Methods for the maturation of cardiomyocytes on amniotic fluid cell-derived ECM, cellular constructs, and uses for cardiotoxicity and proarrhythmic screening of drug compounds

Abstract

To provide methods for forming mature cardiomyocytes.SOLUTION: Disclosed herein are methods of using a cell-derived extracellular matrix derived in-vitro from cells isolated from amniotic fluid (AFC-ECM) for the maturation of immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs) in culture, forming mature cardiomyocytes. Also disclosed herein is a cell construct comprising a monolayer of these mature cardiomyocytes on an AFC-ECM useful for cardiotoxicity and / or proarrhythmic screening assays of drug compounds. Also disclosed herein are methods for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro using such cell constructs.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 808,690, filed February 21, 2019, which is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the use of cellular constructs of human stem cell-derived cardiomyocytes on a cell-derived extracellular matrix, methods of making the constructs, and methods for cardiotoxicity and proarrhythmic screening assays of drug compounds using the constructs. [Background technology]

[0003] Cardiotoxicity, or the perceived potential for cardiotoxicity, is a major cause of toxicity-related drug attrition during new drug investigation and selection. Cardiac safety testing of new chemical entities that are potential lead drug candidates is an important aspect of the drug discovery and development pipeline. A large number of cardiac side effects of cardiac and non-cardiac drugs are caused by drug interactions with one or more cardiac ion channels. Cardiac ion channels regulate cellular excitability, contractility and overall cardiac performance, and alterations in cardiac ion channel function can lead to sudden cardiac death. This contributed to the publication of drug candidate testing guidelines from The International Conference on Harmonization (ICH).

[0004] Current preclinical drug candidate testing guidelines from IHC (ICH S7A and S7B-Pharmacology Studies) are based on genetically modified xenogeneic cells and in It is increasingly recognized that these studies, e.g., hERG assays and QT prolongation studies, do not accurately predict cardiotoxicity and proarrhythmic risk in humans. Since 2005, the cardiac safety of a drug compound has been determined almost exclusively by its effect or potential effect on the QT interval or action potential duration (APD) of the electrocardiogram (ECG) and its potential to lead to a life-threatening arrhythmia called torsades de pointes (TdP). However, QT prolongation is not an ideal indicator of TdP, since drugs that prolong the QT interval do not necessarily cause TdP. It is now recognized that QT prolongation parameters are only surrogate markers of proarrhythmia. Data from preclinical and clinical trials indicate that there is no consistent relationship between the magnitude of QT prolongation and the risk of developing a fatal arrhythmia such as TdP.

[0005] Thus, the US Food and Drug Administration (FDA) and other stakeholders in drug discovery have called for an evolution in preclinical cardiotoxicity testing. The proposed new paradigm is called the Comprehensive in-vitro Proarrhythmia Assay (CiPA). An integral part of the CiPA Initiative (http: / / cipaproject.org / ) involves the incorporation of data collected from human stem cell-derived cardiomyocytes for cardiotoxicity and proarrhythmia assays. The overall goal of these new proposed guidelines is to provide a more accurate and comprehensive mechanism-based assessment of proarrhythmic potential that will more accurately evaluate the risk of new drugs. Reviewing the proposed CiPA Initiative guidelines, the FDA prescribes two advances that must be made before human cardiomyocytes can be incorporated into the new initiative. First, the growth and maturation state of human stem cell-derived cardiomyocytes needs to evolve to more closely resemble the structure and function of adult human cardiomyocytes. Second, reliable high-throughput screening platforms that use these cells need to be developed.

[0006] There are three general types of systems currently utilized to assess 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 are most commonly used in very early research studies to assess the electrophysiology of individual cells. These devices provide accurate and sensitive ionic current measurements, but cannot be used for in vitro cell systems that more closely mimic cell-cell interactions in cardiac tissue. MEA systems contain electrodes that are incorporated into cell culture wells to allow measurement of current across the well. While allowing for high-throughput analysis and the ability to measure impedance in in vitro cell systems, these systems have low spatial resolution and do not provide data on action potential shape, hindering direct visualization of cells and the ability to assess 3-D culture systems that more closely mimic cardiac tissue architecture. VSD systems are of increasing interest because they address many of the shortcomings of current techniques mentioned above, with features including: 1) enabling high throughput analysis; 2) providing high spatial resolution; 3) allowing visualization of impulse propagation across the culture dish; and 4) allowing modification of culture conditions, including the addition of extracellular matrix, leading to a more natural testing environment.

[0007] The use of cardiomyocytes derived from human stem cells, e.g., induced pluripotent stem cells, has had limited success to date in cardiotoxicity and proarrhythmic assay screening. Cardiomyocytes derived from induced human pluripotent stem cells (hiPSC-CMs) are commercially available and can be purchased from several companies in cryopreserved vials that can be thawed and plated as monolayers. These hiPSC-CMs can be created in vitro at large scale. Also, hiPSC-CMs can be derived from patient-specific hiPSCs for specific individuals. However, there are still hurdles to overcome to make hiPSC-CMs a meaningful part of the emerging CiPA Initiative paradigm. 1) Progress needs to be made in the structural and functional maturation of hiPSC-CMs. Notably, Kir2.1 potassium channels are absent in currently available hiPSC-CMs, and sodium channel expression is low. Currently available hiPSC-CMs are functionally and structurally very immature. The majority of currently used hiPSC-CM-based proarrhythmic screening assays rely on immature fetal-like cells that do not resemble either the structure or function of adult cardiomyocytes. 2) Electrical pacing of hiPSC-CM monolayers in a high-throughput electrophysiological screening platform is required. The majority of current hiPSC-CM-based proarrhythmic screens rely exclusively 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) cause fatal TdP arrhythmias. Although certain maturation states of immature hiPSC-CMs have been achieved by using Matrigel™ ECM and bone marrow cell-derived ECM in culture, the use of these hiPSC-CMs in proarrhythmic screening assays has not produced consistent results, especially in the observation of arrhythmic activation patterns consistent with those known to occur in the case of TdP in humans, between low-risk and high-risk drugs.

[0008] Therefore, advanced materials and new methods are needed for accurate assessment and screening of the cardiac safety liability of drug compounds for accurate, reliable and efficient development of new drug candidates. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure provides a solution to at least some of the above-mentioned limitations and deficiencies in the art related to the assessment and screening of cardiac safety liability of drug candidates. The solution is premised on the discovery of an extracellular matrix derived from cells isolated from amniotic fluid (AFC-ECM) that can be used for maturation of human stem cell-derived cardiomyocytes. These mature cardiomyocytes can then be used in cell constructs with the AFC-ECM for cardiotoxicity and proarrhythmic testing of drugs.

[0010] In one aspect, a method is disclosed for maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, 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) contacting the immature hiPSC-CM with the AFC-ECM; and (d) culturing the immature hiPSC-CM with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM, thereby forming mature cardiomyocytes, wherein the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, the mature cardiomyocytes have a morphology similar or the same as the morphology shown in any one of Figures 6-14. In some embodiments, the immature hiPSC-CMs are plated on the AFC-ECM. In some embodiments, mature cardiomyocytes form a monolayer on the AFC-ECM, thereby forming a cell construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, and mature cardiomyocytes are aligned on the AFC-ECM. In some embodiments, immature hiPSC-CMs do not express the inwardly rectifying potassium channel Kir2.1. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with distinct sarcomere structures. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a circular shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (binuclear).

[0011] In another aspect, a cell construct is disclosed that includes a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid, where the mature cardiomyocytes are human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) cultured in the AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, the mature cardiomyocytes have a morphology similar or the same as that shown in any one of Figures 6-14. In some aspects, the mature cardiomyocytes include and / or express the inwardly rectifying Kir2.1 potassium channel. In certain aspects, mature cardiomyocytes can be matured from immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs) in culture on AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomeric structures (striped appearance) similar to adult human cardiac tissue. In some embodiments, a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks (fiber track) 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 their isoforms. 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 comprise decorin, perlecan, and / or collagen (III). In some embodiments, the immature hiPSC-CM does not comprise rod-shaped cells with distinct sarcomere structures. In some embodiments, the immature hiPSC-CM can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a round shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (bilukaryotic).

[0012] In another aspect, a method is disclosed for creating a cellular construct of mature cardiomyocytes on an extracellular matrix in vitro derived from cells isolated from amniotic fluid (AFC-ECM), comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix in vitro derived from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CM on the AFC-ECM; and (d) culturing the plated immature hiPSC-CM on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, the mature cardiomyocytes have a morphology similar or the same as the morphology shown in any one of Figures 6-14. In some embodiments, a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the cell construct. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with distinct sarcomere structures. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a circular shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (binuclear).

[0013] In another aspect, a method for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro is disclosed, comprising contacting the drug compound with a mature cardiomyocyte of any one of the cell constructs disclosed throughout this specification, and observing changes in the electrophysiology of the mature cardiomyocyte to confirm whether the drug compound has a cardiotoxicity and / or proarrhythmic effect on the mature cardiomyocyte. The changes in the electrophysiology of the mature cardiomyocyte confirm that the drug compound has a cardiotoxicity and / or proarrhythmic effect on the mature cardiomyocyte. The changes in the electrophysiology of the mature cardiomyocyte may include, but are not limited to, APD prolongation, APD prolongation plus rotor, and / or various types of arrhythmias, such as tachyarrhythmia (TA), quiescence (Q), delayed afterdepolarization (DAD), and / or early afterdepolarization (EAD). The observations may also include cell viability, cell density, and / or cell morphology. In some embodiments, the changes in the electrophysiology of the mature cardiomyocyte are prolongation of action potential duration (APD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is early after-depolarization (EAD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is delayed after-depolarization (DAD). In some embodiments, the change in the electrophysiology of mature cardiomyocytes is action potential duration prolongation (APD prolongation) plus rotor. In some embodiments, the change in the electrophysiology of mature cardiomyocytes is arrhythmia.In some aspects, the cell construct can be prepared by a process including: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CM on the AFC-ECM; and (d) culturing the plated immature hiPSC-CM on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into mature cardiomyocytes to form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, the mature cardiomyocytes have a morphology similar or the same as the morphology shown in any one of Figures 6-14. In one embodiment, the immature hiPSC-CM does not express the inwardly rectifying potassium channel Kir2.1. In another embodiment, a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In another embodiment, fiber tracks are present on the cell construct. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with distinct sarcomere structures. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a round shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (binuclear).

