Method for maturation of cardiomyocytes on amniotic fluid cell-derived ECM, cell constructs and use thereof for cardiotoxicity and arrhythmogenicity screening of drug compounds

Abstract

Disclosed herein is a method for maturing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells in culture using a cell-derived extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid to form mature cardiomyocytes. Also disclosed herein is a cell construct comprising a monolayer of these mature cardiomyocytes on AFC-ECM for use in cardiotoxicity and / or arrhythmogenic screening assays of drug compounds. Also disclosed herein is a method for determining the cardiotoxicity and / or arrhythmogenic effects of drug compounds in vitro using such a cell construct.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 808,690, filed on February 21, 2019, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present disclosure generally relates to the use of cell constructs of cardiomyocytes derived from human stem cells on a cell-derived extracellular matrix, methods of making the constructs, and methods of using the constructs to perform cardiotoxicity and arrhythmogenic screening assays for drug compounds. Background Art

[0004] Cardiotoxicity, or the perceived potential for cardiotoxicity, is the leading cause of toxicity-related drug attrition during the development and selection of new drugs. Cardiac safety testing of new chemical entities that become lead drug candidates is a critical aspect of the drug discovery and development pipeline. A large number of cardiac side effects of both cardiac and non-cardiac drugs result from drug interactions with one or more cardiac ion channels. Cardiac ion channels regulate cellular excitability, contractility, and overall cardiac function, and alterations in cardiac ion channel function may lead to sudden cardiac death. This has led to the International Conference on Harmonization (ICH) to issue guidelines for testing of candidate drugs.

[0005] Current ICH guidelines for testing preclinical drug candidates (ICH S7A and S7B pharmacology studies) rely on genetically modified xenogeneic cells and in vivo animal models. There is growing recognition that these studies, such as hERG assays and QT prolongation studies, do not accurately predict cardiac toxicity and proarrhythmic risk in humans. Since 2005, the cardiac safety of drug compounds has been almost entirely determined by their effects or potential effects on the QT interval or action potential duration (APD) of the electrocardiogram (ECG) and the potential to cause a life-threatening arrhythmia called torsade de pointes (TdP). However, QT prolongation is not an ideal indicator of TdP because drugs that prolong the QT interval do not always cause TdP. It is now recognized that QT prolongation parameters are only surrogate markers of proarrhythmogenicity. Data from preclinical and clinical trials have shown that there is no fixed relationship between the magnitude of QT prolongation and the risk of developing fatal arrhythmias such as TdP.

[0006] As a result, the U.S. Food and Drug Administration (FDA) and other stakeholders in the drug discovery community have called for the development of preclinical cardiotoxicity testing. The new paradigm proposed is called the Comprehensive In Vitro Arrhythmogenic Assay (CiPA). One component of the CiPA initiative (http: / / cipaproject.org / ) includes the inclusion of data collected from human stem cell-derived cardiomyocytes into cardiotoxicity and arrhythmogenic assays. The overall goal of these new proposed guidelines is to provide more accurate and comprehensive mechanism-based assessments of arrhythmogenic potential, thereby more accurately assessing the risk of new drugs. In reviewing the proposed guidelines for the CiPA initiative, the FDA identified two advances that must be made before human cardiomyocytes can be included in the new initiative. First, the growth and maturation state of human stem cell-derived cardiomyocytes needs to be advanced to more closely resemble the structure and function of adult cardiomyocytes. Second, a reliable high-throughput screening platform using these cells must be developed.

[0007] Three types of systems are currently commonly used to evaluate the electrophysiology of cardiomyocytes in vitro: 1) patch clamp systems; 2) microelectrode array (MEA) systems; and 3) voltage sensitive dye (VSD) visualization methods. Manual patch clamp systems were most commonly used to evaluate the electrophysiology of single cells in very early studies. Although these devices provide accurate and sensitive ion current measurements, they cannot be used in in vitro cell systems that more closely mimic cell-cell interactions in cardiac tissue. MEA systems include electrodes incorporated into cell culture wells, allowing the measurement of current passing through the wells. Although allowing high-throughput analysis in in vitro cell systems and enabling impedance measurement, these systems have low spatial resolution, do not provide data on action potential shape, and hinder the direct visualization of cells and the ability to evaluate 3-D culture systems that more closely mimic cardiac tissue structure. VSD systems are gaining interest because they address many of the shortcomings of the current technologies described above, with features including: 1) allowing high-throughput analysis; 2) providing high spatial resolution; 3) allowing visualization of pulse propagation throughout the culture dish; and 4) allowing modification of culture conditions, including the addition of extracellular matrix, to obtain a more natural testing environment.

[0008] To date, there has been limited success in screening for cardiotoxicity and arrhythmogenic assays using cardiomyocytes derived from human stem cells such as induced pluripotent stem cells. Cardiomyocytes derived from induced human pluripotent stem cells (hiPSC-CMs) are commercially available and can be purchased from several companies as vials that can be thawed and seeded in a monolayer and stored at low temperatures. These hiPSC-CMs are large-scale preparations that can be made in vitro. In addition, hiPSC-CMs can be obtained from patient-specific hiPSCs for specific individuals. However, there are still some obstacles to overcome to make hiPSC-CMs a meaningful part of the new CiPA initiative paradigm. 1) The maturation of hiPSC-CM structure and function must be promoted. It is worth noting that Kir2.1 potassium channels are absent in currently available hiPSC-CMs and sodium channel expression is low. Currently available hiPSC-CMs are very immature in both function and structure. The vast majority of currently used hiPSC-CM-based arrhythmogenic screening assays rely on immature fetal-like cells, which are different from the functional structure of adult cardiomyocytes. 2) There is a need to perform electrical pacing of hiPSC-CM monolayers in high-throughput electrophysiological screening platforms. The vast majority of current hiPSC-CM-based arrhythmogenic screens rely solely on field potential duration (MEA technology) or action potential duration prolongation (VSD technology) as surrogate markers of TdP induction. This is a limitation because not all drugs that prolong the action potential (QT interval) will cause lethal TdP arrhythmias. Although using Matrigel in culture TM ECM and bone marrow cell-derived ECM have achieved some state of maturation of immature hiPSC-CMs, but the use of these hiPSC-CMs in arrhythmogenic screening assays did not produce results consistent with what is known to occur in human TdP cases, particularly between low-risk and high-risk drugs, when observing patterns of arrhythmic activation.

[0009] Therefore, advanced materials and new methods are needed to precisely evaluate and screen the cardiac safety of drug compounds to accurately, reliably, and efficiently develop new drug candidates. Summary of the invention

[0010] The present disclosure provides a solution to at least some of the above limitations and deficiencies in the field related to the assessment and screening of the cardiac safety of candidate drugs. The solution is premised on the discovery of an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid that can be used for the maturation of human stem cell-derived cardiomyocytes. These mature cardiomyocytes can then be used in cell constructs with AFC-ECM for cardiotoxicity and arrhythmogenic testing of drugs.

[0011] In one aspect, a method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells is disclosed, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro; (c) contacting the immature hiPSC-CMs with the AFC-ECM; and (d) culturing the immature hiPSC-CMs with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs to form mature cardiomyocytes; wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult heart tissue. In some embodiments, the mature cardiomyocytes have a similar structure to that of the adult heart tissue. Figures 6 to 14 In some embodiments, immature hiPSC-CMs are seeded on AFC-ECMs. In some embodiments, mature cardiomyocytes are formed as a monolayer on AFC-ECM, thereby forming a cell construct comprising a monolayer of mature cardiomyocytes on AFC-ECM, wherein mature cardiomyocytes are arranged on AFC-ECM. In some embodiments, immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.1. In some embodiments, immature hiPSC-CMs do not include rod-shaped cells with a unique sarcomere structure. In some embodiments, immature hiPSC-CMs may have fewer mitochondria than mature cardiomyocytes, have disordered myofilaments, have a round shape, and / or have a single nucleus. In some embodiments, mature cardiomyocytes may also have a larger amount of mitochondria than immature hiPSC-CMs, have organized and compact myofilaments, and / or have two nuclei (binucleates) as features.

[0012] In another aspect, a cell construct is disclosed, comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from cells isolated in vitro from amniotic fluid (AFC-ECM), wherein the mature cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) cultured in the AFC-ECM, and wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult cardiac tissue. In some embodiments, the mature cardiomyocytes have a structure similar to that of a human heart. Figures 6 to 14In some aspects, mature cardiomyocytes include an inwardly rectifying Kir2.1 potassium channel and / or express an inwardly rectifying potassium channel Kir2.1. In some aspects, mature cardiomyocytes can be matured from immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells on AFC-ECM in culture, wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure (striated appearance) similar to adult cardiac tissue. In some embodiments, a monolayer of mature cardiomyocytes is arranged on AFC-ECM. In some embodiments, fiber tracks are present on the construct. In some embodiments, AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin and collagen alpha-2 (IV) and / or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the AFC-ECM further comprises fibronectin and / or its isoforms. In some embodiments, the AFC-ECM does not contain decorin, perlecan and / or collagen (III). In some embodiments, the immature hiPSC-CM does not include rod-shaped cells with a unique sarcomere structure. In some embodiments, the immature hiPSC-CM may have fewer mitochondria than mature cardiomyocytes, have disordered myofilaments, have a round shape and / or have a single nucleus. In some embodiments, mature cardiomyocytes may also have a larger amount of mitochondria than immature hiPSC-CMs, have organized and compact myofilaments and / or have two nuclei (binucleated) as a feature.

[0013] In another aspect, a method for preparing a cell construct in which mature cardiomyocytes are derived in vitro from an extracellular matrix of cells isolated from amniotic fluid (AFC-ECM) is disclosed, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid; (c) seeding the immature hiPSC-CM on the AFC-ECM; and (d) culturing the seeded immature hiPSC-CM on the AFC-ECM in a culture medium to induce the immature hiPSC-CM to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult heart tissue. In some embodiments, the mature cardiomyocytes have a similar structure to that of the cell construct. Figures 6 to 14In some embodiments, a monolayer of mature cardiomyocytes is arranged on the AFC-ECM. In some embodiments, fiber tracks are present on the cell construct. In some embodiments, the immature hiPSC-CMs do not include rod-shaped cells with a unique sarcomere structure. In some embodiments, the immature hiPSC-CMs may be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a rounded shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes may also be characterized by having a larger amount of mitochondria than immature hiPSC-CMs, having organized, compact myofilaments, and / or having two nuclei (binucleated).

