Differentiation-Dependent Motility-Responses of Developing Neural Progenitors to Optogenetic Stimulation.

During neural tissue genesis, neural stem/progenitor cells are exposed to bioelectric stimuli well before synaptogenesis and neural circuit formation. Fluctuations in the electrochemical potential in the vicinity of developing cells influence the genesis, migration and maturation of neuronal precursors. The complexity of the in vivo environment and the coexistence of various progenitor populations hinder the understanding of the significance of ionic/bioelectric stimuli in the early phases of neuronal differentiation. Using optogenetic stimulation, we investigated the in vitro motility responses of radial glia-like neural stem/progenitor populations to ionic stimuli. Radial glia-like neural stem cells were isolated from CAGloxpStoploxpChR2(H134)-eYFP transgenic mouse embryos. After transfection with Cre-recombinase, ChR2(channelrhodopsin-2)-expressing and non-expressing cells were separated by eYFP fluorescence. Expression of light-gated ion channels were checked by patch clamp and fluorescence intensity assays. Neurogenesis by ChR2-expressing and non-expressing cells was induced by withdrawal of EGF from the medium. Cells in different (stem cell, migrating progenitor and maturing precursor) stages of development were illuminated with laser light (λ = 488 nm; 1.3 mW/mm2; 300 ms) in every 5 min for 12 h. The displacement of the cells was analyzed on images taken at the end of each light pulse. Results demonstrated that the migratory activity decreased with the advancement of neuronal differentiation regardless of stimulation. Light-sensitive cells, however, responded on a differentiation-dependent way. In non-differentiated ChR2-expressing stem cell populations, the motility did not change significantly in response to light-stimulation. The displacement activity of migrating progenitors was enhanced, while the motility of differentiating neuronal precursors was markedly reduced by illumination.

Differentiation-Dependent Energy Production and Metabolite Utilization: A Comparative Study on Neural Stem Cells, Neurons, and Astrocytes.

While it is evident that the metabolic machinery of stem cells should be fairly different from that of differentiated neurons, the basic energy production pathways in neural stem cells (NSCs) or in neurons are far from clear. Using the model of in vitro neuron production by NE-4C NSCs, this study focused on the metabolic changes taking place during the in vitro neuronal differentiation. O2 consumption, H(+) production, and metabolic responses to single metabolites were measured in cultures of NSCs and in their neuronal derivatives, as well as in primary neuronal and astroglial cultures. In metabolite-free solutions, NSCs consumed little O2 and displayed a higher level of mitochondrial proton leak than neurons. In stem cells, glycolysis was the main source of energy for the survival of a 2.5-h period of metabolite deprivation. In contrast, stem cell-derived or primary neurons sustained a high-level oxidative phosphorylation during metabolite deprivation, indicating the consumption of own cellular material for energy production. The stem cells increased O2 consumption and mitochondrial ATP production in response to single metabolites (with the exception of glucose), showing rapid adaptation of the metabolic machinery to the available resources. In contrast, single metabolites did not increase the O2 consumption of neurons or astrocytes. In "starving" neurons, neither lactate nor pyruvate was utilized for mitochondrial ATP production. Gene expression studies also suggested that aerobic glycolysis and rapid metabolic adaptation characterize the NE-4C NSCs, while autophagy and alternative glucose utilization play important roles in the metabolism of stem cell-derived neurons.

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,ß,γ, Rxrα,ß,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.

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.

Survival and differentiation of neuroectodermal cells with stem cell properties at different oxygen levels.

Freeze-lesioned regions of the forebrain cortex provide adequate environment for growth of non-differentiated neural progenitors, but do not support their neuron formation. Reduced oxygen supply, among numerous factors, was suspected to impair neuronal cell fate commitment. In the present study, proliferation and differentiation of neural stem/progenitor cells were investigated at different oxygen levels both in vitro and in vivo. Low (1% atmospheric) oxygen supply did not affect the in vitro viability and proliferation of stem cells or the transcription of "stemness" genes but impaired the viability of committed neuronal progenitors and the expression of proneural and neuronal genes. Consequently, the rate of in vitro neuron formation was markedly reduced under hypoxic conditions. In vivo, neural stem/progenitor cells survived and proliferated in freeze-lesioned adult mouse forebrains, but did not develop into neurons. Hypoperfusion-caused hypoxia in lesioned cortices was partially corrected by hyperbaric oxygen treatment (HBOT). HBOT, while reduced the rate of cell proliferation at the lesion site, resulted in sporadic neuron formation from implanted neural stem cells. The data indicate that in hypoxic brain areas, neural stem cells survive and proliferate, but neural tissue-type differentiation can not proceed. Oxygenation renders the damaged brain areas more permissive for tissue-type differentiation and may help the integration of neural stem/progenitor cells.