Cardiomyocytes and compositions, and methods for producing the same

Abstract

To provide cardiomyocytes and compositions, and methods for producing the same.SOLUTION: Disclosed herein are methods for generating mature cardiomyocytes, and compositions including the mature cardiomyocytes. Also disclosed herein are methods for enhancing maturation of quiescent cardiomyocytes, and compositions including the mature quiescent cardiomyocytes. There is a need for methods or protocols for the generation of mature cardiomyocytes for use in cell therapy and cell screening, among other uses. By leading cardiomyocytes to a quiescent state, maturation of the cardiomyocytes may be enhanced. Disclosed herein are methods of producing a mature cardiomyocyte from an immature cardiomyocyte. The methods comprise contacting the immature cardiomyocyte with an mTOR inhibitor.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 62 / 933,962, filed November 11, 2019, U.S. Provisional Application No. 62 / 884,592, filed August 8, 2019, and U.S. Provisional Application No. 62 / 858,302, filed June 6, 2019, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Current differentiation protocols for generating cardiomyocytes from human induced pluripotent stem cells (iPSCs) are capable of generating highly pure cardiomyocyte populations, as determined by cardiac troponin T expression. However, these cardiomyocytes remain immature and, compared to adult cardiomyocytes, exhibit weaker maximal contractile force, slower upstroke velocity, and immature mitochondrial function, more closely resembling the fetal state. Immature cardiomyocytes exhibit automaticity or pacemaker-like activity, which can result in life-threatening ventricular arrhythmias when delivered to adult animal models. Furthermore, their sarcomere structure is poorly organized, resulting in insufficient contractile force. The immaturity of iPSC-derived cardiomyocytes presents a significant barrier to the clinical translation of cardiomyocyte cell therapy for cardiac disease. During development, cardiomyocytes transition from a fetal proliferative state to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR) signaling pathway plays a key role in nutrient sensing and growth. Transient inhibition of the mTOR signaling pathway may induce cardiomyocyte quiescence and promote cardiomyocyte maturation. There is a need for improved methods to induce cardiomyocyte quiescence during differentiation, thereby promoting cardiomyocyte maturation. Summary of the Invention [Means for solving the problem]

[0003] There is a need for methods or protocols to generate mature cardiomyocytes for use in cell therapy and cell screening, among other applications. By driving cardiomyocytes into a quiescent state, cardiomyocyte maturation can be promoted.

[0004] Disclosed herein is a method for producing mature cardiomyocytes from immature cardiomyocytes, the method comprising contacting the immature cardiomyocytes with an mTOR inhibitor.

[0005] In some embodiments, the mTOR inhibitor is an inhibitor of both mTORC1 and mTORC2. In some embodiments, the mTOR inhibitor inhibits phosphorylation of 4E-BP1. In some embodiments, the mTOR inhibitor inhibits phosphorylation of both ribosomal protein S6 and 4E-BP1. In some embodiments, the mTOR inhibitor is selected from the group consisting of rapamycin, Torin1, Torin2, everolimus, temsirolimus, ridaforolimus, any ATP-competitive mTOR kinase inhibitor (e.g., dactolisib, BGT226, SF1126, PKI-587, or other dual mTOR / PI3K inhibitors, sapanisertib, AZD8055, AZD2014, or other dual mTORC1 / mTORC2 inhibitors), and any analog or derivative of any of the foregoing.

[0006] In some embodiments, the immature cardiomyocytes are derived from iPS cells, ES cells, T cells, and / or fibroblasts. In some embodiments, the immature cardiomyocytes resemble fetal cardiomyocytes.

[0007] In some embodiments, mature cardiomyocytes exhibit increased expression of one or more mature genes (e.g., TNNI3, TNNT2, MYH6, MYH7, NPPB, HCN4, CACNA1c, SERCA2a, PPARGC1, KCNJ2, REST, RyR, and / or SCN5a) compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of one or more sarcomeric proteins (e.g., TNNT2, TNNI3, MYH6, and / or MYH7) compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of one or more ion channel genes (e.g., KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and / or SERCA2a) compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of REST and / or GATA4 compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit a decreased beat rate compared to immature cardiomyocytes.

[0008] In some embodiments, the mature cardiomyocytes are electrically mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are contractilely mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are metabolically mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are one or more of electrically mature cardiomyocytes, contractilely mature cardiomyocytes, and metabolically mature cardiomyocytes.

[0009] In some embodiments, the immature cardiomyocytes are contacted with the mTOR inhibitor after the immature cardiomyocytes begin beating (e.g., 1 to 30 days, 1 to 15 days, or 1 to 3 days). In some embodiments, the immature cardiomyocytes are contacted with the mTOR inhibitor after the immature cardiomyocytes begin to express at least one of troponin T, troponin I, myosin heavy chain 6, or myosin heavy chain 7.

[0010] Also disclosed herein are non-naturally occurring cardiomyocytes, wherein the non-naturally occurring cardiomyocytes are mature cardiomyocytes.

[0011] In some embodiments, the non-naturally occurring cardiomyocytes exhibit increased expression of one or more mature genes (e.g., TNNI3, TNNT2, MYH6, MYH7, NPPB, HCN4, CACNA1c, SERCA2a, PPARGCl, KCNJ2, REST, RyR, and / or SCN5a) relative to immature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes exhibit increased expression of one or more sarcomeric proteins (e.g., TNNT2, TNNI3, MYH6, and / or MYH7) relative to immature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes exhibit increased expression of one or more ion channel genes (e.g., KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and SERCA2a) relative to immature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes exhibit increased expression of REST and / or GATA4 relative to immature cardiomyocytes. In some embodiments, the non-native cardiomyocytes exhibit a reduced beating rate compared to immature cardiomyocytes.

[0012] In some embodiments, the non-naturally occurring cardiomyocytes are electrically mature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes are contractilely mature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes are metabolically mature cardiomyocytes. In some embodiments, the non-naturally occurring cardiomyocytes are one or more of electrically mature cardiomyocytes, contractilely mature cardiomyocytes, and metabolically mature cardiomyocytes.

[0013] Also disclosed herein are methods of treatment comprising administering to a subject in need thereof a composition comprising the non-naturally occurring cardiomyocytes (e.g., mature cardiomyocytes) described herein (e.g., the subject has or is at risk of developing ventricular arrhythmias or reduced systolic cardiac function, e.g., chronic heart failure, or congenital heart disease, or other cardiac disease).

[0014] Also disclosed herein are methods of treatment comprising administering to a subject in need thereof a pharmaceutical composition made with an isolated population of one or more non-naturally occurring cardiomyocytes (e.g., one or more mature cardiomyocytes) described herein (e.g., the subject has or is at risk of developing ventricular arrhythmias or reduced systolic cardiac function, e.g., chronic heart failure, or congenital heart disease, or other cardiac disease).

[0015] Also disclosed herein is a kit (e.g., the kit further includes a component for detecting a marker of a mature cardiomyocyte) comprising immature cardiomyocytes or their precursors, at least one cardiomyocyte maturation factor (e.g., an mTOR inhibitor, e.g., rapamycin, Torin1, Torin2, everolimus, temsirolimus, ridaforolimus, any ATP-competitive mTOR kinase inhibitor (e.g., dactolisib, BGT226, SF1126, PKI-587, or other dual mTOR / PI3K inhibitors, sapanisertib, AZD8055, AZD2014, or other dual mTORC1 / mTORC2 inhibitors), and / or any analog or derivative of any of the foregoing), and instructions for generating cardiomyocytes (e.g., mature cardiomyocytes) using the immature cardiomyocytes or their precursors and the at least one cardiomyocyte maturation factor.

[0016] Also disclosed herein are kits comprising a composition comprising at least one non-naturally occurring cardiomyocyte (e.g., mature cardiomyocyte) described herein and instructions for using the composition in a method of treatment (e.g., treatment of ventricular arrhythmia or reduced systolic cardiac function, e.g., chronic heart failure, or congenital heart disease, or other cardiac disease).

[0017] Also disclosed herein is a method of using the composition in the manufacture of a medicament for the treatment of a cardiac disease (e.g., ventricular arrhythmia or reduced systolic cardiac function, e.g., chronic heart failure, or congenital heart disease), wherein the treatment comprises administering the medicament to a subject in need of such treatment, and the composition comprises at least one non-naturally occurring cardiomyocyte (e.g., mature cardiomyocyte) described herein.

[0018] Also disclosed herein are compositions comprising a conditioned medium, wherein the medium is conditioned by non-naturally occurring cardiomyocytes (e.g., mature cardiomyocytes) described herein.

[0019] Also disclosed herein are compositions comprising one or more non-naturally occurring cardiomyocytes (eg, mature cardiomyocytes) described herein and one or more immature cardiomyocytes.

[0020] Also disclosed herein are populations of cells comprising one or more non-native cardiomyocytes (e.g., mature cardiomyocytes) described herein, wherein at least 10% of the cells in the population are non-native cardiomyocytes (e.g., mature cardiomyocytes) described herein. In some embodiments, the population of cells further comprises immature cardiomyocytes.

[0021] Disclosed herein are cell patches comprising non-native cardiomyocytes (e.g., mature cardiomyocytes) as described herein. In some embodiments, the non-native cardiomyocytes are grown on a membrane.

[0022] Also disclosed herein are three-dimensional structures comprising the non-native cardiomyocytes (eg, mature cardiomyocytes) described herein.

[0023] In some embodiments, the three-dimensional structure is a matrix or scaffold. In some embodiments, the three-dimensional structure is a cell patch. In some embodiments, the three-dimensional structure is a microtissue. In some embodiments, the three-dimensional structure is administered to a subject. In some embodiments, the three-dimensional structure is administered to a subject by implantation.

[0024] Disclosed herein are methods for inducing quiescence in cardiomyocytes, the methods comprising contacting senescent cardiomyocytes with a cardiomyocyte maturation factor, thereby causing the senescent cardiomyocytes to transition to quiescent cardiomyocytes.

[0025] In some embodiments, the cardiomyocyte maturation factor is an upregulator of p53, an inhibitor of the mTOR signaling pathway, or an upregulator of p53 and an mTOR inhibitor. In some embodiments, the cardiomyocyte maturation factor is Torin1 and / or nutlin-3a. In some embodiments, the cardiomyocyte maturation factor is not an mTOR inhibitor, e.g., is not Torin1.

[0026] In some embodiments, quiescent cardiomyocytes exhibit increased expression of one or more quiescent markers (e.g., p16 and p130). In some embodiments, quiescent cardiomyocytes exhibit decreased expression of one or more proliferation markers (e.g., Ki67, cyclin C1, and E2F1). In some embodiments, quiescent cardiomyocytes exhibit increased expression of one or more inhibitory E2F factors (e.g., E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8). In some embodiments, quiescent cardiomyocytes exhibit decreased expression of one or more stimulatory E2F factors (e.g., E2F1, E2F2, and E2F3).

[0027] In some embodiments, the quiescent cardiomyocytes are mature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of one or more sarcomeric proteins (e.g., TNNT2, TNNI3, MYH6, and MYH7) compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of one or more ion channel genes (e.g., KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and SERCA2a) compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit increased expression of REST and / or GATA4 compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes exhibit a decreased beat rate compared to immature cardiomyocytes. In some embodiments, mature cardiomyocytes are electrically mature cardiomyocytes, contractilely mature cardiomyocytes, and / or metabolically mature cardiomyocytes.

[0028] Also disclosed herein are non-native cardiomyocytes. Non-native cardiomyocytes are mature quiescent cardiomyocytes.

[0029] In some embodiments, the non-native cardiomyocytes exhibit increased expression of one or more markers of quiescence (e.g., p16 and p130). In some embodiments, the non-native cardiomyocytes exhibit decreased expression of one or more markers of proliferation (e.g., Ki67, cyclin C1, and E2F1). In some embodiments, the non-native cardiomyocytes exhibit increased expression of one or more inhibitory E2F factors (e.g., E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8). In some embodiments, the non-native cardiomyocytes exhibit decreased expression of one or more stimulatory E2F factors (e.g., E2F1, E2F2, and E2F3).

[0030] Also disclosed herein are methods of treatment comprising administering a composition comprising the non-native cardiomyocytes described herein to a subject in need thereof, in some embodiments, the subject has or is at risk of developing ventricular arrhythmia, reduced systolic cardiac function, chronic heart failure, congenital heart disease or other cardiac disease.

[0031] Also disclosed herein are methods of treatment that include administering a composition comprising quiescent cardiomyocytes produced according to the methods described herein to a subject in need thereof, in some embodiments, the subject has or is at risk of developing ventricular arrhythmia, reduced systolic cardiac function, chronic heart failure, congenital heart disease, or other cardiac disease.

[0032] Also disclosed herein are methods of treatment that include administering to a subject in need thereof a pharmaceutical composition produced using one or more quiescent cardiomyocytes produced according to the methods described herein. In some embodiments, the subject has or is at risk of developing ventricular arrhythmia, reduced systolic cardiac function, chronic heart failure, congenital heart disease, or other cardiac disease.

[0033] The practice of the present invention will typically employ, unless otherwise specified, conventional techniques of cell biology, cell culture, molecular biology, recombinant genetic biology, microbiology, recombinant nucleic acid (e.g., DNA) technology, immunology, and RNA interference (RNAi), which are within the skill of the art. Non-limiting descriptions of some of these techniques can be found in the following publications: Ausubel, F., et al., (eds.), Current Protocols in Molecular Biology,Current Protocols in Immunology,Current Protocols in Protein Science,and Current Protocols in Cell Biology, all John Wiley &Sons, NY, edition as of December 2008, Sambrook, Russell, and Sambrook, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 2001, Harlow, E. and Lane, D., Antibodies-A Laboratory Manual, Cold Spring Harbor Laboratory. Press, Cold Spring Harbor, 1988; Freshney, RI, "Culture of Animal Cells, A Manual of Basic Technique", 5th ed., John Wiley & Sons, Hoboken, NJ, 2005. Non-limiting information on therapeutic agents and human diseases can be found in Goodman and Gilman's The Pharmacological Basis of Therapeutics, 11th Ed., McGraw Hill, 2005, Katzung, B. (ed.) Basic and Clinical Pharmacology, McGraw-Hill / Appleton &Lange;10th ed.(2006) or 11th edition(July Non-limiting information on genes and genetic disorders can be found in McKusick, VA: Mendelian Inheritance in Man. A Catalog of Human Genes and Genetic Disorders. Baltimore: Johns Hopkins University Press, 1998 (12th edition), or the most recent online database: Online Mendelian Inheritance in Man, OMIM™, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University (Baltimore, MD) and National Center for Biotechnology Information, National Library of Medicine (Bethesda, MD), as of May 1, 2010, can be found at the World Wide Web URL: ncbi.nlm.nih.gov / omim / , and in the Online Mendelian Inheritance in Animals (OMIA), a database of genes, genetic diseases, and traits for animal species (other than human and mouse) at omia.angis.org.au / contact.shtml. All patents, patent applications, and other publications (e.g., scientific articles, books, websites, and databases) mentioned herein are incorporated by reference in their entirety. In the event of a conflict between this specification and any of the incorporated references, this specification (including any amendments thereof that may be based on the incorporated references) will control. Standard art-accepted meanings of terms are used herein unless otherwise specified. Standard abbreviations for various terms are used herein.

[0034] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief explanation of the drawings]

