WO2022191171A1 - 細胞クラスターの製造方法 - Google Patents

細胞クラスターの製造方法 Download PDF

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WO2022191171A1
WO2022191171A1 PCT/JP2022/009925 JP2022009925W WO2022191171A1 WO 2022191171 A1 WO2022191171 A1 WO 2022191171A1 JP 2022009925 W JP2022009925 W JP 2022009925W WO 2022191171 A1 WO2022191171 A1 WO 2022191171A1
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cells
pluripotent stem
cell
stem cells
wnt signal
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French (fr)
Japanese (ja)
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貴則 武部
憲和 佐伯
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Takeda Pharmaceutical Co Ltd
Tokyo Medical and Dental University NUC
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Takeda Pharmaceutical Co Ltd
Tokyo Medical and Dental University NUC
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Priority to JP2023505567A priority Critical patent/JPWO2022191171A1/ja
Priority to CN202280019926.8A priority patent/CN116964193A/zh
Priority to EP22767125.2A priority patent/EP4306633A4/en
Priority to US18/281,442 priority patent/US20240158740A1/en
Publication of WO2022191171A1 publication Critical patent/WO2022191171A1/ja
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Definitions

  • the present invention relates to a method for producing cell clusters enriched with desired cells, which are cell clusters having micropatterns specific to embryonic developmental regions formed from pluripotent stem cell colonies by a differentiation-inducing factor. . It also relates to a method for producing a cell population generated by further differentiation of cells in the cell cluster, and an organoid or three-dimensional organ formed by further culturing the cells in the cell cluster or the cells in the cell population.
  • Pluripotent stem cells such as ES cells and iPS cells correspond to the epiblast (epithelial layer) in the definition of embryology, and embryogenesis is initiated from colonies of pluripotent stem cells by differentiation-inducing factors. It is known that cell clusters with region-specific micropatterns are formed.
  • Non-Patent Document 1 describes that two-dimensional culture of human ES cells formed micropatterns containing three germ layers (endoderm, mesoderm and ectoderm) by BMP4 signals.
  • Non-Patent Document 2 describes that differentiation into endoderm and mesoderm was controlled by inhibiting Wnt signals after forming micropatterns from human ES cells.
  • Non-Patent Document 3 describes that when micropatterns were formed from mouse ES cells, yolk sac mesoderm cells also emerged, albeit at a low rate.
  • Patent Document 1 describes a method for producing a matrix composition in which a hierarchical cell network (e.g., blood vessel or nerve network) is formed by air-liquid interface culture. describes the use of vascular endothelial cells as vascular cells.
  • a hierarchical cell network e.g., blood vessel or nerve network
  • lateral plate mesoderm cells are induced from human iPS cells by culturing using a medium containing BMP4, VEGF, CHIR99021, etc., and hematopoietic vascular endothelial cells ( CD34-positive, CD73-negative) were obtained, and a plurality of types of cells including hematopoietic vascular endothelial cells thus obtained were used to produce a matrix composition in which a hierarchical cell network was formed. is stated.
  • the present invention provides desired cells contained in cell clusters formed from pluripotent stem cells, particularly yolk sac mesoderm cells (hereinafter sometimes referred to as "YSMC”) or amniotic membranes.
  • YSMC yolk sac mesoderm cells
  • An object of the present invention is to provide means for enriching ectodermal cells (amniotic ectoderm cells, sometimes referred to as "AEC" in this specification).
  • the present inventors have found that two-dimensional culturing of pluripotent stem cells under the control of Wnt signals can enrich desired cells in cell clusters.
  • By two-dimensionally culturing pluripotent stem cells under conditions that activate Wnt signaling, such as adding an activator to the medium it is possible to produce cell clusters enriched with YSMCs.
  • AEC-enriched cell clusters can be produced by two-dimensionally culturing pluripotent stem cells under conditions in which Wnt signals are suppressed, such as by adding a Wnt signal inhibitor to the medium at the start of culture. .
  • desired cells such as YSMC and AEC and other cells form a layered structure while maintaining a two-dimensional positional relationship and boundary, and the layer containing the desired cells. It is also an important feature that the thickness can be increased.
  • the present invention includes at least the following items.
  • a method for producing cell clusters enriched with desired cells from pluripotent stem cells comprising the step of two-dimensionally culturing pluripotent stem cells under Wnt signal control.
  • the desired cells are yolk sac mesoderm cells, and the step of two-dimensionally culturing the pluripotent stem cells under the control of Wnt signals includes two-dimensionally culturing the pluripotent stem cells under conditions that activate Wnt signals. , Wnt signal activation culture step, the method according to [1].
  • the Wnt signal activation culturing step is a step of two-dimensionally culturing the pluripotent stem cells using a medium supplemented with a Wnt signal activator.
  • the Wnt signal activator is a GSK3 inhibitor.
  • the GSK3 inhibitor is CHIR99021.
  • the Wnt signal activation culture step is performed at the start of culture of the pluripotent stem cells.
