Single cell RNA-seq reveals heterogeneity and hierarchy of mouse mammary epithelia

The mammary epithelia are mainly composed of two distinct lineages, the basal and luminal cells. Due to the limitation of traditional transcriptome analysis of bulk mammary cells, the hierarchy and heterogeneity of mammary cells within these two lineages remain unclear. Hence, we have performed single cell RNA-seq on mammary epithelial cells of virgin and pregnant mice. Based on the respective gene expression profiles, we found mammary cells are highly heterogeneous and can be classified into distinct subgroups with signature markers. Besides, we identified and verified a rare CDH5+ subpopulation of basal lineage as quiescent mammary gland stem cells (MaSCs). Furthermore, knockout of Cdh5 in mouse mammary cells via CRISPR-Cas9 significantly impairs cell stemness and promotes the cell differentiation to luminal lineage. In addition, via pseudo-temporal analysis, we reconstructed the mammary epithelia developmental trajectory and revealed the relevant changes of gene expression and biological functions of mammary cells along the developmental process. Basal and luminal cells form mammary glands of virgin or pregnant (at day 12) FVB mice were FACS-purified for single cell RNA-seq.乳腺上皮主要由两种不同的细胞谱系构成，即基底细胞（basal）与腔上皮细胞（luminal）。由于传统乳腺细胞批量转录组分析存在局限性，这两种谱系内的乳腺细胞层级结构与异质性仍未明确。因此，本研究对未受孕及受孕小鼠的乳腺上皮细胞开展了单细胞RNA测序 (single cell RNA-seq) 实验。基于各自的基因表达谱，我们发现乳腺细胞具有高度异质性，可通过特征标记基因划分为多个不同的亚群。此外，我们鉴定并验证了一类罕见的基底谱系CDH5+亚群，其为静息态乳腺干细胞 (mammary gland stem cells, MaSCs)。进一步，本研究通过成簇规律间隔短回文重复序列相关蛋白9 (CRISPR-Cas9) 敲除小鼠乳腺细胞中的Cdh5基因，结果显示其显著削弱了细胞干性，并促进细胞向腔上皮谱系分化。此外，通过拟时间分析 (pseudo-temporal analysis)，我们重构了乳腺上皮的发育轨迹，并揭示了乳腺细胞在发育过程中基因表达与生物学功能的相关变化。本研究从未受孕及受孕12天的FVB小鼠乳腺中分离的基底细胞与腔上皮细胞，经荧光激活细胞分选 (fluorescence-activated cell sorting, FACS) 纯化后用于单细胞RNA测序实验。