METHOD FOR PRODUCING EXTRACELLULAR VESICLES FROM THREE-DIMENSIONALLY CULTURED STEM CELLS

Abstract

The present disclosure relates to a method for producing extracellular vesicles from three-dimensionally cultured stem cells. The method of the present disclosure can produce stem cell-derived extracellular vesicles with a high yield through orbital shaking culture of stem cell aggregates in the presence of TGF-β and thus can be usefully used in an industrial-scale mass production process of exosomes that can be utilized as a pharmaceutical ingredient substituting for a cell therapeutic agent. Furthermore, the exosomes obtained by the method of the present disclosure have significantly improved immunoregulatory functions as compared to the exosomes produced by the existing method and, therefore, can be applied as a superior therapeutic composition for various inflammations or autoimmune diseases.

Claims

1. A method for producing stem cell-derived extracellular vesicles, comprising: (a) a step of forming cell aggregates by culturing stem cells isolated from a subject; and (b) a step of three-dimensionally culturing the cell aggregates in a culture medium comprising TGF-β (transforming growth factor beta).

2. The method according to claim 1, wherein the stem cells are mesenchymal stem cells.

3. The method according to claim 1, wherein the step (a) is performed by suspension-culturing the stem cells in a multi-well culture plate.

4. The method according to claim 3, wherein the multi-well culture plate is a microwell plate having wells with a size of 300-500 μm.

5. The method according to claim 3, wherein the suspension culture is performed by seeding 300-500 cells per each well of the multi-well culture plate.

6. The method according to claim 1, wherein the TGF-β is TGF-β3.

7. The method according to claim 1, wherein the step (b) is performed by orbital shaking-culturing the cell aggregate in floating state.

8. The method according to claim 7, wherein the orbital shaking culture is performed at 50-70 rpm.

9. The method according to claim 1, which further comprises a step of isolating the extracellular vesicles from the culture medium obtained in the step (b) through multiple times of centrifugation.

10. The method according to claim 1, wherein the extracellular vesicles have an average diameter of 30-150 nm.

11. Stem cell-derived extracellular vesicles produced by the method according to claim 1.

12. A composition for preventing or treating an inflammatory or autoimmune disease, comprising the stem cell-derived extracellular vesicles according to claim 11 as active ingredients.

13. The composition according to claim 12, wherein the inflammatory or autoimmune disease is rheumatoid arthritis, reactive arthritis, type 1 diabetes, type 2 diabetes, systemic lupus erythematosus, multiple sclerosis, cryptogenic fibrosing alveolitis, polymyositis, dermatomyositis, localized scleroderma, systemic scleroderma, colitis, inflammatory bowel disease, Sjorgen's syndrome, Raynaud's phenomenon, Bechet's disease, Kawasaki's disease, primary biliary sclerosis, primary sclerosing cholangitis, ulcerative colitis, graft-versus-host disease (GVHD) or Crohn's disease.

14. Stem cell-derived extracellular vesicles highly expressing one or more protein selected from a group consisting of peroxiredoxin-4, thioredoxin reductase 1 and prostaglandin G/H synthase 2.

15. The stem cell-derived extracellular vesicles according to claim 14, wherein the extracellular vesicles further highly express one or more protein selected from a group consisting of heat shock protein 90-β (HSP90-β), neprilysin, T-complex protein 1 (TCP1) subunit α, filamin A, 40S ribosomal protein S3, myosin-9, transaldolase, fascin, thioredoxin reductase 1 and RuvB-like 2 (RUVBL 2).

16. The stem cell-derived extracellular vesicles according to claim 14, wherein the extracellular vesicles are positive for one or more protein selected from a group consisting of coronin 1A, prolyl 4-hydroxylase subunit α-2 and purine nucleoside phosphorylase.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0057] FIGS. 1a and 1b show three-dimensional culturing of mesenchymal stem cells according to a method of the present disclosure. They show the formation of embryonic bodies (FIG. 1a) and 3D culturing using an orbital shaker (FIG. 1b).

[0058] FIG. 2 shows the yield of exosomes under different culture conditions.

[0059] FIG. 3 shows the change of PDI values depending on the treatment with TGF-β. It can be seen that one peak appears under a 3D shaking culture condition with TGF-β treatment.

