RRC ID 4767
Author Noguchi H, Kobayashi N, Westerman KA, Sakaguchi M, Okitsu T, Totsugawa T, Watanabe T, Matsumura T, Fujiwara T, Ueda T, Miyazaki M, Tanaka N, Leboulch P.
Title Controlled expansion of human endothelial cell populations by Cre-loxP-based reversible immortalization.
Journal Hum. Gene Ther.
Abstract Endothelial cells (ECs) play multiple physiological functions and are central to many pathological processes. Various biological studies as well as cell and gene therapy applications would benefit substantially from a procedure that would result in the expansion in culture of large numbers of highly differentiated human ECs. Here, we report the amplification in vitro of human EC populations, which occurred during the first phase of reversible immortalization resulting from the retroviral transfer of an oncogene that was subsequently excised by Cre-loxP-mediated site-specific recombination. Human umbilical vein endothelial cells (HUVECs) and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector that expresses the simian virus 40 large T (SV40T) gene flanked by positive and negative selectable markers and a pair of loxP recombination targets. Transduced HUVECs and HLSECs yielded clones with greatly extended life spans, referred to as HNNT-1 and HNNT-2 cells, respectively. HNNT-1 and HNNT-2 cells showed morphological characteristics of ECs and were maintained in culture up to population doubling level (PDL) 80 for HNNT-1 and PDL 65 for HNNT-2 cells. HNNT-1 and HNNT-2 cells were not tumorigenic when transplanted into severe combined immunodeficiency mice and were sensitive to ganciclovir as well as G418. Both cell clones expressed EC markers, which include factor VIII, VEGF receptors (Flt-1 and KDR/Flk-1), and CD34, and endocytosed acetylated low-density lipoproteins. Formation of capillary-like structures in a Matrigel assay was observed with HNNT-1 and HNNT-2 cells until at least PDL 50. Complete elimination of the transferred SV40T gene was achieved in virtually 100% of HNNT-1 and HNNT-2 cells after infection with a recombinant adenovirus expressing the Cre recombinase fused to a nuclear localization signal and subsequent selection with G418. Reverted cells maintained their differentiated EC phenotype. This study extends the utility of the reversible immortalization procedure and provides a means to expand primary human ECs of various sources for basic studies and possible cell and gene therapies.
Volume 13(2)
Pages 321-34
Published 2002-1-20
DOI 10.1089/10430340252769833
PMID 11812287
MeSH Animals Antigens, Polyomavirus Transforming / genetics Cell Line Cell Transformation, Neoplastic* Cholesterol, LDL / metabolism Collagen Drug Combinations Endothelium, Vascular / cytology* Endothelium, Vascular / physiology Feasibility Studies Ganciclovir / pharmacology Genetic Vectors Hepatocytes / cytology* Humans Infant, Newborn Integrases / genetics* Laminin Mice Mice, SCID Neovascularization, Physiologic Nuclear Localization Signals / physiology Proteoglycans Simian virus 40 Telomerase Transduction, Genetic Umbilical Veins Viral Proteins / genetics* beta-Galactosidase / metabolism
IF 3.855
Times Cited 29
WOS Category MEDICINE, RESEARCH & EXPERIMENTAL BIOTECHNOLOGY & APPLIED MICROBIOLOGY GENETICS & HEREDITY
Resource
DNA material AxCANCre (RDB01748)