WO2017026838A1 - Cellules souches adhésives améliorées dérivées de cordon ombilical, leur procédé de préparation, et leur utilisation - Google Patents

Cellules souches adhésives améliorées dérivées de cordon ombilical, leur procédé de préparation, et leur utilisation Download PDF

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WO2017026838A1
WO2017026838A1 PCT/KR2016/008887 KR2016008887W WO2017026838A1 WO 2017026838 A1 WO2017026838 A1 WO 2017026838A1 KR 2016008887 W KR2016008887 W KR 2016008887W WO 2017026838 A1 WO2017026838 A1 WO 2017026838A1
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stem cells
umbilical cord
derived
cell
disease
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PCT/KR2016/008887
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English (en)
Korean (ko)
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임재승
신정민
유지민
김지혜
강아름
김혜선
김현주
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주식회사 차바이오텍
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Priority to CN201680057936.5A priority Critical patent/CN108138137A/zh
Priority to JP2018507615A priority patent/JP6648259B2/ja
Priority to ES16835478T priority patent/ES2914692T3/es
Priority to US15/752,057 priority patent/US11690877B2/en
Priority to EP16835478.5A priority patent/EP3336176B1/fr
Priority claimed from KR1020160102721A external-priority patent/KR20170020273A/ko
Publication of WO2017026838A1 publication Critical patent/WO2017026838A1/fr

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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
    • A61K35/48—Reproductive organs
    • A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells

Definitions

  • Cell therapy is a medicine used for the purpose of preventing or treating certain diseases by changing the characteristics of cells by proliferating or selecting cells in vitro to restore the function of cells and tissues.
  • the field is receiving.
  • stem cell therapy can be divided into embryonic stem and adult stem cells.
  • stem cells are isolated and cultured from adipose tissue or cord blood.
  • bone marrow and adipose tissue cells are collected by invasive methods, and stem cells isolated from mature or old age patients have differentiation and proliferation ability.
  • umbilical cord blood collection is easy but the content of stem cells in the umbilical cord blood is low.
  • the umbilical cord unlike bone marrow or adipose-derived cells, is extracted from tissues that have already been separated from the body and thus is non-invasive and facilitates the extraction process. In addition, unlike embryonic stem cells, they are free from ethical aspects. Therefore, it has recently been in the spotlight as a useful material of refractory or regenerative medicine, and as a primitive cell, it can satisfy both proliferative capacity and differentiation capacity, and has the advantage that it can be used after differentiation according to organ characteristics as well as organ regeneration. However, since there are many types of cells inside the umbilical cord, studies to find the optimal cell as a therapeutic agent and to identify new characteristics of cells that can be separated or extracted into a uniform cell population are required.
  • One aspect is to provide an improved umbilical cord derived adherent stem cell or cell population thereof.
  • Another aspect includes culturing by attaching the separated umbilical cord to the culture vessel; Separating the improved umbilical cord-derived adherent stem cells by contacting the cultured umbilical cord with a separation enzyme; Providing a method for producing an improved umbilical cord-derived stem cells comprising the step of passage the separated improved umbilical cord-derived stem cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin will be.
  • FGF-4 fibroblast growth factor-4
  • Another aspect is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising the improved umbilical cord-derived adherent stem cells, their cell populations or their cultures as an active ingredient.
  • One aspect provides improved umbilical cord derived stem cells.
  • the improved umbilical cord-derived attached stem cells may be one or more of the following characteristics selected from (a) to (e):
  • CCND1, SERPINE1, PRNP, and CYP1B1 are less expressed than bone marrow stem cells
  • CD200 + selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.
  • the improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):
  • At least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;
  • one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;
  • one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells
  • one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.
  • the improved umbilical cord-derived attached stem cells may be one of the surface antigen characteristics of e) additionally Oct4-, or Nanog-.
  • CD61 + may be a surface antigen property overexpressed in hypoxic conditions.
  • the term “umbilical cord” may refer to a line that connects the mother and the abdomen to allow the mammalian fetus to grow in the placenta, generally three vessels surrounded by Wharton jelly, ie two umbilical arteries It may mean a tissue consisting of and one umbilical vein.
  • the term “enhanced Umbilical Cord Adherent Stem cells” or “Umbilical Cord Adherent Stem cells” herein refers to wharton's Jelly tissue of the umbilical cord or umbilical cord. It can mean a cell derived from, having the ability to differentiate into a variety of tissue cells and has the property of growing on the surface of the culture vessel.
  • the improved umbilical cord-derived adherent stem cells provided herein provide for at least about 20%, 25%, 30%, 35%, CD200, CD141, CD61, CD87, or SSEA4 positive surface markers for cell markers expressed on the cell surface. 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or about 99% and expresses stem cell marker Oct4 At least about 70%, at least 60%, at least 50%, at least 40%, at least Nanog, Tra-1-60, CD3, CD1a, CD11c, CD16, CD86, CD8a, MIC A / B or CD40 negative markers , At least 30% or less, at least 20% or less, at least 10% or less, at least 5% or less, or at least 1% or less.
  • the term "positive” may refer to a stem cell label, which means that the label is present in a greater amount, or at a higher concentration as compared to other non-stem cells on which it is a reference. That is, a cell is positive for that label because a label is present inside or on the surface of the cell and the label can be used to distinguish the cell from one or more other cell types. It may also mean that the cell has its label in an amount sufficient to give a signal greater than the background value, for example a signal from a cytometry device. For example, if a cell can be detectably labeled with an antibody specific for CD200 and the signal from this antibody is detectably greater than the control (eg background value), then the cell is "CD200 +".
