Patent Application
20220062345
The present invention elates to a pharmaceutical composition for preventing or treating an autoimmune disease, including stem cells treated with a protein kinase C activator or a culture thereof, and more specifically, the present invention relates to a pharmaceutical composition for preventing or treating an autoimmune disease, which can inhibit the function of B cells and have an excellent prophylactic or therapeutic effect for an autoimmune disease.
Stem cells are collectively referred to as undifferentiated cells at a stage before being differentiated into each cell constituting tissues, and differentiation into specific cells proceeds by specific differentiation stimuli (environment). Unlike differentiated cells in which cell division is stopped, stem cells can produce cells which are identical to themselves (self-renewal) by cell division, and thus, they have the property of proliferation (expansion). In addition, when a differentiation stimulus is applied, they differentiate into specific cells, and since they can be differentiated into other cells by different environments or different differentiation stimuli, they are characterized in having plasticity of differentiation.
The pluripotency of these stem cells provides a good in vitro experimental model for the study of human developmental processes. New drug development can he facilitated by performing drug tests and toxicity tests on homogeneous human tissues or cells obtained from stem cells. Furthermore, it is possible to obtain a large amount of cells or tissues that can replace damaged tissues, which can he used to treat incurable diseases.
Mesenchymal stem cells are cells that maintain sternness and self-renewal and have. the capacity to differentiate into various mesenchymal tissues (plasticity). These cells can be extracted from bone marrow, adipose tissue, umbilical cord blood, synovial membrane, trabecular hone, infrapatellar fat pad, and the like. Mesenchymal stem cells inhibit the activity and proliferation of T lymphocytes and B lymphocytes, suppress the activity of natural killer cells (NK cells), and have immunomodulatory capacity to regulate the functions of dendritic cells and macrophages, and thus, these are cells capable of allotransplantation and xenotransplantation. In addition, mesenchymal stem cells have the ability to differentiate into various connective tissues such as cartilage, hone tissue, ligaments, bone marrow matrix, and the like. In particular, adipose-derived mesenchymal stern cells derived from adipose tissue are subdivided into names such as adipose-derived tem/stromal cells (ASC), adipose-derived adult stem cells (ADAS), or the like, and these can be applied to the production of biomaterials to treat soft tissue defects caused by trauma, tumor removal surgery, burns, and the like.
Meanwhile, lupus activates dendritic cells, T cells, and B cells by autoantigens as an abnormal immune response. Therefore, autoantibodies are produced, and symptoms appear by invading various organs by forming immune complexes with autoantigens. Various therapeutic agents have been developed to treat such lupus disease, and in particular, mesenchymal stem cells (MSC) having unornodulatory action are being studied as promising therapeutic agents. Although MSC has been reported to inhibit the function of T cells, studies on MSC capable of inhibiting the function of B cells are insufficient.
Korean Patent Laid-Open No. 10-2015-0144679
The present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, including stem cells treated with a protein kinase C activator or a culture thereof.
In addition, the present invention provides a method for preventing or treating an autoimmune disease, including administering the pharmaceutical composition to a subject other than humans.
In addition, the present invention provides a method for preparing an immunosuppressive agent, including culturing by adding a protein kinase C activator to stem cells.
In addition, the present invention provides a method for inhibiting an immune response of a subject other than humans, including administering stem cells treated with a protein kinase C activator or a culture thereof to the subject.
However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems that are not mentioned will be clearly understood by those skilled in the art from the following description.
The present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, including stem cells treated with a protein kinase C activator or a culture thereof.
The protein kinase C activator may include one or more selected from the group consisting of phorbol myristate acetate, ingenol 3-angelate, bryostatin-1, phorbol-12,13-dibutyrate, prostatin, N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide (SC-9), and 5-chloro-N-heptylnaphthalene-1-sulfonamide (SC-10).
The stem cells may be adult stem cells, pluripotent stem cells, induced pluripotent stem cells, or embryonic stem cells.
