Electrophysiological identification of cardiomyocytes for cardiac cell therapy

Abstract

This disclosure relates generally to methods of screening and preparing cardiac cell therapies having low risk of causing graft-induced arrythmias.

Description

[0001] ELECTROPHYSIOLOGICAL IDENTIFICATION OF CARDIOMYOCYTES FOR CARDIAC CELL THERAPY

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 614,438, filed December 22, 2023, entitled “ELECTROPHYSIOLOGICAL IDENTIFICATION OF CARDIOMYOCYTES FOR CARDIAC CELL THERAPY,” 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 cardiomyocytes assessed for risk of graft- induced arrythmia.

[0006] BACKGROUND

[0007] The ability of stem cells to regenerate injured tissue holds great promise for treatment of cardiac disease and injury; however, several obstacles to their adoption persist. Grafts of pluripotent stem-cell derived cardiomyocytes have been shown to induce arrythmias (e.g., sustained ventricular tachycardia) in large animal models. These graft- induced arrythmias may occur rapidly after administration (e.g., within 48 hours) and are sometimes resistant to antiarrhythmic drugs and cardioversion. Despite being highly desired, methods and compositions which mitigate graft-induced arrythmias have been difficult to identify thus far, posing a significant impediment to treatment of cardiac disease and injury.

[0008] SUMMARY

[0009] Cardiac cell therapies, such as cardiomyocyte grafts, can potentially treat subjects having injured cardiac tissue (e.g., as the result of heart disease or failure). However, large animal model studies have shown that administration of cardiomyocyte grafts can sometimes induce arrythmias with severe consequences. Surprisingly, the inventors of the present disclosure have identified methods and compositions which reduce the risk of graft-induced arrythmias; these methods and compositions are described herein. In some embodiments, the methods disclosed herein are useful for identifying populations of cells suitable for cardiac cell therapy.

[0010] In some aspects, provided herein is a method of preparing a batch of electroresponsive cells for cardiac cell therapy, the method comprising: (a) determining that at least a first batch of electroresponsive cells among a plurality of batches has one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA); and (b) preparing at least the first batch of electroresponsive cells for cardiac cell therapy.

[0011] In some embodiments, the at least first batch of electroresponsive cells comprises 40 million cells. In some embodiments, the one or more electrophysiological properties associated with low risk of GIA are determined from one or more samples of the at least first batch. In some embodiments, the sample comprises one or more electroresponsive cells. In some embodiments, the one or more electrophysiological properties associated with low risk of GIA are determined from 3 or more samples of the at least first batch. In some embodiments, the method comprises determining electrophysiological properties from one or more samples of the at least first batch

[0012] In some embodiments, the one or more electrophysiological properties are obtained by patch clamping isolated cells of the batch. In some embodiments, the patch clamping comprises high throughput patch clamping. In some embodiments, the one or more electrophysiological properties associated with low risk of GIA comprise action potential upstroke velocity, ion channel current, peak ion current density, maximum ion conductance, or any combination thereof. In some embodiments, the method comprises obtaining one or more electrophysiological properties by patch clamping isolated cells of the batch

[0013] In some embodiments, the isolated cells undergo patch clamp recordings under any of the conditions suitable for recording sodium channel properties disclosed herein. In some embodiments, the method comprises obtaining patch clamp recordings from the isolated cells under any of the conditions suitable for recording sodium channel properties disclosed herein. In some embodiments, the conditions suitable for recording sodium channel properties comprise: an internal solution comprising cesium fluoride, sodium chloride, HEPES, magnesium chloride, and sodium- ATP, having a normal physiological pH, and having a normal physiological osmolarity; an external solution comprising TEA-CI, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, HEPES, and D-glucose, having a normal physiological pH, and having a normal physiological osmolarity; and normal physiological temperatures. In some embodiments, the conditions suitable for recording sodium channel properties comprise: an internal solution comprising 108-112 mM cesium fluoride, 9-11 mM cesium chloride, 9- 11 mM sodium chloride, 9-11 mM HEPES, 1-2 mM magnesium chloride, and 1-3 mM sodium- ATP, having a pH of 7.1-7.3, and having an osmolarity of 280-290 mOsm; an external solution comprising 105-115 mM TEA-CI, 38-42 mM sodium chloride3-5 mM potassium chloride, 0.5- 1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 9-11 mM HEPES, and 4-6 mM D- glucose, having a pH of 7.1-7.5, and having an osmolarity of 290-300 mOsm; and a temperature of 22-27 °C.

[0014] In some embodiments, the conditions suitable for recording sodium channel properties comprise: an internal solution comprising llOmM cesium fluoride, lOmM cesium chloride, lOmM sodium chloride, lOmM HEPES, 1.5mM magnesium chloride, and 2mM sodium- ATP, having a pH of 7.2, and having an osmolarity of 285 mOsm; an external solution comprising lOOmM TEA-CI, 40mM sodium chloride, 4mM potassium chloride, ImM magnesium chloride, 2mM calcium chloride, lOmM HEPES, and 5mM D-glucose, having a pH of 7.4, and an osmolarity of 295 mOsm; and a temperature of 25°C.

[0015] In some embodiments, the isolated cells undergo patch clamp recordings under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the method comprises obtaining patch clamp recordings from the isolated cells under conditions suitable for recording calcium channel properties. In some embodiments, the conditions suitable for recording calcium channel properties comprise: an internal solution comprising cesium fluoride, sodium chloride, HEPES, magnesium chloride, and sodium- ATP, having a normal physiological pH, and having a normal physiological osmolarity; an external solution comprising TEA-CI, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, HEPES, and D-glucose, having a normal physiological pH, and having a normal physiological osmolarity; and normal physiological temperatures. In some embodiments, the conditions suitable for recording calcium channel properties comprise: an internal solution comprising 108-112 mM cesium fluoride, 9-11 mM cesium chloride, 9-11 mM sodium chloride, 9-11 mM HEPES, 1.0-2.0 magnesium chloride, and l-3mM sodium-ATP, having a pH of 7.1- 7.5, and having an osmolarity of 280-290 mOsm; an external solution comprising 135-145 mM sodium chloride, 3-5 mM potassium chloride, 0.5-2.0 mM magnesium chloride, 1-3 mM calcium chloride, 9-11 mM HEPES, and 4-6 mM D-glucose, having a pH of 7.1-7.5, and having an osmolarity of 290-300 mOsm; and a temperature of 22-27°C. In some embodiments, conditions suitable for recording calcium channel properties comprise: an internal solution comprising 110 mM cesium fluoride, 10 mM cesium chloride, 10 mM sodium chloride, 10 mM HEPES, 1.5mM magnesium chloride, and 2 mM sodium-ATP, having a pH of 7.2, and having an osmolarity of 285 mOsm; an external solution comprising 140 mM sodium chloride, 4 mM potassium chloride, 1 mM magnesium chloride, 2 mM calcium chloride, 10 mM HEPES, and 5 mM D-glucose, having a pH of 7.4, and an osmolarity of 295 mOsm; and a temperature of 25°C. In some embodiments, the at least first batch of cells comprises excitable cells.

[0016] In some embodiments, at least first batch of cells comprises immature cardiomyocytes or mature cardiomyocytes. In some embodiments, the immature cardiomyocytes or mature cardiomyocytes are derived from pluripotent stem cells. In some embodiments, the immature cardiomyocytes or mature cardiomyocytes are derived from embryonic stem cells. In some embodiments, the pluripotent stem cells express one or more markers selected from: SSCA3 / 4, TRA-160, OCT3 / 4, NANOG, SOX2.

