Electrophysiological maturation of human stem cell derived cardiomyocytes

Abstract

The technology disclosed herein relates to compositions and methods for improving the electrophysiological maturity of in vitro-differentiated cardiomyocytes. Media that promote electrophysiological maturation are described, as well as methods for the use of such media and the electrophysiologically matured cardiomyocytes in e.g., transplant and regenerative medicine procedures. The technology described herein is directed to cardiomyocyte maturation medium. Also described herein are associated compositions, kits, and matured cardiomyocytes. The cardiomyocyte maturation medium disclosed here can be used to modulate at least one electrophysiologic property of a cardiomyocyte. The electrophysiologically matured cardiomyocytes can engraft into cardiac tissue and can reduce or inhibit engraftment arrythmia in a subject.

Description

Attorney Docket No.034186-000102WOP2 ELECTROPHYSIOLOGICAL MATURATION OF HUMAN STEM CELL DERIVED CARDIOMYOCYTES CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No.63 / 528,816 filed July 25, 2023, and U.S. Provisional Application No.63 / 570,513 filed March 27, 2024, the contents of each of which are incorporated herein by reference in their entireties. GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant No. R01 HL148081, awarded by the National Institutes of Health (NIH). The government has certain rights in the invention. TECHNICAL FIELD

[0003] The technology described herein relates to compositions and methods for treating cardiac diseases and disorders and to maturation of cardiomyocytes. BACKGROUND

[0004] Cardiovascular disease remains the leading cause of death for both men and women worldwide, with a rapidly growing impact on developing nations. Cardiomyocyte replacement therapy is an area of active investigation for the treatment of cardiovascular disease, and can restore heart function after myocardial infarction. Human stem cells cultured in vitro can serve as a starting material for producing human cardiomyocytes for engraftment into an injured heart. However, there are complications involved with cardiac engraftment, one of which is the lack of maturity of the in vitro- differentiated cardiomyocytes which can lead to the development of transient cardiac arrhythmias. A method of maturing cardiomyocytes in order to prevent and treat arrhythmias caused by the cardiac graft is needed to improve patient outcomes following cardiomyocyte replacement therapy.

[0005] Cardiovascular disease is a leading cause of mortality worldwide. Therapeutic options for severe heart failure are limited, and therefore new treatments are sought. Regenerative therapy has emerged as an approach for mending damaged hearts. Transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), such as human embryonic stem cell (hESC)- 1 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 CMs or human induced pluripotent stem cell (hiPSC)-CMs, has been recognized as a promising cell source for remuscularization and functional recovery in failing hearts. In considering clinical applications, there are still a few safety hurdles to overcome, particularly the occurrence of transient ventricular arrhythmias following the transplantation of hPSC-CMs, known as engraftment arrhythmias (EAs). Transplanted cardiomyocytes (CMs) have shown the ability to engraft and survive long-term in damaged hearts. These cells can electrically integrate with host cardiomyocytes, allowing the host and graft to beat in sync, thereby potentially increasing cardiac output. However, hPSC-CMs produced in vitro often lack full maturation and display characteristics divergent from adult working CMs, such as spontaneous action potentials. Electrophysiological studies indicate that these action potentials can propagate to the host myocardium and trigger engraftment arrhythmias. Consequently, there is a pressing need to develop methods for the in vitro electrical maturation of hPSC-CMs. See e.g., Chong et al. Nature 510, 273-277 (2014); Liu et al. Nat Biotechnol 36, 597-605 (2018); Shiba et al. Nature 538, 388-391 (2016); Romanguolo et al. Stem Cell Reports 12, 967-981 (2019); Gerbin et al. PLoS One 10, e0131446 (2015); Shiba et al. Nature 489, 322-325 (2012); Karbassi et al. Nat Rev Cardiol 17, 341-359 (2020); the contents of each of which are incorporated by reference herein in their entireties.

[0006] Cardiomyocyte maturation encompasses a multifaceted process that involves various aspects such as metabolism, cell cycle regulation, structural development, electrical function, Ca2+handling, and contractile function. Several in vitro strategies have been explored to mature human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), including extended culture periods, exposure to biochemical cues, co-culture with non-cardiomyocytes, adjustments in substrate stiffness, and biophysical stimulation like pacing. These methods have achieved partial success, contributing to subsets of cardiac maturation and underscoring the complexity of this process, which is characterized by multiple control points. During the perinatal stage, CMs undergo significant changes, including shifts in metabolic hormones, energy substrates, and oxygen supply. This transition initiates a metabolic reprogramming from glycolysis to fatty acid (FA) oxidation, subsequently leading to cell cycle arrest and hypertrophic growth. Recent progress in understanding heart development has revealed that the use of metabolic modulators to shift metabolism towards an adult-like state can significantly enhance the electrophysiological function of hPSC-CMs. See e.g., Guo et al. Circ Res 126, 1086-1106 (2020); Yang et al. J Mol 2 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Cell Cardiol 72, 296-304 (2014); Parikh et al. Circ Res 121, 1323-1330 (2017); Mills et al. Proc Natl Acad Sci U S A 114, E8372-E8381 (2017); Yang et al. Stem Cell Reports 13, 657-668 (2019); Feyen et al. Cell Rep 32, 107925 (2020); Correia et al. Sci Rep 7, 8590 (2017); Zhang et al. Aging (Albany NY) 15, 12551-12569 (2023); Ye et al. Front Cell Dev Biol 9, 644667 (2021); the contents of each of which are incorporated by reference herein in their entireties.

[0007] Metabolic reprogramming is a central driver in the complex maturation process of hPSC- CMs. There is a great need to identify a combination of metabolic modulators that can accelerate electrical maturation to a degree sufficient to eliminate EAs after transplantation. SUMMARY

[0008] The technology described herein relates to media and methods using such media to promote electrophysiological maturation of in vitro-differentiated cardiomyocytes.

[0009] In one aspect, described herein is a cell maturation medium composition for promoting electrophysiological maturity in in-vitro differentiated cardiomyocytes, the medium comprising: a) glucose at 1 mM or less; b) triiodothyronine (T3); and c) dexamethasone.

[0010] In one embodiment, in any of the aspects described herein, the concentration of T3 is between 10nM to 1μM.

[0011] In another embodiment, in any of the aspects described herein, the concentration of T3 is 1 nM.

[0012] In another embodiment, in any of the aspects described herein, the concentration of T3 is 100 nM.

[0013] In another embodiment, in any of the aspects described herein, the concentration of dexamethasone is between 0.1μM to 10μM.

[0014] In another embodiment, in any of the aspects described herein, the concentration of dexamethasone is 1 μM.

[0015] In another embodiment, in any of the aspects described herein, the concentration of dexamethasone is 100 nM.

[0016] In another embodiment, in any of the aspects described herein, the medium comprises RPMI with a glucose concentration of 1 mM or less.

[0017] In another embodiment, in any of the aspects described herein, the medium does not comprise glucose. 3 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0018] In another embodiment, in any of the aspects described herein, the cell maturation medium composition further comprises one or more fatty acids found in mammalian milk. The mammalian milk can be human or bovine milk.

[0019] In another embodiment, in any of the aspects described herein, the fatty acid(s) are selected from the group consisting of linoleic, oleic and palmitic acid.

[0020] In another embodiment, in any of the aspects described herein, the cell maturation medium composition comprises linoleic, oleic and palmitic acids.

[0021] In another embodiment, in any of the aspects described herein, the concentration of linoleic, oleic and palmitic acids is from 0.1μM to 10μM.

[0022] In another embodiment, in any of the aspects described herein, the concentration of linoleic, oleic and palmitic acids is 460 mM.

[0023] In another embodiment, in any of the aspects described herein, the concentration of linoleic acid is from 22.5 – 137 μM.

[0024] In another embodiment, in any of the aspects described herein, the concentration of oleic acid is from 40 – 243 μM.

[0025] In another embodiment, in any of the aspects described herein, the concentration of palmitic acid is from 52.5 – 80 μM.

[0026] In another embodiment, in any of the aspects described herein, the cell maturation medium composition further comprises galactose.

[0027] In another embodiment, in any of the aspects described herein, the galactose concentration is 0.5mM to 10mM.

[0028] In another embodiment, in any of the aspects described herein, the galactose concentration is 0.5 mM.

[0029] In another embodiment, in any of the aspects described herein, the cell maturation medium composition further comprises an AMP-activated protein kinase (AMPK) activator. In various embodiments, the AMPK activator can be 5-aminoimidizole-4-carboxamide-1-β-D- ribofuranoside (AICAR; also referred to as 5-aminoimidazole-4-carboxamide riboside), or an AMPK-activating analog thereof. Analogs include 5-aminoimidazole-4-carboxamide-1-β-D- ribofuranosyl-5′-monophosphate, and others disclosed in U.S. Pat. No.5,777,100, which is hereby incorporated by reference herein, and prodrugs or precursors of AICAR (such as those disclosed in U.S. Pat. No.5,082,829, hereby incorporated by reference herein). 4 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0030] In another embodiment, in any of the aspects described herein, the AMPK activator is 5- aminoimidizole-4-carboxamide riboside (AICAR). In another embodiment, in any of the aspects described herein, the AICAR is present at 0.3mM to 2.0mM. In another embodiment, in any of the aspects described herein, the AICAR is present at 1 mM.

[0031] In another aspect, described herein is a composition comprising in vitro differentiated cardiomyocytes in admixture with a cell maturation medium composition of any one of the embodiments described herein.

[0032] In one embodiment, in any of the aspects described herein, the in vitro differentiated cardiomyocytes are in suspension.

[0033] In another embodiment, in any of the aspects described herein, the in vitro differentiated cardiomyocytes are in monolayer culture.

[0034] In another embodiment, in any of the aspects described herein, the in vitro differentiated cardiomyocytes are in 3D culture.

[0035] In another embodiment, in any of the aspects described herein, the in vitro differentiated cardiomyocytes are in vitro differentiated from a stem cell.

[0036] In another embodiment, in any of the aspects described herein, the stem cell is an iPS cell or an ES cell.

[0037] In another embodiment, in any of the aspects described herein, the iPS cell is derived from a subject with a cardiac disease or disorder.

[0038] In another aspect, described herein is a transplant composition comprising a composition of any one of the embodiments.

[0039] In one embodiment, in any of the aspects described herein, the in vitro-differentiated cardiomyocytes are autologous to a transplant recipient.

[0040] In another embodiment, in any of the aspects described herein, the transplant composition further comprises a pharmaceutically acceptable carrier.

[0041] In another embodiment, in any of the aspects described herein, the transplant composition further comprises a matrix.

[0042] In another embodiment, in any of the aspects described herein, the transplant composition further comprises cardiac endothelial cells.

[0043] In another aspect, described herein is a method of promoting electrophysiological maturation of an in vitro differentiated cardiomyocyte, the method comprising contacting the in 5 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of the embodiments described herein.

[0044] In one embodiment, in any of the aspects described herein, electrophysiological maturity is determined by one or more of the following markers as compared to a reference level: increased expression of SCN5A (encodes Nav1.5, a fast sodium channel), KCNJ2 (encodes Kir2.1 channel, an inward rectifier potassium channel), CACNA1C (encodes Cav1.2, α1C subunit of voltage-dependent L type calcium channel), ATP2A2 (encodes SR calcium ATPase), RYR2 (encodes the ryanodine receptor in SR), Cx43 (Connexin 43, Gap junction α-1) protein and / or Nav1.5 protein, decreased expression of HCN4 (encodes a member of the hyperpolarization-activated cyclic nucleotide-gated sodium channel) and / or CACNA1H (encodes Cav3.2,α1H subunit of voltage-dependent T type calcium channel), and / or decreased field potential frequency, increased field potential duration, increased spike amplitude, increased spike slope, increased conduction velocity, increased calcium transient amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay.

[0045] In another embodiment, in any of the aspects described herein, the cardiomyocytes are contacted with the cell maturation medium composition for at least 10 days.

[0046] In another aspect, described herein is an in vitro-differentiated cardiomyocyte produced by the method of any one of the embodiments.

[0047] In one embodiment, in any of the aspects described herein, the cardiomyocyte has increased electrophysiological maturity relative to an in vitro differentiated cardiomyocyte prepared without contacting with the cell maturation medium composition.

[0048] In another embodiment, in any of the aspects described herein, the in vitro-differentiated and electrophysiologically matured cardiomyocyte, when administered to cardiac tissue of a subject, promotes reduced engraftment arrhythmia relative to an in vitro differentiated cardiomyocyte prepared in the same manner but not contacted with the cell maturation medium composition.

[0049] In another aspect, described herein is a method of transplanting in vitro differentiated cardiomyocytes, the method comprising contacting the in vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of the embodiments described herein, and then administering the cardiomyocytes to a transplant subject. 6 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0050] In one embodiment, in any of the aspects described herein, the method results in reduced engraftment arrhythmia in the recipient.

[0051] In another embodiment, in any of the aspects described herein, the cardiomyocyte is differentiated in vitro from an iPS cell autologous to the recipient.

[0052] In another embodiment, in any of the aspects described herein, the transplant subject has been diagnosed with a cardiac disease or disorder.

[0053] In another aspect, described herein is a method of treating a cardiac disease or disorder, the method comprising administering in vitro differentiated cardiomyocytes in the cell maturation medium composition of any one of the embodiments described herein to a subject in need thereof.

[0054] In another aspect, described herein is a method of treating a cardiac disease or disorder, the method comprising administering a transplant composition of any one of the embodiments described herein or an in vitro differentiated cardiomyocyte of any one of the embodiments described herein to a subject in need thereof.

[0055] In one embodiment, in any of the aspects described herein, the treatment results in reduced engraftment arrhythmia relative to treatment using in vitro-differentiated cells that have not been contacted with cell maturation medium.

[0056] In another embodiment, in any of the aspects described herein, the in-vitro differentiated cardiomyocyte is differentiated in vitro from an iPS cell autologous to the subject.

[0057] In another embodiment, in any of the aspects described herein, the disease or disorder comprises cardiac infarction.

[0058] In another aspect, described herein is a composition of any one of the embodiments, for use in the treatment of a cardiac disease or disorder.

[0059] In another aspect, described herein is a kit for maturing an in vitro-differentiated cardiomyocyte, the kit comprising a cell maturation medium of any one of the embodiments described herein, and instructions for maturing an in vitro-differentiated cardiomyocyte.

[0060] In one embodiment, in any of the aspects described herein, the kit further comprises an in vitro-differentiated cardiomyocyte.

[0061] In another embodiment, in any of the aspects described herein, the kit is for use in transplant to cardiac tissue of a subject in need thereof. 7 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0062] In another embodiment, in any of the aspects described herein, the kit is for use in treatment of a cardiac disease or disorder in a subject in need thereof.

[0063] The technology described herein is directed to cell culture media for electrophysiological maturation of cardiomyocytes and associated compositions and methods. Transplantation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) improves cardiac function post-infarction and can be used to repair injured hearts. A concern for clinical use, however, has been engraftment-related ventricular arrhythmias, which occur for several weeks after transplantation into large animals. Automaticity of graft cells, related to the electrical immaturity of hPSC-CMs, most likely triggers arrhythmic events. Described herein is testing of the hypothesis that modulating metabolism toward an adult-like state can control ion channel gene expression and enhance electrophysiological maturation of hPSC-CMs in vitro. Guided by gene expression and in vitro electrophysiology, described herein is the determination of the concentration and combination of metabolic modulators, including but not limited to triiodothyronine, dexamethasone, glucose, galactose, fatty acids (FA), and AMPK activator. hPSC-CMs treated with the media described herein exhibited slower beat rates, longer field potentials, faster conduction velocities, and enhanced Ca2+kinetics, along with improvement in the expression of electrophysiological genes, which was achieved through the combination of metabolic modulators working synergistically. Moreover, the media described herein increased mitochondrial content, respiratory capacity and FA utilization for larger energy production and storage, promoted cell cycle withdrawal, improved myofibril organization as well as sarcomeric isoform switching from a fetal to an adult type, and increased cell size. The metabolically matured cells engrafted into mouse hearts with efficiency comparable to immature hPSC-CMs. Thus, metabolic reprogramming can accelerate many elements of maturation, including transcriptome, electrophysiology, sarcomere structure, cell cycle, metabolome and capacity for both glycolysis and oxidative phosphorylation.

[0064] Amounts of the “metabolic modulators” (e.g., Gal, FA, AICAR, T3, and / or Dex) expressed here are amounts added to the standard medium formulations, e.g., amounts added to RPMI / B-27 (no glucose) medium.

[0065] Accordingly, in one aspect described herein is a cardiomyocyte maturation medium comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex). 8 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0066] In some embodiments of any of the aspects, the galactose (Gal) is at a concentration selected from: (a) about 0.5 mM; (b) at most 0.5 mM; (c) at most 1.0 mM (d) at most 10.0 mM; (e) 0.5 mM to 1.0 mM; or (f) 0.5 mM to 10.0 mM.

[0067] In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration selected from: (a) about 460 μM; (b) at most 460 μM; (c) at least 230 μM; (d) at least 115 μM; (e) 115 μM to 460 μM; or (f) 230 μM to 460 μM.

[0068] In some embodiments of any of the aspects, the fatty acids (FA) comprise palmitic acids, oleic acids, and / or linoleic acids.

[0069] In some embodiments of any of the aspects, the fatty acids (FA) comprise palmitic acid.

[0070] In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration selected from: (a) about 1.0 mM; (b) at most 1.0 mM; (c) at least 0.3 mM; or (d) 0.25 mM to 1.0 mM.

[0071] In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises about 460 μM fatty acids (FA) and about 1.0 mM AICAR.

[0072] In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration selected from: (a) about 100 nM; (b) at most 100 nM; or (c) 50 nM to 200 nM; or (d) 10 nM to 1 µM.

[0073] In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration selected from: (a) about 1.0 µM; (b) at most 1.0 µM; or (c) 0.5 µM to 2.0 µM; or (d) 10 nM to 10 µM.

[0074] In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises about 100 nM triiodothyronine (T3) and about 1.0 µM dexamethasone (Dex).

[0075] In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises essentially no glucose.

[0076] In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises 0 mM glucose.

[0077] In some embodiments of any of the aspects, the cardiomyocyte maturation medium further comprises comprising: (a) at most 1 mM glucose; (b) at most 5.0 mM glucose; (c) 0 mM to 1 mM glucose; or (d) 0 mM to 5.0 mM glucose.

[0078] In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises about 0.5 mM galactose (Gal) and 0 mM glucose. 9 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0079] In some embodiments of any of the aspects, the cardiomyocyte maturation medium further comprises carnosine.

[0080] In some embodiments of any of the aspects, the carnosine is at a concentration of 2 mM to 10 mM.

[0081] In one aspect described herein is a cardiomyocyte maturation medium comprising: (a) 0.5 mM to 10 mM galactose (Gal); (b) 115 μM to 460 μM fatty acids (FA); (c) 0.25 mM to 1 mM 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) 10 nM to 1 μM triiodothyronine (T3); (e) 10 nM to 10 µM dexamethasone (Dex); and (f) 0 mM to 5 mM glucose.

[0082] In one aspect described herein is a cardiomyocyte maturation medium comprising: (a) about 0.5 mM galactose (Gal); (b) about 460 μM fatty acids (FA); (c) about 1.0 mM 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) about 100 nM triiodothyronine (T3); (e) about 1.0 µM dexamethasone (Dex); and (f) 0 mM glucose.

[0083] In one aspect described herein is a composition comprising a cardiomyocyte maturation medium as described herein, in combination with a cardiomyocyte or population thereof.

[0084] In some embodiments of any of the aspects, the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte.

[0085] In some embodiments of any of the aspects, the cardiomyocyte is electrophysiologically immature.

[0086] In some embodiments of any of the aspects, the cardiomyocyte is electrophysiologically mature.

[0087] In one aspect described herein is a method of electrophysiologically maturing cardiomyocytes, the method comprising contacting electrophysiologically immature cardiomyocytes with a cardiomyocyte maturation medium as described herein for an amount of time sufficient to mature one or more electrophysiological parameters of the cardiomyocytes.

[0088] In one aspect described herein is a method of modulating at least one electrophysiologic property of a cardiomyocyte or population thereof, the method comprising contacting electrophysiologically immature cardiomyocytes with a cardiomyocyte maturation medium as described herein for an amount of time sufficient to modulate one or more electrophysiological parameters of the cardiomyocytes.

[0089] In some embodiments of any of the aspects, the sufficient of amount of time is at least one week. 10 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0090] In some embodiments of any of the aspects, the sufficient of amount of time is one to two weeks.

[0091] In some embodiments of any of the aspects, the electrophysiologically immature cardiomyocytes are differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0092] In some embodiments of any of the aspects, the electrophysiologically immature cardiomyocytes are contacted with the cardiomyocyte maturation medium at least 20 days after differentiation from ESCs or iPSCs.

[0093] In some embodiments of any of the aspects, the ESCs or iPSCs are human cells.

[0094] In some embodiments of any of the aspects, the ESCs or iPSCs are differentiated into electrophysiologically immature cardiomyocytes in vitro or ex vivo.

[0095] In some embodiments of any of the aspects, the maturation method is performed in vitro or ex vivo.

[0096] In some embodiments of any of the aspects, the cardiomyocytes are cultured on MATRIGEL-coated tissue culture plates, wherein the MATRIGEL comprises solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

[0097] In some embodiments of any of the aspects, at least one electrophysiologic property of a cardiomyocyte or population thereof is modulated by the method.

[0098] In some embodiments of any of the aspects, the at least one electrophysiologic property is selected from the group consisting of: beat rate, field potential duration, spike amplitude, spike slope, conduction velocity, Ca2+kinetics, and expression of electrophysiological genes.

[0099] In some embodiments of any of the aspects, the modulation of at least one electrophysiologic property comprises: decreased beat rate, increased field potential duration (FPD), increased spike amplitude, increased spike slope, increased conduction velocity, modulated Ca2+kinetics, and / or modulated expression of electrophysiological genes.

[0100] In some embodiments of any of the aspects, the Ca2+kinetics comprise amplitude, time to peak, time to 50% decay, and / or time to 90% decay.

[0101] In some embodiments of any of the aspects, the modulated Ca2+kinetics comprise increased amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay. 11 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0102] In some embodiments of any of the aspects, the modulated expression of electrophysiological genes comprises increased expression of at least one electrophysiological gene.

[0103] In some embodiments of any of the aspects, the modulated expression of electrophysiological genes comprises increased expression at least two electrophysiological genes.

[0104] In some embodiments of any of the aspects, the electrophysiological genes are selected from the group consisting of: sarcomeric, metabolic, ion-channel, and calcium(Ca2+)-handling genes.

[0105] In some embodiments of any of the aspects, (a) the sarcomeric genes comprise TNNI3 and / or MYL2; (b) the metabolic genes comprise PPARGC1A and / or CPT1B; (c) the ion-channel genes comprise SCN5A, KCNJ2, and / or HCN4; and / or (d) the calcium(Ca2+)-handling genes comprise CACNA1C, CACNA1H, ATPA2, and / or RYR2.

[0106] In one aspect described herein is a kit for cardiomyocyte maturation comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex). In some embodiments of any of the aspects, the kit further comprises RPMI-1640 cell culture medium, B-27™ supplement, lactate, fetal bovine serum (FBS), Y-27632, and / or MATRIGEL.

[0107] In one aspect described herein is a cardiomyocyte or population thereof electrophysiologically matured in a cardiomyocyte maturation medium as described herein.

[0108] In one aspect described herein is a cardiomyocyte or population thereof electrophysiologically matured using a method as described herein.

[0109] In one aspect described herein is a cardiomyocyte or population thereof electrophysiologically matured using a kit as described herein.

[0110] In some embodiments of any of the aspects, the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte.

[0111] In one aspect described herein is a cardiomyocyte transplant composition comprising a cardiomyocyte or population thereof as described herein, in combination with a pharmaceutically acceptable carrier or scaffold.

[0112] In one aspect described herein is a method of reducing or inhibiting engraftment arrythmia, the method comprising administering a cardiomyocyte, or population thereof, as 12 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 described herein or a cardiomyocyte transplant as described herein to a subject in need thereof, wherein the cardiomyocyte, or population thereof, or the cardiomyocyte transplant composition induces a reduced engraftment arrhythmia in the subject relative to an in vitro differentiated or ex vivo differentiated cardiomyocyte, or population thereof, or cardiomyocyte transplant composition that has not been matured using a maturation medium as described herein, a method as described herein, or a kit as described herein.

[0113] In one aspect described herein is a method of treating a cardiac disease, disorder, or injury, the method comprising administering a cardiomyocyte or population thereof as described herein or a cardiomyocyte transplant composition as described herein to a subject in need thereof.

[0114] In some embodiments of any of the aspects, the cardiomyocyte or population thereof or the cardiomyocyte transplant composition is engrafted in or on the heart of the subject.

[0115] In some embodiments of any of the aspects, the subject is a human.

[0116] In some embodiments of any of the aspects, the subject is a non-human mammal.

[0117] In some embodiments of any of the aspects, engraftment arrythmia is reduced by at least 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] FIG.1A-1E shows the supplementation of T3 and Dex augments maturation of hESC-CMs induced by glucose restriction. FIG.1A shows the schema for treatment of hESCs-CMs with regular RPMI, low glucose and T3 / Dex / low glucose and the analysis. FIG.1B depicts QRT-PCR analysis for maturation markers in metabolism (PPARGC1A and ESRRA), cell cycle (CCNA2, CCNB1, and CDK1), electrophysiology (SCN5A, SCN5A fetal isoform, SCN5A adult isoform, KCNJ2, and HCN4), and sarcomere (TNNI1, TNNI3, MYH6, and MYH7). FIG.1C shows immunofluorescence images for α-Actinin, Phalloidin, and Hoechst. Cell area and circularity index were analyzed. FIG.1D examines flux analysis for basal respiration, maximal respiration, spare capacity, and ATP production. FIG.1E shows microelectrode array analyses for spike slope, spike amplitude and conduction velocity. All data are presented as mean ±SEM. *, P<0.05, **, P<0.01 vs. the relevant control (one-way ANOVA). Scale bars represent 20μm. 13 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0119] FIG.2A-2H examines the maturation status of TDLG-CMs in metabolism. FIG.2A shows a PCA plot for metabolomics data from Ctrl-CMs and TDLG-CMs. FIG.2B examines volcano plot of fold change and adjusted P-value in metabolites between Ctrl-CMs and TDLG- CMs. FIG.2C shows heatmaps images of metabolomics analysis of Ctrl-CMs and TDLG-CMs in glycolysis, pentose phosphate pathway, and hexosamine pathway. FIG.2D shows heatmaps images of metabolomics analysis of Ctrl-CMs and TDLG-CMs in purine and pyrimidine metabolism. FIG.2E examines cellular metabolites in energy production (lactate, pyruvate, and lactate / pyruvate ratio). FIGs.2F and 2G examine cellular metabolites in redox state (GSSG, GSH, GSSG / GSH ratio, NADP, NADPH, and NADP / NADPH ratio). FIG.2H examines cellular metabolites in energy storage (creatine, phosphocreatine, ATP, and PCr / ATP fold change). All data are presented as mean ±SEM. *, P<0.05, **, P<0.01 vs. the relevant control (two-tailed, Student t-test).

[0120] FIGs.3A-3G shows the characteristics of TDLG-CMs in transcriptome. FIG.3A shows a PCA plot for RNA-seq data from Ctrl-CMs and TDLG-CMs. FIG.3B shows a heatmap image of differentially expressed genes (DEGs) between Ctrl- and TDLG-CMs. FIG.3C shows volcano plots of upregulated and downregulated DEGs in TDLG-CMs. FIGs.3D-3E examine gene ontology (GO) biological processes terms for upregulated (FIG.3D) or downregulated (FIG.3E) DEGs in TDLG-CMs. See also Tables 1 and 2. FIGs.3F-3G shows pathways for upregulated (FIG.3F) or downregulated (FIG.3G) DEGs in TDLG-CMs. See also Tables 3 and 4. FIG.3H shows the top 5 transcriptional factor binding sites for upregulated or downregulated DEGs in TDLG-CMs.

[0121] FIGs.4A-4J shows single cell RNA-seq of immature and mature CMs. FIG.4A shows a UMAP plot of the combined data set of Ctrl- and TDLG-cells. FIG.4B shows a UMAP clustering analysis by Seurat identified ten clusters. FIGs.4C-4J shows UMAP and violin plots for the expression in cardiac marker (TNNT2) (FIG.4C), metabolic genes (COX6A2 and CKM) (FIG.4D), Ca2+handling genes (ATP2A2 and RYR2) (FIG.4E), ventricular markers (MYL2 and IRX4) (FIG.4F), ion channel genes (SCN5A and GJA1) (FIG.4G), cell cycle gene (CDK1) (FIG.4H), ECM gene (COL1A2) (FIG.4I). See also FIG.4J. Top10 gene ontology biological processes of upregulated DEGs in cluster 0). *, P<0.05, **, P<0.01 vs. the relevant control (two- tailed, Student t-test). 14 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0122] FIGs.5A-5G shows engraftment of mature CMs in pig and arrhythmia burden after transplantation. FIG.5A shows immunohistochemistry images of pig left ventricular sections stained with slow skeletal troponin I (ssTNI). FIG.5B shows arrhythmia burden in TDLG group (n=2), compared to Ctrl group (n=7). All data are presented as mean ±SD.

[0123] FIGs.6A-6G shows single cell RNA-seq of immature and mature CMs. FIGs.6A-6G shows UMAP and violin plots for the expression in in metabolic genes (ESRRA, PPARA, FABP3, and SLC2A4) (FIG.6A), intercalated disc genes (MYZAP and DES) (FIG. 6B), intermediate filament gene (VIM) (FIG.6C), UMAP and violin plots of expression in ventricular marker (FHL2) (FIG.6D), ion channel gene (KCNJ2) (FIG.6E), cell cycle genes (CCNA2 and CCNB1) (FIG.6F), ECM genes (BMP2 and TGFB2) (FIG.6G).

[0124] FIGs.7A-7E shows the optimization of glucose and galactose concentration. FIG.7A shows the time course of maturation markers expression in sarcomere, metabolism, ion channels and calcium handling, comparing in vivo maturation (hESC-CMs transplanted to rat hearts) to in vitro maturation (hESC-CMs cultured on tissue culture plates). FIG.7B shows the scheme for qRT-PCR and microelectrode array (MEA) based screening for electrophysiological maturation (1st round Glu vs. Gal, 2nd round FA vs. AICAR) FIG.7C shows heatmaps of ion channel and Ca2+handling genes expression in response to full factorial Glu (0, 1, 5.5 and 11mM) and Gal (0, 0.5.1 and 10mM). Expression was assessed after 2 weeks of treatment. n=3 from one batch. Glu deprivation induced higher SCN5A, KCNJ2, ATP2A2 and RYR2 expression.0.5mM Gal supplementation in combination with Glu deprivation revealed the robust effect. . # P<0.1, * P<0.05, ** P<0.01 vs. control condition (11mM Glu / 0mM Gal), n=3 technical replicate, data are mean. FIG.7D shows the cell density in 4 candidate conditions of glucose and galactose. n=3 technical replicate, data are mean. FIG.7E demonstrates that the optimal (opti) condition for cell growth and maturation is 0.5mM of galactose and 0mM glucose.

[0125] FIGs.8A-8E shows the optimization of fatty acid and AICAR concentration. FIG. 8A shows heatmaps of ion channel gene expression in response to full factorial FA (0, 115, 230 and 460µM) and AICAR (0, 0.3.1 and 2mM). Expression was assessed after 2 weeks of treatment. AICAR induced higher SCN5A, KCNJ2, ATP2A2 and RYR2 expression. # P<0.1, * P<0.05, ** P<0.01 vs. control condition (0µM FA / 0mM AICAR), n=3 technical replicate, data are mean. FIG.8B shows the heatmap of cell density in response to full factorial FA (0, 115, 230 and 460µM) and AICAR (0, 0.3, 1 and 2mM). High AICAR with either no or low FA resulted in 15 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 less cell number in 2 weeks. FA prevented suppression of cell density induced by high AICAR. # P<0.1, * P<0.05, ** P<0.01 vs. control condition (11mM Glu / 0mM Gal), n=3 technical replicate, data are mean. FIG.8C shows that low cell density conditions are excluded and candidates are selected, based on the expression of ion channel / Ca2+handling genes. FIG.8D shows the MEA data in candidate conditions (4 batches. Each bath includes n=6-8 technical replicate.). Supplementation of 460µM FA and 1mM AICAR promoted electrical maturation at 1week. FIG.8E shows the time course of MEA analysis of maturation media (Glu 0mM RPMI / Gal 0.5mM / FA 460µM / AICAR 1mM / T3 / Dex) treated CMs. The effect was diminished after 2 weeks of treatment and 1 week of maturation media treatment is sufficient for electrical maturation.

[0126] FIGs.9A-9H shows characteristics of MM-treated cardiomyocytes. FIG.9A shows the qRT-PCR data for ion channel, sarcomere and metabolism genes in Ctrl- or MM-CMs. (n=6- 7). Data were normalized to HPRT. FIG.9B shows the representative electron microscope images of mitochondria in Ctrl- or MM-CMs (left). Percentages of mitochondrial volume per cell volume are shown (right, n=3). Scale bars represent 2μm. FIG.9C shows the representative kinetics of the OCR measured with palmitate oxidation stress test (left) in Ctrl- or MM-CMs. OCR parameters of palmitate oxidation stress test are shown (right). FIG.9D shows the representative data of flow cytometer analysis of cTnT and cTnI expression in CMs treated with or without MM (left). cytometer analysis. Percentage of cTnI positive cells in cTnT positive population are shown (right, n=4). FIG.9E shows the assessment of the cell size of cTnT positive cells by flow cytometer. Representative histogram of FSC-A intensity in Ctrl- and MM- CMs is shown (left). Geometrical means of FSC-A intensity are shown (right, n=4). FIG.9F shows the assessment of cell cycle activity by flow cytometer. Representative data of EdU (for S phase) and FxCycle (for DNA content) staining (left). Quantification of %G1, %S or %G2 / M phase are shown (right, n=4). FIG.9G shows the assessment of Ca2+transient by calcium indicator (Fluo-4). Representative Ca2flux recording in Ctrl and MM-CM (left). Ca2+transient parameters from individual Ctrl- (n=65 cells from 3 independent batches) or MM-CMs (n=53 cells from 3 independent batches) (right). CMs were paced at 1Hz at 37 °C. Scale bars represent 0.5s. FIG.9H shows the contraction parameters of engineered heart tissues (EHTs) consist of Ctrl- or MM-CMs (n=4). EHTs were paced at 1.5Hz at 37 °C. * P<0.05, ** P<0.01 vs. Ctrl- CMs, data are mean±SEM 16 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0127] FIGs.10A-10E demonstrates that MM promoted maturation in transcriptome. FIG.10A shows a PCA plot of Ctrl- and MM-CMs in RNA-seq data. FIG.10B shows heatmaps of key genes involved in FA oxidation, sarcomere, and ion channels. FIG.10C shows the gene ontology (GO) analysis of the upregulated and the downregulated differentially expressed genes (DEGs) in MM-CM. Top10 GO terms are shown. FIG.10D shows pathway analysis of the upregulated and the downregulated DEGs in MM-CM. Top 5 pathways are shown. FIG.10E shows transcriptional factors (TFs) enrichment analysis of the upregulated and the downregulated DEGs in MM-CM. Top 5 TFs are shown.

[0128] FIGs.11A-11C demonstrates that engraftment of MM-CMs was comparable to Ctrl-CMs. FIG.11A shows the scheme for mouse engraftment study. FIG.11B shows the number of engrafted cells (human) in 100ng of total DNA including host mouse DNA are analyzed using Alu-PCR (n=10-12). FIG.11C shows the representative immunostaining images of mouse hearts 2 weeks following transplantation of Ctrl or MM-CMs. Images of ssTnI, HE and PAS staining with or without amylase are shown.

[0129] FIGs.12A-12B shows the optimization of glucose and galactose concentration. Related to FIG.7A-7E. FIG.12A shows the schema for in vitro and in vivo maturation of hPSC-CMs in transcriptome using RNAseq data previously published1. FIG.12B shows the bright field images after 2 weeks of indicated treatment.

