Electrophysiological assay for cardiac cell potency

Abstract

This disclosure relates generally to methods of screening and preparing cardiac cell therapies for potency (e.g., cardiac cell function).

Description

[0001] ELECTROPHYSIOLOGICAL ASSAY FOR CARDIAC CELL POTENCY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 663,047, filed June 21, 2024, entitled “ELECTROPHYSIOLOGICAL ASSAY FOR CARDIAC CELL POTENCY,” the entire disclosure of which is hereby incorporated by reference herein in its entirety.

[0004] FIELD

[0005] This disclosure relates to compositions and methods related to stem-cell derived cells assessed for potency.

[0006] BACKGROUND

[0007] The ability of stem cells to regenerate injured tissue holds great promise for treatment of cardiac disease and injury. For example, stem-cell derived cardiomyocytes may be useful for repairing and / or replacing injured cardiac muscle. Assessment of cell potency (e.g., function output) is a critical step in demonstrating stem-cell therapy efficacy in a preclinical setting; however, assays for determining cell potency are highly specific to the type of cell therapy. Assays which predict and / or demonstrate the functional output of cardiac cell therapeutics are highly desirable.

[0008] SUMMARY

[0009] Cardiac cell therapies, such as cardiomyocyte grafts, are useful for treating subjects having injured cardiac tissue (e.g., as the result of heart disease or failure). Described herein, in some aspects, are methods of preparing a plurality of cells (e.g., human cells) for cardiac cell therapy.

[0010] In some aspects, the method of preparing a plurality of cells for cardiac cell therapy comprises:

[0011] (a) determining that at least 50% of the plurality of cells have one or more electrophysiological and / or mechanical properties associated with cardiac cell function under conditions suitable for recording cardiac cell activity; and

[0012] (b) preparing the plurality of cells for cardiac cell therapy.

[0013] In further aspects, the method of preparing a plurality of cells for cardiac cell therapy comprises: (a) determining by extracellular recording that at least 50% of the plurality of cells have one or more of the following properties under conditions suitable for recording cardiac cell activity: i. a median spontaneous beat period of about 1.6s to about 5.0s and / or a median paced beat period with 1 Hz stimulation of about 0.80s to about 1.20s; ii. a median spontaneous beat amplitude of about 0.60% to about 1.20% and / or a median paced beat amplitude with 1 Hz stimulation of about 0.30% to about 0.90%; iii. a median spontaneous excitation contraction delay of about 250ms to about 1100ms and / or a median excitation contraction delay with 1 Hz stimulation of about 100ms to about 600ms; iv. a spontaneous spike amplitude of about 0.5mV to about 1.2mV and / or a paced spike amplitude with 1Hz stimulation of about 1.80mV to about 2.20mV; v. a spontaneous spike slope of about 1.40V / s to about 1.80V / s and / or a paced spike slope with 1Hz stimulation of about 3.9V / s to about 4.7V / s; vi. a spontaneous local field potential (LFP) having a median duration of about 60ms to about 950ms and / or a paced LFP with 1Hz stimulation having a median duration of about 600ms to about 1000ms; and

[0014] (b) preparing the plurality of cells for cardiac cell therapy.

[0015] In some embodiments, the median spontaneous beat period is about 2.5s to about 4.5s. In some embodiments, the median spontaneous beat period is about 3.0s to about 4.0s. In some embodiments, the median spontaneous beat period is about 3.2s to about 3.7s. In some to embodiments, the median spontaneous beat period is about 3.5s.

[0016] In some embodiments, the median paced beat period with 1Hz stimulation is about 0.90s to about 1.10s. In some embodiments, the median paced beat period with 1Hz stimulation is about 0.95s to about 1.05s. In some embodiments, the median paced beat period with 1Hz stimulation is about 1.00s.

[0017] In some embodiments, the median spontaneous beat amplitude is about 0.70% to about 1.10%. In some embodiments, the median spontaneous beat amplitude is about 0.80% to about 1.00%. In some embodiments, the median spontaneous beat amplitude stimulation is about 0.90%.

[0018] In some embodiments, the median paced beat amplitude with 1Hz stimulation is about 0.40% to about 0.80%. In some embodiments, the median paced beat amplitude with 1Hz stimulation is about 0.50% to about 0.70%. In some embodiments, the median paced beat amplitude with 1Hz stimulation is about 0.60%.

[0019] In some embodiments, the median spontaneous excitation-contraction delay is about 350ms to about 800ms. In some embodiments, the median spontaneous excitation-contraction delay is about 450ms to about 700ms. In some embodiments, the median spontaneous excitation-contraction delay is about 500ms to about 600ms. In some embodiments, the median spontaneous excitation-contraction delay is about 540ms to about 550ms. In some embodiments, the median spontaneous excitation-contraction delay is about 545ms.

[0020] In some embodiments, the median paced excitation-contraction delay with 1Hz stimulation is about 240ms to about 550ms. In some embodiments, the median paced excitationcontraction delay with 1Hz stimulation is about 375 to about 465ms. In some embodiments, the median paced excitation-contraction delay with 1Hz stimulation is about 415 to about 425ms. In some embodiments, the median paced excitation-contraction delay with 1Hz stimulation is about 420ms.

[0021] In some embodiments, the median spontaneous spike amplitude is about 0.55mV to about 0.95mV. In some embodiments, the median spontaneous spike amplitude is about 0.70mV to about 0.80mV. In some embodiments, the median spontaneous spike amplitude stimulation is about 0.75mV.

[0022] In some embodiments, the median paced spike amplitude with 1Hz stimulation is about 1.90mV to about 2.10mV. In some embodiments, the median paced spike amplitude with 1Hz stimulation is about 1.95mV to about 2.05mV. In some embodiments, the median paced spike amplitude with 1Hz stimulation is about 2.00m V.

[0023] In some embodiments, the median spontaneous spike slope is about 1.45V / s to about 1.75V / s. In some embodiments, the median spontaneous spike slope is about 1.50V / s to about

[0024] 1.75V / s. In some embodiments, the median spontaneous spike slope is about 1.55V / s to about

[0025] 1.65V / s. In some embodiments, the median spontaneous spike slope is about 1.60V / s.

[0026] In some embodiments, the median paced spike slope is about 4.00V / s to about 4.60V / s.

[0027] In some embodiments, the median paced spike slope is about 4.1V / s to about 4.50V / s. In some embodiments, the median paced spike slope is about 4.25V / s to about 4.35V / s. In some embodiments, the median paced spike slope is about 4.30V / s.

[0028] In some embodiments, the spontaneous LFP has a median duration of about 75ms to about 900ms. In some embodiments, the spontaneous LFP has a median duration of about 100ms to about 700ms. In some embodiments, the spontaneous LFP has a median duration of about 200ms to about 500ms. In some embodiments, the spontaneous LFP has a median duration of about 250ms to about 300ms. In some embodiments, the spontaneous LFP has a median duration of about 265ms to about 275ms. In some embodiments, the spontaneous LFP has a median duration of about 270ms.

[0029] In some embodiments, the paced LFP with 1Hz stimulation has a median duration of about 800ms to about 900ms. In some embodiments, the paced LFP with 1Hz stimulation has a median duration of about 820ms to about 880ms. In some embodiments, the paced LFP with 1Hz stimulation has a median duration of about 845ms to about 855ms. In some embodiments, the paced LFP with 1Hz stimulation has a median duration of about 850ms. In some embodiments, the LFP is determined from the electrical signals of 2 to 30 cells.

[0030] In some embodiments, the plurality of cells comprises about 4 million to about 40 million cells.

[0031] In some embodiments, the plurality of cells is a batch of cells.

[0032] In some embodiments, the one or more properties are determined from 3 or more samples of the plurality of cells. In some embodiments, each sample comprises at least one cardiac cell.

[0033] In some embodiments, the one or more electrophysiological properties are obtained from an extracellular recording of the plurality of cells.

[0034] In some embodiments, the extracellular recording of the batch comprises recording on a multi-electrode array (MEA). In some embodiments, the MEA comprises a coating. In some embodiments, the MEA is at least partially coated with a high-molecular weight glycoprotein. In some embodiments, the high molecular weight glycoprotein is laminin.

[0035] In some embodiments, the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising 0.2mM to 2.0mM glucose, 130- 155mM sodium chloride, 4-6mM potassium chloride, and 0.3-1.3mM calcium chloride; and a pH of 7.4 to 7.8 and temperature of 35°C to 37°C.

[0036] In some embodiments, the cells are cardiomyocytes. In some embodiments, the cardiomyocytes are mature cardiomyocytes. In some embodiments the cells are derived from pluripotent stem cells. In some embodiments, the cells are derived from embryonic stem cells. In some embodiments, the plurality of cells is a batch of cardiomyocytes. In some embodiments, the plurality of cells is a batch of mature cardiomyocytes. In some embodiments, the batch of mature cardiomyocytes comprises 40 million cells. In some embodiments, preparing the plurality of cells for cardiac cell therapy comprises contacting the plurality of cells with a physiologically acceptable medium suitable for administration to a subject. Described herein, in some aspects, are compositions for cardiac cell therapy. In some aspects, a composition for cardiac cell therapy comprises a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof, wherein the plurality of cells are derived from stem cells; and wherein at least 50% of the plurality of cells have one or more of the following electrophysiological properties:

[0037] (a) a median spontaneous beat period of about 1.6s to about 5.0s and / or a median paced beat period with 1 Hz stimulation of about 0.80s to about 1.20s;

[0038] (b) a median spontaneous beat amplitude of about 0.60% to about 1.20% and / or a median paced beat amplitude with 1 Hz stimulation of about 0.30% to about 0.90%;

[0039] (c) a median spontaneous excitation contraction delay of about 250ms to about 1100ms and / or a median excitation contraction delay with 1 Hz stimulation of about 100ms to about 600ms;

[0040] (d) a spontaneous spike amplitude of about 0.5mV to about 1.2mV and / or a paced spike amplitude with 1Hz stimulation of about 1.80mV to about 2.20mV;

[0041] (e) a spontaneous spike slope of about 1.40V / s to about 1.80V / s and / or a paced spike slope with 1Hz stimulation of about 3.9V / s to about 4.7V / s;

[0042] (f) a spontaneous local field potential (LFP) having a median duration of about 60ms to about 950ms and / or a paced LFP with 1Hz stimulation having a median duration of about 600ms to about 1000ms; and / or wherein the one or more electrophysiological properties are measured by extracellular recording via a multi electrode array (MEA) at least partially coated with a high-molecular weight glycoprotein under conditions suitable for recording cardiac cell activity.

[0043] Also described herein, in some embodiments, are methods of treating a subject in need of cardiac cell therapy. In some aspects, the method of treating a subject in need of cardiac cell therapy comprises administering to the subject a cardiac cell therapy described herein.

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] FIGs. 1A-1E show an illustrative example of a method of assessing stem-cell derived cardiomyocytes (“Drug Product”) for potency using a multi electrode array (MEA), with different cell media and substrates. FIG. 1A is a schematic showing a process in which effects of cell media and substrate on cardiac potency are assessed. Frozen cardiomyocytes are thawed and plated in a 24-well plate having one of three substrates: Laminin 521, Fibronectin, or PO+Geltrex. Cardiomyocytes are cultured for 14 days with a cell medium (e.g., iCell Maintenance or StemDiff Maintenance), and assessed for monolayer formation and electrical activity. FIG. IB shows monolayer formation on an MEA coated with Laminin 521, Fibronectin, or PO+ Geltrex across cell culture media (iCell Maintenance (iCM) or StemDiff) at DIV14 using brightfield images at lOx magnification (Scale Bar = 100 pm). FIG. 1C shows immunocytochemistry (ICC) staining of cardiomyocytes cultured in iCell Maintenance or StemDiff media conditions for 14 days (Scale Bar= 100 pm). Cells were stained for Sarcomere- a- Actin or Hoechst identity markers. FIG. ID shows spike amplitude detection of a Laminin 521 coated MEA plate across the conditions shown in FIG. IB at DIV3 and DIV14. FIG. IE shows total active electrodes and beat period for cells cultured with StemDiff or iCell Maintenance and on MEAs coated with Laminin 521 (LN521), Fibronectin, or PO+Geltrex, at DIV 2, DIV 3, DIV 4, DIV 7, DIV 9, and DIV 14.

