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Research Article
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Pericytes promote skin regeneration by inducing epidermal cell polarity and planar cell divisions

View ORCID ProfileLizhe Zhuang, Kynan T Lawlor, Holger Schlueter, Zalitha Pieterse, Yu Yu, View ORCID ProfilePritinder Kaur  Correspondence email
Lizhe Zhuang
1Peter MacCallum Cancer Centre, Melbourne, Australia
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  • ORCID record for Lizhe Zhuang
Kynan T Lawlor
1Peter MacCallum Cancer Centre, Melbourne, Australia
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Holger Schlueter
1Peter MacCallum Cancer Centre, Melbourne, Australia
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Zalitha Pieterse
2School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia
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Yu Yu
2School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia
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Pritinder Kaur
1Peter MacCallum Cancer Centre, Melbourne, Australia
2School of Pharmacy and Biomedical Sciences, Curtin Health Innovation Research Institute, Curtin University, Perth, Australia
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  • For correspondence: pritinder.kaur@curtin.edu.au
Published 24 July 2018. DOI: 10.26508/lsa.201700009
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  • Figure S1.
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    Figure S1. Dermal cells from human neonatal foreskin can be distinguished into fibroblasts and pericytes based on differential VLA-1 expression.

    (A, B) Immunostaining of human neonatal skin with (A) αSMA (green) and FAPα (red) or (B) VLA-1 (green) and FAPα (red) showing mutually exclusive staining of pericytes with αSMA and VLA-1 in capillaries and fibroblasts with FAPα. VLA-1 is undetectable by in situ immunofluorescence staining in fibroblasts because of its significantly lower levels of expression in these cells. Nuclei stained with DAPI (white); c, capillary; e, epidermis; white arrows indicate FAPα-positive dermal fibroblasts. (C) Flow cytometric analysis of VLA-1 (x-axis) versus FAPα (y-axis) expression in freshly isolated CD45−dermal cells from neonatal human skin, revealing that VLA-1dim cells are exclusively FAPα positive, whereas VLA-1bri cells previously shown by us to be pericytes are negative for FAPα.

  • Figure S2.
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    Figure S2. Organotypic culture methodology reveals normalisation of basal cell polarity and epidermal:dermal junction in pericyte co-cultures.

    (A) Schematic of OC technique illustrating establishment of 3D skin regeneration within a six-well insert, where cells are grown initially submerged in culture media to establish a DE consisting of fibroblasts, pericytes, or both within a type 1 collagen gel and then maintained at an air–liquid interface to promote epidermal stratification and differentiation. (B) Histological analysis of OCs seeded with either fibroblasts, pericytes, or both on paraffin sections stained with H&E from two independent experiments—first and second row (supporting data shown from a third experiment in Fig 1A) to verify reproducibility of the observation that pericytes induce basal cell polarization in the basal layer of OCs. (C) Transmission electron microscopy analysis of dermo-epidermal junctional region of OCs from two independent OC experiments seeded with either fibroblasts, pericytes, or both, revealing that hemi-desmosome and basement membrane assembly are consistently observed in pericyte cocultures—images are additional data supporting transmission electron microscopy results shown in Fig 2B. Results in this figure and Fig 2B are representative of 18 random fields from two independent biological replicates. Scale bar = 0.5 μm.

  • Figure 1.
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    Figure 1. Pericytes promote epidermal regeneration in OCs that most closely resembles human skin epidermis.

    (A, C, E, G, H) Human keratinocytes placed in OCs with DEs made up of fibroblasts, pericytes, or a combination of both exhibited differences in morphology. Hematoxylin and eosin staining revealed a polarized basal layer (A) and immunostaining showed increased expression of the proliferative markers Ki67 (C), ΔN-p63 (E), basal layer K15 expression (G), and ordered suprabasal K10 expression (H) in the presence of pericytes. Images representative of four independent OC experiments. (B, D, F) Quantitation of increased thickness of the epithelium (B), increased number of Ki67+ cells (12 random fields/group) (D), and % basal/total ΔN-p63+ (24 random fields/group) (F) in the presence of pericytes from four independent OC experiments. Error bars are mean ± SD. Unpaired t test. **P < 0.01, ***P < 0.001, n.s. not significant. Scale bar = 100 μm.

  • Figure 2.
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    Figure 2. Pericytes enhance LAMA5 deposition and assembly of the basement membrane and hemi-desmosomes in OCs.

    (A) Immunofluorescent staining for LAMA5 (red) and DAPI (blue, nuclei) in OCs populated with fibroblasts, pericytes, or a combination of both demonstrating its uniform deposition in pericyte-populated OCs. Images representative of three independent OC experiments. Scale bar = 100 μm. (B) Transmission electron micrographs of the dermo-epidermal junction of OCs revealing absence of a basement membrane in fibroblast OCs, sporadic initiation of hemi-desmosomes in fibroblast and pericyte-combined OCs, and complete basement membrane assembly with a continuous lamina lucida (LL) and lamina densa (LD) (enlarged boxed area) and regular hemi-desmosomes (white arrows) in pericyte OCs. The higher magnification pericyte OC transmission electron micrograph illustrates the inner plaque (ip) and outer plaque (op) of the hemi-desmosomes. Data representative of 18 random fields from two independent OCs. epi, epidermis; der, dermis. Scale bar = 0.5 μm.

  • Figure 3.
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    Figure 3. Pericytes make close contact with the basal layer and instruct basal keratinocytes to undergo predominantly planar divisions parallel to the basement membrane in OCs.