[0017] In some embodiments, the at least first batch of cells determined to have a low risk of GIA comprises immature cardiomyocytes. In some embodiments, the immature cardiomyocytes comprise positive / high expression of cTNT, MYL2, MYL7, KCNJ2, CACNA1C, CACNA1H, SCN5A, HCN4, and comprise negative / low expression of: MKI67 and CD90.

[0018] In some embodiments, immature cardiomyocytes having a normally distributed maximum sodium conductance are determined to have a low risk of GIA. In some embodiments, the immature cardiomyocytes have a normally distributed maximum sodium conductance under any of the conditions suitable for recording sodium channel properties disclosed herein. In some embodiments, preparing the immature cardiomyocytes for cardiac cell therapy comprises contacting the immature cardiomyocytes with a maturation cocktail under any of the conditions that promote cardiomyocyte maturation disclosed herein.

[0019] In some embodiments, the at least first batch of cells determined to have a low risk of GIA comprises mature cardiomyocytes. In some embodiments, the mature cardiomyocytes comprise positive / high expression of MYL2, cTNT, SCN5A, KCNJ2, CACNA1C and comprise negative / low expression of MLY7, CD90, CACNA1H, HCN4, and MKI67.

[0020] In some embodiments, the mature cardiomyocytes have a spontaneous upstroke velocity of about 45V / s to 60V / s under any of the conditions suitable for recording sodium channel properties disclosed herein or under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have an action potential upstroke velocity of at least about 48V / s to 57V / s under any of the conditions suitable for recording sodium channel properties disclosed herein or under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have an action potential upstroke velocity of 50V / s to 55V / s under any of the conditions suitable for recording sodium channel properties disclosed herein or under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have a peak sodium current density of at least about 135pA / pF within a range of -20mV to -40mV under any of the conditions suitable for recording sodium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have a peak sodium current density of at least about 140pA / pF within a range of -20mV to -40mV under any of the conditions suitable for recording sodium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have a peak sodium current density of >| 145 |pA / pF within a range of -20mV to -40mV under any of the conditions suitable for recording sodium channel properties disclosed herein.

[0021] In some embodiments, the mature cardiomyocytes have a normal (Gaussian) frequency distribution of sodium conductance under any of the conditions suitable for recording sodium channel properties disclosed herein.

[0022] In some embodiments, the mature cardiomyocytes have a peak calcium current density at least |2| pA / pF within a range of lOmV to 20mV under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have a peak calcium current density of at least |3| pA / pF within a range of lOmV to 20mV under any of the conditions suitable for recording calcium channel properties disclosed herein. In some embodiments, the mature cardiomyocytes have a peak calcium current density of >|4| pA / pF within a range of lOmV to 20mV under any of the conditions suitable for recording calcium channel properties disclosed herein.

[0023] In some embodiments, the mature cardiomyocytes have a normally distributed maximum calcium conductance under any of the conditions suitable for recording calcium channel properties disclosed herein.

[0024] In some embodiments, the mature cardiomyocytes have a peak sodium current density of at least -150 pA / pF to -200 pA / pF under any of the conditions suitable for recording sodium channel properties disclosed herein and peak calcium current density of at least -9 pA / pF to -11 pA / pF under any of the conditions suitable for recording calcium channel properties. In some embodiments, the mature cardiomyocytes have a peak sodium current density of >| 145 |pA / pF within a range of -20mV to -40mV under conditions suitable for recording sodium channel properties and peak calcium current density of >|4| pA / pF within a range of lOmV to 20mV under conditions suitable for recording calcium channel properties disclosed herein.

[0025] In some embodiments, preparing the mature cardiomyocytes for cardiac cell therapy comprises contacting the mature cardiomyocytes with a physiologically acceptable medium suitable for administration to a subject. In some aspects, provided herein is a method of differentiating a plurality of cardiomyocyte precursor cells for cardiac cell therapy, the method comprising contacting a plurality of cardiomyocyte precursor cells that have been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a medium comprising a differentiating agent under any of the conditions that promote cardiomyocyte differentiation disclosed herein.

[0026] In some aspects, provided herein is a method of maturing a plurality of immature cardiomyocytes for cardiac cell therapy, the method comprising contacting the plurality of immature cardiomyocytes that been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a maturation cocktail under any of the conditions that promote cardiomyocyte maturation disclosed herein.

[0027] In some aspects, provided herein is a method of preparing a plurality of mature cardiomyocytes for cardiac cell therapy, the method comprising contacting the plurality of mature cardiomyocytes that been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a physiologically acceptable medium suitable for administration to a subject in need of cardiac cell therapy.

[0028] In some aspects, provided herein is 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 comprises mature cardiomyocytes; and wherein no more than 20% of the mature cardiomyocytes have one or more electrophysiological properties associated with a high risk of graft-induced arrythmia (GIA).

[0029] In some aspects, provided herein is a composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells comprises immature cardiomyocytes; and wherein no more than 20% of the immature cardiomyocytes have one or more electrophysiological properties associated with a high risk of graft- induced arrythmia (GIA).

[0030] In some aspects, provided herein is a composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells comprises cardiomyocyte precursors; and wherein no more than 20% of the cardiomyocyte precursors have one or more electrophysiological properties associated with a high risk of graft- induced arrythmia (GIA).

[0031] In some aspects, provided herein is a composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells has been determined to have one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA) have been prepared for cardiac cell therapy. In some aspects, provided herein is a method of treating a subject in need of cardiac cell therapy, the method comprising administering to the subject a cardiac cell therapy comprising: a batch of electroresponsive cells prepared according to a method described herein, or a composition described herein.

[0032] In some aspects, provided herein is a method of treating a subject in need of cardiac cell therapy, the method comprising: (a) determining that at least a first batch of electroresponsive cells among a plurality of batches has one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA); and (b) administering the at least the first batch of electroresponsive cells to the subject. In some embodiments, the determining in (a) comprises a method described herein.

[0033] In some aspects, provided herein is a method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of electroresponsive cells to a subject, wherein the plurality of electroresponsive cells has one or more electrophysiological properties associated with low risk of graft- induced arrythmia (GIA). In some embodiments, the plurality of electroresponsive cells are comprised in a composition described herein.

[0034] In some aspects, provided herein is a method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of electroresponsive cells to a subject, wherein the plurality of electroresponsive cells has been determined to have one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA).

[0035] In some embodiments, the plurality of electroresponsive cells is comprised in a composition described herein.

[0036] In some embodiments, the plurality of electroresponsive cells has one or more electrophysiological properties associated with low risk of GIA (e.g., ventricular tachycardia).

[0037] In some embodiments, a method disclosed herein comprises determining that a plurality of electroresponsive cells have one or more electrophysiological properties associated with low risk of GIA (e.g., ventricular tachycardia).

[0038] In some embodiments, a method disclosed herein comprises administering a composition to the subject, the composition comprising a plurality of electroresponsive cells to a subject, wherein the plurality of electroresponsive cells has been determined to have one or more electrophysiological properties associated with low risk of GIA (e.g., ventricular tachycardia). BRIEF DESCRIPTION OF DRAWINGS

[0039] FIGs. 1A-1C show how treatment of myocardial infarction by implantation of cardiomyocytes can cause sustained GIA post cell delivery. FIG. 1A shows an administration protocol for induction of a myocardial infraction (MI) in a Yorkshire pig and subsequent cell transplantation. FIG. IB shows an EKG recording of an exemplary pig having sustained graft- induced arrythmia (GIA) after cell implantation. Neither administration of an anti- arrhythmic cocktail nor cardioversion reversed the sustained RA. VAP = vascular access port. FIG. 1C shows post-necropsy scoring of the implanted cardiomyocytes in two media preparations by severity of induced GIA and categorization as low GIA (scores under 3) or high GIA (scores above 3).

