Background: Current systems generating lymphatic endothelial cell (LEC) from human induced pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival in vivo. Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel (PA-RGDS). Methods and
Results: hPSCs were cultured with GSK3β inhibitor on collagen-coated plates for 2-3 days to induce differentiation into the mesodermal lineage. The mesodermally differentiated cells were then cultured with VEGFC, VEGFA, EGF, and bFGF for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN+FLT4+ cells (hPSC-derived lymphatic endothelial cells, hPSC-LECs) showed highly purified and fully functional LEC characteristics in vitro. These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs encapsulated with PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. Histological examination demonstrated that the skin thickness was significantly reduced and the density of lymphatic vessels was markedly increased when the hPSC-LECs were encapsulated with PA-RGDS compared to others.
Conclusion: This study demonstrated for the first time that hPSC can be differentiated into LECs in a clinically compatible manner with a high yield. Furthermore, nanomatrix encapsulated hPSC-LECs can substantially improve lymphedema in mouse tail through enhancement of cell survival and lymphatic neovascularization. This engineered hPSC-LEC therapy represents a novel option for treating lymphedema.