|
|
|
|
|
|
|
||
Original Article |
Correspondence to: Dean G. Tang, MRC Laboratory for Molecular Cell Biology, University College London, London, WC1E 6BT, The United Kingdom. Tel:44 171 419 3538 Fax:44 171 380 7805 E-mail:d.tang{at}ucl.ac.uk.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS). They develop from precursor cells (OPCs), some of which persist in the adult CNS. Adult OPCs differ in many of their properties from OPCs in the developing CNS. In this study we have purified OPCs from postnatal rat optic nerve and cultured them in serum-free medium containing platelet-derived growth factor (PDGF), the main mitogen for OPCs, but in the absence of thyroid hormone in order to inhibit their differentiation into oligodendrocytes. We find that many of the cells continue to proliferate for more than a year and progressively acquire a number of the characteristics of OPCs isolated from adult optic nerve. These findings suggest that OPCs have an intrinsic maturation program that progressively changes the cell's phenotype over many months. When we culture the postnatal OPCs in the same conditions but with the addition of basic fibroblast growth factor (bFGF), the cells acquire these mature characteristics much more slowly, suggesting that the combination of bFGF and PDGF, previously shown to inhibit OPC differentiation, also inhibits OPC maturation. The challenge now is to determine the molecular basis of such a protracted maturation program and how the program is restrained by bFGF.
Key Words: oligodendrocyte precursor cells, cell cycle, optic nerve, PDGF, bFGF
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
In many cell lineages the behavior of precursor cells changes as development proceeds, but it is generally not known to what extent the changes reflect changes in the cells' environment, an intracellular program for change built into the cells themselves, or both. We have been addressing this problem in the oligodendrocyte cell lineage.
Oligodendrocytes develop from proliferating precursor cells (OPCs)1 that initially arise in germinal zones and then migrate throughout the CNS (
Various growth factors can promote OPC proliferation in culture, including PDGF (
OPC differentiation is tightly coupled to cell cycle withdrawal and, in cultures of OPCs isolated from the postnatal rat optic nerve at least, it appears to be regulated by a cell-intrinsic timer (
OPCs are present in the adult CNS (ffrench-Constant and Raff, 1986;
In this study, we investigate how purified OPCs from neonatal rat optic nerve behave when cultured continuously for many months in the presence of PDGF, or PDGF and bFGF, but in the absence of TH. We compare these long-term cultured OPCs with OPCs freshly isolated from adult optic nerves of different ages. The results suggest that the long-term cultured OPCs gradually acquire several of the characteristics of adult OPCs, suggesting that OPCs have an intrinsic maturation program that progressively changes the cells' properties over many months.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Animals and Reagents
Sprague-Dawley rats were obtained from the Animal Facility at University College London. Recombinant human PDGF-AA and NT-3 were purchased from Peprotech, bFGF from Promega. NRG (glial growth factor 2, or GGF2) was a gift from Cambridge NeuroScience. The following antibodies were used: monoclonal anti-RAN-2 (
Purification and Culture of OPCs
OPCs were prepared from optic nerves of postnatal day zero (P0), P7, P9, or P14 rats and purified by sequential immunopanning as previously described (
Cultures were maintained at 37°C in an 8% CO2 incubator, and half of the culture medium was changed every 48 h. For high-density cultures, fresh PDGF was added every 8 h. Cells were passaged using 0.0125% trypsin (GIBCO BRL) when they reached 80–90% confluence, which generally took 15–20 d. About 2,000 cells were plated into T25 flasks at each passage. A total of eight independent sets of P7 cultures were initiated and followed through multiple passages. The morphology of the cells was regularly observed by inverted phase microscopy and photography (Tmax 100 B/W film) and sometimes by time-lapse video recording (see below).
