CA2509732C - A method for the discrimination and isolation of mammary epithelial stem and colony-forming cells - Google Patents
A method for the discrimination and isolation of mammary epithelial stem and colony-forming cells Download PDFInfo
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- CA2509732C CA2509732C CA2509732A CA2509732A CA2509732C CA 2509732 C CA2509732 C CA 2509732C CA 2509732 A CA2509732 A CA 2509732A CA 2509732 A CA2509732 A CA 2509732A CA 2509732 C CA2509732 C CA 2509732C
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
- C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/289—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against CD45
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
- C12N5/0631—Mammary cells
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56966—Animal cells
Abstract
The present invention relates to an improved method that permits the differential isolation of mouse mammary stem cells and colony forming cells (CFCs). The method involves depletion of non-epithelial cells from freshly dissociated mouse mammary tissue by incubation with an antibody composition containing antibodies specific for CD45, Ter119, CD31 and optionally CD140a. After formation of conjugates between the non-epithelial mammary cells and the antibodies specific for CD45, Ter119, CD31 and optionally CD140a, the cell conjugates are removed and the remaining epithelial cells are then incubated with an antibody composition containing antibodies specific for CD24 and CD49f. After formation of conjugates between the epithelial cells and the antibodies specific for CD24 and CD49f, the mouse mammary stem and the luminal-restricted CFC cells can be differentially isolated. The invention also relates to kits for carrying out this method and to the cell preparations prepared by this method.
Description
B&P File No: 7771-131 Title: A Method for the Discrimination and Isolation of Mammary Epithelial Stem and Colony-forming Cells.
FIELD OF THE INVENTION
The present invention relates to novel antibody compositions, and processes and kits for preparing cell preparations of different subsets of mouse mammary cells, and the use of the cell preparations in the study of cell biology and cancer.
BACKGROUND OF THE INVENTION
The mammary gland is a compound tubulo-alveolar gland that is composed of a series of branched ducts that, during lactation, drain sac-like alveoli (lobules). In the rodent, the mammary epithelium is embedded within a mammary fat pad rich in stromal (non-epithelial cells). The mammary epithelium is composed of two lineages of epithelial cells: the luminal cells (which make milk during lactation) and basal positioned myoepithelial cells. Generation and maintenance of the mammary epithelium is via the mammary stem cell (MaSC), which is defined here as that cell that can generate both the ductal and lobular structures of the mammary gland, can generate all the cell lineages of the mammary epithelium and can self-renew.
The MaSC is of interest to the breast cancer biologist since cancer theory suggests that it is the stem cell, and possibly some of its more immediate descendants that have decreased stem cell potential but still have proliferative potential that are the targets for malignant transformation. As well, recent publications in the literature demonstrate that malignancies themselves have a stem cell component that propagates the tumor (Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.
Proc Natl Acad Sci U S A. 2003;100:3983-8). This has huge implications in the treatment of cancer since it suggests that in order for cancer to be successfully contained or eradicated, it is the tumor stem cell component that has to be the therapeutic target. The ability to identify and purify mammary stem cells as well as mammary cells with high proliferative capacity but not necessarily having stem cell properties would be invaluable to the study of breast cancer and epithelial cell tumor biology.
FIELD OF THE INVENTION
The present invention relates to novel antibody compositions, and processes and kits for preparing cell preparations of different subsets of mouse mammary cells, and the use of the cell preparations in the study of cell biology and cancer.
BACKGROUND OF THE INVENTION
The mammary gland is a compound tubulo-alveolar gland that is composed of a series of branched ducts that, during lactation, drain sac-like alveoli (lobules). In the rodent, the mammary epithelium is embedded within a mammary fat pad rich in stromal (non-epithelial cells). The mammary epithelium is composed of two lineages of epithelial cells: the luminal cells (which make milk during lactation) and basal positioned myoepithelial cells. Generation and maintenance of the mammary epithelium is via the mammary stem cell (MaSC), which is defined here as that cell that can generate both the ductal and lobular structures of the mammary gland, can generate all the cell lineages of the mammary epithelium and can self-renew.
The MaSC is of interest to the breast cancer biologist since cancer theory suggests that it is the stem cell, and possibly some of its more immediate descendants that have decreased stem cell potential but still have proliferative potential that are the targets for malignant transformation. As well, recent publications in the literature demonstrate that malignancies themselves have a stem cell component that propagates the tumor (Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF.
Proc Natl Acad Sci U S A. 2003;100:3983-8). This has huge implications in the treatment of cancer since it suggests that in order for cancer to be successfully contained or eradicated, it is the tumor stem cell component that has to be the therapeutic target. The ability to identify and purify mammary stem cells as well as mammary cells with high proliferative capacity but not necessarily having stem cell properties would be invaluable to the study of breast cancer and epithelial cell tumor biology.
In 1998 an experiment was performed which definitively demonstrated that a cell exists within the mouse mammary gland that fulfils the criteria of a MaSC
(Kordon EC, Smith GH. Development 1998;125:1921-30). Our own experiments involving transplantation of freshly isolated non-cultured mammary epithelial cells obtained from adult female mice into recipient mice indicates that MaSC occur at a frequency of about 1 cell in 1,300 total mammary cells and that there are approximately 1,400 MaSC per gland in the mouse.
There have been a number of in vitro studies trying to characterize the cells with proliferative potential in the human, mouse and rat mammary glands in a hope to identify the mammary stem cell. These experiments typically involve seeding phenotypically distinct subtypes of mammary cells at clonal densities in culture dishes in order to identify cells with growth potential by their ability to form colonies.
Cells with the potential to form colonies in vitro are termed colony-forming cells (CFCs), and these assays detect all cells that have growth potential, regardless if they are stem cells or not. The inventors research data has demonstrated that the majority (>90%) of CFCs are not stem cells, but cells with growth potential that reside lower in the cellular hierarchy than stem cells. CFCs themselves can be subdivided into different subtypes such as luminal-restricted CFCs (which can only give rise to luminal cells) and bipotent CFCs (which can generate both luminal and myoepithelial cells). In the mouse mammary gland, approximately 90% of all CFCs are of the luminal-restricted type. The phenotypes of mammary CFCs isolated from different species are summarized in Table I:
These in vitro studies to characterize CFCs are limited because colony assays, in their current state, are unable to identify MaSC and to discriminate between MaSC
that generate colonies from other CFCs that are deficient in stem cell properties.
The first insight into the phenotype of MaSC was reported by Welm and colleagues who demonstrated that expression of the cell surface molecule Sca-1 enriches for MaSCs that generate ductal-lobular outgrowths when transplanted into the cleared mammary fat pads of recipient mice (Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Dev Biol 2002;245:42-56). However this is a crude enrichment strategy since approximately 20-60% of all mammary cells express Sca-1, and thus MaSC are far from being purified in Sca-1+ enriched subpopulations.
To date, there has been no description of a method in the prior art that permits the isolation to high purity of stromal, luminal, myoepithelial, CFCs and MaSC
subpopulations of mammary cells. The current method of invention satisfies this need.
SUMMARY OF THE INVENTION
The present invention relates to antibody compositions and methods that can be used to separate non-epithelial cells from epithelial cells in a sample containing mammary cells. The present inventors have developed antibody cocktail compositions and a method that can be used to identify the following mammary cell subtypes:
= Stromal (non-epithelial); and = Epithelial, which comprise the subtypes:
= Luminal;
= Myoepithelial;
= Luminal-restricted CFC; and = MaSC.
The present invention relies on the observation that mammary stem cells (MaSC) express CD24 and high levels of CD49f (a-6 integrin), but do not express the hematopoietic markers CD45, Terl 19 and the endothelial marker CD31 (Stingl J, Ricketson I, Choi D, Eaves CJ. Proceed Am Assoc Cancer Res 2004;45:641-2).
Enrichment of mammary cells on the basis of this strategy (CD45"/Ter119-/CD31-/CD49r/CD24+) as outlined this patent application results in a purity of about MaSC in 20 sorted cells. CD140a is a marker expressed by many of the mammary stromal cells and some mammary cells (Crowey M.R., Bowtell D and Serra R. Dev Biol 279: 58-72). Inclusion of CD140a into the stromal cell-depletion cocktail results in similar MaSC purities following FACS. Although the use of the markers CD45, Terl19, CD31 and CD140a is not essential to isolate MaSCs and CFCs, their use is beneficial since many of these cells types co-express CD24 and CD49f and thus can decrease the purity of stem and CFC cell enriched fractions. This is particularly so in mammary cell preparations with high levels of stromal cell contamination.
