R the reprogramming factors, Oct4, Sox2 and Klf4, discounted transgene expression

R the reprogramming factors, Oct4, Sox2 and Klf4, discounted transgene Rosiglitazone expression in the GG3.1 line (Additional file 1, Fig. S2B). The overall quality of this cell line was further ensured by expression analyses of genes in the Dlk1-Dio3 locusKoehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-2202/12/Page 3 ofFigure 1 iPSC lines subjected to neural induction (Ni) produce populations of neurons with similar morphology to ESC-derived neurons. (A) Ni proceeds in three stages: (1) expansion of undifferentiated cells, (2) formation of embryoid bodies (EB) in a floating serum-free culture and (3) neuronal differentiation on poly-d-lysine/laminin coated plates in the presence of N2-supplement and brain derived neurotrophic factor (BDNF). (B, C) Representative micrographs for the miPS-25 (B), GG3.1 (C) and wild-type (WT) ESC lines at critical time points of neural induction: Undifferentiated, EB day 5 and Ni days 3 and 7. Bars represent 100 m.Koehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-2202/12/Page 4 ofon chromosome 12 (Additional file 1, Fig. S2C and D). Recent reports concluded that repression of this locus, specifically the genes Gtl2 and Rian, is a defining feature of poor quality mouse iPSCs that lack the ability to generate "all-iPSC mice" via tetraploid complementation [31,32]. We analyzed the expression level of Gtl2 and Rian in the GG3.1 line and found no difference in their expression levels when compared to ESCs (Additional file 1, Fig. S2C and D). Moreover, no significant difference in expression levels of Gtl2 and Rian was observed between early- and late-passage iPSCs (Data not shown). Considering the final differentiation performance of the GG3.1 line (i.e. post-extended passaging), this method of iPSC quality assessment should prove useful in future experiments where new iPSCs are derived. To better characterize cellular phenotype, we performed immunocytochemistry on GG3.1 cells at neural induction day 7. Thirty to forty percent (n = 3) of cells stained positive for the early neural marker HuC/D, as well as, the mature neural markers Synaptophysin (Syn), II-tubulin (TuJ1), microtubule associated protein 2 (MAP2) and neural nuclei protein (NeuN). As shown in previous studies, a subset of cells expressed brain-specific homeobox/POU domain protein 3A (Brn3a), indicating the presence of sensory-like neurons (Figure 2A-D). The majority of these cells were also positive for neurofilament and calretinin, consistent with our previous analysis of ESC-derived neurons (Additional file 1, Fig. S3D-F) [29]. Furthermore, we found that Map2, TuJ1, NeuN and neurofilament expression persisted beyond day 15 in iPSC cultures (data not shown). The presence of Syn + puncta and growth cones was indicative of maturing neurons (Figure 2D). This staining profile is consistent with the forebrain-like neurons observed in our and others' previous ESC analysis (See Additional file 1, Fig. S3A-F for further characterization) [29,33]. From this point on, the GG3.1 and miPS-25 lines were chosen for further analysis based on their disparate methods of generation and ability to form spherical EBs with similar abundance ( 0.7-1 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/17139194 ?10 3 /mL, n = 3) as ESCs.Extended passaging enhances pluripotent gene expression in an undifferentiated state and increases the rate/efficiency of neuronal conversionAlthough iPSCs exhibit neural phenotypes similar to ESCs at early-passages, we postulated that the observed morp.