WO2017039251A1 - Cellule adhérente postnatale améliorée, et utilisation de celle-ci - Google Patents

Cellule adhérente postnatale améliorée, et utilisation de celle-ci Download PDF

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WO2017039251A1
WO2017039251A1 PCT/KR2016/009552 KR2016009552W WO2017039251A1 WO 2017039251 A1 WO2017039251 A1 WO 2017039251A1 KR 2016009552 W KR2016009552 W KR 2016009552W WO 2017039251 A1 WO2017039251 A1 WO 2017039251A1
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cells
adherent
postpartum
cell
group
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PCT/KR2016/009552
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English (en)
Korean (ko)
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김혜선
유지민
강아름
김지혜
김현주
임재승
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주식회사 차바이오텍
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Priority to US15/755,848 priority Critical patent/US20180325960A1/en
Priority claimed from KR1020160109504A external-priority patent/KR102011634B1/ko
Publication of WO2017039251A1 publication Critical patent/WO2017039251A1/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

  • the present invention relates to improved postnatal adherent cells (ePACs), a method for preparing the same, and a cell therapy using the same as an active ingredient.
  • ePACs postnatal adherent cells
  • Cell therapies may be used to treat autologous, allogenic, or xenogenic cells in vitro or to alter the biological properties of cells to restore the function of cells and tissues.
  • Cell therapy drugs can be divided into somatic cell therapy and stem cell therapy, depending on the type of cell used and the degree of differentiation, and stem cell therapy can be classified into embryonic stem cell therapy and adult stem cell therapy.
  • Stem cell therapy has attracted attention for the ideal reason that stem cells with self-proliferative and multipotent properties can help regenerate damaged cells to treat disease and improve symptoms.
  • stem cells has difficulty in ethical problems and securing the desired number of cells.
  • Embryonic stem cells have superior performance to adult stem cells but have negative aspects in terms of bioethics.
  • Adult stem cells involve pain in donors during the collection and isolation of stem cells and the efficiency of separation of stem cells. This has the disadvantage of being low.
  • the placenta is a tissue that is abundant and usually discarded as medical waste after childbirth, and since the placenta-derived cells are separated and extracted from the extracted placenta, there is no ethical problem, and it is easy to recover a large amount of desired cells.
  • the placenta may provide a source of cellular therapy that is easily ethically controversial for experimental and clinical applications. Accordingly, if there is no ethical problem and the placental cells capable of obtaining a large amount of cells can perform the functions of the existing stem cells, they may be used as good cell therapy.
  • the present inventors have isolated a novel pluripotent cell from the placenta, which has marker properties different from those of conventional placental stem cells, and is excellent in proliferative and differentiating ability, thereby completing the present invention.
  • One aspect is to provide placental derived cells with novel properties.
  • Another aspect is to provide a cell population comprising placental derived cells having the novel properties.
  • Another aspect is to provide a method for producing placental derived cells having the novel properties.
  • Yet another aspect is to provide a composition comprising a placental derived cell having said novel properties and a cell therapeutic agent.
  • One aspect provides improved postpartum adherent cells.
  • the improved postpartum adherent cells are provided.
  • (c) secrete one or more proteins selected from the group consisting of VEGF, TGF- ⁇ 1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2 and MMP3;
  • the HYOU1 or GLIPR1 protein may have one or more properties selected from the group consisting of properties that are less expressed than bone marrow derived stem cells.
  • the term "enhanced Postnatal adherent cells (ePACs)” is a cell that has the property of separating from the placenta and adhering to the culture vessel surface and growing, having excellent proliferative capacity and ability to differentiate into various tissue cells. Means.
  • the improved postpartum adherent cells of the present invention may also be referred to as 'postpartum adherent cells'.
  • placenta refers to an organ that is made for the fetus during the pregnancy of a mammal and includes an amnion, chorion, or decidual film.
  • Amniotic membrane is a thin, transparent membrane that surrounds the fetus, and contains amniotic fluid.
  • the amniotic membrane may contain fetal stem cells.
  • the decidual membrane is a membrane formed by deforming epithelial cells of the uterus so that the fertilized egg is implanted in the uterus.
  • the chorion is the membrane between the amnion surrounding the fetus or amniotic fluid and the decidual membrane. It occurs in the fertilized egg and forms part of the egg.
  • the placenta may specifically be a human placenta, and specifically, may be a placenta separated into a human in vitro.
  • the placenta may specifically be amnion, chorion, or trophoblast. That is, the improved postpartum adherent cells of the present invention may specifically be adherent cells isolated from amnion, chorion, or trophoblast. More specifically, the cells may be cells isolated from amnion, and most specifically, cells isolated from amnion of human placenta separated in vitro.
  • the enhanced postpartum adherent cells may be derived from the fetus and / or the mother (ie, may be genotype of the fetus or the mother), and may specifically be derived from the fetus.
  • the improved postpartum adherent cells of the present invention may have the form of adherent fibroblasts during passaging.
  • the cells of the present invention may have the properties of cells that require attachment to a surface in order to grow in vitro.
  • the cells of the present invention may exhibit a specific form of fusiform fibroblasts.
  • the cells of the present invention may have an average diameter of 20 ⁇ m or less up to 18 passages, and may have an average diameter of 13 to 14 ⁇ m in the initial passages, for example, 1 to 6 passages.
  • the enhanced postpartum adherent cells may also differentiate into adipocytes, osteocytes or chondrocytes.
