Trophoblast-derived factors drive human mesenchymal stem cell differentiation along an endothelial lineage: A model of early placental vasculogenesis

Mechanisms controlling the process and patterning of blood vessel development in the placenta remain largely unknown. The close physical proximity of early blood vessels observed in the placenta and the cytotrophoblast, as well as the reported production of vasculogenic growth factors by the latter, suggests that signalling between these two niches may be important. Here, we have developed an in vitro model to address the hypothesis that the cytotrophoblast, by the secretion of soluble factors, drives differentiation of resident sub-trophoblastic mesenchymal stem cells (MSCs) along a vascular lineage, thereby establishing feto-placental circulation. BM-MSCs (a readily available model for placental stem cells) were treated with conditioned medium containing the secretome from human BeWo trophoblast cells, or endothelial growth medium (EGM2) supplemented with exogenous growth factors (VEGF, IGF1 and EGF) for 10-12 days. Trophoblast-conditioned media, found to contain detectable concentrations of cytokines including VEGF, uPAR, TIMP-1, TIMP-2, IL6 and placental growth factor, induced the expression of the endothelial genes CD31, von Willibrand factor (vWF), FLT-1, VEGFR2 and VE-Cadherin. Upregulation of vWF protein was also detected following growth in trophoblast-conditioned media, using immunocytochemistry. Wound healing (migration assay) and Matrigel-tube formation assays confirmed that the BM-MSCs cultured in trophoblast-conditioned media exhibited functional measures of endothelial cells in addition to expressing relevant markers. Identification of key trophoblast-secreted factors and their promotion of endothelial differentiation in BM-MSCs helps advance our theories regarding the close relationship of the mesenchymal stem cell-cytotrophoblast niche in coordinating the complex angiogenic events that occur in the placenta. The in vitro model presented here provides an accessible and reproducible tool for further investigations into placental development.