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WO2020209280A1 - Cellule dérivée de l'épithélium destinée à être utilisée dans la production de germes de follicules pileux régénérés - Google Patents

Cellule dérivée de l'épithélium destinée à être utilisée dans la production de germes de follicules pileux régénérés Download PDF

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WO2020209280A1
WO2020209280A1 PCT/JP2020/015788 JP2020015788W WO2020209280A1 WO 2020209280 A1 WO2020209280 A1 WO 2020209280A1 JP 2020015788 W JP2020015788 W JP 2020015788W WO 2020209280 A1 WO2020209280 A1 WO 2020209280A1
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cells
epithelial
derived
itgb5
cell
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Japanese (ja)
Inventor
孝 辻
真 武尾
尚一 岡本
美帆 小川
悠 森
恭介 岸本
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Kyocera Corp
Organ Technologies Inc
RIKEN
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Kyocera Corp
Organ Technologies Inc
RIKEN
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Priority to JP2021513663A priority Critical patent/JP7792089B2/ja
Publication of WO2020209280A1 publication Critical patent/WO2020209280A1/fr
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Priority to JP2025160730A priority patent/JP2025175203A/ja
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    • 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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0666—Mesenchymal stem cells from hair follicles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/119—Other fibroblast growth factors, e.g. FGF-4, FGF-8, FGF-10
    • 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
    • C12N2501/155—Bone morphogenic proteins [BMP]; Osteogenins; Osteogenic factor; Bone inducing factor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to epithelial-derived cells that can be used in the production of regenerated hair follicle primordium.
  • Non-Patent Document 1 In order to realize hair regenerative medicine, it is necessary to acquire epithelial-derived stem cells and mesenchymal stem cells having the ability to induce hair follicles, which are necessary for reconstructing hair follicle primordium, and to proliferate them in vitro.
  • mesenchymal stem cells it is known that dermal papilla cells that can be obtained from dermal papilla have a hair follicle-inducing ability, and it has been shown that they can be obtained by culturing in vitro. (Non-Patent Document 1).
  • Lgr5 Leucine-rich repeat-contining G-protain coupled recipient 5
  • intestinal villi are regenerated from the cultured Lgr5-positive stem cells. It has been shown that it is possible (Non-Patent Document 2).
  • Non-Patent Document 3 stem cells having a hair follicle-inducing ability exist in the hair follicle (Non-Patent Document 3), and for in vitro proliferation, a cell surface marker CD34 (3D culture of mouse hair follicle-derived cells) ( Although there is a report on a method for proliferating CD34 antigen) -positive and CD49f (integrin ⁇ 6) -positive stem cells (Non-Patent Document 4), its organ regeneration ability has not been sufficiently proved.
  • CD34 3D culture of mouse hair follicle-derived cells
  • An object of the present invention is to provide a novel epithelial-derived cell that can be used for producing a regenerated hair follicle primordium.
  • the present invention includes the following features: [1] Epithelial-derived cells Expressing detectable levels of integrin ⁇ 5 (Itgb5), Characterized by Epithelial-derived cells.
  • the epithelial-derived cell according to [1]. Further expresses detectable levels of cell surface markers CD34 and / or CD49f. Characterized by Epithelial-derived cells.
  • the epithelial-derived cell according to [1] or [2]. Further expresses detectable levels of cell surface markers CD34 and CD49f, Characterized by Epithelial-derived cells.
  • the epithelial-derived cell population according to any one of [5] to [8]. It is characterized by being used for the production of regenerated hair follicle primordium, Cell population.
  • a method for producing a regenerated hair follicle primordium A step of obtaining a regenerated hair follicle primordium by culturing the epithelial-derived cell population according to any one of [5] to [8] while contacting the mesenchymal cell-derived cell population. Production method.
  • a novel epithelial-derived cell that can be used for producing a regenerated hair follicle primordium is provided.
  • FIG. 1 shows an analysis diagram of mouse Itgb5-positive cells after separation (right frame) from cultured mouse skin epithelial-derived cells (left frame) by flow cytometry.
  • FIG. 2 shows the organ primordium method (A) used to examine the differentiation potential of mouse Itgb5-positive cells in hair follicles and the histological analysis (B) showing the differentiation potential of mouse Itgb5-positive cells.
  • FIG. 3 shows a cell population (A) expressing CD34 and CD49f of cultured mouse skin epithelial-derived cells and a cell population expressing CD34 and Itgb5 before and after isolation (B) (left frame: before separation, center frame: Itgb5 negative / positive cells).
