Abstract:
BACKGROUND AND AIMS:Liver endothelial cells (LECs) perform an essential role in important pathophysiologic functions in the liver. Establishment of a human LEC line facilitates advances in LEC research. Here, we present immortalization of human LECs using retroviral gene transfer of simian virus 40 large T antigen (SV40T) and human telomerase reverse transcriptase (hTERT). We also demonstrate excision of SV40T and hTERT with TAT-mediated Cre/loxP recombination and subsequent cell sorting. METHODS:First, human LECs were transduced with a retroviral vector somatostatin receptor (SSR)#69 expressing SV40T and hygromycin-resistance genes flanked by a pair of loxA recombination targets. Then, cells were retrovirally superinfected with SSR#197 encoding hTERT and green fluorescent protein (GFP) cDNAs that were intervened by two loxBs. One SV40T-and hTERT-immortalized LEC clone, TMNK-1, was established and analyzed for its biologic characteristics. RESULTS:The cells were hygromycin-resistant and uniformly positive for GFP expression. TMNK-1 expressed EC markers, including factor VIII, vascular endothelial growth factor receptors (flt-1, KDR/Flk-1), and CD34, showed uptake of Di-I-acetylated-low-density lipoprotein and angiogenic potential in Matrigel assays. After lipopolysaccharide treatment, TMNK-1 produced tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited increased expression of intra-cellular adhesive molecule-1, vascular cellular adhesive molecule-1, and VE-cadherin. After treatment with TAT-Cre recombinase fusion protein, approximately 60% of TMNK-1 was negative for GFP expression, and subsequent cell sorting of this population for GFP allowed for collection of the reverted form of TMNK-1. CONCLUSIONS:This study demonstrates the utility and efficiency of the reversible immortalization procedure to expand primary human LECs for basic studies.
journal_name
Transplantationjournal_title
Transplantationauthors
Matsumura T,Takesue M,Westerman KA,Okitsu T,Sakaguchi M,Fukazawa T,Totsugawa T,Noguchi H,Yamamoto S,Stolz DB,Tanaka N,Leboulch P,Kobayashi Ndoi
10.1097/01.tp.0000124286.82961.7esubject
Has Abstractpub_date
2004-05-15 00:00:00pages
1357-65issue
9eissn
0041-1337issn
1534-6080pii
00007890-200405150-00008journal_volume
77pub_type
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