NG2 and Olig2 Expression Provides Evidence for Phenotypic Deregulation of Cultur - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:58 |只看该作者 |倒序浏览 |打印

作者：Cecile Dromarda, Sylvain Bartolamia, Loc Deleyrollea, Hirohide Takebayashib, Chantal Ripolla, Lionel Simonneaua, Sylvie Promea, Sylvie Puecha, Christophe Tran Van Baa, Christophe Duperrayc, Jean Valmiera, Alain Privata, Jean-Philippe Hugnota作者单位：aINSERM U, Physiopathologie et Thrapie des dficits sensoriels et moteurs, Institut des Neurosciences de Montpellier, Hpital St ELOI, Montpellier Cedex , France;bDivision of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan;cService Regional INSE , d7 O' j2 |- A% l
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      【摘要】1 z9 o8 i8 \: e* `) D" x
   Neural stem cells cultured with fibroblast growth factor 2 (FGF2)/epidermal growth factor (EGF) generate clonal expansions called neurospheres (NS), which are widely used for therapy in animal models. However, their cellular composition is still poorly defined. Here, we report that NS derived from several embryonic and adult central nervous system (CNS) regions are composed mainly of remarkable cells coexpressing radial glia markers (BLBP, RC2, GLAST), oligodendrogenic/neurogenic factors (Mash1, Olig2, Nkx2.2), and markers that in vivo are typical of the oligodendrocyte lineage (NG2, A2B5, PDGFR-). On NS differentiation, the latter remain mostly expressed in neurons, together with Olig2 and Mash1. Using cytometry, we show that in growing NS the small population of multipotential self-renewing NS-forming cells are A2B5  and NG2 . Additionally, we demonstrate that these NS-forming cells in the embryonic spinal cord were initially NG2¨C and rapidly acquired NG2 in vitro. NG2 and Olig2 were found to be rapidly induced by cell culture conditions in spinal cord neural precursor cells. Olig2 expression was also induced in astrocytes and embryonic peripheral nervous system (PNS) cells in culture after EGF/FGF treatment. These data provide new evidence for profound phenotypic modifications in CNS and PNS neural precursor cells induced by culture conditions.
      【关键词】 Neural stem cells Oligodendrocytes NG Proteoglycan Peripheral nervous system Neurospheres Cytometry Plasticity
   INTRODUCTION

Neurospheres (NS) are widely used as a model for studying the neural lineage in vitro . According to these criteria, in the adult rodent CNS, only the ventricular subependyma appears to contain NSC, whereas the dentate gyrus contains lineage and differentiation-restricted cells, referred to as progenitors.
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NS have been extensively used as a source of new cells for therapeutic strategies in mouse models of human nervous system diseases. NS cells appear to be endowed with a high capacity to migrate to lesioned sites and to cross the blood-brain barrier .

The identity of the NS-forming cells has been partially elucidated in adult CNS. In the brain, both glial fibrillary acidic protein-positive (GFAP ) cells and type-C cells (NG2 /Dlx-2 ) located in the subependymal zone can form NS . The identity of the NS-forming cells, in contrast to postnatal CNS, in the embryo is largely unknown.+ |4 s$ O: L7 u6 u
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The NS cellular composition is still unclear. They appear to be heterogeneous entities containing a majority of poorly defined neural immature cells, unable to form new NS, and only a small fraction of multipotential self-renewing NS-forming cells (0.1%¨C10%). The majority of NS cells express markers of radial glial cells (RC2, GLAST, BLBP) . Indeed, these cells differ in their expression of growth factors and transcription factors and in the cell types they generate. With regard to this in vivo heterogeneity, very few data are available on the phenotype of radial glial cells contained in NS.
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Finally, there is an ongoing debate about the physiological relevance of the NS model for studying the diversity, phenotype, and fate of neural precursor cells present during CNS development. Several studies indicate that regional specifications and intrinsic differences are maintained in NS cultures even at late passages .1 F8 Y: \8 |. _, r6 x
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Considering their high therapeutic potential and their broad use as a model of neural development, it is essential to better characterize the identity of NS-forming cells (both in vivo and in vitro) and NS cellular composition. Here, we further explore the phenotype of embryonic NS-forming cells and the cellular composition of NS derived from different CNS regions. We focused our analysis on the expression of markers that are relatively specific for the oligodendrocyte lineage in vivo (A2B5, platelet-derived growth factor receptor -, NG2) and on important neural precursor cell fate-determining transcription factors (Olig2, Mash1, Sox 9, Sox10, Nkx2.2). We found that regardless of their origin, NS contain remarkable cells coexpressing A2B5, PDGFR-, NG2, Olig2, Sox9, and radial glia markers (RC2, BLBP, GLAST). Using cytometry and clonal analysis, we investigated whether NS-forming cells contained in NS were NG2  and A2B5  and whether these cells were originally derived from NG2  or NG2¨C cells in the embryonic neural tube. The possibility of the direct implication of the basic helix-loop-helix (bHLH) genes (Olig2 and Mash1) in the expression of NG2 in neural precursors was explored. Our data provide evidence that NS are initially derived from NG2¨C NSC that subsequently generate NG2  NSC and that profound phenotype deregulations are likely to occur in CNS and also in peripheral nervous system (PNS) neural precursors cultured in NS culture conditions.

MATERIALS AND METHODS

Dissection and Cell Isolation

For information regarding dissection and cell isolation, see the supplemental online data.

