WO2016111378A1 - COMPOSITION POUR LE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE COMPRENANT UN SURNAGEANT DE CULTURE DE CELLULES iPS ET SON PROCÉDÉ DE FABRICATION, PROCÉDÉ D'INHIBITION DE L'APPARITION D'UNE MALADIE NEURODÉGÉNÉRATIVE ET PROCÉDÉ DE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE - Google Patents

COMPOSITION POUR LE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE COMPRENANT UN SURNAGEANT DE CULTURE DE CELLULES iPS ET SON PROCÉDÉ DE FABRICATION, PROCÉDÉ D'INHIBITION DE L'APPARITION D'UNE MALADIE NEURODÉGÉNÉRATIVE ET PROCÉDÉ DE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE Download PDF

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WO2016111378A1
WO2016111378A1 PCT/JP2016/050729 JP2016050729W WO2016111378A1 WO 2016111378 A1 WO2016111378 A1 WO 2016111378A1 JP 2016050729 W JP2016050729 W JP 2016050729W WO 2016111378 A1 WO2016111378 A1 WO 2016111378A1
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neurodegenerative disease
composition
treating
cells
culture supernatant
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Japanese (ja)
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上田　実
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上田　実
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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
    • 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
    • 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
    • A61K35/54—Ovaries; Ova; Ovules; Embryos; Foetal cells; Germ cells
    • A61K35/545—Embryonic stem cells; Pluripotent stem cells; Induced pluripotent stem cells; Uncharacterised stem cells

Definitions

  • the present invention relates to a composition for treating a neurodegenerative disease including an iPS cell culture supernatant and a method for producing the same, a method for suppressing the onset of a neurodegenerative disease, and a method for treating a neurodegenerative disease.
  • stem cells have attracted attention as an alternative technique for diseases that are difficult to treat depending on conventional medicine.
  • Regenerative medicine using stem cells is a promising tool in a new clinical platform for all intractable diseases.
  • stem cells including embryonic stem cells (ES cells) and somatic stem cells have been reported.
  • somatic stem cells mesenchymal stem cells (MSC) isolated from various tissues such as bone marrow, adipose tissue, skin, umbilical cord, and placenta have been particularly used for clinical application in skin regeneration.
  • iPS cells induced pluripotent stem cells
  • iPS cells are cells created by introducing several types of reprogramming factors into somatic cells, and can be differentiated into all three germ layers. And “self-replicating” capable of growing while maintaining an undifferentiated state (see, for example, Japanese Patent No. 5098028). Attempts have also been made to differentiate iPS cells and obtain transplant materials using this differentiation pluripotency (see, for example, International Publication No. WO2011-136378).
  • a neurodegenerative disease is a disorder in which a specific group of nerve cells (for example, nerve cells related to cognitive function or cells related to motor function) are gradually damaged among the nerve cells in the central nervous system (for example, the brain and spinal cord). It is a disease that falls off. The cause of its onset is unclear, and the cells that fall off depend on the type of disease.
  • Neurodegenerative diseases include diseases in which motor ability is reduced, diseases in which sense of balance is reduced, diseases in which muscle strength is reduced, and diseases in which cognitive ability is reduced.
  • Diseases with reduced motor ability include Parkinson's disease, Parkinson's syndrome (multiple system atrophy, progressive supranuclear paralysis, etc.).
  • Diseases that reduce balance include spinocerebellar degeneration and some spastic paraplegia.
  • diseases in which muscle strength is reduced include amyotrophic lateral sclerosis.
  • Diseases with reduced cognitive ability include Alzheimer's disease, Lewy body dementia, corticobasal degeneration. Neurodegenerative diseases are more common in the elderly. Moreover, although it is generally sporadic, the case of familial is also seen.
  • a progressive neurodegenerative disease in which only the motor nerve is selectively impaired is called a motor neuron disease (MND).
  • MND motor neuron disease
  • ALS amyotrophic lateral sclerosis
  • ALS often develops in the 50s, and when ALS is involved, the limbs are not stressed and the muscle mass decreases. Typically, force is not applied to the tip of one hand, and this symptom gradually spreads throughout the body. Eventually, the muscles of the whole body became unable to move and became bedridden, and eventually died of respiratory muscle paralysis. There is no fundamental cure yet, and basically only symptomatic treatment can be performed. The period from onset to death is about 2 to 4 years, but the speed of progression varies from individual to individual.
  • iPS cells themselves utilized the pluripotency and self-renewal properties of iPS cells themselves. In this case, for example, transplantation of a transplant material obtained by differentiating iPS cells is necessary, which places a burden on the patient.
  • the present invention has been made in view of the above situation, and a novel composition for treating neurodegenerative diseases using the culture supernatant of iPS cells, a method for producing the same, a method for suppressing the onset of neurodegenerative diseases, and treatment for neurodegenerative diseases It is an object to provide a method.
  • the composition for treating a neurodegenerative disease according to the first aspect of the present invention is a composition for treating a neurodegenerative disease comprising an iPS cell culture supernatant obtained by culturing iPS cells.
  • the method for producing a composition for treating a neurodegenerative disease according to the second aspect of the present invention comprises: (1) culturing iPS cells; and (2) collecting the culture supernatant obtained by culturing the iPS cells; A method for producing a composition for treating a neurodegenerative disease.
  • the use according to the third aspect of the present invention is the use of an iPS cell culture supernatant obtained by culturing iPS cells in the manufacture of a composition for treating a neurodegenerative disease.
  • the method according to the fourth aspect of the present invention is an effective amount of the composition for treating a neurodegenerative disease according to the first aspect, in a subject before the onset of the neurodegenerative disease, to suppress the onset of the neurodegenerative disease.
  • the composition for treating a neurodegenerative disease according to the first aspect is administered to a subject having a neurodegenerative disease in an amount effective for treating the neurodegenerative disease.
  • a method for treating a neurodegenerative disease is administered to a subject having a neurodegenerative disease in an amount effective for treating the neurodegenerative disease.
