Person:

Turcotte, Raphaël

Transplantation of a single hematopoietic stem cell is an important method for its functional characterization, but the standard transplantation protocol relies on cell homing to the bone marrow after intravenous injection. Here, we present a method to transplant single cells directly into the bone marrow of live mice. We developed an optical platform that integrates a multiphoton microscope with a laser ablation unit for microsurgery and an optical tweezer for cell micromanipulation. These tools allow image-guided single cell transplantation with high spatial control. The platform was used to deliver single hematopoietic stem cells. The engraftment of transplants was tracked over time, illustrating that the technique can be useful for studying both normal and malignant stem cells in vivo.

Background: Sirtuin-1 (SirT1), a nicotinamide adenine dinucleotide+–dependent deacetylase, is a key enzyme in the cellular response to metabolic, inflammatory, and oxidative stresses; however, the role of endogenous SirT1 in the vasculature has not been fully elucidated. Our goal was to evaluate the role of vascular smooth muscle SirT1 in the physiological response of the aortic wall to angiotensin II, a potent hypertrophic, oxidant, and inflammatory stimulus. Methods and Results: Mice lacking SirT1 in vascular smooth muscle (ie, smooth muscle SirT1 knockout) had drastically high mortality (70%) caused by aortic dissection after angiotensin II infusion (1 mg/kg per day) but not after an equipotent dose of norepinephrine, despite comparable blood pressure increases. Smooth muscle SirT1 knockout mice did not show any abnormal aortic morphology or blood pressure compared with wild-type littermates. Nonetheless, in response to angiotensin II, aortas from smooth muscle SirT1 knockout mice had severely disorganized elastic lamellae with frequent elastin breaks, increased oxidant production, and aortic stiffness compared with angiotensin II–treated wild-type mice. Matrix metalloproteinase expression and activity were increased in the aortas of angiotensin II–treated smooth muscle SirT1 knockout mice and were prevented in mice overexpressing SirT1 in vascular smooth muscle or with use of the oxidant scavenger tempol. Conclusions: Endogenous SirT1 in aortic smooth muscle is required to maintain the structural integrity of the aortic wall in response to oxidant and inflammatory stimuli, at least in part, by suppressing oxidant-induced matrix metalloproteinase activity. SirT1 activators could potentially be a novel therapeutic approach to prevent aortic dissection and rupture in patients at risk, such as those with hypertension or genetic disorders, such as Marfan’s syndrome.

Clonal heterogeneity and selection underpin many biological processes including development and tumor progression. Combinatorial fluorescent protein expression in germline cells has proven its utility for tracking the formation and regeneration of different organ systems. Such cell populations encoded by combinatorial fluorescent proteins are also attractive tools for understanding clonal expansion and clonal competition in cancer. However, the assignment of clonal identity requires an analytical framework in which clonal markings can be parameterized and validated. Here we present a systematic and quantitative method for RGB analysis of fluorescent melanoma cancer clones. We then demonstrate refined clonal trackability of melanoma cells using this scheme.