CN108841787B - Effect of EPO on exosomes released from mesenchymal Stem cells - Google Patents

Effect of EPO on exosomes released from mesenchymal Stem cells Download PDF

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CN108841787B
CN108841787B CN201810769767.9A CN201810769767A CN108841787B CN 108841787 B CN108841787 B CN 108841787B CN 201810769767 A CN201810769767 A CN 201810769767A CN 108841787 B CN108841787 B CN 108841787B
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CN108841787A (en
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李璇
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Shanghai Lanweisaier Biotechnology Co ltd
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    • C12N2501/14—Erythropoietin [EPO]

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Abstract

The invention relates to a method for improving the number or performance of exosomes secreted by mesenchymal stem cells, which increases the number of exosomes released by MSC by adding EPO during the culture of the mesenchymal stem cells and has stronger endothelial cell proliferation promoting capacity and angiogenesis promoting capacity. The invention provides a powerful tool for applying the MSC exosome to clinic.

Description

Effect of EPO on exosomes released from mesenchymal Stem cells
Technical Field
The present invention relates to the field of exosomes, in particular to the use of Erythropoietin (EPO) to alter the performance of exosomes released from mesenchymal stem cells.
Background
Mesenchymal Stem Cells (MSCs) are a group of adult stem cells with osteogenic, chondrogenic, and adipogenic functions. The MSC can play the functions of regulating immunity, promoting angiogenesis, supporting hematopoiesis and the like in a paracrine mode. MSCs are abundant in source and present in various tissues such as placenta, umbilical cord, bone marrow, fat, and the like. Therefore, MSC has been approved by several countries as a drug for the treatment of graft versus host disease, ulcerative colitis, myocardial infarction convalescence, etc. However, whether administered intravenously, intramuscularly or intramuscularly, exogenously transplanted MSCs died in 72h most. Then how do the dead MSCs perform the above-mentioned functions? Previous studies have shown that MSCs cultured in vitro release large amounts of exosomes under hypoxic and serum deprivation conditions. The exosome can promote endothelial cells to proliferate in vitro, promote the endothelial cells to form a capillary vessel reticular structure and accelerate the recovery of blood flow of an ischemia model. In fact, MSC-derived exosomes have entered clinical trials for the treatment of diabetes and renal insufficiency. Therefore, the MSC-derived exosomes have potential practical value as materials for "cell-free" cell therapy. In order to improve the treatment effect, a way for acquiring exosomes with stronger functions is explored, and the method is necessary for developing clinical test research of the MSC exosomes.
Previous data indicate that Erythropoietin (EPO) can promote MSC to secrete hepatocyte growth factor, which is known to have strong angiogenesis promoting effect. It will be discussed herein whether EPO stimulates MSC to release exosomes and whether such exosomes have a stronger pro-angiogenic function. The results show that EPO promotes MSC to release exosome with stronger function, and provides beneficial data for clinical trial research of the exosome from MSC.
Disclosure of Invention
The invention provides a method for improving the number or performance of exosomes secreted by mesenchymal stem cells, which comprises the following steps: (1) suspending the MSC in an MSC serum-free culture medium, inoculating the MSC in a culture dish, and culturing overnight to allow the cells to adhere to the wall; (2) removing the culture medium, replacing with alpha-MEM culture medium, adding Erythropoietin (EPO), and culturing for 72 h; (3) collecting supernatant, centrifuging to remove cell debris, filtering by a filter membrane, centrifuging to collect precipitate, and suspending the precipitate in buffer solution to obtain the exosome with improved quantity and performance.
Preferably, said properties refer to the endothelial cell proliferation-promoting and angiogenesis-promoting capacity of the exosomes.
Preferably, wherein in step (1) according to 5X 10 6 Cells/dish were seeded on culture dishes.
Preferably, the concentration of EPO in step (2) is 1U/ml.
Preferably, the centrifugation conditions in which the cell debris is removed in step (3) are 1500g for 30min, and the centrifugation conditions after filtration through a filter membrane are 100,000g for 1 h.
Preferably, wherein the precipitate in step (3) is suspended in a buffer containing 5mmol/L KCl, 1mmol/L MgCl2 and 136mmol/L NaCl.
Preferably, the mesenchymal stem cells are human umbilical cord MSCs.
The invention also provides application of Erythropoietin (EPO) in increasing the number or performance of exosomes secreted by mesenchymal stem cells, wherein the performance refers to the endothelial cell proliferation promoting capacity and the angiogenesis promoting capacity of the exosomes.
The invention also provides application of Erythropoietin (EPO) in preparation of a reagent for improving the number or the performance of exosomes secreted by mesenchymal stem cells, wherein the performance refers to the endothelial cell proliferation promoting capacity and the angiogenesis promoting capacity of the exosomes.
