WO2020045547A1 - 線維芽細胞の製造方法及びｇ－ｃｓｆ陽性線維芽細胞集団 - Google Patents

線維芽細胞の製造方法及びｇ－ｃｓｆ陽性線維芽細胞集団 Download PDF

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WO2020045547A1
WO2020045547A1 PCT/JP2019/033835 JP2019033835W WO2020045547A1 WO 2020045547 A1 WO2020045547 A1 WO 2020045547A1 JP 2019033835 W JP2019033835 W JP 2019033835W WO 2020045547 A1 WO2020045547 A1 WO 2020045547A1
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fibroblasts
positive
cells
hcf
csf
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French (fr)
Japanese (ja)
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貴紘 岩宮
友之 大杉
鈴木　雅也
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Metcela Inc
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Metcela Inc
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Priority to SG11202102028SA priority Critical patent/SG11202102028SA/en
Priority to IL281081A priority patent/IL281081B/en
Priority to KR1020217009135A priority patent/KR102883773B1/ko
Priority to CN201980056534.7A priority patent/CN112639082A/zh
Priority to CA3110831A priority patent/CA3110831A1/en
Priority to AU2019332400A priority patent/AU2019332400B2/en
Application filed by Metcela Inc filed Critical Metcela Inc
Priority to JP2020539572A priority patent/JP6883904B2/ja
Priority to EP19856084.9A priority patent/EP3845635A4/en
Publication of WO2020045547A1 publication Critical patent/WO2020045547A1/ja
Priority to US17/187,459 priority patent/US20210254009A1/en
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    • 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
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/53—Colony-stimulating factor [CSF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70542—CD106
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2304—Interleukin-4 (IL-4)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/599—Cell markers; Cell surface determinants with CD designations not provided for elsewhere

Definitions

  • the present invention mainly relates to a method for producing CD106-positive and / or CD90-positive fibroblasts, and further relates to a pharmaceutical composition for treating heart disease containing the fibroblasts.
  • the present invention also relates to a cell population containing G-CSF positive fibroblasts.
  • Fibroblasts are one type of stromal cells present in various organs of a living body. It secretes growth factors and cytokines, produces and degrades extracellular matrix in response to inflammation and injury of organs and tissues, regulates the function of organs and tissues through various cell-cell interactions, and maintains the microenvironment. Has a role to maintain the nature. On the other hand, even in diseases such as fibrosis and cancer, fibroblasts maintain a pathological microenvironment and also have the aspect of promoting disease. Generally, fibroblasts are known as spindle-shaped cells that hold many cytoplasmic processes, but it is known that the genes and proteins expressed by fibroblasts vary greatly depending on the organ where they are located ( Non-Patent Document 1).
  • fibroblasts that are positive for Vascular cell adhesion molecular-1 (VCAM-1, CD106). (See Patent Document 1).
  • Patent Document 1 in order to obtain a sufficient number of CD106-positive fibroblasts, a technique such as cell sorting using CD106 as a marker is used. However, there was a problem that the amount (cell number) of CD106-positive fibroblasts was very small depending on the fibroblasts.
  • fibroblasts effective as an injection are preferably CD90-positive as well as CD106-positive.
  • An object of the present invention is to provide a new method for producing CD106-positive and / or CD90-positive fibroblasts.
  • the present inventors have studied to solve the above problems, and are known to have TNF- ⁇ (Tumor Necrosis Factor- ⁇ ), which is known as a factor that induces hemorrhagic necrosis in tumors, and allergic effects. Culturing fibroblasts in the presence of one or more selected from the group consisting of IL-4 (Interleukin-4), found that CD106-positive and / or CD90-positive fibroblasts increased, The present invention has been completed.
  • the present invention includes the following first aspect (Invention A).
  • a method for producing CD106-positive and / or CD90-positive fibroblasts Culturing the fibroblasts in the presence of one or more selected from the group consisting of TNF- ⁇ and IL-4 to increase CD106-positive and / or CD90-positive fibroblasts; Including, methods.
  • A3 The method according to (A1) or (A2), wherein the CD106-positive and / or CD90-positive fibroblasts are CD106-positive and CD90-positive.
  • (A4) The method according to any one of (A1) to (A3), further comprising the step of enriching CD106-positive and / or CD90-positive fibroblasts.
  • (A5) The method according to any of (A4), wherein the concentration is performed using an anti-CD106 antibody and / or an anti-CD90 antibody.
  • the present invention includes the following second aspect (Invention B).
  • B1 A cell population containing fibroblasts, including CD106-positive adult fibroblasts.
  • B2 The ratio (cell number) of CD106-positive adult fibroblasts to fibroblasts is 3.36% or more, preferably 5% or more, and more preferably 7% or more of the fibroblasts is CD90.
  • the cell population according to (B1) which is positive, CD106 positive, and G-CSF positive.
  • B3 The cell population according to (B1) or (B2), wherein the adult fibroblasts are CD106-positive and CD90-positive.
  • (B4) The cell population according to any of (B1) to (B3), wherein the adult fibroblasts are adult fibroblasts stimulated by TNF- ⁇ and / or IL-4.
