WO2016039278A1 - Procédé de production de glande sécrétoire - Google Patents

Procédé de production de glande sécrétoire Download PDF

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WO2016039278A1
WO2016039278A1 PCT/JP2015/075286 JP2015075286W WO2016039278A1 WO 2016039278 A1 WO2016039278 A1 WO 2016039278A1 JP 2015075286 W JP2015075286 W JP 2015075286W WO 2016039278 A1 WO2016039278 A1 WO 2016039278A1
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cells
tissue
animal
embryoid body
gland
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PCT/JP2015/075286
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Japanese (ja)
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孝 辻
美帆 小川
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株式会社オーガンテクノロジーズ
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Priority to JP2016547423A priority Critical patent/JP6666844B2/ja
Publication of WO2016039278A1 publication Critical patent/WO2016039278A1/fr

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    • 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

Definitions

  • the present invention relates to a method for producing a secretory gland and the produced secretory gland.
  • stem cell technology With the recent development of stem cell technology, a technology for inducing cells constituting various tissues of living bodies from stem cells has been established.
  • biological organs have a three-dimensional arrangement of multiple types of functional cells, and the development proceeds by the interaction of each cell. It is very difficult to induce artificially.
  • a technique for constructing a regenerative organ that can be immediately transplanted has not been developed yet.
  • organ primordia such as hair follicle primordia using epithelial cells and mesenchymal cells derived from organ primordia in vivo ("organ primordium").
  • organ primordium epithelial cells and mesenchymal cells derived from organ primordia in vivo
  • the present inventors prepared secretory gland primordia such as salivary gland and lacrimal gland using the organ primordium method described above, and after culturing after inserting a guide into the primordia, A method for producing an organ primordium having a conduit that can be combined with the conduit is also shown (Patent Document 1).
  • Non-Patent Document 1 and Patent Document 1 described above are very promising techniques for constructing transplantable regenerative organs, but use organ primordium in the living body as a material for regenerative organs. It is difficult to apply when it is difficult to collect materials. Accordingly, the present inventors have attempted to develop a technique for efficiently producing secretory glands from pluripotent stem cells with respect to regeneration of secretory glands.
  • the present inventors have produced an embryoid body using pluripotent stem cells, combined with a scaffold material, and then transplanted to an animal.
  • the inventors have found that secretory glands can be produced, and have completed the present invention.
  • the present invention is a method for producing a secretory gland, The following steps: (A) preparing a conjugate comprising (A) and (B) below; (A) all or part of an embryoid body (B) scaffold material (b) implanting the conjugate prepared in step (a) into an animal; and (C) producing a secretory gland derived from the conjugate in the animal,
  • a method comprising:
  • the animal is a non-human animal.
  • the non-human animal is a non-human immunodeficient animal.
  • the embryoid body is an embryoid body prepared from iPS cells or ES cells.
  • the embryoid body is stimulated with a physiologically active substance capable of activating the Wnt pathway.
  • the Wnt path is a classic Wnt path.
  • the “physiologically active substance capable of activating the Wnt pathway” is Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt6, Wnt7b, Wnt8a, Wnt8b, Wnt10b, and TGF-. It is selected from the group consisting of ⁇ .
  • the “physiologically active substance capable of activating the Wnt pathway” may be, for example, a Wnt receptor agonist.
  • the scaffold material is a collagen gel.
  • the transplant is a transplant under the renal capsule.
  • a secretory gland can be frequently produced in a teratoma derived from a pluripotent stem cell.
  • the secretory gland produced by the method of the present invention is used as an organ for transplantation for restoring its function, for example, when the function of the secretory gland such as salivary gland is reduced or lost innate or acquired in the living body. Can be used. In this case, it is also possible to use a technique as described in Patent Document 1 above.
  • FIG. 1 shows a tissue image of a teratoma formed when iPS cells in a single cell state are transplanted into a living body.