The adult stem cells may be mesenchymal stem cells, mesenchymal stromal cells, or multipotent stem cells.
The protein kinase C activator may increase the expression of CXCL10 in stem cells.
The protein kinase C activator may increase apoptosis of B cells by increasing the expression of programmed death-ligand 1 (PD-L1) in stem cells.
The autoimmune disease may be selected from the group consisting of lupus (systemic lupus erythematosus), rheumatoid arthritis, progressive systemic sclerosis (scleroderma), atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphthous stomatitis, chronic thyroiditis, inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis, polymyositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, Grave's disease, polyarteritis nodosa, ankylosing spondylitis, fibromyalgia syndrome, and temporal arteritis.
In addition, the present invention provides a method for preventing or treating an autoimmune disease, including administering the pharmaceutical composition to a subject other than humans.
In addition, the present invention provides a method for preparing an immunosuppressive agent, including culturing by adding a protein kinase C activator to stem cells.
In addition, the present invention provides a method for inhibiting an immune response of a subject other than humans, including administering stem cells treated with a protein kinase C activator or a culture thereof to the subject.
In addition, the present invention provides a method for preventing or treating an autoimmune disease, including administering or feeding a pharmaceutical composition including stem cells treated with a protein kinase C activator or a culture thereof to a subject.
In addition, the present invention provides a use of a pharmaceutical composition including stem cells treated with a protein kinase C activator or a culture thereof in the prevention or treatment of an autoimmune disease.
The stem cells or a culture thereof according to the present invention may be effectively used for the prevention or treatment of autoimmune diseases. More specifically, since stem cells treated with a protein kinase C activator or a culture thereof can inhibit the function of B cells, they have an excellent prophylactic or therapeutic effect for autoimmune diseases.
Hereinafter, the present invention will be described detail.
The present invention provides a pharmaceutical composition for preventing or treating an autoimmune disease, including stem cells treated with a protein kinase C activator or a culture thereof.
As used herein, the term “stem cells” may be cells having the ability to differentiate into various tissues, that is, ‘undifferentiated cells.’
In the present invention, the stem cells may he adult stent cells, pluripotent stern cells, induced pluripotent stem cells, or embryonic stem cells.
The adult stem cells may he derived from tissue selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane, and placenta. In addition, the adult stem cells may be mesenchymal stem cells, mesenchymal stromal cells, or multipotent stem cells, and preferably, mesenchymal stem cells. The method for obtaining stem cells from each tissue may be by a method known in the art, and it is not limited to the methods of the exemplary embodiments of the present invention.
As used herein, the term “culture” may include a cell culture solution including stem cells, a culture supernatant from which stem cells are removed from a cell culture solution, and a diluted solution thereof. The composition of the culture may additionally include components required for conventional stem cell culture, as well as components that act synergistically on stem cell proliferation, and accordingly, the composition may be easily selected by a person having ordinary skill in the art.
As the medium used for culturing the stem cells, a conventional medium known to be. suitable for stem cell culture in the art may be used, for example, Dulbecco's modified Eagle medium (DMEM) or keratinocyte serum free medium (keratinocyte-SFM) may be used.
The stem cell medium may be supplemented with additives. In general, it may contain a neutral buffer (e.g., phosphate and/or high-concentration bicarbonate) and a protein nutrient (e.g., serum such as FBS, serum substitutes, albumin, or essential amino acids and non-essential amino acids such as glutamine) in isotonic solutions. Furthermore, it may contain lipids (fatty acid, cholesterol, HDL or LDL extracts of serum) and other components found in most preservative media of this kind (e.g., insulin or transferrin, nucleosides or nucleotides, pyruvate, sugars in any ionized form or salt such as glucose, selenium, glucocorticoids, for example, hydrocortisone and/or reducing agents, and for example, β-mercaptoethanol).