[0130] FIGs.13A-13B shows the optimization of fatty acid and AICAR concentration. Related to FIG.8A-8E. FIG.13A shows the bright field images after 2weeks of treatment. Low cell density in FA 0 or 115µM in combination with AICAR 1 or 2mM. FIG.13B shows the MEA analysis of regular RPMI (Ctrl) treated CMs and maturation media (MM) treated CMs at 1week.3 different batches. Each bath includes n=3-10 technical replicates. * P<0.05, ** P<0.01 vs. Ctrl-CMs, data are mean±SEM.

[0131] FIGs.14A-14O demonstrates that MM-CMs displayed more matured cardiac properties. Related to FIG.9A-9H. FIGs.14A and B show the representative immunoblots of Nav1.5, CX43 and VINCULIN protein in Ctrl- or MM-CMs (FIG.14A). Quantitative data are shown in FIG.14B. Normalized to VINCULIN, n=4. FIGs.14C and D show the representative immunoblots of cTNI, α-ACTININ and VINCULIN protein in Ctrl- or MM-CMs (FIG.14C). Quantitative data are shown in FIG.14D. Normalized to VINCULIN, n=4. FIGs.14E and F show the representative kinetics of the OCR measured with palmitate oxidation stress test in 17 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Ctrl- or MM-CMs(E). OCR parameters of palmitate oxidation stress test are shown in (F). FIG. 14G shows the assessment of nile red intensity by flow cytometer. Geometrical means of nile red intensity are shown (n=4). FIG.14H shows the assessment of mitochondrial DNA contents obtained by qRT-PCR for mitochondrial and nuclear DNA (n=3). FIG.14I shows the representative kinetics of the OCR measured with mito stress test in Ctrl- or MM-CMs. OCR parameters of mito stress test are shown in (K). n=4. FIGs.14J and K shows the representative kinetics of the OCR measured with palmitate oxidation stress test in Ctrl- or MM-CMs(E). OCR parameters of palmitate oxidation stress test are shown in (F). FIGs.14L and M shows the representative kinetics of the ECAR measured with mito stress test in Ctrl- or MM-CMs (L). ECAR parameters of mito stress test are shown in (M). n=4. FIG.14O shows the representative electron microscope images of glycogen in Ctrl- or MM-CMs (left). Percentages of glycogen volume per cell volume are shown (right, n=3). Scale bars represent 1μm. * P<0.05, ** P<0.01 vs. Ctrl-CMs, data are mean±SEM.

[0132] FIGs.15A-15D shows the Transcriptional profile of Ctrl- and MM-CMs. Related to FIG.10A-10E. FIG.15A shows the RNA-seq expression heatmaps of genes involved in mitochondrial biogenesis, complex1-5, cardiac subtype (atrial, ventricular, and nodal). FIG.15B shows the heatmaps of TCA cycle gene expression. FIG.15C shows the volcano plots of upregulated and downregulated DEGs. FIG.15D shows the gene set enrichment analysis (GSEA) between Ctrl- and MM-CMs. The top half of the plots show enrichment score (ES) on the Y-axis. The Enrichment profile represented by the line / curve in the top half of the plot. The bottom half of the plots show Ranked list metric (Signal2Noise) on the Y-axis. The line in the bottom half of the plots represents the Ranking metric scores. The left side of the plots signifies the positively correlated terms while the right side of the plots signifies the negatively correlated terms. The axis represents the Rank in Ordered Dataset from 0 to 60,000. Each line in the middle of the plots represents a hit.

[0133] FIG 16A-16B examines conditions in order to optimize the amount of glucose and galactose (FIG.16A) and fatty acids (FA) and AICAR (FIG.16B) in the cell maturation media.

[0134] FIG.17 examines the gene expression profiles of genes specific to cardiomyocyte maturation and cell density after exposure to different concentrations of glucose and galactose, while keeping T3 at 100nM and dexamethasone at 1μM.

[0135] FIG.18 examines the gene expression profiles of genes specific to cardiomyocyte 18 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 maturation and cell density after exposure to different concentrations of fatty acids and AICAR, while keeping glucose at 0 mM, galactose at 0.5mM, T3 at 100nM, and dexamethasone at 1μM.

[0136] FIG.19 examines cell morphology after exposure to different concentrations of fatty acids and AICAR.

[0137] FIG.20 examines the gene expression profiles of genes specific to cardiomyocyte maturation after exposure to different concentrations of glucose (from 0mM to 1mM), while keeping galactose at 0.5mM, T3 at 100 nM, and dexamethasone at 1μM.

[0138] FIG.21 examines the gene expression profiles of genes specific to cardiomyocyte maturation and cell density after exposure to different concentrations of FA (from 0 μM to1840 μM), while keeping AICAR at 1mM, glucose at 0mM, galactose at 0.5mM, T3 at 100nM, and dexamethasone at 1μM.

[0139] FIG.22 examines the gene expression profiles of genes specific to cardiomyocyte maturation and cell density after exposure to different concentrations of T3 (from 10nM to 1μM) and dexamethasone (from 0.1μM to 10μM), while keeping FA at 460 μM, AICAR 1mM, glucose at 0mM and galactose at 0.5mM.

[0140] Amounts of the “metabolic modulators” (e.g., Gal, FA, AICAR, T3, and / or Dex) expressed here are amounts added to the standard medium formulations, e.g., amounts added to RPMI / B-27 (no glucose) medium.

[0141] Fig.23A-23E: Glucose and galactose concentrations. (Fig.23A) Time course comparisons of the expression of sarcomeric (e.g., TNNI3, MYL2), metabolic (e.g., PPARGC1A, CPT1B), ion-channels (e.g., SCN5A, KCNJ2, HCN4), and calcium(Ca2+)-handling (e.g., CACNA1C, CACNA1H, ATPA2, RYR2) maturation markers for in vivo maturation (hPSC-CMs transplanted into rat hearts) and in vitro maturation (hPSC-CMs cultured on tissue culture plates). (Fig.23B) Schematic representation of gene expression and microelectrode array (MEA)-based screening for electrophysiological maturation, including Round 1 [glucose (Glu) vs. galactose (Gal)] and Round 2 [fatty acids (FA) vs.5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)]. (Fig.23C) Heatmaps displaying the expression of ion channel genes (e.g., SCN5A, KCNJ2, HCN4, CACNA1C, CACNA1H, ATPA2, RYR2) in response to various concentrations of Glu (0 mM, 1 mM, 5.5 mM, and 11 mM added) and Gal (0 mM, 0.5 mM, 1 mM, and 10 mM added), assessed after 2 weeks of treatment. Expression levels are relative to the control condition (11 mM Glu / 0mM Gal added). (Fig.23D) Cell density measurements 19 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 under four candidate conditions for Glu and Gal concentrations. (Fig.23E) Selection of a condition (Opti) based on ion channel gene expression, following the exclusion of conditions with low cell density (Low).#P < 0.1, * P < 0.05, ** P < 0.01 relative to the control condition (11mM Glu / 0mM Gal added); n = 3 from one batch; data are means.

[0142] Fig.24A-24E: Fatty acids and AICAR concentrations. (Fig.24A) Heatmaps showing ion channel gene expression (e.g., SCN5A, KCNJ2, HCN4, CACNA1C, CACNA1H, ATPA2, RYR2) in response to a full factorial arrangement of fatty acids (FA; 0 µM, 115 µM, 230 µM, and 460 µM added) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; 0 mM, 0.3 mM, 1 mM, and 2 mM added), assessed after two weeks of treatment. Data normalized to HPRT and relative to the control condition (no FA added, no AICAR added). AICAR induced the higher expression of SCN5A, KCNJ2, ATP2A2, and RYR2. n = 3 technical replicates; data are mean values. (Fig.24B) Heatmap of cell density in response to varying concentrations of FA (0 µM, 115 µM, 230 µM, and 460 µM added) and AICAR (0 mM, 0.3 mM, 1 mM, and 2 mM added). n = 3 from one batch; data are mean values. (Fig.24C) Following the exclusion of low cell density conditions (Low), candidate conditions (Cand) were selected based on ion channel gene expression. (Fig.24D) Multi-electrode array (MEA) data (e.g., beat rate, FPDc, spike amplitude, spike slope, conduction velocity) in candidate conditions (e.g., control, +0.3 mM AICAR, +460 µM FA, +460 µM FA / 1 mM AICAR), from three different batches. Each batch includes n = 6–8 technical replicates; data are presented as mean ± SEM. (Fig.24E) Time course analysis of MEA data (e.g., beat rate, FPDc, spike amplitude, spike slope, conduction velocity) indicating that one week of treatment with maturation media is sufficient for electrical maturation.#P < 0.1, * P < 0.05, ** P < 0.01 relative to control condition (0 µM FA / 0 mM AICAR added).

[0143] Fig.25A-25G: Characteristics of MM-Treated cardiomyocytes. (Fig.25A) qRT- PCR analysis of ion channels / Ca2+handling genes (e.g., SCN5A, adult / fetal SCN5A, KCNJ2, HCN4, CACNA1C, CACNA1H, ATPA2, RYR2), metabolism genes (e.g., PPARGC1, CD36), and sarcomere genes (e.g., TNNI1, TTNI3, MYH6, MYH7, adult / fetal TTN) in cardiomyocytes cultured in control media (Ctrl) or maturation media (MM) for one week (n = 7). Data are presented as fold changes normalized to HPRT. (Fig.25B) Left: Representative transmission electron microscope (TEM) images of cardiomyocytes. Right: Quantitative assessment of mitochondrial volume (n = 3). (Fig.25C) Left: Representative trace of the oxygen consumption 20 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 rate (OCR) measured in a fatty acid (FA) oxidation assay. Right: Quantification of maximal respiration, spare capacity, and ATP production. (Fig.25D) Left: Representative confocal microscope images of Ctrl-CMs and MM-CMs stained with α-Actinin, cTnI, and DAPI. Right: Assessments of cell size (n = 3). (Fig.25E) Left: Flow cytometry analyses of Ctrl-CMs and MM-CMs stained with cTnT and cTnI, and Ctrl-CMs stained with isotype control (iso). Right: Quantification of cells positive for both markers. (Fig.25F) Cell cycle activity assessment. First page: Representative data from EdU flow cytometry. Second page: Quantification of the percentage of cells in G1, S, and G2 / M phases (n = 4). (Fig.25G) Calcium transient assay. Top Left: Representative individual calcium indicator (Fluo-4) recordings. Top Right and Bottom: Analysis of parameters (e.g., amplitude, time to peak, time to 50 decay, time to 90% decay) from individual Ctrl-CMs (n = 65 cells from three independent batches) and MM-CMs (n = 53 cells from three independent batches). CMs were paced at 1 Hz, 37°C. * P < 0.05, ** P < 0.01 relative to Ctrl-CMs; data are mean ± SEM.

[0144] Fig.26A-26I: MM-CMs evidence a mature transcriptome. (Fig.26A) Volcano plot depicting the magnitude of effect size (log2 fold change) against significance [–log10 adjusted P- value (padj)] for differentially expressed genes (DEGs) between MM-CMs and Ctrl-CMs. The purple dots indicate genes that are significantly upregulated (right) or downregulated (left). Significant differences were defined as padj < 0.05 and absolute log2fold change [log2(FC)] > 0.58. (Fig.26B) Gene Ontology (GO) biological process (BP) terms associated with upregulated (left) and downregulated (right) DEGs in MM-CMs. (Fig.26C) Reactome Pathway analysis results for upregulated (left) and downregulated (right) DEGs in MM-CMs. (Fig.26D) Transcription factor binding site (TFBS) enrichment analysis for upregulated (left) and downregulated (right) differentially expressed genes (DEGs) in MM-CMs. (Fig.26E) PCA score plot for RNA-seq data derived from CMs subjected to the following treatments: control (Ctrl), MM, and “MM minus 1” interventions. (Fig.26F) Hierarchically clustered heatmap displaying DEGs between MM-CMs and Ctrl-CMs across all conditions. (Fig.26G) Accompanying the heatmap in Fig.26F are the GO BP terms associated with each of the four identified clusters. (Fig.26H, Fig.26I) UpSet plots illustrating the numbers of upregulated (Fig.26H) and downregulated (Fig.26I) DEGs across comparisons between MM and all other conditions.

[0145] Fig.27A-27F: Targeted metabolomics analysis of Ctrl-CMs versus MM-CMs. (Fig. 27A) PCA plot illustrating the metabolic profiles of Ctrl-CMs vs. MM-CMs based on targeted 21 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 metabolomics analysis (n = 4 for each group). (Fig.27B) Volcano plot comparing the abundance of metabolites between Ctrl-CMs and MM-CMs. Significant differences were defined as a q- value < 0.05 and an absolute log2 fold change > 2. (Fig.27C) Heatmap displaying the abundance of metabolites involved in glycolysis, fatty acid (FA) metabolism, the TCA cycle, and oxidative phosphorylation (OXPHOS). (Fig.27D–27F) Quantitative analyses of metabolites related to energetic status (Fig.27D; creatine, PCr, ATP, PCr / ATP), the pentose phosphate pathway (Fig.27E; glucoronate, ribulose-5-phosphate, ribose-5-P, IMP, NADPH), and β-alanine metabolism (Fig.27F; 5,6-dihydrouracil, histidine, carnosine). Data are represented as fold changes normalized to protein concentration. * P < 0.05, ** P < 0.01 relative to Ctrl-CMs; data are mean ± SEM, n = 4.

[0146] Fig.28A-28C: Engraftment of MM-CMs in adult mouse hearts. (Fig.28A) Schematic representation of the mouse engraftment protocol. (Fig.28B) Quantification of the number of engrafted human cells (either Ctrl-CMs or MM-CMs) in 100 ng of total DNA, including host mouse DNA (n = 10–12). (Fig.28C) Immunostaining [ssTnI and hematoxylin and eosin (HE)] of mouse heart sections two weeks post-transplantation with Ctrl-CMs or MM-CMs. * P < 0.05, ** P < 0.01 relative to Ctrl-CMs; data are mean ± SEM.

[0147] Fig.29A-29B is a series of schematics and images. (Fig.29A) Schematic illustrating the comparison of in vitro and in vivo maturation of hPSC-CMs, based on transcriptome analysis using RNA-seq data (see e.g., Genome Expression Omnibus (GEO) repository: GSE190758, available on the world wide web at 0-www-ncbi-nlm-nih- gov.brum.beds.ac.uk / geo / query / acc.cgi?acc=GSE190758). (Fig.29B) Bright field microscope images captured after two weeks of treatment, highlighting low cell density in the 0 mM glucose (Glu) / 0 mM galactose (Gal) condition.

[0148] Fig.30A-8B is a series of images and bar graphs. (Fig.30A) Bright field microscope images after two weeks of treatment with varying concentrations of fatty acids (FA; 0–460 μM added) and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; 0–2 mM added). (Fig. 30B) Multi-electrode array (MEA) data (e.g., beat rate, FPDc, spike amplitude, spike slope, conduction velocity) for hESC-CMs cultured in control media (Ctrl; high glucose RPMI / B27; cyan) and maturation media (MM; grey). MM is shown to promote electrical maturation within one week. Data from three different batches, each with n = 3–10 technical replicates. * P < 0.05, ** P < 0.01 relative to Ctrl-CMs; data are mean ± SEM. 22 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0149] Fig.31A-31P is a series of blots and bar graphs. (Fig.31A, Fig.31B) Representative immunoblots (Fig.31A) and quantification of Nav1.5, Cx43, and Vinculin protein levels (Fig. 31B) (n = 4). (Fig.31C, Fig.31D) Representative immunoblots (Fig.31C) and quantification of cTnI, α-Actinin, and Vinculin protein levels (Fig.31D) (n = 4). (Fig.31E, Fig.31F) (Fig.31E) Representative traces of the oxygen consumption rate (OCR) measured with a fatty acid (FA) oxidation assay. (Fig.31F) Quantification of maximal respiration, spare capacity, and ATP production (n = 4). (Fig.31G) Quantitative assessment of lipid accumulation using Nile red staining (n = 4). (Fig.31H) Evaluation of mitochondrial DNA content through qRT-PCR for mitochondrial and nuclear DNA (ncDNA) (n = 3). (Fig.31I) Left: Representative transmission electron microscope (TEM) images. Right: Quantification of glycogen-positive (+) areas (n = 3). (Fig.31J, Fig.31K) (Fig.31J) Representative trace of OCR from the mito stress test and (Fig. 31K) quantification of maximal respiration, spare capacity, and ATP production (n=4). (Fig. 31L, Fig.31M) (Fig.31L) Representative trace of extracellular acidification rates (ECAR) from a glycolytic stress test and (Fig.31M) quantification of glycolytic capacity (n = 4). (Fig.31N) Flow cytometry assessment of cell size for cTnI-positive (+) or -negative (-) cells. Geometrical mean intensity of FSC-A was quantified (n = 4). (Fig.31O) Flow cytometry assessment of cell size for Ctrl-CMs and MM-CMs. Left: Representative histogram from each group. Right: Geometrical mean intensity of FSC-A (n = 4). (Fig.31P) Representative periodic acid-Schiff (PAS) staining images with (+) or without (-) amylase treatment.

[0150] Fig.32A-32I is a series of heatmaps and plots. (Fig.32A–32E) Heatmaps showcasing key genes involved in mitochondrial biogenesis (Fig.32A), fatty acid oxidation (Fig.32B), the TCA cycle (Fig.32C), sarcomere structure (Fig.32D), and ion channel / transporter (e.g., Ca2+handling) functions (Fig.32E). (Fig.32F) PCA loading vector plot displaying the top 10 positive and negative loading vectors (genes) for each principal component axis: PC1 and PC2. (Fig.32G, Fig.32H) Gene Ontology (GO) biological process (BP) terms associated with the top 500 PC1 positive loading vectors (top panel of Fig.32G), PC1 negative loading vectors (bottom panel of Fig.32G), PC2 positive loading vectors (top panel of Fig.32H), and PC2 negative loading vectors (bottom panel of Fig.32H). (Fig.32I) Hierarchically clustered heatmap displaying key genes involved in ion channel / transporter functions between MM-CMs and Ctrl- CMs across all conditions. 23 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0151] Fig.33A-33D is a series of graphs. (Fig.33A, Fig.33B) (Fig.33A) Representative mitochondrial complex activity assay and (Fig.33B) corresponding quantitative data (n = 4) of Complex I, Complex II, and Complex V. (Fig.33C) Flow cytometry assessment of mitoSOX™ intensity, indicating mitochondrial reactive oxygen species (ROS) production. Left: Representative histogram for each group. Right: Quantification of the geometrical mean fluorescent intensity (n = 3). (Fig.33D) Dose-dependent improvement in Ca2+ handling (e.g., amplitude, time to peak, time to 50 decay, time to 90% decay) with carnosine treatment (2 mM of carnosine: “Car_2nM”; 10 mM of carnosine: “_10nM”). * P < 0.05, ** P < 0.01 relative to Ctrl-CMs; data are mean ± SEM.

[0152] Fig.34 shows immunostaining (periodic acid-Schiff (PAS) with or without amylase) of mouse heart sections two weeks following transplantation of Ctrl-CMs or MM-CMs.

[0153] Fig.35 shows the experimental plan for identifying working range of each maturation medium component.

[0154] Fig.36 shows cell density after 1 week treatment with the indicated maturation medium.

[0155] Fig.37 shows toxic concentrations for each intervention in the maturation medium.

[0156] Fig.38 shows testing of the T3 working concentration of 10nM-1μM. Based on the expression of HCN4, 10nM-1μM was an effective range for added T3.

[0157] Fig.39 shows testing of the Dexamethasone working concentration of 10nM-10μM. Based on the expression of KCNJ2 and the cell density, 10nM-10μM was an effective range for added dexamethasone.

[0158] Fig.40 shows testing of the glucose working concentration of 0mM-5mM. Based on the expression of HCN4, 0mM-5mM was an effective range for glucose restriction.

[0159] Fig.41 shows testing of the galactose working concentration of 0.5mM-10mM. Based on the expression of HCN4, 0.5mM-10mM was an effective range for added galactose.

[0160] Fig.42 shows testing of the FA working concentration of 115μM-460μM. Based on the expression of HCN4 and KCNJ2, and the cell density, 115μM-460μM was an effective range for added fatty acids.

[0161] Fig.43 shows testing of the AICAR working concentration of 0.25mM-1mM. Based on the expression of KCNJ2 and the cell density, 0.25mM-1mM was an effective range for added AICAR.

[0162] Fig.44 shows the working range for each intervention in the maturation medium. 24 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 DETAILED DESCRIPTION

[0163] The technology described herein relates to compositions and methods for advancing the electrical or electrophysiological maturity of in-vitro differentiated cardiomyocytes in order to be used for treatment of a cardiac injury or disorder, such as a myocardial infarction. Definitions

[0164] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims, are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. 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 technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0165] Definitions of common terms in cellular and molecular biology can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blackwell Science Ltd., 1999-2012 (ISBN 9783527600908); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); Immunology by Wemer Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN- 1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al, Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in 25 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are all incorporated by reference herein in their entireties.

[0166] As used herein, “electrophysiological maturity” when applied to cardiomyocytes refers to the phenotype of a cell that comprises an electrophysiological phenotype similar to adult cardiomyocytes and does not comprise at least one electrophysiological feature of a fetal cardiomyocyte. For the avoidance of doubt, an electrophysiologically mature in vitro- differentiated cardiomyocyte generated as described herein will have at least one of the following phenotypic characteristics as compared to an in vitro-differentiated cardiomyocyte prepared in the same manner but absent incubation in a maturation medium as described herein: increased expression of an ion channel gene, increased sodium current density, increased inwardly-rectifying potassium channel current density, decreased action potential frequency, decreased calcium wave frequency, and decreased field potential frequency, where “increased” and “decreased” are as defined herein below.

[0167] As used herein, the term "cardiomyocyte" or "CM" refers to a cardiac muscle cell. Cardiomyocytes (CMs) generally comprise phenotypic and / or structural features associated with cardiac muscle (e.g., electrophysiological phenotypes, sarcomeres, actin, myosin and cardiac troponin T expression, etc.).

[0168] As used herein, “in vitro-differentiated cardiomyocytes" refers to cardiomyocytes that are generated in culture, typically, but not necessarily via step-wise differentiation from a progenitor cell such as a stem cell or a pluripotent stem cell. Examples of stem cell types include, but are not limited to, a human embryonic stem cell, an induced pluripotent stem cell, an early mesoderm cell, a lateral plate mesoderm cell or a cardiac progenitor cell. Thus, while cardiomyocytes in vivo are ultimately derived from a stem cell, i.e., during development of a tissue or organism, a stem cell-derived cardiomyocyte as described herein has been created by in vitro differentiation from a stem cell. As used herein, a cardiomyocyte differentiated in vitro from a stem cell, e.g., an induced pluripotent stem (iPS) cell or embryonic stem cell ("ES cell" or "ESC"), is a "stem-cell derived cardiomyocyte" or "in vitro- differentiated cardiomyocyte" if it has expression of cardiac troponin T (cTnT). Where the electrophysiological 26 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 disturbances of engraftment arrhythmia are anticipated to occur regardless of the differentiation approach used to generate the cardiomyocytes, it is specifically contemplated herein that any in vitro stem-cell derived cardiomyocyte can benefit from maturation in a maturation medium as described herein. Methods for differentiating stem cells in vitro to cardiomyocytes are known in the art and exemplary approaches are described elsewhere herein.

[0169] The term "pluripotent" as used herein refers to a cell with the capacity, under different conditions, to differentiate to cell types characteristic of all three germ cell layers (endoderm, mesoderm and ectoderm). Pluripotent cells are characterized primarily by their ability to differentiate to all three germ layers, using, for example, a nude mouse and teratoma formation assay. Pluripotency is also evidenced by the expression of embryonic stem (ES) cell markers, although the preferred test for pluripotency is the demonstration of the capacity to differentiate into cells of each of the three germ layers. In some embodiments, a pluripotent stem cell is produced or generated from a cell that is not a pluripotent cell and are referred to herein as "induced pluripotent stem cells."

[0170] As used herein, the terms "induced pluripotent stem cell," "iPS cell," "iPSC," "hiPSC," and "human induced pluripotent stem cell" are used interchangeably herein and refer to a pluripotent cell artificially derived from a parental cell, such as a differentiated somatic cell. iPSCs are capable of self renewal and differentiation into cell fate-committed stem cells, including cells of the cardiac lineages, as well as various types of mature cells. Examples of parental cells useful for the generation of induced pluripotent stem cells include, but are not limited to, somatic cells such as fibroblasts, cardiac progenitor cells, skeletal muscle cells, and the like. Such "iPS" or "iPSC" cells can be created by inducing the expression of certain regulatory genes or by the exogenous application of certain proteins. Methods for the induction of iPSCs are known in the art and are further described below. (See, e.g., Zhou et al. , Stem Cells 27 (11): 2667-74 (2009); Huangfu et al. , Nature Biotechnol.26 (7): 795 (2008); Woltjen etal, Nature 458 (7239): 766-770 (2009); and Zhou et al, Cell Stem Cell 8:381-384 (2009); each of which is incorporated by reference herein in their entirety.) As used herein, "hiPSCs" are human induced pluripotent stem cells. Parental cells can include somatic cells that have been reprogrammed to induce a pluripotent, undifferentiated phenotype by various means.

[0171] The term "derived from," used in reference to a stem cell means the stem cell was 27 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 generated by reprogramming of a differentiated cell to a stem cell phenotype. The term "derived from," used in reference to a differentiated cell means the cell is the result of differentiation, e.g., in vitro differentiation, of a stem cell. As used herein, "iPSC-CMs" or "induced pluripotent stem cell-derived cardiomyocytes" are used interchangeably to refer to cardiomyocytes derived from an induced pluripotent stem cell. As used herein, the term "derived from" when referring to a cell can also encompass the initial cell that is generated and any subsequent progeny thereof.

[0172] As used herein, the term "epicardial cells," in part, refers to epithelial cells of the epicardium, which typically serves as an outer protective layer of the heart and can provide factors for myocardial growth and maturation. Epicardial cells, as described herein, express WT1 and / or TCF21 and can further express one or more of the following markers: TBX18; BNC1; and cytokeratins. Additional functional epicardial cell criteria include, but are not limited to, formation of a polarized epithelial sheet and the ability to undergo epithelial-to-mesenchymal transition (EMT) (in vitro or in vivo) to generate fibroblasts (e.g., cardiac fibroblasts) and vascular smooth muscle lineages. An epicardial cell (or a population thereof) as the term is used herein can help promote engraftment of cardiomyocytes when co-administered to cardiac tissue as described herein. Additional information on how to produce in-vitro-differentiated epicardial cells can be found in PCT / US2018 / 022659, which is incorporated herein by reference in its entirety.

[0173] As used herein, “RPMI”, refers to a cell culture medium that contains around 14.1μM phenol red, 102.7mM sodium chloride, 23.8mM sodium bicarbonate, 10.65mM disodium phosphate, 5.37mM potassium chloride, 0.83mM magnesium sulfate, 0.609mM calcium nitrate, and around 11 mM D-glucose. D-glucose and glucose are used interchangeably throughout this disclosure. RPMI is commercially available from a number of suppliers.

[0174] As used herein, “RPMI without glucose”, refers to a cell medium that contains the components of RPMI noted above, but not D-glucose.

[0175] As used herein, “RPMI with a glucose concentration of 1mM or less” refers to a cell medium that contains the components of RPMI noted above, with 1 mM D-glucose or less instead of the standard 11 mM D-glucose. For the avoidance of doubt, “RPMI with a glucose concentration of 1mM or less” includes a level of glucose, whereas “RPMI without glucose” lacks glucose entirely.

[0176] As used herein, the term "treating" includes reducing or alleviating at least one adverse 28 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 effect or symptom of a condition, disease or disorder. For example, the term "treating" and "treatment" refers to administering to a subject an effective amount of a composition, e.g., an effective amount of a composition comprising a population of e.g., epicardial cells, cardiomyocytes etc. so that the subject has a reduction in at least one symptom of the disease or an improvement in the disease, for example, beneficial or desired clinical results (e.g., improved cardiac function in an infarcted area of the heart, improved engraftment of cardiomyocytes etc.). For purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, diminishment of extent of disease, disease stabilization (e.g., not worsening), delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, treating can refer to prolonging survival as compared to expected survival if not receiving treatment. Thus, one of skill in the art realizes that a treatment can improve the disease condition, but may not be a complete cure for the disease. In some embodiments, treatment can include prophylaxis. However, in alternative embodiments, treatment does not include prophylaxis.

[0177] "Treatment" of a cardiac disorder, a cardiac disease, or a cardiac injury (e.g., myocardial infarction) as referred to herein refers to therapeutic intervention that enhances cardiac function and / or enhances cardiomyocyte engraftment and / or enhances cardiomyocyte transplant or graft vascularization in a treated area, while avoiding or minimizing engraftment arrhythmia, thus improving the function of e.g., the heart. That is, cardiac "treatment" is oriented to the function of the heart (e.g., enhanced function within an infarcted area), and / or other site treated with the compositions described herein. A therapeutic approach that improves the function of the heart, for example as assessed by measuring left-ventricular end-systolic dimension (LVESD)) by at least 10%, and preferably by at least 20%, 30%, 40%, 50%, 75%, 90%, 100% or more, e.g., 2-fold, 5-fold, 10-fold or more, up to and including full function, relative to such function prior to such therapy is considered effective treatment. Effective treatment need not cure or directly impact the underlying cause of the heart disease or disorder to be considered effective treatment.

[0178] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, 29 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 commensurate with a reasonable benefit / risk ratio.

[0179] As used herein, "prevention" or "preventing," when used in reference to a disease, disorder or symptoms thereof, refers to a reduction in the likelihood that an individual will develop a disease or disorder, e.g., heart failure following myocardial infarction, or engraftment arrhythmia following cardiomyocyte transplantation as but two examples. The likelihood of developing a disease or disorder is reduced, for example, when an individual having one or more risk factors for a disease or disorder either fails to develop the disorder or develops such disease or disorder at a later time or with less severity, statistically speaking, relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop symptoms of a disease, or the development of reduced (e.g., by at least 10% on a clinically accepted scale for that disease or disorder) or delayed (e.g., by days, weeks, months or years) symptoms is considered effective prevention.

[0180] The terms "decrease", "reduced", "reduction", or "inhibit" are all used herein to mean a decrease or lessening of a property, level, or other parameter by a statistically significant amount. In some embodiments, "reduce," "reduction" or "decrease" or "inhibit" typically means a decrease by at least 10% as compared to a reference level and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% , or more. As used herein, "reduction" or "inhibition" does not encompass a complete inhibition or reduction as compared to a reference level. "Complete inhibition" is a 100% inhibition as compared to a reference level.

[0181] The terms "increased" 'increase" or "enhance" or "activate" are all used herein to generally mean an increase of a property, level, or other parameter by a statically significant amount; for the avoidance of any doubt, the terms "increased", "increase" or "enhance" or "activate" means an increase of at least 10% as compared to a reference level, for example an increase of 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% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or 30 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 at least about a 5 -fold or at least about a 10-fold increase, at least about a 20-fold increase, at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase or more as compared to a reference level.

[0182] As used herein the term "comprising" or "comprises" is used in reference to compositions, methods, and respective component(s) thereof, that are essential to the invention, yet open to the inclusion of unspecified elements, whether essential or not.

[0183] As used herein the term "consisting essentially of” refers to those 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.

[0184] The term "consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the embodiment.

[0185] 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, references to "the method" includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0186] The compositions and methods described herein provide improvements in cardiomyocyte transplantation using in vitro-differentiated cardiomyocytes. In particular, the compositions and methods provide improved electrophysiological maturity of in vitro- differentiated cardiomyocytes that reduce the severity and / or duration of so-called engraftment arrhythmia in transplant recipients. As used herein, the phrase "engraftment arrhythmia" or "EA" is a novel and aberrant cardiac rhythm or rate that occurs following administration of a graft of cardiac cells or cardiomyocytes, e.g., in vitro differentiated cardiomyocytes, and can be a serious complication of cardiac remuscularization therapy. Without wishing to be bound by theory, it is believed that engraftment arrhythmias are related to automaticity of the transplanted cells, which disrupts the normal electrical rhythm of the host tissue. Engraftment arrhythmias are observed after cardiac graft transplantation and generally persist transiently for days to weeks. While engraftment arrhythmia generally resolves on its own over time, engraftment arrhythmia can cause sudden cardiac death and heart failure in the subject. An abnormal electrocardiogram (EKG) is an indication that the subject has an abnormal cardiac rhythm or 31 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 cardiac arrhythmia.

[0187] A key feature of engraftment arrhythmia is the origination of the stimulus from the site of engraftment, rather than from the sinoatrial node (SAN) or AV node. A disturbance in rhythm is any recurring or prolonged deviation from a normal sinus rhythm. A disturbance in rate includes a deviation of at least 10% ( e.g ., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) up or down in the subject's normal resting heart rate upon induction or introduction of new cardiomyocytes to a subject's cardiac tissue. In some embodiments, engraftment arrhythmia is caused by or related to the introduction of exogenous cardiomyocytes, including, but not limited to in vitro-differentiated cardiomyocytes, to cardiac tissue, e.g., as in a transplant of cardiomyocytes administered, for example, to promote repair of an infarct or to augment cardiac function, e.g., in a cardiomyopathy. In another embodiment, engraftment arrhythmia is a heart rate above 100 beats / minute. “Arrhythmia burden,” or “engraftment arrhythmia burden” refers to the proportion of time in which the subject experiences engraftment arrhythmia. Thus, the severity or duration of engraftment arrhythmia can be measured, e.g., as a fraction or proportion of the day or other observation period in which the graft recipient experiences arrhythmia. Following graft CM administration, the disturbance in cardiac rate or rhythm can be prolonged, e.g., lasting more than 5% of the day (or other observation period). In some embodiments, the reduction in engraftment arrhythmia after transplant of the cardiomyocytes as described herein (including the in vitro-differentiated human cardiomyocytes), includes a reduction of at least 5%, at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more in the proportion of the day (or other observation period) in which the graft recipient experiences arrhythmia relative to arrhythmia experienced by a subject receiving CMs that are not treated with the methods described herein to promote electrophysiological maturity. A reduction in engraftment arrhythmia could also include a reduction of at least 1-fold, at least 2-fold, at least 3- fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, at least 100-fold or more relative to arrhythmia experienced by a subject receiving CMs that are not treated with the methods described herein to promote electrophysiological maturity. 32 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0188] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term “about.” The term “about” when used in connection with percentages can mean ±1%. In Vitro Differentiated Cardiomyocytes

[0189] The methods and compositions described herein use in vitro-differentiated cardiomyocytes. Methods for the differentiation cardiomyocytes from ESCs or iPSCs are known in the art. As one example, see LaFlamme et al., Nature Biotech 25:1015-1024 (2007), which describes the in vitro differentiation of cardiomyocytes and which is incorporated herein by reference in its entirety.

