WO2022191130A1 - Procédé de prolifération de cellules myocardiques, procédé de fabrication de cellules myocardiques, et agent de prolifération de cellules myocardiques - Google Patents

Procédé de prolifération de cellules myocardiques, procédé de fabrication de cellules myocardiques, et agent de prolifération de cellules myocardiques Download PDF

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WO2022191130A1
WO2022191130A1 PCT/JP2022/009718 JP2022009718W WO2022191130A1 WO 2022191130 A1 WO2022191130 A1 WO 2022191130A1 JP 2022009718 W JP2022009718 W JP 2022009718W WO 2022191130 A1 WO2022191130 A1 WO 2022191130A1
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cardiomyocytes
cardiomyocyte
inhibitors
cells
inhibitor
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Japanese (ja)
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潤 山下
美樹 吉岡
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国立大学法人京都大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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

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  • the present invention relates to a method for proliferating and producing cardiomyocytes and a cardiomyocyte proliferating agent used therein.
  • Non-Patent Document 1 by the present inventors has already identified and reported a compound cocktail that induces proliferation of mouse ES cells and human iPS cell-derived cardiomyocytes.
  • Non-Patent Document 1 the present inventors have found a technique for inducing the proliferation of mouse ES cell- and human iPS cell-derived cardiomyocytes.
  • mouse and human myocardial cells differ in their proliferation-arrested state, suggesting that there is a mechanism specific to human cells. Therefore, further elucidation of the human cell-specific mechanism and discovery of the method and necessary factors for proliferation of human cardiomyocytes are expected to greatly contribute to cardiac regenerative medicine.
  • the present invention has been made in view of the above circumstances, and provides a method for proliferating cardiomyocytes, a method for producing cardiomyocytes, and a method for producing cardiomyocytes, which enables induction of proliferation of human cardiomyocytes with a drug and contributes to cardiac regenerative medicine.
  • An object of the present invention is to provide a cardiomyocyte proliferation agent.
  • Embodiments of the invention have the following aspects.
  • proliferation of cardiomyocytes comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors; Method.
  • the above method for proliferation of cardiomyocytes wherein the compound is an ALK inhibitor.
  • the ALK inhibitor is a TGF ⁇ RI (ALK5) inhibitor.
  • ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • cardiomyocyte production comprising the step of culturing cardiomyocytes in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Method.
  • ALK inhibitors cytochrome P450 omega-hydroxylase inhibitors
  • fatty acid synthesis inhibitors fatty acid synthesis inhibitors.
  • ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • the method for producing cardiomyocytes, wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
  • the above production method wherein the pluripotent stem cells are derived from humans.
  • a cardiomyocyte proliferation agent containing, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • the cardiomyocyte proliferation agent, wherein the compound is an ALK inhibitor.
  • the cardiomyocyte proliferation agent wherein the ALK inhibitor is a TGF ⁇ RI (ALK5) inhibitor.
  • the cardiomyocyte proliferation agent wherein the ALK5 inhibitor is one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • the cardiomyocyte proliferation agent wherein the cardiomyocytes are pluripotent stem cell-derived cardiomyocytes.
  • the cardiomyocyte proliferation agent, wherein the pluripotent stem cells are human-derived.
  • a method for proliferating cardiomyocytes a method for producing cardiomyocytes, and a cardiomyocyte-proliferating agent that can induce the proliferation of human cardiomyocytes and contribute to cardiac regenerative medicine can be obtained.
  • FIG. 3 is a schematic diagram showing an operation for inducing differentiation of cardiomyocytes in this example.
  • FIG. 2 is a graph showing cTnT(+)% of cardiomyocytes and a schematic diagram of a cardiomyocyte culture plate in this example.
  • Fig. 2 is a schematic diagram showing the operation of proliferation evaluation for selection of a positive control in this example.
  • FIG. 4 is a photograph showing cell staining of each positive control compound in this example.
  • FIG. 4 is a graph showing the amount of S-phase cells when each positive control compound of this example was added.
  • FIG. 10 is a graph showing the amount of M-phase cells when each positive control compound of this example was added.
  • FIG. 4 is a schematic diagram showing the operation of cardiomyocyte proliferation in this example.
