Breast cancer arises from breast epithelial cells that acquire genetic alterations leading to subsequent loss of tissue homeostasis. Several distinct epithelial subpopulations have been proposed, but complete understanding of the spectrum of heterogeneity and differentiation hierarchy in the human breast remains elusive. Here, we use single-cell mRNA sequencing (scRNAseq) to profile the transcriptomes of 25,790 primary human breast epithelial cells isolated from reduction mammoplasties of seven individuals. Unbiased clustering analysis reveals the existence of three distinct epithelial cell populations, one basal and two luminal cell types, which we identify as secretory L1- and hormone-responsive L2-type cells. Pseudotemporal reconstruction of differentiation trajectories produces one continuous lineage hierarchy that closely connects the basal lineage to the two differentiated luminal branches. Our comprehensive cell atlas provides insights into the cellular blueprint of the human breast epithelium and will form the foundation to understand how the system goes awry during breast cancer.

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SUMMARY After influenza infection, lineage-negative epithelial progenitors (LNEPs) exhibit a binary response to reconstitute epithelial barriers: activating a Notch-dependent ΔNp63/cytokeratin 5(Krt5) remodeling program or differentiating into alveolar type II cells (AEC2s). Here we show that local lung hypoxia, via hypoxia inducible factor (HIF1α), drives Notch signaling and Krt5pos basal-like cell expansion. Single cell transcriptional profiling of human AEC2s from fibrotic lungs revealed a hypoxic subpopulation with activated Notch, suppressed surfactant protein C (SPC), and trans-differentiation toward a Krt5pos basal-like state. Activated murine Krt5pos LNEPs and diseased human AEC2s upregulate strikingly similar core pathways underlying migration and squamous metaplasia. While robust, HIF1α-driven metaplasia is ultimately inferior to AEC2 reconstitution in restoring normal lung function. HIF1α deletion or enhanced Wnt/β-catenin activity in Sox2+ LNEPs blocks Notch and Krt5 activation, instead promoting rapid AEC2 differentiation and migration and improving the quality of alveolar repair.

Summary To investigate the role of immune cells in lung regeneration, we used a unilateral pneumonectomy model that promotes the formation of new alveoli in the remaining lobes. Immunofluorescence and single cell RNA- sequencing found CD115+ and CCR2+ monocytes and M2-like macrophages accumulating in the lung during the peak of type 2 alveolar epithelial stem cell (AEC2) proliferation. Genetic loss of function in mice and adoptive transfer studies revealed that bone marrow-derived macrophages traffic to the lung through a CCL2-CCR2 chemokine axis and are required for optimal lung regeneration, along with Il4ra-expressing leukocytes. Our data suggest that these cells modulate AEC2 proliferation and differentiation. Finally, we provide evidence that group 2 innate lymphoid cells are a source of IL13 that promotes lung regeneration. Together, our data highlight the potential for immunomodulatory therapies to stimulate alveologenesis in adults.

Injury-induced BMP signaling in the midgut negatively regulates intestinal stem cell division, whereas regional constitutive BMP signaling promotes copper cell differentiation.