Cell surface marker signature of cardiomyocytes derived from dysrhythmogenic pluripotent stem cells

Abstract

A method for identifying cardiomyocytes derived from arrhythmogenic pluripotent stem cells (PSC-CMs), comprising: (i) determining whether CD200 is expressed on the surface of PSC-CMs, wherein when CD200 is expressed on the surface of PSC-CMs, the PSC-CMs are arrhythmogenic PSC-CMs, and the method is disclosed herein.

Description

Technical Field

[0001] The present invention relates to a method for identifying subpopulations of cardiomyocytes, particularly human pluripotent stem cell-derived cardiomyocytes, such as arrhythmogenic cardiomyocytes, pacemaker cardiomyocytes or ventricular cardiomyocytes.

Background Art

[0002] Myocardial infarction (MI), the major cause of heart failure, results in the loss of up to one billion highly specialized cardiomyocytes. Although there have been major breakthroughs in cardiology in the last few decades, heart disease continues to be the cause of more deaths worldwide than any other disease, and the problem is exacerbated by the inability of the damaged adult heart to meaningfully regenerate. Despite great interest, the results obtained from many attempts to replace damaged or destroyed cardiomyocytes with exogenous cells have been inconsistent. Most cell types tested clinically lack the certain ability to differentiate into functional cardiomyocytes and exert any beneficial effects via paracrine mechanisms rather than remuscularisation. In contrast, pluripotent stem cells (PSCs) exhibit cardiomyogenic differentiation ability and potentially provide an unlimited source of cardiomyocytes for therapeutic use. Intriguing preclinical data have confirmed that transplanted PSC-derived cardiomyocytes (PSC-CMs) can remuscularize and improve cardiac function in clinically relevant large animal MI models. However, hurdles remain for large-scale clinical translation. The most concerning of these are related to potentially lethal cardiac arrhythmias (hereinafter referred to as engraftment arrhythmia (EA)) that occur after intramyocardial PSC-CM delivery.

[0003] There is little research on the potential mechanisms of EA, and the existing evidence from electrophysiological studies only suggests that EA originates from PSC-CM grafts with abnormal impulse generation and enhanced automaticity characteristics. Similar to the lack of data on the EA mechanism, there is only one study on treatment strategies for alleviating EA, which suggests that drug therapy (using amiodarone and ivabradine) can suppress but not eliminate EA.

[0004] Therefore, a method is needed to reduce or eliminate EA after intramyocardial PSC-CM delivery.

Summary of the Invention

[0005] In a first aspect of the present invention, there is provided a method for identifying cardiomyocytes derived from arrhythmogenic pluripotent stem cells (PSC-CM), comprising: (i) determining whether CD200 is expressed on the surface of PSC-CM, wherein when CD200 is expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM.

[0006] The following options can be used individually or in any combination together with the first aspect of the present invention.

[0007] The method of the first aspect of the present invention further comprises: (ii) determining whether signal regulatory protein α (CD172a) is expressed on the surface of PSC-CM, and / or (iii) determining whether CD90 is expressed on the surface of PSC-CM, wherein when CD200 is expressed on the surface of PSC-CM, CD172a is expressed on the surface of PSC-CM, and / or CD90 is not expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM.

[0008] The method of the first aspect of the present invention further comprises: (ii) determining whether CD172a is expressed on the surface of PSC-CM; (iii) determining whether CD90 is expressed on the surface of PSC-CM, may be included, If CD200 and CD172a are expressed on the surface of PSC-CM and CD90 is not expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM.

[0009] If CD200 is not expressed on the surface of PSC-CM, the PSC-CM may be non-arrhythmogenic PSC-CM.

[0010] Determining whether CD200, CD172a, and / or CD90 are expressed on the surface of PSC-CM may include exposing the PSC-CM to an agent containing a detectable label to provide labeled PSC-CM, and detecting the detectable label, and the agent selectively binds to CD200, CD172a, or CD90. Determining whether CD200, CD172a, and CD90 are expressed on the surface of PSC-CM may include exposing the PSC-CM to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90 to provide labeled PSC-CM, and detecting the detectable label.

[0011] The agent containing a detectable label may be an antibody, and the detectable label may be a fluorophore. Detecting the detectable label may include subjecting the labeled hPSC-CM to flow cytometry.

[0012] The PSC-CM may be human pluripotent stem cell-derived cardiomyocytes (hPSC-CM).

[0013] In a second aspect of the invention, a method for determining whether a dose containing a plurality of PSC-CM is likely to cause arrhythmia upon transplantation in a subject, (i) determining whether the dose contains arrhythmogenic PSC-CM, wherein whether the PSC-CM is arrhythmogenic PSC-CM is determined according to the method of the first aspect of the present invention, including determining; When the dose contains arrhythmogenic PSC-CM, a method is provided in which the dose is likely to cause arrhythmia at the time of transplantation in a subject.

[0014] The following options can be used individually or in any combination with the second aspect of the present invention.

[0015] The method of the second aspect of the present invention further may include (ii) determining the proportion of arrhythmogenic PSC-CM in the dose; When the proportion of PSC-CM in the dose expressing CD200 on the surface exceeds 0.001%, the dose is likely to cause arrhythmia at the time of transplantation in a subject.

[0016] In a third aspect of the present invention, a method for eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CM in a subject, before transplantation of the dose, (i) identifying arrhythmogenic PSC-CM contained in the crude dose according to the method of the first aspect of the present invention; and (ii) removing arrhythmogenic PSC-CM from the crude dose to obtain a purified dose, including the method.

[0017] The following options can be used individually or in any combination with the third aspect of the present invention.

[0018] The method of the third aspect of the present invention further may include (iii) transplanting the purified dose into the subject.

[0019] The crude dose may include pluripotent stem cells (PSC) that have been differentiated to form PSC-CM and have not been subjected to any additional treatment to enrich or deplete cell subpopulations.

[0020] Arrhythmias after transplantation at a refined dose can be reduced compared to arrhythmias after transplantation at a crude dose. Arrhythmias can be reduced when defined by the cumulative time per day spent on arrhythmias over 25 days after transplantation. Arrhythmias can be reduced by at least about 50%, or at least about 60, 70, 80, 90, 95, or 100%.

[0021] Step (ii) can include removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. Step (ii) can include removing at least about 60% of the arrhythmogenic PSC-CMs, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose.

[0022] Step (ii) can be performed by fluorescence-activated cell sorting or magnetic-activated cell sorting.

[0023] In a fourth aspect of the present invention, a method of providing a dose containing a plurality of PSC-CMs substantially free of arrhythmogenic PSC-CMs, (i) identifying arrhythmogenic PSC-CMs contained in a crude dose according to the method of the first aspect of the present invention; and (ii) removing arrhythmogenic PSC-CMs from the crude dose to obtain a dose of PSC-CMs substantially free of arrhythmogenic PSC-CMs, is provided.

[0024] The following options can be used individually or in any combination with the fourth aspect of the present invention.

[0025] The crude dose can include human pluripotent stem cells (PSCs) that have been differentiated to form PSC-CMs and have not been subjected to any additional treatment to enrich or deplete cell subpopulations.

[0026] Step (ii) may include removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. Step (ii) may include removing at least about 60%, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose.

[0027] A dose of PSC-CMs substantially free of arrhythmogenic PSC-CMs may contain less than 0.001% arrhythmogenic PSC-CMs relative to the total number of PSC-CMs in the dose.

[0028] Step (ii) may be performed by fluorescence-activated cell sorting or magnetic-activated cell sorting.

[0029] In a fifth aspect of the invention, a method of identifying pluripotent stem cell-derived cardiac cells (PSC-PM) having pacemaker properties, comprising (i) determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, a method is provided wherein the pluripotent stem cell-derived cardiac cells are PSC-PM.

[0030] The following options can be used individually or in any combination with the fifth aspect of the invention.

[0031] The method of the fifth aspect of the invention further comprises (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are pacemaker PSC-PM.

[0032] The method of the fifth aspect of the present invention further comprises (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells; (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, and may include When CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-PM.

[0033] When CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells cannot be PSC-PM.

[0034] Determining whether CD200, CD172a, and / or CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells may include exposing the pluripotent stem cell-derived cardiac cells to an agent containing a detectable label to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, wherein the agent selectively binds to CD200, CD172a, or CD90. Determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells may include exposing the pluripotent stem cell-derived cardiac cells to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90 to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label.

[0035] The agent containing the detectable label may be an antibody, and the detectable label may be a fluorophore. Detecting the detectable label may include subjecting the labeled pluripotent stem cell-derived cardiac cells to flow cytometry.

[0036] In the sixth aspect of the present invention, a method for providing a substantially pure dose of PSC-PM, (i) Identifying PSC-PM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method of the fifth aspect of the present invention; (ii) Isolating PSC-PM from a plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-PM, A method is provided that includes:

[0037] The following options can be used individually or in any combination together with the sixth aspect of the present invention.

[0038] Step (ii) can be performed by fluorescence-activated cell sorting or magnetic-activated cell sorting.

[0039] A substantially pure dose of PSC-PM can contain less than 0.001% of cells that are not PSC-PM relative to the total number of cells in the dose.

[0040] In the seventh aspect of the present invention, a method for identifying pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VM), (i) Determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, including, When CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, a method is provided in which the pluripotent stem cell-derived cardiac cells are PSC-VM.

[0041] The following options can be used individually or in any combination together with the seventh aspect of the present invention.

[0042] The method of the seventh aspect of the present invention further (ii) Determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or (iii) Determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, can be included, If CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, then the pluripotent stem cell-derived cardiac cells are PSC-VM.

[0043] The method of the seventh aspect of the present invention further (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells; (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, and may include If CD200 and CD90 are not expressed on the surface of pluripotent stem cell-derived cardiac cells and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, then the pluripotent stem cell-derived cardiac cells are PSC-VM.

[0044] If CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells cannot be PSC-VM.

[0045] Determining whether CD200, CD172a, and / or CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells may include exposing the pluripotent stem cell-derived cardiac cells to an agent containing a detectable label to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, where the agent selectively binds to CD200, CD172a, or CD90. Determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells may include exposing the pluripotent stem cell-derived cardiac cells to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90 to provide labeled pluripotent stem cells.

[0046] The agent containing a detectable label can be an antibody, and the detectable label can be a fluorophore. Detecting the detectable label can include subjecting the labeled pluripotent stem cell-derived cardiac cells to flow cytometry.

[0047] In an eighth aspect of the invention, a method of providing a substantially pure dose of PSC-VM, comprising: (i) identifying PSC-VM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method of the seventh aspect of the invention; and (ii) isolating PSC-VM from the plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-VM. A method is provided.

[0048] The following options can be used individually or in any combination with the eighth aspect of the invention.

[0049] Step (ii) can be performed by fluorescence-activated cell sorting or magnetic-activated cell sorting.

[0050] A substantially pure dose of PSC-PM can contain less than 0.001% of cells that are not PSC-PM relative to the total number of cells in the dose.

[0051] In a ninth aspect of the invention, a method of eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CM in a subject, comprising culturing a plurality of PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CM, wherein the arrhythmogenic PSC-CM is a PSC-CM that expresses CD200 on the surface. A method is provided.

[0052] The following options can be used individually or in any combination with the ninth aspect of the invention.

[0053] The arrhythmia after transplantation of a dose subjected to the method of the ninth aspect of the present invention can be reduced as compared with the arrhythmia after transplantation of a dose not subjected to the method of the ninth aspect of the present invention. The arrhythmia can be reduced when defined by the cumulative time per day spent on arrhythmia over 25 days after transplantation. The arrhythmia can be reduced by at least about 50%, or at least about 60, 70, 80, 90, 95, or 100%.

[0054] In a tenth aspect of the present invention, there is provided a method of providing a dose of PSC-CM substantially free of arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CM, wherein the arrhythmogenic PSC-CM is PSC-CM expressing CD200 on the surface.

[0055] The following options can be used individually or in any combination with the tenth aspect of the present invention.

[0056] A dose of PSC-CM substantially free of arrhythmogenic PSC-CM can contain less than 0.001% arrhythmogenic PSC-CM relative to the total number of PSC-CM in the dose.

[0057] The arrhythmogenic PSC-CM can be PSC-CM that expresses CD200 on the surface, expresses CD172a, and / or does not express CD90. The arrhythmogenic PSC-CM can be PSC-CM that expresses CD200 and CD172a on the surface and does not express CD90.

[0058] In an eleventh aspect of the present invention, there is provided a method of providing a dose of PSC-CM containing a high proportion of non-arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to increase the generation of non-arrhythmogenic PSC-CM, wherein the non-arrhythmogenic PSC-CM is PSC-CM that does not express CD200 on the surface.

[0059] The following options can be used individually or in any combination with the eleventh aspect of the present invention.

[0060] The dosage of PSC-CM containing a high percentage of non-arrhythmogenic PSC-CM can contain at least about 5% more non-arrhythmogenic PSC-CM compared to a control dosage obtained by culturing PSCs in a culture medium, relative to the total number of PSC-CMs in the dosage and in the absence of conditions effective to increase the generation of non-arrhythmogenic PSC-CM.

[0061] Non-arrhythmogenic PSC-CM can be PSC-CM that does not express CD200 on the surface, does not express CD90, and / or expresses CD172a. Non-arrhythmogenic PSC-CM can be PSC-CM that does not express CD200 and CD90 on the surface and expresses CD172a.

[0062] Conditions effective to suppress the generation of arrhythmogenic PSC-CM can include the addition of a retinoic acid inhibitor to the culture medium or the removal of vitamin A from the culture medium.

[0063] The retinoic acid inhibitor can be added during cardiomyocyte differentiation. The retinoic acid inhibitor can be added between day 3 and day 7 of cardiomyocyte differentiation.

[0064] The retinoic acid inhibitor can be selected from the group consisting of 4-diethylaminobenzaldehyde (DEAB), 4-[(1E)-2-[5,6-dihydro-5,5-dimethyl-8-(2-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid (BMS-493), (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoic acid (BMS-189453), and disulfiram.

[0065] Vitamin A can be removed from the culture medium during cardiomyocyte differentiation.

Brief Description of the Drawings

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[0067] Definition As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

[0068] As used herein, the term "comprising" means "including". Variations of the word "comprising", such as "comprise" and "comprises", have corresponding varying meanings.

[0069] When the term "about" is used herein with respect to a recited numerical value, it is understood that the recited numerical value and numerical values within ±10% of the recited value are included therewith.

[0070] When the term "between" is used herein in reference to a range of numerical values, it is understood that the numerical values of each endpoint of the range are included therein. For example, a concentration between 2 mg / mL and 10 mg / mL includes the concentrations of 2 mg / mL and 10 mg / mL.

[0071] The terms "subject" and "patient" can be used interchangeably and refer to a human or other mammal that has, is at risk of having, or has suffered from arrhythmia after transplantation of PSC-CM.

[0072] Whether a marker such as CD200, CD90, or CD172a is expressed on the surface of a cell such as PSC-CM can be represented by a + / − notation. For example, to state that a cell is or has signature CD90+ is equivalent to stating that the cell expresses CD90 on its surface.

[0073] The surface marker CD172a refers to signal regulatory protein alpha (SIRPα). In the context of this specification, CD172a may also be referred to as SIRPA. In the context of this specification, the terms CD172a, SIRPα, and SIRPA are used interchangeably.

[0074] In the context of this specification, the expressly stated "arrhythmia" refers to ventricular arrhythmia, and specifically may refer to ventricular tachycardia. Ventricular tachycardia can be defined as a broad complex rhythm accompanied by a QRS duration > 120 ms (measured by electrocardiogram examination) and a heart rate > 100 beats per minute.

[0075] In the context of this specification, an "arrhythmogenic" cell or (sub)population of cells is understood to be a cell or (sub)population of cells that causes a particularly high incidence of arrhythmia upon transplantation in a subject. In particular, this is a cell or (sub)population of cells that causes arrhythmia even when mature (i.e., after a certain period post-transplantation). All PSC-CMs are immature compared to adult myocytes and do not immediately electrically couple to the heart, so they exhibit automaticity first and may cause arrhythmia. However, a specific subpopulation of cells referred to herein as arrhythmogenic cells is thought to not lose its arrhythmogenicity despite the overall maturation of the PSC-CM graft. That is, arrhythmogenic cells can cause arrhythmia in vivo despite the period of maturation. The term "non-arrhythmogenic" has the corresponding opposite meaning. In the context of this specification, a non-arrhythmogenic cell or (sub)population of cells does not cause a high incidence of arrhythmia upon transplantation in a subject. In particular, a non-arrhythmogenic cell or (sub)population of cells does not cause arrhythmia in vivo after the period of maturation. The period of maturation can refer to a period of several weeks, for example, 2 weeks, or a period of 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks.

[0076] A "purified" population of cells refers to a population of cells that is enriched for a selected cell type. For example, a "purified population of non-arrhythmogenic PSC-CMs" refers to a population of PSC-CMs that is enriched for non-arrhythmogenic PSC-CMs. The purified population can be enriched to at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for the desired cell type.

[0077] Any description of the prior art materials in this specification or any opinion derived from or based on such materials in this specification does not admit that such materials or the opinions derived therefrom are part of the common general knowledge in the relevant technical field.

[0078] All materials mentioned in this specification for the purpose of explanation are hereby incorporated by reference in their entirety into this specification, unless otherwise specified. Detailed Description of the Invention

[0079] Current cardiac differentiation protocols yield a heterogeneous cell output. Although ventricular cardiomyocytes are dominant, in addition to other cell types such as atrial and pacemaker-like cells, non-muscle cells also exist. To date, no studies have definitively evaluated how the composition of PSC-CM cell doses can affect EA loading. The inventors have surprisingly found that the presence of PSC-CM with the surface marker signature CD172a+ / CD90- / CD200+ in the cell dose is positively correlated with the incidence of arrhythmias at the time of transplantation in the subject. The presence of PSC-CM with the surface marker signature CD172a+ / CD90- / CD200- in the cell dose has been found to be negatively correlated with the incidence of arrhythmias at the time of transplantation in the subject.

[0080] Since the CD172a+ / CD90- / CD200+ signature can be easily identified by well-established methods such as flow cytometry, the method of the present invention provides a rapid, simple, and convenient means for identifying the presence of arrhythmogenic cells, and if scaled up, removing arrhythmogenic cells from the cell dose and reducing arrhythmias at the time of transplantation.

[0081] 1. Identification of Arrhythmogenic PSC-CM In a first aspect of the present invention, a method for identifying arrhythmogenic pluripotent stem cell-derived cardiomyocytes (PSC-CM) is provided.