  • (B5) A pharmaceutical composition comprising the cell population according to any of (B1) to (B4) and a pharmaceutically acceptable excipient.
  • (B6) The pharmaceutical composition according to (B5), which is a therapeutic agent for ischemic heart disease.
  • (B7) The pharmaceutical composition according to (B5) or (B6), for administration to an organ from which adult fibroblasts are derived.
  • the present invention includes the following third aspect (Invention C).
  • C1 A method for producing G-CSF positive fibroblasts, Culturing the fibroblasts in the presence of one or more selected from the group consisting of TNF- ⁇ and IL-4, increasing the expression level of G-CSF, and increasing the number of G-CSF positive fibroblasts; Including, methods.
  • C2) The method according to (C1), wherein the cultured fibroblasts are fibroblasts obtained from an adult.
  • C3 The method according to (C1) or (C2), further comprising, after the culture, enriching G-CSF-positive fibroblasts.
  • (C4) The method according to any of (C1) to (C3), wherein the enrichment comprises a step of sorting with an anti-CD106 antibody and / or an anti-CD90 antibody.
  • (C5) A cell population containing isolated CD106-positive and G-CSF-positive fibroblasts.
  • (C6) The cell population according to (C5), wherein the ratio (number of cells) of G-CSF-positive fibroblasts to fibroblasts is 6.75% or more.
  • the present invention further includes the following aspects (Invention D).
  • (D1) An injectable composition for treating a heart disease, comprising a G-CSF positive fibroblast.
  • D2 The composition for injection according to (D1), wherein the ratio (the number of cells) of G-CSF-positive fibroblasts to fibroblasts is 6.75% or more in the composition for injection.
  • (D3) preparing a fibroblast population containing G-CSF positive fibroblasts; Injecting the fibroblast population into or around the necrotic heart tissue area and / or injecting it into the coronary artery.
  • (D4) The method according to (D3), wherein the ratio (cell number) of G-CSF-positive fibroblasts to fibroblasts is 6.75% or more in the fibroblast population.
  • a new method for producing CD106-positive and / or CD90-positive fibroblasts can be provided. Furthermore, a G-CSF positive fibroblast population can be provided.
  • FIG. 2 is a flow cytometry analysis of fibroblasts derived from an adult heart when TNF- ⁇ is added.
  • A is a flow cytometry analysis of adult heart-derived fibroblasts upon addition of IL-4.
  • (B) is a flow cytometry analysis of adult heart-derived fibroblasts when two agents, TNF- ⁇ and IL-4, are added.
  • (A) is a control and none of them was added. It is a graph which shows the ratio of (A) CD106 positive cells, (B) the ratio of CD90 positive cells, and (C) the ratio of both positive (DP: double positive) cells at the added concentration of TNF- ⁇ and IL-4.
  • N 3).
  • (A) shows fetal heart-derived fibroblasts (F-HCF) and iPS cardiomyocyte fraction-derived fibroblasts (i) by addition of TNF- ⁇ (50 ng / mL) and IL-4 (2 ng / mL).
  • -HCF is a flow cytometry analysis diagram. Control shows the flow cytometry analysis of F-HCF and i-HCF without addition of TNF- ⁇ (50 ng / mL) and IL-4 (2 ng / mL).
  • (A) is a flow cytometry analysis diagram of fetal heart-derived CD106-negative fibroblasts (F-VNCF).
  • (B) is a flow cytometry analysis diagram of fetal heart-derived CD106-positive fibroblasts (F-VCF).
  • A-HCF adult heart-derived fibroblasts
  • D Flow cytometry analysis of A-HCF (uA-HCF) cultured with the addition of TNF- ⁇ (50 ng / mL) and IL-4 (2 ng / mL).
  • E Flow cytometry analysis of both CD106 and CD90 positive uA-HCF cell groups (uA90 ⁇ 106-HCF) collected by fluorescence-activated ⁇ cell ⁇ sorting (FACS). Only cells in the gate (P4) enclosed in the figure were collected by FACS.
  • uA90-HCF refers to a CD90-positive uA-HCF cell group subjected to cell sorting using CD90 as an index
  • uA106-HCF refers to a CD106-positive uA-HCF cell group subjected to cell sorting using CD106 as an index.
  • (A) Flow cytometry analysis of fibroblasts administered to a chronic heart failure model rat.
  • (B) is a graph showing the ratio of CD106-positive cells, the ratio of CD90-positive cells, and the ratio of both positive (DP: double positive) cells in fibroblasts administered to a chronic heart failure model rat. It is an echocardiogram image (M mode) of a rat heart (drawing substitute photograph).
  • (A) is a graph showing changes in LVEF (%) of rat heart by various types of cardiac fibroblasts.
  • (C) is a graph showing changes in LVFS (%) of rat heart due to various cardiac fibroblasts.
  • uA90.106-HCF is a group of uA-HCF cells positive for both CD106 and CD90 collected by fluorescence-activated ⁇ cell sorting ⁇ (FACS). Only cells in the gate (P3) enclosed in the figure were collected by FACS. It is an echocardiogram (M mode) of a rat chronic heart failure model to which various cardiac fibroblasts were administered (drawing for photograph).
