JP7710754B2 - Exosomes derived from cells treated with endoplasmic reticulum stress inducers and uses thereof - Google Patents

Description

This application claims priority to Korean Patent Application No. 10-2020-0174576, filed on December 14, 2020, the entire specification of which is incorporated herein by reference.

The present invention relates to therapeutic uses of exosomes derived from mesenchymal stem cells that have been induced with endoplasmic reticulum stress.

Mesenchymal stem cells (MSCs) are known to have immunomodulatory properties, which are mediated by immunosuppression, vasoregulation and paracrine factor activity. The known paracrine secretome includes secretion of immunomodulatory cytokines, growth factors and small membrane vesicles. However, the direct use of mesenchymal stem cells presents challenges, such as maintaining their phenotypic and functional stability or high isolation and handling costs.

Among the mesenchymal stem cell secretome (MSC secretome), exosomes mediate the transfer of paracrine factors in intercellular communication and play an important role in immune regulation. Exosomes are small membrane lipid vesicles with a size of 30-150 nm that are released into the extracellular space and deliver proteins, mRNA, microRNA (miRNA) or other components to target cells, promoting the activation of immune regulation pathways. In particular, exosomes secreted from mesenchymal stem cells are known to exhibit the regenerative medicine therapeutic efficacy of stem cells. The functions of exosomes and the factors they transmit vary depending on the cell type, and cellular stress can affect the factors transmitted by exosomes.

The endoplasmic reticulum plays an important role in regulating signaling pathways to ensure cellular homeostasis, where newly synthesized proteins undergo folding and translocation through the ER membrane. However, when the ER is unable to maintain homeostasis under stressful pathological and physiological conditions, the unfolded protein response (UPR) can be activated and affect cellular functions. Long-term ER dysfunction or stress is involved in the pathogenesis of various diseases, including metabolic and inflammation-related diseases. Thapsigargin (TSG) is known to increase cytoplasmic calcium concentration and deplete ER calcium stores, inducing ER stress.

Korean Patent Registration No. 10-1980453 relates to a composition for promoting the production of exosomes derived from stem cells, and discloses a composition and method for increasing the production amount of exosomes derived from stem cells and the content of proteins and RNA in the exosomes using pioglitazone, metformin, or AICAR (5-aminoimidazole-4-carboxamide ribonucleotide). However, no research or description has been disclosed regarding exosomes secreted from stem cells treated with endoplasmic reticulum stress inducers such as thapsigargin.

Kim, W. , Lee, S. K. , Kwon, Y. W. , Chung, S. G. & Kim, S. Pioglitazone-Primed Mesenchymal Stem Cells Stimulate Cell Proliferation, Collagen Synthesis and Matrix Gene Expression in Tenocytes. International journal of molecular sciences 20, doi:10.3390/ijms20030472 (2019). Kim, H. S. et al. Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2. Gastroenterology 145, 1392-1403. e1391-1398, doi:10.1053/j. gastro. 2013.08.033 (2013). Kang, J. Y. et al. Xeno-Free Condition Enhances Therapeutic Functions of Human Wharton’s jelly-Derived Mesenchymal Stem Cells against Experimental Colitis by Upregulated Indoleamine 2,3-Dioxygenase Activity. J Clin Med 9, doi:10.3390/jcm9092913 (2020).

The inventors have made extensive efforts to provide the secretome of mesenchymal stem cells as an alternative to therapies that directly use mesenchymal stem cells. As a result, they have confirmed that exosomes secreted from mesenchymal stem cells in which stress has been induced in the endoplasmic reticulum by endoplasmic reticulum stress inducers such as thapsigargin are involved in the activation and polarization of T cells and macrophages, thereby completing the present invention.

Therefore, an object of the present invention is to provide a pharmaceutical composition for preventing or treating an inflammatory disease, comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer, and use thereof.

The present invention provides a pharmaceutical composition for preventing or treating an inflammatory disease, comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver.

According to a preferred embodiment of the present invention, the size of the exosomes may be between 10 and 200 nm.

According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection and inflammatory bowel disease.

The present invention also provides an immunomodulatory composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the present invention, the immune regulation is characterized by increasing the levels of anti-inflammatory cytokines, regulatory T cells and M2-type macrophages, and decreasing the levels of pro-inflammatory cytokines.
The therapeutic effect may be immunomodulatory, decreasing the levels of T cell cytokines, helper T cells and M1-type macrophages.

The present invention also provides a method for promoting the production of exosomes derived from mesenchymal stem cells, comprising the step of treating mesenchymal stem cells with an endoplasmic reticulum stress inducer.

The present invention also provides a method for treating an inflammatory disease, comprising administering to a patient suffering from the inflammatory disease a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

The present invention also provides a use of a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of an inflammatory disease.

Exosomes derived from mesenchymal stem cells (MSCs) are known to be less immunogenic than parental MSCs due to their low content of membrane-bound proteins, including tetraspanins (CD81, CD63, CD9), heat shock proteins (HSP60, HSP70, HSP90), and TSG 101, and therefore do not cause rejection due to activation of alloimmune responses at MHC-mismatched receptors.

The inventors have confirmed that exosomes (TSG-Exo) secreted from human Wharton's jelly-derived MSCs (WJ-MSCs) treated with thapsigargin (TSG), an endoplasmic reticulum (ER) stress inducer, have improved immunomodulatory properties, in order to apply MSC-derived exosomes to the treatment of immune-mediated inflammatory diseases.

When WJ-MSCs were stimulated with TSG, exosome secretion increased, and the yield and expression of immunomodulatory and anti-inflammatory factors such as IL-10, COX2, or IDO (Indoleamine 2,3-dioxygenase) increased, while the expression levels of pro-inflammatory factors (IFNγ, TNFα, or IL-1β) decreased or remained similar. The above results were also confirmed with TSG-Exo, suggesting that TSG-induced exosome release has a secretion mechanism different from inflammation- or senescence-associated secretion mechanisms (SASPs).

