Márta Jelitai

Simultaneous PKC and cAMP activation induces differentiation of human dental pulp stem cells into functionally active neurons

The plasticity of dental pulp stem cells (DPSCs) has been demonstrated by several studies showing that they appear to self-maintain through several passages, giving rise to a variety of cells. The aim of the present study was to differentiate DPSCs to mature neuronal cells showing functional evidence of voltage gated ion channel activities in vitro. First, DPSC cultures were seeded on poly-l-lysine coated surfaces and pretreated for 48h with a medium containing basic fibroblast growth factor and the demethylating agent 5-azacytidine. Then neural induction was performed by the simultaneous activation of protein kinase C and the cyclic adenosine monophosphate pathway. Finally, maturation of the induced cells was achieved by continuous treatment with neurotrophin-3, dibutyryl cyclic AMP, and other supplementary components. Non-induced DPSCs already expressed vimentin, nestin, N-tubulin, neurogenin-2 and neurofilament-M. The inductive treatment resulted in decreased vimentin, nestin, N-tubulin and increased neurogenin-2, neuron-specific enolase, neurofilament-M and glial fibrillary acidic protein expression. By the end of the maturation period, all investigated genes were expressed at higher levels than in undifferentiated controls except vimentin and nestin. Patch clamp analysis revealed the functional activity of both voltage-dependent sodium and potassium channels in the differentiated cells. Our results demonstrate that although most surviving cells show neuronal morphology and express neuronal markers, there is a functional heterogeneity among the differentiated cells obtained by the in vitro differentiation protocol described herein. Nevertheless, this study clearly indicates that the dental pulp contains a cell population that is capable of neural commitment by our three step neuroinductive protocol.

Translocator protein (TSPO 18 kDa) is expressed by neural stem and neuronal precursor cells

Translocator protein 18 kDa, the peripheral benzodiazepine receptor by its earlier name, is a mitochondrial membrane protein associated with the mitochondrial permeability pore. While the function of the protein is not properly understood, it is known to play roles in necrotic and apoptotic processes of the neural tissue. In the healthy adult brain, TSPO expression is restricted to glial cells. In developing or damaged neural regions, however, TSPO appears in differentiating/regenerating neurons. Using immunocytochemical, molecular biological and cell biological techniques, we demonstrate that TSPO mRNA and protein, while missing from mature neurons, are present in neural stem cells and also in postmitotic neuronal precursors. Investigating some distinct stages of in vitro differentiation of NE-4C neural stem cells, TSPO 18 kDa was found to be repressed in a relatively late phase of neuron formation, when mature neuron-specific features appear. This timing indicates that mitochondria in fully developed neurons display specific characteristics and provides an additional marker for characterising neuronal differentiation.

Role of γ-aminobutyric acid in early neuronal development: Studies with an embryonic neuroectodermal stem cell clone

γ‐Aminobutyric acid (GABA) has been known to function as an autocrine/paracrine signal molecule in addition to its well‐known inhibitory neurotransmitter function. Studies on the developing brain and on primary brain cell cultures provided evidence for a variety of GABA functions in periods preceding the formation of synapses. The exact role of GABA in the early neural development, however, is still not well understood. In this study, one‐cell‐derived NE‐4C neuroectodermal stem cells were induced to form neurons and astrocytes in vitro, and the role of GABA was investigated in defined phases of neurogenesis. Noninduced NE‐4C cells contained GABA, expressed GABA(A)R α subunits, and carried functional GABA(A) ion channels. A moderate cytoplasmic GABA content was detected during the entire period of differentiation. By the time of the formation of differentiated neurons, neuron‐like cells with both high and low GABA content were clearly distinguishable. HPLC analysis indicated that NE‐4C cells released GABA into their fluid environment during all stages of neuronal development. By using the patch‐clamp technique, GABA‐evoked currents were recorded during the entire proliferation/differentiation period, whereas a GABA‐evoked increase in intracellular Ca2+ was detected only during the maturation of postmitotic neuronal precursors. Bicuculline blocked both the ion currents and the [Ca2+]i increase in response to GABA. Neuron formation was facilitated by GABA through GABA(A) ion channels during postmitotic differentiation, but not earlier during the phases of cell fate commitment. Although the data clearly demonstrate an early responsiveness to GABA, understanding the significance of GABA influence in early neural cell fate decisions will require further investigation.

Changes of KCl sensitivity of proliferating neural progenitors during in vitro neurogenesis

The effects of KCl‐treatment on the survival and proliferation of NE‐4C self‐renewing neural progenitor cells were investigated during early phases of in vitro induced neurogenesis. NE‐4C cells, derived from the anterior brain vesicles of embryonic mouse (E9), divided continuously under non‐inducing conditions, but acquired neuronal features within 6 days, if induced by all‐trans retinoic acid (RA). During the first 2 days of induction, the cells went on proliferating and did not show signs of morphological differentiation. In this stage, the resting membrane potential of RA‐induced cells adopted more negative values in comparison to non‐induced ones. Despite the increased membrane polarity and K+ conductance, addition of 20–50 mM KCl failed to elicit inward Na+ currents and did not induce an increase in the intracellular Ca+ level. Long‐term treatment with 25 mM KCl, on the other hand, resulted in a selective loss of cells committed to neuronal fate by both decreasing the rate of cell proliferation and increasing the rate of cell death. The data indicate that the viability and proliferation of neural progenitors are influenced by extracellular K+‐level in a differentiation stage‐dependent manner.