[0014] The following embodiments 1 to 20 are also disclosed in relation to the present invention: Embodiment 1 is a method for maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (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, thereby forming mature cardiomyocytes. wherein mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. Embodiment 2 is the method of embodiment 1, wherein immature hiPSC-CMs are plated on AFC-ECM. Embodiment 3 is a method according to any one of embodiments 1 or 2, wherein mature cardiomyocytes form a monolayer on the AFC-ECM, thereby forming a cellular construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, and the mature cardiomyocytes are aligned on the AFC-ECM. Embodiment 4 is the method according to any one of Embodiments 1 to 3, wherein the immature hiPSC-CMs do not express the inward rectifying potassium channel Kir2.1. Embodiment 5 is a cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), the mature cardiomyocytes being human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) cultured in the AFC-ECM, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. Embodiment 6 is a cell construct according to embodiment 5, wherein a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. Embodiment 7 is a cell construct according to any one of embodiments 5 or 6, wherein fiber tracks are present on the construct. Embodiment 8 is a cell construct described in 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 their isoforms. Embodiment 9 is a cell construct according to embodiment 8, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2 and / or the isoform of agrin is isoform 6. Embodiment 10 is a cell construct according to any one of embodiments 8 or 9, wherein the AFC-ECM further comprises fibronectin and / or its isoforms. Embodiment 11 is a cell construct according to any one of embodiments 5 to 10, wherein the AFC-ECM does not contain decorin, perlecan, and / or collagen(III). Embodiment 12 is a method for producing a cellular construct of mature cardiomyocytes on an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating immature hiPSC-CMs onto AFC-ECM; (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct. wherein mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. Embodiment 13 is the method of embodiment 12, wherein a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. Embodiment 14 is a method according to any one of embodiments 12 or 13, wherein the fiber tracks are present on a cell construct. Embodiment 15 is a method for determining the cardiotoxicity and / or proarrhythmic effects of a drug compound in vitro, comprising the steps of contacting the drug compound with mature cardiomyocytes of any one of the cell constructs described in embodiments 5 to 11, and observing changes in the electrophysiology of the mature cardiomyocytes to determine whether the drug compound has cardiotoxicity and / or proarrhythmic effects on the mature cardiomyocytes. Embodiment 16 is the method of embodiment 15, wherein the change in the electrophysiology of mature cardiomyocytes is a prolongation of action potential duration (APD), and the prolongation of APD indicates that the drug compound has a cardiotoxic and / or proarrhythmic effect on mature cardiomyocytes. Embodiment 17 is the method of embodiment 15, wherein the change in the electrophysiology of mature cardiomyocytes is early after-depolarization, and the early after-depolarization (EAD) confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on mature cardiomyocytes. Embodiment 18 is the method of embodiment 15, wherein the change in the electrophysiology of mature cardiomyocytes is a delayed afterdepolarization, and the delayed afterdepolarization (DAD) confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on mature cardiomyocytes. Embodiment 19 is the method of embodiment 15, wherein the change in electrophysiology of mature cardiomyocytes is action potential duration (APD) plus rotors, and a prolongation of APD plus rotors indicates that the drug compound has a cardiotoxic and / or proarrhythmic effect on mature cardiomyocytes. Embodiment 20 is the method of embodiment 15, wherein the change in the electrophysiology of mature cardiomyocytes is arrhythmia, and the arrhythmia confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on mature cardiomyocytes.

[0015] The term "about" or "approximately" is defined as close to what would be 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%.

[0016] The term "substantially" and variations thereof are defined to include ranges within 10%, within 5%, within 1%, or within 0.5%.

[0017] As used herein, the term "% w / w" or "wt.%" refers to the weight percent of a component based on the total weight of the material (e.g., composition) that includes the component. In a non-limiting example, 10 grams of a component in 100 grams of a composition is 10% w / w of the component in the total weight of the composition. As used herein, the term "% v / v" or "vol.%" refers to the volume percent of a component based on the total volume of the material (e.g., composition) that includes the component. In a non-limiting example, 10 mL of a component in 100 mL of a composition is 10% v / v of the component in the total volume of the composition. As used herein, the term "% w / v" refers to the weight percent of a component based on the total volume of the material (e.g., composition) that includes the component. In a non-limiting example, 10 grams of a component in 100 mL of a composition is 10% w / v of the component in the total volume of the composition. As used herein, the term "% v / w" refers to the volume percent of a component based on the total weight of the material (e.g., composition) that includes the component. In a non-limiting example, 10 mL of a component in 100 grams of composition is 10% v / w of the component by total weight of the composition.

[0018] The terms "inhibit" or "reduce" or "prevent" or "avoid," or any variation of these terms, when used in the claims and / or specification, include any measurable reduction or complete inhibition to achieve a desired result.

[0019] The term "effective," as that term is used in the specification and / or claims, means adequate to accomplish a desired, expected, or intended result.

[0020] The words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0021] The use of the word "a" or "an" when used in conjunction with the terms "comprising," "having," "including," or "containing" (or any variation of these words) can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0022] The compositions and methods for their use may "comprise," "consist essentially of," or "consist of" any of the components or steps disclosed throughout the specification. With respect to the transitional phrase "consist essentially of," in one non-limiting aspect, a fundamental and novel feature of the cellular constructs disclosed herein is their use in cardiotoxicity and / or proarrhythmic screening tests of drug compounds due to their ability to mature stem cell-derived cardiomyocytes to a maturation state resembling that of mature native adult cardiomyocytes and native cardiac tissue.

[0023] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0024] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating particular embodiments of the present invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. The present invention provides, for example, the following items. (Item 1) 1. A method for maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) contacting the immature hiPSC-CM 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, thereby forming mature cardiomyocytes; wherein the mature cardiomyocytes are characterized by rod-shaped cells having distinct sarcomere structures similar to adult human cardiac tissue. (Item 2) 2. The method of claim 1, wherein the immature hiPSC-CMs are plated on the AFC-ECM. (Item 3) The method of any one of items 1 or 2, wherein the mature cardiomyocytes form a monolayer on the AFC-ECM, thereby forming a cellular construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM, and the mature cardiomyocytes are aligned on the AFC-ECM. (Item 4) The method of any one of items 1 to 3, wherein the immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.1. (Item 5) A cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid, the mature cardiomyocytes being human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) cultured in the AFC-ECM, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. (Item 6) 6. The cell construct of item 5, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. (Item 7) 7. The cell construct of any one of items 5 or 6, wherein fiber tracks are present on the construct. (Item 8) The cell construct of any one of items 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 their isoforms. (Item 9) The cell construct described in item 8, wherein the collagen alpha-1 (XVIII) isoform isoform 2 and / or the agrin isoform isoform 6. (Item 10) The cell construct of any one of items 8 or 9, wherein the AFC-ECM further comprises fibronectin and / or its isoforms. (Item 11) The cell construct of any one of items 5 to 10, wherein the AFC-ECM does not contain decorin, perlecan, and / or collagen(III). (Item 12) 1. A method for producing cellular constructs of mature cardiomyocytes on extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM), comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CMs); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs onto the AFC-ECM; (d) culturing the immature hiPSC-CMs plated on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes and form a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cell construct; wherein the mature cardiomyocytes are characterized by rod-shaped cells having distinct sarcomere structures similar to adult human cardiac tissue. (Item 13) 13. The method of claim 12, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. (Item 14) 14. The method according to any one of items 12 or 13, wherein fiber tracks are present on the cell construct. (Item 15) A method for determining in vitro the cardiotoxic and / or proarrhythmic effects of a drug compound, comprising the steps of contacting the drug compound with the mature cardiomyocytes of a cell construct according to any one of items 5 to 11, and observing changes in the electrophysiology of the mature cardiomyocytes to ascertain whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. (Item 16) The method of claim 15, wherein the change in the electrophysiology of the mature cardiomyocyte is a prolongation of the action potential duration (APD), and a prolongation of the APD confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocyte. (Item 17) The method of claim 15, wherein the change in the electrophysiology of the mature cardiomyocyte is an early after-depolarization (EAD), and the early after-depolarization (EAD) confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocyte. (Item 18) 16. The method of claim 15, wherein the change in the electrophysiology of the mature cardiomyocyte is a delayed afterdepolarization (DAD), and the delayed afterdepolarization (DAD) confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocyte. (Item 19) The method of claim 15, wherein the change in the electrophysiology of the mature cardiomyocyte is action potential duration (APD) plus rotor, and a prolongation of APD plus rotor indicates that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocyte. (Item 20) 16. The method of claim 15, wherein the change in the electrophysiology of the mature cardiomyocyte is arrhythmia, and the arrhythmia confirms that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocyte. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 is a photomicrograph of a bright field image of amniotic fluid cell-derived ECM at 100× magnification using a 10× objective.

[0026] [Diagram 2] FIG. 2 shows atomic force micrographs of three representative 40×40 μm sections of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM showing topography, adhesion, and stiffness.

[0027] [Diagram 3] Figure 3 is a scatter plot showing quantification of adhesion and stiffness (elastic modulus) of bone marrow and amniotic fluid cell-derived ECM, each point represents an independent measurement point.

[0028] [Figure 4] FIG. 4 is a set of micrographs showing days 0 and 2 of culture of iPSCs on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0029] [Diagram 5] FIG. 5 is a plot of the growth curves of iPSCs cultured in the presence of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0030] [Figure 6] FIG. 6 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 3.13 mm2.

[0031] [Figure 7] FIG. 7 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 2.15 mm2.

[0032] [Figure 8] FIG. 8 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 1.46 mm2.

[0033] [Figure 9] FIG. 9 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.54 mm2.

[0034] [Figure 10] FIG. 10 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.54 mm2.

[0035] [Figure 11] FIG. 11 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.54 mm2.

[0036] [Figure 12] FIG. 12 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.54 mm2.

[0037] [Figure 13] FIG. 13 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.13 mm2.

[0038] [Figure 14] FIG. 14 is a photomicrograph of mature cardiomyocytes on AFC-ECM in a single well - 0.04 mm2.

