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TISSUE-SPECIFIC STEM CELLS |
aINSERM U583, Physiopathologie et Thérapie des déficits sensoriels et moteurs, Institut des Neurosciences de Montpellier, Hôpital St ELOI, Montpellier Cedex 05, France;
bDivision of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Myodaiji, Okazaki, Japan;
cService Regional INSERM de cytométrie en flux, Montpellier Cedex 05, France
Key Words. Neural stem cells • Oligodendrocytes • NG2 • Proteoglycan • Peripheral nervous system • Neurospheres • Cytometry Plasticity
Correspondence: Jean-Philippe Hugnot, M.D., Ph.D., INSERM U583, Physiopathologie et Thérapie des déficits sensoriels et moteurs Institut des Neurosciences de Montpellier, Hôpital St ELOI, BP 74103 80, avenue Augustin Fliche 34091 Montpellier Cedex 05, France. Telephone: 33 4.99.63.60.08 Fax: 33 4.99.63.60.20 e-mail: hugnot{at}univ-montp2.fr
Received November 10, 2005;
accepted for publication October 10, 2006.
First published online in STEM CELLS EXPRESS October 19, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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). 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– 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.
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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 [6]. Promising results have been obtained in models of multiple sclerosis, Parkinson's disease, brain tumors, and spinal cord lesions. However, the mechanisms whereby NS cells provide therapeutic benefits are still unclear. In addition to replacing lost cells, neural precursor cell transplantation appears to be able to reduce glial scar [6], to rescue dysfunctional neurons probably through the release of trophic factors [7], and to recruit endogenous neural precursor cells for regeneration [6]. Recently, NS cells have also been shown to induce apoptosis of CNS-infiltrating T cells [8].
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 [9–11], but their long-term passageability has been thoroughly studied for GFAP+ cell-derived NS only. Apart from the neurogenic regions, some A2B5+ cells located in the human subcortical white matter are also endowed with the capacity of generating passageable multipotential NS [12]. More recently, postnatal brain microglial cells have been shown to form multipotential NS-like structures, but the very high serum concentration needed to produce these NS, questions the physiological relevance of this observation [13]. The identity of the NS-forming cells, in contrast to postnatal CNS, in the embryo is largely unknown.
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%–10%). The majority of NS cells express markers of radial glial cells (RC2, GLAST, BLBP) [14–16]. The latter are known to provide a transient scaffold for neuronal cell migration during development, and more recent data indicate that they also serve as neuronal progenitors in all regions of the CNS [17]. However, the radial glia cell population appears to be heterogeneous within and between different brain regions [18]. 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.
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 [19–22]. In contrast, other data argue for the existence of deregulations in the differentiation potential and spatial identity of neural precursors grown in NS in the presence of high concentrations of growth factors [23–26].
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 [PDGFR]-, 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– 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– 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.
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MATERIALS AND METHODS |
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Cell Culture
For NS cultures, acutely dissociated cells were cultured at 37°C 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 2–5 cells per microliter (equivalent to 250–600 cells per cm2) to allow clonal expansion [27]. 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°C). 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 300–500 µm) were harvested under the microscope, individually plated on coverslips, and allowed to differentiate for 4–6 days before processing for triple-labeling.
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(PeproTech). For passaging, cells were incubated for 15 minutes with Versene (Invitrogen Corporation). Transfection experiments were carried out without interferon- and at 37°C (nonimmortalized conditions). Plasmid transfection was performed using Effectene Transfection Reagent (Qiagen Inc., Valencia, CA, http://www1.qiagen.com). Plasmids pcDNA3, pcDNA3 mOlig2 (Dr. H. Takebayashi, Kyoto, Japan) [30], 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.
Immunodetection
Immunodetection was carried out as described in [15] 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°C), 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.
RNA Isolation and Reverse Transcription-Polymerase Chain Reaction
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 [15]. Primers were designed using the Primer3 software (Qiagen Inc.). Each PCR was performed in parallel with RT(–) 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
For cell sorting, NS were harvested by centrifugation, incubated in HBSS without Ca2+ and Mg2+ (HBSS–) (15 minutes, 37°C), mechanically dissociated with a yellow tip, and rinsed in HBSS–. The cell suspension (approximately 20 x 106 cells) was incubated (15 minutes, 4°C) with the primary antibodies (NG2 [1:500], A2B5 [1:500]) 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– and then incubated (15 minutes, 4°C) 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– 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– 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.
