Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines

Donai, Kenichiro; Kuroda, Kensuke; Guo, Yi; So, Kyoung Ha; Sone, Hirohito; Kobayashi, Masayuki; Nishimori, Katsuhiko; Fukuda, T.

doi:10.1016/j.ab.2013.08.014

Cited by 17 publications

(25 citation statements)

References 26 publications

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“…The experimental conditions for the established mouse iPS cells are as follows: (1) mouse iPS cells were cultured on MEF feeder cells in mouse iPS cell medium [DMEM with 15 % Knockout Serum Replacement (referred to as KSR, #10828-028, Invitrogen), 0.22 mM 2-mercaptoethanol (#21438-82, Nacalai Tesque), 100 9 MEM Non-essential Amino Acids Solution (#139-15651, Wako Pure Chemical Industries, Osaka, Japan), 1009 antibiotic-antimycotic mixed solution and 1,0009 leukemia inhibitory factor (LIF, Human, recombinant, Culture Supernatant, #125-05603, Wako Pure Chemical Industries)]; (2) mouse iPS cells were cultured on collagen (Cellmatrix Type I-A, Nitta Gelatin, Osaka, Japan)-coated dishes in ESF-C medium (#2004-05, Cell Science and Technology Institute, Sendai, Japan) containing 1,000 9 LIF; (3) the same cell culture of point (2) containing three low-molecular-weight inhibitors [1.5 lM CHIR99021 (#163-24001, Wako Pure Chemical Industries), 0.5 lM PD0325901 (hereinafter the combination of these two inhibitors are referred to as 2i for short, #13034, Cayman Chemical, Ann Arbor, MI, USA), and 0.5 lM Thiazovivin (referred to as Tzv, #04-0017, Stemgent, Cambridge, MA, USA)]. The preparation of MEFs and feeder cells were described in our previous report (Donai et al 2013). …”

Section: Cell Culturementioning

confidence: 99%

“…Detailed method for detection of AP activity and pluripotent markers with immunocytochemistry was previously described in our publication (Donai et al 2013). EBs on gelatin-coated dishes were stained with bIII tubulin monoclonal antibody (1:600 dilution, #MAB1637, Millipore, Billerica, MA, USA), 4 0 ,6-diamidino-2-phenylindole (DAPI) solution (1:1,000 dilution, #340-07971, Wako Pure Chemical Industries) and Alexa Fluor 568 (1:500 dilution, #A21124, Invitrogen).…”

Section: Alkaline Phosphatase (Ap) Staining and Immunocytochemistrymentioning

confidence: 99%

“…Complementary DNA (cDNA) synthesis and PCR were performed in a similar way as in our previous report (Donai et al 2013).…”

Section: Alkaline Phosphatase (Ap) Staining and Immunocytochemistrymentioning

confidence: 99%

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Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Donai

Inagaki

et al. 2014

Cytotechnology

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Embryonic stem cells and induced pluripotent stem (iPS) cells are usually maintained on feeder cells derived from mouse embryonic fibroblasts (MEFs). In recent years, the cell culture of iPS cells under serum-and feeder-free conditions is gaining attention in overcoming the biosafety issues for clinical applications. In this study, we report on the use of multiple small-molecular inhibitors (i.e., CHIR99021, PD0325901, and Thiazovivin) to efficiently cultivate mouse iPS cells without feeder cells in a chemically-defined and serum-free condition. In this condition, we showed that mouse iPS cells are expressing the Nanog, Oct3/4, and SSEA-1 pluripotent markers, indicating that the culture condition is optimized to maintain the pluripotent status of iPS cells. Without these small-molecular inhibitors, mouse iPS cells required the adaptation period to start the stable cell proliferation. The application of these inhibitors enabled us the shortcut culture method for the cellular adaptation. This study will be useful to efficiently establish mouse iPS cell lines without MEF-derived feeder cells.

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Section: Cell Culturementioning

confidence: 99%

Section: Alkaline Phosphatase (Ap) Staining and Immunocytochemistrymentioning

confidence: 99%

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Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Donai

Inagaki

et al. 2014

Cytotechnology

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“…The detailed protocol was described in our previous manuscript (Donai et al 2013). We named the cells transfected with R24C mutant CDK4, Cyclin D, and TERT as K4DT cells, from the last characters of the introduced genes.…”

Section: Viral Vectors and Gene Transfectionmentioning

confidence: 99%

“…The procedure is described in detail in our previous article (Donai et al 2013). Primary antibodies against Cyclin D1 (1:5000, code no.…”

Section: Western Blottingmentioning

confidence: 99%

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators

et al. 2016

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Lowland Anoa has become endangered due to hunting and human activity. Protection and breeding of endangered species in a controlled environment is the best way of conservation. However, it is not possible to adopt this approach for all endangered species because of the cost involved and the everincreasing number of critically endangered species. In consideration of these limitations to the conventional conservation methods, we established a primary cell culture of endangered buffalo (Lowland Anoa, Bubalus quarlesi), for the preservation of this biological resource. In addition, we introduced human derived, mutant cyclin dependent kinase 4 (CDK4), Cyclin D, and telomerase reverse transcriptase (TERT) into the primary cells. The successful introduction of these three genes was confirmed by western blot with specific antibodies, and enzymatic activity. 123 Cytotechnology (2016) 68:1937-1947 DOI 10.1007 We also showed that the expression of mutant CDK4, Cyclin D, and TERT allows us to efficiently establish an immortalized cell line, with an intact chromosome pattern, from Lowland Anoa. To the best of our knowledge, this study is the first investigation that established an immortalized cell line of an endangered wild animal species.

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Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators

Sekine,

Yasunaga,

Kumamoto

et al. 2023

Advanced Biology

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Primary cultured cells cannot proliferate infinite. The overcoming of this limit can be classified as immortalization. Bypass of p16 senescence protein induces efficient immortalization various types of mammalians is previously reported. However, the Cetacea species is not known. Here, that common minke whale‐derived cells can be immortalized with a combination of human genes, mutant cyclin‐dependent kinase 4 (CDK4R24C), cyclin D1, and Telomerase Reverse Transcriptase (TERT) is reported. These results indicate that the function of cell cycle regulators in premature senescence is evolutionarily conserved. This study describes the conserved roles of cell cycle regulators in the immortalization of cells from humans to Cetacea species. Furthermore, using RNA‐seq based on next‐generation sequencing, the gene expression profiles of immortalized cells are compared with parental cells as well as those immortalized with SV40 large T antigen, which is once a popular method for cellular immortalization. The profiling results show that newly established common minke‐whale‐derived immortaliozed cells have completely different profiles from SV40 cells. This result indicates that the expression of mutant CDK4, cyclin D1, and TERT enables to establish immortalized cell lines with different biological nature from SV40 expressing cells.

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Establishment of a reporter system to monitor silencing status in induced pluripotent stem cell lines

Cited by 17 publications

References 26 publications

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Low-molecular-weight inhibitors of cell differentiation enable efficient growth of mouse iPS cells under feeder-free conditions

Cellular conservation of endangered midget buffalo (Lowland Anoa, Bubalus quarlesi) by establishment of primary cultured cell, and its immortalization with expression of cell cycle regulators

Characterization of Common Minke Whale (Balaenoptera Acutorostrata) Cell Lines Immortalized with the Expression of Cell Cycle Regulators

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