Whole transcriptome sequencing of bovine mammary epithelial subpopulations

The bovine mammary gland has a heterogeneous epithelial population which comprises terminally differentiated luminal and myoepithelial cells but also bipotent or lineage restricted progenitors and adult stem cells. The aim of our study was to use a novel surface marker (P-Cadherin) to characterize different mammary subpopulation, to sort adult stem cells with better enrichment and to perform whole RNA-seq among sorted population to identify biological processes or molecular functions associated gene enrichment. We found that only the CD49fhigh/PCadherinneg was enriched for adult mammary stem cells, while in other fractions we could detect luminal progenitors. Analysis of functional enrichments showed that in the stem cell compartment proliferation associated genes were downregulated, while genes relating to adhesion to ECM and to other neighboring cells were upregulated. Overall design: Bovine mammary cells were collected at the slaughter house from 3 animals. Isolated mammary epithelial cells of each animal were sorted into 4 fraction according to their expression of CD49f and P-Cadherin. RNA was extracted from cells for each fraction and sequence using Illumina TruSeq with a depth/coverage of 30M reads per sample. Data from the 3 animals were then used as replicates to identify differentially expressed genes among the 4 mammary subpopulations.

牛乳腺存在异质性上皮细胞群，其不仅包含终末分化的腔上皮细胞（luminal cell）与肌上皮细胞（myoepithelial cell），还含有双潜能或谱系受限的祖细胞以及成体干细胞。本研究旨在利用一种新型表面标志物P-钙黏蛋白（P-Cadherin）对不同乳腺亚群进行表征，通过更高效的富集流程分选得到成体干细胞，并对分选后的细胞群体开展全转录组RNA测序（RNA-seq），以识别与生物学过程或分子功能相关的基因富集特征。研究结果显示，仅CD49f高表达/P-钙黏蛋白阴性（CD49fhigh/P-Cadherinneg）的细胞组分富集了牛乳腺成体干细胞，而其余组分中可检测到腔上皮祖细胞。功能富集分析表明，在干细胞亚群中，与细胞增殖相关的基因表达下调；而与细胞外基质（Extracellular Matrix, ECM）黏附及邻近细胞间黏附相关的基因表达上调。实验整体设计：从屠宰场采集3头实验动物的乳腺组织，分离得到每头动物的乳腺上皮细胞后，依据CD49f与P-钙黏蛋白的表达水平将其分为4个组分；提取各组分细胞的总RNA，采用Illumina TruSeq建库测序方案进行测序，每个样本的测序深度/覆盖度达30M读段（reads）；以3头动物的测序数据作为生物学重复，对4个乳腺亚群间的差异表达基因进行鉴定。