Preparation method of extracellular vesicles derived from myocardial cells derived from human induced pluripotent stem cells as well as product and application of extracellular vesicles

Abstract

The invention relates to the technical field of biological medicines, in particular to a preparation method of extracellular vesicles derived from myocardial cells derived from human induced pluripotent stem cells as well as a product and application of the extracellular vesicles. The method comprises the following steps: pretreating human induced pluripotent stem cell derived myocardial cells (hiPSCs-CMs) by using honokiol, and culturing the treated human induced pluripotent stem cell derived myocardial cells to collect extracellular vesicles (EVs) secreted by the human induced pluripotent stem cell derived myocardial cells. The extracellular vesicles secreted by the myocardial cells derived from the human induced pluripotent stem cells pretreated by honokiol are rich in functional mitochondria, show a better myocardial infarction treatment effect in animal experiments, and have huge clinical potential.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for preparing extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes, and a product and application thereof. Background Art

[0002] Induced pluripotent stem cell (iPSCs) technology refers to the reprogramming of terminally differentiated somatic cells into pluripotent stem cells by introducing specific transcription factors. Due to the advantages of human induced pluripotent stem cells (hiPSCs) such as reduced immune rejection and unlimited reproduction in vitro, cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) provide an ideal cell source for cardiac repair, cardiac drug testing, in vitro cardiac modeling, etc.

[0003] Myocardial infarction (MI) refers to myocardial ischemic necrosis, a global disease with high morbidity and mortality. The adult myocardium is one of the tissues with the weakest regenerative capacity in the human body. Myocardial infarction will lead to a large number of dead myocardial cells that cannot be replenished. Therefore, cell therapy that exogenously supplements healthy myocardial cells is one of the main directions for the treatment of myocardial infarction in the future.

[0004] With the deepening of stem cell research, the exploration of hiPSCs-CMs transplantation for the treatment of myocardial infarction has shown its application prospects (Cao, Nan et al. "Highly efficient induction and long-term maintenance of multipotent cardiovascular progenitors from human pluripotent stem cells under defined conditions." Cell research vol. 23, 9 (2013): 1119-32; Jiang, Xin et al. "Cardiac repair in a murine model of myocardial infarction with humaninduced pluripotent stem cell-derived cardiomyocytes." Stem cell research & therapy vol. 11, 1297. 17 Jul. 2020). In theory, transplanting myocardial lineage cells into the heart of myocardial infarction can replenish myocardial cells that have died due to injury, but in reality, due to the inflammatory microenvironment in the myocardial infarction area, the survival rate of transplanted myocardial cells is low and the efficacy of cell transplantation is limited.

[0005] Extracellular vesicles (EVs) are small membrane vesicles that have been widely studied as a potential alternative to cell therapy, as studies have shown that EVs can provide effects similar to cell therapy while avoiding some of the risks associated with cell therapy applications, such as immune response.

[0006] At present, there is increasing evidence that a small number of cardiomyocytes successfully implanted into the recipient heart cannot directly repair the damaged myocardium, but play a limited role in the form of "paracrine", that is, secretion of EVs (Yang, Lihong et al. "Stemcell-derived extracellular vesicles for myocardial infarction: a meta-analysis of controlled animal studies." Aging vol. 11, 4 (2019): 1129-1150.). Some studies have shown that continuous delivery of EVs secreted by cardiomyocytes differentiated from induced pluripotent stem cells to the heart may help treat heart damage (Liu, Bohao et al. "Cardiac recovery via extended cell-free delivery of extracellular vesicles secreted by cardiomyocytes derived from induced pluripotent stem cells." Nature biomedical engineering vol. 2, 5 (2018): 293-303.).

[0007] Regarding the specific mechanism by which EVs improve cardiac function, it is already known that EVs contain rich bioactive RNA, proteins, lipids and metabolites, and EVs can affect the biological properties of recipient cells through these bioactive substances after being transferred from donor cells. In addition, studies have reported that extracellular vesicles derived from human induced pluripotent stem cells (HiPSC-CMs-EVs) have an improving effect on cardiac function in mice with myocardial infarction, which is achieved through mitochondrial transfer (Ikeda, Gentaro et al. "Mitochondria-Rich Extracellular Vesicles From Autologous Stem Cell-Derived Cardiomyocytes Restore Energetics of IschemicMyocardium." Journal of the American College of Cardiology vol.77,8(2021):1073-1088.).