[0190] A number of protocols for differentiating ESCs and iPSCs to cardiomyocytes are known in the art. For example, agents can be added or removed from cell culture media to direct differentiation to cardiomyocytes in a step-wise fashion. Non-limiting examples of factors and agents that can promote cardiomyocyte differentiation include small molecules (e.g., Wnt inhibitors, GSK3 inhibitors), polypeptides (e.g., growth factors), nucleic acids, vectors, and patterned substrates (e.g., nanopatterns). The addition of growth factors necessary in cardiovascular development, including but not limited to fibroblast growth factor 2 (FGF2), transforming growth factor β (TGFβ) superfamily growth factors- Activin A and BMP4, vascular endothelial growth factor (VEGF), and the Wnt inhibitor DKK-1, can also be beneficial in directing differentiation along the cardiac lineage. Additional examples of factors and conditions that help promote cardiomyocyte differentiation include but are not limited to B27 supplement lacking insulin, cell-conditioned media, external electrical pacing, and nanopatterned substrates, among others. In certain embodiments, the step-wise differentiation of ESCs or iPSCs to cardiomyocytes proceeds in the following order: ESC or iPSC > cardiogenic mesoderm > cardiac progenitor cells > cardiomyocytes (see e.g., Lian X, Zhang J, Azarin SM, Zhu K, Hazeltine LB, Bao X, Hsiao C, Kamp TJ, Palecek SP. Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt / β-catenin signaling under fully defined conditions. Nat Protoc.2013 Jan;8(1):162-75. doi: 10.1038 / nprot.2012.150. Epub 2012 Dec 20. PMID: 23257984; PMCID: PMC3612968, US Application No.2017 / 0058263 A1; 2008 / 0089874 A1; 2006 / 0040389 A1; US Patent No.10,155,927 B2; 9,994,812 B2; and 9,663,764 B2, the contents of each of which are incorporated herein by reference their entireties). 33 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Transplant composition

[0191] As used herein, the term “transplanting” or “transplant” is used in the context of the placement of cells, e.g. cardiomyocytes, as described herein into a subject, by a method or route which results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced. The cells e.g. cardiomyocytes, or cardiomyocytes admixed with epicardial cells, can be implanted directly into the cardiac tissue of the recipient, e.g., at or near a site of a cardiac defect, e.g., an infarction, or into cardiac tissue of a subject with a cardiac disease. As one of skill in the art will appreciate, long-term engraftment of the cardiomyocytes is desired as cardiomyocytes generally do not proliferate to an extent that the heart can heal from an acute injury comprising cell death. In some embodiments, the cells are optionally transplanted on or within a scaffold or biocompatible material that supports viability of the implanted cardiomyocytes, and / or, for example, assists with keeping administered cells in the desired location for engraftment or promotes integration with native cardiac cells in a subject. Preferably, the cardiomyocytes are human stem cell derived-cardiomyocytes or in vitro- differentiated cardiomyocytes as described herein. In some embodiments, the in vitro- differentiated cardiomyocytes are differentiated in vitro from embryonic stem cells or from iPS cells.

[0192] A scaffold is a structure, comprising a biocompatible material including but not limited to a gel, sheet, or lattice that can contain the cells in a desired location but permit the entry or diffusion of factors in the environment necessary for survival and function. A number of biocompatible polymers suitable for a scaffold are known in the art.

[0193] One of skill in the art can determine the number of cardiomyocytes needed for a graft. In some embodiments, about 10 million cardiomyocytes to about 10 billion cardiomyocytes are administered to the subject. For use in the various aspects described herein, an effective amount of human cardiomyocytes can comprise at least 1 X 107, at least 1.1 X 107, at least 1.2 X 107, at least 1.3 X 107, at least 1.4 X 107, at least 1.5 X 107, at least 1.6 X 107, at least 1.7 X 107, at least 1.8 X 107, at least 1.9 X 107, at least 2 X 107, at least 3 X 107, at least 4 X 107, at least 5 X 107, at least 6 X 107, at least 7 X 107, at least 8 X 107, at least 9 X 107, at least 1 X 108, at least 2 X 108, at least 5 X 108, at least 7 X 108, at least 1 X 109, at least 2 X 109, at least 3 X 109, at least 4 X 109, at least 5 X 109, at least 6 X 109, at least 7 X 109, at least 8 X 109, at least 9 X 109, at least 1 X 1010, or more cardiomyocytes for engraftment. 34 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Electrophysiological maturity

[0194] The cell activity and maturation can be determined by a number of parameters such as electrophysiological maturity, metabolic maturity, or contractile maturity of a cardiomyocyte.

[0195] Mature cardiomyocytes have functional ion channels that permit the synchronization of cardiac muscle contraction. The electrophysiological function of cardiomyocytes can be measured by a variety of methods. Non-limiting examples of such methods include whole cell patch clamp (manual or automated), multielectrode arrays, field potential stimulation, calcium imaging and optical mapping, among others. Cardiomyocytes can be electrically stimulated during whole cell current clamp or field potential recordings to produce an electrical and / or contractile response. Furthermore, cardiomyocytes can be genetically modified, for example, to express a channel rhodopsin that allows for optical stimulation of the cells.

[0196] Measurement of field potentials and biopotentials of cardiomyocytes can be used to determine their differentiation stage and cell maturity. Without limitations, the following parameters can be used to determine electrophysiological function of e.g., cardiomyocytes: change in field potential duration (FPD), quantification of FPD, beat frequency, beats per minute, upstroke velocity, resting membrane potential, amplitude of action potential, maximum diastolic potential, time constant of relaxation, action potential duration (APD) of 90% repolarization, interspike interval, change in beat interval, current density, activation and inactivation kinetics, among others.

[0197] Electrophysiological maturity is determined by one or more of the following markers as compared to a reference level: increased gene expression of an ion channel gene, increased sodium current density, increased inwardly-rectifying potassium channel current density, decreased action potential frequency, decreased calcium wave frequency, and decreased field potential frequency as “increased” and “decreased” are defined herein.

[0198] During a disease state, the electrophysiological function of cardiomyocytes can be compromised. For example, a prolonged field potential duration (FPD) and a prolonged action potential duration (APD) as compared to a normal stem cell derived cardiomyocyte is indicative of a cardiomyocyte having arrhythmic potential. As used herein, the term “field potential duration (FPD)” refers to the time from a given depolarizing event to the last depolarizing event while the term “action potential duration (APD)” refers to the time between the opening of the sodium channel (or phase 0) and the opening of potassium channels that bring the voltage 35 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 membrane to resting potential (phase 3). The “normal” or “prolonged” values for FPD and APD depend on the cell line tested, however a FDP of ~200ms and APD of ~100ms can be considered “normal” for hESC-CMs and FPD > 400ms and APD >300ms can be considered “prolonged” for such cells. (see e.g., Stem Cell Res Ther 12, 278 (2021)). Scaffold compositions

[0199] In one aspect, the cardiomyocytes described herein can be admixed with or cultured on a preparation that provides a scaffold or patterned substrate to support the cells. A scaffold is a structure comprising a biocompatible material including but not limited to a gel, sheet, or lattice that can contain the cells in a desired location but permit the entry or diffusion of factors in the environment necessary for survival and function. A number of biocompatible polymers suitable for a scaffold are known in the art. Such a scaffold or patterned substrate can provide a physical advantage in securing the cells in a given location, e.g., after implantation.

[0200] Biocompatible synthetic, natural, as well as semi-synthetic polymers, can be used for synthesizing polymeric particles that can be used as a scaffold material. In general, for the practice of the methods described herein, it is preferable that a scaffold biodegrades such that the cardiomyocytes can be isolated from the polymer prior to implantation or such that the scaffold degrades over time in a subject and does not require removal. Thus, in one embodiment, the scaffold provides a temporary structure or matrix for growth and / or delivery of cardiomyocytes to a subject in need thereof. In some embodiments, the scaffold permits human cells to be grown in a shape suitable for transplantation or administration into a subject in need thereof, thereby permitting removal of the scaffold prior to implantation and reducing the risk of rejection or allergic response initiated by the scaffold itself.

[0201] Examples of polymers which can be used include natural and synthetic polymers, although synthetic polymers are preferred for reproducibility and controlled release kinetics. Synthetic polymers that can be used include biodegradable polymers such as poly(lactide) (PLA), poly(glycolic acid) (PGA), poly(lactide-co-glycolide) (PLGA), and other polyhydroxyacids, poly(caprolactone), polycarbonates, polyamides, polyanhydrides, polyphosphazene, polyamino acids, polyortho esters, polyacetals, polycyanoacrylates and biodegradable polyurethanes; non-biodegradable polymers such as polyacrylates, ethylene-vinyl acetate polymers and other acyl-substituted cellulose acetates and derivatives thereof; polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl fluoride, poly(vinyl imidazole), 36 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 chlorosulphonated polyolefins, and polyethylene oxide. Examples of biodegradable natural polymers include proteins such as albumin, collagen, fibrin, silk, synthetic polyamino acids and prolamines; polysaccharides such as alginate, heparin; and other naturally occurring biodegradable polymers of sugar units. Alternately, combinations of the aforementioned polymers can be used. In one aspect, a natural polymer that is not generally found in the extracellular matrix can be used.

[0202] PLA, PGA and PLA / PGA copolymers are particularly useful for forming biodegradable scaffolds. PLA polymers are usually prepared from the cyclic esters of lactic acids. Both L(+) and D(-) forms of lactic acid can be used to prepare the PLA polymers, as well as the optically inactive DL-lactic acid mixture of D(-) and L(+) lactic acids. Methods of preparing polylactides are well documented in the patent literature. The following U.S. Patents, the teachings of which are hereby incorporated by reference, describe in detail suitable polylactides, their properties and their preparation: U.S. Pat. No.1,995,970 to Dorough; U.S. Pat. No.2,703,316 to Schneider; U.S. Pat. No.2,758,987 to Salzberg; U.S. Pat. No.2,951,828 to Zeile; U.S. Pat. No.2,676,945 to Higgins; and U.S. Pat. Nos.2,683,136; 3,531,561 to Trehu.

[0203] PGA is a homopolymer of glycolic acid (hydroxyacetic acid). In the conversion of glycolic acid to poly (glycolic acid), glycolic acid is initially reacted with itself to form the cyclic ester glycolide, which in the presence of heat and a catalyst is converted to a high molecular weight linear-chain polymer. PGA polymers and their properties are described in more detail in “Cyanamid Research Develops World's First Synthetic Absorbable Suture”, Chemistry and Industry, 905 (1970).

[0204] Fibers can be formed by melt-spinning, extrusion, casting, or other techniques well known in the polymer processing area. Preferred solvents, if used to remove a scaffold prior to implantation, are those which are completely removed by the processing or which are biocompatible in the amounts remaining after processing.

[0205] Polymers for use in a matrix should meet the mechanical and biochemical parameters necessary to provide adequate support for the cells with subsequent growth and proliferation. The polymers can be characterized with respect to mechanical properties such as tensile strength using an Instron tester, for polymer molecular weight by gel permeation chromatography (GPC), glass transition temperature by differential scanning calorimetry (DSC) and bond structure by infrared (IR) spectroscopy. 37 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0206] The substrate or scaffold can optionally be nanopatterned or micropatterned, for example, with grooves and ridges that permit or facilitate growth, arrangement or maturity of cardiac tissues on the scaffold. Scaffolds can be of any desired shape and can comprise a wide range of geometries that are useful for the methods described herein. A non-limiting list of shapes includes, for example, patches, hollow particles, tubes, sheets, cylinders, spheres, and fibers, among others. The shape or size of the scaffold should not substantially impede cell growth, cell differentiation, cell proliferation or any other cellular process, nor should the scaffold induce cell death via e.g., apoptosis or necrosis. In addition, care should be taken to ensure that the scaffold shape permits appropriate surface area for delivery of nutrients from the surrounding medium to cells in the population, such that cell viability is not impaired. The scaffold porosity can also be varied as desired by one of skill in the art.

[0207] In some embodiments, attachment of the cells to a polymer is enhanced by coating the polymers with compounds such as basement membrane components, fibronectin, agar, agarose, gelatin, gum arabic, collagens types I, II, III, IV, and V, laminin, glycosaminoglycans, polyvinyl alcohol, mixtures thereof, and other hydrophilic and peptide attachment materials known to those skilled in the art of cell culture or tissue engineering. Examples of a material for coating a polymeric scaffold include polyvinyl alcohol and collagen. As will be appreciated by one of skill in the art, Matrigel™ is not suitable for administration to a human subject, thus the compositions described herein do not include Matrigel™.

[0208] These factors are known to those skilled in the art and are available commercially or described in the literature. Bioactive molecules can be incorporated into the matrix and released over time by diffusion and / or degradation of the matrix, or they can be suspended with the cell suspension. Pharmaceutically Acceptable Carriers

[0209] The methods of administering human cardiomyocytes to a subject as described herein involve the use of therapeutic compositions comprising such cells. Therapeutic compositions contain a physiologically tolerable carrier together with the cell composition and optionally at least one additional bioactive agent, polypeptide(s), nucleic acid(s) encoding said polypeptide, or factor(s), dissolved or dispersed therein as an active ingredient.

[0210] In a preferred embodiment, the therapeutic composition is not substantially immunogenic when administered to a mammal or human patient for therapeutic purposes, unless 38 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 so desired. As used herein, the terms “pharmaceutically acceptable,” “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, transplant rejection, allergic reaction, and the like. A pharmaceutically acceptable carrier will not promote the raising of an immune response to an agent with which it is admixed, unless so desired. The preparation of a composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically, such compositions are prepared as injectable either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared.

[0211] A transplant composition for humans can include one or more pharmaceutically acceptable carrier or materials as excipients. In contrast, a cell culture composition (not for human transplant) typically will use research reagents like cell culture media as an excipient. Cardiomyocytes could also be administered in an FDA-approved matrix / scaffold or in combination with FDA-approved drugs as described above.

[0212] In general, the compositions comprising cardiomyocytes described herein are administered as suspension formulations where the cells are admixed with a pharmaceutically acceptable carrier. One of skill in the art will recognize that a pharmaceutically acceptable carrier to be used in a cell composition will not include buffers, compounds, cryopreservation agents, preservatives, or other agents in amounts that substantially interfere with the viability of the cells to be delivered to the subject. A formulation comprising cells can include, e.g., osmotic buffers that permit cell membrane integrity to be maintained, and optionally, nutrients to maintain cell viability or enhance engraftment upon administration. Such formulations and suspensions are known to those of skill in the art and / or can be adapted for use with the cells as described herein using routine experimentation.

[0213] A cell composition can also be emulsified or presented as a liposome composition, provided that the emulsification procedure does not adversely affect cell viability. The cells and any other active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. 39 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0214] Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or which contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions. The amount of an active compound used in the cell compositions as described herein that is effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. Cardiovascular Diseases

[0215] In some aspects, provided herein are methods for the treatment and / or prevention of a cardiac injury or a cardiac disease or disorder in a subject in need thereof. The methods described herein can be used to treat, ameliorate, prevent or slow the progression of a number of diseases or their symptoms, such as those resulting in pathological damage to the structure and / or function of the heart.

[0216] A cardiovascular disease is a disease that affects the heart and / or circulatory system of a subject. Such cardiac diseases or cardiac-related disease include, but are not limited to, myocardial infarction, cardiac arrhythmia, heart failure, atherosclerotic heart disease, cardiomyopathy, congenital heart defect (e.g., non-compaction cardiomyopathy, septal defects, hypoplastic left heart), hypertrophic cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, arrhythmogenic right ventricular dysplasia (ARVD), long QT syndrome, catecholaminergic polymorphic ventricular tachycardia (CPVT), Barth syndrome, valvular stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia, and muscular dystrophies such as Duchenne or related cardiac disease, and cardiomegaly. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent or slow the progression of a cardiovascular disease. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent or slow the progression of myocardial infarction, cardiac arrhythmia, heart failure, atherosclerotic heart disease, cardiomyopathy, congenital heart defect (e.g., non-compaction cardiomyopathy, septal defects, hypoplastic left heart), hypertrophic 40 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 cardiomyopathy, dilated cardiomyopathy, cardiac hypertrophy, myocarditis, ARVD, long QT syndrome, CPVT, Barth syndrome, valvular stenosis, regurgitation, ischemia, fibrillation, polymorphic ventricular tachycardia, and muscular dystrophies such as Duchenne or related cardiac disease, and cardiomegaly.

[0217] The term, “cardiac event” refers to an incident of myocardial injury, myocardial infarction, ventricular fibrillation, stenosis, arrhythmia, or the like. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent or slow the progression of a cardiac event.

[0218] Symptoms of cardiovascular disease can include but are not limited to syncope, fatigue, shortness of breath, chest pain, and palpitations. A cardiovascular disease is generally diagnosed by a physical examination, blood tests, and / or an electrocardiogram (EKG). An abnormal EKG is an indication that the subject has an abnormal cardiac rhythm or cardiac arrhythmia. Methods of diagnosing arrhythmias are known in the art. In some embodiments, the methods described herein can be used to treat, ameliorate, prevent or slow the progression of symptoms of cardiovascular disease.

[0219] Cardiac electrophysiological and contractile function is a tightly controlled process. When ion channel regulation or contractile function is disrupted in a cardiac cell or tissue, this can result in cardiac arrhythmias that can sometimes be deadly. Cardiac diseases remain a leading cause of death worldwide.

[0220] Human stem cell derived cardiomyocytes have emerged as a promising treatment for cardiovascular diseases and cardiac injuries sustained from myocardial infarction. However, the functional maturity of in vitro-differentiated cardiomyocytes in existing models is generally lacking and these cardiomyocytes can cause arrhythmias following engraftment. While not wishing to be bound by theory, the introduction of exogenous cardiomyocytes that have their own impulse generating activity has the potential to disturb the closely regulated electrophysiological function of the heart, leading to arrhythmia – while noted for in vitro- differentiated cardiomyocyte grafts, this effect can also occur, for example, when cardiomyocytes derived from other sources are transplanted to cardiac tissue. The media described herein and methods for using them and cells matured in them can ameliorate engraftment arrhythmias induced by such transplants. 41 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Administration and Efficacy

[0221] Cardiomyocyte transplant as described herein can be used to treat a cardiac disease or disorder or to treat cardiac injury. Indicators of cardiac disease or cardiac disorder, or cardiac injury include adverse changes in functional indicators or parameters, e.g., stroke volume, heart rate, left ventricular ejection fraction, heart rate, heart rhythm, blood pressure, heart volume, regurgitation, etc. as well as biochemical indicators, such as a decrease in markers of cardiac injury, such as serum lactate dehydrogenase, or serum troponin, among others. As one example, myocardial ischemia and reperfusion are associated with reduced cardiac function. Subjects that have suffered an ischemic cardiac event and / or ischemia / reperfusion event have reduced cardiac function when compared to that before ischemia. Measures of cardiac function include, for example, ejection fraction and fractional shortening. Ejection fraction is the fraction of blood pumped out of a ventricle with each heartbeat. The term ejection fraction applies to both the right and left ventricles. LVEF refers to the left ventricular ejection fraction (LVEF). Fractional shortening refers to the difference between end- diastolic and end-systolic dimensions divided by end-diastolic dimension. At least one or more of these indicators of cardiac disesase or cardiac disorder can be improved using in-vitro cardiomyocyte transplantation with cardiomyocytes electrophysiologically matured as described herein.

[0222] “Improved”, as used herein, refers to a beneficial change in any one or all of the signs or symptoms of the cardiac disease or disorder, or when one or more other clinically accepted symptoms or markers of disease is / are improved, or ameliorated, e.g., by at least 10% following treatment, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% using transplantation of in- vitro differentiated cardiomyocytes that are electrophysiologically matured as described herein.

[0223] As discussed herein above, improvement in function can also include a reduction in the incidence, severity and / or duration of engraftment arrhythmia caused by transplanted in vitro- differentiated cardiomyocytes when the cardiomyocytes are electrophysiologically matured using compositions and methods as described herein prior to transplant. Thus, in this context, an improvement can refer to at least a 10% reduction in the incidence, severity and / or duration of engraftment arrhythmia, e.g., reduction by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% or more 42 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 relative to that occurring when the transplanted, in vitro differentiated cardiomyocytes are not treated to improve electrophysiological maturity as described herein.

[0224] The administration or transplantation of cardiomyocytes, including transplant of in vitro differentiated electrophysiologically matured cardiomyocytes as described herein, can generally be performed as known in the art and described, for example, in International Application PCT / US2022 / 027382, which is incorporated by reference herein in its entirety. While an important readout will be a reduction in severity or duration of engraftment arrhythmia relative to a similar subject receiving in vitro differentiated cardiomyocytes that were not treated or incubated with maturation-promoting media as described herein, measured or measurable parameters of treatment efficacy include clinically detectable markers of disease, for example, elevated or depressed levels of a clinical or biological marker, as well as parameters related to a clinically accepted scale of symptoms or markers for a given disease or disorder. It will be understood, however, that the usage of the compositions and formulations as described herein will be decided by the attending physician within the scope of sound medical judgment. The exact amount of in vitro-differentiated cardiomyocytes, matured as described herein, that are required for therapeutic efficacy will vary depending on factors such as the severity and type of disease being treated.

[0225] The term "effective amount" as used herein refers to the amount of a population of in vitro-differentiated cardiomyocytes needed to alleviate at least one or more symptoms of a disease or disorder, including but not limited to a cardiac injury or a cardiac disease or disorder. An "effective amount" relates to a sufficient amount of a composition to provide the desired effect, e.g., treat a subject having an infarct zone following myocardial infarction, improve cardiomyocyte engraftment, prevent onset of heart failure following cardiac injury, enhance vascularization of a graft etc. The term "therapeutically effective amount" therefore refers to an amount of in vitro-differentiated human cardiomyocytes or a composition comprising such cells that is sufficient to promote a particular effect when administered to a typical subject, such as one who has, or is at risk for, a cardiac disease or disorder. An effective amount as used herein would also include an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a disease symptom (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate "effective amount" can be determined by one of ordinary 43 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 skill in the art using routine experimentation.

[0226] For use in the various aspects described herein, an effective amount of in vitro- differentiated human cardiomyocytes comprises at least 1 X 103, at least 1 X 104, at least 1 X 105,at least 5 X 105, at least 1 X 106, at least 2 X 106, at least 3 X 106, at least 4 X 106, at least 5 X 106, at least 6 X 106, at least 7 X 106, at least 8 X 106, at least 9 X 106, at least 1 X 107, at least 1.1 X 107, at least 1.2 X 107, at least 1.3 X 107, at least 1.4 X 107, at least 1.5 X 107, at least 1.6 X 107, at least 1.7 X 107, at least 1.8 X 107, at least 1.9 X 107, at least 2 X 107, at least 3 X 107, at least 4 X 107, at least 5 X 107, at least 6 X 107, at least 7 X 107, at least 8 X 107, at least 9 X 107, at least 1 X 108, at least 2 X 108, at least 5 X108, at least 7 X 108, at least 1 X 109, at least 2 X 109, at least 3 X 109, at least 4 X 109, at least 5 X 109or more cardiomyocytes and / or epicardial cells.

[0227] By preventing, reducing or alleviating engraftment arrhythmia, the clinical outcome for the subject can be improved, including reduced risk for heart failure or sudden cardiac death. Methods for improving engraftment or preventing engraftment arrhythmias, such as those described in e.g., US 2020-0085880, WO 2022 / 216800, WO 2022 / 235610, WO 2020 / 190739, or WO 2021 / 163037, the contents of each of which are incorporated herein by reference in their entireties, can be combined with the methods and compositions described herein. While the compositions and methods described herein can reduce engraftment arrhythmia on their own, it is specifically contemplated that anti-arrhythmia drugs known in the art can additionally be administered to the transplant recipient to further reduce the incidence, severity and / or duration of engraftment arrhythmia.

[0228] By “alleviate” in this context is meant a reduction in arrhythmia burden of at least 10% relative to arrhythmia occurring or expected to occur without administration or use of compositions and methods as described herein. Arrhythmic burden can be calculated as described in the working examples herein or as known in the art. Arrhythmic burden without the compositions and methods described herein can be 75% or more. This can be reduced by at least 10% with the administration or use of compositions and methods described herein.

[0229] In some embodiments of any of the aspects, the administration of compositions and methods described herein reduces post-graft accelerated resting heart rate experienced by the graft recipient by at least 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, 44 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 60% or more relative to a subject receiving a graft of the same type of cells in the absence of electrophysiological maturation as described herein.

[0230] Thus, in some embodiments of any of the aspects, the administration of compositions or methods described herein reduces engraftment arrhythmia burden by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or more relative to a subject receiving a graft of the same type of cardiomyocytes in the absence of electrophysiological maturation as described herein. While at least 10% reduction is considered effective treatment, it is contemplated that administration of compositions and methods described herein permit up to complete cessation of engraftment arrhythmia.

[0231] In some embodiments of any of the aspects, the methods described herein comprise administering an effective amount of in vitro differentiated cardiomyocytes treated as described herein to a subject in order to alleviate at least one symptom of a cardiovascular disease or disorder and / or to alleviate at least one symptom of an engraftment arrhythmia. As used herein, "alleviating at least one symptom of the cardiovascular disease" or “alleviating at least one symptom of an engraftment arrhythmia” is ameliorating any condition or symptom associated with the cardiovascular disease (e.g., fatigue, shortness of breath, syncope, chest pain) and includes, for example, reduction of the arrhythmia itself. As compared with an equivalent untreated control, such reduction is by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or more as measured by any standard technique.

[0232] In some embodiments, the in vitro differentiated cardiomyocytes as described herein are administered when there is tachycardia. In some embodiments, the in vitro differentiated cardiomyocytes as described herein are administered to limit or control tachycardia as commonly occurs with engraftment arrhythmia. Adult human resting heart rate is generally 60 to 100 beats per minute (bpm). Tachycardia is a resting heart rate greater than this. However, while a resting heart rate of 60 to 100 bpm is a goal, patients can typically manage with a rate of less than 150 bpms. Thus, in vitro differentiated cardiomyocytes can be administered for tachycardia after grafting with a goal of maintaining resting heart rate between 100 and 150 bpms, preferably less than 140 bpm, less than 130bpm, less than 120 bpm, or less than 110 bpm.

[0233] Exemplary modes of administration for use in the methods described herein include, but are not limited to, injection, intracardiac delivery, systemic administration and implantation (with 45 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 or without a scaffold material). These methods are particularly aimed at therapeutic and prophylactic treatments of human subjects having, or at risk of having, a cardiac disease or disorder. The human in vitro-differentiated cardiomyocytes treated as described herein to promote electrophysiological maturity can be administered to a subject having any cardiac disease or disorder by any appropriate route which results in an effective treatment in the subject. In some embodiments of the aspects described herein, a subject having a cardiac disorder is first selected prior to administration of the cells.

[0234] In some embodiments, an effective amount of cardiomyocytes is administered to a subject by intracardiac administration or delivery. As used herein, "intracardiac" administration or delivery refers to all routes of administration whereby a population of cardiomyocytes is administered in a way that results in direct contact of the cells with the myocardium of a subject, including, but not limited to, direct cardiac injection, intra- myocardial injection(s), intra-infarct zone injection, injection during surgery (e.g., cardiac bypass surgery, during implantation of a cardiac mini-pump or a pacemaker, etc.). In some such embodiments, the cells are injected into the myocardium or into the cavity of the atria and / or ventricles. In some embodiments, intracardiac delivery of cells includes administration methods whereby cells are administered, for example as a cell suspension, to a subject undergoing surgery via a single injection or multiple "mini" injections into the desired region of the heart.

[0235] The choice of formulation will depend upon the specific composition used and the number of cardiomyocytes to be administered; such formulations can be adjusted by the skilled practitioner. However, as an example, where the composition is cardiomyocytes in a pharmaceutically acceptable carrier, the composition can be a suspension of the cells in an appropriate buffer (e.g., saline buffer) at an effective concentration of cells per mL of solution. The formulation can also include cell nutrients, a simple sugar (e.g., for osmotic pressure regulation) or other components to maintain the viability of the cells. Alternatively, the formulation can comprise a scaffold, such as a biodegradable scaffold.

[0236] In some embodiments, additional agents to aid in treatment of the subject can be administered before or following treatment with the in vitro-differentiated cardiomyocytes as described. Such additional agents can be used to prepare the target tissue for administration of the progenitor cells. Alternatively, the additional agents can be administered after the in vitro-differentiated cardiomyocytes to support the engraftment and growth of the 46 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 administered cell into the heart or other desired administration site. In some embodiments, the additional agent comprises growth factors, such as VEGF or PDGF. Other exemplary agents can be used to reduce the load on the heart while the cardiomyocytes are engrafting (e.g., beta blockers, medications to lower blood pressure etc.).

[0237] The efficacy of treatment can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the symptoms, or other clinically accepted symptoms or markers of disease, e.g., heart failure, arrhythmia, and / or cardiac injury are reduced, e.g., by at least 10% following treatment with a composition comprising human cardiomyocytes and / or epicardial cells as described herein.

[0238] Non-limiting examples of clinical tests that can be used to assess cardiac functional parameters include echocardiography (with or without Doppler flow imaging), electrocardiogram (EKG), exercise stress test, Holter monitoring, or measurement of β-natriuretic peptide.

[0239] Where necessary or desired, animal models of cardiac injury or cardiac disease can be used to gauge the effectiveness of a particular composition as described herein. For example, an isolated working rabbit or rat heart model, or a coronary ligation model in either canines or porcines can be used. Animal models of cardiac function are useful for monitoring infarct zones, coronary perfusion, electrical conduction, left ventricular end diastolic pressure, left ventricular ejection fraction, heart rate, blood pressure, degree of hypertrophy, diastolic relaxation function, cardiac output, heart rate variability, and ventricular wall thickness, etc.

[0240] In some embodiments, a composition comprising the in vitro-differentiated cardiomyocytes as described herein is delivered at least 6 hours following the initiation of reperfusion, for example, following a myocardial infarction. During an ischemic insult and subsequent reperfusion, the microenvironment of the heart or that of the infarcted zone can be too "hostile" to permit engraftment of cardiomyocytes administered to the heart. Thus, in some embodiments it is preferable to administer such compositions at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 36 hours, at least 48 hours, at least 60 hours, at least 72 hours, at least 84 hours, at least 96 hours, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days or more following the initiation of reperfusion. In some embodiments, the compositions comprising cardiomyocytes as described herein can be administered to an infarcted zone, peri-infarcted zone, ischemic zone, penumbra, areas of wall thinning, areas of non- compaction, area(s) at risk of maladaptive cardiac remodeling, or the 47 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 border zone of the heart at an extended length of time after a myocardial infarction (e.g., 1 month, 6 months, one year, 2 years, 5 years, 10 years, 20 years, 30 years or more), however as will be appreciated by those of skill in the art, the success of engraftment following a lengthy interval of time after infarct will depend on a number of factors, including but not limited to, amount of scar tissue deposition, density of scar tissue, size of the infarcted zone, degree of vascularization surrounding the infarcted zone, etc. As such, earlier intervention by administration of compositions comprising in vitro-differentiated cardiomyocytes may be more efficacious than administration after e.g., a month or more after infarct.

[0241] Embodiments of the technology described herein include cardiomyocyte maturation medium. Also described herein are associated compositions, kits, and matured cardiomyocytes. The cardiomyocyte maturation medium disclosed here can be used to modulate at least one electrophysiologic property of a cardiomyocyte. The electrophysiologically matured cardiomyocytes can be used to reduce or inhibit engraftment arrythmia in subject. Cardiomyocyte Maturation Media

[0242] In multiple aspects, described herein is a cardiomyocyte maturation medium comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex). Amounts of the “metabolic modulators” (e.g., Gal, FA, AICAR, T3, and / or Dex) expressed here are amounts added to the standard medium formulations, e.g., amounts added to RPMI / B-27 (no glucose) medium.

[0243] In one aspect, described herein is a cardiomyocyte maturation medium comprising: (a) 0.5 mM to 10.0 mM galactose (Gal); (b) 115 μM to 460 μM fatty acids (FA); (c) 0.25 mM to 1.0 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) 10 nM to 1 μM triiodothyronine (T3); and (e) 10 nM to 10.0 µM dexamethasone (Dex). In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises 0 mM to 5 mM glucose. Galactose

[0244] In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of about 0.5 mM. In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of at most 0.5 mM. In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of at most 1.0 mM. In some embodiments of any of the aspects, the 48 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 galactose (Gal) is at a concentration of at most 10.0 mM. In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of 0.5 mM to 1.0 mM. In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of 0.5 mM to 10.0 mM.

[0245] In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, about 5 mM, about 5.1 mM, about 5.2 mM, about 5.3 mM, about 5.4 mM, about 5.5 mM, about 5.6 mM, about 5.7 mM, about 5.8 mM, about 5.9 mM, about 6 mM, about 6.1 mM, about 6.2 mM, about 6.3 mM, about 6.4 mM, about 6.5 mM, about 6.6 mM, about 6.7 mM, about 6.8 mM, about 6.9 mM, about 7 mM, about 7.1 mM, about 7.2 mM, about 7.3 mM, about 7.4 mM, about 7.5 mM, about 7.6 mM, about 7.7 mM, about 7.8 mM, about 7.9 mM, about 8 mM, about 8.1 mM, about 8.2 mM, about 8.3 mM, about 8.4 mM, about 8.5 mM, about 8.6 mM, about 8.7 mM, about 8.8 mM, about 8.9 mM, about 9 mM, about 9.1 mM, about 9.2 mM, about 9.3 mM, about 9.4 mM, about 9.5 mM, about 9.6 mM, about 9.7 mM, about 9.8 mM, about 9.9 mM, or about 10 mM.

[0246] In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 4.1 mM, at least 4.2 mM, at least 4.3 mM, at least 4.4 mM, at least 4.5 mM, at least 4.6 mM, at least 4.7 mM, at least 4.8 mM, at least 4.9 mM, at least 5 mM, at least 5.1 mM, at least 5.2 mM, at least 5.3 mM, at least 5.4 mM, at least 5.5 mM, at least 5.6 mM, at least 5.7 mM, at least 5.8 mM, at least 5.9 mM, at least 6 mM, at least 6.1 mM, at 49 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 least 6.2 mM, at least 6.3 mM, at least 6.4 mM, at least 6.5 mM, at least 6.6 mM, at least 6.7 mM, at least 6.8 mM, at least 6.9 mM, at least 7 mM, at least 7.1 mM, at least 7.2 mM, at least 7.3 mM, at least 7.4 mM, at least 7.5 mM, at least 7.6 mM, at least 7.7 mM, at least 7.8 mM, at least 7.9 mM, at least 8 mM, at least 8.1 mM, at least 8.2 mM, at least 8.3 mM, at least 8.4 mM, at least 8.5 mM, at least 8.6 mM, at least 8.7 mM, at least 8.8 mM, at least 8.9 mM, at least 9 mM, at least 9.1 mM, at least 9.2 mM, at least 9.3 mM, at least 9.4 mM, at least 9.5 mM, at least 9.6 mM, at least 9.7 mM, at least 9.8 mM, at least 9.9 mM, or 10 mM.

[0247] In some embodiments of any of the aspects, the galactose (Gal) is at a concentration of at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 mM, at most 0.9 mM, at most 1 mM, at most 1.1 mM, at most 1.2 mM, at most 1.3 mM, at most 1.4 mM, at most 1.5 mM, at most 1.6 mM, at most 1.7 mM, at most 1.8 mM, at most 1.9 mM, at most 2 mM, at most 2.1 mM, at most 2.2 mM, at most 2.3 mM, at most 2.4 mM, at most 2.5 mM, at most 2.6 mM, at most 2.7 mM, at most 2.8 mM, at most 2.9 mM, at most 3 mM, at most 3.1 mM, at most 3.2 mM, at most 3.3 mM, at most 3.4 mM, at most 3.5 mM, at most 3.6 mM, at most 3.7 mM, at most 3.8 mM, at most 3.9 mM, at most 4 mM, at most 4.1 mM, at most 4.2 mM, at most 4.3 mM, at most 4.4 mM, at most 4.5 mM, at most 4.6 mM, at most 4.7 mM, at most 4.8 mM, at most 4.9 mM, at most 5 mM, at most 5.1 mM, at most 5.2 mM, at most 5.3 mM, at most 5.4 mM, at most 5.5 mM, at most 5.6 mM, at most 5.7 mM, at most 5.8 mM, at most 5.9 mM, at most 6 mM, at most 6.1 mM, at most 6.2 mM, at most 6.3 mM, at most 6.4 mM, at most 6.5 mM, at most 6.6 mM, at most 6.7 mM, at most 6.8 mM, at most 6.9 mM, at most 7 mM, at most 7.1 mM, at most 7.2 mM, at most 7.3 mM, at most 7.4 mM, at most 7.5 mM, at most 7.6 mM, at most 7.7 mM, at most 7.8 mM, at most 7.9 mM, at most 8 mM, at most 8.1 mM, at most 8.2 mM, at most 8.3 mM, at most 8.4 mM, at most 8.5 mM, at most 8.6 mM, at most 8.7 mM, at most 8.8 mM, at most 8.9 mM, at most 9 mM, at most 9.1 mM, at most 9.2 mM, at most 9.3 mM, at most 9.4 mM, at most 9.5 mM, at most 9.6 mM, at most 9.7 mM, at most 9.8 mM, at most 9.9 mM, or at most 10 mM. Fatty Acids

[0248] In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of about 460 μM. In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of at most 460 μM. In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of at least 230 μM. In some embodiments of any of the aspects, the 50 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 fatty acids (FA) are at a concentration of at least 115 μM. In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of 115 μM to 460 μM. In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of 230 μM to 460 μM.