Accordingly, the present invention provides a method of separating non-epithelial cells from epithelial cells in a sample containing mammary cells comprising 1) reacting the sample with an antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens;
2) removing the conjugates; and 3) recovering a cell preparation which is enriched in mammary epithelial cells.
The antibody composition used to isolate non-epithelial cells preferably comprises antibodies that bind to CD45, Terl 19, CD31 and optionally CD140a.
In a preferred embodiment, the method is used to enrich for mammary stem cells or luminal restricted colony forming cells. Accordingly, the present inventions provides a method of enriching for mammary stem cells or luminal restricted colony forming cells in a sample containing mammary cells comprising 1) reacting the sample with a first antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates; 3) recovering a cell preparation which is enriched in mammary epithelial cells; and 4) reacting the sample enriched in epithelial cells with a second antibody composition cable of binding the antigens CD24 and/or CD49f under conditions so that conjugates form between antibodies and the cells in the sample containing the antigens CD24 and/or CD49f; and 5) recovering cells that are bound by the antibodies.
The present invention also relates to a kit useful in performing the process of the invention and instructions for performing the process of the invention.
The invention further relates to cell preparations obtained in accordance with the process of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
(Kordon EC, Smith GH. Development 1998;125:1921-30). Our own experiments involving transplantation of freshly isolated non-cultured mammary epithelial cells obtained from adult female mice into recipient mice indicates that MaSC occur at a frequency of about 1 cell in 1,300 total mammary cells and that there are approximately 1,400 MaSC per gland in the mouse.
There have been a number of in vitro studies trying to characterize the cells with proliferative potential in the human, mouse and rat mammary glands in a hope to identify the mammary stem cell. These experiments typically involve seeding phenotypically distinct subtypes of mammary cells at clonal densities in culture dishes in order to identify cells with growth potential by their ability to form colonies.
Cells with the potential to form colonies in vitro are termed colony-forming cells (CFCs), and these assays detect all cells that have growth potential, regardless if they are stem cells or not. The inventors research data has demonstrated that the majority (>90%) of CFCs are not stem cells, but cells with growth potential that reside lower in the cellular hierarchy than stem cells. CFCs themselves can be subdivided into different subtypes such as luminal-restricted CFCs (which can only give rise to luminal cells) and bipotent CFCs (which can generate both luminal and myoepithelial cells). In the mouse mammary gland, approximately 90% of all CFCs are of the luminal-restricted type. The phenotypes of mammary CFCs isolated from different species are summarized in Table I:
These in vitro studies to characterize CFCs are limited because colony assays, in their current state, are unable to identify MaSC and to discriminate between MaSC
that generate colonies from other CFCs that are deficient in stem cell properties.
The first insight into the phenotype of MaSC was reported by Welm and colleagues who demonstrated that expression of the cell surface molecule Sca-1 enriches for MaSCs that generate ductal-lobular outgrowths when transplanted into the cleared mammary fat pads of recipient mice (Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Dev Biol 2002;245:42-56). However this is a crude enrichment strategy since approximately 20-60% of all mammary cells express Sca-1, and thus MaSC are far from being purified in Sca-1+ enriched subpopulations.
To date, there has been no description of a method in the prior art that permits the isolation to high purity of stromal, luminal, myoepithelial, CFCs and MaSC
subpopulations of mammary cells. The current method of invention satisfies this need.
SUMMARY OF THE INVENTION
The present invention relates to antibody compositions and methods that can be used to separate non-epithelial cells from epithelial cells in a sample containing mammary cells. The present inventors have developed antibody cocktail compositions and a method that can be used to identify the following mammary cell subtypes:
= Stromal (non-epithelial); and = Epithelial, which comprise the subtypes:
= Luminal;
= Myoepithelial;
= Luminal-restricted CFC; and = MaSC.
The present invention relies on the observation that mammary stem cells (MaSC) express CD24 and high levels of CD49f (a-6 integrin), but do not express the hematopoietic markers CD45, Terl 19 and the endothelial marker CD31 (Stingl J, Ricketson I, Choi D, Eaves CJ. Proceed Am Assoc Cancer Res 2004;45:641-2).
Enrichment of mammary cells on the basis of this strategy (CD45"/Ter119-/CD31-/CD49r/CD24+) as outlined this patent application results in a purity of about MaSC in 20 sorted cells. CD140a is a marker expressed by many of the mammary stromal cells and some mammary cells (Crowey M.R., Bowtell D and Serra R. Dev Biol 279: 58-72). Inclusion of CD140a into the stromal cell-depletion cocktail results in similar MaSC purities following FACS. Although the use of the markers CD45, Terl19, CD31 and CD140a is not essential to isolate MaSCs and CFCs, their use is beneficial since many of these cells types co-express CD24 and CD49f and thus can decrease the purity of stem and CFC cell enriched fractions. This is particularly so in mammary cell preparations with high levels of stromal cell contamination.
Accordingly, the present invention provides a method of separating non-epithelial cells from epithelial cells in a sample containing mammary cells comprising 1) reacting the sample with an antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens;
2) removing the conjugates; and 3) recovering a cell preparation which is enriched in mammary epithelial cells.
The antibody composition used to isolate non-epithelial cells preferably comprises antibodies that bind to CD45, Terl 19, CD31 and optionally CD140a.
In a preferred embodiment, the method is used to enrich for mammary stem cells or luminal restricted colony forming cells. Accordingly, the present inventions provides a method of enriching for mammary stem cells or luminal restricted colony forming cells in a sample containing mammary cells comprising 1) reacting the sample with a first antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates; 3) recovering a cell preparation which is enriched in mammary epithelial cells; and 4) reacting the sample enriched in epithelial cells with a second antibody composition cable of binding the antigens CD24 and/or CD49f under conditions so that conjugates form between antibodies and the cells in the sample containing the antigens CD24 and/or CD49f; and 5) recovering cells that are bound by the antibodies.
The present invention also relates to a kit useful in performing the process of the invention and instructions for performing the process of the invention.
The invention further relates to cell preparations obtained in accordance with the process of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: FACS dot plot showing the distribution of CD45"/Ter119"/CD31"/CD140a cells according to their co-expression of CD24 and CD49f. The different cell subpopulations are indicated by circles and arrows.
FIG. 2: Six day-old pure luminal cell colony grown in vitro.
FIG. 3: Whole mount of a mouse mammary fat pad illustrating the outgrowth obtained by implanting a mouse MaSC.
FIG 4: Expression of CD24 among cells of the mouse mammary gland. Arrows indicate CD24+ endothelial cells and arrowheads indicate CD24+ stromal cells.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
The term "differentiated cells" used herein refers to mouse mammary cells which have limited or no proliferative capacity. The differentiated cells represent the specialized end cells that serve a specific function. In the case of the mammary gland, these cells are the luminal cells and the myoepithelial cells.
The term "colony-forming cells" or "CFCs", also known as "progenitor cells"
used herein refers to cells which are the immediate precursors to the differentiated cells. They have extensive proliferative capacity. In the mouse mammary gland, approximately 90% of all CFCs have a luminal cell phenotype and give rise to only pure luminal epithelial cell colonies. Mammary CFCs can be detected by their ability to generate colonies in vitro. FIG. 2 illustrates a pure luminal cell colony generated after 6 days of culture.
The term "stem cells" used herein refers the cells that give rise to the CFCs.
Mammary stem cells (MaSC) are defined as those cells that can generate both the ductal and lobular structures of the mammary gland, can generate all the cell lineages of the mammary epithelium and can self-renew.
The term "antibody" is understood to include monoclonal antibodies and polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2) and recombinantly produced binding partners. Antibodies are understood to be reactive against a selected antigen on the surface of a cell if they bind with an affinity (association constant) of greater than or equal to 107 M"~.
II. METHODS AND COMPOSTIONS
The present invention provides a method of separating non-epithelial cells from epithelial cells in a sample containing mammary cells comprising 1) reacting the sample with an antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates;
and 3) recovering a cell preparation which is enriched in mammary epithelial cells.
