Method Of Cell Isolation

• US 20080038230A1
• Filed: 05/12/2005
• Published: 02/14/2008
• Est. Priority Date: 05/12/2004
• Status: Active Grant

• Current Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Original Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Inventors: Visvader, Jane, Lindeman, Geoffrey, Vaillant, Francois, Shackleton, Mark
• Granted Patent: US 8,741,635 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 2501/01 Modulators of cAMP or cGMP, e.g. non-hydrolysable analogs, phosphodiesterase inhibitors, cholera toxin
• CPC Class Codes: C12N 2501/11 Epidermal growth factor [EGF]
• CPC Class Codes: C12N 2501/33 Insulin together with transferrin C12N2500/25; Insulin-like growth factors C12N2501/105
• CPC Class Codes: C12N 2501/39 Steroid hormones
• CPC Class Codes: C12N 5/0631 Mammary cells
• CPC Class Codes: G01N 33/5073 Stem cells

Abstract

The present invention relates generally to a method for the generation of a substantially homogenous population of undifferentiated cells. More particularly, the present invention relates to a method for isolating a substantially homogenous population of stem cells, and in particular, mammary stem cells (MaSCs). The MaSCs of the present invention are isolated on the basis of differential levels of proteins present on their cell surface. The MaSCs of the present invention are particularly useful as targets for identifying agents which modulate MaSC survival, self-renewal, proliferation and/or differentiation in both normal and diseased tissue such as, but not limited to, tumor tissue, and, also as source of tissue for the regeneration, replacement and/or augmentation of tissue damaged and/or lost after disease or injury.

Claims

1. A method for isolating a substantially homogenous population of MaSCs cells from a biological sample said method comprising subjecting said biological sample to a tissue-disruption means to provide a heterogenous population of cells comprising the MaSCs to be isolated and subjecting said heterogenous population of cells to a cell surface marker discrimination means to isolate a substantially homogenous population of MaSCs.

2. The method of claim 1 wherein the cell surface markers are selected from CD45, Lin (TER119), CD31, CD24 and CD29.

3. The method of claim 2 wherein the cell surface marker discrimination results in a population of cells characterized by being CD45^lo, Lin^lo, CD31^lo, CD24^hi and CD29^hi.

4. The method of claim 3 wherein the cells are derived from a mammal.

5. The method of claim 4 wherein the mammal is a human.

6. The method of claim 4 wherein the mammal is a mouse.

7. The method of claim 1 or 2 or 3 wherein the method of cell sorting is by Fluorescence Activated Cell Sorting (FACS).

8. A method for isolating a substantially homogenous population of CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs from a biological sample said method comprising subjecting said biological sample to a tissue-disruption means to provide a heterogenous population of cells comprising the CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs to be isolated and subjecting said heterogenous population of cells to a cell-surface marker discrimination means to isolate a substantially homogenous population of CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs.

9. The method of claim 8 wherein the cells are derived from a mammal.

10. The method of claim 9 wherein the mammal is a human.

11. The method of claim 9 wherein the mammal is a mouse.

12. The method of claim 8 or 9 or 10 or 11 wherein the method of cell sorting is by Fluorescence Activated Cell Sorting (FACS).

13. A substantially homogenous population of MaSCs selected according to a method comprising subjecting said biological sample to a tissue-disruption means to provide a heterogenous population of cells comprising the CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs to be isolated and subjecting said heterogenous population of cells to a cell-surface marker discrimination means to isolate a substantially homogenous population of CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs.

14. The homogenous population of claim 13 wherein the MaSCs are derived from a mammal.

15. The homogenous population of claim 14 wherein the mammal is a human.

16. The homogenous population of claim 14 wherein the mammal is a mouse.

17. A method for cell replacement therapy in an organism, said method comprising generating a substantially homogenous population of CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs isolated according to a method comprising subjecting a biological sample to a tissue-disruption means to provide a heterogenous population of cells comprising the CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs to be isolated and subjecting said heterogenous population of cells to a cell-surface marker discrimination means to isolate a substantially homogenous population of CD45^loTER119^loCD31^loCD24^hiCD29^hi MaSCs and introducing said homogenous population of MaSCs to said organism or an organism which is capable of receiving said MaSCs.

18. A pharmaceutical composition comprising a modulator of MaSCs and one or more pharmaceutically acceptable carriers and/or diluents.

19. A method for screening for a modulation of an MaSC said method comprising contacting said MaSC with a putative modulator and screening for modulation of MaSC growth or development.