WO2014058080A1 - Procédé de promotion de la reprogrammation d'une cellule somatique, et trousse de préparation cellulaire - Google Patents

Procédé de promotion de la reprogrammation d'une cellule somatique, et trousse de préparation cellulaire Download PDF

Info

Publication number
WO2014058080A1
WO2014058080A1 PCT/JP2013/082246 JP2013082246W WO2014058080A1 WO 2014058080 A1 WO2014058080 A1 WO 2014058080A1 JP 2013082246 W JP2013082246 W JP 2013082246W WO 2014058080 A1 WO2014058080 A1 WO 2014058080A1
Authority
WO
WIPO (PCT)
Prior art keywords
inhibitor
cells
kpa
cell
substrate
Prior art date
Application number
PCT/JP2013/082246
Other languages
English (en)
Japanese (ja)
Inventor
藤田　英明
朋信 渡邊
清香 樋口
Original Assignee
独立行政法人理化学研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 独立行政法人理化学研究所 filed Critical 独立行政法人理化学研究所
Priority to JP2014540925A priority Critical patent/JPWO2014058080A1/ja
Publication of WO2014058080A1 publication Critical patent/WO2014058080A1/fr

Links

Images

Classifications

    • 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
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • 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
    • C12N2506/1307—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from adult fibroblasts
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/30—Synthetic polymers