Furthermore, despite being derived from human MSCs, no obvious immune rejection reaction was observed when TSG-Exo was administered to a mouse colitis model, which meant that TSG-Exo was immune-tolerated by the host even in a xenogeneic system, and is advantageous for future clinical application. TSG-Exo substantially alleviated colitis in a mouse colitis model by reducing inflammatory responses and maintaining intestinal barrier integrity. The significant increase in Tregs and M2-type macrophages in the colon of colitis mice treated with TSG-Exo suggests the anti-inflammatory effect of TSG-Exo.
When treated with A-stimulated MNCs (mononuclear cells), the anti-inflammatory factors of exosomes were efficiently transferred to MNCs, resulting in similar expression in exosomes and exosome-treated MNCs.TSG-Exo showed better modulation of T cell/macrophage polarization than control exosomes, suggesting that exosome secretome may be a reliable indicator for predicting the therapeutic effect of exosomes.

TSG-Exo significantly enhanced the proliferation of human peripheral blood-derived T cells and suppressed Th1 and Th17 differentiation, whereas regulatory T cells and M2 macrophages were enriched compared to the control exosome-treated group. This suggests that intestinal Tregs and macrophages may be mediators for the anti-inflammatory effects of MSC exosomes. The upregulation of Tregs and M2 macrophages maintained intestinal homeostasis as suggested by disease activity score, histological score and reduced neutrophil activity.

These data imply that TSG treatment was sufficient to induce exosome release in WJ-MSCs and promoted the immunomodulatory properties of TSG-Exo, indicating that TSG-treated MSCs or TSG-Exo may provide a new therapeutic approach for the treatment of colitis.

Therefore, the present invention can provide a pharmaceutical composition for preventing or treating an inflammatory disease, comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be between 10 and 200 nm. Preferably, the size of the exosomes may be between 30 and 150 nm, more preferably between 50 and 120 nm.

According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection and inflammatory bowel disease. Preferably, the inflammatory disease may be inflammatory bowel disease, which may be ulcerative colitis, Crohn's disease or Behcet's disease.

The present invention also provides an immunomodulatory composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the present invention, the immune regulation is characterized by increasing the levels of anti-inflammatory cytokines, regulatory T cells and M2-type macrophages, and decreasing the levels of pro-inflammatory cytokines.
The therapeutic effect may be immunomodulatory, decreasing the levels of T cell cytokines, helper T cells and M1-type macrophages.

The present invention can also provide a method for promoting the production of exosomes derived from mesenchymal stem cells, comprising the step of treating mesenchymal stem cells with an endoplasmic reticulum stress inducer.

The promotion of production may refer to an increase in the number of exosomes or an increase in exosomal protein and mRNA production.

The present invention can also provide a method for treating an inflammatory disease, comprising administering to a patient with an inflammatory disease a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer.

According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be between 10 and 200 nm. Preferably, the size of the exosomes may be between 30 and 150 nm, more preferably between 50 and 120 nm.

According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection and inflammatory bowel disease. Preferably, the inflammatory disease may be inflammatory bowel disease, which may be ulcerative colitis, Crohn's disease or Behcet's disease.

The present invention can also provide a use of a composition comprising exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer for use in the prevention or treatment of an inflammatory disease.

According to a preferred embodiment of the present invention, the endoplasmic reticulum stress inducer may be one or more selected from the group consisting of thapsigargin (TSG), tunicamycin, brefeldin A, dithiothreitol (DTT), and MG132.

According to a preferred embodiment of the present invention, the mesenchymal stem cells may be obtained from one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle, and fetal liver. Preferably, the mesenchymal stem cells may be obtained from Wharton's jelly.

According to a preferred embodiment of the present invention, the size of the exosomes may be between 10 and 200 nm. Preferably, the size of the exosomes may be between 30 and 150 nm, more preferably between 50 and 120 nm.

According to a preferred embodiment of the present invention, the inflammatory disease may be any one or more selected from the group consisting of acute or chronic inflammatory diseases, chronic bronchitis, rhinitis, arthritis, autoimmune diseases, transplant rejection and inflammatory bowel disease. Preferably, the inflammatory disease may be inflammatory bowel disease, which may be ulcerative colitis, Crohn's disease or Behcet's disease.

The following definitions apply to the terms used in this specification:

In this invention, "prevention" refers to any action that suppresses inflammatory diseases or delays their onset.

"Improvement" or "treatment" in the present invention means any action that improves or benefits parameters related to inflammatory diseases, such as the severity of symptoms, by using exosomes derived from mesenchymal stem cells treated with the endoplasmic reticulum stress inducer of the present invention.

The pharmaceutical compositions of the present invention may be in various parenteral dosage forms. When the compositions are formulated, they may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickeners, wetting agents, disintegrants or surfactants, diluents or excipients.

Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, and gelatin.

The composition of the present invention can be administered parenterally, and can be formulated by methods known in the art in the form of an injectable solution for parenteral administration by intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intrauterine dura or intracerebrovascular injection.

The injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. In the case of injections, suitable carriers may be, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and/or vegetable oils. More preferably, suitable carriers may be Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injections from contamination by microorganisms, various antibacterial and antifungal agents such as paraben, chlorobutanol, phenol, sorbic acid, thimerosal, etc. may be further included. In addition, the injections may often further include isotonic agents such as sugar or sodium chloride.

