RNA-sequencing of milk cells extracted from pre-partum secretions and longitudinally from mature human milk across the first year of lactation

Changes in mammary cell behavior mediating normal breast development during pregnancy and lactation are poorly understood due to limited availability of breast biopsies during this time. Human milk contains a hierarchy of cells including stem cells, mature milk producing cells (lactocytes) and myoepithelial cells. Here we non-invasively sampled the total epithelial cell population of the lactating mammary gland from mature HM collected from healthy mother/infant dyads during the first year postpartum, and explored temporal changes in the mammary cell transcriptome using RNA sequencing. Comparisons were done with mammary secretions from late pregnancy from the same women and with purchased resting mammary tissue. Distinct gene signatures were found for the different mammary developmental stages examined. Cell adhesion pathways were differentially regulated between the resting gland and pregnancy, whereas immune cell signaling and morphogenesis/cancer pathways differed between lactation and pregnancy or the resting gland, respectively. The transcriptome of lactation remained consistent in the first year postpartum in these successfully lactating women. The gene signatures characteristic of HM cells confirmed lactation genes previously reported in animal models and the HM fat globule. This study identifies key genes and molecular pathways undergoing controlled regulation as the mammary gland transitions from a quiescent into a functional organ, providing experimental targets for the molecular investigation of mammary gland pathologies. Overall design: The human milk cell transcriptome was generated from cells extracted from pre-partum samples and mature milk at months 1, 3, 6 and 12 of lactation.

由于妊娠与泌乳阶段乳腺活检样本获取受限，当前学界对介导孕期及哺乳期正常乳腺发育的乳腺细胞行为变化仍知之甚少。人乳中包含一套细胞谱系，涵盖干细胞、成熟泌乳细胞（lactocytes）及肌上皮细胞。本研究通过无创采样，从产后第一年的健康母婴对中采集的成熟人乳（HM）中分离得到泌乳期乳腺的全部上皮细胞群体，并借助RNA测序（RNA sequencing）技术解析乳腺细胞转录组的时序变化。研究同时以同受试者孕晚期的乳腺分泌物及市售静息态乳腺组织作为对照样本开展比对分析。本研究在不同乳腺发育阶段样本中均检测到独特的基因表达特征。静息态乳腺与妊娠状态的乳腺间，细胞黏附通路呈现差异调控；而泌乳期与妊娠期、静息态乳腺分别在免疫细胞信号通路及形态发生/癌症通路上存在显著差异。在成功完成泌乳的受试者中，产后第一年的泌乳期乳腺转录组整体保持稳定。人乳细胞的特征性基因表达谱验证了此前在动物模型及人乳脂肪球中报道的泌乳相关基因。本研究明确了乳腺从静息态向功能态转变过程中受精准调控的关键基因与分子通路，为乳腺疾病的分子机制研究提供了实验靶点。实验整体设计：本研究的人乳细胞转录组数据来源于孕晚期样本以及泌乳第1、3、6、12个月时采集的成熟人乳中提取的细胞。