  • the cells can be induced to differentiate along specific cell lineages, including 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 be performed using techniques such as flow cytometry or immunocytochemistry to measure cell surface labels (eg, staining cells with tissue-specific or cell-label specific antibodies) and changes in morphology, or by optical microscopy. Or by examining the morphology of the cells using confocal microscopy, or by measuring changes in gene expression using techniques known in the art such as PCR and gene-expression profiles.
  • the enhanced postpartum adherent cells are also selected from the group consisting of VEGF, TGF- ⁇ 1, IL-6, progranulin, follistatin, Angiostatin, MMP2, MMP10, TRAIL R2 and MMP3.
  • One or more proteins may be secreted.
  • the cells may secrete prograulin. More specifically, the cell is at least two, at least three or all of the proteins selected from the group consisting of VEGF, TGF- ⁇ 1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2 and MMP3 Can secrete Most specifically, the cells may secrete progranulin, or secrete one or more of progranulin and the other proteins.
  • the cells of the present invention may be increased secretion amount of VEGF and / or IL-6 as the passage is increased.
  • the improved postpartum adherent cells may be increased secretion amount of VEGF and / or IL-6 as the passage up to six passages.
  • the cells may be more than twice the secretion amount of VEGF and / or IL-6 than the passage 1 from 5 passages.
  • the cells of the present invention may be a constant secretion of TGF- ⁇ 1 and / or progranulin, regardless of increased passage.
  • the improved postpartum adherent cells may further secrete HGF in addition to the VEGF, TGF- ⁇ 1, IL-6 and / or progranulin.
  • the cells of the invention can secrete HGF in a constant amount regardless of donor and / or passage.
  • the improved postpartum adherent cells of the present invention may exhibit negative immunological properties for HLA-G, a surface antigen for antigen compatibility, and / or may show positive immunological properties for CD200, a surface antigen involved in immunosuppression. Can be represented.
  • the improved postpartum adherent cells may exhibit positive immunological properties against CD200, CD61, CD130, CD321, SSEA4, MIC A / B and CD338.
  • the improved postpartum adherent cells CD34 expressed in hematopoietic stem cells may exhibit negative immunological properties.
  • the improved postpartum adherent cells of the present invention may exhibit a high expression rate of CD200 compared to the expression levels of other tissue CD200 analyzed in a previous study (in the case of cord blood-derived mesenchymal stem cells do not express CD200).
  • a previous study in the case of cord blood-derived mesenchymal stem cells do not express CD200.
  • the improved postpartum adherent cells may have superior immunomodulatory capacity compared to other cells.
  • the cells of the present invention can maintain immunological properties positive for CD200 in the number of cells at least 70%.
  • the term “positive” may refer to a cell marker (marker), which means that the label is present in a greater amount, or at a higher concentration, as compared to other cells on which it is based.
  • a cell can be positive for that label because any label is present inside or on the surface of the cell and 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.
  • a cell can be detectably labeled with an antibody specific for CD105, and if the signal from this antibody is detectably greater than the control (eg background value), then the cell is "positive for CD45" or " CD105 + ".
  • the term “negative” means that even when an antibody specific for a particular cell surface label is used, the label cannot be detected in comparison with the background value. For example, if you can not detectably labeled cells with an antibody specific to the CD45 cells or "CD45 -" "negative for CD45” it is.
  • 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.
  • flow cytometry is HLA-G, It can be confirmed that the negative characteristics for CD34 and positive characteristics for CD200, CD61, CD130, CD321, SSEA4, MIC A / B and CD338.
  • the improved postpartum adherent cells of the present invention may exhibit negative immunological properties for Oct4, Nanog, and / or TRA-1-60, markers that further maintain the properties of progenitor cells of embryonic stem cells.
  • the flow cytometry the negative characteristics for the TRA-1-60, and also through the immunocytochemical staining method it can be confirmed that the negative characteristics for Oct4, Nanog.
  • the improved postpartum adherent cells of the present invention are also negative immunity against CD44, CD73, CD90, CD105, and CD3, CD8a, CD11c, CD19, CD45, CD56, which are markers characteristic of mesenchymal stem cells. It may have further physiological properties. It may also have negative immunological properties to CD80, CD86 or / or CD31 expressed in vascular endothelial cells, which are co-stimulatory factors that promote immune activity.
  • the cells may further have positive immunological properties for HLA-ABC, a surface antigen related to antigen compatibility, and CD29 and CD49, which are related to the ability to migrate to damaged tissues. It can also show positive immunological properties for CD9, SSEA4, which represents a non-nutrient membrane.
  • the cells may further have positive or partial positive immunological properties for CD54, CD106, and CD166, which are surface antigens related to immunosuppression.
  • the additional immunological properties may be specifically determined by flow cytometry.
  • the improved postpartum adherent cells of the present invention can express more than one gene selected from the group consisting of COL3A1, IGFBP5, PRNP and MT1A compared to bone marrow derived stem cells. Specifically, at least two, or at least three genes selected from the group consisting of COL3A1, IGFBP5, PRNP and MT1A, or more specifically all genes in the cells can be expressed more than the bone marrow-derived stem cells.
  • the improved postpartum adherent cells may express less than one or more genes or proteins selected from the group consisting of COL1A1, COL1A2, TPM2, TAGLN, CYP1B1, CXCL12, PENK, HYOU1 and GLIPR1 compared to bone marrow-derived stem cells.
  • the cells of the present invention may exhibit a difference in expression levels of at least two times the genes and at least 1.5 times the proteins relative to the bone marrow-derived stem cells.
  • the difference in the expression level may be, for example, comparing the expression levels of genes and proteins at the mRNA level and protein level.
