Neuroscience Letters 370 (2004) 230–235
In vitro differentiated neural stem cells express functional glial glutamate transporters Nicolas Vanhoutte1 , Isabelle de Hemptinne1 , C´eline Vermeiren, Jean-Marie Maloteaux, Emmanuel Hermans∗ Laboratoire de Pharmacologie Exp´erimentale (FARL), Universit´e Catholique de Louvain 54.10, Avenue Hippocrate 54, B-1200 Brussels, Belgium Received 11 June 2004; received in revised form 21 July 2004; accepted 13 August 2004
Abstract The possibility to isolate stem cells from the adult central nervous system and to maintain and propagate these cells in vitro has raised a general interest with regards to their use in cell replacement therapy for degenerative brain diseases. Considering the critical role played by astrocytes in the control of glutamate homeostasis, we have characterised the expression of functional glutamate transporters in neural stem cells exposed to selected culture conditions favouring their differentiation into astrocytes. Commonly, neural stem cells proliferate in suspension as neurospheres in serum-free medium. The addition of serum or a supplement of growth factors (G5) to the culture medium was found to trigger cell adhesion on coated surfaces and to favour their differentiation. Indeed, after 7 days in these conditions, the vast majority of the cells adopted markedly distinct morphologies corresponding to protoplasmic (with serum) or fibrous (with G5 supplement) astrocytes and approximately 35–40% acquired the expression of the glial fibrillary acidic protein (GFAP). Immunocytochemical analysis also revealed that the treatments with serum or with the G5 supplement triggered the expression of the glial glutamate transporters GLT-1 (35 and 21%, respectively) and GLAST (29 and 69%, respectively). This effect was correlated with a robust increase in the Na+ -dependent [3 H]-d-aspartate uptake, which was partially inhibited by dihydrokainate, a selective blocker of GLT-1. Together, these results indicate that in vitro differentiation of cultured neural stem cells can give rise to distinct populations of astrocytes expressing functional glutamate transporters. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Neural stem cells; Glutamate uptake; Glutamate transporters; Astrocytes; GFAP; Differentiation
Studies carried out in the recent years have revealed the unexpected presence of progenitor cells within the central nervous system (CNS) of adult mammalians (for review, see ). As for many other tissues, the existence of CNS-derived stem cells opens new perspectives in the treatment of CNS related disorders [3,9]. In addition, neural stem cells (NSCs) constitute a valuable tool for fundamental research in neurobiology and hence, several techniques have been developed for the isolation, purification and in vitro maintenance of these cells from several regions of the CNS . Commonly, NSCs are grown in defined serum-free medium as a suspension of epidermal growth factor (EGF)-supported proliferating cells ∗ 1
Corresponding author. Tel.: +32 2 764 9339; fax: +32 2 764 5460. E-mail address: [email protected]
(E. Hermans). Contributed equally to this study.
0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.08.039
. The multicellular floating neurospheres can be mechanically dissociated into isolated cells that proliferate into new spheres. Once plated on coated surface and maintained in selected culture conditions, these cells tend to differentiate into mixed cultures of neurones and glial cells [10,31]. The observation that adult neural progenitors can evolve into functional neurones has raised a considerable interest with respect of their use in regenerating damaged brain tissue [11,13]. However, beside their differentiation into neurones, many studies have revealed that a high proportion of NSCs give rise to glial cells clearly identified by the presence of typical markers of oligodendrocytes or astrocytes . For a long time considered as physical and metabolic supports for neurones, glial cells and in particular astrocytes, have received a growing interest as it is now well established that astrocytes directly or indirectly participate in the regulation
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of cell communication in the central nervous system . Indeed, these cells ensure most of the glutamate uptake from the extracellular environment and protect neurones from the excitotoxicity of this amino acid . Hence, promoting glutamate uptake constitutes a putative approach in the treatment of CNS disorders involving glutamate toxicity . In this respect, several authors have reported on the regulation of glutamate uptake in glial cells [25,27]. While much effort was directed toward the characterisation of glutamate transporters expressed in primary cultured astrocytes , less is known about the expression and functional properties of these transporters in glial cells obtained upon differentiation of cultured neural