  • the method of [1], wherein the pluripotent stem cells are induced pluripotent stem cells.
  • a method for producing a cell population further comprising a culturing step of differentiating the obtained yolk sac mesoderm cells into CD34+ vascular endothelial progenitor cells after the Wnt signal activation culturing step of [2].
  • the method of [7] further comprising a culturing step of differentiating the CD34+ vascular endothelial progenitor cells into CD34+CD32+ vitelline vein hematopoietic endothelial cells.
  • a cell cluster obtained by the method described in [1].
  • a method for producing an organoid or a three-dimensional organ comprising three-dimensionally culturing at least part of the cell cluster according to [9] or the cell population according to [10].
  • An organoid or three-dimensional organ obtained by the production method of [11].
  • the desired cells are amniotic ectoderm cells, and the step of two-dimensionally culturing the pluripotent stem cells under the control of Wnt signals includes two-dimensionally culturing the pluripotent stem cells under conditions in which Wnt signals are suppressed.
  • the method according to [13], wherein the Wnt signal suppression culture step is performed using a medium supplemented with a Wnt signal inhibitor.
  • the method of [14], wherein the Wnt signaling inhibitor is IWP-2.
  • YSMC-enriched cell clusters can be produced, and AEC-enriched cell clusters can be produced by a Wnt signal suppression culture step. Furthermore, YSMC-enriched cell clusters can efficiently produce CD34+ vascular endothelial progenitor cells, which can be used, for example, in the production of organoids or three-dimensional organs.
  • FIG. 1 shows examples of [1] “Preparation of human yolk sac mesoderm cells by Wnt signal activation culture process (first embodiment of the present invention)” and [3] “Cell population differentiation markers by immunocell staining About “analysis”. "+CHIR” indicates when CHIR99021 (Wnt signal activator) was added to the medium, and "(-)” indicates when CHIR99021 was not added as a control.
  • Fig. 1A An image observed by an optical microscope. The upper right row is an image of human iPS cells 2 days after the start of culture under Wnt signal activation conditions (Wnt signal activator is added to the medium), and the lower right row is a control condition in which Wnt signals are not activated ( Fig.
  • FIG. 10 is an image of human iPS cells 2 days after initiation of culture under the condition that no Wnt signal activator is added to the medium.
  • FIG. 1B Fluorescence images of Brachyury and GATA6. In the color image, the center of the cell cluster is stained green, indicating Brachyury expression, and the outer edge is stained purple, indicating GATA6 expression.
  • FIG. 1C A graph showing the relationship between the fluorescence intensity (converted to grayscale) of each of Brachyury, GATA6 and FOXF1 in each of +CHIR and (-) fluorescence images and the distance from the center.
  • the center of the cell cluster is stained blue with DAPI, and the outer edge is stained red, indicating the expression of GATA6.
  • Middle row fluorescence images of SOX2 and CDX2.
  • the center of the cell cluster is stained with cyan, indicating SOX2 expression, and the outer edge is stained with pink, indicating CDX2 expression.
  • Bottom fluorescence images of TFAP2A and SOX2.
  • the central part of the cell cluster is stained with cyan indicating SOX2 expression and the outer edge is stained with orange indicating TFAP2A expression (predominantly +IWP-2).
  • FIG. 3 shows examples of [4] “Preparation of human yolk vein hematopoietic endothelial cells”, [5] Differentiation marker analysis of cell populations by immune cell staining, and [6] “Differentiation marker analysis using flow cytometry”.
  • “+CHIR” indicates the case of differentiation induction from cell clusters obtained by adding CHIR99021 (Wnt signal activator) to the medium, and "(-)” indicates cells obtained without the addition of CHIR99021 as a control. This is the case when differentiation is induced from the cluster.
  • FIG. 3A Fluorescence images of Brachyury and CD34.
  • the cell population on the right (+CHIR) has a relatively small green central area showing Brachyury expression and a relatively large red peripheral area showing CD34 expression.
  • the cell population on the left (-) has a relatively large green central area indicating Brachyury expression, and the outer edge is stained with GATA6 and orange indicating lower Brachyury expression than the central area.
  • FIG. 3B Analysis results of flow cytometry based on the expression of CD34 and CD32.
  • the method for producing cell clusters of the present invention is a method for producing cell clusters enriched with desired cells from pluripotent stem cells, and includes a step of two-dimensionally culturing pluripotent stem cells under the control of Wnt signals.
  • the method for producing cell clusters of the present invention typically includes the following two embodiments, a first embodiment and a second embodiment.
  • desired cells are "enriched" by the amount of desired cells in a cell cluster or the ratio of the amount of desired cells to the amount of total cells is enriched Refers to an increase relative to the amount or proportion in a control, such as a previous cell cluster, or a cell cluster obtained without practicing the invention (under conditions that do not modulate the Wnt signal).
  • a control such as a previous cell cluster, or a cell cluster obtained without practicing the invention (under conditions that do not modulate the Wnt signal).
