In vitro model for developmental progression from vasculogenesis to angiogenesis with a murine endothelial precursor cell line, MFLM-4

In the embryo, vascular networks are developed through both vasculogenesis, the assembly of vessels from endothelial progenitor cells or hemangioblasts, and angiogenesis, the sprouting of vessels from preexisting capillaries. Cell culture models using endothelial cells (EC) and various extracellular matrix components have been useful in understanding the cellular and molecular factors involved in angiogenesis. However, there are few models of vasculogenesis. Using a murine endothelial precursor cell line, MFLM-4, derived from e14.5 lung mesenchyme, we have developed a culture system that not only recapitulates many of the characteristics of vasculogenesis but also progresses into angiogenesis. By 8 h, MFLM-4 cultured on the basement membrane preparation Matrigel invade the matrix, coalesce, and assemble into large clusters of cells resembling blood islands. During vascular development, blood islands are the focal areas for coalescence of endothelial precursors. For MFLM-4, this phase of in vitro vasculogenic clustering does not require proliferation. If proliferation is not blocked, MFLM-4 progresses into an angiogenic phase with the clusters forming multicell angiogenic sprouts. Through 3 days of culture, lumens form within the clusters, adjacent clusters are connected with tube-like structures, and eventually an extensive network or plexus of clusters connected by capillary-like tubes is formed. MFLM-4 cultured on Matrigel provides an in vitro system for analysis of the multistage, concurrent processes of vasculogenesis and angiogenesis.