progenitors . Brunet et al. recently reported on the acquisition of astrocytes metabolic features, including GLAST expression, in differentiated mouse neural stem cells . The aim of the present study was to evaluate the functional expression of the glial glutamate transporters GLT-1 and GLAST in cells derived from proliferating neurospheres exposed to culture conditions favouring astrocytes maturation. Morphological and biochemical properties of these cells were examined after exposure to either foetal bovine serum (FBS) or the culture supplement G5. Brains were dissected under sterile conditions from foetus of Wistar rats at 16–18 days of gestation. Experimental protocols meet the guidelines of the local and governmental ethical committees. Striatum from 2 or 3 animals were collected and mechanically dissociated with a Pasteur pipette and diluted (1.5 × 105 cell/ml) in proliferation medium consisting of DMEM-F12 supplemented with 20 ng/ml EGF, 10 ng/ml basic fibroblast growth factor (FGF2 ) and B27 in the absence of serum and antibiotics. This cell suspension was transferred in ventilated non-coated culture flasks (20 ml/75 cm2 ) and maintained at 37 ◦ C in a humidified atmosphere containing 5% CO2 . In these conditions, non-adherent cells divided and formed neurospheres. Every 3–4 days, these neurospheres were collected by centrifugation (274 g for 5 min) and after mechanical dissociation, the cells were resuspended in culture medium and transferred into non-coated culture flasks (2.5 × 104 cells/cm2 ). After three passages (approximately 10 days), dissociated cells were transferred into 6- or 24-well poly-l-lysine (10 g/ml) coated plates in differentiation media and characterised after either 24 h or 7 days of culture. Differentiation medium consisted of DMEM-F12 with 20 ng/ml EGF and 10 ng/ml FGF2 supplemented with either 10% FBS or the culture supplement G5 diluted 1/100, as suggested by the manufacturer (composition: insulin 500 g/ml, human transferrin 5 mg/ml, selenite 0.52 g/ml, biotin 1 g/ml, hydrocortisone 0.36 g/ml, FGF2 0.52 g/ml and EGF 1 g/ml). All the culture media, culture supplements and consumables were from Invitrogen (Merelbeke, Belgium). For immunocytochemistry, cells grown on coated 12 mm round glass coverslips were fixed with paraformaldehyde 4% (v/v) for 15 min at room temperature and permeabilized thereafter with 1% Triton X-100 (v/v) in TBS (Tris–HCl 50mM, NaCl 150 mM, pH 7.4) during 15 min. Non-specific
binding was blocked by incubating the cells in a TBS solution containing non-fat dry milk (30 mg/ml) for 1 h at 37 ◦ C. Cells were then incubated during 1 h with primary antibodies i.e. a rabbit anti-glial fibrillary acidic protein (GFAP) antibody (1:1500, Dako, Heule, Belgium), a guinea pig anti-GLAST antibody (1:1000, Chemicon, Hampshire, UK), a guinea pig anti-GLT-1 antibody (1:1000, Chemicon, Hampshire, UK). Secondary antibodies, applied for 1 h at room temperature were FITC-conjugated anti-rabbit IgG antibody (1:500) and Cy3-conjugated antiguinea pig IgG antibody (1:1000), both from Jackson Immunoresearch Laboratory (de Pinte, Belgium). Nucleus were stained during 30 min with the nuclear dye DAPI (1/5000). After three rinses in TBS, the preparations were mounted in Fluoprep (BioMerieux, Brussels, Belgium) and examined using an Olympus IX70 inverted fluorescent microscope coupled to a CCD camera (T.I.L.L. photonics, Martinsried, Germany). Excitation light (488, 400 and 540 nm for FITC, DAPI and Cy3, respectively) was obtained from a Xenon lamp coupled to a monochromator (T.I.L.L. photonics, Martinsried, Germany). Digital images were acquired using appropriate filters and combined using the TILLvisION software. Functional characterisation of glutamate transporters was realised on cells grown on poly-l-lysine coated 24-well plates as previously published . Briefly, transport velocity was estimated by measuring the uptake of [3 H]-d-aspartate (20 nM) after a 6-min incubation at 37 ◦ C in a buffer containing Na+ . The specific activity of the glutamate transporters (expressed as the uptake velocity per the quantity of protein in mg) was estimated after subtracting the data obtained using Na+ -free Krebs buffer. When indicated, the inhibitors l-trans-pyrrolidine-2,4-dicarboxylic acid (tPDC, 1 mM) and dihydrokainate (DHK, 100 M) were added 6 min before the addition of [3 H]-d-aspartate. Preliminary experiments conducted with distinct structures of the rat foetal CNS revealed that the highest density of proliferating NSCs was obtained from the striatum. As commonly reported , these cells proliferated in suspension as neurospheres on non-adherent surfaces and the majority of them appeared positive when tested for nestin expression by immunocytochemistry (not shown). After three passages, when dissociated cells were transferred into coated flasks and maintained in the proliferation medium lacking the supplement B27, a low proportion of these NSCs