  • FIG. 4 shows the organ primordium method used to examine the functional analysis of Itgb5-positive cells for hair growth persistence, typical hair growth examples after transplantation (white arrowhead: hair growth), and mouse Itgb5-negative cells and mice.
  • the hair growth rate of Itgb5 negative / positive cells and the rate at which the hair cycle is 3 times or more (hair cycle 3 times or more) are shown.
  • FIG. 5 shows a cell population expressing CD34 and CD49f of cultured mouse skin epithelial-derived cells (A), a cell population expressing CD34 and Itgb5 (gating the black frame (1) of A, B), and a cell population expressing CD86 and Itgb5. (The black frame (2) of B is gating, C) is shown.
  • the present invention relates to novel epithelial-derived cells.
  • the epithelial-derived cells of the present invention are epithelial-derived cells prepared from animal (typically human) epithelial cells and are characterized by expressing detectable levels of Itgb5.
  • the term "detectable level” means that when a cell surface marker is detected by a method known to those skilled in the art, its expression is detectable with respect to the negative control in each method. Point to. Therefore, the degree of expression does not matter, and in the present invention, even a small expression level can correspond to a "detectable level" as long as the expression is detected with respect to a negative control. ..
  • methods used for detecting cell surface marker mRNA messenger RNA
  • cDNA complementary DNA
  • protein include color development, luminescence, fluorescence, UV (Ultraviolet) or RI (Radioisotope). ..
  • flow cytometry Specifically, flow cytometry, Northern blotting, Western blotting, RT-PCR (Reverse transcrition-polymerase chain reaction), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), enzyme-linked immunosorbent assay, enzyme-linked immunosorbent assay Immunoassay spot analysis (ELISPOT analysis), competitive enzyme immunoassay (Enzyme multiplicated immunoassay technology), radioallergen adsorption (RAST) test, radioimmunoassay, radiobinding assay, gel filtration chromatography, agarose electrophoresis, acrylamide electrophoresis, etc.
  • ELISPOT analysis enzyme-linked immunosorbent assay spot analysis
  • RAST radioallergen adsorption
  • radioimmunoassay radiobinding assay
  • gel filtration chromatography agarose electrophoresis
  • acrylamide electrophoresis etc.
  • the technique used to detect cell surface markers is flow cytometry.
  • the epithelial-derived cells of the invention further express other epithelial cell markers at detectable levels.
  • Such other epithelial cell markers include, but are not limited to, CD34, CD49f, integrin ⁇ 5, integrin ⁇ 1, integrin ⁇ 6, integrin ⁇ 8, TGF ⁇ (Transforming growth factor beta) receptor1, TGF ⁇ receptor2, TGF ⁇ receptor2.
  • the epithelial-derived cells of the invention further express detectable levels of CD34 and CD49f.
  • the epithelial-derived cells of the invention do not express detectable levels of specific cell surface markers. Examples of such cell surface markers include PDGFR ⁇ , CD86 and the like. In one embodiment, the epithelial-derived cells of the invention do not express detectable levels of PDGFR ⁇ , and CD86.
  • the epithelial-derived cells of the present invention can be obtained by culturing cells derived from animal epithelial tissue, for example, in a basal medium containing a specific expression-inducing substance according to a conventional method.
  • the basal medium refers to a medium containing a carbon source, a nitrogen source, an inorganic salt, etc., which are essential for culturing cells, especially mammalian cells (for example, humans).
  • a medium that is generally used for culturing animal cells can be used, and the medium is not limited to this, such as Eagle's medium.
  • MEM minimal essential medium
  • DMEM Dalbeco's modified Eagle's medium
  • MEM- ⁇ minimum essential medium ⁇
  • Ham's F-12 and F-10 medium DMEM / F12 medium, Williams medium E, RPMI-1640 medium , MCDB medium, 199 medium, Fisher medium, Iscove's modified Dalveco medium (IMDM), McCoy's modified medium, DEF-CS medium, CnT-PR medium, Advanced DMEM medium, Advanced MEM medium, Advanced DMEM / F12 medium, or a mixture thereof.
  • IMDM Iscove's modified Dalveco medium
  • DEF-CS CnT-PR medium
  • Advanced DMEM medium Advanced MEM medium
  • Advanced DMEM / F12 medium or a mixture thereof.
  • the medium and the like can be mentioned.
  • the production of epithelial-derived cells of the invention uses at least the following as said expression inducer: At least one bone morphogenetic protein (BMP) inhibitor, At least one of fibroblast growth factor (FGF); and at least one of Sonic hedgehog (SHH) or SHH agonist.