[0014] In another aspect, a method for determining the cardiotoxicity and / or arrhythmogenic effects of a drug compound in vitro is disclosed, the method comprising contacting the drug compound with mature cardiomyocytes of any one of the cell constructs disclosed throughout the specification, and observing the electrophysiological changes of the mature cardiomyocytes to confirm whether the drug compound has cardiotoxicity and / or arrhythmogenic effects on mature cardiomyocytes. The electrophysiological changes of mature cardiomyocytes confirm that the drug compound has cardiotoxicity and / or arrhythmogenic effects on mature cardiomyocytes. The electrophysiological changes of mature cardiomyocytes may include, but are not limited to, APD prolongation, APD prolongation plus rotors, and / or various types of arrhythmias, such as tachyarrhythmias (TA), quiescence (Q), delayed afterdepolarization (DAD), and / or early afterdepolarization (EAD). Observation may also include cell viability, cell density, and / or cell morphology. In some embodiments, the electrophysiological changes of mature cardiomyocytes are the extension of action potential duration (APD). In some embodiments, the electrophysiological changes of mature cardiomyocytes are early afterdepolarization (EAD). In some embodiments, the electrophysiological change of mature cardiomyocytes is delayed afterdepolarization (DAD). In some embodiments, the electrophysiological change of mature cardiomyocytes is prolonged action potential duration (APD prolongation) plus rotors. In some embodiments, the electrophysiological change of mature cardiomyocytes is arrhythmia. In some aspects, the cell construct can be prepared by a method including the following: (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro; (c) seeding the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the seeded immature hiPSC-CMs on the AFC-ECM in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult heart tissue. In some embodiments, mature cardiomyocytes have similar characteristics to those of Figures 6 to 14. In one embodiment, the immature hiPSC-CM does not express the inward rectifier potassium channel Kir2.1. In another embodiment, a monolayer of mature cardiomyocytes is arranged on the AFC-ECM. In another embodiment, fiber tracks are present on the cell construct. In some embodiments, the immature hiPSC-CM does not include rod-shaped cells with a unique sarcomere structure. In some embodiments, the immature hiPSC-CM may have fewer mitochondria than mature cardiomyocytes, have disordered myofilaments, have a round shape and / or have a single nucleus. In some embodiments, mature cardiomyocytes may also have a larger amount of mitochondria than immature hiPSC-CMs, have organized and compact myofilaments and / or have two nuclei (binucleated) as characteristics.

[0015] Additionally, the following embodiments 1 to 20 are disclosed in the context of the present invention:

[0016] Embodiment 1 is a method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising:

[0017] (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs);

[0018] (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM);

[0019] (c) contacting immature hiPSC-CMs with AFC-ECMs; and

[0020] (d) culturing the immature hiPSC-CMs with AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs to form mature cardiomyocytes;

[0021] The mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult heart tissue.

[0022] Embodiment 2 is the method of embodiment 1, wherein the immature hiPSC-CMs are seeded on AFC-ECM.

[0023] Embodiment 3 is the method of any one of embodiments 1 or 2, wherein the mature cardiomyocytes are formed as a monolayer on the AFC-ECM, thereby forming a cell construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, wherein the mature cardiomyocytes are arranged on the AFC-ECM.

[0024] Embodiment 4 is the method of any one of embodiments 1 to 3, wherein the immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.1.

[0025] Embodiment 5 is a cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from cells isolated in vitro from amniotic fluid (AFC-ECM), wherein the mature cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) cultured in AFC-ECM, and wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult heart tissue.

[0026] Embodiment 6 is the cell construct of embodiment 5, wherein a monolayer of mature cardiomyocytes is arranged on the AFC-ECM.

[0027] Embodiment 7 is the cell construct of any one of embodiments 5 or 6, wherein fiber tracks are present on the construct.

[0028] Embodiment 8 is the cell construct of any one of embodiments 5 to 7, wherein the AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin and collagen alpha-2 (IV) and / or its isoforms.

[0029] Embodiment 9 is the cell construct of embodiment 8, wherein the isoform of collagen α-1 (XVIII) is isoform 2, and / or wherein the isoform of agrin is isoform 6.

[0030] Embodiment 10 is the cell construct of any one of embodiments 8 or 9, wherein the AFC-ECM further comprises fibronectin and / or its isoforms.

[0031] Embodiment 11 is the cell construct of any one of embodiments 5 to 10, wherein the AFC-ECM does not contain decorin, perlecan and / or collagen (III).

[0032] Embodiment 12 is a method for preparing a cell construct of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro, the method comprising:

[0033] (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs),

[0034] (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM);

[0035] (c) seeding the immature hiPSC-CM on the AFC-ECM;

[0036] (d) culturing the seeded immature hiPSC-CMs on the AFC-ECM in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct;

[0037] The mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult heart tissue.

[0038] Embodiment 13 is the method of embodiment 12, wherein a monolayer of mature cardiomyocytes is arranged on the AFC-ECM.

[0039] Embodiment 14 is the method of any one of embodiments 12 or 13, wherein fiber tracks are present on the cell construct.

[0040] Embodiment 15 is a method for determining the cardiotoxic and / or arrhythmogenic effects of a drug compound in vitro, the method comprising contacting the drug compound with mature cardiomyocytes of any one of the cell constructs of Embodiments 5 to 11, and observing electrophysiological changes in the mature cardiomyocytes to confirm whether the drug compound has cardiotoxic and / or arrhythmogenic effects on the mature cardiomyocytes.

[0041] Embodiment 16 is the method of embodiment 15, wherein the change in electrophysiology of the mature cardiomyocytes is a prolongation of action potential duration (APD), and wherein the prolongation of APD confirms that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

[0042] Embodiment 17 is the method of embodiment 15, wherein the change in electrophysiology of the mature cardiomyocytes is early afterdepolarization, and wherein the early afterdepolarization (EAD) confirms that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

[0043] Embodiment 18 is the method of embodiment 15, wherein the change in electrophysiology of the mature cardiomyocytes is a delayed afterdepolarization, and wherein the delayed afterdepolarization (DAD) confirms that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

[0044] Embodiment 19 is the method of embodiment 15, wherein the electrophysiological change in the mature cardiomyocytes is action potential duration (APD) plus rotor, and wherein the prolongation of the APD plus rotor confirms that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

[0045] Embodiment 20 is the method of embodiment 15, wherein the change in electrophysiology of the mature cardiomyocytes is an arrhythmia, and wherein the arrhythmia demonstrates that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

[0046] The term "about" or "approximately" is defined as close to as understood by one of ordinary skill in the art, and in one non-limiting embodiment, the term is defined as within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0047] The term "substantially" and variations thereof are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%.

[0048] As used herein, the term "weight / weight %" or "weight %" refers to the weight percentage of the total weight of the material (e.g., composition) containing the component. In a non-limiting example, 10 grams of components in 100 grams of composition are 10 weight / weight % of the component in the total weight of the composition. As used herein, the term "volume / volume %" or "volume %" refers to the volume percentage of the total volume of the material (e.g., composition) containing the component. In a non-limiting example, 100mL components in 100mL compositions are 10 volume / volume % of the component in the total volume of the composition. As used herein, the term "weight / volume %" refers to the weight percentage of the total volume of the material (e.g., composition) containing the component. In a non-limiting example, 10 grams of components in 100mL compositions are 10 weight / volume % of the component in the total volume of the composition. As used herein, the term "volume / weight %" refers to the volume percentage of the total weight of the material (e.g., composition) containing the component. In one non-limiting example, 10 mL of a component in 100 grams of a composition is 10 volume / weight % of the component in the total volume of the composition.

[0049] When used in the claims and / or specification, the terms "inhibit" or "reduce" or "prevent" or "avoid" include any measurable reduction or complete inhibition to achieve the desired result.

[0050] The term "effective," as that term is used in the specification and / or claims, means sufficient to achieve a desired, expected, or intended result.

[0051] The words "comprising," "having," "including," or "containing" are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0052] When used in conjunction with the terms "comprising," "having," "including," or "containing" (or any variations of these words), without a quantifier may mean "a," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0053] The compositions and methods of use thereof may "comprise," "consist essentially of," or "consist of" any ingredient or step disclosed throughout the specification. With respect to the transitional expression "consist essentially of," in one non-limiting aspect, the basic and novel features of the cell constructs disclosed herein are their use in screening tests for cardiotoxicity and / or proarrhythmogenicity of drug compounds, because they have the ability to mature stem cell-derived cardiomyocytes to a mature state similar to mature native adult cardiomyocytes and native cardiac tissue.

[0054] It is contemplated that any embodiment discussed in this specification can be implemented in conjunction with any method or composition of the invention, and vice versa. In addition, the methods of the invention can be implemented using the compositions of the invention.

[0055] Other purposes, features and advantages of the present invention will become apparent from the following specific embodiments. However, it should be understood that the specific embodiments and specific examples, although indicating specific embodiments of the present invention, are given only as illustrations, because various changes and modifications within the spirit and scope of the present invention will become apparent to those skilled in the art from the specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 : Photomicrograph of bright field image of amniocyte-derived ECM at 100x magnification using a 10x objective.

[0057] Figure 2 : Atomic force micrographs of three representative 40×40 um sections of amniocyte-derived ECM and bone marrow cell-derived ECM showing morphology, adhesion and stiffness.

[0058] Figure 3 : Scatter plots showing quantification of adhesion and stiffness (elastic modulus) of bone marrow cell- and amniocyte-derived ECM. Each dot represents an independent measurement point.

[0059] Figure 4 : Micrographs showing day 0 and day 2 of iPSC culture on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0060] Figure 5: Growth curves of iPSCs cultured in the presence of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0061] Figure 6 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 3.13 mm 2 .

[0062] Figure 7 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 2.15 mm 2 .

[0063] Figure 8 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 1.46 mm 2 .

[0064] Fig. 9 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.54 mm 2 .

[0065] Fig.10 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.54 mm 2 .

[0066] Fig.11 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.54 mm 2 .

[0067] Fig.12 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.54 mm 2 .

[0068] Fig.13 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.13 mm 2 .

[0069] Fig.14 : Photomicrograph of mature cardiomyocytes on AFC-ECM in a single well – 0.04 mm 2 .

[0070] Fig.15 : Standard Matrigel in single well TM Micrograph of cardiomyocytes on ECM – 3.13 mm 2 .

[0071] Fig.16 : Standard Matrigel in single well TM Micrograph of cardiomyocytes on ECM – 2.15 mm 2 .

[0072] Fig.17 : Standard Matrigel in single well TM Micrograph of cardiomyocytes on ECM – 1.46 mm 2 .

[0073] Fig.18 : Standard Matrigel in single well TM Micrograph of cardiomyocytes on ECM – 0.54 mm 2 .

[0074] Fig.19 : Standard Matrigel in single well TM Micrograph of cardiomyocytes on ECM – 0.13 mm 2 .

[0075] Fig. 20 : Photomicrograph of mature cardiomyocytes on BM-ECM in a single well – 2.15 mm 2 .

[0076] Fig.21 : Photomicrograph of mature cardiomyocytes on BM-ECM in a single well – 1.46 mm 2 .

[0077] Fig. 22 : Schematic diagram of the configuration of the instrument for recording action potential or calcium transient measurements using mature cardiomyocytes on AFC-ECM in multiwell plates.

[0078] Fig.23 : Baseline and after drug E4031, from Matrigel TM Spontaneous action potential recordings recorded from cardiomyocytes cultured on ECM, BM-ECM, and AFC-ECM.