Definitions

  • the present invention relates to a method for enhancing reprogramming of somatic cells and a kit for producing reprogrammed cells.
  • iPS cells induced pluriopotent stem cells
  • IPS cells are prepared by allowing a predetermined nuclear reprogramming factor to act on somatic cells. Therefore, iPS cells have fewer ethical problems than embryonic stem cells (ES cells) established from early embryos.
  • ES cells embryonic stem cells
  • Various methods have been reported so far as methods for producing iPS cells. For example, as a representative method, introduction of four transcription factor groups can be mentioned (Patent Document 1). However, in the approach of Patent Document 1, undesired phenomena such as inducing cell canceration have been reported depending on the type of gene to be artificially expressed.
  • an iPS cell or a tissue prepared from the iPS cell is transplanted into a human body for treatment or the like, an artificially expressed gene and a vector in which the gene is incorporated may affect the human body. I cannot deny sex.
  • Non-Patent Document 1 describes the use of a combination of one transcription factor that is artificially expressed and a predetermined compound for the purpose of reprogramming human-derived somatic cells.
  • the present invention has been made to solve the above-described problems, and an object of the present invention is to provide a method for enhancing cell reprogramming without requiring gene introduction, and use thereof.
  • the present invention includes any of the following contents.
  • a method for enhancing reprogramming of somatic cells comprising an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and a 3′-phosphoinositide dependent protein kinase-1
  • somatic cells in contact with an abiotic substrate having an elastic modulus in the range of 0.1 kPa to 200 kPa
  • a method comprising a culture step of culturing.
  • An abiotic substrate having an elastic modulus of 0.1 kPa to 200 kPa, an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and A kit for preparing a reprogrammed cell comprising at least one factor selected from the group of factors consisting of an activator of 3′-phosphoinositide-dependent protein kinase-1.
  • a method for producing a cell with enhanced reprogramming comprising an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and a 3′-phosphoinositide-dependent protein kinase—
  • somatic cells in contact with an abiotic substrate having an elastic modulus in the range of 0.1 kPa to 200 kPa
  • a method comprising culturing a cell.
  • A a substrate for cell culture, (b) an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and 3′- A cell culture medium comprising at least one factor selected from a group of factors consisting of an activator of phosphoinositide-dependent protein kinase-1, and (c) an elastic modulus of 0.1 kPa or more immersed in the cell culture medium And a non-biological substrate that is within a range of 200 kPa or less.
  • the present invention has an advantage that the reprogramming of somatic cells can be promoted with high efficiency and speed, while having the advantage that introduction of foreign genes can be completely removed as compared with the prior art.
  • Example of this invention it is a figure which shows the state by which the iPS-like colony was induced
  • the method according to the present invention is a method for enhancing reprogramming of somatic cells, At least selected from the group consisting of an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and an activator of 3′-phosphoinositide-dependent protein kinase-1.
  • It comprises a culturing step of culturing somatic cells in contact with an abiotic substrate having an elastic modulus of 0.1 kPa or more and 200 kPa or less. Note that this method can also be regarded as a method of producing cells with enhanced reprogramming using somatic cells as starting cells.
  • enhanced reprogramming of somatic cells refers to a state in which the expression level of a gene serving as an undifferentiated marker is enhanced compared to the starting cell (somatic cell). That is, it can be considered that the starting cell is brought into a different stage of differentiation (including an undifferentiated state).
  • the cell morphology is determined using microscopic observation or a cell sorter, a change in cell morphology is observed.
  • genes that are undifferentiation markers include, but are not limited to, Nanog gene, Oct3 / 4 gene, Tbx3 gene, Sox2 gene, SSEA3 gene, Klf4 gene, Rex1 gene, and Stella gene.
  • the undifferentiated marker is arbitrarily selected by those skilled in the art, but is preferably both the Nanog gene and the Oct3 / 4 gene.
  • the Oct3 / 4 gene is preferably in a positive state in which expression is particularly increased.
  • change in cell morphology broadly refers to changes that occur in the morphology of somatic cells as starting cells as somatic cell reprogramming increases.
  • somatic cell that is the starting cell is an adherent cell
  • the target somatic cell is brought into a state showing multipotency or an earlier state, more preferably totipotency ( pluripotency) or an earlier state.
  • multipotency refers to the ability to differentiate into some cell types such as the nervous system or hematopoietic system.
  • totipotency refers to the ability to differentiate into all cells and tissues that constitute an individual, although the individual itself cannot be constituted.
  • the “cell with enhanced reprogramming” obtained by the method of the present invention is a cell in which the expression of the undifferentiated marker is positive, or a cell in which the expression level of the undifferentiated marker is enhanced compared to the starting cell. is there.
  • the “cell with enhanced reprogramming” is preferably a nuclear reprogramming cell. Comparison of presence or absence of teratoma formation ability, presence or absence of ES cell-like colony formation, or positive target (ES cell or iPS cell (induced pluripotent stem cell) prepared by conventional technology)
  • the expression level of the undifferentiated marker can be used as an index, but is not limited thereto.
  • the differentiation ability and undifferentiation proliferation ability of the separated nuclear reprogramming cells can be easily confirmed by using confirmation means widely used for ES cells.
  • An “iPS cell” is a cell having properties close to those of an ES cell (Embryonic® Stem® Cell). More specifically, it is an undifferentiated cell, which has pluripotency and undifferentiated proliferation depending on culture conditions. Including cells having the ability.
  • cells with enhanced reprogramming obtained by the method of the present invention is not particularly limited.
  • the use of tests and research similar to ES cells the use of drug screening (somatic cells of the liver system), It can be used for cell therapy / tissue therapy (autologous cell / autologous tissue transplantation, allogeneic cell / transgenic tissue transplantation) and the like.
  • the base material for culturing somatic cells is abiotic and has an elastic modulus in the range of 0.1 kPa to 200 kPa. More specifically, the substrate is abiotic means that the substrate is not a cell or a cell construct.
  • the cell construct refers to a construct formed as an aggregate of cells such as a cell sheet and a tissue.
  • Examples of the abiotic base material having an elastic modulus of 0.1 kPa or more and 200 kPa or less include hydrous gel (hydrogel), silicone, gelatin, and collagen gel. Is preferred.
  • Water-containing gels are: acrylamide polymer; acrylic acid polymer; methacrylic acid polymer; alginic acid gel such as calcium alginate gel and magnesium alginate gel; kappa type or iota type carrageenan gel; native type or deacyl type gellan gum gel
  • An organic polymer that constitutes the hydrogel such as containing a predetermined amount of an aqueous liquid.