[0040] FIGs. 2A-2B show that neither high nor low GIA batches exhibit Funny current (If), suggesting HCN4, a potassium channel, is unlikely to cause graft-induced arrythmias. FIG. 2A shows current in mature CMs (top) vs immature CMs (bottom) during voltage clamping at - 130mV for 500ms. Immature CMs exhibit Funny current, an HCN4-mediated current, while mature CMs do not. FIG. 2B shows a comparison of the same voltage clamp experiment of FIG. 3A in immature CMs (positive control), low GIA CMs, and high GIA CMs. Though immature CMs display If, neither low GIA nor high GIA batches exhibit If, suggesting HCN4 is unlikely to cause graft- induced arrythmias.

[0041] FIGs. 3A-3C show that high GIA cells and low GIA cells exhibit different action potential (AP) upstroke velocities (e.g., exhibit spiking without injection of current). FIG. 3A shows a representative trace of high GIA and low GIA APs. FIG. 3B shows that spontaneous AP in high GIA cells have a slower upstroke velocity when compared to low GIA cells, suggesting high GIA cells contain sodium channels with altered activation kinetics. FIG. 3C shows that paced upstroke velocity is similar across low and high GIA cells.

[0042] FIGs. 4A-4C show that high GIA cells, on average, have shorter spontaneous AP durations, longer paced AP durations, and comparable resting membrane potentials (RMP). FIG. 4A shows quantification of spontaneous AP waveform AP90 for low GIA and high GIA cells. High GIA cells exhibit shorter spike duration relative to low GIA cells, suggesting underlying sodium and calcium channel dysfunctions. FIG. 4B shows quantification of paced waveform AP90 for low GIA and high GIA cells. High GIA cells exhibit longer spike durations when APs are paced relative to low GIA cells. FIG. 4C shows RMP at 0 current for both low and high GIA cells. RMPs for low and high GIA cells are similar. High GIA batches also had more spontaneously active cells (5 / 18 cells; 27.778%) than low GIA batches (2 / 15 cells; 13.333%), but no correlation between spontaneity and RMP was observed. These findings indicate expression of KCNJ2, a potassium channel, does not contribute to high GIA.

[0043] FIGs. 5A-5F show manual patch clamping or high throughput patch clamping of low and high GIA cells to probe sodium current channel activity. FIG. 5A shows representative traces for manual patch clamping of low GIA cells and high GIA cells. Cells were voltage clamped from -60mV to +60mV (in lOmV steps) for 50ms and peak current was measured. FIG. 5B is a sodium channel current / voltage (IV) plot showing current density from manually patched clamped low and high GIA cells. High GIA cells displayed attenuated sodium channel density, which is produced by the ion channel NaV1.5 (SCN5A), relative to low GIA cells, suggesting sodium channel dysfunctions in high GIA cells. FIG. 5C is a sodium channel IV plot for showing peak from low GIA and high GIA cells patched using high throughput patch clamping (NANION SYNCHROPATCH® 384). High GIA cells displayed similarly attenuated sodium channel density as cells recorded with manual patch clamp. FIG. 5D shows representative current traces of low GIA (Run 10) and high GIA (Run 6) cells at a -30mV voltage step. FIG. 5E is a sodium channel IV plot combining cells recorded with manual patch clamp (Run 4, Run 5) and cells recorded with high throughput patch clamping (Run 6). High GIA and low GIA differences persist regardless of recording method. FIG. 5F shows a summary of peak maximum sodium peak densities from high GIA and low GIA cells.

[0044] FIGs. 6A-6C shows sodium conductance of low GIA (Run 4) and high GIA (Run 5 and Run 6). FIG. 6A shows maximum sodium conductance of cells from low GIA and high GIA batches. FIG. 6B shows predicted versus actual sodium conductance for the same cells; results suggest a lack of normal distribution for high GIA cells only. FIG. 6C shows distribution of the maximum sodium conductance; sodium conductance of low GIA cells was normally distributed, while sodium conductance of high GIA cells was not.

[0045] FIGs. 7A-7E show manual patch clamping or high throughput patch clamping of low and high GIA cells to probe calcium current channel activity. FIG. 7A shows voltage steps used to probe calcium current with manual patch clamping. Low GIA cells and high GIA cells were voltage clamped with an initial pre-pulse (arrow A) at -60mV for 50ms to eliminate sodium current overlap, then subjected to voltage clamp for calcium current induction (arrow B) from - 60mV to +60mV (in 5mV steps). FIG. 7B is a calcium channel IV plot showing peak calcium current density from manually patched clamped low and high GIA cells. High GIA cells displayed attenuated L-type calcium current density relative to low GIA cells, indicating calcium channel dysfunctions in high GIA cells. FIG. 7C is a calcium channel IV plot showing peak calcium current density from low GIA (Run 10) and high GIA (Run 6) cells patched using high throughput patch clamping. High GIA cells displayed similarly attenuated calcium current density as cells recorded with manual patch clamp. FIG. 7D shows a representative trace of sodium vs calcium responses at a +10mV voltage step. FIG. 7E is a calcium channel IV plot combining cells recorded with manual patch clamp (Run 4, Run 5) and cells recorded with high throughput patch clamping (Run 6).

[0046] DETAILED DESCRIPTION

[0047] 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. Though cardiac cell therapies hold great promise for treating cardiac injuries, their use in large animal test subjects (e.g., pigs) has faced a number of obstacles, including the emergence of graft-induced arrythmias. Large animal test subjects to which cardiac grafts are administered often experience arrythmias of varying frequency and severity, some of which cannot be treated with antiarrhythmic drugs or cardioversion. Though many attempts have been made to identify cells having a risk of contributing arrythmias, all have so far have been unsuccessful. The inventors have developed methods of identifying cells for preparation into cardiac cell therapies with a low risk of causing graft-induced arrythmias.

[0048] Described herein, in some embodiments, are compositions and methods related to identifying and / or preparing suitable cells and / or suitable batches of cells for cardiac cell therapies. In some aspects, compositions disclosed herein comprise isolated cardiomyocytes (e.g., precursors, immature, mature) having electrophysiological characteristics associated with low risk of causing graft- induced arrythmia (low GIA). Described herein, in some embodiments, are compositions and methods related to identifying and / or preparing suitable cells and / or suitable batches of cells for cardiac cell therapies.

[0049] Cardiomyocytes

[0050] The present disclosure relates, in some aspects, to cardiac cell therapies (e.g., cardiac grafts) comprising cardiomyocytes having one or more properties associated with low risk causing graft-induced arrythmia when administered to a subject as part of a cardiac cell therapy. In some embodiments, cardiomyocytes are 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.

[0051] In some embodiments, cardiomyocytes are cardiomyocyte precursors. In some embodiments, a cardiomyocyte precursor is any precursor (e.g., progenitor) cell with the capacity to differentiate into a cardiomyocyte cell. In some embodiments, a cardiomyocyte precursor is or is derived from a 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). As described herein, cardiomyocyte precursors may have the following characteristics: (1) positive / high expression of one or more of the following markers: stage-specific embryonic antigen 3 and / or 4 (SSEA3 / 4), podocalyxin (TRA-160), octamer-binding transcription 3 and / or 4 (OCT3 / 4), homeobox protein NANOG (NANOG), and sex-determining region Y-box 2 (SOX2) (e.g., as determined using flowcytometric analysis); (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.

