A novel approach for reliable differentiation of lymphatic endothelial progenitor cells in vitro

The lymphatic system plays critical roles in fluid homeostasis, immune regulation, and lipid transport, making its dysfunction a contributor to numerous pathological conditions. Lymphatic tissue engineering aims to develop therapeutic strategies for lymphatic regeneration and repair, depending on the availability of suitable cell sources for lymphatic endothelial cell (LEC) generation. Key cell sources explored for lymphatic tissue engineering include the dermis, bone marrow and stromal vascular fraction. To our knowledge, no existing differentiation protocols can accurately generate lymphatic endothelial progenitor cells (LEPCs). This study presents a novel in vitro differentiation protocol for generating LEPCs from stromal vascular fraction, dermis and bone marrow-derived mesenchymal stem cells (MSCs). The feasibility of using them as tissue sources was first evaluated leading to dermal cells exclusion due to low yield post-isolation, and highlighting the limited differentiation efficiency of bone marrow MSCs. However, the stromal vascular fraction emerged as the optimal source, exhibiting robust LEPCs differentiation and superior scalability. In vitro characterization confirmed that LEPCs maintain a lymphatic endothelial phenotype, exhibiting high migratory ability, robust angiogenic structure formation, and consistent expression of key lymphatic markers in both 2D and 3D cultures, as validated by RNA-Seq analysis. The protocol provides a consistent platform for studying lymphatic endothelial biology and potential applications in regenerative medicine and therapeutic angiogenesis.