Human placenta perivascular stem cell-derived extracellular vesicles and their therapeutic effects on ischemic diseases

Abstract

The present invention specifically relates to extracellular vesicles of human placental perivascular stem cells and their therapeutic effects on ischemic diseases. The present invention provides an extracellular vesicle, characterized in that the extracellular vesicle is derived from human placental perivascular stem cells, and the human placental perivascular stem cells co-express CD248, CD276 and CD44. Research shows that extracellular vesicles of human placental perivascular stem cells highly express the specific protein ALIX compared with extracellular vesicles of human umbilical cord mesenchymal stem cells, and the number of extracellular vesicles contained in human placental perivascular stem cells is larger. The extracellular vesicles of human placental perivascular stem cells provided by the present invention have more definite and better curative effects on myocardial infarction, lower limb ischemia and ischemic stroke compared with the existing extracellular vesicles of human umbilical cord mesenchymal stem cells, providing theoretical support for subsequent clinical treatment of ischemic diseases and large-scale application of extracellular vesicles of human placental perivascular stem cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to human placenta perivascular stem cell extracellular vesicles and their therapeutic effects on ischemic diseases. More particularly, it relates to human placenta perivascular stem cells and their transplantation for the treatment of acute myocardial infarction and lower limb ischemia. Background Art

[0002] Ischemic diseases are relatively common clinical diseases with certain potential risks. Cardiovascular and lower limb ischemia are more common among them. With the continuous improvement of living standards, their incidence shows an upward trend. Cardiovascular diseases include, for example, coronary heart disease, rheumatic heart disease, etc. Among them, the highest fatality rate is acute myocardial infarction. In ischemic cerebrovascular diseases, the incidence of ischemic stroke is relatively high. Currently, studies have shown that mesenchymal stem cells can treat stroke through paracrine. Lower limb ischemic diseases are a series of diseases caused by arterial stenosis or occlusion, including lower limb arteriosclerosis obliterans, thromboangiitis obliterans, and diabetic foot, etc.

[0003] Extracellular vesicles (EVs) are lipid bilayer membrane vesicles formed by cells through endocytosis, fusion, and exocytosis processes (Nat Rev Cardiol. 2017;14(5):259-272.). They are produced by paracrine secretion of cells and play a key role in cell communication, migration, immune regulation, and signal transduction (Curr Biol. 2018;28(8):R435-R444.; Endocr Rev. 2022;43(3):441-468.). Extracellular vesicles are divided into three types according to their diameter: apoptotic bodies with a diameter greater than 1 μm, microvesicles with a diameter of about 100-1000 nm, and exosomes with the smallest diameter of about 40-100 nm. All three contain proteins, lipids, nuclear substances, and non-coding RNAs (Nat Rev Cardiol. 2017;14(5):259-272.). Research reports have shown that they can play a regenerative role in the treatment of myocardial infarction (Nat Rev Cardiol. 2020 Nov;17(11):685-697.). Direct cardiac injection of exosomes derived from human pluripotent stem cell-derived cardiomyocytes (Induced Pluripotent Stem Cell-Derived Cardiomyocytes, iPSC-CM) (Sci Transl Med. 2020;12(561):eaay1318.) or exosomes derived from allogeneic cardiosphere-derived cells (Cardiosphere-Derived Cells, CDC) (Stem Cell Rev Rep. 2020;16(3):612-625.) can promote the repair of infarcted myocardium in pigs. Transplantation of extracellular vesicles derived from human induced pluripotent stem cell-derived cardiovascular progenitor cells (CPC) can promote the resolution of the inflammatory response after acute myocardial infarction in mice (Cardiovasc Res. 2021;117(1):292-307.). Injection of exosomes rich in mitochondria from human induced pluripotent stem cell-derived cardiomyocytes into the myocardial infarction site in mice can enhance cardiac function (J Am Coll Cardiol. 2021;77(8):1073-1088.). Intramyocardial injection of apoptotic bodies derived from allogeneic bone marrow mesenchymal stem cells (Bone marrow mesenchymal stem cells, BM-MSC) in rats with myocardial infarction can reduce the infarct area and promote angiogenesis (Autophagy. 2020;16(12):2140-2155.).Transplantation of small extracellular vesicles (sEVs) derived from hypoxia-treated bone marrow mesenchymal stem cells into the infarcted area of mouse myocardium enhances angiogenesis in ischemic myocardium by downregulating matrix metalloproteinase 19 (MMP19) in fibroblasts and reducing the degradation of vascular endothelial growth factor A (VEGFA) (Sci Transl Med. 2021;13(584):eabb0202). Injection of exosomes derived from umbilical cord-derived mesenchymal stem cells (UC-MSCs) into the pericardium of mouse myocardial infarction accumulates in the mediastinal lymph nodes and induces the differentiation of regulatory T cells to promote heart repair (Circ Res. 2022;131(10):e135-e150.). Transplantation of exosomes derived from human adipose stem cells (ASCs) into mice with hindlimb ischemia can promote angiogenesis and skeletal muscle protection, which is related to the enrichment of neuregulin-1 (NRG1) / mRNA (Arterioscler Thromb Vasc Biol. 2020;40(1):239-254.). Intramuscular injection of exosomes derived from human umbilical cord mesenchymal stem cells into the ischemic hindlimbs of mice increases the expression of FOXO3a by regulating miR-421 through circHIPK3, thereby inhibiting pyroptosis and the release of IL-1β and IL-18 (Theranostics. 2020;10(15):6728-6742.).

[0004] However, there are many controversies in the treatment of extracellular vesicles: First, the therapeutic effects of extracellular vesicles from different cell sources are different, which may be related to the different contents of extracellular vesicles. Transplantation of human pluripotent stem cell-derived cardiomyocytes (iPSC-CM) into the ischemic hearts of rats alleviated the progression of cardiomyopathy after initial myocardial infarction to the same extent as transplantation of cardiomyocytes derived from human embryonic stem cells (Embryonic Stem Cell-Cardiomyocytes, ESC-CM). At the same time, it was found that the 20 most abundant miRNAs in exosomes derived from ESC-CM and iPSC-CM were the same, and the lncRNA content between the exosomes of the two groups of cells was also highly similar (Stem Cells. 2017; 35(10): 2138-2149.). A comparative study of exosomes derived from cardiospheres and c-kit cardiac progenitor cells (CPC) found that exosomes derived from CPC were superior to exosomes derived from CDC in improving left ventricular function, neovascular density, and reducing fibrosis. At the same time, exosomes derived from CPC contained higher concentrations of hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), stromal cell-derived factor 1α (SDF-1α), angiopoietin-1 (ANG-1), VEGFA, platelet-derived growth factor B (PDGFB), and basic fibroblast growth factor (bFGF) (Sci Transl Med. 2019; 11(493): eaau1168.). A Meta-analysis of preclinical experiments on myocardial infarction diseases found that the effect of extracellular vesicles derived from mesenchymal stem cells in improving the ejection fraction (EF) of sexual function (MD = 11.72% [95% CI: 9.17, 14.48]) was better than that of extracellular vesicles derived from cardiac stem cells (EF: MD = 8.78% [95% CI: 6.11, 11.45]) (Stem Cell Rev Rep. 2022; 18(3): 1143-1167.). Extracellular vesicles derived from human cardiospheres (CDC-EVs) enhanced the Arg1 / Nos2 ratio in macrophages and reduced the area of myocardial infarction more than exosomes derived from human bone marrow-derived mesenchymal stem cells (SciRep. 2021; 11(1): 8666.).Intramyocardial injection of exosomes secreted by CPCs reduced scar size and improved ventricular function more effectively than exosomes secreted by bone marrow-derived mesenchymal stem / progenitor cells (BMCs) after myocardial infarction in rats (Cardiovasc Res. 2018;114(7):992-1005.). Due to differences in tissue source, culture conditions, preparation process, and stem cell content of different mesenchymal stem cells, the effects of their extracellular vesicles on promoting angiogenesis also vary (Cytotherapy. 2019;21(5):497-508.). Compared with exosomes derived from human bone marrow and human umbilical cord mesenchymal cells, intramyocardial injection of exosomes derived from human adipose-derived mesenchymal stem cells (ADMSCs) was more effective in reducing myocardial infarction area and cardiomyocyte apoptosis and enhancing cardiac function (J Cell Biochem. 2020;121(3):2089-2102.). Secondly, since it is difficult to separate vesicle subsets, the relative concentration of each vesicle subtype in the obtained extracellular vesicles is unknown, and currently, the research on extracellular vesicles actually uses a mixed subset of vesicles (Nat Rev Cardiol. 2017;14(5):259-272). The specific mechanism of action of each subset of extracellular vesicles in diseases is still poorly understood (Cytotherapy. 2019;21(5):497-508.). In addition, since the residence time of extracellular vesicles at the injury site is short after injection into the heart, this greatly hinders their efficacy (ACS Nano. 2021;15(7):11099-11111.). There is currently no research record of extracellular vesicles remaining at the injury site for more than 3 hours after myocardial injection (51. Nat Biomed Eng. 2018;2(5):293-303.). Extracellular vesicles are cleared when passing through the mononuclear macrophage system in the blood circulation, which weakens their therapeutic effect (Biomaterials. 2021;275:121000.). Research has shown that the clearance rate of extracellular vesicles isolated from MSCs overexpressing CD47 (CD47 is a transmembrane protein, and cells lacking CD47 will be cleared by macrophages) injected through the tail vein of mice is reduced, improving the therapeutic effect on myocardial ischemia-reperfusion in mice (Biomaterials. 2021;275:121000.).