  • the term “negative” means that even when an antibody specific for a specific cell surface label is used, the label cannot be detected in comparison with the background value. For example, if a cell cannot be detectably labeled with an antibody specific for CD3, the cell is "CD3-".
  • Such immunological properties can be determined by conventional methods known in the art. For example, various methods may be used, such as flow cytometry, immunohistochemical staining, or RT-PCR.
  • An improved umbilical cord-derived adherent stem cell may express more than one gene or protein selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 compared to bone marrow-derived stem cells. Specifically, at least two or three or more genes or proteins selected from the group consisting of COL1A1, IGFBP4, TAGLN, STC1, LRRC17 and IL33 in the cells, or more specifically, all genes or proteins are more than bone marrow-derived stem cells. Can be expressed.
  • the genes expressed more in the improved umbilical cord-derived adherent stem cells may include.
  • the gene has not been reported for its association with enhanced umbilical cord derived stem cells.
  • the improved umbilical cord-derived stem cells may exhibit a difference in expression levels of two or more times for the gene as compared to the bone marrow-derived stem cells.
  • the difference in the expression level may be, for example, comparing the expression level of the gene at the mRNA level.
  • the expression level difference may also be, for example, by microarray analysis.
  • the improved umbilical cord-derived attached stem cells may express less than one or more genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 compared to bone marrow-derived stem cells.
  • at least two, or at least three, genes or proteins selected from the group consisting of CCND1, SERPINE1, PRNP, and CYP1B1 in the cells, or more specifically, all genes or proteins may be less expressed than bone marrow-derived stem cells.
  • Less gene or protein expressed in the improved umbilical cord-derived attached stem cells according to one embodiment compared to the bone marrow-derived stem cells may include MTA2A, TM4SF1, HIST1H4C, and NME1.
  • the gene or protein has not been reported for its association with improved umbilical cord derived stem cells.
  • the improved umbilical cord-derived stem cells may exhibit a difference in expression levels of two or more times for the genes or proteins as compared to the bone marrow-derived stem cells.
  • the difference in the expression level may be, for example, comparing the expression level of the gene at the mRNA level.
  • the expression level difference may also be, for example, by microarray analysis.
  • the improved umbilical cord-derived stem cells may have the form of subcultured fibroblasts.
  • the cell may have a property of a cell that requires attachment to a surface for growth in vitro, and may exhibit a specific form of fusiform fibroblasts.
  • the improved umbilical cord-derived attached stem cells may be one having colony forming ability.
  • the cells may have a high colony forming ability as compared to culture in normal acid conditions.
  • the improved umbilical cord-derived stem cells may be differentiated into adipocytes, osteocytes or chondrocytes.
  • the cells can be induced to differentiate along specific cell lineages, including, for example, adipocyte differentiation, chondrocyte differentiation, osteoblast differentiation, hematopoietic cell differentiation, myocyte differentiation, vascular cell differentiation, neuronal differentiation, hepatocyte differentiation.
  • differentiation refers to a phenomenon in which structures or functions are specialized to each other during cell division, proliferation, and growth, that is, changes in form or function to perform a task given to each cell, tissue, etc. of an organism.
  • Measurement of differentiation into specific cell types can be performed by methods well known in the art, and can induce differentiation into specific cells through known methods.
  • the differentiation may also be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labeling (e.g., staining cells with tissue-specific or cell-labeling specific antibodies) and morphological changes, Or by examining the morphology of the cells using confocal microscopy, or by measuring changes in gene expression using techniques well known in the art such as PCR and gene-expression profiles.
  • the improved umbilical cord-derived stem cells are IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, GRO, IFN ⁇ , IL-1a, IL-1b, IL-1ra, IL- 3, IL-4, IL-7, IL-9, IL-12 (p40), IL12 (P70), IL-13, IL-14, IFN ⁇ 2, MDC, sIL-2Ra, Eotaxin, Flt-3 ligand, MCP Can secrete proteins of ⁇ 1, MIP-1a, MIP1b, RANTE, Fractalkine, IP-10, EGF, FGF-2, IGF-1 SR, EpCAM, IGFBP3 or a combination thereof.
  • the cell is at least two, at least three, at least four, five of the protein selected from the group consisting of IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, and GRO At least 6, at least 7, at least 8, at least 9, at least 10 or all proteins can be secreted.
  • the improved umbilical cord-derived stem cells are S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME, MIR1978, SLC2A3, BHLHB2, in cells cultured in hypoxic conditions compared to normal oxygen culture conditions.
  • the expression level of one or more genes or proteins selected from the group consisting of BNIP3L, IGFBP5, NDUFA4L2, DPYD, and SCARA3 may be increased.
  • the improved umbilical cord-derived stem cells cultured in hypoxic culture conditions were S100A10, BNIP3, IGFBP5, PGK1, TPI1, DCN, PGM1, PFKFB3, LOC644774, MME than the improved umbilical cord-derived stem cells cultured in normal oxygen culture conditions.
  • the gene or protein has not been reported for its association with improved umbilical cord derived stem cells.
  • the difference in expression levels can be two or more times.
  • the difference in expression level may be, for example, a comparison of expression levels of genes and proteins at the mRNA or protein level.
  • the expression level difference can also be, for example, by microarray and proteomics analysis.
  • the improved umbilical cord-derived attached stem cells have an expression level of one or more genes or proteins selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 in cells cultured in hypoxic culture conditions compared to normal oxygen culture conditions. May be decreasing.
  • the improved umbilical cord-derived stem cells cultured in hypoxic culture conditions are at least two selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 than the improved umbilical cord-derived stem cells cultured in normal oxygen culture conditions.
  • the level of expression of three or more or all genes or proteins may decrease.
  • the gene or protein has not been reported for its association with improved umbilical cord derived stem cells.
