ARTICLE |
Correspondence to: Eulalia Bazán, Servicio de Neurobiología, Departamento de Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar Km 9, 28034 Madrid, Spain. E-mail: eulalia.bazan@hrc.es
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Neural stem cells proliferate in liquid culture as cell clusters (neurospheres). This study was undertaken to characterize the epidermal growth factor (EGF)-expanded free-floating neurospheres derived from rat fetal striatum. We examined the ultrastructural and antigenic characteristics of these spheres. They consisted of two cell types, electron-dense and electron-lucent cells. Lucent cells were immunopositive to actin, vimentin, and nestin, whereas dense cells were immunopositive to actin, weakly positive to vimentin, and nestin-negative. Neurospheres contained healthy, apoptotic, and necrotic cells. Healthy cells were attached to each other by adherens junctions. They showed many pseudopodia and occasionally a single cilium. Sphere cells showed phagocytic capability because healthy cells phagocytosed the cell debris derived from dead cells in a particular process that involves the engulfment of dying cells by cell processes from healthy cells. Sphere cells showed a cytoplasmic and a nuclear pool of fibroblast growth factor (FGF) receptors. They expressed E- and N-cadherin, - and ß-catenin, EGF receptor, and a specific subset of FGF receptors. Because sphere cells expressed this factor in the absence of exogenous FGF-2, we propose that they are able to synthesize FGF-2.
(J Histochem Cytochem 51:89103, 2003)
Key Words: stem cells, progenitor cells, ultrastructure, growth factors, cell markers, adhesion molecules, nestin
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
A NUMBER OF STUDIES have analyzed the origin and development of neural stem cells, which are clonogenic cells with self-renewal and multipotentiality. Fundamental problems, such as their localization in the central nervous system, characterization, lineage restriction, and differentiation, have been studied (reviewed in and -ß), and tumor necrosis factor-
(TNF-
)], cell adhesion molecules (E-, N- and P-cadherin,
-, ß-, and
-catenin), cytoskeletal proteins and cell-specific markers [actin, vimentin, desmin, nestin, glial fibrillary acidic protein (GFAP), ß-tubulin III, neuron-specific nuclear protein (NeuN), and A2B5] was also addressed to further characterize these cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Primary Antisera
Polyclonal antibodies against EGFR, FGFR-1, -2, -3, FGF-2, TGF-ß, TNF-, E (epithelial)-, N (neural)-, and P (placental)-cadherin,
-, ß-, and
-catenin (also termed plakoglobin) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA) and the polyclonal anti-GABA antibody was obtained from Chemicon International (Temecula, CA). The monoclonal antibodies used here were anti-nestin (clone rat 401; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), ß-tubulin isotype III and smooth muscle
-actin (both from Sigma; St Louis, MO), actin and vimentin (both from Amersham; Poole, UK), desmin and tyrosine hydroxylase (both from BoehringerMannheim; Mannheim, Germany), GFAP (Dako; Glostrup, Denmark), TGF-
(Santa Cruz Biotechnology), NeuN (Chemicon), and A2B5 from hybridoma supernatant. These antibodies have previously been utilized for immunocytochemical and immunoblotting studies and have been extensively characterized to confirm their specificity (
Cell Cultures
Striata from E15 SpragueDawley rat fetuses were dissected and mechanically dissociated. Cell suspensions were cultured as previously described (
Light Microscopy
After six passages, neurosphere cells were plated at a density of 20,00030,000 cells/cm2 on poly-L-ornithine-coated glass coverslips. Coverslips were maintained in DF12 with EGF for 3 days and then switched to DF12 without EGF for longer culture periods. Immunocytochemical studies were carried out at 2 hr, 3, 6, and 10 days post plating (dpp). Cells were fixed with 4% paraformaldehyde or 2% paraformaldehyde0.5% glutaraldehyde for 10 min and immunostained to detect differentiation into neurons and glia as described in
Electron Microscopy
