Somatic cells can be induced into pluripotent stem cells (iPSCs) with a combination of four transcription factors, Oct4/Sox2/Klf4/c-Myc or Oct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtain patient-specific cells for various therapeutic and research applications. However, several problems remain for this approach to be therapeutically relevant due to drawbacks associated with efficiency and viral genome integration. Recently, it was shown that neural progenitor cells (NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs. However, NPCs express Sox2 endogenously, possibly facilitating reprogramming in the absence of exogenous Sox2. In this study, we identified a small-molecule combination, BIX-01294 and BayK8644, that enables reprogramming of Oct4/Klf4-transduced mouse embryonic fibroblasts, which do not endogenously express the factors essential for reprogramming. This study demonstrates that small molecules identified through a phenotypic screen can compensate for viral transduction of critical factors, such as Sox2, and improve reprogramming efficiency.

Ca(2+) influx through voltage-gated Ca(2+) channels, especially the L-type (Ca(v)1), activates downstream signaling to the nucleus that affects gene expression and, consequently, cell fate. We hypothesized that these Ca(2+) signals may also influence the neuronal differentiation of neural stem/progenitor cells (NSCs) derived from the brain cortex of postnatal mice. We first studied Ca(2+) transients induced by membrane depolarization in Fluo 4-AM-loaded NSCs using confocal microscopy. Undifferentiated cells (nestin(+)) exhibited no detectable Ca(2+) signals whereas, during 12 days of fetal bovine serum-induced differentiation, neurons (beta-III-tubulin(+)/MAP2(+)) displayed time-dependent increases in intracellular Ca(2+) transients, with DeltaF/F ratios ranging from 0.4 on day 3 to 3.3 on day 12. Patch-clamp experiments revealed similar correlation between NSC differentiation and macroscopic Ba(2+) current density. These currents were markedly reduced (-77%) by Ca(v)1 channel blockade with 5 microm nifedipine. To determine the influence of Ca(v)1-mediated Ca(2+) influx on NSC differentiation, cells were cultured in differentiative medium with either nifedipine (5 microm) or the L-channel activator Bay K 8644 (10 microm). The latter treatment significantly increased the percentage of beta-III-tubulin(+)/MAP2(+) cells whereas nifedipine produced opposite effects. Pretreatment with nifedipine also inhibited the functional maturation of neurons, which responded to membrane depolarization with weak Ca(2+) signals. Conversely, Bay K 8644 pretreatment significantly enhanced the percentage of responsive cells and the amplitudes of Ca(2+) transients. These data suggest that NSC differentiation is strongly correlated with the expression of voltage-gated Ca(2+) channels, especially the Ca(v)1, and that Ca(2+) influx through these channels plays a key role in promoting neuronal differentiation.

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.