WO2018088821A1 - Composition favorisant l'activité des cellules souches, comprenant un inhibiteur d'histone désacétylase et un facteur d'amorçage en tant que principes actifs - Google Patents

Composition favorisant l'activité des cellules souches, comprenant un inhibiteur d'histone désacétylase et un facteur d'amorçage en tant que principes actifs Download PDF

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WO2018088821A1
WO2018088821A1 PCT/KR2017/012675 KR2017012675W WO2018088821A1 WO 2018088821 A1 WO2018088821 A1 WO 2018088821A1 KR 2017012675 W KR2017012675 W KR 2017012675W WO 2018088821 A1 WO2018088821 A1 WO 2018088821A1
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disease
stem cells
vpa
stem cell
pulmonary
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PCT/KR2017/012675
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English (en)
Korean (ko)
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신동명
임지선
오연목
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울산대학교 산학협력단
재단법인 아산사회복지재단
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Priority claimed from KR1020170148019A external-priority patent/KR102097191B1/ko
Application filed by 울산대학교 산학협력단, 재단법인 아산사회복지재단 filed Critical 울산대학교 산학협력단
Priority to JP2019520846A priority Critical patent/JP6934516B2/ja
Priority to CN201780069520.XA priority patent/CN110462023B/zh
Publication of WO2018088821A1 publication Critical patent/WO2018088821A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/465—Nicotine; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.
  • MSCs Mesenchymal stem-cells
  • BM bone-marrow
  • UC umbilical-cord
  • UB umbilical cord blood
  • MSCs Multipotent progenitor cells.
  • Administration of MSCs repairs organ tissues of damaged organs to provide a therapeutic effect.
  • the efficacy of MSCs is due to cytoprotective effects regulated by cytokines and paracrine factors such as pro-angiogenic and pro-arteriogenic effects.
  • current MSC treatments are limited due to their low therapeutic effectiveness, particularly in vivo engraftment and survival of transplanted cells. Most ( ⁇ 99%) intravenously inject MSCs near the lungs, of which only 2-3% are released into the circulation. Thus, there is a need to understand the exact mechanisms for the migration and engraftment of MSCs during tissue repair.
  • chemokines such as stromal cell derived factor-1 (SDF-1) and growth factors such as vascular endothelial growth-factor (VEGF), platelet- Platelet-derived growth-factor (PDGF) or hepatocyte growth-factor (HGF) plays an important role in the migration and engraftment of adult MSCs.
  • SDF-1 stromal cell derived factor-1
  • growth factors such as vascular endothelial growth-factor (VEGF), platelet- Platelet-derived growth-factor (PDGF) or hepatocyte growth-factor (HGF) plays an important role in the migration and engraftment of adult MSCs.
  • VEGF vascular endothelial growth-factor
  • PDGF platelet- Platelet-derived growth-factor
  • HGF hepatocyte growth-factor
  • HSPCs After chemotherapy () in the bone marrow.
  • the sensitivity / responsiveness of HSPCs to the SDF-1 gradient is positively influenced (“primed”) by a rich set of molecules in the injured tissue.
  • bioactive lipids eg, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)
  • neutrophil-derived cationic peptide caseycides eg, sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P)
  • neutrophil-derived cationic peptide caseycides eg, neutrophil-derived cationic peptide caseycides.
  • Cathelicidin LL-37
  • sMAC soluble membrane attack complex
  • primed MSCs promote cell migration, colony-forming activity and anti-inflammatory capacity in cell culture conditions, which enhances the effect of treating pulmonary arterial hypertension (PAH).
  • PAH pulmonary arterial hypertension
  • primed MSCs still exhibit limited in vivo engraftment in injured tissue.
  • An object of the present invention is to provide a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient, and a method for promoting stem cell activity through the same.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating hypertension, including a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture solution thereof as an active ingredient.
  • Another object of the present invention to provide a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases, including stem cells treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient. Is in.
  • the present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.