In the composition of the present invention, the protein kinase C activator may include one or more selected from the group consisting of phorbol myristate acetate (PMA), ingenol 3-angelate (I3A), bryostatin-1, phorbol-12,13-dibutyrate (PDBu), prostatin, SC-9 (N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, CAS No. 102649-78-5), and SC-10 (5-chloro-N-heptylnaphthalene-1-sulfonamide, CAS No. 102649-79-6).
The treatment concentration of the protein kinase C activator may be 1 μg/mL to 20 μg/mL. The treatment time of the protein kinase C activator may be 1 hour to 120 hours.
The protein kinase C activator may increase the expression of CXCL10 in stem cells to increase B cell migration., thereby increasing the effect of contact-dependent inhibition. In addition, the protein kinase C activator may increase the expression of programmed death-ligand 1 (PD-L1) in stem cells to increase apoptosis of B cells.
According to the present invention, mesenchymal stem cells that are treated with a protein kinase C activator may inhibit the function of B cells, and the stem cells and a culture thereof according to the present invention may be effectively used in the prevention and treatment of autoimmune diseases.
The autoimmune disease may be selected from the group consisting of lupus (systemic lupus erythematosus), rheumatoid arthritis, progressive systemic sclerosis (scleroderma), atopic dermatitis, alopecia areata, psoriasis, pemphigus, asthma, aphthous stomatitis, chronic thyroiditis, inflammatory enteritis, Behcet's disease, Crohn's disease, dermatomyositis, polymyositis, multiple sclerosis, autoimmune hemolytic anemia, autoimmune encephalomyelitis, myasthenia gravis, Grave's disease, polyarteritis nodosa, ankylosing spondylitis, fibromyalgia syndrome, and temporal arteritis.
As used herein, the term “prevention” refers to all actions that suppress or delay the onset of autoimmune diseases through administration of the composition, and the term “treatment” refers to all actions that alleviate or ameliorate the symptoms of autoimmune diseases through administration of the composition.
In addition, the stem cells or a culture thereof according to the present invention may include 1.0×105 to 1.0×109 cells per 1 mL, preferably, 1.0×106 to 1.0×108 cells per 1 mL, and more preferably, 1.0×107 cells per 1 mL.
The stem cells or a culture thereof according to the invention may be used without freezing or may be frozen for future use. If it is necessary to be frozen, standard cryopreservatives (e.g., DMSO, glycerol, and the EpiLife cell freeze medium (Cascade Biologics)) may be added to a cell population prior to freezing.
In another aspect, the present invention provides a method for preventing or treating an autoimmune disease, including administering the pharmaceutical composition to a subject other than humans.
As used herein, the term “administration” means the introduction of a given substance into a subject by an appropriate method.
As used herein, the term “individual” means all animals, such as mice, rats, livestock, and the like, including humans that have or may develop an autoimmune disease. As a specific example, it may be a mammal, including a human.
In addition, the stem cells or a culture thereof according to the present invention may be formulated and administered into a preparation as a unit dosage form suitable for administration into a patient's body according to a conventional method in the pharmaceutical field, and the preparation includes an effective dose to he administered once or several times. Suitable formulations for this purpose as parenteral administration preparations may preferably be injections such as ampoules for injection, infusions such as infusion bags, sprays such as aerosol preparations, and the like. The ampoule for injection may be mixed with an injection solution immediately before use, and physiological saline, glucose, tnannitol, Ringer's solution, and the like may be used as the injection solution. In addition, the infusion bag may be made of a polyvinyl chloride or polyethylene material, and infusion bags from Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo may be exemplified.
The pharmaceutical preparation may further include one or more pharmaceutically acceptable conventional inert carriers other than the active ingredient, for example, preservatives, painless agents, solubilizers, stabilizers, or the like in the case of injections, and bases, excipients, lubricants, preservatives, or the like in the case of preparations for topical administration.