  • BMP bone morphogenetic protein
  • FGF fibroblast growth factor
  • SHH Sonic hedgehog
  • the BMP inhibitor used for producing the epithelial-derived cell of the present invention is not particularly limited as long as it can block or inhibit the binding of the BMP molecule to the BMP receptor. Whether or not a molecule or compound has BMP inhibitory activity is determined by measuring the transcriptional activity of BMP using a method known to those skilled in the art (Zilberberg et al., BMC Cell Biol, 8:41, 2007.). Can be determined by.
  • BMP inhibitors used in the production of epithelial-derived cells of the present invention are not limited to, for example, Noggin, Dorsomorphin, Chordin, Follistatin. , And Ectodin and the like.
  • one type of BMP inhibitor may be used alone, or a plurality of types may be used in combination.
  • the concentration of the BMP inhibitor used in the production of the epithelial-derived cells of the present invention is 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, more preferably 1 ng / mL to 100 ng / mL. It can be in the range of mL.
  • the fibroblast growth factor (FGF) used in the production of the epithelial-derived cells of the present invention is not limited to, for example, FGF-1, FGF-2, FGF-3, FGF-4, and the like. FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10 and the like can be mentioned.
  • FGF-1, FGF-2, FGF-3, FGF-4, and the like FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10 and the like can be mentioned.
  • one type of fibroblast growth factor may be used alone, or a plurality of types may be used in combination.
  • at least two fibroblast growth factors are used in combination as fibroblast growth factors.
  • At least FGF-7 is used as the fibroblast growth factor.
  • the concentration of fibroblast growth factor used in the production of the epithelial-derived cells of the present invention is 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, more preferably 1 ng / mL to. It can be in the range of 100 ng / mL.
  • the SHH agonist used in the production of epithelial-derived cells of the present invention is not particularly limited as long as it can enhance SHH-mediated signal transduction. Whether or not a molecule or compound has SHH agonist activity can be determined using, for example, a method as suggested in JP-A-2009-213442.
  • SHH agonists used in the production of epithelial-derived cells of the present invention include, but are not limited to, SAG (Smoothened hedgehog) or purmorphamine.
  • the concentration of SHH or SHH agonist used in the production of the epithelial-derived cells of the present invention is 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, more preferably 1 ng / mL to 100 ng. It can be in the range of / mL.
  • At least one receptor tyrosine kinase ligand is further used to produce the epithelial-derived cells of the invention. Further use of a receptor tyrosine kinase ligand for the production of epithelial-derived cells of the present invention is preferable in that the engraftment rate when the produced regenerated hair follicle primordium is transplanted into a living body can be significantly improved.
  • Receptor-type tyrosine kinase ligands used in the production of epidermal-derived cells of the present invention are not limited to, for example, EGF (Epidermal Growth Factor), TGF ⁇ (Transforming growth factor ⁇ ), and amphiregulin. , And heparin-binding EGF-like growth factor (HB-EGF).
  • the concentration of the receptor tyrosine kinase ligand used in the production of the epithelial-derived cells of the present invention is 0.1 ng / mL to 1000 ng / mL, preferably 0.3 ng / mL to 300 ng / mL, more preferably 1 ng / mL to. It can be in the range of 100 ng / mL.
  • At least one TGF ⁇ receptor / ALK5 (Anaplastic lymphoma kinase 5) inhibitor is further used for the production of epithelial-derived cells of the present invention.
  • the TGF ⁇ receptor / ALK5 inhibitor used in the production of the epithelial-derived cells of the present invention is not particularly limited as long as it can inhibit the interaction between the TGF ⁇ receptor and ALK5, and is a TGF ⁇ receptor inhibitor or ALK5 inhibitor. May include agents and the like.
  • the TGF ⁇ receptor / ALK5 inhibitor used in the production of epithelial-derived cells of the present invention is not limited to, for example, SB431542, A83-01, ALK5 Inhibitor, D4476, LY364497, SB525334, SD208. Can be mentioned.
  • the concentration of the TGF ⁇ receptor / ALK5 inhibitor used in the production of the epithelial-derived cells of the present invention is 0.001 ng / mL to 1000 ng / mL, preferably 0.01 ng / mL to 100 ng / mL, more preferably 0. It can be in the range of 1 ng / mL to 10 ng / mL.
  • cell culture media for epithelial-derived cells may be used.
  • Other components for optimizing such cell culture media include, but are not limited to, Glutamax TM , B27 supplement (Thermo Fisher Scientific), N2 supplement (Thermo Fisher Scientific), and the like. Items, Rock inhibitors such as Y-27632, and the like can be mentioned.
