Method of selecting a cardiomyoctye using intracellular mitochondria as an indicator
View Patent ↗The invention relates to methods of selecting a cardiomyocyte from a cell population derived from a whole heart or a differentiated cell population derived from a stem cell without genetic alteration. Specifically, the invention relates to a method of selecting a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell. The invention also relates to methods of enriching a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of a cardiomyocyte, producing a cardiomyocyte without genetic alteration of a cardiomyocyte, and evaluating the ratio of a cardiomyocyte in a cardiomyocyte-containing cell mixture.
1. A method of selecting a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of the cardiomyocyte comprising selecting the cardiomyocyte on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell, wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a fetus whole heart or a cell mixture derived from a cell having an ability to differentiate to the cardiomyocyte.
2. The method of claim 1 , wherein the cardiomyocyte is selected on the basis of a relative content of cellular mitochondria.
3. The method of claim 1 , wherein the cardiomyocyte is selected on the basis of a relative mitochondrial transmembrane potential of the cell.
4. The method of claim 1 , wherein the cardiomyocyte is selected on the basis of both a relative content of cellular mitochondria and transmembrane potential.
5. The method of claim 1 , wherein said method comprises a step of labeling the cardiomyocyte-containing cell mixture with a mitochondrial indicator and a step of measuring a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell.
6. The method of claim 5 , wherein said method further comprises a step of culturing the labeled cell in the absence of the mitochondrial indicator, after the step of labeling the cardiomyocyte-containing cell mixture with the mitochondrial indicator, and before the step of measuring a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell.
7. The method of claim 5 , wherein said mitochondrial indicator is A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 or T3168.
8. The method of claim 7 , wherein said mitochondrial indicator is M7512, T3168, T668 or R302.
9. The method of claim 1 , wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a fetus whole heart.
10. The method of claim 1 , wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a cell having an ability to differentiate to the cardiomyocyte, and the cell having an ability to differentiate to a cardiomyocyte is a stem cell, a progenitor cell or an egg cell.
11. The method of claim 1 , further comprising evaluating the ratio of a cardiomyocyte in a cardiomyocyte-containing cell mixture, comprising:
(1) labeling the cardiomyocyte-containing cell mixture with a mitochondrial indicator; and
(2) measuring the ratio of the cardiomyocyte to a non-cardiomyocyte on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell, whereby the ratio of a cardiomyocyte in a cardiomyocyte-containing cell mixture can be evaluated.
12. The method of claim 11 , wherein said cell having an ability to differentiate to the cardiomyocyte is a stem cell, a progenitor cell, or an egg cell.
13. The method of claim 11 , wherein said mitochondrial indicator is A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 or T3168.
14. The method of claim 13 , wherein said mitochondrial indicator is M7512, T3168, T668 or R302.
15. A method of enriching a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of the cardiomyocyte, comprising:
(1) labeling the cardiomyocyte-containing cell mixture with a mitochondrial indicator; and
(2) selecting the cardiomyocyte on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell,
wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a fetus whole heart or a cell mixture derived from a cell having an ability to differentiate to the cardiomyocyte.
16. The method of claim 15 , wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a fetus whole heart.
17. The method of claim 15 , wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a cell having, an ability to differentiate to the cardiomyocyte. and said cell having an ability to differentiate to a cardiomyocyte is a stem cell, a progenitor cell, or an egg cell.
18. The method of claim 15 , wherein said method further comprises a step of culturing the labeled cell in the absence of the mitochondrial indicator, after step (1), and before step (2).
19. The method of claim 15 , wherein said mitochondrial indicator is A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 or T3168.
20. The method of claim 19 , wherein said mitochondrial indicator is M7512, T3168, T668 or R302.
21. A method of producing a cardiomyocyte without genetic alteration of the cardiomyocyte, comprising:
(1) differentiating and inducing the cardiomyocyte from a cell having an ability to differentiate to the cardiomyocyte to prepare a cardiomyocyte-containing cell mixture;
(2) labeling the cardiomyocyte-containing cell mixture with a mitochondrial indicator; and
(3) selecting the cardiomyocyte on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell.
22. The method of claim 21 , wherein said cell having an ability to differentiate to the cardiomyocyte is a stem cell, a progenitor cell, or an egg cell.
23. The method of claim 21 , wherein said method further comprises a step of culturing the labeled cell in the absence of the mitochondrial indicator, after step (2), before step (3).
24. The method of claim 21 , wherein said mitochondrial indicator is A1372, D273, D288, D308, D378, D426, D632, D633, D22421, D23806, L6868, M7502, M7510, M7511, M7512, M7513, M7514, M22422, M22423, M22425, M22426, R302, R634, R648, R14060, R22420, T639, T668, T669 or T3168.
25. The method of claim 24 , wherein said mitochondria indicator is M7512, T3168, T668 or R302.
26. A method of isolating a cardiomyocyte from a cardiomyocyte-containing cell mixture without genetic alteration of the cardiomyocyte comprising isolating the cardiomyocyte on the basis of a relative content of cellular mitochondria and/or a relative mitochondrial transmembrane potential of the cell, wherein said cardiomyocyte-containing cell mixture is a cell mixture derived from a fetus whole heart or a cell mixture derived from a cell having an ability to differentiate to the cardiomyocyte.