Store-operated calcium entry modulates neuronal network activity in a model of chronic epilepsy.

Store-operated Ca(2+) entry (SOCE) over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and has only recently been shown to play a role in CNS processes like synaptic plasticity. However, the direct effect of SOCE on the excitability of neuronal networks in vitro and in vivo has never been determined. We confirmed the presence of SOCE and the expression of the calcium sensors STIM1 and STIM2, which convey information about the calcium load of the stores to channel proteins at the plasma membrane, in neurons and astrocytes. Inhibition of SOCE by pharmacological agents 2-APB and ML-9 reduced the steady-state neuronal Ca(2+) concentration, reduced network activity, and increased synchrony of primary neuronal cultures grown on multi-electrode arrays, which prompted us to elucidate the relative expression of STIM proteins in conditions of pathologic excitability. Both proteins were increased in brains of chronic epileptic rodents and strongly expressed in hippocampal specimens from medial temporal lobe epilepsy patients. Pharmacologic inhibition of SOCE in chronic epileptic hippocampal slices suppressed interictal spikes and rhythmized epileptic burst activity. Our results indicate that SOCE modulates the activity of neuronal networks in vitro and in vivo and delineates SOCE as a potential drug target.

Residual tumor cells are unique cellular targets in glioblastoma.

Residual tumor cells remain beyond the margins of every glioblastoma (GBM) resection. Their resistance to postsurgical therapy is considered a major driving force of mortality, but their biology remains largely uncharacterized. In this study, residual tumor cells were derived via experimental biopsy of the resection margin after standard neurosurgery for direct comparison with samples from the routinely resected tumor tissue. In vitro analysis of proliferation, invasion, stem cell qualities, GBM-typical antigens, genotypes, and in vitro drug and irradiation challenge studies revealed these cells as unique entities. Our findings suggest a need for characterization of residual tumor cells to optimize diagnosis and treatment of GBM.

Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1/S phase transition and accelerates neuronal differentiation in vitro and in vivo.

The controlled in vitro differentiation of human embryonic stem cells (hESCs) and other pluripotent stem cells provides interesting prospects for generating large numbers of human neurons for a variety of biomedical applications. A major bottleneck associated with this approach is the long time required for hESC-derived neural cells to give rise to mature neuronal progeny. In the developing vertebrate nervous system, Notch signaling represents a key regulator of neural stem cell (NSC) maintenance. Here, we set out to explore whether this signaling pathway can be exploited to modulate the differentiation of hESC-derived NSCs (hESNSCs). We assessed the expression of Notch pathway components in hESNSCs and demonstrate that Notch signaling is active under self-renewing culture conditions. Inhibition of Notch activity by the gamma-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT) in hESNSCs affects the expression of human homologues of known targets of Notch and of several cell cycle regulators. Furthermore, DAPT-mediated Notch inhibition delays G1/S-phase transition and commits hESNSCs to neurogenesis. Combined with growth factor withdrawal, inhibition of Notch signaling results in a marked acceleration of differentiation, thereby shortening the time required for the generation of electrophysiologically active hESNSC-derived neurons. This effect can be exploited for neural cell transplantation, where transient Notch inhibition before grafting suffices to promote the onset of neuronal differentiation of hESNSCs in the host tissue. Thus, interference with Notch signaling provides a tool for controlling human NSC differentiation both in vitro and in vivo.

CD44 and hyaluronan promote invasive growth of B35 neuroblastoma cells into the brain.

Hyaluronan and its receptor CD44 are known to contribute to the invasive growth of different tumors of the central nervous system. It is not known, however, if CD44 is sufficient to activate invasive growth into the brain tissue. This study examines how CD44 regulates the motility and invasive growth of B35 neuroblastoma cells into a hyaluronan-rich environment. A comprehensive experimental approach was used encompassing biochemical techniques, single molecule microscopy, correlative confocal and scanning electron microscopy, morphometry of cellular extensions, live-cell imaging and tracking, transplantation onto organotypic brain slices, two-photon imaging and invasion assays. We found that CD44-GFP fusion protein was localized in filopodia and in focal bleb-like protrusions where it provided binding sites for hyaluronan. Transient expression of CD44-GFP was sufficient to increase the length of filopodia, to enhance cell migration and to promote invasive growth into hyaluronan-rich brain tissue. Thus, CD44 controls molecular devices localized in filopodia and bleb-like specializations of the cell surface that enhance cell migration and invasive growth.

Electrophysiological evaluation of engrafted stem cell-derived neurons.

Recent advances in the neural stem cell field have provided a wealth of methods for generating large amounts of purified neuronal precursor cells. It has become a question of paramount importance to determine whether these cells integrate and interact with established neural circuitry after engraftment. In principle, neurons have to fulfill three basic functions: receive incoming signals via synapses, compute and forward processed information to other neurons or effector cells. It is anticipated that functionally integrating stem cell-derived donor neurons perform accordingly. Here we provide protocols for the efficient electrophysiological evaluation of engrafted cells and highlight current limitations thereof.

Signals from embryonic fibroblasts induce adult intestinal epithelial cells to form nestin-positive cells with proliferation and multilineage differentiation capacity in vitro.

The intestinal epithelium has one of the greatest regenerative capacities in the body; however, neither stem nor progenitor cells have been successfully cultivated from the intestine. In this study, we applied an "artificial niche" of mouse embryonic fibroblasts to derive multipotent cells from the intestinal epithelium. Cocultivation of adult mouse and human intestinal epithelium with fibroblast feeder cells led to the generation of a novel type of nestin-positive cells (intestinal epithelium-derived nestin-positive cells [INPs]). Transcriptome analyses demonstrated that mouse embryonic fibroblasts expressed relatively high levels of Wnt/bone morphogenetic protein (BMP) transcripts, and the formation of INPs was specifically associated with an increase in Lef1, Wnt4, Wnt5a, and Wnt/BMP-responsive factors, but a decrease of BMP4 transcript abundance. In vitro, INPs showed a high but finite proliferative capacity and readily differentiated into cells expressing neural, pancreatic, and hepatic transcripts and proteins; however, these derivatives did not show functional properties. In vivo, INPs failed to form chimeras following injection into mouse blastocysts but integrated into hippocampal brain slice cultures in situ. We conclude that the use of embryonic fibroblasts seems to reprogram adult intestinal epithelial cells by modulation of Wnt/BMP signaling to a cell type with a more primitive embryonic-like stage of development that has a high degree of flexibility and plasticity.

Functional network integration of embryonic stem cell-derived astrocytes in hippocampal slice cultures.

Embryonic stem (ES) cells provide attractive prospects for neural transplantation. So far, grafting strategies in the CNS have focused mainly on neuronal replacement. Employing a slice culture model, we found that ES cell-derived glial precursors (ESGPs) possess a remarkable capacity to integrate into the host glial network. Following deposition on the surface of hippocampal slices, ESGPs actively migrate into the recipient tissue and establish extensive cell-cell contacts with recipient glia. Gap junction-mediated coupling between donor and host astrocytes permits widespread delivery of dye from single donor cells. During maturation, engrafted donor cells display morphological, immunochemical and electrophysiological properties that are characteristic of differentiating native glia. Our findings provide the first evidence of functional integration of grafted astrocytes, and depict glial network integration as a potential route for widespread transcellular delivery of small molecules to the CNS.