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Article |
Address correspondence to Fred H. Gage, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, CA 92037. Tel.: (858) 453-4100. Fax: (858) 597-0824. email: gage{at}salk.edu
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Abstract |
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Key Words: glia; neural stem cell; insulin; BMP; hippocampus
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Introduction |
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Recent work by Song et al. (2002) directly examined the idea that different environments can influence the fate specification of neural stem cells. Astrocytes from neurogenic regions (but not nonneurogenic regions) can preferentially direct multipotent adult neural stem cells to differentiate into neurons. Interestingly, these neural stem cells appear to preferentially differentiate into oligodendrocytes when cultured together with hippocampus-derived neurons. These results suggest that specific signals from different CNS environments exist for lineage-specific differentiation. Examples of extrinsic factors important for neuronal and astroglial differentiation of multipotent adult neural stem cells have already been identified (Takahashi et al., 1999; Lim et al., 2000; Tanigaki et al., 2001). However, little is known regarding the control of oligodendrocyte differentiation of multipotent adult neural stem cells.
Insulin-like growth factors (IGFs) comprise one class of molecules that have effects on oligodendrocyte biology during development (D'Ercole et al., 1996). IGFs (IGF-I, IGF-II) and insulin can all independently promote the survival of purified oligodendrocytes in culture (Barres et al., 1993). Furthermore, IGFs have important roles in the proliferation and differentiation of cells that have already committed to an oligodendroglial lineage during development (McMorris et al., 1986; McMorris and Dubois-Dalcq, 1988). In addition, transgenic and knockout mouse models have revealed in vivo effects of IGFs on oligodendrocyte development and myelination. Overexpression of IGF-I in transgenic mice results in increased brain size and myelin content (Carson et al., 1993; Ye et al., 1995). Conversely, IGF-I knockout mice have smaller brains, reduced oligodendrocyte numbers, and CNS hypomyelination (Baker et al., 1993; Carson et al., 1993; Beck et al., 1995; Ye et al., 2002).
In this paper, we show that IGF-I can preferentially induce the differentiation of multipotent adult neural progenitor cells into oligodendrocytes. Using a modeling approach, we show that the IGF-Iinduced increase in oligodendrocyte numbers is attributable to an instructive differentiation of uncommitted cells to an oligodendroglial fate, not to a selective proliferation or survival of committed oligodendrocyte progenitors. The IGF-Iinduced oligodendrocyte differentiation appears to be mediated, at least in part, by the inhibition of bone morphogenetic protein (BMP) signaling. Furthermore, overexpression of IGF-I in the hippocampus led to an increase in immunoreactivity for oligodendrocyte markers. These results provide evidence that IGF-I is an important regulator of oligodendrocyte differentiation from multipotent adult neural progenitor cells.
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Results |
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Furthermore, 500 ng/ml IGF-II or insulin could also promote the preferential differentiation of neural progenitor cells into oligodendrocytes (Fig. 1 D). The IGF-mediated oligodendrocyte differentiation is likely to occur through an activation of IGF-I receptors; addition of IGF-I, IGF-II, or insulin at different concentrations (201,000 ng/ml) showed a rank order of potency IGF-I > IGF-II > insulin, which is consistent with the pharmacology of the IGF-I receptor (LeRoith et al., 1993). RT-PCR analysis showed IGF-I receptor mRNA expression in neural progenitors (unpublished data). There was also a small percentage of neural progenitor cells that differentiated into Tuj1+ neurons (515%) with IGF-I or -II or insulin treatment (each at 500 ng/ml), with few to no GFAP+ astrocytes (Fig. 1 D). The IGF-Iinduced oligodendrocyte differentiation occurred in a dose-dependent manner, whereas the percentage of Tuj1-positive cells did not appear to change at any of the tested concentrations (Fig. 1 E).
