WO2022218443A1 - 间充质干细胞来源的外泌体治疗脑卒中的方法和组合物 - Google Patents

间充质干细胞来源的外泌体治疗脑卒中的方法和组合物 Download PDF

Info

Publication number
WO2022218443A1
WO2022218443A1 PCT/CN2022/087513 CN2022087513W WO2022218443A1 WO 2022218443 A1 WO2022218443 A1 WO 2022218443A1 CN 2022087513 W CN2022087513 W CN 2022087513W WO 2022218443 A1 WO2022218443 A1 WO 2022218443A1
Authority
WO
WIPO (PCT)
Prior art keywords
stem cells
mesenchymal stem
exosomes
cells
derived
Prior art date
Application number
PCT/CN2022/087513
Other languages
English (en)
French (fr)
Inventor
路钢
陈伟仪
苏献伟
曾金时
刘强
Original Assignee
卓越细胞工程(香港)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 卓越细胞工程(香港)有限公司 filed Critical 卓越细胞工程(香港)有限公司
Publication of WO2022218443A1 publication Critical patent/WO2022218443A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to the fields of cytology and pharmacology. Specifically, the present invention provides a method for treating stroke by using mesenchymal stem cell-derived exosomes and a pharmaceutical composition for treating stroke.
  • Stroke is the second leading cause of death and disability worldwide. Ischemic stroke accounts for about 80% of strokes. Traditional drugs, thrombolytics, can be used to restore blood flow to the brain in acute stroke patients, but only in some patients and must be administered within 4.5 hours of stroke onset. In the past two decades, active and innovative researches have been carried out on the treatment of ischemic stroke, among which stem cell-based neuroreparation therapy has become one of the research directions.
  • mesenchymal stem cells are an important member of the stem cell family. Studies have found that mesenchymal stem cells have the characteristics of strong proliferation ability and multi-directional differentiation potential, immunomodulatory function, no immune rejection and less allograft rejection. Natural mesenchymal stem cells are derived from the mesoderm in early development. Mesenchymal stem cells can be isolated and cultured from various tissues. Mesenchymal stem cells can also be differentiated from pluripotent stem cells. Among them, induced pluripotent stem cells (induced pluripotent stem cells, iPSCs), also known as induced pluripotent stem cells or artificial pluripotent stem cells, are artificially prepared cells with the stemness of embryonic stem cells.
  • induced pluripotent stem cells induced pluripotent stem cells, iPSCs
  • iPSCs induced pluripotent stem cells
  • iPSCs induced pluripotent stem cells
  • artificial pluripotent stem cells are artificially prepared cells with the stemness of embryonic stem cells.
  • Induced pluripotent stem cells were first successfully prepared by Japanese scientist Shinya Yamanaka in 2006, using a viral vector to transfer a combination of four transcription factors (Oct4, Sox2, Klf4 and c-Myc) into differentiated somatic cells, so that A cell type that resembles embryonic stem cells and embryonic APSC pluripotent cells. Induced pluripotent stem cells have been widely used in biotechnology and medical research.
  • Extracellular vesicles are important mediators in cellular communication and have been shown to contribute to the paracrine effect of stem cells on tissue regeneration.
  • Extracellular vesicles are nanoscale (50–1000 nm) lipid bilayer spheres that encapsulate parent cell components such as membrane proteins, RNA, small noncoding RNAs, etc.
  • Extracellular vesicles can be classified into various categories according to their diameter, weight gradient, RNA/protein content, source of derivation and biological function, among which, exosomes are the main type, which are about 30 to 200 nm in diameter, usually With surface markers such as CD9, CD81 and CD63.
  • stem cells or extracellular vesicles of various origins are used in therapy, their roles, including risks, in damaged tissues or organs have not been well documented or demonstrated. Therefore, there is also a need in the art for effective and safe therapies and drugs for the treatment of stroke, especially ischemic stroke, such as new stem cells or their secreted exosome-based therapies and drugs.
  • the present application provides novel therapies and pharmaceutical compositions for the treatment of stroke using mesenchymal stem cell-derived exosomes.
  • the inventors of the present applicant discovered for the first time that mesenchymal stem cell-derived exosomes have the activity of repairing cell damage caused by hypoxia and glucose deficiency, and can effectively treat stroke, especially ischemic stroke.
  • the applicant has also unexpectedly discovered specific preparation methods and conditions for obtaining exosomes from stem cell culture fluids, including stem cell sources, cell culture methods (such as the number of cell passages), etc., as well as specific administration doses and methods, etc. Exosomes are particularly effective in the treatment of stroke, and there are no side effects such as cell damage to the subjects.
  • the present invention provides a pharmaceutical composition for treating stroke, which contains mesenchymal stem cell-derived exosomes.
  • the exosomes in the pharmaceutical composition provided by the present invention have the activity of repairing cell damage, especially the activity of repairing cell damage caused by hypoxia and glucose deficiency.
  • Stroke also known as stroke, is an acute cerebral blood circulation disorder, with sudden fainting, unconsciousness, and hemiplegia as the main symptoms, accompanied by skewed corners of the mouth and unfavorable language. Stroke includes hemorrhagic stroke, ischemic stroke, hypertensive encephalopathy and vascular dementia. Hypertension, arteriosclerosis, cerebrovascular malformations, and cerebral aneurysms often lead to hemorrhagic stroke; rheumatic heart disease, atrial fibrillation, bacterial endocarditis, etc. often lead to ischemic stroke.
  • the pharmaceutical composition is suitable for the treatment of ischemic stroke, including transient ischemic attack, atherosclerotic thrombotic cerebral infarction, lacunar cerebral infarction, and cerebral embolism Wait.
  • mesenchymal stem cells are also referred to as pluripotent mesenchymal cells, which are mainly obtained from fat or bone marrow and can differentiate into various cells of mesodermal origin, such as bone, fat, cartilage, tendon and muscle Wait.
  • Mesenchymal stem cells can be isolated and cultured from a variety of tissues, but their abilities and cell surface markers differ from each other depending on their origin.
  • Mesenchymal stem cells are generally defined by cells that can differentiate into osteocytes, chondrocytes, and muscle cells, and express cell surface markers such as CD73(+), CD105(+), CD34(-), and CD45(-).
  • the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose, umbilical cord blood, teeth or pluripotent stem cells. More preferably, the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, adipose or pluripotent stem cells.
  • stem cells refers to undifferentiated cells that have the ability to self-renew and differentiate to produce progeny cells at the single-cell level.
  • Stem cells are characterized by their ability to differentiate in vitro from a variety of germ layers (endoderm, mesoderm, and ectoderm) into functional cells of various cell lineages, as well as to generate tissue of various germ layers after transplantation and to substantially assist after injection into blastocysts. the ability to form in all or most tissues.
  • pluripotent stem cells refers to stem cells capable of producing all embryonic cell types.
  • Natural pluripotent stem cells include embryonic stem cells.
  • Induced pluripotent stem cells iPSCs
  • induced pluripotent stem cells are artificially prepared cells with the stemness of embryonic stem cells.
  • four transcription factors Oct4, The combination of Sox2, Klf4 and c-Myc was transferred into differentiated somatic cells and reprogrammed.
  • the mesenchymal stem cells are mesenchymal stem cells derived from induced pluripotent stem cells.
  • the mesenchymal stem cells are mesenchymal stem cells obtained by preparing induced pluripotent stem cells from dermal fibroblasts and further inducing their differentiation.
  • Extracellular vesicles are membrane vesicles secreted by cells.
  • Extracellular vesicles can have diameters (in the case of particles other than spheroids, their largest dimension) of between about 10 nm and about 5000 nm.
  • exosomes refer to small secretory vesicles, typically having a diameter (or, in the case of a particle that is not a spheroid, its largest dimension) between about 30 nm and about 180 nm, for example having a diameter of about 30 nm to about 180 nm. diameter between about 150nm.
  • Exosomes contain nucleic acids, proteins, or other biomolecules, or have nucleic acids, proteins, or other biomolecules in their membranes, and can act as carriers between different locations in the body or biological system.
  • Exosomes can be isolated from a variety of biological sources including mammals such as mice, rats, guinea pigs, rabbits, dogs, cats, cattle, horses, goats, sheep, primates, or humans. Exosomes can be isolated from biological fluids such as serum, plasma, whole blood, urine, saliva, breast milk, tears, sweat, synovial fluid, cerebrospinal fluid, semen, vaginal fluid, ascites fluid, and amniotic fluid. Exosomes can also be isolated from experimental samples such as media obtained from cultured cells.
  • the mesenchymal stem cells used for extracting exosomes are mesenchymal stem cells obtained by subculturing mesenchymal stem cells differentiated from bone marrow cells, adipocytes or induced pluripotent stem cells.
  • the mesenchymal stem cells are 1-15 passages, preferably 3-10 passages, and more preferably 5-8 passages.
  • the primary cells used for subculture generally refer to the mesenchymal stem cells that appear first after the mesenchymal stem cell-derived cells are induced and differentiated, that is, the mesenchymal stem cells that appear in the cell population with Cells with mesenchymal stem cell characteristics (for example, having mesenchymal stem cell-specific surface markers, etc.) account for more than 50% of the total number of cells, or preferably more than 75%, or more preferably more than 90%.
  • Primary cells can be directly used for subculture, or they can be used for subculture after being thawed after cryopreservation.
  • the inventors of the present application unexpectedly found that, among the subcultured mesenchymal stem cells, mesenchymal stem cells of earlier passages (such as cells that have been passaged 10 times or less, especially 8 times or less)
  • the repair activity of exosomes produced by cells of the following passages) on damaged neuronal cells was significantly better than that of exosomes produced by mesenchymal stem cells of later passages (such as cells passaged more than 10 times).
  • exosomes used in the present invention can be carried out by various methods known in the art. Methods for isolating exosomes include ultrafiltration, polymer precipitation, size chromatography, or ultracentrifugation. Preferably, the exosomes used in the present invention can be prepared by ultrafiltration and polymer (eg, PEG) precipitation.
  • Methods for isolating exosomes include ultrafiltration, polymer precipitation, size chromatography, or ultracentrifugation.
  • the exosomes used in the present invention can be prepared by ultrafiltration and polymer (eg, PEG) precipitation.
  • the exosomes used in the present invention are prepared from cell culture fluid by ultrafiltration.
  • an ultrafiltration membrane with a molecular weight cut-off of about 100 kDa is used to screen exosomes. Exosomes were present in fractions that were not able to pass through an ultrafiltration membrane with a molecular weight cut-off of about 100 kDa.
  • the ultrafiltration method further comprises the step of filtration using a 4 ⁇ m pore size filter and/or a 0.22 ⁇ m pore size filter before the ultrafiltration membrane having a molecular weight cut-off of about 100 kDa.
  • filtration can be performed stepwise through a 4 ⁇ m pore size cell filter, a 0.22 ⁇ m pore size filter and a filter with a MWCO (molecular weight cut-off value) of 100 kD.
  • the ultrafiltration method used in the present invention may comprise the following main steps:
  • the pharmaceutical composition for preventing or treating stroke provided by the present invention contains the above-mentioned exosomes in a pharmaceutically effective amount.
  • the exosomes can be included in the pharmaceutical composition alone or together with one or more pharmaceutically acceptable carriers, excipients or diluents.
  • a pharmaceutically effective amount means an amount sufficient to prevent, ameliorate or treat the symptoms of stroke, especially ischemic stroke.
  • the dosage of the exosomes in the pharmaceutical composition provided by the present invention is about 1-500ug, preferably about 5-200ug, more preferably about 10-100ug. In yet another aspect of the present invention, the dosage of the exosomes is about 40-5000 ug/kg body weight, preferably about 400-4000 ug/kg body weight.
  • the dosage of the exosomes in the pharmaceutical composition provided by the present invention is about 1 ⁇ 10 9 to 1 ⁇ 10 12 , preferably about 1 ⁇ 10 10 to 1 ⁇ 10 11 . In yet another aspect of the present invention, the dosage of the exosomes is about 1 ⁇ 10 10 to 4 ⁇ 10 13 /kg body weight, preferably about 1 ⁇ 10 11 to 4 ⁇ 10 12 /kg body weight.
  • compositions provided by the present invention can be prepared into unit dosage formulations suitable for administration to a patient according to methods commonly used in the pharmaceutical field, and the formulations contain an administration amount effective by one or several administrations.
  • the pharmaceutical compositions provided by the present invention may be in the form of single or multiple administration.
  • the pharmaceutically effective amount can be appropriately changed according to the severity of the disorder, the age, weight, health and sex of the patient, the route of administration, the treatment period and the like.
  • the exosomes employed in the provided pharmaceutical compositions of the present invention are particularly suitable for use in therapy through multiple administrations.
  • the pharmaceutical compositions provided by the present invention are in the form of multiple administrations.
  • the pharmaceutical composition provided by the present invention is in a dosage form for administration at intervals of about 1 day to 10 days.