The compositions of the present invention are administered in a pharma- tically effective amount. A pharma-tically effective amount means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment duration, co-administered drugs, and other factors well known in the medical field. The compositions of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered in single or multiple doses. That is, the total effective amount of the compositions of the present invention can be administered to a patient in a single dose or by a fractionated treatment protocol in which multiple doses are administered over an extended period of time. Taking all of the above factors into consideration, it is important to administer an amount that can obtain maximum effect at a minimum dose without side effects, which can be easily determined by one of ordinary skill in the art.

The dosage of the pharmaceutical composition of the present invention varies depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage is preferably 0.01 to 50 mg, more preferably 0.1 to 30 mg, per kg of body weight per day based on the exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer when administered parenterally, and is preferably 0.01 to 100 mg, more preferably 0.01 to 10 mg, per kg of body weight per day based on the exosomes derived from mesenchymal stem cells treated with an endoplasmic reticulum stress inducer when administered orally.
The dosage may be administered in an amount of 100 mg once or in several divided doses, however, the dosage may be increased or decreased depending on the route of administration, the severity of obesity, sex, body weight, age, etc., and the above dosage does not limit the scope of the present invention in any way.

The compositions of the present invention can be used alone or in combination with surgery, radiation therapy, hormonal therapy, chemotherapy and methods using biological response modifiers.

Exosomes derived from mesenchymal stem cells treated with endoplasmic reticulum stress inducers according to the present invention (TSG-Exo) have increased immunomodulatory capabilities, including increased levels of anti-inflammatory cytokines, regulatory T cells, and M2 macrophages, and decreased levels of pro-inflammatory cytokines, helper T cells, and M1 macrophages, and effectively alleviated inflammation in a mouse model of colitis. Therefore, the present invention can be effectively used as a composition for preventing or treating inflammatory diseases and as a composition for immunomodulation.

FIG. 1a shows the expression levels of MSC-specific antigens CD29, CD44, CD73, CD105, CD146, and CD34, CD45 by flow cytometry as cell surface markers and exosome (Exos) isolation of WJ-MSCs. FIG. 1b shows cell surface markers of WJ-MSCs and a scheme for exosome (Exos) isolation based on ultracentrifugation. Figure 2 shows the characteristics of exosomes derived from naive (CTL-Exo) or TSG-primed WJ-MSCs (TSG-Exo). (A) Transmission electron microscopy (TEM) images of exosomes (scale bar = 100 nm); (B) Western blot analysis of CD63 and β-actin in WJ-MSCs and exosomes; (C) Exosome size distribution and concentration by qNano; (D) Exosome protein concentration by BCA protein analysis; (*p<0.05, **p<0.01, ***p<0.001). FIG. 3 shows immune modulators of exosomes derived from naive (naive; CTL-Exo) or TSG-primed WJ-MSCs (TSG-Exo). (E) Relative mRNA expression levels of immune regulators by qRT-PCR; (F) levels of TGFβ, IDO and COX2 by Western blot analysis; (G) relative mRNA expression levels of immune regulators in Con A stimulated MNCs co-cultured with CTL-Exo (+CTL-Exo) or TSG-Exo (+TSG-Exo) for 48 hours by qRT-PCR; (H) levels of early or late apoptosis in Con A stimulated MNCs by flow cytometry; (*p<0.05, **p<0.01, ***p<0.001). 4 shows the T cell proliferation regulation of TSG-primed WJ-MSC-derived exosomes (TSG-Exo) on activated peripheral blood mononuclear cells (PBMCs): (A) Representative histogram of total T cell proliferation; (B) Quantitative analysis of total T, CD4+ T and CD8+ T cell proliferation rates; (C) Fluorescence profiles of CFSE-labeled cells in five individual PBMC fractions. Figure 5 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate helper T cell (Th) differentiation and macrophage polarization. (D) Percentages of CD4+IFNγ+Th1, CD4+IL-17A+Th17, and CD4+CD25+Foxp3+Treg by flow cytometry; (E) Expression levels of M1 macrophage (CD80, CD86) or M2 macrophage (CD206, CD163) surface markers by flow cytometry. FIG. 6: TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate helper T cell (Th) differentiation, showing the percentages of Th1, Th17 and Treg cells by flow cytometry. FIG. 7 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate macrophage polarization, and shows representative images of M0, M1 and M2 (scale bar=100 μm). FIG. 8 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) regulate macrophage polarization and demonstrate the expression of M1 and M2 macrophage surface markers by flow cytometry. 9 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) ameliorate DSS-induced colitis in mice. (A) Schematic image of DSS-induced colitis experiment; (B) body weight; and (C) change in Disease Activity Index (DAI) score; are shown (n=3 mice per group; *p<0.05, **p<0.01, ***p<0.001). Figure 10 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) ameliorate DSS-induced colitis in mice. (D,E) Colon length measured at day 10 when mice were sacrificed; (F) H&E staining of colon sections (scale bar = 100 μm); (G) histological score; (H) colonic MPO activity are shown (n = 3 mice per group; *p<0.05, **p<0.01, ***p<0.001). 11 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) induce regulatory T cell and M2 macrophage polarization in vivo. (A) Th1, Th2, Th17 and Treg-associated cytokines in colon tissues by qRT-PCR; (B) expression levels of macrophage phenotype-associated genes; (*p<0.05, **p<0.01, ***p<0.001). 12 shows that TSG-primed WJ-MSC-derived exosomes (TSG-Exo) induce M2 macrophage polarization in the colonic lamina propria in vivo. (C) The percentage of F4/80+CD11b+ macrophages in lamina propria cells of 3% DSS-colitic mice colon analyzed and quantified by flow cytometry (D); (E) CD206 and Arginase 1 expression in F4/80+CD11b+ macrophages analyzed and quantified by flow cytometry; shown as relative mean fluorescence intensity (RelMFI) normalized to the control group (*p<0.05, **p<0.01, ***p<0.001). FIG. 13 shows the secretion patterns of endoplasmic reticulum stress markers, pro-inflammatory and anti-inflammatory cytokines according to the types of endoplasmic reticulum stress inducers (thapsigargin (TSG, Th), tunicamycin (Tu), brefeldin A (BFA), dithiothreitol (DTT), and MG132 (MG)) treated in human WJ-MSCs. It was confirmed that the expression levels of endoplasmic reticulum stress marker (GRP94) and anti-inflammatory cytokines (COX2, IDO, IL-10) increased, while the expression levels of pro-inflammatory cytokines (IL-1β, IFNγ, TNFα) decreased, by treatment with endoplasmic reticulum stress inducers. 14 shows the results of confirming the inhibition of T cell proliferation as an immunomodulatory effect of human WJ-MSCs treated with endoplasmic reticulum stress inducers (TSG, Tu, BFA, DTT, MG). It was confirmed that MSCs treated with endoplasmic reticulum stress inducers had a greater inhibitory effect on the proliferation of total T, CD4+, and CD8+ T cells compared to untreated MSCs. 15 shows the results of confirming the inhibition of T cell proliferation as an immunomodulatory effect of exosomes secreted from human WJ-MSCs treated with endoplasmic reticulum stress inducers (TSG, Tu, BFA, DTT, MG). It was confirmed that MSCs treated with endoplasmic reticulum stress inducers TSG, Tu, or BFA showed a greater inhibitory effect on the proliferation of total T, CD4+, and CD8+ T cells compared to untreated MSCs.