[0039] [Figure 15] FIG. 15 is a photomicrograph of cardiomyocytes on standard Matrigel™ ECM in a single well - 3.13 mm2.

[0040] [Figure 16] FIG. 16 is a photomicrograph of cardiomyocytes on standard Matrigel™ ECM in a single well - 2.15 mm2.

[0041] [Figure 17] FIG. 17 is a photomicrograph of cardiomyocytes on standard Matrigel™ ECM in a single well - 1.46 mm2.

[0042] [Figure 18] FIG. 18 is a photomicrograph of cardiomyocytes on standard Matrigel™ ECM in a single well - 0.54 mm2.

[0043] [Figure 19] FIG. 19 is a photomicrograph of cardiomyocytes on standard Matrigel™ ECM in a single well - 0.13 mm2.

[0044] [Figure 20] FIG. 20 is a photomicrograph of mature cardiomyocytes on BM-ECM in a single well - 2.15 mm2.

[0045] [Figure 21] FIG. 21 is a photomicrograph of mature cardiomyocytes on BM-ECM in a single well - 1.46 mm2.

[0046] [Figure 22] FIG. 22 is a schematic diagram of the instrumentation setup for recording action potentials or calcium transient measurements using mature cardiomyocytes on AFC-ECM in multi-well plates.

[0047] [Figure 23-1] FIG. 23 shows baseline and post-drug E4031 spontaneous action potential recordings from cardiomyocytes cultured on Matrigel™ ECM, BM-ECM, and AFC-ECM. [Figure 23-2] Same as above.

[0048] [Figure 24] FIG. 24 is a bar graph of the number of stable rotors (TdP-like arrhythmias) from cardiomyocytes cultured on Matrigel™ ECM, BM-ECM, and AFC-ECM following contact with the drug E4031.

[0049] [Diagram 25] FIG. 25 shows spontaneous action potential recordings from adult cardiomyocytes cultured on AFC-ECM for various drugs.

[0050] [Figure 26] FIG. 26 is a bar graph of all arrhythmias recorded for all doses of each of the drugs listed.

[0051] [Figure 27] FIG. 27 is a bar graph of the % of wells with arrhythmia at 10× the effective therapeutic plasma concentration (ETPC) of each of the drugs listed.

[0052] [Figure 28]FIG. 28 is a bar graph of action potential triangulation (APD90-APD30) over time (milliseconds) for each of the drugs listed.

[0053] [Figure 29] FIG. 29 is a bar graph of action potential triangulation (APD90-APD30) over time (milliseconds) for each of a portion of the listed drugs comparing cardiomyocyte performance of cardiomyocytes on AFC-ECM (SBS-AF matrix) versus Matrigel™ ECM.

[0054] [Diagram 30] Figure 30 is a bar graph of drug-induced action potential maximum triangulation for the listed drugs comparing cardiomyocyte performance of iCell® hiPSC-CMs from Cellular Dynamics (open circles) vs. Cor.4U® hiPSC-CMs from Ncardia (closed circles) at any concentration of the listed drug. Figure from Blinova et al, 2018, Cell Reports.

[0055] [Diagram 31] FIG. 31 is a photomicrograph of hiPSC-CMs cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for troponin I and using DAPI to mark nuclei.

[0056] [Diagram 32] FIG. 32 is a photomicrograph of hiPSC-CMs cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for α-actinin and using DAPI to mark nuclei.

[0057] [Diagram 33] FIG. 33 is a photomicrograph of hiPSC-CMs cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for cTnT and N-cadherin, and using DAPI to mark nuclei.

[0058] [Diagram 34] FIG. 34 is a photomicrograph of a single hiPSC-CM cell cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for cTnT and using DAPI to mark nuclei.

[0059] [Diagram 35] FIG. 35 is a photomicrograph of a single hiPSC-CM cell cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for α-actinin and using DAPI to mark nuclei.

[0060] [Diagram 36] FIG. 36 is a graph comparing cell circularity of the single cells shown in FIG.

[0061] [Figure 37] FIG. 37 is a photomicrograph of hiPSC-CMs cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescence staining for cTnI expression and using DAPI to mark nuclei.

[0062] [Figure 38] FIG. 38 is a western blotting of hiPSC-CM on Matrigel™ ECM and AFC-ECM for cTnI expression and GAPDH.

[0063] [Figure 39] FIG. 39 is a graph of cTnI expression relative to GAPDH for hiPSC-CMs on Matrigel™ ECM versus AFC-ECM.

[0064] [Diagram 40]FIG. 40 is a photomicrograph of hiPSC-CMs on Matrigel™ ECM versus AFC-ECM stained for mitochondria with MitoTracker Red.

[0065] [Diagram 41] FIG. 41 is a graph showing MitoTraker™ Red fluorescence intensity / cardiomyocytes for hiPSC-CMs on Matrigel™ ECM versus AFC-ECM.

[0066] [Diagram 42] FIG. 42 shows photomicrographs (transmitted light) of hiPSC-CMs cultured on Matrigel™ ECM and AFC-ECM coated on microelectrode array (MEA) plates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] Human stem cell-derived cardiomyocytes, such as those derived from human induced pluripotent stem cells (hiPSC-CM), matured in culture on extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM) have demonstrated better and more consistent cardiotoxicity and proarrhythmic assay results than immature hiPSC-CM or hiPSC-CM matured on other ECMs, such as Matrigel™ ECM and bone marrow cell-derived ECM. Surprisingly, hiPSC-CM matured on AFC-ECM demonstrate a higher state of maturation than hiPSC-CM matured on other ECMs, such as Matrigel™ ECM, and even on other natural cell-derived ECMs, such as bone marrow cell-derived ECM, as shown by their cell morphology and sarcomere structure found in cardiomyocytes of normal adult human cardiac tissue. Furthermore, more robust expression of cTnI (cardiac troponin I) protein, as demonstrated by Western blotting techniques, was seen for hiPSC-CMs cultured on AFC-ECM than for hiPSC-CMs cultured on Matrigel™ ECM. Also, hiPSC-CMs cultured on AFC-ECM have more mitochondria and mitochondria with more polarized inner membrane potential than hiPSC-CMs cultured on Matrigel™ ECM. Thus, AFC-ECM can stimulate mitochondrial biogenesis, maturation, and function in hiPSC-CMs. Normal adult or mature human cardiac human tissue is characterized by rod-shaped cells with sarcomeric structure (striped appearance). Cardiomyocyte morphology and sarcomeric structure 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 with a striped appearance, identified by the arrows in the micrograph in Figure 14. Furthermore, the use of a silicone substrate such as PDMS was not required to achieve these results.Surprisingly, cell constructs including AFC-ECM and monolayers of mature cardiomyocytes matured from immature hiPSC-CMs in culture on AFC-ECM have been shown to be useful, for example, by enabling consistent visualization of drug-induced arrhythmias such as torsade de pointes (TdP) in high-throughput in vitro screening assays. This "TdP in a dish" technology is a major advancement over relying on field potential duration (MEA technology) or action potential duration prolongation as surrogate markers of drug-induced TdP. Thus, the cell constructs and methods disclosed herein go beyond the current CiPA Initiative guidelines by developing a more comprehensive and predictive in vitro arrhythmia assay that allows visualization of arrhythmic events in an in vitro model, rather than simple polarization and depolarization events, which are indirect indicators of arrhythmogenesis. The cell systems and methods outlined in this disclosure allow arrhythmic events to be propagated and visualized in an in vitro human cardiac monolayer model system. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with distinguishable sarcomere structures. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, disorganized myofilaments, a round shape, and / or a single nucleus. In some embodiments, mature cardiomyocytes can be characterized by having rod-shaped cells with distinct sarcomere structures (striped appearance). In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, structured and compact myofilaments, and / or having two nuclei (binuclear).

[0068] Disclosed herein is a method for forming mature cardiomyocytes (mature hiPSC-CMs) using cell-derived, in-vitro derived extracellular matrix (AFC-ECM) from cells isolated from amniotic fluid for maturation of immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells in culture. Also disclosed herein is a cell construct comprising a monolayer of these mature cardiomyocytes on AFC-ECM, which is useful for cardiotoxicity and / or proarrhythmic screening assays of drug compounds. Also disclosed herein is a method for determining the cardiotoxicity and / or proarrhythmic effect of drug compounds in vitro using such cell constructs. A. Amniotic fluid cell-derived extracellular matrix (AFC-ECM)

[0069] 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 fetal amniotic membrane, skin, and cells from the developing fetus that are detached from the digestive, respiratory, and urogenital tracts. The placenta also has several cell sources, including membrane sheets (amnion and chorion), villi, and blood. Umbilical cord cells generally come from two cell sources: umbilical cord blood and Wharton's jelly. Cells from these three perinatal cell sources may include stem cells. The cells used to produce the amniotic fluid cell-derived ECM of the present invention are obtained from amniotic fluid of mammals, including but not limited to 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 sourced from humans who are full-term (greater than about 37 weeks of gestation) or preterm (less than about 37 weeks of gestation). Preterm birth includes late preterm birth (about 33 weeks to about 37 weeks gestation), mid-term preterm birth (about 29 weeks to about 33 weeks gestation), and very preterm birth (about 23 weeks to about 29 weeks gestation). Amniotic fluid can be provided from a human prior to birth at any gestational age where amniotic fluid is present, and can be combined with a source of amniotic fluid at birth. Typically, amniotic fluid is collected prior to birth by the procedure of amniocentesis. In some embodiments, amniotic fluid is provided from a human at term, preterm, late preterm, mid-term, very preterm, or prior to birth, or combinations thereof. In some embodiments, amniotic fluid is provided prenatally and collected from about 10 weeks gestation until birth, or from about 10 weeks gestation until about 23 weeks gestation, or from about 10 weeks gestation until about 16 weeks gestation, or from about 12 weeks gestation until birth, or from about 12 weeks gestation until about 23 weeks gestation, or from about 12 weeks gestation until about 16 weeks gestation. In some embodiments, amniotic fluid provided prenatally is collected by an amniocentesis procedure. 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.

