1% BSA to block in a wet box at room temperature for 30 min. After blocking is completed, remove the blocking solution without washing. 3) Use the primary antibody diluent to dilute the primary antibody to the antibody working solution according to the IHC dilution ratio of the corresponding antibody. After uniformly adding the antibody working solution to the tissue area, place it in a wet box for incubation at 4 °C overnight. 4) The next day, remove the primary antibody working solution, and wash it with PBS solution for 3 times. Use the secondary antibody diluent to prepare the fluorescent secondary antibody working solution, and uniformly add it to the tissue area. Place it in a wet box for incubation at room temperature for 2 h. 5) Remove the secondary antibody working solution, add an appropriate amount of DAPI staining solution, and incubate it at room temperature in the dark for 5 min to label the cell nucleus. 6) After removing the DAPI, wash it with PBS solution for 3 times, air-dry it at room temperature in the dark, and then add an appropriate amount of anti-fluorescence quenching mounting medium. Cover the slide with a coverslip, and try to avoid air bubbles. Use nail polish or resin to seal the edges of the coverslip. 7) Immediately observe it under a fluorescence microscope or a confocal microscope, or store the mounted slide in a 4 °C refrigerator in the dark for no more than 7 days to prevent fluorescence quenching.

[0065] 4. Calculation and analysis scheme Use the Angiogenesis Analyzer of the ImageJ software to perform blood vessel-related calculations and analyses.

[0066] 5. Results The results of immunofluorescence staining are shown in FIG. 1. Figure 2As shown, where the star symbol indicates that the patient is unable to form fine branches of the vascular network, indicating that the vascularized brain organoids constructed in Example 1 of the present application reproduce the pathological feature of the clinical "smoke-like" abnormal collateral formation of Moyamoya disease.

[0067] The results of the analysis of immunofluorescence staining are shown in Figure 3 As shown, the number of junctions, the total number of main junctions and the total number of main segments of the vascularized brain organoids derived from Moyamoya disease patients are significantly reduced compared with healthy controls, indicating a decrease in the ability to form a vascular network, an increase in fine branches (as can be seen from the total length of branches in Figure 3 E, there is no difference in the total length of branches, but Figure 3 the decrease in main segments in D graph in

[0068] Test Example 2 RNA extraction and qPCR detection of the vascularized brain organoids obtained in Example 1 and Comparative Example 1 (the following section describes the sources of the reagents: TRIzol reagent was purchased from Invitrogen, USA, with the item number 15596018; DEPC-water was purchased from Invitrogen, USA, with the item number AM9915G; RNaseZap™ RNase Eliminator was purchased from Invitrogen, USA, with the item number AM9780; Transcriptor High Fidelity cDNA Synthesis Kit was purchased from Roche, Switzerland, with the item number 5091284001) 1. Extract total RNA from cells using TRIzol method 1) Wash the tissue sample with PBS and transfer it to an RNase-free 1.5 mL centrifuge tube, add 500 μL of TRIzol reagent, sonicate to break up, add TRIzol reagent to 1 mL, stand at room temperature for 5 min; 2) Pre-cool the centrifuge, add 1 / 5 volume (200 μL) of chloroform to the above 1.5 mL EP tube, add 200 μL of chloroform (half the amount of cells), shake vigorously for 15 s (do not vortex), stand at room temperature for 3 min, centrifuge at 12000 rpm at 4°C for 15 min; 3) After centrifugation, remove the supernatant and carefully transfer the upper aqueous phase to a clean 1.5 mL EP tube, add an equal volume of isopropanol, mix well and stand at room temperature for 10 min; 4) Centrifuge at 4°C, 12000 rpm for 10 min, discard the supernatant; 5) Add 1 mL of 80% ethanol solution (prepared by mixing anhydrous ethanol and DEPC water) to the EP tube, invert to wash the RNA; 6) 7500rpm, 4℃ centrifugation for 5min, carefully aspirate the supernatant and leave the sheet-shaped precipitate; 7) Clean and ventilated room temperature for 3min to volatilize the residual ethanol; 8) Add appropriate amount of DEPC water to dissolve RNA (tissue 10-30μL) 9) After agarose electrophoresis analysis of total RNA band and spectrophotometer measurement of total RNA concentration, immediately refer to the following steps to reverse transcribe it into cDNA and store it at-20℃ low temperature; 10) The rest of the total RNA is divided and stored at-80℃ ultra-low temperature.