[Example 1]
Confirmation of exosome characteristics derived from TSG-primed WJ-MSCs <1-1> Preparation and culture of WJ-MSCs Wharton's jelly-derived mesenchymal stem cells (WJ-MSCs) were isolated and cultured based on Non-Patent Document 3, and further characterized based on the surface marker expression profile.

Specifically, Wharton's jelly tissue was obtained from the umbilical cord after normal full-term delivery with written consent from the pregnant woman and approval from the Institutional Review Board of Asan Medical Center, Seoul (Protocol No. 2015-3030). WJ-MSCs were isolated according to Non-Patent Document 1 and subjected to a mycoplasma test. Cells were cultured in _DMEM (Gibco BRL, Grand Island, NY) with 10% FBS (Tissue Culture Biologicals, Tulare, CA), 1% Glutamax (Gibco), 25 ng/ml EGF (Biolegend NS, Inc., San Diego, CA), 50 ng/ml bFGF (Peprotech, Rocky Hill, NJ) and 1% antibiotic/antimicrobial (Gibco) at 37°C and 5% CO2. Upon reaching 80-90% confluence, cells were detached with 0.05% Trypsin-EDTA (Gibco) and further subcultured.

Flow cytometry demonstrated that WJ-MSCs positively expressed MSC-specific cell surface markers CD29, CD44, CD73, CD105, and CD146, and negatively expressed hematopoietic stem cell-specific markers CD34 and CD45 (Figure 1a).

<1-2> Isolation and characterization of exosomes derived from WJ-MSCs We sought to confirm the expression of inflammatory genes, along with exosome-specific markers, and pro- and anti-inflammatory cytokine secretion patterns to determine the immunomodulatory properties of MSCs or MSC-derived exosomes.

WJ-MSC-derived exosomes (Exo(s)) were isolated from culture supernatants of naive (CTL-Exo) or TSG-primed WJ-MSC (TSG-Exo) by standard ultracentrifugation (Fig. 1b). WJ-MSC cells (3 x 10 ⁶ ) were stimulated with 1 μM thapsigargin (TSG; Sigma-Aldrich, St. Louis, MO) in 100 mm culture dishes for 24 h. The medium was then replaced with medium supplemented with 10% Exo-depleted FBS. Exo-depleted FBS was obtained by ultracentrifugation at 100,000 x g for 18 h. After 72 hours of culture, the culture medium was collected and centrifuged at 300xg for 10 minutes, 2,500xg for 25 minutes, and 10,000xg for 1 hour at 4°C. The supernatant was filtered using a 0.22 μm filter and ultracentrifuged at 100,000xg for 2 hours. The Exo pellet was then washed with sterile PBS by centrifugation at 100,000xg for 2 hours, suspended in 200 μl of PBS, and stored at -80°C until use. To suppress Exo production, the cells were treated with culture medium containing 10 μM GW4869 (Santa Cruz Biotechnology, Dallas, TX) for 12 hours. The medium was then replaced with medium supplemented with 10% Exo-depleted FBS. After 72 hours, the culture supernatant was ultracentrifuged to separate Exo and stored at -80°C until use (concentrated CM). Concentrated CM derived from GW4869-treated MSCs (suppressing Exo secretion) was used as a vehicle control for all mouse experiments. The amount of Exo was determined by BCA analysis kit (Thermo Fisher Scientific, Waltham, MA) for the measurement of total protein. Exo size and number were determined by qNano (Izon Science, Christchurch, New Zealand) and transmission electron microscope (TEM; Jeol, Tokyo, Japan) according to the manufacturer's guidelines.

For Western blot analysis, WJ-MSC lysates and Exo were dissolved in RIPA buffer (Thermo Fisher) with protease inhibitors. Equal amounts of protein were applied to SDS-PAGE and analyzed with CD63 (SBI), COX2 (Abcam, Cambridge, UK), IDO (Merk Millipore, Darmstadt, Germany), TGFβ, and GAPDH (Santa Cruz) primary antibodies. Bands were detected using enhanced chemiluminescence (Thermo Fisher), and images were captured using the LAS-3000 system (Fujifilm, Tokyo, Japan).