The stem cells according to the present invention, a culture thereof, or a pharmaceutical preparation thereof prepared as described above may he administered with other stein cells used for transplantation and other uses by using an administration method conventionally used in the art, or in the form of a mixture with such stern cells. Preferably, it is possible to directly engraft or implant into the diseased area of a patient in need of treatment, or directly implant or inject into the abdominal cavity, but is not limited thereto. In addition, the administration is possible both by non-surgical administration using a catheter or surgical administration methods such as infusion or implantation after incision of a disease site, but a non-surgical administration method using a catheter is more preferable. Also, in addition to administering parenterally according to a conventional method, for example, directly into the lesion, transplantation by intravascular injection, which is a general method of hematopoietic stem cell transplantation, is also possible.
The daily dose of the stem cells may be administered by dividing into 1.0×104 to 1.0×1010 cells/kg of body weight, preferably, 1.0×105 to 1.0×109 cells/kg of body weight once or several times. However, it should be understood that the actual dosage of the active ingredient should be determined in light of various related factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and sex, and thus, the dosage does not limit the scope of the present invention in any way.
In another aspect, the stem cells treated with a protein kinase C activator and a culture. thereof according to the present invention may he administered to suppress an autoimmune reaction, and the present invention provides a method for inhibiting an immune response in a subject, including administering stem cells treated with a protein kinase C activator or a culture thereof to the subject.
As used herein, the term “subject” refers to mammals including cows, dogs, pigs, chickens, sheep, horses, and humans, but is not limited thereto. Moreover, preferably, the administration of stem cells or a culture thereof to which a protein kinase C activator is added may he intraperitoneal or intravascular administration, direct administration into a lesion, administration into a synovial cavity of the joint, or the like.
The suppression of the immune response may be characterized by preventing or treating an immune disease.
In another aspect, the present invention provides a method for preparing an immunosuppressive agent, including culturing by adding a protein kinase C activator to stem cells.
As used herein, the term “immunosuppressive agent” as described above in the present invention refers to a preparation that can cure autoimmune diseases by suppressing an immune response, as a preparation including stem cells obtained by treating a protein kinase C activator to stem cells or a culture thereof.
Hereinafter, the present invention will be described in more detail through exemplary embodiments. The objects, features, and advantages of the present invention will be readily understood through the following exemplary embodiments. The present invention is not limited to the exemplary embodiments described herein, and may be embodied in other forms. The exemplary embodiments introduced herein are provided to sufficiently convey the spirit of the present invention to those skilled in the art to which the present invention pertains. Therefore, the present invention should not be limited by the following exemplary embodiments.
As an abnormal immune response, lupus activates dendritic cells, T cells, and B cells by autoantigens. Accordingly, autoantibodies are produced, and autoantigens and immune complexes are formed and invade various organs to show symptoms. Various therapeutic agents have been developed to treat such lupus disease, and mesenchymal stem cells (MSC), which have immunomodulatory effects, are being studied as promising therapeutic agents. It has been reported that MSC inhibits the function of T cells. However, it has not been clearly confirmed whether MSC inhibits the function of B cells. Accordingly, the present inventors confirmed whether human MSC inhibits the function of human B cells through the following exemplary embodiments.
The present inventors confirmed that human MSC does not inhibit the production of IgM in human B cells and slightly increases the same through preliminary experiments. The present inventors treated 20 types of chemicals to human MSC to prepare human MSC that inhibits human B cell function. Through screening, it was confirmed that phorbol myristate acetate (PMA), which is a protein kinase C activator, activates human MSC and ultimately inhibits human B cell function, and the mechanism of action of a protein kinase C activator activating human MSC was investigated.
Materials and Methods
Materials
MRL/lpr mice were purchased from Jackson Laboratory (Bar Harbor, Maine, USA). The mice were stored at a specific pathogen-free state under a condition of a temperature at 21° C. to 24° C. and a relative humidity at 40% to 60% under a 12-hour light/dark cycle.
Human mesenchymal stern cells (MSC) were obtained from hone marrow (BM) cells of human tibiae and femurs. BM cells were cultured in α-MEM medium including 10% fetal bovine serum (FBS), 2 mM L-glutamine, and penicillin/streptomycin under a condition of 37° C. and 5% CO2. Non-adherent cells were removed on day 1, and adhered cells were cultured with medium replenishment every 3 days and used between day 17 and day 20.
