Stem Cells
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

First published online June 13, 2005
Stem Cells Vol. 23 No. 8 September 2005, pp. 1044 -1049
doi:10.1634/stemcells.2005-0037; www.StemCells.com
© 2005 AlphaMed Press

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0037v1
23/8/1044    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rowe, E. W.
Right arrow Articles by Jeftinija, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rowe, E. W.
Right arrow Articles by Jeftinija, S.

RAPID COMMUNICATIONS

Development of Functional Neurons from Postnatal Stem Cells In Vitro

Eric W. Rowe, Dusan M. Jeftinija, Ksenija Jeftinija, Srdija Jeftinija

Department of Biomedical Sciences, Iowa State University, Ames, Iowa, USA

Key Words. Adult stem cells • Astrocytes • Long-term cultures • Neural stem cell • Calcium transients

Correspondence: Srdija Jeftinija, D.V.M., Ph.D., Department of Biomedical Sciences, Iowa State University, 1098 Vet Med Bldg, Ames, Iowa 50011, USA. Telephone: 515-294-8494; Fax: 515-294-2315; e-mail: sjeftini{at}iastate.edu


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In order for stem cells to fulfill their clinical promise, we must understand their developmental transitions and it must be possible to control the differentiation of stem cells into specific cell fates. To understand the mechanism of the sequential restriction and multipotency of stem cells, we have established culture conditions that allow the differentiation of multipotential neural stem cells from postnatal stem cells. We used immunocytochemistry, fluorescence microscopy, and calcium imaging to demonstrate that progeny of adult rat neural stem cells develop into functional neurons that release excitatory neurotransmitters. We also found that the nontoxic heavy chain fragment of tetanus toxin, a toxin that targets neurons with high specificity, retained the specificity toward neural stem cell–derived neurons. These studies show that neural stem cells derived from adult tissues retain the potential to differentiate into functional neurons with morphological and functional properties of mature central nervous system neurons.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
In the past decade, there has been an explosion of research related to stem cells. This has led to the identification and isolation of adult stem cells from many organs, including the brain, spinal cord, bone marrow, liver, intestine, retina, skeletal muscle, pancreas, cornea, and skin [1]. Adult and embryonic stem cells differ from each other in certain aspects. Embryonic stem cells are derived from the inner cell mass of a blastocyst and are pluripotent, whereas adult stem cells are derived from differentiated organs and have varying degrees of plasticity [2]. Adult stem cells, like embryonic stem cells, are capable of self-renewal and can give rise to fully differentiated mature cell types [2].

Neural stem cells (NSCs) can generate cells of glial and neuronal lineages and have been identified in the hippocampus, sub-ventricular zone, olfactory bulb, and spinal cord of adult mammals [2, 3]. They are the object of increasing attention for their potential use in therapies of central nervous system (CNS) disorders [3–5]. Recent research demonstrates that fetal bovine serum (FBS) promotes differentiation of optic nerve oligodendrocyte precursor cells (OPCs) to a type 2 astrocyte, and when followed by exposure to basic fibroblast growth factor (bFGF), these cells revert to a state in which they cannot only self-renew but are capable of differentiating into oligodendrocytes, astrocytes, and neurons [6]. The possibility of neural progenitor contamination from other neurogenic regions is remote because the optic nerve was harvested rostral to the optic chiasm, eliminating concerns of any neuronal contamination.

OPCs are widely distributed in the CNS and constitute a major cycling population in the brain and spinal cord [7]. In some regions of the adult rat CNS, 70% of the dividing cells have been found to be OPCs [7]. In humans, OPCs comprise 3% of the cells in the subcortical white matter [8]. The widespread distribution of these cells and their potential to revert into a cell type that can give rise to all three major cell types of the CNS make them very appealing therapeutic candidates [9].

