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WO2025170010A1 - Procédé de production de cellules d'épicarde mûres - Google Patents

Procédé de production de cellules d'épicarde mûres

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Publication number
WO2025170010A1
WO2025170010A1 PCT/JP2025/004014 JP2025004014W WO2025170010A1 WO 2025170010 A1 WO2025170010 A1 WO 2025170010A1 JP 2025004014 W JP2025004014 W JP 2025004014W WO 2025170010 A1 WO2025170010 A1 WO 2025170010A1
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WIPO (PCT)
Prior art keywords
cells
epicardial
torin1
cell
expression
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Pending
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PCT/JP2025/004014
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English (en)
Japanese (ja)
Inventor
善紀 吉田
アントニオ ルセナカカセ
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Kyoto University NUC
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Kyoto University NUC
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Publication of WO2025170010A1 publication Critical patent/WO2025170010A1/fr
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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • 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
    • C12N5/0006—Modification of the membrane of cells, e.g. cell decoration
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to a method for producing mature epicardial cells. More specifically, it relates to a method for producing mature epicardial cells, which includes a step of culturing epicardial cells in a medium containing an mTORC2 inhibitor, and to uses of the epicardial cells obtained by this method.
  • the human epicardium is the outermost layer of the heart and plays a crucial role in cardiac development and regeneration.
  • Epicardial cells are the source of various cells that form the heart, including cardiac fibroblasts, smooth muscle cells, and endothelial cells, and play an important role in cardiac organogenesis. Furthermore, these epicardial cell-derived cells play important roles in maintaining cardiac homeostasis and regulating responses to organ injury. For these reasons, epicardial cells and cells derived from epicardial cells are important cell groups in research on cardiac disease pathology models. Furthermore, recent mouse studies have shown that epicardial cells have the ability to regenerate after myocardial injury during fetal development and the first few days after birth (Non-Patent Document 1).
  • Non-Patent Document 2 the epicardium becomes quiescent and loses its regenerative ability as the tissue functionally matures.
  • Non-Patent Document 3 quiescent epicardial cells remain metabolically and transcriptionally active.
  • Non-Patent Document 4 fetal-type epicardial cells
  • WT1 and TBX18 markers of fetal-type epicardial cells
  • EMT epithelial-mesenchymal transition
  • an objective of the present invention is to provide a method for producing mature epicardial cells, and epicardial cells produced by said method.
  • inducing epicardial cell quiescence might produce adult-type (mature) epicardial cells that lack regenerative capacity.
  • mTOR mechanistic target of rapamycin
  • the mTOR signaling pathway plays an important role in controlling cellular quiescence in mature tissues by sensing and responding to changes in nutrient availability, growth factors, and other environmental cues (e.g., Liu, G. Y. & Sabatini, D. M. Nat. Rev. Mol. Cell Biol. 21, 183-203 (2020)).
  • mTOR mechanistic target of rapamycin
  • the present invention is as follows.
  • a method for producing mature epicardial cells comprising the step of culturing epicardial cells in a medium containing an mTORC2 inhibitor.
  • the method according to [1] or [2], wherein at least one of the mTORC2 inhibitors is selected from the group consisting of Torin1, sapanisertib, WYE-687, AZD8055, and bistusertib.
  • the method according to [1] or [2], wherein at least one of the mTORC2 inhibitors is Torin1.
  • a transplantation therapy agent comprising the mature epicardial cells or cardiac organoids described in [7].
  • a method for producing mature cardiomyocytes comprising a step of co-culturing the mature epicardial cells or cardiac organoids described in [7] with cardiomyocytes.
  • a method for maturing epicardial cells comprising the step of culturing epicardial cells in a medium containing an mTORC2 inhibitor.
  • a method for maturing cardiomyocytes comprising a step of co-culturing the mature epicardial cells or cardiac organoids according to [7] with cardiomyocytes.
  • a method for treating or preventing heart disease comprising administering or transplanting an effective amount of the mature epicardial cells or cardiac organoids described in [7] and/or the cardiomyocytes or cell aggregates described in [10-2] to a mammal.
  • the mature epicardial cells or cardiac organoids according to [7] and/or the cardiomyocytes or cell aggregates according to [10-2] for use in the treatment or prevention of cardiac disease.
  • the present invention provides a novel method for producing mature epicardial cells from immature epicardial cells. Furthermore, it is also possible to produce mature cardiomyocytes using these mature epicardial cells.
  • FIG. 1 Venn diagram for targeted screening and identification of mTORC2-dependent elements also expressed in primary adult epicardial tissue.
  • Venn1 top left shows transcription factors (TFs) from Torin1, excluding those shared with or unique to rapamycin.
  • Venn2 (bottom left) shows TFs from differentially expressed genes (DEGs) after filtering out statistically significant genes in both fetal and adult epicardium.
  • Epicardial cells constitute the outermost layer of an animal's heart and play an important role in cardiac development and regeneration.
  • immature epicardial cells refer to cells that express WT1 and TBX18 and have proliferation and epithelial-mesenchymal transition activity.
  • mature epicardial cells refer to cells that have reduced or absent (in other words, “no expression”) fetal epicardial marker expression levels compared to epicardial cells prior to the step of culturing in a medium containing an mTORC2 inhibitor. These cells typically have further reduced or absent proliferation and epithelial-mesenchymal transition activity compared to epicardial cells prior to the step of culturing in a medium containing an mTORC2 inhibitor.
  • the epicardial cells of the present invention may also have increased expression levels of IGF2 and/or FN1.
  • markers for fetal epicardial cells include WT1, TBX18, ALDH1A2, and HAND2.
  • Cells in which the expression level of at least one of these (e.g., WT1) is reduced or absent can be considered mature epicardial cells, but preferably the expression levels of two or more (e.g., a combination of WT1 and TBX18, a combination of WT1, TBX18, ALDH1A2, and HAND2, etc.) are reduced or absent.
