Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT.
OBJECTIVE
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.
Matsumura
Toshihisa
T
Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan.
Takesue
Michihiko
M
Westerman
Karen A
KA
Okitsu
Teru
T
Sakaguchi
Masakiyo
M
Fukazawa
Takuya
T
Totsugawa
Toshinori
T
Noguchi
Hirofumi
H
Yamamoto
Shinichiro
S
Stolz
Donna B
DB
Tanaka
Noriaki
N
Leboulch
Philippe
P
Kobayashi
Naoya
N
D000818:Animals; D015703:Antigens, CD; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D015415:Biomarkers; D015820:Cadherins / Q000235:genetics; D002461:Cell Line, Transformed / Q000166:cytology* / Q000502:physiology; D002469:Cell Separation; D004268:DNA-Binding Proteins; D019040:E-Selectin / Q000235:genetics; D042783:Endothelial Cells / Q000166:cytology*; D015696:Gene Products, tat / Q000235:genetics; D006801:Humans; D019426:Integrases / Q000235:genetics; D018799:Intercellular Adhesion Molecule-1 / Q000235:genetics; D015850:Interleukin-6 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology*; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D016513:Mice, SCID; D009368:Neoplasm Transplantation; D018919:Neovascularization, Physiologic; D011956:Receptors, Cell Surface / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D019098:Telomerase / Q000235:genetics*; D051193:Toll-Like Receptors; D014161:Transduction, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism; D019010:Vascular Cell Adhesion Molecule-1 / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics; D000099152:Cadherin 5
D015703:Antigens, CD; D000952:Antigens, Polyomavirus Transforming; D015415:Biomarkers; D015820:Cadherins; D004268:DNA-Binding Proteins; D019040:E-Selectin; D015696:Gene Products, tat; D019426:Integrases; D018799:Intercellular Adhesion Molecule-1; D015850:Interleukin-6; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; D019098:Telomerase; D051193:Toll-Like Receptors; D014409:Tumor Necrosis Factor-alpha; D019010:Vascular Cell Adhesion Molecule-1; D014764:Viral Proteins; D000099152:Cadherin 5; C045073:Cre recombinase