In a preferred embodiment, the method is used to enrich for mammary stem cells or luminal restricted colony forming cells. Accordingly, the present inventions provides a method of enriching for mammary stem cells or luminal restricted colony forming cells in a sample containing mammary cells comprising 1) reacting the sample with a first antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates; 3) recovering a cell preparation which is enriched in mammary epithelial cells; and 4) reacting the sample enriched in epithelial cells with a second antibody composition cable of binding the antigens CD24 and/or CD49f under conditions so that conjugates form between antibodies and the cells in the sample containing the antigens CD24 and/or CD49f; and recovering cells that are bound by the antibodies.
The antibody composition used to isolate non-epithelial cells preferably comprises antibodies that bind to CD45, Terl 19, CD31 and optionally CD140a.
CD45 and Ten 19 are cell surface proteins that are preferentially expressed by cells of the hematopoietic system (CD45. In: The Leukocyte Antigen Facts Book.
Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 244-47, 1997; Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL, Katsura Y. Br J Haematol.
2000;109: 280-7). CD31 is a cell surface protein that is preferentially, but not exclusively expressed by endothelial cells (CD31. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 206-8, 1997), whereas CD140a is a cell surface protein that is preferentially, but not exclusively expressed by stromal cells (Orr-Urtreger A, Lonai P. Development. 1992;115:1045-58). It is expressed by some cells of the mammary epithelium (Crowley M.R., Bowtell D.
and Serra R. Dev Biol 279:58-72).
CD49f is a cell surface protein that is widely distributed among cells of different tissues (CD49f. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 267-8, 1997). In the epithelia, it is preferentially expressed by the cells in the basal compartment (Carter,W.G., Kaur,P., Gil,S.G., Gahr,P.J. & Wayner,E.A. J Cell Biol. 1990: 111, 3141-54). CD24 is widely distributed among cells of different tissues. In the mammary gland, it is widely distributed through-out the epithelium and the stroma (FIG. 4). Many CD45+/Ter119+/CD31+/CD 140a+ cells express either or both CD49f and CD24.
FIG. 2: Six day-old pure luminal cell colony grown in vitro.
FIG. 3: Whole mount of a mouse mammary fat pad illustrating the outgrowth obtained by implanting a mouse MaSC.
FIG 4: Expression of CD24 among cells of the mouse mammary gland. Arrows indicate CD24+ endothelial cells and arrowheads indicate CD24+ stromal cells.
DETAILED DESCRIPTION OF THE INVENTION
I. DEFINITIONS
The term "differentiated cells" used herein refers to mouse mammary cells which have limited or no proliferative capacity. The differentiated cells represent the specialized end cells that serve a specific function. In the case of the mammary gland, these cells are the luminal cells and the myoepithelial cells.
The term "colony-forming cells" or "CFCs", also known as "progenitor cells"
used herein refers to cells which are the immediate precursors to the differentiated cells. They have extensive proliferative capacity. In the mouse mammary gland, approximately 90% of all CFCs have a luminal cell phenotype and give rise to only pure luminal epithelial cell colonies. Mammary CFCs can be detected by their ability to generate colonies in vitro. FIG. 2 illustrates a pure luminal cell colony generated after 6 days of culture.
The term "stem cells" used herein refers the cells that give rise to the CFCs.
Mammary stem cells (MaSC) are defined as those cells that can generate both the ductal and lobular structures of the mammary gland, can generate all the cell lineages of the mammary epithelium and can self-renew.
The term "antibody" is understood to include monoclonal antibodies and polyclonal antibodies, antibody fragments (e.g., Fab, and F(ab')2) and recombinantly produced binding partners. Antibodies are understood to be reactive against a selected antigen on the surface of a cell if they bind with an affinity (association constant) of greater than or equal to 107 M"~.
II. METHODS AND COMPOSTIONS
The present invention provides a method of separating non-epithelial cells from epithelial cells in a sample containing mammary cells comprising 1) reacting the sample with an antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates;
and 3) recovering a cell preparation which is enriched in mammary epithelial cells.
In a preferred embodiment, the method is used to enrich for mammary stem cells or luminal restricted colony forming cells. Accordingly, the present inventions provides a method of enriching for mammary stem cells or luminal restricted colony forming cells in a sample containing mammary cells comprising 1) reacting the sample with a first antibody composition capable of binding to antigens on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates; 3) recovering a cell preparation which is enriched in mammary epithelial cells; and 4) reacting the sample enriched in epithelial cells with a second antibody composition cable of binding the antigens CD24 and/or CD49f under conditions so that conjugates form between antibodies and the cells in the sample containing the antigens CD24 and/or CD49f; and recovering cells that are bound by the antibodies.
The antibody composition used to isolate non-epithelial cells preferably comprises antibodies that bind to CD45, Terl 19, CD31 and optionally CD140a.
CD45 and Ten 19 are cell surface proteins that are preferentially expressed by cells of the hematopoietic system (CD45. In: The Leukocyte Antigen Facts Book.
Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 244-47, 1997; Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL, Katsura Y. Br J Haematol.
2000;109: 280-7). CD31 is a cell surface protein that is preferentially, but not exclusively expressed by endothelial cells (CD31. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 206-8, 1997), whereas CD140a is a cell surface protein that is preferentially, but not exclusively expressed by stromal cells (Orr-Urtreger A, Lonai P. Development. 1992;115:1045-58). It is expressed by some cells of the mammary epithelium (Crowley M.R., Bowtell D.
and Serra R. Dev Biol 279:58-72).
CD49f is a cell surface protein that is widely distributed among cells of different tissues (CD49f. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 267-8, 1997). In the epithelia, it is preferentially expressed by the cells in the basal compartment (Carter,W.G., Kaur,P., Gil,S.G., Gahr,P.J. & Wayner,E.A. J Cell Biol. 1990: 111, 3141-54). CD24 is widely distributed among cells of different tissues. In the mammary gland, it is widely distributed through-out the epithelium and the stroma (FIG. 4). Many CD45+/Ter119+/CD31+/CD 140a+ cells express either or both CD49f and CD24.
The present invention also relies on the observation that mammary cell populations enriched for stem cell activity (CD45/Ter119-/CD31"/CD140a"
/CD49r+/CD24+ phenotype) are deficient in CFCs since approximately 90% of all CFCs are luminal-restricted CFCs and have a slightly different phenotype in which CD49f is expressed at lower levels (CD45-/Ter119-/CD31-/CD140a"/CD49r/CD24+).
Accordingly, the present invention allows one to distinguish the MaSC from the CFCs.
The presence of stem cells can be detected by transplanting a cell suspension containing a stem cell into epithelium-free ("cleared") mouse mammary fat pads. Six weeks following implantation and pregnancy, engraftment can be detected by resecting the fat pad from the mouse and staining the fat pad with a dye to highlight the mammary tree. An example of an engrafted fat pad is illustrated in FIG. 3.
When the present invention is used in a cell separation technology such as flow cytometry, the entire mammary epithelial hierarchy spanning from stem cells to differentiated luminal and myoepithelial cells can be visualized on a flow cytometry plot as illustrated in FIG. 1. Consequently, any of the mammary cell subpopulations can be identified and isolated to high purity. This method is superior to any method described to date since it no other method permits the differential isolation of MaSC
and luminal-restricted CFCs. The method is also unique since there is no prior art describing the visualization of the mammary hierarchy.
Two general approaches may be used to differentially isolate the different subsets of mammary cells. The first is to label a portion of the non-epithelial component of the mammary cell suspension with antibodies specific for the cell surface epitopes CD45, Ter119, CD31 and CD140a and then to selectively deplete these cells by a negative selection strategy. Such strategies include directly or indirectly conjugating these antibodies to some type of a matrix such as magnetic beads, a panning surface, dense particles for density centrifugation, and adsorption column or an adsorption membrane. The leftover epithelial enriched population of mammary cells is then labelled with fluorochrome-conjugated antibodies specific for CD24 and CD49f and the different epithelial cell subpopulations (as well as /Ter119"/CD31-/CD140a stromal cells) can be visualized by flow cytometry.
A second alternative method is to use stromal- and hematopoietic-specific (CD45/Terl19/CD31/CD140a) antibodies, CD24 antibodies and CD49f antibodies each conjugated directly or indirectly to a different fluorochrome. As a result, the contaminating CD45+/Ter119+/CD31+/CD140a+ stromal and hematopoietic cells can be gated out from the CD24 and CD49f flow cytometry analysis.