  • enriching and “enrichment” of desired cells refer to the desired amount of cells in a cell cluster or the desired amount of cells relative to the total amount of cells. Refers to increasing the proportion of an amount as compared to the amount or proportion in a control.
  • the amount or proportion of desired cells in cell clusters produced by the invention is at least 20%, 30%, 40%, or 40% compared to the amount or proportion in control cell clusters. %, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, Enriched to increase by 1000%.
  • two-dimensional culture of pluripotent stem cells refers to feeder-free adherent culture of pluripotent stem cells.
  • Culture methods for two-dimensionally culturing pluripotent stem cells such as iPS cells and ES cells (feeder-free adherent culture) are known.
  • methods for culturing human ES/iPS cells feeder-free are described in Rodin S et al., Nat Biotechnol. (2010) 28(6):611-5, Chen et al., Nat Methods (2011) 8(5 ):424-429, Miyazaki, T. et al. Nat Commun (2012) 3, 1236, Okita et al., Stem Cells, (2013) 31(3):458-66, and Nakagawa M et al., Scientific Reports , (2014) 4:3594.
  • cell cluster refers to an aggregate of cells formed from colonies of pluripotent stem cells and having micropatterns specific to embryonic development regions.
  • a "colony” refers to a visible clump formed from a single cell on a solid medium
  • a “pluripotent stem cell colony” refers to a single pluripotent stem cell that is undifferentiated. It refers to a visible cell aggregate that self-renews while maintaining , and that the replicated cells adhere to each other and multiply.
  • micropattern refers to a state in which different cell types are not randomly arranged but arranged according to a certain rule (the rule may be naturally occurring or artificially designed), and "embryonic development area A “specific micropattern” is a geometric arrangement that partially approximates the cell differentiation pattern in living embryogenesis, formed by the differentiation of cell aggregates into multiple types of cells while maintaining radial symmetry.
  • differentiation-inducing factors added to the medium are known (see, for example, Non-Patent Documents 1 to 3 cited above).
  • the same differentiation-inducing factors as in the past can be used in both the first embodiment and the second embodiment described below. Examples of such differentiation-inducing factors include BMP4, TGFb, bFGF, and VEGF, and BMP4 and VEGF are particularly preferred.
  • the amount of the differentiation-inducing factor to be used is not particularly limited, and it depends on the type of the differentiation-inducing factor in the Wnt signal activation culture step or the Wnt signal suppression culture step, the combination of the differentiation-inducing factors, or the combination of the differentiation-inducing factor and the medium.
  • cell clusters having a predetermined micropattern are formed, and particularly in the present invention, cell clusters enriched with desired cells are formed when a Wnt signal activator or Wnt signal inhibitor is further added.
  • the concentration of BMP4 in the medium can be, for example, in the range of 10-100 ⁇ M, preferably 50-80 ⁇ M.
  • the concentration of TGFb in the medium can be, for example, in the range of 2-20 ⁇ M, preferably 2-10 ⁇ M.
  • the concentration of bFGF in the medium can be, for example, within the range of 10-100 ⁇ M, preferably 50-80 ⁇ M.
  • the concentration of VEGF in the medium can be, for example, in the range of 5-100 ⁇ M, preferably 50-80 ⁇ M.
  • a Wnt signal activator may be further added to the "medium" used for two-dimensional culturing of pluripotent stem cells under the control of Wnt signals.
  • general media used for two-dimensional culture of pluripotent stem cells especially two-dimensional culture of pluripotent stem cells It can be basically the same as a known medium used for forming micropatterned cell clusters.
  • a person skilled in the art can prepare an appropriate medium by selecting an appropriate type and amount of basal medium and additives used as necessary according to the pluripotent stem cells to be cultured, and mixing them. be able to.
  • Media for pluripotent stem cells such as iPS cells and ES cells include, for example, DMEM, DMEM/F12 or DME culture medium containing 10-15% FBS (these culture mediums further include LIF, penicillin/ streptomycin, puromycin, L-glutamine, non-essential amino acids, ⁇ -mercaptoethanol, etc.) and commercially available culture media such as mouse ES cell culture media (TX-WES culture media, Thrombo X), primate ES cell culture medium (primate ES/iPS cell culture medium, Reprocell), serum-free medium (mTESR, Stemcell Technology), iPS/ES cell growth medium/for regenerative medicine Medium (StemFit (registered trademark) AK02N, Ajinomoto Healthy Supply Co., Inc.), feeder-free medium for iPS cell culture (Essential 8, Gibco), and serum-free medium can also be used (e.g., Sun N, et al. (2009), Proc Natl Acad Sci
  • the culture vessel is not particularly limited, and dishes, flasks, microplates, cell culture sheets such as the product name "OptiCell” (Nunc), etc. can be used.
  • Culture vessels are surface-treated to improve cell adhesion (hydrophilicity), collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, fibronectin, matrigel (e.g., BD Matrigel (Japan) It is preferably coated with a substrate for cell adhesion such as Becton Deckinson)), vitronectin or the like.