  • a new composition for treating neurodegenerative diseases using a culture supernatant of iPS cells a method for producing the same, a method for suppressing the onset of neurodegenerative diseases, and a method for treating neurodegenerative diseases.
  • the composition for treating a neurodegenerative disease according to the present disclosure includes an iPS cell culture supernatant obtained by culturing iPS cells.
  • iPS cells have pluripotency and self-renewal.
  • paying attention to substances secreted from iPS cells there has been no attempt to obtain a therapeutic effect on neurodegenerative diseases using the culture supernatant of iPS cells. On the contrary, nothing was known about what biological effect the culture supernatant of iPS cells had.
  • the inventor of the present invention has made an unprecedented attempt for the first time to use iPS cell culture supernatant to obtain a therapeutic effect on neurodegenerative diseases.
  • the iPS cell culture supernatant has a broad repair ability with respect to the central nervous tissue in general, and the repair ability is the same as that of somatic stem cells such as mesenchymal stem cells and hematopoietic stem cells.
  • the inventor has discovered that it is outstanding compared to the repair ability shown. This is because, compared with secretions secreted from somatic stem cells (including cytokines and the like), secretions secreted from iPS cells having pluripotency differ greatly in substance composition.
  • the ability to repair is widely shown to general central nervous tissues, and the degree of the repair ability is also high.
  • the content of iPS cells in the culture supernatant is higher than the content of somatic stem cells in the culture supernatant, or the specific growth factors and cytokines are iPS cells. It is considered to be contained in the cell culture supernatant but not substantially contained in the somatic stem cell culture supernatant.
  • the culture supernatant of iPS cells contains an anti-aging substance (for example, a specific type of protein) that is not included in the culture supernatant of somatic stem cells.
  • Kiyoshi is considered to have an excellent anti-aging effect not seen in the culture supernatant of somatic stem cells.
  • the present invention is not restricted by the estimation.
  • “repair” means that part or all of the tissue function lost due to the disease is maintained or restored compared to the function of the tissue before the disease occurs. In addition to recovering the function of the tissue, it also broadly includes regeneration as a functional tissue. The evaluation of whether the function is maintained or restored differs depending on the tissue in which the disease occurs, but may be performed based on an appearance or an assay usually used for evaluating the degree of the target function.
  • an iPS cell culture supernatant obtained by culturing iPS cells is used as an active ingredient of the composition for treating a neurodegenerative disease. Since the iPS cell culture supernatant contains a cytokine mixture and the like, it exhibits a repair ability in various central nervous tissues.
  • the endogenous stem cells can differentiate and proliferate due to the mixture of cytokines in the iPS cell culture supernatant acting as an induction signal for the endogenous stem cells of the central nervous tissue, which is capable of repairing. It can be estimated that the expression of However, the present invention is not restricted by the estimation.
  • the production method is also described in the following description.
  • iPS cells Artificial pluripotent stem cells
  • ES cells which are produced by introducing reprogramming factors into somatic cells, have almost the same characteristics as ES cells, such as differentiation pluripotency and proliferation ability by self-replication.
  • Cell-derived artificial stem cells K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007), Cell, 131: 861-872; J. Yu et al. (2007), Science, 318: 1917-1920; Nakagawa, M. et al., Nat. Biotechnol. 26: 101-106 (2008); International Publication WO 2007/069666).
  • the reprogramming factor can be introduced into somatic cells, for example, in the form of DNA or protein.
  • the reprogramming factor is a gene that is specifically expressed in ES cells, its gene product or noncoding RNA, a gene that plays an important role in maintaining undifferentiation of ES cells, its gene product or noncoding RNA, or a small molecule It may be constituted by a compound.
  • genes included in the reprogramming factor include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, as described in JP2013-247934A, for example.
  • iPS cells can be prepared by various iPS cell preparation methods reported so far. It is naturally assumed that an iPS cell production method developed in the future will be applied.
  • the most basic method for producing iPS cells is to introduce four factors, transcription factors Oct3 / 4, Sox2, Klf4 and c-Myc, into cells using viruses (Takahashi K, Yamanaka S : Cell 126 (4), 663-676, 2006; Takahashi, K, et al: Cell 131 (5), 861-72, 2007).
  • Human iPS cells have been reported to be established by introducing four factors, Oct4, Sox2, Lin28 and Nonog (Yu J, et al: Science 318 (5858), 1917-1920, 2007).
  • Three factors excluding c-Myc (Nakagawa M, et al: Nat. Biotechnol.
  • lentiviruses Yu J, et al: Science 318 (5858), 1917-1920, 2007
  • adenoviruses Stadtfeld M, et al: Science 322 (5903 ), 945-949, 2008
  • plasmid Okita K, et al: Science 322 (5903), 949-953, 2008; Okita K.
  • transposon vector Wang J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009
  • transposon vector Wang J, Hu K, Smuga-Otto K, Tian S, et al: Science 324, 797-801, 2009
  • the somatic cell into which the reprogramming factor is introduced in the preparation of the iPS cell may be any cell other than a germ cell derived from a mammal (eg, human, mouse, monkey, pig, rat, etc.), for example, As described in JP2013-247934A, keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface layer), exocrine gland epithelial cells (eg, mammary cells) , Hormone-secreting cells (eg, adrenal medullary cells), metabolism / storage cells (eg, hepatocytes), luminal epithelial cells that make up the interface (eg, type I alveolar cells), lumens of inner chain vessels Epithelial cells (eg, vascular endothelial cells), cilia cells with transport ability (eg, airway epithelial cells), extracellular matrix secreting cells (eg,
  • the degree of cell differentiation and the age of the animal from which the cells are collected can be used as a source of somatic cells in the present disclosure.
  • undifferentiated progenitor cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • bone marrow-derived mesenchymal stem cells are preferable from the viewpoint of stability.
  • the somatic cell is preferably derived from a human.
  • a culture solution for iPS cell induction for example, DMEM, DMEM / F12 or DME culture solution containing 10 to 15% FBS (in these culture solutions)
  • LIF penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.