[0052] In some embodiments, cardiomyocytes are immature cardiomyocytes. In some embodiments, an immature cardiomyocyte is 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 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: (1) cluster of differentiation 36 (CD36) negative / low (relative to a mature cardiomyocyte), myosin regulatory light chain 2v (MLC2v) negative / low (relative to a mature cardiomyocyte), and myosin regulatory light chain 2a (MLC2a) positive / high (relative to a mature cardiomyocyte), e.g., using flow cytometric analysis; and (2) capable of spontaneous action potentials and contraction. In some embodiments, an immature cardiomyocyte expresses (e.g., is positive / high for) NK2 homeobox 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 / Low” genetic or molecular markers provided in Table 1. In some embodiments, an immature cardiomyocyte has one or more electrophysiological properties according to Table 1.

[0053] 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, the cardiomyocytes are matured in vivo (e.g., in a subject). In some embodiments, the 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: (1) cardiac muscle troponin T (cTNT, also referred to synonymously herein as TNNT2) positive / high, MLC2v positive / high, and downregulated (e.g., reduced expression of) MLC2a (relative to an immature cardiomyocytes), e.g., using flow cytometric analysis; and (2) capable of contraction with application of an external stimulus (e.g., electrical stimulus). In some embodiments, mature cardiomyocytes are TNI 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. In some embodiments, a mature cardiomyocyte has one or more electrophysiological properties according to Table 1. Table 1. Non-limiting characteristics of cardiomyocytes throughout development

[0054] 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.

[0055] Expression of a marker (e.g., a gene, a gene product) 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. A cell may be understood to be positive (+) for a marker which is present in the cell at a sufficient level to be 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 a baseline or threshold (e.g., above a population average, at a higher quantity than a control cell). A cell may be understood to be negative (-) for a marker which is not present in the cell in a sufficient amount to be 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 a baseline or threshold (e.g., below a population average, at a lower quantity relative to a control cell).

[0056] Methods of Identifying Cells Suitable for Cardiac Cell Therapy

[0057] The present disclosure relates, in some aspects, to methods of identifying cells that are suitable for cardiac cell therapy. In some embodiments, the method comprises identifying cells as having one or more properties associated with low risk or high risk of causing graft-induced arrythmia (GIA) when administered to a subject as part of a cardiac cell therapy (e.g., cardiac graft). Generally, the methods disclosed herein can be practiced on any cardiomyocyte (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, an electroresponsive cell is an excitable cell (e.g., one capable of producing an action potential).

[0058] Graft-induced arrythmias (GIAs) are arrythmias in the heart of a subject to whom cardiac cell therapy (e.g., a cardiac graft) has been administered, and which are presumed to be caused, wholly or in part, by the cardiac cell therapy. Arrythmia, also known as dysrhythmia, can refer to an irregularity in cardiac activity (e.g., rate, rhythm). Non-limiting examples of arrythmias include: extrasystole, supraventricular arrhythmia (e.g., paroxysmal supraventricular tachycardia (PSVT), accessory pathway tachycardia, AV nodal re-entrant tachycardia (AVNRT), Atrial tachycardia, Atrial fibrillation, Atrial flutter), ventricular arrhythmia (e.g., Premature ventricular contractions (PVCs), ventricular tachycardia (V-tach), ventricular fibrillation (V-fib), or long QT), and bradyarrhythmia (e.g., Sinus node dysfunction or heart block). In some embodiments, a GIA is tachycardia. In some embodiments, a GIA is ventricular tachycardia, such as sustained ventricular tachycardia. In some embodiments, a cardiac cell therapy disclosed herein reduces the risk of GIA (e.g., sustained ventricular tachycardia). A GIA may occur after a cardiac graft. GIAs (e.g., ventricular tachycardia) may be caused at least in part by a subpopulation of cells (e.g., atrial cells, pacemaker cells, conduction system cells, and / or other cardiac or non-cardiac subpopulation of cells responsible or contributing to graft associated arrhythmia). A GIA may be caused, for example, by a subpopulation of stem cell-derived cardiomyocytes. GIAs may be transient or sustained (e.g., persistent, or chronic). A GIA may be associated without adverse symptoms. In some embodiments, a graft-induced arrythmia is associated with adverse symptoms of which nonlimiting examples include palpitations, lightheadedness, shortness of breath, chest pain, decreased levels of consciousness, stroke, heart failure, or death.

[0059] In some embodiments, a GIA can be treated or managed such that adverse symptoms are reduced or eliminated. In some embodiments, a graft-induced arrythmia can be treated such that normal cardiac activity is restored. In some embodiments, GIAs are temporarily eliminated or reduced, such that the arrythmia recurs even with treatment.

[0060] In some embodiments, GIAs are resistant (e.g., not fully eliminated by) typical treatments for arrhythmia (e.g., administration of antiarrhythmic drugs and / or cardioversion). In some embodiments, GIAs are not eliminated or reduced by administration of antiarrhythmic drugs. In some embodiments, GIAs are not eliminated or reduced by cardioversion (e.g., pharmacological, electrical).

[0061] Table 2 summarizes graft-induced arrythmias observed in Yorkshire pig models of myocardial infarction, as described in Example 1.

[0062] Table 2, Arrythmia Scores in Yorkshire Pig Models of Myocardial Infarction

[0063] GIA = graft-induced arrythmia; BAR = bright, alert, and responsive; PVC = premature ventricular contractions; non-sustained GIA = GIA lasting < 30s; sustained GIA = GIA lasting >30s;

[0064] In some embodiments, cardiomyocytes are identified as being “low GIA”, such that the cardiomyocytes have one or more properties associated with low risk of causing arrythmias when administered to a subject as part of a graft. In some embodiments, cardiomyocytes identified as low GIA are mature cardiomyocytes. In some embodiments, cardiomyocytes identified as low GIA are immature cardiomyocytes. In some embodiments, cardiomyocytes identified as low GIA are cardiomyocyte precursors.

[0065] In some embodiments, cardiomyocytes are identified as being “high GIA”, such that the cardiomyocytes have one or more properties associated with high risk of causing arrythmias when administered to a subject as part of a graft. In some embodiments, cardiomyocytes identified as high GIA are mature cardiomyocytes. In some embodiments, cardiomyocytes identified as high GIA are immature cardiomyocytes. In some embodiments, cardiomyocytes identified as high GIA are cardiomyocyte precursors.

[0066] In some embodiments, methods of identifying cardiomyocytes as high GIA or low GIA comprise determining one or more electrophysiological properties of the cardiomyocytes.

[0067] In some embodiments, an electrophysiological property of cardiomyocytes comprises action potential waveform and / or frequency. Action potentials are rapid changes in voltage (e.g., membrane potential) which trigger electrical, chemical, and / or mechanical changes in a cell (e.g., trigger a contraction in a cardiomyocyte). Action potentials in cardiac cells generally comprise distinct phases of ion channel activity. In phase 0, an initial, rapid inward current (e.g., depolarization phase, “upstroke” phase) driven by sodium channel activity occurs, having a typical duration of about 2ms in ventricular cardiomyocytes. Phase 1 begins as sodium channels inactivate, allowing a brief outward current (e.g., inactivation phase, “notch” phase) driven by potassium channel activity. In phase 2 (e.g., “plateau” phase), L-type calcium channels chloride channels activate, allowing for inward flux of calcium and transient outward flux of potassium ions. In phase 3 (e.g., repolarization phase, “downstroke” phase), L-type calcium channels slowly close; the cell returns to resting membrane potential, entering phase 4. In some embodiments, action potentials are invoked by application of an external stimulus (e.g., current injection, voltage step, activity of electrically coupled cell). In some embodiments, action potentials are spontaneous (e.g., occur without an external stimulus). 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), phase duration (e.g., notch duration), refractory period duration, percent action potential duration (e.g., AP30, AP50, AP70, AP90), and action potential morphology.

