Emerging evidence has suggested a contribution of bone marrow (BM) cells to lymphatic vessel formation; however, the exact phenotype of the cells with lymphatic endothelial progenitor cell (LEPC) function has yet to be identified. Here we investigate the identity of BM-derived LEPCs and their role in lymphatic neovascularization.

Culture of BM-mononuclear cells (MNCs) in the presence of VEGFA, VEGFC and EGF resulted in expression of lymphatic endothelial cell (LEC) markers. Among these cells, podoplanin⁺ cells were isolated by magnetic-labeled cell separation system (MACS) and characterized by FACS and immunocytochemistry. These podoplanin⁺ cells highly express markers for LECs, hematopoietic lineages, and stem/progenitor cells, and upon further cultivation, generate LECs. We further confirmed that podoplanin⁺ cells exist in small numbers in BM and peripheral blood (PB) of normal mice, but are significantly (15 fold) augmented upon lymphangiogenic stimuli such as tumor implantation. Next, to evaluate the potential of podoplanin⁺ cells for the formation of new lymphatic vessels in vivo, we injected culture-isolated or freshly isolated BM-derived podoplanin+ cells into wound and tumor models. Immunohistochemistry demonstrated that the injected cells were incorporated into the lymphatic vasculature, displayed LEC phenotypes, and increased lymphatic vascular density in tissues, suggesting lymphvasculogenesis. Podoplanin⁺ cells also expressed high levels of lymphangiogenic cytokines and increased proliferation of LECs during co-culture, suggesting a lymphangiogenic or paracrine role.

Our results provide compelling evidence that BM-derived podoplanin⁺ cells, a previously unrecognized cell type, function as LEPCs and participate in postnatal lymphatic neovascularization through both lymphvasculogenesis and lymphangiogenesis.