The two hallmark properties of any stem cell population are the ability to differentiate into multiple cell lineages and the ability to self-renew to produce more stem cells 7
. While most adult cells are unable to divide, existing in a stage of terminal differentiation, tissue-specific adult stem cells retain the ability to divide and produce the multiple cell types within the organ from which they are derived. In the mouse mammary gland, the first clues for the existence of an adult stem cell of the mammary gland came from the work of DeOme et al.
in the 1950s when it was shown that small pieces of mammary epithelium, when transplanted into recipient fat pads cleared of their endogenous epithelium, could expand and differentiate into a fully functional reconstituted gland 8
. Cells from nearly any location within the mammary gland, or during any developmental stage, could repopulate a mammary gland 9
. Subsequent experiments in both humans and mice demonstrated that this reconstitution ability was due to the activity of a single cell. Tsai et al.
suggested clonal expansion was responsible for human mammary gland growth based on X-chromosome inactivation patterns 10
, while Kordon and Smith demonstrated through retroviral tagging that mouse mammary glands were the progeny of a single cell 11
. Based on this evidence, a number of experimental approaches were undertaken to identify and purify MaSCs based on their biological or morphological properties.
One strategy to isolate MaSCs relies on a feature believed to be (although not universally accepted as) a key mechanism of DNA replication during stem cell division. As certain adult stem cells divide, they preferentially retain one of their DNA strands throughout multiple divisions in order to protect against the formation of deleterious mutations that occur during DNA replication 12, 13
. By performing pulse-chase experiments with DNA labels, Smith et al.
showed there was a population of cells within the mammary gland which retained their DNA label through asymmetric segregation of DNA strands. These cells were still actively dividing and featured stem cell characteristics 14
. Roughly 30-40% of the cells that retained their DNA label also expressed receptors for the reproductive hormones estrogen and progesterone 15
. As an early alternative approach to label retention, the heterogeneity of morphological characteristics of mammary epithelial cells was exploited to try to enrich for cells with stem cell characteristics. Pale cells with low cellular complexity (i.e. few cytoplasmic organelles) were shown to express the properties of MaSCs in differentiating conditions 9
A major limitation of both the morphological and the label retention methods is that they do not lend themselves to the easy isolation of large numbers of relatively pure MaSC populations for use in in vitro
or in vivo
assays. As such, these methods did not definitively show that a single label-retaining cell or pale cell could reconstitute a fully functional gland in vivo
, which is the gold standard for stem cell assays. A better approach to isolate putative MaSCs involved the simple isolation of different cell populations based on the expression of surface marker proteins from dissociated mammary gland preparations using fluorescence-activated cell sorting (FACS). An initial marker that showed some promise was stem cell antigen-1 or Sca1. Sca1+
cells were shown to be a subpopulation of the label-retaining epithelial cells, and when isolated they showed a degree of in vivo
reconstitution ability. However, subsequent studies would identify markers which could enrich for MaSCs to a much higher degree of purity, and MaSCs identified by other methods have been shown to be Sca1low/− 7, 16
In 2006, mouse MaSCs were identified based on the expression of CD24 (heat-stable antigen) and high expression of either CD29 (β1-integrin) or CD49f (α6-integrin) 7, 17
. A single Lin−
cell was able to reconstitute an entire mammary gland in vivo
. The CD29 protein is not just a surrogate marker for MaSCs but is actually functionally important, as CD29 ablation in the basal compartment reduced MaSC activity 18
. Multi-lineage differentiation of progenitors into luminal and myoepithelial cells was confirmed via histological analysis and a variety of in vitro
differentiation assays. The second hallmark of stem cells, self-renewal, was confirmed via the observation of clonal gland outgrowth during serial gland reconstitution experiments. With respect to previous markers of stemness, Sca1 did not further enrich for the MaSCs, but the newly isolated MaSCs did seem to retain their DNA label 7
. Based on expression profiling and histological staining, the remaining non-stem cell fraction of the Lin−
populations represents basal/myoepithelial cells. Downstream progenitor and mature luminal cells are primarily observed in the CD24+
fraction. A specific luminal progenitor subpopulation of the Lin−
population has been identified based on strong expression of CD61 19
. While the lineage of cells that differentiate to form the mammary gland has not been as well characterized as systems such as the colon, a more detailed description of the hierarchy of cells within the mammary gland can be found in the recent review by Visvader and Smith 20
MaSCs are important for the two main growth phases of the mammary gland: the ductal elongation during pubertal expansion and the lobuloalveolar expansion during pregnancy. However, it is unknown whether the same population of cells with a high degree of plasticity can perform either of these functions depending on the local hormonal and growth cues, or if MaSCs begin to differentiate early and are programmed to perform only one of the two functions. There is evidence in other adult stem cell systems for the existence of two functionally distinct stem cell populations within one tissue 21
. Several lines of evidence support the assertion that this may be the case in the mouse mammary gland as well. Based on label retention studies, it was discovered that putative MaSCs existed in both basal and luminal locations 15
. With the subsequent identification of better surface marker profiles to efficiently purify MaSCs, stronger evidence emerged in parallel for the existence of distinct MaSC populations. In the MaSC fraction based on CD24 and CD49f staining, many cells expressed the basal maker K14. However, other cells expressed the luminal marker K18. It did not seem though that cells expressed both of these markers, suggesting that these cells might reside in distinct locations 17
. A luciferase-based transgenic mouse model for MaSC activity did reveal luciferase-expressing cells in both basal and myoepithelial locations 22
. Notably, when the MaSC marker CD29 is deleted from the basal compartment of the mammary gland, the mammary epithelial cells can no longer reconstitute a new mammary gland, but they can form alveoli late in pregnancy 18
, suggesting a distinct MaSC population. Similar results were also obtained when the Wnt receptor LPR5 was deleted 23
. A recent study showed that using a GFP reporter driven by the s-SHIP promoter, GFP+
replicating “active” MaSCs can be identified in cap cells in puberty and basal alveolar bud cells in pregnancy, but not in adult virgin animals, or in mammary tissues during lactation or involution stages 16
. Future characterizations will help to better understand whether or not distinct MaSC populations exist, and how they are controlled by their local micro-environmental cues.
With respect to the human mammary gland, identification of authentic MaSCs is a greater challenge because of the difficulty in obtaining normal tissue samples and the lack of an ideal in vivo
reconstitution system. Nevertheless, various attempts have been made to characterize human MaSCs both in vitro
and in vivo
. By following similar methods of growing primary neural cells in non-adherent conditions which resulted in the formation of neural stem cell-enriched “neurospheres”, mammary stem/progenitor cells could be enriched by forming “mammospheres” 24
. This method was further refined (for both human and mouse cells) by staining the mammary epithelial cells with the lipophilic dye PKH26 and selecting for cells that were slow dividing and retained this label during mammosphere growth. These cells were shown to have MaSC function in humanized mouse mammary glands in vivo 25
. An alternative isolation method was shown later by sorting cells based on the surface maker profile of Lin−
and suspending these cells with irradiated human fibroblasts in a collagen gel and then implanting them under the kidney capsule of estrogen/progesterone-treated mice 26
. It was subsequently shown that this same population of cells could differentiate into mammary gland structures in mouse mammary glands when transplanted with supporting fibroblasts 27
. Expression profiling of human and mouse MaSCs-enriched populations has shown a significant degree of conservation in gene expression across species 28
, providing validity to using the more readily accessible mouse model. Based on these enrichment methods for both human and mouse MaSCs, subsequent experiments have begun to elucidate the mechanisms by which MaSCs are controlled through various signaling pathways.