The present invention relates to the method of differentially labelling mouse MaSC and luminal-restricted CFCs with antibodies specific for the CD24 and CD49f cell surface epitopes and the subsequent isolation of different subtypes of mammary cells, including MaSC and CFCs to high purities by flow cytometry.
The present invention relates to the antibody composition comprising antibodies specific for the CD24 and CD49f cell surface epitopes to differentially label mouse MaSC and luminal-restricted CFCs and to use this differential labelling strategy to isolate the different subtypes of mammary cells, including MaSC
and CFCs to high purities by flow cytometry.
The antibodies used in the method of the invention may be labelled with a marker or they may be conjugated to a matrix. Examples of markers are biotin, which can be removed by avidin bound to a support, and fluorochromes, e.g.
fluorescein, which provide for separation using fluorescence activated sorters. Examples of matrices are magnetic beads, which allow for direct magnetic separation (Kernshead 1992), panning surfaces e.g. plates, (Lebkowski, J.S, et al., (1994), J. of Cellular Biochemistry supple. 18b:58), dense particles for density centrifugation (Van Vlasselaer, P., Density Adjusted Cell Sorting (DACS), A Novel Method to Remove Tumor Cells From Peripheral Blood and Bone Marrow StemCell Transplants. (1995) 3rd International Symposium on Recent Advances in Hematopoietic Stem Cell Transplantation-Clinical Progress, New Technologies and Gene Therapy, San Diego, CA), dense particles alone (Zwerner et al., Immunol. Meth. 1996 198(2):199-202) adsorption columns (Berenson et al. 1986, Journal of Immunological Methods 91:11-19.), and adsorption membranes.
The antibodies in the antibody compositions may be directly or indirectly 5 coupled to a matrix. For example, the antibodies that bind non-epithelial cells in the compositions may be chemically bound to the surface of magnetic particles for example, using cyanogen bromide. When the magnetic particles are reacted with a sample, conjugates will form between the magnetic particles with bound antibodies specific for antigens on the surfaces of the non-epithelial cells and the cells having the 10 antigens on their surfaces.
Alternatively, the antibodies may be indirectly conjugated to a matrix using antibodies. For example, a matrix may be coated with a second antibody having specificity for the antibodies in the antibody composition. By way of example, if the antibodies in the antibody composition are mouse IgG antibodies, the second antibody may be rabbit anti-mouse IgG.
The antibodies in the antibody compositions may also be incorporated in antibody reagents which indirectly conjugate to a matrix. Examples of antibody reagents are bispecific antibodies, tetrameric antibody complexes, and biotinylated antibodies.
Bispecific antibodies contain a variable region of an antibody in an antibody composition of the invention, and a variable region specific for at least one antigen on the surface of a matrix. The bispecific antibodies may be prepared by forming hybrid hybridomas. The hybrid hybridomas may be prepared using the procedures known in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA) 83: 1453) and Staerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may also be constructed by chemical means using procedures such as those described by Staerz et al., (1985, Nature, 314:628) and Perez et al., (1985 Nature 316:354), or by expression of recombinant immunoglobulin gene constructs.
/CD49r+/CD24+ phenotype) are deficient in CFCs since approximately 90% of all CFCs are luminal-restricted CFCs and have a slightly different phenotype in which CD49f is expressed at lower levels (CD45-/Ter119-/CD31-/CD140a"/CD49r/CD24+).
Accordingly, the present invention allows one to distinguish the MaSC from the CFCs.
The presence of stem cells can be detected by transplanting a cell suspension containing a stem cell into epithelium-free ("cleared") mouse mammary fat pads. Six weeks following implantation and pregnancy, engraftment can be detected by resecting the fat pad from the mouse and staining the fat pad with a dye to highlight the mammary tree. An example of an engrafted fat pad is illustrated in FIG. 3.
When the present invention is used in a cell separation technology such as flow cytometry, the entire mammary epithelial hierarchy spanning from stem cells to differentiated luminal and myoepithelial cells can be visualized on a flow cytometry plot as illustrated in FIG. 1. Consequently, any of the mammary cell subpopulations can be identified and isolated to high purity. This method is superior to any method described to date since it no other method permits the differential isolation of MaSC
and luminal-restricted CFCs. The method is also unique since there is no prior art describing the visualization of the mammary hierarchy.
Two general approaches may be used to differentially isolate the different subsets of mammary cells. The first is to label a portion of the non-epithelial component of the mammary cell suspension with antibodies specific for the cell surface epitopes CD45, Ter119, CD31 and CD140a and then to selectively deplete these cells by a negative selection strategy. Such strategies include directly or indirectly conjugating these antibodies to some type of a matrix such as magnetic beads, a panning surface, dense particles for density centrifugation, and adsorption column or an adsorption membrane. The leftover epithelial enriched population of mammary cells is then labelled with fluorochrome-conjugated antibodies specific for CD24 and CD49f and the different epithelial cell subpopulations (as well as /Ter119"/CD31-/CD140a stromal cells) can be visualized by flow cytometry.
A second alternative method is to use stromal- and hematopoietic-specific (CD45/Terl19/CD31/CD140a) antibodies, CD24 antibodies and CD49f antibodies each conjugated directly or indirectly to a different fluorochrome. As a result, the contaminating CD45+/Ter119+/CD31+/CD140a+ stromal and hematopoietic cells can be gated out from the CD24 and CD49f flow cytometry analysis.
The present invention relates to the method of differentially labelling mouse MaSC and luminal-restricted CFCs with antibodies specific for the CD24 and CD49f cell surface epitopes and the subsequent isolation of different subtypes of mammary cells, including MaSC and CFCs to high purities by flow cytometry.
The present invention relates to the antibody composition comprising antibodies specific for the CD24 and CD49f cell surface epitopes to differentially label mouse MaSC and luminal-restricted CFCs and to use this differential labelling strategy to isolate the different subtypes of mammary cells, including MaSC
and CFCs to high purities by flow cytometry.
The antibodies used in the method of the invention may be labelled with a marker or they may be conjugated to a matrix. Examples of markers are biotin, which can be removed by avidin bound to a support, and fluorochromes, e.g.
fluorescein, which provide for separation using fluorescence activated sorters. Examples of matrices are magnetic beads, which allow for direct magnetic separation (Kernshead 1992), panning surfaces e.g. plates, (Lebkowski, J.S, et al., (1994), J. of Cellular Biochemistry supple. 18b:58), dense particles for density centrifugation (Van Vlasselaer, P., Density Adjusted Cell Sorting (DACS), A Novel Method to Remove Tumor Cells From Peripheral Blood and Bone Marrow StemCell Transplants. (1995) 3rd International Symposium on Recent Advances in Hematopoietic Stem Cell Transplantation-Clinical Progress, New Technologies and Gene Therapy, San Diego, CA), dense particles alone (Zwerner et al., Immunol. Meth. 1996 198(2):199-202) adsorption columns (Berenson et al. 1986, Journal of Immunological Methods 91:11-19.), and adsorption membranes.
The antibodies in the antibody compositions may be directly or indirectly 5 coupled to a matrix. For example, the antibodies that bind non-epithelial cells in the compositions may be chemically bound to the surface of magnetic particles for example, using cyanogen bromide. When the magnetic particles are reacted with a sample, conjugates will form between the magnetic particles with bound antibodies specific for antigens on the surfaces of the non-epithelial cells and the cells having the 10 antigens on their surfaces.
Alternatively, the antibodies may be indirectly conjugated to a matrix using antibodies. For example, a matrix may be coated with a second antibody having specificity for the antibodies in the antibody composition. By way of example, if the antibodies in the antibody composition are mouse IgG antibodies, the second antibody may be rabbit anti-mouse IgG.
The antibodies in the antibody compositions may also be incorporated in antibody reagents which indirectly conjugate to a matrix. Examples of antibody reagents are bispecific antibodies, tetrameric antibody complexes, and biotinylated antibodies.
Bispecific antibodies contain a variable region of an antibody in an antibody composition of the invention, and a variable region specific for at least one antigen on the surface of a matrix. The bispecific antibodies may be prepared by forming hybrid hybridomas. The hybrid hybridomas may be prepared using the procedures known in the art such as those disclosed in Staerz & Bevan, (1986, PNAS (USA) 83: 1453) and Staerz & Bevan, (1986, Immunology Today, 7:241). Bispecific antibodies may also be constructed by chemical means using procedures such as those described by Staerz et al., (1985, Nature, 314:628) and Perez et al., (1985 Nature 316:354), or by expression of recombinant immunoglobulin gene constructs.