  • a basal medium in the production of epithelial-derived cells of the invention, a basal medium, a combination of the following additives, and other components for optimizing the cell culture medium are used: nogin,. EGF, FGF-7, FGF-10, SAG.
  • a basal medium in the production of epithelial-derived cells of the invention, a basal medium, a combination of the following additives, and other components for optimizing the cell culture medium are used: Nogin, FGF-7, FGF-10, SAG.
  • cells derived from epithelial tissue to be cultured can typically be prepared from hair follicles, for example, the outermost layer of the outer root sheath of the bulge region (eg, the bulge region).
  • Cells can be prepared from hair follicles, for example, the outermost layer of the outer root sheath of the bulge region (eg, the bulge region).
  • Cells hair matrix bases, or hair follicle epithelium derived from iPS cells (induced Pruripotent Stem cells) or ES cells (Embryonic stem cells) can be used.
  • Epithelial tissues that can be used in the present invention include mammalian primates (eg humans, monkeys, etc.), ungulates (eg pigs, cows, horses, etc.), and small mammalian rodents (eg mice, rats, etc.). It can be collected from various animals such as dogs and cats in addition to rabbits).
  • the conditions used for tissue collection may be applied as they are, and the epithelial tissue may be taken out in an aseptic state and stored in an appropriate storage solution.
  • Preparation of cells derived from epithelial tissue from hair follicles is performed, for example, by first separating hair follicles isolated from surrounding tissues into epithelial tissue and mesenchymal tissue according to their shape. At that time, an enzyme may be used to facilitate the separation. Examples of the enzyme include known enzymes such as dispase, collagenase, and trypsin, and those skilled in the art can appropriately use preferable enzymes.
  • cells derived from epithelial tissue may be used.
  • Cells derived from other than hair follicles are, but are not limited to, differentiated, eg, keratinized, skin or oral mucosa or gingival epithelial cells, preferably skin or mucosa.
  • immature epithelial-derived precursor cells capable of differentiating into keratinized epithelial-derived cells, such as non-keratinized epithelial-derived cells or stem cells thereof, can be mentioned.
  • an oral epithelial-derived cell or a primary cultured cell thereof is used as an epithelial-derived cell is described in JP-A-2008-29756, and the disclosure thereof is incorporated herein by reference as a whole.
  • 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 animals can be used.
  • ES cells derived from the inner cell mass of humans mice, rats, dogs, cats, rabbits, cows, horses, sheep, goats, pigs, or monkeys can be used.
  • IPS cells generally mean pluripotency that can differentiate into a large number of cells such as ES cells by introducing several kinds of genes and / or drugs into somatic cells, and division and proliferation.
  • the present invention is not limited to the above description, and broadly includes cells recognized by those skilled in the art as "iPS cells”.
  • the origin of the iPS cells that can be used in the present invention is not particularly limited, and animal-derived iPS cells can be used.
  • iPS cells derived from humans, mice, rats, dogs, cats, rabbits, cows, horses, sheep, goats, pigs, or monkeys can be used.
  • somatic cells from which the iPS cells 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 induced by any method can be used as long as the iPS cells can be induced from somatic cells. be able to.
  • single cell formation treatment refers to the process of separating a plurality of cells (typically tissues or organs) that are bound or adhered to each other into individual cells.
  • Such a single cell formation treatment may be carried out by a method known to those skilled in the art, and is not limited to this, and can be carried out by, for example, an enzyme treatment. Enzymes that can be used for the enzyme treatment and conditions for use thereof are also known to those skilled in the art, and for example, enzymes such as dispase, collagenase, and trypsin can be used.
  • Single cell formation is preferable from the viewpoint of promoting efficient proliferation of desired epithelial-derived cells.
  • the epithelial tissue-derived cells are cells derived from the epithelial tissue in the bulge region.
  • Such cells can be prepared by collecting tissue from the bulge region epithelium (eg, the outermost layer of the outer root sheath) and subjecting it to a single cell formation process.
  • conditions generally used for culturing animal cells can be adopted, for example, culturing in an incubator at a temperature of about 37 ° C. and a concentration of 5% CO 2 for 5 to 8 days. Can be applied.
  • an antibiotic such as streptomycin may be added to the culture medium as appropriate.
  • an extracellular matrix known to those skilled in the art may be used as appropriate, and for example, Matrigel (registered trademark) (Corning), Type IV collagen, atelocollagen, Type I collagen, Type III collagen and the like can be used. ..