[0082] Pluripotent stem cell-derived cardiomyocytes refer to cardiomyocytes obtained by differentiating and developing pluripotent stem cells into cardiomyocytes. Generally speaking, this involves mimicking development in vivo by changing signal transduction pathways and the cellular microenvironment. PSC-CMs are typically produced from pluripotent stem cells (PSCs) in a series of steps, namely 2D expansion, 3D expansion, and subsequent cardiomyocyte differentiation (accompanied by mesoderm induction and subsequent cardiac specification). See Figure 1. This forms a heterogeneous mixture of pluripotent stem cell-derived cardiac cells, some of which may be PSC-CMs. PSC-CMs themselves are heterogeneous. PSC-CMs can be produced by any suitable protocol known to those skilled in the art, such as the protocol of Chen et al. (Stem Cell Res 2015, 15(2), p. 365). The PSC can be a human PSC that gives rise to the formation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs), or a non-human PSC that gives rise to the formation of non-human pluripotent stem cell-derived cardiomyocytes. The PSC can be derived from any mammal. The term human pluripotent stem cell-derived cardiomyocytes (PSC-CMs) is understood to encompass both human- and non-human-derived cells.

[0083] Arrhythmogenic PSC-CMs are PSC-CMs that, when transplanted in a subject, cause a particularly high incidence of arrhythmia in the subject, as defined above. In particular, these are cells or (sub)populations of cells that cause arrhythmia even when mature.

[0084] The method of the first aspect of the present invention includes determining whether CD200 is expressed on the surface of PSC-CMs. If CD200 is expressed on the surface of PSC-CMs, the PSC-CMs are arrhythmogenic PSC-CMs. If CD200 is not expressed on the surface of PSC-CMs, the PSC-CMs are not arrhythmogenic PSC-CMs.

[0085] The method of the first aspect of the present invention may also include determining whether other markers are expressed (or absent) on the surface of PSC-CM. In one embodiment, the method includes determining whether CD200 is expressed on the surface of PSC-CM and determining whether CD172a and / or CD90 is expressed on the surface of PSC-CM. In one embodiment, the method may include determining whether CD200 and CD172a are expressed on the surface of PSC-CM. In this case, if both CD200 and CD172a are expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM. Conversely, if CD200 is not expressed on the surface of PSC-CM and CD172a is expressed on the surface of PSC-CM, the CM is not arrhythmogenic PSC-CM. In one embodiment, the method may include determining whether CD200 and CD90 are expressed on the surface of PSC-CM. In this case, if CD200 is expressed on the surface of PSC-CM and CD90 is not expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM. Conversely, if neither CD200 nor CD90 is expressed on the surface of PSC-CM, the PSC-CM is not arrhythmogenic PSC-CM. In another embodiment, the method includes determining whether CD200, CD172a, and CD90 are expressed on the surface of PSC-CM. In this case, if CD200 and CD172a are expressed on the surface of PSC-CM and CD90 is not expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM. Conversely, if CD200 and CD90 are not expressed on the surface of PSC-CM and CD172a is expressed on the surface of PSC-CM, the PSC-CM is not arrhythmogenic PSC-CM.

[0086] The step of determining whether CD200 and optionally other markers are expressed on the surface of PSC-CM can be carried out using any suitable method known to those skilled in the art. In one embodiment, the PSC-CM can be exposed to an agent containing a detectable label, and the agent selectively binds to the marker. Then, the excess agent containing the detectable label is removed, for example, by washing. If the marker is present on the surface of the PSC-CM, the agent binds to the marker, providing labeled PSC-CM. Thus, the labeled PSC-CM is PSC-CM expressing the marker (e.g., CD200, or any marker to which the agent selectively binds). Then, the detectable label can be detected to confirm the presence of the marker. In one embodiment, the marker is CD200, and the agent containing the detectable label selectively binds to CD200. In one embodiment, the marker is CD90, and the agent containing the detectable label selectively binds to CD90. In one embodiment, the marker is CD172a, and the agent containing the detectable label selectively binds to CD172a.

[0087] If the expression of multiple markers is to be determined, the PSC-CM can be treated with a plurality of agents each containing a detectable label, each of which is selective for one of the markers to be determined, and each having a distinct detectable label. In one embodiment, to determine whether CD200, CD172a, and CD90 are expressed on the surface of PSC-CM, the PSC-CM is exposed to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90, to obtain triple-labeled PSC-CM. Then, the excess agent containing the detectable label is removed, for example, by washing, and then each of the detectable labels is detected.

[0088] In one embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be a fluorophore. In this case, the expression of the marker is indicated by the wavelength and intensity of the fluorescence emitted from the labeled PSC-CM. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to a fluorophore. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to fluorophores are known to those skilled in the art and are commercially available, and any of them are suitable for use in the method of the present invention. Examples of suitable antibodies conjugated to fluorophores include anti-CD172a PECy5, anti-CD90 BV650, and anti-CD200 BV421.

[0089] In another embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be magnetic beads. In this case, the expression of the marker is indicated by the response of the PSC-CM to a magnetic field. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to magnetic beads. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to magnetic beads are known to those skilled in the art and are commercially available, and any of them are suitable for use in the method of the present invention.

[0090] In one embodiment, determining whether CD200 and optionally other markers are expressed on the surface of PSC-CM can be performed by genetic modification to incorporate a reporter gene into the PSC-CM. The reporter gene is transcribed in conjunction with the transcription of the marker gene, and the reporter gene encodes a detectable product. Thus, the detectable product is produced when the marker gene is transcribed. For example, the reporter gene can encode green fluorescent protein (GFP) or luciferase. If the expression of multiple markers is to be determined, multiple separate reporter genes may be required. Genetic modification techniques for introducing the reporter gene into cell lines are known to those skilled in the art and can be performed using commercially available kits. Any such technique is suitable for use in the methods of the present invention.

[0091] In one embodiment, the step of determining whether CD200, and optionally other markers, are expressed on the surface of PSC-CM is performed using flow cytometry. Typically, in this case, the agent that selectively binds to the marker is an antibody and the detectable label is a fluorophore. Preliminary processing steps may be performed prior to using flow cytometry to determine whether CD200, and optionally other markers, are expressed on the surface of PSC-CM. This can include compensation, staining with a viability marker (e.g., Zombie NIR), and manual gating to remove artifacts such as necrotic debris, dead cells, and doublets.

[0092] Flow cytometry enables the detection of labeled PSC-CMs among a plurality of PSC-CMs. Typically, an appropriate fluorescence intensity threshold (“gate”) is set by an operator for a control sample, and if the fluorescence level of a cell exceeds these gate levels, that cell is counted as a labeled PSC-CM. The fluorescence intensity values relative to the gates depend on the antibody labeling and vary from flow cytometer to flow cytometer, as well as between individual runs and between different batches of labeled antibodies. A commonly used guideline is to set the gates such that no more than 0.5% of the events in the control sample fall outside each gate. These gates are then applied to the sample to be measured, i.e., the sample containing PSC-CMs that have been exposed to an antibody containing a detectable label that is a fluorophore.

[0093] 2. Screening for arrhythmogenic PSC-CMs In a second aspect of the invention, a method is provided for determining whether a dose containing a plurality of PSC-CMs is likely to cause arrhythmia upon transplantation in a subject.

[0094] Transplantation of a dose of PSC-CMs is a potential treatment for replacement of damaged or destroyed cardiomyocytes after myocardial infarction. According to a second aspect of the invention, such a dose can be screened for the presence of arrhythmogenic PSC-CMs prior to transplantation in order to evaluate whether it is likely to cause implant arrhythmia.

[0095] A dose of PSC-CMs refers to a plurality of PSC-CMs, as described above in Section 1. A dose typically refers to the amount of PSC-CMs intended for transplantation into a subject.

[0096] Transplantation of the dose can involve, for example, injection of the dose into the subject's heart, specifically into the infarct and border zones.

[0097] In the method of the second aspect of the present invention, the dose is subjected to the method of the first aspect of the present invention as described in Section 1 to identify whether the dose contains arrhythmogenic PSC-CM. That is, it is determined whether a dose containing a plurality of PSC-CM contains PSC-CM expressing CD200 (and optionally other markers such as CD172a and / or CD90) on the surface, and PSC-CM expressing CD200 (and optionally expressing CD172a and / or not expressing CD90) on the surface is identified as arrhythmogenic PSC-CM. It is understood that the method of the first aspect of the present invention can be applied to a representative sample taken from the dose to determine whether CD200 (and optionally other markers such as CD172a and / or CD90) is expressed on the surface of a plurality of PSC-CM contained in the dose. If the dose contains arrhythmogenic PSC-CM, the dose is likely to cause arrhythmia upon transplantation into the subject.

[0098] The method of the second aspect of the present invention may further include determining the proportion of arrhythmogenic PSC-CM in the dose, that is, the percentage of a plurality of PSC-CM that are arrhythmogenic PSC-CM contained in the dose. If the proportion of arrhythmogenic PSC-CM in the dose exceeds about 0.001%, the dose is likely to cause arrhythmia upon transplantation in the subject. If the proportion of arrhythmogenic PSC-CM in the dose is more than about 0.005%, or about 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, or 20%, the dose is likely to cause arrhythmia upon transplantation in the subject.

[0099] 3. Cell Sorting for Arrhythmogenic PSC-CM In the third aspect of the present invention, a method is provided for eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CM in a subject.

[0100] Transplantation of PSC-CM doses is a potential treatment for replacement of damaged or destroyed cardiomyocytes after myocardial infarction. A third aspect of the present invention provides a method of removing arrhythmogenic PSC-CM from a dose prior to transplantation to eliminate or reduce arrhythmias.

[0101] The method of the third aspect of the present invention is carried out prior to transplanting a dose of PSC-CM into a subject. First, to identify arrhythmogenic PSC-CM, a crude dose of PSC-CM is subjected to the method of the first aspect of the present invention described in Section 1. That is, it is determined whether any of the plurality of PSC-CM contained in the dose express CD200 (and optionally other markers such as CD172a and / or CD90) on the surface, and PSC-CM that express CD200 (and optionally express CD172a and / or do not express CD90) on the surface are identified as arrhythmogenic PSC-CM.

[0102] A crude dose of PSC-CM means a dose containing a plurality of pluripotent stem cells (PSC) that have been differentiated to form PSC-CM but have not been subjected to any additional treatment intended to enrich or deplete subpopulations of cardiomyocytes. A crude dose is also understood to be a dose that has not been subjected to the method of the third aspect of the present invention.

[0103] Second, in the method of the third aspect of the present invention, arrhythmogenic PSC-CMs are removed from the crude dose to provide a purified dose. The arrhythmogenic PSC-CMs can be removed from the crude dose using any suitable method known to those skilled in the art. In one embodiment, the arrhythmogenic PSC-CMs are removed from the crude dose using fluorescence-activated cell sorting (FACS). In this case, in the method of identifying arrhythmogenic PSC-CMs, the PSC-CMs are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to a fluorophore. Typically, in a FACS system, individual cells are placed in droplets, and depending on the wavelength and intensity of the fluorescence emitted from the cells, a charge is applied to the droplets, and the droplets can be deflected by an electromagnetic field for separate collection if necessary. In one embodiment, the arrhythmogenic PSC-CMs are removed from the crude dose using magnetic-activated cell sorting (MACS). In this case, in the method of identifying arrhythmogenic PSC-CMs, the PSC-CMs are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to magnetic beads. Typically, in a MACS system, the cells are passed through a magnetized column. The cells labeled with magnetic beads are magnetized to the wall of the column, and the unlabeled cells pass through the column and are thereby separated. The step of identifying arrhythmogenic PSC-CMs and the step of removing arrhythmogenic PSC-CMs may be performed sequentially as performed, for example, in a FACS system, or may be performed simultaneously as performed, for example, in a MACS system.

[0104] After transplantation of the purified dose in a subject, arrhythmias can be eliminated or reduced compared to arrhythmias after transplantation of the crude dose. This can actually be evaluated by comparing the arrhythmias that occur in test subjects receiving the purified dose with the arrhythmias that occur in control subjects receiving the crude dose. Arrhythmias can be reduced when defined by the cumulative time per day spent on arrhythmias over 25 days after transplantation. Arrhythmias can be reduced by at least about 10%, or at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 99 or at least about 100%. Arrhythmias can be eliminated.

[0105] Step (ii) of the method of the third aspect of the present invention may include removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. It may include removing at least about 60%, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose.

[0106] In a fourth aspect of the present invention, there is provided a method of providing a dose containing a plurality of PSC-CMs substantially free of arrhythmogenic PSC-CMs.

[0107] In the method of the fourth aspect of the present invention, first, a crude dose of PSC-CMs is subjected to the method of the first aspect of the present invention described in Section 1 to identify arrhythmogenic PSC-CMs. That is, it is determined whether any of the plurality of PSC-CMs contained in the dose express CD200 (and optionally other markers such as CD172a and / or CD90) on the surface, and PSC-CMs that express CD200 (and optionally express CD172a and / or do not express CD90) on the surface are identified as arrhythmogenic PSC-CMs. The crude dose is as described above with respect to the third aspect of the present invention.

[0108] Second, in the method of the fourth aspect of the present invention, the arrhythmogenic PSC-CMs are removed from the crude dose to provide a dose of PSC-CMs substantially free of arrhythmogenic PSC-CMs. Step (ii) can be performed by any suitable method known to those skilled in the art as described above for the corresponding step of the method of the third aspect of the present invention.

[0109] Step (ii) of the method of the fourth aspect of the present invention may include removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. It may include removing at least about 60%, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose.

[0110] A dose of PSC-CM that substantially does not contain arrhythmogenic PSC-CM may contain less than 0.001% arrhythmogenic PSC-CM relative to the total number of PSC-CM in the dose. A dose of PSC-CM that substantially does not contain arrhythmogenic PSC-CM may contain less than 0.005% or less than about 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, or 20% arrhythmogenic PSC-CM relative to the total number of PSC-CM in the dose.

[0111] 4. Identification of Pacemaker Cells and Provision of a Population of Pacemaker Cells In a fifth aspect of the invention, a method for identifying pluripotent stem cell-derived cardiac cells (PSC-PM) having pacemaker properties is provided.

[0112] A pure or substantially pure population of pacemaker cells can provide a useful substrate for biological research and drug testing. For example, a population of pacemaker cells can be useful in an assay for testing drugs for cardiac side effects.

[0113] Pluripotent stem cell-derived cardiac cells (PSC-PM) with pacemaker properties refer to cells obtained from pluripotent stem cells differentiated and developed into cardiomyocytes. Generally speaking, this involves mimicking development in vivo by changing signaling pathways and the cellular microenvironment. Pluripotent stem cell-derived cardiac cells are typically produced from pluripotent stem cells (PSC) in a series of steps, namely 2D proliferation, 3D proliferation, and subsequent myocardial differentiation (accompanied by mesoderm induction and subsequent cardiac specification). See Figure 1. This forms a heterogeneous mixture of pluripotent stem cell-derived cardiac cells, some of which may be PSC-PM. PSC-PM can be produced by any suitable protocol known to those skilled in the art, such as the protocol of Chen et al. (Stem Cell Res 2015, 15(2), p. 365). The PSC may be human PSC that gives rise to the formation of human pluripotent stem cell-derived cardiac cells, or non-human PSC that gives rise to the formation of non-human pluripotent stem cell-derived cardiac cells. The PSC can be derived from any mammal. The terms pluripotent stem cell-derived cardiac cells and PSC-PM are understood to encompass both human-derived and non-human-derived cells.

[0114] The method of the fifth aspect of the present invention includes determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells. If CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-PM. If CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are not PSC-PM.

[0115] The method of the fifth aspect of the present invention may also include determining whether other markers are expressed (or not present) on the surface of pluripotent stem cell-derived cardiac cells. In one embodiment, the method includes determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells and determining whether CD172a and / or CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells. In one embodiment, the method may include determining whether CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, if both CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-PM. Conversely, if CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-PM. In one embodiment, the method may include determining whether CD200 and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, if CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-PM. Conversely, if neither CD200 nor CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-PM. In another embodiment, the method includes determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, if CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-PM. Conversely, if CD200 and CD90 are not expressed on the surface of pluripotent stem cell-derived cardiac cells and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-PM.

[0116] The step of determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells can be carried out using any suitable method known to those skilled in the art. In one embodiment, the pluripotent stem cell-derived cardiac cells may be exposed to an agent containing a detectable label, which selectively binds to the marker. Then, the excess agent containing the detectable label is removed, for example, by washing. If the marker is present on the surface of the pluripotent stem cell-derived cardiac cells, the agent binds to the marker, providing labeled pluripotent stem cell-derived cardiac cells. Thus, the labeled pluripotent stem cell-derived cardiac cells are cells that express the marker (e.g., CD200, or any marker to which the agent selectively binds). Then, the detectable label can be detected to confirm the presence of the marker. In one embodiment, the marker is CD200 and the agent containing the detectable label selectively binds to CD200. In one embodiment, the marker is CD90 and the agent containing the detectable label selectively binds to CD90. In one embodiment, the marker is CD172a and the agent containing the detectable label selectively binds to CD172a.

[0117] When the expression of multiple markers is to be determined, the pluripotent stem cell-derived cardiac cells can be treated with multiple agents each containing a detectable label, each of which is selective for one of the markers to be determined and each has a distinct detectable label. In one embodiment, to determine whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are exposed to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90, providing triply labeled pluripotent stem cell-derived cardiac cells. Then, the excess agents containing the detectable labels are removed, for example, by washing, and then each of the detectable labels is detected.

[0118] In one embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be a fluorophore. In this case, the expression of the marker is indicated by the wavelength and intensity of the fluorescence emitted from the labeled pluripotent stem cell-derived cardiac cells. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to a fluorophore. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to fluorophores are known to those skilled in the art, are commercially available, and any of them are suitable for use in the methods of the present invention. Examples of suitable antibodies conjugated to fluorophores include anti-CD172a PECy5, anti-CD90 BV650, and anti-CD200 BV421.

[0119] In another embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be magnetic beads. In this case, the expression of the marker is indicated by the response of the pluripotent stem cell-derived cardiac cells to a magnetic field. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to magnetic beads. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to magnetic beads are known to those skilled in the art, are commercially available, and any of them are suitable for use in the methods of the present invention.

[0120] In one embodiment, determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells can be performed by genetic modification to integrate a reporter gene into the pluripotent stem cell-derived cardiac cells. The reporter gene is transcribed in conjunction with the transcription of the marker gene, and the reporter gene encodes a detectable product. Thus, the detectable product is produced when the marker gene is transcribed. For example, the reporter gene can encode green fluorescent protein (GFP) or luciferase. If the expression of multiple markers is to be determined, multiple separate reporter genes may be required. Genetic modification techniques for introducing reporter genes into cell lines are known to those skilled in the art and can be carried out using commercially available kits. Any such technique is suitable for use in the method of the present invention.

[0121] In one embodiment, the step of determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells is carried out using flow cytometry. Typically, in this case, the agent that selectively binds to the marker is an antibody and the detectable label is a fluorophore. Preliminary processing steps may be performed before determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells. This can include compensation, staining with a viability marker (e.g., Zombie NIR), and manual gating to remove artifacts such as necrotic tissue fragments, dead cells, and doublets.

