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Functional and molecular characterisation of mammary side population cells.

Alvi AJ, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco Md, Dale TC, Smalley MJ - Breast Cancer Res. (2002)

Bottom Line: Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo.Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker.This work therefore has implications for mammary stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breakthrough Toby Robins Breast Cancer Centre, Institute of Cancer Research, London, UK.

ABSTRACT

Background: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown.

Methods: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP.

Results: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures.

Conclusion: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.

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Analysis of mouse side population (SP) cells. Flow cytometric analysis of mouse cells stained with Hoechst 33342: (a) SP in mouse mammary epithelial cells (box), (b) SP in mouse bone marrow cells. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4). (c) RT-PCR analysis of ABC transporter cassette proteins in SP and non-SP cells from mouse mammary epithelial cells. M, molecular weight marker; Brcp1, breast cancer resistance protein; Mrp, multidrug resistance-associated protein. Sizes of bands: Brcp1, 327 bp; Mrp1, 701 bp; Mrp3, 301 bp; Mrp4, 222 bp.
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Figure 2: Analysis of mouse side population (SP) cells. Flow cytometric analysis of mouse cells stained with Hoechst 33342: (a) SP in mouse mammary epithelial cells (box), (b) SP in mouse bone marrow cells. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4). (c) RT-PCR analysis of ABC transporter cassette proteins in SP and non-SP cells from mouse mammary epithelial cells. M, molecular weight marker; Brcp1, breast cancer resistance protein; Mrp, multidrug resistance-associated protein. Sizes of bands: Brcp1, 327 bp; Mrp1, 701 bp; Mrp3, 301 bp; Mrp4, 222 bp.

Mentions: We next examined whether the SP was present in mouse mammary epithelial cells. Results from 17 independent experiments showed that 0.45 ± 0.22% of mouse mammary epithelial cells had the SP phenotype (Fig. 2a). This proportion of cells exhibiting the mammary SP phenotype was similar to that observed in similarly stained mouse bone marrow samples (0.14 ± 0.11%) (Fig. 2b) and was in line with previous studies [5]. Representative flow cytometry traces of Hoechst-stained cells from mouse mammary epithelium and mouse bone marrow are shown in Figure 2a,2b. The increased availability of SP cells from the mouse mammary epithelium encouraged us to move to a mouse model for subsequent studies.


Functional and molecular characterisation of mammary side population cells.

Alvi AJ, Clayton H, Joshi C, Enver T, Ashworth A, Vivanco Md, Dale TC, Smalley MJ - Breast Cancer Res. (2002)

Analysis of mouse side population (SP) cells. Flow cytometric analysis of mouse cells stained with Hoechst 33342: (a) SP in mouse mammary epithelial cells (box), (b) SP in mouse bone marrow cells. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4). (c) RT-PCR analysis of ABC transporter cassette proteins in SP and non-SP cells from mouse mammary epithelial cells. M, molecular weight marker; Brcp1, breast cancer resistance protein; Mrp, multidrug resistance-associated protein. Sizes of bands: Brcp1, 327 bp; Mrp1, 701 bp; Mrp3, 301 bp; Mrp4, 222 bp.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC154129&req=5

Figure 2: Analysis of mouse side population (SP) cells. Flow cytometric analysis of mouse cells stained with Hoechst 33342: (a) SP in mouse mammary epithelial cells (box), (b) SP in mouse bone marrow cells. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4). (c) RT-PCR analysis of ABC transporter cassette proteins in SP and non-SP cells from mouse mammary epithelial cells. M, molecular weight marker; Brcp1, breast cancer resistance protein; Mrp, multidrug resistance-associated protein. Sizes of bands: Brcp1, 327 bp; Mrp1, 701 bp; Mrp3, 301 bp; Mrp4, 222 bp.
Mentions: We next examined whether the SP was present in mouse mammary epithelial cells. Results from 17 independent experiments showed that 0.45 ± 0.22% of mouse mammary epithelial cells had the SP phenotype (Fig. 2a). This proportion of cells exhibiting the mammary SP phenotype was similar to that observed in similarly stained mouse bone marrow samples (0.14 ± 0.11%) (Fig. 2b) and was in line with previous studies [5]. Representative flow cytometry traces of Hoechst-stained cells from mouse mammary epithelium and mouse bone marrow are shown in Figure 2a,2b. The increased availability of SP cells from the mouse mammary epithelium encouraged us to move to a mouse model for subsequent studies.

Bottom Line: Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo.Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker.This work therefore has implications for mammary stem cell biology.

View Article: PubMed Central - HTML - PubMed

Affiliation: Breakthrough Toby Robins Breast Cancer Centre, Institute of Cancer Research, London, UK.

ABSTRACT

Background: Breast cancer is thought to arise in mammary epithelial stem cells. However, the identity of these stem cells is unknown.

Methods: Studies in the haematopoetic and muscle systems show that stem cells have the ability to efflux the dye Hoechst 33342. Cells with this phenotype are referred to as the side population (SP). We have adapted the techniques from the haematopoetic and muscle systems to look for a mammary epithelial SP.

Results: Of mammary epithelial cells isolated from both the human and mouse mammary epithelia, 0.2-0.45% formed a distinct SP. The SP was relatively undifferentiated but grew as typical differentiated epithelial clones when cultured. Transplantation of murine SP cells at limiting dilution into cleared mammary fat pads generated epithelial ductal and lobuloalveolar structures.

Conclusion: These data demonstrate the existence of an undifferentiated SP in human and murine mammary epithelium. Purified SP cells are a live single-cell population that retain the ability to differentiate in vitro and in vivo. Studies of haematopoetic cells have suggested that the SP phenotype constitutes a universal stem cell marker. This work therefore has implications for mammary stem cell biology.

Show MeSH
Related in: MedlinePlus