Method for producing mature epicardial cell

Abstract

The present invention provides a method for producing mature epicardial cells, the method involving a step for culturing epicardial cells in a medium containing a mTORC2 inhibitor.

Description

Method for producing mature epicardial cells

[0001] The present invention relates to a method for producing mature epicardial cells, more specifically to a method for producing mature epicardial cells, which comprises a step of culturing epicardial cells in a medium containing an mTORC2 inhibitor, and uses of the epicardial cells obtained by the method.

[0002] The human epicardium is the outermost layer of the heart and plays a crucial role in cardiac development and regeneration. Epicardial cells are the source of various cells that form the heart, including cardiac fibroblasts, smooth muscle cells, and endothelial cells, and play an important role in cardiac organogenesis. Furthermore, epicardial cell-derived cells play important roles in maintaining cardiac homeostasis and regulating responses to organ injury. For these reasons, epicardial cells and their derived cells are important cell groups in cardiac disease pathology model research. Furthermore, recent mouse studies have shown that epicardial cells have the ability to regenerate after myocardial injury during fetal development and the first few days after birth (Non-Patent Document 1). However, beyond this time frame, the epicardium becomes quiescent and loses its regenerative potential as the tissue functionally matures (Non-Patent Document 2). Despite their non-proliferative nature, quiescent epicardial cells remain metabolically and transcriptionally active (Non-Patent Document 3).

[0003] Although several methods for generating epicardial cells from human induced pluripotent stem cells have been reported (e.g., Non-Patent Document 4, Non-Patent Document 5), these cells are fetal-type epicardial cells, express WT1 and TBX18, which are markers of fetal-type epicardial cells, and possess proliferative activity and epithelial-mesenchymal transition (EMT) activity. Although adult-type (mature) epicardial cells are necessary for cardiac disease research, no method for generating epicardial cells has been established.

[0004] Cai, W. et al. Cell Rep. 28, 190-201.e3 (2019)Cao, Y., Duca, S. & Cao, J. Cold Spring Harb. Perspect. Biol. 12, a037192 (2020)Hesse, J. et al. eLife 10, e65921 (2021)Witty, AD et al. Nat. Biotechnol. 32, 1026-1035 (2014)Junghof, J. et al. Npj Regen. Med. 7, 1-14 (2022)

[0005] Therefore, an objective of the present invention is to provide a method for producing mature epicardial cells, and epicardial cells produced by the method.

[0006] While studying the molecular mechanisms regulating epicardial quiescence and loss of regenerative capacity in adulthood, the present inventors conceived the idea of ​​producing adult-type (mature) epicardial cells that lack regenerative capacity by inducing epicardial cell quiescence. As one method for inducing epicardial cell quiescence, we focused on the mechanistic target of rapamycin (mTOR) signaling pathway. The mTOR signaling pathway plays a key role in controlling cellular quiescence in mature tissues by sensing and responding to changes in nutrient availability, growth factors, and other environmental cues (e.g., Liu, GY & Sabatini, DM Nat. Rev. Mol. Cell Biol. 21, 183-203 (2020)). However, the extent to which mTOR signaling regulates human epicardial development remains largely unknown.

[0007] After extensive research, the inventors decided to treat epicardial cells with the mTOR inhibitor Torin1. As a result, they succeeded for the first time in generating WT1- and TBX18-negative adult epicardial cells from human iPS cells. Surprisingly, the use of the mTORC1 kinase inhibitor rapamycin failed to reduce WT1 levels in epicardial cells. Therefore, they concluded that epicardial cell maturation depends on a mechanism dependent on mTORC2 within the mTOR signaling pathway. Based on these findings, the inventors conducted further research and completed the present invention.

[0008] That is, the present invention provides the following: [1] A method for producing mature epicardial cells, comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor. [2] The method according to [1], wherein the medium contains an mTORC1 inhibitor. [3-1] The method according to [1] or [2], wherein at least one mTORC2 inhibitor is selected from the group consisting of Torin1, sapanisertib, WYE-687, AZD8055, and bistusertib. [3-2] The method according to [1] or [2], wherein at least one mTORC2 inhibitor is Torin1. [4] The method according to any one of [1] to [3-2], wherein the mature epicardial cells are cells that do not express WT1 and / or TBX18. [5-1] The method according to any one of [1] to [4], wherein the epicardial cells are derived from pluripotent stem cells. [5-2] The method according to any one of [1] to [5-1], wherein the epicardial cells are derived from humans. [6] The method according to any one of [1] to [5-2], wherein the epicardial cells are contained in cardiac organoids. [7] Mature epicardial cells or cardiac organoids comprising said cells, obtained by the method according to any one of [1] to [6]. [8] A transplantation therapy agent comprising the mature epicardial cells or cardiac organoids according to [7]. [9] A method for producing mature cardiomyocytes, comprising a step of co-culturing the mature epicardial cells or cardiac organoids according to [7] with cardiomyocytes. [10-1] The method according to [9], wherein the cardiomyocytes are contained in cell aggregates. [10-2] Mature cardiomyocytes or cell aggregates comprising said cells, obtained by the method according to [9] or [10-1].

[11] A method for maturing epicardial cells, comprising a step of culturing epicardial cells in a medium containing an mTORC2 inhibitor.

[12] A method for maturing cardiomyocytes, comprising a step of co-culturing the mature epicardial cells or cardiac organoids according to [7] with cardiomyocytes.

[13] A method for treating or preventing heart disease, comprising administering or transplanting an effective amount of the mature epicardial cells or cardiac organoids described in [7] and / or the cardiomyocytes or cell aggregates described in [10-2] to a mammal.

[14] The mature epicardial cells or cardiac organoids according to [7] and / or the cardiomyocytes or cell aggregates according to [10-2] for use in the treatment or prevention of cardiac disease.

[15] Use of the mature epicardial cells or cardiac organoids according to [7] and / or the cardiomyocytes or cell aggregates according to [10-2] in the manufacture of a drug for the treatment or prevention of cardiac disease.

[0009] According to the present invention, a novel method for producing mature epicardial cells from immature epicardial cells is provided, and it also becomes possible to produce mature cardiomyocytes using these mature epicardial cells.

