Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

Tanaka, Akihito; Woltjen, Knut; Miyake, Katsuya; Hotta, Akitsu; Ikeya, Makoto; Yamamoto, Takuya; Nishino, Tokiko; Shoji, Emi; Sehara-Fujisawa, Atsuko; Manabe, Yasuko; Fujii, Nobuharu; Hanaoka, Kazunori; Era, Takumi; Yamashita, Satoshi; Isobe, Ken-ichi; Sakurai, Hidetoshi

doi:10.1371/journal.pone.0061540

Cited by 180 publications

(219 citation statements)

References 44 publications

(69 reference statements)

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“…Previous studies have employed FuGENE HD, lipofectamine PLUS, and lipofectamine 2000 reagents for transfection. 17,21,22 For the first time, we compared FuGENE, LTX, X-tremeGENE, and TransIT-2020 reagents to identify a method that would achieve higher transfection efficiency in iPS cells. Transfections are typically performed on cells that are growing and attaching onto plates or dishes.…”

Section: Discussionmentioning

confidence: 99%

“…In the suspension method, cells were harvested with Accutase, suspended, and incubated in the transfection reagent for 5 min at room temperature in a hood. 17 The transfection reagents, FuGENE HD Transfection Reagent (Promega, Madison, WI), Lipofectamine LTX (Life Technologies, Grand Island, NY), X-tremeGENE Transfection Reagent (Roche, Basel, Switzerland), and TransIT-2020 Transfection Reagent (Mirus Bio, Madison, WI), were used following the manufacturer's instructions.…”

Section: Transfection Of 201b7 Cellsmentioning

confidence: 99%

“…17,18 Introduction of multiple transcription factors to iPS cells promote differentiation to target somatic cells. 19 Transcription factors are introduced into iPS cells through plasmids or viral vectors.…”

Section: Introductionmentioning

confidence: 99%

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Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

Tomizawa¹,

Shinozaki²,

Motoyoshi³

et al. 2014

Tissue Engineering Part A

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Transcription factors are essential for the differentiation of human induced pluripotent stem cells (iPS) into specialized cell types. Embryoid body (EB) formation promotes the differentiation of iPS cells. We sought to establish an efficient method of transfection and rotary culture to generate EBs that stably express two genes. The pMetLuc2-Reporter vector was transfected using FuGENE HD (FuGENE), Lipofectamine LTX (LTX), X-tremeGENE, or TransIT-2020 transfection reagents. The media was analyzed using a Metridia luciferase (MetLuc) assay. Transfections were performed on cells adherent to plates/dishes (adherent method) or suspended in the media (suspension method). The 201B7 cells transfected with episomal vectors were selected using G418 (200 mg/mL) or hygromycin B (300 mg/mL). Rotary culture was performed at 2.5 or 9.9 rpm. Efficiency of EB formation was compared among plates and dishes. Cell density was compared at 1.6 · 10 3 , · 10 4 , and · 10 5 cells/ mL. The suspended method of transfection using the FuGENE HD reagent was the most efficient. The expression of pEBMulti/Met-Hyg was detected 11 days posttransfection. Double transformants were selected 6 days posttransfection with pEBNK/EGFP-Neo and pEBNK/Cherry-Hyg. Both EGFP and CherryPicker were expressed in all of the surviving cells. EBs were formed most efficiently from cells cultured at a density of 1.6 · 10 5 cells/mL in six-well plates or 6 cm dishes. The selected cells formed EBs. FuGENE-mediated transfection of plasmids using the suspension method was effective in transforming iPS cells. Furthermore, the episomal vectors enabled us to perform a stable double transfection of EB-forming iPS cells.

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Section: Discussionmentioning

confidence: 99%

Section: Transfection Of 201b7 Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

Tomizawa¹,

Shinozaki²,

Motoyoshi³

et al. 2014

Tissue Engineering Part A

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show abstract

“…Therefore, supplementation of functional myogenic progenitors to repair damaged tissues through transplantation may represent an effective treatment against MDs. Successful generation of myogenic progenitors from hPSCs has been achieved by exogenous expression of transcription factors PAX7 or MYOD1 [1][2][3][4][5] (Table 1). Upon ectopic expression of PAX7 in hPSCs-derived embryoid bodies (EBs), the derived myogenic progenitors can differentiate to multinucleated myofibers in vitro, and develop into myocytes after transplantation into mdx mice (a mouse model of MDs) [2].…”

mentioning

confidence: 99%

“…Moreover, PAX7+ progenitors can improve muscle contractility in the mdx mouse and supplement satellite cells. As an alternative approach, exogenous expression of MYOD1 in hPSCs differentiates them into terminal multinucleated myogenic cells [1,[3][4][5]. Myogenic cells generated using MYOD1 usually fail to maintain a progenitor status under current culture conditions, and thus are not an ideal cell type for transplantation.…”

mentioning

confidence: 99%

Regeneration: making muscle from hPSCs

Zhu

et al. 2014

Cell Res

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Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

Cited by 180 publications

References 44 publications

Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

Dual Gene Expression in Embryoid Bodies Derived from Human Induced Pluripotent Stem Cells Using Episomal Vectors

Regeneration: making muscle from hPSCs

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