  • the present invention provides a method for promoting stem cell activity comprising the step of treating a histone deacetylation inhibitor and priming factor to the isolated stem cells.
  • the present invention also provides a pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
  • the present invention also provides the use of histone deacetylation inhibitors and priming factors for promoting stem cell activity.
  • a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof there is provided the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof.
  • the present invention also provides the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof in the manufacture of a medicament for preventing or treating an inflammatory disease or an immune disease.
  • the present invention relates to a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.
  • a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.
  • the activity of the stem cells is increased. Excellent improvement.
  • primed MSCs promote cell migration, colony-forming activity, and anti-inflammatory capacity in cell culture conditions, which may be useful therapeutic strategies because they enhance the effect of treating hypertension, inflammatory or immunological diseases. do.
  • FIG. 2 shows the results for the effect of VPA on UC-MSCs primed with S1P.
  • a and B Flow cytometry and pluripotency (B) for surface antigen (A) of UC-MSCs primed for 24 hours with 0.5-mM VPA alone (VPA) or in combination with 50-nM S1P (VPA + S1P) The analysis results are shown.
  • FIG. 3 shows the results for improved reactivity to SDF-1 in UC-MSCs primed with VPA + S1P.
  • a and B 150-ng / ml SDF-1 of MSCs exposed for 24 hours to 0.5-mM VPA alone (VPA), 50-nM S1P alone (S1P) or combination with 50-nM S1P (VPA + S1P) The results of the chemotaxis analysis for.
  • A Representative images of transwell insets are shown in the migration assay.
  • (B) Relative amounts for migration were quantified as a change in magnification for the number of migrated cells and expressed as means ⁇ SEM (n 6).
  • VPA + S1P upregulated genes involved in the therapeutic effect of MSCs VPA + S1P upregulated genes involved in the therapeutic effect of MSCs.
  • MMP12 MMP12 in UC-MSCs primed for 24 h with 0.5-mM VPA alone (VPA), 50-nM S1P alone (S1P) or in combination with 50-nM S1P (VPA + S1P) (A), growth factors (B), angiogenesis (C) and anti-inflammatory (D) related gene expression levels. Expression levels were calculated as the numerical ratio of primed UC-MSCs to unprimed UC-MSCs (NT).
  • FIG. 7 shows the results of improving the treatability of VPA + S1P primed UC-MSCs with respect to bladder function recovery in the HCl-IC rat model.
  • (a) Results of awake cystometry under non-anesthetic.
  • FIG. 9 shows the effect of UCB-MSCs and VPA + S1P primed MSCs (VPA + S1P-MSCs) in MCT induced PAH rat model.
  • MCT induced an increase in RVSP levels and significantly reduced this elevation as a result of administration of VPA + S1P primed UC-MSC two weeks after MCT infusion (left panel).
  • MCT increased the weight ratio of RV / (LV + S) and VPA + S1P-UC-MSC significantly reduced this effect (right panel).
  • MCT increased lung tissue inflammation and medial wall thickness index (vascular wall thickness per vessel diameter), and VPA + S1P-MSC significantly reduced this increase (top panel).
  • Unprimed UCB-MSC showed little improvement in RV / (LV + S) and media thickness index. It shows an immunofluorescence staining of ⁇ -SMA + smooth muscle cells in the pulmonary artery (lower panel) (magnification ⁇ 400).
  • RVSP right ventricular systolic pressure
  • CTL control
  • MSC human cord blood mesenchymal stem cell
  • S1P-MSC sphingosine-1-phosphate primed human umbilical cord mesenchymal stem cells
  • RV right ventricle
  • LV + S left ventricle and interventricular septum.
  • CXCR4 a major target of stem cell priming, is upregulated by DNA-demethylating agent 5-azacytidine (5-Aza) and histone deacetylation inhibitor valproic acid (VPA). do.
  • 5-azacytidine 5-Aza
  • VPA histone deacetylation inhibitor valproic acid
  • the present invention provides a composition for promoting stem cell activity comprising a histone deacetylation inhibitor and a priming factor as an active ingredient.