  • the expression level difference can be determined, for example, by microarray or proteomics array.
  • the enhanced postpartum adherent cells have increased expression levels of one or more genes selected from the group consisting of PGK1, BNIP3, TPI1, ERRFI1, LOC644774, SLC2A3, PLIN2, and TUBB2B in cells cultured in hypoxic culture conditions compared to normal oxygen culture conditions. It may be.
  • improved postpartum adherent cells cultured in hypoxic culture conditions are at least two selected from the group consisting of PGK1, BNIP3, TPI1, ERRFI1, LOC644774, SLC2A3, PLIN2 and TUBB2B than enhanced postpartum adherent cells cultured in normal oxygen culture conditions.
  • the expression level of at least 3, at least 4, at least 5, at least 6, or all genes may be increased.
  • the difference in expression levels can be two or more times.
  • the cells may exhibit a difference in expression levels of three or more times in normal oxygen culture conditions and low oxygen culture conditions in the case of ERRFI1, LOC644774 or SLC2A3.
  • the enhanced postpartum adherent cells may have reduced expression levels of one or more genes or proteins selected from the group consisting of SERPINE1, TAGLN, TGM2, IL8, ALDH1A3, and DPP4 in cells cultured at low oxygen culture conditions compared to normal oxygen culture conditions. It may be.
  • enhanced postpartum adherent cells cultured in hypoxic culture conditions are at least 2, 3 or more selected from the group consisting of SERPINE1, TAGLN, TGM2, IL8 and ALDH1A3 than enhanced postpartum adherent cells cultured in normal oxygen culture conditions.
  • the expression level of all genes may be reduced. The difference in expression levels can be two or more times.
  • the cells may exhibit a difference in expression level of 2.5 times or more in the case of SERPINE1, TAGLN, IL8, or ALDH1A3 specifically in normal oxygen culture conditions and low oxygen culture conditions, and may express a difference in expression level of 4 times or more in the case of IL8.
  • the difference in the expression level of the DPP4 protein may be 1.5 times or more.
  • the difference in the expression level in the normal oxygen culture conditions and hypoxic culture conditions may be a comparison of the expression levels of genes and proteins at the mRNA level and protein level, for example.
  • the expression level difference can also be determined, for example, by microarray or proteomics array.
  • Another aspect provides the improved postpartum adherent cell population of the present invention.
  • the enhanced postpartum adherent cells included in the cell population are as described above.
  • the cell population may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% enhanced postpartum adherent cells.
  • the cell population of the present invention is a cell population of the present invention.
  • (c) secrete one or more proteins selected from the group consisting of VEGF, TGF- ⁇ 1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2 and MMP3;
  • HYOU1 or GLIPR1 protein may be characterized as being less expressed than bone marrow derived stem cells.
  • the cells in the cell population may be specifically derived from amnion.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells in the cell population of the invention can exhibit negative immunological properties to HLA-G. have.
  • at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells in the cell population may exhibit negative immunological properties to CD34.
  • cells in the cell population may have up to 50%, up to 40%, up to 30%, up to 20%, up to 10%, up to 5%, or up to 1% of cells with positive immunological properties relative to CD34. Can be.
  • At least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells in the cell population are positive for CD200, CD61, CD130, CD321, SSEA4 and CD338. It can show the immunological characteristics of. Specifically, in the cell population of the present invention, at least about 70%, 80%, 85%, 90% or more of the cells may exhibit positive immunological characteristics with respect to CD200, CD61, CD130, CD321, SSEA4, and CD338.
  • the cell populations of the present invention additionally comprise at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the cells in Oct4, Nanog, and / or TRA. Negative for I-60.
  • the cells in the cell population are about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Or at least 90% of the cells can express more than one gene or protein selected from the group consisting of COL3A1, IGFBP5, PRNP, MT1A, LIN28B, FERMT3, RAB27B and PEG10 compared to bone marrow derived stem cells.
  • the cells in the cell population are about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Or at least 90% of the cells may express less than one gene or protein selected from the group consisting of COL1A1, COL1A2, TPM2, TAGLN, CYP1B1, CXCL12, PENK, HYOU1 and GLIPR1 compared to bone marrow derived stem cells.
  • the cells in the cell population are about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, Or 90% or more of the cells express progranulin.
  • PGK1, BNIP3, TPI1, ERRFI1 in cells of about 50%, 60%, 70%, 80%, 90%, 95% or 98% or more in the cell population cultured in hypoxic conditions compared to normal oxygen culture conditions.
  • MRNA or protein of one or more genes selected from the group consisting of, LOC644774, SLC2A3, PLIN2, TUBB2B, PODXL, HIST1H1B and PLEK2 can be expressed at higher levels.
  • At least about 50%, 60%, 70%, 80%, 90%, 95%, or 98% of the cells in the cell population are cultured in hypoxic conditions compared to normal oxygen culture conditions, PSERPINE1, TAGLN, TGM2, IL8 MRNAs or proteins of one or more genes selected from the group consisting of ALDH1A3 and DPP4 can be expressed at lower levels.
  • Another embodiment provides a method for producing improved postpartum adherent cells comprising the step of reacting amnion tissue isolated from the chorionic valve of the placenta with an enzyme mixture.
  • the enhanced postpartum adherent cells are as described above.
  • (c) secrete one or more proteins selected from the group consisting of VEGF, TGF- ⁇ 1, IL-6, progranulin, follistatin, angiostatin, MMP2, MMP10, TRAIL R2 and MMP3;
  • HYOU1 or GLIPR1 protein is less expressed than bone marrow derived stem cells.
