Derivation, Characterization, and Stable Transfection of Induced Pluripotent Stem Cells from Fischer344 Rats

Liskovykh, Mikhail; Chuykin, Ilya; Ranjan, Ashish; Safina, Dina; Popova, Elena; Tolkunova, Elena; Mosienko, Valentina; Minina, J.M.; Жданова, Н. С.; Mullins, John J.; Bäder, Michael; Alenina, Natalia; Tomilin, Alexey

doi:10.1371/journal.pone.0027345

Cited by 26 publications

(33 citation statements)

References 38 publications

(64 reference statements)

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“…293T cells were transfected with envelope-encoding pMD2G (5 mg), packaging pCMV-dR8.74PAX2 (5 mg), and either Ascl1 (Addgene #27150), Brn2 (Addgene #27151), Myt1l (Addgene #27152), M2RTta (Addgene #20342), cMyc 35 or OKSM (Addgene #20328)-encoding LVTHMbased plasmids (20 mg) by the calciumphosphate method. Lentiviruses were collected from the cell culture supernatant and processed as described elsewhere.…”

Section: Plasmids and Lentivirusesmentioning

confidence: 99%

“…35 Briefly, 10 5 OG2 MEFs were infected with OKSM and M2RTta lentiviruses and cultured in DMEM supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 5% KSR, 1,000 U/ mL LIF, 100 U/ml penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, 1x non-essential amino acids, 50 mM b-mercaptoethanol. The culture medium was supplemented with DOX (2ug/ ml) and aphidicolin (5 ug/ml) when indicated.…”

Section: Fibroblast Isolation and Viral Transductionmentioning

confidence: 99%

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Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells

et al. 2015

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These authors equally contributed to this work.Keywords: cell division, direct conversion, fibroblast, neuron, reprogramming, transdifferentiation Abbreviations: iPS cells, induced pluripotent stem cells; ES cells, embryonic stem cells; BAM, Brn2, Ascl1 and Myt1l; BAMCM, Brn2, Ascl1, Myt1l and cMyc; DOX, doxycycline; BrdU, bromodeoxyuridine; MEF, mouse embryonic fibroblasts.Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

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Section: Plasmids and Lentivirusesmentioning

confidence: 99%

Section: Fibroblast Isolation and Viral Transductionmentioning

confidence: 99%

Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells

et al. 2015

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“…Additional report suggests that the mechanism of hepatocyte differentiation from hepatoblasts is distinct from that of cholangiocytes [73,77] . Transcription factors are also essential for hepatocyte differentiation, and transfection reagents or electroporation are conventionally used to introduce genes of interest into iPS cells [78,79] ; however, it remains difficult to effectively introduce transcription factors into hiPS cells using these methods [80] . Therefore, methods for hepatocyte differentiation using adenoviral vector-mediated expression of transcription factors will be discussed.…”

Section: Protocols For Differentiation Of Hips Cells Into Hepatocytesmentioning

confidence: 99%

Induction of Hepatocyte Differentiation in Human Pluripotent Stem Cells

Tomizawa

Shinozaki

Motoyoshi

et al. 2015

Journal of Gastroenterology and Hepatology Research

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be advantageous in that it could resolve the ethical problems associated with stem cell research and could eliminate the need for administration of immunosuppresants in hepatocyte transplantation. To establish a method for the production of hepatocytes from hiPS cells, it is necessary to understand both the mechanism of hepatocyte differentiation as well as the current techniques for culturing of primary hepatocytes. Meanwhile, hepatocytes can also be differentiated from endodermal cells upon stimulation with growth factors that induce expression of specific transcriptional regulators. Current protocols for the generation of hepatocytes from hiPS cells include the sequential application of growth factors to mimic the environment of fetal liver. Initially, cells are treated with activin and bone morphogenetic proteins, followed by the application of hepatocyte growth factor and, at the final differentiation stage, Oncostatin M. Expression of transcription factors is necessary for hepatocyte differentiation. However, because the efficiency at which vectors expressing these factors can be transfected into hiPS cells is disappointingly low, adenoviral vectors have been used, which have a transduction efficiency of 100%. This method is used to sequentially introduce sex-determining region Y (Sry) HMG box (Sox) 17, hematopoietically expressed homeobox, and hepatocyte nuclear factor-4α into hiPS cells. These factors, in concert with the stimulation with growth factors, promote differentiation of these cells to hepatocytes. Despite the development of these procedures, however, the resulting cells remain immature and only perform certain hepatocyte functions. In this review, we describe each of the above points, and then discuss novel approaches and future directions for the In vitro production of hepatocytes. ABSTRACTCulturing of hepatocytes is used in both experimental and clinical procedures. Human induced pluripotent stem (hiPS) cells can be established from the somatic cells of individual patients. The In vitro production of hepatocytes from these hiPS cells would

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“…Recent progress in derivation of induced pluripotent stem (iPS) cells, which are likely the functional equivalents of ES cells, from somatic cells by forced expression of exogenous transcription factors (e.g., Oct4, Sox2, c-Myc, and Klf4) will further facilitate transgenic technology. Rat iPS cell lines have been derived from a variety of somatic cells, including fibroblasts, neural precursor cells, bone marrow cells, and liver progenitor cells (Chang et al, 2010;Li et al, 2009;Liao et al, 2009;Liskovykh et al, 2011;Merkl et al, 2013). The status of pluripotency of extant rat iPS cell lines is still under investigation (Bui et al, 2013).…”

Section: Ips Cellsmentioning

confidence: 99%