  • FIG. 1A shows a weakly enlarged photograph of an HE image of a tissue section. The same lot tissue is divided into a plurality of tissues, and two sections are shown. The position of a region showing typical histological characteristics in this tissue is indicated by af.
  • FIG. 1B shows a strongly magnified image of the af region shown in FIG. 1A.
  • a is the respiratory system
  • b is the neuroepithelial system
  • c is the mesodermal connective tissue
  • d is the respiratory system (bronchi)
  • e is the upper digestive system
  • f was distinguished from the ectodermal deciduous system partially containing transitional epithelium. Histological analysis of all three cases was performed, and only one very small secretory gland was observed (d).
  • FIG. 2 shows a tissue image of teratoma formed when iPS cells are in an embryoid body and transplanted in vivo.
  • FIG. 2A shows a weakly enlarged photograph of an HE image of a tissue section. The same lot tissue is divided into a plurality of tissues, and two sections are shown. The representative histological features of this tissue are shown, and the position of the region where the exocrine gland-like structure is recognized is indicated by ad.
  • FIG. 2B shows a strongly magnified image of the a-d region shown by FIG. 2A. It was found that exocrine gland-like tissue structures (dot gland box) were concentrated in all transplants to be analyzed.
  • FIG. 3 shows an HE-stained image of a teratoma formed by transplanting an embryoid body subjected to Wnt10b stimulation into a living body.
  • the left figure shows skin-like tissue containing ectodermal cyst hair follicles, and the right figure shows mucosal-like tissue.
  • FIG. 4A shows a schematic diagram of a method for forming a teratoma enclosing various organs, which is an embodiment of the present invention.
  • Embryoid bodies were formed from iPS cells, arranged three-dimensionally in a collagen gel, and then transplanted into adults to form teratomas.
  • FIG. 4B shows the classification of the various organs formed in the teratoma.
  • Each of the figures a to d shows typical histological images of a: skin-like tissue, b: mucosal-like tissue, c: cyst tissue consisting of transitional epithelium, and d: cyst tissue consisting of endoderm epithelium.
  • Cyst cyst cavity
  • Epi epithelial tissue
  • Der dermal tissue
  • Ad adipose tissue
  • LP lamina intestinal
  • SM smooth muscle tissue.
  • the cystic epithelial tissue of c is composed of a single-layered columnar epithelium including squamous stratified epithelium (*) and goblet cells (arrow).
  • FIG. 4C shows the frequency of organs induced by teratoma derived from embryoid bodies subjected to Wnt10b stimulation (Dark bar), and the frequency of organs induced by teratomas derived from embryoid bodies not stimulated by Wnt10b (Light bar). It is the graph which compared.
  • FIG. 4D shows the number of hair follicle formation (Dark bar) induced per gram of teratoma derived from embryoid bodies subjected to Wnt10b stimulation, and the hair follicle induced per gram of teratoma derived from embryoid bodies not subjected to Wnt10b stimulation. It is the graph which compared the number of formation (Light bar).
  • FIG. 5 shows a histological image of a secretory gland-like structure induced by a teratoma derived from an embryoid body subjected to Wnt10b stimulation.
  • the left figure shows an HE-stained image
  • the middle figure shows an immunostained image with an anti-amylase antibody
  • the right figure shows an immunostained image with an anti-E-cadherin antibody.
  • the arrow in the figure indicates the cytoplasmic granular amylase in the acinar-like tissue.
  • the dotted line in the figure indicates the area of the acinar tissue, and “D” in the figure indicates the adjacent duct-like structure.
  • pluripotent stem cell refers to a cell having both the pluripotency capable of differentiating into any cell of the living body and the self-replicating ability capable of maintaining the pluripotency even after differentiation and proliferation, for example, ES cell And iPS cells.
