Compositions of cardiomyocyte subpopulations

Patent metadata

Field	Value
Publication number	US-9717762-B2
Application number	US-201414547994-A
Country	US
Kind code	B2
Filing date	Nov 19, 2014
Priority date	Nov 20, 2013
Publication date	Aug 1, 2017
Grant date	Aug 1, 2017

Abstract

The present invention provides the use of the antigens CD49e and/or CD49f as selection markers for enrichment, isolation, detection and/or analysis of atrial and ventricular cardiomyocytes and a method for enrichment, isolation, detection and/or analysis of these cells from a sample comprising cardiomyocytes. In addition substantially pure compositions of these cardiomyocyte subpopulations are provided.

First claim

The invention claimed is:

1. A method for determining atrial and/or ventricular cardiomyocytes in a sample comprising cardiomyocytes, the method comprising the steps:
a) contacting said sample:
i) with an antigen-binding moiety specific for the CD49e antigen coupled to a fluorophore, and/or
ii) with an antigen-binding moiety specific for the CD49f antigen coupled to a fluorophore,

b) contacting said sample with an agent that is distinguishably distinct from the antigen binding moieties used in step (a), whereby the agent specifically labels cardiomyocytes in the sample,
c) determining atrial and/or ventricular cardiomyocytes in the sample by detecting and comparing the intensity of labeling of cells in the sample for the CD49e antigen and/or the CD49f antigen with the intensity of labeling of the same cells by the agent, wherein
ventricular cardiomyocytes are determined as being labeled with the agent and having more intense labeling of CD49e and/or less intense labeling of CD49f; and/or
atrial cardiomyocytes are determined as being labeled with the agent and having more intense labeling of CD49f and/or less intense labeling of CD49e.

2. The method of claim 1 , wherein said agent is an antigen-binding moiety specific for the cardiomyocyte cell surface marker CD340 coupled to a fluorophore.

3. A method of determining atrial cardiomyocytes according to claim 1 , comprising the steps:
a) contacting said sample with the antigen-binding moiety specific for the CD49f antigen coupled to a fluorophore,
b) contacting said sample with the agent, thereby co-labeling the cardiomyocytes,
c) determining atrial cardiomyocytes in the sample by detecting and comparing the intensity of labeling of cardiomyocytes in the sample for the CD49f antigen with the intensity of labeling of the same cells by the agent, wherein atrial cardiomyocytes are determined as having more intense labeling of CD49f.

4. A method isolating or purifying atrial cardiomyocytes in a sample comprising cardiomyocytes, the method comprising the steps:
I) determining atrial cardiomyocytes in the sample according to the method of claim 3 , and
II) separating atrial cardiomyocytes determined in step (I) from other cells in the sample.

5. A method of determining ventricular cardiomyocytes according to claim 1 , comprising the steps:
a) contacting said sample with the antigen-binding moiety specific for the CD49f antigen coupled to a fluorophore,
b) contacting said sample with the agent, thereby co-labeling the cardiomyocytes,
c) determining ventricular cardiomyocytes in the sample by detecting and comparing the intensity of labeling of cardiomyocytes in the sample for the CD49f antigen with the intensity of labeling of the same cells by the agent, wherein ventricular cardiomyocytes are determined as having less intense labeling of CD49f.

6. A method isolating or purifying ventricular cardiomyocytes in a sample comprising cardiomyocytes, the method comprising the steps:
I) determining ventricular cardiomyocytes in the sample according to the method of claim 5 , and
II) separating ventricular cardiomyocytes determined in step (I) from other cells in the sample.

7. A method of determining ventricular cardiomyocytes according to claim 1 , comprising the steps:
a) contacting said sample with the antigen-binding moiety specific for the CD49e antigen coupled to a fluorophore,
b) contacting said sample with the agent, thereby co-labeling the cardiomyocytes,
c) determining ventricular cardiomyocytes in the sample by detecting and comparing the intensity of labeling of cardiomyocytes in the sample for the CD49e antigen with the intensity of labeling of the same cells by the agent, wherein ventricular cardiomyocytes are determined as having more intense labeling of CD49e.