tended to adhere. These cells failed to divide and stayed almost quiescent for up to 7 days, with some showing small processes. In contrast, the addition of either FBS or the culture supplement G5 considerably increased the cell adherence within 24 h of plating and favourised their proliferation. Microscopic observation revealed that the cells exposed to FBS or to the supplement G5 adopted markedly distinct morphologies (Fig. 1). Indeed, in the presence of the supplement G5, cells developed long and ramified processes, leading to a typical fibrous astrocytes morphology (type II astrocytes). In contrast, when cultivated with FBS, cells displayed the characteristics of protoplasmic astrocytes (type I or so-called velate astrocytes) as cell nuclei
N. Vanhoutte et al. / Neuroscience Letters 370 (2004) 230–235
Fig. 1. Morphology and GFAP expression in NSCs during in vitro differentiation. NSCs isolated from the rat striatum were exposed to differentiation medium alone (A, D) or supplemented with either the supplement G5 (B, E) or FBS (C, F) for 24 h (A–C) or 7 days (D–F). Cell morphology was examined by phase contrast microscopy and GFAP expression was detected by immunofluorescence using a specific polyclonal antibody visualised with a FITC-coupled secondary antibody (green). Cell nuclei were visualised after DNA staining with DAPI (blue). Bar: 20 m.
were surrounded by a large flat cytoplasm with short and tiny processes. Such difference in cell morphology upon differentiation was also observed after immunostaining of GFAP (Fig. 1). Indeed, while cells maintained in the absence of culture supplement appeared GFAP-negative after 24 h or 7 days, both FBS and G5 treatments were found to considerably increase the number of GFAP positive cells, reaching up to 35 and 38% after 7 days, respectively. Labelling was concentrated along the long processes of the fibrous astrocytes obtained in G5-containing medium while a dense network of GFAP positive filaments was detected in the large cytoplasm of protoplasmic astrocytes obtained with FBS. The phenotypic nature of the GFAP-negative cells in culture differentiated during 7 days remains unresolved as we failed to detect any microtubule associated protein (MAP-2) positive cells (not shown). The expression of the glial glutamate transporters GLT-1 and GLAST in adherent cells obtained from NSCs was examined by immunocytochemistry. The anti-GLT-1 antibody systematically showed intense and putatively non-specific labelling of nuclei which appeared in pink after DAPI staining of nuclear DNA. After either 24 h or 7 days of culture in the absence of G5 or FBS, a very low percentage of cells showed a discrete GLAST immunoreactivity while GLT-1 expression could not be detected (Fig. 2; Table 1). In contrast, exposure of the cells to FBS or the supplement G5 considerably increased the number of positive cells as well as the intensity of GLAST and/or GLT-1 labellings. As summarised in Table 1, a robust expression of GLAST was observed in cells differentiated for 7 days in the presence of G5, while the GLT-1 expression appeared higher in cells exposed to FBS as compared
to those exposed to G5. Examination of double labelling experiments indicated that the expression of the transporters was not restricted to GFAP positive cells. Thus, depending on the conditions tested, 30–70% of the cells expressing the transporters were also positive for GFAP. Considering the limited percentage of GFAP-positive cells, particularly after exposure to G5, the quantitative analysis of immunostaining suggested that some cells could express both types of glutamate transporters simultaneously. The functional properties of the glial glutamate transporters expressed in NSCs were evaluated by measuring their ability to take up [3 H]-d-aspartate (20 nM). Adhering cells cultured in the absence of the supplement G5 or FBS for either 24 h or 7 days exerted a rather low uptake activity (approximately 0.2–0.5 pmol/mg protein/min). In contrast, while exposure of the cells to FBS or the supplement G5 for 24 h had little influence on the uptake, cells differentiated for 7 days showed a robust and significant increase in the specific (Na+ -dependent) [3 H]-d-aspartate uptake, this effect being more pronounced when using the culture supplement G5. As expected, the [3 H]-d-aspartate uptake measured in these cells involves glutamate transporters, as it was entirely inhibited in the presence of tPDC (1 mM). A pharmacological characterisation of the glutamate transporters involved in the uptake of aspartate was obtained using DHK (100 M), a specific blocker of GLT-1. As shown in Fig. 3B, DHK significantly decreased the uptake velocity by approximately 55 and 25%, for cells differentiated in the presence of FBS or G5, respectively. These data demonstrate that functional GLT-1 transporters are at least partially involved in the Na+ dependent [3 H]-d-aspartate uptake measured in these cells.
