WO2023210713A1 - 心外膜細胞再生促進剤および心外膜細胞の再生促進方法 - Google Patents

心外膜細胞再生促進剤および心外膜細胞の再生促進方法 Download PDF

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WO2023210713A1
WO2023210713A1 PCT/JP2023/016537 JP2023016537W WO2023210713A1 WO 2023210713 A1 WO2023210713 A1 WO 2023210713A1 JP 2023016537 W JP2023016537 W JP 2023016537W WO 2023210713 A1 WO2023210713 A1 WO 2023210713A1
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epicardial
cells
inhibitor
cell
cdkn1a
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善紀 吉田
カカセ アントニオ ルセナ
雨 田
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Kyoto University NUC
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Definitions

  • the present invention relates to an agent for promoting epicardial cell regeneration and a medicament containing the same for treating cardiac damage.
  • the present invention also relates to a method for promoting regeneration of epicardial cells and a method for producing epicardial cells with enhanced regenerative potential.
  • the ability of the heart to regenerate in response to injury differs depending on the species. Neonatal mice are able to regenerate their hearts and restore function after injury, but this regenerative ability is lost after birth. Furthermore, this regenerative ability is completely lacking in humans.
  • the epicardium which surrounds the heart, has functions essential for heart regeneration. In species capable of heart regeneration during the neonatal period, the epicardium undergoes a transition from non-proliferation to reactivation upon cardiac injury. However, the adult human epicardium permanently has little proliferative capacity.
  • Non-Patent Document 1 there are many approaches for differentiating epicardial cells derived from human pluripotent stem (iPS) cells (for example, Non-Patent Document 1).
  • these approaches do not result in accelerated epicardial regeneration, nor do they explore the regenerative potential of human iPS cell-derived epicardial cells for clinical purposes.
  • iPS pluripotent stem
  • cyclin-dependent kinase inhibitor p21 affects epimorphic regeneration and is involved in liver regeneration.
  • the effect of p21 on the regenerative ability of epicardial cells has not been reported. Because p21 is intricately involved in many cellular processes, it is difficult to predict what effect a decrease in p21 protein levels will have in a particular tissue or cell.
  • An object of the present invention is to provide a drug for promoting regeneration of epicardial cells.
  • the present invention aims to provide a drug for promoting epicardial cell regeneration and for treating cardiac damage.
  • a further object of the present invention is to provide a method for promoting regeneration of epicardial cells.
  • the present inventors conducted extensive research to solve the above problems and found that p21 is an important factor that inhibits the reactivation of epicardial cells, and by using a p21 inhibitor, The present invention was completed based on the discovery that the present invention can promote the regeneration of epicardial cells and exert therapeutic effects such as repair of damaged heart tissue.
  • An epicardial cell regeneration promoter containing a p21 inhibitor [2] The epicardial cell regeneration promoter according to [1], wherein the p21 inhibitor is a CDKN1A gene expression inhibitor. [3] The epicardial cell regeneration promoter according to [2], wherein the CDKN1A gene expression inhibitor is a nucleic acid containing an siRNA sequence targeting the CDKN1A gene. [4] The epicardial cell regeneration promoter according to [3], wherein the siRNA sequence includes a sense strand having the sequence set forth in SEQ ID NO: 1 and an antisense strand having the sequence set forth in SEQ ID NO: 2.
  • the p21 inhibitor includes a guide RNA for a CRISPR-Cas system
  • the guide RNA for the CRISPR-Cas system is a guide RNA designed to target the CDKN1A gene.
  • a pharmaceutical composition for treating cardiac damage comprising the epicardial cell regeneration promoter according to any one of [1] to [5].
  • the method according to [7], wherein the step of inhibiting p21 is performed by inhibiting expression of the CDKN1A gene.
