Lipidomics of mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs), such as adipose-derived stem cells (ADSCs) and skeletal muscle-derived stem cells (MDSCs), are potential sources for cell-based therapeutic strategies. However, there is little knowledge about the lipid composition of these stem cells and the mechanisms of their differentiation. Lipids have important biological and physiological functions that are critical for understanding the regulation and control of stem cell fate. This study sought to analyze the lipidome of rabbit ADSCs and MDSCs and their adipogenic and osteogenic differentiation. The MSCs were isolated and were characterized by flow cytometry. Lipids were extracted from both MSCs and differentiated cells, and the lipids were subsequently analyzed with a hybrid triple quadrupole time-of-flight mass spectrometer. The results showed a total of 1687 lipid species. MSCs exhibited different lipid profiles as well as changes in lipid composition after differentiation. Furthermore, the expression levels of N-acyl-phosphatidylethanolamine (NAPE) 54:7+NH4 (-FA 17:0(NH4)) and phosphatidylcholine (PC) 42:6+Na were higher in the adipogenic lineages in of both MSC types, and NAPE 58:2+NH4 (-FA 17:0 (NH4)) and NAPE 56:2+NH4 (-FA 17:0 (NH4)) had higher levels in the osteogenic lineages, suggesting lipid similarities in cells differentiated from different stem cell sources.

Cell kinetics, DNA integrity, differentiation, and lipid fingerprinting analysis of rabbit adipose-derived stem cells.

Human adipose tissue has been described as a potential alternative reservoir for stem cells. Although studies have been performed in rabbits using autologous adipose-derived stem cells (ADSC), these cells have not been well characterized. The primary objectives of this study were to demonstrate the presence of adipose-derived stem cells isolated from rabbit inguinal fat pads and to characterize them through osteogenic and adipogenic in vitro differentiation and lipid fingerprinting analysis. The secondary objective was to evaluate cell behavior through growth kinetics, cell viability, and DNA integrity. Rabbit ADSCs were isolated to determine the in vitro growth kinetics and cell viability. DNA integrity was assessed by an alkaline Comet assay in passages 0 and 5. The osteogenic differentiation was evaluated by Von Kossa, and Alizarin Red S staining and adipogenic differentiation were assessed by Oil Red O staining. Lipid fingerprinting analyses of control, adipogenic, and osteogenic differentiated cells were performed by MALDI-TOF/MS. We demonstrate that rabbit ADSC have a constant growth rate at the early passages, with increased DNA fragmentation at or after passage 5. Rabbit ADSC viability was similar in passages 2 and 5 (90.7% and 86.6%, respectively), but there was a tendency to decreased cellular growth rate after passage 3. The ADSC were characterized by the expression of surface markers such as CD29 (67.4%) and CD44 (89.4%), using CD 45 (0.77%) as a negative control. ADSC from rabbits were successfully isolated form the inguinal region. These cells were capable to differentiate into osteogenic and adipogenic tissue when they were placed in inductive media. After each passage, there was a trend towards decreased cell growth. On the other hand, DNA fragmentation increased at each passage. ADSC had a different lipid profile when placed in control, adipogenic, or osteogenic media.

Cyclosporine A and NAC on the inducible nitric oxide synthase expression and nitric oxide synthesis in rat renal artery cultured cells.

BACKGROUND: The immunosuppressor cyclosporine A (CsA) presents the nephrotoxicity as its major side effect that is mostly attributed to a renal vasoconstriction. This may be due to an excessive generation of vasoconstrictors like reactive oxygen species (ROS), or due to a reduction of vasodilators such as the nitric oxide, which in turn, can be caused by increased amounts of ROS. We evaluated the effect of CsA and the antioxidant N-acetylcysteine (NAC) on inducible nitric oxide synthase (iNOS) mRNA expression and nitric oxide synthesis, in rat renal artery vascular smooth muscle cells (rVSMCs) primary culture. METHODS: In cells treated during 72 hours with CsA (10 microg/mL), its vehicle (control) (10 microL/mL), Escherichia coli lipopolysaccharide (LPS) (100 microg/mL), CsA + LPS, NAC (6.13 mmol/L), or CsA + NAC, we determined the nitric oxide synthesis (Griess and chemiluminescence methods), iNOS expression [reverse transcription-polymerase chain reaction (RT-PCR)] and cell viability (acridine orange method). RESULTS: In rVSMCs, LPS increased nitric oxide and iNOS expression; CsA decreased basal and LPS-induced nitric oxide and iNOS expression; NAC increased nitric oxide and blunted the nitric oxide reduction caused by CsA, with no effect on iNOS. CsA reduced cell viability. CONCLUSION: In this study, CsA reduced nitric oxide synthesis in rVSMCs, both through iNOS down-regulation and reduction of cell viability, which could be responsible for the vasoconstrictive effect of the CsA. In the effect of CsA on nitric oxide, probably a role is also played by free radical production, as this effect was blunted by NAC.