Fig 2: Lgr5 function is not required for mammary transplantation capacity. a Single-cell suspensions were prepared from Lgr5KIHET and Lgr5KIHOM littermates at stage E18. Live, single, EpCAMHIGH; CD49fHIGH; GFPPOS cells were sorted and injected into cleared fat pads of recipient mice. Eight weeks later, glands were isolated and carmine stained to visualize outgrowth and take rates were calculated as the fraction of positive outgrowths per number of injections. b Three thousand fMaSCs from Lgr5KIHET and Lgr5KIHOM littermates were transplanted into four fat pads each and outgrowths were produced as described in a. Outgrowths were dissociated and single cells were pooled and measured for GFP, EpCAM, and CD49f expression using FACS. c Four thousand cells from the EpCAMMED; CD49fHIGH population described in Fig. 4b were re-transplanted into two recipient, cleared fat pads for each genotype. Eight weeks later, secondary transplants were carmine stained to visualize epithelium of secondary outgrowths. Images are representatives of three positive outgrowths for each genotype

Fig 3: Lineage plasticity of basal and mesenchymal tumor cell populations isolated from mouse and human bladder tumors.a Cytokeratin 5 tomato reporter (KRT5ERT2-LSL) mice showing that CK5-positive basal cells are enriched for CD49f expression in normal bladders (top). Subsequent to OHBBN-induced carcinogenesis, Tdt cells show heterogeneity in CD49f and Epcam expression (bottom). Right bar graphs, distribution of Tdt-positive;Epcam-CD49f-positive cells in normal and OHBBN-induced bladder cancer. b In-vivo transplantation of CD49f-high and CD49f-low tumor populations. Sorted and transplanted basal (GFP+EpcamhighCD49fhigh) cells or mesenchymal cell populations (GFP+EpcamlowCD49flow) were implanted to the bladder muscle wall of FVB mice and evaluated for tumor formation. Formed tumors were dissociated to single cells and assessed for redistribution of Epcam-CD49f expression according to the defined quadrants (Q1, Q2, Q3, and Q4) after progression. c CD49f-low cells were isolated by flow cytometry from an early (P0) passage PDX model of MIBC (tumor 15934) followed by transplantation to immune compromised mice. Expression analysis of derived tumors under the same flow cytometry settings, shows marked redistribution and increased expression in CD49f (n = 5). Lower, representative histology of resulting tumor outgrowths derived from CD49flowEpcamlow cells (bar = 100 µM). Individual data points are represented by circles, the error bars represent mean ± SD of the three independent replicates. Each experiment was performed at least six times.

Fig 4: Single-cell RNA-seq analysis of interfollicular keratinocytes, related to Figure 3WT mice were treated daily with either vehicle control (S. epi + vehicle) or antiretroviral (S. epi + antiretroviral) beginning at 1 week before S. epidermidis association for a total of 3 weeks. At day 14 post association, interfollicular keratinocytes were sorted by FACS from the epidermis of ear pinnae and analyzed by single-cell RNA-seq. (A) UMAP projection plots showing the expression profiles of keratinocytes. Colors represent cells clustered together based on similarity of global gene expression. Cells identity was assigned based on the expression level of specific genes: Interfollicular epidermal basal cells: Krt14hiMt2hiMt1hiPostnlow; Interfollicular epidermal differentiated cells subcluster 1 (D1): Krt14midKrt10midMt4hi; Interfollicular epidermal differentiated cells subcluster 2 (D2): Krt14lowKrt10hiSbsnhi;Interfollicular epidermal keratinized cells subcluster 2 (K2): Krt14lowKrt10hiLorhiFlg2hi; Upper hair follicle: Krt79hiKrt17hiDefbhi; and Infundibular basal cells: Krt14midMt2midMt1midPostn+. (B) Schematic illustrating the anatomical localization of distinct populations of keratinocytes. (C) UMAP projection plots depicting expression of the indicated genes that are involved in antigen presentation on MHC class II. Data are representative of one experiment using 5 mice per group. (D) MLV SU expression (shown as integrated MFI) detected by flow cytometry on the surface of Sca-1+CD49f+MHCII+ and Sca-1+CD49f+MHCII– keratinocytes from unassociated or S. epidermidis-associated mice at day 7 post-association. ****P ? 0.0001 as calculated with one-way ANOVA with Holm-Šidák multiple comparison test (D). Data are representative of one (A and C) or three (D) independent experiments using 5 mice per group.

Fig 5: Cell culture and characterization between robotic bioprinting and manual implantation. (a and b) Cell viability of epidermal stem cells (Epi-SCs) and skin-derived precursors (SKPs) cultured for 7 days after robotic bioprinting and hand implantation. (c and d) Cell proliferation of Epi-SCs and SKPs cultures for 7 days after robotic bioprinting and hand implantation. ****p < 0.0001, ***p < 0.001, **p < 0.01. (e) Flow cytometry analysis showed high expression of CD29 and CD49f in Epi-SCs after normal culture, robotic bioprinting, and hand implantation. (f) Real-time polymerase chain reaction analysis of SKPs for their expression of genes involved with stemness and hair genesis.* p < 0.05. (g) Alkaline phosphatase activities of SKPs in normal culture, robotic bioprinting, and hand implantation. (h–j) Immunofluorescence analysis of fibronectin (red), nestin (green), Bmp6 (red) expression in SKPs in normal culture, robotic bioprinting, and hand implantation. (k) CD49f expression of Epi-SCs in normal culture, robotic bioprinting and hand implantation (scale bar: 100 µm)