  • p21 inhibitor for promoting epicardial cell regeneration [13] p21 inhibitor for promoting epicardial cell regeneration. [14] Use of a p21 inhibitor in the manufacture of an epicardial cell regeneration promoter. [15] p21 inhibitors to treat heart damage. [16] Use of p21 inhibitors in the manufacture of medicaments for treating heart damage. [17] A method for promoting epicardial cell regeneration in a subject, comprising the step of administering an effective amount of a p21 inhibitor to a subject in need thereof. [18] A method of treating cardiac damage in a subject comprising administering an effective amount of a p21 inhibitor to a subject in need thereof.
  • regeneration of epicardial cells can be promoted. This promotes the regeneration of epicardial cells in damaged heart tissue, and also induces repair and regeneration of the myocardium, increasing the regenerative ability of the damaged heart and making it possible to treat heart damage. shall be.
  • Epicardial cell regeneration promoter relates to an epicardial cell regeneration promoter containing a p21 inhibitor.
  • Epicardial cells are cells that cover the surface of the myocardium and are characterized by the expression of markers such as WT1 (Wilms Tumor 1), TBX18 (T-Box Transcription Factor 18), and ALDH1A2 (Aldehyde Dehydrogenase 1 Family Member A2). .
  • Promoting epicardial cell regeneration means, for example, improving the survival rate of epicardial cells, improving the proliferation ability of epicardial cells, improving the epicardial tissue repair ability, and/or regenerating the epicardium. It is characterized by increased gene expression associated with reactivation of brain function.
  • the survival rate of epicardial cells can be shown, for example, by culturing epicardial cells and measuring the survival rate (percentage of living cells) after a certain period of time, by adding a p21 inhibitor to the medium. Sometimes, it can be determined that the survival rate of epicardial cells has improved when the survival rate of epicardial cells increases compared to when the p21 inhibitor is not added to the culture medium. The period for culturing the epicardial cells at this time may be, for example, 14 days.
  • the proliferation ability of epicardial cells can be shown, for example, by culturing epicardial cells, measuring the number of cells over time over a certain period of time, and drawing a growth curve. It can be determined that the proliferation ability of epicardial cells has improved when the slope of the proliferation curve of epicardial cells increases compared to the case where no p21 inhibitor is added to the medium. At this time, the period for culturing the epicardial cells may be, for example, 7 days. Further, the cell number may be measured over time, for example, on a daily basis.
  • the tissue repair capacity of the epicardium is demonstrated, for example, by wound healing assays.
  • a wound healing assay involves, for example, culturing epicardial cells until they reach confluence, and then physically scraping some of the cells to create a simulated wound that is repaired after a certain period of time. If the degree of repair of epicardial cells is improved when a p21 inhibitor is added to the medium compared to when a p21 inhibitor is not added to the medium, the epicardial tissue repair ability is It can be judged that it has improved.
  • genes related to reactivation of epicardial regeneration ability include the transcription factors WT1 and TBX18, and the aldehyde dehydrogenase ALDH1A2, and when cultured with a p21 inhibitor added to the medium, and when cultured with a p21 inhibitor added to the medium, In addition, when the expression level of these genes increased compared to when culturing without adding p21 inhibitor to the medium, the expression of genes related to reactivation of epicardial regenerative ability was improved. Can be judged. Gene expression level can be evaluated, for example, by a known method such as RT-PCR.
  • p21 is a protein known as a cyclin-dependent kinase inhibitor, also called CDKN1A, and is expressed from the CDKN1A gene locus.
  • CDKN1A cyclin-dependent kinase inhibitor
  • the p21 protein is also simply referred to as p21, and the gene that expresses p21 is sometimes referred to as the CDKN1A gene or CDKN1A.
  • p21 is known to have the function of binding to a complex of cyclin and cyclin-dependent kinase 2 or 4, inhibiting its activity, and controlling cycle progression in the G1 phase of the cell cycle.
  • the p21 protein and CDKN1A gene are not particularly limited, they can be selected based on epicardial cells that promote regeneration, and when promoting the regeneration of human epicardial cells, human p21 protein or human CDKN1A gene Preferably, inhibitors are used.