  • the pharmaceutical composition is in a dosage form for administration at intervals of about 5-7 days. More preferably, the pharmaceutical composition is in a dosage form that is administered 7 days apart.
  • the dosage of each administration of the exosomes is about 1-500ug, preferably about 5 ug. -200ug, more preferably about 10-100ug.
  • the dosage of each administration of the exosomes is about 40-5000ug/kg Body weight, preferably about 400-4000 ug/kg body weight.
  • the dosage of each administration of the exosomes is about 1 ⁇ 10 9 to 1 ⁇ 10 12 , preferably about 1 ⁇ 10 10 to 1 ⁇ 10 11 .
  • the dosage of each administration of the exosomes is about 1 ⁇ 10 10 to 4 ⁇ 10 13 /kg body weight, preferably about 1 ⁇ 10 11 to 4 ⁇ 10 12 /kg body weight.
  • compositions provided by the present invention are physiologically acceptable and, when administered to humans, generally do not cause allergic reactions, such as gastroenteritis or dizziness or the like.
  • carriers, excipients and diluents may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, Calcium phosphate, calcium silicate, cellulose, methylcellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.
  • fillers, deflocculants, lubricants, humectants, flavoring agents, emulsifiers, preservatives, and the like may also be included.
  • the pharmaceutical preparations may also contain one or more common pharmaceutically acceptable inert carriers, such as preservatives, analgesics, solubilizers, stabilizers, etc. for injection or bases, excipients for topical preparations agents, lubricants or preservatives.
  • inert carriers such as preservatives, analgesics, solubilizers, stabilizers, etc. for injection or bases, excipients for topical preparations agents, lubricants or preservatives.
  • compositions or pharmaceutical formulations of the present disclosure prepared as described above can be administered to mammals, eg, rats, mice, livestock, humans, and the like, by a variety of routes including parenteral and oral routes. Any mode of administration commonly used in the art can be used. For example, administration can be by intracerebroventricular administration or the like.
  • the present invention also provides a method for treating stroke, comprising administering mesenchymal stem cell-derived exosomes to a patient.
  • the patient may be a mammal such as a rat, mouse, livestock, human, and the like. Any mode of administration commonly used in the art can be used. For example, administration can be by intracerebroventricular administration or the like.
  • the mesenchymal stem cells are bone marrow-derived, adipose-derived, umbilical cord blood-derived, tooth-derived or pluripotent stem cell-derived mesenchymal stem cells.
  • the mesenchymal stem cells are mesenchymal stem cells derived from induced pluripotent stem cells.
  • the mesenchymal stem cells are cells that have been passaged for 1-15 times, preferably cells that have been passaged 3-10 times, and preferably cells that have been passaged 5-8 times passaged cells.
  • the exosomes are prepared by an ultrafiltration method, and in the ultrafiltration method, an ultrafiltration membrane with a molecular weight cut-off of about 100 kDa is used to screen the exosomes.
  • the ultrafiltration method further comprises the step of filtration using a filter with a pore size of 4 ⁇ m and/or a filter with a pore size of 0.22 ⁇ m before the ultrafiltration membrane with a molecular weight cut-off of about 100 kDa.
  • the method for treating stroke is administered to a patient about 1-500ug, preferably about 5-200ug, more preferably about 10-100ug of exosomes.
  • 40-5000 ug/kg body weight, preferably about 400-4000 ug/kg body weight of exosomes are administered to the patient.
  • about 1 ⁇ 10 9 to 1 ⁇ 10 12 preferably about 1 ⁇ 10 10 to 1 ⁇ 10 11 exosomes are administered to a patient in the method for treating stroke.
  • the method for treating stroke is administered to a patient about 1 ⁇ 10 10 to 4 ⁇ 10 13 /kg body weight, preferably about 1 ⁇ 10 11 to 4 ⁇ 10 12 /kg Body weight exosomes.
  • the exosomes are administered to a patient one or more times.
  • the exosomes are administered to the patient at intervals of about 1 day to 10 days, preferably, the exosomes are administered to the patient at intervals of about 5 to 7 days, more preferably at intervals of 7 days given to the patient.
  • the dosage of each administration of the exosomes is about 1-500ug, preferably About 5-200ug, more preferably about 10-100ug.
  • the dosage of each administration of the exosomes is about 40- 5000ug/kg body weight, preferably about 400-4000ug/kg body weight.
  • the dosage of the exosomes per administration is about 1 ⁇ 10 9 to 1 ⁇ 10 12 , preferably about 1 ⁇ 10 10 to 1 ⁇ 10 11 .
  • the dosage for each administration is about 1 ⁇ 10 10 to 4 ⁇ 10 13 pieces/kg body weight, preferably about 1 ⁇ 10 11 to 4 ⁇ 10 12 pieces/kg body weight.
  • the method for treating stroke is a method for treating hemorrhagic stroke and ischemic stroke, preferably a method for treating ischemic stroke.
  • Figure 1 shows the preparation of iPSCs.
  • Figure 1A shows the naive cells used to prepare induced pluripotent stem cells (iPSCs) - human neonatal dermal fibroblasts. Scale bar, 100 ⁇ m.
  • Figure 1B shows iPSCs obtained after reprogramming.
  • Figure 2 shows the identification of mesenchymal stem cells obtained by differentiating iPSCs using an exemplary embodiment of the method of the present invention.
  • Differentiated mesenchymal stem cells were detected by flow cytometry, more than 95% of cells expressed mesenchymal stem cell positive markers (CD73, CD90 and CD105), and less than 1% of cells detected negative markers (CD45/CD34/ CD11b/CD19/HLA-DR PE).
  • Fig. 3 shows the morphology of mesenchymal stem cells from different sources (induced pluripotent stem cell-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and adipose-derived mesenchymal stem cells).
  • Figure 4 shows the identification and comparison of exosomes isolated from mesenchymal stem cells at different passages.
  • Figure 4(A) shows the identification of the expression of exosome surface markers CD9, CD63 and CD81 by western blotting.
  • Figure 4(B) shows transmission electron microscopy images showing exemplary photographs of the morphology of exosomes isolated from mesenchymal stem cells at different passages.
  • FIG. 5 shows the results of an oxygen glucose deprivation/reoxygenation (OGD/R) experiment. It is used to detect the activity of exosomes isolated from mesenchymal stem cells of different passages.
  • CRL control group, normal growing cells without OGD treatment;
  • OGD-2hr-FBS normal medium after OGD treatment for 2 hours, adding 10% FBS (positive control after OGD treatment);
  • OGD-2hr-PBS OGD After 2 hours of treatment, normal medium was restored, and 10% volume of PBS was added (negative control after OGD treatment);
  • OGD-2hr-EV normal medium was restored after 2 hours of treatment, and 10% volume of exosomal EV was added.
  • Figure 6 shows the expression of protein markers CD9, CD63 and CD81 detected by Western blotting on exosomes of iPS-MSCs (P8 passage) prepared by PEG precipitation, ultracentrifugation and ultrafiltration, respectively.
  • Figure 7 shows that the positive marker CD63 was detected by flow cytometry on exosomes of iPS-MSCs (P8 passage) prepared by PEG precipitation method, ultracentrifugation method and ultrafiltration method, respectively.
  • FIG. 8 shows the results of oxygen glucose deprivation/reoxygenation (OGD/R) experiments. It is used to detect the activity of exosomes prepared by PEG precipitation method, ultracentrifugation method and ultrafiltration method respectively.
  • PBS group 10% volume PBS was added after OGD treatment as a negative control.
  • FBS control group normal medium was restored after OGD treatment, and 10% FBS was added (positive control after OGD treatment).
  • the experimental groups of each preparation method After OGD treatment for 2 hours, the normal medium was restored, and 10% volume of exosomal EV was added.
  • Figure 9 shows the results of an oxygen glucose deprivation/reoxygenation (OGD/R) experiment. It is used to detect the activity of exosomes isolated from different sources of mesenchymal stem cells (induced pluripotent stem cell-derived mesenchymal stem cells (P8 passage), bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells).
  • OGD/R oxygen glucose deprivation/reoxygenation
  • CRL control group, normal growing cells without OGD treatment
  • OGD-2hr-FBS normal medium after OGD treatment for 2 hours, adding 10% FBS (positive control after OGD treatment)
  • OGD-2hr-PBS OGD After 2 hours of treatment, normal medium was restored, and 10% volume of PBS was added (negative control after OGD treatment);
  • OGD-2hr-EV normal medium was restored after 2 hours of treatment, and 10% volume of exosomal EV was added.
  • Figure 10 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen is divided into single-injection EV and multiple-injection EV.
  • Treatment regimen 1 (A1): 30ug EV (tail vein injection of exosomes on day 1).
  • Treatment regimen 2 (A2): 10ug x3 EV (exosomes were injected via tail vein on days 1, 3, and 5, respectively). On days 0, 3, 14, and 28, their behaviors were scored with the Modified Neurological Deficiency Score (mNSS) system.
  • mNSS Modified Neurological Deficiency Score
  • Figure 11 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen is divided into single-injection EV and multiple-injection EV.
  • Treatment regimen 1 (A1): 30ug EV (tail vein injection of exosomes on day 1).
  • Treatment regimen 2 (A2): 10ug x3 EV (exosomes were injected via tail vein on days 1, 3, and 5, respectively).
  • the expression of vascular endothelial growth factor VEGF was detected by immunohistochemical staining.
  • the left panel of Figure 11 is a typical staining pattern of each treatment group.
  • the right panel of Fig. 11 is a statistical graph of the number of VEGF-positive cells in each treatment group.
  • Figure 12 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen was divided into multiple injections of EV on different intervals.
  • Treatment regimen 1 (A1): 10ug x 3 EVs (exosomes injected via tail vein on days 1, 3, and 5).
  • Treatment regimen 2 (A2): 10ug x 3 EVs (tail vein injection on days 0, 7, and 14).
  • mNSS Modified Neurological Deficiency Score
  • Figure 13 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen was divided into multiple injections of EV on different intervals.
  • Treatment regimen 1 (A1): 10ug x 3 EVs (exosomes injected via tail vein on days 1, 3, and 5).
  • Treatment regimen 2 (A2): 10ug x 3 EVs (tail vein injection on days 0, 7, and 14).
  • the expression of vascular endothelial growth factor VEGF was detected by immunohistochemical staining.
  • the left panel of Figure 13 is a typical staining pattern of each treatment group.
  • the right panel of Fig. 14 is a statistical graph of the number of VEGF-positive cells in each treatment group.
  • Figure 14 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen is divided into multiple injections of low-dose or high-dose EV.
  • Treatment plan 1 (A1): 10ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21).
  • Treatment plan 2 (A2): 100ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21).
  • mNSS Modified Neurological Deficiency Score
  • Figure 15 shows the effect of administering exosomes to mice in a mouse arterial occlusion model.
  • the dosing regimen is divided into multiple injections of low-dose or high-dose EV.
  • Treatment plan 1 (A1): 10ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21).
  • Treatment plan 2 (A2): 100ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21).
  • the expression of vascular endothelial growth factor VEGF was detected by immunohistochemical staining.
  • the left panel of Figure 15 is a typical staining pattern of each treatment group.
  • the right panel of Fig. 15 is a statistical graph of the number of VEGF-positive cells in each treatment group.
  • Figure 16 shows the results of H&E staining to assess potential lesions or tumorigenesis in mice following exosome treatment.
  • A Brain;
  • B Heart;
  • C Lung;
  • D Liver;
  • E Kidney.
  • the left side of each figure shows tissue (or a magnification including a portion thereof) of an EV-injected individual, and the right side shows tissue (or a magnification of a portion including a portion thereof) of a PBS-injected individual.
  • Human neonatal dermal fibroblasts (ATCC, PCS-201-010 TM ) were used as starting cells for the preparation of iPSCs.
  • the medium for culturing fibroblasts was DMEM (GIBCO, 11965-092).
  • the training process mainly includes:
  • 106 fibroblasts were transfected with episomal plasmids (Addgene, 27077, 27078, 27080 and 27082) by NEPA21 electrotransfection instrument (Nepa Gene). Transfected cells were seeded into Matrigel (CORNING, 354277)-coated 4-well plates.
  • the obtained induced pluripotent stem cells can be used directly for further experiments or cryopreserved in liquid nitrogen.
  • Example 2 Take out the cryopreserved human iPSCs prepared in Example 1 from liquid nitrogen, place them in a 37°C water bath to thaw quickly, and transfer the cryopreserved solution containing cells to a medium containing 2ml of mTeSR1 medium (STEMCELL TECHNOLOGIES, 85850). In a centrifuge tube, centrifuge at 300 ⁇ g for 3 min, remove the supernatant, add new mTeSR1 to resuspend the cells, and transfer to a coated six-well plate.
  • iPSCs were differentiated into mesenchymal stem cells using a commercially available mesenchymal stem cell differentiation kit (STEMCELL TECHNOLOGIES, 05240).
  • the main steps include
  • the iPSCs prepared in Example 2 were seeded in a 12-well plate, and cultured in 1 ml of mTeSR1 or TeSR-E8 medium.
  • mesenchymal stem cell identification kit (BD Stemflow, 562245).
  • the prepared mesenchymal stem cells were identified by detecting the specific surface antigen profile of the mesenchymal stem cells by flow cytometry (FACS). As shown in Figure 2, the results show that more than 95% of the prepared cells have the positive expression markers (CD73, CD90 and CD105) of mesenchymal stem cells defined by ISCT (Mesenchymal and Tissue Stem Cell Committee of the International Society); Meanwhile, negative expression markers (CD45/CD34/CD11b/CD19/HLA-DR PE) were detected in less than 1% of cells.
  • FACS flow cytometry
  • the adult bone marrow fluid samples from healthy people were mixed with the culture medium to directly plate and adhere to the primary culture.