  • FIG. 1 is a graph showing changes in LVEF (%) in a rat model of chronic heart failure caused by various cardiac fibroblasts (*** P ⁇ 0.01 vs. A-HCF, *** P ⁇ 0.05).
  • vs. uA-HCF ** P ⁇ 0.01 vs. Control (group to which only medium (high-glucose DMEM + 10% neonatal calf serum (NBCS)) was administered), Sham (thoracotomy similar to control and cell-administered group)
  • (B) is a graph showing the difference in increase and decrease of LVEF 18 weeks after injection of various cardiac fibroblasts (LVEF (18W) -LVEF (0W), *** P ⁇ 0.01 vs. A-HCF, *** P ⁇ 0.01 vs. uA-HCF, ** P ⁇ 0.01 vs. Control, * P ⁇ 0.05 vs. Control).
  • (C) is a graph showing changes in LVFS (%) in a rat model of chronic heart failure caused by various cardiac fibroblasts (*** P ⁇ 0.01 ⁇ vs. A-HCF, *** P ⁇ 0.05). vs. @ uA-HCF, ** P ⁇ 0.01 ⁇ vs. @ Control).
  • (D) is a graph showing the difference in LVFS increase / decrease at 18 weeks after injection of various cardiac fibroblasts (LVFS (18W) -LVFS (0W), *** P ⁇ 0.01 vs. A-HCF, *** P ⁇ 0.05 @ vs. @ uA-HCF, ** P ⁇ 0.01 @ vs. @ Control).
  • One embodiment of the present invention is a method for producing CD106-positive (also referred to as CD106 +) and / or CD90-positive (also referred to as CD90 +) fibroblasts, comprising fibroblasts, TNF- ⁇ and IL-4. Culturing in the presence of one or more selected from the group consisting of: increasing CD106-positive and / or CD90-positive fibroblasts. By culturing in the presence of one or more members selected from the group consisting of TNF- ⁇ and IL-4, a population of fibroblasts having increased CD106 + and / or CD90 + fibroblasts can be produced.
  • enrichment refers to an operation of separating cells in which the ratio of the number of specific cells to the total number of cells increases.
  • culture is not included in “concentration”.
  • isolated means that one component is separated from tissue, and “purified” means that one component is separated from at least one or more other components.
  • “comprising” means that an unspecified third component may be included.
  • “consisting of” means essentially free of an unspecified third component. The term “essentially free” is used in the sense that it does not exclude the inclusion of an amount of a third component mixed in the production process that cannot be technically removed.
  • Fibroblasts are a type of stromal cell that produces collagen and other extracellular matrices. Fibroblasts are a cell type that exists in connective tissues in the body and is a major component thereof. Fibroblasts can be positive for one or more selected from the group consisting of the stromal cell markers vimentin and DDR2. In some embodiments, the fibroblasts are not cells that are positive for the cardiomyocyte-specific markers cardiac troponin and ⁇ -actinin. In some embodiments, the fibroblast is not a cell that is positive for VE-cadherin. For example, fibroblasts are not vascular endothelial cells. For example, fibroblasts are not vascular smooth muscle cells.
  • Fibroblasts can be myofibroblasts or they can be non-myofibroblasts. Fibroblasts can be fibroblasts derived from heart tissue, for example, fibroblasts isolated from heart tissue. Fibroblasts can be fibroblasts isolated from epicardium or endocardium. Fibroblasts can be fibroblasts isolated from fetal epicardium or endocardium. Fibroblasts can be fibroblasts isolated from adult epicardium or endocardium. The fibroblasts can be collagen-producing cells that are positive for one or more selected from the group consisting of vimentin and DDR2.
  • the fibroblast in all aspects of the present invention may be preferably fibroblasts derived from cardiac tissue (hereinafter sometimes referred to as “cardiac fibroblasts”), for example, epicardium or intracardiac It may be a fibroblast isolated from a membrane.
  • cardiac fibroblasts any cell that differentiates into fibroblasts in a living body may be used as a material for fibroblasts.
  • the fibroblasts used for transplantation into the heart may preferably be fibroblasts derived from cardiac tissue, epicardium or endocardium.
  • the fibroblasts may be enriched, isolated or purified fibroblasts.
  • fibroblasts there is no limitation on the origin of fibroblasts, and pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and Muse cells, and adult (somatic) stem cells such as mesenchymal stem cells May be used after differentiation.
  • pluripotent stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) and Muse cells
  • adult (somatic) stem cells such as mesenchymal stem cells May be used after differentiation.
  • primary cells collected from animals (including humans) may be used, or established cells may be used.
  • fibroblasts derived from epicardium or endocardium are used, and fibroblasts obtained from an adult human heart are preferred, but not limited thereto.
  • fibroblasts have been described above, in all embodiments the same applies to fibroblasts derived from cardiac tissue and fibroblasts isolated from epicardium or endocardium. . In the following examples, the present invention will be described in experiments using cardiac fibroblasts as fibroblasts.