[0249] In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of about 115 μM, about 120 μM, about 125 μM, about 130 μM, about 135 μM, about 140 μM, about 145 μM, about 150 μM, about 155 μM, about 160 μM, about 165 μM, about 170 μM, about 175 μM, about 180 μM, about 185 μM, about 190 μM, about 195 μM, about 200 μM, about 205 μM, about 210 μM, about 215 μM, about 220 μM, about 225 μM, about 230 μM, about 235 μM, about 240 μM, about 245 μM, about 250 μM, about 255 μM, about 260 μM, about 265 μM, about 270 μM, about 275 μM, about 280 μM, about 285 μM, about 290 μM, about 295 μM, about 300 μM, about 305 μM, about 310 μM, about 315 μM, about 320 μM, about 325 μM, about 330 μM, about 335 μM, about 340 μM, about 345 μM, about 350 μM, about 355 μM, about 360 μM, about 365 μM, about 370 μM, about 375 μM, about 380 μM, about 385 μM, about 390 μM, about 395 μM, about 400 μM, about 405 μM, about 410 μM, about 415 μM, about 420 μM, about 425 μM, about 430 μM, about 435 μM, about 440 μM, about 445 μM, about 450 μM, about 455 μM, or about 460 μM.

[0250] In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of at least 115 μM, at least 120 μM, at least 125 μM, at least 130 μM, at least 135 μM, at least 140 μM, at least 145 μM, at least 150 μM, at least 155 μM, at least 160 μM, at least 165 μM, at least 170 μM, at least 175 μM, at least 180 μM, at least 185 μM, at least 190 μM, at least 195 μM, at least 200 μM, at least 205 μM, at least 210 μM, at least 215 μM, at least 220 μM, at least 225 μM, at least 230 μM, at least 235 μM, at least 240 μM, at least 245 μM, at least 250 μM, at least 255 μM, at least 260 μM, at least 265 μM, at least 270 μM, at least 275 μM, at least 280 μM, at least 285 μM, at least 290 μM, at least 295 μM, at least 300 μM, at least 305 μM, at least 310 μM, at least 315 μM, at least 320 μM, at least 325 μM, at least 330 μM, at least 335 μM, at least 340 μM, at least 345 μM, at least 350 μM, at least 355 μM, at least 360 μM, at least 365 μM, at least 370 μM, at least 375 μM, at least 380 μM, at least 385 μM, at least 390 μM, at least 395 μM, at least 400 μM, at least 405 μM, at least 410 μM, at least 415 μM, at least 420 μM, at least 425 μM, at least 430 μM, at least 435 μM, at least 440 μM, at least 445 μM, at least 450 μM, at least 455 μM, or 460 μM. 51 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0251] In some embodiments of any of the aspects, the fatty acids (FA) are at a concentration of at most 115 μM, at most 120 μM, at most 125 μM, at most 130 μM, at most 135 μM, at most 140 μM, at most 145 μM, at most 150 μM, at most 155 μM, at most 160 μM, at most 165 μM, at most 170 μM, at most 175 μM, at most 180 μM, at most 185 μM, at most 190 μM, at most 195 μM, at most 200 μM, at most 205 μM, at most 210 μM, at most 215 μM, at most 220 μM, at most 225 μM, at most 230 μM, at most 235 μM, at most 240 μM, at most 245 μM, at most 250 μM, at most 255 μM, at most 260 μM, at most 265 μM, at most 270 μM, at most 275 μM, at most 280 μM, at most 285 μM, at most 290 μM, at most 295 μM, at most 300 μM, at most 305 μM, at most 310 μM, at most 315 μM, at most 320 μM, at most 325 μM, at most 330 μM, at most 335 μM, at most 340 μM, at most 345 μM, at most 350 μM, at most 355 μM, at most 360 μM, at most 365 μM, at most 370 μM, at most 375 μM, at most 380 μM, at most 385 μM, at most 390 μM, at most 395 μM, at most 400 μM, at most 405 μM, at most 410 μM, at most 415 μM, at most 420 μM, at most 425 μM, at most 430 μM, at most 435 μM, at most 440 μM, at most 445 μM, at most 450 μM, at most 455 μM, or at most 460 μM. AICAR

[0252] In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration of about 1.0 mM. In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration of at most 1.0 mM. In some embodiments of any of the aspects, the 5-aminoimidazole-4- carboxamide ribonucleoside (AICAR) is at a concentration of at least 0.3 mM. In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration of 0.25 mM to 1.0 mM.

[0253] In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration of about 0.25 mM, about 0.26 mM, about 0.27 mM, about 0.28 mM, about 0.29 mM, about 0.3 mM, about 0.31 mM, about 0.32 mM, about 0.33 mM, about 0.34 mM, about 0.35 mM, about 0.36 mM, about 0.37 mM, about 0.38 mM, about 0.39 mM, about 0.4 mM, about 0.41 mM, about 0.42 mM, about 0.43 mM, about 0.44 mM, about 0.45 mM, about 0.46 mM, about 0.47 mM, about 0.48 mM, about 0.49 mM, about 0.5 mM, about 0.51 mM, about 0.52 mM, about 0.53 mM, about 0.54 mM, about 0.55 mM, about 0.56 mM, about 0.57 mM, about 0.58 mM, about 0.59 mM, about 0.6 mM, about 0.61 mM, about 0.62 mM, about 0.63 mM, about 0.64 mM, about 0.65 mM, about 0.66 mM, about 52 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 0.67 mM, about 0.68 mM, about 0.69 mM, about 0.7 mM, about 0.71 mM, about 0.72 mM, about 0.73 mM, about 0.74 mM, about 0.75 mM, about 0.76 mM, about 0.77 mM, about 0.78 mM, about 0.79 mM, about 0.8 mM, about 0.81 mM, about 0.82 mM, about 0.83 mM, about 0.84 mM, about 0.85 mM, about 0.86 mM, about 0.87 mM, about 0.88 mM, about 0.89 mM, about 0.9 mM, about 0.91 mM, about 0.92 mM, about 0.93 mM, about 0.94 mM, about 0.95 mM, about 0.96 mM, about 0.97 mM, about 0.98 mM, about 0.99 mM, or about 1 mM.

[0254] In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration of at least 0.25 mM, at least 0.26 mM, at least 0.27 mM, at least 0.28 mM, at least 0.29 mM, at least 0.3 mM, at least 0.31 mM, at least 0.32 mM, at least 0.33 mM, at least 0.34 mM, at least 0.35 mM, at least 0.36 mM, at least 0.37 mM, at least 0.38 mM, at least 0.39 mM, at least 0.4 mM, at least 0.41 mM, at least 0.42 mM, at least 0.43 mM, at least 0.44 mM, at least 0.45 mM, at least 0.46 mM, at least 0.47 mM, at least 0.48 mM, at least 0.49 mM, at least 0.5 mM, at least 0.51 mM, at least 0.52 mM, at least 0.53 mM, at least 0.54 mM, at least 0.55 mM, at least 0.56 mM, at least 0.57 mM, at least 0.58 mM, at least 0.59 mM, at least 0.6 mM, at least 0.61 mM, at least 0.62 mM, at least 0.63 mM, at least 0.64 mM, at least 0.65 mM, at least 0.66 mM, at least 0.67 mM, at least 0.68 mM, at least 0.69 mM, at least 0.7 mM, at least 0.71 mM, at least 0.72 mM, at least 0.73 mM, at least 0.74 mM, at least 0.75 mM, at least 0.76 mM, at least 0.77 mM, at least 0.78 mM, at least 0.79 mM, at least 0.8 mM, at least 0.81 mM, at least 0.82 mM, at least 0.83 mM, at least 0.84 mM, at least 0.85 mM, at least 0.86 mM, at least 0.87 mM, at least 0.88 mM, at least 0.89 mM, at least 0.9 mM, at least 0.91 mM, at least 0.92 mM, at least 0.93 mM, at least 0.94 mM, at least 0.95 mM, at least 0.96 mM, at least 0.97 mM, at least 0.98 mM, at least 0.99 mM, or 1 mM.

[0255] In some embodiments of any of the aspects, the 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration at most 0.25 mM, at most 0.26 mM, at most 0.27 mM, at most 0.28 mM, at most 0.29 mM, at most 0.3 mM, at most 0.31 mM, at most 0.32 mM, at most 0.33 mM, at most 0.34 mM, at most 0.35 mM, at most 0.36 mM, at most 0.37 mM, at most 0.38 mM, at most 0.39 mM, at most 0.4 mM, at most 0.41 mM, at most 0.42 mM, at most 0.43 mM, at most 0.44 mM, at most 0.45 mM, at most 0.46 mM, at most 0.47 mM, at most 0.48 mM, at most 0.49 mM, at most 0.5 mM, at most 0.51 mM, at most 0.52 mM, at most 0.53 mM, at most 0.54 mM, at most 0.55 mM, at most 0.56 mM, at most 0.57 mM, at most 0.58 mM, at most 0.59 mM, at most 0.6 mM, at most 0.61 mM, at most 0.62 mM, at most 0.63 mM, at most 53 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 0.64 mM, at most 0.65 mM, at most 0.66 mM, at most 0.67 mM, at most 0.68 mM, at most 0.69 mM, at most 0.7 mM, at most 0.71 mM, at most 0.72 mM, at most 0.73 mM, at most 0.74 mM, at most 0.75 mM, at most 0.76 mM, at most 0.77 mM, at most 0.78 mM, at most 0.79 mM, at most 0.8 mM, at most 0.81 mM, at most 0.82 mM, at most 0.83 mM, at most 0.84 mM, at most 0.85 mM, at most 0.86 mM, at most 0.87 mM, at most 0.88 mM, at most 0.89 mM, at most 0.9 mM, at most 0.91 mM, at most 0.92 mM, at most 0.93 mM, at most 0.94 mM, at most 0.95 mM, at most 0.96 mM, at most 0.97 mM, at most 0.98 mM, at most 0.99 mM, or at most 1 mM. Triiodothyronine (T3)

[0256] In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of about 100 nM. In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of at most 100 nM. In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of 50 nM to 200 nM. In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of 10 nM to 1 µM.

[0257] In some embodiments of any of the aspects, the triiodothyronine (T3) is in the cardiomyocyte maturation medium at a concentration above physiological concentrations (e.g., greater than 1 ng / mL or 1.54 nM T3, greater than 2 ng / mL or 3.07 nM T3, greater than 10 ng / mL or 15.4 nM T3; see e.g., Larsen et al., J Clin Invest.1972 Aug;51(8):1939-49, Sterling et al., J Clin Invest.1969;48(6):1150-1158).

[0258] In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 51 nM, about 52 nM, about 53 nM, about 54 nM, about 55 nM, about 56 nM, about 57 nM, about 58 nM, about 59 nM, about 60 nM, about 61 nM, about 62 nM, about 63 nM, about 64 nM, about 65 nM, about 66 nM, about 67 nM, about 68 nM, about 69 nM, about 70 nM, about 71 nM, about 72 nM, about 73 nM, about 74 nM, about 75 nM, about 76 nM, about 77 nM, about 78 nM, about 79 nM, about 80 nM, about 81 nM, about 82 nM, about 83 nM, about 84 nM, about 85 nM, about 86 nM, about 87 nM, about 88 nM, about 89 nM, about 90 nM, about 91 nM, about 92 nM, about 93 nM, about 94 nM, about 95 nM, about 96 nM, about 97 nM, about 98 nM, about 99 nM, about 100 nM, about 101 nM, about 102 nM, about 103 nM, about 104 nM, about 105 nM, about 106 nM, about 107 nM, about 108 nM, about 109 nM, about 110 nM, about 111 nM, about 112 nM, about 113 nM, about 114 nM, about 115 nM, about 116 nM, about 117 nM, about 118 nM, about 119 nM, about 120 nM, about 121 nM, about 122 nM, about 123 54 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 nM, about 124 nM, about 125 nM, about 126 nM, about 127 nM, about 128 nM, about 129 nM, about 130 nM, about 131 nM, about 132 nM, about 133 nM, about 134 nM, about 135 nM, about 136 nM, about 137 nM, about 138 nM, about 139 nM, about 140 nM, about 141 nM, about 142 nM, about 143 nM, about 144 nM, about 145 nM, about 146 nM, about 147 nM, about 148 nM, about 149 nM, about 150 nM, about 151 nM, about 152 nM, about 153 nM, about 154 nM, about 155 nM, about 156 nM, about 157 nM, about 158 nM, about 159 nM, about 160 nM, about 161 nM, about 162 nM, about 163 nM, about 164 nM, about 165 nM, about 166 nM, about 167 nM, about 168 nM, about 169 nM, about 170 nM, about 171 nM, about 172 nM, about 173 nM, about 174 nM, about 175 nM, about 176 nM, about 177 nM, about 178 nM, about 179 nM, about 180 nM, about 181 nM, about 182 nM, about 183 nM, about 184 nM, about 185 nM, about 186 nM, about 187 nM, about 188 nM, about 189 nM, about 190 nM, about 191 nM, about 192 nM, about 193 nM, about 194 nM, about 195 nM, about 196 nM, about 197 nM, about 198 nM, about 199 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, about 300 nM, about 310 nM, about 320 nM, about 330 nM, about 340 nM, about 350 nM, about 360 nM, about 370 nM, about 380 nM, about 390 nM, about 400 nM, about 410 nM, about 420 nM, about 430 nM, about 440 nM, about 450 nM, about 460 nM, about 470 nM, about 480 nM, about 490 nM, about 0.5 µM, about 0.51 µM, about 0.52 µM, about 0.53 µM, about 0.54 µM, about 0.55 µM, about 0.56 µM, about 0.57 µM, about 0.58 µM, about 0.59 µM, about 0.6 µM, about 0.61 µM, about 0.62 µM, about 0.63 µM, about 0.64 µM, about 0.65 µM, about 0.66 µM, about 0.67 µM, about 0.68 µM, about 0.69 µM, about 0.7 µM, about 0.71 µM, about 0.72 µM, about 0.73 µM, about 0.74 µM, about 0.75 µM, about 0.76 µM, about 0.77 µM, about 0.78 µM, about 0.79 µM, about 0.8 µM, about 0.81 µM, about 0.82 µM, about 0.83 µM, about 0.84 µM, about 0.85 µM, about 0.86 µM, about 0.87 µM, about 0.88 µM, about 0.89 µM, about 0.9 µM, about 0.91 µM, about 0.92 µM, about 0.93 µM, about 0.94 µM, about 0.95 µM, about 0.96 µM, about 0.97 µM, about 0.98 µM, about 0.99 µM, or about 1 µM.

[0259] In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 51 nM, at least 52 nM, at least 53 nM, at least 54 nM, at least 55 nM, at least 56 nM, at least 57 nM, at least 58 nM, at least 59 nM, at least 60 nM, at least 61 nM, at least 62 nM, at least 63 nM, at least 64 nM, at least 65 nM, at least 66 nM, at least 67 nM, at least 68 nM, at least 69 nM, 55 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 at least 70 nM, at least 71 nM, at least 72 nM, at least 73 nM, at least 74 nM, at least 75 nM, at least 76 nM, at least 77 nM, at least 78 nM, at least 79 nM, at least 80 nM, at least 81 nM, at least 82 nM, at least 83 nM, at least 84 nM, at least 85 nM, at least 86 nM, at least 87 nM, at least 88 nM, at least 89 nM, at least 90 nM, at least 91 nM, at least 92 nM, at least 93 nM, at least 94 nM, at least 95 nM, at least 96 nM, at least 97 nM, at least 98 nM, at least 99 nM, at least 100 nM, at least 101 nM, at least 102 nM, at least 103 nM, at least 104 nM, at least 105 nM, at least 106 nM, at least 107 nM, at least 108 nM, at least 109 nM, at least 110 nM, at least 111 nM, at least 112 nM, at least 113 nM, at least 114 nM, at least 115 nM, at least 116 nM, at least 117 nM, at least 118 nM, at least 119 nM, at least 120 nM, at least 121 nM, at least 122 nM, at least 123 nM, at least 124 nM, at least 125 nM, at least 126 nM, at least 127 nM, at least 128 nM, at least 129 nM, at least 130 nM, at least 131 nM, at least 132 nM, at least 133 nM, at least 134 nM, at least 135 nM, at least 136 nM, at least 137 nM, at least 138 nM, at least 139 nM, at least 140 nM, at least 141 nM, at least 142 nM, at least 143 nM, at least 144 nM, at least 145 nM, at least 146 nM, at least 147 nM, at least 148 nM, at least 149 nM, at least 150 nM, at least 151 nM, at least 152 nM, at least 153 nM, at least 154 nM, at least 155 nM, at least 156 nM, at least 157 nM, at least 158 nM, at least 159 nM, at least 160 nM, at least 161 nM, at least 162 nM, at least 163 nM, at least 164 nM, at least 165 nM, at least 166 nM, at least 167 nM, at least 168 nM, at least 169 nM, at least 170 nM, at least 171 nM, at least 172 nM, at least 173 nM, at least 174 nM, at least 175 nM, at least 176 nM, at least 177 nM, at least 178 nM, at least 179 nM, at least 180 nM, at least 181 nM, at least 182 nM, at least 183 nM, at least 184 nM, at least 185 nM, at least 186 nM, at least 187 nM, at least 188 nM, at least 189 nM, at least 190 nM, at least 191 nM, at least 192 nM, at least 193 nM, at least 194 nM, at least 195 nM, at least 196 nM, at least 197 nM, at least 198 nM, at least 199 nM, 200 nM, at least 210 nM, at least 220 nM, at least 230 nM, at least 240 nM, at least 250 nM, at least 260 nM, at least 270 nM, at least 280 nM, at least 290 nM, at least 300 nM, at least 310 nM, at least 320 nM, at least 330 nM, at least 340 nM, at least 350 nM, at least 360 nM, at least 370 nM, at least 380 nM, at least 390 nM, at least 400 nM, at least 410 nM, at least 420 nM, at least 430 nM, at least 440 nM, at least 450 nM, at least 460 nM, at least 470 nM, at least 480 nM, at least 490 nM, at least 0.5 µM, at least 0.51 µM, at least 0.52 µM, at least 0.53 µM, at least 0.54 µM, at least 0.55 µM, at least 0.56 µM, at least 0.57 µM, at least 0.58 µM, at least 0.59 µM, at least 0.6 µM, at least 0.61 µM, at least 0.62 µM, at least 0.63 µM, at least 0.64 µM, at least 0.65 µM, at least 0.66 µM, at least 0.67 µM, at least 0.68 µM, at least 0.69 µM, at 56 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 least 0.7 µM, at least 0.71 µM, at least 0.72 µM, at least 0.73 µM, at least 0.74 µM, at least 0.75 µM, at least 0.76 µM, at least 0.77 µM, at least 0.78 µM, at least 0.79 µM, at least 0.8 µM, at least 0.81 µM, at least 0.82 µM, at least 0.83 µM, at least 0.84 µM, at least 0.85 µM, at least 0.86 µM, at least 0.87 µM, at least 0.88 µM, at least 0.89 µM, at least 0.9 µM, at least 0.91 µM, at least 0.92 µM, at least 0.93 µM, at least 0.94 µM, at least 0.95 µM, at least 0.96 µM, at least 0.97 µM, at least 0.98 µM, at least 0.99 µM, or 1 µM.

[0260] In some embodiments of any of the aspects, the triiodothyronine (T3) is at a concentration of at most 10 nM, at most 20 nM, at most 30 nM, at most 40 nM, at most 50 nM, at most 51 nM, at most 52 nM, at most 53 nM, at most 54 nM, at most 55 nM, at most 56 nM, at most 57 nM, at most 58 nM, at most 59 nM, at most 60 nM, at most 61 nM, at most 62 nM, at most 63 nM, at most 64 nM, at most 65 nM, at most 66 nM, at most 67 nM, at most 68 nM, at most 69 nM, at most 70 nM, at most 71 nM, at most 72 nM, at most 73 nM, at most 74 nM, at most 75 nM, at most 76 nM, at most 77 nM, at most 78 nM, at most 79 nM, at most 80 nM, at most 81 nM, at most 82 nM, at most 83 nM, at most 84 nM, at most 85 nM, at most 86 nM, at most 87 nM, at most 88 nM, at most 89 nM, at most 90 nM, at most 91 nM, at most 92 nM, at most 93 nM, at most 94 nM, at most 95 nM, at most 96 nM, at most 97 nM, at most 98 nM, at most 99 nM, at most 100 nM, at most 101 nM, at most 102 nM, at most 103 nM, at most 104 nM, at most 105 nM, at most 106 nM, at most 107 nM, at most 108 nM, at most 109 nM, at most 110 nM, at most 111 nM, at most 112 nM, at most 113 nM, at most 114 nM, at most 115 nM, at most 116 nM, at most 117 nM, at most 118 nM, at most 119 nM, at most 120 nM, at most 121 nM, at most 122 nM, at most 123 nM, at most 124 nM, at most 125 nM, at most 126 nM, at most 127 nM, at most 128 nM, at most 129 nM, at most 130 nM, at most 131 nM, at most 132 nM, at most 133 nM, at most 134 nM, at most 135 nM, at most 136 nM, at most 137 nM, at most 138 nM, at most 139 nM, at most 140 nM, at most 141 nM, at most 142 nM, at most 143 nM, at most 144 nM, at most 145 nM, at most 146 nM, at most 147 nM, at most 148 nM, at most 149 nM, at most 150 nM, at most 151 nM, at most 152 nM, at most 153 nM, at most 154 nM, at most 155 nM, at most 156 nM, at most 157 nM, at most 158 nM, at most 159 nM, at most 160 nM, at most 161 nM, at most 162 nM, at most 163 nM, at most 164 nM, at most 165 nM, at most 166 nM, at most 167 nM, at most 168 nM, at most 169 nM, at most 170 nM, at most 171 nM, at most 172 nM, at most 173 nM, at most 174 nM, at most 175 nM, at most 176 nM, at most 177 nM, at most 178 nM, at most 179 nM, at most 180 nM, at most 181 nM, at most 182 nM, at most 183 nM, at 57 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 most 184 nM, at most 185 nM, at most 186 nM, at most 187 nM, at most 188 nM, at most 189 nM, at most 190 nM, at most 191 nM, at most 192 nM, at most 193 nM, at most 194 nM, at most 195 nM, at most 196 nM, at most 197 nM, at most 198 nM, at most 199 nM, at most 200 nM, at most 210 nM, at most 220 nM, at most 230 nM, at most 240 nM, at most 250 nM, at most 260 nM, at most 270 nM, at most 280 nM, at most 290 nM, at most 300 nM, at most 310 nM, at most 320 nM, at most 330 nM, at most 340 nM, at most 350 nM, at most 360 nM, at most 370 nM, at most 380 nM, at most 390 nM, at most 400 nM, at most 410 nM, at most 420 nM, at most 430 nM, at most 440 nM, at most 450 nM, at most 460 nM, at most 470 nM, at most 480 nM, at most 490 nM, at most 0.5 µM, at most 0.51 µM, at most 0.52 µM, at most 0.53 µM, at most 0.54 µM, at most 0.55 µM, at most 0.56 µM, at most 0.57 µM, at most 0.58 µM, at most 0.59 µM, at most 0.6 µM, at most 0.61 µM, at most 0.62 µM, at most 0.63 µM, at most 0.64 µM, at most 0.65 µM, at most 0.66 µM, at most 0.67 µM, at most 0.68 µM, at most 0.69 µM, at most 0.7 µM, at most 0.71 µM, at most 0.72 µM, at most 0.73 µM, at most 0.74 µM, at most 0.75 µM, at most 0.76 µM, at most 0.77 µM, at most 0.78 µM, at most 0.79 µM, at most 0.8 µM, at most 0.81 µM, at most 0.82 µM, at most 0.83 µM, at most 0.84 µM, at most 0.85 µM, at most 0.86 µM, at most 0.87 µM, at most 0.88 µM, at most 0.89 µM, at most 0.9 µM, at most 0.91 µM, at most 0.92 µM, at most 0.93 µM, at most 0.94 µM, at most 0.95 µM, at most 0.96 µM, at most 0.97 µM, at most 0.98 µM, at most 0.99 µM, or at most 1 µM. Dexamethasone

[0261] In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of about 1.0 µM. In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of at most 1.0 µM. In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of 0.5 µM to 2.0 µM. In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of 10 nM to 10 µM.

[0262] In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of about 10 nM, about 20 nM, about 30 nM, about 40 nM, about 50 nM, about 60 nM, about 70 nM, about 80 nM, about 90 nM, about 100 nM, about 110 nM, about 120 nM, about 130 nM, about 140 nM, about 150 nM, about 160 nM, about 170 nM, about 180 nM, about 190 nM, about 200 nM, about 210 nM, about 220 nM, about 230 nM, about 240 nM, about 250 nM, about 260 nM, about 270 nM, about 280 nM, about 290 nM, about 300 nM, about 310 nM, about 320 nM, about 330 nM, about 340 nM, about 350 nM, about 360 nM, about 370 nM, about 58 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 380 nM, about 390 nM, about 400 nM, about 410 nM, about 420 nM, about 430 nM, about 440 nM, about 450 nM, about 460 nM, about 470 nM, about 480 nM, about 490 nM, about 0.5 µM, about 0.51 µM, about 0.52 µM, about 0.53 µM, about 0.54 µM, about 0.55 µM, about 0.56 µM, about 0.57 µM, about 0.58 µM, about 0.59 µM, about 0.6 µM, about 0.61 µM, about 0.62 µM, about 0.63 µM, about 0.64 µM, about 0.65 µM, about 0.66 µM, about 0.67 µM, about 0.68 µM, about 0.69 µM, about 0.7 µM, about 0.71 µM, about 0.72 µM, about 0.73 µM, about 0.74 µM, about 0.75 µM, about 0.76 µM, about 0.77 µM, about 0.78 µM, about 0.79 µM, about 0.8 µM, about 0.81 µM, about 0.82 µM, about 0.83 µM, about 0.84 µM, about 0.85 µM, about 0.86 µM, about 0.87 µM, about 0.88 µM, about 0.89 µM, about 0.9 µM, about 0.91 µM, about 0.92 µM, about 0.93 µM, about 0.94 µM, about 0.95 µM, about 0.96 µM, about 0.97 µM, about 0.98 µM, about 0.99 µM, about 1 µM, about 1.01 µM, about 1.02 µM, about 1.03 µM, about 1.04 µM, about 1.05 µM, about 1.06 µM, about 1.07 µM, about 1.08 µM, about 1.09 µM, about 1.1 µM, about 1.11 µM, about 1.12 µM, about 1.13 µM, about 1.14 µM, about 1.15 µM, about 1.16 µM, about 1.17 µM, about 1.18 µM, about 1.19 µM, about 1.2 µM, about 1.21 µM, about 1.22 µM, about 1.23 µM, about 1.24 µM, about 1.25 µM, about 1.26 µM, about 1.27 µM, about 1.28 µM, about 1.29 µM, about 1.3 µM, about 1.31 µM, about 1.32 µM, about 1.33 µM, about 1.34 µM, about 1.35 µM, about 1.36 µM, about 1.37 µM, about 1.38 µM, about 1.39 µM, about 1.4 µM, about 1.41 µM, about 1.42 µM, about 1.43 µM, about 1.44 µM, about 1.45 µM, about 1.46 µM, about 1.47 µM, about 1.48 µM, about 1.49 µM, about 1.5 µM, about 1.51 µM, about 1.52 µM, about 1.53 µM, about 1.54 µM, about 1.55 µM, about 1.56 µM, about 1.57 µM, about 1.58 µM, about 1.59 µM, about 1.6 µM, about 1.61 µM, about 1.62 µM, about 1.63 µM, about 1.64 µM, about 1.65 µM, about 1.66 µM, about 1.67 µM, about 1.68 µM, about 1.69 µM, about 1.7 µM, about 1.71 µM, about 1.72 µM, about 1.73 µM, about 1.74 µM, about 1.75 µM, about 1.76 µM, about 1.77 µM, about 1.78 µM, about 1.79 µM, about 1.8 µM, about 1.81 µM, about 1.82 µM, about 1.83 µM, about 1.84 µM, about 1.85 µM, about 1.86 µM, about 1.87 µM, about 1.88 µM, about 1.89 µM, about 1.9 µM, about 1.91 µM, about 1.92 µM, about 1.93 µM, about 1.94 µM, about 1.95 µM, about 1.96 µM, about 1.97 µM, about 1.98 µM, about 1.99 µM, about 2 µM, about 2.5 µM, about 3 µM, about 3.5 µM, about 4 µM, about 4.5 µM, about 5 µM, about 5.5 µM, about 6 µM, about 6.5 µM, about 7 µM, about 7.5 µM, about 8 µM, about 8.5 µM, about 9 µM, about 9.5 µM, or about 10 µM. 59 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0263] In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of at least 10 nM, at least 20 nM, at least 30 nM, at least 40 nM, at least 50 nM, at least 60 nM, at least 70 nM, at least 80 nM, at least 90 nM, at least 100 nM, at least 110 nM, at least 120 nM, at least 130 nM, at least 140 nM, at least 150 nM, at least 160 nM, at least 170 nM, at least 180 nM, at least 190 nM, at least 200 nM, at least 210 nM, at least 220 nM, at least 230 nM, at least 240 nM, at least 250 nM, at least 260 nM, at least 270 nM, at least 280 nM, at least 290 nM, at least 300 nM, at least 310 nM, at least 320 nM, at least 330 nM, at least 340 nM, at least 350 nM, at least 360 nM, at least 370 nM, at least 380 nM, at least 390 nM, at least 400 nM, at least 410 nM, at least 420 nM, at least 430 nM, at least 440 nM, at least 450 nM, at least 460 nM, at least 470 nM, at least 480 nM, at least 490 nM, at least 0.5 µM, at least 0.51 µM, at least 0.52 µM, at least 0.53 µM, at least 0.54 µM, at least 0.55 µM, at least 0.56 µM, at least 0.57 µM, at least 0.58 µM, at least 0.59 µM, at least 0.6 µM, at least 0.61 µM, at least 0.62 µM, at least 0.63 µM, at least 0.64 µM, at least 0.65 µM, at least 0.66 µM, at least 0.67 µM, at least 0.68 µM, at least 0.69 µM, at least 0.7 µM, at least 0.71 µM, at least 0.72 µM, at least 0.73 µM, at least 0.74 µM, at least 0.75 µM, at least 0.76 µM, at least 0.77 µM, at least 0.78 µM, at least 0.79 µM, at least 0.8 µM, at least 0.81 µM, at least 0.82 µM, at least 0.83 µM, at least 0.84 µM, at least 0.85 µM, at least 0.86 µM, at least 0.87 µM, at least 0.88 µM, at least 0.89 µM, at least 0.9 µM, at least 0.91 µM, at least 0.92 µM, at least 0.93 µM, at least 0.94 µM, at least 0.95 µM, at least 0.96 µM, at least 0.97 µM, at least 0.98 µM, at least 0.99 µM, at least 1 µM, at least 1.01 µM, at least 1.02 µM, at least 1.03 µM, at least 1.04 µM, at least 1.05 µM, at least 1.06 µM, at least 1.07 µM, at least 1.08 µM, at least 1.09 µM, at least 1.1 µM, at least 1.11 µM, at least 1.12 µM, at least 1.13 µM, at least 1.14 µM, at least 1.15 µM, at least 1.16 µM, at least 1.17 µM, at least 1.18 µM, at least 1.19 µM, at least 1.2 µM, at least 1.21 µM, at least 1.22 µM, at least 1.23 µM, at least 1.24 µM, at least 1.25 µM, at least 1.26 µM, at least 1.27 µM, at least 1.28 µM, at least 1.29 µM, at least 1.3 µM, at least 1.31 µM, at least 1.32 µM, at least 1.33 µM, at least 1.34 µM, at least 1.35 µM, at least 1.36 µM, at least 1.37 µM, at least 1.38 µM, at least 1.39 µM, at least 1.4 µM, at least 1.41 µM, at least 1.42 µM, at least 1.43 µM, at least 1.44 µM, at least 1.45 µM, at least 1.46 µM, at least 1.47 µM, at least 1.48 µM, at least 1.49 µM, at least 1.5 µM, at least 1.51 µM, at least 1.52 µM, at least 1.53 µM, at least 1.54 µM, at least 1.55 µM, at least 1.56 µM, at least 1.57 µM, at least 1.58 µM, at least 1.59 µM, at least 1.6 µM, at least 1.61 µM, at least 1.62 µM, at least 1.63 µM, at least 1.64 µM, at least 1.65 µM, at least 1.66 µM, at least 1.67 µM, at 60 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 least 1.68 µM, at least 1.69 µM, at least 1.7 µM, at least 1.71 µM, at least 1.72 µM, at least 1.73 µM, at least 1.74 µM, at least 1.75 µM, at least 1.76 µM, at least 1.77 µM, at least 1.78 µM, at least 1.79 µM, at least 1.8 µM, at least 1.81 µM, at least 1.82 µM, at least 1.83 µM, at least 1.84 µM, at least 1.85 µM, at least 1.86 µM, at least 1.87 µM, at least 1.88 µM, at least 1.89 µM, at least 1.9 µM, at least 1.91 µM, at least 1.92 µM, at least 1.93 µM, at least 1.94 µM, at least 1.95 µM, at least 1.96 µM, at least 1.97 µM, at least 1.98 µM, at least 1.99 µM, 2 µM, at least 2.5 µM, at least 3 µM, at least 3.5 µM, at least 4 µM, at least 4.5 µM, at least 5 µM, at least 5.5 µM, at least 6 µM, at least 6.5 µM, at least 7 µM, at least 7.5 µM, at least 8 µM, at least 8.5 µM, at least 9 µM, at least 9.5 µM, or 10 µM.

