WO2020262531A1 - Inducteur de cellule souche d'endoderme primitif - Google Patents

Inducteur de cellule souche d'endoderme primitif Download PDF

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WO2020262531A1
WO2020262531A1 PCT/JP2020/025018 JP2020025018W WO2020262531A1 WO 2020262531 A1 WO2020262531 A1 WO 2020262531A1 JP 2020025018 W JP2020025018 W JP 2020025018W WO 2020262531 A1 WO2020262531 A1 WO 2020262531A1
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cells
stem cells
medium
serum
primitive endoderm
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康秀 大日向
明彦 古関
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国立研究開発法人理化学研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0604—Whole embryos; Culture medium therefor
    • 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
    • C12N2501/40—Regulators of development
    • C12N2501/415—Wnt; Frizzeled
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the present invention relates to a primitive endoderm stem cell inducer and a method for producing primitive endoderm stem cells using the same.
  • a technique for producing a trophoblast ectoderm-like structure using TS cells is known (Patent Document 1).
  • fertilized eggs repeat cleavage to form early embryos called blastocysts.
  • blastocysts epiplasts (stem cells called ES cells have been established), trophoblasts (stem cells called TS cells have been established), and primitive endoderm (strains called XEN cells have been established).
  • this strain is a collection of dozens of three types of cells that do not have traits as stem cells. All tissues of the conception product are derived from these cells. If stem cells of each of these three types of cells can be established, there is a possibility of artificial blastocyst reconstruction by combining them.
  • a method for establishing primitive endoderm stem cells has not been known.
  • the present invention provides a primitive endoderm stem cell inducer and a method for producing primitive endoderm stem cells using the same.
  • the present inventor has completed the present invention by discovering for the first time that a Wnt activator or GSK3 inhibitor can induce primitive endoderm stem cells.
  • a primitive endoderm stem cell inducer containing a Wnt activator or a GSK3 inhibitor [2] The inducer of [1], wherein the Wnt activator or GSK3 inhibitor is CHIR99021.
  • a medium for inducing primitive endoderm stem cells which contains the inducer of [1] or [2] and does not contain serum.
  • the medium of [3] which does not contain PD0325901.
  • a method for producing primitive endoderm stem cells which comprises culturing blastocysts in a medium containing a Wnt activator or a GSK3 inhibitor and not containing serum.
  • [6] The method of [5], wherein the medium does not contain PD0325901.
  • [7] The method of [5] or [6], wherein the cells are derived from humans.
  • [8] Primitive endoderm stem cells obtained by culturing blastocysts in a medium containing a Wnt activator or GSK3 inhibitor and not containing serum.
  • a construct comprising the stem cells of [8] or [9] and epiblast stem cells, ES cells and / or trophoblast stem cells.
  • [12] Use of Wnt activator or GSK3 inhibitor in the induction of primitive endoderm stem cells. [13] Use of [12] where the Wnt activator or GSK3 inhibitor is CHIR99021. [14] Use of a serum-free medium containing the inducer of [1] or [2] in inducing primitive endoderm stem cells. [15] Use of [12] to [14], excluding cells derived from humans. [16] Primordial endoderm stem cells, epiblast stem cells and / or ES cells obtained by culturing blastocysts in a medium containing a Wnt activator or GSK3 inhibitor and not containing serum are mixed with trophoblast stem cells to form an embryo. Obtaining a blastocyst-like construct, A method comprising transplanting the resulting blastocyst-like construct into the uterus and determining the implantation ability of the transplanted blastocyst-like construct.
  • the present invention is capable of inducing primordial endoderm stem cells, producing artificial blastocyst-like constructs comprising the primordial endoderm stem cells, epiblast stem cells, ES cells and / or trophoblast stem cells. Implantation ability can be determined.
  • FIG. 1 is a chimera of ES cells and TS cells and tetraploid embryos.
  • FIG. 2 shows a conventional XEN cell and a PrES cell obtained by the present invention.
  • FIG. 3 shows a chimera using XEN cells and a chimera using PrES cells.
  • FIG. 4 shows the injection of ES cells and PrES cells into a blastocyst or a trophocyst.