Purification and Culture of Adult OPCs
Adult OPCs were purified from P50, P300, P390, or P510 rat optic nerves, using a collagenase-trypsin protocol initially described by
Determination of Cell Cycle Time by BrdU Incorporation
To label OPCs in S phase, cells that had been in culture for different periods of time were plated at a density of 3,000 cells/13-mm PDL-coated glass coverslip and cultured for 2 d in medium containing either PDGF or PDGF plus bFGF. Then 2.5 µM BrdU (Boehringer Mannheim) was added to the culture medium for 2–144 h, as indicated in Results. Half of the media was changed every 3 d and fresh BrdU was added. Finally, the cells were fixed in 4% paraformaldehyde (5 min, room temperature), permeabilized in 70% ethanol (in PBS, -20°C) for 10 min, incubated in 6N HCl and 1% Triton X-100 (15 min, room temperature) to denature nuclear DNA, and then incubated in 0.1 M sodium borate (in PBS and 1% Triton X-100) for 10 min. The cells were subsequently blocked with 50% normal goat serum and 1% Triton X-100 for 30 min and then incubated with monoclonal anti-BrdU antibody (supernatant; used at 1:5 dilution) for 1 h, followed by a goat anti–mouse IgG-biotin and streptavidin-FITC (diluted 1:100, 30 min each; Amersham). Finally, the nucleus of all cells was labeled with bisbenzamide (Hoechst 33342 dye). In some experiments, cells were first surface-labeled with the A2B5 monoclonal antibody, before ethanol fixation (see below), using Texas red–coupled goat anti-mouse Ig antibodies. To label the proliferating OPCs freshly isolated from adult animals, cells growing on coverslips for 2–3 d were pulsed with 2.5 µM BrdU for 8 h and then processed for immunofluorescence staining as described above. All coverslips were mounted with Citifluor (Chemistry Lab) on glass slides and sealed with nail vanish. Cells were examined in a Zeiss Axioplan-2 fluorescence microscope and photographed on either Tmax-400 black and white film or Kodak Ektochrome 100 color slide film.
A total of 1,000–1,500 cells (identified by bisbenzamide nuclear staining) were counted per coverslip to determine the proportion of BrdU+ cells. Two coverslips were counted for each condition. The labeling index was plotted against the BrdU pulse time to obtain a cumulative labeling curve. The estimate of cell cycle time (Tc) was determined from the cumulative labeling index (
2 was >0.95. All the experiments were repeated at least twice.
Determination of Population Doubling Time, Cell Death, and Spontaneous Differentiation by Clonal Analysis
Purified OPCs that were in culture for different periods of time were plated at clonal density (2,000 cells/flask) into PDL-coated T25 flasks and cultured in either PDGF or PDGF plus bFGF for 8 d. Duplicate or triplicate flasks were set up for each time point. OPCs and oligodendrocytes were identified by their characteristic morphologies (
Induced Differentiation
Differentiation of OPCs was induced by either the addition of TH (T3 at 30 ng/ml;
In some experiments cells were plated at clonal density (1,000 cells/flask) in PDL-coated slide flasks and treated as described above. The number of differentiated oligodendrocytes in each clone was counted every 2 d, and clones were scored as precursor clones or oligodendrocyte clones according to the predominant (i.e., >50%) cell type present in the clone (
Immunofluorescence Staining
Cells of different ages were plated onto either PDL-coated 13-mm glass coverslips (3,000 cells/coverslip) or slide flasks (10,000 cells/flask) and cultured for 2–3 d before they were immunostained. For cell surface staining with A2B5, O1, O4, or anti-GC antibodies, cells were fixed in 3.7% paraformaldehyde for 5 min at room temperature. For staining for NG2 antigen, MBP, and PLP, cells were fixed and permeabilized for 5 min in acid alcohol (95% ethanol and 5% glacial acetic acid) at room temperature. For intracellular labeling of vimentin, nestin, and GFAP, cells were fixed and permeabilized for 10 min in methanol:acetone (1:1) at –20°C. Following washing, cells were blocked for nonspecific binding sites with either 50% horse serum (for anti-vimentin antibody) or goat serum (for the other antibodies) for 30 min at room temperature. Primary antibodies were used at either 1:10 (for supernatants) or 1:100 (for the others), diluted in 25% blocking serum in PBS. The incubations were generally 1 h at room temperature. After extensive washing, coverslips were incubated (30 min, room temperature) in goat anti-mouse IgG-biotin, goat anti–rabbit IgG-biotin, or donkey anti–goat IgG-biotin, all diluted 1:100 in 25% blocking serum in PBS. Finally, cells were incubated with a mixture of either FITC-streptavidin or Texas red–streptavidin (both at 1:100) and either bisbenzamide (1:200) or propidium iodide (PI, 1 µg/ml) for 30 min at room temperature. Coverslips were mounted and observed as described for BrdU staining. The fluorescent and biotin-coupled reagents were all obtained from Amersham.
