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) |