[0122] Flow cytometry enables the detection of labeled pluripotent stem cell-derived cardiac cells from among a large number of cells. Typically, appropriate fluorescence intensity thresholds (“gates”) are set by an operator against a control sample, and if the fluorescence level of a cell exceeds these gate levels, the cell is counted as a labeled pluripotent stem cell-derived cardiac cell. The fluorescence intensity values against the gates are antibody-labeling dependent and vary from flow cytometer to flow cytometer, as well as between individual runs and between different batches of antibodies. A commonly used guideline is to set the gates such that no more than 0.5% of the events in the control sample fall outside each gate. These gates are then applied to the sample to be measured, i.e., a sample containing pluripotent stem cell-derived cardiac cells that have been exposed to an antibody containing a detectable label that is a fluorophore.

[0123] In a sixth aspect of the invention, a method is provided for providing a substantially pure dose of pluripotent stem cell-derived cardiac cells (PSC-PM) having pacemaker properties.

[0124] In the method of the sixth aspect of the invention, a plurality of pluripotent stem cell-derived cardiac cells are subjected to the method of the fifth aspect of the invention described above to identify PSC-PM. That is, it is determined which pluripotent stem cell-derived cardiac cells express CD200 (and optionally other markers such as CD172a and / or CD90) on the surface, and pluripotent stem cell-derived cardiac cells that express CD200 (and optionally express CD172a and / or do not express CD90) on the surface are identified as PSC-PM.

[0125] Second, in the method of the sixth aspect of the present invention, PSC-PM is isolated from a plurality of pluripotent stem cell-derived cardiac cells to provide a substantially pure dose of PSC-PM. PSC-PM can be isolated from a plurality of pluripotent stem cell-derived cardiac cells using any suitable method known to those skilled in the art. In one embodiment, PSC-PM is isolated from a plurality of pluripotent stem cell-derived cardiac cells using fluorescence-activated cell sorting (FACS). In this case, in the method of identifying PSC-PM, the pluripotent stem cell-derived cardiac cells are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to a fluorophore. Typically, in a FACS system, individual cells are placed in droplets, and depending on the wavelength and intensity of the fluorescence emitted from the cells, a charge is applied to the droplets, and the droplets can be deflected by an electromagnetic field for separate collection as needed. In one embodiment, PSC-PM is isolated from a plurality of pluripotent stem cell-derived cardiac cells using magnetic-activated cell sorting (MACS). In this case, in the method of identifying PSC-PM, the pluripotent stem cell-derived cardiac cells are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to magnetic beads. Typically, in a MACS system, the cells are passed through a magnetized column. The cells labeled with magnetic beads are magnetized to the wall of the column, and the unlabeled cells pass through the column and are thereby separated. The step of identifying PSC-PM and the step of isolating PSC-PM may be performed sequentially as performed, for example, in a FACS system, or may be performed simultaneously as performed, for example, in a MACS system.

[0126] The method of the sixth aspect of the present invention provides a substantially pure dose of PSC-PM. Substantially pure means that, relative to the total number of cells in the dose, no more than 0.001% of the cells in the dose are not PSC-PM, or no more than about 0.005%, or about 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, or 20% of the cells in the dose are not PSC-PM. Whether a cell is PSC-PM is determined as described above with respect to the fifth aspect of the present invention.

[0127] 5. Identification of ventricular cells and provision of a population of ventricular cells In a seventh aspect of the present invention, there is provided a method for identifying pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VM).

[0128] A pure or substantially pure population of ventricular cardiomyocytes can provide a useful substrate for biological research and drug testing. For example, a pure population of ventricular cardiomyocytes can be useful in assays for testing drugs for cardiac side effects.

[0129] Pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VM) refer to cells obtained from pluripotent stem cells that have been differentiated and developed into cardiomyocytes. Generally speaking, this involves mimicking development in vivo by changing signaling pathways and the cellular microenvironment. Pluripotent stem cell-derived cardiac cells are typically produced from pluripotent stem cells (PSC) in a series of steps, namely 2D proliferation, 3D proliferation, and subsequent myocardial differentiation (involving mesoderm induction and subsequent cardiac specification). See Figure 1. This forms a heterogeneous mixture of pluripotent stem cell-derived cardiac cells, some of which can be PSC-VM. PSC-VM can be produced by any suitable protocol known to those skilled in the art, such as the protocol of Chen et al. (Stem Cell Res 2015, 15(2), p. 365). The PSC can be a human PSC that gives rise to the formation of human pluripotent stem cell-derived cardiac cells, or a non-human PSC that gives rise to the formation of non-human pluripotent stem cell-derived cardiac cells. The PSC can be derived from any mammal. The terms pluripotent stem cell-derived cardiac cells and PSC-VM are understood to encompass both human-derived and non-human-derived cells.

[0130] The method of the seventh aspect of the present invention includes determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells. If CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-VM. If CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are not PSC-VM.

[0131] The method of the seventh aspect of the present invention may also include determining whether other markers are expressed (or not present) on the surface of pluripotent stem cell-derived cardiac cells. In one embodiment, the method includes determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells and determining whether CD172a and / or CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells. In one embodiment, the method may include determining whether CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, when CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-VM. Conversely, when both CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-VM. In one embodiment, the method may include determining whether CD200 and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, when neither CD200 nor CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-VM. Conversely, when CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-VM. In another embodiment, the method includes determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells. In this case, when CD200 and CD90 are not expressed on the surface of pluripotent stem cell-derived cardiac cells and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are PSC-VM. Conversely, when CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the cells are not PSC-VM.

[0132] The step of determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells can be carried out using any suitable method known to those skilled in the art. In one embodiment, the pluripotent stem cell-derived cardiac cells may be exposed to an agent containing a detectable label, which selectively binds to the marker. Subsequently, the excess agent containing the detectable label is removed, for example, by washing. If the marker is present on the surface of the pluripotent stem cell-derived cardiac cells, the agent binds to the marker, providing labeled pluripotent stem cell-derived cardiac cells. Thus, the labeled pluripotent stem cell-derived cardiac cells are cells that express the marker (e.g., CD200, or any marker to which the agent selectively binds). Then, the detectable label can be detected to confirm the presence of the marker. In one embodiment, the marker is CD200 and the agent containing the detectable label selectively binds to CD200. In one embodiment, the marker is CD90 and the agent containing the detectable label selectively binds to CD90. In one embodiment, the marker is CD172a and the agent containing the detectable label selectively binds to CD172a.

[0133] If the expression of multiple markers is to be determined, the pluripotent stem cell-derived cardiac cells can be treated with a plurality of agents containing detectable labels, each of which is selective for one of the markers to be determined and each has a distinct detectable label. In one embodiment, to determine whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are exposed to a first agent containing a first detectable label that selectively binds to CD200, a second agent containing a second detectable label that selectively binds to CD172a, and a third agent containing a third detectable label that selectively binds to CD90, providing triply labeled pluripotent stem cell-derived cardiac cells. Subsequently, the excess agents containing the detectable labels are removed, for example, by washing, and then each of the detectable labels is detected.

[0134] In one embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be a fluorophore. In this case, the expression of the marker is indicated by the wavelength and intensity of the fluorescence emitted from the labeled pluripotent stem cell-derived cardiac cells. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to a fluorophore. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to fluorophores are known to those skilled in the art, are commercially available, and any of them are suitable for use in the methods of the present invention. Examples of suitable antibodies conjugated to fluorophores include anti-CD172a PECy5, anti-CD90 BV650, and anti-CD200 BV421.

[0135] In another embodiment, the agent that selectively binds to the marker can be an antibody, and the detectable label can be magnetic beads. In this case, the expression of the marker is indicated by the response of the pluripotent stem cell-derived cardiac cells to a magnetic field. In some embodiments, the antibody can be an anti-CD200, anti-CD172a, or anti-CD90 antibody conjugated to magnetic beads. Many anti-CD200 antibodies, anti-CD172a antibodies, and anti-CD90 antibodies conjugated to magnetic beads are known to those skilled in the art, are commercially available, and any of them are suitable for use in the methods of the present invention.

[0136] In one embodiment, determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells can be performed by genetic modification to incorporate a reporter gene into the pluripotent stem cell-derived cardiac cells. The reporter gene is transcribed in conjunction with the transcription of the marker gene, and the reporter gene encodes a detectable product. Thus, the detectable product is produced when the marker gene is transcribed. For example, the reporter gene can encode green fluorescent protein (GFP) or luciferase. If the expression of multiple markers is to be determined, multiple separate reporter genes may be required. Genetic modification techniques for introducing reporter genes into cell lines are known to those skilled in the art and can be carried out using commercially available kits. Any such technique is suitable for use in the method of the present invention.

[0137] In one embodiment, the step of determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells is carried out using flow cytometry. Typically, in this case, the agent that selectively binds to the marker is an antibody and the detectable label is a fluorophore. Preliminary processing steps may be performed before determining whether CD200 and optionally other markers are expressed on the surface of pluripotent stem cell-derived cardiac cells. This can include compensation, staining with a viability marker (e.g., Zombie NIR), and manual gating to remove artifacts such as necrotic tissue fragments, dead cells, and doublets.

[0138] Flow cytometry enables the detection of labeled pluripotent stem cell-derived cardiac cells from among a large number of cells. Typically, appropriate fluorescence intensity thresholds ("gates") are set by an operator against a control sample, and if the fluorescence level of a cell exceeds these gate levels, the cell is counted as a labeled pluripotent stem cell-derived cardiac cell. The fluorescence intensity values against the gates depend on the antibody labeling and vary from flow cytometer to flow cytometer, as well as between individual runs and between different batches of antibodies. A commonly used guideline is to set the gates such that no more than 0.5% of the events in the control sample fall outside each gate. These gates are then applied to the sample to be measured, i.e., a sample containing pluripotent stem cell-derived cardiac cells that have been exposed to an antibody containing a detectable label that is a fluorophore.

[0139] In an eighth aspect of the present invention, a method is provided for providing a substantially pure dose of pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VMs).

[0140] In the method of the eighth aspect of the present invention, a plurality of pluripotent stem cell-derived cardiac cells are subjected to the method of the seventh aspect of the present invention described above to identify PSC-VMs. That is, it is determined which pluripotent stem cell-derived cardiac cells express CD200 (and optionally other markers such as CD172a and / or CD90) on the surface, and pluripotent stem cell-derived cardiac cells that do not express CD200 (and optionally express CD172a and / or do not express CD90) on the surface are identified as PSC-VMs.

[0141] Second, in the method of the eighth aspect of the present invention, the PSC-VMs are isolated from a plurality of pluripotent stem cell-derived cardiac cells to provide a substantially pure dose of PSC-VMs. The PSC-VMs can be isolated from a plurality of pluripotent stem cell-derived cardiac cells using any suitable method known to those skilled in the art. In one embodiment, the PSC-VMs are isolated from a plurality of pluripotent stem cell-derived cardiac cells using fluorescence-activated cell sorting (FACS). In this case, in the method of identifying PSC-VMs, the pluripotent stem cell-derived cardiac cells are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to a fluorophore. Typically, in a FACS system, individual cells are placed in droplets, and depending on the wavelength and intensity of the fluorescence emitted by the cells, a charge is applied to the droplets, and the droplets can be deflected by an electromagnetic field for separate collection as needed. In one embodiment, the PSC-VMs are isolated from a plurality of pluripotent stem cell-derived cardiac cells using magnetic-activated cell sorting (MACS). In this case, in the method of identifying PSC-VMs, the pluripotent stem cell-derived cardiac cells are exposed to an agent containing a detectable label, and the agent selectively binds to a marker that is an antibody conjugated to magnetic beads. Typically, in a MACS system, the cells are passed through a magnetized column. The cells labeled with magnetic beads are magnetized to the wall of the column, and the unlabeled cells pass through the column and are thereby separated. The step of identifying PSC-VMs and the step of removing PSC-VMs may be performed sequentially as in, for example, a FACS system, or may be performed simultaneously as in, for example, a MACS system.

[0142] The method of the eighth aspect of the present invention provides a substantially pure dose of PSC-VMs. Substantially pure means that 0.001% or less of the pluripotent stem cell-derived cardiac cells in the dose are not PSC-VMs, or about 0.005% or less, or about 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, or 20% or less of the pluripotent stem cell-derived cardiac cells in the dose are not PSC-VMs.

[0143] 6. Reduction of arrhythmia by modifying cell culture conditions In a ninth aspect of the invention, there is provided a method of eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CMs in a subject by modifying the conditions under which the PSC-CMs are cultured.

[0144] In a tenth aspect, there is provided a method of providing a dose of PSC-CM that is substantially free of arrhythmogenic PSC-CM by modifying the conditions under which the PSC-CM is cultured.

[0145] In an eleventh aspect of the invention, there is provided a method of providing a dose of PSC-CM that contains a high proportion of non-arrhythmogenic PSC-CM by modifying the conditions under which the PSC-CM is cultured.

[0146] The inventors have surprisingly found that by adding retinoic acid to the culture medium during PSC differentiation, a plurality of PSC-CMs enriched in the arrhythmogenic CD172a+ / CD90- / CD200+ subpopulation are produced. Conversely, adding a retinoic acid inhibitor during PSC differentiation, or performing PSC differentiation in the absence of vitamin A, can advantageously suppress the generation of the arrhythmogenic CD172a+ / CD90- / CD200+ subpopulation and / or increase the generation of the non-arrhythmogenic CD172a+ / CD90- / CD200- subpopulation.

[0147] The culture medium can be any culture medium suitable for the differentiation of PSCs into PSC-CMs, and various such media are known and commercially available in the art. The culture medium can be a chemically defined medium, a serum-based medium, a serum-free medium, or a xenofree medium. Examples of suitable media include mTeSR medium (e.g., mTeSR Plus basal medium supplemented with mTeSR Plus 5X supplement), TeSR-2 medium, Essential 8 medium, RPMI (Roswell Park Memorial Institute) medium (e.g., RPMI 1640 supplemented with B27 supplement).

[0148] In the method according to the 9th and / or 10th aspect of the present invention, a plurality of PSCs are cultured and differentiated to obtain PSC-CM. The differentiation can be carried out in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CM, and the arrhythmogenic PSC-CM is PSC-CM that expresses CD200 on the surface. The culture medium can be a standard culture medium as described in differentiation protocols known to those skilled in the art, and a drug can be added thereto to suppress the generation of arrhythmogenic PSC-CM or remove vitamin A therefrom.

[0149] In one embodiment, the arrhythmogenic PSC-CM expresses CD200 on the surface. In one embodiment, the arrhythmogenic PSC-CM expresses CD200 on the surface, expresses CD172a, and / or does not express CD90. In one embodiment, the arrhythmogenic PSC-CM expresses CD200 on the surface and expresses CD172a. In one embodiment, the arrhythmogenic PSC-CM expresses CD200 on the surface and does not express CD90. In one embodiment, the arrhythmogenic PSC-CM expresses CD200 on the surface, expresses CD172a, and does not express CD90.

[0150] In the method according to the 11th aspect of the present invention, a plurality of PSCs are cultured and differentiated to obtain PSC-CM. The differentiation can be carried out in a culture medium under conditions effective to increase the generation of non-arrhythmogenic PSC-CM, and the non-arrhythmogenic PSC-CM is PSC-CM that does not express CD200 on the surface. The culture medium can be a standard culture medium as described in differentiation protocols known to those skilled in the art, and a drug can be added thereto to enhance the generation of arrhythmogenic PSC-CM or remove vitamin A therefrom.

[0151] In one embodiment, non-arrhythmogenic PSC-CMs do not express CD200 on the surface. In one embodiment, non-arrhythmogenic PSC-CMs do not express CD200 on the surface, express CD172a, and / or do not express CD90. In one embodiment, non-arrhythmogenic PSC-CMs do not express CD200 on the surface and express CD172a. In one embodiment, non-arrhythmogenic PSC-CMs do not express CD200 on the surface and express CD90. In one embodiment, non-arrhythmogenic PSC-CMs do not express CD200 on the surface, express CD172a, and do not express CD90.

[0152] In the 9th to 11th aspects of the present invention, the conditions effective for suppressing the generation of arrhythmogenic PSC-CMs may include the addition of a retinoic acid inhibitor to the culture medium. A retinoic acid inhibitor is a compound that inhibits the function of retinoic acid. The retinoic acid inhibitor can be, for example, an antagonist or inverse agonist of a retinoic acid receptor, or an inhibitor of an enzyme involved in the production of retinoic acid (e.g., aldehyde dehydrogenase). The retinoic acid inhibitor can be selected from the group consisting of 4-diethylaminobenzaldehyde (DEAB), 4-[(1E)-2-[5,6-dihydro-5,5-dimethyl-8-(2-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid (BMS-493), (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoic acid (BMS-189453), and disulfiram. The retinoic acid inhibitor can be DEAB. The retinoic acid inhibitor can be BMS-493. The retinoic acid inhibitor can be BMS-189453. The retinoic acid inhibitor can be disulfiram. In one embodiment, the retinoic acid inhibitor is added to the culture medium during cardiac mesoderm induction. In one embodiment, the retinoic acid inhibitor is added to the culture medium during cardiomyocyte differentiation. Specifically, the retinoic acid inhibitor can be added between day 3 and day 7 of cardiomyocyte differentiation. The retinoic acid inhibitor can be added between day 3 and day 5 of cardiomyocyte differentiation. The retinoic acid inhibitor can be added between day 3 and day 7 of cardiomyocyte differentiation. The retinoic acid inhibitor can be added between day 5 and day 7 of cardiomyocyte differentiation. The exact timing can vary depending on the differentiation protocol. In one embodiment, in 2D culture, the retinoic acid inhibitor is added on day 3 or day 5 together with a Wnt inhibitor (IWP-2) and left in culture until day 7 or day 9. In one embodiment, in 3D culture, the retinoic acid inhibitor can be added 3 days after Wnt inhibition.

[0153] In the 9th to 11th aspects of the present invention, the conditions effective for suppressing the generation of arrhythmogenic PSC-CMs may include the removal of vitamin A from the culture medium. The removal of vitamin A can be a complete removal such that the culture medium does not contain vitamin A, or a partial removal such that the culture medium contains a lower concentration of vitamin A than under standard conditions. The removal of vitamin A can be performed during cardiomyocyte differentiation, specifically, from the 5th day to the 7th day after Wnt inhibition. In one embodiment, the conditions effective for suppressing the generation of arrhythmogenic PSC-CMs may include the addition of a retinoic acid inhibitor to the culture medium and the removal of vitamin A from the culture medium, either simultaneously or sequentially. For example, a retinoic acid inhibitor can be added to the standard medium on the 3rd day after mesoderm induction, followed by replacement with a chemically defined medium without vitamin A on the 5th day. In one embodiment, the retinoic acid receptor may have ligands other than vitamin A, and thus, the removal of vitamin A may be accompanied by the addition of a retinoic acid inhibitor that is an antagonist or inverse agonist of the retinoic acid receptor, such as BMS-493 or BMS-189453.

