Epicardial cell regeneration promoter and method for promoting epicardial cell regeneration

Abstract

Provided is a drug usable as pharmaceutical for promoting the regeneration of epicardial cells and treating heart damage. A p21 inhibitor is used as the epicardial cell regeneration promoter.

Description

Epicardial cell regeneration promoter and method for promoting epicardial cell regeneration

[0001] The present invention relates to an epicardial cell regeneration promoter and a pharmaceutical composition for treating cardiac damage, including the same. The present invention also relates to a method for promoting epicardial cell regeneration and a method for producing epicardial cells with enhanced regenerative ability.

[0002] The regenerative capacity of the heart following injury varies among species. Neonatal mice are able to regenerate their hearts and recover function after injury, but this regenerative capacity is lost after birth. Furthermore, this regenerative capacity is completely lacking in humans. The epicardium, which surrounds the heart, possesses essential functions for cardiac regeneration. In species capable of cardiac regeneration during the neonatal period, the epicardium undergoes a transition from non-proliferation to reactivation following cardiac injury. However, the epicardium in adult humans permanently lacks almost no proliferative capacity.

[0003] To date, many approaches have been developed for differentiating human pluripotent stem (iPS) cell-derived epicardial cells (e.g., Non-Patent Document 1). However, these approaches have not promoted epicardial regeneration or explored the regenerative potential of human iPS cell-derived epicardial cells for clinical purposes. That is, most approaches focus on differentiation and preserve the ability of human iPS cell-derived epicardial cells for cell characterization, but there have been no reports on promoting epicardial cell regeneration.

[0004] The cyclin-dependent kinase inhibitor p21 has been suggested to affect epimorphic regeneration and to be involved in liver regeneration. However, the effect of p21 on the regenerative capacity of epicardial cells has not been reported. Because p21 is intricately involved in many cellular processes, it is difficult to predict how reduced levels of this protein will affect specific tissues and cells.

[0005] Junghof J, et al. NPJ Regen Med. 2022 Feb 2;7(1):14. doi: 10.1038 / s41536-022-00207-w.

[0006] An objective of the present invention is to provide a drug for promoting epicardial cell regeneration. In particular, an objective of the present invention is to provide a drug for promoting epicardial cell regeneration and treating cardiac damage. Another objective of the present invention is to provide a method for promoting epicardial cell regeneration.

[0007] As a result of intensive research to solve the above-mentioned problems, the inventors have discovered that p21 is an important factor that inhibits the reactivation of epicardial cells, and that the use of p21 inhibitors can promote the regeneration of epicardial cells and exert therapeutic effects such as repairing damaged cardiac tissue, thereby completing the present invention.

[0008] [1] An epicardial cell regeneration promoter comprising a p21 inhibitor. [2] The epicardial cell regeneration promoter according to [1], wherein the p21 inhibitor is an inhibitor of CDKN1A gene expression. [3] The epicardial cell regeneration promoter according to [2], wherein the CDKN1A gene expression inhibitor is a nucleic acid comprising an siRNA sequence targeting the CDKN1A gene. [4] The epicardial cell regeneration promoter according to [3], wherein the siRNA sequence comprises 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. [5] The epicardial cell regeneration promoter according to [1] or [2], wherein the p21 inhibitor comprises a guide RNA for a CRISPR-Cas system, and the guide RNA for the CRISPR-Cas system is a guide RNA designed to target the CDKN1A gene. [6] A pharmaceutical composition for treating cardiac damage, comprising the epicardial cell regeneration promoter according to any one of [1] to [5]. [7] A method for promoting epicardial cell regeneration in vitro, comprising a step of inhibiting p21 in the epicardial cells. [8] The method according to [7], wherein the step of inhibiting p21 is carried out by inhibiting expression of the CDKN1A gene. [9] The method according to [8], wherein the inhibition of CDKN1A gene expression is achieved by introducing siRNA into the epicardial cells.

[10] The method according to [8], wherein the step of inhibiting p21 is carried out by introducing a guide RNA designed to target the CDKN1A gene and a CRISPR enzyme into the epicardial cells.

[11] 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). ​​

[12] The method according to

[11] , wherein the epicardial cells of step A) are epicardial cells induced to differentiate from pluripotent stem cells.

[13] A p21 inhibitor for promoting epicardial cell regeneration.

[14] Use of a p21 inhibitor in the manufacture of an epicardial cell regeneration promoter.

