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Plementary Table 1 in Additional File 1) with Moloney murine leukemia viruses (MMLVs) carrying the coding regions of mouse Oct4, Sox2, Klf4 and/or Nanog or human Oct4, Sox2 and Klf4. R1 mouse embryonic stem cells and iPSCs were maintained in culture as described previously (Figure 1A) [29]. Briefly, iPS and ES cells were plated on gelatin-coated tissue culture plates and grown in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) (Invitrogen) supplemented with 15 FBS (Invitrogen), 1.0 mM sodium pyruvate (Stemcell Technologies), 10 mM nonessential amino acids (Stemcell Technologies), 0.01 penicillin streptomycin (Stemcell Technologies), 2.0 mM L-glutamine (Stemcell Technologies), 1,000 units/ml leukemia inhibiting factor (Chemicon), and 0.055 mM 2-mercaptoethanol. Cells were passaged by dissociation with 0.25 trypsin-EDTA every 2-3 days. Two days after passaging the health and phenotypic stability of the cells was assessed. Five to ten representative DIC images were taken and then analyzed on MetaMorph software. Dissociation of tightly packed clones and/or the appearance of enlarged and flattened cells were indicators of spontaneous differentiation.Neural inductionsingle cells using 0.25 trypsin-EDTA and resuspended in differentiation medium containing Glasgow's Minimum Essential Medium (GMEM) (GIBCO/Invitrogen), 5 Knockout serum replacement (Invitrogen), 2.0 mM L-glutamine, 1.0 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.01 penicillin streptomycin, and 0.1 mM 2-mercaptoethanol. Cells were plated on gelatincoated plates for 40 minutes to remove any residual feeder cells or partially differentiated cells. Cells were then cultured in low adherence 100 mm bacterial plates for 5 days at a density of 5-10 ?104 (iPSC) or 5 ?104 (ESC) PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/10956013 cells per ml to allow embryoid body Gemcitabine (hydrochloride) (EB) formation. Differentiation medium was changed at day 3. On day 5, EBs were plated en bloc on tissue culture plates or chamber slides double-coated with poly-D-lysine (200 g/ml) and mouse laminin (10 g/ml) at a density of 1-2 ?102 EBs per cm2 in fresh medium. Before plating, EB were imaged to assess size and shape. At least 50 EBs were analyzed using MetaMorph software to determine the average EB diameter for each biological replicate. Twenty-four-thirty-six hours post plating, the medium was changed to neural induction medium containing GMEM, 1 N2, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids, 0.1 mM 2mercaptoethanol, 0.01 penicillin streptomycin and 10 ng/ml brain-derived neurotrophic factor (BDNF) (PeproTech). Neural induction cultures were maintained for 3, 7 or 15 days before cells were harvested for RNA extraction, electrophysiological recordings, flow cytometry analysis, or fixation with 4 paraformaldehyde for immunocytochemistry.Quantitative RT-PCRAfter 6-8 (early) and 20-30 (late) passages, iPSC and latepassage (30-40) ESCs were subjected to neural differentiation according to a previously established procedure for ESCs (Figure 1A) [29,33]. Cells were dissociated intoThe relative expression levels of pluripotency markers and early/mature neural markers were assessed by conventional reverse transcriptase PCR (RT-PCR) or quantitative real-time RT-PCR (qRT-PCR) using a previously described procedure [41]. At various time points of cell culture and neural induction (undifferentiated day 5-7, EB day 5, and days 3, 7 and 15 of neural induction), total RNA was isolated using the RNeasy Minikit (Qiagen) and then treated with.