[0060] FIGS. 4a-4c show the effect of TGF-β on proliferation of T cells. After inducing the proliferation of PBMCs using PHA, T cell-inhibiting effect was investigated for the culture media of a negative control group (untreated group), a positive control group (MSC treatment group), exosomes obtained by 3D shaking culture only (3D-EV), and exosomes obtained by 3D shaking culture with addition of TGF-β3 (T-3D-EV) (FIG. 4a). As a result, it was confirmed that the exosomes obtained by 3D shaking culture with addition of TGF-β3 had the most noticeable T cell-inhibiting effect (FIGS. 4b and 4c). This shows that the exosomes obtained by the method of the present disclosure have improved function as well as high yield.

[0061] FIG. 5a shows a result of investigating the size of exosomes through dynamic light scattering (DLS) analysis. FIG. 5b shows a result of observing the shape and structure of exosomes by transmission electron microscopy (TEM). FIG. 5c shows a result of western blotting analysis for investigating the expression of CD9, CD63, flotillin-1 and Alix. FIG. 5d shows a result of immunophenotyping analysis for the surface of exosomes through flow cytometry.

[0062] FIGS. 6a and 6b show a result of investigating the wound-healing ability of keratinocytes (HaCaT cells) by the exosomes of the present disclosure.

[0063] FIG. 7 shows proteins changing specifically in T-3D-EV samples, which are exosomes obtained by a method of the present disclosure, among four clusters identified through clustering analysis.

[0064] FIG. 8 shows a comparative analysis result of the biological characteristics of proteins showing specific expression profiles in exosomes of the present disclosure.

[0065] FIG. 9 shows the biological functions and immune-related characteristics of proteins of three identified clusters.

[0066] FIG. 10 shows the degree of discrimination between groups identified through principal component analysis using discrimination indices.

[0067] FIG. 11 shows a result of gene set enrichment analysis, showing enriched gene sets, normalized enrichment scores (NES) and p-values.

[0068] FIG. 12 shows gene groups enriched in the present disclosure.

[0069] FIGS. 13 and 14 show proteins whose expression has increased or decreased 2 times or more in exosomes obtained by a method of the present disclosure as compared to a 3D culture group (FIG. 13) and proteins expressed only in the exosomes obtained by the method of the present disclosure (FIG. 14).

[0070] FIG. 15 shows the expression profile of inflammation-associated proteins in exosomes obtained by a method of the present disclosure.

BEST MODE

[0071] Hereinafter, the present disclosure will be described in more detail through examples. The examples are only for describing he present disclosure specifically, and it will be obvious to those having ordinary knowledge in the art that the scope of the present disclosure is not limited by the examples.

EXAMPLES

Example 1: Three-Dimensional Culture of Mesenchymal Stem Cells

[0072] For 3D culturing of stem cells, AggreWell™400 (STEMCELL Technologies; #34425) having about 7000microwells with a size of 400 μm treated with an F127 solution and uniform spheroids having a diameter of 120-200 μm were produced by seeding about 400 umbilical cord-derived mesenchymal stem cells per well (Seoul National University Hospital, Konkuk University Bioethics Committee: 001355-201705-BR-181). The spheroids were seeded in a culture medium containing TGF-β3 on a non-absorbent culture dish and then shaking-cultured for 3 days at 60 rpm and 37° C. using an orbital shaker (INFORS HT Celtron; #69455). 3 days later, exosomes were isolated from the culture medium.

Example 2: Isolation and Quantification of Exosomes

[0073] After removing cell debris by centrifuging the culture medium at 300 g for 10 minutes, followed by centrifugation at 2000 g for 10 minutes, the supernatant was transferred to a fresh tube and centrifuged again at 10,000 g for 30 minutes. After centrifuging the supernatant again at 187,000 g for 2 hours, exosomes were obtained from the resulting pellets. After isolating the exosomes from the culture medium, the number and peaks of the exosomes were investigated by conducting nanoparticle tracking analysis (NTA; NS300, NanoSight System) according to the manufacturer's instructions. As a result, when 3D culture was applied without shaking (60 rpm), the yield was not significantly different from that of general 2D culture, suggesting that the effect of the three-dimensional culturing itself is not significant. Significant increase in the yield was confirmed for the group to which all of 3D culture, shaking (60 rpm) and TGF-β3 addition were applied as compared to when only TGF-β33 was added during 3D culture without shaking (60 rpm) or when only 3D shaking culture was applied without addition of TGF-β3 (FIG. 2). In addition, one peak appeared when 3D shaking culture was performed with TGF-β3 treatment, suggesting that homogenous exosomes were produced (FIG. 3).