N. Vanhoutte et al. / Neuroscience Letters 370 (2004) 230–235
Fig. 2. Expression of the glutamate transporters after in vitro differentiation of NSCs. Rat neural stem cells (passage 3) were exposed for 7 days to differentiation medium alone (A, D) or supplemented with either the supplement G5 (B, E) or FBS (C, F). Immunodetections of GLAST (A–C) and GLT-1 (D–F) were performed using specific polyclonal antibodies visualised with a Cy3-coupled secondary antibody (red) while GFAP expression was detected using a specific polyclonal antibody visualised with a FITC-coupled secondary antibody (green). In each panel, left image shows immunodetection of the transporters (GLAST or GLT-1) whereas right image shows merged immunodetection of the transporter and GFAP. Cell nuclei were visualised after DNA staining with DAPI (blue). Bar: 20 m.
In summary, the present study indicates that exposing NSCs obtained from the embryonic rat striatum to FBS or to the supplement G5 (defined culture conditions known to favour maturation of primary cultured astrocytes) , promotes their differentiation into astrocytes expressing functional glial glutamate transporters. Interestingly, the two differentiation conditions examined gave rise to markedly different astroglial cultures. Most evidently, after 7 days of in vitro differentiation, cells adopted distinct morphologies. Combined with immunocytochemical detection of GFAP, our observations revealed that the culture was mainly constituted of fibrous-like astrocytes when maintained in the presence of the supplement G5 while a typical protoplasmic phenotype predominated in FBS supported cultures. Accordingly, Chiang et
al. previously reported that astroglial progenitor cells turned into either protoplasmic or fibrous astrocytes when exposed to FBS or FGF2 , respectively . Indeed, the combination of FGF2 and EGF which are both present in the G5 supplement is known to potently promote the in vitro maturation of glial progenitors into fibrous astrocytes [19,21]. Noteworthy, although both differentiation protocols tested gave rise to morphologically homogenous populations of cells, a relatively high percentage of these were negative for GFAP staining. This contrasts with primary cultured astrocytes where the majority of the cells express GFAP, in particular when exposed to growth factors . Heterogeneous GFAP expression could indicate that the cells did not reach full maturation after 7 days. Indeed, immature astrocytes do not readily express GFAP, but express
Table 1 Quantitative analysis of immunocytochemical detection of GFAP, GLAST and GLT-1 in differentiated NSCs 24 h
3.8 ± 3.1
16.2 ± 5.6
22.1 ± 10.9
37.9 ± 9.7
34.5 ± 13.1
5.2 ± 1.0 N.D.
15.3 ± 2.1 5.4 ± 3.3
18.7 ± 1.0 5.3 ± 1.2
4.2 ± 3.4 N.D.
69.1 ± 9.5 28.8 ± 7.1
29.3 ± 1.6 14.9 ± 2.7
15.8 ± 0.9 12.9 ± 2.7
6.3 ± 0.6 4.3 ± 1.3
21.1 ± 3.3 15.9 ± 0.3
35.2 ± 3.0 10.6 ± 2.2
Rat neural stem cells (passage 3) were exposed for 7 days to differentiation medium alone or supplemented with either the supplement G5 or FBS. Cells were analysed by immunofluorescence microscopy as shown in Figs. 1 and 2 and the number of positive cells expressing each antigen was evaluated by examination of five fields from three independent cultures. The total number of cells in these samples was determined by counting DAPI stained cell nuclei and results are expressed as percentages of positive cells (mean ± S.E.M.). Double labelling experiments allowed to determine the percentage of cells co-expressing the transporters and GFAP. N.D., non detected.
