WO2012053718A9 - Méthode pour induire la différenciation de cellules souches adultes et de cellules nerveuses à l'aide d'un champ magnétique - Google Patents

Méthode pour induire la différenciation de cellules souches adultes et de cellules nerveuses à l'aide d'un champ magnétique Download PDF

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WO2012053718A9
WO2012053718A9 PCT/KR2011/004190 KR2011004190W WO2012053718A9 WO 2012053718 A9 WO2012053718 A9 WO 2012053718A9 KR 2011004190 W KR2011004190 W KR 2011004190W WO 2012053718 A9 WO2012053718 A9 WO 2012053718A9
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stem cells
cells
differentiation
mesenchymal stem
neurons
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PCT/KR2011/004190
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Korean (ko)
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WO2012053718A1 (fr
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박정극
김성민
김수찬
윤문영
조현진
서영권
윤희훈
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동국대학교 산학협력단
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Priority to US13/702,975 priority Critical patent/US8673605B2/en
Publication of WO2012053718A1 publication Critical patent/WO2012053718A1/fr
Publication of WO2012053718A9 publication Critical patent/WO2012053718A9/fr

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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
    • C12N5/0618—Cells of the nervous system
    • 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
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • 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
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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)
    • 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
    • C12N2506/1361—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 dental pulp or dental follicle 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
    • C12N2529/00—Culture process characterised by the use of electromagnetic stimulation