By using a phenomenon in which gene expression is inhibited sequence-specifically by 12 to 21 mer dsRNA called small interfering RNA (siRNA) (manufactured by Bioneer, Korea), CXCL10, CXCL12, and CCL2-knockdown hMSCs were cultured for 48 hours to induce knockdown reactions.
As substances used for the pretreatment condition for hMSC, PMA (10 ng/mL, 24-hour treatment, Sigma), I3A (10 μg/mL, 24-hour treatment, Sigma), and IFN-γ (10 ng/mL, 7-day treatment, Sigma) were used.
ELISA
IFN-γ measurement'as performed according to the test method provided by R&D (#DY285-05), and for IgM, the IgM ELISA kit (eBioscience, Vienna, Austria) was used for measurement. The capture antibody and the coating buffer were diluted at a ratio of 1:250 in 96 wells for ELISA and coated at 4° C. for 18 hours. After washing twice using a 200 μL of a wash solution, 250 μL of a blocking buffer was placed in a well, and then, it was reacted at room temperature for 2 hours. Afterwards, washing was performed twice using 200 μL of a wash solution, and the sample was prepared by diluting a specimen in an assay buffer (1×). In each well, 90 μL of an assay buffer (1×), 50 μL of a detection antibody, and 10 μL of a standard or the sample were added, and it was reacted at room temperature for 3 hours. After the reaction was completed, washing was performed using a washing solution. 100 μL of a substrate solution was added to each well, followed by reacting for 5 minutes, and 100 μL of a stop solution was added to stop the reaction. Finally, the value was measured at 450 nm to 570 nm.
Western Blot
Cells were lysed in ice using a cell lysis buffer (Cell Signaling Technology, Danvers, Mass., USA). Proteins were obtained by centrifugation at 12,000 rpm for 15 minutes at 4° C. The proteins were quantified using the Bradford reagent, and electrophoresis was performed at 75V with 20 μg of the proteins by using an SDS-polyacrylamide gel. After the SDS-polyacrylamide gel was transferred to a PVDF membrane at 95V for 90 minutes, 5% nonfat dry milk was added to TBS (TTBS) including 0.5% Tween 20 and blocked for 1 hour. After blocking, the membrane was placed in 5% BSA/TTBS including a primary antibody and shaken at 4° C. for one day. The next day, after washing the membrane with TTBS, the membrane was placed in 5% BSA/TTBS including a secondary antibody, and the secondary antibody was attached at room temperature for 90 minutes. After washing the membrane with TTBS and reacting the membrane with enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech, Piscataway, N.J., USA), the degree of protein expression was measured using a ChemiDoc™ XRS+ machine (Bio-Rad, CA, USA).
RNA Isolation Using the Trizol Method
Cells were treated with Trypsin-EDTA (Gibco) at 0.1:25% at a time point when the confluency of 70% to 80% was reached, followed by culturing under a condition of 5% CO2 and 37° C. After 24 hours of material treatment, the cells were recovered, and the total RNA was isolated from the cells using the TRIZOL reagent (invitrogen, MD, USA), and the total RNA was quantified by absorbance measurement at 260 nm.
Reverse Transcriptase-PCR
A total RNA concentration of 0.3 μg was used, and it was synthesized at 42° C. for 1 hour and at 95° C. for 5 minutes. Polymerase chain reaction was performed using 3 μL of synthesized cDNA and 10 pM of a primer. For the basic process of this reaction, the pre-denaturing phase was performed at 94° C. for 5 minutes, the denaturing phase was performed at 94° C. for 30 seconds, the annealing phase was performed at 56° C. for 30 seconds, the elongation phase was performed at 72° C. for 1 minute, and the post-elongation phase was performed at 72° C. for 5 minutes. Electrophoresis was performed by making a 1% agarose gel.
