JP-7857030-B2 - Methods for maturation of cardiomyocytes on the ECM derived from amniotic fluid cells, cell constructs, and their use in screening for cardiotoxicity and proarrhythmic effects of drug compounds.

Inventors

• トラヴィス ブロック
• エドワード エス． グリフィー
• トッド ヘロン

Assignees

• ステムバイオシス インコーポレイテッド

Dates

Publication Date

20260512

Application Date

20240722

Priority Date

20190221

Claims (20)

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; A method comprising (c) contacting the immature hiPSC-CM with the AFC-ECM; and (d) culturing the immature hiPSC-CM together with the AFC-ECM in a culture medium to induce maturation of the immature hiPSC-CM into two-nucleated mature cardiomyocytes.
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 1 , wherein the period of 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 method comprising the steps 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 forming a monolayer of the mature cardiomyocytes on the AFC-ECM, thereby forming the cellular construct.

Description

Cross-reference 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. This disclosure generally relates to the use of cellular constructs of human stem cell-derived cardiomyocytes on a cell-derived extracellular matrix, methods for creating such constructs, and methods for using these constructs for cardiotoxicity and proarrhythmic screening assays of drug compounds. Cardiotoxicity, or perceived potential cardiotoxicity, is a major cause of toxicity-related drug 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). Current preclinical drug candidate trial guidelines from the IHC (ICH S7A and S7B - Pharmacology Studies) describe genetically modified xenocellular and in This depends 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), and 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. Therefore, the U.S. 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-Vitro Proarrhythmic Assay (CiPA). An integral part of the CiPA Initiative (http://cipaproject.org/) includes 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 to 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. Secondly, a reliable, high-throughput screening platform using these cells needs to be developed. 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 current 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, including: 1) enabling high-throughput analysis; 2) providing high spatial resolution; 3) enabling visualization of impulse propagation across culture dishes; and 4) allowing modification of culture conditions, including the addition of extracellular matrix, leading to a more natural test environment. The use of cardiomyo