Tài liệu Báo cáo khoa học: Development of a new method for isolation and long-term culture of

Development of a new method for isolation and long-term

culture of organ-specific blood vascular and lymphatic

endothelial cells of the mouse

Takashi Yamaguchi, Taeko Ichise, Osamu Iwata, Akiko Hori, Tomomi Adachi, Masaru Nakamura,

Nobuaki Yoshida and Hirotake Ichise

Laboratory of Gene Expression and Regulation, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Japan

As an indispensable component of the vascular system,

endothelial cells (ECs) have pivotal roles in develop￾ment and in health and disease [1]. Their properties

have been studied by a combination of in vitro analy￾ses of human primary ECs and in vivo analyses of

genetically modified mice exhibiting vascular pheno￾types. Human primary ECs are well-established

resources and are suitable for studying signal transduc￾tion and cellular physiology in vitro. However, it is still

difficult to control their gene expression strictly by

current overexpression and knockdown procedures. In

addition, they are not representative of all types of

ECs at various developmental stages and in vascular

beds [2]. On the other hand, the use of genetically

Keywords

Cre ⁄ loxP recombination; endothelial cell

culture; endothelial heterogeneity; SV40

tsA58 large T antigen; transgenic mouse

Correspondence

H. Ichise, Laboratory of Gene Expression

and Regulation, Center for Experimental

Medicine, Institute of Medical Science,

University of Tokyo, 4-6-1 Shirokanedai,

Minato-ku, Tokyo 108-8639, Japan

Fax: +81 3 5449 5455

Tel: +81 3 5449 5754

E-mail: [email protected]

(Received 27 November 2007, revised 13

February 2008, accepted 22 February 2008)

doi:10.1111/j.1742-4658.2008.06353.x

Endothelial cells are indispensable components of the vascular system, and

play pivotal roles during development and in health and disease. Their

properties have been studied extensively by in vivo analysis of genetically

modified mice. However, further analysis of the molecular and cellular phe￾notypes of endothelial cells and their heterogeneity at various developmen￾tal stages, in vascular beds and in various organs has often been hampered

by difficulties in culturing mouse endothelial cells. In order to overcome

these difficulties, we developed a new transgenic mouse line expressing the

SV40 tsA58 large T antigen (tsA58T Ag) under the control of a binary

expression system based on Cre ⁄ loxP recombination. tsA58T Ag-positive

endothelial cells in primary cultures of a variety of organs proliferate con￾tinuously at 33 C without undergoing cell senescence. The resulting cell

population consists of blood vascular and lymphatic endothelial cells,

which could be separated by immunosorting. Even when cultured for two

months, the cells maintained endothelial cell properties, as assessed by

expression of endothelium-specific markers and intracellular signaling

through the vascular endothelial growth factor receptors VEGFR–2 and

VEGFR-3, as well as their physiological characteristics. In addition, lym￾phatic vessel endothelial hyaluronan receptor-1 (Lyve-1) expression in liver

sinusoidal endothelial cells in vivo was retained in vitro, suggesting that an

organ-specific endothelial characteristic was maintained. These results show

that our transgenic cell culture system is useful for culturing murine endo￾thelial cells, and will provide an accessible method and applications for

studying endothelial cell biology.

Abbreviations

BEC, blood vascular endothelial cell; DiI, 1,1’-dioctadecyl-3,3,3’,3’-tetramethylindocarbocyanine perchlorate; EC, endothelial cell; ESC,

embryonic stem cell; HRP, horseradish peroxidase; LDL, low-density lipoprotein; LEC, lymphatic endothelial cell; Lyve-1, lymphatic vessel

endothelial hyaluronan receptor-1; MACS, magnetic-activated cell separation; MAPK, mitogen-activated protein kinase; PFA,

paraformaldehyde; Prox-1, prospero-related homeobox-1; SV40T Ag, SV40 large T antigen; tsA58T Ag, large T antigen of SV40 mutant strain

tsA58; VEGF, vascular endothelial growth factor; VEGFR, vascular endothelial growth factor receptor.

1988 FEBS Journal 275 (2008) 1988–1998 ª 2008 The Authors Journal compilation ª 2008 FEBS