[0008] As we all know, mitochondria are the center of cellular energy metabolism and signal transduction, and play a key role in regulating material and energy metabolism, redox state, signal transduction, cell survival and death, and are considered to be important organelles for regulating immune response, inflammation and tissue regeneration. EVs-mediated mitochondrial transfer has also been shown to have the function of repairing the metabolic function of damaged recipient cells (Zhou, Xiyue et al. "MitoEVs: A new player in multiple disease pathology and treatment." Journal of extracellular vesicles vol. 12, 4 (2023): e12320.).

[0009] Therefore, preparing mitochondria-rich hiPSCs-CMs-derived EVs and utilizing mitochondrial dynamics to regulate energetics and promote metabolic remodeling of EVs, thereby improving the quality of EVs, has good application prospects and research significance for the treatment of myocardial infarction. Summary of the invention

[0010] Technical issues

[0011] Existing hiPSCs-CMs-derived EVs have the disadvantages of low quality, poor effect in treating myocardial infarction, and the need for large-scale and continuous delivery, which are not conducive to actual clinical applications.

[0012] Technical Solution

[0013] The first aspect of the present application provides a method for preparing extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes, the method comprising: pretreating human induced pluripotent stem cell-derived cardiomyocytes with honokiol (HKL), and culturing the treated human induced pluripotent stem cell-derived cardiomyocytes to collect the extracellular vesicles secreted by them.

[0014] In some embodiments, the specific preparation steps of the preparation method are:

[0015] a. Cultivate human induced pluripotent stem cells using basal medium until the cell confluence reaches 85% to 95%;

[0016] b. inducing the human induced pluripotent stem cells in step a into cardiomyocytes using an induction differentiation medium;

[0017] c. culturing the cardiomyocytes of step b for 3 to 5 days using a purified medium containing sodium L-lactate;

[0018] d. pretreating the cardiomyocytes in step c with honokiol for 12 to 48 hours;

[0019] e. Aspirate the conditioned medium of the cardiomyocytes in step d, perform gradient centrifugation and collect the extracellular vesicles.

[0020] In some embodiments, the concentration of magnolol in step d is 3-5 μM, preferably 5 μM.

[0021] In some embodiments, the basal culture medium in step a is PGM1 culture medium.

[0022] In some embodiments, the induction differentiation medium in step b is divided into a first-stage induction differentiation medium and a second-stage induction differentiation medium; wherein the components of the first-stage induction differentiation medium are mainly composed of 1640 / B27 insulin-free medium and CHIR99021 with a final concentration of 5 to 6 μM; and the components of the second-stage induction differentiation medium are mainly composed of 1640 / B27 insulin-free medium and IWP2 with a final concentration of 4 to 5 μM.

[0023] Preferably, the components of the first stage induction differentiation medium mainly consist of 1640 / B27 insulin-free medium and CHIR99021 with a final concentration of 6 μM; the components of the second stage induction differentiation medium mainly consist of 1640 / B27 insulin-free medium and IWP2 with a final concentration of 5 μM.

[0024] In some embodiments, the purification medium in step c is mainly composed of 2% by volume of B27 additive, sugar-free RPMI 1640 medium and L-sodium lactate with a final concentration of 40-50 nM.

[0025] In some embodiments, the specific conditions of the gradient centrifugation in step e are: first centrifuge at 300g for 10-15 min to remove cells; centrifuge at 2000g for 20-25 min to remove cell debris and apoptotic bodies; centrifuge at 18000g for 60-70 min to collect the precipitate and resuspend it; and centrifuge at 18000g for another 60-70 min.

[0026] Preferably, the specific conditions of the gradient centrifugation in step e are: first centrifuge at 300g for 10 minutes to remove cells; centrifuge at 2000g for 20 minutes to remove cell debris and apoptotic bodies; centrifuge at 18000g for 60 minutes to collect the precipitate and resuspend it; and centrifuge at 18000g for another 60 minutes.