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Fig. 3. Characterisation of Na+ -dependent [3 H]-d-aspartate uptake in NSCs after in vitro differentiation. Panel A shows the velocity of Na+ -dependent [3 H]-d-aspartate uptake (20 nM) measured on rat NSCs (passage 3) exposed for 24 h or 7 days to differentiation medium alone or supplemented with either G5 or FBS. Panel B shows the effect of the glutamate transporters blockers DHK (100 M) and tPDC (1 mM) on the uptake measured on cells exposed for 7 days to the differentiation media. Data are the mean with S.E.M. from at least three independent experiments performed in duplicate. One-way ANOVA followed by Newman–Keul’s test for multiple comparisons was used in order to compare short and long term exposure to the culture media, to analyse the influence of G5 and FBS or to evaluate the effect of the transporter blockers. In panel A, ## p < 0.01 and ### p < 0.001 denote significant difference between 24 h and 7 days while ** p < 0.01 and *** p < 0.001 indicate significant influence of the growth factors. In panel B, ** p < 0.01 and *** p < 0.001 indicate significant differences with the corresponding values measured in the absence of blockers.
nestin, a common marker of neuroglial progenitors . On the other hand, it is well documented that a high percentage of astrocytes do not express GFAP in the brain [6,29], while its induction has been frequently associated with gliosis processes in vivo [23,30]. Hence, a recent study revealed that a high proportion of NSCs-derived cells showing an astrocyte morphology were GFAP negative . Considering glutamate transporters, it is generally accepted that GLAST expression remains constant during development while GLT-1 expression increases postnatally . However, it is generally admitted that the expression of GLT-1 is absent when culturing cortical astrocytes. Thus, in vitro, primary cultured astrocytes mainly express the GLAST while GLT-1 induction requires optimised culture conditions and/or contacts with neuronal cells [24,27]. Furthermore, regarding glutamate uptake, the question of whether cultured astrocytes constitute an appropriate model to study the properties of GLT-1 has been raised as induction of the GLT-1 expression failed to correlate with functionality . In this respect, a major observation of the present study concerns the possibility to induce the expression of the glial GLT-1 transporter in astrocytes derived from NSCs cultures. Furthermore, our data also indicate that the two distinct populations of astrocytes obtained after in vitro
differentiation of the NSCs differ in their glutamate handling properties. Indeed, the low concentration of substrate used in the uptake experiments would certainly favour the detection of high affinity functional transporters. Therefore our results indicate that cells exposed to the supplement G5 express rather low levels of functional GLT-1 (Km of approximately 2 M) and that our data mostly reflects GLAST-mediated uptake (Km of approximately 70 M). In contrast, the uptake measured in FBS supported cultures clearly involves both GLT-1 and GLAST, as further demonstrated using the specific blocker DHK. Interestingly, fibrous and protoplasmic astrocytes were reported to differ biochemically and developmentally [2,16]. In addition, previous studies conducted on primary cultured astrocytes also revealed that induction of glutamate transporters is concomitant with a morphological differentiation [24,27]. In our differentiated NSCs, functional expression of GLT-1 is more evident in protoplasmic-like astrocytes (FBS supported differentiation) where immunocytochemical studies revealed a modest correlation with GFAP expression. In contrast, in primary cultured astrocytes exposed to cyclic AMP, enhanced GLT-1 expression is frequently associated with cells showing a robust GFAP labelling and a stellate morphology . While such discrepancy remains unresolved, these data suggest that mature astrocytes obtained from primary cultures of newborn animals differ from those obtained from the NSCs. In this respect, and in contrast with the former model, it is noteworthy that GLT-1 expressed by protoplasmic astrocytes derived from NSCs significantly participates in substrate uptake. In conclusion, this work provides evidence that manipulating the chemical environment of neural stem cells isolated from late rat embryo may drive their differentiation into morphologically and biochemically distinct types of astrocytes. These cells may constitute an appropriate model for the study of functional astroglial glutamate transporters.
Acknowledgements We thank S. Wislet-Gendebien and Professor B. Rogister (Universit´e de Li`ege, Belgium) for their help in setting up the NSCs culture and the ICC technique. This work was supported by the National Fund for Scientific Research (F.N.R.S., Belgium, Conventions FRSM 3.4.610.01.F and T´el´evie) and by the Belgian Queen Elisabeth Medical Foundation. I.d.H. and E.H. are scientific collaborator and Senior Research Associate of the F.N.R.S., respectively. C.V. is supported by the F.R.I.A.
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