Method of cell isolation

• US 8,741,635 B2
• Filed: 05/12/2005
• Issued: 06/03/2014
• Est. Priority Date: 05/12/2004
• Status: Expired due to Fees

• Current Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Original Assignee: The Walter & Eliza Hall Institute Of Medical Research
• Inventors: Lindeman, Geoffrey John, Shackleton, Mark, Vaillant, Francois, Visvader, Jane Ellen
• Primary Examiner(s): DRISCOLL, LORA E BARNHART
• Application Number:
  US11/596,397
• Publication Number:
  US 20080038230A1
• Time in Patent Office: 3,309 Days
• Field of Search: None
• US Class Current: 435/325
• 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 homogeneous population of mammary stem (MaSCs) cells comprising:

obtaining mammary tissue;

disrupting said mammary tissue to provide a heterogenous population of cells comprising the MaSCs to be isolated;

contacting the heterogeneous population of cells with specific binding partners for each of the following markers;

CD45, TER119, CD31, CD24 and CD29; and

selecting cells in the heterogeneous population that demonstrate a low amount of binding to the specific binding partners for CD45, TER119, CD31 and a high amount of binding to the specific binding partners for the CD24 and CD29 markers such that said cells are CD45^loTER119^loCD31^loCD24^hiCD29^hi,

thereby obtaining a substantially homogeneous population of MaSCs.

2. The method of claim 1 wherein the cells are from a human.

3. The method of claim 1 wherein the cells are from a mouse.

4. The method of claim 1 wherein the selecting is by fluorescence activated cell sorting (FACS).

8. A method for cell replacement therapy in an organism, said method comprising:

generating a substantially homogenous population of MaSCs according to the method of claim 1, and

introducing said homogenous population of MaSCs to said organism or an organism which is capable of receiving said MaSCs.

5. A substantially homogenous population of MaSCs selected according to a method comprising:

obtaining mammary tissue;

disrupting said mammary tissue to provide a heterogenous population of cells comprising the MaSCs to be isolated;

contacting the heterogeneous population of cells with specific binding partners for each of the following markers;

CD45, TER119, CD31, CD24 and CD29; and

selecting cells in the heterogeneous population that demonstrate a low amount of binding to the specific binding partners for CD45, TER119, CD31 and a high amount of binding to the specific binding partners for the CD24 and CD29 markers, wherein said cells are CD45^loTER119^loCD31^loCD24^hiCD29^hi, thereby obtaining the substantially homogeneous population of MaSCs,

wherein said substantially homogenous population of MaSCs is at least 50% pure with regard to the CD45^loTER119^loCD31^loCD24^hiCD29^hi phenotype and is depleted of Hoechst₃₃₃₄₂-effluxing side population (SP) cells compared to the number of SP cells in an overall CD45^loTER119^loCD31^lo population isolated from said mammary tissue.

6. The homogenous population of claim 5 wherein the cells are from human.

7. The homogenous population of claim 5 wherein the cells are from mouse.

9. A method for screening for a modulation of a substantially homogeneous population of MaSCs according to claim 5, said method comprising contacting said MaSCs with a putative modulator and screening for modulation of MaSC growth or development.

11. A substantially homogenous population of MaSCs according to claim 5 that is at least 90% pure with regard to the CD45^loTER119^loCD31^loCD24^hiCD29^hi phenotype.

10. A substantially homogenous population of MaSCs, wherein at least 50% of the cells in the population are CD45^loTER119^loCD31^loCD24^hiCD29^hi, and wherein said substantially homogenous population of MaSCs is depleted of Hoechst₃₃₃₄₂-effluxing side population (SP) cells compared to the number of SP cells in an overall CD45^loTER119^loCD31^lo population isolated from said mammary tissue.