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First published online 5 February 2008
doi: 10.1242/jcs.025502

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A distinct population of clonogenic and multipotent murine follicular keratinocytes residing in the upper isthmus

Uffe Birk Jensen^1,2,*, Xiaohong Yan^3,*, Charlotte Triel¹, Seung-Hyun Woo⁴, Rikke Christensen¹ and David M. Owens^3,4,

¹ Institute of Human Genetics, University of Aarhus
² Department of Clinical Genetics, Aarhus University Hospital, DK8000 Aarhus C., Denmark
³ Department of Dermatology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA
⁴ Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, NY 10032, USA

View larger version (141K): [in this window] [in a new window]   Fig. 1. Sca-1 cell surface marker expression in adult murine skin. Epidermal whole mounts prepared from tail skin (A-F) and dorsal skin cryosections (G-J) were labeled with antibodies against the cell surface markers 6 integrin (A,G,E), Sca-1 (B,F,H) and CD34 (C,I). Fluorescent images were taken with a Zeiss LSM 510 confocal microscope. To visualize colocalization, A-C and G-I were merged to highlight UI cells in tail (D) or dorsal (J) skin. High-magnification images of A,B show continuous staining of 6 integrin (E) and Sca-1 (F) in the basal layer of the IFE. bg, bulge; IFD, infundibulum; IFE, interfollicular epidermis; sg, sebaceous gland. Scale bars: 50 µm (A-D and G-I); 10 µm (E,F).

View larger version (60K): [in this window] [in a new window]   Fig. 2. High-magnification view of the hair follicle UI region and FACS analysis of UI epidermal keratinocytes from murine skin. Epidermal whole mounts were probed with antibodies to 6 integrin (A), Sca-1 (B) and CD34 (C). Merged panel (D) shows high magnification image of three HF compartments including 6L+HCD34–Sca-1+ IFE + IFD (P1), 6LCD34–Sca-1– UI (P2) and 6L+HCD34+Sca-1– (P3) regions isolated by FACS below (E-M). (E-M) Keratinocytes were isolated from adult dorsal skin as previously described (Morris, 1994), labeled with 6 integrin, CD34 and Sca-1 antibodies and subjected to flow cytometry. Viable 6-positive cells were identified and further gated based on their surface expression of CD34 and Sca-1. Scale bar: 20 µm.

View larger version (73K): [in this window] [in a new window]   Fig. 3. Colony-forming efficiency of UI epidermal keratinocytes. (A-C) All six FACS-sorted cell populations from Fig. 2 were plated at equal density and cultured for 2 weeks. Cultures were stained with Rhodamine and the average (n=3 plates per group) total colony number (B) and the percent of colonies >4 mm in size (C) were quantified using NIH ImageJ software and statistically compared using a Student's t-test (*P < 0.05, statistically different compared with UI cells; error bars represent s.d.). (D-F) Higher-magnification phase-contrast images of three FACS-sorted populations shown in A.

View larger version (115K): [in this window] [in a new window]   Fig. 4. Hair reconstitution assay showing multipotent capacity of UI keratinocytes. (A-H) FACS-sorted keratinocyte populations were mixed with dermal fibroblasts and injected into silicon graft chambers. Chambers were removed 1 week after cell injection and images of reconstituted skin and hair were captured 6 weeks post grafting. The number of FACS-sorted keratinocytes implanted in each graft is indicated. (B,D,F,H) Side view of each panel above. Dashed circle designates the graft perimeter (G,H). (I-U) H-2Kb (I,L,M,O,T), H-2Kq (J-K,N,P,T) and GFP (Q-S,U) immunofluorescent detection in C57Bl/6 (I,K) and nude (J,L) skin and a skin graft reconstituted from UI (M-S) or IFE + IFD (T-U) cells. Arrowheads indicate positive immunofluorescence and arrow points to boundary between grafted epidermis (H-2Kb-positive) and endogenous nude epidermis (H-2Kq-positive) at the graft periphery (T). DF, dermal-fibroblast-only graft (control); DP, dermal papilla; bl, basal layer; sl, suprabasal layer. Dashed line delineates epidermal basal layer (K,L,R) or encircles dermal papilla cells (M-N). Scale bars: 20 µm.

View larger version (33K): [in this window] [in a new window]   Fig. 5. Cell cycle status and label retention in UI keratinocytes. (A-F) FACS dot blot showing DNA content (y-axis) vs pulse width (x-axis) from FACS-sorted keratinocytes isolated from telogen (A,C,E) and anagen (B,D,F) skin. (G) Quantification of G0-G1, S or G2-M phase cells. (H) Cells retaining label are represented as the average percentage of BrdU-positive cells for each FACS-sorted population within the total number of BrdU-positive cells in the entire epidermis per experiment. 2000 cells per population were counted in each experiment and data represent the average of two experiments. *P < 0.05, statistically significant from UI and IFE + IFD cells (Student's t-test). Error bars denote s.d.

View larger version (57K): [in this window] [in a new window]   Fig. 6. Verification of enriched transcripts in UI cells by RT-PCR analysis. (A) Total RNA from FACS sorted UI, bulge and IFE + IFD epidermal cells was used to generate first-strand cDNA for semi-quantitative RT-PCR analysis using primers for genes enriched in UI cells (Table 2). RT-PCR for GAPDH was conducted as an internal control. Each RT-PCR reaction was generated from undiluted (1), 1:5 dilution (1/5) or a 1:25 dilution (1/25) of each cDNA sample. No amplification products were observed in the water control (C) samples. (B) Quantification of RT-PCR transcript levels generated from undiluted cDNA samples using NIH ImageJ software. Data are expressed as arbitrary densitometric units relative to GAPDH levels.

View larger version (118K): [in this window] [in a new window]   Fig. 7. Immunofluorescent labeling of FACS-sorted UI cells and murine HFs. (A-D) 6 integrin (A,C) and K14 (B,D) immunofluorescence in cytospun UI and IFE + IFD FACS-sorted cells. Arrowheads indicate polarized surface 6 or cytoplasmic K14 expression. (E) 6 integrin and CD44 immunofluorescence in a HF. (F-H) Tbc1d10c immunofluorescence in cryosections of adult mouse skin. Three separate HFs are shown. Arrowheads indicate positive Tbc1d10c labeling in the UI region and a hashed line demarcates the basal layer of the IFE (I,K). Staining in the cornified IFE was also observed in control sections stained with secondary antibodies alone (data not shown). BG, bulge; hs, hair shaft; sg, sebaceous gland. Scale bars: 100 µm.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Schematic of UI and MTS24 HF compartments. Distinguishing surface marker expression profiles for 6 integrin are shown as high (H, bright green) or low (L, pale green). Two possible compartments for 6HCD34–Sca-1– cells, bordering either side of the UI region (left panel) or distributed throughout the basal layer of the IFE (right panel), are shown. sd, sebaceous duct; hs, hair shaft; dp, dermal papilla.