Lymphatic malformations are prevalent in a myriad of diseases, including RASopathies such as Noonan syndrome. Although common, such malformations remain largely understudied due to a lack of cell or animal models with which to study them. Existing differentiation protocols for lymphatic endothelial cells (LECs) from human pluripotent stem cells have poor yield and require feeder cells. Here, we sought to develop a novel strategy to robustly differentiate human pluripotent stem cells (hPSCs) into cells with a lymphatic phenotype in feeder-free, serum-free conditions. We hypothesized that recapitulating lymphangiogenesis in vitro will allow induction of hPSCs to adopt a lymphatic phenotype. We further demonstrated that VEGF-C/VEGFR3 signaling alone was not sufficient in driving lymphatic fate. Additionally, we implicate a synergistic role for hepatocyte growth factor (HGF) and angiopoietin-1 in directing lymphatic cell phenotypes. With the use of immunohistochemistry, qPCR, as well as flow cytometry, we were able to indicate expression of lymphatic specific markers in our differentiated population. These findings aim to gain more insight into methods by which lymphangiogenesis can be recapitulated in an in vitro system, and in doing so increase the efficiency with which LECs are differentiated as well as the accessibility to that cell population. Establishing a feeder-free and serum-free differentiation protocol for LECs will provide a robust model to study the cellular and molecular mechanisms that govern lymphatic development in vivo and disease.
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