[0035] [Figure 1A]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1B]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1C]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1D]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1E]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1F]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1G]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1H]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 1I]Figures 1A-1I show that Torin1 treatment increases cellular quiescence in induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs). Figure 1A shows a schematic diagram of the differentiation protocol with Torin1 treatment, which was performed over 7 days, starting approximately 2 days after the onset of beating, unless otherwise noted. Figure 1B shows Western analysis of phospho-S6 and phospho-Akt for BJRiPS-CMs at baseline and 30 minutes and 2, 4, 10, 24, and 48 hours after a single treatment of iPSC-derived CMs with Torin1 (200 nM). Figure 1C shows cell counts per well of a 12-well plate during differentiation. For BJRiPS-CMs, Torin1 treatment began on day 9, approximately 2 days after the onset of CM beating, with n = 3 per group per time point. Figure 1D shows the percentage of TNNT2+CMs in the viable cell population by flow cytometry for Gibco iPS-CMs (n = 3 per condition, non-significant one-way ANOVA). Figure 1E shows qPCR of selected quiescent markers (TP53, RB1, RBL2(p130), CDKN1a(p21), CDKN1b(p27), CDKN2a(p16), and HES1) and proliferation markers (MKI67, CCNA1, CCNB1, CCNC1, CCND1, CDK3, and E2F1) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days starting approximately 2 days after the onset of beating. n = 3 per condition; *p<0.05, ****p<0.001 by Holm-Sidak multiple t-test compared to control for each gene; BJR iPS-CM. Figure 1F shows representative flow cytometry plots of iPSC-derived CMs stained with Hoechst 33342 and pyronin Y to distinguish G0, G1, and S / G2 / M phases for control, 10 nM Torin1-treated, or 200 nM Torin1-treated Gibco iPS-CMs. Figure 1G shows the percentage of TNNT2+ cardiomyocytes in G0, G1, or S / G2 / M phases after 1 week of Torin1 treatment (200 nM) starting approximately 2 days after the onset of beating.n = 3 per condition; ***p<0.001, ****p<0.0001 by two-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 1H shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase in control CM after 1 week of 0.02% DMSO treatment followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group; **p<0.01 by two-way ANOVA with Sidak's multiple comparison test; Gibco iPSC-CM. Figure 1I shows the percentage of TNNT2+ CM in G0, G1, or S / G2 / M phase after 1 week of Torin1 treatment (200 nM) followed by 10% FBS starting approximately 2 days after the onset of beating, or in serum-free control. n = 3 per group, **p < 0.01 by two-way ANOVA with Sidak's multiple comparison test, Gibco iPSC-CM. DMSO is dimethyl sulfoxide, IWP-4 is Wnt production inhibitor-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 2A]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition; *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test; UCSD-CM. DMSO is dimethyl sulfoxide; TBP is TATA-binding protein. [Figure 2B]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2C]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2D]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2E]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2F]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2G]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2H]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition. *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. UCSD-CM. DMSO is dimethyl sulfoxide, and TBP is TATA-binding protein. [Figure 2I]Figures 2A-2I show that Torin1 treatment increases the expression of sarcomeric genes and enhances the contractility of iPSC-derived cardiomyocytes. Figure 2A shows qPCR analysis of selected sarcomeric genes (MYH6, MYH7, TNNT2, and TNNI3) in iPSC-derived cardiomyocytes after 7 days of treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO), starting approximately 2 days after the onset of beating. n = 3 per condition; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test; BJR iPS-CM. Figure 2B shows a representative Western blot analysis of TNNT2 and TNNI3 in Gibco iPSC-CMs after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. β-Tubulin is shown as a loading control. Figure 2C shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells. n = 3 per condition; **p < 0.01, ****p < 0.0001 by one-way ANOVA with Tukey's multiple comparison test; Gibco iPS-CM. Figure 2D shows representative images of MTF during diastole and systole, along with lateral schematics of MTF during diastole and systole. Figure 2E shows representative force vs. time plots of myofibrillar membranes (MTF) seeded with iPSC-derived cardiomyocytes treated with Torin1 (200 nM) or vehicle for 7 days, starting approximately 2 days after the onset of beating. Figure 2F shows the relative maximum contractile force (normalized to the control) quantified using myofibrillar membranes. n = 8–9 per group; **p < 0.01 by Kruskal-Wallis test; 3 batches of BJRiPS-CM (2 batches) plus 1 batch of Gibco iPS-CM (3 batches). Figure 2G shows representative immunostaining images of cardiomyocytes treated or not with Torin1 (200 nM x 7 days). Blue = DAPI, green = alpha-actinin, yellow = F-actin, magenta = TNNT2. BJRiPS-CM. Scale bar = 10 µm. Figure 2H shows sarcomere length in control (n = 13 cells) and Torin1-treated (n = 17 cells) BJRiPS-CM. Non-significant by unpaired t-test.Figure 2I shows the mean fluorescence intensity of TNNT2-Alexa Fluor 647 in TNNT2+ cells after treatment with DMSO for 7 days (control), DMSO for 7 days followed by 10% fetal bovine serum (FBS) for 2 days, Torin1 (200 nM) for 7 days, or Torin1 (200 nM) for 7 days followed by 10% FBS for 2 days. n = 3 per condition; *p < 0.01, **p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test; UCSD-CM. DMSO is dimethyl sulfoxide; TBP is TATA-binding protein. [Figure 3A]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 3B]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 3C]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 3D]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 3E]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 3F]Figures 3A–3F show that Torin1 increases oxygen consumption rate and mitochondrial polarization in iPSC-derived cardiomyocytes. Figure 3A shows the time profile of mean oxygen consumption rate normalized to baseline, as assessed by the Seahorse mitochondrial stress test. Open circles = control (n = 70 wells); closed squares = Torin1 (69 wells, 200 nM x 7 days; BJRiPS-CMs were reseeded onto Seahorse plates 24–48 h prior to the assay). Data are from two independent experiments combined. Figure 3B shows the oxygen consumption rate normalized to baseline for maximum OCR, spare expiratory capacity, and non-mitochondrial OCR, comparing control (open bars, n = 70 wells) with closed bars (open bars, n = 69 wells). *p<0.05, ***p<0.001 by two-way ANOVA with Sidak's multiple comparison test, BJRiPS-CMs. Figure 3C shows the mean Mitotracker Green FM fluorescence intensity of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3D shows the mitochondrial (ND1) to nuclear (B2M) DNA ratio of BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. *p<0.05 by two-group independent t-test, n=3 per group, BJRiPS cell lines. Figure 3E shows the relative ratio of red to green MitoProbe JC-1 fluorescence in BJRiPS-CMs after treatment with Torin1 (200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 6 per group, **p < 0.01 by Kruskal-Wallis test.Figure 3F shows qPCR analysis of selected genes related to fatty acid metabolism (PPARGC1α (PGC1α), CD36, SLC27A1 (FATP1), SLC27A6 (FATP6), and LPIN1) or glucose metabolism (GLUT1, GLUT4, PFK, and PYGM) in BJRiPS-CMs after treatment with Torin1 (10 nM, 50 nM, or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, n=3 per group. DMSO is dimethyl sulfoxide, and FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]propanedinitrile. [Figure 4A]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4B]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4C]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4D]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4E]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4F]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4G]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4H]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4I]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 4J]Figures 4A-4J show that Torin1 treatment increases the expression of selected ion channels and increases the peak rise time and fall rate of action potential profiles. Figure 4A shows qPCR analysis of selected ion channels (KCNJ2, CACNA1c, RY2, ATP2a2, SCN5a, and HCN4) in iPSC-derived cardiomyocytes after treatment with Torin1 (10 nM or 200 nM) or vehicle control (0.02% DMSO) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.05, ****p < 0.001 by two-way ANOVA with Tukey's multiple comparison test, BJR iPS cell line. Figure 4B shows flow cytometry analysis of the mean fluorescence intensity of Kir2.1 (encoded by KCNJ2) after 7 days of Torin1 treatment, starting approximately 2 days after the onset of beating. n = 3 per group; *p < 0.01 by independent two-group t-test; BJRiPS strain. Figure 4C shows the spontaneous beating rate of Torin1-treated or untreated cardiomyocytes; n = 6-10 wells per condition; *p < 0.05, ***p < 0.001 by one-way ANOVA with Tukey's multiple comparison test. Figure 4D shows a representative image demonstrating cell segmentation performed automatically by CyteSeer software. Blue = Hoechst dye; green = FluoVolt. Figure 4E shows a representative action potential profile of Torin1-treated cardiomyocytes with a longer plateau period compared to controls. CTD25 (25% duration of calcium transient, or duration at 25% decrease from maximum amplitude), CTD75 (75% duration of calcium transient, or duration at 75% decrease from maximum amplitude), T75-25 (time required for voltage to decay from 75% to 25% of maximum). Figure 4F shows peak rise time (msec) for UCSD-CM (****p<0.0001). Figure 4G shows fall rate (msec) for UCSD-CM (****p<0.0001). Figure 4H shows CTD25 time (msec) for UCSD-CM (non-significant). Figure 4I shows CTD75 time (msec) for UCSD-CM (*p<0.05).Figure 4J shows T75-25 time (msec) for UCSD-CM; ***p<0.001. Fluovolt data: n=531 cells for control and n=315 for Torin1; analysis by independent paired t-test. [Figure 5A] Figures 5A-5D show that Torin1 increases p53 expression, an effect inhibited by pifithrin-α. Figure 5A shows representative Western blots of p53, phospho-53, p21(CDKN1α), GATA4, NKX2.5, and β-tubulin from Gibco iPS-CM lysates treated with 0 (DMSO), 10, 50, or 200 nM Torin1 for 7 days, starting approximately 2 days after the onset of beating. Cells were harvested on the final day of treatment. [Figure 5B] Figures 5A-5D show that Torin1 increases p53 expression, an effect inhibited by pifithrin-α. Figure 5B shows cell cycle analysis showing the percentage of TNNT2+BJRiPS-CMs in G0, G1, or S / G2 / M phases after treatment with vehicle (DMSO, dimethyl sulfoxide), pifithrin-α (10 μM) for 1 week, Torin1 (200 nM) for 7 days, or co-treatment with pifithrin-α (10 μM) and Torin1 (200 nM) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group. Two-way ANOVA with Tukey's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 5C] Figures 5A-5D show that Torin1 increases p53 expression, an effect inhibited by pifithrin-α. Figure 5C shows representative Western blots of p53, TNNI3, p21, and β-tubulin from BJRiPS-derived cardiomyocyte lysates treated with control (DMSO), Torin1 (200 nM), pifithrin-α (10 μM), or Torin1 (200 nM) + pifithrin-α (10 μM) for 7 days, starting approximately 2 days after the onset of beating. [Figure 5D]Figures 5A-5D show that Torin1 increases p53 expression, an effect inhibited by pifithrin-α. Figure 5D shows densitometry of the TNNI3 band by Western analysis of BJRiPS-CMs, n = 3 per group, **p < 0.01 by two-way ANOVA with Tukey's multiple comparison test. [Figure 6A] Figures 6A-6E show that upregulation of p53 by nutlin-3a enhances TNNI3 expression and has a synergistic effect with Torin1. Figure 6A shows a schematic diagram illustrating the proposed mechanism of cardiomyocyte maturation. T3 is triiodothyronine. [Figure 6B] Figures 6A-6E show that upregulation of p53 by nutlin-3a enhances TNNI3 expression and has a synergistic effect with Torin1. Figure 6B shows cell cycle analysis showing the percentage of TNNT2+BJRiPS-CMs in G0, G1, or S / G2 / M phases after treatment with vehicle (DMSO, dimethyl sulfoxide), nutlin-3a (10 μM) for 24 hours, Torin1 (200 nM) for 7 days, or co-treatment with nutlin-3a (10 μM for the first 24 hours of Torin1 treatment) and Torin1 (200 nM) for 7 days, starting approximately 2 days after the onset of beating. n = 3 per group. *p < 0.05, ***p < 0.001, ****p < 0.0001 by two-way ANOVA with Tukey's multiple comparison test. [Figure 6C] Figures 6A-6E show that upregulation of p53 by nutlin-3a enhances TNNI3 expression and has a synergistic effect with Torin1. Figure 6C shows representative Western blots of MDM2, p53, TNNI3, and β-tubulin from BJRiPS-derived cardiomyocyte lysates treated with control (DMSO), nutlin-3a for 24 hours (10 μM), Torin1 for 7 days (200 nM), or Torin1 for 7 days (200 nM) + nutlin-3a (10 μM) for 24 hours, starting approximately 2 days after the onset of beating (nutlin-3a was only administered for the first 24 hours of Torin1 treatment). Cells were harvested on the final day of treatment, and BJRiPS-CMs were obtained. [Figure 6D] Figures 6A-6E show that upregulation of p53 by nutlin-3a enhances TNNI3 expression and has a synergistic effect with Torin1. Figure 6D shows densitometry of p53 bands by Western analysis; **p<0.01 by two-way ANOVA with Tukey's multiple comparison test, n=3 per condition. [Figure 6E] Figures 6A-6E show that upregulation of p53 by nutlin-3a enhances TNNI3 expression and has a synergistic effect with Torin1. Figure 6E shows densitometry of TNNI3 bands by Western analysis; *p<0.05, **p<0.01 by two-way ANOVA with Tukey's multiple comparison test, n=3 per condition, BJRIPS-CM. [Figure 7A] Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7B]Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7C] Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7D]Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7E] Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7F]Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7G] Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 7H]Figures 7A-7H show NanoString gene expression analysis using the PanCancer pathway panel for Gibco iPS-CMs, comparing cells treated with or without Torin1 and then treated with or without 10% fetal bovine serum (FBS). Figure 7A shows unsupervised hierarchical clustering analysis of cell cycle pathway genes. Figure 7B shows unsupervised hierarchical clustering analysis of protein metabolism pathway genes. Figures 7C-7E show volcano plots illustrating differential gene expression analysis of cell cycle pathway genes (Figure 7C: control vs. control + FBS, Figure 7D: control vs. Torin1, Figure 7E: Torin1 vs. Torin1 + FBS). Figures 7F-7H show volcano plots illustrating differential gene expression analysis of protein metabolism pathway genes (Figure 7F: control vs. control + FBS, Figure 7G: control vs. Torin1, Figure 7H: Torin1 vs. Torin1 + FBS). QC is a quality control. [Figure 8A] Figures 8A-8C show assessment of proliferation in non-dissociated cells. Cardiomyocytes were differentiated in 12-well plates, then fixed and stained in their original plates to minimize the possibility of selection of cells surviving dissociation. Figure 8A shows representative images of cells stained with Ki67 / TNNT2 / DAPI or phospho-H3 / TNNT2 / DAPI. Scale bar = 100 μm. Blue = DAPI, yellow = Ki67, green = pH3, magenta = TNNT2. Figure 8B shows pie charts depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are Ki67+ or Ki67-, comparing control (n = 1088 cells) with Torin1-treated cells (n = 837 cells). ****p < 0.0001 by chi-square analysis. Figure 8C shows a pie chart depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are pH3+ or pH3- in control (n=962 cells) compared with Torin1-treated cells (n=799 cells). DMSO is dimethyl sulfoxide. [Figure 8B]Figures 8A-8C show assessment of proliferation in non-dissociated cells. Cardiomyocytes were differentiated in 12-well plates, then fixed and stained in their original plates to minimize the possibility of selection of cells surviving dissociation. Figure 8A shows representative images of cells stained with Ki67 / TNNT2 / DAPI or phospho-H3 / TNNT2 / DAPI. Scale bar = 100 μm. Blue = DAPI, yellow = Ki67, green = pH3, magenta = TNNT2. Figure 8B shows pie charts depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are Ki67+ or Ki67-, comparing control (n = 1088 cells) with Torin1-treated cells (n = 837 cells). ****p < 0.0001 by chi-square analysis. Figure 8C shows a pie chart depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are pH3+ or pH3- in control (n=962 cells) compared with Torin1-treated cells (n=799 cells). DMSO is dimethyl sulfoxide. [Figure 8C] Figures 8A-8C show assessment of proliferation in non-dissociated cells. Cardiomyocytes were differentiated in 12-well plates, then fixed and stained in their original plates to minimize the possibility of selection of cells surviving dissociation. Figure 8A shows representative images of cells stained with Ki67 / TNNT2 / DAPI or phospho-H3 / TNNT2 / DAPI. Scale bar = 100 μm. Blue = DAPI, yellow = Ki67, green = pH3, magenta = TNNT2. Figure 8B shows pie charts depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are Ki67+ or Ki67-, comparing control (n = 1088 cells) with Torin1-treated cells (n = 837 cells). ****p < 0.0001 by chi-square analysis. Figure 8C shows a pie chart depicting the percentage of total DAPI / TNNT+ cardiomyocytes that are pH3+ or pH3- in control (n=962 cells) compared with Torin1-treated cells (n=799 cells). DMSO is dimethyl sulfoxide. [Figure 9A]Figures 9A-9I show qPCR analysis of selected genes (TNNT2 in Figure 9A, TNNI3 in Figure 9B, PPARGC1a in Figure 9C, RYR2 in Figure 9D, KCNJ2 in Figure 9E, CACNA1c in Figure 9F, TP53 in Figure 9G, CDKN1a(p21) in Figure 9H, and GATA4 in Figure 9I) compared with commercially available human fetal and adult heart RNA reference samples. N = 6-12 per group, data are from two to four independent experiments combined. *p<0.05, **p<0.01 by Kruskal-Wallis test, BJRiPS-CM. Vertical lines indicate that fetal and adult heart RNA were not included in the statistical analysis due to the lack of biological replicates (both were from a single tube purchased from the distributor). Error bars are not shown for fetal and adult heart RNA due to the lack of biological replicates. DMSO is dimethyl sulfoxide, and TBP is TATA binding protein. [Figure 9B] Same as above. [Figure 9C] Same as above. [Figure 9D] Same as above. [Figure 9E] Same as above. [Figure 9F] Same as above. [Figure 9G] Same as above. [Figure 9H] Same as above. [Figure 9I] Same as above. [Figure 10A] Figures 10A-10B show the evaluation of different Torin1 treatment periods. Figure 10A shows the mean fluorescence intensity of TNNT2-AlexaFluor647 after treatment with control (DMSO vehicle) or 200 nM Torin1 on differentiation days 8-11, 8-15, 9-11, 9-16, 11-14, or 11-18. n=3 per group, *p<0.05 compared to control by one-way ANOVA with Dunnett's multiple comparison test. [Figure 10B]Figures 10A-10B show the evaluation of different Torin1 treatment periods. Figure 10B shows the mean fluorescence intensity of Kir2.1 (extracellular) after treatment with control (DMSO vehicle) or 200 nM Torin1 on differentiation days 8-11, 8-15, 9-11, 9-16, 11-14, or 11-18. n=3 per group; *p<0.05, **p<0.01, ****p<0.0001 compared to control by one-way ANOVA with Dunnett's multiple comparison test. [Figure 11A] Figures 11A-11D show that delayed Torin1 treatment increases TNNI3 expression and decreases p21 expression. Figure 11A shows a schematic of the protocol used for delayed Torin1 treatment. Figure 11B shows representative Western blot images of BJRiPS-derived cardiomyocytes treated with 0, 10, 50, or 200 nM Torin1 for 4 days, harvested on day 22 of differentiation, and then assessed with antibodies against TNNI3, p21, or b-tubulin (loading control). Figure 11C shows relative band intensity densitometry analysis of the TNNI3 band (n = 3 per group, *p < 0.05 by one-way ANOVA with Tukey's multiple comparison test). Figure 11D shows relative band intensity densitometry analysis of the p21 band (n = 3 per group, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's multiple comparison test). DMSO is dimethyl sulfoxide, IWP-4 is inhibitor of Wnt production-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), and RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 11B]Figures 11A-11D show that delayed Torin1 treatment increases TNNI3 expression and decreases p21 expression. Figure 11A shows a schematic of the protocol used for delayed Torin1 treatment. Figure 11B shows representative Western blot images of BJRiPS-derived cardiomyocytes treated with 0, 10, 50, or 200 nM Torin1 for 4 days, harvested on day 22 of differentiation, and then assessed with antibodies against TNNI3, p21, or b-tubulin (loading control). Figure 11C shows relative band intensity densitometry analysis of the TNNI3 band (n = 3 per group, *p < 0.05 by one-way ANOVA with Tukey's multiple comparison test). Figure 11D shows relative band intensity densitometry analysis of the p21 band (n = 3 per group, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's multiple comparison test). DMSO is dimethyl sulfoxide, IWP-4 is inhibitor of Wnt production-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), and RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 11C]Figures 11A-11D show that delayed Torin1 treatment increases TNNI3 expression and decreases p21 expression. Figure 11A shows a schematic of the protocol used for delayed Torin1 treatment. Figure 11B shows representative Western blot images of BJRiPS-derived cardiomyocytes treated with 0, 10, 50, or 200 nM Torin1 for 4 days, harvested on day 22 of differentiation, and then assessed with antibodies against TNNI3, p21, or b-tubulin (loading control). Figure 11C shows relative band intensity densitometry analysis of the TNNI3 band (n = 3 per group, *p < 0.05 by one-way ANOVA with Tukey's multiple comparison test). Figure 11D shows relative band intensity densitometry analysis of the p21 band (n = 3 per group, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's multiple comparison test). DMSO is dimethyl sulfoxide, IWP-4 is inhibitor of Wnt production-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), and RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 11D]Figures 11A-11D show that delayed Torin1 treatment increases TNNI3 expression and decreases p21 expression. Figure 11A shows a schematic of the protocol used for delayed Torin1 treatment. Figure 11B shows representative Western blot images of BJRiPS-derived cardiomyocytes treated with 0, 10, 50, or 200 nM Torin1 for 4 days, harvested on day 22 of differentiation, and then assessed with antibodies against TNNI3, p21, or b-tubulin (loading control). Figure 11C shows relative band intensity densitometry analysis of the TNNI3 band (n = 3 per group, *p < 0.05 by one-way ANOVA with Tukey's multiple comparison test). Figure 11D shows relative band intensity densitometry analysis of the p21 band (n = 3 per group, *p < 0.05, **p < 0.01 by one-way ANOVA with Tukey's multiple comparison test). DMSO is dimethyl sulfoxide, IWP-4 is inhibitor of Wnt production-4, RI is ROCK (Rho-associated coiled-coil-containing protein kinase) inhibitor (Y-27632), and RPMI is Roswell Park Memorial Institute 1640 medium. [Figure 12A]Figures 12A-12C show the extracellular acidification rate (ECAR) in Torin1-treated cells. Figure 12A shows the mean ECAR profiles normalized to baseline during the Seahorse mitochondrial stress test. Data are from two independent experiments: control (open circles, n = 70 wells), Torin1 (200 nM) × 7 days (closed squares, n = 69 wells), and BJRiPS cell line. Figure 12B shows the mean ECAR profiles normalized to baseline during the Seahorse glycolysis stress test for BJRiPS cell line (n = 4-6 per condition). Figure 12C shows ECAR values ​​for glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification for control (DMSO) vs. Torin1 (200 nM × 7 days) (n = 4-6 per group). ****p<0.0001 by two-way ANOVA with Sidak's multiple comparison test. FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile. [Figure 12B] Figures 12A-12C show the extracellular acidification rate (ECAR) in Torin1-treated cells. Figure 12A shows the mean ECAR profiles normalized to baseline during the Seahorse mitochondrial stress test. Data are from two independent experiments: control (open circles, n = 70 wells), Torin1 (200 nM) × 7 days (closed squares, n = 69 wells), and BJRiPS cell line. Figure 12B shows the mean ECAR profiles normalized to baseline during the Seahorse glycolysis stress test for BJRiPS cell line (n = 4-6 per condition). Figure 12C shows ECAR values ​​for glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification for control (DMSO) vs. Torin1 (200 nM × 7 days) (n = 4-6 per group). ****p<0.0001 by two-way ANOVA with Sidak's multiple comparison test. FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile. [Figure 12C]Figures 12A-12C show the extracellular acidification rate (ECAR) in Torin1-treated cells. Figure 12A shows the mean ECAR profiles normalized to baseline during the Seahorse mitochondrial stress test. Data are from two independent experiments: control (open circles, n = 70 wells), Torin1 (200 nM) × 7 days (closed squares, n = 69 wells), and BJRiPS cell line. Figure 12B shows the mean ECAR profiles normalized to baseline during the Seahorse glycolysis stress test for BJRiPS cell line (n = 4-6 per condition). Figure 12C shows ECAR values ​​for glycolysis, glycolytic capacity, glycolytic reserve, and non-glycolytic acidification for control (DMSO) vs. Torin1 (200 nM × 7 days) (n = 4-6 per group). ****p<0.0001 by two-way ANOVA with Sidak's multiple comparison test. FCCP is 2-[2-[4-(trifluoromethoxy)phenyl]hydrazinylidene]-propanedinitrile. [Figure 13A] Figures 13A-13C show evaluation of mitochondrial gene and protein expression and the effects of fatty acids. Figure 13A shows qPCR analysis of selected mitochondrial genes (TFAM, DNML1, MFN1, MFN2, OPA1, PHB2). n = 3 per group. Figure 13B shows a representative Western blot analysis of selected mitochondrial proteins (MFN1, OPA1, DRP1, TFAM1) from Gibco iPS-CMs after 7 days of Torin1 treatment starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. Figure 13C shows a representative Western blot analysis of selected metabolic proteins (OPA1, phosphor-AMPK, CD36) from BJR iPS-CMs after 7 days of Torin1 treatment + / - fatty acids (chemically defined lipid concentrate (Gibco) diluted 1:100) starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. DMSO is dimethyl sulfoxide, and TBP is TATA binding protein. [Figure 13B]Figures 13A-13C show evaluation of mitochondrial gene and protein expression and the effects of fatty acids. Figure 13A shows qPCR analysis of selected mitochondrial genes (TFAM, DNML1, MFN1, MFN2, OPA1, PHB2). n = 3 per group. Figure 13B shows a representative Western blot analysis of selected mitochondrial proteins (MFN1, OPA1, DRP1, TFAM1) from Gibco iPS-CMs after 7 days of Torin1 treatment starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. Figure 13C shows a representative Western blot analysis of selected metabolic proteins (OPA1, phosphor-AMPK, CD36) from BJR iPS-CMs after 7 days of Torin1 treatment + / - fatty acids (chemically defined lipid concentrate (Gibco) diluted 1:100) starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. DMSO is dimethyl sulfoxide, and TBP is TATA binding protein. [Figure 13C]Figures 13A-13C show evaluation of mitochondrial gene and protein expression and the effects of fatty acids. Figure 13A shows qPCR analysis of selected mitochondrial genes (TFAM, DNML1, MFN1, MFN2, OPA1, PHB2). n = 3 per group. Figure 13B shows a representative Western blot analysis of selected mitochondrial proteins (MFN1, OPA1, DRP1, TFAM1) from Gibco iPS-CMs after 7 days of Torin1 treatment starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. Figure 13C shows a representative Western blot analysis of selected metabolic proteins (OPA1, phosphor-AMPK, CD36) from BJR iPS-CMs after 7 days of Torin1 treatment + / - fatty acids (chemically defined lipid concentrate (Gibco) diluted 1:100) starting approximately 2 days after the onset of beating, with b-tubulin shown as a loading control. DMSO is dimethyl sulfoxide, and TBP is TATA binding protein. [Figure 14A] Figures 14A-14C show calcium transient analysis using a Vala Kinetic Image Cytometer. Calcium transfer was assessed using Fluo-4 AM dye. Isoproterenol (1 μM, "iso") was added to some wells to assess isoproterenol responsiveness. Figure 14A shows calcium peak rise time. Figure 14B shows calcium full width at half maximum (FWHM, the width of the profile at which the calcium amplitude is half its maximum) time. Figure 14C shows calcium peak decay time. Control, n = 129 cells; control + 1 μM iso, n = 41 cells; Torin1, n = 274 cells; Torin1 + 1 μM iso, n = 65 cells. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. [Figure 14B]Figures 14A-14C show calcium transient analysis using a Vala Kinetic Image Cytometer. Calcium transfer was assessed using Fluo-4 AM dye. Isoproterenol (1 μM, "iso") was added to some wells to assess isoproterenol responsiveness. Figure 14A shows calcium peak rise time. Figure 14B shows calcium full width at half maximum (FWHM, the width of the profile at which the calcium amplitude is half its maximum) time. Figure 14C shows calcium peak decay time. Control, n = 129 cells; control + 1 μM iso, n = 41 cells; Torin1, n = 274 cells; Torin1 + 1 μM iso, n = 65 cells. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. [Figure 14C] Figures 14A-14C show calcium transient analysis using a Vala Kinetic Image Cytometer. Calcium transfer was assessed using Fluo-4 AM dye. Isoproterenol (1 μM, "iso") was added to some wells to assess isoproterenol responsiveness. Figure 14A shows calcium peak rise time. Figure 14B shows calcium full width at half maximum (FWHM, the width of the profile at which the calcium amplitude is half its maximum) time. Figure 14C shows calcium peak decay time. Control, n = 129 cells; control + 1 μM iso, n = 41 cells; Torin1, n = 274 cells; Torin1 + 1 μM iso, n = 65 cells. *p < 0.05, **p < 0.01, ****p < 0.0001 by one-way ANOVA with Sidak's multiple comparison test. [Figure 15A]NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15B] NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15C]NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15D] NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15E]NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15F] NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15G]NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15H] NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 15I]NanoString gene expression analysis with the PanCancer pathway panel comparing cells treated or not with Torin1, followed by treatment or not with 10% fetal bovine serum (FBS). Figure 15A shows unsupervised hierarchical clustering analysis of all genes. Figure 15B shows principal component analysis of all genes. Figures 15C-15E show volcano plots showing differential gene expression analysis of all genes (Figure 15C: control vs. control + FBS, Figure 15D: control vs. Torin1, Figure 15E: Torin1 vs. Torin1 + FBS). Figure 15F shows principal component analysis of cell cycle pathways. Figure 15G shows cell cycle pathway scores determined by nSolver software analysis. Figure 15H shows principal components of protein metabolism pathways. Figure 15I shows protein metabolism pathway scores determined by nSolver software analysis. [Figure 16] Figure 16 shows that cellular quiescence is a temporary non-proliferative state that can last for the life of an organism. Temporary mTOR inhibition of cell cycle arrest can result in cellular quiescence. Cell quiescence can be a variety of states with varying degrees of quiescence and other molecular states that fit well into the overall concept of quiescence. [Figure 17A] 17A-17B show that Torin1 inhibits phosphorylation of S6K and Akt and enhances phosphorylation of AMPK. Figure 17A shows a schematic diagram of the downstream pathways of mTORC1 and mTORC2. [Figure 17B] Figures 17A-17B show that Torin1 inhibits the phosphorylation of S6K and Akt and enhances the phosphorylation of AMPK. Figure 17B shows Western analysis demonstrating the phosphorylation and expression of selected downstream pathways of mTORC1 and mTORC2 0-48 hours after administration of Torin1 (200 nM) to BJRiPSC-derived human cardiomyocytes. [Figure 18A]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18B]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18C]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18D]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18E]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18F]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 18G]Figures 18A-18G show that Torin1 enhances the expression of selected markers of maturation in cardiomyocytes derived from three different cell lines. Figure 18A shows a schematic of the cardiomyocyte differentiation and maturation protocol. Torin1 is added approximately 2 days after the initiation of beating. Figures 18B-18D show quantitative PCR showing dose-dependent increases in TNNI3 (Figure 18B), CACNA1c (Figure 18C), and ATP2a2 (SERCA2a) (Figure 18D) following treatment with vehicle (0.02% DMSO), 10, 50, or 200 nM Torin1 on day 9 of differentiation (BJRiPS-derived cardiomyocytes). *p<0.05, **p<0.01 by Kruskal-Wallis test with Dunn's multiple comparison test. Figure 18E shows that the mean fluorescence intensity of TNNT2 increased after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines, suggesting increased TNNT2 expression. *p<0.05, **p<0.01 by two-way ANOVA. Figures 18F-18G show Western blots (Figure 18F) and densitometric analysis (Figure 18G) demonstrating increased expression of TNNI3 following 4 days of treatment with vehicle or Torin1 (10, 50, 200 nM) in BJRiPS-derived cardiomyocytes. *p<0.05 by one-way ANOVA. [Figure 19] A schematic diagram of the regulation of cap-dependent protein translation by EIF4E is shown. Phosphorylation of 4E-BP1 by mTORC1 releases EIF4E from 4E-BP1 and promotes the interaction of EIF4E with EIF4G, resulting in mRNA translation. Torin1 treatment can reduce EIF4E-dependent protein translation. [Figure 20A]Figures 20A-20D show qPCR of selected genes in BJRIPS-derived cardiomyocytes. Figure 20A shows a trend toward higher TNNI3 expression when Torin1 initiation occurs approximately 2 days after the onset of beating. Figures 20B-20D show that control (untreated) cells exhibit peak EIFG1 mRNA expression around day 9 of differentiation (Figure 20B), peak EIF4E mRNA expression around days 7-9 (Figure 20C), and peak 4EBP1 expression around day 11 (Figure 20D). These data suggest that the EIF4E / 4EBP1 stoichiometry fluctuates throughout differentiation, which may render cells more or less responsive to mTOR inhibition. [Figure 20B] Figures 20A-20D show qPCR of selected genes in BJRIPS-derived cardiomyocytes. Figure 20A shows a trend toward higher TNNI3 expression when Torin1 initiation occurs approximately 2 days after the onset of beating. Figures 20B-20D show that control (untreated) cells exhibit peak EIFG1 mRNA expression around day 9 of differentiation (Figure 20B), peak EIF4E mRNA expression around days 7-9 (Figure 20C), and peak 4EBP1 expression around day 11 (Figure 20D). These data suggest that the EIF4E / 4EBP1 stoichiometry fluctuates throughout differentiation, which may render cells more or less responsive to mTOR inhibition. [Figure 20C] Figures 20A-20D show qPCR of selected genes in BJRIPS-derived cardiomyocytes. Figure 20A shows a trend toward higher TNNI3 expression when Torin1 initiation occurs approximately 2 days after the onset of beating. Figures 20B-20D show that control (untreated) cells exhibit peak EIFG1 mRNA expression around day 9 of differentiation (Figure 20B), peak EIF4E mRNA expression around days 7-9 (Figure 20C), and peak 4EBP1 expression around day 11 (Figure 20D). These data suggest that the EIF4E / 4EBP1 stoichiometry fluctuates throughout differentiation, which may render cells more or less responsive to mTOR inhibition. [Figure 20D]Figures 20A-20D show qPCR of selected genes in BJRIPS-derived cardiomyocytes. Figure 20A shows a trend toward higher TNNI3 expression when Torin1 initiation occurs approximately 2 days after the onset of beating. Figures 20B-20D show that control (untreated) cells exhibit peak EIFG1 mRNA expression around day 9 of differentiation (Figure 20B), peak EIF4E mRNA expression around days 7-9 (Figure 20C), and peak 4EBP1 expression around day 11 (Figure 20D). These data suggest that the EIF4E / 4EBP1 stoichiometry fluctuates throughout differentiation, which may render cells more or less responsive to mTOR inhibition. [Figure 21] A schematic diagram of EIF4E-dependent and EIF4E2-dependent mRNA translation is shown. While 4E-BP1 bound to EIF4E inhibits protein translation, phosphorylation of 4E-BP1 by mTORC1 liberates EIF4E, allowing it to bind to EIF4G instead. The EIF4E / EIF4G complex initiates EIF4E-dependent mRNA translation. In contrast, EIF4E2 binds to HIF2α and initiates EIF4E2-dependent translation. Rapamycin increases HIF2a expression. [Figure 22A] A-B show Seahorse mitochondria stress assays of Gibco iPSC-derived cardiomyocytes. A shows the oxygen consumption rate (OCR) at baseline and after 7 days of treatment with oligomycin, FCCP, and rotenone / antimycin A with or without Torin1. B shows that Torin1 treatment for 7 days significantly increases maximal OCR. **p<0.01 by Student's t-test. These results suggest that Torin1 treatment increases the oxidative capacity of iPSC-derived cardiomyocytes. [Figure 22B] Same as above. [Figure 23]A schematic representation of the quiescent G0 state, where cells can cycle between deeper and shallower quiescence rather than an immutable phenotype, is shown. mTOR controls whether cells remain quiescent or progress to senescence, as cell cycle arrest without mTOR inhibition leads to senescence, whereas simultaneous cell cycle arrest and mTOR inhibition leads to quiescence (31). [Figure 24] A schematic diagram of the Rb-E2F quiescence regulator is shown. Shallow quiescence requires a low level of growth stimulus to activate E2F and re-initiate the cell cycle, whereas deep quiescence requires a higher level of growth stimulus. [Figure 25A] A–F show qPCR analysis of mRNA expression of (A) E1F2, (B) E2F4, (C) RBL2 (retinoblastoma-like protein 2, also known as p130), (D) MDM2 (mouse double mutant 2 homolog, an E3 ubiquitin-protein ligase), (E) CDKN1a (cyclin-dependent kinase inhibitor 1a, also known as p21), and (F) CDK2 (cyclin-dependent kinase 2) with or without Torin1 treatment. Cardiomyocytes derived from BJRiPS cells began beating approximately at day 8, while Torin1 treatment (200 nM) began at day 9. These results suggest that Torin1 regulates the expression of cell cycle regulators. [Figure 25B] Same as above. [Figure 25C] Same as above. [Figure 25D] Same as above. [Figure 25E] Same as above. [Figure 25F] Same as above. [Figure 26A] Panels A and B show GCaMP-derived cardiomyocytes differentiated in 3D suspension culture. Panel A shows a representative spheroid on day 0 of differentiation, demonstrating that the ideal size at the start of differentiation is approximately 300 μm. Panel B shows the % TNNT2+ cardiomyocytes on day 10 of differentiation. Torin1 treatment was performed for 24 hours starting on day 9, followed by assay, and the % TNNT2+ cells tended to be higher in Torin1-treated wells. [Figure 26B]Same as above. [Figure 27] A schematic diagram of the differentiation protocol for producing cardiomyocytes is shown. The baseline differentiation protocol (top panel) was based on Lian et al., Nature Protocols 2013, and was modified to include inhibition of the mTOR pathway after the initiation of cardiomyocyte beating. Small molecules were used to sequentially activate and then inhibit the Wnt pathway to generate human cardiomyocytes from human iPS cells. Using the previously published protocol, the cells remained immature, more similar to fetal cardiomyocytes. After the cells began beating (around day 8–10 of differentiation), the mTOR pathway was inhibited to enhance cardiomyocyte maturation. [Figure 28] We show that mTOR complex 1 (mTORC1) is inhibited by Torin1, as indicated by decreased phosphorylation of the ribosomal protein S6. The mTORC1 complex results in the phosphorylation of the ribosomal protein S6, which results in decreased phosphorylation of S6 when mTOR is inhibited. Western blots treated with Torin1 (10 nM, 50 nM, and 200 nM) on days 25–29 (after lactate enrichment) show decreased phosphorylation of S6 when mTOR is inhibited. [Figure 29] We demonstrate that mTOR inhibition reduces cardiomyocyte beating rate. Normal mature adult human cardiomyocytes naturally beat 20–30 times per minute, and the human heart rate is faster because pacemaker tissue drives the heart rate. Immature human cardiomyocytes beat at a faster rate, potentially leading to life-threatening arrhythmias when delivered to animal models of heart failure. Cardiomyocytes with reduced automaticity (i.e., reduced driving force for spontaneous beating) may reduce the risk of arrhythmia in cell therapy approaches. A slower intrinsic beating rate may indicate reduced automaticity. We evaluated the mTOR inhibitors Torin1 (10 nM, 50 nM, and 200 nM) and Torin2 (200 nM), and demonstrated that mTOR inhibition reduced cardiomyocyte beating rate. BJRiPS-CMs were treated in duplicate. Treatment began on day 11 of differentiation and continued for 4–5 days. N = 4–10 wells per group. [Figure 30] We demonstrate that Torin1 increases the RNA expression of contractile proteins in cardiomyocytes. Cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain protein 6 (MYH6), and myosin heavy chain protein 7 (MYH7) are components of the contractile apparatus of cardiomyocytes, and mature cardiomyocytes possess higher levels of these proteins. Torin1 increased the RNA expression of these proteins in a dose-dependent manner (10 nM, 50 nM, and 200 nM). Increased expression of these proteins may enhance cardiomyocyte contractility. qPCR was performed to measure relative mRNA expression. BJRiPS-derived cardiomyocytes were treated with RBI containing Torin1 on days 11 to 18 of the differentiation protocol. N = 3 per group, single cell dose. [Figure 31A] Increased TNNI3 protein expression after Torin1 treatment. Western blot (A) and densitometric analysis (B) show increased protein expression of TNNI3, an essential cardiac sarcomeric protein, in BJRiPS-derived cardiomyocytes after 4 days of Torin1 treatment (10 nM, 50 nM, and 200 nM). Lactate purification occurred over 3 days. Torin1 was administered on days 25–29, with 50 μg of protein given. [Figure 31B] Same as above. [Figure 32] This figure shows an increase in ion channel expression after Torin1 treatment. A more mature ion channel expression profile is thought to be necessary for the electrical maturity of cardiomyocytes. Ion channel gene expression increased with Torin1 treatment (10 nM, 50 nM, and 200 nM). The ion channel gene expression examined included KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and SERCA2a (ATP2a2). Relative mRNA expression was measured using qPCR. BJRiPS-derived cardiomyocytes were treated with RBI containing Torin1 on days 11 to 18 of the differentiation protocol. N = 3 per group, single cell run. [Figure 33]Increased expression of NPPB (BNP) after Torin1 treatment. Brain natriuretic peptide (BNP) is produced by cardiomyocytes during development. Brain natriuretic peptide (BNP) expression in cardiomyocytes increased after treatment with Torin1 (10 nM, 50 nM, and 200 nM). Relative mRNA expression was measured using qPCR. BJRiPS-derived cardiomyocytes were treated with RBI containing Torin1 on days 11 to 18 of the differentiation protocol. N=3 / group, single cell batch. [Figure 34] This figure shows increased expression of the transcription factor REST after Torin1 treatment. REST is a transcription factor that regulates the expression of certain ion channels in cardiac cells. Torin1 (10 nM, 50 nM, and 200 nM) increases REST gene expression. Increased REST gene expression by mTOR inhibition may stimulate cardiomyocyte maturation. Relative mRNA expression was measured using qPCR and flow cytometry. qPCR: 1 replicate, n=3 / group; flow cytometry: 1-3 replicates. [Figure 35] Increased expression of PGG1-alpha (PPARGC1a), a regulator of the oxidative phosphorylation pathway, is shown after Torin1 treatment (10 nM, 50 nM, and 200 nM). Mature cardiomyocytes switch their energy source from glycolysis to oxidative phosphorylation. Upregulation of PGC1alpha may enhance pathways related to oxidative phosphorylation, suggesting a more mature phenotype. Relative mRNA expression was measured using qPCR. BJRiPS-derived cardiomyocytes were treated with RBI containing Torin1 on days 11-18 of the differentiation protocol. N=3 / group, single cell dose. [Figure 36] Mean fluorescence intensity of TNNT2 after treatment with vehicle or Torin1 in cardiomyocytes derived from BJRiPS, GCaMP, or Gibco cell lines is shown, suggesting increased TNNT2 expression in cardiomyocytes derived from multiple cell lines. *p<0.05, **p<0.01 by two-way ANOVA. [Figure 37A]A-B show that Torin1 treatment can increase the oxidative capacity of iPSC-derived cardiomyocytes. Seahorse mitochondria stress assays were performed on Gibco iPSC-derived cardiomyocytes. A shows the oxygen consumption rate (OCR) at baseline and after 7 days of treatment with oligomycin, FCCP, and rotenone / antimycin A with or without Torin1. B shows that Torin1 treatment over 7 days significantly increases maximal OCR. **p<0.01 by Student's t-test. [Figure 37B] Same as above. [Figure 38A] Figures A and B show that Torin1 inhibits phosphorylation of S6K and Akt and enhances phosphorylation of AMPK. Figure A shows a schematic diagram of downstream pathways of mTORC1 and mTORC2. Figure B shows Western analysis showing the phosphorylation and expression of selected downstream pathways of mTORC1 and mTORC2 0 to 48 hours after administration of Torin1 (200 nM) to BJRiPS iPS-derived human cardiomyocytes. [Figure 38B] Same as above. [Figure 39A] Panels A and B show GCaMP-derived cardiomyocytes differentiated in 3D suspension culture. Panel A shows a representative spheroid on day 0 of differentiation, demonstrating that the ideal size at the start of differentiation is approximately 300 μm. Panel B shows the % TNNT2+ cardiomyocytes on day 10 of differentiation. Torin1 treatment was initiated on day 9 and continued for 24 hours before assay, and the % TNNT2+ cells tended to be higher in Torin1-treated wells. [Figure 39B] [Figure 40] Exemplary structures of mTOR inhibitors. DETAILED DESCRIPTION OF THE INVENTION

