Von Willebrand Disease (VWD) is a bleeding disorder in which von Willebrand Factor (VWF), a polymeric blood protein, is either completely absent or in a dysfunctional state within the circulatory system. VWF’s function is to respond to shear force through an unfolding conformational change, and it is this unfolding and consequential exposure of platelet binding sites that initiates the blood clotting cascade. Affecting 1% of the entire population, VWD is the most common inherited bleeding disorder. However, few effective treatments exist. A major barrier to understanding VWF function is the absence of human cellular and vascular models that can accurately reconstitute complex phenotypes and molecular mechanisms. Studies of VWF at the cellular and vascular scale can provide important insight into physiological factors that regulate VWF. Human cells are particularly attractive and provide a highly accessible, species-specific model that can be more flexible than mouse models. Current cellular models for VWF functional studies are primarily limited to endogenous VWF secreted from human umbilical vein endothelial cells (HUVECs), which are not immortal, making it difficult to engineer disease models. This project serves to directly address these barriers through the creation and characterization of stable VWF knock-out human pluripotent stem cell lines (hPSC). These cells are immortal and can differentiate into numerous lineages including endothelia. VWF knock-out cells differentiated normally into endothelial cells, as expected, based on cell morphology and endothelial marker expression. The VWF knock-out cells have been confirmed through immunoblot and immunofluorescence to be deficient in various VWF-associated proteins, such as Factor VIII and Angiopoietin-2. We are currently investigating the mechanisms behind these deficiencies and how they relate to the absence of VWF. These findings will enable us to better understand the function of VWF, which will ultimately guide us to discover effective treatments for VWD.

Autophagy is a fundamental biological pathway that facilitates the degradation and recycling of intracellular components. While it is known to be activated in response to cellular stress, it may also have critical roles in developmental processes. To date, studies in mice have shown that deletion of core autophagy proteins impairs the production of many essential hematopoietic lineages, suggesting that autophagy is critical for blood cell differentiation. In particular red cell production is known to be dependent on autophagy however (1) when does autophagy occur? and (2) how is it regulated? In human erythropoiesis is unknown. Preliminary data from the Doulatov lab has identified a novel negative regulator of autophagy, ATG4A, in human erythropoiesis. The ATG4 family of proteins are cysteine proteases known to regulate LC3B a critical molecule which decorates the outside of the autophagosome. However, whether its expression is confined to erythropoiesis or broadly applies to other hematopoietic lineages is unknown. Therefore, I performed a bioinformatic analysis on a large RNA microarray dataset which profiles gene expression in 38 distinct cell populations in the 7 major hematopoietic lineages. Only ATG4A and ATG4B were detected in the dataset, and when mapped across the hematopoietic hierarchy had differing patterns of expression. In contrast to ATG4B the expression of autophagy protein ATG4A was selectively upregulated in the erythroid lineage. Previous studies have described a critical role BNIP3L(NIX) in erythropoiesis, therefore I compared the expression pattern of ATG4A to BNIP3L. ATG4A and BNIP3L had similar patterns of expression suggesting multiple autophagy proteins may be upregulated during erythroid differentiation. Taken together these data suggest that ATG4A ia a unique regulator in the human erythroid lineage.