Department of Pediatrics, Section of Nutrition, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
Department of Pediatrics, Section of Hematology, Oncology, and Bone Marrow Transplant, University of Colorado Anschutz Medical Campus, Aurora, CO, USA.
J Mammary Gland Biol Neoplasia. 2020 Dec;25(4):367-387. doi: 10.1007/s10911-020-09466-z. Epub 2020 Nov 20.
Cells in human milk are an untapped source, as potential "liquid breast biopsies", of material for investigating lactation physiology in a non-invasive manner. We used single cell RNA sequencing (scRNA-seq) to identify milk-derived mammary epithelial cells (MECs) and their transcriptional signatures in women with diet-controlled gestational diabetes (GDM) with normal lactation. Methodology is described for coordinating milk collections with single cell capture and library preparation via cryopreservation, in addition to scRNA-seq data processing and analyses of MEC transcriptional signatures. We comprehensively characterized 3740 cells from milk samples from two mothers at two weeks postpartum. Most cells (>90%) were luminal MECs (luMECs) expressing lactalbumin alpha and casein beta and positive for keratin 8 and keratin 18. Few cells were keratin 14 basal MECs and a small immune cell population was present (<10%). Analysis of differential gene expression among clusters identified six potentially distinct luMEC subpopulation signatures, suggesting the potential for subtle functional differences among luMECs, and included one cluster that was positive for both progenitor markers and mature milk transcripts. No expression of pluripotency markers POU class 5 homeobox 1 (POU5F1, encoding OCT4) SRY-box transcription factor 2 (SOX2) or nanog homeobox (NANOG), was observed. These observations were supported by flow cytometric analysis of MECs from mature milk samples from three women with diet-controlled GDM (2-8 mo postpartum), indicating a negligible basal/stem cell population (epithelial cell adhesion molecule (EPCAM)/integrin subunit alpha 6 (CD49f), 0.07%) and a small progenitor population (EPCAM/CD49f, 1.1%). We provide a computational framework for others and future studies, as well as report the first milk-derived cells to be analyzed by scRNA-seq. We discuss the clinical potential and current limitations of using milk-derived cells as material for characterizing human mammary physiology.
人乳中的细胞是一种未被开发的潜在“液体乳腺活组织检查”资源，可用于以非侵入性方式研究哺乳期的生理学。我们使用单细胞 RNA 测序 (scRNA-seq) 鉴定了饮食控制的妊娠期糖尿病 (GDM) 且正常哺乳女性的乳汁来源乳腺上皮细胞 (MEC) 及其转录特征。本文描述了一种方法，该方法可通过冷冻保存协调乳汁收集、单细胞捕获和文库制备，以及 scRNA-seq 数据处理和 MEC 转录特征分析。我们全面描述了两位母亲产后两周的 2 个奶样中的 3740 个细胞。大多数细胞 (>90%)是表达乳白蛋白α和酪蛋白β、角蛋白 8 和角蛋白 18 的腔上皮细胞 (luMEC)。少数细胞是角蛋白 14 基底上皮细胞，还有一小部分免疫细胞 (<10%)。对聚类之间差异基因表达的分析确定了 6 个潜在的不同 luMEC 亚群特征，这表明 luMEC 之间可能存在微妙的功能差异，其中一个簇既表达祖细胞标记物，也表达成熟乳汁转录物。未观察到多能性标记物 POU 类 5 同源框 1 (POU5F1，编码 OCT4)、SRY 盒转录因子 2 (SOX2) 或 Nanog homeobox (NANOG) 的表达。这一观察结果得到了对来自三位饮食控制的 GDM 女性 (产后 2-8 个月) 的成熟乳样本中的 MEC 进行的流式细胞术分析的支持，表明基底/干细胞群体 (上皮细胞黏附分子 (EPCAM)/整合素亚基α 6 (CD49f) 极小 (0.07%)，祖细胞群体也很小 (EPCAM/CD49f，1.1%)。我们为其他人以及未来的研究提供了一个计算框架，并报告了首次通过 scRNA-seq 分析的乳汁衍生细胞。我们讨论了将乳汁衍生细胞用作表征人类乳腺生理学的材料的临床潜力和当前限制。
