Growth and differentiation of progenitor/stem cells derived from the human mammary gland

Abstract

Estrogen is necessary for the full development of the mammary gland and it is also involved in breast cancer development. We set out to identify and characterise progenitor/stem cells in the human mammary gland and to explore the role of estrogen in their proliferation and differentiation. Three candidate stem cell populations were isolated: double positive (DP) cells co-expressed the luminal and myoepithelial markers, EMA and CALLA, respectively, whereas double negative (DN) cells did not express these cell surface markers; side population (SP) cells were characterised by their differential ability to efflux the dye Hoechst 33342. The ABC transporter, breast cancer resistance protein (BCRP) was more highly expressed in SP cells than in non-SP cells and a specific BCRP inhibitor, Ko143, reduced SP formation, suggesting that BCRP confers the SP phenotype in mammary epithelial cells, as has been demonstrated in other tissues. Interestingly, SP cells were double negative for the EMA and CALLA antigens and therefore represent a separate and distinct population to DP cells. Single cell multiplex RT-PCR indicated that the SP and DN cells do not express detectable levels of ERα or ERβ, suggesting that estrogen is not involved in their proliferation. DP cells expressed ERα but at a lower level than differentiated luminal cells. These findings invoke a potential strategy for the breast stem/progenitor cells to ignore the mitogenic effects of estrogen. All three cell populations generated mixed colonies containing both luminal and myoepithelial cells from a single cell and therefore represent candidate multipotent stem cells. However, DN cells predominately generated luminal colonies and exhibited a much higher cloning efficiency than differentiated luminal cells. Further characterisation of these candidate progenitor/stem cells should contribute to a better understanding of normal mammary gland development and breast tumorigenesis.

Introduction

The adult human mammary gland consists of a branching system of ducts surrounded by a collagenous and fatty stroma. An inner layer of luminal epithelial cells lines the system of ducts and lobules and an outer layer of contractile myoepithelial cells surrounds the luminal cells. Although the adult breast is often called ‘resting’ (outside pregnancy and lactation), proliferation and apoptosis occur during each menstrual cycle. Thymidine labelling has shown that proliferation occurs mainly in the luminal cells, is maximal during the second half of the cycle and declines with age [1]. In addition, most breast cancers arise from luminal epithelial cells [2].

Luminal and myoepithelial cells can be identified by their expression of various surface and cytoskeletal proteins. Luminal cells are characterised by their expression of epithelial membrane antigen (EMA) [3], epithelial specific antigen (ESA) [4] and cytokeratins (K) 7, 8, 18 and 19 [5]. Myoepithelial cells express common acute lymphoblastic leukaemia antigen (CALLA) [6], α-smooth muscle actin (α-SMA) [7], α6 integrin [8], vimentin [9] and K5 and K14 [10]. However, only EMA and CALLA represent exclusive luminal and myoepithelial cell surface markers, respectively, and can be used to separate the cell types to 98% purity using fluorescent-activated cell sorting (FACS) [3]. Furthermore, EMA and CALLA are the only known luminal and myoepithelial markers exclusively expressed at the RNA level as well as at the protein level in the human breast [11].

The steroid hormone estrogen is necessary for the normal development of the mammary gland, where it is responsible for epithelial cell proliferation and ductal morphogenesis [12]. Estrogen has also been implicated in the induction and progression of mammary carcinomas [13]. The diverse effects of estrogen are mediated by the estrogen receptor (ER), a ligand inducible transcription factor that belongs to the super-family of steroid hormone and nuclear receptors [14]. Two isoforms of the receptor exist, ERα [15] and ERβ [16]. In the human mammary gland, ERα is expressed in approximately 10–30% of luminal cells but is not expressed in myoepithelial cells [17]. In contrast, ERβ is expressed in both luminal and myoepithelial cells [18]. ERα-positive carcinomas express significantly more ERα than the normal mammary epithelium [19]. Despite the clear role of estrogen in proliferation of the mammary epithelium, the way in which it exerts its effect is not fully understood. During the estrous cycle, the majority of proliferating cells, both in the terminal end buds and in the ducts, do not seem to contain ERα [20], suggesting that estrogen acts indirectly in the mammary epithelium by inducing ERα-positive cells to produce growth factors that stimulate neighbouring ERα-negative cells to divide.