RT-PCR
The expression levels of mRNA for FasL, PD-L1, PD-L2, ICAM1, VCAM1, β-actin, IFN-γ, TNF-α, IL-12, COX-2, iNOS, IDO, TGF-β, CCL2, CCL3, CXCL10, and CXCL12 were analyzed by quantitative real-time PCR (qPCR). The relative mRNA amount of each sample was calculated based on a threshold cycle (Ct) compared to the threshold cycle (Ct) of β-actin, which is a housekeeping gene.
Annexin V Staining
After washing the cells twice with cold PBS, the cells were released in a binding buffer at a concentration of 1×106 cells/mL. Then, after transferring an amount of 100 μL to a 5 mL culture tube, the cells were stained by passing through an incubation process for 15 minutes at 25° C. room temperature, and under a dark condition using 5 μL, of the Annexin V Apoptosis Detection Kit (BD Pharmingen, USA). Then, after adding 400 μL of a binding buffer solution, it was measured using flow cytometry (Canton, BD Bioscience).
Primer Sequence Information
The information of the used primers is as follows. CCL2 forward primer 5-′ATG AAA GTC TCT GCC GCC CTT CTG T-3′ (SEQ ID NO: 1), CCL2 reverse primer 5-′AGT CTT CGG AGT TTG GGT TTG CTT G-3′ (SEQ ID NO: 2), CCL3 forward primer 5′-ATG CAG GTC TCC ACT GCT GCC CTT-3′(SEQ ID NO: 3), CCL3 reverse primer 5′-GCA CTC AGC TCC AGG TCG CTG ACA T-3′ (SEQ ID NO: 4), CXCL10 forward primer 5′-CCT GCT TCA AAT ATT TCC CT-3′ (SEQ ID NO: 5), CXCL10 reverse primer 5′-CCT TCC TGT ATG TGT TTG GA-3′ (SEQ ID NO: 6), CXCL12 forward primer 5′-ATG AAC GCC AAG GTC GTG GTC G-3′ (SEQ ID NO: 7), CXCL12 reverse primer 5′-TGT TGT TGT TCT TCA GCC G-3′ (SEQ ID NO: 8), COX-2 forward primer 5′-TCC TTG CTG TTC CCA CCC AT-3′ (SEQ ID NO: 9), COX-2 reverse primer 5′-CAT CAT CAG ACC AGG CAC CA-3′ (SEQ ID NO: 10), iNOS forward primer 5′-ACG TGC GTT ACT CCA CCA AC-3′ (SEQ ID NO: iNOS reverse primer 5′-CAT AGC GGA TGA GCT GAG CA-3′ (SEQ ID NO: 12), IDO forward primer 5′-AGCC TGA TCT CAT AGA GTC TG-3′ (SEQ ID NO: 13), IDO reverse primer 5′-TTA CTG CAG TCT CCA TCA CG-3′ (SEQ ID NO: 14), TGF-β forward primer 5′-CAG ATC CTG TCC AAG CTG-3′ (SEQ ID NO: 15), TGF-β reverse primer 5′-TCG GAG CTC TGA TGT (SEQ ID NO: 16), FasL forward primer 5′-GGA TTG GGC CTG GGG ATG TTT CA-3′ (SEQ ID NO: 17), FasL reverse primer 5′-TTG TGG CTC AGG GGC AGG TTG TTG-3′ (SEQ ID NO: 18), PD-L1 forward primer 5 -TTG GGA AAT GGA GGA TAA GA-3′ (SEQ ID NO: 19), PD-L1 reverse