  • mature epicardial cells do not express WT1 and/or TBX18, and more preferably do not express WT1 and TBX18.
  • epicardial cells can be matured by culturing them in a medium containing an mTORC2 inhibitor, and epicardial cells within cardiac organoids can also be matured.
  • the epicardial cells used in the present invention are contained in cardiac organoids, and the epicardial cells are subjected to the production method of the present invention in the form of cardiac organoids. Therefore, the present invention also provides a method for producing cardiac organoids containing mature epicardium, which includes the step of culturing cardiac organoids containing epicardial cells in a medium containing an mTORC2 inhibitor.
  • cells includes “cell populations.” Furthermore, unless otherwise specified, “cells” refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Furthermore, unless otherwise specified, “cell populations” also include “organoids” such as “cell aggregates” and “artificial tissues.”
  • organoid refers to a structure (typically a spheroid) containing multiple types of cells.
  • artificial tissue typically refers to a structure that has a structure and/or function similar to that of tissue in a living organism. Whether a structure is an organoid or an artificial tissue can be determined, for example, by observing a sample that has been stained as necessary (e.g., immunostaining, hematoxylin-eosin (HE) staining, etc.) under a microscope to confirm the localization of cells and the presence or absence of layered structure formation.
  • HE hematoxylin-eosin
  • cardiac organoid refers to an organoid containing epicardial cells, and includes artificial cardiac tissue.
  • artificial cardiac tissue refers to artificial tissue containing epicardial cells.
  • cardiac tissue will be simply referred to as "cardiac tissue.”
  • cardiac organoids of the present invention contain TNNT2-positive cardiomyocytes exhibiting pulsatile activity, CD31-positive endothelial cells, NFATC1-positive endocardial cells, and VIM-positive cardiac fibroblasts. Cardiac organoids may also have tight junctions as indicated by ZO-1 expression.
  • the epicardial cells used in the present invention can be obtained by known methods. Examples include isolation from the heart using known techniques, differentiation induction of pluripotent stem cells, and procurement from companies such as ATCC. Midgut cells can be isolated from the heart using, for example, flow cytometry or mass cytometry using a surface antigen (e.g., CDH18) as an indicator, magnetic cell separation, or affinity columns immobilized with the desired antigen.
  • the epicardial cells used in the present invention are preferably obtained by differentiation induction of pluripotent stem cells.
  • the origin of the epicardial cells is not particularly limited, and may be from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, and sheep; carnivores such as dogs and cats; humans; and primates such as monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. Humans are the preferred origin.
  • the production method of the present invention may comprise at least one of the following steps (A) to (C).
  • EBs embryonic bodies
  • B A step of inducing cell aggregates containing mesodermal cells by subjecting the cell aggregates obtained in step (A) to suspension culture in the presence of BMP4, activin A, and bFGF.
  • C A step of dispersing the cell aggregates obtained in step (B) and culturing them in the presence of CHIR99021, BMP4, VEGF, and SB431542 to induce differentiation into epicardial cells.
  • the epicardial cells obtained in step (C) can also be maintained for a long period of time in a maintenance medium.
  • maintenance medium include, but are not limited to, DMEM containing 10% FBS and 10 ⁇ M SB431542.
  • step (A) The formation of cell aggregates in step (A) is typically carried out by the following method.
  • pluripotent stem cells are recovered from subculture and dispersed into single cells or a state close to single cells.
  • the pluripotent stem cells are dispersed using an appropriate cell dissociation solution.
  • cell dissociation solutions include EDTA; trypsin, collagenase IV, metalloproteases, and other proteolytic enzymes, which can be used alone or in appropriate combinations.
  • Commercially available cell dissociation solutions include Accutase (MILLIPORE), Dispase (EIDIA), and TrypLE (Invitrogen).
  • the dispersed pluripotent stem cells are suspended in a medium in a low-adhesion culture vessel.
  • the duration of step (A) can be determined as appropriate by those skilled in the art, and is usually 0.5 to 5 days (particularly 1 day).
  • Step (B) is a step of inducing differentiation of pluripotent stem cells into mesodermal cells, and the medium used in this step preferably contains an extracellular matrix (e.g., 0.5% Matrigel) in addition to the above-mentioned additives.
  • the duration of step (B) can be appropriately determined by those skilled in the art, and is usually 1 to 6 days, with 2 to 4 days (particularly 2.5 days) being more preferred.
  • Step (C) is a step of inducing differentiation of mesodermal cells into epicardial cells, and is typically carried out by dispersing cell aggregates into single cells or a state close to single cells, as in step (A), and then culturing the cells in an adherent culture.
  • the duration of step (C) can be appropriately determined by those skilled in the art, and is usually 3 to 10 days, with 4 to 7 days (particularly 5.5 days) being more preferable.
  • cardiac organoids can be induced by, for example, culturing the cell aggregates obtained in step (A) above in suspension in a medium containing CHIR99021, BMP4, and Activin A, then treating the cell aggregates with Wnt-C59 for approximately two days, and continuing the suspension culture until the 15th day.
  • step (A) It is also possible to produce artificial cardiac tissue from cardiac organoids by dispersing cell aggregates into single cells or a state close to single cells, as in step (A), and then culturing the cells in an adhesive culture medium for artificial cardiac tissue.
  • pluripotent stem cells refer to stem cells that can differentiate into tissues and cells with a variety of different morphologies and functions in the body, and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm).
  • pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells), with iPS cells (more preferably human iPS cells) being preferred.
  • iPS cells induced pluripotent stem cells
  • ES cells embryonic stem cells
  • mGS cells multipotent germline stem cells
  • EG cells embryonic germ stem cells
  • iPS cells more preferably human iPS cells
  • ES cells are stem cells that are pluripotent and have the ability to proliferate through self-renewal and are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice.