The cells used for electron microscopic analysis were EGF-expanded over six passages. Free-floating neurospheres were fixed in situ with 2.5% paraformaldehyde0.5% glutaraldehyde in PBS for 2 hr at 4C. After rinsing in PBS, some cells were postfixed for 30 min with 1% osmium tetroxide in PBS, dehydrated through graded ethanol series, en bloc-stained during dehydration with a saturated solution of uranyl acetate in 70% ethanol, and embedded in Araldite for the ultrastructural study of cells. For immunocytochemical studies, cells were fixed as described above, dehydrated through graded ethanol series and embedded in Lowicryl K4M. Semithin sections (1 µm thick) were stained with 1% toluidine blue and examined under a light microscope. Immunogold assays were performed as previously described (
Western Blotting Protein Analysis
Additional cell cultures (sixth passage floating neurospheres and cultures at 3 dpp) were homogenized in 0.5 M Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 12 mM 2-mercaptoethanol, 1 mM benzamidine, 0.5% NP-40, and 1 mM phenylmethylsulfonyl fluoride. Homogenates were centrifuged at 10,000 x g for 30 min. Aliquots of 20 µg protein were separated by electrophoresis in 6%, 9%, or 15% SDS-polyacrylamide minigels and transferred to nitrocellulose filters. The filters were soaked in blocking solution (5% Blotto0.05% Tween-20 in TBS, pH 7.4) for 2 hr at 37C and then incubated with the primary antibodies diluted in the same blocking solution: anti-E-, N-, and P-cadherin, -, ß-, and
-catenin, FGFR-1, -2, -3, EGFR, FGF-2, TGF-
and -ß, TNF-
, actin, smooth muscle
-actin, vimentin, desmin, nestin, GFAP, and NeuN. After extensive washing with TBSTween 20, the filters were incubated with the peroxidase-labeled second antibodies (Chemicon) diluted 1:5000 in blocking solution. The filters were developed with an enhanced chemiluminescence (ECL) Western blotting analysis, following the procedure described by the manufacturer (Amersham). Three different Western blot analyses per sample and primary antibody were carried out.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sphere Cell Differentiation
Cells were isolated from rat fetal striatum and propagated in vitro. After six passages, floating neurospheres (Fig 1A1E) were seeded onto adherent substrate and treated with EGF for 3 more days to enhance proliferation of cells. Afterwards the mitogen was withdrawn and cells grew in a defined medium, which promoted the differentiation process. At 2 hr after plating, most cells in undifferentiated spheres were immunopositive to nestin (Fig 1F) and EGFR (Fig 1G) and were shown to incorporate BrdU, indicative of cell division. However, a few neurosphere cells were immunopositive to GFAP (a marker of astrocytes) and A2B5 (a marker of early glial precursors). At 3 dpp, most cells were stained for nestin and a small percentage of cells showed ß-tubulin III immunoreactivity and a clear neuronal morphology. Between 6 and 10 dpp, the percentage of nestin-positive cells decreased and the cell population immunopositive to ß-tubulin III and GFAP increased, as previously described (
|
Ultrastructural Characterization of Floating Spheres
Free-floating spheres were EGF-expanded over six passages and characterized by conventional electron microscopy (Fig 2 and Fig 3A3F) and immunocytochemical methods (Fig 3G, Fig 3H, Fig 4, and Fig 5). Floating neurospheres showed a round or oval morphology (Fig 1A) and were formed by a variable number of cells (between four or five and several hundred cells). Neurospheres had a noncompact cytoarchitecture with large intercellular spaces (Fig 1B and Fig 2). Cells were randomly distributed within these aggregates, with no apparent organization. None of the cells we studied had ultrastructural features of neurons, glial, or ependymal cells. Although they showed a variable appearance in both morphology and size, two subpopulations of cells were distinguished: dark and light cells (Fig 2A). The proportion of both cell types varied in the different neurospheres. Light cells showed lower electron density and had larger mitochondria than dark cells (Fig 2A). Light cells also showed intermediate filaments (1012 nm thick) that were randomly distributed (Fig 3D, Fig 3E, and Fig 4E) and arranged in bundles (Fig 3B and Fig 5A), whereas dark cells did not have filament bundles. Light cells also