  • the difference in expression levels can be two or more times.
  • the difference in expression level may be, for example, a comparison of expression levels of genes and proteins at the mRNA or protein level.
  • the expression level difference can also be, for example, by microarray and prote
  • Another aspect provides an improved umbilical cord derived stem cell population.
  • the umbilical cord-derived attached stem cells are as described above.
  • Another aspect includes culturing by attaching the separated umbilical cord to the culture vessel; Separating the improved umbilical cord-derived adherent stem cells by contacting the cultured umbilical cord with a separation enzyme; It provides a method for producing an improved umbilical cord-derived attached stem cells comprising the step of passage the separated improved umbilical cord-derived attached stem cells in a medium containing fibroblast growth factor-4 (FGF-4) and heparin. .
  • FGF-4 fibroblast growth factor-4
  • the umbilical cord may use a placenta isolated after childbirth from a healthy mother (eg, HIV, HCV, HBV negative mother). That is, the term "isolated umbilical cord” may refer to an umbilical cord separated after giving birth from a mother's mother. The separated umbilical cord may be stored in a sterilized container and ice quickly after being separated.
  • a healthy mother eg, HIV, HCV, HBV negative mother.
  • the method of obtaining separating the umbilical cord from the placenta includes, for example, separating the umbilical cord from the separated placenta; Removing blood outside the separated umbilical cord; Removing the arteries and veins of the umbilical cord from which the blood is removed; And / or severing the blood from which the arteries and veins have been removed to a predetermined size (eg, 1 to 20 mm).
  • a predetermined size eg, 1 to 20 mm.
  • the step of separating the stem cells from the fragmented umbilical cord may be performed. Separating the improved umbilical cord-derived attached stem cells may be attached to the cultured umbilical cord attached to the culture vessel for 5 to 20 days, for example, 10 to 20 days, for example, 10 to 15 days; Confirming that the cells extend from the cultured umbilical cord tissue; And / or treating the sequestration enzyme in the umbilical cord tissue.
  • the separating enzyme may include collagenase.
  • the collagenase may refer to an enzyme that breaks down the peptide bonds of collagen, and may include collagenase type I, type II, type III, type IV, or a combination thereof.
  • the degrading enzyme may comprise 5 to 30 U / ml, for example 5 to 25 U / ml, 10 to 25 U / ml, or 20 U / ml of collagenase.
  • the separation enzyme may include trypsin, and / or dispase, and the solution containing the separation enzyme may also include collagenase, trypsin, and / or dispase. Water, saline, for example, HBSS (Hank's Balanced Salt Solution) may be included.
  • the treatment time of the separation enzyme may be, for example, 1 hour to 20 hours, 2 hours to 10 hours, 4 hours to 9 hours, or 5 hours to 6 hours.
  • the reaction of the tissue and the separation enzyme may be a reaction by performing shaking, the shaking is about 20 to 40 °C, about 30 to 40 °C, or 35 to 40 °C, for example, 37 It may be carried out at °C, may be carried out for about 5 to 60 minutes or 10 to 30 minutes, it may also be carried out twice, for example 10 to 30 minutes.
  • a process for inactivating the separation enzyme may be further performed.
  • FBS may be added to stop the enzyme reaction.
  • the method for separating tissue cells for example, improved umbilical cord-derived stem cells from the enzyme reaction solution can be carried out by a method known in the art, for example, after centrifugation, the cell body ( cells can be isolated using a strainer.
  • the term "isolation of improved umbilical cord-derived adherent stem cells” refers to at least 20%, 30%, 40%, 50%, 60%, 70% of cells normally associated with stem cells in an untreated mammalian umbilical cord. May mean removing 80%, 90%, 95% or 99%.
  • a population of cells containing stem cells from one organ can be said to be “isolated” when the other cell that is normally associated with the stem cells in the organ is untreated.
  • the isolated improved umbilical cord-derived attached stem cells may include a step of passage culture with P0.
  • the passaging step may further include the treatment of an Animal Component Free (ACF) recombinant enzyme prior to cell transplantation for passaging.
  • ACF Animal Component Free
  • the term "Animal Component Free Enzyme” is of non-animal origin, which may mean that the enzyme is not purified from an animal source.
  • the enzyme without the animal derived component may be of recombinant origin, for example bacterial, yeast or plant origin. Enzymes of recombinant origin may refer to any enzyme produced by recombinant DNA technology, including the use of microorganisms such as bacteria, viruses, yeast, plants, etc. for their production.
  • the enzyme can be recombinant trypsin without animal derived components, for example recombinant trypsin produced in corn.
  • Recombinant trypsin without the animal-derived component is commercially available, for example TrypLE TM Select (GIBCO Invitrogen), TrypLE TM Express (GIBCO Invitrogen), TrypZean TM (Sigma Aldrich) or Recombinant Trypsin Solution TM (Biological Industries) Can be.
  • the passaging step includes culturing in a stem cell culture medium, for example, fibroblast growth factor-4 (FGF-4) and heparin added medium.
  • FGF-4 in the medium may be added at a concentration of about 10 ng / ml to about 40 ng / ml, or about 20 ng / ml to about 30 mg / ml, for example 25 ng / ml.
  • Heparin in the medium may be added at a concentration of about 0.5 ⁇ g / ml to 2 ⁇ g / ml, or 0.5 ⁇ g / ml to 1.5 ⁇ g / ml, for example 1 ⁇ g / ml.
  • the medium may further include, for example, fetal bovine serum, and antibiotics (eg, penicillin, streptomycin, gentamicin, etc.).
  • antibiotics eg, penicillin, streptomycin, gentamicin, etc.
