Differentiation dynamics of mammary epithelial cells revealed by single-cell RNA-sequencing

The mammary gland is a unique organ as it undergoes most of its development during puberty and adulthood. Characterising the hierarchy of the various mammary epithelial cells and how they are regulated in response to gestation, lactation and involution is important for understanding how breast cancer develops. Recent studies have used numerous markers to enrich, isolate and characterise the different epithelial cell compartments within the adult mammary gland. However, in all of these studies only a handful of markers were used to define and trace cell populations. Therefore, there is a need for an unbiased and comprehensive description of mammary epithelial cells within the gland at different developmental stages. To this end we used single cell RNA sequencing (scRNAseq) to determine the gene expression profile of individual mammary epithelial cells across four adult developmental stages; nulliparous, mid gestation, lactation and post weaning (full natural involution). Our data from 25,010 individual cells identifies 8 distinct mammary epithelial cell populations and allows their hierarchical structure across development to be charted. Interestingly, the effect of gestation and lactation appeared to be more pronounced for some cell types. For example, our analysis revealed a cluster of luminal progenitor cells in post involution glands, which is distinct from progenitors found in nulliparous glands. The data also showed that few clusters could be fully characterized by a single marker gene. We argue instead that the epithelial cells – especially in the luminal compartment – should rather be conceptualized as being part of a continuous spectrum of differentiation. This view highlights the plasticity of the tissue and might help to explain some of the conflicting results from lineage tracing studies. Overall design: We compared the gene expression profile at single-cell level of four adult developmental time-points of the murine mammary gland. Tissues were harvested at gestation day 14.5 (G), lactation day 6 (L) and day 11 post natural weaning of the pups (PI). Tissue from NP females was harvested at 8 weeks of age. Two individual mice per developmental time point were used in the study. Lymph node divested mammary glands (excluding the cervical pair) were dissected from the mice and mechanically dissociated. The finely minced tissue was transferred to a digestion mix consisting of DMEM/F12 (Gibco) + 10 mM HEPES (Gibco) + collagenase (Roche) + 200 U ml-1 hyaluronidase (Sigma) + gentamicin (Gibco) and incubated for 3 hours at 37° C. From the single-cell suspensions, epithelial cells were sorted based on EpCam. Cells that were positive for CD31, CD45 or Ter119 were excluded.