  • FIG. 5 shows the fate of each factor and stem cell to be derived.
  • FIG. 6 shows the fate of each factor and stem cell to be derived.
  • the present invention provides a primitive endoderm stem cell inducer, including a Wnt activator or GSK3 inhibitor.
  • the present invention also provides a medium for inducing primitive endoderm stem cells, which contains this inducer and does not contain serum.
  • the present invention also provides a method for producing primitive endoderm stem cells, which comprises culturing blastocysts in a medium containing a Wnt activator or GSK3 inhibitor and not containing serum.
  • the present invention also provides primordial endoderm stem cells obtained by culturing blastocysts in a medium containing a Wnt activator or GSK3 inhibitor and not containing serum.
  • the present invention also provides constructs comprising the stem cells, epiblast stem cells, ES cells and / or trophoblast stem cells.
  • the present invention also provides the use of Wnt activators or GSK3 inhibitors in the induction of primitive endoderm stem cells.
  • the present invention mixes primordial endoderm stem cells, epiblast stem cells and / or ES cells with trophoblast stem cells obtained by culturing blastocysts in a medium containing Wnt activator or GSK3 inhibitor and not serum. And get a blastocyst-like construct.
  • Wnt (Wingless + Int-1) is a gene that affects cell proliferation, differentiation and migration. So far, 19 types of Wnt genes have been identified in mammals (Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, Wnt16).
  • Wnt activates at least three different signaling pathways, including the "classical” ⁇ -catenin pathway and the “non-canonical” in-plane cell polarity (PCP) and Ca pathways. It acts by activating cell surface receptors in responder cells.
  • a Wnt activator is a substance that activates a Wnt signal. Examples of the Wnt activator include GSK3 inhibitors such as CHIR99021 (CAS [252917-06-9]) and Wnt receptors, in addition to the expression products of the Wnt gene described above.
  • Glycogen synthase kinase 3 is one of the proline-directed serine / threonine phosphorylating enzymes.
  • GSK3 inhibitors include GSK-3 ⁇ inhibitors I, VI, VII, VIII, XI, XII, CHIR99021, valproic acid, TDZD-8, SB-216763, 6-bromoinsilvin-3'-oxime, and Kenpaullone. Can be mentioned.
  • Primitive endoderm is a cell that is formed by blastocysts and mainly derives the endoderm layer of the yolk sac.
  • Primitive endoderm stem cells are stem cell lines that are established from the primordial endoderm of blastocysts and retain the primordial endoderm-like properties.
  • the primitive endoderm stem cell inducer is a medium containing CHIR99021 or a Wnt signaling promoter (for example, a Wnt ligand or a GSK3 inhibitor), and MEF feeder cells.
  • a medium for ES cells / iPS cells of mammalian cells can be used.
  • StemFit (registered trademark) Basic02 (Ajinomoto) can be mentioned.
  • the medium can be a serum-containing medium or a serum-free medium.
  • the serum-free medium means a medium containing no prepared or unpurified serum, and a medium containing purified blood-derived components or animal tissue-derived components (for example, growth factors) corresponds to a serum-free medium. It shall be. From the viewpoint of avoiding contamination of chemically undetermined components, a serum-free medium is preferably used in the present invention.
  • the medium is preferably a chemically defined medium.
  • the “chemically defined medium” means a medium composed of only chemically defined factors, such as serum and conditioned medium, which do not contain chemically undetermined factors.
  • the medium can contain the primitive endoderm stem cell inducer of the present invention.
  • the medium preferably contains the primitive endoderm stem cell inducer of the present invention, but does not contain PD0325901.
  • the method for producing primitive endoderm stem cells includes culturing blastocysts in a medium containing a Wnt activator or a GSK3 inhibitor and not containing serum.
  • the medium preferably contains the primitive endoderm stem cell inducer of the present invention, but does not contain PD0325901.
  • a blastocyst is an early mammalian embryo, which is initially composed of a trophoblast and an inner cell mass. After the middle stage, the inner cell mass separates and is composed of trophoblast, early epiblast, and primitive endoderm. It is obtained by collecting from a living body or culturing a fertilized egg. In one embodiment, the blastocysts or cells of the invention are derived from mammals other than humans.
