Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Abstract

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34 Video LinkThe video component of this article can be found at

Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly

“…The unbound cells were removed into a sterile Falcon tube, pelleted, and resuspended in EGM-2MV supplemented with vascular endothelial growth factor–C before being seeded in fibronectin-coated plastic. For the isolation of the GLA054-LM-LECs, a FACS strategy was applied, as described before (Lokmic et al, 2015), using anti-human podoplanin (Sigma-Aldrich)–Alexa Fluor 488 (1:200; Cell Signaling), Brilliant Violet 421 anti-human CD34 antibody (1:200; BioLegend), PE mouse anti-human CD31 (1:50; Becton Dickinson), and 7AAD PerCP (1:20; BioLegend). For the isolation of GLA061-LM-LECs, a CD31-positive, podoplanin-positive selection strategy was employed using antibody-coated magnetic beads (Dynabeads; Invitrogen, Life Technologies) to separate LECs from other cell types (Osborn et al, 2015).…”

The role of lymphangiogenesis and angiogenesis in tumor metastasis

“…Likewise, it has been shown that integration of circulating cells that exhibit lymphendothelial features may initiate a pathologic outgrowth of the lymphatic system. Thus, a third source of tumor LECs comes from transdifferentiation of non-endothelial cells [ 92 ]. Although bone marrow-derived cells (BMDCs), including endothelial progenitor cells, are essential for the formation of new blood vessels, it is conceivable that they may also contribute to neo-lymphangiogenesis in tumors.…”

Revascularization of decellularized lung scaffolds: principles and progress

“…Lymphatic ECs lack tight barrier function, and exclusively respond to lymphangiogenic factors, e.g., VEGF-C (4). The availability of protocols for the isolation/purification of ECs from different vascular beds (32) or the lymphatics (54,109) may enable their targeted use in bioengineering different segments of the pulmonary vascular, the bronchial circulation, or the lymphatic tree. A review of the lymphatic endothelium, however, as important as it is for lung function during development (39) and in the mature organ, is beyond the scope of this review.…”