[15] A p21 inhibitor for treating cardiac damage.

[16] Use of a p21 inhibitor in the manufacture of a medicament for treating cardiac 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 for treating cardiac damage in a subject, comprising the step of administering an effective amount of a p21 inhibitor to a subject in need thereof.

[0009] The present invention can promote the regeneration of epicardial cells, thereby promoting the regeneration of epicardial cells in damaged cardiac tissue, thereby inducing repair and regeneration of the myocardium, enhancing the regenerative capacity of the damaged heart, and enabling the treatment of cardiac injury.

[0010] Graph showing the relative mRNA expression level of CDKN1A in human epicardial cells differentiated from a human iPS cell line after an siRNA transfection assay. Analysis was performed in biological triplicate. **: P<0.01 compared to the control condition. Photograph showing the results of Western blot analysis of p21 protein and WT1 protein after an siRNA transfection assay in human epicardial cells. Graph showing the cell growth curve for human epicardial cells over 7 days after an siRNA transfection assay. The photograph on the right shows a photo of cells on a dish on day 7. Analysis was performed in biological triplicate. *: P<0.05, ***: P<0.001 compared to the control condition. Graph showing the results of a colony formation assay in human epicardial cells, in which colony formation was performed 14 days after an siRNA transfection assay. The photograph on the right shows a photo of colonies on a dish on day 14. Analysis was performed in biological triplicate. *: P<0.05 compared to the control condition. A graph showing the wound closure rate 0 to 24 hours after scratching human epicardial cells in a wound healing assay, and photographs of the wound closure after 24 hours are shown. Analysis was performed in biological triplicates. *: P<0.05, ***: P<0.001 compared to the control condition. Figure showing transcriptome analysis to detect gene signatures of cellular quiescence using RNA-Seq analysis. Comparisons between human iPS cell-derived fetal epicardium and adult epicardium (GSE84085), and between human iPS cell-derived fetal epicardium and the siCDKN1A group, are shown in heat maps displaying relative mRNA expression levels (log FC) to detect cellular quiescence. 1 shows the results of Western blot analysis of p21 protein and WT1 protein in epicardial explants derived from 12-day-old (E12) embryos of CDKN1A knockout mice and control mice (C57BL / 6J mice). Figure 2 shows photographs of the results of ex vivo explant assays in which epicardial explants derived from the hearts of E12 embryos of CDKN1A knockout mice and control mice were observed under a microscope for epiecardial cell proliferation and explant length after 7 days of culture.

[0011] The present invention will be described in detail below.

[0012] <1> Agent for Promoting Epicardial Cell Regeneration The present invention relates to an agent for promoting epicardial cell regeneration, which comprises a p21 inhibitor.

[0013] Addition of a p21 inhibitor to epicardial cells can reactivate the regenerative ability that is halted in adult epicardial cells, thereby promoting epicardial cell regeneration.

[0014] 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).

[0015] "Promotion of epicardial cell regeneration" is characterized, for example, by improved epicardial cell viability, improved epicardial cell proliferation, improved epicardial tissue repair capacity, and / or enhanced gene expression associated with reactivation of epicardial regenerative capacity.

[0016] The viability of epicardial cells can be determined, for example, by culturing epicardial cells and measuring the viability (proportion of viable cells) after a certain period of time. When the viability of epicardial cells increases when a p21 inhibitor is added to a medium compared to when a p21 inhibitor is not added to the medium, the viability of epicardial cells can be determined to be improved. In this case, the period for culturing epicardial cells may be, for example, 14 days.

[0017] The proliferation ability of epicardial cells can be shown, for example, by culturing epicardial cells, measuring the cell number over a certain period of time, and plotting a growth curve. When the slope of the growth curve of epicardial cells increases when a p21 inhibitor is added to the medium compared to when the p21 inhibitor is not added to the medium, it can be determined that the proliferation ability of epicardial cells has been improved. The period for culturing epicardial cells may be, for example, 7 days. Furthermore, the measurement of the cell number over time may be, for example, daily.

[0018] The tissue repair ability of the epicardium can be demonstrated, for example, by a wound healing assay. Specifically, the wound healing assay involves culturing epicardial cells until they become confluent, then physically scraping off some of the cells to create a simulated wound, and measuring the degree of repair after a certain period of time. If the addition of a p21 inhibitor to a medium improves the degree of repair of epicardial cells compared to when the p21 inhibitor is not added to the medium, it can be determined that the tissue repair ability of the epicardium has been improved.