[0069] 2. Reverse transcription of RNA to cDNA The reaction system is shown in Table 2 and Table 3.

[0070] Table 2 Reaction system 1

[0071] Table 3 Reaction system 2

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

[0073] 2) Prepare reaction system 2, gently mix, do not vortex, then add to step 1) tube, mix again gently; 3) In the PCR instrument, 25℃ reaction for 10min, then 55℃ reaction for 30min, then 85℃ reaction for 5min to inactivate the enzyme; 4) The reverse-transcribed cDNA needs to be stored at-20℃ for standby.

[0074] 3. qPCR detection of related gene expression Using the forward and reverse primers of the commercially available kit, GFAP, MAP2, PDGFRβ, and GAPDH were detected.

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

[0076] The qPCR reaction program is: 95℃ pre-denaturation for 10min; enter amplification cycle: 95℃ thermal denaturation for 10s, 60℃ annealing for 15s, 72℃ extension for 30s, cycle 40 times; melting curve: 95℃ 10s, slowly cool to 65℃.

[0077] GAPDH as an internal reference, respectively, measured Ct value of each gene amplification to the baseline level, the final experiment with △△Ct method to calculate the relative expression level of the gene.

[0078] Results as Figure 4 shown, compared with healthy controls, patients with moyamoya disease PDGFRβ expression decreased, indicating that the number of pericytes and possible vascular dysfunction; MAP2 expression decreased, suggesting that the number of mature neurons decreased or delayed development; GFAP expression increased, suggesting that the number of astrocytes increased, consistent with the clinical and pathological phenotype.

[0079] Test Example 3 Nitric oxide content detection experiment (using total nitric oxide detection kit, item number S0023, company Biyun Tian Biotechnology) 1. Preparation of experimental reagents 1) Preparation of standard Dilute 1M NaNO2 standard into 2, 5, 10, 20, 40, 60, 80 μM (in this experiment, use the vascularized brain organ maintenance medium (a mixture containing neural maturation medium and vascular maturation medium)) with the solution used to prepare or dilute the sample, and place it on ice for standby.

[0080] 2) Preparation of NADPH solution Add 1ml Milli-Q grade ultrapure water to 5mg NADPH, invert to mix and dissolve, then dilute to 3mL, prepare 2mM NADPH working solution, take out the part used for this experiment, and store the rest of the NADPH solution at -80℃ refrigerator.

[0081] 2. Nitric oxide detection 1) Collect 400 μL of cell supernatant for each sample and place it on ice for standby; 2) Take out FAD, Nitrate Reductase and LDH and place them on ice; at the same time, restore Griess Reagent I and Griess Reagent II to room temperature before use; all reactions need to 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 turn, mix well, then incubate at 37℃ for 30 min, pay attention to avoid bubbles; replace 60 μL of sample with 60 μL of culture medium and standard for blank control and standard; 4) After incubation, add 10 μL of LDH Buffer and 10 μL of LDH in turn, mix gently, then incubate at 37℃ for 30 min; 5) After the incubation, 50 μL Griess Reagent I and 50 μL Griess Reagent II were added in turn, mixed gently, and then incubated at room temperature for 10 min before measuring A540.

[0082] Results are shown in Figure 5 As shown in the results, the amount of NO secretion in the culture supernatant of the vascularized brain organoids derived from the patient with moyamoya disease was significantly decreased compared with the healthy control, indicating abnormal vascular function, damaged endothelium, or abnormal vasculogenesis.