[0046] FIGs. 2A-2H show an illustrative example of a method of assessing stem-cell derived cardiomyocytes (“Drug Product”) for potency using a multi electrode array (MEA), with different cell culture media and different seeding densities. FIG. 2A is a schematic showing a process in which effects of cell culture media and seeding densities on cardiac potency of cells from 3 different batches (Batch 1, Batch 2, and Batch 3) are assessed. Frozen cardiomyocytes are thawed and plated in a 24-well plate having Laminin 521. Cardiomyocytes are cultured for 14 days with a cell medium (e.g., iCell Maintenance or StemDiff Maintenance), and assessed for monolayer formation and electrical activity. FIG. 2B shows monolayer formation on an MEA coated with Laminin 521, across cell culture media (iCell Maintenance (iCM) or StemDiff) and seeding density (40,000 (40K), 50,000 (50K), 60,000 (60K), or 100,000 (100K) cells) at DIV14 using brightfield images at lOx magnification (Scale Bar = 100 pm). FIGs. 2C-2D show beat period, spike amplitude, and field potential duration for Batch 1 cultured with StemDiff (FIG. 2C) or iCell Maintenance culture media (FIG. 2D) at 40K, 60K, 80K, or 100K density. FIGs. 2E-2F show beat period, spike amplitude, and field potential duration for Batch 2 cultured with StemDiff (FIG. 2E) or iCell Maintenance culture media (FIG. 2F) at 40K, 60K, 80K, or 100K density. FIGs. 2G-2H show beat period, spike amplitude, and field potential duration for Batch 3 cultured with StemDiff (FIG. 2G) or iCell Maintenance culture media (FIG. 2H) at 40K, 60K, 80K, or 100K density.

[0047] FIGs. 3A-3F show illustrative examples of measures of electrophysiological characteristics associated with cardiac potency. FIG. 3A shows paced spike waveform plots for cardiomyocytes at DIV 3, cultured with iCell Maintenance culture or StemDiff media and stimulated repeatedly at 1Hz with an 800mV, 200ps electrical pulse .Stimulation initiation is indicated with a purple line. FIG. 3B shows contractility waveforms recorded from cardiomyocytes at DIV3, cultured with StemDiff media. Spontaneous contractions are shown on the left and paced contractions from cells stimulated at 1Hz with an 800mV, 200ps electrical pulse (right). FIG. 3C shows spike amplitudes recorded from the cardiomyocytes of FIG. 3B under spontaneous and paced conditions. FIG. 3D shows spontaneous and paced field potential durations from the cardiomyocytes of FIGs. 3B and 3C. FIG. 3E shows spontaneous and paced beat amplitude from the cardiomyocytes of FIGs. 3B-3D. FIG. 3F shows spontaneous and paced beat period from the cardiomyocytes of FIGs. 3B-3D.

[0048] DETAILED DESCRIPTION

[0049] Described herein, in some aspects, are methods and compositions useful for preparing cardiac cell therapies. Cardiac cell therapies generally refer to stem-cell derived exogenous cells that are administered to the heart of a subject (e.g., in the form of a graft) in order to repair cardiac tissue in need thereof. However, cardiac cell therapies often demonstrate variability in, inter alia, electrophysiological and / or mechanical characteristics which may limit the therapeutic potential of cell therapies in a clinical setting. It is believed that variability in cardiac function arises from variability in differentiation and maturation protocols, as well as field-wide lack of knowledge on the mechanisms underlying certain features of cardiac cell function. Accordingly, methods of identifying cells having certain electrophysiological and / or mechanical qualities predictive and / or indicative of cardiac potency are highly desirable for cardiac regenerative therapies. The inventors have surprisingly developed several methods of assessing cardiac function and potency in stem cell-derived cells (e.g., stem cell-derived human cells).

[0050] Described herein, in some aspects, are methods and compositions related to stem-cell derived cells assessed for potency.

[0051] Characteristics of Cells Having Cardiac Potency

[0052] The present disclosure relates, in some aspects, to methods of identifying cells that are suitable for cardiac cell therapy (e.g., that have cardiac potency). In some embodiments, the method comprises identifying cells as having one or more characteristics associated with (e.g., predictive of) cardiac potency. “Potency” refers to the ability of a drug product and / or cell therapy to achieve an intended goal (e.g., therapeutic effect, mechanism of action); it is generally desirable for a potency assay to reflect clinical efficacy of an assessed product. Potency tests may also be used to assess manufacturing consistency and product stability. The term “cardiac potency” refers to the ability of a stem-cell derived cardiac cell to integrate with (e.g., form a colony within), repair, replace, regenerate, restore, or otherwise alter cardiac tissue or function of a subject when administered to the subject as part of a cardiac cell therapy (e.g., cardiac graft). In some embodiments, an electroresponsive cell is an excitable cell (e.g., one capable of producing an action potential).

[0053] Generally, the methods disclosed herein can be practiced on any cardiac cell (e.g., mature, immature, precursor) which is electroresponsive (e.g., electrically, chemically, and / or mechanically responsive to induced changes in transmembrane current and / or voltage). In some embodiments, a cardiac cell identified as having cardiac potency is a cardiomyocyte. In some embodiments, a cardiac cell identified as having cardiac potency is a mature cardiomyocyte. In some embodiments, a cardiac cell identified as having cardiac potency is an immature cardiomyocyte. In some embodiments, a cardiac cell identified as having cardiac potency is a cardiomyocyte precursor.

[0054] In some embodiments, cardiomyocytes are identified as lacking cardiac potency, such that the cardiomyocytes have one or more properties associated with an inability to integrate with (e.g., form a colony within), repair, replace, regenerate, restore, or otherwise alter cardiac tissue or function of a subject when administered to the subject as part of a cardiac cell therapy (e.g., cardiac graft). In some embodiments, cardiomyocytes identified as lacking cardiac potency are mature cardiomyocytes. In some embodiments, cardiomyocytes identified as lacking cardiac potency are immature cardiomyocytes. In some embodiments, cardiomyocytes identified as lacking cardiac potency are cardiomyocyte precursors.

[0055] In some embodiments, methods of identifying cells (e.g., cardiomyocytes) as having (or lacking) cardiac potency comprise determining one or more electrophysiological and / or mechanical properties of the cells. In some embodiments, the electrophysiological and / or mechanical properties are spontaneous properties, such that they are exhibited in the absence of external stimuli. In some embodiments, the electrophysiological and / or mechanical properties are stimulated properties, such that they are exhibited as a result of application of an external stimulus. In some embodiments, a stimulated property is a paced property, such that it is exhibited repeatedly and at regular (or near-regular) intervals by a cardiomyocyte as a result of a single or repeated (e.g., paced) stimulus.

[0056] Quantities of certain electrophysiological and / or mechanical properties or characteristics provided herein may, in some instances, be provided as an approximate value, for example, when provided as “about” a certain value. As used herein, the term “about” means approximately. In the context of numerical ranges, the term “about” refers to ±10% of the numerical values cited. For example, “about 1.95mVs to about 2.05mV” should be understood to include any value between 1.95mV and 2.05mV (inclusive), but also any value between 1.75mV and 2.05-2.21mV. “About” does not refer to percentages that are above 100% (e.g., above 100%) unless describing % change relative to another quantity (e.g., “an increase of about 105%”).

[0057] In some embodiments, an electrophysiological and / or mechanical property of cells comprises one or more characteristics of excitation-contraction coupling. “Excitationcontraction coupling” or “beating”, as used synonymously herein, refer to the physiological process through which a cell converts an electrical stimulus (e.g., external) into a mechanical response (e.g., contraction). In some embodiments, an isolated cardiac cell (e.g., cardiomyocyte) is capable of beating independently, such that it exhibits beating in the absence of functional (e.g., molecular, or electrical) or physical (e.g., via a junction, such as a gap junction) connection with another isolated cardiac cell (e.g., another isolated cardiomyocyte). In some embodiments, a plurality of cells (e.g., cardiomyocytes) are capable of beating as a unit (e.g., in a monolayer), such that the cells of the plurality beat together (e.g., in unison). Non-limiting characteristics of excitation-contraction coupling include beat period (e.g., beat cycle), beat amplitude, excitationcontraction delay, and ability to beat in the presence or absence of a stimulus.

[0058] In some embodiments, one or more cardiomyocytes are “self-beating,” such that the one or more cardiomyocytes exhibits spontaneous excitation-contraction coupling (e.g., in the absence of external stimuli). In some embodiments, one or more cardiomyocytes exhibits “rhythmic beating,” such that the one or more cardiomyocytes exhibit continuous excitationcontraction coupling with a regular beat period. In some embodiments, rhythmic beating occurs spontaneously, such that it occurs in the absence of an external stimulus. In some embodiments, rhythmic beating is induced via an external stimulus.

[0059] In some embodiments, a characteristic of excitation-contraction coupling comprises a beat period. A “beat period” or “beat cycle,” as used synonymously herein, refer to the timing between peak amplitudes of sequential beats of one or more cardiomyocytes. Beat periods may differ depending on a variety of factors, for example, the developmental stage of a cardiomyocyte, cell culture media, or recording substrate.

[0060] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 1.6s to about 5.0s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 1.6s, about 1.7s, about 1.8s, about 1.9s, about 2.0s, about 2.1s, about 2.2s, about 2.3s, about 2.4s, about 2.5s, about 2.6s, about 2.7s, about 2.8s, about 2.9s, about 3.0s about 3.1s, about 3.2s, about 3.3s, about 3.4s, about 3.5s, about 3.6s, about 3.7s, about 3.8s, about 3.9s, about 4.0s, about 4.1s, about 4.2s, about 4.3s, about 4.4s, about 4.5s, about 4.6s, about 4.7s, about 4.8s, about 4.9s, or about 5.0s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 2.5s to about 4.5s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 3.0s to about 4.0s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 3.2s and about 3.7s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat period of about 3.5s.

[0061] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat period between about 0.80ms and about 1.20s with 1Hz stimulation. Typically, a cardiac cell having cardiac potency which is stimulated with an N Hz electrical stimulation (e.g., via a cathode), wherein N is any number, will exhibit a paced beat period of about N seconds. For example, if the cardiac cell is stimulated with a 1Hz electrical pulse, a median paced beat period of about Is can be observed (e.g., during extracellular recording). In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat period of about 0.80s, about 0.85s, about 0.90s, about 0.95s, about 1.00s, about 1.05s, about 1.10s, about 1.15s, or about 1.20s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat period between about 0.90s and about 1.10s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat period between about 0.95s and about 1.05s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat period of about 1.00s with a 1Hz stimulation.

[0062] In some embodiments, a characteristic of excitation-contraction coupling comprises a beat amplitude. A beat amplitude, as used herein, refers to percent myocardial strain relative to length of a cardiac cell. Myocardial strain is measured as a change in myocardial fiber length over a beat cycle (e.g., between beats). In some embodiments, a beat amplitude is measured during spontaneous beating. In some embodiments, a beat amplitude is measured during paced (e.g., externally stimulated) beating.

[0063] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat amplitude of about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, about 0.90%, about 0.95%, about 1.00%, about 1.05%, about 1.10%, about 1.15%, or about 1.20%. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat amplitude of about 0.70% to about 1.10%. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat amplitude of about 0.80% to about 1.00%. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous beat amplitude of about 0.90%.

[0064] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat amplitude of about 0.30% to about 0.90% with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat amplitude of about 0.30%, about 0.35%, about 0.40%, about 0.45%, about 0.50%, about 0.55%, about 0.60%, about 0.65%, about 0.70%, about 0.75%, about 0.80%, about 0.85%, or about 0.90% with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat amplitude of about 0.40% to about 0.80% with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat amplitude of about 0.50% to about 0.70% with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced beat amplitude of about 0.60% with 1Hz stimulation.