[0264] In some embodiments of any of the aspects, the dexamethasone (Dex) is at a concentration of at most 10 nM, at most 20 nM, at most 30 nM, at most 40 nM, at most 50 nM, at most 60 nM, at most 70 nM, at most 80 nM, at most 90 nM, at most 100 nM, at most 110 nM, at most 120 nM, at most 130 nM, at most 140 nM, at most 150 nM, at most 160 nM, at most 170 nM, at most 180 nM, at most 190 nM, at most 200 nM, at most 210 nM, at most 220 nM, at most 230 nM, at most 240 nM, at most 250 nM, at most 260 nM, at most 270 nM, at most 280 nM, at most 290 nM, at most 300 nM, at most 310 nM, at most 320 nM, at most 330 nM, at most 340 nM, at most 350 nM, at most 360 nM, at most 370 nM, at most 380 nM, at most 390 nM, at most 400 nM, at most 410 nM, at most 420 nM, at most 430 nM, at most 440 nM, at most 450 nM, at most 460 nM, at most 470 nM, at most 480 nM, at most 490 nM, at most 0.5 µM, at most 0.51 µM, at most 0.52 µM, at most 0.53 µM, at most 0.54 µM, at most 0.55 µM, at most 0.56 µM, at most 0.57 µM, at most 0.58 µM, at most 0.59 µM, at most 0.6 µM, at most 0.61 µM, at most 0.62 µM, at most 0.63 µM, at most 0.64 µM, at most 0.65 µM, at most 0.66 µM, at most 0.67 µM, at most 0.68 µM, at most 0.69 µM, at most 0.7 µM, at most 0.71 µM, at most 0.72 µM, at most 0.73 µM, at most 0.74 µM, at most 0.75 µM, at most 0.76 µM, at most 0.77 µM, at most 0.78 µM, at most 0.79 µM, at most 0.8 µM, at most 0.81 µM, at most 0.82 µM, at most 0.83 µM, at most 0.84 µM, at most 0.85 µM, at most 0.86 µM, at most 0.87 µM, at most 0.88 µM, at most 0.89 µM, at most 0.9 µM, at most 0.91 µM, at most 0.92 µM, at most 0.93 µM, at most 0.94 µM, at most 0.95 µM, at most 0.96 µM, at most 0.97 µM, at most 0.98 µM, at most 0.99 µM, at most 1 µM, at most 1.01 µM, at most 1.02 µM, at most 1.03 µM, at most 1.04 µM, at most 1.05 µM, at most 1.06 µM, at most 1.07 µM, at most 1.08 µM, at most 1.09 µM, at most 1.1 µM, at most 1.11 µM, at most 1.12 µM, at most 1.13 µM, at most 1.14 µM, at most 1.15 µM, at most 1.16 61 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 µM, at most 1.17 µM, at most 1.18 µM, at most 1.19 µM, at most 1.2 µM, at most 1.21 µM, at most 1.22 µM, at most 1.23 µM, at most 1.24 µM, at most 1.25 µM, at most 1.26 µM, at most 1.27 µM, at most 1.28 µM, at most 1.29 µM, at most 1.3 µM, at most 1.31 µM, at most 1.32 µM, at most 1.33 µM, at most 1.34 µM, at most 1.35 µM, at most 1.36 µM, at most 1.37 µM, at most 1.38 µM, at most 1.39 µM, at most 1.4 µM, at most 1.41 µM, at most 1.42 µM, at most 1.43 µM, at most 1.44 µM, at most 1.45 µM, at most 1.46 µM, at most 1.47 µM, at most 1.48 µM, at most 1.49 µM, at most 1.5 µM, at most 1.51 µM, at most 1.52 µM, at most 1.53 µM, at most 1.54 µM, at most 1.55 µM, at most 1.56 µM, at most 1.57 µM, at most 1.58 µM, at most 1.59 µM, at most 1.6 µM, at most 1.61 µM, at most 1.62 µM, at most 1.63 µM, at most 1.64 µM, at most 1.65 µM, at most 1.66 µM, at most 1.67 µM, at most 1.68 µM, at most 1.69 µM, at most 1.7 µM, at most 1.71 µM, at most 1.72 µM, at most 1.73 µM, at most 1.74 µM, at most 1.75 µM, at most 1.76 µM, at most 1.77 µM, at most 1.78 µM, at most 1.79 µM, at most 1.8 µM, at most 1.81 µM, at most 1.82 µM, at most 1.83 µM, at most 1.84 µM, at most 1.85 µM, at most 1.86 µM, at most 1.87 µM, at most 1.88 µM, at most 1.89 µM, at most 1.9 µM, at most 1.91 µM, at most 1.92 µM, at most 1.93 µM, at most 1.94 µM, at most 1.95 µM, at most 1.96 µM, at most 1.97 µM, at most 1.98 µM, at most 1.99 µM, at most 2 µM, at most 2.5 µM, at most 3 µM, at most 3.5 µM, at most 4 µM, at most 4.5 µM, at most 5 µM, at most 5.5 µM, at most 6 µM, at most 6.5 µM, at most 7 µM, at most 7.5 µM, at most 8 µM, at most 8.5 µM, at most 9 µM, at most 9.5 µM, or at most 10 µM. Glucose

[0265] In some embodiments of any of the aspects, the medium comprises essentially no glucose. In some embodiments of any of the aspects, the medium comprises 0 mM glucose. In some embodiments of any of the aspects, the medium comprises at most 1 mM glucose. In some embodiments of any of the aspects, the medium comprises at most 5.0 mM glucose. In some embodiments of any of the aspects, the medium comprises 0 mM to 1 mM glucose. In some embodiments of any of the aspects, the medium comprises 0 mM to 5.0 mM glucose.

[0266] In some embodiments of any of the aspects, the glucose is at a concentration of 0 mM, about 0.1 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.4 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2 mM, about 2.1 mM, about 2.2 mM, about 2.3 mM, about 2.4 mM, about 2.5 mM, 62 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 about 2.6 mM, about 2.7 mM, about 2.8 mM, about 2.9 mM, about 3 mM, about 3.1 mM, about 3.2 mM, about 3.3 mM, about 3.4 mM, about 3.5 mM, about 3.6 mM, about 3.7 mM, about 3.8 mM, about 3.9 mM, about 4 mM, about 4.1 mM, about 4.2 mM, about 4.3 mM, about 4.4 mM, about 4.5 mM, about 4.6 mM, about 4.7 mM, about 4.8 mM, about 4.9 mM, or about 5 mM.

[0267] In some embodiments of any of the aspects, the glucose is at a concentration of at least 0.1 mM, at least 0.2 mM, at least 0.3 mM, at least 0.4 mM, at least 0.5 mM, at least 0.6 mM, at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at least 1 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at least 1.4 mM, at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at least 1.8 mM, at least 1.9 mM, at least 2 mM, at least 2.1 mM, at least 2.2 mM, at least 2.3 mM, at least 2.4 mM, at least 2.5 mM, at least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at least 3 mM, at least 3.1 mM, at least 3.2 mM, at least 3.3 mM, at least 3.4 mM, at least 3.5 mM, at least 3.6 mM, at least 3.7 mM, at least 3.8 mM, at least 3.9 mM, at least 4 mM, at least 4.1 mM, at least 4.2 mM, at least 4.3 mM, at least 4.4 mM, at least 4.5 mM, at least 4.6 mM, at least 4.7 mM, at least 4.8 mM, at least 4.9 mM, or 5 mM.

[0268] In some embodiments of any of the aspects, the glucose is at a concentration of 0 mM, at most 0.1 mM, at most 0.2 mM, at most 0.3 mM, at most 0.4 mM, at most 0.5 mM, at most 0.6 mM, at most 0.7 mM, at most 0.8 mM, at most 0.9 mM, at most 1 mM, at most 1.1 mM, at most 1.2 mM, at most 1.3 mM, at most 1.4 mM, at most 1.5 mM, at most 1.6 mM, at most 1.7 mM, at most 1.8 mM, at most 1.9 mM, at most 2 mM, at most 2.1 mM, at most 2.2 mM, at most 2.3 mM, at most 2.4 mM, at most 2.5 mM, at most 2.6 mM, at most 2.7 mM, at most 2.8 mM, at most 2.9 mM, at most 3 mM, at most 3.1 mM, at most 3.2 mM, at most 3.3 mM, at most 3.4 mM, at most 3.5 mM, at most 3.6 mM, at most 3.7 mM, at most 3.8 mM, at most 3.9 mM, at most 4 mM, at most 4.1 mM, at most 4.2 mM, at most 4.3 mM, at most 4.4 mM, at most 4.5 mM, at most 4.6 mM, at most 4.7 mM, at most 4.8 mM, at most 4.9 mM, or at most 5 mM. Cell Culture Media

[0269] In some embodiments of any of the aspects, the cardiomyocyte maturation medium further comprises Roswell Park Memorial Institute™ (RPMI) 1640 cell culture medium. In some embodiments of any of the aspects, the RPMI 1640 comprises inorganic salts, amino acids, vitamins, glutathione, and / or Phenol red. 63 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0270] In some embodiments of any of the aspects, the inorganic salts comprise: calcium nitrate • 4H2O; magnesium sulfate (anhydrous); potassium chloride; sodium bicarbonate; sodium chloride; and / or sodium phosphate dibasic (anhydrous).

[0271] In some embodiments of any of the aspects, the amino acids comprise: L-alanyl-L- glutamine; L-arginine; L-asparagine (anhydrous); L-aspartic acid; L-cystine • 2HCl; L-glutamic acid; L-glutamine; glycine; L-histidine; hydroxy-L-proline; L-isoleucine; L-leucine; L-lysine • HCl; L-methionine; L-phenylalanine; L-proline; L-serine; L-threonine; L-tryptophan; L-tyrosine • 2Na • 2H2O; and / or L-valine.

[0272] In some embodiments of any of the aspects, the vitamins comprise: D-biotin; choline chloride; folic acid; myo-inositol; niacinamide; p-aminobenzoic acid; D-pantothenic acid (hemicalcium); pyridoxine • HCl; riboflavin; thiamine • HCl; and / or vitamin B12.

[0273] In some embodiments of any of the aspects, the cardiomyocyte maturation medium further comprises B-27™ supplement. In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises a constant amount of B-27™ supplement and its components. In some embodiments of any of the aspects, the cardiomyocyte maturation medium comprises B-27™ supplement at a final concentration of 1x, e.g., 1 / 50 of a 50x B-27™ supplement.

[0274] In some embodiments of any of the aspects, the B-27™ supplement comprises approximately physiological concentrations of: biotin; DL alpha tocopherol acetate; DL alpha- tocopherol; vitamin A (acetate); BSA , fatty acid free fraction V; catalase; human recombinant insulin; human transferrin; superoxide dismutase; corticosterone; D-galactose; ethanolamine HCl; glutathione (reduced); L-carnitine HCl; linoleic acid; linolenic acid; progesterone; putrescine 2 HCl; sodium selenite; and / or T3 (triiodothyronine) and / or analogs thereof. As used herein, the term “physiological concentration” refers to the concentration found in a mammalian subject at a normal physiological condition or the concentration found in cell culture necessary and / or sufficient to maintain cultured cells at a normal physiological condition.

[0275] All patents and other publications; including literature references, issued patents, published patent applications, and co-pending patent applications; cited throughout this application are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in 64 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 connection with the technology described herein. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicants and does not constitute any admission as to the correctness of the dates or contents of these documents.

[0276] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.

[0277] Specific elements of any of the foregoing embodiments can be combined or substituted for elements in other embodiments. Furthermore, while advantages associated with certain embodiments of the disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments need necessarily exhibit such advantages to fall within the scope of the disclosure.

[0278] Some embodiments of the technology described herein can be defined according to any of the following numbered paragraphs: 1. A cell maturation medium composition for promoting electrophysiological maturity in in-vitro differentiated cardiomyocytes, the medium comprising: a) glucose at 1 mM or less; b) triiodothyronine (T3); and c) dexamethasone. 65 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 2. The cell maturation medium composition of paragraph 1, wherein the concentration of T3 is between 10nM-1μM. 3. The cell maturation medium composition of any one of paragraphs 1-2, wherein the concentration of T3 is 1 nM. 4. The cell maturation medium composition of any one of paragraphs 1-3, wherein the concentration of T3 is 100 nM. 5. The cell maturation medium composition of any one of paragraphs 1-4, wherein the concentration of dexamethasone is between 0.1μM-10μM. 6. The cell maturation medium composition of any one of paragraphs 1-5, wherein the concentration of dexamethasone is 1 μM. 7. The cell maturation medium composition of any one of paragraphs 1-5, wherein the concentration of dexamethasone is 100 nM. 8. The cell maturation medium composition of any one of paragraphs 1-7, wherein the medium comprises RPMI with a glucose concentration of 1 mM or less. 9. The cell maturation medium composition of any one of paragraphs 1-8, wherein the medium does not comprise glucose. 10. The cell maturation medium composition of any one of paragraphs 1-9, further comprising one or more fatty acids found in mammalian milk. 11. The cell maturation medium composition of paragraph 10, wherein the fatty acid(s) are selected from the group consisting of linoleic, oleic and palmitic acid. 12. The cell maturation medium composition of paragraph 11, which comprises linoleic, oleic and palmitic acids. 13. The cell maturation medium composition of paragraph 12, wherein the concentration of linoleic, oleic and palmitic acids is from 115 – 460 μM. 14. The cell maturation medium composition of paragraph 13, wherein the concentration of linoleic, oleic and palmitic acids is 460 μM. 15. The cell maturation medium composition of paragraph 13, wherein the concentration of linoleic acid is from 22.5 – 137 μM. 16. The cell maturation medium composition of paragraph 13, wherein the concentration of oleic acid is from 40 – 243 μM. 17. The cell maturation medium composition of paragraph 13, wherein the concentration of 66 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 palmitic acid is from 52.5 – 80 μM. 18. The cell maturation medium composition of any one of paragraphs 1-17, further comprising galactose. 19. The cell maturation medium composition of any one of paragraphs 1-18, wherein the galactose concentration is 0.5 – 10 mM. 20. The cell maturation medium composition of paragraph 19, wherein the galactose concentration is 0.5 mM. 21. The cell maturation medium composition of any one of paragraphs 1-20, further comprising an AMP-activated protein kinase (AMPK) activator. 22. The cell maturation medium composition of paragraph 21, wherein the AMPK activator is 5-aminoimidizole-4-carboxamide riboside (AICAR). 23. The cell maturation medium composition of paragraph 22, wherein the AICAR is present at 0.3 to 2.0 mM. 24. The cell maturation medium composition of paragraph 23, wherein the AICAR is present at 1 mM. 25. A composition comprising in vitro differentiated cardiomyocytes in admixture with a cell maturation medium composition of any one of paragraphs 1-24. 26. The composition of paragraph 25, wherein the in vitro differentiated cardiomyocytes are in suspension. 27. The composition of paragraph 25, wherein the in vitro differentiated cardiomyocytes are in monolayer culture. 28. The composition of paragraph 25, wherein the in vitro differentiated cardiomyocytes are in 3D culture. 29. The composition of any one of paragraphs 25-28, wherein the in vitro differentiated cardiomyocytes are in vitro differentiated from a stem cell. 30. The composition of paragraph 29, wherein the stem cell is an iPS cell or an ES cell. 31. The composition of paragraph 30, wherein the iPS cell is derived from a subject with a cardiac disease or disorder. 32. A transplant composition comprising a composition of any one of paragraphs 25-31. 33. The transplant composition of paragraph 32, wherein the in vitro-differentiated cardiomyocytes are autologous to a transplant recipient. 67 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 34. The transplant composition of paragraph 32 or 33, further comprising a pharmaceutically acceptable carrier. 35. The transplant composition of any one of paragraphs 32-34, wherein the composition further comprises a matrix. 36. The transplant composition of any one of paragraphs 32-35, further comprising cardiac endothelial cells. 37. A method of promoting electrophysiological maturation of an in vitro differentiated cardiomyocyte, the method comprising contacting the in vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of paragraphs 1- 24. 38. The method of paragraph 37, wherein electrophysiological maturity is determined by one or more of the following markers as compared to a reference level: increased expression of SCN5A (encodes Nav1.5, a fast sodium channel), KCNJ2 (encodes Kir2.1 channel, an inward rectifier potassium channel), CACNA1C (encodes Cav1.2, α1C subunit of voltage-dependent L type calcium channel), ATP2A2 (encodes SR calcium ATPase), RYR2 (encodes the ryanodine receptor in SR), Cx43 (Connexin 43, Gap junction α-1) protein and / or Nav1.5 protein, decreased expression of HCN4 (encodes a member of the hyperpolarization-activated cyclic nucleotide-gated sodium channel) and / or CACNA1H (encodes Cav3.2,α1H subunit of voltage-dependent T type calcium channel), and / or decreased field potential frequency, increased field potential duration, increased spike amplitude, increased spike slope, increased conduction velocity, increased calcium transient amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay. 39. The method of paragraph 37 or 38, wherein the cardiomyocytes are contacted with the cell maturation medium composition for at least 7 days. 40. An in vitro-differentiated cardiomyocyte produced by the method of any one of paragraphs 37-39. 41. The in vitro-differentiated cardiomyocyte of paragraph 40, wherein the cardiomyocyte has increased electrophysiological maturity relative to an in vitro differentiated cardiomyocyte prepared without contacting with the cell maturation medium composition. 68 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 42. The in vitro-differentiated cardiomyocyte of paragraph 40 or 41, which, when administered to cardiac tissue of a subject, promotes reduced engraftment arrhythmia relative to an in vitro differentiated cardiomyocyte prepared in the same manner but not contacted with the cell maturation medium composition. 43. A method of transplanting in vitro differentiated cardiomyocytes, the method comprising contacting the in vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of paragraphs 1-24, and then administering the cardiomyocytes to a transplant subject. 44. The method of paragraph 43, wherein the method results in reduced engraftment arrhythmia in the recipient. 45. The method of paragraph 43 or 44, wherein the in vitro cardiomyocyte is differentiated in vitro from iPS cell autologous to the recipient. 46. The method of any one of paragraphs 43-45, wherein the transplant subject has been diagnosed with a cardiac disease or disorder. 47. A method of treating a cardiac disease or disorder, the method comprising administering in vitro differentiated cardiomyocytes in the cell maturation medium composition of any one of paragraphs 1-24 to a subject in need thereof. 48. A method of treating a cardiac disease or disorder, the method comprising administering a transplant composition of any one of paragraphs 32-36 or an in vitro differentiated cardiomyocyte of any one of paragraphs 40-42 to a subject in need thereof. 49. The method of paragraph 48, wherein the treatment results in reduced engraftment arrhythmia relative to treatment using in vitro-differentiated cells that have not been contacted with cell maturation medium. 50. The method of any one of paragraphs 47-49, wherein the in-vitro differentiated cardiomyocyte is differentiated in vitro from an iPS cell autologous to the subject. 51. The method of any one of paragraphs 47-50, wherein the disease or disorder comprises cardiac infarction. 52. A composition of any one of paragraphs 25-36 or 40-42, for use in the treatment of a cardiac disease or disorder. 53. A kit for maturing an in vitro-differentiated cardiomyocyte, the kit comprising a cell 69 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 maturation medium of any one of paragraphs 1-24 and instructions for maturing an in vitro-differentiated cardiomyocyte. 54. The kit of paragraphs 53, further comprising an in vitro-differentiated cardiomyocyte. 55. The kit of any one of paragraphs 53-54, for use in preparation of a transplant to cardiac tissue of a subject in need thereof. 56. The kit of any one of paragraphs 53-55, for use in preparation of a treatment of a cardiac disease or disorder in a subject in need thereof. 57. A cardiomyocyte maturation medium comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex). 58. The cardiomyocyte maturation medium of paragraph 57, wherein the galactose (Gal) is at a concentration selected from: (a) about 0.5 mM; (b) at most 0.5 mM; (c) at most 1.0 mM (d) at most 10.0 mM; (e) 0.5 mM to 1.0 mM; or (f) 0.5 mM to 10.0 mM. 59. The cardiomyocyte maturation medium of any one of paragraphs 57-58, wherein the fatty acids (FA) are at a concentration selected from: (a) about 460 μM; (b) at most 460 μM; (c) at least 230 μM; (d) at least 115 μM; (e) 115 μM to 460 μM; or (f) 230 μM to 460 μM. 60. The cardiomyocyte maturation medium of paragraph 59, wherein the fatty acids (FA) comprise palmitic acids, oleic acids, and / or linoleic acids. 70 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 61. The cardiomyocyte maturation medium of paragraph 59, wherein the fatty acids (FA) comprise palmitic acid. 62. The cardiomyocyte maturation medium of any one of paragraphs 57-61, wherein the 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration selected from: (a) about 1.0 mM; (b) at most 1.0 mM; (c) at least 0.3 mM; or (d) 0.25 mM to 1.0 mM. 63. The cardiomyocyte maturation medium of any one of paragraphs 57-62, comprising about 460 μM fatty acids (FA) and about 1.0 mM AICAR. 64. The cardiomyocyte maturation medium of any one of paragraphs 57-63, wherein the triiodothyronine (T3) is at a concentration selected from: (a) about 100 nM; (b) at most 100 nM; or (c) 50 nM to 200 nM; or (d) 10 nM to 1 µM. 65. The cardiomyocyte maturation medium of any one of paragraphs 57-64, wherein the dexamethasone (Dex) is at a concentration selected from: (a) about 1.0 µM; (b) at most 1.0 µM; or (c) 0.5 µM to 2.0 µM; or (d) 10 nM to 10 µM. 66. The cardiomyocyte maturation medium of any one of paragraphs 57-65, comprising about 100 nM triiodothyronine (T3) and about 1.0 µM dexamethasone (Dex). 67. The cardiomyocyte maturation medium of any one of paragraphs 57-66, comprising essentially no glucose. 68. The cardiomyocyte maturation medium of any one of paragraphs 57-67, comprising 0 mM glucose. 69. The cardiomyocyte maturation medium of any one of paragraphs 57-68, further comprising: 71 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 (a) at most 1 mM glucose; (b) at most 5.0 mM glucose; (c) 0 mM to 1 mM glucose; or (d) 0 mM to 5.0 mM glucose. 70. The cardiomyocyte maturation medium of any one of paragraphs 57-69, comprising about 0.5 mM galactose (Gal) and 0 mM glucose. 71. The cardiomyocyte maturation medium of any one of paragraphs 57-70, further comprising carnosine. 72. The cardiomyocyte maturation medium of paragraph 71, wherein the carnosine is at a concentration of 2 mM to 10 mM. 73. A cardiomyocyte maturation medium comprising: (a) 0.5 mM to 10 mM galactose (Gal); (b) 115 μM to 460 μM fatty acids (FA); (c) 0.25 mM to 1 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) 10 nM to 1 μM triiodothyronine (T3); (e) 10 nM to 10 µM dexamethasone (Dex); and (f) 0 mM to 5 mM glucose. 74. A cardiomyocyte maturation medium comprising: (a) about 0.5 mM galactose (Gal); (b) about 460 μM fatty acids (FA); (c) about 1.0 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) about 100 nM triiodothyronine (T3); (e) about 1.0 µM dexamethasone (Dex); and (f) 0 mM glucose. 75. A composition comprising the cardiomyocyte maturation medium of any one of paragraphs 57-74, in combination with a cardiomyocyte or population thereof. 76. The composition of paragraph 75, wherein the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte. 77. The composition of paragraph 75 or 76, wherein the cardiomyocyte is electrophysiologically immature. 72 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 78. The composition of paragraph 75 or 76, wherein the cardiomyocyte is electrophysiologically mature. 79. A method of electrophysiologically maturing cardiomyocytes, the method comprising contacting electrophysiologically immature cardiomyocytes with the cardiomyocyte maturation medium of any one of paragraphs 57-74 for an amount of time sufficient to mature one or more electrophysiological parameters of the cardiomyocytes. 80. A method of modulating at least one electrophysiologic property of a cardiomyocyte or population thereof, the method comprising contacting electrophysiologically immature cardiomyocytes with the cardiomyocyte maturation medium of any one of paragraphs 57-74 for an amount of time sufficient to modulate one or more electrophysiological parameters of the cardiomyocytes. 81. The method of any one of paragraphs 79-80, wherein the sufficient of amount of time is at least one week. 82. The method of any one of paragraphs 79-81, wherein the sufficient of amount of time is one to two weeks. 83. The method of any one of paragraphs 79-82, wherein the electrophysiologically immature cardiomyocytes are differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs). 84. The method of any one of paragraphs 79-83, wherein the electrophysiologically immature cardiomyocytes are contacted with the cardiomyocyte maturation medium at least 20 days after differentiation from ESCs or iPSCs. 85. The method of any one of paragraphs 83-84, wherein the ESCs or iPSCs are human cells. 86. The method of any one of paragraphs 83-85, wherein the ESCs or iPSCs are differentiated into electrophysiologically immature cardiomyocytes in vitro or ex vivo. 87. The method of any one of paragraphs 79-86, wherein the maturation method is performed in vitro or ex vivo. 88. The method of any one of paragraphs 79-87, wherein the cardiomyocytes are cultured on MATRIGEL-coated tissue culture plates, wherein the MATRIGEL comprises solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells. 73 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 89. The method of any one of paragraphs 79-88, wherein at least one electrophysiologic property of a cardiomyocyte or population thereof is modulated by the method. 90. The method of any one of paragraphs 79-89, wherein the at least one electrophysiologic property is selected from the group consisting of: beat rate, field potential duration, spike amplitude, spike slope, conduction velocity, Ca2+kinetics, and expression of electrophysiological genes. 91. The method of any one of paragraphs 79-90, wherein the modulation of at least one electrophysiologic property comprises: decreased beat rate, increased field potential duration (FPD), increased spike amplitude, increased spike slope, increased conduction velocity, modulated Ca2+kinetics, and / or modulated expression of electrophysiological genes. 92. The method of any one of paragraphs 90-91, wherein the Ca2+kinetics comprise amplitude, time to peak, time to 50% decay, and / or time to 90% decay. 93. The method of any one of paragraphs 90-92, wherein the modulated Ca2+kinetics comprise increased amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay. 94. The method of any one of paragraphs 90-93, wherein the modulated expression of electrophysiological genes comprises increased expression of at least one electrophysiological gene. 95. The method of any one of paragraphs 90-94, wherein the modulated expression of electrophysiological genes comprises increased expression at least two electrophysiological genes. 96. The method of any one of paragraphs 90-96, wherein the electrophysiological genes are selected from the group consisting of: sarcomeric, metabolic, ion-channel, and calcium(Ca2+)-handling genes. 97. The method of paragraph 96, wherein: (a) the sarcomeric genes comprise TNNI3 and / or MYL2; (b) the metabolic genes comprise PPARGC1A and / or CPT1B; (c) the ion-channel genes comprise SCN5A, KCNJ2, and / or HCN4; and / or 74 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 (d) the calcium(Ca2+)-handling genes comprise CACNA1C, CACNA1H, ATPA2, and / or RYR2. 98. A kit for cardiomyocyte maturation comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex). 99. A cardiomyocyte or population thereof electrophysiologically matured in the cardiomyocyte maturation medium of any one of paragraphs 57-74. 100. A cardiomyocyte or population thereof electrophysiologically matured using the method of any one of paragraphs 79-97. 101. A cardiomyocyte or population thereof electrophysiologically matured using the kit of paragraph 98. 102. The cardiomyocyte or population thereof of any one of paragraphs 99-101, wherein the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte. 103. A cardiomyocyte transplant composition comprising the cardiomyocyte or population thereof of any one of paragraphs 99-102, in combination with a pharmaceutically acceptable carrier or scaffold. 104. A method of reducing or inhibiting engraftment arrythmia, the method comprising administering the cardiomyocyte, or population thereof, of any one of paragraphs 99- 102 or the cardiomyocyte transplant composition of paragraph 103 to a subject in need thereof, wherein the cardiomyocyte, or population thereof, or the cardiomyocyte transplant composition induces a reduced engraftment arrhythmia in the subject relative to an in vitro differentiated or ex vivo differentiated cardiomyocyte, or population thereof, or cardiomyocyte transplant composition that has not been matured using the maturation medium of any one of paragraphs 57-74, the method of any one of paragraphs 79-97, or the kit of paragraph 98. 105. A method of treating a cardiac disease, disorder, or injury, the method comprising administering the cardiomyocyte or population thereof of any one of paragraphs 99-102 75 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 or the cardiomyocyte transplant composition of paragraph 103 to a subject in need thereof. 106. The method of any one of paragraphs 104-105, wherein the cardiomyocyte or population thereof or the cardiomyocyte transplant composition is engrafted in or on the heart of the subject. 107. The method of any one of paragraphs 104-106, wherein the subject is a human. 108. The method of any one of paragraphs 104-106, wherein the subject is a non- human mammal. 109. The method of any one of paragraphs 104-108, wherein engraftment arrythmia is reduced by at least 10%. EXAMPLES EXAMPLE 1: Introduction

[0279] Regenerative capacity of adult cardiomyocytes (CMs) is limited in human hearts, therefore after myocardial infarction cardiomyocytes are lost and replaced by non-contractile fibrotic scar, resulting in cardiac dysfunction. Transplantation of human pluripotent stem cell derived cardiomyocytes (hPSC-CMs), such as human embryonic stem cell (hESC)-CMs or human induced pluripotent stem cell (hiPSC)-CMs, have proven to be a promising therapeutic approach for remuscularization and functional improvement in failing hearts1-3. Transplanted hPSC-CMs have been shown to engraft and electrophysiologically couple to host myocardium4. Electrophysiological connection between host and graft cardiac muscles enables synchronous contraction for generating larger stroke volume1,5,6. While engrafted hPSC-CMs provides functional benefits to heal injured hearts, some concerns in safety have emerged. Engraftment arrhythmias (EAs), transient ventricular arrhythmias following transplantation observed not in small animals, but in large animals such as non-human primates and pigs, are considered a major roadblock for clinical application1-3,7. Culture of hPSC-CMs generally represents an immature phenotype resembling embryonic or fetal cardiomyocytes, including spontaneous action potentials8. Electrophysiological studies conducted by the group and others indicated that the automaticity of graft CMs triggered EAs in large animals2,7. To eliminate arrhythmic events after 76 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 transplantation, production of electrophysiologically matured hPSC-CMs has been highly demanded.

[0280] Lessons from heart development suggest that the alteration in metabolism may trigger immature cardiomyocytes to acquire more adult like properties, including myofibril, cell cycle, size, and electrophysiological and contractile functions9. RPMI media commonly used for culturing hPSC-CMs contains higher glucose (11mM) than physiological fasting level (approximately 5mM), which can delay the metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS)10,11. It has been reported that glucose restriction (1mM) or deprivation (0mM) promotes not only metabolic reprogramming to aerobic respiration, but also transcriptional, structural, electrophysiological maturation of hPSC-CMs10,12,13. Thyroid hormone and glucocorticoid are metabolic hormones proven to improve the maturity of hPSC-CMs14-17. Triiodothyronine (T3), an active form of thyroid hormone, accelerates maturation in hPSC-CMs, improving cell size, sarcomere length, contractile force, Ca2+handling, and mitochondrial respiration14. Dexamethasone (Dex), a class of glucocorticoid, enhances T-tubule formation and improves Ca2+kinetics and electrophysiological function in the presence of T3 treatment15-17. While restriction of carbon sources and supplementation of metabolic hormones affect maturation differently, both share the beneficial effect on electrophysiology. We hypothesized that a combination of metabolic hormones with glucose starvation would enhance maturation of hPSC-CMs, including improved electrophysiological property, and the electrophysiologically matured cells could contribute to reduction of arrhythmic complications after transplantation. To address this, we tested if supplementation of T3 and Dex to low glucose media (1mM) can promote a wide range maturation process synergistically. Subsequently, the matured CMs were transplanted to porcine hearts to evaluate engraftment and arrhythmogenicity in large animals.

[0281] Here, it has been demonstrated that metabolic hormones in combination with glucose restriction enhanced maturation of hPSC-CMs in vitro. For example, an increased mitochondrial oxidation capacity was shown with larger energy storage and efficient transfer system, improved electrophysiological function, alteration in global gene expression pattern, reduced nucleotide synthesis and larger cell size. Transplantation to pig hearts revealed that the matured CMs engraft comparably to immature PSC-CMs in size and have reduced EA burden, which was accomplished without administration of antiarrhythmic drugs or editing ion channel genes18. Thus, metabolic intervention accelerated hPSC-CMs maturation broadly in vitro. Importantly, 77 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 this is the first study to reveal that matured cells were able to engraft to host pig hearts and with reduced arrhythmic events. Results TDLG promotes cardiac metabolism, contractile isoform switch, cell cycle and electrophysiology maturation

[0282] To investigate the additive effect of metabolic hormones to glucose restriction on maturation properties, hESC-CMs were treated with regular RPMI (glucose 11mM: Ctrl), low glucose RPMI (glucose 1mM: LG) or T31nM and Dex 100μM(TD) in the presence of LG (TDLG) for 14 days (FIG.1A). While both glucose 0mM and 1mM have been reported to promote maturation of hPSC-CMs, cell density was significantly reduced by glucose deprivation, which was restored by supplementation of glucose 1mM (data not shown)10,12,13. Mitochondrial respiratory function was assessed by flux analyzer. Compared to Ctrl-CMs, LG-CMs exhibited higher mitochondrial respiration including maximal and spare oxidative consumption rate, which were further enhanced by supplementation of TD, consistent with the upregulation of PPARGC1A and ESRRA, key drivers of mitochondrial biogenesis (FIG.1B-1C). Regarding sarcomere composition, TDLG induced isoform switching of troponin I from TNNI1 (fetal) toward TNNI3 (adult) (FIG.1B). In contrast, ratio of MYH7 (adult isoform) to MYH6 (fetal isoform) was decreased, contributed by T3 treatment (FIG.1B)14. CCNA2, CCNB1 and CDK1, cell cycle genes responsible for entry into DNA synthesis and mitosis phase followed by cytokinesis, are significantly suppressed by TDLG (FIG.1B). Immunostaining with α-Actinin and phalloidin revealed that TDLG increased the cell area of α-Actinin positive hPSC-CMs by 2- fold (from 1576μm2in Ctrl to 3058μm2in TDLG), while LG alone did not affect the cell size (Fig.1D). Despite the change in cell area, circularity index was not affected by either LG or TDLG treatment (FIG.1D). HCN4, a pacemaker channel responsible for hyperpolarization- activated funny current, was strongly suppressed by TDLG, while KCNJ2, an outward potassium channel essential for maintain resting membrane potential, was upregulated by TDLG. Microelectrode array (MEA) indicated that LG had a minimal effect on electrophysiology, increasing only corrected field potential duration (cFPD) (FIG.1E). Hence, TDLG-CMs exhibited greater spike amplitude and spike slope than Ctrl-CMs and LG-CMs, which, while not wishing to be bound by theory is possibly explained by the higher total SCN5A expression with 78 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 improved isoform switching from fetal to adult type (FIG.1B, FIG.1E).

[0283] Thus, TD supplementation augmented the maturation process of hPSC-CMs induced by glucose restriction, including enhanced expression of maturation markers, active OXPHOS state, cell cycle arrest with hypertrophic change and improved electrophysiological function. TDLG- CMs were then focused on to evaluate the global maturation status in metabolism and transcription. Maturation in metabolic pathway

[0284] To understand metabolic phenotype and identify pathways possibly contributing to maturation of hPSC-CMs, targeted metabolomics analysis of Ctrl-CMs and TDLG-CMs was conducted.137 metabolites were measured in both groups using liquid chromatography with tandem mass spectrometry. Principal components analysis (PCA) showed a distinct separation of 2 groups (FIG.2A). Consistent with the minimal glucose supply, heatmap analysis displayed that glucose-derived metabolites, including glycolysis, pentose phosphate pathway (PPP) and hexosamine pathway, were suppressed in TDLG-CMs (FIG.2B-2C). When pyruvate is actively converted into acetyl-CoA for entering TCA cycle, there is less accumulation of lactate, an end product of glycolysis derived from pyruvate19. TDLG-CMs exhibited decrease in lactate and the ratio of lactate to pyruvate, indicating more glucose metabolites entering TCA cycle (FIG.2E). Purine and pyrimidine nucleotide metabolism, fueled by PPP, were suppressed by TDLG, possibly leading to less cell proliferation (FIG.2C-2D). There were 42 differentially abundant metabolites between 2 groups, including 12 increased metabolites and 30 decreased metabolites in TDLG-CMs (FIG.2B). Interestingly, ubiquinol, a component of mitochondrial inner membrane and crucial for electron transport in the mitochondrial respiration chain, was the most significantly increased metabolite, suggesting active OXPHOS state in TDLG-CMs, which was consistent with the result of extracellular flux analysis (FIG.1D, FIG.2B). Compared to glycolysis, OXPHOS generates larger energy in concert with reactive oxygen species as byproduct. The ratio of GSSG (oxidized glutathione) / GSH (reduced glutathione) and NADP (oxidized form) / NADPH (reduced form), indicators for oxidative stress state, were significantly increased by TDLG treatment (FIGs.2F-2G). Creatine and Phosphocreatine (PCr), metabolites on creatine kinase (CK)-PCr shuttle which is an efficient energy transfer system predominant in adult CMs, were more abundant in TDLG-CMs (FIG.2B, FIG.2H). Moreover, the ratio of PCr 79 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 to ATP was significantly increased, indicating the greater energy storage in TDLG-CMs (FIG. 2H). Metabolites relate to carnitine biosynthesis, including 6-N-trimethyllysine, deoxycarnitine, and carnitine, were significantly enriched in TDLG-CMs, suggesting larger capacity to transfer fatty acids from the cytoplasm to mitochondria for β-oxidation (FIG.2B).

[0285] Collectively, TDLG-CMs exhibited a metabolic switch from glycolysis to OXPHOS, leading to greater energy reserve, which was possibly supplied directly to consumers such as myosin and sarcoplasmic reticulum ATPases using sophisticated energy delivery system. Moreover, lower glycolytic state contributed to less nucleotide synthesis through the suppression of PPP. Transcriptional maturation in TDLG-CMs

[0286] Next, bulk RNA-seq was performed to elucidate the difference in global transcriptome profile between Ctrl-CMs and TDLG-CMs. PCA showed that PC1 clearly separates TDLG samples from Ctrl samples (FIG.3A).4127 differentially expressed genes (DEGs) were identified between two groups, including 1876 up-regulated genes and 2251 down-regulated genes in TDLG-CMs (FIG.3B). Volcano plots revealed that TDGL treatment improved the expression of maturation related genes in metabolism, electrophysiology (FIG.3B). TDGL upregulated mitochondrial components (TOMM40 and TIMM21), lipid metabolism genes (ACSS1 and PDK4), cytoplasmic muscle type CK (CKM) and sarcomeric mitochondrial CK (CKMT2), along with enhanced estrogen-related receptors (ESRRA and ESRRB) expression, indicating maturation in energetic state. Ion channels or Ca2+handling related genes were highly expressed in TDLG-CMs, including SCN5A, KCNJ2, KCND3 and ATP2A2, in concert with downregulation of HCN4 (FIG.3C). Gene ontology (GO) analysis revealed that upregulated DEGs in TDLG-CMs were significantly enriched for GO terms associated with electrophysiological and contractile function, such as transmembrane transport, ion transport and heart contraction, in addition to metabolism, such as carboxylic acid metabolic process (FIG. 3D, Table 1). In contrast, GO terms for downregulated DEGs were enriched for GO terms associated with extracellular matrix, such as cell adhesion and extracellular matrix organization (FIG.3E, Table.2). Pathway analysis revealed the activation of transmembrane transport and lipid metabolism in TDLG-CMs, whereas ECM production was significantly reduced (FIGs.3F- 3G, Table 3, Table 4). Intriguingly, transcriptional factor (TF) binding site analysis in 80 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 upregulated DEGs identified E12(E2A), ablation of which has been reported to promote differentiation of hESCs to nodal like CMs, as a top TF20, indicating that TDLG treatment potentially suppresses a pacemaker transcriptome signature, consistent with suppression of HCN4, TBX2, TBX18 and GJC1 (FIG.3B, FIG.3H).

