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Cold Atmospheric Plasma (CAP) is a novel promising tool developed in several biomedical applications such as cutaneous wound healing or skin cancer. Nevertheless, in vitro studies are lacking regarding to CAP effects on cellular actors involved in healthy skin healing and regarding to the mechanism of action. In this study, we investigated the effect of a 3 minutes exposure to CAP-Helium on human dermal fibroblasts and Adipose-derived Stromal Cells (ASC) obtained from the same tissue sample. We observed that CAP treatment did not induce cell death but lead to proliferation arrest with an increase in p53/p21 and DNA damages. Interestingly we showed that CAP treated dermal fibroblasts and ASC developed a senescence phenotype with p16 expression, characteristic morphological changes, Senescence-Associated β-galactosidase expression and the secretion of pro-inflammatory cytokines defined as the Senescence-Associated Secretory Phenotype (SASP). Moreover this senescence phenotype is associated with a glycolytic switch and an increase in mitochondria content. Despite this senescence phenotype, cells kept in vitro functional properties like differentiation potential and immunomodulatory effects. To conclude, we demonstrated that two main skin cellular actors are resistant to cell death but develop a senescence phenotype while maintaining some functional characteristics after 3 minutes of CAP-Helium treatment in vitro.

Non-revascularizable critical limb ischemia (CLI) is the most severe stage of peripheral arterial disease, with no therapeutic option. Extensive preclinical studies have demonstrated that adipose-derived stroma cell (ASC) transplantation strongly improves revascularization and tissue perfusion in ischemic limbs. This study, named ACellDREAM, is the first phase I trial to evaluate the feasibility and safety of intramuscular injections of autologous ASC in non-revascularizable CLI patients.Seven patients were consecutively enrolled, on the basis of the following criteria: (i) lower-limb rest pain or ulcer; (ii) ankle systolic oxygen pressure 50 or 70 mm Hg for non-diabetic and diabetic patients, respectively, or first-toe systolic oxygen pressure 30 mm Hg or 50 mm Hg for non-diabetic and diabetic patients, respectively; (iii) not suitable for revascularization. ASCs from abdominal fat were grown for 2 weeks and were then characterized.More than 200 million cells were obtained, with almost total homogeneity and no karyotype abnormality. The expressions of stemness markers Oct4 and Nanog were very low, whereas expression of telomerase was undetectable in human ASCs compared with human embryonic stem cells. ASCs (10(8)) were then intramuscularly injected into the ischemic leg of patients, with no complication, as judged by an independent committee. Trans-cutaneous oxygen pressure tended to increase in most patients. Ulcer evolution and wound healing showed improvement.These data demonstrate the feasibility and safety of autologous ASC transplantation in patients with objectively proven CLI not suitable for revascularization. The improved wound healing also supports a putative functional efficiency.

Background Adipose-derived mesenchymalstromal cells (ASC) are currently tested in regenerative medicine to promote tissue reconstruction after injury. Regardingautologous purpose, the possible loss of therapeutic function and cell properties during aging have been questioned in adults. To date no reliable information is available concerning ASC from pediatric patients and a better knowledge is required for clinical applications. Methods Subcutaneous adipose tissue was collected from 27 donors (0–1 years old) and 50 donors (1–12 years old) and compared with adult ASC for in vitro characteristics. ASC were then tested in a mouse model of limb ischemia. Results Cells from the stromal vascular fraction (SVF) and subsequent cultured ASC were prepared. Only a greater amount in SVF cell number and ASC proliferative rate were found. Cell phenotype, colony formingunit-fibroblast (CFU-F) content, immunomodulation effect and adipogenic, osteoblastic and angiogenic potentials were not significantly different. In vivo , pediatric ASC induced an increase in microangiographic score in a mouse model of limb ischemia, even though improvement in vascular density was not significantly correlated to limb rescue. Finally messengerRNA (mRNA) analysis using a microarray approach identified that only 305 genes were differentially expressed (217 down- and 88 up-regulated) in pediatric versus adult ASC, confirming that ASC from both age groups shared very close intrinsic properties. Conclusion This is the first study reporting a comparative analysis of ASC from a large number of donors and showing that their in vitro and in vivo properties were similar and maintained during aging.