[0065] In some embodiments, a characteristic of excitation-contraction coupling comprises a measure of excitation-contraction delay. Excitation-contraction delay refers to the period between which the spike of an action potential (e.g., as recorded via an extracellular recording electrode) and the peak amplitude of a cardiac cell contraction (e.g., as recorded via an extracellular impedance electrode) occurs in a beating cell. In some embodiments, an excitationcontraction delay is measured during spontaneous beating. In some embodiments, an excitationcontraction delay is measured during paced (e.g., externally stimulated) beating.

[0066] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous excitation-contraction delay of about 250ms to about 1100ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous excitation-contraction delay of about 250ms, about 255ms, about 260ms, about 265ms, about 270ms, about 275ms, about 280ms, about 285ms, about 290ms, about 295ms, about 300ms, about 305ms, about 310ms, about 315ms, about 320ms, about 325ms, about 330ms, about 335ms, about 340ms, about 345ms, about 350ms, about 355ms, about 360ms, about 365ms, about 370ms, about 375ms, about 380ms, about 385ms, about 390ms, about 395ms, about 400ms, about 405ms, about 410ms, about 415ms, about 420ms, about 425ms, about 430ms, about 435ms, about 440ms, about 445ms, about 450ms, about 455ms, about 460ms, about 465ms, about 470ms, about 475ms, about 480ms, about 485ms, about 490ms, about 495ms, about 500ms, about 505ms, about 510ms, about 515ms, about 520ms, about 525ms, about 530ms, about 535ms, about 540ms, about 545ms, about 550ms, about 555ms, about 560ms, about 565ms, about 570ms, about 575ms, about 580ms, about 585ms, about 590ms, about 595ms, about 600ms, about 605ms, about 610ms, about 615ms, about 620ms, about 625ms, about 630ms, about 635ms, about 640ms, about 645ms, about 650ms, about 655ms, about 660ms, about 665ms, about 670ms, about 675ms, about 680ms, about 685ms, about 690ms, about 695ms, about 700ms, about 705ms, about 710ms, about 715ms, about 720ms, about 725ms, about 730ms, about 735ms, about 740ms, about 745ms, about 750ms, about 755ms, about 760ms, about 765ms, about 770ms, about 775ms, about 780ms, about 785ms, about 790ms, about 795ms, about 800ms, about 805ms, about 810ms, about 815ms, about 820ms, about 825ms, about 830ms, about 835ms, about 840ms, about 845ms, about 850ms, about 855ms, about 860ms, about 865ms, about 870ms, about 875ms, about 880ms, about 885ms, about 890ms, about 895ms, about 900ms, about 905ms, about 910ms, about 915ms, about 920ms, about 925ms, about 930ms, about 935ms, about 940ms, about 945ms, about 950ms, about 955ms, about 960ms, about 965ms, about 970ms, about 975ms, about 980ms, about 985ms, about 990ms, about 995ms, about 1000ms, about 1005ms, about 1010ms, about 1015ms, about 1020ms, about 1025ms, about 1030ms, about 1035ms, about 1040ms, about 1045ms, 1050ms, about 1055ms, about 1060ms, about 1065ms, 1070ms, about 1075ms, about 1080ms, about 1085ms, 1090ms, about 1095ms, or about 1100ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous excitation-contraction delay of about 350ms to about 800ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous excitation-contraction delay of about 450ms to about 700ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous excitation-contraction delay of about 500ms to about 600ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous excitation-contraction delay of about 540ms to about 550ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous excitation-contraction delay of about 545ms.

[0067] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 100ms to about 600ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 100ms, about 105ms, about 110ms, about 115ms, about 120ms, about 125ms, about 130ms, about 135ms, about 140ms, about 145ms, about 150ms, about 250ms, about 255ms, about 260ms, about 265ms, about 270ms, about 275ms, about 280ms, about 285ms, about 290ms, about 295ms, about 300ms, about 305ms, about 310ms, about 315ms, about 320ms, about 325ms, about 330ms, about 335ms, about 340ms, about 345ms, about 350ms, about 355ms, about 360ms, about 365ms, about 370ms, about 375ms, about 380ms, about 385ms, about 390ms, about 395ms, about 400ms, about 405ms, about 410ms, about 415ms, about 420ms, about 425ms, about 430ms, about 435ms, about 440ms, about 445ms, about 450ms, about 455ms, about 460ms, about 465ms, about 470ms, about 475ms, about 480ms, about 485ms, about 490ms, about 495ms, about 500ms, about 505ms, about 510ms, about 515ms, about 520ms, about 525ms, about 530ms, about 535ms, about 540ms, about 545ms, about 550ms, about 555ms, about 560ms, about 565ms, about 570ms, about 575ms, about 580ms, about 585ms, about 590ms, about 595ms, or about 600ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 240ms to about 550ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 375ms to about 465ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 415ms to about 425ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced excitation-contraction delay of about 420ms with 1Hz stimulation.

[0068] In some embodiments, an electrophysiological and / or mechanical property of cardiomyocytes comprises one or more characteristics of a cardiomyocyte spike or action potential. The terms “spike” and “action potential,” used synonymously herein, refer to rapid changes in voltage (e.g., membrane potential) which trigger electrical, chemical, and / or mechanical changes in a cell (e.g., trigger beating). Action potentials in cardiac cells generally comprise distinct phases of channel activity. In phase 0, an initial, rapid inward current (e.g., depolarization, “upstroke”) driven by sodium channel activity, typically between 2ms in ventricular cardiomyocytes; phase 1, which begins as sodium channels inactivate, allowing a brief outward current (e.g., inactivation, “notch”) by potassium channel activity; in this phase, L-type calcium channels chloride channels activate, allowing for inward flux of calcium and chloride ions respectively. In phase 3 (e.g., repolarization, “downstroke”), L-type calcium channels slowly close and the cell returns to resting membrane potential, entering phase 4. During extracellular recordings, changes in ionic current in a cell may be recorded as changes to ionic current in a cell culture, medium, or other solution supporting the cell during recording.

[0069] In some embodiments, spikes are spontaneous (e.g., occur in the absence of an external stimulus). In some embodiments, spikes are paced, such that they are continuously invoked by application of an external stimulus (e.g., via a stimulation electrode, current injection, voltage step, activity of electrically coupled cell). Actional potentials are generally characterized by their waveforms (e.g., change in current over time) and frequency. Non-limiting examples of waveform characteristics include resting membrane potential, phase velocity (e.g., upstroke velocity), spike slope (e.g., upstroke slope), phase duration (e.g., notch duration), refractory period duration, percent action potential duration (e.g., AP30, AP50, AP70, AP90), and action potential morphology.

[0070] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike amplitude of about 0.5mV to about 1.2mV. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike amplitude of about 0.50mV, about 0.55mV, about 0.60mV, about 0.65mV, about 0.70mV, about 0.7 ImV, about 0.72mV, about 0.73mV, about 0.74mV, about 0.75mV, about 0.80mV, about 0.85mV, 0.90mV, about 0.95mV, about l.OOmV, about 1.05mV, about l.lOmV, about 1.15mV, or about 1.20mV. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike amplitude of about 0.55mV to about 0.95mV. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous spike amplitude of about 0.70mV to about 0.80mV. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike amplitude of about 0.75mV.

[0071] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike amplitude of about 1.80mV to about 2.20mV with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike amplitude of about 1.80mV, about 1.85mV, about 1.90mV, about 1.95mV, about 2.00mV, about 2.05mV, about 2.10mV, about 2.15mV, or about 2.20mV with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike amplitude of about 1.90mV to about 2.10mV with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike amplitude of about 1.95mV to about 2.05mV with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike amplitude of about 2.00mV with 1Hz stimulation.

[0072] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.40 V / s to about 1.80V / s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.40 V / s, about 1.45V / s, about 1.50V / s, about 1.55V / s, about 1.60V / s, about 1.65V / s, about 1.70V / s, about 1.75V / s, or about 1.80V / s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.45V / s to about 1.75V / s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.50V / s to about 1.75V / s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.55V / s to about 1.65V / s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median spontaneous spike slope of about 1.60V / S.

[0073] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 3.90V / s to about 4.7 V / s. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 3.90V / s, about 3.95V / s, about 4.00V / s, about 4.05V / s, about 4.10V / s, about 4.15V / s, about 4.20V / s, about 4.25V / s, about 4.30V / s, about 4.35V / s, about 4.40V / s, about 4.45V / s, about 4.50V / s, about 4.55V / s, about 4.60V / s, about 4.65V / s, or about 4.70 with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 4.00V / s to about 4.60V / s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 4.1 V / s to about 4.50V / s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 4.25V / s to about 4.35V / s with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a median paced spike slope of about 4.30 V / s with 1Hz stimulation.

[0074] In some embodiments, an electrophysiological and / or mechanical property of cardiomyocytes comprises one or more characteristics of a local field potential. A local field potential (LFP) is a transient electrical signal generated by the summed and synchronous activity of a plurality of cells. Characteristics of LFPs include, for example, duration and amplitude. In some embodiments, an LFP is determined from the electrical signals of 2 to 30 cells. In some embodiments, an LFP is determined from the electrical signals of 3 cells, of 4 cells, of 5 cells, of 6 cells, of 7 cells, of 8 cells, of 9 cells, of 10 cells, of 11 cells, of 12 cells, of 13 cells, of 14 cells, of 15 cells, of 16 cells, of 17 cells, of 18 cells, of 19 cells, of 20 cells, of 21 cells, of 22 cells, of 23 cells, of 24 cells, of 25 cells, of 26 cells, of 27 cells, of 28 cells, of 29 cells, or of 30 cells. In some embodiments, an LFP is determined from 15 cells to 25 cells. In some embodiments, an LFP is determined from about 20 cells.

[0075] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 60ms to about 950ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 60ms, about 65ms, about 70ms, about 75ms, about 80ms, about 85ms, about 90ms, about 95ms, about 100ms, about 105ms, about 110ms, about 115ms, about 120ms, about 125ms, about 130ms, about 135ms, about 140ms, about 145ms, about 150ms, about 155ms, about 160ms, about 165ms, about 170ms, about 175ms, about 180ms, about 185ms, about 190ms, about 195ms, about 200ms, about 205ms, about 210ms, about 215ms, about 220ms, about 225ms, about 230ms, about 235ms, about 240ms, about 245ms, about 250ms, about 255ms, about 260ms, about 265ms, about 270ms, about 275ms, about 280ms, about 285ms, about 290ms, about 295ms, about 300ms, about 305ms, about 310ms, about 315ms, about 320ms, about 325ms, about 330ms, about 335ms, about 340ms, about 345ms, about 350ms, about 355ms, about 360ms, about 365ms, about 370ms, about 375ms, about 380ms, about 385ms, about 390ms, about 395ms, about 400ms, about 405ms, about 410ms, about 500ms, about 505ms, about 510ms, about 515ms, about 520ms, about 525ms, about 530ms, about 535ms, about 540ms, about 545ms, about 550ms, about 555ms, about 560ms, about 565ms, about 570ms, about 575ms, about 580ms, about 585ms, about 590ms, about 595ms, about 600ms, about 605ms, about 610ms, about 615ms, about 620ms, about 625ms, about 630ms, about 635ms, about 640ms, about 645ms, about 650ms, about 655ms, about 660ms, about 665ms, about 670ms, about 675ms, about 680ms, about 685ms, about 690ms, about 695ms, about 700ms, about 705ms, about 710ms, about 715ms, about 720ms, about 725ms, about 730ms, about 735ms, about 740ms, about 745ms, about 750ms, about 755ms, about 760ms, about 765ms, about 770ms, about 775ms, about 780ms, about 785ms, about 790ms, about 795ms, about 800ms, about 805ms, about 810ms, about 815ms, about 820ms, about 825ms, about 830ms, about 835ms, about 840ms, about 845ms, about 850ms, about 855ms, about 860ms, about 865ms, about 870ms, about 875ms, about 880ms, about 885ms, about 890ms, about 895ms, about 900ms, about 905ms, about 910ms, about 915ms, about 920ms, about 925ms, about 930ms, about 935ms, about 940ms, about 945ms, or about 950ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 75ms to about 900ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 100ms to about 700ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 200ms to about 500ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 250ms to about 300ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 265ms to about 275ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 270ms.