[0010] Dynamics of mTOR signaling during epicardial formation. (a) Retrospective analysis of GSE51483, a dataset of bulk samples of mouse embryonic hearts at various developmental stages, showing the multistep inactivation of the mTOR signaling switch during development. (b) Schematic of the mTOR signaling network in the developing heart, highlighting downstream signaling of complexes 1 and 2 regulating phospho-Akt. (c) Western blot analysis of mouse hearts for characterization of mTOR targets during development [pAkt, pS6k, p4E-BP1, p27; β-actin protein levels are used as an endogenous housekeeping control]. (d) Retrospective analysis of GSE51483, a dataset for evaluation of cell cycle dynamics during cardiac development and maturation. (e) Immunocytochemical analysis of ZO-1 and WT1 expression in the epicardium of the left atrium at E12 [Scale bar: 500 μm; inset: 100 μm]. (f) Immunocytochemical analysis of ZO-1 and WT1 expression in left ventricular epicardium at E12 and E14. (g) Phase-contrast photographs of epicardial proliferation from E12. (h) Relative mRNA expression (relative to E12) of Wt1, Tbx18, Cdh18, and Nbl1 genes during epicardial formation [n=3; t-test, *p<0.05, **p<0.005, ***p<0.0005]. (i) Western blot analysis of epicardial explants for characterization of mTOR target inhibition over time [pAkt, pS6k, p4E-BP1, WT1; β-actin protein levels are used as an endogenous housekeeping control]. Effect of Torin1 on MECs. (a) Cell viability and IC in MECs after 96-hour treatment with decreasing concentrations of Torin1 at a fixed 1:3 ratio. 50(b) MTT assay showing transcriptome differences between control, untreated, and Torin1-treated MEC1 cells. (c) Relative mRNA expression analysis derived from RNA-seq data for genes related to epicardial maturation, the mTOR pathway, proliferation, and the EMT program (Wt1, Sema3d, Scx, Gata4, Rptor, Rictor, E2f1, Ccnb1, Cdk4, Cdk6, Mki67, Snai1, Cdh2, and Cdh1). (d) Phase-contrast photographs of untreated and MEC1 cells treated with 50 nM Torin1 and 5 ng / mL TGF-β for 48 hours. (e) mRNA expression analysis of the cadherin1 / 2 switch to assess EMT initiation [n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005]. Characterization of Torin1 effects on mouse epicardial maturation. (a) Immunocytochemistry was used to analyze WT1 expression in E12 cardiac tissue after 24 hours of treatment with 200 nM Torin1 to induce ectopic maturation. (b) Western blot analysis of E12 and E14 epicardial explants to characterize targeted inhibition of mTOR and cardiac maturation upon Torin1 treatment [cTnI (TNNI3), pS6k, pAkt, SNAI1, WT1; β-actin protein levels are used as an endogenous housekeeping control]. (c) Western blot analysis of E12 epicardial explants to characterize targeted inhibition of mTOR and epicardial maturation upon Torin1 treatment [pAkt, pS6k, TNNI1, NBL1, WT1; β-actin protein levels are used as an endogenous housekeeping control]. (d) Phase-contrast imaging was used to characterize the effect of Torin1 on epicardial growth in mouse epicardium. The epicardium was also treated with 10 μM of the ALK5 / TGF-β inhibitor SB431542 to prevent spontaneous differentiation. (e) PCA of Torin1-treated and untreated E12-derived mouse epicardial explants [black circles: control, n = 2; gray circles: Torin1, n = 2]. (f) Differentially enriched gene plot (DE plot) highlighting 898 up- and down-regulated genes upon Torin1 treatment.(g) PANTHER-based analysis of statistically significant biological processes in E12 explants ectopically matured with the mTOR inhibitor Torin1. (h) PANTHER-based analysis of statistically significant compromised pathways in E12 explants ectopically matured with the mTOR inhibitor Torin1. (i) mRNA expression analysis of genes associated with proliferation (MKi67) and the mouse embryonic epicardial program (Wt1, Tbx18, Raldh1a2, Hand2, Gata4) (black bars: control; gray bars: Torin1). (j) mRNA expression analysis of genes associated with cardiac maturation (Tnni3, Myh6) (black bars: control; gray bars: Torin1). (k) mRNA expression analysis of genes associated with epicardial quiescence (Hes1, Rb1) (black bars: control; gray bars: Torin1). Differentiation of human iPSCs to the epicardium. (a) Schematic diagram of human iPSC differentiation into epicardium. (b) Phase-contrast micrographs showing hiPSC line 409B2 (left) and EPI cells at day 24 of differentiation (right). (c) qRT-PCR analysis of WT1, TBX18, and ALDH1A2 during induction compared to day 12 of epicardial differentiation [day 12 n=3, day 24 n=3; *p<0.05, **p<0.005, ***p<0.0005]. (d) Flow cytometry-derived histogram of WT1 protein expression. (e) Immunocytochemistry analysis of hiPSC-derived epicardial cells showing increased expression of ZO1 and WT1 at day 24 (from day 6, Figure 4d). Generation of mature hiPSC-derived epicardial monolayers by mTOR inhibition. (a) Schematic diagram of the workflow for monitoring the effects of Torin1 on hiPSC-derived epicardial monolayers. (b) Western blot analysis of hiPSC-derived epicardial cells treated with Torin1 for mTOR target inhibition and assessment of quiescence-associated markers [pAkt, pS6k, p53, p16, p21; β-actin protein levels are used as an endogenous housekeeping control].(c) Phase-contrast micrograph showing Torin1-treated hiPSC-derived epicardium at day 31, which retained its cobblestone morphology [Scale bar: 100 μm]. (d) 7-day growth curve of Torin1-treated epicardium (Torin1, gray circles) [n=3; **p=0.0038, ****p<0.0001]. (e) Immunocytochemical analysis of Torin1-treated hiPSC-derived epicardial cells for the expression of WT1, TBX18, and ZO-1 [Scale bar: 500 μm; Inset: 100 μm]. (f) Immunocytochemical analysis of Torin1-treated hiPSC-derived epicardial cells for the expression of Ki67 (left) and their quantification (right) [Scale bar: 500 μm; Inset: 100 μm]. (g) Immunocytochemical analysis (left) and respective quantification (right) of phospho-histone 3 (pH3) expression in Torin1-treated hiPSC epicardial cells [n=3, *p<0.05, **p<0.005, ***p<0.0005] [Scale bar: 500 μm; inset: 100 μm]. (h) β-galactosidase assay (dark gray) showing senescent cells in mature hiPSC-derived epicardium (left) and respective quantification (%, right). (i) Relative mRNA expression analysis of quiescence-related genes (TP53, CDKN2A, HES1, RB1) and proliferation-related genes (MKI67, CCNA1, CCNB1, E2F1) in hiPSC-derived epicardium after Torin1 treatment (gray bars: Torin1). (j) FACS analysis based on pyrrolin Y and Hoechst 33342 staining to identify cells in G0. (k) Quantification and statistical analysis of quiescent hiPSC-derived epicardial cells after Torin1 treatment (gray bars: Torin1) [n=3, *p<0.05, **p<0.005, ***p<0.0005]. Efficacy and dose adjustment of Torin1 on hiPSC-derived epicardium. (a) Cell viability and IC in hiPSC-derived epicardium after 96 h treatment with decreasing concentrations of Torin1 at a fixed 1:3 ratio. 50(b) Western blot analysis of hiPSC-derived epicardium treated with decreasing concentrations of Torin1 from 2000 nM to the target optimal concentration for reducing WT1 protein levels [WT1, p21 protein; β-actin protein levels are used as endogenous housekeeping controls]. (c) Western blot analysis of hiPSC-derived epicardium treated with 200 nM Torin1 over a 2-day time period to target the effect of Torin1 in inhibiting mTOR signaling during the optimal time frame for replacing the medium with fresh Torin1 [pS6k, pAkt, p4E-BP1, p21 protein; β-actin protein levels are used as endogenous housekeeping controls]. (d) Western blot analysis of hiPSC-derived epicardium treated with 200 nM Torin1 over a 10-day time period, targeting the maximal effect of Torin1 in inducing epicardial maturation, defined as the decrease in WT1 and TBX18 proteins and the gain of NBL1 [WT1, TBX18, NBL1; β-actin protein levels are used as an endogenous housekeeping control]. Loss of spontaneous EMT development in mature hiPSC-derived epicardium. (a) Immunocytochemical analysis of hiPSC-derived epicardial cells treated with 200 nM Torin1 for SNAI1 expression [Scale bar: 500 μm; inset: 100 μm]. Characterization of cellular senescence and SASP in Torin1-treated epicardial cells. (a) Phase-contrast images of epicardium treated with various concentrations of Torin1 to detect senescent cells. (b) Quantification of β-galactosidase assay in mature hiPSC-derived epicardium as a percentage (%) [n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005]. (c) Relative mRNA expression analysis of SASP genes CXCL8 (IL-8) and SPP1 in mature hiPSC-derived epicardium [n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005]. Characterization of the effect of mTOR inhibition in hiPSC-derived cells using the GiWiGi protocol.(a) Phase-contrast image of d16 hiPSC-derived epicardial cells differentiated from the feeder-free iPS cell line 1390C1 according to the GiWiGi protocol. (b) Western blot analysis of 200 nM Torin1-treated hiPSC-derived epicardium to characterize the degree of mTOR target inhibition and epicardial maturation upon 7 days of treatment with 200 nM Torin1 [WT1, p21, p16, p53, p4E-BP1; β-actin protein levels served as an endogenous housekeeping control]. Total RNA transcriptome analysis of Torin1-treated hiPSC-derived epicardium. (a) PCA of d31 hiPSC-derived epicardial monolayers treated with Torin1 and untreated [black circles: control, n=3; gray circles: Torin1, n=3]. (b) Differentially enriched gene plot (DE plot) highlighting up- and down-regulated genes upon Torin1 treatment. (c) mRNA expression analysis for proliferation-related genes (MKI67) and fetal epicardial program-related genes (WT1, TBX18, ALDH1A2, HAND2) (black bars: control; gray bars: Torin1) (n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005). (d) mRNA expression analysis for EMT-related genes (SNAI1, SNAI2) (black bars: control; gray bars: Torin1) (n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005). (e, f) Heatmap showing the up- and down-regulated gene signature required for epicardial quiescence activation enriched in hiPSC-derived epicardium treated with Torin1 compared to primary human adult epicardium (GSE84085). Characterization of Torin1 and other rapalogs in maturing human cardiac organoids. (a) mRNA expression levels from RNA-Seq data (Figure 12a) of MKI67 in hiPSC-derived epicardium in response to Torin1, tacrolimus, and rapamycin.(b) Immunocytochemical analysis of WT1 and TNNI3 expression in human cardiac organoids (hHO) treated with Torin1 (200 nM), tacrolimus (1 μM), or rapamycin (200 nM) [Scale bar: 500 μm; inset: 100 μm]. (c) Immunocytochemical analysis of SNAI1 and ZO-1 expression in hHO treated with Torin1 (200 nM), tacrolimus (1 μM), or rapamycin (200 nM) [Scale bar: 500 μm; inset: 100 μm]. Characterization of the influence of mTORC2 on epicardial maturation. (a) PCA of d31 hiPSC-derived epicardial monolayers treated with Torin1, tacrolimus, and rapamycin, and untreated [black circles: control, n = 2; gray circles: Torin1, n = 2; dark gray circles: rapamycin, n = 2; light gray circles: tacrolimus, n = 1]. (b) Venn diagram for targeted screening and identification of mTORC2-dependent elements also expressed in primary adult epicardial tissue. Venn1 (top left) shows transcription factors (TFs) from Torin1, excluding those shared with or unique to rapamycin. Venn2 (bottom left) shows TFs from differentially expressed genes (DEGs) after filtering out statistically significant genes in both fetal and adult epicardium. Venn3 (right) filters out shared elements between Torin1 TFs (from Venn1) and shared TFs identified in both Venn1 and Venn2. (c) Relative mRNA levels of the top five transcription factors (TFs) most upregulated upon Torin1 treatment in hiPSC-derived epicardial monolayers. (d) Schematic diagram of the workflow for transfection of siRNA against YBX3 and MAFF. (e) mRNA expression analysis for YBX3, MAFF, WT1, TBX18, TCF21, and MKI67 genes in Torin1-treated hiPSC-derived epicardial monolayers overexpressing siRNA against YBX3 (siYBX3: gray bars) and siRNA against MAFF (siMAFF: dark gray bars) [n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005].(f) Immunocytochemical analysis of hiPSC-derived epicardial cells treated with siYBX3 and Torin1 for WT1 and ZO-1 expression [Scale bar: 500 μm; inset: 100 μm]. (g) Western blot analysis of hiPSC-derived epicardial cells treated with Torin1 overexpressing siRNA against YBX3 and siRNA against MAFF individually for characterization of proteins characteristic of fetal programming [WT1, TBX18; β-actin protein levels are used as an endogenous housekeeping control]. (h) Western blot analysis of hiPSC-derived epicardial cells treated with Torin1 overexpressing siRNA against YBX3 and siRNA against MAFF individually for characterization of proteins characteristic of cell proliferation [KI67; β-actin protein levels are used as an endogenous housekeeping control]. Paracrine effects of mature hiPSC-derived epicardium on fetal-like hiPSC-derived ventricular cardiomyocytes. (a) Schematic of the co-culture experiment to assess the paracrine signaling of mature hiPSC-derived epicardium to hiPSC-derived ventricular cardiomyocytes. (b) mRNA expression levels of epicardial IGF2 and FN1 expression 1 week after mTOR inhibition with Torin1 (gray bars: Torin1) [n=3, *p<0.05, **p<0.005, ***p<0.0005]. (c) Adult isoform of cardiac troponin I (mCherry) in beating EBs composed of ventricular hiPSC-derived cardiomyocytes co-cultured for 10 days with Torin1-treated mature hiPSC-derived epicardium (arrows in phase contrast). + , TNNI3) and fetal cardiac troponin (eGFP +(d) mRNA expression levels of cardiac TNNI3 in hiPSC-derived EBs after 10 days of coculture with Torin1-treated hiPSC-derived epicardium (dark gray bars: mature epicardium) [n=3, *p<0.05, **p<0.005, ***p<0.0005]. (e) mRNA expression levels of genes associated with left ventricular compression (TBX5, HEY2, PRDM16, and NPPA) in hiPSC-derived EBs after 10 days of coculture with Torin1-treated hiPSC-derived epicardium (dark gray bars: mature epicardium) [n=3, t-test, *p<0.05, **p<0.005, ***p<0.0005]. Generation of mature human iPSC-derived cardiac organoids. (a) Schematic of the protocol to generate self-organized mature human cardiac organoids (hHO) by downregulating mTOR signaling. (b) hHO cell viability and IC after 96 h treatment with decreasing concentrations of Torin1 at a fixed 1:3 ratio. 50 (c) Size (mm ) of human cardiac organoids before (untreated, black circles) and after 1 week of Torin1 treatment (Torin1, gray circles). 2) [n ≥ 11, two-tailed unpaired t-test]. (d) Phase-contrast micrographs of untreated hHOs and hHOs treated with Torin1 [Scale bar: 500 μm]. (e) Immunocytochemical analysis of WT1 and TNNT2 expression in hHOs at developmental day 15 [Scale bar: 500 μm; inset: 50 μm]. (f) Immunocytochemical analysis of SNAI1 and ZO-1 expression in hHOs at developmental day 15 [Scale bar: 500 μm; inset: 100 μm]. (g) Immunocytochemical analysis of WT1 and TNNT2 expression in hHOs treated with Torin1 (200 nM) [Scale bar: 500 μm; inset: 100 μm]. (h) Immunocytochemical analysis of WT1 and TNNI3 expression in hHO treated with Torin1 (200 nM), tacrolimus (1 μM), or rapamycin (200 nM) [Scale bar: 500 μm; inset: 100 μm]. (i) Immunocytochemical analysis of SNAI1 and ZO-1 expression in hHO treated with Torin1 (200 nM), tacrolimus (1 μM), or rapamycin (200 nM) [Scale bar: 500 μm; inset: 100 μm].

[0011] 1. Method for Producing Epicardial Cells The present invention provides a method for producing mature epicardial cells (hereinafter, sometimes referred to as the "production method of the present invention"), which comprises culturing epicardial cells in a medium containing an mTORC2 inhibitor. Epicardial cells are broadly classified into immature (i.e., "fetal") epicardial cells and mature (i.e., "adult") epicardial cells. Hereinafter, unless otherwise specified, "epicardial cells" refers to immature epicardial cells. Furthermore, in this specification, the phrase "culturing epicardial cells in a medium containing an mTORC2 inhibitor" can be appropriately interpreted as "contacting epicardial cells with an mTORC2 inhibitor."

[0012] Epicardial cells constitute the outermost layer of an animal's heart and play an important role in cardiac development and regeneration. As used herein, "immature epicardial cells" refer to cells that express WT1 and TBX18 and have proliferation and epithelial-mesenchymal transition (EMT) activity. As used herein, "mature epicardial cells" refer to cells that have reduced or absent (i.e., "no expression") of fetal epicardial markers compared to epicardial cells prior to culturing in a medium containing an mTORC2 inhibitor. These cells typically have reduced or absent proliferation and EMT activity compared to epicardial cells prior to culturing in a medium containing an mTORC2 inhibitor. The epicardial cells of the present invention may also have increased expression levels of IGF2 and / or FN1. Examples of markers for fetal epicardial cells include WT1, TBX18, ALDH1A2, and HAND2. Cells in which the expression level of at least one of these (e.g., WT1) is reduced or absent can be considered mature epicardial cells, but preferably cells in which the expression levels of two or more (e.g., a combination of WT1 and TBX18, a combination of WT1, TBX18, ALDH1A2, and HAND2) are reduced or absent. In one embodiment, mature epicardial cells do not express WT1 and / or TBX18, more preferably, they do not express WT1 and TBX18.

[0013] The proliferation activity may be evaluated based on a proliferation assay such as that described in the Examples below, or based on the expression levels of cell division-related genes (e.g., MKI67, CCNA1, CCNB1, E2F1, etc.). Alternatively, it may be evaluated based on the expression levels of genes associated with the cell cycle arrest phase (e.g., TP53, CDKN2A, HES1, RB1, etc.). In one embodiment, mature epicardial cells are cells that have lower expression levels of MKI67, CCNA1, CCNB1, and E2F1 and / or higher expression levels of TP53, CDKN2A, HES1, and RB1 compared to epicardial cells before the step of culturing in a medium containing an mTORC2 inhibitor.

[0014] Epithelial-mesenchymal transition (EMT) in epicardial cells refers to the differentiation of epicardial cells into smooth muscle cells, and EMT activity can be assessed by the expression of EMT-related genes (e.g., SNAI1, SNAI2, TCF21, etc.). Herein, cells expressing at least one of SNAI1, SNAI2, and TCF21 can be assessed as having EMT activity. In one embodiment, mature epicardial cells have a lower expression level of at least one of SNAI1, SNAI2, and TCF21, preferably all of these genes, compared to epicardial cells before the step of culturing them in a medium containing an mTORC2 inhibitor.

[0015] As shown in the examples below, epicardial cells can be matured by culturing them in a medium containing mTORC2 inhibitor, and can further mature the epicardial cells in cardiac organoid.Therefore, in one aspect of the present invention, the epicardial cells used in the present invention are contained in cardiac organoid, and epicardial cells are subjected to the method of the present invention in the form of cardiac organoid.Therefore, the present invention also provides a method for producing cardiac organoids comprising mature epicardium, comprising the step of culturing cardiac organoids comprising epicardial cells in a medium containing mTORC2 inhibitor.

[0016] In this specification, unless otherwise specified, "cells" includes "cell populations". Furthermore, unless otherwise specified, "cells" refers to those obtained by cell culture. A cell population may be composed of one type of cell, or may be composed of two or more types of cells. Furthermore, unless otherwise specified, "cell populations" also include "organoids" such as "cell aggregates" and "artificial tissues".

[0017] In this specification, "organoid" refers to a structure (typically a spheroid) containing multiple types of cells. Also, "artificial tissue" typically refers to a structure having a structure and / or function similar to that of tissue in vivo. Whether a structure is an organoid or an artificial tissue can be determined, for example, by observing a sample stained as needed (e.g., immunostaining, hematoxylin-eosin (HE) staining, etc.) under a microscope to confirm the localization of cells and the presence or absence of layer structure formation.

[0018] In this specification, " cardiac organoid " refers to the organoid that comprises epicardial cells, and this organoid also includes artificial cardiac tissue. " Artificial cardiac tissue " refers to the artificial tissue that comprises epicardial cells. Hereinafter, unless otherwise specified, artificial cardiac tissue will simply be referred to as " cardiac tissue ". Typically, the cardiac organoid of the present invention comprises TNNT2-positive cardiomyocytes that exhibit pulsating activity, CD31-positive endothelial cells, NFATC1-positive endocardial cells, and VIM-positive cardiac fibroblasts. In addition, cardiac organoid can have tight junctions that are expressed by ZO-1 expression.

