Fig 2: D-galactose alters luminal progenitor cell differentiation in vitro. (A) Representative immunofluorescence images showing distinct colonies formed by luminal progenitor cells on plates pre-seeded with irradiated NIH-3T3 fibroblasts; (B) Bar graph shows the distribution of different types of colonies formed by luminal progenitor from control and D-galactose-treated mice (> 20 colonies per animal × 3 animals). K14: basal cell marker keratin 14; K8: luminal cell marker keratin 8.

Fig 3: A distinct transcriptomic landscape drives the establishment of early patagium phenotypes.(A) Heatmap displaying eigengene expression of WGCNA modules across early patagium development. Columns represent individuals, sorted by increasing weight. Rows represent modules, sorted by their correlation with sample age. Cell color represents module eigengene expression. (B and C) Genes differentially expressed between the patagium primordium and dorsal (B) or shoulder skin (C). Labeled genes are those that were both differentially expressed in the patagium primordium relative to comparison skin regions (up-regulated shown in green, and down-regulated shown in purple) and belonged to the enriched Wnt-signaling Gene Ontology term (GO:0030111). (D) In situ hybridizations of Wnt5a, Wnt11, and Dkk2. (E) Schematic of tissue recombination experiments. (F) Explants stained for KRT4 and DAPI. Asterisk denotes the mesenchymal condensate. (G) Quantification of epidermal thickness in recombination experiments. (H) H&E- and KRT14-stained, bead-implanted explants. Asterisk shows implanted bead. (I) Quantification of epidermal thickness in bead-implanted explants. Statistical significance in (G) (****P < 0.0001; n = 5) and (I) (****P < 0.0001; n = 4) was assessed using a general mixed effects model ANOVA test. Dotted lines in (D) and (H) delineate the dermis-epidermis boundary. Scale bars, 400 μm (zoomed out) and 100 μm (zoomed in) (D), 100 μm (F), and 200 μm (H).

Fig 4: Immunostaining of hair follicloids. (a) Histological analysis of hair follicloids. Hair follicles were sectioned and stained with fluorescently labeled antibodies against K14, AE13, and versican. (b) Schematics of K14, AE13, and versican expression in hair follicloids.

Fig 5: Characterization of a microfluidic human vagina-on-a-chip model. A A diagram of a two-channel organ chip viewed from above (left) and a higher magnification micrograph through a cross section of a vagina chip showing a stratified human vaginal epithelial cells cultured in the top channel atop a 50-μm thick porous membrane (dashed lines indicate top and bottom surface) with human uterine fibroblasts cultured on the opposed side of the membrane in the lower channel (right). B Representative cross-sectional immunofluorescence micrographs of human vagina chips showing squamous stratified vaginal epithelium immunostained for CK14, CK15, CK5, CK13, Involucrin, ZO-1, E-cadherin, DSG-3, DSG-1, and F-actin (to show all cells). Dashed line, upper boundary of the porous membrane; yellow, different markers; magenta, DAPI-stained nuclei. C A graph showing the changes in apparent permeability (P app) of the vaginal tissue barrier measured on-chip for two human donors 05328 (Hispanic) and 04033 (Caucasian) measured by quantifying Cascade Blue transport. Data are presented as mean ± s.d.; n = 4. D RT-qPCR results showing relative mRNA expression of ESR1, PGR, PCK1, GCGR, KRT15, CLDN17, and ZO-1 in the vagina chip before (D0) and after differentiation on day 10 in the presence or absence of 0.4 nM (blue) or 4 nM (green) β-estradiol (D10)