[0005] Therefore, for extracellular vesicles from specific sources, it is crucial to clarify their efficacy and treatment mechanism for ischemic diseases. Summary of the Invention

[0006] Problems to be Solved by the Invention

[0007] In view of the problems existing in the prior art, for example, due to different stem cell types, the inclusion substances in their secreted extracellular vesicles are different, and the curative effects and mechanisms for ischemic diseases are different, resulting in disputes in the treatment of extracellular vesicles. The present invention extracts extracellular vesicles of human placental perivascular stem cells (hPPSCs-EVs), analyzes the differences between hPPSCs-EVs and extracellular vesicles of human umbilical cord mesenchymal stem cells (hUCMSCs-EVs) through proteomics and metabolomics, discovers the superiority of hPPSCs-EVs in treating ischemic diseases such as myocardial infarction, lower limb ischemia, and ischemic stroke through comparative analysis, and simultaneously explores and analyzes its treatment mechanism.

[0008] Solutions for Solving the Problems

[0009] To solve the problems existing in the above prior art, the present invention provides the following technical solutions:

[0010] In a first aspect of the present invention, there is provided an extracellular vesicle, which is derived from human placental perivascular stem cells, and the human placental perivascular stem cells co-express CD248, CD276, and CD44.

[0011] In some embodiments of the present invention, the method for preparing the extracellular vesicle comprises the steps shown in (i) to (ii) below:

[0012] (i) Culturing human placental perivascular stem cells in a medium with or without serum for 36 - 72 h, and collecting the cell culture supernatant;

[0013] (ii) Processing the cell culture supernatant successively as follows: centrifuging to obtain a supernatant, filtering to obtain a filtrate, and centrifuging to obtain a precipitate, thus obtaining the extracellular vesicle.

[0014] In some embodiments of the present invention, the serum is fetal bovine serum without extracellular vesicles;

[0015] Preferably, the method for preparing the fetal bovine serum without extracellular vesicles comprises the step of ultracentrifuging the fetal bovine serum;

[0016] Preferably, the conditions for the ultracentrifugation include: centrifuging at 80000 - 120000 g for 11 - 13 h;

[0017] More preferably, the conditions for the ultracentrifugation include: centrifuging at 90000 - 110000 g for 11 - 13 h.

[0018] In some embodiments of the present invention, in step (i), the culture conditions of the human placental perivascular stem cells include: the culture temperature is 36.5 - 37.5 °C, and the culture environment contains 4.8% - 5.2% CO2.

[0019] In some preferred embodiments of the present invention, in step (i), the culture conditions of the human placental perivascular stem cells include: the culture temperature is 37 °C, and the culture environment contains 5% CO2.

[0020] In some embodiments of the present invention, in step (ii), the cell culture supernatant is sequentially processed as follows: centrifuged at 250 - 350 g for 5 - 15 min to obtain a supernatant, centrifuged at 1700 - 2300 g for 10 - 20 min to obtain a supernatant, centrifuged at 8500 - 11500 g for 25 - 35 min to obtain a supernatant, filtered through a microporous membrane to obtain a filtrate, and centrifuged at 80000 - 120000 g for 60 - 80 min to obtain a precipitate.

[0021] In some preferred embodiments of the present invention, in step (ii), the cell culture supernatant is sequentially processed as follows: centrifuged at 280 - 320 g for 8 - 12 min to obtain a supernatant, centrifuged at 1900 - 2100 g for 13 - 17 min to obtain a supernatant, centrifuged at 9000 - 11000 g for 28 - 32 min to obtain a supernatant, filtered through a microporous membrane to obtain a filtrate, and centrifuged at 90000 - 110000 g for 65 - 75 min to obtain a precipitate.

[0022] In some embodiments of the present invention, the pore size of the microporous membrane is 40 nm - 1000 nm.

[0023] In some embodiments of the present invention, the extracellular vesicles highly express ALIX protein compared to the extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

[0024] The second aspect of the present invention provides the use of the extracellular vesicles described in the first aspect of the present invention in the preparation of a drug for preventing and / or treating ischemic diseases.

[0025] In some embodiments of the present invention, the extracellular vesicles can be used for preventing and / or treating ischemic diseases. In some embodiments, the extracellular vesicles described in the present invention prevent and / or treat ischemic diseases by promoting angiogenesis.

[0026] In some embodiments of the present invention, the present invention provides a method for preventing and / or treating ischemic diseases, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0027] In some embodiments of the present invention, the ischemic disease has any one or more of the following symptoms: reduced angiogenesis ability of ischemic tissue, reduced blood vessel density of ischemic tissue, reduced muscle fibers in ischemic tissue, increased fibrosis degree of ischemic tissue, and mitochondrial damage in ischemic tissue.

[0028] In some preferred embodiments of the present invention, the ischemic disease includes any one or more of myocardial infarction, lower limb ischemia, and ischemic stroke.

[0029] In some embodiments of the present invention, the extracellular vesicles can be used for preventing and / or treating any one or more of myocardial infarction, lower limb ischemia, and ischemic stroke.

[0030] In some embodiments of the present invention, the present invention provides a method for preventing and / or treating any one or more of myocardial infarction, lower limb ischemia, and ischemic stroke, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0031] The third aspect of the present invention provides the use of the extracellular vesicles described in the first aspect of the present invention in any one or more of the following (a) to (f):

[0032] (a) Preparing a preparation or drug for enhancing the angiogenesis ability of ischemic tissue;

[0033] (b) Preparing a preparation or drug for enhancing the blood vessel density of ischemic tissue;

[0034] (c) Preparing a preparation or drug for increasing the muscle fiber content of ischemic tissue;

[0035] (d) Preparing a preparation or drug for inhibiting or reducing the fibrosis degree of ischemic tissue;

[0036] (e) Preparing a preparation or drug for promoting the repair of mitochondrial damage in ischemic tissue;

[0037] (f) Preparing a preparation or drug for promoting fatty acid oxidation in ischemic tissue.

[0038] In some embodiments of the present invention, the extracellular vesicles can be used for enhancing the angiogenesis ability of ischemic tissue.

[0039] In some embodiments of the present invention, the extracellular vesicles can be used for enhancing the blood vessel density of ischemic tissue.

[0040] In some embodiments of the present invention, the extracellular vesicles can be used for increasing the muscle fiber content of ischemic tissue.

[0041] In some embodiments of the present invention, the extracellular vesicles can be used to inhibit or reduce the degree of fibrosis in ischemic tissues.

[0042] In some embodiments of the present invention, the extracellular vesicles can be used to promote the repair of mitochondrial damage in ischemic tissues.

[0043] In some embodiments of the present invention, the extracellular vesicles can be used to promote the fatty acid oxidation capacity of ischemic tissues.

[0044] In some embodiments of the present invention, the present invention provides a method for enhancing the angiogenesis ability of ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0045] In some embodiments of the present invention, the present invention provides a method for increasing the vascular density of ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0046] In some embodiments of the present invention, the present invention provides a method for increasing the myofiber content of ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0047] In some embodiments of the present invention, the present invention provides a method for inhibiting or reducing the degree of fibrosis in ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0048] In some embodiments of the present invention, the present invention provides a method for promoting the repair of mitochondrial damage in ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0049] In some embodiments of the present invention, the present invention provides a method for promoting the fatty acid oxidation capacity of ischemic tissues, which may include administering an effective amount of the extracellular vesicles to an individual in need thereof.

[0050] Effects of the Invention

[0051] By implementing the above technical solutions, the present invention has achieved the following technical effects:

[0052] Experimental data showed that the number of vesicles contained in human placental perivascular stem cells (hPPSCs-EVs) was higher than that in human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs). The former highly expressed the extracellular vesicle-specific protein ALIX. The results of proteomic analysis showed that 281 proteins were up-regulated and 346 proteins were down-regulated in hPPSCs-EVs compared with hUCMSCs-EVs. The metabolic regulation pathway was up-regulated, and the proteasome-related proteins were highly expressed. The results of metabolomic analysis showed that 145 metabolites were up-regulated in hPPSCs-EVs compared with hUCMSCs-EVs. The highly expressed metabolites included amino acids, organic acids, fatty acids, and hormones, etc. The highly expressed metabolites were N-Methylalanine, N-Ethylglycine, phosphatidylcholine (PC), 21-Deoxycortisol. The combined analysis of differential proteins and differential metabolites showed that the regulatory metabolic pathway proteins and metabolite enrichment were up-regulated in hPPSCs-EVs compared with hUCMSCs-EVs. Further, the research showed that hPPSCs-EVs had a stronger repair and treatment effect than hUCMSCs-EVs in myocardial infarction model and lower limb ischemia model transplantation. hPPSCs-EVs promoted myocardial infarction treatment by activating the PPAR metabolic pathway to promote fatty acid metabolism compared with hUCMSCs-EVs. hPPSCs-EVs activated PGC1α to promote mitochondrial biogenesis in infarcted myocardium and ischemic lower limbs, and promoted the repair and regeneration of infarcted myocardium and ischemic lower limbs compared with hUCMSCs-EVs.

[0053] Compared with the existing human umbilical cord mesenchymal stem cell extracellular vesicles, the human placental perivascular stem cell extracellular vesicles provided by the present invention have more definite and good curative effects on ischemic diseases such as myocardial infarction, lower limb ischemia, and ischemic stroke, providing theoretical support for subsequent clinical treatment of ischemic diseases and large-scale application of human placental perivascular stem cell extracellular vesicles. Brief Description of the Drawings

[0054] Figure 1A It is the result of differential comparative analysis between human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the electron microscopy detection result of hPPSCs-EVs and hUCMSCs-EVs, b is the nanoparticle tracking analysis result of extracellular vesicles, c is the WB detection result of extracellular vesicle marker proteins, and d is the gray scale analysis result of extracellular vesicle marker proteins.

[0055] Figure 1BProteomic analysis results of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the Volcano Plot analysis result, b is the Heatmap analysis result, c is the Domain Enrichment analysis result, d is the BP enrichment analysis result, e is the CC enrichment analysis result, and f is the KEGG enrichment analysis result.

[0056] Figure 1C Metabolomic analysis results of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the Volcano Plot analysis result, b is the Heatmap analysis result, c is the differential metabolite analysis result, and d is the KEGG enrichment analysis result.