  • the preparation method may be more specifically for producing improved postpartum adherent cells having the characteristics of progranulin protein secretion.
  • the cells may further exhibit negative immunological properties for Oct4, Nanog, TRA-1-60 and / or CD80.
  • the amniotic tissue may be obtained by separating the chorion membrane from the separated placenta, scraping off the chorion membrane to remove the chorion.
  • the chorionic membrane can be separated by, for example, a method of pulling it off from the placenta.
  • the amniotic membrane may be obtained after blood is removed by Ca / Mg free DPBS containing gentamicin from the chorionic valve.
  • the chorionic valve can be obtained by washing blood with Ca / Mg free DPBS containing gentamicin, followed by chorionic removal by scraping.
  • the amnion tissue obtained can be directly reacted with the enzyme mixture or finely chopped using sterile scissors or the like and then reacted with the enzyme mixture.
  • the amnion tissue may be finely sliced (eg, about 5 mm or less) using sterile scissors or the like, and then the enzyme mixture may be added to the sliced cells.
  • the enzyme mixture may refer to an enzyme mixture obtained by mixing various enzymes, or a reaction enzyme solution.
  • the enzyme mixture can lyse the tissue to separate enhanced postpartum adherent cells from the tissue.
  • the enzyme mixture may specifically include one or more selected from the group consisting of trypsin, dispase, and collagenase. More specifically the enzyme mixture may comprise all of trypsin, dispase, and collagenase.
  • the enzyme mixture may further include a DNase.
  • the enzyme mixture may comprise water, saline, such as Hank's Balanced Salt Solution (HBSS), including collagenase, trypsin and dispase.
  • HBSS Hank's Balanced Salt Solution
  • 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, specifically collagenase type I Can be.
  • the DNase may be a hydrolase (DN) I or / and II.
  • the concentration of collagenase in the enzyme mixture solution may be, for example, 0.5 mg / ml to 5 mg / ml, 0.5 mg / ml to 3 mg / ml, 0.8 mg / ml to 2 mg / ml, or 0.8 mg / ml To 1.5 mg / ml, and in one embodiment, 1.2 mg / ml.
  • the concentration of trypsin in the enzyme mixture solution is for example 1 mg / ml to 5 mg / ml, 1 mg / ml to 3 mg / ml, 1.5 mg / ml to 2.5 mg / ml, or 1.5 mg / ml to 2 mg / ml, and in one embodiment, may be 1.8 mg / ml.
  • the concentration of dispase in the enzyme mixture solution may be, for example, 0.1 U / ml to 5 U / ml, 0.1 U / ml to 3 U / ml, 0.5 U / ml to 2.5 U / ml, or 0.5 U / ml To 1.5 U / ml, and in one embodiment, 1 U / ml.
  • the concentration of DNA hydrolase in the enzyme mixture solution may be, for example, 0.001 mg / ml to 1 mg / ml, 0.001 mg / ml to 0.5 mg / ml, 0.01 mg / ml to 0.25 mg / ml, or 0.01 mg / ml to 0.05 mg / ml, and in one embodiment 25 ⁇ g / ml.
  • the reaction of the amniotic tissue and the enzyme mixture solution 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 ° C., and may be performed for about 5 to 60 minutes or 10 to 30 minutes.
  • tissue cells for example, amniotic cells (ie adherent cells) from the enzyme reaction solution can be carried out by methods known in the art, for example, after centrifugation, Cell bodies can be used to separate cells.
  • the yield of improved postpartum adherent cells can be increased, for example, by 50 times or more compared with those without. have.
  • the improved postpartum adherent cell production method of the present invention may further comprise the step of reacting the cells collected after the reaction of the amnion tissue and the enzyme mixture by adding a recombinant enzyme without an animal-derived component.
  • Attaching culture (eg, P0) to the isolated cell population in a container may comprise a stem cell culture medium, such as Fibroblast Growth Factor-4 (FGF-4). And culturing in a medium to which heparin is added.
  • 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.
  • PS-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 culture may be performed under normal oxygen conditions or hypoxic conditions.
  • the term “normoxia” may refer to a state where the oxygen partial pressure is 21%.
  • the term “hypoxia” may refer to a low oxygen partial pressure condition as compared to conventional normal oxygen conditions.
  • 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 culturing may be carried out, for example, for 2 to 7 days, or for 3 to 5 days, and may be treated with a recombinant enzyme without an animal-derived component after the culturing.
  • 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 may 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.
  • TrypLE TM Select Gibco Invitrogen
  • TrypLE TM Express GIBCO Invitrogen
  • TrypZean TM Sigma Aldrich
  • Recombinant Trypsin Solution TM Biological Industries
  • the passage number of the passage is not particularly limited and may be appropriately selected according to the number of desired proliferating cells. For example, by carrying out 1 to 20 passages, 1 to 6 passages, 1 passage, 3 passages, or 6 passages, a clinically necessary number of cumulative proliferating cells can be obtained. Specifically, it may be at least 1 passage to 10 passages.
  • the improved postpartum adherent cells obtained as described above may be obtained from a stem cell culture medium, for example, Fibroblast Growth Factor-4 (FGF-F). 4) and passage in a medium to which heparin is added. Fibroblast Growth Factor-4 (FGF-4) and heparin added medium and culture conditions are as described above.
  • FGF-F Fibroblast Growth Factor-4
  • the treatment of a recombinant enzyme without an animal-derived component may be additionally performed. That is, the purity of adherent cells can be improved by collecting the cells by treating a recombinant enzyme without an animal-derived component before passing the cells to the next step in 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.