[0070] Amniotic fluid contains cells that have the capacity to differentiate into cell types derived from all three germ layers (ectoderm, endoderm, mesoderm), either spontaneously or as a result of treatment with certain growth factors or combinations of growth factors known to those skilled in the art. That is, a single cell has the capacity to be induced to express genes specific to any of the three germ layers. Amniotic fluid also contains a mixture of different cell types, including cells from the developing fetus detached from the fetal amniotic membrane, skin, and digestive, respiratory, and urogenital tracts. Due to their origin from the amniotic fluid and placental membranes, these cells maintain a high degree of pluripotent differentiation potential and can include cell populations that include cells of all three germ layers. Amniotic fluid cells can include stem cells. In some embodiments, amniotic fluid cells are isolated stem cells. In some embodiments, amniotic fluid cells can include cell types derived from all three germ layers (ectoderm, endoderm, mesoderm) and / or multipotent stem cells and / or pluripotent stem cells. These include stem cells that have the ability to differentiate into erythrocytes.

[0071] The amniotic fluid cell-derived ECM disclosed herein may comprise a variety of proteins. The proteins of the ECM may be identified by techniques known in the art, including mass spectrometry and immunohistochemical staining. The ECM may comprise, 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 comprise any combination of any of the components in Table 2 and any variants, derivatives, or isoforms thereof. In some embodiments, the combination may comprise, consist essentially of, or consist of 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 cell-derived ECM further comprises, consists essentially of, or consists of fibronectin and / or its isoforms. In some embodiments, the amniotic fluid cell-derived ECM does not include any one or all of decorin, perlecan, and collagen(III).Some notable differences between the proteins of the amniotic fluid cell-derived ECM of the present invention and bone marrow cell-derived matrix are listed in Table 1. Table 1 Differences between amniotic fluid cell-derived ECM (AFC matrix) and bone marrow cell-derived matrix (BM matrix) [Table 1]

[0072] Amniotic fluid cell-derived ECM can be produced by the following process: (a) isolating cells from amniotic fluid; (b) seeding the isolated cells onto a cell culture vessel or a cell culture vessel coated with a substrate; (c) adding culture medium to the cell culture vessel; and (d) culturing the cells, thereby producing the cell-derived ECM; and (e) Decellularize cell-derived ECM as needed.

[0073] Any cell seeding density may be used that allows the cells to form a confluent monolayer immediately or after a period of culture. In some embodiments, the seeding density is about 10 cells / cm 2 ~about 100,000 cells / cm 2 , or approximately 100 cells / cm 2 ~about 75,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 50,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 10,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 5,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 2,500 cells / cm 2 , or approximately 1,000 cells / cm 2 ~about 25,000 cells / cm 2 , or approximately 2,000 cells / cm 2 ~about 10,000 cells / cm 2 , or approximately 3,000 cells / cm 2 ~about 5,000 cells / cm 2 It is.

[0074] For the purposes of the present invention, any type of vessel suitable for culturing cells can be used. Examples include, but are not limited to, cell culture flasks, T-flasks, spinner flasks, fermenters, and bioreactors. Rocker bottles, shake flasks, tubes, and other vessels are also suitable vessels when placed on a rocking platform or shaker. Cell culture vessels can be coated with a substrate to improve cell attachment. A non-limiting example of a suitable substrate for coating cell vessels is fibronectin.

[0075] Various commercially available cell culture media, such as alpha minimal essential medium (α-MEM) culture medium (Thermo Fisher Scientific, Grand Island, NY), are suitable for culturing amniotic fluid cells. Commercially available culture media can be modified by adding various supplementary substances, such as sodium bicarbonate, L-glutamine, penicillin, streptomycin, amphotericin B, and / or serum to the medium. The serum can be fetal bovine serum. The medium can be serum-free. In addition, substances such as L-ascorbic acid can be added to the medium or modified medium to induce the production of cells in the ECM.

[0076] The initial culture medium can be exchanged and / or replaced with another medium at various times during the culture process. For example, the initial medium can be a "complete medium" and then replaced with an "induction medium" during the culture process. A non-limiting example of a "complete medium" includes (α-MEM) plus 2 mM L-glutamine plus antibiotic-antimycotic plus 15% fetal bovine serum. A non-limiting example of an "induction medium" includes "complete medium" plus 50 mM L-ascorbic acid.

[0077] Amniotic fluid cell culture can be performed in an incubator at 37°C with 5% CO2 and 90% humidity. Culture can be performed under a variety of environmental conditions, including, but not limited to, normoxia, i.e., 20-21% atmospheric oxygen, or hypoxic conditions.

[0078] Decellularization of amniotic fluid cells from amniotic fluid cell-derived ECM may include removal of live amniotic fluid cells or rendering amniotic fluid cells non-viable. Amniotic fluid cells may be decellularized from ECM using methods known in the art, including but not limited to lysing amniotic fluid cells and then removing the lysed amniotic fluid cells by washing. Various substances may be used to remove amniotic fluid cells from ECM. Non-limiting examples include "extraction buffer" containing TRITON® X-100 and ammonium hydroxide in PBS buffer. After ECM is decellularized from amniotic fluid cells, the resulting ECM is substantially cell-free or free of live amniotic fluid cells. When feeder cells are used, the decellularization method is applied to any live feeder cells present on the ECM, and thus the ECM is substantially free or free of live feeder cells. The decellularization method is applied to any live cells present on the ECM, and thus the ECM is substantially free or free of any live cells. Thus, decellularized ECM means that the ECM is acellular, meaning that the ECM does not contain any living cells.

[0079] In some embodiments, the amniotic fluid cell-derived ECM (AFC-ECM) is a three-dimensional (3D) ECM.

[0080] The methods described above also apply to the production of cell-derived ECM from other perinatal cells, such as cells from the umbilical cord, including umbilical cord blood and Wharton's jelly, as well as cells from placental tissues, including membrane sheets (amnion and chorion), villi, and blood.

[0081] In one embodiment, the perinatal cell-derived ECM is produced by the following process. (a) isolating cells from the umbilical cord; (b) seeding the isolated cells onto a cell culture vessel or a cell culture vessel coated with a substrate; (c) adding culture medium to the cell culture vessel; and (d) culturing the cells, thereby producing the cell-derived ECM; and (e) Decellularize cell-derived ECM as needed. In some embodiments, the cells isolated from the umbilical cord are from cord blood and / or Wharton's jelly.

[0082] In another embodiment, the perinatal cell-derived ECM is produced by the following process: (a) isolating cells from placental tissue; (b) seeding the isolated cells onto a cell culture vessel or a cell culture vessel coated with a substrate; (c) adding culture medium to the cell culture vessel; and (d) culturing the cells, thereby producing the cell-derived ECM; and (e) Decellularize cell-derived ECM as needed. In some embodiments, cells isolated from placental tissue are from membrane sheets (amnion and / or chorion), villi, and / or blood.

[0083] In one aspect, a cell-derived extracellular matrix (ECM) is disclosed that is derived in vitro from cells isolated from the umbilical cord. In some embodiments, the cells isolated from the umbilical cord are from umbilical cord blood and / or Wharton's jelly.

[0084] In another aspect, a cell-derived extracellular matrix (ECM) is disclosed that is derived in vitro from cells isolated from placental tissue. In some embodiments, the cells isolated from placental tissue are from membrane sheets (amnion and / or chorion), villi, and / or blood. B. Cellular Constructs and Methods for Maturation of Immature hiPSC-CMs

[0085] Disclosed herein is a method for maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, 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) contacting the immature hiPSC-CM with the AFC-ECM; and (d) culturing the immature hiPSC-CM with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM, thereby forming mature cardiomyocytes (mature hiPSC-CM), wherein the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human heart tissue. Normal adult human heart tissue is characterized by rod-shaped cells with sarcomere structures (striped appearance). The morphology and sarcomere structures of cardiomyocytes can be visually identified by a microscope. 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 with a striped appearance, identified by the arrows in the micrograph of FIG. 14.

[0086] Also disclosed herein is a cellular construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived in vitro from cells isolated from amniotic fluid, the mature cardiomyocytes being human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) cultured in the AFC-ECM, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In certain aspects, mature cardiomyocytes can be matured from immature cardiomyocytes (immature hiPSC-CM) derived from human induced pluripotent stem cells in culture on the AFC-ECM, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some aspects, the mature cardiomyocytes comprise and / or express the inwardly rectifying Kir2.1 potassium channel. In some embodiments, a monolayer of mature cardiomyocytes is aligned on the 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 their isoforms. In some embodiments, the isoform of collagen alpha-1 (XVIII) is isoform 2, and / or 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 comprise decorin, perlecan, and / or collagen (III). In some embodiments, the immature hiPSC-CM does not comprise rod-shaped cells with distinct sarcomere structures. In some embodiments, the immature hiPSC-CM can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a round shape, and / or having a single nucleus.In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (bilukaryotic).

[0087] Also disclosed herein is a method for the preparation of a cellular construct comprising mature cardiomyocytes on an extracellular matrix in vitro derived from cells isolated from amniotic fluid (AFC-ECM), comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix in vitro derived from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CM on the AFC-ECM; and (d) culturing the plated immature hiPSC-CM on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the construct.

[0088] Immature hiPSC-CMs can be obtained from commercial sources such as Cellular Dynamics International-FUJI under the trade name iCell®, and from commercial sources such as Takara Bio under the trade name Cellartis®. iCell® Cardiomyocytes, iCell® Cardiomyocytes 2, and Cellartis® Cardiomyocytes are cryopreserved live cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs) available in vials. Immature hiPSC-CMs can also be generated from patient-specific hiPSCs in a laboratory setting for a particular individual. In this case, differentiation of hiPSCs can be achieved using a small molecule protocol to obtain beating cardiomyocytes by day 8-10 using a GSK3 inhibitor, RPMI / B27 minus insulin, a Wnt inhibitor and RPMI / B27 plus insulin at various days during the 7-day differentiation period. Suitable non-limiting examples of methods for generating immature hiPSC-CMs are disclosed in U.S. Patent Application Publication No. 2015 / 0329825, which is incorporated herein by reference. In some embodiments, immature hiPSC-CMs do not express the inwardly rectifying potassium channel Kir2.1. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with distinguishable sarcomere structures. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized myofilaments, having a round shape, and / or having a single nucleus.

[0089] The AFC-ECM can be obtained using the production methods disclosed herein and can have the characteristics described herein. In some embodiments, the AFC-ECM is decellularized prior to contact with the immature hiPSC-CM.