A tetrameric immunological complex may be prepared by mixing a first monoclonal antibody which is capable of binding to at least one antigen on the surface of a matrix, and a second monoclonal antibody from the antibody composition of the invention. The first and second monoclonal antibody are from a first animal species.
The first and second antibody are reacted with an about equimolar amount of monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. The first and second antibody may also be reacted with an about equimolar amount of the F(ab')2 fragments of monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. (See U.S. Patent No.
4,868,109 to Lansdorp for a description of tetrameric antibody complexes and methods for preparing same).
The antibodies of the invention may be biotinylated and indirectly conjugated to a matrix which is labelled with (strept) avidin. For example, biotinylated antibodies contained in the antibody composition of the invention may be used in combination with magnetic iron-dextran particles that are covalently labelled with (strept) avidin (Miltenyi, S. et al., Cytometry 11:231, 1990). Many alternative indirect ways to specifically cross-link the antibodies in the antibody composition and matrices would also be apparent to those skilled in the art.
In an embodiment of the invention, the cell conjugates are removed by magnetic separation using magnetic particles. Suitable magnetic particles include particles in ferrofluids and other colloidal magnetic solutions. "Ferrofluid"
refers to a colloidal solution containing particles consisting of a magnetic core, such as magnetite (Fe304) coated or embedded in material that prevents the crystals from interacting.
Examples of such materials include proteins, such as ferritin, polysaccharides, such as dextrans, or synthetic polymers such as sulfonated polystyrene cross-linked with divinylbenzene. The core portion is generally too small to hold a permanent magnetic field. The ferrofluids become magnetized when placed in a magnetic field.
Examples of ferrofluids and methods for preparing them are described by Kemshead J.T.
(1992) in J. Hematotherapy, 1:35-44, at pages 36 to 39, and Ziolo et al. Science (1994) 257:219. Colloidal particles of dextran-iron complex are preferably used in the process of the invention. (See Molday, R.S. and McKenzie, L.L. FEBS Lett. 170:232, 1984;
Miltenyi et al., Cytometry 11:231, 1990; and Molday, R.S. and MacKenzie, D., J.
Immunol. Methods 52:353, 1982; Thomas et al., J. Hematother. 2:297 (1993); and U.S.
Patent No. 4,452,733).
In accordance with the magnetic separation method, the sample containing the epithelial cells to be recovered, is reacted with the above described antibody reagents, preferably tetrameric antibody complexes, so that the antibody reagents bind to the non-epithelial cells present in the sample to form cell conjugates of the targeted non-epithelial cells and the antibody reagents. The reaction conditions are selected to provide the desired level of binding of the targeted non-epithelial cells and the antibody reagents. Preferably the sample is incubated with the antibody reagents for a period of 5 to 60 minutes at either 4 or ambient room temperature. The concentration of the antibody reagents is selected depending on the estimated concentration of the targeted differentiated cells in the sample. Generally, the concentration is between about 0.1 to 50 g/ml of sample. The magnetic particles are then added and the mixture is incubated for a period of about 5 minutes to 30 minutes at the selected temperature. The sample is then ready to be separated over a magnetic filter device. Preferably, the magnetic separation procedure is carried out using the magnetic filter and methods described in U.S. Patent No. 5,514,340 to Lansdorp and Thomas.
The sample containing the magnetically labelled cell conjugates is passed through the magnetic filter in the presence of a magnetic field. In a preferred embodiment of the invention, the magnet is a dipole magnet with a gap varying from 0.3 to 3.0 inches bore and having a magnetic field of 0,5-2 Tesla. The magnetically labelled cell conjugates are retained in the high gradient magnetic column and the materials which are not magnetically labelled flow through the column after washing with a buffer.
The first and second antibody are reacted with an about equimolar amount of monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. The first and second antibody may also be reacted with an about equimolar amount of the F(ab')2 fragments of monoclonal antibodies of a second animal species which are directed against the Fc-fragments of the antibodies of the first animal species. (See U.S. Patent No.
4,868,109 to Lansdorp for a description of tetrameric antibody complexes and methods for preparing same).
The antibodies of the invention may be biotinylated and indirectly conjugated to a matrix which is labelled with (strept) avidin. For example, biotinylated antibodies contained in the antibody composition of the invention may be used in combination with magnetic iron-dextran particles that are covalently labelled with (strept) avidin (Miltenyi, S. et al., Cytometry 11:231, 1990). Many alternative indirect ways to specifically cross-link the antibodies in the antibody composition and matrices would also be apparent to those skilled in the art.
In an embodiment of the invention, the cell conjugates are removed by magnetic separation using magnetic particles. Suitable magnetic particles include particles in ferrofluids and other colloidal magnetic solutions. "Ferrofluid"
refers to a colloidal solution containing particles consisting of a magnetic core, such as magnetite (Fe304) coated or embedded in material that prevents the crystals from interacting.
Examples of such materials include proteins, such as ferritin, polysaccharides, such as dextrans, or synthetic polymers such as sulfonated polystyrene cross-linked with divinylbenzene. The core portion is generally too small to hold a permanent magnetic field. The ferrofluids become magnetized when placed in a magnetic field.
Examples of ferrofluids and methods for preparing them are described by Kemshead J.T.
(1992) in J. Hematotherapy, 1:35-44, at pages 36 to 39, and Ziolo et al. Science (1994) 257:219. Colloidal particles of dextran-iron complex are preferably used in the process of the invention. (See Molday, R.S. and McKenzie, L.L. FEBS Lett. 170:232, 1984;
Miltenyi et al., Cytometry 11:231, 1990; and Molday, R.S. and MacKenzie, D., J.
Immunol. Methods 52:353, 1982; Thomas et al., J. Hematother. 2:297 (1993); and U.S.
Patent No. 4,452,733).
In accordance with the magnetic separation method, the sample containing the epithelial cells to be recovered, is reacted with the above described antibody reagents, preferably tetrameric antibody complexes, so that the antibody reagents bind to the non-epithelial cells present in the sample to form cell conjugates of the targeted non-epithelial cells and the antibody reagents. The reaction conditions are selected to provide the desired level of binding of the targeted non-epithelial cells and the antibody reagents. Preferably the sample is incubated with the antibody reagents for a period of 5 to 60 minutes at either 4 or ambient room temperature. The concentration of the antibody reagents is selected depending on the estimated concentration of the targeted differentiated cells in the sample. Generally, the concentration is between about 0.1 to 50 g/ml of sample. The magnetic particles are then added and the mixture is incubated for a period of about 5 minutes to 30 minutes at the selected temperature. The sample is then ready to be separated over a magnetic filter device. Preferably, the magnetic separation procedure is carried out using the magnetic filter and methods described in U.S. Patent No. 5,514,340 to Lansdorp and Thomas.
The sample containing the magnetically labelled cell conjugates is passed through the magnetic filter in the presence of a magnetic field. In a preferred embodiment of the invention, the magnet is a dipole magnet with a gap varying from 0.3 to 3.0 inches bore and having a magnetic field of 0,5-2 Tesla. The magnetically labelled cell conjugates are retained in the high gradient magnetic column and the materials which are not magnetically labelled flow through the column after washing with a buffer.
The preparation containing non-magnetically labeled MaSC or CFC cells may be analyzed using procedures such as flow cytometry. The activity of the MaSC
cells in the preparation may also be assessed for example by transplanting into mice as described previously.
III. USES OF THE COMPOSITIONS AND METHODS OF THE INVENTION
The invention may be used in the isolation of different subsets of mouse mammary cells including MaSC and luminal-restricted progenitor cells. These cells are of interest to breast cancer biologists, mammary gland biologists and developmental biologists. In particular, the ability to purify mammary progenitors and stem cells free of stromal cell contamination is important in determining the gene expression profiles of these cells and the factors that regulate their cell behaviour.
The invention includes kits for preparing samples enriched in mammary epithelial cells comprising antibodies that bind to non-epithelial cells in a mammary sample and instructions for the use thereof. The antibody composition for use in the kit preferably comprises antibodies that bind to CD45, Ter 119, CD31 and CD
140a.