Cell Culture
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For NS cultures, acutely dissociated cells were cultured at 37¡ãC at 100 cells per microliter (equivalent to 20,000 cells per cm2) in 75-cm2 tissue culture flasks (NUNC A/S, Roskilde, Denmark, http://www.nuncbrand.com) coated with 400 µg/cm2 poly-2-hydroxyethyl-methacrylate (poly-HEME) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) to prevent cell attachment. To examine the phenotype and fate of clonal NS after passaging, the cells were seeded at 2¨C5 cells per microliter (equivalent to 250¨C600 cells per cm2) to allow clonal expansion . The medium consisted of N2 supplement (Invitrogen Corporation, Carlsbad, CA, http://www.invitrogen.com), 2 mM L-glutamine (Invitrogen Corporation), 0.6% glucose (Sigma-Aldrich), 20 µg/ml bovine insulin (Sigma-Aldrich), and 2 µg/ml ciprofloxacine (United States Biological Inc., Swampscott, MA, http://www.usbio.net) in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen Corporation) supplemented with 20 ng/ml epidermal growth factor (EGF) (PeproTech, Rocky Hill, NJ, http://www.peprotech.com) and 10 ng/ml fibroblast growth factor 2 (FGF2) (PeproTech). NS were passaged every 7 days by incubation in 0.25% trypsin/1 mM EDTA (Invitrogen Corporation) (3 minutes, 37¡ãC). The cells were mechanically dissociated in the presence of 2 mM CaCl2, 0.01% DNase I, and 0.5% soybean trypsin inhibitor and then rinsed once with medium. Embryonic dorsal spinal cord cells (Fig. 1C; supplemental online Fig. S3) were plated with or without FGF2/EGF at 50,000 cells per cm2 either on poly-D-lysine (PDL)-coated (adherent condition) or in poly-HEME-coated (non adherent condition) coverslips. For differentiation experiments, NS were rinsed twice with Hanks' balanced salt solution (HBSS) (Invitrogen Corporation), enzymatically dissociated, and plated on coverslips coated with 40 µg/ml PDL (Sigma-Aldrich) for 4 days at 250,000 cells per cm2 in 24-well dishes without FGF2 and EGF. Alternatively, to analyze the multipotency of clonally expanded NS, single spheres (size 300¨C500 µm) were harvested under the microscope, individually plated on coverslips, and allowed to differentiate for 4¨C6 days before processing for triple-labeling.
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Figure 1. Induction of Olig2 by culture condition. (A): Olig2 immunodetection on 12-µm-thick cryostat section of embryonic day 13.5 (E13.5) thoracic spinal cord and dorsal root ganglia (DRG). Left photograph: low-magnification image of section stained with Hoechst (gray). Scale bar = 100 µm. Middle and right photographs: High-magnification images of DRG (red square on left photograph) and ventral part of the neural tube (yellow square on left photograph). Olig2 (green) is expressed only in ventrally located cells in the spinal cord. Scale bar = 50 µm. (B): Time-course expression of Olig2 in E13.5 embryonic spinal cord cultures. Protocol is identical to that described in Figure 4F legend. The percentage of Olig2  cells ¡À SEM (n = 3 independent experiments) in the cellular suspension is indicated from days 0 to 7. (C): Time-course expression of Olig2 in dorsal E13.5 embryonic spinal cord cultures. Cells were cultured on poly-D-lysine with ( ) or without (¨C) growth factor (GF) at a density of 50,000 cells per square centimeter. The number of Olig2  cells per field (x20 objective) is indicated ¡À SEM (n = 3 independent experiments). (D): Expression of Olig2 in astrocytes. Control: Upon NS differentiation, GFAP  (green) cells (arrow) are Olig2¨C (red). After 24-hour reintroduction of FGF2/EGF, the vast majority of GFAP  cells display a strong Olig2 staining. Scale bar = 10 µm. (E): RC2 immunodetection on 12-µm-thick cryostat section of E13.5 thoracic spinal cord (SP) and DRG. Left photograph: section stained with Hoechst (gray). Right upper photograph: RC2 staining (green). Note the elongated radial glia cells (arrow). Green staining in the DRG (arrowheads) is not specific and is also detected in red with cyanine 3-filtered light (supplemental online Fig. S7). Scale bar = 100 µm. (F): Detection of Olig2 (upper photograph, red) and RC2 (lower photograph, green) in E13.5 peripheral nervous system (PNS) cells cultured 10 days in NS media. Nuclei are stained with Hoechst (right photographs). Scale bar = 10 µm. (G): Phenotype of NS derived from E13.5 embryonic PNS. Photographs are confocal images of NS immunolabeled with the indicated marker. Scale bars = 10 µm. Dotted circle indicates NS outline. (H): Immunodetection of cells expressing smooth-muscle actin (green, arrows on right photographs) in differentiated embryonic NS (upper photographs) and PNS NS (lower photographs). Nuclei are stained with Hoechst (blue). (I): Triple-immunostaining of MHP36 cells transfected with both Olig2 and Mash1 plasmids revealed that Mash1 (red) and Olig2 (green) are not sufficient to induce NG2 expression (far-red). Nuclei are labeled with Hoechst (blue). Scale bar = 10 µm. Abbreviations: EGF, epidermal growth factor; FGF2, fibroblast growth factor 2; GFAP, glial fibrillary acidic protein.  i( d% I* }% L5 y; Z" Q* L8 b

MHP36 Cell Culture and Transfection

This NSC line (a gift from Dr. J. Sinden, London) , peGFP (Dr. S.F. Heinemann, San Francisco, California), and pcDNA1 Mash1 (Dr. F. Guillemot, London) were prepared using the EndoFree Plasmid Maxiprep Kit (Qiagen Inc.). Cells were tested for NG2 and PDGFR- expression 48 hours after transfection.