  • a commercially available culture solution for example, a culture solution for mouse ES cell culture (TX- WES culture medium, Thrombo X), culture medium for primate ES cell culture (culture medium for primate ES / iPS cells, Reprocell), serum-free medium (mTeSR, Stemcell Technology)
  • TX- WES culture medium Thrombo X
  • primate ES cell culture culture medium for primate ES cell culture
  • Reprocell serum-free medium
  • serum-free medium mTeSR, Stemcell Technology
  • Examples of the culture method include somatic cells on DMEM or DMEM / F12 culture medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C., for example, as described in JP2013-247934A.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • the cells can be cultured in a bFGF-containing primate ES cell culture medium, and iPS-like colonies can be generated about 30 to about 45 days or more after the contact.
  • 10% FBS-containing DMEM culture medium including LIF, penicillin / streptomycin, etc.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • somatic cells to be initialized themselves are used (Takahashi K, et al. (2009), PLoS One. 4: e8067 or WO2010 / 137746), or an extracellular matrix (for example, Laminin ( WO2009 / 123349) and Matrigel (BD)) are exemplified.
  • iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y, et al. (2009), Cell Stem Cell. 5: 237 -241 or WO2010 / 013845).
  • hypoxic conditions oxygen concentration of 0.1% or more and 15% or less
  • the culture medium is exchanged with a fresh culture medium once a day from the second day onward.
  • the number of somatic cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
  • IPS cells can be selected according to the shape of the formed colonies as described in JP2013-247934A.
  • pluripotent stem cell markers such as Fbxo15, Nanog, Oct / 4, Fgf-4, Esg-1 and Cript can be selected as an index.
  • a drug resistance gene that is expressed in conjunction with a gene that is expressed when somatic cells are initialized for example, Oct3 / 4, Nanog
  • a culture solution containing the corresponding drug selection
  • the established iPS cells can be selected by culturing with the culture medium.
  • iPS cells are selected by observing with a fluorescence microscope, in the case of a luminescent enzyme gene, by adding a luminescent substrate, and in the case of a chromogenic enzyme gene, by adding a chromogenic substrate. can do. Selected cells are collected as iPS cells.
  • the culture medium and culture conditions for producing iPS cells include the culture medium and culture conditions described in the above description.
  • the use of feeder cells is not essential for maintaining iPS cells.
  • feeder cells can be omitted by using laminin 511 (A novel efficient feeder-free culture system for the derivation of human induced).
  • pluripotent stem cells Masato Nakagawa, et al. Scientific Reports 4, Article number: 3594doi: 10.1038 / srep03594 Received 09 October 2013 Accepted 06 December 2013 Published 08 January 2014 (Nature)).
  • the composition for treating a neurodegenerative disease according to the present disclosure may not contain serum. Safety may be improved by not containing serum.
  • a culture supernatant not containing serum can be prepared by culturing iPS cells in a serum-free medium (serum-free medium).
  • a serum-free culture supernatant can also be obtained by performing subculture once or a plurality of times and culturing the last or last several subcultures in a serum-free medium.
  • serum-free culture supernatant can also be obtained from the collected culture supernatant by removing the serum using dialysis or solvent replacement using a column.
  • a serum-free medium may be used throughout the entire process or for the last or several subcultures from the end.
  • Iscov modified Dulbecco medium IMDM
  • Ham F12 medium HamF12
  • SIGMA GIBCO
  • RPMI1640 medium RPMI1640 medium
  • Two or more basic media may be used in combination.
  • IMDM and HamF12 are mixed in equal amounts (for example, commercially available as trade name: IMDM / HamF12 (GIBCO)) can be mentioned.
  • ingredients that can be added to the medium include serum (fetal calf serum, human serum, sheep serum, etc.), serum substitutes (Knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various Vitamins and various minerals can be mentioned.
  • serum fetal calf serum, human serum, sheep serum, etc.
  • serum substitutes Kernockout serum replacement (KSR), etc.
  • bovine serum albumin BSA
  • antibiotics various Vitamins and various minerals can be mentioned.
  • the culture time for obtaining the culture supernatant is, for example, 5 hours to 7 days, and may be 1 day to 6 days.
  • the culture temperature is, for example, 36 ° C. to 38 ° C., for example 37 ° C., and the CO 2 concentration is 4 to 6%, for example 5%.
  • the culture may be performed, for example, by three-dimensional culture under non-adhesive conditions, for example, suspension culture (for example, dispersion culture, aggregated suspension culture, etc.).
  • the culture supernatant of iPS cells can be obtained by separating and removing cell components.
  • the culture supernatant is not only the supernatant itself from which the cell components are separated and removed from the culture solution, but also various treatments (for example, centrifugation, concentration, solvent replacement, dialysis, freezing, drying, freeze-drying, dilution) , Desalted, preserved, etc.) are also included in the range. Details of the culture supernatant treatment method will be described later.
  • the culture supernatant does not contain cell components. For this reason, the culture supernatant in this indication does not contain the iPS cell used for culture.
  • the composition for treating a neurodegenerative disease includes the culture supernatant of iPS cells obtained as described above as an active ingredient.
  • the composition for treating a neurodegenerative disease is a composition.
  • the iPS cells are not included as a whole.
  • the composition for treating a neurodegenerative disease does not contain cells (regardless of cell type) as a whole composition. That is, it is cell-free.
  • the composition for treating a neurodegenerative disease of this embodiment is clearly distinguished from various compositions containing iPS cells as a matter of course by iPS cells per se.
  • a typical example of this embodiment is a composition for treating a neurodegenerative disease that does not contain iPS cells and is composed only of the culture supernatant of iPS cells.
  • composition for treating a neurodegenerative disease may further include other components on the condition that the expected therapeutic effect is maintained depending on the state of the subject to be applied.
  • components that can be additionally included is as follows.
  • Bioabsorbable material As the organic bioabsorbable material, hyaluronic acid, collagen, fibrinogen (for example, Bolheel (registered trademark)) and the like can be used.