[0154] In the method of the 9th aspect of the present invention, arrhythmia can be eliminated or reduced after transplantation of a dose of PSC-CMs as compared to arrhythmia after transplantation of a dose of PSC-CMs not subjected to the method of the 9th aspect of the present invention. This can actually be evaluated by comparing the arrhythmia that a test subject receiving a purified dose will have with the arrhythmia that a control subject receiving a crude dose will have. Arrhythmia can be reduced when defined by the cumulative time per day spent on arrhythmia over 25 days after transplantation. Arrhythmia can be reduced by at least about 10%, or at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 99 or at least about 100%. Arrhythmia can be eliminated.

[0155] The method of the tenth aspect of the present invention provides a dose of PSC-CM substantially free of arrhythmogenic PSC-CM. A dose of PSC-CM substantially free of arrhythmogenic PSC-CM may contain less than 0.001% arrhythmogenic PSC-CM relative to the total number of PSC-CM in the dose. A dose of PSC-CM substantially free of arrhythmogenic PSC-CM may contain less than 0.005% or less than about 0.01, 0.05, 0.1, 0.5, 1, 2, 2.5, 5, 10, 15, or 20% arrhythmogenic PSC-CM relative to the total number of PSC-CM in the dose.

[0156] In the method of the eleventh aspect of the present invention, a dose of PSC-CM containing a high proportion of non-arrhythmogenic PSC-CM is provided. The non-arrhythmogenic PSC-CM is as described above. An increase in the proportion of non-arrhythmogenic PSC-CM means that the proportion of non-arrhythmogenic PSC-CM contained in the dose is at least about 5% greater than the proportion of non-arrhythmogenic PSC-CM contained in the control dose. The proportion of non-arrhythmogenic PSC-CM contained in the dose may be at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 95% greater than the proportion of non-arrhythmogenic PSC-CM contained in the control dose. The proportion is calculated relative to the total number of PSC-CM in the dose or control dose as required. The increase means an increase in the proportion. For example, if the control dose contains 10% non-arrhythmogenic PSC-CM, a dose of PSC-CM provided according to the eleventh aspect of the present invention having 5% more non-arrhythmogenic PSC-CM contains at least 15% non-arrhythmogenic PSC-CM, i.e., a 5% increase. The control dose is a dose prepared using standard culture medium, i.e., in the absence of conditions effective to increase the production of non-arrhythmogenic PSC-CM. For example, if the conditions effective to increase the production of non-arrhythmogenic PSC-CM include the addition of a retinoic acid inhibitor to the culture medium, the control dose is prepared without the addition of the retinoic acid inhibitor.

Examples

[0157] Cell heterogeneity in PSC-CM grafts is associated with treatable arrhythmias. Current cardiac differentiation protocols yield heterogeneous cell outputs. While ventricular cardiomyocytes are dominant, other cell types, such as atrial and pacemaker-like cells, as well as non-muscle cells, are also present. Despite the widely assumed view that purified ventricular cardiomyocytes could be the most desirable cell product for transplantation applications, to date, no study has definitively evaluated how the composition of PSC-CM cell doses can affect EA loading. In this study, the inventors sought to understand the important relationship between cell heterogeneity and arrhythmogenesis. The inventors also sought to test effective clinically available pharmacological and procedural anti-arrhythmic treatments that could eliminate EA when it occurs in clinical trials. The inventors used a porcine MI model to phenotype the composition of the input cells with particular focus on myocardial cell subpopulations and then test the transplanted PSC-CM. The inventors aimed to identify cell properties that predict arrhythmogenesis and hypothesized that this would provide useful data for the safe production of cardiomyocytes for future clinical use.

[0158] Methods Cell production Bioreactor differentiation protocol The H9 cell line containing the gCaMP6f fluorescent calcium reporter was used in all experiments (provided by the University of Queensland StemCore facility). Each production run started from a frozen working cell bank (WCB) cryovial, which was grown as a monolayer for 8 days on Matrigel (Corning) using commercially available medium mTeSR™ Plus (Stem Cell Technologies) (Figure 1). On day -3, the cells were transferred into mTeSR 3D supplemented with 10 μM Y-27632 (TOCRIS Bioscience) and 10 μg / mL of the thermoresponsive polymer PNiPAM conjugated to fibronectin (Chen, X. et al. Tissue Eng Part C Methods 24, 146-157) at a density of 2.5-3.0×10 5Inoculated at a density of cells / mL to form aggregates. Using a DASbox (Eppendorf) stirred tank bioreactor system, the suspension was controlled at 37.2 °C, pH 7.2, and 30% DO. On day -1, rapamycin (Merck) was added to a final concentration of 5 nM to enhance survival during differentiation. On day 0, the pluripotent aggregates were washed twice with RPMI 1640 and then cultured in insulin - free RPMI - B27 (Thermo Fisher Scientific) containing 6 μM of CHIR 99021 (TOCRIS Bioscience) and 5 nM of rapamycin. On day 1, 24 hours after mesoderm induction, the aggregates were washed once with RPMI 1640 and transferred back to RPMI - B27 without insulin but containing 5 nM of Rapamycin. On day 2, the aggregates were washed and transferred back to RPMI - B27 without insulin but containing 2 μM of IWP - 2 (Stem Cell Technologies). On day 4, the aggregates were transferred to RPMI - B27 containing insulin, and the medium was changed every other day until cryopreservation on day 15. Before cryopreservation, H9 - gCaMP6f - derived cardiomyocytes were given a heat shock at 42 °C for 30 minutes and treated with a survival - promoting cocktail (100 ng / mL IGF - 1, 0.6 μM of cyclosporine A) to enhance their survival after transplantation. The cardiomyocyte aggregates were incubated in 4 mg / mL collagenase IV for 6 hours, washed, and dissociated into single cells using TrypLE (Thermo Fisher). The cardiomyocytes were cryopreserved at 10×10 6 cells / mL.

[0159] Flow cytometry The expression of cTnT was measured with a Cytoflex Flow Cytometer (Beckman Coulter). Briefly, 1×10 6The cells were fixed with 2% paraformaldehyde for 10 minutes at room temperature. The cells were stored in FACS wash buffer (0.5% BSA in PBS) at 4°C until staining. The samples were permeabilized in 0.1% Triton X-100 for 10 minutes and then stained with CTNT-FITC 1:50 (Miltenyi Biotec) for 30 minutes at room temperature. The cells were washed twice before analysis. The excitation laser and emission filter used were as follows: excitation: 488, emission: 525 / 40.

[0160] Quantitative PCR After dissociation of the aggregates, 1×10 6 cells were collected in RNAprotect Cell Reagent (Qiagen) and stored at 4°C until RNA extraction. RNA was extracted using the Qiagen RNeasy Mini Kit according to the manufacturer's instructions. Using the RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific), 1 μg of RNA was converted to cDNA in a 20 μL reaction according to the manufacturer's instructions. The cDNA was diluted 1:10 with DNase / RNase-free water, and 1 μL of the diluted cDNA was used in each 10 μL reaction with 4 μL of 1 μM F / R primer and 5 μL of Fast SYBR Green qPCR master mix (ThermoFisher Scientific). An overview of the genes used and their respective primers is shown in Table 1.

[0161]

Table 1

[0162] The reactions were performed in technical triplicate. Quantitative PCR was performed on a BioRad CFX96 Real-Time PCR Detection System using the standard cycling parameters described in the master mix protocol. A dissociation curve was obtained at the end of each run. The fold difference in expression was calculated using the comparative CT method using GAPDH cDNA from a reference sample. Undifferentiated hPSCs or fetal heart RNA were used as reference samples depending on the gene and time point.

[0163] Single-cell RNA sequencing Hash tagging and sequencing protocol Frozen PSC-CMs were thawed in a water bath and the contents of each vial were transferred to a 50 mL Falcon tube containing RPMI-B27 medium supplemented with insulin (ThermoFisher Scientific), 5% FBS (ThermoFisher Scientific), and 10 uM ROCK inhibitor Y-27632 (STEMCELL Technologies). Each sample was spun down at 1000 rpm for 5 minutes and the cell pellet was resuspended in filtered PBS + 2% BSA. The cells were then incubated in blocking buffer containing 5 uL of Human TruStain FcX solution (BioLegend) + 100 uL of staining buffer (2% BSA + 0.01% Tween20 in filtered PBS) for 10 minutes at room temperature. Next, 1 uL of hash tag antibody (BioLegend) was added to each sample and incubated on ice for 20 minutes. The hash tagging protocol used was one that allowed multiple samples to be pooled together in a single sequencing run. The cells were then washed twice with staining buffer and 4 x 10 5Individual cells were collected and pooled in 2 mL Eppendorf tubes. Prior to sequencing, a viability test was performed, suggesting a viability of 80% of the captured cells. The cell suspension was loaded onto 10× Genomics Single Cell 3’ Chips to form single cell gel beads (GEMs) in emulsion. A Chromium library pool containing 90% gene expression library with 10% hashing library spike-in was generated and sequenced on an Illumina NextSeq 500 instrument. The sequencing data was further processed to generate FASTQ files and raw count matrices using the CellRanger pipeline at the sequencing facility of the University of Queensland. Multiplexed samples were demultiplexed to their original identities by mapping sample reads to the GRCh38-3.0.0 human reference genome and hashing the sequences.

[0164] Bioinformatics analysis The gene expression count matrix was loaded into Seurat. Before creating the Seurat object, the inventors removed genes expressed in less than 1% of the total cells. The hashtag matrix was added to the Seurat object as a new assay separate from RNA. The RNA data was normalized by log normalization, and the cell hashing data was normalized by centered log ratio (CLR) transformation. After normalization, the cells were demultiplexed and mapped to their original sample identities or assigned as doublets or negatives based on the Seurat HTODemux algorithm. Furthermore, the inventors converted the Seurat object to a SingleCellExperiment and ran single-cell doublet scoring (scds) for doublet annotation. The Scds method returns three scores, while HTODemux returns only one mark. Cells were considered doublets and excluded if annotated as doublets by at least two of the classifications. The inventors further filtered the cells according to the standard quality control (QC) workflow in Seurat. After preprocessing the data, the inventors removed low-quality cells and retained 11,307 cells for further analysis. After data normalization and scaling, the inventors performed dimensional reduction and selected 50 principal component (PC) dimensions as input for unsupervised clustering and uniform manifold approximation and projection (UMAP) plots. By examining the expression levels of marker genes, the cell types that distinguish each cluster were annotated. The Nebulosa package was used to enhance the visualization of marker gene expression.

[0165] High-parameter flow cytometry Antibody staining Thawed PSC-CM (about 1.5×10 per sample tube) 6Individual cells were stained with an amine-reactive viability dye (Zombie NIR, Biolegend) in PBS for 30 minutes. The samples were washed twice with PBS + 2% FBS and centrifuged at 225 G each time. The samples were then stained with fluorescent dye-conjugated membrane marker antibodies as shown in Table 2 below in PBS + 2% FBS for 30 minutes and washed twice.

[0166]

Table 2

[0167] For the measurement of cardiomyocyte troponin T-positive cells after thawing, separate samples of PSC-CM were fixed with 4% PFA for 20 minutes and permeabilized in PBS + 0.5% Tween-20. The cells were incubated with an anti-human troponin T antibody in permeabilization buffer for 30 minutes and then washed twice with PBS + 2% FBS.

[0168] Single-color compensation controls were prepared using compensation beads as follows: CompBead Plus, BD, for mouse antibodies; AbC Total Antibody, ThermoFisher, for rabbit antibodies; ArC amine-reactive beads, ThermoFisher, for Zombie NIR, unstained PSC-CM, for endogenous GcAMP / GFP.

[0169] Data acquisition and analysis PSC-CM and compensation controls were analyzed using a BD FACSymphony A5 cytometer and FACS Diva software with application settings.

[0170] Data analysis was performed using FlowJo software (Treestar, version 10.7.2). An overview of the manual gating and data processing methods is shown in Figure 2. The corrected data was manually gated to remove necrotic tissue fragments, non-viable cells, and doublets. To identify PSC-CM subpopulations based on surface marker expression, dimensionality reduction was performed using t-distributed stochastic neighbor embedding (tSNE), and unsupervised clustering was carried out using FlowSOM (opt-SNE parameters: gradient algorithm - Barnes-Hut, learning configuration - opt-SNE, KNN algorithm - exact vantage point tree, Iterations - 1000, Perplexity - 30; FlowSOM parameters: number of metaclusters - 25, SOM grid size - 10×10, node scale - 100%, set seed - 3). To compare the dose compositions between animals and identify potentially arrhythmogenic subpopulations, the inventors downsampled and concatenated the data prior to dimensionality reduction and clustering.

[0171] In Vitro Electrophysiological Assays iPSC-CM Monolayer Differentiation iPSC-CMs (SCVI-8, Stanford Cardiovascular Institute) were used in in vitro electrophysiology experiments and differentiated under 2D monolayer culture. Two days prior to differentiation, PSCs were seeded at 1.5×10 per well on Matrigel-coated 6-well culture plates. 6Cells were seeded at a density of in mTeSR Plus (STEMCELL Technologies) + 10 μM Y-27632. On the first day of differentiation (day 0), the medium was changed from mTeSR Plus to RPMI 1640 + B27 without insulin + Glutamax + penicillin / streptomycin + 6 μM of the GSK3 inhibitor, CHIR-99021 (Tocris Bioscience). After 24 hours, the cells were washed with PBS to remove CHIR-99021, and the medium was changed to RPMI 1640 + B27 without insulin + Glutamax + penicillin / streptomycin. On day 3, the medium was supplemented with the Wnt signaling inhibitor, IWP-2 (5 μM). On day 5, the medium was changed to RPMI 1640 + B27 without insulin + Glutamax + penicillin / streptomycin. For atrial / pacemaker cell enrichment differentiation, 1 μM retinoic acid (Sigma-Aldrich) was added at the time of medium change on day 3 and also on day 5. On day 7, the medium was changed to RPMI 1640 + B27 containing insulin + Glutamax + penicillin / streptomycin. Thereafter, the medium was changed every 2 - 3 days. Cardiomyocyte beating started between days 6 and 7.

[0172] Patch clamp For electrophysiological measurements using high-throughput patch clamp, on day 15, beating iPSC-CMs were dissociated with TrypLE (ThermoFisher) and re-seeded at a density of 1.5×10 6 cells per well onto Matrigel-coated 12-well plates and maintained in culture until used for patch clamp experiments between days 30 and 35. On the day of patch clamp recording, the cells were washed with PBS, dissociated with TrypLE, and centrifuged at 300G for 3 minutes. The cells were then resuspended in a solution of 80% RPMI-1640 without phenol red + Ca 2+ + 10% FBS and 20% divalent cation-free buffer (pH 7.4 with NaOH) containing 140 NaCl, 4 KCl, 5 glucose, 10 HEPES. The cells were counted, and using the divalent-free solution, approximately 2×10 5Diluted to a concentration of cells / mL.

[0173] Patch-clamp recordings were collected using a Syncropatch 384 PE (Nanion Technologies) in voltage-clamp mode. Sodium currents (I Na ) were recorded using internal (pH 7.2 with CsOH) and external (pH 7.4 with NaOH) solutions containing, respectively: 110 CsF, 10 NaCl, 10 CsCl, 10 HEPES, 10 CsOH, 10 EGTA, and 140 NaCl, 4 KCl, 5 glucose, 10 HEPES, 2 CaCl2, 1 MgCl2. Sodium currents were evoked from a holding potential of -80 mV with 5 mV voltage steps in the range of -120 to +40 mV, with a 200 ms prepulse to -120 mV.

[0174] Optoelectrophysiological examination Action potentials were recorded using a kinetic imaging cytometer (KIC, Vala Sciences, San Diego, CA, USA) and a voltage-sensitive fluorescent indicator (FluoVolt, ThermoFisher). On day 14 of differentiation, beating cardiomyocytes were dissociated with TrypLE (ThermoFisher) and reseeded at a density of 1.5×10 4 cells per well on a Matrigel-coated flat-bottom 96-well plate (Greiner CELLSTAR, Sigma-Aldrich). The cells were maintained for an additional 5 - 7 days. On the day of recording, the cells were washed with 100 μL of RPMI-1640 without phenol red + Ca 2+ . After removing the medium, the cells were exchanged with 50 μL of fresh RPMI without phenol red + Ca 2+ and incubated at 37 °C for 60 minutes. The cells were then incubated for 20 minutes in RPMI-1640 without phenol red supplemented with FluoVolt, Powerload pluronic solution, and Hoechst dye. Then, the FluoVolt solution was removed and replaced with fresh RPMI-1640 without phenol red + Ca 2+ .

[0175] The cells were stimulated with KIC for 10 seconds at 1.5 Hz, following which they were not stimulated for 5 seconds and then recorded without further stimulation for another 5 seconds. Cell segmentation and single-cell transient extraction were performed using CyteSeer Scanner software (Vala Sciences, San Diego, CA, USA). Arbitrarily selected action potential measurement results were analyzed using custom KIC data analysis software (KIC DAT) 70 for analysis.

[0176] Porcine experiment Acclimation / housing All experiments were conducted on female Landrace pigs (2 to 4 months old, 25 - 30 kg) obtained from the same regional source. All measures in this study were approved by the Western Sydney Local Health District Animal Ethics Committee (Protocol ID: 4262.03.17). The animals were housed in a dedicated large animal research facility and brought there 1 - 2 weeks before the first scheduled measure for acclimation.

[0177] Myocardial infarction Myocardial infarction was induced percutaneously by the method reported previously (Thavapalachandran, S. et al. Science Translational Medicine 12, eaay2140 (2020)). Briefly, animals were premedicated with intramuscular ketamine (10 mg / kg), methadone (0.3 mg / kg), and midazolam (0.3 mg / kg), intubated, ventilated, and maintained with inhaled isoflurane. A 6F hockey stick guide catheter (Medtronic, Minnesota, U.S.A) was used to engage the left coronary artery percutaneously via the right femoral artery. A 0.36 mm coronary guide wire (Sion Blue, Asahi Intecc, Aichi) was delivered to the left anterior descending (LAD) branch. Myocardial infarction was induced by occluding the central LAD distal to the first diagonal branch for 90 minutes with an inflated 2.0 - 3.0 mm angioplasty balloon (Boston Scientific, Massachusetts, U.S.A). Coronary angiography performed after reperfusion confirmed vessel patency and resolution of ST elevation. Ventricular arrhythmias were treated with antiarrhythmic drugs and defibrillation as needed.

[0178] Implantation of the remote measurement device All animals received an implant of a remote measurement transmitter (easyTEL +, Emka technologies) under general anesthesia. The transmitter was implanted in a subcutaneous pocket formed on the left flank. The apex and base of the heart were captured through subcutaneous leads. Signal quality was evaluated before securing the device and leads in place.

[0179] Remote measurement analysis Remote measurement ECG and accelerometer data were continuously monitored from the time of device implantation. The semi - automatic quantification of heart rate, arrhythmia burden, and accelerometer data was performed offline by a cardiologist using the EcgAUTO 3.5.5.16 software package (Emka technologies, Paris, France). Arrhythmias were defined as ectopic beats (e.g., ventricular premature contractions) or dysrhythmias. The entire dataset recorded for each subject was analyzed, and the data were presented as daily averages (mean ± S.E.M).

