Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT

Toshihisa Matsumura, Michihiko Takesue, Karen A. Westerman, Teru Okitsu, Masakiyo Sakaguchi, Takuya Fukazawa, Toshinori Totsugawa, Hirofumi Noguchi, Shinichiro Yamamoto, Donna B. Stolz, Noriaki Tanaka, Philippe Leboulch, Naoya Kobayashi

Research output: Contribution to journalArticle

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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)-α and interleukin (IL)-6 and exhibited increased expression of intracellular 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.

Original languageEnglish
Pages (from-to)1357-1365
Number of pages9
JournalTransplantation
Volume77
Issue number9 SUPPL.
DOIs
Publication statusPublished - May 15 2004

Fingerprint

Simian virus 40
Viral Tumor Antigens
Endothelial Cells
Cell Line
Liver
Green Fluorescent Proteins
Somatostatin Receptors
Adhesives
Genetic Recombination
Vascular Endothelial Growth Factor Receptor-1
human TERT protein
Factor VIII
Genes
Blood Vessels
Lipopolysaccharides
Interleukin-6
Complementary DNA
Clone Cells
Tumor Necrosis Factor-alpha
Research

ASJC Scopus subject areas

  • Transplantation
  • Immunology

Cite this

Matsumura, T., Takesue, M., Westerman, K. A., Okitsu, T., Sakaguchi, M., Fukazawa, T., ... Kobayashi, N. (2004). Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT. Transplantation, 77(9 SUPPL.), 1357-1365. https://doi.org/10.1097/01.TP.0000124286.82961.7E

Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT. / Matsumura, Toshihisa; Takesue, Michihiko; Westerman, Karen A.; Okitsu, Teru; Sakaguchi, Masakiyo; Fukazawa, Takuya; Totsugawa, Toshinori; Noguchi, Hirofumi; Yamamoto, Shinichiro; Stolz, Donna B.; Tanaka, Noriaki; Leboulch, Philippe; Kobayashi, Naoya.

In: Transplantation, Vol. 77, No. 9 SUPPL., 15.05.2004, p. 1357-1365.

Research output: Contribution to journalArticle

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, N 2004, 'Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT', Transplantation, vol. 77, no. 9 SUPPL., pp. 1357-1365. https://doi.org/10.1097/01.TP.0000124286.82961.7E
Matsumura, Toshihisa ; Takesue, Michihiko ; Westerman, Karen A. ; Okitsu, Teru ; Sakaguchi, Masakiyo ; Fukazawa, Takuya ; Totsugawa, Toshinori ; Noguchi, Hirofumi ; Yamamoto, Shinichiro ; Stolz, Donna B. ; Tanaka, Noriaki ; Leboulch, Philippe ; Kobayashi, Naoya. / Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT. In: Transplantation. 2004 ; Vol. 77, No. 9 SUPPL. pp. 1357-1365.
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T1 - Establishment of an immortalized human-liver endothelial cell line with SV40T and hTERT

AU - Matsumura, Toshihisa

AU - Takesue, Michihiko

AU - Westerman, Karen A.

AU - Okitsu, Teru

AU - Sakaguchi, Masakiyo

AU - Fukazawa, Takuya

AU - Totsugawa, Toshinori

AU - Noguchi, Hirofumi

AU - Yamamoto, Shinichiro

AU - Stolz, Donna B.

AU - Tanaka, Noriaki

AU - Leboulch, Philippe

AU - Kobayashi, Naoya

PY - 2004/5/15

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N2 - 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)-α and interleukin (IL)-6 and exhibited increased expression of intracellular 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.

AB - 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)-α and interleukin (IL)-6 and exhibited increased expression of intracellular 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.

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