Immunodetection3 X- p4 K6 E. d8 r1 F+ h
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Immunodetection was carried out as described in  on cells fixed for 15 minutes at room temperature with 4% paraformaldehyde. For surface antigen labeling (O4, GalC, A2B5), Triton was omitted; for multilabeling including both surface and intracellular markers, 0.01% Triton was used. Cells freshly dissociated from tissues or small NS were stuck on poly-L-ornithine-coated coverslips by centrifugation (10 minutes, 1,000 rpm) in 24-well dishes and immediately fixed. For large NS, 12-µm-thick frozen sections were made from fixed NS, incubated in 25% sucrose (overnight 4¡ãC), and embedded in OCT (Tissue-Tek, Torrance, CA, http://www.sakura-americas.com). For embryonic frozen sections, E13.5 embryos were fixed in 4% paraformaldehyde for 6 hours and cryoprotected by incubation successively for 2 hours in 12%, 15%, and 18% sucrose solutions in phosphate-buffered saline (PBS), overnight in 25%, and for 2 hours in 25% sucrose in OCT. They were embedded in pure OCT and frozen in cold isopentane in liquid nitrogen. The primary antibodies used are provided in the supplemental online data. Negative controls were performed using isotype-matched immunoglobulins (Sigma-Aldrich). Nuclei were stained with either 5 µg/ml Hoechst 33242 solution (Sigma-Aldrich) or 1 µg/ml propidium iodide solution (Sigma-Aldrich). Slides were observed using a Leica DMR fluorescence microscope (Leica, Wetzlar, Germany, http://www.leica.com) or a confocal scanning Zeiss axiovert 100TV inverted microscope (Carl Zeiss, Jena, Germany, http://www.zeiss.com) equipped with a Bio-Rad MRC 1024 laser (Bio-Rad Laboratories, Inc., Hercules, CA, http://www.bio-rad.com). At least 300 cells were counted for each staining.' P$ |$ ?; h' T9 T
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RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
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Total RNAs were extracted from murine undifferentiated NS using the RNeasy Mini Kit (Qiagen Inc.). Reverse transcription-polymerase chain reaction (RT-PCR) was performed as described in . Primers were designed using the Primer3 software (Qiagen Inc.). Each PCR was performed in parallel with RT(¨C) samples in which the reverse transcriptase was omitted to check that the amplified product was not derived from genomic DNA. ß-Actin amplification was used as an internal positive control. A PCR amplification was considered positive if a unique band at the exact size was obtained. Primer sequences, expected size, and details of amplification conditions are provided in the supplemental online data.

Flow Cytometry$ r. c1 U& A' z$ }' c. O
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For cell sorting, NS were harvested by centrifugation, incubated in HBSS without Ca2  and Mg2  (HBSS¨C) (15 minutes, 37¡ãC), mechanically dissociated with a yellow tip, and rinsed in HBSS¨C. The cell suspension (approximately 20 x 106 cells) was incubated (15 minutes, 4¡ãC) with the primary antibodies (NG2 ) or control antibodies to obtain the background fluorescence (rabbit IgG for NG2; IgM for A2B5) diluted in 0.5% PBS-bovine serum albumin (BSA) (crystalline bovine albumin; Invitrogen Corporation). Cells were rinsed in HBSS¨C and then incubated (15 minutes, 4¡ãC) with secondary antibodies (1:400; goat anti-rabbit IgG Alexa Fluor 488 or goat anti-mouse IgM Alexa Fluor 488; Invitrogen Corporation). Cells were rinsed in HBSS¨C and resuspended in 2 µg/ml propidium iodide. To sort cells from embryonic day 13.5 (E13.5) spinal cord, approximately 20 embryos were dissected. Spinal cord cells were mechanically dissociated using a yellow tip, passed through a 40-µm strainer to obtain a single cell suspension, and rinsed with HBSS¨C to eliminate cell debris. Labeling was performed as described above. Cell sorting was performed using a FACSVantage SE Turbosort (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) equipped with a 488 nm Laser Sapphire 488-20. A red fluorescence (for propidium iodide) and a size threshold were used to eliminate dead cells and cellular debris. Cells were collected in PBS or in NS medium. After cytometry, cells were directly seeded in 25-cm2 flasks to allow NS formation at clonal density (five cells per microliter, equivalent to 600 cells per cm2). The total number of NS formed was assessed 10 days later by carefully scanning the entire flask with a binocular microscope.