The invention further includes kits for preparing samples enriched in MaSC or CFC cells comprising antibodies that bind to CD24 and/or CD49f and instructions for the use thereof.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1 Method for Determining the In Vivo and In Vitro Growth Properties of Different Subsets of Mammary Cells Mammary glands from young adult female mice were removed and digested for 8 hours at 37 C in EpiCult-BTM (StemCell Technologies, Vancouver, BC, Canada) supplemented with 5% fetal bovine serum (FBS) and 300 U/mL collagenase and 100 U/mL hyaluronidase (StemCell Technologies). At the end of this time, the preparation is vortexed and centrifuged at 450g for 5 minutes. The supernatant is discarded and contaminating red blood cells lysed with an ammonium chloride wash.
Following centrifugation, the cells are suspended in 2 mL of 0.25% trypsin prewarmed to 37 C and further dissociated by gentle pipetting for 1-2 minutes.
The cells are then washed once with 10 mL of Hank's Balanced Salt Solution supplemented with 2% FBS (HF) and incubated with 5 mg/mL dispaseTM II
(StemCell Technologies) and 0.1 mg/mL deoxyribonuclease I (StemCell Technologies) for 2 min at 37 C. The resultant cell suspension is diluted with HF and then filtered through a 40 m mesh to obtain a single cell suspension.
Hematopoietic (CD45 - and Ter119'), endothelial (CD31 -) and stromal (CD140a) cells are removed by pre-incubating freshly dissociated cells in 2 ghnL
Fc receptor antibody 2.4G2 (American Type Culture Collection, Rockville, MD, USA) followed by a 10 minute incubation with antibodies specific for CD45 (clone 30-Fll from BD Pharmingen, San Diego, USA), Terl19 (clone Terl19 from BD
Pharmingen), CD31 (clone MEC 13.3 from BD Pharmingen) and with CD I40a (clone APA5 from eBioscience, San Diego, CA, USA). Antibody concentrations during the incubation process typically ranges from 0.5-2.0 g/mL. These CD45, Terl 19, and CD140a antibodies can be directly or indirectly conjugated to a fluorochrome or biotin such that the antibody labelled cells can be selectively detected by flow cytometry or selectively depleted by binding to some type of matrix (e.g., magnetic beads, a panning surface, dense particles for density centrifugation, an adsorption column, or an adsorption membrane). If a negative selection strategy is implemented, the remaining CD45/Terl 19/CD31/CDI40a depleted mammary cells are then labelled with antibodies recognizing epitopes of the cell surface proteins CD24 and CD49f. A
suitable antibody clone specific for mouse CD24 is the M 1 /69 clone (BD
Pharmingen). A suitable antibody clone specific for mouse CD49f is the GoH3 clone (BD Pharmingen). Both antibodies are used at a concentration of 1 g/mL during the incubation process. The CD24 and CD49f antibodies are directly conjugated to different fluorochromes that are also distinct from that used to identify the CD45+/Terl19+/CD31+/CDI40a+ cells if no depletion step was used prior to CD24 5 and CD49f antibody labelling. Following treatment of the cell suspension with an agent to discriminate cells with non-intact plasma membranes (e.g., propidium iodide) the labelled cells can be analyzed by flow cytometry and cells at any stage of differentiation from MaSC to differentiated luminal and myoepithelial cells can be selectively isolated using fluorescence-activated cell sorting (FACS).
A suspension of freshly dissociated mouse mammary cells were labelled with biotin-conjugated monoclonal antibodies specific for the CD45/Ter119/CD31/CD140a cell surface epitopes and the labelled cells removed by immunomagnetic cell depetion (EasySepTM Biotin Selection Kit from StemCell Technologies) and the remaining cells were incubated with monoclonal antibodies specific for CD24 and CD49f conjugated to R-phycoerythrin and fluorescein isothiocyanate (FITC), respectively. The MaSC enriched and CFC enriched cell fractions as outlined in FIG. 1 were then isolated by FACS. The two cell populations were then assessed for MaSC and CFC content by transplantation into cleared fat pads (Kordon EC, Smith GH. Development 1998;125:1921-30) and by in vitro colony assays (Stingl J, Zandieh I, Eaves CJ, Emerman JT.. Breast Cancer Res Treat 2001:67:93-109), respectively. The MaSC fraction was found to be highly enriched for stem cells since approximately 75% of all stem cells present in the mouse mammary gland were in this subpopulation at a highly enriched frequency of I
stem cell in 50 sorted cells. This represents an approximately 25-fold enrichment over non-sorted cells. Less than 10% of all CFCs were localized to the MaSC sorted population of cells. The CFC fraction was found to be highly purified for luminal-restricted CFCs with approximately 1 CFC for every 6 sorted cells. This represented approximately 90% of all CFCs present in the mammary gland. The MaSC frequency in the CFC fraction is <1 in 750 sorted cells. This example is the first description of the recognition, differential isolation and functional characterization of mammary stem and progenitor cells.
Example 2 Method for Determining the Gene Expression Profile of Mammary Stem and Progenitor Cells A suspension of freshly dissociated mouse mammary cells were labelled with biotin-conjugated monoclonal antibodies specific for the CD45/Terl19/CD31/CD140a cell surface epitopes and the labelled cells removed by immunomagnetic cell depetion (EasySepTM Biotin Selection Kit from StemCell Technologies) and the remaining cells were incubated with monoclonal antibodies specific for CD24 and CD49f conjugated to R-phycoerythrin and fluorescein isothiocyanate (FITC) respectively. The MaSC enriched and CFC enriched cell fractions as outlined in FIG. 1 were then isolated by FACS. Total riboxynucleic acid (RNA) was then isolated and the gene expression profiles of the different subsets of cells were then analyzed by microarray analysis and genes preferentially expressed by the different subsets of cells identified. This example is the first description of the gene expression profiles of highly purified mammary stem and progenitor cell fractions.
TABLE I
Species CFC Phenotype References Lum inal -restricted B i p o t e n t CF C
CFC Phenotype phenotype Human EpCAM+/CD49f'/ EpCAM+/CD49f/ Stingl J, Eaves CJ, Kuusk U, Emerman JT.
MUCI+/CD133+/ MUCI-/CD133-/ Differentiation 1998;63:201-13.
C D I O-/Thy-1- CD 10+/Thy-1 +
Stingl J, Zandieh 1, Eaves CJ, Emerman IT.. Breast Cancer Res Treat 2001:67:93-109.
Stingl J, Raouf A, Emerman JT and Eaves CJ. J Mammary Gland Biology Neoplasia 2005;10:49-59.
O'Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA. Differentiation 1991;46:209-21.
Kao CY, Nomata K, Oakley CS, Welsch CW and Chang CC. Carcinogenesis 1995;16:531-38.
Kao CY, Oakley CS, Welsch CW, Chang CC. In Vitro Cell Dev Biol Anim 1997;33:282-88.
Stingl J, Eaves CJ, Emerman JT. In: Ip M
and Asch BB, editors. Methods in Mammary Gland Biology and Breast Cancer Research. New York (NY): Kluwer Academic/Plenum Publishers; 2000. p.
177-93.
Clarke C, Titley J, Davies S and O'Hare MJ. Epithelial Cell Biol 1994;3: 38-46.
Rat Peanut Agglutinin binding+ Thy-1" Kim ND and Clifton KH. Experimental Cell Res 1993;207: 74-85.
MUC1+ CD10 Dundas SR, Ormerod MG, Gusterson BA, O'Hare MJ. J Cell Sci 1991;100:459-71.
Mouse CD45-/Ter119'/CD31"/CD24+/CD49f*/ Stingl J, Ricketson I, Choi D, Eaves CJ.
Sca-flow Proceed Am Assoc Cancer Res 2004;45:641-2.
mMFGM+ JB6 epitope+ Smalley MJ, Titley J and O'Hare MJ. In Vitro Cell Dev Biol-Animal 1998; 34;
711-721.
mMFGM = mouse milk fat globule membrane JB6 = antibody of unknown epitope specificity References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Proc Natl Acad Sci U S A. 2003;100:3983-8.
CD24. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 192-3, 1997.
CD3 1. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 206-8, 1997.
CD45. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 244-47, 1997.
CD49f. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 267-8, 1997.
Carter,W.G., Kaur,P., Gil,S.G., Gahr,P.J. & Wayner,E.A. J Cell Biol. 1990:
111, 3141-54.
Clarke C, Titley J, Davies S and O'Hare MJ. Epithelial Cell Biol 1994;3: 38-46.
