Exosomal Micro RNAs Derived from Dermal Papilla Cells Mediate Hair Follicle Stem Cell Proliferation and Differentiation

Abstract

Recent studies have demonstrated that dermal papilla cell-derived exosomes (DPC-Exos) promote the anagen stage of hair follicle (HF) growth and delay the catagen stage. However, the roles of DPC-Exos in regulating hair follicle stem cell (HFSC) quiescence and activation remain unknown. Here, we found that HFSC differentiation was induced by co-culture with DPCs, and that DPC-Exos attached to the surface of HFSCs. Using micro RNA (miRNA) high-throughput sequencing, we identified 111 miRNAs that were significantly differentially expressed between DPC-Exos and DPCs, and the predicted target genes of the top 34 differentially expressed miRNAs indicated that DPC-Exos regulate HFSCs proliferation and differentiation via genes involved in cellular signal transduction, fatty acid expression regulation, and cellular communication. The overexpression of miR-22-5p indicated that it negatively regulates HFSC proliferation and LEF1 was revealed as the direct target gene of miR-22-5p. We therefore propose the miR-22-5p-LEF1 axis as a novel pathway regulating HFSC proliferation.

Keywords: dermal papilla; exosome; hair follicle stem cells; proliferation.

Figure 1

Isolation and characterization of hair follicle stem cells (HFSCs) from the Shaanbei White cashmere goat. A. Adhesion culture of hair follicles. Scale bar, 1000 μm. B. Fluorescence-activated cell sorting of CD34. C. HFSCs after purification. Scale bar, 1000 μm. D. Observation of HFSCs by transmission electron microscopy. Scale bar, 2 μm. E. Growth curve of HFSCs. Passage five HFSCs were seeded in 24-well plates at a density of 1 × 10⁴ cells/mL and cell counts were performed at 24 h intervals using a hemocytometer. Three wells were counted for each time point, and the mean cell number was calculated to plot a cell growth curve. F. Protein expression of HFSC markers. G. Immunocytochemical staining of HFSCs markers. Scale bar, 100 μm. H. Relative mRNA expression levels of HFSC markers.

Figure 2

Identification and functional analysis of exosomes derived from dermal papilla cells (DPCs). A. Detection of CD63 and Canx in exosomes. B. Analysis of exosome size by nanoparticle tracking analysis. C. Observation of DPC-derived exosomes by transmission electron microscopy. Scale bar, 1 μm. D. Transport of extracellular vesicles (EVs) in co-culture without physical contact between different cell types. Transwell systems were used to separate recipient cells from DiI-labeled DPCs, with DiI-labeled DPCs in the upper filter and hair follicle stem cells (HFSCs) in the lower chamber. Scale bar, 100 μm. E. DiI-labeled exosomes derived from DPCs (DPC-Exos) were taken up by HFSCs (DiI label is shown in red, 4′,6-diamidino-2-phenylindole label is shown in blue). Scale bar, 200 μm. F. Hematoxylin and eosin staining results of goat hair cells in the anagen and telogen phases. Red arrows show the location of dermal papillae. Scale bar, 200 μm.

Figure 3

miRNA expression profiles of exosomes derived from dermal papilla cells (DPC-Exos). A. Schematic diagram of relative miRNA expression between samples. X- and Y-axes represent the sample log10 (TPM+1) and the square of Pearson correlation coefficients (R2), respectively. B. Venn diagram showing the unique and overlapping miRNAs between DPCs and DPC-Exos. Numbers in parentheses are the numbers of differentially expressed miRNAs in DPCs and DPC-Exos. C. Heatmap diagram of differential miRNA expression between DPCs and DPCs-Exos. Red, blue, and white colors indicate increased expression, decreased expression, and mean values, respectively. Each row in the figure represents one miRNA and each column shows one sample. Each cell shows the differential expression of each miRNA. D. Volcano plot of known differential miRNAs. The X-axis represents the miRNA fold change in different groups or samples, whereas the Y-axis represents the statistical significance of the change in miRNA expression. Circles represent miRNA: green circles denote miRNAs that were not significantly expressed, red circles denote significantly up-regulated miRNAs, blue circles denote significantly down-regulated miRNAs. E. Volcano plot of novel differential miRNAs. F. Quantitative real-time PCR validation of miRNAs expression between DPCs and DPC-Exos. The abundances of miR-1, miR-122, miR-378-3p, miR-145-5p, let-7f-5p, miR-21-5p, miR-592, and miR-106a-5 were normalized to the abundance of miRNA 39 (* P <0.05).

Figure 4

Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. A. Histogram depicting candidate target gene GO enrichment. The X-axis represents the number of target genes associated with a unique term (and corresponding sub-terms). The Y-axis denotes the GO terms. The three different classifications represent the three basic GO term classes (from top to bottom, biological process, cellular component, and molecular function). B. Candidate target gene KEGG enrichment scatter plot. The X-axis represents pathways and the Y-axis represents the enrichment factor. The size of dots indicates the number of target genes in a pathway, and the color of dots indicates the different range of q-values. C. Enrichment map of biological processes targeted by the top 34 differentially expressed miRNAs in DPC-Exos. Nodes represent the 29 genes associated with HFSCs and hair shaft growth and the nine candidate miRNAs that target them, which are connected by the edges.

Figure 5

miR-22-5p inhibits hair follicle stem cell (HFSC) proliferation by targeting LEF1. A. CCK-8 was used to detect the effect of miR-22-5p on HFSCs. B-C. The statistical results and representative figures of the effect of miR-22-5p on the proliferation of HFSCs as detected by EdU. ** P <0.01; Scale bar, 400 μm. D-E. Effect of miR-22-5p on the cell cycle of HFSCs. * P <0.05. F. Dual-luciferase reporter assay. * P <0.05, ** P <0.01.