IGF-I–induced oligodendrocyte differentiation
First, we tested the action of exogenous IGF-I on the differentiation of hippocampus-derived adult neural progenitor cells. These neural progenitor cells have stem cell properties in vitro: (1) they self-renew in the presence of basic FGF-2; (2) single genetically marked clones can differentiate into all three main CNS cell types in vitro (neurons, oligodendrocytes, and astrocytes) and when grafted back to adult hippocampus in vivo; and (3) they express progenitor cell markers such as nestin, but lack markers of lineage-specific differentiation ( A; Gage et al., 1995
; Palmer et al., 1997
). To confirm that the adult neural progenitor cell population in our model system is indeed multipotent, we first used various standard differentiation paradigms and evaluated the expression of lineage markers. Differentiation with 1 μM retinoic acid (RA) and 1% FBS resulted in mixed numbers of Tuj1+
astrocytes, and RIP+
oligodendrocytes ( A). Neuron-enriched differentiation can be achieved with 1 μM RA and 5 μM forskolin (Chu, V., personal communication), and astrocyte-enriched differentiation with 50 ng/ml leukemia inhibitory factor and 50 ng/ml BMP2 ( A; Nakashima et al., 1999
). The quantification of lineage-specific differentiation is also shown ( C). These results are in agreement with previous reports (Palmer et al., 1997
), and indicate that the majority of the adult neural progenitor cells within the bulk population are multipotent progenitors, and not restricted neuronal or glial progenitors.
Figure 1. IGF-I–induced differentiation of multipotent neural progenitor cells. (A) Adult hippocampus-derived neural progenitor cells cultured in insulin-containing N2 media with 20 ng/ml FGF-2 (undifferentiated), 1 μM RA, and 1% FBS for 4 d (mixed), (more ...)
To determine the effects of IGF-I on neural progenitor cell differentiation, we compared cells treated with and without IGF-I over a 4-d period. All experiments were performed in insulin-free N2 medium to avoid its actions on insulin and/or IGF-I receptors. When FGF-2 was withdrawn from neural progenitor cells and the cells were cultured in the absence of insulin for 4 d, lineage-specific differentiation was not seen ( B). There was also a dramatic decrease in cell survival, consistent with the important effects of IGFs for preventing cell death (Vincent and Feldman, 2002
). We found that neural progenitor cells cultured in the presence of 500 ng/ml IGF-I differentiated into a large percentage of RIP+
cells (~45–55%; ). RIP is a marker for both immature and mature oligodendrocytes, in that it stains pre-ensheathing oligodendrocytes through myelinating stages and associated myelin (Friedman et al., 1989
). In addition to RIP staining, these cells exhibited small and round somata with the characteristic weblike oligodendrocyte morphology within 4 d ( B). Approximately 23% of the total cells were also positive for myelin basic protein (MBP), a late-appearing marker for myelinating oligodendrocytes (Akiyama et al., 2002
), at the 4-d time point ( B).
Furthermore, 500 ng/ml IGF-II or insulin could also promote the preferential differentiation of neural progenitor cells into oligodendrocytes ( 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 (20–1,000 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 (~5–15%) with IGF-I or -II or insulin treatment (each at 500 ng/ml), with few to no GFAP+
astrocytes ( D). The IGF-I–induced 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 ( 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 (, A–C). 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 (). 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 (~2–4%) exhibited both staining and morphological criteria indicative of a differentiated oligodendrocyte in Q-VD-OPh–treated cultures. This finding is presumably due to spontaneous differentiation upon FGF-2 withdrawal. In addition, we did not detect MBP staining in Q-VD-OPH–treated 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 (). The quantification of oligodendrocyte differentiation is also shown ( C). The presence of NG2+
cells was not observed in Q-VD-OPh–treated 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.
Figure 2. IGF-I–mediated increase in oligodendrocyte differentiation is independent of effects on neural progenitor cell survival in 2-d cultures. (A) Treatment of cells with 2 μM Q-VD-OPh resulted in a general absence of apoptosis in short-term (more ...)
The oligodendrocyte-promoting effects of IGF-I were confirmed with an independently derived rat hippocampus progenitor cell line, as well as a clonally derived line (unpublished data). Furthermore, early-passaged cultures (<5) of multipotent neural progenitor cells derived from the whole brain of P10 ICR mice also exhibited a preferential differentiation into oligodendrocytes upon IGF-I treatment (unpublished data). These data suggest that IGFs (IGF-I and -II) and insulin, through an activation of IGF-I receptors, are important in stimulating the differentiation and maturation of multipotent adult neural progenitor cells into oligodendrocytes.
IGF-I–induced 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 ( A). The amount of cell death in IGF-I–treated 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-I–treated 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.
Figure 3. Effect of IGF-I on the survival, proliferation, and instructive oligodendrocyte differentiation of adult neural progenitor cells. (A) Quantification of cell death in insulin-free (no IGF-I) and IGF-I–treated (500 ng/ml) cultures, as determined (more ...)