  • Another aspect provides improved postpartum adherent cells produced by the above methods of manufacture.
  • the enhanced postpartum adherent cells are as described above.
  • the improved postpartum adherent cells prepared by the method for producing an improved postpartum adherent cell according to one embodiment are transformed into a vascular endothelial growth factor (VEGF), transforming growth factor (VEGF) that is particularly effective for neurological diseases.
  • MMP2 matrix metalloproteinase 2
  • MMP10
  • Another aspect provides a composition comprising an improved postpartum adherent cell of the present invention or an improved postpartum adherent cell population of the present invention.
  • Another aspect is to provide a cell therapy, pharmaceutical composition or formulation comprising the improved postpartum adherent cells of the present invention, a cell population thereof or a culture thereof as an active ingredient.
  • Another aspect provides the use of the improved postpartum adherent cells, populations thereof, or cultures thereof of the present invention for use in the preparation of cell therapies, pharmaceutical compositions or formulations.
  • the improved postpartum adherent cells and the cell population comprising the enhanced postpartum adherent cells are as described above, respectively.
  • the composition may be a composition for enhanced proliferation of adherent cells or for differentiation into specific cells.
  • the composition may be a pharmaceutical composition for the prevention or treatment of various diseases.
  • the composition may be a pharmaceutical composition for the prevention or treatment of neurodegenerative diseases.
  • the neurodegenerative diseases may also include, for example, Alzheimer's disease, concussion, stroke, Parkinson's disese, Pick's disease, Huntington's disease, and progressive supranuclear palsy. ), Multiple sclerosis, Amyotrophic lateral sclerosis (ALS), dementia, and traumatic brain injury (TBI).
  • Another aspect also provides the use of said improved postpartum adherent cells, cell populations thereof or cultures thereof for use in the manufacture of a medicament for use in the treatment or prevention of diseases such as neurodegenerative diseases.
  • Still another aspect provides a method of treating or preventing a disease, eg, a neurodegenerative disease, comprising administering the improved postpartum adherent cells, a population of cells thereof, or a culture thereof to an individual in need thereof. to provide.
  • a disease eg, a neurodegenerative disease
  • the dosage of the 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 10 8 cells / kg body weight based on adherent cells. ) Or an 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.
  • the pharmaceutical composition according to one embodiment may include the adherent cells and a pharmaceutically acceptable carrier and / or additive 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 therapy or pharmaceutical composition according to one embodiment is formulated in a formulation suitable for injection, the cell aggregate may be dissolved in a pharmaceutically acceptable carrier or frozen in solution.
  • the pharmaceutical composition according to one embodiment may be a suspension, dissolution aid, stabilizer, tonicity agent, preservative, adsorption agent, surfactant, diluent, excipient, pH adjuster, painless agent, Buffers, reducing agents, antioxidants and the like may be included as appropriate.
  • 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.
  • a pharmaceutical composition according to one embodiment is in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which may be readily implemented by one of ordinary skill in the art. It can be prepared by 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.
  • Another aspect provides an agent comprising an enhanced postpartum adherent cell or a population of cells comprising the same.
  • the improved postpartum adherent cells and the cell population comprising the enhanced postpartum adherent cells are as described above.
  • the formulation may be a pharmaceutical formulation.
  • the agent may comprise a cell population comprising the enhanced postpartum adherent cells or the same, in addition to these may include fat, bone, muscle, nerve, cartilage and cardiomyocytes differentiated from them.
  • the formulation may be for oral administration or parenteral administration.
  • Each formulation may include conventional pharmaceutically acceptable carriers, for example, in the case of injectables, there may be preservatives, analgesics, solubilizers or stabilizers, and in the case of topical administration, bases, excipients, lubricants or Preservatives and the like.
  • Another aspect provides a cell therapy agent comprising an enhanced postpartum adherent cell or a cell population comprising the same as an active ingredient.
  • the improved postpartum adherent cells and the cell population including the enhanced postpartum adherent cells are as described above.
  • the term 'cell therapeutic agent' refers to a pharmaceutical product used for the purpose of treatment, diagnosis, and prevention as cells and tissues prepared by isolation, culture and special methods from humans. Or for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferating and screening living autologous, allogeneic, or heterologous cells in vitro or by altering the biological characteristics of the cells in order to restore the function of the cells or tissues. May refer to a drug.
  • the improved postpartum adherent cells of the present invention can be applied to various types of therapeutic protocols in which the tissues or organs of the body are enriched, treated or replaced by the engraftment, transplantation or infusion of the desired cell population, such as stem cells or derived cell populations. Can be used.
  • the enhanced postpartum adherent cells can replace or enhance existing tissue, resulting in new or altered tissue, or combined with biological tissue or structure.
  • stem cells may be replaced with the improved postpartum adherent cells of the present invention in therapeutic protocols where typically stem cells other than placenta are used.
  • the cell therapeutic agent may be formulated in an injectable formulation.
  • known conventional ingredients for formulating can be used and can be formulated in conventional manner.
  • Improved postpartum adherent cells are easy to ethical and secured because of the donation of tissue discarded from the mother after childbirth, and has excellent characteristics of proliferation due to the characteristics of cells derived from the fetus, as well as neurological and immune diseases It is suitable as a therapeutic composition because it secretes a large amount of proteins known to be useful for vascular diseases, or the like.
  • Figure 2 is an image observed at 100X magnification using an inverted microscope (Eclipse TS100 (Nikon)), showing the enhanced morphology of postnatal adherent cells.
