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WO2014209013A1 - Nouvelles cellules souches présentant des caractéristiques de cellules endothéliales et dérivées de cellules souches mésenchymateuses - Google Patents

Nouvelles cellules souches présentant des caractéristiques de cellules endothéliales et dérivées de cellules souches mésenchymateuses Download PDF

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WO2014209013A1
WO2014209013A1 PCT/KR2014/005634 KR2014005634W WO2014209013A1 WO 2014209013 A1 WO2014209013 A1 WO 2014209013A1 KR 2014005634 W KR2014005634 W KR 2014005634W WO 2014209013 A1 WO2014209013 A1 WO 2014209013A1
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stem cells
ischemic
cells
stem cell
cell
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Korean (ko)
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김동익
김애경
김민희
김도형
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Samsung Life Public Welfare Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/069—Vascular Endothelial cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1353—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from bone marrow mesenchymal stem cells (BM-MSC)

Definitions

  • the present invention relates to a method for producing the stem cells from the stem cells and mesenchymal stem cells comprising the characteristics of endothelial cells, derived from mesenchymal stem cells.
  • the present invention also relates to a pharmaceutical composition for the prevention or treatment of ischemic diseases comprising the stem cells or cultures thereof.
  • Ischemia is a state of reduced blood flow to body organs, tissues or sites, which ultimately leads to necrosis of cells and tissues, which are irreversible damage.
  • the brain and heart are the most sensitive organs of the bloodstream, for example, when ischemic tissues are caused by stroke or head injury, triggering a process called ischemic cascade, which permanently damages brain tissue. .
  • Ischemic disease is a disease related to ischemia caused by an organic disorder of the blood supply. Cardiac diseases (ischemic cardiomyopathy, myocardial infarction, ischemic heart failure, etc.).
  • Cell therapy products including stem cell therapies, can be used to proliferate, select, or otherwise invigorate living autologous, allogeneic, or xenogeneic cells in vitro to restore cell and tissue function. It is defined as a drug that is used for the purpose of treatment, diagnosis and prevention through a series of actions, such as changing the biological characteristics.
  • Stem cell therapies are particularly the case of using stem cells, and the current application field is essential, but it does not naturally work well to recover and regenerate lost cells such as neurological disease, heart disease, lung disease, liver disease, cancer If not, development is actively underway.
  • Stem cells have great potential in cell therapy in that they have differentiation potential and can induce differentiation from damaged tissues to necessary cells.However, at the present development level, the survival rate after transplantation in the body is not high. It is hard to find a successful example.
  • fat, bone marrow or umbilical cord blood-derived stem cell therapy can be used to treat ischemic diseases, and it has been found that blood vessels can be regenerated.
  • most of the stem cells transplanted into the ischemic site by two-dimensional culture are killed to treat cell therapy. There has been a problem that the efficacy is not great.
  • Rehman J et al. Also reported that adipose derived stem cells secrete factors related to blood vessel regeneration (Circulation 2004; 109 (10): 1292-8). The survival rate of the cells transplanted to the ischemic site was extremely low. Furthermore, Nakagami H et al.
  • the present inventors have made intensive efforts for the invention of a new stem cell line that can be used as a cell therapy, and as a result, invented a novel stem cell including the characteristics of endothelial cells, and confirmed the effect in its ischemic disease model. Has come to completion.
  • One aspect of the present invention relates to a method for producing the stem cells from the mesenchymal stem cells and stem cells comprising the characteristics of endothelial cells, derived from mesenchymal stem cells.
  • one aspect of the present invention relates to a pharmaceutical composition for preventing or treating ischemic disease comprising the stem cells or a culture thereof.
  • One aspect of the present invention provides a stem cell comprising the characteristics of endothelial cells, derived from Mesenchymal stem cells.
  • the present inventors cultured mesenchymal stem cells under low oxygen partial pressure conditions, and produced a novel cell line containing both new characteristics, that is, endothelial cell characteristics and stem cell characteristics.
  • MTSC Messenthelial stem cell
  • the stem cells including the characteristics of the endothelial cells of the present invention, CD 44, CD 105, CD 90 and CD 73, which are markers of stem cells, are positive, compared with CD 45, CD 144, CD 31 and mesenchymal stem cells. Characterized by an increased expression of CD 34.
  • stem cell is an undifferentiated cell having the ability to differentiate into various body tissues, which are totipotent stem cells, pluripotent stem cells, and multipotent stem cells. stem cell).