  • An example of the amino acid sequence of the human p21 protein is shown in SEQ ID NO: 10
  • an example of the nucleotide sequence of the human CDKN1A gene is shown in SEQ ID NO: 9.
  • inhibitortion of p21 includes inhibition of p21 expression and activity.
  • Inhibition of p21 expression means decreasing the amount of transcription (mRNA amount) or translation amount (amount of protein) of the gene encoding p21 protein (CDKN1A). Inhibition of p21 expression can be confirmed by comparing the expression level of p21 protein or CDKN1A gene per a certain number of epicardial cells before and after addition of the p21 inhibitor.
  • the expression level of the p21 protein or CDKN1A gene may be reduced compared to the expression level before addition of the p21 inhibitor, for example, 50% or less, 20% or less, or 10% or less of the expression level before addition of the p21 inhibitor. It is preferable that the expression level decreases to below the detection limit level, and the expression level may disappear below the detection limit level.
  • Expression of p21 protein or the gene encoding p21 protein can be measured by Western blotting, RT-PCR, etc.
  • Inhibition of p21 expression can be achieved, for example, by an operation that reduces the expression of the gene encoding the p21 protein (CDKN1A gene).
  • Inhibition of CDKN1A gene expression is not particularly limited, but includes, for example, introducing mutations into the CDKN1A gene that reduce transcription efficiency or translation efficiency, manipulating small molecules involved in transcription and translation control, and using nucleic acids such as siRNA that cause RNA interference. This is accomplished through manipulation, etc.
  • CDKN1A gene expression can be achieved by disrupting the CDKN1A gene.
  • Disruption of a gene means that the gene is modified so that it no longer produces a protein that functions normally. Failure to produce a protein that functions normally includes cases in which no protein is produced from the same gene, or cases in which a protein with reduced or lost function per molecule is produced from the same gene.
  • Gene disruption or mutation introduction may be performed, for example, by using the CRISPR-Cas system. Specifically, for example, it may be carried out using a guide RNA designed for gene modification or mutation introduction and a CRISPR enzyme. Moreover, at the same time, DNA fragments for homologous recombination may be used.
  • the p21 inhibitor is a substance for "p21 inhibition" described above. Specifically, it may be, for example, a p21 activity inhibitor, a nucleic acid such as siRNA that targets the CDKN1A gene and causes RNA interference, a guide RNA for a CRISPR-Cas system that targets the CDKN1A gene, and a CRISPR enzyme.
  • nucleic acids such as siRNA that cause RNA interference include siRNA, shRNA, miRNA, and their precursors. That is, the p21 inhibitor may be a nucleic acid such as siRNA, shRNA, miRNA, or a precursor thereof that targets the CDKN1A gene, or a DNA containing a sequence for expressing these RNAs.
  • a sequence of a nucleic acid such as siRNA that causes inhibition of CDKN1A gene expression can be designed by a known method based on the sequence of the CDKN1A gene.
  • the sequence of guide RNA that causes inhibition of CDKN1A gene expression by the CRISPR-Cas system can be designed by a known method based on the sequence of the CDKN1A gene.
  • the p21 inhibitor is an siRNA comprising a sense strand comprising the nucleotide sequence set forth in SEQ ID NO: 1 and an antisense strand comprising the nucleotide sequence set forth in SEQ ID NO: 2, or contained in these sequences. It may be shRNA containing the sequence as a core sequence, or DNA containing the sequence for expressing these RNAs.
  • Examples of methods for introducing nucleic acids into cells or tissues include viral vectors and lipofection. Therefore, when the p21 inhibitor is a nucleic acid such as RNA or DNA, it may exist in the form of a viral vector or a complex for lipofection.
  • the virus vector any known vector can be used, and examples thereof include adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors.
