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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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Formation of iPSC colonies.A) The ESC-like colonies were generated from rat MSCs by lentiviral transfection. These colonies were positive for alkaline phosphatase (ALP) staining (upper panel), whereas the non-transfected rat MSCs could not form any colonies (lower panel). B) Generation of riPSCs was confirmed by the expression of SSEA-1 (red) and EGFP (green in the upper panels). These riPSC colonies also expressed Nanog (red in the lower panels). C) Both MSCs derived from DS/obese (a) and DS/lean (d) rats showed a bipolar shape, while colonies of both o-riPSCs (b, c) and l-iPSCs (e, f) formed clusters expressing EGFP, showing a similar appearance to ESCs. D) The ratio of clones expressing pluripotency markers (SSEA-1 or Nanog) was demonstrated by counting the number of total pluripotency marker-positive colonies per the total number of EGFP-positive colonies. Almost all established EGFP-positive clones expressed both of pluripotency markers, respectively.
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pone-0104462-g002: Formation of iPSC colonies.A) The ESC-like colonies were generated from rat MSCs by lentiviral transfection. These colonies were positive for alkaline phosphatase (ALP) staining (upper panel), whereas the non-transfected rat MSCs could not form any colonies (lower panel). B) Generation of riPSCs was confirmed by the expression of SSEA-1 (red) and EGFP (green in the upper panels). These riPSC colonies also expressed Nanog (red in the lower panels). C) Both MSCs derived from DS/obese (a) and DS/lean (d) rats showed a bipolar shape, while colonies of both o-riPSCs (b, c) and l-iPSCs (e, f) formed clusters expressing EGFP, showing a similar appearance to ESCs. D) The ratio of clones expressing pluripotency markers (SSEA-1 or Nanog) was demonstrated by counting the number of total pluripotency marker-positive colonies per the total number of EGFP-positive colonies. Almost all established EGFP-positive clones expressed both of pluripotency markers, respectively.

Mentions: Morphologically ESC-like colonies appeared 10 days after transfection. They expressed EGFP, stage-specific embryonic antigen (SSEA)-1, and Nanog (Fig. 2B). EGFP-positive colonies expressed ALP (Fig. 2A). Colonies from DS/obese rats and DS/lean rats showed similar appearance (Fig. 2C). Furthermore, more than 95% of established EGFP-positive colonies showed distinct key features of rESCs, such as expression of pluripotency markers (Fig. 2D). However, MSCs that were not transfected with the reprogramming factors could not generate any colonies expressing EGFP, even though they were cultured under the same conditions for 10 days. These results indicate that the cells generated from DS/obese and DS/lean MSCs by lentiviral transfection were iPSCs, i.e., riPSCs.


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)

Formation of iPSC colonies.A) The ESC-like colonies were generated from rat MSCs by lentiviral transfection. These colonies were positive for alkaline phosphatase (ALP) staining (upper panel), whereas the non-transfected rat MSCs could not form any colonies (lower panel). B) Generation of riPSCs was confirmed by the expression of SSEA-1 (red) and EGFP (green in the upper panels). These riPSC colonies also expressed Nanog (red in the lower panels). C) Both MSCs derived from DS/obese (a) and DS/lean (d) rats showed a bipolar shape, while colonies of both o-riPSCs (b, c) and l-iPSCs (e, f) formed clusters expressing EGFP, showing a similar appearance to ESCs. D) The ratio of clones expressing pluripotency markers (SSEA-1 or Nanog) was demonstrated by counting the number of total pluripotency marker-positive colonies per the total number of EGFP-positive colonies. Almost all established EGFP-positive clones expressed both of pluripotency markers, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104462-g002: Formation of iPSC colonies.A) The ESC-like colonies were generated from rat MSCs by lentiviral transfection. These colonies were positive for alkaline phosphatase (ALP) staining (upper panel), whereas the non-transfected rat MSCs could not form any colonies (lower panel). B) Generation of riPSCs was confirmed by the expression of SSEA-1 (red) and EGFP (green in the upper panels). These riPSC colonies also expressed Nanog (red in the lower panels). C) Both MSCs derived from DS/obese (a) and DS/lean (d) rats showed a bipolar shape, while colonies of both o-riPSCs (b, c) and l-iPSCs (e, f) formed clusters expressing EGFP, showing a similar appearance to ESCs. D) The ratio of clones expressing pluripotency markers (SSEA-1 or Nanog) was demonstrated by counting the number of total pluripotency marker-positive colonies per the total number of EGFP-positive colonies. Almost all established EGFP-positive clones expressed both of pluripotency markers, respectively.
Mentions: Morphologically ESC-like colonies appeared 10 days after transfection. They expressed EGFP, stage-specific embryonic antigen (SSEA)-1, and Nanog (Fig. 2B). EGFP-positive colonies expressed ALP (Fig. 2A). Colonies from DS/obese rats and DS/lean rats showed similar appearance (Fig. 2C). Furthermore, more than 95% of established EGFP-positive colonies showed distinct key features of rESCs, such as expression of pluripotency markers (Fig. 2D). However, MSCs that were not transfected with the reprogramming factors could not generate any colonies expressing EGFP, even though they were cultured under the same conditions for 10 days. These results indicate that the cells generated from DS/obese and DS/lean MSCs by lentiviral transfection were iPSCs, i.e., riPSCs.

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