Definitions

  • the present invention relates to a method for differentiating mesenchymal stem cells or adult stem cells. More specifically, the present invention relates to a method of differentiating the cells into neurons by treating electromagnetic waves of a specific frequency with mesenchymal stem cells or adult stem cells.
  • bone marrow-derived mesenchymal stem cells were induced to differentiate into neurons by chemical differentiation method. Differentiated neurolabeling factors were identified by lot method (B3T, GFAP, MAP-2, NeuN) and ELISA (neural growth factor (NGF), brain-derived neuronal cell factor (BDNF)), but they showed electrophysiological characteristics. Did. Studies on the use of mesenchymal stem cells in neurotherapy by mixed cultures with neurons or neural progenitors (Croft AP, Exp. Neurol. , 216 (2): 329-41 (2009)) It is practically impossible to obtain enough human neurons or neural precursor cells to use.
  • GDNF glial cell line-derived neurotrophic factor
  • Conventionally known neurotherapy technology using wave is a device for applying low frequency energy of less than about 10 Hz to brain tissue. It is a device that induces magnetic field by electric flow by applying electric stimulation directly after implanting electrode in the brain of patient.
  • US20060205993 a device that induces magnetic field by electric flow by applying electric stimulation directly after implanting electrode in the brain of patient.
  • Zheng developed a technique (JP 2008-543388) to improve brain function by combining high frequency or multiple frequency components to give magnetic stimulation to the central nervous system.
  • Riken manufactures nerve cells by electropulsing embryonic stem cells.
  • Technology (US200740065941) has been developed.
  • Gliner et al. Developed a technique for producing neurons by treating the cells with electric pulses (US20050075679).
  • the above techniques have been applied to implant electrodes by directly implanting electrodes, which is accompanied by pain in the patient, and in the case of embryonic stem cells, the possibility of tumor formation is limited, and thus it is limited to be applied to clinical
  • This study is to use mesenchymal stem cells and adult stem cells as a cell therapy for treating various neurological diseases, and it is a non-invasive method to treat mesenchymal stem cells and adult stem cells by neurons. There is a demand for developing new technologies that differentiate the system.
  • the present inventors have recognized the above problems and necessities, and have intensively tried to induce differentiation of mesenchymal stem cells and adult stem cells, which are relatively easy to obtain, to obtain neural or neural stem cells that are difficult to obtain.
  • the present invention has been completed by confirming that electromagnetic fields of a specific frequency can differentiate stem cells.
  • an object of the present invention is to provide a method for differentiating mesenchymal stem cells or adult stem cells into neurons.
  • Another object of the present invention to provide a composition for treating neurological diseases.
  • the present invention provides a method for differentiating stem cells into neurons by treating an electromagnetic field to mesenchymal stem cells or adult stem cells.
  • the nerve cells include astrocytes, oligodendrocytes.
  • the electromagnetic field is preferably treated at a frequency of 1 to 1000 Hz.
  • the electromagnetic field is preferably treated to a strength of 1 to 5 mT.
  • the mesenchymal stem cells of the present invention may be derived from bone marrow, adipose derived, umbilical cord.
  • the present invention also provides a composition for treating neurological diseases comprising neurons differentiated by the above method.
  • the neurological disease may include Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage or spinal cord injury.
  • Stem cell differentiation method and differentiation apparatus using the electromagnetic field according to the present invention by using the low-frequency wave induced differentiation of adult stem cells into neurons, it is possible to easily obtain the neurons or neural stem cells difficult to obtain cells, Although it is possible to induce neuronal differentiation media, it is possible to induce neuronal differentiation even in low-cost general medium conditions, not expensive neuronal induction media by adding growth factors, so Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage, spinal cord injury, etc. It can be useful for the treatment of brain nerve disease.
  • Figure 2 shows the results of neuronal mRNA expression after induction of neural differentiation of adult stem cells.
  • Figure 3 shows that the proliferation of mesenchymal stem cells after exposure to electromagnetic fields of 50Hz, 100Hz, 200Hz using BrdU assay and cell number measurement.
  • Figure 4 shows the results of morphological changes observed with an optical microscope.
  • A, C is a control group
  • B, D is 50Hz
  • E is 100Hz
  • F is 200Hz exposed to bone marrow-derived mesenchymal stem cells
  • A, B is EGM medium
  • C, D, E, F is LDMEM medium It shows the case of culture.
  • Figure 6 shows the expression of neuronal markers Nestin, MAP2, tau, NeuroD1, DCX, NF-L by 50Hz electromagnetic field.
  • Figure 7 shows the expression of neuronal markers MAP2 and NeuroD1 by 100, 200Hz electromagnetic field.
  • Figure 9 shows the expression of neuronal markers MAP2 and DCX by 50Hz, 1mT electromagnetic field in umbilical cord (A), adipose derived (B) stem cells.
  • Figure 10 shows the implementation of the electromagnetic field generating device for adult stem cell differentiation according to an embodiment of the present invention.
  • 11 is a result of analyzing the expression of neuronal markers by mRNA after irradiating electromagnetic waves of 50 Hz (1 mT), 100 Hz (1 mT) to the fourth pulp stem cell in subculture.
  • FIG. 13 shows the results of observing morphological changes of cells after irradiating electromagnetic waves of 50 Hz (1 mT) and 100 Hz (4 mT) to the seventh pulp stem cell in subculture.
