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WO2019093047A1 - Procédé de production in vitro d'une glande exocrine fonctionnelle, et glande exocrine ainsi produite - Google Patents

Procédé de production in vitro d'une glande exocrine fonctionnelle, et glande exocrine ainsi produite Download PDF

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Publication number
WO2019093047A1
WO2019093047A1 PCT/JP2018/037678 JP2018037678W WO2019093047A1 WO 2019093047 A1 WO2019093047 A1 WO 2019093047A1 JP 2018037678 W JP2018037678 W JP 2018037678W WO 2019093047 A1 WO2019093047 A1 WO 2019093047A1
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Prior art keywords
pharmaceutical composition
salivary gland
cell
gene
cells
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Ceased
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English (en)
Japanese (ja)
Inventor
田中　準一
健二 美島
孝 辻
美帆 小川
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Showa University
Organ Technologies Inc
RIKEN
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Showa University
Organ Technologies Inc
RIKEN
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Priority to JP2019552666A priority Critical patent/JPWO2019093047A1/ja
Publication of WO2019093047A1 publication Critical patent/WO2019093047A1/fr
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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
    • A61K35/37—Digestive system
    • A61K35/38—Stomach; Intestine; Goblet cells; Oral mucosa; Saliva
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/40—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material
    • A61L27/44—Composite materials, i.e. containing one material dispersed in a matrix of the same or different material having a macromolecular matrix
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method of inducing functional exocrine glands from pluripotent stem cells, and exocrine glands induced by the method.
  • regenerative medicine which replaces the diseased or damaged organ or tissue that has become dysfunctional with age, with a fully functional regenerated organ or tissue.
  • regeneration of organs or tissues having the same function and structure as organs or tissues in a living body has not been achieved.
  • stem cell technology has established a technique for inducing cells that constitute various tissues of a living body from stem cells.
  • organs of a living body take on a three-dimensional arrangement of multiple types of functional cells and their development progresses through the interaction of each cell, it is necessary to compare specific organs in comparison with the case of inducing each cell. It is very difficult to artificially guide.
  • construction techniques for rapidly transplantable regenerative organs have not yet been developed.
  • the present inventors artificially reconstruct an organ primordia such as a hair follicle primordial or the like using epithelial cells and mesenchymal cells derived from organ primordia in a living body ("Organ primordia Law) was established (Non-Patent Document 1). Furthermore, the present inventors use the above-described organ primordia method to prepare a primordia of a secretory gland such as a salivary gland and a lacrimal gland, insert a guide into the primordia, and culture the living body. Also shown is a method of manufacturing an organ primordia having a conduit connectable with the conduit (US Pat.
  • the methods described in these documents use epithelial cells and / or mesenchymal cells derived from a tissue of a living body at least in part as a source of regenerated organ primordia. Therefore, as a more versatile method for producing organs, development of a method for producing organs in vitro, which is completely derived from pluripotent stem cells, is required.
  • Non-patent document 2 for eye cup
  • Non-patent document 3 for anterior segment tissue
  • non-patent document 4 for pituitary
  • the present inventors diligently studied about a method of inducing differentiation of functional exocrine gland derived from pluripotent stem cells. As a result, it has been surprisingly found that, by adding predetermined steps to oral ectoderm derived from pluripotent stem cells, functional exocrine glands (eg, salivary glands) can be produced in vitro. , Came to complete the present invention.
  • functional exocrine glands eg, salivary glands
  • the present invention is a method of producing a functional exocrine gland in vitro, comprising (I): culturing cell aggregates containing oral ectoderm under conditions that induce the expression of Sox9 and Foxc1; (Ii): further culturing all or part of the cell aggregate cultured in the above step (i) in a medium containing FGF7 and FGF10, On the way, including.
  • the oral ectoderm is characterized by being derived from ES cells or iPS cells.
  • the exocrine gland is a salivary gland.
  • the “condition for inducing the expression of Sox9 and Foxc1” in the step (i) is (A) A condition that induces the expression of Sox9 and Foxc1 by introducing the Sox9 expression gene and the Foxc1 expression gene into the cell aggregate containing the above-mentioned oral ectoderm, or (B) conditions for inducing the expression of the introduced Sox9 gene and Foxc1 gene in a cell aggregate containing oral ectoderm into which Sox9 expression gene and Foxc1 expression gene have been introduced in advance; It is characterized by being.
