Standardized generation and differentiation of neural precursor cells from human pluripotent stem cells.

Precise, robust and scalable directed differentiation of pluripotent stem cells is an important goal with respect to disease modeling or future therapies. Using the AggreWell™400 system we have standardized the differentiation of human embryonic and induced pluripotent stem cells to a neuronal fate using defined conditions. This allows reproducibility in replicate experiments and facilitates the direct comparison of cell lines. Since the starting point for EB formation is a single cell suspension, this protocol is suitable for standard and novel methods of pluripotent stem cell culture. Moreover, an intermediate population of neural precursor cells, which are routinely >95% NCAM(pos) and Tra-1-60(neg) by FACS analysis, may be expanded and frozen prior to differentiation allowing a convenient starting point for downstream experiments.

Akt-mediated survival of oligodendrocytes induced by neuregulins.

Neuregulins have been implicated in a number of events in cells in the oligodendrocyte lineage, including enhanced survival, mitosis, migration, and differentiation. At least two signaling pathways have been shown to be involved in neuregulin signaling: the phosphatidylinositol (PI)-3 kinase and the mitogen-activated protein kinase pathways. In the present studies, we examined the signaling pathway involved in the survival function of heregulin, focusing on heregulin-induced changes in Akt activity in cultured glial cells, and the consequences of Akt activation in cells in the oligodendrocyte lineage. Heregulin binds erbB receptors, and in our studies, primary cultures of both oligodendrocyte progenitor cells and differentiating oligodendrocytes expressed erbB2, erbB3, and erbB4 receptors. In C6 glioma cells and primary cultures of oligodendrocytes, heregulin induced time- and dose-dependent Akt phosphorylation at Ser(473) in a wortmannin-sensitive manner. To investigate further the signaling pathway for heregulin in glial cells, BAD was overexpressed in C6 glioma cells. In these cells, heregulin induced phosphorylation of BAD at Ser(136). Apoptosis of oligodendrocyte progenitor cells induced by growth factor deprivation was effectively blocked by heregulin in a wortmannin-sensitive manner. Overexpression of dominant negative Akt but not of wild-type Akt by adenoviral gene transfer in primary cultures of both oligodendrocytes and their progenitors induced significant apoptosis through activation of the caspase cascade. The present data suggest that the survival function of heregulin is mediated through the PI-3 kinase/Akt pathway in cells in the oligodendrocyte lineage and that the Akt pathway may be quite important for survival of cells in this lineage.

Rat brain glial cells in primary culture and subculture contain the delta, epsilon and zeta subspecies of protein kinase C as well as the conventional subspecies.

We raised polyclonal antibodies against the C-terminal peptides of protein kinase C (PkC) subspecies alpha, beta 1, beta 2, gamma, delta, epsilon, and zeta and checked their specificity against brain extracts using Western immunoblot analysis. With equal amounts of protein applied to gels PkC subspecies beta 1, delta, epsilon and zeta were detected in primary cultures of mixed glial cells: bands for the alpha and beta 2 subspecies were less prominent. PkC gamma was not detected in primary glial cultures. The epsilon and zeta subspecies of PkC were detected in subcultures of type 1 astrocytes with weaker bands for the alpha, beta 1 and beta 2 subspecies. Blots of O-2A-lineage glia contained PkCs delta and zeta as prominent bands: the alpha, beta 1 and epsilon subspecies were also present. All PkC subspecies including PkC gamma were detected in C6 glioma cells.

Effects of sodium butyrate on the expression of sodium channels by neuronal cell lines derived from the rat CNS.

We have studied the effects of sodium butyrate on cell morphology and the expression of mRNAs encoding voltage-gated sodium channels in five neuronal cell lines, B35, B50, B65, B103 and B104, all derived from the rat CNS. The cells were grown in medium supplemented with 2.5 mM sodium-n-butyrate and examined daily by phase-contrast microscopy. Sodium butyrate caused slowing of cell division and the formation of longer and more highly branched cytoplasmic processes than were present in untreated cells. Expression of sodium channel mRNA was analysed by PCR with primers that allow the transcripts encoding the different types of sodium channel to be distinguished according to the lengths of the PCR products. The identity of the PCR products was confirmed by restriction enzyme digestion. Southern blotting and hybridization with internal radiolabelled probes. Prior to sodium butyrate treatment, expression of sodium channel mRNA was largely restricted to B50 and B104 cells: B50 cells showed expression of rat brain types I and II sodium channel and B104 cells expressed rat brain type III sodium channel. After treatment for 5 days with sodium butyrate, sodium channel mRNA was detected in all five cell lines. In addition to type I and type II sodium channel, B50 cells expressed rat brain type III sodium channel. These three types of sodium channel were also expressed by B35, B65 and B103 cells. Even after butyrate treatment, B104 cells expressed only type III sodium channel. The treatment also induced expression of rat skeletal muscle SkM1 sodium channel in B35 cells but only trace amounts in the other neuronal cell lines.