  • CS-CM medium with 10% fetal bovine serum, 50 ⁇ g / ml gentamicin, 1 ⁇ g / ml heparin, and 25 ng / ml FGF-4 added can be used.
  • the passage culture may be performed at about 20 to 40 °C, about 30 to 40 °C, or 35 to 40 °C, for example, 37 °C, the incubation time for each passage is, for example, 2 to 7 days, Or 3 to 5 days.
  • the passage number of the passage is not particularly limited, and may be appropriately selected according to the number of desired proliferating cells. Typically, at least one passage or more than 10 passages may be used. For example, by performing 1 to 20 passages and 3 to 15 passages, a clinically necessary number of cumulative proliferating cells can be obtained.
  • the treatment of a recombinant enzyme without an animal-derived component may be additionally performed. That is, the purity of the cells can be improved by collecting the cells by treating the enzymes without animal-derived components before passing the cells to the next step for each passaging step. For example, in a step from P1 to P2, the recombinant enzyme without the animal-derived component may be treated before transplanting the cells for P2.
  • the passaging step may be to passaging under low oxygen conditions compared to the normal oxygen conditions 21%.
  • the term "hypoxia” may refer to a low oxygen partial pressure condition compared to 21% oxygen partial pressure, which is a normal normal oxygen condition.
  • the low oxygen condition may be a state having an oxygen partial pressure of 1 to 15%, 1 to 12%, 1 to 10%, or 1 to 5%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9%.
  • the expression of any one or more selected from the group consisting of DPYD and SCARA3 may be increased or the expression of any one or more selected from the group consisting of IL8, ALDH1A1, NQO1, DLC1, CTHRC1 and CPA4 may be decreased.
  • the improved umbilical cord-derived stem cells produced by the production method have the characteristics as described above.
  • the prepared umbilical cord-derived stem cells may be selected from the following (a) to (e). It may have the following characteristics:
  • CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells
  • CD200 + selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.
  • the improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):
  • At least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;
  • one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;
  • one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells
  • one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.
  • the improved umbilical cord-derived attached stem cells may be one of the surface antigen characteristics of e) additionally Oct4-, or Nanog-.
  • CD61 + may be a surface antigen property overexpressed in hypoxic conditions.
  • Another aspect is to provide a cell therapy, pharmaceutical composition or formulation comprising the improved umbilical cord-derived adherent stem cells, their cell populations or their cultures as an active ingredient.
  • Another aspect provides the use of said improved umbilical cord-derived adherent stem cells, populations thereof or cultures thereof for use in the manufacture of cell therapies, pharmaceutical compositions or formulations.
  • an improved umbilical cord-derived adherent stem cell having one or more of the following properties selected from (a) to (e), a cell therapy, pharmaceutical composition, or agent comprising a cell population thereof may be provided:
  • CCND1, SERPINE1, PRNP and CYP1B1 are less expressed than bone marrow stem cells
  • CD200 + selected from the group consisting of CD200 +, Tra-1-60-, CD3-, CD1a-, CD11c-, CD16-, CD86-, CD8a-, CD40-, CD141 +, CD61 +, CD87 +, MIC A / B- and SSEA4 + One or more surface antigen properties.
  • the improved umbilical cord-derived attached stem cells may further have one or more properties selected from the following (f) to (i):
  • At least one selected from the group consisting of S100A10, BNIP3, IGFBP5, NDUFA4L2, DPYD and SCARA3 is expressed more than in culture under normal oxygen conditions;
  • one or more selected from the group consisting of IL8, ALDH1A1, DLC1, CTHRC1, and CPA4 is less expressed compared to culture in normal oxygen conditions;
  • one or more selected from the group consisting of SNCA, DSG2, NRP2, and PLAT is more expressed compared to bone marrow stem cells
  • one or more selected from the group consisting of TPMT, NAGK, and ANXA4 is less expressed compared to bone marrow stem cells.
  • the aspect also includes a pharmaceutical composition comprising a culture of the improved umbilical cord-derived adherent stem cells.
  • a pharmaceutical composition comprising a culture of the improved umbilical cord-derived adherent stem cells.
  • the pharmaceutical composition for the treatment or prevention of inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases comprising the improved umbilical cord-derived attached stem cells, their cell population, or a culture medium thereof as an active ingredient. to provide.
  • umbilical cord-derived adherent stem cells for use in the manufacture of a medicament for use in the treatment or prevention of diseases such as inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases, cell populations thereof. Or the use of the culture solution thereof.
  • Also another aspect is a disease, eg, inflammatory disease, ischemic disease, and / or administration comprising administering the improved umbilical cord-derived attached stem cells, populations thereof, or cultures thereof to an individual in need thereof. Or methods of treating or preventing neurodegenerative diseases.
  • the improved umbilical cord-derived attached stem cells are as described above.
  • An improved umbilical cord-derived adherent stem cell is a protein that is beneficial for treating a disease as described above (eg, IL-6, IL-8, G-CSF, GM-CSF, MCP-3, VEGF, or GRO) and its ability to migrate to damaged tissues are not only remarkable, but also have anti-inflammatory, blood vessel regeneration, and nerve regeneration effects, thus preventing various diseases including inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases. Or may be usefully used in cell therapeutics or pharmaceutical compositions for treatment.
  • Examples of such diseases may include inflammatory diseases, ischemic diseases, and / or neurodegenerative diseases.
  • Examples of the inflammatory disease may include bronchitis, gastritis, arteriosclerosis, arthritis, inflammatory bowel disease (IBD), hepatitis, cholecystitis, fungal infection, gastric ulcer, asthma, atopic dermatitis, tendinitis or nephritis .
