Isolation, culture, and characterization of endothelial cells from mouse glomeruli

Abstract

Cloned glomerular endothelial cells (GENC) have many potential uses and applications in immunologic and physiologic studies. Propagation of GENC has been difficult and available homogeneous GENC, particularly from mice, are limited. Herein we report isolation, cloning, propagation, and characterization of GENC from mice. tsA58 immorto mice were used to isolate glomerular cells. Glomeruli were isolated by differential sieving, and decapsulated explants were cultured in permissive and optimal conditions for endothelial cells. The primary cells from glomerular outgrowths were expanded, taking advantage of the temperature-sensitive tsA58 gene, and then the cells were allowed to undergo spontaneous transformation. The cells were then sorted using anti-CD31 antibodies and their capacity to uptake acetylated-low-density lipoprotein (LDL). Individual subclones isolated by patch cloning were characterized using multiple markers. One of the homogeneous clones was morphologically endothelial-like, positive for CD31, CD106, CD62E, CD54, and acetylated-LDL uptake, formed tubes, and was negative for epithelial and mesangial cell markers. The functional properties of this GENC clone appeared to be intact, and signaling pathway was not altered. Two of the clones displayed the characteristics of either visceral epithelial or mesangial cells. The identified clones should have utility in multiple areas of investigation.