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, 6 (1), 74-84

Identification of Drugs That Regulate Dermal Stem Cells and Enhance Skin Repair

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Identification of Drugs That Regulate Dermal Stem Cells and Enhance Skin Repair

Sibel Naska et al. Stem Cell Reports.

Abstract

Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs), a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Figures

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Figure 1
Figure 1
Identification of Compounds that Enhance Self-Renewal and Proliferation of Cultured SKPs (A–C) Number of SKP spheres generated from secondary human SKPs grown for 7 days in varying concentrations of alprostadil (Alp) (A) or TM (B) or in 100 nM alprostadil, TM, kaempferol (Kae), MG-624, or pramoxine (Pram) (C). In (C) numbers are expressed relative to DMSO alone. (D–F) Number of SKP spheres generated from secondary neonatal rat SKPs grown for 7 days in 100 nM of each of the five drugs (D), or in varying concentrations of alprostadil (E) or TM (F). (G and H) Number (G) and size (H) of rat SKP spheres generated over 7 days in 100 nM alprostadil, TM, or both. (I) Number of rat SKP spheres generated in 14-day clonal methylcellulose assays with 100 nM alprostadil, TM, or latanoprost (Latan). (J and K) Secondary rat SKP spheres were grown for 4 days, and 100 nM alprostadil or TM was added for two additional days. (J) shows spheres immunostained for Ki67 (top) and counterstained with DAPI to show nuclei (bottom). (K) shows the percentage of Ki67-positive cells. Scale bar represents 100 μm. (L) NIH 3T3 cells were treated for 24 hr with 100 nM alprostadil or TM, immunostained for Ki67, and the percentage of positive cells determined. N.S., not significant. In all panels, results were pooled from 3 to 4 independent experiments with, in the human experiments, 3–4 different human SKP lines. Error bars indicate SEM, and in all cases p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with multiple comparison post hoc tests. See also Figure S1.
Figure 2
Figure 2
Topical Alprostadil or TM Promotes Skin Repair in Middle-Aged Mice (A and B) Scatterplots showing wound closure at 7 days (A) and 9 days (B) after injury for 9-month-old mice treated daily with alprostadil (Alp), TM, or vehicle alone (Control). n = 10 or 14 mice per group, from three independent experiments. (C–F) H&E-stained sections through the center of the wound bed 9 days after injury (C) were analyzed for wound width (D), epithelial gap (E), and new dermal tissue (F). n = 18, 14, and 17, vehicle-, alprostadil-, and TM-treated mice total, from three independent experiments. NE indicates the new epithelium, RD the regenerating dermis, and OW the borders of the wound. Scale bar represents 1 mm. (G–J) Sections from the center of the wound bed of mice 7 days after injury were immunostained for Ki67 (G and H) or CD31 (I and J), counterstained with hematoxylin, and analyzed for the percentage of Ki67-positive cells (H) or the relative number of CD31-positive blood vessels (J) at the leading edge of the regenerating dermis. n = at least nine mice per group in (H) and three per group in (J). Insets in (G and I) show higher magnification images, with arrows denoting positive cells (black or dark brown). LE indicates the leading edge of the new epidermis, and RD the regenerating dermis. Scale bar represents 125 μm. Error bars indicate SEM, and in all cases p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with multiple comparison post hoc tests except for (E), where comparisons were made by pairwise Student's t tests.
Figure 3
Figure 3