  • acrylamide polymer hydrogels are preferred.
  • R 1 , R 2 , and R 3 are structures outside the basic skeleton and are not particularly limited.
  • a hydrocarbyl group such as a hydrogen atom, a halogen atom, a hydroxyl group, an amino group, an alkyl group, and an alkoxy group Group.
  • the number of carbon atoms in the hydrocarbyl group and alkoxy group is preferably in the range of 1 to 10, and more preferably in the range of 1 to 5.
  • Examples of monomers having a basic skeleton of acrylamide include acrylamide (AAm), N, N′-methylenebisacrylamide, N-isopropylacrylamide, hydroxyethylacrylamide, N, N-dimethylacrylamide, N, N-diethylacrylamide, and dimethyl. Examples thereof include aminopropylacrylamide and dimethylaminopropylacrylamide quaternized products or salts thereof.
  • the abiotic substrate is a hydrogel as a monomer homopolymer or copolymer
  • a crosslinking agent that crosslinks the polymers, and polymerization initiation An agent or the like may be included.
  • the elastic modulus of the abiotic substrate may be in the range of 0.1 kPa or more and 200 kPa or less, but from the viewpoint of reprogramming efficiency, it is preferably in the range of 0.1 kPa or more and 100 kPa or less, More preferably, it is in the range of 0.1 kPa to 10 kPa, more preferably in the range of 0.1 kPa to 4 kPa, 0.1 kPa to 3 kPa to 3.5 kPa, or More preferably, it is in the range of 0.1 kPa or more and less than 3 kPa, particularly preferably in the range of 0.1 kPa or more and 1 kPa to 1.5 kPa, or more preferably 0.2 kPa or more and 1 kPa or less.
  • the elastic modulus of the substrate is less than 0.1 kPa, or exceeds 200 kPa, the efficiency of somatic cell reprogramming is significantly reduced, or reprogramming does not proceed at all.
  • a base material is a water-containing gel, it is easy to obtain the base material of a desired elasticity modulus by adjusting the water content or the crosslinking degree between polymers.
  • the elastic modulus of the abiotic substrate may be measured by a method according to the type of the substrate.
  • measurement may be performed by, for example, a method of calculating from the sinking amount of the weight or a method of stress measurement using an AFM (atomic force microscope).
  • P is the weight load (unit N)
  • a is the radius (unit m) of the bottom surface of the weight (contact surface with the hydrous gel)
  • ⁇ h is the sinking amount of the weight (unit m)
  • ⁇ is the sample. This is the Poisson's ratio of the water-containing gel. In the case of the water-containing gel used in Example 1 described later, ⁇ is 0.45.
  • the hydrogel In the case of stress measurement by AFM, after placing the hydrogel in water or an aqueous solution and sufficiently swelling (substantial saturated swelling state), the hydrogel is pushed by an AFM cantilever whose elastic modulus is known, Measure the deflection of the AFM cantilever.
  • the elastic modulus E (unit Pa) of the water-containing gel is determined from the value of the stress F (unit m) determined from the deflection of the AFM cantilever and the distance ⁇ (unit m) by which the water-containing gel is pushed in, using the following equation.
  • ⁇ is the angle of the tip of the AFM cantilever (opening angle: unit degree)
  • ⁇ is the Poisson's ratio of the sample (0.45 in the example).
  • ⁇ is 1 ⁇ m or less when measuring the elastic modulus.
  • the thickness of the abiotic substrate is not particularly limited, but is preferably in the range of 10 ⁇ m or more and 5 mm or less, and preferably in the range of 200 ⁇ m or less from the viewpoint of fluorescence observation.
  • the abiotic substrate may be fixed in the form of a film on the surface of the cell culture substrate, for example.
  • the substrate for cell culture include a dish for cell culture, a well plate for cell culture, and the like, and a configuration in which the base material is fixed to the bottom of the dish or the bottom of the well of the well plate is exemplified. .
  • the surface of the abiotic substrate may be provided with a coating that serves as a scaffold for somatic cells, such as a gelatin coat, a collagen coat, and a laminin coat.
  • a crosslinker that crosslinks the non-biological substrate and the coating material may be provided.
  • the type of the crosslinker may be appropriately selected according to the material of the base material. However, when the base material is an acrylamide polymer, crosslinkers such as Sulfo-SANPAH (Pierce), Sulfo-SMCC, and GMBS are listed. It is done.
  • histone deacetylase inhibitor is not particularly limited, and specific examples include trichostatin A, butyric acid, apicidin, valproic acid, and NaB. Of these, trichostatin A may be preferred.
  • the said inhibitor may use only 1 type and may be used in combination of 2 or more type.
  • the amount of the inhibitor used is not particularly limited as long as the effect of enhancing reprogramming of somatic cells is obtained, but the concentration in the cell culture medium in the culturing step is, for example, in the range of 0.1 nM to 10 mM, and 1 nM Above, it is preferably in the range of 1 mM or less, more preferably in the range of 2 nM or more and 0.5 mM or less.
  • NaB is used as the inhibitor, it is, for example, within a range of 10 ⁇ M or more and 10 mM or less, preferably within a range of 0.1 mM or more and 1 mM or less, and within a range of 0.2 mM or more and 0.5 mM or less.
  • trichostatin A is used as the inhibitor, it is, for example, in the range of 1 nM to 100 nM, preferably in the range of 2 nM to 50 nM, preferably in the range of 5 nM to 30 nM. More preferred.
  • TGF- ⁇ receptor inhibitor is not particularly limited. Specifically, for example, A-83-01 (3- (6-Methylpyridin-2-yl) -1-phenylthiocarbamoyl-4-quinolin-4- ylpyrazole), SB431542 (manufactured by Stemgent), and LY2157299 (manufactured by Wako Pure Chemical Industries, Ltd.). Of these, A-83-01 may be preferred.
  • the inhibitor may preferably be a type I inhibitor. The said inhibitor may use only 1 type and may be used in combination of 2 or more type.
  • the amount of the inhibitor used is not particularly limited as long as the effect of enhancing somatic cell reprogramming is obtained, but the concentration in the cell culture medium in the culturing step is, for example, in the range of 0.1 nM to 1 mM, and 1 nM Above, it is preferably within the range of 100 ⁇ M or less, and more preferably within the range of 10 nM or more and 10 ⁇ M or less.
  • A-83-01 is used as the inhibitor, it is preferably in the range of 10 nM to 1 mM, preferably in the range of 50 nM to 200 ⁇ M, and preferably in the range of 100 nM to 100 ⁇ M. Is more preferable.
  • SB431542 When SB431542 is used as the inhibitor, for example, it is in the range of 100 nM to 100 ⁇ M, preferably in the range of 200 nM to 50 ⁇ M, more preferably in the range of 500 nM to 20 ⁇ M. .
  • LY2157299 When LY2157299 is used as the inhibitor, it is preferably in the range of 1 nM to 10 ⁇ M, preferably in the range of 10 nM to 1 ⁇ M, and more preferably in the range of 50 nM to 200 nM. .
  • the type of the MAPK / ERK pathway inhibitor is not particularly limited. Specifically, for example, PD0325901 (N-[(2R) -2,3-dihydroxypropoxy] -3,4-difluoro-2-[(2-fluoro -4-iodophenyl) amino] -benzamide), U0126 (Promega), 5-Iodotubercidin (Merck) and the like. Among these, PD0325901 may be preferable.
  • the said inhibitor may use only 1 type and may be used in combination of 2 or more type.
  • the amount of the inhibitor used is not particularly limited as long as the effect of enhancing somatic cell reprogramming is obtained, but the concentration in the cell culture medium in the culturing step is, for example, in the range of 0.1 nM to 1 mM, and 1 nM Above, it is preferably within the range of 100 ⁇ M or less, and more preferably within the range of 10 nM or more and 10 ⁇ M or less.
