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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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The iMEFs express most mesenchymal stem cell markers.The iMEF cells were seeded at subconfluency for 24 h and stained with MSC marker antibodies as indicated. The consensus MSC markers include CD44, CD90/Thy-1, CD73, CD105/Endoglin and CD166/ALCAM [11]. Cell nuclei were stained with DAPI. Respective IgG Isotypes were used as immunostaining control.
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pone-0032428-g003: The iMEFs express most mesenchymal stem cell markers.The iMEF cells were seeded at subconfluency for 24 h and stained with MSC marker antibodies as indicated. The consensus MSC markers include CD44, CD90/Thy-1, CD73, CD105/Endoglin and CD166/ALCAM [11]. Cell nuclei were stained with DAPI. Respective IgG Isotypes were used as immunostaining control.

Mentions: To assert if iMEFs possess MSC-like multipotency, we characterized the expression of MSC markers using immunofluorescence staining. It has been reported that the consensus human MSC markers include CD90/Thy-1, CD73, CD105/Endoglin, CD166/ALCAM and CD44 [11]. Although mouse MSCs do not necessarily express all the same molecules as those on human cells, we found that all of these markers were readily detectable in iMEFs by immunofluorescence staining (Fig. 3). Flow cytometry indicated that more than 90% of MSC population expresses these markers (data not shown). Furthermore, we found that the expression of CD45 and CD34 was not detectable (data not shown). Thus, these results demonstrate that the iMEFs express most if not all of the conventional MSC markers, suggesting that these cells may possess MSC-like phenotypes.


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)

The iMEFs express most mesenchymal stem cell markers.The iMEF cells were seeded at subconfluency for 24 h and stained with MSC marker antibodies as indicated. The consensus MSC markers include CD44, CD90/Thy-1, CD73, CD105/Endoglin and CD166/ALCAM [11]. Cell nuclei were stained with DAPI. Respective IgG Isotypes were used as immunostaining control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0032428-g003: The iMEFs express most mesenchymal stem cell markers.The iMEF cells were seeded at subconfluency for 24 h and stained with MSC marker antibodies as indicated. The consensus MSC markers include CD44, CD90/Thy-1, CD73, CD105/Endoglin and CD166/ALCAM [11]. Cell nuclei were stained with DAPI. Respective IgG Isotypes were used as immunostaining control.
Mentions: To assert if iMEFs possess MSC-like multipotency, we characterized the expression of MSC markers using immunofluorescence staining. It has been reported that the consensus human MSC markers include CD90/Thy-1, CD73, CD105/Endoglin, CD166/ALCAM and CD44 [11]. Although mouse MSCs do not necessarily express all the same molecules as those on human cells, we found that all of these markers were readily detectable in iMEFs by immunofluorescence staining (Fig. 3). Flow cytometry indicated that more than 90% of MSC population expresses these markers (data not shown). Furthermore, we found that the expression of CD45 and CD34 was not detectable (data not shown). Thus, these results demonstrate that the iMEFs express most if not all of the conventional MSC markers, suggesting that these cells may possess MSC-like phenotypes.

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