Clusterin-positive heart organoid and use thereof

Abstract

Provided in the present invention are a clusterin-positive heart organoid and the use thereof. The clusterin-positive heart organoid prepared by means of the method of the present invention has more diversified cellular compositions, and can better simulate the environment of the heart in a human body. The clusterin-positive heart organoid can better improve cardiac functions.

Description

Clusterin-positive cardiac organoids and their applications

[0001] This application claims priority to patent application No. CN 202410347723.2, filed on March 25, 2024; the entire contents of which are incorporated herein. Technical Field

[0002] The present invention belongs to the field of cardiovascular diseases, and more specifically, relates to Clusterin-positive cardiac organoids, preparation methods and applications thereof. Background Art

[0003] Heart disease caused by myocardial cell necrosis due to myocardial infarction has become one of the most important causes of harm to human life and health. After myocardial infarction, a large number of myocardial cells die and are replaced by fibrotic tissue, resulting in a decline in myocardial contractile function, which eventually develops into heart failure or even death. Among the current treatment methods, thrombolysis, percutaneous coronary intervention or coronary artery bypass grafting are usually used to clear the infarcted blood vessels, and corresponding drugs are given to slow down myocardial remodeling, but they cannot replenish the dead myocardial cells from the root. The patient's survival period after treatment is still short and the effect is limited. Therefore, promoting the proliferation of original myocardial cells or artificially supplementing myocardial cells is an important means to effectively solve the problem of short survival of patients after myocardial infarction.

[0004] Studies have found that in the early stages of mammalian mouse birth, its heart has a certain proliferation capacity. After the mouse heart is damaged at this stage, the cardiomyocytes can proliferate on their own and repair the damaged area. Through the study of the regeneration process of newborn mice, researchers have discovered several new targets that promote myocardial proliferation. However, these targets could not be clinically transformed due to some technical problems and adverse reactions in subsequent studies. At the same time, some studies have attempted to inject in vitro differentiated cardiomyocytes into the heart. Although this method can replenish lost cardiomyocytes and improve heart function to a certain extent, the survival rate of transplanted cardiomyocytes and their integration with the original cardiomyocytes are not satisfactory.

[0005] Currently, in vivo cardiac organoid models derived from stem cells have been established, but these organoids contain a limited number of cell types and cannot effectively simulate the in vivo environment. Furthermore, the effectiveness of using these organoids to improve cardiac function remains suboptimal.

[0006] Therefore, there is an urgent need in this field to solve the problem that existing cardiac organoids cannot simulate the in vivo environment well and the effect of improving cardiac function is not ideal. Summary of the Invention

[0007] The present invention aims to provide a Clusterin-positive cardiac organoid, a preparation method, and applications thereof. Clusterin-positive cardiac organoids prepared using the methods of the present invention have a more diverse cellular composition and can better simulate the environment of the human heart. Application of these Clusterin-positive cardiac organoids can improve cardiac function.

[0008] In a first aspect of the present invention, a method for preparing cardiac organoids is provided, comprising: inducing cardiac cells to differentiate into Clusterin-positive (CLU + ) Heart organoids.

[0009] In one or more embodiments, the cardiac cells are obtained by using pluripotent stem cells as starting cells; preferably, the pluripotent stem cells are differentiated into cardiac cells to form cardiac cells (including mature cardiomyocytes), and VPA is used to stimulate the cells to obtain Clusterin positive (CLU + ) Heart organoids.

[0010] In one or more embodiments, VPA is added to the culture medium; preferably, the concentration of VPA in the culture medium is 60-2000 μM, more preferably 100-1500 μM, 150-1200 μM, 180-800 μM or 190-600 μM.

[0011] In one or more embodiments, after VPA is added to the culture medium, the culture is continued for 5 to 16 days, preferably 6 to 14 days (more preferably 7 to 13 days).

[0012] In one or more embodiments, VPA is contained in a culture medium D for culturing, wherein the culture medium D comprises B27 and VPA.

[0013] In one or more embodiments, the method comprises: (a) culturing pluripotent stem cells with culture media A, B, and C to obtain cardiac cells (including cardiomyocytes); preferably, the culture is performed for 5 to 10 days; preferably, 6 to 9 days; (b) culturing with culture media D containing VPA to obtain the Clusterin-positive cardiac organoids; wherein, the culture media A comprises B27, FGF2, LY294002, Activin A, BMP4, CHIR99021, and insulin; the culture media B comprises B27, BMP4, FGF2, IWP2, and retinoic acid; the culture media C comprises B27, BMP4, FGF2, and insulin; and the culture media D comprises: B27 and VPA.

[0014] In one or more embodiments, in step (a), culture medium A is cultured for 1 to 2 days, preferably 30 to 42 hours, and more preferably 36 to 40 hours.

[0015] In one or more embodiments, in step (a), culture medium B is cultured for 2 to 6 days, preferably 2.5 to 5.5 days, preferably 3 to 5 days, and more preferably 3.5 to 4.5 days.

[0016] In one or more embodiments, in step (a), the culture medium C is cultured for 1 to 3 days, preferably 1.5 to 2.5 days.

[0017] In one or more embodiments, the concentration of B27 in the culture medium D is 5-40 μl / ml (preferably 7-30 μl / ml or 8-25 μl / ml or 9-20 μl / ml, for example, 10 μl / ml).

[0018] In one or more embodiments, in the culture medium A, the concentration of B27 is 5-40 μl / ml (preferably 7-30 μl / ml or 8-25 μl / ml or 9-20 μl / ml, for example 10 μl / ml); the concentration of FGF2 is 2-50 ng / ml (preferably 5-40 ng / ml, more preferably 8-30 ng / ml); the concentration of LY294002 is 0.5-10 μM (preferably 1-8 μM or 1.5-5 μM); Activin The concentration of A is 10-100 ng / ml (preferably 15-80 ng / ml or 20-70 ng / ml); the concentration of BMP4 is 1-30 ng / ml (preferably 3-20 ng / ml or 5-15 ng / ml); the concentration of CHIR99021 is 1-15 μM (preferably 2-10 μM or 3-8 μM); the concentration of insulin is 0.1-5 μg / ml (preferably 0.5-4 μg / ml or 0.5-2 μg / ml).

[0019] In one or more embodiments, in the culture medium B, the concentration of B27 is 5-40 μl / ml (preferably 7-30 μl / ml or 8-25 μl / ml or 9-20 μl / ml, for example 10 μl / ml); the concentration of BMP4 is 3-50 ng / ml (preferably 5-40 ng / ml or 10-30 ng / ml); the concentration of FGF2 is 2-50 ng / ml (preferably 5-40 ng / ml, more preferably 8-30 ng / ml); the concentration of IWP2 is 1-15 μM (preferably 2-10 μM or 3-8 μM); and the concentration of retinoic acid is 0.1-5 μM (preferably 0.2-3 μM, 0.3-2 μM or 0.4-1 μM).

[0020] In one or more embodiments, in the culture medium C, the concentration of B27 is 5-40 μl / ml (preferably 7-30 μl / ml or 8-25 μl / ml or 9-20 μl / ml, for example 10 μl / ml); the concentration of BMP4 is 1-30 ng / ml (preferably 3-20 ng / ml or 5-15 ng / ml); the concentration of FGF2 is 2-50 ng / ml (preferably 5-40 ng / ml, more preferably 8-30 ng / ml); and the concentration of insulin is 5-15 μg / ml (preferably 8-13 μg / ml or 9-12 μg / ml).

[0021] In one or more embodiments, culture medium A, B, C or D further comprises a basal culture medium.

[0022] In one or more embodiments, the basal culture medium is selected from RPMI1640 culture medium, Advanced DMEM / F12 culture medium, DMEM / F12 culture medium, and CDM culture medium; preferably, it is RPMI1640 culture medium.

[0023] In one or more embodiments, the method comprises the following steps:

[0024] 1) Induced pluripotent stem cells;

[0025] 2) One day after induction in step 1), counted as day 0, cultured in medium A;

[0026] 3) After day 1-2, continue culturing with medium B (e.g., for 4 days);

[0027] 4) On days 5-6, continue culturing with medium C (e.g., for 2 days);

[0028] 5) On days 7 to 8, culture was continued with culture medium D, and the Clusterin-positive cardiac organoid model was obtained on days 14 to 20.