  • the cell population proliferated according to the above is an in vitro culture containing cells expressing Itgb5 at a constant ratio, and if necessary, positive selection based on Itgb5 is performed to separate cells expressing Itgb5. It may be separated or enriched.
  • Negative and positive selection techniques based on surface markers are also known to those of skill in the art, and any antibody-based technique can be used, including, for example, fluorescence activated cell sorting (FACS) and magnetic bead separation.
  • FACS fluorescence activated cell sorting
  • the epithelial-derived cells of the present invention can typically be used in the production of regenerated hair follicle primordiums used in hair follicle regenerative medicine.
  • the hair follicle primordium is the tissue from which the hair follicle is derived and is composed of epithelial-derived cells and mesenchymal cells.
  • the hair follicle primordium is formed by thickening a part of the epidermis during the embryonic period and aggregating the facing mesenchymal cells.
  • the "regenerated hair follicle primordium" refers to a hair follicle primordium artificially derived or regenerated from epithelial-derived cells and mesenchymal cells.
  • the epithelial-derived cells of the present invention can typically be used in the production of regenerated hair follicle primordiums used in hair follicle regenerative medicine.
  • the epithelial-derived cells of the present invention are used for producing a regenerated hair follicle primordium
  • the regenerated hair follicles are cultured by culturing the cell population containing the epithelial-derived cells in contact with the cell population derived from mesenchymal cells.
  • the primordium can be produced.
  • a "cell population containing epithelial-derived cells” has the ability to produce regenerated hair follicle primordia, preferably sustained hair growth (ie, hair cycle 1 or more, preferably 2, 3, 4, or 5).
  • the culture conditions for contacting and culturing a cell population containing epithelial-derived cells and a cell population derived from mesenchymal cells can be the conditions used for culturing general animal cells, for example, at least 12 hours. , Preferably at least 16 hours, more preferably at least 24 hours, still more preferably at least 40 hours, and the medium and culture conditions may be changed during the culture.
  • Example 1 Analysis of differentiation ability of Itgb5-positive cells by cell lineage tracking 1) Preparation of reagents 1-1) Preparation of anatomical medium FBS (Bioest, final concentration: 10%), HEPES in DMEM (Thermo Fisher Scientific) (Thermo Fisher Scientific, final concentration: 10 mM) and Pencillin-Streptomycin (Thermo Fisher Scientific, final concentration: 1%) were mixed.
  • FBS Bioest, final concentration: 10%
  • HEPES in DMEM Thermo Fisher Scientific
  • Pencillin-Streptomycin Thermo Fisher Scientific, final concentration: 1%) were mixed.
  • HEPES 1M HEPES was prepared by dissolving HEPES (Dojin Kagaku Kenkyusho) in Milli-Q water and then adjusting the pH to 7.4 with NaOH (Fujifilm Wako Pure Chemical Industries, Ltd.).
  • DMEM 10xDMEM was prepared by dissolving DMEM powder (Thermo Fisher Scientific) for 1 L in 100 mL of Milli-Q water.
  • 1x PBS KCl Flujifilm Wako Pure Chemical Industries, Ltd., final concentration 27 mM
  • KH 2 PO 4 Fluji Film Wako Pure Chemical Industries, Ltd., final concentration 15 mM
  • Na 2 HPO 4 Fluji Film Wako Pure Chemical Industries, Ltd., final concentration 80 mM
  • NaCl in milli-Q water Feujifilm Wako Pure Chemical Industries, Ltd., final concentration 1.37M
  • the extracellular fluid containing the detached cells was passed through a 70 ⁇ m cell strainer (Corning) and centrifuged at 250 xg, 4 ° C., and 5 minutes in a micro high-speed centrifuge (Hitachi Industry). After centrifugation, the supernatant was removed, and cells derived from mouse skin epithelium were collected by adding and mixing Advanced DMEM / F-12 (Thermo Fisher Scientific). After measuring the cell concentration of mouse skin epithelial-derived cells, the cell suspension was separated into tubes. The tube is centrifuged at 310 xg at 4 ° C.
  • 1xGlutamax (Thermo Fisher Scientific), 1xB27 supplement (Thermo Fisher Scientific), 1xN2 supplement (Thermo Fisher Scientific), 1xN2 supplement (Thermo Fisher Scientific), 10 ⁇ M mL FGF-7 (R & D), 50 ng / mL FGF-10 (R & D), 50 ng / mL SHH agent (SAG, cayman), and 50 ng / mL BMP medium (Noggin, Peprotech), and 1% PencilliteS.
  • Medium containing Scientific) was added at 3 mL / well, and three-dimensional culture was carried out in a CO 2 incubator (Thermo Fisher Scientific) at 37 ° C. and a CO 2 concentration of 5% for 6 days.
