Transcriptional changes in the mammary gland during lactation revealed by single cell sequencing of cells from human milk

Abstract

Under normal conditions, the most significant expansion and differentiation of the adult mammary gland occurs in response to systemic reproductive hormones during pregnancy and lactation to enable milk synthesis and secretion to sustain the offspring. However, human mammary tissue remodelling that takes place during pregnancy and lactation remains poorly understood due to the challenge of acquiring samples. We report here single-cell transcriptomic analysis of 110,744 viable breast cells isolated from human milk or non-lactating breast tissue, isolated from nine and seven donors, respectively. We found that human milk largely contains epithelial cells belonging to the luminal lineage and a repertoire of immune cells. Further transcriptomic analysis of the milk cells identified two distinct secretory cell types that shared similarities with luminal progenitors, but no populations comparable to hormone-responsive cells. Taken together, our data offers a reference map and a window into the cellular dynamics that occur during human lactation and may provide further insights on the interplay between pregnancy, lactation and breast cancer.

Cellular and transcriptional diversity over the course of human lactation

“…These subsets agree with other datasets describing scRNA-seq on hBM in smaller cohorts on the basis of component cell clusters and genes expressed in these clusters with some exceptions ( SI Appendix , Fig. S3 A – E ) ( 15 , 27 , 30 ). Due to increased cell numbers in this dataset, we identified more immune clusters (seven) than previous studies, which identified two or three clusters, adding dendritic cells, a second macrophage cluster, and eosinophils to these previously identified.…”

“…4 B and SI Appendix , Figs. S3 B , C , and F and S8 F ) ( 15 , 27 ). We found that all epithelial subclusters expressed genes related to milk synthesis—such as LALBA , CSN2 , XDH , and FASN , as well as canonical luminal cell markers ( EPCAM , KRT18 , KRT19 )—suggesting a clear luminal lineage and role in milk production ( Fig.…”

“…hBM contains live cells, which are thought to enter the breast through exfoliation during the process of breastfeeding, thereby providing an opportunity to study lactational cells ( 12 ). Cells from hBM are viable and can be cultured, and hBM immune cells were shown to transfer to offspring bloodstream and tissues in animal models ( 12 , 15 – 17 ). The investigation of these live cells provides both noninvasive surveillance of the cells in the mammary mucosa and allows for a more detailed understanding of their roles in infant development ( 12 , 17 , 18 ).…”

“…To date, several studies have used either bulk ( 12 , 23 – 26 ) or single-cell RNA-sequencing (scRNA-seq) to study the transcriptome of hBM in small cohorts ( 15 , 27 ). These studies have revealed subsets of epithelial cells in hBM, including progenitor luminal cells, and genes that change in bulk over the course of lactation.…”

“…Bulk analysis, however, limits our ability to delineate key cell states and uncover specialized cell phenotypes ( 28 , 29 ). Previous scRNA-seq analyses, meanwhile, havebeen limited by low sample numbers, small donor pools, and few time points, thereby decreasing the ability to characterize the cross-donor heterogeneity of breast milk longitudinally ( 15 , 26 , 30 ). Longitudinal studies of other milk factors have revealed dynamic shifts in hormone concentrations, cytokine levels, and overall protein content up to 3 mo postpartum, and suggest that most components decrease in concentration early in lactation ( 31 – 36 ).…”