  • CD106 also called VCAM-1 (VCAM1)
  • VCAM1 VCAM-1
  • CD90 is also called Thy-1 (Thy1)
  • GPI glycosyl-phosphatidylinositol
  • CD90 + fibroblasts do not contain cardiomyocytes is a concept that it is acceptable that some of them are contained, and 5% or less, 4% or less, 3% or less of all cells, It may be 2% or less, 1% or less, 0.5% or less, 0.1% or less, 0.01% or less.
  • the ratio of CD90 + fibroblasts may be 1% or more, 10% or more, 20% or more, or 30% or more. May be 40% or more, may be 50% or more, may be 60% or more, may be 70% or more, may be 80% or more, may be 90% or more, It may be 95% or more, 98% or more, or 100%.
  • the fibroblasts may be Connexin43 positive (Connexin43 +) fibroblasts.
  • Connexin 43 is a transmembrane protein that is known to be expressed along with atherosclerotic plaque on the surface of blood vessels and to bind to adjacent cells as gap junctions in cardiomyocytes and to transmit electrical excitation of the heart. .
  • the present inventors believe that Connexin 43+ enables the exchange of electrical signals in heart tissue and improves the therapeutic effect when applied to heart disease.
  • the ratio of Connexin43 + fibroblasts may be 1% or more, 10% or more, 20% or more, or 30% or more. , 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more %, 98% or more, and 100%.
  • the method of contacting fibroblasts with one or more factors selected from the group consisting of TNF- ⁇ and IL-4 is not particularly limited, and typically, fibroblasts Adding the factor to the medium containing the cells, but is not limited to this.
  • factors selected from the group consisting of TNF- ⁇ and IL-4
  • they may be separately contacted with fibroblasts, respectively, or may be contacted simultaneously.
  • a plurality of factors may be mixed once and then added to fibroblasts.
  • the fibroblasts can be cultured in the medium to which the factor is added, the fibroblasts can be cultured while maintaining the expression levels of CD106 and / or CD90 in the fibroblasts. Therefore, fibroblasts having high CD106 and / or CD90 expression levels can be produced.
  • the amount of TNF- ⁇ added is not particularly limited, but is usually 0.1 ng / mL or more, may be 0.5 ng / mL or more, and may be 1 ng / mL or more. And may be 10 ng / mL or more.
  • the upper limit is not particularly limited, and is usually 500 ng / mL or less, and may be 100 ng / mL or less.
  • the amount of IL-4 added is not particularly limited, but is usually 0.1 ng / mL or more, may be 0.5 ng / mL or more, and may be 1 ng / mL or more.
  • the upper limit is not particularly limited, and is usually 10 ng / mL or less, may be 5 ng / mL or less, and may be 1 ng / mL or less.
  • the ratio (weight ratio) of TNF- ⁇ to IL-4 to be added is usually 10,000: 1 to 1: 1 for TNF- ⁇ : IL-4, and 50000: 1 to 10: 1. : 1 may be used. Further, the ratio is 1: 1 to 1: 10000, and may be 1:10 to 1: 50000.
  • CD106 + and / or CD90 + fibroblasts can be produced by further culturing the fibroblasts contacted with these factors.
  • the culture of the fibroblasts is not particularly limited as long as it can be CD106 + and / or CD90 +, or may be performed under a condition suitable for the culture, and may be performed by a known cell culture method.
  • the medium used for the culture can be appropriately set depending on the type of the cells to be cultured and the like. For example, DMEM, ⁇ -MEM, RPMI-1640, HFDM-1 (+) and the like can be used. Nutrients such as FCS and FBS, growth factors, cytokines, antibiotics and the like may be added to the medium.
  • the cells may be cultured in a medium containing TNF- ⁇ and / or IL-4.
  • the number of days can be appropriately set for the culture period depending on the purpose, such as until a desired number of cells is obtained and / or a desired function is provided. For example, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 2 weeks, 1 month, 2 months, 3 months, 6 months, etc. can give.
  • the culture temperature can be appropriately set according to the type of cells to be cultured, and may be, for example, 10 ° C or higher, 15 ° C or higher, 20 ° C or higher, 25 ° C or higher, 30 ° C or higher, C. or lower, 50 C. or lower, 45.degree.
  • Cultured fibroblasts may be collected by a collection step.
  • the cells may be separated and collected by a protease such as trypsin, or the cells may be separated by temperature change and collected using a temperature-responsive culture dish.
  • the antibody may be collected or concentrated by an automatic magnetic cell separator (eg, autoMACS), or may be collected or concentrated by a magnetic cell separator (eg, MACS), It may be collected or concentrated by a closed magnetic cell separation device (for example, Prodigy), or may be collected or concentrated by a cell sorter (for example, FACS).
  • a drug resistance gene may be introduced under the promoter of the gene encoding the CD90 protein and / or CD106 protein, and selection may be made with a drug.
  • the production method of the present embodiment may include a step of sorting CD90 + fibroblasts using an anti-CD90 antibody, and a step of sorting CD106 + fibroblasts using an anti-CD106 antibody, These two steps may be combined.
  • the step of sorting may be performed at any timing. It may be before the contact with the above factors, after the contact with the factors, before the culture, or after the culture.
