Cell Hierarchy and Lineage Commitment in the Bovine Mammary Gland

The bovine mammary gland is a favorable organ for studying mammary cell hierarchy due to its robust milk-production capabilities that reflect the adaptation of its cell populations to extensive expansion and differentiation. It also shares basic characteristics with the human breast, and identification of its cell composition may broaden our understanding of the diversity in cell hierarchy among mammals. Here, Lin− epithelial cells were sorted according to expression of CD24 and CD49f into four populations: CD24medCD49fpos (putative stem cells, puStm), CD24negCD49fpos (Basal), CD24highCD49fneg (putative progenitors, puPgt) and CD24medCD49fneg (luminal, Lum). These populations maintained differential gene expression of lineage markers and markers of stem cells and luminal progenitors. Of note was the high expression of Stat5a in the puPgt cells, and of Notch1, Delta1, Jagged1 and Hey1 in the puStm and Basal populations. Cultured puStm and Basal cells formed lineage-restricted basal or luminal clones and after re-sorting, colonies that preserved a duct-like alignment of epithelial layers. In contrast, puPgt and Lum cells generated only luminal clones and unorganized colonies. Under non-adherent culture conditions, the puPgt and puStm populations generated significantly more floating colonies. The increase in cell number during culture provides a measure of propagation potential, which was highest for the puStm cells. Taken together, these analyses position puStm cells at the top of the cell hierarchy and denote the presence of both bi-potent and luminally restricted progenitors. In addition, a population of differentiated luminal cells was marked. Finally, combining ALDH activity with cell-surface marker analyses defined a small subpopulation that is potentially stem cell- enriched.牛乳腺是研究乳腺细胞层级的适宜器官，因其具备强大的泌乳能力，该能力反映了其细胞群体对大规模扩增与分化的适应性。它与人类乳腺具有基本相似的特征，对其细胞组成的解析或可拓宽我们对哺乳动物细胞层级多样性的认知。本研究中，研究人员依据CD24（分化簇24, cluster of differentiation 24）与CD49f（分化簇49f, cluster of differentiation 49f）的表达水平，将Lin⁻（谱系阴性，lineage negative）上皮细胞分为四个群体：CD24medCD49fpos（推定干细胞，putative stem cells, puStm）、CD24negCD49fpos（基底细胞，Basal）、CD24highCD49fneg（推定祖细胞，putative progenitors, puPgt）以及CD24medCD49fneg（腔上皮细胞，luminal, Lum）。上述四群细胞均维持了谱系标记物、干细胞标记物及腔上皮祖细胞标记物的差异表达特征。值得注意的是，Stat5a在puPgt细胞中呈高表达，而Notch1、Delta1、Jagged1及Hey1则在puStm与Basal群体中高表达。体外培养的puStm与Basal细胞可形成谱系限制性的基底或腔上皮克隆，且在重新分选后，可形成保持上皮层导管样排列的集落。与之相反，puPgt与Lum细胞仅能形成腔上皮克隆与无组织结构的集落。在非贴壁培养条件下，puPgt与puStm群体形成的悬浮集落数量显著更多。培养过程中细胞数量的增长可反映细胞的增殖潜能，其中puStm细胞的增殖潜能最高。综上，上述分析结果表明puStm细胞位于细胞层级的顶端，并证实了双潜能祖细胞与腔限制性祖细胞的存在。此外，本研究还鉴定出一群已分化的腔上皮细胞。最后，结合乙醛脱氢酶（aldehyde dehydrogenase, ALDH）活性检测与细胞表面标记物分析，可定义出一小群潜在富集干细胞的亚群。