  • ES cells were discovered in mice in 1981 (M.J. Evans and M.H. Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (J.A. Thomson et al. (1998), Science 282:1145-1147; J.A. Thomson et al. ( (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; J.A. Thomson et al. (1996), Biol.
  • ES cells can be established by extracting the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on fibroblast feeders.
  • ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from developmentally arrested embryos (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).
  • nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology, and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502).
  • nt ES (nuclear transfer ES) cells are ES cells established from the inner cell mass of a blastocyst derived from a cloned embryo obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell.
  • nt ES cells To create nt ES cells, a combination of nuclear transfer technology (Cibelli J.B. et al. (1998), Nature Biotechnol., 16:642-646) and ES cell production technology (mentioned above) is used (Wakayama Kiyoka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52).
  • nuclear transfer the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, which can then be initialized by culturing for several hours.
  • ES cell lines used in the present invention include, for example, various mouse ES cell lines established by inGenious targeting laboratory, RIKEN (Institute of Physical and Chemical Research), and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis.
  • human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.
  • iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors).
  • iPSCs established by Yamanaka et al. by introducing four factors, Oct3/4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al.
  • Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a 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.), etc. can also be used.
  • induced pluripotent stem cells established by introducing four factors, OCT3/4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.
  • all published papers e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol.
  • iPSC lines established by the NIH, RIKEN, Kyoto University, and other institutions are available as induced pluripotent stem cell lines.
  • human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, as well as Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 1390B1, 1390C1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 lines.
  • the induced pluripotent stem cells used in the production methods of the present invention may be cells derived from patients with a genetic disease (e.g., a patient with a genetic heart disease).
  • a genetic disease e.g., a patient with a genetic heart disease
  • Cells induced to differentiate from pluripotent stem cells derived from a patient with a genetic heart disease can serve as a disease model that reflects the pathology of the disease, and are therefore suitable for screening therapeutic or preventive drugs for the disease.
  • pluripotent stem cells derived from a patient with a genetic heart disease can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into mature epicardial cells or cardiac organoids containing such cells, making it possible to use the cells or organoids as a therapeutic agent for the heart disease.
  • mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Like ES cells, these cells can be induced to differentiate into cells of various lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be produced (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture media containing glial cell line-derived neurotrophic factor (GDNF), and germline stem cells can be obtained by repeated passage under culture conditions similar to those for ES cells (Takebayashi Masanori et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 41-46, Yodosha, Tokyo, Japan).
  • GDNF glial cell line-derived neurotrophic factor
  • mTORC2 inhibitors used in the present invention include small molecule compounds such as Torin1, Torin2, omipalisib (GSK2126458), KU-0063794, OSI-027, XL388, Palomid 529 (P529), WYE-354, torkinib (PP242), sapanisertib (TAK-228), WYE-687, AZD8055, and vistusertib (AZD2014), with Torin1 being preferred. Sapanisertib, WYE-687, AZD8055, and vistusertib may also be suitably used.
  • the medium contains an mTORC1 inhibitor, and the mTORC1 inhibitor may be the same as the mTORC2 inhibitor used in the present invention (when the mTORC2 inhibitor also has mTORC1 inhibitory activity) or may be a different mTORC1 inhibitor.
  • the term "compound” encompasses not only the free form but also its pharmacologically acceptable salts and hydrates.
  • pharmacologically acceptable salts vary depending on the type of compound, examples include inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salts, and ammonium salts; base addition salts such as organic base salts of trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, and N,N'-dibenzylethylenediamine; inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, and phosphate; and organic acid salts such as citrate, oxalate, acetate, formate, propionate
  • proteins or peptides their origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.). Furthermore, the proteins or peptides used in the production methods of the present invention include not only wild-type proteins or peptides, but also mutants thereof that have similar functions.
  • the gene can be said to be expressed or to be positive.
  • the production of mRNA encoded by the gene is not detected (i.e., below the detection limit) by the method (RT-qPCR) described in the Examples below, or if it is at background levels, the gene can be said to be not expressed or to be negative.
  • mTORC2 expression inhibitor when the mTORC2 expression inhibitor is a nucleic acid, such nucleic acid may be referred to as an "mTORC2 expression-inhibiting nucleic acid.”
  • a single mTORC2 expression inhibitor may be used, or multiple mTORC2 expression inhibitors may be used.
  • an mTORC2 expression inhibitor may be used in combination with another mTORC1 expression inhibitor.
  • the mTORC1 expression inhibitor may be a substance capable of inhibiting the expression of any of the proteins that make up mTORC1. Examples of such proteins include mTOR, raptor, and PRAS40.
  • the protein targeted by the mTORC2 expression-inhibiting nucleic acid When the protein targeted by the mTORC2 expression-inhibiting nucleic acid has multiple isoforms, it is typically capable of inhibiting the expression of a transcript encoding the full-length protein, but other isoforms may also be used as long as they are capable of inhibiting Akt phosphorylation.
  • the base sequence targeted by the mTORC2 expression-inhibiting nucleic acid can be appropriately designed, for example, based on information from a publicly known database (e.g., the NCBI database).
  • the length of the target sequence is not particularly limited as long as the expression-inhibiting nucleic acid can specifically recognize and bind to it, but is preferably 12 nucleotides or longer, more preferably 15 nucleotides or longer, and even more preferably 17 nucleotides or longer.
  • the upper limit of the length is also not particularly limited, but is, for example, 30 nucleotides or shorter, preferably 25 nucleotides or shorter, and more preferably 22 nucleotides or shorter.
  • complementary refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, wobble base pairs, etc.) through hydrogen bonds. Therefore, “complementary sequence” is used to mean not only a sequence that is completely complementary to a target RNA sequence or target DNA sequence (i.e., hybridizes without mismatches), but also a sequence containing one to several (e.g., 2, 3, 4, 5 or more) mismatches, as long as it can hybridize with the target sequence under stringent conditions or under the physiological conditions of mammalian cells.