  • the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.
  • VPA valproic acid
  • NaB sodium butyrate
  • NAD nicotinamide
  • sirtinol sirtinol
  • the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.
  • the VPA may be included in a concentration of 0.1 to 1.0 mM
  • the S1P may be included in a concentration of 10 to 50 nM, but is not limited thereto.
  • the term "priming” refers to a phenomenon in which reactivity (activity) is improved to enhance the therapeutic efficacy of stem cells, and in the present invention, a histone deacetylation inhibitor and a priming factor that induce the priming. (priming factor) to promote the activity of the stem cells.
  • stem cell activity refers to cell mobility, cell proliferation ability, pluripotency (in vitro differentiation into chondrocytes, osteocytes or adipocyte lineage), colony forming ability or anti-inflammatory activity of stem cells, etc. .
  • the activity promotion or priming induction is not only induced priming of stem cells when the stem cells are directly treated with histone deacetylation inhibitors and priming factors, but also by the priming treatment (pre-treatment). Other differentiation may be induced by using stem cells with improved stem cell activity.
  • composition for promoting activity can be enhanced by injecting in vivo by mixing with a cell therapy agent for treatment, to enhance the in vivo effect of the cell therapy, and after treatment of the present composition to the stem cells themselves cell therapy agent increased function It can also be used as a method for implanting in vivo.
  • the priming of the present invention is to enhance stem cell function, including cell mobility, colony forming ability, and anti-inflammatory activity of stem cells.
  • stem cell refers to a cell having the ability to differentiate into two or more cells while having a self-replicating ability, totipotent stem cells, pluripotent stem cells It can be classified into multipotent stem cells.
  • Stem cells of the present invention may be selected without appropriate limitation depending on the purpose, and may be derived from adult cells, such as all known tissues, cells, etc. derived from mammals, including humans, preferably humans, for example, Bone marrow, umbilical cord blood, placenta (or placental tissue cells), fat (or adipose tissue cells) and the like.
  • the stem cells are restricted from bone marrow, adipose tissue, muscle tissue, ex vivo cultured autologous mesenchymal stem cells, allogeneic mesenchymal stem cells, umbilical cord blood, embryonic yolk sac, placenta, umbilical cord, periosteum, fetal and adolescent skin, and blood It may be a stem cell obtained without, and may be a stem cell derived from the fetus or shortly after birth or adult.
  • the stem cells are neural stem cells, liver stem cells, hematopoietic stem cells, cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells and It is selected from the group consisting of pancreatic stem cells, and more preferably may be selected from the group consisting of liver stem cells, hematopoietic stem cells, cord blood stem cells and mesenchymal stem cells, but is not limited thereto.
  • the present invention provides a method for promoting stem cell activity comprising the step of treating a histone deacetylation inhibitor and priming factor to the isolated stem cells.
  • the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.
  • VPA valproic acid
  • NaB sodium butyrate
  • NAD nicotinamide
  • sirtinol sirtinol
  • the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.
  • the VPA may be included in a concentration of 0.1 to 1.0 mM
  • the S1P may be included in a concentration of 10 to 50 nM, but is not limited thereto.
  • the present invention also provides a pharmaceutical composition for preventing or treating hypertension, comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory diseases or immune diseases comprising a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture medium thereof as an active ingredient.
  • stem cells when stem cells are activated through priming, they can be applied to various inflammatory diseases and immune diseases by participating in immune regulation and inflammatory response, and there are various reports on the mechanisms of immune control and inflammatory response of primed stem cells. (J Orthop Res. 2016 Apr 6, 1-12; Stem Cell Rev and Rep (2014) 10: 351-375; Stem Cells International 2016 Article ID 9364213).
  • the histone deacetylation inhibitor may be valproic acid (VPA), sodium butyrate (NaB), nicotinamide (NAD) or sirtinol, but is not limited thereto. It doesn't happen.