  • the first embodiment is a method for producing a cell cluster enriched with "yolk sac mesoderm cells” (YSMC) as “desired cells",
  • YSMC yolk sac mesoderm cells
  • Wnt signal activation culture step a step of two-dimensionally culturing pluripotent stem cells under conditions that activate Wnt signals.
  • YSMC are cells that differentiate into blood cells that form blood islands and cells that form blood vessels in the yolk sac, which is an extraembryonic hematopoietic organ in the early stages of development.
  • FOXF1, GATA3, GATA4, fibronectin (FN1), collagen (e.g., COL1A1, COL1A2, COL3A1, COL4A1, COL6A1, COL6A3), laminin-111 (e.g., LAMA1, LAMB1, LAMC1), KDR and HAND1, etc. may also be positive.
  • YSMC are enriched in cell clusters and to what extent they are enriched can be determined, for example, by analyzing fluorescence images obtained by immunostaining targeting such cell markers and comparing them with controls. can be evaluated or determined quantitatively or qualitatively.
  • the Wnt signal activation culture step is performed at the start of culture (early culture) for inducing differentiation from pluripotent stem cells to cell clusters having a predetermined micropattern, more specifically, from pluripotent stem cells to extraembryonic cells.
  • this occurs before differentiation into the embryonic epiblast, which contains the embryonic ectoderm and the primitive streak, and the extraembryonic ectoderm.
  • Wnt signaling is activated.
  • a Wnt signal activator (together with a predetermined differentiation-inducing factor) to the medium on Day 0, and culture pluripotent stem cells such as iPS cells in the medium. In such embodiments, it is not essential to add the Wnt signal activator to the medium on Day 1 and/or the medium on Day 2 as well.
  • Conditions for activating Wnt signaling are not particularly limited as long as the effect of the present invention is that YSMC-enriched cell clusters are obtained, and known means are used. be able to.
  • conditions for activating Wnt signaling include the addition of a type and amount of a substance that has the effect of activating Wnt signaling, ie, a “Wnt signaling activator” to the medium.
  • Wnt signal activator is not particularly limited, for example, a GSK3 inhibitor is preferable.
  • GSK3 inhibitors include compounds such as CHIR99021, SB-216763, BIO (6-bromoindirubin-3'-oxime), and LY2090314.
  • Wnt signal activators other than GSK3 inhibitors include SFRP inhibitors (eg, WAY-316606), Notum inhibitors (eg, ABC99), PP2A activators (eg, IQ1), ARFGAP1 activation (e.g. QS11), ⁇ -catenin activator (e.g.
  • DCA (hetero)arylpyrimidine, 2-amino-4-[3,4-(methylenedioxy)benzyl-amino]-6-(3- and compounds such as methoxyphenyl)pyrimidines.
  • two or more, eg, two, three, or four Wnt signaling activators may be used in combination.
  • the amount of Wnt signal activator to be used is not particularly limited. can be adjusted as appropriate so as to be enriched in
  • a GSK3 inhibitor such as CHIR99021
  • the concentration of the GSK3 inhibitor in the medium can be, for example, within the range of 0.5-3 ⁇ M, preferably 1-2 ⁇ M.
  • the growth factor BMP4 which is commonly used for culturing iPS cells and the like (essential for induction of almost all differentiated cells from epiblasts), is known to be related to Wnt signals.
  • Wnt signal activator in the first embodiment When used in a general amount for use in the culture of, i.e., in an amount in which the effect of the first embodiment of the present invention of enriching YSMC is not observed (e.g., used as a control in the examples below) shall not be regarded as the Wnt signal activator in the first embodiment.
  • the Wnt signal activator in the present invention is the first embodiment of the present invention that alone enriches YSMCs even if BMP4 is present in the medium in a general amount. Refers to substances other than BMP4 of a type and amount that are found (such as by comparison with a control) to have the effect of
  • the second embodiment is a method for producing a cell cluster enriched with "amniotic ectoderm cells (AEC)" as “desired cells”, , as a “step of two-dimensionally culturing pluripotent stem cells under the control of Wnt signals", perform a "step of two-dimensionally culturing pluripotent stem cells under conditions in which Wnt signals are suppressed" (Wnt signal-suppressed culture step).
  • AEC amniotic ectoderm cells
  • AECs are ectodermal cells that differentiate into amniotic epithelial cells that cover the fetal embryo.
  • cell markers for AEC CDX2, TFAP2A, GATA3, TFAP2B, E-cadherin, etc. are known, and can be identified by whether one or more of them are positive (if positive, AEC, other than AEC if negative).
  • cell markers positive markers
  • whether or not one or more of NANOG, SOX2, etc. which are cell markers of undifferentiated cells or embryonic ectoderm are negative, is also used to identify AECs. Available (AEC if negative, undifferentiated cells or ectoderm if positive).
  • Whether AEC is enriched in cell clusters can be quantitatively or qualitatively evaluated or determined, for example, by analyzing fluorescence images obtained by immunostaining targeting such cell markers and comparing with controls. can do.