Crowley M.R., Bowtell D. and Serra R. Dev Biol 279:58-72.
Dundas SR, Ormerod MG, Gusterson BA, O'Hare MJ. J Cell Sci 1991;100:459-71.
Kao CY, Nomata K, Oakley CS, Welsch CW and Chang CC. Carcinogenesis 1995;16:531-38.
Kao CY, Oakley CS, Welsch CW, Chang CC. In Vitro Cell Dev Biol Anim 1997;33:282-88.
5 Kim ND and Clifton KH. Experimental Cell Res 1993;207: 74-85.
Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL, Katsura Y. Br J
Haematol. 2000;109: 280-7.
10 Kordon EC, Smith GH. Development 1998;125:1921-30.
O'Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA. Differentiation 1991;46:209-21.
15 Orr-Urtreger A, Lonai P. Development. 1992;115:1045-58.
Smalley MJ, Titley J and O'Hare MJ. In Vitro Cell Dev Biol-Animal 1998; 34;
721.
20 Stingl J, Eaves CJ, Kuusk U, Emerman JT. Differentiation 1998;63:201-13.
Stingl J, Eaves CJ, Emerman JT. In: Ip M and Asch BB, editors. Methods in Mammary Gland Biology and Breast Cancer Research. New York (NY): Kluwer Academic/Plenum Publishers; 2000. p. 177-93.
Stingl J, Zandieh I, Eaves CJ, Emerman JT.. Breast Cancer Res Treat 2001:67:93-109.
Stingl J, Ricketson I, Choi D, Eaves CJ. Proceed Am Assoc Cancer Res 2004;45:641-2.
Stingl J, Raouf A, Emerman JT and Eaves CJ. J Mammary Gland Biology Neoplasia 2005; 10:49-59.
Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Dev Biol 2002;245:42-56.
cells in the preparation may also be assessed for example by transplanting into mice as described previously.
III. USES OF THE COMPOSITIONS AND METHODS OF THE INVENTION
The invention may be used in the isolation of different subsets of mouse mammary cells including MaSC and luminal-restricted progenitor cells. These cells are of interest to breast cancer biologists, mammary gland biologists and developmental biologists. In particular, the ability to purify mammary progenitors and stem cells free of stromal cell contamination is important in determining the gene expression profiles of these cells and the factors that regulate their cell behaviour.
The invention includes kits for preparing samples enriched in mammary epithelial cells comprising antibodies that bind to non-epithelial cells in a mammary sample and instructions for the use thereof. The antibody composition for use in the kit preferably comprises antibodies that bind to CD45, Ter 119, CD31 and CD
140a.
The invention further includes kits for preparing samples enriched in MaSC or CFC cells comprising antibodies that bind to CD24 and/or CD49f and instructions for the use thereof.
The following non-limiting examples are illustrative of the present invention:
EXAMPLES
Example 1 Method for Determining the In Vivo and In Vitro Growth Properties of Different Subsets of Mammary Cells Mammary glands from young adult female mice were removed and digested for 8 hours at 37 C in EpiCult-BTM (StemCell Technologies, Vancouver, BC, Canada) supplemented with 5% fetal bovine serum (FBS) and 300 U/mL collagenase and 100 U/mL hyaluronidase (StemCell Technologies). At the end of this time, the preparation is vortexed and centrifuged at 450g for 5 minutes. The supernatant is discarded and contaminating red blood cells lysed with an ammonium chloride wash.
Following centrifugation, the cells are suspended in 2 mL of 0.25% trypsin prewarmed to 37 C and further dissociated by gentle pipetting for 1-2 minutes.
The cells are then washed once with 10 mL of Hank's Balanced Salt Solution supplemented with 2% FBS (HF) and incubated with 5 mg/mL dispaseTM II
(StemCell Technologies) and 0.1 mg/mL deoxyribonuclease I (StemCell Technologies) for 2 min at 37 C. The resultant cell suspension is diluted with HF and then filtered through a 40 m mesh to obtain a single cell suspension.
Hematopoietic (CD45 - and Ter119'), endothelial (CD31 -) and stromal (CD140a) cells are removed by pre-incubating freshly dissociated cells in 2 ghnL
Fc receptor antibody 2.4G2 (American Type Culture Collection, Rockville, MD, USA) followed by a 10 minute incubation with antibodies specific for CD45 (clone 30-Fll from BD Pharmingen, San Diego, USA), Terl19 (clone Terl19 from BD
Pharmingen), CD31 (clone MEC 13.3 from BD Pharmingen) and with CD I40a (clone APA5 from eBioscience, San Diego, CA, USA). Antibody concentrations during the incubation process typically ranges from 0.5-2.0 g/mL. These CD45, Terl 19, and CD140a antibodies can be directly or indirectly conjugated to a fluorochrome or biotin such that the antibody labelled cells can be selectively detected by flow cytometry or selectively depleted by binding to some type of matrix (e.g., magnetic beads, a panning surface, dense particles for density centrifugation, an adsorption column, or an adsorption membrane). If a negative selection strategy is implemented, the remaining CD45/Terl 19/CD31/CDI40a depleted mammary cells are then labelled with antibodies recognizing epitopes of the cell surface proteins CD24 and CD49f. A
suitable antibody clone specific for mouse CD24 is the M 1 /69 clone (BD
Pharmingen). A suitable antibody clone specific for mouse CD49f is the GoH3 clone (BD Pharmingen). Both antibodies are used at a concentration of 1 g/mL during the incubation process. The CD24 and CD49f antibodies are directly conjugated to different fluorochromes that are also distinct from that used to identify the CD45+/Terl19+/CD31+/CDI40a+ cells if no depletion step was used prior to CD24 5 and CD49f antibody labelling. Following treatment of the cell suspension with an agent to discriminate cells with non-intact plasma membranes (e.g., propidium iodide) the labelled cells can be analyzed by flow cytometry and cells at any stage of differentiation from MaSC to differentiated luminal and myoepithelial cells can be selectively isolated using fluorescence-activated cell sorting (FACS).
A suspension of freshly dissociated mouse mammary cells were labelled with biotin-conjugated monoclonal antibodies specific for the CD45/Ter119/CD31/CD140a cell surface epitopes and the labelled cells removed by immunomagnetic cell depetion (EasySepTM Biotin Selection Kit from StemCell Technologies) and the remaining cells were incubated with monoclonal antibodies specific for CD24 and CD49f conjugated to R-phycoerythrin and fluorescein isothiocyanate (FITC), respectively. The MaSC enriched and CFC enriched cell fractions as outlined in FIG. 1 were then isolated by FACS. The two cell populations were then assessed for MaSC and CFC content by transplantation into cleared fat pads (Kordon EC, Smith GH. Development 1998;125:1921-30) and by in vitro colony assays (Stingl J, Zandieh I, Eaves CJ, Emerman JT.. Breast Cancer Res Treat 2001:67:93-109), respectively. The MaSC fraction was found to be highly enriched for stem cells since approximately 75% of all stem cells present in the mouse mammary gland were in this subpopulation at a highly enriched frequency of I
stem cell in 50 sorted cells. This represents an approximately 25-fold enrichment over non-sorted cells. Less than 10% of all CFCs were localized to the MaSC sorted population of cells. The CFC fraction was found to be highly purified for luminal-restricted CFCs with approximately 1 CFC for every 6 sorted cells. This represented approximately 90% of all CFCs present in the mammary gland. The MaSC frequency in the CFC fraction is <1 in 750 sorted cells. This example is the first description of the recognition, differential isolation and functional characterization of mammary stem and progenitor cells.
Example 2 Method for Determining the Gene Expression Profile of Mammary Stem and Progenitor Cells A suspension of freshly dissociated mouse mammary cells were labelled with biotin-conjugated monoclonal antibodies specific for the CD45/Terl19/CD31/CD140a cell surface epitopes and the labelled cells removed by immunomagnetic cell depetion (EasySepTM Biotin Selection Kit from StemCell Technologies) and the remaining cells were incubated with monoclonal antibodies specific for CD24 and CD49f conjugated to R-phycoerythrin and fluorescein isothiocyanate (FITC) respectively. The MaSC enriched and CFC enriched cell fractions as outlined in FIG. 1 were then isolated by FACS. Total riboxynucleic acid (RNA) was then isolated and the gene expression profiles of the different subsets of cells were then analyzed by microarray analysis and genes preferentially expressed by the different subsets of cells identified. This example is the first description of the gene expression profiles of highly purified mammary stem and progenitor cell fractions.