  • the gel in which cells were colonized was peeled off from the culture dish with a cell scraper.
  • the peeled gel was collected in a 1.5 mL tube (maximum 3 gels / tube), and 1 mL of the culture supernatant was added.
  • Collagenase I (Worthington) was added to a final concentration of 100 U / mL, and the gel was dissolved by reacting at 37 ° C. for 60 to 90 minutes. After centrifugation at 590 xg at 4 ° C. for 3 minutes, the supernatant was removed, and PBS ( ⁇ ) (Nacalai Tesque) was washed once with 1 mL.
  • a cell suspension was prepared by adding and mixing 1 mL of 35 U / mL DNase Type I (Sigma) -containing anatomical medium. The cell suspension was passed through a 35 ⁇ m cell strainer (Corning) and centrifuged at 590 xg at 4 ° C. for 3 minutes. After centrifugation, the supernatant was removed, and cells derived from cultured mouse skin epithelium were recovered by suspending in Advanced DMEM / F-12 medium containing 1% Pencillin-Streptomycin, and the cell concentration was measured.
  • collected cultured mouse skin epithelial-derived cells (number of cells: 1x10 5 or 2x10 7 ) were mixed with 0.2 mM EDTA (Fujifilm Wako Pure Drug) and 0.05% BSA (BM bio Japan) -containing PBS (-).
  • 0.2 mM EDTA and 0 containing eFluor660-labeled CD34 monoclonal antibody Invitrogen, 50-fold dilution
  • PE-labeled human / mouse CD49f monoclonal antibody BioLegend, 50-fold dilution
  • Itgb5 antibody R & D, 50-fold dilution
  • mouse Itgb5-positive cells were isolated from CD34 and CD49f-positive cells. The results are shown in FIG. It was confirmed that mouse Itgb5-positive cells were isolated from the cultured mouse skin epithelial-derived cells (Fig. 1).
  • regenerated hair follicle primordium was prepared using mouse Itgb5-positive cells according to the organ primordium method (Patent No. 5932671). did.
  • skin mesenchymal cells were prepared from mouse fetuses according to a conventional method. That is, the skin containing the hair primordium was collected from a C57BL / 6 mouse fetus (SLC) aged 18.0 to 18.5 days on a 35 mm dish (Becton Dickinson), and the method reported by Nakao et al.
  • the epithelial layer was washed twice with PBS ( ⁇ ) and reacted with 1 mL of Actase (Thermo Fisher Scientific) at room temperature for 45 minutes. After the reaction, 2 mL of 70 U / mL DNase Type I-containing anatomical medium was added and mixed. After mixing, it was passed through a 35 ⁇ m cell strainer and centrifuged at 590 xg at 4 ° C. for 3 minutes. After centrifugation, the supernatant was removed, and cells derived from mouse fetal skin epithelium were collected by adding and mixing anatomical medium.
  • the dermis layer was transferred to a 50 mL tube (Becton Dickinson) and reacted with 910 U / mL Collagenase I diluted with anatomical medium at 37 ° C. for 1 hour at 55 rpm with shaking. After the reaction, 10 mL of anatomical medium was added, passed through a 10 ⁇ m cell strainer (Sysmex), and centrifuged at 590 xg at 4 ° C. for 3 minutes. After centrifugation, the supernatant was removed, and mouse fetal skin mesenchymal cells were collected by adding and mixing 70 U / mL DNase Type I-containing anatomical medium.
  • mouse mixed epithelial-derived cells were prepared by mixing mouse Itgb5-positive cells and collected mouse fetal skin epithelial-derived cells at a ratio of 1: 9.
  • Mice mixed epithelial-derived cells and mouse fetal skin mesenchymal cells were separately transferred to 1.5 mL microtubes (Eppendorf) coated with silicone grease (Tole Dow Corning) and centrifuged (600 xg, 4 ° C, 3 ° C.). It was collected as a precipitate by (minutes), and the supernatant of the culture solution after centrifugation was removed using 0.5-20 ⁇ L (Eppendorf) of GELoader Tip.
  • the mouse mixed epithelial-derived cells prepared above are brought into close contact with the aggregates of mouse fetal skin mesenchymal cells using a 0.1-10 ⁇ L pipette tip (Quality Scientific Plastics). As described above, about 0.2 ⁇ L was injected to prepare a cell aggregate (number of cells: 1x10, 4 cells / primordium). Furthermore, from the mouse mixed epithelium-derived cell side of the cell aggregate of mouse fetal skin mesenchymal cells and mouse mixed epithelium-derived cells, a nylon thread (Matsuda Medical Industry) with a total length of 5 mm was applied to the structure of the cell aggregate (particularly mouse mixed epithelium).
