Functional Characterization of Melanocyte Stem Cells in Hair Follicles

Abstract

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.

Sox9-Positive Progenitor Cells Play a Key Role in Renal Tubule Epithelial Regeneration in Mice

“…Clonogenicity and multilineage differentiation capacity in vitro have been widely used and considered classic methods to determine stemness. This method has helped to identify stem cells in various organs, including bone marrow, adipose, skin and liver (Angelotti et al, 2012; Arrigoni et al, 2009; Nishikawa-Torikai et al, 2011; Pittenger et al, 1999; Suzuki et al, 2008). As stem cells rarely divide, in vivo long-term label retention assays (Bruno et al, 2009; Maeshima et al, 2003) has been another frequently used method.…”

Conditional Immortalization Establishes a Repertoire of Mouse Melanocyte Progenitors with Distinct Melanogenic Differentiation Potential

“…In hair follicles, melanoblasts are segregated into hair matrix MCs (for hair pigmentation) and melanocyte stem cells (MCSCs). The discovery of MCSCs and induced pluripotent (iPS) cells provides important resources to elucidate mechanisms underlying melanogenesis and pathogenesis of MC-related disorders (Lin and Chuong, 2011; Nishikawa-Torikai et al ., 2011; Nishimura, 2011; Ohta et al ., 2011; Yang et al ., 2011). Although MCSCs and iPS cells are important MC precursors for mechanistic studies, their isolation and expansion are technically challenging.…”

The Clonal Growth Behavior of Melanocytes Derived From Melanocyte Stem Cells in Glabrous Skin

“…According to the published literature, during embryonic development, melanocytic precursors, called melanoblasts, emerge from pluripotent cells, migrate from the neural crest and then colonize in the skin [8]. The MSC system has many unique advantages not only for the study of mechanisms involved in pigmentary diseases but also to provide a sufficient supply of MCs for cell-based treatments [9].…”