[0083] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. A method of constructing a model of moyamoya disease, characterized by, The method comprises the following steps: CD34+ hematopoietic progenitor cells in peripheral blood of a patient with moyamoya disease are taken, reprogrammed to generate hiPSCs, the hiPSCs are cultured to 70-80% confluence, and the differentiation degree is not more than 10%; The hiPSCs are cultured using EB inoculation medium for 45-51 hours to obtain first-stage embryoid bodies; The first-stage embryoid bodies are further cultured using EB inoculation medium for 45-51 hours to obtain second-stage embryoid bodies, and the second-stage embryoid bodies are cultured using ectoderm induction medium for 44-52 hours to obtain primary brain organoids; The first-stage embryoid bodies are cultured using mesoderm induction medium for 44-52 hours, then cultured using endothelial cell induction medium for 44-52 hours, and then cultured using blood vessel maturation culture medium for 48-96 hours to obtain primary blood vessel organoids; The primary brain organoids and the primary blood vessel organoids are fused to obtain mixed organoids; The mixed organoids are cultured using nerve induction culture medium and blood vessel maturation culture medium for 68-76 hours to obtain preliminary blood-vesselized brain organoids; The preliminary blood-vesselized brain organoids are cultured using nerve maturation culture medium and blood vessel maturation culture medium for more than 28 days to obtain a moyamoya disease model; The EB inoculation medium is composed of basal medium 1, 2-mercaptoethanol with a final concentration of 0.1 mM, bFGF with a final concentration of 4 ng / mL, and Y-27632 with a final concentration of 50 μM, and the basal medium 1 is composed of the following raw materials in terms of volume fraction: 75% DMEM / F12, 20% KnockOut™ Serum Replacement, 3% fetal bovine serum special for embryonic stem cells, 1% glutamine, and 1% MEM non-essential amino acid solution.

2. The method of claim 1, wherein, The reprogramming is performed using a Sendai virus reprogramming kit.

3. The method of claim 1, wherein, The ectoderm induction medium is composed of basal medium 2 and heparin with a final concentration of 1 μg / mL, and the basal medium 2 is composed of the following raw materials in terms of volume fraction: 97% DMEM / F12, 1% N-2 supplement, 1% glutamine, and 1% MEM non-essential amino acid solution.

4. The method of claim 1, wherein, The mesoderm induction medium is composed of APEL2 and CHIR-99021 with a final concentration of 6 μM; the endothelial cell induction medium is composed of APEL2, VEGF165 with a final concentration of 50 ng / mL, BMP4 with a final concentration of 25 ng / mL, and bFGF with a final concentration of 10 ng / mL; and the blood vessel maturation culture medium is composed of Endothelial Cell Growth Medium MV 2 and VEGF165 with a final concentration of 50 ng / mL.

5. The method of claim 1, wherein, Matrigel is used when the fusion is performed, and 15-20 μL of Matrigel is needed to wrap one primary brain organoid and one primary blood vessel organoid.

6. The method of claim 1, wherein, The volume ratio of the neural induction culture medium and the vascular maturation culture medium is 1:1, the neural induction culture medium is composed of basal medium 3, 2-mercaptoethanol with a final concentration of 50 μM and insulin with a final concentration of 2.5 μg / mL, and the basal medium 3 is composed of the following raw materials in 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.

7. The method of claim 1, wherein, The volume ratio of the neural maturation culture medium and the vascular maturation culture medium is (1.5:1)~(1:1.5), the neural maturation culture medium is composed of the basal medium 3, 2-mercaptoethanol with a final concentration of 50 μM, insulin with a final concentration of 2.5 μg / mL, VEGF165 with a final concentration of 20 ng / mL, brain-derived neurotrophic factor with a final concentration of 20 ng / mL and hydrocortisone with a final concentration of 0.2 µg / mL.

8. The method of claim 1, wherein, The temperature of the culture is 36.5~37.5 ℃.

9. A model of moyamoya disease, characterized by, Obtained by the method of any one of claims 1~8.

10. The use of the method of any one of claims 1~8 or the moyamoya disease model of claim 9 in the screening of drugs for preventing and treating moyamoya disease.

Images