[0029] In one or more embodiments, the step 1) comprises: incubating pluripotent stem cells (e.g., embryonic stem cells) with mTeSR medium containing ROCKi, followed by digestion and centrifugation of the pluripotent stem cells, re-seeding, and culturing for 1 day; more preferably, the ROCKi is Y27632; more preferably, the concentration of ROCKi is 1-10 μM, more preferably 2-8 μM or 4-6 μM, for example, 5 μM; more preferably, the embryonic stem cells after digestion and centrifugation are seeded at a density of 5,000-10,000 cells / 96 wells (e.g., 8,000 cells / 96 wells) and cultured for 1 day.

[0030] In one or more embodiments, in step 5), after adding culture medium D, fresh culture medium D is replaced once a day, and the Clusterin-positive cardiac organoid model is obtained on day 14 to 18, preferably day 14 to 17, day 14 to 16, or day 14 to 15.

[0031] In one or more embodiments, the pluripotent stem cells include embryonic stem cells and induced pluripotent stem cells.

[0032] In one or more embodiments, the pluripotent stem cells are derived from mammals; more preferably, the mammals include humans, rabbits, mice, sheep, pigs, and monkeys; more preferably, the pluripotent stem cells are human pluripotent stem cells, more preferably human embryonic stem cells (e.g., human H9 ES cells) or human induced pluripotent stem cells.

[0033] In another aspect of the present invention, a Clusterin-positive cardiac organoid (including a cell culture or organoid culture containing the same) is provided, which is prepared by any of the methods described above.

[0034] In one or more embodiments, the Clusterin-positive cardiac organoids include: endothelial cells, epicardial cells, vascular smooth muscle cells, epicardial cells, ventricular cardiomyocytes, and atrial cardiomyocytes.

[0035] In one or more embodiments, the markers expressed by the Clusterin-positive cardiac organoids include: cTNT, CD31, α-SMA, MYL7 / MLC 2A, WT1 and / or MLC 2V.

[0036] In another aspect of the present invention, a pharmaceutical composition or a drug kit is provided, comprising the Clusterin-positive cardiac organoid.

[0037] In another aspect of the present invention, VPA is provided for inducing cardiac cells to differentiate into Clusterin-positive (CLU + ) cardiac organoids, or for the preparation of cardiac cells induced to differentiate into Clusterin positive (CLU + )Compositions of cardiac organoids (e.g., culture medium).

[0038] In another aspect of the present invention, a culture medium composition for preparing Clusterin-positive cardiac organoids is provided, comprising: culture media A, B, C and D, wherein culture medium A comprises B27, FGF2, LY294002, Activin A, BMP4, CHIR99021 and insulin; culture medium B comprises B27, BMP4, FGF2, IWP2 and retinoic acid; culture medium C comprises B27, BMP4, FGF2 and insulin; and culture medium D comprises B27 and VPA.

[0039] In another aspect of the present invention, there is provided a use of the Clusterin-positive cardiac organoid in preparing a pharmaceutical composition for improving cardiac function or treating cardiac damage.

[0040] In one or more embodiments, the improving cardiac function includes: improving electrocardiographic abnormalities, improving cardiac contractility, improving cardiac ejection capacity, increasing blood flow velocity, or increasing heart beat rate;

[0041] In one or more embodiments, the cardiac injury comprises myocardial infarction, cardiac fibrosis, ventricular wall thickening, or myocardial inflammation.

[0042] In one or more embodiments, the myocardial infarction includes: acute myocardial infarction, myocardial ischemia, subacute myocardial infarction, and old myocardial infarction.

[0043] In one or more embodiments, the treatment comprises administering Clusterin-positive cardiac organoids to a subject in need, thereby improving the subject's cardiac function; more preferably, the treatment comprises: increasing cardiac shortening fraction (FS), increasing cardiac ejection fraction (EF), R wave recovery, and strengthening interventricular conduction (PR interval).

[0044] In another aspect of the present invention, there is provided a use of a Clusterin upregulator in the preparation of a drug for treating cardiac inflammation; wherein the Clusterin upregulator is an expression vector or Clusterin-positive cardiac organoid that can express Clusterin after being transferred into cells; preferably, the expression vector includes: AAV vector, adenovirus vector, lentivirus vector; more preferably, the expression vector is an AAV vector.

[0045] In one or more embodiments, the AAV includes or is selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0046] In one or more embodiments, according to the mechanism of inflammation, the cardiac inflammation includes: cardiac fibrosis caused by inflammation, cardiac inflammation caused by infection, myocardial cell death, and decreased cardiac function; according to the site of inflammation, the cardiac inflammation includes: myocarditis, endocarditis, and pericarditis; more preferably, the cardiac inflammation is LPS-induced cardiac inflammation.

[0047] In another aspect of the present invention, the use of serum Clusterin protein in the preparation of a detection system for evaluating myocardial infarction is provided; wherein, the evaluation comprises: detecting the level of Clusterin protein in the serum of a subject; if the level of Clusterin protein is high (e.g., higher than 180, 185, 190, 200 μg / ml), it indicates that the severity of myocardial infarction in the subject is relatively low; conversely, if the level of Clusterin protein is low (e.g., lower than 175, 170, 165, 160, 155, 150 μg / ml), it indicates that the severity of myocardial infarction in the subject is relatively high.

[0048] In one or more embodiments, the threshold value for determining the Clusterin protein content is a value or a value range selected from 170 to 185 μg / ml.

[0049] In another aspect of the present invention, a detection system for evaluating myocardial infarction is provided, comprising:

[0050] (1) a detection component or module for analyzing Clusterin protein in a serum sample to be tested and determining the amount of the protein;

[0051] (2) an analysis component or module that analyzes the value obtained in (1), and sets a threshold value; when the value obtained in (1) is higher than the threshold value, it indicates that the patient has a good prognosis (the severity of the disease is relatively low); when the value obtained in (1) is lower than the threshold value, it indicates that the patient has a poor prognosis (the severity of the disease is relatively high); preferably, the threshold value is a value or a range of values ​​selected from 170 to 185 μg / ml.

[0052] In one or more embodiments, the method for detecting the Clusterin protein content in the serum of a subject comprises an immunological method; more preferably, the method comprises ELISA.

[0053] In one or more embodiments, the myocardial infarction includes: acute myocardial infarction, myocardial ischemia, subacute myocardial infarction, and old myocardial infarction.

[0054] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1. CLU+ cardiac organoid differentiation;

[0056] (A)CLU + Schematic diagram of cardiac organoid differentiation;

[0057] (B)CLU + Brightfield images of cardiac organoids and normal cardiac organoids, magnification: 4x;

[0058] (C)CLU + CLU antibody staining and statistical results of cardiac organoids and normal cardiac organoids, where cTNT: red fluorescence, a cardiomyocyte marker; CLU: green fluorescence; DAPI: blue fluorescence; scale bar = 100 μm; scale bar of the enlarged image in Figure C = 100 μm.

[0059] Figure 2. Identification of CLU + Fluorescence images of cardiomyocytes, endothelial cells, and vascular smooth muscle cells in cardiac organoids; cTNT: cyan fluorescence; CD31: yellow fluorescence; α-SMA: purple fluorescence; Merge indicates the superposition of all fluorescence images; Scale bar = 50 μm.

[0060] Figure 3. Identification of CLU + Fluorescent images of cardiomyocytes, endothelial cells, and epicardial cells in cardiac organoids; DAPI: blue fluorescence; MYL7: purple fluorescence; WT1: yellow fluorescence; CD31: cyan fluorescence; Merge indicates the superposition of all fluorescence images; Scale bar = 50 μm.

[0061] Figure 4. Identification of CLU + Fluorescent images of ventricular and atrial cardiomyocytes in cardiac organoids; DAPI: blue fluorescence; MLC 2V: green fluorescence; MLV 2A: purple fluorescence; scale bar = 50 μm.

[0062] Figure 5. Fluorescent images showing the identification of cardiomyocytes, endothelial cells, and epicardial cells in normal cardiac organoids; DAPI: blue fluorescence; MYL7: purple fluorescence; WT1: yellow fluorescence; CD31: cyan fluorescence; Merge indicates the image after all fluorescence images are superimposed; Scale bar = 50 μm.