showed cytoplasmic areas without ribosomes where many scattered filaments accumulated. These areas were surrounded by ribosomes, the Golgi complex, rough endoplasmic reticulum, and mitochondria (Fig 3D and Fig 3E). Occasionally, electron-dense cells showed some scattered filaments, in lower amounts than light cells. All other ultrastructural features were similar for both cell types. Moreover, cells in the different stages of mitotic division (Fig 2F), in interphase (Fig 2A and Fig 2H), and in the different phases of the apoptotic and necrotic processes (Fig 2B2E) were observed. Healthy light and dark cells were irregularly shaped and contained many free ribosomes and mitochondria (Fig 2A). The rough and smooth endoplasmic reticula were moderately developed. The Golgi complex was located next to the nucleus and the Golgi stacks were small and included three to five cisternae (Fig 3D and Fig 3E). Some of these cells showed centrioles and about 12% of the cells presented a single cilium (over 870 cells were examined). Each cilium (Fig 3G) consisted of the basal body, the ciliary roots, and the free part (about 2.1 µm long and 0.10.2 µm thick). Both coated and uncoated vesicles were often observed in these cells. They had large nuclei that frequently showed deep indentations of the nuclear envelope (Fig 2E). These nuclei exhibited a dispersed chromatin pattern with small aggregates of condensed chromatin masses along the nuclear envelope and in the form of inner patches (Fig 2). Light and dark cells had one or two large nucleoli, with a reticulated structural configuration (Fig 2F and Fig 3C). Healthy cells were attached to each other by adherens junctions 64120 nm in length (Fig 3F). At the adherens junctions, cell membranes ran parallel and were separated by a gap of 1020 nm. These junctions showed a characteristic 10-nm-thick, electron-dense reinforcement at the cytoplasmic sides of the apposed plasma membranes (Fig 3F). Desmosomes, gap junctions, and tight junctions were not observed. No cell junctions were found between healthy cells and apoptotic/necrotic cells. With the light microscope and toluidine blue staining, neurosphere cells showed many pseudopodia or cilium-like structures (Fig 1C). With the electron microscope, these structures were identified as cell processes from healthy cells that spread through the intercellular space (Fig 3A). These processes showed a variable morphology both in width and length (compare the cell processes shown in Fig 2H and Fig 3A). They usually contained ribosomes and cytoskeletal filaments. The thicker processes also contained mitochondria and rough endoplasmic reticulum (Fig 2H). Furthermore, apoptotic and necrotic cells were totally engulfed by cell processes of healthy cells (Fig 1D, Fig 1E, and Fig 2B2E).
|
|
|
|
Because ultrastructural characterization remains the most reliable method for identifying apoptosis and necrosis, we analyzed the sequence of ultrastructural changes that accompany cell death in sphere cells (Fig 2). Early apoptosis was characterized by segregation and condensation of the chromatin in sharply delineated masses that abut on the nuclear envelope (Fig 2B and Fig 2C). The increase in cytoplasmic electron density that usually accompanies apoptosis was not evident in apoptotic sphere cells (compare the apoptotic cell shown in Fig 2B with adjacent healthy cells). The later apoptotic phase, characterized by the formation of apoptotic bodies, was occasionally observed. Finally, apoptotic bodies were not phagocytosed by nearby cells. Instead, they underwent degenerative changes similar to those found in necrosis and to those described in apoptotic bodies that escape phagocytosis in tissues and cultures (
Expression of Growth Factors and Their Receptors
FGF-2 is a heparin-binding polypeptide that exerts mitogenic effects on stem cells and functions through FGFRs with intrinsic tyrosine kinase activity. We have previously studied the expression of FGFR-1, -2, and -3 in the progeny of neural stem cells (
As a consequence of alternative mRNA splicing, a number of FGFR isoforms have been described for FGFR-1, -2, and -3, which are specific for each tissue or cell type ( and -ß, and to TNF-
did not recognize any band in both lysates (not shown).