Before stem cells can be used successfully as replacement therapies for neurodegenerative disorders, we must understand the functional properties of these cells. For a stem cell–derived neuron to be considered functional, it must be stably differentiated, polarized showing a single axon and multiple dendrites, capable of generating an action potential, and not only be able to release neurotransmitters but also possess receptors for them [10]. In this paper, we examine whether cells that are differentiated from OPCs and display morphological characteristics of neurons also have functional properties similar to neurons from other brain regions in culture. Specifically, we demonstrate that NSC-derived neurons release glutamate in a calcium-dependent manner and express a set of glutamate receptors.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Stem Cell Cultures
As described previously [11–13], optic nerves were harvested rostral to the optic chiasm from Sprague-Dawley rats 21 days postnatal. The tissues were minced and digested in a solution of papain (0.01%) and DNase (0.01%) dissolved in Earl’s basal salt solution for 50 to 60 minutes at 37°C. Cell and tissue fragments were washed three times with Dulbecco’s modified Eagle’s medium (DMEM) containing 10% FBS. The tissues were disassociated with a glass pipette and maintained for 24 hours in high-glucose DMEM/F12 supplemented with 2.5 ml L-glutamine and 10% FBS in a tissue culture flask kept at 37°C and 5% CO2. After 24 hours, the medium was changed to a growing medium made up of DMEM/F-12 and 20 ng/ml bFGF. To compensate for instability of growth factors, the freshly prepared growing medium was changed every 2 days. The cells were split by gently washing the flask. Cells were seeded to poly-L-lysine–coated coverslips for 3–5 days before being used in experiments. In experiments in which the NSCs were differentiated, the medium consisted of DMEM/F-12, 1% FBS, all-transretinoic acid (100 ng/ml), bFGF (1 ng/ml), and 0.5 ml of penicillin and streptomycin per 50 ml. All of the cells that were used in experiments were in culture for at least 3 weeks.

Intracellular Calcium Imaging
Intracellular calcium concentrations ([Ca2+]i) were evaluated by ratiometric imaging techniques. Cells were loaded with Fura 2-AM for 40 to 60 minutes at room temperature. The loading solution contained 1 µl of 25% (wt/wt) Pluronic F-127 and 4 nM of Fura 2-AM diluted in 1 ml of HEPES buffer. The loading solution was removed and the culture was incubated another 10 minutes in HEPES-buffered solution to allow for deesterification of Fura 2-AM. The coverslips were then placed onto a perfusion chamber and connected to a micro pump with a flow rate of 200 µl per minute. The test substances were placed in syringes on a six-valve manifold and applied into the perfusion chamber by the same perfusion system. As a result of the spatial distance between the syringe and the culture in the chamber, there was a time delay between the turning on the valve and onset of the response.

All image processing and analysis was performed using an Attoflour system with an inverted microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com). Wavelengths of 340 and 380 nm were used to excite the Fura-2, and the emitted light was collected at 520 nm. Background subtraction and ratio images were used to calculate the [Ca2+]i according to Equation 5 by Grynkiewitz et al. [14].

Release Methods and High-Performance Liquid Chromatography
The release of excitatory amino acids was determined using the procedure previously described [15]. Briefly, the coverslips with NSC cultures were mounted into a 50-µl perfusion chamber and perfused at 200 µl per minute with normal HEPES buffer (in mM: NaCl 140, KCl 5, MgCl2 2, CaCl2 2, glucose 5, and HEPES 10; pH 7.4) for a period of 30–40 minutes to allow equilibration. After equilibration, 200-µl samples were collected every minute. Four control samples were collected for determination of basal concentrations of amino acids. Test substances were dissolved in the recording solution and delivered in known concentrations to the cultures. The amino acid content in the samples was determined by high-performance liquid chromatography (HPLC) with fluorescence detection. Before injection, aliquots of the samples were derivatized with o-phthalaldehyde 2-mercaptoethanol reagent (Pierce, Rockford, IL, http://www.piercenet.com). Chromatography was performed on a 15-cm Microsorb-MV HPLC column (Rainin Instrument Co., Oakland, CA, http://www.rainin.com) using a pH 5.9 sodium acetate methanol gradient. Basal rates of amino acids released were determined as the mean of the amino acids in four samples collected just before stimulation.