[0063] Figure 6. CLU + Schematic diagram of cardiac organoid transplantation, in vivo imaging results, and ultrasound examination results;

[0064] (A)CLU +Schematic diagram of cardiac organoid transplantation; LAD: left anterior descending coronary artery (LAD) ligation; ECO: echocardiogram;

[0065] (B)CLU + Representative images of in vivo imaging of cardiac organoids 1 day after transplantation;

[0066] (C)CLU + Representative images of in vivo imaging of cardiac organoids 5 days after transplantation;

[0067] (D) Mice transplanted with CLU + Echocardiographic results of a cardiac organoid 7 days after implantation. B-mode displays information reflected from interfaces at different depths as grayscale, i.e., brightness mode. M-mode can display the trajectory of a motion pattern, which primarily shows how the position of tissue echo interfaces changes over time and is primarily used in the cardiac field.

[0068] (EF) Changes in EF value (E) and FS value (F) of continuous cardiac ultrasound before and after surgery in mice.

[0069] Figure 7. In situ injection of CLU + Cardiac organoids significantly improved ECG abnormalities in mice;

[0070] (A) In situ injection of CLU + Schematic diagram of a cardiac organoid;

[0071] (B) In situ injection of CLU + electrocardiogram (ECG) after cardiac organoids;

[0072] (C) In situ injection of CLU + The effects of cardiac organoids on heart rate;

[0073] (D) In ​​situ injection of CLU + Effects of cardiac organoids on PR interval;

[0074] Among them, in situ injection of organoids, matrix gel, and blank corresponded to the LAD+organoid group, LAD+matrigel group, and LAD group, respectively, and the sham operation group served as the control group.

[0075] Figure 8. Effects of in vivo injection of AAV-9-Clusterin on cardiac function in mice with myocardial infarction;

[0076] (A) Flowchart of AAV injection in mice;

[0077] (B) Changes in proliferating cardiomyocytes (PH3+ cardiomyocytes) in the hearts of mice injected with AAV-9 control virus (AAV-Control) and AAV-9-Clusterin virus (AAV-CLU);

[0078] (C) Immunofluorescence images of proliferating cardiomyocytes (PH3+ cardiomyocytes) in the hearts of mice injected with AAV-9 control virus (AAV-Control) and AAV-9-Clusterin virus (AAV-CLU), where DAPI: blue fluorescence; PH3: pink fluorescence; TNNI1: yellow fluorescence; scale bar = 20 μm;

[0079] (D) Representative images of cardiac fibrosis detection after LAD in mice injected with AAV-9 control virus (AAV-Control) and AAV-9-Clusterin virus (AAV-CLU), scale bar = 20 μm;

[0080] (E) Statistical results of fibrotic scar tissue in (D);

[0081] (F) Cardiac ultrasound results of mice in the sham group (Sham), injected with AAV-9 control virus (AAV-Control), and AAV-9-Clusterin virus (AAV-CLU) 21 days after LAD;

[0082] (GH) Changes in EF (G) and FS (H) values ​​of continuous cardiac ultrasound before and after surgery in mice injected with AAV-9 control virus (AAV-Control) and AAV-9-Clusterin virus (AAV-CLU) in the sham group (Sham).

[0083] Figure 9. Effects of in vivo injection of AAV-9-Clusterin on cardiac function in inflammatory mice;

[0084] (A) Cardiac ultrasound results of mice injected with AAV-9 control virus (AAV-Control) and AAV-9-Clusterin virus (AAV-CLU) 1 day after LPS injection;

[0085] (B) Statistical results of ejection fraction (EF), fractional shortening (FS), ejection volume (SV), cardiac output (CO), aortic blood flow velocity, and heart rate in (A).

[0086] Figure 10. Correlation analysis between Clusterin levels in serum of patients with myocardial infarction and healthy controls and disease;

[0087] (A) Flowchart of the experimental design for collecting serum from patients with myocardial infarction and healthy subjects;

[0088] (B) Clusterin levels in serum of patients with myocardial infarction and healthy controls;

[0089] (CG) Correlation analysis between the Clusterin level in the serum of patients with myocardial infarction and the serum TnI (C), BNP (D), inflammatory factor TNF-α (F), IL6 levels (G), and ejection fraction (E) in patients with myocardial infarction.

[0090] Figure 11. Myocardial infarction patients were grouped according to the KILIP classification to explore their relationship with serum Clusterin (CLU) levels. Among them, the severity of patients from grade 1 to grade 4 increases gradually, with grade 1 being the mildest and grade 4 being the most severe.

[0091] Figure 12. Relationship between ejection fraction (EF) and serum Clusterin (CLU) content in patients with myocardial infarction. DETAILED DESCRIPTION

[0092] After extensive research, the inventors have developed a method for preparing Clusterin-positive cardiac organoids, as well as the Clusterin-positive cardiac organoids produced by this method. These organoids have a more diverse cellular composition, including endothelial cells, epicardial cells, vascular smooth muscle cells, epicardial cells, ventricular cardiomyocytes, and atrial cardiomyocytes, better simulating the environment of the human heart. Application of these Clusterin-positive cardiac organoids can improve cardiac function.

[0093] As used herein, "Clusterin" includes the Clusterin gene (CLU) and the Clusterin protein. The Clusterin gene can be referenced by NCBI GeneID: 1191; the Clusterin protein can be referenced by NCBI: NP_001822.3.

[0094] As used herein, "organoid" refers to a three-dimensional cellular structure composed of various cells that make up a tissue and can mimic the environment of living tissue. In this article, organoids particularly refer to cardiac organoids.

[0095] Producing cardiac organoids

[0096] The method for preparing cardiac organoids of the present invention comprises: inducing cardiac cells to differentiate into Clusterin positive (CLU + ) Heart organoids.

[0097] As a preferred embodiment of the present invention, the cardiac cells are obtained by using pluripotent stem cells as starting cells; preferably, the pluripotent stem cells are differentiated into cardiac cells to form cardiac cells (including mature cardiomyocytes), and stimulated with VPA (preferably, also including B27) to obtain Clusterin positive (CLU + ) cardiac organoids. In one or more embodiments, VPA is added to the culture medium. The concentration of VPA in the culture medium can be 60-2000 μM, 100-1500 μM, 150-1200 μM, 180-800 μM, or 190-600 μM, for example, 200 μM. As a preferred embodiment of the present invention, after VPA is added to the culture medium, the culture is carried out for 5-16 days, preferably 6-14 days (e.g., 7-13 days).

[0098] As a preferred embodiment of the present invention, the cardiac cells (including cardiomyocytes) can be obtained by culturing pluripotent stem cells in culture media A, B, and C, wherein culture media A includes B27, FGF2, LY294002, Activin A, BMP4, CHIR99021, and insulin; culture media B includes B27, BMP4, FGF2, IWP2, and retinoic acid; and culture media C includes B27, BMP4, FGF2, and insulin. In one or more embodiments, the culture is performed for 5 to 10 days, for example, 6 to 9 days.

[0099] In a preferred embodiment of the present invention, a method for preparing Clusterin-positive cardiac organoids is provided, comprising sequentially inducing pluripotent stem cells using culture media A, B, C, and D to obtain the Clusterin-positive cardiac organoid model; wherein the culture media A comprises basal culture medium, B27, FGF2, LY294002, Activin A, BMP4, CHIR99021, and insulin; the culture media B comprises basal culture medium, B27, BMP4, FGF2, IWP2, and retinoic acid; the culture media C comprises basal culture medium, B27, BMP4, FGF2, and insulin; and the culture media D comprises: basal culture medium, B27, and VPA.

[0100] It should be understood that before adding a new culture medium for cultivation, a basal culture medium can be used for washing to remove the influence of the previous culture medium. When adding a new culture medium for cultivation, fresh culture medium can be regularly (e.g., daily) replaced according to the cell state to maintain a good cell growth state.

[0101] The present invention also provides Clusterin-positive cardiac organoids (including cell cultures or organoid cultures containing the same) prepared according to any embodiment of the present invention.

[0102] Culture medium composition

[0103] The present invention also provides a culture medium composition suitable for preparing Clusterin-positive cardiac organoids, comprising: culture media A, B, C and D, wherein the culture medium A comprises a basal culture medium, B27, FGF2, LY294002, Activin A, BMP4, CHIR99021 and insulin; the culture medium B comprises a basal culture medium, B27, BMP4, FGF2, IWP2 and retinoic acid; the culture medium C comprises a basal culture medium, B27, BMP4, FGF2 and insulin; and the culture medium D comprises a basal culture medium, B27 and VPA.

[0104] In one or more embodiments, the medium composition further comprises a medium E comprising a basal medium and ROCKi.