  • the anti-CD90 antibody and the anti-CD106 antibody known antibodies can be used, and commercial products can be obtained and used.
  • Sorting with an anti-CD90 antibody and / or an anti-CD106 antibody increases the proportion of CD106 + and / or CD90 + fibroblasts in fibroblasts, and has a high therapeutic effect on heart disease CD106 + and / or CD90 + fibroblasts Can be obtained.
  • fibroblasts with improved expression of CD106 and / or CD90 can be obtained, and the fibroblast population containing the fibroblasts is another of the present invention. It is an embodiment.
  • a cell population of fibroblasts including CD106 + and / or CD90 + adult fibroblasts has enhanced expression of CD106 and / or CD90 by being stimulated by TNF- ⁇ and / or IL-4, It can be suitably used as a pharmaceutical composition.
  • the ratio of CD106 + fibroblasts to total fibroblasts in the cell population is not particularly limited, but may be 3.36% or more, 5% or more, or 10% or more based on the number of cells. 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more , 80% or more, 90% or more, and 95% or more.
  • the ratio of CD90 + fibroblasts to total fibroblasts is not particularly limited, but may be 5% or more, 10% or more, or 20% or more based on the number of cells. , 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% % Or 95% or more.
  • the ratio of CD106 + and CD90 + fibroblasts to total fibroblasts is not particularly limited, but may be 5% or more, 10% or more, or 20% or more based on the number of cells. 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more , 90% or more, and 95% or more.
  • G-CSF-positive fibroblasts can be produced by contacting fibroblasts with one or more factors selected from the group consisting of TNF- ⁇ and IL-4. ing. It has been reported that G-CSF suppresses myocardial cell death associated with heart failure and suppresses progression of myocardial remodeling (Harada, M. et al. G-CSF presents cardiac remodeling after myocardial infection activation-activating action-activating joint-action). Stat pathway in cardiomyocytes. Nat. Med. 11, 305-311 (2005)). It has also been clarified that cell proliferation of cardiomyocytes is promoted (Shimoji, K. et al.
  • G-CSF Promotes the Proliferation of Developing Cardiomyocytes InVivo and in-derivation from CE2 CromS romance Descrip- tion of the Derivatives from Derivatives from Derivatives of the Derivatives of the Cardiomyocytes. (2010)).
  • a method of producing G-CSF-positive fibroblasts which comprises the step of culturing under conditions that enhance CSF expression.
  • the produced G-CSF-positive fibroblasts may be enriched with an automatic magnetic cell separator (eg, autoMACS) using an anti-CD90 antibody and / or an anti-CD106 antibody, or a magnetic cell separator (eg, MACS).
  • a drug resistance gene may be introduced under the promoter of the G-CSF protein-encoding gene, and selection may be made with a drug.
  • G-CSF-positive fibroblasts are obtained, and a fibroblast population containing the G-CSF-positive fibroblasts is another embodiment of the present invention. And injecting the cell population containing the G-CSF-positive fibroblasts into or around the necrotic heart tissue region and / or injecting it into the coronary artery. It can also be applied to methods for treating heart disease.
  • the ratio of G-CSF positive fibroblasts to total fibroblasts is not particularly limited, but may be 1% or more, or 5% or more, based on the number of cells, 6.75% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, It may be 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more.
  • the expression level of the gene encoding the G-CSF protein in fibroblasts may be 0.01 or more, 0.02 or more, or 0.03 or more, with FPKM normalized with ⁇ -actin. May be present, may be 0.04 or more, may be 0.05 or more, and may be 0.1 or more.
  • the expression level of the gene encoding the G-CSF protein in the fibroblast is 1.1 times or more, and may be 2 times or more, as compared with the expression level of the natural (living body) fibroblast. May be 5 times or more, may be 10 times or more, may be 20 times or more, may be 50 times or more, may be 100 times or more, may be 200 times or more. , 500 times or more.
  • G-CSF positive fibroblasts may be positive for CD106 and / or CD90, and the proportion of fibroblasts (based on the number of cells) may be 1% or more, or 10% or more; 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% Or more, 90% or more, 95% or more, 98% or more, and 100%.
  • the proportion (based on the number of cells) of G-CSF-positive fibroblasts among CD106 and / or CD90-positive fibroblasts may be 1% or more, It may be 3% or more, 5% or more, 10% or more, 25% or more, or 50% or more.
  • G-CSF-positive, CD106-positive, and CD90-positive fibroblasts may be 1% or more, 5% or more, and 7% or more in the cell population containing fibroblasts, based on the number of cells. % Or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more 70% or more, 80% or more, 90% or more, 95% or more, 98% or more, 100% Good.
  • the cell population of fibroblasts as a pharmaceutical composition may contain other components that are physiologically acceptable as a pharmaceutical composition, in addition to the cell population of fibroblasts.
  • the function of the heart can be improved.
