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.

Fig. 1. Exploring the diversity between non-lactating mammary cells (NMCs) and lactation derived mammary cells (LMCs).

a Cells from non-lactating tissue (above) and human milk (below) were isolated using either mechanical dissociation and enzymatic digestion or centrifugation, for downstream analysis. b Mammary cells from both non-lactating breast tissue (top) or lactating milk cells (bottom) were cultured either in i 2D (n = 10, see Supplementary Fig. 2) or ii 3D, scale bar represents 250 μm. c Representative flow cytometric profiles of immune/stromal (Draq5⁺/CD45⁺), luminal (Draq5⁺/EpCAM⁺/CD49f^+/−) and myoepithelial cells (Draq5⁺/EpCAM⁻/CD49f⁺) from i NMCs and ii LMCs. d Schematic diagram for the scRNA-seq experimental set-up for cell samples from seven non-lactating participants and nine lactating females. e Uniform manifold approximation and projection (UMAP) dimensional reduction of the mammary cells reveals distinct clusters arising from NMCs and LMCs.

Fig. 2. Clustering analysis of non-lactating (NMC) and lactation-associated (LMCs) mammary epithelial cells reveals different subpopulations arising from different developmental stages.

a Five major epithelial clusters were identified in our data set consisting of NMC myoepithelial (MY), luminal hormone-responsive (HR) and luminal progenitor (LP) clusters and LMC major luminal clusters 1 and 2 (LC1 and LC2). b Uniform manifold approximation and projections (UMAPs) coloured by marker genes characterizing the various clusters. c Volcano plot displaying the findings of the differential gene expression analysis revealed 1640 genes more highly expressed in LC1 compared to 1782 genes highly expressed in LC2. Significant genes are in red with the top 10 being annotated. d Top 10 regulons significantly upregulated in each luminal cell cluster. e Significant regulons found in LCs. i UMAP of STAT5A regulon activation in luminal cells and ii genes associated with STAT5A regulon. iii UMAP of SOX10 regulon activation in luminal cells and iv genes associated with SOX10 regulon.

Fig. 3. Investigation of the stromal compartment of non-lactating and lactating mammary epithelial cells.

a Stromal cells were classified into vascular accessory (VA), endothelial (EN), fibroblasts (FB) and immune (IM) cells. b Canonical stromal markers were used to classify the different stromal subtypes where LMC only contained IM cells. c Sub-setting and re-clustering of IM cells revealed that both myeloid and lymphocytic lineages were sequenced from both NMC and LMCs. d CellChat analysis between luminal and immune cell subtypes identified in milk (i). ii Observed interactions between the cell types. iii All immune cell subtypes signal to LC1 via the EGF, MK and SPP1 signalling pathway (selected receptor–ligand pairs shown), whereas LC1 and LC2 luminal clusters from milk are signalling to milk immune cells via MHC-II, MHC-I, CSF and GRN signalling pathways (selected receptor–ligand pairs shown).

Fig. 4. Comparing lactation-derived mammary cell (LMC) luminal clusters (LCs) with all other non-lactating mammary cell (NMC) types reveals similarity to non-lactating luminal progenitor (LP) cells.

a Violin plots of the mammary cell scores for i hormone-responsive (HR, mature luminal), ii luminal progenitor (LP), iii stromal or iv myoepithelial cells across the major cell clusters identified in this study. b Differential gene expression analysis derived using quasi-likelihood negative binomial generalized log-linear models revealed 1146 genes highly expressed in LC LMCs compared to 922 genes more highly expressed in LP NMCs, as displayed by a volcano plot (for a full list, see Supplementary Dataset 7). c Important biological process gene ontology pathways that were annotated by the top 10% of genes significantly differentially expressed (false discovery rate corrected p value <1 × 10⁻⁸) and upregulated in either LC (left) or LP (right); for a full list, see Supplementary Dataset 8 and 9. d Key LP (left) or LC (right) genes expressed in NMC-LP, LMC-LC or all other NMC clusters; colours represent overall normalized gene expression and size equals cell proportions.