[0287] Thus, the findings from bulk RNA-seq analysis showed that TDLG treatment altered the global gene expression profiles of hPSC-CMs to more matured patterns in energy metabolism, electrophysiology and contraction along with the suppression of pacemaker markers.

[0288] Next, scRNA-seq was conducted for identifying matured cell population in metabolism, sarcomere, cell cycle and electrophysiology. Single cell RNA-seq (scRNA-seq) allows us to elucidate the cellular heterogeneity which can be masked in bulk RNA-seq. UMAP analysis revealed clear separation of Ctrl and TDLG cells, identifying 10 clusters expressing TNNT2 in common (FIG.4A-4C). TDLG-CMs is composed of 5 clusters (cluster 0, 1, 5, 7 and 9), highly expressing adult CM-related genes in metabolism (COX6A2, CKM, ESRRA, PPARA, FABP3, and SLC5A4), Ca handling (ATP2A2 and RYR2) and intercalated disc (MYZAP and DES), while Ctrl-CMs consisted of other 5 clusters (cluster 2, 3, 4, 6 and 8), abundantly expressing VIM, an intermediate filament enriched in fetal CMs (FIGs.4D-4E, FIG.5A-5C)21,22. Three clusters (cluster 0, 2 and 7) represented ventricular subtype, expressing MYL2, IRX4 and FHL2 (FIG.4F, FIG.5D). Furthermore, cluster 0, one of those 3 clusters, was characterized by enhanced expression of SCN5A, KCNJ2, and GJA1, indicating electrophysiologically matured population (FIG.4G, FIG.5E). GO term analysis revealed that gene markers for cluster 0 were enriched for GO terms associated with contraction and electrophysiological function, such as striated muscle contraction and cardiac muscle cell action potential involved in contraction (FIG. 4J). Whereas both cluster 6 and 7 represented proliferative CMs, indicated by enriched cell cycle genes (CDK1, CCNA2, and CCNB1), the expression was more prominent in cluster 6 (FIG.4H, FIG.5F). Interestingly, cluster 3 and 4 are characterized by extracellular matrix production (BMP2, TGFB2, COL1A2) (FIG.4I, FIG.5G).

[0289] Taken together, cluster 0, exhibiting wide-ranging mature transcriptional profile in metabolism, myofibril, and electrophysiology, was composed of TDLG-CMs, while clusters which were characterized by fetal CMs related genes, were predominantly comprised of Ctrl- CMs, suggesting the robust effect of TDLG treatment on maturation. 81 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Mature CMs reduce EA burden after transplantation to porcine hearts

[0290] Next, it was asked whether the matured cells could engraft and ameliorate the arrhythmias. To answer this question, 150 million CMs that were treated with or without TDLG were transplanted to porcine hearts. Histological analysis of ssTnI staining revealed that TDLG- CMs engrafted comparably to Ctrl-CMs in graft size at 4weeks post-transplantation (FIG.5A). All subjects exhibited EAs by post-transplant day 4. One of the Ctrl subjects experienced unstable EA with heart rate in excess of 350 bpm, necessitating euthanasia. All other subjects remained clinically stable and survived until the endpoint. Peak arrhythmia burden was significantly reduced in TDLG-CMs recipients at day 7 (FIG.5B). TDLG-CMs recipients also experienced spontaneous resolution of arrhythmia (burden <10% of day) by 2 weeks, whereas Ctrl recipients had a high arrhythmia burden (>35%) at 2 weeks (Fig.5B). These results demonstrate that the matured cells displayed comparable engraftability to immature CMs in porcine hearts and suppressed arrhythmogenicity. References

[0291] The following references are incorporated herein by reference in their entireties. 1 Chong, J. J. et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non- human primate hearts. Nature 510, 273-277 (2014). https: / / doi.org:10.1038 / nature13233 2 Liu, Y. W. et al. Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nat Biotechnol 36, 597-605 (2018). https: / / doi.org:10.1038 / nbt.4162 3 Shiba, Y. et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature 538, 388-391 (2016). https: / / doi.org:10.1038 / nature19815 4 Gerbin, K. A., Yang, X., Murry, C. E. & Coulombe, K. L. Enhanced Electrical Integration of Engineered Human Myocardium via Intramyocardial versus Epicardial Delivery in Infarcted Rat Hearts. PLoS One 10, e0131446 (2015). https: / / doi.org:10.1371 / journal.pone.0131446 5 Shiba, Y. et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489, 322-325 (2012). https: / / doi.org:10.1038 / nature11317 6 Gerbin, K. A., Mitzelfelt, K. A., Guan, X., Martinson, A. M. & Murry, C. E. Delta-1 Functionalized Hydrogel Promotes hESC-Cardiomyocyte Graft Proliferation and Maintains Heart Function Post-Injury. Mol Ther Methods Clin Dev 17, 986-998 (2020). 82 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 https: / / doi.org:10.1016 / j.omtm.2020.04.011 7 Romagnuolo, R. et al. Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias. Stem Cell Reports 12, 967-981 (2019). https: / / doi.org:10.1016 / j.stemcr.2019.04.005 8 Karbassi, E. et al. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol 17, 341-359 (2020). https: / / doi.org:10.1038 / s41569-019- 0331-x 9 Piquereau, J. & Ventura-Clapier, R. Maturation of Cardiac Energy Metabolism During Perinatal Development. Front Physiol 9, 959 (2018). https: / / doi.org:10.3389 / fphys.2018.00959 10 Nakano, H. et al. Glucose inhibits cardiac muscle maturation through nucleotide biosynthesis. Elife 6 (2017). https: / / doi.org:10.7554 / eLife.29330 11 Hoeg-Jensen, T. Review: Glucose-sensitive insulin. Mol Metab 46, 101107 (2021). https: / / doi.org:10.1016 / j.molmet.2020.101107 12 Hu, D. et al. Metabolic Maturation of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Inhibition of HIF1α and LDHA. Circ Res 123, 1066-1079 (2018). https: / / doi.org:10.1161 / CIRCRESAHA.118.313249 13 Mills, R. J. et al. Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proc Natl Acad Sci U S A 114, E8372-E8381 (2017). https: / / doi.org:10.1073 / pnas.1707316114 14 Yang, X. et al. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes- derived from induced pluripotent stem cells. J Mol Cell Cardiol 72, 296-304 (2014). https: / / doi.org:10.1016 / j.yjmcc.2014.04.005 15 Parikh, S. S. et al. Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circ Res 121, 1323-1330 (2017). https: / / doi.org:10.1161 / CIRCRESAHA.117.311920 16 Birket, M. J. et al. Contractile Defect Caused by Mutation in MYBPC3 Revealed under Conditions Optimized for Human PSC-Cardiomyocyte Function. Cell Rep 13, 733-745 (2015). https: / / doi.org:10.1016 / j.celrep.2015.09.025 17 Wang, L. et al. Triiodothyronine and dexamethasone alter potassium channel expression and promote electrophysiological maturation of human-induced pluripotent stem cell-derived cardiomyocytes. J Mol Cell Cardiol 161, 130-138 (2021). 83 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 https: / / doi.org:10.1016 / j.yjmcc.2021.08.005 18 Nakamura, K. et al. Pharmacologic therapy for engraftment arrhythmia induced by transplantation of human cardiomyocytes. Stem Cell Reports 16, 2473-2487 (2021). https: / / doi.org:10.1016 / j.stemcr.2021.08.005 19 Li, X. et al. Lactate metabolism in human health and disease. Signal Transduct Target Ther 7, 305 (2022). https: / / doi.org:10.1038 / s41392-022-01151-3 20 Li, X. et al. E2A ablation enhances proportion of nodal-like cardiomyocytes in cardiac- specific differentiation of human embryonic stem cells. EBioMedicine 71, 103575 (2021). https: / / doi.org:10.1016 / j.ebiom.2021.103575 21 Kim, H. D. Expression of intermediate filament desmin and vimentin in the human fetal heart. Anat Rec 246, 271-278 (1996). https: / / doi.org:10.1002 / (SICI)1097- 0185(199610)246:2<271::AID-AR13>3.0.CO;2-L 22 Zuppinger, C. et al. Characterization of cytoskeleton features and maturation status of cultured human iPSC-derived cardiomyocytes. Eur J Histochem 61, 2763 (2017). https: / / doi.org:10.4081 / ejh.2017.2763

[0292] Table 1: Top 20 GO BP terms for upregulated DEGs in TDLG-CMs 84 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 q-value85 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 GO:0009991 response to extracellular stimulus 6.53E-04 GO:0031669 cellular response to nutrient levels 8.11E-04 s

[0294] Table 3: Top 20 pathways for upregulated DEGs in TDLG-CMs 86 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 q-value ID Pathway FDRq-value ID Pathwa FDR87 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 M3005 Genes encoding collagen proteins 5.21E-18 83105 Pathways in cancer 6.07E-18Example 2: Introduction

[0296] Cardiovascular disease is a leading cause of mortality worldwide. Therapeutic options for severe heart failure are limited, and therefore a new treatment is sought. Regenerative therapy has emerged as a novel approach for mending damaged hearts. Transplantation of hPSC-CMs, such as human embryonic stem cell (hESC)-CMs or human induced pluripotent stem cell (hiPSC)-CMs, has been recognized as promising cell source for remuscularization and functional recovery in failing hearts1-3. Considering clinical application, a few hurdles still remain in safety, especially a transient period of ventricular arrhythmias after transplantation of hPSC-CMs, called engraftment arrhythmias (EAs)1-4. Transplanted CMs are able to engraft and survive long term in damaged hearts. They are electrically connected to host cardiomyocytes, which permits host and graft to beat synchronously for producing greater cardiac output1,5,6. However, hPSC-CMs in vitro lack maturation and exhibit characteristics far from adult working CMs, including spontaneous action potentials, which the electrophysiological studies indicate then propagate to host myocardium and initiate engraftment arrhythmias7. Thus, it is desirable to produce electrically matured hPSC-CMs in vitro2,4.

[0297] Maturation of cardiomyocytes is a complicated process covering various features, including metabolism, cell cycle, structure, electrical function, Ca handling, and contractile function. Multiple approaches have been proposed to mature hPSC-CMs in vitro, such as prolonged culture, biochemical cues, co-culture with non-cardiomyocytes, substrate stiffness and biophysical stimulation such as pacing. These have led to complementary subsets of cardiac maturation, suggesting maturation is a complex process with multiple control points7,8. In the perinatal stage, CMs experience alteration in metabolic hormones, energy substrates, and oxygen 88 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 supply, initiating metabolic reprogramming from glycolysis to FA oxidation, which is followed by cell cycle exit and hypertrophic growth. Given the knowledge in heart development, manipulation of adult-like metabolism with metabolic modulators enables hPSC-CMs to improve the electrophysiological function. The identified triiodothyronine (T3) as the first metabolic factor to enhance maturation in hPSC-CMs, improving cell size, sarcomere length, contractile force, Ca handling, and mitochondrial respiration9. Dexamethasone (Dex), a glucocorticoid metabolic hormone, promoted T-tube formation and improved Ca kinetics, when added in combination with T310. Manipulation of energy source, by switching from glucose (Glu) to FA, improves morphology, Ca kinetics, and electrophysiological function in addition to metabolism11-14. While long term culture in FA enriched media induces cell damage, called lipotoxicity, which is mitigated by galactose (Gal) supplementation14. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, is activated in response to the increase in cellular AMP or ADP / ATP ratio and regulate the metabolism in glucose, lipids, and proteins. AICAR, an activator of AMPK signaling, enhanced transcriptional maturation in expression of metabolic, structural, and electrophysiological genes15. While each metabolic modulator affects maturation differently, they have positive effect on electrophysiology / Ca handling in common. Taken together, metabolic reprogramming plays a key role as a central driver for this complexed maturation process and combination of metabolic modulators potentially accelerate electrical maturation to higher extent sufficient to eliminate EAs after transplantation.

[0298] In this study, an optimal combination of 3 major metabolic elements was investigated, metabolic hormones (T3 and Dex), energy source (Glu, Gal, and FA) and energy sensing (AMPK) for electrical maturation, by assessing ion channel gene expression and electrophysiological phenotype under systematical modulation of the concentration of each factor. It was demonstrated that one week’s culture in this optimized media not only increases mitochondrial biogenesis and fatty acid oxidation, it also induces matured properties in cell cycle, global transcriptome, electrophysiology, and Ca kinetics. Critically, the matured cells retain the ability to engraft in the adult heart. Thus, metabolic regulators can be optimized to reprogram hPSC-CM toward a more adult-like phenotype. This may be useful in preventing complications related to immaturity, such as engraftment arrhythmias. 89 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Results Establishing the Screen Design

[0299] Although hPSC-CMs are immature in vitro, once transplanted into rodent or non-human primate hearts they mature close to adult cardiomyocytes transcriptionally and morphologically2,16. Following in vivo maturation, EA burden wanes over a time course of several weeks post-transplantation. A recently established RNAseq dataset (GEO: GSE190758) was used to document transcriptional maturation of hESC-CMs in the in vivo rat heart using laser-capture microdissection followed by bulk RNAseq, comparing this also to in vitro maturation over one year in monolayer culture (FIG.12A)17. As example of established markers of maturation, FIG.7A demonstrates up-regulation of myofibril (TNNI3 and MYL2) and metabolic transcripts (PPARGC1A and CPT1B) (FIG.7A), with in vivo rates substantially exceeding in vitro rates. Next, dynamics of ion channel / transporter genes were studied as electrical maturation markers to use in a subsequent screening process (FIG.7A). In vivo maturation was associated with substantial upregulation of transcripts for SCN5A (encodes Nav1.5, a fast Na channel), KCNJ2 (encodes Kir2.1 channel, an inward rectifier K channel), CACNA1C (encodes Cav1.2, α1C subunit of voltage-dependent L type Ca channel), ATP2A2 (encodes SR Ca ATPase), and RYR2 (encodes the ryanodine receptor in SR). Conversely, transcripts associated with pacemaking were suppressed, including HCN4 (encodes a member of the hyperpolarization-activated cyclic nucleotide-gated Na channels, important for pacemaking) and CACNA1H (encodes Cav3.2,α1H subunit of voltage-dependent T type Ca channel, important in pacemaking) during the maturation in vivo.

[0300] Next was to systematically screen conditions to induce ion channel gene expression toward a more adult-like phenotype. To minimize variability, one large batch of cardiomyocytes derived from RUES2 hESC line was generated, frozen at differentiation day 10, and used for all studies. After thawing the hESC-CMs were cultured in RPMI / B27 media from for 4 days, and then switched them to glucose-free media supplemented with lactate from day 14 to day 18 to eliminate non-cardiomyocytes. The lactate-purified cardiomyocytes then were replated on each well format in same cell density and subjected to the maturation intervention from day 20 through day 3418(FIG.7B). It is noted that RPMI includes the amino acids glutamine, arginine, asparagine, cystine, leucine, isoleucine, lysine, serine, aspartic acid, glutamic acid, hydroxyproline, proline, threonine, tyrosine, valine, histidine, methionine, phenylalanine, 90 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 glycine, tryptophan and reduced glutathione, as well as i-inositol, choline chloride, para- aminobenzoic acid, folic acid, nicotinamide, pyridoxine hydrochloride, thyamine hydrochloride, calcium pantothenate, biotin, riboflavin and cyanocobalamin. RPMI was originally described in Moore et al., JAMA 199 (8): 519-524 (1967). Optimizing carbohydrate sources

[0301] The screening was broken into 2 sequential rounds (FIG.7B). Round 1 was a 4x4 comparison of glucose vs. galactose concentrations, using RPMI / B27 supplemented with T3100 nM and dexamethasone 1 µM as a basal media as optimized by Parikh et al10. The optimal concentration was selected based on expression of ion channel or Ca handling gene as well as cell viability and used as a basal condition for next round. Round 2 was fatty acid vs. AICAR concentrations. Based on expression of key electrophysiological genes, 16 conditions from the second round were narrowed down to 3 conditions, and these conditions moved on to phenotypic analysis with multi-electrode array (MEA) system to identify an optimal concentration of each modulators to enhance electrical maturation. As high glucose RPMI (glucose 11mM and galactose 0mM) with B27 is a commonly used culture media for hPSC-CMs, glucose 11mM without galactose supplementation is defined as a control condition for round 1.

[0302] Compared to control, glucose deprivation significantly upregulated the expression of KCNJ2, and RYR2, however Ca channel genes, such as CACNA1C and CACNA1H were not affected by glucose reduction (FIG.7C). Galactose supplementation (0.5 mM) increased the expression of SCN5A when glucose was removed (FIG.7C). HCN4 expression was suppressed in no glucose and no galactose condition but notably, complete absence of both carbohydrates negatively affected ATP2A2 and RYR2 expression (FIG.7C, FIG.12B). Glucose removal significantly reduced the cell density, which was rescued by galactose supplementation. Based on the expression of SCN5A, KCNJ2, and RYR2 and also cell density after treatment, no glucose supplemented with galactose 0.5 mM was selected as a basal condition for next round screening (FIG.7E). Optimizing FA and AICAR

[0303] Following round 1 screening, full factorial optimization of fatty acid and AICAR was performed. Compared to control (FA 0 μM and AICAR 0 mM), high concentration of AICAR (1 91 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 or 2 mM) upregulated the expression of SCN5A, ATP2A2 and RYR2. HCN4 expression was not suppressed by any combination of AICAR or FA treatment (FIG.8A). KCNJ2 was elevated by AICAR 1 mM supplemented with high FA (230 or 460 μM), and also by either FA 460 μM or AICAR 0.3 mM (FIG.8A). There was a negative correlation between AICAR concentration and cell density (FIG.8B, FIG.13A). On the other hand, the addition of FA rescued the low cell count induced by AICAR treatment (FIG.8B, FIG.13A). Based on the low cell density, high AICAR with no or low FA conditions are excluded from candidates. The expression of electrophysiological genes, especially KCNJ2, SCN5A, and ATP2A2, led us to select 3 conditions for electrophysiological screening (FA 0μM / AICAR 0.3mM, FA 460μM / AICAR 0mM, FA 460μM / AICAR 1mM) (FIG.8C). Maturation media improves electrophysiological features

[0304] To identify an optimal media based on electrophysiological phenotype, 4 conditions were set up, including the above 3 candidates, using the optimized media from round 1 as a base media control (glucose 0 mM / galactose 0.5 mM; no FA or AICAR). Cultures were screened on MEA plates. Compared to control, FA 460 μM / AICAR 1 mM reduced beat rate by 32.6-42.5% increased corrected field potential duration (FPDc) by 74.1-154.9%, and increased conduction velocity by 25.5-71.2% in all 3 batches at 1 week (FIG.8D). Surprisingly, the effect was diminished after 2 weeks of treatment (FIG.8E, FIG.9F). Since hPSC-CMs exhibited the most matured electrical phenotype when cultured in glucose 0 mM / galactose 0.5mM / FA 460 μM / AICAR 1 mM / T3 / Dex / RPMI / B27 for 1 week, this was defined as maturation media (MM).

[0305] Next, another series of MEA experiments was carried out to confirm if MM treated CMs exhibit more mature features than hPSC-CMs grown in regular cardiac media (high glucose RMPI / B27; Ctrl-CMs). In agreement with the hypothesis, MM-CMs displayed a 23.5-42.5% slower beat rate (significant in 3 out of 3 batches), a 30.1-79.3% longer FPDc (significant in 3 out of 3 batches) and a 52.2-289.3% faster conduction velocity (significant in 3 out of 3 batches) (FIG.13B). MM induced higher total SCN5A expression and isoform switch from fetal to adult type (FIG.9A). Further, MM, promoted protein expression of Nav1.5 and CX43, which likely contributed to the faster conduction velocity in the matured cells (FIG.13B, FIG.14A). MM upregulated the expression of KCNJ2 in concert with suppression of HCN4, which likely underlies the reduced automaticity in the matured cells (FIG.9A, FIG.13B). Taken together, an 92 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 optimal media was identified and treatment period for electrical maturation, evidenced by ion channel gene expression and electrophysiological activities. MM-CMs promote maturation in metabolism, cell cycle, morphology, and Ca handling

[0306] In addition to electrophysiological properties, it was investigated whether MM activated maturation programs more broadly. MM upregulated PPARGC1A (PGC1-α) expression, an activator of mitochondrial biogenesis, and this was associated with a 1.5-fold increase in the ratio of mitochondrial DNA to nuclear DNA (p<0.05; FIG.9A, FIG.14H). Transmission electron microscope (TEM) identified the increase in mitochondrial volume from 6.4% to 12.3% with MM (p<0.05; FIG.9B). To assess whether these structural features were accompanied by increased aerobic capacity, metabolic flux was measured in a Seahorse instrument. Cardiomyocytes cultured in MM had higher basal respiration (4.6-fold), maximal respiration (3.2-fold), spare capacity (3.0-fold), and ATP production (6.0-fold) in mitochondrial stress test (FIGs.14J-14K). Along with mitochondrial maturation, CD36, a transporter required for FA uptake, was upregulated by MM (FIG.9A). To assess the capacity of FA utilization, oxygen consumption rate (OCR) was measured using Seahorse flux analyzer in the presence of palmitate-BSA, BSA-only, or BSA with etomoxir, an inhibitor of an inhibitor of FAO through irreversible inhibition of CPT1. This showed that MM-CMs had a 2.4-fold greater ability to oxidize palmitate compared to Ctrl-CMs, indicating enhanced usage of exogenous FA for oxidation (FIG.9C). Intracellular lipid storage was evaluated by Nile red stain using a flow cytometer. MM-CMs displayed a 84.1% higher mean fluorescent intensity of Nile red, suggesting efficient FA uptake, compared to Ctrl-CMs (FIG.14G). To test for the ability to metabolize endogenous FAs, CMs in FA-free medium (BSA-only) were studied and measured maximal respiration in the absence and presence of etomoxir. The etomoxir-sensitive maximal respiration and spare capacity were 2.2-fold and 2.7-fold greater, respectively, in MM-CMs compared to controls, while absolute OCR rates were similar with etomoxir treatment. This indicates larger accumulation and utilization of endogenous FA in MM-CMs (FIGs.14E-14F). Taken together, MM-CMs uptake and store FA more efficiently with greater capacity of FA oxidation for energy production.

[0307] PSC-cardiomyocytes have much larger stores of glycogen, a stored form of polymeric glucose, than adult cardiomyocytes. Glycogen content was measured histochemically in MM 93 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 and control PSC-CMs by performing periodic acid-Schiff (PAS) stains in the presence or absence of amylase to digest glycogen. In control cells, 23±2.9% of the area was stained with amylase- sensitive PAS. The matured cells exhibited a 98% reduction in glycogen content, to 0.4% of their surface area (p<0.05), reflecting the removal of glucose in MM (FIG.14H). Despite the minimal glycogen stores, MM-CMs exhibited 2.3-fold higher maximal extracellular acidification rate (ECAR; p<0.01), indicating the capacity to activate anaerobic glycolysis in the presence of exogenous glucose (FIGs.14L-14M).

[0308] Next, the effects of MM were explored on Ca2+dynamics. Since MM increased the expression of both RYR2, which is crucial for Ca2+-induced Ca2+release from SR, and ATP2A2, which takes Ca2+back up into the SR, it was hypothesized that MM would increase the magnitude and rate of Ca2+cycling. In support of this hypothesis, Ca2+transient analysis in single cells revealed that MM-CMs exhibited 24.0% higher amplitude (Fmax / F0) and 17.1% shorter time to peak. Moreover, the MM-CMs showed a faster 50% (reduction by 29.1%) and 90% decay (reduction by 19.6%), indicating enhanced Ca2+kinetics (FIG.9G).

[0309] MM treatment promoted fetal type to adult type isoform switching of multiple myofilament in mRNAs or proteins. For example, there was a ~4-fold upregulation in cTnI mRNA and protein levels, accompanied by a nearly 10-fold increase in the ratio of adult:fetal TTN transcripts (N2B:N2BA; FIG.9A, FIG.9D, FIG.14C). A notable exception was in the cardiac myosins, which showed increased MHC6:MHC7 ratios, opposite to the developmental trajectory. It was previously shown that this results from T3 treatment, which directly activates transcription of MYH69. TEM revealed that MM-CMs possessed organized sarcomere structure with clear A bands, I bands, and Z discs, while Ctrl-CMs displayed disarrayed striation (FIG. 14I). The difference in cell size was evaluated with Forward Scatter (FSC) using flow cytometry. FSC intensity, which corresponds to cell diameter, was increased 10.6% by MM treatment (p<0.05; FIG.9E).

[0310] Finally, the hypothesis was tested that MM would promote cell cycle withdrawal, an important step in postnatal maturation. To study the cell cycle of hESC-CMs, flow cytometer was performed after staining with EdU for S phase and FxCycle for DNA content. At one week the percentage of control cells in S phase was 8.8%, which was reduced to 2.7% in MM. At two weeks there was a further reduction in S phase cells, from 5.2% in controls to 0.6% in MM. MM 94 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 did not affect the percentage of cells in G1 or G2 / M phases. These data indicate that MM induces a significant cell cycle arrest at G1 / S transition.

[0311] Taken together, these data demonstrate that, in addition to promoting electrical maturation, MM increased mitochondrial volume, increased cellular respiratory capacity and FA oxidation capacity, reduced glycogen stores, enhanced Ca2+handling, increased myofibril isoform switching, sarcomere structure and cell size, and reduced cell cycle activity. All of these parameters indicate a more adult-like phenotype. Transcriptional maturation in MM-CMs

[0312] To understand the global change in cardiomyocyte transcriptome during metabolic reprogramming, RNA-seq analysis was performed on Ctrl- and MM-CMs. Principal component analysis clustered in two clearly separated groups, Ctrl-CMs and MM CMs, indicating reproducibility and robustness of MM treatment (FIG.10A). Heatmap showed higher expression of mitochondrial biogenesis genes, resulting in enriched mitochondrial component (FIG.10B, FIG.15A). Metabolically, FAO and TCA cycle genes were globally upregulated in MM-CMs (FIG.10B, FIG.15B). Ion transporters which associated with action potential or Ca handling were highly expressed in MM-CMs, including SCN5A, KCNJ2, KCNJ12, RYR2 and PLN, in concert with suppression of HCN4 (FIG.10B, FIG.15C). In addition, GJA1 (encoding connexin43), which is predominant in ventricular CMs, was upregulated in MM-CMs, while GJA5 (connexin 40), which is expressed not in ventricular CMs but in atrial CMs, and GJC1 (connexin 45), which are primary gap junction genes in pacemaker cells or conduction system, were downregulated (FIG.10B, FIG.15A-15B). AP-1 targeted genes such as JUN and ATF3 were significantly upregulated in MM, which were recently shown to induce postnatal cardiac maturation in metabolism and binucleation, was upregulated in MM (FIG.15C)19. Gene ontology (GO) analysis was conducted based on the list of differentially expressed genes (DEGs). GO terms for upregulated DEGs in MM-CMs were related to electrophysiology, such as ion transmembrane transport, as well as metabolism, such as generation of precursor metabolites and energy, and sarcomere such as muscle structure development and striated muscle contraction (FIGs.10C-10D). On the other hand, GO terms for downregulated DEGs in MM-CMs included extracellular matrix, neuron, and vascular development (FIGs.10C-10D). Gene Set Enrichment Analysis (GSEA) revealed that MM upregulated genes which correlate with resting membrane 95 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 potential, gap junction and muscle contraction, while WNT signaling, which has been reported to reduce the maturity of hPSC-CMs, and ECM related genes, were suppressed (FIG.15B)20. Transcriptional factor (TF) binding site analysis in upregulated DEGs showed that ERR1, adult isoform of estrogen-related receptors which regulate metabolic homeostasis and necessary for postnatal maturation, was ranked as a top TF, suggesting that metabolic alteration plays a central role in wide-ranged maturation process in MM-CMs (FIG.10E). On the other hand, LEF1, which has been shown to induce proliferation and delay maturation process in hPSC-CMs, was included in top5 TFs in downregulated DEGs.20

[0313] Collectively, transcriptome analysis revealed that MM broadly reprogrammed cardiomyocyte gene expression to include not only metabolic networks, but also including ion channels / transporters and myofibril genes, in concert with suppression of pacemaker markers. Engraftment and survival of MM-CMs in adult mouse hearts

[0314] Since adult cardiomyocytes do not survive the stress of cell transplantation21, it was asked whether MM might have an adverse impact on iPSC-CM engraftability. Ctrl-CMs or MM-CMs were transplanted into the uninjured hearts of adult NOD-SCID mice (FIG.11A). Engraftment was quantified at 1week post-transplantation with Alu based qPCR (Alu-qPCR), a method allows to quantify the number of human genomes in a background of mouse genomic DNA. The 1w-MM-CMs showed human genome copy numbers to Ctrl-CMs, indicating that MM had no adverse impact on engraftment in the adult mouse heart (FIG.11B). Next, the survival and characteristics of engrafted cells were assessed histologically at 2 weeks post- transplantation. Engrafted human CMs, positive for ssTnI, were readily identified in both MM- CM and Ctrl-CM groups, confirming survival for at least 2 weeks in host mouse hearts. Metabolically, glycogen was stored in MM-CMs as well as in Ctrl-CMs, which is different from the phenotype confirmed in vitro condition, indicating the capability to adapt to nutrition supply, such as capacity to increase glucose utilization (FIG.11C, FIG.14L-14M). Collectively, MM- CMs survived transplantation and engrafted equivalently to Ctrl-CMs, indicating that the maturation regimen is compatible with remuscularization therapy. References

[0315] The following references are incorporated herein by reference in their entireties. 96 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 1 Chong, J. J. et al. Human embryonic-stem-cell-derived cardiomyocytes regenerate non- human primate hearts. Nature 510, 273-277 (2014). doi.org:10.1038 / nature13233 2 Liu, Y. W. et al. Human embryonic stem cell-derived cardiomyocytes restore function in infarcted hearts of non-human primates. Nat Biotechnol 36, 597-605 (2018). doi.org:10.1038 / nbt.4162 3 Shiba, Y. et al. Allogeneic transplantation of iPS cell-derived cardiomyocytes regenerates primate hearts. Nature 538, 388-391 (2016). doi.org:10.1038 / nature19815 4 Romagnuolo, R. et al. Human Embryonic Stem Cell-Derived Cardiomyocytes Regenerate the Infarcted Pig Heart but Induce Ventricular Tachyarrhythmias. Stem Cell Reports 12, 967-981 (2019). doi.org:10.1016 / j.stemcr.2019.04.005 5 Gerbin, K. A., Yang, X., Murry, C. E. & Coulombe, K. L. Enhanced Electrical Integration of Engineered Human Myocardium via Intramyocardial versus Epicardial Delivery in Infarcted Rat Hearts. PLoS One 10, e0131446 (2015). doi.org:10.1371 / journal.pone.0131446 6 Shiba, Y. et al. Human ES-cell-derived cardiomyocytes electrically couple and suppress arrhythmias in injured hearts. Nature 489, 322-325 (2012). doi.org:10.1038 / nature11317 7 Karbassi, E. et al. Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine. Nat Rev Cardiol 17, 341-359 (2020). doi.org:10.1038 / s41569-019- 0331-x 8 Guo, Y. & Pu, W. T. Cardiomyocyte Maturation: New Phase in Development. Circ Res 126, 1086-1106 (2020). doi.org:10.1161 / CIRCRESAHA.119.315862 9 Yang, X. et al. Tri-iodo-l-thyronine promotes the maturation of human cardiomyocytes- derived from induced pluripotent stem cells. J Mol Cell Cardiol 72, 296-304 (2014). doi.org:10.1016 / j.yjmcc.2014.04.005 10 Parikh, S. S. et al. Thyroid and Glucocorticoid Hormones Promote Functional T-Tubule Development in Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes. Circ Res 121, 1323-1330 (2017). doi.org:10.1161 / CIRCRESAHA.117.311920 11 Mills, R. J. et al. Functional screening in human cardiac organoids reveals a metabolic mechanism for cardiomyocyte cell cycle arrest. Proc Natl Acad Sci U S A 114, E8372-E8381 (2017). doi.org:10.1073 / pnas.1707316114 12 Yang, X. et al. Fatty Acids Enhance the Maturation of Cardiomyocytes Derived from Human Pluripotent Stem Cells. Stem Cell Reports 13, 657-668 (2019). doi.org:10.1016 / j.stemcr.2019.08.013 13 Feyen, D. A. M. et al. Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes. Cell Rep 32, 107925 (2020). doi.org:10.1016 / j.celrep.2020.107925 14 Correia, C. et al. Distinct carbon sources affect structural and functional maturation of cardiomyocytes derived from human pluripotent stem cells. Sci Rep 7, 8590 (2017). doi.org:10.1038 / s41598-017-08713-4 15 Ye, L. et al. Activation of AMPK Promotes Maturation of Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells. Front Cell Dev Biol 9, 644667 (2021). doi.org:10.3389 / fcell.2021.644667 16 Kadota, S., Pabon, L., Reinecke, H. & Murry, C. E. In Vivo Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Neonatal and Adult Rat Hearts. Stem Cell Reports 8, 278-289 (2017). doi.org:10.1016 / j.stemcr.2016.10.009 17 Marchiano, S. et al. Gene editing to prevent ventricular arrhythmias associated with cardiomyocyte cell therapy. Cell Stem Cell 30, 741 (2023). doi.org:10.1016 / j.stem.2023.04.010 97 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 18 Tohyama, S. et al. Distinct metabolic flow enables large-scale purification of mouse and human pluripotent stem cell-derived cardiomyocytes. Cell Stem Cell 12, 127-137 (2013). doi.org:10.1016 / j.stem.2012.09.013 19 Zhang, H. et al. AP-1 activation mediates postnatal cardiomyocyte maturation. Cardiovasc Res (2022). doi.org:10.1093 / cvr / cvac088 20 Buikema, J. W. et al. Wnt Activation and Reduced Cell-Cell Contact Synergistically Induce Massive Expansion of Functional Human iPSC-Derived Cardiomyocytes. Cell Stem Cell 27, 50-63.e55 (2020). doi.org:10.1016 / j.stem.2020.06.001 21 Reinecke, H., Zhang, M., Bartosek, T. & Murry, C. E. Survival, integration, and differentiation of cardiomyocyte grafts: a study in normal and injured rat hearts. Circulation 100, 193-202 (1999). doi.org:10.1161 / 01.cir.100.2.193 Example 3:

[0316] Tables 5 and 6: Working Range for Each Modulator Met Lo Rationale Hi Rationale(differ Sing Refere Range in Refere abol we (differentially gh entially le nce combinat nce D t 9 4 6 5 5 5 7 7 9 44870-6522-3379.4Attorney Docket No.034186-000102WOP2 Fatt 11 upregulation of 46 Downregulation 67- 315646 100- 28819 y 5μ RYR2 at 115μM 0μ of HCN4 at 115 45 200μM 274 7 7 5 5 6 5Rationale diff t e D t 4 44870-6522-3379.4Attorney Docket No.034186-000102WOP2 n of HCN4 0 2 9 4 4 4 0 2 9 4Example 4: Enhanced Electrophysiological Maturation of Human Pluripotent Stem Cell Derived Cardiomyocytes by a Combination of Metabolic Modulators

[0317] Described herein is the combination of three key metabolic elements for electrical maturation of cardiomyocytes: metabolic hormones (T3 and Dex), energy sources (Glu, Gal, and FA), and energy sensing (AMPK). Through the systematic modulation of each factor's concentration, the expression of ion channel / transporter genes and the electrophysiological phenotype were assessed. One week of culture in the disclosed medium not only enhanced mitochondrial biogenesis, fatty acid oxidation, and the overall metabolome, but it also induced characteristics of maturation in terms of the cell cycle, cell size, the transcriptome, electrophysiology, and Ca2+kinetics. Each factor contributed uniquely to the maturation process, and synergistic effects were strongly evident when the factors were combined. The matured cells maintained the ability to engraft into the adult heart, indicating that metabolic regulators can 100 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 effectively reprogram hPSC-CMs towards a more adult-like phenotype. This maturation can help prevent immaturity-related complications such as engraftment arrhythmias. Results

[0318] Establishing the experimental design for the screening of mature cardiomyocytes.