[0068] In some embodiments, cardiomyocytes identified as being low GIA have a resting membrane potential (e.g., an average resting membrane potential) of between about -40mV to about -55mV, inclusive. In some embodiments, cardiomyocytes identified as being low GIA have a resting membrane potential of about -45mV to about -50mV, inclusive.

[0069] In some embodiments, cardiomyocytes identified as being high GIA have a resting membrane potential (e.g., an average resting membrane potential) of between about -45mV to about -60mV, inclusive. In some embodiments, cardiomyocytes identified as being high GIA have a resting membrane potential of between about -50mV to about -55mV. In some embodiments, cardiomyocytes identified as being putative high GIA cells have an increased membrane potential (e.g., hyperpolarized; further negative) relative to cardiomyocytes identified as being putative low GIA cells.

[0070] In some embodiments, cardiomyocytes identified as being high GIA have a spontaneous upstroke (e.g., depolarization) velocity of about 50V / s to 70V / s, inclusive, under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a spontaneous upstroke (e.g., depolarization) velocity of about 50V / s to 55V / s, inclusive, under conditions shown in Table 3.

[0071] In some embodiments, cardiomyocytes identified as being putative high GIA have increased variability in average spontaneous upstroke velocity relative to cardiomyocytes identified as putative low GIA.

[0072] In some embodiments, cardiomyocytes identified as being low GIA have a peak amplitude of about 75mV to about 95mV, inclusive. In some embodiments, cardiomyocytes identified as being low GIA have a peak amplitude of about 80mV to about 90mV, inclusive. In some embodiments, cardiomyocytes identified as being low GIA have a peak amplitude of about 85mV.

[0073] In some embodiments, cardiomyocytes identified as being high GIA have a peak amplitude of about 80mV to about l lOmV. In some embodiments, cardiomyocytes identified as being high GIA have a peak amplitude of about 85mV to about 105mV. In some embodiments, cardiomyocytes identified as being high GIA have a peak amplitude of about 90mV to about lOOmV, inclusive. In some embodiments, cardiomyocytes identified as being high GIA have a peak amplitude of about 95mV. In some embodiments, cardiomyocytes identified as being putative high GIA cells have an increased peak amplitude relative to the peak amplitude of cardiomyocytes identified as being putative low GIA cells.

[0074] In some embodiments, cardiomyocytes identified as being high GIA have a spontaneous AP90 (e.g., time to reach 90% of total spontaneous action potential duration) between about 350ms to about 400ms, inclusive, under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a spontaneous AP90 between about 320ms to about 370ms, inclusive, under conditions shown in Table 3.

[0075] In some embodiments, cardiomyocytes identified as being high GIA have a spontaneous AP70 (e.g., time to reach 70% of total spontaneous action potential duration) between about 205ms to about 250ms, inclusive, under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a spontaneous AP70 between about 210ms to about 240ms under conditions shown in Table 3.

[0076] In some embodiments, cardiomyocytes identified as being high GIA have a spontaneous AP50 (e.g., time to reach 50% of total spontaneous action potential duration) of about 130ms to about 170ms under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a spontaneous AP50 of about 140ms to about 160ms under conditions shown in Table 3.

[0077] In some embodiments, cardiomyocytes identified as being high GIA have a spontaneous AP30 (e.g., time to reach 30% of total spontaneous action potential duration) of about 90ms to about 115ms under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a spontaneous AP30 of about 90ms to about 100ms under conditions shown in Table 3.

[0078] In some embodiments, an electrophysiological property of cardiomyocytes comprises ion current density. Current density is a measure of flux of an ion across a cell membrane as a function of membrane potential, and intracellular and extracellular concentrations of the ion. In some embodiments, current density is calculated as peak ion current divided by capacitance, as a function of voltage input. Current density can be measured for any ion for which a cardiomyocyte has a corresponding channel (e.g., sodium, calcium, chlorine, potassium) and can be calculated at one or voltage steps. In some embodiments, cardiomyocytes identified as being low GIA have a maximum peak sodium current density of >| 145 |p A / pF within a range of -20mV to -40mV under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being low GIA have a maximum peak L-type calcium current density of >|4|pA / pF within a range of lOmV to 20mV under conditions shown in Table 3.

[0079] In some embodiments, cardiomyocytes identified as being high GIA have a maximum peak sodium current density of [>|65|pA / pF within a range of -20mV to -40mV under conditions shown in Table 3. In some embodiments, cardiomyocytes identified as being high GIA have a maximum peak L-type calcium current density of >|2|pA / pF within a range of lOmV to 20mV under conditions shown in Table 3. In some embodiments, an electrophysiological property of cardiomyocytes comprises ion conductance. Ion conductance refers to the permeability of a cell membrane to an ion, typically mediated by expression of ion channels, transporters and / or pumps, as well as intracellular and extracellular concentrations of the ion. Conductance can be measured for any ion for which a cardiomyocyte has a corresponding channel, transports, and / or pump (e.g., sodium, calcium, chlorine, potassium).

[0080] In some embodiments, an electrophysiological property of cardiomyocytes comprises a channel rectification characteristic.

[0081] In some embodiments, an electrophysiological property of cardiomyocytes comprises an electrophysiological profile comprising action potential waveform characteristics, ion current density, ion channel activation and inactivation curve, ion conductance, or any combination thereof.

[0082] In some embodiments, one or more electrophysiological properties of cardiomyocytes are determined by patch clamping cardiomyocytes. Patch clamping refers to techniques for measuring electrophysiological properties (e.g., ion channel function) of a cell; a cell which has undergone patch clamping has been “patched.” Patch clamping allows for the assessment of ion channel function in a cell (e.g., cardiomyocyte) by recording transmembrane currents through a specific ion channel expressed by the cell. Traditionally, patch clamping comprises manual patch clamping, in which a glass pipette filled with an internal solution is positioned against the membrane of an isolated cell in an external solution to form a high resistance seal. Voltage (e.g., via voltage clamping), current (e.g., via current clamping), internal solutions, and / or external solutions can be altered to further probe ion channel function. Methods for manual patch clamp recordings and analysis are known in the art, see e.g., Kornreich, J. Vet. Cardiol. 2007, 9.1, 25- 37, Liem et al., Neurosurgery. 1995, 36(2), 382-392.

[0083] In some embodiments, patch clamping comprises automated patch clamping, such that cells are patched using automated methods (e.g., robotic, software-driven) which reduce or eliminate human involvement in procedure (e.g., in forming the high resistance seal, in recording the electrophysiological properties). In some embodiments, automated patch clamping comprises high throughput patch clamping, in which multiple cells are recorded (e.g., patched) in parallel during the same recording session. In some embodiments, 2 or more cells are patched (e.g., recorded) in parallel. In some embodiments, 10 or more cells are patched in parallel. In some embodiments, 20 or more cells are patched in parallel. In some embodiments, 40 or more cells are patched in parallel. In some embodiments, 50 or more cells are patched in parallel. In some embodiments, lOOor more cells are patched in parallel. In some embodiments, 200 or more cells are patched in parallel. In some embodiments, about 300 or more cells are patched in parallel. In some embodiments, 400 or more cells are patched in parallel. In some embodiments, 500 or more cells are patched in parallel. In some embodiments, 1000 or more cells are patched in parallel. Methods for automated patch clamp recordings are known in the art, e.g., using the Nanion SynchroPatch 384 automated patch clamp system as described on the world wide web at nanion.de / products / syncropatch-384 / specifications / ).

[0084] 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 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 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.

[0085] 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, properties of a batch are determined from three or more samples of the batch. Preferably, a sample is representative of a batch.