  • Gelling material As the gelling material, a material having high biocompatibility is preferably used, and hyaluronic acid, collagen, fibrin glue, or the like can be used. Various types of hyaluronic acid and collagen can be selected and used, but those suitable for the purpose of application (application tissue) of the composition for treating neurodegenerative diseases according to the present disclosure are preferably employed.
  • the collagen used is preferably soluble (acid-soluble collagen, alkali-soluble collagen, enzyme-soluble collagen, etc.).
  • compositions for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
  • excipient lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used.
  • disintegrant starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer.
  • emulsifier gum arabic, sodium alginate, tragacanth and the like can be used.
  • suspending agent glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used.
  • soothing agent benzyl alcohol, chlorobutanol, sorbitol and the like can be used.
  • stabilizer propylene glycol, ascorbic acid or the like can be used.
  • preservatives phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used.
  • preservatives benzalkonium chloride, paraoxybenzoic acid, chlorobutanol, and the like can be used.
  • Antibiotics, pH adjusting agents, growth factors for example, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)
  • growth factors for example, epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (
  • the final form of the composition for treating a neurodegenerative disease according to the present disclosure is not particularly limited. Examples of forms are liquid (liquid, gel, etc.) and solid (powder, fine granules, granules, etc.).
  • the composition for treating a neurodegenerative disease according to the present disclosure may have a form suitable for inhalation, and for example, may have a liquid form that can be sprayed in a nebulizer or a diffuser.
  • the composition for treating the neurodegenerative disease according to the present disclosure is in a form capable of nasal administration in consideration of the presence of the blood brain barrier (BBB). It is desirable. For example, nasal administration may be performed in the form of a spray or powder.
  • BBB blood brain barrier
  • the culture supernatant of iPS cells is more advantageous than the case of using iPS cells themselves in terms of prior preparation and storage, and can be said to be particularly suitable for the treatment of the acute stage or subacute stage of neurodegenerative diseases. Moreover, the usefulness of the culture supernatant of iPS cells is extremely high in that it does not contain cellular components and can overcome the problem of immune rejection.
  • the composition for treating a neurodegenerative disease according to the present disclosure is a composition using iPS cells as an active ingredient, paying attention to the usefulness of iPS cells themselves, in that the culture supernatant of iPS cells is included. Etc., as well as in terms of wording, are substantially distinguished.
  • the composition for treating a neurodegenerative disease according to the present disclosure may include iPS cells in addition to the culture supernatant of iPS cells.
  • iPS cells it is preferable to use iPS cells that have not been induced to differentiate after production (in other words, maintained in an undifferentiated state).
  • the therapeutic effect may be improved by additionally using iPS cells.
  • the composition for treating a neurodegenerative disease according to the present disclosure has an effect of repairing a tissue against a neurodegenerative disease by containing a culture supernatant of iPS cells. The degree of this effect far surpasses the effect obtained by using the culture supernatant of conventional somatic stem cells.
  • the composition for treating a neurodegenerative disease according to the present disclosure surprisingly has a wide and general repair effect on general nerve tissues of the central nervous system. Therefore, the composition for treating a neurodegenerative disease according to the present disclosure is not limited to a specific neurodegenerative disease, and can be used as a composition for treating a neurodegenerative disease for a wide range of neurodegenerative diseases.
  • the composition for treating a neurodegenerative disease of the present invention is before the onset of the neurodegenerative disease, even if the affliction to the neurodegenerative disease is unknown, the neurodegenerative disease within the subject can be administered by administration to the subject. Can be effectively suppressed. For this reason, it is possible to treat various neurodegenerative diseases before onset by habitually using the composition for treating neurodegenerative diseases according to the present disclosure.
  • a method for producing a composition for treating a neurodegenerative disease is also provided.
  • the collected culture supernatant is subjected to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting. May further be included.
  • the production method may further include a step of adding an additional component to the collected culture supernatant.
  • the production method may further include a step of creating the iPS cell by introducing an reprogramming factor into a somatic cell.
  • a step of creating the iPS cell by introducing an reprogramming factor into a somatic cell.
  • an additional component the matter described in description of the composition for neurodegenerative disease treatment concerning this indication is applied as it is. Subjecting the collected culture supernatant to one or more treatments selected from centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, and desalting; When the culture supernatant includes both steps of adding additional components, both steps may be performed first, and if possible, may be performed simultaneously.
  • the culture supernatant of iPS cells is collected.
  • the culture solution can be collected by suction with a dropper or pipette.
  • the collected culture supernatant is used as an active ingredient of the composition for treating neurodegenerative diseases according to the present disclosure as it is or after undergoing one or more treatments.
  • the treatment include centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (eg, 4 ° C., ⁇ 80 ° C.).
  • the culture supernatant of iPS cells exhibits the desired action without complicated and sophisticated purification.
  • the composition for treating a neurodegenerative disease according to the present disclosure can be produced by a simple process.
  • the fact that a complicated purification step is not required is also advantageous in that a decrease in activity associated with purification can be avoided.
  • the composition for treating a neurodegenerative disease according to the present disclosure may be formulated.
  • a method for concentrating the iPS cell culture supernatant for formulation a method usually used for concentrating the culture supernatant can be applied.
  • the concentration method include the following two methods. 1. Spin column concentration method The culture supernatant is concentrated using Amicon Ultra Centrifugal Filter Units-10K (Millipore) (maximum 75-fold concentration). The specific operation procedure is as follows.
  • the culture supernatant (up to 15 ml) is put into Amicon Ultra Centrifugal Filter Units-10K, centrifuged at 4000 g for about 60 minutes, and concentrated to 200 ⁇ l.
  • (ii) Put the same amount of sterile PBS as the culture supernatant into the above tube, and centrifuge again at ⁇ 4000g for about 60 minutes to replace the base solution with PBS.
  • (iii) Collect 200 ⁇ l of the obtained solution into a micro test tube to obtain a concentrated iPS cell culture supernatant.