Next, we determined whether proliferation of progenitors might contribute to the observed increase in oligodendrocyte differentiation. 2.5 μM BrdU was added to parallel cultures for 12 h at 0, 12, 24, and 36 h after plating, followed by fixation and quantification of total cell numbers and BrdU+ cells. We counted the progenitor cells (evidenced by a lack of RIP staining) that were labeled with BrdU at each time point. For cells grown in the absence of IGF-I, we observed a progressive decrease in the percentage of cells that were proliferating (BrdU+) during the 48-h experiments ( B). There was a marked decrease in proliferation during the 36- and 48-h periods, probably due to limited cell survival in the absence of IGF-I. In cultures treated with IGF-I, the percentage of RIP− cells that incorporated BrdU also exhibited a decreasing trend in the first three time periods, but to a lesser extent compared with the cells grown without IGF-I ( B, blue columns). This initial decrease in proliferation is most likely due to FGF-2 mitogen withdrawal. The fact that we could already observe the generation of RIP+ oligodendrocytes at the 24-h time point ( C), coupled with the observation that there was a decrease in BrdU uptake of RIP− cells, suggests that IGF-I did not have a major proliferative effect on progenitor cells.
Figure 4. IGF-I instructs neural progenitor cells to commit to an oligodendroglial lineage and increases the proliferation of committed oligodendrocytes. (A) Adult neural progenitor cell states in culture: α, proliferation rate of progenitors (RIP− (more ...)
These data taken alone may not be sufficient to fully evaluate the effects of IGF-I on proliferation of RIP−
progenitors because if there is also a differentiation effect (conversion of RIP−
cells), a substantial decrease in the percentage of RIP−
cells that were BrdU+
may be seen. Therefore, to separate the IGF-I effects on proliferation of progenitors from the instructive differentiation of progenitors to oligodendrocytes, we developed a mathematical model ( A). This modeling approach has previously been used to quantify the effects of hippocampal astrocytes on the proliferation and neuronal differentiation of adult neural progenitor cells (Song et al., 2002
). To distinguish the proliferation and differentiation rates, the model required that we count the number of RIP+
cells that were either positive or negative for BrdU at each time point. There was a progressive increase in the overall number of RIP+
cells over time in the presence of IGF-I ( C). We also stained the cultures at each time point for Tuj1+
neurons and GFAP+
astrocytes to determine if the model should account for the differentiation to alternative cell types; because neuronal differentiation was minimal and astrocyte differentiation was undetectable, these possibilities did not merit inclusion into the model. Interestingly, after 24 h, there was also a significant increase in RIP+
cells positive for BrdU, suggesting that committed oligodendrocytes may still be proliferating in the presence of IGF-I ( B, red columns). Because of this finding, we decided to separate the proliferation rate of RIP−
(defined as α) and RIP+
cells (γ), as well as the differentiation rate (β) of RIP−
cells. The overall death rate measured by Hoechst dye in live cultures could not be experimentally separated into a RIP−
death rate (δ) and a RIP+
death rate (
; Song et al., 2002
). Therefore, the model was bounded by the two possibilities that all cell death is from only RIP−
or only RIP+
cells. We also considered the more probable assumption that the death rates for both RIP−
cells were equal. Six equations were used to derive values for these rates. The first equation describes the differential gain in RIP−
is the number of RIP−
cells, α is the proliferation rate of RIP−
cells (per 12-h interval), β is the differentiation rate of RIP−
cells, and δ is the death rate of RIP−
cells. Similarly for RIP+
is the number of RIP+
cells, γ is a proliferation rate of RIP+
is the RIP+
death rate. The sum of (
) and (
) is the equation defining total cell count:
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; αi
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:
where #BrdUi + 1
is the number of cells that incorporated BrdU between time “i” and “i+1.” Inspection of BrdU+
cells that are negative or positive for RIP gives the equations:
where the βi
/(1 + αi
term represents the number of RIP−
cells that may have divided then differentiated within the same 12-h interval [probability of differentiation βi
times the 2αi
/(1 + αi
) term for probability that the differentiating cell is BrdU+
, with a 1/2 correction for the order of events]. Because dead cells cannot be accurately separated into RIP+
cells, the model is fitted under three possible assumptions: all death is RIP+
(δ = 0); all death is RIP−
= 0); and the death rates are equal (δ =
). The proliferation
were solved with the differentiation equation 2
to find values for αi
, and γi
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 (, columns) and predicted values (; lines). The model-derived values indicated that there was no increase in the proliferation rate of RIP−
cells (α) over time, reinforcing the idea that the increased proliferation rate of oligodendrocyte progenitors was not the cause for the net increase in oligodendrocyte numbers ( D, blue line). The model does predict an increase in differentiation rate (β; E, blue line); together with the lack of change in the proliferation rate (α), this can only be taken to mean an increase in instructive differentiation of 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 (γ) of RIP+ cells ( D, red line). If the γ 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-I–induced 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-I–induced 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 (, A–C). Similar results were observed with BMP4 (unpublished data). This inhibition of oligodendrocyte differentiation is dose-dependent; higher concentrations of BMP2 (5–50 ng/ml) resulted in a greater suppression of oligodendrocyte differentiation, whereas partial differentiation could still be observed at lower BMP2 concentrations (0.05–0.5 ng/ml; C). These results are consistent with the ability of BMPs to repress oligodendrocyte differentiation.
