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Figure 1. Historical model of the mammary epithelial cell differentiation hierarchy. Multipotent mammary stem cells (MaSCs) have the
ability to self-renew and give rise to all downstream lineages. Bipotent stem cells give rise to committed unipotent progenitor populations
that maintain basal/myoepithelial and luminal lineages. These unipotent cells are responsible for maintaining mature basal/myoepithelial
and luminal lineages in the postnatal gland. Two luminal progenitor populations have been reported, one giving rise to alveolar cells during
pregnancy and the other responsible for maintaining the mature ductal cells

Although lineage tracing allows for the visualization of cellular differentiation within the context of the
normal tissue environment, it is not without its own set of limitations. The ability to detect rare heterogeneous
populations of cells using genetic lineage tracing is highly dependent on recombination efficiency. High
variability in the extent of recombination not only exists between mouse models (i.e., K5-Cre versus K14-Cre)
but also between individual mice from the same strain . In addition, labeling efficiency varies significantly
[42]
throughout the ductal tree of a single mammary gland . Several explanations for this variability have been
[42]
postulated [8,10,42] . Inadequate strength and specificity of the promoter driving recombination could result
in inconsistent labeling. This may be particularly relevant to quiescent stem cells that are transcriptionally
uncommitted to a certain lineage. Additionally, inducible creERT2 models are reliant on accurate dosing of
Tamoxifen, a selective estrogen receptor (ER) modulator, to induce sufficient recombination. Since mammary
gland development is highly dependent on ER signaling, tamoxifen administration, even at low does, can
alter normal mammary gland biology and lead to false readouts .
[43]
Taken together, the results from both transplantation and lineage tracing studies have led to a highly
compartmentalized view of the mammary epithelial hierarchy in which fetal MaSCs give rise to a common
bipotent progenitor which differentiates into lineage-restricted and unipotent luminal and myoepithelial
progenitors. Unipotent progenitors then give rise to mature myoepithelial and luminal lineages (for detailed
reviews see Visvader and Stingl , 2014, and Yang et al. , 2017) [Figure 1]. Although transplantation and
[44]
[10]
lineage tracing methods have been fundamental to our understanding of mammary epithelial biology, it
has become increasingly evident that these methods are insufficient to fully characterize rare or highly
heterogeneous cell populations. Gene expression signatures on bulk isolates of the major epithelial
compartments including luminal progenitors, mature luminal, and MaSC-enriched basal epithelial cells,
have been generated . However, the utility of these signatures to identify critical molecular regulators and
[45]
markers of epithelial subsets has been limited due to the heterogeneity of each epithelial population in which
only a small fraction of cells expresses the same set of transcripts (as in the case of stem populations within
the basal fraction) [22,32,46] . As a single cell can give rise to an entire mammary gland, it is imperative that the