The severe and rapid death of neural progenitor cells cultured in the absence of IGF-I left open the possibility that FGF-2 withdrawal alone could be promoting oligodendrocyte differentiation and that the addition of IGF-I merely promoted oligodendrocyte survival. To address this issue, we kept neural progenitor cells alive with the broad caspase inhibitor Q-VD-OPh (Caserta et al., 2003), and assessed neural progenitor differentiation with and without IGF-I in 2-d cultures (Fig. 2, AC). Neural progenitors that differentiated into definitive oligodendrocytes were scored on the basis of morphological criteria (elaboration of weblike processes), as well as immunoreactivity with various oligodendrocyte markers (O4, NG2, RIP, and MBP). In the absence of Q-VD-OPh in insulin-free N2 medium, there was decreased cell survival, evidenced by the presence of fragmented DAPI-stained nuclei and a general disappearance of cells. Addition of 2 µM Q-VD-OPh to the cells promoted the majority of the cells to survive, as seen by smooth DAPI-stained nuclei and a general persistence of cells in 2-d cultures, without obvious effects on proliferation or differentiation (Fig. 2, A and B). The absolute number of cells does not significantly change in Q-VD-OPh-treated cultures compared with cells just after plating (unpublished data). Although the cells exhibited some degree of O4 and RIP staining in the soma, only a small number of cells (24%) exhibited both staining and morphological criteria indicative of a differentiated oligodendrocyte in Q-VD-OPhtreated cultures. This finding is presumably due to spontaneous differentiation upon FGF-2 withdrawal. In addition, we did not detect MBP staining in Q-VD-OPHtreated cultures, further confirming that FGF-2 withdrawal alone could not promote the robust differentiation and maturation of a multipotent neural progenitor cell into a definitive oligodendrocyte. Only when neural progenitors were cultured with 500 ng/ml IGF-1 did we observe a large increase in O4 and RIP+ cells with the characteristic weblike morphology in 2-d cultures (Fig. 2, A and B). The quantification of oligodendrocyte differentiation is also shown (Fig. 2 C). The presence of NG2+ cells was not observed in Q-VD-OPhtreated cultures, with or without IGF-I, suggesting that adult multipotent neural progenitor cells do not transition through an NG2+ cell upon IGF-I stimulation. These data suggest that IGF-1 can directly induce multipotent neural progenitor cells to differentiate into oligodendrocytes, instead of merely promoting the survival of differentiated oligodendrocytes.
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IGF-Iinduced increase in oligodendrocytes is attributable to an instructive differentiation and a subsequent proliferation of committed oligodendrocytes
Although these results establish IGF-I as an inducer of oligodendrocyte differentiation for a population of multipotent neural progenitor cells, it is more complicated to quantitatively assess the instructive versus selective effects of IGF-I. Therefore, we determined whether the increase of oligodendrocytes after IGF-I treatment could be due to a combination of selective oligodendrocyte survival, increased proliferation of progenitors, and/or instructive differentiation of progenitor cells to an oligodendroglial lineage. First, we directly measured the effect of IGF-I on cell survival. We asked whether a selective decrease in the death of oligodendrocytes (and/or oligodendrocyte progenitors) could explain the net increase in number of oligodendrocytes (defined by the expression of RIP, a marker of both immature and mature oligodendrocytes). If IGF-I had a selective survival effect on oligodendrocytes and/or their progenitors, we would expect to see a change in cell death due to the increased death of other cell types. To quantify cell death of the progeny of neural progenitor cells, living cultures were stained with 1 µg/ml propidium iodide, which stains dead cells, and 1 µg/ml Hoechst 33342, which stains live and dead cells. Staining of live (rather than fixed) cultures was used to avoid underestimating cell death due to possible detachment of dying and/or dead cells from culture substrates. This assay revealed a relatively small percentage of dead/dying cells, and one-way ANOVA analysis revealed no significant change (P = 0.3930) in the percentage of cells that died at any of the time points (Fig. 3 A). The amount of cell death in IGF-Itreated cultures was similar to the amount of cell death under standard proliferating conditions with FGF-2 (unpublished data). Cultures grown in the absence of IGF-I exhibited a progressive increase in overall cell death, reinforcing the role of IGF-I as an important factor for cell survival. Because there was minimal death and no significant difference in the percentage of dead/dying cells at each of the time points in the IGF-Itreated cultures, a selective survival of oligodendrocyte progenitors or oligodendrocytes does not appear to have a significant role in the increased net oligodendrocyte differentiation with IGF-I treatment.
