Tosoni Daniela, Di Fiore Pier Paolo, Pece Salvatore
Istituto Europeo di Oncologia, Milan, Italy.
Methods Mol Biol. 2012;916:59-79. doi: 10.1007/978-1-61779-980-8_6.
Normal and tumor stem cells are present in rare quantities in tissues and this has historically represented a major hurdle to in-depth investigations of their biology. In the case of the mammary gland, the relative promiscuity of the immunophenotypical markers described in several studies for the isolation of human and mouse mammary stem cells limits their usefulness, in particular when highly purified mammary stem cell fractions are required for an in-depth molecular and functional characterization (Stingl et al. Nature 439:993-997, 2006; Shackleton et al. Nature 439:84-88, 2006; Liao et al. Cancer Res 67:8131-8138, 2007; Eirew et al. Nat Med 14:1384-1389, 2008; Raouf et al. Cell Stem Cell 3:109-118, 2008; Lim et al. Nat Med 15:907-913, 2009). In fact, most so-called stem cell markers are not selectively expressed by mammary stem cells, but are instead also expressed by terminally differentiated luminal and/or myoepithelial cells or by bipotent progenitors within the mammary gland (Stingl et al. Nature 439:993-997, 2006; Eirew et al. Nat Med 14:1384-1389, 2008; Raouf et al. Cell Stem Cell 3:109-118, 2008; Stingl et al. Differentiation 63:201-213, 1998; Jones et al. Cancer Res 64:3037-3045, 2004). Here, we describe a new methodology that does not require the use of immunophenotypical markers to obtain highly pure populations of mammary stem cells. This approach exploits two functional properties of mammary stem cells: (1) their quiescent or slowly proliferative phenotype, as compared to their progeny; and (2) their ability to survive and proliferate in anchorage-independent conditions, giving rise to clonal spheroids, commonly known as "mammospheres" (Dontu et al. Genes Dev 17:1253-1270, 2003; Pece et al. Cell 140:62-73, 2010; Cicalese et al. Cell 138:1083-1095, 2009). In the context of mammospheres, stem cells, which perform one or two rounds of division and then reenter quiescence, are identified based on their ability to retain a lipophilic fluorescent dye, PKH26, that is by contrast progressively lost by dilution in the actively proliferating progeny of precursors (Pece et al. Cell 140:62-73, 2010; Cicalese et al. Cell 138:1083-1095, 2009). Following mammosphere dissociation, the differential degree of PKH26 epifluorescence displayed by stem cells compared to precursor cells is exploited for their purification by FACS sorting. As a result, the scarcely represented PKH26-labeled mammary stem cells are purified to near homogeneity and can be used for further molecular and biological studies.
正常干细胞和肿瘤干细胞在组织中的含量稀少，这在历史上一直是深入研究其生物学特性的主要障碍。就乳腺而言，多项研究中描述的用于分离人和小鼠乳腺干细胞的免疫表型标志物相对混杂，限制了它们的实用性，特别是当需要高度纯化的乳腺干细胞组分进行深入的分子和功能表征时（斯汀格尔等人，《自然》439:993 - 997，2006年；沙克尔顿等人，《自然》439:84 - 88，2006年；廖等人，《癌症研究》67:8131 - 8138，2007年；艾尔鲁等人，《自然医学》14:1384 - 1389，2008年；拉乌夫等人，《细胞干细胞》3:109 - 118，2008年；林等人，《自然医学》15:907 - 913，2009年）。事实上，大多数所谓的干细胞标志物并非由乳腺干细胞选择性表达，而是也由终末分化的腔上皮细胞和/或肌上皮细胞或乳腺内的双能祖细胞表达（斯汀格尔等人，《自然》439:993 - 997，2006年；艾尔鲁等人，《自然医学》14:1384 - 1389，2008年；拉乌夫等人，《细胞干细胞》3:109 - 118，2008年；斯汀格尔等人，《分化》63:201 - 213，1998年；琼斯等人，《癌症研究》64:3037 - 3045，2004年）。在此，我们描述了一种新方法，该方法无需使用免疫表型标志物即可获得高度纯化的乳腺干细胞群体。这种方法利用了乳腺干细胞的两种功能特性：（1）与它们的子代相比，其静止或缓慢增殖的表型；（2）它们在非锚定依赖条件下存活和增殖的能力，从而形成克隆球体，通常称为“乳腺球”（东图等人，《基因与发育》17:1253 - 1270，2003年；佩切等人，《细胞》140:62 - 73，2010年；奇卡莱塞等人，《细胞》138:1083 - 1095，2009年）。在乳腺球的背景下，进行一轮或两轮分裂然后重新进入静止状态的干细胞是根据它们保留亲脂性荧光染料PKH26的能力来识别的，相比之下，这种染料在前体细胞的活跃增殖子代中会因稀释而逐渐丢失（佩切等人，《细胞》140:62 - 73，2010年；奇卡莱塞等人，《细胞》138:1083 - 1095，2009年）。在乳腺球解离后，利用干细胞与前体细胞相比显示出的PKH26落射荧光的差异程度，通过荧光激活细胞分选（FACS）进行纯化。结果，含量稀少的PKH26标记的乳腺干细胞被纯化至接近同质状态，可用于进一步的分子和生物学研究。
