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WO2009066817A1 - Procédé pour induire la différenciation de cellules souches mésenchymateuses humaines en neurones moteurs - Google Patents

Procédé pour induire la différenciation de cellules souches mésenchymateuses humaines en neurones moteurs Download PDF

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Publication number
WO2009066817A1
WO2009066817A1 PCT/KR2007/005951 KR2007005951W WO2009066817A1 WO 2009066817 A1 WO2009066817 A1 WO 2009066817A1 KR 2007005951 W KR2007005951 W KR 2007005951W WO 2009066817 A1 WO2009066817 A1 WO 2009066817A1
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Prior art keywords
mesenchymal stem
stem cells
differentiation
forskolin
retinoic acid
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PCT/KR2007/005951
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English (en)
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Mi-Sook Chang
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Seoul National University Industry Foundation
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Priority to PCT/KR2007/005951 priority Critical patent/WO2009066817A1/fr
Publication of WO2009066817A1 publication Critical patent/WO2009066817A1/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
    • C12N5/0619—Neurons
    • 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
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/44—Thiols, e.g. mercaptoethanol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/01—Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
    • 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 inducing differentiation of bone marrow-derived mesenchymal stem cells into mature neurons and motor neurons by culturing them in an optimum medium supplemented with necessary compositions.
  • mesenchymal stem cells used to be known to be differentiated only into connective tissues could also be differentiated into neurons.
  • Sanchez et al reported that when mesenchymal stem cells were cultured in the presence of retinoic acid and BDNF (brain-derived neurotrophic factor) , the cells were differentiated into neurons and astrocytes (Sanchez-Ramos et al., Exp Neurology 164:247-256, 2000).
  • the present inventors introduced two-phase pre-induction system and tried to optimize the condition of the neuronal induction medium.
  • the present inventors finally completed this invention by confirming the expression of a marker for differentiated mature neurons and reproducible differentiation of mesenchymal stem cells into neurons and confirming the expression of a marker for motor neurons.
  • the present invention provides a method for differentiation into motor neurons comprising the following steps:
  • step 2) inducing the first differentiation of the mesenchymal stem cells pre-induced in step 1) in the first motor neuron differentiation induction medium containing retinoic acid and forskolin for 1.5 - 2.5 days;
  • step 3 inducing the second differentiation of the mesenchymal stem sells first-differentiated in step 2) in the second motor neuron differentiation induction medium containing basic fibroblast growth factor (bFGF) , retinoic acid and forskolin for 1.5 - 2.5 days; and,
  • bFGF basic fibroblast growth factor
  • step 4) inducing the third differentiation of the mesenchymal stem cells second-differentiated in step 3) in the third motor neuron differentiation induction medium containing bFGF, retinoic acid, forskolin and SHH (sonic hedgehog) for 3 - 5 days.
  • the present invention also provides the first motor neuron differentiation induction medium containing retinoic acid and forskolin to induce differentiation of the pre- induced mesenchymal stem cells into motor neurons, the second motor neuron differentiation induction medium containing bFGF, retinoic acid and forskolin and the third motor neuron differentiation induction medium containing bFGF, retinoic acid, forskolin and SHH.
  • the present invention further provides motor neurons differentiated by the method of the present invention.
  • the present invention also provides a therapeutic agent for neurodegenerative disease containing the motor neurons.
  • the present invention also provides a method for treating neurodegenerative disease containing the step of administering the motor neurons to a patient with neurodegenerative disease.
  • the present invention provides a use of the motor neurons for the production of a therapeutic agent for neurodegenerative disease.
  • the method of the present invention can provide neurons or motor neurons for the cell therapy of neurodegenerative diseases by inducing differentiation of mesenchymal stem cells into neurons or motor neurons.
  • Fig. 1 is a set of photographs illustrating the morphological changes of mesenchymal stem cells treated with various media: a: Untreated control; b: MNIM-treated group; and, c: MNIM+CM-treated group.
  • Fig. 2 is a set of photographs illustrating the expression levels of neuronal marker genes (NF-L, NF-M and NF-H) in mesenchymal stem cells treated with various media.
  • Fig. 3 is a set of photographs illustrating the expression levels of RA signal transduction genes (RARa and RARb) in mesenchymal stem cells treated with various media.
