Developmental Cell, Volume 31
Supplemental Information
Hair Follicle Dermal Stem Cells Regenerate
the Dermal Sheath, Repopulate the Dermal
Papilla, and Modulate Hair Type
Waleed Rahmani, Sepideh Abbasi, Andrew Hagner, Eko Raharjo, Ranjan Kumar, Akitsu
Hotta, Scott Magness, Daniel Metzger, and Jeff Biernaskie
Supplemental Data
Figure S1 – Cell division in HF mesenchyme is restricted largely to dermal sheath at
early anagen, related to Figure 1. A) Low magnification images of adult dorsal skin at
various stages of the hair cycle stained for the proliferative marker Ki67 (green), Keratin
5 (red). Nuclei are stained with Hoechst (blue). B) High magnification images of aSMA
(red) dermal sheath cells co-localizing with Ki67 (green, arrowheads) and located
adjacent to epithelial outer root sheath (Keratin 5; blue). Scale bars are 50µm.
C) Quantification of dermal sheath (DS) proliferation over the hair cycle. *** p<0.001
compared to all other groups ; †† p<0.01, compared to catagen. D) Quantification of cell
proliferation in the dermal papilla. For D, E, n= 3 mice per time-point, 30 follicles/mouse
were quantified for each timepoint.
Figure S2 – Dermal sheath cells are retained over multiple regenerative cycles,
related to Figure 2.
Tamoxifen was administered at Postnatal day 23/24. Low magnification images of adult
αSMACreERT2:YFP backskin. YFP+ve DSC progeny (green) are observed in the DS and
DP at 3rd anagen (A), and 4th anagen (B). Arrows indicate follicles containing YFP+ve
cells in the DP.
(C) YFP+ve hfDSC progeny (green, arrows) at 2nd telogen and (D) 8 months of age. Note
that >90% of follicles have retained YFP+ve cells surrounding the telogen DP.
E) αSMACreERT2:YFP+ve hfDSCs (green, arrows) express α8-integrin (red) in telogen.
Scale bars are 100µm and 20µm.
Figure S3 – hfDSC progeny recruited to DP exhibit expression of DP markers,
related to Figure 3. αSMACreERT2:YFP+ve DSC progeny (green) residing in the DP colocalize with DP markers (shown in red) A) versican, B) Lef1, C) α9−integrin, D)
Noggin and E) Sox2. High magnification images of boxed regions are shown in insets at
right. DP are outlined by dashed lines.
Scale bars are 10µm. Insets are 5µm.
Figure S4 - hfDSCs self renew and are activated to proliferate at early anagen,
related to Figure 3.
(A) Image of an early anagen HF showing Ki67+ve HF mesenchymal cells reside in the
dermal sheath that surrounds the DP, but never within the ERα+ve DP.
(B) Images of early anagen HF (P22) following tamoxifen administration at P3-4.
Animals were pulsed with EdU at P20-22 and sacrificed 24hrs later. YFP+ve dermal
sheath cells were actively dividing, but no EdU incorporation was observed in DP.
C) Newly regenerated αSMACreERT2:YFP+ve dermal sheath cells are derived from cells
that are activated to proliferate at early anagen (P20-22), but not later (P23-25). EdU is
shown in red.
D) Examination of follicles at the subsequent 2nd telogen shows that
αSMACreERT2:YFP+ve cells (green) that are retained within the follicle are derived from
cells that mitotically (EdU+ve, red) active during early anagen (P20-22) and not at later
times (P23-25).
(E) Temporal origin of αSMACreERT2:YFP+ve dermal sheath cells was quantified. There
was a 2-fold increase in αSMACreERT2:YFP+ve EdU+ve cells when pulsed at early anagen
versus mid anagen suggesting a restricted period of activation (n=4 mice, 60 follicles per
time point).
F,G) αSMACreERT2:YFP+ve Edu+ve (red) cells were exclusively observed in the dermal
cup of 3rd anagen follicles, demonstrating long term retention and self renewal within this
niche. Scale bars are 50µm except (D) 25µm.
Figure S5 – hfDSCs differentiate and exit the DSC pool, related to Figure 4.
A small subset of αSMACreERT2:ROSAconfetti clones had vacated the DS cup region and
consequently all progeny committed to either (A) DS or (B) DP fates. In (A) progeny
from two different clones (yellow, arrows) and (green, arrowheads) were only observed
in the proximal DS. (B) A single clone (yellow, arrows) showing two daughter cells
residing in the DP. Scale bar is 50µm.
Figure S6 – Diphtheria toxin-mediated killing of hfDSCs, related to Figure 7.
A) Telogen follicle from DT-treated αSMACreERT2:ROSAYFP:+/+ skin graft showing an
absence of DTR (red, arrow) expression in YFP+ cells. Scale is 10µm.