  • ES cell Embryonic Stem cells
  • stem cell line made from an inner cell mass belonging to a part of an embryo at the blastocyst stage, which is an early stage of animal development, and is extremely numerous. It has the pluripotency capable of differentiating into cells and the self-replicating ability that can maintain pluripotency even after dividing proliferation.
  • the origin of ES cells that can be used in the present invention is not particularly limited, and ES cells derived from the inner cell mass of any animal can be used.
  • ES cells derived from the inner cell mass of humans, mice, rats, pigs, monkeys can be used as the origin of ES cells.
  • iPS cells induced Pluripotent Stem cells
  • ES cells ES cells
  • somatic cells A cell that has sex and self-replicating ability to maintain pluripotency even after dividing proliferation.
  • the origin of iPS cells that can be used in the present invention is not particularly limited, and iPS cells derived from any animal can be used.
  • iPS cells derived from humans, mice, rats, pigs, and monkeys can be used as the origin of iPS cells.
  • somatic cells derived from iPS cells that 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 iPS cell induction method that can be used in the present invention is not particularly limited, and any method can be used as long as it 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 culture environment or medium known in the art or a culture environment or medium equivalent thereto.
  • a culture environment or medium known in the art or a culture environment or medium equivalent thereto For example, mouse embryo fibroblasts (MEF) can be used as feeder cells for culturing pluripotent stem cells.
  • MEF mouse embryo fibroblasts
  • 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.
  • teratoma is a well-differentiated germ cell tumor having a bi- or tri-germ component, and is also referred to as a malformed species.
  • the “teratoma” in the present invention also includes a structure histologically similar to a deformed species that can be generated when pluripotent stem cells are transplanted into a living body. Teratomas can occur naturally in vivo, but can also be generated artificially by transplanting pluripotent stem cells into animals.
  • the site for transplanting cells into an animal is not particularly limited, and for example, it can be transplanted under the kidney capsule, subcutaneous, or testis of an animal.
  • the method for transplanting cells into an animal is not particularly limited.
  • the kidney capsule of a mouse an incision of 2 to 3 mm is made in the kidney capsule, and the kidney capsule and the kidney parenchyma are peeled off. In the meantime, cells can be transplanted.
  • a method for confirming that teratoma is formed in an animal transplanted with pluripotent stem cells is not particularly limited.
  • a laparotomy is performed 3 to 4 weeks after transplantation to form a tumor in appearance. This can be confirmed by visual inspection.
  • analyzing the tumor histologically it is possible to confirm the formation of trigerminal tissue that is characteristic of teratomas.
  • the “secretory gland” in the present invention may be an exocrine gland or an endocrine gland.
  • exocrine glands include lacrimal gland, salivary gland, cardia gland, pyloric gland, gastric gland, intestinal crypt, prostate, sweat gland, sebaceous gland, etc.
  • endocrine gland include pituitary gland, thyroid gland, parathyroid , Adrenal glands, gonadal and the like.
  • pancreatic cell since the pancreatic cell has a function as an exocrine gland (secretion of trypsin, amylase, etc.) and a function as an endocrine gland (secretion of insulin, glucagon, somatostatin, etc.), it is included in the secretory gland in the present invention.
  • organ primordium refers to a region of an embryo or a structure of an embryo that is determined to develop in a specific organ as the developmental stage proceeds in a living body, and is simply referred to as “primitive primordium”. There is also. Almost all organs in a living body are generated from organ primordia derived from epithelial stem cells and mesenchymal stem cells by a fetal development program, and develop into a predetermined position and a predetermined number.
  • the method for confirming that the target secretory gland is formed in the teratoma is not particularly limited.
  • the teratoma is opened and the skin appendages (for example, hair follicles, nails, sebaceous glands, sweat glands, It can be confirmed by looking for a structure that appears to be full-thickness skin with mammary glands).
  • a tissue section of teratoma can be prepared and confirmed to be a predetermined organ from the tissue structure.
  • it can be analyzed by an in situ hybridization method that a gene expressed in each organ is expressed at an appropriate site.
