Fig 2: Fibrin hydrogels support stem cell for de novo hair genesis. (A) Representative images of hair growth observed 4 weeks after transplantation (N = 3). (B) HE staining of hair genesis tissue shows the hair follicle, epidermis and dermis regenerated. Scale bar: 100 µm. (C and D) Representative IF images of K1 and K14 in regenerated tissue of fibrin hydrogels and Matrigel. Scale bar: 100 µm. (E) The numbers of hair shafts per wound in each wound and each group (N = 3).

Fig 3: The genetic loss of epidermal Pgc-1a reduces cell growth and enhances differentiation after physiologic skin damage. (A) Schematic of cre-lox recombination to delete Pgc-1a in keratinocytes. (B) Epidermal Pgc-1a mRNA (Ppargc1a) in an n of 7–9 WT and epiPgc-1a KO mice. (C), representative images of keratin 14 (K14), keratin 10 (K10), and loricrin (Lor). (D) The UVB-induced fold-change in EdU+ epidermal cells (n = 5–6) and (E) K10 layer thickness (n = 4–7) relative to sham skin in WT and epiPgc-1a KO mice. (F), quantification of ?H2AX and thymine dimers in UVB exposed epidermis with representative images at right from an n of 5 WT and epiPgc-1a KO mice. (G) TPA-induced fold-change in EdU+ epidermal cells (n = 5–7) and (H) K10 layer thickening (n = 6–9) relative to control skin in WT and epiPgc-1a KO mice. (I) The open wound area after 9 days in an n of 7–8 WT and epiPgc-1a KO mice. All data are plotted as mean ± SEM. All data were analyzed using an unpaired two-tailed students t-test. *Significantly different (p < 0.05) from WT. Scale = 50 µm for C–H.

Fig 4: Ectopic IL-24 induction in homeostatic skin epithelium elicits a wound-like response without injury(A) Schematic of the generation of TRE-IL-24 mice. Selective targeting to skin EpdSCs was achieved by packaging the transgene in a lentivirus and in utero injection into the amniotic sacs of E9.5 mouse embryos genetic for the Krt14-rtTA doxycycline inducible transcriptional activator. The lentivirus also contained a constitutively expressed Pgk-H2BGFP to monitor integration efficiency. Skins were harvested after mice were fed Dox food for 2, 3, or 4 days.(B) Left: images of mice at postnatal days 1 and 4. Note flaky skin phenotype, evident by day-4. Right: Images of hematoxylin and eosin (H&E) staining and trichrome staining performed on sagittal sections of homeostatic skins from Dox-fed WT and Tre-Il24 mice. Quantifications are of epidermal thickness and intensity of trichrome staining to evaluate dermal collagen deposition (n = 3 mice per genotype).(C) Sagittal sections of homeostatic skins from WT and Tre-Il24 mice immunolabeled for Ki67, GFP and CD31. Quantifications are of percentages of proliferating (Ki67+) EpdSCs (top), and underlying endothelial cells (Ki67+CD31+) (middle) and non-endothelial dermal cells (Ki67+CD31-) (bottom) (n = 3 mice per genotype).(D) Sagittal sections of homeostatic skins from WT and Tre-Il24 mice immunolabeled for GFP and CD31. Quantifications are of percentage of interfollicular epidermis close to CD31+ endothelial cells (top), and the distance (mm) between epidermis and CD31+ vasculature (bottom) (n = 3 mice per genotype per time point).(E) Sagittal sections of homeostatic skins from WT and Tre-Il24 mice were immunolabeled for GFP and p-STAT3. Prior to collecting skins, mice were given Dox food for 2 days. Quantifications are of percentage of p-STAT3+ epidermal, endothelial, and fibroblast cells. Quantifications of dermal cell types were made by performing similar immunofluorescence as for epidermis, but using antibodies against CD31 and PDGFa, respectively (n = 3 mice per genotype).White dotted lines: epidermal-dermal border. DAPI, nuclei; scale bars, 100 µm. Data in (B)–(E) are presented as mean ± SEM. Experiments were performed R =3×. Statistical significance was determined using two-tailed unpaired Student’s t tests; **** p < 0.0001; *** p < 0.001; ** p < 0.01; and * p < 0.05.

Fig 5: Characterization of GPP53 and GPN1 macroscopic tumors(A) Histologic classification of macroscopic tumors (top panel) and immunohistochemistry for NICD (green, middle panels) and a smooth muscle actin (SMA) (green, lower panels). Red, K14 staining. All examples shown are from GPP53 mice.(B) Table showing tumors with somatic Gli1 or Gli2 copy number gains (+++, amplified). Asterisk, GPP53-Het tumor.(C) Representative DNA copy number plot of a GPP53 tumor that amplified a region of chromosome 10 containing Gli1.(D) Same as (C) but of a different GPP53 tumor that amplified a region of chromosome 1 containing Gli2.(E) mRNA in situ staining for Gli1 (top panels), Gli2 (middle panels), and Ptch2 (lower panels) in GPP53 tumors (microscopic or Gli1- or Gli2-amplified) and in macroscopic GPN1 tumors.Scale bars, 50 µm. See also Figures S4 and S5.