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Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential.

Huang E, Bi Y, Jiang W, Luo X, Yang K, Gao JL, Gao Y, Luo Q, Shi Q, Kim SH, Liu X, Li M, Hu N, Liu H, Cui J, Zhang W, Li R, Chen X, Shen J, Kong Y, Zhang J, Wang J, Luo J, He BC, Wang H, Reid RR, Luu HH, Haydon RC, Yang L, He TC - PLoS ONE (2012)

Bottom Line: Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications.The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion.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.

View Article: PubMed Central - PubMed

Affiliation: School of Bioengineering, Chongqing University, Chongqing, China.

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.

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Morphology of primary and immortalized mouse embryonic fibroblasts (MEFs).(A) Schematic representation of the reversible immortalization vector SSR #69 [28]. This retroviral vector contains the hygromycin and SV40 T antigen expression cassette flanked by loxP sites. (B) Morphology of primary MEFs in cell culture. Primary MEFs were seeded at 20–30% confluence and passed consecutively for five passages (P5). (C) Morphology of the reversibly immortalized MEFs (iMEFs) in cell culture. The iMEF cells were seeded at low density and passed consecutively for 15 passages (P15).
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pone-0032428-g001: Morphology of primary and immortalized mouse embryonic fibroblasts (MEFs).(A) Schematic representation of the reversible immortalization vector SSR #69 [28]. This retroviral vector contains the hygromycin and SV40 T antigen expression cassette flanked by loxP sites. (B) Morphology of primary MEFs in cell culture. Primary MEFs were seeded at 20–30% confluence and passed consecutively for five passages (P5). (C) Morphology of the reversibly immortalized MEFs (iMEFs) in cell culture. The iMEF cells were seeded at low density and passed consecutively for 15 passages (P15).

Mentions: To establish the immortalized MEFs (iMEFs), early passage MEFs (<3 passages) were seeded in 25 cm2 flasks and infected with packaged retrovirus SSR #69, which expresses SV40 T Ag flanked with Cre/loxP sites (Fig. 1A) [28]. Stable iMEF cell pools were established by selecting the infected cells with hygromycin B (at 4 µg/ml) for one week. Aliquots of the iMEFs were kept in liquid nitrogen tanks. Human bone marrow stromal stem cells were immortalized in a similar fashion.


Conditionally immortalized mouse embryonic fibroblasts retain proliferative activity without compromising multipotent differentiation potential.

Huang E, Bi Y, Jiang W, Luo X, Yang K, Gao JL, Gao Y, Luo Q, Shi Q, Kim SH, Liu X, Li M, Hu N, Liu H, Cui J, Zhang W, Li R, Chen X, Shen J, Kong Y, Zhang J, Wang J, Luo J, He BC, Wang H, Reid RR, Luu HH, Haydon RC, Yang L, He TC - PLoS ONE (2012)

Morphology of primary and immortalized mouse embryonic fibroblasts (MEFs).(A) Schematic representation of the reversible immortalization vector SSR #69 [28]. This retroviral vector contains the hygromycin and SV40 T antigen expression cassette flanked by loxP sites. (B) Morphology of primary MEFs in cell culture. Primary MEFs were seeded at 20–30% confluence and passed consecutively for five passages (P5). (C) Morphology of the reversibly immortalized MEFs (iMEFs) in cell culture. The iMEF cells were seeded at low density and passed consecutively for 15 passages (P15).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285668&req=5

pone-0032428-g001: Morphology of primary and immortalized mouse embryonic fibroblasts (MEFs).(A) Schematic representation of the reversible immortalization vector SSR #69 [28]. This retroviral vector contains the hygromycin and SV40 T antigen expression cassette flanked by loxP sites. (B) Morphology of primary MEFs in cell culture. Primary MEFs were seeded at 20–30% confluence and passed consecutively for five passages (P5). (C) Morphology of the reversibly immortalized MEFs (iMEFs) in cell culture. The iMEF cells were seeded at low density and passed consecutively for 15 passages (P15).
Mentions: To establish the immortalized MEFs (iMEFs), early passage MEFs (<3 passages) were seeded in 25 cm2 flasks and infected with packaged retrovirus SSR #69, which expresses SV40 T Ag flanked with Cre/loxP sites (Fig. 1A) [28]. Stable iMEF cell pools were established by selecting the infected cells with hygromycin B (at 4 µg/ml) for one week. Aliquots of the iMEFs were kept in liquid nitrogen tanks. Human bone marrow stromal stem cells were immortalized in a similar fashion.

Bottom Line: Although MSCs have attracted significant attention for basic and translational research, primary MSCs have limited life span in culture which hampers MSCs' broader applications.The removal of SV40 large T reduces the differentiation potential of iMEFs possibly due to the decreased progenitor expansion.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.

View Article: PubMed Central - PubMed

Affiliation: School of Bioengineering, Chongqing University, Chongqing, China.

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.

Show MeSH
Related in: MedlinePlus