P85	Proteomic analysis of stem cell-derived smooth muscle cells.
X.Yin, Q.Xiao, U.Mayr, M.Mayr, Q.Xu
St Georges University of London, London, GB.

Background Embryonic stem cells (ES cells) can differentiate into a variety of mature cells, such as vascular smooth muscle (SMCs), which could be a tissue source for repairing damaged organs. However, the molecular mechanisms of stem cell differentiation into SMCs are not fully understood, and associated changes in protein expression remain to be characterized. Methods and Results Mouse ES cells were cultivated on collagen-IV-coated flasks, and Sca-1+ progenitor cells were isolated with magnetic beads. The isolated cells were placed into gelatin-coated flasks and cultured in DM medium with or without PDGF-BB (10ng/ml) for 3 to 90 days. Both immunostaining and FACS analyses revealed that more than 95% of these cells were positive for smooth muscle α-actin (SMA), SM22, calponin, and smooth muscle myosin heavy chain (SMMHC). Protein extracts of ES cells, Sca-1+ progenitor cells, ES cell-derived SMCs (ES-SMCs) and aortic SMCs were separated by two-dimensional gel electrophoresis. 300 protein species of each cell line were analyzed by mass spectrometry. Protein maps are available on our website http://www.vascular-proteomics.com. Notably, antioxidants were upregulated in ES-SMCs. Enzymatic assays confirmed a 3-fold increase in glutathione reductase activity compared to aortic SMCs. Despite this rise in anti-oxidative defense capacity, ES-SMCs displayed increased oxidation of redox-sensitive proteins and were more sensitive to oxidative stimuli: depletion of intracellular glutathione by diethylmaleate or inhibition of glutathione reductase by carmustine resulted in a remarkable loss of cell viability in ES-SMCs, but not in aortic SMCs. Conclusion We present the first protein profiles of murine aortic SMC, ES-SMC and their progenitors. Our proteomic comparison revealed that differentiation of ES cells to SMCs is associated with increased oxidative stress and consequently ES-SMCs require additional antioxidant protection for survival. These results indicate that treatment with anti-oxidants could be beneficial for tissue repair from ES cells.

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