Preferably, the mesenchymal stem cells are human umbilical cord MSCs.
The invention has the following positive effects:
the research shows that after stimulation by EPO, the number of exosomes released by MSC is increased, and the exosomes have stronger endothelial cell proliferation promoting capacity and angiogenesis promoting capacity. The research provides necessary information and powerful tools for applying the MSC exosome to clinic.
Drawings
Fig. 1 is a morphological observation of microparticles released from human mesenchymal stem cells: and (4) carrying out ultra-high speed centrifugation on the MSC culture supernatant to obtain particles, and observing the particles by a projection electron microscope. Bar is 200nm.
Figure 2 is a surface molecular expression analysis of microparticles from human mesenchymal stem cells: microparticles in the mesenchymal stem cell supernatant were collected and reacted with CTR, CD9, CD63, CD81 antibodies. The X-axis is relative fluorescence intensity and the Y-axis is counts.
Figure 3 is the effect of MSC-secreted exosomes on endothelial cell proliferation for different culture conditions: exosomes secreted by MSC under different culture conditions are added into a culture medium of human umbilical cord endothelial cells, and the proliferation condition of the exosomes is observed by an MTT test. Y-axis: OD490 nm. Results were from two independent experiments. Untraded is the experimental group with unstimulated exosomes added, EPO is the experimental group with MSC exosomes stimulated with EPO.
Figure 4 is a graph of the effect of MSC-secreted exosomes on endothelial cell network formation in different culture conditions: untratated is the experimental group with unstimulated exosomes added, and EPO is the experimental group with EPO stimulating MSC exosomes added.
Detailed Description
The present invention is described in further detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.
Reagent and apparatus
Human MSC serum-free medium was purchased from the company, mitsui biotechnology, beijing. Acetylated latex beads (3.9 μm diameter) were manufactured by Invitrogen corporation, USA. FITC-labeled mouse monoclonal antibodies against human CD9, CD63, and CD81 were all available from Becton Dicbnson, USA. The electron microscope is Hitachi H.7650, and the ultra-high speed centrifuge is Beckman corporation, USA. The flow cytometer FACSCalibur is a BD company in usa.
Example 1: culture and identification of human umbilical cord MSC
The human umbilical cord specimen is derived from a normal delivery fetus in a general hospital of the liberation force and is voluntarily donated by a parturient, and the application of the specimen accords with the application specification of a human specimen experiment made by the ethical committee. The procedure was as provided in the human MSC serum-free medium protocol. Briefly, human umbilical cord was harvested, the outer membrane and the artery and vein removed, minced, and digested overnight at 37 deg.C with the addition of collagenase type I at a final concentration of 0.2%. Then, trypsin was added to a final concentration of 0.05% and the reaction was carried out at room temperature for 1 hour. The digested cells were collected by centrifugation and plated on a 150mm diameter petri dish. Non-adherent cells were removed after 1 week and passaged by digestion. Passage 3 cells were used for the following experiments. According to the conventional method in the laboratory, the cells are subjected to morphological observation, surface molecular characteristic detection and in-vitro osteogenesis and adipogenic differentiation identification, and the obtained cells are proved to be in line with the characteristics of the human MSC.
Example 2: preparation and identification of MSC-derived exosomes
1. Preparation of exosomes
Collecting third generation human umbilical cord MSC by trypsinization, counting cell number, suspending in human MSC serum-free culture medium according to 5 × 10 6 Cells/dish were seeded in 150mm diameter culture dishes at 37 ℃ with 5% CO 2 Cells were allowed to adhere by overnight incubation at 95% humidity. The medium was removed, replaced with alpha-MEM, and EPO (1U/ml) was added and the culture was continued for 72 hours. The supernatant was collected and centrifuged (1500g, 30min) to remove cell debris. The culture supernatant was filtered through a 0.22 μm-diameter filter to remove any apoptotic bodies and other particles. Centrifuging at 4 deg.C for 100,000g for 1 hr, and collecting precipitate. Suspending the precipitate in a suspension containing 5mmol/L KCl, 1mmol/L MgCl 2 136mmol/L NaCl solution, and centrifuged and washed 2 times as above to remove the remaining protein. The pellet was suspended in the above buffer and the protein concentration was measured by the Bradford method. Subpackaging and storing at-80 deg.C for use. The exosomes used in this study were derived from MSCs cultured in three samples.
As a result: the supernatant of MSC culture was filtered through a 0.22 μm filter and then centrifuged at ultra high speed to obtain microparticles. The observation of a projection electron microscope shows that the average size of the obtained particles is about 80 mu m, the particles are in a hollow cystic structure and accord with the morphological characteristics of exosomes (figure 1). Indicating that the exosome from the MSC can be obtained by using the ultra-high speed centrifugation method.
