Application of mesenchymal stem cell exosome subdivision subgroup in cerebral apoplexy treatment

Abstract

The invention relates to the technical field of stem cells, and provides application of a mesenchymal stem cell exosome subdivision subgroup in cerebral apoplexy treatment. Compared with undivided subgroups and positive drugs, the exosome subgroups S1 and S2 have the advantages that the effects of improving post-stroke neurological functions and reducing infarct volumes are more remarkable, the purity is high, large-scale preparation can be realized, and good preparations or drug products can be formed. The invention provides a new treatment scheme for cerebral apoplexy treatment, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of stem cell biomedical technology, specifically relating to the application of a subpopulation of mesenchymal stem cell exosomes in the treatment of stroke. Background Technology

[0002] Stroke, the second leading cause of death worldwide and the leading cause of disability in adults, presents significant challenges in treatment. Ischemic stroke accounts for 60-80% of all cases. While intravenous thrombolysis (rt-PA) and endovascular thrombectomy are effective treatments in the acute phase, they are limited by a strict time window (<4.5 hours) and specific indications. Over 90% of ischemic stroke patients suffer irreversible neurological damage due to missing the treatment window. Repeated failures in clinical trials of neuroprotective agents highlight the urgent need to develop novel treatment strategies.

[0003] Exosomes are nanoscale vesicles (30-150 nm) secreted by cells, carrying bioactive molecules such as proteins, nucleic acids, and lipids, and possessing intercellular communication and regulatory functions. In recent years, exosomes derived from mesenchymal stem cells (MSCs) have been found to have anti-inflammatory, neuroprotective, synaptic remodeling, and angiogenesis effects. Although the application of stem cell exosomes in various diseases is becoming increasingly widespread, limitations such as poor therapeutic efficacy and reproducibility remain. Further research into the subpopulation of exosomes is necessary to improve their therapeutic effects. Summary of the Invention

[0004] This application provides the use of subgroups S1 and S2 of mesenchymal stem cell exosomes in the treatment of stroke. By subdividing stem cell exosomes into subgroups, the uniformity of the product is improved, while achieving a more efficient and stable stroke treatment effect.

[0005] This invention provides the application of mesenchymal stem cell exosome subpopulations in the preparation of formulations for the treatment or prevention of stroke, wherein the formulations contain exosome subpopulations S1 and / or S2 as active ingredients; the exosome subpopulation S1 has a peak particle size range of 105–135 nm and an average particle size of 115–145 nm, and surface markers including HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A; the exosome subpopulation S2 has a peak particle size range of 75–105 nm and an average particle size of 85–115 nm, and surface markers including B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

[0006] As an optional feature, the surface markers of the exosome subgroup S1 also include CD63, wherein CD63 is expressed at low levels, and CD9 and ALIX are expressed at high levels, and the exosome purity is not less than 1×10⁻⁶. 8 Particle count / μg protein; Surface markers for exosome subpopulation S2 also include ALIX and CD9, with high expression of CD63 and low expression of CD9 and ALIX, and exosome purity not less than 1×10⁻⁶. 8 Particle count / μg protein.

[0007] As an optional method, the preparation of the exosome subpopulations S1 and S2 includes: isolating and culturing human umbilical cord-derived mesenchymal stem cells, collecting their culture supernatant; filtering; using a tangential flow filtration system to obtain a concentrated solution from the filtered supernatant, and separating and purifying it by size exclusion chromatography and / or reverse affinity chromatography to obtain exosome subpopulations S1 and S2.

[0008] As an alternative, when using size exclusion chromatography to separate and purify the concentrate, the component corresponding to the first UV absorption peak in the eluent is collected as exosome subgroup S1, and the component corresponding to the second UV absorption peak is collected as exosome subgroup S2.

[0009] As an optional method, the preparation of the culture supernatant includes: selecting and isolating mesenchymal stem cells derived from umbilical cords from healthy mothers who are in the logarithmic growth phase and within the seventh generation; seeding the mesenchymal stem cells in a serum-free, well-defined mesenchymal stem cell-specific culture medium and culturing them to the 5th to 7th generation until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, replacing the culture medium with fresh medium, continuing the culture, and collecting the cell culture supernatant containing exosomes.

[0010] As an optional approach, the filtration, concentration, and separation and purification steps in the preparation method include: deep filtration of the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentration of the pretreatment solution by a tangential flow ultrafiltration system, separation and purification by size exclusion chromatography, and collection of the components corresponding to the first and second ultraviolet absorption peaks in the eluent to obtain exosome subpopulations S1 and S2 respectively.