[0019] The epicardial cells used in the present invention can be obtained by known methods. Examples include isolation from the heart using known techniques, differentiation induction of pluripotent stem cells, and procurement from companies such as ATCC. Midgut cells can be isolated from the heart using, for example, flow cytometry or mass cytometry using surface antigens (e.g., CDH18) as an indicator, magnetic cell separation, or affinity columns immobilized with the desired antigen. The epicardial cells used in the present invention are preferably obtained by differentiation induction of pluripotent stem cells. The origin of the epicardial cells is not particularly limited, and may be from rodents such as rats, mice, hamsters, and guinea pigs; lagomorphs such as rabbits; ungulates such as pigs, cows, goats, and sheep; carnivores such as dogs and cats; and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. Humans are the preferred origin.

[0020] Known methods can be used to induce differentiation of pluripotent stem cells into epicardial cells. Examples of such methods include those described in Non-Patent Document 4 or Non-Patent Document 5, including the following steps (A) to (C). Therefore, the production method of the present invention may include at least one of the following steps (A) to (C): (A) a step of culturing pluripotent stem cells in suspension to form cell aggregates (embryoid bodies (EBs)); (B) a step of culturing the cell aggregates obtained in step (A) in suspension in the presence of BMP4, activin A, and bFGF to induce cell aggregates containing mesodermal cells; and (C) a step of dispersing the cell aggregates obtained in step (B) and culturing them in the presence of CHIR99021, BMP4, VEGF, and SB431542 to induce differentiation into epicardial cells.

[0021] The epicardial cells obtained in step (C) can also be maintained for a long period of time in a maintenance medium, such as, but not limited to, DMEM containing 10% FBS and 10 μM SB431542.

[0022] The formation of cell aggregates in step (A) is typically carried out by the following method. First, pluripotent stem cells are recovered from subculture and dispersed into single cells or a state close to single cells. Dissociation of pluripotent stem cells is carried out using an appropriate cell dissociation solution. Examples of cell dissociation solutions include EDTA; proteolytic enzymes such as trypsin, collagenase IV, and metalloproteases, which can be used alone or in appropriate combinations. Commercially available cell dissociation solutions include Accutase (MILLIPORE), Dispase (EIDIA), and TrypLE (Invitrogen). The dispersed pluripotent stem cells are suspended in a medium in a low-adhesion culture vessel. The duration of step (A) can be determined appropriately by those skilled in the art and is typically 0.5 to 5 days (particularly, 1 day).

[0023] Step (B) is a step of inducing differentiation of pluripotent stem cells into mesodermal cells, and the medium used in this step preferably contains an extracellular matrix (e.g., 0.5% Matrigel) in addition to the above-mentioned additives. The period of step (B) can be appropriately determined by those skilled in the art and is usually 1 to 6 days, with 2 to 4 days (particularly 2.5 days) being more preferred.

[0024] Step (C) is a step of inducing differentiation of mesodermal cells into epicardial cells, and can typically be performed by dispersing cell aggregates into single cells or a state close to single cells, as in step (A), and then culturing the cells in an adherent culture. The period of step (C) can be appropriately determined by those skilled in the art and is usually 3 to 10 days, with 4 to 7 days (particularly 5.5 days) being more preferable.

[0025] Cardiac organoids can also be induced by, for example, culturing the cell aggregates obtained in step (A) above in suspension in a medium containing CHIR99021, BMP4, and Activin A, then treating the cell aggregates with Wnt-C59 for approximately 2 days, and continuing the suspension culture until the 15th day.

[0026] It is also possible to produce artificial cardiac tissue from cardiac organoids by dispersing cell aggregates into single cells or a state close to this, as in step (A), and then culturing the cells in an adhesive culture medium for artificial cardiac tissue culture.

[0027] The term "pluripotent stem cells" refers to stem cells that can differentiate into various tissues and cells with different morphologies and functions in the body and have the ability to differentiate into cells of any of the three germ layers (endoderm, mesoderm, and ectoderm). Examples of pluripotent stem cells used in the present invention include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), embryonic stem cells derived from cloned embryos obtained by nuclear transfer (ntES cells), multipotent germline stem cells (mGS cells), and embryonic germ stem cells (EG cells). Preferably, iPS cells (more preferably, human iPS cells) are used. When the pluripotent stem cells are ES cells or any cells derived from human embryos, they may be produced by or without the destruction of the embryo. However, from an ethical standpoint, cells produced without the destruction of the embryo are preferred.

[0028] ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman (1981), Nature 292:154-156), and subsequently, ES cell lines were established in humans, monkeys, and other primates (JA Thomson et al. (1998), Science 282:1145-1147; JA Thomson et al. (1995), Proc. Natl. Acad. Sci. USA, 92:7844-7848; JA Thomson et al. (1996), Biol. Reprod., 55:254-259; JA Thomson and VS Marshall (1998), Curr. Top. Dev. Biol., 38:133-165). ES cells can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Alternatively, ES cells can be established using only a single blastomere from an embryo at the cleavage stage prior to the blastocyst stage (Chung Y. et al. (2008), Cell Stem Cell 2: 113-117), or from a developmentally arrested embryo (Zhang X. et al. (2006), Stem Cells 24: 2669-2676).

[0029] nt ES cells are ES cells derived from cloned embryos produced by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (Wakayama T. et al. (2001), Science, 292:740-743; S. Wakayama et al. (2005), Biol. Reprod., 72:932-936; Byrne J. et al. (2007), Nature, 450:497-502). Specifically, nt ES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. To generate nt ES cells, nuclear transfer technology (Cibelli JB et al. (1998), Nature Biotechnol., 16:642-646) is combined with ES cell generation technology (see above) (Wakayama Sayaka et al. (2008), Experimental Medicine, Vol. 26, No. 5 (Special Issue), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, and the egg is then cultured for several hours to reprogram the embryo.

[0030] Examples of ES cell lines that can be used in the present invention include mouse ES cell lines established by, for example, inGenious targeting laboratory, Inc., RIKEN (Riken), etc., and human ES cell lines established by, for example, the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, and Cellartis, Inc. Specific examples of human ES cell lines include CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 strains distributed by ESI Bio, H1 and H9 strains distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 strains distributed by RIKEN.

[0031] iPS cells are cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of iPS cells, including iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al. Cell, (2007) 131: 861-872), Nanog-iPSCs established by selecting using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317), and iPSCs created using a method that does not include c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May;8(5):409-12, Okita K et al. Stem Cells. 31(3):458-66.), etc. can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells created by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells created by Sakurada et al. (JP Patent Publication No. 2008-307007) can also be used.In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7, 795-797), or patent publications (e.g., JP 2008-307007 A, JP 2008-283972 A, US 2008-2336610 A, US 2009-047263 A, WO 2007-069666 A, WO 2008-118220 A, WO 2008-124133 A, WO 2008-151058 A, WO 2009-006930 A, WO 2009-006997 A, WO 2009-007852 A) and known in the art can be used.

[0032] Available induced pluripotent stem cell lines include various iPSC lines established by the NIH, RIKEN, Kyoto University, etc. Examples of human iPSC lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 strains, and Kyoto University's 253G1, 253G4, 1201C1, 1205D1, 1210B2, 1383D2, 1383D6, 1390B1, 1390C1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, and FfI-01s04 strains.

[0033] The induced pluripotent stem cells used in the production method of the present invention may be cells derived from patients with hereditary diseases (e.g., patients with hereditary heart disease). Cells induced to differentiate from pluripotent stem cells derived from patients with hereditary heart disease can serve as disease models that reflect the pathology of the disease, making them suitable for screening therapeutic or preventive drugs for the disease. Alternatively, pluripotent stem cells derived from patients with hereditary heart disease can be genetically repaired by genome editing using the CRISPR-Cas system or the like, and then differentiated into mature epicardial cells or cardiac organoids containing these cells, making it possible to use these cells or organoids as therapeutic agents for heart disease.

[0034] mGS cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to ES cells, these cells can be induced to differentiate into various cell lineages, e.g., when transplanted into mouse blastocysts, chimeric mice can be generated (Kanatsu-Shinohara M. et al. (2003) Biol. Reprod., 69:612-616; Shinohara K. et al. (2004) Cell, 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, germline stem cells can be obtained by repeated passage under similar culture conditions to ES cells (Takebayashi M. et al. (2008) Experimental Medicine, Vol. 26, No. 5 (Supplementary Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0035] EG cells are derived from embryonic primordial germ cells (PGCs) and have pluripotency similar to that of ES cells. They can be established by culturing PGCs in the presence of LIF, bFGF, stem cell factor, and other substances (Matsui Y. et al. (1992), Cell, 70:841-847; JL Resnick et al. (1992), Nature, 359:550-551).

[0036] The species from which the pluripotent stem cells are derived is not particularly limited, and may be cells from, for example, rodents such as rats, mice, hamsters, and guinea pigs, lagomorphs such as rabbits, ungulates such as pigs, cows, goats, and sheep, carnivores such as dogs and cats, and primates such as humans, monkeys, rhesus monkeys, marmosets, orangutans, and chimpanzees. The preferred species is human.

[0037] mTOR exists in cells as two complexes: mTORC1 (mTOR Complex 1) and mTORC2 (mTOR Complex 2). mTORC1 contains raptor, and mTORC2 contains rictor. These complexes are differentially regulated and have different substrate specificities and rapamycin sensitivity. For example, mTORC1 phosphorylates S6 kinase (S6K) and 4EBP1, thereby promoting increased protein translation and ribosome synthesis, thereby promoting cell growth and cell cycle progression. S6K also acts in a feedback pathway to attenuate PI3K / Akt activity. mTORC2 is thought to regulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases, such as Akt.

[0038] The mTORC2 inhibitor used in the present invention is not particularly limited as long as it can inhibit the phosphorylation of Akt by mTORC2 (typically, phosphorylation at the S473 site). Preferably, the mTORC2 inhibitor of the present invention also has mTORC1 inhibitory activity. Specific examples of mTORC2 inhibitors used in the present invention include small molecule compounds such as Torin1, Torin2, omipalisib (GSK2126458), KU-0063794, OSI-027, XL388, Palomid 529 (P529), WYE-354, torkinib (PP242), sapanisertib (TAK-228), WYE-687, AZD8055, and vistusertib (AZD2014), with Torin1 being preferred. Sapanisertib, WYE-687, AZD8055, and vistusertib can also be suitably used. Also preferred are antibodies, peptides, or aptamers that inhibit the phosphorylation of Akt in mTORC2. Only one type of mTORC2 inhibitor may be used, or multiple types may be used. Furthermore, the mTORC2 inhibitor of the present invention may be used in combination with other mTORC1 inhibitors (especially when the mTORC2 inhibitor used is a specific inhibitor of mTORC2). Thus, in one embodiment, the method of the present invention comprises a medium containing an mTORC1 inhibitor, and the mTORC1 inhibitor may be the same as the mTORC2 inhibitor used in the present invention (when the mTORC2 inhibitor also has mTORC1 inhibitory activity), or may be a different mTORC1 inhibitor.

[0039] When each substance used in the present invention is a low molecular weight compound, the compound includes not only the free form but also its pharmacologically acceptable salt and hydrate.Pharmacologically acceptable salts vary depending on the type of compound, but include, for example, inorganic base salts such as alkali metal salts (sodium salt, potassium salt, etc.), alkaline earth metal salts (calcium salt, magnesium salt, etc.), aluminum salt, ammonium salt, etc., and organic base salts such as trimethylamine, triethylamine, pyridine, picoline, ethanolamine, diethanolamine, triethanolamine, dicyclohexylamine, N,N'-dibenzylethylenediamine, etc., base addition salts, or inorganic acid salts such as hydrochloride, hydrobromide, sulfate, hydroiodide, nitrate, phosphate, etc., and organic acid salts such as citrate, oxalate, acetate, formate, propionate, benzoate, trifluoroacetate, maleate, tartrate, methanesulfonate, benzenesulfonate, paratoluenesulfonate, etc., acid addition salts.

[0040] When the substances used in the present invention are proteins or peptides, their origin is not particularly limited, but is preferably mammalian (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.). Furthermore, the proteins or peptides used in the production methods of the present invention include not only wild-type proteins or peptides but also their mutants having similar functions. Examples of mutants include proteins or peptides that have a high degree of identity (e.g., 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more) to the amino acid sequence of a specific wild-type protein or peptide.

[0041] Substances capable of suppressing the expression of any of the proteins that make up mTORC2 (hereinafter sometimes referred to as "mTORC2 expression inhibitors") are also suitable as mTORC2 inhibitors for use in the present invention. Proteins that make up mTORC2 include mTOR, rictor, mSIN1, mLST8, deptor, and procto. It is sufficient to suppress the expression of at least one of these proteins. Preferred targets are mTOR, deptor, and mLST8, which are also targets of mTORC1, with mTOR being more preferred.

[0042] As used herein, unless otherwise specified, the term "gene expression" is used to mean at least "production of a transcription product," but preferably also "production of a functional protein." Therefore, suppression of gene expression may include not only a decrease in the amount of a transcription product transcribed from the gene in a cell upon contact with an expression inhibitor of the target gene, but also a decrease in the amount of a functional protein encoded by the gene in a cell. Furthermore, as used herein, transcription products typically include mRNA and pre-mRNA, but are preferably mRNA.

[0043] Furthermore, when the production of mRNA encoded by the gene is detected at least by the method (RT-qPCR) described in the Examples below, the gene can be said to be expressed or positive. On the other hand, when the production of mRNA encoded by the gene is not detected (i.e., below the detection limit) by the method (RT-qPCR) described in the Examples below, or when it is at background levels, the gene can be said to be not expressed or negative.

