Methods for isolating human cardiac ventricular progenitor cells
The present invention provides methods for isolating human cardiac ventricular progenitor cells (HVPs), wherein cultures of day 5-7 cardiac progenitor cells are negatively selected for one or more first markers expressed on human pluripotent stem cells, such as TRA-1-60, to thereby isolate HVPs. The methods can further include positive selection for expression of a second marker selected from the group consisting of JAG1, FZD4, LIFR, FGFR3 and TNFSF9. Large populations, including clonal populations, of isolated HVPs that are first marker negative/second marker positive are also provided. Methods of in vivo use of the HVPs for cardiac repair or to improve cardiac function are also provided. Methods of using the HVPs for cardiac toxicity screening of test compounds are also provided.
1. A method for isolating a cell population comprising engraftable human cardiac ventricular progenitor cells (HVPs), the method comprising: subjecting human pluripotent stem cells to activation of Wnt/P-catenin signaling on day 0, followed by inhibition of Wnt/p-catenin signaling from day 3 to day 5 to obtain a culture comprising HVPs; on day 5-7, contacting the culture comprising HVPs with one or more first agents reactive with at least one first marker selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, E-cadherin, Podocalyxin, and combinations thereof; and isolating first marker-nonreactive negative cells to thereby isolate a substantially pure cell population comprising engraftable HVPs positive for at least one of surface marker selected from the group consisting of JAG1, FZD3, LIFR, FGFR3 and TNFSF9.
2. The method of claim 1 , wherein the first agent is an antibody that binds the first marker.
3. The method of claim 1 , wherein the first marker-nonreactive negative cells are isolated by fluorescence activated cell sorting (FACS) or by magnetic activated cell sorting (MACS).
4. The method of claim 1 , wherein the culture comprising HVPs is contacted with the one or more first agents on day 6.
5. The method of claim 1 , which further comprises administering the cell population comprising engraftable HVPs to a subject;
wherein the cell population comprising engraftable HVPs forms a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix in the subject.
6. The method of claim 5 , which further comprises detecting cardiac function in the subject indicative of engraftment of the isolated cell population comprising engraftable HVPs.
7. A method for isolating a cell population comprising engraftable human cardiac ventricular progenitor cells (HVPs), the method consisting essentially of: subjecting human pluripotent stem cells to activation of Wnt/P-catenin signaling on day 0, followed by inhibition of Wnt/p-catenin signaling from day 3 to day 5 to obtain a culture comprising HVPs; on day 5-7, contacting the culture comprising HVPs with one or more first agents reactive with at least one first marker selected from the group consisting of TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, E-cadherin, Podocalyxin, and combinations thereof; and isolating first marker-nonreactive negative cells to thereby isolate a substantially pure cell population comprising engraftable HVPs positive for at least one of surface marker selected from the group consisting of JAG1, FZD3, LIFR, FGFR3 and TNFSF9.
8. The method of claim 7 , wherein the first agent is an antibody that binds the first marker.
9. The method of claim 7 , wherein the first marker-nonreactive negative cells are isolated by fluorescence activated cell sorting (FACS) or by magnetic activated cell sorting (MACS).
10. The method of claim 7 , wherein the culture comprising HVPs is contacted with the one or more first agents on day 6.
11. The method of claim 7 , which further comprises administering the cell population comprising engraftable HVPs to a subject;
wherein the cell population comprising engraftable HVPs forms a vascularized, electrically responsive ventricular muscle patch that secretes an extracellular matrix in the subject.
12. The method of claim 11 , which further comprises detecting cardiac function in the subject indicative of engraftment of the isolated cell population comprising engraftable HVPs.