The mammary stem cell conundrum: is it unipotent or multipotent?

Exploring the normal biology and regulation of stem cells has the promise to yield insights into the etiological roots and survival of breast cancer cells. Many studies have supported the existence of a multipotent mammary stem cell that regenerates all aspects of glandular development. However, Van Keymeulen and colleagues (2011) illustrated the presence of lineage-restricted unipotent stem cells that self-renew and collaborate in postnatal mammary development, whereas multipotent stem cells were found only during embryonic mammogenesis. This prompts a re-evaluation of currently accepted mammary stem cell dynamics and conceivably its impact on the evolution of different breast cancer subtypes.

Th e capacity of the mammary gland for rapid growth and regeneration is attributed to mammary stem cells (MaSCs). Mammopoiesis initiates in the embryo but the majority of glandular development occurs during puberty. Recurrent reproductive cycles elicit transient but signi ficant alveolar epithelial expansion, whereas pregnancy involves heightened lobuloalveologenesis and lactational diff erentiation. Deome and colleagues [1] were the fi rst to show that tissue transplants from several portions of the gland form an entire gland in vivo in epithelium-divested fat pads, suggesting the existence of MaSCs. Trans plantation of tissue fragments or dis persed cells has since become a routine functional assay in the mammary gland fi eld. Limiting-dilution trans plants of cells have demonstrated the capacity of single cells to clonally expand to form a functional mammary gland and self-renew in serial transplants, supporting the existence of a multipotent MaSC [2].
Bilayered mammary ducts are composed of inner luminal epithelial and outer basal/myoepithelial cells. When mammary cells are purifi ed by fl uorescenceactivated cell sorting (FACS) using surface markers that segregate luminal (CD24 med/+ CD49f lo /CD29 lo ) and basal (CD24 med/+ CD49f hi /CD29 hi ) cells and are transplanted, basal cells generate robust functional mammary outgrowths whereas luminal cells lack this capacity [3,4]. Indeed, a single sorted basal cell has been shown to generate an entire mammary gland constituting ducts and alveoli and serially transplant in vivo. Th is illustrates the multipotent and self-renewal capacity of the basal MaSC. Injections of sorted/unsorted cells in limiting dilutions allow deductions of MaSC numbers in varying experimental conditions [5,6]. Interestingly, human breast stem cells capable of in vivo reconstitution were also found in basal cells (CD49f + EpCAM neg-low ) but not in luminal cells [7].
Van Keymeulen and colleagues [8] reported the existence of lineage-restricted unipotent stem cells in both luminal and basal epithelial populations and challenged prevailing work on the contribution of multipotent MaSCs to postnatal gland development. Th e authors performed lineage-tracing experiments of embry onic, pubertal, adult, pregnant, and involuting mammary glands, employing K14-rtTA/TetO-Cre/Rosa-YFP and K5-CreER/Rosa-YFP mice for tracking basal cells and K8-CreER/Rosa-YFP and K18-CreER/Rosa-YFP mice for tracking luminal cells. Induction of K14-driven YFP in embryos led to labeling of both luminal and basal cells at puberty, implying that embry onic K14 + cells are multipotent. Postnatal YFP induction showed exclusive labeling of basal cells that clonally expanded during puberty and pregnancy. Similar results were obtained with K5-CreER, indicating that K14 + /K5 + cells are unipotent and do not contribute to luminal cell progeny. In contrast, YFP driven by K8/K18 luminal promoters labeled only luminal cells that clonally expanded in K8-CreER mice but not in K18-CreER mice.
Th e diff erentiation potential of both epithelial lineages was determined by using transplantation assays. Unsorted cells from basal-specifi c Cre lines generated outgrowths in which YFP + cells were predominantly

Abstract
Exploring the normal biology and regulation of stem cells has the promise to yield insights into the etiological roots and survival of breast cancer cells. Many studies have supported the existence of a multipotent mammary stem cell that regenerates all aspects of glandular development. However, Van Keymeulen and colleagues (2011) illustrated the presence of lineage-restricted unipotent stem cells that self-renew and collaborate in postnatal mammary development, whereas multipotent stem cells were found only during embryonic mammogenesis. This prompts a re-evaluation of currently accepted mammary stem cell dynamics and conceivably its impact on the evolution of diff erent breast cancer subtypes.