B) Telogen follicle from DT-treated αSMACreERT2:ROSAYFP: iDTR skin graft showing coexpression of DTR (red, arrows) in YFP+ve hfDSC. Scale is 10µm.
A) Higher magnification inset of cell in B. Note the membrane localization of DTR
(arrowheads). Scale is 5 µm.
D, E) αSMACreERT2:ROSAYFP: iDTR full thickness skin grafts following treatment with
PBS or Diphtheria toxin (DT; bottom). Note the large number of follicles that no longer
contain YFP+ (green) cells following DT treatment (arrows). Scale bars are 500µm.
Nuclei are stained with Hoechst (blue).
Supplementary Table 1, related to Figure 2 - Percentage of αSMACreERT2:YFP+ve
pelage hair follicles over naturally occurring or depilation-induced hair cycles.
Mouse 2nd Anagen 2nd Telogen 1 98.84% 99.06% 84.57% 2 87.01% 3rd 3rd Anagen 4th Anagen 5th Anagen Anagen (dep) (dep) (dep) 3 94.53% 92.58% 4 96.50% 98.20% 99.00% 5 96.63% 91.60% 98.44% 6 89.90% 86.04% 7 97.60% 99.47 Supplemental Experimental Procedures Hair follicle mesenchymal cell proliferation kinetics. Proliferation of mesenchymal cells within the DP and DS compartments was done in adult male C57Bl/6 and PDGFRα:H2BGFP mice (Hamilton et al., 2003) (Jax Mice). Backskin was harvested at ages P16, 19, 21, 24, 27, 30 and 35, mounted in OCT media, snap frozen and sectioned at a thickness of 20-‐50µm. To assess proliferation across the hair cycle, 50 hairs at each time-‐point (n= 2 mice per timepoint) were characterized according to follicle phenotype and Ki67+ve cells within the DP or DS compartments were counted. Hair follicle formation assay For transplants, prospectively isolated αSMAdsRed+ve cd34-‐ve ITGα8-‐ve DS cells were grown in the presence of lentiviral particles expressing green fluorescent protein under the EF-‐1α promoter (Hotta et al., 2009). Polybrene (8µg/ml) was included in the medium and cells were washed extensively after 48 hours. GFP+veαSMAdsRed+ve DS cells (2-‐4 × 105 cells) were combined with 104 newborn epidermal aggregates and injected subcutaneously into adult nude mice (nu/nu; Charles River) for 12 days as previously described (Biernaskie et al., 2009; Zheng et al., 2005). Antibodies for Immunofluorescence and cell sorting The following primary antibodies were used for immunofluorescence: chicken and rabbit GFP (1:500; Millipore), K14 (1:500; Covance), Versican (1:250; a gift from R. LeBaron), LEF1 (1:100, Cell Signaling), noggin (1:50, Abcam), Sox2 (1:100, Stemgent), estrogen receptor alpha (1:50; Epitomics), rabbit αSMA (1:500; Millipore), Ki-‐67 (1:200 Dako), αSMA (1:500, Millipore), Keratin-‐5 (1:500, Covance). For cell sorting we used antibodies raised against cd34 (1:100, eBioscience), ITGα9 and ITGα8 (both 1:100, R&D Systems). Secondary antibodies used were Alexa488-‐conjugated goat anti-‐chicken or –rabbit and Alexa 555 goat anti-‐rabbit and Alexa647 goat anti-‐rabbit, -‐mouse or -‐rat (1:1000; all from Invitrogen). For detection of EdU, a copper-‐catalyzed Click-‐iT reaction kit was used per the manufacturer instructions (Invitrogen). Titrating the copper sulfate concentration to 1/8th the suggested amount was necessary to preserve the YFP signal. Supplemental References Biernaskie, J., Paris, M., Morozova, O., Fagan, B.M., Marra, M., Pevny, L., and Miller, F.D. (2009). SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells. Cell Stem Cell 5, 610-‐623. Hamilton, T.G., Klinghoffer, R.A., Corrin, P.D., and Soriano, P. (2003). Evolutionary divergence of platelet-‐derived growth factor alpha receptor signaling mechanisms. Mol Cell Biol 23, 4013-‐4025. Hotta, A., Cheung, A.Y.L., Farra, N., Vijayaragavan, K., Seguin, C.A., Draper, J.S., Pasceri, P., Maksakova, I.A., Mager, D.L., Rossant, J., et al. (2009). Isolation of human iPS cells using EOS lentiviral vectors to select for pluripotency. Nat Methods 6, 370-‐376. Zheng, Y., Du, X., Wang, W., Boucher, M., Parimoo, S., and Stenn, K. (2005). Organogenesis from dissociated cells: generation of mature cycling hair follicles from skin-‐derived cells. J Invest Dermatol 124, 867-‐876.