Application of exosome derived from human induced pluripotent stem cells in preparation of medicine for treating Kawasaki disease

Abstract

The invention belongs to the field of biological medicine, and particularly relates to application of exosome derived from human induced pluripotent stem cells to preparation of medicine for treating Kawasaki disease, and the exosome is prepared by secreting and purifying human induced pluripotent stem cells. The human induced pluripotent stem cell-derived exosome (iPS-EXO) provided by the invention can achieve a certain treatment effect on a Candida albicans water-soluble component (CAWS)-induced coronary artery vasculitis Kawasaki disease (KD) model mouse, so that a new method is provided for treating KD by adopting the induced pluripotent stem cell-derived exosome in the future.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to the use of exosomes derived from human induced pluripotent stem cells for preparing a drug for treating Kawasaki disease. Background Art

[0002] Kawasaki disease (KD) is an acute, self-limited vasculitis that primarily affects children, particularly those under 5 years of age, and is characterized by persistent fever, rash, cervical lymphadenopathy, and conjunctivitis [Agarwal S, Agrawal DK: Kawasaki disease: etiopathogenesis and novel treatment strategies. Expert Review of Clinical Immunology 2016, 13(3): 247-258.]. The etiology of KD is still unclear. It can lead to serious complications such as coronary artery aneurysms and acquired heart disease. If not diagnosed and treated early, it can even lead to death [Ae R, Makino N, Kosami K, Kuwabara M, Matsubara Y, Nakamura Y: Epidemiology, Treatments, and Cardiac Complications in Patients with Kawasaki Disease: The Nationwide Survey in Japan, 2017-2018. The Journal of Pediatrics 2020, 225: 23-29.e22.]. Currently, the main clinical treatment for KD is timely intravenous immunoglobulin (IVIG) treatment, but many patients still do not respond to IVIG treatment [Kuo HC: Diagnosis, Progress, and Treatment Update of Kawasaki Disease. International Journal of Molecular Sciences 2023, 24(18).]. Therefore, there is an urgent need to explore more KD treatment options.

[0003] Exosomes are nanoscale vesicles released by cells, with a diameter of approximately 30-150 nanometers. They contain bioactive molecules such as proteins, nucleic acids (such as mRNA, microRNA, lncRNA, CirRNA, etc.), lipids and metabolites, which can be internalized by target cells and affect their functions [Abels ER, Breakefield XO: Introduction to Extracellular Vesicles: Biogenesis, RNA Cargo Selection, Content, Release, and Uptake. Cellular and Molecular Neurobiology 2016, 36(3): 301-312.]. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the existing technology and to provide an application of exosomes derived from human induced pluripotent stem cells for the preparation of a drug for the treatment of Kawasaki disease.

[0005] The technical solution adopted by the present invention is as follows: the use of exosomes derived from human induced pluripotent stem cells for the preparation of a drug for treating Kawasaki disease, wherein the exosomes are secreted and purified from human induced pluripotent stem cells.

[0006] The method for preparing exosomes derived from human induced pluripotent stem cells comprises the following steps: (1) Cultivate human induced pluripotent stem cells and collect the culture supernatant; (2) The culture supernatant was separated and purified to obtain exosomes derived from human induced pluripotent stem cells.

[0007] In step (1), human induced pluripotent stem cells were placed in PSCeasy ® Culture the cells in 1% EDTA Human pluripotent stem cell medium at 37°C in a 5% CO2 incubator. Replace the medium daily. Subculture the cells with EDTA when the cell density reaches 80-90%.

[0008] In step (2), the collected supernatant was centrifuged at 300 × g for 10 min at 4°C to remove floating cells and cell debris; the supernatant was centrifuged at 2000 × g for 10 minutes and then at 10000 × g for 1 hour at 4°C to further eliminate cell residues; the supernatant was then filtered through a 0.22 μm sterile filter into a new tube in a 32 Ti ultracentrifuge rotor; and ultracentrifuged at 31000 rpm for 90 min at 4°C to obtain exosomes derived from human induced pluripotent stem cells.

[0009] The present invention provides human induced pluripotent stem cell-derived exosomes (iPS-EXO), which can have a certain therapeutic effect on the Kawasaki disease (KD) model mouse model of coronary artery vasculitis induced by Candida albicans water-soluble fraction (CAWS), thereby providing a new method for the future treatment of KD using exosomes derived from induced pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying any creative work, other drawings obtained based on these drawings still fall within the scope of the present invention.

