Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, Kobe, Japan.
J Invest Dermatol. 2011 Dec;131(12):2358-67. doi: 10.1038/jid.2011.195. Epub 2011 Jul 14.
In mice, coat pigmentation requires a stem cell (SC) system in which the survival, proliferation, and differentiation of melanocytes (MCs) are regulated by microenvironments in hair follicles (HFs). In vitro systems are required to analyze the behavior of single melanocyte stem cells (MCSCs) and their potential to form SC systems in vivo. We describe here an experimental system for the isolation, self-renewal, and differentiation of MCSCs, as well as an in vivo reconstitution assay for assessing their potential. Using Dct(tm1(Cre)Bee)/CAG-CAT-GFP mice, we show that, in the presence of stem cell factor and basic fibroblast growth factor and the XB2 feeder cell line, purified MCSCs can undergo clonogenic proliferation, resulting in c-Kit(low) side scatter(low) cells. In culture, these cells maintain their capacity to differentiate and reconstitute an MCSC system in HFs. As these cells are present in the upper part of the HF near the bulge region, express only low levels of housekeeping genes, and are resistant to neonatal treatment with ACK2, it is likely that only MCSCs that are quiescent in vivo have clonogenic activity in vitro. We also found that MCSCs can be purified from wild-type mice by fluorescent cell sorting and can be characterized in vitro.
在小鼠中，毛色需要一个干细胞（SC）系统来调节，其中黑素细胞（MCs）的存活、增殖和分化受毛囊（HFs）的微环境调节。需要体外系统来分析单个黑素干细胞（MCSCs）的行为及其在体内形成 SC 系统的潜力。我们在这里描述了一种用于分离、自我更新和分化 MCSCs 的实验系统，以及一种用于评估其潜力的体内重建测定法。使用 Dct(tm1(Cre)Bee)/CAG-CAT-GFP 小鼠，我们表明，在干细胞因子和碱性成纤维细胞生长因子以及 XB2 饲养细胞系存在的情况下，纯化的 MCSCs 可以进行克隆增殖，导致 c-Kit(low)侧散射(low)细胞。在培养中，这些细胞保持分化和重建 HF 中 MCSC 系统的能力。由于这些细胞存在于 HF 的上部靠近隆起区域，仅表达低水平的管家基因，并且对新生鼠用 ACK2 处理具有抗性，因此可能只有体内静止的 MCSCs 在体外具有克隆活性。我们还发现，MCSCs 可以通过荧光细胞分选从野生型小鼠中纯化出来，并可以在体外进行表征。
