Application of exosomes derived from human induced pluripotent stem cells in the preparation of drugs for treating Kawasaki disease

Abstract

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, wherein the exosomes are secreted and purified from human induced pluripotent stem cells. The present invention provides exosomes derived from human induced pluripotent stem cells (iPS-EXO), which can play a certain therapeutic role in the coronary artery vasculitis Kawasaki disease (KD) model mice induced by Candida albicans water-soluble fraction (CAWS), thereby providing a new method for treating KD using exosomes derived from induced pluripotent stem cells 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, especially 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 aneurysm and acquired heart disease. If it is not diagnosed and treated early, it may 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 method for clinical treatment of 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 about 30-150 nanometers. They contain biologically active 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 prior art and to provide an application of exosomes derived from human induced pluripotent stem cells for preparing a drug for treating 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 preparing 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:

[0007] (1) Cultivating human induced pluripotent stem cells and collecting culture supernatant;

[0008] (2) The culture supernatant is separated and purified to obtain exosomes derived from human induced pluripotent stem cells.

[0009] In step (1), human induced pluripotent stem cells were placed in PSCeasy ® II Human pluripotent stemCell 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.

[0010] 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.

[0011] The human induced pluripotent stem cell-derived exosomes (iPS-EXO) provided by the present invention can play a certain therapeutic role in the coronary vasculitis Kawasaki disease (KD) model mice induced by the water-soluble fraction of Candida albicans (CAWS), thereby providing a new method for the future treatment of KD with exosomes derived from induced pluripotent stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, without paying creative labor, other drawings obtained based on these drawings still belong to the scope of the present invention.

[0013] Figure 1 In the figure, (A) Bright field image of cloned cluster iPS cells under microscope, scale bar 100 μm; (B) Immunofluorescence staining of iPS cell biomarkers such as NANOG, OCT4, SOX2, and SSEA4. Scale bar 25 μm; (C) Tracer fluorescence image of DiI-labeled iPS-EXO after being absorbed by mouse lung, kidney, liver, spleen and other organ tissues;

[0014] Figure 2 In the figure, (A) Schematic diagram of the effect of iPS-EXO on CAWS-induced KD mice; (B) Size and shape of iPS-EXO under transmission electron microscopy, scale bar, 500 nm; (C) Nanoparticle tracking analyzer (NTA) detected the particle size distribution of iPS-EXO; (D) Western blot analysis of the expression of exosome-related surface markers CD9, CD63 and CD81 of iPS-EXO; (E) Fluorescence microscopy confirmed the location of iPS-EXO labeled with DiI (red fluorescence) in human umbilical vein endothelial cells (HUVECs) positively stained with CD31, scale bar, 100 μm; (F) Quantification of the fluorescence intensity of DiI-labeled iPS-EXO in HUVECs (n = 3); (G) The location of DiI-labeled iPS-EXO in the aortic root and proximal coronary artery regions of mouse heart tissue was detected by in vivo imaging; (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;

[0015] Figure 3(A) TUNEL staining was used to detect apoptosis of HUVECs with different treatments, scale bar 100 μm; (B) Quantification results of TUNEL staining in each group (n = 3); (C) Flow cytometry was used to detect the apoptosis rate of HUVECs with different treatments; (D) Quantification of Q1-LR (early apoptosis) and Q1-UR (late apoptosis) apoptosis rates of cells in each group (n = 3); (E) Western blot was used to detect the expression of BAX, BCL-2, and GADPH proteins; (F) Quantification of the expression levels of each protein (n = 3); mean ± SD. * p <0.05, ** p <0.01 indicates a significant difference, ns>0.05 indicates an insignificant difference;

[0016] Figure 4 (A) HE staining of the aortic root and proximal coronary artery of mouse heart tissue; (B) Quantitative results of HE staining in each group (n = 5); (C) CD45 immunohistochemical staining around the coronary artery of mouse heart tissue; (D) Quantitative results of CD45 immunohistochemical staining in each group (n = 5); (E) TNF-α immunohistochemical staining around the coronary artery 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 artery 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;