[0057] Figure 1D Combined analysis results of differential proteins and differential metabolites of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs).

[0058] Figure 2A Echocardiogram and statistical analysis results of the transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs) for the treatment of myocardial infarction. Among them, a is the echocardiogram of each group, b is the statistical analysis result of LVFS of each group, c is the statistical analysis result of LVEF of each group, d is the statistical analysis result of LVDS of each group, and e is the statistical analysis result of LVAWS of each group.

[0059] Figure 2B Masson staining analysis results of myocardial infarction tissues after the transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the Masson staining map of myocardial infarction tissues of each group, b is the statistical analysis result of the collagen fiber area of each group, and c is the statistical analysis result of the ventricular wall thickness of the left ventricular infarction area of each group.

[0060] Figure 2CAnalysis results of CD31+ and α-SMA+ vascular density in myocardial infarction tissues after transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the immunofluorescence detection results of CD31 and α-SMA expression in each group, b is the statistical analysis results of CD31+ vascular density in each group, c is the statistical analysis results of α-SMA+ vascular density in each group, and d is the qPCR detection results of angiogenesis genes VEGF-A, VEGFR2, Dll4, and PDGF-B in each group.

[0061] Figure 2D Histological analysis results of lower limb ischemia model after transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the HE and Masson staining results of each group, b is the statistical analysis results of collagen fiber area in muscle tissues of each group, and c is the statistical analysis results of the average cross-sectional area of lower limb muscle fibers in each group.

[0062] Figure 2E Analysis results of CD31+ and α-SMA+ vascular density in lower limb ischemia tissues after transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the immunofluorescence detection results of CD31 and α-SMA expression in each group, b is the statistical analysis results of CD31+ vascular density in each group, c is the statistical analysis results of α-SMA+ vascular density in each group, and d is the qPCR detection results of angiogenesis genes VEGF-A, VEGFR2, Dll4, and PDGF-B in each group.

[0063] Figure 3A Analysis results of the activation of the PPAR fatty acid metabolism pathway on D14 in ischemic myocardial infarction treated with human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell extracellular vesicles (hUCMSCs-EVs); among them, a is the KEGG enrichment analysis results of transplanted hPPSCs-EVs compared with hUCMSCs-EVs, b is the WB detection results of p-AMPK, AMPK, PPARα / β, and PGC-1α after transplantation treatment in each group, c is the protein gray scale analysis results of p-AMPK, PPARα / β, and PGC-1α after transplantation treatment in each group, d is the enrichment analysis results of transplanted hPPSCs-EVs compared with hUCMSCs-EVs, and e is the qPCR verification results of the expression of genes related to the fatty acid β-oxidation pathway after transplantation treatment in each group.

[0064] Figure 3BAnalysis results of the activation of the PPAR fatty acid metabolism pathway by human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUCMSCs-EVs) in the treatment of ischemic myocardial infarction on day 3; among them, a and b are the KEGG enrichment analysis results of transplanted hPPSCs-EVs compared with hUCMSCs-EVs, c is the WB detection results of p-AMPK, AMPK, PPARα / β, and PGC-1α after transplantation treatment in each group, d is the protein gray scale analysis results of p-AMPK, PPARα / β, and PGC-1α after transplantation treatment in each group, e is the enrichment analysis results of transplanted hPPSCs-EVs compared with hUCMSCs-EVs, and f is the qPCR verification results of the expression of genes related to the fatty acid β-oxidation pathway after transplantation treatment in each group.

[0065] Figure 4A Verification results of the repair of mitochondrial damage in acute myocardial infarction in mice promoted by human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs) through the activation of PPAR / PGC-1α; among them, a is the transmission electron microscope detection results of each group on day 14 after transplantation treatment, b is the RNA-seq analysis results of the up-regulated expression of mitochondrial-related genes in transplanted hPPSCs-EVs compared with hUCMSCs-EVs in myocardial infarction, c is the qPCR detection results of the expression of mitochondrial complex-related genes in each group on day 14 after transplantation treatment, and d is the qPCR detection results of the expression of mitochondrial complex-related genes in each group on day 3 after transplantation treatment.

[0066] Figure 4B Verification results of the repair of mitochondrial damage in lower limb ischemia in mice promoted by human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs) through the activation of PPAR / PGC-1α; among them, a is the transmission electron microscope detection results of each group on day 15 after transplantation treatment, and b is the qPCR detection results of the expression of mitochondrial complex-related genes in each group on day 3 after transplantation treatment. Detailed implementation manners

[0067] In the present invention, the term "a", "an", or "the" may mean "one", or may mean "one or more", "at least one", and "one or more than one".

[0068] In the present invention, the terms "comprising", "having", "including", or "containing" may mean inclusive or open-ended, and do not exclude additional, unrecited elements or method steps. At the same time, "comprising", "having", "including", or "containing" may also mean closed-ended, excluding additional, unrecited elements or method steps.

[0069] In the present invention, the meaning represented by the term "may" includes both the meaning of performing a certain treatment and the meaning of not performing a certain treatment.

[0070] In the present invention, the terms "individual", "donor", "patient" or "subject" include mammals. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs and horses), primates (e.g., humans and non-human primates such as monkeys), rabbits, and rodents (e.g., mice and rats).

[0071] In the present invention, the term "myocardial infarction" refers to ischemic necrosis of the myocardium, which is based on coronary artery lesions, with a sharp reduction or interruption of coronary blood flow, resulting in severe and persistent acute ischemia of the corresponding myocardium, and ultimately leading to ischemic necrosis of the myocardium. Myocardial infarction includes primary acute myocardial infarction, secondary acute myocardial infarction, sudden cardiac death, myocardial infarction related to PCI, and acute myocardial infarction related to coronary artery bypass grafting.

[0072] In the present invention, the term "lower limb ischemia" refers to a class of diseases caused by various reasons leading to stenosis or occlusion of the lower limb arteries and insufficient blood perfusion, resulting in ischemic manifestations such as intermittent claudication, ulcers, and gangrene of the lower limbs. Lower limb ischemia includes acute arterial embolism, arteriosclerosis obliterans, and thromboangiitis obliterans.

[0073] In the present invention, the term "ischemic stroke" refers to the general term for necrosis of brain tissue caused by stenosis or occlusion of the brain's blood supply arteries (carotid artery and vertebral artery) and insufficient cerebral blood supply, which is also known as ischemic stroke.

[0074] In the present invention, "treatment" means that after a subject suffers from a disease, the subject is exposed to (e.g., administered) the extracellular vesicles derived from human placental perivascular stem cells described in the present invention, so that the symptoms of the disease are alleviated compared with when not exposed, and it does not necessarily mean complete suppression of the symptoms of the disease. Suffering from a disease means that the body shows symptoms of the disease.

[0075] In the present invention, "prevention" means that before a subject suffers from a disease, by exposing the subject to the extracellular vesicles derived from human placental perivascular stem cells described in the present invention, the probability of suffering from the disease and / or the symptoms after suffering from the disease are reduced compared with when not exposed, and it does not necessarily mean complete suppression of the disease.

[0076] In the present invention, "D3", "D14", etc. can represent the number of days after a biological material has undergone a certain treatment, such as the 3rd day, the 14th day, etc. after a mouse has received extracellular vesicle transplantation.

[0077] Unless otherwise defined, all other technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise specified, the units used in this specification are international standard units, and the numerical values and numerical ranges appearing in the present invention should be understood to include the inevitable systematic errors in industrial production.

[0078] Example

[0079] The specific examples listed in the present invention are only examples of the present invention, and the present invention is not limited to the specific examples described below. For those skilled in the art, any equivalent modifications and substitutions to the examples described below are also within the scope of the present invention. Therefore, equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention. Those not specified in the examples are carried out under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments not indicated by the manufacturer can be purchased commercially as conventional products. To better illustrate the present invention, numerous specific details are given in the following detailed description. Those skilled in the art should understand that the present invention can be implemented without some specific details. In other examples, methods, means, equipment and steps well known to those skilled in the art are not described in detail to highlight the gist of the present invention.

[0080] Example 1: Isolation, identification and transplantation treatment of human placental perivascular stem cell-derived extracellular vesicles for acute myocardial infarction and lower limb ischemia

[0081] 1. Experimental methods

[0082] 1.1 Isolation and identification of human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs)

[0083] (1) Obtaining human placental perivascular stem cells (hPPSCs)

[0084] After full-term delivery of a human placenta without infectious and genetic diseases, it was added to a human perivascular stem cell medium for culture. The components of the special medium for human perivascular stem cells are serum-free medium (100×GlutaMAX TM , 100×Pen Strep, EGF, N2, B27, BDNF, TGF-β, PDGF-AA, PDGF-BB). The cells were inoculated into a sterile culture dish and cultured in an incubator at 37.5°C and 5% CO2. The medium was changed every 2 days, and subculture was carried out every 3 - 5 days.

[0085] Upon identification, hPPSCs co-expressed CD248, CD276, and CD44. Therefore, the isolated human placental perivascular stem cells were defined as CD248+ / CD276+ / CD44+ hPPSCs (hereinafter referred to as human placental perivascular stem cells or hPPSCs). Meanwhile, hPPSCs also expressed α-SMA, NG2, CD140b, CD105, CD90, and CD73, but did not express CD34, CD31, and vWF.

[0086] (2) Obtaining human placental perivascular stem cell-derived extracellular vesicles and human umbilical cord mesenchymal stem cell-derived extracellular vesicles

[0087] ① Preparation of exosome-free fetal bovine serum (FBS): Ultracentrifugation at 100,000 g for 12 h to remove serum exosomes;

[0088] ② When the cell density of hPPSCs and hUCMSCs reached 70%, the medium was replaced with exosome-free serum medium, and the cells were cultured at 37 °C and 5% CO2 for 48 h, and then the cell culture supernatant was collected.