  • Ethanol precipitation concentration method Concentrate the culture supernatant using the ethanol precipitation method (concentration up to 10 times). The specific operation procedure is as follows. (i) Add 45 ml of 100% ethanol to 5 ml of the culture supernatant, mix and leave at -20 ° C for 60 minutes. (ii) Centrifuge for 15 minutes at 4 ° C and 15000g. (iii) Remove the supernatant, add 10 ml of 90% ethanol and stir well. (iv) Centrifuge at 4 ° C and 15000g for 5 minutes. (v) The supernatant is removed, and the resulting pellet is dissolved in 500 ⁇ l of sterilized water, collected in a micro test tube, and used as a concentrated iPS cell culture supernatant.
  • the iPS cell culture supernatant in the composition for treating a neurodegenerative disease according to the present disclosure may be lyophilized. Thereby, good storage stability is obtained.
  • a lyophilization method of the iPS cell culture supernatant a method generally used for lyophilization of the culture supernatant can be applied. Examples of the lyophilization method include the following methods.
  • the iPS cell culture supernatant or concentrated iPS cell culture supernatant obtained by the above method is frozen at ⁇ 80 ° C. for 2 hours to half a day.
  • the obtained sample is used as a lyophilized iPS cell culture supernatant (can be stored at -80 ° C).
  • the subject comprising administering the composition for treating a neurodegenerative disease according to the present disclosure to the subject before the onset of the neurodegenerative disease in an amount effective for suppressing the onset of the neurodegenerative disease.
  • a method of suppressing the onset of the neurodegenerative disease before the onset of the neurodegenerative disease is also provided.
  • the subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ).
  • the subject may be a subject determined to have a risk of developing a neurodegenerative disease. Such a risk can be determined by genetic diagnosis, family analysis or the like. For example, in some cases, it has been statistically revealed that the presence of a specific allele in a specific gene correlates with the probability of suffering a specific disease.
  • neurodegenerative diseases become more difficult to treat as they progress. For this reason, early detection of such neurodegenerative diseases is important, but early detection is not always reliable. Considering this point, it is desirable to be able to start treatment in advance before the onset of neurodegenerative disease. However, since the drugs used for such treatment are continuously used, the burden on the administration subject is small. In addition, it is desirable that the neurodegenerative disease is generally effective in that the progression of the neurodegenerative disease that is not yet clear at that stage must be suppressed. Satisfying these requirements simultaneously has been difficult with conventional knowledge.
  • the composition for neurodegenerative disease treatment according to the present disclosure can be administered without performing invasive treatment such as transplantation or injection, there is no risk of tumor formation, and the composition for treating neurodegenerative disease is Widely effective in neurodegenerative diseases in general. From this, the said composition for neurodegenerative disease treatment has high effectiveness in the early treatment (progression suppression) of the neurodegenerative disease before onset as mentioned above. Such early treatment before onset is referred to as preemptive medicine in the present disclosure.
  • the dose of the composition for treating a neurodegenerative disease is, for example, 0.1 mg / kg / day to 1000 mg / kg / day in terms of the amount of untreated culture supernatant, and 1 mg / kg / day. It may be ⁇ 100 mg / kg / day.
  • the administration method is not particularly limited.
  • the administration of the composition for treating a neurodegenerative disease is preferably parenteral administration, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying.
  • Examples of the administration method of the composition for treating neurodegenerative diseases include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary administration) Absorption) and nasal administration.
  • nasal administration, transpulmonary administration and the like are preferable because they are minimally invasive.
  • nasal administration is particularly effective in suppressing the onset of neurodegenerative diseases related to the brain because there is no need to worry about the possibility of passage through the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • As the administration schedule for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc. of the subject (recipient) can be considered.
  • the administration method can be selected by those skilled in the art based on the type of tissue targeted for onset suppression, the type of neurodegenerative disease, and the like. For example, when the tissue is on the head, it is not necessary to consider passage through the blood-brain barrier, and since it is minimally invasive, it is particularly preferable to apply nasal administration or the like. For example, when the tissue is the brain, nasal administration can be preferably applied.
  • iPS cells can also be administered in addition to the neurodegenerative disease treatment composition according to the present disclosure.
  • the composition for treating a neurodegenerative disease and iPS cells can be administered to a subject simultaneously or at different timings. In such a case, it is preferable to use iPS cells that have not been differentiated after collection (in other words, maintained in an undifferentiated state).
  • a kit comprising a first component containing the neurodegenerative disease treatment composition and a second component containing iPS cells is used. Administration may be performed.
  • Each component may be a separate capsule, for example, or a separate ampoule or vial.
  • the neurodegenerative disease is not particularly limited. For example, amyotrophic lateral sclerosis (ALS), Parkinson's syndrome (Parkinson's disease, etc.), Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA) (substantia nigra) Somatic degeneration (SND), Shy-Drager syndrome (Shy-Drager syndrome), olive bridge cerebellar atrophy (OPCA), spinal cerebellar degeneration (SCD) (spinal cerebellar ataxia (SCA3, commonly known as Machado-Joseph disease) ), Friedreich's ataxia (Friedreich's ataxia, etc.), etc.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's syndrome Parkinson's syndrome
  • PPP progressive supranuclear palsy
  • MSA multiple system atrophy
  • SND substantially nigra
  • Somatic degeneration SND
  • Shy-Drager syndrome Shy-Drager
  • the neurodegenerative disease may be a motor neuron disease (MND).
  • MND motor neuron disease
  • the site where the disease occurs is not particularly limited. Examples of the site where the disease occurs include the brain and spinal cord, the brain includes the forebrain and brainstem, the forebrain includes the cerebrum and diencephalon, and the brainstem includes the midbrain and the lobe brain.
  • the cerebrum includes the olfactory brain, pelvis, striatum, hippocampus and cerebral neocortex, and the diencephalon includes the thalamus, thalamus, hypothalamus, thalamus abdomen, pituitary gland, pineal gland, and third ventricle There is.
  • the mesencephalon includes the midbrain, cerebral peduncle, pretectal region, and midbrain aqueduct, and the hindbrain includes the pons, cerebellum, and medulla.