  • substances that inhibit p21 protein activity include antibodies (including partial fragments) against p21 protein and compounds that bind to p21 protein and inhibit p21 protein activity. Inhibition of p21 protein activity can be confirmed by comparing the p21 protein activity per certain number of epicardial cells before and after addition of a p21 inhibitor. The activity of p21 protein only needs to be decreased compared to the cells before addition of the p21 inhibitor, but for example, the activity of p21 protein is 50% or less compared to the cells before addition of the p21 inhibitor, It is preferable that the activity is reduced to 20% or less or 10% or less, and the activity may be completely lost.
  • An antibody against p21 protein can be obtained by a known method using p21 protein or a partial peptide thereof (for example, a partial peptide of the C-terminal region of p21) as an antigen.
  • a commercially available anti-p21 antibody may be used.
  • the p21 inhibitor can be added at a concentration that can inhibit the expression and function of p21.
  • the p21 inhibitor is not particularly limited as long as it inhibits p21 expression or function, but specific examples include p21 antibodies, p21 siRNA, p21 shRNA, p21 antisense compounds, and 2-(2-chlorophenyl)- 5,7-dihydroxy-8-[(3S,4R)-3-hydroxy-1-methylpiperidin-4-yl]-4H-chromen-4-one (flavopiridol), (1R,2R,4S)- 4-[(2R)-2-[(1R, 9S, 12S, 15R, 16E, 18R, 19R, 21R, 23S, 24E, 26E, 28E, 30S, 32S, 35R)-1,18-dihydroxy-19, 30-dimethoxy-15,17,21,23,29,35-hexamethyl-2,3,10,14,20-pentaoxo-11,36-dioxa-4-azatricyclo[30.3.1.0 ⁇ 4 ,9 ⁇ ]hexatriacont
  • Epicardial cells have essential functions in the heart's regeneration in response to injury. Therefore, the epicardial cell regeneration promoting agent of the present invention reactivates the regenerative ability of the epicardium and acquires the ability of the heart to regenerate in response to damage, so that it can be used as a medicine for treating heart damage. Therefore, the present invention provides a medicament for treating heart damage that contains a p21 inhibitor as an active ingredient.
  • the above-mentioned p21 inhibitor can be used as a medicine for treating heart damage, but the p21 inhibitor can also be used as a pharmaceutical composition by combining it with a pharmacologically acceptable carrier.
  • pharmacologically acceptable carriers carriers used in pharmaceuticals such as solvents, buffers, stabilizers, excipients, etc. can be used, and they are selected as appropriate depending on the type of p21 inhibitor and the dosage form of the pharmaceutical. sell.
  • a medicament for treating cardiac damage may include reagents for delivering the nucleic acid to cells, such as lipofection reagents, and reagents for stabilizing the nucleic acid.
  • the target of the medicament of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but it is preferably a mammal, more preferably a primate such as a human or a rodent such as a mouse, and a human Even more preferably.
  • the medicament of the present invention can be administered orally or parenterally, it is preferably administered locally to the damaged site of the heart.
  • the dosage form of the pharmaceutical is not particularly limited, but it can be administered, for example, in the form of an injection solution or a drip preparation.
  • the dosage of the medicament of the present invention varies depending on the type of p21 inhibitor, the age, sex, symptoms and administration method of the subject, and is selected as appropriate. 0.01 mg to 1000 mg per day, preferably 0.1 mg to 100 mg. Administration may be in a single dose or in multiple doses.
  • the medicament of the present invention may be used in combination with other cardiac damage therapeutics.
  • One embodiment of the method of the present invention is a method for promoting regeneration of epicardial cells in vitro, the method comprising the step of inhibiting p21 in the epicardial cells. It is.
  • Another aspect of the method of the present invention is a method for producing epicardial cells with enhanced regenerative potential, comprising: A) providing epicardial cells; and B) inhibiting p21 in the epicardial cells of step A). Regarding the method.
  • promoting the regeneration of epicardial cells means, for example, improving the survival rate of epicardial cells, improving the proliferation ability of epicardial cells, improving the ability of epicardial tissue repair, and and/or characterized by an increase in gene expression associated with reactivation of epicardial regenerative potential.