  • Wnt signal inhibitor is not particularly limited, for example, a porcupine (porcupin, PORCN) inhibitor is preferable.
  • Porcupine inhibitors include, for example, compounds such as IWP-2, LGK974, Wnt-C59, ETC-159, IWP-O1, IWP L6, GNF-6231, Porcn-IN-1.
  • Wnt signal inhibitors include, for example, (non-membrane-bound) free Wnt inhibitors (e.g., Ant1.4Br/Ant1.4Cl), Frizzled inhibitors (e.g., Niclosamide), Vacuolar ATPase inhibitors.
  • agents e.g., apicularen and bafilomycin
  • tankyrase 1/Axin activators e.g., XAV939
  • Axin activators e.g., IWR
  • tankyrase e.g., Axin activators
  • Axin activators e.g., G007-LK and G244-LM
  • CK1 inhibitors e.g. pyrvinium
  • Dsh inhibitors e.g. NSC668036
  • TCF/ ⁇ -catenin inhibitors e.g. 2,4-diamino-quinazoline, PKF115-584
  • TCF inhibitors e.g.
  • CREB Compounds such as binding protein inhibitors (eg ICG-001), ⁇ -catenin TBL interaction inhibitors (eg BC2059), Shizokaol D and the like.
  • binding protein inhibitors eg ICG-001
  • ⁇ -catenin TBL interaction inhibitors eg BC2059
  • Shizokaol D e.g., Shizokaol D and the like.
  • two or more, for example, two, three, or four Wnt signal inhibitors may be used in combination.
  • the amount of the Wnt signal inhibitor to be used is not particularly limited, and depending on the type of the Wnt signal inhibitor, the desired degree of action and effect, that is, the desired degree of enrichment of AECs in cell clusters, can be obtained. can be adjusted as appropriate.
  • a porcupine inhibitor such as IWP-2
  • the concentration of the porcupine inhibitor in the medium is, for example, 0.5-2 ⁇ M, preferably 0.5-1 ⁇ M. can be done.
  • BMP4 can be added to the medium in a general amount for use in culturing iPS cells and the like. However, it refers to the types and amounts of substances found (such as by comparison with a control) to have the effect of the second embodiment of the present invention of enriching AECs.
  • the cell clusters obtained by the method for producing cell clusters of the present invention contain cells other than desired cells such as YSMC and AEC (herein referred to as "undesired cells”). can be done. Undesired cells include cells other than YSMC and AEC contained in known micropatterns, such as SOX2-positive embryonic ectoderm, T (brachyury)-positive cells (primitive streak), definitive mesoderm derived therefrom, and and definitive endoderm.
  • desired cells such as YSMC and AEC and undesired cells form a layered structure while maintaining two-dimensional positional relationships and boundaries. It is also one of the features of the present invention that the thickness of the layer containing desired cells can be increased.
  • CD34+ vascular endothelial progenitor cells In the method for producing a cell population of the present invention, CD34+ vascular endothelial progenitor cells (EPCs) (EPC differentiation step), and may further include other steps as necessary.
  • EPCs endothelial progenitor cells
  • the "cell population” particularly referred to in relation to the production method is the differentiation from the desired cells (YSMC, AEC, etc.) by further culturing after the above-mentioned “cell cluster” is formed. It refers to an aggregate of cells that contains the cells that have been treated.
  • EPCs are cells that can be identified as CD34-positive cells and have the ability to differentiate into vascular endothelial cells (EC).
  • ECs differentiated from EPCs include, for example, vitelline venous hemogenic endothelial cells (VVHEC, CD34-positive and CD32-positive) described later, microvessel endothelial cells (MVEC), umbilical vein Endothelial cells (umbilical-vein endothelial cells: UVEC) and the like are included.
  • ECs differentiated from EPCs include hematopoietic endothelial cells (hemogenic endothelial cells; HEC, CD34-positive and CD73-negative), which refer to vascular endothelial cells with hematopoietic potential, and vascular endothelial cells without hematopoietic potential. Both non-hemogenic endothelial cells (non-HEC, CD31 positive, CD73 positive and CD144 positive) are included.
  • the medium in the method for producing a cell population of the present invention may be a medium for pluripotent stem cells in the above-described method for producing a cell cluster of the present invention, or a medium for target cells differentiated from pluripotent stem cells or the like, or Mixtures can be used.
  • Basic media for EPC include, for example, DMEM/F-12 (Gibco), Stempro-34 SFM (Gibco), Essential 6 medium (Gibco), Essential 8 medium (Gibco), EGM (Lonza), BulletKit (Lonza) , EGM-2 (Lonza), BulletKit (Lonza), EGM-2 MV (Lonza), VascuLife EnGS Comp Kit (LCT), Human Endothelial-SFM Basal Growth Medium (Invitrogen), Human Microvascular Endothelial Cell Growth Medium (TOYOBO) etc.