  • the incubator used for culturing is not particularly limited, but for example, a flask, a tissue culture flask, a dish, a petri dish, a tissue culture dish, a multi-dish, a microplate, a microwell plate, a micropore, a multiplate, and a multiwell.
  • Examples include plates, flask slides, petri dishes, tubes, trays, culture bags, and roller bottles.
  • One embodiment of the present invention is a primitive endoderm stem cell obtained by culturing a blastocyst in a medium containing a Wnt activator or a GSK3 inhibitor and not containing serum.
  • the medium preferably contains the primitive endoderm stem cell inducer of the present invention, but does not contain PD0325901.
  • One aspect of the present invention is i) Obtained by culturing blastocysts in a medium containing a Wnt activator or GSK3 inhibitor and without serum, preferably a medium containing the primitive endoderm stem cell inducer of the present invention but not PD0325901. , Primitive endoderm stem cells, and ii) a construct containing epiblast stem cells, ES cells and / or trophoblast stem cells.
  • the construct of the present invention preferably does not contain cells derived from humans.
  • One aspect of the present invention is the use of a Wnt activator or GSK3 inhibitor in the induction of primordial endoderm stem cells.
  • the Wnt activator or GSK3 inhibitor is CHIR99021.
  • One aspect of the present invention is Use in the induction of primordial endoderm stem cells in a serum-free medium containing a Wnt activator or GSK3 inhibitor.
  • the Wnt activator or GSK3 inhibitor is CHIR99021.
  • the cells of the invention do not contain cells derived from humans.
  • One aspect of the invention is primordial endoderm stem cells, epiblast stem cells and / or ES cells and trophoblast stem cells obtained by culturing blastocysts in a serum-free medium containing a Wnt activator or GSK3 inhibitor.
  • a method for determining the implantation ability of a blastocyst-like structure which comprises transplanting the obtained blastocyst-like structure into the uterus and determining the implantation ability of the transplanted blastocyst-like structure. Is.
  • ES cells and TS cells C57B6N ⁇ 129X1 F1 (# 1M, # 6M) ES cells were placed on a gelatin-coated dish in standard ES medium [Neurobasal: DMEMF12 (1: 1), 0.5 ⁇ N2 supplement (Thermo Fisher Scientific).
  • a chimera When a chimera is prepared using ES cells and TS cells, it is possible to prepare an individual in which most of the conception product, including almost all cells of the offspring, is composed of stem cells.
  • the tissues derived from the primitive endoderm visceral endoderm (VE), mural endoderm (PE), borderline endoderm (MZE) are these stem cells. It can be seen that it is not derived from. Therefore, it is understood that in order to produce an embryo using only stem cells, at least stem cells that derive primordial endoderm tissue are required (Fig. 1).
  • XEN cells have been established from the primitive endoderm.
  • XEN cells are used when establishing ES cells (established or cultured in a serum-containing medium supplemented with LIF) or TS cells (established or cultured in a serum-containing MEF condition medium supplemented with FGF4).
  • ES cells established or cultured in a serum-containing medium supplemented with LIF
  • TS cells established or cultured in a serum-containing MEF condition medium supplemented with FGF4
  • Primitive endoderm is derived from visceral endoderm (VE), mural endoderm (PE), and borderline endoderm (MZE), but even if XEN cells are injected into the blastocyst, the mural endoderm It formed only a part of (PE) and was not a stem cell that sufficiently retained the properties of the primitive endoderm.
  • VE visceral endoderm
  • PE mural endoderm
  • MZE borderline endoderm
  • CD1-Tg CAG-EGFP
  • CAG-EGFP CAG-EGFP
  • PWK BRC, RIKEN
  • C57BL6N Charles River F1
  • PrESCM for each blastocyst, PrESCM [StemFit® Basic02, 1 x penicillin-streptomycin (Thermo Fisher Scientific), 10 ⁇ M CHIR99021 (Reprocell), 50 ng / ml human GFG4 (Sigma), 1 ng / ml heparin (Sigma), 25 ng. 48-well MMC-treated MEF feeder plate in [/ ml human PDGF-AA (R & D)] was transferred to one well. Embryos were cultured in PrES medium for 5 days until they formed growth. Propagation was dissociated with 1 ⁇ TrypLE® Select (Thermo Fisher Scientific) and transferred to a new 48-well MEF feeder plate.
