Methods for maturation of cardiomyocytes on the ECM derived from amniotic fluid cells, cell constructs, and their use for screening of cardiotoxicity and proarrhythmic effects 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-references to related applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 808,690, filed on 21 February 2019, which is incorporated herein by reference in its entirety.

[0002] This disclosure generally relates to the use of cell constructs of human stem cell-derived cardiomyocytes on cell-derived extracellular matrix, methods for creating such constructs, and methods for using these constructs for cardiotoxicity and proarrhythmic screening assays of drug compounds. [Background technology]

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

[0004] Current preclinical drug candidate trial guidelines from the IHC (ICH S7A and S7B - Pharmacology Studies) are for genetically modified xenocellular and in These studies rely on vivo animal models. It is becoming increasingly recognized that these studies, such as hERG assays and QT prolongation studies, do not accurately predict the risk of cardiotoxicity and proarrhythmia in humans. Since 2005, the cardiac safety of drug compounds has been determined almost exclusively by their effect or potential effect on the QT interval or action potential duration (APD) on the electrocardiogram (ECG), as well as their potential to lead to life-threatening arrhythmias known as torsades de pointes (TdP). However, QT prolongation is not an ideal indicator of TdP, as drugs that prolong the QT interval do not necessarily cause TdP. It is now recognized that the QT prolongation parameter is merely a surrogate marker for proarrhythmia. Data from preclinical and clinical trials show that there is no consistent relationship between the magnitude of QT prolongation and the risk of developing fatal arrhythmias such as TdP.

[0005] Therefore, the US Food and Drug Administration (FDA) and other stakeholders in drug discovery have called for advancements in preclinical cardiotoxicity testing. The proposed new paradigm is called the Comprehensive In-Vitamin Proarrhythmic Assay (CiPA). An integral part of the CiPA Initiative (http: / / cipaproject.org / ) is the incorporation of data collected from human stem cell-derived cardiomyocytes for cardiotoxicity and proarrhythmic assays. The overall objective of these new proposed guidelines is to provide a more accurate and comprehensive mechanism-based assessment of proarrhythmic potential, which will more accurately assess the risks of new drugs. Considering the proposed CiPA Initiative guidelines, the FDA specifies two advancements that must be made before human cardiomyocytes can be incorporated into the new initiative. First, the growth and maturation states of human stem cell-derived cardiomyocytes need to evolve to more closely resemble the structure and function of adult human cardiomyocytes. Second, a reliable high-throughput screening platform using these cells needs to be developed.

[0006] There are generally three types of systems currently used to evaluate the electrophysiology of cardiomyocytes in vitro: 1) patch-clamp systems; 2) microelectrode array (MEA) systems; and 3) voltage-sensitive dye (VSD) visualization methods. Manual patch-clamp systems are most commonly used in very early investigative studies to evaluate the electrophysiology of individual cells. While these devices provide accurate and highly sensitive ion current measurements, they cannot be used in in vitro cell systems that more closely mimic cell-cell interactions in cardiac tissue. MEA systems include electrodes that are incorporated into cell culture wells to enable measurement of currents across the wells. While enabling high-throughput analysis and the ability to measure impedance in in vitro cell systems, these systems have low spatial resolution, do not provide data on action potential shape, and hinder the ability to evaluate direct cell visualization and 3-D culture systems that more closely mimic cardiac tissue architecture. The VSD system is attracting increasing attention because it addresses many of the shortcomings of the current technologies mentioned above, with features including: 1) enabling high-throughput analysis; 2) providing high spatial resolution; 3) enabling visualization of impulse propagation across culture dishes; and 4) enabling modification of culture conditions, including the addition of extracellular matrix, leading to a more natural test environment.

[0007] The use of human stem cell-derived cardiomyocytes, such as induced pluripotent stem cells, has had limited success to date in cardiotoxicity and proarrhythmic assay screening. Induced human pluripotent stem cell-derived cardiomyocytes (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 produced in large quantities in vitro. Furthermore, hiPSC-CMs can be obtained from patient-specific hiPSCs for specific individuals. However, there are still hurdles to overcome in order to make hiPSC-CMs a meaningful part of the new CiPA Initiative paradigm. 1) The structural and functional maturation of hiPSC-CMs needs to be advanced. In particular, the Kir2.1 potassium channel is absent in currently available hiPSC-CMs, and sodium channel expression is low. Currently available hiPSC-CMs are functionally and structurally very immature. The vast majority of currently used hiPSC-CM-based proarrhythmic screening assays rely on immature fetal-like cells that do not resemble adult cardiomyocytes in structure or function.2) Electrical pacing of hiPSC-CM monolayers is required in high-throughput electrophysiological screening platforms. The vast majority of current hiPSC-CM-based proarrhythmic screenings rely solely on electric field potential duration (MEA technology) or action potential duration prolongation (VSD technology) as surrogate markers for TdP induction. This is a limitation, as not all drugs that prolong the action potential (QT interval) cause lethal TdP arrhythmias. Certain maturation states of immature hiPSC-CMs have been achieved by using Matrigel® ECM and bone marrow cell-derived ECMs in culture, but the use of these hiPSC-CMs in proarrhythmic screening assays has not yielded consistent results, particularly in observing arrhythmia activation patterns consistent with those known to occur in humans for TdP, between low-risk and high-risk drugs.

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

[0009] This disclosure provides solutions to at least some of the aforementioned limitations and shortcomings in the present technology related to the evaluation and screening of the cardiosafety liability of drug candidates. The solutions presuppose the discovery of an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid, which can be used for the maturation of human stem cell-derived cardiomyocytes. These mature cardiomyocytes can then be used in cell constructs with the AFC-ECM for drug cardiotoxicity and proarrhythmic testing.

[0010] In one embodiment, a method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells is disclosed, comprising the steps of (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix (AFC-ECM) induced 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 having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, the mature cardiomyocytes have a morphology similar to or identical to that shown in any one of Figures 6-14. In some embodiments, the immature hiPSC-CM are plated onto the AFC-ECM. In some embodiments, mature cardiomyocytes form a monolayer on the AFC-ECM, thereby forming a cellular construct containing a monolayer of mature cardiomyocytes on the AFC-ECM, and the 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 a distinguishable sarcomere structure. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a round shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having more mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (buniculous).

[0011] In another embodiment, a cell construct is disclosed comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) in vitro induced from cells isolated from amniotic fluid, wherein 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 having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, the mature cardiomyocytes have a morphology similar to or identical to that shown in any one of Figures 6-14. In some embodiments, the mature cardiomyocytes contain and / or express the inwardly rectifying potassium channel Kir2.1. In certain embodiments, mature cardiomyocytes may 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 a distinctive sarcomere structure (striped appearance) similar to that of adult human heart tissue. In some embodiments, a monolayer of mature cardiomyocytes is aligned on AFC-ECM. In some embodiments, fiber tracks (fiber A track is present on the construct. In some embodiments, AFC-ECM comprises laminin, collagen alpha-1(XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2(IV), and / or 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, AFC-ECM further comprises fibronectin and / or its isoforms. In some embodiments, AFC-ECM does not contain decorin, perlecan, and / or collagen(III). In some embodiments, immature hiPSC-CM does not contain rod-shaped cells having a distinguishable sarcomere structure. In some embodiments, immature hiPSC-CM can be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a round shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having a greater number of mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (buniculous).

[0012] In another embodiment, a method is disclosed for creating a cellular construct of mature cardiomyocytes on an extracellular matrix (AFC-ECM) in vitro induced from cells isolated from amniotic fluid, comprising the steps of (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) in vitro induced from cells isolated from amniotic fluid; (c) plating the immature hiPSC-CMs onto the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in culture medium to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes, thereby forming a monolayer of mature cardiomyocytes on the AFC-ECM and thus forming a cellular construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, the mature cardiomyocytes have a morphology similar to or identical to that 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 cellular 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 disordered myofilaments, having a rounded shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be further characterized by having more mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (buniculous).

[0013] In another embodiment, a method is disclosed for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro, comprising the steps of: contacting the drug compound with mature cardiomyocytes of any one of the cell constructs disclosed herein; and observing electrophysiological changes in the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. The electrophysiological changes in the mature cardiomyocytes support the fact that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. The electrophysiological changes in the mature cardiomyocytes 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 post-depolarization (DAD), and / or early post-depolarization (EAD). Observations may also include cell viability, cell density, and / or cellular morphology. In some embodiments, the electrophysiological change in the mature cardiomyocytes is an extension of the action potential duration (APD). In some embodiments, the electrophysiological change in mature cardiomyocytes is early after depolarization (EAD). In some embodiments, the electrophysiological change in mature cardiomyocytes is delayed after depolarization (DAD). In some embodiments, the electrophysiological change in mature cardiomyocytes is action potential duration prolongation (APD prolongation) plus rotor. In some embodiments, the electrophysiological change in mature cardiomyocytes is arrhythmia.In one aspect, the cell construct can be prepared by a process comprising: (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix induced in vitro from cells isolated from amniotic fluid (AFC-ECM); (c) plating the immature hiPSC-CMs on the AFC-ECM; and (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 the cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to adult human heart tissue. In some embodiments, the mature cardiomyocytes have a morphology similar or identical to that shown in any one of FIGS. 6-14. In one embodiment, the immature hiPSC-CMs do not express the inward rectifier potassium channel Kir2.1. In another embodiment, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In another embodiment, a fibrous track is present on the cell construct. In some embodiments, the immature hiPSC-CMs do not include rod-shaped cells having a distinguishable sarcomere structure. In some embodiments, the immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disorganized muscle filaments, having a circular shape, and / or having a single nucleus. In some embodiments, the mature cardiomyocytes can be further characterized by having a greater amount of mitochondria than immature hiPSC-CMs, having structured dense muscle filaments, and / or having two nuclei (binucleation).