  • the heart disease includes diseases caused by disorders, defects, dysfunction, and the like of heart tissue, and includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, and dilated cardiomyopathy. Examples include, but are not limited to: That is, according to the present invention, there is provided a pharmaceutical composition comprising CD106 + and / or CD90 + fibroblasts, or G-CSF + fibroblasts, which is used for improving cardiac function. Pharmaceutical compositions are provided for use in proliferating and / or for improving cardiac ejection fraction. According to the present invention, there is also provided a pharmaceutical composition containing CD106 + and / or CD90 + fibroblasts or G-CSF + fibroblasts, which is used for suppressing progression of fibrosis of heart tissue. Is provided.
  • CD106 + and / or CD90 + fibroblasts or G-CSF + fibroblasts secrete cytokines and the like to maintain organ homeostasis and regulate inflammatory responses, and the secreted cytokines / chemokines regenerate myocardial tissue. It can form a microenvironment suitable for the growth of the myocardial cells and regulate the pulsation of the myocardial cells.
  • CD106 + and / or CD90 + fibroblasts, or G-CSF + fibroblasts can inhibit the progression of fibrosis.
  • the injection may contain other cells and other components in addition to CD106 + and / or CD90 + fibroblasts or G-CSF + fibroblasts.
  • the ratio of CD106 + and / or CD90 + fibroblasts or G-CSF + fibroblasts to the total amount of fibroblasts contained in the injection is 0.03% or more based on the number of cells.
  • the number of CD106 + and / or CD90 + and G-CSF + fibroblasts contained in the injection may be 1 ⁇ 10 6 cell or more, and may be 5 ⁇ 10 6 cell or more. It may be 1 ⁇ 10 7 cells or more.
  • the CD106 + and / or CD90 + fibroblasts contained in the injectable composition, or the G-CSF + fibroblasts may be cells co-cultured with other cells, such as cardiomyocytes.
  • the injection may contain other components that are physiologically acceptable as an injection.
  • Such other components include physiological saline, phosphate buffered saline (PBS), cell preservation solution, cell culture solution, hydrogel, extracellular matrix, cryopreservation solution, and the like.
  • injectables are obtained by injecting adult fibroblasts into heart tissue, for example, by injecting into or around necrotic heart tissue area, and / or in coronary arteries, or into veins, arteries, lymph nodes, lymph vessels, Can treat heart disease.
  • autologous transplantation may be performed by using the tissue of the patient with heart disease as the origin of the adult fibroblast.
  • the fibroblast population may be used as a culture scaffold for other cells and may be used to form organs or tissues, for example, to construct planar or three-dimensional cellular tissue.
  • the fibroblast population may be a planar or three-dimensional tissue after co-culturing other cells, for example, cardiomyocytes, but can function effectively as a planar or three-dimensional tissue without co-culturing.
  • Examples of the flat or three-dimensional tissue include, but are not limited to, a cell sheet, a cell fiber, and a tissue formed by a 3D printer.
  • Heart disease can be treated by applying such planar or three-dimensional tissue to a necrotic heart tissue region or replacing it with necrotic heart tissue as an artificial organ.
  • BV421 mouse anti-human CD106 antibody (BD Biosciences, San Jose, CA); BV421 ⁇ ⁇ ⁇ mouse IgG1; ⁇ isotype control (BD Biosciences); human CD90-PE ( Miltenyi Biotech, Bergisch Gladbach, Germany; REA control (S) -PE (Miltenyi Biotec); mouse monoclonal anti-cardiac troponin T (cTnT) ⁇ (Thermo, Scientific, Waltham, UK; Rabbit, Korea, K.K.
  • F-VNCF CD106-negative fetal heart-derived fibroblasts
  • F-VCF CD106-positive fetal-heart-derived fibroblasts
  • uA106-HCF adult heart-derived fibroblasts were supplemented with TNF- ⁇ and IL-4.
  • CD106-positive adult heart-derived fibroblasts cell-sorted using CD106 as an index were subjected to primary immunostaining with human CD106 (VCAM-1) -biotin (Miltenyi Biotec). And secondary immunostaining with anti-biotin microbeads (Miltenyi Biotec).
  • uA90-HCF CD90-positive adult heart-derived fibroblasts obtained by adding TNF- ⁇ and IL-4 to adult heart-derived fibroblasts to increase the proportion of CD90- and CD106-positive cells, followed by cell sorting using CD90 as an index Cells
  • uA90-HCF CD90-positive adult heart-derived fibroblasts obtained by adding TNF- ⁇ and IL-4 to adult heart-derived fibroblasts to increase the proportion of CD90- and CD106-positive cells, followed by cell sorting using CD90 as an index Cells
  • BV421 mouse anti-human CD106 antibody (BD Biosciences, San Jose, Calif.); BV421 mouse IgG1; kappa isotype control (BD Biosciences); Immunostaining was performed with (S) -PE (Miltenyi Biotec), and the cells were collected by cell sorting with BD FACS ARIA III (BD Biosciences).
  • Adult cardiac fibroblasts (uA90 ⁇ 106-HCF) with a high proportion of CD90 and CD106 positive cells can be obtained by adding TNF- ⁇ and IL-4 to adult heart-derived fibroblasts and increasing the proportion of CD90 and CD106 positive cells.
  • the cells were collected by cell sorting using BD FACS ARIA III (BD Biosciences).
