Abstract
Human pluripotent stem cells (hPSCs) have emerged as an important source for cell therapy. However, to date, no studies demonstrated generation of purified hPSC-derived lymphatic endothelial cells (LECs) and tested their therapeutic potential in disease models. Here we sought to differentiate hPSCs into the LEC lineage, purify them with LEC markers, and evaluate their therapeutic effects. We found that an OP9-assisted culture system reinforced by addition of VEGF-A, VEGF-C, and EGF most efficiently generated LECs, which were then isolated via FACS-sorting with LYVE-1 and PODOPLANIN. These hPSC-derived LYVE-1+PODOPLANIN+cells showed a pure committed LEC phenotype, formed new lymphatic vessels, and expressed lymphangiogenic factors at high levels. These hPSC-derived LECs enhanced wound healing through lymphangiogenesis and lymphvasculogenesis. Here we report, for the first time, that LECs can be selectively isolated from differentiating hPSCs, and that these cells are potent for lymphatic vessel formation in vivo and wound healing. This system and the purified hPSC-derived LECs can serve as a new platform for studying LEC development as well as for cell therapy.
Highlights
Human pluripotent stem cells have emerged as an important source for cell therapy
To determine whether human embryonic stem cells (hESCs) were differentiated into lymphatic endothelial cells (LECs), we performed gene expression analysis with an emphasis on the expression of key LEC markers such as PROX1, LYVE1, VEGFR3 and PODOPLANIN
We found that hESCs (H1 and H9) began to express the LEC markers at day 7, peaked around day 20, and maintained expression up to day 30 (Fig. 1a)
Summary
Human pluripotent stem cells (hPSCs) have emerged as an important source for cell therapy. Macrophages derived from diabetic mice failed to improve wound repair, but upon activation with IL-1β promoted the recovery of the tissue injury with enhanced lymphatic regeneration, suggesting a critical role of lymphatic vessels in wound healing[18] Despite this emerging knowledge of the importance of lymphatic vessels in wound healing, there are no studies available regarding the effects of stem cell therapy targeting lymphatic neovascularization on wound repair. We for the first time developed an efficient culture system to differentiate hESCs and hiPSCs into the lymphatic endothelial lineage and isolated LYVE-1+PODOPLANIN+cells as functional LECs. we demonstrated the contribution of these hPSC-derived LECs to in vivo lymphatic vascular commitment and their therapeutic potential in wound healing
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