  • embryoid body refers to a cell mass formed when pluripotent stem cells such as ES cells and iPS cells are cultured in suspension. Embryoid bodies may take the form of embryos and may be composed of various tissues.
  • the method for producing an embryoid body that can be used in the present invention is not particularly limited. For example, a method of seeding pluripotent stem cells on a low-adhesion plate, or hanging a droplet of a cell suspension of pluripotent stem cells. The hanging drop method according to, and the method of suspension culture while shaking the culture dish of pluripotent stem cells can be used.
  • iPS cells when an embryoid body is prepared by seeding iPS cells on a low adhesion plate, iPS cells are 1500 cells to 10000 cells / 200 ⁇ l / well, more preferably 2000 cells to 4000 cells / 200 ⁇ l / well.
  • An embryoid body can be produced by seeding on an adhesive plate and culturing. If the number of cells to be seeded is less than 1500 cells / 200 ⁇ l / well, the embryoid body may not be formed properly, and if it exceeds 10,000 cells / 200 ⁇ l / well, necrosis due to lack of nutrition may occur inside the embryoid body.
  • the embryoid bodies used in the present invention are from the start of suspension culture, but, for example, those from 5 to 9 days from the start of suspension culture can be suitably used. .
  • an embryoid body when used for transplantation, all or part of the embryoid body can be used for transplantation.
  • the embryoid body can be used for transplantation as it is, or only part of the embryoid body can be used for transplantation.
  • the method for separating only the surface tissue from the embryoid body is not particularly limited.
  • the surface tissue of the embryoid body can be physically collected by microsurgery using a syringe under a stereomicroscope.
  • “scaffold material” expresses various cell functions such as cell adhesion, proliferation, differentiation, activation, migration, migration, and morphological change by contacting the material with the cell on or within the material. It refers to all materials that can be promoted and is not particularly limited as long as it is suitable for transplanting pluripotent stem cells.
  • a collagen gel can be used as the scaffold material, and preferably, a type I collagen gel, a type III collagen gel, a type IV collagen gel, and a matrigel can be used.
  • Transplanting pluripotent stem cells in a scaffold material prevents pluripotent stem cells from dissipating in the transplanted tissue and becomes a scaffold for tissue survival
  • Secretory glands can be produced in the teratoma.
  • the embryoid body can be transplanted in a collagen gel while maintaining a desired three-dimensional arrangement.
  • transplantation is performed in a state where the surface tissues of the respective embryoid bodies are in contact with each other, whereby a secretory gland can be more efficiently produced in the teratoma.
  • a method for producing a conjugate including “all or part of an embryoid body” and a scaffold material It may be used for transplantation after the scaffold material is combined with “all or part of the embryoid body” in vitro.
  • the scaffold material is introduced into the body and then “all or part of the embryoid body”. May be combined by injection and transplantation may be performed.
  • the collagen gel and the embryoid body are put into a sol-like collagen gel and solidified. A combination with all or a part of can be produced.
  • the “physiologically active substance capable of activating the Wnt pathway” may be, for example, a physiologically active substance capable of activating the classic Wnt pathway (also referred to as ⁇ -catenin pathway). It may be a physiologically active substance that can activate the pathway (in-plane cell polarity pathway; also referred to as PCP pathway or Ca2 + pathway). Examples of physiologically active substances that can activate the classical Wnt pathway include Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt6, Wnt7b, Wnt8a, Wnt8b, Wnt10b, and TGF- ⁇ .
  • physiologically active substances that can activate the classical Wnt pathway include Wnt4, Wnt5a, and Wnt11. That Wnt10b can activate the classical Wnt pathway ( ⁇ -catenin pathway) is described in, for example, Maksim V. et al. Prikus et al. (Science 332, 586 (2011)), Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt6, Wnt7b, Wnt8a, Wnt8b can activate the classic Wnt pathway ( ⁇ -catenin pathway), Wnt4, The fact that Wnt5a and Wnt11 can activate the non-classical Wnt pathway is described in, for example, Kemp et al. (Functional Development and Embrology 1 (1), 1-13 (2007)), TGF- ⁇ stabilizes the expression of ⁇ -catenin in derma fibroblast, for example, Sato (Acta derm6; : 300-307).