[0076] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 60ms to about 950ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 60ms, about 65ms, about 70ms, about 75ms, about 80ms, about 85ms, about 90ms, about 95ms, about 100ms, about 105ms, about 110ms, about 115ms, about 120ms, about 125ms, about 130ms, about 135ms, about 140ms, about 145ms, about 150ms, about 155ms, about 160ms, about 165ms, about 170ms, about 175ms, about 180ms, about 185ms, about 190ms, about 195ms, about 200ms, about 205ms, about 210ms, about 215ms, about 220ms, about 225ms, about 230ms, about 235ms, about 240ms, about 245ms, about 250ms, about 255ms, about 260ms, about 265ms, about 270ms, about 275ms, about 280ms, about 285ms, about 290ms, about 295ms, about 300ms, about 305ms, about 310ms, about 315ms, about 320ms, about 325ms, about 330ms, about 335ms, about 340ms, about 345ms, about 350ms, about 355ms, about 360ms, about 365ms, about 370ms, about 375ms, about 380ms, about 385ms, about 390ms, about 395ms, about 400ms, about 405ms, about 410ms, about 500ms, about 505ms, about 510ms, about 515ms, about 520ms, about 525ms, about 530ms, about 535ms, about 540ms, about 545ms, about 550ms, about 555ms, about 560ms, about 565ms, about 570ms, about 575ms, about 580ms, about 585ms, about 590ms, about 595ms, about 600ms, about 605ms, about 610ms, about 615ms, about 620ms, about 625ms, about 630ms, about 635ms, about 640ms, about 645ms, about 650ms, about 655ms, about 660ms, about 665ms, about 670ms, about 675ms, about 680ms, about 685ms, about 690ms, about 695ms, about 700ms, about 705ms, about 710ms, about 715ms, about 720ms, about 725ms, about 730ms, about 735ms, about 740ms, about 745ms, about 750ms, about 755ms, about 760ms, about 765ms, about 770ms, about 775ms, about 780ms, about 785ms, about 790ms, about 795ms, about 800ms, about 805ms, about 810ms, about 815ms, about 820ms, about 825ms, about 830ms, about 835ms, about 840ms, about 845ms, about 850ms, about 855ms, about 860ms, about 865ms, about 870ms, about 875ms, about 880ms, about 885ms, about 890ms, about 895ms, about 900ms, about 905ms, about 910ms, about 915ms, about 920ms, about 925ms, about 930ms, about 935ms, about 940ms, about 945ms, or about 950ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 75ms to about 900ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 100ms to about 700ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 200ms to about 500ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 250ms to about 300ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 265ms to about 275ms. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a spontaneous LFP having a median duration of about 270ms.

[0077] In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 600ms to about 1000ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 600ms, about 605ms, about 610ms, about 615ms, about 620ms, about 625ms, about 630ms, about 635ms, about 640ms, about 645ms, about 650ms, about 655ms, about 660ms, about 665ms, about 670ms, about 675ms, about 680ms, about 685ms, about 690ms, about 695ms, about 700ms, about 705ms, about 710ms, about 715ms, about 720ms, about 725ms, about 730ms, about 735ms, about 740ms, about 745ms, about 750ms, about 755ms, about 760ms, about 765ms, about 770ms, about 775ms, about 780ms, about 785ms, about 790ms, about 795ms, about 800ms, about 805ms, about 810ms, about 815ms, about 820ms, about 825ms, about 830ms, about 835ms, about 840ms, about 845ms, about 850ms, about 855ms, about 860ms, about 865ms, about 870ms, about 875ms, about 880ms, about 885ms, about 890ms, about 895ms, about 900ms, about 905ms, about 910ms, about 915ms, about 920ms, about 925ms, about 930ms, about 935ms, about 940ms, about 945ms, about 950ms, about 955ms, about 960ms, about 965ms, about 970ms, about 975ms, about 980ms, about 985ms, about 990ms, about 995ms, or about 1000ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 620ms to about 910ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 800ms to about 900ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 820ms to about 880ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 845ms to about 855ms with 1Hz stimulation. In some embodiments, a cardiac cell identified as having cardiac potency exhibits a paced LFP having a median duration of about 850ms with 1Hz stimulation.

[0078] Cardiomyocytes

[0079] The present disclosure relates, in some aspects, to cardiac cell therapies (e.g., cardiac grafts) comprising cells having one or more properties associated with cardiac potency. In some embodiments, the cells are cardiomyocytes. The term “cardiomyocyte,” as used herein, refers to cells having cardiomyocyte lineage, including, but not limited to ventricular cardiomyocytes, atrial cardiomyocytes, and / or cardiac smooth muscle cells. Cardiomyocytes can be understood to be cells at any stage of cardiomyocyte development without restriction, unless stated otherwise.

[0080] In some embodiments, cardiomyocytes are cardiomyocyte precursors. A “cardiomyocyte precursor” is any precursor (e.g., progenitor) cell with the capacity to differentiate into a cardiomyocyte. In some embodiments, a cardiomyocyte precursor is or is derived from a stem cell (e.g., a human stem cell). In some embodiments, a cardiomyocyte precursor is or is derived from an induced pluripotent stem cell (iPSC). In some embodiments, a cardiomyocyte precursor is or is derived from a human pluripotent stem cell (hPSC). In some embodiments, a cardiomyocyte precursor is or is derived from an embryonic stem cell (ESC) (e.g., a human embryonic stem cell). As described herein, cardiomyocyte precursors may have the following characteristics: (1) positive / high expression of stage- specific embryonic antigen 3 and / or 4 (SSEA3 / 4), podocalyxin ERA- 1-60), octamer-binding transcription 3 and / or 4 (OCT3 / 4). homeobox protein NANOG (NANOG), and sex-determining region Y-box 2 (.8'0X2); (2) capable of self-replication. In some embodiments, cardiomyocyte precursors are capable of differentiation into all three germ layers (e.g., endoderm, mesoderm, ectoderm) or derivatives thereof. In some embodiments, cardiomyocyte precursors are mesoderm cells induced from stem cells which have been contacted by induction media (e.g., a Wnt agonist). In some embodiments, cardiomyocyte precursors are cardiac progenitors differentiated from mesoderm cells which have been contacted by cardiomyocyte differentiation media (e.g., contacted by a Wnt antagonist). In some embodiments, cardiomyocyte precursor cells are not excitable.

[0081] In some embodiments, cardiomyocytes are immature cardiomyocytes. The term “immature cardiomyocyte,” as used herein, refers to any cell which is in any stage of differentiation to become a mature cardiomyocyte and comparable to cardiac muscle cells in early stages of fetal development. In some embodiments, an immature cardiomyocyte is an immature human cardiomyocyte. In some embodiments, an immature cardiomyocyte is derived from a stem cell (e.g., a human stem cell). In some embodiments, an immature cardiomyocyte has been contacted with one or more differentiation agents (e.g., RPMI-1640 supplemented with B27). In some embodiments, an immature cardiomyocyte has been contacted with a maturation media (e.g., RPMI-1640 supplemented with B27 without insulin]). Immature cardiomyocytes are generally not quiescent (e.g., have not entered cell cycle arrest). In some embodiments, cardiomyocytes are immature ventricular cardiomyocytes. In some embodiments, cardiomyocytes are immature atrial cardiomyocytes. In some embodiments, cardiomyocytes are immature cardiac smooth muscle cells. As described herein, immature cardiomyocytes may exhibit the following characteristics within about 14 days from mesoderm induction: cluster of differentiation 36 (CD36) negative / low (relative to a mature cardiomyocyte), myosin regulatory light chain 2v (MLC2v, also known as MYL2) negative / low (relative to a mature cardiomyocyte), and myosin regulatory light chain 2a MLC2a, also known as MYL7) positive / high (relative to a mature cardiomyocyte), e.g., using flow cytometric analysis. In some embodiments, an immature cardiomyocyte expresses homeobox protein Nkx2-5 (NKX2-5). In some embodiments, an immature cardiomyocyte has positive / high expression of one or more positive / high genetic or molecular markers provided in Table 1 and has negative / low expression of one or more negative genetic or molecular markers provided in Table 1.

[0082] In some embodiments, cardiomyocytes are mature cardiomyocytes. In some embodiments, a mature cardiomyocyte is any terminally differentiated (e.g., quiescent) cardiac muscle cell. In some embodiments, a mature cardiomyocyte is derived from an immature cardiomyocyte which has been contacted by a maturation cocktail (e.g., RPMI-1640 supplemented with b27 minus insulin). In some embodiments, a mature cardiomyocyte is derived from a stem cell (e.g., a human stem cell). In some embodiments, cardiomyocytes are matured in vivo (e.g., in a subject). In some embodiments, cardiomyocytes are matured in vitro. In some embodiments, cardiomyocytes are mature ventricular cardiomyocytes. In some embodiments, cardiomyocytes are mature atrial cardiomyocytes. In some embodiments, cardiomyocytes are mature cardiac smooth muscle cells. In some embodiments, mature cardiomyocytes exhibit the following characteristics within about 51 days from mesoderm induction: CTNT2 positive / high, MLC2v positive / high, and downregulated MLC2a (relative to an immature cardiomyocytes), e.g., using flow cytometric analysis. In some embodiments, mature cardiomyocytes are cardiac muscle troponin T (CTNT, also referred to synonymously herein as TNNT2) positive / high. In some embodiments, a mature cardiomyocyte has positive / high expression of one or more positive / high genetic or molecular markers provided in Table 1 and has negative / low expression of one or more negative genetic or molecular markers provided in Table 1.

[0083] Table 1, Non-limiting characteristics of cardiomyocytes throughout development

[0084] Non-limiting examples of cardiomyocyte differentiation and maturation procedures can be found in PCT Publication No.: WO 2014 / 200339, PCT Publication No.: WO 2017 / 039445, PCT Publication No.: WO 2020 / 227232, U.S. Publication No.: US 2020 / 0407687, each of which are incorporated herein by reference.

[0085] Expression of a marker by a cell can be measured any number of ways, but generally refers to quantification of the presence (or absence) of a distinct signal corresponding to the marker in or on the cell, compared to a control and / or baseline. In some instances, expression of a marker is measured as an absolute quantity (e.g., weight, molar amount, concentration). In some instances, expression of a marker is expressed as a relative quantity (e.g., fold-increase, fold-decrease, percent). In some instances, expression of a marker in a cell is measured in a semi-quantitative manner (e.g., “high” or “low” fluorescence, as used in flow cytometry).

[0086] Cells can be understood to be “positive” or have “high expression” of one or more markers if they express the marker at or above a baseline level. For example, a cell may be understood to be positive (+) for a marker which is detected in the cell. A cell may be understood to have “high” expression for a marker when the quantity (e.g., absolute, relative, semi- quantitative) of the marker in the cell is above some baseline or threshold (e.g., population average, control cell). Cells can be understood to be “negative” or have “low expression” of one or more markers if they do not express the marker at or above a baseline level. For example, a cell may be understood to be negative (-) for a marker which is not detected in the cell. A cell may be understood to have “low” expression for a marker when the quantity (e.g., absolute, relative, semi-quantitative) of the marker in the cell is below some baseline or threshold (e.g., population average, control cell).

[0087] Methods of determining expression of a marker by a cell are known to those of skill in the art and are dependent on the marker of interest and / or its form (e.g., surface protein encoded by a gene, transcript of a gene). In a non-limiting example, presence of a marker is determined using flow cytometry, immunohistochemistry, immunofluorescence, polymerase chain reaction (e.g., reverse transcription PCR), fluorescence-activated cell sorting (FACS), microarrays, and / or ribonucleic acid (RNA) sequencing, such as single cell RNA sequencing (scRNA-seq).

[0088] Methods for Identifying Cells Having Cardiac Potency

[0089] Methods described herein relate to, in some aspects, methods of identifying cells having cardiac potency (e.g., by determining one or more electrophysiological and / or mechanical property of a cell).

[0090] In some embodiments, the method comprises performing extracellular recordings on one or more cells to determine one or more electrophysiological and / or mechanical properties of the one or more cells. In some embodiments, the one or more cardiac cell comprises one or more electroresponsive cells. In some embodiments, the electroresponsive cells in the method are excitable cells.

