| 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 |
4.51
|
| Times Cited |
30
|
|
WOS Category
|
MEDICINE, RESEARCH & EXPERIMENTAL
BIOTECHNOLOGY & APPLIED MICROBIOLOGY
GENETICS & HEREDITY
|
| Resource |
| DNA material |
AxCANCre (RDB01748) |