  • it includes a sequence that has 80% or more identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more), and most preferably 100%, with a sequence that is completely complementary to a target RNA sequence or target DNA sequence.
  • siRNA is double-stranded RNA composed of RNA (i.e., the antisense strand) with a sequence complementary to the target RNA sequence and its complementary strand.
  • a preferred form of siRNA is small hairpin RNA (shRNA), which is single-stranded RNA in which a sequence complementary to the target RNA sequence (first sequence) is linked to its complementary sequence (second sequence) via a hairpin loop portion, and in which the first sequence forms a double-stranded structure with the second sequence by adopting a hairpin loop structure.
  • shRNA may be in the form of a nucleic acid (e.g., an expression vector, etc.) that encodes the shRNA.
  • each nucleic acid strand of the double-stranded nucleic acid is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides, preferably 15 to 30 nucleotides, and more preferably 20 to 27 nucleotides.
  • miRNA refers to a single-stranded or double-stranded RNA (e.g., miRNA/miRNA * ) that does not cleave target RNA like siRNA, but rather recognizes the 3' untranslated region (UTR) of target RNA and controls translation.
  • miRNA refers to an endogenous non-coding RNA (ncRNA) of approximately 20 to 25 bases that is originally encoded on the genome.
  • ncRNA non-coding RNA
  • Pri-miRNA is expressed from the miRNA gene, followed by the generation of pre-miRNA, which then produces mature-miRNA. The mature-miRNA is then incorporated into RISC to produce single-stranded miRNA.
  • the miRNA used in the present invention may be in the form of pri-miRNA, pre-miRNA, mature-miRNA (miRNA/miRNA * ), or even single-stranded RNA.
  • the miRNA may be in the form of a nucleic acid (e.g., an expression vector) encoding the miRNA.
  • the length of the miRNA is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides in length, preferably 15 to 30 nucleotides in length, and more preferably 20 to 27 nucleotides in length.
  • the sense and antisense strands of the target RNA sequence can be synthesized using a commercially available automated DNA/RNA synthesizer (e.g., Applied Biosystems, Beckman, etc.), denatured in an appropriate annealing buffer at about 90°C to about 95°C for about 1 minute, and then annealed at about 30°C to about 70°C for about 1 to about 8 hours.
  • HDO can also be produced, for example, by the method described in WO2013/089283.
  • StemFit registered trademark
  • AK02 medium Alignomoto Co., Inc.
  • StemFit registered trademark
  • AK03 medium Alignomoto Co., Inc.
  • StemFit registered trademark
  • Basic03 medium CTS (registered trademark) KnockOut SR XenoFree Medium (Gibco)
  • mTeSR1 medium TeSR1 medium
  • Iscove's modified Dulbecco's medium GE Healthcare
  • Improved MEM Thermo Fisher Scientific
  • suspension culture refers to culture carried out under conditions that maintain cells or cell clumps suspended in the culture medium, i.e., culture under conditions that do not allow strong cell-substratum junctions to form between the cells or cell clumps and the culture vessel. Furthermore, when performing suspension culture, cells are typically in the form of cell clumps before and after suspension culture. As used herein, “adhesion culture” refers to culture under conditions that allow strong cell-substratum junctions to form between the cells and cultureware, etc.
  • the culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture,” and can be appropriately determined by one of ordinary skill in the art.
  • Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles.
  • Another example of a vessel for suspension culture is a bioreactor. These culture vessels are preferably non-cell-adhesive to enable suspension culture.
  • non-cell-adhesive culture vessels include those whose surfaces have not been artificially treated (e.g., coated with an extracellular matrix) to improve cell adhesion.
  • the well bottom shape of these culture vessels is not particularly limited, and examples include flat-bottomed, U-shaped, and V-shaped vessels.
  • Culture vessels used in adherent culture include those whose surfaces have been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine and polyornithine, or surface treatments such as positive charging).
  • basement membrane preparations include, for example, Matrigel and Geltrex.
  • laminin or a fragment thereof is also preferred.
  • laminin or a fragment thereof include laminin-111 or a fragment thereof containing the E8 region, laminin-211 or a fragment thereof containing the E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof containing the E8 region, laminin-221 or a fragment thereof containing the E8 region, laminin-332 or a fragment thereof containing the E8 region, laminin-3A11 or a fragment thereof containing the E8 region, laminin-411 or a fragment thereof containing the E8 region (e.g., iMatrix-411), and laminin-421.
  • laminin-511 or fragments thereof containing its E8 region laminin-511 or fragments thereof containing its E8 region (e.g., iMatrix-511, iMatrix-511 silk), laminin-521 or fragments thereof containing its E8 region, laminin-213 or fragments thereof containing its E8 region, laminin-423 or fragments thereof containing its E8 region, laminin-523 or fragments thereof containing its E8 region, laminin-212/222 or fragments thereof containing its E8 region, and laminin-522 or fragments thereof containing its E8 region.
  • laminin-511 or fragments thereof containing its E8 region e.g., iMatrix-511, iMatrix-511 silk
  • laminin-521 or fragments thereof containing its E8 region laminin-213 or fragments thereof containing its E8 region
  • laminin-423 or fragments thereof containing its E8 region laminin-523 or fragments thereof containing
  • cells may be cultured under feeder-free conditions and/or xeno-free conditions.
  • all steps may be performed under feeder-free and xeno-free conditions.
  • feeder-free refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to prepare the culture conditions for the cells to be cultured.
  • xeno-free refers to a medium or culture conditions that do not contain components derived from organisms other than the biological species of the cells to be cultured.
  • the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2- containing air, with the CO2 concentration preferably being about 2 to 5%.
  • the culture period for the steps of the production method of the present invention is not particularly limited, but is typically 1 to 20 days, preferably 3 to 15 days, and more preferably 5 to 10 days (particularly 7 days).