[0319] Although hPSC-CMs exhibit immaturity in vitro, they undergo significant maturation, both transcriptionally and morphologically, when transplanted into rodent or non-human primate hearts, approaching the state of adult cardiomyocytes. Following in vivo maturation, engraftment arrhythmia (EA) burden wanes over a time course of several weeks post-transplantation. To evaluate combinations of metabolic modulators for the electrical maturation of cardiomyocytes, marker genes were identified for use in screening conditions. For this purpose, an analysis was performed on an RNA-seq dataset (GEO: GSE190758), which documents the transcriptional maturation of hPSC-CMs following transplantation into rat hearts; see e.g., Kadota et al., Stem Cell Reports 8, 278-289 (2017). This in vivo maturation, achieved using laser-capture microdissection followed by bulk RNA sequencing, was compared to the in vitro maturation of hPSC-CMs over one year in monolayer culture (see e.g., Fig.29A). As markers of maturation, Fig.23A shows the upregulation of myofibril (TNNI3 and MYL2) and metabolic genes (PPARGC1A and CPT1B), with in vivo rates of expression surpassing those of in vitro cultured cells. The dynamics of ion channel / transporter genes, which are key determinants of electrical maturation, were also examined (see e.g., Fig.23A). In vivo maturation correlated with a marked increase in transcripts for SCN5A (Nav1.5, a fast sodium channel), KCNJ2 (Kir2.1 channel, an inward rectifier potassium channel), CACNA1C (Cav1.2, the α1C subunit of the voltage- dependent L type calcium channel), ATP2A2 (SR calcium ATPase), and RYR2 (ryanodine receptor in SR). In contrast, transcripts associated with pacemaking, including HCN4 (a hyperpolarization-activated cyclic nucleotide-gated sodium channel critical for pacemaking) and CACNA1H (Cav3.2, the α1H subunit of the voltage-dependent T type calcium channel important for pacemaking), were suppressed during in vivo maturation.

[0320] With this information, a systematic screening was designed and implemented to induce more adult-like ion channel gene expression in cardiomyocytes. A large batch of cardiomyocytes from the RUES2 human embryonic stem cell (hESC) line was prepared, frozen on differentiation day 10, and used for all transcriptional screening studies. Upon thawing, the hESC-CMs were cultured in RPMI / B27 media for 4 days; then, the cells were cultured in glucose-free media 101 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 supplemented with lactate from days 14 to 18 to selectively eliminate non-cardiomyocytes. After this lactate purification, the cardiomyocytes were replated at a consistent density in each well and subjected to maturation interventions from days 20 to 34 (see e.g., Fig.23B).

[0321] The screening was divided into two sequential rounds (see e.g., Fig.23B). In Round 1, a 4 × 4 comparison of glucose (Glu) versus galactose (Gal) concentrations was conducted, using RPMI / B27 media supplemented with 100 nM triiodothyronine (T3) and 1 µM dexamethasone (Dex).

[0322] A specific concentration was determined based on the expression of ion channel or Ca2+handling genes, alongside cell viability, and this concentration was used as the baseline for the next round. Round 2 focused on comparing fatty acids (FA) versus AICAR concentrations. Then, based on the expression of key electrophysiological genes, the 16 conditions were narrowed down to three. These conditions were further analyzed using a multi-electrode array (MEA) system to determine the concentrations of each modulator in the medium for enhancing electrical maturation.

[0323] Carbohydrate sources

[0324] Relative to the control condition—high-glucose RPMI (11 mM Glu and 0 mM Gal) with B27, a culture medium for hPSC-CMs—glucose deprivation resulted in the upregulation of KCNJ2 and RYR2 expression, although Ca2+channel genes like CACNA1C and CACNA1H were unaffected by the reduction in glucose (see e.g., Fig.23C). Gal supplementation (0.5 mM) in the absence of glucose resulted in the elevation of SCN5A expression (see e.g., Fig.23C). The absence of both glucose and galactose suppressed HCN4 expression but, this complete carbohydrate deprivation adversely impacted the expression of ATP2A2 and RYR2 (see e.g., Fig. 23D, Fig.29B). Removing glucose significantly decreased cell density, which was ameliorated by Gal supplementation (see e.g., Fig.23D). Considering the expression levels of SCN5A, KCNJ2, and RYR2, as well as cell density post-treatment, a no-Glu condition supplemented with 0.5 mM Gal was selected as the baseline for the subsequent round of screening (see e.g., Fig. 23E).

[0325] FA and AICAR

[0326] After completing the first round of screening, full factorial testing of FA and de ribonucleoside (AICAR) was undertaken. When compared to the control condition (0 μM FA / 0 mM AICAR), higher concentrations of AICAR (1 mM or 2 mM) resulted in the significant 102 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 upregulation of SCN5A, ATP2A2, and RYR2 expression; however, HCN4 expression remained unaffected by any combination of AICAR or FA treatment (see e.g., Fig.24A). KCNJ2 expression was enhanced by 1 mM AICAR in combination with higher levels of FA (230 μM or 460 μM), as well as by either 460 μM FA or 0.3 mM AICAR alone (see e.g., Fig.24A). A negative correlation was observed between AICAR concentration and cell density (see e.g., Fig. 24B, Fig.30A). Conversely, the addition of FA counteracted the reduction in cell count induced by AICAR (see e.g., Fig.24B, Fig.30A). Due to the low cell density, conditions with high AICAR and no or low FA were not considered further. The expression profiles of key electrophysiological genes, particularly SCN5A, KCNJ2, and ATP2A2, guided selection of three conditions for further electrophysiological screening: 0μM FA / 0.3 mM AICAR, 460 μM FA / 0 mM AICAR, and 460 μM FA / 1 mM AICAR (see e.g., Fig.24C).

[0327] Maturation media improves electrophysiological features.

[0328] To determine the media based on electrophysiological phenotype, four conditions were tested, including the three candidate conditions identified earlier, using the media from Round 1 as a baseline control (0 mM Glu / 0.5 mM Gal; no FA or AICAR). These cultures were analyzed on MEA plates. Relative to the control condition, the 460 μM FA / 1 mM AICAR condition resulted in a 32.6% to 42.5% reduction in beat rate, a 74.1% to 154.9% increase in corrected field potential duration (FPDc), and a 25.5% to 71.2% increase in conduction velocity across all three batches after one week (see e.g., Fig.24D). This effect diminished after two weeks of treatment (see e.g., Fig.24E, Fig.25F). Given that hPSC-CMs exhibited the most mature electrophysiological phenotype under the condition 0 mM Glu / 0.5mM Gal / 460 μM FA / 1 mM AICAR / 100 nM T3 / 1 µM Dex / RPMI / B27 for one week, this condition is herein designated as “maturation media” (MM).

[0329] Next, another series of MEA experiments was conducted to compare MM-treated CMs (“MM-CMs”) with control hPSC-CMs grown in standard cardiac media (high-glucose RPMI / B27; “Ctrl-CMs”). Consistent with the hypothesis, MM-CMs evidenced a 23.5% to 42.5% slower beat rate (significant across all three batches), a 30.1% to 79.3% longer FPDc (significant across all three batches), and a 52.2% to 289.3% faster conduction velocity (significant across all three batches) (see e.g., Fig.30B). MM treatment led to an increase in total SCN5A expression and an isoform switch from fetal to adult types (see e.g., Fig.25A). Additionally, MM enhanced the protein expression of Nav1.5 and Cx43, contributing to the faster conduction velocity in 103 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 matured cells (see e.g., Fig.30B, Fig.31A-31B). MM also upregulated KCNJ2 expression and suppressed the expression of HCN4, contributing to reduced automaticity in matured cells (see e.g., Fig.25A, Fig.30B). Taken together, the disclosed media and treatment duration can be used for electrical maturation of cardiomyocytes, as evidenced by changes in ion channel gene expression and electrophysiological activity.

[0330] MM-CMs promote maturation in metabolism, cell cycle, morphology, and Ca2+handling.

[0331] In addition to improving electrophysiological properties, MM's broader impact on maturation programs was investigated. MM upregulated PPARGC1A (PGC1-α), a key activator of mitochondrial biogenesis, leading to a 1.5-fold increase in the mitochondrial to nuclear DNA ratio (p < 0.05; see e.g., Fig.25A, Fig.31H). Transmission electron microscopy (TEM) showed an increase in mitochondrial volume from 6.4% to 12.3% (p < 0.05; see e.g., Fig.25B). To determine if these structural changes corresponded with enhanced aerobic capacity, metabolic flux was measured with a Seahorse instrument. In the mitochondrial stress test, MM-CMs exhibited significantly higher basal respiration (4.6-fold), maximal respiration (3.2-fold), spare capacity (3.0-fold), and ATP production (6.0-fold) (see e.g., Fig.31J–31K). Furthermore, MM upregulated the expression of CD36, which codes for a crucial transporter in fatty acid (FA) uptake and is a recognized marker of maturation (see e.g., Fig.25A).

[0332] Using the Seahorse flux analyzer, the cells’ capacity for FA utilization was assessed by measuring the oxygen consumption rate (OCR) in the presence of palmitate-BSA, BSA-only, or BSA with etomoxir, an inhibitor of CPT1 that promotes fatty acid oxidation (FAO). The results showed that MM-CMs had a 2.4-fold greater ability to oxidize palmitate compared to Ctrl-CMs, indicating an enhanced use of exogenous FAs (see e.g., Fig.25C). Intracellular lipid storage, evaluated using Nile red staining and flow cytometry, was 84.1% higher in MM-CMs, indicating an efficient uptake of FA (see e.g., Fig.31G). Additionally, in FA-free medium (BSA-only), MM-CMs showed 2.2-fold and 2.7-fold increases in etomoxir-sensitive maximal respiration and spare capacity, respectively. The absolute OCR rates remained similar with etomoxir (“Etom”) treatment, indicating a higher accumulation and utilization of endogenous FAs in MM-CMs (see e.g., Fig.31E, 31F). Overall, MM-CMs demonstrate more efficient uptake and storage of FA, alongside an enhanced capacity for FA oxidation, contributing to their energy production. 104 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0333] Pluripotent stem cell-derived cardiomyocytes are known to have substantially larger glycogen stores, a form of stored polymeric glucose, compared to adult cardiomyocytes. Thus, to evaluate glycogen content, histochemical analyses were performed using periodic acid-Schiff (PAS) staining in Ctrl-CMs and MM-CMs, both with and without amylase to digest glycogen. Between Ctrl-CMs and MM-CMs, a significant difference was noted in the area stained with amylase-sensitive PAS, which was quantified using transmission electron microscopy (TEM) (see e.g., Fig.31I, Fig.31P). In control cells, 23% ± 2.9% of the surface area was occupied by glycogen; conversely, MM-treated cells displayed a dramatic 98% reduction in glycogen content: only 0.4% of their surface area was occupied by glycogen (p < 0.05), thereby mirroring the effect of glucose removal in MM (see e.g., Fig.31I). Despite these minimal glycogen stores, MM-CMs showed a 1.4-fold increase in maximal extracellular acidification rate (ECAR; p < 0.01) during a Seahorse glycolysis stress test. This indicates an enhanced capacity for activating anaerobic glycolysis in the presence of exogenous glucose (see e.g., Fig.31L-31M).

[0334] Next, the impact of MM on Ca2+dynamics was examined. Given that MM upregulated the expression of RYR2, which is crucial for Ca2+-induced Ca2+release from the sarcoplasmic reticulum (SR), and ATP2A2, which is responsible for Ca2+reuptake into the SR, it was hypothesized that MM enhances both the magnitude and rate of Ca2+cycling. Supporting this hypothesis, Ca2+transient analysis in individual cells demonstrated that MM-CMs display a 24.0% increase in amplitude (Fmax / F0) and a 17.1% reduction in time to peak (see e.g., Fig. 25G). Additionally, MM-CMs demonstrated faster Ca2+decay rates, with a 29.1% reduction in time to 50% decay and a 19.6% reduction in time to 90% decay, both indicative of improved Ca2+kinetics (see e.g., Fig.25G).

[0335] MM treatment facilitated the switch from fetal to adult isoforms in various myofilament mRNAs and proteins. There was an approximately 4-fold increase in cardiac troponin I (cTnI) mRNA and protein levels, along with a nearly 10-fold rise in the transcript ratio adult to fetal titin (TTN) (N2B:N2BA; see e.g., Fig.25A, 25D; Fig.31C-31D). An exception was observed in cardiac myosins, where the ratio of MHY6 to MYH7 increased, contrary to the usual developmental trajectory (see e.g., Fig.25A). This change is attributed to T3 treatment, which directly activates MYH6 transcription. Immunostaining for cTnI and α-Actinin revealed that MM treatment induced hypertrophic growth, increasing cell size from 1598 cm² to 1781 cm² and elevating the proportion of cTnI-positive [cTnI(+)] cardiomyocytes from 11.3% to 59.0%, as 105 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 confirmed by flow cytometry (see e.g., Fig.25D, Fig.31N; Fig.25E). Forward Scatter (FSC) intensity, indicative of cell diameter, was higher in cTnI(+) cardiomyocytes compared to the cTnI(-) population. This finding indicates a correlation between sarcomeric isoform switching and increased cell size (see e.g., Fig.31N). Corresponding with the rise in the cTnI(+) population, MM treatment led to a 10.6% increase in FSC intensity, further supporting this association (see e.g., Fig.25E, Fig.31O). Through TEM analysis of MM-CMs, organized sarcomere alignment were observed with distinct A bands, I bands, and Z discs (see e.g., Fig. 31I); this was in contrast to the disorganized striation in Ctrl-CMs (see e.g., Fig.31I).

[0336] Finally, the hypothesis was tested that MM promotes cell cycle withdrawal, a critical aspect of postnatal maturation. The cell cycle status of hESC-CMs was assessed using flow cytometry, applying EdU staining to mark the S phase and FxCycle for overall DNA content. One week after treatment, the percentage of control cells in S phase was recorded at 8.8%, while only 2.7% MM-treated cells were in S phase (see e.g., Fig.25F). After two weeks, a more pronounced reduction was observed, with 5.2% of control cells and 0.6% of MM-CMs in S phase (see e.g., Fig.25F). MM did not significantly alter the proportion of cells in the G1 or G2 / M phases (see e.g., Fig.25F). These findings indicate that MM effectively induces cell cycle arrest at the G1 / S transition, further supporting its role in promoting cardiomyocyte maturation.

[0337] The above findings collectively demonstrate that MM not only promotes electrical maturation but also significantly enhances various other aspects of cardiomyocyte functionality. These include an increase in mitochondrial volume, enhanced cellular respiratory, fatty acid oxidation, and glycolytic capacities, alongside a reduction in glycogen stores. Additionally, MM improves Ca2+handling, facilitates myofibril isoform switching, augments sarcomere structure and cell size, and induces cell cycle arrest. Together, these changes demonstrate a transition towards a more adult-like phenotype in cardiomyocytes.

[0338] MM promotes the transcriptional maturation of cardiomyocytes.

[0339] To investigate how the transcriptome changes as hPSC-CMs undergo metabolic reprogramming, RNA-seq datasets were prepared and analyzed from three biologically independent samples for control (Ctrl) and MM condition. Volcano plots, which visualize the differential gene expression between Ctrl-CMs and MM-CMs as derived from DESeq2 analysis, highlighted an upregulation of mitochondrial biogenesis genes in MM-CMs (see e.g., Fig.26A), and complementary heatmaps further illustrated this gene expression pattern (see e.g., Fig.32A). 106 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Additionally, FA oxidation and TCA cycle genes were generally upregulated in MM-CMs, demonstrating their metabolic maturation (see e.g., Fig.26A; Fig.32B, 32C). Sarcomere genes, including maturation markers TNNI3 and MYL2, were also upregulated under MM treatment (see e.g., Fig.26A; Fig.32D). Ion transporters related to action potentials and Ca2+handling, including SCN5A, KCNJ2, KCNJ12, RYR2, and PLN, exhibited high expression in MM-CMs, and this was coupled with the downregulation of pacemaker genes HCN4 and TBX18 (see e.g., Fig.26A; Fig.32E). GJA1, which encodes Connexin 43 and is predominant in ventricular CMs, was upregulated in MM-CMs, while GJA5, which codes for Connexin 40 and is expressed in atrial CMs to the exclusion of ventricular CMs, was downregulated (see e.g., Fig.26A). Furthermore, AP-1 target genes such as JUN and ATF3, known to induce postnatal cardiac maturation in metabolism and binucleation, were significantly upregulated in MM-CMs (see e.g., Fig.26A). Finally, Wnt signaling genes, including WNT5A, WNT5B, and FZD5, were suppressed by MM treatment (see e.g., Fig.26A), a process linked to the maturation of hPSC-CMs.

[0340] Continuing to evaluate the DESeq2-derived differentially expressed genes (DEGs) from the comparison of Ctrl-CMs and MM-CMs, enrichment analyses was conducted for Gene Ontology (GO) terms (see e.g., Fig.26B), Reactome Pathways (see e.g., Fig.26C), and Transcription Factor Binding Sites (see e.g., TFBS; Fig.26D). Upregulated DEGs in MM-CMs were associated with GO terms and Reactome Pathways related to electrophysiology, including inorganic ion transmembrane transport and connexon plasma membrane transport, as well as metabolic processes like oxidative phosphorylation and lipid metabolism, and sarcomere functions such as muscle structure development and striated muscle contraction (see e.g., Fig. 26C, 26D). Conversely, downregulated DEGs in MM-CMs were linked to GO terms and pathways involving the extracellular matrix and neuronal and vascular development, as well as Wnt signaling (see e.g., Fig.26C, 26D). TFBS analysis revealed ERR1, an adult isoform of Estrogen-Related Receptor Alpha crucial for metabolic homeostasis and postnatal maturation, as a top TF associated with DEGs upregulated MM-CMs, indicating a significant role for metabolic alteration in the MM-CM maturation process (see e.g., Fig.26D). In contrast, LEF1, known to promote proliferation and delay maturation in hPSC-CMs1, emerged as a top TF in downregulated DEGs (see e.g., Fig.26D).

[0341] Each metabolic modulator in MM exerted a distinct impact on cardiac maturation, and “the whole is greater than the sum of its parts”. 107 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0342] To evaluate the effect of each metabolic modulator on transcriptional changes induced by MM, transcriptome analyses were conducted of Ctrl-CMs, MM-CMs, and a range of “MM minus 1” intervention CMs. This set includes MM-treated CMs subjected to glucose deprivation (termed “wGlu”), CMs treated with the MM cocktail excluding galactose (“noGal”), those without dexamethasone (“noDex”), those minus fatty acids (“noFA”), those without AICAR (“noAIC”), and those without triiodothyronine (“noT3”).

[0343] First, to evaluate relationships among the different conditions, principal component analysis (PCA) was performed. A score plot for PC1 and PC2 revealed the clustering of samples in distinct groups (see e.g., Fig.26E). The wGlu, noGal, and noDex samples clustered close to MM, whereas the noFA, noAIC, and noT3 samples were distinctly separate, underscoring the substantial impact of FA, AICAR, and T3 on the capacity of MM to mature CMs. To understand the genes influencing sample projection in PC space, the top 500 loading vectors (genes) were analyzed on both the positive and negative axes of PC1 and PC2 (see e.g., Fig.32F, 32G, 32H). On the PC1 positive axis, which corresponds with the noAIC, noT3, and Ctrl samples, there was an enrichment of GO terms related to neuronal development and extracellular organization (see e.g., Fig.32G). In contrast, the loading vectors on the PC1 negative axis, aligning with the wGlu, MM, noGal, noDex, and noFA samples, were significantly associated with oxidative metabolism, sarcomere structure, and electrophysiology (see e.g., Fig.32G). For the PC2 positive axis, aligning with the MM, noAIC, noDex, noGal, noT3, and wGlu samples, there was an enrichment in terms for muscle development (see e.g., Fig.32H). Meanwhile, loading vectors on the PC2 negative axis, associated with the noFA and Ctrl samples, showed enrichment in terms related to early development and lipid biosynthesis (see e.g., Fig.32H).

[0344] To dissect the specific effects of each metabolic modulator within the MM treatment spectrum, the DEGs from MM-CMS and Ctrl-CMs (see e.g., Fig.26A) were used to construct a hierarchically clustered heatmap (see e.g., Fig.26F). This heatmap showcases the gene expression patterns across various conditions, highlighting the specific impacts of the modulators. Significant roles were observed for AICAR, T3, and Dex in suppressing cluster 2 genes, which are associated with GO terms like extracellular matrix organization, neuronal development, vascular development, and Wnt signaling (see e.g., Fig.26F, 26G). AICAR, T3, and Dex were also involved in upregulating cluster 1 genes, which are linked to oxidative phosphorylation, cellular lipid metabolism, inorganic ion transmembrane transport, and heart 108 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 contraction (see e.g., Fig.26F, 26G). FA predominantly suppressed cluster 3 genes, which are related to embryonic morphogenesis and SA node action potential membrane depolarization; FA also promoted the expression of cluster 4 genes, which are associated with muscle development and fatty acid transport (see e.g., Fig.26F, 26G). Glucose deprivation played a partial role in the suppression of cluster 1 genes, whereas Gal evidenced minimal influence on MM-mediated transcriptional changes (see e.g., Fig.26F, 26G).

[0345] To delineate the unique and overlapping transcriptional changes elicited by each metabolic modulator in MM, the intersections of DESeq2-derived DEGs were analyzed, comparing MM-CMs against CMs treated with individual “MM minus 1” interventions (see e.g., Fig.26H, 26I). The findings revealed that over 70% of DEGs in the MM-versus-Ctrl comparison are unique to this pair. Specifically, this represents 550 out of 777 upregulated DEGs and 329 out of 434 downregulated DEGs (see e.g., Fig.26H, 26I). This observation indicates that the metabolic interventions in the MM treatment acted synergistically, resulting in a more profound impact on transcriptional reprogramming than the combination of their individual effects.

[0346] Taken together, the transcriptome analyses demonstrated that MM significantly reprograms cardiomyocyte gene expression. This reprogramming extends beyond metabolic networks to encompass ion channels / transporters and myofibril genes, while also suppressing pacemaker markers. Notably, glucose deprivation and galactose emerged as minor contributors to the MM-mediated maturation process, in contrast to the major roles played by AICAR, T3, Dex, and FA. Crucially, the combined effect of these metabolic interventions markedly enhanced cardiac maturation in a synergistic manner, underscoring the importance of metabolic alteration in driving comprehensive cardiac development.

[0347] MM induced metabolic reprogramming and improved energetic state.

[0348] To investigate the metabolic phenotype and identify key pathways involved in the maturation of hPSC-CMs, targeted metabolomics analyses were conducted of Ctrl-CMs and MM-CMs. 158 metabolites were measured in both groups using liquid chromatography-tandem mass spectrometry. PCA plots revealed a distinct separation between the two groups (see e.g., Fig.27A). Consistent with carbohydrate restriction, volcano plots and heatmap analysis demonstrated a suppression of glycolytic intermediates in MM-CMs, including DHAP / D-GA3P, glycerol-3-P, 2 / 3-phosphoglyceric acid, and lactate (see e.g., Fig.27B-27C). Reflecting 109 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 carbohydrate restriction, a volcano plot (see e.g., Fig.27B) and heatmap (see e.g., Fig.27C) revealed suppressed levels of glycolytic intermediates in MM-CMs, including DHAP / D-GA3P, glycerol-3-P, 2 / 3-phosphoglyceric acid, and lactate. Conversely, MM-CMs showed elevated levels of lipids and acylcarnitines, essential for FA oxidation; these include acetylcarnitine, isovalerylcarnitine, succinylcarnitine, and glutarylcarnitine, indicating an enhanced uptake and utilization of fatty acids (see e.g., Fig.27B-27C). TCA cycle intermediates, including cis- aconitate, succinate, and oxaloacetate, were also more abundant in MM-CMs, indicating an active oxidative phosphorylation (OXPHOS) state and aligning with the Seahorse flux analysis results (see e.g., Fig.25C; Fig.27B, 27C).

[0349] Out of 61 differentially abundant metabolites between the two groups, 53 were increased and 8 were decreased in MM-CMs (see e.g., Fig.27C). Compared to glycolysis, OXPHOS generates more energy alongside reactive oxygen species (ROS) byproducts. In MM- CMs, creatine (Cr) and phosphocreatine (PCr)—key components of the creatine kinase- phosphocreatine (CK-PCr) system predominant in adult CMs—were more abundant (see e.g., Fig.27D). The ratio of PCr to ATP was significantly higher in MM-CMs, indicating an improved cardiac energy status (see e.g., Fig.27D). Despite limited carbohydrate availability, pentose phosphate pathway metabolites, such as glucoronates and ribulose 5-phosphate, were elevated in MM-CMs, leading to increased NADPH production vital for the balancing of cellular oxidation-reduction (redox) (see e.g., Fig.27E).

[0350] The metabolic shift to OXPHOS in MM-CMs, particularly at the active complex 1 of the electron transport chain—one of the major sites of mitochondrial ROS generation—was evidenced by a significant increase in mitoSOX™ intensity (see e.g., Fig.27A, Fig.33C). β- alanine metabolites, which have been reported to increase during maturation, were enriched in MM-CMs (see e.g., Fig.27F). Carnosine, a dipeptide of β-alanine and L-histidine known for enhancing Ca2+kinetics and the redox system in muscles, was one of the most significantly increased metabolites in MM-CMs (see e.g., Fig.27B, 27F). To evaluate its effect on Ca2+kinetics, hPSC-CMs were treated with carnosine for one week in standard culture media. This treatment led to a dose-dependent improvement in amplitude, contributing to the enhanced Ca2+kinetics observed in MM-CMs (see e.g., Fig.33D).

[0351] In summary, MM-CMs demonstrated a significant shift in energy substrates, moving from glucose to FA, and underwent metabolic reprogramming from cytoplasmic glycolysis to 110 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 mitochondrial oxidation. This transition resulted in increased energy storage and ROS production, coupled with an enhanced antioxidant system.

[0352] Engraftment and survival of MM-CMs in adult mouse hearts.

[0353] Adult cardiomyocytes do not survive the stress of cell transplantation. Thus, to assess whether the maturation regimen of MM might negatively impact the engraftability of hPSC- CMs, either Ctrl-CMs or MM-CMs were transplanted into the uninjured hearts of adult NOD- SCID mice (see e.g., Fig.28A). Engraftment was quantified one week post-transplantation using Alu-based quantitative PCR (Alu-qPCR), a method that allows for the quantification of human genome copies against a background of mouse genomic DNA. The results showed that MM- CMs had human genome copy numbers comparable to those of Ctrl-CMs, indicating that MM treatment did not adversely affect engraftment in the adult mouse heart (see e.g., Fig.28B). Two weeks post-transplantation, the survival and characteristics of the engrafted cells were histologically evaluated. Human cardiomyocytes, marked by slow skeletal TnI (ssTnI), were observed in both Ctrl-CMs and MM-CM transplanted groups, confirming their survival for at least two weeks in the host mouse hearts (see e.g., Fig.28C). Despite the in vitro phenotype (see e.g., Fig.31I, 31P), glycogen storage was noted in both MM-CMs and Ctrl-CMs, indicating an in vivo adaptive capacity to the nutritional supply, including increased glucose utilization (see e.g., Fig.34). Taken together, MM-CMs not only survived transplantation but also engrafted at levels equivalent to Ctrl-CMs, indicating that the described maturation regimen is compatible with remuscularization therapy. Discussion

[0354] Transplantation of hPSC-CMs is an approach to mend the damaged heart through remuscularization. Graft cells are able to electrically couple to host myocardium for synchronous contraction, leading to the improvement in cardiac function. While studies in large animals have shown long-term survival and functional recovery, transient ventricular arrhythmias emerged shortly after transplantation. Since this engraftment arrhythmia (EA) is a major concern in safety for clinical application, intensive efforts have been conducted on electrophysiological analysis of EA. Results implied that ectopic electrical impulses from the graft cells propagated to host myocardium, thereby triggering EA. hPSC-CMs display immature cardiac traits such as automaticity, transitioning to a mature state more rapidly within the in vivo setting compared to the long-term culture in vitro. This can explain the decrease in EA burden within a few weeks 111 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 post-transplantation. Thus, the electrical immaturity of transplanted hPSC-CMs is a critical issue that needs to be addressed.

[0355] Multiple approaches have been attempted to enhance the maturation process mimicking the in vivo environment. Cardiac metabolism drastically shifts from glycolysis to OXPHOS during perinatal period through the alteration in energy substrates and oxygen availability and plays a pivotal role in driving the complex maturation process. Using the lessons from cardiac development, biochemical cues have been proven to contribute not only to the manipulation of adult CMs-like metabolism, but also to the improvement in other features such as cell cycle and growth, sarcomere structure, contractile and electrical function in hPSC-CMs. It was hypothesized that, through the combination of six key metabolic modulators (T3, Dex, Glu, Gal, FA, and AICAR)—each of which exhibits beneficial effects on electrophysiology—immature hPSC-CMs can be driven to attain an electrical profile that resembles adult working cardiomyocytes.

[0356] The effective dose of T3 for maturing hPSC-CMs is about 31 nM when used alone, in contrast to the higher concentration of 100 nM employed in conjunction with Dex. Similarly, fatty acids (FA) treatment alone can range from 67 µM to 115 µM, but can be increased to 200 µM when combined with other metabolic reprogramming factors. This indicates that metabolic interventions interact with each other, leading to varying impactful concentrations depending on the combination used. In this study, combinations and concentrations of metabolic hormones (T3, Dex), carbon sources (Glu, Gal, FA), and an energy-sensing enzyme (AMPK) were tested to enhance the electrical properties of hPSC-CMs.

[0357] To develop a culture medium, each intervention was systematically screened to evaluate the transcriptional level of ion channel / transporter genes and to assess electrophysiological characteristics using a multi-electrode array (MEA). hPSC-CMs cultured in 0 mM Glu RPMI / B27 media supplemented with 0.5 mM Gal, 460 μM FA, 1 mM AICAR, 100 nM T3, and 1 µM Dex for one week exhibited a mature electrophysiological phenotype. This culture medium is referred to herein as “maturation media” or “maturation medium.” (MM). Such a combination of metabolic interventions allows for the generation of electrically matured hPSC-CMs in vitro. The contribution of individual metabolic component to the observed maturation effects was investigated. 112 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0358] Various previous culture conditions have been developed to assess cardiac maturation across multiple properties, rather than focusing on a single aspect. The combination of conditions often varies depending on the specific outcomes measured during screenings. The goal herein was to cultivate hPSC-CMs that are both engraftable and electrically mature, thus capable of eliminating irregular heart rhythms post-transplantation. However, the culture conditions for enhancing morphological, structural, or contractile features can differ from those required for electrophysiological maturation. This is due to the involvement of multiple signaling pathways that regulate distinct aspects of cardiac maturation. Indeed, while the maturation media described herein did not significantly enhance contractility (data not shown), it markedly improved electrophysiology and Ca2+kinetics.

[0359] Compared to standard RPMI / B27 media, the MM described herein altered key cardiac features in hPSC-CMs. MM significantly reduced beat rate, increased field potential duration (FPDc), and enhanced conduction velocity across all batches—phenotypes typically seen in long-term cultured hPSC-CMs undergoing wide-ranging maturation. Transcriptomics analyses revealed a decrease in the expression of nodal cell markers associated with pacemaking activity, such as HCN4 and CACNA1G (encoding Cav3.1, the α1G subunit of voltage-dependent T type calcium channels). Conversely, MM upregulated transcripts of essential electrophysiological genes—including KCNJ2, KCNJ12, SCN5A, ATP2A2, RYR2, and GJA1—that are predominantly expressed in adult ventricular cardiomyocytes.

[0360] Reflecting the increased mRNA and protein levels of Cx43 and Nav1.5, along with an SCN5A isoform switch from its fetal to adult type, MM-CMs showed a significant increase in conduction velocity, regulated by fast inward sodium current (INa) and gap junctional electrical coupling. Spike amplitude and slope, correlated with INa, were also enhanced in most batches. Similar to adult cardiomyocytes, which rely heavily on SERCA for Ca2+transport, the upregulation of ATP2A2 and abundant RYR2 in MM-CMs improved Ca2+flux. Carnosine, markedly increased by MM, also enhanced Ca2+kinetics, via improved release from ryanodine receptors.

[0361] Each metabolic intervention in MM distinctly regulated various electrophysiological gene sets (see e.g., Fig.32I, Table 7). T3 primarily upregulated KCNJ2, ATP2A2, and RYR2; Dex mainly increased KCNJ12; FA contributed to the upregulation of KCNJ2 and GJA1, and the suppression of HCN4. AICAR predominantly upregulated SCN5A and KCNJ2 while suppressing 113 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 CACNA1G. Glucose deprivation chiefly enhanced KCNJ2, while Gal modestly increased SCN5A and KCNJ12, also improving cell density.

[0362] Table 7: Role of each modulator on MM induced improvement in electrophysiological genes expression (Generated from Fig.32I). Contribu�on of each modulator

[0363] Beyond electrical properties, MM-treated hPSC-CMs showed accelerated maturation in other cardiac features. Reflecting the FA-rich and glucose-depleted culture environment, MM- treated cells actively up-took and utilized FAs for oxidation, storing minimal glycogen and low amounts of glucose derivatives. These matured cells exhibited enhanced glycolytic capacity in the presence of glucose, indicative of metabolic flexibility—a characteristic of adult hearts under various stimuli, including ischemia and hypertrophic changes.

[0364] Enhanced mitochondrial content, upregulated creatine kinase expression, and an increased ratio of PCr to ATP facilitated efficient energy transfer to energy consumers like 114 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 myofibrils and SERCA. Additionally, MM-CMs displayed well-defined sarcomere structure with isoform switching from sTnI to cTnI, undergoing hypertrophic growth concurrent with cell cycle withdrawal.

[0365] Experiments involving MM with the removal of one component at a time—i.e., “MM minus one” experiments—shed light on the significant impact of T3, Dex, AICAR, and FA on the broad maturation process induced by MM. T3 was found to enhance the expression of ion transporter and sarcomere genes while suppressing genes related to the extracellular matrix and Wnt signaling. AICAR activated lipid metabolism and concurrently inhibited ECM organization. FA treatment boosted the expression of genes involved in fatty acid transport and muscle processes, and it suppressed genes associated with neural development. Glucose deprivation and galactose supplementation had a limited effect on this maturation process. The pretreatment of hPSC derivatives in media devoid of glucose but rich in lactate for cardiomyocyte purification might have minimized the benefits offered by the modulation of carbohydrates.

[0366] While immature cardiomyocytes have demonstrated the ability to engraft into host myocardium, there have been concerns regarding the engraftment of more mature cardiomyocytes. MM-CMs were capable of successfully engrafting into mouse hearts, showing graft sizes and cell numbers comparable to those of Ctrl-CMs. This successful engraftment can be attributed to their metabolic adaptability in the in vivo environment, such as their capacity for glucose storage and activation of glycolysis. Rodents have been commonly used to assess the engraftment of hPSC-CMs. Engraftment arrhythmias have been predominantly observed in larger animals, including porcine models and non-human primates (see e.g., Romagnuolo et al., Stem Cell Reports 12, 967-981 (2019); Marchiano et al. Cell Stem Cell 30, 741 (2023)), rather than in rodent models. Therefore, transplanting electrically matured hPSC-CMs into larger animals provides valuable insights into the origins of engraftment arrhythmias and explore the relationship between the levels of electrical maturity and the extent of arrhythmia burden. Engrafting hPSCs-CMs matured using the disclosed maturation media into the hearts of large mammalian subjects (e.g., pigs, non-human primates, humans) will result in reduced engraftment arrhythmias compared to negative controls (e.g., hPSCs-CMs not matured using the disclosed maturation media).