[0086] 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 low or high risk of causing graft-induced arrythmia (e.g., being low GIA or high GIA) based on electrophysiological properties of a sample of the batch.

[0087] Cardiac Cell Therapies

[0088] Cells identified and / or prepared 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 cardiac cells. In some embodiments, cardiac cell therapies (e.g., cardiac grafts) further comprise endothelial cells, conduction cells, pacemaker cells, and / or fibroblasts. 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.

[0089] 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) <?.#., non-human primates and humans).

[0090] 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.

[0091] 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.

[0092] In some embodiments, a cardiac cell therapy (e.g., a cardiac graft) disclosed herein, when administered to a subject, is associated with a reduced risk of GIA compared to other cardiac cell therapies.

[0093] 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 no more than about 50% of the mature cardiomyocytes are high GIA mature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes wherein no more than about 40% of the mature cardiomyocytes are high GIA mature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes wherein no more than about 30% of the mature cardiomyocytes are high GIA mature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes wherein no more than about 20% of the mature cardiomyocytes are high GIA mature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes wherein no more than about 10% of the mature cardiomyocytes are high GIA mature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA).

[0094] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises mature cardiomyocytes (e.g., low GIA mature cardiomyocytes) produced from a batch of immature cardiomyocytes identified as being low GIA. In some embodiments, a batch of low GIA immature cardiomyocytes 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 (e.g., low GIA mature cardiomyocytes) are made by culturing low GIA immature cardiomyocyte 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, low GIA mature cardiomyocytes are made by culturing low GIA immature cardiomyocyte 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.

[0095] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises a plurality of cells comprising immature cardiomyocytes. In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes wherein no more than about 50% of the immature cardiomyocytes are high GIA immature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes wherein no more than about 40% of the immature cardiomyocytes are high GIA immature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes wherein no more than about 30% of the immature cardiomyocytes are high GIA immature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes wherein no more than about 20% of the immature cardiomyocytes are high GIA immature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA). In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes wherein no more than about 10% of the immature cardiomyocytes are high GIA immature cardiomyocytes (e.g., have one or more electrophysiological properties associated with a high risk of GIA).

[0096] In some embodiments, a cardiac cell therapy (e.g., cardiac graft) comprises immature cardiomyocytes produced from a batch of low GIA cardiomyocyte precursors. In some embodiments, a batch of low GIA cardiomyocyte precursors is contacted with a differentiating agent under conditions that promote cardiomyocyte differentiation. Differentiating agents for cardiac cells are known by those of skill in the art. In some embodiments, immature cardiomyocytes (e.g., low GIA immature cardiomyocytes) are made by culturing low GIA cardiomyocyte precursors 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 differentiating agent. In some embodiments, low GIA immature cardiomyocytes are made by culturing low GIA cardiomyocyte precursors 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 differentiating agent.

[0097] 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.

[0098] EXAMPLES

[0099] Example 1. Identification of electrophysiological properties of cardiomyocytes for transplantation which lower risk of graft-induced arrythmias

[0100] Graft-induced arrythmia observed in myocardial infarction model pigs Graft-induced arrythmia (GIA) poses a significant barrier to administration of cardiac stem cell therapies to human subjects. To identify structural and functional differences between high and low GIA cardiomyocyte (CM) batches, human pluripotent stem cells (hPSCs) were differentiated and matured into mature CMs, then transplanted into pig models of myocardial infarction (MI). FIG. 1A shows a 2-3-month protocol for cardiomyocyte implantation and recovery. MI was induced in Yorkshire pigs between 25-40kg through balloon occlusion of the mid left anterior descending coronary artery for 90-120 minutes, followed by reperfusion. CM were delivered 3-5 weeks post- MI. Vascular access ports (VAP) were implanted during MRI- guided surgery 7 days prior to CM graft transplant. Prepared hPSC-derived cardiomyocytes were implanted along with a telemetry device, after which electrocardiographic activity was recorded until the end of the protocol; pigs were also administered immunosuppressants through the end of the protocol. Typically, pigs were monitored for 28-56 days, after which they were sacrificed and underwent necropsy; however, several pigs displayed sustained graft-induced arrythmia (GIA). Table 2 shows criterion for determining sustained GIA in MI pigs.

[0101] FIG. IB shows an EKG recording of an exemplary MI pig displaying anti- arrhythmic drug-resistant and cardioversion-resistant sustained GIA as early as 48 hours post transplantation. As shown in FIG. 1C, after necropsy, CMs associated with GIA scores of 4 or 5 were deemed “high GIA” and CMs associated with GIA scores of 1-3 were designated “low GIA”.

[0102] Lack of HCN4-driven Funny current (If), in high and low GIA CMs

[0103] One widely held hypothesis for the cause of GIA is that CMs in transplanted high GIA grafts are insufficiently mature. Induction of Funny current (If), a mixed hyperpolarization activated depolarizing current mediated by the gene HCN4, can be measured in immature ventricular CMs by voltage clamping cells to hyperpolarize them at -130mV from a holding voltage of -40mV. Unlike mature CMs, which do not exhibit If, hyperpolarized immature CMs display a stereotypical slow inward current (FIG. 2A). FIG. 2B shows If in batches of putatively mature low GIA and high GIA CMs, compared to immature CMs. Surprisingly, both low GIA and high GIA CMs did not display If, indicating transplanted cells are similarly mature, or at least that HCN4 channel dysfunction is unlikely to cause sustained GIA.

[0104] Functional differences in spontaneous activity of high GIA and low GIA cells Spontaneous activity of low GIA mature CM01 cells (Run 4) and high GIA mature CM01 cells (Run 5) was recorded during manual patch clamping; recording conditions are summarized in Table 3 below.

[0105] Table 3. Manual / High-throughput Patch Clamp Solutions

[0106] Surprisingly, the inventors observed differences in several features of spontaneous activity. As shown in FIGs. 3A-3C, high GIA cells exhibit slower upstroke velocities in spontaneous action potentials, but not paced action potentials, than low GIA cells; given that the initial phase of an action potential (AP) is driven by sodium currents, this change in upstroke velocity suggests that sodium channels in high GIA cells are deficient or otherwise have altered activation kinetics.

[0107] Similarly, FIG. 4A shows duration of spontaneous AP90 is reduced in high GIA cells relative to low GIA cells, suggesting an underlying dysfunction of sodium and / or calcium channels in high GIA cells. Despite these differences, both low and high GIA cells display a similar paced upstroke velocity (FIG. 4B) and resting membrane potential (RMP) of around - 50mV (FIG. 4C). Of note, high GIA batches also had more spontaneously active cells (5 / 18 cells; 27.778%) than low GIA batches (2 / 15 cells; 13.333%), but no correlation between spontaneity and RMP was observed. These findings suggest that while expression and / or activity of sodium and calcium channels may be altered, expression of KCNJ2, a potassium channel, does not contribute to high GIA. Example 2. Altered sodium current density and conductance in high GIA Cells

[0108] To further probe possible sodium channel dysfunctions, low and high GIA cells underwent manual patch clamp or high-throughput patch clamp under the conditions provided in Table 3 (“Sodium Channel Recordings”). Cells were voltage clamped and subjected to lOmV steps spanning-60mV to +60mV for 50ms per step; a representative trace is shown in (FIG. 5A).

