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First published online 3 July 2006
doi: 10.1242/dev.02443

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The cell-surface marker MTS24 identifies a novel population of follicular keratinocytes with characteristics of progenitor cells

Joanne G. W. Nijhof^1,*, Kristin M. Braun^2,*,, Adam Giangreco², Carina van Pelt¹, Hiroshi Kawamoto³, Richard L. Boyd⁴, Rein Willemze¹, Leon H. F. Mullenders⁵, Fiona M. Watt², Frank R. de Gruijl¹ and Willem van Ewijk^6,

¹ Department of Dermatology, Leiden University Medical Center (LUMC), Leiden, The Netherlands.
² Keratinocyte Laboratory, Cancer Research UK, London Research Institute, London, UK.
³ Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan.
⁴ Department of Immunology, Central and Eastern Clinical School, Monash University, Melbourne, Australia.
⁵ Department of Toxicogenetics, LUMC, Leiden, The Netherlands.
⁶ Department of Immunology and the Center for Electron Microscopy/Department of Molecular and Cellular Biology, LUMC, Leiden, The Netherlands.

View larger version (137K): [in a new window]   Fig. 1. MTS24 reactivity in mouse epidermis. Formalin-fixed frozen section of C57Bl/6 mouse tail skin (A,B) and Balb/c mouse dorsal skin (C-E) showing MTS24-Cy3 staining (red) in the hair follicle and merged with DAPI (B,D,E; blue) to highlight nuclei. Inset (E) shows that MTS24-staining was found on the membrane of follicular cells. (F,G) Wholemounts from C57Bl/6 mouse tail skin showing MTS24-Alexa 488 labelling (green) within the hair follicle, counterstained with ToPro3 (blue). (H,I) MTS24-Cy3 staining in hairless SKH-1 mouse dorsal skin and merged with DAPI (I). The asterisk marks the smear-like appearance of MTS24 within the inner hair shaft. (J) MTS24-Cy3 staining in 2-day-old hairless SKH-1 mouse dorsal skin. The green staining of the hair was caused by autofluorescence. (K-N) Immunoelectronmicroscopic pictures of MTS24-labelling. (M) Cross-sectional overview of murine hair follicle from 2-day-old SKH-1 mouse dorsal skin. K,L,N are higher magnifications of certain regions (indicated by the arrow) within M. (K,M) Active cycling cells were found in the outer root sheath (ORS, asterisks). Within the inner root sheath (IRS), apoptotic cells were found (L,M; crosshatches). Detail (K) of the ORS showing three neighbouring cells (cells 1-3). MTS24 was found on the cell membrane of these cells (arrowheads). (L) Apoptotic cell (crosshatch) within the IRS. MTS24 was found on the cell membrane (arrowheads). (N) Membranes of dead cells within the IRS positive for MTS24 (arrowheads). SG, sebaceous gland; HF, hair follicle; IFE, interfollicular epidermis; BG; bulge, HS, hair shaft. Scale bars: 50 µm (in A,B,F,G), 25 µm (in C,D,H-J), 10 µm (in E) and 1 µm (in K-N).

View larger version (122K): [in a new window]   Fig. 2. MTS24 and CD34 reactivity during hair follicle development. (A) Staining of MTS24-Cy3 (red) and keratin 17-FITC (green) in skin obtained from Balb/c mice at E20.5 during embryonic development and at day 2 and day 8 after birth. Keratin 17 was selectively expressed within the developing hair follicles. Note the interfollicular staining of MTS24 and its co-localisation with keratin 17 expression in 2-day-old Balb/c mice (arrowhead). (B,C) Expression of CD34-Cy3 (red), keratin 17-FITC (green) in dorsal skin from Balb/c mice at day 4 (B) and day 6 (C). Note that CD34 was expressed from day 6 after birth but not at day 4 after birth. Red staining of stratum corneum is caused by autofluorescence. (D,E) Frozen sections of dorsal epidermis from K14Nß-cateninER transgenic mice treated with 4OHT for 21 days were immunolabelled for MTS24 (green; D,E) and keratin 14 (red; D) or keratin 17 (red; E). MTS24-positive regions of ectopic follicles are demarcated with brackets (D,E). Nuclei were counterstained (blue) with DAPI (A-C) or ToPro3 (E). HF, hair follicle; IFE, interfollicular epidermis; BG, bulge. Scale bars: 25 µm (A, upper and second panel); 50 µm (A, third panel; B-E).