As a result, transmission electron microscopy analysis showed that the purified exosomes contained round vesicles with diameters of 50-120 nm (Fig. 2A). The exosome-specific marker CD63 was expressed in CTL-Exo and TSG-Exo, whereas the cytoplasmic marker β-actin was not expressed in any of the exosomes (Fig. 2B). qNano analysis showed that the average particle size of exosomes was 110-120 nm in both CTL-Exo and TSG-Exo (Fig. 2C). The production of exosomes from TSG-primed WJ-MSCs was 50% higher than that from naive WJ-MSCs (Fig. 2C, D). TSG-Exo had significantly higher expression levels of IL-10, TGFβ, COX2, and IDO, but lower expression of proinflammatory cytokines, including IFNγ, TNFα, and IL-1β, compared to CTL-Exo (Figure 3E). Consistent with the mRNA expression results, TGFβ, COX2, and IDO protein expression levels were significantly increased in TSG-Exo (Figure 3F).

[Example 2]
Confirmation of the immunomodulatory effect of TSG-primed exosomes - suppression of T cell proliferation To evaluate the immunomodulatory potential of TSG-primed exosomes, expression levels of anti- and pro-inflammatory cytokines were measured in activated mononuclear cells (MNCs).

For quantitative RT-PCR, total RNA was extracted from cell, Exos or tissue samples using TRizol Reagent (Invitrogen, Waltham, MA), and cDNA was synthesized from total RNA using Superscript ^™ reverse transcriptase (Invitrogen). Quantitative PCR was measured using SYBR Green Master Mix (Applied Biosystems, Foster City, CA) in an MX300P thermal cycler (Stratagene, San Diego, CA). mRNA expression levels were normalized to the levels of GAPDH. Primer sequences for qRT-PCR are listed in Table 1 below.

Mononuclear cells (MNCs) for apoptosis analysis were isolated from hUCB using Ficoll-Hypaque (GE Healthcare, Chicago, IL) gradient centrifugation based on Non-Patent Document 2 and resuspended in RPMI 1640 medium supplemented with 10% Exo-depleted FBS. Umbilical cord blood (UCB) units were acquired from Korea Catholic Hematopoietic Stem Cell Bank (CHSCB) from April 2019 to June 2020 with Institutional Review Board approval (IRB No. 2019-0467-0003). MNCs were stimulated with 5 μg/ml Con A (Concanavalin A) and seeded in 96-well plates (1 × 10 ^cells /well) in growth medium with or without Exos (4 μg) derived from naive or TSG-primed WJ-MSCs for 72 h. Annexin V/7AAD (BD Biosciences, Franklin Lakes, NJ) staining was performed to determine cell apoptosis according to the manufacturer's guidelines and analyzed by flow cytometry.

Next, to test the immunosuppressive effect of TSG-Exo on the proliferation of activated peripheral blood-derived mononuclear cells (PBMCs), human peripheral blood from healthy donors was provided by the Korean Red Cross (Seoul, Korea) with the approval of the Institutional Review Board of The Catholic University of Japan (IRB No. 2019-2891-0003). PBMCs were separated by centrifugation through a Ficoll-Hypaque density gradient and resuspended in RPMI 1640 medium supplemented with 10% Exo-depleted FBS.

T cell proliferation was quantified by CFSE dilution. Isolated PBMCs were labeled with 2 μM CFSE (CellTrace CFSE Cell Proliferation Kit, Thermo Fisher Scientific). CFSE-labeled PBMCs (1×10 ⁵ /well) were co-cultured in 96-well plates in the presence of anti-CD3/CD28 microbeads (Gibco) and recombinant human IL-2 (30 U/ml, Peprotech) with or without Exos (4 μg) derived from naive or TSG-primed WJ-MSCs. After 6 days of culture, cells were stained with fluorescently labeled human monoclonal antibodies to CD45, CD3, CD4, or CD8 (BD Biosciences) and analyzed by flow cytometry. All cells were gated on 7AAD-negative cells.

As a result, exosome treatment of MNCs stimulated with Con A (concanavalin A) increased the expression levels of COX2, NOS2, IDO, IL-10 and TGFβ, whereas it decreased the expression levels of TNFα and IL-1β. This was more prominent in activated MNCs treated with TSG-Exo than those treated with CTL-Exo (Fig. 3G). TSG-Exo slightly reduced early apoptosis in MNCs but had no effect on late apoptosis, suggesting that exosome treatment does not induce cytotoxicity (Fig. 3H). CFSE dilution analysis showed that the addition of TSG-Exo more potently suppressed the proliferation of total T, CD4+ T and CD8+ T cells than CTL-Exo (Fig. 4A, B). In TSG-Exo-treated PBMCs, the percentage of cells in early division cycle 1 was significantly increased, and the percentage of cells in division cycles 4 and 5 was significantly decreased (Figure 4C). These results suggest that TSG-primed exosomes increase the production of anti-inflammatory cytokines and have a significant inhibitory effect on T cell proliferation.

[Example 3]
Confirmation of immunomodulatory effect of TSG-primed exosomes - Enhancement of regulatory T cells and M2 macrophage polarization <3-1> Confirmation of T helper cell (Th) differentiation of TSG-primed exosomes It has been reported that MSC exosomes can mediate immunomodulatory effects through differentiation of regulatory T cells (Tregs). Therefore, the present inventors investigated the differentiation of T helper cells (Th) to confirm the effect of TSG-Exo on specific T cell subsets.

