Culture Of Neural Precursor Cells Research Articles

Static and dynamic 3D culture of neural precursor cells on macroporous cryogel microcarriers

Neural precursor cells have been much studied to further our understanding of the far-reaching and controversial question of adult neurogenesis. Currently, differentiation of primary neural precursor cells from the mouse dentate gyrus via 2-dimentional in vitro culture yields low numbers of neurons, a major hindrance to the field of study. 3-dimentional “neurosphere” culture allows better 3D cell-cell contact, but control over cell differentiation is poor because nutrition and oxygen restrictions at the core of the sphere causes spontaneous differentiation, predominantly to glial cells, not neurons. Our group has developed macroporous scaffolds, which overcome the above-mentioned problems, allowing long-term culture of neural stem cells, which can be differentiated into a much higher yield of neurons. Herein we describe a method for culturing neural precursor cells on RGD peptide functionalized-heparin containing cryogel scaffolds, either in standard non-adherent well-plates (static culture) or in spinner flasks (dynamic culture). This method includes:•The synthesis and characterization of heparin based microcarriers.•A “static” 3D culture method for that does not require spinner flask equipment.•“Dynamic” culture in which cell loaded microcarriers are transferred to a spinner flask.

Macroporous heparin-based microcarriers allow long-term 3D culture and differentiation of neural precursor cells

Adult neurogenesis and the neurogenic niche in the dentate gyrus are subjects of much research interest. Enhancing our knowledge of this niche process and the role played by this unique microenvironment would further our understanding of plasticity and its relevance for cognition in health and disease. The complex three-dimensional (3D) nature of the niche microenvironment is poorly recapitulated in current cell culture experimental procedures. Neural precursor cells (NPCs) are cultured either on two-dimensional (2D) surfaces, where cells quickly reach confluency and passaging is required, or as 3D neurospheres, with the limitation of poor diffusion of nutrients and thus partial differentiation of cells over time. Herein, we culture NPCs on microscale scaffolds termed microcarriers, composed of poly(ethylene glycol) and heparin, designed to more closely represent the 3D environment of the neurogenic niche. The interconnected macroporous structure of the microcarriers allows NPCs to attach to their pore walls with subsequent continuous proliferation (analyzed up to 28 days) without formation of a necrotic core. Removal of basic fibroblast growth factor and epidermal growth factor from the culture medium results in differentiation of the NPCs. Unlike 2D culture, a high percentage of neurons was achieved on the microcarriers (22% MAP2 positive cells) indicating that these 3D microscale scaffolds give a more conducive environment for neuronal differentiation. Microcarrier culture of NPCs allows long-term cell expansion and better differentiation, which provides superior culture conditions for studying/modelling the neurogenic niche.

Involvement of caspase-3/PTEN signaling pathway in isoflurane-induced decrease of self-renewal capacity of hippocampal neural precursor cells

Evidence has shown that children exposed to isoflurane anesthesia in early childhood display long-term cognitive abnormalities, and decreased self-renewal capacity of hippocampal neural precursor cells (NPCs), which are associated with cognition impairment. Caspase-3 has long been considered as a mediator in isoflurane-induced neuroapoptosis. However, accumulating data indicate that caspase-3 also plays a non-apoptotic negative regulatory role in NPCs self-renewal. In this study we used in vitro NPC cultures to test whether caspase-3 and its downstream signaling effectors were involved in isoflurane-induced impairment of the self-renewal capacity of hippocampal NPCs. We showed that isoflurane exposure induced a decrease in the self-renewal capacity of hippocampal NPCs by decreasing proliferation and increasing neuronal differentiation. Furthermore, we found that isoflurane exposure significantly increased the levels of active caspase-3 and decreased the levels of phospho-PTEN under both proliferation and differentiation conditions. Inhibition of either caspase-3 with Z-DEVD-fmk or PTEN with BPV (phen) in NPCs, attenuated the isoflurane-induced decrease of their proliferation and increase of neuronal differentiation. Application of Z-DEVD-fmk also attenuated isoflurane-induced decrease in phospho-PTEN expression. Taken together, our in vitro results reveal a previously uncharacterized involvement of caspase-3/PTEN signaling in the isoflurane-induced impairment of NPCs self-renewal, and contribute to the identification of novel targets for maintaining NPCs self-renewal in isoflurane-induced cognitive dysfunction.