  • ischemic diseases include ischemic stroke, myocardial infarction, ischemic heart disease, ischemic brain disease, ischemic heart failure, ischemic enteritis, ischemic vascular disease, ischemic eye disease, ischemic retinopathy, ischemic glaucoma, ischemic renal failure, or ischemic leg disease
  • ischemic stroke or “stroke” may mean a disease caused by necrosis of brain tissue or cells due to a decrease in cerebral blood flow for a predetermined time, and "cerebral infarction”. Can be used interchangeably.
  • neurodegenerative diseases include spinal cord injury, multiple sclerosis, Alzheimer's disease, Frontotemporal dementia, progressive supranuclear palsy, cortical basal degeneration, and Pick's disease. ), Or boxer dementia (Dementia pugilistica, DP).
  • the dosage of the cell therapy or pharmaceutical composition is 1.0 X 10 3 to 1.0 X 10 10 cells / kg body weight or individual, or 1.0 X 10 7 to 1.0 X, based on the improved umbilical cord-derived adherent stem cells. 10 8 cells / kg body weight or individual.
  • the dosage may be variously prescribed by such factors as the formulation method, the mode of administration, the age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and reaction sensitivity of the patient. These factors can be taken into account to properly adjust the dosage.
  • the number of administrations may be one or two or more times within the range of clinically acceptable side effects, and may be administered to one or two or more sites of administration.
  • the amount converted into the amount can be administered.
  • the target animal for the treatment according to one embodiment include humans and mammals for other purposes, and specifically, humans, monkeys, mice, rats, rabbits, sheep, cattle, dogs, horses, pigs, and the like. Included.
  • Cell therapy or pharmaceutical composition may include the enhanced umbilical cord-derived attached stem cells and a pharmaceutically acceptable carrier and / or additives as an active ingredient.
  • a pharmaceutically acceptable carrier examples include sterile water, physiological saline, conventional buffers (phosphate, citric acid, other organic acids, etc.), stabilizers, salts, antioxidants (ascorbic acid, etc.), surfactants, suspending agents, isotonic agents, or preservatives. can do.
  • organic substances such as biopolymers, inorganic substances such as hydroxyapatite, specifically collagen matrix, polylactic acid polymers or copolymers, polyethylene glycol polymers or copolymers, and chemical derivatives thereof.
  • the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in solution.
  • Improved umbilical cord-derived adherent stem cells are various types in which the tissues or organs of the body are fortified, treated or replaced by engraftment, transplantation or infusion of the desired cell population, for example stem cells or derived cell populations. Can be used in the treatment protocol.
  • the improved umbilical cord-derived adherent stem cells may replace or enhance existing tissue, resulting in new or altered tissue or combined with biological tissue or structures.
  • stem cells may be replaced with the improved umbilical cord-derived stem cells of the present disclosure in treatment protocols where typically tissue-derived stem cells other than the umbilical cord are used.
  • the cell therapeutic agent or pharmaceutical composition according to one embodiment may be prepared according to the administration method or dosage form thereof, and as necessary, suspending agents, dissolution aids, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, A passivating agent, a buffer, a reducing agent, an antioxidant, etc. can be included suitably.
  • suspending agents, dissolution aids, stabilizers, isotonic agents, preservatives, anti-adsorption agents, surfactants, diluents, excipients, pH adjusters, A passivating agent, a buffer, a reducing agent, an antioxidant, etc. can be included suitably.
  • Pharmaceutically acceptable carriers and formulations suitable for the present invention including those exemplified above, are described in detail in Remington's Pharmaceutical Sciences, 19th ed., 1995.
  • the cell therapeutic agent or pharmaceutical composition according to one embodiment is formulated with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by one of ordinary skill in the art. It may be prepared in unit dose form or incorporated into a multi-dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oil or aqueous media or in the form of powders, granules, tablets or capsules. In addition, cell therapy agents may be formulated into injectable formulations. In this case, known conventional ingredients for formulating can be used and can be formulated in conventional manner.
  • an anti-inflammatory effect there is an anti-inflammatory effect, a blood vessel regeneration effect, or a nerve regeneration effect, and thus may be usefully used in pharmaceutical compositions or cell therapeutics for the treatment or prevention of various diseases.
  • 1A is a diagram showing the cell morphology before and after the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment.
  • Figure 1b is a diagram showing the cell morphology according to the treatment time of the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment: G1: Col I treatment group, G2: Col I treatment group after tissue attachment.
  • FIG. 2 is a diagram illustrating a comparison between hypoxic and normal oxygen conditions in improved umbilical cord-derived attached stem cell culture according to one embodiment: 3%: low oxygen partial pressure (3% O 2), 21%: normal oxygen Partial pressure (21% O2).
  • FIG. 3 is a view showing the results of performing karyotype analysis of the genetic stability of the improved umbilical cord-derived stem cells according to one embodiment.
  • Figure 4 is a view showing the results of analyzing the surface protein of the umbilical cord-derived attached stem cells according to one embodiment.
  • Figure 5 is a view showing the results of analyzing the multipotency of the umbilical cord-derived attached stem cells according to one embodiment.
  • Figure 6 is a view showing a result of comparing the analysis of protein expression of the improved umbilical cord-derived stem cells according to one embodiment.
  • Figure 7a is a view showing the results of analyzing the anti-inflammatory effect of the umbilical cord-derived attached stem cells according to one embodiment
  • Figure 7b is a view showing the results of analyzing the blood vessel regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment
  • Figure 7c is a view showing the result of analyzing the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.
  • Example 1 Preparation, Characterization of Enhanced Umbilical Cord-derived Attached Stem Cells, and Analysis of Anti-inflammatory, Neuronal and Vascular Regeneration Effects
  • Informed consent was obtained from a healthy mother who had delivered normally and informed consent, and the cord was isolated from placental tissue collected at normal placental delivery.