[0019] Examples of genes associated with the reactivation of epicardial regeneration include the transcription factors WT1 and TBX18, and the aldehyde dehydrogenase ALDH1A2. When cultured with the addition of a p21 inhibitor to the medium, the expression levels of these genes increase compared to cultured without the addition of a p21 inhibitor, and this can be considered to indicate that the expression of genes associated with the reactivation of epicardial regeneration is improved. Gene expression levels can be evaluated by known methods such as RT-PCR.

[0020] p21 is known as a cyclin-dependent kinase inhibitor and is a protein also called CDKN1A, which is expressed from the CDKN1A gene locus. In the present invention, the p21 protein is also referred to simply as p21, and the gene that expresses p21 may be referred to as the CDKN1A gene or CDKN1A. p21 is known to bind to a complex of cyclin and cyclin-dependent kinase 2 or 4, inhibiting their activity and thereby controlling cell cycle progression in the G1 phase of the cell cycle.

[0021] The p21 protein and CDKN1A gene are not particularly limited, but can be selected based on the epicardial cells for which they promote regeneration. When promoting the regeneration of human epicardial cells, it is preferable to use an inhibitor of the human p21 protein or the human CDKN1A gene. An example of the amino acid sequence of the human p21 protein is shown in SEQ ID NO: 10, and an example of the nucleotide sequence of the human CDKN1A gene is shown in SEQ ID NO: 9.

[0022] In the present invention, "inhibition of p21" includes inhibition of p21 expression and activity.

[0023] Inhibition of p21 expression means a decrease in the transcription level (mRNA level) or translation level (protein level) of the gene (CDKN1A) encoding the p21 protein. Inhibition of p21 expression can be confirmed by comparing the expression levels of p21 protein or CDKN1A gene per a certain number of epicardial cells before and after the addition of a p21 inhibitor. The expression level of p21 protein or CDKN1A gene may be reduced compared to the expression level before the addition of a p21 inhibitor, and is preferably reduced to, for example, 50% or less, 20% or less, or 10% or less of the expression level before the addition of a p21 inhibitor. The expression level may also be reduced to below the detection limit level.

[0024] The expression of p21 protein or the gene (CDKN1A) encoding p21 protein can be measured by Western blotting, RT-PCR, or the like.

[0025] Inhibition of p21 expression can be achieved, for example, by reducing the expression of the gene encoding the p21 protein (CDKN1A gene).

[0026] Inhibition of CDKN1A gene expression is not particularly limited, and can be achieved, for example, by introducing a mutation into the CDKN1A gene that reduces transcription or translation efficiency, manipulating small molecules involved in transcription or translation control, or manipulating nucleic acids such as siRNA that causes RNA interference.

[0027] Furthermore, inhibition of CDKN1A gene expression can be achieved by disrupting the CDKN1A gene. Disruption of the gene means that the gene is modified so that it does not produce a protein that functions normally. Not producing a protein that functions normally includes cases where no protein is produced from the gene at all, or cases where a protein with reduced or no function per molecule is produced from the gene.

[0028] Gene disruption or introduction of a mutation may be carried out, for example, by using the CRISPR-Cas system. Specifically, for example, it may be carried out by using a guide RNA designed for gene modification or introduction of a mutation and a CRISPR enzyme. Alternatively, a DNA fragment for homologous recombination may be used at the same time.

[0029] The p21 inhibitor is a substance for the above-mentioned "p21 inhibition." Specifically, it may be, for example, a p21 activity inhibitor, a nucleic acid such as siRNA that targets the CDKN1A gene and induces RNA interference, or a guide RNA and CRISPR enzyme for the CRISPR-Cas system that targets the CDKN1A gene.

[0030] Specific examples of nucleic acids such as siRNA that bring about RNA interference include siRNA, shRNA, miRNA, precursors thereof, etc. That is, the p21 inhibitor may be a nucleic acid such as siRNA, shRNA, miRNA, or precursors thereof that target the CDKN1A gene, or DNA containing a sequence for expressing these RNAs.

[0031] The sequence of a nucleic acid such as an siRNA that causes inhibition of CDKN1A gene expression can be designed by a known method based on the sequence of the CDKN1A gene. Furthermore, the sequence of a 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.

