Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate

Abstract

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

Fig. 1.

Analyses of stem/progenitor cell markers in parental and hTERT immortalized human mammary epithelial cells isolated and cultured in DFCI-1 medium. (A) A total of 50 μg of cell lysates were Western blotted using indicated antibodies. Breast cancer cell lines MDA-MB-231 and T47D are used as controls. β-Actin was used as loading control. (B) Immunofluorenscence staining of normal and immortal hMECs using indicated markers (red).

Fig. 2.

Isolation of K5⁺/K19⁻ and K5⁺/K19⁺ cell types from 70N.TERT cell line. (A) Morphology of K5⁺/K19⁻ and K5⁺/K19⁺ cell type cultured in 2-D culture in DFCI-1 medium is shown. (B) Immunofluorenscence staining of K5⁺/K19⁻ andK5+/ K19⁻ is shown. (C) Western blotting of K5⁺/K19⁻ and K5⁺/K19⁺ cell types using indicated antibodies (D) Immunofluorescence staining of human mammary tissue specimen with anti-K5 (green), and anti-K19 (red) double staining. K5⁺/K19⁻ (shown by arrow) and K5⁺/K19⁺ (shown by arrowhead).

Fig. 3.

In vitro self-renewal and myoepithelial cell differentiation of K5⁺/K19⁻ and K5⁺/K19⁺ cell types in MEGM medium. (A) Morphology of cells before and after start of differentiation; loose cells with fibroblastic morphology represent myoepithelial cells and center tight colony represent undifferentiated cells. (B) Immunostaining of cells with rabbit-anti human K5 (green) and mouse anti-human CD49f, K8, E-cadherin (red). (C) Immunofluorescence staining of myoepithelial cells. The cells were costained with rabbit-anti human K5 (green) and three myoepithelial cell markers: mouse anti-human α-SMA, Thy-1, and CD10 (red).

Fig. 4.

Analyses of luminal cell differentiation. K5⁺/K19⁻ and K5⁺/K19⁺ cell types cultured in MEGM and DFCI-2 media were stained with luminal cell marker MUC1. Both cell types cultured in MEGM (A) or DFCI-1 (B) were stained with anti-MUC1 antibody followed by costaining with rabbit-anti human K5 (green) and mouse anti-human MUC1 (red) and anti-vimentin (green). VIM, vimentin.