[0089] (3) Isolation of extracellular vesicles

[0090] ① Centrifuge the cell culture supernatant at 300 g for 10 min, 2000 g for 15 min, and 10,000 g for 30 min, and filter through a 0.22 μm filter membrane;

[0091] ② Take the filtrate and centrifuge at 100,000 g for 70 min to obtain extracellular vesicle precipitate, namely human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs) and human umbilical cord mesenchymal stem cell-derived extracellular vesicles (hUCMSCs-EVs), resuspend with PBS and centrifuge again, and store at -80 °C.

[0092] (4) Identification of extracellular vesicles

[0093] ① Nanoparticle tracking analysis (NTA): Use the Zeta View system for nanoparticle tracking analysis to detect the particle size and potential of extracellular vesicles;

[0094] ② Observation by transmission electron microscopy (TEM): Observe whether the obtained sample shows the typical bilayer membrane "cup-shaped" vesicle structure of extracellular vesicles under transmission electron microscopy;

[0095] ③ Protein immunoblotting (WB) to identify the expression of specific proteins such as Alix and CD63 on the surface of extracellular vesicles.

[0096] 1.2 Proteomic and metabolomic analysis of human placental perivascular stem cell-derived extracellular vesicles (hPPSCs-EVs)

[0097] hPPSCs-EVs and hUCMSCs-EVs were subjected to ultra-high performance liquid chromatography (UPLC) and tandem mass spectrometry (MS / MS) analysis, and sample data were collected. KEGG pathway analysis and differential correlation analysis were performed based on proteomic and metabolomic data.

[0098] 1.3 Preparation of mouse acute myocardial infarction and lower limb ischemia models

[0099] Male C57BL / 6J and BalbC mice aged 8 - 10 weeks (body weight 20 - 25 g) were purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd., China. The mice were housed at an environmental temperature of 22 - 24 °C, humidity of 50 - 60%, a 12 h light / dark cycle, and fed with sterile mouse food and fresh water. Animal experiments were approved by the Ethics Committee of Wenzhou Medical University, with the ethics number (wydw2022 - 0759).

[0100] The method for establishing an acute myocardial infarction model in male C57BL / 6J mice was referred to the report by Michael LH et al. (Am J Physiol. 1995; 269: H2147 - H2154.). The C57BL / 6J mice were placed in an induction chamber (RWD, R510) and anesthetized by inhaling 3.0% isoflurane (RWD, R510 - 22 - 16). After losing the righting reflex, tracheal intubation was performed. The mice were fixed in the right lateral position, and the intubation was connected to a ventilator (Chengdu Taimeng Software Co., Ltd., HX - 100E) with a tidal volume of 2.3 - 2.5 mL and a frequency of 125 beats per minute. The surgery was maintained under 1.5% isoflurane anesthesia. The chest cavity was opened through the fourth intercostal space to expose the heart, the pericardium was torn, and the left anterior descending branch was located 1 - 2 mm below the left atrial appendage under a dissecting microscope and ligated with 7 - 0 suture. The whitening of the anterior wall of the left ventricle indicated successful ligation.

[0101] The method for establishing a lower limb ischemia model in male BalbC mice was referred to the report by Niiyama H et al. (J VisExp. 2009; (23): 1035.). The mice were anesthetized by inhaling 1.5% isoflurane, and the surgery was performed under a dissecting microscope. The skin on the inner thigh was incised from the knee joint to the midpoint of the groin, and the adipose tissue around the vascular nerve bundle was bluntly dissected. The vascular nerve bundle was exposed by puncturing the femoral fascia sheath. The femoral artery and femoral vein were separated distally to the femoral artery near the knee joint, and the distal femoral artery was ligated with a double knot, with a distance of 2 mm between the two knots. The femoral artery, femoral vein, and femoral nerve were separated proximally to the femoral artery near the groin, and the proximal femoral artery was ligated with a double knot. The femoral artery between the distal and proximal double knots of the femur was transected.

[0102] 1.4 Transplantation of human placental perivascular stem cell extracellular vesicles (hPPSCs-EVs)

[0103] The mice were randomly divided into groups: Sham group, PBS group, hUCMSCs-EVs group, and hPPSCs-EVs group, and were raised separately after modeling. For the acute myocardial infarction model, 40 μL of PBS, 40 μL of 100 μg hUCMSCs-EVs, and 40 μL of 100 μg hPPSCs-EVs were injected at three points in the infarct area; for the acute ischemic gastrocnemius muscle of the lower limb, 60 μL of PBS, 60 μL of 300 μg hUCMSCs-EVs, and 60 μL of 300 μg hPPSCs-EVs were injected at three points.

[0104] 1.5 Immunohistochemistry and fluorescence detection

[0105] C57BL / 6J mice with acute myocardial infarction and BALB / c mice with lower limb ischemia were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.05 mL / 10 g). After sequential cardiac perfusion with 0.9% sodium chloride (Chenxin, 2201190721) and 4% paraformaldehyde (Shanghai Lingfeng Chemical Reagent Co., Ltd., 20100601), the hearts and gastrocnemius muscles were removed and placed in 4% paraformaldehyde for fixation for 48 h, rinsed with running water for 2 h, dehydrated with gradient ethanol (Luquan, 20220401) for 30 min each (70%, 80%, 90%, 95%, 100% ethanol), mixed 50% absolute ethanol with 50% xylene, and made transparent with xylene (Jiani, 20130115) for 10 min each. Then, 50% xylene was mixed with 50% paraffin (Macklin, P815423), and infiltrated with 100% paraffin (melting point 58 - 60 °C) for 1 h each, followed by paraffin embedding.

[0106] 4-μm paraffin sections of the mouse hearts and gastrocnemius muscles were baked at 60 °C for 1 h, and then dewaxed and hydrated in xylene, 100% alcohol, 95% alcohol, 90% alcohol, 80% alcohol, 70% alcohol, and distilled water for 15 min each, and then subjected to HE staining (Solarbio, G1120), Masson's trichrome staining (fast green method) (Solarbio, G1343), and immunofluorescence staining respectively.

[0107] HE staining (Solarbio, G1120): The nuclear was stained with hematoxylin solution for 9 min, differentiated with acidic ethanol for 2 - 3 s, blued with tap water for 15 min, stained with eosin for 2 min, and sealed with neutral gum (Solarbio, G8590) after natural air drying.

[0108] Masson staining (Solarbio, G1343): After mixing solution A and solution B of hematoxylin iron staining solution at a ratio of 1:1 and staining the cell nuclei for 6 min, differentiate with acidic ethanol differentiation solution for 2 - 3 s, and blue with blueing solution for 4 min; stain with ponceau staining solution for 8 min, wash with a weak acid working solution prepared by mixing distilled water and weak acid solution at a ratio of 2:1 for 1 min; stain with phosphomolybdic acid solution for 2 min to fade the collagen fibers to red, wash with the prepared weak acid working solution for 1 min, and finally stain with fast green staining solution for 3 min to stain the collagen fibers blue. After the tissue is naturally air-dried, mount with neutral balsam (Solarbio, G8590).

[0109] Immunofluorescence of paraffin sections: After dewaxing and hydrating paraffin sections to distilled water, repair antigens at high temperature on an induction cooker (ɑ-SMA, CD31 citrate buffer (Solarbio, C1010) repair (pH = 6.0); incubate with 0.2% TritonX-100 (SIGMA, 066K0089) for 15 min, wash with PBST 3 times, 5 min each time; block antigens with 10% goat serum for 1 h, add primary antibodies (antibodies diluted with PBS, α-SMA (1:150, Abcam, ab7817), CD31 (1:100, Abcam, ab281583)) and incubate overnight at 4℃. After rewarming for 1 h, wash with PBST 4 times, 15 min each time; incubate the secondary antibody (1:1500, Goat Anti-Mouse IgG H&L(Alexa 488), Abcam, ab150113) for 1 h, wash with PBST 4 times, 15 min each time; finally add DAPI (Solarbio, S2110) to stain the cell nuclei, mount the slides, seal with nail polish, observe and take pictures with a Zeiss inverted fluorescence microscope (Axio Observer 5, Germany), and process and analyze with ZEN 2.6 imaging software for subsequent statistics.

[0110] Immunofluorescence of frozen sections: After removing the mouse heart and gastrocnemius muscle of the lower limb, place them in a mold containing OCT embedding medium (SAKURA, 4583), freeze them on the quick-freezing table of a cryostat for 1 h, and then make 6-μm frozen sections. Soak them in PBS for 10 min to wash away the embedding medium, and then immunofluorescence staining can be carried out. First, fix with 4% paraformaldehyde for 10 min, wash with PBS phosphate buffer 3 times, 5 min each time; then perform antigen repair, and the method is the same as that of paraffin immunofluorescence.

[0111] 1.6 Echocardiogram detection

[0112] After establishing the acute myocardial infarction model in C57BL / 6J mice, cardiac echocardiography was performed on days 7, 14, and 28 using a high-resolution small animal ultrasound imaging system (Canada / VisualSonics, Vevo 3100). One day before the examination, the chest hair of the mice was removed. On the day of echocardiography, the mice were induced with 2% isoflurane (RWD, R510-22) by inhalation anesthesia and placed in a supine position with their feet closely attached to the electrodes and fixed on the mouse board; 0.5% isoflurane was used to maintain anesthesia, and the heart rate was 470±30 bmp. The MX400: 30MHz probe was placed at an angle of 20°-30° to the midline of the sternum to obtain the B-mode cardiac long-axis view of the heart and the apical pulsation; the probe was rotated clockwise by 90° to obtain the B-mode short-axis view of the left ventricle and the M-mode ventricular motion curve. Vevo lab (v3.1) was used for data analysis, and cardiac function indexes such as left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), end-diastolic left ventricular posterior wall (LVPWd), end-systolic left ventricular posterior wall (LVPWs), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), left ventricular diastolic diameter (LVDS), and left ventricular anterior wall thickness during systole (LVAWS) were calculated from the M-mode of the left ventricular short axis.