  • mesenchymal stem cell is a multipotent that has the ability to differentiate into ectoderm cells, such as bone, cartilage, fat, muscle cells, or even ectoderm cells such as neurons. It is a multipotent stem cell.
  • the mesenchymal stem cells may be derived from those selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerves, skin, amniotic membrane, chorion, decidual membrane, and placenta.
  • the mesenchymal stem cells may be derived from mammals other than humans, fetuses or humans. Mammals other than humans may be more preferably canine, feline, ape, animal, cow, sheep, pig, horse, rat, mouse or guinea pig, and the like, without limitation.
  • Stem cells comprising the characteristics of the endothelial cells of the present invention can be expressed angiogenesis factors and stem cell factors
  • the angiogenesis factors are AAMP (angio-associated migratory protein), bFGF (basic fibroblast growth factor) or ANG -1 (Angiopoietin-2)
  • the stem cell factor may be Sall4 (Sal-like protein, KLF-4 (Kruppel-likefactor, oct (octamer-binding transcription factor) or Nanog.
  • the endothelial of the present invention Stem cells comprising the characteristics of the cell may be expressed apoptosis factor, the apoptosis factor may be TNF-b (Tumor necrosis factor-b), BAX, BCL2 (B-cell lymphoma or P53. Or both expression of a protein.
  • the characteristics of the stem cells of the present invention may be improved cell proliferation, cell mobility or DNA integrity.
  • stem cells containing the characteristics of the endothelial cells has been deposited with the Korea Research Institute of Bioscience and Biotechnology, accession number may be KCTC 12404BP.
  • the stem cells of the present invention can be induced by culturing mesenchymal stem cells at low oxygen partial pressure as described above.
  • the low oxygen partial pressure may be 0.1 to 10%. Preferably from 0.5% to 5%, more preferably 1%.
  • the culture may be performed without the addition of a separate growth factor.
  • Another aspect of the present invention provides a method for producing a stem cell comprising the characteristics of endothelial cells, comprising culturing the mesenchymal stem cells under low oxygen partial pressure conditions.
  • the low oxygen partial pressure may be 0.1 to 10%. Preferably from 0.5% to 5%, more preferably 1%.
  • the culturing can be performed without treatment of other growth factors.
  • Another aspect of the present invention provides a pharmaceutical composition for preventing or treating ischemic disease, comprising the stem cells or a culture thereof.
  • the stem cells have both endothelial and stem cell characteristics, and have high cell proliferation rate, cell migration ability, and DNA preservation ability, and thus exhibit an active neovascularization ability in ischemic diseases, and thus are effectively used for preventing or treating ischemic diseases. Can be.
  • the ischemic disease may be ischemic heart disease, myocardial infarction, angina, lower limb ischemic disease, extremity ischemic disease, ischemic neurosis, ischemic pulmonary disease, ischemic colitis, ischemic heart failure, obstructive arteriosclerosis or ischemic cerebrovascular disease.
  • Ischemic cerebral disease can be, for example, thrombosis, embolism, transient ischemic attack, cerebral infarction, cerebral hemorrhage, stroke, subarachnoid hemorrhage, white matter disorder or small infarction.
  • the composition may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the composition are conventionally used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like.
  • the pharmaceutical composition may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
  • the pharmaceutical composition for preventing or treating the ischemic disease may be administered orally or parenterally.
  • parenteral administration it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, pulmonary administration and rectal administration.
  • oral administration because proteins or peptides are digested, oral compositions should be formulated to coat the active agent or to protect it from degradation in the stomach.
  • the composition may be administered by any device in which the active substance may migrate to the target cell.
  • Suitable dosages of the pharmaceutical compositions for the prevention or treatment of ischemic diseases include factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to response. It can be prescribed in various ways.
  • the preferred dosage of the composition is within the 100-100,000,000 (10 2 -10 8) cell / kg range, based on an adult.
  • pharmaceutically effective amount means an amount sufficient to prevent or treat cancer or to prevent or treat a disease due to angiogenesis.
  • the composition may be prepared in unit dose form or formulated into a multi-dose container by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods readily available to those skilled in the art.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the composition may be administered as a separate therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered once or additionally if necessary.
