Methods for Cardiac Fibroblast Differentiation of Human Pluripotent Stem Cells

Abstract

Methods for generating high-yield high-purity cardiac fibroblasts are described. Differentiation methods comprising thematically defined culture conditions and methods for in vitro maintenance of human pluripotent stern cell-derived cardiac fibroblasts are also provided.

1 - 10 . (canceled) 
     
     
         11 . A method for generating a human arrhythmia model, the method comprising the steps of:
 co-culturing a population of cardiac fibroblast cells in the presence of a population of cardiomyocytes in a chemically defined culture medium.   
     
     
         12 . The method of  claim 11 , wherein the cardiac fibroblast cells are generated by a method comprising culturing human cardiac mesoderm progenitor cells in a chemically defined culture medium comprising a fibroblast growth factor, whereby a cell population comprising human cardiac fibroblast cells is obtained after about 20 days in culture. 
     
     
         13 . The method of  claim 11 , wherein the co-culture comprises 10%, 50%, 70%, or 90% cardiac fibroblast cells. 
     
     
         14 . The method of  claim 11 , wherein the co-culture is seeded at a density between about 60,000 cells/cm 2  and about 120,000 cells/cm 2 . 
     
     
         15 . A method of screening a test agent, the method comprising:
 co-culturing a population of cardiac fibroblast cells in the presence of a population of cardiomyocytes in a chemically defined culture medium;   contacting the co-culture with a test agent;   measuring a functional parameter of the contacted co-culture; and   comparing the functional parameter to that parameter measured in a co-culture which has not been contacted with the test agent, wherein modulation of the functional parameter after contact with the test agent indicates the test agent is a candidate therapeutic agent.   
     
     
         16 . The method of  claim 15 , wherein the test agent is selected from the group consisting of (i) an organic compound; (ii) a nucleic acid; (iii) a peptide; (iii) a polypeptide; and
 (iv) an antibody.   
     
     
         17 . The method of  claim 15 , wherein the functional parameter is selected from the group consisting of electrical impulse propagation pattern, conduction velocity, and action potential duration. 
     
     
         18 . The method of  claim 18 , wherein the electrical impulse propagation pattern is measured using a fluorescent membrane potential dye. 
     
     
         19 . The method of  claim 17 , wherein acceleration of the conduction velocity after contact with the test agent indicates the test agent is a candidate therapeutic agent. 
     
     
         20 . The method of  claim 17 , wherein prolongation of the action potential duration after contact with the test agent indicates the test agent is a candidate therapeutic agent. 
     
     
         21 . The method of  claim 17 , wherein the electrical impulse propagation pattern is measured as the fibrillatory or reentry pattern and an increase in the pattern after contact with the test agent indicates the test agent is a candidate therapeutic agent. 
     
     
         22 . The method of  claim 15 , wherein the cardiac fibroblast cells are generated by a method comprising culturing human cardiac mesoderm progenitor cells in a chemically defined culture medium comprising a fibroblast growth factor, whereby a cell population comprising human cardiac fibroblast cells is obtained after about 20 days in culture. 
     
     
         23 . A kit for differentiating human pluripotent stem cells into cardiac fibroblasts, the kit comprising: (i) a chemically defined culture medium suitable for differentiating human cardiac progenitor cells into cardiac fibroblasts; (ii) an agent that activates Wnt signaling in human cardiac progenitor cells; (iii) a fibroblast growth factor; and (iv) instructions describing a method for generating human CFs, the method employing the culture medium, the agent, and the fibroblast growth factor.