APPLICATION OF ADIPOCYTE-DERIVED STEM CELLS IN TREATMENT OF CUTANEOUS RADIATION SYNDROME

Diane Riccobono*, Diane Agay*, Harry Scherthan^$, Fabien Forcheron*, Mylène Vivier*, Bruno Ballester*, Viktor Meineke^$, Michel Drouet*.

*: Département de Radiobiologie, IRBA-antenne La Tronche-CRSSA, 24 Avenue des Maquis du Grésivaudan, 38702 La Tronche, France.

$

: Institut für Radiobiologie der Bundeswehr, Neuherbergstraße 11, 80937 München, Germany.

Corresponding author: Diane Riccobono: Département de Radiobiologie, IRBA-antenne La

Tronche-CRSSA, 24 Avenue des Maquis du Grésivaudan, 38702 La Tronche, France. Email: diane.riccobono@gmail.com, Tel: +33476636857; Fax: +33476636922

E-mails for the other authors:

Diane Agay*: diane.agay@crssa.net Fabien Forcheron*: fforcheron@yahoo.fr Bruno Ballester*: brunoballester@sfr.fr Michel Drouet*: micheldrouet@crssa.net Harry Schertan^$: schert@web.de

Mylène Vivier*: vivier.crssa@yahoo.fr

Viktor Meineke^$: Viktor.Meineke@uni-ulm.de

Acknowledgements: This work was supported by a grant from Direction Générale de

l’Armement (Paris, France). We thank H. Chaussard for his technical assistance.

Keywords: Cutaneous Radiation Syndrome, Adipocyte Derived Stem Cells, Minipigs, Wound

ABSTRACT

Cutaneous radiation syndrome caused by local high dose irradiation is characterized by delayed outcome and incomplete healing. Recent therapeutic management of accidentally irradiated burn patients has suggested the benefit of local cellular therapy using mesenchymal stem cells grafting. According to the proposed strategy of early treatment, large amounts of stem cells would be necessary in the days following exposure and hospitalization, which would require allogeneic stem cells banking. In this context, we compared in a large animal model the benefit of local autologous and allogeneic adipocyte-derived stem cell injection.

Minipigs were locally irradiated using a ⁶⁰Co gamma source at the dose of 50 Gy and divided in three groups. Two groups were grafted with autologous (n=5) or allogeneic (n=5) adipocyte derived stem cells four times after the radiation exposure whereas the control group received the vehicle without cells (n=8). A clinical score was elaborated to compare the efficiency of the three treatments.

All controls exhibited local inflammatory injuries leading to a persistent painful necrosis thus mimicking the clinical evolution in human victims. In the autologous adipocyte-derived stem cells group, skin healing without necrosis or uncontrollable pain was observed. In contrast, the clinical outcome was not significantly different in the adipocyte-derived stem cell allogeneic group when compared with controls.

This study suggests that autologous adipocyte-derived stem cell grafting improves cutaneous radiation syndrome wound healing whereas allogeneic adipocyte derived stem cells do not. Further studies will establish whether manipulation of allogeneic stem cells will improve their therapeutic potential.

INTRODUCTION

The skin is a complex radiosensitive organ that is frequently irradiated in accidental or therapeutic context (Hopewell 1990; Peter 1996). The wound evolution in ionizing radiation (IR) exposed skin results from its physical barrier role, differentiation program and its immunologic and thermoregulation functions, and is unique for this tissue. Exposure to a high irradiation dose (> 20Gy) induces the Cutaneous Radiation Syndrome (CRS). This syndrome is clinically characterised by a delayed clinical outcome and diagnosis. CRS is characterized by an early erythema, followed by hair loss, dry, then wet desquamation several weeks later. It is associated with recurrent and extensive inflammatory processes leading to irreversible necrotic waves and followed by a poorly spontaneous revascularization and an incomplete healing (Lefaix and Delanian 2005; Drouet et al. 2011). CRS treatment remains complex: following the evaluation of irradiated area extension and excision of the exposed necrotic skin and subcutaneous tissues, a transient skin flap is associated with autologous skin graft (Lefaix and Delanian 2005; Agay et al. 2010).

Mesenchymal Stem Cells (MSCs) may represent a new therapeutic approach in this context as Bone Marrow-MSCs (BM-MSCs) have been shown to enhance wound healing (Wu et al. 2007). However, the mechanisms involved are still poorly understood (Chen et al. 2008) but experiments from different groups indicate that BM-MSCs secrete numerous cytokines and chemokines well known to be important to stimulate and achieve wound healing, such as Vascular Endothelial Growth Factor alpha (VEGF alpha), Epidermal Growth Factor-alpha (EGF alpha), Insulin Growth Factor-1 (IGF1), Keratinocyte Growth Factor (KGF), Angiopoietin-1 (Ang1), Stromal derived factor-1 (SDF1), Macrophage Inflammatory Protein-1 alpha and beta (MIPProtein-1 alpha and beta), and erythropoietin in greater amounts than dermal fibroblasts (Estrada et al. 2009). Based on these experiments, BM-MSCs local injection and skin surgery have performed in a few cases of accidental CRS (Lataillade et al. 2007). Moreover, Adipocyte-derived Stem Cells (ASCs) located in adult fat tissues may represent an alternative multipotent stem cell source in adult. ASCs have an equal potential as BM-MSCs to differentiate into cells and tissues of mesodermal origin, such as adipocytes, cartilage, bone, and skeletal muscle (Schaffler and Buchler 2007). As BM-MSCs, ASCs secrete angiogenesis-related cytokines, such as VEGF alpha and anti-apoptotic factors, which might be suitable for regenerative cell therapy (Rehman et al. 2004;

Nakagami et al. 2006). Moreover, the easiness of access (iterative if required) to subcutaneous adipose tissue and the simplicity of isolation procedures provide a clear advantage (Gimble et al. 2007).

Nowadays, the autologous MSCs strategy for CRS treatment is hampered by the delay induced by cell culture, which is aggravated by the fact that CRS diagnosis is frequently delayed many weeks after accidental IR exposure. In this context the development of allogeneic MSCs cell banks may represent an interesting approach and may be required to develop an early cell therapy strategy; indeed stem cell collection from unirradiated area early after accidental irradiation in order to rapidly produce a graft of clinical size appears more problematic than harvesting cells in radiotherapy patients (Akita et al. 2010).

Our group has recently proposed a new minipig model of stem cell therapy to treat CRS (Agay et al. 2010). Using this model, we are now evaluating the therapeutic potential of allogeneic ASCs. We present here the first results of clinical score testing. Globally the animals grafted with allogeneic ASCs exhibited a very low clinical score when compared with autologously grafted animals, which suggests the limit of allogeneic multipotent stem cells use and banking strategy.