  • FIG. 3 is a photographic diagram showing immunostaining of cardiomyocyte proliferation in this example.
  • FIG. 4 is a graph showing cell number evaluation of cardiomyocyte proliferation in this example.
  • FIG. 4 is a graph showing the EdU+/cTnT+ nuclei counted for the compounds picked up in the 1st screening of this example.
  • FIG. 10 is a graph showing the number of EdU+/cTnT+ nuclei counted for another compound picked up in the 1st screening of this example.
  • FIG. 10 is a graph showing counts of EdU+/cTnT+ nuclei for yet another compound picked up in the 1st screening of this example.
  • FIG. 3 is a photographic diagram showing a part of immunostaining that was the basis for counting in this example.
  • FIG. 2 is a photographic diagram showing a bright field after culturing for 1 day and 1+5 days in the cell number evaluation test of this example.
  • FIG. 4 is a graph showing evaluation of total cell count in tests using various ALK5 inhibitors of this example.
  • FIG. 4 is a graph showing evaluation of cTnT(+)% in tests using various ALK5 inhibitors of this example.
  • FIG. 4 is a graph showing evaluation of cTnT(+) cell numbers in tests using various ALK5 inhibitors of this example.
  • FIG. 2 is a schematic diagram showing one mode of operation for inducing differentiation of cardiomyocytes.
  • cardiomyocyte proliferation method the cardiomyocyte production method, and the cardiomyocyte proliferation agent according to the present invention will be described with reference to embodiments.
  • the present invention is not limited to the following embodiments.
  • cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process.
  • ALK inhibitor broadly refers to a component that inhibits the function of ALK (activin receptor-like kinase).
  • ALK inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • the ALK inhibitor is preferably a TGF ⁇ RI (TGF- ⁇ receptor type-1) (ALK5) inhibitor.
  • the ALK5 inhibitor is preferably one or more compounds selected from the group consisting of LY364947, SB431542, A83-01, and RepSox.
  • a cytochrome P450 omega-hydroxylase inhibitor broadly refers to a component that inhibits the function of cytochrome P450 omega-hydroxylase.
  • Cytochrome P450 omega-hydroxylase inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • a preferred cytochrome P450 omega-hydroxylase inhibitor is 12(S)-hydroxy-16-heptadecinoic acid.
  • Fatty acid synthesis inhibitors broadly refer to ingredients that inhibit fatty acid synthesis.
  • Fatty acid synthesis inhibitors are, for example, nucleic acids (such as siRNA, miRNA, and antisense oligonucleotides), proteins, or small compounds.
  • the fatty acid synthesis inhibitor is C75 or cerulenin.
  • ALK inhibitors cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • concentration of the medium is 0.5 to 40 ⁇ M, preferably 2 to 10 ⁇ M.
  • cardiomyocytes broadly refer to myocardial cells, specifically cells having self-pulsating properties. Cardiomyocytes also include myocardial progenitor cells. Cardiomyocytes also broadly include cells that have the potential to give rise to cardiomyocytes and vascular smooth muscle that form beating muscle and electrically conductive tissue. Cardiomyocytes and cardiomyocyte progenitor cells may be mixed with each other or may be individual. Cardiomyocytes and myocardial progenitor cells broadly include cells positive for the myocardial marker cardiac troponin (cTNT) or ⁇ -myosin heavy chain ( ⁇ MHC). Cardiomyocytes also include cell populations that contain a high percentage of cardiomyocytes relative to other cell types. A cell population containing 50% or more cardiomyocytes is preferred, preferably 60% or 70% or more, more preferably 80% or more, and particularly preferably 90% or more.
  • cTNT myocardial marker cardiac troponin
  • ⁇ MHC ⁇ -myosin heavy chain
  • the cardiomyocytes are preferably cardiomyocytes derived from pluripotent stem cells.
  • ES cells, ntES cells, GS cells, EG cells, Muse cells, iPS cells, and the like are known as pluripotent stem cells.
  • iPS cells are preferably used as the pluripotent stem cells of the present embodiment.
  • iPS cells are pluripotent stem cells derived from somatic cells and have pluripotency and self-renewal potential.