[0027] In some embodiments, step b is specifically as follows: taking the time when the cells are at a confluence of 85% to 95% as day 0, using the first stage induction differentiation medium to culture cardiomyocytes on day 0, changing to maintenance medium I to culture cardiomyocytes on day 2, changing to the second stage induction differentiation medium to culture cardiomyocytes for 48 hours on day 3; changing to maintenance medium I to culture cardiomyocytes for 48 hours on day 5; and changing to maintenance medium II to culture cardiomyocytes for 6 days on day 7.

[0028] In some embodiments, the maintenance medium I is 1640 / B27 insulin-free medium, mainly composed of 2% by volume of B27 insulin-free additive and RPMI 1640 medium; the maintenance medium II is 1640 / B27 medium, mainly composed of 2% by volume of B27 additive and RPMI 1640 medium.

[0029] In some embodiments, step c is specifically as follows: on the 13th day, the cardiomyocytes are cultured in a purified medium for 3 days; on the 16th day, the cardiomyocytes are cultured in a maintenance medium II for 48 hours to obtain cardiomyocytes with a purity of more than 98%.

[0030] In some embodiments, step d is specifically as follows: on the 18th day, the purified cardiomyocytes are re-seeded and the cardiomyocytes are continued to be cultured using maintenance medium II for 48 hours, and on the 20th day, magnolia bark phenol is added to maintenance medium II to pretreat the cardiomyocytes, and after 12 to 48 hours, the cardiomyocytes are rinsed with phosphate buffered saline 2 to 3 times, and then maintenance medium II is added to continue culturing the cardiomyocytes for 48 hours.

[0031] Preferably, step d is specifically as follows: on the 18th day, the purified cardiomyocytes are re-seeded and the cardiomyocytes are continuously cultured using maintenance medium II for 48 hours; on the 20th day, honokiol with a final concentration of 5 μM is added to maintenance medium II to pretreat the cardiomyocytes; 48 hours later, the cardiomyocytes are rinsed 3 times with phosphate buffered saline, and maintenance medium II is added to continue culturing the cardiomyocytes for 48 hours.

[0032] In some embodiments, the phosphate buffered saline is DPBS.

[0033] The second aspect of the present application provides extracellular vesicles prepared by any of the preparation methods described above.

[0034] The third aspect of the present application provides the use of the extracellular vesicles in the preparation of drugs for treating or improving myocardial infarction.

[0035] Technical Effects

[0036] The present study found that honokiol can significantly increase the number, quality and function of mitochondria in HiPSCs-CMs and the EVs they secrete, thereby improving the quality of EVs. The EVs secreted by HiPSCs-CMs pretreated with honokiol prepared in the present application can significantly reduce the area of ​​myocardial infarction, reduce cardiomyocyte apoptosis, and improve the cardiac function of mice with myocardial infarction in animal experiments; and can effectively reduce the release of inflammatory factors, increase the release of anti-inflammatory factors, and improve the inflammatory microenvironment. Compared with the EVs secreted by untreated HiPSCs-CMs, the EVs secreted by HiPSCs-CMs pretreated with honokiol showed better therapeutic effects on myocardial infarction. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Shown is the expression level of cTnT in purified cardiomyocytes derived from human induced pluripotent stem cells detected by flow cytometry;

[0038] Figure 2 Shown is the expression of extracellular vesicle marker proteins such as CD9, CD63, and CD81 in extracellular vesicles derived from human induced pluripotent stem cells-derived cardiomyocytes;

[0039] Figure 3A Shown is the morphological structure of extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes under electron microscope;

[0040] Figure 3B The figure shows the particle size distribution of extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes analyzed by nanoparticle tracking analysis technology;

[0041] Figure 3C Shown are the results of Zeta potential of extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes;

[0042] Figure 4A Shown is the morphological structure of mitochondria in extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes under an electron microscope;

[0043] Figure 4B The figure shows the proportion of functional mitochondria contained in extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes detected by flow cytometry;

[0044] Figure 4C The figure shows the ATP detection kit to detect the ATP content in the extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes;

[0045] Figure 4D The figure shows the expression of mitochondrial electron respiratory chain complexes in extracellular vesicles derived from human induced pluripotent stem cell-derived cardiomyocytes detected by WB;