  • Fig. 4 is a set of photographs illustrating the expression levels of SHH signal transduction genes (Gli2, Gli3 and Smo) in mesenchymal stem cells treated with various media.
  • Fig. 5 is a set of photographs illustrating the expression level of the motor neuronal marker gene (Isletl).
  • Fig. 6 is a set of photographs illustrating the expression of neuronal marker protein (NF-M) in mesenchymal stem cells treated with various media, confirmed by immunofluorescence assay (Red: NF-M, Green: GFP) .
  • NF-M neuronal marker protein
  • the present invention provides a method for differentiation into motor neurons comprising the following steps:
  • step 2) inducing the first differentiation of the mesenchymal stem cells pre-induced in step 1) in the first motor neuron differentiation induction medium containing retinoic acid and forskolin for 1.5 - 2.5 days;
  • step 3 inducing the second differentiation of the mesenchymal stem sells first-differentiated in step 2) in the second motor neuron differentiation induction medium containing basic fibroblast growth factor (bFGF) , retinoic acid and forskolin for 1.5 - 2.5 days; and,
  • bFGF basic fibroblast growth factor
  • step 4) inducing the third differentiation of the mesenchymal stem cells second-differentiated in step 3) in the third motor neuron differentiation induction medium containing bFGF, retinoic acid, forskolin and SHH (sonic hedgehog) for 3 - 5 days.
  • the mesenchymal stem cells are preferably isolated from human bone marrow. Particularly, mononuclear cells are isolated from bone marrow, which are cultured for 1 ⁇ 2 weeks. Then, differentiation-ready hematopoietic stem cells are all differentiated to generate mature blood cells and remaining stem cells are isolated, by which mesenchymal stem cells are obtained. In addition to the separation of mesenchymal stem cells from mononuclear cells isolated from bone marrow, the whole mononuclear cells containing mesenchymal stem cells can be cultured according to the method of the present invention to mass-produce neurons.
  • the pre-induction of step 1) is preferably performed twice with increasing the concentration of ⁇ - mercaptoethanol .
  • the second pre-induction time is preferably shortened to 1/4 ⁇ 1/8 of the first pre-induction time and 1/8 time is more preferred.
  • the first motor neuron differentiation induction medium of step 2) preferably contains 0.8 - 12 ⁇ M retinoic acid and 4 - 6 ⁇ M forskolin, and more preferably 1 ⁇ M retinoic acid and 5 ⁇ M forskolin.
  • the first differentiation induction of step 2) is preferably performed for 1.5 -2.5 days and more preferably for 1.8 - 2.2 days and most preferably for 2 days.
  • the second motor neuron differentiation induction medium of step 3) preferably contains 8 - 12 ng/m# of bFGF, 0.8 - 1.2 ⁇ M retinoic acid and 4-6 ⁇ M forskolin, and more preferably 10 ng/m-£ of bFGF, 1 ⁇ M retinoic acid and 5 ⁇ M forskolin.
  • the second differentiation induction of step 3) is preferably performed for 1.5 - 2.5 days and more preferably for 1.8 - 2.2 days and most preferably for 2 days.
  • the third motor neuron differentiation induction medium of step 4) preferably contains 8 - 12 ng/m# of bFGF, 0.8 - 1.2 ⁇ M retinoic acid, 4-6 ⁇ M forskolin and 4 - 6 nM SHH (sonic hedgehog), and more preferably 10 ng/m# of bFGF, 1 ⁇ M retinoic acid, 5 ⁇ M forskolin and 5 nM SHH.
  • the third differentiation induction of step 4) is preferably- performed for 3 - 5 days and more preferably for 3.5 - 4.5 days and most preferably for 4 days.
  • the present inventors succeeded in differentiating mesenchymal stem cells into motor neuron-like cells by using the method of the invention and confirmed that the differentiated cells remained in that phase even after 4 days from the culture in the basic medium not supplemented with the above compounds (see Fig. 1) .
  • the differentiation was also examined by RT-PCR using various neuronal markers, signal transduction markers and motor neuronal markers.