[0014] Also, the following Embodiments 1-20 are disclosed in connection with 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 (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) Providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) Contacting immature hiPSC-CMs with AFC-ECM; and (d) Culturing immature hiPSC-CMs with AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CMs, thereby forming mature cardiomyocytes comprising, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to adult human heart tissue. Embodiment 2 is the method according to Embodiment 1, wherein the immature hiPSC-CMs are plated on AFC-ECM. Embodiment 3 is the method according to any one of Embodiments 1 or 2, wherein the mature cardiomyocytes form a monolayer on AFC-ECM, thereby forming a cell construct comprising a monolayer of mature cardiomyocytes on AFC-ECM, and the mature cardiomyocytes are aligned on 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 rectifier potassium channel Kir2.1. Embodiment 5 is a cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid, wherein the mature cardiomyocytes are cardiomyocytes derived from human induced pluripotent stem cells (hiPSC-CMs) cultured with AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to adult human heart tissue . Embodiment 6 is the cell construct according to Embodiment 5, wherein the monolayer of mature cardiomyocytes is aligned on AFC-ECM. Embodiment 7 is the cell construct according to any one of Embodiments 5 or 6, wherein a fiber track is present on the construct. Embodiment 8 is a cell construct according to 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 isoforms thereof. Embodiment 9 is the cell construct according to Embodiment 8, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2 and / or the isoform of agryn 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 creating a cell construct of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid, (a) A step of providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells, (b) A step of providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) A step of plating immature hiPSC-CM onto AFC-ECM; (d) A step in which immature hiPSC-CMs plated on AFC-ECM are cultured in culture medium to induce maturation of immature hiPSC-CMs into mature cardiomyocytes, forming a monolayer of mature cardiomyocytes on AFC-ECM, thereby forming a cell construct. This method includes a feature in which mature cardiomyocytes are characterized by rod-shaped cells that have a distinctive sarcomere structure similar to that of adult human heart tissue. Embodiment 13 is the method of Embodiment 12, wherein a monolayer of mature cardiomyocytes is aligned on the AFC-ECM. Embodiment 14 is the method according to either Embodiment 12 or 13, wherein the fiber tracks are present on the cell construct. Embodiment 15 is a method for determining the cardiotoxicity and / or proarrhythmic effect 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 electrophysiological changes in the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. Embodiment 16 is the method of Embodiment 15, wherein the electrophysiological change in mature cardiomyocytes is an extension of the action potential duration (APD), and the extension of the APD supports the idea that the drug compound has cardiotoxic and / or proarrhythmic effects on mature cardiomyocytes. Embodiment 17 is the method of Embodiment 15, wherein the electrophysiological change in mature cardiomyocytes is early afterdepolarization, and early afterdepolarization (EAD) supports the idea that the drug compound has cardiotoxic and / or proarrhythmic effects on mature cardiomyocytes. Embodiment 18 is the method of Embodiment 15, wherein the electrophysiological change in mature cardiomyocytes is delayed depolarization, and delayed depolarization (DAD) supports the idea that the drug compound has cardiotoxic and / or proarrhythmic effects on mature cardiomyocytes. Embodiment 19 is the method of Embodiment 15, wherein the electrophysiological changes in mature cardiomyocytes are action potential duration (APD) plus rotor, and the APD prolongation plus rotor supports the conclusion that the drug compound has cardiotoxic and / or proarrhythmic effects on mature cardiomyocytes. Embodiment 20 is the method of Embodiment 15, wherein the electrophysiological changes in mature cardiomyocytes are arrhythmias, and the arrhythmias support the conclusion that the drug compound has cardiotoxic and / or proarrhythmic effects on mature cardiomyocytes.

[0015] The terms “about” or “near” are defined as being close to what is understood by those skilled in the art, and in one non-limiting embodiment, the term is defined as being within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.

[0016] The term “substantially” and its variations are defined as including a range of 10%, 5%, 1%, or 0.5%.

[0017] As used herein, the terms "%w / w" or "wt.%" mean a weight percentage of an ingredient based on the total weight of the material (e.g., a composition) containing the ingredient. In a non-limiting example, 10 grams of an ingredient in 100 grams of a composition constitutes 10% w / w of the ingredient in the total weight of the composition. As used herein, the terms "%v / v" or "vol.%" mean a volume percentage of an ingredient based on the total volume of the material (e.g., a composition) containing the ingredient. In a non-limiting example, 10 mL of an ingredient in 100 mL of a composition constitutes 10% v / v of the ingredient in the total volume of the composition. As used herein, the terms "%w / v" mean a weight percentage of an ingredient based on the total volume of the material (e.g., a composition) containing the ingredient. In a non-limiting example, 10 grams of an ingredient in 100 mL of a composition constitutes 10% w / v of the ingredient in the total volume of the composition. As used herein, the terms "%v / w" mean a volume percentage of an ingredient based on the total weight of the material (e.g., a composition) containing the ingredient. In a non-limiting example, 10 mL of a component in 100 grams of a composition represents 10% v / w of the component in the total weight of the composition.

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

[0019] The term “effective” means, where used in the specification and / or claims, appropriate for achieving the 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 comprehensive or open-ended and do not exclude further elements or methodological steps that are not mentioned.

[0021] The use of the word “a” or “an” can mean “one” when used in conjunction with the terms “comprising,” “having,” “including,” or “containing” (or any variation of these words), but it also coincides 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 "contain," "essentially consist of," or "consist of" any of the components or steps disclosed throughout this Spec. With respect to the transitional phrase "essentially consisting of," in one non-limiting aspect, the fundamental and novel features of the cell constructs disclosed herein are their use in screening tests for the cardiotoxicity and / or proarrhythmia of drug compounds, due to their ability to mature stem cell-derived cardiomyocytes to a mature state similar to that of mature natural adult cardiomyocytes and natural cardiac tissue.

[0023] Any embodiment discussed herein is intended to be implementable with respect to any method or composition of the present invention, and vice versa. Furthermore, the compositions of the present invention can be used to achieve the methods of the present invention.

[0024] Other objects, features, and advantages of the present invention will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples illustrate specific embodiments of the present invention, but are provided for illustrative purposes only. This is because various modifications and alterations within the spirit and scope of the present 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) A method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells, (a) A step of providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) A step of providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) the step of bringing the immature hiPSC-CM into contact with the AFC-ECM; and (d) A step of culturing the immature hiPSC-CM together with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM and thereby form mature cardiomyocytes; A method comprising, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. (Item 2) The method according to item 1, wherein the immature hiPSC-CM is plated onto the AFC-ECM. (Item 3) The method according to either item 1 or 2, wherein the mature cardiomyocytes form a monolayer on the AFC-ECM, thereby forming a cell construct on the AFC-ECM that includes a monolayer of mature cardiomyocytes, and the mature cardiomyocytes are aligned on the AFC-ECM. (Item 4) The method according to any one of items 1 to 3, wherein the immature hiPSC-CM does not express the inward rectifying potassium channel Kir2.1. (Item 5) A cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid, wherein the mature cardiomyocytes are human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) cultured in AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. (Item 6) The cell construct according to item 5, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. (Item 7) A cell construct according to either item 5 or 6, wherein fiber tracks are present on the construct. (Item 8) The cell construct according to 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 isoforms thereof. (Item 9) The cell construct according to item 8, wherein the isoform of collagen alpha-1(XVIII) is isoform 2, and / or the isoform of agryn is isoform 6. (Item 10) The cell construct according to any one of item 8 or 9, wherein the AFC-ECM further comprises fibronectin and / or its isoforms. (Item 11) The AFC-ECM is a cell construct according to any one of items 5 to 10, wherein the AFC-ECM does not contain decorin, perlecan, and / or collagen(III). (Item 12) A method for creating a cell construct of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid, (a) A step of providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells, (b) A step of providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) Plating the immature hiPSC-CM onto the AFC-ECM; (d) Culturing the immature hiPSC-CM plated on the AFC-ECM in a culture medium to induce the maturation of the immature hiPSC-CM into mature cardiomyocytes, forming a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cell construct; A method comprising, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. (Item 13) The method according to item 12, wherein the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. (Item 14) The method according to either item 12 or 13, wherein fiber tracks are present on the cell construct. (Item 15) A method for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro, comprising the steps of: contacting the drug compound with mature cardiomyocytes of a cell construct described in any one of items 5 to 11; and observing electrophysiological changes in the mature cardiomyocytes to determine whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. (Item 16) The method according to item 15, wherein the electrophysiological change in the mature cardiomyocytes is an extension of the action potential duration (APD), and the extension of the APD supports the finding that the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes. (Item 17) The method according to item 15, wherein the electrophysiological change in the mature cardiomyocytes is early afterdepolarization (EAD), and the early afterdepolarization (EAD) supports the idea that the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes. (Item 18) The method according to item 15, wherein the electrophysiological change in the mature cardiomyocytes is delayed depolarization (DAD), and the delayed depolarization (DAD) supports the finding that the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes. (Item 19) The method according to item 15, wherein the electrophysiological change in the mature cardiomyocytes is action potential duration (APD) plus rotor, and the APD prolongation plus rotor supports that the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes. (Item 20) The method according to item 15, wherein the electrophysiological changes in the mature cardiomyocytes are arrhythmias, and the arrhythmias support that the drug compound has cardiotoxic and / or proarrhythmic effects on the cardiomyocytes. [Brief explanation of the drawing]

[0025] [Figure 1] Figure 1 is a bright-field micrograph of amniotic fluid cell-derived ECM at a magnification of 100x using a 10x objective lens.