[0032] Specifically, for example, the p21 inhibitor may be an siRNA comprising a sense strand containing the nucleotide sequence set forth in SEQ ID NO: 1 and an antisense strand containing the nucleotide sequence set forth in SEQ ID NO: 2, or an shRNA comprising a sequence contained in these sequences as a core sequence, or a DNA comprising a sequence for expressing these RNAs.

[0033] Methods for introducing nucleic acids into cells or tissues include, for example, viral vectors and lipofection. Therefore, when the p21 inhibitor is a nucleic acid such as RNA or DNA, it may be present in the form of a viral vector or a complex for lipofection. Any known viral vector can be used, including, for example, adenovirus vectors, adeno-associated virus vectors, and Sendai virus vectors.

[0034] Examples of substances that inhibit the activity of p21 protein include antibodies against p21 protein (including partial fragments) and compounds that bind to p21 protein and inhibit its activity. Inhibition of p21 protein activity can be confirmed by comparing the activity of p21 protein per certain number of epicardial cells before and after the addition of a p21 inhibitor. The activity of p21 protein may be reduced compared to the cells before the addition of a p21 inhibitor, but for example, the activity of p21 protein is preferably reduced to 50% or less, 20% or less, or 10% or less compared to the cells before the addition of a p21 inhibitor, and the activity may be completely lost.

[0035] Antibodies against p21 protein can be obtained by known methods using p21 protein or a partial peptide thereof (for example, a partial peptide of the C-terminal region of p21) as an antigen. Alternatively, commercially available anti-p21 antibodies may be used.

[0036] The p21 inhibitor can be added at a concentration that can inhibit the expression and function of p21.

[0037] The p21 inhibitor is not particularly limited as long as it inhibits the expression or function of p21. Specific examples of the p21 inhibitor 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}]hexatriaconta-16,24,26,28-tetraen-12-yl]propyl]-2-methoxycyclohexyl-3-hydroxy-2-(hydroxymethyl)-2-methylpropanoate (temsirolimus), (3R,4S,5S,6R,7R,9R,11S,12R,13S,14R)-6-{[(2S,3R , 4S,6R)-4-(dimethylamino)-3-hydroxy-6-methyloxan-2-yl]oxy}-14-ethyl-7,12,13-trihydroxy-4-{[(2R,4R,5S,6S)-5-hydroxy-4-methoxy-4,6-dimethyloxan-2-yl]oxy}-3,5,7,9,11,13-hexamethyl-10-(2,4,7-trioxa-1-azaoctan-1-ylidene)-1-oxacyclotetradecan-2-one (roxithromycin), 6-hydroxy-2-(4-hydroxyphenyl)benzo[b]thien-3-yl] [4-[2-(1-piperidinyl)ethoxy]phenyl]methanone hydrochloride (raloxifene hydrochloride), (7S,9E,11S,12R,13S,14R,15R,16R,17S,18S,19E,21Z)-2,15,17,27,29-pentahydroxy-11-methoxy-3,7,12,14,16,18,22-heptamethyl-26-{(E)-[(4-methylpiperazin-1-yl)imino]methyl}-6,23-dioxo-8,30-dioxa-24-azatetracyclo[23.3.1.14'7.05'28]triaconta-1(28), 2,4,9,19,21,25(29),26-octaen-13-yl acetate (rifampicin), [(8R,9S,10R,13S,14S,17R)-17-acetyl-6,10,13-trimethyl-3-oxo-2,8,9,11,12,14,15,16-octahydro-1H-cyclopenta[a]phenanthren-17-yl]acetate (megestrol acetate), 8-(4-amino-1-methylbutylamino)-6-methoxyquinoline diphosphate (primaquine diphosphate), potassium; [2-butyl-5-chloro-3-[[4-[2-(1,2,3-triaza-4-azanilide-2, Examples include (5-dien-5-yl)phenyl]phenyl]methyl]imidazol-4-yl]methanol (losartan potassium), (2S)-3-methyl-2-[pentanoyl-[[4-[2-(2H-tetrazol-5-yl)phenyl]phenyl]methyl]amino]butanoic acid (valsartan), (Z)-but-2-enedioic acid; 2-(2,2-dicyclohexylethyl)piperidine (perhexiline maleate), 3-O-methyl-5-O-(2-methylpropyl)-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate (nisoldipine), and the like.