  • neuronal markers such as NF-L
  • NF-L neurofilament 68 kDa
  • NF-M neuroofilament 160 kDa
  • NF-H neuroofilament 200 kDa
  • NF-M neuronal marker
  • Retinoic acid is expressed in paraxial mesoderm during development and regulates the expressions of class I and class II homeodomains and accelerates the differentiation of ventral neuronal subtype.
  • Forskolin (7 beta-acetoxy-8, 13-epoxy-l alpha, 6 beta, 9 alpha-trihydroxy-labd-14-ene-ll-one) activates adenylcyclase and increases the level of cyclic AMP to stimulate cell receptors.
  • cAMP is an important signal transducer, which is necessary for the cell to respond to hormone and plays an important role in activating gene transcription necessary for cell survival and differentiation.
  • Basic fibroblast growth factor (bFGF) is expressed in node and presomitic mesoderm during development and regulates the specification of neurons along with retinoic acid.
  • SHH sonic hedgehog
  • SHH is expressed in notochord and floor plate during development and is necessary for organogenesis in vertebrates and acts as a morphogen in the brain and spinal cord during development.
  • SHH also acts as an axon guidance cue pulling commissural axon during development of the spinal cord and regulates adult stem cell division and contributes to the motor neuron differentiation along with bFGF and retinoic acid.
  • the present invention also provides the first motor neuron differentiation induction medium containing retinoic acid and forskolin to induce differentiation of the pre- induced mesenchymal stem cells into motor neurons, the second motor neuron differentiation induction medium containing basic bFGF, retinoic acid and forskolin and the third motor neuron differentiation induction medium containing bFGF, retinoic acid, forskolin and SHH.
  • the first - third motor neuron differentiation induction media facilitate the differentiation of the pre- induced mesenchymal stem cells into motor neurons if they are used stepwise properly. That is, if the media are used by the wrong order or one or more media among them are not used, the differentiation into motor neurons will be disturbed.
  • the present invention further provides motor neurons differentiated by the method of the invention.
  • the differentiation of mesenchymal stem cells was induced according to the method of the invention.
  • the level of Islet-1 the motor neuronal marker
  • the level of Islet-1 was significantly increased.
  • the level of Islet-1 was maintained, suggesting that the mesenchymal stem cells were differentiated into motor neurons (see Fig. 5) .
  • the present invention also provides a therapeutic agent comprising the motor neurons for neurodegenerative disease.
  • the neurodegenerative disease is caused by the damage and apoptosis of motor neurons, which is exemplified by amyotrophic lateral sclerosis, spinal cord injury, Parkinson's disease, Alzheimer's disease, Pick's disease, Huntington's disease and traumatic central nervous system diseases, etc.
  • the therapeutic agent of the present invention can be formulated by the conventional method well known to those in the art.
  • the therapeutic agent can be formulated as injectable solutions for parenteral administration such as sterilized solutions or suspensions by mixing with water or other pharmaceutically acceptable liquids.
  • the motor neurons can be mixed with pharmaceutically acceptable carriers or mediums, such as sterilized water, saline, vegetable oil, emulsifiers, suspensions, surfactants, stabilizers, excipients, vehicles, antiseptics and binders, and then formulated as a unit dosage authorized by manufacture of medicines.
  • the effective dose of the therapeutic agent of the invention means the dose inducing the target properly under designated conditions.
  • SteriLe composition for the injection can be provided by using an excipient such as distilled water for injection.
  • an excipient such as distilled water for injection.
  • saline, isotonic solution including glucose or other adjuvants which is exemplified by D-sorbitol, D-mannose, D-mannitol, and sodium chloride can be co-used with solubilizing agents, for example alcohol particularly ethanol, polyalcohol such as propylene glycol, polyethylene glycol and non-ionic surfactant polysorbate 80 (TM), HCO-50.
  • solubilizing agents for example alcohol particularly ethanol, polyalcohol such as propylene glycol, polyethylene glycol and non-ionic surfactant polysorbate 80 (TM), HCO-50.
  • the oil is exemplified by sesame oil and soybean oil, which can be used together with solubilizing agents such as benzyl benzoate and benzyl alcohol.
  • the oil can also be mixed with buffers such as phosphate buffer, sodium acetate buffer; painkillers such as procaine hydrochloride; stabilizers such as benzyl alcohol and phenol; and antioxidants.
  • the prepared injectable solution is filled in proper ampoules.