  • VPA valproic acid
  • NaB sodium butyrate
  • NAD nicotinamide
  • sirtinol sirtinol
  • the priming factor is sphingosine-1-phosphate (S1P), ceramide-1-phosphate (C1P), cathelicidin (LL-37) or It may be a pioglitazone, but is not limited thereto.
  • the "culture medium” includes a culture medium capable of supporting stem cell growth and survival in vitro, secretion of cultured stem cells contained in the medium, and the like.
  • the medium used for culturing includes all conventional mediums used in the art suitable for culturing stem cells. Depending on the type of cells, medium and culture conditions can be selected.
  • the medium used for the culturing is preferably a cell culture minimum medium (CCMM), and generally includes a carbon source, a nitrogen source and a trace element component.
  • CCMM cell culture minimum medium
  • Such cell culture minimal media include, for example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI1640, F-10, F-12, ⁇ Minimal Essential Medium (GMEM) (Glasgow's Minimal essential Medium) and Iscove's Modified Dulbecco's Medium, but are not limited to these.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basic Medium Eagle
  • RPMI1640 RPMI1640
  • F-10 F-12
  • GMEM ⁇ Minimal Essential Medium
  • Iscove's Modified Dulbecco's Medium Iscove's Modified Dulbecco's Medium
  • the medium may include antibiotics such as penicillin, streptomycin, gentamicin, and the like.
  • the present invention is the form containing all of the stem cells, their secretions, media components, forms containing only secretions and media components, only secretion to separate or used in combination with stem cells, or by administering only stem cells It is also possible to use it in a form that produces secretions in the body.
  • the stem cells can be obtained using any method known in the art.
  • Stem cells treated with histone deacetylation inhibitors and priming factors of the present invention can be used as a cell therapy for the treatment of certain diseases, and the treatment can be direct or pre-treatment of the molecules. .
  • cell therapy means proliferating, screening, or otherwise altering the biological properties of cells in vitro to autologous, allogenic, and xenogenic cells to restore the function of cells and tissues. It refers to medicines used for the purpose of treatment, diagnosis and prevention through a series of actions.
  • the cell therapeutic agent may be administered to the human body via any general route as long as it can reach the desired tissue.
  • the hypertension is idiopathic pulmonary arterial hypertension; Familial pulmonary arterial hypertension; Pulmonary arterial hypertension associated with collagen vascular disease, congenital pulmonary shortness, portal hypertension, HIV infection, drugs or toxins; Pulmonary hypertension associated with thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic capillary dilatation, hemochromatosis, myeloproliferative disorder or splenectomy; Pulmonary arterial hypertension associated with pulmonary capillary angiomatosis; Persistent pulmonary hypertension in newborns; Pulmonary hypertension associated with chronic obstructive pulmonary disease, interstitial lung disease, hypoxia induced alveolar hypoventilatory disorder, hypoxia induced sleep disorder breathing or chronic exposure to high altitudes; Pulmonary hypertension associated with developmental abnormalities; And pulmonary hypertension by thromboembolic obstruction of distal pulmonary artery, more preferably idiopathic pulmonary arterial hypertension, familial
  • compositions of the present invention may be prepared using pharmaceutically suitable and physiologically acceptable auxiliaries in addition to the active ingredients, which may include excipients, disintegrants, sweeteners, binders, coatings, swelling agents, lubricants, lubricants. Or solubilizers such as flavoring agents can be used.
  • the pharmaceutical composition of the present invention may be preferably formulated into a pharmaceutical composition by containing one or more pharmaceutically acceptable carriers in addition to the active ingredient for administration.
  • Acceptable pharmaceutical carriers in compositions formulated in liquid solutions are sterile and physiologically compatible, including saline, sterile water, Ringer's solution, buffered saline, albumin injectable solutions, dextrose solution, maltodextrin solution, glycerol, ethanol and One or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers and bacteriostatic agents may be added as necessary. Diluents, dispersants, surfactants, binders and lubricants may also be added in addition to formulate into injectable formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like.