|
Expression of Cytoskeletal Components and Cell-specific Markers
The electron microscopic study revealed that sphere cells were immunonegative for neuronal (ß-tubulin III, NeuN) and astrocyte (GFAP) markers (not shown), and immunopositive for vimentin. However, dark cells differ from light cells in the intensity of the immunoreaction to vimentin and in the distribution of this cytoskeletal protein. In this sense, dark cells showed a weak immunoreaction that was randomly distributed through the cytoplasm (Fig 4D) and light cells showed an intense vimentin immunoreaction that was found in scattered filaments (Fig 4E) and in filament bundles (Fig 5A). Surprisingly, light cells were immunostained for nestin (Fig 5B), whereas dark cells were immunonegative (Fig 5C). In light cells, nestin was mainly found in scattered short filaments and at random in the cytoplasm (Fig 5B). Both cell types were immunoreactive for actin that was mainly distributed in cytoplasmic processes and all along the cell periphery, adjacent to the plasma membrane (Fig 5D and Fig 5E). No dark cell was stained for A2B5, and a small proportion of light cells (about 12%) showed a weak immunoreaction to A2B5. In these cells, some isolated gold particles were found in the plasma membrane and in the adjacent cytoplasm (Fig 5F).
No immunostaining appeared in negative controls (Fig 5G). For all the antibodies, background staining was very low. Therefore, the labeling density in the resin outside the cell, in mitochondria, and in the cell nuclei for the antibodies against cytoskeletal proteins was extremely low in all cases (Fig 3G, Fig 3H, Fig 4, and Fig 5).
The results of Western blotting analyses (Fig 7) showed a single band at the corresponding molecular weight of 45 kD for actin in both lysates (spheres and cultures at 3 dpp). The anti-vimentin antibody recognized a band of 55 kD in both lysates and a weak band of lower molecular weight in cells at 3 dpp (Fig 7). Immunoblotting analyses were able to detect low levels of GFAP in floating spheres and higher levels in cultures at 3 dpp (Fig 7). These results indicate that GFAP may be present in some neurosphere cells but at low levels that are undetectable by immunocytochemistry. The anti-nestin antibody recognized bands of 240 and 220 kD in neurospheres and in cultures at 3 dpp (Fig 7). The anti-NeuN antibody did not stain any band in neurospheres, but it recognized two bands of approximately 48 and 60 kD in cultures at 3 dpp (Fig 7). No immunoreactive bands for smooth muscle -actin and desmin were observed in both lysates (not shown).
|
Expression of Cell Adhesion Molecules
Homotypic cellcell adhesion molecules (which hold cells of the same type together) can be divided into two main groups: (a) the immunoglobulin superfamily, showing calcium-independent cellcell adhesion, and (b) the calcium-dependent cadherin superfamily (-, ß-, and
-catenin in floating spheres and in cultures at 3 dpp. Cadherins are transmembrane adhesion receptors localized in specialized cell-to-cell adhesion sites, such as adherens junctions, and catenins are a group of intracellular proteins that link the cadherin molecules to the actin microfilaments. A representative Western blot of adhesion molecules is shown in Fig 7. The antibodies recognized bands at approximately 120, 138, 102, and 92 kD for E- and N-cadherin,
- and ß-catenin, respectively, in floating spheres (Fig 7) and in cell cultures at 3 dpp (not shown). No bands were observed for P-cadherin and
-catenin in both lysates (Fig 7). The results obtained in both lysates (floating spheres and cultures at 3 dpp) demonstrated that the levels of ß-catenin were higher than those of
-catenin.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ultrastructural appearance of the cells accurately reflects their physiological state. For example, neurons with a high metabolic activity have large nucleoli with a reticulated structural configuration, whereas neuronal cell types with low protein synthesis activity, such as cerebellar granule cells, show micronucleoli with a ring-shaped configuration (