[0090] The immature hiPSC-CMs may be in suspension when in contact with the AFC-ECM, or the cells may be plated directly on the AFC-ECM in or on a suitable cell culture vessel, or in a multi-well plate. Non-limiting examples of suitable multi-well plates include 6-well, 12-well, 24-well, 48-well, 96-well, and 384-well plates. In some embodiments, the contact surface of the cell culture vessel or multi-well plate is coated with polydimethylsiloxane (PDMS) before the formation of the AFC-ECM. In some embodiments, the contact surface of the cell culture vessel or multi-well plate is not coated with PDMS before the formation of the AFC-ECM. Any cell seeding density of immature hiPSC-CMs may be used. In some embodiments, a cell seeding density of immature hiPSC-CMs is used that allows the cells to form a confluent monolayer immediately or after a period of culture. The cell density may be modified as desired to improve monolayer formation. In multi-well plates, immature hiPSC-CM cells are placed in the center of each well.Non-limiting examples of cell seeding density of immature hiPSC-CM in various multi-well plates are as follows: in 6-well plates, about 200,000 cells can be plated per well; in 12-well plates, about 150,000 cells can be plated per well; in 24-well plates, about 175,000 cells can be plated per well; in 48-well plates, about 100,000 cells can be plated per well; in 96-well plates, about 50,000 cells can be plated per well; in 384-well plates, about 15,000 cells can be plated per well.In some embodiments, the cell seeding density of immature hiPSC-CM is about 50,000 cells per well in 96-well plates. 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, e.g., RPMI medium or Media 199 medium, is added to the immature hiPSC-CMs in contact with the AFC-ECM, and the cells are cultured with the AFC-ECM using standard cell culture techniques for a period of time, typically 7 days, until the cells mature into mature cardiomyocytes with morphology similar to native adult cardiomyocytes. During the first 3-4 days, the immature hiPSC-CMs are adherent and form a continuous monolayer, initiating the maturation process. Surprisingly, the period for cardiomyocyte maturation can take 7 days or less, although generally much longer periods, e.g., up to 100 days, are typical with other substrates. The morphology of these mature cardiomyocytes can be characterized by rod-shaped cells with distinct sarcomere structures (striped appearance), which are the fundamental contractile units of muscle, that can be visualized using conventional light microscopy techniques. In some embodiments, mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured and compact myofilaments, and / or having two nuclei (binuclear). Also, the AFC-ECM can naturally produce fiber tracks that mature cardiomyocytes follow. This creates a degree of anisotropy in the monolayer that more closely mimics the native heart. Thus, cardiomyocytes can naturally align on the AFC-ECM after the alignment of the AFC-ECM in the native anisotropic configuration defined by amniotic fluid cells during the formation of the AFC-ECM. In various embodiments, the period for immature hiPSC-CMs to mature in culture can be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, 110, 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-CMs to mature in culture is 7 days. In some embodiments, immature hiPSC-CMs are plated on AFC-ECM.In some embodiments, the mature cardiomyocytes form a confluent monolayer on the AFC-ECM during culture, thereby forming a cellular construct comprising a monolayer of mature cardiomyocytes on the AFC-ECM. In some embodiments, the mature cardiomyocytes are aligned on the AFC-ECM. In some embodiments, the immature hiPSC-CMs are plated on the AFC-ECM in a multi-well plate. In some embodiments, the multi-well plate has a polydimethylsiloxane (PDMS) insert. In some embodiments, the multi-well plate does not have a PDMS insert. C. Methods for determining the cardiotoxic and / or proarrhythmic effects of drug compounds

[0091] Disclosed herein is a method for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro, comprising contacting the drug compound with a mature cardiomyocyte of any one of the cell constructs disclosed throughout the present specification, and observing one or more changes in the electrophysiology of the mature cardiomyocyte to confirm whether the drug compound has a cardiotoxicity and / or proarrhythmic effect on the mature cardiomyocyte. The one or more changes in the electrophysiology of the mature cardiomyocyte indicate and support that the drug compound has a cardiotoxicity and / or proarrhythmic effect on the mature cardiomyocyte. The one or more changes in the electrophysiology of the mature cardiomyocyte may include, but are not limited to, APD prolongation, APD prolongation plus rotor, and / or various types of arrhythmias, such as tachyarrhythmia (TA), quiescence (Q), delayed afterdepolarization (DAD), and / or early afterdepolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is a prolongation of the action potential duration (APD). In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is an early afterdepolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is delayed afterdepolarization (DAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is action potential duration (APD) plus rotors. In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is arrhythmia. In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is tachyarrhythmia (TA). In some embodiments, the change in the electrophysiology of the mature cardiomyocyte is quiescence (Q). The observations may also include cell viability, cell density, and / or cell morphology.In some aspects, the cell construct can be prepared by a process including: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CM on the AFC-ECM; and (d) culturing the plated immature hiPSC-CM on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into mature cardiomyocytes, forming a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In other aspects, the method can include contacting a drug compound with any one of the cell constructs disclosed throughout the present specification, and observing cardiotoxicity and / or proarrhythmic events. In one particular instance, the method includes the steps of: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix derived in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CM on the AFC-ECM; (d) culturing the plated immature hiPSC-CM on the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into mature cardiomyocytes and form a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct; (e) contacting a drug compound with the monolayer of mature cardiomyocytes of the construct; and (f) observing changes in electrophysiology of the mature cardiomyocytes to determine whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes, the mature cardiomyocytes being characterized by rod-shaped cells with distinct sarcomere structures similar to adult human cardiac tissue. In some embodiments, immature hiPSC-CMs are plated onto the AFC-ECM in a multi-well plate, hi some embodiments, the multi-well plate has a polydimethylsiloxane (PDMS) insert.In some embodiments, the multi-well plate does not have a PDMS insert. In some embodiments, the immature hiPSC-CMs do not express the inwardly rectifying potassium channel Kir2.1. In some embodiments, a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fiber tracks are present on the cell construct.

[0092] Cardiotoxicity and / or proarrhythmia testing may be performed using any type of device 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 (generally 7 days or less), the electrophysiology of each well is observed using a plate reader. A suitable voltage-sensitive or calcium-sensitive fluorescent dye is loaded into each well. Non-limiting voltage-sensitive dyes include FluoVolt™ dyes available from ThermoFisher. In some embodiments, the plate reader may rely on a suitable high-spatiotemporal CCD camera combined with suitable illumination, such as light-emitting diodes (LEDs) of appropriate wavelengths to excite each dye. Such high-spatiotemporal CCD cameras are 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 to allow simultaneous visualization of all wells of a multi-well plate with sufficient resolution to observe action potentials and calcium wave propagation. Each plate is centered under the camera system, the lights are switched on, camera acquisition is started, and electrophysiological activity is recorded. The experiment is performed at about 37°C. After taking a baseline reading, the drug to be tested is added to the wells and the effects are recorded. Spontaneous activity is recorded for a period of time sufficient to obtain images, for example, at least 10 seconds. The images can be stored on a computer. The images can be analyzed and action potential duration, conduction velocity, beat rate, and activation pattern can be quantified using image analysis software. Visualization of electrical wave patterns is important to determine the effect of drug compounds in causing potentially fatal arrhythmias, such as torsades de pointes (TdP). Thus, in addition to providing information about the effect of compounds on spontaneous action potential duration, the methods disclosed herein can also provide information about impulse conduction velocity and activation patterns, depending on the type of device used. D. Methods for Expanding / Proliferating Mammalian Stem Cells

[0093] Also disclosed herein is a method of using cell-derived, amniotic fluid-isolated cell-derived in vitro extracellular matrix (AFC-ECM) for the isolation, maintenance, and expansion / growth of mammalian cells. In vitro cell culture is perhaps the most ubiquitous, important, and least well understood aspect of all cell biology and the developing fields of regenerative medicine and tissue engineering. First, it allows the observation of cell behavior, which allows various aspects of cell function to be studied in detail. Second, it allows the number of specific cell populations to be increased. For basic research as well as many clinical applications, it is necessary to obtain large amounts of relatively rare cells from small biological samples. In-vitro cell culture allows small numbers of cells to be expanded outside the body to obtain more suitable numbers of cells. Finally, it allows the cells to be stored for later use. By expanding cell numbers in-vitro and freezing viable cells for later use, cells for multiple experiments can be obtained from relatively small biological samples over a period of days, months, or even years.

[0094] Despite the ubiquity of cell culture, the effects of in vitro culture on the natural characteristics of cells are still relatively poorly understood. Much of the current practice does not result from deliberate thought, planning, and experimentation, but instead from chance observations. Mammalian cell culture began in the early 1900s, when Alexis Carrel and Montrose Burrows first published a scientific paper on culturing mammalian tissue in vitro in 1911. They were studying the physiology and anatomy of mammalian tissues by cutting slices and placing them on microscope slides. They noticed that some cells migrated from the tissue onto the slide. They subsequently described a method for culturing cells permanently. Although some of their observations now seem invalid, their work paved the way for modern cell culture.

[0095] Following the discovery of hematopoietic stem cells (HSCs), groups around the world were studying them. During culture (in suspension) of HSCs, a subpopulation of bone marrow cells was observed to adhere to the bottom of plastic flasks and begin to proliferate. These cells were later recognized as distinct from HSCs and were eventually named mesenchymal stem cells (MSCs). Because of this chance observation leading to the discovery of MSCs, adhesion to plastic is still widely used as the defining attribute of MSCs and many other mammalian cell types.

[0096] The practice of culturing cells on plastic substrates can be problematic because there is substantial evidence, now widely accepted in the literature, that demonstrates the critical role of the microenvironment in regulating cellular function. The microenvironment has been shown to help direct the differentiation of stem and progenitor cells and regulate the behavior of mature cell types.

[0097] When cells are removed from their native environment for expansion in-vitro, they may lose important cues from their surrounding extracellular matrix or microenvironment that relay important information to the cells about the composition and state of their surroundings. Changes in the cell's microenvironment can have profound effects on the behavior of these cells. The current standard method for isolating and expanding most adherent cells in vitro is to place the cells in culture vessels made of polystyrene (plastic). The polystyrene may be treated in some manner to promote cell attachment and growth, but in many cases the surface is completely foreign to the cells. In other cases, the surface may be coated with individual matrix proteins (e.g., fibronectin or collagen) or some combination of proteins. These simple substrates ignore the complexity of the native microenvironment and the important role of the microenvironment in normal cell function. The cells immediately begin to respond to this foreign environment in a manner that is very different from when the cells are in their native environment.