  • compositions of the present invention may be granules, powders, coated tablets, tablets, capsules, suppositories, syrups, juices, suspensions, emulsions, drops or injectable solutions and sustained release formulations of the active compounds, and the like.
  • the pharmaceutical compositions of the present invention may be administered in a conventional manner via intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, sternum, transdermal, nasal, inhalation, topical, rectal, oral, intraocular or intradermal routes.
  • An effective amount of the active ingredient of the pharmaceutical composition of the present invention means an amount required to prevent or treat a disease.
  • the type of disease the severity of the disease, the type and amount of the active and other ingredients contained in the composition, the type of formulation and the age, weight, general health, sex and diet, sex and diet, time of administration, route of administration and composition of the patient. It can be adjusted according to various factors including the rate of secretion, the duration of treatment, and the drug used concurrently.
  • the present invention also provides the use of histone deacetylation inhibitors and priming factors for promoting stem cell activity.
  • a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof there is provided the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof.
  • the present invention also provides the use of a stem cell treated with a histone deacetylation inhibitor and a priming factor or a culture thereof in the manufacture of a medicament for preventing or treating an inflammatory disease or an immune disease.
  • UC-derived MSCs are 2-mM L-glutamine, 20-mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES) (pH 7.3), minimal-required medium ( minimum-essential medium (MEM) non-essential amino acid solution, penicillin / streptomycin (Corning Cellgro, Pittsburgh, PA), 1-mg / ml ascorbic acid (Sigma-Aldrich), 10% heat-inactivated fetal bovine serum ( fetal bovine serum; FBS) (HyClone), 5-ng / mL human epidermal growth factor (Sigma-Aldrich, St.
  • DMEM low-glucose Dulbecco's modified Eagle's medium
  • UC-MSCs extended to 5 times or less were used. Expression of surface proteins was analyzed as previously reported (Stem Cells and Dev. 24 (2015) 1658-1671).
  • UC-MSCs were primed for 1 day with VPA (0.5-mM; Sigma-Aldrich) or 5-Aza (1- ⁇ M; Sigma-Aldrich) alone or mixed with 50-nM S1P.
  • VPA 0.5-mM
  • Sigma-Aldrich 5-Aza (1- ⁇ M; Sigma-Aldrich
  • UC-MSCs are TrypLE solution (Thermo Scientific, Pittsburgh PA) as was separated, washed and resuspended in DMEM containing 0.5% bovine serum albumin (BSA) , 3 X 10 4 trans-well inserts with cells / well density ( Transwell inserts) (Corning Costar, Pittsburgh, Pa.) Were inoculated into the upper chamber.
  • BSA bovine serum albumin
  • the lower chamber was filled with 150-ng / ml SDF-1 (R & D Systems) in DMEM containing 0.5% BSA. After 1 day, the inserts were removed from the transwell plates. Cells remaining in the upper chamber were scraped with cotton wool, and the migrated cells were fixed in 4% paraformaldehyde (PFA) solution dissolved in phosphate-buffered saline (PBS), followed by 0.5% crystal violet ( Sigma-Aldrich). Digital images were analyzed with Image Pro 5.0 software (Media Cybernetics, Rockville, MD, USA) to quantify the stained cells on the underside of the membrane.
  • PFA paraformaldehyde
  • PBS phosphate-buffered saline
  • crystal violet Sigma-Aldrich
  • UC-MSCs primed with 50-nM S1P and 0.5-mM VPA were starvated for 12 hours at 37 ° C. in DMEM containing 0.5% BSA and 5, 10, 20 with 150-ng / ml SDF-1. Or after 30 min of stimulation, phosphorylation of mitogen-activated protein kinase (MAPK) p42 / 44 and AKT (Ser473) using a 30 ⁇ g-cell extract was analyzed via Western blot. For gene expression analysis, total RNA from indicated cells was reverse transcribed and the indicated transcripts were quantified via RQ-PCR.