  • the preferable administration method to a patient is parenteral administration, precisely intravenous injection once to three times, but more frequent administrations can be applied. Administration time can be short or long. Examples of formulations for preferred administration are injections and percutaneously administrable preparations.
  • Injection is exemplified by intravenous injection, intraarterial injection, selective intra-arterial injection, intramuscular injection, intraperitoneal injection, hypodermic injection, intracerebroventricular injection, intracerebral injection and bone marrow injection, and intravenous injection is preferred.
  • the intravenous injection is carried out by general blood transfusion process which does not require any surgery or local anesthesia and is easy to manipulate in a hospital, which makes both patient and doctor more comfortable. In the near future, considering the advancement of emergency medicine, administration during emergency sending or in the onset place will be possible.
  • the present invention provides a treatment method for neurodegenerative disease containing the step of administering the motor neurons to a patient with neurodegenerative disease.
  • the effective dose of the motor neurons is l ⁇ l ⁇ 4 cell/ kg - IxIO 8 cell/kg, more preferably IxIO 5 cell/kg - IxIO 7 cell/kg, and most preferably 5> ⁇ 10 5 cell/kg - 5> ⁇ 10 6 cell/kg, but not always limited thereto.
  • the administration method is not limited but any parenteral administration method can be applied.
  • Systemic administration or local administration can be used but systemic administration is preferred and particularly intravenous administration is more preferred.
  • the present invention provides a use of the motor neurons for the production of a therapeutic agent for neurodegenerative disease.
  • the use of the motor neurons differentiated from mesenchymal stem cells originated from bone marrow can solve the problems of cell supply and immune rejection response.
  • Example 1 Recurrence of differentiation of mesenchymal stem cells into neurons
  • Poietics Normal Human Mesenchymal Stem Cells were purchased from Cambrex (USA) .
  • the above mesenchymal stem cells were sub-cultured two times in a MSC growth medium (mesenchymal cell growth supplement, 10 m# 200 iriM L- glutamine, 0.5 mi penicillin-streptomycin mixture; Cambrex, USA) comprising a basic medium (MSCGM, 500 m#; Cambrex, USA) and a growth supplement, and then transferred into DMEM (Gibco, USA) supplemented with 10% FBS (Gibco, USA) , 100 nq/mi penicillin and 100 U/ift£ streptomycin, followed by further culture in a 37 ° C , 5% CO2 incubator. After three days of culture in the incubator, the medium was removed and the cells were washed with phosphate-buffered saline
  • Mesenchymal stem cells were sub-cultured in DMEM supplemented with 10% FBS and penicillin-streptomycin. To induce neuronal differentiation, the stem cells were cultured in a general medium containing 1 mM ⁇ - mercaptoethanol (Sigma, USA) for 24 hours and then the medium was replaced with another general medium containing 2 mM ⁇ -mercaptoethanol, followed by further culture for 3 hours (pre-induction) . Differentiation of the pre-induced mesenchymal stem cells was induced in the medium containing 1 ⁇ M retinoic acid (Sigma, USA) and 5 ⁇ M forskolin (Sigma, USA) for 2 days.
  • a general medium containing 1 mM ⁇ - mercaptoethanol Sigma, USA
  • pre-induction Differentiation of the pre-induced mesenchymal stem cells was induced in the medium containing 1 ⁇ M retinoic acid (Sigma, USA) and 5 ⁇ M forskolin (Sigma, USA) for 2 days
  • differentiation of the cells was induced in the medium containing 1 ⁇ M retinoic acid, 5 ⁇ M forskolin and 10 ng/vd of bFGF (Peprotech, USA) for 2 days.
  • differentiation of the cells into motor neurons was induced in the medium containing 1 ⁇ M retinoic acid, 5 ⁇ M forskolin, 10 ng/m£ of bFGF, 5 nM SHH (sonic hedgehog; R&D systems, USA) for 4 days.
  • the differentiated cells were further cultured in the basic medium (complete medium, CM) not supplemented with any compound for the motor neuron differentiation, followed by examination whether the differentiation status was maintained (MNIM + CM) .
  • the untreated cells cultured in the basic medium (CM) were used as a control.