[0079] Fig.24 :After contact with drug E4031, from Matrigel TM Bar graph of the number of stable rotors (TdP-like arrhythmias) of cardiomyocytes cultured on ECM, BM-ECM, and AFC-ECM.

[0080] Fig.25 : Spontaneous action potential recordings from mature cardiomyocytes cultured on AFC-ECM in response to various drugs.

[0081] Fig.26 : Bar graph of total arrhythmias recorded for all doses of each drug listed.

[0082] Fig. 27 : Bar graph of the % of wells with arrhythmias at 10 times the effective therapeutic plasma concentration (ETPC) of each listed drug.

[0083] Fig.28 : Bar graph of action potential triangulation (APD90-APD30) expressed in time (milliseconds) for each drug listed.

[0084] Fig.29 : Bar graph of action potential triangulation (APD90-APD30) expressed in time (milliseconds) for each of some of the listed drugs, comparing on AFC-ECM (SBS-AF matrix) vs. on Matrigel TM Cardiomyocyte performance of cardiomyocytes on ECM.

[0085] Fig.30 : Bar graph of triangulated maximum drug-induced action potentials for the listed drugs, compared to the data from Cellular Dynamics hiPSC-CMs (open circles) and Cor. Cardiomyocyte performance of hiPSC-CMs (black circles) at any concentration of the listed drugs. Figure from Blinova et al, 2018, Cell Reports.

[0086] Fig.31 : In Matrigel TM Photomicrographs of hiPSC-CMs cultured on ECM and AFC-ECM with immunofluorescence staining for troponin I and DAPI used to label cell nuclei.

[0087] Fig.32 : In Matrigel TM Micrographs of hiPSC-CMs cultured on ECM and AFC-ECM, with immunofluorescence staining for α-actinin and DAPI used to label cell nuclei.

[0088] Fig.33 : In Matrigel TM Micrographs of hiPSC-CMs cultured on ECM and AFC-ECM, with immunofluorescence staining for cTnT and N-cadherin and DAPI used to label cell nuclei.

[0089] Fig.34 : In Matrigel TM Micrographs of individual hiPSC-CM cells cultured on ECM and AFC-ECM, with immunofluorescence staining for cTnT and DAPI used to label cell nuclei.

[0090] Fig.35 : In Matrigel TMMicrographs of individual hiPSC-CM cells cultured on ECM and AFC-ECM, with immunofluorescence staining for α-actinin and DAPI used to label cell nuclei.

[0091] Fig.36 : Fig.35 A comparison of the cell circularity of individual cells is shown in FIG.

[0092] Fig.37 : In Matrigel TM Micrographs of hiPSC-CMs cultured on ECM and AFC-ECM, with immunofluorescence staining for cTnI expression and DAPI used to label cell nuclei.

[0093] Fig.38 : In Matrigel TM Western blotting for cTnI expression and GAPDH of hiPSC-CMs on ECM and AFC-ECM.

[0094] Fig.39 : In Matrigel TM Plot of cTnI expression relative to GAPDH for hiPSC-CMs on ECM and AFC-ECM.

[0095] Fig.40 :Matrigel stained with MitoTracker Red for mitochondria TM Micrographs of hiPSC-CMs on ECM and AFC-ECM.

[0096] Fig.41 : This figure shows Matrigel TM MitoTraker of hiPSC-CMs on ECM and AFC-ECM TM Red fluorescence intensity / cardiomyocyte.

[0097] Fig.42 : Matrigel coated on microelectrode array (MEA) plates TM Micrographs of hiPSC-CMs cultured on ECM and AFC-ECM (transmitted light). DETAILED DESCRIPTION

[0098] Human stem cell-derived cardiomyocytes, such as those derived from human induced pluripotent stem cells (hiPSC-CMs), that have been matured in culture on extracellular matrices derived in vitro from cells isolated from amniotic fluid (AFC-ECM), have been shown to be more robust than immature hiPSC-CMs or on other ECMs such as Matrigel. TMhiPSC-CMs matured on ECM and bone marrow-derived ECM had better and more consistent results in cardiotoxicity and arrhythmogenicity assays. hiPSC-CMs matured on AFC-ECM unexpectedly showed better cardiotoxicity and arrhythmogenicity than other ECMs such as Matrigel. TM hiPSC-CMs cultured on AFC-ECM and even on other natural cell-derived ECMs such as bone marrow-derived ECMs showed a higher state of maturity, as indicated by their cell morphology and sarcomere structure seen in cardiomyocytes of normal adult heart tissue. In addition, as confirmed by Western blotting, hiPSC-CMs cultured on AFC-ECM showed a higher level of maturation than those cultured on Matrigel. TM More robust expression of cTnI (cardiac troponin I) protein was observed in hiPSC-CMs cultured on ECM. TM hiPSC-CMs cultured on AFC-ECM have more mitochondria and mitochondria have more polarized inner membrane potentials compared to hiPSC-CMs cultured on ECM. Therefore, AFC-EMC can stimulate mitochondrial biogenesis, maturation, and functional operation in hiPSC-CMs. Human tissue of a normal adult or mature human heart is characterized by rod-shaped cells with a sarcomere structure (striated appearance). The morphology and sarcomere structure of cardiomyocytes can be visually identified by microscopy (transmitted light or by immunofluorescence staining). As a non-limiting example, the morphology and sarcomere structure of cardiomyocytes matured on AFC-ECM can be clearly seen by the presence of rod-shaped cells, which have a striated appearance. Fig.14The appearance of stripes marked by arrows in the micrographs of . In addition, there is no need to use silicone substrates such as PDMS to achieve these results. Surprisingly, cell constructs comprising AFC-ECM and a monolayer of mature cardiomyocytes that have been matured from immature hiPSC-CMs on AFC-ECM in culture have proven useful, for example, in in vitro screening assays with high throughput to allow consistent visualization of drug-induced arrhythmias such as torsade de pointes (TdP). This "TdP in a dish" technology is a major advance over relying on field potential duration (MEA technology) or action potential duration extension as an alternative marker for drug-induced TdP. Therefore, the cell constructs and methods disclosed herein go beyond the guidelines of the current CiPA initiative by developing more comprehensive and predictable in vitro arrhythmia assays, allowing visualization of arrhythmic events in in vitro models rather than simple polarization and depolarization events that are indirect indicators of arrhythmogenicity. The cell systems and methods outlined in the present disclosure allow propagation and visualization of arrhythmic events in an in vitro human cardiac monolayer model system. In some embodiments, immature hiPSC-CMs do not include rod-shaped cells with a unique sarcomere structure. In some embodiments, immature hiPSC-CMs may be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a rounded shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes may be characterized by having rod-shaped cells with a unique sarcomere structure (striated appearance). In some embodiments, mature cardiomyocytes may also be characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having organized, compact myofilaments, and / or having two nuclei (binucleated).

[0099] Disclosed herein is a method for maturing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells to form mature cardiomyocytes (mature hiPSC-CMs) in culture using a cell-derived extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid. Also disclosed herein is a cell construct comprising a monolayer of these mature cardiomyocytes on AFC-ECM, which can be used for cardiotoxicity and / or arrhythmogenic screening assays of drug compounds. Also disclosed herein is a method for determining the cardiotoxicity and / or arrhythmogenic effects of drug compounds in vitro using such a cell construct.

[0100] A. Amniotic fluid cell-derived extracellular matrix (AFC-ECM)

[0101] Perinatal cells can be divided into three groups: cells from amniotic fluid; cells from the placenta; and cells from the umbilical cord. Amniotic fluid has several cell sources, including cells shed from the fetal amnion, skin, and digestive, respiratory, and urogenital tracts of the developing fetus. The placenta also has several cell sources, including membranes (amniotic and chorionic), villi, and blood. Umbilical cord cells usually come from two sources: cord blood and Wharton's jelly. Cells from these three perinatal sources may include stem cells. Cells for producing the amniotic fluid cell-derived ECM of the present invention are obtained from the amniotic fluid of mammals, including but not limited to humans (Homo sapiens), mice, rabbits, cats, dogs, pigs, horses, or primates. In a preferred embodiment, the cells are from human amniotic fluid. Amniotic fluid can be derived from humans who are born at term (greater than about 37 weeks of gestational age) or premature (less than about 37 weeks of gestational age). Premature birth includes late premature birth (about 33 weeks to about 37 weeks gestational age), moderate premature birth (about 29 weeks to about 33 weeks gestational age) and very early premature birth (about 23 weeks to about 29 weeks gestational age). Amniotic fluid can be derived from people before delivery of any gestational age in which there is amniotic fluid, and can be combined with the amniotic fluid source during delivery. Usually, before delivery, amniotic fluid is collected by amniocentesis. In some embodiments, amniotic fluid is derived from people or combinations thereof before full-term delivery, premature delivery, late premature delivery, moderate premature delivery, very early premature delivery or delivery. In some embodiments, amniotic fluid is derived from before delivery and is collected from about 10 weeks gestational age until delivery, or from about 10 weeks to about 23 weeks gestational age, or from about 10 weeks to about 16 weeks gestational age, or from about 12 weeks gestational age until delivery, or from about 12 weeks to about 23 weeks gestational age or from about 12 weeks to about 16 weeks gestational age. In some embodiments, the amniotic fluid derived from before delivery is collected by amniocentesis. Cells can be obtained and isolated from amniotic fluid by techniques known in the art, such as those disclosed in Murphy et al., Amniotic Fluid Stem Cells, Perinatal Stem Cells, Second Ed. 2013.

[0102] Amniotic fluid is made up of cells that can spontaneously or due to treatment with specific growth factors or a combination of growth factors known to those skilled in the art be differentiated into the cell type derived from all 3 embryonic germ layers (ectoderm, endoderm, mesoderm). That is, a single cell has the ability to be induced to express a gene that is specific to any one of the three germ layers. Amniotic fluid also contains a mixture of different cell types, including cells that come from the fetal amnion, skin and digestive tract, respiratory tract and urogenital tract of the developing fetus. Due to the origin of amniotic fluid and placental membranes, these cells can maintain a highly pluripotent differentiation potential and include a cell colony containing cells of all three germ layers. Amniotic fluid cells may include stem cells. In some embodiments, amniotic fluid cells are isolated stem cells. In some embodiments, amniotic fluid cells include stem cells with the ability to differentiate into the cell type derived from all 3 embryonic germ layers (ectoderm, endoderm, mesoderm) and / or pluripotent stem cells and / or pluripotent stem cells.

[0103] The amniotic fluid cell-derived ECM disclosed herein may include various proteins. The proteins of the ECM can be identified by techniques known in the art, and may include mass spectrometry and immunohistochemical staining. The ECM may include, but is not limited to, the components listed in Table 2 (see Example 1 below) and any variants, derivatives or isoforms thereof. The amniotic fluid cell-derived ECM may include any combination of any components in Table 2 and any variants, derivatives or isoforms thereof. In some embodiments, the combination may include, consist essentially of, or consist of: laminin, collagen α-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen α-2 (IV) and / or its isoforms. In some embodiments, the isoform of collagen α-1 (XVIII) is isoform 2. In some embodiments, the isoform of agrin is isoform 6. In some embodiments, the cell-derived ECM also includes, consists essentially of, or consists of fibronectin and / or its isoforms. In some embodiments, the amniocyte-derived ECM does not contain any or all of decorin, perlecan, and collagen (III). Table 1 describes some notable differences in proteins between the amniocyte-derived ECM of the present invention and bone marrow cell-derived matrix.