View larger version (88K): [in a new window]   Fig. 3. MTS24 co-staining with described markers for epidermal stem cells. (A-C) Frozen section of dorsal skin showing expression of keratin 14-Cy3 (A; red) and MTS24-FITC (B; green) and merged (C) in hair follicle. Arrowhead highlights co-localisation of keratin 14 expression and MTS24 labelling. (D-F) Expression of CD34-Cy3 (D; red) and MTS24-FITC (E; green) and merged (F) in dorsal skin. Note that CD34 expression (arrowhead) was found in a different location within the hair follicle than MTS24-staining (asterisk). (G-I) Wholemount of tail epidermis showing no co-localisation between keratin 15-FITC (G; green) and MTS24-Cy3 (H; red) labelling within the hair follicle. Merged image is shown in (I). (J) Labelling with MTS24-Cy3 (red) and FITC anti-mouse (green) secondary antibody alone, shows that staining of sebaceous gland is non-specific in (G,I). (K) Wholemount of tail epidermis showing 6 integrin-FITC (green) and MTS24-Cy3 (red) co-staining (arrowhead). SKH-1 (L,M) or wild-type (N,O) neonatal mice received repeated injections with BrdU to generate label-retaining cells. Frozen sections (L-N) or tail wholemounts (O) were collected at 1 day (L), 6 weeks (M) or 10 weeks (N,O) after the last injection with BrdU. Tissue was labelled for BrdU-FITC (green) and MTS24-Cy3 (red). Nuclear counterstain was DAPI (L,M,N; blue). Arrowheads indicate BrdU label-retaining cells (L-O). In each panel the bulge area is bracketed. SG, sebaceous gland; HF, hair follicle; IFE, interfollicular epidermis. Scale bars: 50 µm.

View larger version (45K): [in a new window]   Fig. 4. 6 integrin+/MTS24+ keratinocytes form large colonies with high efficiency in culture. Keratinocytes harvested from dorsal epidermis of adult C57Bl/6 mice were analysed by FACS (A,B) or were sorted (C-F) under sterile conditions. (A,B) Flow cytometric analysis of (A) isotype control versus (B) MTS24-labelled keratinocytes indicates that there is a subpopulation of MTS24+ keratinocytes. (C-F) Sorted 6 single+, 6+/MTS24+ or the unseparated mixture (all sorted) keratinocytes were grown for 14 days, fixed in 4% formal saline, and stained with Rhodamine B to visualise colony growth. Data are shown from a representative experiment, which was repeated several times with similar results. (C) FACS selection of keratinocytes. (D) Representative culture dishes with stained colonies. (E,F) Graphs of colony-forming efficiency (E, black bars) and size of colonies (E, white bars; F). Bars represent the mean of at least four replicate culture wells±s.e.m. Asterisks indicate significant differences of 6+/MTS24-relative to 6+/MTS24+ (P<0.05; unpaired two-tailed t test).

View larger version (73K): [in a new window]   Fig. 5. CD34 and MTS24 identify distinct subpopulations of basal keratinocytes with high in vitro replicative capacity. (A,B) Keratinocytes harvested from dorsal epidermis of adult C57Bl/6 mice were sorted under sterile conditions. (A) Flow cytometric analysis shows that CD34+ basal keratinocytes are members of a different population of cells than the MTS24+ keratinocytes. Sorted 6 single+, 6+/MTS24+, 6+/CD34+ or the unseparated mixture (all sorted) keratinocytes colonies were grown for 14 days to visualise the colonies (B) and compare relative colony-forming efficiency (C). (D) Individual colonies from 6+/MTS24+ and 6+/CD34+ keratinocytes were passaged and replated at clonal density for an additional 14 days. (E) A graphical comparison of the size of colonies derived following passage. Bars represent the mean of at least four replicate culture wells±s.e.m. Data are shown from representative experiments that were repeated with similar results.

View larger version (16K): [in a new window]   Fig. 6. MTS24+ and CD34+ basal keratinocytes show similar gene expression profiles. FACS-sorted 6+/MTS24+ and 6+/CD34+ keratinocytes were analysed by Q-PCR for expression of a selected group of genes. 6+/MTS24+ and 6+/CD34+ show the same pattern for genes that are supposed to be lower expressed (red bars) in hair follicle stem cells or whose expression is enriched (green bars) in hair follicle stem cells. Expression is normalized to the reference gene (ß-actin) and fold changes for 6+/MTS24+ and 6+/CD34+ keratinocytes are in comparison to 6+/MTS24- and 6+/CD34-keratinocytes respectively. Average data given are from two independent isolations and Q-PCR was performed in triplicate per sorted population.

View larger version (29K): [in a new window]   Fig. 7. Models for the relationship between the bulge and MTS24-positive hair follicle subpopulations. The phenotype of key markers is noted for highlighted regions. Several possibilities for the relationship between these subpopulations are indicated in the figure. K15, keratin 15; LRC, label-retaining cells; 6, 6-integrin; K14, keratin 14.

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