[0046] Figure 5A Shown are representative images collected by cardiac color Doppler ultrasound showing cardiac function at various stages of each group of mice in the animal experiment;

[0047] Figure 5B Shown are the statistical results of left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) of each group of myocardial infarction mice in the animal experiment;

[0048] Fig. 6A Shown is HE staining showing the infiltration of inflammatory cells in the myocardial infarction area of ​​each group of myocardial infarction mice in the animal experiment;

[0049] Figure 6B Shown is Masson staining showing that the infarct area of ​​the heart was reduced in each group of myocardial infarction mice in the animal experiment;

[0050] Figure 6C The figure shows the hypertrophy of myocardial cells in each group of myocardial infarction mice in the animal experiment by WGA staining;

[0051] Figure 7 Shown is apoptotic cell staining to show the apoptosis of myocardial cells in each group of myocardial infarction mice in the animal experiment;

[0052] Fig. 8A The figure shows the release of inflammatory factor IL-6 and anti-inflammatory factor IL-10 in myocardial cells of each group of myocardial infarction mice in animal experiments detected by ELISA;

[0053] Figure 8B The figure shows the mRNA expression levels of inflammatory factor IL-6 and anti-inflammatory factor IL-10 in the cardiomyocytes of each group of myocardial infarction mice in the animal experiment detected by qPCR. DETAILED DESCRIPTION

[0054] To facilitate the technical solution of the application, the following first provides a general description and definition of the terms and expressions involved in the application.

[0055] The terms "comprises", "includes" or any other variation thereof are intended to cover non-exclusive inclusion, such that a process, method, article or apparatus that includes a list of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article or apparatus. In the absence of further limitations, an element defined by the phrase "comprises a ..." does not exclude the presence of additional identical elements in the process, method, article or apparatus that includes the element.

[0056] The term "conditioned medium" refers to the supernatant remaining after removing the medium in which cells have been cultured.

[0057] In each group of comparative experiments provided in this application, unless otherwise specified, except for the differences indicated in each group, other experimental conditions, materials, etc. are kept consistent to ensure comparability.

[0058] The reagents, instruments, equipment and experimental methods used in the embodiments of the present invention, unless otherwise specified, are all commercially available. Experimental methods without specific conditions are conventional methods and conventional conditions well known in the art, or are based on the conditions recommended by the instrument manufacturer.

[0059] The following further describes the extracellular vesicles derived from human induced pluripotent stem cells-derived cardiomyocytes pretreated with honokiol provided in the present application, as well as the preparation method and application thereof.

[0060] Example 1: Preparation of extracellular vesicles from human induced pluripotent stem cell-derived cardiomyocytes pretreated with honokiol

[0061] Human induced pluripotent stem cells (purchased from Beijing Saibei Biotechnology Co., Ltd.) were cultured with PGM1 medium (purchased from Beijing Saibei Biotechnology Co., Ltd.), and when the cell confluence reached 85% to 95%, differentiation was performed using an induced differentiation medium. The specific steps are as follows: on day 0 (the default setting is that the cells are at 85% to 95% confluence as day 0), add 2 mL of the first stage induction differentiation medium to each well of the 12-well plate and culture the cells for 48 hours; on day 2, replace the medium with maintenance medium I (1640 / B27 insulin-free medium) and culture the cells for 24 hours; on day 3, replace the medium with the second stage induction differentiation medium and culture the cells for 48 hours; on day 5, replace the medium with maintenance medium I (1640 / B27 insulin-free medium) and culture the cells for 48 hours; on day 7, replace the cells with maintenance medium II (1640 / B27 medium) and culture the cells for 6 days; on day 13, add purification medium and culture the cells for 3 days; on day 16, replace the medium with maintenance medium II and continue culturing for 48 hours; the purity of the purified cardiomyocytes detected by flow cytometry reached more than 98% ( Figure 1 ), and then re-seeded in the well plate on the 18th day and cultured for another 48 hours using maintenance medium II. On the 20th day, 5 μM HKL (purchased from Selleck, purity>99%) was added to the maintenance medium II to pretreat the cells for 48 hours, and then the cells were rinsed with DPBS 2 to 3 times (3 times in this embodiment) and replaced with maintenance medium II (1640 / B27 medium) without HKL to continue culturing the cells. After 48 hours, the conditioned medium was collected and separated by differential centrifugation to obtain extracellular vesicles (HiPSC-CMs-EVs) secreted by HiPSC-CMs pretreated with honokiol. The components of each culture medium are detailed in Table 1. The specific steps of the differential centrifugation method include: after collecting the conditioned medium, centrifuge at 300g for 10 minutes to remove cells; centrifuge at 2000g for 20 minutes to remove cell debris and apoptotic bodies; high-speed centrifuge at 18000g for 60 minutes to collect the precipitate and resuspend it, and then ultracentrifuge at 18000g for 60 minutes to obtain the extracellular capsules secreted by HiPSC-CMs pretreated with HKL (HiPSC-CMs HKL -EVs).