[0036] Current differentiation protocols for producing cardiomyocytes from human induced pluripotent stem cells (iPSCs) can yield highly pure cardiomyocyte populations, as determined by the expression of cardiac troponin T. However, these cardiomyocytes remain immature, closer to the fetal state, exhibiting lower maximal contractile force, slower rise rates, and immature mitochondrial function compared to adult cardiomyocytes. The immaturity of iPSC-derived cardiomyocytes may pose a significant barrier to the clinical translation of cardiomyocyte therapy for cardiac disease. During development, cardiomyocytes undergo a transition from a proliferative state in the fetus to a more mature but quiescent state after birth. The mechanistic target of rapamycin (mTOR) signaling pathway plays a crucial role in nutrient sensing and growth. Temporary inhibition of the mTOR signaling pathway can induce cardiomyocyte quiescence and enhance cardiomyocyte maturation.

[0037] Small molecules that modulate the Wnt pathway were used to differentiate cardiomyocytes from iPSC lines. In some embodiments, inhibitors of the mTOR pathway were used at various time points to quantify the contractile, metabolic, and electrophysiological properties of mature iPSC-derived cardiomyocytes. In some embodiments, small molecule inhibitors were used to inhibit p53 signaling, and in some embodiments, different small molecule inhibitors were used to upregulate p53 and enhance its activation.

[0038] In some embodiments, mTOR inhibitors increased the percentage of quiescent (G0) cells (e.g., 24-48%, p<0.05) compared to vehicle controls. Additionally, when mTOR inhibitors were initiated after the onset of cardiomyocyte beating, mTOR inhibitors could dose-dependently increase the expression of select sarcomeric proteins (including TNNI3) and ion channels (including Kir2.1). In some embodiments, treated cells exhibited increased relative maximum contractile force, increased maximum oxygen consumption rate, decreased peak rise time, and increased fall rate. Treatment with mTOR inhibitors could increase p53 protein expression, which could be inhibited by administering p53 inhibitors. In contrast, p53 activators could independently upregulate p53, resulting in increased TNNI3 expression, and could act synergistically with mTOR inhibitors to further increase both p53 and TNNI3 expression.

[0039] Aspects of the present disclosure relate to compositions, methods, kits, and agents for generating cardiomyocytes (referred to herein as non-native cardiomyocytes, non-native cardiomyocytes, quiescent cardiomyocytes, or mature cardiomyocytes) from at least one stem cell, and the mature or quiescent cardiomyocytes produced by those compositions, methods, kits, and agents for use in cell therapies, assays, and various therapeutic methods.

[0040] In vitro produced cardiomyocytes generated according to the methods described herein exhibit many advantages, for example, they are electrically mature (e.g., exhibit reduced automaticity), contractilely mature, and metabolically mature. Additionally, the generated cardiomyocytes may provide a new backbone for cell therapy (e.g., transplantation into subjects requiring additional and / or functional cardiomyocytes) and research.

[0041] definition For convenience, some terms employed in the present specification, examples, and appended claims are collected here. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0042] As used herein, the term "somatic cell" refers to any cell that makes up the body of an organism, as opposed to a germline cell. In mammals, germline cells (also known as "gametes") are sperm and eggs that fuse during fertilization to produce a cell called a zygote, from which an entire mammalian embryo develops. All other cell types in a mammal's body—although unlike sperm and eggs, whose cells of origin (gametocytes) are undifferentiated stem cells—are somatic cell types, and internal organs, skin, bone, blood, and connective tissue are all made up of somatic cells. In some embodiments, a somatic cell is a "non-embryonic somatic cell," which refers to a somatic cell that is not present in or obtained from an embryo or that cannot be obtained by propagation of such a cell in a test tube. In some embodiments, a somatic cell is an "adult somatic cell," which refers to a cell that is present in or obtained from an organism other than an embryo or fetus, or that can be obtained by propagation of such a cell in a test tube.

[0043] As used herein, the term "adult cell" refers to a cell found throughout the body after embryonic development.

[0044] The terms "precursor" or "precursor" cells are used interchangeably herein and refer to cells that have a more primitive cellular phenotype (i.e., that is earlier along a developmental pathway or progress than a fully differentiated cell) than the cells they can develop from by differentiation. Precursor cells often also have significant or very high proliferative capacity. A progenitor cell can give rise to multiple different differentiated cell types or a single differentiated cell type, depending on the developmental pathway and the environment in which the cell develops and differentiates.

[0045] The term "phenotype" refers to the overall biological trait or traits that characterize a cell or organism under a particular set of environmental conditions and factors, regardless of its actual genotype.

[0046] As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into more than one differentiated cell type, preferably into cell types characteristic of all three germ cell layers. Pluripotent cells are primarily characterized by their ability to differentiate into more than one cell type, preferably into all three germ layers, using, for example, a nude mouse teratoma formation assay. While pluripotency can also be demonstrated by the expression of embryonic stem (ES) cell markers, the preferred test for pluripotency is the demonstration of the ability to differentiate into cells of each of the three germ layers. It should be noted that simply culturing such cells does not render them pluripotent. Reprogrammed pluripotent cells (e.g., iPS cells, as that term is defined herein) also have the characteristic of being able to be passaged for extended periods without losing growth potential, compared to their primary cell parents, which typically have the ability to divide only a limited number of times in culture.