  • Example 2 Detection of neuronal differentiation ⁇ 2-l> Detection of neuronal differentiation markers by RT- PCR RNA was extracted from untreated control, MNIM- treated group and MNIM+CM-treated group mesenchymal stem cells obtained in Example ⁇ l-2> by using Trizol solution (Invitrogen, USA) . Reverse transcription (RT) was performed with 2 ⁇ g of the extracted RNA by using MMLV reverse transcriptase (MMLV RTase; Promega, USA) .
  • MMLV RTase MMLV reverse transcriptase
  • RT-PCR was performed with 50 ⁇ i of volume by using 0.5 ⁇ g of oligo (dT) primer, 2.5 mM dNTPs, 5 ⁇ MMLV buffer, RNase inhibitor and MMLV RTase. Then, the RT-PCR product was used as template and PCR amplification was performed using a PCR machine (Bio-Rad, USA) as follows; predenaturation at 94 °C for 5 minutes, denaturation at 94 ° C for 45 seconds, annealing at 55-65 ° C for 45 seconds, polymerization at 72 ° C for 45 seconds, 35-40 cycles from denaturation to polymerization, and final extension at 74 ° C for 7 minutes.
  • a PCR machine Bio-Rad, USA
  • PCR was performed with water instead of the RT product.
  • a master mix composed of Taq polymerase, 2.5 mM dNTPs, the primer sets of Table 1 and 10x buffer were loaded in each reaction tube, to which RT product or water was added as a template, followed by PCR.
  • the PCR product was electrophoresed on 1.5%-2% agarose gel, and the band size was measured by using a transilluminator .
  • the expressions of NF-M and NF-L, mature neuronal markers depended on the induction time by MNIM in hMCS. Particularly, the expressions of NF-M and NF-L were hardly detected in the untreated control group. After treating hMSC with MNIM, the expressions of NF-M and NF-L were increased (Fig. 2). And, the expression of motor neuronal marker (Islet-1) was clearly increased in the MNIM-treated group compared with the untreated control group (Fig. 5). After inducing differentiation, the cells were cultured in the basic medium used for the culture before differentiation.
  • a cover slip (Fisher Scientific, USA) was coated with 20 ⁇ g/ni of PDL (Sigma, USA) for a day, which was then re- coated with 10 ⁇ g/ ⁇ i of laminin (Sigma, USA) for three hours, followed by washing with distilled water 5 times.
  • the growing mesenchymal stem cells cultured by the method of Example ⁇ l-2> were detached from the culture vessel using 0.1% trypsin/EDTA, which were seeded on the prepared PDL- laminin coated cover slip and then cultured in DMEM
  • the mesenchymal stem cells were incubated with the primary antibody NF-M (neurofilament 150 kDa; Chemicon, USA) diluted in the blocking buffer overnight at 4 ° C , followed by washing with 1* PBS three times. Then, Alexa546- conjugated anti rabbit secondary antibody (Molecular probe, USA) diluted in the blocking buffer (1:500) was bound to the stem cells for one hour at room temperature in a dark room. Histological analysis was performed by using a confocal microscope (FluoViewTM Confocal Microscope; Olympus, Japan).
  • neuronal marker NF-M was not detected in the untreated control group, but the expression was increased in the MNIM-treated group. From the observation of morphology of the above experimental group was confirmed that the mesenchymal stem cells cultured in the MNIM was differentiated into motor neurons or neurons.
  • SEQ. ID. NO: 1 is a human NF-L forward primer for the amplification of human NF-L.
  • SEQ. ID. NO: 2 is a human NF-L reverse primer for the amplification of human NF-L.
  • SEQ. ID. NO: 3 is a human NF-M forward primer for the amplification of human NF-M.
  • SEQ. ID. NO: 4 is a human NF-M reverse primer for the amplification of human NF-M.
  • SEQ. ID. NO: 5 is a human NF-H forward primer for the amplification of human NF-H.
  • SEQ. ID. NO: 6 is a human NF-H reverse primer for the amplification of human NF-H.
  • SEQ. ID. NO: 7 is a human RAR alpha forward primer for the amplification of human RAR alpha.
  • SEQ. ID. NO: 8 is a human RAR alpha reverse primer for the amplification of human RAR alpha.