  • Figure 3 shows the immunomodulatory capacity of ePACs, which is a graph of the inhibition rate of each T cell proliferation when monocyte cells isolated from peripheral blood were co-cultured directly or indirectly with ePACs.
  • Figure 4 shows the immunomodulatory capacity of ePACs according to passage culture, 1 passage (P1), three passages (P3), and six passages (P6) cultured ePACs directly or indirectly with monocytes isolated from peripheral blood After co-culture, the rate of T cell proliferation inhibition for each is shown.
  • ePACs of the present invention show the multipotency of the ePACs of the present invention. It was confirmed that ePACs of the present invention can differentiate into adipocytes, bone cells, or chondrocytes under both hypoxic and normal oxygen conditions.
  • A is the cumulative population doubling level (CPDL) of ePACs
  • B is the population doubling level (PDL) of ePACs
  • C is the cell size of ePACs.
  • Figure 9 shows the results of analyzing the secreted proteins of ePACs through the Ab array, the secretion of ePACs passages for VEGF, TGF- ⁇ 1, progranulin, and IL-6 among them by ELISA.
  • Figure 10 shows the amount of HGF secretion by ePACs passages analyzed using ELISA.
  • FIG. 11 is a graph showing the results of immunoassay and other surface proteins of HLA-ABC, HLA-DR, and HLA-G of enhanced postpartum adherent cells by flow cytometry.
  • FIG. 12 is a view showing a result of comparative analysis of protein expression of enhanced postpartum adherent cells according to one embodiment.
  • Figure 13 shows the results of analyzing the T cell proliferation inhibitory effect (a) and neuronal cell proliferation effect (b) of the improved postpartum adherent stem cells according to one embodiment.
  • Example 1.1 After centrifugation of the amnion cells isolated in Example 1.1, the supernatant was removed, the cell precipitate was suspended in PS-CM medium and inoculated in a T-flask, and then cultured at 37 ° C. under hypoxia (CO 2 5%, O 2 3%). Incubated). The cells were colonized and cultured until they occupy 50-80% of the T-flask bottom area. Every 3 to 4 days, PS-CM medium was replaced to remove non-stick cells from the bottom of the flask. The components of the PS-CM medium are shown in Table 2 below.
  • Example 1.2. ( 1) The culture was carried out in the same manner as the hypoxic condition. Oxygen concentration was 21% in culture, and the same PC-CM medium as in Example 1.2. (1) was used.
  • Example 1 The shape and culture characteristics of the improved postpartum adherent cells cultured in Example 1 were observed at 100 ⁇ magnification using an inverted microscope (Eclipse TS100 (Nikon)).
  • Figure 2 is a micrograph of the cell, the improved postpartum adherent cells of the present invention had a specific form of fibroblasts with irregular projections, it was confirmed that the property of proliferation attached to the plastic wear.
  • the improved postpartum adherent cells of the present invention were smaller in size than the conventionally known placental-derived mesenchymal stem cells, and confirmed to have a uniform cell morphology.
  • Oct4 and Nanog were confirmed through ICC (Immunocytochemistry) on the cells cultured according to Example 1.
  • Embryonic stem cells (ES) were used as a positive control, and the expression patterns of OCT4 and Nanog at P1 and P6 were analyzed.
  • each harvested cell was washed three times with DPBS and fixed for 10 minutes with PBS containing 4% paraformaldehyde. After washing three times with DPBS, a solution containing 0.2% Triton-X100 was added and reacted for 10 minutes at room temperature, then washed three times with DPBS. 10% normal goat serum was added and blocked at room temperature for 30 minutes, and then the stem cell markers Oct4 and Nanog, which are the primary antibodies, were added to block light and reacted at 4 ° C. overnight. After washing three times with DPBS, the secondary antibody was added to the Got antibody and reacted in the dark for 1 hour. Finally washed three times with DPBS, then observed under fluorescence microscopy (FIG. 1).
  • the improved postpartum adherent cells of the present invention compared to ES showed negative characteristics for Oct4 and Nanog, which maintain the characteristics of progenitor cells of embryonic stem cells in both hypoxic and normal oxygen conditions (FIG. 1). This shows that the formation of teratoma has been lost, and thus, the cells of the present invention can be expected to have high safety when used as a cell therapy.
  • Secretory protein profiling was performed to analyze the secreted proteins of the cells prepared in Example 1. Specifically, the adherent cells cultured until 90% confluence under normal and hypoxic conditions were secreted by MEM alpha GlutaMAX (Invitrogen), which is a serum-free medium. The secreted protein was then concentrated to 1 mg / ml. The concentrated solution was reacted with a membrane (human antibody array, Raybio) to which 504 secreted proteins were implanted, followed by fluorescence to identify proteins secreted from adherent cells. As a result, as shown in Table 3 it was confirmed that the secretion of a total of 54 proteins.
  • MEM alpha GlutaMAX Invitrogen
  • VEGF vascular endothelial growth factor
  • TGF- ⁇ 1 vascular endothelial growth factor
  • progranulin vascular endothelial growth factor-6
  • HGF vascular endothelial growth factor-6
  • the secretion amount of the five proteins was analyzed by ELISA (enzyme immunostaining) method.
  • Cells of each passage were inoculated in 6-well plates with the same number and cultured for 1 day, and then replaced with MEM alpha GlutaMAX (Invitrogen), a medium without serum. After incubation, this culture was used as a sample.