  • Stem cells according to the present invention have both endothelial and stem cell characteristics, and have high cell proliferation rate, cell migration capacity and DNA preservation ability, and thus exhibit an active neovascularization ability in ischemic disease, thereby preventing or treating ischemic disease. Can be used effectively.
  • FIG. 1 is a diagram showing the cell proliferation of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia) by the number of passages.
  • Figure 2 is a diagram comparing the cell proliferation of MSC cultured at normal oxygen partial pressure (Normoxia) of passage number 6 and MTSC cultured at hypoxia (Hypoxia).
  • FIG 3 is a diagram showing the morphological characteristics of MSC cultured at normal oxygen partial pressure (Normoxia) at passage number 4 and MTSC cultured at hypoxia (Hypoxia).
  • FIG. 4 is a diagram illustrating the cell migration capacity of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia).
  • FIG. 5 is a diagram comparing the cell migration capacity of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at low oxygen partial pressure (Hypoxia).
  • FIG. 6 is a diagram comparing gene expression amounts of MSC cultured at normal oxygen partial pressure (Normoxia) and MTSC cultured at hypoxia (Hypoxia).
  • FIG. 7 is a diagram comparing DNA damage of MSCs cultured at normal oxygen partial pressure (Normoxia) and MTSCs cultured at hypoxia (Hypoxia).
  • FIG. 8 is a diagram confirming the angiogenic capacity of the MSC and MTSC in the ischemia of the lower limbs.
  • FIG. 9 is a diagram illustrating the angiogenic capacity of MSC and MTSC in the ischemic animal model using the immunohistochemistry.
  • FIG. 10 is a diagram comparing gene expression patterns of MSC and MTSC in the ischemia of the lower limbs.
  • FIG. 11 is a diagram confirming that CD31, a surface protein of endothelial cells, is expressed in MTSC cultured at hypoxia (1%) and not expressed in MSC cultured at normoxia (20%).
  • FIG. 12 is a diagram confirming the high expression of VEGF affecting cell growth and ischemia treatment in MTSC cultured at Hypoxia (1%).
  • FIG. 13 shows that the differentiation experiments showed that MTSC was suppressed from adipocyte differentiation compared to MSC, but differentiation into osteoblasts was enhanced.
  • hUCB-MSC2 human Umbilical Cord blood derived-MSC 2 (purchased from PromoCell (USA): order number: C12972, Lot Number: 1080102.3) in a 100 cm 2 Petri dish with DMEM (Dulbecco's modified Eagle medium) Plated in Petri dishes containing low glucose with addition of 10% fetal bovine serum, 100 U / mL penicillin, 100 ⁇ g / mL streptomycin.
  • MTSC was obtained by incubating MSC under hypoxia conditions (1, 5 or 10% oxygen partial pressure, in particular 1% oxygen partial pressure and 5% CO 2 ). Cultures were incubated in low glucose DMEM (Dulbecco's modified Eagle medium) containing low glucose / 10% fetal bovine serum, 100 U / mL penicillin, 100 ⁇ g / mL streptomycin. At 80% density, cells under each condition were harvested using 0.25% trypsin and replated at 2,000 cells / cm 2 in the medium.
  • DMEM Dynamic Eagle medium
  • the MTSC was deposited to KRIBB (Korea Research Institute of Bioscience and Biotechnology) dated May 2, 2013, accession number KCTC 12404BP.
  • hypoxia conditions were the same as in Example 1, normal oxygen partial pressure (Normoxia) conditions were hUCB-MSC2 (human Umbilical Cord blood derived-MSC 2) in a Petri dish of 100 cm 2 2.0X10 5 cells / 5 plates were plated for each culture period (Day-1, 3, 5, 7) at the concentration of the plate. Incubate at normal and low oxygen partial pressure, and isolate cells from Petri dishes using 0.25% trypsin for each culture period, and count the number of cells three times using a hemocytometer to determine the average cell number.
  • Normal oxygen partial pressure normal oxygen partial pressure
  • MTSC cultured at low oxygen partial pressure of MSC showed higher cell proliferation ability than MSC regardless of the number of passages.
  • MTSC at low oxygen partial pressure showed significantly higher cell migration capacity than MSC at normal oxygen partial pressure.
  • FIG. 5 is a photograph analyzing the migration of cells, and as shown in FIG. 5, in a space from which culture-insertion is removed, an empty space without cells is calculated after a predetermined time and is normal. As a result of comparing the cell migration capacity of the cells cultured at the oxygen partial pressure and the low oxygen partial pressure, it was confirmed that the low oxygen partial pressure environmental culture cells show a high mobility.
  • RNAs of MSC and MTSC were extracted.
  • Stem cell markers Sall4 (Sal-like protein 4), KLF-4 (Kruppel-likefactor4), OCT-4 (octamer-binding transcription factor4) Nanog and HIF-1 (Hypoxia-inducible factor-1), and blood vessels Angiogenic markers such as AAMP (angio-associated migratory cell protein), bFGF (basic fibroblast growth factor) and ANG-1 (Angiopoietin-1) and apoptosis markers TNF- ⁇ (Tumor necrosis factor- ⁇ ), BAX (Bcl 2-associated X qRT-PCR was performed using protein), BCL2 (B-cell lymphoma 2 and P53 (protein 53 or tumor protein 53)).
  • mRNA was extracted from cells cultured at normal and low oxygen partial pressure and converted to cDNA, followed by qRT-PCR.