  • the site where the disease occurs may be any of these sites.
  • the composition for treating neurodegenerative diseases according to the present disclosure contains various components including cytokines, and these components stimulate the ability of endogenous stem cells to suppress the onset of neurodegenerative diseases. Because it is possible.
  • a neurodegenerative disease treatment composition according to the present disclosure is administered to a subject having a neurodegenerative disease in an amount effective for treating the neurodegenerative disease,
  • a method of treating a degenerative disease is also provided.
  • the subjects include humans or non-human mammals (pet animals, domestic animals, laboratory animals. Specifically, for example, mice, rats, guinea pigs, hamsters, monkeys, cows, pigs, goats, sheep, dogs, cats, etc. ).
  • the dosage of the composition for treating neurodegenerative diseases is, for example, 0.1 mg / kg / day to 1000 mg / kg / day in terms of the amount of untreated culture supernatant. Yes, it may be 1 mg / kg / day to 100 mg / kg / day.
  • the administration method is not particularly limited.
  • the administration of the composition for treating a neurodegenerative disease is preferably parenteral administration, and the parenteral administration may be systemic administration or local administration. Examples of topical administration include injection into the target tissue, application or spraying.
  • Examples of the administration method of the composition for treating neurodegenerative diseases include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, transpulmonary administration (transpulmonary administration) Absorption) and nasal administration.
  • nasal administration, transpulmonary administration and the like are preferable because they are minimally invasive.
  • nasal administration is particularly effective in suppressing the onset of neurodegenerative diseases related to the brain because there is no need to worry about the possibility of passage through the blood-brain barrier (BBB).
  • BBB blood-brain barrier
  • As the administration schedule for example, once to several times a day, once every two days, or once every three days can be adopted. In preparing the administration schedule, the gender, age, weight, disease state, etc.
  • the range of “treatment” includes not only a treatment to cure a disease or tissue abnormality, but also stops the progression of the disease or tissue abnormality until it is not completely cured, or delays compared to a case in which no treatment is performed. Treatment is also included. Further, in the present disclosure, the start time of treatment may be after onset is observed, or may be performed before onset as described above.
  • the administration method can be selected by those skilled in the art based on the type of neurodegenerative disease tissue, the type of neurodegenerative disease, and the like. For example, when the tissue is on the head, it is not necessary to consider passage through the blood-brain barrier, and since it is minimally invasive, it is particularly preferable to apply nasal administration or the like. For example, when the tissue is the brain, nasal administration can be preferably applied.
  • iPS cells can also be administered in addition to the neurodegenerative disease treatment composition according to the present disclosure.
  • the composition for treating a neurodegenerative disease and iPS cells can be administered to a subject simultaneously or at different timings. In such a case, it is preferable to use iPS cells that have not been differentiated after collection (in other words, maintained in an undifferentiated state).
  • a kit comprising a first component containing the neurodegenerative disease treatment composition and a second component containing iPS cells is used. Administration may be performed.
  • Each component may be a separate capsule, for example, or a separate ampoule or vial.
  • the second component may be administered to the treatment target administered with the first component simultaneously with or after the administration of the first component.
  • the method of administering the first component and the second component simultaneously is particularly suitable for application to neurodegenerative diseases in the acute phase and subacute phase.
  • “simultaneous” does not require strict simultaneity. Therefore, when both elements are administered under the condition that there is no time difference, for example, both elements are mixed and then administered to the subject, both elements are immediately administered after one is administered. The case where the administration is performed under conditions without substantial time difference is also included in the concept of “simultaneous” herein.
  • the neurodegenerative disease to be treated by the method for treating a neurodegenerative disease is not particularly limited.
  • amyotrophic lateral sclerosis ALS
  • Parkinson's syndrome Parkinson's syndrome
  • Alzheimer's dementia progressive supranuclear palsy
  • PSP progressive supranuclear palsy
  • Huntington's disease multiple system atrophy (MSA) (substantia nigra) Somatic degeneration (SND), Shy-Drager syndrome (Shy-Drager syndrome), olive bridge cerebellar atrophy (OPCA), spinal cerebellar degeneration (SCD) (spinal cerebellar ataxia (SCA3, commonly known as Machado-Joseph disease) ), Friedreich ataxia (Friedreich ataxia, etc.) and the like.
  • ALS amyotrophic lateral sclerosis
  • Parkinson's syndrome Parkinson's syndrome
  • Alzheimer's dementia progressive supranuclear palsy
  • PSP progressive supranuclear palsy
  • MSA multiple system at
  • the site of the disease is not particularly limited. Examples of the site of the disease include the brain and spinal cord.
  • the brain includes the forebrain and brainstem, the forebrain includes the cerebrum and diencephalon, and the brainstem includes the midbrain and the lobe brain.
  • the cerebrum includes the olfactory brain, pelvis, striatum, hippocampus and cerebral neocortex, and the diencephalon includes the thalamus, thalamus, hypothalamus, thalamus abdomen, pituitary gland, pineal gland, and third ventricle There is.
  • the mesencephalon includes the midbrain, cerebral peduncle, pretectal region, and midbrain aqueduct, and the hindbrain includes the pons, cerebellum, and medulla.
  • the site of the disease may be any of these sites.
  • the treatment of a neurodegenerative disease may be achieved based on the ability of endogenous stem cells.
  • the composition for treating neurodegenerative diseases according to the present disclosure includes various components including cytokines, and these components stimulate the ability of endogenous stem cells to achieve treatment of neurodegenerative diseases. Because it is possible.
  • an iPS cell culture supernatant obtained by culturing iPS cells in the production of a composition for treating a neurodegenerative disease is also provided.
  • the iPS cell culture supernatant, the composition for treating a neurodegenerative disease, and a method for using the iPS cell culture supernatant See the description of the method).
  • Embodiments of the present invention include the following.
  • a composition for treating a neurodegenerative disease comprising an iPS cell culture supernatant obtained by culturing iPS cells.
  • the composition for treating a neurodegenerative disease according to ⁇ 1> which does not contain the iPS cell.