[0047] As used herein, the terms "iPS cells" and "induced pluripotent stem cells" are used interchangeably and refer to pluripotent stem cells that have been artificially derived (e.g., induced or by complete reversion) from non-pluripotent cells, typically adult somatic cells, for example, by inducing forced expression of one or more genes.

[0048] As used herein, the term "stem cell" refers to an undifferentiated cell that is capable of proliferation and the generation of many more progenitor cells capable of giving rise to a large number of mother cells, which in turn can give rise to differentiated or differentiable daughter cells. The daughter cells themselves can be induced to proliferate and generate progeny that subsequently differentiate into one or more mature cell types, while also retaining one or more cell types possessing the parent developmental potential. The term "stem cell" refers to a subset of progenitor cells that, under certain circumstances, have the ability or potential to differentiate into a more specialized or specialized phenotype and, under certain circumstances, retain the ability to proliferate largely without differentiation. In one embodiment, the term stem cell generally refers to a naturally occurring mother cell that causes its progeny (grandchildren) to specialize, sometimes in various directions, by differentiation, e.g., the acquisition of entirely individual characteristics, as occurs in the gradual diversification of embryonic cells and tissues. Cell differentiation is a complex process that typically occurs through many cell divisions. Differentiated cells can be derived from pluripotent cells, which themselves are derived from pluripotent cells, etc. Although each of these pluripotent cells can be considered a stem cell, the range of cell types each can give rise to can vary considerably. Some differentiated cells also have the ability to give rise to cells with greater developmental potential. Such ability can be natural or artificially induced by treatment with various factors. In many biological cases, stem cells are also "pluripotent" because they can generate progeny of more than one distinct cell type, but this is not required for "stemness." Self-renewal is another classic part of the definition of stem cells, which, as used herein, is essential. In theory, self-renewal can occur by either of two primary mechanisms. Stem cells can divide asymmetrically into one daughter that retains its stem state and another daughter that expresses some distinct, otherwise specific function and phenotype. Alternatively, some stem cells in a population can divide symmetrically into two stems, thus maintaining some stem cells as a whole while other cells in the population give rise only to differentiated progeny.Although cells that start as stem cells may progress to a differentiated phenotype, it is technically possible for the stem cell phenotype to subsequently "reverse" and re-emerge, a term often referred to by those skilled in the art as "dedifferentiation," "reprogramming," or "reverse differentiation." As used herein, the term "pluripotent stem cells" includes embryonic stem cells, induced pluripotent stem cells, placental stem cells, and the like.

[0049] As used herein, "quiescence" or "cellular quiescence" refers to a cellular resting state induced by nutrient deprivation and characterized by the ability to resume the cell cycle in response to appropriate stimuli. Quiescent cells retain metabolic and transcriptional activity. Cells undergo a transient initiation period of G0, a deep G0, and a shallower state of quiescence, G, in which cells are more responsive to stimuli that induce reentry into the cell cycle. alert Quiescent cardiomyocytes may have varying depths of quiescence, including the expression of one or more quiescent markers, including p16 and p130.

[0050] The terms "intrinsic cardiomyocyte" or "intrinsic mature cardiomyocyte" are used herein to refer to mature cardiomyocytes. Mature cardiomyocytes may exhibit electrical maturity, contractile maturity, and / or metabolic maturity. Cardiomyocyte phenotypes are well known to those skilled in the art and include, for example, the ability to beat spontaneously, expression of markers such as cardiac troponin, TNNT2, TNNI3, myosin heavy chain, MYH6, MYH7, ryanodine receptor (RyR), sodium channel protein SCN5a, voltage-gated potassium channel KCNJ2, ATP2A2, PPARGC1a, Cx43, and distinct morphological traits, such as having organized sarcomeres, rod-shaped cells, and having T-tubules.

[0051] As used herein, the terms "cardiomyocytes," "non-native cardiomyocytes," "non-native cardiomyocytes," "quiescent cardiomyocytes," and "mature cardiomyocytes" all refer to cardiomyocytes produced by the methods described herein. Cardiomyocytes can be ventricular, atrial, and / or nodal cardiomyocytes, or a mixed population of cardiomyocytes. Cardiomyocytes can exhibit one or more characteristics that may be common to endogenous cardiomyocytes, including, but not limited to, the ability to beat spontaneously, electrical maturity, metabolic maturity, contractile maturity, appropriate expression of one or more genetic markers (e.g., TNNI3, TNNT1, MYH6, MYH7, KCNJ2, RyR, and REST), appropriate expression of one or more quiescent markers (e.g., p16 and p130), appropriate morphological characteristics (e.g., rod-shaped cells and organized sarcomeres), and proliferation in culture. However, non-native cardiomyocytes are not identical to and can be distinguished from endogenous cardiomyocytes described herein, including distinction based on gene expression.

[0052] The term "cardiomyocyte marker" refers to, but is not limited to, proteins, peptides, nucleic acids, protein and nucleic acid polymorphisms, splice variants, protein or nucleic acid fragments, elements, and other analytes that are specifically expressed or present in endogenous cardiac myocytes. Exemplary cardiac myocyte markers include, but are not limited to, cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), potassium channel KCNJ2, repressor element 1 transcriptional silencing factor (REST), ryanodine receptor (RyR), sodium channel (SCN5a), and those described in Yang et al. Circ. Res. 2014;114(3):511-23.

[0053] As used herein, the term "immature cardiomyocyte" refers to a cardiomyocyte that is immature (e.g., electrically, metabolically, and / or contractilely). Immature cardiomyocytes exhibit automaticity, or pacemaker-like activity, have a higher resting membrane potential and a slower rate of rise, have a less well-organized sarcomere structure and a lower maximal contractile force, lack T-tubules, obtain energy primarily through glycolysis (rather than oxidative phosphorylation), and may be in a senescent rather than quiescent state.

[0054] As used herein, the term "proliferation" refers to the growth and division of cells. In some embodiments, the term "proliferation" as used herein with respect to cells refers to a group of cells that can increase in number over a period of time.

[0055] In the context of cellular ontogeny, the adjectives "differentiated" or "differentiating" are relative terms, meaning that a "differentiated cell" is one that has progressed further down the developmental pathway than the cell to which it is being compared. Thus, stem cells can differentiate into lineage-committed progenitor cells (e.g., mesodermal stem cells), which in turn can differentiate into other types of progenitor cells further down the pathway (e.g., cardiomyocyte precursors), and then into terminally differentiated cells that fulfill characteristic roles in a given tissue type and may or may not retain the capacity for further proliferation.

[0056] As used herein, the term "agent" refers to any compound or substance, including, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. An "agent" can be any chemical entity or moiety, including, but not limited to, synthetic and natural proteinaceous and non-proteinaceous entities. In some embodiments, an agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, an oligomer of nucleic acid, an amino acid, or a carbohydrate, including, but not limited to, a protein, an oligonucleotide, a ribozyme, a DNAzyme, a glycoprotein, an siRNA, a lipoprotein, an aptamer, and modifications and combinations thereof. In certain embodiments, an agent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties, including macrolides, leptomycin, and related natural products or their analogs. The compound may be known to have a desired activity and / or property, or may be selected from a diverse library of compounds.

[0057] As used herein, the term "contacting" (i.e., contacting at least one immature cardiomyocyte or its precursor with a maturation factor or combination of maturation factors) is intended to include incubating cells with differentiation medium and / or agents in vitro (e.g., adding maturation factors to cells in culture). In some embodiments, the term "contacting" is not intended to include exposing cells in vivo to a compound disclosed herein that may be naturally present in a subject (i.e., exposure that may occur as a result of a natural physiological process). The step of contacting at least one immature cardiomyocyte or its precursor with a maturation factor as in the embodiments described herein can be carried out in any suitable manner. For example, cells may be treated in adherent or suspension culture. In some embodiments, cells are treated under conditions that promote the formation of cardiomyocytes. The present disclosure contemplates any conditions that promote the formation of mature cardiomyocytes. Examples of conditions that promote the formation of mature cardiomyocytes include, but are not limited to, suspension culture in low-adherent tissue culture plates, spinner flasks, and Aggrewell plates. In some embodiments, the inventors observed that mature cardiomyocytes remained stable in culture. In some embodiments, serum (eg, heat-inactivated fetal bovine serum) is added before the cells are dissociated and replated.

[0058] It is understood that cells contacted with a maturation factor (e.g., a cardiomyocyte maturation factor) may be simultaneously or subsequently contacted with another agent, e.g., a differentiation agent, or an environment that stabilizes the cells or further differentiates or matures the cells.

[0059] Similarly, at least one immature cardiomyocyte or its precursor may be contacted with at least one cardiomyocyte maturation factor and then with at least one other cardiomyocyte maturation factor. In some embodiments, the cells are contacted with at least one cardiomyocyte maturation factor but the contacting is temporally separated; in some embodiments, the cells are contacted with at least one cardiomyocyte maturation factor substantially simultaneously. In some embodiments, the cells are contacted with at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten cardiomyocyte maturation factors.

[0060] The term "cell culture medium" (also referred to herein as "culture medium" or "culture medium") referred to herein is a medium for culturing cells, containing nutrients that maintain cell viability and support growth. Cell culture media may contain any of the following in appropriate combinations: salt(s), buffer(s), amino acids, glucose or other sugar(s), antibodies, serum or serum replacements, and other components, such as peptide growth factors. Cell culture media commonly used for specific cell types are known to those skilled in the art.

[0061] The term "cell line" refers to a population of generally or substantially identical cells, typically derived from a single ancestral cell or from a defined and / or substantially identical population of ancestral cells. A cell line may be one that has been or can be maintained in culture for an extended period of time (e.g., months, years, indefinite period). It may have undergone a spontaneous or induced process of transformation that confers indefinite culture life to the cells. Cell lines include all cell lines recognized as such in the art. It will be recognized that over time, cells undergo mutations and possibly epigenetic changes such that at least some properties of individual cells of a cell line may differ from one another. In some embodiments, a cell line comprises cardiomyocytes as described herein.

[0062] The term "exogenous" refers to a substance present in a cell or organism other than its natural source. For example, the terms "exogenous nucleic acid" or "exogenous protein" refer to a nucleic acid or protein that has been introduced by a process involving manipulation into a biological system, such as a cell or organism, in which it is not normally found or is found in reduced amounts. A substance would be considered foreign if it was introduced into a cell or an ancestor of a cell that inherits the substance. In contrast, the term "endogenous" refers to a substance that is naturally present in a biological system.

[0063] As used herein, the term "genetically modified" or "engineered" cell refers to a cell into which foreign nucleic acid has been introduced by a manual process (or the progeny of such a cell that inherit at least a portion of the nucleic acid). The nucleic acid may contain, for example, a sequence foreign to the cell, a native sequence (i.e., a sequence found naturally in the cell) but in an arrangement that is not naturally occurring (e.g., the coding region is linked to a promoter from a different gene), or an altered form of a native sequence, etc. The process of introducing a nucleic acid into a cell can be accomplished by any suitable technique. Suitable techniques include calcium phosphate or lipid-mediated transfection, electroporation, and transduction or infection using viral vectors. In some embodiments, a polynucleotide, or a portion thereof, is integrated into the genome of the cell. The nucleic acid may be subsequently removed or excised from the genome, provided that such removal or excision results in a detectable alteration in the cell compared to an otherwise equivalent, unmodified cell. It should be recognized that the term genetically modified is intended to include the direct introduction of modified RNA (e.g., synthetic modified RNA) into a cell. Such synthetic modified RNAs include modifications to prevent rapid degradation by endo- and exonucleases and to avoid or reduce cellular innate immune or interferon responses to RNA. Modifications include, but are not limited to, (a) terminal modifications, such as 5'-terminal modifications (phosphorylation / dephosphorylation, conjugation, reverse linkage, etc.), 3'-terminal modifications (conjugation, DNA nucleotides, reverse linkage, etc.), (b) base modifications, such as replacement with modified bases, base stabilization, base destabilization, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' or 4' position) or sugar replacement, and (d) internucleoside linkage modifications, including modification or replacement of phosphodiester linkages.To the extent that such modifications disrupt translation (i.e., result in a 50% or greater reduction in translation compared to the absence of the modification in an in vitro translation assay, e.g., in rabbit reticulocytes), the modifications are not suitable for the methods and compositions described herein.

[0064] The term "expression" refers to the cellular processes involved in the production of RNA and proteins, including, but not limited to, transcription, translation, folding, modification, and processing, as applicable, and the secretion of proteins, as appropriate. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene.

[0065] As used herein, the terms "isolated" or "partially purified," in the case of a nucleic acid or polypeptide, refer to a nucleic acid or polypeptide that has been separated from at least one other component (e.g., a nucleic acid or polypeptide) that is present with the nucleic acid or polypeptide as found in its natural source and / or that would be present with the nucleic acid or polypeptide when expressed or, in the case of a secreted polypeptide, secreted by a cell. Chemically synthesized nucleic acids or polypeptides or those synthesized using in vitro transcription / translation are considered "isolated."

[0066] As used herein, the term "isolated cell" refers to a cell that has been removed from the organism in which it was originally found, or the progeny of such a cell. Optionally, the cell has been cultured in a test tube, e.g., in the presence of other cells. Optionally, the cell is later introduced into a second organism, or reintroduced into the organism from which it (or its ancestor cells) was isolated.

[0067] As used herein, the term "isolated population," with respect to an isolated cell population, refers to a population of cells that has been removed and separated from a mixed or heterogeneous population of cells. In some embodiments, an isolated population is a substantially pure population of cells relative to the heterogeneous population from which the cells were isolated or enriched.

[0068] The term "substantially pure" with respect to a particular cell population refers to a cell population that is at least about 75%, preferably at least about 85%, more preferably at least about 90%, and most preferably at least about 95% pure with respect to the cells that make up the entire cell population. Stated differently, the terms "substantially pure" or "essentially purified" with respect to a population of cardiomyocytes refer to a cell population that contains less than about 20%, more preferably less than about 15%, less than 10%, less than 8%, less than 7%, and most preferably less than about 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 1% of cells that are not cardiomyocytes as defined herein. In some embodiments, the present invention encompasses methods of expanding a population of cardiomyocytes, wherein the expanded cardiomyocyte population is a substantially pure cardiomyocyte population.

[0069] The terms "enriching" or "enriched" are used interchangeably herein and mean that the yield (fraction) of a certain type of cell is increased by at least 10% over the fraction of that type of cell at the start of the culture or preparation.

[0070] The terms "renewal" or "self-renewal" or "proliferation" are used interchangeably herein to refer to the ability of stem cells to self-renew over an extended period of time and / or over a period of months to years by dividing into the same unspecialized cell type. In some cases, proliferation refers to the proliferation of cells by repeated division of a single cell into two identical daughter cells.

[0071] The term "modulate" is used consistent with its use in the art, i.e., to bring about or facilitate a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest. Without limitation, such a change can be an increase, decrease, or change in the relative intensity or activity of various components or components of the process, pathway, or phenomenon. A "modulator" is an agent that brings about or facilitates a qualitative or quantitative change, alteration, or modification in a process, pathway, or phenomenon of interest.

[0072] As used herein, the term "DNA" is defined as deoxyribonucleic acid.

[0073] As used herein, the term "marker" refers to a cellular characteristic and / or phenotype. Markers can be used for the selection of cells containing a characteristic of interest. Markers will vary depending on the specific cell. A marker is a characteristic, whether morphological, functional, or biochemical (enzymatic) characteristic of cells of a particular cell type, or a molecule expressed by the cell type. Preferably, such a marker is a protein, more preferably having an epitope for an antibody or other binding molecule available in the art. However, markers may consist of any molecule found in cells, including, but not limited to, proteins (peptides and polypeptides), lipids, polysaccharides, nucleic acids, and steroids. Examples of morphological characteristics or traits include, but are not limited to, shape, size, and nucleus-to-cytoplasm ratio. Examples of functional characteristics or traits include, but are not limited to, the ability to adhere to a particular substrate, the ability to take up or exclude a particular dye, the ability to migrate under certain conditions, and the ability to differentiate or dedifferentiate along a particular lineage. Markers can be detected by any method available to those skilled in the art. A marker can also be the absence of a morphological trait, or the absence of a protein, lipid, etc. A marker can also be a panel of unique traits for the presence and absence of polypeptides in combination with other morphological traits.

[0074] The term "selectable marker" refers to a gene, RNA, or protein that, when expressed, confers a selectable phenotype on a cell, such as resistance to cytotoxic or cytostatic agents (e.g., antibiotic resistance), nutritional prototrophy, or expression of a specific protein that can be used to distinguish expressing from non-expressing cells. Proteins that are easily detectable, such as fluorescent or luminescent proteins or enzymes that act on a substrate to produce a colored, fluorescent, or luminescent substance (a "detectable marker"), constitute a subset of selectable markers. The presence of a selectable marker linked to expression control elements native to genes normally selectively or exclusively expressed in pluripotent cells allows for the identification and selection of somatic cells that have been reprogrammed to the pluripotent state. Various selectable marker genes are available, including the neomycin resistance gene (neo), puromycin resistance gene (puro), guanine phosphoribosyltransferase (gpt), dihydrofolate reductase (DHFR), adenosine deaminase (ada), puromycin-N-acetyltransferase (PAC), hygromycin resistance gene (hyg), multidrug resistance gene (mdr), thymidine kinase (TK), hypoxanthine-guanine phosphoribosyltransferase (HPRT), and hisD gene. Detectable markers include green fluorescent protein (GFP), blue, sapphire, yellow, red, orange, and cyan fluorescent proteins, and variants of any of these. Light-emitting proteins, such as luciferase (e.g., firefly or Renilla luciferase), are also used. As will be apparent to those skilled in the art, the term "selectable marker," as used herein, can refer to either a gene or the expression product of a gene, e.g., an encoded protein.

[0075] In some embodiments, a selectable marker confers a growth and / or survival advantage to cells that express the marker over cells that do not express the marker or over cells that express the marker but at a significantly reduced level. Such a growth and / or survival advantage typically occurs when the cells are maintained under certain conditions, i.e., "selective conditions." To ensure effective selection, a population of cells can be maintained under certain conditions for a sufficient length of time so that cells that do not express the marker do not grow and / or survive and are eliminated from the cell population or are reduced in number to only a small percentage of the population. The process of selecting for cells that express a marker by maintaining a population of cells under selective conditions that largely or completely eliminates cells that do not express the marker that confers a growth and / or survival advantage is referred to herein as "positive selection," and the marker is said to be "useful for positive selection." Negative selection and markers that are useful for negative selection are also of interest in some of the methods described herein. Expression of such a marker confers a growth and / or survival disadvantage on cells expressing the marker relative to cells that do not express the marker or that express the marker at a significantly reduced level (i.e., put another way, cells that do not express the marker have a growth and / or survival advantage relative to cells that do express the marker). Thus, cells expressing the marker may be largely or completely eliminated from a population of cells if maintained under selection conditions for a sufficient length of time.

[0076] The terms "subject" and "individual" are used interchangeably herein to refer to an animal, e.g., a human, from which cells can be obtained and / or to which treatment, including prophylactic treatment, using cells as described herein is provided. In the case of treatment of an infection, condition, or disease state specific to a particular animal, e.g., a human subject, the term subject refers to that particular animal. The terms "non-human animal" and "non-human mammal" are used interchangeably herein and include mammals, e.g., rats, mice, rabbits, sheep, cats, dogs, cows, pigs, and non-human primates. The term "subject" also encompasses any vertebrate, including, but not limited to, mammals, reptiles, amphibians, and fish. Advantageously, however, the subject is a mammal, such as a human, or other mammal, such as a domesticated mammal, e.g., a dog, cat, horse, or a production mammal, e.g., a cow, sheep, pig, or the like.

[0077] The terms "treat," "treating," "treatment," and the like, when applied to isolated cells, include subjecting the cells to any type of process or condition, or performing any type of manipulation or procedure on the cells. When applied to a subject, the terms "treat," "treating," "treatment," and the like refer to providing medical or surgical consultation, care, or management to an individual. The individual is typically ill or injured, or at increased risk of developing a disease compared to the average member of the population, and is in need of such consultation, care, or management. The terms may also include administering an effective amount of a composition to a subject such that the subject exhibits a reduction in at least one symptom of the disease or an improvement in the disease, e.g., a beneficial or desired clinical result. For purposes of the present invention, a beneficial or desired clinical result includes, but is not limited to, alleviation of one or more symptoms, a reduction in the extent of the disease, a stabilization (i.e., not worsening) of the disease state, a delay or slowing of disease progression, a remission or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. Treating can also refer to longer survival than expected if not treated. That is, as one of skill in the art will appreciate, treatment may ameliorate the disease state, but may not completely cure the disease. The term "treatment" includes prophylaxis. Those in need of treatment include those who have symptoms and have already been diagnosed (e.g., a muscle disorder or disease), as well as those who are likely to develop symptoms due to genetic susceptibility or other factors.

[0078] The term "tissue" refers to a group or layer of specialized cells that together perform a particular specialized function. The term "tissue-specific" refers to the source of cells from a particular tissue.

[0079] "Decrease," "reduced," "reduction," "decrease," or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. However, for the avoidance of doubt, "reduced," "reduction," or "decrease," or "inhibit" means a decrease of at least 10% when compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% decrease, or up to and including 100% decrease (i.e., absent levels when compared to a reference sample), or any decrease between 10-100% when compared to a reference level.

[0080] The terms "increased," "increase," or "improve," or "activate" are all used herein to generally mean an increase by a statistically significant amount. For the avoidance of doubt, the terms "increased," "increase," or "improve," or "activate" mean an increase of at least 10% compared to a reference level, for example, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or more compared to a reference level.

[0081] The term "statistically significant" or "significantly" refers to statistical significance and generally means two standard deviations (2SD) or less from the normal concentration of the marker. The term provides statistical evidence of a difference. The term is defined as the likelihood of making a decision to reject the null hypothesis when the null hypothesis is in fact true. The decision is often made using a P-value.