primer 5′-GGA TGT GCC AGA GGT AGT TCT-3′(SEQ ID NO: 20), PD-L2 forward primer 5′-ACA CCG TGA AAG AGC C-3′ (SEQ ID NO: 21), PD-L2 reverse primer 5′-AAT GTG AAG CAG CCA AG-3′ (SEQ ID NO: 22), ICAM1 forward primer 5′-CGT GCC GCA CTG AAG TGG AC-3′ (SEQ ID NO: 23), VCAM1 reverse primer 5′-CCT CAC ACT TCA CTG TCA CCT-3′ (SEQ ID NO: 24), VCAM1 forward primer 5′-ATG ACA TGC TTG AGC CAG G-3′ (SEQ ID NO: 25), VCAM1 reverse primer 5′-GTG TCT CCT TCT TTG ACA CT-3′ (SEQ ID NO: 26), β-actin forward primer 5′-GTG GGG CGC CCC AGG CAC CA-3′ (SEQ ID NO: 27), β-actin reverse primer 5′ CTC CTT AAT GTC ACG CAC GA-3′ (SEQ ID NO: 28). The mouse primers used were INF-γ forward primer 5′-AGC GGC TGA CTG AAC TCA GAT TGT AG-3′ (SEQ ID NO: 29), INF-γ reverse primer 5′-GTC ACA GTT TTC AGC TGT ATA GGG-3′ (SEQ ID NO: 30), TNF-α forward primer 5′-AGG TTC TGT CCC TTT CAC TCA CTG -3′ (SEQ ID NO: 31), TNF-α reverse primer 5′-AGA GAA CCT GGG AGT CAA GGT A-3′ (SEQ ID NO: 32), IL-12 forward primer 5′-AGA GGT GGA CTG GAC TCC CGA -3′ (SEQ ID NO: 33), IL-12 reverse primer 5′-TTT GGT GCT TCA CAC TTC AG-3′ (SEQ ID NO: 34), β-actin forward primer 5′-TGG AAT CCT GTG GCA TCC ATG AAA C-3′ (SEQ ID NO: 35), β-actin reverse primer 5′-TAA AAC GCA GCT CAG TAACAG TCC G-3′ (SEQ ID NO: 36).
Time-Lapse Imaging
By using Culture-insert m-dish35 mm (ibidi GmbH, Martinsried, Germany) for image capturing to confirm cell migration, MSCs (70 mL, 0.3×106 cells/mL) were seeded on the left side, and B cells (70 mL, 3×106 cells/mL) were seeded on the right side. After the cells were stabilized, inserts were removed and photographed using Biostation IM-Q (Nikon, Tokyo. Japan). In this experiment, photographing was performed for 3 hours to 6 hours at 2-minute intervals, and data analysis was performed by using the Imaris 7.2 software (Bitplane Inc., South Windsor, Conn., USA).
Chemotaxis Assay
For the migration of B cells, 24-transwell plates (Costar, Corning, N.Y., USA) with a 5 μm insert upper well were used. B cells (1×105 cells/well, upper chamber) isolated from PBMC and 13A-treated hMSCs (1×103 cells/well, lower chamber) were co-cultured with ODN for 72 hours using a transwell. The level of IgM in the supernatant was measured by ELISA.
Statistical Analysis
Statistical analysis of experimental data was performed using the GraphPad Prism 5.0 (GraphPad, San Diego, Calif., USA) software.