  • the qRT-PCR method is as follows. In each muscle tissue, modified cDNA 40ng / ul, primer 10ng, and SYBR Green were added to each muscle tissue, using a qRT-PCR machine for 1 cycle at 95 ° C for 10 seconds, at 5 ° C at 95 ° C and for 34 seconds at 60 ° C. 40 cycles, 15 seconds at 95 degrees, 60 seconds at 60 degrees and 1 cycle at 95 degrees for 15 seconds to amplify and confirm the expression, and the results were compared with the MSC cultured at normal oxygen partial pressure, This is shown in FIG. 6.
  • angiogenesis factors such as AAMP, bFGF, ANG-1 in the cells cultured at low oxygen partial pressure compared to normal oxygen partial pressure is 1.02 times, 2.04 times, 2.66 times higher, respectively, TNF-b, BAX, Apoptosis factor gene expression, such as BCL2 and P53, was also high, confirming high safety.
  • Sall4, KLF-4, Oct, Nanog was confirmed that the high stem cell high expression amount.
  • the expression level of HIF-1 was confirmed to increase the expression level of HIF-1 in cells cultured in hypoxia.
  • HIF-1 hyperoxia-inducible factor-1) is a transcription factor that is expressed in a hypoxic environment. Highly expressed in hypoxia-induced cells is evidence that the cells were cultured in hypoxic state. When HIF-1 is activated, angiogenesis is activated.
  • DNA integrity of MSC at normal oxygen partial pressure and MTSC at low oxygen partial pressure was confirmed using p-H2AX (S139).
  • ⁇ p-H2AX is a major indicator of DNA's integrity, and the induction of the genetic stability marker ⁇ p-H2AX (S139) means that it is not genetically stable.
  • ⁇ p-H2AX (S139) expression was confirmed as the passage of the cell progressed, indicating that the stability of the cell was reduced.
  • ⁇ p-H2AX (S139) is stably maintained even when the number of passages increases, which means that DNA damage was not confirmed despite the increase in the number of passages. High genetic stability could be confirmed.
  • Flow cytometry was performed to confirm cellular characteristics.
  • Cell surfaces were marked with antibodies to CD45, CD73, CD44, CD105, CD90, CD31, CD34 and CD144.
  • CD45, CD73, CD44, CD105, CD90, CD31, CD34, and CD144 were attached to the surface of each cell, using a flow cytometer (FACS: Fluorescence-activated cell sorting). Analyzed.
  • MTSC is more positive than the MSC for the stem cell markers CD44, CD105, CD90 and CD73, and the positive rate is higher for the endothelial cell markers CD144, CD31, and CD34 than the MSC.
  • the positive ratio was also high in CD45.
  • MTSC cultured at low oxygen partial pressure includes endothelial cell characteristics while maintaining stem cell characteristics, unlike cell characteristics of conventional MSCs, and cell proliferation ability, cell migration capacity, and DNA preservation. It was confirmed that the capacity is a novel stem cell line containing increased properties compared to MSC.
  • Example 6 Characterization of MTSC (Mesenthelial stem cell) in the ischemic mouse model
  • mice Three to six passages of 1 ⁇ 10 6 MSC and MTSC cells were suspended in 60 ⁇ l physiological saline and injected into the ischemic lower extremity of mice. Mice were purchased from Orient Bio Co., Ltd., and strain was induced using the 6-week-old Balb-C / nude limb ischemia model.
  • the method of inducing the lower limb ischemia was a method of tying and cutting the lower limb artery of the mouse. Both cells were implanted intramuscularly into the lower limb muscle (femoral artery ligation site) immediately after induction of the ischemia. Four weeks after injecting the cells, the lower extremity muscle was harvested and the angiogenic effect was confirmed.
  • the MTSC-implanted group showed significantly higher neovascularization ability than the ischemic model and the MSC-implanted group.
  • the neovascularization ability of MTSC was confirmed through immunohistochemistry (IHC). Specifically, the harvested lower limb muscle tissue was made of paraffin blocks, and then sectioned to 4 ⁇ m and hardened on a slide. The slide was labeled with a CD31 or vWF marker that specifically binds to vascular endothelial cells to confirm the count in muscle tissue. The results are shown in FIG.
  • qRT-PCR Quantitative Rean-Time Polymerase Chain Reaction
  • mRNA was isolated from muscle tissue harvested 4 weeks after cell treatment, and mixed with CD31, Ang-1 and PIGF primers to perform qRT-PCR.
  • the test method is as follows. In each muscle tissue, modified cDNA 40ng / ul, primer 10ng, and SYBR Green were added to each muscle tissue for 1 cycle at 95 degrees for 10 seconds, 5 seconds at 95 degrees, and 34 seconds at 60 degrees using a qRT-PCR machine. 40 cycles and 15 seconds at 95 degrees, 60 seconds at 60 degrees, and 1 cycle at 95 degrees for 15 seconds were amplified and confirmed. The results are shown in FIG.
  • Example 7 Confirmation of expression of CD31, an endothelial cell marker in MTSC (Mesenthelial stem cell)
  • CD31 which is used as a marker of endothelial cells, was expressed in MTSC (Mesenthelial stem cell) cultured at low oxygen partial pressure (1%), but MSC cultured at normal oxygen partial pressure (20%) Expression of CD31 was not confirmed at all.
  • VEGF vascular endothelial growth factor
  • MTSC Messenthelial stem cell
  • Adipogenesis was stained at 14 days and Osteogenesis at 21 days after induction of differentiation. Staining proceeded as follows.
  • the medium was removed, washed with PBS, the cells were fixed with 10% formalin for 30 minutes, and treated with 2% Alzarin Red S for 5 minutes, washed and observed under a microscope.