[0113] 1.7 Western blot detection

[0114] (1) Protein extraction and concentration determination of tissues, cells, and extracellular vesicles:

[0115] After lysing tissues, cells, and extracellular vesicles with RIPA (Beyotime, P1048), they were ground at 60HZ for 120 s (Shanghai Jingxin Industrial Co., Ltd.); after standing on ice for 10 min, the supernatant was taken, centrifuged at 4℃ and 13,000 rpm for 10 min, and the supernatant was collected. Working solution and standard product were prepared using Bicinchoninic Acid Assay (BCA) (Thermo Scientific, 23225). 100 μL of the working solution and 10 μL of the standard product or sample were added to each well of a 96-well plate (JET, TCP011096), incubated at 37℃ for 30 min, and the OD value was measured using a microplate reader (Thermo Scientific, USA) at a wavelength of 562 nm. A standard curve was plotted and the concentration of each sample was calculated. 5× DualColor Protein Loading Buffer (the volume ratio of the sample to the Loading Buffer was 4:1) (Fude Biotech, FD002) was added to each sample, boiled at 99℃ for 10 min, and stored at -80℃.

[0116] (2) Electrophoresis

[0117] 1× Electrophoresis buffer preparation: 3.02 g of glycine (Solarbio, Cat#G8200), 18.8 g of tris(hydroxymethyl)aminomethane (Solarbio, Cat#T8060), 1 g of sodium dodecyl sulfate (Sigma, Lot#SLCG4661), diluted to 1 L with deionized water; Preparation of PAGE gel (Yamei, PG21), samples and protein Marker (PageRuler Prestained Protein Ladder) (ThermoScientific, REF26617) were added in sequence; Electrophoresis was carried out at a constant voltage of 80 V (BIO-RAD, USA) until the bromophenol blue reached the junction of the stacking gel and the separating gel, and then the voltage was adjusted to 100 V until the bromophenol blue reached the bottom.

[0118] (3) Membrane transfer

[0119] 1× Membrane transfer buffer preparation: 3.03 g of glycine (Solarbio, Cat#G8200), 144 g of tris(hydroxymethyl)aminomethane (Solarbio, Cat#T8060), 200 mL of methanol, diluted to 1 L with deionized water; The PVDF (Polyvinylidene Fluoride) membrane (Immobilon, ISEQ00010) was soaked in methanol (Changshu Hongsheng Fine Chemical Co., Ltd., CY20211010) for 5 min of activation, and then placed in the membrane transfer tank in the order of blackboard - sponge - two layers of filter paper - gel - PVDF membrane - two layers of filter paper - sponge - whiteboard. It was placed in an ice-water mixture and transferred at a constant current of 300 mA for 1.75 h.

[0120] (4) Antibody incubation

[0121] Block with 5% defatted milk (Beyotime, P0216) at room temperature for 1 h; wash three times with 1×TBST buffer prepared by diluting 8.7 g of NaCl (Zhejiang Zhongxing Chemical Reagent Co., Ltd., 20160107), 10 mL of 1 M Tris-HCl buffer (pH 8.0) (Solarbio, Cat#T1150), 1 mL of Tween-20 (Solarbio, Cat#T8220) to 1 L with deionized water. Dilute the primary antibody with the primary antibody diluent (Beyotime, P0023B) and use the primary antibodies as follows: AnTi-ALIX antibody (Abcam, ab275377), CD81 Rabbit pAb (ABclonal, A5270), CD63 Rabbit mAb (ABclonal, A19023), AMPKα Rabbit pAb (ABclonal, A11184), Anti-AMPK alpha 1 (phospho T183) / alpha2 (phospho T172) Antibody (Abcam, ab133448), Anti-PPAR alpha / delta Antibody (Abcam, ab178865), PGC-1α Monoclonal antibody (Proteintech, 66369), β-Tubulin Rabbit pAb (ABclonal, AC008), incubate overnight at 4°C; wash four times with 1×TBST. Dilute the secondary antibody with 1×TBST and use the secondary antibodies as follows: Anti-rabbit IgG, HRP-linked Antibody (CST, 7074), Anti-mouse IgG, HRP-linked Antibody (CST, 7076), incubate at room temperature for 1 h; wash four times with 1×TBST. Mix the developing solution A and B solution (BIO-RAD, 170-5061) and drop it on the membrane, image with a multifunctional gel imaging system (BIO-RAD, USA), expose with tubulin as the internal reference, and analyze and calculate the gray value of the band using Image Lab and ImageJ 3.0 software (n = 3).

[0122] 1.8 qPCR detection

[0123] (1) RNA extraction

[0124] 10 - 30 mg of tissue was placed into an RNase-free EP tube (Axygen, MCT-150-C), frozen in liquid nitrogen, and stored at -80 °C. 1 mL of TRIzol (Ambion, 15596026) was added, and homogenization was performed using a grinder (Shanghai Jingxin, Jxfsptpr-24) at 60 Hz for 50 seconds. After incubation at room temperature for 5 minutes, the mixture was transferred to an EP tube, 200 μL of chloroform (Aldrich, 485403-500MG) was added, and the mixture was vigorously shaken for 15 seconds and then incubated at room temperature for 3 minutes. After centrifugation at 4 °C and 12000 g for 15 minutes, the upper aqueous phase was transferred to an EP tube. 5 μL of nucleic acid precipitant (Solarbio, SA1020) and 500 μL of isopropanol (Titan, G75885B) were added successively, and the mixture was gently inverted up and down for mixing and then allowed to stand at room temperature for 10 minutes. After centrifugation at 4 °C and 12000 g for 10 minutes, RNA was seen to adhere to the bottom of the tube.

[0125] 75% ethanol was prepared using DEPC-H2O (Generay, D1007-500mL) and absolute ethanol (Titan, G73537B). The supernatant was discarded, 1 mL of 75% ethanol was added to wash the precipitate, and after vortexing for 30 seconds, centrifugation was performed at 4 °C and 7500 g for 2 minutes. The supernatant was discarded, and the washing operation was repeated once. The RNA precipitate was air-dried at room temperature for 5 minutes until there was no liquid residue in the tube, and 30 - 50 μL of DEPC-H2O was added to dissolve the RNA. The RNA concentration was measured using a ultra-micro nucleic acid and protein analyzer (Thermo Fisher, ND-ONEC-W) and stored at -80 °C.

[0126] (2) Reverse transcription of total RNA into cDNA

[0127] The reverse transcription kit (Takara, Code No.RR047A) was used to remove gDNA. A mixture was prepared on ice at a ratio of 5×gDNA Eraser Buffer:gDNA Eraser (Takara, Code No.RR047A) = 2:1. RNase Free dH2O and Total RNA were successively added to an EP tube (Biofil, PCR-000-200). After low-speed centrifugation, the mixture was incubated at 42 °C for 2 minutes using a thermostatic mixing incubator (Eppendorf, 5386000079).

[0128] Mix PrimeScript RT Enzyme Mix I (Takara, Code No. RR047A), RT Primer Mix (Takara, Code No. RR047A), 5×PrimeScript Buffer 2 (Takara, Code No. RR047A) and RNase Free dH2O (Takara, Code No. RR047A) in a ratio of 1:1:4:4. Add 10 μL of the mixture per EP tube to the total RNA with gDNA removed, and perform gradient PCR (Applied Biosystems, ProFlex) reaction at 37 °C for 15 min, 85 °C for 5 Sec, and hold at 4 °C forever. After termination, add 200 μL of DEPC-H2O (Generay, D1007-500 mL) and store at -20 °C.

[0129] (3) qPCR

[0130] Perform qPCR reaction with 2×RealStar Power Dye qPCR Premix (GenStar, A311-10). Add 6.9 μL of cDNA to each well of an optical 96-well reaction plate (Applied Biosystems, 4346906). Prepare a mixture of primers and dyes (GenStar, A311-10) in the dark, vortex for 30 Sec, add 8.1 μL to each well, tightly cover with an optical film (Applied Biosystems, 4360954), and centrifuge at 2000 g for 2 min. Perform PCR reaction using a Real-Time PCR System (Applied Biosystems, 4376600). Pre-denature at 95 °C for 10 min; denature at 95 °C for 15 Sec, anneal / extend at 60 °C for 1 min, for a total of 40 cycles; the instrument automatically sets the melting curve.

[0131] For qPCR result analysis, use the 2^ (-ΔΔCt) method, with β-actin as the internal reference gene, and calculate the relative expression level of the target gene. Each gene has 3 replicates, and the experiment is repeated 3 times. ΔCt = Ct value of the target gene - Ct value of the internal reference gene, ΔΔCt = ΔCt value of the experimental group - ΔCt value of the sham operation group. Use GraphPad Prism to compare the differences in the target genes between groups, and P < 0.05 indicates statistical significance.

[0132] 1.9 Transmission electron microscopy detection

[0133] Cut the myocardial tissue into 1 mm 3, the tissue was washed with 0.1% sodium chloride solution (Zuo Ba Health Technology Co., Ltd., C19091502), immersed in 2.5% glutaraldehyde (EMS, 16020) at 4°C for 2 h; after being washed 3 times with 0.1 M phosphate buffer (Yubo Biotech, YB160232), it was fixed in the dark with 1% osmium tetroxide (Tedpella, 18459) for 1 h, and washed 2 times with phosphate buffer and pure water respectively; stained with 1% uranyl acetate (Syntechem, 541-09-3) for 2 h, dehydrated successively with 70% acetone, 80% acetone, 90% acetone, and 100% acetone. The tissue block was infiltrated in a 1:1 mixture of acetone and epoxy resin (SPI, 1220324) in an oven at 37°C for 1 h, then placed in a 1:4 mixture of acetone and epoxy resin in an oven at 37°C overnight; the epoxy resin was placed in an oven at 45°C for 2 h, and finally, it was embedded directionally, placed in an oven at 45°C for 3 h, and polymerized in an oven at 65°C for 48 h. The tissue block was cut into <100 nm thin slices using an ultramicrotome (USA), and images were collected at 15,000 times and 30,000 times respectively using a transmission electron microscope (HITACHI, Japan).