[0017] Figure 5 (A) RT-qPCR was used to detect the expression levels of inflammatory factors and chemokines in each group (n = 6); (B) TUNEL staining was used to detect the apoptosis of endothelial cells around coronary arteries in mouse heart tissue, scale bar 100 μm; (C) Quantification of TUNEL staining results in each group (n = 3); (D) Western blot was used to detect the expression of BAX and BCL-2 proteins in the heart tissue of each group of mice; (E) Quantification of protein expression in each group (n = 3); mean ± SD, * p <0.05, ** p <0.01 indicated that the difference was statistically significant, and ns>0.05 indicated that the difference was not statistically significant. DETAILED DESCRIPTION

[0018] In order to make the objectives, technical solutions and advantages of the present invention more clear, the present invention will be further described in detail below with reference to the accompanying drawings.

[0019] Example 1, Preparation of exosomes derived from induced pluripotent stem cells:

[0020] (1) Culture of human induced pluripotent stem cells (iPS):

[0021] Human induced pluripotent stem cells (iPS) were purchased from CELLAPY Biotechnology Co., Ltd. (hiPSC-U1, Beijing, China) and cultured in PSCeasy ® II Human pluripotent stem Cell Medium (CA1014500, CELLAPY) at 37°C in a 5% CO2 incubator. Replace the medium with fresh medium every day. When the cell density reaches 80-90%, subculture the cells with EDTA. Collect the culture supernatant and store at -80°C.

[0022] (2) Preparation of exosomes (iPS-EXO):

[0023] 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 h 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. iPS-derived exosomes (iPS-EXO) were obtained by ultracentrifugation at 31000 rpm for 90 min at 4°C. The protein concentration of iPS-EXO was detected using a BCA protein detection kit (Beyotime, Shanghai, China). The expression of cell exosome surface marker proteins CD9, CD63, and CD81 was detected by Western blot. The morphology of iPS-EXO was observed using a Japanese transmission electron microscope (TEM). The particle size of iPS-EXO was measured using a nanoparticle tracking analyzer (NTA).

[0024] The iPS cells used in this study were densely arranged and grew in colonies ( Figure 1 A), these iPS cells can positively express NANOG, OCT4, SOX2, SSEA4 and other pluripotent stem cell markers ( Figure 1 B). The resulting 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).

[0025] Example 2, Preparation of DiI-labeled iPS-EXO:

[0026] iPS-EXO was labeled with DiI (Beyotime, Shanghai, China), which is a lipophilic fluorescent dye widely used for cell membrane labeling and cell tracking. 2.5 μL DiI (400×) was mixed with 2.5 μL staining enhancement solution, and then 500 μL staining buffer was added and mixed. Incubate at room temperature for 10 min, add 50 μL iPS-EXO, incubate at 37°C for 20 min, add 1 mL PBS, and incubate at room temperature for 5 min to stop the reaction and prevent nonspecific binding of DiI dye. Subsequently, DiI-labeled iPS-EXO was collected and ultracentrifuged at 120,000 g for 90 min at 4°C. DiI-labeled iPS-EXO was used for tracing experiments of iPS-EXO uptake by cells in vitro and tissues in vivo in the following examples.

[0027] Example 3, in vitro cell iPS-EXO uptake experiment:

[0028] In vitro human umbilical vein endothelial cells (HUVECs) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). HUVECs were cultured in high-glucose DMEM medium (Gibco, NY, USA) containing 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 different concentrations (0.01 ng / mL to 10000 ng / mL) of tumor necrosis factor-α (TNF-α) using a CCK-8 assay kit (YEASEN, Shanghai, China) to explore the optimal working concentration of TNF-α. At the same time, HUVECs were treated with iPS-EXO at different concentrations (0 μg / mL to 200 μg / mL) to evaluate their cytotoxicity. Based on the TNF-α-induced KD cell model, HUVECs were treated with iPS-EXO at different concentrations (5 μg / mL ~ 100 μg / mL) to explore the optimal concentration. Then, 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. First, HUVECs were pretreated with TNF-α to establish a KD cell model, and then TNF-α-induced HUVECs were treated with iPS-EXO and iPS-EXO+CC mixtures for 24 h.