  • the gene transfer is characterized by the transfer of a gene by a viral vector, the transfer of a gene by a transfection reagent, or the transfer of a gene by electroporation.
  • the virus vector is an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, or a Sendai virus vector.
  • the step (ii) is a step of further culturing a part of the outer layer of the cell aggregate cultured in the step (i) in a medium containing FGF7 and FGF10. It is characterized by
  • the concentration of FGF7 in the medium is 1 to 1000 ng / ml
  • the concentration of FGF10 in the medium is 1 to 1000 ng / ml.
  • the step (i) is characterized in that it is carried out using a growth factor-free medium.
  • the culture period of the step (i) is 0 to 7 days, and the culture period of the step (ii) is 10 to 32 days.
  • Another embodiment of the present invention relates to a pharmaceutical composition for use in the treatment of a disease accompanied by tissue disorder or dysfunction of salivary gland, wherein said pharmaceutical composition is regenerated by any of the above methods It is characterized by containing a salivary gland.
  • the pharmaceutical composition comprises a complex comprising a cell assembly substantially composed of mesenchymal cells, and a regenerating salivary gland produced by any of the above methods. It is characterized by including.
  • a filamentous structure is inserted so as to penetrate the cell assembly substantially composed of the mesenchymal cells and the regenerated salivary gland. It features.
  • all or part of the regenerated salivary gland or all or part of the complex is characterized in being embedded in a cell culture scaffold material.
  • the scaffold material is a collagen gel.
  • the disease is Sjogren's syndrome, litholithiasis, LADDO (Lacrimo-auriculo-dento-digital syndrome), ALSG (aplasia of lacrimal and salivary glands), Mikulicz disease (IgG4 associated sclerosability) Disease), other salivary gland inflammatory diseases, or salivary gland dysfunction due to the side effect of radiation treatment.
  • Another embodiment of the present invention relates to the use of the regenerated salivary gland produced by any of the above methods for the production of a pharmaceutical composition for use in the treatment of a disease associated with tissue disorder or dysfunction of the salivary gland.
  • Another embodiment of the present invention relates to a method of treating a disease associated with tissue disorder or dysfunction of salivary gland, wherein the method comprises regenerating salivary gland produced by the above method, or the pharmaceutical composition as described above. Applying to a subject in need of treatment of a disease.
  • the subject is a mammal other than human.
  • FIG. 1 shows the procedure of an embodiment of the present invention.
  • ES Embryonic Stem Cell (embryonic stem cells)
  • ME Mesendoderm (embryonic stem cells)
  • DE Definitive Ectoderm (embryonic ectoderm)
  • NNE Non-neural Ectoderm (non-neural ectoderm)
  • NE Neural Ectoderm (neural ectoderm)
  • EPI Epidermis (epidermis)
  • OE Oral Ectoderm (oral ectoderm)
  • OESG Oral Epithelial Salivary Gland (oral epithelial salivary gland)
  • SG Salivary Gland (salivary gland)
  • FIG. 2 is a diagram showing that Pan-cytokeratin (PK) -positive primordial oral mucosa (oral ectoderm) positive is formed in the outer layer of the aggregate on the 8th day after the induction of differentiation induction.
  • FIG. 3 shows that Sox9-expressing gene and Foxc1-expressing gene are introduced into the aggregate on the 8th day of differentiation induction initiation using an adenovirus vector, then the outer layer of the aggregate is separated and further cultured in the presence of FGF7 and FGF10 It is the figure which showed that.
  • FIG. 3 shows that Sox9-expressing gene and Foxc1-expressing gene are introduced into the aggregate on the 8th day of differentiation induction initiation using an adenovirus vector, then the outer layer of the aggregate is separated and further cultured in the presence of FGF7 and FGF10 It is the figure which showed that.
  • FIG. 4 is a diagram showing that a budding-like structure similar to the embryonic salivary gland primordia is formed from the clumps on day 23 of differentiation induction, and branching morphogenesis is observed on day 28 of differentiation induction.
  • FIG. 5 shows that ES cell-derived salivary glands on day 23 and 28 of differentiation induction are composed of Pan-cytokeratin (PK) -positive epithelial cells, and Keratin 18 (K18) -positive ductal epithelium is placed on the mesial side, AQP5 It is a figure which shows that the positive acinar cell and the (alpha)-SMA positive myoepithelial cell are arrange
  • PK Pan-cytokeratin
  • K18 Keratin 18
  • FIG. 6 is a diagram showing that, as a result of component analysis of gene expression profile by RNA-sequence, gene expression of ES cell-derived salivary gland (i-SG) is similar to that of salivary gland source at 15-18 days of embryonic day .
