Abstract 1064: Creation Of Lymphatic Endothelial Cells From Induced Pluripotent Stem Cells For Use In Regenerative Therapy For Lymphedema

Abstract

Background/Objective: Lymphedema is a condition characterized by impaired flow of interstitial fluid through the lymphatic system and chronic obliteration of distal lymphatic collecting vessels. Despite advances in our knowledge of the development of lymphatic vessels, there has been little progress translating this knowledge into clinically relevant regenerative therapies. Although lymphatic endothelial cells (LECs) share a lineage with arterial and venous endothelial cell progenitors, currently described processes to derive LECs from stem cells in vitro take up to a month, require embryoid body formation, and have been minimally characterized. Methods: We have developed a protocol for the generation of lymphatic endothelial cells from induced pluripotent stem cells (iPSCs) over 11 days. First, iPSCs are directed to become endothelial cell progenitors over 6 days and purified with CD144 magnetic beads. Then, ECs are directed to differentiate into LECs using a 5-day treatment with 10 ng/mL Ang1 alone or in combination with 200 ng/mL VEGFc. Human primary LECs were used as a positive control and iPSC-ECs without Ang1 or VEGFc treatment as well as fibroblasts were negative controls. Results: Treating iPSC-derived endothelial cells for 5 days with Ang1 +/- VEGFc yields iPSC-LECs with morphological similarity to primary lymphatic controls and expression of multiple lymphatic markers including Podoplanin, Lyve1, Prox1, and VEGFR3, verified by immunofluorescent staining, western blotting, and flow cytometry across two independent cell lines. In flow cytometry studies, 75% of LECs treated with Ang1 and 72% of LECs treated with Ang1 and VEGFc co-express Podoplanin and Lyve1. There was more than a three-fold increase compared to CD144+ endothelial progenitors (20.4%, p<0.001) while fibroblast expression was negligible (0.6%, p<0.001). Finally, the overall transcriptome of LECs treated with Ang1 was compared to primary human lymphatic cells using single cell RNA sequencing. Conclusions: Treatment with Ang1 with or without VEGFc allows fast and efficient generation of LECs from iPSCs. This allows LEC differentiation in vitro from a wide variety of patient specific cell types and improves the practicality of regenerative therapy for lymphedema.