NFAT transcription factors regulate survival, proliferation, migration, and differentiation of neural precursor cells.

The study of factors that regulate the survival, proliferation, and differentiation of neural precursor cells (NPCs) is essential to understand neural development as well as brain regeneration. The Nuclear Factor of Activated T Cells (NFAT) is a family of transcription factors that can affect these processes besides playing key roles during development, such as stimulating axonal growth in neurons, maturation of immune system cells, heart valve formation, and differentiation of skeletal muscle and bone. Interestingly, NFAT signaling can also promote cell differentiation in adults, participating in tissue regeneration. The goal of the present study is to evaluate the expression of NFAT isoforms in NPCs, and to investigate its possible role in NPC survival, proliferation, migration, and differentiation. Our findings indicate that NFAT proteins are active not only in neurogenic brain regions such as hippocampus and subventricular zone (SVZ), but also in cultured NPCs. The inhibition of NFAT activation with the peptide VIVIT reduced neurosphere size and cell density in NPC cultures by decreasing proliferation and increasing cell death. VIVIT also decreased NPC migration and differentiation of astrocytes and neurons from NPCs. In addition, we identified NFATc3 as a predominant NFAT isoform in NPC cultures, finding that a constitutively-active form of NFATc3 expressed by adenoviral infection reduces NPC proliferation, stimulates migration, and is a potent inducer of NPC differentiation into astrocytes and neurons. In summary, our work uncovers active roles for NFAT signaling in NPC survival, proliferation and differentiation, and highlights its therapeutic potential for tissue regeneration.

Neural Precursor Cells Cultured at Physiologically Relevant Oxygen Tensions Have a Survival Advantage Following Transplantation

Traditionally, in vitro stem cell systems have used oxygen tensions that are far removed from the in vivo situation. This is particularly true for the central nervous system, where oxygen (O2) levels range from 8% at the pia to 0.5% in the midbrain, whereas cells are usually cultured in a 20% O2 environment. Cell transplantation strategies therefore typically introduce a stress challenge at the time of transplantation as the cells are switched from 20% to 3% O2 (the average in adult organs). We have modeled the oxygen stress that occurs during transplantation, demonstrating that in vitro transfer of neonatal rat cortical neural precursor cells (NPCs) from a 20% to a 3% O2 environment results in significant cell death, whereas maintenance at 3% O2 is protective. This survival benefit translates to the in vivo environment, where culture of NPCs at 3% rather than 20% O2 approximately doubles survival in the immediate post-transplantation phase. Furthermore, NPC fate is affected by culture at low, physiological O2 tensions (3%), with particularly marked effects on the oligodendrocyte lineage, both in vitro and in vivo. We propose that careful consideration of physiological oxygen environments, and particularly changes in oxygen tension, has relevance for the practical approaches to cellular therapies.

Highly Pure and Expandable PSA-NCAM-Positive Neural Precursors from Human ESC and iPSC-Derived Neural Rosettes