  • Additives for EPC include, for example, B27 Supplements (GIBCO), BMP4 (bone morphogenetic factor 4), GSK ⁇ inhibitor (e.g., CHIR99021), VEGF (vascular endothelial cell growth factor), FGF2 (Fibroblast Growth Factor (bFGF ( basic fibroblast growth factor)), Folskolin, SCF (Stem Cell Factor), TGF ⁇ receptor inhibitor (e.g., SB431542), Flt-3L (Fms-related tyrosine kinase 3 ligand), IL-3 (Interleukin 3) , IL-6 (Interleukin 6), TPO (thrombopoietin), hEGF (recombinant human epidermal growth factor), hydrocortisone, ascorbic acid, IGF1, FBS (fetal bovine serum), antibiotics (e.g. gentamicin, amphotericin B ), heparin, L-glutamine, phenol red and BBE.
  • the EPC differentiation step is a predetermined period (e.g., about 2 days) following the Wnt signal activation culture step, in other words, it is calculated from the start of differentiation induction from the above-described pluripotent stem cells into cell clusters having a predetermined micropattern. It is appropriate to perform it on the 2nd to 4th day (Day2-Day4).
  • YSMCs Culture methods for differentiating YSMCs into EPCs, medium composition (types and amounts of basal medium, differentiation-inducing factors, other additives, etc.), culture conditions, and other culture-related matters are described, for example, by Ohta, R. See et al, J. Vis. Exp. (148), e59823, doi:10.3791/59823 (2019).
  • a differentiation-inducing factor such as VEGF, FGF2 (bFGF), SCF, TGFb inhibitor (eg, SB431542) can be added to the medium for the EPC differentiation step.
  • the method for producing a cell population of the present invention can further include other steps as necessary.
  • Such optional steps include, for example, a culturing step in which EPCs obtained by the EPC differentiation step are further differentiated into other cells.
  • the method for producing a cell population of the present invention further comprises a culture step (VVHEC differentiation step) of differentiating CD34 + CD32 + vitelline venous hemogenic endothelial cells (VVHEC) from EPCs.
  • VVHEC is a HEC derived from the yolk sac mesoderm, is known as a cell responsible for hematopoiesis in the yolk sac in the early fetal period, and is a cell that can be differentiated from YSMC via EPC in the present invention.
  • VVHEC cell markers for VVHEC
  • FN1 ACTA2 and LYVE1
  • vein markers such as NR2F2, APLNR and PROX1, and one or more (preferably all) of them are known. It can be identified by whether it is positive (VVHEC if positive, other than VVHEC if negative).
  • the VVHEC differentiation step is performed for a predetermined period (e.g., about 2 days) following the EPC culture step, in other words, 4 days from the start of differentiation induction from the above-described pluripotent stem cells to cell clusters having a predetermined micropattern. It is suitable to perform on the 6th day (Day 4-Day 6).
  • the culture method for differentiating EPCs into VVHEC, medium composition (types and amounts of basal medium, differentiation-inducing factors, other additives, etc.), culture conditions, and other culture-related matters include, for example, Matsybara et al. , Biochemical and Biophysical Research Communications, 515(1), 2019.
  • the medium for the VVHEC differentiation step can be supplemented with appropriate amounts of differentiation inducers such as VEGF, SCF, Flt-3L, IL-3, IL-6 and TPO.
  • the method for producing a cell population of the present invention includes a culture step of differentiating VVHEC obtained by the VVHEC differentiation step into other cells such as monocytes/macrophages (CD14 positive), erythroid/myelocyte progenitor cells (CD33 positive), and the like. It can also contain more.
  • the culture method for differentiating VVHEC into these cells, medium composition (types and amounts of basal medium, differentiation-inducing factors, other additives, etc.), culture conditions, and other culture-related matters are the same as known methods. can be When differentiating VVHEC into monocytes/macrophages (CD14 positive), erythroid/myelocyte progenitor cells (CD33 positive), etc., see, for example, Gene I. Uenishi et al, Nature Communications (2018) 9:1828. can.
  • the method for producing an organoid or three-dimensional organ of the present invention involves three-dimensionally culturing the cell cluster obtained by the method for producing a cell cluster of the present invention or at least a part of the cell population obtained by the method for producing a cell cluster of the present invention. including the step of
  • three-dimensional culture refers to culturing cells in a state of being embedded in a medium containing scaffolding components such as extracellular matrix.
  • Various methods and embodiments are common or publicly known as methods for producing organoids or three-dimensional organs by three-dimensional culture (see, for example, Patent Document 1 cited above).
  • the same methods and embodiments as conventional general or known production methods are used. , organoids or three-dimensional organs can be produced.
  • embryoids and gastruloids which are a type of organoid and are three-dimensionally constructed from the structure of early embryos.
  • the cell clusters or cell populations according to the present invention which contain cells located in the boundary region between the embryo and the ectoderm, such as YSMC and AEC, it is possible to produce embryoids or gastruloids whose structures can be more precisely reproduced. .