Immunocytochemistry
Immunocytochemistry was performed using monoclonal antibodies raised against glial fibrilary acidic protein (1:5,000; Valeant, Costa Mesa, CA, http://www.valeant.com/index.jspf) and microtubule-associated protein 2 (MAP2) (1:2,000; Boehringer Ingelheim, Ingelheim, Germany, http://www.boehringer-ingelheim.com/corporate/home/home.asp) and polyclonal antibody raised against synaptotegmin (1:5,000; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). Inactivation of endogenous hydrogen peroxidase was achieved by rinsing the culture for 15 minutes in 0.3% H2O2 in 50-mM potassium phosphate–buffered saline. Normal horse serum (1:1,000, for monoclonal antibodies; Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and normal goat serum (1:1,000, for polyclonal antibody; Vector Laboratories) were used to block endogenous antibodies. Primary antisera was diluted at above dilution and incubated overnight at room temperature. Secondary antibodies for monoclonal primaries (horse anti-mouse, 1:10,000; Vector Laboratories) and polyclonal primaries (goat anti-rabbit; 1:10,000; Vector Laboratories) and ABC kit were applied at room temperature using dilutions recommended by the supplier (Vectastain, Vector Laboratories). Negative controls were processed by omitting the specific antiserum.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Morphological Characterization of NSCs
To determine whether there were progenitors in the optic nerve with a latent ability to generate neurons, the optic nerve was harvested, dissociated, and fractionated. As described by Kondo and Raff [6], short-term exposure of acutely dissociated OPCs to FBS followed by culturing in serum-free medium with bFGF predictably resulted in cells expressing neuronal markers. In contrast, less than 5% of cells cultured in platelet-derived growth factor or FBS for 1 month or longer were MAP2-positive [6]. These results demonstrate that OPCs cultured sequentially in FBS and bFGF will generate NSCs as a result of treatment rather than the effect of time in culture. Using this protocol, but in the tissue culture flasks without poly-L-lysine coating, the cells proliferated in bFGF and created neurosphere bodies for many weeks and months. After several weeks in culture, a small but significant minority of cells were MAP2-positive neurons when induced to differentiate. Neurons were often found in small clusters, suggesting a clonal derivation. Figure 1AGo illustrates MAP2 antibody–stained cells that were exposed to differentiation media, indicating that the cells were neurons and that few if any non-neuronal cells were present. The high percentage of MAP2-positive cells grown under these conditions is consistent with the findings of Kondo and Raff [6]. In addition to expressing neuronal microtubule marker, most of the cells (more than 90%) reacted with secretory protein synaptotegmin antibody (Fig. 1BGo). Most of the synaptotegmin immunoproduct labeling was confined to the cell bodies of the differentiated cells, which is a characteristic of young neurons. The fact that these neurons were generated from cells isolated from the optic nerve dispels any concerns of contamination from other known neurogenic zones within the CNS.



View larger version (98K):
[in this window]
[in a new window]
  		Figure 1. Neuronal marker expression in optic nerve–derived NSCs cultured for 6 months. (A): Ninety percent of the cells grown in differentiation media show immunoreactivity for MAP2 antibody. Both individual and cell clusters were stained. (B): In addition to expressing MAP2, more than 90% of the cells reacted with secretory protein synaptotagmin. Note the higher concentration of immunoproduct in the cell body, which is characteristic of young neurons. Arrowheads indicate cells outside the cluster that expressed immunoproduct in bodies only. (C): Fluorescein isothiocyanate–labeled heavy chain tetanus toxin has the same tropism for the NSC-differentiated neurons as it does for neurons. Scale bar = 20 µm. Abbreviations: MAP2, microtubule-associated protein 2; NSC, neural stem cell.

 
To further assess the morphological characteristics of neurons derived from NSCs, we exposed these cells to the heavy chain (HC) component of tetanus toxin. The C-terminal portion of the HC is responsible for the neurospecificity of tetanus toxin. Recent studies demonstrated that the HC of the tetanus toxin is a multivalent oligosaccharide-binding protein, and the sugar-binding site in the C-terminal domain ensures specific binding of tetanus toxin to carbohydrate-containing receptors on the neuronal membrane (for review, see [16]). Differentiated NSCs were exposed to the HC component of tetanus toxin that was labeled with fluorescein isothiocyanate (FITC) (Fig. 1CGo). The cultures were then examined using confocal microscopy, and more than 90% of the cells in culture were FITC-labeled. This demonstrates that the tetanus toxin has a high affinity for the differentiated NSC neurons and further illustrates that these cells are morphologically similar to neurons.

Functional Identification of NSCs
Several independent criteria were used to functionally identify NSCs as cells that possess functional properties similar to those of functional neurons. These included the presence of voltage-gated calcium channels, ligand-gated calcium channels, and ionotropic glutamate AMPA ({alpha}-amino-3-hydroxy-5-methyl isoxazole propionic acid) receptors. Coverslips containing NSCs were mounted onto a fast-rate exchange perfusion chamber for imaging experiments. Fura-2 calcium imaging was used to examine the stimulatory effects of a nonselective depolarizing stimulus, 50 mM K+, and two neurotransmitters, ATP and AMPA.