8. A method of determining atrial cardiomyocytes according to claim 1 , comprising the steps:
a) contacting said sample with the antigen-binding moiety specific for the CD49e antigen coupled to a fluorophore,
b) contacting said sample with the agent, thereby co-labeling the cardiomyocytes,
c) determining atrial cardiomyocytes in the sample by detecting and comparing the intensity of labeling of cardiomyocytes in the sample for the CD49e antigen with the intensity of labeling of the same cells by the agent, wherein atrial cardiomyocytes are determined as having less intense labeling of CD49e.

9. The method of claim 1 , wherein the sample is contacted with both an antigen binding moiety specific for CD49e labeled with a first fluorophore, and a second antigen binding moiety specific for CD49f labeled with a second fluorophore that is different from the first fluorophore.

10. The method of claim 1 , wherein the agent that specifically labels cardiomyocytes is an antigen binding moiety specific for CD61, CD146, CD112 or CD340.

11. A method isolating or purifying atrial and/or ventricular cardiomyocytes in a sample comprising cardiomyocytes, the method comprising the steps:
I) determining atrial and/or ventricular cardiomyocytes in the sample according to the method of claim 1 , and
II) separating cardiomyocytes determined in step (I) from other cells in the sample.

12. A method for separating atrial cardiomyocytes and/or ventricular cardiomyocytes in a sample that is pre-enriched for cardiomyocytes, the method comprising the steps:
a) contacting said pre-enriched sample with an antigen-binding moiety specific for the CD49e antigen coupled to a tag, and
b) separating cardiomyocytes based on intensity of labeling of the cardiomyocytes by the tag;
wherein atrial cardiomyocytes in the sample are less strongly tagged by the antigen binding moiety specific for CD49e antigen, and are thereby separated from ventricular cardiomyocytes which are more strongly tagged by the antigen binding moiety specific for the CD49e antigen.

13. The method of claim 12 , wherein said sample has been pre-enriched for cardiomyocytes by a cell depleting method.

14. The method of claim 13 , wherein said cell depleting method comprises the steps:
I) contacting a non-enriched sample comprising non-cardiomyocytes and cardiomyocytes with combinations of antigen-binding moieties specific for cell surface markers of non-cardiomyocytes coupled to tags, thereby labeling the non-cardiomyocytes,
II) isolating the non-labeled cardiomyocytes from said non-enriched sample.

15. The method of claim 14 , wherein said cell surface markers of non-cardiomyocytes are selected from the group consisting of surface markers Sca-1, CD15, CD31, CD38, CD45, CD49b, CD49d, CD54, CD66a, CD73, CD90.1, CD90.2, CD105, CD117, CD138, CD140a, CD140b, CD184, CD326, with the proviso that for PSC-derived non-cardiomyocytes at least one cell surface marker is CD31, CD66a, CD38, CD49b, Sca-1, or CD105 and at least one cell surface marker is CD326 or CD15, or with the proviso that for neonatal non-cardiomyocytes at least one cell surface marker is CD31, CD105, or CD146 and at the other cell surface markers are CD45, CD51 and CD90.2.

16. The method of claim 14 , wherein in said cell depletion method said tag is a magnetic particle, and wherein said isolation of said non-labeled cardiomyocytes of said non-enriched sample is performed by magnetic cell sorting.

17. The method of claim 12 , wherein in said method comprises separation of cells by flow cytometry.

18. The method of claim 12 , wherein said antigen binding moiety is an antibody or fragment thereof.

19. The method of claim 12 , wherein said atrial and/or ventricular cardiomyocytes are human or murine cells.

Assignees

• Miltenyi Biotec Gmbh
• Miltenyi Biotec

Inventors

• Wiencierz Anne Maria
• Eckardt Dominik
• Bosio Andreas

Classifications

• G01N33/56966

  Animal cells · CPC title

• C12N5/00

  Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor; (plant reproduction by tissue culture techniques A01H4/00) · CPC title

• A61K35/34 Primary

  Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44) · CPC title

• C12N5/0657

  Cardiomyocytes; Heart cells · CPC title

• G01N2333/7055

  Integrin beta1-subunit-containing molecules, e.g. CD29, CD49 · CPC title