[0109] As shown in FIG. 5B, manual patch clamping of low GIA (Run 4) mature CM01 cells reveals that sodium current density is significantly attenuated in high GIA cells relative to low GIA cells, suggesting a dysfunction in NaV1.5 (SCN5A) sodium channels. High-throughput patch clamping (NANION SYNCHROPATCH® 384) of low GIA (Run 10) and high GIA (Run 6) mature CM01 cells finds similar attenuation of sodium current density (FIG. 5C). A representative trace of CM01 cells recorded using high throughput patch clamping is shown in FIG. 5D. These findings are consistent across recording schemes, as shown in FIG. 5E. Peak sodium current density for low GIA (Run 4) and high GIA (Run 5, Run 6) CMOls are summarized in FIG. 5F. Maximum sodium conductance in high GIA cells was also found to be altered.

[0110] Though average maximum sodium conductance was similar across low GIA (Run 4) and high GIA (Run 5, Run 6) CMOls, as shown in FIG. 6A, distribution of sodium conductance across cells differed. FIG. 6B demonstrates that actual sodium conductance of high GIA CMOls (Run 5, Run 6) deviated from predicted values, while actual and predicted low GIA CM01 sodium conductance aligned. This deviation of high GIA CM01 sodium conductance suggests abnormal distribution of cells. Accordingly, FIG. 6C shows maximum sodium conductance of high GIA cells is positively skewed, more cells demonstrating lower maximum sodium conductance. Overall, these findings demonstrate that sodium channels are dysfunctional in high GIA cells, and suggest that sodium channel kinetics may be a useful metric for differentiating between putative high GIA cells.

[0111] Example 3. Attenuated Calcium current density in High GIA Cells

[0112] To further probe possible calcium channel dysfunctions, low and high GIA CMOls underwent manual patch clamp or high-throughput patch clamp under the conditions provided in Table 3 (“Calcium Channel Recordings”). To eliminate sodium current overlap with calcium current, CMs were driven with an initial pre-pulse at -60mV for 50ms, then subjected to 5mV steps spanning -60mV to +60mV for 50ms per step (FIG. 7A). As shown in FIG. 7B, manual patch clamping revealed attenuation of L-type calcium current density in high GIA (Run 5) cells relative to low GIA (Run 4) cells. Similar findings were made in low GIA (Run 10) and high GIA (Run 6) CMOls measured using high-throughput patch clamping (FIG. 7C). A representative trace from high-throughput patch clamping of low sodium vs calcium current at a +10mV step is shown in FIG. 7D. Manual and high-throughput patch clamping traces are summarized in FIG. 7E; unlike the robust differences observed in Example 2, peak calcium current density differences are smaller between low GIA and high GIA cells.

[0113] EQUIVALENTS AND SCOPE

[0114] 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.

[0115] 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 it 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.

[0116] 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, e.g., 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, e.g., “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.

[0117] 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, e.g., 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 (e.g., “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.

[0118] 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. As used herein in the specification and claims, the phrase “about” refers to ±10% of the numerical values cited. For example, “about 10ms to about 20ms” should be understood to include any value between 10ms and 20ms (inclusive), but also any value between 9ms and 22ms.

[0119] 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.

[0120] 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, e.g., 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.”

[0121] 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.

[0122] 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.

[0123] 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 batch of electroresponsive cells for cardiac cell therapy, the method comprising:(a) determining that at least a first batch of electroresponsive cells among a plurality of batches has one or more electrophysiological properties associated with low risk of graft- induced arrythmia (GIA); and(b) preparing at least the first batch of electroresponsive cells for cardiac cell therapy.

2. The method of claim 1, wherein the at least first batch of electroresponsive cells comprises 40 million cells.

3. The method of claim 1 or 2, wherein the one or more electrophysiological properties associated with low risk of GIA are determined from one or more samples of the at least first batch.

4. The method of claim 3, wherein the sample comprises one or more electroresponsive cells.

5. The method of claim 3 or 4, wherein the one or more electrophysiological properties associated with low risk of GIA are determined from 3 or more samples of the at least first batch.

6. The method of any one of claims 1 to 5, wherein the one or more electrophysiological properties are obtained by patch clamping isolated cells of the batch.

7. The method of claim 6, wherein the patch clamping comprises high throughput patch clamping.

8. The method of any one of claim 1 to 7, wherein the one or more electrophysiological properties associated with low risk of GIA comprise action potential upstroke velocity, ion channel current, peak ion current density, maximum ion conductance, or any combination thereof.

9. The method of any one of claims 6 to 8, wherein the isolated cells undergo patch clamp recordings under conditions suitable for recording sodium channel properties.

10. The method of claim 9, wherein the conditions suitable for recording sodium channel properties comprise: an internal solution comprising cesium fluoride, sodium chloride, HEPES, magnesium chloride, and sodium- ATP, having a normal physiological pH, and having a normal physiological osmolarity; an external solution comprising TEA-CI, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, HEPES, and D-glucose, having a normal physiological pH, and having a normal physiological osmolarity; and normal physiological temperatures.

11. The method of claim 10, wherein the conditions suitable for recording sodium channel properties comprise: an internal solution comprising 108-112mM cesium fluoride, 9-llmM cesium chloride, 9-11 mM sodium chloride, 9-11 mM HEPES, 1-2 mM magnesium chloride, and l-3mM sodium- ATP, having a pH of 7.1-7.3, and having an osmolarity of 280-290 mOsm; an external solution comprising 105-115 mM TEA-CI, 38-42 mM sodium chloride3-5 mM potassium chloride, 0.5- 1.5 mM magnesium chloride, 1.5-2.5 mM calcium chloride, 9-11 mM HEPES, and 4-6 mM D-glucose, having a pH of 7.1-7.5, and having an osmolarity of 290- 300 mOsm; and a temperature of 22-27 °C.

12. The method of claim 11, wherein the conditions suitable for recording sodium channel properties comprise: an internal solution comprising llOmM cesium fluoride, lOmM cesium chloride, lOmM sodium chloride, lOmM HEPES, 1.5mM magnesium chloride, and 2mM sodium- ATP, having a pH of 7.2, and having an osmolarity of 285 mOsm; an external solution comprising lOOmM TEA-CI, 40mM sodium chloride, 4mM potassium chloride, ImM magnesium chloride, 2mM calcium chloride, lOmM HEPES, and 5mM D-glucose, having a pH of 7.4, and an osmolarity of 295 mOsm; and a temperature of 25 °C.

13. The method of any one of claims 6 to 8, wherein the isolated cells undergo patch clamp recordings under conditions suitable for recording calcium channel properties.

14. The method of claim 13, wherein the conditions suitable for recording calcium channel properties comprise: an internal solution comprising cesium fluoride, sodium chloride, HEPES, magnesium chloride, and sodium- ATP, having a normal physiological pH, and having a normal physiological osmolarity; an external solution comprising TEA-CI, sodium chloride, potassium chloride, magnesium chloride, calcium chloride, HEPES, and D-glucose, having a normal physiological pH, and having a normal physiological osmolarity; and normal physiological temperatures.

15. The method of claim 14, wherein the conditions suitable for recording calcium channel properties comprise: an internal solution comprising 108-112 mM cesium fluoride, 9-11 mM cesium chloride, 9-11 mM sodium chloride, 9-11 mM HEPES, 1.0-2.0 magnesium chloride, and l-3mM sodium- ATP, having a pH of 7.1-7.5, and having an osmolarity of 280-290 mOsm; an external solution comprising 135-145 mM sodium chloride, 3-5 mM potassium chloride, 0.5- 2.0 mM magnesium chloride, 1-3 mM calcium chloride, 9-11 mM HEPES, and 4-6 mM D- glucose, having a pH of 7.1-7.5, and having an osmolarity of 290-300 mOsm; and a temperature of 22-27 °C.