[0104] Table 1

[0105] Differences between amniotic fluid cell-derived ECM (AFC-stroma) and bone marrow cell-derived stroma (BM-stroma)

[0106]

[0107] Amniotic fluid cell-derived ECM can be generated by the following methods:

[0108] (a) Isolation of cells from amniotic fluid,

[0109] (b) seeding the separated cells onto a cell culture container or onto a cell culture container coated with a substrate,

[0110] (c) adding culture medium to the cell culture vessel, and

[0111] (d) culturing the cells to produce cell-derived ECM, and

[0112] (e) Optionally decellularizing the cell-derived ECM.

[0113] Any cell seeding density that allows the cells to form a confluent monolayer immediately or over a period of time in culture can be used. In some embodiments, the seeding density is about 10 cells / cm 2 About 100,000 cells / cm 2 , or about 100 cells / cm 2 About 75,000 cells / cm 2 , or about 500 cells / cm 2 About 50,000 cells / cm 2 , or about 500 cells / cm 2 About 10,000 cells / cm 2 , or about 500 cells / cm 2 About 5000 cells / cm 2 , or about 500 cells / cm 2 About 2500 cells / cm 2 , or about 1000 cells / cm 2 About 25,000 cells / cm 2 , or about 2000 cells / cm 2 About 10,000 cells / cm 2 , or about 3000 cells / cm 2 About 5000 cells / cm 2 .

[0114] Any type of container suitable for cell culture can be used in the present invention. Examples include, but are not limited to, cell culture bottles, T-flasks, stirred bottles, spinner bottles, fermenters, and bioreactors. Shake bottles, vibrating flasks, test tubes, and other containers are also suitable containers when placed on a rocking platform or vibrator. The cell culture container can be coated with a substrate to allow better cell adhesion. A non-limiting example of a suitable substrate for coating a cell container is fibronectin.

[0115] Various commercially available cell culture media, for example, alpha minimum essential medium (α-MEM) (Thermo Fisher Scientific, Grand Island, New York), are suitable for culturing amniocytes. Commercially available culture media can be modified by adding various supplements to the culture media, for example, sodium bicarbonate, L-glutamine, penicillin, streptomycin, amphotericin B and / or serum. The serum can be fetal bovine serum. The culture media can also be serum-free. In addition, substances such as L-ascorbic acid can be added to the culture media or modified culture media to induce the cells to produce ECM.

[0116] The initial culture medium can be changed and / or replaced with another culture medium at different times during the culture process. For example, the initial culture medium can be a "complete culture medium" and then replaced with an "induction culture medium" during the culture process. A non-limiting example of a "complete culture medium" contains (α-MEM) plus 2mM L-glutamine plus antibiotic-antimycotic plus 15% fetal bovine serum. A non-limiting example of an "induction culture medium" contains a "complete culture medium" plus 50mM L-ascorbic acid.

[0117] The amniotic fluid cells can be cultured in an incubator at 37°C, 5% CO2 and 90% humidity. The culture can be carried out under various environmental conditions, including but not limited to normoxic conditions, i.e., 20% to 21% oxygen in the atmosphere, or hypoxic conditions.

[0118] De-amniocyteing the ECM of amniocyte origin may include removing viable amniocytes or making the amniocytes non-viable. De-amniocytes may be obtained from the ECM using methods known in the art, and may include but is not limited to lysing the amniocytes and then removing the lysed amniocytes by washing. Various substances may be used to remove amniocytes from the ECM. Non-limiting examples include "extraction buffer" containing TRITON X-100 and ammonium hydroxide in PBS buffer. After the ECM has been de-amniocyteed, the resulting ECM is thus substantially free of cells or free of viable amniocytes. If feeder cells are used, the decellularization method is also applicable to any viable feeder cells present on the ECM, thereby producing an ECM that is substantially free of or free of viable feeder cells. The decellularization method is also applicable to any viable cells present on the ECM, thereby producing an ECM that is substantially free of or free of any viable cells. Therefore, the decellularized ECM means that the ECM is acellular, which means that the ECM does not contain any viable cells.

[0119] In some embodiments, the amniotic fluid cell-derived ECM (AFC-ECM) is a three-dimensional (3D) ECM.

[0120] The methods described above are also applicable to the production of cell-derived ECM from other perinatal cells, such as cells from the umbilical cord, including cord blood and Wharton's jelly; and cells from placental tissue, including membranes (amnion and chorion), villi, and blood.

[0121] In one embodiment, perinatal cell-derived ECM is produced by the following method:

[0122] (a) isolating cells from the umbilical cord,

[0123] (b) seeding the separated cells onto a cell culture container or onto a cell culture container coated with a substrate,

[0124] (c) adding culture medium to the cell culture vessel, and

[0125] (d) culturing the cells to produce cell-derived ECM, and

[0126] (e) Optionally decellularizing the cell-derived ECM.

[0127] In some embodiments, the cells isolated from the umbilical cord are from cord blood and / or Wharton's jelly.

[0128] In another embodiment, perinatal cell-derived ECM is produced by:

[0129] (a) isolating cells from placental tissue,

[0130] (b) seeding the separated cells onto a cell culture container or onto a cell culture container coated with a substrate,

[0131] (c) adding culture medium to the cell culture vessel, and

[0132] (d) culturing the cells to produce cell-derived ECM, and

[0133] (e) Optionally decellularizing the cell-derived ECM.

[0134] In some embodiments, cells isolated from placental tissue are from membranes (amniotic and / or chorionic), villi, and / or blood.

[0135] In one aspect, a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from an umbilical cord is disclosed. In some embodiments, the cells isolated from the umbilical cord are from umbilical cord blood and / or Wharton's jelly.

[0136] In another aspect, a cell-derived extracellular matrix (ECM) derived in vitro from cells isolated from placental tissue is disclosed. In some embodiments, the cells isolated from placental tissue are from membranes (amniotic membrane and / or chorionic membrane), villi and / or blood.

[0137] B. Cellular Constructs and Methods for Maturating Immature hiPSC-CMs

[0138] Disclosed herein is a method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid; (c) contacting the immature hiPSC-CMs with the AFC-ECM; and (d) culturing the immature hiPSC-CMs with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs to form mature cardiomyocytes (mature hiPSC-CMs); wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult cardiac tissue. Normal adult cardiac tissue is characterized by rod-shaped cells having a sarcomere structure (striated appearance). The morphology and sarcomere structure of the cardiomyocytes can be visually identified by microscopy. As a non-limiting example, the morphology and sarcomere structure of the cardiomyocytes matured on the AFC-ECM can be clearly seen by the presence of rod-shaped cells having a sarcomere structure composed of Fig.14 The arrows in the micrographs identify the appearance of the stripes.

[0139] Also disclosed herein is a cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived from cells isolated in vitro from amniotic fluid, wherein the mature cardiomyocytes are cardiomyocytes (hiPSC-CM) derived from human induced pluripotent stem cells cultured in AFC-ECM, and wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult cardiac tissue. In certain aspects, mature cardiomyocytes can be matured from immature cardiomyocytes (immature hiPSC-CM) derived from human induced pluripotent stem cells on AFC-ECM in culture, wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult cardiac tissue. In some aspects, mature cardiomyocytes include inwardly rectifying Kir2.1 potassium channels and / or express inwardly rectifying potassium channels Kir2.1. In some embodiments, a monolayer of mature cardiomyocytes is arranged on AFC-ECM. In some embodiments, fiber tracks are present on the construct. In some embodiments, the AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV) and / or isoforms thereof. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2, and / or wherein the isoform of agrin is isoform 6. In some embodiments, the AFC-ECM further comprises fibronectin and / or isoforms thereof. In some embodiments, the AFC-ECM does not contain decorin, perlecan, and / or collagen (III). In some embodiments, the immature hiPSC-CM does not include rod-shaped cells having a unique sarcomere structure. In some embodiments, the immature hiPSC-CM may be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a rounded shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes may also be characterized as having a greater number of mitochondria than immature hiPSC-CMs, having organized, compact myofilaments, and / or having two nuclei (binucleated).

[0140] Also disclosed herein is a method for preparing a cell construct containing mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro, the method comprising: (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro; (c) seeding immature hiPSC-CMs on AFC-ECMs; and (d) culturing the seeded immature hiPSC-CMs on AFC-ECMs in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on AFC-ECMs, thereby forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult heart tissue. In some embodiments, a monolayer of mature cardiomyocytes is arranged on AFC-ECM. In some embodiments, fiber tracks are present on the construct.

[0141] Immature hiPSC-CMs can be obtained from commercial sources, such as Cellular Dynamics International-FUJI under the trade name Obtained and marketed from Takara Bio under the trade name get. Cardiomyocytes, Cardiomyocytes 2 and Cardiomyocytes are cryopreserved surviving cardiomyocytes derived from human induced pluripotent stem cells (hiPSC) available in vials. Immature hiPSC-CMs can also be generated from patient-specific hiPSCs for specific individuals in a laboratory environment. In this case, the differentiation of hiPSCs can be achieved using a small molecule solution to obtain beating cardiomyocytes by the 8th to 10th day, wherein GSK3 inhibitors, RPMI / B27 minus insulin, Wnt inhibitors and RPMI / B27 plus insulin are used on different days during the 7-day differentiation period. Suitable non-limiting examples of methods for generating immature hiPSC-CMs are disclosed in U.S. Publication 2015 / 0329825, which is incorporated herein by reference. In some embodiments, immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.1. In some embodiments, immature hiPSC-CMs do not include rod-shaped cells with unique sarcomere structures. In some embodiments, immature hiPSC-CMs may be characterized as having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a rounded shape, and / or having a single nucleus.

[0142] AFC-ECM can be obtained using the preparation methods disclosed in the present disclosure and can have the characteristics as described in the present disclosure. In some embodiments, the AFC-ECM is decellularized prior to contacting with the immature hiPSC-CMs.

