Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Abstract

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34(Neg)CD31(Pos) LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34(Neg)CD31(Pos) lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel™-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplanin(Pos) vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.

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

“…However, we have found that the LM LECs proliferate faster and are more resistant to apoptosis than foreskin LECs 15 suggesting that there is a primary defect in the LM LECs. When LM LECs are implanted in a mouse xenograft model, they form structures reminiscent of lymphatic malformations 15 . This supports a hypothesis that lymphatic malformations may be caused by one or more somatic mutations arising in LM LECs during fetal development.…”

“…Kaposiform lymphangiomatosis is an example of an LEC tumor 14 . Lymphatic malformations are thought to arise during embryonic development and grow in proportion to the child 15,16 . They rarely regress but can remain asymptomatic until trauma or infection precipitates rapid growth leading to clinical complications.…”

“…The orderly structure of lymphatic network and conduction of lymph from the tissue to venous circulation described above is perturbed in lymphatic malformations which consist of localized collections of abnormal cystic structures filled with lymphatic fluid. While there is no clinical or experimental evidence that these cystic vessels are connected to the lymphatic circulation or that they contain functional lymphatic valves, their lymphatic identity is confirmed by expression of range of lymphatic cell markers such as PODOPLANIN, CD31, Lymphatic Vessel Endothelial Receptor 1 (LYVE-1), Prospero homeobox protein 1 (PROX-1) and VEGFR-3 15,17,18 . These cystic structures can be either small (microcystic) or large (macrocystic), but most lymphatic malformations contain both microcystic and macrocystic components (Figure 1) …”

“…The isolation of viable LECs would facilitate comparisons between abnormal and normal LECs in assays such as migration, proliferation, tube forming ability and survival in response to reduced nutrient availability or pro-apoptotic agents 15 . Isolated LECs would further enable us to perform cell-specific gene expression and proteomic studies, to delineate new LEC subpopulations and discover novel pharmacological agents suitable for clinical management of lymphatic malformations.…”

“…We have previously published a LEC isolation method based on magnetic bead separation of LECs from neonatal foreskin and lymphatic malformations 15 . We reported a strategy of separating normal and diseased LECs from vascular endothelial cells based on the absence of CD34 expression, followed by subjecting CD34…”