[0098] Five main approaches are currently being adopted to address this issue in culturing cells on plastic substrates:

[0099] 1. Ignoring the problem. Instead of attempting to achieve a desired function that matches the expected function in vivo, one can test numerous cell types in different media to find cells that exhibit a particular desired function even in the absence of a suitable matrix substrate. This approach is inelegant and often fails to obtain the desired results due to the complex interactions between variables and the wide range of interactions between cells and the extracellular matrix.

[0100] 2. Identify the key components. Many academic laboratories and some companies have taken the approach of considering the tissue from which the cells were isolated and looking for unique elements within 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 in nature, the matrix is ​​an extremely complex environment containing in some cases over 100 different proteins. Cells respond just as strongly to signals that they need but cannot accept as they do to signals that they do not need and can accept.

[0101] 3. Shotgun approach. The use of protein gels such as MATRIGEL™ employs a kind of shotgun approach. A gel is created that contains many different matrix proteins in the hope that the gel will contain the necessary binding motifs for many different cell types. This approach can fail by providing cues that push the cells in a particular direction or by failing to provide all the cues that the cells are expecting.

[0102] 4. Tissue-derived matrices. This is a biomimetic approach that typically involves isolating the tissue of interest 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 disintegrated tissue. For example, if one wishes to culture satellite cells, one may harvest the muscle, homogenize the tissue, and then coat the culture vessel with the homogenized muscle and seed the cells. This method often fails for several reasons. First, even within a particular tissue type, the stem / progenitor cell niche may be different from the rest of the tissue. Simply homogenizing the muscle does not guarantee that a suitable niche has been created. Second, the niche consists of structural and physical cues in addition to biochemical cues. Even if many / most of the biochemical cues are present in the tissue homogenate, the structure has been disrupted and 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 is a source of lot-to-lot variation.

[0103] 5. Cell-derived matrix. Cells in culture can be induced to secrete a matrix into the culture vessel. This matrix is ​​the best available approximation of the in vivo niche and can be manufactured in vitro. Cells can be induced in vitro to produce the matrix, and then the cells can be removed from the matrix, for example, by using a non-ionic detergent that preserves the structure and chemistry of the matrix. This approach has several important advantages: (1) the matrix structure can be recreated and left unperturbed; (2) the matrix can be customized based on the tissue / cell type of interest; (3) the matrix can be specific to the stem and progenitor cell niche; and (4) the matrix can be manufactured in large quantities.

[0104] With regard to cell-derived matrices, not all cell types can be efficiently isolated and expanded on any given cell-derived matrix. Indeed, pluripotent stem cells (PSCs) appear to have very different requirements for a supportive growth substrate than other cell types. It is known that the specific cell type used to produce a matrix affects the composition of the matrix and therefore the response of different cell types to that 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 studies disclosed in U.S. Pat. No. 8,084,023 have described the production and composition of extracellular matrix produced by bone marrow stromal cells or mesenchymal stem cells (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 was shown to support the expansion of other MSCs, but was not effective for the attachment and growth of other types of stem cells, specifically induced pluripotent stem cells (iPSCs). iPSCs have shown the expanded potential to form cells and tissues from a much broader category than MSCs. This represents a particularly interesting challenge, since it is impractical to produce cell-derived matrices from iPSCs due to the difficulty of growing confluent monolayers of iPSCs under standard culture conditions. A major limitation of previous cell-derived matrices is that to create tissue-specific matrices (e.g. bone marrow matrix from bone marrow MSCs, adipose matrix from adipose MSCs, or endothelial matrix from hUVECs), the target population of cells must already be able to adhere to the starting substrate. The difficulty with iPSCs, embryonic stem cells (ES), and many other cell types is that they do not readily adhere to simple substrates.

[0105] The present disclosure provides a solution to at least some of the above-mentioned limitations and deficiencies in the art related to cell-derived extracellular matrix (ECM) that supports the isolation, expansion, and proliferation of pluripotent stem cells (PSCs), including but not limited to induced pluripotent stem cells (iPSCs) and embryonic stem cells (ES). The solution is premised on the use of amniotic fluid cell-derived extracellular matrix. The use of neutral, easily adherent, highly proliferative perinatal cells found in amniotic fluid allows the creation of an extracellular matrix (ECM) that surprisingly supports the adhesion, isolation, expansion, and proliferation of these PSCs. This technical achievement was not possible with the cell-derived ECM of the prior art.

[0106] The function of mammalian cells is determined primarily by the environment in which they reside, e.g., the extracellular matrix. Cells respond to signals present in the environment (positive signals) and to signals that are necessary but not present (negative signals). Indeterminate stem cells can produce a matrix that contains niche motifs necessary to maintain stem cell viability and stemness, but may lack many lineage-specific signals that more mature cells may secrete to direct stem cells to specific fates. Without being bound by theory, it is suggested that immature cells, e.g., perinatal cells or perinatal stem cells, may produce an ECM that is different from the ECM previously disclosed in the art, such as bone marrow stromal cell-derived ECM, and may allow for better isolation and expansion / growth of stem cells, such as pluripotent stem cells (PSCs), which have higher potential than mesenchymal stem cells (MSCs). Mass spectrometry analysis showed that, compared to ECMs derived from previously known cells, the amniotic fluid cell-derived ECM of the present invention contains matrix proteins found in all three germ layers and lacks certain proteins strongly associated with osteogenic cell lineages. Additionally, the ECM of the present disclosure contains specific motifs, such as laminin, that are known to promote the attachment and spreading of pluripotent cells.

[0107] Pluripotent stem cells (PSCs) 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 currently the only known naturally occurring pluripotent stem cells. Induced pluripotent stem (iPSC) cells are also PSCs. iPSCs are generally derived from cells taken from adult tissues or cells and reprogrammed to the level of embryonic stem cells. Methods for producing iPSCs are known in the art.

[0108] The method of expanding / growing pluripotent stem cells (PSCs) includes obtaining PSCs and culturing them in the presence of amniotic fluid cell-derived ECM of the present invention. The PSCs can be iPSCs or ES. Any seeding density that allows the cells to form a confluent monolayer immediately or after a period of culture may be used. In some embodiments, the seeding density is about 10 cells / cm. 2 ~about 100,000 cells / cm 2 , or approximately 100 cells / cm 2 ~about 75,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 50,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 10,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 5,000 cells / cm 2 , or approximately 500 cells / cm 2 ~about 2,500 cells / cm 2 , or approximately 1,000 cells / cm 2 ~about 25,000 cells / cm 2 , or approximately 2,000 cells / cm 2 ~about 10,000 cells / cm 2 , or approximately 3,000 cells / cm 2 ~about 5000 cells / cm 2 It is.

[0109] In some embodiments, the PSCs are maintained in an undifferentiated state to maintain their stemness. Suitable cell culture techniques for expanding PSCs in culture are known in the art. Suitable commercially available culture media for stem cell expansion include, but are not limited to, StemMACS™ iPS-Brew XF available from Miltenyl Biotec. In some embodiments, no Rock inhibitors are used. Once the cells begin to approach confluence (e.g., as determined by bright field microscopy), the cells can be manually passaged by cutting larger colonies into smaller colonies, which are then physically lifted from the dish and placed on a new plate of amniotic fluid cell-derived ECM for reseeding. This procedure can be repeated indefinitely. In some embodiments, a method is disclosed for expanding pluripotent stem cells (PSCs) in culture, comprising culturing the PSCs in a culture medium in the presence of cell-derived extracellular matrix (ECM), thereby expanding the PSCs, where the cell-derived ECM is derived in vitro from cells isolated from amniotic fluid.

[0110] The above methods of expanding / growing PSCs are also applicable for use in expanding / growing PSCs in culture in the presence of ECM from other perinatal cells. In some embodiments, a method of expanding pluripotent stem cells (PSCs) in culture is disclosed, comprising culturing the PSCs in a culture medium in the presence of extracellular matrix (ECM) from the cells, thereby expanding the PSCs, where the ECM from the cells is derived in-vitro from cells isolated from umbilical cord or placental tissue. In some embodiments, the cells isolated from umbilical cord are from umbilical cord blood and / or Wharton's jelly. In other embodiments, the cells isolated from placental tissue are from membrane sheets (amnion and / or chorion), villi, and / or blood. EXAMPLES

[0111] The following examples are included to demonstrate certain non-limiting aspects of the invention. Those of skill in the art will recognize that the procedures disclosed in the examples which follow represent procedures discovered by applicants to work well in the practice of the invention. However, those of skill in the art will recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention. Example 1 Production of amniotic fluid cell-derived ECM (AFC-ECM)

[0112] Four amniotic fluid cell-derived ECMs (Matrix A, Matrix B, Matrix C, and Matrix D) were generated using the following procedure. Cells were aseptically isolated from amniotic fluid collected from four full-term donors (>37 weeks gestation) and seeded onto fibronectin-coated tissue culture-treated flasks and cultured in complete medium at 37°C, 5% CO2, and 90% RH in an incubator. The complete medium was alpha minimal essential medium (aMEM) plus 2 mM L-glutamine plus antibiotic-antimycotic plus 15% fetal bovine serum.

[0113] On days 3-4, half of the complete medium was aspirated from the flasks and replaced with half of fresh complete medium. The flasks were returned to the incubator under the same conditions as above.

[0114] On days 7-8, the complete medium was aspirated from the culture flasks and replenished with induction medium. The flasks were returned to the incubator under the same conditions as above. Induction medium was complete medium plus 50 mM L-ascorbic acid.

[0115] On days 10–11, the induction medium was aspirated from the culture flasks and the ECM formed in the flasks was washed once with phosphate-buffered saline (PBS). The PBS was then aspirated from the flasks. Extraction buffer was added to the flasks and incubated at room temperature for 7–10 min to remove cells from the respective ECM, and then the extraction buffer was aspirated from the flasks. The extraction buffer was PBS containing 0.5% (v / v) TRITON®-X100 and 20 mM ammonium hydroxide (NH4OH).

