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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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Flow-cytometric analysis of human breast epithelial cells stained with 5 μM Hoechst 33342. (a) Trace demonstrating the presence of a side population (box). (b) Addition of 20 μM verapamil (+v), resulting in a 12-fold reduction in the side population. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4).
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Figure 1: Flow-cytometric analysis of human breast epithelial cells stained with 5 μM Hoechst 33342. (a) Trace demonstrating the presence of a side population (box). (b) Addition of 20 μM verapamil (+v), resulting in a 12-fold reduction in the side population. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4).

Mentions: Epithelial cells isolated from nine independent, normal human breast samples were stained with Hoechst and analysed by FACS. Each preparation contained a small fraction of cells (0.18 ± 0.23%) that exhibited a SP (Fig. 1a). The formation of the SP population was blocked by 20 μM verapamil, consistent with previous studies showing a requirement for ABC transporter family function in the SP phenotype (Fig. 1b) [5]. The SP was found in all samples regardless of age, parity, contraceptive status or day of menstrual cycle (Table 1).


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)

Flow-cytometric analysis of human breast epithelial cells stained with 5 μM Hoechst 33342. (a) Trace demonstrating the presence of a side population (box). (b) Addition of 20 μM verapamil (+v), resulting in a 12-fold reduction in the side population. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Flow-cytometric analysis of human breast epithelial cells stained with 5 μM Hoechst 33342. (a) Trace demonstrating the presence of a side population (box). (b) Addition of 20 μM verapamil (+v), resulting in a 12-fold reduction in the side population. X axis, Hoechst red fluorescence intensity (FL5); Y axis, Hoechst blue fluorescence intensity (FL4).
Mentions: Epithelial cells isolated from nine independent, normal human breast samples were stained with Hoechst and analysed by FACS. Each preparation contained a small fraction of cells (0.18 ± 0.23%) that exhibited a SP (Fig. 1a). The formation of the SP population was blocked by 20 μM verapamil, consistent with previous studies showing a requirement for ABC transporter family function in the SP phenotype (Fig. 1b) [5]. The SP was found in all samples regardless of age, parity, contraceptive status or day of menstrual cycle (Table 1).

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