  • the embryoid body is stimulated by a “bioactive substance capable of activating the Wnt pathway”, but in addition to the “bioactive substance capable of activating the Wnt pathway”,
  • the embryoid body can be additionally stimulated by various cytokines known to be involved in development and various factors used for induction of embryo differentiation. Examples of such cytokines and factors include Hox protein, Dlx protein, TGF ⁇ , SAG, insulin, BMP4, Cyclopamine, FGF2, ⁇ FGF, SB431542, BMP4, Nodal, apotransferrin, FGF8, Endotheline, LDN193189, EFN9902, EFN9902 , Activin, retinoic acid, or any combination thereof.
  • cytokines and factors may be administered at the same timing as the “bioactive substance capable of activating the Wnt pathway” when stimulating the embryoid body, and “physiological activation capable of activating the Wnt pathway” It may be administered at a different timing from the “substance”.
  • a protein whose expression is increased in the differentiation process from an embryo to the tissue in a living body for example, the protein exemplified above
  • the induction efficiency to the target tissue can be increased.
  • the type of animal into which cells are transplanted is not particularly limited, and any animal can be used for transplantation.
  • non-human animals such as pigs, cows, monkeys, baboons, dogs, cats, rats, and mice can be used for transplantation to avoid ethical problems that arise when cells are transplanted into humans.
  • rejection due to the immune function of the living body can be prevented, and a teratoma can be produced efficiently.
  • a teratoma derived from a cell of another species can be produced in the living body of the non-human immunodeficient animal.
  • a human cell-derived teratoma can be produced in the living body of the non-human immunodeficient animal.
  • an “immune-deficient animal” refers to an animal that lacks part or all of the immune function of a living body, and the type of immune function that is deficient is not particularly limited, but is derived from other species of animals transplanted into the living body. Animals lacking immune function are preferred so as not to exclude the cells or tissues.
  • SCID mice, nude mice, NOD mice, NOD-SCID mice, IL-2Rg knockout mice, RAG2 knockout mice, NOG mice, RAG2 / IL-2Rg double knockout mice can be used as long as they are immunodeficient mice.
  • an immunodeficient rat can be used as a SCID rat.
  • IL-2rg knockout pigs can be used for immunodeficient pigs.
  • a method for extracting a desired organ from the teratoma is not particularly limited, but for example, it can be extracted by microsurgery.
  • first, second, etc. may be used to represent various elements, but these elements should not be limited by those terms. These terms are only used to distinguish one element from another, for example, the first element is referred to as the second element, and similarly, the second element is the first element. Can be made without departing from the scope of the present invention.
  • Example 1 Formation of secretory gland by in vivo transplantation of a conjugate containing an embryoid body 1.
  • Materials and Methods (1) Experimental animals C.I. B-17 / lcr-scid / scid Jcl mice were purchased from Claire (Tokyo, Japan). Further, scid / scid hr / hr (SHO) mice were purchased from Charles River (Kanagawa, Japan). Mouse management and manipulation was in accordance with NIH laboratory animal guidelines. All experiments were conducted with the approval of the Laboratory Animal Care Committee of Tokyo University of Science.
  • the medium was changed every day, and subculture was performed using a solution obtained by adding 0.25% Trypsin-1 mM EDTA (Invitrogen) to D-PBS ( ⁇ ) (Nacalai Tesque) on the second day after subculture.
  • SNLP76.7-4 feeder cells are cultured on a gelatin-coated dish (referred to as gelatin-coated dish in this example) at 37 ° C. for 2 hours or more with a 0.1% gelatin aqueous solution. did.