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Abstract

La présente invention concerne des cellules souches présentant des caractéristiques de cellules endothéliales et dérivées de cellules souches mésenchymateuses, et un procédé de production des cellules souches à partir de cellules souches mésenchymateuses. En outre, la présente invention concerne une composition pharmaceutique de prévention ou de traitement des maladies ischémiques, la composition contenant les cellules souches ou un liquide de culture de celles-ci. Les cellules souches selon la présente invention présentent des caractéristiques à la fois des cellules endothéliales et des cellules souches, et conservent un taux de prolifération cellulaire, une capacité de migration des cellules, et une capacité de conservation d'ADN élevés, faisant preuve de là d'une capacité active d'angiogenèse dans les maladies ischémiques, et ainsi peuvent être efficacement utilisées pour prévenir ou traiter les maladies ischémiques.
PCT/KR2014/005634 2013-06-25 2014-06-25 Nouvelles cellules souches présentant des caractéristiques de cellules endothéliales et dérivées de cellules souches mésenchymateuses Ceased WO2014209013A1 (fr)

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EP4352207A4 (fr) * 2021-05-23 2025-04-09 Bonus Therapeutics Ltd. Compositions de cellules ayant une capacité thérapeutique améliorée

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Publication number Priority date Publication date Assignee Title
EP4352207A4 (fr) * 2021-05-23 2025-04-09 Bonus Therapeutics Ltd. Compositions de cellules ayant une capacité thérapeutique améliorée
CN118406642A (zh) * 2023-04-04 2024-07-30 温州医科大学 人胎盘血管周干细胞及其对缺血性疾病的治疗作用

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