Cell cultures isolated from the adult mouse brain and treated with mercaptoethanol, which inhibits cellcell contact, yield two types of neurospheres: I and II (
Some studies indicate that EGF-expanded stem cells generate two subpopulations of cells responsive to FGF-2, one restricted to neuronal differentiation and the other to a bipotential cell that develops into neurons and astrocytes (, which exists in the E15 rat fetal brain (
Although it has been shown that migrating neuroblasts (- and ß-catenin leads us to suggest that sphere cell compaction could be mediated by adherens junctions through specific cadherin/catenin/cytoskeleton complexes. Moreover, during chain migration, neural precursors move as chains of closely apposed cells connected by adherens junctions (
-catenin, which associate with
-catenin, and that
-catenin binds this ternary complex to the actin cytoskeleton (reviewed in
- and ß-catenin but not
-catenin in neurosphere cells, the complex expressed in these cells should be cadherin/ß-/
-catenin/actin. In addition we found higher levels of ß-catenin than those required to bind
-catenin on a 1:1 molar ratio. This suggests that ß-catenin could have other roles in sphere cells in addition to its function in cell adhesion. In this sense, it has been shown that ß-catenin plays a central role in the regulation of gene expression via the wingless-Wnt pathway that affects the embryonic axis specification (reviewed in
Apoptosis is an active mode of programmed cell death characterized by calcium influx, endonuclease activation, oligonucleosomal DNA fragmentation, chromatin condensation, and rearrangement of the plasma membrane with exposure of phosphatidylserine residues at the extracellular face. Within tissues, residual bodies of apoptotic cells are rapidly phagocytosed by macrophages, microglia, or other nearby cells (reviewed in
This study is the first to demonstrate the ultrastructural characteristics of EGF-expanded free-floating neurospheres derived from rat fetal striatum, the presence of two types of cells in these neurospheres, the phagocytic capability of sphere cells, and the expression by these cells of actin, vimentin, EGFR, a specific subset of cadherins, catenins, and FGFR isoforms. Moreover, we propose that neurosphere cells synthesize detectable levels of FGF-2. However, this study raises new research problems dealing with several aspects of neurosphere cells' structure and function, such as the roles of ß-catenin and endogenous FGF-2 in these cells and the molecular mechanisms underlying the recognition of dying cells and the phagocytotic processes.
![]() |
Acknowledgments |
---|
Supported in part by grants from the Fondo de Investigaciones Sanitarias (FIS 97/269 to E. Bazán and FIS 97/254 to C.L. Paíno) and Comunidad Autonoma de Madrid (CAM 8.5/4.1/99 to E. Bazán). Drs Lobo and Caso are recipients of postdoctoral fellowships from the Comunidad Autonoma de Madrid (CAM 8.5/4.1/99) and FIO foundation (Fundación para la Investigación en Oncología) respectively.
The two first authors have participated equally in this work.
Received for publication March 20, 2002; accepted July 24, 2002.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arenas MI, Bethencourt FR, De Miguel MP, Fraile B, Romo E, Paniagua R (1997) Immunocytochemical and quantitative study of actin, desmin and vimentin in the peritubular cells of the testes from elderly men. J Reprod Fertil 110:183-193[Abstract]
Arenas MI, Romo E, Royuela M, Fraile B, Paniagua R (2000) E-, N- and P-cadherin, and -, ß- and
-catenin protein expression in normal, hyperplastic and carcinomatous human prostate. Histochem J 32:659-667[Medline]
Arsenijevic Y, Weiss S (1998) Insulin-like growth factor-I is a differentiation factor for postmitotic CNS stem cell-derived neural precursors: distinct actions from those of brain-derived neurotrophic factor. J Neurosci 18:2118-2128