Time-Lapse Video Recording
OPCs of different ages were cultured at clonal density (1,000 cells/flask) in PDL-coated slide flasks in medium containing either PDGF or PDGF plus bFGF. After 2–3 d, the flask was placed on the stage of a Zeiss inverted phase-contrast microscope and maintained at 37°C. Time-lapse video recordings were made using a Sony CCD black and white video camera and a Sony video cassette recorder. Cell cycle times were determined by measuring the time between mitotic telophases. Cell motility was measured by determining the distance that the cell body had moved during a specific period of time, and the results were expressed as µm/h. Generally, clones of 2–10 cells were chosen for analysis.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Establishment of Long-Term OPC Cultures and Changes in Morphology
When purified P7 OPCs were cultured in serum-free B-S medium containing either PDGF or PDGF plus bFGF, but in the absence of TH, most of the cells initially had a bipolar or unipolar morphology with unbranched processes (Fig 1A and Fig B). When cultured at high density (15,000–20,000 cells/T25 flask) in PDGF alone, the cells divided rapidly and reached near confluence (~300,000 cells/flask) in 7–8 d; when cultured at clonal density (2,000 cells/T25 flask), they reached near confluence in ~2 wk. When cultured in PDGF plus bFGF, the cells reached near confluence later (10–12 d at high density and ~20 d at low density), suggesting that bFGF inhibited PDGF-stimulated OPC proliferation (see below).
|
We repeatedly passaged the cells when they reached near confluence, replating 2,000 cells per T25 flask at each passage. Many cells died in the initial few passages, and among the surviving cells the rate of spontaneous differentiation and apoptotic cell death, which occurred randomly in most clones (Fig 1C and Fig D; see below), increased progressively, as previously described (
When OPCs cultured in PDGF alone for 75 d were switched to PDGF plus for bFGF for 60 d, they had a bipolar morphology. When OPCs cultured in PDGF alone for 300 d were switched to PDGF plus bFGF for 30 d, cell death increased, but most cells maintained a complex morphology (not shown).
Analysis of Cell Cycle Time, Spontaneous Differentiation, and Cell Death
To quantitate the changes that occurred with time in culture, we carried out clonal analyses to determine population doubling times and the amount of spontaneous differentiation and cell death. We did BrdU labeling experiments to estimate Tc. As shown in Table 1, P7 OPCs cultured in PDGF for 8 d had a population doubling time of ~27 h, while cells cultured in PDGF plus bFGF for the same time period had a population doubling time of ~47 h. Using cumulative BrdU labeling to estimate Tc, we found that the inhibitory effect of bFGF on the population doubling time in the first 2–3 wk culture resulted at least partly from an increase in Tc (Fig 2): at 2 d, for example, OPCs cultured in PDGF had a Tc of ~25 h, whereas the same preparation of cells cultured in PDGF plus bFGF had a Tc of ~30 h (Fig 2 C). bFGF also enhanced the rate of cell death at 8 d: the rate was ~7% in cells cultured in PDGF alone and ~12% in cells cultured in PDGF plus bFGF (Table 2). At these early times, the rate of spontaneous differentiation was not statistically different in cells cultured in PDGF plus bFGF compared with cells cultured in PDGF alone (Table 2).
|
|
|
By 2–3 wk after initial plating, the population doubling times had increased and were still much higher in PDGF plus bFGF than in PDGF alone (Table 1). Cumulative BrdU labeling experiments (Fig 2A and Fig B) indicated that part of the increase with time was due to an increase in Tc, while clonal analyses indicated that increased spontaneous differentiation and cell death also contributed (Table 2). Nonetheless, the difference between PDGF alone and PDGF plus bFGF was mainly due to the difference in Tcs (Fig 2A and Fig B).