Example 3: PBMC Proliferation Assay

[0074] It was investigated whether the exosomes obtained from the culture medium treated with TGF-β obtained in Example 2 have T cell-inhibiting effect as compared to a control group (normal cell culture) by conducting PBMC proliferation assay according to a previously reported method (Hsu, P. J., et al. J. Vis. Exp. (106), e53265, doi:10.3791/53265(2015)). After isolating PBMCs from blood (Konkuk University Hospital, Konkuk University Bioethics Committee: 7001355-201705-BR-181) using ficoll and culturing for 5 days, the PBMCs were stained with CFSE (carboxyfluorescein succinimidyl ester, Invitrogen; #C34554) and the proliferation of the PBMCs was investigated by flow cytometry. After inducing an inflammatory environment accompanied by T cell proliferation by treating with PHA (phytohemagglutinin, Sigma; #L1668), the PBMC-inhibiting effect of exosomes obtained from a normal cell culture (EV), the exosomes obtained by 3D shaking culture only (3D-EV) and the exosomes obtained by 3D shaking culture with addition of TGF-β3 (T-3D-EV) was investigated with respect to the PBMC-inhibiting effect of mesenchymal stem cells as a positive control group (FIG. 4a). As a result, the exosomes of the present disclosure obtained by 3D shaking culture with TGF-β treatment showed the best T cell-inhibiting effect close to 80% with respect to that of the positive control group by decreasing the proliferative T cells of the MSC treatment group from 43.1% to 9.6% (FIGS. 4b and 4c). Therefore, it can be seen that the exosomes obtained by the method of the present disclosure have improved function as well as high yield.

Example 4: Characterization of Exosomes

[0075] The size of the exosomes was investigated by dynamic light scattering (DLS) using Nano Zetasizer (Malvern Instruments, Malvern, UK), and the number of the EVs was counted with the nanoparticle tracking analyzer NS300 (Nanosight, Amesbery, UK). The shape and structure of the exosomes were analyzed by at transmission electron microscopy (TEM, JEM-1010, Nippon Denshi, Tokyo, Japan) 80 kV. As a result, the exosomes were cup- or sphere-shaped (FIG. 5b).

[0076] After adhering the exosomes onto a grid (formvar/carbon 300 mesh, copper, FCF300-CU 50/pk), negative staining was performed using 1% phosphotungstic acid hydrate (Sigma, P4006). For identification of exosome-associated positive markers, the expression of CD9 (ab263023, Abcam), CD63 (ab134045, Abcam), flotillin-1 (#18634, CST) and Alix (#2171, CST) proteins was investigated by immunoblotting. As a result of western blotting, the positive markers for the exosomes were expressed, whereas the expression of the exosome negative marker GM130 (#12480, CST) protein was not observed.

[0077] Immunophenotyping analysis was conducted on the surface of the exosomes by flow cytometry. First, because the size of the exosomes is not appropriate for analysis by flow cytometry, 2.7 μm Dynabeads (10620D, Invitrogen, exosome-human CD9 flow detection reagent (from cell culture)) to which CD9 antibody, a positive marker of exosomes, is conjugated were attached to the exosomes to increase the size, which were then labeled with CD9-BV421 (BD Bioscience, 743047), CD63-PE (BD Bioscience, 556020) and CD81-APC (MACS Miltenyi Biotec, M130-119-787) antibodies. Then, the intensity of fluorescence generated by the labeled antibodies were measured using a flow cytometer (Beckman Coulter, CytoFlex flow cytometry analyzer). As a result, it was confirmed that the fluorescence intensity of CD9, CD63 and CD81 was 96% or higher. From the fact that the isolated exosomes express 96-98% of exosome positive markers, it can be seen that homogenous exosomes were isolated.

Example 5: Wound Healing Assay of Exosomes

[0078] For evaluation of the wound healing capacity of the exosomes in keratinocytes (HaCaT cells), HaCaT cells were inoculated onto a culture dish and, after culturing to 90% confluency, a long scratch was made using a 1000 μL tip end. Then, after replacing with a cell culture medium (Dulbecco's modified Eagle's medium-high glucose, D6429, Sigma) containing the exosomes (1E+10 particles/mL), the wound site was imaged with given time intervals.