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

[0027] [Figure 3] Figure 3 is a scatter plot showing the quantitative analysis of adhesion and stiffness (elastic modulus) of ECM derived from bone marrow cells and amniotic fluid cells. Each point represents an independent measurement point.

[0028] [Figure 4] Figure 4 shows micrographs of iPSC cultures on day 0 and day 2 on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

[0029] [Figure 5] Figure 5 shows plots of growth curves for iPSCs cultured in the presence of amniotic fluid cell-derived ECM and bone marrow cell-derived ECM.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0046] [Figure 22] Figure 22 is a schematic diagram of the instrument configuration for recording transient action potentials or calcium measurements using mature cardiomyocytes on AFC-ECM in a multiwell plate.

[0047] [Figure 23-1] Figure 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] Figure 24 is a bar graph showing the number of stable rotors (TdP-like arrhythmias) from cardiomyocytes cultured on Matrigel® ECM, BM-ECM, and AFC-ECM after contact with the drug E4031.

[0049] [Figure 25] Figure 25 shows recordings of spontaneous action potentials from mature cardiomyocytes cultured on AFC-ECM for various drugs.

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

[0051] [Figure 27] Figure 27 is a bar graph showing the percentage of wells with arrhythmias at 10 × effective therapeutic plasma concentration (ETPC) for each of the listed drugs.

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

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

[0054] [Figure 30] Figure 30 is a bar graph of the maximum drug-induced action potential triangulation for the listed drugs, comparing the cardiomyocyte performance of iCell® hiPSC-CM (white circles) from Cellular Dynamics versus Cor.4U® hiPSC-CM (black circles) from Ncardia at arbitrary concentrations of the listed drugs. Figure from Blinova et al, 2018, Cell Reports.

[0055] [Figure 31] Figure 31 is a micrograph of hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for troponin I and DAPI to mark the nuclei.

[0056] [Figure 32] Figure 32 shows micrographs of hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for α-actinin and DAPI to mark the nuclei.

[0057] [Figure 33] Figure 33 is a micrograph of hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for cTnT and N-cadherin, with DAPI used to mark the nuclei.

[0058] [Figure 34] Figure 34 is a micrograph of a single hiPSC-CM cell cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for cTnT and DAPI to mark the nucleus.

[0059] [Figure 35] Figure 35 is a micrograph of a single hiPSC-CM cell cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for α-actinin and DAPI to mark the nucleus.

[0060] [Figure 36] Figure 36 is a graph comparing the cell roundness of single cells shown in Figure 35.

[0061] [Figure 37] Figure 37 shows micrographs of hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for cTnI expression and DAPI to mark the nuclei.

[0062] [Figure 38] Figure 38 shows Western blotting of hiPSC-CMs on Matrigel® ECM and AFC-ECM for cTnI expression and GAPDH.

[0063] [Figure 39] Figure 39 shows a graph of cTnI expression in Matrigel® ECM versus hiPSC-CM on AFC-ECM, compared to GAPDH.

[0064] [Figure 40]Figure 40 shows micrographs of hiPSC-CMs on Matrigel® ECM versus AFC-ECM, stained for mitochondria with MitoTracker Red.

[0065] [Figure 41] Figure 41 is a graph showing MitoTraker® Red fluorescence intensity / cardiomyocyte for Matrigel® ECM versus hiPSC-CM on AFC-ECM.

[0066] [Figure 42] Figure 42 shows micrographs (transmitted light) of hiPSC-CM cultured on Matrigel® ECM and AFC-ECM coated on microelectrode array (MEA) plates. [Modes for carrying out the invention]

[0067] Human stem cell-derived cardiomyocytes (hiPSC-CMs) matured in vitro on an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid, such as those derived from human induced pluripotent stem cells, demonstrate better and more consistent cardiotoxicity and proarrhythmic assay results than immature hiPSC-CMs or hiPSC-CMs matured on other ECMs, such as Matrigel® ECM and myeloid cell-derived ECMs. Remarkably, hiPSC-CMs matured on AFC-ECM demonstrate a higher state of maturation than hiPSC-CMs matured on other ECMs, such as Matrigel® ECMs, and even on other native cell-derived ECMs, such as myeloid cell-derived ECMs, as indicated by their cellular morphology and sarcomere structure, which are found in cardiomyocytes of normal adult human heart tissue. Furthermore, more robust expression of the cTnI (cardiac troponin I) protein, as demonstrated by Western blotting, was observed in hiPSC-CMs cultured on AFC-ECM compared to hiPSC-CMs cultured on Matrigel® ECM. Additionally, hiPSC-CMs cultured on AFC-ECM had more mitochondria and mitochondria with more polarized inner membrane potentials than hiPSC-CMs cultured on Matrigel® ECM. Therefore, AFC-ECM can stimulate mitochondrial biosynthesis, maturation, and function in hiPSC-CMs. Normal adult or mature human heart tissue is characterized by rod-shaped cells with sarcomere structures (a striped appearance). The morphology and sarcomere structure of cardiomyocytes can be visually identified by microscopy (by transmitted light or immunofluorescence staining). As a non-limiting example, the morphology and sarcomere structure of mature cardiomyocytes on AFC-ECM can be clearly observed 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 silicone substrates such as PDMS was not required to achieve these results.Remarkably, cell constructs containing a monolayer of mature cardiomyocytes matured from immature hiPSC-CMs in culture on an AFC-ECM have been shown to be useful, for example, in high-throughput in vitro screening assays, by enabling consistent visualization of drug-induced arrhythmias such as torsades de pointes (TdPs). This “TdP in the Dish” technique represents a significant advance beyond relying on electric field potential duration (MEA technique) or action potential duration prolongation as surrogate markers for drug-induced TdPs. Thus, the cell constructs and methods disclosed herein surpass current CiPA Initiative guidelines by developing more comprehensive and predictive in vitro arrhythmia assays that enable visualization of arrhythmic events in an in vitro model, rather than simple polarization and depolarization events, which are indirect indicators of proarrhythmia. The cell systems and methods outlined herein enable the propagation and visualization of arrhythmic events in an in vitro human heart monolayer model system. In some embodiments, the 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 disordered myofilaments, having a round shape, and / or having a single nucleus. In some embodiments, mature cardiomyocytes can be characterized by having rod-shaped cells with a distinguishable sarcomere structure (a striped appearance). In some embodiments, mature cardiomyocytes can be further characterized by having more mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (bifurcation).

[0068] A method for forming mature cardiomyocytes (mature hiPSC-CMs) from immature human induced pluripotent stem cells (immature hiPSC-CMs) in culture using an extracellular matrix (AFC-ECM) derived from cells isolated from amniotic fluid and induced in vitro is disclosed herein. A cell construct containing a monolayer of these mature cardiomyocytes on an AFC-ECM, useful for drug compound cardiotoxicity and / or proarrhythmic screening assays, is also disclosed herein. A method for determining the cardiotoxic and / or proarrhythmic effects of drug compounds in vitro using such a cell construct is also disclosed herein. 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 contains several cell sources, including the amniotic membrane, skin, and developing fetal cells detached from the digestive tract, respiratory organs, and urogenital organs. The placenta also contains several cell sources, including membrane sheets (amnion and chorionic membrane), chorionic 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 the amniotic fluid of mammals, including but not limited to humans (homo sapiens), mice, rabbits, cats, dogs, pigs, horses, or primates. In preferred embodiments, the cells are from human amniotic fluid. Amniotic fluid can be supplied from full-term (more than approximately 37 weeks of gestation) or premature (less than approximately 37 weeks of gestation) humans. Preterm births include late preterm births (approximately 33 to 37 weeks of gestation), mid-preterm births (approximately 29 to 33 weeks of gestation), and very preterm births (approximately 23 to 29 weeks of gestation). Amniotic fluid can be supplied from a human before birth at any stage of gestation in which amniotic fluid is present, and can be combined with the source of amniotic fluid at birth. Generally, amniotic fluid is collected prior to birth by amniocentesis. In some embodiments, amniotic fluid is supplied from a human during full-term, preterm, late preterm, mid-preterm, very preterm births, or before birth, or in combination thereof. In some embodiments, amniotic fluid is supplied prenatally and collected between approximately 10 weeks of gestation and birth, or between approximately 10 weeks and 23 weeks of gestation, or between approximately 10 weeks and 16 weeks of gestation, or between approximately 12 weeks and 23 weeks of gestation, or between approximately 12 weeks and 16 weeks of gestation. In some embodiments, amniotic fluid supplied prenatally is collected by amniocentesis. Cells can be obtained and isolated from amniotic fluid by methods known in this art, for example, by the method disclosed in Murphy et al., Amniotic Fluid Stem Cells, Perinatal Stem Cells, Second Ed. 2013.