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The time-course experiments were performed in 12-h intervals, allowing for measurement of the proliferation and differentiation rates at each 12-h time interval; i is the proliferation rate of RIP- cells at time "i" and so forth. BrdU uptake experiments allow for direct measurements of the number of new cells that were generated within the time interval:
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To ensure that the model for oligodendrocyte differentiation accurately represented the biological system, we compared the values predicted by the model for total cell counts () and cell differentiation (
) to direct measurements at the time points between 12 h and 2 d. No significant differences were found between direct measurements (Fig. 4, B and C, columns) and predicted values (Fig. 4, B and C; lines). The model-derived values indicated that there was no increase in the proliferation rate of RIP- cells (
) over time, reinforcing the idea that the increased proliferation rate of oligodendrocyte progenitors was not the cause for the net increase in oligodendrocyte numbers (Fig. 4 D, blue line). The model does predict an increase in differentiation rate (ß; Fig. 4 E, blue line); together with the lack of change in the proliferation rate (
), this can only be taken to mean an increase in instructive differentiation of RIP- to RIP+ cells by IGF-I.
The increase in instructive differentiation by itself is not enough to account for the conversion of RIP- to RIP+ cells. The net increase in RIP+ cells must also include in the explanation an increase in the proliferation rate () of RIP+ cells (Fig. 4 D, red line). If the
term was set to zero (no RIP+ proliferation), the model predicted significantly fewer RIP+ oligodendrocytes than were observed experimentally. Therefore, the only way to resolve the net increase in oligodendrocyte numbers according to the model is an instructive differentiation from adult multipotent neural progenitors to oligodendrocytes and a subsequent proliferation of committed oligodendrocytes in the presence of IGF-I.
IGF-Iinduced oligodendrocyte differentiation is mediated through an inhibition of BMP signaling
In the developing vertebrate telencephalon and spinal cord, BMPs have been shown to act as inhibitory signals for oligodendrocyte fate specification (Gross et al., 1996; Mekki-Dauriac et al., 2002). Additional analyses have shown that oligodendrocyte lineage progression requires an active inhibition of BMP signaling (Mabie et al., 1999; Mehler et al., 2000). To gain insight into the molecular mechanism of IGF-Iinduced oligodendrocyte differentiation, we asked whether IGF-I effects are mediated through an inhibition of BMP signaling. First, we tested whether BMPs could inhibit adult neural progenitor cell oligodendrocyte differentiation. Addition of BMP2 in the presence of 500 ng/ml IGF-I resulted in a suppression of oligodendrocyte differentiation, as evidenced by a reduction in RIP+ cells compared with cultures treated with IGF-I alone (Fig. 5, AC). Similar results were observed with BMP4 (unpublished data). This inhibition of oligodendrocyte differentiation is dose-dependent; higher concentrations of BMP2 (550 ng/ml) resulted in a greater suppression of oligodendrocyte differentiation, whereas partial differentiation could still be observed at lower BMP2 concentrations (0.050.5 ng/ml; Fig. 5 C). These results are consistent with the ability of BMPs to repress oligodendrocyte differentiation.