  • the concentration and purity of the recovered RNA were analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif.) And a NanoDrop One (Thermo Fisher Scientific). 1 ⁇ g of RNA having a RIN value of 7 or more was used for library preparation. Preparation of the library for the next-generation sequencer was performed according to the manufacturer's protocol of NENBNext Ultra RNA Library Prep Kit for Illumina.
  • mRNA was isolated using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB) and Ribo-Zer rRNA removal Kit (Illumina). For the mRNA fragmentation and priming, NEBNext First Strand Synthesis Reaction Buffer and NEBNext Random Primers were used. First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase. The second strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix.
  • NEBNext Poly A
  • NEB mRNA Magnetic Isolation Module
  • Ribo-Zer rRNA removal Kit Illumina
  • First strand cDNA was synthesized using ProtoScript II Reverse Transcriptase.
  • the second strand cDNA was synthesized using Second Strand Synthesis Enzyme Mix.
  • Double-stranded cDNA purified by AxyPrep Mag PCR Clean-up (Axygen, Union City, Calif.) was treated with End Prep Enzyme Mix to repair both ends and add dA to the end of the amplified product, and then perform TA cloning.
  • AxyPrep Mag PCR Clean-up (Axygen).
  • Each sample was amplified for 11 cycles by PCR using P5 and P7 primers.
  • the PCR product was washed with AxyPrep Mag PCR Clean-up (Axygen) and evaluated with an Agilent 2100 Bioanalyzer (Agilent Technologies). For quantification of the PCR product, Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) was used.
  • Expression difference analysis used DESeq Bioconductor package. Correction of false discovery rate (FDR) was performed by Benjamini and Hochberg's, and P-value ⁇ 0.05 was considered significant.
  • IN Cell Analyzer 2200 GE Healthcare, Buckinghamshire, UK
  • IN Cell Developer Toolbox 1.92 GE Healthcare
  • inhalation anesthesia gas was connected to a ventilator to perform anesthesia.
  • the patient Under artificial respiratory management, the patient was fixed in the supine position, and two or three ribs were cut from the left third rib to the fifth rib at the position of the costal cartilage, and the chest was opened. The pericardium was exfoliated to expose the heart, and the left atrium was lifted, and the depth of the left ventricle was measured using a weakly curved round needle with a vascular thread (6-0: Nescosture).
  • the yarn was passed through a length of about 2 mm and a length of 4 to 5 mm.
  • the thread was squeezed using an arterial cleanser (snare method), and the coronary artery was subjected to ischemia for 30 minutes.After 30 minutes, reperfusion was performed, and when the condition was stabilized, it was confirmed that there was no bleeding, and thoracic drainage was performed.
  • the muscle layer and the skin were sutured.Intracutaneous suture was performed on the skin, and when the normal suture was performed, thread removal was performed while observing the postoperative condition.
  • LVEF left ventricular ejection fraction
  • fibroblasts ⁇ administered to rats with chronic heart failure>
  • the various fibroblasts cryopreserved on the day of the administration test were thawed, diluted with high-glucose DMEM + 10% NBCS, and 2.0 ⁇ 10 6 cells / 50 ⁇ L of a cell suspension was determined as the number of viable cells for each individual.
  • the animals were maintained under anesthesia in the same manner as when the model was prepared, and under the control of artificial respiration, the infarct was divided into two sites and a total of 50 ⁇ L of the cell suspension was administered using a catheter with a 30G needle.
  • TNF- ⁇ and IL-4 were added as a mixture of two drugs to fibroblasts (A-HCF) derived from an adult heart, and cultured in HFDM-1 (+) medium for 3 days.
  • the percentage of CD106-positive cells (%) and the percentage of CD90-positive cells (%) were evaluated using a meter.
  • FIGS. 3-1 and 3-2 From FIG. 3A, when the two agents TNF- ⁇ and IL-4 shown in B were mixed and added as compared with the control shown in A (no addition), the ratio (%) of CD106-positive cells and CD90-positive cells It can be understood that the ratio (%) of the two agents TNF- ⁇ and IL-4 shown in B were mixed and added as compared with the control shown in A (no addition), the ratio (%) of CD106-positive cells and CD90-positive cells It can be understood that the ratio (%) of the
  • TNF- ⁇ 50 ng / mL
  • IL-4 2 ng / mL
  • F-HCF fetal heart-derived fibroblasts
  • i-HCF iPS cell-derived cardiac fibroblasts
  • the ratio (%) of CD106-positive cells and the percentage of CD90-positive cells were obtained when two agents, TNF- ⁇ and IL-4, were mixed and added, even if the origin of cardiac fibroblasts was different. It can be understood that the ratio (%) of the
  • fibroblasts obtained by cell-sorting F-HCF with an anti-CD106 antibody to greatly increase the ratio of CD106 and CD90-positive cells, and fibroblasts having a greatly reduced ratio of CD106-positive cells (F-VNCF) was added to control, A-HCF, and A-HCF were mixed with TNF- ⁇ (50 ng / mL) and IL-4 (2 ng / mL) in a mixture of two agents.