[0091] Extracellular recording refers to techniques for monitoring and recording electrophysiological and / or mechanical properties of a cell non-invasively (e.g., without piercing the cell membrane), typically of two or more cells suspended in an extracellular medium or solution, using two or more electrodes (e.g., a recording electrode and a reference electrode). Extracellular recording may be performed through a number of means, for example, by amplifying and sampling (e.g., recording) electrical voltages signals between a reference electrode and at least one recording electrode. Extracellular recordings may be especially useful for potency assays, as they generally preserve the integrity of the cellular membrane of assayed cells, can be repeated, and allow for assessment of thousands of cells at once.

[0092] During extracellular recordings, reference electrodes generally do not make contact with any cells, such that the reference electrode amplifies and / or samples electrical voltages in the extracellular medium or solution; at the same time, the surface of at least one recording electrode makes contact with one or more cells. Electrical voltages are detected on the two or more electrodes as a result of ionic current in the cells (e.g., as the result of an action potential). Electrodes may have a sampling rate of 5.0kHz / channel to lOOkHz / channel. In some embodiments, an electrode has a sampling rate of at least 5.0kHz / channel, at least 6.0kHz / channel, at least 7.0kHz / channel, at least 8.0kHz / channel, at least 9.0kHz / channel, at least lO.OkHz / channel, at least 15kHz / channel, at least 20kHz / channel, at least 3OkHz / channel, at least 40kHz / channel, at least 5OkHz / channel, at least 60kHz / channel, at least 70kHz / channel, at least 8OkHz / channel, at least 90kHz / channel, or lOOkHz / channel.

[0093] In some embodiments, extracellular recordings are performed using a plurality of recording electrodes. In some embodiments, extracellular recordings are performed using one or more microelectrodes. Microelectrodes are electrodes having a tip area commensurate with a cross-sectional area of a cell, for example, between 1pm2and 1000pm2(e.g., 10pm2tolOOpm2, 100pm2to 200pm2, 200pm2to 300pm2, 300pm2to 400pm2, 400pm2to 500pm2). In some embodiments, a microelectrode comprises an electrically conductive material; for example, a microelectrode may be formed of an inert metal (e.g., tungsten, platinum, gold, indium tin oxide) and / or any other conductive material compatible with the electrostimulation and / or electro recording experiments described herein. In some embodiments, extracellular recordings may be performed using an array of microelectrodes, for example, to enhance sampling or increase detection ranges. Accordingly, multielectrode arrays (MEAs) may comprise 2 to 36000 electrodes. In some embodiments, an MEA comprises 2 or more, 4 or more, 6 or more, 12 or more, 16 or more, 20 or more, 24 or more, 28 or more, 32 or more, 48 or more, 64 or more, 96 or more, 128 or more, 240 or more, 480 or more, 600 or more, 1200 or more, 2400 or more, 4800 or more, 6000 or more, 12000 or more, 24000 or more, or 36000 microelectrodes. In some embodiments, extracellular recordings are performed using a plurality of MEAs. In some embodiments, extracellular recordings are performed using a plate (e.g., a well-plate) comprising 2 or more MEAs. In some embodiments, extracellular recordings are performed on a well-plate comprising 2, 3, 4, 5, 6, 8, 12, 16, 24, 32, 48, 64, 96, 128 or more MEAs. In some embodiments, extracellular recordings are performed on a well-plate comprising 6, 12, 16, 24, 32, 48, 64, 96, 128, or more MEAs, wherein each MEA comprises 24 or more, 32 or more, 48 or more, 64 or more, 96 or more, or 128 or more microelectrodes. In some embodiments, each MEA is positioned in a well of a well-plate. In some embodiments, at least one MEA is positioned in each well of a well-plate. MEAs suitable for use in these methods are known in the art, e.g., MaxTwo High-Density Microelectrode Array (HD-MEA) System (See: www.mxwbio.com / products / maxtwo-multiwell-microelectrode-array), Axion Biosystems Maestro Pro (See: www.axionbiosystems.com / technology / cardiac-mea).

[0094] In some embodiments, one or more electrophysiological and / or mechanical property of cells (e.g., cardiomyocytes) is determined by an extracellular recording of one or more cells per recording electrode. In some embodiments, one or more electrophysiological and / or mechanical property of cells (e.g., cardiomyocytes) is determined by an extracellular recording of 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, or 20 or more cells per electrode. In some embodiments, one or more electrophysiological and / or mechanical property of cells (e.g., cardiomyocytes) is determined by an extracellular recording of 1 to 5 cells per electrode. In some embodiments, one or more electrophysiological and / or mechanical property of cells (e.g., cardiomyocytes) is determined by an extracellular recording of 2 to 4 cells per electrode.

[0095] In some embodiments, extracellular recordings are performed in parallel with impedance monitoring. Impedance monitoring, as used herein, refers to a monitoring of changes to a ratio of voltage / current while weak alternating current (AC) is applied between two or more impedance electrodes, wherein cell culture media acts as an electrolyte. Changes to impedance may be reflective of one or more properties, including, but not limited to cell number, morphology, adhesion, cell movement, and cell-cell contact. Impedance monitoring may be useful for the detection of, for example, contractile activity of cardiomyocytes (e.g., measures related to excitation-contraction / beating). Methods of monitoring impedance for use in cardiomyocyte assays are known in the art, see e.g., Lamore, S. D. et al. (2015). “Cardiomyocyte Impedance Assays.” Assay Guidance Manual (Internet). Eli Lilly & Company and the National Center for Advancing Translational Sciences. In some embodiments, extracellular recordings are conducted using at least two impedance electrodes.

[0096] In some embodiments, extracellular recordings are performed in parallel with electrical stimulation of the same cells and / or after electrical stimulation of the same cells. “Electrical stimulation,” as used herein, refers to the introduction of an electrical stimulus to a cell or plurality of cells in an extracellular medium or solution using a stimulation electrode (e.g., cathode). An electrical stimulus may comprise a singular pulse or paced pulses (e.g., repeated pulses continuously invoked by application of an external stimulus).

[0097] In some embodiments, a pulse of an electrical stimulus (e.g., each pulse of a paced electrical stimulus) is a square pulse. A square pulse comprises a waveform consisting of instantaneous transitions between two levels, wherein each level has a certain duration. In some embodiments, a square pulse comprises a first level and a second level, wherein each level has the same duration. In some embodiments, a square pulse comprises a first level and a second level, wherein each level has different durations. In some embodiments, a first level and / or second level have a duration of about 50ps to 350ps. In some embodiments, a first level and / or second level have a duration of about lOOps to 300ps. In some embodiments, a first level and / or second level have a duration of about 150|j s to 250|as. In some embodiments, a first level and / or second level have a duration of about 200|as.

[0098] In some embodiments, a pulse of an electrical stimulus (e.g., each pulse of a paced electrical stimulus) has an amplitude of 200-1000mV. In some embodiments, a pulse of an electrical stimulus (e.g. each pulse of a paced electrical stimulus) has an amplitude of about 200mV, about 210mV, about 220mV, about 230mV, about 240mV, about 250mV, about 260mV, about 270mV, about 280mV, about 290mV, about 300mV, about 310mV, about 320mV, about 330mV, about 340mV, about 350mV, about 360mV, about 370mV, about 380mV, about 390mV, about 400mV, about 410mV, about 420mV, about 430mV, about 440mV, about 450mV, about 460mV, about 470mV, about 480mV, about 490mV, about 500mV, about 510mV, about 520mV, about 530mV, about 540mV, about 550mV, about 560mV, about 570mV, about 580mV, about 590mV, about 600mV, about 610mV, about 620mV, about 630mV, about 640mV, about 650mV, about 660mV, about 670mV, about 680mV, about 690mV, about 700mV, about 710mV, about 720mV, about 730mV, about 740mV, about 750mV, about 760mV, about 770mV, about 780mV, about 790mV, about 800mV, about 810mV, about 820mV, about 830mV, about 840mV, about 850mV, about 860mV, about 870mV, about 880mV, about 890mV, about 900mV, about 910mV, about 920mV, about 930mV, about 940mV, about 950mV, about 960mV, about 970mV, about 980mV, about 990mV, or about lOOOmV. In some embodiments, a pulse of an electrical stimulus (e.g., each pulse of a paced electrical stimulus) has an amplitude of about 600mV to about lOOOmV. In some embodiments, a pulse of an electrical stimulus (e.g., each pulse of a paced electrical stimulus) has an amplitude of about 700mV to about 900mV. In some embodiments, a pulse of an electrical stimulus (e.g., each pulse of a paced electrical stimulus) has an amplitude of about 800mV.

[0099] In some embodiments, a paced electrical stimulus comprises a frequency of about 0.5Hz to about 2.0Hz. In some embodiments, a paced electrical stimulus comprises a frequency of about 0.5Hz, about 0.6Hz, about 0.7Hz, about 0.8Hz, about 0.9Hz, about 1Hz, about 1.2Hz, about 1.3Hz, about 1.4Hz, about 1.5Hz, about 1.6Hz, about 1.7Hz, about 1.8Hz, about 1.9Hz, or about 2.0Hz. In some embodiments, a paced electrical stimulus comprises a frequency of about 0.5Hz to about 1.5Hz. In some embodiments, a paced electrical stimulus comprises a frequency of about 0.8Hz to about 1.2Hz. In some embodiments, a paced electrical stimulus comprises a frequency of about 0.9Hz to about 1.1Hz. In some embodiments, a paced electrical stimulus a frequency of about 1.0Hz. In some embodiments, extracellular recordings are performed using electrodes (e.g., microelectrodes of an MEA) which comprise a coating with surface features that facilitate binding with cells. The coatings may range from a monolayer in thickness (e.g., a self-assembled monolayer) to a thicker coating for retaining the cells. In non-limiting embodiments, electrodes may have surface features including, but not limited to, cationic amine groups, cationic charge groups, silica, recombinant fibronectin, hydroxyl groups, positive charge groups, positively charged resin groups (e.g., diethylaminoethyl), negative charge groups, combinations thereof, and / or any other suitable surface features to help retain the cells in place during the extracellular recording process. In some embodiments, the coating is a naturally occurring biological molecule or biochemical, or a natural or synthetic derivative thereof. In some embodiments, a coating comprises an extracellular matrix component (e.g., fibronectin, collagen), a glycoprotein (e.g., laminin), and / or a polymeric molecule derived from a naturally occurring biochemical (e.g., polylysines, such as poly-L-lysine or poly-D-lysine; polyomithine, such as poly-L- omithine). In one non-limiting example, electrodes are coated with a high-molecular weight glycoprotein such as laminin (e.g., LN521). It should be appreciated that in some embodiments, each of a plurality of electrodes may substantially have the same coating, while in other embodiments, different electrodes (e.g., each electrode, a subset of electrodes) may have different coatings. Coatings that are both compatible with cell adhesion and retain properties through numerous cycles of electrical stimulus are also contemplated.

[0100] In some embodiments, the method comprises obtaining extracellular recordings from isolated cells under conditions suitable for recording cardiac cell activity (e.g., one or more electrophysiological and / or mechanical properties described herein). In some embodiments, the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution. In some embodiments, the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising glucose, sodium chloride, potassium chloride, and calcium chloride; and comprise a normal physiological to basic pH and normal physiological temperatures. In some embodiments, the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising O.lmM to 2.2mM glucose, 100-165mM sodium chloride, 3-7mM potassium chloride, and 0.2-1.5mM calcium chloride; and comprise a pH of 7.2 to 8.0 and temperature of 32°C to 38°C. In some embodiments, the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising 0.2mM to 2.0mM glucose, 130-155mM sodium chloride, 4-6mM potassium chloride, and 0.3-1.3mM calcium chloride; and a pH of 7.4 to 7.8 and temperature of 35°C to 37°C. In some embodiments, the method comprises a plurality of cells comprising a plurality of immature cardiomyocytes. In some embodiments, the immature cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells express one or more of the following markers: SSCA3 / 4, TRA-160, OCT3 / 4, NANOG, SOX2. In some embodiments, the immature cardiomyocytes are derived from embryonic stem cells.