To determine the presence of voltage-gated calcium channels in cultured cells, 50 mM K+ was used. Elevated potassium concentrations are commonly used to functionally identify neurons [17]. In resting conditions, the cytoplasmic calcium level of NSCs was 91 ± 2 nM (n = 201). Brief perfusion application of 50 mM K+ (2 minutes) produced an increase in the level of calcium in 97% of the cells studied (n = 194; Fig. 2Go). This increase of intracellular calcium reached the peak level of 207 ± 7 nM (n = 189; Fig. 2Go) approximately 80 seconds (mechanical delay) after the initiation of 50 mM K+ application and was sustained for several minutes. Removal of external calcium from the bathing medium abolished the potassium-induced calcium transients in NSC-derived neurons (Fig. 3Go), indicating that the increase in intracellular calcium was dependent on external calcium sources. It has been well documented that perfusion application of 50 mM K+ on astrocytes was without effect on intracellular calcium concentration [15, 17]. These data demonstrate that NSC-derived neurons express voltage-dependent calcium channels similar to those of functional neurons.



View larger version (28K):
[in this window]
[in a new window]
  		Figure 2. Kinetic changes of [Ca2+]i and glutamate release in differentiated neural stem cells (NSCs) in response to perfusion application of 50 mM K+ and ATP. (A): Perfusion application of 50 mM K+ and 100 µM ATP in the presence of low Ca2+ was without effect. (B): The increase in intracellular calcium induced by perfusion application of ATP coincided with the increase in the release of glutamate from differentiated NSCs.

 


View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. Kinetic changes of [Ca2+]i in a mixed neural stem cell astrocyte culture in response to perfusion application of 50 mM K+ and 100 µM ATP. The group of cells in the upper set of graphs responded to both K+ and ATP under normal conditions but failed to respond in the presence of a low extracellular calcium solution. This type of response is characteristic of neurons. The cell kinetic tracing in the lower set of graphs failed to respond to 50 mM K+ under both conditions but responded to ATP, which is characteristic of an astrocyte. The separation of the graphs is done artificially using software to make the effect more obvious.

 
Activation of purinoceptors leads to an increase in intracellular calcium in a variety of excitable and unexcitable cells [18]. Two mechanisms are involved in ATP-evoked intracellular calcium increase. First, ATP can activate ATP-gated cation channels [19]. This first mechanism is consistent with the P2X subtype of receptor [20]. Second, extracellular ATP can stimulate the breakdown of inositol phospholipids, and the resulting increase in inositol 1,4,5,-triphosphate (IP3) is responsible for the elevation of cytosolic Ca2+ [15]. This mechanism is coupled to the P2Y type of receptor. Astrocytes have a relatively small population of P2X receptors but a large population of P2Y receptors that mediate increases in intracellular calcium [15, 20]. Neurons also display both the P2X and P2Y receptor, but the P2Y receptor mediates slow changes in membrane potential [20]. This evidence prompted us to use ATP in combination with low extracellular calcium to functionally discriminate astrocyte-like from neuron-like cells.

Figure 2Go illustrates a typical response of differentiated NSC cultures to perfusion application of 50 mM K+ followed by perfusion application of 100 µM ATP for 1 minute. Application of ATP induced an increase in calcium level in 83% of cells (180 of 216 cells in eight experiments), and this effect was completely abolished in 87% of the cells bathed in low calcium (121 of 139 cells, six independent cultures). This finding strongly suggests that the differentiated cells have a P2X type of purinergic receptor that is consistent with neurons and that few non-neuronal cells are present.

To further assess the functional characteristics of differentiated cells, we investigated whether these cells were capable of releasing glutamate in response to ATP stimulation. The release of glutamate from NSC cultures was assayed using HPLC on the superfusate. The basal release of glutamate into the superfusate was 19 ± 2 nM (p < .01). Addition of 100 µM ATP caused an increase in release of glutamate from differentiated NSC cultures to 30 ± 7 nM (p < .01) (Fig. 2BGo).

To confirm that optic nerve–derived stem cells were mainly of neuronal phenotype, we plated NSCs onto established cortical astrocyte cultures and stimulated them with potassium and ATP. As can be seen in Figure 3Go, there were two kinds of cells. One group of cells responded to potassium and ATP in normal calcium but failed to respond to either in low calcium (Fig. 3Go; Ca2+ transients of cells in upper set of tracings). The second group of cells did not respond to potassium but responded to ATP in both normal (2 mM) calcium and low (26 nM) calcium HEPES buffer (Fig. 3Go; lower set of tracings). For functional identification, the cells in upper tracings were identified as neuron-like cells and lower tracing cells were identified as astrocytes.