[0044] The mTORC2 expression inhibitor is not limited to any substance capable of suppressing the expression of a target protein in epicardial cells, but is preferably a nucleic acid. Examples of such nucleic acids include antisense nucleic acids (e.g., antisense oligonucleotides (ASOs) and the like) (including nucleic acids encoding such nucleic acids), siRNA (including nucleic acids encoding such siRNA), heteroduplex oligonucleotides (HDO), shRNA (including nucleic acids encoding such shRNA), and miRNA (microRNA) (including nucleic acids encoding such miRNA). Hereinafter, when the mTORC2 expression inhibitor is a nucleic acid, such a nucleic acid may be referred to as an "mTORC2 expression-inhibiting nucleic acid." A single mTORC2 expression inhibitor may be used, or multiple mTORC2 expression inhibitors may be used. Furthermore, the mTORC2 expression inhibitor may be used in combination with other mTORC1 expression inhibitors. The mTORC1 expression inhibitor may be a substance capable of suppressing the expression of any protein that constitutes mTORC1, such as mTOR, raptor, and PRAS40.

[0045] When the protein targeted by the mTORC2 expression-inhibiting nucleic acid has multiple isoforms, it is typically capable of inhibiting the expression of a transcript encoding the full-length protein, but other isoforms may also be used as long as they are capable of inhibiting Akt phosphorylation. The base sequence targeted by the mTORC2 expression-inhibiting nucleic acid (target sequence) can be appropriately designed, for example, based on information from a known database (e.g., the NCBI database). The length of the target sequence is not particularly limited as long as the expression-inhibiting nucleic acid can specifically recognize and bind to it, but is preferably 12 nucleotides or longer, more preferably 15 nucleotides or longer, and even more preferably 17 nucleotides or longer. The upper limit of the length is also not particularly limited, but is, for example, 30 nucleotides or shorter, preferably 25 nucleotides or shorter, and more preferably 22 nucleotides or shorter.

[0046] As used herein, "complementary" refers to a relationship in which nucleic acid bases can form so-called Watson-Crick base pairs (natural base pairs) or non-Watson-Crick base pairs (Hoogsteen base pairs, wobble base pairs, etc.) through hydrogen bonds. Therefore, the term "complementary sequence" refers not only to sequences that are completely complementary to a target RNA sequence or target DNA sequence (i.e., hybridize without mismatches), but also to sequences containing one to several (e.g., 2, 3, 4, 5, or more) mismatches, as long as they can hybridize with the target sequence under stringent conditions or under the physiological conditions of mammalian cells. For example, the term "complementary sequence" refers to a sequence that has 80% or more (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more), and most preferably 100%, identity with a sequence that is completely complementary to a target RNA sequence or target DNA sequence.

[0047] Antisense nucleic acids are single-stranded nucleic acids containing a sequence complementary to a target RNA sequence. The antisense nucleic acid forms a double-stranded region with the target RNA sequence due to its complementary sequence, and this double-stranded region is cleaved by ribonuclease H (RNase H), thereby suppressing KLHL32 gene expression. The length of the antisense nucleic acid is not particularly limited, but is typically 10 to 30 nucleotides, more preferably 13 to 30 nucleotides.

[0048] siRNA is a double-stranded RNA consisting of an RNA having a sequence complementary to a target RNA sequence (i.e., an antisense strand) and its complementary strand. Also, a preferred embodiment of siRNA is a single-stranded RNA in which a sequence complementary to the target RNA sequence (a first sequence) is linked to its complementary sequence (a second sequence) via a hairpin loop, and the first sequence forms a double-stranded structure with the second sequence by adopting a hairpin loop structure (small hairpin RNA: shRNA). Furthermore, shRNA may be in the form of a nucleic acid (e.g., an expression vector) encoding the shRNA.

[0049] HDO refers to a double-stranded nucleic acid composed of a DNA backbone (i.e., an antisense strand) and an RNA (cRNA) strand complementary to the DNA. When HDO is taken up into cells, the cRNA strand is cleaved by RNase H inside the cell. The HDO backbone DNA becomes a single-stranded antisense nucleic acid through cleavage of the cRNA strand, and it is presumed that the antisense effect is exerted by binding to the target RNA, where RNase H again cleaves the target RNA.

[0050] The length of each nucleic acid strand of the double-stranded nucleic acid is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides, preferably 15 to 30 nucleotides, more preferably 20 to 27 nucleotides.

[0051] As used herein, "miRNA" refers to a single-stranded or double-stranded RNA (e.g., miRNA / miRNA) that does not cleave target RNA like siRNA, but recognizes the 3' untranslated region (UTR) of target RNA and controls translation. * miRNA refers to endogenous non-coding RNA (ncRNA) of about 20 to 25 bases that is originally encoded on the genome, and pri-miRNA is expressed from the miRNA gene, followed by the generation of pre-miRNA, which then produces mature-miRNA. The mature-miRNA is then incorporated into RISC to produce single-stranded miRNA. The miRNA used in the present invention may be in the form of pri-miRNA or pre-miRNA, and may be in the form of mature-miRNA (miRNA / miRNA * ) or even in the form of single-stranded RNA. Furthermore, miRNA may be in the form of a nucleic acid (e.g., an expression vector) that encodes the miRNA. The length of the miRNA (when the miRNA is double-stranded, the length of each nucleic acid strand) is not particularly limited as long as it can exert an antisense effect, but is, for example, 10 to 50 nucleotides long, preferably 15 to 30 nucleotides long, and more preferably 20 to 27 nucleotides long.

[0052] mTORC2 expression-inhibiting nucleic acids can be obtained by chemical synthesis using conventional methods or by production using recombinant DNA technology. Alternatively, commercially available nucleic acids can be used. For example, nucleic acids can be designed based on the target RNA sequence using commercially available software (e.g., RNAiDesigner; Invitrogen). In the case of double-stranded nucleic acids, the sense and antisense strands of the target RNA sequence can be synthesized using a commercially available automated DNA / RNA synthesizer (e.g., Applied Biosystems, Beckman, etc.), denatured in an appropriate annealing buffer at about 90°C to about 95°C for about 1 minute, and then annealed at about 30°C to about 70°C for about 1 to about 8 hours. HDO can also be prepared, for example, by the method described in WO2013 / 089283.

[0053] The basal medium used in the present invention is not particularly limited, but includes RPMI-1640 medium, EagleMEM (EMEM), Dulbecco's modified MEM, Glasgow's MEM (GMEM), α-MEM, 199 medium, IMDM, DMEM, Hybridoma serum-free medium, KnockOut TMDMEM, Advanced TM medium (e.g. Advanced MEM, Advanced RPMI, Advanced DMEM / F-12), Ham's Medium F-12, Ham's Medium F-10, Ham's Medium F12K, DMEM / F-12, ATCC-CRCM30, DM-160, DM-201, BME, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, MCDB105, MCDB107, MCDB131, MCDB153, MCDB201, NCTC109, NCTC135, Waymouth's Medium (e.g. Waymouth's MB752 / 1), CMRL medium (e.g. CMRL-1066), Williams' medium E, Brinster's BMOC-3 Medium, Essential 8(E8) Medium, Essential 8 Flex Medium, StemPro 34, MesenPRO RS (Thermo Fisher Scientific), ReproFF2, Primate ES Cell Medium, ReproStem (ReproCELL Co., Ltd.), ProculAD (Rohto Pharmaceutical Co., Ltd.), MSCBM-CD, MSCGM-CD (Lonza), EX-CELL302 medium (SAFC) or EX-CELL-CD-CHO (SAFC), ReproMed TM Examples of suitable media include, but are not limited to, iPSC Medium (ReproCELL Corporation) and mixtures thereof. In the method of the present invention, DMEM is preferred. Furthermore, StemPro 34 is preferred for inducing differentiation of pluripotent stem cells into epicardial cells.

[0054] In particular, for culture under feeder-free and xeno-free conditions, StemFit (registered trademark) AK02 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) AK03 medium (Ajinomoto Co., Inc.), StemFit (registered trademark) Basic03 medium, CTS (registered trademark) KnockOut SR XenoFree Medium (Gibco), mTeSR1 medium, TeSR1 medium (Stem Cell Technologies), Iscove's modified Dulbecco's medium (GE Healthcare), Improved MEM (Thermo Fisher Scientific), and the like can also be used.

[0055] If necessary, the medium may contain serum, such as fetal bovine serum, horse serum, or human serum, or may contain one or more serum substitutes such as Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), albumin, transferrin, apotransferrin, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, or 3'-thiolglycerol, or may also contain one or more substances such as lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvate, buffers, inorganic salts, selenate, progesterone, and putrescine.

[0056] As used herein, "suspension culture" refers to culture performed under conditions that maintain cells or cell clumps suspended in a culture medium, i.e., culture under conditions that do not allow the formation of strong cell-substratum junctions between the cells or cell clumps and the culture vessel. Furthermore, when performing suspension culture, cells typically remain in the form of cell clumps before and after suspension culture. As used herein, "adhesion culture" refers to culture under conditions that allow the formation of strong cell-substratum junctions between the cells and the cultureware, etc.

[0057] The culture vessel used for suspension culture is not particularly limited as long as it is capable of "suspension culture," and can be appropriately determined by one skilled in the art. Examples of such culture vessels include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, micropores, multi-plates, multi-well plates, chamber slides, Petri dishes, tubes, trays, culture bags, and roller bottles. Furthermore, bioreactors are an example of vessels for suspension culture. These culture vessels are preferably non-cell-adhesive to enable suspension culture. Examples of non-cell-adhesive culture vessels include those whose surfaces have not been artificially treated (e.g., coated with extracellular matrix) to improve cell adhesion. The well bottom shape of these culture vessels is not particularly limited, and examples include flat-bottomed, U-shaped, and V-shaped vessels.

[0058] Culture vessels used in adhesion culture include those whose surfaces have been artificially treated to improve cell adhesion (e.g., coating with basement membrane preparations, extracellular matrices such as fibronectin, laminin or fragments thereof, entactin, collagen, gelatin, synthemax, vitronectin, etc., or polymers such as polylysine and polyornithine, or surface treatments such as positive charge treatment). Commercially available basement membrane preparations include, for example, Matrigel and Geltrex.

[0059] Alternatively, from the viewpoint of xeno-free, laminin or a fragment thereof is also preferred. Examples of laminin or a fragment thereof include laminin-111 or a fragment thereof comprising its E8 region, laminin-211 or a fragment thereof comprising its E8 region (e.g., iMatrix-211), laminin-121 or a fragment thereof comprising its E8 region, laminin-221 or a fragment thereof comprising its E8 region, laminin-332 or a fragment thereof comprising its E8 region, laminin-3A11 or a fragment thereof comprising its E8 region, laminin-411 or a fragment thereof comprising its E8 region (e.g., iMatrix-411), laminin-421 or a fragment thereof comprising its E8 region, and laminin-511 or a fragment thereof comprising its E8 region (e.g., iMatrix-511, iMatrix-511). silk), laminin-521 or a fragment thereof containing its E8 region, laminin-213 or a fragment thereof containing its E8 region, laminin-423 or a fragment thereof containing its E8 region, laminin-523 or a fragment thereof containing its E8 region, laminin-212 / 222 or a fragment thereof containing its E8 region, and laminin-522 or a fragment thereof containing its E8 region.

[0060] In at least one of the steps of the production method of the present invention and other steps (e.g., steps (A) to (C) above), cells may be cultured under feeder-free conditions and / or xeno-free conditions. In the production method of the present invention, all steps may be performed under feeder-free and xeno-free conditions. As used herein, "feeder-free" refers to a medium or culture conditions that do not contain other cell types (i.e., feeder cells) that play a supporting role and are used to establish culture conditions for the cells to be cultured. Furthermore, "xeno-free" refers to a medium or culture conditions that do not contain components derived from organisms other than the biological species of the cells to be cultured.

[0061] The culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is carried out in an atmosphere of CO2-containing air, with the CO2 concentration preferably being about 2 to 5%.

[0062] The seeding density of cells is not particularly limited as long as the cells can grow. Typically, the seeding density is 1.0 × 10 2 ~1.0×10 7 cells / cm2 , preferably 1.0 x 10 3 ~1.0×10 6 cells / cm 2 , more preferably 1.0 × 10 4 ~1.0×10 5 cells / cm 2 is.

[0063] The concentration of the mTORC2 inhibitor used in step (1) of the production method of the present invention in the medium is typically 1 nM to 10 μM, preferably 10 nM to 2 μM, and more preferably 50 nM to 600 nM (particularly 200 nM) when Torin1 is used. When an mTORC2 inhibitor other than Torin1 is used, the concentration of mTORC2 in the medium is appropriately selected.

[0064] The culture period in the steps of the production method of the present invention is not particularly limited, but is typically 1 to 20 days, preferably 3 to 15 days, and more preferably 5 to 10 days (particularly 7 days).

[0065] The method of the present invention may include a step of isolating mature epicardial cells obtained by the method of the present invention. The isolated cells may be cryopreserved using a cell cryopreservation solution. The collected cells may be counted using a cell counter, or may be labeled with an antibody against a cell surface marker and selected by flow cytometry, mass cytometry, magnetic cell sorting, or the like.

[0066] In yet another aspect, the present invention provides a method for maturing epicardial cells, comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor. The definitions and specific examples of epicardial cells and mTORC2, as well as the culture method, are all incorporated herein by reference in the description of the production method of the present invention.

[0067] 2. Mature epicardial cells, cardiac organoids comprising said cells and their uses By the method of the present invention, mature epicardial cells or cardiac organoids such as cardiac tissue comprising said cells can be obtained.Therefore, in another aspect of the present invention, also provide the mature epicardial cells or cardiac organoids comprising said cells or cardiac tissue (hereinafter sometimes referred to as "epicardial cells of the present invention") obtained by the method of the present invention ("obtained" can be appropriately read as "obtained").