Tissue-specific stem cells appear to be in most organs of the body and can be defined as cells that have the capacity to self-renew and to generate all of the differentiated cell lineages appropriate to their location. However, the thorough identification and isolation of these somatic stem cells has been a challenge in most cases. During the last few years, evidence has been accumulating concerning the existence of mammary stem cells that can cause both epithelial cell lineages. Through the use of retroviral-tagging, it has been shown that an entire mammary gland may comprise the progeny from a single cell and that this process can be repeated by serial transplantation [21], however, the identity of these cells is still unknown. The existence of three types of human breast epithelial progenitors has been proposed based on the marker expression of colonies grown at low clonal density in in vitro cultures [4]. However, the identified bipotent progenitors only generate colonies with a central core of cells expressing the luminal marker ESA, surrounded by cells expressing the myoepithelial marker K14 [22]. Evidence that a subset of ESA-positive cells may represent stem cells or progenitor cells in the human breast was also inferred by studies in which EMA⁺ESA⁺ and EMA⁻ESA⁺ luminal cells were immortalised with the E6/E7 genes from human papilloma virus type 16 and both were shown to maintain a luminal epithelial phenotype [23]. In clonal cultures at limiting dilution, the EMA⁻ESA⁺ cell line was able to generate itself, as well as EMA⁺ESA⁺ luminal cells and Thy-1⁺ α-SMA⁺ myoepithelial cells. The finding that a subset of luminal cells can give rise to myoepithelial cells suggests that myoepithelial cells may be derived from luminal cells, rather than from a lineage uncommitted stem cell. Indeed, culture of luminal and myoepithelial has revealed that a subset of luminal cells gradually converted to myoepithelial cells, based on the expression of lineage-specific markers, but not vice versa [24], further supporting the hypothesis that myoepithelial cells are derived from luminal precursors.

Recently, an in vitro cultivation system that has been developed supports the propagation of human mammary epithelial cells able to generate non-adherent mammospheres that were enriched for early progenitor/stem cells [25]. The gene expression profile of undifferentiated mammary epithelial cells grown as mammospheres was compared to that of differentiated cells [25]. Several genes upregulated in mammosphere-derived cells were identified, and a significant overlap was observed with the genetic programs of embryonic, neural and haematopoietic stem cells [26], [27].

The hypothesis that cancer originates from mutations in the stem cell population is well established [28], [29]. There is considerable evidence that certain types of leukaemia arise from mutations that accumulate in haematopoietic stem cells (HSCs) [29], [30], [31]. However, less definitive evidence exists suggesting that solid cancers arise from mutations in stem cells, although the clonal origin of many cancers and the similar properties exhibited by stem cells and cancer cells suggest that solid cancers are derived from mutated stem cells [28]. Recently, breast cancer stem cells able to generate tumours in the mammary fat pad of immunocompromised NOD/SCID mice have been identified [32], supporting the hypothesis that mammary tumours are initiated in mutated stem cells. Several cancers, including breast cancer, have been shown to be clonal in origin [33], [34], [35], suggesting that they represent the progeny of a single mutated stem cell. However, polyclonal breast carcinomas have also been identified, suggesting the mutation of more than one cell [36]. Most breast cancers exhibit a luminal cell phenotype, while myoepithelial cells are often absent in invasive breast carcinomas [2], [37]. The low incidence of myoepithelial cells in breast tumours has led to the suggestion that stem cells are pushed toward the luminal lineage thereby bypassing the myoepithelial pathway [38].

The aim of this study is to identify and characterise stem cells in the human mammary gland. Although there is mounting evidence for the existence of human mammary stem cells, they have yet to be definitively identified. To investigate the function and regulation of stem cells in a particular tissue, it is necessary to be able to identify them and design assays to functionally characterise them. The rarity of stem cells, and the absence of specific stem cell markers in the human mammary epithelium, poses a hurdle for their identification. We adopted two parallel approaches to identify stem cells in the human breast based on the knowledge of HSCs. Multipotent cells in the haematopoietic system co-express erythroid and myeloid genes before exclusive lineage commitment and differentiation [39], [40]. EMA and CALLA are exclusively expressed at the protein level in the breast on luminal cells and myoepithelial cells, respectively. If multilineage gene expression is a universal feature of stem cells, breast epithelial cells co-expressing EMA and CALLA may represent an enriched population of breast stem cells or progenitor cells and therefore, we have investigated the presence of mammary epithelial cells co-expressing both antigens. The ability of primitive HSCs to efflux the fluorescent dye, Hoechst 33342, is often used to isolate these cells [41]. The finding that SP cells exist in several different tissues, including in the mammary gland, as reported by our group [42], suggests that the ability to efflux this dye may be a common stem cell property, and therefore we have studied SP cells in the human mammary gland. The putative stem cell populations identified were characterised by analysing their gene expression profile at the single cell level, their ability to generate epithelial cell lineages in in vitro growth assays and the influence of estrogen in their proliferation and differentiation.