RESULTS
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NS Are Composed Mainly of Radial Glial Cells Expressing NG2, A2B5, PDGFR-, and Sox9
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NS derived from adult and embryonic CNS are composed of cells expressing radial glial markers (BLBP, RC2, and GLAST). On differentiation, these NS generate mainly astroglial cells, whereas neuronal cells and oligodendrocytes account for only a small percentage of cells (typically 1%¨C10%) . We further quantified the broad expression of the A2B5 and NG2 on NS cell surfaces by flow cytometry analysis (Fig. 2C) (NG2  cells: 78% ¡À 4%, n = 4; A2B5  cells: 94% ¡À 3%, n = 6). The presence of NG2 and PDGFR- was confirmed at the mRNA level by RT-PCR (Fig. 2D). NS contained a small proportion of GFAP  cells (9.8% ¡À 4%, n = 5) and most of them (89% ¡À 4%, n = 5) were found to strongly express NG2 (Fig. 2E).
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Figure 2. Characterization of neurospheres (NS) cells. (A): Immunolabeling of 4-day-expanded clonal NS derived from embryonic day 13.5 (E13.5) mouse spinal cord. All NS are stained for NG2, A2B5, and PDGFR- (green). Nuclei (blue) are stained with Hoechst. Scale bar = 100 µm. (B): At higher magnification, A2B5 (red), NG2 (green), and PDGFR- (green) antibody stained the majority of cells within NS. Nuclei are labeled with Hoechst (blue) or propidium iodide (red). NG2 and PDGFR- stainings are confocal photographs. Scale bar = 10 µm. (C): Flow cytometry analysis of NS cells revealed that most NS cells express NG2 and A2B5. Diagrams represent the number of cells (counts) versus the levels of fluorescence (FL1-H) using control nonimmune antibodies (immunoglobulin G , IgM) or NG2/A2B5 antibodies. Green arrow delimits the highly fluorescent cell fraction used for assessing NS formation from A2B5  and NG2  cells (Fig. 4A and text). Red arrow delimits the NG2¨C fraction used for cytometry in Figure 4A. (D): Detection of NG2 and PDGFR- mRNA expression in NS cells by reverse transcription-polymerase chain reaction analysis. Reverse transcriptase was omitted (¨C) in control experiment. (E): A GFAP  (green) cell (arrow) expressing NG2 (red). In this experiment, small NS (10¨C20 cells) were mechanically dissociated before plating and fixation. Scale bar = 10 µm. (F): Percentage of embryonic or adult-derived NS expressing the indicated markers. This experiment is representative of two experiments. (G): NG2 (green) and GFAP (red) double-labeling of 12-µm-thick cryostat sections of 2-week-expanded NS. Stainings were analyzed by confocal microscopy. Scale bar = 50 µm. (H): Double-labeling of dissociated NS cells with NG2 (green) and radial glial cell markers (red): GLAST, RC2, and BLBP. Scale bar = 10 µm. (I): Percentage of NG2  cells immunoreactive for BLBP, RC2, and GLAST in growing NS. Values are means ¡À SEMs of three independent cultures. Abbreviations: bp, base pair; GFAP, glial fibrillary acidic protein; PDGFR, platelet-derived growth factor receptor.: x6 z9 {) d  i! ~, c

As a comparison, we derived NS from E13 embryonic cortex, striatum, and adult spinal cord and examined their phenotype. Regardless of their origin, all NS strongly expressed A2B5, NG2, and PDGFR- (Fig. 2F). Fluorescence-activated cell-sorting analysis of NS cells derived from embryonic striatum confirmed that more than 80% of cells were NG2 .

The phenotype of larger embryonic spinal cord NS (100¨C500 µm), which had been expanded for 2 weeks and contained several hundred cells, was also examined (Fig. 2G). To avoid artifacts due to limited antibody penetration, the spheres were embedded and sliced before staining. Like small spheres, all large spheres contained a majority of cells expressing NG2, A2B5, and PDGFR- (data not shown). However, heterogeneity was evident as patches of NG2¨C cells were apparent in some NS (Fig. 2G). These NG2¨C cells often colocalized with GFAP staining, suggesting that astroglial differentiation occurs in the large spheres. To ascertain that the wide expression of the markers NG2, A2B5, and PDGFR- was not transient, E13.5 spinal cord-derived NS were cultured for up to 16 passages (approximately 3 months). The phenotype of these long-term cultured NS cells appeared to be identical to that of NS that had been passaged four times (data not shown). To more precisely define the identity of cells expressing NG2, colabeling was carried out with markers typical for radial glial cells (BLBP, RC2, GLAST). As shown in Figure 2H and 2I, we found that in embryonic spinal cord NS, the vast majority of cells expressing NG2 were also positive for BLBP, GLAST, and RC2. Identical results were obtained with NS derived from adult spinal cord and embryonic striatum (data not shown). In summary, this phenotypic analysis indicates that NS are composed mainly of remarkable radial glial cells expressing NG2, A2B5, PDGFR-, and Sox9 and partly Nkx2.2.9 v% `' M* P3 F

A2B5, NG2, and PDGFR- Expression Persists in Neuronal Cells after NS Differentiation) J) D6 G: j5 G* {9 F4 z! V
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We next examined the evolution of the NG2, A2B5, and PDGFR- markers when embryonic spinal cord NS were allowed to differentiate. After plating on an adhesive substrate and in absence of growth factors, these NS typically generate 5%¨C10% neurons, 80% astroglial cells, and 1%¨C5% oligodendrocytes, which were detected by ß3-tubulin, GFAP, and O4 stainings, respectively . Double-labelings were carried out with these markers and A2B5, PDGFR-, and NG2. Surprisingly, most NG2  cells (approximately 90%) coexpressed ß3-tubulin (Fig. 3A, 3C), whereas less than 1% of NG2  cells coexpressed the astrocytic marker GFAP or the oligodendrogenic markers O4 and CNPase (Fig. 3B, 3C). The same NG2 profile was observed with differentiated NS derived from embryonic striatum (Fig. 3C). The neuronal phenotype of NG2  cells was confirmed by colabeling these cells with additional neuronal markers, namely Map2ab, NeuN, and -internexin (Fig. 3A, 3C). Similarly to NG2 detection, PDGFR- and A2B5 detection was associated mainly with cells expressing neuronal markers such as ß3-tubulin and Map2ab after differentiation and not with cells expressing the glial markers GFAP, O4, or GalC (Fig. 3D¨C3F). Of note, in addition to neurons, strong staining for A2B5 was observed in apoptotic cells that are common after NS differentiation (Fig. 3E, arrowhead). Given that in the undifferentiated state, NG2, A2B5, and PDGFR- were found to be expressed by the majority of NS cells, whereas after differentiation their expression is restricted to the small neuronal population (less than 10%), these results are indicative of their downregulation in glial cells after differentiation.% \% D2 ^5 e* s/ O) j4 O7 w" M8 M0 f

