Application of extracellular vesicles from mesenchymal stem cells promotes hair growth by regulating human dermal cells and follicles

Figure 1 Isolation and characterization of human mesenchymal stem cell-derived extracellular vesicles. A: The morphology of human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) was confirmed using transmission electron microscopy (scale bars: 500 and 100 nm); B: hMSC-EV size was determined using nanoparticle tracking analysis (n = 5); C: Western blot analysis using Alix, cytochrome C, GM130, PCNA, and Wnt3a antibodies on hMSCs and hMSC-EVs; D and E: Flow cytometry count graphs of only beads, control 1 (beads + hMSC-EVs), control 2 [beads + hMSC-EVs + Secondary fluorescein isothiocyanate (FITC) antibody], and Wnt3a (beads + hMSC-EVs + Wnt3a antibody + Secondary FITC antibody) (n = 3). The values obtained from experiments are shown mean ± SD. FITC: Fluorescein isothiocyanate; hMSC-EVs: Human mesenchymal stem cell-derived extracellular vesicles, NTA: Nanoparticle tracking analysis.

Figure 2 Interaction of human mesenchymal stem cell-derived extracellular vesicles with dermal papillae cells leads to cell proliferation and activation of Wnt/β-catenin signaling. A: Dermal papillae (DP) cells incubated for 2 h with non-labeled human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) (10 μg/mL) and DiD-labeled hMSC-EVs (5 and 10 μg/mL; hMSC-EVs/DiD) (scale bar: 20 μm); B: DP cell proliferation was determined using a CCK8 assay 24 h after treatment with 0–10 μg hMSC-EVs (n = 5); C: β-catenin immunofluorescence assay in DP cells after 24 h of treatment with hMSC-EVs (10 μg/mL) (scale bar: 20 μm); D: The levels of β-catenin in the nuclear fraction of DP cells treated with hMSC-EVs (5 and 10 μg/mL) with histone H3 used as a loading control for nuclear fraction; E: Quantitative real-time polymerase chain reaction results of mRNA expression of Axin2, EP2 and LEF1 in DP cells treated with hMSC-EVs (5 and 10 μg/mL) for 24 h (n = 3). The values obtained from experiments are shown mean ± SD (^bP < 0.01; ^cP < 0.001. Student’s t-test was used for comparison). hMSC-EVs: Human mesenchymal stem cell-derived extracellular vesicles; DP: Dermal papillae.

Figure 3 Interaction of human mesenchymal stem cell-derived extracellular vesicles with outer root sheath cells leads to cell proliferation, migration, and differentiation. A: Outer root sheath (ORS) cells incubated for 2 h with non-labeled human mesenchymal stem cell-derived extracellular vesicles (hMSC-EVs) (5 μg/mL) and DiD-labeled hMSC-EVs (2.5 and 5 μg/mL; hMSC-EVs/DiD) (scale bar: 20 μm); B: ORS cell proliferation was determined using a CCK8 assay 24 h after treatment with 0–5 μg/mL hMSC-EVs (n = 4); C and D: Phase-contrast microscopy images of migrated ORS cells 24 h after treatment with hMSC-EVs (2.5 and 5 μg/mL; scale bar: 50 μm); the quantified data of migrated cells are shown in (A) (n = 3); E: Quantitative real-time polymerase chain reaction results of mRNA expressions of keratin (K) 6, K16, K17, and K75 in ORS cells treated with hMSC-EVs (5 and 10 μg/mL) for 24 h (n = 3). The values obtained from experiments are shown mean ± SD (^aP < 0.05; ^bP < 0.01; ^cP < 0.001. Student’s t-test was used for comparison). NS: Not significant; hMSC-EVs: Human mesenchymal stem cell-derived extracellular vesicles; ORS: Outer root sheath.

Figure 4 Human mesenchymal stem cell-derived extracellular vesicles treatment promoted human hair follicle shaft elongation. A: Representative images of human hair follicles of an individual after human mesenchymal stem cell-derived extracellular vesicles (0, 0.05, and 0.01 μg/mL) and XAV939 (5 μM) treatments; B: Quantified data of hair shaft elongation on day 6 (n = 6). (^aP < 0.05; ^cP < 0.001. Student’s t-test was used for comparison). hMSC-EVs: Human mesenchymal stem cell-derived extracellular vesicles.