  • PrES cell colonies reached 50-80% confluence and cells were passaged to a 12-well MEF feeder plate and then expanded on a 6-well plate.
  • PrES cells were frozen in Cellbanker® 1plus (Zenoaq) and stored in an ultra-low temperature freezer (Panasonic) at ⁇ 150 ° C.
  • the use of serum-free cryoprotectants significantly reduced cell viability.
  • Other growth factors used were 10 3 U / ml ESGRO LIF (Merck), 20 mg / ml human activin A (R & D), 100 ng / ml mouse Cripto (R & D), 20 ng / ml human BMP4 (R & D), 100 ng / ml. It was mouse Nodal (R & D).
  • the other compounds used were 10 ⁇ M XAV939 (Merck) and 1 ⁇ M PD0325901 (Reprocell).
  • XEN cells were derived according to a standard protocol with minor modifications. Blastocysts of CD1-Tg (CAG-EGFP) female x CD1-Tg (CAG-EGFP) male were recovered at E3.5.
  • Serum-containing ES medium on MEF feeder plate [RPMI1640 (Thermo Fisher Scientific), 20% fetal bovine serum (FBS, biosera), 1 x penicillin-streptomycin (Thermo Fisher Scientific), 1 x non-essential amino acids (NEAA, Thermo Fisher Scientific), 0.1 mM ⁇ -mercaptoethanol ( ⁇ -ME, Sigma), transferred to one of the wells of a 48-well MEF feeder plate in 10 3 U / ml ESGRO LIF. Embryos were cultured for 7 days until they formed a growth. Proliferators were removed by pipetting and the remaining adherent cells were cultured for an additional 7 consecutive days.
  • Proliferated cells were dissociated with 0.05% trypsin-EDTA (Thermo Fisher Scientific), transferred to a new 48-well MEF feeder plate, and then excited on a 6-well plate.
  • XEN cells were frozen in Cellbanker® 1plus (Zenoaq) and stored in an ultra-low temperature freezer (Panasonic) at ⁇ 150 ° C. Removal of the primary culture was not necessary when CHIR99021 (3 ⁇ M) was added.
  • XEN cells In the chimera using XEN cells, in the E14.5 embryo, the XEN cells could not form primitive endoderm-derived tissues (VE, PE, MZE) only by leaving engraftment in a part of PE. .. In contrast, PrES cells were able to contribute to VE, PE, and MZE with high efficiency. In addition, PrES cells did not contribute to the Epblast lineage and the trophoblast lineage. (Fig. 3)
  • a three-dimensional structure very similar to the trophoblast can be produced (nutrient cells, trophocyst). Since the present invention has succeeded in establishing and culturing primordial endoderm stem cells (PrES cells), not only the conventional trophoblast-like three-dimensional structure is produced, but also ES cells and PrES cells are injected into the trophoblast to blastocyst. It has become possible to produce a blastocyst-like three-dimensional structure. (Fig. 4)
  • XEN cells were established from the remaining adherent cells by culturing blastocysts under ES cell (or TS cell) conditions and removing the formed outgrowth.
  • ES cell or TS cell
  • PrES cells, TS cells and XEN cells morphologically different from ES cells were induced in CHIR99021-containing StemFit® Basic02 serum-free medium on MEF. The induction of PrES cells was dramatically suppressed by the addition of PD0325901, but ES cells were induced instead. (Fig. 5)
  • CD1-Tg CAG-EGFP
  • CD1-Tg CAG-EGFP
  • CD1-Tg CAG-EGFP
  • CD1 (Clea) x CD1 ES cells were induced, and the Basic02 ES medium [StemFit® Basic02, 1 on the MEF feeder dish. ⁇ Penicillin-streptomycin (Thermo Fisher Scientific), 3 ⁇ M CHIR99021, 1 ⁇ M PD0325901, and 10 3 U / ml ESGRO LIF].