[0116] Each of the ECMs in the flasks from which the cells had been removed was washed three times with PBS and once with sterile water, which was then aspirated from the flasks. The four ECMs in the flasks from which the cells had been removed were allowed to dry at room temperature and then stored at 4°C.

[0117] A bright field image micrograph of amniotic fluid cell-derived ECM (Matrix B) at 100x magnification using a 10x objective is shown in Figure 1. Atomic force micrographs of three representative 40x40um sections of amniotic fluid cell-derived ECM (Matrix B) and bone marrow cell-derived ECM showing topography, adhesion, and stiffness are shown in Figure 2. Bone marrow cell-derived and amniotic fluid cell-derived ECM are structurally and physically distinct. Quantification of adhesion and stiffness (elastic modulus) of bone marrow cell-derived and amniotic fluid cell-derived ECM shows that bone marrow ECM is 10x stiffer and 3x non-adhesive than amniotic fluid ECM, as shown in the scatter plots in Figure 3a (adhesion) and Figure 3b (stiffness), where BM is bone marrow cell-derived ECM and AD is amniotic fluid cell-derived ECM (Matrix B). Each point represents an independent measurement point. Example 2 Composition of amniotic fluid cell-derived ECM

[0118] The composition of each of the amniotic fluid cell-derived ECMs produced in Example 1 was determined by mass spectrometry. The components are listed in Table 2 along with their spectral counts and molecular weights. Table 2 Amniotic fluid cell-derived ECM (AFC-ECM) components [Table 2-1]

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Table 2-19

Table 2-20

Table 2-21

Table 2-22

Table 2-23

Table 2-24

Table 2-25

Table 2-26

Table 2-27

Table 2-28

Table 2-29

Table 2-30

Table 2-31

Table 2-32

Table 2-33

Table 2-34

Table 2-35

Table 2-36

Table 2-37

Table 2-38

Table 2-39

Table 2-40

Table 2-41

Table 2-42

Table 2-43

Table 2-44

Table 2-45

Table 2-46

Table 2-47

Table 2-48

[0119] Induced pluripotent stem cells (iPSCs) were cultured and expanded on the amniotic fluid cell-derived ECM (Matrix B) of Example 1 using the following procedure: Commercially available cryopreserved iPSCs were thawed using a water bath at 37°C. The cell suspension was then cultured in a commercially available medium for stem cell proliferation (Miltenyi The cells were diluted in iPSCs (MACS iPS Brew, Biotec) and plated at approximately 1,000 cells / cm on the ECM in 6-well plates. 2, and seeded with 2 mL of medium / well. 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. Every 24 hours, the entire medium was replaced with fresh medium. Once the cells began to approach confluence (as judged by bright field microscopy), the cells were manually passaged by cutting the large colonies into approximately 100 smaller colonies with a sterile needle, which were then physically lifted out of the dish with a sterile needle and placed into a new plate of ECM for reseeding. This procedure can be repeated indefinitely.

[0120] Micrographs showing days 0 and 2 of culture of iPSCs on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM are shown in FIG.

[0121] FIG. 5 shows plots of iPSC colony growth curves for iPSCs cultured in the presence of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0122] As can be seen in Figures 4 and 5, iPSCs expanded in culture in the presence of amniotic fluid cell-derived ECM, whereas iPSCs cultured in the presence of bone marrow cell-derived ECM did not grow. Example 4 Preparation of cell constructs of mature cardiomyocytes on AFC-ECM and maturation of immature hiPSC-CMs on AFC-ECM

[0123] Cellular constructs comprising a monolayer of mature cardiomyocytes on in-vitro amniotic fluid derived cell-derived extracellular matrix (AFC-ECM) were prepared using the following method.

[0124] AFC-ECM was prepared in 96-well plates (no silicone inserts were used) using the methodology outlined in Example 1. Immature hiPSC-CMs (iCell® Cardiomyocytes), commercially available from Cellular Dynamics International-FUJI, were plated onto the AFC-ECM using standard cell culture techniques. The immature hiPSC-CMs were plated at a density of 50,000 cells per well (96-well plates) or 200,000 cells per well (6-well plates) and cultured in RPMI medium for 7 days to form a confluent monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cellular construct of a monolayer of mature cardiomyocytes on the AFC-ECM.

[0125] Over a period of 7 days, immature hiPSC-CMs were observed to mature into the morphology and alignment of mature native adult cardiomyocytes characterized by rod-shaped cells with distinct sarcomere structures. For comparative purposes, iCell® immature hiPSC-CMs were also plated in 96-well plates (silicone inserts were not used) and cultured in a similar manner on standard Matrigel™ ECM and on bone marrow cell-derived ECM (BM-ECM) prepared by the method disclosed in U.S. Patent No. 8,084,023, which is incorporated herein by reference. The results of this study are shown in micrographs of cardiomyocytes on different ECMs in Figures 6-21.

[0126] As can be seen in Figures 6-14, cardiomyocytes on AFC-ECM are mature rod-shaped cells with distinct sarcomere structures similar to native adult cardiomyocytes. The morphology and sarcomere structures of cardiomyocytes matured on AFC-ECM can be clearly seen by the presence of rod-shaped cells with a striped appearance, identified by the arrows in the micrograph of Figure 14. These figures also show the presence of fiber tracks on AFC-ECM, and that the monolayer of mature cardiomyocytes, aligned with AFC-ECM, closely resembles features found in native adult cardiomyocytes and native cardiac muscle tissue. In contrast, as can be seen in Figures 15-19 and Figures 20-21, cardiomyocytes on standard Matrigel™ ECM and BM-ECM, respectively, resemble fetal-like cardiomyocytes and have neither features of native adult cardiomyocytes nor features of native cardiac muscle tissue.

[0127] Thus, immature hiPSC-CMs cultured on AFC-ECM achieved a more mature state than immature hiPSC-CMs cultured on standard Matrigel™ ECM or native cell-derived ECM from bone marrow cells. It is clear that hiPSC-CM morphology was affected differently by the different ECMs. Example 5 High-throughput cardiotoxicity screening of drugs using cell constructs of mature cardiomyocytes on AFC-ECM

[0128] Cell constructs of a monolayer of mature cardiomyocytes on AFC-ECM were prepared as described in Example 4 using 96-well plates (no silicone inserts were used). 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™ dye in Hanks balanced salt solution was loaded into each well. A high-spatiotemporal CCD camera (SciMeasure DaVinci camera) was combined with a light-emitting diode (LED) as shown in the schematic diagram of FIG. 22. The camera and lens combination was designed to allow for simultaneous visualization of all wells of a 96-well plate with sufficient resolution to observe action potentials and calcium wave propagation. Each plate was centered under the camera system, the illumination was switched on, camera acquisition was started, and electrophysiological activity was recorded. The experiment was performed at approximately 37° C. Spontaneous activity was recorded for at least 10 seconds, and images were stored on a computer. After obtaining baseline readings, 500nM of the drug E4031 (hERG channel blocker) was added to each well. Images were analyzed, and action potential duration, conduction velocity, beat rate, and activation pattern were quantified using image analysis software. For comparison purposes, immature hiPSC-CMs were cultured on standard Matrigel™ ECM and bone marrow cell-derived ECM (BM-ECM) in 96-well plates (no silicone inserts were used) as described in Example 4, and 500nM of the drug E4031 (hERG channel blocker) was analyzed in a similar manner to the AFC-ECM study. The results of the E4031 study are shown in Figure 23 and Figure 24. As can be seen in FIG. 23, spontaneous action potential recordings from cardiomyocytes on Matrigel™ ECM and BM-ECM showed that only the drug E-4031 caused action potential duration (APD) prolongation; however, for mature cardiomyocytes on AFC-ECM, the drug E-4031 caused APD prolongation plus rotors (TdP-like arrhythmias), an arrhythmia activation pattern consistent with that known to occur with TdP in humans.Thus, while all three ECMs produced a monolayer of cardiomyocytes that responded with the expected APD prolongation, only the AFC-ECM produced a monolayer of cardiomyocytes that demonstrated the transition of APD prolongation to tachyarrhythmia characteristic of TdP. As can be seen in Figure 24, 100% of cardiomyocytes on the AFC-ECM responded to the drug E4031 with rotors (TdP-like arrhythmias).

[0129] In further studies, the drugs Domperidone (3 μM), Disopyramide (100 μM), Azimilide (10 μM), d,l Sotalol (D,1 Sotalol) (100 μM), Ibutilide (0.10 μM), and Bepridil (10 μM) were tested with mature cardiomyocytes on AFC-ECM (without silicone insert) prepared in Example 4 in a 96-well plate using the plate reader method described above. The results of the test are shown in FIG. 25. As can be seen in these results, different types of arrhythmias were caused by the different drugs noted in the recordings, namely tachyarrhythmia (TA), quiescence (Q), and early afterdepolarization (EAD). These are the ranges of arrhythmias observed in response to drugs classified by the FDA as high risk for causing TdP fatal arrhythmias in patients.

[0130] Various drugs shown in Tables 3 and 4 below were also tested using the plate reader method described above with mature cardiomyocytes on AFC-ECM (without silicone insert) prepared in Example 4 in a 96-well plate, and observations were noted for any arrhythmias detected and APD90 prolongation at 10 times the effective therapeutic plasma concentration (ETPC). The drugs were selected from the CiPA Initiative list of compounds for CiPA validation and testing. These are classified as high, intermediate or low risk for 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. Table 3 [Table 3] Table 4 [Table 4]

[0131] Further analysis of the data from the drugs tested in Tables 3 and 4 is shown in Figures 26-29. Figure 26 graphically illustrates the total number of arrhythmias observed for each drug compound at any dose. Figure 27 graphically illustrates the relative incidence of arrhythmias for each drug at specific clinically relevant doses of 10× the effective therapeutic plasma concentration (ETPC).

[0132] FIG. 28 graphically illustrates action potential triangulation (APD90-APD30) over time (milliseconds) for each drug. Triangulation is defined as the repolarization time from APD30 to APD90. Action potential triangulation (APD90-APD30) is used as a predictor for proarrhythmic effects of drugs. FIG. 29 illustrates action potential triangulation over time (milliseconds) for select drugs comparing cardiomyocyte assay performance of cardiomyocytes on AFC-ECM (SBS-AF Matrix) vs. Matrigel™ ECM.