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RESULTS |
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, and Sox9 [33]), although these markers have also been reported to be expressed by other cell types, especially in vitro. In addition, we examined the expression of the oligodendrocytic lineage markers CNPase, Nkx2.2 [34], Sox10 [35], and Sox9. The latter is expressed by neuroepithelial cells at the onset of gliogenesis and is a major molecular component in determining their glial fate [35]. NS were derived from E13.5 embryonic spinal cord and passaged four times to avoid any potential contamination with nonproliferative cells. The phenotype of NS expanded for 4 days under clonal conditions (sphere size: approximately 10 cells) was then examined by immunofluorescence. Surprisingly, we found that all spheres derived from embryonic spinal cord displayed a broad expression of the three markers NG2, A2B5, and PDGFR- (Fig. 2A, 2F). A more detailed examination indicated that within spheres, nearly all cells were positive for these three markers (Fig. 2B). Immunodetection performed on dissociated NS revealed that 95% ± 5% (n = 3) cells expressed PDGFR-, which (as observed in other cell types [36]) appeared to be mainly cytoplasmic. Sox 10 and CNPase expression was not detected in undifferentiated NS. In contrast, Nkx2.2 was expressed by a significant number of cells (between 10% and 30%) in all NS (supplemental online Fig. S1) and the majority of NS cells were strongly positive for Sox9 (supplemental online Fig. S1). Interestingly, in NS cells, this transcription factor was confined mainly to the cytoplasm as observed by others during gonad development [37]. We further quantified the broad expression of the A2B5 and NG2 on NS cell surfaces by flow cytometry analysis (Fig. 2C) (NG2+ cells: 78% ± 4%, n = 4; A2B5+ cells: 94% ± 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% ± 4%, n = 5) and most of them (89% ± 4%, n = 5) were found to strongly express NG2 (Fig. 2E).
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(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 (100–500 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– cells were apparent in some NS (Fig. 2G). These NG2– 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.
A2B5, NG2, and PDGFR- Expression Persists in Neuronal Cells after NS Differentiation
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%–10% neurons, 80% astroglial cells, and 1%–5% oligodendrocytes, which were detected by ß3-tubulin, GFAP, and O4 stainings, respectively [15]. 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–3F). 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.
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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) [30, 45–47]. By means of RT-PCR analysis, mRNAs for these genes were detected in embryonic spinal cord NS (Fig. 5A). By means of immunodetection in NS cells, no convincing Olig1 staining was obtained, but Olig2 and Mash1 proteins were readily detected. In small NS (2–4 days of growth), most if not all cells were found to be positive for Olig2 and Mash1 (Fig. 5B). As spheres became larger (10 days of growth), Olig2 was still widely expressed whereas Mash1 expression appeared heterogeneous and negative cells were apparent (Fig. 5B). Olig2 and Mash1 expression was also examined after NS differentiation (Fig. 5C, 5E). The GFAP+ astrocytes were negative for Olig2, whereas (as expected) 75% of the O4+ oligodendrogenic cells strongly expressed Olig2 (Fig. 5C). Surprisingly, the vast majority of Map2ab+ and ß3-tubulin+ neuronal cells were positive for Olig2+ (Fig. 5C). This was also observed with differentiated NS derived from embryonic striatum (Fig. 5D). Regarding Mash1 (Fig. 5E), approximately 20% of doublecortin+ cells (a marker of young neurons [48]) 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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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 [25] and the promotion of radial glial identity of embryonic neural precursor cells [42]. The observed induction of NG2 and Olig2 in cells derived from embryonic spinal cord argues for some deregulation occurring in culture. To obtain further insight into this issue, we examined the expression of the radial glial marker RC2 and of Olig2 in cells derived from E13.5 PNS (dorsal root ganglia [DRG]). 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% ± 4.3% (n = 5) and 89.7% ± 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 (<0.01% of seeded cells). These NS have a phenotype comparable with CNS NS (i.e., expression of the radial glial marker RC2 together with NG2, A2B5, and Olig2 markers) (Fig. 1G). They also express BLBP and GLAST (data not shown). On differentiation, these NS generate ß3-tubulin+ neurons, GFAP+ cells, and CNPase+ cells morphologically indistinguishable from those derived from CNS-derived NS (data not shown). The generated neurons did not express the marker peripherin as would be expected for PNS-derived precursor cells (not shown). As expected, these NS also generated smooth-muscle actin+ (SMA) cells after differentiation (Fig. 1H), but surprisingly, such cells were also observed at a similar abundance after differentiation of E13.5 spinal cord NS (Fig. 1H).
Mash1 and Olig2 Are Not Sufficient to Induce NG2 in a Neural Precursor Cell Line
Mash1 expression was recently reported to be induced in neural precursor cells by FGF2 and EGF [24]. Because both expressions of the Olig2/Mash1 bHLH genes and NG2 seemed to be under the influence of the growth factors, we investigated whether Olig2 and Mash1 overexpression would be able to trigger NG2 expression in a neural precursor cell line. For that, we used a conditionally immortalized NSC line (MHP36) derived from the embryonic hippocampus, which generates glial and neuronal cells on differentiation in vitro and in vivo [28]. 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–. 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.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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, 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– 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.
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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 [54]. 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– cells, this indicates that a phenotypic transition of multipotential self-renewing NS-forming cells from NG2– 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– cells into NG2+ cells or by production of NG2+ cells from NG2– 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.
