Methods for isolating human cardiac ventricular progenitor cells
First Claim
1. A method for generating human ventricular muscle cells in vivo in a subject using engraftable human cardiac ventricular progenitor cells (HVPs), the method comprising:
- contacting a culture of day 5-7 cardiac progenitor cells (CPCs) 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 with one or more second agents reactive with a second marker selected from the group consisting of JAG1, LIFR, FGFR3, TNFSF9 and combinations thereof, wherein the culture is contacted with the one or more second agents either before, simultaneously with or after contacting with the one or more first agents,wherein the culture of day 5-7 CPCs has been obtained by subjecting human pluripotent stem cells to activation of Wnt/β
-catenin signaling on day 0, followed by inhibition of Wnt/β
-catenin signaling from day 3 to day 5;
isolating first marker negative/second marker positive cells to thereby isolate a cell population comprising engraftable HVPs,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; and
detecting cardiac function in the subject indicative of engraftment of the isolated cell population comprising engraftable HVPs.
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Abstract
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.
32 Citations
31 Claims
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1. A method for generating human ventricular muscle cells in vivo in a subject using engraftable human cardiac ventricular progenitor cells (HVPs), the method comprising:
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contacting a culture of day 5-7 cardiac progenitor cells (CPCs) 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 with one or more second agents reactive with a second marker selected from the group consisting of JAG1, LIFR, FGFR3, TNFSF9 and combinations thereof, wherein the culture is contacted with the one or more second agents either before, simultaneously with or after contacting with the one or more first agents, wherein the culture of day 5-7 CPCs has been obtained by subjecting human pluripotent stem cells to activation of Wnt/β
-catenin signaling on day 0, followed by inhibition of Wnt/β
-catenin signaling from day 3 to day 5;isolating first marker negative/second marker positive cells to thereby isolate a cell population comprising engraftable HVPs, 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; and detecting cardiac function in the subject indicative of engraftment of the isolated cell population comprising engraftable HVPs. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 29, 30, 31)
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15. A composition comprising an isolated population of at least 1×
- 106 purified engraftable human cardiac ventricular progenitor cells (HVPs), wherein the population of HVPs is derived from human embryonic stem (ES) cells or induced pluripotent stem cells (iPSC) and is;
(i) negative for 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 (ii) positive for at least one second marker selected from the group consisting of JAG1, LIFR, FGFR3 and TNFSF9, wherein the HVPs are complexed with at least one antibody that binds JAG1, LIFR, FGFR3 or TNFSF9.
- 106 purified engraftable human cardiac ventricular progenitor cells (HVPs), wherein the population of HVPs is derived from human embryonic stem (ES) cells or induced pluripotent stem cells (iPSC) and is;
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16. A method for isolating a cell population comprising engraftable human cardiac ventricular progenitor cells (HVPs), the method comprising:
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contacting a culture of day 5-7 cardiac progenitor cells (CPCs) comprising HVPs with one or more first agents reactive with a 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 with one or more second agents reactive with a second marker selected from the group consisting of JAG1, LIFR, FGFR3, TNFSF9 and combinations thereof, wherein the culture is contacted with the one or more second agents either before, simultaneously with or after contacting with the one or more first agents; wherein the culture of day 5-7 CPCs has been obtained by subjecting human embryonic stem (ES) cells or induced pluripotent stem cells (iPSC) to activation of Wnt/β
-catenin signaling on day 0, followed by inhibition of Wnt/β
-catenin signaling from day 3 to day 5; andisolating first marker negative/second marker positive cells to thereby isolate a cell population comprising engraftable HVPs. - View Dependent Claims (17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
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Specification