Single cell transcriptome sequencing of mammary stem cells in the pubertal mammary gland

The mammary gland is a highly dynamic organ that mainly develops during puberty. Based on morphology and proliferation analysis, mammary stem cells (MaSCs) are thought to be close to or reside in the terminal end buds (TEBs) during pubertal development. However, exclusive stem cell markers are lacking, and therefore the true identity of MaSCs, including their location, multiplicity, dynamics and fate during branching morphogenesis, has yet to be defined. To gain more insights into the molecular identity and heterogeneity of the MaSC pool, we performed single cell transcriptome sequencing of mammary epithelial cells micro-dissected from ducts and TEBs during puberty. These data show that the behaviour of MaSCs cannot be directly linked to a single expression profile. Instead, morphogenesis of the mammary epithelium relies upon a heterogeneous population of MaSCs that functions long-term as a single equipotent pool of stem cells. Ducts and terminal end buds were micro-dissected from the 4th and the 5th murine mammary gland at 5 weeks-of-age, dissociated into single cells, and FACS sorted. Single-cell transcriptomics was performed on live cells using an automated version of CEL-seq2 on live, FACS sorted cells. The StemID algorithm was used to identify clusters of cells corresponding to basal and luminal cells types derived from ducts and terminal end buds.乳腺是一种高度动态的器官，其发育主要集中于青春期阶段。结合形态学与增殖分析结果，青春期发育过程中，乳腺干细胞（mammary stem cells, MaSCs）被认为靠近或定位于末端芽基（terminal end buds, TEBs）。然而目前缺乏特异性的干细胞标记物，因此乳腺干细胞的真实身份——包括其在分支形态发生过程中的定位、多态性、动态变化及细胞命运——仍有待明确。为深入解析乳腺干细胞群体的分子特征与异质性，我们对青春期时期从导管和末端芽基显微解剖分离的乳腺上皮细胞开展了单细胞转录组测序。本研究数据表明，乳腺干细胞的行为无法直接通过单一表达谱进行表征。相反，乳腺上皮的形态发生依赖于异质性的乳腺干细胞群体，该群体作为一个等潜能的干细胞池长期发挥功能。我们从5周龄小鼠的第4和第5乳腺中显微解剖分离导管与末端芽基，将其解离为单细胞后通过荧光激活细胞分选（FACS）进行分选。我们采用自动化版本的CEL-seq2技术对经FACS分选的活细胞开展单细胞转录组分析，并借助StemID算法鉴定了源自导管和末端芽基的基底细胞与管腔细胞类型的细胞簇。