[0068] Furthermore, in the Examples described below, it was demonstrated that the paracrine effect of the epicardial cells of the present invention can promote the maturation of cardiomyocytes. Therefore, in yet another aspect, a method for producing mature cardiomyocytes, or a method for maturing cardiomyocytes, comprising the step of co-culturing the epicardial cells of the present invention with cardiomyocytes, is provided. In one embodiment, the cardiomyocytes used in such a method are cardiomyocytes contained in cell aggregates (typically, embryoid bodies). These methods may also comprise the step of isolating mature cardiomyocytes using flow cytometry or the like. Hereinafter, cardiomyocytes obtained by the above-described method for producing mature cardiomyocytes may be referred to as "cardiomyocytes of the present invention." Cardiomyocytes are broadly divided into immature (i.e., "fetal") cardiomyocytes and mature (i.e., "adult") cardiomyocytes; however, unless otherwise specified, "cardiomyocytes" refers to immature cardiomyocytes.

[0069] Cardiomyocytes are cells that constitute the myocardium of animals. As used herein, immature and mature cardiomyocytes refer to cells that repeatedly contract and relax (exhibit pulsatile activity). Immature and mature cardiomyocytes typically express at least one cardiomyocyte marker selected from the group consisting of cardiac troponin T (cTNT), αMHC (α myosin heavy chain, MYH6), and βMHC (MYH7). Immature cardiomyocytes refer to cells that express TNNI1. Furthermore, "mature cardiomyocytes" refer to cells that express TNNI3 and in which the TNNI1 expression level has decreased or disappeared and the TNNI3 expression level has increased compared to cardiomyocytes before the step of co-culturing with the epicardial cells of the present invention, preferably cells that do not express TNNI1 but express TNNI3.

[0070] The cardiomyocytes used in the present invention can be obtained by known methods. Examples include isolation from the heart using known techniques, differentiation induction of pluripotent stem cells, and procurement from companies such as ATCC. Midgut cells can be isolated from the heart using, for example, flow cytometry or mass cytometry using a surface antigen (e.g., CD82) as an indicator, magnetic cell separation, or affinity columns immobilized with the desired antigen. The epicardial cells used in the present invention are preferably obtained by differentiation induction of pluripotent stem cells.

[0071] To induce differentiation into epicardial cells, cell aggregates containing mesodermal cells obtained in step (B) above can be cultured in suspension in a medium containing epicardial cell-inducing factors to induce cell aggregates containing epicardial cells. This step can be performed, for example, by culturing cell aggregates containing mesodermal cells in suspension in the presence of VEGF, IWP-3, Dorsomorphin, and SB431542. The duration of this step can be determined appropriately by those skilled in the art, and is typically 6 to 25 days, with 10 to 20 days being more preferred. For all other culture methods, the same description of the production method of the present invention is incorporated herein by reference.

[0072] The period for co-culturing the epicardial cells of the present invention with cardiomyocytes is not particularly limited, but is typically 5 to 20 days, preferably 6 to 15 days, and more preferably 7 to 14 days (particularly 10 days). The culture may be suspension culture or adherent culture, with suspension culture being preferred. The co-culture method is not particularly limited as long as the epicardial cells and cardiomyocytes are cultured in the same medium. For all other culture methods, the same description as for the production method of the present invention is applicable.

[0073] Because the epicardial cells and cardiomyocytes of the present invention can be used in medical treatments such as regenerative medicine, in another aspect, a transplantation therapeutic agent (hereinafter, sometimes referred to as the "transplantation therapeutic agent of the present invention") containing the epicardial cells and / or cardiomyocytes of the present invention is provided. The present invention also encompasses a method for treating or preventing heart disease, in which an effective amount of the epicardial cells and / or cardiomyocytes of the present invention is administered or transplanted into a mammal (e.g., human, mouse, rat, monkey, cow, horse, pig, dog, etc.) that is the target of treatment or prevention. Unless otherwise specified, in this specification, a therapeutic or preventive agent (or therapeutic or preventive method) for a disease also encompasses a pharmaceutical agent (or method) that can both treat and prevent the disease.

[0074] Examples of cardiac diseases include heart failure, chronic heart failure, severe heart failure, ischemic heart disease, myocardial infarction, acute myocardial infarction, chronic myocardial infarction, cardiomyopathy, ischemic cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, dilated cardiomyopathy, amyloid cardiomyopathy, alcoholic cardiomyopathy, viral cardiomyopathy, stress-induced cardiomyopathy, myocarditis, congenital disorders, genetic disorders, left ventricular noncompaction, mitral valve prolapse, arrhythmogenic right ventricular dysplasia, various valvular stenosis diseases, various valvular regurgitation diseases, etc. The epicardial cells and cardiomyocytes of the present invention exhibit therapeutic or preventive effects against cardiac diseases when transplanted into the affected area, for example.

[0075] The transplantation therapy agent of the present invention can be administered or transplanted into the body of a subject in need thereof. The cells or organoids to be transplanted should be administered in a therapeutically or prophylactically effective amount, which may vary depending on factors such as the age, weight, size of the transplant site, and severity of the disease of the transplant subject. The number of cells is not particularly limited, but for example, 10 × 104 Cell ~10×10 11 It can be as small as a cell.

[0076] When the epicardial cells and / or cardiomyocytes of the present invention are used as transplantation therapy agents, it is desirable to use cells derived from iPS cells established from somatic cells with the same or substantially the same HLA genotype as the recipient individual, in order to avoid rejection. Here, "substantially the same" means that the HLA genotype is identical to that of the transplanted cells to an extent that immune responses can be suppressed with immunosuppressants, e.g., somatic cells with an HLA type that matches the three HLA loci (HLA-A, HLA-B, and HLA-DR) or the four HLA loci (HLA-C). If sufficient cells cannot be obtained due to age, constitution, or other reasons, they can be transplanted in a state that avoids rejection by embedding them in capsules or porous containers made of polyethylene glycol or silicone.

[0077] The epicardial cells and cardiomyocytes of the present invention are prepared as parenteral preparations such as injections, suspensions, and infusions by mixing with a pharmaceutically acceptable carrier according to conventional methods. Therefore, in one embodiment, a method for producing a transplantation therapy agent is also provided, which includes a step of formulating the epicardial cells and / or cardiomyocytes of the present invention. Such a method may include a step of preparing the epicardial cells and / or cardiomyocytes of the present invention. Furthermore, it may also include a step of preserving the epicardial cells and / or cardiomyocytes of the present invention.

[0078] Pharmaceutically acceptable carriers that can be contained in such parenteral formulations include aqueous solutions for injection, such as physiological saline, isotonic solutions containing glucose or other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.), etc. The epicardial cells and cardiomyocytes of the present invention may be formulated with, for example, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0079] The transplantation therapy agent of the present invention is provided in a cryopreserved state under conditions typically used for cryopreserving cells and can be thawed immediately before use. In this case, it may further contain serum or a serum substitute, an organic solvent (e.g., DMSO), etc. In this case, the concentration of the serum or serum substitute is not particularly limited, but may be about 1 to about 30% (v / v), preferably about 5 to about 20% (v / v). The concentration of the organic solvent is not particularly limited, but may be 0 to about 50% (v / v), preferably about 5 to about 20% (v / v).

[0080] The epicardial cells and cardiomyocytes of the present invention can also be used in methods for screening candidate drugs that are useful for treating or preventing heart disease. Thus, in yet another aspect of the present invention, a method for screening therapeutic or preventive drugs for disease is provided, comprising culturing the epicardial cells and / or cardiomyocytes of the present invention in the presence or absence of a test substance. Such cardiac diseases include those similar to those targeted for treatment or prevention by the transplantation therapy agents of the present invention. Using cells derived from patients with heart disease as the epicardial cells and / or cardiomyocytes of the present invention used for screening may enable more accurate screening.

[0081] Test substances used in the present invention include, for example, cell extracts, cell culture supernatants, microbial fermentation products, extracts derived from marine organisms, plant extracts, purified or crude proteins, peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.

[0082] The test substance can also be obtained using any of the many approaches to combinatorial library technology known in the art, including (1) biological libraries, (2) synthetic library technology using deconvolution, (3) "one-bead one-compound" library technology, and (4) synthetic library technology using affinity chromatography selection. While the biological library technology using affinity chromatography selection is limited to peptide libraries, the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des. 12:145-67). Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90:6909-13; Erb et al. (1994) Proc. Natl. Acad. Sci. USA 91:11422-6; Zuckermann et al. (1994) J. Med. Chem. 37:2678-85; Cho et al. (1993) Science 261:1303-5; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061; Gallop et al. (1994) J. Med. Chem. 37:1233-51).Compound libraries can be stored in solution (see Houghten (1992) Bio / Techniques 13:412-21) or on beads (Lam (1991) Nature 354:82-4), chips (Fodor (1993) Nature 364:555-6), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al. (1992) Proc. Natl. Acad. Sci. USA 89:1865-9), or phage (Scott and Smith (1990) Science 249:386-90; Devlin (1990) Science 249:404-6; Cwirla et al. (1990) Proc. Natl. Acad. Sci. USA 87:6378-82; Felici (1991) J. Mol. Biol. 222:301-10; U.S. Patent Application No. 2002103360).

[0083] The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples in any way.

[0084] Materials and Methods: Cell Lines and Culture Conditions. 201B, a hiPSC cell line reprogrammed using Yamanaka factors via retroviral methods, was propagated in ReproCell ES solution containing 4 ng / ml bFGF, supported by an irradiated MEF feeder layer. CTK was administered to the cells to remove the feeder layer. The feeder-free 1390C1 and T1 / T3 reporter (PMID: 34155205) cell lines were maintained in complete StemFit® AK02N solution on dishes coated with iMatrix-511 (obtained from Matrixome). The MEC1 mouse epicardial cell line (Merck, SCC187) was obtained and grown according to the manufacturer's guidelines. Routine testing for mycoplasma was performed to ensure the absence of contamination.

[0085] <Cell Transfection> For gene silencing tests, Silencer® Select siRNA (obtained from ThermoFisher), specifically siYBX3 (s107202) and siMAFF (s115936), were prepared at 10 μmol concentrations. To silence cells, 10 μl of the siRNA solution was mixed with 50 μl of Opt-MEM, followed by 10 μl of RNAiMax® to create a silencing mixture. After allowing to stand at room temperature for 10 minutes, the mixture was slowly added to a 6-well plate containing approximately 20,000–30,000 cells in 2 ml of maintenance medium, which had been stored the previous day. The medium was refreshed one day later, and cells were cultured according to standard protocols. For immunocytochemistry tests, 260 μl of the silencing mixture was slowly added to a 12-well plate containing approximately 5,000 cells seeded the previous day in 1 ml of maintenance medium.

[0086] Cell Culture and Human Cardiac Organoid Induction: Feeder-independent hiPSCs (1390C1) were cultured on iMatrix-511 (Nippi)-coated dishes using StemFit® AK02N medium (Reprocell). The process for generating human cardiac organoids from PSCs followed established protocols. Briefly, using Accumax, cells were suspended in Essential 8 Flex medium (Gibco) containing 10 μM of the ROCK inhibitor Y-27632. To generate embryoid bodies (EBs), 10,000 cells were dispersed into hemispherical, low-attachment HEMA-coated 96-well plates in a total volume of 100 μl per well on day -2. Fresh Essential 8 Flex medium was added the following day. On the initial day (day 0), Essential 8 Flex medium was discarded, and differentiation was initiated using RPMI 1640 / B-27 medium without insulin (Gibco) and supplemented with CHIR99021, BMP4, and Activin A. On day 1, the existing medium was replaced with fresh RPMI 1640 / B-27 without insulin. By day 2, the solution was transferred to RPMI 1640 / B-27 without insulin and containing Wnt-C59. On days 4 and 6, the medium was refreshed with insulin-free RPMI 1640 / B-27 and standard RPMI 1640 / B-27, respectively. On day 7, organoids were briefly treated (1 hour) with CHIR99021 in RPMI 1640 / B-27. From day 7 onward, the medium was changed every other day until day 15 was reached.

[0087] Differentiation of hiPSCs into Epicardial Cells Epicardial cells were differentiated as previously reported (PMID: 35110584). hiPSCs were converted into a single-cell mixture using Accutase. These cells were transferred to HEMA-coated plates designed for low attachment (ranging from 6,000 to 8,000 cells per 96-well plate) and allowed to form embryoid bodies (EBs). The differentiation solution contained fully enriched StemPro®-34 medium, further enriched with components such as 50 μg / ml ascorbic acid, 2 mM L-glutamine, 0.4 μM monothioglycerol, and 150 mg / ml transferrin. Differentiation was initiated by adding 0.5% Matrigel, 10 μM Y-27632, and 2 ng / ml human recombinant (hr) BMP4 to the medium. After the first 24 hours, EBs were immersed in differentiation solution containing 10 ng / ml hrBMP4, 2 ng / ml activin A, and 5 ng / ml hrbFGF. After 84 hours, EBs were collected, digested using Accutase, and plated (0.3 x 10 cells) on 0.1% gelatin-coated plates. 5 cells / cm 2 The hiPSCs were then repopulated (at a density of 100 μM). This new environment contained differentiation medium containing 3 mM CHIR99021, 30 ng / ml hrBMP4, 5 ng / ml hrVEGF, and 10 μM SB431542. From day 7, the resulting hiPSC-derived epicardial-like (EPI) cells were maintained in a maintenance solution, essentially DMEM supplemented with 10% FBS and 10 μM SB431542. When subculturing EPI cells, they were removed using a 5-minute Accutase treatment and then repopulated as described previously.