The increased rates of spontaneous differentiation and cell death at 2–3 wk were not related to the passaging of the cells, as cells cultured for 17 d with or without passaging had very similar rates of spontaneous differentiation and cell death (Table 2). This observation raised the possibility that the increase in Tc, spontaneous differentiation, and cell death may reflect the normal maturation of OPCs. To test this possibility, we purified OPCs from P0, P9, and P14 rat optic nerves and performed cumulative BrdU labeling experiments to estimate Tc and clonal analyses to determine population doubling time, spontaneous differentiation, and cell death. As shown in Fig 3, P14 OPCs had longer Tcs and produced smaller clones than P9 OPCs, which in turn had longer Tcs and produced smaller clones than P0 OPCs; furthermore, P9 and P14 OPCs had significantly higher rates of spontaneous differentiation and cell death than P0 OPCs. The results were remarkably similar to those obtained with cultured OPCs of equivalent ages (Fig 2; Table 1 and Table 2), supporting the possibility that the changes with time in culture reflect normal OPC cell maturation, rather than a cell culture artefact.
|
After 17 d in culture, spontaneous differentiation and cell death continued to increase (Table 2), reaching plateau levels by 4–6 wk (Table 2 and data not shown). By 55 d in culture, the Tcs stabilized at ~35 h (Fig 2). Spontaneous differentiation and cell death rates stabilized at <10%, and now cells in PDGF plus bFGF showed lower spontaneous differentiation and death rates than cells in PDGF alone (Table 2 and not shown). Once established after 40–60 d in culture, the OPCs appeared to self renew indefinitely. This was the case for eight of eight separate long-term experiments. In each case, the established OPCs in either PDGF or PDGF plus bFGF had Tcs of ~35 h (Fig 2 C). The cells in PDGF alone had longer population doubling times than the same set of cells in PDGF plus bFGF (Table 1), mainly because the spontaneous differentiation and death rates were lower in bFGF (Table 2).
Induced Differentiation
When cells in PDGF were induced to differentiate by the addition of TH after various periods of time in culture, the rate at which oligodendrocytes developed progressively slowed as the time before TH addition increased, although the rate of oligodendrocyte development when PDGF was withdrawn slowed much less. After 2 d in culture, ~100% of P7 OPCs differentiated into morphologically typical oligodendrocytes within 6 d when PDGF was withdrawn (Fig 4 A). Although the response of such cells to TH addition was slower than to PDGF withdrawal, >80% differentiated within 7 d (Fig 4 A). By contrast, only ~50% of OPCs cultured in PDGF for 75 d differentiated within 1 wk when induced by TH, although their response to PDGF withdrawal was comparable to that of cells cultured in PDGF for 2 d (not shown). After 150 d in PDGF, the response to TH addition was even slower, although the response to PDGF withdrawal was still just as fast as after 2 d in PDGF (Fig 4 C). After 390 d, it took 18 d for 40% of the cells to differentiate after TH addition and 10 d before almost all of the cells differentiated after PDGF removal (Fig 4 E). In all cases, differentiation was confirmed by RT-PCR analysis of MBP and PLP mRNAs (Fig 5 and not shown); for 390-d cultures, differentiation was also confirmed by clonal analysis and MBP staining (not shown). In summary, the response of OPCs to TH decreases with time in culture, whereas the response to PDGF withdrawal decreases much less.
|
|
OPCs cultured in PDGF plus bFGF showed much less slowing in their differentiation response with time in culture. Almost all of these cells differentiated within 1 wk when deprived of both PDGF and bFGF, even after they had been in culture for 390 d (Fig 4B, Fig D, and Fig F) or 510 d (not shown). The cells responded somewhat more slowly when deprived only of PDGF (Fig 4B, Fig D, and Fig F), suggesting that bFGF weakly suppresses PDGF withdrawal–induced differentiation. bFGF had a much stronger inhibitory effect on TH-induced differentiation: in the presence of bFGF and PDGF, only ~16% of OPCs that had been in culture for 2 d differentiated after 6 d of TH treatment (Fig 4 B), but a similar rate of differentiation was seen when cells cultured in PDGF plus bFGF for 390 d were treated with TH.