[0105] In one or more embodiments, the ROCKi is Y27632.

[0106] In one or more embodiments, the concentration of ROCKi is 1-10 μM, more preferably 2-8 μM or 4-6 μM.

[0107] In one or more embodiments, the basal medium in the culture medium E is mTeSR medium.

[0108] Clusterin-positive cardiac organoids and their applications

[0109] In a specific embodiment of the present invention, the inventors prepared Clusterin-positive cardiac organoids, coated them in matrix gel and injected them into the border area of ​​the left anterior descending artery ligation in mice with acute myocardial infarction. The results showed that Clusterin-positive cardiac organoids significantly improved the cardiac function of mice with myocardial infarction.

[0110] Therefore, the present invention provides the use of Clusterin-positive cardiac organoids in the preparation of a pharmaceutical composition for improving cardiac function or treating cardiac damage.

[0111] The "treatment" includes administering Clusterin-positive cardiac organoids to subjects in need, thereby improving cardiac function or treating cardiac damage. In one or more embodiments, the improvement of cardiac function includes but is not limited to: improving ECG abnormalities, improving cardiac contractility, improving cardiac ejection capacity, increasing blood flow rate or increasing heart rate. In one or more embodiments, the cardiac damage includes but is not limited to: myocardial infarction, cardiac fibrosis, ventricular wall thickening or myocardial inflammation. In one or more embodiments, the "myocardial infarction" includes but is not limited to: acute myocardial infarction, myocardial ischemia, subacute myocardial infarction, and old myocardial infarction.

[0112] After myocardial infarction, a large number of myocardial cells die and are replaced by fibrotic tissue, resulting in a decline in myocardial contractile function and weakened interventricular conduction, which is specifically manifested as a decrease in cardiac shortening fraction (FS) and ejection fraction (EF). The electrocardiogram of subjects with myocardial infarction also has a certain evolution pattern, mainly manifested as the appearance of pathological Q waves from none to none, a decrease or disappearance of R wave amplitude, ST segment elevation to gradual return to normal, and T wave high peak to upright and inverted. In one or more embodiments, the "treatment" includes but is not limited to: increasing cardiac shortening fraction (FS), increasing cardiac ejection fraction (EF), recovery of R waves, and strengthening of interventricular conduction (PR interval).

[0113] The present invention also provides a pharmaceutical composition or a medicine kit, which contains an effective amount of the Clusterin-positive cardiac organoid and a pharmaceutically acceptable carrier.

[0114] The pharmaceutical composition or kit of the present invention can be used directly to treat myocardial infarction, and can also be used in combination with other therapeutic agents or adjuvants.

[0115] Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, typically at a pH of about 5-8, preferably about 6-8. To prevent cell migration within Clusterin-positive cardiac organoids after injection, the drug may also include Matrigel. The concentration of Matrigel may be 40% to 60%, for example, 50%, based on the total volume of the drug composition or kit.

[0116] The pharmaceutical composition or kit of the present invention contains a safe and effective amount of Clusterin-positive cardiac organoids and a pharmaceutically acceptable carrier. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical preparation should match the mode of administration. The drug of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. The drug is preferably manufactured under sterile conditions. The amount of active ingredient administered is a therapeutically effective amount. The pharmaceutical preparation of the present invention can also be prepared as a sustained-release preparation.

[0117] The present invention also provides a method for treating myocardial infarction, comprising administering an effective amount of Clusterin-positive cardiac organoids to a subject.

[0118] The effective amount of the Clusterin-positive cardiac organoids described herein can vary depending on the mode of administration and the severity of the disease being treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). These factors include, but are not limited to, pharmacokinetic parameters of the Clusterin-positive cardiac organoids, such as bioavailability, metabolism, and half-life; the severity of the disease being treated, the patient's weight, the patient's immune status, and the route of administration.

[0119] The Clusterin-positive cardiac organoids of the present invention can be administered in situ. The Clusterin-positive cardiac organoids of the present invention can be administered by, but are not limited to, injection (e.g., intracavitary (non-intravenous), intravenous, intramuscular, subcutaneous, intradermal, and acupuncture injection).

[0120] Pharmaceutical applications

[0121] In a specific embodiment of the present invention, the inventors analyzed the serum of patients with myocardial infarction and healthy controls and found that Clusterin levels were negatively correlated with the severity of myocardial infarction and with inflammatory factors (such as TNF-α and IL-6). Furthermore, upregulating Clusterin levels in mice with myocardial infarction significantly reduced the area of ​​cardiac fibrosis and restored cardiac function.

[0122] Therefore, the present invention provides the use of a Clusterin upregulator in preparing a medicament for treating cardiac inflammation.

[0123] In one or more embodiments, the Clusterin upregulator includes: a Clusterin overexpression agent, a Clusterin activation or promotion agent. More specifically, the Clusterin upregulator may include: an expression vector or expression construct capable of expressing Clusterin after transfer into cells, a regulatory factor that specifically promotes Clusterin expression, a CRISPR gene editing agent or homologous recombination agent targeting Clusterin.

[0124] In one or more specific embodiments, the expression vector includes but is not limited to: AAV vector, adenovirus vector, lentivirus vector.

[0125] In one or more specific embodiments, the AAV includes but is not limited to AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and AAV9.

[0126] In one or more specific embodiments, the upregulator of Clusterin is AAV-Clusterin, such as AAV9-Clusterin.

[0127] In one or more embodiments, the upregulation, enhancement, promotion refers to a significant upregulation, enhancement, promotion, such as upregulation, enhancement, promotion by 5%, 10%, 20%, 40%, 60%, 80%, 90% or more.

[0128] It should be understood that, depending on the mechanism of inflammation, cardiac inflammation includes, but is not limited to, inflammation-induced cardiac fibrosis, infection-induced cardiac inflammation, myocardial cell death, and decreased cardiac function. Depending on the site of inflammation, cardiac inflammation includes, but is not limited to, myocarditis, endocarditis, and pericarditis. In one or more specific embodiments, infection-induced cardiac inflammation refers to LPS-induced cardiac inflammation.

[0129] The present invention also provides a method for treating cardiac inflammation, comprising administering an effective amount of a Clusterin upregulator to a subject.

[0130] The present invention also provides the use of Clusterin in the preparation of a detection system for evaluating myocardial infarction. The severity of a patient's myocardial infarction can be determined by measuring the level of Clusterin protein / gene in serum. In one or more embodiments, the evaluation comprises: measuring the level of Clusterin protein in the subject's serum; a high level of Clusterin protein indicates a low severity of myocardial infarction in the subject; conversely, a low level of Clusterin protein indicates a high severity of myocardial infarction in the subject.

[0131] The present invention also provides a method for detecting the severity of myocardial infarction, comprising: detecting the level of Clusterin protein / gene in the serum of a subject; if the level of Clusterin is high, it indicates that the severity of myocardial infarction in the subject is low; conversely, if the level of Clusterin is low, it indicates that the severity of myocardial infarction in the subject is high.

[0132] The present invention also provides a detection system for evaluating myocardial infarction, comprising:

[0133] (1) a detection component or module for analyzing Clusterin protein in a serum sample to be tested and determining the amount of the protein;

[0134] (2) an analysis component or module that analyzes the value obtained in (1), and sets a threshold value; when the value obtained in (1) is higher than the threshold value, it indicates that the patient has a good prognosis (the severity of the disease is relatively low); when the value obtained in (1) is lower than the threshold value, it indicates that the patient has a poor prognosis (the severity of the disease is relatively high); preferably, the threshold value is a value or a range of values ​​selected from 170 to 185 μg / ml.

[0135] The detection method includes, but is not limited to, PCR and ELISA. Those skilled in the art can select a suitable detection method as needed and use relevant reagents and detection instruments suitable for the detection method based on their general technical knowledge.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the invention. The experimental methods in the following examples, for which specific conditions are not specified, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.

[0137] Experimental Materials and Methods

[0138] Experimental Materials

[0139] mTeSR: Stemcell, catalog number 85850.

[0140] ROCKi: Y-27632, Stem Cell Company, catalog number 72308.

[0141] B27: Thermofisher-Invitrogen, catalog number 17504044.

[0142] FGF2: PEPROTECH, catalog number 100-18B-50UG.

[0143] LY294002: Selleck, product number S1105.

[0144] Activin A: R&D company, product number 338-AC-050.

[0145] BMP4: R&D company, product number 314-BP-01M.

[0146] CHIR99021: Stem cell company, product number 72054.