  • the present invention provides a method for maturing epicardial cells, comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor.
  • a method for maturing epicardial cells comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor.
  • Mature epicardial cells, cardiac organoids comprising said cells and their uses By the method of the present invention, mature epicardial cells or cardiac organoids such as cardiac tissue comprising said cells can be obtained.Therefore, in another aspect of the present invention, also provide the mature epicardial cells or cardiac organoids comprising said cells or cardiac tissue (hereinafter sometimes referred to as "epicardial cells of the present invention") obtained by the method of the present invention ("obtained” can be appropriately read as “obtained”).
  • a method for producing mature cardiomyocytes, or a method for maturing cardiomyocytes including this step is provided, which includes the step of co-culturing the epicardial cells of the present invention with cardiomyocytes.
  • the cardiomyocytes used in such a method are cardiomyocytes contained in cell aggregates (typically, embryoid bodies). These methods may also include the step of isolating mature cardiomyocytes using flow cytometry or the like.
  • cardiomyocytes obtained by the above-described method for producing mature cardiomyocytes may be referred to as "cardiomyocytes of the present invention.
  • Cardiomyocytes are broadly divided into immature (i.e., “fetal") cardiomyocytes and mature (i.e., "adult") cardiomyocytes; however, unless otherwise specified, “cardiomyocytes” refers to immature cardiomyocytes.
  • Cardiomyocytes are cells that make up the myocardium of animals, and as used herein, immature cardiomyocytes and mature cardiomyocytes refer to cells that repeatedly contract and relax (have pulsatile activity). Immature cardiomyocytes and mature cardiomyocytes typically express at least one cardiomyocyte marker selected from the group consisting of cardiac troponin T (cTNT), ⁇ MHC ( ⁇ myosin heavy chain, MYH6), and ⁇ MHC (MYH7). Immature cardiomyocytes refer to cells that express TNNI1.
  • cTNT cardiac troponin T
  • ⁇ MHC ⁇ myosin heavy chain
  • MYH7 ⁇ MHC
  • mature cardiomyocytes refer to cells that express TNNI3 and in which the expression level of TNNI1 has decreased or disappeared and the expression level of TNNI3 has increased compared to cardiomyocytes prior to the step of co-culturing with the epicardial cells of the present invention, and preferably cells that do not express TNNI1 but express TNNI3.
  • the cardiomyocytes used in the present invention can be obtained by known methods. Examples include methods of isolating cardiomyocytes from the heart using known techniques, methods of inducing differentiation of pluripotent stem cells, and methods of obtaining them from companies such as ATCC. Midgut cells can be isolated from the heart using, for example, methods that use flow cytometry or mass cytometry using a surface antigen (e.g., CD82) as an indicator, magnetic cell separation, or an affinity column on which the desired antigen is immobilized.
  • the epicardial cells used in the present invention are preferably obtained by methods of inducing differentiation of pluripotent stem cells.
  • Cell aggregates containing epicardial cells can also be induced to differentiate into epicardial cells by subjecting the cell aggregates containing mesodermal cells obtained in step (B) above to suspension culture in a medium containing epicardial cell-inducing factors.
  • This step can be performed, for example, by subjecting cell aggregates containing mesodermal cells to suspension culture in the presence of VEGF, IWP-3, Dorsomorphin, and SB431542.
  • the duration of this step can be determined appropriately by those skilled in the art, and is typically 6 to 25 days, with 10 to 20 days being more preferable. For all other culture methods, the descriptions of the production method of the present invention are incorporated by reference.
  • the period for co-culturing the epicardial cells of the present invention with cardiomyocytes is not particularly limited, but is typically 5 to 20 days, preferably 6 to 15 days, and more preferably 7 to 14 days (particularly 10 days).
  • the culture may be either suspension culture or adherent culture, with suspension culture being preferred.
  • the co-culturing method is not particularly limited, as long as the epicardial cells and cardiomyocytes are cultured in the same medium. For all other culture methods, the same description of the production method of the present invention is applicable.
  • a transplantation therapy agent (hereinafter sometimes referred to as the "transplantation therapy agent of the present invention") containing the epicardial cells and/or cardiomyocytes of the present invention is provided.
  • the present invention also encompasses a method for treating or preventing heart disease in which an effective amount of the epicardial cells and/or cardiomyocytes of the present invention is administered to or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) that is the target of treatment or prevention.
  • a mammal e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.
  • the term "drug for treating or preventing a disease also encompasses a pharmaceutical agent (or method) that can both treat and prevent the disease.
  • the transplantation therapy agent of the present invention can be administered or transplanted into the body of a subject in need thereof.
  • the cells or organoids to be transplanted should be administered in a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the transplant subject, and is not particularly limited.
  • the number of cells can be about 10 x 10 cells to 10 x 10 cells.
  • the epicardial cells and/or cardiomyocytes of the present invention are used as transplantation therapy agents, from the standpoint of avoiding rejection.
  • substantially the same means that the HLA genotype matches the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants; for example, somatic cells with an HLA type that matches the three gene loci HLA-A, HLA-B, and HLA-DR, or four gene loci including HLA-C. If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.
  • the epicardial cells and cardiomyocytes of the present invention are manufactured as parenteral preparations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, there is also provided a method for manufacturing a transplant therapy agent, which includes the step of formulating the epicardial cells and/or cardiomyocytes of the present invention. Such a manufacturing method may also include the step of preparing the epicardial cells and/or cardiomyocytes of the present invention. It may also further include the step of preserving the epicardial cells and/or cardiomyocytes of the present invention.
  • compositions include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.).
  • aqueous solutions for injection such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.).
  • the epicardial cells and cardiomyocytes of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.
  • buffers e.g., phosphate buffer, sodium acetate buffer
  • soothing agents e.g., benzal
  • the transplantation therapy agent of the present invention is provided in a frozen state stored under conditions typically used for cryopreserving cells, and can be thawed at the time of use.