[0079] As a result, the groups treated with the exosomes showed faster wound closure than the negative control group, and the group to which TGF-β3 and 3D shaking culture were applied (T-3D-EV) showed faster wound closure than the group to which only 3D shaking culture was applied (3D-EV) (FIG. 6).

Example 6: Analysis of Proteins Expressed in Exosomes

[0080] Extraction and Quantitative Analysis of Proteins

[0081] Peptides were prepared and expressed from proteins stained in gels by the in-gel digestion method. Specifically, after destaining the proteins using a 50 mM ammonium bicarbonate/50% acetonitrile solution and a 100% acetonitrile solution, disulfide bonds were reduced at 37° C. using 50 mM dithiothreitol. Then, after conducting alkylation under light-shaded condition using 55 mM iodoacetamide, dehydration was conducted using a 100% acetonitrile solution. Then, peptides were prepared using LysS/trypsin mixed protease dissolved in 50 mM ammonium bicarbonate. The extracted peptides were dissolved in 0.1% formic acid and subjected to liquid chromatography (LC) and mass spectrometry. The mass spectrometry was performed using Q-Exactive Plus (Thermo, USA), and the liquid chromatography was performed using UltiMate™ 3000 RSLCnano System (Thermo, USA). The analysis was conducted by injecting 5 μL of the peptide sample to an ion trap mass spectrometer coupled with NanoLC at a flow rate of 250 μL/min. The LC was performed for a total of 200 minutes including 150 minutes of a concentration gradient of solution A (5% dimethyl sulfoxide, 0.1% formic acid) and solution B (95% acetonitrile, 0.1% formic acid, 5% dimethyl sulfoxide). A fused silica capillary column filled with C18 (2 μm, 100 Å), with an inner diameter of 75 μm, an outer diameter of 360 μm and a length of 50 cm, was used to separate the peptide mixture. The separated peptides were introduced to the mass spectrometer to obtain spectrum data. Orbitrap MS analysis was performed by repeating 20 cycles of MS/MS (resolution 17,500) using Orbitrap MS according to the HCD (higher energy collision dissociation, 27% energy level) method following one survey scan (resolution 70,000) in a range of 350-1800 m/z using an ion trap MS. The detection of overlapping peptide ions was minimized by setting the dynamic exclusion option to 20 seconds. The auto gain control target setting of the ion trap was 3E06 for full MS and 1E5 for FT MS/MS. Qualitative analysis and label-free quantitative analysis were performed for the acquired RAW file using the Andromeda algorithm-based data analysis software MaxQuant (version 1.6.10.43, https://www.maxquant.org/). For cysteine, cysteine carbamidomethylation was set as a fixed modification, while methionine oxidation was set as a variable modification. The Human SwissProt database published in October 2019 was used as a protein sequence database and the MaxLFQ algorithm was used for the label-free quantitative analysis of proteins. The identified proteome was subjected to heat mapping, clustering analysis and principal component analysis using Perseus (http://www.perseus-framework.org). The ClueGO, ShinyGO v0.60 and GSEA programs were used for characterization of the protein clusters.

[0082] The difference in the protein expression pattern of each sample was quantified by label-free quantitative analysis using the data obtained from the quantitative analysis, and the proteins were divided into four clusters depending on the difference in patterns. From the four clusters, the proteins showing specific difference in the T-3D-EV sample, i.e., the exosomes obtained by the method of the present disclosure, are shown in FIG. 7.

[0083] Comparative Analysis of Biological Characteristics of Proteins

[0084] For screening of proteins having specific characteristics in T-3D-EV, the protein expression profile was compared with those of a 3D shaking culture group (3D-EV) and a 2D culture group (2D-EV) without TGF-β3 treatment. As shown in FIG. 8, the proteins showing specific characteristics in T-3D-EV showed the characteristics in glycolytic process, connective tissue replacement involved in inflammatory response wound healing, platelet formation, pentose phosphate pathway, oxidative branch, etc., and showed distinct difference from other control groups. As shown in FIG. 9, immune-associated characteristics were also observed in T-3D.