After B cells (1×105 cells/well) isolated from peripheral blood mononuclear cells (PBMC) and PMA-treated hMSC (1×103 cells/well) were co-cultured for 72 hours with CpG oligodeoxynucleotides (ODN, 5 μg/mL), the level of IgM in the supernatant was measured by ELISA (A in
Whereas naive MSC did not inhibit IgM production in B cells, PMA-treated MSC inhibited IgM production in B cells (A in
The chemokines of MSC were analyzed by RT-PCR (A in
As PMA-treated MSCs inhibited IgM of B cells by cell-cell contact, migration and contact dynamics between the two cells were studied, and research was primarily conducted on chemokine signaling which is important for the migration of cells. It was confirmed that MSC expressed CCL2 and CXCL12, and when treated with PMA, the expression of CXCL10 increased (A and B in
By using Culture-Dish35 mm, MSCs (0.2×106 cells/mL) and B cells (2×106 cells/ml,) were added and photographed for 3 hours using time-lapse imaging, and then, the movement of representative B cells was marked with a green track (A in
In order to confirm the contact between the PMA-treated MSCs and B cells at a single cell level, the two cells were directly mixed and analyzed. After mixing the PMA-treated MSCs with the B cells, imaging was performed for 6 hours at 2-minute intervals, and it was confirmed that compared to the control group PMA-treated MSCs, the contact of B cells with respect to PMA-treated MSCs in which CXCL10 was knock down (KD) was reduced (A in
PMA was treated to MSC, and after 24 hours, proteins were obtained, and signaling was analyzed through western blot (A in
PMA is a PKC activator that activates PKC in the cytoplasm and moves it to the plasma membrane. When PMA was treated to MSC, it was confirmed that PKC-α and δ in the cytoplasm were transferred to the cell membrane, and phosphorylation of ERK was increased (A in
After PDLL siRNA was treated to PMA-treated MSC, it was co-cultured for 24 hours, and the cells were stained using annexin V-APC and PI-PE and analyzed by FACS (A in
PD-L1 induces the death of cells expressing PD1. In this experiment, it was verified whether PD-L1 of the PMA-treated MSC induces B cell death by binding to PD1 on the B cell surface. When PD-L1 binds to PD1, caspase in the B cells is activated, and apoptosis is induced. When apoptosis proceeds, phosphatidic serine (PS) inside the cell membrane is exposed to the outside of the cell membrane, and as annexin V binds thereto, apoptosis can be measured, PMA-treated. MSC strongly increased the expression of caspase in B cells (B in
In order to proceed with the therapeutic effect experiment on PMA-treated MSC, the therapeutic effect was verified through MRL/lpr mice, which are a naturally occurring lupus animal model. MSC was injected intravenously into the mice at a concentration of 4×104 cells/mouse, and the time of administration was injected once from 12 weeks of age when the onset of disease began in the mice (the control group ((diluted solution) and MSC 4×104 cells/injection were administered at 12 weeks of age of the MRL/Ipr mice,). As a measurement index of animal experiments, the survival rate and body weight of the mice were checked every week, and anti-dsDNA and IgG were measured every two weeks. Survival rate (A in
The mice in the PMA-treated MSC administration group survived 100% until the age. of 30 weeks, but the mice in the IFN-γ-treated MSC and MSC administration groups survived only 0% and 33%, respectively (A in
After administering the control group (diluted solution) and MSC (4×104 cells/injection) to MRL/lpr mice at 12 weeks of age, kidneys were separated from 24-week-old MRL/lpr mice and fixed with formalin, and the degree of infiltration of immune cells was measured using DAB staining.
In the lupus disease, inflammation occurs at the kidneys by autoantibodies and immune complexes, and the infiltration of immune cells increases. Thus, the kidneys of the mice were separated, and the degree of cell infiltration was confirmed through DAB staining. PMA-treated MSC reduced kidney infiltration of T cells, B cells, macrophages, and dendritic cells compared to the control group, and increased the infiltration of Treg (A and B in
The above description of the present invention is for illustration only, and those skilled in the art to which the present invention pertains can understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. Therefore, it should be understood that the exemplary embodiments described above are illustrative in all respects and not restrictive.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2018-0137578 | Nov 2018 | KR | national |
This application is a 37 U.S.C. 371 National Phase Entry Application from PCT/KR2019/014960, filed Nov. 6, 2019, which claims priority to and the benefit of Korean Patent Application No. 10-2018-0137578, filed on Nov. 9, 2018, the disclosures of which are incorporated herein by reference in their entireties. The instant application contains a Sequence Listing which has been submitted via EFS-Web and is hereby incorporated by reference in its entirety. Said Sequence Listing, created on Sep. 27, 2021, is named U.S. Ser. No. 17/292,315_Sequencelisting_Revised.TXTand is 8,000 bytes in size.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2019/014960 | 11/6/2019 | WO | 00 |
