CO2 laser increases the regenerative capacity of human adipose-derived stem cells by a mechanism involving the redox state and enhanced secretion of pro-angiogenic molecules.

CO2 laser has a beneficial effect on stem cells by mechanisms that are not clearly elucidated. We hypothesize that the effect of fractional CO2 laser on human adipose-derived stem cells (ADSC) could be due to changes in redox homeostasis and secretion of factors contributing to cellular proliferation and angiogenic potential. ADSC incubated in medium containing 0.5 or 10 % FBS were exposed to a single irradiation of a 10,600-nm fractional CO2 laser; non-irradiated ADSC were used as control. Viability/proliferation of ADSC was assessed by MTT assay; the intracellular reactive oxygen species (ROS) levels and the mitochondrial membrane potential (∆Ψm) were determined with DCFH-DA and JC-1 fluorescent probes, respectively. Molecules secreted by ADSC in the medium were determined by ELISA assay, and their capacity to support endothelial tube-like formation by the Matrigel assay. The results showed that compared to controls, ADSC kept in low FBS medium and irradiated with CO2 laser at 9 W exhibited: (a) increased proliferation (∼20 %), (b) transient increase of mitochondrial ROS and the capacity to restore Δψm after rotenone induced depolarization, and (c) augmented secretion in the conditioned medium of MMP-2 (twofold), MMP-9 (eightfold), VEGF (twofold), and adiponectin (∼50 %) that have the capacity to support angiogenesis of endothelial progenitor cells. In conclusion, the mechanisms underlying the benefic effect of CO2 laser on ADSC are the activation of the redox pathways which increases cell proliferation and enhances secretion of angiogenic molecules. These results explain, in part, the mechanisms involved in the increased regenerative potential of CO2 laser-exposed ADSC that could be exploited for clinical applications.

Testosterone stimulates proliferation and preserves stemness of human adult mesenchymal stem cells and endothelial progenitor cells.

Human adult stem and progenitor cells are promising cell types widely studied for their clinical benefits. A reduced number of stem cells present in the human body are associated with numerous dysfunctions. Since androgens have a profound effect on different cell types, we questioned whether testosterone (T), one of the main androgens, influence and are involved in the proliferation of stem cells and÷or affect their stemness potential. Isolated mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) were cultured and then stimulated with different concentration of testosterone (10-100 nM). The cellular proliferation rate, adhesion, and viability were measured in real-time using xCELLigence system and DNA-cell proliferation assay. The immunophenotype of the stimulated cells versus non-stimulated cells was determined by flow cytometry. The maximal effect on MSCs and EPCs proliferation was obtained at 40 nM testosterone; this concentration was used in further experiments. The cellular index measured in real-time by impedance-based dynamic measurements revealed that 40 nM testosterone had a proliferative effect on both MSCs and EPCs, having a proliferative index of ˜50% above the control (non-stimulated) cells. Furthermore, flow cytometry assay indicated that testosterone stimulation did not alter the phenotype of MSCs and EPCs, both cell types preserving the expression of the characteristic surface markers. Testosterone stimulation increases the proliferation and preserves stemness of MSCs and EPCs suggesting that, besides other factors, the hormone may engineer these cells and increase their therapeutic potential.

Isolation method and xeno-free culture conditions influence multipotent differentiation capacity of human Wharton's jelly-derived mesenchymal stem cells.

INTRODUCTION: Human Wharton's jelly (WJ) has become a preferred source of mesenchymal stem cells (MSCs) whose clinical applications are limited by the use of adequate xeno-free (XF), in vitro manipulation conditions. Therefore, the objective of our study was to characterize WJ-derived MSCs (WJ-MSCs), isolated by different methods and cultured in a commercially available, MSC XF medium, not least of all by investigating their endothelial differentiation capacity. METHODS: WJ explants and enzymatically dissociated WJ cells were cultured in a defined, XF medium for MSCs. Adherent cells at passages 2 and 5 were characterized as MSCs by flow cytometry, MTT, real-time quantitative reverse transcription PCR, and functional multipotent differentiation assays. The endothelial differentiation capacity of MSCs isolated and expanded until passage 2 in the MSC XF medium, and then subcultured for five passages in a commercially available endothelial growth medium (group A), was assessed over serial passages, as compared to adherent WJ-derived cells isolated and expanded for five consecutive passages in the endothelial medium (group B). RESULTS: The MSC phenotype of WJ explant- and pellet-derived cells, isolated and expanded in the MSC XF medium, was proven based on the expression of CD44/CD73/CD90/CD105 surface markers and osteo-/adipo-/chondrogenic multipotent differentiation potential, which differed according to the isolation method and/or passage number. Upon exposure to endothelial differentiation cues, cells belonging to group A did not exhibit endothelial cell characteristics over serial passages; by contrast, WJ pellet-derived cells belonging to group B expressed endothelial characteristics at gene, protein and functional levels, potentially due to culture conditions favoring the isolation of other stem/progenitor cell types than MSCs, able to give rise to an endothelial progeny. CONCLUSIONS: The use of defined, MSC XF media for isolation and expansion of human WJ-MSCs is a prerequisite for the establishment of their real endothelial differentiation capacity, as candidates for clinical therapy applications. Thus, the standardization of WJ-MSCs isolation and culture expansion techniques in defined, MSC XF media, for their accurate characterization, would be a priority in the stem cell research field.