Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells.

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Increasing evidence suggests that early neurodevelopmental defects in Huntington's disease (HD) patients could contribute to the later adult neurodegenerative phenotype. Here, by using HD-derived induced pluripotent stem cell lines, we report that early telencephalic induction and late neural identity are affected in cortical and striatal populations. We show that a large CAG expansion causes complete failure of the neuro-ectodermal acquisition, while cells carrying shorter CAGs repeats show gross abnormalities in neural rosette formation as well as disrupted cytoarchitecture in cortical organoids. Gene-expression analysis showed that control organoid overlapped with mature human fetal cortical areas, while HD organoids correlated with the immature ventricular zone/subventricular zone. We also report that defects in neuroectoderm and rosette formation could be rescued by molecular and pharmacological approaches leading to a recovery of striatal identity. These results show that mutant huntingtin precludes normal neuronal fate acquisition and highlights a possible connection between mutant huntingtin and abnormal neural development in HD.

SUMMARYMedium-sized spiny neurons (MSNs) are the only neostriatum projection neurons, and their degeneration underlies some of the clinical features of Huntington's disease. Using knowledge of human developmental biology and exposure to key neurodevelopmental molecules, human pluripotent stem (hPS) cells were induced to differentiate into MSNs. In a feeder-free adherent culture, ventral telencephalic specification is induced by BMP/TGF inhibition and subsequent SHH/DKK1 treatment. + MSNs. Similar to mature MSNs, these neurons carry dopamine and A2a receptors, elicit a typical firing pattern and show inhibitory postsynaptic currents, as well as dopamine neuromodulation and synaptic integration ability in vivo. When transplanted into the striatum of quinolinic acid-lesioned rats, hPS-derived neurons survive and differentiate into DARPP-32 + neurons, leading to a restoration of apomorphine-induced rotation behavior. In summary, hPS cells can be efficiently driven to acquire a functional striatal fate using an ontogeny-recapitulating stepwise method that represents a platform for in vitro human developmental neurobiology studies and drug screening approaches.

The mechanisms that control the periodicity of spontaneous epileptiform cortical potentials were investigated in the in vitro isolated guinea pig brain preparation. A brief intracortical application of bicuculline in the piriform cortex induced spontaneous interictal spikes (sISs) that recurred with high periodicity (8.5 Ϯ 3.1 sec, mean Ϯ SD). Intracellular recordings from principal neurons showed that the early phase of the inter-sIS period is caused by a GABAb receptor-mediated inhibitory potential. The late component of the interspike period correlated to a slowly decaying depolarization abolished at membrane potentials positive to Ϫ32.1 Ϯ 5.3 mV and was not associated with membrane conductance changes. Specific pharmacological tests excluded the contribution of synaptic and intrinsic conductances to the late inter-sIS interval. Recordings with ion-sensitive electrodes demonstrated that sISs determined both a rapid increase in extracellular K ϩ concentration (0.5-1 mM) and an extracellular alkalinization (0.05-0.08 pH units) that slowly decayed during the inter-sIS period and returned to control values just before a subsequent sIS was generated. These observations were not congruous with the presence of a silent period, because both extracellular increase in K ϩ and alkalinization are commonly associated with an increase in neuronal excitability. Extracellular alkalinization could be correlated to an sIS-induced intracellular acidification, a phenomenon that reduces cell coupling by impairing gap junction function. When intracellular acidification was transiently prevented by arterial perfusion with NH 4 Cl (10-20 mM), spontaneous ictal-like epileptiform discharges were induced. In addition, the gap junction blockers octanol (0.2-2 mM) and 18-␣-glycyrrethinic acid (20 M) applied either via the arterial system or locally in the cortex completely and reversibly abolished the sIS. The results reported here suggest that the massive cell discharge associated with an sIS induce a strong inhibition, possibly secondary to a pH-dependent uncoupling of gap junctions, that regulates sIS periodicity.