Fast excitatory transmission between the neurons of the CNS occurs when glutamate directly activates AMPA and kainate receptors. AMPA receptors lacking the GluR2 subunit are permeable to Ca2+ [8]. To further functionally characterize NSCs, we applied 10 µM AMPA for 1 minute. Figure 4Go illustrates the typical response of differentiated NSCs to applications of potassium and AMPA. Stimulatory effects of AMPA were abolished in low calcium, indicating that AMPA receptors in NSCs are permeable to extracellular Ca2+. Perfusion applications of potassium and 10 µM AMPA were without effect on enriched cortical astrocyte cultures (Fig. 4BGo).



View larger version (28K):
[in this window]
[in a new window]
  		Figure 4. Kinetic changes of [Ca2+]i in a neural stem cell culture and enriched astrocyte culture in response to perfusion application of 50 mM K+ and 10 µM AMPA ({alpha}-amino-3-hydroxy-5-methyl isoxazole propionic acid). (A): The AMPA response is abolished in low-calcium HEPES, indicating that the increase in [Ca2+]i is dependent on extracellular calcium. (B): Both 50 mM K and AMPA failed to induce calcium increase in enriched astrocyte culture, whereas cells responded to application of ATP in low calcium.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our data demonstrate that rat OPCs grown under the proper conditions are capable of differentiating into cells that have both morphological and functional characteristics similar to neurons. The morphological findings are consistent with the observations of Kondo and Raff [6] and Nunes et al. [8]. Our findings that differentiated cells respond to both depolarizing stimuli (50 mM K) and glutamate receptor agonist (AMPA) are consistent with the previously described work, showing that a large percentage of NSCs have voltage-dependent Ca2+ channels and widespread presence of glutamate receptors [8]. In addition, we demonstrated that the differentiated cells have ATP receptors that are ligand-gated Ca2+ channels, as indicated by the lack of increase in [Ca2+]i in the presence of a low Ca2+ extracellular environment.

Functional differentiation of neurons is a critical first step in assessing their potential for therapeutic use. Part of the criteria used to assess neuronal functional differentiation includes not only the ability to respond to neurotransmitters but also the ability to release them [10]. To do this, we demonstrated that these cells have immunoreactivity to synaptotegmin, which indicates that at least part of the machinery necessary for synaptic release is present. The ability of differentiated cells to release glutamate in response to ATP exposure was confirmed by measuring the glutamate levels in the superperfusate using HPLC analysis. This further demonstrates that these differentiated cells have functional capabilities similar to those of neurons. To our knowledge, this is the first demonstration that neurons derived from OPCs are capable of releasing glutamate.

This is an example of a glial-restricted progenitor cell taking on the characteristics of a neural stem cell. This raises the question of cell plasticity and transdifferentiation [21, 22]. In theory, this is a population of somewhat restricted cells, but with the proper external cues, they are capable of breaking out of their glial progenitor niche and generating cells with both the morphological and functional characteristics of neurons. These data suggest that environmental cues are important in maintaining and determining the fate of progenitor cells and that it is not solely due to the lineage commitment of the cell [22–24]. It is known that cells in culture for periods of time have the potential for mutation, which would allow for more plasticity in vitro than in vivo [25]. However, it has been shown that progenitor cells acutely isolated from non-neurogenic regions can generate neurons [25]. These results could also be explained by the isolation of another, yet unidentified, neural stem cell that was co-isolated with the OPC.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Kirschstein R, Skirboll L. Stem Cells: Scientific Progress and Future Research Direction. Washington, DC: Department of Health and Human Services, 2001:23–42.