Homogeneous culture of neural precursor cells (NPCs) derived from human pluripotent stem cells (hPSCs) would provide a powerful tool for biomedical applications. However, previous efforts to expand mechanically dissected neural rosettes for cultivation of NPCs remain concerns regarding non-neural cell contamination. In addition, several attempts to purify NPCs using cell surface markers have not demonstrated the expansion capability of the sorted cells. In the present study, we show that polysialic acid-neural cell adhesion molecule (PSA-NCAM) is detected in neural rosette cells derived from hPSCs, and employ PSA-NCAM as a marker for purifying expandable primitive NPCs from the neural rosettes. PSA-NCAM-positive NPCs (termed hNPCPSA-NCAM+) were isolated from the heterogeneous cell population of mechanically harvested neural rosettes using magnetic-based cell sorting. The hNPCPSA-NCAM+ extensively expressed neural markers such as Sox1, Sox2, Nestin, and Musashi-1 (80∼98% of the total cells) and were propagated for multiple passages while retaining their primitive characteristics in our culture condition. Interestingly, PSA-NCAM-negative cells largely exhibited characteristics of neural crest cells. The hNPCPSA-NCAM+ showed multipotency and responsiveness to instructive cues towards region-specific neuronal subtypes in vitro. When transplanted into the rat striatum, hNPCPSA-NCAM+ differentiated into neurons, astrocytes, and oligodendrocytes without particular signs of tumorigenesis. Furthermore, Ki67-positive proliferating cells and non-neural lineage cells were rarely detected in the grafts of hNPCPSA-NCAM+ compared to those of neural rosette cells. Our results suggest that PSA-NCAM-mediated cell isolation provides a highly expandable population of pure primitive NPCs from hPSCs that will lend themselves as a promising strategy for drug screening and cell therapy for neurodegenerative disorders.

Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation

The use of allogeneic fetal neural precursor cells (NPCs) as a cell replacement therapy in neurodegenerative disorders holds great promise. However, previous studies concerning the possibility of alloimmune rejection of the transplanted cells have been inconclusive. Here, we used flow cytometry to quantify the expression of major histocompatibility complex (MHC) molecules by human NPCs, obtained from the cortex or ventral mesencephalon of fetuses with gestational ages between 7 and 11 weeks. MHC class I was undetectable on the surface of freshly isolated primary fetal tissue from either location, but increased over time in proliferating NPC cultures; after 7 days in vitro, MHC class I was detectable on most cells. Following differentiation, MHC class I expression persisted on non-neuronal cells. MHC class II levels remained low at all time points but were inducible by pro-inflammatory cytokines, whereas the co-stimulatory molecules, CD80 and CD86, remained undetectable. Nonetheless, CD4+ and CD8+ T cells proliferated when peripheral blood mononuclear cells (PBMCs) were cultured with allogeneic NPCs. Weaker responses were obtained when NPCs were co-cultured with purified allogeneic responder T cells, suggesting that indirect allorecognition contributed significantly to PBMC responses. In conclusion, differentiating human NPCs are immunogenic in vitro, suggesting that they may trigger immune rejection unless transplant recipients are immunosuppressed.

Lysosome Dysfunction Triggers Atg7-dependent Neural Apoptosis

Macroautophagy (autophagy) is a process wherein bulk cytosolic proteins and damaged organelles are sequestered and degraded via the lysosome. Alterations in autophagy-associated proteins have been shown to cause neural tube closure defects, neurodegeneration, and tumor formation. Normal lysosome function is critical for autophagy completion and when altered may lead to an accumulation of autophagic vacuoles (AVs) and caspase activation. The tumor suppressor p53 is highly expressed in neural precursor cells (NPCs) and has an important role in the regulation of both autophagy and apoptosis. We hypothesized that altered lysosome function would lead to NPC death via an interaction between autophagy- and apoptosis-associated proteins. To test our hypothesis, we utilized FGF2-expanded NPCs and the neural stem cell line, C17.2, in combination with the lysosomotropic agent chloroquine (CQ) and the vacuolar ATPase inhibitor bafilomycin A1 (Baf A1). Both CQ and Baf A1 caused concentration- and time-dependent AV accumulation, p53 phosphorylation, increased damage regulator autophagy modulator levels, caspase-3 activation, and cell death. Short hairpin RNA knockdown of Atg7, but not Beclin1, expression significantly inhibited CQ- and Baf A1-induced cell death, indicating that Atg7 is an upstream mediator of lysosome dysfunction-induced cell death. Cell death and/or caspase-3 activation was also attenuated by protein synthesis inhibition, p53 deficiency, or Bax deficiency, indicating involvement of the intrinsic apoptotic death pathway. In contrast to lysosome dysfunction, starvation-induced AV accumulation was inhibited by either Atg7 or Beclin1 knockdown, and Atg7 knockdown had no effect on starvation-induced death. These findings indicate that Atg7- and Beclin1-induced autophagy plays a cytoprotective role during starvation but that Atg7 has a unique pro-apoptotic function in response to lysosome dysfunction.

