Integrin β1

The main reagents include DMEM/F12 (1:1) medium, knockout serum replacement (KSR), valproic acid, fetal bovine serum, Essential 8™ Flex Medium, Geltrex™, KSFM medium, BMP‐4, bovine pituitary extract (BPE), (Gibco), NANOG, OCT4, SOX2, SSEA‐4, TRA‐1‐81, TRA‐1‐60, Krt19, Integrinβ1, CD200, CD31 and VEGF‐A antibodies (Abcam), PDGF‐B and Ang2 antibodies (Santa Cruz), recombinant human EGF (R&D), RA, valproic acid, sodium alginate (Sigma), Matrigel^® Matrix (Corning), mTeSRTM1 medium (Stem Cell), Astragalus polysaccharide (Solarbio), silk fibroin and collagen (Hefei Bomei Bio), and Dextran Texas red™ (Invitrogen). Primers were designed using Primier 6.0 software, and all primers were synthesized as shown in Table 1.

rHFSCs (P3) were inoculated on a slide and cultured for 2 days. Cells were rinsed with PBS–Tween, fixed with 4% (w/v) paraformaldehyde (Kelong Chemical Reagent Company, Chengdu, China) and blocked with 5% (w/v) bovine serum albumin at room temperature. Integrin β1 (1:100; Abcam, Cambridge, UK), integrin α6 (1:50; Abcam) and CK15 (1:100; Abcam) antibodies were then added separately. PBS–Tween was added instead of primary antibody in the control group. After incubation at room temperature and washing with PBS–Tween, fluorescein-labeled secondary antibody was added (1:100; Jackson, San Francisco, CA, USA) and incubated in the dark for 30 minutes. 4′,6-Diamidino-2-phenylindole (1:2,000 dilution; Roche, La Roche, Switzerland) was then added and incubated for 5 minutes for nuclear staining. Cells were then air dried in the dark, mounted with Mounting Solution and observed using fluorescence microscopy (Olympus, Tokyo, Japan).

Cell surface antigens on cells were evaluated with FACS. The cells were dissociated with 0.05% trypsin/EDTA (Highclone), washed with PBS, fixed with 4% paraformaldehyde, permeabilized with Triton X‐100, and blocked with a BSA mixture. The samples were then stained with antibodies against human octamer‐binding transcription factor 4 (OCT4; R&D systems, 1:200), stage specific embryonic antigen 1 (SSEA1; R&D systems, 1:200), cluster of differentiation 31 (CD31‐FITC; Miltenyi Biotec, 1:100), CD34 (CD34‐PerCP, BioLegend, 1:100), human leukocyte antigen ‐ DR isotype (HLA‐DR, HLA‐DR‐APC, BioLegend, 1:100), CD73 (CD73‐PE, BioLegend, 1:100), CD90 (CD90‐APC, BioLegend, 1:200), CD105 (CD105‐FITC, BioLegend, 1:150), integrin α5 (Santa Cruz, 1:200), integrin α11 (Abcam, 1:200), integrin β1(Abcam, 1:200), and integrin β5 (BioLegend, 1:200) for 30 min or 1 h at 4 °C. Samples were subsequently stained with fluorescently labeled secondary antibodies (Alexa Fluor‐488 or 594 conjugated goat antirabbit (Abcam, 1:400), Alexa Fluor‐488 or 594 conjugated goat antimouse (Abcam, 1:400)) for 30 min at 4 °C. The corresponding mouse/rabbit isotype antibodies (Abcam, 1:200) were used as controls. Cell immunotypes were determined with the Accuri C6 flow cytometer (BD Biosciences) and the percentage of expressed cell surface antigens was calculated for 10 000 gated‐cell events.