[0143] When in contact with AFC-ECM, the immature hiPSC-CM may be in suspension, or the cells may be seeded directly onto the AFC-ECM in or on a suitable cell culture container or in a multi-well plate. Non-limiting examples of suitable multi-well plates include 6-, 12-, 24-, 48, 96- and 384-well plates. In some embodiments, before forming the AFC-ECM, the contact surface of the cell culture container or multi-well plate is coated with polydimethylsiloxane (PDMS). In some embodiments, before forming the AFC-ECM, the contact surface of the cell culture container or multi-well plate is not coated with PDMS. Any cell seeding density of immature hiPSC-CM can be used. In some embodiments, a cell seeding density of immature hiPSC-CM that allows cells to form a confluent monolayer immediately or after a period of time in culture is used. The cell density can be changed as needed to improve the formation of a monolayer. In a multi-well plate, immature hiPSC-CM cells are placed in the center of each well. Non-limiting examples of cell seeding densities of immature hiPSC-CMs in various multi-well plates are as follows: about 200,000 cells can be seeded per well in a 6-well plate; about 150,000 cells can be seeded per well in a 12-well plate; about 175,000 cells can be seeded per well in a 24-well plate; about 100,000 cells can be seeded per well in a 48-well plate; about 50,000 cells can be seeded per well in a 96-well plate; and about 15,000 cells can be seeded per well in a 384-well plate. In some embodiments, the cell seeding density of immature hiPSC-CMs is about 50,000 cells per well in a 96-well plate. In some embodiments, the cell seeding density is about 200,000 cells per well in a 6-well plate. To induce maturation of immature hiPSC-CMs, a suitable culture medium such as RPMI culture medium or Media 199 culture medium is added to the immature hiPSC-CMs in contact with AFC-ECM, and the cells are cultured with AFC-ECM using standard cell culture techniques for a period of time, usually 7 days, until the cells mature into mature cardiomyocytes with a similar morphology to natural adult cardiomyocytes. Within the first 3 to 4 days, immature hiPCS-CMs will adhere to form a continuous monolayer and begin the maturation process. Surprisingly, the time period for cardiomyocyte maturation may be 7 days or less, while other substrates are typically much longer time periods, such as up to 100 days. The morphology of these mature cardiomyocytes can be characterized by rod-shaped cells with a unique sarcomere structure (striated appearance), which is the basic contractile unit of muscle and can be visualized using conventional optical microscopy techniques. In some embodiments, mature cardiomyocytes can also be characterized by having a larger amount of mitochondria than immature hiPSC-CMs, having organized and compact myofilaments, and / or having two nuclei (binucleates).Moreover, AFC-ECM can naturally produce fiber tracks followed by mature cardiomyocytes. This makes the monolayer that more closely simulates the natural heart produce a certain degree of anisotropy. Therefore, after the AFC-ECM has been arranged by the amniotic fluid cells in a natural anisotropic configuration during the formation of AFC-ECM, the cardiomyocytes can be naturally arranged on the AFC-ECM. In various embodiments, the time period for immature hiPSC-CMs to mature in culture can be 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 25 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days or 120 days. Preferably, the time period for cardiomyocyte maturation is 14 days, or more preferably 10 days, or even more preferably 7 days. In some embodiments, the time period for immature hiPSC-CM to mature in culture is 7 days. In some embodiments, immature hiPSC-CM is seeded on AFC-ECM. In some embodiments, during the culture process, mature cardiomyocytes are formed as a fusion monolayer on AFC-ECM, thereby forming a cell construct containing a monolayer of mature cardiomyocytes on AFC-ECM. In some embodiments, mature cardiomyocytes are arranged on AFC-ECM. In some embodiments, immature hiPSC-CM is seeded on AFC-ECM in a multi-well plate. In some embodiments, the multi-well plate has an insert of polydimethylsiloxane (PDMS). In some embodiments, the multi-well plate does not have an insert of PDMS.

[0144] C. Methods for Determining Cardiotoxic and / or Proarrhythmic Effects of Drug Compounds

[0145] Disclosed herein is a method for determining the cardiotoxicity and / or arrhythmogenic effects of a drug compound in vitro, the method comprising contacting the drug compound with mature cardiomyocytes of any of the cell constructs disclosed throughout the specification, and observing one or more changes in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has cardiotoxic and / or arrhythmogenic effects on mature cardiomyocytes. One or more changes in the electrophysiology of mature cardiomyocytes indicate and confirm that the drug compound has cardiotoxic and / or arrhythmogenic effects on mature cardiomyocytes. One or more changes in the electrophysiology of mature cardiomyocytes may include, but are not limited to, APD prolongation, APD prolongation plus rotors, and / or various types of arrhythmias, such as tachyarrhythmias (TA), quiescence (Q), delayed afterdepolarization (DAD), and / or early afterdepolarization (EAD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is an extension of the action potential duration (APD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is early afterdepolarization (EAD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is delayed afterdepolarization (DAD). In some embodiments, the electrophysiological change of mature cardiomyocytes is action potential duration (APD) plus rotors. In some embodiments, the electrophysiological change of mature cardiomyocytes is arrhythmia. In some embodiments, the electrophysiological change of mature cardiomyocytes is tachyarrhythmia (TA). In some embodiments, the electrophysiological change of mature cardiomyocytes is quiescence (Q). Observations may also include cell viability, cell density and / or cell morphology. In some aspects, the cell construct can be prepared by a method comprising: (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid in vitro; (c) seeding the immature hiPSC-CMs on the AFC-ECM; and (d) culturing the seeded immature hiPSC-CMs on the AFC-ECM in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a unique sarcomere structure similar to adult cardiac tissue. In other aspects, the method may include contacting a drug compound with any of the cell constructs disclosed throughout the specification and observing cardiotoxicity and / or arrhythmogenic events.In a particular case, the method comprises: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid; (c) seeding the immature hiPSC-CMs on the AFC-ECM; (d) culturing the seeded immature hiPSC-CMs on the AFC-ECM in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct; (e) contacting a drug compound with the monolayer of mature cardiomyocytes of the construct; and (f) observing changes in the electrophysiology of mature cardiomyocytes to confirm whether the drug compound has cardiotoxic and / or arrhythmogenic effects on mature cardiomyocytes; wherein the mature cardiomyocytes are characterized by rod-shaped cells with a unique sarcomere structure similar to adult heart tissue. In some embodiments, the immature hiPSC-CMs are seeded on the AFC-ECM in a multi-well plate. In some embodiments, the multiwell plate has an insert of polydimethylsiloxane (PDMS). In some embodiments, the multiwell plate does not have an insert of PDMS. In some embodiments, the immature hiPSC-CM does not express the inward rectifier potassium channel Kir2.1. In some embodiments, a monolayer of mature cardiomyocytes is arranged on the AFC-ECM. In some embodiments, there are fiber tracks on the cell construct.

[0146] Cardiotoxicity and / or arrhythmogenic testing can be performed using any type of equipment suitable for measuring such activity. In some embodiments, a cell construct of a monolayer of mature cardiomyocytes on AFC-ECM is prepared as described above. After the maturation process (usually 7 days or less), the electrophysiology of each well is observed using a plate reader. Appropriate voltage-sensitive or calcium-sensitive fluorescent dyes are loaded into each well. Non-limiting voltage-sensitive dyes include FluoVolt ELISA, which is commercially available from ThermoFisher. TMDye. In some embodiments, the plate reader may rely on a suitable high spatiotemporal CCD camera combined with suitable illumination, such as a light emitting diode (LED) that excites a wavelength suitable for each dye. Such a high spatiotemporal CCD camera is commercially available from SciMeasure. In some embodiments, the camera image acquisition rate is greater than or equal to 150 frames per second. In some embodiments, the camera and lens combination is designed so that it allows visualization of all holes of the multi-well plate at sufficient resolution to observe action potentials and calcium wave propagation. Center each plate below the camera system, turn on the illumination, start camera acquisition and record electrophysiological activity. The experiment is carried out at about 37°C. After obtaining a baseline reading, the drug to be tested is added to the hole and the effect is recorded. Record spontaneous activity for a long enough time to obtain an image, such as at least 10 seconds. The image can be stored on a computer. Analyze the image and can use image analysis software to quantify action potential duration, conduction velocity, beat rate and activation pattern. Visualization of the wave pattern is important for determining the effect of drug compounds causing potentially fatal arrhythmias such as torsade de pointes (TdP). Thus, in addition to providing information about the effects of a compound on the duration of spontaneous action potentials, the methods disclosed herein may also provide information about impulse conduction velocity and activation patterns, depending on the type of device used.

[0147] D. Methods for Expanding / Proliferating Mammalian Stem Cells

[0148] Also disclosed herein is a method for the separation, maintenance and expansion / proliferation of mammalian cells using a cell-derived extracellular matrix (AFC-ECM) derived from cells separated from amniotic fluid in vitro. In all cell biology and the developing fields of regenerative medicine and tissue engineering, in vitro cell culture may be the most common, most important but lacking understanding. First, it allows the observation of cell behavior so that various aspects of cell function can be studied in detail. Secondly, it allows the increase of the number of specific cell clusters. For basic research and many clinical applications, a large number of relatively rare cells need to be obtained from a small amount of biological samples. In vitro cell culture allows a small amount of cells to be expanded outside the body to obtain more relevant quantities. Finally, it allows cells to be stored for later use. By expanding the number of cells in vitro and freezing living cells for later use, relatively small biological samples can produce cells for multiple experiments within a few days, months or even years.

[0149] Despite the ubiquity of cell culture, the effects of in vitro culture on the natural properties of cells are relatively poorly understood. Many current practices stem not from careful thought, planning, and experimentation, but from serendipitous observations. Mammalian cell culture began in the early 20th century, with the first academic paper on in vitro mammalian tissue culture published in 1911 by Alexis Carrel and Montrose Burrows. They studied the physiology and anatomy of tissues by cutting sections of mammalian tissue and placing the sections on microscope slides. They then noticed that some cells migrated from the tissue onto the slide. They went on to describe techniques for culturing cells in perpetuity. Some of their observations may now appear invalid, but their work paved the way for modern cell culture.

[0150] Following the discovery of hematopoietic stem cells (HSCs), groups around the world began studying (HSCs). During their culture (in suspension), a subpopulation of bone marrow cells was observed to adhere to the bottom of a plastic flask and begin to proliferate. It was later recognized that these cells were distinct from HSCs and were eventually called mesenchymal stem cells (MSCs). Because this serendipitous observation led to the discovery of MSCs, plastic adhesion is still widely used as a defining property of MSCs and many other mammalian cell types.

[0151] The practice of growing cells on plastic substrates can be problematic because of the overwhelming evidence now well established in the literature that the microenvironment plays a critical role in regulating cell function. The microenvironment has been shown to help direct the differentiation of stem and progenitor cells and regulate the behavior of mature cell types.

[0152] When cells are taken out of their natural environment for in vitro expansion, they lose important clues from their surrounding extracellular matrix or microenvironment, which convey important information about the composition and state of their surrounding environment to the cells. Changes in the cell microenvironment can have a profound effect on the behavior of these cells. The current standard for isolating and expanding most adherent cells in vitro is to place the cells in a culture container composed of polystyrene (plastic). The polystyrene may have been treated in some way to promote cell attachment and growth, but in most cases, the surface is completely unfamiliar to the cells. In other cases, the surface may be coated with a single matrix protein (e.g., fibronectin or collagen) or a combination of certain proteins. These simple substrates ignore the complexity of the natural microenvironment and the key role of the microenvironment in normal cell function. The cells will immediately begin to respond to this strange environment in a way that is very different from when the cells are in their natural environment.