Impact of lactic acid on cell proliferation and free radical‐induced cell death in monolayer cultures of neural precursor cells

Biomaterials prepared from polyesters of lactic acid and glycolic acid, or a mixture of the two, degrade in the presence of water into the naturally occurring metabolites, lactic acid and glycolic acid. While the lactic acid degradation product that is released from biomaterials is well tolerated by the body, lactic acid can influence the metabolic function of cells; it can serve as an energy substrate for cells, and has been shown to have antioxidant properties. Neural precursor cells, a cell population of considerable interest as a source of cells for neural tissue regeneration strategies, generate a high amount of reactive oxygen species, and when associated with a degradable biomaterial, may be impacted by released lactic acid. In this work, the effect of lactic acid on a neural cell population containing proliferative neural precursor cells was examined in monolayer culture. Lactic acid was found to scavenge exogenously added free radicals produced in the presence of either hydrogen peroxide or a photoinitiator (I2959) commonly utilized in the preparation of photopolymerizable biomaterials. In addition to its effect on exogenously added free radicals, lactic acid reduced intracellular redox state, increased the proliferation of the cell population, and modified the cell composition. The findings of this study provide insight into the role that lactic acid plays naturally on developing neural cells and are also of interest to biomaterials scientists that are focused on the development of degradable lactic-acid-based polymers for cell culture devices. The effect of lactic acid on other cell populations may differ and should be characterized to best understand how cells function in degradable cell culture devices.

Combined extrinsic and intrinsic manipulations exert complementary neuronal enrichment in embryonic rat neural precursor cultures: An in vitro and in vivo analysis

Numerous central nervous system (CNS) disorders share a common pathology in dysregulation of gamma-aminobutyric acid (GABA) inhibitory signaling. Transplantation of GABA-releasing cells at the site of disinhibition holds promise for alleviating disease symptoms with fewer side effects than traditional drug therapies. We manipulated fibroblast growth factor (FGF)-2 deprivation and mammalian achaete-scute homolog (MASH)1 transcription factor levels in an attempt to amplify the default GABAergic neuronal fate in cultured rat embryonic neural precursor cells (NPCs) for use in transplantation studies. Naïve and MASH1 lentivirus-transduced NPCs were maintained in FGF-2 or deprived of FGF-2 for varying lengths of time. Immunostaining and quantitative analysis showed that GABA- and beta-III-tubulin-immunoreactive cells generally decreased through successive passages, suggesting a loss of neurogenic potential in rat neurospheres expanded in vitro. However, FGF-2 deprivation resulted in a small, but significantly increased population of GABAergic cells derived from passaged neurospheres. In contrast to naïve and GFP lentivirus-transduced clones, MASH1 transduction resulted in increased bromodeoxyuridine (BrdU) incorporation and clonal colony size. Western blotting showed that MASH1 overexpression and FGF-2 deprivation additively increased beta-III-tubulin and decreased cyclic nucleotide phosphodiesterase (CNPase) expression, whereas FGF-2 deprivation alone attenuated glial fibrillary acidic protein (GFAP) expression. These results suggest that low FGF-2 signaling and MASH1 activity can operate in concert to enrich NPC cultures for a GABA neuronal phenotype. When transplanted into the adult rat spinal cord, this combination also yielded GABAergic neurons. These findings indicate that, even for successful utilization of the default GABAergic neuronal precursor fate, a combination of both extrinsic and intrinsic manipulations will likely be necessary to realize the full potential of NSC grafts in restoring function.

