Table 1.
Antibodies used for immunohistochemistry.
Fig 1.
Localization of Merkel cells in cross-sections of ESS after grafting to mice.
Immunohistochemistry was performed using antibodies against KRT20 (green) and KRT18 (red); DAPI was used to counterstain nuclei (blue). Shown are representative sections of ESS excised from mice at week 2 (A), week 4 (B), week 6 (C), week 8 (D), week 10 (E), week 12 (F), and week 14 (G) after grafting. H, Section of normal human skin (control). Dashed lines indicate locations of dermal-epidermal junctions. Rare KRT20-positive cells were observed at 2 weeks after grafting (arrow in A); co-localization of KRT20 and KRT18 was observed at later time points (B-G). Examples of oval (red arrow) and dendritic (yellow arrows) Merkel cell shapes are indicated (C). Scale bar in A (50 μm) is same for all panels.
Fig 2.
Localization of Merkel cells in ESS after grafting suggests random organization.
Epidermal sheets of ESS excised from mice at 2 weeks (A), 4 weeks (B), 6 weeks (C), 8 weeks (D), 10 weeks (E), 12 weeks (F), and 14 weeks (G) after grafting, and normal human skin (H) are shown following immunostaining with antibodies against KRT20 (green) and KRT18 (red). DAPI was used to counterstain nuclei (blue). Representative en face images are shown such that the basal epidermis is viewed from the dermal side. Examples of oval (red arrow) and dendritic (yellow arrows) Merkel cell shapes are indicated (C). Scale bar in A (50 μm) is same for all panels.
Fig 3.
Merkel cell density in ESS is highly variable.
Shown are en face images of epidermis from ESS at 12 weeks (A-B) and 14 weeks (C-D) after grafting, and normal human skin (E-F), immunostained with antibodies against KRT20 (green) and KRT18 (red); DAPI was used to counterstain nuclei (blue). Tissue was photographed at low magnification to illustrate variability in density of Merkel cells and relatively random distribution. Scale bar in A (200 μm) is same for all panels.
Fig 4.
Demonstration of human cell engraftment in ESS in vivo.
Immunohistochemistry was performed using an antibody against HLA-ABC (green) to localize human keratinocytes in ESS after grafting to mice. Merkel cells were visualized by staining with an antibody against KRT20 (red). Nuclei were counterstained using DAPI (blue; C, F, I). Sections are oriented with the epidermis at the top of each image. Note that images in each row depict a single section. A-C, ESS at 4 weeks after grafting. D-F, ESS at 8 weeks after grafting. G-I, ESS from 12 weeks after grafting. Arrows indicate examples of KRT20-positive Merkel cells in grafted human ESS. Scale bar in A is same for all sections (50 μm).
Fig 5.
Merkel cells in grafted ESS are derived from human epidermal cells.
Immunohistochemistry was performed using a human-specific antibody against E-cadherin (ECAD; red); Merkel cells were localized by KRT20 immunostaining (green). A-B, ESS at 6 weeks after grafting; C-D, ESS at 12 weeks after grafting. Sections are oriented with the epidermis at the top of each image; in A-D, each row depicts images of the same tissue section. Arrows depict examples of KRT20-positive Merkel cells in ESS. E-F, Controls for native human skin (E) and immunodeficient mouse skin (F) demonstrating specificity of anti-human E-cadherin antibody (red). Section shown in F depicts the border (arrowhead) between grafted human ESS (right) and flanking mouse skin (left). Nuclei were counterstained with DAPI (B, D, E, F; blue); scale bar in A is for all sections (50 μm).
Fig 6.
Co-localization of KRT20 and basal keratinocyte marker KRT15 in Merkel cells in grafted ESS.
Immunohistochemistry was performed using antibodies against KRT20 (green) and KRT15 (red). Shown are sections of ESS excised from mice at 4 weeks (A-C), 6 weeks (D-F), 8 weeks (G-I), 12 weeks (J-L), and 14 weeks (M-O) after grafting. Sections are oriented with the epidermis at the top of each image. Each row of three images depicts the same section. Arrows indicate examples of KRT20-positive Merkel cells in grafted ESS. Nuclei were counterstained with DAPI (C, F, I, O; blue). Scale bar in A is for all sections (50 μm).
Fig 7.
Co-localization of KRT20 and neuroendocrine marker synaptophysin (SYP) in grafted ESS.
Immunohistochemistry was performed using antibodies against KRT20 (green) and SYP (red); nuclei were counterstained with DAPI (blue). A-F, Cross sections of ESS at 4 weeks (A), 6 weeks (B), 8 weeks (C), 10 weeks (D), 12 weeks (E), and 14 weeks (F) after grafting; sections are oriented with the epidermis at the top of each image. Arrows indicate examples of KRT20/SYP-positive Merkel cells. G-L, Epidermal sheets of ESS at 4 weeks (G), 6 weeks (H), 8 weeks (I), 10 weeks (J), 12 weeks (K), and 14 weeks (L) after grafting, photographed en face following immunostaining. Scale bar in A is for all images (50 μm).
Fig 8.
Merkel cells in grafted ESS are associated with neurons expressing neurofilament medium (NF-M).
Immunochemistry with antibodies against NF-M (red) and KRT20 (green) was used to localize neurons and Merkel cells, respectively, in ESS after grafting to mice. Nuclei were counterstained with DAPI (blue; B, C, E, F, H, I, K, L). Shown are cross sections of ESS at 4 weeks (A-C), 6 weeks (D-F), 8 weeks (G-I), and 12 weeks (J-L) after grafting; each row contains images of the same section. Images shown in the right-hand column (C, F, I, L) are 3-fold magnified images of boxed areas in center column (B, E, H, K). Scale bars in panels in first row are same for all images in column. White arrows indicate examples of NF-M-positive nerves associated with or in proximity to Merkel cells; yellow arrows indicate NF-M-positive nerves not associated with Merkel cells.
Fig 9.
Merkel cells in grafted ESS are associated with neurons expression neurofilament heavy (NF-M) by eight weeks after grafting.
Immunochemistry with antibodies against NF-H (red) and KRT20 (green) was used to localize neurons and Merkel cells, respectively, in ESS after grafting to mice. Nuclei were counterstained with DAPI (blue; B, D, F, H). Shown are cross sections of ESS at 4 weeks (A-B), 8 weeks (C-D), 12 weeks (E-F), and 14 weeks (G-H) after grafting; each row contains images of the same section. Scale bars in A is same for all images. White arrows indicate examples of NF-H-positive nerves associated with or in proximity to Merkel cells; yellow arrows indicate NF-H-positive nerves not associated with Merkel cells.