  • DMEM Dulbecco's Modified Eagle's Medium
  • 7% fetal bovine serum 50 units / ml penicillin, 50 ⁇ s / ml penicillin Medium was used.
  • Mitomycin C was added to the above medium to a final concentration of 12 ⁇ g / ml, and SNLP76.7-4 feeder cells were reacted at 37 ° C.
  • iPS cells 1 ⁇ 10 6 cells from which feeder cells have been removed by sorting are suspended in 30 ⁇ l type I collagen gel (Nitta Gelatin), and 10 minutes or more in a CO 2 incubator at 37 ° C. Cells were encapsulated in the collagen gel by solidifying the collagen gel. These iPS cells encapsulated in collagen gel were used for transplantation.
  • embryoid bodies When embryoid bodies are used for transplantation, 48 embryoid bodies prepared by the method described in (3) from 3000 cells of iPS cells are included in 30 ⁇ l type I collagen gel (Nitta gelatin). The collagen gel was solidified in a CO 2 incubator at 37 ° C. for 10 minutes or longer to encapsulate the embryoid body in the collagen gel. These embryoid bodies encapsulated in collagen gel were used for transplantation.
  • mice 7-10 weeks old C.I. B-17 / lcr-scid / scid Jcl mice were anesthetized by intraperitoneal injection of 5 ng / ml pentobarbital, and iPS cells encapsulated in the aforementioned collagen gel, or embryoid bodies prepared from iPS cells, Implanted under the skin. On the 28th day after transplantation, the formed teratomas were extracted.
  • ectodermal tissues such as epidermis and neural rosette structure, mesodermal tissues such as muscle, bone and cartilage, and endoderm tissues such as intestinal tract-like epithelium was observed in the teratoma (Fig. 1). .
  • secretory glands only a very small secretory gland was observed in one case among all three cases subjected to histological analysis.
  • an embryoid body that is an aggregate of iPS cells is prepared by the method described in (3), and the kidney capsule of SCID mice is formed by the method described in (4). Transplanted. Teratomas formed 28 days after transplantation were analyzed histologically by HE staining. As a result, in the teratoma formed in the group transplanted with embryoid bodies, secretory gland-like tissue structures were confirmed with high frequency (FIG. 2). Furthermore, a luminal structure consisting of columnar epithelium was found adjacent to the secretory gland-like structure, suggesting that these are part of the ductal structure adjacent to the glandular tissue. In other words, by mixing and transplanting the iPS cells in the embryoid body state with the scaffold material, the secretory glands can be induced more efficiently than in the case of transplanting the single-cell iPS cells. Indicated.
  • Example 2 Formation of a secretory gland by in vivo transplantation of a conjugate containing an embryoid body 1.
  • Materials and Methods (1) Experimental animals C.I. B-17 / lcr-scid / scidJcl mice were purchased from Claire (Tokyo, Japan) and scid / scid hr / hr (SHO) mice were purchased from Charles River (Kanagawa, Japan). Mouse management and manipulation was in accordance with NIH laboratory animal guidelines. All experiments were conducted with the approval of the Laboratory Animal Care Committee of Tokyo University of Science.
  • the medium was changed every day, and on the second day after subculture, subculture was performed using a solution in which 0.25% Trypsin-1 mM EDTA (Invitrogen) was added to D-PBS ( ⁇ ) (Nacalai Tesque).
  • SNLP76.7-4 feeder cells were cultured on gelatin-coated dishes in 0.1% gelatin aqueous solution at 37 ° C. for 2 hours or more.
  • Dulbecco's Modified Eagle's Medium (DMEM without sodium pyruvate; Nacalai Tesque), 7% fat bovine serum, 50 units / mL penicillin, 50 units / mL penicillin Medium was used.