[0082] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and each component or components thereof that are essential to the invention, but also embrace the inclusion of elements not expressly specified, whether essential or not.

[0083] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term permits the presence of additional elements that do not materially affect the basic and novel or functional characteristic(s) of that embodiment of the invention.

[0084] The term "consisting of" refers to compositions, methods, and each component thereof as described herein, and does not include any element not recited in the description of that embodiment.

[0085] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein and / or that will become apparent to those skilled in the art upon reading this disclosure, etc.

[0086] stem cells Stem cells are cells that retain the ability to self-renew through cell division and can differentiate into a variety of specialized cell types. There are two main types of mammalian stem cells: embryonic stem (ES) cells, found in blastocysts, and adult stem cells, found in adult tissues. In the developing embryo, stem cells are capable of differentiating into all specialized embryonic tissues. In the adult organism, stem and progenitor cells act as the body's repair system, not only replenishing specialized cells but also maintaining the normal turnover of regenerating organs, such as blood, skin, or intestinal tissue. Pluripotent stem cells can differentiate into cells derived from any of the three germ layers.

[0087] Although certain embodiments are described below with respect to the use of stem cells to generate cardiomyocytes (e.g., mature cardiomyocytes) or their precursors, germ cells can be used instead of, or in addition to, stem cells to provide at least one type of cardiomyocyte using protocols similar to the exemplary protocols described herein. Suitable germ cells can be prepared, for example, from primordial germ cells present in human fetal material collected approximately 8-11 weeks after the last menstrual period. Exemplary germ cell preparation methods are described, for example, in Shamblott et al. al., Proc. Natl. Acad. Sci. USA 95:13726,1998, and U.S. Patent No. 6,090,622.

[0088] ES cells, e.g., human embryonic stem cells (hESCs) or mouse embryonic stem cells (mESCs), have virtually limitless replicative capacity and the potential to differentiate into most cell types, making them, in principle, an unlimited starting material for generating differentiated cells for clinical therapy (stemcells.nih.gov / info / scireport / 2006report.htm, 2006). One potential application of ES cells is to generate new cardiomyocytes for cell replacement therapy of heart failure (e.g., chronic heart failure) by first generating cardiac progenitor cells, e.g., from hESCs, and then further differentiating the cardiac progenitor cells into at least one immature cardiomyocyte or its precursor, and then further differentiating the at least one immature cardiomyocyte or its precursor into a cardiomyocyte (e.g., a mature cardiomyocyte).

[0089] hESC cells have been described, for example, by Cowan et al. (N Engl. J. Med. 350:1353, 2004) and Thomson et al. (Science 282:1145, 1998). Embryonic stem cells from other primates, such as rhesus monkey stem cells (Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995), marmoset stem cells (Thomson et al., Biol. Reprod. 55:254, 1996), and human embryonic germ (hEG) cells (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998), can also be used in the methods disclosed herein. mESCs are described, for example, by Tremml et al. (Curr Protoc Stem Cell Biol. Chapter 1:Unit 1C.4, 2008). Stem cells can be, for example, unipotent, totipotent, multipotent, or pluripotent. In some instances, any cell of primate origin that can generate progeny that are derivatives of at least one germ layer, or all three germ layers, can be used in the methods disclosed herein.

[0090] In certain instances, ES cells can be isolated as described, for example, in Cowan et al. (N Engl. J. Med. 350:1353, 2004) and U.S. Pat. No. 5,843,780, and Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844, 1995. For example, hESC cells can be isolated as described in Thomson et al. (U.S. Pat. No. 6,200,806; Science hESCs can be prepared from human blastocyst cells using techniques described in (Bongso et al., Nature Biotech. 282:1145, 1998; Curr. Top. Dev. Biol. 38:133ff., 1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Equivalent cell types to hESCs include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined, for example, in WO 01 / 51610 (Bresagen). hESCs can also be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or single-cell human embryos can be developed to the blastocyst stage (Bongso et al., Hum Reprod 4:706, 1989). Embryos are cultured in G1.2 and G2.2 medium to the blastocyst stage (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developing blastocysts by brief exposure to pronase (Sigma). The inner cell mass can be isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, followed by three 5-minute washes in DMEM and a 3-minute exposure to a 1:5 dilution of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two additional washes in DMEM, lysed trophectoderm cells are carefully removed from the intact inner cell mass (ICM) by pipetting, and the ICM is plated onto mEF feeder layers. After 9-15 days, the inner cell mass-derived products can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) without calcium or magnesium and containing 1 mM EDTA, by exposure to or dispersion with trypsin, or by mechanical dissociation with a micropipette, and then replated onto mEFs in fresh medium. Outgrowing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated.ES-like morphology is characterized by compact colonies with a high apparent nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting hESCs can then be split periodically every 1–2 weeks, for example, by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or individual colony selection with a micropipette. In some cases, clump sizes of approximately 50–100 cells are optimal. mESCs can be prepared, for example, using the technique described by Conner et al. (Curr. Prot. in Mol. Biol. Unit 23.4, 2003).

[0091] Embryonic stem cells can be isolated from blastocysts of primate species (U.S. Patent No. 5,843,780; Thomson et al., Proc. Natl. Acad. Sci. USA 92:7844,1995). Human embryonic stem (hES) cells have been described by Thomson et al. (U.S. Patent No. 6,200,806; Science 282:1145,1998; Curr. Top. Dev. Biol. 38:133 ff.,1998) and Reubinoff et al., Nature Biotech. 18:399, 2000. Equivalent cell types to hES cells include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as generally described, for example, in WO 01 / 51610 (Bresagen).

[0092] Alternatively, in some embodiments, hES cells can be obtained from human preimplantation embryos. Alternatively, in vitro fertilized (IVF) embryos can be used, or single-cell human embryos can be developed to the blastocyst stage (Bongso et al., Hum Reprod. 4:706, 1989). Embryos are cultured in G1.2 and G2.2 medium to the blastocyst stage (Gardner et al., Fertil. Steril. 69:84, 1998). The zona pellucida is removed from developing blastocysts by brief exposure to pronase (Sigma). The inner cell mass can be isolated by immunosurgery, in which blastocysts are exposed to a 1:50 dilution of rabbit anti-human spleen cell antiserum for 30 minutes, followed by three 5-minute washes in DMEM and a 3-minute exposure to a 1:5 dilution of guinea pig complement (Gibco) (Solter et al., Proc. Natl. Acad. Sci. USA 72:5099, 1975). After two more washes in DMEM, the lysed trophectoderm cells are carefully removed from the intact inner cell mass (ICM) by pipetting, and the ICM is plated onto an mEF feeder layer.

[0093] After 9–15 days, the inner cell mass-derived products can be dissociated into clumps by exposure to phosphate-buffered saline (PBS) without calcium or magnesium and containing 1 mM EDTA, by exposure to or dispersion with trypsin, or by mechanical dissociation with a micropipette, and then replated onto mEFs in fresh medium. Outgrowing colonies with undifferentiated morphology can be individually selected with a micropipette, mechanically dissociated into clumps, and replated. ES-like morphology is characterized by compact colonies with an apparent high nucleus-to-cytoplasm ratio and prominent nucleoli. The resulting ES cells can then be split periodically every 1–2 weeks by brief trypsinization, exposure to Dulbecco's PBS (containing 2 mM EDTA), exposure to type IV collagenase (approximately 200 U / mL; Gibco), or by selecting individual colonies with a micropipette. Clump sizes of approximately 50–100 cells are optimal.

[0094] In some embodiments, human embryonic germ (hEG) cells are pluripotent stem cells that can be used in the methods disclosed herein to differentiate into primitive endoderm cells. hEG cells can be prepared from primordial germ cells present in human fetal material collected approximately 8-11 weeks after the last menstrual period. Suitable preparation methods are described in Shamblott et al. al., Proc. Natl. Acad. Sci. USA 95:13726,1998, and U.S. Patent No. 6,090,622, which are incorporated by reference in their entirety.

[0095] Briefly, genital ridges were processed to form dispersed cells. EG growth medium was prepared from DMEM supplemented with 4500 mg / L D-glucose, 2200 mg / L mM NaHCO3, 15% ES maintenance (qualified) fetal bovine serum (BRL), 2 mM glutamine (BRL), 1 mM sodium pyruvate (BRL), 1000–2000 U / mL human recombinant leukemia inhibitory factor (LIF, Genzyme), 1–2 ng / mL human recombinant bFGF (Genzyme), and 10 μM forskolin in 10% DMSO. A 96-well tissue culture plate is prepared with a subconfluent layer of feeder cells (e.g., STO cells, ATCC No. CRL1503) cultured for 3 days in modified EG growth medium without LIF, bFGF, or forskolin. The cells are then inactivated with 5000 rad of gamma irradiation, and approximately 0.2 mL of primary germ cell (PGC) suspension is added to each well. The first passage is completed after 7–10 days in EG growth medium, and each well is transferred individually to a well of a 24-well culture dish previously prepared with irradiated STO mouse fibroblasts. The cells are cultured with daily medium changes until a cell morphology consistent with EG cells is observed. This typically occurs after 7–30 days, or 1–4 passages.

[0096] In certain instances, stem cells can be undifferentiated (e.g., the cells are not committed to a particular lineage) before being exposed to at least one cardiomyocyte maturation factor according to the methods disclosed herein, while in other instances, it may be desirable to differentiate the stem cells into one or more intermediate cell types before exposure to at least one cardiomyocyte maturation factor(s) described herein. For example, stem cells may exhibit morphological, biological, or physical characteristics that can be used to distinguish undifferentiated cells from differentiated cells of embryonic or adult origin. In some instances, undifferentiated cells may appear in colonies of cells with a high nuclear-to-cytoplasmic ratio and prominent nucleoli in a two-dimensional microscopic view. Stem cells may be stem cells themselves (e.g., substantially free of any undifferentiated cells) or may be used in the presence of differentiated cells. In certain instances, stem cells can be cultured in the presence of appropriate nutrients and, optionally, other cells, to allow the stem cells to grow and, optionally, differentiate. For example, embryonic fibroblasts or fibroblast-like cells may be present in the culture to support stem cell growth. Fibroblasts may be present during one stage of stem cell growth, but not necessarily during all stages. For example, fibroblasts may be added to stem cell cultures during the initial culture stage, but not during one or more subsequent culture stages.

[0097] The stem cells used in all aspects of the invention can be any cell derived from any type of tissue (e.g., embryonic tissue, such as fetal or prefetal tissue, or adult tissue), but are characterized by their ability, under appropriate conditions, to generate progeny of different cell types, including all derivatives of at least one of the three germ layers (endoderm, mesoderm, and ectoderm). These cell types can be provided in the form of established cell lines or can be obtained directly from primary embryonic tissue and immediately used for differentiation. Included are cells registered with the NIH Human Embryonic Stem Cell Registry, e.g., hESBGN-01, hESBGN-02, hESBGN-03, hESBGN-04 (BresaGen, Inc.); HES-1, HES-2, HES-3, HES-4, HES-5, HES-6 (ES Cell International); Miz-hES1 (MizMedi Hospital-Seoul National University); HSF-1, HSF-6 (University of California at San Francisco); and H1, H7, H9, H13, H14 (Wisconsin Alumni Research Foundation (WiCell Research Institute)). In some embodiments, the source of human stem cells or pluripotent stem cells used for chemically induced differentiation into mature cardiomyocytes did not involve the destruction of human embryos.

[0098] In another embodiment, stem cells can be isolated from tissues, including solid tissues. In some embodiments, the tissue is skin, adipose tissue (e.g., adipose tissue), muscle tissue, heart, or cardiac tissue. In other embodiments, the tissue is, for example, but not limited to, umbilical cord blood, placenta, bone marrow, or cartilage.

[0099] Stem cells of interest also include various embryonic cells, such as human embryonic stem (hES) cells, as described in Thomson et al. (1998) Science 282:1145; embryonic stem cells of other primates, such as rhesus monkey stem cells (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844); marmoset stem cells (Thomson et al. (1996) Biol. Reprod. 55:254); and human embryonic germ (hEG) cells (Shambloft et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Also of interest are lineage-committed stem cells, such as mesodermal stem cells and other early cardiogenic cells (see, e.g., Reyes et al. (2001) Blood 98:2615-2625; Eisenberg & Bader (1996) CircRes. 78(2):205-16). Stem cells can be obtained from any mammalian species, e.g., human, equine, bovine, porcine, canine, feline, rodent, e.g., mouse, rat, hamster, primate, etc. In some embodiments, human embryos have not been destroyed to provide a source of pluripotent cells for use in the methods and compositions as disclosed herein.

[0100] ES cells are considered undifferentiated when they are not committed to a specific lineage. Such cells exhibit morphological characteristics that distinguish them from differentiated cells of embryonic or adult origin. Undifferentiated ES cells are easily recognized by those skilled in the art and typically appear in colonies of cells with a high nuclear-to-cytoplasmic ratio and prominent nucleoli under two-dimensional microscopic view. Undifferentiated ES cells express genes that can be used as markers to detect the presence of undifferentiated cells, and the polypeptide products of these genes can be used as negative selection markers. See, e.g., U.S. Application No. 2003 / 0224411 A1; Bhattacharya (2004) Blood 103(8):2956-64; and Thomson (1998), supra, each of which is incorporated herein by reference. Human ES cell lines express cell surface markers characteristic of undifferentiated non-human primate ES cells and human EC cells, including stage-specific embryonic antigen (SSEA)-3, SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The globo-series glycolipid GL7, which carries the SSEA-4 epitope, is formed by the addition of sialic acid to the globo-series glycolipid GbS, which carries the SSEA-3 epitope. Thus, GL7 reacts with both SSEA-3 and SSEA-4 antibodies. Undifferentiated human ES cell lines did not stain with SSEA-1 staining, whereas differentiated cells stained strongly with SSEA-1 staining. Methods for propagating hES cells in an undifferentiated form are described in WO 99 / 20741, WO 01 / 51616, and WO 03 / 020920.

[0101] A cell mixture from a suitable source of endothelial, muscle, and / or neural stem cells can be collected from a mammalian donor by methods known in the art. A suitable source is the hematopoietic microenvironment. For example, circulating peripheral blood, preferably mobilized (i.e., replenished), can be removed from a subject. Alternatively, bone marrow can be obtained from a mammal, such as a human patient undergoing an autologous transplant. In some embodiments, stem cells can be obtained from a subject's adipose tissue, e.g., using Cytori's CELUTION™ SYSTEM, as described in U.S. Patent Nos. 7,390,484 and 7,429,488, which are incorporated herein by reference in their entirety.

[0102] In some embodiments, human umbilical cord blood cells (HUCBCs) are useful in the methods disclosed herein. Human Umbilical Cord Blood Cells (HUCBCs) are recognized as a rich source of hematopoietic and mesenchymal progenitor cells (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113). Previously, umbilical cord and placental blood was considered waste and typically discarded at the birth of an infant. Umbilical cord blood cells are used as a source of transplantable stem and progenitor cells, as well as a source of bone marrow-repopulating cells for the treatment of malignant diseases (i.e., acute lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, myelodysplastic syndromes, and neuroblastoma) and non-malignant diseases, such as Fanconi anemia and aplastic anemia (Kohli-Kumar et al., 1993). Br.J. Haematol. 85:419-422;Wagner et al.,1992 Blood 79;1874-1881;Lu et al.,1996 Crit. Rev. Oncol. Hematol 22:61-78; Lu et al., 1995 Cell Transplantation 4:493-503). A distinct advantage of HUCBC is that the immature immune properties of these cells closely resemble those of fetal cells, significantly reducing the risk of rejection by the host (Taylor & Bryson, 1985 J. Immunol. 134:1493-1497). Human umbilical cord blood contains mesenchymal and hematopoietic progenitor cells, as well as endothelial cell precursors that can be expanded in tissue culture (Broxmeyer et al., 1992 Proc. Natl. Acad. Sci. USA 89:4109-4113; Kohli-Kumar et al., 1993 Br. J. Haematol. 85:419-422;Wagner et al.,1992 Blood 79;1874-1881;Lu et al.,1996 Crit. Rev. Oncol. Hematol 22:61-78;Lu et al.,1995 Cell Transplantation 4:493-503;Taylor & Bryson,1985 J. Immunol.134:1493-1497;Broxmeyer,1995 Transfusion 35:694-702;Chen et al.,2001 Stroke 32:2682-2688;Nieda et al.,1997 Br. J. Haematology 98:775-777;Erices et al.,2000 Br. J. Haematology 109:235-242). The total hematopoietic progenitor cell content in umbilical cord blood equals or exceeds that of bone marrow, and in addition, the highly proliferative hematopoietic cells in HUCBCs are eight times more abundant than those in bone marrow and express hematopoietic markers such as CD14, CD34, and CD45 (Sanchez-Ramos et al., 2001 Exp. Neur. 171:109-115; Bicknese et al., 2002 Cell Transplantation 11:261-264; Lu et al., 1993 J. ExpMed. 178:2089-2096).

[0103] In another embodiment, the pluripotent cells are cells in the hematopoietic microenvironment, e.g., circulating peripheral blood, preferably derived from mammalian peripheral blood, umbilical cord blood, bone marrow, fetal liver, or the mononuclear fraction of the yolk sac. Stem cells, particularly neural stem cells, may also be derived from the central nervous system, including the meninges.

[0104] In another embodiment, the pluripotent cells are present in embryoid bodies formed by harvesting ES cells by brief protease digestion and growing small aggregates of undifferentiated human ESCs in suspension culture. Differentiation is induced by withdrawal of conditioned medium. The resulting embryoid bodies are plated onto a semi-solid substrate. Formation of differentiated cells may be observed after about 7 days to about 4 weeks. Viable differentiated cells from in vitro cultures of stem cells are selected by partially dissociating the embryoid bodies or similar structures to provide cell aggregates. Aggregates containing cells of interest are selected for phenotypic characteristics using methods that substantially maintain cell-to-cell contact within the aggregates.

[0105] In alternative embodiments, the stem cells can be reprogrammed stem cells, e.g., stem cells derived from somatic or differentiated cells. In such embodiments, the dedifferentiated stem cells can be, for example, but not limited to, neoplastic, tumor, and cancer cells, or alternatively, induced reprogrammed cells, e.g., induced pluripotent stem cells or iPS cells.

[0106] Cloning and cell culture Exemplary methods of molecular genetics and genetic engineering that can be utilized in the techniques described herein can be found, for example, in current editions of Molecular Cloning: A Laboratory Manual, (Sambrook et al., Cold Spring Harbor), Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.), and Current Protocols in Molecular Biology (F.M. Ausubel et al. eds., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques are discussed in, for example, Current Protocols in Protein Science (J.E. Colligan et al. eds., Wiley & Sons), Current Protocols in Cell Biology (J.S. Bonifacino et al., Wiley & Sons), and Current Protocols in Immunology (J.E. Colligan et al. al. eds., Wiley & Sons. Exemplary reagents, cloning vectors, and kits for genetic manipulation are commercially available from, for example, BioRad, Stratagene, Invitrogen, ClonTech, and Sigma-Aldrich Co.

[0107] Suitable cell culture methods can be found, for example, in the current edition of Culture of Animal Cells: A Manual of Basic Technique (RI Freshney ed., Wiley & Sons); General Techniques of Cell Culture (MA Harrison & IF Rae, Cambridge University Press); and Embryonic Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Suitable tissue culture consumables and reagents are commercially available from, for example, Gibco / BRL, Nalgene-Nunc International, Sigma Chemical Co., and ICN Biomedicals.

[0108] Pluripotent stem cells can be continuously propagated in culture by those skilled in the art using culture conditions that promote proliferation without promoting differentiation. An example of a serum-containing ES medium is made up of 80% DMEM (e.g., Knockout DMEM, Gibco), 20% defined fetal bovine serum (FBS, Hyclone) or serum replacement (WO 98 / 30679), 1% non-essential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Immediately before use, human bFGF is added to a concentration of 4 ng / mL (WO 99 / 20741, Geron Corp.). Traditionally, ES cells are cultured on a layer of feeder cells, typically fibroblasts derived from embryonic or fetal tissue.

[0109] Alternatively, pluripotent SCs can be maintained in an undifferentiated state even in the absence of feeder cells. The environment for feeder-free culture includes an appropriate culture substrate, particularly an extracellular matrix such as MATRIGEL® (a gelatinous protein mixture) or laminin. Typically, enzymatic digestion is stopped (approximately 5 minutes using collagenase IV) before the cells are completely dispersed. Then, clumps of approximately 10–2,000 cells are seeded directly onto the substrate without further dispersion.

[0110] Cardiomyocyte generation Aspects of the present disclosure relate to the generation of cardiomyocytes (e.g., mature, quiescent cardiomyocytes). Generally, cardiomyocytes generated by the methods disclosed herein exhibit multiple characteristics of functionally mature, quiescent cardiomyocytes, including, but not limited to, being electrically mature (e.g., exhibiting reduced automaticity), contractilely mature, and metabolically mature.

[0111] Cardiomyocytes can be generated by any suitable culture protocol or series of culture protocols that differentiate stem cells or pluripotent cells to a desired differentiation stage. In some embodiments, cardiomyocytes or precursors thereof are generated by culturing at least one pluripotent cell for a length of time and under conditions appropriate for the at least one pluripotent cell to differentiate into a cardiomyocyte or precursor thereof. In some embodiments, cardiomyocytes are generated by transitioning immature cardiomyocytes from a senescent to a quiescent state, thereby rendering the cardiomyocytes more mature.

[0112] In some embodiments, the cardiomyocytes are a substantially pure population of cardiomyocytes. In some embodiments, the population of cardiomyocytes or their precursors comprises a mixture of pluripotent cells or differentiated cells. In some embodiments, the population of cardiomyocytes or their precursors is substantially free of or devoid of embryonic stem cells or pluripotent cells or iPS cells.