Enriched Monolayer Precursor Cell Cultures from Micro-Dissected Adult Mouse Dentate Gyrus Yield Functional Granule Cell-Like Neurons

BackgroundStem cell cultures are key tools of basic and applied research in Regenerative Medicine. In the adult mammalian brain, lifelong neurogenesis originating from local precursor cells occurs in the neurogenic regions of the hippocampal dentate gyrus. Despite widespread interest in adult hippocampal neurogenesis and the use of mouse models to study it, no protocol existed for adult murine long-term precursor cell cultures with hippocampus-specific differentiation potential.Methodology/Principal FindingsWe describe a new strategy to obtain serum-free monolayer cultures of neural precursor cells from microdissected dentate gyrus of adult mice. Neurons generated from these adherent hippocampal precursor cell cultures expressed the characteristic markers like transcription factor Prox1 and showed the TTX-sensitive sodium currents of mature granule cells in vivo. Similar to granule cells in vivo, treatment with kainic acid or brain derived neurotrophic factor (BDNF) elicited the expression of GABAergic markers, further supporting the correspondence between the in vitro and in vivo phenotype. When plated as single cells (in individual wells) or at lowest density for two to three consecutive generations, a subset of the cells showed self-renewal and gave rise to cells with properties of neurons, astrocytes and oligodendrocytes. The precursor cell fate was sensitive to culture conditions with their phenotype highly influenced by factors within the media (sonic hedgehog, BMP, LIF) and externally applied growth factors (EGF, FGF2, BDNF, and NT3).Conclusions/SignificanceWe report the conditions required to generate adult murine dentate gyrus precursor cell cultures and to analyze functional properties of precursor cells and their differentiated granule cell-like progeny in vitro.

Neural differentiation from human embryonic stem cells in a defined adherent culture condition

Understanding how to direct human embryonic stem cells (hESCs) toward a specific lineage pathway and generate appropriate cell types robustly is very important, not only for the study of developmental biology but also for potentially using these cells to treat human diseases. In this study, hESCs were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor. Our protocol seems to recapitulate the early steps of nervous system development in vivo in that undifferentiated hESCs organized into rosettes and then neural tube-like structures are formed. Differentiating cells expressed neuroectodermal and mature neuron markers during neural plate and tube formation and maturation, as shown by reverse transcriptase-PCR. More than 90% of differentiated cells expressed additional neuron-specific antigens (i.e., tubulin-III, MAP-2, synaptophysin and neurofilament protein). Ultrastructural analysis of differentiating neural tube-like structures in three dimensional collagen scaffolds showed an ependymal-like layer and neural structure with typical synapses. These results provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons with typical cellular, molecular and ultrastuctureal markers. The culture of neural precursor cells in a collagen scaffold may provide a new approach for the repair of spinal cord injury.

Neural differentiation from human embryonic stem cells in a defined adherent culture condition

Understanding how to direct human embryonic stem cells (hESCs) toward a specific lineage pathway and generate appropriate cell types robustly is very important, not only for the study of developmental biology but also for potentially using these cells to treat human diseases. In this study, hESCs were differentiated to the neural lineage in defined adherent culture by retinoic acid and basic fibroblast growth factor. Our protocol seems to recapitulate the early steps of nervous system development in vivo in that undifferentiated hESCs organized into rosettes and then neural tube-like structures are formed. Differentiating cells expressed neuroectodermal and mature neuron markers during neural plate and tube formation and maturation, as shown by reverse transcriptase-PCR. More than 90% of differentiated cells expressed additional neuron-specific antigens (i.e., tubulin-III, MAP-2, synaptophysin and neurofilament protein). Ultrastructural analysis of differentiating neural tube-like structures in three dimensional collagen scaffolds showed an ependymal-like layer and neural structure with typical synapses. These results provide a simple and relatively defined system for differentiation of hESCs to neural lineages, particularly neurons with typical cellular, molecular and ultrastuctureal markers. The culture of neural precursor cells in a collagen scaffold may provide a new approach for the repair of spinal cord injury.

