Autologous adipocyte derived stem cells favour healing in a minipig model of cutaneous radiation syndrome

Abstract

Cutaneous radiation syndrome (CRS) is the delayed consequence of localized skin exposure to high doses of ionizing radiation. Here we examined for the first time in a large animal model the therapeutic potential of autologous adipose tissue-derived stroma cells (ASCs). For experiments, Göttingen minipigs were locally gamma irradiated using a (60)Co source at the dose of 50 Gy and grafted (n = 5) or not (n = 8). ASCs were cultured in MEM-alpha with 10% fetal calf serum and basic fibroblast growth factor (2 ng.mL(-1)) and post irradiation were intradermally injected on days 25, 46, 67 and finally between days 95 and 115 (50 × 10(6) ASCs each time) into the exposed area. All controls exhibited a clinical evolution with final necrosis (day 91). In grafted pigs an ultimate wound healing was observed in four out of five grafted animals (day 130 +/- 28). Immunohistological analysis of cytokeratin expression showed a complete epidermis recovery. Grafted ASCs accumulated at the dermis/subcutis barrier in which they attracted numerous immune cells, and even an increased vasculature in one pig. Globally this study suggests that local injection of ASCs may represent a useful strategy to mitigate CRS.

Figure 1. Study design.

Figure 2. Characterization of ASCs.

(A): Flow cytometric analysis after the 3^rd passage of porcine ASCs labelled with FITC- or PE-conjugated control isotype IgG (bold curves) or antibodies against indicated cell surface proteins (dark curves). (B): Differentiation of ASCs cultured in specific media.

Figure 3. ASCs favoured wound healing after local irradiation.

Evolution of clinical score (A). Clinic and thermal camera imaging from representative animals on day 1 (B), then in the entry/exit area of PBS-treated (C/D) or ASC-grafted minipigs (E/G). Water content in entry area skin (F). Final examination of skin thickness and subcutaneous tissue (H). Complete burn healing in one ASC-grafted minipig (I).

Figure 4. Engraftment of ASCs into the irradiated skin.

Location of transplanted ASCs (in red) in the epidermis (A), in the dermis (B), and in the subcutis (C) of skin biopsies centred on injection plots, performed three days after the 3rd graft, on day 70. Brightly red fluorescing quantum dots marked ASCs (arrows) around blue nuclei (DAPI) near the cutis/subcutis border and among adipose tissue. The immunostaining of CD3 and lambda light chains (in pink) in the subcutis of healthy (D), PBS-treated (F), and ASC-grafted (F) skin underlined the infiltration by T and B lymphocytes three days after the ASC graft. Magnification: ×100.”

Figure 5. Epidermal and dermal radio-induced injuries in the skin of PBS-treated minipigs.

Kinetic study from four iterative biopsies performed few days after each PBS-treatment in representative controls (magnification ×100). Progressive disorganisation of the epidermal layers, keratinocytes degeneration, vacuolation and barrier disruption. Numerous dermal cavities two weeks after the 4^th PBS-treatment.

Figure 6. Proliferation of keratinocytes in grafted animals.

After the third graft, the proliferation of keratinocytes in the epidermis-dermis junction is revealed by the Ki67 immunostaining (in green) in the irradiated skin of grafted animals (A) versus controls (B).

Figure 7. After local irradiation ASCs favoured reepithelialisation and enhanced lymphocyte infiltration and angiogenesis in dermis.

Kinetic study from four iterative biopsies performed few days after each ASC-graft in representative animals (magnification in the original ×100). Early cleaning of damaged epidermis was followed by complete epidermis recovery/hyperproliferative keratinocyte activity. Two weeks after the 4^th ASC-graft, early T and B lymphocyte infiltration in dermis near the subcutis, with a strong increase in vascularisation.

Figure 8. ASC grafting stimulated cutaneous vascularization.

The red immunofluorescent staining of Von Willebrand factor revealed the increase of the vasculature in the dermis of the ASC-grafted minipig (A) in comparison with PBS-treated control (B), two weeks after the fourth treatment (day119). Magnification: ×100.