Expression of Antigenic Markers and Migration
We next used immunofluorescence to examine the expression of a number of antigenic markers in OPCs after various times in culture. We stained cells for A2B5, O1, O4, GC, NG2, MBP, PLP, vimentin, nestin, and GFAP, most of which are expressed by oligodendrocyte lineage cells at various developmental stages (
|
|
Most interestingly, OPCs cultured in PDGF alone progressively acquired staining by O4, O1, and anti-GC antibodies: whereas cells in culture for 3 d did not stain with any of these antibodies (Table 3), those cultured for 50 d showed some staining with all three antibodies (Fig 6E, Fig I, and Fig M). Cells acquired O4 staining first: ~30% of the OPCs in PDGF for 50 d were stained strongly by O4 antibody (Fig 6 E, and Table 3), whereas most cells of this age showed only weak staining with O1 and anti-GC antibodies (Fig 6I and Fig M; Table 3). The staining of these antibodies increased with time, and by 300 d, most of the cells showed strong staining with all three antibodies, comparable to that observed for fully differentiated oligodendrocytes (Fig 6F, Fig J, and Fig N).
OPCs cultured in PDGF and bFGF also acquired these antigens but with a much slower time course. After 50 d the cells did not stain with the O1 antibody (Fig 6 K) and stained only very weakly with the O4 and anti-GC antibodies (Fig 6G and Fig O; Table 3). The staining with the anti-GC antibody (
Nestin is an intermediate filament protein characteristic of neural stem cells (
Although the long-term OPCs cultured in PDGF alone strongly stained with O4, O1, and anti-GC antibodies (Fig 6F, Fig J, and Fig N), the cells were still clearly precursors rather than oligodendrocytes as they also expressed NG2 (Fig 7 A), an antigen expressed on OPCs that is lost when OPCs differentiate into oligodendrocytes (
|
We also examined the migration of OPCs after various times in culture. As shown in Table 4, the migration rate of OPCs cultured in PDGF alone progressively slowed, while that of OPCs cultured in PDGF plus bFGF slowed much less, further suggesting that the presence of bFGF slows down OPC maturation in culture.
|
Freshly Purified Adult OPCs
Taken together, our findings suggested that OPCs cultured in PDGF for an extended period of time came to resemble adult OPCs in a number of their properties (
P150 rats. The addition of neuregulin greatly enhanced the survival of the older cells, as we report elsewhere (Fernandez, P.-A., D.G. Tang, L. Cheng, A.W. Mudge, A. Prochiantz, and M.C. Raff, manuscript submitted for publication).
We first compared the ability of purified adult OPCs and age-matched long-term cultured OPCs to incorporate BrdU after an 8-h pulse of BrdU. As shown in Fig 8 A, ~45% of OPCs maintained in PDGF for 50 d and ~35% maintained for 300 d or 390 d incorporated BrdU, whereas <10% of the adult OPCs did so under the same conditions, and the proportion decreased with increasing age of the adult OPCs. The OPCs from P300 and P390 rats were maintained in PDGF and NRG, but NRG did not enhance BrdU incorporation, either by itself or with PDGF (not shown).
|
Although adult OPCs proliferated much more slowly than long-term cultured OPCs, the two types of OPCs shared a number of other characteristics. Both populations differentiated equally slowly in response to TH addition, although P50 OPCs also differentiated slowly in response to PDGF withdrawal, whereas P7 OPCs that had been in culture for 50 d differentiated quickly when PDGF was withdrawn (Fig 8 B). The two types of OPCs were also antigenically similar. Adult OPCs of all ages examined, just like long-term cultured OPCs, stained strongly for A5B5 (Fig 9A and Fig B), but they did not stain for MBP, PLP GFAP, or filamentous vimentin (not shown). Moreover, P50 adult OPCs stained only weakly with O1, O4, and anti-GC antibodies, whereas P300 OPCs stained strongly with all these antibodies (Fig 9C and Fig D, and not shown). A notable difference, however, was that adult OPCs of all ages stained strongly for filamentous nestin (Fig 9E and Fig F), whereas the longterm cultured OPCs in PDGF progressively lost such staining (see Fig 6 R).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
Most OPCs in the developing optic nerve divide a limited number of times before they stop and terminally differentiate into postmitotic oligodendrocytes (reviewed in
By manipulating the culture medium appropriately one can keep most perinatal OPCs dividing in culture beyond the time when they would normally withdraw from the cell cycle and terminally differentiate. One way is to omit TH from the culture medium (Barres et al., 1994;
Noble and his colleagues (
Why Do So Many P7 OPCs Differentiate and/or Die in Our Cultures?