[0101] In some embodiments, the method comprises a plurality of cells comprising a plurality of mature cardiomyocytes. In some embodiments, the mature cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the pluripotent stem cells express one or more of the following markers: SSCA3 / 4, TRA-160, OCT3 / 4, NANOG, SOX2. In some embodiments, the mature cardiomyocytes are derived from embryonic stem cells. In some embodiments, the mature cardiomyocytes comprise positive / high expression of TNNT2. In some embodiments, the mature cardiomyocytes comprise positive / high expression of MLC2v, TNNT2, SCN5A, KCNJ2, CACNA1C and comprise negative / low expression of MLC2a, CD90, CACNA1H, HCN4, and MKI67.

[0102] In some embodiments, the method comprises determining one or more electrophysiological and / or mechanical property of a batch of cells. As used herein, a “batch” of cells (e.g., cardiomyocytes) comprises a plurality of cells belonging to a same treatment group. In some embodiments, a batch comprises cells having about the same chronological age (e.g., days since contact with a differentiation or maturation agent). In some embodiments, a batch comprises cells having about the same developmental age (e.g., expressing cell markers within a particular range). In some embodiments, a batch of cells comprises at least 100 million cells. In some embodiments, a batch of cells comprises about 200 million cells. In some embodiments, a batch of cells comprises about 300 million cells. In some embodiments, a batch of cells comprises about 400 million cells. In some embodiments, a batch of cells comprises about 500 million cells. In some embodiments, a batch of cells comprises about 600 million cells. In some embodiments, a batch of cells comprises about 700 million cells. In some embodiments, a batch of cells comprises about 800 million cells. In some embodiments, a batch of cells comprises about 900 million cells. In some embodiments, a batch of cells comprises about 1 billion cells. In some embodiments, a batch of cells comprises about 2 billion cells. In some embodiments, a batch of cells comprises about 3 billion cells. In some embodiments, a batch of cells comprises about 4 billion cells. In some embodiments, a batch of cells comprises about 5 billion cells. In some embodiments, a batch of cells comprises about 10 billion cells. In some embodiments, a batch of cells comprises about 20 billion cells. In some embodiments, a batch of cells comprises about 100 million cells to about 1 billion cells. In some embodiments, a batch of cells comprises about 200 million cells to about 800 billion cells. In some embodiments, a batch of cells comprises about 400 million cells to about 600 billion cells. In some embodiments, a batch of cells comprises about 1 billion cells to about 25 billion cells. In some embodiments, a batch of cells comprises about 10 billion to about 20 billion cells. In some embodiments, a batch of cells comprises about 1 billion, about 2 billion , about 3 billion, about 4 billion, about 5 billion, about 6 billion, about 7 billion, about 8 billion, about 9 billion, about 10 billion, about 11 billion, about 12 billion, about 13 billion, about 14 billion, about 15 billion, about 16 billion, about 17 billion, about 18 billion, about 19 billion, about 20 billion cells.

[0103] A sample is a portion of a batch and comprises one or more cells of the batch. In some embodiments, a sample comprises lxl0'9% to 1% of a batch. In some embodiments, a sample comprises about 1 million or more cells. In some embodiments, a sample comprises about 2 million or more cells. In some embodiments, a sample comprises about 3 million or more cells. In some embodiments, a sample comprises about 4 million or more cells. In some embodiments, a sample comprises about 5 million or more cells. In some embodiments, a sample comprises about 6 million or more cells. In some embodiments, a sample comprises about 7 million or more cells. In some embodiments, a sample comprises about 8 million or more cells. In some embodiments, a sample comprises about 9 million or more cells. In some embodiments, a sample comprises about 10 million or more cells. In some embodiments, a sample comprises about 20 million or more cells. In some embodiments, a sample comprises about 30 million or more cells. In some embodiments, a sample comprises about 40 million or more cells. In some embodiments, a sample comprises about 50 million or more cells. In some embodiments, a sample comprises about 60 million or more cells. In some embodiments, a sample comprises about 70 million or more cells. In some embodiments, a sample comprises about 80 million or more cells. In some embodiments, a sample comprises about 90 million or more cells.

[0104] In some embodiments, a subset of a batch (e.g., a sample) may be used to characterize the batch. For example, properties of a batch may be determined from three or more samples of the batch. Preferably, a sample is representative of a batch.

[0105] In some embodiments, a batch comprises cells derived from the same ancestral cells. In some embodiments, a batch comprises cells derived from the same cell line. In some embodiments, a batch comprises cells from two or more different cell lines. In some embodiments, a batch comprises cells having similar genetic profiles (e.g., developmental, functional, cell type). In some embodiments, a batch of cells (e.g., cardiomyocytes) is identified as having one or more electrophysiological and / or mechanical properties associated with cardiac potency. Cardiac Cell Therapies

[0106] Compositions

[0107] Cells identified by a method disclosed herein may be used for cardiac cell therapy. Cardiac cell therapies (e.g., cardiac grafts) of the disclosure include, in some embodiments, cellular compositions comprising a plurality of cardiomyocytes and physiologically acceptable medium. Cardiac cell therapies may comprise one or more types of cardiomyocytes (e.g., ventricular cardiomyocytes, atrial cardiomyocytes, and / or smooth muscle cells) at one or more stages of development (e.g., mature cardiomyocytes and / or immature cardiomyocytes). In some embodiments, the cardiomyocytes are ventricular cardiomyocytes. In some embodiments, cardiomyocytes are immune evading or hypoimmune. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise vascular cells and / or cells. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise endothelial cells, conduction cells, pacemaker cells, and / or fibroblasts. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) comprise cells derived from human stem cells. Cardiac cell therapies may comprise any physiologically acceptable medium helpful for administering, adhering, growing, and / or maintaining the cardiac cell therapy (e.g., cardiac graft) in a subject. Physiologically acceptable media for administration of tissues are known in the art.

[0108] In embodiments, cardiac cell therapies are administered to a subject having experienced cardiac injury, including, but not limited to, damage to cells and tissue of the heart, including cardiomyocytes. Cardiac injury may be caused by a number of factors, including, but not limited to, heart disease (e.g., coronary heart disease, cardiomyopathy, endocarditis, congenital cardiovascular defects, congestive heart failure), medications, and non-cardiac diseases (e.g., high blood pressure, diabetes, viruses). In some embodiments, cardiac injury is caused by heart failure (e.g., HFrEF). In some embodiments, cardiac injury is caused by myocardial infarction. In some embodiments, cardiac injury is caused by ischemia. In some embodiments, cardiac injury comprises an injured ventricle (e.g., left ventricle). In some embodiments, a cardiac cell therapy (e.g., a cardiac graft) is administered to a subject in need thereof, such as a subject having experienced cardiac injury. A subject may be any mammal, including, but not limited to, mice, rats, guinea pigs, hamsters, pigs, cows, sheep, goats, horses, and primates, including humans.

[0109] In some embodiments, a cardiac graft is administered to a subject which may have or may receive mechanical circulatory support before, after, or at the time of receiving the cardiac graft. In some embodiments the mechanical circulatory support may be a Left Ventricular Assist Device (LVAD), a Right Ventricular Assist Device (RVAD), a Bi Ventricular Assist Device (BiVAD), Extra Corporeal Membrane Oxygenation (ECMO), or Implantable Cardiac Defibrillator (ICD), or a combination thereof. In certain embodiments, the ICD is linked by a biventricular pacer. In some embodiments, mechanical circulatory support may be an intravascular, microaxial blood pump (such as Impella 5.0). In some embodiments, this mechanical circulatory support is ceased after the subject receives a cardiac graft. In some embodiments, the mechanical circulatory support is used or implanted on or to the subject prior to the administration of the cardiac graft.

[0110] Cardiac cell therapies (e.g., cardiac grafts) are typically administered directly to the heart of a subject, and may be administered to a subject through any suitable method; non-limiting examples include open surgical approaches (e.g., direct administration to the heart during open heart surgery), minimally invasive approaches (e.g., administration via a cardiac catheter or needle), or percutaneous / intravascular approaches.

[0111] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) is a cellular composition comprising a plurality of mature cardiomyocytes. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of mature cardiomyocytes wherein about 50% or more, about 55% or more, about 60% or more, about 65% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 96% or more, about 97% or more, about 98% or more, about 99% or more, or 100% of plurality of cells are mature cardio myocytes. In some embodiments, the mature cardiomyocytes comprise positive / high expression of TNNT2. In some embodiments, the mature cardiomyocytes comprise positive / high expression of one or more of: MLC2v, TNNT2, SCN5A, KCNJ2, CACNA1C. In some embodiments, the mature cardiomyocytes comprise negative / low expression of one or more of: MLC2a, CD90, CACNA1H, HCN4, and MKI67. In some embodiments, the mature cardiomyocytes comprise positive / high expression of one or more of: MLC2v, TNNT2, SCN5A, KCNJ2, CACNA1C and negative / low expression of one or more of: MLC2a, CD90, CACNA1H, HCN4, and MKI67. In some embodiments, the mature cardiomyocytes comprise positive / high expression of MLC2v, TNNT2, SCN5A, KCNJ2, CACNA1C and negative / low expression of MLC2a, CD90. CACNA1H, HCN4, and MKI67.

[0112] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes identified as having cardiac potency, wherein a plurality of the mature cardiomyocytes have one or more properties (e.g., have one or more electrophysiological and / or mechanical properties) associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 50% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 55% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 60% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 65% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 70% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 75% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 80% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 85% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 90% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 95% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 96% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 97% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 98% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein at least 99% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising mature cardiomyocytes, wherein 100% of the mature cardiomyocytes have been identified as having one or more properties associated with cardiac potency.

[0113] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes produced from a batch of immature cardiomyocytes identified as having one or more properties associated with cardiac potency. In some embodiments, a batch of immature cardiomyocytes identified as having one or more properties associated with cardiac potency is contacted with a maturation cocktail under conditions that promote cardiomyocyte maturation. Standard methods for maturing cardiomyocytes are described in Guo et al. , Circulation Research, 2020, 126, 1086-1106, incorporated herein by reference in its entirety. In some embodiments, mature cardiomyocytes are made by culturing immature cardiomyocyte identified as having one or more properties associated with cardiac potency on a substrate (e.g., adherent culture) for at least 2 weeks (e.g., between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, between 6 and 8, between 8 and 10, or between 10 and 12 weeks, inclusive, or more than 12 weeks) in culture media comprising a maturation cocktail. In some embodiments, mature cardiomyocytes identified as having one or more properties associated with cardiac potency are made by culturing immature cardiomyocyte identified as having one or more properties associated with cardiac potency on a substrate (e.g., adherent culture) for at least 2 weeks (e.g., between 2 and 3, between 3 and 4, between 4 and 5, between 5 and 6, between 6 and 8, between 8 and 10, or between 10 and 12 weeks, inclusive, or more than 12 weeks) in culture media that promotes oxidative phosphorylation.

[0114] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes identified as having cardiac potency, wherein a plurality of the immature cardiomyocytes have one or more properties (e.g., have one or more electrophysiological and / or mechanical properties) associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 50% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 55% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 60% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 65% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 70% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 75% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 80% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 85% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 90% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 95% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 96% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 97% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 98% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein at least 99% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency. In some embodiments, the cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes, wherein 100% of the immature cardiomyocytes have been identified as having one or more properties associated with cardiac potency.

[0115] In some embodiments, a cardiac cell therapy is a cardiac graft. In some embodiments, a cardiac graft comprises about 100 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 200 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 300 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 400 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 500 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 600 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 700 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 800 million to about 1 billion cardiomyocytes. In some embodiments, a cardiac graft comprises about 900 million to about 1 billion cardiomyocytes.

