Fig 1.
CD45-positive hematopoietic cells in skin wounds of mice.
(A) Immunofluorescent staining in normal and wounded skin of mice with CD45 antibody. Balb/C mice with eight-millimeter punch biopsy excisional wounds for 14 days were euthanized and wounded skin and normal dorsal skin were harvested and fixed. Skin sections were prepared for immunofluorescent staining with CD45 antibody (green). DAPI (blue) was used as a nuclear counterstain. Scale bars in all images was 50 μm. A representative of wounded skin of 16 wounds from 8 mice was shown. (B) The statistical analysis of CD45 positive cell percentage in skin sections is depicted. Ten HPF of five normal and five wounded skin were counted (n = 5).
Fig 2.
Conversion of CD45-positive hematopoietic cells to keratinocytes in re-epithelialization skin of mice.
(A) Double immunofluorescent staining with keratin-14 (K14) and CD45 antibodies in normal skin (Top panels) and wounded skin (Bottom panels) of Balb/C mice. Wounded skin was taken from mice with 8 mm punch biopsy of excisional wounds after wounding for 14 days. (B) Double immunofluorescent staining with K14 and CD45 antibodies in cultured skin cells isolated from wound edge of Balb/C mouse skin. Skin cells were isolated from the edge of wounded skin of mice by collagenase digestion. Cells were cultured for 48 hours before fixation and performing an immunofluorescent staining. DAPI (blue) was used as a nuclear counterstain. Scale bars in all images was 50 μm.
Fig 3.
Myeloid cells are the cellular origin of hematopoietic cell-converting keratinocytes.
(A) Double immunofluorescent staining with antibodies of CD45 and myeloid cell marker CD11b in wounded skin of mice. (B) Double immunofluorescent staining with antibodies of CD11b and K14 in wounded skin of mice. (C) Double immunofluorescent staining with antibodies of CD11b and K14 in cultured skin cells. Skin cells were isolated by collagenase digestion from wounded edge skin of Balb/C mice after injury for 14 days and cultured for 48 hours in vitro before fixation and staining. DAPI (blue) was used as a nuclear counterstain. Scale bars in all images was 50 μm.
Fig 4.
Distribution of injected splenocyte-derived myeloid cells in healed mouse skin.
(A) Schematic figure of Dil-labeled or GFP-expressing mouse myeloid cell lineage tracing experiments. Splenocytes were isolated from spleens of Balb/C or C57BL/6-Tg (UBC-GFP) mice and cultured in a medium containing 5 ng/ml of M-CSF to generate myeloid cells. One million labeled cells were dermally injected into the healthy skin at one spot where was one centimeter far from the edge of wounds at the same time of skin injury. (B) Dil-labeled cells in wounded skin of mice received nothing (Left) or one million Dil-labeled splenocyte-derived myeloid cells (Right). (C) GFP-positive cells in wounded skin of mice received nothing (Left) or one million GFP-expressing splenocyte-derived myeloid cells (Right). Skins were stained with GFP antibody (green). DAPI (blue) was used as a nuclear counterstain. Scale bars in all images were 100 μm.
Fig 5.
Conversion of GFP-expressing myeloid cells to keratinocytes in re-epithelialized wounded skin.
(A) Double immunofluorescent staining with antibodies of GFP (green) and K14 (red) in healed skin from wounds of C57BL/6 mice received one million GFP-expressing splenocyte-derived myeloid cells. (B) Double immunofluorescent staining with K14 (red) and GFP (green) antibodies in cultured skin cells. Skin cells were isolated from wound edge of mice received GFP-expressing myeloid cells and injury for 14 days. Cells were cultured for 48 hours before fixation and staining. DAPI (blue) was used as a nuclear counterstain. Scale bars in all images was 50 μm.
Fig 6.
Hair follicle-like structures are seen in wounded skin of mice received GFP-expressing myeloid cells for 4 weeks.
Eight millimeters punch biopsies of excisional wounds were created in C57BL/6 mice received one million GFP-expressed splenocyte-derived myeloid cells simultaneously. Four weeks after wounding, mice were euthanized, wounded skin were collected for examination of new hair follicle regeneration. (A) Images were taken under the fluorescent microscope in normal and wounded skin of the same mice received one million cells for 4 weeks. (B) H & E staining in wounded skin of mice received one million cells for 4 weeks. Arrows indicate wounding area. Top panels, magnification of 20 ×; Low panels, magnification of 100 ×. Scale bars, 100 μm.
Fig 7.
Detection of GFP-expressing myeloid cells in new forming hair follicle in wounded skin of mice.
(A) Double immunofluorescent staining with keratin-14 (K14) (red) and GFP (green) antibodies in wounded skin of mice which received one million GFP-expressing splenocyte-derived myeloid cells for 4 weeks. (B) Double immunofluorescent staining with keratin-14 (K14) (red) and GFP (green) antibodies in cultured hair follicles isolated from the edge of wounds in mice received one million GFP-expressing myeloid cells for 4 weeks. DAPI (blue) was used as a nuclear counterstain. Scale bars in all images was 50 μm.