Method of identifying and/or isolating stem cells and prognosing responsiveness to leukemia treatment

• US 20020102244A1
• Filed: 05/29/2001
• Published: 08/01/2002
• Est. Priority Date: 05/28/1998
• Status: Active Grant

• Current Assignee: St Jude Children's Research Hospital Incorporated (Abbott Laboratories Incorporated)
• Original Assignee: St Jude Children's Research Hospital Incorporated (Abbott Laboratories Incorporated)
• Inventors: Sorrentino, Brian, Schuetz, John
• Granted Patent: US 7,622,557 B2
• Time in Patent Office: Days
• US Class Current: 424/93.21
• CPC Class Codes: A61K 2035/124 the cells being hematopoietic, bone marrow derived or blood cells
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C12N 2799/022 where the vector is derived from an adenovirus
• CPC Class Codes: C12N 2799/027 where the vector is derived from a retrovirus
• CPC Class Codes: G01N 33/5005 involving human or animal cells immunoassay G01N33/56966; immunoassays of protozoa G01N33/56905; protozoa in screening assays C12Q1/025
• CPC Class Codes: G01N 33/56966 Animal cells
• CPC Class Codes: G01N 33/57426 leukemia

Abstract

The present invention includes methods of identifying and/or isolating stem cells based on expression of BCRP. The present invention also describes methods of obtaining and/or using cell populations enriched for stem cells. In addition, methods are provided for diagnosing and/or prognosing leukemia, particularly human acute myelogenous leukemia (AML), through assaying for BCRP expression in leukemic cells.

Claims

1. A method of identifying a stem cell comprising:

(a) obtaining a cell sample which contains a stem cell; and

(b) detecting the expression of BCRP by a cell in the cell sample;

wherein a cell that expresses BCRP is identified as a stem cell.

2. The method of claim 1 wherein said detecting is performed with an antibody that binds to BCRP;

and wherein a stem cell is identified if it binds to said antibody.

3. The method of claim 1 wherein said detecting is performed with a PCR probe for the nucleic acid that expresses BCRP.

4. The method of claim 1 wherein said detecting is performed by measuring the pumping activity of the cell;

wherein a stem cell is identified if it can remove Hoechst 33342 dye, but not Rhodamine 123 dye.

5. The method of claim 1 further comprising:

(c) detecting the expression of a cell marker associated with stem cells;

wherein a cell that expresses BCRP and expresses the cell marker is identified as a stem cell.

6. The method of claim 5 wherein said cell marker is selected from the group consisting of EM10, CD34, Thy-1, P-gp, Ac133, and c-kit.

7. The method of claim 5 wherein said cell marker is CD34 and wherein the cell is also CD38-.

8. The method of claim 1 further comprising

(c) detecting the expression of a lineage specific marker;

wherein a cell that expresses BCRP but does not express the lineage specific marker is identified as a stem cell.

9. The method of claim 8 wherein said stem cell is a hematopoietic stem cell and the lineage specific marker is selected from the group consisting of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD20, CD33, CD38, CD56, CD71, HLA-DR, and glycophorin A.

10. A method of isolating a cell that expresses BCRP comprising:

(a) obtaining a cell sample which contains a cell that expresses BCRP;

(b) detecting the expression of BCRP by a cell in the cell sample; and

(c) isolating the cell that expresses BCRP.

11. A method of isolating a cell that expresses BCRP comprising:

(a) obtaining a cell sample which contains a cell that expresses BCRP;

(b) contacting the cell sample with an antibody that binds to BCRP; and

(c) isolating a cell from the cell sample that binds to said antibody;

wherein the isolated cell is a cell that expresses BCRP.

12. The method of claim 11, wherein said isolating is performed by flow cytometry.

13. The method of claim 12, wherein said antibody has a fluorescent label and said isolating is performed by fluorescence-activated cell sorting.

14. An isolated stem cell obtained by the method of claim 11.

17. The antibody of claim 16 wherein the BCRP is on the surface of a viable stem cell.

19. The method of claim 18, wherein the leukemia is acute myelogenous leukemia (AML).

20. The method of claim 19 wherein the leukemic cell is a blast cell.

15. A cell population enriched for stem cells obtained by a method comprising:

(a) obtaining a mixed population of cells which comprises both stem cells and cells that are not stem cells; and

(b) separating the cells of the mixed population of cells into two individual groups of cells based on whether they do or do not express BCRP;

wherein the group of cells that express BCRP is a cell population enriched for stem cells.

16. An antibody that recognizes an extracellular portion of a BCRP;

wherein said extracellular portion of the BCRP is in its natural conformation.

18. A method for determining the amount of BCRP expressed in a human subject having or suspected of having leukemia comprising:

(a) obtaining a leukemic cell from the subject; and

(b) determining the amount of BCRP expressed in the leukemic cell.