Th cells were induced by stimulation with family-specific cytokines on CD4+ cells purified from human PBMCs in the presence of exosomes. Specifically, CD4+ T cells were isolated from PBMC lymphocytes using a human CD4 T cell isolation kit (Miltenyi Biotec, Marburg, Germany) according to the manufacturer's protocol. CD4+ T cells were stimulated with anti-CD3/CD28 microbeads and IL-2 (20 ng/ml), and specific cytokines were added for differentiation of specific Th cells: IFNγ (25 ng/ml) and IL-12 (25 ng/ml) for Th1; IL-6 (50 g/ml) and TGFβ (25 ng/ml) for Th17; TGFβ (25 ng/ml) and retinoic acid (10 nM) for Treg. Cytokine-treated CD4+ T cells ( ^5x105 /well) were cultured in 24-well plates with Exos derived from naive or TSG-primed WJ-MSCs for 5 days. For intracellular staining, cells were stimulated with cell stimulation cocktail (Invitrogen) for 5 hours before staining. Cells were stained with cell surface markers anti-CD3, anti-CD4, and anti-CD25 antibodies, then fixed/permeabilized and stained with fluorescently labeled anti-IFNγ, anti-IL-17A, and anti-Foxp3 antibodies (BD Biosciences). Stained cells were analyzed by flow cytometry.

As a result, coculture with TSG-Exo significantly reduced the induction of IFNγ+CD4+Th1 and IL-17A+CD4+Th17 cells and increased Foxp3+CD25+CD4+Treg cells compared to CTL-Exo (Fig. 5D and Fig. 6). M1 macrophages showed a round morphology, whereas M2 macrophages showed a spindle-shaped morphology (Fig. 7).

<3-2> Confirmation of enhancement of M2 macrophage polarization by TSG-primed exosomes It has been reported that MSC exosomes can mediate immunomodulatory effects through induction of M2 macrophage polarization. Therefore, to confirm the effect of TSG-Exo on specific T cell subsets, the present inventors investigated whether TSG-Exo can regulate the phenotype of macrophages.

GM-CSF (for M1 type) or M-CSF (for M2 type) stimulated primary human macrophages were cultured with exosomes in the presence of M1 type or M2 type cytokines. Specifically, CD14+ monocytes were isolated from hUCB-derived MNCs using CD14-conjugated microbeads (Miltenyi Biotec). To generate macrophages, monocytes ( ^5x105 /well) were cultured with GM-CSF (50ng/ml; Peprotech) or M-CSF (100ng/ml) in RPMI 1640 medium (Gibco) containing 10% FBS for 6 days. For M1 polarization in 24-well plates, GM-CSF-derived macrophages were stimulated with IFNγ (ng/ml) plus LPS (1 μg/ml) together with Exo derived from naive or TSG-primed WJ-MSCs for 48 h. For M2 polarization, M-CSF-derived macrophages were stimulated with IL-4 (20 ng/ml) and IL-13 (20 ng/ml) together with Exo derived from naive or TSG-primed WJ-MSCs for 48 h. After co-culture, cells were stained with fluorescently labeled human monoclonal antibodies against CD14, CD80, CD86, CD206, and CD163 (BD Biosciences) and analyzed by flow cytometry.

As a result, the M1 cell surface markers CD80+ and CD86+ were significantly decreased in M1 macrophages cultured with TSG-Exo, whereas those cultured with CTL-Exo. However, the expression of M2 cell surface markers CD206+ and CD163+ was increased when cultured with exosomes, and this effect was even greater in the TSG-Exo-treated group (Figures 5E and 8). These results suggest that TSG-Exo significantly reduced the expression of M1 macrophages cultured with TSG-Exo.
These results suggest that TSG-treated MSCs induces the M2 macrophage phenotype more effectively than naive exosomes. Thus, TSG treatment of MSCs can generate exosomes with improved immunomodulatory properties.

[Example 4]
Confirmation of the Alleviating Effect of TSG-Primed Exosomes on DSS-Induced Colitis The present inventors confirmed the potential protective effect of TSG-Exo in a 3% DSS-induced colitis mouse model (FIG. 9A).

Specifically, to prepare a DSS-induced colitis mouse model, 3% (w/v) DSS (Dextran sulfate) was added to the drinking water of 7-week-old C57BL/6 mice for 7 days.
The mice were induced by administration of 200 μg of Exo diluted in 200 μl PBS (MP Biomedicals, Santa Ana, CA). Mice were divided into 4 groups of 10 mice each: (1) control group (negative control group), (2) DSS administered with concentrated CM extracted from GW4869-treated MSCs (solvent control group, 200 μl) (positive control group), (3) DSS administered with Exo derived from naive MSCs, and (4) DSS administered with Exos derived from TSG-primed MSCs. Mice in groups (3) and (4) were intraperitoneally injected with 200 μg of Exo diluted in 200 μl PBS on days 1, 3, and 5. Mice were euthanized on day 10. The severity of colitis was assessed daily using a disease activity index (DAI) including weight loss (0-4), stool consistency (0-4), blood in stool (0-4), coat roughness (0-4), rectal prolapse (0-3), curved posture (0-3), bedding soiling (0-2), and not inquisitive/alert (0-2). All animal experimental procedures were approved by the Animal Research Ethics Committee of The Catholic University of Japan (IACUC no. 2019-0301-03). As a vehicle control group, a culture supernatant of MSCs treated with GW4869 (10 μM), an inhibitor of neutral sphingomyelinase supernatant 2 (nSMase2) that regulates exosome secretion (GW-CM) was used.

Mice were sacrificed on day 10 and the colon lengths were measured. Colon tissues were fixed in 4% formaldehyde (Wako, Osaka, Japan), embedded in paraffin, and sectioned at 5 μm. For histological analysis, colon sections were stained with hematoxylin and eosin (H&E). Histopathological scores were determined in a blinded manner according to the degree of inflammatory cell infiltration (0-4) and the degree of tissue damage (0-4).