  • the present invention relates to a method of differentiating stem cells into neurons by treating electromagnetic fields with mesenchymal stem cells or adult stem cells.
  • the electromagnetic wave used in the present invention is a phenomenon in which an electromagnetic field whose intensity changes periodically propagates into space, and the same meaning as an electromagnetic wave, and a low frequency electromagnetic wave refers to a wave having a low frequency, which usually means 10 kHz or less.
  • Messenchymal stem cell may be derived from an embryo or adult tissue, may be derived from bone marrow, adipose derived, umbilical cord derived. Stem cells of the present invention also include adult stem cells, preferably the adult stem cells may be pulp stem cells.
  • Stem cells are undifferentiated cells, which can divide and self-renewal for a long time, and can differentiate into various kinds of cells given a certain condition. Stem cells are divided into embryonic stem cells and adult stem cells according to the origin of the tissue. Potential has limited disadvantages than embryonic stem cells, but many therapeutics are studied for adult stem cells without ethical problems and no side effects. have.
  • adult stem cells are used, which may use stem cells commercially available or stem cells separated from biological tissues.
  • Neurons include astrocytes, oligodendrocytes, and neurons.
  • the electromagnetic field of the present invention can be differentiated into neurons only when treated with mesenchymal stem cells or adult stem cells at a specific frequency.
  • a technique for inducing neural differentiation of adult stem cells using an electromagnetic field in vitro is preferably performed at an intensity of 1 to 1000 Hz, more preferably 1 to 1 to 10 mT, more preferably to 1 to 5 mT. It may be a low frequency electromagnetic wave having a frequency of from 200Hz.
  • mesenchymal stem cells express markers of various types of neurons (MAP2), astrocytic cells (GFAP), and oligodendrocytes (O4) after electromagnetic field exposure (FIG. 8). .
  • the treatment of the electromagnetic field to the pulp stem cells can be differentiated into neurons.
  • the cells treated with 50 Hz (1 mT) and 100 Hz (4 mT) expression of neuronal differentiation markers and neurites were confirmed.
  • the method of the present invention relates to a method of inducing differentiation of adult stem cells and adult stem cells into neurons by using an electromagnetic field, which has the advantage of inducing nerve differentiation even in growth medium conditions.
  • nonhematopoitic stem cell media (Milteny) was incubated for 10 days. All mesenchymal stem cells used in the experiment can be cultured in 37 °C, 5% carbon dioxide environment.
  • Mesenchymal stem cell differentiation apparatus of the present invention may be preferably in the form illustrated in FIG.
  • it may further include a technique well known to those skilled in the art of the present invention, which is included in the scope of the technical idea of the present invention.
  • the present invention also relates to a composition for treating neurological diseases comprising neurons differentiated by the above method.
  • composition of the present invention is based on neurons differentiated from mesenchymal stem cells, it is nontoxic and safe.
  • the neurological disease treatment composition may include a pharmaceutical composition well known to those skilled in the art in addition to the neuron of the present invention, and may be modified in various dosage forms, which is a technical idea of the present invention. Obviously included in the category of.
  • composition may be formulated and administered in a unit dosage form suitable for administration in the body of a patient according to conventional methods in the pharmaceutical field, and the preparation may be effective to develop alveoli by one or several administrations. Dosage.
  • Formulations suitable for this purpose are preferably parenteral preparations such as injections such as ampoules for injection.
  • the injection ampoule may be mixed with the injection solution immediately before use, and physiological saline, glucose, mannitol, and Ringer's solution may be used as the injection solution.
  • one or more pharmaceutically acceptable inert carriers such as preservatives, analgesic agents, solubilizers or stabilizers in the case of injections, etc. It may further include a base, excipients, lubricants or preservatives.
  • composition or pharmaceutical formulation of the present invention thus prepared may be administered in the form of a mixture with or in combination with other stem cells used for transplantation and other uses, using administration methods commonly used in the art.
  • administration methods commonly used in the art.
  • the administration may be both non-surgical administration using a catheter and surgical administration methods such as infusion or transplantation after thoracic incision, but non-surgical administration using a catheter is more preferable.
  • Neurological diseases of the present invention refers to all of the neurological diseases including Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage, spinal cord injury, etc.
  • the differentiated neurons or neural stem cells according to the present invention is the function of nerve cells in neurological diseases It can function as a therapeutic agent for neurological diseases by restoring.
  • the surgically removed tooth of the human body is placed in 20 ml medium of ⁇ -MEM (Welljin) containing 200 ⁇ l of antibiotic / antifungal and 10 ⁇ l of gentamycin. Transported to the laboratory in a short time, stored in a box.
  • ⁇ -MEM Welljin
