title: Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential.
abstract: Mesenchymal stem cells (MSCs) are multipotent cells which reside in many tissues and can give rise to multiple lineages including bone, cartilage and adipose. Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications. Here, we investigate if mouse mesenchymal progenitors can be conditionally immortalized with SV40 large T antigen and maintain long-term cell proliferation without compromising their multipotency. Using the system which expresses SV40 large T antigen flanked with Cre/loxP sites, we demonstrate that mouse embryonic fibroblasts (MEFs) can be efficiently immortalized by SV40 large T antigen. The conditionally immortalized MEFs (iMEFs) exhibit an enhanced proliferative activity and maintain long-term cell proliferation, which can be reversed by Cre recombinase. The iMEFs express most MSC markers and retain multipotency as they can differentiate into osteogenic, chondrogenic and adipogenic lineages under appropriate differentiation conditions in vitro and in vivo. The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion. Furthermore, the iMEFs are apparently not tumorigenic when they are subcutaneously injected into athymic nude mice. Thus, the conditionally immortalized iMEFs not only maintain long-term cell proliferation but also retain the ability to differentiate into multiple lineages. Our results suggest that the reversible immortalization strategy using SV40 large T antigen may be an efficient and safe approach to establishing long-term cell culture of primary mesenchymal progenitors for basic and translational research, as well as for potential clinical applications.
authors.affiliation: School of Bioengineering, Chongqing University, Chongqing, China.
authors.forename: Enyi
authors.initials: E
authors.lastname: Huang
authors.forename: Yang
authors.initials: Y
authors.lastname: Bi
authors.forename: Wei
authors.initials: W
authors.lastname: Jiang
authors.forename: Xiaoji
authors.initials: X
authors.lastname: Luo
authors.forename: Ke
authors.initials: K
authors.lastname: Yang
authors.forename: Jian-Li
authors.initials: JL
authors.lastname: Gao
authors.forename: Yanhong
authors.initials: Y
authors.lastname: Gao
authors.forename: Qing
authors.initials: Q
authors.lastname: Luo
authors.forename: Qiong
authors.initials: Q
authors.lastname: Shi
authors.forename: Stephanie H
authors.initials: SH
authors.lastname: Kim
authors.forename: Xing
authors.initials: X
authors.lastname: Liu
authors.forename: Mi
authors.initials: M
authors.lastname: Li
authors.forename: Ning
authors.initials: N
authors.lastname: Hu
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Jing
authors.initials: J
authors.lastname: Cui
authors.forename: Wenwen
authors.initials: W
authors.lastname: Zhang
authors.forename: Ruidong
authors.initials: R
authors.lastname: Li
authors.forename: Xiang
authors.initials: X
authors.lastname: Chen
authors.forename: Jikun
authors.initials: J
authors.lastname: Shen
authors.forename: Yuhan
authors.initials: Y
authors.lastname: Kong
authors.forename: Jiye
authors.initials: J
authors.lastname: Zhang
authors.forename: Jinhua
authors.initials: J
authors.lastname: Wang
authors.forename: Jinyong
authors.initials: J
authors.lastname: Luo
authors.forename: Bai-Cheng
authors.initials: BC
authors.lastname: He
authors.forename: Huicong
authors.initials: H
authors.lastname: Wang
authors.forename: Russell R
authors.initials: RR
authors.lastname: Reid
authors.forename: Hue H
authors.initials: HH
authors.lastname: Luu
authors.forename: Rex C
authors.initials: RC
authors.lastname: Haydon
authors.forename: Li
authors.initials: L
authors.lastname: Yang
authors.forename: Tong-Chuan
authors.initials: TC
authors.lastname: He
mesh_terms: D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000378:metabolism*; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002460:Cell Line; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D002478:Cells, Cultured; D005347:Fibroblasts / Q000166:cytology*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D055427:Growth Differentiation Factor 2 / Q000378:metabolism; D006801:Humans; D019426:Integrases / Q000378:metabolism; D059630:Mesenchymal Stem Cells / Q000166:cytology; D051379:Mice; D008819:Mice, Nude; D047495:PPAR gamma / Q000378:metabolism; D013234:Stem Cells
chemical_list: D000952:Antigens, Polyomavirus Transforming; D055427:Growth Differentiation Factor 2; D047495:PPAR gamma; D049452:Green Fluorescent Proteins; C045073:Cre recombinase; D019426:Integrases