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JP-7854808-B2 - Treatment of heart disorders

JP7854808B2JP 7854808 B2JP7854808 B2JP 7854808B2JP-7854808-B2

Inventors

  • ベルナルド，アンドレイア

Assignees

  • ザ フランシス クリック インスティチュート リミティッド

Dates

Publication Date
20260507
Application Date
20200605
Priority Date
20190607

Claims (20)

  1. A cell population containing at least approximately 60% of cells that are double-positive for markers HAND1 and MLC2v, wherein the cells are left ventricular cardiomyocytes.
  2. A population of cells according to claim 1, comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are double-positive for markers HAND1 and MLC2v.
  3. A population of cells according to claim 1 or 2 , comprising at least about 65, 70, 75, 80, 85, 90, 95, or 100% of cells that are positive for the marker TBX5, as well as for HAND1 and MLC2V.
  4. A population of cells according to any one of claims 1 to 3, comprising at least about 85% of cells that are double-positive for markers HAND1 and MLC2v.
  5. A population of cells according to any one of claims 1 to 4, comprising at least about 85% of cells positive for the markers TBX5, HAND1, and MLC2v.
  6. A population of cells according to any one of claims 1 to 5, comprising at least about 85% of cells positive for the markers IRX4, TBX5, HAND1, and MLC2v.
  7. A population of cells according to any one of claims 1 to 6, including mature cardiomyocytes.
  8. The aforementioned mature cells exhibit characteristics of maturity, The aforementioned characteristics of maturity are, (i) Ventricular action potential shape; (ii) Low heart rate and/or periodicity; (iii) Conduction velocity characteristic of neonatal ventricular cells; (iv) The average value of the pulse amplitude higher than (iv); (v) Field potential intervals that match the field potential intervals of ventricular cardiomyocytes; (vi) Fast action potential rise time (Trise); (vii) CaT similar to the calcium transient (CaT) found in adult human ventricular cardiomyocytes; and (viii) Improved CaT rise time (time to peak, Tpeak) and CaT Selected from, A population of cells according to claim 7.
  9. The aforementioned mature cells show improved maturity markers. The aforementioned maturity marker is (i) Organization, length, and function of sarcomeres; (ii) Indication of the presence of connexin-43, a mature gap junction marker; sarcomere alpha-actinin; telesonin, a mature Z-disk marker; M-protein, a mature M-band marker; and desmin, a cardiomyocyte-associated intermediate filament in the Z-disk; (iii) Display of an extensive, interconnected mitochondrial network typical of neonatal cardiomyocytes; and (iv) Selected from increased mitochondrial DNA activation, A population of cells according to claim 7 or 8.
  10. A population of cells according to any one of claims 1 to 8, wherein the cells can be paced.
  11. A population of cells according to any one of claims 1 to 10, for use in the treatment or prevention of left ventricular disorders.
  12. A population of cells for use according to claim 11, wherein the left ventricular disorder is selected from myocardial infarction, heart failure, left ventricular hypertrophy, hypoplastic left heart syndrome, and left ventricular noncompaction cardiomyopathy (LVNC).
  13. A population of cells for use according to claim 12, wherein the left ventricular disorder is myocardial infarction.
  14. A population of cells for use according to claim 12, wherein the left ventricular dysfunction is heart failure.
  15. A method for preparing a cell population containing at least about 60% left ventricular cardiomyocytes that are double-positive for markers HAND1 and MLC2v, comprising the step of culturing pluripotent stem cells selected from embryonic stem cells or induced pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or inverse agonist , Chiron, BMP4, activin A, and FGF2 , The culture medium is as follows : (i) Chiron in concentrations of approximately 2–6 μM, 2–4 μM, or 2–3 μM; (ii) BMP4 in a concentration of approximately 1 to 6 ng/ml, or approximately 3 to 5 ng/ml; (iii) Activin A at approximately 3 to 10 ng/ml; (iv) FGF2 at approximately 3 to 10 ng/ml Includes , A method further comprising the step of culturing the cells in a medium containing a retinoic acid receptor antagonist or inverse agonist, a Wnt inhibitor, and L -ascorbic acid.
  16. It has a duration of 15 to 25 days, and the cell population exhibits characteristics of maturity . The aforementioned characteristics of maturity are, (i) Ventricular action potential shape; (ii) Low heart rate and/or periodicity; (iii) Conduction velocity characteristic of neonatal ventricular cells; (iv) The average value of the pulse amplitude higher than (iv); (v) Field potential intervals that match the field potential intervals of ventricular cardiomyocytes; (vi) Fast action potential rise time (Trise); (vii) CaT similar to the calcium transient (CaT) found in adult human ventricular cardiomyocytes; and (viii) Improved CaT rise time (time to peak, Tpeak) and CaT Selected from , The method according to claim 15.
  17. It has a duration of 15 to 25 days, and the cell population shows markers of maturity. The aforementioned maturity marker is (i) Organization, length, and function of sarcomeres; (ii) Indication of the presence of connexin-43, a mature gap junction marker; sarcomere alpha-actinin; telesonin, a mature Z-disk marker; M-protein, a mature M-band marker; and desmin, a cardiomyocyte-associated intermediate filament in the Z-disk; (iii) Display of an extensive, interconnected mitochondrial network typical of neonatal cardiomyocytes; and (iv) Increased activation of mitochondrial DNA Selected from, The method according to claim 15 or 16.
  18. The method according to any one of claims 15 to 17, further comprising the step of culturing the cells in a medium containing a retinoic acid receptor antagonist or inverse agonist, a Wnt inhibitor and L-ascorbic acid , and then culturing the cells in a medium that does not contain vitamin A but contains L-ascorbic acid.
  19. The method according to any one of claims 15 to 18 , comprising the protocol shown in Table 1.
  20. A population of cells obtained or obtainable by the method described in any one of claims 15 to 19 .