Figure 3. Expression of A2B5, NG2, and PDGFR- in neuronal cells after differentiation. (A): Double-immunostaining of differentiated neurospheres (NS) revealed that NG2  cells (green) coexpressed (arrows) several neuronal markers ß3-tubulin (red), Map2ab (red), NeuN (red), and -internexin (red). (B): Double-immunostaining of differentiated NS revealed that NG2  cells (green) did not coexpress GFAP (red) or O4 (red). (C): Percentage of NG2  cells ¡À SEM (n = 3) coexpressing astrocytic marker (GFAP), oligodendrogenic markers (O4 and CNPase), and neuronal marker (ß3-tubulin, Map2ab, NeuN, and -internexin) in differentiated embryonic spinal cord (upper histograms) and embryonic striatum (lower histograms) NS cells. (D): Double-immunostaining of differentiated spinal cord NS cells revealed that PDGFR-  cells (green) coexpressed (arrows) ß3-tubulin (red) and Map2ab (red), but not GFAP (red) or O4 (red). Nuclei are labeled with Hoechst (blue). (E): Double-immunostaining of differentiated NS cells revealed that A2B5  cells (green) coexpressed (arrows) ß3-tubulin (red) and Map2ab (red), but not GFAP (red) or GalC (red). Note that most dead cells, displaying a typical apoptotic nucleus, also stained for A2B5 (arrowhead). Nuclei are labeled with Hoechst (blue). (F): Percentage of A2B5  cells and PDGFR-  cells ¡À SEM (n = 3) coexpressing differentiated cell markers. Scale bars = 10 µm. Abbreviations: GFAP, glial fibrillary acidic protein; PDGFR, platelet-derived growth factor receptor.6 z7 }: a; F" a  T

NS-Forming Cells in NS Expressed NG2  and A2B5 : p8 d( \1 D4 A7 H

NS typically contain only a small population (1%¨C10% for embryonic spinal cord NS  (supplemental online Fig. S2). After four passages, cells were dissociated, mixed 50:50 with nontransgenic NS cells, and allowed to grow for 7 days. Cells were then sorted for negative and positive GFP expression (supplemental online Fig. S2) and allowed to form NS at clonal density for 10 days. All observed NS (129/129) derived from GFP -sorted cells were GFP , and 151/152 NS derived from the GFP¨C fraction were GFP¨C. This demonstrates that the cellular contamination between fractions in our cytometric analysis was below 1%, thus validating this approach for analyzing the phenotype of NS-forming cells. We then established that dissociated NS cells, incubated with control nonimmune IgG and processed for cytometric analysis, generated new NS at an NS-forming rate (nsfr) of 7.6% ¡À 0.2% (n = 5) (Fig. 4A). In a parallel experiment, dissociated NS cells were incubated with NG2 and strongly NG2  fluorescent (55% of cells; green arrow-delimited cell fraction on Fig. 2C) and NG2¨C cells (2.6% of cells; red arrow-delimited cell fraction) were sorted and allowed to form NS at clonal density. NG2  cells formed NS at a rate equivalent to IgG control: nsfr = 7.0% ¡À 0.3% (n = 5) (Fig. 4A). In contrast, NG2¨C cells showed an nsfr of only 0.6% ¡À 0.2% (n = 2). To confirm that NS generated from the NG2  fraction are truly derived from NG2  cells, we submitted the NG2  fraction to a second round of cytometry. These NG2  double-sorted cells show the same nsfr as that of double-sorted control IgG-incubated cells (Fig. 4A). The similar decrease of the nsfr observed in both double-sorted NG2- and IgG-incubated cells is likely due to a reduction of cellular viability induced by the second cytometry. To check that the NG2  cell-derived NS were passageable and multipotent, these were cultured for five passages, allowed to differentiate, and processed for triple-labeling. Of the more than 50 NS tested, all were multipotent as indicated by the presence of Map2ab  neurons, GFAP  astrocytes, and O4  oligodendrocytes (Fig. 4B). Because NS are composed of a majority of NG2  cells and because these cells have an nsfr equivalent to that of the whole NS cell population (in contrast to NG2¨C cells), we conclude that the majority of multipotential and self-renewing NS-forming cells in NS are NG2 . Using a similar analysis with A2B5 marker (green arrow-delimited cell fraction on Fig. 2C), we found that that the vast majority of NS-forming cells express this marker (not shown).