[0070] Amniotic fluid contains cells that have the ability to differentiate spontaneously or as a result of treatment with certain growth factors or combinations of growth factors known to those skilled in the art into cell types derived from all three germ layers (ectoderm, endoderm, and mesoderm). That is, a single cell has the ability 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 of the developing fetus detached from the amniotic membrane, skin, and the digestive tract, respiratory organs, and urogenital organs. Due to their origin in the amniotic fluid and placental membranes, these cells may retain a high degree of pluripotent differentiation potential and may contain cell populations containing cells from all three germ layers. Amniotic fluid cells may contain stem cells. In some embodiments, amniotic fluid cells are isolated stem cells. In some embodiments, amniotic fluid cells are cell types derived from all three germ layers (ectoderm, endoderm, and mesoderm) and / or multipotent stem cells and / or pluripotent stem cells. Includes stem cells that have the ability to differentiate into (cells).

[0071] The amniotic fluid cell-derived ECM disclosed herein may comprise a variety of proteins. Proteins in the ECM can be identified by methods 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 comprises, essentially consists of, or may consist of, 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 cell-derived ECM further comprises, essentially consists of, or consists of fibronectin and / or its isoforms. In some embodiments, the amniotic fluid cell-derived ECM does not contain one or all of decorin, perlecan, and collagen(III). Some notable differences between the amniotic fluid cell-derived ECM and the bone marrow cell-derived matrix proteins of the present invention 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] ECM derived from amniotic fluid cells can be produced through the following process: (a) Isolate cells from amniotic fluid, (b) Seed the isolated cells into a cell culture vessel or a cell culture vessel coated with a substrate. (c) Add culture medium to the cell culture vessel, and (d) Culturing the cells to thereby produce cell-derived ECM, and (e) Decellularizing the cell-derived ECM as needed.

[0073] Any seeding density of cells that allows the cells to form a confluent monolayer either immediately or after a period of culture may be used. In some embodiments, the seeding density is from about 10 cells / cm

[0075] , to about 100,000 cells / cm 2 , or from about 100 cells / cm 2 to about 75,000 cells / cm 2 , or from about 500 cells / cm 2 to about 50,000 cells / cm 2 , or from about 500 cells / cm 2 to about 10,000 cells / cm 2 , or from about 500 cells / cm 2 to about 5,000 cells / cm 2 , or from about 500 cells / cm 2 to about 2,500 cells / cm 2 , or from about 1,000 cells / cm 2 to about 25,000 cells / cm 2 , or from about 2,000 cells / cm 2 to about IO,000 cells / cm 2 , or from about 3,000 cells / cm 2 to about 5,000 cells / cm 2 .

[0074] For the present invention, any type of container suitable for culturing cells can be used. Examples include, but are not limited to, cell culture flasks, T flasks, stirred flasks, rotating flasks, fermenters, and bioreactors. Shaking bottles, shaking flasks, tubes, and other containers are also suitable when placed on a shaking platform or shaker. The cell culture container can be coated with a substrate to improve cell adhesion. Non-limiting examples of suitable substrates for coating the cell container are fibronectin.

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

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

[0077] Amniotic fluid cells can be cultured in an incubator at 37°C with 5% CO2 and 90% humidity. Culture can be carried out under a variety of environmental conditions, including but not limited to normal oxygen (20-21% oxygen in the atmosphere) or hypoxic conditions.

[0078] Decellularization of amniotic fluid cell-derived ECM from amniotic fluid cells may involve the removal or non-viability of living amniotic fluid cells. Decellularization of amniotic fluid cells from the ECM may involve, but are not limited to, lysing the amniotic fluid cells and then removing the lysed cells by washing, methods known in this technique. Various substances can be used to remove amniotic fluid cells from the ECM. Non-limiting examples include "extraction buffers" containing TRITON® X-100 and ammonium hydroxide in a PBS buffer. After the ECM is decellularized from amniotic fluid cells, the resulting ECM is substantially cell-free or does not contain living amniotic fluid cells. If feeder cells are used, the decellularization method applies to any living feeder cells present on the ECM, and therefore the ECM is substantially cell-free or does not contain living feeder cells. Therefore, a decellularized ECM means that the ECM is cell-free, and 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 above method is also applicable to the production of cell-derived extracorporeal membranes (ECM) from cells from the umbilical cord, including umbilical cord blood and Wharton's jelly, as well as from other perinatal cells such as cells from placental tissue, including membrane sheets (amnion and chorionic membrane), chorionic villi, and blood.

[0081] In one embodiment, perinatal cell-derived ECM is produced by the following process. (a) Isolate cells from the umbilical cord, (b) Seed the isolated cells into a cell culture vessel or a cell culture vessel coated with a substrate. (c) Add culture medium to the cell culture vessel, and (d) Culturing cells to produce cell-derived ECM, and (e) Decellularize the cell-derived ECM as needed. In some embodiments, the cells isolated from the umbilical cord are from umbilical cord blood and / or Wharton's jelly.

[0082] In another embodiment, perinatal cell-derived ECM is produced by the following process: (a) Isolate cells from placental tissue, (b) Seed the isolated cells into a cell culture vessel or a cell culture vessel coated with a substrate. (c) Add culture medium to the cell culture vessel, and (d) Culturing cells to produce cell-derived ECM, and (e) Decellularize the cell-derived ECM as needed. In some embodiments, cells isolated from placental tissue are from membrane sheets (amnion and / or chorion), chorionic villi, and / or blood.

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

[0084] In another embodiment, an extracellular matrix (ECM) derived from cells isolated from placental tissue in vitro is disclosed. 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 maturing immature hiPSC-CM

[0085] A method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells is disclosed herein, comprising the steps of (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing extracellular matrix (AFC-ECM) induced 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 having a distinguishable sarcomere structure similar to that of adult human heart tissue. Normal adult human heart tissue is characterized by rod-shaped cells having a sarcomere structure (a striped appearance). The morphology and sarcomere structure of cardiomyocytes can be visually identified by microscopy. As a non-limiting example, the morphology and sarcomere structure of mature cardiomyocytes on AFC-ECM can be clearly observed by the presence of rod-shaped cells with a striped appearance, identified by the arrows in the micrograph in Figure 14.

[0086] Also disclosed herein is a cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid, wherein the mature cardiomyocytes are human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) cultured on the AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In certain embodiments, the mature cardiomyocytes may be matured from immature human induced pluripotent stem cell-derived cardiomyocytes (immature hiPSC-CM) in culture on the AFC-ECM, and the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, the mature cardiomyocytes contain and / or express the inwardly rectifying potassium channel Kir2.1. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, fibrous tracks are present on the construct. In some embodiments, AFC-ECM comprises laminin, collagen alpha-1(XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2(IV), and / or 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, AFC-ECM further comprises fibronectin and / or its isoforms. In some embodiments, AFC-ECM does not contain decorin, perlecan, and / or collagen(III). In some embodiments, immature hiPSC-CM does not contain rod-shaped cells having a distinguishable sarcomere structure. In some embodiments, immature hiPSC-CM can be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a round shape, and / or having a single nucleus.In some embodiments, mature cardiomyocytes can be further characterized by having a greater number of mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (buniculous).

[0087] A method for preparing a cell construct comprising mature cardiomyocytes on an extracellular matrix (AFC-ECM) in vitro induced from cells isolated from amniotic fluid, comprising the steps of (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) in vitro induced from cells isolated from amniotic fluid; (c) plating the immature hiPSC-CMs onto the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in culture medium to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes, thereby forming a monolayer of mature cardiomyocytes on the AFC-ECM and thus forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, the monolayer of mature cardiomyocytes is aligned on the AFC-ECM. In some embodiments, the fiber track is located on a structure.

[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 2Cellartis® Cardiomyocytes are cryopreserved living 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 by days 8–10 using small molecule protocols to obtain beating cardiomyocytes, with GSK3 inhibitors, RPMI / B27 minus insulin, Wnt inhibitors, and RPMI / B27 plus insulin at various days during a 7-day differentiation period. Preferred non-limiting examples of methods for generating immature hiPSC-CMs are disclosed in U.S. Patent Application Publication 2015 / 0329825, which is incorporated herein by reference. In some embodiments, immature hiPSC-CMs do not express the inward rectifying potassium channel Kir2.1. In some embodiments, immature hiPSC-CMs do not contain rod-shaped cells with a distinguishable sarcomere structure. In some embodiments, immature hiPSC-CMs can be characterized by having fewer mitochondria than mature cardiomyocytes, having disordered myofilaments, having a rounded shape, and / or having a single nucleus.

