Considering the increasing interest in adipose-derived stem cells (ASCs) in regenerative medicine, optimization of methods aimed at isolation, characterization, expansion and evaluation of differentiation potential is critical to ensure (a) the quality of stem cells also in terms of genetic stability; (b) the reproducibility of beneficial effects; and (c) the safety of their use. Numerous studies have been conducted to understand the mechanisms that regulate ASC proliferation, growth and differentiation, however standard protocols about harvesting and processing techniques are not yet defined. It is also important to note that some steps in the procedures of harvesting and/or processing have been reported to affect recovery and/or the physiology of ASCs. Even considering the great opportunity that the ASCs provide for the identification of novel molecular targets for new or old drugs, the definition of homogeneous preparation methods that ensure adequate quality assurance and control, in accordance with current GMPs (good manufacturing practices), is required. Here, we summarize the literature reports to provide a detailed overview of the methodological issues underlying human ASCs isolation, processing, characterization, expansion, differentiation techniques, recalling at the same time their basilar principles, advantages and limits, in particular focusing on how these procedures could affect the ASC quality, functionality and plasticity.

Aberrant nitric oxide synthase 2 (NOS2) expression has been suggested as an interesting therapeutic target that is being implicated as a component of the molecular profile of several human malignant tumors, including glioblastoma, which is the most aggressive brain tumor with limited therapeutic options and poor prognosis. The aim of the present work was to evaluate the effect of 1400W, a specific NOS2 inhibitor, on human glioma cells in terms of clonogenic potential, proliferation, migration rate, and neurosphere generation ability. NOS2 expression was determined by Western blotting. Nitric oxide (NO) production was measured through nitrite level determination. The trypan blue exclusion test and the plate colony formation assay were performed to evaluate cell proliferation and clonogenic potential. Cell proliferation and migration ability was assessed by the in vitro wound-healing assay. Neurosphere generation in a specific stemcell medium was investigated. NOS2 was confirmed to be expressed in both the glioma cell line and a human glioma primary culture, and overexpressed in relative derived neurospheres. Experiments that aimed to evaluate the influence of 1400W on U-87 MG, T98G (glioblastoma cell lines) and primary glioma cells sustained the crucial role played by NOS2 in proliferation, colony formation, migration, and neurosphere generation, thus supporting the emerging relevance of a NOS2/NO system as a prognostic factor for glioma malignancy and recurrence.