[0011] Figure 1 (A) Bright-field microscopic image of clonal iPS cell clusters, scale bar 100 μm; (B) Immunofluorescence staining of iPS cell biomarkers such as NANOG, OCT4, SOX2, and SSEA4. Scale bar 25 μm; (C) Fluorescent images of DiI-labeled iPS-EXOs taken up by mouse lung, kidney, liver, spleen, and other organs. Figure 2 (A) Schematic diagram of the effects of iPS-EXO on CAWS-induced KD mice; (B) Size and shape of iPS-EXO under transmission electron microscopy, scale bar, 500 nm; (C) Size distribution of iPS-EXO detected by nanoparticle tracking analyzer (NTA); (D) Western blot analysis of the expression of exosome-associated surface markers CD9, CD63, and CD81 in iPS-EXO; (E) Fluorescence microscopy confirmed the location of DiI (red fluorescence)-labeled iPS-EXO in CD31-positive human umbilical vein endothelial cells (HUVECs), scale bar, 100 μm; (F) Quantification of the fluorescence intensity of DiI-labeled iPS-EXO in HUVECs (n = 3); (G) In vivo imaging detected the location of DiI-labeled iPS-EXO in the aortic root and proximal coronary artery regions of mouse heart tissue; (H) Quantification of the fluorescence intensity of DiI-labeled iPS-EXO in mouse heart tissue (n = 3); mean ± SD. ** p <0.01, ** p <0.001 indicated that the difference was statistically significant; Figure 3(A) TUNEL staining was used to detect apoptosis in HUVECs treated with different methods, scale bar: 100 μm; (B) Quantification of TUNEL staining results in each group (n = 3); (C) Flow cytometry was used to detect apoptosis rates in HUVECs treated with different methods; (D) Quantification of Q1-LR (early apoptosis) and Q1-UR (late apoptosis) apoptosis rates in each group (n = 3); (E) Western blot analysis of BAX, BCL-2, and GADPH protein expressions; (F) Quantification of protein expression levels (n = 3); mean ± SD. * p <0.05, ** p <0.01 indicates a significant difference, ns>0.05 indicates an insignificant difference; Figure 4 (A) HE staining of the aortic root and proximal coronary arteries of mouse heart tissue; (B) Quantitative results of HE staining in each group (n = 5); (C) CD45 immunohistochemical staining around the coronary arteries of mouse heart tissue; (D) Quantitative results of CD45 immunohistochemical staining in each group (n = 5); (E) TNF-α immunohistochemical staining around the coronary arteries of mouse heart tissue; (F) Quantitative results of TNF-α immunohistochemical staining in each group (n = 5); (G) Masson staining of the aortic root and proximal coronary arteries of mouse heart tissue; (H) Quantitative results of Masson staining in each group (n = 5); mean ± SD, * p <0.05, ** p <0.01 indicates statistically significant differences, scale bars 100 μm, 300 μm; Figure 5 Figure 2. (A) RT-qPCR detection of the expression levels of inflammatory factors and chemokines in each group (n = 6); (B) TUNEL staining detection of apoptosis of endothelial cells around coronary arteries in mouse heart tissues, scale bar 100 μm; (C) Quantification of TUNEL staining results in each group (n = 3); (D) Western blot detection of BAX and BCL-2 protein expression in mouse heart tissues in each group; (E) Quantification of protein expression in each group (n = 3); mean ± SD, * p <0.05, ** p <0.01 indicates that the difference is statistically significant, and ns>0.05 indicates that the difference is not statistically significant. DETAILED DESCRIPTION

[0012] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be described in further detail below with reference to the accompanying drawings.

[0013] Example 1, Preparation of Exosomes from Induced Pluripotent Stem Cells: (1) Culture of human induced pluripotent stem cells (iPS): Human induced pluripotent stem cells (iPS) were purchased from CELLAPY Biotechnology Co., Ltd. (hiPSC-U1, Beijing, China) and cultured in PSCeasy ® Culture the cells in 1% EDTA-2 Human pluripotent stem cell medium (CA1014500, CELLAPY) at 37°C in a 5% CO2 incubator. Replace the medium daily. Subculture the cells with EDTA when the cell density reaches 80-90%. Collect the culture supernatant and store at -80°C.

