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. 2009 Sep;119(9):2795-806.
doi: 10.1172/JCI38535. Epub 2009 Aug 3.

A Role for Pericytes as Microenvironmental Regulators of Human Skin Tissue Regeneration

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Free PMC article

A Role for Pericytes as Microenvironmental Regulators of Human Skin Tissue Regeneration

Sophie Paquet-Fifield et al. J Clin Invest. .
Free PMC article

Abstract

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

Figures

Figure 1
Figure 1. mAb HD-1 recognizes a subpopulation of pericyte-like dermal cells in close contact with the epithelium.
(A) In situ immunostaining with mAb HD-1 on 3-μm-thick frozen sections of neonatal foreskin. HD-1–positive cells (green, FITC) were detected in the dermis underlying basal epithelial keratinocytes. Nuclei are stained red with propidium iodide. This image is representative of more than 50 independent samples. Original magnification, ×10. (B) Single color flow cytometric analysis of a typical human dermal cell suspension stained with mAb HD-1 (green line) or an isotype-matched control (IgG1, red line), illustrating the presence of HD-1dim and HD-1bri fractions. A total of 10,000 7AAD-negative (i.e., living) cells were analyzed. (C and D) In situ immunostaining of frozen sections of human upper dermis costained with mAb HD-1 (green) and the endothelial marker vWF (red, C) illustrating that mAb HD-1 recognizes pericytes surrounding the vWF-positive endothelial cells lining the blood vessel. (D) Colocalization of the pericyte marker ACTA2, or SMA (red, D), and the HD-1 antigen (green). Nuclei are stained blue with DAPI. Original magnification, ×60. (E and F) Cytospins of the HD-1dim and HD-1bri fractions of human neonatal foreskin dermal cell isolates collected by FACS and stained for the pericyte marker ACTA2. The HD-1bri cells (E) were positive for ACTA2, in contrast to the HD-1dim cells (F). These pictures are representative of 3 independent sorting and staining experiments. Original magnification, ×20.
Figure 2
Figure 2. HD-1dim cells and HD-1bri cells display a distinct molecular profile.
Microarray analyses of neonatal human dermal HD-1bri and HD-1dim populations from 4 independent FACS experiments, visualized using Treeview software, obtained by displaying a hierarchical cluster with average linkage analysis of normalized gene expression (>2-fold change, adjusted P values less than 0.05). These data illustrate the 2,288 probe sets differentially overexpressed (red) or underexpressed (green) in the HD-1bri cells compared with the HD-1dim population. The gene expression patterns are consistent among the 4 replicate experiments.
Figure 3
Figure 3. The inclusion of HD-1bri pericytes in the dermal equivalent of OCs reconstituted with P7 HFFs stimulates epidermal regeneration capacity of α6dim differentiating keratinocytes.
Sections of OCs stained with H&E showing reconstituted skin tissue from α6bri (stem and TA) keratinocytes (AC) or α6dim early differentiating keratinocytes (DF) grown on dermal equivalents containing either P7 HFFs alone (A and D), P7 HFFs plus 10% HD-1bri cells (B and E), or fresh HFFs depleted of HD-1bri cells (C and F). The intrinsically low epidermal tissue-regenerative capacity of α6dim keratinocytes is substantially enhanced by coculture with dermal equivalents containing HD-1bri cells (E; n = 3), while depletion of HD-1bri cells diminishes their tissue-regenerative capacity (F; n = 2). Notably, the α6bri stem and TA–containing fraction exhibits excellent tissue regeneration irrespective of the presence of pericytes (AC; n = 3). (GI) Ki67 staining of OCs of α6bri (stem and TA) keratinocytes (G) or α6dim early differentiating keratinocytes (H and I) grown on dermal equivalents containing either P7 HFFs alone (G and H) or P7 HFFs plus 10% HD-1bri cells (I). Note the increase in cellularity and number of Ki67-positive cells in the α6dim epithelium upon coculture with the HD-1bri cells compared with P7 HFFs alone (I versus H), restoring epithelial regeneration to levels comparable to those obtained with the α6bri stem and TA compartment (G). Scale bars: 100 μm.
Figure 4
Figure 4. Human dermal pericytes synthesize and secrete LAMA5 in the skin.
(A) Quantification of LAMA5 mRNA expression by RT-PCR in freshly sorted epidermal cell populations from human neonatal foreskin. LAMA5 mRNA was highly expressed in unfractionated human foreskin keratinocytes (HFK), KSCs, and TA keratinocytes; and downregulated in early differentiating keratinocytes (ED). LAMA5 was undetectable in HFFs. (B) LAMA5 mRNA expression by RT-PCR in HD-1bri cells, HD-1dim cells, and HFFs, illustrating overexpression in the HD-1bri pericytes. In A and B, data represent results from 3 independent sorting experiments; mean ± SEM is shown. (CI) Immunogold electron microscopic localization of human LAMA5 with mAb 4C7 in skin sections. LAMA5 was detected in the basement membrane between the endothelial cells (En) of blood vessels and the surrounding pericytes (P) (C and F); in the basement membrane region between the epidermis (E) and dermis (D) (G); and at hemidesmosomes (HD) (I). In addition, immunogold particles were also localized abluminally, i.e., secreted into the dermal space at the periphery of pericytes (D). Labeling with isotype-matched negative control mAb 1D4.5 showed the absence of staining in skin sections, as shown for blood vessels (E) and the epidermal-dermal junction (H). (D and F) Higher-magnification views of regions indicated in C. Scale bars in CF: 10 μm; G and H: 0.5 μm; I: 0.1 μm.
Figure 5
Figure 5. Increased deposition of LAMA5 by α6dim keratinocytes cocultured with HD-1bri pericytes in OCs.
Immunofluorescence staining for LAMA5 (mAb 4C7, red) and HD-1 antigen (green) performed on 3-μm frozen sections of OCs, showing epithelial sheets regenerated by α6bri (combined stem and TA; A and B) or α6dim early differentiating keratinocytes (C and D) seeded on a dermal equivalent containing either P7 HFFs alone (A and C) or P7 HFFs plus HD-1bri pericytes (B and D). Nuclei were counterstained with DAPI (blue). LAMA5 immunostaining in the epidermal-dermal junction was observed in all OCs reconstituted with α6bri keratinocytes, irrespective of the presence of pericytes (A and B). In contrast, OCs reconstituted with α6dim keratinocytes cocultured with HD-1bri pericytes (D) displayed largely increased LAMA5 levels compared with controls (C). HD-1 staining revealed the localization of pericytes in close proximity to the epidermal cells in OCs, where they were added back to the dermal equivalents (B and D). Original magnification, ×20.

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