[0153] There are five main approaches currently used to address this problem of growing cells on plastic substrates:

[0154] 1. Ignoring the problem - Rather than trying to achieve a desired function that matches the expected function in vivo, multiple cell types can be tested in various culture media to find cells that exhibit the specific desired function in the absence of an appropriate matrix substrate. This approach is not complex, but it often fails to produce the desired results due to the complex interplay of variables and the breadth of interactions between cells and the extracellular matrix.

[0155] 2. Finding out the key components - Many academic labs and several companies have taken the approach of considering the tissue from which the cells were isolated and looking for unique elements in that tissue that may be important for cell function. The cells are then cultured on a simple substrate consisting of only one or a few matrix components. This approach often fails because the matrix is ​​naturally a very complex environment, containing more than one hundred different proteins in some cases. Cells respond just as strongly to signals they need but cannot receive as to signals they do not need but do receive.

[0156] 3. Shotgun method - protein gel such as MATRIGEL TM The use of genomic DNA takes a shotgun approach. A gel is created that contains many different matrix proteins in the hope that it will contain the binding motifs that are needed by many different cell types. This approach can fail by providing cues that push cells in a specific direction or by failing to provide all the cues that the cells expect.

[0157] 4. Tissue-derived matrices - This is a biomimetic approach that typically involves isolating the target tissue from a genetically similar animal, physically disrupting or chemically digesting the tissue to obtain a solution or homogenous suspension, and then coating the culture vessel with the deconstructed tissue. For example, one who wishes to culture satellite cells might collect muscle, homogenize the tissue, and then coat the culture vessel in the homogenized muscle before seeding the cells. This approach often fails for a few reasons. First, even within a specific tissue type, the stem / progenitor niche may be different from the rest of the tissue. Simply homogenizing muscle does not guarantee the creation of a suitable niche. Second, in addition to biochemical cues, the niche also includes structural and physical cues. Even if many / most biochemical cues are present in a tissue homogenate, the structure has been disrupted, so the cells may sense very different mechanical cues. Finally, the manufacturability of tissue-derived matrices depends on the availability of the tissue. This affects the total amount of cell culture possible and leads to batch-to-batch variability.

[0158] 5. Cell-derived matrix - Cells in culture can be induced to secrete a matrix in their culture vessel. This matrix is ​​the best approximation available for the in vivo niche and can be manufactured in vitro. Cells can be induced to form a matrix in vitro and then the cells can be subsequently removed from the matrix, for example by using a non-ionic detergent to preserve the structure and chemistry of the matrix. This approach has several key advantages. (1) The matrix structure can be recreated and not disturbed. (2) The matrix can be customized based on the target tissue / cell type. (3) The matrix can be specific for the stem cell and progenitor cell niche. (4) The matrix can be manufactured in large quantities.

[0159] Regarding cell-derived matrices, not all cell types can be effectively separated and amplified on any given cell-derived matrix. In fact, compared with other types of cells, pluripotent stem cells (PSCs) seem to have very different requirements for supportive growth substrates. It is known that specific cell types used to produce matrix will affect the composition of the matrix and thus the response of various cell types to the matrix (see Marinkovic, M. et al., One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior. Matrix Biol. 54–55, 426–441 (2016)). Previous work disclosed in US 8084023 describes the production and composition of extracellular matrices produced by bone marrow stroma or mesenchymal stem cells (see also Chen, X. et al., Extracellular Matrix Made by Bone Marrow Cells Facilitates Expansion of Marrow-Derived Mesenchymal Progenitor Cells and Prevents Their Differentiation into Osteoblasts. Journal of Bone and Mineral Research 22, 1943–1956 (2007) and Lai, Y. et al., Reconstitution of marrow-derived extracellular matrix ex vivo: a robust culture system for expanding large-scale highly functional human mesenchymal stem cells. Stem cells and development 19, 1095–107 (2010)). This bone marrow cell-derived matrix has been shown to support the expansion of other MSCs, but is ineffective for the attachment and growth of other types of stem cells, particularly induced pluripotent stem cells (iPSCs). iPSCs have shown greater potential to form cells and tissues from a much wider range of classes than MSCs. This is a particularly interesting challenge because the difficulty of growing confluent monolayers of iPSCs under standard culture conditions makes it impractical to generate cell-derived matrices from iPSCs.A major limitation of previous cell-derived matrices is that in order to make tissue-specific matrices (e.g., bone marrow matrices from bone marrow MSCs, adipose matrices from adipose MSCs, or endothelial matrices from hUVECs), it is necessary that the target cell population is already able to adhere to the starting substrate. The difficulty with iPSCs, embryonic stem (ES) cells, and many other cell types is that they do not readily adhere to simple substrates.

[0160] The present disclosure provides a solution to at least some of the above-mentioned limitations and defects in the field related to the cell-derived extracellular matrix (ECM) for separation, amplification and proliferation of supporting pluripotent stem cells (PSC), including but not limited to induced pluripotent stem cells (iPSC) and embryonic stem cells (ES). The solution is based on the use of an extracellular matrix derived from amniotic fluid cells. The use of unformed, easily adherent and highly proliferative perinatal cells found in amniotic fluid allows the production of an extracellular matrix (ECM) that unexpectedly supports the adhesion, separation, amplification and proliferation of these PSCs. It is impossible to achieve this technical achievement using the cell-derived ECM of the prior art.

[0161] The function of mammalian cells depends largely on the environment in which they are located, such as the extracellular matrix. They respond to signals present in their environment (positive signals) as well as signals that are needed but not present (negative signals). Uncommitted stem cells are likely to produce such a matrix that contains the niche motifs necessary to maintain stem cell viability and stem cell properties, but lacks many lineage-specific signals that more mature cells will secrete, which will drive stem cells toward a specific fate. Without being bound by theory, it is said that less mature cells, such as perinatal cells or perinatal stem cells, may produce ECMs different from ECMs previously disclosed in the art, such as ECMs derived from bone marrow stromal cells, and may allow for better separation and expansion / proliferation of stem cells such as pluripotent stem cells (PSCs) with higher potential than mesenchymal stem cells (MSCs). Mass spectrometry analysis shows that compared with previously known cell-derived ECMs, the amniotic fluid cell-derived ECM of the present invention contains matrix proteins found in all three germ layers and lacks specific proteins closely related to the osteogenic lineage. Moreover, the ECM disclosed herein contains specific motifs known to promote the adhesion and expansion of pluripotent cells, such as laminin.

[0162] Pluripotent stem cells (PSC) can self-renew and differentiate into any of the three germ layers: ectoderm, endoderm and mesoderm, from which all tissues and organs develop. Embryonic stem cells (ES) are the only known natural pluripotent stem cells. Induced pluripotent stem cells (iPSC) are also PSC. iPSCs are derived from cells or adult cells that are usually taken from adult tissues and are reprogrammed to the level of embryonic stem cells. Methods for producing iPSCs are known in the art.

[0163] The method of expanding / proliferating pluripotent stem cells (PSCs) comprises obtaining PSCs and culturing them in the presence of an amniotic fluid cell-derived ECM of the present invention. The PSCs may be iPSCs or ES cells. Any seeding density that allows the cells to form a confluent monolayer immediately or after a period of time in culture may be used. In some embodiments, the seeding density is about 10 cells / cm 2 About 100,000 cells / cm 2 , or about 100 cells / cm 2 About 75,000 cells / cm 2 , or about 500 cells / cm 2 About 50,000 cells / cm 2 , or about 500 cells / cm 2 About 10,000 cells / cm 2 , or about 500 cells / cm 2 About 5000 cells / cm 2 , or about 500 cells / cm 2 About 2500 cells / cm 2 , or about 1000 cells / cm 2 About 25,000 cells / cm 2 , or about 2000 cells / cm 2 About 10,000 cells / cm 2 , or about 3000 cells / cm 2 About 5000 cells / cm 2 .

[0164] In some embodiments, the PSCs are maintained in an undifferentiated state and retain their stem cell properties. Cell culture techniques suitable for the proliferation of PSCs in culture are known in the art. Suitable commercially available culture media for stem cell proliferation include, but are not limited to, StemMACS available from Miltenyl Biotec. TMiPS-Brew XF. In some embodiments, Rock inhibitor is not used. After the cells begin to approach fusion (e.g., as determined by brightfield microscopy), cells can be passaged manually by cutting large colonies into smaller colonies and then re-seeding them by physically removing them from the culture dish and placing them on a new plate of amniotic fluid cell-derived ECM. This procedure can be repeated indefinitely. In some embodiments, a method for proliferating pluripotent stem cells (PSCs) in culture is disclosed, the method comprising culturing PSCs in the presence of a cell-derived extracellular matrix (ECM) in a culture medium, thereby proliferating the PSCs, wherein the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid.

[0165] The method for amplifying / proliferating PSC described above is also applicable to the use of amplifying / proliferating PSC in culture in the presence of ECM from other perinatal cell sources. In some embodiments, a method for proliferating pluripotent stem cells (PSC) in culture is disclosed, the method comprising culturing PSC in the presence of a cell-derived extracellular matrix (ECM) in a culture medium, thereby proliferating PSC, wherein the cell-derived ECM is derived in vitro from cells isolated from umbilical cord or placental tissue. In some embodiments, the cells isolated from the umbilical cord are from umbilical cord blood and / or Wharton's jelly. In other embodiments, the cells isolated from placental tissue are from membranes (amniotic membrane and / or chorionic membrane), villi and / or blood.

[0166] Example

[0167] The following examples are included to illustrate certain non-limiting aspects of the present invention. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the applicant has discovered to work well in the practice of the present invention. However, in light of this disclosure, it will be appreciated by those skilled in the art that many changes may be made to the disclosed specific embodiments and still similar or similar results may be obtained without departing from the spirit and scope of the present invention.

[0168] Example 1 - Production of amniotic fluid cell-derived ECM (AFC-ECM)

[0169] Four amniocyte-derived ECMs (matrix A, matrix B, matrix C, and matrix D) were prepared using the following procedure: cells aseptically isolated from amniotic fluid collected from term deliveries (>37 weeks gestational age) of 4 donors were seeded onto tissue culture treated flasks coated with fibronectin and cultured in complete medium at 37°C, 5% CO2, and 90% RH in an incubator. The complete medium was alpha minimum essential medium (aMEM) plus 2 mM L-glutamine plus antibiotic-antimycotic plus 15% fetal bovine serum.

[0170] On day 3 to 4, aspirate half of the complete medium from the flask and replace with half of new complete medium. Return the flask to the incubator under the same conditions as above.

[0171] On day 7 to 8, the complete medium was aspirated from the culture flask and supplemented with induction medium. The flask was returned to the incubator under the same conditions as above. The induction medium was complete medium plus 50 mM L-ascorbic acid.

[0172] On day 10 to day 11, the induction medium was aspirated from the culture flask, and the ECM formed in the flask was washed once with phosphate buffered saline (PBS). Then the PBS was aspirated from the flask. Extraction buffer was added to the flask and incubated at room temperature for 7 to 10 minutes to decellularize each ECM, and then the extraction buffer was aspirated from the flask. The extraction buffer was PBS containing 0.5% (vol / vol) TRITON-X100 and 20 mM ammonium hydroxide (NH4OH).

