The existence of stem and progenitor cells in the human mammary gland has been widely postulated; however, until now, neither the location nor the candidate cellular entities have been definitively identified and functionally characterized. Previously, we had described two cell lines that were derived from the human breast using magnetic sorting and HPV16 E6/E7 immortalization (Gudjonsson et al., 2002b
). One of the immortalized clones, MUC1−
, was able to generate itself as well as luminal epithelial and myoepithelial cells; it further expressed keratin K19 and formed TDLU-like structures inside a 3D lrECM. The other, MUC1+
, was lineage restricted, keratin K19 negative, and formed acinus-like spheres within the 3D lrECM. Although this was an important advance, several essential questions remained: did these virally transduced cells have identical counterparts in vivo, or were these a result of immortalization? If counterparts existed, where were they located in situ? Were there additional progenitor cells we had not been able to immortalize? Could we develop a procedure whereby one could isolate the untransduced counterparts reproducibly? Were there differences between ducts and lobules, and, intriguingly, was there a hierarchy?
In this study, we have answered all of the aforementioned questions: we have demonstrated the existence of four distinct human mammary epithelial cell types in situ, two of which most certainly are precursors to the two immortalized cell lines we had isolated previously using E6/E7. The cells are distributed in a stem cell zone in ducts and outside this zone in lobules. The size of the stem cell zone varies somewhat with the marker used, the most restricted and presumably most specific being the presence of keratin K15 and SSEA-4. Ducts and lobules were dissected, collected under the microscope, cloned, and characterized. We believe it is critical to meticulously dissect lobules distal to the intralobular terminal duct. Failure to do so could explain previously published data on the reported absence of a difference in growth patterns between ducts and lobules (O'Hare et al., 1991
). Permanent cell lines were established, cloned, and characterized further with respect to the profiles defined in situ and in primary cultures. The procedures we have developed, while painstaking and time consuming by necessity because they are for normal human tissues, are nevertheless robust. We show that these four cell types are hierarchically connected such that only one cell type can give rise to all others, which themselves are lineage-restricted progenitors.
An important implication of these findings is that for the first time, a stem cell zone containing one or more mammary stem cells was identified in the human breast. Mouse mammary stem cells have recently been isolated prospectively based on various FACS strategies (Shackleton et al., 2006
; Stingl et al., 2006a
). However, so far, FACS profiles have not translated into the cell of origin or location in situ. In this study, by the use of anatomical markers, we demonstrate that the previously described gates for bipotent progenitors, colony-forming cells, and mammary repopulating units in mice (Shackleton et al., 2006
; Stingl et al., 2006a
) enrich specifically for cells of ductal origin in the human breast. Older studies had suggested that the mammary ducts of rodents may harbor such a stem cell niche. For instance, in the mammary gland from virgin mouse, candidate stem cells (stained with antibodies JB6 and JsE3) were found in ducts rather than in alveoli, and it was postulated that these served to regenerate ductal epithelium as well as forming new alveolar buds (Sonnenberg et al., 1986
). This postulate was born out recently in an experiment with rudimentary ducts from postgestational mice transplanted to cleared fat pads of TGF-β1 transgenic mice, which retained the capacity to reactivate lobular structures at late pregnancy (Boulanger et al., 2005
). Apparently, the mouse mammary gland ductal niche responds specifically to the MMTV–c-myc transgene by amplification of the stem cell compartment (Chepko et al., 2005
). This implied strongly that in mice, an entire TDLU at any time represents the progeny from a single early ductal progenitor. Indeed, seminal studies of X chromosome inactivation in the human breast had demonstrated the presence of contiguous patches of normal mammary epithelium suggestive of being derived from single stem cells (Tsai et al., 1996
). These patches were explained to be the result of some developmentally important long-term primitive stem cells, which were presumed to be estrogen receptor negative, as opposed to shorter term estrogen receptor–positive stem cells important for adult tissue homeostasis (Clarke, 2005
). The hierarchy we describe here is essentially compatible with the aforementioned findings. However, it is important to recognize that the present identification of a stem cell zone in ducts does not necessarily exclude the existence of stem cells at other locations, although despite an extensive search, we have not observed such additional candidates as of yet.
The possibility of one stemlike cell population in an organ, which precedes all of the other stemlike cells during development, appears to be a general phenomenon because such multiple cell-type niches were also previously described for the hair follicle (Blanpain et al., 2004
). The division of labor into that of the maintenance of tissue homeostasis and the more elaborate development or regenerative remodelling between stem cell compartments may also be a general phenomenon because in the skin, homeostasis is maintained exclusively by the epidermal proliferative unit, but wound repair depends on bulge cells from hair follicles (Ito et al., 2005
). In analogy to hair follicles, we would propose that mammary tissue homeostasis is maintained by proliferative units in the TDLUs, whereas more elaborate structures, including the formation of new TDLUs from ductal alveolar buds, depend on the recruitment of cells from ducts.