  • the separated umbilical cord was washed 2 to 5 times with Ca / Mg free DPBS to remove blood. Thereafter, the outer amnion was not peeled off, and two arteries and one vein were removed, and the umbilical cord was cut to a size of 1 to 5 mm. Thereafter, the umbilical cord was attached to the culture vessel and cultured for 10 to 15 days. After confirming that the cells were stretched from the cultured tissue, the treated umbilical cord was treated with 200 U / ml collagenase I for 5 to 6 hours. Derived adherent stem cells were isolated. Before and after the collagenase I treatment, the cell morphology was confirmed at 40x and 100x magnification under an optical microscope in order to confirm that the cells extend from the umbilical cord attached tissues, and the results are shown in FIG. 1A.
  • 1A is a diagram showing the cell morphology before and after the separation enzyme in improved umbilical cord-derived adherent stem cell separation according to one embodiment.
  • the separated cells were P0, and were treated with MEM alpha GlutaMAX (CS-CM medium) containing 25 ng / ml FGF4, 1 ug / ml heparin, and 10% FBS at 37 ° C. under hypoxic culture conditions (O 2 3%). Incubated. Every 3 to 4 days thereafter, the CS-CM medium was replaced to remove cells that did not adhere to the bottom of the flask. (3 min) treatment and subculture.
  • CS-CM medium MEM alpha GlutaMAX
  • the umbilical cord-derived attached stem cells were isolated and cultured in the same manner as in (1.1) except that the treatment of collagenase I in (1.1) was performed before attaching the umbilical cord to the culture vessel.
  • the stem cells isolated in (1.2) and the stem cell group isolated in (1.1) are named G1 and G2, respectively.
  • the morphology of the cells was confirmed at 40 ⁇ and 100 ⁇ magnification under the light microscope, and the results are shown in FIG. 1B.
  • Figure 1b is a diagram showing the cell morphology according to the treatment time of the separation enzyme in the improved umbilical cord-derived adherent stem cell separation according to one embodiment.
  • tissue weight of the G1 and G2 the number of cells after treatment with the enzyme and cell number (P0) was compared.
  • FIG. 2 is a diagram illustrating a comparison between hypoxic conditions and normal oxygen conditions in an improved umbilical cord-derived attached stem cell culture according to one embodiment.
  • GTG-Banding assay was performed to analyze the genetic safety of the improved umbilical cord-derived stem cells prepared in (1.1) and (1.3).
  • DNA was extracted from P7 and P14 cells using a Promega DNA Extraction Kit and used as a sample.
  • An Illumina HumanOmni1-Quad Chip was used and measured using an iSCAN® scanner.
  • 400 ng of each DNA sample is amplified by whole genome amplification, randomly fragmented by chemical method, purified by 2-propanol precipitation, and the chip is buffered before loading the DNA sample.
  • DNA samples were added to the chips pretreated with the solution. Following incubation for about 16 hours, staining, allele specific primer extension (ASPE), hybridization, target removal, and washing were performed. Then, scanning was performed with IlluminaScan, and the data was analyzed using GenomeStudio® software, and the results are shown in FIG. 3.
  • FIG. 3 is a view showing the results of performing karyotype analysis of the genetic stability of the improved umbilical cord-derived stem cells according to one embodiment.
  • the improved umbilical cord-derived attached stem cells produced by the manufacturing method according to one embodiment can be seen that no genetic variation occurs until P14.
  • the cells were washed using DPBS, and then placed in DPBS containing 2% FBS, followed by Tra-1-60, CD3, CD1a, CD11c, CD16, CD14, CD86, CD8a, CD19, CD40, CD80. , CD200, CD141, CD61, CD87, MIC A / B, SSEA4 markers were reacted for 20 minutes on ice. Then, the surface antigen was analyzed by flow cytometry (FACS Calibur, Becton Bickinson), the results are shown in Figure 4a.
  • Figure 4 is a view showing the results of analyzing the surface protein of the umbilical cord-derived attached stem cells according to one embodiment.
  • improved umbilical cord-derived adherent stem cells is a cell selectively positive for CD200, CD141, CD61, CD87 SSEA4, TRA-1, CD3, CD1a, CD11c, CD16, CD86 , CD8a, CD40, and MIC A / B cells that are selectively negative, and in addition, CD61 is a cell selectively positive in hypoxic conditions.
  • the improved umbilical cord-derived attached stem cells according to one embodiment does not express the embryonic stem cell specific markers Oct4, Nanog proteins.
  • Adipocyte differentiation capacity analysis of the improved umbilical cord-derived adherent stem cells was performed by the following method.
  • the improved umbilical cord-derived stem cells prepared in (1.1) and (1.3) above were put into Adipogenesis differentiation media (StemPro® Adipogenesis Differentiation Kit, Life Technology), and the medium was exchanged every three days for two weeks. While culturing. After that, the culture solution was removed, washed with Ca / Mg free DPBS, 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After washing with 60% isopropanol and then adding Oil Red O and reacting for 10 minutes, washing with purified water and observing fat cells under a microscope, the results are shown in FIG. 5.
  • Bone cell differentiation assay of the improved umbilical cord-derived stem cells was performed by the following method.
  • the improved umbilical cord-derived stem cells prepared in (1.1) and (1.3) above were put into osteogenic differentiation media (StemPro® Osteogenesis Differentiation Kit, Life Technology) and the medium was exchanged every three days for two weeks. While culturing. Thereafter, the culture medium was prepared, washed with Ca / Mg free DPBS, and then 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. After the reaction, add purified water, wash, add 1% silver nitrate solution, react for 5 minutes at room temperature, wash with purified water, and add 5% sodium thiosulfate solution at room temperature. The reaction was carried out for 5 minutes. Next, after washing with purified water, 0.1% Nuclear Fast Red Solution was added and reacted at room temperature for 5 minutes. Thereafter, the sample was washed with purified water and analyzed for calcium accumulated samples under a microscope, and the results are shown in FIG. 5.