  • PD0325901 is used as the inhibitor, it is, for example, in the range of 0.01 ⁇ M or more and 1 mM or less, preferably in the range of 0.05 ⁇ M or more and 100 ⁇ M or less, and in the range of 0.1 ⁇ M or more and 10 ⁇ M or less.
  • U0126 is used as the inhibitor, it is, for example, in the range of 1 nM or more and 10 ⁇ M or less, preferably in the range of 10 nM or more and 1 ⁇ M or less, and more preferably in the range of 50 nM or more and 200 or less. .
  • the type of activator of 3′-phosphoinositide-dependent protein kinase-1 is not particularly limited. Specifically, for example, PS48 ((2Z) -5- (4-Chlorophenyl) -3-phenyl -2-pentenoic acid).
  • the activator may preferably be one that specifically binds to the HM / PIF pocket of PDK-1 and activates PDK-1.
  • the activator may be used alone or in combination of two or more.
  • the amount of the activator used is not particularly limited as long as the effect of enhancing somatic cell reprogramming is obtained, but the concentration in the cell culture medium in the culturing step is, for example, in the range of 0.01 ⁇ M or more and 1 mM or less, The range is preferably 0.1 ⁇ M or more and 100 ⁇ M or less, more preferably 1 ⁇ M or more and 10 ⁇ M or less.
  • Combination of inhibitors and activators One from at least two of an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and an activator of 3′-phosphoinositide-dependent protein kinase-1. It is preferable to select and use two or more types of factors in combination, more preferably to select and use one or more types of factors from at least three, and more preferably to select one or more types of factors from four. It may be more preferable to use in combination. When these are used in combination, an example of a preferable combination is a combination of NaB, A-83-01, PD0325901, and PS48. Another example of a preferred combination is a combination of trichostatin A, A-83-01, PD0325901, and PS48. Yet another example of a preferred combination is a combination of NaB, PD0325901, and PS48.
  • it preferably contains at least an inhibitor of TGF- ⁇ receptor, more preferably contains at least an inhibitor of MAPK / ERK pathway, and 3′-phosphoinositide-dependent protein kinase— More preferably, it contains at least one activator, and particularly preferably contains at least an inhibitor of histone deacetylase. In another embodiment, it preferably comprises an inhibitor of histone deacetylase, more preferably further comprises an activator of 3′-phosphoinositide-dependent protein kinase-1, and the MAPK / ERK pathway It is further preferable to further include an inhibitor of the above, and it is particularly preferable to further include an inhibitor of TGF- ⁇ receptor. In still another embodiment, it is preferable that at least trichostatin A is included.
  • the following condition 1) When one or more factors are selected from one, two, or three of the above, it is most preferable to satisfy at least the following condition 1), and at least satisfy the following 2) condition: Preferably, it may be further preferable to satisfy at least the following condition 3). That is, an embodiment in which the following conditions 1) and 2) are simultaneously satisfied, or an aspect in which the following conditions 1) to 3) are simultaneously satisfied may be particularly preferable embodiments.
  • One or more factors are selected from histone deacetylase inhibitors. 2) Select one or more factors from the activator of 3′-phosphoinositide-dependent protein kinase-1. 3) Select one or more factors from inhibitors of the MAPK / ERK pathway.
  • the “somatic cell” to be reprogrammed that is, the type and origin of the starting cell is not particularly limited, and any somatic cell can be used.
  • the type of somatic cell may be any cell other than germ cells, for example, keratinized epithelial cells (eg, keratinized epidermal cells), mucosal epithelial cells (eg, epithelial cells of the tongue surface layer), exocrine secretion Glandular epithelial cells (eg, mammary cells), hormone-secreting cells (eg, adrenal medullary cells), metabolism / storage cells (eg, hepatocytes), luminal epithelial cells that constitute the interface (eg, type I alveoli) Cells), lumenal epithelial cells (eg, vascular endothelial cells), cilia cells with carrying ability (eg, airway epithelial cells), cells for extracellular matrix secretion (eg, fibroblasts), contraction Sex
  • somatic stem cells include tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells.
  • fibroblasts In a preferred embodiment, fibroblasts, synovial cells, keratinocytes, amniotic cells, endometrial cells, glial cells, astrocytes, meningeal cells, bone marrow derived myeloids that have been reported to establish induced pluripotent stem cells Cells, peripheral blood-derived CD34 positive blood cells, bone marrow mononuclear cells, T cells, natural killer T cells, natural killer cell lymphoma (NK cell lymphoma), B lymphocytes, umbilical vein endothelial cells, pancreatic ⁇ cells, testis-derived cells, Examples include natural killer tooth embryo-derived cells, mesentery-derived cells, neural stem cells, hematopoietic stem cells, stem cells, and adipose stem cells. Somatic cells may be primary culture cells or subculture cells. The origin of somatic cells may be derived from a mature individual in addition to those derived from a fetus (fetal) individual, for example.
  • the animal species from which the somatic cell is derived is not particularly limited, but a somatic cell derived from a mammal (mammal) is preferable in consideration of industrial use such as cell therapy / tissue therapy.
  • a somatic cell derived from a mammal is preferable in consideration of industrial use such as cell therapy / tissue therapy.
  • the kind of mammal is not specifically limited, Experimental animals such as primates excluding mice, rats, rabbits, guinea pigs and humans; Pet animals such as dogs and cats; Domestic animals such as cows and horses; . Particularly in clinical application, human-derived tissue or cells are preferred.
  • somatic cells differentiated from cells whose reprogramming has been enhanced by the present invention or somatic cells differentiated from iPS cells established by a conventional technique, “somatic cells” that are targets of enhanced reprogramming by this method.
  • somatic cells that are targets of enhanced reprogramming by this method.
  • Can be treated as A preferred embodiment of the somatic cell is the same as described above.
  • somatic cells obtained by enhancing reprogramming of somatic cells preferably nuclear reprogramming cells
  • somatic cells isolated from the patient it may be desirable to use somatic cells isolated from the patient in some aspects, For example, somatic cells involved in disease or somatic cells involved in disease treatment can be used.
  • Methods for enhancing reprogramming of somatic cells include inhibitors of histone deacetylase, inhibitors of TGF- ⁇ receptor, inhibitors of MAPK / ERK pathway, and 3′-phosphoinositide-dependent protein kinase -1 (PDK-1) activator in the presence of at least one factor selected from the factor group, somatic cells are cultured in contact with the abiotic substrate having a predetermined elastic modulus.
  • the method includes a culturing step.
  • the predetermined elasticity described above A method comprising a culturing step of culturing somatic cells in contact with a non-biological substrate.
  • the non-biological substrate is placed in a liquid cell culture medium, and culturing is performed with somatic cells in contact with the surface of the substrate.
  • a cell culture medium for iPS cells or a cell culture medium for ES cells can be appropriately used.
  • ESGRO trade name of Millipore
  • ReproFF trade name manufactured by Reprocell
  • the culture conditions in the culture step are not particularly limited, and may be set according to, for example, a known method for producing iPS cells (Reference Document 1 etc.).
