Novel cytometry-based characterization of lysosomal storage disease affected patient's cells

Wolman disease (WD) is a severe lysosomal storage disorder characterized by fatal lipid accumulation caused by the deficiency of a lipid metabolic enzyme, Lysosomal Acid Lipase (LAL), involved in the lysosomal hydrolysis of cholesterols and triglycerides. Due to the imbalance of lipids homeostasis, WD patients suffer from severe hepatosplenomegaly, hepatic failure and adrenal calcification resulting in a premature infant death within the first year of age. In this work, we explored multiple imaging analyses to fully characterize the phenotype of LAL deficient cells. In particular, we stained WD patients' fibroblasts for intracellular lipid droplets (LD) and lysosomes and we analysed staining intensity and granularity as well as an increased number of LD and lysosomes using fluorescence wide field microscopy, confocal microscopy, conventional and image flow cytometry. Noteworthy, we showed that lipid homeostasis was restored upon delivery of a functional LAL transgene. Finally, since fibroblasts cannot be used as routine clinical test as they are difficult to collect from WD patients, we confirmed our observations in LAL deficient human blood cell lines and in peripheral blood mononuclear cells (PBMC) from LAL deficient (LAL-D) mouse model, as a proxy for easily accessible WD PBMC. Overall, we expect that this novel imaging analysis pipeline will help to diagnose WD, follow its progression and evaluate the success of enzyme replacement therapy or gene correction strategies for WD as well as other lysosomal storage disorders.