[0180] Cardiac MRI Before intubation and mechanical ventilation, animals were pre - administered intramuscular ketamine (10 mg / kg), methadone (0.3 mg / kg), and midazolam (0.3 mg / kg). General anesthesia was induced with intravenous propofol (2 - 5 mg / kg) and maintained with 2% inhaled isoflurane. In all image acquisitions, respiration was arrested at end - expiration. All CMR examinations were performed on a Siemens 3T Prisma system (Siemens Medical Systems) using a spinal array coil and an 18 - channel body array with 4 - lead electrocardiogram (ECG) gating. Axial and coronal TrueFISP (true fast imaging with steady - state precession) sequences through the heart were acquired to plan preliminary 2 - chamber, short - axis, and 4 - chamber single - slice gradient images. Next, true 2 - chamber, 3 - chamber, 4 - chamber, and right ventricular outflow tract 8 - mm single - slice TRUFI (true fast imaging) cine were planned and acquired, and further, a short - axis stack of 14 consecutive 8 - mm slices starting just distal to the apex and extending into the atrium was planned and acquired from 2 - chamber and 4 - chamber cine images at end - diastole.

[0181] TrueFISP imaging was used for all acquisitions with the following parameters: TE (echo time) 1.3 ms, TR (repetition time) at the R - R interval of the individual animal, FOV (field of view) 370 mm, slice thickness 8 mm, in - plane resolution 1.4 mm×1.4 mm, flip angle 10 degrees, 25 computed phases.

[0182] Image analysis was performed offline by two independent blinded cardiac specialists using dedicated software (Medis Suite MR 3.2, Medis Medical Imaging, Schuttersveld 9, the Netherlands). Volume measurement evaluation was performed according to the Society for Cardiovascular Magnetic Resonance guidelines. Briefly, short-axis end-diastolic and end-systolic images were selected as the maximum and minimum intermediate ventricular cross-sectional areas. The endocardial and epicardial boundaries at end-diastole were manually traced for each slice together with the endocardial boundary at end-systole. Vendor-specific automatic contouring algorithms were not used due to suboptimal performance in non-human subjects. Papillary muscles were included in the volume and excluded from the mass calculation. The difference between the end-diastolic endocardial boundary and the end-systolic endocardial boundary represents the left or right ventricular stroke volume, and the ejection fraction was calculated as stroke volume / end-diastolic volume. All parameters were analyzed by two independent blinded observers with excellent inter-observer variability. Intra-observer variability was determined by having one observer, blinded to previous measurements, repeat the measurements for all subjects on two separate occasions at least two weeks apart.

[0183] ADAS-3D CMR Reconstruction Following CMR imaging, a separate offline segmentation software, ADAS-3D (SpaMedical, Barcelona, Galgos) was utilized to process 3D reconstructions of the left ventricle (LV) and fibrosis identified by LGE for integration into an electroanatomical mapping system (EAM). To assist in alignment to the EAM reconstruction, the complete anatomical structure of the heart including the coronary arteries was also exported. Endocardial and epicardial boundaries were delineated in all slices and scar evaluation was performed based on the pixel signal intensity (PSI) between the two boundaries. Values for identification of high density fibrosis and border zone regions using the maximum PSI have previously been correlated with low voltage regions and conduction channels in the EAM. Fibrosis area was calculated by averaging the PSI from the endocardial layer to the mid-myocardial layer and the mean PSI from the mid-myocardial layer to the epicardial layer. The calculated values were then projected onto the LV reconstruction of the endocardial and epicardial surfaces using trilinear interpolation.

[0184] Thoracotomy, epicardial mapping and cell injection On day 0, the animals were sedated and returned to the operating room for thoracotomy and epicardial injection. Following cannulation and intubation, an arterial line was inserted into the auricular artery or distal limb artery to enable continuous blood pressure monitoring. A 100 mcg fentanyl patch was applied and intravenous antibiotics (cefazolin, 1 g) were administered. Intercostal nerve blocks from the second to the sixth rib spaces were performed using bupivacaine and lignocaine.

[0185] Thoracotomy was performed by making an incision in the left lateral chest wall at the 4th / 5th intercostal space. A self-retaining rib retractor was used to open the incision under direct vision. The pericardium was opened anteriorly, and the posterior mediastinum was wrapped with moist gauze to gently expose the cardiac apex and anterior left ventricle. Hemodynamics were carefully monitored, and metaraminol boluses were administered as needed to maintain systolic blood pressure above 100 mmHg. The epicardial surface of the left ventricle was electroanatomically mapped using an electrophysiological mapping catheter (Navistar Thermocool Smarttouch, Biosense Webster). Cardiac MRI was imported into the EAM system, and alignment was performed by placing the mapping catheter at the reference landmarks under direct visualization. The reference landmarks used were the cardiac apex, mitral annulus, and left anterior descending artery. EAM points were acquired with the mapping catheter in the EAM system, and the same landmarks were selected on cardiac MRI. Initial alignment was performed using the landmarks, and then secondary alignment by best fit of all anatomical structures was applied using the EAM system software (CartoMerge, Biosense Webster). Confirmation of alignment accuracy was rechecked using the EAM catheter. After alignment, the electroanatomical voltage map was evaluated to identify scars, borders, and remote zones. Then, either vehicle (8 × 300 μL of RPMI B27 injection) or cells (750 × 10 6 cell distribution) were injected epicardially into the infarct and border zones under direct vision using a 27-gauge insulin syringe.

[0186] Central venous catheter insertion and immunosuppression All subjects received an immunosuppressive regimen of three drugs to prevent xenograft rejection. Oral cyclosporine A (10 - 15 mg / kg, twice daily) was administered to the animals starting 5 days before cell injection, aiming to maintain trough levels above 250 ng / mL. On day 0, a 2-lumen 5 French central venous catheter (Teleflex) was implanted into the right jugular vein under ultrasound guidance to facilitate continuous administration of intravenous immunosuppressants and regular blood sampling. After central venous line placement and before cell injection, 500 mg of abatacept (CTLA4-Ig, Bristol-Myers Squibb), 30 mg / kg of methylprednisolone, and 3 - 5 mg / kg of cyclosporine A were administered intravenously. From day 1 after transplantation until euthanasia, the subjects received oral cyclosporine A (10 - 15 mg / kg) twice daily along with 100 mg of intravenous methylprednisolone daily to maintain trough levels above 250 ng / mL. An additional 250 mg dose of intravenous abatacept was given 2 weeks after transplantation. Prophylactic oral amoxicillin / clavulanic acid was given daily to all subjects to prevent central venous line infections and thoracotomy infections, and 30 mg of oral lansoprazole was given daily for gastrointestinal protection.

[0187] Antiarrhythmic treatment Animals randomized to antiarrhythmic treatment received an IV bolus of 150 mg of amiodarone at the time of cell injection. From day 1 after transplantation until euthanasia, they were treated with a regimen of 200 mg of oral amiodarone twice daily and 10 mg of oral ivabradine twice daily.

[0188] Electrophysiological testing Before euthanasia, all subjects were subjected to an electrophysiological study under general anesthesia to determine the inducibility, mechanism, and electroanatomical origin of any ventricular arrhythmias. An 8.5 Fr Agilis steerable introducer (Abbott Medical) was inserted into the right common femoral vein under ultrasound guidance, through which an Advisor HD Grid (Abbott Medical) multi-electrode electrophysiological catheter was advanced into the right ventricle. A 6 Fr 10-pole electrophysiological catheter was placed into the coronary sinus via the right femoral vein. Using the Ensite Precision EAM system (Abbott Medical), substrate maps depicting scars, boundaries, and remote zones were generated using bipolar and unipolar voltage cutoffs of 0.5 - 1.5 mV and 3 - 8.3 mV, respectively. Subsequently, the left ventricle was mapped via a retrograde aortic approach. If the subject was in sinus rhythm, the substrate map was created first. Following substrate mapping, ventricular tachycardia (VT) induction by programmed electrical stimulation (PES) was performed. After an 8-beat drive train at 400 ms, up to 4 additional stimuli delivered one at a time followed. The first extrastimulus was delivered at a coupling interval of 300 ms and then decreased by 10 ms to ventricular refractoriness. A modified "Michigan" protocol was further implemented. The Michigan protocol uses only 4 additional stimuli. At each drive train cycle length of 350 ms, the programmed stimuli are initiated at coupling intervals of 290, 280, 270, and 260 ms for the 1st - 4th additional stimuli. The coupling intervals of the additional stimuli are simultaneously shortened in 10 ms steps until the additional stimuli become refractory or an arrhythmia is induced. Both PES protocols were repeated at 2 sites. Following PES, isoproterenol bolus (20 mcg) and infusion (6 - 10 mcg / min) were administered by burst pacing starting at 300 ms and decreased in 20 ms steps until refractoriness during isoproterenol delivery and washout. The mechanism of any spontaneous or induced ventricular arrhythmia was elucidated by standard pacing maneuvers and an EAM to identify the origin of the arrhythmia thereafter.

[0189] Catheter ablation Subjects who underwent catheter ablation for the treatment of EA were premedicated with intramuscular ketamine (10 mg / kg), methadone (0.3 mg / kg), and midazolam (0.3 mg / kg), intubated, ventilated, and maintained with inhaled isoflurane. An arterial line was inserted into the auricular artery or distal limb artery to enable continuous blood pressure monitoring. All subjects had spontaneous arrhythmias at the time of ablation procedures. The arrhythmia origin was mapped electroanatomically as described above. At the site of the earliest activation mapping, high-frequency ablation lesions were delivered using a 4-mm tip open irrigation catheter (Flexability, Abbott Medical). A single ground patch was placed on each animal, and ablation was performed using a power of 30 - 40 W and an irrigation flow rate of normal saline at 13 mL / min in power control mode. Delivery of each lesion was attempted for 30 - 60 seconds, unless terminated prematurely due to catheter movement or an increase in impedance. Ablation was terminated after sinus rhythm recovered, and subsequently VT induction was attempted as described above.

[0190] Euthanasia and tissue collection After the final EPS procedure, the subjects were euthanized with potassium chloride (75 - 150 mg / kg), the hearts were excised, and fixed in 10% neutral buffered formalin for subsequent analysis.

[0191] Histological examination Tissue processing The entire porcine heart was fixed in 10% neutral buffered formalin for at least 48 hours. The ventricles were then sliced into transverse sections approximately 1 cm thick from the apex (level 1) to the base (level 7). After fixation, the tissue was exchanged into 70% ethanol, processed, and paraffin-embedded. Processing, embedding, and sectioning of level 1 blocks were performed at the Westmead Institute for Medical Research. Processing, embedding, and sectioning of larger blocks (level 2 and above) were performed by the Veterinary Pathology Diagnostics Services at the University of Sydney.

[0192] Immunohistochemical examination Immunohistochemical analysis was performed on 4-μm sections taken from levels 1, 2, or 3. The sections were deparaffinized in xylene and hydrated by sequential incubation in 100%, 95%, 70%, 50% ethanol, and water. Antigen retrieval was performed using heated sodium citrate buffer, and then the sections were washed in PBS + 0.1% Tween 20, blocked with 5% goat serum in PBS + 0.5% Tween-20, and stained with the primary antibody overnight at 4°C. The next day, the sections were washed and incubated with the secondary antibody for 1 hour at room temperature in the dark. The sections were then washed again, incubated with DAPI (1 μg / mL, Sigma-Aldrich / Merck) for 10 minutes, rinsed with PBS, and mounted with PBS:glycerol. For the gross description of the PSC-CM grafts and high-frequency ablation sites, whole-mount sections from level 2 tissue blocks were stained with primary antibodies targeting human Ku80 and cardiac troponin T as described above. For the bright-field detection of secondary staining, the inventors used the ImmPRESS Duet double staining polymer kit HRP / AP (Vector Laboratories, MP-7714), and then counterstained the sections with aniline blue (1% aniline blue in MilliQ water, 1 minute).

[0193] Imaging and analysis Immunofluorescence and bright-field microscopy were performed using an Olympus VS120 Slide Scanner with a 20× objective lens (UPLSAPO 20X / NA 0.75, WD 0.6 / CG thickness 0.17) or a 40× objective lens (UPLSAPO 40X / NA 0.95, WD 0.18 / CG thickness 0.11–0.23). Images were acquired using Olympus VS-ASW 2.92 software and processed using Olympus VS-DESKTOP 2.9.

[0194] Confocal images of GFP, Cx43, and cTnT immunostained porcine tissues were taken with a Leica SP8 microscope using adjustable white light and a 405 nm laser. A two-channel scanning approach was used to adjust the filters for optimal signal detection of Alexa Fluor 488, Alexa Fluor 594, and Alexa Fluor 647. Z-stacks (7 μm with a step size of 0.5 mm) were imaged using a confocal pinhole set to 1 airy unit.

[0195] Spatial transcriptomics Tissue preparation Formalin-fixed paraffin-embedded tissues from level 1 blocks were used for spatial transcriptomic analysis of transplanted PSC-CMs. Using tissue microarray punches, the inventors collected 3 mm diameter tissue samples from regions confirmed to contain PSC-CM grafts. The samples were thawed and re-embedded in four 6 mm × 6 mm paraffin blocks, each containing three samples.

[0196] RNA quality assessment For RNA extraction using the RNeasy FFPE Kit (#73504, Qiagen), 3 - 5 7-μm sections were collected per sample. The RNA integrity number (RIN) and DV200 were determined by a BioAnalyzer using the RNA 6000 Pico Kit (#5067-1513, Agilent). DV200 is the percentage of total RNA fragments longer than 200 bp. All samples had a DV200 of 46 - 57%.

[0197] Tissue optimization To find the optimal permeation treatment time and the thickness of tissue sections, the inventors adapted the Visium Spatial Tissue Optimization User Guide to fresh frozen ST (CG000238 Rev A, 10X Genomics). FFPE blocks were sectioned to 7 μm by a rotary microtome. Paraffin sections were floated on nuclease-free water on a 43°C water bath for 2 minutes per section and captured while floating on each assigned array on a Visium tissue optimization slide (#3000394). Subsequently, the slides with FFPE sections were dehydrated at room temperature for 1 hour using silica bead desiccant and then stored overnight at 4°C in a sealed slide box containing silica beads. The next day, the slides were dried at 37°C for 15 minutes to be completely dehydrated, then the wax was melted at 60°C for 60 minutes and deparaffinized with xylene (5 minutes, 2 times). Rehydration of the tissue was performed using an ethanol gradient (100% for 2 minutes, 2 times; 90% for 2 minutes, 2 times; 85% for 2 minutes). The above temperatures and times were optimized to ensure that the tissue adhered to the slide throughout the process.

[0198] Tissue sections were stained with hematoxylin and eosin (H&E) according to a standard protocol and imaged at 20–40× magnification with a Zeiss AxioScan Z1 slide scanner. Next, the tissue was decrosslinked by incubating with collagenase for 20 min at 37 °C and then for 60 min at 70 °C in TE buffer (pH 8.0). Immediately after decrosslinking, the tissue sections were permeabilized by incubating with pepsin (0.1%) for various times (5–40 min). In this step, RNA is released from the tissue onto the slide glass and the poly(A) tail hybridizes to the slide-bound poly(dT) oligo. After permeabilization, a reverse transcription reaction was performed to synthesize cDNA labeled with cyanine 3 (Cy3). Next, the tissue sections were removed from the slides using tissue removal buffer. The cDNA products on the slides were visualized with a Leica DMi8 inverted wide-field microscope. By comparing the fluorescence intensities and evaluating the spread of the signals, the inventors determined the optimal permeabilization conditions to avoid over- or under-permeabilization.

[0199] Preparation of sequencing libraries The FFPE block was sectioned, placed on a Visium Spatial Gene Expression Slide (#2000233, accommodating four capture areas), dried, dewaxed, deparaffinized, and stained as in tissue optimization procedures. After permeabilization, cDNA was synthesized using standard unlabeled nucleotides, followed by second-strand synthesis. The spatial barcodes and unique molecular identifiers (UMIs) are part of the oligos printed on the slide glass, and this sequence is incorporated into the first-strand cDNA. The double-stranded DNA product was denatured, and the released cDNA was PCR amplified for 18 cycles, end-repaired, A-tailed, and size-selected by SPRIselect (0.8X bead clean-up). Adapter ligation, PCR amplification, and sample indexing were performed according to the Illumina TruSeq protocol. After final PCR amplification (10 - 15 cycles) and size selection (0.55× and 0.8× double-sided clean-up), QC was performed by BioAnalyzer. The library was sequenced using the NovaSeq SP100 V1.5 kit (138 cycles), paired-end protocol, as follows: Read 1 - 28 bp, Index 1 - 10 bp, Index 2 - 10 bp, Read 2 - 90 bp.

[0200] Sequencing data analysis The raw NovaSeq BCL output files were converted to fastq files using SpaceRanger mkfastq V1.3.0 and bcl2fastq2-V2.17.1. The fastq files were further processed using cutadapt V3.2 to remove adapter sequences and polyA sequences in Read2. Using SpaceRanger mkref, a hybrid reference genome was created from the combination of Scrofa 11.1 (reference annotation Scrofa 11.1.105.gtf) and GRCh38-3.0.0 (genome build GRCh38.p12). The trimmed fastq reads were mapped to the hybrid genome using SpaceRanger count based on STAR splicing recognition alignment. The gene expression matrix contains uniquely and confidently mapped UMIs. A UMI is counted only if it is mapped to a single exon locus (at least 50% of the reads cross an exon), or if multiple mappings occur, this UMI must have MAPQ255 (uniquely mapped), have bases that are 100% compatible with the exons of the annotated transcript, and be aligned to the same strand. High-resolution H&E images were used for mapping gene expression to spatial spots. Gene expression was used for downstream analysis.

[0201] Quality control Capturing interspecies gene expression from fixed transplanted tissues has been a challenge so that both human and porcine-derived mRNAs can be measured. The inventors developed a protocol that enables unbiased capture of two species. The inventors noted that the overall quality of the data was not optimal. Therefore, a quality control process was applied to select the top two samples with the highest quality among all samples. Prior to downstream analysis, genes and spots that were likely to be noise or outliers were identified and removed. Across all samples, the inventors established thresholds for the number of detected genes (80) and the number of spots containing a sufficient number of genes (10). This process yielded two samples that passed the quality threshold, one with RA treatment (RA-PSC-CM#3) and the other with standard treatment (PSC-CM+CA#2). Each sample contained 1229 and 1107 human genes and 9105 and 5501 porcine genes from 808 and 567 spots for RA and standard treatment, respectively.

[0202] Identification of Human and Porcine Spots For each spot, the inventors calculated its human score as the total number of reads mapped to the human genome and its porcine score as that mapped to the porcine genome. In the RA-treated tissue, the inventors assigned a spot as a human spot if its human score > 30 and its porcine score < 700. In the standard-treated tissue, the inventors assigned a spot as a human spot if its human score > 30 and its porcine score < 350. In total, the inventors identified 62 human spots in sample DZ24 and 50 human spots in sample DZ22.