[0147] Insulin: Thermofisher-Invitrogen, catalog number 12585014.

[0148] RPMI1640: Thermofisher-Invitrogen, catalog number C11875500BT.

[0149] IWP2: Selleck, product number S7086.

[0150] Retinoic acid: R&D company, product number 338-AC-050.

[0151] Valproic acid (VPA): Sigma, product number PHR1061.

[0152] Matrigel: Corning-Biocoat, catalog number 354277.

[0153] D-luciferin bioluminescent enzyme kit: Yeasen, catalog number 40901ES01.

[0154] LPS: Sigma, product number L4391.

[0155] Human CLU ELISA kit: R&D company, catalog number DCLU00.

[0156] Human TNF ELISA kit: Abclonal, catalog number RK00027.

[0157] Human IL6 ELISA kit: Abclonal, catalog number RK00008.

[0158] Experimental methods

[0159] 1. Immunofluorescence detection

[0160] Fixation and permeabilization:

[0161] (1) Place the sections at room temperature for approximately 30 minutes, blow dry to prevent detachment, scrape off the OCT surrounding the tissue, and outline the tissue area with a hydrophobic pen. (2) Wash the sections with 10-40 μl / ml PBSTx three times, 5 minutes each wash. (3) Treat with pre-cooled acetone for 5 minutes. (4) Remove the sections and dry them at room temperature for 10 minutes. (5) Wash the sections with 10-40 μl / ml PBSTx three times, 5 minutes each wash.

[0162] Closed:

[0163] (1) Add 4% BSA in 10-40 μl / ml PBSTx to block for 60 min at room temperature. (2) After blocking, discard the blocking solution without washing and directly add the primary antibody.

[0164] Primary antibody incubation:

[0165] (1) The primary antibodies were prepared in 0.2% PBSTx containing 4% BSA, anti-CD31 (R&D Systems, Catalog No. AF806-SP) to label endothelial cells; anti-WT1 (Abcam, Catalog No. ab89901) to label epicardial cells; anti-MLC-2V (Proteintech, Catalog No. 10906-1-AP) to label ventricular cardiomyocytes; anti-MLC-2A (Synaptic Systems, Catalog No. 311011) to label atrial cardiomyocytes; anti-cTNT (abcam, Catalog No. ab56357) to label cardiomyocytes; anti-α-SMA (Abcam, Catalog No. ab5694) to label smooth muscle cells; anti-MYL7 (Synaptic Systems, Catalog No. 311011) to label atrial cardiomyocytes; anti-anti-H3S10P (Cell Signaling Technology, Cat. No. 4499) to mark proliferating cells, i.e., PH3+ staining; anti-Clusterin alpha chain (Abcam, Cat. No. ab184100). (2) Incubate with primary antibody overnight at 4°C. (3) Wash six times in PBS with 0.1% Tween 20, 5 min each wash.

[0166] Secondary antibody and DAPI incubation:

[0167] (1) Dissolve the secondary antibody in 0.2% PBSTx according to the ratio recommended in the antibody manual. Dissolve DAPI in the secondary antibody at a ratio of 1:1000. (2) Incubate with the secondary antibody at room temperature for 1 hour, protected from light. (3) Wash and wash six times in 0.1% Tween-20 PBS after staining, each wash for 5 minutes.

[0168] Seal the slides and count the results:

[0169] CLU + The percentage of cardiomyocytes was analyzed using imageJ software, and the number of cells co-expressing CLU and cardiomyocytes was counted, and the number of all cells was calculated using DAPI.

[0170] 2. Preparation of Mouse Myocardial Infarction Model and Dosage Regimen

[0171] Preparation of mouse myocardial infarction model: After anesthesia, the mice were subjected to an acute myocardial infarction model by ligating the left anterior descending coronary artery (LAD). Adult mice were anesthetized with isoflurane. Check the tail pinch and hind foot reflex to ensure that the mouse was fully anesthetized. Secure the limbs with medical tape and make a surgical incision in the skin and ribs (between the third and fourth ribs) using surgical scissors. Gently open the chest cavity using a chest stent and firmly ligate the left anterior descending coronary artery with an 8-0 non-retractable suture. The ligation was judged to be successful when the heart showed signs of cyanosis. Close the incision with a 6-0 retractable suture and expel the air from the chest. In the sham control group, the same operation was performed except that the ligation of the LAD coronary artery was omitted. After ligation, the mice were provided with pure oxygen for 4-5 minutes until they resumed spontaneous breathing.

[0172] (1) Dosage regimen 1: CLU + Cardiac organoid drug delivery

[0173] Ten 6-week-old male C57 mice (Lingchang Co., Ltd.) were anesthetized and the acute myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD). Ten minutes after ligation, a 50% Matrigel-coated CLU was injected into the mouse body using a 22G syringe. + Cardiac organoids and normal cardiac organoids (obtained using method 1) were injected into the border area of ​​the left anterior descending artery ligation (4 organoids per mouse, injected at two sites in the border area of ​​the left anterior descending artery ligation. The organoids were placed in 20 μl of Matrigel diluted in PBS to prevent cell migration within the organoids after injection). Five mice were injected with CLU+ cardiac organoids and five mice were injected with normal cardiac organoids. All mice were intraperitoneally injected with cyclosporine A (5 mg / kg / day) three days before surgery and every other day after surgery to suppress mouse immune rejection and promote organoid colonization. Starting from the completion of the ligation surgery (LAD), ultrasound examination and bioluminescence enzyme tracking in vivo were performed on days 1 and 5, and ultrasound examination was performed on days 1, 3, 5, and 7 to observe changes in mouse cardiac function. A schematic diagram is shown in Figure 6A.

[0174] (2) Dosage regimen 2: CLU + Cardiac organoids were administered, with LAD, LAD+Matrigel, and LAD+organoids as controls

[0175] Sixteen 8-week-old male C57 mice (Lingchang Company) were anesthetized and subjected to the method of ligating the left anterior descending coronary artery (LAD) to prepare an acute myocardial infarction model. Four mice were anesthetized and the chest cavity was opened but no ligation was performed, and they were used as the sham operation (Sham) group. Among the mice with myocardial infarction, four mice were anesthetized and the chest cavity was opened and ligated, and they were used as the LAD group. Ten minutes after ligation, four mice were injected with Matrigel into the border area of ​​the left anterior descending coronary artery ligation using a 22G syringe, and they were used as the LAD+Matrigel group. Ten minutes after ligation, the other four mice were injected with CLU coated with 50% Matrigel using a 22G syringe. + Cardiac organoids were injected into the border area of ​​the left anterior descending artery ligation (4 organoids were injected into each mouse, respectively, at 2 sites in the border area of ​​the left anterior descending artery ligation. The organoids were placed in 20 microliters of PBS-diluted matrigel to prevent cell migration within the organoids after injection) as the LAD+ organoid group. All mice were intraperitoneally injected with cyclosporine A (5 mg / Kg / day) three days before surgery, and the drug was given every other day after surgery to suppress mouse immune rejection and help organoid colonization. Starting from the completion of the ligation surgery (LAD), cardiac electrophysiological testing was performed on the 10th day to observe changes in the mouse cardiac electrophysiology. The schematic diagram is shown in Figure 7A.

[0176] (3) Dosage regimen three: AAV-Clusterin administration

[0177] Twenty-four 6-week-old male C57 mice (Lingchang Company) were randomly divided into sham operation group (Sham), AAV-Control group (injected with AAV-9 control virus) and AAV-CLU group (injected with AAV-9-Clusterin virus), with 8 mice in each group. The animals were acclimated to the animal room for a period of time after arrival. Six days after arrival, the AAV-Control group and AAV-CLU group were injected (1×10 13 A MI model was established at 10 weeks of age using LAD ligation using AAV-9 control and AAV-9-Clusterin viruses (both viruses). Ultrasound examinations were performed on the mice the day after surgery and at 11, 12, and 13 weeks of age. On day 3, three mice from each group were randomly selected and sacrificed, and their hearts were harvested. Subsequent ultrasound examinations were performed on the remaining mice to observe changes in cardiac function. A schematic diagram is shown in Figure 8A.

[0178] 3. Bioluminescent enzyme detection

[0179] Mice injected with organoids were anesthetized with isoflurane. Bioluminescent detection substrate (15 mg / ml) purchased from Yeasen was injected intraperitoneally at a concentration of 10 μl / g body weight. After 15 minutes, the cells were detected using a PE small animal imaging system. Organoids were prepared from H9 cells expressing bioluminescent enzymes according to the method for preparing cardiac organoids described herein.