  • Kits according to the present invention include, for example, kits for the production of cell clusters enriched in yolk sac mesoderm cells (YSMC) comprising one or more Wnt signal activators, or one or more Wnt signal inhibitors, A kit for the production of cell clusters enriched for amniotic ectodermal cells (AEC). Matters related to such kits, such as Wnt signal activators, Wnt signal inhibitors, culture methods using the above kits, other components that can be included in the kit, etc., are described in this specification. The matters described in relation to the method for producing a cluster, the method for producing a cell population, etc. are the same, and the matters described therein can be referred to.
  • pluripotent stem cell refers to a cell that can differentiate into various tissues and cells with different morphologies and functions in the body, ectoderm) refers to stem cells that have the ability to differentiate into cells of any lineage. Pluripotent stem cells that can be used in the present invention are not particularly limited. Embryonic stem cells derived from cloned embryos obtained, spermatogonial stem cells, embryonic germ cells and the like can be mentioned.
  • iPS cells Induced pluripotent stem cells
  • iPS cells refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors).
  • iPSCs induced pluripotent stem cells, and iPSCs established by Yamanaka et al. (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), and human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. , et al.
  • Nanog-iPS cells were established by selecting Nanog expression as an indicator (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells generated by c-Myc-free method (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101 - 106), iPS cells established by introducing 6 factors by virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31( 3):458-66.) can also be used.
  • induced pluripotent stem cells established by introducing the four factors of OCT3/4, SOX2, NANOG, and LIN28 produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Unexamined Patent Application Publication No. 2008-307007) and the like can also be used.
  • iPS cell lines established by NIH, RIKEN (RIKEN), Kyoto University, etc. can be used as induced pluripotent stem cells (iPS cells).
  • iPS cells induced pluripotent stem cells
  • human iPS cell lines RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains, Kyoto University's 201B7, 253G1, and 253G4 strains, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 625A4 strain, 648A1 strain, 1201C1 strain, 1205D1 strain, 1210B2 strain, 1231A3 strain, 1383D2 strain, 1383D6 strain, and the like.
  • clinical grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines produced using these cell lines may be used.
  • mouse ESCs can use various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (RIKEN), etc., and human ES cells , NIH, RIKEN, Kyoto University, and various human ES cell lines established by Cellartis are available.
  • human ES cell lines include NIH CHB-1 to CHB-12, RUES1, RUES2, HUES1 to HUES28 strains, WisCell Research H1 and H9 strains, RIKEN KhES-1 and KhES- 2 strains, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. can be used.
  • clinical grade cell lines and research and clinical cell lines generated using those cell lines may be used.
  • the term “cell marker” refers to a gene that is specifically expressed (positive marker) or not expressed (negative marker) in a given cell type, specifically as mRNA by transcription of the gene in the genome , or a substance that is produced (positive marker) or not produced (negative marker) as a protein by translation of its mRNA.
  • the cell marker is preferably a protein expressed on the cell surface (cell surface marker) that can be labeled (stained) with a fluorescent substance, and can easily detect, concentrate, isolate, etc. cells expressing the cell marker. is.
  • a marker gene is "positive” means that the expression level of the mRNA or protein of the gene is detectable by a technique commonly used or known to those skilled in the art, or is lower than a predetermined threshold (background level, etc.) means high.
  • a predetermined threshold background level, etc.
  • Whether a cell marker is positive or negative can be determined with qualitative or quantitative results by methods commonly known to those skilled in the art.
  • a cell marker as a protein can be detected or its expression level can be measured using an immunological assay using an antibody specific to the protein, such as ELISA, immunostaining, flow cytometry, and the like.
  • Cell markers as mRNA are detected or the expression level is measured using assays using nucleic acids specific to the mRNA, such as RT-PCR (including quantitative PCR), microarrays, biochips, etc. be able to.
  • Organoid refers to a three-dimensional structure that is artificially created by combining multiple types of cells and resembles various organs and tissues. Organoids include organ organoids, cancer organoids, etc. In a broad sense, embryoids that are three-dimensionally constructed from the structure of an early embryo (eg, Science 07 Jun 2019: Vol. 364, Issue 6444, pp. 948-951 DOI: 10.1126/science.aax0164) and gastruloids (eg, Nature volume 582, pages 410-415 (2020)).
  • Organ organoids include not only those at a relatively mature stage as organoids that have similar functions and structures to various organs or tissues, but also "organ buds” at an early stage of such complexity, Structures called “primordia” and the like are also included.
  • organ organoids such as liver, pancreas, kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonad, brain, spinal cord, etc. (e.g.
  • the three-dimensional structure of organoids can be confirmed with the naked eye or by microscopic observation.
  • proteins of these markers are more preferably secreted into the culture supernatant. It can be determined by whether
  • three-dimensional organ refers to a structure comprising a more mature cell population or structure than an organ organoid, which can also be called a mature organ organoid.