[0173] Each decellularized ECM in the flask was washed three times with PBS and then once with sterile water, and then the sterile water was aspirated from the flask.The four decellularized ECMs in the flask were allowed to dry at room temperature and then stored at 4°C.

[0174] Figure 1 A photomicrograph of a bright field image of amniocyte-derived ECM (matrix B) at 100x magnification using a 10x objective is shown in FIG. Figure 2 Atomic force micrographs of three representative 40×40 μm sections of amniocyte-derived ECM (matrix B) and bone marrow-derived ECM are shown in Figure 1, showing morphology, adhesion, and stiffness. Bone marrow-derived and amniocyte-derived ECM are structurally and physically different. Quantification of adhesion and stiffness (elastic modulus) of bone marrow-derived and amniocyte-derived ECMs showed that bone marrow ECMs were 10-fold stiffer and 3-fold less adherent relative to amniotic fluid ECMs, as shown in Figure 2. Figure 3 a (adhesion) and Figure 3 b (stiffness) in a scatter plot, where BM = bone marrow-derived ECM, AD = amniotic fluid-derived ECM (matrix B). Each point represents an independent measurement point.

[0175] Example 2 - Composition of amniocyte-derived ECM

[0176] The composition of each amniocyte-derived ECM produced in Example 1 was determined by mass spectrometry. The components are listed in Table 2 along with their spectral counts and molecular weights.

[0177] Table 2

[0178] Components of amniotic fluid cell-derived ECM (AFC-ECM)

[0179]

[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239] Example 3 - Proliferation of iPSCs on amniotic fluid cell-derived ECM

[0240] Induced pluripotent stem cells (iPSCs) were propagated on the amniocyte-derived ECM (matrix B) from Example 1 using the following procedure: Commercially available cryopreserved iPSCs were thawed using a 37°C water bath. The cell suspension was diluted into a commercially available medium for stem cell proliferation (Miltenyi Biotec MACS iPS Brew) and plated at approximately 1000 cells / cm in a 6-well plate with 2 mL of medium / well. 2 Seed onto ECM. No Rock inhibitor was used. On day 1, the entire volume of medium was gently aspirated from the cells in culture and replaced with fresh medium. The entire medium was replaced with fresh medium every 24 hours. Once the cells began to approach confluence (as determined by bright field microscopy), cells were passaged manually by cutting large colonies into approximately 100 smaller colonies using a sterile needle, which were then physically removed from the culture dish with a sterile needle and placed on a new plate of ECM for reseeding. This procedure can be repeated indefinitely.

[0241] Figure 4 , photomicrographs showing day 0 and day 2 of iPSC culture on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM are shown.

[0242] Figure 5 Graphs showing iPSC colony growth curves of iPSCs cultured in the presence of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0243] like Figure 4 and Figure 5As can be seen in the Figure 3, iPSCs proliferated in culture in the presence of amniotic fluid cell-derived ECM, while iPSCs cultured in the presence of bone marrow cell-derived ECM did not grow.

[0244] Example 4 - Preparation of cell constructs of mature cardiomyocytes on AFC-ECM and maturation of immature hiPSC-CMs on AFC-ECM

[0245] Cell constructs comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived from in vitro amniotic fluid-derived cells (AFC-ECM) were prepared using the following method.

[0246] AFC-ECMs were prepared in 96-well plates using methods as outlined in Example 1 (without using silicone inserts). Immature hiPSC-CMs ( Immature hiPSC-CMs were seeded at a density of 50,000 cells per well (96-well plate) or 200,000 cells per well (6-well plate) and cultured in RPMI medium for 7 days to form a confluent monolayer of mature cardiomyocytes on AFC-ECM, thereby forming a cell construct of a mature cardiomyocyte monolayer on AFC-ECM.

[0247] During the 7-day period, immature hiPSC-CMs were observed to mature into the morphology and arrangement of mature native adult cardiomyocytes, characterized by rod-shaped cells with a unique sarcomere structure. Immature hiPSC-CMs (without silicone inserts) were grown in a similar manner on standard Matrigel TM The results are shown in Figure 8. Figures 6 to 21 Micrographs of cardiomyocytes on different ECMs.

[0248] like Figures 6 to 14 As can be seen in the figure, the cardiomyocytes on AFC-ECM are mature rod-shaped cells with a unique sarcomere structure similar to that of natural adult cardiomyocytes. The morphology and sarcomere structure of the mature cardiomyocytes on AFC-ECM can be clearly seen by the presence of rod-shaped cells, which have a unique structure composed of Fig.14The arrows in the micrographs of FIG. 4 show the presence of fiber tracks on the AFC-ECM and also show that the monolayer of mature cardiomyocytes is aligned with the AFC-ECM, which is very similar to the features found in native adult cardiomyocytes and native myocardial tissue. In contrast, as Figures 15 to 19 and Figure 20 to Figure 21 As can be seen in the standard Matrigel TM The cardiomyocytes on ECM and BM-ECM resemble fetal-like cardiomyocytes and do not have the characteristics of native adult cardiomyocytes or native myocardial tissue, respectively.

[0249] Therefore, immature hiPSC-CMs cultured on AFC-ECM were more sensitive than those cultured on standard Matrigel. TM Immature hiPSC-CMs cultured on ECM or native cell-derived ECM from bone marrow cells reached a higher state of maturity. It is clear that hiPSC-CM morphology is affected differently by different ECMs.

[0250] Example 5 - High-throughput cardiotoxicity screening of drugs using cell constructs of mature cardiomyocytes on AFC-ECM

[0251] As described in Example 4, a 96-well plate (without silicone inserts) was used to prepare a monolayer of mature cardiomyocytes on AFC-ECM. After a 7-day maturation process, the electrophysiology of each well was observed using a plate reader using the following high-throughput screening method. FluoVolt in Hanks balanced salt solution was added. TM Dye was loaded into each well. A high-resolution spatiotemporal CCD camera (SciMeasure DaVinci camera) coupled with a light-emitting diode (LED) was used, such as Fig. 22 As shown in the schematic diagram in . The camera and lens combination is designed to allow simultaneous visualization of all wells of a 96-well plate with sufficient resolution to observe action potentials and calcium wave propagation. Each plate is centered under the camera system, the lighting is turned on, the camera acquisition is started and electrophysiological activity is recorded. The experiment was carried out at about 37°C. Spontaneous activity was recorded for at least 10 seconds and the images were stored on a computer. After obtaining a baseline reading, 500nM of the drug E4031 (hERG channel blocker) was added to each well. The images were analyzed and image analysis software was used to quantify action potential duration, conduction velocity, beat rate, and activation pattern. For comparison purposes, standard Matrigel was used in a 96-well plate (without using silicone inserts). TMImmature hiPSC-CMs were cultured on ECM and bone marrow-derived ECM (BM-ECM) as described in Example 4 and analyzed at 500 nM of the drug E4031 (hERG channel blocker) in a similar manner to the AFC-ECM study. The results of the E4031 assay are shown in Fig.23 and Fig.24 In. Fig.23 As can be seen, the records are from Matrigel TM Spontaneous action potential recordings from cardiomyocytes grown on ECM and BM-ECM revealed that the drug E-4031 caused only a prolongation of the action potential duration (APD); however, in mature cardiomyocytes grown on AFC-ECM, the drug E-4031 caused APD prolongation plus rotors (TdP-like arrhythmias), a pattern of arrhythmic activation consistent with that known to occur in human cases of TdP. Thus, all three ECMs produced cardiomyocyte monolayers that responded with the predicted APD prolongation, but only AFC-ECM produced cardiomyocyte monolayers that displayed a transition from APD prolongation to the tachyarrhythmias characteristic of TdP. Fig.24 As can be seen in the figure, 100% of the cardiomyocytes on AFC-ECM responded to the drug E4031 with rotors (TdP-like arrhythmias).

[0252] In additional studies, the drugs domperidone (3 μM), disopyramide (100 μM), azimilide (10 μM), D,1 sotalol (100 μM), ibutilide (0.10 μM), and bepridil (10 μM) were tested in 96-well plates using the plate reader method described above with mature cardiomyocytes on AFC-ECM prepared as in Example 4 (without silicone inserts). The results of the testing are shown in Fig.25 As can be seen from the results, various types of arrhythmias were induced by various drugs, namely tachyarrhythmias (TA), static (Q), early afterdepolarizations (EAD), as indicated by symbols in the records. These are the range of arrhythmia types observed as a response to drugs classified by the FDA as having a high risk of causing fatal arrhythmias in patients with TdP.

[0253] Various drugs shown in Tables 3 and 4 below were also tested using mature cardiomyocytes on AFC-ECM as prepared in Example 4 (without silicone inserts) in 96-well plates using the plate reader method described above, and the observations for any arrhythmias detected and APD90 prolongation at 10 times the effective therapeutic plasma concentration (ETPC) are indicated in symbols. These drugs were selected from the CiPA initiative's list of compounds for validation and testing of CiPA and were classified as having a high risk, medium risk, or low risk of causing fatal arrhythmias (TdP) in patients. A complete list of CiPA compounds can be found at http: / / cipaproject.org / wp-content / uploads / sites / 24 / 2016 / 05 / CiPA-Compounds.pdf.

[0254] Table 3

[0255]

[0256]

[0257] Table 4

[0258]

[0259] Further analysis of the data from the drugs tested in Tables 3 and 4 is presented in Figure 26 to Figure 29 Shown in. Fig.26 The total number of arrhythmias observed at any dose for each drug compound is shown graphically. Fig. 27 The relative incidence of arrhythmias for each drug at specific clinically relevant doses, 10 times the effective therapeutic plasma concentration (ETPC), is graphically presented.

[0260] Fig.28 The action potential triangulation (APD90-APD30) is shown graphically for each drug in terms of time (milliseconds). Triangulation is defined as the repolarization time from APD30 to APD90. Action potential triangulation (APD90-APD30) is used as a predictor of drug-induced arrhythmogenicity. Fig.29 Action potential triangulation expressed as time (milliseconds) for some drugs is shown, comparing cardiomyocytes grown on AFC-ECM (SBS-AF matrix) versus on Matrigel TM Cardiomyocyte assay performance on ECM.

[0261] Fig.30 The maximum drug-induced action potential triangulation for the listed drugs is shown graphically, compared to the data from Cellular Dynamics. hiPSC-CMs (open circles) and Ncardia Cardiomyocyte performance of hiPSC-CM (black circle) at any concentration of the listed drugs. This figure is from the publication Blinova et al, International Multisite Study of Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Drug Proarrhythmic Potential Assessment, 2018, Cell Reports 24, 3582-3592. Fig.28 In contrast, Fig.30 In the dataset shown in Figure 2, there is little stratification between high-risk and medium-risk compounds. TM Compared to WT ECM, the AFC-ECM of the present disclosure provides for the generation of more mature cardiomyocytes with more realistic functionalities and drug responsiveness.

