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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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Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs.(A) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. (B) and (C) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). (D) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. (E) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining (panels a and b), or stained for ALP activity, followed by Oil-Red O staining (panels c and d). (F) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 (a) while Oil-red O staining was done at day 14 (b). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.
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pone-0032428-g004: Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs.(A) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. (B) and (C) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). (D) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. (E) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining (panels a and b), or stained for ALP activity, followed by Oil-Red O staining (panels c and d). (F) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 (a) while Oil-red O staining was done at day 14 (b). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.

Mentions: Given the fact that MSC markers are not specific and unique, MSCs are by definition required to give rise to at least osteogenic, chondrogenic and adipogenic lineages [1]–[5]. We tested if the iMEFs were able to differentiate into these lineages. We have demonstrated that BMP9 is one of the most potent factors that can induce osteogenic and adipogenic, to a lesser extent, chondrogenic differentiation [29]–[33]. When the iMEFs were transduced with BMP9 or GFP adenoviral vector, the three key lineage-specific regulators, Runx2 (osteogenic), Sox9 (chondrogenic) and PPARγ2 (adipogenic), were significantly up-regulated at as early as day 3 (Fig. 4A). We further analyzed the early osteogenic marker alkaline phosphatase (ALP) in these cells. As shown in Fig. 4B, BMP9 induced much higher ALP activity at day 5 in the iMEFs than that in primary MEFs. The ALP activity induced by BMP9 in iMEFs and MEFs was also confirmed quantitatively (Fig. 4C). The BMP9-transduced iMEFs were further shown to effectively undergo late stage of osteogenic differentiation as evidenced by matrix mineralization assessed with Alizarin Red S staining (Fig. 4D).


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)

Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs.(A) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. (B) and (C) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). (D) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. (E) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining (panels a and b), or stained for ALP activity, followed by Oil-Red O staining (panels c and d). (F) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 (a) while Oil-red O staining was done at day 14 (b). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285668&req=5

pone-0032428-g004: Induction of osteogenic, chondrogenic, and adipogenic lineage markers in iMEFs.(A) Expression of lineage-specific regulators in iMEFs stimulated by BMP9. Subconfluent iMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. (B) and (C) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and iMEFs. Subconfluent primary MEFs and iMEFs were infected with AdBMP9 or AdGFP. ALP activity was histochemically stained on day 5 (B) or quantitatively determined at days 3, 5 and 7 (C). The ALP activity was normalized with total cellular protein (TCP) (C). (D) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and iMEFs were infected with AdBMP9 or AdGFP for 14 days. Cells were fixed and stained with Alizarin Red S. (E) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent iMEFs were infected with AdBMP9, AdPPARγ2, or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining (panels a and b), or stained for ALP activity, followed by Oil-Red O staining (panels c and d). (F) Osteogenic and adipogenic differentiation of immortalized human bone marrow stromal stem cells. Subconfluent cells were infected with AdBMP9 or AdGFP. ALP staining was carried out at day 7 (a) while Oil-red O staining was done at day 14 (b). Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.
Mentions: Given the fact that MSC markers are not specific and unique, MSCs are by definition required to give rise to at least osteogenic, chondrogenic and adipogenic lineages [1]–[5]. We tested if the iMEFs were able to differentiate into these lineages. We have demonstrated that BMP9 is one of the most potent factors that can induce osteogenic and adipogenic, to a lesser extent, chondrogenic differentiation [29]–[33]. When the iMEFs were transduced with BMP9 or GFP adenoviral vector, the three key lineage-specific regulators, Runx2 (osteogenic), Sox9 (chondrogenic) and PPARγ2 (adipogenic), were significantly up-regulated at as early as day 3 (Fig. 4A). We further analyzed the early osteogenic marker alkaline phosphatase (ALP) in these cells. As shown in Fig. 4B, BMP9 induced much higher ALP activity at day 5 in the iMEFs than that in primary MEFs. The ALP activity induced by BMP9 in iMEFs and MEFs was also confirmed quantitatively (Fig. 4C). The BMP9-transduced iMEFs were further shown to effectively undergo late stage of osteogenic differentiation as evidenced by matrix mineralization assessed with Alizarin Red S staining (Fig. 4D).

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