  • the collected bone marrow was resuspended in 10ml of complete medium, then inoculated into a 10cm cell culture dish, and cultured in an incubator at 37°C, 5% CO 2 and saturated humidity. After 3 days, half of the medium was changed, and after 7 days, the full amount of medium was changed, the original medium was discarded, and the cells were gently washed with sterile PBS. . Change the fluid every other day. After about 2 weeks, passage was performed to obtain mesenchymal stem cells.
  • Adipose-derived mesenchymal stem cells were isolated from adult adipose tissue of healthy people by enzymatic digestion, and primary culture was carried out by adherence method.
  • adipose tissue obtained by liposuction, digest it with 0.075% collagenase for 30 minutes, filter the digested liquid through a 200-mesh cell screen, and wash the filtered cells twice with culture medium at 1000 rpm at room temperature. Centrifuge for 10 minutes, collect the cells, resuspend the centrifuged cells in complete medium, inoculate them into culture flasks at a density of 2x104/cm2, and place them in an incubator at 37°C, 5% CO2, and saturated humidity conditions. Cultivated below.
  • Figure 3 shows the morphology of MSC cells (induced pluripotent stem cell-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells) prepared according to Example 3, Example 4 and Example 5 .
  • MSC cells induced pluripotent stem cell-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells
  • the EVs produced by MSCs were identified and observed, including the use of electron microscopy and the observation of EV morphology and size, and the use of Western blotting technology to identify the expression of EV surface markers CD9, CD63, and CD81.
  • Exosomes were extracted from the iPSC-derived MSCs obtained in Example 3 by the ultrafiltration method described in Example 8, and EVs produced by different generations of MSCs were identified.
  • Figure 4A the expression of EV surface markers CD9, CD63, and CD81 was first identified by western blotting. It was found that exosomes were prepared in different passages of MSCs, and the expression of surface markers was higher in EVs produced in the earlier passages P5 and P8 cells, while the 10 passages later (P13) The expression of surface markers of EVs generated by MSCs was lower.
  • EVs collected at passages P5 and P8 were uniform in size, and the average diameter of the particles was less than 100 nm.
  • the EVs collected from the P13 generation had lower size uniformity and a larger average diameter than the EVs collected from the P5 and P8 generations.
  • Oxygen-glucose deprivation/reoxygenation (OGD/R) experiments were performed on EVs generated from MSCs of different passages.
  • the mouse hippocampal neuron cell line HT22 in the OGD/R experiment was treated with exosome groups prepared at different passages.
  • exosomes in the exosome-PBS solution were quantified using the Pierce TM BCA Protein Assay Kit.
  • Particle counters such as NanoSight Nanoparticle Tracking Analyzer can be used for detection and calculation.
  • exosomal protein is equivalent to about 2 ⁇ 10 9 exosome particles.
  • Oxygen-glucose deprivation/reoxygenation (OGD/R) experiment was performed using OGD/R
  • the culture medium is removed and washed twice with 2 ml of PBS. Add an appropriate amount of trypsin to digest the cells, collect the digested cells, count the cells, and finally put them in a 96-well plate, with 2x10 3 cells per well, and culture them overnight. The next day, the complete medium was removed, washed twice with PBS, and replaced with DMEM glucose free medium.
  • the Billups-Rothenberg hypoxia chamber was pre-inflated with a mixed gas (95%N 2 +5%CO 2 ), the pressure of the pressure reducing valve was adjusted to 0.15MPa, and the mixed gas was inflated into the hypoxia chamber at a rate of 40L/min for 10min, that is, The gas in the chamber can be completely replaced with the mixed gas.
  • the chamber is clamped and placed in a CO 2 incubator, that is, the cells are treated with hypoxia and hypoglycemia. After 2 hours, the chamber was opened, the cultured cells were taken out, and the normal culture medium DMEM High Glucose was replaced.
  • the positive control group was added with 10% volume of FBS and 1% penicillin/streptomycin; the treatment group was added with the same volume of exosomes after OGD treatment; the negative control group was added with the same volume of PBS.
  • MTS (20ul/well/96-well plate) to detect cell proliferation, and detect the absorption value of each well at 490nm.
  • the final concentration of exosomes produced by adding MSCs of different generations was 100 ug/mL.
  • the results are shown in Figure 5.
  • the CRL group was a normal growing cell, and the whole experiment was under normal culture conditions, so the cell state was the best. Cells treated with OGD will show different degrees of apoptosis.
  • the exosomes prepared with different passages have increased cell proliferation, which can significantly promote the proliferation of damaged neuronal cells. effect.
  • EVs prepared at P5 and P8 had a more pronounced effect on promoting the proliferation of damaged neuronal cells than EVs prepared at P13.
  • the results showed that the properties of EVs extracted from the culture medium of early-stage MSCs and their repairing effects on damaged neuronal cells were significantly better than those of late-stage MSCs.
  • EVs obtained using different preparation methods were identified. As shown in Figure 6, the EVs of iPS-MSCs (P8 generation) prepared by PEG precipitation, ultracentrifugation and ultrafiltration in Example 6, Example 7 and Example 8 were detected for their protein markers. The expressions of CD9, CD63, CD81, and protein markers of EV prepared by ultrafiltration were significantly higher than those by ultracentrifugation and precipitation.
  • the resulting EVs were subjected to FACS analysis (using CD9 antibody-coated beads and PE fluorescently labeled CD63 antibody conjugates that capture the CD9-expressing EV population).
  • FACS analysis using CD9 antibody-coated beads and PE fluorescently labeled CD63 antibody conjugates that capture the CD9-expressing EV population.
  • the results showed that the proportions of CD63 expressed in the products separated by ultrafiltration, ultracentrifugation and PEG precipitation were 95.7%, 76.2% and 81.6%, respectively.
  • the average diameter of the particles purified by ultrafiltration, ultracentrifugation and PEG precipitation were all less than about 100 nm.
  • the EVs prepared by the above different methods were further subjected to the oxygen glucose deprivation/reoxygenation (OGD/R) experiment as described in Example 9.
  • the final concentration of added exosomes was 100 ug/mL.
  • PBS group negative control group
  • the cell proliferation in the exosome groups prepared by different methods was significantly increased.
  • EV prepared by ultrafiltration method has significantly higher effect on promoting proliferation of damaged neuronal cells than PEG precipitation method and ultracentrifugation method.
  • Example 9 The effect of EVs produced by mesenchymal stem cells from different sources on cell proliferation in mouse neuronal cells after oxygen-glucose deprivation/reoxygenation and reglycemia (OGD/R) experiments
  • Oxygen-glucose deprivation/reoxygenation (OGD/R) experiments were performed using the exosomes prepared by the ultrafiltration method described in Example 9. The final concentration of added exosomes was 100 ug/mL. The results are shown in Figure 9.
  • the mouse middle cerebral artery occlusion (cerebral ischemia-reperfusion) model namely the MCAO model, is used to simulate the human cerebral ischemia model.
  • the mNSS scoring system evaluates the postoperative recovery status of cerebral ischemia model mice, and detects the expression of vascular endothelial growth factor VEGF by immunohistochemical staining, and observes that the exosomes provided by the present invention can promote the growth of vascular endothelial cells. Proliferation and angiogenesis improve the situation of blood supply to ischemic tissue.
  • Exosome sample use the exosomes derived from iPS-MSC (P8 generation) prepared by ultrafiltration method obtained in Example 8, and use Pierce TM BCA Protein Assay Kit to exosome-PBS solution. Quantitative determination and formulation to specified concentrations are performed.
  • mice MCAO cerebrospinal ischemic artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass artery bypass a vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena vena artery artery artery artery artery artery artery
  • the common carotid artery, external carotid artery and internal carotid artery were ligated in sequence.
  • the upper part of the ligation of the external carotid artery was cauterized with a coagulation pen, and the incision was made below the external carotid artery with microscissors.
  • Insert a pre-prepared suture (Guangzhou Jialing Biotechnology Co., Ltd.: product model L1800) from the notch of the external carotid artery, and adjust the insertion angle of the suture so that it is in a straight line with the internal carotid artery.
  • the ligature of the internal carotid artery Untie the ligature of the internal carotid artery, and slowly insert the suture into the skull along the internal carotid artery until a slight resistance is felt to stop the thread. At this time, the insertion depth of the suture is about 0.8-1.0 cm (distance from the bifurcation). Secure the external carotid artery with a ligature. After 45 minutes of ischemia, the suture was slowly withdrawn. Restoration of internal carotid artery blood flow begins reperfusion. Mice in the sham-operated group underwent the same surgical procedure, but no suture was inserted. All animal models were completed by one person to reduce experimental error. After the operation, the mice were housed in single cages with free food and water. And on the 0th, 3rd, 14th, 28th day, the modified neurological deficit score (mNSS) system was used to score the behavior, and the observation end point was 28th day.
  • mNSS modified neurological deficit score
  • Immunohistochemistry was used to detect the changes of vascular endothelial growth factor A (VEGFA) in the penumbra of cerebral infarction (hippocampus) after MCAO model.
  • VEGFA vascular endothelial growth factor A
  • Paraffin sections were deparaffinized and rehydrated with xylene, alcohol (100%, 95%, 80%, 75%), sodium citrate antigen retrieval (boiled for 30 minutes) and endogenous peroxidase (VECTOR, SP) -6000) after blocking, stained with VECTASTAIN Elite ABC kit (VECTOR, PK-6100). After blocking at room temperature for 30 min, the blocking solution was removed by suction, VEGFA antibody (Abeam, ab51745) was added dropwise, and the cells were incubated at 4°C overnight.
  • VEGFA antibody Abeam, ab51745
  • the biotinylated antibody and secondary antibody were then incubated sequentially according to the kit instructions. After cleaning, pre-prepared DAB was added dropwise for color development for 5 minutes, hematoxylin counterstaining for 4 minutes, hydrochloric acid-ethanol differentiation for 30 seconds, alcohol dehydration (concentrations were 75%, 80%, 95%, 100% in sequence) and xylene. Use neutral gum to seal the film after transparency. The expression of VEGF was detected by immunohistochemistry. To evaluate the recovery status of the MCAO/R model after different EV treatment regimens.
  • Treatment 1 30ug EV (injection of exosomes via tail vein on day 1)
  • Treatment regimen 2 (A2): 10ug x 3 EVs (exosomes injected via tail vein on days 1, 3, and 5).
  • mNSS Modified Neurological Deficiency Score
  • the results of immunohistochemical staining showed that the expression of VEGF was significantly increased in mice treated with injection every other day and every week, proving that it promotes the proliferation and angiogenesis of vascular endothelial cells and effectively improves ischemia tissue blood supply. Moreover, the results showed that the average expression of VEGF in mice treated with weekly injection regimen was higher than that in mice treated with every other day injection regimen.
  • Treatment plan 1 10ug x 3 EVs (injection of exosomes via tail vein on days 1, 3, and 5)
  • Treatment regimen 2 (A2): 10ug x 3 EVs (tail vein injection on days 0, 7, and 14).
  • mNSS Modified Neurological Deficiency Score
  • the results of immunohistochemical staining showed that the expression of VEGF was significantly increased in mice treated with alternate days and weekly injections, proving that it promotes the proliferation and angiogenesis of vascular endothelial cells, and effectively improves the deficiency of vascular endothelial cells. Blood supply to blood tissue. Moreover, the results showed that the average expression of VEGF in mice treated with weekly injection regimen was higher than that in mice treated with every other day injection regimen.
  • Treatment plan 1 10ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21)
  • Treatment plan 2 100ug EV x4 (injection of exosomes via tail vein on days 0, 7, 14, and 21).
  • the results of immunohistochemical staining showed that the expression of VEGF was significantly increased in mice injected with different doses of exosomes per week, proving that it promotes the proliferation and angiogenesis of vascular endothelial cells and effectively improves the Blood supply to ischemic tissue. Moreover, the results showed that the average expression of VEGF in mice treated with the higher injection dose regimen was higher than that in the mice treated with the lower injection dose regimen.
  • Exosome sample use the exosomes derived from iPS-MSC (P8 generation) prepared by ultrafiltration method obtained in Example 8, and use Pierce TM BCA Protein Assay Kit to exosome-PBS solution. Quantitative determination and formulation to specified concentrations are performed.
  • mice Healthy mice were injected with a solution containing 10 ⁇ g of exosomes to inject PBS as a control.
  • "about” means ⁇ 10%, preferably ⁇ 5%, more preferably ⁇ 2%, such as ⁇ 1%, ⁇ 0.5% or ⁇ 0.1%.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Cell Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Zoology (AREA)
  • Public Health (AREA)
  • Organic Chemistry (AREA)
  • Epidemiology (AREA)
  • Virology (AREA)
  • Urology & Nephrology (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Wood Science & Technology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Vascular Medicine (AREA)
  • Cardiology (AREA)
  • Reproductive Health (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Microbiology (AREA)
  • Gynecology & Obstetrics (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

