L status of iPSC-derived neurons, we performed whole cell patch clamp

L status of iPSC-derived neurons, we performed whole cell patch clamp experiments Rosiglitazone between days 7-14 of neural induction (Figure 6). For consistent analysis, we chose cells with a distinct bipolar or multipolar morphology (Figure 6A). The average resting membrane potentials were similar between early and late-passage iPSCs at 55 mV, which was more depolarized than those recorded in ESCs (Figure 6B). Using a current step protocol, 90 of patched ESC-derived neurons elicited repeated action potentials and robust inward and outward currents (Figure 6C). By contrast, early-passage iPSC-derived neurons, although morphologically similar to PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/6833145 ESC-derived cells, produced only solitary or paired action potentials with comparatively weak inward and outward currents (Figure 6C). Action potentials were recorded from only 23 of cells. Hyperpolarizing the cells (to -70 mV) typically did not substantially enhance the ability of early-passage iPSC-derived neurons to generate repetitive action potentials. Moreover, these cells displayed poor membrane integrity, as indicated by low input resistances that tended to get even lower fairly rapidly, which made recording difficult. Late-passage iPSC-derived neurons were capable of producing action potentials of similar amplitude and frequency asKoehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-2202/12/Page 9 ofFigure 6 Electrophysiological assessment of GG3.1 iPSC-derived neurons. (A) Micrograph showing a representative neuronal cell that was targeted for recording. (B) Average resting membrane potentials for one set of experiments shows no discernable difference between EP and LP neurons. (C) Representative traces for whole cell-patch clamp recordings in EP, LP and WT ESCs. Action potentials were elicited with 500 ms long current injections of 2-340 pA. Current recordings were generated by stepping up membrane potential from -90 mV to +60 mV in 10 mV increments.ESC-derived neurons. Robust action potentials were recorded from 58 of cells (Figure 6C). Accordingly, the inward and outward currents (most likely sodium and potassium currents, respectively, although this was not empirically determined) were equivalent with those detected in ESC neurons (Figure 6C).Discussion To our knowledge, this is the first study to specifically compare the neural differentiation capacity between early- and late-passage murine iPSCs. Of our four iPSC lines, three (GG3.1/3 and miPS-25) generated neuronal populations greater than 30 (n = 3 per line) of the total cell populations in early-passage culture when weapplied an ESC-based neuronal induction protocol. Our group and others have previously shown that this protocol yields neuronal population of greater than 80 purity using murine ESCs [29,33]. Quantitative gene expression analysis revealed a similar, but temporally delayed pattern of neural lineage gene expression between ESCs and one iPSC line (GG3.1). We found that serial passaging improved the stability and maintenance of two newly derived iPSC lines in an undifferentiated state (Figure 3). Moreover, upon neural induction, late-passage iPSCs and ESCs undergo nearly identical temporal changes in gene expression (Figures 4 and 5). These results strongly suggest that sufficient cellular divisions are necessary to generated stable iPSCs clonesKoehler et al. BMC Neuroscience 2011, 12:82 http://www.biomedcentral.com/1471-2202/12/Page 10 ofthat can achieve directed differentiation efficiencies com.