SOCS3 negatively regulates LIF signaling in neural precursor cells

Cytokines that signal through the LIFRβ/gp130 receptor complex, including LIF and CNTF, promote the self-renewal of embryonic and adult neural precursor cells (NPCs). In non-CNS tissues, the protein suppressor of cytokine signaling-3 (SOCS3) negatively regulates signaling through gp130. Here, we analyze the role of SOCS3 in inhibiting LIF signaling in NPCs in vitro. SOCS3 is rapidly expressed by NPCs in response to LIF stimulation, with this expression largely dependent on recruitment of STAT proteins to the activated gp130 receptor. Proliferating NPC cultures can be generated from SOCS3 knockout (SOCS3 KO/KO) embryos and display prolonged STAT3 phosphorylation and induction of the GFAP gene in response to LIF. In comparison with SOCS3 wild-type (SOCS3 WT/WT) NPCs, SOCS3 KO/KO cultures display enhanced self-renewal capacity. However, the clonal potential of SOCS3 WT/WT but not SOCS3 KO/KO NPCs is enhanced by exogenous LIF. Thus, SOCS3 acts as a negative regulator of LIF signaling in NPCs.

Properties of cortical precursor cells cultured long term are similar to those of precursors at later developmental stages

In vitro cultures of neural precursor cells are useful experimental tools for studies on the mechanisms of brain development, as well as for generating renewable sources in cell therapy for neurodegenerative disorders. The systematic characterization of cultured neural precursors is a prerequisite for obtaining basic information on brain development. Here, we examine the cell survival, proliferation, and differentiation potential of cultured neural precursors from different embryonic ages and those of the precursors expanded in vitro for different periods of time. Precursor cells were isolated at rat embryonic days 14 (E14) and 19 (E19) and cultured in the presence of a mitogen basic fibroblast growth factor (bFGF). The numbers of TUNEL+ and BrdU+ cells in E19 cortical precursor cultures were significantly lower than those in E14 cultures, indicating that the programmed cell death and proliferation potential of neural precursors are reduced during the progression of brain development. E14 cells tended to differentiate into neurons, and E19 cells into astrocytes. To determine whether the intrinsic properties of neural precursors are similarly altered during in vitro culture, E14 precursor cells were expanded for different periods. Precursor cells expanded for longer periods displayed lower apoptotic and proliferation indices, as well as astrogenic developmental potential. Clonal analysis data confirmed the transition of precursor differentiation potential from neurogenic to astrogenic over the culture period. Our findings collectively suggest that neural precursor cells undergo time-dependent changes in properties via an intrinsic program, both in vivo and in vitro.

The Wnt/beta-catenin pathway directs neuronal differentiation of cortical neural precursor cells.

Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Here, we show that Wnt signaling inhibits the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of beta-catenin in mouse cortical NPC cultures induced neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling led to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the beta-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized beta-catenin did not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development.

Increased Activity and Involvement of Caspase-3 in Radiation-Induced Apoptosis in Neural Cells Precursors from Developing Rat Brain

Using primary cultures of neural precursor cells of cortex from developing rat brain, we demonstrated the involvement of caspase-3 in the apoptotic process induced by gamma irradiation. The precursor nature of cells was confirmed by nestin and GFAP immunoreactivity and by the capacity of differentiation in neuronal and glial cells after 5 days in culture. Neural precursors were irradiated with single doses ranging from 0.1 to 4 Gy. Cellular death, determined 24 h post-irradiation (pi) was dose-dependent and the induction of apoptosis was confirmed by nuclear condensation, DNA fragmentation and hypodiploid DNA peak represented by the “sub G1” region. For the higher doses, apoptosis was evident after 4–6 h pi and increased during 24 h. Caspase-3 activity increased with doses and was maximal at 4–6 h pi with 3 Gy and remained similar with 4 Gy. The protection from radiation-induced apoptosis by caspase-3 inhibitor, zDEVD-fmk, confirmed that this enzyme is involved in the apoptotic mechanism in this system. The possibility of using this tissue culture system for studying the effects of ionizing radiation on morphological and molecular differentiation was considered.

Neurotrophic factor in the treatment of Parkinson disease.