© 2010 BioMed Central Ltd
The mammary stem cell conundrum: is it unipotent or multipotent?

V I E W P O I N T
*Correspondence: rkhokha@uhnresearch.ca 1 basal, although YFP + clones were also rarely seen in luminal cells. YFP + cells from the luminal-specifi c Cre line remained restricted to the luminal layer similar to that observed in the intact gland. Th is YFP chimerism was preserved in secondary grafts that the authors cite as evidence of self-renewal in these unipotent cells. When FACS-purifi ed YFP + basal or luminal cells were transplanted, basal, but not luminal, cells were capable of reconstituting a mammary gland, recapitulating previous fi ndings that originally defi ned multipotent basal MaSCs. In mixing experiments, reducing the luminal/basal cell ratio increased the potential of basal cells to generate luminal cells. Th e study argues that the experimental setting of the transplantation assay forces diff erentiation of basal MaSCs into both epithelial lineages while the intact gland relies on lineage-restricted unipotent stem cells.
Lineage-tracing experiments have proven valuable in uncovering new insights into stem cells in other tissue contexts such as the skin and intestine, and Van Keymeulen and colleagues [8] have harnessed this technique to address a fundamental question about stem cell diff eren tiation potential. Th eir fi ndings bring new perspective to the MaSC fi eld. Previous work has adhered to transplantation as a reliable gold-standard stem cell assay. Th e lack of detection of a multipotent basal MaSC in lineagetracing experiments of the intact postnatal gland now thrusts scepticism on the contribution of basal multipotent stem cells to postnatal gland regeneration. Although the study broadens our conceptualization of the MaSC and raises questions about the relevance of transplantation as a functional stem cell readout, facets of this study warrant further discussion. Stem cells in other tissues such as the hematopoietic system have been shown to possess varying degrees of self-renewal, including short-term, long-term, and even intermediate repopulat ing potentials [9]. Th e basal and luminal unipotent cells were shown to maintain their chimerism in secondary grafts that the authors demonstrate to be selfrenewal but whether these cells have short-or long-term potential is unclear. Second, luminal cells, though unipotent, were unable to regenerate a gland independent of basal cells in transplantations, whereas basal cells possess this intrinsic property, implying a hierarchy of stem cell capacity. Luminal cells clearly rely on basal cells for cues that are likely paracrine to facilitate their unipotency in the intact gland. Furthermore, several groups have identifi ed dual-positive cells (K14 + with K8 + / K18 + /K19 + ) in the human breast, suggesting the existence of bipotent progenitors and candidate stem cell zones [10][11][12]. Dual-positive cells, as well as markers that tag basal cells, have also been noted in situ in the murine gland within the luminal compartment of terminal end buds and alveoli [13,14]. Van Keymeulen and colleagues [8] found Lgr5 + cells, an intestinal stem cell marker, predominantly in basal mammary cells but additionally in luminal cells. It is conceivable that these are indicative of a bipotent cell population but this possibility has been overlooked in their lineage-tracing experiments, perhaps because of low K14/K5 expression levels in dual-positive cells.
Identifying epithelial populations that have regenerative capacity and defi ning their extrinsic and intrinsic regulatory mechanisms are relevant to understanding not only the normal development but also the etiology of breast cancers in which transformed cells share properties akin to those of stem cells, notably self-renewal [15]. Given the heterogeneity of breast cancers, it is surmised that specifi c mammary cells are cells of origin for diff erent cancer subtypes. It will be important to understand whether the likely targets of transformation are initially unipotent or multipotent and whether the mutation repertoire infl uences their diff erentiation potential and self-renewal capacity.

Competing interests
The authors declare that they have no competing interests.