The Role of GABA in the Early Neuronal Development

Publisher Summary This chapter describes the developmental changes in the composition and distribution of the gamma-aminobutyric acid (GABA) signaling system and discusses the effects of GABA on the distinct steps of neuronal differentiation. A large body of experimental evidence demonstrates that GABA-signaling possess an inherent capability for potent regulation of almost all steps of neuronal differentiation and neural tissue formation. Several data also suggest that a transient GABA-production is an inherent feature of many differentiating neuronal populations, regardless of the future neurotransmitter phenotype. The apparently normal prenatal brain development in GABA-deficient animals, however, indicates that GABA can be replaced by other regulatory factors in modulating neural-cell production, neuronal-precursor migration, process outgrowth, path finding, and neurite elongation. The most intriguing developmental role that has been attributed to GABA is the generation and maintenance of activity waves in the period of functional network formation.

Isolation of Radial Glia-Like Neural Stem Cells from Fetal and Adult Mouse Forebrain via Selective Adhesion to a Novel Adhesive Peptide-Conjugate

Preferential adhesion of neural stem cells to surfaces covered with a novel synthetic adhesive polypeptide (AK-cyclo[RGDfC]) provided a unique, rapid procedure for isolating radial glia-like cells from both fetal and adult rodent brain. Radial glia-like (RGl) neural stem/progenitor cells grew readily on the peptide-covered surfaces under serum-free culture conditions in the presence of EGF as the only growth factor supplement. Proliferating cells derived either from fetal (E 14.5) forebrain or from different regions of the adult brain maintained several radial glia-specific features including nestin, RC2 immunoreactivity and Pax6, Sox2, Blbp, Glast gene expression. Proliferating RGl cells were obtained also from non-neurogenic zones including the parenchyma of the adult cerebral cortex and dorsal midbrain. Continuous proliferation allowed isolating one-cell derived clones of radial glia-like cells. All clones generated neurons, astrocytes and oligodendrocytes under appropriate inducing conditions. Electrophysiological characterization indicated that passive conductance with large delayed rectifying potassium current might be a uniform feature of non-induced radial glia-like cells. Upon induction, all clones gave rise to GABAergic neurons. Significant differences were found, however, among the clones in the generation of glutamatergic and cathecolamine-synthesizing neurons and in the production of oligodendrocytes.

NMDA receptor NR2B subunit over‐expression increases cerebellar granule cell migratory activity

Glutamate acting on NMDA receptors (NMDARs) is known to influence cerebellar granule cell migration. Subunit composition of NMDARs in granule cells changes characteristically during development: NR2B subunit containing receptors are abundant during migration towards the internal granule cell layer but are gradually replaced by NR2A and/or NR2C subunits once the final position is reached. Cerebellar granule cell migration was investigated using mutant mouse lines either with a deletion of the NR2C gene (NR2C−/− mice) or expressing NR2B instead of the NR2C subunit (NR2C‐2B mice). BrdU‐labeling revealed that over‐expression of NR2B increased granule cell translocation in vivo, while the lack of NR2C subunit did not have any detectable effects on cell migration. Cellular composition of wild‐type and mutant dissociated cerebellar granule cell cultures isolated from 10‐day‐old cerebella were similar, but NR2C‐2B cultures had elevated level of NR2B subunits and intracellular Ca2+ imaging revealed higher sensitivity towards the addition of NR2B‐selective antagonist in vitro. Time‐lapse videomicroscopic observations revealed that average migratory velocity and the proportion of translocating cell bodies were significantly higher in NR2C‐2B than in wild‐type cultures. Our results provide evidence that NR2B‐containing NMDARs can have specialized roles during granule cell migration and can increase migratory speed.

Retinoid Machinery in Distinct Neural Stem Cell Populations with Different Retinoid Responsiveness

Retinoic acid (RA) is present at sites of neurogenesis in both the embryonic and adult brain. While it is widely accepted that RA signaling is involved in the regulation of neural stem cell differentiation, little is known about vitamin A utilization and biosynthesis of active retinoids in the neurogenic niches, or about the details of retinoid metabolism in neural stem cells and differentiating progenies. Here we provide data on retinoid responsiveness and RA production of distinct neural stem cell/neural progenitor populations. In addition, we demonstrate differentiation-related changes in the expression of genes encoding proteins of the retinoid machinery, including components responsible for uptake (Stra6) and storage (Lrat) of vitamin A, transport of retinoids (Rbp4, CrbpI, CrabpI-II), synthesis (Rdh10, Raldh1-4), degradation of RA (Cyp26a1-c1) and RA signaling (Rarα,β,γ, Rxrα,β,γ). We show that both early embryonic neuroectodermal (NE-4C) stem cells and late embryonic or adult derived radial glia like progenitors (RGl cells) are capable to produce bioactive retinoids but respond differently to retinoid signals. However, while neuronal differentiation of RGl cells can not be induced by RA, neuron formation by NE-4C cells is initiated by both RA and RA-precursors (retinol or retinyl acetate). The data indicate that endogenous RA production, at least in some neural stem cell populations, may result in autocrine regulation of neuronal differentiation.