  • Cartilage cell differentiation assay of enhanced umbilical cord-derived adherent stem cells was performed by the following method.
  • Figure 5 is a view showing the results of analyzing the multipotency of the umbilical cord-derived attached stem cells according to one embodiment.
  • the improved umbilical cord-derived attached stem cells according to one embodiment prepared by the method according to (1.1) and (1.3) is differentiated into adipocytes, bone cells, chondrocytes, it can be seen that there is a multipotent ability have.
  • the culture medium in which the improved umbilical cord-derived stem cells were cultured for 24 hours was incubated with antibody-coated capture beads for 2 hours at room temperature, and washed.
  • the beads are then incubated with biotin-labeled anti-human cytokine and chemokine antibody for 1 hour and streptavidin phycoerythrin for 30 minutes. Incubated.
  • the secretion protein expression level was analyzed using the Luminex 200 program to wash and quantify the beads, and the results are shown in Table 2.
  • Cytokine (pg / ml) 3% oxygen partial pressure Inflammation IFNr 46 IL-1a 19 IL-1b 5 IL-1ra 30 IL-2 0 IL-3 5 IL-4 8 IL-5 0 IL-6 744 IL-7 20 IL-8 > 10,000 IL-9 One IL-10 0 IL-12 (p40) 14 IL-12 (p70) 2 IL-13 One IL-15 One IL-17 0 TNFa 0 TNFb 0 IFNa2 50 MDC 2 sCD40L 0 sIL-2Ra 4 Chemotaxis / Recruitment / Hematopoiesis Eotaxin 117 Flt-3 Ligand 6 G-CSF 3,001 GM-CSF 46 MCP-1 > 10,000 MCP-3 1,033 MIP-1a 13 MIP-1b 2 RANTES 7 Fractalkine 116 IP-10 10 Angiogenesis / Tissue remodeling VEGF 170 Growth factor / Fibrosis EGF 16 GRO > 10,000 PDGF-AA 0 PD
  • the improved umbilical cord-derived attached stem cells is IL-6, IL-8, G-CSF, GM-CSF, MCP-1, MCP-3, VEGF, GRO, IGF It can be seen that the secretion of -1 SR, EpCAM, IGFBP3.
  • COL1A1, IGFBP4, TAGLN, S100A10, SQSTM1, DSTN, DCN, PHGDH, FBLN1, MFGE8, HLA-A, VASN, KIAA1199, STC1, LRRC17, IL33, SNCA, DSG2, NRP2 , PLAT is expressed in enhanced umbilical stem-derived stem cells and CCND1, SERPINE1, PRNP, MT2A, TM4SF1, HIST1H4C, NME1, CXCL6, NTSR1, PTGS2, CYP1B1, TPMT, NAGK, and ANXA4 are found in enhanced umbilical stem-derived stem cells. It can be seen that the expression is lower than the bone marrow stem cells.
  • COL1A1 an increasing gene, is known to be expressed in collagen of connective tissue including cartilage as alpha-1 type I collagen.
  • the gene has not been reported for its relationship with improved umbilical cord-derived adherent stem cells.
  • IGFBP4 an increase gene, is an insulin-like growth factor binding protein and is known to inhibit various cancer cells. It has been reported that IGFBP4 is detected in the serum of umbilical cord blood, but the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.
  • TAGLN an increasing gene of the genes, is a gene expressed in fibroblasts and smooth muscles, and its function has not been identified yet. Although expression in bone marrow stem cells has been reported, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.
  • CCND1 a decreasing gene
  • Cyclin D1 When CCND1 is overexpressed, GND accelerates the cell cycle from G1 to S phase, thereby promoting cell growth. It has been reported to be expressed mainly in cancer cells.
  • Umbilical cord blood stem cells have been reported to inhibit C6 glioma proliferation by decreasing CCND1. However, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.
  • SERPINE1 a reduction gene of the genes, is known as an endothelial plasminogen activator inhibitor and functions as an inhibitor of tissue plasminogen activator (tPA).
  • tPA tissue plasminogen activator
  • PRNP a reduction gene of the genes, is known to be expressed in various tissues as well as the nervous system as a major prion protein (CD230). Abnormalities in the PRNP gene have been reported to cause neurological diseases. However, the gene has not been reported for its relationship with improved umbilical cord-derived adherent stem cells.
  • S100A10 an increasing gene, is a S100 calcium-binding protein A10 that regulates cell cycle and differentiation. It is also known to function as exocytosis and endocytosis. Although bone marrow stem cells have been studied as one of the proteins that are highly expressed when differentiated into bone, the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.
  • BNIP3 an increase gene of the genes, is known as an increased gene in UCB-MSC when comparing gene expression at the mRNA levels of UCB-MSC (cord blood derived stem cells) and UCB-MNC (cord blood derived blood cells).
  • UCB-MSC cord blood derived stem cells
  • UCB-MNC cord blood derived blood cells
  • IGFBP5 an increasing gene of the genes, is an insulin-like growth factor binding protein 5, which plays a role in development and is located in the extracellular space.
  • IGFBP5 an insulin-like growth factor binding protein 5
  • This gene has not been reported for its association with enhanced umbilical cord-derived adherent stem cells.
  • IL8 a decreasing gene of the gene, is secreted from phagocytes and mesenchymal cells when exposed to an inflammatory environment to activate neutrophils that induce chemotaxis.
  • the gene has not been reported for its association with improved umbilical cord-derived adherent stem cells.