[0203] Differential Expression Analysis Only one biological sample was present for each condition, but the inventors utilized spots measured separately from each other for performing pseudo-bulk differential gene expression analysis. For each sample, the inventors randomly pooled the spots into three equally sized pools. For each pool, the average gene expression was calculated spot-wise. By doing this, the inventors created three pseudo-replicates of RA treatment versus standard treatment for each condition. Using the pseudo-replicates, the inventors performed differential expression analysis using library size normalization and quasi-likelihood test according to the edgeR pipeline. Genes with an adjusted p-value less than 0.05 were considered significant.

[0204] Statistical analysis Continuous data were represented as mean ± standard error of the mean (SEM). Normality was evaluated using the Shapiro–Wilk test, and appropriate parametric or non-parametric tests were performed according to the distribution of the data. Statistical comparisons of normally distributed data were performed using unpaired t-tests or ordinary one-way analysis of variance, followed by Sidak's post hoc test, adjusted for multiple comparisons. Non-normally distributed data were statistically compared using the Mann–Whitney test or Kruskal–Wallis test, followed by Dunn's test for adjustment for multiple comparisons. Correlations were represented using Pearson or Spearman correlation coefficients. Inter- and intra-observer variability in CMR analysis was represented using Bland–Altman plots. Survival analysis was performed using the Kaplan–Meier method, and the log-rank test was applied to determine the significance between overall survivals among groups. A P-value < 0.05 was considered statistically significant. All analyses were performed using GraphPad Prism Version 9.3.1 software.

[0205] Results Bioreactor production of PSC-CM To generate the necessary number of cardiomyocytes for the porcine transplantation experiment, the inventors used a stirred tank bioreactor system for PSC expansion and differentiation. Using the H9 human embryonic stem cell line containing the gCaMP6f fluorescent calcium reporter, PSC-CMs were generated according to a three-step process (Figure 1). Each batch of cardiomyocytes was generated from the same working cell bank (WCB) material. This was done to limit batch variability arising from fluctuations in the input population. The WCB was characterized for pluripotency markers, genetic stability, and differentiation potential. After monolayer culture, cells were seeded into a controlled environment stirred tank reactor (30% DO, pH 7.2, 37.2 °C, 0.2–1.5 L volume) for aggregate formation and pluripotent expansion over 3 days, and aggregates with a diameter of 100–200 μm were consistently obtained. The thermoresponsive polymer, pNIPAAM, chemically linked to the recombinant produced extracellular fragment of fibronectin (domains 7–10) was used as previously reported (Chen, X. et al. Tissue Eng Part C Methods 24, 146–157) to assist in aggregate formation. After heat shock and pretreatment of the aggregates with IGF-1 and cyclosporine A, the aggregates were dissociated into single cells and cryopreserved on day 15. Evaluation of cardiac troponin (cTnT) expression demonstrated evidence of >80% cTnT + cells and cytoskeletal formation on average. Additionally, genes for pluripotency, early mesoderm induction, and cardiac specification were evaluated via quantitative polymerase chain reaction (qPCR) over the course of differentiation. These showed expression trends comparable to previously published PSC-CM production methods.

[0206] PSC-CM-related engraftment arrhythmias are essentially focal and automatic After generating sufficient PSC-CMs, the inventors conducted a three-stage transplantation experiment in 23 Landrace pigs. Of these, 2 subjects died after induction of myocardial infarction, and 1 died due to complications following thoracotomy.

[0207] The first stage of the large animal experiment was designed to gain insights into the electrophysiological properties of PSC-CM-related engraftment arrhythmia (EA) and to evaluate the therapeutic efficacy of clinically available antiarrhythmic agents (AA). Transplantation tests were performed in 15 subjects two weeks after percutaneous-induced ischemia-reperfusion myocardial infarction (Figure 3a). The animals were randomized into one of four treatment groups: PSC-CM (n = 4), PSC-CM + AA (n = 4), vehicle (n = 5), or sham infarction with vehicle injection (n = 2). 750,000,000 PSC-CMs or vehicle were delivered to the infarct and border zones via epicardial injection after left thoracotomy. To enable accurate targeting and annotation of cell injection, the inventors developed a novel technique that combined epicardial voltage mapping with reconstructed cardiac magnetic resonance imaging (CMR) using a dedicated cardiac imaging platform, ADAS 3D (Figure 3b). Spontaneous arrhythmias were not observed in any of the vehicle-treated subjects, but all cell-treated subjects developed EA within one week of PSC-CM transplantation (Figure 3c). In a follow-up electroanatomical mapping study performed four weeks after transplantation, the EA origin was localized to the lesion site of cell injection (Figure 3d). Subsequent histological analysis confirmed that these were the cell engraftment sites.

[0208] In the same end - mapping procedure, the EA mechanism was elucidated by observing electrophysiological characteristics and evaluating the arrhythmic response to pacing maneuvers. All EAs occurred either spontaneously or following administration of the catecholamine - agonist drug isoproterenol. Termination was also generally spontaneous, and EAs did not terminate with rapid ventricular pacing (Figure 3e) or after electrical defibrillation by application of direct - current (DC) current to the external chest wall. Variations in the cycle length of EAs (the duration between consecutive electrocardiogram QRS complexes) were observed without changes in electrocardiogram (ECG) morphology. Collectively, these findings indicate enhanced automaticity as the EA mechanism. This suggests that PSC - CM grafts independently beat more rapidly than the sinoatrial node of the recipient heart. In contrast, only vehicle - treated animals developed arrhythmias that could be induced by rapid ventricular pacing, had a fixed cycle length, and were terminable by rapid pacing or DC electrical defibrillation. This suggests a scar - mediated reentry circuit as the mechanism of arrhythmias induced in vehicle - treated animals, which is a typical cause of post - MI ventricular arrhythmias.

[0209] Effective pharmacological suppression of PSC - CM - related engraftment arrhythmias Considering the automatic mechanism for PSC-CM EA, the inventors hypothesized that suppressing the rate of graft automaticity below the rate of sinus node would reduce the arrhythmia burden. Ivabradine, a selective inhibitor of the pacemaker current involved in myocardial automaticity, and amiodarone, a widely used antiarrhythmic agent that blocks multiple ion channels, were selected as antiarrhythmic agents. The inventors hypothesized that the distinct mechanisms of action for each of these drugs would provide effective rates and rhythm control in the treatment of focal automatic EA. From in vitro experiments, it was confirmed that both of these drugs induced a dose-dependent decrease in the spontaneous beating rate of PSC-CM cultured in monolayers (Figure 3f). Pigs treated with PSC-CM+AA received an intravenous bolus of amiodarone at the time of cell injection and subsequently received daily oral administration of amiodarone and ivabradine from day 1 to day 28 after cell injection. Arrhythmia detection was performed through blinded analysis of continuous ECG data transmitted from an implanted remote measurement unit. All animals developed transient arrhythmias that could be attributed to reperfusion injury immediately after myocardial infarction, but sinus rhythm was maintained for several days until epicardial injection. Amiodarone-ivabradine treatment was extremely effective in suppressing EA and was associated with clinically and statistically significant decreases in all remote measurement parameters observed in drug-treated pigs (Figures 3g - 3k): total time of arrhythmia (166.4 ± 66.4 vs. 28.6 ± 8.6; p < 0.05), days with arrhythmia (23.3 ± 0.8 vs. 12.0 ± 2.2; p < 0.005), and peak arrhythmia heart rate (228.0 ± 23.9 beats per minute (b.p.m) vs. 146.5 ± 6.4 b.p.m; p < 0.05). To further investigate this finding, the beating rates of sinus rhythm and EA were analyzed for each animal. EA was found to be slower in drug-treated subjects (143.4 ± 2.9 b.p.m vs. 103.7 ± 2.4; p < 0.0001), and the difference between the EA beating rate and the SR beating rate was significantly decreased in PSC-CM+AA subjects (39.2 ± 2.1 b.p.m vs. 19.7 ± 2.3 b.p.m; p < 0.0001). In summary, these findings suggest that amiodarone-ivabradine treatment can successfully reduce PSC-CM graft automaticity and result in a decrease in EA burden at a slower maximum rate.

[0210] In PSC-CM using antiarrhythmic pharmacological treatment, left ventricular function after myocardial infarction is improved. The primary aim of this study was not to demonstrate a definite and beneficial effect of PSC-CM therapy. Nevertheless, the inventors evaluated the cardiac structure and function using serial CMR in recipients treated with cells and vehicle. All subjects underwent CMR 2 days before and 4 weeks after epicardial PSC-CM injection (Figure 4a). Image analysis was performed with excellent inter- and intra-observer variability according to standard reporting guidelines by two blinded observers. Three animals were excluded from the functional analysis due to failed infarction formation, and all scar sizes were less than 1.5% of the left ventricular volume. Left ventricular ejection fraction (LVEF) was maintained in sham subjects (58 ± 1%) and decreased similarly in all infarcted animals before epicardial injection (vehicle: 43 ± 2%, PSC-CM: 38 ± 3%, PSC-CM+AA: 38 ± 6%. p = 0.44, ns). At 4-week follow-up, no significant change in scar size expressed as a percentage of total left ventricular volume was shown between groups (change in scar size - vehicle: 2 ± 1%, PSC-CM: -0.6 ± 2%, PSC-CM+AA: 0.8 ± 2%. p < 0.05; p = 0.99) (Figure 4b). Nevertheless, a statistically significant improvement in LVEF was observed (change in LVEF - vehicle: 0.3 ± 0.3%, PSC-CM: 4 ± 2%, PSC-CM+AA: 8 ± 2%, p < 0.05), and post hoc analysis showed that this was brought about by the PSC-CM+AA group (Figures 4c - 4d). To further investigate this finding, the effect of the intervention on left ventricular volume was evaluated. Although stroke volume (LVSV) improved in PSC-CM+AA subjects (change in LVSV - vehicle: 10 ± 2 mL, PSC-CM: 13 ± 1 mL, PSC-CM+AA: 24 ± 4 mL, p < 0.05), the change in end-diastolic left ventricular volume (LVEDV) was similar between groups and was rather larger in PSC-CM+AA animals (change in LVEDV - vehicle: 21 ± 4 mL, PSC-CM: 21 ± 3 mL, PSC-CM+AA: 35 ± 13 mL, p = 0.38, ns) (Figures 4e - 4f).In summary, these data suggest that the improvement in function observed in PSC-CM recipients may be due to greater left ventricular contractility rather than detrimental remodeling and decreased left ventricular dilation after MI, and the greatest benefit is evident in animals with arrhythmias suppressed by drug therapy.

[0211] The therapeutic efficacy on right ventricular function was also evaluated. Despite not targeting right ventricular infarction, a slight decline in right ventricular ejection fraction was observed in all infarcted animals (Sham: 57±1%, Vehicle: 53±5%, PSC-CM: 54±3%, PSC-CM+AA: 53±3%; p = 0.96, ns). A trend towards improvement was evident in all cell recipients, and this difference approached but did not reach statistical significance (change in RVEF - Vehicle: -2±4%, PSC-CM: 10±3%, PSC-CM±AA: 5±3%; p = 0.08, ns).

[0212] The PSC-CM cell dose is heterogeneous in the arrhythmogenic subpopulation Next, the inventors sought to gain phenotypic insights into the input PSC-CM compositions to identify arrhythmogenic cell properties that could be strategically targeted. Representative samples from the cell doses of each PSC-CM recipient were retained prior to transplantation for use in single-cell RNA sequencing (scRNA-seq) and high-dimensional flow cytometry experiments. Uniform manifold approximation and projection (UMAP) plots of the clustered scRNA-seq data identified 10 distinct cell subpopulations, the identities of which were inferred based on differential gene expression (Figures 5a-c). Most cells expressed markers of the involved cardiac lineage such as NKX2-5, SIRPA, and cTnT corresponding to clusters 0-4. Further heterogeneity was observed within these cardiomyocyte populations. Compact ventricular markers such as MYL2, IRX4, MYH7, and HEY2 were most abundantly expressed in cluster 0, while atrial and pacemaker markers including SHOX2, VSNL1, NPPA, and NR2F1 were closely localized to cluster 1. Markers of trabecular myocardium such as KCNJ3, SEMA3A, IRX3, and SCN5A were mainly expressed in cluster 2, and proliferation markers such as CDK1 and MKI67 identified cluster 3. Cluster 4 showed strong expression of the glycolytic marker HK2, distinguishing an early-stage glycolytic cardiomyocyte population. Non-cardiomyocyte populations were also present, together with clusters 5-9 containing fibroblasts, epithelial cells, endodermal cells, epicardial cells, and endothelial cells. Collectively, this data confirms the dynamic transcriptional heterogeneity of the profiled cells used in the described animal studies.

[0213] Cell heterogeneity was also confirmed by cell dose characterization by high-parameter flow cytometry (Figures 5d-5e). The inventors designed an antibody panel containing 12 surface markers to examine the cell composition of the delivered PSC-CM (Table 2). t-distributed stochastic neighbor embedding (tSNE) plots obtained as a result overlaid with 25 FlowSOM metaclusters were annotated where possible based on previously reported surface marker signatures. Notably, cardiomyocytes were CD172a + / CD90- classified as and CD172a + / CD90 - / CD77 + cells were considered to be involved ventricular cardiomyocytes. A non - myocyte subpopulation also existed, and fibroblasts were defined as CD172a - / CD90 + and endothelial cells were defined as CD34 + / CD31 + A small subpopulation of non - myocytes expressing CD13, which may represent mesodermal progenitor cells, was identified. Due to limitations in the surface marker panel when determining cell fate, some subpopulations could not be definitively labeled and were annotated as lineage - unspecified cells. The results of the single - cell RNA sequencing above (Figure 5a) indicate that these populations are likely to represent cells derived from epithelial and endodermal lineages. + To compare the relative abundance of specific subpopulations within and between cell doses, data from all doses were concatenated and analyzed. This enabled quantification of the subpopulations, which was then correlated with the total arrhythmia burden of each cell recipient. PSC - CM + AA subjects were excluded from this analysis due to confounding effects on arrhythmia suppression. Interestingly, the strongest correlation between subpopulation quantification and arrhythmia burden occurred in a previously undefined CD172a

[0214] / CD90 + / CD90 - / CD200 + population (r = 0.80). Conversely, CD172a + / CD90 - / CD200 - cells maintained a negative association with arrhythmia burden (r = - 0.77). To further define this surface marker signature, scRNA - seq data were examined. CD172a + / CD90 - / CD200 + cells were isolated into cluster 1 (atrial and pacemaker cardiomyocytes), and CD172a + / CD90 - / CD200 -Isolated in all remaining cardiomyocyte clusters (Figure 5f). In summary, from these data, cell heterogeneity of transplanted PSC-CMs was confirmed, and a possible causal relationship between atrial and pacemaker-like cardiomyocytes and arrhythmogenicity in PSC-CM-treated subjects was identified.

[0215] Early activation of the retinoic acid signaling pathway during PSC-CM differentiation concentrates atrial and pacemaker-like subpopulations To further investigate the arrhythmogenic potential of atrial and pacemaker-like cardiomyocytes, we developed a novel bioreactor differentiation protocol to enrich these subpopulations. By modifying the standard protocol to activate the retinoic acid (RA) signaling pathway from day 2 to day 6 (Figure 1), cardiac progenitor cells were directed towards atrial and pacemaker-like fates (RA-PSC-CM). During CM production, there was a decrease in cTnT expression when the RA treatment condition was compared to control differentiation performed in parallel (62.4 ± 33.1% vs. 83.6 ± 7.0%, n = 12). Importantly, the expression of atrial and nodal genes such as NPPA, MYL2a, SHOX2, and HCN4 was upregulated at the early time point of day 8 in RA-treated cultures and significantly upregulated by day 15, accompanied by inverse suppression of ventricular markers such as IRX4 and MLC2v. This transcriptional pattern was confirmed by scRNA-seq analysis, and RA-PSC-CM showed a marked increase in atrial and pacemaker-like cardiomyocytes and a decrease in ventricular cardiomyocytes (Figures 6a-6b). Using the GCAMPf6$ reporter integrated into PSCs, the beating frequency was evaluated on day 15 before aggregate dissociation. Retinoic acid-treated aggregates showed a significantly increased spontaneous contraction rate compared to controls (14.2 ± 5.5 b.p.m. vs. 50.7 ± 16.8 b.p.m., n = 6 biological replicates, with more than 20 aggregates counted per replicate). Electrophysiological analysis confirmed phenotypic differences between standard cell preparations and RA-PSC-CM cell preparations, and RA-PSC-CM showed a faster spontaneous firing frequency, decreased action potential duration, and lower sodium current density (Figures 6c-6d), all characteristics consistent with atrial and pacemaker cardiomyocytes rather than ventricular cardiomyocytes.

[0216] RA-PSC-CM is highly arrhythmogenic after transplantation into infarcted myocardium To determine whether RA-PSC-CM transplantation increases the in vivo arrhythmia burden, transplantation studies were performed in three additional infarcted pigs in the second stage of the large animal experiment. All of these additional animals received an equivalent dose of 750,000,000 RA-PSC-CM two weeks after myocardial infarction. A marked increase in arrhythmia burden and rate was observed in all three animals that developed rapid and almost continuous EA by day 8 after cell delivery (Figs. 6e - 6f). Higher percentages of the putative arrhythmogenic CD172a+ / CD90- / CD200+ subpopulation and a decrease in the non-arrhythmogenic CD172a+ / CD90- / CD200- subpopulation were shown from high-parameter flow cytometry analysis of each cell dose (Figs. 6g - 6h). Importantly, these additional animals significantly strengthened the positive (r = 0.92, p < 0.005) and negative (r = -0.90, p < 0.05) arrhythmia correlations of these surface marker signatures (Figs. 6i - 6j), confirming the potential utility of these signatures in arrhythmia prediction. The elevated arrhythmia rate and burden were less favorably tolerated by animals that showed a significantly reduced activity level as quantified by accelerometer data (Fig. 6k). Unfortunately, two of the three RA-PSC-CM recipients died either from heart failure-related death or arrhythmia-related death before completing their experimental time course (Fig. 6l), and the third survived only by timely intervention with catheter ablation as further described below.

[0217] RA-PSC-CM grafts contain a rich arrhythmogenic subpopulation and have reduced sarcomeric proteins and inserted intercalated disc tissue compared to PSC-CM grafts The fate of the transplanted cells was also examined in histological and spatial transcriptome experiments. Human grafts were identified by staining with an antibody against the human nuclear antigen Ku80 in the cell line's GCAMP indicator or with green fluorescent protein (GFP). For the detection of ventricles, atria, and pacemaker-like CMs within the grafts, we stained sections with antibodies targeting cardiac troponin T, MLC2v, and MLC2a. Interestingly, the RA-PSC-CM grafts contained significantly more MLC2a + cardiomyocytes, suggesting greater atrial cardiomyocyte engraftment (Figure 7a). Furthermore, these grafts also had a greater proportion of troponin + / MLC2v - cardiomyocytes compared to standard PSC-CM grafts, which was a signature suggesting larger graft pacemaker cardiomyocytes. We also examined the expression of CD172a + / CD200 + cardiomyocytes in standard versus RA-PSC-CM grafts (Figure 7b). Again, a significant difference was observed between the two groups with RA-PSC-CM grafts having more arrhythmogenic CD172a + / CD200 + cardiomyocytes (Figure 7b).