Bazán E, LópezToledano MA, Mena MA, Martín del Río R, Paino CL, Herranz AS (1996) Endogenous amino acid profile during in vitro differentiation of neural stem cells. In Fiskum G, ed. Neurodegenerative Diseases. New York, Plenum Press, 225-234
Bazán E, LópezToledano MA, Redondo C, Alcazar A, Mena MA, Paino CL, Herranz AS (1998) Characterization of rat neural stem cells from embryonic striatum and mesencephalon during in vitro differentiation. In Castellano B, González B, NietoSampedro M, eds. Understanding Glial Cells. Dordrecht, Kluwer Academic Publishers, 133-147
Bernard O, Li M, Reid HH (1991) Expression of two different forms of fibroblast growth factor receptor 1 in different mouse tissues and cell lines. Proc Natl Acad Sci USA 88:7625-7629[Abstract]
Bjorklund A, Stenevi U, Dunnett SB, Gage FH (1982) Cross-species neural grafting in a rat model of Parkinson's disease. Nature 298:652-654[Medline]
Blakemore WF, Franklin RJ (2000) Transplantation options for therapeutic central nervous system remyelination. Cell Transplant 9:289-294[Medline]
ChuLaGraff G, Doe CQ (1993) Neuroblast specification and formation regulated by wingless in the Drosophila CNS. Science 261:1594-1597[Medline]
Ciccolini F (2001) Identification of two distinct types of multipotent neural precursors that appear sequentially during CNS development. Mol Cell Neurosci 17:895-907[Medline]
Ciccolini F, Svendsen CN (1998) Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth factor (EGF) responsiveness in mouse striatal precursor cells: identification of neural precursors responding to both EGF and FGF-2. J Neurosci 18:7869-7880
Daadi MM, Weiss S (1999) Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain. J Neurosci 19:4484-4497
Davis AA, Temple S (1994) A self-renewing multipotential stem cell in embryonic rat cerebral cortex. Nature 372:263-266[Medline]
Decker L, AvellanaAdalid V, NaitOumesmar B, Durbec P, BaronVan Evercooren A (2000) Oligodendrocyte precursors migration and differentiation: combined effects of PSA residues, growth factors, and substrates. Mol Cell Neurosci 16:422-439[Medline]
Doetsch F, Caillé I, Lim DA, GarcíaVerdugo JM, AlvarezBuylla A (1999a) Subventricular zone astrocytes are neural stem cells in the adult mammalian brain. Cell 97:703-716[Medline]
Doetsch F, GarcíaVerdugo JM, AlvarezBuylla A (1997) Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J Neurosci 17:5046-5061
Doetsch F, GarcíaVerdugo JM, AlvarezBuylla A (1999b) Regeneration of a germinal layer in the adult mammalian brain. Proc Natl Acad Sci USA 96:11619-11624
Edelman GM, Crossin KL (1991) Cell adhesion molecules: implications for a molecular histology. Annu Rev Biochem 60:155-190[Medline]
Gage FH, Bjorklund A (1998) Intracerebral grafting of neuronal cell suspensions into the adult brain. Cent Nerv Syst Trauma 1:45-56
Gage FH, Ray J, Fisher LJ (1995) Isolation, characterization, and use of stem cells from CNS. Annu Rev Neurosci 18:159-192[Medline]
Geiger B, Ayalon O (1992) Cadherins. Annu Rev Cell Biol 8:307-332
Ghosh A, Greenberg ME (1995) Distinct roles for bFGF and NT-3 in the regulation of cortical neurogenesis. Neuron 15:89-103[Medline]
Gould SJ, Howard S, Papadaki L (1990) The development of ependyma in the human fetal brain: an immunohistological and electron microscopic study. Dev Brain Res 55:255-267[Medline]
Gritti A, Frölichsthal P, Galli R, Parati EA, Cova L, Pagano SF, Bjornson CR et al. (1999) Epidermal and fibroblast growth factors behave as mitogenic regulators for a single multipotent stem cell-like population from the subventricular region of the adult mouse forebrain. J Neurosci 19:3287-3297
Gritti A, Parati EA, Cova L, Frölichsthal P, Galli R, Wanke E, Faravelli L et al. (1996) Multipotent stem cells from the adult mouse brain proliferate and self-renew in response to basic fibroblast growth factor. J Neurosci 16:1091-1100[Abstract]
Gritti A, Vescovi AL, Galli R (2002) Adult neural stem cells: plasticity and developmental potential. J Physiol 96:81-90