  • the reduction gene ALDH1A1 is an aldehyde dehydrogenase 1 family, member A1, which is an enzyme responsible for the major oxidation pathway of alcohol metabolism.
  • the gene has not been reported for its association with enhanced umbilical cord derived stem cells.
  • PBMC growth inhibition assay was performed as follows. First, the improved umbilical cord-derived adherent stem cells were inoculated in 24 well plates by concentration, and then cultured for 24 hours. Then, PHA was stimulated by adding PHA to CFSE stained PBMC and co-cultured with the improved umbilical cord-derived stem cells for 5 days. Thereafter, according to the presence or absence of the transwell, whether the umbilical cord-derived stem cells secreted by the cytokine secretion is inhibited or whether the inflammation suppressed by direct contact with the cells were distinguished, the results are shown in Figure 7a.
  • Figure 7a is a diagram showing the results of analyzing the anti-inflammatory effect of the umbilical cord-derived attached stem cells according to one embodiment.
  • the improved umbilical cord-derived attached stem cells can be confirmed that the proliferative capacity of PBMC is inhibited compared to the control.
  • the umbilical cord-derived attached stem cell: PBMC ratio is 1:10, it can be seen that up to about 30.51 ⁇ 1.74% of the proliferation inhibitory effect is obtained in indirect coculture.
  • activated PBMCs secrete anti-inflammatory cytokines (IL-10), and enhanced umbilical cord-derived stem cells play a role in PBMCs increasing IL-10 secretion. It can be seen that.
  • the improved umbilical cord-derived attached stem cells can be usefully used for the treatment of inflammatory diseases.
  • Vascular endothelial cell proliferation assay was performed to analyze the vascular regeneration effect of the improved umbilical cord-derived attached stem cells prepared in (1.1).
  • a sample was prepared by collecting a culture medium (conditioned medium) of EBM-2 and enhanced umbilical cord-derived attached stem cells. Subsequently, when inoculated with vascular endothelial cells (HUVEC) in a 96 well plate and grown for 1 day, EBM-2 and cultured cells of enhanced umbilical cord-derived adherent stem cells were dispensed and cultured for 4 days. Cyto X TM Cell viability assay kit (WST-1) reagent was added in 10% of the medium and reacted in an incubator for 2-3 hours. Then, the proliferation rate of endothelial cells was analyzed by measuring at 450 nm using a microreader, and the results are shown in FIG. 7B.
  • WST-1 Cyto X TM Cell viability assay kit
  • Figure 7b is a view showing the results of analyzing the blood vessel regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.
  • the proliferation ability of the vascular cells cultured in EBM-2 medium is 100%
  • the vascular cells cultured in the conditioned medium of the improved umbilical cord-derived stem cells proliferate 172 ⁇ 15.22%. You can check it.
  • the improved umbilical cord-derived stem cells has a blood vessel regeneration effect.
  • Neuronal cell proliferation analysis was performed to analyze the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells prepared in (1.1).
  • a sample was prepared by collecting a culture medium (conditioned medium) of MEM and improved umbilical cord-derived attached stem cells. Then, when inoculated neurons (SH-SY5Y) in 96 well plate, and when grown for about one day, the culture medium of MEM and improved umbilical cord-derived attached stem cells were each dispensed and incubated for 4 days. Cyto X TM Cell viability assay kit (WST-1) was added in 10% aliquots of the medium and allowed to react in an incubator for 2-3 hours. The proliferation rate of neurons was analyzed by measuring at 450 nm using a microreader, and the results are shown in FIG. 7C.
  • WST-1 Cyto X TM Cell viability assay kit
  • Figure 7c is a view showing the result of analyzing the neuronal regeneration effect of the improved umbilical cord-derived attached stem cells according to one embodiment.
  • the neurons cultured in the conditioned medium of the improved umbilical cord-derived attached stem cells proliferate 302 ⁇ 15.97%. .
  • the improved umbilical cord-derived stem cells has a neuronal regeneration effect.

Abstract

La présente invention concerne des cellules souches adhésives améliorées dérivées de cordon ombilical, leur procédé de préparation, et leur utilisation. Les cellules souches adhésives améliorées dérivées de cordon ombilical présentent un effet anti-inflammatoire, un effet de régénération de vaisseaux sanguins, ou un effet de régénération nerveuse, pouvant ainsi être utilisées dans une composition pharmaceutique ou un agent thérapeutique à base de cellules pour le traitement ou la prévention de diverses maladies.
PCT/KR2016/008887 2015-08-12 2016-08-12 Cellules souches adhésives améliorées dérivées de cordon ombilical, leur procédé de préparation, et leur utilisation WO2017026838A1 (fr)

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CN201680057936.5A CN108138137A (zh) 2015-08-12 2016-08-12 增强的脐带来源的粘着干细胞、其制备方法及其用途
JP2018507615A JP6648259B2 (ja) 2015-08-12 2016-08-12 向上された臍帯由来付着型幹細胞、その製造方法及びその用途
ES16835478T ES2914692T3 (es) 2015-08-12 2016-08-12 Células madre adhesivas derivadas de cordón umbilical mejoradas, método de preparación para las mismas, y uso de las mismas
US15/752,057 US11690877B2 (en) 2015-08-12 2016-08-12 Umbilical cord-derived adherent stem cells, preparation method therefor, and use thereof
EP16835478.5A EP3336176B1 (fr) 2015-08-12 2016-08-12 Cellules souches adhésives améliorées dérivées de cordon ombilical, leur procédé de préparation, et leur utilisation

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KR20140008749A (ko) * 2011-07-11 2014-01-22 (주)차바이오메드 탯줄 추출물의 제조방법 및 그의 용도
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