  • a nylon thread Matsuda Medical Industry
  • a stereoscopic microscope (contact surface between the derived cells and the mouse fetal skin mesenchymal cells) so as to vertically penetrate the contact surface between the mouse fetal skin mesenchymal cell fraction and the mouse mixed epithelium-derived cell fraction.
  • Cell Culture Insert (Corning) of 0.4 ⁇ m pore size set on a 6-well plate (Corning) containing 1 mL of DMEM containing 10% FBS and 1% Penicillin-Streptomycin, inserted after confirmation under Cell Zeiss). The whole reconstitution gel was transferred onto the cells, and the cells were cultured for 16 to 40 hours in a CO 2 incubator set at 37 ° C. and a CO 2 concentration of 5% to prepare regenerated hair follicle primordia.
  • mice Intradermal transplantation of regenerated hair follicle primordium in nude mice
  • the regenerated hair follicle primordium was transplanted into the skin of mice according to a conventional method. That is, 5 to 8 week old Balb / c nu / nu mice (SLC) were anesthetized according to a conventional method, and the back was disinfected with Isodine and then placed in a natural lying position. A puncture was performed using a V-lance micromes (Nippon Archon) to form a transplant wound from the epidermal layer of the skin to the lower layer of the dermis layer.
  • V-lance micromes Neippon Archon
  • the transplanted wound was set to a depth of about 400 ⁇ m in the vertical direction from the body surface, and about 1 mm in the horizontal direction. Insert the regenerated hair follicle primordium into which the nylon thread guide is inserted, and use the sharp tweezers No. so that the epithelial-derived cell component faces the body surface side of the transplanted wound. It was inserted using 5 (Natsume Seisakusho). The transplantation depth was adjusted so that the upper end of the epithelial-derived cell component of the regenerated hair follicle primordium was exposed at the upper end of the transplant wound, and the nylon thread guide was positioned so as to be exposed on the body surface. The nylon thread guide was fixed to the skin surface close to the transplant wound with a steri strip (3M), and then the transplant wound was protected with a nurse van (sample net) and surgical tape (3M). The protective tape was removed 5 to 7 days after transplantation.
  • the prepared tissue sections were transferred to a 24-well plate (Becton Dickinson) and placed in 500 ⁇ L of wash buffer (0.1% Triton X-100 prepared by diluting Triton X-100 (Alfa Aesar) with 1 x PBS) at room temperature. It was washed by infiltrating for 10 minutes (washing times 2 times). After washing, it was resuspended in washing buffer containing Itgb5 antibody (R & D, diluted 50-fold) and shaken at 4 ° C. overnight. The supernatant was removed, and the mixture was washed by infiltrating 500 ⁇ L of a washing buffer solution at room temperature for 1 hour (washing times 3 times).
  • wash buffer 0.1% Triton X-100 prepared by diluting Triton X-100 (Alfa Aesar) with 1 x PBS
  • mice Itgb5-positive cells were localized in the sebaceous glands (white arrowheads), stem cell niches (white arrowheads), hair follicle variable parts (white arrowheads) and hair bulbs (white arrowheads) that make up the hair follicles. From this (Fig. 2), it is expected that Itgb5-positive cells have the ability to differentiate into various tissues and regions.
  • Example 2 Functional analysis of Itgb5-positive cells for hair growth persistence 1) Preparation of reagents, 2) Experimental animals and 3) Seeding, culturing and recovery of mouse skin epithelial-derived cells were carried out by the methods described above.
  • mice Itgb5-negative / positive cells and mouse Itgb5-negative cells Itgb5-negative and Itgb5-positive fractions of cultured mouse skin epithelial-derived cells that are CD34 and CD49f-positive using a flow cytometer large black in the left frame of FIG. 3B.
  • Mouse Itgb5 negative / positive cells were prepared by separating the squares) (center frame of FIG. 3B).
  • mouse Itgb5-negative cells were prepared by separating (cell sorting) the Itgb5-negative fraction (small black square in the left frame of FIG. 3B) (right frame of FIG. 3B).