Figure 5. IGF-I–mediated oligodendrocyte differentiation involves an inhibition of BMP signaling. (A and B) Cells treated with 500 ng/ml IGF-I alone (A) or in combination with 50 ng/ml BMP2 (B). Cells were stained for an oligodendrocyte marker (RIP) and (more ...)
BMP signaling can be inhibited by extracellular proteins, such as Noggin, that bind BMP ligands in a competitive manner, interfering with the ability of ligand binding to cognate cell surface receptors (Balemans and Van Hul, 2002
). If the IGF-I induction of oligodendrocyte differentiation is mediated through an inhibition of BMP signaling, the function of BMP antagonists could be involved. To test the involvement of BMP antagonists, we treated neural progenitor cells with different concentrations of IGF-I in conjunction with exogenous Noggin and compared the extent of oligodendrocyte differentiation to cultures treated with IGF-I alone (, D–F). When 500 ng/ml Noggin was added to cultures treated with 20–100 ng/ml IGF-I, there was a significant increase in the percentage of oligodendrocytes compared with cultures treated with IGF-I alone ( F; P < 0.05, t
test). Addition of 500 ng/ml Noggin together with highest concentrations of IGF-I (500–1,000 ng/ml) did not result in a significant change in the percentage of RIP+
oligodendrocytes compared with IGF-I alone ( F), presumably due to saturating effects of IGF-I. Next, we wanted to rule out if Noggin by itself had any effects on oligodendrocyte differentiation and/or enhanced survival. Neural progenitor cells treated with 500 ng/ml Noggin alone in insulin-free media did not display any enhanced differentiation or survival effects. Finally, addition of 500 ng/ml Noggin together with 50 ng/ml BMP2 and 500 ng/ml IGF-I efficiently reversed the inhibitory effects of BMP signaling on oligodendrocyte differentiation ().
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 (; 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-I–treated cultures included 20 ng/ml FGF-2, the up-regulation of BMP antagonists was not observed, suggesting that FGF-2 can suppress the IGF-I–mediated up-regulation of Noggin and Smad6. Together, these results suggest that IGF-I–instructive effects on oligodendrocyte differentiation are mediated, at least in part, through an inhibition of BMP signaling.
Figure 6. IGF-I induction of oligodendrocyte differentiation is associated with an up-regulation of BMP antagonists Noggin, Smad6, and Smad7. Q-PCR analysis and quantification of relative fold change (normalized to expression levels under FGF-2 conditions) of Noggin, (more ...)
Effects of IGF-I on oligodendrocytes in vivo
Our findings raised the question of whether IGF-I also has effects on oligodendrocyte differentiation in vivo in the region where adult hippocampal neural progenitor cells normally reside. Therefore, we used adeno-associated virus (AAV) to overexpress IGF-I in the adult rat hippocampus. To evaluate effects on the endogenous oligodendrocyte population due to IGF-I overexpression in vivo, we used the oligodendrocyte marker RIP. We focused our analyses on the hilar region of the dentate gyrus because there is a higher concentration of oligodendrocytes in this region. An increase of RIP staining in the hilus of rAAV-IGF-I–infected animals compared with rAAV-β-gal–infected controls was apparent (). Quantification of RIP immunofluorescence in the hilus of rAAV-β-gal–infected control animals resulted in an average pixel intensity of 45.49 ± 2.01 (n = 3 rats), compared with an average pixel intensity of 99.95 ± 18.27 (n = 4 rats) in rAAV-IGF-I–infected animals (P < 0.05, t test). Similar results were seen in at least three adjacent fields of the same section.
Figure 7. IGF-I overexpression in the hilus promotes oligodendrocyte differentiation in vivo. (A–J) Representative images of brain sections focusing in on the hilar region in animals injected with rAAV-β-gal controls (A–D and I) and rAAV-IGF-I (more ...)
A similar trend was observed using MBP immunofluorescence as a marker for mature oligodendrocytes. The extent of MBP staining appeared more intense in the hilus of IGF-I–infected animals compared with control animals (). The average pixel intensity of MBP immunofluorescence in controls was 30.8 ± 6.84 (n = 3 rats) compared with an average pixel intensity of 53.53 ± 5.72 (n = 4 rats) in rAAV-IGF-I–infected animals (P < 0.05, t test). There was no observable difference in GFAP staining between control and IGF-I–infected animals, suggesting that there is not a general increase in all glial cell lineages in animals overexpressing IGF-I ().
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 π isoform of GST (GST-π). GST-π 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-π–positive cells per section was 8.08 ± 2.43 (n
= 3 rats) for rAAV-β-gal control animals and 29.14 ± 5.04 (n
= 4 rats) for rAAV-IGF-I animals, which is a threefold difference (, I–K; P < 0.001, t
test). These results support that IGF-I is a regulator of oligodendrocyte differentiation in vivo as well as in vitro.