[0134] 1.10 Statistical methods

[0135] The statistical values were expressed as mean ± standard deviation. The data type of this study was quantitative data. IBM SPSS Statistics 25 was used for statistical analysis of the experimental data: First, the normality of the sample data was tested. When Skewness / S.E and Kurtosis / S.E both satisfied P < 1.96, the data conformed to normality; second, Levene's test was used to detect the homogeneity of variance of the sample data. When P > 0.1, it was determined that the variance was homogeneous; third, for the comparison of multiple independent samples, α = 0.05. On the basis that the data satisfied normality, independence (no mutual correlation between data was determined as independence), and homogeneity of variance (P > 0.1), one-way analysis of variance and Turkey's multiple analysis (one-way ANOVA with Turkey's post hoc test) were used. If the data satisfied independence and normality but did not satisfy homogeneity of variance, the Tamhane's T2 test was used to test the data. For the comparison of two independent samples, α = 0.05. When the data satisfied normality, independence (no mutual correlation between data was determined as independence), and homogeneity of variance (P > 0.1), the two independent samples t-test was used; if the data satisfied independence and normality but did not satisfy homogeneity of variance, Welch's test was used. The P value was expressed as a numerical value. P < 0.05 was considered to be statistically significant, and GraphPad Prism 8.0.1 was used for plotting.

[0136] 1.11 Histological analysis

[0137] HE staining was used for the statistical analysis of the cross-sectional area of lower limb ischemic muscle fibers, and Masson staining was used for the statistical analysis of the degree of lower limb ischemic fibrosis, the degree of left ventricular fibrosis in myocardial infarction tissues, and the ventricular wall thickness of the left ventricular infarction area. ImageJ 1.8.0.172 software was used to calculate the average cross-sectional area of lower limb muscle fibers. There were 6 mice in each group, and 5 fields of view of 200× were selected from each mouse. 30 muscle fibers were randomly selected from each field of view for statistical analysis; ImageJ 1.8.0.172 software was used to measure and calculate the degree of fibrosis of lower limb muscles and left ventricles in myocardial infarction tissues; the degree of fibrosis of lower limb tissues was expressed as the ratio of the area of blue collagen fibers to the area of red non-collagen fibers in the tissue (6 mice in each group, and 5 fields of view of 200× were selected from each mouse); the degree of left ventricular fibrosis in myocardial infarction tissues was expressed as the ratio of the area of blue collagen fibers to the area of red non-collagen fibers in the left atrium (removing the papillary muscles); ImageJ 1.8.0.172 software was used to calculate the ventricular wall thickness of the left ventricular infarction area. The ventricular wall thickness of the left ventricular infarction area was the ventricular wall thickness at the midpoint of the collagen fiber circumference, which was the average value of the ventricular wall thickness at the junction of collagen fibers and normal tissues on both sides. The data were measured three times and the average value was taken.

[0138] 1.12 Statistical analysis of vascular density

[0139] Immunofluorescence staining of α-SMA and CD31 was performed on the normal group, PBS group, hUCMSCs-EVs group, and hPPSCs-EVs group respectively. Images of the infarction area and the infarction border area were collected with an inverted fluorescence microscope (Axio Observer 5, Germany). There were 6 samples in each group, and 8-15 images were collected from each sample; ImageJ image analysis software was used to measure and count the area of the infarction area and the infarction border area and the number of positive fluorescence of α-SMA and CD31, and calculate the vascular density of the infarction area and the infarction border area. The vascular density of the infarction area and the infarction border area (unit: number / μm 2 ) = total number of blood vessels in the infarction area and the infarction border area / total area of the field of view of the infarction area and the infarction border area.

[0140] 2. Experimental results

[0141] 2.1 Differential analysis of protein groups and metabolomes of hPPSCs-EVs and hUCMSCs-EVs

[0142] hPPSCs-EVs and hUCMSCs-EVs were detected by electron microscopy and showed a disc shape, as Figure 1A , a. The number of vesicles contained in human placental perivascular stem cells (hPPSCs-EVs) was higher than that of human umbilical cord mesenchymal stem cell exosomes (hUCMSCs-EVs), as Figure 1A , b. hPPSCs-EVs and hUCMSCs-EVs expressed exosome-specific proteins CD81, CD63, and ALIX, as Figure 1A, c. ALIX was highly expressed in hPPSCs-EVs compared with hUCMSCs-EVs, and the statistics showed a significant difference (P = 0.0007, n = 3), but there was no significant difference in CD81 and CD63. As shown in Figure 1A , d.

[0143] Proteomic analysis showed that 281 proteins were upregulated and 346 proteins were downregulated in hPPSCs-EVs compared with hUCMSCs-EVs. As shown in Figure 1B , a; Heatmap of differentially expressed proteins, as shown in Figure 1B , b. Domain Enrichment was enriched in protein regions such as Collagen, Proteasome, and hydrolases (p < 0.05). As shown in Figure 1B , c. The metabolic regulation pathway was highly expressed in GO of BP. As shown in Figure 1B , d. GO of CC was enriched in the Proteasome pathway. As shown in Figure 1B , e. KEGG was also enriched in the Proteasome pathway. As shown in Figure 1B , f. The above proteomic analysis results indicated that hPPSCs-EVs were different from hUCMSCs-EVs, and the metabolic regulation pathway was upregulated. Further, metabolomic analysis was performed to analyze the metabolite differences between hPPSCs-EVs and hUCMSCs-EVs. 145 metabolites were upregulated in hPPSCs-EVs compared with hUCMSCs-EVs. As shown in Figure 1C , a. Heatmap analysis showed that the metabolites highly expressed in hPPSCs-EVs compared with hUCMSCs-EVs included amino acids, organic acids, fatty acids, and hormones. As shown in Figure 1C , b. Further analysis of the differentially expressed metabolites showed that N-Methylalanine, N-Ethylglycine, PC, and 21-Deoxycortisol were highly expressed in hPPSCs-EVs compared with hUCMSCs-EVs. As shown in Figure 1C , c. The KEGG enrichment metabolic pathway of the metabolites upregulated in hPPSCs-EVs compared with hUCMSCs-EVs. As shown in Figure 1C , d. Further, a combined analysis of the differentially expressed proteins and metabolites was performed. The proteins and metabolites regulating the metabolic pathway were most enriched in hPPSCs-EVs compared with hUCMSCs-EVs. As shown in Figure 1D .

[0144] 2.2 Therapeutic effects of hPPSCs-EVs and hUCMSCs-EVs on acute myocardial infarction and lower limb ischemia

[0145] To verify the therapeutic effect of hPPSCs-EVs on acute myocardial infarction, in this experiment, PBS, 100 μg hUCMSCs-EV, and 100 μg hPPSCs-EVs were injected at three points in the myocardial infarction area of mice. Echocardiography was performed on D14. Compared with the PBS group and the hUCMSCs-EVs group, the left ventricular wall thickness increased in the hPPSCs-EVs transplantation group on D14, and cardiac function was restored. As Figure 2A , a. Statistical analysis of LVFS showed no significant difference between the hUCMSCs-EVs group and the PBS group, but there was a highly significant difference between the hPPSCs-EVs group and the PBS group (P < 0.0001, n = 12) and between the hPPSCs-EVs group and the hUCMSCs-EVs group (P < 0.0001, n = 12). As Figure 2A , b; Statistical analysis of LVEF showed no significant difference between the hUCMSCs-EVs group and the PBS group. There was a highly significant difference between the hPPSCs-EVs group and the PBS group (P < 0.00001, n = 12) and between the hPPSCs-EVs group and the hUCMSCs-EVs group (P = 0.013, n = 12). As Figure 2A , c; Statistical analysis of LVDS showed no significant difference between the hUCMSCs-EVs group and the PBS group. There was a highly significant difference between the hPPSCs-EVs group and the PBS group (P = 0.000002, n = 12) and between the hPPSCs-EVs group and the hUCMSCs-EVs group (P = 0.001, n = 12). As Figure 2A , d; Statistical analysis of LVAWS showed no significant difference between the hUCMSCs-EVs group and the PBS group. There was a highly significant difference between the hPPSCs-EVs group and the PBS group (P < 0.0001, n = 12) and between the hPPSCs-EVs group and the hUCMSCs-EVs group (P < 0.0001, n = 12). As Figure 2A , e; The above results indicate that transplantation of hPPSCs-EVs promotes the improvement of cardiac function in the mouse myocardial infarction model compared with transplantation of PBS and hUCMSCs-EVs, and is helpful for myocardial repair and regeneration.

[0146] The Masson staining results on D14 showed that the collagen fiber area decreased and the left ventricular wall thickness increased in the hPPSCs-EVs group compared with the PBS group and the hUCMSCs-EVs group. As Figure 2B , a. Statistical analysis showed no significant difference in myocardial fiber area between the hUCMSCs-EVs group and the PBS group. The collagen area decreased significantly in the hPPSCs-EVs transplantation group compared with the PBS group (P = 0.000411, n = 10) and the hUCMSCs-EVs group (P = 0.049, n = 10). As Figure 2B, b. There was no significant difference in the left ventricular wall thickness between the hUCMSCs-EVs group and the PBS group. The left ventricular wall thickness in the hPPSCs-EVs transplantation group was increased compared with that in the PBS group (P = 0.006, n = 10) and the hUCMSCs-EVs group (P = 0.012, n = 10), showing a highly significant difference as Figure 2B , c. The above experiments confirmed that transplantation of hPPSCs-EVs could promote a reduction in the area of collagen fibers and an increase in the left ventricular wall thickness compared with transplantation of PBS and hUCMSCs-EVs.