  • FIG. 7 shows that ES cell-derived salivary glands show an increase in intracellular Ca 2+ concentration by carbachol stimulation, and that the action is suppressed by atropine.
  • FIG. 8 left is a stereomicroscopic image of ES cell-derived salivary glands 30 days after transplantation. In FIG.
  • FIG. 8 shows that the excretory duct and ES of the recipient are obtained by transplanting the ES cell-derived salivary gland (i-SG) into the parotid ablated mouse.
  • FIG. 5 is a diagram showing that cell-derived salivary gland structures are connected and engrafted mature acinar cells having mucus-producing ability.
  • FIG. 9A is a diagram showing an outline of a method of transplanting ES cell-derived salivary glands in the present example.
  • FIG. 9B shows a histologic image of the case where fetal derived salivary gland mesenchymal tissue is added to ES cell-derived salivary gland and transplanted (FIG.
  • FIG. 10 is a diagram showing that the recipient-derived nerve invades into the transplanted ES cell-derived salivary gland structure. Arrowheads in the figure represent nerve fibers, arrows represent calponin-positive myoepithelial cells, and myoepithelial cells are arranged to wrap ES cell-derived acinar cells (A, B, C), and nerve fibers close to myoepithelial cells. Is oriented.
  • FIG. 10 is a diagram showing that the recipient-derived nerve invades into the transplanted ES cell-derived salivary gland structure. Arrowheads in the figure represent nerve fibers, arrows represent calponin-positive myoepithelial cells, and myoepithelial cells are arranged to wrap ES cell-derived acinar cells (A, B, C), and nerve fibers close to myoepithelial cells. Is oriented.
  • FIG. 10 is a diagram showing that the recipient-derived nerve invades into the transplanted ES cell-derived salivary gland structure. Arrow
  • FIG. 11A is a diagram showing that the amount of salivary secretion is increased by taste stimulation by citric acid to a recipient transplanted with ES cell-derived salivary gland obtained by laminating salivary gland mesenchymal tissue of 13.5 days of gestational age.
  • FIG. 11B is a diagram showing an increase in the amount of saliva secretion by stimulating the transplanted ES cell-derived salivary gland with the parasympathomimetic agent pilocarpic acid.
  • pluripotent stem cell refers to a cell having both pluripotency capable of differentiating into any cell of a living body and self-replication ability capable of maintaining pluripotency even after differentiation and proliferation. Cells and iPS cells.
  • ES cells Embryonic Stem cells
  • stem cell lines produced from an inner cell mass belonging to a part of blastocyst stage embryo which is an early developmental stage of an animal, and very many And pluripotency capable of differentiating into cells, and self-replication ability capable of maintaining pluripotency even after division and proliferation.
  • ES cells derived from the inner cell mass of any animal can be used.
  • ES cells derived from the inner cell mass of human, mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig and monkey can be used as a source of ES cells.
  • iPS cells induced Pluripotent Stem cells
  • ES cells ES cells
  • somatic cells ES cells
  • the present invention is not limited to the above description, and broadly includes cells that those skilled in the art recognize as "iPS cells”.
  • the origin of the iPS cells that can be used in the present invention is not particularly limited, and any animal-derived iPS cells can be used.
  • iPS cells derived from human, mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig and monkey can be used as a source of iPS cells.
  • somatic cells from which iPS cells can be derived which can be used in the present invention are not particularly limited, and iPS cells derived from cells derived from any tissue can be used.
  • the method for inducing iPS cells that can be used in the present invention is not particularly limited, and iPS cells derived using any method can be used as long as they can induce iPS cells from somatic cells. be able to.
  • the method for culturing pluripotent stem cells without differentiation is not particularly limited, and a person skilled in the art can appropriately select a known culture environment or culture medium.
  • adhesion culture may be performed using feeder cells or the like, or suspension culture may be performed without using feeder cells or the like.
  • a medium for culturing pluripotent stem cells a medium generally used for culturing pluripotent stem cells can be used, and the composition thereof is not particularly limited, and a person skilled in the art is an expert in the field. It can be suitably prepared based on common sense.