Parkinson disease (PD) is a well-known degenerative disease resulting in the depletion of dopamine-producing neurons in the pars compacta of the substantia nigra. Adenoviral vector delivery of neurotrophic factors may provide a potential therapy for PD. The authors examined whether glial cell line-derived neurotrophic factor (GDNF) delivered via adenoviral vector (Ad-GDNF) could promote functional recovery in a rat model of PD. Additionally, they examined whether neural precursor cells (NPCs) provide the therapeutic potential of cultured neural cells for cell regeneration and replacement in PD. All animals underwent stereotactic injection of 6-hydroxydopamine into the right substantia nigra. Eight weeks later, the rats were tested for apomorphine-induced rotational asymmetry and evaluation of explanted grafts infected with the complementary DNA for GDNF containing NPCs and NPCs alone. In the NPC cultures of embryonic rat striata, the authors found that basic fibroblast growth factor induced the proliferation of stem cells, which give rise to spheres of undifferentiated cells that generate neurons and glia. In this study the authors found that the reduction of apomorphine-induced rotation was more prominent in parkinsonian rats that received Ad-GDNF-treated grafts containing NPCs (61%) than in those that received grafts of NPCs alone (16%).

Telomerase activity in the subventricular zone of adult mice

The subventricular zone (SVZ) is the most active site for the production of new neurons in the adult mouse brain. Neural stem cells in the adult SVZ give rise to neuroblasts that travel via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into interneurons. The enzyme telomerase has been identified in other population of stem cells and is necessary for the synthesis of telomeric DNA to prevent chromosomal shortening, end-to-end fusions, and apoptosis during successive rounds of cell division. However, previous studies have failed to detect telomerase in the adult mammalian brain. Here we demonstrate that telomerase is expressed by all brain regions shortly after birth, but becomes restricted to the SVZ and olfactory bulb in the adult mouse brain. Cultures of neural precursor cells or of migratory neuroblasts purified from the SVZ were each found to possess telomerase activity. After elimination of migrating neuroblasts and immature precursor cells in vivo by treatment with cytosine-β- d-arabinofuranoside (Ara-C), telomerase activity was still detectable in the remaining SVZ, which includes a population of neural stem cells. Following withdrawal of Ara-C, telomerase activity subsequently increased with a time course that parallels regeneration of the SVZ network and RMS. Finally, intracranial surgery alone, which has previously been shown to increase the number of cells in the SVZ, produced higher telomerase levels in the SVZ. We conclude that telomerase is active in neural precursor cells of the adult mouse and suggest that its regulation is an important parameter for cellular proliferation to occur in the mammalian brain.

Astroglial differentiation from neuroepithelial precursor cells of amphibian embryos: an in vivo and in vitro analysis.

Initial development of astroglial phenotype has been studied in vitro in an amphibian embryo (Pleurodeles waltI), to document the differentiation potentialities acquired by neural precursor cells isolated at the early neurula stage. In particular, we sought to determine whether interactions between neuroepithelial cells and the inducing tissue, the chordamesoderm, are required beyond this stage to specify precursor cells along glial lineages. Glial cell differentiation was documented by examining the appearance of glial fibrillary acidic protein (GFAp), a specific marker of astroglial lineages. Cells expressing GFAp-immunoreactivity differentiated rapidly, after 48 hours of culture, from cultivated neural plate cells, irrespective of the presence or absence of the inducing tissue. The widespread expression of Pleurodeles GFAp protein in neural plate cultures, in which CNS precursor cells develop alone in a simple saline medium, showed that prolonged contact with chordamesodermal cells was not necessary for the emergence of the astroglial phenotype. In addition, the initial development of astroglial phenotype has been defined in vivo. The first detectable GFAp-immunoreactivity was visualized in the neural tube of stage-24 embryos, a stage corresponding to 2-3 days in culture, defining radial glial cell end-feet. Thus, dissociation and culture of neural precursor cells did not appear to modify the onset of astroglial differentiation. At stage 32, GFAp-immunoreactivity was observed over the entire length of radial glial fibers and was also evidenced in mitotic cells located in the ventricular zone, suggesting that radial glial cells were not all post-mitotic.