  • the iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting and storage.
  • composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 3> The composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 4>, wherein the iPS cell is derived from a bone marrow-derived mesenchymal stem cell.
  • composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 5> which does not contain serum.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinal cerebellum
  • ALS amyotrophic lateral sclerosis
  • PSP progressive supranuclear palsy
  • MSA multiple system atrophy
  • SCD degeneration
  • ⁇ 10> The composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 9>, which is administered by nasal administration.
  • ⁇ 11> (1) a step of culturing iPS cells; and (2) a step of collecting a culture supernatant obtained by culturing the iPS cells, A method for producing a composition for treating a neurodegenerative disease.
  • the method according to ⁇ 11> further comprising the step of introducing the reprogramming factor into the somatic cell to produce the iPS cell.
  • the collected culture supernatant is subjected to at least one treatment selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage.
  • ⁇ 14> The method according to any one of ⁇ 11> to ⁇ 13>, further comprising a step of adding an additional component to the collected culture supernatant.
  • ⁇ 15> Use of an iPS cell culture supernatant obtained by culturing iPS cells in the production of a composition for treating a neurodegenerative disease.
  • ⁇ 16> The use according to ⁇ 15>, wherein the composition for treating a neurodegenerative disease does not contain the iPS cell.
  • ⁇ 17> The use according to ⁇ 15> or ⁇ 16>, wherein the composition for treating a neurodegenerative disease is cell-free.
  • ⁇ 18> The iPS cell culture supernatant is treated with one or more selected from the group consisting of centrifugation, concentration, solvent replacement, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage.
  • ⁇ 19> The use according to any one of ⁇ 15> to ⁇ 18>, wherein the iPS cell is derived from a bone marrow-derived mesenchymal stem cell.
  • ⁇ 20> The use according to any one of ⁇ 15> to ⁇ 19>, wherein the composition for treating a neurodegenerative disease does not contain serum.
  • ⁇ 21> The composition for treating neurodegenerative disease according to any one of ⁇ 15> to ⁇ 20>, wherein the composition for treating a neurodegenerative disease is used to suppress the onset of the neurodegenerative disease before the onset of the neurodegenerative disease in a subject. use.
  • ⁇ 22> The use according to ⁇ 21>, wherein the subject is a subject determined to have a risk of developing the neurodegenerative disease.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinal cerebellum
  • ALS amyotrophic lateral sclerosis
  • PSP progressive supranuclear palsy
  • MSA multiple system atrophy
  • spinal cerebellum The use according to any one of ⁇ 15> to ⁇ 22>, which is selected from the group consisting of degeneration (SCD) and Friedreich ataxia.
  • SCD degeneration
  • ⁇ 24> The use according to any one of ⁇ 15> to ⁇ 23>, wherein the composition for treating a neurodegenerative disease is administered by nasal administration.
  • composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 6> is applied to a subject before the onset of the neurodegenerative disease in an amount effective for suppressing the onset of the neurodegenerative disease.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinal cerebellum ⁇ 25> or ⁇ 26>, wherein the method is selected from the group consisting of degeneration (SCD) and Friedreich ataxia.
  • SCD degeneration
  • ⁇ 28> The method according to any one of ⁇ 25> to ⁇ 27>, wherein the onset suppression of the neurodegenerative disease is achieved based on the ability of endogenous stem cells.
  • ⁇ 29> The composition for treating a neurodegenerative disease according to any one of ⁇ 1> to ⁇ 6> is administered to a subject having a neurodegenerative disease in an amount effective for treating the neurodegenerative disease.
  • the neurodegenerative disease is amyotrophic lateral sclerosis (ALS), Parkinson's syndrome, Alzheimer's dementia, progressive supranuclear palsy (PSP), Huntington's disease, multiple system atrophy (MSA), spinal cerebellum ⁇ 29>
  • the method according to ⁇ 29> selected from the group consisting of degeneration (SCD) and Friedreich ataxia.
  • ⁇ 31> The method according to ⁇ 29> or ⁇ 30>, wherein the treatment of the neurodegenerative disease is achieved based on the ability of endogenous stem cells.
  • ⁇ 32> The method according to any one of ⁇ 25> to ⁇ 31>, wherein the composition for treating a neurodegenerative disease is administered by nasal administration.
  • Example 1 Verification of ALS progression inhibitory effect of iPS cell culture supernatant using ALS model mouse APS model culture mouse according to the present disclosure was administered using a mutant SOD1 (G93A) -introduced mouse which is an ALS model mouse. The difference in the progression of ALS between the cases with and without administration was examined.
  • a culture supernatant of human iPS cells was obtained as follows. In the following, culture was performed at 37 ° C. (IPS cells) Except for using human bone marrow-derived mesenchymal cells as a source of iPS cells, Takahashi K, et al. "Induction of Pluripotent Stem Cells from Adult Human Fibroblasts by Defined Factors" Cell.
  • iPS cells prepared according to the method described in the above were obtained from Kyoto University. Specifically, a retrovirus (pMX vector) containing human Oct3 / 4, Sox2, Klf4 and c-Myc was introduced into human bone marrow-derived mesenchymal cells, 6 days after the introduction, the cells were trypsinized, and mitomycin C Were plated on SNL feeder cells treated with (McMahon and Bradley, 1990). On the next day, the medium was changed from 10% FBS-containing DMEM to a primate ES cell culture medium (primate ES cell culture, manufactured by Reprocell) supplemented with 4 ng / ml bFGF.
  • iPS cell medium a human basic FGF (bFGF) (serum-free; manufactured by Reprocell Corporation; hereinafter, this bFGF-added primate ES cell medium is referred to as iPS cell medium. Used).
  • bFGF human basic FGF
  • the human iPS cells maintained as described above were detached from the adhesion culture petri dish using an ES cell dissociation solution (manufactured by Reprocell Co., Ltd.), and suspended in the iPS cell culture medium placed in the non-adhesion culture petri dish for 1 week. .
  • an embryoid body (EB) was formed.
  • the formed embryoid bodies (EBs) were seeded on adherent culture dishes and grown for 1 week in DMEM containing 10% FBS and 1% anti-anti (registered trademark, antifungal agent) ( For cell outgrowth, see Stem Cells Dev. 22, 102-113, 2013).
  • human iPS cells were detached from the adhesion culture dish using a 0.05% trypsin-EDTA solution, and seeded in a new adhesion culture dish. As a result, human iPS cells became single cells.
  • DMEM containing 10% FBS and 1% anti-anti (registered trademark, antifungal agent) as a medium the cells were cultured for one week. After confirming that human iPS cells became 70-80% or more confluent, the medium was replaced with serum-free medium (DMEM containing 1% anti-anti (registered trademark, antifungal agent) without FBS). After culturing for one day (48 hours), the supernatant was collected.
  • the collected supernatant was centrifuged at 1500 rpm for 5 minutes, and the supernatant was collected again and then centrifuged at 3000 rpm for 3 minutes. The supernatant was collected again and used as a human iPS cell culture supernatant in subsequent experiments.
  • the centrifugation was performed using a tabletop multi-centrifuge LC-120 manufactured by Tommy Seiko.
  • FIG. 2 shows the survival time in each of the experimental group and the control group. While the average survival time of the control group was 150.5 days, the average survival time was significantly increased to 170.4 days in the experimental group in which the iPS cell culture supernatant was administered nasally. This indicates that the progression of ALS was remarkably suppressed by administration of the iPS cell culture supernatant.
  • the inflammation markers IFN- ⁇ , IL-17 and TNF- ⁇ , the M1 macrophage marker iNOS and the M2 macrophage marker arginase 1, and the inflammatory cytokine in T cells The expression of BrdU, IL-2, IFN- ⁇ and IL-17 was measured. The results are shown in FIG. As can be seen from FIG. 4, in the control group, the expression of the inflammatory marker was high and the amount of M1 macrophages was high, while the amount of the M2 macrophage was low, whereas in the experimental group in which the iPS cell culture supernatant was administered nasally, the expression of the inflammatory marker was high. It was suppressed, and there were few M1 macrophages and conversely many M2 macrophages. This shows that inflammation in the spinal cord is remarkably suppressed by administration of the iPS cell culture supernatant.
  • the above gastrocnemius muscle (180 days after birth) was further subjected to histological observation by HE staining. The results are shown in FIG. As can be seen from FIG. 6, extensive destruction of muscle fibers was observed in the control group, whereas in the experimental group in which the iPS cell culture supernatant was administered nasally, the same structure as that of wild-type muscle fibers was maintained. It was. In FIG. 4 and FIG. 5, iPS-CM represents an experimental group to which the iPS cell culture supernatant was administered.
  • Human iPS cells prepared using plasmid Human iPS cells prepared using plasmid
  • the p53 shRNA expression cassette driven by the mouse U6 promoter was inserted into the BamHI site of plasmid pCXLE-hOCT3 / 4-shp53 (pCXLE-hOCT3 / 4 with OCT3 / 4 (Addgene accession code 27076)) ), PCXLE-hSK (with Addgene accession code 27078, SOX2 and KLF4) and pCXLE-hUL (with Addgene accession code 27080, L-MYC and LIN28), and a microporator (Invitrogen) ) was introduced into 6 ⁇ 10 5 human bone marrow-derived mesenchymal cells by electroporation.
  • the introduction conditions were 1800 V, 20 ms, and one pulse.
  • Cells were trypsinized 7 days after transfection and 1 ⁇ 10 5 cells were re-plated into 100 mm dishes covered with SNL or MEF feeder layers.
  • the medium was replaced with the medium for iPS cells.
  • Colonies were counted 26-32 days after plating. Colonies similar to human ES cells were selected.
  • the culture supernatant was prepared in the same manner as described above.
  • the effect when this culture supernatant was administered to a mutant SOD1 (G93A) -introduced mouse was examined in the same manner as described above.
  • the iPS cell culture supernatant had excellent properties for suppressing neurodegenerative diseases.
  • the neurodegenerative disease treatment composition according to the present disclosure is used due to the inhibitory effect of the iPS cell culture supernatant according to the present disclosure, the preemptive medical treatment or treatment (progression suppression of the neurodegenerative disease according to the present disclosure). Can be achieved).

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Abstract

Une composition pour le traitement d'une maladie neurodégénérative selon la présente invention comprend un surnageant de culture de cellules iPS qui est obtenu par la culture de cellules iPS. La présente invention concerne en outre : un procédé de fabrication de ladite composition pour le traitement d'une maladie neurodégénérative; un procédé d'inhibition de l'apparition d'une maladie neurodégénérative; et un procédé de traitement d'une maladie neurodégénérative.
PCT/JP2016/050729 2015-01-09 2016-01-12 COMPOSITION POUR LE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE COMPRENANT UN SURNAGEANT DE CULTURE DE CELLULES iPS ET SON PROCÉDÉ DE FABRICATION, PROCÉDÉ D'INHIBITION DE L'APPARITION D'UNE MALADIE NEURODÉGÉNÉRATIVE ET PROCÉDÉ DE TRAITEMENT D'UNE MALADIE NEURODÉGÉNÉRATIVE WO2016111378A1 (fr)

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JP2015003494A JP2016128397A (ja) 2015-01-09 2015-01-09 ｉＰＳ細胞培養上清を含む神経変性疾患治療用組成物およびその製造方法、神経変性疾患発症抑制方法ならびに神経変性疾患治療方法
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WO2011118795A1 (fr) * 2010-03-26 2011-09-29 国立大学法人名古屋大学 Composition pour le traitement d'une partie lésée

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011118795A1 (fr) * 2010-03-26 2011-09-29 国立大学法人名古屋大学 Composition pour le traitement d'une partie lésée

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YOO, J. ET AL.: "Stem cells as promising therapeutic options for neurological disorders", J. CELL BIOCHEM., vol. 114, no. 4, 2013, pages 743 - 753 *

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