  • exocrine gland in the present invention refers to a gland in which secretory granules from exocrine cells are released directly or through a conduit to the surface of the body surface or lumenal epithelium.
  • examples of the "exocrine gland” in the present invention include lacrimal gland, salivary gland, cardiac gland, pyloric gland, gastric gland, intestinal crypt, prostate, sweat gland, sebaceous gland and the like, preferably salivary gland.
  • the confirmation that salivary gland was formed can use suitably the method which can be utilized for persons skilled in the art, such as analysis by a tissue image and functional analysis by stimulation.
  • oral ectoderm in the present invention refers to a single-layer cell sheet that forms the mucous membrane epithelium of the oral cavity during embryonic development.
  • the oral ectoderm develops from the ectoderm adjacent to the interbrain in the early embryo, and a portion of it forms the ratoke sac underlying the pituitary.
  • the presence of oral ectoderm in tissues can be confirmed, for example, by expression of pan-cytokeartin (PK).
  • PK pan-cytokeartin
  • the buccal ectoderm used in the methods of the present invention may be derived from pluripotent stem cells (eg, ES cells or iPS cells).
  • pluripotent stem cells eg, ES cells or iPS cells.
  • the method for inducing buccal ectoderm from pluripotent stem cells is not limited, and methods known to those skilled in the art can be used.
  • the induction of buccal ectoderm from ES cells in the examples of the present invention is described by K. K. et al. R. Koehler, A .; M. Mikosz, A .; I. Molosh D. Patel, E., et al. Reference is made to the method described in Hashino, Nature 500, 217 (Aug 08, 2013). Suga et al. , Nature 480, 57 (Nov 09, 2011), and Ochiai H et al. , Stem Cell Research 15: 290-298, 2015, and the like
  • SFEBq method serum agglutination suspension culture: S erum-free F loating culture of E mbryoid B ody-like aggregates with q uick reaggregation
  • pluripotent stem cells are single-celled with an enzyme and reaggregated into a cluster of about 3000 cells is used as a material for differentiation culture.
  • culturing this cell aggregate in a method of adhering cells to a culture petri dish and conducting culture as in the usual cell culture, three-dimensional tissue formation is impaired, and a clean structure can not be formed.
  • the culture vessel by coating the culture vessel with non-cell-adhesive polymer to prevent cells and tissues from adhering to the vessel, three-dimensional tissue formation is enabled by the culture method in which the cell mass is suspended in the culture solution. .
  • the cells are cultured for several days while suspended in a special culture solution that does not contain any component having a neural differentiation inhibitory effect such as serum or transcription factor. This method makes it possible to differentiate some cells into ectodermal cells.
  • the description of WO2009 / 148170 can be referred to.
  • the “cell aggregate containing buccal ectoderm” in the present invention may be a cell clump containing the number of buccal ectoderm required for induction to exocrine glands, and can be prepared, for example, by the method described above.
  • cell aggregates containing oral ectoderm are cultured under conditions that induce the expression of Sox9 and Foxc1.
  • the culture in the first step of the present invention may preferably be suspension culture.
  • the Sox9 expression gene and the Foxc1 expression gene are introduced into a cell aggregate containing oral ectoderm by gene transfer with a viral vector, gene transfer with a transfection reagent, or gene transfer with electroporation. You may When a gene is introduced by a viral vector, for example, an adenovirus vector, an adeno-associated virus vector, a lentivirus vector, a retrovirus vector, a Sendai virus vector, etc. can be used.
  • Sox9 and Foxc1 As another “condition for inducing the expression of Sox9 and Foxc1”, for example, in the cell aggregate containing the oral ectoderm in which Sox9 expression gene and Foxc1 expression gene have been introduced in advance, the Sox9 gene and the Foxc1 gene Conditions may be mentioned which induce (start) expression. Specifically, for example, by using a Tet expression induction system, expression is induced by inducing Sox9 expression gene and Foxc1 expression gene introduced at the ES cell stage with a drug in the first culture step of the present invention. It can be started. In addition, it is not limited to the above-mentioned example, but various gene expression induction systems available to those skilled in the art can be used in the present invention.
  • condition for inducing the expression of Sox9 and the “condition for inducing the expression of Foxc1” may be the same or different.