  • Whether or not a three-dimensional organ was obtained from an organ organoid is determined, for example, by the density of cells in the structure (whether it exceeds a predetermined standard, etc.), the three-dimensional shape of the structure (whether it is more three-dimensional than a certain level, etc.), Functions and traits (whether a predetermined function or trait such as a metabolic function is acquired), cell markers (whether the gene or protein expression of the cell marker is positive, whether the density of positive cells exceeds a predetermined standard, whether the amount of marker protein secreted in the culture supernatant exceeds a predetermined standard, etc.).
  • the above cell density, three-dimensional shape, function and trait, cell markers, etc. can be appropriately set according to the organ organoid and three-dimensional organ. Or whether or not a level close to it has been achieved can be used as the basis for the above determination.
  • the cells contained in colonies, cell clusters, cell populations, organoids, three-dimensional organs, etc. of pluripotent stem cells of the present invention may be derived from humans or non-human animals such as mice, rats, and dogs. , pigs, monkeys, and other mammals.
  • the cells are preferably of human origin.
  • “Comprise, include, contain, etc.” means the inclusion of elements following the phrase, but is not limited to this. Thus, the inclusion of the elements following the phrase, but not the exclusion of any other element, is suggested.
  • “consisting of,” means including and limited to any and all elements following the phrase. Thus, the phrase “consisting of” indicates that the listed element is required or required and that other elements are substantially absent.
  • “consisting essentially of, etc.” includes any element following the phrase and is limited to other elements that do not affect the activity or action specified in this disclosure for that element. means to be Thus, the phrase “consisting essentially of” indicates that the listed elements are required or required but other elements are optional and that they affect the activity or action of the listed elements. indicates that it may or may not be present, depending on whether it exerts
  • Day represents the number of days from the initiation of differentiation induction of human iPS cell colonies.
  • Day 0 is the time of initiation of culture under Wnt signal control.
  • Example 1 Preparation of yolk sac mesoderm cells and amniotic ectoderm cells under Wnt signal control
  • Human iPS cells (625A4; iPS Research Institute, Kyoto University) were cultured in AK02N (Ajinomoto) (10cm dish; 8ml, 24-well plate; 0.5ml) at 5% CO 2 and 37°C for 6-7 days. , formed iPS cell colonies with a diameter of 500-700 ⁇ m.
  • the resulting colonies were added to Essential 8 medium (Gibco) (10 cm dish; 8 ml, 24-well plate; 0.5 ml) with BMP4 (80 ng/ml), VEGF (80 ng/ml) and CHIR99021 (2 ⁇ M). It was cultured in a medium at 5% CO 2 and 37° C. for 2 days (Day 0-2).
  • Essential 8 medium Gibco
  • BMP4 80 ng/ml
  • VEGF 80 ng/ml
  • CHIR99021 2 ⁇ M
  • Fig. 1 shows the analysis results of the cell markers Brachyury, GATA6 and FOXF1 for the cell clusters on Day 2 of [1].
  • the Wnt signal activator CHIR99021 was added to the medium (+CHIR), compared to the case where it was not added (-), Brachyury-negative, GATA6-positive and FOXF1-positive
  • YSMC was enriched, in other words, the peripheral YSMC region was increased
  • FIGGS. 1B and 1C the peripheral YSMC region was increased
  • Fig. 2 shows the analysis results of the cell markers GATA6, SOX2, CDX2 and TFAP2A for the cell clusters on Day 2 of [1] and [2].
  • IWP-2 which is a Wnt signal inhibitor
  • - when IWP-2, which is a Wnt signal inhibitor, was added to the medium (+IWP-2), compared to when it was not added (-), CDX2 positive (middle row) and It was confirmed that cell clusters enriched with TFAP2A-positive (bottom row) AECs, in other words, having an AEC region at the outer edge, were formed.
  • Stempro-34 SFM (Gibco) (10 cm dish; 8 ml) was added with VEGF (80 ng/ml), SCF (50 ng/ml), Flt-3L (50 ng/ml), IL-3 (50 ng/ml), IL- 6 (50 ng/ml) and TPO (5 ng/ml) were added to the medium, cultured at 5% CO 2 at 37° C. for 2 days, and then the medium with the above composition was replaced with a medium excluding VEGF.
  • CD34-positive and CD32-positive yolk vein hematopoietic endothelial cells were induced by culturing at 37° C. in 5% CO 2 for 2 days.
  • FIG. 3(A) shows the analysis results of cell markers Brachyury and CD34 for the cell population on Day 6 of [4]. It was confirmed that abundant CD34-positive vascular endothelial progenitor cells emerged from the outer YSM region of the cell cluster (+CHIR) obtained according to the first embodiment of the present invention.
  • Figure 3(B) shows the results of flow cytometry for the cell population on Day 8 of [4]. From the YSM region at the outer edge of the cell cluster (+CHIR) obtained according to the first embodiment of the present invention, more CD34-positive and CD32-positive vitelliform hematopoietic endothelial cells compared to the control (-) In other words, CD34-positive vascular endothelial progenitor cells in the YSM region were confirmed to have the ability to differentiate into CD32-positive yolk vein hematopoietic endothelial cells.

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