[0038] <Drug for treating cardiac injury> Epicardial cells have an essential function for cardiac regeneration following injury. Therefore, the epicardial cell regeneration promoter of the present invention can be used as a pharmaceutical for treating cardiac injury by reactivating the regenerative ability of the epicardium and thereby acquiring the cardiac regenerative ability following injury. Therefore, the present invention provides a pharmaceutical for treating cardiac injury, which contains a p21 inhibitor as an active ingredient.

[0039] The pharmaceutical for treating cardiac damage may use the above-mentioned p21 inhibitor as it is, or may be used as a pharmaceutical composition by combining the p21 inhibitor with a pharmacologically acceptable carrier. The pharmacologically acceptable carrier may be a carrier used in pharmaceuticals, such as a solvent, buffer, stabilizer, or excipient, and may be appropriately selected depending on the type of p21 inhibitor and the dosage form of the pharmaceutical. When the p21 inhibitor is a nucleic acid, the pharmaceutical for treating cardiac damage may include a reagent for delivering the nucleic acid into cells, such as a lipofection reagent, or a reagent for stabilizing the nucleic acid.

[0040] The subject of the pharmaceutical of the present invention is not particularly limited as long as the effects of the present invention can be obtained, but is preferably a mammal, more preferably a primate such as a human or a rodent such as a mouse, and even more preferably a human.

[0041] The pharmaceutical composition of the present invention can be administered orally or parenterally, but is preferably administered locally to the damaged site of the heart. The dosage form of the pharmaceutical composition is not particularly limited, and it can be administered, for example, in the form of an injection or an infusion preparation.

[0042] The dosage of the pharmaceutical 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 appropriately, but is, for example, 0.01 mg to 1000 mg, preferably 0.1 mg to 100 mg, per day as the amount of the p21 inhibitor as an active ingredient. Administration may be in a single dose or multiple doses.

[0043] The medicament of the present invention may be used in combination with other therapeutic agents for cardiac damage.

[0044] <2> Method of the Present Invention One aspect of the method of the present invention is a method for promoting epicardial cell regeneration in vitro, which comprises the step of inhibiting p21 in the epicardial cells.

[0045] Another aspect of the method of the present invention relates to a method for producing epicardial cells with enhanced regenerative potential, comprising the steps of: A) providing epicardial cells; and B) inhibiting p21 in the epicardial cells of step A).

[0046] Here, "promoting epicardial cell regeneration" refers to, for example, improving epicardial cell viability, improving epicardial cell proliferation, improving epicardial tissue repair capacity, and / or enhancing gene expression associated with reactivation of epicardial regenerative capacity. Furthermore, "enhanced regenerative capacity" may refer to, for example, improving epicardial cell viability, improving epicardial cell proliferation, improving epicardial tissue repair capacity, and / or enhancing gene expression associated with reactivation of epicardial regenerative capacity.

[0047] 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.

[0048] Here, pluripotent stem cells include, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells ("GS cells"), embryonic germ cells ("EG cells"), pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells), etc. 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, and even more preferably from humans.

[0049] Methods for producing iPS cells are known in the art, and iPS cells can be produced by, for example, introducing a reprogramming factor into any somatic cell. Examples of reprogramming factors include genes or gene products such as Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO 2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 / 0689 55, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO 2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Huangfu D, et al. (2008), Nat. Biotechnol. , 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3,568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9.

[0050] Somatic cells used to obtain iPS cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature healthy or diseased somatic cells, as well as primary culture cells, passaged cells, and established cell lines. Specific examples of 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) differentiated cells such as blood cells (peripheral blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, liver cells, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (exocrine pancreatic cells, etc.), brain cells, lung cells, kidney cells, and adipocytes.

[0051] Specifically, epicardial cells derived from pluripotent stem cells may be epicardial cells induced to differentiate from pluripotent stem cells in vitro. Any known method can be used to induce differentiation from pluripotent stem cells into epicardial cells, and for example, differentiation may be induced by adding an appropriate differentiation-inducing factor to the culture medium.