    (A–F) Whole mount confocal microscopy of OCs populated with fibroblasts (A–C) or pericytes (D–F) stained with rhodamine-conjugated phalloidin (red, actin) and DAPI (blue, nuclear); z-stacks (C, F) reveal irregular dermo-epidermal junctions in fibroblast-populated OCs (A, C) and their bipolar morphology (B). Pericyte-populated OCs show stellate pericyte morphology (E) and their proximity at the dermo-epidermal interface (D); z-stack reveals a well-organized and even basal layer and close contact with pericytes (F). Images representative of six random fields from one of three OC experiments. White dotted line = interface between the epidermis (epi) and dermis (der). Scale bar = 100 μm. (G–I) Triple immunofluorescent staining for DAPI (G: blue, DNA), pH 3 (H: green, mitotic cells), and γ-tubulin (I: red, centrosomes) in OCs populated with fibroblasts (F), pericytes (P), or a combination of both (F + P), showing representative cell divisions. Cell division orientation relative to the basement membrane was determined by measuring the angle between the dermo-epidermal junction and the centrosomal axis in pH 3+ cells. J. Quantitation of the angle of cell division (0o–90o) as % of total mitotic events in OCs populated with fibroblasts (F), pericytes (P), or both (F + P), from 43, 23, and 23 mitoses per group from three independent experiments displayed as a radial bar chart showing a tendency for mitoses parallel to the basement membrane in the presence of pericytes. Scale bar = 20 μm.

  • Figure S3.
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    Figure S3. Validation of GFP and BMP-2 expression in lentivirus-transduced neonatal human skin fibroblasts.

    (A) Immunofluorescence analysis for GFP in vector-transduced control fibroblasts (F_GFP) and BMP-2–transduced fibroblasts (F_BMP2) in culture. Scale bar = 50 μm. (B) qPCR analysis of BMP-2 expression in control GFP (F_GFP) and BMP-2 (F_BMP2)–transduced fibroblasts, confirming expression of BMP-2 transcripts in the latter. Data expressed as mean ± SD from triplicate experiments. Statistical analysis performed using unpaired t test, **P < 0.01. (C–F) Immunofluorescence staining for BMP-2 in brefeldin-treated pericytes (C, D) or untreated pericytes (E, F), stained with anti-BMP-2 polyclonal antibody (C, E) or isotype control polyclonal antibody (D, F), confirming BMP-2 expression in pericytes (C). (G–J) Immunofluorescence staining with anti-BMP-2 (G, I) or isotype control antibody (H, J) in brefeldin A–treated F_GFP fibroblasts (G, H) and F_BMP2 fibroblasts (I, J). (K) Quantitation of BMP-2 immunofluorescence signal in F_BMP2 (n = 43 cells) and F_GFP (n = 83 cells) fibroblasts versus pericytes (n = 28 cells) in a representative experiment from a total of three experiments. Statistical significance was established by one-way ANOVA Tukey's multiple comparison test **P < 0.01, ****P < 0.0001. (L) OCs generated with F_GFP control or F_BMP2 fibroblasts stained for K15, demonstrating its absence. Dotted line indicates dermo-epidermal junction. Staining representative of three independent OC experiments. Scale bar = 100 μm.

  • Figure 4.
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    Figure 4. Fibroblasts expressing BMP-2 increase epithelial thickness, restore cell polarity, increase planar cell divisions, and ordered differentiation to human keratinocytes in OCs.

    (A, C, E, G, H) OCs populated with control (F_GFP) or BMP-2–transduced (F_BMP2) fibroblasts were analyzed after hematoxylin and eosin staining (A) or immunostaining with Ki67 (C), ΔN-p63 (E), LAMA5 (G), and K10 (H) to reveal improved basal cell polarity, increased expression of Ki67, ΔN-p63, and LAMA5 and better suprabasal K10 expression in the presence of F_BMP-2 compared with F_GFP controls. Images are representative of three independent experiments. (B, D, F) Quantitation of epithelial thickness (B), number of Ki67+ (D), and ΔN-p63+ (F) cells in OCs populated with control (F_GFP) or BMP-2–transduced fibroblasts (F_BMP2). Error bars represent mean ± SD; statistical analysis performed using unpaired t test; n.s. not significant; *P < 0.05; ***P < 0.00. All quantitative data are derived from three independent experiments. (I, J, L, M) Immunostaining of OCs for pH 3 (green) and γ-tubulin (red) to determine keratinocyte cell division orientation in the presence of BMP-2–transduced (I, J: F_GFP, n = 28 mitoses) and control fibroblasts (L, M: F_BMP2, n = 27 mitoses). (K, N) Radial bar charts illustrating angle of keratinocyte mitoses obtained with F_BMP2 (K) and F_GFP control (N) demonstrating increased planar divisions in the latter.

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Pericytes improve epidermal renewal
Lizhe Zhuang, Kynan T Lawlor, Holger Schlueter, Zalitha Pieterse, Yu Yu, Pritinder Kaur
Life Science Alliance Jul 2018, 1 (4) e201700009; DOI: 10.26508/lsa.201700009

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Pericytes improve epidermal renewal
Lizhe Zhuang, Kynan T Lawlor, Holger Schlueter, Zalitha Pieterse, Yu Yu, Pritinder Kaur
Life Science Alliance Jul 2018, 1 (4) e201700009; DOI: 10.26508/lsa.201700009
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Volume 1, No. 4
August 2018
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