Topical Treatment of Mouse Skin with Alprostadil or TM In Vivo Alters the Self-Renewal of SKPs Cultured from the Treated Skin in the Absence of Drug, and Acute Drug Treatment of Cultured SKPs Causes Changes in Gene Expression (A–D) Primary SKPs were isolated from 9-month-old mice that had received punch wounds (Inj Skin; A and B) or that had intact skin (Uninj Skin; C and D) and that were then treated with topical vehicle (Control), alprostadil (Alp), or TM for 7 days. The primary SKPs were passaged into medium without drugs and the number (A and C) and size (B and D) of secondary spheres were quantified. n = at least three independent experiments each and error bars indicate SEM. p < 0.05, ∗∗p < 0.01, ∗∗∗p < .001, one-way ANOVA with multiple comparison post hoc tests except for (B), where pairwise comparisons of control versus alprostadil or TM were made with Student's t test. (E and F) mRNA from secondary SKP spheres generated as in (A–D) were analyzed on Affymetrix GeneChip Mouse Gene 2.0 ST arrays. (E) Heatmap and hierarchical clustering of all the samples based on probesets that differed by p < 0.01 (unadjusted) regardless of the fold change when comparing SKPs from vehicle-treated, injured skin versus alprostadil-treated injured skin. Three independent replicates of SKPs from vehicle-treated injured skin (Con Inj), alprostadil- or TM-treated injured skin (Alp Inj or TM Inj), and vehicle-treated uninjured skin (Con Uninj) were compared. (F) Venn diagram comparing the overlap of genes that differed by p < 0.01 (unadjusted) from pairwise comparisons of SKPs from vehicle (Control) versus TM- or alprostadil-treated injured skin. (G–K) Three independent preparations of primary neonatal rat SKPs were dissociated and cultured for 24 hr in alprostadil, TM, or vehicle (Con), and mRNA was isolated and analyzed on an Affymetrix GeneChip Rat Gene 2.0 ST array. (G) Heatmaps of pairwise comparisons of vehicle versus alprostadil- or TM-treated SKPs showing the average of the raw expression (log2) data for probesets that differed by p < 0.01 (unadjusted) with a fold change of greater than 1.1. (H and I) Venn diagrams comparing the overlap of all genes that differed by p < 0.01 (unadjusted) (H) or the top 50 most significantly changed genes (I) from pairwise comparisons of vehicle versus TM- or alprostadil-treated SKPs. The 23 overlapping genes in (I) are shown underneath. (J and K) Gene ontology (GO) enrichment analysis for gene categories that were significantly different in the comparisons of vehicle (Ctrl) versus TM- or alprostadil-treated SKPs showing highly significant enrichment for categories involving cell proliferation (J) and the MAP kinase (MAPK) pathway (K). See also Figure S2 and Tables S1, S2, S3, S4, S5, S6, S7, S8 and S9.
Figure 4
Figure 4
Signaling via the MEK-ERK Pathway Is Necessary for Basal and Drug-Induced SKPs Self-Renewal (A–C) SKPs were treated with alprostadil (Alp), TM, or vehicle (Control) for 10 min, and lysates probed on western blots with anti-phosphorylated ERK1/2 T202/Y204 (pERK) (A) or anti-phosphorylated STAT3 (C) and reprobed for total ERK1/2 or total STAT3. (B) shows quantification by scanning densitometry of three experiments as in (A), where pERK was normalized to total ERK1/2. (D) Western blot of SKPs treated with varying concentrations of trametinib (MEKi) or DMSO (Control) for 30 min, probed for pERK1/2 and reprobed for total ERK1/2. (E and F) Number (E) and size (F) of SKP spheres generated in clonal methylcellulose assays from secondary neonatal rat SKPs grown 14 days in 100 nM trametinib or DMSO. (G and H) Secondary rat SKPs were cultured for 4 days, and 10 or 100 nM trametinib added for 2 more days. (G) shows spheres immunostained for Ki67 (top) and counterstained with DAPI (bottom). (H) shows the percentage of Ki67-positive cells. Scale bar represents 100 μm. (I) Western blot of SKPs treated with 100 nM trametinib for 30 min and coincidently stimulated with 100 nM alprostadil or TM for the final 10 min, probed for pERK1/2, and reprobed for total ERK1/2. (J) Number of rat SKP spheres generated over 7 days in 100 nM alprostadil or TM with or without 100 nM trametinib. (K) Heatmap comparing microarray data for three independent preparations of dissociated rat SKPs cultured for 24 hr in TM with or without trametinib, focusing on 29 of the top 50 genes that were altered by TM relative to control (see Figure 3), and that were significantly normalized by MEK inhibition. The heatmap shows the average of the raw expression (log2) data for probesets that differed by p < 0.01 (unadjusted). In all cases, results are pooled from at least three independent experiments. Error bars indicate SEM, and ∗∗p < 0.01, ∗∗∗p < 0.001, one-way ANOVA with multiple comparison post hoc tests, except (E and F), which were analyzed by Student's t test.

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