A novel approach for reliable differentiation of lymphatic endothelial precursor 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 generation. Key cell sources explored for lymphatic tissue engineering include the dermis, bone marrow (BM) and stromal vascular fraction (SVF). To our knowledge, no existing differentiation protocols can accurately generate lymphatic endothelial precursor cells (LEPCs). This study presents a novel in vitro differentiation protocol for generating murine LEPCs from stromal SVF, dermis and BM-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 BM-MSCs. SVF-derived LEPCs showed the most prominent results, exhibiting robust LEPCs differentiation and superior scalability. In vitro characterization confirmed that LEPCs maintain a distinct lymphatic endothelial phenotype, exhibiting high migratory capacity, robust capillary-like network formation, and consistent expression of key lymphatic markers across 2D and 3D cultures, supported by strong transcriptomic alignment as validated by RNA-Seq. Furthermore, in vivo modelling demonstrated their preclinical efficacy, as transplanted LEPCs integrated seamlessly into pre-existing lymphatic vessels within a wound-healing model. The protocol provides a consistent platform for studying lymphatic endothelial biology and potential applications in regenerative medicine and therapeutic angiogenesis.