[0147] To further detect the angiogenesis-promoting effect of hPPSCs-EVs, CD31 and α-SMA immunofluorescence staining was performed. The number of blood vessels in the hPPSCs-EVs transplantation group was increased compared with that in the PBS group and the hUCMSCs-EVs group, as Figure 2C , a; Statistical analysis of the CD31+ blood vessel density showed no significant difference between the PBS group and the hUCMSCs-EVs group. The blood vessel density in the hPPSCs-EVs transplantation group was increased compared with that in the PBS group (P = 0.002, n = 4) and the hUCMSCs-EVs group (P = 0.002, n = 4), showing a significant difference, as Figure 2C , b. Statistical analysis of the α-SMA+ blood vessel density showed no significant difference between the PBS group and the hUCMSCs-EVs group. The blood vessel density in the hPPSCs-EVs transplantation group was increased compared with that in the PBS group (P = 0.043, n = 6) and the hUCMSCs-EVs group (P = 0.041, n = 6), showing a significant difference, as Figure 2C , c. qPCR further verified that the treatment with hPPSCs-EVs induced upregulation of the expression of genes related to angiogenesis. The expressions of VEGF-A (P = 0.0005), VEGFR2 (P = 0.0360), and Dll4 (P = 0.0041) in the hPPSCs-EVs group were significantly upregulated, and the expression of PDGF-B (P = 0.0002) was significantly downregulated compared with those in the PBS group; compared with those in the hUCMSCs-EVs group, the expressions of VEGF-A (P = 0.0014), VEGFR2 (P = 0.0335), and Dll4 (P = 0.0004) in the hPPSCs-EVs group were significantly upregulated, and there was no significant difference in the expression of PDGF-B; compared with those in the PBS group, except for the downregulation of the expression of PDGF-B (P = 0.0045) in the hUCMSCs-EVs group, there was no significant difference in the expressions of VEGFR2, Dll4, and PDGF-B; n = 3, as Figure 2C , d. This experiment showed that transplantation of hPPSCs-EVs promoted an increase in the blood vessel density in the infarcted myocardium of mice; In summary, transplantation of hPPSCs-EVs improved the cardiac function of mice, reduced the area of collagen fibers in the infarcted area, increased the ventricular wall thickness, and promoted angiogenesis.

[0148] To further verify the therapeutic and reparative effects of hPPSCs-EVs in ischemic diseases, we used a mouse model of hindlimb ischemia for further verification. The results of HE and Masson staining showed that the muscle bundle area of the hPPSCs-EVs transplanted mouse hindlimb ischemia model on D14 was increased and the collagen fiber area was decreased compared with that of the hUCMSCs-EVs transplanted mice, as Figure 2D , a. Through statistical analysis, there was no significant decrease in the collagen fiber area in the hUCMSCs-EVs group compared with the PBS group, and there was no statistical difference; while the collagen fiber area in the hPPSCs-EVs group was decreased compared with the PBS group (P = 0.000002, n = 6, each n had 5 fields of 200×), and the collagen fiber area in the hPPSCs-EVs group was decreased compared with the hUCMSCs-EVs group (P = 0.000117, n = 6, each n had 5 fields of 200×), as Figure 2D , b. Statistical analysis of the average cross-sectional area of HE-stained muscle fibers showed that compared with the mice in the PBS treatment group, the average cross-sectional area of muscle fibers in the hPPSCs-EVs group of mice was significantly increased, and the difference was statistically significant (P = 0.003, n = 6, each n had 5 fields of 200×, and 30 muscle fibers were randomly selected in each field). There was no significant difference in the average cross-sectional area of muscle fibers in the hUCMSCs-EVs group of mice compared with the PBS group, and the average cross-sectional area of muscle fibers in the hPPSCs-EVs group was significantly increased compared with the hUCMSCs-EVs group, and the difference was statistically significant (P = 0.005, n = 6, each n had 5 fields of 200×, and 30 muscle fibers were randomly selected in each field) as Figure 2D , c. The results showed that hPPSCs-EVs had a more significant therapeutic effect on hindlimb ischemia than hUCMSCs-EVs. Immunofluorescence CD31 and α-SMA staining showed that the positive vascular density in the hPPSCs-EVs group was increased compared with the PBS group and the hUCMSCs-EVs group, as Figure 2E , a. Statistical analysis showed that there was no significant difference in the CD31+ vascular density between the hUCMSCs-EVs group and the PBS group, while the CD31+ vascular density in the hPPSCs-EVs group was significantly increased compared with the PBS group, and the difference was significant (P = 0.000651, n = 4). The CD31+ vascular density in the hPPSCs-EVs group was also significantly increased compared with the hUCMSCs-EVs group (P = 0.003, n = 4), as Figure 2E, b. Statistical analysis of α-SMA positive vessel density showed no significant difference between the hUCMSCs-EVs group and the PBS group. The α-SMA positive vessel density in the hPPSCs-EVs group was significantly increased compared with that in the PBS group, with a statistically significant difference (P < 0.00001, n = 4). The α-SMA positive vessel density in the hPPSCs-EVs group was significantly increased compared with that in the hUCMSCs-EVs group, with a statistically significant difference (P < 0.00001, n = 4), as Figure 2E , c; Further verification by q-PCR showed that the expressions of VEGF-A (P < 0.0001), VEGFR2 (P = 0.0007), and PDGF-B (P = 0.0030) in the hPPSCs-EVs group were significantly up-regulated compared with those in the PBS group; the expressions of VEGFR2 (P < 0.0001) and PDGF-B (P < 0.0001) in the hPPSCs-EVs group were significantly up-regulated compared with those in the hUCMSCs-EVs group; there were no significant differences in the expressions of VEGFR2, PDGF-B, and PDGFRβ between the hUCMSCs-EVs group and the PBS group except for the up-regulated expression of VEGF-A (P = 0.0030); n = 3, as Figure 2E , d. The above results indicate that transplantation of hPPSCs-EVs in lower limb ischemia can significantly promote the repair and regeneration of mouse lower limb ischemia, reduce the fibrotic area, increase the muscle bundle area, and promote angiogenesis.

[0149] 2.3 hPPSCs-EVs regulate the repair and regeneration of acute myocardial infarction by activating the PPAR metabolic pathway

[0150] To further explore the mechanism of hPPSCs-EVs promoting myocardial infarction repair in mice, this study performed sequencing analysis on the myocardial infarction tissues of transplanted D14 mice. RNA-seq KEGG enrichment analysis showed that the PPAR and AMPK signaling pathways were highly expressed in the hPPSCs-EVs group transplanted at D14 compared with the hUCMSCs-EVs group, as Figure 3A , a. This result suggests that hPPSCs-EVs regulate the repair and regeneration of mouse myocardial infarction by activating the PPAR signaling pathway. Further verification by WB showed high expression of p-AMPK, PPARα / β, and PGC-1α proteins in the hPPSCs-EVs group, as Figure 3A, b. The results of protein gray scale analysis showed that compared with the PBS group, the expression levels of p-AMPK (P = 0.0494), PPARα / β (P = 0.0283), and PGC-1α (P = 0.0342) metabolic regulatory proteins were up-regulated in the hPPSCs-EVs group, with statistically significant differences; however, there were no statistically significant differences in p-AMPK (P = 0.2490), PPARα / β (P = 0.9130), and PGC-1α (P = 0.9940) between the hUCMSCs-EVs group and the PBS group; compared with the hUCMSCs-EVs group, the protein expression levels of p-AMPK (P = 0.0034), PPARα / β (P = 0.0118), and PGC-1α (P = 0.0487) were increased in the hPPSCs-EVs group, with significant statistical differences; n = 3, as Figure 3A , c. The genes highly expressed in the hPPSCs-EVs group were further enriched in the PPARα-regulated lipid metabolism pathway compared with the hUCMSCs-EVs group, such as Figure 3A , d. qPCR verified the activation of the fatty acid β-oxidation pathway downstream of PPARα. The expression levels of Acaa2 (P = 0.0340), Acads (P = 0.0435), Acadvl (P = 0.0058), Acsl6 (P = 0.2497), Cpt1b (P = 0.0187), and Cpt2 (P = 0.4121) were higher in the hPPSCs-EVs group than in the hUCMSCs-EVs group; the expression levels of Acaa2 (P = 0.0014), Acads (P = 0.0058), Acadvl (P = 0.0018), Acsl6 (P = 0.0006), Cpt1b (P = 0.0021), and Cpt2 (P = 0.0049) were higher in the hPPSCs-EVs group than in the PBS group; n = 3, as Figure 3A , e. The above results indicated that transplantation of hPPSCs-EVs activated the AMPK and PPAR pathways, further activated the mitochondrial biogenesis gene PGC-1α and the fatty acid β-oxidation pathway, and thus promoted myocardial infarction repair and regeneration.