  • enhanced regenerative ability means, for example, improved survival rate of epicardial cells, improved proliferative ability of epicardial cells, improved ability to repair epicardial tissue, and/or improved ability to repair epicardial tissue. It may also mean an increase in gene expression associated with reactivation of membrane regeneration capacity.
  • the epicardial cells may be epicardial cells collected from a mammalian subject such as a human or a mouse, or may be epicardial cells induced to differentiate from pluripotent stem cells.
  • pluripotent stem cells include, but are not particularly limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, and embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES). cells, spermatogonial stem cells ("GS cells”), embryonic germ cells (“EG cells”), cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells).
  • ES embryonic stem
  • iPS induced pluripotent stem
  • GS cells spermatogonial stem cells
  • EG cells embryonic germ cells
  • cultured fibroblasts and pluripotent cells derived from bone marrow stem cells (Muse cells).
  • Preferred pluripotent stem cells are iPS cells and ES cells.
  • the origin of the pluripotent stem cells is not particularly limited as long as the effects of the present invention can be obtained, but they are preferably derived from mammals, more preferably from primates such as humans or rodents such as mice, Even more preferably it is of human origin.
  • the initialization factors are, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15.
  • Examples include genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors may be used alone or in combination. Also good.
  • Combinations of initialization factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, W O2009/091659, WO2009/101084, WO2009/ 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO 2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/14 7395, WO2010/147612,
  • Somatic cells used to obtain iPS cells include, but are not limited to, fetal somatic cells, neonatal somatic cells, and mature healthy or diseased somatic cells. It also includes primary cultured cells, subcultured cells, and established cell lines. Specifically, somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) blood cells (peripheral stem cells).
  • tissue stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells
  • tissue progenitor cells tissue progenitor cells
  • blood cells peripheral stem cells
  • lymphocytes epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells) etc.), differentiated cells such as brain cells, lung cells, kidney cells, and fat cells.
  • epicardial cells derived from pluripotent stem cells may be, for example, epicardial cells induced to differentiate from pluripotent stem cells in vitro. Any known method can be used to induce differentiation of epicardial cells from pluripotent stem cells. For example, differentiation may be induced by adding an appropriate differentiation-inducing factor to the culture medium.
  • the method for inducing differentiation of epicardial cells from pluripotent stem cells is not particularly limited, and any known method may be used. Specifically, for example, embryoid bodies (EBs) are formed from pluripotent stem cells, and the EBs are treated with a GSK3 (Glycogen synthase kinase 3) inhibitor such as CHIR99021 or a TGF (Transforming growth factor) ⁇ inhibitor. Examples include monolayer culture in the presence of certain SB431542, BMP4 (Bone morphogenetic protein 4), and VEGF (Vascular endothelial growth factor). In addition, methods described in the following documents can also be used. 1. Witty A., et al. Nat Biotechnol.
  • epicardial cells induced to differentiate from pluripotent stem cells may be used after isolation and purification, or a cell population containing epicardial cells may be used as is.
  • the epicardial cells thus provided are treated to inhibit p21.
  • inhibiting p21 means inhibiting p21 using the above-mentioned "p21 inhibitor”.
  • the step of inhibiting p21 in epicardial cells may be carried out, for example, by adding the above p21 inhibitor to epicardial cells.
  • An example of a method for treatment with a p21 inhibitor is a method in which a p21 inhibitor is added to a culture solution of epicardial cells and cultured for a sufficient time for regeneration of epicardial cells, for example, 5 hours to 10 days. Ru.
  • the concentration of the p21 inhibitor added may be a concentration sufficient for the regeneration of epicardial cells, and can be appropriately set depending on the type of p21 inhibitor.
  • a p21 inhibitor may be additionally administered. Furthermore, after the p21 inhibitory effect is exerted, it may be removed from the culture medium. Epicardial cells with enhanced regenerative capacity may be purified or concentrated before use.
  • the epicardial cells produced by the method of the present invention may be used to treat cardiac damage by transplanting them into a subject as appropriate.
