Abstract 465: Successful Culture Isolation of Bone Marrow-derived Lymphatic Endothelial Progenitor Cells
Background. Recent studies demonstrated that bone marrow (BM)-derived cells can be incorporated into lymphatic vasculature in inflammatory foci. However, the existence and identify of lymphatic endothelial progenitor cells (LEPCs) are unclear. In this study, we investigated whether putative LEPCs can be culture-isolated from BM mononuclear cells (MNCs), which have a phenotype of hematopoietic BM cells and have the potential to differentiate into lymphatic endothelial cells (LECs) in vitro and in vivo.
Methods and Results. First, to determine whether BM-MNCs can differentiate into LECs, we tried various culture conditions which include cytokines known to induce lymphangiogenesis. BM mononuclear cells (BM-MNCs) from FVB mice were cultured in 6 different conditions using EBM media and various combinations of VEGF-A (A), VEGF-C (C), and EGF (E). We checked the expression of LEC-specific markers such as Prox1, LYVE-1, VEGFR-3 and podoplanin at day 1, 4, 7, 10 and 14 using quantitative RT-PCR, immunocytochemistry and FACS. Uncultured BM-MNCs express LEC markers minimally (By FACS, LYVE-1, 3.3 %; VEGFR-3, < 0.5%; podoplanin, 2.1%, no expression of Prox-1 by qRT-PCR). In all the culture conditions, LEC markers were expressed within a week. Especially, BM-MNCs cultured in EBM plus A, C, and E revealed the highest expression of all LEC markers at day 4 (> 30 to 40% by FACS). The expression of podoplanin, a more specific LEC maker among others, was continuously increased over 2 wks. We further isolated podoplanin+ cells from 4-day cultured cells by a magnetic bead separation technique, and confirmed that >98% of these cells express CD45, suggesting that podoplanin+ cells are not contaminated LECs residing in BM. We next injected these purified podoplanin+ cells intravenously in a cornea angiogenesis model and directly into the skin in a tail lymphedema model, and observed colocalization of these cells with LECs and augmentation of lymphatic vessel growth.
In Conclusion. We for the first time successfully culture-isolated putative LEPCs using a defined culture condition, which express podoplanin and CD45, and found that LEPCs could augment lymphangiogenesis. These findings suggest that LEPCs can be used for treating various lymphatic diseases