[0133] FIG. 30 graphically depicts maximum drug-induced action potential triangulation for the listed drugs comparing cardiomyocyte performance of iCell® hiPSC-CMs from Cellular Dynamics (open circles) versus Cor.4U® hiPSC-CMs from Ncardia (closed circles) 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. In contrast to FIG. 28, there is little stratification between high and intermediate risk compounds in the data set shown in FIG. 30. Thus, the AFC-ECM of the present disclosure provides for the production of more mature cardiomyocytes with more realistic function and drug responsiveness than other native cell-derived ECMs or Matrigel™ ECM. Example 6 Observation of cell-based constructs of mature cardiomyocytes on AFC-ECM

[0134] Cellular constructs of mature cardiomyocytes on AFC-ECM were prepared according to the procedure described in Example 4 above, with the following modifications noted below:

[0135] AFC-ECM was deposited onto Thermanox coverslips to allow for immunostaining of cells and imaging using a laser scanning confocal microscope (Nikon A1R).

[0136] Matrigel™ ECM was applied to a separate subset of Thermanox coverslips for comparison.

[0137] Cells (iCell® Cardiomyocytes) from Cellular Dynamics International-FUJI were plated as monolayers on these ECM-coated Thermanox coverslips at a density of 200,000 cells per well in 6-well plates.

[0138] After 7 days of incubation in cell culture medium, cells were fixed in 3% paraformaldehyde and processed for immunocytochemistry using application of commercially available primary antibodies to determine cellular expression and localization in hiPSC-CMs. The following primary antibodies against important cardiac filament 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 staining was used to mark nuclei.

[0139] All procedures for cell labeling and visualization are as described in the following references, which are 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).

[0140] Cell shape was quantified using fluorescent images analyzed in NIS Elements Software. Cell area and perimeter were used to quantitate cell circularity using a well-established mathematical equation; circularity index = 4π * area / perimeter. 2 .

[0141] In some cases, mitochondria were stained using MitoTracker™ Red CMXRos (Thermo Fisher).

[0142] Results: Consistent with the results seen in Example 4 above, data using fluorescent labeling and imaging demonstrate that AFC-ECM promotes rapid (7 day) maturation of hiPSC-CMs, showing that hiPSC-CMs cultured on Matrigel™ ECM are round in shape and have disorganized sarcomeres, whereas the same batch of hiPSC-CMs cultured on AFC-ECM (syngeneic control) are rod-shaped and have tight compaction / organization of sarcomeres and myofilaments.

[0143] The photomicrograph in Figure 31 shows hiPSC-CMs cultured on Matrigel™ ECM vs. AFC-ECM using immunofluorescent staining for troponin I and using DAPI to mark the nuclei. The photomicrograph in Figure 32 shows hiPSC-CMs cultured on Matrigel™ ECM vs. AFC-ECM using immunofluorescent staining for α-actinin and using DAPI to mark the nuclei. The photomicrograph in Figure 33 shows hiPSC-CMs cultured on Matrigel™ ECM vs. AFC-ECM using immunofluorescent staining for cTnT and N-cadherin and using DAPI to mark the nuclei. As can be seen in Figures 31-33, hiPSC-CMs cultured on Matrigel™ ECM are round in shape and have disorganization of sarcomeres, while the same batch of hiPSC-CMs cultured on AFC-ECM (syngeneic control) are rod-shaped and have tight compaction / structuring of sarcomeres and myofilaments. In Figures 31-33, some examples of cells are identified with arrows as either round or rod-shaped cells, and some examples of sarcomeres are identified with arrows. The photomicrograph in Figure 34 shows a single hiPSC-CM cell cultured on Matrigel™ ECM vs. AFC-ECM using immunofluorescent staining for cTnT and DAPI to mark the nucleus, showing that the cells cultured on Matrigel™ ECM are round, while the cells cultured on AFC-ECM are rod-shaped. The micrographs in Figure 35 show single hiPSC-CM cells cultured on Matrigel™ ECM versus AFC-ECM using immunofluorescent staining for α-actinin and DAPI to mark nuclei, showing that cells cultured on Matrigel™ ECM are round, while cells cultured on AFC-ECM are rod-shaped. The graph in Figure 36 shows a comparison of cell circularity of the single cells shown in Figure 35, showing a higher circularity index for cells cultured on Matrigel™ ECM, indicating greater roundness.

[0144] The photomicrographs in Figure 37 show hiPSC-CMs cultured on Matrigel™ ECM vs. AFC-ECM using immunofluorescence staining for cTnI expression, using DAPI to mark nuclei. Figure 38 shows Western blotting of hiPSC-CMs on Matrigel™ ECM and AFC-ECM for cTnI expression and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Analysis of the Western blotting in Figure 38 is shown in the graph in Figure 39, which shows cTnI expression compared to GAPDH for hiPSC-CMs on Matrigel™ ECM vs. AFC-ECM. This analysis shows a higher cTnI expression / GAPDH ratio for hiPSC-CMs on AFC-ECM than on Matrigel™ ECM, indicating more robust cTnI expression from hiPSC-CMs on AFC-ECM than on Matrigel™ ECM.

[0145] The micrographs in Figure 40 show hiPSC-CMs on Matrigel™ ECM vs. AFC-ECM stained for mitochondria with MitoTracker Red, indicating that cells on AFC-ECM have more mitochondria and mitochondria with a more polarized inner membrane potential, as evidenced by the larger red signal. The graph in Figure 41 shows MitoTraker™ Red fluorescence intensity / cardiomyocytes for hiPSC-CMs on Matrigel™ ECM vs. AFC-ECM, indicating that hiPSC-CMs on AFC-ECM have higher MitoTraker™ Red fluorescence intensity, indicating that these cells have more mitochondria and mitochondria with a more polarized inner membrane potential than hiPSC-CMs on Matrigel™ ECM.

[0146] The photomicrographs (transmitted light) in Figure 42 show hiPSC-CMs cultured on Matrigel™ ECM and AFC-ECM coated on a microelectrode array (MEA) plate. As can be seen in Figure 42, hiPSC-CMs cultured on Matrigel™ ECM are round in shape, while hiPSC-CMs cultured on AFC-ECM are rod-shaped.

Claims

1. A method for maturation of immature cardiomyocytes derived from human induced pluripotent stem cells, said method comprising: (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM), wherein the immature cardiomyocytes exhibit a single nucleus; (b) providing an in vitro derived extracellular matrix (AFC-ECM) by culturing cells isolated from amniotic fluid obtained from a human of greater than 37 weeks gestation, said AFC-ECM comprising laminin, collagen alpha-1 (XVIII), basement membrane specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), or isoforms thereof; (c) contacting the immature hiPSC-CM with the AFC-ECM; and (d) culturing the immature hiPSC-CM with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into binucleated mature cardiomyocytes. A method comprising:

2. The method of claim 1, wherein the step of contacting the immature hiPSC-CM with the AFC-ECM includes plating the immature hiPSC-CM on the AFC-ECM.

3. The method of claim 2, wherein the AFC-ECM is contained in a cell culture vessel or a multi-well plate during plating.

4. A method according to any one of claims 1 to 3, wherein the mature cardiomyocytes form as a monolayer on the AFC-ECM.

5. The method of claim 4, wherein the monolayer of mature cardiomyocytes is a confluent monolayer.

6. A method according to any one of claims 1 to 5, wherein the immature hiPSC-CM does not express the inward rectifier potassium channel Kir2.

1.

7. A method according to any one of claims 1 to 6, wherein the mature cardiomyocytes are characterized by rod-shaped cells with a distinct sarcomere structure similar to adult human cardiac tissue.

8. A method according to any one of claims 1 to 7, wherein the mature cardiomyocytes are anisotropically aligned on an anisotropic track on which the AFC-ECM is structured.

9. The method of claim 1, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2 or the isoform of agrin is isoform 6.

10. The method of claim 1, wherein the AFC-ECM further comprises fibronectin or an isoform thereof.

11. The method of claim 1, wherein the AFC-ECM does not contain decorin, perlecan, and collagen(III).

12. The method of any one of claims 1 to 11, wherein the cells isolated from amniotic fluid include fetal cells from the amniotic membrane, skin, or the gastrointestinal, respiratory, or genitourinary tract.

13. The method according to any one of claims 1 to 12, wherein the period of maturation of the immature cardiomyocytes into the mature cardiomyocytes during step (d) is between 4 days and 14 days.

14. A cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived in vitro by culturing cells isolated from amniotic fluid obtained from a human of gestational age greater than 37 weeks, 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 isoforms thereof; the mature cardiomyocytes are AFC-ECM-cultured cardiomyocytes derived from immature human induced pluripotent stem cells (hiPSC-CM); A cell construct, wherein the mature cardiomyocytes are characterized by two nuclei.

15. The cell construct described in claim 14, wherein the monolayer of mature cardiomyocytes is a confluent monolayer.

16. A cell construct described in claim 14 or 15, wherein the immature hiPSC-CM does not express the inward rectifier potassium channel Kir2.

1.

17. A cell construct described in any one of claims 14 to 16, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinct sarcomere structure similar to adult human cardiac tissue.

18. The cell construct of claim 14, wherein the monolayer of mature cardiomyocytes is anisotropically aligned on an anisotropic track on which the AFC-ECM is structured.

19. A cell construct described in any one of claims 14 to 18, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2 and / or the isoform of agrin is isoform 6.

20. A cell structure described in any one of claims 14 to 19, wherein the AFC-ECM further contains fibronectin and / or its isoforms.

21. A cell structure described in any one of claims 14 to 20, wherein the AFC-ECM does not contain decorin, perlecan, and / or collagen (III).

22. A cell construct according to any one of claims 14 to 21, wherein the cells isolated from amniotic fluid include fetal cells from the amniotic membrane, skin, or the gastrointestinal, respiratory, or genitourinary tract.

23. A method for producing a cellular construct of mature cardiomyocytes, the method comprising: Maturating immature cardiomyocytes derived from human induced pluripotent stem cells according to the method of any one of claims 1 to 13, wherein the mature cardiomyocytes are characterized by two nuclei; and forming a monolayer of said mature cardiomyocytes on said AFC-ECM, thereby forming said cell construct; A method comprising:

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