Figure 4. Metabolites that undergo mitochondrial β-oxidation, and neuroprotectin D1 promote cardiac and neuronal differentiation.

(a) Role of acyl-carnitines in fatty acid metabolism in the mitochondria. (b) Saturated fatty acids and acyl-carnitines enhance neuronal and cardiomyocyte differentiation, respectively. Map2ab/βIII-tubulin double positive neuron count increases with higher concentrations of palmitic acid (0.8 µM to 8µM) and capric acid (0.1 µM to 3 µM). An increase in CT-3 positive clusters was observed with increasing concentrations of palmityl-carnitine. Differentiation media was supplemented with 4, 8, or 16 µM concentrations of palmityl-carnitine for the first 9 days and the metabolite removed for the last day of differentiation. Cardiomyocyte differentiation was evaluated with CT3 staining, an antibody for the cardiac cell marker cardiotroponin. Marker positive counts in ES cells differentiated in the absence of DMSO or ethanol were not different from the DMSO or ethanol controls. (c) Neuroprotectin D1 (NPD1) enhances neuronal differentiation. Differentiation media was supplemented with 50 nM of NPD1, leukotriene B4 or C4 throughout differentiation. ** indicates p-value < 0.001 as compared to that of ethanol treated cells. Photographs show neuronal differentiation evaluated with βIII-tubulin (red) staining. Nuclear staining was performed with DAPI (blue). Four independently prepared cell culture replicates were analyzed for each metabolite concentration. Data points and error bars represent mean values and s.d.