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paths represented throughout development, underscoring the immense diversity and flexibility that likely
underlies mammary gland development and gives rise to breast cancer heterogeneity. Here we discuss the
recent studies utilizing scRNA-sequencing of mammary epithelium and how these new findings have shifted
our current perspectives of mammary gland development and breast cancer.

HISTORICAL PERSPECTIVE OF THE MAMMARY EPITHELIAL HIERARCHY
In 1959, DeOme and colleagues developed the mammary transplantation assay . Using this technique,
[15]
transplantation of murine ductal fragments [16,17] or dissociated suspensions of normal mammary epithelium [18]
into the cleared fat pad of a recipient mouse resulted in the regeneration of a complete ductal structure. Smith
and Medina then reported that morphologically distinct cells with regenerating capacity could be found
throughout the entire mammary tree and persisted throughout pregnancy, lactation, and involution . Both
[19]
the frequency and regenerative ability of these cells was not affected by age or reproductive history [19,20] .
Later studies demonstrated that mammary gland reconstitution was achievable upon transplantation of a
single cell. This was first reported by Kordon and Smith, using serial transplantation and limiting dilution
assays of mammary epithelium labeled with unique viral insertions generated by mouse mammary tumor
virus (MMTV) . Similarly, by labeling cells with a LacZ transgene, Shackleton et al. confirmed this
[21]
[22]
finding and demonstrated that stem cell populations were enriched in the basal epithelial fraction (defined
by Lin CD29 CD24 cell surface markers). Additionally, cells from reconstituted glands harbored the same
+
hi
-
repopulating activity of the original and were capable of fully differentiating into milk-producing alveolar
cells during pregnancy. These studies implicated the existence of a long lived multipotent mammary stem
cell population (MaSC), capable of giving rise to all downstream lineages within the gland.
Following the disclosure of MaSCs, the search for molecular markers specific to the stem cell population
became a major focus within the field. Transplantation of luminal vs. basal epithelial populations revealed
that cells exhibiting stem-like properties were located within the basal fraction and could be enriched by
FACS sorting using CD49f CD29 CD24 +/mod EpCAM Sca1 expression as markers [22-25] . Additionally,
+
hi
hi
low
expression of s-SHIP , LGR5 [27-29] , Lrp5/6 , and Axin2 enriched for populations with repopulating
[26]
[30]
[31]
activity upon transplantation; however, stem cell activity was not exclusive to these subsets and the degree
to which these markers may enrich for overlapping stem cell populations was not clear . Difficulty
[10]
surrounding the identification of MaSC markers was due, in part, to the rarity of MaSCs in the adult gland.
The frequency of repopulating cells was found to be higher during embryogenesis (~7% of total cells by E18
compared to ~2%-5% frequency in the adult gland) [22,23,32,33] ; however, enriched populations of fetal (f)MaSCs
displayed altered gene expression profiles from their adult counterparts further complicating the isolation
and characterization of a multipotent population .
[32]
Transplantation studies demonstrated the existence of a stem cell entity that was capable of multi-lineage
differentiation within the adult gland. Yet, various studies employing lineage tracing analyses led to confusion
surrounding these findings. While some groups provided evidence to support multipotent cells, others reported
that only unipotent cells remain after birth. Early lineage tracing experiments by Van Keymeulen et al. , used
[34]
the promoters of the keratin 14, 5, and 8 (K14, K5, K8) genes, to drive tracer expression and track both luminal
(K8) and myoepithelial (K14, K5) lineages. This analysis demonstrated that prior to birth, K14+ cells were
multipotent; however, after birth, these cells became unipotent, only giving rise to myoepithelial cells. Cells
labeled by the K8 promoter revealed a second unipotent population responsible for maintaining the luminal
lineage after birth. These unipotent populations supported epithelial expansion during puberty and pregnancy
underscoring their long lived capacity within the gland. Additionally, lineage tracing at the single cell level
revealed that unipotent populations were dispersed throughout the ductal tree, estimating that within each major
duct, ~20 luminal and ~15 basal unipotent cells contribute to ductal expansion during puberty . Furthermore,
[35]
by employing clonal analyses at saturation using DOX-inducible systems to trace luminal (K8rtTA-CreTetO/