Metabolically Driven Differentiation of Human Pluripotent Stem Cells to Generate Robust and Functional Lymphatic Endothelial Cells.

The lymphatic system plays various crucial but underappreciated roles in fluid transport and immune response in numerous organs and tissue types. Consequently, generation of lymphatic vessels has been postulated as an innovative therapeutic strategy for various diseases. However, there is a lack of efficient and reliable method to differentiate human pluripotent stem cells into lymphatic endothelial cells (LEC) for lymphatic regeneration. Current differentiation methods suffer from poor yield and low lymphatic marker expression, while also having limited clinical applicability due to its reliance on either the embryoid body intermediates or xenogenic supporting cells. Here, we develop a stepwise differentiation method that relies on fully defined treatment with sodium acetate, a metabolite resulting from fatty acid oxidation, along with VEGF-C, that yields high expression of lymphatic marker and retain the functionality of native LECs. We show that differentiated LECs (dLEC) express comparable levels of lymphatic markers compared to native LECs, which is up to 15 times higher than the current leading standard of dLECs. We also show that the dLECs can form lymphatic vascular networks in 2D and 3D and secrete important lymphangiocrine for organ maturation. Upon implantation into murine lymphedema models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. In conclusion, our functional dLECs can be incorporated into novel tissue engineering applications in personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders.