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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

mRNA expressions.The expression of mRNAs was analyzed by RT-PCR. A) Introduced four genes (EGFP, Sox2, Klf4 and Oct3/4) were expressed in both o-riPSCs and l-riPSCs, but not in both MSCs. Transgenes (mouse mRNA) were detected only in undifferentiated riPSCs cultured in medium containing Dox. Leptin receptor was not detected in the o-riPSCs, however, it was expressed in thel-riPSCs. B) After differentiation in embryoid bodies (EB), Sox17, SM-22a, and Ncam were clearly demonstrated in both o-riPSCs and l-riPSCs. Introduced genes (Sox2, Klf4 and Oct3/4) and Transgenes (mouse mRNA) were not detected in differentiated embryoid bodies (EB) of both o-riPSCs and l-riPSCs.
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pone-0104462-g003: mRNA expressions.The expression of mRNAs was analyzed by RT-PCR. A) Introduced four genes (EGFP, Sox2, Klf4 and Oct3/4) were expressed in both o-riPSCs and l-riPSCs, but not in both MSCs. Transgenes (mouse mRNA) were detected only in undifferentiated riPSCs cultured in medium containing Dox. Leptin receptor was not detected in the o-riPSCs, however, it was expressed in thel-riPSCs. B) After differentiation in embryoid bodies (EB), Sox17, SM-22a, and Ncam were clearly demonstrated in both o-riPSCs and l-riPSCs. Introduced genes (Sox2, Klf4 and Oct3/4) and Transgenes (mouse mRNA) were not detected in differentiated embryoid bodies (EB) of both o-riPSCs and l-riPSCs.

Mentions: RT-PCR showed that the riPSCs expressed many undifferentiated ESC-marker genes, including Sox2, Oct3/4, Klf4, and Eras. Although Sox2, Klf4, and Eras were not detected in non-induced MSCs, low-level expression of Oct3/4 was detected in non-induced MSCs. The primers used to amplify the sequence between T2A and Sox2 were used to detect transgene expression. Transgenes were detected only in conditions with Dox and were not detected in non-induced MSCs or differentiated riPSCs, indicating that expression via the lentiviral vector was controlled by a tetracycline-responsive element. Leptin receptor was not detected in the riPSCs from DS/obese rats (o-riPSCs), although it was expressed in the riPSCs from DS/lean rats (l-riPSCs) (Fig. 3A).


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)

mRNA expressions.The expression of mRNAs was analyzed by RT-PCR. A) Introduced four genes (EGFP, Sox2, Klf4 and Oct3/4) were expressed in both o-riPSCs and l-riPSCs, but not in both MSCs. Transgenes (mouse mRNA) were detected only in undifferentiated riPSCs cultured in medium containing Dox. Leptin receptor was not detected in the o-riPSCs, however, it was expressed in thel-riPSCs. B) After differentiation in embryoid bodies (EB), Sox17, SM-22a, and Ncam were clearly demonstrated in both o-riPSCs and l-riPSCs. Introduced genes (Sox2, Klf4 and Oct3/4) and Transgenes (mouse mRNA) were not detected in differentiated embryoid bodies (EB) of both o-riPSCs and l-riPSCs.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104462-g003: mRNA expressions.The expression of mRNAs was analyzed by RT-PCR. A) Introduced four genes (EGFP, Sox2, Klf4 and Oct3/4) were expressed in both o-riPSCs and l-riPSCs, but not in both MSCs. Transgenes (mouse mRNA) were detected only in undifferentiated riPSCs cultured in medium containing Dox. Leptin receptor was not detected in the o-riPSCs, however, it was expressed in thel-riPSCs. B) After differentiation in embryoid bodies (EB), Sox17, SM-22a, and Ncam were clearly demonstrated in both o-riPSCs and l-riPSCs. Introduced genes (Sox2, Klf4 and Oct3/4) and Transgenes (mouse mRNA) were not detected in differentiated embryoid bodies (EB) of both o-riPSCs and l-riPSCs.
Mentions: RT-PCR showed that the riPSCs expressed many undifferentiated ESC-marker genes, including Sox2, Oct3/4, Klf4, and Eras. Although Sox2, Klf4, and Eras were not detected in non-induced MSCs, low-level expression of Oct3/4 was detected in non-induced MSCs. The primers used to amplify the sequence between T2A and Sox2 were used to detect transgene expression. Transgenes were detected only in conditions with Dox and were not detected in non-induced MSCs or differentiated riPSCs, indicating that expression via the lentiviral vector was controlled by a tetracycline-responsive element. Leptin receptor was not detected in the riPSCs from DS/obese rats (o-riPSCs), although it was expressed in the riPSCs from DS/lean rats (l-riPSCs) (Fig. 3A).

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