  • iPS cell line (409B2) was used to induce differentiation of human epicardial cells.
  • Human iPS cell lines are grown on radiation-treated mouse embryonic fibroblasts (MEFs) in ES cell medium (Primate ES Cell Medium (REPROCELL, Cat. RCHEMD001) supplemented with 4 ng/mL fibroblast growth factor (bFGF). )) was maintained.
  • ES cell medium Primary ES Cell Medium (REPROCELL, Cat. RCHEMD001) supplemented with 4 ng/mL fibroblast growth factor (bFGF).
  • bFGF fibroblast growth factor
  • StemPro-34 medium (Gibco) supplemented with 2mM L-glutamine, 50 ⁇ M/mL ascorbic acid, 0.4 ⁇ M monothioglycerol, and 150mg/mL transferrin was used.
  • 10 ⁇ M Y27632 Rock inhibitor
  • 2 ng/mL human recombinant BMP4 0.5% Matrigel (Corning) were added to the differentiation medium.
  • cell culture medium supplemented with 4 ng/mL activin A, 10 ng/mL human recombinant bFGF, 20 ng/mL human recombinant BMP4 was added 100% by volume.
  • EBs were dissociated into single cells and replated onto gelatin-coated dishes using basal medium containing 3mM CHIR99021, 10 ⁇ M SB431542, 30ng/mL human recombinant BMP4, and 5ng/mL human recombinant VEGF. (0.3x10 5 cells/cm 2 ), and differentiation of human epicardial cells was induced.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • siRNA transfection assay 0.8 x 106 human iPS cell-derived fetal epicardium cells were seeded per 10 cm dish, and transfection was performed using RNAiMax (Invitrogen). The medium was replaced with fresh medium 24 hours after transfection.
  • Small interfering RNA (siRNA) targeting CDKN1A sense strand: CAAGGAGUCAGACAUUUUAtt (SEQ ID NO: 1), antisense strand: UAAAAUGUCUGACUCCUUGtt (SEQ ID NO: 2)
  • negative control No. 1 derived from Ambion Silencer Select. 1 siRNA (4390843) was used according to the manufacturer's instructions.
  • Quantitative RT-PCR RNA was extracted using QIAzol lysis reagent (QIAGEN), and cDNA was synthesized from the total amount of RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Next, quantitative RT-PCR was performed using Thunderbird SYBR qPCR Mix (TOYOBO) in One Step Real-Time PCR System (Applied Biosystems). Gene expression results were quantified by the ⁇ Ct method and normalized with GAPDH.
  • the primers used are as follows.
  • CDKN1A Forward 5'-AGGGGACAGCAGAGGAAG-3' (SEQ ID NO: 3); Reverse 5'-GCGTTTGGAGTGGTAGAAATCTG-3' (SEQ ID NO: 4), WT1: Forward 5'-CAGCTTGAATGCATGACCTG-3' (SEQ ID NO: 5); Reverse 5'- GATGCCGACCGTACAAGAGT-3' (SEQ ID NO: 6), GAPDH: forward 5'-TGATGACATCAAGAAGGTGGTGAAG-3' (SEQ ID NO: 7); reverse 5'-TCCTTGGAGGCCATGTGGCCAT-3' (SEQ ID NO: 8).
  • Cell growth Curve Assay Cell growth time course curves were evaluated on 6-well plates. Cells were seeded at a density of 4 ⁇ 10 4 per well, and total cell counts were performed with trypan blue at 24-hour intervals for 8 days. Cell growth curves were generated by plotting cell number versus time. After seeding, cells were fixed with 4% paraformaldehyde (PFA) and stained with crystal violet on the day of counting. Values were expressed as cell proliferation rate of cells grown in the presence of 10% FBS. 0% means the number of cells on day 0.
  • PFA paraformaldehyde
  • Colony Formation Assay For the cell colony formation assay, 5000 cells were seeded in a 10 cm dish and cultured for 10 days. Colonies were then fixed with 4% PFA, stained with crystal violet and counted by Image-J.