16. The method of claim 15, wherein the conditions suitable for recording calcium channel properties comprise: an internal solution comprising 110 mM cesium fluoride, 10 mM cesium chloride, 10 mM sodium chloride, 10 mM HEPES, 1.5mM magnesium chloride, and 2 mM sodium- ATP, having a pH of 7.2, and having an osmolarity of 285 mOsm; an external solution comprising 140 mM sodium chloride, 4 mM potassium chloride, 1 mM magnesium chloride, 2 mM calcium chloride, 10 mM HEPES, and 5 mM D-glucose, having a pH of 7.4, and an osmolarity of 295 mOsm; and a temperature of 25 °C.

17. The method of any one of claims 1 to 16, wherein the at least first batch of cells comprise excitable cells.

18. The method of any one of claims 1 to 17, wherein the at least first batch of cells comprises immature cardiomyocytes or mature cardiomyocytes.

19. The method of claim 18, wherein the immature cardiomyocytes or mature cardiomyocytes are derived from pluripotent stem cells.

20. The method of claim 18, wherein the immature cardiomyocytes or mature cardiomyocytes are derived from embryonic stem cells.

21. The method of claim 19, wherein the pluripotent stem cells express one or more markers selected from: SSCA3 / 4, TRA-160, OCT3 / 4, NANOG, SOX2.

22. The method of any one of claims 1 to 17, wherein the at least first batch of cells determined to have a low risk of GIA comprises immature cardiomyocytes.

23. The method of claim 22, wherein the immature cardiomyocytes comprise positive / high expression of cTNT, MYL2, MYL7, KCNJ2, CACNA1C, CACNA1H, SCN5A, HCN4, and comprise negative / low expression of: MKI67 and CD90.

24. The method of claim 22 or 23, wherein immature cardiomyocytes having a normally distributed maximum sodium conductance is determined to have a low risk of GIA.

25. The method of claim 22 or 23, wherein the immature cardiomyocytes have a normally distributed maximum sodium conductance under the conditions of any one of claims 10 to 12.

26. The method of claim 22 or 23, wherein preparing the immature cardiomyocytes for cardiac cell therapy comprises contacting the immature cardiomyocytes with a maturation cocktail under conditions that promote cardiomyocyte maturation.

27. The method of any one of claims 1 to 17, wherein the at least first batch of cells determined to have a low risk of GIA comprises mature cardiomyocytes.

28. The method of claim 27, wherein the mature cardiomyocytes comprise positive / high expression of MYL2, cTNT, SCN5A, KCNJ2, CACNA1C and comprise negative / low expression of MLY7, CD90, CACNA1H, HCN4, and MKI67.

29. The method of claim 27 or 28, wherein the mature cardiomyocytes have a spontaneous upstroke velocity of about 45V / s to 60V / s under the conditions of claim 12 or 16.

30. The method of claim 29, wherein the mature cardiomyocytes have an action potential upstroke velocity of at least about 48V / s to 57V / s under the conditions of claim 12 or 16.

31. The method of claim 30, wherein the mature cardiomyocytes have an action potential upstroke velocity of 50V / s to 55V / s under the conditions of claim 12 or 16.

32. The method of claim 27 or 28, wherein the mature cardiomyocytes have a peak sodium current density of at least about 135pA / pF within a range of -20mV to -40mV under the conditions of claim 12.

33. The method of claim 32, wherein the mature cardiomyocytes have a peak sodium current density of at least about 140pA / pF within a range of -20mV to -40mV under the conditions of claim 12.

34. The method of claim 33, wherein the mature cardiomyocytes have a peak sodium current density of >| 145 |pA / pF within a range of -20mV to -40mV under the conditions of claim 12.

35. The method of claim 27 or 28, wherein the mature cardiomyocytes have a normal (Gaussian) frequency distribution of sodium conductance under the conditions of any one of claim 12.

36. The method of claim 27 or 28, wherein the mature cardiomyocytes have a peak calcium current density at least |2| pA / pF within a range of lOmV to 20mV under the conditions of claim 16.

37. The method of claim 36, wherein the mature cardiomyocytes have a peak calcium current density of at least |3| pA / pF within a range of lOmV to 20mV under the conditions of claim 16.

38. The method of claim 37, wherein the mature cardiomyocytes have a peak calcium current density of >|4| pA / pF within a range of lOmV to 20mV under the conditions of claim 16.

39. The method of claim 38, wherein the mature cardiomyocytes have a normally distributed maximum calcium conductance under the conditions of claim 16.

40. The method of claim 27 or 28, wherein the mature cardiomyocytes have a peak sodium current density of at least -150 pA / pF to -200 pA / pF under the conditions of claim 12 and peak calcium current density of at least -9 pA / pF to -11 pA / pF under the conditions of claim 16.

41. The method of claim 27 or 28, wherein preparing the mature cardiomyocytes for cardiac cell therapy comprises contacting the mature cardiomyocytes with a physiologically acceptable medium suitable for administration to a subject.

42. A method of differentiating a plurality of cardiomyocyte precursor cells for cardiac cell therapy, the method comprising contacting a plurality of cardiomyocyte precursor cells that have been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a medium comprising a differentiating agent under conditions that promote cardiomyocyte differentiation.

43. A method of maturing a plurality of immature cardiomyocytes for cardiac cell therapy, the method comprising contacting the plurality of immature cardiomyocytes that been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a maturation cocktail under conditions that promote cardiomyocyte maturation.

44. A method of preparing a plurality of mature cardiomyocytes for cardiac cell therapy, the method comprising contacting the plurality of mature cardiomyocytes that been determined to have one or more electrophysiological properties associated with a low risk of graft-induced arrythmia with a physiologically acceptable medium suitable for administration to a subject in need of cardiac cell therapy.

45. 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 comprises mature cardiomyocytes; andwherein no more than 20% of the mature cardiomyocytes have one or more electrophysiological properties associated with a high risk of graft- induced arrythmia (GIA).

46. A composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells comprises immature cardiomyocytes; and wherein no more than 20% of the immature cardiomyocytes have one or more electrophysiological properties associated with a high risk of graft- induced arrythmia (GIA).

47. A composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells comprises cardiomyocyte precursors; and wherein no more than 20% of the cardiomyocyte precursors have one or more electrophysiological properties associated with a high risk of graft- induced arrythmia (GIA).

48. A composition for cardiac cell therapy comprising a plurality of cells, wherein the plurality of cells has been determined to have one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA) have been prepared for cardiac cell therapy.

49. A method of treating a subject in need of cardiac cell therapy, the method comprising administering to the subject a cardiac cell therapy comprising a batch of electroresponsive cells prepared according to the method of any one of claims 1-41 or the composition of any one of claims 45-48.

50. A method of treating a subject in need of cardiac cell therapy, the method comprising:(a) determining that at least a first batch of electroresponsive cells among a plurality of batches has one or more electrophysiological properties associated with low risk of graft- induced arrythmia (GIA); and(b) administering the at least the first batch of electroresponsive cells to the subject.

51. The method of claim 50, wherein determining in (a) comprises the method of any one of claims 1-41.

52. A method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of electroresponsive cells to a subject, wherein the plurality of electroresponsive cells has one or more electrophysiological properties associated with low risk of graft-induced arrythmia (GIA).

53. The method of claim 52, wherein the plurality of electroresponsive cells are comprised in the composition of any one of claims 45-48.

54. A method of treating a subject in need of cardiac cell therapy, the method comprising administering a plurality of electroresponsive cells to a subject, wherein the plurality of electroresponsive cells has been determined to have one or more electrophysiological properties associated with low risk of graft- induced arrythmia (GIA).

55. The method of claim 54, wherein the plurality of electroresponsive cells is comprised in the composition of any one of claims 45-48.

Images