  • the culture period is, for example, 2 to 30 days, The period is preferably 3 to 20 days, and more preferably 4 to 10 days.
  • the culture temperature is, for example, 35 ° C to 39 ° C, preferably 35.5 ° C to 38.5 ° C, and more preferably 36 ° C to 38 ° C.
  • Reference 1 Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) Induction of pluripotent stem cells from adult human fibroblastsby defined factors. Cell 131 (5): 861-72.
  • At least one selected factor is added to be present in the cell culture medium. More specifically, the “additive” may be added to the cell culture medium and mixed, and then provided to the abiotic substrate, or the cell culture medium may be supplied to the abiotic substrate. After that, the “additive” may be added, or the “additive” is contained in the abiotic base material, and the “additive” is supplied to the cell culture medium through the base material. You may be made to do.
  • the culture step can be performed without introducing any gene involved in reprogramming into the somatic cell as the starting cell.
  • a culture system for performing the culture step is as described above: 1) a substrate for cell culture (for example, a culture container such as a dish), 2) a liquid cell culture medium disposed in the substrate, 3 A) a non-biological substrate immersed in the cell culture medium. Somatic cells to be cultured are arranged on the surface of a non-biological substrate during the culture. Moreover, you may add suitably the factor which improves the undifferentiation ability of a cell (undifferentiation ability improvement factor) to a culture system as needed. CCL2 (chemokine (CC motif) ligand 2) and the like can be mentioned as an undifferentiated ability improving factor.
  • CCL2 chemokine (CC motif) ligand 2
  • the factor for improving undifferentiation may be added directly to the cell culture medium, or it is incorporated into a carrier such as protein beads (registered trademark), cross-linked gelatin hydrogel (hydrogel obtained by cross-linking gelatin), atelocollagen, and nanofiber. You may make it release slowly by the method of making it.
  • the carrier incorporating the undifferentiation ability improving factor may be provided to the culture system in a state of being placed on a carrier fixing member (a disk or the like that can be placed in a cell culture substrate). Alternatively, the factor may be gradually released by including the undifferentiated ability improving factor in the abiotic base material.
  • cells with enhanced reprogramming obtained by the method according to the present invention may be separated from cells other than those desired by performing a separation and recovery step as necessary.
  • the method for performing the separation and recovery step is not particularly limited, and for example, desired cells are separated using morphological characteristics as an index, for example, desired cells are separated using the presence or absence or expression level of a predetermined marker as an index, etc. These means can be adopted as appropriate.
  • the kit according to the present invention comprises 1) an abiotic substrate having an elastic modulus of 0.1 kPa to 200 kPa, and 2) an inhibitor of histone deacetylase, inhibition of TGF- ⁇ receptor. And at least one factor selected from the group consisting of an agent, an inhibitor of the MAPK / ERK pathway, and an activator of 3′-phosphoinositide-dependent protein kinase-1. Regarding the factor 2), an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and activation of 3′-phosphoinositide-dependent protein kinase-1 It is preferable to comprise an agent.
  • the configurations 1) to 2) are the same as those described in [1. This is the same as that described in the section “Method for enhancing reprogramming of somatic cells and cells with enhanced reprogramming”.
  • the abiotic substrate is fixed to the surface of the cell culture substrate, for example, in a film form.
  • the substrate for cell culture include a dish for cell culture, a well plate for cell culture, and the like, and a configuration in which the base material is fixed to the bottom of the dish or the bottom of the well of the well plate is exemplified.
  • the substrate for cell culture may be a flat plate member (disk or plate or the like), and an abiotic substrate may be fixed to the surface of the substrate (see FIG. 5).
  • the method for fixing the substrate to the cell culture substrate is not particularly limited, and the method may be appropriately selected according to the material of the substrate and the substrate.
  • both may be fixed using the adhesive force of the water-containing gel, and if necessary Both may be fixed after treating the surface of the substrate such as glass with an appropriate silane coupling agent.
  • the surface on the side in contact with the somatic cell among the surfaces of the non-biological substrate is protected with a removable protective layer.
  • the protective layer is, for example, a glass thin film or a plastic thin film.
  • these protective layers are removed in order to seed somatic cells (see also (b) in FIG. 5).
  • the non-biological base material is a hydrogel
  • a protective layer can be attached using the adhesive property of the gel, thereby preventing drying and alteration of the hydrogel. .
  • the protective layer indirectly covers and protects the substrate.
  • a configuration for example, a configuration in which a protective layer covers the well plate
  • a configuration in which a base material is stored in a highly airtight bag may be used.
  • the structure contained in the said non-biological base material may be sufficient as at least one selected. If these “additives” are contained in the base material in advance, it is preferable from the viewpoint of improving convenience and making the production kit compact.
  • the substrate is a hydrogel, these “additives” can be incorporated into the substrate in a state dissolved or suspended in water.
  • the “additives” are dissolved or suspended in water or the like and stored in a storage container with a lid or a storage bag. What is being done is mentioned.
  • the additive when the “additive” is solid at normal temperature and pressure, the additive may be stored in a storage container with a lid or a storage bag in the solid state.
  • the preparation kit may include an instruction manual for the kit.
  • the instructions for use of the kit include the above [1.
  • a method of enhancing reprogramming of somatic cells as described in the column of “Methods of enhancing reprogramming of somatic cells and cells with enhanced reprogramming” is recorded.
  • the instructions for use may be printed on a recording medium such as paper, or electronically recorded on an electronic recording medium such as a floppy disk, a compact disk (CD), an MD, or a flash memory. It may be a thing.
  • This preparation kit can be suitably used for the construction of a method for enhancing reprogramming of somatic cells according to the present invention and a culture system for carrying out the method.
  • the present invention includes any of the following contents.
  • a method for enhancing somatic cell reprogramming which is an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and a 3′-phosphoinositide-dependent protein kinase
  • a method comprising a culture step of culturing cells.
  • the water-containing gel is a water-containing gel of a homopolymer or copolymer of a monomer having a basic skeleton of acrylamide.
  • the above culturing step involves the presence of an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and an activator of 3′-phosphoinositide-dependent protein kinase-1.
  • the concentration is 10 ⁇ M or more and 10 mM or less, and when an inhibitor of TGF- ⁇ receptor is included, the concentration is 0.01 ⁇ M or more.