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

[0090] Immature hiPSC-CM may be in suspension when in contact with AFC-ECM, or the cells may be plated directly onto 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 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 AFC-ECM. Immature hiPSC-CM can be used at any cell seeding density. In some embodiments, a cell seeding density of immature hiPSC-CM 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 immature hiPSC-CM cell seeding densities in various multi-well plates are as follows: in a 6-well plate, approximately 200,000 cells may be plated per well; in a 12-well plate, approximately 150,000 cells may be plated per well; in a 24-well plate, approximately 175,000 cells may be plated per well; in a 48-well plate, approximately 100,000 cells may be plated per well; in a 96-well plate, approximately 50,000 cells may be plated per well; and in a 384-well plate, approximately 15,000 cells may be plated per well. In some embodiments, the cell seeding density of immature hiPSC-CM is approximately 50,000 cells per well in a 96-well plate. In some embodiments, the cell seeding density is approximately 200,000 cells per well in a 6-well plate.To induce maturation of immature hiPSC-CMs, a suitable culture medium, such as RPMI medium or Media 199 medium, is added to the immature hiPSC-CMs in contact with AFC-ECMs, and the cells are cultured with the AFC-ECMs using standard cell culture techniques for a period, typically 7 days, until the cells mature into mature cardiomyocytes with morphology similar to that of native adult cardiomyocytes. During the first 3-4 days, the immature hiPSC-CMs are adhesive, forming continuous monolayers, and initiating the maturation process. Surprisingly, the period for cardiomyocyte maturation can take 7 days or less, but generally, much longer periods, such as up to 100 days, are typical with other substrates. The morphology of these mature cardiomyocytes can be characterized by rod-shaped cells with distinguishable sarcomere structures (a striped appearance), which are the basal contractile units of muscle, and can be visualized using conventional light microscopy techniques. In some embodiments, mature cardiomyocytes can be further characterized by having a greater number of mitochondria than immature hiPSC-CMs, having structured, dense myofilaments, and / or having two nuclei (binuclear). Furthermore, the AFC-ECM can spontaneously produce the fibrous tracks that mature cardiomyocytes follow. This results in a degree of anisotropy in the monolayer, which more closely mimics the natural heart. Thus, cardiomyocytes can spontaneously align on the AFC-ECM after the alignment of the AFC-ECM, which is determined by amniotic cells in a naturally anisotropic configuration during AFC-ECM formation. In various embodiments, the period for immature hiPSC-CMs to mature in culture may 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 period for cardiomyocyte maturation is 14 days, more preferably 10 days, or even more preferably 7 days. In some embodiments, the period for immature hiPSC-CM to mature in culture is 7 days. In some embodiments, immature hiPSC-CM are plated onto AFC-ECM.In some embodiments, mature cardiomyocytes form a confluent monolayer on the AFC-ECM during culture, thereby forming a cell construct containing a monolayer of mature cardiomyocytes on the AFC-ECM. In some embodiments, the mature cardiomyocytes are aligned on the AFC-ECM. In some embodiments, immature hiPSC-CMs are plated on the AFC-ECM in a multiwell plate. In some embodiments, the multiwell plate has a polydimethylsiloxane (PDMS) insert. In some embodiments, the multiwell plate does not have a PDMS insert. C. Methods for determining the cardiotoxicity and / or proarrhythmic effects of drug compounds

[0091] A method for determining the cardiotoxicity and / or proarrhythmic effect of a drug compound in vitro is disclosed herein, comprising the steps of: contacting the drug compound with mature cardiomyocytes of any one of the cell constructs disclosed herein; and observing one or more changes in the electrophysiology of the mature cardiomyocytes to confirm whether the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. One or more changes in the electrophysiology of the mature cardiomyocytes indicate and support that the drug compound has a cardiotoxic and / or proarrhythmic effect on the mature cardiomyocytes. One or more changes in the electrophysiology of the mature cardiomyocytes may include, but are not limited to, APD prolongation, APD prolongation plus rotor, and / or various types of arrhythmias, such as tachyarrhythmia (TA), resting state (Q), delayed after depolarization (DAD), and / or early after depolarization (EAD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is prolongation of the action potential duration (APD). In some embodiments, the change in the electrophysiology of the mature cardiomyocytes is early after depolarization (EAD). In some embodiments, the electrophysiological change in mature cardiomyocytes is delayed depolarization (DAD). In some embodiments, the electrophysiological change in mature cardiomyocytes is action potential duration (APD) plus rotor. In some embodiments, the electrophysiological change in mature cardiomyocytes is arrhythmia. In some embodiments, the electrophysiological change in mature cardiomyocytes is tachyarrhythmia (TA). In some embodiments, the electrophysiological change in mature cardiomyocytes is quiescent state (Q). Observations may also include cell viability, cell density, and / or cellular morphology.In some embodiments, the cell construct can be prepared by a process comprising: (a) providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells; (b) providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) plating the immature hiPSC-CMs onto the AFC-ECM; and (d) culturing the plated immature hiPSC-CMs on the AFC-ECM in culture medium to induce maturation of the immature hiPSC-CMs into mature cardiomyocytes, thereby forming a monolayer of mature cardiomyocytes on the AFC-ECM and thus forming a cell construct, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In other embodiments, the method may include the steps of contacting a drug compound with one of the cell constructs disclosed herein, as well as observing cardiotoxic and / or proarrhythmic events. In one particular case, the method comprises the steps of (a) providing immature cardiomyocytes derived from human induced pluripotent stem cells (immature hiPSC-CM); (b) providing an extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid; (c) plating the immature hiPSC-CM onto the AFC-ECM; (d) culturing the plated immature hiPSC-CM on the AFC-ECM in 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 cellular construct; (e) contacting a drug compound with the monolayer of mature cardiomyocytes of the construct; and (f) observing electrophysiological changes in the mature cardiomyocytes to determine whether the drug compound has cardiotoxic and / or proarrhythmic effects on the mature cardiomyocytes, characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue. In some embodiments, immature hiPSC-CM is plated onto AFC-ECM in a multiwell plate. In some embodiments, the multiwell plate has a polydimethylsiloxane (PDMS) insert.In some embodiments, the multiwell plate does not have a PDMS insert. In some embodiments, immature hiPSC-CMs do not express the inward 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 proarrhythmic testing may be performed using any type of apparatus suitable for measuring such activity. In some embodiments, a monolayer cell construct of mature cardiomyocytes on an 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, commercially 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 commercially available from SciMeasure. In some embodiments, the camera image acquisition rate is greater than or equal to 150 frames per second. In some embodiments, the camera and lens combination is designed to allow simultaneous visualization of all wells of a multiwell plate with sufficient resolution to observe action potentials and calcium wave propagation. Each plate is centered under the camera system, the illumination is switched on, camera acquisition is initiated, and electrophysiological activity is recorded. The experiment is conducted at approximately 37°C. After baseline readings are made, the drug to be tested is added to the wells and its effect is recorded. Spontaneous activity is recorded for a sufficient period to obtain images, e.g., at least 10 seconds. Images may be stored on a computer. The images are analyzed, and action potential duration, conduction velocity, heart rate, and activation pattern may be quantified using image analysis software. Visualization of the electrical wave pattern is important for determining the effect of drug compounds on potentially lethal arrhythmias, e.g., 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 pattern, depending on the type of apparatus used. D. Methods for expanding / proliferating mammalian stem cells

[0093] Methods for the isolation, maintenance, and expansion / proliferation of mammalian cells using cell-derived extracellular matrix (AFC-ECM) induced in vitro from cells isolated from amniotic fluid are also disclosed herein. In vitro cell culture is perhaps the most universal, important, and yet poorly understood aspect of all cell biology and the developing fields of regenerative medicine and tissue engineering. Firstly, it allows for the observation of cell behavior, thereby enabling detailed study of various aspects of cellular function. Secondly, it allows for the increase of the number of specific cell populations. For basic research and many clinical applications, it is necessary to obtain a large number of relatively rare cells from a small amount of biological sample. In vitro cell culture makes it possible to expand a small number of cells in vitro to obtain a more appropriate number of cells. Finally, it makes it possible to preserve cells for future use. By expanding the number of cells in vitro and freezing the viable cells for later use, cells for multiple experiments can be obtained from a relatively small amount of biological sample over a period of days, months, or even years.

[0094] Despite the widespread existence of cell culture, the effects of in vitro culture on the innate characteristics of cells remain relatively underdeveloped. Much of current practice arises not from deliberate ideas, plans, and experiments, but rather from accidental observations. Mammalian cell culture began in the early 1900s, with Alexis Carrel and Montrose Burrows first publishing a scientific paper on in vitro culture of mammalian tissue in 1911. They were studying the physiology and anatomy of tissues by cutting sections of mammalian tissue and placing them on microscope slides. In doing so, they noticed that some cells migrated from the tissue onto the slides. They subsequently described a method for permanently culturing cells. Although some of their observations now appear invalid, their research paved the way for modern cell culture.