[0088] <Differentiation of hiPSCs into Cardiac Cells> Ventricular cardiomyocytes were differentiated as follows. hiPSCs were dissociated into individual cells using a half-concentrated solution of TrypLE select (available from Thermo Fisher Scientific) mixed with 0.5 mM EDTA. These cells were then added to 1.5 ml / well of StemPro-34 medium (Invitrogen). This medium contains additional components such as L-glutamine, MTG, ascorbic acid (AA), transferrin, ROCK inhibitor (Y-27632), Matrigel (available from Corning), and BMP4. 2 x 10 cells were cultured in the same medium. 6 The cells were cultured at a concentration of 1000 cells / well for the purpose of embryoid body (EB) generation. The next day, an additional 1.5 ml of the same StemPro-34 medium containing more bFGF, activin A, and BMP4 was added to the wells. By day 3, the EBs were washed with IMDM (Invitrogen version) and transferred to another batch of enriched StemPro-34 medium, also containing VEGF, IWP-3, Dorsomorphin, and SB431542. On day 6, the medium was replaced with the same StemPro-34 mix, and this medium was subsequently refreshed every 2–3 days. The plates were initially maintained in a hypoxic setting (5% O2) for 10 days, after which they were transferred to a normal oxygen atmosphere.

[0089] <Proliferation assay> 4.5 x 10 cells in a 6 cm diameter dish 3Growth timelines were plotted for both untreated cells (control) and Torin1-treated cells (Torin1) by seeding 1000 cells per well. After 1 day, the old medium was replaced with fresh medium, and the cells were set as a baseline for proliferation comparison (designated as day 0). Proliferation was assessed every 2 days by fixing the cells with 2% PA and staining them with 1% CV (crystal violet, Sigma; C6158-50G). After thoroughly washing the cells, crystal violet was dissolved in 20% acetic acid (Sigma), and the absorbance at 595 nm was measured to estimate relative cell number (using a PerkinElmer EnVision 2104 Multilabel Reader). Relative numbers indicate cell proliferation in the designated medium. Zero percent (0%) indicates the initial growth benchmark set on day 0.

[0090] Isolation of Mouse Fetal Heart and Whole Heart Lysates. E12, E14, and E18 embryos were collected in preheated PBS and carefully removed surrounding foreign material using tweezers. The embryo's head was separated, and the anterior thoracic cavity was torn open to access the heart. The fetal heart was then mechanically isolated and placed in a separate dish filled with preheated PBS. To prepare cardiac protein lysates, the hearts were first minced and then exposed to trypsin at 37°C for 20 minutes. The cell mixture was again vortexed and treated at 37°C for another 20 minutes. After centrifugation at 300g for 5 minutes, the clear liquid (supernatant) was collected.

[0091] Ex vivo epicardial explants: Hearts harvested at E12 or E14 were placed on 0.1% gelatin-coated dishes in DMEM (low glucose) enriched with 15% FBS. Growth cultures began to appear within 24 hours after placement. After 72 hours, the heart slices were carefully lifted using tweezers. The next day, the explant cells were trypsinized and then cultured in DMEM (low glucose) containing 10% FBS, further enriched with 10 μM SB431542.

[0092] Cytotoxicity Assay in hiPSC-derived Epicardial Cells: For all experiments, Torin1 was freshly prepared in DMSO. Human iPSC-derived epicardial cells were individually plated in 96-well plates at a density of 5,000 cells per well. To evaluate treatment effects, decreasing concentrations of Torin1 were administered to rapidly dividing cells at a consistent 1:3 ratio. One day after seeding, the efficacy of all treatments was evaluated with decreasing doses of Torin1 alone. After 96 hours, cell viability was measured using crystal violet. To measure cytotoxic effects, absorbance at 595 nm was recorded using a microplate reader. Based on these results, IC values ​​were calculated using GraphPad Prism 7 software. 50 values ​​were calculated.

[0093] Cardiotoxicity Assay in iPSC-Derived Human Cardiac Organoids (hHO) For all tests, Torin1 was freshly mixed with DMSO. Cardiac organoids (hHO) were differentiated and individually plated in 96-well low-attachment plates. Treatment effects were assessed after administering decreasing concentrations of Torin1 at a fixed 1:3 ratio to organoids at day 15 of growth. Each treatment used a decreasing dose of Torin1 as monotherapy. After 4 days (or 96 hours), the proliferation capacity of organoids was assessed using an MTT assay. Cytotoxicity levels were assessed by measuring absorbance at 595 nm using a dedicated microplate reader, followed by IC values ​​using GraphPad Prism 7 software. 50 values ​​were calculated.

[0094] <Detection of SA-βGal activity> β-galactosidase activity was measured using Senescence β-Galactosidase Staining Kit (Cell Signaling TECHNOLOGY, #9860) according to the manufacturer's protocol.

[0095] Immunocytochemistry (ICC) Cells were fixed with 4% PFA for 15 minutes and stored in PBS at 4°C. Blocking was performed for 30–45 minutes with a solution containing 1% BSA, 0.5% Triton X-100, and 0.1 M glycine in PBS. After three washes with PBS, primary antibodies were added (excluding glycine): anti-TBX18 (1:200, ab15262, Abcam); anti-WT1 (1:200, ab89901, Abcam); anti-ZO-1 (1:200, 33-9100, Invitrogen); anti-pH3 (1:200, C36B11, CST); and anti-Ki67 (1:200, BioLegend, 350502) in the same solution overnight at 4°C. The next day, after washing with PBS, the secondary antibody, donkey anti-rabbit Alexa Fluro 594 (1:1000, A21207, Invitrogen), was introduced in 1% BSA-PBS for 2 hours at room temperature. After three PBS washes, the cells were counterstained with Hoechst 33342 at a ratio of 1:10,000. Imaging was performed using a Keyence microscope.

[0096] Immunofluorescence staining of hHO cells was performed as previously described. hHO cells were stabilized using 4% PFA solution at room temperature for 45–60 minutes, followed by overnight incubation at 4°C in a solution containing 10% normal donkey serum (Abcam), 0.5% Triton X-100 (Nacalai Tesque), and 0.5% BSA (Nacalai Tesque). Subsequently, hHO cells were stained with primary and secondary antibodies in a PBS mixture containing 1% normal donkey serum, 0.5% Triton X-100, and 0.5% BSA. Nuclear staining was performed using Hoechst 33342 at a concentration of 1:1000 before mounting. Fluorescence observation was performed using an Olympus FV3000 confocal laser scanning microscope. Primary antibodies included anti-cardiac troponin T (1:200, ab8295, Abcam) and anti-TNNI3 (1:200, ab10231, Abcam). Secondary antibodies were donkey anti-mouse Alexa-Fluor 488 (1:200, A21202, Invitrogen), donkey anti-rabbit Alexa-Fluor 594 (1:200, A21207, Invitrogen), donkey anti-goat Alexa-Fluor 488 (1:200, A11055, Invitrogen), and donkey anti-goat Alexa-Fluor 647 (1:200, A21447, Invitrogen).

[0097] Flow cytometry analysis (FACS) Cells were dissociated into individual cells using Accutase, washed twice, and fixed with 4% PFA for 15 minutes. To identify quiescent cells in G0, Hoechst 33342 and Pyronin Y staining were employed as previously described. Analysis was performed using BD FACSDiva v6 or v8 and FlowJo v10. Cell populations were recognized by FSC / SSC gating, and doublets were excluded. Negative gates were set using unstained samples or isotype controls, and the absence of signal from negatives in the positive gate was confirmed.

[0098] Immunoblotting: Cells were lysed using M-PER Mammalian Protein Extraction Reagent buffer (78501, Thermo Scientific), and hHO was lysed with T-PER Tissue Protein Extraction Reagent (78510, Thermo Scientific). Protein content was measured using a Bradford assay with BSA as a reference. Primary antibodies were commercially available. The primary antibodies used in the examples were anti-phospho-S6 ribosomal protein antibody (5364, CST), anti-phospho-4E-BP1 antibody (2855, CST), anti-4E-BP1 antibody (9644, CST), anti-phospho-AKT antibody (9271, CST), anti-β-actin antibody (A5441, Sigma), anti-WT1 antibody (ab89901), anti-cTnI TNNI3 antibody (ab10231), anti-SNAI1 antibody (ab63371), anti-NBL1 antibody (ab174843), anti-p53 antibody (12790S, D963E, CST), anti-p16 antibody (12D1, CST), anti-p21 antibody (D7C1M, CST), anti-TBX18 antibody (ab15262), and anti-KI67 antibody (Biolegend; The secondary antibodies used were goat anti-rabbit IgG antibody with HRP (sc-2054, Santa Cruz) and anti-mouse IgG antibody with HRP (7076, CST), both used at a dilution of 1:5000.

[0099] Image Acquisition, Processing, and Analysis: Images from the microscope were captured using a BZ-X710 and processed with Keyence BZ-X Analyzer software. Color representations are as specified in the figure captions. Selected portions of images were selected, cropped, and enlarged to display both phase contrast and fluorescent details. Western blot data were collected using Cytiva's ImageQuant 800. Extensive image evaluation was performed using Microsoft PowerPoint and ImageJ version 1.52.

[0100] Bioinformatics and RNA-Seq: Retrospective bioinformatics analysis performed in this study was performed using the R2: Genome Analysis and Visualization Platform (http: / / r2.amc.nl / ). The publicly available dataset used for the retrospective analysis in this study was GSE51483. NOISeq was used for data normalization. Processed and raw fastq files generated during this study were stored in Gene Expression Omnibus (GEO). To gain deeper insights into gene expression, raw data was analyzed using RStudio. The NOISeq pipeline from the Bioconductor package was used to read, explore, and preprocess gene expression data. This was useful for RNA-Seq data and differential expression analysis. Hierarchical cluster dendrograms were created using the hclust, stats, and agnes packages from the cluster package. Distances were determined using the Manhattan city block distance algorithm. The kmeans function was used to calculate Kmeans. The fatoextra package provided tools to calculate and plot distance and correlation matrices using get_dist and fviz_dist. Cluster scatter plots were created using the fviz_cluster function. An R script was used to group expression data of differentially expressed genes (DEGs) and draw heat maps. DOSE and clusterProfiler were used for statistical analysis and visualization of gene functional profiles and GO term clusters. Ingenuity Pathway Analysis (IPA) was employed to map upstream pathways and identify trends in pathway activity. Chord diagrams were created using GOPlot.

[0101] <Quantitative RT-PCR> Total RNA was extracted using QIAzol® Lysis Reagent (Qiagen) according to the manufacturer's instructions. 1 μg of RNA was converted to cDNA using the ReverTra Ace system (Toyobo Biotech). Quantitative RT-PCR (qPCR) was performed using the StepOnePlus Real-Time PCR System and Next SYBR® qPCR Mix (Thunderbird). GAPDH served as an internal reference control. Primer sequences are listed in Table 1 below.

[0102]

[0103]

[0104] Example 1: Validation of the Relationship between Reduced Activity of the mTOR Signaling Pathway and Physiological Cardiac Maturation The mechanistic target of rapamycin (mTOR) is a central regulator of cell proliferation and metabolism and plays an important role in tissue homeostasis. mTOR signaling is particularly intertwined with the insulin-like growth factor 1 (IGF-1) pathway (Feng, Z. & Levine, AJ Trends Cell Biol. 20, 427-434 (2010); Floyd, S. et al. Mol. Biol. Cell 18, 3545-3555 (2007); Schiaffino, S. & Mammucari, C. Skelet. Muscle 1, 4 (2011)). IGF-1 promotes activation of the mTOR pathway, which promotes cell proliferation, inhibits cell apoptosis during development and regeneration, and regulates overall cellularity within tissues. By controlling the balance between cell proliferation and cell death through the IGF-1 pathway, mTOR maintains tissue integrity and function, and reduced mTOR signaling may be involved in the fundamental trigger for initiating functional maturation of tissues during adulthood.

[0105] First, to assess the dynamics of mTOR signaling during mouse cardiogenesis, we retrospectively analyzed publicly available transcriptome data over time intervals to monitor gene expression changes associated with cardiac maturation (Li, X. et al. Genomics 46, 482-495 (2014)). We identified a transcriptome switch that gradually inactivates mTOR signaling starting at E121.5 (Figure 1a). We performed experiments to examine the downregulation of genes involved in the mTOR pathway, particularly those involved in the assembly of the two major mTOR complexes, complex 1 (C1) and complex 2 (C2) (Figure 1b), namely, MTOR, RPTOR, and RICTOR. These complexes play a key role in phosphorylating downstream targets upon pathway activation. mTORC1 primarily regulates protein synthesis, cell proliferation, and autophagy through the ribosomal protein S6 kinase (S6k) and eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) (Liu, GY & Sabatini, DM Nat. Rev. Mol. Cell Biol. 21, 183-203 (2020)). In contrast, mTORC2 is primarily involved in cell survival and cytoskeletal organization through the phosphorylation of complex 1-associated protein kinase B (also known as Akt) (Figure 1b) (Sen, B. et al. J. Bone Miner. Res. (2014)).

[0106] To determine the developmental switch toward cardiac maturity, we examined phosphorylation targets of mTORC1 / 2 in protein lysates obtained from fetal hearts at different time intervals. At E18, we observed a significant decrease in pAkt, pS6k, and p4E-BP1 levels compared with their younger E12 counterparts, indicating a more proliferative state at E12 and confirming the progressive inactivation of mTOR over time. Furthermore, at postnatal week 7 (P7), we observed upregulation of p27, a key cell cycle inhibitor associated with cell quiescence (Figure 1c). Increased p27 levels arrest cell cycle progression, maintaining cells in the non-dividing but metabolically active G0 phase, preventing excessive cell proliferation and allowing cells to acquire adult functions and specialization. Furthermore, we observed a time-dependent upregulation of essential cyclin-dependent kinase inhibitors (Figure 1d), correlating with cell quiescence as part of the trigger for cardiac development toward maturity.