[0011] As an option, the stroke includes ischemic stroke and hemorrhagic stroke.

[0012] Alternatively, the concentration of the exosome subpopulations in the formulation is 1×10⁻⁶. 8 ~1×10¹¹ particles / mL.

[0013] As an alternative, the formulation comprises a combination of mesenchymal stem cell exosome subgroups S1 and / or S2 and a drug for treating stroke; the drug for treating stroke includes intravenous thrombolytic drugs, drugs that improve cerebral collateral circulation, anticoagulants, anti-inflammatory drugs, and neuroprotective agents.

[0014] As an option, the dosage form of the formulation includes lyophilized powder, hydrogel, or sterile solution; the administration method of the formulation includes intravenous injection, nasal administration, or in situ administration to brain tissue.

[0015] This invention provides the application of subgroups of exosomes in the treatment of stroke. The technical solution of this invention discovers and confirms that subgroups of exosomes S1 and S2 are significantly more effective than unsubgrouped exosomes and the positive control drug, septoplastin, in both cell and animal experiments. Compared to unsubgroups and septoplastin, subgroups of exosomes show more significant effects in improving post-stroke neurological function and reducing infarct volume. Furthermore, subgroups S1 and S2 have high purity, can be prepared on a large scale, and can be formulated into good pharmaceutical products, which is of great significance for the application of exosomes in the field of stroke treatment. Attached Figure Description

[0016] Figure 1 The study demonstrated the effects of subgroups of exosomes S1, S2, and unsubgrouped exosomes, as well as the positive drug serotonin, on the excitotoxicity of HT22 in neurons, the proliferation of bEnd.3 vascular endothelial cells, and the inflammatory response of BV2 microkeratocytes.

[0017] Figure 2 The results of efficacy evaluation of exosomes subgroups S1, S2 and unsubgrouped exosomes and the positive drug succinate in a rat model of ischemic stroke. Detailed Implementation

[0018] The technical solution of the present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] This invention provides novel medical applications for exosome subsets S1 and S2 derived from mesenchymal stem cells. For the first time, the present invention has demonstrated their potential use as active ingredients in the preparation of formulations for the treatment of stroke. These formulations may include pharmaceutical or other product forms.

[0020] Exosomes derived from mesenchymal stem cells (MSCs) possess anti-inflammatory, neuroprotective, synaptic remodeling, and angiogenesis effects, making them potential applications in stroke treatment. However, stem cell exosomes still face limitations such as poor therapeutic efficacy and low reproducibility. Omics analysis has revealed that exosomes contain both beneficial and harmful components for stroke treatment. Our research team has subdivided MSC exosomes into subpopulations, improving the homogeneity of exosome products while discovering and validating the significant efficacy of this subpopulation in stroke treatment. Its high purity, scalability, and ability to form effective formulations or pharmaceutical products are of great significance for the application of exosomes in stroke treatment.

[0021] Specifically, the mesenchymal stem cell-derived exosome subsets S1 and S2 described in this invention are defined and defined by the following characteristics or the following preparation methods.

[0022] The peak particle size range of the mesenchymal stem cell exosome subpopulation S1 is 120±15 nm, with an average particle size of 130±15 nm. Surface markers include HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A. More specifically, in some protocols, the surface markers of exosome subpopulation S1 also include CD63, with low expression of CD63 and high expression of CD9 and ALIX, and exosome purity not less than 1×10⁻⁶. 8 Particle count / μg protein. The peak particle size range of the S2 subpopulation of mesenchymal stem cell exosomes is 90nm±15nm, with an average particle size of 100±15nm. Surface markers include B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN. More specifically, in some protocols, the surface markers of the S2 subpopulation of mesenchymal stem cell exosomes also include ALIX and CD9, with high expression of CD63 and low expression of CD9 and ALIX. The exosome purity is not less than 1×10⁻⁶. 8 Particle count / μg protein.

[0023] The method for preparing the subpopulations S1 and S2 of mesenchymal stem cell exosomes includes: isolating and culturing mesenchymal stem cells derived from human umbilical cord, collecting the supernatant of the culture medium; filtering; obtaining a concentrate from the filtered supernatant using a tangential flow filtration system, and separating and purifying the concentrate by size exclusion chromatography and / or reverse affinity chromatography to obtain the subpopulations S1 and S2 of exosomes.