  • SEQ. ID. NO: 9 is a human RAR beta forward primer for the amplification of human RAR beta.
  • SEQ. ID. NO: 10 is a human RAR beta reverse primer for the amplification of human RAR beta.
  • SEQ. ID. NO: 11 is a human Gli2 forward primer for the amplification of human Gli2.
  • SEQ. ID. NO: 12 is a human Gli2 reverse primer for the amplification of human Gli2.
  • SEQ. ID. NO: 13 is a human Gli3 forward primer for the amplification of human Gli3.
  • SEQ. ID. NO: 14 is a human Gli3 reverse primer for the amplification of human Gli3.
  • SEQ. ID. NO: 15 is a human Smo forward primer for the amplification of human Smo.
  • SEQ. ID. NO: 16 is a human Smo reverse primer for the amplification of human Smo.
  • SEQ. ID. NO: 17 is a human Isletl forward primer for the amplification of human Isletl.
  • SEQ. ID. NO: 18 is a human Isletl reverse primer for the amplification of human Isletl.
  • SEQ. ID. NO: 19 is a human beta-actin forward primer for the amplification of human beta-actin.
  • SEQ. ID. NO: 20 is a human beta-actin reverse primer for the amplification of human beta-actin.

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Abstract

La présente invention porte sur un procédé d'induction d'une différenciation de cellules souches mésenchymateuses issues de moelle osseuse en neurones matures et neurones moteurs par culture de celles-ci dans un milieu optimal supplémenté par une composition nécessaire. Le procédé de la présente invention peut fournir des neurones ou des neurones moteurs pour la thérapie cellulaire de maladies neurodégénératives par induction d'une différenciation de cellules souches mésenchymateuses en neurones ou neurones moteurs.
PCT/KR2007/005951 2007-11-23 2007-11-23 Procédé pour induire la différenciation de cellules souches mésenchymateuses humaines en neurones moteurs WO2009066817A1 (fr)

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CN116640727A (zh) * 2023-07-26 2023-08-25 成都高新绮澳医疗美容诊所有限公司 一种提高细胞活力的营养液及其制备方法、应用
EP4331593A4 (fr) * 2021-04-27 2025-04-23 Univ Nat Chonnam Ind Found Composition pharmaceutique pour la prévention ou le traitement de la maladie de parkinson, et son procédé de préparation

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020039724A1 (en) * 2000-05-17 2002-04-04 Carpenter Melissa K. Neural progenitor cell populations
US20050003544A1 (en) * 2003-06-11 2005-01-06 Goldman Steven A. Enriched or purified population of motor neurons and its preparation from a population of embryonic stem cells
US20050064586A1 (en) * 2001-11-06 2005-03-24 Huizhen Sheng Somatic cell derived embryonic stem cells and its differentiated cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020039724A1 (en) * 2000-05-17 2002-04-04 Carpenter Melissa K. Neural progenitor cell populations
US20050064586A1 (en) * 2001-11-06 2005-03-24 Huizhen Sheng Somatic cell derived embryonic stem cells and its differentiated cells
US20050003544A1 (en) * 2003-06-11 2005-01-06 Goldman Steven A. Enriched or purified population of motor neurons and its preparation from a population of embryonic stem cells

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WICHTERLE, H. ET AL.: "Directed Differentiation of Embryonic Stem Cells into Motor Neurons", CELL, vol. 110, 2002, pages 385 - 397, XP002537463, DOI: doi:10.1016/S0092-8674(02)00835-8 *
WOODBURY, D. ET AL.: "Adult Rat and Human Bone Marrow Stromal Cells Differentiate Into Neurons", J. NEUROSCI. RES., vol. 61, 2000, pages 364 - 370 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4331593A4 (fr) * 2021-04-27 2025-04-23 Univ Nat Chonnam Ind Found Composition pharmaceutique pour la prévention ou le traitement de la maladie de parkinson, et son procédé de préparation
CN116640727A (zh) * 2023-07-26 2023-08-25 成都高新绮澳医疗美容诊所有限公司 一种提高细胞活力的营养液及其制备方法、应用
CN116640727B (zh) * 2023-07-26 2023-09-22 成都高新绮澳医疗美容诊所有限公司 一种提高细胞活力的营养液及其制备方法、应用

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