Gumbiner BM (1996) Cell adhesion: the molecular basis of tissue architecture and morphogenesis. Cell 84:345-357[Medline]
Hadjiolov AA (1985) The Nucleolus and Ribosome Biogenesis. Vienna, Springer-Verlag
Handler A, Lobo MVT, Alonso FJM, Paíno CL, Mena MA (2000) Functional implications of the noradrenergic-cholinergic switch induced by retinoic acid in NB69 neuroblastoma cells. J Neurosci Res 60:311-320[Medline]
Jacobson MD, Weil M, Raff MC (1997) Programmed cell death and animal development. Cell 88:347-354[Medline]
Jacques TS, Relvas JB, Nishimura S, Pytela R, Edwards GM, Streuli CH, Constant CF (1998) Neural precursor cell chain migration and division are regulated through different beta1 integrins. Development 125:3167-3177
Johansson CB, Momma S, Clarke DL, Risling M, Lendahl U, Frisen J (1999) Identification of a neural stem cell in the adult mammalian central nervous system. Cell 96:25-34[Medline]
Kalyani AJ, Mujtaba T, Rao MS (1999) Expression of EGF receptor and FGF receptor isoforms during neuroepithelial stem cell differentiation. J Neurobiol 38:207-224[Medline]
Keegan K, Meyer S, Hayman MJ (1991) Structural and biosynthetic characterization of the fibroblast growth factor receptor 3 (FGFR3) protein. Oncogene 6:2229-2236[Medline]
Kerr JFR, Winterford CM, Harmon BV (1994) Morphological criteria for identifying apoptosis. In Celis JE, ed, Cell Biology. A Laboratory Handbook. Vol 1, pp. London, Academic Press, 319329
Kilpatrick TJ, Bartlett PF (1993) Cloning and growth of multipotential neural precursors: requirements for proliferation and differentiation. Neuron 10:255-265[Medline]
Kilpatrick TJ, Bartlett PF (1995) Cloned multipotential precursors from the mouse cerebrum require FGF-2, whereas glial restricted precursors are stimulated with either FGF-2 or EGF. J Neurosci 15:3653-3661[Abstract]
Kornblum HI, Hussain RJ, Bronstein JM, Gall CM, Lee DC, Seroogy KB (1997) Prenatal ontogeny of the epidermal growth factor receptor and its ligand, transforming growth factor alpha, in the rat brain. J Comp Neurol 380:243-261[Medline]
Kukekov VG, Laywell ED, Suslov O, Davies K, Scheffler B, Thomas LB, O'Brien TF et al. (1999) Multipotent stem/progenitor cells with similar properties arise from two neurogenic regions of adult human brain. Exp Neurol 156:333-344[Medline]
Kukekov VG, Laywell ED, Thomas LB, Steindler DA (1997) A nestin-negative precursor cell from the adult mouse brain gives rise to neurons and glia. Glia 21:399-407[Medline]
Laywell ED, Rakic P, Kukekov VG, Holland EC, Steindler DA (2000) Identification of a multipotent astrocytic stem cell in the immature and adult mouse brain. Proc Natl Acad Sci USA 97:13883-13888
Lee S-H, Lumelsky N, Studer L, Auerbach JM, McKay RD (2000a) Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nature Biotechnol 18:675-679[Medline]
Lee JC, MayerProschel M, Rao MS (2000b) Gliogenesis in the central nervous system. Glia 30:105-121[Medline]
Lendahl U, Zimmermann LB, McKay RDG (1990) CNS stem cells express a new class of intermediate filament protein. Cell 60:585-595[Medline]
Lobo MVT, Alonso FJM, Rodríguez S, Alcázar A, Martín E, Muñoz F, Santander R et al. (1997) Localization of eukaryotic initiation factor 2 in neuron primary cultures and established cell lines. Histochem J 29:453-468[Medline]
Lois C, GarcíaVerdugo J-M, AlvarezBuylla A (1996) Chain migration of neuronal precursors. Science 271:978-981[Abstract]
Ma W, Maric D, Li BS, Hu Q, Andreadis JD, Grant GM, Liu QY et al. (2000) Acetylcholine stimulates cortical precursor cell proliferation in vitro via muscarinic receptor activation and MAP kinase phosphorylation. Eur J Neurosci 12:1227-1240[Medline]
Martens DJ, Tropepe V, van der Kooy D (2000) Separate proliferation kinetics of fibroblast growth factor-responsive and epidermal growth factor-responsive neural stem cells within the embryonic forebrain germinal zone. J Neurosci 20:1085-1095
MayerProschel M, Kalyani AJ, Mujtaba T, Rao MS (1997) Isolation of lineage-restricted neural precursors from multipotent neuroepithelial stem cells. Neuron 19:773-785[Medline]