[0095] Following the discovery of hematopoietic stem cells (HSCs), groups around the world were studying them. During the culture of HSCs (in suspension), 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 accidental observation that led to the discovery of MSCs, adhesion to plastics is still widely used today as a defining characteristic of MSCs and many other mammalian cell types.

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

[0097] When cells are isolated from their natural environment to be enlarged in vitro, they may lose crucial clues that relay important information about the composition and condition of their surroundings from the surrounding extracellular matrix or microenvironment. Changes in the cellular microenvironment can have significant effects on the behavior of these cells. The current standard method for isolating and enlarging most adherent cells in vitro is to place the cells in a culture vessel made of polystyrene (plastic). While the polystyrene may be treated in some way to promote cell adhesion and growth, 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 certain combinations of proteins. These simple substrates ignore the complexity of the natural microenvironment and its crucial role in normal cellular function. Cells immediately begin to respond to this foreign environment in ways that are significantly different from when the cells are in their natural environment.

[0098] To address this problem in culturing cells on plastic substrates, five main approaches are currently being employed:

[0099] 1. Ignore the problem. Instead of attempting to achieve the desired function that matches the function expected in vivo, numerous cell types can be tested in various culture media to discover cells that exhibit a specific desired function even without a suitable matrix substrate. This approach is unsophisticated and often fails to yield the desired results due to the complex interplay between variables and the wide range of interactions between cells and the extracellular matrix.

[0100] 2. Identify key components. Many academic laboratories and some companies have taken an approach of considering the original tissue from which the cells were isolated and searching for specific elements within that tissue that may be important for cellular function. They then culture the cells 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, more than 100 different proteins. Cells respond just as strongly to signals they need but cannot accept as they do to signals they do not need but accept.

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

[0102] 4. Tissue-derived matrix. 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 homogeneous suspension, and then coating culture vessels with the disrupted tissue. For example, if one were to culture satellite cells, one might collect muscle, homogenize the tissue, then coat culture vessels with the homogenized muscle and seed the cells. This method often fails for several reasons. Firstly, even within a particular tissue type, the niche of stem cells / progenitor cells may differ from the rest of the tissue. Simply homogenizing muscle does not guarantee that a suitable niche is created. Secondly, 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 is disrupted, and cells may sense very different mechanical cues. Finally, the manufacturability of tissue-derived matrix depends on the availability of the tissue. This affects the total amount of cell culture that can be produced and is a source of lot-to-lot variability.

[0103] 5. Cell-derived matrix. Cells in culture can be induced to secrete a matrix into their culture vessel. This matrix is ​​the best available approximation of the in vivo niche and can be produced 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-ionized surfactant that preserves the structure and chemistry of the matrix. This approach has several important advantages: (1) The matrix structure can be recreated and left undisturbed. (2) The matrix can be customized based on the target tissue / cell type. (3) The matrix can be specific to the stem cell and progenitor cell niche. (4) The matrix can be produced in large quantities.

[0104] Regarding cell-derived matrices, it is not possible to efficiently isolate all cell types and expand them on any given cell-derived matrix. In practice, pluripotent stem cells (PSCs) appear to have very different requirements for supporting growth substrates than other cell types. It is known that the specific cell type used to produce the matrix influences the matrix's composition and, therefore, the response of various 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. Patent No. 8,084,023 describe 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. See also Stem cells and development 19, 1095-107 (2010). While this bone marrow cell-derived matrix has been shown to support the expansion of other MSCs, it has not been effective for the adhesion and growth of other types of stem cells, specifically induced pluripotent stem cells (iPSCs). iPSCs have shown an extended potential to form cells and tissues from a much broader category than MSCs. This represents a particularly interesting challenge, as it is impractical to produce cell-derived matrices from iPSCs because it is difficult to grow confluent monolayers of iPSCs under standard culture conditions. The main limitation of previous cell-derived matrices is that, in order 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] This disclosure provides solutions to at least some of the aforementioned limitations and shortcomings in the present technology relating 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 solutions presuppose the use of amniotic fluid cell-derived extracellular matrix. The use of neutral, readily adhering, and highly proliferative perinatal cells found in amniotic fluid enables the creation of an extracellular matrix (ECM) that remarkably supports the adhesion, isolation, expansion, and proliferation of these PSCs. This technological achievement was not possible with conventional cell-derived ECMs.

[0106] The function of mammalian cells is primarily determined by the environment in which they exist, such as the extracellular matrix. Cells respond to signals present in that environment (positive signals) and to signals that are necessary but absent (negative signals). Neutral stem cells can produce a matrix containing niche motifs necessary to maintain stem cell viability and stem cell properties, but may lack many cell lineage-specific signals that more mature cells may secrete and guide stem cells toward specific fates. While not bound by theory, it is suggested that immature cells, such as perinatal cells or perinatal stem cells, may produce an ECM different from those previously disclosed in this technology, such as bone marrow stromal cell-derived ECM, which may enable better isolation and expansion / proliferation of stem cells, such as pluripotent stem cells (PSCs) with a higher potential than mesenchymal stem cells (MSCs). Mass spectrometry has shown that, compared to previously known cell-derived ECMs, the amniotic fluid cell-derived ECM of this invention contains matrix proteins found in all three germ layers and lacks specific proteins strongly associated with osteogenic cell lineages. Furthermore, the ECM of this disclosure includes certain motifs, such as laminin, which are known to promote the adhesion and expansion of pluripotent cells.

[0107] Pluripotent stem cells (PSCs) can regenerate and differentiate into one of the three germ layers: the 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 induced from cells taken from adult tissue or adult cells and reprogrammed to the embryonic stem cell level. Methods for producing iPSCs are known in this technology.

[0108] A method for expanding / proliferating pluripotent stem cells (PSCs) comprises the steps of obtaining PSCs and culturing them in the presence of amniotic fluid cell-derived ECM according to the present invention. The PSCs may be iPSCs or ES. Any 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 / cm². 2 ~about 100,000 cells / cm 2 , or approximately 100 cells / cm² 2 ~about 75,000 cells / cm 2 , or approximately 500 cells / cm² 2 ~about 50,000 cells / cm 2 , or approximately 500 cells / cm² 2 ~about 10,000 cells / cm 2 , or approximately 500 cells / cm² 2 ~about 5,000 cells / cm 2 , or approximately 500 cells / cm² 2 ~about 2,500 cells / cm 2 , or approximately 1,000 cells / cm² 2 ~about 25,000 cells / cm 2 , or approximately 2,000 cells / cm² 2 ~about 10,000 cells / cm 2 , or approximately 3,000 cells / cm² 2 ~about 5000 cells / cm 2 That is the case.

[0109] In some embodiments, PSCs are maintained in an undifferentiated state to preserve their stem cell properties. Suitable cell culture techniques for the proliferation of PSCs in culture are known in this art. Suitable commercially available culture media for stem cell proliferation include, but are not limited to, StemMACS® iPS-Brew XF, available from Miltenyl Biotec. In some embodiments, Rock inhibitors are not used. Once the cells begin to approach confluence (for example, as determined by bright-field microscopy), the cells can be manually subcultured by cutting larger colonies into smaller colonies, then physically lifting them from the dish and reseeding them on a fresh plate of amniotic fluid cell-derived ECM. This procedure can be repeated indefinitely. In some embodiments, a method for growing pluripotent stem cells (PSCs) in culture is disclosed, comprising the step of culturing the PSCs in culture medium in the presence of cell-derived extracellular matrix (ECM), thereby growing the PSCs, the cell-derived ECM being induced in vitro from cells isolated from amniotic fluid.

[0110] The above method for expanding / proliferating PSCs is also applicable to the expansion / proliferation of PSCs in culture in the presence of other perinatal cell-derived ECMs. In some embodiments, a method for proliferating pluripotent stem cells (PSCs) in culture is disclosed, comprising the step of culturing the PSCs in culture medium in the presence of a cell-derived extracellular matrix (ECM), thereby proliferating the PSCs, wherein the cell-derived ECM is induced in vitro from cells isolated from umbilical cord or placental tissue. In some embodiments, the cells isolated from the umbilical cord are from umbilical cord blood and / or Wharton's jelly. In other embodiments, the cells isolated from placental tissue are from membrane sheets (amnion and / or chorionic membrane), chorionic villi, and / or blood. [Examples]

[0111] The following embodiments are included to demonstrate certain non-limiting aspects of the present invention. Those skilled in the art will recognize that the methods disclosed in the following embodiments represent methods discovered by the applicant to function well in carrying out the present invention. However, those skilled in the art will recognize that many modifications are possible in the specific embodiments disclosed without departing from the spirit and scope of the present invention, and that similar or comparable results can be obtained. (Example 1) Production of amniotic fluid cell-derived extracellular membrane (ECM) (AFC-ECM)

[0112] Four types of amniotic fluid cell-derived extracellular matrix (ECM) (Matrix A, Matrix B, Matrix C, and Matrix D) were prepared using the following procedure. Cells aseptically isolated from amniotic fluid collected from four full-term donors (over 37 weeks of gestation) were seeded into tissue culture-treated flasks coated with fibronectin and cultured in an incubator at 37°C, 5% CO2, and 90% RH in complete medium. The complete medium consisted of alpha minimal basal medium (aMEM) plus 2 mM L-glutamine plus antibiotic-antifungal agent plus 15% fetal bovine serum.