[0024] In some schemes, the preparation method of the subpopulations S1 and S2 of mesenchymal stem cell exosomes includes the following steps: 1) Collection of cell culture supernatant: Culture umbilical cord mesenchymal stem cells to passage 5-7 and collect the cell culture supernatant within this passage range; 2) Pretreatment: The supernatant was filtered to remove large vesicles and apoptotic bodies to obtain a pretreated solution; 3) Exosome subpopulation isolation: The pretreated solution was concentrated by a tangential flow ultrafiltration system and then purified by size exclusion chromatography. The components corresponding to the first and second UV absorption peaks in the eluent were collected to obtain the target exosome subpopulations S1 and S2.

[0025] More specifically, the preparation method includes: selecting and isolating mesenchymal stem cells derived from umbilical cords from healthy mothers within the seventh generation and in the logarithmic growth phase; seeding the mesenchymal stem cells in a serum-free, well-defined mesenchymal stem cell-specific culture medium and culturing them to the 5th to 7th generation until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS and replacing it with fresh culture medium, continuing the culture, and collecting the cell culture supernatant containing exosomes; subjecting the cell culture supernatant to deep filtration to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentrating the pretreatment solution using a tangential flow ultrafiltration system, separating and purifying it by size exclusion chromatography, collecting the components corresponding to the first and second ultraviolet absorption peaks in the eluent to obtain the exosome subpopulations S1 and S2.

[0026] Furthermore, in some schemes, the preparation methods and operational details of the aforementioned exosome subgroups S1 and S2 are the same as or substantially the same as the relevant schemes in patent application text CN118222495A.

[0027] In some embodiments, the exosome subpopulations S1 and / or S2 derived from mesenchymal stem cells are the sole active components of the formulation or drug of the present invention. In other embodiments, the formulation may be used in combination with other drugs for treating stroke, in addition to the exosome subpopulations; these other drugs for treating stroke include intravenous thrombolytic drugs, drugs that improve cerebral collateral circulation, anticoagulants, anti-inflammatory drugs, neuroprotective agents, and other drugs that improve stroke symptoms.

[0028] In a preferred embodiment, the dosage form of the formulation of the present invention can be prepared as a lyophilized powder, hydrogel, or injection, with a concentration of 1×10⁻⁶. 8 It contains approximately 1×10¹¹ particles / mL and is used for the treatment of stroke. The formulation can be administered via intravenous injection, nasal administration, or in situ delivery to the brain tissue.

[0029] In some embodiments of the stroke described in this invention, the stroke includes hemorrhagic stroke or ischemic stroke; more preferably, the stroke is ischemic stroke. More specifically, the stroke also includes: acute ischemic stroke, sequelae of ischemic stroke, acute hemorrhagic stroke, or hemorrhagic stroke.

[0030] Example 1: Preparation and identification of subpopulations S1 and S2 of mesenchymal stem cell exosomes (1) Preparation of mesenchymal stem cell culture supernatant and pretreatment solution: In a CO2 incubator, umbilical cord mesenchymal stem cells were cultured to P5-7 generation with cell viability greater than 80%. The cell culture medium was filtered through a deep filter to remove large vesicles and apoptotic bodies, thus obtaining the mesenchymal stem cell culture pretreatment solution.

[0031] (2) Concentration of mesenchymal stem cell culture supernatant: The mesenchymal stem cell culture supernatant was concentrated 20 times by a tangential flow filtration concentration system to obtain a concentrate. The tangential flow concentration system used a 300kDa hollow fiber filter, washed with PBS 16-25 times, with a transmembrane pressure (TMP) of 0.5 bar and a flow rate of 385LMH.

[0032] (3) Size exclusion chromatography: The above products are further purified by the following steps: Equilibration: Equilibrate the Sephacryl S-400 HR gel filter column 2CV using PBS buffer.

[0033] Sample loading: 0.5–30 mg / mL.

[0034] Elution: Wash with PBS buffer. Start collecting when the UV signal rises and stop collecting when it approaches the baseline level. Collect the first and second UV absorption peaks, which are the exosome subpopulations S1 and S2, respectively.

[0035] Clean in place (CIP): Rinse 2CV with 0.5M NaOH solution.

[0036] (4) Exosome identification: Transmission electron microscopy (TEM) showed that exosome subsets S1 and S2 both had a classic teacup-like morphology; nanoparticle tracking (NTA) analysis showed that the peak particle size range of S1 particles was 120±15 nm, and the average particle size was 130±15 nm. Western blotting showed that surface markers of S1 included HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, Filamin-A, etc. The peak particle size range of S2 particles was 90 nm±15 nm, and the average particle size was 100±15 nm. Western blotting showed that surface markers of S2 included B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

[0037] Examples 2-5 below investigate and validate the function and activity of exosome subgroups S1 and S2. The exosome subgroups S1 and S2 used are the exosome subgroups S1 and S2 prepared in Example 1.