  • it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc.
  • the concentration of serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v/v), preferably about 5 to about 20% (v/v).
  • the concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v/v), preferably about 5 to about 20% (v/v).
  • Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low-molecular-weight compounds, and natural compounds.
  • test substances can also be obtained using any of the many approaches to combinatorial library methods known in the art, including (1) biological libraries, (2) synthetic library methods using deconvolution, (3) "one-bead one-compound” library methods, and (4) synthetic library methods using affinity chromatography selection. While the biological library method using affinity chromatography selection is limited to peptide libraries, the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al.
  • Compound libraries can be stored in solution (see Houghten (1992) Bio/Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Patent No. 5,223,409), spores (U.S. Patent Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Ac. ad. Sci. USA 89:1865-9) or as phages (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).
  • Cell lines and culture conditions 201B a hiPSC cell line reprogrammed using Yamanaka factors via retroviral methods, was propagated in ReproCell ES solution containing 4 ng/ml bFGF, supported by an irradiated MEF feeder layer. CTK was administered to the cells to remove the feeder layer.
  • the feeder-free 1390C1 and T1/T3 reporter (PMID: 34155205) cell lines were maintained in complete StemFit® AK02N solution on dishes coated with iMatrix-511 (obtained from Matrixome).
  • the MEC1 mouse epicardial cell line (Merck, SCC187) was obtained and grown according to the manufacturer's guidelines. Routine testing for mycoplasma was performed to ensure the absence of contamination.
  • siRNA solution was prepared with 50 ⁇ l of Opt-MEM, followed by 10 ⁇ l of RNAiMax to create a silencing mixture. After allowing to stand at room temperature for 10 minutes, the mixture was slowly added to a 6-well plate containing approximately 20,000–30,000 cells in 2 ml of maintenance medium, which had been set aside the previous day. The medium was refreshed one day later, and cells were cultured according to standard protocols. For immunocytochemistry tests, 260 ⁇ l of the silencing mixture was slowly added to a 12-well plate containing approximately 5,000 cells seeded the previous day in 1 ml of maintenance medium.
  • Feeder-independent hiPSCs (1390C1) were cultured on iMatrix-511 (Nippi)-coated dishes using StemFit® AK02N medium (Reprocell).
  • the process for generating human cardiac organoids from PSCs followed established protocols. Briefly, cells were suspended in Essential 8 Flex medium (Gibco) containing 10 ⁇ M of the ROCK inhibitor Y-27632 using Accumax. To generate embryoid bodies (EBs), 10,000 cells were dispersed into hemispherical, low-attachment HEMA-coated 96-well plates on day -2 in a total volume of 100 ⁇ l per well. Fresh Essential 8 Flex medium was added the following day.
  • hiPSCs were differentiated as previously reported (PMID: 35110584).
  • hiPSCs were converted into a single-cell mixture using Accutase. These cells were transferred to HEMA-coated plates designed for low attachment (ranging from 6,000 to 8,000 cells per 96-well plate) to form embryoid bodies (EBs).
  • the differentiation solution contained fully enriched StemPro®-34 medium, further enriched with components such as 50 ⁇ g/ml ascorbic acid, 2 mM L-glutamine, 0.4 ⁇ M monothioglycerol, and 150 mg/ml transferrin.
  • Differentiation was initiated by adding 0.5% Matrigel, 10 ⁇ M Y-27632, and 2 ng/ml human recombinant (hr) BMP4 to the medium. After the first 24 hours, EBs were immersed in a differentiation solution containing 10 ng/ml hrBMP4, 2 ng/ml activin A, and 5 ng/ml hrbFGF. After 84 hours, EBs were collected, disaggregated using Accutase, and replated (at a density of 0.3 x 10 cells/ cm ) onto 0.1% gelatin-coated plates.
  • This new environment contained differentiation medium containing 3 mM CHIR99021, 30 ng/ml hrBMP4, 5 ng/ml hrVEGF, and 10 ⁇ M SB431542.
  • a maintenance solution essentially DMEM supplemented with 10% FBS and 10 ⁇ M SB431542.
  • Ventricular cardiomyocytes were differentiated as follows: hiPSCs were dissociated into individual cells using a half-concentrated solution of TrypLE select (Thermo Fisher Scientific) mixed with 0.5 mM EDTA. These cells were then added to 1.5 ml/well of StemPro-34 medium (Invitrogen). This medium contains additional components, such as L-glutamine, MTG, ascorbic acid (AA), transferrin, ROCK inhibitor (Y-27632), Matrigel (Corning), and BMP4, at a concentration of 2 x 10 cells/well for embryoid body (EB) generation.
  • TrypLE select TrypLE select
  • AA ascorbic acid
  • ROCK inhibitor Y-27632
  • Matrigel Matrigel
  • BMP4 BMP4
  • E12, E14, and E18 embryos were collected in preheated PBS and carefully removed surrounding foreign material using tweezers. The embryo's head was separated, and the anterior thoracic cavity was torn open to access the heart. The fetal heart was then mechanically isolated and placed in a separate dish filled with preheated PBS. To prepare cardiac protein lysates, the hearts were first minced and then exposed to trypsin at 37°C for 20 minutes. The cell mixture was again vortexed and treated at 37°C for another 20 minutes. After centrifugation at 300g for 5 minutes, the clear liquid (supernatant) was collected.
  • Torin1 Cytotoxicity assay in hiPSC epicardial cells
  • Torin1 was freshly prepared in DMSO.
  • Human iPSC-derived epicardial cells were individually plated in 96-well plates at a density of 5,000 cells per well.
  • concentrations of Torin1 were administered to rapidly dividing cells at a consistent 1:3 ratio.
  • cell viability was measured using crystal violet.
  • absorbance at 595 nm was recorded using a microplate reader. Based on these results, IC50 values were calculated using GraphPad Prism 7 software.