[0029] 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-EXO can be successfully taken up by HUVECs.

[0030] To explore 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 could significantly reduce the number of apoptotic cells, while AMPK inhibitor compound C (CC) could significantly reverse the effect of iPS-EXO ( Figure 3 B). Flow cytometry was used to detect the apoptosis rate of each group ( 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 apoptotic protein BAX and anti-apoptotic 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 the reversal effect of iPS-EXO ( Figure 3 F). This suggests that iPS-EXO can alleviate TNF-α-induced HUVEC apoptosis.

[0031] Example 4, in vivo tissue iPS-EXO uptake experiment:

[0032] First, Candida albicans water-soluble fraction (CAWS) was extracted from Candida albicans strain NBRC1385. Then, CAWS was dissolved in phosphate-buffered saline (PBS), sterilized by autoclave, and stored at -80°C after measuring the concentration.

[0033] Four-week-old male C57BL / 6 mice were purchased from the Experimental Animal Center of Wenzhou Medical University. The mice were kept at a temperature of 23 ± 2°C and a relative humidity of 45% to 55% with a 12-h dark / light cycle, and water and food were available to the mice 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.

[0034] 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 A, the CAWS group of mice was injected intraperitoneally with 4 mg / kg of CAWS every day for five consecutive days to induce the establishment of a KD mouse model, while the control group of mice was injected intraperitoneally with an equal volume of PBS buffer. In this study, the survival rate of mice injected with CAWS was about 90%. Based on the successfully established CAWS-induced KD mouse model, mice in the CAWS + iPS-L-EXO group and CAWS + iPS-H-EXO group were injected with 5 μg / kg iPS-EXO and 50 μg / kg iPS-EXO through the tail vein every day for 3 consecutive weeks, respectively. After stopping the iPS-EXO injection and feeding for another week, the mice in each group were euthanized by excessive anesthesia. The heart, liver, spleen, lung, and kidney organs of the mice were obtained for small animal in vivo imaging, and the heart tissue with aortic root and coronary artery area was obtained for subsequent experiments.

[0035] Small animal in vivo imaging was used to evaluate the uptake of DiI-labeled iPS-EXO in mouse heart, lung, liver, spleen, and kidney organ tissues. The results showed that iPS-EXO could be successfully taken up by these organ 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 region of the coronary artery ( 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 there was almost no fluorescence signal in the control group ( Figure 2 H). These results indicate that iPS-EXO can be successfully taken up by cardiac tissue.

[0036] 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 of the mouse heart were stained with HE to evaluate the inflammatory infiltration around the coronary artery ( Figure 4 A). 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 could significantly improve the inflammatory infiltration, among which the CAWS + iPS-H-EXO group had the best effect ( Figure 4 B).

[0037] 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).

[0038] 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 level of TNF-α 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).

[0039] 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 could significantly reduce 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 reduce inflammation and fibrosis caused by CAWS and has a protective effect on the coronary arteries of KD mice.

[0040] The mRNA extracted from the aortic root and proximal coronary artery regions of the heart tissue of each group of mice was used for subsequent RT-qPCR analysis. RT-qPCR results showed that iPS-EXO could significantly reduce 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).

[0041] 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 each group of mice ( Figure 5B). The 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, but iPS-L-EXO or iPS-H-EXO treatment could effectively reduce the TUNEL-positive cells in CAWS-induced mice, and iPS-H-EXO treatment was more effective than iPS-L-EXO treatment in reducing the percentage of TUNEL-positive cells ( Figure 5 C). Western blot was used to detect 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, and the BAX level and BAX / BCL-2 ratio were effectively upregulated, but this trend could be reversed by iPS-L-EXO or iPS-H-EXO treatment ( Figure 5 E). These data further demonstrate that iPS-EXO has a protective effect against inflammation and apoptosis in CAWS-induced mice.

[0042] The above disclosure is only the preferred embodiment of the present invention, which 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; 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) Isolating and purifying the culture supernatant to obtain exosomes derived from human induced pluripotent stem cells; 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; 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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