  • the respective conditions can be appropriately selected by those skilled in the art based on the common technical knowledge in this field.
  • the first culture step of the present invention may be carried out for 0 to 7 days (preferably 0 to 3 days, more preferably 0 to 1 day).
  • the first step on day 0 means, for example, starting the second step of the present invention the day the Sox 9 expression gene and the Foxc 1 expression gene were introduced into a cell aggregate containing oral ectoderm.
  • the medium used in the first culture step of the present invention is a medium substantially free of growth factor (eg, growth-factor-free Chemically Defined Medium (gfCDM medium) used in the SFEBq method)
  • gfCDM medium growth-factor-free Chemically Defined Medium
  • various other media may be used as long as they do not cause unintended differentiation induction in oral ectoderm.
  • substances generally added to the cell culture medium for example, supplements such as amino acids and transferrin, antibiotics, etc. may be added to the culture medium as long as they do not cause unintended differentiation induction in oral ectoderm. Good.
  • all or part of the cell aggregate prepared in the first step is further cultured in a medium containing FGF7 and FGF10.
  • the culture in the second step of the present invention may preferably be suspension culture.
  • the concentration of FGF7 in the culture medium and FGF10 may be contained at a concentration necessary for inducing exocrine glands (eg, salivary glands) from oral ectoderm, and can be appropriately adjusted by those skilled in the art.
  • the concentration of FGF7 in the medium may be in the range of 1 to 1000 ng / ml, preferably 5 to 750 ng / ml, 10 to 500 ng / ml, 20 to 400 ng / ml, 30 to 300 ng / ml, 40 It may be in the range of ⁇ 200 ng / ml or 50 ⁇ 150 ng / ml.
  • the concentration of FGF10 in the medium may be in the range of 1 to 1000 ng / ml, preferably 5 to 750 ng / ml, 10 to 600 ng / ml, 30 to 500 ng / ml, 50 to 400 ng / ml , 100-300 ng / ml, or 150-250 ng / ml.
  • various media can be used as long as they do not cause unintended differentiation induction in oral ectoderm.
  • BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM Zinc Option medium, IMDM medium, Medium 199 medium, Eagle MEM medium, ⁇ MEM medium, ⁇ MEM medium, DMEM medium, advanced DMEM / F12 medium, Ham medium, Ham ' s F-12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal medium, mixed medium thereof and the like can be mentioned.
  • substances eg, supplements such as amino acids and transferrin, antibiotics, etc.
  • supplements such as amino acids and transferrin, antibiotics, etc.
  • the cell aggregate cultured in the first culture step may be used as it is, but using only a part of the cell aggregate cultured in the first culture step It is also good.
  • a part of the outer layer of the cell aggregate cultured in the first culture step can be isolated and applied to the second culture step to increase the induction efficiency of exocrine glands (eg, salivary glands) .
  • the method of isolating a part of the outer layer of the cell aggregate is not limited, and may be mechanically isolated using, for example, a pipette, an injection needle, a medical scalpel or the like.
  • the second culture step of the present invention is carried out for 10 to 32 days (preferably 12 to 20 days, more preferably 15 to 18 days).
  • the regenerated salivary glands produced by the method of the present invention can be used, for example, in the treatment of diseases involving tissue damage or dysfunction of the salivary glands.
  • diseases include Sjogren's syndrome, litholithiasis, Lacrimo-auriculo-dento-digital syndrome, ALSG (aplasia of lacrimal and salivary glands), Mikulicz's disease (IgG4 related sclerosability) Disease), other salivary gland inflammatory diseases, or salivary gland dysfunction due to the side effect of radiation treatment.
  • the treatment of diseases utilizing the present invention can be used for any animal, for example, mammals (mouse, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig, monkey) , Humans, etc.).
  • mammals mae, rat, dog, cat, rabbit, cow, horse, sheep, goat, pig, monkey
  • Humans etc.
  • humans can be excluded from the subject.
  • the regenerated salivary glands produced by the method of the present invention are used to treat a disease, it is preferable to transplant the regenerated salivary glands produced by the method of the present invention to a subject in need of treatment.
  • the method for transplanting the regenerating salivary gland to the subject is not limited, but for example, referring to WO 2012/108069, after inserting a guide (filamentary structure) for promoting the formation of the duct of the secretory gland into the regenerating salivary gland, It can be ported.