[0218] To visualize sarcomere organization and gap junction formation between cardiomyocytes, we stained the grafts with antibodies against cardiac troponin T (cTnT) and connexin 43 (Cx43) (Figure 7c). High-magnification confocal images showed organized sarcomeres in standard PSC-CM grafts, and Cx43 was appropriately localized to the inserted intercalated discs (Figure 7d). In stark contrast, RA-PSC-CM grafts had a disorganized cTnT expression with sporadic Cx43 and a lateral expression pattern compared to standard PSC-CM grafts. We also stained the tissues with antibodies against CD31 and α-smooth muscle actin to identify new blood vessel growth within the grafts (Figure 7e). Both standard PSC-CM and RA-PSC-CM grafts contained abundant recipient-derived microvessels (capillaries and arterioles) to support long-term graft survival.

[0219] Using a non-targeted spatial transcriptomics protocol, we captured polyA-tailed RNA from the transplanted myocardium of one PSC-CM-treated pig and one RA-PSC-CM-treated pig. By capturing both human RNA and pig RNA, we detected 1,107 and 1,229 human genes and 5,501 and 9,105 pig genes from 567 and 808 spatial spots in PSC-CM tissue and RA-PSC-CM tissue, respectively. The spatial spots were uniquely barcoded hexagonal regions 55 μm in diameter containing an average of 1 - 9 cells. Based on the expression values of these genes, we defined regions containing only pig cells (red dots) and spots containing only human cells (cyan dots) (Figure 7f). The data-driven approach could automatically label human spots, which was independently confirmed by matching with tissue regions containing the human nuclear antigen Ku80 (Figure 7f). This is understood to be the first successful gene mapping of human- or pig-derived cells within the transplanted region. This mapping formed the basis for our comparative analysis of the transcriptional profiles between PSC-CM and RA-PSC-CM grafts.

[0220] Differential expression analysis of human spots identified three upregulated genes in RA-PSC-CM grafts compared to PSC-CM. Two of these genes, NPPA (atrial natriuretic peptide) and MYH6 (myosin heavy chain, α isoform), are markers of atrial-like cardiomyocytes (Figure 7f) and were also highly expressed in the atrial and pacemaker PSC-CM clusters identified using scRNAseq (Figure 5a - c). The third upregulated gene, ELN (elastin), is an important component of the extracellular matrix. Genes for ventricular myosin isoforms (MYL2 and MYH7) and desmosome components (DES, PKP2) were also found to be significantly downregulated in RA-PSC-CM grafts.

[0221] In summary, these data confirm that RA-PSC-CM grafts have a high arrhythmogenic potential in the atria and pacemaker-like cardiomyocytes, and also identify CD172a+ / CD90- / CD200+ and CD172a+ / CD90- / CD200- as novel surface marker signatures for selecting arrhythmogenic or non-arrhythmogenic cell preparations, respectively.

[0222] Catheter ablation is a viable alternative treatment strategy for PSC-CM-related implant arrhythmias. Despite the demonstration of amiodarone-ivabradine in suppressing EA burden, fallback strategies in cases of invasive or refractory EA are essential for safe PSC-CM clinical translation. Therefore, in the third and final stages of the large animal study, the inventors sought to determine the feasibility and efficacy of catheter ablation (CA) as an alternative EA treatment strategy. CA is an advanced treatment option for treating cardiac arrhythmias, where the arrhythmia foci are electroanatomically identified and then typically destroyed using high-frequency energy delivered through specialized catheters. Additionally, two pigs were intended to proceed to CA two weeks after cell injection and received PSC-CM delivery after myocardial infarction. Only one of these subjects showed sufficient EA to facilitate electroanatomical mapping and ablation, while the second had an insufficient burden. ScRNA-seq for the latter showed minimal contribution of atrial and pacemaker-like cardiomyocytes at the input cell dose, and flow cytometry showed a high percentage of non-arrhythmogenic CD172a + / CD90 - / CD200 -Cardiomyocytes were shown. Together, these findings account for the low arrhythmia burden in this subject. In the first subject, the EA burden trended upward until day 13, at which point electroanatomical mapping and CA were performed. This localized EA origin was the inferolateral apex, and this area was the target of a series of ablations to terminate EA and restore sinus rhythm (Figs. 8a–b). EA did not recur during the operation despite the monitoring period and invasive arrhythmia induction strategies (programmed electrical stimulation, isoproterenol injection, burst pacing). Remote measurement analysis from the following 2 weeks showed a significant decrease in the EA burden (Fig. 8c), and only isolated ventricular ectopic beats were observed instead of sustained arrhythmia.

[0223] After this successful proof-of-concept experiment, the inventors sought to evaluate the efficacy of CA in pigs treated with highly arrhythmogenic RA-PSC-CM. Only 1 of the 3 pigs treated with RA-PSC-CM received CA, and the remaining 2 animals died before the planned ablation procedure could be performed. In the treated animal, CA was a life-saving intervention that facilitated survival until the final time point. However, compared to standard PSC-CM pigs, two procedures were required with a greater number of ablation lesions and higher radiofrequency doses required to terminate the arrhythmia. Furthermore, EA recurred within 12 hours after each procedure (Figure 8c), despite the apparent initial success of the procedure. Interestingly, the recurrent EA always originated from new sites and was found to correlate with separate cell injection locations. In total, 4 different origins of EA were identified, and 3 of them (EA1-3) were successfully excised. The final arrhythmia (EA4) was mapped during the final procedure but was not ablated due to reaching the predetermined endpoint, and euthanasia following the mapping procedure was performed as planned. Examination of the heart after tissue harvest demonstrated excellent anatomical correlation between the prior endocardial activation map with visible transmural ablation lesions identifying EA1-3 and islands of viable cell grafts identifying EA4 (Figure 8d). Interestingly, despite the recurrence after each ablation, the new arrhythmias were of a lower heart rate (Figures 8e-8f) and were better tolerated by the animals in clinical evaluation. Histological analysis confirmed the success of the destruction of the engraftment area by each ablation, and intact viable grafts were the cause of the residual arrhythmia (Figure 8g).

[0224] In summary, these results indicate that CA is a viable treatment strategy for EA, but PSC-CM grafts can exhibit hierarchical pacemaker-like properties where ectopic EA activity returns to alternative graft sites when the dominant graft is excised. In particular, the success of treatment with highly arrhythmogenic cell doses may require complete excision of all transplanted areas if these cell populations are not removed prior to transplantation.

[0225] Discussion The inventors have shown that PSC-CM-related EA can be suppressed and potentially eliminated using clinically available pharmacological and interventional therapies. The inventors have further identified the cellular characteristics of arrhythmogenic PSC-CM through definitive phenotyping of the infused and transplanted cells. These data can inform cell production strategies to provide safe and effective PSC-CM for future clinical trials.

[0226] The inventors have shown that the combination of ivabradine and amiodarone is highly effective in suppressing graft automaticity and reducing the EA rate and burden. Ivabradine was selected considering its specific inhibition of the I f current that causes pacemaker automaticity, and the characteristics hypothesized by the inventors may be effective in suppressing automatic EA. Conversely, amiodarone was selected considering that it is an established antiarrhythmic agent that can be empirically administered in future PSC-CM clinical trials. This is related to K + , Na + and Ca 2+They have a wide range of mechanisms of action that antagonize the channel. Combinations of these drugs effectively suppressed EA but did not eradicate it, and in particular, in the case of highly arrhythmogenic cell doses, alternative treatment strategies needed to be evaluated. Therefore, the inventors have subsequently shown that catheter ablation (CA) using high-frequency energy application to arrhythmogenic foci identified by electroanatomical mapping is a viable strategy for treating and eliminating EA. The results of this proof-of-concept experiment have important practical implications since the first patient is being treated with PSC-CM. Interestingly, the ablation experiments also provided important mechanistic insights into EA, demonstrating for the first time that the pacemaker activity can regress to a lower intrinsic pacemaker rate graft when the dominant graft is excised. A caveat for CA therapy for EA is that excising multiple graft regions risks loss of the contractile benefit exerted by PSC-CM transplantation. Therefore, the inventors propose that CA should only be pursued in cases of invasive and pharmacologically refractory EA, and more carefully, the generation of less arrhythmogenic PSC-CM cell products should be investigated. For this purpose, the inventors also sought to gain mechanistic insights into the cell compositions that may contribute to EA.

[0227] Substantial variability in EA load has been reported within and among PSC-CM large animal studies. This may reflect several factors such as differences in animal species or cell delivery techniques, but is likely primarily determined by the molecular composition of the cell product. A decrease in arrhythmia load has been observed after the period of in vivo graft maturation, suggesting that PSC-CM immaturity may be an important determinant of arrhythmia occurrence. However, it is suggested that not all PSC-CM recipients achieve "electrical maturation" and that immaturity may not be the only important cell property. Current PSC-CM differentiation protocols are known to generate heterogeneous cell populations containing mixtures of ventricular, atrial, and pacemaker-like cardiomyocytes. The inventors herein for the first time outline the importance of this heterogeneity in arrhythmia occurrence, identify atrial cells and pacemaker-like cells as causative subpopulations, and describe a unique surface marker signature predictive of arrhythmogenicity. Cells enriched for atrial and pacemaker-like cardiomyocytes were generated via addition of retinoic acid to the bioreactor differentiation protocol and shown to be highly arrhythmogenic, supporting a causal relationship between these subpopulations and EA. The inventors show that the enhanced automaticity of these subpopulations in vitro is directly converted in vivo to a more abundant and rapid EA. Furthermore, by definitively phenotyping the input cell dose and correlating these to the resulting arrhythmia load, the inventors identify CD172a + / CD90 + / CD200 + and CD172a + / CD90 - / CD200 - cells as arrhythmogenic and non-arrhythmogenic cardiomyocytes, respectively. By doing so, the inventors provide not only a simple quality control tool for assessing the arrhythmogenic potential of the cell dose, but also a means for removing arrhythmogenic cells by cell sorting prior to transplantation. Intriguingly, scRNA-seq data show that these arrhythmogenic CD172a + / CD90 + / CD200 +It is shown that cardiomyocytes have a transcriptome signature that coincides with atrial and pacemaker subpopulations, further supporting the recognition that these cell types are important in arrhythmogenesis. Currently, there is great interest in generating chamber-specific cardiomyocytes for various therapeutic or drug discovery applications. This data further endorses the pursuit of transplanting purified ventricular cardiomyocytes that lack atrial and pacemaker-like cells for the purpose of therapeutic cardiac remuscularization.

[0228] Finally, although not a primary intention and despite the small sample size, the inventors were able to demonstrate an improvement in left ventricular function in all cell recipients, except those in whom EA was improved with drug therapy. The PSC-CM grafts induced direct contractility, and the data suggest that this beneficial effect may be further enhanced by addressing arrhythmogenicity. Interestingly, a tendency for improvement was also observed in the non-transplanted right ventricle, suggesting that the influence of immunomodulatory paracrines may also be a factor.

[0229] In conclusion, the repopulation of infarcted myocardium with functional, force-generating cardiomyocytes is an exciting therapeutic prospect that positions PSC-CMs as a major candidate for cardiac regeneration. Although associated with transient arrhythmia occurrence, here the inventors state that a mechanistic understanding of this predictable complication is likely to be addressable via modifications to the cardiomyocyte production protocol. Furthermore, the inventors show that PSC-CM engraftment arrhythmias can be suppressed with clinically available pharmacological and procedural anti-arrhythmia strategies, which are important safety considerations given several impending clinical trials.

[0230] DEAB PSC-CM Culture Methods Culture of hPSC SCVI8 hPSC SCVI8 was cultured at 37 °C and 5% CO2 in a Petri dish coated with Matrigel® (Corning, catalog number 354277) in complete mTeSR® Plus basal medium (StemCell Technologies, catalog number 100-0276) supplemented with 20% (v / v) mTeSR® Plus 5X supplement (StemCell Technologies) and 0.5% (v / v) streptomycin and penicillin (ThermoFisher, catalog number 15140163). The medium was changed daily, and the cells were passaged when the confluence reached approximately 80%.

[0231] Differentiation of cardiomyocytes Differentiation of cardiomyocytes was performed using small molecule modulators of the classical Wnt signaling as described by Lian et al. (Lian et al. Nature. 20138(1):162-175), except that 1 nM, 1 μM, or 100 μM of 4-diethylaminobenzaldehyde (DEAB, Sigma Merck, Cat. D86256) was added (Figure 9a). Briefly, cells were added on either day 3 or day 5 of hPSC SCVI differentiation from confluent dishes, washed once with calcium- and magnesium-free Dulbecco's phosphate-buffered saline (DPBS) (Lonza, Cat. No. 17-512F), harvested with TrypLE® Express Enzyme (ThermoFisher, Cat. No. 12604021), and seeded at 3.75×10 in a Matrigel®-coated 12-well plate with 2 mL volume of complete mTeSR Plus basal medium and 10 μM of Y-27632 (RHO / ROCK) inhibitor (StemCell Technologies, catalog number 072304). 5Seeded at a concentration of cells / mL. On day 0 of differentiation, the cells were washed once with DPBS and transferred to a mesoderm induction medium consisting of 6 μM CHIR99021 (GSK3 inhibitor, Tocris, catalog number 4423) in Roswell Park Memorial Institute (RPMI) 1640 complete medium (without glutamine, ThermoFisher, catalog number 21870092), supplemented with 2% (v / v) B-27 supplement, insulin-free (ThermoFisher, catalog number A1895601), 0.5% (v / v) streptomycin and penicillin, and 1% (v / v) GlutaMAX™ supplement (ThermoFisher, catalog number 35050061). On day 1, CHIR99021 was removed and the cells were maintained in complete RPMI without insulin. On day 3, the cells were transferred to a heart induction medium consisting of 5 μM IWP-2 (Tocris, catalog number 3533) in complete RPMI without insulin and removed on day 5. On day 7 of differentiation, the cells were maintained in complete RPMI supplemented with 2% (v / v) B-27™ Supplement (50X) (ThermoFisher, catalog number 17504-001), 0.5% (v / v) streptomycin and penicillin, and 1% (v / v) GlutaMAX™ supplement. The medium was changed every 2 - 3 days. The cells were harvested on day 14 for quantitative real-time polymerase chain reaction (RT-PCR).

[0232] Quantitative reverse transcription polymerase chain reaction (RT-PCR) Total cellular RNA was extracted using the Isolate II RNA Mini Kit (Bioline, catalog number BIO-52073) according to the manufacturer's instructions. Reverse transcription was performed using M-MLV reverse transcriptase (Promega, catalog number M1701) according to the manufacturer's protocol. mRNA gene expression was evaluated by qPCR using the SensiFAST™ SYBR® No-ROX kit (Meridian Bioscience, catalog number BIO-98050) on a Bio-Rad CFX384 real-time PCR detection system. The following PCR conditions were 40 cycles of 2 min at 95°C, 5 s at 95°C, 10 s at 60°C, and 15 s at 72°C, followed by analysis of the melting curve. An overview of all primers used for the detection of mRNA expression is shown in Table 3. All data were normalized to the reference gene 18S and also to control cells (untreated PSC-CM).

[0233]

Table 3

[0234] Statistical analysis All statistical tests were performed using Prism 9.00 software (GraphPad Software Inc). Details of the tests performed are shown in the figure legends.

[0235] Results Differentiating hPSCs were treated with DEAB at concentrations ranging from 1 nM to 100 μM on days 3 to 7 or days 5 to 7 of differentiation (Figure 9a). RT-PCR analysis of PSC-CM on day 14 demonstrated no significant differences in the expression levels of the cardiac-specific gene cTnT (Figure 9b) and the ventricular markers MYH7, MYL2v, and IRX4 (Figures 9c - 9e) across different treatment groups. However, in PSC-CM cells treated with DEAB, the expression level of SHOX2, a gene related to the SAN pacemaker, was significantly lower (Figure 9f), and the expression level of KCNJ3, a pacemaker ion channel gene, was also significantly lower (Figure 9g).

[0236] The non-significant trends in cTnT, MYH7, and MLC2v expression indicate that ventricular cardiomyocytes are enriched while atrial / pacemaker cells are depleted (see KCNJ3 and SHOX2 data). From Figure 5, it can be seen that the CD172a+ / CD90- / CD200- subpopulation does not express SHOX2 and KCNJ3. Therefore, PSC-CMs treated with DEAB during differentiation are expected to be enriched in the CD172a+ / CD90- / CD200- subpopulation. Also, from Figure 5, it can be seen that the CD172a+ / CD90- / CD200+ subpopulation has increased expression of SHOX2, and thus it can be understood that PSC-CMs treated with DEAB during differentiation are expected to be depleted in the CD172a+ / CD90- / CD200+ subpopulation.

[0237] PSC-CM cultures with vitamin A removed PSC-CM cultures are prepared in culture media with low vitamin A and culture media without vitamin A. Removal of vitamin A may be important 2 days after Wnt inhibition.

[0238] An exemplary protocol is as follows. Chemically defined medium (CDM) is RPMI 1640 basal medium, L-ascorbic acid 2-phosphate, and bovine serum albumin (BSA). CDM is supplemented with B27, which is available with both the presence and absence of vitamin A. Day 0 = CHIR99021 addition (CDM B27, insulin-free, with vitamin A). Day 1 = Medium change (CDM B27, insulin-free, with vitamin A). Day 3 = IWP-2 addition (CDM B27, insulin-free, with vitamin A). Day 5 = Medium change (CDM B27 + insulin, without vitamin A). Day 7 = Medium change (CDM B27 + insulin, without vitamin A).

[0239] An alternative exemplary protocol is as follows. Day 0 = Addition of CHIR99021 (RPMI B27, without insulin, containing vitamin A). Day 3 = Addition of IWP-2 (, RPMI B27, without insulin, containing vitamin A, retinoic acid inhibitor). Day 5 = Medium change (CDM B27 + insulin, without vitamin A).

[0240] An alternative exemplary protocol is as follows. Day 0 = Addition of CHIR99021 (CDM B27, without insulin, containing vitamin A). Day 3 = Addition of IWP-2 (CDM B27, without insulin, containing vitamin A). Day 5 = Medium change (CDM B27 + insulin, without vitamin A, + BMS inhibitor). Day 7 = Medium change (CDM B27 + insulin, without vitamin A, + BMS inhibitor).