  • Pluripotency means the ability to differentiate into all three germ layer lineages.
  • the self-renewal ability means the ability to proliferate while maintaining an undifferentiated state.
  • the pluripotent stem cells are more preferably human-derived pluripotent stem cells.
  • human-derived iPS cells Human iPS cells, HiPS cells, human iPS cells
  • a method for obtaining iPS cells can be performed by a conventionally known method.
  • K. Takahashi and S.; Yamanaka (2006) Cell, 126:663-676, or the method described in WO2007/069666 or the like can be used.
  • it can be produced by introducing a specific reprogramming factor into a somatic cell in the form of DNA or protein.
  • Reprogramming factors include Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERAs, ECAT15-2, Tcl1, Genes such as beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1, or proteins encoding them can be used singly or in combination.
  • the reprogramming factor is DNA, it can be introduced into cells using vectors such as viruses, plasmids or artificial chromosomes, lipofection, liposomes, or microinjection.
  • the reprogramming factor is a protein, it can be introduced into cells by techniques such as lipofection, fusion with a cell membrane permeable peptide, or microinjection.
  • a method for obtaining pluripotent stem cell-derived cardiomyocytes that is, induction of differentiation of cardiomyocytes from pluripotent stem cells can be performed by a known method. See, for example, Toyama, S.; , et al. (2013). Cell Stem Cell 12(1):127-137, Laflamme et al. , Nat Biotech. 2007 Sep;25(9):1015-24, Uosaki et al. , Plos ONE. 2011; 6(8): e23657 can be used.
  • a ready-made myocardial induction medium, an existing kit containing the medium, or the like can also be used.
  • various myocardial differentiation-inducing factors can be used to induce the differentiation of pluripotent stem cells into cardiomyocytes.
  • Cardiac differentiation inducers include mesoderm-inducing factors (activin A, BMP4, bFGF, VEGF, or SCF, etc.), cardiac determinants (VEGF, or DKK1, etc.), Wnt signal inhibitors (XAV939, IWR-1, IWP- 2, or IWP-4, etc.), BMP signal inhibitors (NOGGIN, etc.), TGF ⁇ /activin/NODAL signal inhibitors (SB431542, etc.), retinoic acid signal inhibitors, or cardiac differentiation factors (e.g., VEGF, bFGF, or DKK, etc.). These myocardial differentiation-inducing factors can be used singly or in combination.
  • pluripotent stem cells are cultured to induce differentiation into mesoderm.
  • Differentiation into mesoderm can be induced by culturing for 1 to 7 days in the presence of the mesoderm-inducing factor.
  • culture is performed for 1 to 4 days in the presence of the cardiomyocyte differentiation-inducing factor.
  • culture is preferably performed for 2 to 3 days in the presence of cardiomyocyte differentiation-inducing factors.
  • Dkk-1 which is an endogenous Wnt inhibitor
  • XAV939 and IWP-4 which are Wnt signal inhibitors
  • myocardial differentiation-inducing factors may be used in combination, for example, XAV939 and IWP-4 may be used in combination.
  • culture is performed in the presence of matrigel for 1 to 2 days as a pretreatment before inducing differentiation into mesoderm, and culture is further performed in the presence of bFGF when inducing differentiation into mesoderm. is also preferred. By this operation, cardiomyocyte differentiation is induced with higher efficiency, and pulsating cells appear from around 4 to 10 days after the initiation of culture for inducing differentiation into mesoderm.
  • the cardiomyocyte proliferation method of the present embodiment includes the step of culturing the cardiomyocytes in the presence of any one or more of the compounds.
  • the medium may contain other components in addition to the above components.
  • Cultivation for proliferation of cardiomyocytes may be carried out by separating the cardiomyocytes obtained by the aforementioned induction of differentiation, transplanting them to a plate, and culturing them. Alternatively, culture for proliferation may be performed again on the plate that has been cultured for differentiation induction.
  • the cardiomyocytes obtained by the induction of differentiation are preferably seeded again at a low density and cultured to proliferate the cardiomyocytes.