  • iPSC-derived human cardiac organoids hHO
  • Torin1 was freshly mixed with DMSO.
  • Cardiac organoids (hHO) were differentiated and individually plated into 96-well low-attachment plates. Treatment effects were assessed after administering decreasing concentrations of Torin1 at a fixed 1:3 ratio to organoids at day 15 of growth. Each treatment included a decreasing dose of Torin1 as monotherapy. After 4 days (or 96 hours), the organoids' proliferation capacity was assessed using an MTT assay. Cytotoxicity levels were assessed by measuring absorbance at 595 nm using a dedicated microplate reader, and IC50 values were then calculated using GraphPad Prism 7 software.
  • ICC Immunocytochemistry
  • Flow cytometry analysis/FACS> Cells were dissociated into individual cells using Accutase, washed twice, and fixed in 4% PFA for 15 minutes. To identify quiescent cells in G0 , Hoechst 33342 and Pyronin Y staining were employed, as previously described. Analysis was performed using BD FACSDiva v6 or v8 and FlowJo v10. Cell populations were recognized by FSC/SSC gating, and doublets were excluded. A negative gate was set using unstained samples or isotype controls to ensure the absence of signal from negatives in the positive gate.
  • the primary antibodies used in the examples were anti-phospho-S6 ribosomal protein antibody (5364, CST), anti-phospho-4E-BP1 antibody (2855, CST), anti-4E-BP1 antibody (9644, CST), anti-phospho-AKT antibody (9271, CST), anti- ⁇ -actin antibody (A5441, Sigma), anti-WT1 antibody (ab89901), anti-cTnI TNNI3 antibody (ab10231), anti-SNAI1 antibody (ab63371), anti-NBL1 antibody (ab174843), anti-p53 antibody (12790S, D963E, CST), anti-p16 antibody (12D1, CST), anti-p21 antibody (D7C1M, CST), anti-TBX18 antibody (ab15262), and anti-KI67 antibody (Biolegend;
  • the secondary antibodies used were goat anti-rabbit IgG antibody with HRP (sc-2054, Santa Cruz) and anti-mouse IgG antibody with HRP (7076,
  • Example 1 Validation of the Relationship between Reduced Activity of the mTOR Signaling Pathway and Physiological Cardiac Maturation
  • mTOR mechanistic target of rapamycin
  • IGF-1 insulin-like growth factor 1 pathway
  • IGF-1 promotes activation of the mTOR pathway, which promotes cell proliferation, inhibits cell apoptosis during development and regeneration, and regulates overall cellularity within tissues.
  • mTOR maintains tissue integrity and function, and reduced mTOR signaling may be involved in the fundamental trigger for initiating functional maturation of tissues during adulthood.
  • mTORC1 primarily regulates protein synthesis, cell proliferation, and autophagy through the ribosomal protein S6 kinase (S6k) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (Liu, G. Y. & Sabatini, D. M. Nat. Rev. Mol. Cell Biol. 21, 183-203 (2020)).
  • S6k ribosomal protein S6 kinase
  • eIF4E eukaryotic translation initiation factor 4E-binding protein 1
  • 4E-BP1 eukaryotic translation initiation factor 4E-binding protein 1
  • mTORC2 is primarily involved in cell survival and cytoskeletal organization through phosphorylation of complex 1-associated protein kinase B (also known as Akt) ( Figure 1b) (Sen, B. et al. J. Bone Miner. Res. (2014)).
  • Example 2 Effect of Chemical Inhibition of mTOR During Embryonic Development on In Vivo Epicardial Maturation.
  • mTORKi an mTOR kinase inhibitor that can block a wide range of cellular activities controlled by mTOR.
  • Torin1 interacts with the ATP-binding pocket of both mTOR complexes (Liu, Q. et al. J. Med. Chem. 53, 7146-7155 (2010)), and therefore can inhibit the function of mTORC1 and mTORC2.
  • Example 3 Examination of the effects of dual mTORC1/2 inhibition on epicardial quiescence and maturation in hiPSC-derived epicardium To fully understand the contribution of cell quiescence in human fetal epicardial maturation, we used epicardium derived from human induced pluripotent stem cells and manipulated them to systematically study the effects of cell cycle withdrawal via complete inhibition of the mTOR pathway.
  • EPI epicardial-like cells from hiPSCs
  • Non-Patent Document 5 Cardiac mesoderm was formed using activin A and BMP4 (days 1–3/4), and epicardial fate was subsequently determined by activating WNT signaling (days 3/4–6).
  • WNT WNT signaling
  • TGF- ⁇ signaling SB431542
  • Torin1 treatment (Fig. 5a) significantly reduced the phosphorylation levels of both S6K and Akt (Fig. 5b). This highlights its effect on both mTORC1 (S6K) and mTORC2 (Akt) while preserving epicardial structure (Fig. 5c). Torin1-treated cells rapidly arrested cell proliferation (Fig. 5d). Growth arrest is a key phenotype of the adult human epicardium, a finding similar to that observed in mouse counterparts.
  • Torin1 treatment effectively reduced nuclear WT1 and TBX18 levels and preserved tight junctions, as indicated by ZO-1 expression, while maintaining epicardial morphology (Fig. 5e).
  • ⁇ -galactosidase ⁇ -gal
  • Torin1-treated epicardium significantly reduced ⁇ -gal + cells (Fig. 5h, Fig. 8a, b), suggesting that dual inhibition of mTORC1 and mTORC2 can shift epicardium away from the senescent phenotype, as confirmed by reduced expression of SASP-related genes IL-8 and SPP1 (Fig. 8c).