2. Identification of exosomes
The exosome surface molecule expression analysis was performed using flow cytometry as provided in the literature. Briefly, latex beads were washed with MES buffer and bound to exosomes overnight. After washing, glycine was added for blocking, and after washing again, FITC-labeled mouse anti-human CD9, CD63, and CD81 monoclonal antibodies were added. After 20 minutes reaction at room temperature, the reaction was washed and at least 10,000 data points were collected by flow cytometry (FACS-Calibur, BD). The results were analyzed using winmdi2.9 software. The morphology of the obtained exosomes was observed using a transmission electron microscope and photographed at 10,000 times magnification according to the method provided in the above document.
Analysis by WinMdi software showed that microparticles obtained by this method uniformly expressed CD9, CD63 and CD 81. As a result, it was confirmed that the microparticles obtained by ultracentrifugation were exosomes (FIG. 2).
Example 3: effect of growth factors on exosomes secreted by MSCs
The growth factors treated MSCs to influence their secreted Exosomes (EPO) were as in example 2.
1. Protein content determination
To initially understand the changes in the number of exosomes released by human umbilical cord MSCs after EPO treatment. The supernatant was collected for exosome isolation and purification, and the cell number was counted after trypsin digestion. The protein content in exosomes was determined by the Bradford method and calculated 10 8 The number of exosomes released by each cell. The results showed that the exosome protein content of the unstimulated group and that of the EPO group were 256. + -. 124. mu.g/10, respectively 8 Cells, 1021. + -. 392. mu.g/10 8 Cells, after EPO stimulation, the number of exosomes released by human umbilical cord MSCs was significantly increased (P)<0.01). The results suggest that EPO treatment may prompt MSC to release exosomes.
2. MTT assay
The MTT experiment is utilized to observe the influence of MSC exosomes of different sources on endothelial cell proliferation. Human umbilical cord endothelial cells are provided by the laboratory hematology research laboratory of the institute for radiology and radiology, institute of military medical sciences. MTT experiments were performed according to the conventional method. Briefly, human umbilical cord endothelium was seeded into 96-well plates at 2000 cells/well. Exosomes which are not treated by the cell factors and exosomes which are treated by EPO are respectively added into a culture system, the concentration of the exosomes is 10 mu g/ml, and each group comprises 3 multiple wells. After 72h of incubation, MTT was added and incubation continued for 4 h. Thereafter, dimethyl sulfoxide was added. The optical density at 490nm was determined.
The results are shown in FIG. 3. After EPO and treatment, MSC releases exosomes with stronger endothelial cell proliferation activity compared with an untreated control group. The OD values of EPO group were significantly higher than those of untreated group (P < 0.05). The above results suggest that the exosomes released from MSC have stronger endothelial cell proliferation promoting ability after EPO treatment.
3. Capillary-like structure formation experiment
Matrigel experiments are a classical technique for observing endothelial cell function, especially the ability of capillary networks to form, and the results represent in vivo neovascularization activity. To investigate the angiogenesis promoting effect of exosomes from different sources, endothelial cells were seeded in Matrigel-plated culture plates, 10 μ g/ml of exosomes were added to the system, and the number of reticulocytes per high-power field was counted after 24 h. Specifically, the method is reported in the literature, in which the umbilical cord endothelial cells are suspended in DMEM medium containing 1% fetal bovine serum and cultured in the presence of 10% fetal bovine serum 5 Perwell was seeded in Matrigel-plated 24-well plates. The experiment is divided into a negative control group without exosome, a group without exosome stimulation and an MSC exosome stimulation group stimulated by EPO, and each group has 3 multiple holes. After 24h incubation, the counts were counted according to literature methods.
As a result, the numbers of the reticular structures per visual field of the unstimulated group and the EPO group are respectively 2.6 +/-0.84 and 4.6 +/-1.57, and the cell growth factor treatment group is obviously higher than that of the untreated group (P < 0.01). The above results suggest that the exosomes released by the MSCs have a stronger angiogenesis promoting ability after stimulation by EPO.
While embodiments of the invention have been disclosed above, it is not intended that they be limited to the applications set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. Therefore, the invention is not to be limited to the specific details and illustrations shown and described herein, without departing from the general concept as defined by the claims and their equivalents.

