Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties
Isolation, immortalization, and characterization of a human breast epithelial cell line with stem cell properties
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DOI：
10.1101/gad.952602
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发表时间：
2002-03-15
影响因子：
10.5
通讯作者：
Petersen, OW
中科院分区：
文献类型：
--
作者：
Gudjonsson, T;Villadsen, R;Petersen, OW
人类乳房的上皮室由两个不同的谱系组成：腔上皮谱系和肌上皮谱系。我们之前已经证明，在培养中，腔上皮细胞的一部分可以转化为肌上皮细胞，这意味着可能存在祖细胞。因此，我们着手鉴定和分离腔上皮室中可能的前体。利用细胞表面标志物和免疫磁性分选，我们从原代培养的缩乳性乳腺肿瘤中分离出两种腔上皮细胞群。主要群体共表达唾液粘蛋白(MUC+)和上皮特异性抗原(ESA(+))，而少数群体位于基底以上，表达上皮特异性抗原，但不表达唾液粘蛋白(MUC-/ESA(+))。通过转导人乳头瘤病毒16型E6/E7基因，进一步建立了两个细胞系。这两个细胞系都保持了腔上皮细胞的表型，表现为紧密连接蛋白claudin-1和occludin的表达，并在半透性滤器上产生了高的跨上皮电阻。而在克隆培养中，MUC+/ESA(+)上皮细胞系为腔上皮细胞，其分化受限制，基底层以上来源的MUC-/ESA(+)上皮细胞系能够产生MUC+/ESA(+)上皮细胞和Thy-1(+)/α-平滑肌肌动蛋白(+)(Asma(+))肌上皮细胞。MUC-/ESA(+)上皮细胞系与MUC+/ESA(+)上皮细胞系进一步不同于MUC+/ESA(+)上皮细胞系的角蛋白K19的表达，这是体内末梢导管小叶单位上皮细胞亚群的特征。在重组的基底膜内，MUC+/ESA(+)上皮细胞系形成腺泡样球。相反，MUC-/ESA(+)上皮细胞系在形态和标记表达上形成了类似于未培养的末端导管小叶单位的精细分支结构。将细胞外基质包埋的细胞接种到裸鼠皮下，获得了类似的结构。因此，腔上皮谱系中的MUC-/ESA(+)上皮细胞可能是人类乳房末梢导管小叶单位的前体细胞。
The epithelial compartment of the human breast comprises two distinct lineages: the luminal epithelial and the myoepithelial lineage. We have shown previously that a subset of the luminal epithelial cells could convert to myoepithelial cells in culture signifying the possible existence of a progenitor cell. We therefore set out to identify and isolate the putative precursor in the luminal epithelial compartment. Using cell surface markers and immunomagnetic sorting, we isolated two luminal epithelial cell populations from primary cultures of reduction mammoplasties. The major population coexpresses sialomucin (MUC+) and epithelial-specific antigen (ESA(+)) whereas the minor population has a suprabasal position and expresses epithelial specific antigen but no sialomucin (MUC-/ESA(+)). Two cell lines were further established by transduction of the E6/E7 genes from human papilloma virus type 16. Both cell lines maintained a luminal epithelial phenotype as evidenced by expression of the tight junction proteins, claudin-1 and occludin, and by generation of a high transepithelial electrical resistance on semipermeable filters. Whereas in clonal cultures, the MUC+/ESA(+) epithelial cell line was luminal epithelial restricted in its differentiation repertoire, the suprabasal-derived MUC-/ESA(+) epithelial cell line was able to generate itself as well as MUC+/ESA(+) epithelial cells and Thy-1(+)/alpha-smooth muscle actin(+) (ASMA(+)) myoepithelial cells. The MUC-/ESA(+) epithelial cell line further differed from the MUC+/ESA(+) epithelial cell line by the expression of keratin K19, a feature of a subpopulation of epithelial cells in terminal duct lobular units in vivo. Within a reconstituted basement membrane, the MUC+/ESA(+) epithelial cell line formed acinus-like spheres. In contrast, the MUC-/ESA(+) epithelial cell line formed elaborate branching structures resembling uncultured terminal duct lobular units both by morphology and marker expression. Similar structures were obtained by inoculating the extracellular matrix-embedded cells subcutaneously in nude mice. Thus, MUC-/ESA(+) epithelial cells within the luminal epithelial lineage may function as precursor cells of terminal duct lobular units in the human breast.