[0038] Example 2: Neuronal cell excitotoxicity experiment Cell viability was assessed using the CCK8 assay: HT22 cells in the logarithmic growth phase were subjected to a concentration of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 1 / mL into 96-well cell culture plates. After cell attachment, the culture supernatant was discarded. The culture medium for both the model and experimental groups was replaced with fresh medium containing 2.5 mM glutamate. The control group contained no glutamate, while the experimental groups were supplemented with 1×10⁻⁶ mM glutamate. 10 Undifferentiated exosome subgroups, 1×10 10 Subgroups S1 and S2 were further subdivided and incubated with 5 μg / mL of succinate. Each group was divided into 6 replicates. After culturing for 48 h, 100 μL of DMEM basal medium and 10 μL of CCK8 solution were added to each well of the cell culture plate and incubated for another 30 min. The absorbance value (A) was measured at 450 nm. Cell viability % = OD experimental group / OD control group × 100%.

[0039] Example 3: Vascular endothelial cell proliferation experiment Cell proliferation capacity was detected using the CCK8 assay: bEnd.3 cells in logarithmic growth phase were subjected to a 3×10⁻⁶ saturation solution. 4 Cells were seeded at a density of 1 / mL into 96-well cell culture plates. After cell attachment, the culture supernatant was discarded, and the medium was replaced with DMEM basal medium. 1×10⁶ cells were added to the experimental group. 10 Undifferentiated exosome subgroups, 1×10 10Subgroups S1 and S2 were subdivided and treated with 5 μg / mL of succinate. The blank group was treated with an equal amount of PBS. Each group was divided into 6 replicates. After culturing for 72 h, the old culture medium was discarded, and 100 μL of DMEM basal medium and 10 μL of CCK8 solution were added to each well for 30 min of incubation. The absorbance (A) was measured at 450 nm. The proliferation rate % = OD experimental group / OD blank group × 100%.

[0040] Example 4 Microglia Inhibition Validation Experiment BV2, which is in the logarithmic growth phase, will be used at 4 × 10 4 Cells were seeded at a density of 1 × 10⁶ mL into 24-well cell culture plates. After cell attachment, the culture supernatant was discarded and replaced with fresh culture medium. Experimental groups received 1 × 10⁶ cells / mL. 10 Undifferentiated exosome subgroups, 1×10 10 Subgroups S1 and S2 were subdivided and treated with 5 μg / mL of succinate. An equal amount of PBS was added to the blank group. At the same time, 1 μg / mL of LPS was added to each group. Each group was divided into 3 replicates. After culturing for 48 hours, the cell culture supernatant was collected, centrifuged at 2000×g for 5 minutes, and the supernatant was used for enzyme-linked immunosorbent assay to detect TNF-α expression.

[0041] from Figure 1 The results show that, for the HT22 excitotoxicity model of Example 2, although exosome, S1, S2, and sempervir all significantly inhibited glutamate-induced excitotoxic apoptosis in HT22 cells, the inhibitory effects of subsets S1 and S2 were significantly better than those of exosome and sempervir. Figure 1 A). In the bEnd.3 cell proliferation experiment of Example 3, exosomes, S1, and S2 all showed promoting effects, while succinate showed slight inhibition but no statistical difference. This is consistent with previous scientific studies that exosomes have cell proliferation-promoting activity. Although the effect of the S1 subset was slightly weaker than that of exosomes, there was no statistical difference, while the effect of the S2 subset was significantly better than that of exosomes. Figure 1 B). Regarding the BV2 inflammation suppression experiment in Example 4, exosome, S1, S2 and sempervirine all significantly inhibited LPS-induced TNF-α overexpression, but S1 and S2 were significantly better than exosome and sempervirine.

[0042] Example 5: Construction and drug administration of a transient middle cerebral artery occlusion model To establish a transient middle cerebral artery occlusion (tMCAO) model, rats were anesthetized with 5% chloral hydrate, fixed in a supine position, and the midline of the neck was incised. The right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were separated. The proximal end of the CCA and the origin of the ECA were ligated, and the ICA was clamped with a hemostatic clip. A small incision was made in the CCA, and a suture was inserted into the ICA until resistance was felt, indicating that the suture had passed through the origin of the MCA and reached the proximal end of the anterior cerebral artery (ACA). After 90 minutes, the suture was slowly removed to restore blood flow to the MCA. The skin was sutured, and the incision was disinfected. After model establishment, drugs were administered according to the experimental design. After the surgical procedure, the experimental animals were kept warm on a warming mat until they were fully recovered, at which point the model was evaluated.