[0113] In some embodiments, somatic cells, e.g., fibroblasts, can be isolated from a subject, e.g., as a tissue biopsy, such as a skin biopsy, and reprogrammed for further differentiation into induced pluripotent stem cells to generate cardiomyocytes or their precursors for use in the compositions and methods described herein. In some embodiments, the somatic cells, e.g., fibroblasts, are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded and then converted into cardiomyocytes by the methods disclosed herein.

[0114] In some embodiments, cardiomyocytes or their precursors are maintained in culture by methods known to those of skill in the art, and in some embodiments, expanded, and then converted into cardiomyocytes by the methods disclosed herein.

[0115] Furthermore, the cardiomyocytes or their precursors, e.g., immature cardiomyocytes, can be derived from any mammalian species, including, but not limited to, murine, bovine, simian, porcine, equine, ovine, or human cells. For clarity and brevity, the methods described herein are with respect to mammalian cardiomyocytes or their precursors, but it should be understood that all methods described herein are readily applicable to other cell types of cardiomyocytes or their precursors. In some embodiments, the cardiomyocytes or their precursors are derived from a human individual.

[0116] Aspects of the present disclosure involve immature, senescent cardiomyocytes. Immature cardiomyocytes, as used herein, can be derived from any source and generated according to any suitable protocol. In some aspects, pluripotent stem cells, e.g., iPSCs or hESCs, are differentiated into immature cardiomyocytes. In some aspects, the immature cardiomyocytes are further matured into mature cardiomyocytes. In some embodiments, the pluripotent stem cells are differentiated into immature cardiomyocytes using the differentiation protocol described in Lian et al. (Nat Protoc. 2012;8(1):162-175), which is incorporated herein by reference. In some embodiments, the differentiation protocol described in Lian is modified as described herein. In some embodiments, the pluripotent stem cells are contacted with one or more small molecules to manipulate the Wnt pathway, thereby differentiating the pluripotent stem cells into immature cardiomyocytes. In some aspects, the one or more small molecules are selected from the group consisting of CHIR 99021 and IWP4. In some embodiments, the population of pluripotent stem cells is contacted with a first Wnt pathway modifier (eg, CHIR 99021) and then with a second Wnt pathway modifier (eg, IWP4).

[0117] Aspects of the present disclosure involve cardiomyocytes (e.g., mature cardiomyocytes). Cardiomyocytes as used herein can be derived from any source and generated according to any suitable protocol. In some embodiments, senescent cardiomyocytes (e.g., immature cardiomyocytes) are induced to become quiescent cardiomyocytes (e.g., mature cardiomyocytes). The quiescent state of the cells can promote cardiomyocyte maturation. In some embodiments, immature cardiomyocytes are induced to mature into mature cardiomyocytes.

[0118] In some aspects, the present disclosure provides methods of inducing quiescence in cardiomyocytes (e.g., transitioning cardiomyocytes into quiescence). In certain aspects, inducing quiescence in cardiomyocytes promotes cardiomyocyte maturation. In some embodiments, cardiomyocytes are transitioned into quiescence by upregulating expression of a cell cycle regulator in the cardiomyocyte. In certain embodiments, cardiomyocytes are transitioned into quiescence by upregulating expression of p53 in the cardiomyocyte. Expression of p53 in cardiomyocytes can be upregulated by contacting the cardiomyocyte with a p53 activator. In some embodiments, the p53 activator is a small molecule. In certain embodiments, the p53 activator is Torin1. In certain embodiments, the p53 activator is not Torin1. In certain embodiments, the p53 activator is nutlin-3a. In some embodiments, the p53 activator is a combination of two or more agents. In some embodiments, cardiomyocytes are transitioned into quiescence by inhibiting mTOR signaling and upregulating expression of p53. In some embodiments, upregulating p53 expression without inhibiting mTOR signaling causes cardiomyocytes to transition to quiescence. In some aspects, mTOR signaling in cardiomyocytes can be inhibited by contacting the cardiomyocytes with an inhibitor of mTORC1 and mTORC2. In some embodiments, the inhibitor of mTORC1 and mTORC2 is Torin1. In some embodiments, the inhibitor of mTORC1 and mTORC2 is other than Torin1. In certain embodiments, contacting the cardiomyocytes with a single agent upregulates p53 expression and inhibits mTOR signaling. In certain embodiments, the single agent is Torin1. In other embodiments, the single agent is other than Torin1.

[0119] In some aspects, the present disclosure provides methods for generating mature cardiomyocytes (e.g., electrically mature, contractilely mature, and / or metabolically mature) from immature cardiomyocytes, the methods comprising inducing quiescence in the cardiomyocytes (i.e., transitioning the cardiomyocytes toward quiescence, i.e., a mature stage). In some embodiments, cardiomyocytes are transitioned toward quiescence (e.g., a mature stage) by upregulating p53 expression. Cardiomyocytes can be transitioned toward quiescence by contacting immature cardiomyocytes with a p53 activator (e.g., nutlin-3a). In some embodiments, cardiomyocytes are transitioned toward quiescence (e.g., a mature stage) by upregulating p53 expression and downregulating mTOR signaling. In some embodiments, cardiomyocytes are transitioned toward quiescence by upregulating p53 expression and not downregulating mTOR signaling. In some embodiments, cardiomyocytes are transitioned toward quiescence by contacting immature, senescent cardiomyocytes with Torin1. In some embodiments, cardiomyocytes are transitioned toward quiescence by contacting immature, senescent cardiomyocytes with an agent that is not Torin1.

[0120] In some aspects, the present disclosure provides methods for generating mature cardiomyocytes (e.g., electrically mature, contractilely mature, and / or metabolically mature) from immature cardiomyocytes, the methods comprising contacting a population of cells comprising immature cardiomyocytes with at least one cardiomyocyte maturation factor comprising a p53 activator and an mTOR inhibitor, to induce maturation (e.g., in vitro maturation) of at least one immature cardiomyocyte in the population. In some embodiments, the population of cells comprising immature cardiomyocytes is contacted with at least one cardiomyocyte maturation factor (e.g., a p53 activator and an mTOR inhibitor). In some aspects, upregulation of p53 expression in conjunction with mTOR inhibition results in more mature stem cell-derived cardiomyocytes.

[0121] The present disclosure contemplates the use of any p53 activator that promotes the transition of immature cardiomyocytes from senescence to quiescence (e.g., differentiation and / or maturation into cardiomyocytes (e.g., alone or in combination with another cardiomyocyte maturation factor, e.g., an mTOR inhibitor)). In some embodiments, the p53 activator is an upregulator of p53 expression. Upregulation of p53 can include an increase in total p53 and phosphorylated p53 protein. Examples of p53 activators or upregulators include small molecules, nucleic acids, amino acids, metabolites, polypeptides, antibodies and antibody-like molecules, aptamers, macrocycles, and other molecules. In some aspects, the p53 upregulator is nutlin-3a. In some aspects, the p53 upregulator is Torin1. In some aspects, the p53 upregulator is an agent that is not Torin1. In some aspects, the p53 upregulator is an agent that is not an mTOR inhibitor. In some embodiments, the upregulator of p53 is a combination of nutlin-3a and Torin1.

[0122] In some aspects, the present disclosure provides methods for generating mature cardiomyocytes (e.g., electrically mature, contractilely mature, and / or metabolically mature) from immature cardiomyocytes, the methods comprising contacting a population of cells comprising immature cardiomyocytes with at least one cardiomyocyte maturation factor, including an mTOR inhibitor, to induce at least one immature cardiomyocyte in the population to mature into a cardiomyocyte (e.g., in vitro maturation). In some embodiments, the population of cells comprising immature cardiomyocytes is contacted with at least one cardiomyocyte maturation factor (e.g., an mTOR inhibitor, a PI3K inhibitor, or an Akt inhibitor). In some aspects, the PI3K / Akt / mTOR pathway is manipulated (e.g., inhibited) to result in more mature stem cell-derived cardiomyocytes.

[0123] The present disclosure contemplates the use of any mTOR inhibitor (e.g., alone or in combination with another cardiomyocyte maturation factor (e.g., a p53 upregulator)) that promotes differentiation and / or maturation of immature cardiomyocytes into cardiomyocytes. In some embodiments, mTOR includes mTORC1 and / or mTORC2. In some embodiments, the mTOR inhibitor is an inhibitor of mTORC1 and / or mTORC2. In some embodiments, the mTOR inhibitor inhibits phosphorylation of 4E-BP1. Inhibiting phosphorylation of 4E-BP1 can affect regulation of the oxidative phosphorylation pathway. Inhibiting phosphorylation of 4E-BP1 can degrade p21, thereby upregulating p53. Non-limiting examples of modifiers of the oxidative phosphorylation pathway include 4EGI-1, JR-AB2-011 (an mTORC2 inhibitor), AICAR (an AMPK activator), metformin (an AMPK activator and mTORC1 / 2 inhibitor), and HLM006474 (an E2F inhibitor). In some embodiments, the mTOR inhibitor inhibits the phosphorylation of 4E-BP1 and ribosomal protein S6. In some embodiments, the mTOR inhibitor comprises Torin1, Torin2, rapamycin, everolimus, and / or temsirolimus. In certain embodiments, a population of cells comprising immature cardiomyocytes is contacted with Torin1 to induce maturation of at least one immature cardiomyocyte in the population into a cardiomyocyte (e.g., a mature cardiomyocyte). In some embodiments, a population of cells comprising immature cardiomyocytes is contacted with Torin2 to induce maturation of at least one immature cardiomyocyte in the population into a cardiomyocyte (e.g., a mature cardiomyocyte).

[0124] In some embodiments, the contacting can be achieved by maintaining at least one immature cardiomyocyte or its precursor in a culture medium containing one or more cardiomyocyte maturation factors. In some embodiments, the at least one immature cardiomyocyte or its precursor can be genetically engineered. In some embodiments, the at least one immature cardiomyocyte or its precursor can be genetically engineered to express one or more cardiomyocyte (e.g., mature cardiomyocyte) markers as disclosed herein, e.g., at least one polypeptide selected from TNNI3, KCNJ2, REST / NRSF, Ryr, and SCN5a, or an amino acid sequence substantially homologous thereto, or a functional fragment or functional variant thereof.

[0125] When immature cardiomyocytes or their precursors are maintained under in vitro conditions, conventional tissue culture conditions and methods can be used and are known to those skilled in the art. Isolation and culture methods for various cells are well within the capabilities of one of ordinary skill in the art.

[0126] In the methods of the present disclosure, at least one cardiomyocyte or its precursor can generally be cultured under standard temperature, pH, and other environmental conditions, e.g., for adherent cells, at 37°C in a tissue culture plate in an atmosphere containing 5-10% CO. The cells and / or culture medium are appropriately modified to achieve conversion to cardiomyocytes as described herein.

[0127] In certain instances, cardiomyocyte maturation factors can be used to induce differentiation of at least one immature cardiomyocyte or precursor thereof by exposing or contacting the at least one immature cardiomyocyte or precursor thereof to an effective amount of a cardiomyocyte maturation factor described herein to differentiate the at least one immature cardiomyocyte or precursor thereof into at least one cardiomyocyte (e.g., a mature cardiomyocyte).

[0128] Thus, included herein are cells and compositions produced by the methods described herein. The exact amount and type of cardiomyocyte maturation factor can vary depending on the number of immature cardiomyocytes or their precursors, the desired differentiation step, and the number of prior differentiation steps that have been performed.

[0129] In certain instances, the cardiomyocyte maturation factor is present in an effective amount. As used herein, "effective amount" refers to the amount of compound that must be present to cause at least 10%, or at least 20%, or at least 30% of the cells in a population of immature cardiomyocytes or their precursors to differentiate into cardiomyocytes.

[0130] In a further example, the cardiomyocyte maturation factor can be present in the culture medium of the at least one immature cardiomyocyte or its precursor, or the cardiomyocyte maturation factor can be added to the at least one immature cardiomyocyte or its precursor during a certain stage of growth.

[0131] In some embodiments, the immature cardiomyocytes are contacted with a cardiomyocyte maturation factor (e.g., an mTOR inhibitor and / or a p53 upregulator) after the immature cardiomyocytes have begun beating. In some embodiments, the immature cardiomyocytes have been beating for 1-5 days, 1-4 days, 1-3 days, 1-2 days, 1 day, 2 days, 3 days, 4 days, or 5 days before being contacted with the cardiomyocyte maturation factor. In some embodiments, the immature cardiomyocytes have been beating for 1-40 days, 2-35 days, 3-30 days, 4-25 days, 5-20 days, 7-35 days, 14-30 days, or 21-28 days before being contacted with the cardiomyocyte maturation factor. In some embodiments, if the immature cardiomyocytes have not begun beating, the immature cardiomyocytes are not contacted with the cardiomyocyte maturation factor (e.g., an mTOR inhibitor and / or a p53 upregulator).

[0132] In some embodiments, the immature cardiomyocytes are contacted with the cardiomyocyte maturation factor (e.g., an mTOR inhibitor and / or a p53 upregulator) after the immature cardiomyocytes have begun to express troponin T (TNNT2) and myosin heavy chain 6 (MYH6). In some embodiments, the immature cardiomyocytes are contacted with the cardiomyocyte maturation factor (e.g., an mTOR inhibitor and / or a p53 upregulator) after the immature cardiomyocytes have begun to express troponin T (TNNT2), troponin I (TNNI3), myosin heavy chain 6 (MYH6), and myosin heavy chain 7 (MYH7).

[0133] When at least one type of immature cardiomyocyte or its precursor is maintained under in vitro conditions, conventional tissue culture conditions and methods can be used and are known to those skilled in the art. Isolation and culture methods for various cells are well within the capabilities of those skilled in the art.

[0134] In some embodiments, pluripotent stem cells are cultured in RPMI+B27 and contacted with a GSK3 inhibitor / WNT activator (e.g., CHIR 99021) on day 0 of the differentiation protocol. On day 2 of the protocol, the WNT activator (e.g., CHIR 99021) is removed. On day 3 of the protocol, a WNT inhibitor (e.g., IWP4) is added, and on day 5, the WNT inhibitor (e.g., IWP4) is removed. On day 7, insulin is added to the cultures, and the medium is changed every 2-3 days. On day 11, an mTOR inhibitor (e.g., Torin1) is added and maintained in the medium until day 18. At the completion of the differentiation protocol, mature cardiomyocytes are obtained from the culture medium.

[0135] In some embodiments, pluripotent stem cells are cultured in RPMI+B27 and contacted with a GSK3 inhibitor / WNT activator (e.g., CHIR 99021) on days 0-2 of the differentiation protocol. A WNT inhibitor (e.g., IWP4) is added on days 2-4 of the protocol. On day 7, insulin is added to the cultures, and the medium is changed every 2-3 days. Once beating, cardiomyocytes are treated with Torin1. Torin1 treatment began approximately 2 days after the onset of beating and continued for a period of 7 days. Upon completion of Torin1 treatment, the medium was returned to RPMI / B27 / insulin and maintained with medium changes every 2-3 days. Upon completion of the differentiation protocol, mature cardiomyocytes are obtained from the culture medium.

[0136] In some embodiments, the differentiation protocol to obtain cardiomyocytes from immature cardiomyocytes or their precursors is performed in a two-dimensional culture system. In some embodiments, the differentiation protocol to obtain cardiomyocytes from immature cardiomyocytes or their precursors is performed in a three-dimensional culture system (e.g., using a 3D bioreactor system).

[0137] Aspects of the present disclosure involve the generation of cardiomyocytes that resemble endogenous mature cardiomyocytes in morphology and function, yet are distinct from native cardiomyocytes. In some embodiments, the cardiomyocyte morphology resembles that of endogenous cardiomyocytes. In some embodiments, the cardiomyocytes are quiescent. In some embodiments, the cardiomyocytes exhibit increased expression of quiescent markers (e.g., p16 and p130). In some embodiments, the cardiomyocytes exhibit increased expression of proliferation markers (e.g., cyclin C1, E2F1, and Ki67). In some embodiments, the cardiomyocytes exhibit increased expression of inhibitory E2F factors (e.g., E2F3b-8). In some embodiments, the cardiomyocytes exhibit decreased expression of stimulatory E2F factors (e.g., E2F1-3a).

[0138] In some embodiments, the cardiomyocytes are mature. In some embodiments, the cardiomyocytes exhibit increased expression of sarcomeric proteins (e.g., TNNT2, TNNI3, MYH6, and / or MYH7). In some embodiments, the cardiomyocytes exhibit a decreased beat rate when compared to fetal or immature cardiomyocytes. In some embodiments, the cardiomyocytes exhibit increased expression of ion channels (e.g., KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and / or SERCA2a (ATP2a2)). In some embodiments, the cardiomyocytes exhibit increased expression of brain natriuretic peptide. In some embodiments, the cardiomyocytes exhibit increased expression of nuclear transcription factors (e.g., REST / NRSF, GATA4, TBX20, BRG1, YAP, ERBB2, PITX2, MEIS1, ATA4, Nkx2-5, TBX5, NFAT, TEAD, HAND, CHF1, FoxO3 / FoxO4, CHF1 / Hey2, CHF1 / Hey2, FoxO1, Mef2C, SRF, p53, NFkB, and combinations thereof). In some embodiments, the cardiomyocytes exhibit increased expression of regulators of the oxidative phosphorylation pathway (e.g., PGC1-alpha).

[0139] The generation of cardiomyocytes by converting or maturing at least one type of immature cardiomyocyte or its precursor using the method of the present disclosure has many advantages. First, the method of the present disclosure allows for the generation of autologous cardiomyocytes. Autologous cardiomyocytes are cells that are specific to and genetically matched with an individual. Generally, autologous cells are less likely to be subject to immune rejection than non-autologous cells. The cells are derived from at least one type of immature cardiomyocyte or its precursor, e.g., cardiac progenitor cells, obtained by reprogramming somatic cells (e.g., fibroblasts) from an individual to induce a pluripotent state, then culturing the pluripotent cells to differentiate at least a portion of the pluripotent cells into at least one type of immature cardiomyocyte or its precursor, and subsequently inducing in vitro maturation of the at least one immature cardiomyocyte into a cardiomyocyte (e.g., a mature cardiomyocyte).

[0140] In some embodiments, the subject from which at least one immature cardiomyocyte or precursor thereof is obtained is a mammalian subject, e.g., a human subject. In some embodiments, the subject suffers from a cardiac disorder. In some embodiments, the subject suffers from chronic heart failure. In some embodiments, the subject suffers from ventricular arrhythmia. In such embodiments, at least one immature cardiomyocyte or precursor thereof can be differentiated ex vivo into a cardiomyocyte by a method as described herein, and then administered to the subject from which the cells were collected in a method of treating the subject's cardiac disorder (e.g., heart failure).

[0141] In some embodiments, at least one immature cardiomyocyte or precursor thereof is located in a subject (in vivo) and converted in vivo to a cardiomyocyte by a method as disclosed herein. In some embodiments, the in vivo conversion of at least one immature cardiomyocyte or precursor thereof to a cardiomyocyte can be achieved by administering to the subject a composition comprising at least one, at least two, at least three, at least four, or more cardiomyocyte maturation factors as described herein. In some embodiments, the in vivo conversion of at least one immature cardiomyocyte or precursor thereof to a cardiomyocyte can be achieved by administering to the subject a composition comprising at least one, at least two, at least three, or at least four cardiomyocyte maturation factors as described herein.

[0142] Cardiomyocytes In some embodiments, the present disclosure provides mature cardiomyocytes. The cardiomyocytes disclosed herein share many distinctive features with native cardiomyocytes but differ in certain aspects (e.g., gene expression profiles). In some embodiments, the cardiomyocytes are non-native or non-naturally occurring. As used herein, "non-native" or "non-naturally occurring" means that the cardiomyocytes differ significantly in certain aspects from naturally occurring cardiomyocytes, i.e., native cardiomyocytes. However, it should be recognized that such significant differences typically relate to structural features that may result in cardiomyocytes exhibiting certain functional differences; for example, even if the gene expression pattern of a cardiomyocyte differs from that of a native cardiomyocyte, the cardiomyocyte may behave in a manner similar to a native cardiomyocyte, although certain functions may be altered (e.g., improved) compared to a native cardiomyocyte.

[0143] The cardiomyocytes of the present disclosure share many unique characteristics with native cardiomyocytes that are important for normal cardiomyocyte function. The characteristics of mature cardiomyocytes are described in Yang et al. Circ. Res. 2014;114(3):511-23.

[0144] In some embodiments, the cardiomyocytes are quiescent. In some embodiments, the cardiomyocytes retain quiescent metabolic and transcriptional activity. In some embodiments, quiescence promotes cardiomyocyte maturation. In some embodiments, the cardiomyocytes express or express elevated levels (i.e., compared to controls) of certain quiescent markers, including p16 and p130. In some embodiments, the cardiomyocytes do not express or express reduced levels (i.e., compared to controls) of proliferation markers, such as Ki67, cyclin C1, and E2F1. In some embodiments, the cardiomyocytes exhibit increased expression of inhibitory E2F factors (e.g., E2F3b, E2F4, E2F5, E2F6, E2F7, and E2F8). In some embodiments, the cardiomyocytes exhibit decreased expression of stimulatory E2F factors (e.g., E2F1, E2F2, and E2F3).

[0145] In some embodiments, the cardiomyocytes are electrically mature cardiomyocytes. In some embodiments, the cardiomyocytes exhibit reduced automaticity. Native, mature, adult human cardiomyocytes naturally beat at 20-30 beats per minute. In some aspects, the cardiomyocytes described herein exhibit a slower intrinsic beat rate. In some embodiments, the cardiomyocytes beat at 15-35 beats per minute, 15-20 beats per minute, or 30-35 beats per minute. A slower intrinsic beat rate may be indicative of reduced automaticity, and cardiomyocytes with reduced automaticity (i.e., reduced spontaneous beating drive) may reduce the risk of arrhythmias in cell therapy.