Claims (3)

1. A method of increasing the number or performance of exosomes secreted by mesenchymal stem cells, the method comprising the steps of: (1) suspending the MSC in an MSC serum-free culture medium, inoculating the MSC in a culture dish, and culturing overnight to allow the cells to adhere to the wall; (2) removing the culture medium, replacing with alpha-MEM culture medium, adding Erythropoietin (EPO), and culturing for 72 h; (3) collecting supernatant, centrifuging to remove cell debris, filtering by a filter membrane, centrifuging to collect precipitate, and suspending the precipitate in buffer solution to obtain exosome with improved quantity and performance, wherein the performance refers to the endothelial cell proliferation promoting capacity and angiogenesis promoting capacity of the exosome, the concentration of EPO in the step (2) is 1U/ml, the centrifugation condition for removing the cell debris in the step (3) is 1500g and 30min, the centrifugation condition after filtering by the filter membrane is 100,000g, centrifuging for 1h, and suspending the precipitate in a buffer solution containing 5mmol/L KCl and 1mmol/L MgCl in the step (3) 2 And 136mmol/L NaCl in buffer; the mesenchymal stem cells are human umbilical cord MSCs.
2. The method of claim 1, wherein the ratio of 5 x 10 in step (1) is 5 x 10 6 Cells/dish were seeded on culture dish.
3. Use of Erythropoietin (EPO) for increasing the number or performance of exosomes secreted by mesenchymal stem cells, wherein the performance is the endothelial cell proliferation-promoting ability and the angiogenesis-promoting ability of the exosomes, characterised in that the step of claim 1 is employed in increasing the number or performance of exosomes secreted by mesenchymal stem cells, which are human umbilical cord MSCs, using Erythropoietin (EPO).
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CN111172103A (en) * 2019-12-05 2020-05-19 伯仕利生物科技发展（盐城）有限公司 Preparation method and application of stem cell exosome stimulated by angelica sinensis extract
CN113215100B (en) * 2021-06-10 2022-07-12 浙江大学 Application of small molecule compound MLN4924 in promoting secretion of exosome
CN117586955B (en) * 2024-01-19 2024-04-19 吉林大学 Preparation and application of EPO-stimulated macrophage-derived exosome

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WO2011010966A1 (en) * 2009-07-23 2011-01-27 Agency For Science, Technology And Research (A*Star) Pre-natal mesenchymal stem cells
CN106282107A (en) * 2016-08-30 2017-01-04 章毅 Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body
CN106913583A (en) * 2017-04-20 2017-07-04 深圳市赛欧细胞生物科技有限公司 The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents
CN107217034A (en) * 2017-04-20 2017-09-29 深圳市赛欧细胞生物科技有限公司 A kind of human umbilical cord mesenchymal stem cells source excretion body and its acquisition methods and application

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011010966A1 (en) * 2009-07-23 2011-01-27 Agency For Science, Technology And Research (A*Star) Pre-natal mesenchymal stem cells
CN106282107A (en) * 2016-08-30 2017-01-04 章毅 Human plactnta mescenchymal stem cell source separates outer method and the application thereof secreting body
CN106913583A (en) * 2017-04-20 2017-07-04 深圳市赛欧细胞生物科技有限公司 The preparation method and application of human mesenchymal stem cell source excretion body biologically active agents
CN107217034A (en) * 2017-04-20 2017-09-29 深圳市赛欧细胞生物科技有限公司 A kind of human umbilical cord mesenchymal stem cells source excretion body and its acquisition methods and application

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