Use of Neuropilin-1 (NRP1) as a cell surface marker for isolating human cardiac ventricular progenitor cells
The present invention provides NRP1 as a cell surface marker for isolating human cardiomyogenic ventricular progenitor cells (HVPs), in particular progenitor cells that preferentially differentiate into cardiac ventricular muscle cells. Additional HVP cell surface markers identified by single cell sequencing are also provided. The invention provides in vitro methods of the separation of NRP1+ ventricular progenitor cells, and the large scale expansion and propagation thereof. Large clonal populations of isolated NRP1+ ventricular progenitor cells are also provided. Methods of in vivo use of NRP1+ ventricular progenitor cells for cardiac repair or to improve cardiac function are also provided. Methods of using the NRP1+ 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:
subjecting human pluripotent stem cells to Wnt/β-catenin signaling activation on day 0 of culture, followed by inhibition of Wnt/β-catenin signaling activation on day 3 to day 5 of culture to obtain a culture of human cells containing cardiac progenitor cells;
contacting the culture of human cells containing cardiac progenitor cells with one or more agents reactive with neuropilin-1 (NRP1), wherein the culture is contacted with the one or more agents reactive with NRP1 on day 5-7 of culture; and
separating NRP1 reactive positive cells from non-reactive cells to thereby isolate NRP1+ human cardiac ventricular progenitor cells.
2. The method of claim 1 , wherein the culture of human cells is further contacted with at least one second agent reactive with human cardiac ventricular progenitor cells; and NRP1 reactive/second agent reactive positive cells are separated from non-reactive cells to thereby isolate NRP1+ human cardiac ventricular progenitor cells.
3. The method of claim 2 , wherein the at least one second agent is reactive with at least one of Jagged 1 (JAG1), Frizzled 4 (FZD4), Leukemia Inhibitory Factor Receptor (LIFR), Fibroblast Growth Factor Receptor 3 (FGFR3) and Tumor Necrosis Factor Ligand Superfamily Member 9 (TNFSF9).
4. The method of claim 2 , wherein the culture of human cells is contacted with the agent reactive with NRP1 before contacting with the at least one second agent.
5. The method of claim 2 , wherein the culture of human cells is contacted with the at least one second agent before contacting with the agent reactive with NRP1.
6. The method of claim 2 , wherein the culture of human cells is contacted simultaneously with the agent reactive with NRP1 and with the at least one second agent.
7. The method of claim 1 , which further comprises contacting the human cardiac ventricular progenitor cells with one or more agents reactive with at least one marker that is expressed on the surface of human pluripotent stem cells but is not expressed on the surface of human cardiac ventricular progenitor cells and separating marker-nonreactive negative cells from reactive cells to thereby further isolate human cardiac ventricular progenitor cells.
8. The method of claim 7 , wherein the at least one marker that is expressed on the surface of human pluripotent stem cells is selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, E-cadherin and Podocalyxin, and combinations thereof.
9. The method of claim 7 , wherein the at least one marker that is expressed on the surface of human pluripotent stem cells is TRA-1-60.
10. The method of claim 9 , wherein the agent reactive with TRA-1-60 is an anti-TRA-1-60 antibody.
11. The method of claim 1 , wherein the agent reactive with NRP1 is an anti-NRP1 antibody.
12. The method of claim 1 , wherein the agent reactive with NRP1 comprises a soluble NRP1 ligand.
13. The method of claim 12 , wherein the NRP1 ligand is VEGF-A or Sema3A.
14. The method of claim 1 , wherein the NRP1 reactive positive cells are separated from the non-reactive cells by fluorescent activated cell sorting (FACS) or magnetic activated cell sorting (MACS).
15. The method of claim 1 , wherein the NRP1+ human cardiac ventricular progenitor cells are cultured and differentiated such that they are MLC2v positive.
16. The method of claim 1 , wherein the human pluripotent stem cells are human embryonic stem cells or human induced pluripotent stem cells.