Metabolically driven differentiation of human pluripotent stem cells to generate robust and functional lymphatic endothelial cells

Abstract

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. Given that LECs exclusively rely on anaerobic and fatty acid metabolism due to the hypoxic environment of the lymph, here we report that the unique lymphatic-specific metabolic pathways can be exploited to promote lymphatic identity in differentiated LECs (dLECs). We show that dLECs express elevated levels of lymphatic markers compared to native endothelial cells, which is up to 15 times higher than the current leading standard of dLECs. Moreover, dLECs can form lymphatic vascular networks in both 2D and 3D, as well as secrete important lymphangiocrine for organ maturation. Upon implantation into double-ligation tail lymphedema and mammary fat pad models, dLECs were able to integrate with the host lymphatic vessels, restore fluid flow, and reduce swelling. Collectively, we show that metabolite supplementation can drive stem cell differentiation into dLECs, which can be incorporated into new alternative methods for personalized therapies and disease modeling, as well as provide a direct therapeutic option for lymphedema and lymphatic disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=179 SRC="FIGDIR/small/686405v2_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1c4caa5org.highwire.dtl.DTLVardef@d42b08org.highwire.dtl.DTLVardef@1554ffcorg.highwire.dtl.DTLVardef@1f64ae6_HPS_FORMAT_FIGEXP M_FIG C_FIG A xeno-free and stepwise differentiation protocol with VEGF-C and sodium acetate induces lymphatic identity in iPSC-derived endothelial cells. Differentiated LECs (dLECs) express key lymphatic markers as verified using bulk RNA-sequencing, qPCR, FACS, and immunostaining. These dLECs secrete important cytokines for lympangiocrine signaling, able to form robust 2D and 3D lymphatic networks, as well as demonstrate in vivo functionality and host integration in murine models.