Generation of virus-free induced pluripotent stem cell clones on a synthetic matrix via a single cell subcloning in the naïve state.
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.
Riken Center for Developmental Biology, Minatojima-Minamimachi, Chuo-ku, Kobe, Japan.
Naoki
N
Nishishita
Masayuki
M
Shikamura
Chiemi
C
Takenaka
Nozomi
N
Takada
Noemi
N
Fusaki
Noemi
N
Fusak
Shin
S
Kawamata
D000818:Animals; D002478:Cells, Cultured; D002999:Clone Cells / Q000166:cytology* / Q000276:immunology; D003470:Culture Media; D005822:Genetic Vectors; D006801:Humans; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D051379:Mice; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000276:immunology; D011323:Primates; D020133:Reverse Transcriptase Polymerase Chain Reaction; D029082:Sendai virus / Q000235:genetics
D003470:Culture Media; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4