提供了一种用于治疗脑卒中的药物组合物，其含有间充质干细胞来源的外泌体。还提供了用间充质干细胞来源的外泌体治疗脑卒中的方法。该方法和药物组合物中采用的外泌体通过特定的制备方法和条件从间充质干细胞中获得，具有修复因缺氧缺糖引起的细胞损伤的活性，同时，通过特定的给药剂量和方式，能够有效地治疗缺血性脑卒中，并且不产生细胞损伤的副作用。

Description

间充质干细胞来源的外泌体治疗脑卒中的方法和组合物
本申请为2021年4月16日提交的、申请号为202110411486.8、发明名称为“间充质干细胞来源的外泌体治疗脑卒中的方法和组合物”的分案申请，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及细胞学和药物学领域。具体的，本发明提供了一种采用间充质干细胞来源的外泌体治疗脑卒中的方法和用于治疗脑卒中的药物组合物。
背景技术
脑卒中(stroke)是全球第二大死亡原因和致残的主要原因。缺血性脑卒中约占脑卒中的80％。传统的药物——溶栓剂可在急性脑卒中患者中用于恢复脑部血流，但只能在部分患者中发挥作用，而且必须在中风发作4.5小时内使用。在过去的二十年中对缺血性脑卒中的治疗进行了积极的创新性的研究，其中，基于干细胞的神经修复疗法成为其中一种研究方向。
间充质干细胞是干细胞家族的重要成员。研究发现，间充质干细胞具有强大的增殖能力和多向分化潜能、具有免疫调节功能、不存在免疫排斥的特性和异体移植排异性较轻等特性。天然的间充质干细胞来源于发育早期的中胚层。间充质干细胞可从各种组织分离出来并培养。间充质干细胞还可从万能干细胞分化得到。其中，诱导万能干细胞(induced pluripotent stem cell，iPSC)，也称为诱导多能干细胞或人工万能干细胞，是人工制备的具有胚胎干细胞的干性的细胞。诱导万能干细胞由日本科学家山中伸弥(Shinya Yamanaka)于2006年首次制备成功，是利用病毒载体将四个转录因子(Oct4,Sox2,Klf4和c-Myc)的组合转入分化的体细胞中，使其重编程而得到的类似胚胎干细胞和胚胎APSC多能细胞的一种细胞类型。诱导万能干细胞已广泛在生物技术和医学研究领域得到应用。
细胞外囊泡(extracellular vesicles，EVs)是细胞通讯中的重要介体，并被证明有助于干细胞旁分泌对组织再生的作用。细胞外囊泡是纳米级(50–1000nm)的脂质双层球，包裹着母细胞的成分，例如膜蛋白，RNA，小的非编码RNA等。根据直径，重量梯度，RNA/蛋白质的含量，衍生来源和生物学功能的不同，细胞外囊泡可分为多种类别，其中，外泌体是主要类型，其直径为约30至200nm，通常带有表面标记，例如CD9，CD81和CD63。
然而，无论是干细胞或各种来源的细胞外囊泡在用于治疗时，它们在受损 伤的组织或器官中的作用包括风险都还没有充分地记录或证明。因此，本领域还需要治疗脑卒中特别是缺血性脑卒中的有效和安全的疗法和药物，例如新的基于干细胞或其分泌的外泌体的疗法和药物。
发明内容
本申请提供了新的采用间充质干细胞来源的外泌体治疗脑卒中的疗法和药物组合物。本申请人的发明人首次发现了间充质干细胞来源的外泌体具有修复因缺氧缺糖引起的细胞损伤的活性，能够有效地治疗脑卒中，特别是缺血性脑卒中。本申请人还出乎意料地发现了特定从干细胞培养液中获得外泌体的制备方法和条件，包括干细胞来源、细胞培养方法(如细胞传代次数)等，以及特定的给药剂量和方式等对外泌体治疗脑卒中特别有效，同时没有对受试者产生细胞损伤等的副作用。
具体的，在本发明的其中一个方面，本发明提供了一种用于治疗脑卒中的药物组合物，其含有间充质干细胞来源的外泌体。本发明提供的药物组合物中的外泌体具有修复细胞损伤的活性，特别是修复因缺氧缺糖引起的细胞损伤的活性。
脑卒中(Stroke)，也称为中风，是一种急性脑血液循环障碍性疾病，以猝然昏倒，不省人事，伴发口角歪斜、语言不利而出现半身不遂为主要症状。脑卒中包括出血性脑卒中、缺血性脑卒中、高血压脑病和血管性痴呆。高血压、动脉硬化、脑血管畸形、脑动脉瘤常可导致出血性中风；风湿性心脏病、心房颤动、细菌性心内膜炎等常导致缺血性中风。
在本发明的其中一个方面，所述药物组合物适于用于治疗缺血性脑卒中，包括短暂性脑缺血发作、动脉粥样硬化性血栓性脑梗塞、腔隙性脑梗塞、脑栓塞等。
在本发明中，术语间充质干细胞也被称作多潜能间质细胞，主要是从脂肪或者骨髓中获得，能够分化为中胚层起源的多种细胞，例如骨、脂肪、软骨、肌腱和肌肉等。间充质干细胞可从各种组织分离出来并培养，但它们的能力和细胞表面标记依据其来源而互不相同。间充质干细胞通常由可分化为骨细胞、软骨细胞和肌细胞的细胞界定，并且表达CD73(+)、CD105(+)、CD34(-)和CD45(-)等细胞表面标记。
在本发明的其中一个方面，所述间充质干细胞为骨髓来源、脂肪来源、脐带血来源、牙齿来源或万能干细胞来源的间充质干细胞。更优选的，所述间充质干细胞为骨髓来源、脂肪来源或万能干细胞来源的间充质干细胞。
在本发明中，术语干细胞是指在单细胞水平上具有自我更新和分化产生子代细胞的能力的未分化细胞。干细胞的特征在于：其具有在体外由多种胚层(内胚层、中胚层和外胚层)分化成各种细胞谱系的功能细胞以及移植后产生多种胚层的组织和注入胚泡后基本上有助于所有或大部分组织形成的能力。
在本发明中，术语万能干细胞指能够产生所有的胚胎细胞类型的干细胞。天然的万能干细胞包括胚胎干细胞。诱导万能干细胞(induced pluripotent stem cell，iPSC)，也称为诱导多能干细胞或人工万能干细胞，是人工制备的具有胚胎干细胞的干性的细胞，例如可以利用病毒载体将四个转录因子(Oct4,Sox2,Klf4和c-Myc)的组合转入分化的体细胞中，使其重编程而得到。
在本发明的其中一个方面，所述间充质干细胞为诱导万能干细胞来源的间充质干细胞。在本发明的其中又一个方面，所述间充质干细胞是以真皮成纤维细胞为起源制备诱导万能干细胞，并进一步诱导其分化得到的间充质干细胞。
细胞外囊泡是由细胞分泌的膜囊泡。细胞外囊泡可以具有在约10nm至约5000nm之间的直径(在颗粒不是球体的情况下，指其最大尺寸)。在本发明中，外泌体指的是小的分泌型囊泡，通常具有在约30nm至约180nm之间的直径(在颗粒不是球体的情况下，指其最大尺寸)，例如具有约30nm至约150nm之间的直径。外泌体包含核酸、蛋白或其他生物分子，或在其膜中具有核酸、蛋白或其他生物分子，并且可以在身体或生物系统中的不同位置之间充当载体。
外泌体可以从包括哺乳动物诸如小鼠、大鼠、豚鼠、兔、狗、猫、牛、马、山羊、绵羊、灵长类动物或人类的多种生物来源分离。外泌体可以从生物流体诸如血清、血浆、全血、尿液、唾液、母乳、泪液、汗液、关节液、脑脊液、精液、阴道液、腹水液和羊水分离。外泌体也可以从实验样品诸如从培养的细胞取得的培养基分离。
在本发明的其中一个方面，用于提取外泌体的间充质干细胞为从骨髓细胞、脂肪细胞或诱导万能干细胞等分化形成的间充质干细胞进行传代培养后得到的间充质干细胞。例如，所述间充质干细胞为经1-15次传代的细胞，优选为经3-10次传代的细胞，更优选为经5-8次传代的细胞。在本发明中，用于传代培养的初代细胞，即P0细胞，通常是指将所述间充质干细胞来源细胞在经诱导和分化后最早出现的间充质干细胞，即在细胞种群中出现具有间充质干细胞特性(例如具有间充质干细胞特异性表面标记等)细胞占细胞总数的50％以上，或优选75％以上，或更优选超过90％以上。初代细胞可直接用于传代培养，也可在冷冻保藏后经复苏后用于传代培养。本申请的发明人出乎意料地发现，经过传代培养的间充质干细胞中，较早期代次间充质干细胞(如经10次或10次以下传代的细胞，特别是经8次或8次以下传代的细胞)产生的外泌体对受损神经元细胞的修复活性要明显优于较晚期代次间充质干细胞(如经10次以上传代的细胞)产生的外泌体的活性。
用于本发明的外泌体可以通过本领域已知的各种方法进行。分离外泌体的方法包括超滤法、聚合物沉淀法、尺寸色谱法或超速离心法等。优选的，用于本发明的外泌体可以通过超滤法和聚合物(例如PEG)沉淀法制备得到。
在本发明的其中一个方面，用于本发明的外泌体通过超滤法从细胞培养液 中制备得到。在用于本发明的超滤法中，采用截留分子量为约100kDa的超滤膜对外泌体进行筛选。外泌体存在于未能够通过截留分子量为约100kDa的超滤膜的组分中。
在用于本发明的超滤法中，还可以采用多过滤系统和方法，即通过不同孔径的过滤器逐步过滤。在本发明的其中又一个方面，所述超滤法在截留分子量为约100kDa的超滤膜之前还包括采用4μm孔径的过滤器和/或0.22μm孔径的过滤器进行过滤的步骤。例如，可以通过4μm孔径的细胞过滤器，0.22μm孔径的过滤器和具MWCO(分子量截止值)为100kD的过滤器逐步进行过滤。
更具体的，用于本发明的超滤法可包括以下主要步骤：
a.将间充质干细胞培养24-42小时，收集上清液；
b.低速离心(如2000g)除去细胞和/或细胞碎片；
c.取上清用0.22μm孔径过滤器过滤；
d.将滤液用截留分子量为100kDa的超滤离心管经3000g离心；
e.收集被超滤离心管截留的组分，溶于溶液。
本发明提供的用于预防或治疗脑卒中的药物组合物中含有药学有效量的上述外泌体。所述外泌体可单独或与一种或更多种可药用载体、赋形剂或稀释剂一起包含在所述药物组合物中。药学有效量意指足以预防、改善或治疗脑卒中，特别是缺血性脑卒中的症状的量。
在本发明的其中一个方面，本发明提供的药物组合物中所述外泌体的剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug。在本发明的其中又一个方面，所述外泌体的剂量为约40-5000ug/kg体重，优选为约400-4000ug/kg体重。
在本发明的其中一个方面，本发明提供的药物组合物中所述外泌体的剂量为约1×10 9至1×10 12个，优选为约1×10 10至1×10 11个。在本发明的其中又一个方面，所述外泌体的剂量为约1×10 10至4×10 13个/kg体重，优选为约1×10 11至4×10 12个/kg体重。
此外，本发明提供的药物组合物可制备成适合于根据药学领域中常用的方法施用至患者体内的单位剂量制剂，并且所述制剂含有通过一次或数次施用有效的施用量。本发明提供的药物组合物可为单次或多次给药的剂型。药学有效量可根据病症的严重程度，患者的年龄、体重、健康状况和性别，施用途径，治疗期等适当地改变。
本发明的提供的药物组合物中采用的外泌体特别适合通过多次给药用于治疗。在其中一个方面，本发明提供的药物组合物为多次给药的剂型。在本发明的其中又一个方面，本发明提供的药物组合物为间隔约1天-10天给药的剂型。优选的，所述药物组合物为间隔约5-7天给药的剂型。更优选的，所述药物组合物为间隔7天给药的剂型。