  • the seeding density can be appropriately selected from approximately 0.1 to 5 ⁇ 10 4 cells/cm 2 . Preferably, it is 0.5 to 2.0 ⁇ 10 4 cells/cm 2 .
  • Culture for proliferation of cardiomyocytes can be performed by conventionally known means and conditions.
  • the culture temperature is 30 to 40° C., preferably about 37° C.
  • the CO 2 concentration is about 2% to 5%
  • the O 2 concentration is so-called hypoxic conditions, eg about 5% to 20%.
  • the medium used for culturing cardiomyocytes is not particularly limited, and can be appropriately selected according to the purpose.
  • a basal medium can be used.
  • basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, and RPMI 1640 medium. , Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 (Invitrogen), and a mixed medium containing these may be used.
  • the basal medium can be serum-containing or serum-free. When containing serum, a basal medium containing 5% to 20% by weight of serum can be used.
  • the medium may contain other ingredients.
  • Other ingredients include nutrients and other additives.
  • Nutrients include, for example, serum replacement.
  • Serum substitutes include albumin, transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, collagen precursors, trace amounts It may contain one or more elements such as 2-mercaptoethanol or 1-thiolglycerol.
  • Other ingredients include lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, low-molecular-weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, or inorganic salts. It may contain one or more kinds.
  • RPMI medium is used as a medium and cultured in the presence of insulin as an antibiotic.
  • the cardiomyocytes obtained by the cardiomyocyte proliferation method of the present embodiment can be used for treatment of human heart disease (cardiac disease).
  • the heart disease broadly includes diseases in the heart or diseases requiring treatment of the heart.
  • the obtained cardiomyocytes may be administered to a patient, or the obtained cardiomyocytes may be used as a therapeutic agent for heart disease.
  • a method for administering the myocardial cells a means of suspending the cells in a liquid and administering them to the myocardium, or attaching the cells via a sheet, bandage, or the like can be used.
  • the heart disease includes heart failure, ischemic heart disease, myocardial infarction, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, diastolic phase hypertrophic cardiomyopathy, dilated cardiomyopathy, etc., or defects due to disorders. It is not limited as long as it is a disease affecting the heart.
  • cardiomyocytes are cultured in the presence of one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors. Including process.
  • the compound and the step of culturing cardiomyocytes can be selected from those described in the cardiomyocyte proliferation method. That is, the method for producing cardiomyocytes of the present embodiment preferably includes the steps in the method for growing cardiomyocytes. In other words, by adding the steps in the method for growing cardiomyocytes to cultured cardiomyocytes, the cardiomyocytes proliferate, and the cardiomyocytes for the proliferation can be produced.
  • the cardiomyocyte proliferation agent of this embodiment contains, as an active ingredient, one or more compounds selected from the group consisting of ALK inhibitors, cytochrome P450 omega-hydroxylase inhibitors, and fatty acid synthesis inhibitors.
  • the compound can be selected from those described in the cardiomyocyte proliferation method.
  • the cardiomyocyte-proliferating agent of the present embodiment may be used in the above-described cardiomyocyte-proliferating method and cardiomyocyte-producing method. That is, it may be added to proliferate cultured cardiomyocytes.
  • the cardiomyocyte-proliferating agent of the present embodiment is used in a cardiomyocyte-proliferating method and a cardiomyocyte-producing method
  • the cardiomyocyte-proliferating agent of the present embodiment is added to the culture system of cardiomyocytes in culture. can be used.
  • the cardiomyocyte proliferation agent may be administered to the patient.
  • the cardiomyocytes are preferably those in the patient's body.
  • Cardiac diseases or conditions requiring treatment of the heart include the examples described in the cardiomyocyte proliferation method.
  • the cardiomyocyte proliferation agent may contain other ingredients as appropriate.
  • it may contain ingredients conventionally known as those used in the treatment of diseases of the heart and other circulatory systems.
  • Cardiomyocyte proliferation agents including ALK inhibitors, may also be used for anti-cancer therapeutic effects.
  • an ALK inhibitor In the cardiomyocyte proliferation method, cardiomyocyte production method, and cardiomyocyte proliferation agent of the present embodiment, an ALK inhibitor, a cytochrome P450 omega-hydroxylase inhibitor, or a fatty acid synthesis inhibitor is used. These compounds have the effect of inducing proliferation of human cardiomyocytes.