[0146] In some embodiments, the cardiomyocytes are contractile mature cardiomyocytes. In some embodiments, the cardiomyocytes exhibit increased RNA and protein expression of contractile proteins (e.g., sarcomeric contractile proteins) (i.e., compared to immature cardiomyocytes). In some aspects, the cardiomyocytes exhibit increased RNA and protein expression of at least one of cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain protein 6 (MYH6), and myosin heavy chain protein 7 (MYH7). In some embodiments, the cardiomyocytes exhibit increased protein RNA expression in a dose-dependent manner (e.g., upon treatment of immature cardiomyocytes with an mTOR inhibitor). Increased expression of one or more sarcomeric proteins may improve cardiomyocyte contractility. In some aspects, the cardiomyocytes exhibit increased total content or amount of TNNI3, and in some aspects, increased TNNI3 content or amount compared to slow skeletal muscle-type TNNI3. In some aspects, cardiomyocytes exhibit increased MYH7 content or protein relative to MYH6 content or protein (e.g., in humans). In some embodiments, TNNI3 expression in cardiomyocytes is increased by approximately 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold when compared to immature cardiomyocytes. In some aspects, cardiomyocyte contractility can be measured by measuring cell movement (e.g., using one or more imaging methods). A soft substrate that bends with cardiomyocyte contraction can be used in conjunction with one or more imaging methods to quantitate contractility. In some aspects, cardiomyocytes have increased contractility when compared to immature cardiomyocytes.

[0147] In some embodiments, the cardiomyocytes are metabolically mature cardiomyocytes. In some aspects, the cardiomyocytes have increased metabolic activity when compared to immature cardiomyocytes. In some embodiments, the cardiomyocytes have increased oxygen consumption and / or extracellular acidification rates when compared to immature cardiomyocytes. Metabolic maturity can be quantified using the Seahorse Mitostress Metabolic Assay (Agilent). This assay can be used to measure oxygen consumption rate and extracellular acidification rate in response to one or more compounds (e.g., small molecule compounds) that affect mitochondrial function.

[0148] In some embodiments, the cardiomyocytes exhibit a morphology similar to that of endogenous mature cardiomyocytes. In some embodiments, the cardiomyocytes form rod-shaped cells. In some embodiments, the cardiomyocytes exhibit an organized sarcomere structure. In some aspects, the average sarcomere length is 1.0-4.0 μm, 1.5-3.5 μm, or 2.0-3.0 μm. In some aspects, the average sarcomere length is about 1.5 μm, 1.6 μm, 1.7 μm, 1.8 μm, 1.9 μm, 2.0 μm, 2.1 μm, 2.2 μm, 2.3 μm, 2.4 μm, 2.5 μm, 2.6 μm, 2.7 μm, 2.8 μm, 2.9 μm, or 3.0 μm.

[0149] In some aspects, cardiomyocytes exhibit a mature ion channel expression profile. In some embodiments, cardiomyocytes exhibit increased ion channel expression (i.e., compared to immature cardiomyocytes). Ion channel expression may be increased for one or more of KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, or SERCA2a (ATP2a2). In some aspects, KCNJ2 expression in cardiomyocytes increases in a dose-dependent manner (e.g., upon treatment of immature cardiomyocytes with an mTOR inhibitor). In some embodiments, cardiomyocytes exhibit increased expression of SCN5a, KCNJ2, and RYR2 (i.e., compared to immature cardiomyocytes). In some embodiments, the resting membrane potential of cardiomyocytes is within the range of -70 to -150 mV, -75 to -125 mV, -80 to -100 mV, or -85 to -95 mV. In some embodiments, the resting membrane potential of the cardiomyocytes is about -85 mV, -86 mV, -87 mV, -88 mV, -89 mV, -90 mV, -91 mV, -92 mV, -93 mV, -94 mV, or -95 mV. In some embodiments, the rise rate of the cardiomyocytes is in the range of 150-350 V / sec, 175-325 V / sec, 200-300 V / sec, or 225-275 V / sec. In some embodiments, the rise rate of the cardiomyocytes is about 200 V / sec, 210 V / sec, 220 V / sec, 230 V / sec, 240 V / sec, 250 V / sec, 260 V / sec, 270 V / sec, 280 V / sec, 290 V / sec, or 300 V / sec.

[0150] In some embodiments, cardiomyocytes exhibit increased expression of transcription factors (e.g., nuclear transcription factors). In some embodiments, cardiomyocytes exhibit increased expression of one or more transcription factors. In some embodiments, cardiomyocytes exhibit increased expression of the nuclear transcription factor repressor element-1 silencing transcription factor (REST) ​​(i.e., compared to immature cardiomyocytes). REST is also known as neural-specific silencer factor. In some aspects, expression of REST represses expression of HCN4. In some embodiments, cardiomyocytes exhibit increased expression of REST in a dose-dependent manner (e.g., upon treatment of immature cardiomyocytes with an mTOR inhibitor). In some embodiments, the cardiomyocytes exhibit increased expression of a transcription factor selected from the group consisting of REST / NRSF, GATA4, TBX20, BRG1, YAP, ERBB2, PITX2, MEIS1, ATA4, Nkx2-5, TBX5, NFAT, TEAD, HAND, CHF1, FoxO3 / FoxO4, CHF1 / Hey2, CHF1 / Hey2, FoxO1, Mef2C, SRF, p53, NFkB, and combinations thereof.

[0151] In some embodiments, cardiomyocytes exhibit increased expression of regulators of the oxidative phosphorylation pathway (i.e., compared to immature cardiomyocytes). In some embodiments, cardiomyocytes exhibit increased expression of the oxidative phosphorylation pathway regulator PGC1-alpha (PPARGC1a). Mature cardiomyocytes switch energy extraction from glycolysis to oxidative phosphorylation. Upregulation of PGC1alpha may promote pathways related to oxidative phosphorylation. In some embodiments, metabolism occurs preferentially from fatty acids, e.g., fatty acid β-oxidation (e.g., instead of glycolysis).

[0152] In some embodiments, the cardiomyocytes exhibit increased expression of brain natriuretic peptide (BNP) (i.e., compared to immature cardiomyocytes). In some embodiments, the cardiomyocytes exhibit increased expression of natriuretic peptide B (NPPB).

[0153] In some embodiments, the cardiomyocytes exhibit increased mitochondrial content (i.e., compared to immature cardiomyocytes). For example, the cardiomyocytes may have increased mitochondrial length and elevated mitochondrial membrane potential. In some embodiments, mitochondria occupy approximately 5-70%, 10-60%, 15-50%, or 20-40% of the cardiomyocyte by volume. In some aspects, mitochondria are distributed throughout the cardiomyocyte in a crystal-like lattice pattern.

[0154] In some embodiments, the cardiomyocytes have a conduction velocity of about 0.15-2.5 meters / second, 0.2-2.0 meters / second, 0.25-1.5 meters / second, or 0.3-1.0 meters / second. In some embodiments, the cardiomyocytes have a conduction velocity of about 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0 meters / second.

[0155] In some embodiments, the cardiomyocytes exhibit at least one marker characteristic of endogenous mature cardiomyocytes selected from the group consisting of TNNT2, TNNI3, MYH7, MYH6, KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, SERCA2a (ATP2a2), NPPB (BNP), REST, and PPARGC1a (PGC1a). In some embodiments, the cardiomyocytes exhibit at least one marker characteristic of endogenous mature cardiomyocytes selected from the group consisting of TNNT2, TNNI3, KCNJ2, REST, RyR, and SCN5a.

[0156] As the present invention is not intended to be limited by the starting cells from which the cardiomyocytes are derived, cardiomyocytes can be differentiated in vitro from any starting cell. Examples of starting cells include, without limitation, immature cardiomyocytes or any of their precursors, such as cardiac progenitor cells, pluripotent stem cells, embryonic stem cells, and induced pluripotent stem cells. In some embodiments, cardiomyocytes are differentiated in vitro from initialized cells, partially initialized cells (i.e., somatic cells, e.g., fibroblasts that have been partially initialized to exist in an intermediate state between the induced pluripotent cells and the somatic cells from which they are derived), or transdifferentiated cells. In some embodiments, the cardiomyocytes disclosed herein can be differentiated in vitro from immature cardiomyocytes or their precursors. In some embodiments, cardiomyocytes are differentiated in vitro from precursors selected from the group consisting of immature cardiomyocytes, cardiac progenitor cells, and pluripotent stem cells. In some embodiments, the pluripotent stem cells are selected from the group consisting of embryonic stem cells and induced pluripotent stem cells. In some embodiments, the cardiomyocytes or the pluripotent stem cells from which the cardiomyocytes are derived are human. In some embodiments, the cardiomyocytes are human.

[0157] In some embodiments, the cardiomyocytes are not genetically modified. In some embodiments, the cardiomyocytes acquire characteristics in common with native cardiomyocytes in the absence of genetic modification of the cells. In some embodiments, the cardiomyocytes are genetically modified.

[0158] In some aspects, the present disclosure provides cell lines comprising the cardiomyocytes described herein. In some embodiments, the cardiomyocytes are capable of being frozen, thawed, and passaged. The cardiomyocytes can be passaged at least five times without significant morphological changes.

[0159] Aspects of the present disclosure relate to isolated populations of cardiomyocytes produced by the methods described herein. In some embodiments, the cardiomyocyte population is produced by contacting at least one immature cardiomyocyte with at least one cardiomyocyte maturation factor described herein.

[0160] Aspects of the present disclosure involve microcapsules comprising an isolated population of cells (e.g., cardiomyocytes) described herein. Microcapsules are well known in the art. Suitable examples of microcapsules have been described in the literature (e.g., Orive et al., "Application of cell encapsulation for controlled delivery of biological therapeutics", Advanced Drug Delivery Reviews (2013), dx.doi.org / 10.1016 / j.addr.2013.07.009; Hernandez et al., "Microcapsules and microcarriers for in situ cell delivery", Advanced Drug Delivery Reviews 2010;62:711-730; Murua et al., "Cell microencapsulation technology: Towards clinical application", Journal of Controlled Release 2008;132:76-83; and Zanin et al., "The development of encapsulated cell technologies as therapies for neurological and sensory diseases”, Journal of Controlled Release 2012;160:3-13). Microcapsules can be formulated in a variety of ways. Exemplary microcapsules consist of an alginate core surrounded by a polycation layer, which is then coated with an outer alginate membrane. The polycation membrane forms a semipermeable membrane, thereby conferring stability and biocompatibility. Examples of polycations include, but are not limited to, poly-L-lysine, poly-L-ornithine, chitosan, lactose-modified chitosan, and photopolymerized biomaterials. In some embodiments, the alginate core is modified, e.g., to generate a scaffold comprising an alginate core covalently linked to an oligopeptide at the RGD sequence (arginine, glycine, aspartic acid). In some embodiments, the alginate core is modified, e.g., to generate covalently reinforced microcapsules with chemoenzymatically modified alginate for improved stability. In some embodiments, the alginate core is modified, for example, to generate a membrane-mimetic film assembled by in situ polymerization of acrylate-functionalized phospholipids. In some embodiments, the microcapsules are comprised of alginate enzymatically modified using epimerase. In some embodiments, the microcapsules have covalent bonds between adjacent layers of the microcapsule membrane. In some embodiments, the microcapsules comprise subsieve-sized capsules comprising alginate coupled with phenolic moieties. In some embodiments, the microcapsules comprise a scaffold comprising alginate-agarose. In some embodiments, cardiomyocytes are modified with PEG and then encapsulated within alginate. In some embodiments, an isolated population of cells, e.g., cardiomyocytes, is encapsulated in photoreactive liposomes and alginate.It should be appreciated that the alginate compounds used in the microcapsules can be replaced with other suitable biomaterials, including, but not limited to, PEG, chitosan, PES hollow fibers, collagen, hyaluronic acid, dextran with RGD, EHD and PEGDA, PMBV and PVA, PGSAS, agarose, agarose with gelatin, PLGA, and multilayered embodiments thereof.

[0161] In some embodiments, compositions comprising a population of cardiomyocytes produced by the methods described herein can also be used as a functional component of a medical device. For example, the device can contain a population of cardiomyocytes (e.g., produced from a population of immature cardiomyocytes or their precursors) behind a semipermeable membrane that prevents the passage of the cell population, thereby retaining the cell population within the device. Other examples of devices include those intended for either implantation into cardiac patients or extracorporeal therapy.

[0162] Aspects of the present disclosure involve assays comprising an isolated population of cardiomyocytes (e.g., mature cardiomyocytes) described herein. In some embodiments, the assay can be used to identify one or more candidate agents that promote or inhibit the fate of a mature cardiomyocyte. In some embodiments, the assay can be used to identify one or more candidate agents that promote the differentiation of at least one immature cardiomyocyte or its precursor into a cardiomyocyte. In some embodiments, the assay can be used to identify one or more candidate agents that promote the transition from senescent immature cardiomyocytes to quiescent mature cardiomyocytes.

[0163] The present disclosure contemplates methods in which cardiomyocytes, generated by the methods described herein from iPS cells derived from cells extracted or isolated from an individual suffering from a disease (e.g., heart failure or a cardiac-related disorder), are compared to normal cardiomyocytes from a healthy individual without the disease to identify differences between the cardiomyocytes and normal cardiomyocytes that may serve as markers of the disease (e.g., epigenetic and / or genetic markers). In some embodiments, cardiomyocytes are obtained from an individual suffering from heart failure and compared to normal cardiomyocytes. The cardiomyocytes are then reprogrammed into iPS cells, and the iPS cells are analyzed for genetic and / or epigenetic markers present in cardiomyocytes obtained from individuals suffering from heart failure but absent from normal cardiomyocytes to identify markers (e.g., pre-heart failure). In some embodiments, patient-derived iPS cells and / or cardiomyocytes are used to screen for agents (e.g., agents capable of modulating genes that contribute to the heart failure phenotype).

[0164] Confirmation and identification of the presence of cardiomyocytes Any means generally known to those skilled in the art can be used to confirm the presence of quiescent cardiomyocytes compared to the presence of senescent cardiomyocytes. Any means generally known to those skilled in the art can be used to confirm the presence of cardiomyocytes, for example, mature cardiomyocytes that are produced by exposing at least one immature cardiomyocyte or its precursor to at least one cardiomyocyte maturation factor as described herein.

[0165] In some embodiments, the presence of quiescence markers can be determined by detecting the presence or absence of one or more markers indicative of quiescence. In some embodiments, the method can include detecting positive expression of quiescence markers p16 and / or p130. In some embodiments, the method can include detecting negative expression of proliferation markers Ki67, cyclin C1, and / or E2F.

[0166] In some embodiments, confirmation of the presence of cardiomyocyte markers, e.g., chemically induced cardiomyocytes, can be achieved by detecting the presence or absence of one or more markers indicative of endogenous cardiomyocytes. In some embodiments, the method can include detecting positive expression (e.g., presence) of a cardiomyocyte marker. In some embodiments, the method can include detecting positive expression of one or more of the following: sarcomeric proteins (e.g., cardiac troponin T (TNNT2), cardiac troponin I (TNNI3), myosin heavy chain protein 6 (MYH6), and myosin heavy chain protein 7 (MYH7). In some embodiments, the method can include detecting positive expression of one or more ion channels (e.g., KCNJ2, HCN4, SCN5a, RYR2, CACNA1c, and SERCA2a (ATP2a2)). In some embodiments, the method can include detecting positive expression of brain natriuretic peptide (BNP). In some embodiments, the method can include detecting positive expression of one or more transcription factors (e.g., In some embodiments, the method can include detecting positive expression of one or more regulators of the oxidative phosphorylation pathway (e.g., PGC1-alpha (PPARGC1a)). In some embodiments, the markers can be detected using reagents, such as reagents for detecting TNNI3 and / or KCNJ2. Specifically, cardiomyocytes herein express TNNI3 and KCNJ2 and do not express significant levels of other markers that may be indicative of immature cardiomyocytes. Cardiomyocytes can also be characterized by downregulation of certain markers. For example, cardiomyocytes may be characterized by statistically significant downregulation of HCN4.

[0167] The reagent for the marker can be, for example, an antibody against the marker, or a primer for RT-PCR or PCR reaction, for example, semi-quantitative or quantitative RT-PCR or PCR reaction.Such a marker can be used to evaluate whether cardiomyocytes have been generated.Antibodies or other detection reagents can be linked to labels, for example, radioactive, fluorescent (e.g., GFP), or colorimetric labels for detection.When the detection reagent is a primer, it can be provided in a dry preparation, for example, in a lyophilized form, or in a solution.

[0168] The progression of at least one type of immature cardiomyocyte from senescence to quiescence can be monitored by determining the expression of the marker that characterizes quiescent cardiomyocyte.In some processes, the expression of a certain marker is determined by detecting the presence or absence of this marker.Alternatively, the expression of a certain marker can be determined by measuring the level of the marker present in the cells of cell culture or cell population.In some processes, the expression of the marker that characterizes quiescent cardiomyocyte and the significant lack of expression of the marker that characterizes senescent cardiomyocyte from which quiescent cardiomyocyte is derived are determined.

[0169] The progression of at least one immature cardiomyocyte or its precursor to cardiomyocyte can be monitored by determining the expression of the marker that characterizes mature cardiomyocyte.In some processes, the expression of a certain marker is determined by detecting the presence or absence of this marker.Alternatively, the expression of a certain marker can be determined by measuring the level of marker present in the cells of cell culture or cell population.In some processes, the expression of the marker that characterizes mature cardiomyocyte and the significant lack of expression of the marker that characterizes immature cardiomyocyte or its precursor that mature cardiomyocyte originates are determined.

[0170] As described in connection with monitoring the generation of cardiomyocytes (e.g., mature cardiomyocytes) from immature cardiomyocytes, quantitative or semi-quantitative techniques, such as blot transfer and immunocytochemistry, can be used to measure marker expression, and methods commonly known to those skilled in the art are used. Alternatively, marker expression can be accurately quantified by methods commonly known in the art through the use of techniques such as quantitative PCR. Techniques for measuring extracellular marker content, such as ELISA, may also be used.

[0171] Of course, the present invention is not limited to the markers listed herein as cardiomyocyte markers, but also encompasses markers such as cell surface markers, antigens, and other gene products such as ESTs, RNA (including microRNA and antisense RNA), DNA (including genes and cDNA), and portions thereof.

[0172] Cardiomyocyte enrichment, isolation, and purification Another aspect of the present invention relates to isolating a population of cardiomyocytes (e.g., mature cardiomyocytes) from a heterogeneous cell population, e.g., a mixed cell population including mature cardiomyocytes and immature cardiomyocytes or their precursors from which the mature cardiomyocytes were derived. In some embodiments, a population of quiescent cardiomyocytes is isolated from a heterogeneous cell population, e.g., a mixed cell population including senescent cardiomyocytes and quiescent cardiomyocytes. A population of cardiomyocytes produced by any of the above processes can be enriched, isolated, and / or purified using any cell surface marker that is present on cardiomyocytes but not on the immature cardiomyocytes or their precursors from which the cardiomyocytes were derived. Such cell surface markers are also referred to as affinity tags specific for cardiomyocytes (e.g., mature cardiomyocytes). Examples of cardiomyocyte-specific affinity tags include marker molecules, e.g., antibodies, ligands, or other binding agents specific for polypeptides, that are present on the cell surface of cardiomyocytes but are substantially absent from other cell types (e.g., immature cardiomyocytes). In some processes, antibodies that bind to cell surface antigens of cardiomyocytes are used as affinity tags for enrichment, isolation, or purification of chemically derivatized (e.g., by contacting with at least one cardiomyocyte maturation factor as described herein) cardiomyocytes produced by the methods described herein. Such antibodies are known and commercially available.

[0173] Those skilled in the art will readily appreciate the process of using antibodies to enrich, isolate, and / or purify cardiomyocytes. For example, in some embodiments, a reagent, such as an antibody, is incubated with a cell population containing cardiomyocytes, where the cell population has been treated to reduce cell-cell and substrate adhesion. The cell population is then washed, centrifuged, and resuspended. In some embodiments, if the antibody is not already labeled, the cell suspension is then incubated with a secondary antibody, e.g., an FITC-conjugated antibody capable of binding to the primary antibody. The cardiomyocytes are then washed, centrifuged, and resuspended in a buffer solution. The cardiomyocyte suspension is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). The antibody-bound, fluorescently reprogrammed cells are collected separately from unbound, non-fluorescent cells, resulting in the isolation of cardiomyocytes from other cells present in the cell suspension, such as immature cardiomyocytes or their precursors.

[0174] In another embodiment of the processes described herein, the isolated cell composition comprising cardiomyocytes can be further purified by using alternative affinity-based methods or by additional rounds of sorting using the same or different cardiomyocyte-specific markers. For example, in some embodiments, FACS sorting is used to first isolate cardiomyocytes expressing TNNT2, either alone or in combination with kCNJ2 expression, or alternatively, using a cardiomyocyte marker disclosed herein, from cells in the cell population that do not express one of these markers (e.g., negative cells). In some embodiments, TNNI3 and / or MYH7 are also used as markers for FACS sorting, either alone or in combination with TNNT2 and / or KCNJ2. A second FACS sort, e.g., sorting positive cells using FACS again to isolate cells positive for a different marker than the first sort, enriches the cell population for reprogrammed cells.

[0175] In an alternative embodiment, FACS sorting is used to separate cells by negatively sorting for markers that are present in most immature cardiomyocytes but not in cardiomyocytes (eg, mature cardiomyocytes).

[0176] In some embodiments of the process described herein, cardiomyocytes are fluorescently labeled without the use of antibodies, and then isolated from unlabeled cells by using a fluorescence activated cell sorter (FACS). In such embodiments, the reprogrammed cells are labeled using the above-described method with a nucleic acid encoding GFP, YFP, or another nucleic acid encoding an expressible fluorescent labeling gene, such as a gene encoding luciferase.

[0177] In addition to the procedure just described, chemically induced cardiomyocytes can also be isolated by other cell isolation techniques. Cardiomyocytes can also be enriched or isolated by serial subculture in growth conditions that promote the selective survival or proliferation of cardiomyocytes. Such methods are known to those skilled in the art.