[0052] The method for inducing differentiation of pluripotent stem cells into epicardial cells is not particularly limited, and known methods can be used. Specifically, for example, embryoid bodies (EBs) can be formed from pluripotent stem cells, and the EBs can be cultured in monolayers in the presence of a GSK3 (glycogen synthase kinase 3) inhibitor such as CHIR99021, a TGF (transforming growth factor) β inhibitor SB431542, BMP4 (bone morphogenetic protein 4), and VEGF (vascular endothelial growth factor). Other methods described in the following literature can also be used. 1. Witty A., et al. Nat Biotechnol. 2014 Oct;32(10):1026-35. doi: 10.1038 / nbt.3002. PMID: 25240927. 2. Iyer D, et al. Development. 2015 Apr 15;142(8):1528-41. doi: 10.1242 / dev.119271. PMID: 25813541. 3. Bao X, et al. Nat Biomed Eng. 2016;1:0003. doi: 10.1038 / s41551-016-0003. PMID: 28462012. 4. Guadix JA, et al. Stem Cell Reports. Dec 2017 12;9(6):1754-1764. doi: 10.1016 / j.stemcr.2017.10.023. PMID: 29173898.

[0053] The epicardial cells induced to differentiate from pluripotent stem cells may be isolated and purified before use, or a cell population containing epicardial cells may be used as is.

[0054] The epicardial cells thus provided are subjected to a treatment for inhibiting p21. Here, "inhibiting p21" refers to 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-mentioned p21 inhibitor to the epicardial cells.

[0055] An example of a method for treating with a p21 inhibitor is to add a p21 inhibitor to a culture medium for epicardial cells and culture the cells for a time sufficient for epicardial cell regeneration, for example, 5 hours to 10 days. The concentration of the p21 inhibitor added may be sufficient for epicardial cell regeneration and may be appropriately set depending on the type of p21 inhibitor. Additional p21 inhibitors may be administered. Alternatively, the p21 inhibitor may be removed from the culture medium after the p21 inhibitory effect has been exerted. Epicardial cells with enhanced regeneration capacity may be purified or concentrated before use.

[0056] The epicardial cells produced by the method of the present invention may be used to treat cardiac damage, for example by transplanting them into a subject, as appropriate.

[0057] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following embodiments.

[0058] (Procedure) Induction of Differentiation into Human Epicardial Cells. The human iPS cell line (409B2) was used to induce differentiation into human epicardial cells. The human iPS cell line was maintained on irradiated mouse embryonic fibroblasts (MEFs) in Primate ES Cell Medium (REPROCELL, Cat. RCHEMD001) supplemented with 4 ng / mL fibroblast growth factor (bFGF). To form embryoid bodies (EBs), human iPS cells were seeded as a single-cell suspension onto low-adhesion poly-2-hydroxyethyl methacrylate (HEMA)-coated dishes. The primary differentiation medium used was StemPro-34 medium (Gibco) supplemented with 2 mM L-glutamine, 50 μM / mL ascorbic acid, 0.4 μM monothioglycerol, and 150 mg / mL transferrin. To trigger differentiation, 10 μM Y27632 (Rock inhibitor), 2 ng / mL human recombinant BMP4, and 0.5% Matrigel (Corning) were added to the differentiation medium.

[0059] Twenty-four hours after EB formation, 100% volume of cell culture medium supplemented with 4 ng / mL activin A, 10 ng / mL human recombinant bFGF, and 20 ng / mL human recombinant BMP4 was added. After 72 to 96 hours, EBs were dissociated into single cells and replated (0.3 x 10 cells) onto gelatin-coated dishes using basal medium containing 3 mM CHIR99021, 10 μM SB431542, 30 ng / mL human recombinant BMP4, and 5 ng / mL human recombinant VEGF. ５ cells / cm ２ ) and induced differentiation of human epicardial cells. From day 7 onwards, the differentiated human epicardial cells were maintained in DMEM (Dulbecco's modified Eagle's medium) containing 10% fetal bovine serum (FBS) and 10 μM SB431542. These human epicardial cells induced to differentiate from human iPS cell lines are also called human iPS cell-derived fetal epicardium.

[0060] For siRNA transfection assays, human iPS cell-derived fetal epicardium was cultured at 0.8 × 10 cells per 10-cm dish. ６Cells were seeded individually and transfected using RNAiMax (Invitrogen). Fresh medium was replaced 24 hours after transfection. Small interfering RNA (siRNA) targeting CDKN1A (sense strand: CAAGGAGUCAGACAUUUUAtt (SEQ ID NO: 1), antisense strand: UAAAAUGUCUGACUCCUUGtt (SEQ ID NO: 2)) and negative control siRNA No. 1 from Ambion Silencer Select (4390843) were used according to the manufacturer's instructions.