[0107] Based on the idea that epicardial maturation likely involves a similar quiescent pattern as an essential mechanism for cardiac maturation, we examined the epicardium, a single layer of mesothelium known for its remarkable regenerative potential due to a proliferative subpopulation with migratory capacity defined by Wilms' tumor 1 (WT1) expression in the fetal heart (Figure 1e). This population contributes to the regeneration of non-cardiomyocytes. Here, we confirmed the cortical distribution of the tight junction protein ZO-1 and observed expression of the epicardium-specific transcription factor WT1 at E12 (Figure 1f). However, by E14, we observed a decrease in WT1 expression, suggesting a degradation of the primary fetal epicardial network. This decrease in WT1 expression indicates the gradual initiation of the maturation program.

[0108] To better understand epicardial maturation in a specific context, we performed experiments on overgrown epicardial explants from various developmental stages (Fig. 1g). Expression and protein dynamics were monitored over time until the neonatal stage. Our findings confirmed a gradual decline in fetal markers over time (Fig. 1h). Furthermore, we observed that the acquisition of epicardial maturation correlated with a decline in mTOR signaling (Fig. 1i), suggesting a transition to quiescence and tissue homeostasis in adulthood.

[0109] Example 2: Effect of Chemical Inhibition of mTOR During Embryonic Development on In Vivo Epicardial Maturation. To elucidate the physiological significance of the observations in Example 1, we examined the response of E12 hearts exposed to ectopic maturation stimuli. To completely inhibit mTOR signaling, we examined the effects of Torin1, an mTOR kinase inhibitor (mTORKi) that can block a wide range of cellular activities controlled by mTOR. Torin1 interacts with the ATP-binding pocket of both mTOR complexes (Liu, Q. et al. J. Med. Chem. 53, 7146-7155 (2010)), and therefore can inhibit the function of mTORC1 and mTORC2. Freshly isolated hearts were obtained and cultured under anchorage-independent conditions using chemical conditions that ensure cell viability and long-term sustained cardiac contraction. To determine the optimal dose that balances efficacy and cellular safety, we measured the inhibitor's inhibitory potency on mouse epicardial cells. Using monolayers of an established mouse epicardial cell line (MEC1), we measured the IC of Torin1. 50 IC 50 Our results revealed that Torin1 at a concentration of 60 nM was able to inhibit 50% of cell proliferation within 96 h (Fig. 2a).

[0110] We next chose to analyze mTOR inhibition by short-term, acute exposure to Torin1 at a concentration of 200 nmol / L for 24 hours, a condition previously proven to enhance the maturation characteristics of hiPSC-derived cardiomyocytes. Our results confirmed that a single dose of Torin1 significantly reduced WT1 protein expression in mouse epicardium without observed cell death (Fig. 3a). This reinforces the notion that mTOR pathway inhibition can improve epicardial maturation and promote developmental transitions essential for terminating the embryonic scheme.

[0111] To confirm the extent of Torin1-induced maturation in fetal mouse hearts, we systematically assessed and contrasted the effects of Torin1 on mouse hearts from both E12 and E14 stages. Initial results in untreated hearts confirmed greater expression of the markers WT1, phospho-AKT, and phospho-S6K at E12 compared with E14 (Fig. 3b). After Torin1 treatment, WT1 levels were significantly reduced. Concurrently, TNNI3, the adult isoform of cardiac troponin I, was significantly increased, suggesting an accelerated synchronized onset of maturation indices in both the epicardium and myocardium (Fig. 3b).

[0112] Given the potential lack of epicardial marker expression in bulk cardiac tissue, we re-expanded epicardial explants to focus on epicardial maturation in primary culture. After a 7-day treatment period, we observed a consistent decrease in WT1 and other mTOR downstream targets, further confirming the specificity of mTOR downregulation in contributing to cell and tissue maturation (Figure 3c). Torin1-treated epicardial explants also showed the absence of cardiac TNNI1 traces and enhanced presence of NBL1 protein, a maturation marker recognized to counteract the Wt1-mediated Bmp4 signaling pathway during mouse epicardial maturation (Figure 3c).

[0113] Next, epicardial explant cultures were extended for 1 week in the presence of SB431542. This ALK5 inhibitor prevents spontaneous activation of the TGF-β signaling pathway and subsequent epithelial-to-mesenchymal transition (EMT). Results revealed that Torin1-treated samples exhibited reduced proliferation and epicardial outgrowth. Notably, no significant morphological changes were observed. This suggests that the migratory capacity of cells, a hallmark of mature epicardium, was impaired (Fig. 3d).

[0114] To understand the extent of the role of mTOR inhibition in epicardial maturation at the transcriptome level, we subjected Torin1-treated epicardial explants to RNA sequencing. Principal component analysis revealed that the principal component (PC1) accounted for 98.72% of the variance, highlighting the significant impact of Torin1 on epicardial biology (Figure 3e). Global gene expression analysis revealed 898 genes with significant differential expression across conditions (Figure 3f). Notably, a PANTHER-based (Mi, H. et al. Nucleic Acids Res. 49, D394-D403 (2021)) biological process enrichment study identified processes influenced by Torin1 that are crucial for cardiac development, such as cardiomyocyte differentiation and regulation of the BMP signaling pathway, highlighting their role in both epicardial and myocardial maturation (Figure 3g). Subsequent pathway analysis using PANTHER reaffirmed the importance of several cardiogenesis-related pathways (Figure 3h), highlighting cadherin, Wnt, and TGF-β signaling pathways that inform epicardial biology (Dronkers, E., Wauters, MMM, Goumans, MJ & Smits, AM Biomolecules 10, 404 (2020)). We also confirmed Torin1-driven fluctuations in PC1 in MEC1 monolayers (Figure 2b).

[0115] Finally, we observed a decrease in the mRNA levels of fetal epicardial genes, including Wt1, Tbx18, and Raldh1a2 (Fig. 3i). Furthermore, a decrease in MKi67 levels, further confirmed in MEC1s (Fig. 2c–e), along with suppression of EMT capacity, indicated cellular proliferation arrest (Fig. 3i). Remaining traces of cardiomyocytes were confirmed by a significant increase in the levels of Tnni3 and Myh6, markers of mouse cardiomyocyte maturity (Fig. 3j). Taken together, our data suggest that simultaneous inhibition of mTORC1 / 2 via Torin1 is effective in triggering fetal cardiac maturation, as demonstrated in both the epicardium and myocardium. Enhanced mRNA expression of Hes1 and Rb1 also supports the hypothesis of induced proliferation arrest followed by epicardial quiescence (Fig. 3k).

[0116] Example 3: Examination of the effects of dual mTORC1 / 2 inhibition on epicardial quiescence and maturation in hiPSC-derived epicardium To fully understand the contribution of cell quiescence in human fetal epicardial maturation, we used epicardium derived from human induced pluripotent stem cells and manipulated them to systematically study the effects of cell cycle withdrawal via complete inhibition of the mTOR pathway.

[0117] First, we differentiated epicardial-like (EPI) cells from hiPSCs (Non-Patent Document 5) (Fig. 4a). Cardiac mesoderm was formed using activin A and BMP4 (days 1–3 / 4), and epicardial fate was subsequently determined by activating WNT signaling (days 3 / 4–6). To maintain epicardial characteristics and enable long-term EPI cell culture without spontaneous differentiation, we inhibited TGF-β signaling from day 3 / 4 (SB431542). Consistently, EPI cells differentiated from hiPSC line 409B2 exhibited a typical cobblestone-like morphology at day 24 (Fig. 4b) and upregulated WT1, TBX18, and ALDH1A2 during differentiation (Fig. 4c). At day 24, EPI cells expressed nuclear WT1, which was confirmed by flow cytometry and immunofluorescent costaining with ZO1 (Fig. 4d, e). Next, we evaluated the effect of Torin1 to induce epicardial quiescence and its ability to promote complete cell cycle withdrawal by dually inhibiting mTORC1 and mTORC2 (Figure 5a). First, we evaluated the cytotoxic effects of Torin1 at various dose levels in vitro over a 96-hour period using the MTT assay (Figure 6a). Following the results of these refinement steps to assess cytotoxicity (Figure 6b), cells were treated with 200 nmol / L Torin1 for 7 days. This treatment began after verification of peak WT1 protein levels at day 24 of differentiation.

[0118] When administered to differentiated epicardial cells, Torin1 treatment (Figure 5a) significantly reduced the phosphorylation levels of both S6K and Akt (Figure 5b). This highlights its effect on both mTORC1 (S6K) and mTORC2 (Akt) while preserving epicardial structure (Figure 5c). Torin1-treated cells rapidly arrested cell proliferation (Figure 5d). Growth arrest is a key phenotype of the adult human epicardium, a finding similar to that observed in mouse counterparts. Next, we verified that Torin1 treatment effectively reduced nuclear WT1 and TBX18 levels and preserved tight junctions, as indicated by ZO-1 expression, while maintaining epicardial morphology (Figure 5e). Inhibition of the mTOR pathway in epicardial cells significantly reduced the proliferation protein Ki67 (Figure 5f) and maintained the inactivation of the EMT marker SNAI1 (Figure 7a). Furthermore, we found that phosphohistone H3 (pH3), which stains condensed chromosomes in mitosis (M) phase, was a more accurate indicator for measuring the mitotic index, and confirmed the regular decrease in proliferation rate in Torin-1 epicardial cells (Fig. 5g).

[0119] To distinguish between senescent and quiescent phenotypes, we performed a β-galactosidase (β-gal) assay due to its specificity for senescent cells. Its quantitative nature allows us to monitor the dynamics of senescent fate upon complete inhibition of the mTOR pathway. Torin1-treated epicardium exhibited a significant increase in β-gal + The number of cells significantly decreased (Fig. 5h, Fig. 8a, b), suggesting that dual inhibition of mTORC1 and mTORC2 can shift the epicardium away from a senescent phenotype, as confirmed by the decreased expression of SASP-related genes IL-8 and SPP1 (Fig. 8c). We confirmed that the transcription of markers related to cell proliferation (MKI67, CCNA1, CCNB1, and E2F1) was significantly decreased (Fig. 5i), while a trend toward increased expression of markers related to cell quiescence, including p16, HES1, Rb1, and p53, was observed.

[0120] To eliminate potential variability arising from donor- or protocol-specific differences in iPS cell origin and culture conditions, we validated our key findings using epicardial monolayers obtained from feeder-free colonies of the 1390C1 strain, following the established epicardial GiWiGi protocol (Witty, AD et al. Nat. Biotechnol. 32, 1026-1035 (2014)) (Figure 9a). Furthermore, we confirmed our initial observations and corroborated our previous protein analysis by demonstrating that Torin1 effectively reduced WT1 protein expression (Figure 9b).

[0121] To further investigate the level of quiescence, we utilized pyronin Y staining. Pyronin Y staining has proven to be a reliable method for detecting G0 cells because it selectively stains the RNA content within cells (Eddaoudi, A., Canning, SL & Kato, I. Methods Mol. Biol. Clifton NJ 1686, 49-57 (2018)). To demonstrate that G0 cells exhibit reduced RNA synthesis compared to actively cycling cells, we combined this technique with Hoechst 33342 or propidium iodide staining to simultaneously label DNA content and identify distinct cell populations in various phases of the cell cycle. Treatment of epicardial cells with Torin1 resulted in a substantial exit from the cell cycle, as demonstrated by FACS dynamics (Figure 5j). This exit was evidenced by a significant increase in the number of quiescent (G0) cells from 14.2% to 82.5% (Figure 5k), along with a decrease in G1 cells from 75.9% to 1.83% (Figure 5k).

[0122] To investigate the transcriptional impact of Torin1 on iPSC-derived epicardium, we performed a detailed RNA-sequencing analysis of cells treated with Torin1 (Lucena-Cacace, A. & Yoshida, Y. Methods Mol. Biol. Clifton NJ 2320, 219-232 (2021)). Consistent with previous observations regarding mTOR inhibition in mouse epicardium, Torin1 significantly altered the transcriptome of hiPSC-derived epicardium, with principal component 1 (PC1) accounting for 99.5% of the PCA variance (Figure 10a). Analysis identified 379 differentially expressed genes specifically attributed to Torin1 treatment (Figure 10b). While expression of TJP1, the gene responsible for ZO-1 protein, was unaffected by Torin1 treatment, we confirmed significant reductions in the fetal epicardial genes WT1, TBX18, and ALDH1A2 (Figure 10c). This downregulation occurred simultaneously with a significant decrease in MKI67 mRNA levels, consistent with the protein analysis. Furthermore, genes driving epithelial-mesenchymal transition (EMT) were significantly downregulated (Figure ​(Figure10d),10), as was CDH18, a cadherin associated with epicardial development. Increased transcription of RB1 and HES1 reaffirmed the quiescent transcriptome (Sang, L., Coller, HA & Roberts, JM Science 321, 1095-1100 (2008); Harris, L. & Guillemot, F. Genes Dev. 33, 479-481 (2019); Sueda, R. & Kageyama, R. Dev. Growth Differ. 62, 59-66 (2020)) (Figure 10e). More broadly, we aligned the RNA-seq data from this study with GSE84085 (Bao, X. et al. Nat. Biomed. Eng. 1, 0003 (2016)), a dataset containing primary human adult epicardial samples. Heatmap comparison highlighted that adult epicardium and Torin1-treated, iPSC-derived epicardium exhibit global features consistent with quiescent, mature human epicardium (Figure ​(Figure10f).