  • a guide filamentary structure
  • the regenerated salivary gland produced by the method of the present invention may be transplanted to a subject alone, but when it is adhered to a cell assembly substantially composed of mesenchymal cells and transplanted as a complex, The growth efficiency of salivary gland tissue at the transplantation destination is improved.
  • the origin of the mesenchymal cells used in the present invention is not limited, and may be, for example, mesenchymal cells derived from pluripotent stem cells (eg, ES cells, iPS cells) by a known method, or a living tissue It may be a mesenchymal cell of origin (eg, a fetal mesenchymal cell).
  • the whole or part of the regenerated salivary gland produced by the method of the present invention or the complex of the regenerated salivary gland and the mesenchymal cells is embedded in a scaffold for cell culture and then transplanted to the subject. And increase the survival rate in the target tissue.
  • scaffolds for cell culture which can be used in the present invention include collagen gels (more specifically, type I collagen gel, type III collagen gel, type IV collagen gel, matrigel, etc.) .
  • the material of the thread-like structure used as a guide for promoting the formation of the conduit of regenerated salivary gland in the present invention is not limited, for example, fibers made of synthetic or natural bioabsorbable polymers, metal fibers such as stainless steel, Chemical fibers such as carbon fibers and glass fibers, natural animal fibers, plant fibers and the like can be mentioned, and more specifically, nylon yarn, stainless steel wire and the like can be mentioned.
  • the guide may have various shapes as long as it is thread-like, for example, it may be in the form of hollow fiber.
  • the diameter and length of the thread-like structure used as a guide for promoting the formation of the conduit of the regenerating salivary gland can be appropriately designed according to the subject.
  • the diameter may be 5 to 100 ⁇ m, more preferably 10 to 70 ⁇ m, and still more preferably 20 to 50 ⁇ m.
  • the length of the guide may be, for example, 1 mm to 10 mm, more preferably 2 to 8 mm, and still more preferably 2 mm to 5 mm.
  • the method of inserting the filamentous structure into the regenerating salivary gland is not limited as long as it is inserted so as not to destroy the tissue of the regenerating salivary gland.
  • the filamentous structure is inserted so as to penetrate the regenerating salivary gland and the aggregate of mesenchymal cells. Preferably.
  • the regenerated salivary gland may be cultured for a certain period of time with the filamentous structure inserted, and then subjected to transplantation.
  • the culture period after guide insertion can be set appropriately depending on the subject, but can be cultured, for example, for 1 to 7 days.
  • FIG. 1 shows an outline of the procedure of this embodiment.
  • the procedure for inducing from the oral ectoderm (OE) to the salivary gland (SG) is the subject of the present invention.
  • Mouse ES cells are dispersed into single cells by trypsinization, and clumps are formed according to the SFEBq method (Nakano et al, Cell Stem Cell, 10 (6): 771-785, 2012) to induce differentiation Flocculation culture for 37 ° C. in the presence of 5% CO 2.
  • the specific procedure is shown below.
  • the culture medium for differentiation induction is a growth factor-free chemically-defined medium (gfCDM; Wataya et al, Proc Natl Acad Sci USA, 105 (33): 11796-11801, 2008). Using.
  • the induction from ES cells to oral ectoderm in this example is as described in K. et al. R. Koehler, A .; M. Mikosz, A .; I. Molosh D. Patel, E., et al. It implemented with reference to the method as described in Hashino, Nature 500, 217 (Aug 08, 2013).
  • SB-431542 TGF- ⁇ inhibitor
  • FGF2 thickens the epithelium of the outer layer of cell mass, so LDN-193189 ( BMP inhibitors are added respectively to suppress differentiation into the epidermis (see FIG. 1).
  • Second culture step The outer layer of the aggregate was mechanically isolated under a stereomicroscope and transferred to a 24-well plate coated with a low cell adsorptive surface.
  • the medium used was one obtained by adding 1% matrigel to a medium (advanced DMEM / F12 supplemented with N2 supplement, GlutaMax, penicillin, streptomycin) to which FGF7 (100 ng / ml) and FGF10 (200 ng / ml) were added. Medium replacement was continued once every two days.
  • This structure was composed of Pan-cytokeratin-positive epithelial cells, and Keratin 18-positive ductal epithelium, AQP5-positive acinar cells, and ⁇ -SMA-positive myoepithelial cells were arranged with polarity (FIG. 5). Moreover, the result of gene expression profile by RNA-sequence was also similar to the gene expression profile in the salivary gland source at 15 to 18 days of gestation (FIG. 6).