Neutrophil infiltration into colonic tissue was measured using the myeloperoxidase (MPO) Colorimetric Activity Assay Kit (Sigma). The colon was homogenized in MPO assay buffer, and the supernatant was collected by centrifugation at 13,000 x g for 10 min. The supernatant was mixed with MPO assay buffer and MPO substrate, and incubated at 25°C for 120 min, after which a tetramethylbenzidine (TNB) probe was added. Absorbance was measured at 412 nm using a spectrophotometer (Biotek, Winooski, WT).

As a result, GW4869 successfully inhibited exosome release in MSCs. GW-CM-treated colitic mice showed persistent weight loss and significantly elevated disease activity index (DAI), such as stool consistency, bloody diarrhea, and general activity. However, exosome treatment improved weight loss and DAI, especially in the TSG-Exo-treated group (Fig. 9B, C). Furthermore, the shortening of colon length by DSS administration was significantly improved in TSG-Exo-treated mice compared with CTL-Exo-treated mice (Fig. 10D, E). Histological examination showed that TSG-Exo-treated mice maintained colonic tissue integrity; significant structural destruction, loss of crypt cells, and reduced infiltration of inflammatory cells; and had even lower histological scores compared with CTL-Exo-treated mice (Fig. 10F, G). Furthermore, MPO (myeloperoxidase) activity, which indicates neutrophil infiltration, was significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice in mice sacrificed on day 10 (Figure 10H). These results indicate that TSG-Exo has improved protective activity against intestinal inflammation in DSS-induced colitis compared to naive exosomes.

[Example 5]
Confirmation of the effect of TSG-primed exosomes on enhancing polarization of regulatory T cells and M2 macrophages in the inflamed colon Immune cell responses play an important role in the pathogenesis of inflammatory bowel disease (IBD). In particular, the balance between Tregs and other T cells in the intestinal microenvironment plays an important role in alleviating colitis, and macrophages mediate inflammatory responses through M1/M2 polarization in the colon and play an important role in the pathogenesis of IBD. Therefore, we attempted to confirm the immunomodulatory effect of TSG-primed exosomes by enhancing polarization of regulatory T cells and M2 macrophages in the inflamed colon. We then investigated the effect of TSG-primed exosomes on the immune cell profile of mice with DSS-induced colitis.

Specifically, 3% DSS-colitis mice (n=2-4 mice per group) were sacrificed on day 10, colon tissues were harvested, and the levels of Th1, Th2, Th17, and Treg cells in the colon tissues were evaluated. Lamina propria cells were isolated from the intestine. Colon tissues were cut into small pieces (3mm x 3mm) and incubated with HBSS buffer containing 2mM EDTA (Sigma) in a shaking incubator at 220 rpm for 30 minutes. After removing the epithelium at 37°C, the tissue was digested in RPMI containing 5% FBS, 1 mg/ml Collagenase D (Sigma) and 0.1 mg/ml DNase I (Sigma) at 220 rpm for 60 min at 37°C in a shaking incubator. The digested sample was filtered through a 40 μm cell strainer and then centrifuged at 300 x g for 5 min. The isolated cells were stained with fluorescently labeled antibodies against CD11b, F4/80, CD206 and arginase 1 (BD Biosciences) and analyzed by flow cytometry.

As a result, the expression levels of Th1 (T-bet), Th2 (GATA3) and Th17 (RORγ family transcription factors) were increased in colonic tissues of GW-CM-treated mice, but decreased in exosome-treated mice. In particular, GATA3 expression was further significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice. Foxp3 and Treg transcription factors were significantly increased in TSG-Exo-treated mice compared to CTL-Exo-treated mice (Figure 11A).

In addition, the expression of M1-type markers CXCL9, MCP1, and iNOS was decreased in CTL-Exo- and TSG-Exo-treated mice compared to GW-CM-treated mice. CXCL9 levels were significantly decreased in TSG-Exo-treated mice compared to CTL-Exo-treated mice. The expression of M2-type markers Arg1 and CD206 was increased with exosome treatment, and this effect was significantly greater in the TSG-Exo-treated group (Figure 11B).

The proportion of F4/80+CD11b+ cells (macrophages) in the lamina propria of the colon was significantly increased in DSS-induced colitis mice compared to negative control mice (Fig. 12C, D). Exosome treatment did not affect the proportion of F4/80+CD11b+ cells (Fig. 12C, D), but the expression levels of CD206 and arginase 1 in colonic macrophages were significantly elevated in TSG-primed exosome-treated mice (Fig. 12E). These data suggest that TSG-primed exosomes attenuate mucosal inflammation by inducing regulatory T cells and polarizing M2-type macrophages.

[Example 6]
Analysis of ER stress-induced WJ-MSC or exosome <6-1> Confirmation of characteristics of ER stress-induced WJ-MSC To investigate the effect of endoplasmic reticulum stress (ER-stress) on the immunoregulatory properties of human MSC, ER-stress inducers, thapsigargin (TSG, Th, apsigargin), tunicamycin (Tu), brefeldin A (BFA), dithiothreitol (DTT, dithiothreitol) or MG132 (MG), were treated in the same manner as in Example <1-2> above, and the expression of ER stress markers and inflammatory genes was confirmed in ER stress-induced WJ-MSC.

As a result, as shown in Figure 13, the ER-stress markers GRP94 and CHOP were significantly increased in human MSCs after treatment with an ER-stress inducer. In addition, it was confirmed that MSCs treated with an ER-stress inducer had significantly higher expression levels of anti-inflammatory cytokines COX2, IDO, and IL-10 than untreated MSCs, but had lower expression of pro-inflammatory cytokines including IL-1β, IFNγ, and TNFα.

