Abstract 5167: Notch1 is required for endothelial cell lineage differentiation from adult bone marrow stem cells in vitro

Abstract

Abstract Background and Objectives. Adult bone-marrow stem cells (BMSCs) are able to differentiate into endothelial progenitor cells (EPCs) and subsequently endothelial cells (ECs) under appropriate conditions. BMSCs-derived EPCs/ECs contribute to post-natal neovascularization and are important tools for regenerative medicine. However, the molecular mechanism determining specific differentiation of endothelial lineage remains unknown. The Notch signaling pathway plays an integral role in the differentiation of a variety of cell lineages. We hypothesize that Notch signaling is involved in controlling endothelial lineage differentiation from BMSCs and examined the role of Notch1 signaling in this process. Methods and Results. An in vitro method for differentiation of endothelial cell lineage from murine BMSCs was developed. Mononuclear cells were isolated from mouse whole BM cells by light density separation using Ficoll-Paque® Plus, and cultured in the differentiation medium to differentiate endothelial cells. The endothelial phenotype of differentiated cells was characterized by immunostaining and flow cytometry analyses. Those fractions of cells with VEGFR2+/CD31+ or Tie2+/CD31+ were isolated and tested. VEGFR2+/CD31+ cells could form networks on the MatrigelTM, indicating that these cells are ECs. mRNA expression of Notch pathway components in VEGFR2+/CD31+ cells was examined by real-time RT-PCR. Multiple components of the Notch signaling pathway were expressed in the differentiated ECs. The effect of loss-of-function in Notch1 on the differentiation of endothelial cell lineage was examined using BMSCs from Notch1Flox/Flox mice. Cells expressing VEGFR2 were significantly decreased when Notch1 is deleted. Conclusions. A novel and reliable method has been developed to differentiate murine adult bone marrow stem cells into endothelial cell lineage in vitro. The role of Notch1 in determining endothelial cell differentiation was tested on this platform. Our results implicated that Notch1 is required for the differentiation of endothelial cell lineage from BMSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5167.