1. Lineage Identity and Location within the Dermis Determine the Function of Papillary and Reticular Fibroblasts in Human Skin 
Ana Korosec, Sophie Frech, Bernhard Gesslbauer, Martin Vierhapper, Christine Radtke, Peter Petzelbauer, Beate M. Lichtenberger 
Journal of Investigative Dermatology 
Volume 139, Issue 2, Pages (February 2019)
DOI: /j.jid
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2. Figure 1
Isolation of papillary and reticular fibroblasts via FACS. (a) Representative images of skin sections stained for the indicated antigens. (b) Higher magnification of a and CD36 staining. Orange and yellow arrowheads point toward positive cells in the papillary/reticular dermis. Dashed lines separate upper reticular from papillary (300 μm) or lower reticular dermis (1,000 μm). (c, d) Gating strategy. After negative selection (CD45+, LIN+, CD31+, CD235ab+, CD106+, DAPI+, ITGA6+, E-cadherin+), four populations were FACS-sorted. ITGA6+E-cadherin+ keratinocytes are controls. (e) Relative expression of pan-fibroblast markers, LYVE1 and KRT14 (n = 3). Values of each experiment were normalized to FAP+CD90– cells for VIM, FSP1, and COL1A1; to FAP–CD90– for LYVE1; and to keratinocytes for KRT14. ∗P ≤ 0.05, ∗∗P ≤ 0.005, ∗∗∗P ≤ compared with FAP–CD90– (double-negative) cells. (f) Representative bright-field image of FAP–CD90– cells after 2 weeks in culture. Scale bars = 100 (in a and b) or 1,000 μm (in f). Epid., epidermis.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

3. Figure 2
Expression of commonly accepted markers for papillary and reticular fibroblasts in FACS-sorted fibroblast subsets. (a, b) Representative FACS plots showing expression of (a) CD39 and CD26 and (b) CD36 in the three fibroblast subsets. (c–e) Bar graphs showing relative expressions of (c) the pan-fibroblast marker vimentin and (d) alleged papillary (CD26, NTN1, PDPN) and (e) reticular fibroblast markers (ACTA2, PPARγ, CNN1, COL11A1), normalized to the FAP+CD90– fibroblast subset. Expression of (d) FAP and (e) CD90 confirm that the subpopulations were FACS-sorted with high purity. Data represent mean ± standard error of the mean (n = 8–9). ∗P ≤ 0.05, ∗∗P ≤ 0.005, ∗∗∗P ≤ compared with FAP+CD90– cells; #P ≤ 0.05 compared with FAP–CD90+ cells. VIM, vimentin.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

4. Figure 3
FACS-sorted fibroblast subsets display functional differences in vitro. (a–c) Representative images of FACS-sorted fibroblast subsets after 1 week in culture (bright field) (a), of 5-ethynyl-2′-deoxyuridine–positive (EdU+) (b) and Oil Red O-positive cells after performing an adipogenesis assay (c). (d) Quantification of EdU+ cells. (e) Quantification of the absorbance of Oil Red O in stained fibroblast subsets (in c) after dye extraction. OD values from controls without adipogenesis cocktail were subtracted from the ODs shown. Data represent mean of (d) three or (e) six independent experiments ± standard error of the mean. (f–h) Bar graphs showing relative expression of the respective genes after performing an adipogenesis assay (n = 3). Values were normalized to the FAP+CD90– fibroblast subset. ∗P ≤ 0.05, ∗∗P ≤ compared with the FAP+CD90– subset. Scale bars = 1,000 μm (in a) and 10 μm (in c), respectively. OD, optical density.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

5. Figure 4
Expression of commonly accepted markers for papillary and reticular fibroblasts in FACS-sorted fibroblast subsets changes in vitro. (a, b) Bar graphs showing relative expression of the respective genes after 1 or 2 weeks in culture. Both FAP and CD90 are expressed in all fibroblast subsets shortly after seeding in vitro. (a) NTN1 and PDPN have previously been shown to be expressed in fibroblasts within the papillary dermis. (b) ACTA2, CNN1, COL11A1, TGM2, and MGP were reported to be expressed in fibroblasts from the reticular dermis. Data represent mean of three independent experiments ± standard error of the mean. ∗P ≤ 0.05, ∗∗P ≤ compared with the FAP+CD90– subset. w, week.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
FAP+CD90– fibroblasts are enriched in the papillary dermis. (a) Representative images of hematoxylin and eosin-stained sections of full-thickness skin and dermatomed slices of papillary (including epidermis, 0–300 μm), upper reticular (300–1,000 μm), and lower reticular dermis (>1,000 μm). Scale bars = 100 μm. (b) Representative dot plots of fibroblasts isolated from whole skin, papillary dermis, and upper and lower reticular dermis. Gating strategy is described in Figure 1. (c–e) Summary of six independent experiments showing percentages of the indicated fibroblast subsets in the different layers of the dermis or whole skin. Percentages were calculated after removal of the double-negative population. (f) Ratio of FAP+CD90– to FAP–CD90+ fibroblasts. Data have been normalized to the papillary dermis. ∗P ≤ 0.05, ∗∗P ≤ 0.005, ∗∗∗P ≤ compared with FAP+CD90– cells. d., dermis; FB, fibroblast.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions

7. Figure 6
The dermal niche affects the expression of enzymes like DPPIV/CD26 and FAT/CD36 and the function of fibroblast subsets. (a, b) Representative FACS plots showing expression of the indicated antigens in fibroblast subsets isolated from whole skin, papillary dermis, and upper and lower reticular dermis. (c) Representative images of Oil Red O stainings after performing adipogenesis assays. Scale bars = 500 μm. (d–f) Bar graphs showing relative expression of the respective genes in fibroblast subsets isolated from papillary or reticular dermis, respectively (n = 1). Values were normalized to the FAP+CD90– subset isolated from the reticular dermis. (g) Scheme showing that human skin dermis comprises an opposing gradient of papillary and reticular fibroblasts from the skin surface to the deep dermis. Papillary and reticular fibroblasts display distinct expression patterns and functions. Pap., papillary; ret., reticular.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2018 The Authors Terms and Conditions