[0241] The present invention is further illustrated in the following numbered paragraphs: 1. A method for identifying cardiomyocytes derived from arrhythmogenic pluripotent stem cells (PSC-CM), comprising: (i) determining whether CD200 is expressed on the surface of PSC-CM, wherein when CD200 is expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM. 2. Further, (ii) determining whether signal regulatory protein α (CD172a) is expressed on the surface of PSC-CM, and / or (iii) determining whether CD90 is expressed on the surface of PSC-CM, wherein the method according to paragraph 1, wherein when CD200 is expressed on the surface of PSC-CM, CD172a is expressed on the surface of PSC-CM, and / or CD90 is not expressed on the surface of PSC-CM, the PSC-CM is arrhythmogenic PSC-CM. 3. Further, (ii) determining whether CD172a is expressed on the surface of PSC-CM, and (iii) determining whether CD90 is expressed on the surface of the PSC-CM, wherein CD200 and CD172a are expressed on the surface of the PSC-CM, and when CD90 is not expressed on the surface of the PSC-CM, the PSC-CM is arrhythmogenic PSC-CM, the method according to paragraph 1. 4. The method according to any one of paragraphs 1 to 3, wherein when CD200 is not expressed on the surface of the PSC-CM, the PSC-CM is non-arrhythmogenic PSC-CM. 5. Determining whether CD200, CD172a and / or CD90 is expressed on the surface of the PSC-CM includes exposing the PSC-CM to an agent comprising a detectable label to provide a labeled PSC-CM, and detecting the detectable label, wherein the agent selectively binds to CD200, CD172a or CD90, the method according to any one of paragraphs 1 to 4. 6. Determining whether CD200, CD172a and CD90 are expressed on the surface of the PSC-CM includes exposing the PSC-CM to a first agent comprising a first detectable label that selectively binds to CD200, a second agent comprising a second detectable label that selectively binds to CD172a, and a third agent comprising a third detectable label that selectively binds to CD90 to provide a labeled PSC-CM, and detecting the detectable label, the method according to paragraph 5. 7. The method according to paragraph 5 or paragraph 6, wherein the agent comprising a detectable label is an antibody and the detectable label is a fluorophore. 8. The method according to any one of paragraphs 5 to 7, wherein detecting the detectable label includes subjecting the labeled hPSC-CM to flow cytometry. 9. The method according to any one of paragraphs 1 to 8, wherein the PSC-CM is a human pluripotent stem cell-derived cardiomyocyte (hPSC-CM). 10. A method for determining whether a dose containing a plurality of PSC-CM is likely to cause arrhythmia upon transplantation in a subject, (i) determining whether the dose contains arrhythmogenic PSC-CM, wherein whether the PSC-CM is arrhythmogenic PSC-CM is determined according to the method described in any one of paragraphs 1 to 9, including the determination, When the dose contains arrhythmogenic PSC-CM, the method wherein the dose is likely to cause arrhythmia upon transplantation in a subject. 11. Further, (ii) determining the proportion of arrhythmogenic PSC-CM in the dose, including the determination, When the proportion of PSC-CM in the dose expressing CD200 on the surface exceeds 0.001%, the method described in paragraph 10, wherein the dose is likely to cause arrhythmia upon transplantation in a subject. 12. A method for eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CM in a subject, before transplantation of the dose (i) identifying arrhythmogenic PSC-CM contained in the crude dose according to the method described in any one of paragraphs 1 to 9, and (ii) removing arrhythmogenic PSC-CM from the crude dose to obtain a purified dose, including the method. 13. Further, (iii) transplanting the purified dose into the subject, including the method described in paragraph 12. 14. The method according to paragraph 12 or paragraph 13, wherein the crude dose contains pluripotent stem cells (PSC) that have been differentiated to form PSC-CM and have not been subjected to any additional treatment to enrich or deplete cell subpopulations. 15. The method according to any one of paragraphs 12 to 14, wherein the arrhythmia after transplantation of the purified dose is reduced compared to the arrhythmia after transplantation of the crude dose. 16. The method according to paragraph 15, wherein the arrhythmia is reduced when defined by the cumulative time per day spent on arrhythmia over 25 days after transplantation. 17. The method according to paragraph 16, wherein the arrhythmia is reduced by at least about 50%, or at least about 60, 70, 80, 90, 95, or 100%. 18. The method according to any one of paragraphs 12-17, wherein step (ii) comprises removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. 19. The method according to any one of paragraphs 12-18, wherein step (ii) comprises removing at least about 60%, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose. 20. The method according to any one of paragraphs 12-19, wherein step (ii) is performed by fluorescence-activated cell sorting or magnetic-activated cell sorting. 21. A method of providing a dose containing a plurality of PSC-CMs substantially free of arrhythmogenic PSC-CMs, comprising: (i) identifying the arrhythmogenic PSC-CMs contained in the crude dose according to the method according to any one of paragraphs 1-9; and (ii) removing the arrhythmogenic PSC-CMs from the crude dose to obtain a dose of PSC-CMs substantially free of arrhythmogenic PSC-CMs. 22. The method according to paragraph 21, wherein the crude dose comprises human pluripotent stem cells (PSCs) that have been differentiated to form PSC-CMs and have not been subjected to any additional treatment to enrich or deplete cell subpopulations. 23. The method according to paragraph 21 or paragraph 22, wherein step (ii) comprises removing at least about 50% of the arrhythmogenic PSC-CMs from the crude dose. 24. The method according to any one of paragraphs 21-23, wherein step (ii) comprises removing at least about 60%, or at least about 70, 80, 90, 95, or 100% of the arrhythmogenic PSC-CMs from the crude dose. 25. The method according to any one of paragraphs 21-24, wherein the dose of PSC-CMs substantially free of arrhythmogenic PSC-CMs contains less than 0.001% arrhythmogenic PSC-CMs relative to the total number of PSC-CMs in the dose. 26. The method according to any one of paragraphs 21-25, wherein step (ii) is performed by fluorescence-activated cell sorting or magnetic-activated cell sorting. 27. A method for identifying pluripotent stem cell-derived cardiac cells (PSC-PM) having pacemaker characteristics, comprising: (i) determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-PM. 28. Further, (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are pacemaker PSC-PM, according to the method of paragraph 27. 29. Further, (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 and CD172a are expressed on the surface of pluripotent stem cell-derived cardiac cells and CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-PM, according to the method of paragraph 27 or paragraph 28. 30. When CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are not PSC-PM, according to the method of any one of paragraphs 27 to 29. 31. Determining whether CD200, CD172a, and / or CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, comprising exposing the pluripotent stem cell-derived cardiac cells to an agent comprising a detectable label to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, wherein the agent selectively binds to CD200, CD172a, or CD90, the method according to any one of paragraphs 27-30. 32. Determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, comprising exposing the pluripotent stem cell-derived cardiac cells to a first agent comprising a first detectable label that selectively binds to CD200, a second agent comprising a second detectable label that selectively binds to CD172a, and a third agent comprising a third detectable label that selectively binds to CD90 to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, the method according to paragraph 31. 33. The method according to paragraph 31 or paragraph 32, wherein the agent comprising a detectable label is an antibody and the detectable label is a fluorophore. 34. The method according to any one of paragraphs 31-33, wherein detecting the detectable label comprises subjecting the labeled pluripotent stem cell-derived cardiac cells to flow cytometry. 35. A method of providing a substantially pure dose of PSC-PM, comprising: (i) identifying PSC-PM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method of any one of paragraphs 27-34; and (ii) isolating PSC-PM from the plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-PM. 36. The method according to paragraph 35, wherein step (ii) is performed by fluorescence-activated cell sorting or magnetic-activated cell sorting. 37. The method according to paragraph 35 or paragraph 36, wherein the substantially pure dose of PSC-PM contains less than 0.001% of cells that are not PSC-PM relative to the total number of cells in the dose. 38. A method for identifying pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VM), comprising: (i) determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-VM. 39. Further (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and / or CD90 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-VM, according to the method described in paragraph 38. 40. Further (ii) determining whether CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, and (iii) determining whether CD90 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 and CD90 are not expressed on the surface of pluripotent stem cell-derived cardiac cells, and CD172a is expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-VM, according to the method described in paragraph 38 or paragraph 39. 41. When CD200 is not expressed on the surface of pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are not PSC-VM, according to the method described in any one of paragraphs 38 to 40. 42. Determining whether CD200, CD172a, and / or CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, comprising exposing the pluripotent stem cell-derived cardiac cells to an agent comprising a detectable label to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, wherein the agent selectively binds to CD200, CD172a, or CD90, the method according to any one of paragraphs 37 to 41. 43. Determining whether CD200, CD172a, and CD90 are expressed on the surface of pluripotent stem cell-derived cardiac cells, comprising exposing the pluripotent stem cell-derived cardiac cells to a first agent comprising a first detectable label that selectively binds to CD200, a second agent comprising a second detectable label that selectively binds to CD172a, and a third agent comprising a third detectable label that selectively binds to CD90 to provide labeled pluripotent stem cell-derived cardiac cells, and detecting the detectable label, the method according to paragraph 42. 44. The method according to paragraph 42 or paragraph 43, wherein the agent comprising a detectable label is an antibody and the detectable label is a fluorophore. 45. The method according to any one of paragraphs 42 to 44, wherein detecting the detectable label comprises subjecting the labeled pluripotent stem cell-derived cardiac cells to flow cytometry. 46. A method of providing a substantially pure dose of PSC-VM, comprising: (i) identifying PSC-VM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method according to any one of paragraphs 37 to 44; and (ii) isolating PSC-VM from the plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-VM. 47. The method according to paragraph 46, wherein step (ii) is performed by fluorescence-activated cell sorting or magnetic-activated cell sorting. 48. The method according to paragraph 46 or paragraph 47, wherein the substantially pure dose of PSC-PM contains less than 0.001% of cells that are not PSC-PM relative to the total number of cells in the dose. 49. A method of eliminating or reducing post-transplant arrhythmias in a subject with a dose containing a plurality of PSC-CMs, comprising culturing a plurality of PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CMs, wherein the arrhythmogenic PSC-CM is a PSC-CM that expresses CD200 on the surface. 50. The method according to paragraph 49, wherein the post-transplant arrhythmia of the dose subjected to the method according to paragraph 49 is reduced as compared to the post-transplant arrhythmia of a dose not subjected to the method according to paragraph 49. 51. The method according to paragraph 50, wherein the arrhythmia is reduced when defined by the cumulative time per day spent on arrhythmia over 25 days after transplantation. 52. The method according to paragraph 51, wherein the arrhythmia is reduced by at least about 50%, or at least about 60, 70, 80, 90, 95, or 100%. 53. A method of providing a dose of PSC-CM substantially free of arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CMs, wherein the arrhythmogenic PSC-CM is a PSC-CM that expresses CD200 on the surface. 54. The method according to paragraph 53, wherein the dose of PSC-CM substantially free of arrhythmogenic PSC-CM contains less than 0.001% arrhythmogenic PSC-CM relative to the total number of PSC-CMs in the dose. 55. The method according to any one of paragraphs 49 to 54, wherein the arrhythmogenic PSC-CM is a PSC-CM that expresses CD200 on the surface, expresses CD172a, and / or does not express CD90. 56. The method according to any one of paragraphs 49 to 55, wherein the arrhythmogenic PSC-CM is a PSC-CM that expresses CD200 and CD172a on the surface and does not express CD90. 57. A method of providing a dose of PSC-CM containing a high proportion of non-arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to increase the generation of non-arrhythmogenic PSC-CMs, wherein the non-arrhythmogenic PSC-CM is a PSC-CM that does not express CD200 on the surface. 58. The method according to paragraph 57, wherein the dose of PSC-CM containing a high proportion of non-arrhythmogenic PSC-CM is at least about 5% more non-arrhythmogenic PSC-CM compared to a control dose obtained by culturing PSCs in a culture medium, relative to the total number of PSC-CMs during the dose and in the absence of conditions effective to increase the generation of non-arrhythmogenic PSC-CM. 59. The method according to paragraph 57 or paragraph 58, wherein the non-arrhythmogenic PSC-CM is a PSC-CM that does not express CD200 on the surface, does not express CD90, and / or expresses CD172a. 60. The method according to any one of paragraphs 57 to 59, wherein the non-arrhythmogenic PSC-CM is a PSC-CM that does not express CD200 and CD90 on the surface and expresses CD172a. 61. The method according to any one of paragraphs 49 to 60, wherein the conditions effective to suppress the generation of arrhythmogenic PSC-CM include the addition of a retinoic acid inhibitor to the culture medium or the removal of vitamin A from the culture medium. 62. The method according to paragraph 61, wherein the retinoic acid inhibitor is added during cardiomyocyte differentiation. 63. The method according to paragraph 62, wherein the retinoic acid inhibitor is added between day 3 and day 7 of cardiomyocyte differentiation. 64. The method according to any one of paragraphs 61 to 63, wherein the retinoic acid inhibitor is selected from the group consisting of 4-diethylaminobenzaldehyde (DEAB), 4-[(1E)-2-[5,6-dihydro-5,5-dimethyl-8-(2-phenylethynyl)-2-naphthalenyl]ethenyl]benzoic acid (BMS-493), (E)-4-[2-(5,6-dihydro-5,5-dimethyl-8-phenyl-2-naphthalenyl)ethenyl]-benzoic acid (BMS-189453), and disulfiram. 65. The method according to paragraph 61, wherein vitamin A is removed from the culture medium during cardiomyocyte differentiation. 66. A purified population of non-arrhythmogenic pluripotent stem cell-derived cardiomyocytes (PSC-CMs), wherein the population of non-arrhythmogenic PSC-CMs does not express CD200 on the cell surface. 67. The purified population according to paragraph 66 that does not express CD172a on the cell surface. 68. The purified population according to paragraph 66 or 67 that expresses CD90 on the cell surface. 69. A purified population of non-arrhythmogenic PSC-CM, (i) providing a population of PSC-CM, (ii) determining whether CD200 is expressed on the cell surface of the PSC-CM in step (i), (iii) optionally, determining whether CD172a and / or CD90 is expressed on the cell surface of the PSC-CM in step (i), (iv) removing the PSC-CM from step (i) that expresses CD200, and optionally removing the PSC-CM that expresses CD172 and / or does not express CD90, thereby preparing a purified population of non-arrhythmogenic PSC-CM, a population prepared by. 70. A method of performing it in a patient in need of a PSC-CM graft, comprising administering to the patient the population of non-arrhythmogenic PSC-CM according to any one of paragraphs 66 to 69, wherein the patient has a lower risk of engraftment arrhythmia (EA) than a patient receiving a mixed population of PSC-CM. 71. A method of improving engraftment arrhythmia (EA) in a patient who has received a PSC-CM transplant, comprising treating the patient with amiodarone and / or ivabradine. 72. A method of improving EA in a patient who has received a PSC-CM transplant, comprising performing at least one catheter ablation on the patient.

Claims

1. A purified population of non-arrhythmogenic pluripotent stem cell-derived cardiomyocytes (PSC-CMs), wherein the population of non-arrhythmogenic PSC-CMs does not express CD200 on the cell surface.

2. A purified population of non-arrhythmogenic PSC-CMs, comprising: (i) providing a population of PSC-CMs; (ii) determining whether CD200 is expressed on the cell surface of the PSC-CMs in step (i); (iii) optionally, determining whether CD172a and / or CD90 is expressed on the cell surface of the PSC-CMs in step (i); (iv) removing the PSC-CMs from step (i) that express CD200, and optionally removing PSC-CMs that express CD172 and / or do not express CD90, thereby preparing a purified population of non-arrhythmogenic PSC-CMs.

3. A method of performing it in a patient in need of a PSC-CM graft, comprising administering to the patient the population of non-arrhythmogenic PSC-CMs according to claim 1 or 2, wherein the patient has a lower risk of engraftment arrhythmia (EA) than a patient receiving a mixed population of PSC-CMs.

4. A method of identifying arrhythmogenic pluripotent stem cell-derived cardiomyocytes (PSC-CMs), comprising: (i) determining whether CD200 is expressed on the surface of the PSC-CMs, wherein if CD200 is expressed on the surface of the PSC-CMs, the PSC-CMs are arrhythmogenic PSC-CMs.

5. A method of determining whether a dose containing a plurality of PSC-CMs is likely to cause arrhythmia upon transplantation in a subject, comprising: (i) determining whether the dose contains arrhythmogenic PSC-CMs, wherein whether the PSC-CMs are arrhythmogenic PSC-CMs is determined according to the method of claim 4. If the dose contains arrhythmogenic PSC-CMs, the dose is likely to cause arrhythmia upon transplantation in the subject.

6. A method of eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CMs in a subject, before transplantation of the dose, (i) identifying arrhythmogenic PSC-CMs contained in the crude dose according to the method of claim 4. A method comprising: (ii) removing the arrhythmogenic PSC-CM from the crude dose to obtain a purified dose. **Claim 7** A method of providing a dose containing a plurality of PSC-CMs substantially free of arrhythmogenic PSC-CM, comprising: (i) identifying arrhythmogenic PSC-CM contained in a crude dose according to the method described in Claim 4; (ii) removing the arrhythmogenic PSC-CM from the crude dose to obtain a dose of PSC-CM substantially free of arrhythmogenic PSC-CM. **Claim 8** A method of identifying pluripotent stem cell-derived cardiac cells (PSC-PM) having pacemaker properties, comprising: (i) determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is expressed on the surface of the pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-PM. **Claim 9** A method of providing a substantially pure dose of PSC-PM, comprising: (i) identifying PSC-PM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method described in Claim 8; (ii) isolating the PSC-PM from the plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-PM. **Claim 10** A method of identifying pluripotent stem cell-derived ventricular cardiomyocytes (PSC-VM), comprising: (i) determining whether CD200 is expressed on the surface of pluripotent stem cell-derived cardiac cells, wherein when CD200 is not expressed on the surface of the pluripotent stem cell-derived cardiac cells, the pluripotent stem cell-derived cardiac cells are PSC-VM. **Claim 11** A method of providing a substantially pure dose of PSC-VM, comprising: (i) identifying PSC-PM contained in a plurality of pluripotent stem cell-derived cardiac cells according to the method described in Claim 10; (ii) isolating the PSC-VM from the plurality of pluripotent stem cell-derived cardiac cells to obtain a substantially pure dose of PSC-VM. **Claim 12** A method of eliminating or reducing arrhythmia after transplantation of a dose containing a plurality of PSC-CM in a subject, comprising culturing a plurality of PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CM, wherein the arrhythmogenic PSC-CM is PSC-CM expressing CD200 on the surface. **Claim 13** A method for providing a dose of PSC-CM substantially free of arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to suppress the generation of arrhythmogenic PSC-CM, wherein the arrhythmogenic PSC-CM is PSC-CM expressing CD200 on the surface.

14. A method for providing a dose of PSC-CM containing a high proportion of non-arrhythmogenic PSC-CM, comprising culturing PSCs in a culture medium under conditions effective to increase the generation of non-arrhythmogenic PSC-CM, wherein the non-arrhythmogenic PSC-CM is PSC-CM not expressing CD200 on the surface.

15. A method for improving engraftment arrhythmia (EA) in a patient who has received a transplant of PSC-CM, comprising treating the patient with amiodarone and / or ivabradine.

16. A method for improving EA in a patient who has received a transplant of PSC-CM, comprising performing at least one catheter ablation on the patient.

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