[0116] EXAMPLES

[0117] Example 1. Cell media, but not ME A coating, alter monolayer formation and spontaneous activity of cardiomyocytes

[0118] In this Example, the role of cell media and coating substrates on spontaneous activity as recorded by multielectrode arrays (MEAs) was assessed (FIG. 1A). Coating solutions of Laminin 521, Fibronectin, or PO+ Geltrex were prepared according to manufacturer instruction and transferred onto an MEA in a well plate. 5pL of cardiomyocytes (l.OxlO5cells / 5pL) were transferred onto each well, covering the MEAs and incubated at 37°C for 14 days. Suspension media was supplemented with iCell Maintenance (iCM; FujiFilm Cellular Dynamics) or StemDiff Maintenance (StemDiff; STEMCELL Technologies) medium.

[0119] Brightfield images (lOx magnification) were taken of each well to assess monolayer formation (FIG. IB). A monolayer was observed across all experimental conditions, with improved monolayer formation apparent for iCM conditions. Immunocytochemistry (ICC) staining of cells for Sarcomere-a-Actin or Hoechst identity markers was used to identify cardiomyocyte identity and cell nuclei, respectively (FIG. 1C), further confirming monolayer formation.

[0120] Extracellular recordings of cells at DIV 3 and DIV 4 were performed at 37°C and 5% CO2; cells were equilibrated for 10 minutes, and spontaneous activity was recorded for 180 seconds. Cells showed variable spike amplitudes (FIG. ID) at different DIVs. As shown in FIG. IE, cells suspended in iCM media showed increased conductivity (active electrodes) over time, while cells suspended in StemDiff displayed strong activity at DIV 4, with reduced activity thereafter, regardless of coating solution (FIG. IE). Beat period was approximately 2.5s regardless of condition or DIV.

[0121] Together, these results indicate that cell media may affect monolayer formation and electrophysiological and / or mechanical activity of cardiomyocytes over time, while MEA coating does not appear to affect cell properties.

[0122] Example 2. Cell media, but not seeding density or batch origin, alter monolayer formation and spontaneous activity of cardiomyocytes

[0123] In this Example, the role of cell seeding density on assay parameters was assessed. As shown in FIG. 2A, cells from 3 batches (Batch 1, Batch 2, Batch 3) were seeded at 40K, 60K 80K, or 100K cells per plate according to the method described in Example 1, and suspended in either iCM or StemDiff. Extracellular recordings of the spontaneous activity of cells on MEAs coated with Laminin 514 were performed at DIV 2-DIV 14, and monolayer formation was assessed as in Example 1.

[0124] As in Example 1, cells suspended in iCM showed improved monolayer formation compared to cells suspended in StemDiff (FIG. 2B). Cells in iCM also showed more consistent beat period, spike amplitudes, and field potentials within and across batches and seeding densities, with spike amplitudes generally increasing over time, but field potential durations and beat periods remaining constant (FIGs. 2D, 2F, 2H). Cells in StemDiff media varied in their spontaneous activity in a batch-dependent manner with little effect of seeding density. Spike amplitudes and field potentials for the same cells were initially increased relative to cells in iCM media, indicating higher excitability; however, this effect diminished after DIV 6 (FIGs. 2C, E, G). These results suggest that cell media affect cardiomyocyte activity and function over time. Example 3. Paced electrical stimulation induces uniform beating activity in DIV 4 cardiomyocytes

[0125] In this Example, extracellular recordings of cardiomyocytes in both spontaneous (no stimulus) or paced stimulus conditions were performed. As in Example 1, cells were thawed, plated, and prepared for recording on MEAs positioned in well plates. Spikes were successfully induced in cells suspended in StemDiff media (DIV 3) using a 1Hz, 800mV, 200ps duration electrical pulse (FIG. 3A). Both spontaneous and induced contractions of cardiomyocytes was observed (FIG. 3B) in the same cells. While spontaneous beating was observed with variable beat period, 1Hz beats could be reliably induced with a paced 1Hz electrical pulse (FIG. 3C). Paced spike amplitudes were noticeably higher (about 4x) than spontaneous spikes, and field potential durations were noticeably longer than spontaneous field potentials (FIG. 3D); in both instances, cardiomyocyte activity was more consistent when paced stimuli were used applied. Beat amplitude and beat period were both reduced in paced conditions relative to spontaneous activity, but also had less variability. Overall, these results indicate that despite individual variability in spontaneous activity, cells cultured in StemDiff media are capable of uniform activity when provided external stimuli.

[0126] EQUIVALENTS AND SCOPE

[0127] In the claims, articles such as “a,” “an,” and “the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include “or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes aspects in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes aspects in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

[0128] Furthermore, the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Where elements are presented as lists, e.g., in Markush group format, each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is / are referred to as comprising particular elements and / or features, certain aspects of the invention or aspects of the invention consist, or consist essentially of, such elements and / or features. For purposes of simplicity, those aspects have not been specifically set forth in haec verba herein.

[0129] The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one aspect, to A only (optionally including elements other than B); in another aspect, to B only (optionally including elements other than A); in yet another aspect, to both A and B (optionally including other elements); etc.

[0130] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e., “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0131] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one aspect, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another aspect, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another aspect, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0132] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

[0133] In the claims, as well as in the specification above, all transitional phrases such as “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03. It should be appreciated that aspects described in this document using an open-ended transitional phrase (e.g., “comprising”) are also contemplated, in alternative aspects, as “consisting of’ and “consisting essentially of’ the feature described by the open-ended transitional phrase. For example, if the application describes “a composition comprising A and B,” the application also contemplates the alternative aspects “a composition consisting of A and B” and “a composition consisting essentially of A and B.”

[0134] Where ranges are given, endpoints are included. Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the stated ranges in different aspects of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0135] This application refers to various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. If there is a conflict between any of the incorporated references and the instant specification, the specification shall control. In addition, any particular aspect of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such aspects are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular aspect of the invention can be excluded from any claim, for any reason, whether or not related to the existence of prior art. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific aspects described herein. The scope of the present aspects described herein is not intended to be limited to the above Description, but rather is as set forth in the appended claims. Those of ordinary skill in the art will appreciate that various changes and modifications to this description may be made without departing from the spirit or scope of the present invention, as defined in the following claims.

[0136] The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an aspect for a variable herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof. The recitation of an aspect herein includes that aspect as any single aspect or in combination with any other aspects or portions thereof.

Claims

CLAIMSWhat is claimed is:

1. A method of preparing a plurality of cells for cardiac cell therapy, the method comprising:(a) determining that at least 50% of the plurality of cells have one or more electrophysiological and / or mechanical properties associated with cardiac cell function under conditions suitable for recording cardiac cell activity; and(b) preparing the plurality of cells for cardiac cell therapy.

2. A method of preparing a plurality of cells for cardiac cell therapy, the method comprising:(a) determining by extracellular recording that at least 50% of the plurality of cells have one or more of the following properties under conditions suitable for recording cardiac cell activity: i. a median spontaneous beat period of about 1.6s to about 5.0s and / or a median paced beat period with 1 Hz stimulation of about 0.80s to about 1.20s; ii. a median spontaneous beat amplitude of about 0.60% to about 1.20% and / or a median paced beat amplitude with 1 Hz stimulation of about 0.30% to about 0.90%; iii. a median spontaneous excitation contraction delay of about 250ms to about 1100ms and / or a median excitation contraction delay with 1 Hz stimulation of about 100ms to about 600ms; iv. a spontaneous spike amplitude of about 0.5mV to about 1.2mV and / or a paced spike amplitude with 1Hz stimulation of about 1.80mV to about 2.20mV; v. a spontaneous spike slope of about 1.40V / s to about 1.80V / s and / or a paced spike slope with 1Hz stimulation of about 3.9V / s to about 4.7V / s; and / or vi. a spontaneous local field potential (LFP) having a median duration of about 60ms to about 950ms and / or a paced LFP with 1Hz stimulation having a median duration of about 600ms to about 1000ms; and(b) preparing the plurality of cells for cardiac cell therapy.

3. The method of claim 2, wherein the median spontaneous beat period is about 2.5s to about 4.5s.

4. The method of claim 2 or 3, wherein the median spontaneous beat period is about 3.0s to about 4.0s.

5. The method of any one of claims 2 to 4, wherein the median spontaneous beat period is about 3.2s to about 3.7s.

6. The method of any one of claims 2 to 5, wherein the median spontaneous beat period is about 3.5s.

7. The method of any one of claims 2 to 6, wherein the median paced beat period with 1Hz stimulation is about 0.90s to about 1.10s.

8. The method of any one of claims 2 to 7, wherein the median paced beat period with 1Hz stimulation is about 0.95s to about 1.05s.

9. The method of any one of claims 2 to 8, wherein the median paced beat period with 1Hz stimulation is about 1.00s.

10. The method of any one of claims 2 to 9, wherein the median spontaneous beat amplitude is about 0.70% to about 1.10%.

11. The method of any one of claims 2 to 10, wherein the median spontaneous beat amplitude is about 0.80% to about 1.00%.

12. The method of any one of claims 2 to 11, wherein the median spontaneous beat amplitude stimulation is about 0.90%.

13. The method of any one of claims 2 to 12, wherein the median paced beat amplitude with 1Hz stimulation is about 0.40% to about 0.80%.

14. The method of any one of claims 2 to 13, wherein the median paced beat amplitude with 1Hz stimulation is about 0.50% to about 0.70%.

15. The method of any one of claims 2 to 14, wherein the median paced beat amplitude with 1Hz stimulation is about 0.60%.

16. The method of any one of claims 2 to 15, wherein the median spontaneous excitationcontraction delay is about 350ms to about 800ms.

17. The method of any one of claims 2 to 16, wherein the median spontaneous excitationcontraction delay is about 450ms to about 700ms.

18. The method of any one of claims 2 to 17, wherein the median spontaneous excitationcontraction delay is about 500ms to about 600ms.

19. The method of any one of claims 2 to 18, wherein the median spontaneous excitationcontraction delay is about 540ms to about 550ms.

20. The method of any one of claims 2 to 19, wherein the median spontaneous excitationcontraction delay is about 545ms.

21. The method of any one of claims 2 to 20, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 240ms to about 550ms.

22. The method of any one of claims 2 to 21, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 375 to about 465ms.

23. The method of any one of claims 2 to 22, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 415 to about 425ms.

24. The method of any one of claims 2 to 23, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 420ms.

25. The method of any one of claims 2 to 24, wherein the median spontaneous spike amplitude is about 0.55mV to about 0.95mV.

26. The method of any one of claims 2 to 25, wherein the median spontaneous spike amplitude is about 0.70mV to about 0.80mV.

27. The method of any one of claims 2 to 26, wherein the median spontaneous spike amplitude stimulation is about 0.75mV.

28. The method of any one of claims 2 to 27, wherein the median paced spike amplitude with 1Hz stimulation is about 1.90mV to about 2.10mV.

29. The method of any one of claims 2 to 28, wherein the median paced spike amplitude with 1Hz stimulation is about 1.95mV to about 2.05mV.

30. The method of any one of claims 2 to 29, wherein the median paced spike amplitude with 1Hz stimulation is about 2.00mV.

31. The method of any one of claims 2 to 30, wherein the median spontaneous spike slope is about 1.45V / s to about 1.75V / s.

32. The method of any one of claims 2 to 31, wherein the median spontaneous spike slope is about 1.50V / s to about 1.75V / s.

33. The method of any one of claims 2 to 32, wherein the median spontaneous spike slope is about 1.55V / s to about 1.65V / s.

34. The method of any one of claims 2 to 33, wherein the median spontaneous spike slope is about 1.60V / s.

35. The method of any one of claims 2 to 34, wherein the median paced spike slope is about 4.00V / s to about 4.60V / s.

36. The method of any one of claims 2 to 35, wherein the median paced spike slope is about 4.1V / s to about 4.50V / s.

37. The method of any one of claims 2 to 36, wherein the median paced spike slope is about 4.25V / s to about 4.35V / s.

38. The method of any one of claims 2 to 37, wherein the median paced spike slope is about 4.30V / S.

39. The method of any one of claims 2 to 38, wherein the spontaneous LFP has a median duration of about 75ms to about 900ms.

40. The method of any one of claims 2 to 39, wherein the spontaneous LFP has a median duration of about 100ms to about 700ms.

41. The method of any one of claims 2 to 40, wherein the spontaneous LFP has a median duration of about 200ms to about 500ms.