  • Activator of 3′-phosphoinositide-dependent protein kinase-1 having a concentration of 0.01 ⁇ M or more and 1 mM or less when an MAPK / ERK pathway inhibitor is included.
  • kits Selected from the group of factors consisting of the above-mentioned histone deacetylase inhibitors, TGF- ⁇ receptor inhibitors, MAPK / ERK pathway inhibitors, and 3′-phosphoinositide-dependent protein kinase-1 activators
  • the kit according to 9) wherein at least one of the factors is contained in a form exhibiting sustained release.
  • a method for producing a cell with enhanced reprogramming comprising an inhibitor of histone deacetylase, an inhibitor of TGF- ⁇ receptor, an inhibitor of MAPK / ERK pathway, and a 3′-phosphoinositide-dependent protein In the presence of at least one factor selected from the factor group consisting of kinase-1 activators, in contact with an abiotic substrate having an elastic modulus in the range of 0.1 kPa to 200 kPa.
  • a method comprising a culture step of culturing somatic cells.
  • a method for producing a cell with enhanced reprogramming comprising the step of enhancing reprogramming of somatic cells by the method according to any one of 1) to 8) above.
  • a culture system comprising a non-biological substrate in a range of 1 kPa to 200 kPa. 15) The system according to 14), wherein CCL2 is added to the cell culture medium and / or the abiotic substrate as an undifferentiated ability improving factor.
  • Example 1 (Preparation of foundation) A circular glass plate (manufactured by Fisherscientific) with a diameter of 25 mm was washed with 100% ethanol and dried. This circular glass plate was immersed in an aqueous solution containing 0.5% 3-methacryloxypropyltriethoxysilane and 1% acetone for 1 hour, washed again with 100% ethanol and dried. An aqueous solution containing 8.5 ⁇ l of 3-4% Acrylamide, 0.03-0.225% N, N′-Methylenebisacrylamide, 0.1% Tetramethylethylenediamine, and 0.1% ammonium persulfate on the circular glass plate. And a circular glass plate having a diameter of 22 mm was layered (see (a) in FIG. 5).
  • the firmness of the gel was adjusted by changing the ratio of Acrylamide and N, N'-Methylenebisacrylamide. After 1 hour, after the gel solidified, the circular glass plate having a diameter of 22 mm was removed (see (b) in FIG. 5) and washed with a 50 mM HEPES-containing aqueous solution (PH8.5). In this way, a base (gel base) to which a polyacrylamide-based hydrogel was attached was prepared.
  • D-PBS Dulbecco's phosphate buffered saline
  • Human fibroblasts (ScienCell) were cultured in Dulbecco's modified Egel medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. The cultured cells were collected and then seeded with 5 ⁇ 10 4 cells per gel substrate, 10% fetal bovine serum, 1% penicillin, 1% streptomycin, 0.25 mM NaB, 5 ⁇ M PS48, 0.5 ⁇ M The cells were cultured for 4 days in Dulbecco's modified Egel medium containing A-83-01 and 0.5 ⁇ M PD0325901. When the cells were cultured, the gel substrate was placed in a cell culture dish having a size that can be stored, and the gel substrate was completely immersed in the medium.
  • human fibroblasts were cultured under the same conditions except that they were cultured on hard plastic (BD Falcon cell culture dish) or feeder cells instead of the gel base.
  • Nanog positive, Oct3 / 4 positive appearance of colonies When human fibroblasts were cultured on a soft substrate (gel substrate), ES-like colonies appeared after 4 days of culture, and most cells were Nanog and Oct3 / 4 was expressed. The number of colonies expressing both Nanog and Oct3 / 4 was significantly larger than when cultured on hard plastic (shown as Rigid in the figure) (see FIGS. 1 and 2).
  • Example 2 (Preparation of foundation) A circular glass plate (manufactured by Fisherscientific) with a diameter of 25 mm was washed with 100% ethanol and dried. This circular glass plate was immersed in an aqueous solution containing 0.5% 3-methacryloxypropyltriethoxysilane and 1% acetone for 1 hour, washed again with 100% ethanol and dried. On this circular glass plate, 8.5 ⁇ l of an aqueous solution containing 3% Acrylamide, 0.1% N, N′-Methylenebisacrylamide, 0.1% Tetramethylethylenediamine, and 0.1% ammonium persulfate was dropped, and the diameter was further reduced. A 22 mm circular glass plate was layered.
  • the round glass plate having a diameter of 22 mm was removed and washed with a 50 mM HEPES-containing aqueous solution (PH 8.5).
  • a base (gel base) having a hardness of 1 kPa to which a polyacrylamide-based hydrogel was adhered was prepared.
  • Human fibroblasts (ScienCell) were cultured in Dulbecco's modified Egel medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. The cultured cells are collected, then 5 ⁇ 10 4 cells are seeded per gel substrate, 10% fetal bovine serum, 1% penicillin, 1% streptomycin, and any of the following (1) to (4) The cells were cultured for 4 days in Dulbecco's modified Egel medium containing these combinations of reagents.
  • human fibroblasts were cultured in a medium (4i) containing 0.25 mM NaB, 5 ⁇ M PS48, 0.5 ⁇ M A-83-01 and 0.5 ⁇ M PD0325901 and a medium (0i) not containing a drug. .
  • Nanog and Oct3 / 4 were compared by immunostaining.
  • human fibroblasts were cultured on a base of 1 kPa hardness (gel base) with different combinations of drugs, ES-like colonies appeared after 4 days of culture.
  • most cells expressed Nanog (red).
  • Oct3 / 4 green was highly expressed under the conditions of 4i and ⁇ A8301 (FIG. 6).
  • the fluorescence intensity of the immunostained images was compared. As a result, about 90% of Nanog expression and Oct3 / 4 expression of 4i were observed under the condition of -A8301. (FIG. 7).
  • Example 3 (Preparation of foundation) The same operation as in Example 2 was performed.
  • Human fibroblasts (ScienCell) were cultured in Dulbecco's modified Egel medium containing 10% fetal bovine serum, 1% penicillin, and 1% streptomycin. The cultured cells are collected, then 5 ⁇ 10 4 cells are seeded per gel substrate, 10% fetal bovine serum, 1% penicillin, 1% streptomycin, and any of the following (1) to (4) The cells were cultured for 4 days in Dulbecco's modified Egel medium containing these combinations of reagents.
  • human fibroblasts were cultured in a medium (4i) containing 0.25 mM NaB, 5 ⁇ M PS48, 0.5 ⁇ M A-83-01 and 0.5 ⁇ M PD0325901 and a medium (0i) not containing a drug. .
  • Nanog and Oct3 / 4 were compared by immunostaining.
  • human fibroblasts were cultured on a base of 1 kPa hardness (gel base) with different combinations of drugs, ES-like colonies appeared after 4 days of culture.
  • most cells expressed Nanog (red).
  • Oct3 / 4 green was highly expressed under the conditions of 4i and TriA (FIG. 8).
  • the fluorescence intensity of immunostained images was compared. As a result of TriA, the expression of Nanog was about 80% of the condition of 4i, and Oct3 / increased by 10%. 4 expression was seen.
  • the present invention provides a method for enhancing cell reprogramming without requiring gene introduction.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Microbiology (AREA)
  • Transplantation (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé de promotion de la reprogrammation cellulaire somatique comprenant une étape de culture pour la culture de cellules somatiques dans un état de contact avec un substrat non biologique dont le module d'élasticité se situe dans la plage de 0,1 kPa à 200 kPa, en présence d'au moins un facteur choisi dans le groupe de facteurs consistant en un inhibiteur d'histone désacétylase, un inhibiteur du récepteur de TGF-β, un inhibiteur de la voie MAPK/ERK et un activateur de la protéine kinase 1 dépendante de 3'-phosphoinositide.
PCT/JP2013/082246 2012-11-30 2013-11-29 Procédé de promotion de la reprogrammation d'une cellule somatique, et trousse de préparation cellulaire WO2014058080A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014540925A JPWO2014058080A1 (ja) 2012-11-30 2013-11-29 体細胞のリプログラミングを亢進させる方法、及び細胞作製キット