  • MEM alpha GlutaMAX Invitrogen
  • TGF- ⁇ 1 includes the pretreatment of the sample. All were measured using a microplate reader Epoch (BioTek Inc.) at 450 nm and analyzed using Gen5 (2.00) software and the results are shown in FIGS. 9 and 10.
  • VEGF increased with passage
  • TGF-B1 was secreted without change depending on donor or passage.
  • progranulin a good protein for the neurological disorders, did not show significant differences between passages and donors.
  • IL-6 expression was increased with passage, and the difference according to donor was observed. HGF secretion was small and no difference was observed between donor and passage.
  • Example 1 In seven passages of cells cultured by the 1.2 (1) and 1.2 (2) methods of Example 1, they were placed in Adipogenesis differentiation media (StemPro® Adipogenesis Differentiation Kit, Life Technologny) at 3 day intervals for 2 weeks. The culture was incubated while the medium was changed. Thereafter, the culture solution was removed, washed with Ca / Mg Free DPBS, 4% paraformaldehyde was added and reacted at room temperature for 15 minutes. 60% isopropanol was added and washed, and then oil red O was added thereto, reacted for 10 minutes, washed with purified water, and then observed with fat cells under a microscope (FIG. 5).
  • Example 1 The cells prepared in Examples 1.2 (1) and 1.2 (2) of Example 1 were placed into osteogenic differentiation media (StemPro® Osteogenesis Differeniation Kit, Life Technology), and the medium was exchanged every three days for two weeks. Incubated. 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 the reaction, add purified water, wash, add 1% silver nitrate solution, react for 5 minutes at room temperature, wash with whole 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 thereto and reacted at room temperature for 5 minutes. Thereafter, the cells were washed with purified water and analyzed for calcium accumulated samples under a microscope (FIG. 5).
  • Osteogenesis Differeniation Kit Life Technology
  • the improved postpartum adherent cells of the present invention was observed to differentiate into adipocytes, bone cells, chondrocytes under both hypoxic and normal oxygen conditions.
  • the adherent cells were isolated and cultured from the amniotic membrane in the same manner as the culture method of the hypoxic condition of Example 1 above.
  • the same number of cells were inoculated into 6 well plates, and the cells were collected by measuring 70 to 80% of the plate bottom area. A total of three measurements were performed. The total cell number was mixed with 10 ⁇ l of the cell suspension and 10 ⁇ l of trypan blue, and then 10 ⁇ l was counted using a hemocytometer. Doubling time was calculated by using the total number of cells and the time when the number of cells reached twice the number.
  • adherent cells were cultured up to 24 passages to measure the size of the cells. Three cell samples were used, and the cell size was measured using an Autocell counter. All three adherent cells showed a similar proliferation pattern. As can be seen in FIG. 8C, the cell size was about 20 ⁇ m or less up to p18 and was observed to be 13 to 14 ⁇ m at initial passage. On the other hand, as the number of passages increased, the cell size increased.
  • transwells were used to confirm the mobility of cells.
  • the transwell was coated with 0.1% gelatin at 37 ° C., and then the cells cultured in the hypoxic and normal oxygen conditions prepared in Example 1 were suspended in 5 ⁇ 10 5 cells and serum premedium to insert the transwell.
  • the medium was added to the upper chamber and a medium PS-CM (FGF4 and Heparin containing medium) containing chemokine (chemokine) was added to the lower cell culture. After culturing for one day in the incubator, the cells transferred through the transwell were stained with Giemsa, and the cell number was counted under an optical microscope to confirm cell migration. The results are shown in FIG. 7.
  • the colony-forming ability of enhanced postpartum adherent cells cultured under normal and hypoxic conditions was confirmed by CFU analysis.
  • CFU analysis In order to analyze the colony forming ability of the cells, the presence of cell colony was confirmed under a microscope after 10 to 14 days of incubation in a 100 mm culture dish. Next, after washing with DPBS, 2 to 3 mL of the mixed solution of glutaaldehyde and crystal violet was added to the dish containing cells and stained for 30 minutes. Carefully washed with sterile water and counting the number of colonies under the microscope, the results were analyzed by quantifying the mean value.
  • T cells proliferate when monocytes isolated from peripheral blood are activated with PHA.
  • the immunomodulatory function can be confirmed in the adherent cells of the present invention.
  • Donated peripheral blood was used to isolate monocyte blood cells using Ficoll.
  • the cells prepared in Example 1 were inoculated with 50,000 cells in a 24-well plate and attached to the plate for one day in a 37 ° C. incubator. Then, 10 ug / ml PHA was added to the culture medium to activate mononuclear blood cells stained with CFSE. 4 x 10 5 pieces were added and co-cultured directly or indirectly for 5 days. Direct coculture was cocultured with enhanced postpartum adherent cells in 24 wells. Indirect coculture was indirectly cocultured with monocyte blood cells in the Transwell upper chamber. Monocytes were then recovered and analyzed for proliferation of T cells using a flow cytometer (FACS Caliber, BD science).
  • the adherent cells according to the present invention had an immunomodulatory function and were found to be immunomodulatory when cocultured with blood cells directly and indirectly. Specifically, when analyzed by donor, it was confirmed that all donor-derived adherent cells had an inhibitory function of 40% or more (FIG. 3). In addition, when passaged by direct co-culture of peripheral blood mononuclear cells and adherent cells, it was observed that the immunosuppressive mechanism is improved as the passage is increased (FIG. 4).
  • Example 1 The neuronal regeneration effect analysis of enhanced postpartum adherent cells isolated and cultured in Example 1 was performed.