[0367] In summary, described herein is a culture media for generating hPSC-CMs that exhibit electrophysiological properties more akin to adult ventricular cardiomyocytes, as compared to 115 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 cells cultured in standard RPMI / B27 media. This disclosed media is rich in T3, Dex, AICAR, and FA, with a limited supplementation of galactose and no glucose. Each component uniquely modulated the expression of specific electrophysiological genes and, when combined, the components demonstrated synergistic effects that enhanced overall cardiomyocyte maturation. Beyond electrophysiology, the matured cardiomyocytes showed improvements in mitochondrial content and function, metabolism, cell cycle regulation, and cell size, as well as in sarcomere isoform switching and structure, and Ca2+kinetics. These matured cells were able to adapt effectively to the in vivo environment, as evidenced by graft size and cell numbers comparable to those observed with immature hPSC-CMs following transplantation into adult hearts. Therefore, the disclosed maturation media not only advances the field of regenerative medicine but also has applications in disease modeling and drug screening, particularly in studies related to arrhythmias. Material and Methods

[0368] Differentiation of hESC to CMs and treatment of CMs with metabolic modulators.

[0369] Human embryonic stem cells (RUESe002-A, Rockefeller University, USA) were maintained and differentiated into cardiomyocytes, as previously described, see e.g., Palpant et al. Nat Protoc 12, 15-31, 2017. Briefly, hESCs were cultured in mTeSR Plus (STEMCELL, 100- 0276) on MATRIGEL-coated plates (0.17 mg / mL) and passaged with 10 μM Y-27632 using VERSENE upon reaching 80% confluency. hESCs were primed with mTeSR Plus supplemented with 1 mM CHIR99021 for 24 hours. On day 0 of differentiation, mesoderm was induced using a cell line-optimized concentration of CHIR990214 μM in RPMI 1640 supplemented with 500 mg / mL BSA and 213 mg / mL ascorbic acid (RBA media) for 48 hours. On day 2 of differentiation, cells were washed with DPBS, and cardiac progenitors were induced with 2 mM Wnt-C59 in RBA media for 48 hours. On day 4, cells were washed with DPBS and incubated with RBA media for another 48 hours. From days 6 to 14, hESC-CMs were cultured in RPMI 1640 supplemented with B-27 media (RPMI / B27 media; B-27 is supplied as a 50X concentrate – RPMI / B-27 medium includes B-27 at 1X), which was changed every 48 hours. Metabolic selection was performed from days 14 to 18 by culturing cells in RPMI 1640 without glucose supplemented with 4 mM lactate as previously described (see e.g., Tohyama et al., Cell Stem Cell 12, 127-137 (2013)). Following the selection, hESC-CMs were trypsinized and re-plated on MATRIGEL coated tissue culture plates in RPMI / B27 supplemented with 5% FBS and 10 μM 116 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 Y-27632. From day 20, CMs were cultured in RPMI / B-27 media supplemented with or without added metabolic modulators, including T3 (100 nM), dexamethasone (1 µM), galactose (0-10 mM), glucose (0-11 mM), AICAR (0-2 mM), fatty acids (0-460 μM), and BSA with L-carnitine (0-460 μM), which was changed every other day. Unless otherwise noted, analyses were performed on days 27 to 34.

[0370] Quantitative real-time PCR

[0371] Total RNA was extracted from Ctrl-CMs and MM-CMs between days 27 and 34 using the QUICK-RNA MINIPREP Kit (ZYMO RESEARCH, R1055) as per the manufacturer's instructions. cDNA was synthesized from the RNA using the M-MLV RT Kit (THERMO FISHER, 28025013). This cDNA was then combined with SYBR Select Master Mix (THERMO FISHER, 4472913) and 400 nM of each primer for the target genes, and quantitative real-time PCR was performed on a CFX384 REAL-TIME SYSTEM (BIO-RAD), with HPRT1 as the normalization control. Details of primers and probes are provided in Table 8.

[0372] Table 8: Primer lists Forward Reverse primer primer117 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 TGACACTGGCAAA 11 GGTCCTTTTCACCAGC 31 HPRT1 ACAATGCA AAGCT

[0373] Micro Electrode Array (MEA) assay

[0374] D18 hESC-CMs were plated on 24-well MEA plates at 100,000 cells / well. Between days 20 and 34, they were exposed to metabolic modulators, with media changes every other day. For beat recording, the media was replaced with RPMI / B27 at least 2 hours before recordings at 37°C and 5% CO2 using the MAESTRO PRO system (AXION BIOSYSTEMS), following the CiPA protocol (see e.g., link.springer.com / protocol / 10.1007 / 978-1-4939-6661- 5_5). Metrics such as beat rate, field potential duration, spike slope, spike amplitude, and conduction velocity were analyzed using AXIS NAVIGATOR software (AXION BIOSYSTEMS).

[0375] Transmission Electron Microscope (TEM) analyses

[0376] D27 hESC-CMs on six-well plates were fixed with Karnovsky's fixative and sectioned using a UC6 Ultramicrotome (LEICA). Sections were examined with a JEM-1230 Transmission Electron Microscope (JEOL). Mitochondrial and glycogen content analyses were conducted across three independent experiments.

[0377] Flux analyzer measurement 118 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0378] Ctrl- and MM-CMs were seeded on MATRIGEL-coated cell culture microplates (AGILENT SEAHORSE XF24) at a cell density of 100,000 cells / well. Metrics such as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured with a SEAHORSE BIOSCIENCE XF24 Flux Analyzer and normalized to protein concentrations acquired via BCA assay.

[0379] Mitostress test

[0380] Before performing the experiment, the culture medium was changed to 525 µL of RPMI-based SEAHORSE medium (Agilent Seahorse XF RPMI Medium, pH 7.4, 103576-100) supplemented with 1 mM glutamine, 1 mM pyruvate, and 11 mM glucose; then, cells were incubated at 37°C in a non-CO2incubator for 1 hour. The assay involved sequential injections to final concentrations of 1 μM each of oligomycin (SIGMA, 75351), FCCP (SIGMA, C2920), rotenone (SIGMA, R8875), and antimycin A (SIGMA, A8674).

[0381] Palmitate-BSA FAO assay

[0382] One day before the assay, cells were incubated overnight in 250 µL of substrate-limited growth medium (AGILENT SEAHORSE XF RPMI Medium with 0.5 mM glucose, 1 mM glutamine, 1.0% FBS, and 0.5 mM L-carnitine). On the day of the assay, the medium was replaced with 500 µL of substrate-limited assay medium (AGILENT SEAHORSE XF RPMI Medium with 2 mM glucose and 0.5 mM L-carnitine) and incubated for 1 hour at 37°C in a non- CO2incubator. Subsequently, the medium was swapped for 415 µL of the same assay medium, with 85 µL of either palmitate-BSA or BSA control added to designated wells. The assay included sequential injections to achieve final concentrations of 4 μM Etomoxir, 1.5 μM oligomycin, 1 μM FCCP, 0.5 μM rotenone, and 0.5 μM antimycin A. The assay involved the sequential injection of final concentration of 4μM Etomoxir, 1.5μM oligomycin, 1μM FCCP, 0.5μM rotenone, and 0.5μM antimycin A (AGILENT, 103672-100).

[0383] Glycolytic stress test

[0384] Prior to the experiment, the culture medium was switched to 525 µL of RPMI-based SEAHORSE medium (AGILENT SEAHORSE XF RPMI Medium) enriched with 1 mM glutamine, 1 mM pyruvate, and 11 mM glucose. Cells were then incubated at 37°C in a non-CO2incubator for 1 hour. The assay sequence included injections to achieve final concentrations of 10 mM glucose (SIGMA, G7528), 1 μM oligomycin (SIGMA, 75351), and 50 mM 2-DG (CAYMAN, 14325). 119 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0385] Mitochondrial complex activity assay

[0386] Prior to the experiment, the culture medium was switched to 675 µL RPMI-based Seahorse medium (AGILENT SEAHORSE XF RPMI Medium, pH 7.4, 103576-100) with 1 mM glutamine, 1 mM pyruvate, and 11 mM glucose. Cells were then incubated at 37°C in a non-CO2incubator for 1 hour. The assay included sequential injections to reach final concentrations of 2 μM FCCP (SIGMA, C2920), 10 μM rotenone (SIGMA, R8875), 10 μM antimycin A (SIGMA, A8674), 10 mM ascorbate (SIGMA, 95209), and 3 mM TMPD (SIGMA, T7394).

[0387] Flow cytometer

[0388] Live cell analyses

[0389] For live cell analyses, cells were dissociated and incubated for 30 minutes at 37°C in RPMI-B27 with 5 μM MitoSOX™ Red (INVITROGEN, M36008), followed by washing in PBS and resuspension in PBS with 5% FBS. Flow cytometry data were collected using a FACS CANTO II (BD BIOSCIENCES), and mean fluorescent intensity (MFI) was analyzed with FLOWJO software.

[0390] Intracellular staining analyses

[0391] For intracellular staining analyses, cells in suspension were fixed with 4% paraformaldehyde for 15 minutes at room temperature and then incubated for 1 hour at room temperature with either mouse anti-cTnT (THERMOFISHER, MAS-12960) or rabbit anti-cTnI (ABCAM, ab25862) in PBS containing 5% FBS and 0.75% saponin. Following centrifugation and washing, cells were incubated with fluorophore-conjugated secondary antibodies of either goat anti-mouse 488 (INVITROGEN, A11001) or goat anti-rabbit 647 (INVITROGEN, A21244) in PBS with 5% FBS and 0.75% saponin for 1 hour at room temperature. Flow cytometry data were collected with a FACS CANTO II (BD BIOSCIENCES) and analyzed using FLOWJO software.

[0392] EdU staining analyses

[0393] For EdU staining analyses, cells underwent 12 hours of EdU treatment before being fixed with 4% paraformaldehyde and processed using the CLICK-IT PLUS EdU ALEXA FLUOR 488 Flow Cytometry Kit (INVITROGEN C10425), following the manufacturer's instructions. For DNA staining, after a PBS-with-0.01% TRITON-X wash, cells were resuspended in 1:1000 diluted FXCYCLE VIOLET (THERMOFISHER, F10347) in PBS with 0.01% TRITON-X. Flow cytometry data were collected using an LSR II Flow Cytometer (BD 120 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 BIOSCIENCES) and analyzed with FLOWJO software. Lipid droplets were measured using the Lipid Droplets Fluorescence Assay Kit (CAYMAN, 500001) as per the protocol: Cells fixed with Fixative Solution for 10 minutes at room temperature, washed with Assay Buffer, stained with Nile Red Staining Solution for 15 minutes, washed again, and analyzed using the LSR II Flow Cytometer (BD BIOSCIENCES), with mean fluorescent intensity (MFI) evaluated through FLOWJO software.

[0394] Calcium imaging

[0395] hESC-CMs at day 27 were plated at 5,000 cells / cm2on MATRIGEL-coated six-well plates in phenol red-free RPMI 1640 supplemented with B-27. The cells were nourished every other day for three days, followed by a 30-minute incubation with 1 mM Fluo-4 AM (THERMOFISHER, F14201) at 37°C. Calcium transients were recorded under electrical pacing at 1.5 Hz (s pulse duration, 10 mV) using a Culture Pacing System (IONOPTIX), and analyzed as described previously, see e.g., Bertero et al. J Cell Biol 218, 2919-2944 (2019).

[0396] RNA-seq

[0397] Sample preparation, library generation, and sequencing:

[0398] Cardiomyocyte samples (D27) under control (Ctrl), maturation media (MM), and the various “MM minus 1” conditions were collected from three independent differentiations. Total RNA was extracted using the QUICK-RNA Miniprep kit (ZYMO RESEARCH R1055) following the manufacturer's instructions.

[0399] The RNA-seq library preparation was performed, and standard directional libraries were prepared. Starting with total RNA (≥200 nucleotides), poly(T) oligo-attached magnetic beads were used for purification. The RNA was then fragmented and reverse-transcribed into first- strand cDNA using random hexamer primers, followed by second-strand cDNA synthesis using dUTP. The cDNA underwent poly(A) tailing and adapter ligation. After size selection and USER enzyme digestion, PCR amplification was performed, and the resulting DNA was purified. Library concentrations were quantified using QUBIT fluorometers and assessed by real-time quantitative reverse transcription PCR. To determine fragment size distributions, a BIOANALYZER (AGILENT) was employed. Index-coded libraries were pooled and sequenced on a NOVASEQ 6000 system (ILLUMINA) in a high-output run with 300 cycles, 150 cycles for each end.

[0400] Pre-processing and alignment of reads: 121 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0401] Using the program Atria (version 3.2.2; see e.g., Chuan et al. GigaByte 2021, gigabyte31 (2021)), raw paired-end reads in .fastq format were processed to trim away adapters, poly(N) sequences, and low-quality bases. The trimming process is illustrated by the following command line instruction in Formula I below: Formula I: atria \ -t "${threads}" \ -r "${fastq_1}" \ -R "${fastq_2}" \ -o "${directory_out}"

[0402] Trimmed paired-end reads were aligned using the program STAR (version 2.7.10b; see e.g., Dobin et al. Bioinformatics 29, 15-21 (2013)) against an H. sapiens GRCh38 reference genome (ENSEMBL version 110), illustrated as follows in Formula II: Formula II: STAR \ --runThreadN "${threads}" \ --genomeDir "${genome_directory}" \ --readFilesIn "${trimmed_fastq_1}" "${trimmed_fastq_2}" \ --readFilesCommand zcat \ --limitGenomeGenerateRAM "${ram}" \ --outFileNamePrefix "${outfile_prefix}" \ --outSAMtype BAM SortedByCoordinate \ --sjdbGTFfile "${gtf_file}" \ --sjdbOverhang 149 \ --twopassMode Basic \ --outSAMunmapped Within \ --limitOutSJcollapsed 1000000 \ --limitSjdbInsertNsj 1000000 \ --outFilterMultimapNmax 100 \ --outFilterMismatchNmax 33 \ --outFilterMismatchNoverLmax 0.3 \ 122 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 --seedSearchStartLmax 12 \ --alignSJoverhangMin 15 \ --alignEndsType Local \ --outFilterMatchNminOverLread 0 \ --outFilterScoreMinOverLread 0.3 \ --winAnchorMultimapNmax 50 \ --alignSJDBoverhangMin 1 \ --outFilterType BySJout

[0403] .bam output files were sorted and indexed using SAMTOOLS (version 1.16.1; see e.g., Danecek et al. Gigascience 10 (2021)).

[0404] Gene-level quantification of alignments:

[0405] Counts matrices, detailing the numbers of reads aligned to features in Gene Transfer Format (.gtf) files for each sample, were generated from the .bam files using the htseq-count utility within the HTSeq software package (version 2.0.2) (see e.g., Anders et al., Bioinformatics 31, 166-169 (2015); Putri et al., Bioinformatics 38, 2943-2945 (2022)). An example command line invocation follows in Formula III: Formula III: htseq-count \ --nprocesses "${threads}" \ --order "${order}" \ --stranded "${strand}" \ --nonunique "${non_unique}" \ --type "${type}" \ --idattr "${attribute}" \ --with-header \ --add-chromosome-info \ --counts_output "${tsv_out}" \ ${bam_files[*]} \ "${gtf_file}" 123 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0406] Batch-effect correction:

[0407] To address technical variations, or “batch effects”, present in the RNA-seq datasets sampled from three independent differentiations, COMBAT-SEQ was utilized (see e.g., Zhang et al. NAR Genom Bioinform 2, lqaa078 (2020)), which is a module within the SVA package (version 3.48.0; see e.g., Leek et al. Bioinformatics 28, 882-883 (2012)). COMBAT-SEQ was run with vectors specifying the replicates and conditions inputted as arguments for “batch” and “group” parameters, respectively; all other parameters were set to default values. The following snippet of R code illustrates this in Formula IV: Formula IV: corrected_data <- sva::ComBat_seq( as.matrix(counts_only), batch = column_data$batch, group = column_data$group ) %>% tibble::as_tibble()

[0408] Principal component analysis:

[0409] Principal component analysis (PCA) was conducted using the PCAtools package in R (version 2.12.0). Before PCA, the batch effect-corrected counts matrix underwent filtering to remove rows with sums below 10. Subsequently, sample columns in the matrix were normalized and regularized using the DESeq2 (version 1.40.2) rlog method (see e.g., Love et al. Genome Biol 15, 550 (2014)). To identify the most influential loading vectors (genes) for a given principal component (PC), the matrices of component loading vectors were sorted by their sign and magnitude. From this sorting, the top 10 or 500 positive and the top 10 or 500 negative loading vectors were selected.

[0410] Gene expression heatmaps:

[0411] To assess the expression of various gene groups under control (Ctrl), MM, and different “MM minus 1” conditions, the batch effect-corrected RNA-seq counts matrix was first normalized and regularized using the DESeq2 rlog method (version 1.40.2; see e.g., Love et al. Genome Biol 15, 550 (2014)). The resulting normalized matrix served as the input for generating heatmaps using the pheatmap (version 1.0.12) package in R. 124 4870-6522-3379.4Attorney Docket No.034186-000102WOP2

[0412] Differential gene expression analysis:

[0413] DESeq2 (version 1.40.2; see e.g., Love et al. Genome Biol 15, 550 (2014)) was employed for differential gene expression analysis. Before using DESeq2, raw (uncorrected) counts matrices were filtered to remove rows with sums below 10. To account for batch effects from RNA-seq datasets derived from three independent differentiations, Formula V "~ batch + group" was used for general linear modeling. Wald tests were conducted to compare observed results against a null distribution, with an effect size threshold of log2fold change greater than 0.58 (equivalent to a linear fold change greater than approximately 1.5). P-values were adjusted for the false discovery rate using the Benjamini-Hochberg method (see e.g., Benjamini et al., Journal of the Royal Statistical Society: Series B (Methodological) 57, 289-300 (1995)). Results were visualized in volcano plots using the EnhancedVolcano package (version 1.16.0) in R.

[0414] Gene Ontology, Reactome Pathway, and transcription factor binding site enrichment analyses:

[0415] Gene Ontology (see e.g., Ashburner et al., The Gene Ontology Consortium. Nat Genet 25, 25-29 (2000); Chen et al., Nucleic Acids Res 37, W305-311 (2009); Gene Ontology Consortium, Nucleic Acids Res 49, D325-D334 (2021)) term enrichment analyses were performed using the TOPPGENE SUITE TOPPFUN web application (toppgene.cchmc.org / enrichment.jsp; see e.g., Chen et al., Nucleic Acids Res 37, W305-311 (2009)), as were transcription factor binding site (TFBS) enrichment analyses (see e.g., Matys et al., Nucleic Acids Res 34, D108-110 (2006)) and Reactome Pathway analyses (see e.g., Wu et al., Methods Mol Biol 1558, 235-253 (2017); Milacic et al., Nucleic Acids Res 52, D672-D678 (2024)). Default settings we...

Claims

Attorney Docket No.034186-000102WOP2 CLAIMS 1. A cell maturation medium composition for promoting electrophysiological maturity in in-vitro differentiated cardiomyocytes, the medium comprising: a) glucose at 1 mM or less; b) triiodothyronine (T3); and c) dexamethasone.

2. The cell maturation medium composition of claim 1, wherein the concentration of T3 is between 10nM-1μM.

3. The cell maturation medium composition of any one of claims 1-2, wherein the concentration of T3 is 1 nM.

4. The cell maturation medium composition of any one of claims 1-3, wherein the concentration of T3 is 100 nM.

5. The cell maturation medium composition of any one of claims 1-4, wherein the concentration of dexamethasone is between 0.1μM-10μM.

6. The cell maturation medium composition of any one of claims 1-5, wherein the concentration of dexamethasone is 1 μM.

7. The cell maturation medium composition of any one of claims 1-5, wherein the concentration of dexamethasone is 100 nM.

8. The cell maturation medium composition of any one of claims 1-7, wherein the medium comprises RPMI with a glucose concentration of 1 mM or less.

9. The cell maturation medium composition of any one of claims 1-8, wherein the medium does not comprise glucose.

10. The cell maturation medium composition of any one of claims 1-9, further comprising one or more fatty acids found in mammalian milk.

11. The cell maturation medium composition of claim 10, wherein the fatty acid(s) are selected from the group consisting of linoleic, oleic and palmitic acid. 144 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 12. The cell maturation medium composition of claim 11, which comprises linoleic, oleic and palmitic acids.

13. The cell maturation medium composition of claim 12, wherein the concentration of linoleic, oleic and palmitic acids is from 115 – 460 μM.

14. The cell maturation medium composition of claim 13, wherein the concentration of linoleic, oleic and palmitic acids is 460 μM.

15. The cell maturation medium composition of claim 13, wherein the concentration of linoleic acid is from 22.5 – 137 μM.

16. The cell maturation medium composition of claim 13, wherein the concentration of oleic acid is from 40 – 243 μM.

17. The cell maturation medium composition of claim 13, wherein the concentration of palmitic acid is from 52.5 – 80 μM.

18. The cell maturation medium composition of any one of claims 1-17, further comprising galactose.

19. The cell maturation medium composition of any one of claims 1-18, wherein the galactose concentration is 0.5 – 10 mM.

20. The cell maturation medium composition of claim 19, wherein the galactose concentration is 0.5 mM.

21. The cell maturation medium composition of any one of claims 1-20, further comprising an AMP-activated protein kinase (AMPK) activator.

22. The cell maturation medium composition of claim 21, wherein the AMPK activator is 5- aminoimidizole-4-carboxamide riboside (AICAR).

23. The cell maturation medium composition of claim 22, wherein the AICAR is present at 0.3 to 2.0 mM.

24. The cell maturation medium composition of claim 23, wherein the AICAR is present at 1 mM. 145 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 25. A composition comprising in vitro differentiated cardiomyocytes in admixture with a cell maturation medium composition of any one of claims 1-24.

26. The composition of claim 25, wherein the in vitro differentiated cardiomyocytes are in suspension.

27. The composition of claim 25, wherein the in vitro differentiated cardiomyocytes are in monolayer culture.

28. The composition of claim 25, wherein the in vitro differentiated cardiomyocytes are in 3D culture.

29. The composition of any one of claims 25-28, wherein the in vitro differentiated cardiomyocytes are in vitro differentiated from a stem cell.

30. The composition of claim 29, wherein the stem cell is an iPS cell or an ES cell.

31. The composition of claim 30, wherein the iPS cell is derived from a subject with a cardiac disease or disorder.

32. A transplant composition comprising a composition of any one of claims 25-31.

33. The transplant composition of claim 32, wherein the in vitro-differentiated cardiomyocytes are autologous to a transplant recipient.

34. The transplant composition of claim 32 or 33, further comprising a pharmaceutically acceptable carrier.

35. The transplant composition of any one of claims 32-34, wherein the composition further comprises a matrix.

36. The transplant composition of any one of claims 32-35, further comprising cardiac endothelial cells.

37. A method of promoting electrophysiological maturation of an in vitro differentiated cardiomyocyte, the method comprising contacting the in vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of claims 1-24. 146 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 38. The method of claim 37, wherein electrophysiological maturity is determined by one or more of the following markers as compared to a reference level: increased expression of SCN5A (encodes Nav1.5, a fast sodium channel), KCNJ2 (encodes Kir2.1 channel, an inward rectifier potassium channel), CACNA1C (encodes Cav1.2, α1C subunit of voltage-dependent L type calcium channel), ATP2A2 (encodes SR calcium ATPase), RYR2 (encodes the ryanodine receptor in SR), Cx43 (Connexin 43, Gap junction α-1) protein and / or Nav1.5 protein, decreased expression of HCN4 (encodes a member of the hyperpolarization-activated cyclic nucleotide- gated sodium channel) and / or CACNA1H (encodes Cav3.2,α1H subunit of voltage-dependent T type calcium channel), and / or decreased field potential frequency, increased field potential duration, increased spike amplitude, increased spike slope, increased conduction velocity, increased calcium transient amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay.

39. The method of claim 37 or 38, wherein the cardiomyocytes are contacted with the cell maturation medium composition for at least 7 days.

40. An in vitro-differentiated cardiomyocyte produced by the method of any one of claims 37-39.

41. The in vitro-differentiated cardiomyocyte of claim 40, wherein the cardiomyocyte has increased electrophysiological maturity relative to an in vitro differentiated cardiomyocyte prepared without contacting with the cell maturation medium composition.

42. The in vitro-differentiated cardiomyocyte of claim 40 or 41, which, when administered to cardiac tissue of a subject, promotes reduced engraftment arrhythmia relative to an in vitro differentiated cardiomyocyte prepared in the same manner but not contacted with the cell maturation medium composition.

43. A method of transplanting in vitro differentiated cardiomyocytes, the method comprising contacting the in vitro differentiated cardiomyocyte with a cell maturation medium composition of any one of claims 1-24, and then administering the cardiomyocytes to a transplant subject.

44. The method of claim 43, wherein the method results in reduced engraftment arrhythmia in the recipient. 147 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 45. The method of claim 43 or 44, wherein the in vitro cardiomyocyte is differentiated in vitro from an iPS cell autologous to the recipient.

46. The method of any one of claims 43-45, wherein the transplant subject has been diagnosed with a cardiac disease or disorder.

47. A method of treating a cardiac disease or disorder, the method comprising administering in vitro differentiated cardiomyocytes in the cell maturation medium composition of any one of claims 1-24 to a subject in need thereof.

48. A method of treating a cardiac disease or disorder, the method comprising administering a transplant composition of any one of claims 32-36 or an in vitro differentiated cardiomyocyte of any one of claims 40-42 to a subject in need thereof.

49. The method of claim 48, wherein the treatment results in reduced engraftment arrhythmia relative to treatment using in vitro-differentiated cells that have not been contacted with cell maturation medium.

50. The method of any one of clams 47-49, wherein the in-vitro differentiated cardiomyocyte is differentiated in vitro from an iPS cell autologous to the subject.

51. The method of any one of claims 47-50, wherein the disease or disorder comprises cardiac infarction.

52. A composition of any one of claims 25-36 or 40-42, for use in the treatment of a cardiac disease or disorder.

53. A kit for maturing an in vitro-differentiated cardiomyocyte, the kit comprising a cell maturation medium of any one of claims 1-24 and instructions for maturing an in vitro- differentiated cardiomyocyte.

54. The kit of claim 53, further comprising an in vitro-differentiated cardiomyocyte.

55. The kit of any one of claims 53-54, for use in preparation of a transplant to cardiac tissue of a subject in need thereof.

56. The kit of any one of claims 53-55, for use in preparation of a treatment of a cardiac disease or disorder in a subject in need thereof. 148 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 57. A cardiomyocyte maturation medium comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex).

58. The cardiomyocyte maturation medium of claim 57, wherein the galactose (Gal) is at a concentration selected from: (a) about 0.5 mM; (b) at most 0.5 mM; (c) at most 1.0 mM (d) at most 10.0 mM; (e) 0.5 mM to 1.0 mM; or (f) 0.5 mM to 10.0 mM.

59. The cardiomyocyte maturation medium of any one of claims 57-58, wherein the fatty acids (FA) are at a concentration selected from: (a) about 460 μM; (b) at most 460 μM; (c) at least 230 μM; (d) at least 115 μM; (e) 115 μM to 460 μM; or (f) 230 μM to 460 μM.

60. The cardiomyocyte maturation medium of claim 59, wherein the fatty acids (FA) comprise palmitic acids, oleic acids, and / or linoleic acids.

61. The cardiomyocyte maturation medium of claim 59, wherein the fatty acids (FA) comprise palmitic acid.

62. The cardiomyocyte maturation medium of any one of claims 57-61, wherein the 5- aminoimidazole-4-carboxamide ribonucleoside (AICAR) is at a concentration selected from: (a) about 1.0 mM; (b) at most 1.0 mM; (c) at least 0.3 mM; or 149 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 (d) 0.25 mM to 1.0 mM.

63. The cardiomyocyte maturation medium of any one of claims 57-62, comprising about 460 μM fatty acids (FA) and about 1.0 mM AICAR.

64. The cardiomyocyte maturation medium of any one of claims 57-63, wherein the triiodothyronine (T3) is at a concentration selected from: (a) about 100 nM; (b) at most 100 nM; or (c) 50 nM to 200 nM; or (d) 10 nM to 1 µM.

65. The cardiomyocyte maturation medium of any one of claims 57-64, wherein the dexamethasone (Dex) is at a concentration selected from: (a) about 1.0 µM; (b) at most 1.0 µM; or (c) 0.5 µM to 2.0 µM; or (d) 10 nM to 10 µM.

66. The cardiomyocyte maturation medium of any one of claims 57-65, comprising about 100 nM triiodothyronine (T3) and about 1.0 µM dexamethasone (Dex).

67. The cardiomyocyte maturation medium of any one of claims 57-66, comprising essentially no glucose.

68. The cardiomyocyte maturation medium of any one of claims 57-67, comprising 0 mM glucose.

69. The cardiomyocyte maturation medium of any one of claims 57-68, further comprising: (a) at most 1 mM glucose; (b) at most 5.0 mM glucose; (c) 0 mM to 1 mM glucose; or (d) 0 mM to 5.0 mM glucose.

70. The cardiomyocyte maturation medium of any one of claims 57-69, comprising about 0.5 mM galactose (Gal) and 0 mM glucose.

71. The cardiomyocyte maturation medium of any one of claims 57-70, further comprising carnosine. 150 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 72. The cardiomyocyte maturation medium of claim 71, wherein the carnosine is at a concentration of 2 mM to 10 mM.

73. A cardiomyocyte maturation medium comprising: (a) 0.5 mM to 10 mM galactose (Gal); (b) 115 μM to 460 μM fatty acids (FA); (c) 0.25 mM to 1 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) 10 nM to 1 μM triiodothyronine (T3); (e) 10 nM to 10 µM dexamethasone (Dex); and (f) 0 mM to 5 mM glucose.

74. A cardiomyocyte maturation medium comprising: (a) about 0.5 mM galactose (Gal); (b) about 460 μM fatty acids (FA); (c) about 1.0 mM 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) about 100 nM triiodothyronine (T3); (e) about 1.0 µM dexamethasone (Dex); and (f) 0 mM glucose.

75. A composition comprising the cardiomyocyte maturation medium of any one of claims 57- 74, in combination with a cardiomyocyte or population thereof.

76. The composition of claim 75, wherein the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte.

77. The composition of claim 75 or 76, wherein the cardiomyocyte is electrophysiologically immature.

78. The composition of claim 75 or 76, wherein the cardiomyocyte is electrophysiologically mature.

79. A method of electrophysiologically maturing cardiomyocytes, the method comprising contacting electrophysiologically immature cardiomyocytes with the cardiomyocyte maturation medium of any one of claims 57-74 for an amount of time sufficient to mature one or more electrophysiological parameters of the cardiomyocytes.

80. A method of modulating at least one electrophysiologic property of a cardiomyocyte or population thereof, the method comprising contacting electrophysiologically immature cardiomyocytes with the cardiomyocyte maturation medium of any one of claims 57-74 for 151 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 an amount of time sufficient to modulate one or more electrophysiological parameters of the cardiomyocytes.

81. The method of any one of claims 79-80, wherein the sufficient of amount of time is at least one week.

82. The method of any one of claims 79-81, wherein the sufficient of amount of time is one to two weeks.

83. The method of any one of claims 79-82, wherein the electrophysiologically immature cardiomyocytes are differentiated from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

84. The method of any one of claims 79-83, wherein the electrophysiologically immature cardiomyocytes are contacted with the cardiomyocyte maturation medium at least 20 days after differentiation from ESCs or iPSCs.

85. The method of any one of claims 83-84, wherein the ESCs or iPSCs are human cells.

86. The method of any one of claims 83-85, wherein the ESCs or iPSCs are differentiated into electrophysiologically immature cardiomyocytes in vitro or ex vivo.

87. The method of any one of claims 79-86, wherein the maturation method is performed in vitro or ex vivo.

88. The method of any one of claims 79-87, wherein the cardiomyocytes are cultured on MATRIGEL-coated tissue culture plates, wherein the MATRIGEL comprises solubilized basement membrane matrix secreted by Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells.

89. The method of any one of claims 79-88, wherein at least one electrophysiologic property of a cardiomyocyte or population thereof is modulated by the method.

90. The method of any one of claims 79-89, wherein the at least one electrophysiologic property is selected from the group consisting of: beat rate, field potential duration, spike amplitude, spike slope, conduction velocity, Ca2+kinetics, and expression of electrophysiological genes.

91. The method of any one of claims 79-90, wherein the modulation of at least one electrophysiologic property comprises: decreased beat rate, increased field potential duration (FPD), increased spike amplitude, increased spike slope, increased conduction velocity, modulated Ca2+kinetics, and / or modulated expression of electrophysiological genes. 152 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 92. The method of any one of claims 90-91, wherein the Ca2+kinetics comprise amplitude, time to peak, time to 50% decay, and / or time to 90% decay.

93. The method of any one of claims 90-92, wherein the modulated Ca2+kinetics comprise increased amplitude, decreased time to peak, decreased time to 50% decay, and / or decreased time to 90% decay.

94. The method of any one of claims 90-93, wherein the modulated expression of electrophysiological genes comprises increased expression of at least one electrophysiological gene.

95. The method of any one of claims 90-94, wherein the modulated expression of electrophysiological genes comprises increased expression at least two electrophysiological genes.

96. The method of any one of claims 90-96, wherein the electrophysiological genes are selected from the group consisting of: sarcomeric, metabolic, ion-channel, and calcium(Ca2+)- handling genes.

97. The method of claim 96, wherein: (a) the sarcomeric genes comprise TNNI3 and / or MYL2; (b) the metabolic genes comprise PPARGC1A and / or CPT1B; (c) the ion-channel genes comprise SCN5A, KCNJ2, and / or HCN4; and / or (d) the calcium(Ca2+)-handling genes comprise CACNA1C, CACNA1H, ATPA2, and / or RYR2.

98. A kit for cardiomyocyte maturation comprising: (a) galactose (Gal); (b) fatty acids (FA); (c) 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR); (d) triiodothyronine (T3); and (e) dexamethasone (Dex).

99. A cardiomyocyte or population thereof electrophysiologically matured in the cardiomyocyte maturation medium of any one of claims 57-74.

100. A cardiomyocyte or population thereof electrophysiologically matured using the method of any one of claims 79-97. 153 4870-6522-3379.4Attorney Docket No.034186-000102WOP2 101. A cardiomyocyte or population thereof electrophysiologically matured using the kit of claim 98.

102. The cardiomyocyte or population thereof of any one of claims 99-101, wherein the cardiomyocyte is an in vitro differentiated or ex vivo differentiated cardiomyocyte.

103. A cardiomyocyte transplant composition comprising the cardiomyocyte or population thereof of any one of claims 99-102, in combination with a pharmaceutically acceptable carrier or scaffold.

104. A method of reducing or inhibiting engraftment arrythmia, the method comprising administering the cardiomyocyte, or population thereof, of any one of claims 99-102 or the cardiomyocyte transplant composition of claim 103 to a subject in need thereof, wherein the cardiomyocyte, or population thereof, or the cardiomyocyte transplant composition induces a reduced engraftment arrhythmia in the subject relative to an in vitro differentiated or ex vivo differentiated cardiomyocyte, or population thereof, or cardiomyocyte transplant composition that has not been matured using the maturation medium of any one of claims 57-74, the method of any one of claims 79-97, or the kit of claim 98.

105. A method of treating a cardiac disease, disorder, or injury, the method comprising administering the cardiomyocyte or population thereof of any one of claims 99-102 or the cardiomyocyte transplant composition of claim 103 to a subject in need thereof.

106. The method of any one of claims 104-105, wherein the cardiomyocyte or population thereof or the cardiomyocyte transplant composition is engrafted in or on the heart of the subject.

107. The method of any one of claims 104-106, wherein the subject is a human.

108. The method of any one of claims 104-106, wherein the subject is a non-human mammal.

109. The method of any one of claims 104-108, wherein engraftment arrythmia is reduced by at least 10%. 154 4870-6522-3379.4

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