Abstract 5601: Podoplanin Expressing Cells Isolated From Bone Marrow and Peripheral Blood Function as Lymphatic Endothelial Progenitor Cells

Abstract

Background: Previously, we demonstrated that podoplanin expressing (pod+) cells can be culture-isolated from bone marrow (BM) mononuclear cells (MNCs) and that these cells have a potential to differentiate into lymphatic endothelial cells (LECs). In this study, we investigated whether pod+ cells exist in BM and peripheral blood (PB), and they can function as lymphatic endothelial progenitor cells (LEPCs). Methods and Results. First we performed FACS analysis and found that pod+ cells are present in BM and PB less than 0.5% in C57/BL6 mice. Thus, next to investigate whether the number of pod+ cells would be increased in the BM or PB under conditions which promote lymphatic vessel growth, mice were injected with B16-F1 melanoma cells into the back skins. FACS analysis conducted 7 days later showed that the number of pod+ cells in BM and PB were about 15 fold increased in mice with melanoma growth (normal vs tumor, BM: 0.2±0.5% vs. 3.2±1.0%, PB: 0.4±0.3% vs. 6.7±2.0%, P < 0.01). FACS analysis further revealed that the BM pod+ cells derived from the tumor-bearing mice exhibited high levels of VEGFR-3+ and c-KIT+, but not LYVE-1+ cells, but PB pod+ cells derived from the same mice displayed VEGFR-3+ and LYVE-1+, but not c-KIT+ cells. These data suggest that more undifferentiated LEPC phenotypes exist in BM and undergo differentiation during mobilization into PB. Next, to determine the lymphvasculogenic potential of the isolated BM pod+ cells in vivo, we injected the pod+ cells (Dil-labeled or GFP mouse derived) isolated from BM of tumor-bearing mice into skin and ear wound models. Immunohistochemistry showed that the injected cells were incorporated into lymphatic vessels and expressed LEC-specific markers, suggesting LEC differentiation. We also found that these pod+ cells express CD45 in BM but lost CD45 expression after incorporation into lymphatic vessels, suggesting that pod+ cells are hematopoietic in origin but lost hematopoietic properties during differentiation into LEC. In Conclusion. This study for the first time demonstrated that pod+ cells are present in BM and PB, are augmented under lymphatic vascular growth conditions, and contributed to lymphvasculogenesis, suggesting a role of pod+ cells as LEPCs.