  • MAPK mitogen-activated protein kinase
  • An object of the present invention is to develop an umbilical cord-derived MSCs derived from human umbilical cord-derived MSCs primed with V1P + S1P to treat interstitial cystitis / bladder pain syndrome (IC / BPS) in a rat model.
  • V1P + S1P primed UC-MSCs were administered to rats with bladder injuries and affected the effects on bladder urination function, urothelium denudation, mast cell infiltration, tissue fibrosis, apoptosis and tumor formation. Measured. For all experiments, two independent experiments were performed on 5 independent animals per group.
  • HCl infused IC / BPS rat models have been previously reported (Stem Cells and Development 24, 2015, 1648-1657).
  • HCl week after injury the abdomen was cut, using a 500 ⁇ m syringe and 26-gauge needle and a 2.5 ⁇ 10 5 (250 K) UC-MSCs or PBS was administered directly to the front wall outer layer and the ceiling of the bladder.
  • indomethacin PMG Pharm Co., Ltd.
  • gefitinib to block Wnt or IGF-mediated signaling
  • Santa Cruz Biotechnology Santa Cruz, CA, USA; every day at 5 mg / kg was injected subcutaneously.
  • Cystometrograms were performed in non-anesthesia and non-suppressed mice in metabolic cages. Catheter was inserted simultaneously 3 days prior to bladder pressure measurement for measurement of intravesical pressure (IVP) and intra-abdominal pressure (IAP) recordings. Briefly, after anesthesia induction, a cuffed polyethylene catheter (PE-50; Becton-Dickinson, Parsippany, NJ, USA) was inserted into the bladder ceiling via an intraperitoneal incision. To record the IAP, an abdominal balloon (Latex; Daewoo Medical, Incheon, Korea) near the cuff of the catheter tip was placed proximal to the bladder and tied to another catheter with a silk thread.
  • PE-50 Becton-Dickinson, Parsippany, NJ, USA
  • the polyethylene catheter (PE-50) was heated in warm water and the tip of the insert was extended to ⁇ 1.5 times its original length and filled with heparinized saline (100 IU / mL).
  • the extended catheter was inserted into the femoral vein. The catheter then penetrated through the subcutaneous space, exited through the back of the animal, and attached to the back skin. After surgery, each rat was raised individually and maintained in the same way.
  • a T-tube connected to a pressure transducer (Research Grade Blood Pressure Transducer; Harvard Apparatus, Holliston, Mass., USA) and a microinjection pump (PHD22 / 2000 pump; Harvard Apparatus)
  • An indwelling catheter was connected to the bladder via a double valve.
  • another indwelling catheter with a fluid-filled abdominal balloon was connected to another pressure transducer.
  • Sterile saline was injected into the bladder at a rate of 0.4 mL / min and urine volume was continuously recorded through a fluid collector connected to a force displacement transducer (Research Grade Isometric Transducer; Harvard Apparatus).
  • IVP, IAP and urination volume were continuously recorded using an MP150 data acquisition system (Biopac Systems, Goleta, CA, USA) equipped with Acq Knowledge 3.8.1 software at a sampling rate of 50 Hz. All urination cycle values measured for 8 minutes from each animal were used for the evaluation.
  • NVC non-voiding contraction
  • MCT Monocrotaline
  • Male specific-pathogen-free Lewis rats (8 weeks old, 250-280 g) were reared without restriction of food and water, under the control of breeding temperature and light (12 hour contrast cycle).
  • PAH was induced through subcutaneous administration of MCT (60 mg / kg, Sigma).
  • Mice in the control group received the same volume of PBS.
  • priming-induced UC-MSCs or UC-MSCs pretreated with VPA + S1P were administered via the tail vein at a density of 2.5 ⁇ 10 5 cells per 200 ⁇ l PBS.
  • a solvent-only vehicle control was administered with 200 ⁇ l PBS without cells. Prior to injection, cells were washed twice with warm PBS and viability of the administered cells was observed using FACS analysis via 7-AAD (BD Biosciences) exclusion staining.