[0085] Principal Component Analysis

[0086] In principal component analysis, the degree of discrimination between sample groups is identified by discrimination indices. The data shown in FIG. 10 were obtained by repeating mass spectroscopy 3 times with different sample treatment. The contribution of the first principal component was 60.2%, and the contribution of the second principal component was 28.9%. As a result, significant difference in patterns was identified in the qualitative and quantitative characteristics of proteins depending on the sample treatment method. Because the distance between 3D-EV and T-3D-EV is smaller than those from 2D-EV, it is thought that the difference in proteins arises from the difference in the 2D method and the 3D method.

[0087] Gene Set Enrichment Analysis

[0088] Gene set enrichment analysis was conducted using the GSEA module. The enrichment score allocates a pre-specified class when a gene included in the corresponding set is expressed discriminately according to the phenotype. The normalized enrichment score was calculated by normalizing the enrichment score to the number of genes in the gene set. The nominal p-value is re-calculating the normalized enrichment score in order to replace susceptibility and resistance indices and generate a null distribution. FIG. 11 shows a result of the gene set enrichment analysis and shows the enriched gene sets, normalized enrichment scores (NES) and p-values. The chance that detection is significant is higher as the NES is higher and the p-value is lower. The gene groups enriched in the analysis are shown in FIG. 12.

[0089] Identification of T-3D-EV-Specific Proteins

[0090] In order to identify the proteins which show significant difference in the group to which TGF-β was added, comparison was performed for sample pairs. The proteins which showed 2 times or more increase or decrease in 3D-EV as compared to T-3D-EV and had Benjamini-Hochberg FDR of 0.05 or lower were screened. In addition, the proteins detected only in the T-3D sample were included. The screened proteins are shown in FIG. 13 and FIG. 14.

[0091] As a result of the analysis of proteins existing in EV shown in FIG. 15, antioxidant proteins and inflammation-related factors were expressed.

[0092] The representative cellular stress responses are ROS generation and activation of the cellular redox regulatory network thereby. ROS are free radicals which induce the change in the structure and activity of proteins and DNAs by attacking them and, thereby, are involved in DNA repair, cell cycle regulation and cellular growth/apoptosis.

[0093] Six types of peroxiredoxins are present in mammals. They are widely distributed in various cells and tissues and function as important antioxidant proteins that remove H.sub.2O.sub.2 generated in tissues. The thioredoxin system is a representative cellular defense system which resolves stresses induced by ROS, etc. It acts as an ROS scavenger of recovering proteins oxidized by ROS through reversible redox reactions and is involved in various cellular defense systems. It is known as a key protein in charge of the cellular defense system acting not only as a survival-inducing factor of cells but also as an anti-apoptotic, anti-inflammatory and immuno-regulatory factor. It was confirmed that the antioxidant proteins peroxiredoxin-4 and thioredoxin reductase are highly expressed in T-3D-EV, i.e., the exosomes obtained by the method of the present disclosure (FIG. 15).

[0094] In addition, it was found out that COX-2 is also highly expressed in T-3D-EV (FIG. 15). In general, the inflammatory response is part of the defensive response of body tissues to physical or chemical stimulation or bacterial infection from outside. It is a mechanism for repairing or regenerating damaged tissues. When inflammatory response occurs in the body, inflammatory cells such as macrophages secrete inflammatory mediators such as nitrogen oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), etc. The synthesis of PGE2 begins with the generation of arachidonic acid from the membrane phospholipid by phospholipase A2. Arachidonic acid is converted to prostaglandin G2 by enzymatic action and then to the unstable metabolite prostaglandin H2. These two processes are facilitated by cyclooxygenase (COX). COX exists as two or more isoenzymes. Among them, COX-1 is expressed consistently and is involved in physiological functions such as platelet aggregation, protection of the gastric mucous membrane, regulation of renal function, etc. COX-2 is expressed in response to stimulation such as inflammation and it is known that prostaglandin generated by COX-2 is involved in inflammatory response and cellular proliferation. Therefore, it is though that the high expression of COX-2 and T-3D-EV is because the exosomes of the present disclosure are involved in the regulation of inflammatory response.

[0095] While specific exemplary embodiments of the present disclosure have been described in detail, it will be obvious to those having ordinary knowledge in the art that they are mere specific exemplary embodiments and the scope of the present disclosure is not limited by them. It is to be understood that the substantial scope of the present disclosure is defined by the appended claims and their equivalents.