Recently work has been focused on

Recently, work has been focused on the adipose tissue-derived stromal cell (ASCs) population, since retrieval is straight forward and non-harmful coupled to low-risk surgical procedures. However, biological properties of ASCs have only been assessed by indiscriminate isolation from mostly white fat depots. Little has been done to evaluate potential intrinsic differences between anatomic depot regions. By only considering all ASC populations as functionally and biologically equivalent, this has consequently led to incomparable and sometimes contradictory results. Yet, several reports, while supporting intrinsic differences between fat depots, indirectly suggest divergences in ASCs. Abdominal ASCs demonstrate diminished susceptibility to apoptosis (Toyoda et al., 2009), whereas those derived from pericardium, omentum and groin display different Phos-tag (Russo et al., 2014). According to the anatomical location of the fat, ASCs could differently exhibit surface epitopes as well as cell yield, ability to differentiate and morphological properties. This may have important consequence on altered response to pharmaceutical treatments (Russo et al., 2014; Perrini et al., 2008; Tchkonia et al., 2002; Gibellini et al., 2015; Schweizer et al., 2015; Elahi et al., 2016; Luna et al., 2014). Differences between fat depots grown under different conditions are not well known. We have investigated the potential of human mediastinal depot as a novel ASC pool (Siciliano et al., 2015a,b,c), including the treatment with hemoderivate agents, such as platelet lysate (PL) (Siciliano et al., 2015a,b,c). In this study, we aimed to compare ASCs derived from the mediastinal region (fat inside the rib cage) to ASCs derived from the subcutaneous adipose tissue (fat outside the rib cage). Two different supplements, PL and FBS were used to unveil their biological features. Our study revealed functional differences in ASCs derived from the mediastinal region respect to the subcutaneous, showing enhanced pro-angiogenic features, whereas subcutaneous displayed higher proliferative and clonogenic abilities and a greater degree of autophagy associated structures.
Materials and methods
Results
Discussion Finally, although observed only in a single donor, we verified if differences in the two ASC pools mirrored dissimilar intracellular composition and organization. Ultrastructural differences have been reported to reflect differentiative potential and mechanical properties (Pasquinelli et al., 2007; Gonzalez-Cruz et al., 2012). Interestingly, we found that mediastinal ASCs showed a higher cell diameter than subcutaneous ASCs regardless of the treatment, likely coherent with a different anatomic profile. However, the combination of the supplement and the different origin of the fat depot seemed to influence certain ultrastructural characteristics of ASCs. Accordingly, we observed changes in mitochondrial cristae morphology in both ASC pools (suggesting different oxidative states, as well as a preferential anaerobic/aerobic metabolism on the basis of the treatment), but also increased microvilli and microvesicles in presence of PL. These are hallmarks of a more active autophagic flux in ASCs, and confirming the known ability of PL to boost metabolic growth and differentiation compared to FBS (2, 38). A major prevalence of primary lysosomes (suggesting early autophagy) in mediastinal ASCs, versus abundantly and accumulated autophagosomes in the subcutaneous counterpart (markers of advanced autophagy) was found, indicating enhanced, and perhaps faster turnover of this latter in recycling proteins or organelles. As FBS represents a cellular distress compared to PL, it is also plausible that changes in the autophagic process observed upon FBS may reflect an attempt of the cultures to survive in harsher microenvironments, as recently shown upon other treatments (Molaei et al., 2015). To date, autophagy in ASCs represents a relevant topic. It significantly contributes to the dysregulation of the adipose tissue by controlling lipid accumulation, and balancing white and brown fat deposition (Singh et al., 2009; Maixner et al., 2016). Intriguingly, autophagy is enhanced in obesity and associated to visceral fat distribution (Kovsan et al., 2011), thus encouraging further investigations on the ability of the stromal subset during adipogenic commitment.