[0123] Example 4: Searching for Factors Promoting Quiescence and Maturation of hiPSC-Derived Epicardial Cells. mTOR functions as the central kinase in two unique multiprotein complexes, mTORC1 and mTORC2. These complexes differ in their component proteins and sensitivity to the drug rapamycin. Specifically, mTORC1 incorporates raptor as a primary subunit, whereas rictor is essential for mTORC2. mTORC1 activity is rapidly suppressed by rapamycin, whereas mTORC2 activity is largely unaffected during short-term rapamycin exposure. To determine the role of these mTOR complexes in epicardial maturation, human induced pluripotent stem cell-derived epicardial cells were exposed to rapamycin or Torin1, which inhibit both mTOR complexes. Results showed that rapamycin did not inhibit epicardial cell proliferation, suggesting a failure to achieve quiescence, likely due to residual phospho-AKT activity (Figure 11a). This suggests that mTORC2, but not mTORC1, plays a crucial role in epicardial maturation and quiescence.

[0124] Tacrolimus, commonly known as FK506 (Dumont, FJ Curr. Med. Chem. 7, 731-748 (2000)), is primarily used as an immunosuppressant for solid organ transplantation (Wallemacq, PE & Reding, R. Clin. Chem. 39, 2219-2228 (1993)). Its primary mechanism of action is calcineurin inhibition. However, it is noteworthy that both tacrolimus and rapamycin bind to FKBP12 and can affect the epicardial maturation phenotype. However, tacrolimus treatment also failed to reduce epicardial proliferation (Figure 11a), suggesting that inhibition of calcineurin or mTORC1 alone is insufficient to promote epicardial maturation of hiPSC-derived epicardial cells.

[0125] Next, we examined how each treatment affected the dynamics of principal component analysis (PCA). Both rapamycin and tacrolimus induced transcriptome variations that could indicate partial epicardial maturation. However, Torin1 showed the most significant deviation on the Y-axis among all compounds. This striking difference between rapamycin and Torin1 highlights the impact of mTORC2 inhibition and suggests that Torin1 induces a comprehensive maturation phenotype (Figure 12a). We identified 66 transcription factors (TFs) that were uniquely altered by Torin1 treatment and named them mTORC2-responsive transcription factors (mTORC2-TFs). In contrast, when comparing the effects of rapamycin, these were classified as mTORC1-responsive TFs (mTORC1-TFs). Comparative transcriptome analysis of fetal and adult epicardium derived from pluripotent stem cells (PSCs) revealed 104 differentially expressed TFs, which we labeled as mature epicardial TFs. Notably, of these, 24 TFs were located within Torin1-responsive elements (Fig. 12b).

[0126] We then refined the list to include only transcription factors with at least a twofold higher fold change in expression in the mature epicardium compared to the fetal epicardium (GSE84085), which narrowed the list to five transcription factors: MAFF, RELB, MAFG, HIVEP2, and YBX3 (Fig. ​(Fig.12c). 12c).

[0127] Among these five factors, MAFF (Brundage, ME et al. Oncogene 33, 5626-5636 (2014)) and YBX3 (Cooke, A. et al. Cell Rep. 27, 3097-3106.e5 (2019)) have been described to function in regulating signaling of the PI3K / Akt / mTOR axis and were used in the following experiments. To demonstrate the causal relationship and biological relevance of the individual activation of MAFF and YBX3 during epicardial maturation, we systematically investigated the effects of Torin1 treatment in epicardial cells, which ectopically downregulated MAFF and YBX3, achieved by expression of small interfering RNA (siRNA) (siMAFF and siYBX3, respectively) (Figure ​(Figure12d). 12d).

[0128] We verified the selective downregulation of each gene individually and confirmed that Torin1 treatment did not promote their expression (Fig. 12e). We then proceeded to evaluate the expression of two fetal epicardial markers, WT1 and TBX18, as well as the proliferation marker MKi67. Interestingly, the results confirmed that YBX3 and MAFF may play different roles in epicardial maturation. Although downregulation of MAFF did not reduce the proliferation level of epicardial cells (as evidenced by MKi67 mRNA expression), cells with downregulated YBX3 were unable to exit the fetal epicardial program after Torin1 treatment. Notably, WT1 protein levels remained consistent by immunocytochemistry (ICC), indicating that YBX3 is crucial in controlling WT1 and, therefore, the embryonic program (Fig. 12f). Finally, we also examined the protein levels of TBX18 (Fig. S12g) and MKI67 (Fig. S12h) by Western blot to further verify the distinct roles of YBX3 and MAFF in human epicardial maturation.

[0129] Example 5: Validation of the Paracrine Effect of IGF2- and FN1-Expressing Mature hiPSC-Derived Epicardium IGF2 / IGF1R signaling plays a crucial role in human cardiogenesis. In rodent models, the interaction between epicardial IGF2 and myocyte IGF1R has been identified as a key catalyst for myocardial compaction (Meier, AB et al. Nat. Biotechnol. 1-14 (2023)). Furthermore, a recent study identified epicardial fibronectin (FN1) secretion as a driver of accelerated cardiac maturation in 3D engineered tissues (Ong, LP et al. Stem Cell Rep. 18, 936-951 (2023)). Another study using epicardioids investigated the interaction between the myocardium and the epicardium and found that this myocardial compression process in humans mirrors that in rodents (Meier, AB et al. Nat. Biotechnol. 1-14 (2023)).

[0130] We hypothesized that a mature artificial epicardium could potentially amplify the paracrine effects of IGF2 and FN1 expression, promoting the stabilization and subsequent maturation and compaction of hiPSC-derived cardiomyocytes. To investigate this, we independently initiated differentiation of ventricular cardiomyocytes and epicardial cells. For cardiac differentiation, we employed a reporter iPS cell line (Miki, K. et al. Nat. Commun. 12, 3596 (2021)) to monitor the transition switch from the fetal to the adult isoform of cardiac troponin I, a critical developmental switch that can distinguish fetal, neonatal, or adult epicardium (Bedada, F.B. et al. Stem Cell Rep. 3, 594-605 (2014)) (Figure 13a). After differentiation and maturation of epicardial cells as a monolayer, we used a reporter iPS cell line (TNNI1) to monitor the expression of TNNI3 in the presence of mature epicardium. + ) and proceeded to co-culture with beating EBs.

[0131] Previous studies have outlined various interactions between hiPSC-derived epicardial cells and cardiomyocytes, primarily highlighting their role in regenerative capacity and cardiomyocyte cell cycle activation (Bargehr, J. et al. Nat. Biotechnol. 37, 895-906 (2019)). After treatment with Torin1, epicardial cells exhibited significant overexpression of IGF2 and FN1 (Figure 13b). Notably, a 10-day coculture period revealed increased expression of TNNI3 and a corresponding decrease in TNNI1, suggesting that EBs began to adopt certain features of structural maturation during paracrine contact with mature epicardial cells (Figure 13c, d). Furthermore, mRNA expression analysis demonstrated that when EBs were cocultured with mature epicardial cells, they exhibited enhanced expression of genes important for left ventricular compression, such as TBX5 (Ross, S.B. et al. Hum. Genome Var. 7, 1-8 (2020)), HEY2 (Miao, L. et al. Sci. Rep. 8, 2678 (2018)), PRDM16 (Wu, T. et al. Circulation 145, 586-602 (2022)), and NPPA (Tian, ​​X. et al. Nat. Commun. 8, 87 (2017)) (Figure 13e). These findings confirm that mTOR inhibition can promote cardiac maturation in a cell-autonomous manner and exogenously via the paracrine IGF2 and FN1 pathways mediated by the mature epicardium.

[0132] Example 6: Generation of Mature iPSC-Derived Cardiac Organoids with Quiescent Epicardium To evaluate the efficacy of mTOR inhibition in a context that more closely reflects cardiac development, we generated self-organizing human cardiac organoids (hHOs) derived from human iPS cell lines (Lewis-Israeli, YR et al. Nat. Commun. 12, 5142 (2021); Tian, ​​Y. et al. Front. Cell Dev. Biol. 10, 1001453 (2022)). We aimed to examine the effects of Torin1 treatment on epicardial and myocardial maturation in these human cardiac organoids. Specifically, we sought to identify the maturation process of the human epicardium in a complex multicellular environment in which multiple non-autonomous signaling pathways are activated. Using three levels of Wnt signaling regulation, we differentiated the feeder-free human iPS cell line 1390C1 into hHOs (Figure 14a) as previously reported (Tian, ​​Y. et al. Front. Cell Dev. Biol. 10, 1001453 (2022)). By day 6, 80%–90% of these organoids exhibited beating activity, which subsequently increased to 100% by day 10. At day 15, we analyzed the multicellular composition and degree of vascularization within the hHO model.

[0133] Immunocytochemistry (ICC) identified various cell populations. As previously characterized by the present inventors (Tian, ​​Y. et al. Front. Cell Dev. Biol. 10, 1001453 (2022)), TNNT2 + The population corresponds to beating cardiac muscle cells, and WT1 + The cells show human epicardial and CD31 + The cells show endothelial and NFATC1 + are endocardial cells, and VIM +highlights the cardiac fibroblast population. Consistent with previous findings, we noted minimal differences in cell composition ratios between different differentiation batches. Subsequently, we used the MTT assay to characterize the cardiac cytotoxicity profile associated with various Torin1 doses over a 96-hour in vitro period. Human cardiac-derived organoids exhibited resistance to Torin1 comparable to monolayer human epicardial cells, with an IC of 2.51 μM. 50 The values ​​were shown (Fig. 14b).

[0134] Therefore, for further studies, a concentration of 200 nmol / L, IC 50 We decided to continue using a dose of Torin1 below 1000kJ / mL, which proved to be optimal for promoting epicardial quiescence and maturation in hiPSC-derived epicardial monolayers. Treatment of day 15 hHOs with Torin1 for 7 days resulted in a significant effect on overall cell density by day 22. While untreated organoids consistently expanded in size, Torin1-treated organoids exhibited growth arrest (Figure 14c). This indicates that Torin1 can induce quiescence in a 3D environment without causing changes in organoid morphology (Figure 14d).

[0135] To gain a more comprehensive understanding of the effects of mTOR inhibition on human cardiac organoids, we first confirmed the following two conditions before Torin1 treatment: (1) WT1-positive cells are in close proximity to TNNT2-positive cells and independently maintain distinct cellular identities (Fig. 14e). (2) EMT is actively mediated by SNAI1 due to the inherent growth dynamics of developing organoids (Fig. 14f). These observations suggest that epicardial cells are actively differentiating into EPDCs, contributing to the expansion of cardiac organoid cell populations. Next, WT1-positive cells are assumed to be in a state of continuous self-renewal and undergo EMT. Consequently, the distribution of WT1 throughout the organoids is not static but dynamic, exhibiting a fluctuating expression pattern. After Torin1 treatment, we observed a decrease in WT1 expression, whereas the TNNT2 population was unaffected (Fig. 14g, Fig. 11b). This suggests that the epicardium can mature effectively, a finding consistent with our in vivo observations in mouse hearts and the in vitro maturation of hiPSC-derived epicardial monolayers.

[0136] To more rigorously examine mTORC2-dependent phenotypes associated with organoid maturation, we systematically evaluated the effects of tacrolimus and rapamycin on growing cardiac organoids. Remarkably, the results demonstrated that among these compounds, Torin1 was the only one capable of reducing epicardial WT1 protein levels (Figure 14h), confirming that mTORC1 inhibition remains insufficient to induce maturational effects. Furthermore, as confirmed in previous studies in hiPSC-derived cardiomyocytes, we confirmed that Torin1 significantly enhanced the expression of adult cardiac troponin 3 (TNNI3) (Figure 14h), thus suggesting that complete mTOR inhibition, including the epicardium and myocardium, may play a crucial role in global cardiac maturation.

[0137] Finally, we also verified the mTORC2 dependence of the EMT program suppression through the observed reduction in SNAI1 levels (Figure ​(Figure14i,11c),11), confirming the loss of EMT initiation ability and the greater resemblance of these mature-like cardiac organoids to their adult counterparts. In summary, our pharmacological approach aimed at maturing the hiPSC-derived epicardium demonstrated efficacy in generating mature human iPSC-derived cardiac organoids expressing markers of adult cardiomyocytes and epicardial cells. This lays the foundation for establishing a platform for investigating adult epicardial reactivation and provides insights into its fundamental role in the regenerative response.

[0138] The present invention provides a novel method for producing mature epicardial cells from immature epicardial cells. The epicardial cells produced by this method are similar to adult epicardial cells and can be used in adult-level two-dimensional and three-dimensional cardiac cell models, drug discovery screening, regenerative medicine, and the like. Furthermore, since the method can also promote the maturation of cardiomyocytes, it can also be used in methods for maturation of cardiomyocytes.

[0139] This application is based on patent application No. 2024-017166 filed in Japan (filing date: February 7, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing mature epicardial cells, comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor.

2. The method of claim 1, wherein the culture medium contains an mTORC1 inhibitor.

3. The method of claim 1 or 2, wherein at least one of the mTORC2 inhibitors is Torin1.

4. The method according to any one of claims 1 to 3, wherein the mature epicardial cells are cells that do not express WT1 and / or TBX18.

5. The method of any one of claims 1 to 4, wherein the epicardial cells are derived from pluripotent stem cells.

6. The method according to any one of claims 1 to 5, wherein the epicardial cells are contained in cardiac organoids.

7. Mature epicardial cells obtained by the method according to any one of claims 1 to 6, or cardiac organoids comprising said cells.

8. A transplantation therapy comprising the mature epicardial cells or cardiac organoids described in claim 7.

9. A method for producing mature cardiomyocytes, comprising a step of co-culturing the mature epicardial cells or cardiac organoids described in claim 7 with cardiomyocytes.

10. The method of claim 9, wherein the cardiomyocytes are contained in a cell aggregate.

11. A method for maturing epicardial cells, comprising culturing epicardial cells in a medium containing an mTORC2 inhibitor.

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