  • G-CSF granulocyte-colony-stimulating factor
  • G-CSF can significantly increase the expression level by adding two agents, TNF- ⁇ and IL-4, to A-HCF and increasing the ratio of CD106 and CD90 positive cells. It was also found that the expression level of G-CSF can be further improved by performing cell sorting of uA-HCF and increasing the ratio of CD90 and CD106 positive cells. Previous studies have reported that G-CSF suppresses cardiomyocyte death associated with heart failure and suppresses progression of myocardial remodeling, and has also been shown to promote cardiomyocyte cell proliferation.
  • TNF- ⁇ 50 ng / mL
  • IL-4 2 ng / mL
  • the prepared cardiac fibroblasts were prepared as described in Table 1.
  • cardiac fibroblasts cultured without adding TNF- ⁇ and IL-4 were prepared as a control.
  • the prepared cardiac fibroblasts having an improved ratio of CD90 and CD106 positive cells are subjected to cell sorting with an anti-CD90 antibody or a CD106 antibody, so that both CD90 and CD106 positive (DP: double positive) fibroblasts can be efficiently treated.
  • DP double positive
  • iPS-CM iPS-derived cardiomyocytes
  • FIG. 6 shows that the number of cardiomyocytes in a proliferating state was increased under the co-culture conditions of uA-HCF to which TNF- ⁇ and IL-4 were added, as compared with A-HCF, and uA90-HCF and uA106-HCF were observed. Under the co-culture conditions, an increase in the number of proliferating cardiomyocytes was observed.
  • a comparative analysis of the increase / decrease rate (Delta-LVEF, Delta-LVFS) of LVEF and LVFS by various fibroblasts showed that uA-HCF and uA90-HCF improved Delta-LVEF and Delta-LVFS from before cell transplantation.
  • uA90-HCF can significantly improve Delta-LVEF and Delta-LVFS as compared to A-HCF.
  • TNF- ⁇ and IL-4 ⁇ Improvement of the proportion of G-CSF positive cells in human fibroblasts and the proportion of G-CSF positive cells by adding TNF- ⁇ and IL-4> TNF- ⁇ (50 ng / mL) and IL-4 (2 ng / mL) were added as a mixture of two agents, and cultured in HFDM-1 (+) medium for 3 days to improve the ratio of CD90 and CD106 positive cells.
  • Cardiac fibroblasts (uA-HCF) were prepared as described in Table 2. Further, cardiac fibroblasts (uA90 ⁇ 106-HCF) cell-sorted with an anti-CD90 antibody and an anti-CD106 antibody were prepared.
  • A-HCF, uA-HCF, and uA90-HCF were administered intracardiacly to a rat model of chronic heart failure prepared by ischemia-reperfusion using a syringe, and long-term therapeutic effects of various fibroblasts on rat heart failure were evaluated by echocardiography. did.
  • the treatment effect of heart failure could not be recognized by administration of A-HCF, and the LVEF at 18 weeks after transplantation showed 44.1 ⁇ 2.8% and the LVFS showed 17.8 ⁇ 1.4%.
  • uA90-HCF which had been subjected to cell sorting using the CD90 antigen as an index, was found to have an effect of improving LVEF and LVFS over a long period of time (12 weeks after transplantation), and the LVEF at 18 weeks after transplantation was 61.0 ⁇ 2.2. %, And the LVFS showed 27.2 ⁇ 1.4%.
  • uA90-HCF-administered group showed a Delta-LVEF value of 9.2 ⁇ 2.6% and a Delta-LVFS value of 5.6 ⁇ 1.6% at 18 weeks after transplantation. Therefore, it is understood that uA90-HCF can significantly improve the function of failing myocardium over a long period of time.
  • the breakdown of the main items (LVEF, LVFS) and the breakdown of the sub items (LVEDV, LVESV, LVIDd, LVIDs, LVAWd, LVPWTd, HR) are shown in Tables 3 to 11.

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Cited By (6)

* Cited by examiner, † Cited by third party
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WO2021177387A1 (ja) * 2020-03-04 2021-09-10 株式会社メトセラ エリスロポエチン産生能が亢進された線維芽細胞
WO2022186349A1 (ja) 2021-03-04 2022-09-09 株式会社メトセラ リンパ管新生促進因子発現線維芽細胞及びそれを含む医薬組成物
JPWO2022186349A1 (https=) * 2021-03-04 2022-09-09
JP7233147B2 (ja) 2021-03-04 2023-03-06 株式会社メトセラ リンパ管新生促進因子発現線維芽細胞及びそれを含む医薬組成物
KR20230154177A (ko) 2021-03-04 2023-11-07 가부시키가이샤 메토세라 림프관 신생촉진인자 발현 섬유아세포 및 이를 포함하는의약 조성물
WO2023037868A1 (ja) 2021-09-08 2023-03-16 株式会社メトセラ 線維芽細胞を含む腎疾患治療用医薬組成物

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AU2019332400A1 (en) 2021-04-01
SG11202102028SA (en) 2021-03-30
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EP3845635A1 (en) 2021-07-07
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JP6883904B2 (ja) 2021-06-09
AU2019332400B2 (en) 2025-06-26
CN112639082A (zh) 2021-04-09
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US20210254009A1 (en) 2021-08-19

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