RRC ID 36642
著者 Nishishita N, Shikamura M, Takenaka C, Takada N, Fusaki N, Kawamata S.
タイトル Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state.
ジャーナル PLoS One
Abstract CD34+ cord blood cells can be reprogrammed effectively on dishes coated with a synthetic RGD motif polymer (PronectinF®) using a temperature sensitive Sendai virus vector (SeV TS7) carrying reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC. Dish-shaped human ES cell-like colonies emerged in serum-free primate ES cell medium (supplemented with bFGF) in 20% O2 culture conditions. The copy numbers of SeV TS7 vectors in the cytoplasm were drastically reduced by a temperature shift at 38°C for three days. Then, single cells from colonies were seeded on PronectinF®-coated 96-well plates and cultured under naïve culture conditions (N2B27-based medium supplemented with LIF, forskolin, a MAPK inhibitor, and a GSK inhibitor in 5% O2) for cloning purpose. Dome-shaped mouse ES cell-like colonies from single cells emerged on PronectinF®-coated dishes. These cells were collected and cultured again in primate ES cell medium supplemented with bFGF in 20% O2 and maintained on PronectinF®-coated dishes. Cells were assessed for reprogramming, including the absence of residual SeV and their potential for three germ layer differentiation. Generation of virus-free induced pluripotent stem cell (iPSC) clones from single cells under feeder-free conditions will solve some of the safety concerns related to use of xeno- or allogeneic-material in culture, and contribute to the characterization and the standardization of iPS cells intended for use in a clinical setting.
巻・号 7(6)
ページ e38389
公開日 2012-1-1
DOI 10.1371/journal.pone.0038389
PII PONE-D-12-04345
PMID 22719883
PMC PMC3374798
MeSH Animals Cells, Cultured Clone Cells / cytology* Clone Cells / immunology Culture Media Genetic Vectors Humans Karyotyping Kruppel-Like Factor 4 Mice Pluripotent Stem Cells / cytology* Pluripotent Stem Cells / immunology Primates Reverse Transcriptase Polymerase Chain Reaction Sendai virus / genetics
IF 2.74
引用数 24
WOS 分野 MULTIDISCIPLINARY SCIENCES
リソース情報
研究用ヒト臍帯血幹細胞 研究用ヒト臍帯血幹細胞
ヒト・動物細胞 khES-1(HES0001)