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

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

[0115] On days 10-11, the induction medium was aspirated from the culture flask, and the extracellular matrix (ECM) formed in the flask was washed once with phosphate-buffered saline (PBS). Then the PBS was aspirated from the flask. The extraction buffer was added to the flask and incubated at room temperature for 7-10 minutes to remove cells from each ECM, and then the extraction buffer was aspirated from the flask. 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, and then the sterile water was aspirated from the flask. The four ECMs in the flasks from which the cells had been removed were dried at room temperature and then stored at 4°C.

[0117] Figure 1 shows a bright-field micrograph of amniotic fluid cell-derived ECM (Matrix B) at 100x magnification using a 10x objective lens. Figure 2 shows atomic force micrographs of three representative 40×40 μm sections of amniotic fluid cell-derived ECM (Matrix B) and bone marrow cell-derived ECM, showing topography, adhesion, and stiffness. Bone marrow cell-derived and amniotic fluid cell-derived ECM are structurally and physically distinguishable. Quantification of adhesion and stiffness (elastic modulus) of bone marrow cell-derived and amniotic fluid cell-derived ECM shows that bone marrow ECM is 10 times stiffer and 3 times less 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 component of the amniotic fluid cell-derived ECM produced in Example 1 was determined by mass spectrometry. The components are listed in Table 2 along with their spectral counts and molecular weights. 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

Table 2-49

Table 2-50

Table 2-51

Table 2-52

Table 2-53

Table 2-54

[0119] Induced pluripotent stem cells (iPSCs) were cultured and proliferated on the amniotic fluid cell-derived ECM (Matrix B) of Example 1 using the following procedure. Commercially available iPSCs stored frozen were thawed using a 37°C water bath. The cell suspension was diluted in a commercially available medium for stem cell proliferation (Miltenyi Biotec, MACS iPS Brew) and seeded onto the ECM in a 6-well plate at approximately 1,000 cells / cm 2Cells were seeded in 2 mL / well of culture medium. No Rock inhibitors were used. On day 1, the entire volume of medium was gently aspirated from the cultured cells and replaced with fresh medium. The entire medium was replaced with fresh medium every 24 hours. When the cells began to approach confluence (as determined by bright-field microscopy), the cells were manually subcultured by using a sterile needle to cut the large colonies into approximately 100 smaller colonies, then physically lifting these from the dish with a sterile needle and reseeding them on a new plate of ECM. This procedure can be repeated indefinitely.

[0120] Micrographs of iPSCs cultured on amniotic fluid cell-derived ECM and bone marrow cell-derived ECM at day 0 and day 2 are shown in Figure 4.

[0121] Figure 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 proliferated when cultured in the presence of amniotic fluid cell-derived ECM, while iPSCs cultured in the presence of bone marrow cell-derived ECM did not grow. (Example 4) Preparation of cellular constructs of mature cardiomyocytes on AFC-ECM, and maturation of immature hiPSC-CMs on AFC-ECM.

[0123] A cell construct containing a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) derived from cells induced in vitro from amniotic fluid was prepared using the following method.

[0124] AFC-ECM was prepared in a 96-well plate (without using a silicone insert) using the methodology outlined in Example 1. Immature hiPSC-CM (iCell® Cardiomyocytes), commercially available from Cellular Dynamics International-FUJI, were plated onto the AFC-ECM using standard cell culture techniques. The immature hiPSC-CM were plated at a density of 50,000 cells per well (96-well plate) or 200,000 cells per well (6-well plate) and cultured in RPMI medium for 7 days to form a confluent monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming a cell construct of mature cardiomyocyte monolayer on the AFC-ECM.

[0125] Over a 7-day period, immature hiPSC-CMs were observed to mature into the morphology and alignment of mature native adult cardiomyocytes, characterized by rod-shaped cells with distinguishable sarcomere structures. For comparative purposes, immature hiPSC-CMs from iCell® were also cultured in a similar manner, plated in 96-well plates (without using silicone inserts) 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, incorporated herein by reference. The results of this study are shown in Figures 6–21 with micrographs of cardiomyocytes on different ECMs.

[0126] As can be seen in FIGS. 6-14, the cardiomyocytes on the AFC-ECM are mature rod-shaped cells with a distinguishable sarcomere structure similar to that of native adult cardiomyocytes. The morphology and sarcomere structure of the cardiomyocytes matured on the AFC-ECM can be clearly seen by the presence of rod-shaped cells with a striated appearance identified by the arrows in the micrograph of FIG. 14. These figures show the presence of fiber tracks on the AFC-ECM and also show that the monolayer of mature cardiomyocytes is aligned with the AFC-ECM and closely resembles the features found in native adult cardiomyocytes and native heart muscle tissue. In contrast, as can be seen in FIGS. 15-19 and FIGS. 20-21, the cardiomyocytes on the standard Matrigel™ ECM and BM-ECM resemble fetal-like cardiomyocytes, respectively, and do not have the characteristics of native adult cardiomyocytes or native heart muscle tissue.

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

[0128] A monolayer cell construct of mature cardiomyocytes on AFC-ECM was prepared using a 96-well plate (without a silicone insert) as described in Example 4. After a 7-day maturation process, the electrophysiology of each well was observed using a plate reader with the following high-throughput screening method. FluoVolt® dye in Hanks equilibrium salt solution was loaded into each well. A high spatiotemporal CCD camera (SciMeasure DaVinci camera) was combined with light-emitting diodes (LEDs) as shown in the schematic diagram in Figure 22. The camera and lens combination was designed to allow simultaneous visualization of all wells in the 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 initiated, and electrophysiological activity was recorded. The experiment was performed at approximately 37°C. Spontaneous activity was recorded for at least 10 seconds, and the images were saved on a computer. After obtaining baseline reads, 500 nM of the drug E4031 (hERG channel blocker) was added to each well. Images were analyzed, and action potential duration, conduction velocity, heart rate, and activation pattern were quantified using image analysis software. For comparison, immature hiPSC-CMs were cultured on standard Matrigel® ECM and bone marrow cell-derived ECM (BM-ECM) in 96-well plates (without silicone inserts) as described in Example 4, and 500 nM of the drug E4031 (hERG channel blocker) was analyzed in a manner similar to the AFC-ECM study. The results of the E4031 study are shown in Figures 23 and 24. As can be seen in Figure 23, recordings of spontaneous action potentials from cardiomyocytes on Matrigel® ECM and BM-ECM showed that only the drug E-4031 caused action potential duration (APD) prolongation; however, in mature cardiomyocytes on AFC-ECM, the drug E-4031 caused APD prolongation plus rotor (TdP-like arrhythmia), an arrhythmia activation pattern consistent with that known to occur in TdP in humans.Therefore, all three ECMs produced a monolayer of cardiomyocytes in response to the predicted APD prolongation, but only the AFC-ECM produced a monolayer of cardiomyocytes that revealed the transition of APD prolongation to tachyarrhythmia characteristic of TdP. As can be seen in Figure 24, 100% of the cardiomyocytes on the AFC-ECM responded to drug E4031 with rotor (TdP-like arrhythmia).

[0129] In further studies, the drugs domperidone (3 μM), disopyramide (100 μM), azimilide (10 μM), d,l-sotalol (100 μM), ibutilide (0.10 μM), and bepridil (10 μM) were tested using the plate reader method described above with mature cardiomyocytes on AFC-ECM (without silicone inserts) prepared in Example 4 in 96-well plates. The results of the tests are shown in Figure 25. As can be seen in these results, various types of arrhythmias, namely tachyarrhythmias (TA), quiescent dysarthria (Q), and early post-depolarization (EAD), were induced by the various drugs indicated in the recording. These represent the range of arrhythmias observed in response to drugs classified by the FDA as high risk for causing TdP-lethal arrhythmias in patients.

[0130] The 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 inserts) prepared in Example 4 in 96-well plates, and observations regarding any detected arrhythmias and APD90 prolongation at 10 times the effective therapeutic plasma concentration (ETPC) are noted. The drugs were selected from the CiPA Initiative list of compounds for CiPA validation and testing. These are classified as high-risk, intermediate-risk, 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 data from the tested drugs in Tables 3 and 4 is shown in Figures 26–29. Figure 26 graphically shows the total number of arrhythmias observed for each drug compound at any given dose. Figure 27 graphically shows the relative incidence of arrhythmias for each drug at specific clinically appropriate doses of 10 × effective therapeutic plasma concentration (ETPC).

[0132] Figure 28 graphically shows the action potential triangulation (APD90-APD30) over time (milliseconds) for each drug. Triangulation is defined as the repolarization time from APD30 to APD90. The action potential triangulation (APD90-APD30) is used as a predictor of the proarrhythmic effect of a drug. Figure 29 shows the action potential triangulation over time (milliseconds) for some drugs, comparing the cardiomyocyte assay performance of cardiomyocytes on AFC-ECM (SBS-AF Matrix) versus Matrigel® ECM.

