Method for the isolation of stem cells by immuno-labeling with HLA/MHC gene product marker

• US 20020119564A1
• Filed: 05/09/2001
• Published: 08/29/2002
• Est. Priority Date: 05/10/2000
• Status: Active Grant

• Current Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Original Assignee: Cedars-Sinai Medical Center (Cedars Sinai Health System)
• Inventors: Avital, Itzhak, Arnaout, Walid, Inderbitzen, Daniel
• Granted Patent: US 6,828,145 B2
• Time in Patent Office: Days
• US Class Current: 435/366
• CPC Class Codes: C12N 5/0623 Stem cells
• CPC Class Codes: C12N 5/0672 Stem cells; Progenitor cells; Precursor cells; Oval cells
• 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 33/6863 Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors

Abstract

Disclosed herein is the discovery that mammalian stem cells do not express β₂microglobulin (β₂m). The invention discloses a method of isolating stem cells comprising sorting, from a sample of cells, cells that express β₂m from cells that do not express β₂m. One then selects stem cells from the population of cells that does not express β₂m. This is accomplished by selecting cells that express a known marker, such as proteins expressed by genes encoding the major histocompatibility complex. An isolated stem cell is disclosed, as is a method for identifying it and other stem cells.

Claims

1. A method for the isolation of stem cells of a mammal, the method comprising:

obtaining a sample of cells from the mammal;

sorting, from the sample, cells that express β

₂-microglobulin from cells that do not express β

₂-microglobulin;

selecting stem cells from the sample of cells that do not express β

₂-microglobulin.

2. The method of claim 1, wherein the step of sorting comprises sorting by fluorescent activated cell sorting.

3. The method of claim 1, wherein the step of sorting comprises sorting by magnetic bead cell sorting.

4. The method of claim 1, wherein the step of sorting comprises sorting by double magnetic bead cell sorting.

5. The method of claim 1, wherein the step of selecting stem cells further comprises sorting, from the sample of cells that do not express β

₂-microglobulin, cells that express a stem cell marker from cells that do not express a stem cell marker.

6. The method of claim 1, wherein the step of selecting stem cells from the sample further comprises selecting pluripotent stem cells from the sample.

7. The method of claim 1, wherein the step of selecting stem cells from the sample further comprises selecting embryonal stem cells from the sample.

8. The method of claim 5, wherein the stem cell marker is a protein expressed by one or more genes encoding the major histocompatibility complex.

9. The method of claim 8, wherein the one or more genes encode human leukocyte antigens.

10. The method of claim 8, wherein the marker is Thy-1.

11. The method of claim 8, wherein the marker is selected from the group consisting of RT1A, RT1B, and RT1D.

12. The method of claim 8, wherein the marker is selected from the group consisting of flt-3, CD 34, c-Kit, and CD38.

13. The method of claim 5, wherein the step of selecting stem cells further comprises sorting by fluorescent activated cell sorting.

14. The method of claim 5, wherein the step of selecting stem cells further comprises sorting by magnetic bead cell sorting.

15. The method of claim 5, wherein the step of selecting stem cells further comprises sorting by double magnetic bead cell sorting.

16. The method of claim 1, wherein the sample of cells is obtained from an adult mammal.

17. The method of claim 1, wherein the sample of cells is obtained from a fetus.

18. The method of claim 1, wherein the sample of cells is obtained from bone marrow.

19. The method of claim 1, wherein the sample of cells is obtained from the liver of a mammal.

20. The method of claim 1, wherein the sample of cells is obtained from the brain of a mammal.

21. A method for the isolation of stem cells of a mammal, the method comprising:

obtaining a sample of cells from the mammal;

sorting, from the sample, cells that express β

₂-microglobulin from cells that do not express β

₂-microglobulin;

sorting, from the sample of cells that do not express β

₂-microglobulin, cells that express a stem cell marker from cells that do not express a stem cell marker.

22. The method of claim 21, wherein the step of sorting comprises sorting by fluorescent activated cell sorting.

23. The method of claim 21, wherein the step of sorting comprises sorting by magnetic bead cell sorting.

24. The method of claim 21, wherein the step of sorting comprises sorting by double magnetic bead cell sorting.

25. The method of claim 21, wherein the stem cell marker is a protein expressed by one or more genes encoding the major histocompatibility complex.

26. The method of claim 25, wherein the one or more genes encode human leukocyte antigens.

27. The method of claim 25, wherein the marker is Thy-1.

28. The method of claim 25, wherein the marker is selected from the group consisting of RT1A, RT1B, and RT1D.

29. The method of claim 25, wherein the marker is selected from the group consisting of flt-3, CD 34, c-Kit, and CD38.

30. The method of claim 21, wherein the sample of cells is obtained from an adult mammal.

31. The method of claim 21, wherein the sample of cells is obtained from a fetus.

32. The method of claim 21, wherein the sample of cells is obtained from bone marrow.

33. The method of claim 21, wherein the sample of cells is obtained from the liver of a mammal.

34. The method of claim 21, wherein the sample of cells is obtained from the brain of a mammal.

35. The method of claim 21, wherein the cells that express a stem cell marker are pluripotent stem cells.

36. The method of claim 21, wherein the cells that express a stem cell marker are embyonal stem cells.

38. The isolated stem cell of claim 37, wherein the cell expresses Thy-1.

39. The isolated stem cell of claim 38, wherein the cell is derived from bone marrow of a mammal.

40. The isolated stem cell of claim 38, wherein the cell is derived from the liver of a mammal.

41. The isolated stem cell of claim 37, wherein the stem cell is a pluripotent stem cell.

42. The isolated stem cell of claim 37, wherein the stem cell is an embryonal stem cell.

37. An isolated stem cell that does not express β

₂m.