在其中一个方面，本发明提供的药物组合物的剂型中，特别是在所述多次给药的剂型中，所述外泌体的每次给药剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug。在本发明的另一个方面，本发明提供的药物组合物的剂型中，特别是在所述多次给药的剂型中，所述外泌体的每次给药剂量为约40-5000ug/kg体重，优选为约400-4000ug/kg体重。
在其中一个方面，本发明提供的药物组合物的剂型中，特别是在所述多次给药的剂型中，所述外泌体的每次给药剂量为约1×10 9至1×10 12个，优选为约1×10 10至1×10 11个。在本发明的另一个方面，本发明提供的药物组合物的剂型中，特别是在所述多次给药的剂型中，所述外泌体的每次给药剂量为约1×10 10至4×10 13个/kg体重，优选为约1×10 11至4×10 12个/kg体重。
本发明提供的药物组合物是生理学上可接受的，并且当施用于人时，通常不会引起变态反应，例如胃肠病或头晕或类似反应。载体、赋形剂和稀释剂的实例可包括乳糖、右旋糖、蔗糖、山梨糖醇、甘露醇、木糖醇、赤藓糖醇、麦芽糖醇、淀粉、阿拉伯树胶、藻酸盐、明胶、磷酸钙、硅酸钙、纤维素、甲基纤维素、聚乙烯吡咯烷酮、水、羟基苯甲酸甲酯、羟基苯甲酸丙酯、滑石、硬脂酸镁和矿物油。此外，还可包含填充剂、抗絮凝剂、润滑剂、保湿剂、食用香料、乳化剂、防腐剂等。
除活性成分外，药物制剂还可含有一种或更多种可药用常见惰性载体，例如用于注射的防腐剂、止痛剂、增溶剂、稳定剂等或者用于表面制剂的基质、赋形剂、润滑剂或防腐剂等。
如上所述制备的本公开内容的组合物或药物制剂可通过包括肠胃外和经口途径的多种途径施用于哺乳动物，例如大鼠、小鼠、家畜、人等。可使用本领域常用的任何施用方式。例如，施用可以以脑室内给药等方式进行。
本发明还提供了治疗脑卒中的方法，其包括将间充质干细胞来源的外泌体给予患者。患者可以为哺乳动物，例如大鼠、小鼠、家畜、人等。可使用本领域常用的任何施用方式。例如，施用可以以脑室内给药等方式。
在本发明的其中一个方面，所述治疗脑卒中的方法中所述间充质干细胞为骨髓来源、脂肪来源、脐带血来源、牙齿来源或万能干细胞来源的间充质干细胞。
在本发明的其中一个方面，所述治疗脑卒中的方法中所述间充质干细胞为诱导万能干细胞来源的间充质干细胞。
在本发明的其中一个方面，所述治疗脑卒中的方法中所述间充质干细胞为经1-15次传代的细胞，优选为经3-10次传代的细胞，优选为经5-8次传代的细胞。
在本发明的其中一个方面，所述治疗脑卒中的方法中所述外泌体通过超滤法制备得到，所述超滤法中采用截留分子量为约100kDa的超滤膜对外泌体进行 筛选。优选的，所述超滤法在截留分子量为约100kDa的超滤膜之前还包括采用4μm孔径的过滤器和/或0.22μm孔径的过滤器进行过滤的步骤。
在本发明的其中一个方面，所述治疗脑卒中的方法中给予患者约1-500ug，优选为约5-200ug，更优选为约10-100ug的外泌体。在本发明的其中又一个方面，所述治疗脑卒中的方法中给予患者40-5000ug/kg体重，优选为约400-4000ug/kg体重的外泌体。
在本发明的其中一个方面，所述治疗脑卒中的方法中给予患者约1×10 9至1×10 12个，优选为约1×10 10至1×10 11个外泌体。在本发明的其中又一个方面，所述治疗脑卒中的方法中给予患约者1×10 10至4×10 13个/kg体重，优选为约1×10 11至4×10 12个/kg体重的外泌体。
在本发明的其中一个方面，所述治疗脑卒中的方法中将所述外泌体一次或多次给予患者。
在本发明的其中一个方面，所述治疗脑卒中的方法中将所述外泌体间隔约1天-10天给予患者，优选的，间隔约5-7天给予患者，更优选的间隔7天给予患者。
在本发明的其中一个方面，所述治疗脑卒中的方法中，特别是在所述多次给药的治疗方法中，所述外泌体的每次给药剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug。在本发明的另一个方面，本发明提供的所述治疗脑卒中的方法中，特别是在所述多次给药的治疗方法中，所述外泌体的每次给药剂量为约40-5000ug/kg体重，优选为约400-4000ug/kg体重。
在本发明的其中一个方面，所述治疗脑卒中的方法中，所述外泌体的每次给药剂量为约1×10 9至1×10 12个，优选为约1×10 10至1×10 11个。在本发明的另一个方面，本发明提供的所述治疗脑卒中的方法中，特别是在所述多次给药的治疗方法中，每次给药剂量为约1×10 10至4×10 13个/kg体重，优选为约1×10 11至4×10 12个/kg体重。
在本发明的其中一个方面，所述治疗脑卒中的方法为治疗出血性脑卒中和缺血性脑卒中的方法，优选为治疗缺血性脑卒中的方法。
附图说明
图1显示iPSC的制备。图1A显示用于制备诱导万能干细胞(iPSC)的初始细胞-人新生儿真皮成纤维细胞。比例尺，100μm。图1B显示经过重编程后获得的iPSCs。
图2显示采用本发明的方法的示例性实施方式将iPSCs分化得到的间充质干细胞的鉴定。采用流式细胞术检测分化的间充质干细胞，超过95％的细胞表达间充质干细胞阳性标记(CD73，CD90和CD105)，只有不到1％的细胞中检测到阴性标记(CD45/CD34/CD11b/CD19/HLA-DR PE)。
图3显示不同来源的间充质干细胞细胞(诱导万能干细胞来源的间充质干 细胞、骨髓来源间充质干细胞和脂肪来源间充质干细胞)的形态。
图4显示对不同代次的间充质干细胞分离得到的外泌体的鉴定和比较。图4(A)显示利用蛋白质印迹技术对外泌体表面标志物CD9,CD63和CD81的表达进行鉴定。图4(B)显示投射电镜图像，其显示不同代次的间充质干细胞分离得到的外泌体的形态的示例性照片。
图5显示氧糖剥夺/复糖复氧(OGD/R)实验结果。用于检测不同代次的间充质干细胞分离得到的外泌体的活性。CRL:对照组，未经OGD处理正常生长的细胞；OGD-2hr-FBS:OGD处理2小时后恢复正常培养基,加入10％FBS(OGD处理后的阳性对照)；OGD-2hr-PBS:OGD处理2小时后恢复正常培养基,加入10％体积PBS(OGD处理后的阴性对照)；OGD-2hr-EV:处理2小时后恢复正常培养基,加入10％体积外泌体EV。
图6显示对分别采用PEG沉淀法、超速离心法和超滤法制备得到的iPS-MSC(P8代次)的外泌体用蛋白质印迹技术检测蛋白标志物CD9,CD63,CD81的表达。
图7显示对分别采用PEG沉淀法、超速离心法和超滤法制备得到的iPS-MSC(P8代次)的外泌体用流式细胞术检测阳性标记CD63。
图8显示氧糖剥夺/复糖复氧(OGD/R)实验结果。用于检测分别利用PEG沉淀法、超速离心法和超滤法制备得到的外泌体的活性。PBS组：OGD处理后加入10％体积PBS作为阴性对照。FBS对照组:OGD处理后恢复正常培养基，加入10％FBS(OGD处理后的阳性对照)。各制备方法实验组:OGD处理2小时后恢复正常培养基,加入10％体积外泌体EV。
图9显示氧糖剥夺/复糖复氧(OGD/R)实验结果。用于检测不同来源的间充质干细胞(诱导万能干细胞来源的间充质干细胞(P8代次)、骨髓来源间充质干细胞和脂肪来源间充质干细胞)分离得到的外泌体的活性。CRL:对照组，未经OGD处理正常生长的细胞；OGD-2hr-FBS:OGD处理2小时后恢复正常培养基,加入10％FBS(OGD处理后的阳性对照)；OGD-2hr-PBS:OGD处理2小时后恢复正常培养基,加入10％体积PBS(OGD处理后的阴性对照)；OGD-2hr-EV:处理2小时后恢复正常培养基,加入10％体积外泌体EV。
图10显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为单次注射EV和多次注射EV。治疗方案1(A1)：30ug EV(在第1天尾静脉注射外泌体)。治疗方案2(A2)：10ug x3 EV(分别在第1，3，5天尾静脉注射外泌体)。在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分。
图11显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为单次注射EV和多次注射EV。治疗方案1(A1)：30ug EV(在第1天尾静脉注射外泌体)。治疗方案2(A2)：10ug x3 EV(分别在第1，3，5天尾静脉注射外 泌体)。采用免疫组织化学染色法检测血管内皮生成因子VEGF的表达。图11左图为各处理组的典型染色图。图11右图为各处理组中VEGF阳性细胞的数量统计图。
图12显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为不同间隔日期多次注射EV。治疗方案1(A1)：10ug x 3次EV(第1，3，5天尾静脉注射外泌体)。治疗方案2(A2)：10ug x 3次EV(第0，7，14天尾静脉注射)。在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分。
图13显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为不同间隔日期多次注射EV。治疗方案1(A1)：10ug x 3次EV(第1，3，5天尾静脉注射外泌体)。治疗方案2(A2)：10ug x 3次EV(第0，7，14天尾静脉注射)。采用免疫组织化学染色法检测血管内皮生成因子VEGF的表达。图13左图为各处理组的典型染色图。图14右图为各处理组中VEGF阳性细胞的数量统计图。
图14显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为低剂量或高剂量EV的多次注射。治疗方案1(A1)：10ug EV x4(第0,7,14,21天尾静脉注射外泌体)。治疗方案2(A2)：100ug EV x4(第0,7,14,21天尾静脉注射外泌体)。在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分。
图15显示在小鼠动脉阻塞模型中对小鼠给药外泌体的效果。给药方案分为低剂量或高剂量EV的多次注射。治疗方案1(A1)：10ug EV x4(第0,7,14,21天尾静脉注射外泌体)。治疗方案2(A2)：100ug EV x4(第0,7,14,21天尾静脉注射外泌体)。采用免疫组织化学染色法检测血管内皮生成因子VEGF的表达。图15左图为各处理组的典型染色图。图15右图为各处理组中VEGF阳性细胞的数量统计图。