  • the present inventors conducted compound screening using human iPS cell-derived cardiomyocytes in order to discover compounds that can induce proliferation of human cardiomyocytes.
  • compound library screening using human myocardial cells the above compound group having myocardial proliferation activity was found.
  • the same compound was able to induce mitosis twice or more even in human cardiomyocytes cultured for a long period of about 3 months.
  • some cardiomyocytes do not show reactivity to the same compound, and we are currently clarifying the difference between the two by comprehensive gene expression analysis.
  • the same compound did not exhibit a significant myocardial proliferative effect on mouse ES cell-derived cardiomyocytes, suggesting the existence of a human cardiomyocyte-specific growth control mechanism.
  • ALK inhibitors increased the number of human cardiomyocytes by about 1.5-2 fold. Three divisions have been confirmed in cardiomyocytes that respond well to ALK inhibitors. That is, it corresponds to proliferation to eight times the number of cells before division. The effects of ALK inhibitors are human cell-specific and weak in mouse myocardial cells. There has been no report on cardiomyocyte proliferation control by ALK inhibitors. In addition, among ALK inhibitors, there are compounds that have already been tested as anticancer agents, and their use as pharmaceuticals is possible.
  • the method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the cardiomyocyte-proliferating agent of the present embodiment elucidate the molecular mechanism of myocardial proliferation and responsiveness to proliferation stimuli, and enable human cardiomyocytes to proliferate. It is expected to contribute to the development of regenerative medicine.
  • the present inventors added matrigel on day-1 and bFGF on day 1-5 to the two-dimensional cardiomyocyte differentiation induction method DD-protocol (Laflamme et al, Nat Biotech, 2007, etc. described above), A system for more efficient myocardial differentiation (Uosaki et al, Plos ONE, 2011, etc. mentioned above) has been established (Fig. 19). In this method, Dkk-1 was added in the presence of RPMI medium+B27 on days 5-7 to differentiate into cardiomyocytes.
  • This differentiation system can be further manipulated to purify only cells expressing a certain marker ( ⁇ -type platelet-derived growth factor receptor; PDGFR ⁇ ) (Fukushima et al, PLoS One. 2020 Nov 2; 15 ( 11): e0241287.), this purification procedure was carried out in the examples below.
  • a certain marker ⁇ -type platelet-derived growth factor receptor; PDGFR ⁇
  • Fig. 1 shows an outline of the operation for inducing the differentiation of cardiomyocytes used in this example in chronological order.
  • Human iPS cells (201B6) provided by Professor Shinya Yamanaka of Kyoto University were used as iPS cells.
  • bFGF addition amount: 4 ng/mL, Final Conc.
  • matrigel addition amount: 1/1 of the medium was added from day -1 to day 0. 60 was further added.
  • FIG. 2 shows the cardiac troponin T expression rate (cTnT(+)%) of cells obtained by this system. It was shown that this differentiation system yields cardiomyocytes with a cTnT(+)% of over 97% (graph on the left side of FIG. 2). The cardiomyocytes were plated again at a lower density and used for subsequent analysis (schematic diagram on the right side of FIG. 2).
  • FIG. 3 shows a schematic diagram of proliferation evaluation (from Patent Document 1) for selection of a positive control.
  • a positive control for proliferation evaluation a compound found by the present inventors in a mouse cardiomyocyte proliferation screening system was set as a reference.
  • CHIR means CHIR99021.
  • FIG. 4 shows cell staining of DMSO (control), BIO, SU1498, and KN93-administered cultures.
  • BIO, SU1498, and KN93 cardiomyocytes (cTnT) dark in the photograph were stained with EdU and DAPI, which were bright in the photograph.
  • FIG. 5 shows the cell amount in S phase (EdU-positive CM), and
  • FIG. 6 shows the cell amount in M phase (pH 3, ie, phospho-histone positive).
  • BIO, SU1498 and KN93 were found to have myocardial proliferation ability.
