Developmental changes in the in vitro activated regenerative activity of primitive mammary epithelial cells

Many normal adult tissues contain rare stem cells with extensive self-maintaining regenerative potential. During development, the stem cells of the hematopoietic and neural systems undergo intrinsically specified changes in their self-renewal potential. In the mouse, mammary stem cells with transplantable regenerative activity are first detectable a few days before birth. They share some phenotypic properties with their adult counterparts but are enriched in a subpopulation that displays a distinct gene expression profile. Here we show that fetal mammary epithelial cells have a greater direct and inducible growth potential than their adult counterparts. The latter feature is revealed in a novel culture system that enables large numbers of mammary stem cells with serially transplantable activity as well as in vitro clonogenic progenitors to be produced within 7 days from single fetal or adult input cells. We further show that these responses are highly dependent on novel factors produced by fibroblasts. These findings provide new avenues for elucidating mechanisms that regulate normal mammary epithelial stem cell properties at the single-cell level, how these change during development, and how their perturbation may contribute to transformation. We used microarrays to compare the transcriptome of E18.5 fetal and adult MRU-enriched mammary cells. Three biological replicates each of CD31-CD45-Ter119-BP-1-EpCAM+CD49f+ adult basal cells and CD31-CD45-Ter119-EpCAM++CD49f+ fetal cells were sorted. RNA was extracted and hybridized to the Agilent One-Color Gene Expression Arrays .

多数正常成人组织中均存在一类罕见干细胞，具备极强的自我维持与再生潜能。在个体发育进程中，造血系统与神经系统的干细胞会发生自我更新潜能的内在程序化改变。在小鼠模型中，具备可移植再生活性的乳腺干细胞最早可在产前数日被检测到；这类细胞与成体同源乳腺干细胞共享部分表型特征，但富集于具有独特基因表达谱的特定亚群中。本研究证实，胎儿乳腺上皮细胞的直接生长潜能与可诱导生长潜能均高于成体同源细胞。我们通过一种新型体外培养体系揭示了其可诱导生长潜能的相关特性：该体系可从单个胎儿或成体起始细胞出发，在7天内扩增获得大量具备连续移植活性的乳腺干细胞与体外克隆形成祖细胞。本研究进一步发现，上述细胞应答高度依赖成纤维细胞分泌的新型活性因子。本研究结果为阐明单细胞水平下正常乳腺上皮干细胞特性的调控机制、发育过程中这些特性的动态变化，以及其功能紊乱如何促进肿瘤转化提供了全新研究路径。本研究使用基因芯片（microarrays）比较了胚胎发育第18.5天（E18.5）胎鼠与成体富集乳腺重建单位（Mammary Repopulating Unit, MRU）的乳腺细胞转录组。我们分别分选了三组生物学重复的成体基底细胞（表型为CD31⁻CD45⁻Ter119⁻BP-1⁻EpCAM⁺CD49f⁺）与胎鼠细胞（表型为CD31⁻CD45⁻Ter119⁻EpCAM⁺⁺CD49f⁺）。提取总RNA后，将其与安捷伦（Agilent）单通道基因表达芯片进行杂交。