  • RVSP right ventricular systolic pressure
  • RVH right ventricular hypertrophy
  • ⁇ -SMA ⁇ -smooth muscle actin
  • UC-MSCs primed with 5-Aza + S1P in transwell migration assays reduced migration activity for SDF-1 responses by about 30% compared to unprimed cells.
  • FIG. 1E 5-Aza + S1P priming severely impaired the activity of clonal CFU-F, which indicates the frequency of self-renewal into clonal progenitor cells (FIG. 1F).
  • Cell proliferation of MSCs was little changed by the priming of 5-Aza + S1P (FIG. 1D).
  • the present inventors investigated the effect of VPA on the priming of UC-MSCs at low concentrations of S1P treatment.
  • 50-nM S1P four times less than the optimal dose (200-nM) used for priming of fat- and UCB-derived MSCs.
  • UC-MSCs priming increased VT + S1P treatment two to three times higher chemotactic activity for SDF-1 than unprimed cells (FIGS. 3A and 3A). 3B).
  • the response to SDF-1 enhanced by VPA + S1P priming appeared completely different from that of 5-Aza + S1P priming.
  • UC-MSCs exposed to VPA + S1P increased the phosphorylation of MAPK p42 / 44 and AKT proteins, indicating activation of signaling pathways (FIG. 3C).
  • VPA + S1P priming we tested the effect of VPA + S1P priming on other cellular activities related to the therapeutic effect of MSCs. Unlike 5-Aza + S1P (FIG. 1), UC-MSCs primed with VPA + S1P improved both cell proliferation (FIG. 4A) and clonal CFU-F activity (FIG. 4B). However, no increase in cell proliferation and CFU-F activity was observed in UC-MSCs treated with VPA alone and S1P alone (FIGS. 4A and 4B). Since MSCs affect anti-inflammatory and immunomodulatory capacity, we tested whether VPA + S1P priming affected the anti-inflammatory effects of UC-MSCs.
  • the present inventors prepared a conditioned medium collected from unprimed UC-MSCs or VPA alone or from UC-MSCs primed with VPA + S1P, and the conditioned medium stimulated with lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • TNF- ⁇ Tumor necrosis factor- ⁇
  • Conditional medium collected from UC-MSCs was effective in reducing the secretion of TNF- ⁇ from LPS stimulated MH-S cells (FIG. 4C).
  • the conditioned media collected from VPA + S1P primed UC-MSCs inhibited TNF- ⁇ secretion more than unprimed UC-MSCs or UC-MSCs primed with VPA alone.
  • CM collected with IMR90, a human lung fibroblast, as a control which was shown to further increase TNF- ⁇ secretion in vitro anti-inflammatory analysis.
  • UC-MSCs primed with VPA + S1P showed growth factors and their receptors [eg, PDGFB, PDGFRB, cMET] (FIG. 5B), pre-angiogenesis- [eg , VEGFB, VEGFC, ANGPT1, ANGPT2] (FIG. 5C), anti-inflammatory- [eg, LIF, TSG6, IDO1, IDO2] (FIG.
  • the present inventors attempted to determine the lowest concentration of VPA and S1P by performing a chemotaxis analysis on SDF-1a according to various VPA and S1P concentrations.
  • VPA was fixed at 0.5 mM, and the S1P concentration was tested at 5-50 nM. As a result, the lowest concentration of S1P was determined to be 10 nM (FIG. 6A). In addition, S1P was fixed at 50 nM and the VPA concentration was 0.05-0.5 mM. As a result, the lowest concentration of VPA was determined to be 0.1 nM (FIG. 6B).
  • RVSP increased significantly after 4 weeks of MCT administration.
  • administration of UC-MSC primed with VPA + S1P significantly reduced the elevation of MCT-induced RVSP (FIG. 9a).