[0014] (2) Preparation of exosomes (iPS-EXO): The collected supernatant was centrifuged at 300 × g for 10 minutes at 4°C to remove floating cells and cell debris. The supernatant was centrifuged at 2000 × g for 10 minutes and then at 10,000 × g for 1 hour at 4°C to further eliminate residual cells. The supernatant was then filtered through a 0.22 μm sterile filter into a fresh tube in a 32 Ti ultracentrifuge rotor. iPS-derived exosomes (iPS-EXOs) were obtained by ultracentrifugation at 31,000 rpm for 90 minutes at 4°C. The protein concentration of iPS-EXOs was determined using a BCA protein assay kit (Beyotime, Shanghai, China). The expression of exosome surface markers CD9, CD63, and CD81 was assessed by Western blot. The morphology of iPS-EXOs was observed using a transmission electron microscope (TEM). The particle size of iPS-EXOs was measured using a nanoparticle tracking analyzer (NTA).

[0015] The iPS cells used in this study were densely arranged and grew in colony-like patterns ( Figure 1 A), these iPS cells can positively express NANOG, OCT4, SOX2, SSEA4 and other pluripotent stem cell markers ( Figure 1 B). The obtained iPS-EXO showed a typical exosome cup-like structure under transmission electron microscopy (TEM) ( Figure 2 B). Nanoparticle tracking analysis (NTA) showed that the average diameter of iPS-EXO was 100.00 ± 25.00 nm ( Figure 2C). Western blot results showed that iPS-EXO can express exosome-specific surface markers CD63, CD81 and CD9 ( Figure 2 D). These results indicate that we have successfully extracted iPS cell-derived exosomes (iPS-EXO).

[0016] Example 2, Preparation of DiI-labeled iPS-EXO: iPS-EXO were labeled with DiI (Beyotime, Shanghai, China), a lipid-soluble fluorescent dye widely used for cell membrane labeling and cell tracking. 2.5 μL of DiI (400×) was mixed with 2.5 μL of staining enhancement solution and then 500 μL of staining buffer. The mixture was incubated at room temperature for 10 minutes, and 50 μL of iPS-EXO was added. The mixture was incubated at 37°C for 20 minutes. The reaction was stopped by adding 1 mL of PBS and incubated at room temperature for 5 minutes to prevent nonspecific binding of the DiI dye. Subsequently, the DiI-labeled iPS-EXO were collected and ultracentrifuged at 120,000 g for 90 minutes at 4°C. DiI-labeled iPS-EXO were used to track iPS-EXO uptake in cells in vitro and tissues in vivo in the following examples.

[0017] Example 3, in vitro cell iPS-EXO uptake experiment: Human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured in high-glucose DMEM (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, NY, USA) and 1% penicillin / streptomycin (P / S) at 37°C in a 5% CO2 incubator. To establish an in vitro KD cell model, HUVECs were induced with tumor necrosis factor-α (TNF-α) at ​​various concentrations (0.01 ng / mL to 10,000 ng / mL) using a CCK-8 assay kit (YEASEN, Shanghai, China) to determine the optimal working concentration of TNF-α. Simultaneously, HUVECs were treated with iPS-EXO at various concentrations (0 μg / mL to 200 μg / mL) to assess their cytotoxicity. Based on a TNF-α-induced KD cell model, HUVECs were treated with iPS-EXO at varying concentrations (5 μg / mL to 100 μg / mL) to explore the optimal concentration. A control group, a TNF-α (100 ng / mL) group, a TNF-α + iPS-EXO (50 ng / mL) group, and a TNF-α + iPS-EXO + CC (AMPK inhibitor Compound C, 5 μM, Sigma, MO, USA) group were set up. The KD cell model was established by pre-treating HUVECs with TNF-α. Then, TNF-α-induced HUVECs were treated with iPS-EXO or the iPS-EXO + CC mixture, respectively, and cultured for 24 hours.

[0018] CD31-positive HUVECs showed green fluorescence. In vitro tracking results showed that 50 μg / mL iPS-EXO labeled with DiI red fluorescence was successfully taken up by HUVECs ( Figure 2 E). Quantitative results showed that the number of red fluorescent labeled cells in the iPS-EXO group was significantly higher than that in the control group ( Figure 2 F). These results indicate that iPS-EXOs can be successfully taken up by HUVECs.

