Use of jagged 1/frizzled 4 as a cell surface marker for isolating human cardiac ventricular progenitor cells

• US 10,597,637 B2
• Filed: 08/21/2015
• Issued: 03/24/2020
• Est. Priority Date: 08/22/2014
• Status: Active Grant

• Current Assignee: Procella Therapeutics AB
• Original Assignee: Procella Therapeutics AB
• Inventors: Chien, Kenneth R., Lian, Xiaojun
• Primary Examiner(s): Humphrey, Louise W
• Assistant Examiner(s): Perkins, Stephen A
• Application Number:
  US14/832,324
• Publication Number:
  US 20160053229A1
• Time in Patent Office: 1,677 Days
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61P 13/12 of the kidneys
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2501/415 Wnt; Frizzeled
• CPC Class Codes: C12N 2501/42 Notch; Delta; Jagged; Serrate
• CPC Class Codes: C12N 2503/02 Drug screening
• CPC Class Codes: C12N 2506/02 from embryonic cells
• CPC Class Codes: C12N 2506/03 from non-embryonic pluripotent stem cells
• CPC Class Codes: C12N 2506/45 from artificially induced pluripotent stem cells
• CPC Class Codes: C12N 2513/00 3D culture
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The present invention provides Jagged 1 and Frizzled 4 as cell surface markers for isolating human cardiomyogenic ventricular progenitor cells, in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Thus, the invention provides human ventricular progenitor (HVP) cells. The invention provides in vitro methods of the separation of Islet 1+ Jagged 1+ ventricular progenitor cells and/or Islet 1+/Frizzled 4+ ventricular progenitor cells and/or Islet 1+/Jagged 1+/Frizzled 4+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated Jagged 1+ and/or Frizzled 4+ventricular progenitor cells are also provided. Methods of in vivo use of Jagged 1+ and/or Frizzled 4+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the Jagged 1+ and/or Frizzled 4+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.

Claims

1. A method for isolating human cardiac ventricular progenitor cells, the method comprising:

1) providing a culture of human embryonic stem (ES) cells or induced pluripotent stem cells (iPSCs);

2) at day 0, activating Wnt/β

-catenin signaling in said culture from step 1;

3) at day 3-5, inhibiting Wnt/β

-catenin signaling in said culture from step 2 to generate human cardiac progenitor cells (CPCs);

4) at day 5-7, contacting said human CPCs from step 3 with one or more agents reactive with Jagged 1;

5) separating Jagged 1+ cells from negative cells; and

6) isolating the Jagged 1+ cells as Jagged 1+ human cardiac ventricular progenitor cells.

2. The method of claim 1, wherein at step 4) the human CPCs are further contacted with an agent reactive with Islet 1;

at step

     5) Jagged 1+/Islet 1+ cells are separated from negative cells; and

at step

     6) Jagged 1+/Islet 1+ cells are isolated as Jagged 1+/Islet 1+ human cardiac ventricular progenitor cells.

3. The method of claim 1, wherein the agent reactive with Jagged 1 is an anti-Jagged 1 antibody.

4. The method of claim 1, wherein the Jagged 1+ cells are separated from the negative cells by fluorescence activated cell sorting or magnetic activated cell sorting.

5. The method of claim 1, wherein the human cardiac ventricular progenitor cells are further cultured and differentiated such that they are Myosin Light Chain 2v (MLC2v) positive.