Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair

Abstract

The bone marrow provides inflammatory cells and endothelial progenitor cells to healing cutaneous wounds. To further explore the bone marrow contribution to skin and healing wounds, we used a chimeric mouse model in which the bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice is transplanted into normal C57BL mice. We found that normal skin is a target organ for bone marrow-derived cells from both the hematopoietic and the mesenchymal stem cell pool. We present evidence that the bone marrow contribution to normal skin and the healing cutaneous wound is substantially greater than the previously recognized CD45+ subpopulation, where 15%-20% of the spindle-shaped dermal fibroblasts were bone marrow-derived (EGFP+). Furthermore, the bone marrow-derived cells were able to contract a collagen matrix and transcribe both collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. Whereas endothelial progenitor cells were found early during the wound repair process, bone marrow-derived endothelial cells were not seen after epithelialization was complete. Our data show that wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow-derived cells and the adjacent uninjured dermal mesenchymal cells for reconstituting the dermal fibroblast population.

Figure 1

(A): Flow cytometry histogram of circulating cells from a control mouse (left graph) and an EGFP bone marrow–transplanted mouse (middle graph). Note the shift in emission spectra in the FITC channel, with 86% of the circulating cells fluorescing green. To determine degree of autofluorescence, cells were dispersed from normal skin of C57BL mice and EGFP⁺ mice and analyzed by flow cytometry. The right graph shows the emission characteristics in the FITC channel and the degree of overlap. (B):At 10 weeks, flow cytometry of the normal skin of EGFP bone marrow–transplanted mice shows that 14.1% of the cells in the skin are bone marrow–derived. (C): At 10 weeks, fluorescent microscopy of the normal skin shows large numbers of EGFP-expressing bone marrow cells, especially around the hair follicle. The inset shows integration of bone marrow–derived cells around the base of the hair follicle. (D):At 8 months, flow cytometry of the normal skin confirms a significant number (11.3%) of EGFP⁺ cells, which persist in the skin at 8 months after transplant. (E): Fluorescent microscopy of the normal skin confirms EGFP⁺ cells in the dermis at 8 months. Abbreviations: EGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate.

Figure 2

Fluorescent microscopy of various tissues isolated from 8- to 12-month-old chimeras. (A): Brain. (B): Spleen. (C): Small intestine. (D): Normal skin. (E): Lung. (F): Kidney. (G): Cross section of aorta; note the presence of enhanced green fluorescent protein–positive cells in the media. (H): Heart.

Figure 3

(A): Cumulative graph of flow cytometry data obtained from the control mice, including normal skin (day 0), and during wound healing. The y-axis represents the percentage of cells in the wound that stains with the respective antibodies. (B): Cumulative graph of flow cytometry data obtained from the EGFP-expressing chimeras. The percentages of each cell population in the control and experimental groups are very similar; however, bone marrow–derived cells remain in the healed wound and account for more than 15% of all cells in the healed wound (day 28). On fluorescent microscopy, EGFP⁺ cells were seen throughout the healing dermis during the early inflammatory phase on days 14 (C) and 21 (D) after wounding. (E): EGFP⁺ cells persisted in the dermis during the remodeling phase, as seen here on day 42 after wounding. Note the histological resemblance of the EGFP-positive cells to fibroblasts. (F): Microvessel deep in the wound shows EGFP-expressing cells either within the lumen (endothelial cells) or adjacent to the luminal cells (pericytes). Abbreviation: EGFP, enhanced green fluorescent protein.

Figure 4

(A): Normal skin 8 months after transplantation shows EGFP⁺ (green) cells as well as PE-conjugated anti-CD45–labeled cells (red). (B): Day-42 wound stained with PE-conjugated anti-CD45 antibody showing that many of the EGFP⁺ bone marrow–derived cells within the wound do not coexpress CD45. The EGFP⁺CD45⁻ cells appear as spindle-shaped cells resembling fibroblasts. (C): Day-42 wound counterstained with anti-heavy chain myosin antibodies. As with the anti-CD45 antibody, most of the EGFP⁺ cells fail to colocalize anti-myosin and show numerous spindle-shaped EGFP⁺ bone marrow–derived cells in the dermis that lack heavy chain myosin expression. The yellow framed inset shows a higher-power view of the dermal cells. (D): Manual counts of high-powered histological cross sections of day-42 wounds that are counterstained with PE-conjugated anti-CD45 antibody show that only 2.5% of the dermal cells coexpress EGFP and CD45. More than 7% of the dermal cells are of bone marrow origin (EGFP⁺) but lack CD45 expression (p < .05). Abbreviations: EGFP, enhanced green fluorescent protein; PE, phycoerythrin.

Figure 5

(A): Normal skin of HC chimeric mice shows EGFP⁺ cells in the dermis confirmed by fluorescent microscopy. (B): Day-42 wound in an HC chimeric mouse; on fluorescent microscopy, EGFP⁺ cells were seen throughout the healing dermis in the remodeling phase. (C): Normal skin of MC chimeric mice shows EGFP⁺ cells in the dermis confirmed by fluorescent microscopy. (D): Day-42 wound in an MC chimeric mouse; on fluorescent microscopy, EGFP⁺ cells were also seen throughout the healing dermis in the remodeling phase. Abbreviations: EGFP, enhanced green fluorescent protein; HC, hematopoietic cell; MC, mesenchymal cell.

Figure 6

Reverse transcription polymerase chain reaction analysis of cells collected from normal skin via fluorescence-activated cell sorter. Bone marrow–derived EGFP⁺ cells and skin-resident EGFP^– cells were examined for collagen (Col) type I, collagen type III, and β-actin mRNA. Lane 1 represents the DNA molecular standards. In all four chimeras examined, only the EGFP⁺ and, hence, bone marrow–derived cells in the skin expressed collagen type III. Abbreviation: EGFP, enhanced green fluorescent protein.