[0019] To investigate the anti-apoptotic effect of iPS-EXO on TNF-α-induced HUVECs, TUNEL staining was used to evaluate the apoptosis of HUVECs under different treatments ( Figure 3 A). The staining results showed that compared with the control group, the TNF-α group had the most TUNEL-positive (green fluorescence) cells. iPS-EXO treatment significantly reduced the number of apoptotic cells, while AMPK inhibitor compound C (CC) significantly reversed the effects of iPS-EXO ( Figure 3 B). The apoptosis rate of each group was detected by flow cytometry ( Figure 3 C). The results showed that compared with the control group, the apoptosis rate of cells in the TNF-α group was significantly increased. iPS-EXO could significantly reduce TNF-α-induced HUVEC cell apoptosis, but CC could significantly eliminate the inhibitory effect of iPS-EXO ( Figure 3 D). Western blot was used to detect the expression of apoptosis protein BAX and anti-apoptosis protein BCL-2 in cells of each group ( Figure 3 E), the results showed that compared with the control group, the BAX level and BAX / BCL-2 ratio in the TNF-α group were significantly upregulated, and the BCL-2 expression was significantly downregulated. iPS-EXO treatment could reverse this trend, but CC could offset this reversal effect of iPS-EXO ( Figure 3 F). This suggests that iPS-EXO can alleviate TNF-α-induced HUVEC apoptosis.

[0020] Example 4, in vivo tissue iPS-EXO uptake experiment: First, Candida albicans water-soluble fraction (CAWS) was extracted from Candida albicans strain NBRC1385. CAWS was then dissolved in phosphate-buffered saline (PBS), sterilized by autoclaving, and stored at -80°C after concentration measurement.

[0021] Four-week-old male C57BL / 6 mice were purchased from the Experimental Animal Center of Wenzhou Medical University. Mice were housed at 23 ± 2°C and a relative humidity of 45% to 55% on a 12-h dark / light cycle, with water and food available ad libitum. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and approved by the Animal Ethics Committee of Wenzhou Medical University.

[0022] After one week of acclimatization, all mice were randomly divided into four groups: control group (n = 8), CAWS group (n = 8), CAWS + iPS-L-EXO group (n = 8), and CAWS + iPS-H-EXO group (n = 8). Figure 2As shown in Figure A, mice in the CAWS group were intraperitoneally injected with 4 mg / kg of CAWS daily for five consecutive days to induce a KD mouse model, while mice in the control group were intraperitoneally injected with an equal volume of PBS buffer. In this study, the survival rate of mice injected with CAWS was approximately 90%. Based on the successfully established CAWS-induced KD mouse model, mice in the CAWS + iPS-L-EXO group and the CAWS + iPS-H-EXO group were injected daily via the tail vein with 5 μg / kg iPS-EXO and 50 μg / kg iPS-EXO, respectively, for three consecutive weeks. After cessation of iPS-EXO injections and feeding for another week, mice in each group were euthanized by excessive anesthesia. The hearts, livers, spleens, lungs, and kidneys of the mice were examined using a small animal live imaging system, and heart tissue with aortic root and coronary artery regions was obtained for subsequent experiments.

[0023] Small animal in vivo imaging was used to evaluate the uptake of DiI-labeled iPS-EXO in mouse heart, lung, liver, spleen, and kidney tissues. The results showed that iPS-EXO could be successfully taken up by these organs and tissues, showing red fluorescence, and the fluorescence intensity gradually increased with the increase of iPS-EXO concentration. The red fluorescence in the heart tissue mainly accumulated in the aortic root and the proximal coronary artery region ( Figure 2 G and Figure 4 C). Fluorescence quantification results showed that the fluorescence of the 50 mg / kg iPS-EXO group was significantly stronger than that of the 5 mg / kg iPS-EXO group, while the control group had almost no fluorescence signal ( Figure 2 H). These results indicate that iPS-EXOs can be successfully taken up by cardiac tissue.

[0024] CAWS-induced KD mice were treated with low-dose (5 μg / kg, iPS-L-EXO) or high-dose (50 μg / kg, iPS-H-EXO) iPS-EXO. Tissue sections of the aortic root and proximal coronary artery regions of the mice were stained with HE to assess the inflammatory infiltration around the coronary arteries ( Figure 4 HE staining results showed that the inflammatory infiltration around the coronary arteries was the most severe in the CAWS group. iPS-L-EXO or iPS-H-EXO treatment significantly improved the inflammatory infiltration, with the CAWS + iPS-H-EXO group showing the best effect ( Figure 4 B).

[0025] CD45 is known as the common leukocyte antigen and is one of the most important biomarkers in inflammatory response. Figure 4C) The results showed that compared with the control group, the CD45-positive cells (leukocytes) in the CAWS group increased significantly, while the CD45-positive cells in the CAWS + iPS-L-EXO group and the CAWS + iPS-H-EXO group decreased significantly. Among them, the iPS-H-EXO treatment had a better effect on inhibiting leukocytes ( Figure 4 D).