Description

This invention relates to a population of cells with an increased proportion of left ventricular cardiomyocytes, and its use, for example, in the treatment of left ventricular disorders and in the screening of drugs that may be used to treat left ventricular disorders. Cardiovascular disease is a leading cause of death in developed industrial nations. Myocardial infarction (MI), which causes a dramatic loss of contractile myocardium in the left ventricle (LV), is the most common cause of cardiac injury. When the infarction is large, or when a patient suffers from multiple infarctions, it often leads to heart failure, for which the only effective treatment is a heart transplant. The life expectancy for patients with heart failure is up to 5 years, and as many as 60% of patients die within one year of diagnosis. Therefore, there is an urgent need for a safe and sustainable treatment that can replace the damaged myocardium in MI/heart failure. The key to improving long-term patient outcomes is LV regrowth by functional cardiomyocytes. The inventors have developed a novel method for providing a cell population in which the number or percentage of cardiomyocytes expressing left ventricular cell-specific markers is remarkably high. This method results in the rapid generation (20 days) of left ventricular cardiomyocytes that can exhibit a more mature identity than those achievable using previous differentiation methods and long-term culture (60 days or more). Therefore, the inventors provide, for the first time, a population of left ventricular cardiomyocytes with a high degree of homogeneity that may have a higher level of maturity, and a method for generating such a population of cells in an effective and rapid manner. Such a population of cells may be used for various purposes, for example, as cell therapy to treat left ventricular dysfunction, to model left ventricular dysfunction, or for drug screening to identify drugs that may be used to treat left ventricular dysfunction, or for cardiotoxicity testing. This invention provides a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v. Such cells are left ventricular cardiomyocytes. The coexistence of the two markers, HAND1 and MLC2v, is beneficial as information about the left ventricular cardiomyocyte phenotype. This has not been previously recognized in the art. This invention enables, for the first time, the efficient production of such a population of left ventricular cardiomyocytes. This provides a significant contribution to the art, such as therapeutic uses, as will be considered below. As described in this embodiment, evidence is provided that cells according to the present invention are more mature at day 20 than cells that have reached day 60 of long-term culture using monolayer differentiation methods known in the art. For example, immunofluorescence analysis for sarcomere markers is provided. Functional evidence is also provided from electrophysiology that the cells possess ventricular action potential shapes (using three different assays). Furthermore, functional data from calcium imaging regarding calcium response are provided. The inventors further provide evidence that these cells can successfully generate engineered heart tissue (EHT). Finally, the inventors provide evidence that EHT generated using left ventricular-like cardiomyocytes loses spontaneous pacemaker capability, consistent with what is expected in more mature ventricular cardiomyocytes. In one embodiment, the present invention provides a method for treating left ventricular dysfunction in a subject, comprising administering to the subject a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v. In one embodiment, the present invention provides a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for use in the treatment or prevention of left ventricular disorders. In one embodiment, the present invention provides a population of cells containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for use in the manufacture of agents for the treatment or prevention of left ventricular disorders. In one embodiment, the present invention provides the use of a cell population containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v for the treatment or prevention of left ventricular dysfunction. In one embodiment, the present invention provides a method for preparing a cell population containing at least about 60% of cells that are double-positive for the markers HAND1 and MLC2v, the method comprising the step of culturing pluripotent stem cells in a medium containing a retinoic acid receptor antagonist or an inverse agonist. In one embodiment, the above method may include, for example, a step of culturing the cells or cell pop