  • ES cell-derived salivary glands were transplanted into mice from which parotid glands had been excised, they were joined with existing excretory ducts, and production of saliva containing salivary secretory proteins was observed (Fig. 8 left: ES cell-derived 30 days after transplantation)
  • the transplantation of regenerated salivary gland was performed according to the method described in WO 2012/108069.
  • the isolated regenerated salivary gland was embedded in a scaffold material (collagen gel), and a guide (a filamentous structure) for promoting formation of a duct of a secretory gland was inserted, and then transplanted to a mouse (FIG. 9A).
  • a scaffold material collagen gel
  • a guide a filamentous structure
  • FIG. 9B Left: Stereomicroscopic image of ES cell-derived salivary gland 30 days after transplantation, in FIG. 9B: H & E stained image, FIG. 9B right: PAS stained image).
  • the transplanted ES cell-derived salivary gland contained nerves and blood vessels from the recipient (FIG. 10). That is, it has been shown that the regenerated salivary glands produced by the method of the present invention properly engraft the recipient's tissue and cooperate with the surrounding tissue.
  • FIG. 11B when the transplanted ES cell-derived salivary gland was stimulated with the parasympathomimetic agent pilocarpic acid, an increase in salivary secretion was observed (FIG. 11B). That is, it has been shown that the regenerated salivary glands produced by the method of the present invention properly engraft and function in recipient tissues.

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Abstract

Le problème décrit par la présente invention est de fournir un procédé d'induction d'une glande exocrine fonctionnelle à partir de cellules souches pluripotentes. La solution selon l'invention porte sur un procédé de production in vitro d'une glande exocrine fonctionnelle, ledit procédé comprenant : (i) une étape de culture d'un agrégat cellulaire contenant un ectoderme oral dans des conditions telles que l'expression de Sox9 et Foxc1 est induite ; et (ii) une étape de culture supplémentaire de tout ou partie de l'agrégat cellulaire, qui a été cultivé à l'étape (i), dans un milieu contenant FGF7 et FGF10.
PCT/JP2018/037678 2017-11-10 2018-10-10 Procédé de production in vitro d'une glande exocrine fonctionnelle, et glande exocrine ainsi produite Ceased WO2019093047A1 (fr)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012108069A1 (fr) * 2011-02-09 2012-08-16 株式会社オーガンテクノロジーズ Procédé pour produire une ébauche d'organe régénératrice pourvue d'un guide pour transplantation, composition contenant une ébauche d'organe régénératrice pourvue d'un guide pour transplantation produite par celui-ci, et procédé pour transplanter une ébauche d'organe régénératrice pourvue d'un guide pour transplantation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012108069A1 (fr) * 2011-02-09 2012-08-16 株式会社オーガンテクノロジーズ Procédé pour produire une ébauche d'organe régénératrice pourvue d'un guide pour transplantation, composition contenant une ébauche d'organe régénératrice pourvue d'un guide pour transplantation produite par celui-ci, et procédé pour transplanter une ébauche d'organe régénératrice pourvue d'un guide pour transplantation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHATZELI L. ET AL.: "Fgf10 and Sox9 are essential for the establishment of distal progenitor cells during mouse salivary gland development", DEVELOPMENT, vol. 144, no. 12, 15 June 2017 (2017-06-15), pages 2294 - 2305, XP055607733 *
MATTISKE D. ET AL.: "The role of the forkhead transcription factor, Foxcl, in the development of the mouse lacrimal gland", DEV. DYN., vol. 235, no. 4, April 2006 (2006-04-01), pages 1074 - 1080, XP055315749, DOI: doi:10.1002/dvdy.20702 *
OCHIAI H. ET AL.: "BMP4 and FGF strongly induce differentiation of mouse ES cells into oral ectoderm", STEM CELL RESEARCH, vol. 15, no. 2, September 2015 (2015-09-01), pages 290 - 298, XP029291313, DOI: doi:10.1016/j.scr.2015.06.011 *
TANAKA J. ET AL.: "Generation of orthotopically functional salivary gland from embryonic stem cells", NATURE COMMUNICATIONS, vol. 9, no. 1, 4216, 11 October 2018 (2018-10-11), pages 1 - 13, XP055607738 *

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