[0026] TNF-α plays a key role in inflammatory activation and is a potent inflammatory cytokine that can cause damage to KD vascular endothelial cells. TNF-α immunohistochemical staining ( Figure 2 E) The results showed that compared with the control group, the level of TNF-α protein around the coronary arteries in the CAWS group was significantly upregulated, but iPS-L-EXO or iPS-H-EXO could downregulate the TNF-α level in the heart tissue of CAWS-induced mice, and the TNF-α level in the CAWS + iPS-H-EXO group was lower than that in the CAWS + iPS-L-EXO group ( Figure 4 F).

[0027] Masson trichrome staining was used to evaluate the pericoronary fibrosis in each group ( Figure 2 G). The results showed that compared with the control group, there was a large amount of collagen deposition around the coronary arteries in the CAWS group. Both iPS-L-EXO and iPS-H-EXO treatments significantly reduced collagen deposition in CAWS-induced mice, and the collagen deposition in the CAWS + iPS-H-EXO group was lower ( Figure 4 H). This indicates that iPS-EXO can alleviate inflammation and fibrosis caused by CAWS and has a protective effect on the coronary arteries of KD mice.

[0028] mRNA extracted from the aortic root and proximal coronary artery regions of the heart tissue of each group of mice was subsequently used for RT-qPCR analysis. RT-qPCR results showed that iPS-EXO significantly reduced the expression levels of inflammatory factors TNF-α, IL-1β, IL-6 and chemokines CCL5, CCL7, CCL17, CXCL5, and CXCL10 in CAWS-induced mice, and the inhibitory effect of iPS-H-EXO was better than that of iPS-L-EXO ( Figure 5 A).

[0029] Previous studies have shown that cellular inflammation is an important cause of cell apoptosis. To evaluate the anti-apoptotic effect of iPS-EXO on CAWS-induced KD mice, we performed TUNEL staining on the heart tissues of mice in each group ( Figure 5B). Staining results showed that compared with the control group, the number of TUNEL-positive cells around the coronary arteries in the CAWS group increased significantly. However, iPS-L-EXO or iPS-H-EXO treatment effectively reduced the TUNEL-positive cells in CAWS-induced mice, and iPS-H-EXO treatment was more effective in reducing the percentage of TUNEL-positive cells than iPS-L-EXO treatment ( Figure 5 C). Western blot analysis of the expression of apoptosis-related protein BAX and anti-apoptosis-related protein BCL-2 ( Figure 5 D). The results showed that compared with the control group, the BCL-2 level in the CAWS group was significantly downregulated, while the BAX level and the BAX / BCL-2 ratio were effectively upregulated. However, this trend could be reversed by iPS-L-EXO or iPS-H-EXO treatment ( Figure 5 E). These data further demonstrate that iPS-EXOs have a protective effect against inflammation and apoptosis in CAWS-induced mice.

[0030] The above disclosure is merely a preferred embodiment of the present invention and certainly cannot be used to limit the scope of the present invention. Therefore, equivalent changes made according to the claims of the present invention are still within the scope of the present invention.

Claims

1. Use of exosomes derived from human induced pluripotent stem cells for preparing a drug for treating Kawasaki disease, characterized in that: The exosomes are secreted and purified from human induced pluripotent stem cells.

2. Use of the exosomes derived from human induced pluripotent stem cells according to claim 1 for preparing a drug for treating Kawasaki disease, characterized in that: The method for preparing exosomes derived from human induced pluripotent stem cells comprises the following steps: (1) Cultivating human induced pluripotent stem cells and collecting culture supernatant; (2) The culture supernatant is separated and purified to obtain exosomes derived from human induced pluripotent stem cells.

3. Use of the exosomes derived from human induced pluripotent stem cells according to claim 2 for preparing a drug for treating Kawasaki disease, characterized in that: In step (1), human induced pluripotent stem cells were placed in PSCeasy ® II Human pluripotent stem Cell Medium at 37°C in a 5% CO2 incubator; fresh medium was replaced every day; when the cell density reached 80-90%, cells were subcultured with EDTA.

4. Use of the exosomes derived from human induced pluripotent stem cells according to claim 2 for preparing a drug for treating Kawasaki disease, characterized in that: In step (2), the collected supernatant was centrifuged at 300 × g for 10 min at 4°C to remove floating cells and cell debris; the supernatant was centrifuged at 2000 × g for 10 min and then at 10000 × g for 1 hour at 4°C to further eliminate cell residues; the supernatant was then filtered through a 0.22 μm sterile filter into a new tube in a 32 Ti ultracentrifuge rotor; and ultracentrifuged at 31000 rpm for 90 min at 4°C to obtain exosomes derived from human induced pluripotent stem cells.

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