[0061] Quantitative RT-PCR: RNA was extracted using QIAzol lysis reagent (QIAGEN), and cDNA was synthesized from the total RNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO). Quantitative RT-PCR was then performed using Thunderbird SYBR qPCR Mix (TOYOBO) in a One Step Real-Time PCR System (Applied Biosystems). Gene expression results were quantified using the ΔΔCt method and normalized to GAPDH. The primers used were 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).

[0062] Western blot analysis. Cells were lysed with M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific) to obtain total protein, which was then quantified using a Protein Assay BCA Kit (Nacalai Tesque). For Western blot analysis, 8 μg of total protein was loaded per well of an SDS-PAGE gel. The antibodies used and their dilutions were as follows: p21 (1:1000, Cell Signaling Technology, Cat#2947), WT1 (1:1000, Abcam; ab89901), and β-actin (1:1000, Sigma, A5441).

[0063] Cell proliferation curve assay Cell proliferation time course curves were assessed in 6-well plates. Cells were grown at 4 × 10 per well. ４ Cells were seeded at a density of 1000 cells / ml and total cell counts were performed using 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 each counting day. Values ​​were expressed as the cell proliferation rate of cells grown in the presence of 10% FBS. 0% indicates the cell count on day 0.

[0064] For cell colony formation assay, 5000 cells were seeded onto a 10 cm dish and cultured for 10 days, after which the colonies were fixed with 4% PFA, stained with crystal violet, and counted using Image-J.

[0065] Wound healing assay: Cells were seeded in 6-well plates and cultured until 100% confluent. The cell monolayer was then scratched manually with a pipette tip to create a physical wound. The scratched area was imaged at 0, 12, 16, 20, and 24 hours after scratching. The initial cell-free area and the remaining areas at 12, 16, 20, and 24 hours were quantified using Image-J. The percentage of the scratch area repopulated by migrating cells (percent wound closure area) was then calculated relative to the initial scratch area.

[0066] RNA-Seq Analysis. Data normalization for RNA-Seq analysis was performed using NOISeq. Adult epicardial data are available for analysis under the GSE code GSE8484085. Primary RNA-Seq data were processed using RStudio for mapping and gene expression analysis. Gene expression data for searching, reading, and preprocessing were processed using the Bioconductor package NOISeq for data analysis and differential expression analysis of RNA-Seq data. Heatmaps for clustering differentially expressed genes (DEGs) were generated using R scripts.

[0067] CDKN1A knockout (KO) mice were obtained from the Jackson Laboratory (#016565 strain). The second exon of the Cdkn1a gene in this knockout mouse strain was replaced with a neomycin resistance cassette (neo cassette). C57BL / 6J mice with a similar genetic background were used as isogenic controls.

[0068] Epicardial explants. Ex vivo explant assays were performed using 12-day-old mouse embryonic (E12) hearts. First, E12 mouse hearts were placed on gelatin-coated dishes in low-glucose DMEM supplemented with 15% FBS. Proliferative cultures emerged within 24 hours, and after 7 days, the length of the explants was observed under a microscope. Explant cells were maintained in medium supplemented with 10 μM SB431542 to prevent spontaneous epithelial-mesenchymal transition (EMT).

[0069] (Results) Example 1: Human iPS cell-derived fetal epicardium was subjected to an siRNA transfection assay using siRNA targeting CDKN1A (siCDKN1A) or a negative control siRNA (control), and the relative mRNA expression level of CDKN1A was measured by quantitative RT-PCR. The results are shown in Figure 1. Furthermore, the levels of p21 protein and WT1 protein were measured by Western blot for human epicardial cells transfection assayed with each siRNA. The results are shown in Figure 2. siCDKN1A reduced the expression level of CDKN1A at the mRNA level in human iPS cell-derived fetal epicardium to less than 50% compared to the control, and a similar reduction was observed at the p21 protein level.

[0070] Next, a cell proliferation assay was performed on the human iPS cell-derived fetal epicardium that had been subjected to a transfection assay using each siRNA, and a cell proliferation curve was generated. The results are shown in Figure 3. A significant increase in the proliferation potential of human epicardial cells was observed by reducing CDKN1A expression. Similarly, a colony formation assay was performed to measure the viability of human epicardial cells. The results are shown in Figure 4. A significant increase in the viability of human epicardial cells was observed by reducing CDKN1A expression.

