Rapid and efficient generation of human iPSC-derived lymphatic endothelial cells for functional assays

The lymphatic system plays a crucial role in fluid homeostasis, immune responses, and lipid metabolism. Comprising specialized lymphatic endothelial cells (LECs), it enables interstitial fluid uptake and transit to lymph nodes. Investigations of lymphatic vasculature are typically performed in vivo due to the complex nature of this system. However, in vitro models, particularly those using induced pluripotent stem cells (iPSCs), offer a promising alternative by providing a renewable, animal-free, patient-specific, human cell source that can be easily manipulated. Here, we describe a robust and efficient 10-day protocol to differentiate human iPSCs into functional LECs, validated by immunostaining, flow cytometry, and RNA-sequencing. The iPSC-derived LECs (iLECs) exhibit comparable functionality to in vivo lymphatics, including their response to inflammatory and lymphangiogenic stimuli and ability to form 3D lumenized lymphatic networks. The protocol's reproducibility across multiple iPSC lines underscores its reliability and utility for studying lymphatic biology. Overall design: To confirm a lymphatic endothelial cell fate of iPSC-derived lymphatic endothelial cells (LECs) we performed gene expression profiling from three iPSC lines or matched iPSC-derived lymphatic endothelial cell differentiations.