图16显示H&E染色评估外泌体处理后对小鼠的潜在的病变或肿瘤发生的结果。(A)脑；(B)心脏；(C)肺；(D)肝脏；(E)肾脏。其中各个图的左边显示注射EV的个体的组织(或包括其部分放大)，右边显示注射PBS的个体的组织(或包括其部分放大)。各组织中，注射EV的个体(左)与注射PBS的个体(右)的检测无差异。
具体实施方式
下面将结合实施例进一步说明本发明的实质内容和有益效果，该实施例仅用于说明本发明而非对本发明的限制。
实施例1 诱导万能干细胞iPSCs的制备
人新生儿真皮成纤维细胞(ATCC，PCS-201-010 TM)被用来作为制备 iPSCs的初始细胞。培养成纤维细胞的培养基为DMEM(GIBCO，11965-092)。
培养过程主要包括：
第0天，episomal质粒(Addgene，27077、27078、27080和27082)通过NEPA21电转染仪器(Nepa Gene)转染10 6个成纤维细胞。转染的细胞种板到Matrigel(CORNING，354277)包被的4孔板。
第1天，将细胞培养基换成mTeSR1(STEMCELL TECHNOLOGIES，85850)。
然后隔天更换新鲜的mTeSR1培养基，持续到第27-30天左右，出现iPSCs。结果如图1所示。
得到的诱导万能干细胞可直接用于进一步实验或在液氮中冻存。
实施例2 诱导万能干细胞的复苏/培养
以六孔板的一个孔为例。
1.添加1ml Matrigel(CORNING，354277)包被培养板，室温放置半个小时后，去除Matrigel，用PBS清洗一次。
2.把冻存的实施例1制备得到的人iPSCs从液氮中取出，放于37℃水浴中快速解冻，把含有细胞的冻存液转移至含有2ml mTeSR1培养基(STEMCELL TECHNOLOGIES，85850)的离心管中，300×g,3min离心，去除上清液，添加新的mTeSR1重悬细胞，转移至包被好的六孔板中。
3.当细胞长至70％左右，添加1ml中性蛋白酶Dispase(STEMCELL TECHNOLOGIES，07923)消化，细胞培养箱中放置5分钟。
4.吸走Dispase，用PBS清洗一次，添加1ml mTeSR1，将细胞轻柔地刮下来，以最小幅度用枪头轻轻吹打，以适当密度种于Matrigel包被的培养板上，放于37℃细胞培养箱中培养，每天更换新鲜培养基。
实施例3 诱导万能干细胞来源的间充质干细胞的制备
采用商购的间充质干细胞分化试剂盒(STEMCELL TECHNOLOGIES,05240)将iPSCs分化为间充质干细胞。
主要步骤包括
1.将实施例2制备得到的iPSC种于12孔板，1ml mTeSR1或者TeSR-E8培养基培养。
2.第0天，预热STEMdiff-ACF Mesenchymal Induction Medium，将原先培养细胞的mTeSR1或者TeSR-E8替换为1ml STEMdiff-ACF Mesenchymal Induction Medium。第1-3天每天更换新鲜培养液。
3.第4天，预热Mesencult-ACF Plus Medium，去除原先培养细胞的STEMdiff-ACF Mesenchymal Induction Medium，1×PBS清洗一次，添加预热好的Mesencult-ACF Plus Medium。
4.第5天，更换新鲜的Mesencult-ACF Plus Medium。
5.第6天，用Animal Component-free Cell Attachment Substrate包被细胞培养板，预热Mesencult-ACF Plus Medium，并添加ROCK inhibitor Y-27632(Selleckchem，Y-27632)。
6.去除细胞培养基，添加Stempro Accutase(Life Technologies，A1110501)，置于37℃培养箱中，5分钟，将细胞消化成单细胞。
7.终止消化，将细胞收集于15ml离心管中，300×g，5min。用含有Y-27632的Mesencult-ACF Plus Medium重悬细胞，种于包被好的培养板中。每天半量换液，持续3-6天。
8.细胞长至80％密度，进行传代，培养3-6天。
9.在大概21天左右，收集细胞，通过间充质干细胞鉴定试剂盒(BD Stemflow,562245)鉴定证明得到的为间充质干细胞。
通过流式细胞荧光分选技术(FACS)检测间充质干细胞的特定表面抗原谱来鉴定制备得到的间充质干细胞。如图2所示，结果显示制备得到的细胞中有超过95％的细胞具备ISCT(Mesenchymal and Tissue Stem Cell Committee of the International Society)定义的间充质干细胞阳性表达标记(CD73，CD90和CD105)；同时，只有不到1％的细胞中检测到阴性表达标记(CD45/CD34/CD11b/CD19/HLA-DR PE)。
实施例4 骨髓来源间充质干细胞的制备
将健康人的成人骨髓液样本与培养液混合直接铺盘贴壁法作原代培养。
将采集的骨髓重悬于10ml完全培养基,然后接种到10cm细胞培养皿中,放到培养箱内于37℃、5％CO 2、饱和湿度条件下进行培养。3天后半量换液,7天后全量换液，弃去原有的培养基,用无菌的PBS轻轻荡洗，轻轻荡洗,把没有贴壁的细胞除去,换上新鲜的完全培养基。以后每隔一天换液一次。大约2周后进行传代，得到间充质干细胞。
实施例5 脂肪来源间充质干细胞的制备：
将健康人的成人脂肪组织，以酶消化法分离脂肪间充质干细胞，贴壁法做原代培养。
取20ml抽脂术获得的脂肪组织,先用0.075％的胶原酶消化30分钟,消化后的混合液体经200目细胞筛网过滤，过滤后的细胞用培养基洗涤两次,分别在室温下1000rpm离心10分钟，收集细胞，将离心后细胞重悬于完全培养基中,按2x104/cm2,的密度将其接种于培养瓶中,放到培养箱内于37℃、5％CO2、饱和湿度条件下进行培养。3天后半量换液,7天后全量换液,弃去原有的培养基,用 无菌的PBS轻轻荡洗,把没有贴壁的细胞除去,换上新鲜的完全培养基。以后每隔一天换液一次。约2周后进行传代，得到间充质干细胞。
图3显示根据实施例3、实施例4和实施例5制备得到的不同来源的MSC细胞(诱导万能干细胞来源的间充质干细胞、骨髓来源间充质干细胞和脂肪来源间充质干细胞)的形态。
实施例6 外泌体的制备-超速离心法
实验材料：
50ml离心管(Beckman#357003)，超速离心机Beckman Optima(XPN-100)ULTRA-centrifuge
实验步骤：
1)将实施例3制备得到的间充质干细胞进行传代培养，在第8代(P8)细胞密度达到80％～90％时，去培养基，2ml PBS清洗2次。换为无FBS的MSC培养基，培养48小时；2)将收集到的细胞上清液约100mL分装于2个50mL离心管中；3)300g/4℃离心10min除去残余细胞；4)2000g/4℃离心20min除去细胞碎片；5)小心收集上清并用0.22μm孔径过滤器进行过滤；6)继续离心500g,5min；7)2000g/4℃离心30分钟；8)将上清转移至50ml离心管中，配平；9)100000rpm/4℃离心2小时，轻轻去除上清；10)加入200ul PBS溶解管底的沉淀，得到外泌体溶液。
实施例7 外泌体的制备-PEG沉淀法
实验材料：
2xPEG溶液(PEG8000,30％,NaCl 1M)，冷冻离心机(Eppendorf 5804 R)
实验步骤：
1)将实施例3制备得到的间充质干细胞进行传代培养，在第8代(P8)细胞密度达到80％～90％时，去培养基，2ml PBS清洗2次。换为无FBS的MSC培养基，培养48小时；2)将收集到的细胞上清液约100mL分装于2个50mL离心管中；3)300g/4℃离心10min除去残余细胞；4)2000g/4℃离心20min除去细胞碎片；5)小心收集上清并用0.22μm孔径过滤器进行过滤；6)继续离心500g,5min；7)2000g/4℃离心30分钟；8)将上清转移至50ml离心管中，加入等体积2xPEG溶液，颠倒混匀，置于4℃冰箱过夜沉淀；9)3000rpm/4℃离心2小时，轻轻去除上清；10)每管加入100ul PBS溶解管底的沉淀，得到外泌体溶液。
实施例8 外泌体的制备-超滤法
实验材料：冷冻离心机(Eppendorf 5804R)，
Figure PCTCN2022087513-appb-000001
Ultra-15 Centrifugal  Filter Unit(Millipore，100KD#UFC910096)。
实验步骤：
1)将实施例3制备得到的间充质干细胞进行传代培养，分别在第5代(P5)、第8代(P8)和第13代(P13)的间充质干细胞的细胞密度达到80％～90％时，去培养基，2ml PBS清洗2次。换为无FBS的MSC培养基，培养48小时；
2)将收集到的细胞上清液约100mL分装于2个50mL离心管中；
3)300g，4℃离心10min除去残余细胞；
4)2000g离心20min除去细胞碎片；
5)小心收集上清并用0.22μm孔径过滤器进行过滤；
6)将收集到的上清液加入
Figure PCTCN2022087513-appb-000002
Ultra-15中，3000g离心；
7)待上清液超滤完毕后，加入PBS并再次超滤，重复2次洗涤，最终用200μlPBS溶解留着滤膜上的外泌体，得到外泌体溶液；
8)将外泌体溶液转移至1.5mL的Eppendorf管中，-80℃冰箱中保存备用。
对MSC产生的EV进行鉴定和观察，包括利用电镜和对EV形态和尺寸进行观察，以及利用蛋白质印迹技术对EV表面标志物CD9，CD63，CD81的表达进行鉴定。
实施例9 不同代次间充质干细胞产生的外泌体的比较
对实施例3得到的iPSC来源的MSC采用实施例8记载的超滤法提取外泌体(EV)，对不同代次MSC产生的EV进行鉴定。如图4A所示，首先利用蛋白质印迹技术对EV表面标志物CD9,CD63，CD81的表达进行鉴定。结果发现不同代次MSC中都制备得到了外泌体，而在较早期的代次P5和P8细胞产生的EV中表达的表面标志物的量更高，而10代之后的代次(P13)的MSC所产生的EV的表面标志物的表达较低。
同时利用投射电镜技术对EV的形态和尺寸进行了观测。如图4B所示，P5和P8代次所收集的EV尺寸均一，颗粒的平均直径为小于100nm。而P13代次所收集的EV，尺寸均匀度较低，且平均直径比P5和P8代次所收集的EV大。
将不同代次MSC所产生的EV进行氧糖剥夺/复糖复氧(OGD/R)实验。利用不同代次制备的外泌体组对OGD/R实验的小鼠海马神经元细胞系HT22进行处理。
外泌体浓度测定
利用蛋白总量测量试剂盒Pierce TM BCA Protein Assay Kit对外泌体-PBS溶液中的外泌体进行定量测定。
外泌体颗粒个数测定：
可采用NanoSight纳米颗粒跟踪分析仪等颗粒计数器进行检测和计算。
也可按照1μg的外泌体蛋白相当于约2x10 9个外泌体颗粒进行计算。
氧糖剥夺/复糖复氧(OGD/R)实验：
小鼠海马神经元细胞系HT22细胞密度达到80％～90％时，去培养基，2ml PBS清洗2次。加入适量胰酶消化细胞，收集消化后细胞，细胞计数，终于96孔板中，每孔2x10 3细胞，过夜培养。第二天去除完全培养基，PBS清洗2遍，换为DMEM glucose free培养基。Billups-Rothenberg缺氧小室预先使用混合气体(95％N 2+5％CO 2)充气，减压阀压力调整为0.15M Pa，混合气体以40L/min的速度向缺氧小室中充气10min，即可使小室中的气体完全置换为混合气体。
夹闭小室并置入CO 2培养箱，即为细胞缺氧缺糖处理。2h后打开小室，取出培养的细胞，更换为正常培养液DMEM High Glucose。阳性对照组加入10％体积的FBS和1％青霉素/链霉素；处理组为OGD处理后加入同体积的外泌体；阴性对照组加入等体积PBS。继续在CO 2培养箱中复氧培养24h，加入MTS(20ul/每孔/96孔板)检测细胞增殖状况，490nm检测每孔的吸收值。
在本实施例中，加入不同代次MSC所产生的外泌体的终浓度为100ug/mL。结果如图5所示，CRL组是正常生长的细胞，实验全程均在正常培养条件下，因此细胞状态最好。经过OGD处理的细胞均会出现不同程度的凋亡，利用不同代次制备的外泌体组相比加入PBS的OGD处理组,细胞增殖出现升高，对于受损的神经元细胞具有明显促进增殖作用。同时还发现，其中P5和P8制备的EV相对于P13代次制备的EV，对受损的神经元细胞的促进增殖作用更明显。结果表明从早期代次MSC的培养液中提取得到的EV的性质和对于受损神经元细胞的修复作用要明显优于晚期代次MSC的EV。
实施例10 不同制备方法得到的外泌体的比较
对利用不同制备方法得到的EV进行鉴定。如图6所示，对实施例6、实施例7和实施例8分别利用PEG沉淀法、超速离心法和超滤法制备得到的iPS-MSC(P8代次)的EV，检测其蛋白标志物CD9,CD63,CD81,利用超滤法制备EV的蛋白标志物的表达明显高于超速离心法和沉淀法。
另外，如图7所示，对得到的EV进行了FACS分析(采用可捕获表达CD9的EV群体的CD9抗体包被的珠子和PE荧光标记的CD63抗体缀合物)。结果表明，通过超滤法，超速离心法和PEG沉淀分离的产物中表达CD63的比例分别为95.7％，76.2％和81.6％。
同时利用投射电镜技术对EV的形态和尺寸进行了观测。超滤法、超速离心法和PEG沉淀纯化得到的颗粒的平均直径均为小于约100nm。
结果表明，通过超滤法分离的EV比超速离心法和PEG沉淀分离的EV具有更优的性质。
进一步将上述不同方法制备得到的EV进行如实施例9描述的氧糖剥夺/复糖复氧(OGD/R)实验。加入的外泌体的终浓度为100ug/mL。如图8所示，利用不同方法制备的外泌体组相比OGD处理后的负对照组(PBS组),细胞增殖都出现明显升高。其中超滤法制备的EV相对PEG沉淀法、超速离心法对于 受损的神经元细胞的促进增殖作用显著较高。
实施例9 不同来源间充质干细胞产生的EV对小鼠神经元细胞氧糖剥夺/复氧复糖(OGD/R)实验后细胞增殖影响
对实施例3、实施例4和实施例5制备得到的不同来源的MSC(诱导万能干细胞来源的间充质干细胞(P8代次)、骨髓来源间充质干细胞和脂肪来源间充质干细胞)分别采用实施例9描述的超滤法制备得到的外泌体进行氧糖剥夺/复糖复氧(OGD/R)实验。加入的外泌体的终浓度为100ug/mL。结果如图9所示，不同来源外泌体组(100ug)相比OGD处理后的负对照组(PBS组)，细胞增殖出现明显上升，说明不同来源MSC产生的EV对于神经元细胞OGD/R后的损伤具有修复作用。
实施例10 动物实验-小鼠动脉阻塞模型(MCAO模型)
小鼠大脑中动脉阻塞(脑缺血-再灌注)模型，即MCAO模型用于模拟人脑缺血模型，通过使用本发明提供的外泌体的不同治疗方案对MCAO小鼠进行治疗，并通过mNSS评分系统对脑缺血模型小鼠术后的恢复状况进行评估，以及通过免疫组织化学染色方法对血管内皮生成因子VEGF的表达进行检测，观察本发明提供的外泌体对促进血管内皮细胞的增殖和血管生成，改善缺血组织的血液供应的情况。
外泌体样品：采用实施例8得到的采用超滤法制备得到的iPS-MSC(P8代次)来源的外泌体，利用Pierce TM BCA Protein Assay Kit对外泌体-PBS溶液中的外泌体进行定量测定和调配到指定浓度。
小鼠MCAO模型制备：通过改良的Longa方法用于建立小鼠MCAO(脑缺血-再灌注)模型。在手术前将小鼠禁食6h，以减少术中返流和误吸等并发症。用水合氯醛麻醉小鼠，待小鼠麻醉后，仰卧在手术台上，固定其四肢。在颈部作一长约1cm的纵行手术切口。依次切开皮肤和筋膜，纵向撕开颈部肌肉暴露颈部血管。小心分离出颈总动脉，颈内动脉和颈外动脉。依次结扎颈总动脉、颈外动脉和颈内动脉。用电凝笔烧断颈外动脉结扎处的上方，并用显微剪在颈外动脉的下方剪口。从颈外动脉的剪口处插入预先准备好的线栓(广州佳灵生物技术有限公司：产品型号L1800)，调节线栓插入角度，使其于颈内动脉成一直线。解开颈内动脉的结扎线，顺着颈内动脉缓慢的向颅内插入线栓，直至感觉到轻微阻力停止进线。此时，线栓的插入深度约为0.8-1.0cm(距离分叉处)。用结扎线固定住颈外动脉。缺血45分钟后，将线栓缓缓撤出。恢复颈内动脉血流开始再灌注。假手术组小鼠进行相同的手术操作，但未插入线栓。所有动物模型均由一人完成，以减少实验误差。术后将小鼠单笼饲养，自由进食和饮水。并在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分，以28天为观测终点。
采用免疫组织化学方法(immunohistochemistry，IHC)进行染色检测MCAO模型后脑梗死半影区(海马区)血管内皮生长因子A(VEGFA)的变化。石蜡切片经二甲苯、酒精(浓度依次为100％、95％、80％、75％)脱蜡复水、柠檬酸钠抗原修复(煮沸30分钟)和内源性过氧化物酶(VECTOR，SP-6000)封闭后，用VECTASTAIN Elite ABC试剂盒(VECTOR，PK-6100)进行染色。室温封闭30min后，吸去封闭液，滴加VEGFA抗体(Abcam，ab51745)，4℃孵育过夜。然后按照试剂盒说明依次孵育生物素化抗体和二抗。清洗后滴加预先配制好的DAB进行显色5min，进行苏木精复染4分钟、盐酸乙醇分化30秒、酒精脱水(浓度依次为75％、80％、95％、100％)和二甲苯透明后使用中性树胶封片。利用免疫组化方法检测VEGF的表达量。用以评估MCAO/R模型经过不同EV治疗方案后的恢复状况。
动物实验1单次注射EV和多次注射EV(注射EV总量相同)的治疗方案的比较。
治疗方案1(A1)：30ug EV(第1天尾静脉注射外泌体)
治疗方案2(A2)：10ug x 3次EV(第1，3，5天尾静脉注射外泌体)。
在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分。
如图10所示，结果表明隔天注射和每周注射方案治疗小鼠评分随时间推移逐渐下降，神经功能逐渐恢复，每周注射方案的平均评分逐渐低于隔天注射方案。
另外，如图11所示，免疫组织化学染色法结果表明VEGF在隔天注射和每周注射方案治疗小鼠中表达量显著上升，证明其促进血管内皮细胞的增殖和血管生成，有效改善缺血组织的血液供应。而且结果显示每周注射方案处理的小鼠VEGF平均表达量高于隔天注射方案处理的小鼠。
动物实验2不同间隔日期多次注射EV的治疗方案的比较。
治疗方案1(A1)：10ug x 3次EV(第1，3，5天尾静脉注射外泌体)
治疗方案2(A2)：10ug x 3次EV(第0，7，14天尾静脉注射)。
在第0，3，14，28天对其行为学进行改良神经功能缺陷评分(mNSS)系统评分。
如图12所示，结果表明隔天注射和每周注射方案治疗小鼠评分随时间推移逐渐下降，神经功能有所恢复，每周注射方案的平均评分逐渐低于隔天注射方案。
另外，如图13所示，免疫组织化学染色法结果表明VEGF在隔天注射和每周注射方案治疗小鼠中小鼠表达量显著上升，证明其促进血管内皮细胞的增殖和血管生成，有效改善缺血组织的血液供应。而且结果显示每周注射方案处理的小鼠VEGF平均表达量高于隔天注射方案处理的小鼠。
动物实验3注射剂量的比较。
治疗方案1(A1)：10ug EV x4(第0,7,14,21天尾静脉注射外泌体)
治疗方案2(A2)：100ug EV x4(第0,7,14,21天尾静脉注射外泌体)。
如图14所示，结果表明每周注射方案不同剂量的外泌体治疗小鼠评分随时间推移逐渐下降，神经功能逐渐恢复。较高注射剂量方案的平均评分明显高于较低注射剂量方案。
另外，如图15所示，免疫组织化学染色法结果表明VEGF在每周注射方案不同剂量的外泌体的小鼠中表达量显著上升，证明其促进血管内皮细胞的增殖和血管生成，有效改善缺血组织的血液供应。而且结果显示较高注射剂量方案处理的小鼠VEGF平均表达量高于较低注射剂量方案处理的小鼠。
实施例11 动物实验-安全性实验
外泌体样品：采用实施例8得到的采用超滤法制备得到的iPS-MSC(P8代次)来源的外泌体，利用Pierce TM BCA Protein Assay Kit对外泌体-PBS溶液中的外泌体进行定量测定和调配到指定浓度。
向健康小鼠注射含10μg外泌体的溶液，以注射PBS作为对照。在注射后第28天收集了五个主要器官，包括脑，心脏，肺，肝，肾，经H&E染色评估潜在的病变或肿瘤发生。结果如图16所示，注射EV的个体(左)与注射PBS的个体(右)的组织检测无差异，在光学显微镜下无明显病变或肿瘤发生。
上面是对本发明进行的说明，不能将其看成是对本发明进行的限制。除非另外指出，本发明的实践将使用有机化学、聚合物化学、生物技术等的常规技术，显然除在上述说明和实施例中所特别描述之外，还可以别的方式实现本发明。其它在本发明范围内的方面与改进将对本发明所属领域的技术人员显而易见。根据本发明的教导，许多改变和变化是可行的，因此其在本发明的范围之内。
在本发明中，“约”表示±10％，优选为±5％，更优选为±2％，例如为±1％、±0.5％或±0.1％。