  • RNA-Seq analysis Data normalization in RNA-Seq analysis was performed using NOISeq. Adult epicardial data are released for analysis under GSE code GSE8484085. Primary data from RNA-Seq analysis was processed using RStudio for mapping and gene expression analysis. Gene expression data for exploration, loading, and preprocessing were processed using the Bioconductor package NOISeq to perform data analysis and differential expression analysis of RNA-Seq analysis data. A heat map for clustering differentially expressed genes (DEGs) was drawn using R script.
  • DEGs differentially expressed genes
  • CDKN1A knockout (KO) mouse CDKN1A knockout mouse was obtained from Jackson laboratory (strain #016565). The second exon of the Cdkn1a gene of the knockout mouse strain has been replaced with a neomycin resistance cassette (neo cassette). In addition, C57BL/6J mice were used as isogenic controls with similar genetic backgrounds.
  • E12 epicardial Explants
  • E12 mouse 12 day embryonic mice.
  • mouse E12 hearts were placed on gelatin-coated dishes in low glucose DMEM supplemented with 15% FBS. Proliferating cultures appeared within 24 hours, and after 7 days, the length of the explants was observed under the microscope.
  • Cells of explants were maintained in medium supplemented with 10 ⁇ M SB431542 to prevent spontaneous epithelial-mesenchymal transition (EMT).
  • Example 1 An siRNA transfection assay was performed on human iPS cell-derived fetal epicardium using siRNA targeting CDKN1A (siCDKN1A) or negative control siRNA (control), and the relative mRNA expression level of CDKN1A was quantitatively determined. Measured by RT-PCR. The results are shown in Figure 1. In addition, the amounts of p21 protein and WT1 protein were measured by Western blotting in human epicardial cells subjected to transfection assay using each siRNA. The results are shown in Figure 2.
  • siCDKN1A With siCDKN1A, the expression level of CDKN1A at the mRNA level in human iPS cell-derived fetal epicardium was reduced to 50% or less compared to the control, and a similar decrease was observed at the p21 protein level.
  • a wound healing assay was performed on human iPS cell-derived fetal epicardium that had been subjected to a transfection assay using each siRNA.
  • the percentage of the wound closure area over time is shown on the left of FIG. 5, and the photographs of the wound area after 0 and 24 hours are shown on the right of FIG.
  • transcriptome analysis was performed to detect gene signatures of cell quiescence using RNA-Seq analysis ( Figure 6).
  • the figure on the left shows a comparison between human iPS cell-derived fetal epicardium and adult epicardium. (Suppression of CDKN1A expression: siCDKN1A group) comparison is shown in the figure on the right.
  • the gene groups whose expression decreased in the cell quiescence state the gene expression was lowest in the order of adult epicardium, human iPS cell-derived fetal epicardium, and siCDKN1A group.
  • gene expression was highest in the order of adult epicardium, human iPS cell-derived fetal epicardium, and siCDKN1A group. Therefore, the adult epicardium, the human iPS cell-derived fetal epicardium, and the siCDKN1A group are strongly in a state of cell quiescence in that order.In other words, the siCDKN1A group has escaped the state of cell quiescence compared to the human epicardium. This was revealed at the gene expression level.
  • Example 2 Ex vivo explant assays of CDKN1A knockout mice were performed. First, epicardial explants were prepared and cultured ex vivo using E12 hearts of CDKN1A knockout mice and control mice (C57BL/6J mice). The CDKN1A knockout mouse and control mouse are as described in "CDKN1A knockout (KO) mouse” above. In addition, epicardial explants were prepared and cultured using the method described in "Epicardial Explants" above. Next, the amounts of p21 protein and WT1 protein were measured for each epicardial explant by Western blotting. The results are shown in FIG.
  • CDKN1A can reactivate the regenerative ability of epicardial cells and improve wound healing ability. It was also confirmed in explants.

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