TABLE I
Species CFC Phenotype References Lum inal -restricted B i p o t e n t CF C
CFC Phenotype phenotype Human EpCAM+/CD49f'/ EpCAM+/CD49f/ Stingl J, Eaves CJ, Kuusk U, Emerman JT.
MUCI+/CD133+/ MUCI-/CD133-/ Differentiation 1998;63:201-13.
C D I O-/Thy-1- CD 10+/Thy-1 +
Stingl J, Zandieh 1, Eaves CJ, Emerman IT.. Breast Cancer Res Treat 2001:67:93-109.
Stingl J, Raouf A, Emerman JT and Eaves CJ. J Mammary Gland Biology Neoplasia 2005;10:49-59.
O'Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA. Differentiation 1991;46:209-21.
Kao CY, Nomata K, Oakley CS, Welsch CW and Chang CC. Carcinogenesis 1995;16:531-38.
Kao CY, Oakley CS, Welsch CW, Chang CC. In Vitro Cell Dev Biol Anim 1997;33:282-88.
Stingl J, Eaves CJ, Emerman JT. In: Ip M
and Asch BB, editors. Methods in Mammary Gland Biology and Breast Cancer Research. New York (NY): Kluwer Academic/Plenum Publishers; 2000. p.
177-93.
Clarke C, Titley J, Davies S and O'Hare MJ. Epithelial Cell Biol 1994;3: 38-46.
Rat Peanut Agglutinin binding+ Thy-1" Kim ND and Clifton KH. Experimental Cell Res 1993;207: 74-85.
MUC1+ CD10 Dundas SR, Ormerod MG, Gusterson BA, O'Hare MJ. J Cell Sci 1991;100:459-71.
Mouse CD45-/Ter119'/CD31"/CD24+/CD49f*/ Stingl J, Ricketson I, Choi D, Eaves CJ.
Sca-flow Proceed Am Assoc Cancer Res 2004;45:641-2.
mMFGM+ JB6 epitope+ Smalley MJ, Titley J and O'Hare MJ. In Vitro Cell Dev Biol-Animal 1998; 34;
711-721.
mMFGM = mouse milk fat globule membrane JB6 = antibody of unknown epitope specificity References Al-Hajj M, Wicha MS, Benito-Hernandez A, Morrison SJ, Clarke MF. Proc Natl Acad Sci U S A. 2003;100:3983-8.
CD24. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 192-3, 1997.
CD3 1. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 206-8, 1997.
CD45. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 244-47, 1997.
CD49f. In: The Leukocyte Antigen Facts Book. Barclay AN, Brown MH, Law SKA, McKnight AJ, Tomlinson MG van der Merwe PA (Eds.) Academic Press Inc., San Diego, USA, pp 267-8, 1997.
Carter,W.G., Kaur,P., Gil,S.G., Gahr,P.J. & Wayner,E.A. J Cell Biol. 1990:
111, 3141-54.
Clarke C, Titley J, Davies S and O'Hare MJ. Epithelial Cell Biol 1994;3: 38-46.
Crowley M.R., Bowtell D. and Serra R. Dev Biol 279:58-72.
Dundas SR, Ormerod MG, Gusterson BA, O'Hare MJ. J Cell Sci 1991;100:459-71.
Kao CY, Nomata K, Oakley CS, Welsch CW and Chang CC. Carcinogenesis 1995;16:531-38.
Kao CY, Oakley CS, Welsch CW, Chang CC. In Vitro Cell Dev Biol Anim 1997;33:282-88.
5 Kim ND and Clifton KH. Experimental Cell Res 1993;207: 74-85.
Kina T, Ikuta K, Takayama E, Wada K, Majumdar AS, Weissman IL, Katsura Y. Br J
Haematol. 2000;109: 280-7.
10 Kordon EC, Smith GH. Development 1998;125:1921-30.
O'Hare MJ, Ormerod MG, Monaghan P, Lane EB, Gusterson BA. Differentiation 1991;46:209-21.
15 Orr-Urtreger A, Lonai P. Development. 1992;115:1045-58.
Smalley MJ, Titley J and O'Hare MJ. In Vitro Cell Dev Biol-Animal 1998; 34;
721.
20 Stingl J, Eaves CJ, Kuusk U, Emerman JT. Differentiation 1998;63:201-13.
Stingl J, Eaves CJ, Emerman JT. In: Ip M and Asch BB, editors. Methods in Mammary Gland Biology and Breast Cancer Research. New York (NY): Kluwer Academic/Plenum Publishers; 2000. p. 177-93.
Stingl J, Zandieh I, Eaves CJ, Emerman JT.. Breast Cancer Res Treat 2001:67:93-109.
Stingl J, Ricketson I, Choi D, Eaves CJ. Proceed Am Assoc Cancer Res 2004;45:641-2.
Stingl J, Raouf A, Emerman JT and Eaves CJ. J Mammary Gland Biology Neoplasia 2005; 10:49-59.
Welm BE, Tepera SB, Venezia T, Graubert TA, Rosen JM, Goodell MA. Dev Biol 2002;245:42-56.
Claims (17)
1. A method of enriching mammary stem cells and/or luminal restricted colony forming cells in a sample containing mammary cells comprising 1) reacting the sample with first antibody composition comprising antibodies that bind to the antigens CD45, Ter119 and CD31 on non-epithelial cells under conditions so that conjugates are formed between the antibodies and the cells in the sample containing the non-epithelial antigens; 2) removing the conjugates; 3) recovering a cell preparation which is enriched in mammary epithelial cells and 4) reacting the sample enriched in epithelial cells with a second antibody composition capable of binding the antigens CD24 and/or CD49f under conditions so that conjugates form between antibodies and the cells in the sample containing the antigens CD24 and/or CD49f; and recovering cells that are bound by the antibodies, wherein said recovered cells comprise mammary stem cells and/or luminal restricted colony forming cells.
2. The method of claim 1 wherein the mammary cells are mouse cells.
3. The method of claim 1 wherein the mammary cells are normal cells.
4. The method according to any one of claims 1 to 3 wherein the epithelial cells consist of differentiated cells, CFCs or stem cells.
5. The method according to any one of claims 1 to 4 wherein the first antibody composition further comprises antibodies that bind to CD140a.
6. The method according to any one of claims 1 to 5 for recovering mammary stem cells, further comprising determining the levels of CD49f on the cells wherein high levels of CD49f indicates that the cells are mammary stem cells.
7. The method according to any one of claims 1 to 5 for recovering luminal-restricted colony forming cells (CFCs) further comprising determining the levels of CD49f on the cells wherein low levels of CD49f indicates that the cells are luminal restricted CFCs.
8. The method according to any one of claims 1 to 7 wherein the second antibody composition comprises antibodies that bind to CD24.
9. The method according to any one of claims 1 to 7 wherein the second antibody composition comprises antibodies that bind to CD49f.
10. The method according to any one of claims 1 to 7 wherein the second antibody composition comprises antibodies that bind to CD24 and CD49f.
11. The method according to any one of claims 1 to 10 wherein the antibodies in the antibody compositions are monoclonal antibodies.
12. The method according to claim 11 wherein the antibodies in the antibody compositions are labeled with a marker or the antibodies are conjugated to a matrix.
13. A method according to claim 11 wherein the antibodies in the antibody compositions are labeled with biotin or a fluorochrome.
14. A method according to claim 12 wherein the matrix is magnetic beads, a panning surface, dense particles for density centrifugation, an adsorption column, or an adsorption membrane.
15. The method according to claim 12, wherein each of the monoclonal antibodies in the first antibody composition is incorporated in a tetrameric antibody complex which comprises a first monoclonal antibody of a first animal species from the first antibody composition and a second monoclonal antibody of the first animal species which is capable of binding to at least one antigen on the surface of a matrix, which have been conjugated to form a cycle tetramer with two monoclonal antibodies of a second animal species directed against the Fc-fragments of the antibodies of the first animal species.
16. The method according to any one of claims 1 to 15 wherein a fluorescence-activated cell sorter can be used to identify and purify cell-antibody conjugates in the second antibody composition.
17. The method according to claim 2 wherein the mouse mammary cells are obtained from freshly dissociated mouse mammary glands.
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