Figure 1. Multicolor cell sorting of stem cell precursors, transit amplifying progenitors, and their niche from developing hair follicles.

(A) Schematic of postnatal day (P)5 back skin with hair follicles (HF) from the three HF developmental waves. Right: list of cell populations isolated from six different fluorescent reporter mouse lines in four double-transgenic combinations.

(B) Isolation of eight main skin/HF populations from K14-H2BGFP;Lef1-RFP back skin. Top: P5 skin section shows strong H2BGFP expression in epithelial Epi and ORS cells, and RFP expression in upper DFs, the DP and Mc. Mx expresses low levels of H2BGFP. The Shh expressing subpopulation of TAC progenitors and few differentiating cells co-express H2BGFP and RFP. Bottom: FACS plots and gates for cell sorting from HF-enriched dermal preparations. Seven gates mark Mx, ORS, TAC, Mc, and DP from HFs, and DF and a mixture of negative cells (Neg) from the upper dermis. Right: qRT-PCR of known markers confirms TAC and DP enrichment. Data are mean ± SD from two measurements.

(C) Isolation of HFSC precursors from K14-RFP;Sox9-GFP P5 back skin. Top: P5 skin section shows GFP expression in the upper ORS of the future bulge area. All epithelial cells are RFP. Bottom: FACS plots and gates for isolation of HFSC precursors and the remaining HF-ORS, and HF-Mx.

(D,E) Isolation of pure DP subpopulations from P5 back skin. (D) Top: section of P5 Sox2^GFP;Lef1-RFP back skin and GFP quantification shows GFP expression in G-DP and AA-DP cells, compared to ZZ-DP. (E) Top: section of P5 Crabp1-GFP;Lef1-RFP back skin and GFP quantification shows GFP expression in AA-DP and ZZ-DP cells, but not in G-DP. Bottom: FACS plots and gates for sorting. Note that all DP subpopulations are highly enriched as RFP⁺ and ITGA9⁺ cells.

Scale bars are 100 μm (B, C), 20 μm (D, E). See also Figure S1.