[0062] Table 1: Different culture medium components and their contents

[0063]

[0064] Example 2: Preparation of extracellular vesicles from human induced pluripotent stem cell-derived cardiomyocytes pretreated with honokiol

[0065] The difference from Example 1 is that on the 20th day, HKL with a final concentration of 3 μM was added to the B27 medium to pretreat the cells for 48 hours.

[0066] Example 3: Preparation of extracellular vesicles from human induced pluripotent stem cell-derived cardiomyocytes pretreated with honokiol

[0067] The difference from Example 1 is that: when preparing the first stage induction differentiation medium, 25 μL 10mM CHIR99021 is used instead of 30 μL 10mM CHIR99021; when preparing the first stage induction differentiation medium, 20 μL 10mMIWP is used instead of 25 μL 10mM IWP; when preparing the purification medium, 250 μL 1M sodium L-lactate is used instead of 200 μL 1M sodium L-lactate.

[0068] Comparative Example 1: Preparation of extracellular vesicles from human induced pluripotent stem cell-derived cardiomyocytes without pretreatment

[0069] The difference from Example 1 is that the cells were not pretreated with HKL on day 20 and the extracellular vesicles (HiPSC-CMs-EVs) secreted by HiPSC-CMs without pretreatment were finally obtained.

[0070] Example 4: Qualitative identification of extracellular vesicles

[0071] CD9, CD63, and CD81 are marker proteins of extracellular vesicles. Western-Blot was used to analyze the expression of these marker proteins in the final extracts of cardiomyocytes and Example 1 / Comparative Example 1.

[0072] like Figure 2 As shown, the Western-Blot results show that compared with cardiomyocytes, Example 1 highly expresses extracellular vesicle marker proteins such as CD9, CD63, and CD81, which can be determined that extracellular vesicles are successfully obtained from Example 1 / Comparative Example 1.

[0073] Example 5: Extracellular vesicle morphology identification

[0074] The morphological structure of the extracellular vesicles of Example 1 and Comparative Example 1 was analyzed and observed using an electron microscope (FEI, Tecnai G2 Spirit BioTwin); the particle size distribution and Zeta potential of the extracellular vesicles of Example 1 and Comparative Example 1 were analyzed using a nanoparticle tracking analyzer (PARTICLE METRIXs, ZetaVIEW S / N 17-310).

[0075] The results showed that there was no significant difference in morphology, size distribution, and Zeta potential between the extracellular vesicles secreted by HiPSC-CMs pretreated with honokiol and those without pretreatment ( Figure 3A-3CSpecifically, the morphological structure of HiPSC-CMs-EVs was observed under an electron microscope. The extracellular vesicles were spherical or disc-shaped, with a size of about 300 nm. The morphology of the extracellular vesicles secreted by HiPSC-CMs after pretreatment with honokiol did not change ( Figure 3A ); the particle size distribution (Concentration) of the two groups of EVs was analyzed using nanoparticle tracking analysis (NTA). Figure 3B As shown, HiPSC-CMs-EVs and HiPSC-CMs HKL -EVs particle size distribution is in the range of 100-800nm; NTA was used to analyze the Zeta potential of the two groups of EVs, such as Figure 3C As shown, there is no significant difference between the two.

[0076] Example 6: Functional identification of extracellular vesicles

[0077] Functional characterization of EVs secreted by HiPSC-CMs pretreated with or without honokiol was performed, including flow cytometry and ATP content detection kit to prove the secretion of EVs by HiPSC-CMs. HKL -EVs are enriched with more functional mitochondria.