42. The method of any one of claims 2 to 41, wherein the spontaneous LFP has a median duration of about 250ms to about 300ms.

43. The method of any one of claims 2 to 42, wherein the spontaneous LFP has a median duration of about 265ms to about 275ms.

44. The method of any one of claims 2 to 43, wherein the spontaneous LFP has a median duration of about 270ms.

45. The method of any one of claims 2 to 44, wherein the paced LFP with 1Hz stimulation has a median duration of about 800ms to about 900ms.

46. The method of any one of claims 2 to 45, wherein the paced LFP with 1Hz stimulation has a median duration of about 820ms to about 880ms.

47. The method of any one of claims 2 to 46, wherein the paced LFP with 1Hz stimulation has a median duration of about 845ms to about 855ms.

48. The method of any one of claims 2 to 47, wherein the paced LFP with 1Hz stimulation has a median duration of about 850ms.

49. The method of any one of claims 2 to 48, wherein the LFP is determined from the electrical signals of 2 to 30 cells.

50. The method of any one of claims 2 to 49, wherein the plurality of cells comprises about 4 million to about 40 million cells.

51. The method of claim 2, wherein the plurality of cells is a batch of cells.

52. The method of any one of claims 2 to 51, wherein the one or more properties are determined from 3 or more samples of the plurality of cells.

53. The method of claim 52, wherein each sample comprises at least one cardiac cell.

54. The method of any one of claims 2 to 53, wherein the one or more electrophysiological properties are obtained from an extracellular recording of the plurality of cells.

55. The method of claim 54, wherein the extracellular recording of the batch comprises recording on a multi-electrode array (MEA).

56. The method of claim 55, wherein the MEA comprises a coating.

57. The method of claim 55 or 56, wherein the MEA is at least partially coated with a high- molecular weight glycoprotein.

58. The method of claim 57, wherein the high molecular weight glycoprotein is laminin.

59. The method of any one of claims 2 to 58, wherein the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising 0.2mM to 2.0mM glucose, 130-155mM sodium chloride, 4-6mM potassium chloride, and 0.3-1.3mM calcium chloride; and a pH of 7.4 to 7.8 and temperature of 35°C to 37°C.

60. The method of any one of claims 2 to 59, wherein the cells are cardiomyocytes.

61. The method of claim 60, wherein the cardiomyocytes are mature cardiomyocytes.

62. The method of any one of claims 2 to 61, wherein the cells are derived from pluripotent stem cells.

63. The method of any one of claims 2 to 61, wherein the cells are derived from embryonic stem cells.

64. The method of any one of claims 2 to 63, wherein the plurality of cells is a batch of cardiomyocytes.

65. The method of claim 64, wherein the plurality of cells is a batch of mature cardiomyocytes.

66. The method of claim 65, wherein the batch of mature cardiomyocytes comprises 40 million cells.

67. The method of any one of claims 2 to 66, wherein preparing the plurality of cells for cardiac cell therapy comprises contacting the plurality of cells with a physiologically acceptable medium suitable for administration to a subject.

68. A composition for cardiac cell therapy comprising a plurality of cells in a physiologically acceptable medium suitable for administration to a patient in need thereof, wherein the plurality of cells are derived from stem cells; and wherein at least 50% of the plurality of cells have one or more of the following electrophysiological properties:(a) a median spontaneous beat period of about 1.6s to about 5.0s and / or a median paced beat period with 1 Hz stimulation of about 0.80s to about 1.20s;(b) a median spontaneous beat amplitude of about 0.60% to about 1.20% and / or a median paced beat amplitude with 1 Hz stimulation of about 0.30% to about 0.90%;(c) a median spontaneous excitation contraction delay of about 250ms to about 1100ms and / or a median excitation contraction delay with 1 Hz stimulation of about 100ms to about 600ms;(d) a spontaneous spike amplitude of about 0.5mV to about 1.2mV and / or a paced spike amplitude with 1Hz stimulation of about 1.80mV to about 2.20mV;(e) a spontaneous spike slope of about 1.40V / s to about 1.80V / s and / or a paced spike slope with 1Hz stimulation of about 3.9V / s to about 4.7V / s; and / or(f) a spontaneous local field potential (LFP) having a median duration of about 60ms to about 950ms and / or a paced LFP with 1Hz stimulation having a median duration of about 600ms to about 1000ms; wherein the one or more electrophysiological properties are measured by extracellular recording via a multi electrode array (MEA) at least partially coated with a high-molecular weight glycoprotein under conditions suitable for recording cardiac cell activity.

69. The composition of claim 68, wherein the median spontaneous beat period is about 2.5s to about 4.5s.

70. The composition of claim 68 or 69, wherein the median spontaneous beat period is about 3.0s to about 4.0s.

71. The composition of any one of claims 68 to 70, wherein the median spontaneous beat period is about 3.2s to about 3.7s.

72. The composition of any one of claims 68 to 71, wherein the median spontaneous beat period is about 3.5s.

73. The composition of any one of claims 68 to 72, wherein the median paced beat period with 1Hz stimulation is about 0.90s to about 1.10s.

74. The composition of any one of claims 68 to 73, wherein the median paced beat period with 1Hz stimulation is about 0.95s to about 1.05s.

75. The composition of any one of claims 68 to 74, wherein the median paced beat period with 1Hz stimulation is about 1.00s.

76. The composition of any one of claims 68 to 75, wherein the median spontaneous beat amplitude is about 0.70% to about 1.10%.

77. The composition of any one of claims 68 to 76, wherein the median spontaneous beat amplitude is about 0.80% to about 1.00%.

78. The composition of any one of claims 68 to 77, wherein the median spontaneous beat amplitude stimulation is about 0.90%.

79. The composition of any one of claims 68 to 78, wherein the median paced beat amplitude with 1Hz stimulation is about 0.40% to about 0.80%.

80. The composition of any one of claims 68 to 79, wherein the median paced beat amplitude with 1Hz stimulation is about 0.50% to about 0.70%.

81. The composition of any one of claims 68 to 80, wherein the median paced beat amplitude with 1Hz stimulation is about 0.60%.

82. The composition of any one of claims 68 to 81, wherein the median spontaneous excitation-contraction delay is about 350ms to about 800ms.

83. The composition of any one of claims 68 to 82, wherein the median spontaneous excitation-contraction delay is about 450ms to about 700ms.

84. The composition of any one of claims 68 to 83, wherein the median spontaneous excitation-contraction delay is about 500ms to about 600ms.

85. The composition of any one of claims 68 to 84, wherein the median spontaneous excitation-contraction delay is about 540ms to about 550ms.

86. The composition of any one of claims 68 to 85, wherein the median spontaneous excitation-contraction delay is about 545ms.

87. The composition of any one of claims 68 to 86, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 240ms to about 550ms.

88. The composition of any one of claims 68 to 87, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 375 to about 465ms.

89. The composition of any one of claims 68 to 88, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 415 to about 425ms.

90. The composition of any one of claims 68 to 89, wherein the median paced excitationcontraction delay with 1Hz stimulation is about 420ms.

91. The composition of any one of claims 68 to 90, wherein the median spontaneous spike amplitude is about 0.55mV to about 0.95mV.

92. The composition of any one of claims 68 to 91, wherein the median spontaneous spike amplitude is about 0.70mV to about 0.80mV.

93. The composition of any one of claims 68 to 92, wherein the median spontaneous spike amplitude stimulation is about 0.75mV.

94. The composition of any one of claims 68 to 93, wherein the median paced spike amplitude with 1Hz stimulation is about 1.90mV to about 2.10mV.

95. The composition of any one of claims 68 to 94, wherein the median paced spike amplitude with 1Hz stimulation is about 1.95mV to about 2.05mV.

96. The composition of any one of claims 68 to 95, wherein the median paced spike amplitude with 1Hz stimulation is about 2.00m V.

97. The composition of any one of claims 68 to 96, wherein the median spontaneous spike slope is about 1.45V / s to about 1.75V / s.

98. The composition of any one of claims 68 to 97, wherein the median spontaneous spike slope is about 1.50V / s to about 1.75V / s.

99. The composition of any one of claims 68 to 98, wherein the median spontaneous spike slope is about 1.55V / s to about 1.65V / s.

100. The composition of any one of claims 68 to 99, wherein the median spontaneous spike slope is about 1.60V / s.

101. The composition of any one of claims 68 to 100, wherein the median paced spike slope is about 4.00V / s to about 4.60V / s.

102. The composition of any one of claims 68 to 101, wherein the median paced spike slope is about 4.1V / s to about 4.50V / s.

103. The composition of any one of claims 68 to 102, wherein the median paced spike slope is about 4.25V / s to about 4.35V / s.

104. The composition of any one of claims 68 to 103, wherein the median paced spike slope is about 4.30V / s.

105. The composition of any one of claims 68 to 104, wherein the spontaneous LFP has a median duration of about 75ms to about 900ms.

106. The composition of any one of claims 68 to 105, wherein the spontaneous LFP has a median duration of about 100ms to about 700ms.

107. The composition of any one of claims 68 to 106, wherein the spontaneous LFP has a median duration of about 200ms to about 500ms.

108. The composition of any one of claims 68 to 107, wherein the spontaneous LFP has a median duration of about 250ms to about 300ms.

109. The composition of any one of claims 68 to 108, wherein the spontaneous LFP has a median duration of about 265ms to about 275ms.

110. The composition of any one of claims 68 to 109, wherein the spontaneous LFP has a median duration of about 270ms.

111. The composition of any one of claims 68 to 110, wherein the paced LFP with 1Hz stimulation has a median duration of about 800ms to about 900ms.

112. The composition of any one of claims 68 to 111, wherein the paced LFP with 1Hz stimulation has a median duration of about 820ms to about 880ms.

113. The composition of any one of claims 68 to 112, wherein the paced LFP with 1Hz stimulation has a median duration of about 845ms to about 855ms.

114. The composition of any one of claims 68 to 113, wherein the paced LFP with 1Hz stimulation has a median duration of about 850ms.

115. The composition of any one of claims 68 to 114, wherein at least 50% of the plurality of cells have two or more of the electrophysiological properties of (a)-(f).

116. The composition of any one of claims 68 to 115, wherein at least 50% of the plurality of cells have three or more of the electrophysiological properties of (a)-(f).

117. The composition of any one of claims 68 to 116, wherein at least 50% of the plurality of cells have four or more of the electrophysiological properties of (a)-(f).

118. The composition of any one of claims 68 to 117, wherein at least 50% of the plurality of cells have all of the electrophysiological properties of (a)-(f).

119. The composition of any one of claims 68 to 118, wherein at least 60% of the plurality of cells have one or more of the electrophysiological properties of (a)-(f).

120. The composition of any one of claims 68 to 119, wherein at least 70% of the plurality of cells have one or more of the electrophysiological properties of (a)-(f).

121. The composition of any one of claims 68 to 120, wherein at least 80% of the plurality of cells have one or more of the electrophysiological properties of (a)-(f).

122. The composition of any one of claims 68 to 121, wherein at least 90% of the plurality of cells have one or more of the electrophysiological properties of (a)-(f).

123. The composition of any one of claims 68 to 122, wherein the high molecular weight glycoprotein is laminin.

124. The composition of any one of claims 68 to 123, wherein the conditions suitable for recording cardiac cell activity comprise an extracellular medium or solution comprising 0.2mM to 2.0mM glucose, 130-155mM sodium chloride, 4-6mM potassium chloride, and 0.3-1.3mM calcium chloride; and a pH of 7.4 to 7.8 and temperature of 35°C to 37°C.

125. The composition of any one of claims 68 to 124, wherein the cells are cardiomyocytes.

126. The composition of claim 125, wherein the cardiac cells are mature cardiomyocytes.

127. The composition of any one of claims 68 to 126, wherein the cells are derived from pluripotent stem cells.

128. The composition of any one of claims 68 to 127, wherein the cells are derived from embryonic stem cells.

129. The composition of any one of claims 68 to 128, wherein the plurality of cells comprises about 40,000 cells.

130. A method of treating a subject in need of cardiac cell therapy, the method comprising administering to the subject a cardiac cell therapy comprising the composition of any one of claims 68 to 129.

Images