Figure 4. Acquisition of NG2 expression in vitro. (A): Neurospheres (NS)-forming rate (nsfr) of NG2 -, NG2¨C-, and immunoglobulin G (IgG)-sorted cells derived from embryonic day 13.5 (E13.5) embryonic spinal cord NS after one or two cycles of cytometry. The collected NG2  and NG2¨C fractions are indicated in Figure 2C. This experiment is representative of three independent experiments. (B): Example of a triple-immunostaining carried out on a differentiated NS generated from clonal expansion of NG2  cells sorted from NS. Multipotentiality is demonstrated by the presence of Map2ab  (red), GFAP  (green), and O4  (blue) cells. Scale bar = 10 µm. (C): Immunostaining of 12-µm-thick cryostat sections of E13.5 mouse thoracic spinal cord with NG2 (left, green) and RC2 (right, green). NG2 staining is associated mainly with vessels, whereas RC2 stained radial glia cells. Nuclei are labeled with Hoechst (blue). Scale bar = 100 µm. (D): Flow cytometric analysis of freshly dissociated E13.5 spinal cord cells labeled with IgG and NG2 antibody. Diagrams represent the number of cells (counts) versus the levels of fluorescence (FL1-H). The NG2¨C cell fraction (area delimited by red arrows) and the NG2  cell fraction (delimited by blue arrows) were sorted out separately and used in (G). Fluorescence background was set using spinal cord cells labeled with nonimmune IgG (left). (E, F): Time-course expression of NG2 in E13.5 embryonic spinal cord culture. Cells from freshly dissected spinal cords were directly plated and fixed (day 0 ) or seeded at 400 cells per microliter in FGF2/EGF-containing NS media on poly-2-hydroxyethyl-methacrylate-coated T25. An aliquot of the cellular suspension was collected at days 1, 4, and 7, mechanically dissociated, plated, and stained for NG2. (E): Example of NG2  cells (green) present in the cellular suspension at D0 and D7. Nuclei are labeled with Hoechst (blue). Scale bar = 20 µm. (F): Percentage of NG2  cells ¡À SEM (n = 3 independent experiments) in the cellular suspension at the indicated day. (G): nsfr of NG2¨C- and IgG-sorted cells (one and two cycles of cytometry) derived from freshly dissociated E13.5 embryonic spinal cord. The NG2¨C and NG2  fractions are delimited in (D). This experiment is representative of three experiments. (H): NG2 immunolabeling (green) of NS derived from E10 (left) or E13.5 (right) spinal cord NG2¨C-sorted cells. Most, if not all, cells are NG2 . Nuclei are labeled with Hoechst (blue). Scale bar = 10 µm. Abbreviations: EGF, epidermal growth factor; FGF2, fibroblast growth factor 2; GFAP, glial fibrillary acidic protein.; Y8 P% Q0 J) @) E, T! g- S

NS Are Derived from NG2¨C Cells in the Embryonic Spinal Cord

Having established in NS the expression of NG2 in radial glia cells and NS-forming cells, we then examined the in vivo situation. The expressions of RC2 and NG2 markers were examined on E13.5 spinal cord slices (Fig. 4C). As expected, RC2 mainly stained cells with a radial morphology. In contrast, as observed by others at this stage of development, NG2 was barely expressed and the staining was associated mainly with vessels ). After 5 days, E10 NS were examined for NG2 expression and 34/34 showed intense NG2 labeling (Fig. 4H, left). These results indicate that at E10 and E13.5, NS are derived from NG2¨C embryonic NSC that rapidly acquire this marker in vitro.

Expression of Olig2 and Mash1 in Growing and Differentiated NS

The detection in NS cells of markers typically associated with the oligodendrogenic lineage in vivo prompted us to analyze the expression of bHLH transcription factors known to be important for oligodendrocyte formation (Olig1, Olig2, and Mash1) ) were positive, whereas GFAP  and O4  cells were negative. These results demonstrate the wide expression of some bHLH oligodendrogenic/neurogenic factors in NS cells and their persistence in neuronal cells after differentiation.
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Figure 5. Expression of oligodendrogenic/neurogenic transcription factors in growing and differentiated neurospheres (NS). (A): Olig1, Olig2, and Mash1 mRNA expressions were detected in NS cells by reverse transcription-polymerase chain reaction analysis. Reverse transcriptase was omitted (¨C) for negative control. (B): Immunostaining of Mash1 (green) and Olig2 (green) in NS (confocal microscopy). Nuclei are stained with propidium iodide (PI) (red). Upper photographs show small NS (4 days old). Most, if not all, NS cells expressed Olig2 (green) and Mash1 (green). Scale bar = 10 µm. Lower photographs show large NS (2 weeks old). Mash1 and Olig2 stainings are merged with red nucleus staining. Olig2 staining appears homogenous whereas Mash1 is expressed by a subset of cells. Scale bar = 25 µm. (C): Double-immunostaining of differentiated NS cells revealed that Olig2  cells (red) coexpressed O4 (green), Map2ab (green), and ß3-tubulin (green) but not glial fibrillary acidic protein (GFAP) (green). For the ß3-tubulin staining, left and right photographs show Hoechst and Olig2 stainings, respectively. Scale bar = 20 µm. (D): Example of a Map2ab  (green) cell (arrow, left panel), derived from embryonic striatum NS, with an Olig2  (red, right) nucleus. (E): After differentiation of embryonic day-13.5 spinal cord NS, Mash1  cells (red) do not express GFAP (arrow, upper photograph) or O4 (middle photograph). Approximately 20% of Mash1  cells expressed doublecortine (lower photograph). Scale bar = 20 µm. Abbreviations: bp, base pair; MW, molecular weight.

