Use of LIFR or FGFR3 as a cell surface marker for isolating human cardiac ventricular progenitor cells
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. 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 Leukemia Inhibitory Factor Receptor (LIFR) and/or Fibroblast Growth Factor Receptor 3 (FGFR3);
5) separating LIFR+ cells and/or FGFR3+ cells from negative cells; and
6) isolating the LIFR+ cells and/or FGFR3+ cells as LIFR+ and/or FGFR3+ 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) LIFR+ and/or FGFR3+/Islet 1+ cells are separated from negative cells; and at step 6) LIFR+ and/or FGFR3+/Islet 1+ cells are isolated as LIFR+ and/or FGFR3+/Islet 1+ human cardiac ventricular progenitor cells.
3. The method of claim 2 , wherein at step 4) the human CPCs are simultaneously contacted with one or more agents reactive with LIFR and/or FGFR3 and the agent reactive with Islet 1.
4. The method of claim 2 , wherein at step 4) the human CPCs are contacted with the agent reactive with Islet 1 before contacting with the one or more agents reactive with LIFR and/or FGFR3.
5. The method of claim 2 , wherein at step 4) the human CPCs are contacted with the one or more agents reactive with LIFR and/or FGFR3 before contacting with the agent reactive with Islet 1.
6. The method of claim 1 , wherein at step 4) the human CPCs are contacted with an anti-LIFR antibody.
7. The method of claim 1 , wherein at step 4) the human CPCs are contacted with a soluble LIFR ligand fusion protein.
8. The method of claim 7 , wherein the LIFR ligand is LIF.
9. The method of claim 1 , wherein the LIFR+ cells and/or FGFR3+ cells are separated from negative cells by fluorescence activated cell sorting or magnetic activated cell sorting.
10. 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.
11. The method of claim 1 , wherein at step 4) the human CPCs are contacted with an anti-FGFR3 antibody.
12. The method of claim 1 , wherein at step 4) the human CPCs are contacted with a soluble FGFR3 ligand fusion protein.