Until recently, very few markers were assigned directly to a candidate mammary stem cell pool in humans or, indeed, even in rodents, but scattered single cells within the ducts of adult mice were shown to be positive for keratin K6a (Buono et al., 2006
), a marker originally found in putative stem cells in the terminal end bud (Smith et al., 1990
). In the present study, we found that the ductal stem cell zone was characterized by the accumulation of K19+
cells. If these traits were even indirectly related to stem cell activity, we would expect them to fluctuate with the size of the stem cell compartment. One of the most important pathways responsible for mammary stem cell activity is canonical Wnt signaling (Lindvall et al., 2006
). Interestingly, Wnt1- or β-catenin–induced mammary hyperplasia and tumorigenesis in mice correlate with the accumulation of K6-positive cells even though K6 in itself does not appear to be essential for mammary gland development, at least in embryonic knockouts (Grimm et al., 2006
). It has been shown that caveolin-1 deficiency in mice, which conveys mammary hyperplasia and tumorigenesis along with an increased stem cell activity, is characterized by the accumulation of keratin K6-positive cells (Sotgia et al., 2005
). However, the mammary glands of mice that are unable to signal through the Notch pathway most closely mimic the profile of the human mammary stem cell zone (Buono et al., 2006
; Wang et al., 2006
). Under these conditions, there is a remarkable ductal accumulation of cells expressing luminal keratins coordinately with keratins K14 and K6 (Buono et al., 2006
). A similar stem cell–related profile has been recorded in the prostate (Hudson et al., 2001
; Wang et al., 2006
). The appearance of +/+ cells in the suprabasal position has been interpreted as an accumulation of intermediate immature luminal cells (Buono et al., 2006
). Thus, it cannot be excluded that the stem cell zone described here contains bona fide ductal basal cells, which, under the current available culture conditions, are not colony forming.
In mass cultures derived from reduction mammoplasty, we and others had shown previously that a subpopulation of the primary breast luminal epithelial compartment was capable of giving rise to both luminal epithelial and myoepithelial cells (Kao et al., 1995
; Kang et al., 1997
; Péchoux et al., 1999
; Clarke et al., 2005
). Furthermore, in culture and occasionally in suprabasal cells in the epithelial layer in situ, single cells could exhibit dual staining for these two cell types (Péchoux et al., 1999
). Cells in this position were shown later to be K19 positive and could be immortalized occasionally in mass culture with HPV E6/E7 (Gudjonsson et al., 2002b
). The discovery in the present study of a ductal stem cell zone in the human breast opens the possibility of the existence of a hierarchy among differentiated progeny at other locations. In the mammary gland, the ultimate level of differentiation is reached during lactation. Although we have not addressed the consequence of lactation on ductal differentiation, a previous study strongly suggested that ducts are indeed protected from hormone-induced differentiation (Bocker et al., 2002
). This is in agreement with the presumed function of candidate stem cells in mature ducts of humans and rodents as a source of lateral branching during the formation of milk-producing lobules (Taylor-Papadimitriou et al., 1983
; Cardiff and Wellings, 1999
). Functionally, one of the hallmarks of stem cells is the ability to self-renew (for reviews see Watt, 1998
; Mackenzie, 2006
). A culture assay for self-renewal in human breast epithelial cells was successfully adopted from the field of neurobiology combined with the use of 3D laminin-rich gels (Petersen et al., 1992
). Thus, cells prevented from attachment grew in suspension to form the so-called mammospheres (Dontu et al., 2003
) reminiscent of the originally described neurospheres. Self-renewing stem cells were characterized by the ability to form new multipotent mammospheres even after passaging (Dontu et al., 2003
). The other widely used culture assay of self-renewal in epithelial tissues is growth after plating at clonal density (for reviews see Watt, 1998
; Mackenzie, 2006
). We used both assays in the present study and showed that both mammospheres and multipotent clones could self-renew, although we do not yet know how the fraction of the self-renewing clones compare between the assays.
Our findings are pertinent to two poorly understood aspects of breast cancer evolution. For example, we know that breast cancers comprise at least two well-defined subtypes with distinct molecular profiles reminiscent of the luminal and basal lineages (Perou et al., 2000
). This has led to a resurgence of speculation that breast cancers may arise in the different compartments within a stem cell hierarchy (Taylor-Papadimitriou et al., 1983
; Rudland, 1987
; Al-Hajj et al., 2003
; Behbod and Rosen, 2005
; Clarke, 2005
). The hierarchy described here may guide the design of experiments to test the possible role of these progenitors in the origin of breast cancer subtypes, a possibility that is under investigation.