Effects of human pericytes in a murine excision model of wound healing Stuart J. Mills 1,2 , Lizhe Zhuang 1 , Philip Arandjelovic 1 , Allison J. Cowin 2 and Pritinder Kaur 1,3 1 Epithelial Stem Cell Biology Laboratory, Research Division, Peter MacCallum Cancer Centre, Melbourne, Vic., Australia; 2 Regenerative Medicine, Mawson Institute, University of South Australia, Mawson Lakes, SA, Australia; 3 Sir Peter MacCallum Department of Oncology/Department of Anatomy & Neuroscience, University of Melbourne, Melbourne, Vic., Australia Correspondence: Pritinder Kaur, Epithelial Stem Cell Biology Laboratory, Research Division, Peter MacCallum Cancer Centre, St Andrew’s Place, Melbourne, Vic. 3002, Australia, Tel.: +61 3 9656 3714, Fax: +61 3 9656 1411, e-mail: pritinder.kaur@petermac.org Key words: pericytes – skin – stem cells – wound healing Accepted for publication 6 May 2015 Background We previously reported that dermal pericytes profoundly enhance the regenerative capacity of human keratinocytes, above that of dermal fibroblasts in an angiogenesis-free organotypic model (1), a property quite distinct from their well-known role in regulating blood vessel structural stability. Given the mesenchymal stem cell/ MSC-like multilineage differentiation capacity of pericytes (1,2), we hypothesised that pericytes represent a potent stem cell population with a pro-proliferative role in organ growth, repair and regeneration. Questions addressed Do dermal pericytes promote healing in an excision wound model? Experimental design Four mm punch ‘biopsies’ of human neonatal foreskin dermal CD45 À VLA-1/a1/CD49a bri pericytes, CD45 À VLA-1 dim fibroblasts or both cell types (1), implanted into collagen type 1 gels, were placed directly onto 4 mm excisional wounds on the dorsal skin of BALB/c SCID male mice and secured in place with gauze and 3M Tegaderm TM . Wounds were left to heal and harvested at 3 and 7 days (d3/7) postwounding. Results Wound healing assessed by measuring wound widths at d3 (Fig. 1a,c) or wound areas and rate of re-epithelialization showed no statistically significant differences when inoculated with fibro- blasts, pericytes or a combination of both, compared to collagen controls (n = 3 independent experiments). By d7, although all wounds showed an overall decrease in wound width compared to d3, and complete re-epithelialisation, the pericyte-inoculated wounds displayed the greatest wound width of all conditions (Fig. 1b,d), suggesting a delay in dermal wound resolution in their presence. Notably, Ki67 + epidermal cells were abundant in all the wounds, with no significant differences in their numbers in the presence of pericytes (data not shown). Importantly, we confirmed that the inoculated human fibroblasts and pericytes were still pres- ent in the collagen gels at d7 (not detected in collagen controls), albeit at low frequency, using in situ hybridization for the human ALU element, (Fig. 2a). Given the importance of decreased inflammation in the later stages of wound healing, we stained and quantified the number of murine neutrophils (Ly-6G) and macrophages (Mac-3) in the wounds. Nota- bly, pericyte-inoculated wounds showed an increase in neutrophil numbers as early as d3 (P = 0.04), as did fibroblast-treated wounds (P = 0.014) compared to collagen controls, but not in the combined presence of pericytes and fibroblasts. Interestingly, by d7, pericyte- treated wounds continued to display a trend towards higher numbers of neutrophils compared to pericyte + fibroblast (Fig. 2b) not achiev- ing statistical significance (P = 0.08). Concomitantly, macrophage numbers remained elevated in the pericyte-treated wounds compared to fibroblast (P = 0.002) and combined pericyte-/fibroblast-treated wounds (P = 0.0013) at d7 (Fig. 2c). One possibility is that pericytes induce a prolonged inflamma- tory response – we sought to confirm this by determining whether the expression of a central regulator of wound healing, that is TGF-b1 and Smad3 – one of its major downstream targets affect- ing key tissue remodelling processes (3). Although the TGF-b (a) (b) (c) (d) Figure 1. Pericytes delay dermal wound resolution in an excision model. (a, b) Representative H&E staining of day 3 (a) & day 7 (b) wounds treated with collagen alone (COL), collagen gels containing fibroblasts (HFF), pericytes (PERI) or fibroblasts + pericytes (HFF + PERI at 10:1 ratio) with 7.5 9 10 3 cells/gel. Arrows in lower magnification top panels indicate wound margins, and boxed areas are shown at higher magnification images (left and right lower panels) indicating incomplete re-epithelialisation at day 3 (a), and indicated areas showing complete re-epithelialisation by day 7 (b) in all conditions. (a, b) Top panels: scale bar = 100 lm; lower high magnification panels: scale bar = 50 lm. (c, d) Quantitation of wound widths at day 3 (c) and day 7 (d) from all experimental groups shown as mean Æ SEM of 2 replicate experiments, 10 animals/group. ª 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd Experimental Dermatology, 2015, 24, 881–901 881 DOI: 10.1111/exd.12755 www.wileyonlinelibrary.com/journal/EXD Letter to the Editor