  • Mitomycin C was added to the medium to a final concentration of 12 ⁇ g / ml, and SNLP76.7-4 feeder cells were reacted at 37 ° C. for 2 hours and 15 minutes to treat mitomycin of SNLP76.7-4 feeder cells. .
  • the reacted cells were seeded on a gelatin-coated dish at 2.5 ⁇ 10 4 cells / cm 2 . What was cultured for 24 hours or more after mitomycin treatment was used as a feeder cell for co-culture with iPS cells.
  • Wnt10b 0.1 mg / mL Wnt10b (R & D) in PBS was added as a stock to the medium for iPS cells to 1 ⁇ g / mL.
  • Half of the Well medium in which embryoid bodies were formed was discarded, and half of the medium was replaced with a medium for iPS cells containing Wnt10b (final concentration 500 ng / mL).
  • Embryoid bodies in Wnt10b-added medium were cultured for 24 hours in a CO 2 incubator.
  • the epidermis layer of the cyst is a typical skin because the basal cell layer, curved cell layer, granule layer, and stratum corneum are regularly arranged, and the stratum corneum is separated into a clear layer toward the cyst cavity. It was shown to be epidermal tissue. It was shown that the hair shaft grew from the opening of the hair follicle toward the cyst lumen (FIG. 3, black arrow). The connection site between the sebaceous gland and the hair follicle is the hair follicle funnel or the opening of the pore (FIG. 3, left white arrowhead), suggesting that sebum can be secreted into the outer epidermis through the pore.
  • cyst epithelium From the histological characteristics of the formed cyst epithelium and surrounding stromal tissue, skin-like (squamous keratinized epithelial layer / dermis layer / fatty or striated muscle), mucosal-like (squamous stratified horn epithelium / Classified into mucosal lamina (striated muscle), transitional epithelium (transitional form of flat stratified keratinized epithelium and monolayer columnar epithelium), endoderm epithelial (monolayer columnar epithelium / mucosal lamina / striated muscle) The composition ratio was measured (FIG. 4B).
  • iPS cells in embryoid state can be stimulated with a physiologically active substance that can activate the Wnt pathway, and then transplanted to animals to induce secretory glands more efficiently. It was shown that.

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Abstract

L'invention concerne un procédé pour produire efficacement une glande sécrétoire à partir de cellules souches pluripotentes. Ce procédé de production d'une glande sécrétoire se caractérise en ce qu'il comprend les étapes suivantes (a) à c) : (a) une étape de préparation d'un conjugué qui comprend la totalité ou une partie d'un corps embryoïde et un matériau d'échafaudage, (b) une étape de transplantation du conjugué préparé dans l'étape (a) dans un animal, et (c) une étape de production, dans l'animal, d'une glande sécrétoire dérivée de ce conjugué.
PCT/JP2015/075286 2014-09-08 2015-09-07 Procédé de production de glande sécrétoire WO2016039278A1 (fr)

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Cited By (1)

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WO2019039376A1 (fr) * 2017-08-22 2019-02-28 国立大学法人横浜国立大学 Procédé de production de multiples primordia de follicules pileux régénérés, procédé de production de feuille contenant un tissu de follicules pileux, kit de régénération des cheveux et procédé pour la recherche par criblage d'un promoteur de la pousse des cheveux ou d'un inhibiteur de la pousse des cheveux

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WO2011071085A1 (fr) * 2009-12-08 2011-06-16 国立大学法人東京大学 Procédé pour produire des cellules induites pour être différenciées à partir de cellules souches pluripotentes
JP2012120486A (ja) * 2010-12-08 2012-06-28 Kinki Univ 免疫不全動物を用いた細胞の製法

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Publication number Priority date Publication date Assignee Title
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JP7092310B2 (ja) 2017-08-22 2022-06-28 国立大学法人横浜国立大学 複数の再生毛包原基の製造方法、毛包組織含有シートの製造方法、毛髪再生用キット及び発毛促進又は抑制物質をスクリーニングする方法

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