[0133] Figure 30 graphically shows the maximum drug-induced action potential triangulations of the listed drugs, comparing the cardiomyocyte performance of iCell® hiPSC-CM (white circles) from Cellular Dynamics versus Cor.4U® hiPSC-CM (black circles) from Ncardia 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 Figure 28, the dataset shown in Figure 30 shows little stratification between high-risk and intermediate-risk compounds. Therefore, the AFC-ECM of this disclosure provides the production of more mature cardiomyocytes with more realistic function and drug responsiveness than other native cell-derived ECMs or Matrigel® ECMs. (Example 6) Observation of cytoconstructs 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:

[0135] AFC-ECM was deposited onto Thermox coverslips, enabling immunostaining of cells and imaging using a laser scanning confocal microscope (Nikon A1R).

[0136] For comparison, Matrigel® ECM was applied to separate subsets of Thermanox coverslips.

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

[0138] After 7 days of incubation in cell culture medium, cells were fixed in 3% paraformaldehyde and treated for immunocytochemistry using commercially available primary antibodies to determine cell expression and localization in hiPSC-CM. The following primary antibodies were used against key cardiac filament proteins: 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. Nuclei were marked using DAPI (4',6-diamidino-2-phenylindole) fluorescence staining.

[0139] All procedures for cell labeling and visualization are described in the following references incorporated herein by reference: Herron, TJ et al. Extracellular Matrix-Mediated Maturation of Human Pluripotent Stem Cell-Derived Cardiac Monolayer Structure and Electrophysiological Function. Circulation: Arrhythmia and Electrophysiology 9 (2016). da Rocha, MA et al. Deficient cMyBP-C protein expression during cardiomyocyte differentiation underlies human hypertrophic cardiomyopathy cellular phenotypes in disease specific human ES cell derived Cardiomyocytes. J. Mol. Cell. Cardiol. 99, 197-206 (2016). da Rocha, AM et al. hiPSC-CM Monolayer Maturation State Determines Drug Responsiveness in High Throughput Pro-Arrhythmia Screen. Sci. Rep. 7, 13834 (2017).

[0140] Cell morphology was quantified using fluorescence images analyzed in NIS Elements Software. Cell roundness was quantified using cell area and perimeter with an established mathematical equation: roundness index = 4π * area / perimeter 2 .

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

[0142] Results: Consistent with the results observed in Example 4 above, data using fluorescence 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 disordered sarcomeres, while the same batch of hiPSC-CMs cultured on AFC-ECM (inogeneic comparison) are rod-shaped and have tightly densified / structured sarcomeres and myofilaments.

[0143] The micrograph in Figure 31 shows hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for troponin I and DAPI to mark the nuclei. The micrograph in Figure 32 shows hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for α-actin and DAPI to mark the nuclei. The micrograph in Figure 33 shows hiPSC-CM cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for cTnT and N-cadherin and 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 disordered sarcomeres, while the same batch of hiPSC-CMs cultured on AFC-ECM (ingeneic comparison) are rod-shaped and have tightly densified / structured 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 micrograph in Figure 34 shows a single hiPSC-CM cell cultured on Matrigel® ECM vs. AFC-ECM using immunofluorescence staining for cTnT and DAPI to mark the nucleus, showing that cells cultured on Matrigel® ECM are round, while cells cultured on AFC-ECM are rod-shaped. The micrograph in Figure 35 shows a single hiPSC-CM cell cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for α-actinin 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 graph in Figure 36 shows a comparison of the cell roundness of the single cells shown in Figure 35, indicating a higher roundness index for cells cultured on Matrigel® ECM, which exhibits greater roundness.

[0144] The micrograph in Figure 37 shows hiPSC-CMs cultured on Matrigel® ECM versus AFC-ECM, using immunofluorescence staining for cTnI expression and DAPI to mark the nuclei. Figure 38 shows Western blotting of hiPSC-CMs on Matrigel® ECM and AFC-ECM for cTnI expression and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). The 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 versus AFC-ECM. This analysis shows a higher cTnI expression / GAPDH ratio for hiPSC-CMs on AFC-ECM than for hiPSC-CMs on Matrigel® ECM, indicating more robust cTnI expression from hiPSC-CMs on AFC-ECM than from hiPSC-CMs on Matrigel® ECM.

[0145] The micrograph in Figure 40 shows hiPSC-CMs on Matrigel® ECM versus AFC-ECM stained with MitoTracker Red for mitochondria, indicating that cells on AFC-ECM have more mitochondria and mitochondria with more polarized inner membrane potentials, as evidenced by the larger red signal. The graph in Figure 41 shows the MitoTracker® Red fluorescence intensity / cardiomyocellular for Matrigel® ECM versus hiPSC-CMs on AFC-ECM, indicating that hiPSC-CMs on AFC-ECM have a higher MitoTracker® Red fluorescence intensity, suggesting that these cells have more mitochondria and mitochondria with more polarized inner membrane potentials than hiPSC-CMs on Matrigel® ECM.

[0146] The micrograph (transmitted light) in Figure 42 shows hiPSC-CM cultured on Matrigel® ECM and AFC-ECM coated on microelectrode array (MEA) plates. As can be seen in Figure 42, hiPSC-CM cultured on Matrigel® ECM are circular in shape, while hiPSC-CM cultured on AFC-ECM are rod-shaped.

Claims

1. A method for maturing immature cardiomyocytes derived from human induced pluripotent stem cells, wherein the method is (a) A step of providing immature cardiomyocytes (immature hiPSC-CMs) derived from human induced pluripotent stem cells, wherein the immature cardiomyocytes exhibit a single nucleus; (b) A step of providing an in vitro induced extracellular matrix (AFC-ECM) by culturing cells isolated from amniotic fluid obtained from a human being with a gestation period of more than 37 weeks, wherein the AFC-ECM comprises laminin, collagen alpha-1 (XVIII), basement membrane-specific heparan sulfate proteoglycan core protein, agrin, vimentin, and collagen alpha-2 (IV), or isoforms thereof; (c) The step of bringing the immature hiPSC-CM into contact with the AFC-ECM; and (d) A step of culturing the immature hiPSC-CM together with the AFC-ECM in a culture medium to induce the maturation of the immature hiPSC-CM into mature cardiomyocytes having two nuclei. Methods that include...

2. The method according to claim 1, wherein the step of bringing the immature hiPSC-CM into contact with the AFC-ECM includes plating the immature hiPSC-CM onto the AFC-ECM.

3. The method according to claim 2, wherein the AFC-ECM is contained in a cell culture vessel or multiwell plate during plating.

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

5. The method according to claim 4, wherein the monolayer of the mature cardiomyocyte is a confluent monolayer.

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

1.

7. The method according to any one of claims 1 to 6, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue.

8. The 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 according to any one of claims 1 to 8, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, or the isoform of agryn is isoform 6.

10. The method according to any one of claims 1 to 9, wherein the AFC-ECM further comprises fibronectin or an isoform thereof.

11. The method according to any one of claims 1 to 10, wherein the AFC-ECM does not contain decorin, perlecan, and collagen (III).

12. The method according to any one of claims 1 to 11, wherein the period for maturation of the immature cardiomyocytes to mature cardiomyocytes during step (d) is 4 to 14 days.

13. A cell construct comprising a monolayer of mature cardiomyocytes on an extracellular matrix (AFC-ECM) induced in vitro by culturing cells isolated from amniotic fluid obtained from a human with a gestation period of more 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 cardiomyocytes (hiPSC-CM) cultured from AFC-ECM derived from immature human induced pluripotent stem cells. The aforementioned mature cardiomyocyte is characterized by two nuclei, A cell construct in which the AFC-ECM is structured into anisotropic fiber tracks, and the mature cardiomyocytes are anisotropically aligned on the anisotropic fiber tracks of the AFC-ECM.

14. The cell construct according to claim 13, wherein the monolayer of the mature cardiomyocytes is a confluent monolayer.

15. The cell construct according to claim 13 or 14, wherein the immature hiPSC-CM does not express the inward rectifying potassium channel Kir2.

1.

16. The cell construct according to any one of claims 13 to 15, wherein the mature cardiomyocytes are characterized by rod-shaped cells having a distinguishable sarcomere structure similar to that of adult human heart tissue.

17. The cell construct according to any one of claims 13 to 16, wherein the isoform of collagen alpha-1 (XVIII) is isoform 2, and / or the isoform of agryn is isoform 6.

18. The cell construct according to any one of claims 13 to 17, wherein the AFC-ECM further comprises fibronectin and / or its isoform.

19. The cell construct according to any one of claims 13 to 18, wherein the AFC-ECM does not contain decorin, perlecan, and / or collagen (III).

20. A method for producing a cell construct of mature cardiomyocytes, wherein the method is A step of maturing immature cardiomyocytes derived from human induced pluripotent stem cells according to any one of claims 1 to 12, wherein the mature cardiomyocytes are characterized by two nuclei; and The step of forming a monolayer of mature cardiomyocytes on the AFC-ECM, thereby forming the cell construct. Methods that include...

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