<6-2> Confirmation of immunomodulatory effect of ER stress-induced WJ-MSCs - inhibition of T cell proliferation To evaluate the immunomodulatory properties of MSCs treated with endoplasmic reticulum stress (ER-stress) inducers on T cell proliferation, PBMCs and MSCs were co-cultured in the same manner as in Example <1-2> above.

As a result, as shown in Figure 14, it was confirmed that MSCs treated with ER-stress inducers (TSG, Tu, BFA, DTT, MG) had a greater inhibitory effect on the proliferation of total T, CD4+, and CD8+ T cells compared to untreated MSCs.

These results indicate that MSCs subjected to ER stress by ER-stress inducers increase the production of anti-inflammatory cytokines and effectively function in strengthening the suppression of T cell proliferation.

<6-3> Confirmation of immunomodulatory effect of exosomes secreted from ER stress-induced WJ-MSCs - inhibition of T cell proliferation To confirm the immunosuppressive effect of exosomes secreted from MSCs treated with ER stress inducers (TSG, Tu, BFA, DTT, MG), PBMCs and exosomes were co-cultured in the same manner as in Example 2.

As a result, as shown in Figure 15, it was confirmed that exosomes treated with TSG, BFA, and Tu more strongly inhibited the proliferation of total T, CD4+ T, and CD8+ T cells compared to the control group (cont-exo).

These results indicate that ER-stress inducers can generate exosomes with improved immunomodulatory properties in MSCs.

[Statistical analysis]
Statistical analysis was performed using one-way analysis of variance (ANOVA) and Tukey's multiple comparison test with GraphPad Prism v8.0.1 (GraphPad Software, San Diego, CA). All data were expressed as mean ± S.D. A P value <0.05 was considered statistically significant.

Exosomes derived from mesenchymal stem cells treated with endoplasmic reticulum stress inducers according to the present invention (TSG-Exo) have increased immunomodulatory capabilities, including increased levels of anti-inflammatory cytokines, regulatory T cells, and M2 macrophages, and decreased levels of pro-inflammatory cytokines, helper T cells, and M1 macrophages, and effectively alleviated inflammation in a mouse model of colitis. Therefore, the present invention has industrial applicability since it can be effectively used as a composition for preventing or treating inflammatory diseases and as a composition for immunomodulation.

Sequence List Free Text
SEQ ID NO:1 corresponds to the forward primer sequence of hCOX-2.
SEQ ID NO:2 corresponds to the reverse primer sequence for hCOX-2.
SEQ ID NO:3 corresponds to the forward primer sequence of hIDO.
SEQ ID NO: 4 corresponds to the reverse primer sequence of hIDO.
SEQ ID NO:5 corresponds to the forward primer sequence of hIFNγ.
SEQ ID NO:6 corresponds to the reverse primer sequence for hIFNγ.
SEQ ID NO:7 corresponds to the forward primer sequence of hIL-10.
SEQ ID NO:8 corresponds to the reverse primer sequence for hIL-10.
SEQ ID NO:9 corresponds to the forward primer sequence of hIL-1β.
SEQ ID NO: 10 corresponds to the reverse primer sequence for hIL-1β.
SEQ ID NO: 11 corresponds to the forward primer sequence of hNOS2.
SEQ ID NO: 12 corresponds to the reverse primer sequence of hNOS2.
SEQ ID NO: 13 corresponds to the forward primer sequence of hTGFβ.
SEQ ID NO: 14 corresponds to the reverse primer sequence for hTGFβ.
SEQ ID NO: 15 corresponds to the forward primer sequence for hTNFα.
SEQ ID NO: 16 corresponds to the reverse primer sequence for hTNFα.
SEQ ID NO: 17 corresponds to the forward primer sequence of mArg1.
SEQ ID NO: 18 corresponds to the reverse primer sequence of mArg1.
SEQ ID NO: 19 corresponds to the forward primer sequence of mCD206.
SEQ ID NO:20 corresponds to the reverse primer sequence of mCD206.
SEQ ID NO:21 corresponds to the forward primer sequence of mCXCL9.
SEQ ID NO:22 corresponds to the reverse primer sequence of mCXCL9.
SEQ ID NO: 23 corresponds to the forward primer sequence of mFoxp3.
SEQ ID NO:24 corresponds to the reverse primer sequence of mFoxp3.
SEQ ID NO:25 corresponds to the forward primer sequence of mGATA3.
SEQ ID NO:26 corresponds to the reverse primer sequence of mGATA3.
SEQ ID NO:27 corresponds to the forward primer sequence of miNOS.
SEQ ID NO:28 corresponds to the reverse primer sequence of miNOS.
SEQ ID NO:29 corresponds to the forward primer sequence of mMCP1.
SEQ ID NO:30 corresponds to the reverse primer sequence of mMCP1.
SEQ ID NO:31 corresponds to the forward primer sequence of mRORγ.
SEQ ID NO:32 corresponds to the reverse primer sequence of mRORγ.
SEQ ID NO:33 corresponds to the forward primer sequence of mT-bet.
SEQ ID NO:34 corresponds to the reverse primer sequence for mT-bet.

Claims (3)

A method for promoting the production of exosomes derived from mesenchymal stem cells, comprising the step of treating mesenchymal stem cells collected from a human with an endoplasmic reticulum stress inducer, wherein the endoplasmic reticulum stress inducer is thapsigargin (TSG). The method according to claim 1, wherein the mesenchymal stem cells are obtained from one or more tissues selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, amniotic fluid, Wharton's jelly, placenta, peripheral blood, fallopian tube, corneal stroma, lung, muscle and fetal liver. 2. The method of claim 1, wherein the exosomes are in the range of 10-200 nm in size.