Use of lifr or fgfr3 as a cell surface marker for isolating human cardiac ventricular progenitor cells

Abstract

The present invention provides LIFR and FGFR3 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+ LIFR+ ventricular progenitor cells and/or Islet 1+/FGFR3+ ventricular progenitor cells and/or Islet 1+/LIFR+/FGFR3+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated LIFR+ and/or FGFR3+ ventricular progenitor cells are also provided. Methods of in vivo use of LIFR+ and/or FGFR3+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the LIFR+ and/or FGFR3+ ventricular progenitor cells for cardiac toxicity screening of test compounds are also provided.

1 .- 38 . (canceled) 
     
     
         39 . 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) on day 5, day 6 or day 7, contacting said human CPCs from step 3 with one or more agents reactive with a cardiac progenitor marker selected from the group consisting of PDGFRA, TNFSF9 and FZD4;   5) separating cardiac progenitor marker positive cells from negative cells; and   6) isolating the cardiac progenitor marker positive cells to thereby isolate the human cardiac ventricular progenitor cells.   
     
     
         40 . The method of  claim 39 , wherein at step 4) the human CPCs are contacted with an antibody that binds the cardiac progenitor marker. 
     
     
         41 . The method of  claim 39 , wherein at step 4) the human CPCs are contacted with a soluble ligand that binds the cardiac progenitor marker. 
     
     
         42 . The method of  claim 39 , wherein the cardiac progenitor marker is PDGFRA. 
     
     
         43 . The method of  claim 42 , wherein the human CPCs are contacted with an anti-PDGFRA antibody. 
     
     
         44 . The method of  claim 39 , wherein the cardiac progenitor marker is TNFSF9. 
     
     
         45 . The method of  claim 44 , wherein the human CPCs are contacted with an anti-TNFSF9 antibody. 
     
     
         46 . The method of  claim 39 , wherein the cardiac progenitor marker is FZD4. 
     
     
         47 . The method of  claim 46 , wherein the human CPCs are contacted with an anti-FZD4 antibody. 
     
     
         48 . The method of  claim 39 , wherein the cardiac progenitor marker+ cells are separated from negative cells by fluorescence activated cell sorting or magnetic activated cell sorting. 
     
     
         49 . The method of  claim 39 , wherein at step 1), a culture of human ES cells is provided. 
     
     
         50 . The method of  claim 39 , wherein at step 1), a culture of human iPSCs is provided. 
     
     
         51 . The method of  claim 39 , wherein Wnt/β-catenin signaling is activated in the CPCs by culture with a Gsk3 inhibitor. 
     
     
         52 . The method of  claim 39 , wherein Wnt/β-catenin signaling is inhibited in the CPCs by culture with a Porcn inhibitor. 
     
     
         53 . The method of  claim 39 , wherein step 4) is conducted on day 5. 
     
     
         54 . The method of  claim 39 , wherein step 4) is conducted on day 6. 
     
     
         55 . The method of  claim 39 , wherein step 4) is conducted on day 7. 
     
     
         56 . The method of  claim 39 , wherein the human cardiac ventricular progenitor cells are further differentiated such that they are Myosin Light Chain 2v (MLC2v) positive. 
     
     
         57 . The method of  claim 39 , which further comprises administering the human cardiac ventricular progenitor cells directly into the heart of a subject. 
     
     
         58 . The method of  claim 39 , wherein the human cardiac ventricular progenitor cells are administered directly into a ventricular region of the heart of the subject. 
     
     
         59 . The method of  claim 39 , which further comprises contacting the human cardiac ventricular progenitor cells with a test compound and measuring toxicity of the test compound for the cells, wherein toxicity of the test compound for the cells indicates cardiac toxicity of the test compound.