[0071] Furthermore, we performed a wound healing assay on human iPS cell-derived fetal epicardium transfected with each siRNA. The percentage of wound closure over time is shown in Figure 5 (left), and photographs of the wound area at 0 and 24 hours are shown in Figure 5 (right). Reducing CDKN1A expression in human epicardial cells significantly increased wound healing ability between 20 and 24 hours after scratching.

[0072] Furthermore, transcriptome analysis was performed to detect gene signatures of cell quiescence using RNA-Seq analysis (Figure 6). The left figure shows a comparison between human iPS cell-derived fetal epicardium and adult epicardium, while the right figure shows a comparison between human iPS cell-derived fetal epicardium and human iPS cell-derived fetal epicardium (CDKN1A expression suppression: siCDKN1A group) transfection assayed using siCDKN1A. Gene expression levels decreased in the cell quiescent state in the adult epicardium, followed by human iPS cell-derived fetal epicardium and the siCDKN1A group. Furthermore, gene expression levels increased in the cell quiescent state in the adult epicardium, followed by human iPS cell-derived fetal epicardium and the siCDKN1A group. Therefore, the gene expression level revealed that the adult epicardium, human iPS cell-derived fetal epicardium, and siCDKN1A group were in a state of stronger cytoquiescence, in that order; in other words, the siCDKN1A group had escaped from a state of cytoquiescence compared to human epicardium.

[0073] These findings demonstrate that reducing CDKN1A expression in human epicardial cells can reactivate their regenerative potential and improve their wound healing ability.

[0074] (Example 2) An ex vivo explant assay of CDKN1A knockout mice was performed. First, epicardial explants were prepared and cultured ex vivo using E12 hearts from CDKN1A knockout mice and control mice (C57BL / 6J mice). CDKN1A knockout mice and control mice were prepared as described above in "CDKN1A knockout (KO) mice." Furthermore, epicardial explants were prepared and cultured using the method described above in "Epicardial explants." Next, the amounts of p21 protein and WT1 protein in each epicardial explant were measured by Western blotting. The results are shown in Figure 7.

[0075] In epicardial explants obtained from CDKN1A knockout mice, p21 protein was barely observed, consistent with the effects of CDKN1A knockout.

[0076] The appearance of each epicardial explant prepared after 7 days of culture, as observed under a microscope, is shown in Figure 8. Compared with the control, epicardial explants obtained from CDKN1A knockout mice showed a faster proliferation rate and nearly doubled the explant length, suggesting a high regenerative potential.

[0077] From the above, it was confirmed that reducing or eliminating CDKN1A expression can reactivate the regenerative ability of epicardial cells and improve wound healing ability, even in epicardial explants prepared from mouse E12 hearts.

Claims

1. An epicardial cell regeneration promoter containing a p21 inhibitor.

2. The epicardial cell regeneration promoter according to claim 1, wherein the p21 inhibitor is a CDKN1A gene expression inhibitor.

3. The epicardial cell regeneration promoter according to claim 2, wherein the CDKN1A gene expression inhibitor is a nucleic acid containing an siRNA sequence that targets the CDKN1A gene.

4. The epicardial cell regeneration promoter according to claim 3, wherein the siRNA sequence comprises a sense strand having the sequence described in SEQ ID NO: 1 and an antisense strand having the sequence described in SEQ ID NO:

2.

5. The epicardial cell regeneration promoter according to claim 1, wherein the p21 inhibitor comprises a guide RNA for the CRISPR-Cas system, and the guide RNA for the CRISPR-Cas system is a guide RNA designed to target the CDKN1A gene.

6. A pharmaceutical composition for treating cardiac injury, comprising an epicardial cell regeneration promoter according to any one of claims 1 to 5.

7. A method for promoting the regeneration of epicardial cells in vitro, A method comprising the step of inhibiting p21 in the epicardial cells.

8. The method according to claim 7, wherein the step of inhibiting p21 is performed by inhibiting the expression of the CDKN1A gene.

9. The method according to claim 8, wherein the inhibition of the expression of the CDKN1A gene is achieved by introducing siRNA into the epicardial cells.

10. The method according to claim 8, wherein the step of inhibiting p21 is carried out by introducing a guide RNA and a CRISPR enzyme designed to target the CDKN1A gene into the epicardial cells.

11. A method for producing epicardial cells with enhanced regenerative capacity, A) A step of providing epicardial cells, and B) A method comprising the step of inhibiting p21 in the epicardial cells of step A).

12. The method according to claim 11, wherein the epicardial cells in step A) are epicardial cells differentiated from pluripotent stem cells.