[0180] 4. Ultrasonic testing

[0181] Mice undergoing ultrasound examination were anesthetized with isoflurane, and images were acquired using a Vevo 3100 LT (VisualSonics). B-mode is a brightness mode, displaying information reflected from interfaces at different depths in grayscale. M-mode is a motion mode, primarily showing the temporal changes in the position of tissue echo interfaces. EF = (EDV - ES) / EDV × 100%, where EF is ejection fraction, EDV is end-diastolic volume, and ES is end-systolic volume.

[0182] 5. Cardiac electrophysiological monitoring

[0183] Mice undergoing electrocardiogram (ECG) testing were anesthetized with isoflurane, and lead I was measured for five minutes using an iWorx-IX-BIO4 ultrasound instrument. The PR interval and R wave peak were calculated using LabScribe software.

[0184] 6. Preparation of AAV-Clusterin (taking 10 cm cell culture dish as an example)

[0185] (1) Cell plating. Ensure that the confluence of AAV293 cells reaches 70%-80% before transfection. The AAV293 cell culture medium is DMEM + 10% FBS. Replace with 8 ml of fresh FBS-free culture medium 1-2 hours before transfection.

[0186] (2) Take a 15 ml centrifuge tube and add 1 ml of 0.3 M CaCl2 solution. Take 10 μg of pAAV-GOI, 10 μg of pHelper, and 10 μg of pAAV2-8 and add them to the CaCl2 solution and mix well.

[0187] (3) Take another 15 ml centrifuge tube and add 1 ml of 2xHBS. Add the DNA / CaCl2 mixture dropwise to the 2xHBS solution and mix by inverting or repeatedly pipetting.

[0188] (4) Immediately add the DNA / CaCl2 / HBS suspension dropwise to the 293T cells and gently shake the culture dish to evenly distribute the DNA suspension in the culture medium;

[0189] (5) Return the cells to the 37°C incubator;

[0190] (6) After 6 h, the culture medium was replaced with complete DMEM medium;

[0191] (7) Continue culturing for 66h-72h.

[0192] 7. AAV purification (taking 10 cm cell culture dish, n=3 as an example)

[0193] (1) Gently pipette the cells into a 50 ml centrifuge tube and centrifuge at 3000 g for 10 min. Transfer the supernatant to another 50 ml centrifuge tube, filter through a 0.22 μM filter membrane, and sample 100 μl for titer (sample 1). The cell pellet can be discarded directly or resuspended in 10 ml PBS and centrifuged at 3000 g for 10 min. Discard the supernatant and keep the cells aside. If not purified immediately, store the filtered supernatant and cells at -20°C.

[0194] (2) Add PEG800 and NaCl powder to the supernatant filtered in (1) to a final concentration of 10% and 1 M / L, respectively. Shake well. Incubate on ice for at least 1 hour (or overnight at 4°C). Centrifuge at 12,000 g for 15 minutes.

[0195] (3) Discard the supernatant from step (2) and resuspend the pellet in Lysis buffer (1 ml per 10 cm dish). Add benzonase (1 μl / 20 ml) and incubate at 37°C for 30 min. Add an equal volume of chloroform and shake on a shaker for 1 h. Centrifuge at 12,000 g for 15 min. Collect the supernatant.

[0196] (4) Concentrate using an ultrafiltration column. Replace with lysis buffer (5000 g / 10 min, 10 min) to a residual volume of 500 μL.

[0197] 8. Preparation of Mouse Inflammation Model and AAV-Clusterin Administration

[0198] Twenty-four 6-week-old male C57 mice (Lingchang Company) were injected with AAV-9 control (intrathoracic injection, virus at 1×10 13 The researchers then co-administered AAV-9-Clusterin virus (100 viruses / mouse) and AAV-9-Clusterin virus. After two weeks of overexpression, the mice were injected intraperitoneally with LPS at 20 mg / kg to simulate in vivo infection. Twenty-four hours after LPS injection, cardiac function was assessed by ultrasound.

[0199] 9. Cardiac fibrosis detection

[0200] Cardiac fibrosis was detected according to the recommended method of Masson's trichrome staining kit (Solerbo, Cat. No. G1340), as follows:

[0201] (1) Air-dry the frozen sections for 10 minutes; (2) Soak in PBS for 3 minutes (to remove OCT); (3) Place in fixative at 60 degrees for 1 hour; (4) Use the prepared iron hematoxylin staining reagents 1 and 2 (A) Iron hematoxylin staining solution at RT in the dark for 7 minutes; (5) Rinse gently with ultrapure water, immerse up and down 2 times, and immerse for 3 minutes for the third time; (6) Differentiate with acidic ethanol solution (B) for 1 minute; (7) Gently immerse with ultrapure water 1 to 2 times; (8) Return to blue with Masson blue solution (C) for 1 minute; (9) Gently immerse with ultrapure water 1 to 2 times, and immerse for 3 minutes for the third time; (1 0) Ponceau acid fuchsin staining solution (D) at room temperature, shielded from light for 10 minutes; (11) Gently rinse with ultrapure water 1 to 3 times; (12) Rinse with weak acid working solution (E) for 1 minute; (13) Phosphomolybdic acid solution (F) for 2 minutes at room temperature, shielded from light; (14) Rinse with weak acid working solution (E) for 1 minute; (15) Directly immerse in aniline blue staining solution (G) for 5 minutes at room temperature, shielded from light; (16) Rinse with weak acid working solution (E) for 1 minute; (17) Dehydrate with 95% ethanol for 30 seconds; (18) Dehydrate with anhydrous ethanol for 1 minute; (19) Soak in xylene for 10 minutes; (20) Mount with neutral gum. Statistical analysis was performed using imageJ software, and the fibrosis area and the area of ​​the left ventricle (excluding the cardiac cavity) were circled. The fibrosis ratio = the area of ​​the fibrosis area ÷ the area of ​​the left ventricle (excluding the cardiac cavity).

[0202] 10. ELISA detection of Clusterin content

[0203] (1) Remove the unused microwell strips from the plate frame, put the remaining strips back into the aluminum foil bag containing desiccant, and then reseal for storage; (2) Add 350 μL of 10-40 μl / ml washing buffer to each well, let it stand for 40 seconds and then discard the liquid. This step is washed 3 times in total; (3) Add 100 μL of standard / sample diluent (R1) to the blank well; (4) Add 100 μL of standard or sample of different concentrations to the other wells, seal the wells with the provided sealing film, and incubate at 37°C for 2 hours; (5) Prepare the biotinylated antibody (100x) working solution 15 minutes before use; (6) Discard the liquid in the wells and repeat step 2 Washing steps; (7) Add biotinylated antibody working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 1 hour; (8) Prepare streptavidin-HRP (100x) working solution 15 minutes before use; (9) Discard the liquid in the well and repeat the washing steps in step (2); (10) Add streptavidin-HRP working solution (100 μL / well) to each well, cover with a new sealing film, and incubate at 37°C for 30 minutes; (11) Preheat the microplate reader; (12) Discard the liquid in the well and repeat the washing steps in step (2); (13) Add TMB substrate (100 μL / well) to the well. Incubate at 37°C in the dark for 15-20 minutes; (14) Add stop solution (50 μL / well) and immediately place in the microplate reader. Measure the OD value of each well at 450 nm within 5 minutes. If you can choose to calibrate the wavelength, set it to 570nm or 630nm. Subtract the 570nm or 630nm reading from the 450nm reading. This method can correct and remove the OD value of non-coloring substances, thereby obtaining more accurate test results.

[0204] Example 1, CLU + Producing cardiac organoids

[0205] 1. Preparation of cardiac organoids (Method 1)

[0206] Human H9 ES cells were resuspended in mTeSR+ROCKi (mTeSR as medium, ROCKi concentration was 5 μM), seeded at a density of 8,000 cells / 96-well, and cultured for 24 hours. At this stage, the cells aggregated into clusters.

[0207] Then, the cells were cultured with RPMI1640 containing B27 (10 μl / ml), FGF2 (10 ng / ml), LY294002 (2 μM), Activin A (20 ng / ml), BMP4 (10 ng / ml), CHIR99021 (3 μM) and 1 μg / ml insulin for 36-40 h.