  • PrES cells are novel stem cells that have acquired the properties of PrE in vivo.
  • LIF Leukemia inhibitory factor
  • CHIR99021 is an inhibitor of GSK3, promotes ⁇ -catenin accumulation and nuclear translocation, and activates canonical Wnt signaling.
  • Activin A is a growth factor belonging to the Tgf ⁇ superfamily, and has a function in both the undifferentiated state and the differentiation process of cells.
  • FGF4 is a growth factor belonging to the FGF family and activates the MEK / MAPK pathway via the FGF receptor.
  • Heparin promotes FGF cell proliferation activity by interacting with FGF.
  • XAV939 is an inhibitor of tankyrace, which promotes the degradation of ⁇ -catenin and inhibits Wnt signaling through the stabilization of Axin.
  • Cripro is a co-receptor for Nodal and several other Tgf ⁇ family ligands and regulates their signaling.
  • PD0325091 is a MEK inhibitor and suppresses FGF signals.
  • PDGF Plate-Derived Growth factor
  • the present invention provides induction of primordial endoderm stem cells, preparation of artificial blastocyst-like constructs comprising the primordial endoderm stem cells, epiblast stem cells, ES cells and / or trophoblast stem cells, and determination of the implantation ability of the constructs. Can be used for.

Abstract

La présente invention concerne : un inducteur de cellule souche d'endoderme primitif qui contient un activateur Wnt ou un inhibiteur de GSK3 ; un milieu qui est destiné à induire une cellule souche d'endoderme primitif, qui contient ledit inducteur, et ne contient pas de sérum ; et un procédé qui est destiné à produire une cellule souche d'endoderme primitif et comprend la culture d'un blastocyste dans un milieu exempt de sérum contenant un activateur Wnt ou un inhibiteur de GSK3.
PCT/JP2020/025018 2019-06-26 2020-06-25 Inducteur de cellule souche d'endoderme primitif WO2020262531A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
EP4029932A1 (fr) * 2021-01-13 2022-07-20 IMBA-Institut für Molekulare Biotechnologie GmbH Agrégat de cellule de type blastocyste et procédés
WO2022152774A1 (fr) 2021-01-13 2022-07-21 Imba - Institut Für Molekulare Biotechnologie Gmbh Agrégat cellulaire de type blastocyste et procédés

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WO2006128681A1 (fr) * 2005-05-31 2006-12-07 Abcys S.A. Souches endodermiques primitives, leurs procede de preparation et leur utilisation, en particulier pour l'obtention de cellules epitheliales hepatiques primitives
JP2013066495A (ja) * 2006-03-30 2013-04-18 Univ Court Of The Univ Of Edinburgh キナーゼインヒビターを含む培養培地およびその使用
WO2015125926A1 (fr) * 2014-02-21 2015-08-27 国立研究開発法人理化学研究所 Procédé permettant la création et le maintien de cellules souches trophoblastiques
WO2019093340A1 (fr) * 2017-11-07 2019-05-16 国立大学法人京都大学 Procédé d'induction d'endoderme primitif à partir de cellules souches pluripotentes naïves

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WO2006128681A1 (fr) * 2005-05-31 2006-12-07 Abcys S.A. Souches endodermiques primitives, leurs procede de preparation et leur utilisation, en particulier pour l'obtention de cellules epitheliales hepatiques primitives
JP2013066495A (ja) * 2006-03-30 2013-04-18 Univ Court Of The Univ Of Edinburgh キナーゼインヒビターを含む培養培地およびその使用
WO2015125926A1 (fr) * 2014-02-21 2015-08-27 国立研究開発法人理化学研究所 Procédé permettant la création et le maintien de cellules souches trophoblastiques
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4029932A1 (fr) * 2021-01-13 2022-07-20 IMBA-Institut für Molekulare Biotechnologie GmbH Agrégat de cellule de type blastocyste et procédés
WO2022152774A1 (fr) 2021-01-13 2022-07-21 Imba - Institut Für Molekulare Biotechnologie Gmbh Agrégat cellulaire de type blastocyste et procédés

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