  • BIO and CHIR99021 which are GSK3 ⁇ inhibitors
  • SB203580 which is a p38MAPK inhibitor (p38i)
  • SU1498 which is an ERK activator
  • FIG. 7 shows the flow of proliferation of cardiomyocytes.
  • Cardiomyocytes (d19) induced from human iPS cells were seeded under low-density conditions (1.33 ⁇ 10 4 cells/cm 2 ). After culturing in the presence of serum for 24 hours, adhesion of cardiomyocytes to the plate was confirmed, and each compound of the positive control was added at a desired concentration. After culturing for 2 days, 20 ⁇ M of EdU was added. After culturing for another day, the cells were fixed and immunostained with cardiac troponin T (cTnT), which is a cardiomyocyte marker.
  • cTnT cardiac troponin T
  • FIG. EdU and DAPI light color in the photograph
  • cTnT dark color in the photograph
  • DAPI signals arrows, respectively
  • Addition of the positive control showed a more than 1.5-fold increase in the number of EdU+/cTnT+ cell nuclei.
  • Bio-active lipid screening library I & II were analyzed.
  • Bio-active lipid screening library I used more than 785 compounds to be screened stored in 96-well matrix tubes in 1.0 mM DMSO.
  • Bio-active lipid screening library II used 96-well matrix tubes containing >185 compounds to be screened in 0.1 mM DMSO.
  • Cell seeding conditions ⁇ Myocardial muscle induced to differentiate from hiPS cells was used. ⁇ Cell seeding density 1.33 ⁇ 10 4 cells/cm 2 - Reagent was added on the day after cell seeding (20 hrs later). Evaluation conditions: ⁇ cTnT(+)/EdU(+) nuclear numbers -Fixed and stained 3 days (72 hrs) after the addition of the compound. -EdU uptake was evaluated at 24 hrs.
  • Compound conditions ⁇ Cayman Bioactive lipid I & II screening library plate 1-11; 10 ⁇ M, 2 ⁇ M (stock; 1.0 mM) plate 12-14; 1 ⁇ M, 0.2 ⁇ M (stock; 0.1 mM)
  • FIG. 13 shows typical immunostained photographs from which the EdU+/cTnT+ cell nuclei were counted above. From the photograph, addition of each compound (C75, LY364947, 12(S)-hydroxy-16-Hepatadecynoic acid, Cerulenin, SB431542) tends to increase the EdU+/cTnT+ nuclear numbers (bright signal) compared to the negative control. was shown to be in
  • EdU+/cTnT+ nuclear numbers were used as evaluation indices.
  • EdU+/cTnT+ indicates cardiomyocytes undergoing DNA synthesis during the period of EdU addition (24 hrs).
  • human cardiomyocytes are mostly mononuclear cells, but there are also binuclear cells that are temporarily dividing and have binuclear cells. Therefore, an increase in EdU+/cTnT+nuclear numbers does not necessarily correspond to an increase in the number of myocardial cells. Therefore, we set up conditions for a system that evaluates the number of myocardial cells.
  • Cardiomyocytes induced to differentiate from hiPS were seeded at a low density (0.93 ⁇ 10 4 cells/cm 2 ) in the same manner as during screening, and after confirming cell adhesion, each of the above compounds was added. Cardiomyocyte numbers were counted after 1 day and 1+5 days. In addition, cTNT FACS analysis was performed.
  • Fig. 15 shows bright field photographs after 1 day and 1+5 days of culture.
  • the negative control in the figure had a cTnT(+) value of 93.8%, and the positive control had a cTnT(+) value of 97.5%. It can be seen that cardiomyocytes tend to increase with the addition of the positive control.
  • ALK5 inhibitors were added to analyze whether the cardiomyocyte number increased.
  • Both LY364947 and SB431542 are ALK5 inhibitors and, as mentioned above, increase the number of myocardial cells. This increase in cardiomyocyte number may be due to inhibition of ALK5 signaling. Therefore, analysis was performed using other ALK5 inhibitors, A83-01 and RepSox.
  • A83-01 is a potent inhibitor of ALK5 and is also known to inhibit ALK4,7.
  • RepSox (2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine) (denoted as “TGFbR KI” in FIGS. 16-18) ALK5 selective inhibitor.