[0208] The cells were then induced for 4 days with RPMI1640 containing B27 (10 μl / ml), BMP4 (20 ng / ml), FGF2 (10 ng / ml), IWP2 (4 μM), and retinoic acid (0.5 μM), with the medium replaced daily. After this induction, a culture system containing cardiac cells (including cardiomyocytes) was obtained.

[0209] Then, the culture medium was replaced with RPMI1640 medium containing B27 (10 μl / ml), BMP4 (10 ng / ml), FGF2 (10 ng / ml), and insulin (10 μg / ml) for 2 consecutive days, with the culture medium changed daily to obtain a system containing stable, mature cardiac cells (including cardiomyocytes).

[0210] The culture medium was replaced with RPMI1640 culture medium containing B27 (10 μl / ml), and the culture medium was changed every day for 7 consecutive days to obtain cardiac organoids (hereinafter referred to as normal cardiac organoids).

[0211] 2. Preparation of CLU + Cardiac organoids (Method 2)

[0212] Human H9 ES cells were resuspended in mTeSR+ROCKi (mTeSR as medium, ROCKi concentration was 5 μM), seeded at a density of 8,000 cells / 96-well, and cultured for 24 hours. At this stage, the cells aggregated into clusters.

[0213] Then, the cells were cultured with RPMI1640 containing B27 (10 μl / ml), FGF2 (10 ng / ml), LY294002 (2 μM), Activin A (20 ng / ml), BMP4 (10 ng / ml), CHIR99021 (3 μM) and 1 μg / ml insulin for 36-40 h.

[0214] The cells were then induced for 4 days with RPMI1640 containing B27 (10 μl / ml), BMP4 (20 ng / ml), FGF2 (10 ng / ml), IWP2 (4 μM), and retinoic acid (0.5 μM), with the medium replaced daily. After this induction, a culture system containing cardiac cells (including cardiomyocytes) was obtained.

[0215] Then, the culture medium was replaced with RPMI1640 medium containing B27 (10 μl / ml), BMP4 (10 ng / ml), FGF2 (10 ng / ml), and insulin (10 μg / ml) for 2 consecutive days, with the culture medium changed daily to obtain a system containing stable, mature cardiac cells (including cardiomyocytes).

[0216] The culture medium was replaced with RPMI1640 medium containing B27 (10 μl / ml) and VPA (200 μM). The culture medium was changed every day for 7 to 13 consecutive days to obtain cardiac organoids.

[0217] Analysis showed that the cardiac organoids highly expressed Clusterin, which was called Clusterin positive (CLU + ) cardiac organoids (Figure 1A).

[0218] Example 2, CLU + Cellular composition of cardiac organoids

[0219] The surface markers of cardiomyocytes, endothelial cells and smooth muscle cells (cTNT, CD31 and α-SMA) were used to analyze CLU + Immunofluorescence staining of cardiac organoids revealed CLU + Cardiac organoids contain cardiomyocytes, endothelial cells, and smooth muscle cells (Figure 2). + Immunofluorescence staining of cardiac organoids revealed CLU + Cardiac organoids contain cardiomyocytes, epicardial cells, and endothelial cells (Figure 3). + Immunofluorescence staining of cardiac organoids revealed CLU + Heart organoids contain both ventricular and atrial cardiomyocytes (Figure 4).

[0220] It can be seen that the CLU prepared by the present invention + Cardiac organoids have a diverse cellular composition, including endothelial cells, epicardial cells, vascular smooth muscle cells, epicardial cells, ventricular cardiomyocytes and atrial cardiomyocytes, and their structure is similar to that of the human heart.

[0221] Example 3, CLU + Functional analysis of cardiac organoids

[0222] (1) Morphology

[0223] Observation CLU + The morphology of the cardiac organoids shows that compared with the normal cardiac organoids (obtained by method 1), CLU + The cardiac organoids had a smoother appearance (Figure 1B).

[0224] (2) Clusterin content

[0225] For CLU +The Clusterin content in cardiac organoids was observed by immunofluorescence staining, and the percentage of Clusterin-positive (CLU-positive) cardiomyocytes was counted. The results showed that compared with ordinary cardiac organoids, CLU + Cardiac organoids contained more CLU-positive cardiomyocytes (Figure 1C).

[0226] (3) Cell composition

[0227] Analyze CLU + The presence of cardiomyocyte surface markers cTNT, endothelial cell surface marker CD31, smooth muscle cell surface marker α-SMA, atrial cardiomyocyte surface marker MYL7 / MLC-2A, ventricular cardiomyocyte surface marker MLC-2V, and epicardial cell surface marker WT1 in cardiac organoids was evaluated. + Immunofluorescence staining of cardiac organoids revealed that CLU + The fluorescence of cTNT, CD31, α-SMA, MYL7 / MLC-2A, MLC-2V and WT1 can be clearly observed in cardiac organoids (Figures 2-4), indicating that CLU + Cardiac organoids contain a rich cellular composition, including cardiomyocytes, endothelial cells, smooth muscle cells, atrial cardiomyocytes, ventricular cardiomyocytes, and epicardial cells. However, WT1 and CD31 fluorescence were almost undetectable in standard cardiac organoids, indicating that they lack epicardial and endothelial cells (Figure 5).

[0228] The above results show that the CLU + Cardiac organoids have a more diverse cell composition than ordinary heart organoids.

[0229] (4) Promotes cardiac function recovery in mice with myocardial infarction

[0230] The mouse acute myocardial infarction model was established by ligating the left anterior descending coronary artery (LAD). According to the dosing regimen 1, 10 minutes after LAD ligation, CLU was administered to the mice coated with 50% Matrigel using a 22G syringe. + Cardiac organoids and normal heart organoids were injected into the border area of ​​the left anterior descending artery ligation. All mice were intraperitoneally injected with cyclosporine A (5 mg / kg body weight / day).

[0231] In vivo organoid tracking was performed using a bioluminescent enzyme. The results showed that transplanted cardiac organoids successfully colonized the heart, and cardiac organoid survival was detectable even on day five (Figure 6C). Fractional shortening (FS) and ejection fraction (EF) of the mice were measured.

[0232] The results showed that compared with ordinary heart organoids, CLU + Cardiac organoids could restore cardiac function more strongly (Figure 6D-F).

[0233] Example 4, CLU + Cardiac organoids promote cardiac function recovery in mice with myocardial infarction

[0234] An acute myocardial infarction model was established in mice by ligating the left anterior descending coronary artery (LAD). Cardiac electrophysiological changes were observed after administration of cardiac organoids using the second dosing regimen (Figure 7A). ECG data were recorded using a monitoring instrument, and the PR interval and R wave peak were calculated using LabScribe software.

[0235] The results showed that LAD can cause a phenotype of cardiac electrical disturbance similar to that after myocardial infarction (Figure 7B). + Cardiac organoids significantly improved the ECG abnormalities in mice, as demonstrated by the recovery of the R wave (Figure 7B) and the enhancement of interventricular conduction (PR interval) (Figure 7D).

[0236] Example 5: AAV-mediated Clusterin expression promotes cardiomyocyte proliferation and cardiac function recovery in mice with myocardial infarction

[0237] An acute myocardial infarction model was established in mice by ligating the left anterior descending coronary artery (LAD). The effects of AAV-mediated Clusterin expression on cardiomyocyte proliferation and cardiac function were observed using the third dosing regimen (Figure 8A). The results showed that AAV-9-Clusterin injection promoted cardiomyocyte proliferation in the infarct margin (Figures 8B-C).

[0238] Cardiac fibrosis was then assessed in mice injected with either the AAV-9 control virus (AAV-Control) or the AAV-9-Clusterin virus (AAV-CLU). Following LAD, cardiac fibrosis was assessed, and fibrotic scar tissue was analyzed. The results showed a significant reduction in cardiac fibrosis area and increased ventricular wall thickness in the AAV-9-Clusterin group (Figures 8D-E).

[0239] Finally, cardiac function was assessed in mice: 21 days after LAD, cardiac ultrasound was performed in the sham group, mice injected with AAV-9 control virus (AAV-Control), and mice injected with AAV-9-Clusterin virus (AAV-CLU). The results showed that AAV-9-Clusterin injection rescued the decreased cardiac contractility (EF and FS) induced by LAD surgery (Figures 8F-H).

[0240] CLU in Example 2 + Cardiac organoids have diverse cell compositions. After injection of AAV-9-Clusterin, cardiomyocyte proliferation was observed, but no more cell types were observed. This indicates that CLU + The improvements in cardiac organoids would be even more pronounced.

