Mauro Toselli

Long-term tripotent differentiation capacity of human neural stem (NS) cells in adherent culture.

Stem cell lines that provide a renewable and scaleable supply of central nervous system cell types would constitute an invaluable resource for basic and applied neurobiology. Here we describe the generation and long-term expansion of multiple human foetal neural stem (NS) cell lines in monolayer culture without genetic immortalization. Adherent human NS cells are propagated in the presence of epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), under which conditions they stably express neural precursor markers and exhibit negligible differentiation into neurons or glia. However, they produce astrocytes, oligodendrocytes, and neurons upon exposure to appropriate differentiation factors. Single cell cloning demonstrates that human NS cells are tripotent. They retain a diploid karyotype and constant neurogenic capacity after over 100 generations. In contrast to human neurospheres, we observe no requirement for the cytokine leukaemia inhibitory factor (LIF) for continued expansion of adherent human NS cells. Human NS cells can be stably transfected to provide reporter lines and readily imaged in live monolayer cultures, creating the potential for high content genetic and chemical screens.

Developmentally coordinated extrinsic signals drive human pluripotent stem cell differentiation toward authentic DARPP-32(+) medium-sized spiny neurons

Medium-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/TGFβ inhibition and subsequent SHH/DKK1 treatment. The emerging FOXG1+/GSX2+ telencephalic progenitors are then terminally differentiated, resulting in the systematic line-independent generation of FOXP1+/FOXP2+/CTIP2+/calbindin+/DARPP-32+ 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.

ST14A cells have properties of a medium-size spiny neuron.

The ST14A cell line was previously derived from embryonic day 14 rat striatal primordia by retroviral transduction of the temperature-sensitive SV40 large T antigen. We showed that cell division and expression of nestin persists at 33 degrees C, the permissive temperature, whereas cell division ceases, nestin expression decreases, and MAP2 expression increases at the nonpermissive temperature of 39 degrees C. In this study, we further characterized the cells and found that they express other general and subtype-specific neuronal characteristics. ST14A cells express enolase and beta III-tubulin. Furthermore, they express the striatal marker DARPP-32, which is up-regulated upon differentiation of the cells by growth in serum-free medium. Stimulation with dopamine, the D2-dopamine receptor agonist quinpirole, or the D1-dopamine receptor agonist SKF82958 results in phosphorylation of CREB. Treatment of the cells with a mixture of reagents which stimulate the MAPK and adenylyl cyclase pathways radically changes the morphology of the ST14A cells. The cells develop numerous neurite-like appearing processes which stain with beta III-tubulin. Moreover, under these conditions, intracellular injection of rectangular depolarizing current stimuli elicits overshooting action potentials with a relatively fast depolarization rate when starting from a strongly hyperpolarized membrane potential. Taken together, these data imply that the ST14A cell line displays some of the characteristics of a medium-size spiny neuron subtype and provides a new tool to elucidate the pathways and molecules involved in medium-size spiny neuron differentiation and disease.

An optimized experimental strategy for efficient conversion of embryonic stem (ES)-derived mouse neural stem (NS) cells into a nearly homogeneous mature neuronal population

NS cells are a homogeneous population of neural stem cells which were previously derived from embryonic stem cells as well as from the fetal and adult brain. Our previous reports have described a 21 day long neuronal differentiation protocol able to reproducibly convert adult SVZ-derived NS (aNS) cells into a population composed of 65% mature neurons and 35% glial cells. Here we have developed a different procedure specifically applicable to ES-derived NS cells in order to fully explore their neurogenic capacity. Differently from the aNS differentiation procedure, optimized neuronal output from ES-derived NS cells requires replating of the cells on appropriate substrates followed by sequential exposure to modified media. In these conditions, ES-derived NS cells differentiate into neurons with a barely appreciable quota of astrocytes and occasional oligodendrocytes. In particular, 21 days after the beginning of the treatment, 85% of the cells has differentiated into molecularly and electrophysiologically mature neurons belonging to the GABAergic lineage. The procedure, which is applicable with no considerable differences to different ES-derived NS cell lines and to NS cells at different passages, opens to the possibility of molecular and biochemical studies on close-to-uniform stem cell derived neurons.

A study of stretch‐activated channels in the membrane of frog oocytes: interactions with Ca2+ ions.

