Isolation and in vitro expansion of Lgr6-positive multipotent hair follicle stem cells.

Hair follicles (HFs) are a well-known niche of multipotent stem cells. Recently, HF cells expressing leucine-rich orphan G protein-coupled receptors (Lgr) have been described as multipotent adult stem cells with a high potential for regenerative therapies. We have now established the conditions for the isolation and long-term expansion of stem cells from HFs (HFSCs) and analyzed their characteristics (reverse transcription with the polymerase chain reaction, immunohistochemistry) and multipotent capacity (differentiation assays). HFSCs possess a high self-renewal capacity and share characteristics of putative HF epithelial stem cells, such as the expression of Lgr6, cytokeratins (Ck18, Ck19), and multipotent stem cell markers (Sca-1, Bcrp1, nestin, P75NTR). Under defined cell culture conditions, HFSCs are able to differentiate into mesenchymal lineages (adipocytes, chondrocytes, muscle cells) or neurons (βIII-tubulin). We report, for the first time, an in vitro culture method to isolate Lgr6-positive stem cells from HFs. HFSCs represent a unique tool for studying the cell characteristics of Lgr6-positive cells and should provide a novel easily accessible source for regenerative therapies.