[0241] Example 6: AAV-mediated Clusterin expression improves inflammation-induced cardiac dysfunction

[0242] Six-week-old mice were injected with either an AAV-9 control virus or an AAV-9-Clusterin virus. After two weeks of overexpression, the mice were injected with LPS to simulate in vivo infection. Cardiac function was assessed 24 hours after LPS injection.

[0243] The results showed that compared with mice injected with the AAV-9 control virus, injection of AAV-9-Clusterin improved the contractility (EF, FS), ejection fraction (SV, CO), and blood flow rate of the hearts of mice injected with LPS. Furthermore, AAV-9-Clusterin injection increased the heart rate (Figures 9A-B).

[0244] These results suggest that AAV-9-Clusterin can improve cardiac function in mice following inflammatory infection.

[0245] Example 7: Clusterin levels in serum are negatively correlated with the severity of myocardial infarction

[0246] To examine the relationship between serum Clusterin levels and myocardial infarction, the inventors collected serum samples from 100 myocardial infarction patients and 11 healthy individuals from Shanghai First People's Hospital and tested Clusterin levels using ELISA ( FIG10A ).

[0247] The results showed that Clusterin levels in the serum of patients with myocardial infarction were significantly lower than those in healthy controls (Figure 10B). Clusterin levels were negatively correlated with TnI and BNP, clinical indicators used to assess the severity of myocardial infarction (Figures 10C-D), and positively correlated with EF, a post-myocardial function marker (Figure 10E), indicating that Clusterin levels can indicate the occurrence and severity of myocardial infarction.

[0248] Furthermore, the inventors found that Clusterin was negatively correlated with the levels of inflammatory factors (TNF-α and IL6) in patient serum ( Figure 10F-G ), indicating that patients with high Clusterin levels had a weaker inflammatory response in the body.

[0249] Myocardial infarction patients were grouped according to the KILIP classification (Grade I: no obvious heart failure; Grade II: left heart failure, lung rales <50% of the lung field; Grade III: lung rales, and the range of rales is greater than 1 / 2 lung field (acute pulmonary edema); Grade IV: cardiogenic shock, with hemodynamic changes of varying stages and degrees). The inventors found that when the average serum Clusterin level was 162±81μg / ml, the severity of myocardial infarction in patients was the highest, indicating a poor prognosis for myocardial infarction. When the average serum Clusterin level was 196±65μg / ml, the severity of myocardial infarction in patients was less (Figure 11). Based on the data information for the tested patients, the threshold value can be a value within the range of 175-180μg / ml.

[0250] Further analysis of the relationship between Clusterin levels and the cardiac function marker EF revealed that when the average serum Clusterin level was 140±42 μg / ml, the EF level decreased most significantly after myocardial infarction. Meanwhile, when the average serum Clusterin level was 213±57 μg / ml, the patients' cardiac function was least impaired (Figure 12).

[0251] The above results show that Clusterin is closely related to myocardial infarction, and the patient's heart health can be evaluated based on the serum Clusterin content.

[0252] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. A method for preparing a cardiac organoid, comprising: Induce cardiac cells to differentiate into Clusterin-positive (CLU + ) Heart organoids.

2. The method according to claim 1, wherein The cardiac cells are obtained using pluripotent stem cells as starting cells; preferably, the pluripotent stem cells are differentiated into cardiac cells to form cardiac cells, and then stimulated with VPA to obtain Clusterin-positive cardiac organoids.

3. The method according to claim 2, wherein VPA is added to the culture medium; preferably, the concentration of VPA in the culture medium is 60-2000 μM, more preferably 100-1500 μM.

4. The method according to claim 3, wherein After VPA is added to the culture medium, the culture is continued for 5 to 16 days, preferably 6 to 14 days.

5. The method according to claim 2, wherein include: (a) culturing pluripotent stem cells with culture medium A, B, or C to obtain cardiac cells; preferably, the culturing is performed for 5 to 10 days, more preferably 6 to 9 days; (b) culturing with medium D containing VPA to obtain the Clusterin-positive cardiac organoids; The culture medium A comprises B27, FGF2, LY294002, Activin A, BMP4, CHIR99021 and insulin; the culture medium B comprises B27, BMP4, FGF2, IWP2 and retinoic acid; the culture medium C comprises B27, BMP4, FGF2 and insulin; and the culture medium D comprises B27 and VPA.

6. The method according to claim 4, wherein In the culture medium D, the concentration of B27 is 5-40 μl / ml; or In the culture medium A, the concentration of B27 is 5-40 μl / ml; the concentration of FGF2 is 2-50 ng / ml; the concentration of LY294002 is 0.5-10 μM; the concentration of Activin A is 10-100 ng / ml; the concentration of BMP4 is 1-30 ng / ml; the concentration of CHIR99021 is 1-15 μM; and the concentration of insulin is 0.1-5 μg / ml; or In the culture medium B, the concentration of B27 is 5-40 μl / ml; the concentration of BMP4 is 3-50 ng / ml; the concentration of FGF2 is 2-50 ng / ml; the concentration of IWP2 is 1-15 μM; the concentration of retinoic acid is 0.1-5 μM; or In the culture medium C, the concentration of B27 is 5-40 μl / ml; the concentration of BMP4 is 1-30 ng / ml; the concentration of FGF2 is 2-50 ng / ml; and the concentration of insulin is 5-15 μg / ml.

7. A Clusterin-positive cardiac organoid prepared by the method of any one of claims 1 to 6; Preferably, the Clusterin-positive cardiac organoids include: Endothelial cells, epicardial cells, vascular smooth muscle cells, epicardial cells, ventricular cardiomyocytes, atrial cardiomyocytes; Preferably, the markers expressed by the Clusterin-positive cardiac organoids include: cTNT, CD31, α-SMA, MYL7 / MLC 2A, WT1 and / or MLC 2V.

8. A pharmaceutical composition or kit comprising the Clusterin-positive cardiac organoid according to claim 7.

9. Application of VPA for inducing cardiac cells to differentiate into Clusterin-positive cardiac organoids, or for preparing a composition for inducing cardiac cells to differentiate into Clusterin-positive cardiac organoids.

10. A culture medium composition for preparing Clusterin-positive cardiac organoids, comprising: Culture media A, B, C and D, wherein culture media A includes B27, FGF2, LY294002, Activin A, BMP4, CHIR99021 and insulin; culture media B includes B27, BMP4, FGF2, IWP2 and retinoic acid; culture media C includes B27, BMP4, FGF2 and insulin; and culture media D includes B27 and VPA.

11. Use of the Clusterin-positive cardiac organoid according to claim 7 in the preparation of a pharmaceutical composition for improving cardiac function or treating cardiac damage; Preferably, the improving cardiac function comprises: Improve ECG abnormalities, enhance cardiac contractility, enhance cardiac ejection capacity, increase blood flow velocity or increase heart rate; Preferably, the heart damage includes: myocardial infarction, cardiac fibrosis, ventricular wall thickening or myocardial inflammation.

12. Use of a Clusterin upregulator in the preparation of a drug for treating cardiac inflammation; wherein: The Clusterin upregulator is an expression vector or Clusterin-positive cardiac organoid that can express Clusterin after being transferred into cells; preferably, the expression vector includes: AAV vector, adenovirus vector, lentivirus vector; more preferably, the expression vector is an AAV vector.

13. Application of serum Clusterin protein in the preparation of a detection system for evaluating myocardial infarction; wherein, The evaluation includes: detecting the level of Clusterin protein in the serum of the subject; if the level of Clusterin protein is high, it indicates that the severity of myocardial infarction in the subject is low; conversely, if the level of Clusterin protein is low, it indicates that the severity of myocardial infarction in the subject is high; preferably, the threshold value for determining the level of Clusterin protein is a value or a range of values ​​selected from 170 to 185 μg / ml.

14. A detection system for evaluating myocardial infarction, comprising: (1) a detection component or module for analyzing Clusterin protein in a serum sample to be tested and determining the amount of the protein; (2) an analysis component or module, which analyzes the value obtained in (1), and sets a threshold value; when the value obtained in (1) is higher than the threshold value, it indicates that the patient has a good prognosis; when the value obtained in (1) is lower than the threshold value, it indicates that the patient has a poor prognosis; preferably, the threshold value is a value or a range of values ​​selected from 170 to 185 μg / ml.

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