  • the recovered cultured mouse skin epithelial-derived cells (number of cells: 5x10, 7 cells) were reacted with three types of antibodies by the method described above. Resuspended cell suspension were analyzed by BD FACSAria TM III, Itgb5 negative cells from CD34 and CD49f-positive cells:: (1x10 6 cells cell count) (number of cells 1x10 6 cells) and mouse ITGB5 negative / positive cells separated. The results are shown in FIG. It was confirmed that the cultured mouse skin epithelial-derived cells were CD34 and CD49f positive (Fig. 3A). It was also confirmed that Itgb5 negative cells and mouse Itgb5 negative / positive cells were separated from each other (FIG. 3B).
  • a regenerated hair follicle primordium was prepared using the organ primordium method, and hair growth ability by animal transplantation was performed. The function of Itgb5-positive cells with respect to the persistence of hair growth was analyzed using the above as an index. In addition, mouse Itgb5 negative cells were used as a control group. The regenerated hair follicle primordium was prepared according to the organ primordium method (Patent No. 5932671). Mouse fetal skin mesenchymal cells were collected by the method described above, and the number of collected cells was measured.
  • Mouse Itgb5 negative / positive cells, mouse Itgb5 negative cells and mouse fetal skin mesenchymal cells were separately transferred to 1.5 mL microtubes (Eppendorf) coated with silicone grease and centrifuged (600 xg, 4 ° C., 3 minutes). ), And the supernatant of the culture solution after centrifugation was removed using 0.5-20 ⁇ L (Eppendorf) of GELoider Tip. The operation of centrifugation and removal of the supernatant was repeated until the supernatant was removed as much as possible.
  • cell agglomerates (number of cells: 1x10, 4 cells / primordium).
  • a nylon thread having a total length of 5 mm from the mouse Itgb5 negative / positive cell or mouse Itgb5 negative cell side of a cell aggregate of mouse fetal skin mesenchymal cells and mouse Itgb5 negative / positive cells or mouse Itgb5 negative cells (Matsuda Medical Industry)
  • the cell fraction of mouse fetal skin mesenchymal cells and mouse Itgb5 without destroying the structure of the cell aggregate (particularly the contact surface between mouse Itgb5 negative / positive cells or mouse Itgb5 negative cells and mouse fetal skin mesenchymal cells).
  • the cells were inserted after confirmation under a stereoscopic microscope so as to penetrate the contact surface with the negative / positive cells or the mouse Itgb5 negative cell fraction, and set on a 6-well plate (Becton Dickinson) containing 1 mL of DMEM 10% FBS. Transfer the collagen gel onto Cell Culture Inserts (Becton Dickinson) of 4 ⁇ m pore size, and perform organ culture for 16 to 40 hours in a CO 2 incubator set at 37 ° C and 5% CO 2 concentration, and regenerate hair follicles. was produced. The regenerated hair follicle primordium was transplanted by the method described above. Engraftment of the transplant was performed with a fluorescence stereomicroscope SteREO Lumar.

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Abstract

Le problème décrit par la présente invention est de fournir une nouvelle cellule dérivée de l'épithélium qui peut être utilisée pour fabriquer des germes de follicules pileux régénérés. La solution selon l'invention porte sur : une cellule dérivée de l'épithélium exprimant l'intégrine β5 (Itgb5) à un niveau détectable ; une population de cellules comprenant ladite cellule ; et un procédé de production de germes de follicules pileux régénérés faisant appel à ladite population de cellules.
PCT/JP2020/015788 2019-04-12 2020-04-08 Cellule dérivée de l'épithélium destinée à être utilisée dans la production de germes de follicules pileux régénérés Ceased WO2020209280A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Project Area Number 16H07454, 1) Research Results, 3) Research Methods (TAKEO, Makoto. Identification of organ inductive hair follicle stem cells and its regulation mechanism", GRANT-IN-AID FOR SCIENTIFIC RESEARCH, FINAL RESEARCH REPORT., 2018 *
ORDONEZ, P. ET AL.: "Human limbal epithelial progenitor cells express alphavbeta5-integrin and the interferon-inducible chemokine CXCL10/IP-10", STEM CELL RES., vol. 11, 2013, pages 888 - 901, XP028686610, DOI: 10.1016/j.scr.2013.05.013 *
TAKEO, MAKOTO ET AL.: "1AW-20-10(2P- 0350)] Identification of Hair Follicle Stem Cells Necessary for Permanent Hair Growth Cycles in Adult Mice", 42ND ANNUAL MEETING OF THE MOLECULAR BIOLOGY SOCIETY OF JAPAN, 19 November 2019 (2019-11-19) *
TOYOSHIMA, KHO-EI ET AL.: "Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches", NAT. COMMUN., vol. 3, no. 784, 2012, pages 1 - 12, XP055557492, DOI: 10.1038/ncomms1784 *

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