[0078] like Figure 4A-4D As shown, compared with HiPSC-CMs-EVs without pretreatment, HiPSC-CMs HKL -EVs contained more functional mitochondria, higher ATP content, and significantly higher expression levels of subunits encoding mitochondrial respiratory chain complexes. Figure 4A As shown in Figure 2, mitochondria can be observed in both groups of extracellular vesicles under an electron microscope, and there is no obvious difference in the morphology of the mitochondria contained. Figure 4B As shown, Mitotracker Deep-Red dye was used to label the functional mitochondria in the vesicles, and flow cytometry was used to detect the functional mitochondria in the vesicles. The results showed that compared with HiPSC-CMs-EVs without pretreatment, HiPSC-CMs HKL -EVs contain more functional mitochondria, Figure 4C-4D As shown, the ATP content of vesicles in each group was detected using an ATP detection kit. The results showed that compared with HiPSC-CMs-EVs without pretreatment, HiPSC-CMs HKL -EVs contained higher ATP content, and Western-Blot experiments showed that HiPSC-CMs-EVs had higher ATP content than HiPSC-CMs-EVs without pretreatment. HKL-EVs also expressed significantly higher levels of mitochondrial respiratory chain complex subunits. The above results indicate that after pretreatment with honokiol, HiPSC-CMs-EVs contain more and more powerful mitochondria.

[0079] Example 7: Animal Experiment

[0080] The mouse myocardial infarction model was established by ligating the left anterior descending branch of the mouse coronary artery. The mice were divided into the experimental group (HiPSC-CMs HKL -EVs), control group (HiPSC-CMs-EVs), blank control group (PBS), sham operation group (Sham), 10 mice (C57) in each group, HiPSC-CMs were transplanted into the myocardium of the experimental group and the control group mice, respectively. HKL -EVs and HiPSC-CMs-EVs (20μL 50μg / 20μL PBS, randomly injected at three points in the infarct area, for a total of 3 times). Eye blood was collected from each group of mice, and the hearts of the four groups of mice were dissected and collected. The heart tissues were ground and collected, and paraffin sections and frozen sections were prepared. The HiPSC-CMs were tested by cardiac color Doppler ultrasound, HE staining, Masson staining, WGA staining, cell apoptosis experiment, ELISA test, and qPCR experiment. HKL -EVs were functionally evaluated (*: P < 0.05, **: P < 0.01, ***: P < 0.001, ****: P < 0.0001). The results showed that compared with HiPSC-CMs-EVs prepared without honokiol pretreatment, HiPSC-CMs HKL -EVs can significantly improve cardiac function, reduce myocardial infarction area, alleviate cardiomyocyte apoptosis and reduce the release of inflammatory factors in mice with myocardial infarction (see Figures 5A-8B ).

[0081] Specifically, Figure 5A-Figure 5B As shown, cardiac ultrasound showed that transplanted HiPSC-CMs HKL -EVs, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) of myocardial infarction mice were increased, and the cardiac function of myocardial infarction mice was significantly improved.

[0082] like Figure 6A-6C As shown, HE staining showed that HiPSC-CMs HKL -EVs transplantation significantly reduced the infiltration of inflammatory cells in mice with myocardial infarction ( Fig. 6A ), Masson staining showed HiPSC-CMs HKL -EVs transplantation reduced the infarct area in myocardial infarction mice ( Figure 6B ), WGA staining showed that myocardial cell hypertrophy was significantly improved ( Figure 6C ).

[0083] like Figure 7 As shown, apoptotic cell staining indicated that HiPSC-CMs HK L-EVs transplantation can significantly reduce cardiomyocyte apoptosis.

[0084] like Figure 8A-B As shown, ELISA and qPCR results suggest that HiPSC-CMs HKL -EVs transplantation can effectively reduce the release of inflammatory factor IL-6 and increase the release of anti-inflammatory factor IL-10, thereby improving the transplant inflammatory microenvironment.

[0085] The above specific implementation methods further illustrate the purpose, technical solutions and beneficial effects of the present application in detail. It should be understood that the above are only specific implementation methods of the present application and are not used to limit the protection scope of the present application. Any modifications, equivalent substitutions, improvements, etc. made on the basis of the technical solutions of the present application should be included in the protection scope of the present application.