  2. Gage FH. Mammalian neural stem cells. Science 2000;287:1433–1438.[Abstract/Free Full Text]

  3. Temple S. The development of neural stem cells. Nature 2001;414:112–117.[CrossRef][Medline]

  4. Shihabuddin LS, Ray J, Gage FH. Stem cell technology for basic science and clinical applications. Arch Neurol 1999;56:29–32.[Free Full Text]

  5. Rossi F, Cattaneo E. Neural stem cell therapy for neurological diseases: dreams and reality. Nat Rev Neurosci 2002;3:401–409.[CrossRef][Medline]

  6. Kondo T, Raff M. Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 2000;289:1754–1757.[Abstract/Free Full Text]

  7. Dawson MRL, Polito A, Levine JM et al. NG2-epressing glial progenitor cells: an abundant and widespread population of cycling cells in the adult rat CNS. Mol Cell Neurosci 2003;24:476–488.[CrossRef][Medline]

  8. Nunes MC, Roy NS, Keyoung M et al. Identification and isolation of multipotential neural progenitor cells from the subcortical white matter of the adult human brain. Nat Med 2003;9:439–447.[CrossRef][Medline]

  9. Kondo T, Raff M. Chromatin remodeling and histone modification in the conversion of oligodendrocyte precursors to neural stem cells. Genes Dev 2004;18:2963–2972.[Abstract/Free Full Text]

  10. Reh TA. Neural stem cells: form and function. Nat Neurosci 2002;5:392–394.[CrossRef][Medline]

  11. Gage FH, Coates PW, Palmer TD et al. Survival and differentiation of adult neuronal progenitor cells transplanted to the adult brain. Proc Natl Acad Sci U S A 1995;92:11879–11883.[Abstract/Free Full Text]

  12. Palmer TD, Takahashi J, Gage FH. The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 1997;8:389–404.[CrossRef][Medline]

  13. Ahlgren SC, Wallace H, Bishop J et al. Effects of thyroid hormone on embryonic oligodendrocyte precursor cell development in vivo and in vitro. Mol Cell Neurosci 1997;9:420–432.[CrossRef][Medline]

  14. Grynkiewitz G, Poenie M, Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 1985;260:3440–3450.[Abstract/Free Full Text]

  15. Jeremic A, Jeftinija K, Stevanovic J et al. ATP stimulates calcium-dependent glutamate release from cultured astrocytes. J Neurochem 2001;77:664–675.[CrossRef][Medline]

  16. Lalli G, Bohnert S, Deinhardt K et al. The journey of tetanus and botulinum neurotoxins in neurons. Trends Microbiol 2003;11:431–437.[CrossRef][Medline]

  17. Bezzi P, Carmignoto G, Pasti L et al. Prostaglandins stimulate calcium-dependent glutamate release in astrocytes. Nature 1998;391:281–285.[CrossRef][Medline]

  18. O’Connor SE, Dainty IA, Leff P. Further subclassification of ATP receptors based on agonist studies. Trends Pharmacol Sci 1991;12:137–141.[CrossRef][Medline]

  19. Benham CD, Tsien RW. A novel receptor-operated Ca2+-permeable channel activated by ATP in smooth muscle. Nature 1987;328:275–278.[CrossRef][Medline]

  20. Illes P, Ribeiro JA. Neuronal P2 receptors of the central nervous system. Curr Top Med Chem 2004;4:831–838.[Medline]

  21. Weissman I, Anderson DJ, Gage F. Stem and progenitor cells: origins, phenotypes, lineage commitments, and transdifferentiations. Annu Rev Cell Dev Biol 2001;17:387–403.[CrossRef][Medline]

  22. Goldman S. Glia as neural progenitor cells. Trends Neurosci 2003;26:590–596.[CrossRef][Medline]

  23. Song J, Stevens C, Gage F. Neural stem cells from adult hippocampus develop essential properties of functional CNS neurons. Nat Neurosci 2002;5:438–445.[Medline]

  24. Song J, Stevens C, Gage F. Astroglia induce neurogenesis from adult neural stem cells. Nature 2002;417:39–44.[CrossRef][Medline]

  25. Palmer TD, Markakis EA, Willhoite AR et al. Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 1999;19:8487–8497.[Abstract/Free Full Text]

Received on January 27, 2005; accepted for publication on June 9, 2005.




This article has been cited by other articles:


Home page
 Proc. Natl. Acad. Sci. USAHome page
J.-Y. Sul, C.-w. K. Wu, F. Zeng, J. Jochems, M. T. Lee, T. K. Kim, T. Peritz, P. Buckley, D. J. Cappelleri, M. Maronski, et al.
Transcriptome transfer produces a predictable cellular phenotype
PNAS, May 5, 2009; 106(18): 7624 - 7629.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2005-0037v1
23/8/1044    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Reprints/Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rowe, E. W.
Right arrow Articles by Jeftinija, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rowe, E. W.
Right arrow Articles by Jeftinija, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
STEM CELLS THE ONCOLOGIST CME ALPHAMED PRESS JOURNALS