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012263735 2012-11-30
JP2012-263735 2012-11-30

Publications (1)

Publication Number Publication Date
WO2014058080A1 true WO2014058080A1 (fr) 2014-04-17

Family

ID=50477536

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2013/082246 WO2014058080A1 (fr) 2012-11-30 2013-11-29 Procédé de promotion de la reprogrammation d'une cellule somatique, et trousse de préparation cellulaire

Country Status (2)

Country Link
JP (1) JPWO2014058080A1 (fr)
WO (1) WO2014058080A1 (fr)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017119512A1 (fr) * 2016-01-08 2017-07-13 国立研究開発法人国立がん研究センター Procédé de production de cellules souches/précurseurs hépatiques à partir de cellules hépatiques matures à l'aide d'un composé de faible poids moléculaire
JPWO2016117510A1 (ja) * 2015-01-20 2017-10-26 京都府公立大学法人 神経系細胞の製造方法
US11266647B2 (en) 2016-10-26 2022-03-08 Icahn School Of Medicine At Mount Sinai Method for increasing cell proliferation in pancreatic beta cells, treatment method, and composition
US11547712B2 (en) 2017-11-20 2023-01-10 Icahn School Of Medicine At Mount Sinai Kinase inhibitor compounds and compositions and methods of use
US11788064B2 (en) 2018-01-05 2023-10-17 Icahn School Of Medicine At Mount Sinai Method of increasing proliferation of pancreatic beta cells, treatment method, and composition
US11866427B2 (en) 2018-03-20 2024-01-09 Icahn School Of Medicine At Mount Sinai Kinase inhibitor compounds and compositions and methods of use

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011047300A1 (fr) * 2009-10-16 2011-04-21 The Scripps Research Institute Induction de cellules pluripotentes
WO2011072461A1 (fr) * 2009-12-18 2011-06-23 Shanghai He Chen Biotechnology Co., Ltd. Matériels et méthodes pour la production de cellules souches pluripotentes
WO2011123572A1 (fr) * 2010-03-31 2011-10-06 The Scripps Research Institute Nouvelle programmation de cellules

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009005769A2 (fr) * 2007-06-29 2009-01-08 The Trustees Of The University Of Pennsylvania Systèmes de gels souples dans la modulation d'un développement de cellules souches
CN106978393A (zh) * 2007-06-29 2017-07-25 船木真理 用于进行间充质干细胞（msc）生长调节的低硬度凝胶