[0178] Using the methods described herein, enriched, isolated, and / or purified cardiomyocyte populations can be generated in vitro from immature cardiomyocytes or their precursors, which have been differentiated from pluripotent stem cells by the methods described herein. In some embodiments, a suitable enrichment, isolation, and / or purification method relates to the in vitro generation of human cardiomyocytes from human immature cardiomyocytes or their precursors, which have been differentiated from human pluripotent stem cells or from human induced pluripotent stem (iPS) cells. In embodiments in which the cardiomyocytes have been differentiated from immature cardiomyocytes, which were previously derived from iPS cells, the cardiomyocytes can be autologous to the subject from whom the cells were obtained to generate the iPS cells.

[0179] Using the methods described herein, an isolated cell population of cardiomyocytes is enriched for cardiomyocyte (e.g., mature cardiomyocyte) content by at least about 1 to about 1000-fold when compared to the cell population prior to chemical induction of the immature cardiomyocyte or progenitor population. In some embodiments, the cardiomyocyte population is induced, improved, enriched, or increased by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, or more when compared to the cell population prior to chemical induction of the immature cardiomyocyte or progenitor population.

[0180] Compositions containing cardiomyocytes Some embodiments of the present invention relate to cell compositions, e.g., cell cultures or cell populations, comprising cardiomyocytes, wherein the cardiomyocytes are derived from at least one type of immature cardiomyocyte. In some embodiments, the cell composition comprises immature cardiomyocytes.

[0181] In some embodiments, the cell composition comprises quiescent cardiomyocytes, wherein the quiescent cardiomyocytes are derived from at least one senescent cardiomyocyte. In some embodiments, the cell composition comprises senescent cardiomyocytes.

[0182] According to certain embodiments, the chemically induced cardiomyocytes are mammalian cells, and in preferred embodiments, such cardiomyocytes are human cardiomyocytes. In some embodiments, the immature cardiomyocytes are derived from pluripotent stem cells (e.g., human pluripotent stem cells).

[0183] Other embodiments of the invention are compositions, e.g., isolated cell populations or cell cultures, comprising cardiomyocytes produced by the methods as disclosed herein. In such embodiments, the cardiomyocytes comprise less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 12%, less than about 10%, less than about 8%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the cardiomyocyte population. In some embodiments, the composition comprises a population of cardiomyocytes, which constitutes more than about 90% of the total cells in the cell population, e.g., at least about 95%, or at least 96%, or at least 97%, or at least 98%, or at least about 99%, or at least about 100% of the total cells in the cell population are cardiomyocytes.

[0184] Certain other embodiments of the invention relate to compositions, e.g., isolated cell populations or cell cultures, comprising a combination of cardiomyocytes (e.g., mature cardiomyocytes) and immature cardiomyocytes or their precursors from which the cardiomyocytes were derived. In some embodiments, the immature cardiomyocytes from which the cardiomyocytes were derived comprise less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of the total cells in the isolated cell population or culture.

[0185] Further embodiments of the present invention relate to compositions, e.g., isolated cell populations or cell cultures, produced by the processes described herein and comprising chemically induced cardiomyocytes as the majority cell type. In some embodiments, the methods and processes described herein produce cardiomyocytes that are at least about 99%, at least about 98%, at least about 97%, at least about 96%, at least about 95%, at least about 94%, at least about 93%, at least about 92%, at least about 91%, at least about 90%, at least about 89%, at least about 88%, at least about 87%, at least about 86%, at least about 85%, at least about 84%, at least about 83%, at least about 82%, at least about 81%, at least about 80%, at least about 79%, at least about 78%, at least about 77%, at least about 76%, at least about Isolated cell cultures and / or cell populations are generated that comprise 75%, at least about 74%, at least about 73%, at least about 72%, at least about 71%, at least about 70%, at least about 69%, at least about 68%, at least about 67%, at least about 66%, at least about 65%, at least about 64%, at least about 63%, at least about 62%, at least about 61%, at least about 60%, at least about 59%, at least about 58%, at least about 57%, at least about 56%, at least about 55%, at least about 54%, at least about 53%, at least about 52%, at least about 51%, or at least about 50% of the total cell mass.

[0186] In another embodiment, the isolated cell population or composition of cells (or cell culture) comprises human cardiomyocytes. In other embodiments, the methods and processes described herein can produce an isolated cell population comprising at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 24%, at least about 23%, at least about 22%, at least about 21%, at least about 20%, at least about 19%, at least about 18%, at least about 17%, at least about 16%, at least about 15%, at least about 14%, at least about 13%, at least about 12%, at least about 11%, at least about 10%, at least about 9%, at least about 8%, at least about 7%, at least about 6%, at least about 5%, at least about 4%, at least about 3%, at least about 2%, or at least about 1% cardiomyocytes. In a preferred embodiment, the isolated cell population comprises human cardiomyocytes. In some embodiments, the percentage of cardiomyocytes in a cell culture or population is calculated without taking into account any feeder cells remaining in the culture.

[0187] Yet other embodiments of the present invention relate to compositions, e.g., isolated cell populations or cell cultures, comprising a mixture of cardiomyocytes and immature cardiomyocytes or their precursors from which the cardiomyocytes differentiate or mature. For example, cell cultures or cell populations can be generated that comprise at least about 5 cardiomyocytes for every about 95 immature cardiomyocytes or their precursors. In other embodiments, cell cultures or cell populations can be generated that comprise at least about 95 cardiomyocytes for every about 5 immature cardiomyocytes or their precursors. Additionally, cell cultures or cell populations comprising other ratios of cardiomyocytes to immature cardiomyocytes or their precursors are contemplated. For example, compositions can be generated that comprise at least about 1 cardiomyocyte for every about 1,000,000 cells, or at least 100,000 cells, or at least 10,000 cells, or at least 1000 cells, or 500, or at least 250, or at least 100, or at least 10 immature cardiomyocytes or their precursors.

[0188] Further embodiments of the present invention relate to compositions, eg, cell cultures or cell populations, comprising human cells, including human cardiomyocytes, that exhibit at least one characteristic of endogenous cardiomyocytes.

[0189] In preferred embodiments of the present invention, the cardiomyocyte cell cultures and / or cell populations comprise non-recombinant human cardiomyocytes. In such embodiments, the cell cultures and / or cell populations are free or substantially free of recombinant human cardiomyocytes.

[0190] Cardiomyocyte maturation factor Aspects of the present disclosure involve contacting immature cardiomyocytes or their precursors with, for example, a cardiomyocyte maturation factor to induce maturation of the immature cardiomyocytes or differentiation of their precursors into cardiomyocytes (e.g., mature cardiomyocytes). The term "cardiomyocyte maturation factor" refers to an agent that promotes or contributes to the conversion of at least one immature cardiomyocyte or its precursor to a cardiomyocyte. In some embodiments, the cardiomyocyte maturation factor induces differentiation of pluripotent cells (e.g., iPSCs or hESCs) into immature cardiomyocytes, for example, by methods described herein. In some embodiments, the cardiomyocyte maturation factor induces maturation of immature cardiomyocytes into cardiomyocytes, for example, by methods described herein. In some embodiments, the cardiomyocyte maturation factor induces the transition of senescent cardiomyocytes to quiescent cardiomyocytes.

[0191] Generally, at least one cardiomyocyte maturation factor described herein can be used alone or in combination with other cardiomyocyte maturation factors to generate cardiomyocytes by the methods as disclosed herein. In some embodiments, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten cardiomyocyte maturation factors described herein are used in the methods for generating cardiomyocytes (e.g., mature cardiomyocytes).

[0192] In some embodiments, the cardiomyocyte maturation factor comprises a modulator (e.g., an inhibitor) of the phosphoinositide 3-kinase (PI3K) / Akt / mTOR pathway. In some embodiments, the cardiomyocyte maturation factor comprises an inhibitor of the mTOR pathway. In some embodiments, the cardiomyocyte maturation factor comprises an inhibitor of PI3K and / or Akt. In some embodiments, the cardiomyocyte maturation factor comprises a small molecule, nucleic acid, amino acid, metabolite, polypeptide, antibody and antibody-like molecule, aptamer, macrocycle, or other molecule. In some embodiments, the cardiomyocyte maturation factor is selected from the group consisting of Torin1, Torin2, rapamycin, everolimus, and temsirolimus. In some embodiments, the cardiomyocyte maturation factor is Torin1. In some embodiments, the cardiomyocyte maturation factor is Torin2. In some embodiments, the cardiomyocyte maturation factor is rapamycin. In some embodiments, the cardiomyocyte maturation factor is everolimus. In some embodiments, the cardiomyocyte maturation factor is temsirolimus.

[0193] In some embodiments, the cardiomyocyte maturation factor comprises a modulator (e.g., an upregulator) of the cell cycle regulator p53. In some embodiments, the cardiomyocyte maturation factor comprises an upregulator or activator of p53. In some embodiments, the cardiomyocyte maturation factor comprises a small molecule, nucleic acid, amino acid, metabolite, polypeptide, antibody and antibody-like molecule, aptamer, macrocycle, or other molecule. In some embodiments, the cardiomyocyte maturation factor is selected from the group consisting of Torin1 and nutlin-3a. In some embodiments, the cardiomyocyte maturation factor is Torin1. In some embodiments, the cardiomyocyte maturation factor is nutlin-3a. In some embodiments, p53 is upregulated (e.g., synergistically) by administering a combination of nutlin-3a and Torin1. In some embodiments, the cardiomyocyte maturation factor is not Torin1. In some embodiments, the cardiomyocyte maturation factor is not an mTOR inhibitor.

[0194] Compositions and Kits Described herein are compositions comprising the cardiomyocytes described herein (e.g., mature and / or quiescent cardiomyocytes). In some embodiments, the compositions also comprise the maturation factors and / or cell culture medium described herein. Also described herein are compositions comprising the compounds described herein (e.g., cell culture medium containing one or more of the compounds described herein).

[0195] Also described herein are kits for practicing the methods disclosed herein and for generating the cardiomyocytes (e.g., mature and / or quiescent cardiomyocytes) disclosed herein. Also described herein are kits for treating chronic heart failure and reducing the incidence of ventricular arrhythmias. In one aspect, the kit comprises at least one immature and / or senescent cardiomyocyte or precursor thereof and at least one maturation factor as described herein, and optionally, the kit can further comprise instructions for converting the at least one immature cardiomyocyte or precursor thereof into a population of mature cardiomyocytes using the methods described herein. In some embodiments, the kit comprises at least two maturation factors. In some embodiments, the kit comprises at least three maturation factors. In some embodiments, the kit comprises any combination of maturation factors.

[0196] In some embodiments, the compounds in the kit can be provided in a waterproof or airtight container, which in some embodiments is substantially free of other components in the kit. The compounds can be provided in more than one container, e.g., a container containing sufficient reagents for a predetermined number of separate reactions, e.g., one, two, three, or more, to induce immature and / or senescent cardiomyocytes, or their precursors, into mature and / or quiescent cardiomyocytes. The maturation factors can be provided in any form, e.g., liquid, dried, or lyophilized. The compound(s) described herein (e.g., maturation factors) are preferably substantially pure and / or sterile. When the compound(s) described herein are provided in a liquid solution, the liquid solution is preferably an aqueous solution, and preferably a sterile aqueous solution. When the compound(s) described herein are provided in a dry form, reconstitution is generally accomplished by the addition of a suitable solvent. A solvent, e.g., sterile water or a buffer solution, can optionally be provided in the kit.

[0197] In some embodiments, the kit further optionally includes informational literature, which can be descriptive, instructional, marketing, or other documentation regarding the methods described herein and / or the use of the compound(s) described herein for the methods described herein.

[0198] The kit's informational documentation is not limited to being instructional or informational. In one embodiment, the informational documentation can include information about the compound's manufacture, the compound's molecular weight, concentration, expiration date, batch or manufacturing site information, etc. In one embodiment, the informational documentation relates to the administration of the compound. The kit's informational documentation is also not limited in its form. Often, the informational documentation, e.g., instructions, is provided in printed form, e.g., a printed document, drawing, and / or photograph, e.g., a label or printed sheet. However, the informational documentation can also be provided in other formats, e.g., Braille, computer-readable material, video recording, or audio recording. In another embodiment, the kit's informational documentation is contact information, e.g., a physical address, email address, website, or phone number, where a kit user can obtain substantial information about the compounds described herein and / or their use in the methods described herein. Of course, the informational documentation can be provided in any combination of formats.

[0199] In one embodiment, the informational documentation can include instructions for administering a compound(s) as described herein (e.g., a maturation factor) in a manner suitable for carrying out a method described herein, e.g., at an appropriate dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein) (e.g., to a cell in vitro or to a cell in vivo). In another embodiment, the informational documentation can include instructions for administering a compound(s) described herein to a suitable subject, e.g., a human, e.g., a human having or at risk for a disorder described herein, or to a cell in vitro.

[0200] The compositions of the kit can include, in addition to the compound(s) described herein, other ingredients, such as solvents or buffers, stabilizers, preservatives, flavoring agents (e.g., bitter taste antagonists or sweetening agents), fragrances or other cosmetic ingredients, and / or additional agents for treating a condition or disorder described herein. Alternatively, the other ingredients can be included in the kit, but in different compositions or containers than the compounds described herein. In such embodiments, the kit can include instructions for mixing the compound(s) described herein with the other ingredients, or instructions for using the compound(s) described herein with the other ingredients, e.g., instructions for combining the two agents prior to use.

[0201] The kit can include one or more containers for a composition containing at least one maturation factor as described herein. In some embodiments, the kit includes separate containers (e.g., two separate containers for two agents), dividers, or compartments for the composition(s) and informational documentation. For example, the composition can be contained in a bottle, vial, or syringe, and the informational documentation can be contained in a plastic envelope or pouch. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial, or syringe, to which the informational documentation is attached in the form of a label. In some embodiments, the kit includes multiple individual containers (e.g., a pack), each containing one or more unit dosage forms (e.g., dosage forms described herein) of a compound described herein. For example, the kit includes multiple syringes, ampoules, foil pouches, or blister packs, each containing a single unit dose of a compound described herein. The containers of the kits can be airtight, waterproof (eg, impervious to changes in moisture or evaporation), and / or light-tight.

[0202] The kit optionally includes a device suitable for administering the composition, e.g., a syringe, inhaler, pipette, tweezers, measuring spoon, dropper (e.g., eye dropper), swab (e.g., cotton swab or wooden swab), or any such delivery device. In a preferred embodiment, the device is a medical implant device packaged, e.g., for surgical insertion.

[0203] The kit can also include components for detecting markers for cardiomyocytes, such as those described herein, e.g., reagents for detecting mature cardiomyocytes. Alternatively, in some embodiments, the kit can include reagents for detecting negative markers for cardiomyocytes for the purpose of negatively selecting mature cardiomyocytes, or for identifying cells (e.g., cardiomyocytes) that do not express these negative markers. The reagents can be, for example, antibodies against markers or primers for RT-PCR or PCR reactions, e.g., semi-quantitative or quantitative RT-PCR or PCR reactions. Such markers can be used to evaluate whether iPS cells have been generated. If the detection reagent is an antibody, it can be provided, for example, in a lyophilized dry preparation or in a solution. The antibody or other detection reagent can be linked to a label for use in detection, e.g., an X-ray diagnostic, fluorescent (e.g., GFP), or colorimetric label. If the detection reagent is a primer, it can be provided, for example, in a lyophilized dry preparation or in a solution.

[0204] The kit may include cardiomyocytes derived from the same type of immature and / or senescent cardiomyocytes or their precursors, e.g., mature and / or quiescent cardiomyocytes, for use, e.g., as a positive cell type control.

[0205] Methods of administering cells In one embodiment, the cells described herein, e.g., a population of mature cardiomyocytes, are transplantable, e.g., can be administered to a subject as a population of cardiomyocytes. In some embodiments, the cells described herein, e.g., a population of mature quiescent cardiomyocytes, are transplantable, e.g., can be administered to a subject as a population of cardiomyocytes. In some embodiments, the subject to which the population of cardiomyocytes is administered is the same subject from which the pluripotent stem cells used to differentiate into cardiomyocytes were obtained (e.g., for autologous cell therapy). In some embodiments, the subject is a different subject. In some embodiments, the subject is suffering from chronic heart failure or is a healthy subject. For example, the cells for transplantation (e.g., a composition comprising a population of cardiomyocytes) can be in a form suitable for transplantation.

[0206] The method may further include administering the cells to a subject in need thereof, e.g., a mammalian subject, e.g., a human subject. The source of the cells may be a mammal, preferably a human. The source or recipient of the cells may be a non-human subject, e.g., an animal model. The term "mammal" includes organisms including mice, rats, cows, sheep, pigs, rabbits, goats, horses, monkeys, dogs, cats, and preferably humans. Similarly, transplantable cells can be obtained from any of these organisms, including non-human transgenic organisms. In one embodiment, the transplantable cells are genetically engineered, e.g., the cells are genetically engineered to include a foreign gene or to inactivate or alter an endogenous gene.

[0207] A composition comprising a population of cardiomyocytes can be administered to a subject using an implantable device. Implantable devices and related technologies are known in the art and are useful as delivery systems for compounds or compositions described herein where continuous or sustained delivery is desired. In addition, implantable device delivery systems are useful for targeting specific points of compound or composition delivery (e.g., localized sites, organs). Negrin et al., Biomaterials, 22(6):563(2001). Sustained release technologies involving alternative delivery methods can also be used in the present invention. For example, sustained release formulations based on polymer technology, sustained release technology, and encapsulation technology (e.g., polymers, liposomes) can also be used to deliver the compounds and compositions described herein.

[0208] Pharmaceutical Composition For administration to a subject, a cell population produced by a method as disclosed herein, e.g., a population of cardiomyocytes (produced by contacting at least one immature cardiomyocyte with at least one maturation factor (e.g., any one, two, three, or more of the maturation factors as described herein), can be administered to a subject, e.g., in a pharmaceutically acceptable composition. These pharmaceutically acceptable compositions comprise a therapeutically effective amount of a population of mature cardiomyocytes as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents. In some aspects, a pharmaceutically acceptable composition comprises a therapeutically effective amount of a population of mature quiescent cardiomyocytes as described above, formulated together with one or more pharmaceutically acceptable carriers (additives) and / or diluents.

[0209] As described in more detail below, the pharmaceutical compositions of the present invention can be specially formulated for administration in solid or liquid form, including those adapted for oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), lozenges, dragees, capsules, pills, tablets (e.g., buccal, sublingual, and those targeted for systemic absorption), boluses, powders, granules, or lingual pastes; (2) parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, e.g., as a sterile solution or suspension, or sustained-release formulation; (3) topical administration, such as a cream, ointment, or controlled-release patch or spray applied to the skin; (4) vaginal or rectal administration, e.g., as a suppository, cream, or foam; (5) sublingual; (6) intraocular; (7) transdermal; (8) transmucosal; or (9) nasal administration. In addition, the compounds can be implanted into a patient or injected using a drug delivery system. For example, Urquhart, et al., Ann. Rev. Pharmacol. Toxicol. 24:199-236 (1984); Lewis, ed. “Controlled Release of Pesticides and Pharmaceuticals" (Plenum Press, New York, 1981); U.S. Patent No. 3,773,919; and U.S. Patent No. 353,270,960.

[0210] As used herein, the term "pharmaceutically acceptable" refers to compounds, substances, compositions, and / or dosage forms that are suitable, within the scope of sound medical judgment, for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0211] As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable substance, composition, or vehicle, such as a liquid or solid filler, diluent, additive, manufacturing aid (e.g., lubricant, magnesium talc, calcium or zinc stearate, or stearic acid), or solvent encapsulating material, for carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) additives, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffers; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C3 12Alcohols, e.g., ethanol; and (25) other non-toxic, compatible substances used in pharmaceutical formulations. Wetting agents, coloring agents, releasing agents, coating agents, sweetening agents, flavoring agents, fragrances, preservatives, and antioxidants may also be present in the formulation. The terms "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably.

[0212] The phrase "therapeutically effective amount," as used herein with respect to a population of cells, refers to an amount of cells, e.g., a population of mature cardiomyocytes, or related cells in a composition comprising the mature cardiomyocytes of the present invention, that is effective to produce some desired therapeutic effect in at least a subpopulation of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment. For example, the amount of a population of mature cardiomyocytes administered to a subject is sufficient to produce a statistically significant measurable change in at least one symptom, such as the occurrence of chronic heart failure, e.g., systolic heart failure or ventricular arrhythmia. Determining a therapeutically effective amount is well within the capabilities of one skilled in the art. Generally, a therapeutically effective amount may vary depending on the subject's history, age, condition, and sex, as well as the severity and type of medical condition in the subject, and the administration of other pharmaceutically active agents.

[0213] "Treating," "preventing," or "ameliorating" a disease or disorder means delaying or preventing the onset of such disease or disorder, or reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of the conditions associated with such disease or disorder. In one embodiment, symptoms of the disease or disorder are alleviated by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, or at least 50%.

[0214] As used herein, the term "administering" refers to placing a composition in a subject by a method or route that results in at least partial localization of the composition at a desired site to produce a desired effect. Suitable administration routes for the methods of the present invention include both local and systemic administration. Generally, local administration delivers more of the administered cardiomyocytes to a specific location compared to the entire body of the subject, while systemic administration delivers cardiomyocytes essentially throughout the subject's entire body.

[0215] In the context of administering compound-treated cells, the term "administering" also includes transplantation of such cells in a subject. As used herein, the term "transplantation" refers to the process of transplanting or transferring at least one cell into a subject. The term "transplantation" includes, for example, autologous transplantation (removing cell(s) from one location in a patient and transferring them to the same or another location in the same patient), allogeneic transplantation (transplantation between members of the same species), and xenogeneic transplantation (transplantation between membe...

Claims

[Claim 1] The invention described in the specification.

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