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If IGF-I induction of oligodendrocyte differentiation involves the inhibition of BMP signaling, a change in the expression of BMP antagonists upon IGF-I treatment might be observed. In addition to extracellular antagonists of BMP signaling, such as Noggin, intracellular proteins that can interfere with downstream BMP receptor signaling, called inhibitory Smads (Smad6, Smad7), have also been identified (Christian and Nakayama, 1999). Therefore, we performed Q-PCR analysis of cultures treated with 500 ng/ml IGF-I for 24 h and examined the relative fold change of Noggin, Smad6, and Smad7 (Fig. 6; all values were normalized to expression levels under FGF-2 conditions). Expression of Noggin and Smad6 increased by at least four- and sixfold, respectively, after IGF-I treatment. There was only a slight increase in Smad7 levels with IGF-I treatment (1.2-fold). In each case, if IGF-Itreated cultures included 20 ng/ml FGF-2, the up-regulation of BMP antagonists was not observed, suggesting that FGF-2 can suppress the IGF-Imediated up-regulation of Noggin and Smad6. Together, these results suggest that IGF-Iinstructive effects on oligodendrocyte differentiation are mediated, at least in part, through an inhibition of BMP signaling.
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Because we observed an increase in overall staining for various oligodendrocyte markers (RIP and MBP) after IGF-I overexpression in the adult hippocampus, we next determined if there was also a change in the number of oligodendrocytes. For quantification of oligodendrocytes, sections were labeled for the isoform of GST (GST-
). GST-
has been determined in many experiments to label both immature and mature myelinating oligodendrocytes (Tansey and Cammer, 1991; Mason et al., 2001), and facilitates cell counting due to its predominant localization in oligodendrocyte cell bodies as well as in a few of the processes. The average number of GST-
positive cells per section was 8.08 ± 2.43 (n = 3 rats) for rAAV-ß-gal control animals and 29.14 ± 5.04 (n = 4 rats) for rAAV-IGF-I animals, which is a threefold difference (Fig. 7, IK; P < 0.001, t test). These results support that IGF-I is a regulator of oligodendrocyte differentiation in vivo as well as in vitro.
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Discussion |
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To address whether IGF-I effects are instructive and/or selective in nature, we used both experimental and modeling approaches. First, we addressed the question of whether IGF-I might have selective effects on cell survival. Because the frequency of cell death is low in IGF-Itreated cultures and there is no significant difference in the percentage of cells that are dying/dead at each of the time points analyzed, it is unlikely that the net increase in oligodendrocytes by IGF-I comes from the selective survival of committed oligodendrocyte progenitors that then go on to differentiate. Although there is massive cell death in the absence of IGF-I, this cell death applies widely across all types of neural progenitor cells. Treatment of cells with the broad caspase inhibitor Q-VD-OPh further reinforced the finding that IGF-I is not merely acting on cell survival. The addition of Q-VD-OPh is enough for the cells to survive, and not to proliferate or differentiate into definitive oligodendrocytes in short-term cultures; only upon addition of IGF-I is there a massive increase in oligodendrocyte differentiation.
Next, we addressed the question of selective effects on cell proliferation by IGF-I. The BrdU analyses indicate that there is not an increase in proliferation at time points where oligodendrocyte differentiation is already occurring, suggesting that a selective proliferation of progenitors does not appear to make a major contribution to the net oligodendrocyte increase. To address the possibility that a robust differentiation effect by IGF-I might mask a proliferation effect, a model was used to separate the independent contributions of IGF-I on proliferation and differentiation. The model-derived data showed an increase in the rate of oligodendrocyte differentiation and not in the proliferation rate of RIP- progenitors. The model also showed an increase in the proliferation rate of committed oligodendrocytes in IGF-Itreated cultures. Therefore, the simplest interpretation of our data is that IGF-I acts to control the fate choice of multipotent adult neural progenitor cells to an oligodendroglial lineage, and that the net increase in oligodendrocytes by IGF-I is due to an instructive differentiation and an additional proliferation of committed oligodendrocytes.