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011047300A1 (fr) * 2009-10-16 2011-04-21 The Scripps Research Institute Induction de cellules pluripotentes
WO2011072461A1 (fr) * 2009-12-18 2011-06-23 Shanghai He Chen Biotechnology Co., Ltd. Matériels et méthodes pour la production de cellules souches pluripotentes
WO2011123572A1 (fr) * 2010-03-31 2011-10-06 The Scripps Research Institute Nouvelle programmation de cellules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HASSANI SEYEDEH-NAFISEH ET AL.: "Simultaneous Suppression of TGF-P and ERK Signaling Contributes to the Highly Efficient and Reproducible Generation of Mouse Embryonic Stem Cells from Previously Considered Refractory and Non-permissive Strains", STEM CELL REV. AND REP., vol. 8, no. 2, 2011, pages 472 - 481 *
KUNIO HIRANO ET AL.: "Human and Mouse Induced Pluripotent Stem Cells Are Differentially Reprogrammed in Response to Kinase Inhibitors", STEM CELLS AND DEVELOPMENT, vol. 21, no. 8, 2011, pages 1287 - 1298 *
YU JUNYING ET AL.: "Efficient feeder-free episomal reprogramming with small molecules. art. e17557", PLOS ONE, vol. 6, no. 3, 2011, pages 1 - 10 *
ZHU SAIYONG ET AL.: "Reprogramming of Human Primary Somatic Cells by OCT4 and Chemical Compounds", CELL STEM CELL, vol. 7, no. 6, 2010, pages 651 - 655 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPWO2016117510A1 (ja) * 2015-01-20 2017-10-26 京都府公立大学法人 神経系細胞の製造方法
WO2017119512A1 (fr) * 2016-01-08 2017-07-13 国立研究開発法人国立がん研究センター Procédé de production de cellules souches/précurseurs hépatiques à partir de cellules hépatiques matures à l'aide d'un composé de faible poids moléculaire
CN108779439A (zh) * 2016-01-08 2018-11-09 国立研究开发法人国立癌症研究中心 利用低分子化合物的由成熟肝细胞制作肝干细胞/前体细胞的制作方法
JPWO2017119512A1 (ja) * 2016-01-08 2018-11-15 Cynity株式会社 低分子化合物による成熟肝細胞からの肝幹／前駆細胞の作製方法
US10961507B2 (en) 2016-01-08 2021-03-30 Cynity Co., Ltd. Method for producing hepatic stem/precursor cells from mature hepatic cells using low-molecular-weight compound
JP7063624B2 (ja) 2016-01-08 2022-05-09 エヴィア ライフ サイエンシズ インコーポレイテッド 低分子化合物による成熟肝細胞からの肝幹／前駆細胞の作製方法
US11266647B2 (en) 2016-10-26 2022-03-08 Icahn School Of Medicine At Mount Sinai Method for increasing cell proliferation in pancreatic beta cells, treatment method, and composition
US11547712B2 (en) 2017-11-20 2023-01-10 Icahn School Of Medicine At Mount Sinai Kinase inhibitor compounds and compositions and methods of use
US11788064B2 (en) 2018-01-05 2023-10-17 Icahn School Of Medicine At Mount Sinai Method of increasing proliferation of pancreatic beta cells, treatment method, and composition
US11866427B2 (en) 2018-03-20 2024-01-09 Icahn School Of Medicine At Mount Sinai Kinase inhibitor compounds and compositions and methods of use

Also Published As

Publication number Publication date
JPWO2014058080A1 (ja) 2016-09-05

Similar Documents

Publication Publication Date Title
WO2014058080A1 (fr) Procédé de promotion de la reprogrammation d'une cellule somatique, et trousse de préparation cellulaire
US10584316B2 (en) Compostition for reprogramming cells
Assinck et al. Myelinogenic plasticity of oligodendrocyte precursor cells following spinal cord contusion injury
Nagoshi et al. Human spinal oligodendrogenic neural progenitor cells promote functional recovery after spinal cord injury by axonal remyelination and tissue sparing
Eroshenko et al. Effect of substrate stiffness on early human embryonic stem cell differentiation
US11124770B2 (en) Myocardial cell sheet
JP7418754B2 (ja) 腸オルガノイド及びその作製方法
JP5863639B2 (ja) 椎間板の状態に関する指標を得る方法、椎間板障害の治療または予防方法、および髄核細胞集団のポテンシャルまたは品質の評価方法
KR102368751B1 (ko) 모양체 주연부 간세포의 제조 방법
Asmani et al. Three‐dimensional culture of differentiated endometrial stromal cells to oligodendrocyte progenitor cells (OPC s) in fibrin hydrogel
WO2019151386A1 (fr) Procédé de fabrication de cellules
TW201522638A (zh) 多能性幹細胞的製備方法、使用該製備方法而製備出多能性幹細胞、改善劑、以及該多能性幹細胞之分化誘導方法
KR20180025817A (ko) 탈세포화된 세포외 기질을 포함하는 직접교차분화 촉진용 조성물 및 이의 용도
JP6151097B2 (ja) 腸構造体を分化誘導する方法
Abbasi et al. Mesenchymal stem cell behavior on soft hydrogels with aligned surface topographies
JP2011155945A (ja) 組織培養用培養器、及び三次元組織培養物の製造方法
US20190185816A1 (en) Cardiac microtissue and uses thereof
JP6363401B2 (ja) 人工多能性幹細胞の分化誘導方法
Wilson et al. Mechanical characterization of hydrogels and its implications for cellular activities
Zamponi Engineering of Ideal Systems for the Study and Direction of Stem Cell Asymmetrical Division and Fate Determination
JP7501051B2 (ja) 小腸上皮細胞層を含む細胞構造物、その用途、及び、その製造方法
JP6393368B2 (ja) 腸構造体を分化誘導する方法
JP2010136655A (ja) 三次元組織培養物の製造方法
JP2021132584A (ja) シート状細胞培養物の剥離に関する評価方法
Yang Exploiting material softness to regulate mesenchymal stem cells secretome production for" cell-free" regenerative medicine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13846187

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2014540925

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13846187

Country of ref document: EP

Kind code of ref document: A1