Murphy M, Reid K, Ford M, Furness JB, Bartlett PF (1994) FGF2 regulates proliferation of neural crest cells, with subsequent neuronal differentiation regulated by LIF or related factors. Development 120:3519-3528
Nakagawa S, Takeichi M (1995) Neural crest cell-cell adhesion controlled by sequential and subpopulation-specific expression of novel cadherins. Development 121:1321-1332
Palmer TD, Markakis EA, Willhoite AR, Safar F, Gage FH (1999) Fibroblast growth factor-2 activates a latent neurogenic program in neural stem cells from diverse regions of the adult CNS. J Neurosci 19:8487-8497
Palmer TD, Takahashi J, Gage FH (1997) The adult rat hippocampus contains primordial neural stem cells. Mol Cell Neurosci 8:389-404[Medline]
Partanen J, Makela TP, Alitalo R, Lehvaslaiho H, Alitalo K (1990) Putative tyrosine kinases expressed in K-562 human leukemia cells. Proc Natl Acad Sci USA 87:8913-8917[Abstract]
Raff MC (1992) Social controls on cell survival and cell death. Nature 356:397-400[Medline]
Reimers D, LópezToledano MA, Mason I, Cuevas P, Redondo C, Herranz AS, Lobo MVT et al. (2001) Developmental expression of fibroblast growth factor (FGF) receptors in neural stem cell progeny. Modulation of neuronal and glial lineages by basic FGF treatment. Neurol Res 23:612-621[Medline]
Reynolds BA, Tetzlaff W, Weiss S (1992) A multipotent EGF responsive striatal embryonic progenitor cell produces neurons and astrocytes. J Neurosci 12:4565-4574[Abstract]
Reynolds BA, Weiss S (1992) Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255:1707-1710[Medline]
Rousselot P, Lois C, AlvarezBuylla A (1995) Embryonic (PSA) N-CAM reveals chains of migrating neuroblasts between the lateral ventricle and the olfactory bulb of adult mice. J Comp Neurol 351:51-61[Medline]
Seri B, GarcíaVerdugo J-M, McEwen BS, AlvarezBuylla A (2001) Astrocytes give rise to new neurons in the adult mammalian hippocampus. J Neurosci 21:7153-7160
Stensaas LJ, Gilson BC (1972) Ependymal and subependymal cells of the caudato-pallial junction in the lateral ventricle of the neonatal rabbit. Z Zellforsch Mikrosk Anat 132:297-322[Medline]
Tannheimer SL, Rehemtulla A, Ethier SP (2000) Characterization of fibroblast growth factor receptor 2 overexpression in the human breast cancer cell line SUM-52PE. Breast Cancer Res 2:311-320[Medline]
Taupin P, Ray J, Fischer WH, Suhr ST, Hakansson K, Grubb A, Gage FH (2000) FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 28:385-397[Medline]
Temple S, AlvarezBuylla A (1999) Stem cells in the adult mammalian central nervous system. Curr Opin Neurobiol 9:135-141[Medline]
Tennyson VM, Papas GD (1962) An electron microscope study of ependymal cells of the fetal, early postnatal and adult rabbit. Z Zellforsch Mikrosk Anat 56:595-618[Medline]
Tropepe V, Hitoshi S, Sirard C, Mak TW, Rossant J, van der Kooy D (2001) Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism. Neuron 30:65-78[Medline]
Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, van der Kooy D (1999) Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse telencephalon. Dev Biol 208:166-188[Medline]
Tucker EL, Pignatelli M (2000) Catenins and their associated proteins in colorectal cancer. Histol Histopathol 15:251-260[Medline]
Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS et al. (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 97:14720-14725
Vescovi AL, Reynolds BA, Fraser DD, Weiss S (1993) bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11:951-966[Medline]
Vitry S, AvellanaAdalid V, Lachapelle F, Evercooren AB (2001) Migration and multipotentiality of PSA-NCAM+ neural precursors transplanted in the developing brain. Mol Cell Neurosci 17:983-1000[Medline]
Weiss S, Dunne C, Hewson J, Wohl C, Wheatly M, Peterson AC, Reynolds BA (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599-7609
Zhou FC, Kelley MR, Chiang YH, Young P (2000) Three to four-year-old nonpassaged EGF-responsive neural progenitor cells: proliferation, apoptosis, and DNA repair. Exp Neurol 164:200-208[Medline]