  • the method for proliferating cardiomyocytes, the method for producing cardiomyocytes, and the agent for proliferating cardiomyocytes of the present invention enable the induction of human cardiomyocyte proliferation with drugs, and can contribute to cardiac regenerative medicine.

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Abstract

L'invention fournit un procédé de prolifération de cellules myocardiques permettant d'induire une prolifération des cellules myocardiques humaines au moyen d'un médicament, et pouvant contribuer à un traitement médical de régénération cardiaque, et fournit également un procédé de fabrication de cellules myocardiques et un agent de prolifération de cellules myocardiques. Plus précisément, l'invention concerne un procédé de prolifération de cellules myocardiques qui inclut une étape au cours de laquelle les cellules myocardiques sont cultivées en présence d'au moins un composé choisi dans un groupe constitué d'un agent inhibiteur de kinase du lymphome anaplasique, d'un agent inhibiteur d'oméga hydroxylase du cytochrome P450 et un agent inhibiteur de synthèse d'acide gras, et concerne également un procédé de fabrication de cellules myocardiques et un agent de prolifération de cellules myocardiques.
PCT/JP2022/009718 2021-03-08 2022-03-07 Procédé de prolifération de cellules myocardiques, procédé de fabrication de cellules myocardiques, et agent de prolifération de cellules myocardiques WO2022191130A1 (fr)

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US20130331389A1 (en) * 2012-06-11 2013-12-12 National Cheng Kung University Methods and Compositions for Cardiomyocyte Replenishment by Endogenous and Progenitor Stem Cells

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130331389A1 (en) * 2012-06-11 2013-12-12 National Cheng Kung University Methods and Compositions for Cardiomyocyte Replenishment by Endogenous and Progenitor Stem Cells

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
COHEN ETHAN DAVID, YEE MIN, PORTER GEORGE A., RITZER ERIN, MCDAVID ANDREW N., BROOKES PAUL S., PRYHUBER GLORIA S., O’REILLY MICHAE: "Neonatal hyperoxia inhibits proliferation and survival of atrial cardiomyocytes by suppressing fatty acid synthesis", JCI INSIGHT, vol. 6, no. 5, 8 March 2021 (2021-03-08), XP055967804, DOI: 10.1172/jci.insight.140785 *
JIANG SHAN, HUO DAN, WANG XUEYING, ZHAO HUAN, TAN JIANG, ZENG QINGHUA, O'ROURKE STEPHEN, SUN CHENGWEN: "beta-adrenergic Receptor-stimulated Cardiac Myocyte Apoptosis: Role of Cytochrome P450 w-hydroxylase", JOURNAL OF CARDIOVASCULAR PHARMACOLOGY, RAVEN PRESS, NEW YORK, NY, US, vol. 70, no. 2, 1 August 2017 (2017-08-01), US , pages 94 - 101, XP009539641, ISSN: 0160-2446, DOI: 10.1097/FJC.0000000000000499 *
MATTHEW P. PAPPAS; LINDSAY N. PEIFER; SUNNY S. K. CHAN: "Dual TGFβ and Wnt inhibition promotes Mesp1‐mediated mouse pluripotent stem cell differentiation into functional cardiomyocytes", DEVELOPMENT GROWTH AND DIFFERENTIATION., RICHMOND, VIC. : BLACKWELL PUBL. ASIA, US, vol. 62, 4 November 2020 (2020-11-04), US , pages 487 - 494, XP071144350, ISSN: 0012-1592, DOI: 10.1111/dgd.12694 *
MOHAMED TAMER M.A., ANG YEN-SIN, RADZINSKY ETHAN, ZHOU PING, HUANG YU, ELFENBEIN ARYE, FOLEY AMY, MAGNITSKY SERGEY, SRIVASTAVA DEE: "Regulation of Cell Cycle to Stimulate Adult Cardiomyocyte Proliferation and Cardiac Regeneration", CELL, ELSEVIER, AMSTERDAM NL, vol. 173, no. 1, 1 March 2018 (2018-03-01), Amsterdam NL , pages 104 - 116.e12, XP055967738, ISSN: 0092-8674, DOI: 10.1016/j.cell.2018.02.014 *

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