Olig2 Expression Is Induced by Cell Culture in CNS and PNS Cells/ g, f3 b: v  [) J! ?
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The wide expression of Olig2 in embryonic spinal cord NS contrasts with the small number of cells expressing this marker in the ventral E13.5 spinal cord (Fig. 1A). This suggested that, as for NG2, Olig2 expression may be induced under culture conditions. To examine this possibility, E13.5 spinal cord cells were cultured nonadherently in the presence of FGF2/EGF growth factors, and Olig2 expression was monitored for 4 days. As shown on Figure 1B, immediately after the seeding, less than 1% of cells were positive, whereas after 7 days, approximately 80% of the cultured cells displayed strong nuclear Olig2 staining. The vast majority of these olig2  cells expressed the radial glial marker RC2 together with NG2 and A2B5 (not shown). This strong and rapid increase in expression is indicative of induction of the Olig2 bHLH protein in neural cells under cell culture conditions. It was recently reported that the FGF2 growth factor used in the media could be responsible for this effect . To definitively establish that Olig2 could be induced by culture conditions in embryonic spinal cord cells, we carefully dissected the dorsal part of E13.5 spinal cord, which is devoid of Olig2  cells (Fig. 1A), and seeded cells in adherent conditions with and without growth factors. One hour after plating (day 0), not a single Olig2  cell could be detected by immunofluorescence (Fig. 1C). However, after 1 and 2 days of culture, Olig2  cells were readily detected in the condition with FGF2 EGF but not in the control condition (Fig. 1C and Fig. S3A), even though the number of cells was comparable for the two conditions (supplemental online Fig. S3). The same result was obtained when the cells were grown on a nonadherent substrate, and NS formation was observed (data not shown). These Olig2  cells expressed A2B5 (100%) and the glial radial marker RC2 (89% ¡À 3%, n = 3) (supplemental online Fig. S4)." R! a; R3 C4 p; |0 p
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To obtain further insight into potential links between growth factor stimulation and Olig2 expression, we tested the consequences of reintroducing FGF2 and EGF in differentiated NS, considering that Olig2 is not expressed by GFAP  cells (Fig. 5C). Notably, after NS differentiation, GFAP  cells never expressed nestin (supplemental online Fig. S5) but approximately 30% of them expressed vimentin (supplemental online Fig. S5). This indicates that NS-derived astrocytes have various degrees of immaturity. After 4 days of differentiation without growth factors, FGF2 and/or EGF were added again and Olig2 expression was monitored by immunofluorescence. Surprisingly, within 24 hours, almost 100% of GFAP  (n = 3) cells displayed a strong Olig2 nuclear staining (Fig. 1D). This was observed with each of the growth factors added separately or in combination (not shown).

FGF signaling has recently been implicated in the deregulation of dorsoventral patterning ). An immunofluorescence analysis carried out on E13.5 slices indicated the absence of Olig2  cells and RC2  in DRG (Fig. 1A, 1E). This absence of Olig2  or RC2  cells was also confirmed in acutely dissociated cells from E13.5 DRG. We then cultured acutely dissociated PNS cells nonclonally in NS media containing FGF2 and EGF. Extra precautions were taken during PNS dissection to avoid contamination with CNS cells (see the protocol in the supplemental online data and supplemental online Fig. S6). After 10 days, 83.6% ¡À 4.3% (n = 5) and 89.7% ¡À 4.7% (n = 4) of cells expressed RC2 and Olig2, respectively (Fig. 1F). This indicates an induction of these two markers in PNS cells. Remarkably, after reseeding these cells at clonal density, few NS were observed (  i2 Z# @3 }8 D: {9 g" ~

Mash1 and Olig2 Are Not Sufficient to Induce NG2 in a Neural Precursor Cell Line4 X/ R4 z2 Y9 d9 l" j, _  V
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Mash1 expression was recently reported to be induced in neural precursor cells by FGF2 and EGF . Immunodetection revealed that native MHP36 cells were negative for PDGFR-, Olig2, and Mash1 and very few cells were NG2  (data not shown). MHP36 cells were transfected in the nonimmortalizing conditions to eliminate expression of the oncoprotein SV40 TAg, with mouse Olig2 and Mash1 expression plasmids, and then were examined 48 hours later for Olig2, Mash1, and NG2 or PDGFR- expression by triple-labeling. As shown in Figure 1I, double-transfected cells expressing Olig2 and Mash1 were NG2¨C. They did not express PDGFR- either (data not shown). The same results were obtained with cells transfected with Olig2 or Mash1 separately. These results indicate that Olig2 and Mash1 bHLH proteins appear not to be sufficient to trigger the expression of NG2 and PDGFR- in the MHP36 neural precursor cell line.

DISCUSSION

Here, we report new data on neural precursor cells cultured as NS. We observed that NS derived from several adult and embryo regions contain cells with a striking phenotype in which radial glia markers (RC2, BLBP, and GLAST) are coexpressed with markers typical of the oligodendrocyte lineage (A2B5, PDGFR-, NG2, and Olig2). After differentiation, the latter remain principally expressed by neuronal cells. Using cytometry, we showed that in growing NS, the small population of multipotential self-renewing NS-forming cells are A2B5  NG2 . However, we demonstrated that NSC in the embryonic spinal cord were initially NG2¨C but acquired this marker in vitro. NG2 and Olig2 were found to be rapidly induced by cell culture conditions in neural precursor cells, and Olig2 expression was also induced in astrocytes and PNS cells after EGF/FGF treatment. However, Olig2 by itself, or in combination with Mash1, did not appear to be sufficient to induce NG2 expression in a multipotent neural cell line. These data are summarized on Figure 6.- u" E2 l5 f5 f  {! r6 A/ ]

Figure 6. Schematic summary of findings. In E13.5 embryo spinal cord, neural stem cells are initially NG2¨C. When placed in culture, these cells could be directly converted into NG2  cells. Alternatively, they could generate new NG2  neural stem cells by asymmetric division. After several passages, multipotential self-renewing neurospheres (NS)-forming cells are mainly NG2  in vitro. These cells generate NS composed mainly of radial glia cells expressing NG2, A2B5, PDGFR-, Sox9, and Olig2. A fraction of these cells (1%¨C10%) are able to form new multipotential passageable NS after dissociation and replating. After growth factor removal and adhesion, NS cells differentiate into glial cells and neurons. These latter still express NG2, A2B5, PDGFR-, and Olig2. Reintroduction of FGF2/EGF in the media induces Olig2 expression in GFAP  cells and provokes formation of new multipotential passageable NS . Abbreviations: CNS, central nervous system; EGF, epidermal growth factor; FGF2, fibroblast growth factor 2; GFAP, glial fibrillary acidic protein.