[0043] Figure 2 A is the experimental design flowchart for this animal study. Drug administration was completed within 2 hours after model establishment. Results showed that exosomes had a slight but no significant effect on infarct size improvement and neurological function scores. Similac significantly improved infarct size but had no significant effect on neurological function scores. Only the exosome subgroups S1 and S2 of this invention showed significant effects on both indicators, and the effects of S1 and S2 were comparable. Figure 2 BD).

[0044] In summary, the exosome subgroups S1 and S2 exhibit better activity in the treatment of stroke compared to the unsubgrouped exosomes and the marketed drug Sempervir, and have greater potential for future market applications.

[0045] The present invention has been described in detail above with reference to specific embodiments and exemplary examples. However, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the present invention, and all such modifications and improvements fall within the scope of the present invention.

Claims

1. The application of subpopulations of mesenchymal stem cell exosomes in the preparation of agents for the treatment or prevention of stroke, characterized in that, The formulation uses exosome subgroups S1 and / or S2 as active ingredients; the peak particle size range of exosome subgroup S1 is 105–135 nm, the average particle size is 115–145 nm, and the surface markers include HRS, HSPA4, RAS1B, A2M, Syntenin-1, CD9, GPC1, DPP4, HSP90AA1, HSP90AB1, ALIX, CD81, TSG101, and Filamin-A; the peak particle size range of exosome subgroup S2 is 75–105 nm, the average particle size is 85–115 nm, and the surface markers include B2M, PRDX2, FLOT1, FLOT2, MSN, CD63, and GSN.

2. The application according to claim 1, characterized in that, The surface markers of the exosome subpopulation S1 also include CD63, with low expression of CD63 and high expression of CD9 and ALIX, and the exosome purity is not less than 1×10⁻⁶. 8 Particle count / μg protein; Surface markers for exosome subpopulation S2 also include ALIX and CD9, with high expression of CD63 and low expression of CD9 and ALIX, and exosome purity not less than 1×10⁻⁶. 8 Particle count / μg protein.

3. The application according to claim 1, characterized in that, The preparation method of the exosome subpopulation S1 and exosome subpopulation S2 includes: isolating and culturing mesenchymal stem cells derived from human umbilical cord, collecting their culture supernatant; filtering; using a tangential flow filtration system to obtain a concentrated solution from the filtered supernatant, and separating and purifying it by size exclusion chromatography and / or reverse affinity chromatography to obtain exosome subpopulations S1 and S2.

4. The application according to claim 3, characterized in that, When size exclusion chromatography is used to separate and purify the concentrate, the component corresponding to the first UV absorption peak in the eluent is collected as exosome subgroup S1, and the component corresponding to the second UV absorption peak is collected as exosome subgroup S2.

5. The application according to claim 3, characterized in that, The method for preparing the culture supernatant includes: selecting and isolating mesenchymal stem cells derived from umbilical cords from healthy mothers who are in the logarithmic growth phase and within the seventh generation; seeding the mesenchymal stem cells in a serum-free, well-defined mesenchymal stem cell-specific culture medium and culturing them to the 5th to 7th generation until the cell confluence reaches 70%; discarding the culture medium, washing the cells three times with PBS, replacing with fresh culture medium, continuing the culture, and collecting the cell culture supernatant containing exosomes.

6. The application according to claim 3 or 5, characterized in that, The filtration, concentration, and separation and purification steps in the preparation method include: deep filtration of the culture supernatant to remove large vesicles and apoptotic bodies to obtain a pretreatment solution; concentration of the pretreatment solution by a tangential flow ultrafiltration system, separation and purification by size exclusion chromatography, and collection of the components corresponding to the first and second ultraviolet absorption peaks in the eluent to obtain exosome subpopulation S1 and exosome subpopulation S2 respectively.

7. The application according to claim 1, characterized in that, The stroke includes ischemic stroke and hemorrhagic stroke.

8. The application according to claim 1, characterized in that, The concentration of the exosome subpopulations in the formulation is 1×10⁻⁶. 8 ~1×10¹¹ particles / mL.

9. The application according to claim 1, characterized in that, The formulation comprises a combination of mesenchymal stem cell exosome subpopulations S1 and / or S2 and drugs for treating stroke; the drugs for treating stroke include intravenous thrombolytic drugs, drugs for improving cerebral collateral circulation, anticoagulants, anti-inflammatory drugs, and neuroprotective agents.

10. The application according to claim 1, characterized in that, The dosage form of the formulation includes lyophilized powder, hydrogel, or sterile solution; the administration methods of the formulation include intravenous injection, nasal administration, or in situ administration to brain tissue.

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