  • samples were prepared by collecting MEM and a conditioned medium of enhanced postpartum adherent cells. Thereafter, when inoculated into the neuronal cells (SH-SY5Y) in 96 well plate and propagated for about 1 day, the culture solution of MEM and enhanced postpartum adherent cells were each dispensed and incubated for 4 days. Cyto XTM Cell viability assay kit (WST-1) reagent was added in 10% of medium and reacted in an incubator for 2-3 hours. The proliferation rate of SH-SY5Y cells was analyzed by measuring at 450 nm using a microreader, and the results are shown in FIG. 13.
  • FIG. 13 is a view showing the results of analyzing the neuronal regeneration effect of the improved post-natal adherent cells according to one embodiment.
  • the neuronal cell proliferation capacity in the MEM medium is 100%
  • the neurons cultured in the culture of enhanced postpartum adherent cells (ePACs) are about 280%. It can be seen that the growth rate of about 2.5 times more than the control group.
  • the improved postpartum adherent cells according to one embodiment have an immune disease improvement or neuronal regeneration effect. Accordingly, the postpartum adherent cells of the present invention can be used to treat neurodegenerative diseases.
  • BMMSC Bone marrow Mesenchymal Stem Cells
  • Placental-derived adherent cells in the present invention were analyzed by comparing the difference between BMMSC and gene expression.
  • RNA and protein were extracted from enhanced postpartum adherent cells and bone marrow-derived mesenchymal stem cells (BMMSC) (Cambridge Univ).
  • Biotin-labeled cRNA was prepared by synthesizing cDNA using T7 oligo (dT) primers and performing in vitro transcription using biotin-UTP. The prepared cRNA was quantified using NanoDrop. The prepared cRNA was hybridized to HT-12 v4.0 expression beadchip. After hybridization, DNA chips were washed using Illumina Gene Expression System wash (Illumina) to remove nonspecific hybridization, and the washed DNA chips were labeled with streptavidin-Cy3 (Amersham) fluorescent dye.
  • Fluorescently labeled DNA chips were scanned using a confocal laser scanner (Illumina, Inc.) to obtain fluorescence data present in each spot and stored as image files in TIFF form.
  • TIFF image files were quantified with BeadStudio version 3 (Illumina) to quantify the fluorescence values of each spot. Quantitative results were analyzed by Gene-Enrichment and functional analysis using DAVID (http://david.abcc.ncifcrf.gov/home.jsp) program.
  • 2635 genes showed a difference when a gene having a difference of more than 2 times was selected.
  • 1305 genes were increased in postpartum adherent cells of the present invention than BMMSC, and 1330 genes were decreased.
  • 23 genes having the most difference in expression amount were selected and shown in Table 5 below.
  • COL3A1, IGFBP5, PRNP, MT1A, and CCND1 are genes with increased expression in postpartum adherent cells compared to BMMSC.
  • COL1A2, COL1A1, TPM2, TAGLN, CALD1, COL6A3, IGFBP7, SPARC, EFEMP1, CYP1B1, CXCL12 and PENK are genes with reduced expression in postpartum adherent cells compared to BMMSC.
  • ProbeID ACCESSION SYMBOL fold change One ILMN_1785272 NM_000089.3 COL1A2 -2.152358 2 ILMN_1701308 NM_000088.3 COL1A1 -2.238551 3 ILMN_1757604 NM_213674.1 TPM2 -2.183863 4 ILMN_1773079 NM_000090.3 COL3A1 2.13323 5 ILMN_1778668 NM_003186.3 TAGLN -3.064249 6 ILMN_1803429 NM_001001391.1 CD44 2.548315 7 ILMN_1671703 NM_001613.1 ACTA2 -3.620056 8 ILMN_1730487 NM_033140.2 CALD1 -2.928108 9 ILMN_1750324 NM_000599.2 IGFBP5 2.981225 10 ILMN_1699829 NM_001901.1 CTGF -3.536551 11 ILMN_
  • the extracted total protein was peptide (peptide) using FASP which is a tryptic digestion method using a filter. Isotope labeling was then performed for relative quantification, and mass analysis was performed using Easy nLC-1000 / Q-Exactive equipment to perform LC-MS analysis.
  • the protein expression of the enhanced postpartum adherent cells was compared with that of BMMSC, and the postpartum adherent cells expressed more LIN28B, FERMT3, RAB27B and PEG10 than the bone marrow-derived stem cells, and HYOU1. , And GLIPR1 can be found to express less.
  • Example 6.1 For comparison, a microarray and a proteomics array were used, and the detailed method is the same as in Example 6.1. In addition, improved postpartum adherent cells were isolated and cultured in the same manner as in Example 1.
  • 514 genes showed a difference when the genes with more than twofold difference were selected.
  • 229 genes were increased in postpartum adherent cells under hypoxic conditions and 285 genes were decreased.
  • 21 genes having the most difference in expression amount were selected and shown in Table 6 below.
  • PGK1, BNIP3, TPI1, ERRFI1, LOC644774, SLC2A3, PLIN2, and TUBB2B were increased, and ALDH1A3, SERPINE1, IL6, AKR1B1, NQO1, PAPPA, TAGLN, TGM2, IL1MP1, IL8 and EFE.

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Abstract

La présente invention concerne une cellule adhérente postnatale améliorée (ePAC), un procédé de fabrication de celle-ci, ainsi qu'une composition et un agent thérapeutique cellulaire contenant celle-ci en tant que principe actif.
PCT/KR2016/009552 2015-08-28 2016-08-26 Cellule adhérente postnatale améliorée, et utilisation de celle-ci WO2017039251A1 (fr)

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