NS is a widely used model for the expansion and study of neural precursor cells in vitro. NS have also been extensively used for cellular therapy of neurodegenerative and demyelinating diseases. However, both the identity of cells that initiate NS and cells that compose NS are still poorly defined. We and others have reported that NS are composed of radial glial cell-like expressing GLAST, RC2, and BLBP . Thus, it is plausible that, like NG2, NS-forming cells may acquire A2B5 in vitro.

Although NS appear phenotypically homogenous, only a small fraction of the cells they contain are able to form new NS. The identity of this NS-forming subpopulation is still largely unknown. Only recently, Hoechst-based cell sorting (side population method) has allowed purification of these cells . After four passages, our cytometric analysis showed that NS-forming cells are mostly, if not all, NG2  A2B5 , thus they do not differ from the bulk of NS cells for these markers. Because these cells are initially derived from NG2¨C cells, this indicates that a phenotypic transition of multipotential self-renewing NS-forming cells from NG2¨C to NG2  is likely to occur in vitro (Fig. 6). It remains to be established whether this process occurs by direct conversion of the NG2¨C cells into NG2  cells or by production of NG2  cells from NG2¨C cells by asymmetric division (Fig. 6). The above data argue for the existence of a diversity of NSC displaying several phenotypes, including cell surface molecules. As observed in vitro, it is likely that the phenotype of NSC changes during CNS development.! P5 T6 y) ~  `+ J8 k1 e# y! F' x

After NS differentiation, we observed that A2B5, PDGFR-, NG2, and Olig2 remained expressed by neuronal cells whereas glial cells were largely negative (except for a fraction of the oligodendrocytes which remains Olig2 ) (Fig. 6). Numerous in vitro studies have used these markers to identify cells that belong to the oligodendrocyte lineage; however, our data together with other studies clearly indicate that these markers label a variety of cell types, including NS-forming cells, radial glia, and neurons. Thus, care must be taken when interpreting their presence, especially in vitro. Astrocytes derived from NS lack A2B5 expression and thus may be considered as type I astrocytes .( ^& d: H: g3 Z* s' B7 X
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What are the mechanisms by which embryonic spinal cord cells and NS-forming cells acquire NG2 in vitro? As for NG2, the Olig2 bHLH gene was found to be induced in culture. After differentiation, Olig2 was also expressed in NG2  neurons but not in NG2¨C astrocytes. We thus tested whether forced expression of Olig2 was sufficient to trigger the expression of NG2 gene in a readily transfectable multipotent neural cell line (MHP36). However, we found that in these cells, Olig2 alone, or in combination with another cell-culture induced .: Y1 x1 i3 p5 y, n! u4 a+ x; ^# Y
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While this study was in progress, the growth factor FGF2 used in the medium was shown to be involved in the induction of Olig2 in neural precursor cells . The induction of Olig2 in NS-derived GFAP  cells may be related to their immature state given that approximately 30% of these cells express vimentin. It would be interesting to see whether astrocytes derived from adult CNS also express Olig2 when treated with growth factors.

These data indicate that several types of mitogenic factors have an influence on the expression on bHLH proteins that are known to play crucial roles in governing cell fate. It is well known that in addition to being mitogenic factors, at least some members of the FGF family are endowed with morphogenetic properties .; R# }1 d$ Q. \" ~* g! ~" s1 Q

As recently reported by others  may be implicated in this deregulation./ o9 }( U! O. t3 W3 r  S/ X
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In conclusion, our results provide additional support for the emerging idea that culture conditions modify NSC fate and phenotype. This is probably due to the inherent plasticity of stem cells. This might first appear as an obstacle for developing cellular biotherapies targeted at treating diseases in which specific types of neurons are affected. Yet it seems that these modifications are not irreversible because, in vitro, the use of different culture conditions or morphogens can direct stem cells to generate specific types of neurons (for instance , that could help NS cell survival in the lesioned tissue., q( t; v2 i! v! u9 K/ o
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DISCLOSURES5 x4 Y2 s/ `4 {! Z" b8 s

The authors indicate no potential conflicts of interest.! X7 ^/ \7 |; e8 \& k2 O

ACKNOWLEDGMENTS& J: t3 ~( B/ r# q8 V& J- T

This work was supported by the "Association Demain Debout" (Saint G¨¦rand le Puy, France), the "Association Française contre les Myopathies" (Evry, France), the "Institut pour la Recherche sur la Moelle Epini¨¨re," the "Vertical association," the "Association pour la Recherche sur le Cancer," the "Fondation pour la Recherche M¨¦dicale," and the European FRP6 STREP Rescue. We thank Dr. Keith Langley and Dr. H. Hirbec for critical reading of the manuscript, the "Minist¨¨re de la recherche et des nouvelles technologies" for supporting the imaging platform, Dr. F. Guillemot for Mash1 reagents, Dr. N. Heintz for BLBP antibody, P. Jay and J. Blache for Sox9 antibody, and I. Acquatella for technical help for primary cultures of dorsal root ganglia.
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