Claims (20)

  1. 一种用于治疗脑卒中的药物组合物，其含有间充质干细胞来源的外泌体，所述间充质干细胞为经3-10次传代的细胞。
  2. 根据权利要求1所述的药物组合物，其中所述间充质干细胞为骨髓来源、脂肪来源、脐带血来源、牙齿来源或万能干细胞来源的间充质干细胞，优选为诱导万能干细胞来源的间充质干细胞。
  3. 根据权利要求1所述的药物组合物，其中所述间充质干细胞为经5-8次传代的细胞。
  4. 根据权利要求1所述的药物组合物，其中所述外泌体通过超滤法、超速离心法或聚合物(例如PEG)沉淀法制备得到。
  5. 根据权利要求1所述的药物组合物，其中所述外泌体的剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug。
  6. 根据权利要求1所述的药物组合物，其为单次或多次给药的剂型。
  7. 根据权利要求6所述的药物组合物，其为多次给药的剂型。
  8. 根据权利要求7所述的药物组合物，其为间隔约1天-10天给药的剂型，优选的，其为间隔约5-7天给药的剂型，更优选的为间隔7天给药的剂型。
  9. 根据权利要求6-8中任一项所述的药物组合物，其中每次给药剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug，
    例如为约40-5000ug/kg体重，优选为约400-4000ug/kg体重。
  10. 根据权利要求1所述的药物组合物，其中所述脑卒中为出血性脑卒中和缺血性脑卒中，优选为缺血性脑卒中。
  11. 治疗脑卒中的方法，其包括将间充质干细胞来源的外泌体给予患者，所述间充质干细胞为经3-10次传代的细胞。
  12. 根据权利要求11所述的方法，其中所述间充质干细胞为骨髓来源、脂肪来源、脐带血来源、牙齿来源或万能干细胞来源的间充质干细胞，优选为诱导万能干细胞来源的间充质干细胞。
  13. 根据权利要求11所述的方法，其中所述间充质干细胞为经5-8次传代的细胞。
  14. 根据权利要求11所述的方法，其中所述外泌体通过超滤法、超速离心法或聚合物(例如PEG)沉淀法制备得到。
  15. 根据权利要求11所述的方法，其中给予患者约1-500ug，优选为约5-200ug，更优选为约10-100ug的外泌体。
  16. 根据权利要求11所述的方法，其中将所述外泌体单次给予患者。
  17. 根据权利要求11所述的方法，其中将所述外泌体多次给予患者。
  18. 根据权利要求17所述的方法，其中将所述外泌体间隔约1天-10天给予患者，优选的，间隔约5-7天给予患者，更优选的间隔7天给予患者。
  19. 根据权利要求16-18中任一项所述的方法，其中每次给药剂量为约1-500ug，优选为约5-200ug，更优选为约10-100ug，
    例如，其中每次给药剂量为约40-5000ug/kg体重，优选为约400-4000ug/kg体重。
  20. 根据权利要求11所述的方法，其中所述脑卒中为出血性脑卒中和缺血性脑卒中，优选为缺血性脑卒中。
PCT/CN2022/087513 2021-04-16 2022-04-18 间充质干细胞来源的外泌体治疗脑卒中的方法和组合物 WO2022218443A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110411486 2021-04-16
CN202110411486.8 2021-04-16

Publications (1)

Publication Number Publication Date
WO2022218443A1 true WO2022218443A1 (zh) 2022-10-20

Family

ID=83606111

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/087513 WO2022218443A1 (zh) 2021-04-16 2022-04-18 间充质干细胞来源的外泌体治疗脑卒中的方法和组合物

Country Status (2)

Country Link
CN (1) CN115212232A (zh)
WO (1) WO2022218443A1 (zh)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116999465A (zh) * 2023-09-19 2023-11-07 河北中琨生物工程有限责任公司 脐带间充质干细胞来源的外泌体在制备预防和/或治疗缺血性脑卒中药物中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160310534A1 (en) * 2013-12-12 2016-10-27 Samsung Life Public Welfare Foundation Pharmaceutical composition for treating cerebrovascular diseases, containing stem cell-derived exosome as active ingredient
CN108883138A (zh) * 2015-12-30 2018-11-23 加利福利亚大学董事会 增强细胞衍生的囊泡的生产和分离的方法
CN110755450A (zh) * 2019-11-12 2020-02-07 山东大学齐鲁医院 间充质干细胞来源的细胞外囊泡在治疗蛛网膜下腔出血中的应用
CN111647554A (zh) * 2019-05-27 2020-09-11 广州达康基因技术有限公司 从脐带间充质干细胞制备的外泌体制剂及其方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160310534A1 (en) * 2013-12-12 2016-10-27 Samsung Life Public Welfare Foundation Pharmaceutical composition for treating cerebrovascular diseases, containing stem cell-derived exosome as active ingredient
CN108883138A (zh) * 2015-12-30 2018-11-23 加利福利亚大学董事会 增强细胞衍生的囊泡的生产和分离的方法
CN111647554A (zh) * 2019-05-27 2020-09-11 广州达康基因技术有限公司 从脐带间充质干细胞制备的外泌体制剂及其方法
CN110755450A (zh) * 2019-11-12 2020-02-07 山东大学齐鲁医院 间充质干细胞来源的细胞外囊泡在治疗蛛网膜下腔出血中的应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HONGQI XIN, YI LI, YISHENG CUI, JAMES J YANG, ZHENG GANG ZHANG, MICHAEL CHOPP: "Systemic administration of exosomes released from mesenchymal stromal cells promote functional recovery and neurovascular plasticity after stroke in rats", JOURNAL OF CEREBRAL BLOOD FLOW & METABOLISM, vol. 33, no. 11, 1 November 2013 (2013-11-01), Nature Publishing Group, US, pages 1711 - 1715, XP055226654, ISSN: 0271-678X, DOI: 10.1038/jcbfm.2013.152 *
XIA YUGUO, LING XIAOZHENG, HU GUOWEN, ZHU QINGWEI, ZHANG JUNTAO, LI QING, ZHAO BIZENG, WANG YANG, DENG ZHIFENG: "Small extracellular vesicles secreted by human iPSC-derived MSC enhance angiogenesis through inhibiting STAT3-dependent autophagy in ischemic stroke", STEM CELL RESEARCH & THERAPY, vol. 11, no. 1, 1 December 2020 (2020-12-01), XP055977358, DOI: 10.1186/s13287-020-01834-0 *
ZHANG SHANSHAN, WANG RUI, WANG YIFEI, FU JIN: "Research progress of exosomes and ischemic stroke", ZHONGFENG YU SHENJING JIBING ZAZHI = JOURNAL OF APOPLEXY AND NERVOUS DISEASES, JILIN DAXUE BAIQIUEN DI-1 YIYUAN ZHUANHUA YIXUE YANJIUYUAN, CN, vol. 36, no. 2, 28 February 2019 (2019-02-28), CN , pages 187 - 189, XP055977816, ISSN: 1003-2754 *

Also Published As

Publication number Publication date
CN115212232A (zh) 2022-10-21

Similar Documents

Publication Publication Date Title
JP7441079B2 (ja) 神経変性を治療するための方法及び組成物
JP6105846B2 (ja) 虚血組織の細胞療法
JP2009500297A (ja) 眼の変性についての細胞療法
US20110158969A1 (en) Compositions and methods for using stromal cells to enhance treatment of central nervous system injuries
JPWO2008018450A1 (ja) 脂肪組織由来多分化能幹細胞を含有する細胞製剤
CN111182890A (zh) 用于治疗大疱性表皮松解症的方法和组合物
US20180104186A1 (en) Methods and compositions for the treatment of epidermolysis bullosa
CN109893541B (zh) 经血干细胞来源的外泌体在制备治疗宫腔粘连的药物中的应用
US8318485B2 (en) Stem cell therapy for the treatment of diabetic retinopathy and diabetic optic neuropathy
CN113712995A (zh) 神经干细胞联合脐带间充质干细胞在脊髓损伤中的应用
KR101357851B1 (ko) 낭성구조물로부터 망막색소상피세포의 분화를 유도하는 방법
WO2022218443A1 (zh) 间充质干细胞来源的外泌体治疗脑卒中的方法和组合物
US20180280446A1 (en) Agent for Treating Urinary Incontinence Including Stem Cells Derived from Amniotic Fluid
Liang et al. Intravenous infusion of small umbilical cord mesenchymal stem cells could enhance safety and delay retinal degeneration in RCS rats
WO2017147941A1 (zh) 重组间充质干细胞及其制备方法和应用
US20110274670A1 (en) Mesenchymal stem cells which express human hepatic growth factor,manufacturing method thereof, and use thereof as therapeutic agent for liver diseases
WO2012115298A1 (ko) 양수 유래 줄기세포를 함유하는 요실금 치료제
Peng et al. Hyperbaric oxygen therapy combined with Schwann cell transplantation promotes spinal cord injury recovery
KR101423659B1 (ko) 초기 분화된 양수 줄기세포를 포함하는 요실금 치료제
TWI836572B (zh) 從疝氣囊分離純化間質幹細胞的方法及源自於疝氣囊的間質幹細胞用於組織修復的用途
US20230220336A1 (en) Fibroblasts as a regenerative cellular source for the treatment of blindness
WO2014075634A1 (zh) 间质血管层细胞和间充质祖细胞在预防或治疗骨性关节炎中的应用
US20230075134A1 (en) Method for isolating and purifying mesenchymal stem cells from hernia sac and method for tissue repair by using mesenchymal stem cells from hernia sac
WO2023040938A1 (zh) 间充质干细胞来源的外泌体治疗ilk信号通路相关疾病的方法和药物组合物
CN116004531A (zh) 一种经血干细胞培养基及其制备方法和用途

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22787665

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22787665

Country of ref document: EP

Kind code of ref document: A1