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Generation of rat-induced pluripotent stem cells from a new model of metabolic syndrome.

Takenaka-Ninagawa N, Kawabata Y, Watanabe S, Nagata K, Torihashi S - PLoS ONE (2014)

Bottom Line: The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas).Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone.These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Rehabilitation Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

ABSTRACT
We recently characterized DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive rats and Zucker rats, as a new animal model of metabolic syndrome (MetS). Although the phenotype of DS/obese rats is similar to that of humans with MetS, the pathophysiological and metabolic characteristics in each cell type remain to be clarified. Hence, the establishment of induced pluripotent stem cells (iPSCs) derived from MetS rats is essential for investigations of MetS in vitro. Reports of rat iPSCs (riPSCs), however, are few because of the difficulty of comparing to other rodents such as mouse. Recently, the advantage of using mesenchymal stromal cells (MSCs) as a cell source for generating iPSCs was described. We aimed to establish riPSCs from MSCs in adipose tissues of both DS/obese rats and their lean littermates, DahlS.Z-Lepr+/Lepr+ (DS/lean) rats using lentivirus vectors with only three factors Oct4, Klf4, and Sox2 without c-Myc. The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas). Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone. Real-time PCR analysis also revealed that both riPSCs and the adipose tissue from DS/obese and DS/lean rats possess similar expression patterns of adipocyte differentiation-related genes. We succeeded in generating riPSCs effectively from MSCs of both DS/obese and DS/lean rats. These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

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Related in: MedlinePlus

Time schedule of iPSC generation.The experiment was composed of three main processes. The first process was isolation of MSCs from the adipose tissue of either DS/obese or DS/lean rats. The second process was infection of lentivirus. The final process was the selection of iPSC colonies. The collected MSCs were cultured for about 7days. When the MSCs increased in number, we infected them with a lentiviral vector carrying three mouse reprogramming factors, and termed this time point as day 0. The MSCs were then cultured in medium for 2 days, and the cells were replated on SNL feeder cell layers. Finally, EGFP-positive riPSC colonies were picked up at day 10 and some of clones were selected for further analysis.
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pone-0104462-g001: Time schedule of iPSC generation.The experiment was composed of three main processes. The first process was isolation of MSCs from the adipose tissue of either DS/obese or DS/lean rats. The second process was infection of lentivirus. The final process was the selection of iPSC colonies. The collected MSCs were cultured for about 7days. When the MSCs increased in number, we infected them with a lentiviral vector carrying three mouse reprogramming factors, and termed this time point as day 0. The MSCs were then cultured in medium for 2 days, and the cells were replated on SNL feeder cell layers. Finally, EGFP-positive riPSC colonies were picked up at day 10 and some of clones were selected for further analysis.

Mentions: To generate riPSCs, we initially infected MSCs isolated from the adipose tissues of DS/obese rats and DS/lean rats, respectively, with a lentiviral vector carrying three mouse reprogramming factors (Oct3/4, Sox2, and Klf4). They were controlled by a tetracycline-responsive regulatory element and a Ubc promoter-driven reverse tetracycline transactivator containing EGFP. We added Dox to the culture medium from the day of infection. Both infected and non-infected MSCs were seeded onto mitomycin C-treated SNL feeder cell layers (Fig. 1).


Generation of rat-induced pluripotent stem cells from a new model of metabolic syndrome.

Takenaka-Ninagawa N, Kawabata Y, Watanabe S, Nagata K, Torihashi S - PLoS ONE (2014)

Time schedule of iPSC generation.The experiment was composed of three main processes. The first process was isolation of MSCs from the adipose tissue of either DS/obese or DS/lean rats. The second process was infection of lentivirus. The final process was the selection of iPSC colonies. The collected MSCs were cultured for about 7days. When the MSCs increased in number, we infected them with a lentiviral vector carrying three mouse reprogramming factors, and termed this time point as day 0. The MSCs were then cultured in medium for 2 days, and the cells were replated on SNL feeder cell layers. Finally, EGFP-positive riPSC colonies were picked up at day 10 and some of clones were selected for further analysis.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128712&req=5

pone-0104462-g001: Time schedule of iPSC generation.The experiment was composed of three main processes. The first process was isolation of MSCs from the adipose tissue of either DS/obese or DS/lean rats. The second process was infection of lentivirus. The final process was the selection of iPSC colonies. The collected MSCs were cultured for about 7days. When the MSCs increased in number, we infected them with a lentiviral vector carrying three mouse reprogramming factors, and termed this time point as day 0. The MSCs were then cultured in medium for 2 days, and the cells were replated on SNL feeder cell layers. Finally, EGFP-positive riPSC colonies were picked up at day 10 and some of clones were selected for further analysis.
Mentions: To generate riPSCs, we initially infected MSCs isolated from the adipose tissues of DS/obese rats and DS/lean rats, respectively, with a lentiviral vector carrying three mouse reprogramming factors (Oct3/4, Sox2, and Klf4). They were controlled by a tetracycline-responsive regulatory element and a Ubc promoter-driven reverse tetracycline transactivator containing EGFP. We added Dox to the culture medium from the day of infection. Both infected and non-infected MSCs were seeded onto mitomycin C-treated SNL feeder cell layers (Fig. 1).

Bottom Line: The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas).Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone.These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Rehabilitation Sciences, Nagoya University Graduate School of Medicine, Nagoya, Japan.

ABSTRACT
We recently characterized DahlS.Z-Leprfa/Leprfa (DS/obese) rats, derived from a cross between Dahl salt-sensitive rats and Zucker rats, as a new animal model of metabolic syndrome (MetS). Although the phenotype of DS/obese rats is similar to that of humans with MetS, the pathophysiological and metabolic characteristics in each cell type remain to be clarified. Hence, the establishment of induced pluripotent stem cells (iPSCs) derived from MetS rats is essential for investigations of MetS in vitro. Reports of rat iPSCs (riPSCs), however, are few because of the difficulty of comparing to other rodents such as mouse. Recently, the advantage of using mesenchymal stromal cells (MSCs) as a cell source for generating iPSCs was described. We aimed to establish riPSCs from MSCs in adipose tissues of both DS/obese rats and their lean littermates, DahlS.Z-Lepr+/Lepr+ (DS/lean) rats using lentivirus vectors with only three factors Oct4, Klf4, and Sox2 without c-Myc. The morphology, gene expression profiles, and protein expression of established colonies showed embryonic stem cell (ESCs)-like properties, and the differentiation potential into cells from all three germ layers both in vitro and in vivo (teratomas). Both riPSCs became adipocytes after induction of adipogenesis by insulin, T3, and dexamethasone. Real-time PCR analysis also revealed that both riPSCs and the adipose tissue from DS/obese and DS/lean rats possess similar expression patterns of adipocyte differentiation-related genes. We succeeded in generating riPSCs effectively from MSCs of both DS/obese and DS/lean rats. These riPSCs may well serve as highly effective tools for the investigation of MetS pathophysiology in vitro.

Show MeSH
Related in: MedlinePlus