[0262] Example 6 - Observation of cell constructs of mature cardiomyocytes on AFC-ECM

[0263] Cell constructs of mature cardiomyocytes on AFC-ECM were prepared according to the procedure described above in Example 4, with the following modifications:

[0264] AFC-ECM was deposited onto Thermanox coverslips so that cells could be immunostained and imaged using laser scanning confocal microscopy (Nikon A1R).

[0265] Matrigel TM ECM was applied to a separate group of Thermanox coverslips for comparison.

[0266] Cells from Cellular Dynamics International-FUJI ( Cardiomyocytes (CMCs) were seeded as monolayers on these Thermanox coverslips coated with each ECM at a density of 200,000 cells per well in 6-well plates.

[0267] After 7 days of incubation in cell culture medium, cells were fixed in 3% paraformaldehyde and processed for immunocytochemistry using commercially available primary antibodies to determine cellular expression and localization in hiPSC-CMs. The following primary antibodies against key cardiac myofilament proteins were used: troponin I, α-actinin, cardiac troponin T (cTnT), cardiac troponin I (cTnI), and N-cadherin. Commercially available fluorescently labeled secondary antibodies were used for detection. DAPI (4',6-diamidino-2-phenylindole) fluorescent stain was used to label cell nuclei.

[0268] All procedures for cell labeling and visualization were as described in the following references incorporated herein by reference. Herron, TJ et al. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function. Circulation: Arrhythmia and Electrophysiology 9 (2016). da Rocha, MA et al. Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived cardiomyocytes. J. Mol. Cell. Cardiol. 99, 197-206 (2016). da Rocha, AM et al. hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen. Sci. Rep. 7, 13834 (2017).

[0269] Cell shape was quantified using fluorescent images analyzed in NIS Elements software. Cell circularity was quantified using an established mathematical formula using cell area and perimeter; circularity index = 4π*area / perimeter 2 .

[0270] In some cases, MitoTracker TM Mitochondria were stained with Red CMXRos (Thermo Fisher).

[0271] Results: Consistent with the results seen above in Example 4, data using fluorescent labeling and imaging indicated that AFC-ECM promoted rapid (7 days) maturation of hiPSC-CMs and showed that, in Matrigel TM hiPSC-CMs cultured on ECM had a round shape and disorganized sarcomeres, whereas hiPSC-CMs from the same batch cultured on AFC-ECM (isogenic coparator) had a rod shape with compact / tightly organized sarcomeres and myofilaments.

[0272] Fig.31 The micrograph shows the TM hiPSC-CMs cultured on ECM and AFC-ECM were immunofluorescently stained for troponin I and DAPI was used to label cell nuclei. Fig.32 The micrograph shows the TM hiPSC-CMs cultured on ECM and AFC-ECM, with immunofluorescence staining for α-actinin and DAPI used to label cell nuclei. Fig.33 The micrograph shows the TM hiPSC-CMs cultured on ECM and AFC-ECM, where cTnT and N-cadherin were immunofluorescently stained, and DAPI was used to label cell nuclei. Figure 31 to Figure 33 As can be seen in Matrigel TM hiPSC-CMs cultured on ECM showed rounded shapes with disorganized sarcomeres, whereas hiPSC-CMs from the same batch cultured on AFC-ECM (isogenic coparator) showed rod-like shapes with compact / tightly organized sarcomeres and myofilaments. Figure 31 to Figure 33 Some examples of cells that are round or rod-shaped are identified with arrows and some examples of sarcomeres are identified with arrowheads. Fig.34 The micrograph shows the TM Single hiPSC-CM cells cultured on ECM and AFC-ECM, with cTnT immunofluorescence staining, DAPI used to label cell nuclei, and shown on Matrigel TM Cells cultured on ECM were round, whereas cells cultured on AFC-ECM were rod-shaped. Fig.35 The micrograph shows the TMSingle hiPSC-CM cells cultured on ECM and AFC-ECM, with immunofluorescence staining for α-actinin, DAPI used to label nuclei, and shown on Matrigel TM Cells cultured on ECM were round, whereas cells cultured on AFC-ECM were rod-shaped. Fig.36 The diagram in Fig.35 The cell roundness comparison of individual cells is shown in Figure 2 and shows that the cell roundness of the cells in Matrigel TM Cells cultured on ECM had a higher circularity index, indicating that they were rounder.

[0273] Fig.37 The micrograph shows the TM hiPSC-CMs cultured on ECM and AFC-ECM were immunofluorescently stained for cTnI expression, with DAPI used to label cell nuclei. Fig.38 Shown in Matrigel TM Western blotting of hiPSC-CMs on ECM and AFC-ECM for cTnI expression and GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Fig.39 The chart shows Fig.38 Western blot analysis showing Matrigel TM cTnI expression relative to GAPDH in hiPSC-CMs on ECM and AFC-ECM. TM The cTnI expression / GAPDH ratio of hiPSC-CMs on ECM was high, indicating that hiPSC-CMs on AFC-ECM were more sensitive to cTnI than those on Matrigel. TM hiPSC-CMs on ECM expressed cTnI more robustly.

[0274] Fig.40 The micrograph in Figure 2 shows Matrigel stained with MitoTracker Red for mitochondria. TM ECM versus hiPSC-CM on AFC-ECM and showed that cells on AFC-ECM had more mitochondria and that the mitochondria had a more polarized inner membrane potential, as evidenced by the larger red signal. Fig.41 The diagram shows Matrigel TM MitoTraker of hiPSC-CM on ECM vs AFC-ECM TM Red fluorescence intensity / cardiomyocyte, and showed that hiPSC-CMs on AFC-ECM had higher MitoTrakerTM Red fluorescence intensity indicates that these cells are larger than Matrigel TM hiPSC-CMs on ECM had more mitochondria and the mitochondria had more polarized inner membrane potential.

[0275] Fig.42 The micrograph (transmitted light) shows the Matrigel coated on a microelectrode array (MEA) plate. TM hiPSC-CMs cultured on ECM and AFC-ECM. Fig.42 As can be seen in Matrigel TM hiPSC-CMs cultured on ECM were round in shape whereas hiPSC-CMs cultured on AFC-ECM were rod-like in shape.

Claims

1. A method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs), wherein the immature cardiomyocytes exhibit single nuclei; (b) providing an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from human amniotic fluid of greater than 37 weeks of gestation, wherein the cells isolated from the amniotic fluid include fetal cells from the amniotic membrane, skin, and digestive tract, respiratory tract, and urogenital tract, and wherein the AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV) or an isoform of laminin, an isoform of collagen alpha-1 (XVIII), an isoform of basement membrane-specific heparan sulfate proteoglycan core protein, an isoform of agrin, an isoform of vimentin, and an isoform of collagen alpha-2 (IV); (c) Immature hiPSC-CMs were seeded on AFC-ECM; and (d) culturing immature hiPSC-CMs with AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs to form mature cardiomyocytes; Mature cardiomyocytes are characterized by the presence of two nuclei.

2. The method of claim 1, wherein the mature cardiomyocytes are formed as a monolayer on the AFC-ECM, thereby forming a cell construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, wherein the mature cardiomyocytes are arranged on the AFC-ECM.

3. The method of claim 1, wherein the immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.

1.

4. A cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived from cells isolated in vitro from human amniotic fluid greater than 37 weeks of gestation, wherein the cells isolated from the amniotic fluid include fetal cells from the amniotic membrane, skin, and digestive tract, respiratory tract, and urogenital tract, and wherein the mature cardiomyocytes are immature cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CM) cultured in the AFC-ECM, wherein the immature cardiomyocytes exhibit a single nucleus, and wherein AFC-ECM contains laminin, collagen α-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin and collagen α-2 (IV) or isoforms of laminin, isoforms of collagen α-1 (XVIII), isoforms of basement membrane-specific heparan sulfate proteoglycan core protein, isoforms of agrin, isoforms of vimentin and isoforms of collagen α-2 (IV), and wherein mature cardiomyocytes are characterized by exhibiting two nuclei. The cell construct according to claim 4 , wherein a monolayer of mature cardiomyocytes is arranged on the AFC-ECM.

6. The cell construct according to claim 4 or 5, wherein fiber tracks are present on the construct. 7 . The cell construct according to claim 4 , wherein the isoform of collagen α-1 (XVIII) is isoform 2, and / or wherein the isoform of agrin is isoform 6. The cell construct according to claim 7 , wherein the AFC-ECM further comprises fibronectin and / or its isoforms.

9. The cell construct according to claim 4 or 5, wherein the AFC-ECM does not contain decorin, perlecan and / or collagen (III).

10. A method for preparing a cell construct of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from human amniotic fluid greater than 37 weeks of gestation, the method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs), wherein the immature cardiomyocytes exhibit a single nucleus, (b) providing an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from human amniotic fluid of greater than 37 weeks of gestation, wherein the cells isolated from the amniotic fluid include fetal cells from the amniotic membrane, skin, and digestive tract, respiratory tract, and urogenital tract, and wherein the AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV) or an isoform of laminin, an isoform of collagen alpha-1 (XVIII), an isoform of basement membrane-specific heparan sulfate proteoglycan core protein, an isoform of agrin, an isoform of vimentin, and an isoform of collagen alpha-2 (IV); (c) Immature hiPSC-CMs were seeded on AFC-ECM; (d) culturing the seeded immature hiPSC-CMs on the AFC-ECM in a culture medium to induce the immature hiPSC-CMs to mature into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct; Mature cardiomyocytes are characterized by the presence of two nuclei. The method of claim 10 , wherein a monolayer of mature cardiomyocytes is arranged on the AFC-ECM.

12. The method of claim 10 or 11, wherein a fiber track is present on the construct.

13. A method for determining the cardiotoxic and / or arrhythmogenic effects of a drug compound in vitro, the method comprising contacting the drug compound with a monolayer of mature cardiomyocytes of the cell construct according to any one of claims 4 to 9, and observing electrophysiological changes in the mature cardiomyocytes to confirm whether the drug compound has cardiotoxic and / or arrhythmogenic effects on the mature cardiomyocytes.

14. The method of claim 13, wherein the electrophysiological change in the mature cardiomyocytes is a prolongation of action potential duration (APD), and wherein the prolongation of APD confirms that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

15. The method of claim 13, wherein the electrophysiological change of the mature cardiomyocytes is early afterdepolarization (EAD), and wherein the early afterdepolarization (EAD) confirms that the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes.

16. The method of claim 13, wherein the change in electrophysiology of mature cardiomyocytes is delayed after depolarization (DAD), and wherein delayed after depolarization (DAD) confirms that the drug compound has cardiotoxic and / or arrhythmogenic effects on mature cardiomyocytes.

17. The method of claim 13, wherein the change in electrophysiology of mature cardiomyocytes is action potential duration (APD) plus rotor, and wherein the prolongation of APD plus rotor confirms that the drug compound has cardiotoxic and / or arrhythmogenic effects on mature cardiomyocytes.

18. The method of claim 13, wherein the electrophysiological changes in the mature cardiomyocytes are arrhythmias, and wherein the arrhythmias demonstrate that the drug compound has a cardiotoxic and / or arrhythmogenic effect on the mature cardiomyocytes.

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