  • RV / (LV + S) was also increased, with VPA + S1P-MSC significantly reducing RV hypertrophy, and unpriming UC-MSC was ineffective (FIG. 9A).
  • VPA + S1P-MSC administration reduced lung tissue inflammation, medial thickness index, and ⁇ -SMA + smooth muscle cell induced by MCTs (FIG. 9B).
  • VPA + S1P-MSC administration reduced lung tissue inflammation, medial thickness index, and ⁇ -SMA + smooth muscle cell induced by MCTs (FIG. 9B).

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Abstract

La présente invention concerne une composition pour favoriser l'activité de cellules souches, comprenant un inhibiteur d'histone désacétylase et un facteur d'amorçage en tant que principes actifs. Lorsque des cellules souches ont été traitées avec un inhibiteur d'histone désacétylase à faible concentration et un facteur d'amorçage, l'activité des cellules souches est considérablement améliorée. En particulier, les CSM amorcées favorisent un mouvement cellulaire, une activité de formation de colonies et une capacité anti-inflammatoire en ce qui concerne les conditions de culture cellulaire et, de ce fait, améliorent l'hypertension, les maladies inflammatoires ou les effets de traitement de maladies immunes et on s'attend donc à ce qu'elles deviennent une stratégie de traitement utile.
PCT/KR2017/012675 2016-11-10 2017-11-09 Composition favorisant l'activité des cellules souches, comprenant un inhibiteur d'histone désacétylase et un facteur d'amorçage en tant que principes actifs WO2018088821A1 (fr)

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JP2019520846A JP6934516B2 (ja) 2016-11-10 2017-11-09 ヒストン脱アセチル化阻害剤及びプライミング因子を有効成分として含む幹細胞活性促進用組成物
CN201780069520.XA CN110462023B (zh) 2016-11-10 2017-11-09 作为有效成分包括组蛋白去乙酰化酶抑制剂和激活因子的干细胞活性促进用组合物

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11266696B2 (en) * 2019-01-18 2022-03-08 Americord Registry Llc Method of treating stem cells with NAD

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023567A2 (fr) * 1998-10-16 2000-04-27 Novartis Ag Procede pour favoriser l'autorenouvellement et transduction genique amelioree des cellules souches hematopoietiques par des inhibiteurs de desacetylase de l'histone

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000023567A2 (fr) * 1998-10-16 2000-04-27 Novartis Ag Procede pour favoriser l'autorenouvellement et transduction genique amelioree des cellules souches hematopoietiques par des inhibiteurs de desacetylase de l'histone

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
KANG, H. ET AL.: "The Therapeutic Effects of Human Mesenchymal Stem Cells Primed with Sphingosine-1 Phosphate on Pulmonary Artery Hypertension", STEM CELLS DEV., vol. 24, no. 14, 2015, pages 1658 - 1671, XP055493135 *
LIM, J. ET AL.: "Priming with Ceramide-l Phosphate Promotes the Therapeutic Effect of Mesenchymal Stern/stromal Cells on Pulmonary Artery Hypertension", BIOCHEM. BIOPHYS. RES. COMMUN., vol. 473, 2016, pages 35 - 41, XP055493127, DOI: doi:10.1016/j.bbrc.2016.03.046 *
LIM, J. ET AL.: "Valproic Acid Enforces the Priming Effect of Sphingosine-1 Phosphate on Human Mesenchymal Stem Cells", INT. J. MOL. MED., vol. 40, 3 July 2017 (2017-07-03), pages 739 - 747, XP055493143 *
MARQUEZ-CURTIS, L. A. ET AL.: "Migration, Proliferation, and Differentiation of Cord Blood Mesenchymal Stromal Cells Treated with Histone Deacetylase Inhibitor Valproic Acid", STEM CELLS INT., vol. 2014, 16 March 2014 (2014-03-16), pages 1 - 14, XP055493128 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11266696B2 (en) * 2019-01-18 2022-03-08 Americord Registry Llc Method of treating stem cells with NAD

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