Research ArticleCELL BIOLOGY

Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling

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Science Advances  24 Jul 2020:
Vol. 6, no. 30, eaba1685
DOI: 10.1126/sciadv.aba1685
  • Fig. 1 The preparation and characterization of 3D DP spheroids.

    (A) Isolation of mouse dermal papilla (DP) cells from vibrissae. Scale bar, 500 μm. (B) Conventional culture enables the growth of 2D DP cells. Scale bar, 50 μm. (C) Growth of DP spheroids in ultralow cell culture flasks. Scale bar, 100 μm. (D) Double staining of CD133 (green) and β-catenin (red) in spheroids. Scale bar, 100 μm. (E and F) Scanning electron microscopy (SEM) images of keratin (E) and 3D spheroid–loaded keratin. (F) One obvious spheroid is highlighted in yellow. Scale bars, 100 μm. (G) Schematic illustrating the injection sites on the back of a mouse for the cell retention study. (H) The mouse was shaved and injected on the dorsal skin with different formulations as illustrated in (G). Cells were labeled by DiD and then resuspended in PBS or keratin for intradermal injection. In vivo imaging system (IVIS) images were taken at different time points. (I) Quantification of IVIS images. Data are shown as means ± SD, n = 3 mice. The 3D spheroids/keratin showed the longest retention time.

  • Fig. 2 Comparison of the hair follicle phase with topical treatment of 5% minoxidil against injecting 2D DP cells or 3D spheroids, respectively.

    (A) Illustration of the treatment side (left) and untreated side (right) of a depilated mouse. (B) The expression of β-catenin after different treatments at both the treated site and untreated site. (C) Representative images showing the expression of CD133 after different treatments at both the treated site and untreated site. (D) Representative images showing the expression of Ki67 after different treatments at both the treated site and untreated site. Scale bars, 100 μm. (E to G) Quantification of β-catenin–positive (β-catenin+) (E), CD133+ (F), and Ki67+ (G) cells. Pink indicates the treated site, and gray indicates the untreated site. n = 5; n.s., no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 3 Dorsal hair growth experiment on C57BL/6 mice.

    (A) Observation of hair coverage. Mice were divided into five groups (n = 5) and treated on their left halves. Mice were imaged on days 0, 10, 15, and 20, respectively. (B) Quantification of hair coverage on days 10, 15, and 20. Both the left (treated) and right (untreated) sites were recorded. n = 5; n.s., no significant difference; *P < 0.05, **P < 0.01, and ***P < 0.001. Photo credit: S.H., North Carolina State University.

  • Fig. 4 Western blotting and immunohistology with various treatments.

    (A) Western blot analyzing β-catenin, p-Erk1/2, Erk1/2, SFRP2, and GAPDH protein content in the dorsal skin on day 20. (B) Quantification of Western blot protein levels by group. n = 3. (C) Immunofluorescence costaining of SFRP2 and β-catenin on skin samples from different treatment groups. Scale bars, 100 μm. (D) Quantification of the relative expression of SFRP2. (E) Quantification of relative expression of β-catenin. n = 5. n.s., no significant difference; *P < 0.05, **P < 0.01, and ****P < 0.0001.

  • Fig. 5 Secretome from 2D DP cells and 3D DP spheroids.

    (A) Schematic illustrating how DP spheroids promote the hair cycle transition from catagen to telogen via the migration and secretion of factors and exosomes. In anagen, an abundant source of growing DP cells is inside the follicle bulge. DP cells drop during catagen. The replenishment of DP cells promotes the onset of anagen. (B to E) Characterization of 2D DP-XOs and 3D DP-XOs. (B) TEM images. Exosomes are indicated by yellow arrows. Scale bars, 500 μm. (C) Size distribution by NanoSight. (D) Top 10 up-regulated and 10 down-regulated miRNAs of 3D DP-XOs compared with 2D DP-XOs. (n = 3 biological replicate and n = 3 technical replicates for each biological replicate). (E) Schematic illustrating FGF2- and TIMP2-driven hair follicle regulation and miR-218-5p–induced promotion of hair follicle development.

  • Fig. 6 Effects of exosome treatment on dorsal hair regrowth.

    (A) Mice were divided into three groups (n = 4) and treated on their left sides. Mice were imaged on days 10 and 15, respectively. (B) Corresponding hair coverage analysis of different groups. Both the left and right sides were recorded. 3D DP-XOs promoted hair follicle growth more effectively than minoxidil and 2D DP-XOs. n = 4; *P < 0.05, **P < 0.01, and ***P < 0.001. (C) Immunofluorescence costaining of SFRP2 (green) and β-catenin (red) on skin samples from different treatment groups. Nuclei were stained by DAPI (4′,6-diamidino-2-phenylindole) (blue). Scale bars, 100 μm. (D) Quantification of the relative expression of SFRP2. n = 5; ****P < 0.0001. (E) Representative mice imaged on day 20 after injection of negative control, miR-218-5p mimics, and inhibitors, respectively. Red circles indicate injection spots. There is a small bald spot on the injection site, which means that the delivery approach needs further modification. (F) Quantification of hair coverage level (%) of three groups on day 15. n = 4; *P < 0.05 and **P < 0.01. (G) miR-218-5p up-regulated exosomes and miR-218-5p mimics delivered via in vivo-jetPEI can transfect miR-218-5p to target SFRP2 and thus up-regulate the WNT/β-catenin pathway; miR-218-5p inhibitor will block this signaling to a certain degree. Photo credit: S.H., North Carolina State University.

Supplementary Materials

  • Supplementary Materials

    Dermal exosomes containing miR-218-5p promote hair regeneration by regulating β-catenin signaling

    Shiqi Hu, Zhenhua Li, Halle Lutz, Ke Huang, Teng Su, Jhon Cores, Phuong-Uyen Cao Dinh, Ke Cheng

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