  • Torin1 To investigate the transcriptional impact of Torin1 on iPSC-derived epicardium, we performed detailed RNA-sequencing analysis of cells treated with Torin1 (Lucena-Cacace, A. & Yoshida, Y. Methods Mol. Biol. Clifton NJ 2320, 219-232 (2021)). Consistent with previous observations regarding mTOR inhibition in mouse epicardium, Torin1 significantly altered the transcriptome landscape of hiPSC-derived epicardium, with principal component 1 (PC1) accounting for 99.5% of the PCA variance (Fig. 10a). Analysis identified 379 differentially expressed genes specifically attributed to Torin1 treatment (Fig. 10b).
  • PC1 principal component 1
  • TJP1 the gene responsible for ZO-1 protein
  • WT1, TBX18, and ALDH1A2 fetal epicardial genes
  • ALDH1A2 fetal epicardial genes
  • This downregulation occurred concomitantly with a marked decrease in MKI67 mRNA levels, consistent with protein analysis.
  • genes driving epithelial-mesenchymal transition showed significant downregulation (Fig. 10d), as did CDH18, a cadherin associated with epicardial development (Fig. 10d). Elevated transcription of RB1 and HES1 reconfirmed the quiescent transcriptome signature (Sang, L., Coller, H. A. & Roberts, J. M.
  • mTOR functions as the central kinase in two unique multiprotein complexes, mTORC1 and mTORC2. These complexes differ in their component proteins and sensitivity to the drug rapamycin. Specifically, mTORC1 incorporates raptor as a primary subunit, whereas rictor is essential for mTORC2. mTORC1 activity is rapidly suppressed by rapamycin, whereas mTORC2 activity is largely unaffected during short-term rapamycin exposure.
  • Tacrolimus commonly known as FK506 (Dumont, F. J. Curr. Med. Chem. 7, 731-748 (2000)), is primarily used as an immunosuppressant for solid organ transplantation (Wallemacq, P. E. & Reding, R. Clin. Chem. 39, 2219-2228 (1993)). Its primary mechanism of action is calcineurin inhibition, but it is noteworthy that both tacrolimus and rapamycin bind to FKBP12 and can affect the epicardial maturation phenotype. However, tacrolimus treatment also failed to reduce epicardial proliferation (Figure 11a), suggesting that inhibition of calcineurin or mTORC1 alone is insufficient to promote epicardial maturation of hiPSC-derived epicardial cells.
  • Example 5 Validation of the paracrine effect of epicardium derived from mature hiPSCs expressing IGF2 and FN1.
  • IGF2/IGF1R signaling plays a crucial role in human cardiogenesis.
  • the interaction between epicardial IGF2 and myocyte IGF1R has been identified as a key catalyst for myocardial compaction (Meier, A. B. et al. Nat. Biotechnol. 1-14 (2023)).
  • epicardial fibronectin (FN1) secretion as a driver of accelerated cardiac maturation in 3D engineered tissues (Ong, L. P. et al. Stem Cell Rep. 18, 936-951 (2023)).
  • mRNA expression analysis demonstrated that when EBs were co-cultured with mature epicardial cells, they showed enhanced expression of genes important for left ventricular compression, such as TBX5 (Ross, S. B. et al. Hum. Genome Var. 7, 1-8 (2020)), HEY2 (Miao, L. et al. Sci. Rep. 8, 2678 (2016)), PRDM16 (Wu, T. et al. Circulation 145, 586-602 (2022)), and NPPA (Tian, X. et al. Nat. Commun. 8, 87 (2017)) ( Figure 13e). Together, these findings confirm that mTOR inhibition can promote cardiac maturation in a cell-autonomous manner and exogenously via paracrine IGF2 and FN1 mediated by the maturing epicardium.
  • Example 6 Generation of Mature iPSC-Derived Cardiac Organoids with Quiescent Epicardium To evaluate the efficacy of mTOR inhibition in a context that more closely reflects cardiac development, we generated self-organizing human cardiac organoids (hHOs) from human iPS cell lines (Lewis-Israeli, Y. R. et al. Nat. Commun. 12, 5142 (2021); Tian, Y. et al. Front. Cell Dev. Biol. 10, 1001453 (2022)). We aimed to examine the effects of Torin1 treatment on epicardial and myocardial maturation in these human cardiac organoids.
  • hHOs human cardiac organoids
  • Immunocytochemistry identified various cell populations.
  • the TNNT2 + population corresponds to beating cardiomyocytes
  • WT1 + cells represent human epicardial
  • CD31 + cells represent endothelial
  • NFATC1 + represent endocardial cells
  • VIM + highlights cardiac fibroblast populations. Consistent with previous findings, we noted minimal differences in cell composition between different differentiation batches. Subsequently, we used the MTT assay to characterize the cardiac cytotoxicity profile associated with various Torin1 doses over a 96-hour in vitro period. Human cardiac-derived organoids exhibited resistance to Torin1 comparable to monolayer human epicardial cells, exhibiting an IC50 value of 2.51 ⁇ M ( Figure 14b).
  • the present invention provides a novel method for producing mature epicardial cells from immature epicardial cells.
  • the epicardial cells produced by this method are similar to adult epicardial cells and can be used in adult-level two-dimensional and three-dimensional cardiac cell models, drug discovery screening, regenerative medicine, and more. Furthermore, because they may also have the effect of promoting the maturation of cardiomyocytes, they can also be used in methods for maturation of cardiomyocytes.

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Abstract

La présente invention fournit un procédé destiné à produire des cellules d'épicarde mûries, lequel procédé inclut une étape au cours de laquelle des cellules d'épicarde sont cultivées dans un milieu de culture contenant un inhibiteur de mTORC2.
PCT/JP2025/004014 2024-02-07 2025-02-06 Procédé de production de cellules d'épicarde mûres Pending WO2025170010A1 (fr)

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Citations (4)

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JP2023518255A (ja) * 2020-03-20 2023-04-28 イーエムベーアー－インスティテュート フュール モレクラレ バイオテクノロジー ゲゼルシャフト ミット ベシュレンクテル ハフツング 心臓組織モデル
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