MAB5326C3 Sigma-AldrichAnti-Nestin Antibody, clone 10C2, Cy3 conjugate

The AKT family, comprising three highly homologous kinases, is an essential mediator of the PTEN/PI3K pathway, which is deregulated in many human cancers. A thorough understanding of the specific activities of each isoform in normal and disease tissues is lacking. We evaluated the role of each Akt isoform in gliomagenesis by using a model system driven by common glioma abnormalities, loss of function of p53 and Pten, and expression of EGFRvIII. Both Pten deletion and EGFRvIII expression accelerated the proliferation of p53-null primary murine astrocytes. All three Akt isoforms were expressed and phosphorylated in astrocytes, with significantly higher activation in Pten-null cells. Despite substantial compensation in many contexts when individual Akt isoforms were inhibited, isoform-specific effects were also identified. Specifically, loss of Akt1 or Akt2 decreased proliferation of Pten wild-type astrocytes, whereas combined loss of multiple isoforms was needed to inhibit proliferation of Pten-null astrocytes. In addition, Akt3 was required for anchorage-independent growth of transformed astrocytes and human glioma cells, and Akt3 loss inhibited invasion of transformed astrocytes. EGFRvIII expression transformed p53-null astrocytes with or without Pten deletion, causing rapid development of high-grade astrocytoma on intracranial transplantation. Furthermore, tumorigenesis of Pten;p53-null astrocytes expressing EGFRvIII was delayed by Akt1 loss and accelerated by Akt2 loss. Taken together, these results indicate context-dependent roles for individual Akt isoforms and suggest that there may be heterogeneous tumor response to isoform-specific inhibitors.

A subpopulation of glial fibrillary acidic protein (GFAP)-expressing cells located along the length of the lateral ventricles in the subventricular zone (SVZ) have been identified as the multipotent neural stem cells of the adult mammalian brain. We have previously found that, in the adult human brain, a splice variant of GFAP, termed GFAPdelta, was expressed specifically in these cells. To investigate whether GFAPdelta is also present in the precursors of SVZ astrocytes during development and whether GFAPdelta could play a role in the developmental process, we analyzed GFAPdelta expression in the normal developing human cortex and in the cortex of foetuses with the migration disorder lissencephaly type II. We demonstrated for the first time that GFAPdelta is specifically expressed in radial glia and SVZ neural progenitors during human brain development. Expression of GFAPdelta in radial glia starts at around 13 weeks of pregnancy and disappears before birth. GFAPdelta is continuously expressed in the SVZ progenitors at later gestational ages and in the postnatal brain. Co-localization with Ki67 proved that these GFAPdelta-expressing cells are able to proliferate. Furthermore, we showed that the expression pattern of GFAPdelta was disturbed in lissencephaly type II. Overall, these results suggest that the adult SVZ is indeed a remnant of the foetal SVZ, which develops from radial glia. Furthermore, we provide evidence that GFAPdelta can distinguish resting astrocytes from proliferating SVZ progenitors.

Endocannabinoids exert an important neuromodulatory role via presynaptic cannabinoid CB1 receptors and may also participate in the control of neural cell death and survival. The function of the endocannabinoid system has been extensively studied in differentiated neurons, but its potential role in neural progenitor cells remains to be elucidated. Here we show that the CB1 receptor and the endocannabinoid-inactivating enzyme fatty acid amide hydrolase are expressed, both in vitro and in vivo, in postnatal radial glia (RC2+ cells) and in adult nestin type I (nestin(+)GFAP+) neural progenitor cells. Cell culture experiments show that CB1 receptor activation increases progenitor proliferation and differentiation into astroglial cells in vitro. In vivo analysis evidences that, in postnatal CB1(-/-) mouse brain, progenitor proliferation and astrogliogenesis are impaired. Likewise, in adult CB1-deficient mice, neural progenitor proliferation is decreased but is increased in fatty acid amide hydrolase-deficient mice. In addition, endocannabinoid signaling controls neural progenitor differentiation in the adult brain by promoting astroglial differentiation of newly born cells. These results show a novel physiological role of endocannabinoids, which constitute a new family of signaling cues involved in the regulation of neural progenitor cell function.

The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.