Recently, it has been reported that IGF-I can stimulate neurogenesis in the dentate gyrus (Aberg et al., 2000), as well as increase the proliferation and neuronal differentiation of EGF-responsive multipotent neural stem cells derived from E14 mouse striatum (Arsenijevic and Weiss, 1998; Arsenijevic et al., 2001). Although it is not clear whether oligodendrocyte differentiation was examined in these experiments, the possibility still remains that IGF-I promotes the differentiation of multipotent neural progenitor cells to both neuronal and oligodendrocyte lineages. In fact, our work shows that, in IGF-Itreated cultures, although the majority of the cells differentiate into oligodendrocytes, a small number of cells can differentiate into neurons. Because small numbers of cells differentiate into neurons, it was difficult to determine if the effects of IGF-I on neuronal differentiation were instructive or selective in nature, or whether a population of lineage-restricted neuronal progenitors exists that could survive and differentiate in the presence of IGF-I. Because we did not observe an increase in astrocyte differentiation with IGF-I treatment in vitro, or any apparent effects on the existing astrocyte population in vivo, it would appear that IGF-I effects on neural progenitor cells are restricted, at least in part, to oligodendrocyte and neuronal lineages. The possibility remains that IGF-I could have instructive effects on astroglial fate commitment from a multipotent progenitor cell population in vivo.
We hypothesized that oligodendrocyte differentiation of multipotent adult neural progenitor cells might use similar mechanisms as oligodendrocyte progenitors derived from the embryonic brain and spinal cord, and that IGF-I induction of oligodendrocyte differentiation might involve an inhibition of BMP signaling. Our data suggest that BMPs can repress IGF-Iinduced oligodendrocyte differentiation from adult neural progenitor cells. Furthermore, addition of Noggin together with lower concentrations of IGF-I results in a greater percentage of cells that differentiate into oligodendrocytes compared with IGF-I alone, suggesting that IGF-I induction of oligodendrocyte differentiation involves the activation of BMP antagonists such as Noggin. This finding, together with the observation that there is an up-regulation of Noggin, Smad6, and Smad7 with IGF-I treatment, further reinforces the role of IGF-I in the inhibition of BMP signaling to promote oligodendrocyte differentiation. A proposed model is shown in Fig. 8. BMP signaling has been shown to alter the fate of neural progenitor cells by stimulating astroglial differentiation while inhibiting neuronal and oligodendroglial differentiation (Fig. 8 A; Mehler et al., 1997; Mabie et al., 1999; Nakashima et al., 2001). IGFs (IGF-I and -II) and/or insulin activate IGF-I receptors located on multipotent neural progenitor cells, which leads to the up-regulation of BMP antagonists such as Noggin, Smad6, and Smad7. Because Noggin, Smad6, and Smad7 inhibit BMP signaling, the net effects of IGF signaling are a decrease or absence of astrocyte differentiation and an increase in neuronal and oligodendroglial differentiation (Fig. 8 B). Future analyses are needed to determine whether all of the IGF-instructive effects on oligodendrocyte differentiation occur through an inhibition of BMP signaling, or if IGFs can directly promote oligodendroglial lineage commitment in a Noggin/Smad6, Smad7-independent manner.
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Materials and methods |
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Immunocytochemistry and in vitro quantification
Cells were fixed with 4% PFA, followed by immunocytochemical staining as described previously (Palmer et al., 1999). Labeled cells were visualized using an upright microscope (model E800; Nikon) or an inverted microscope (model E600; Nikon) and a CCD camera (Spot RT; Diagnostic Instruments). DAPI was used to identify individual cells. Quantification of cell phenotypes was with StereoInvestigator (MicroBrightfield Inc.); for each marker, 5001,000 cells were sampled systematically from standardized fields at 40x. For quantification of live/dead cells, images were taken of cultures live stained with 1 µg/ml propidium iodide (Molecular Probes, Inc.) and 1 µg/ml Hoescht 33342 (Sigma-Aldrich) at 10x, and cells were counted using Image-Pro (Media Cybernetics). Primary antibodies were used as follows: rabbit anti-Tuj1 (1:7,500; Covance), mouse anti-RIP (1:251:75; Developmental Studies Hybridoma Bank, Iowa City, IA); guinea pig anti-GFAP (1:5001:2,500; Advanced Immunochemical, Inc.); rabbit anti-MBP (1:500; CHEMICON International, Inc.), mouse anti-O4 (1:2; hybridoma supernatant from cells provided by O. Boegler, University of California, San Diego, San Diego, CA), rat anti-BrdU (1:400; Accurate). Secondary antibodies were all purchased from Jackson ImmunoResearch Laboratories and used at 1:250 dilution. The detection of BrdU in cultured cells required treatment in 2N HCL at 37°C for 30 min (Palmer et al., 1999). All experiments were independently replicated at least three times.