[0151] To verify whether hPPSCs-EVs activate the AMPK and PPAR pathways in the early stage of myocardial infarction, we performed sequencing analysis on day 3 after transplantation of hPPSCs-EVs into myocardial infarction mice. RNA-seq analysis of myocardial infarction tissues on day 3 showed that the genes up-regulated by transplantation of hPPSCs-EVs were enriched in the AMPK signaling pathway compared with hUCMSCs-EVs, such as Figure 3B , a. At the same time, they were enriched in the PPAR signaling pathway, such as Figure 3B, b. To verify that the p-AMPK and PPAR signaling pathways are activated, WB experiments determined that the hPPSCs-EVs group had higher expression of p-AMPK, PPARα / β, and PGC-1α than the hUCMSCs-EVs group and the PBS group, n = 3, as Figure 3B , c. The results of gray-scale analysis showed that compared with the PBS group, the hPPSCs-EVs group had up-regulated expression of the metabolic regulatory proteins p-AMPK (P < 0.0494), PPARα / β (P < 0.0283), and PGC-1α (P < 0.0487), with significant statistical differences; compared with the hUCMSCs-EVs group, the hPPSCs-EVs group had increased protein expression of p-AMPK (P < 0.0034), PPARα / β (P < 0.0118), and PGC-1α (P < 0.0342), with significant statistical differences; however, there were no statistical differences in p-AMPK (P < 0.2490), PPARα / β (P < 0.9130), and PGC-1α (P < 0.9940) between the hUCMSCs-EVs group and the PBS group; n = 3, as Figure 3B , d. The genes up-regulated in the hPPSCs-EVs group were further enriched in the PPARα-regulated lipid metabolism pathway compared with the hUCMSCs-EVs group, as Figure 3B , e. qPCR verified that PPARα activated its downstream fatty acid β-oxidation pathway. The hPPSCs-EVs group had higher expression of Acaa2 (P = 0.0014), Acads (P = 0.0058), Acadvl (P = 0.0018), Acsl6 (P = 0.0006), Cpt1b (P = 0.0021), and Cpt2 (P = 0.0049) than the hUCMSCs-EVs group; the hPPSCs-EVs group had higher expression of Acaa2 (P = 0.0340), Acads (P = 0.0435), Acadvl (P = 0.0058), Acsl6 (P = 0.2497), Cpt1b (P = 0.0187), and Cpt2 (P = 0.4121) than the PBS group, n = 3, as Figure 3B , f. These results further indicated that hPPSCs-EVs transplantation at an early stage (D3) activated the AMPK and PPAR in the mouse myocardial metabolic pathway, further activating the mitochondrial biogenesis PGC-1α and the fatty acid β-oxidation pathway.

[0152] 2.4 hPPSCs-EVs promote mitochondrial damage repair in ischemic tissues of mice by activating PPAR / PGC-1α

[0153] To further verify the mechanism by which hPPSCs-EVs regulate mitochondrial biogenesis in mouse myocardial infarction by activating PPAR / PGC-1α. Transmission electron microscopy was performed on D14 after transplantation of hPPSCs-EVs. The mitochondrial structure in the PBS group showed obvious damage compared with the Sham group, including broken and fused cristae, partial absence of cristae edges and cristae membranes, formation of concentric onion-like rings, accompanied by vacuolar changes and mitochondrial membrane loss; compared with the PBS group, the mitochondrial structure in the hUCMSCs-EVs group was improved, but there were still unclear cristae, local dissolution accompanied by partial vacuoles; the mitochondrial structure in the hPPSCs-EVs group was significantly improved, and mitochondrial damage was repaired; as Figure 4A , a. RNA-seq GO enrichment showed that genes upregulated in the hPPSCs-EVs group compared with the hUCMSCs-EVs group were highly expressed genes related to mitochondrial mechanisms, such as Figure 4A , b. qPCR further verified that on D14 after transplantation, the expressions of mitochondrial biogenesis genes PGC-1α (P = 0.0089) and TFAM (P = 0.0004), electron transport chain complex genes NDUFS8 (Complex Ⅰ) (P < 0.0001), SDHB (Complex Ⅱ) (P < 0.0001), UQCRC1 (Complex Ⅲ) (P < 0.0001), and ATPase-6 (Complex Ⅴ) (P = 0.0013) in the hPPSCs-EVs group were significantly upregulated compared with the PBS group, while the expression of mitochondrial fusion gene MFN2 (P = 0.0019) was downregulated; compared with the hUCMSCs-EVs group, the expressions of mitochondrial biogenesis gene TFAM (P = 0.0004), mitochondrial complex genes NDUFS8 (Complex Ⅰ) (P < 0.0001), SDHB (Complex Ⅱ) (P < 0.0001), and UQCRC1 (Complex Ⅲ) (P < 0.0001) in the hPPSCs-EVs group were upregulated, while there were no statistical differences in PGC-1α, MFN2, and ATPase-6 (Complex Ⅴ); n = 3, as Figure 4A, c. To further analyze whether hPPSCs-EVs have a mitochondrial repair effect in the early stage (D3) of myocardial infarction in transplanted mice, we performed qPCR detection on the myocardial tissue transplanted at D3: compared with the PBS group, the expressions of mitochondrial biogenesis genes PGC-1α (P<0.0001), TFAM (P<0.0001), mitochondrial fusion gene MFN2 (P = 0.0302), electron transport chain complex gene NDUFS8 (ComplexⅠ) (P = 0.0442), SDHB (ComplexⅡ) (P<0.0001), UQCRC1 (ComplexⅢ) (P<0.0001), and ATPase-6 (ComplexⅤ) (P = 0.0142) were significantly up-regulated in the hPPSCs-EVs group; compared with the hUCMSCs-EVs group, the expressions of PGC-1α (P = 0.0005), TFAM (P<0.0001), SDHB (ComplexⅡ) (P = 0.0019), UQCRC1 (ComplexⅢ) (P = 0.0017), and ATPase-6 (ComplexⅤ) (P = 0.0257) were significantly up-regulated in the hPPSCs-EVs group, and there was no significant difference in the expressions of MFN2 and NDUFS8 (ComplexⅠ); compared with the PBS group, the expressions of mitochondrial-related genes TFAM (P<0.0001), SDHB (ComplexⅡ) (P = 0.0089), and UQCRC1 (ComplexⅢ) (P = 0.0070) were significantly up-regulated in the hUCMSCs-EVs group, and there was no significant difference in the expressions of PGC-1α, MFN2, NDUFS8, and ATPase-6 genes; as Figure 4A , d. The above results confirmed that transplantation of hPPSCs-EVs had a repair effect on mitochondrial damage in ischemic myocardial infarction at D14 and D3.

[0154] To verify the repair of mitochondrial damage in the lower limbs of mice by hPPSCs-EVs, transmission electron microscopy was performed at D15 after transplantation of hPPSCs-EVs. Compared with the Sham group, the mitochondria in the PBS group were swollen and formed larger vacuoles; compared with the PBS group, the mitochondrial structure and morphology in the hUCMSCs-EVs group were improved to some extent, but there were still mitochondrial vacuoles; the mitochondrial structure and morphology in the hPPSCs-EVs group were significantly improved, the area of mitochondrial vacuoles decreased, and mitochondrial damage was repaired; as Figure 4B, a. qPCR was used to verify the effect of hPPSCs-EVs on the repair of mitochondrial damage in ischemic lower limbs. Mitochondrial detection was performed at an early stage after transplantation (D3): compared with the PBS group, the expressions of mitochondrial biogenesis genes PGC-1α (P<0.0001), TFAM (P = 0.0041), mitochondrial fusion gene MFN2 (P = 0.0071), electron transport chain complex gene NDUFS8 (Complex Ⅰ) (P = 0.0496), SDHB (Complex Ⅱ) (P = 0.0095), UQCRC1 (Complex Ⅲ) (P = 0.0038), and ATPase-6 (Complex Ⅴ) (P<0.0001) were significantly upregulated in the hPPSCs-EVs group; compared with the hUCMSCs-EVs group, the expressions of PGC-1α (P<0.0001), TFAM (P = 0.0011), MFN2 (P = 0.0093), SDHB (Complex Ⅱ) (P = 0.0084), UQCRC1 (Complex Ⅲ) (P = 0.0055), and ATPase-6 (Complex Ⅴ) (P<0.0001) were significantly upregulated in the hPPSCs-EVs group, and there was no significant difference in the expression of NDUFS8 (Complex Ⅰ); there was no significant difference in mitochondrial-related genes between the hUCMSCs-EVs group and the PBS group; n = 3, as Figure 4B , b. The above results indicate that transplantation of hPPSCs-EVs has a reparative effect on mitochondrial damage in the ischemic lower limbs of mice at both D3 and D15.

Claims

1. An extracellular vesicle, characterized in that, The extracellular vesicles are derived from human placental perivascular stem cells, and the human placental perivascular stem cells co-express CD248, CD276, CD44, α-SMA, NG2, CD140b, CD105, CD90 and CD73; the human placental perivascular stem cells do not express CD34, CD31 and vWF; The method for preparing the extracellular vesicles comprises the steps shown in the following (i)-(ii): (i) Culturing human placental perivascular stem cells in a medium with or without serum for 36-72 h, and collecting the cell culture supernatant; (ii) Treating the cell culture supernatant successively as follows: centrifuging at 250-350 g for 5-15 min to obtain a supernatant, centrifuging at 1700-2300 g for 10-20 min to obtain a supernatant, centrifuging at 8500-11500 g for 25-35 min to obtain a supernatant, filtering through a microporous membrane to obtain a filtrate, and centrifuging at 80000-120000 g for 60-80 min to obtain a precipitate, thus obtaining the extracellular vesicles.

2. The extracellular vesicle according to claim 1, wherein The serum is fetal bovine serum free of extracellular vesicles.

3. The extracellular vesicle according to claim 2, wherein The method for preparing the fetal bovine serum free of extracellular vesicles comprises the step of ultracentrifuging the fetal bovine serum.

4. The extracellular vesicle according to claim 3, wherein The conditions of the ultracentrifugation include: centrifuging at 80000-120000 g for 11-13 h.

5. The extracellular vesicle according to claim 1, characterized in that, In the step (i), the culture conditions of the human placental perivascular stem cells include: the culture temperature is 36.5-37.5 °C, and the culture environment contains 4.8%-5.2% CO2.

6. The extracellular vesicle according to claim 1, wherein The pore size of the microporous membrane is 40 nm-1000 nm.

7. The extracellular vesicle according to any one of claims 1 to 6, characterized in that, The extracellular vesicles highly express ALIX protein as compared with the extracellular vesicles derived from human umbilical cord mesenchymal stem cells.

8. Use of the extracellular vesicles according to any one of claims 1-7 in the preparation of a medicament for preventing and / or treating myocardial infarction or lower limb ischemia.

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