The ability to self-renew while simultaneously generating specialized cells is the critical property of adult stem cells. As currently understood, the choice of a dividing stem cell to self-renew or to differentiate is regulated by factors coming from the niche, or stem cell micro-environment. Implicit in all models of stem cell control is that differentiation is the default choice and that self-renewal depends on the presence of extracellular signals released by the niche. Among the signals implicated in the control of various types of stem cells, the Wnt pathway is prominent (Barker et al., 2007
; Jaks et al., 2008
; Kalani et al., 2008
; Reya et al., 2003
; Willert et al., 2003
). For example, Wnt target genes, such as lgr5, are expressed in stem cells of the intestine and the hair follicle, and there are stem cell phenotypes associated with loss of Wnt signaling in these tissues (Barker et al., 2007
; Jaks et al., 2008
). Likewise, in the case of the mammary gland, there is genetic support for a role for Wnt signaling in the maintenance of MaSCs, such as defects in mammary gland development in mice mutant for Wnt pathway components (Boras-Granic et al., 2006
; Brisken et al., 2000
; Chu et al., 2004
; Lindvall et al., 2006
). The experiments described in this paper suggest that Wnt signaling, as monitored by the expression of the Wnt target genes Axin2, is a marker for enriching and identifying MaSCs in vivo. Compared to the Axin2-lacZ−
cells reconstitute at higher frequencies. These data are compatible with the recent report that high expression of the Wnt co-receptor Lrp5 marks MaSCs (Badders et al., 2009
). It is likely that Axin2-lacZ+
cells overlap with Lrp5hi
cells and that Lrp5 expression would allow these cells to be capable of Wnt reception in the first place. Indeed, we detected higher levels of Lrp5 expression in freshly isolated Axin2-lacZ+
cells (). Reduced expression of hey1 in Axin2-lacZ+
cells in vivo is consistent with a previous report of Bouras et al., and supports the notion that inhibition of the Notch pathway in MaSCs enhances their stem cell potential.
In spite of the evidence for a role of Wnt pathway in MaSC regulation, it remained unknown whether Wnt proteins acted directly on mammary stem cells and controlled their self-renewal properties. In this study, we provide multiple lines of evidence that this is indeed the case. In cell culture, we find that MaSCs are dependent on Wnt proteins for long-term expansion and maintenance of self-renewal under defined conditions. In these experiments, Wnt does not act as a mitogenic growth factor, as the proliferation of the cells was not influenced by its presence. Rather, our data support a model of other known growth factors, such as EGF, as proliferative factors, while the Wnt protein prevents differentiation of the dividing cells and thereby leads to self-renewal. In line with this proposal, we find that the expression of Gata-3, a transcription factor that promotes differentiation of mammary stem cells into luminal cells, is suppressed by the Wnt signal (Figure S3C
). The combined use of Wnt, EGF and Matrigel has allowed us, for the first time, to expand MaSCs in cell culture, with full retention of their competence to regenerate an organ. This finding may have important implications for attempts to expand breast cancer stem cells (Al-Hajj et al., 2003
) in a clonal fashion, as well as stem cells from other tissues. Expanding adult stem cells in culture holds great promise for regenerative medicine but has in general been difficult due to the lack of stem cell markers and knowledge about the required growth factors.
The second line of evidence for a role of Wnt signals as stem cell factors comes from the use of Axin2LacZ/LacZ mutant cells. Axin2 encodes a negative regulator of the Wnt pathway. Unlike other repressors of Wnt signaling, such as Axin1, APC or GSK3, Axin2 is a target gene of Wnt signaling, implying that its expression and signal-dampening function are restricted to Wnt-exposed cells. Any consequence of Axin2 loss is therefore dependent on the presence of active Wnt proteins and indicates that cells showing a phenotype are actively receiving Wnt signals. In competitive repopulation experiments with wild type cells, MaSCs isolated from Axin2LacZ/LacZ mice show a marked increase in repopulating the mammary fat pad and regenerating a functional mammary gland, indicating that the propagation of MaSC in vivo is stimulated by external Wnt signals. While this result leaves open the possibility that Wnt acts as a proliferative signal, we suggest that it reflects the self-renewing ability of the Wnt protein, in part based on the increased ability of the Axin2lacZ/lacZ cells to generate colonies in cell culture (). The Axin2lacZ/lacZ hypermorphic phenotype is unique compared to mutations in other Wnt signaling components, which mostly lead to hyperplasia or even tumors. Moreover, mammary glands originating from Axin2lacZ/lacZ MaSCs have normal ratios of basal and luminal cells. In addition, MaSCs colonies cultured in the presence of Wnt appeared to have normal expression levels of the basal lineage marker K14 and the luminal marker K8, suggesting that Wnt signals promote the self-renewal of MaSCs without altering cell fate specification.
The role of Wnt in the mammary gland is in agreement with current models of stem cell regulation, where the ability to sustain a limited pool of stem cells is a hallmark of the niche (Morrison and Spradling, 2008
). This constraint is important in preventing unrestrained expansion of stem cells, which may otherwise lead to cancer (Clarke and Fuller, 2006
). Our study suggests that local Wnt signals act as the self-renewal factors in the adult mammary gland. Indeed, previous studies reported that several Wnts, including Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt6 and Wnt7b, are expressed in the epithelium and/or the mesenchyme in the matured virgin mice (Buhler et al., 1993
; Gavin and McMahon, 1992
; Huguet et al., 1994
; Lane and Leder, 1997
; Olson and Papkoff, 1994
; Weber-Hall et al., 1994
). At present, the precise location of the physiological niche for MaSCs remains unknown. It could be that the niche itself is dynamic as exemplified by the hematopoietic system (reviewed in (Kaplan et al., 2007
). MaSCs may have several niches as they progress through various developmental stages.