IGF-I overexpression in vivo and quantification
cDNA encoding the human IGF-I gene was cloned into a recombinant AAV vector and the virus was prepared as described previously (Kaspar et al., 2002). Expression of IGF-I and ß-gal (control AAV) was first confirmed in human embryonic kidney (HEK-293) cells by RT-PCR and Western analysis and subsequently in the hippocampus by RT-PCR analysis (unpublished data). Recombinant AAV-IGF-I (rAAV-IGF-I) or AAV-ß-gal (rAAV-ß-gal) was stereotaxically injected into the hippocampus (anteroposterior axis, -4.0 mm; mediolateral axis, +2.0 mm; dorsoventral axis, -3.0 from skull, with nose bar at 3.3 mm up) of anesthetized female Fischer-344 rats (150175 g; n = 6). After 4 wk to allow for virus processing and transgene expression, animals were killed. Animals were perfused with 4% PFA and the brains were excised, stored in fixative overnight, and transferred to 30% sucrose. 40-µm coronal sections were cut on a sliding microtome, and sections were processed for standard immunohistochemical staining as described previously (Gage et al., 1995). Sections were triple labeled with mouse anti-RIP (1:50), rabbit anti-MBP (1:500), and guinea pig anti-GFAP (1:1,000). In some cases, sections were labeled with mouse anti-GST- (1:100; BD Biosciences). Images were acquired using a confocal microscope (Radiance 2100; Bio-Rad Laboratories) and an inverted microscope (model TE2000; Nikon) equipped with a 40x NA 1.3 Plan Fluor objective lens. Images were post processed using Adobe PhotoShop®.
All comparative analyses were focused on the hilus of the injected side on matched hippocampal sections. For RIP or MBP staining, immunofluorescence average pixel intensity of equivalent-sized fields (four from each animal) in control and experimental groups was determined from digital images using Adobe Photoshop®. To quantify the number of oligodendrocytes, sections were immunostained with GST-. Only immunolabeled cells with a clear DAPI-positive nucleus were scored. For quantification of GST-
cells, area counts of the hilus within the dentate gyrus of the hippocampus were performed. In each section, three adjacent fields were sampled. Cells in the uppermost focal plane were ignored, and we focused through the thickness of the section to avoid errors caused by oversampling. The results are expressed as the average number of cells per section.
Quantitative real-time PCR analysis
Total RNA was isolated from cell cultures using RNeasy columns (QIAGEN). For real-time quantitative PCR, reactions were performed essentially as described previously (Zhao et al., 2001). The relative amount of the tested message was normalized to the level of the internal control message, GAPDH. Furthermore, independent experiments were performed with a different internal control message, ß-actin, and showed similar results. Primer sequences are available upon request.
Statistics
Results were analyzed for statistical significance using t test or by ANOVA, and all error bars (except in Fig. 3, D and E) are expressed as SDs. Post-hoc analysis was done using Bonferroni corrected planned comparison.
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Acknowledgments |
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This work was supported in part by grants from the National Institutes of Health (AG020938), the George E. Hewitt Foundation for Medical Research, Project ALS, the Christopher Reeve Paralysis Foundation, and the Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad.
Submitted: 19 August 2003
Accepted: 17 November 2003
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