  • pulp tissue was taken and placed in 3 ml of a 1500 U collagenase solution containing 1% (v / v) serum. Stir for 90 minutes in a CO 2 incubator maintained at 37 ° C to loosen cell-tissue livers, take both tissues and enzymes, pipet 30 times in 20 ml of ⁇ -MEM medium containing 10% FBS, and centrifuge at 800 rpm for 5 minutes. Separated. Discard the supernatant and inoculate the remaining cells in a 100 mm dish containing 10 ml of 10% (v / v) FBS-containing ⁇ -MEM medium and incubate for 10 days in a CO 2 incubator maintained at 37 ° C with medium replacement every three days. It was.
  • Example 1 Induction of Differentiation of Mesenchymal Stem Cells into Neurons Using Electromagnetic Waves of 50-200 Hz 1mT Intensity
  • the present invention relates to a method of inducing differentiation of mesenchymal stem cells into neurons using an electromagnetic field, and the present method has an advantage of inducing neuronal differentiation even under growth medium conditions.
  • Nonhematopoietic stem cell media was used to maintain mesenchymal stem cell differentiation.
  • the bone marrow-derived mesenchymal stem cells used in the experiment were Lonza's cells, the adipose derived mesenchymal stem cells were Invitrogen's cells, and the umbilical cord-derived mesenchymal stem cells were used according to the method of Reference Example 1 above. All were incubated in a 37 ° C., 5% carbon dioxide environment.
  • the mesenchymal stem cells were exposed to electromagnetic waves of 1 mT at 50 Hz for 7 days, and immunostained by a known method to observe the results. As confirmed in FIG. 1, it was confirmed that the expression of MAP-2, tau, and syntaxin-1 was significantly increased at 1 mT of 50 Hz as a result of immunostaining. However, it was observed that expression was not possible at 10 mT even at the same frequency. It was determined that the cells differentiate only within certain frequencies and magnetic field densities.
  • BM-MSC bone marrow-derived mesenchymal stem cells
  • LDMEM low glucose DMEM
  • fetal bovine serum 25 mM ascorbic acid
  • EBM medium containing growth factors was used as a positive control, RT-PCR and immunohistochemistry (immunohistochemistry) tests were performed by methods known in the art to confirm gene expression.
  • 10 is an electromagnetic field generating device for adult stem cell differentiation according to an embodiment of the present invention.
  • the medium was removed, and washed once with 10 ml of PBS (Phosphate Buffered Saline).
  • the washed cells were added with 1 ml of 0.05% (w / v) trypsin and 0.01% (w / v) EDTA-containing solution and treated at 37 ° C. for 5 minutes to float the cells at the bottom of the dish and float in the solution.
  • the cell solution was mixed with 15 ml of ⁇ -MEM medium containing 10% (v / v) FBS and centrifuged at 1,000 rpm for 5 minutes to recover only cells. These cells were cultured up to passage 5 in a CO 2 incubator and used for the experiment.
  • the cells were inoculated at a concentration of 1 ⁇ 10 5 cells / dish in a 60 mm culture dish in ⁇ -MEM medium containing 10% (v / v) FBS and replaced in medium every 3 days. 5 in a CO 2 incubator maintained at 37 ° C. Incubated daily.
  • the electromagnetic wave device is mounted in an incubator, and the 60 mm dish is placed in the electromagnetic field, and then electromagnetic waves of 0 Hz (non-irradiated group), 50 Hz (1 mT), and 100 Hz (1 mT) are irradiated to form an electromagnetic field. While culturing.
  • the cultured pulp cells were passaged to passage number 7, and then inoculated at a concentration of 1 ⁇ 10 5 cells / dish in a 60 mm culture dish in ⁇ -MEM medium containing 10% (v / v) FBS and exchanged medium every 3 days. Incubated for 5 days in a CO 2 incubator maintained at 37 °C.
  • the electromagnetic wave device is mounted in an incubator, and the 60 mm dish is placed in the electromagnetic field, and then irradiated with electromagnetic waves of 0 Hz (non-irradiated group), 50 Hz (1 mT), and 100 Hz (4 mT), respectively, to form an electromagnetic field. It was.
  • the cultured cells were recovered and mRNA analysis results showed that the expression of neuronal differentiation markers MAP2, NeuroD1, DCX was increased at 50 Hz (1mT) and 100Hz (4mT) compared to the non-irradiated group, and differentiated into neurons.
  • MAP2 neuronal differentiation markers
  • NeuroD1 DCX
  • Stem cell differentiation method and differentiation apparatus using the electromagnetic field according to the present invention by using the low-frequency wave induced differentiation of adult stem cells into neurons, it is possible to easily obtain the neurons or neural stem cells difficult to obtain cells, As it can induce neuronal differentiation in low-cost general medium conditions, not expensive neural differentiation-inducing medium by adding growth factors, it is useful for the treatment of cerebral neurological diseases such as Alzheimer's disease, depression, Parkinson's disease, cerebral infarction, cerebral hemorrhage and spinal cord injury. Can be.

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Abstract

Cette invention concerne une méthode de différenciation de cellules souches mésenchymateuses ou de cellules souches de pulpe dentaire. Plus spécifiquement, cette invention concerne une méthode de différenciation de cellules souches en cellules nerveuses par traitement des cellules souches mésenchymateuses ou des cellules souches de pulpe dentaire avec un champ magnétique basse fréquence. La méthode de différenciation selon la présente invention peut induire la différenciation, même avec des milieux à bas coûts plutôt que les milieux de différenciation neuraux induits qui sont onéreux en raison de l'ajout de facteurs de croissance, et les cellules nerveuses différenciées selon la présente invention peuvent être utiles pour traiter les maladies neurologiques du cerveau.
PCT/KR2011/004190 2010-06-08 2011-06-08 Méthode pour induire la différenciation de cellules souches adultes et de cellules nerveuses à l'aide d'un champ magnétique WO2012053718A1 (fr)

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KR10-2010-0101651 2010-10-19
KR1020100101651A KR101210988B1 (ko) 2010-06-08 2010-10-19 전자기장을 이용한 성체 줄기세포의 신경세포 분화유도 방법

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