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 [55]. However, given that these GFAP+ cells were derived from NS radial glia cells coexpressing A2B5, PDGFR-, NG2, and Olig2, one can infer that these markers are downregulated on astrocytic differentiation. Their persistence, and in particular that of Olig2, in neuronal cells is really intriguing. Their morphology suggests that these cells are probably immature. These neurons may be related to the A2B5+ ß3-tubulin+ cells identified in the E12.5 mouse neural tube [56], to young NG2+ neurons described in the mouse neocortex, hippocampus, and subventricular zone [11, 57, 58], or to Olig2+ neurons recently observed in fetal human CNS [59].
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– 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 [24] bHLH factor (Mash1), did not induce NG2 expression. Although the MHP36 cell context is undoubtedly different from that of embryonic spinal cord cells, this analysis indicates that Olig2 is not sufficient by itself and that other transcription factors are probably necessary for NG2 activation in neural precursor cells. This is further demonstrated by the Olig1/2 double-knockout mice, which lead to a loss of oligodendrocytes with preservation of NG2 expression [60], and thus a direct role of Olig bHLH genes in NG2 expression is unlikely. As observed in neocortical cultures, the acquisition of NG2 by Olig2+ cells may require coactivation by mitogen-activated protein kinase and phosphatidylinositol-3 kinases [43].
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 [24, 25, 43, 49]. Olig2 factor was found to play a role in NS self-renewal as well as in their differentiation into neurons and oligodendrocytes [24]. FGF2 is also used for promoting Olig2 and PDGFR- expression in embryoid body derived from mouse embryonic stem cells [61]. We extended these observations by showing that Olig2 can be induced in E13.5 embryonic dorsal spinal cord and in PNS cells cultured in NS media. We also made the striking observation that Olig2 is also rapidly expressed in NS-derived GFAP+ astrocytes treated by FGF2. Because Olig2 protein was not observed in the cytoplasm of nontreated astrocytes, it is unlikely that the strong nuclear Olig2 staining we detected after the cytokine treatment was due to a change of localization of the protein as recently reported [62]. EGF treatment of GFAP+ cells also induced strong Olig2 expression. The similar effects of FGF2 and EGF are likely to arise from the vast overlap of their signaling pathways [63]. 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 [64]. This appears to be achieved to some extent by repressing other morphogens such as bone morphogenetic proteins (BMP) [65]. It has been recently proposed that oligodendrocytes can be derived from dorsal spinal cord independently of sonic hedgehog signaling by evasion from dorsal BMP-inhibiting activities [66–68]. The addition of a BMP inhibitor (noggin) promotes the formation of Olig2+ cells from dorsal spinal cord cells in culture [66]. We recently reported that in the embryonic spinal cord NS, growth factors have a profound effect on the expression of endogenous BMPs, notably by repressing BMP4/6 [15]. It is thus tempting to speculate that FGF2 promotes Olig2+ expression partly by inhibiting endogenous BMPs that otherwise might provoke bHLH protein degradation [69].
As recently reported by others [70], NS could also be obtained from embryonic PNS. We found that these NS exhibit a phenotype comparable with CNS NS. Although we cannot absolutely exclude contamination from adjacent CNS, this is unlikely considering the dissecting protocol we used. As expected for PNS precursor cells, these NS give rise to cells expressing SMA, as also observed previously [70]. However, such cells were also obtained from E13.5 embryonic spinal cord NS. SMA+ cells have also been observed after differentiation of embryonic CNS stem cells derived from several regions and from adult subventricular zone cells [71, 72]. Their presence appears to be linked to a CNS/neural crest transition due to BMP/FGF2 signaling or low-density culture conditions. The generation of SMA+ cells may thus represent an additional aspect of deregulation of CNS precursor cells cultured under NS conditions. The high level of endogenous BMP7 detected in embryonic spinal cord NS [15] may be implicated in this deregulation.
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 [73]). The strong expression of NG2 by NS cells could raise concern on the use of these cells for transplantation purposes. In fact, NG2 is a chondroitin sulfate proteoglycan that has been described as one of the main inhibitory molecules for axon growth present in the glial scar after CNS lesions. It appears to be obstructive to axonal regrowth by inducing growth cone collapse [74]. However, given that after in vitro differentiation, NS mainly generate astrocytes that do not express NG2, it is likely that on transplantation, a similar downregulation of NG2 expression occurs. Alternatively, it is possible that NG2 has a positive influence on the therapeutic effects observed with NS cell transplantation. Actually, NG2 is necessary for PDGFR- receptor signaling [75], and both NG2 and PDGFR- have been shown to play an important role in the migration of several cell types [44, 76]. Thus, their expression in NS cells may participate in the remarkable capacity of these cells to migrate in vitro and in vivo. Moreover, NG2 binds several molecules, including cytokines (FGF2, platelet-derived growth factor, and angiostatin) and extracellular cellular matrix molecules (collagen, tenascin, and laminin) [77], that could help NS cell survival in the lesioned tissue.
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