1. We have carried out patch‐clamp measurements on a cationic channel in the plasma membrane of the frog oocyte, which can be specifically activated by membrane stretch. The kinetics of this channel also display a distinct dependence upon membrane potential, the probability of the channel being open increasing with membrane depolarization. 2. When the patch‐clamp pipette filling solution was standard Ringer solution, the single‐channel current‐voltage (I‐V) relationship was linear, the elementary conductance being 38 pS and the reversal potential +7 mV, suggesting very poor selectivity of the channel for the various cations. 3. The I‐V relationship was highly non‐linear having a strong inward‐going rectification when Ca2+‐free solutions were used to fill the patch pipette. These solutions also resulted in a selective, inward cationic permeability through the membrane, with K+ being more permeable than Na+ greater than Li+ greater than Ba2+ greater than Ca2+. 4. Though permeant through the stretch‐activated channel, Ca2+ inhibited in a concentration‐dependent manner the currents carried by other cations. La3+ (0.1 mM) was also an effective channel blocker. 5. The inward current carried by individual cations at a given membrane potential increased with increasing external cation concentration up to a saturating level, this level being maximal for K+ and minimal for Ca2+. Also the half‐saturating concentration was maximal for K+ and minimal for Ca2+ at all membrane potentials. 6. In the presence of a constant Ca2+ concentration (50 microM) increasing [K+] did not change the absolute level at which the current saturated; however the half‐saturating K+ concentration was greatly increased, indicating competitive inhibition between Ca2+ and K+ for the same site. 7. The data are consistent with a model based on Eyring rate theory for current conduction through ionic channels, in which we assume that the ions capable of entering the channel compete for a binding site that they must first occupy before proceeding on. The possible energy profile of the stretch‐activated channel was defined by optimizing the model parameters to obtain the best fit of the experimental data. Ca2+ was found to have a smaller dissociation constant and much longer occupancy time than Na+ or K+, thus accounting for its lower permeability and inhibitory effect on current conduction by other cations through the stretch‐activatable channel.

Age-dependent expression of high-voltage activated calcium currents during cerebellar granule cell development in situ.

Ca2+ currents play a crucial role during neuronal growth. In this paper we describe the development of Ca2+ currents using whole-cell patch-clamp recordings in granule cells of cerebellar slices obtained from 7- to 24day-old rats. Granule cells expressed high-voltage-activated (HVA) Ca2+ currents in different proportions. The percentage of cells with a measurable HVA current, and the size of HVA current increased in parallel with granule cell maturation. At less than 14 days HVA currents consisted of a fast- and slow-inactivating component, while at more than 19 days only the slow-inactivating component remained. The fast-inactivating component had faster activation and inactivation kinetics, a more negative threshold for activation, and steeper steady-state inactivation than the slow-inactivating component. Nifedipine (5 μM) partially blocked both components.ω-Conotoxin (5 μM,ω-CgTx) blocked the slow-inactivating component rather selectively. These results indicate that HVA currents change their gating and pharmacological properties during development. Although the mechanism at the molecular level remains speculative, the developmental changes of the HVA current are relevant to the processes of granule cell maturation and excitability.

Characterization of a Voltage-dependent Calcium Current in the Human Neuroblastoma Cell Line SH-SY5Y During Differentiation.

In the presence of retinoic acid, cultured human neuroblastoma SH‐SY5Y cells grow processes indicative of neuronal differentiation. A voltage‐gated Ca current is already present in undifferentiated cells. A gradual increase of the Ca current density occurs during cell differentiation. According to kinetic and pharmacological properties, Ca currents in differentiated cells are indistinguishable from those elicitable in undifferentiated cells and resemble features of the high‐voltage activated currents present in mammalian neuronal cells. ω‐conotoxin strongly depresses high‐voltage activated currents, both in undifferentiated and in differentiated SH‐SY5Y cells. Interestingly, the Ca agonist Bay K 8644 is effective, albeit with great variability from cell to cell, only in differentiated cells and only when barium is the current carrier through the Ca channels. A diversity of high‐voltage activated Ca channels of distinct pharmacology has been recently observed in other kinds of neurons. This requires a redefinition of the role that voltage‐dependent Ca channel subtypes can play in mammalian neurons.

Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials.

A population of mouse embryonic stem (ES)-derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na(+) currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na(+) channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na(+) currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na(+) current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na(+) channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturation.

Dual modulation of inward rectifier potassium currents in olfactory neuronal cells by promiscuous G protein coupling of the oxytocin receptor

J. Neurochem. (2010) 114, 1424–1435.

Kinetic and pharmacological properties of high- and low-threshold calcium channels in primary cultures of rat hippocampal neurons.

The kinetic, permeability and pharmacological properties of Ca currents were investigated in primary cultures of rat hippocampal neurons. The low-voltage-activated (LVA) Ca current turned on positive to −60mV and fully inactivated in a voltage-dependent way. This current was depressed by nickel (Ni, 40 μM) and amiloride (500 μM) and was insensitive to ω-conotoxin (ω-CgTx) (4 μM) and to the Ca agonist Bay K 8644 (5 μM). The high-voltage-activated (HVA) Ca current turned on positive to −40 mV and inactivated slowly and incompletely. This current was much less sensitive than the LVA current to Ni and amiloride but more sensitive to cadmium. ωCgTx blocked only partially this current (about 50%) in an irreversible way. Bay K 8644 had a clear agonistic action almost exclusively on the ω-CgTx-resistant HVA current component. The present results suggest that the HVA channels, quite homogeneous for their kinetic properties and sensitivity to holding potentials, can be pharmacologically separated in two classes: (i) ω-CgTx-sensitive and Bay-K-8644-insensitive (ω-S/BK-I) and (ii) ω-CgTx-insensitive and Bay-K-8644-sensitive (ω-I/BK-S), the latter displaying a stronger Cadependent inactivation.