Claims

1. A method for preparing extracellular vesicles derived from human induced pluripotent stem cells and cardiomyocytes, characterized in that: The method comprises: pre-treating human induced pluripotent stem cell-derived cardiomyocytes with honokiol, and culturing the treated human induced pluripotent stem cell-derived cardiomyocytes to collect the extracellular vesicles secreted by the cardiomyocytes; the specific preparation steps are as follows: a. Cultivate human induced pluripotent stem cells using basal medium until the cell confluence reaches 85% to 95%; b. inducing the human induced pluripotent stem cells in step a into cardiomyocytes using an induction differentiation medium; c. culturing the cardiomyocytes of step b for 3 to 5 days using a purified medium containing sodium L-lactate; d. pretreating the cardiomyocytes in step c with honokiol for 12 to 48 hours; e. Aspirate the conditioned medium of the cardiomyocytes in step d, perform gradient centrifugation and collect the extracellular vesicles.

2. The preparation method according to claim 1, characterized in that: The final concentration of honokiol in step d is 3-5 μM.

3. The preparation method according to claim 2, characterized in that: The basal culture medium in step a is PGM1 culture medium; the induction differentiation culture medium in step b is divided into a first-stage induction differentiation culture medium and a second-stage induction differentiation culture medium; wherein the first-stage induction differentiation culture medium mainly comprises 1640 / B27 insulin-free culture medium and CHIR99021 with a final concentration of 5 to 6 μM; and the second-stage induction differentiation culture medium mainly comprises 1640 / B27 insulin-free culture medium and IWP2 with a final concentration of 4 to 5 μM.

4. The preparation method according to claim 2, characterized in that: The purification medium in step c mainly consists of 2% B27 additive, sugar-free RPMI 1640 medium and L-sodium lactate with a final concentration of 40-50 nM.

5. The preparation method according to claim 4, characterized in that: The step b is specifically as follows: taking the time when the cells are at a confluence of 85% to 95% as day 0, using the first stage induction differentiation medium to culture cardiomyocytes on day 0, replacing it with maintenance medium I to culture cardiomyocytes on day 2, and replacing it with the second stage induction differentiation medium to culture cardiomyocytes for 48 hours on day 3; replacing it with maintenance medium I to culture cardiomyocytes for 48 hours on day 5; and replacing it with maintenance medium II to culture cardiomyocytes for 6 days on day 7; wherein the maintenance medium I is a 1640 / B27 insulin-free medium, mainly composed of a 2% volume fraction of B27 insulin-free additive and RPMI 1640 medium; and the maintenance medium II is a 1640 / B27 medium, mainly composed of a 2% volume fraction of B27 additive and RPMI 1640 medium.

6. The preparation method according to claim 5, characterized in that: The step c specifically includes: using the purified culture medium to culture the cardiomyocytes for 3 days on the 13th day; and changing to the maintenance culture medium II to culture the cardiomyocytes for 48 hours on the 16th day to obtain cardiomyocytes with a purity of more than 98%.

7. The preparation method according to claim 6, characterized in that: The step d is specifically as follows: on the 18th day, the purified cardiomyocytes are re-seeded and the cardiomyocytes are continuously cultured using maintenance medium II for 48 hours; on the 20th day, honokiol is added to maintenance medium II to pretreat the cardiomyocytes; 12 to 48 hours later, the cardiomyocytes are rinsed 2 to 3 times using phosphate buffered saline, and then maintenance medium II is added to continue culturing the cardiomyocytes for 48 hours.

8. The preparation method according to claim 7, characterized in that: The specific conditions of the gradient centrifugation in step e are: first centrifuge at 300g for 10-15 minutes to remove cells; centrifuge at 2000g for 20-25 minutes to remove cell debris and apoptotic bodies; centrifuge at 18000g for 60-70 minutes to collect the precipitate and resuspend it; and centrifuge at 18000g for another 60-70 minutes.

9. The extracellular vesicles prepared by the preparation method according to any one of claims 1 to 8.

10. Use of the extracellular vesicles according to claim 9 in preparing a drug for treating or improving myocardial infarction.

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