We showed previously that when purified P7 OPCs were cultured for weeks in PDGF without TH their proliferation slowed, their rate of spontaneous differentiation into oligodendrocytes gradually increased, and by 30 d many cells had differentiated and/or died (
We favor the second explanation for several reasons. At the time that our cells become difficult to maintain and passage, they show few of the changes that are characteristic of replicative senescence: they do not stain (unpublished observations), at low pH, with senescence-associated ß-galactosidase (P150 rats but not from P50 rats; finally, bFGF increases the death of neonatal OPCs, whereas it significantly inhibits the spontaneous differentiation and death of long-term cultured OPCs. If we are correct that many of the P7 OPCs die during the first few weeks in our culture because the culture conditions are suboptimal for oligodendrocytes and more mature OPCs, we should be able to find conditions where most neonatal OPCs survive through this critical period in culture without differentiation. If this proves possible, we should then be able to determine whether most P7 OPCs can develop into adult OPCs or whether only a subpopulation of P7 OPCs have this potential.
What Distinguishes the OPCs That Survive?
Despite the significant amount of cell death, many OPCs always seem to survive and continue to proliferate indefinitely, or at least for 18 mo, which is as long as we have followed them. We do not know what is special about these cells that enables them to live while many others differentiate and/or die. One possibility is that they preexist as a subpopulation of stem-like cells with exceptional capacity for survival and self renewal. Another is that they arise as immortalized mutants. Although we cannot exclude this latter possibility, we think it unlikely for four reasons: (a) OPCs with the same phenotype developed in eight out of eight experiments. (b) The cells that survive seem to mature over months and acquire at least some of the properties of adult OPCs on much the same schedule as their normal counterparts in vivo, as we discuss below. (c) Even after 15 mo, the cells growing in either PDGF or PDGF plus bFGF show normal cell cycle checkpoint and apoptotic responses (Tang et al., manuscript in preparation). (d) The cells that survive during the critical period are found in most clones.
Yet another possible explanation for what distinguishes the OPCs that survive in our cultures is that they represent the statistical tail of the OPC population with the best survival capabilities. Whatever their origins, it is clear that these cells have the ability for long-term self renewal in culture, in the absence of other cell types.
The Long-Term Survivors in PDGF Progressively Acquire Some of the Properties of Adult OPCs
The most important finding in this study is that in all eight experiments where we established long-term cultures of purified P7 OPCs in PDGF, we find that the cells progressively acquire, over months, some of the properties that are characteristic of OPCs in the adult optic nerve. The cells become larger and their morphology becomes more complex, so that by 5–6 mo they have multiple branching processes. One has to be cautious, however, in interpreting changes in morphology, as cell morphology is very sensitive to environmental influence. Although OPCs in the adult optic nerve have a similar complex morphology (
More importantly, the OPCs cultured in PDGF progressively change their antigenic phenotype, mimicking many of the antigenic changes that normally occur in vivo. Most remarkably, the cells gradually acquire the glycolipids, including GC, that are recognized by the O1 (
Noble and colleagues (
It was also shown previously that adult OPCs differentiate into oligodendrocytes in culture more slowly than do perinatal OPCs (
Taken together, these findings strongly suggest that the surviving OPCs in our cultures continue to mature in vitro for many months, even though they are growing in isolation from other cell types.
P7 OPCs cultured for many months in PDGF without TH do not acquire all of the characteristics of adult OPCs. They do not slow their proliferation rate to nearly the same extent, for example. Whereas the cultured cells have a Tc of ~35 h from 50 d onward, freshly isolated adult OPCs cultured in the same medium divide much more slowly, at a rate that is inversely correlated with the age of the animal from which they are obtained. It is interesting that adult OPCs can be induced to divide rapidly if they are stimulated in culture with the right combination of factors (
Basic FGF Inhibits the Maturation of OPCs in Culture
Whereas the OPCs growing in PDGF progressively mature and acquire a number of the properties of adult OPCs, those maintained in PDGF plus bFGF mature much more slowly and maintain many of their youthful characteristics. They retain their simple bipolar morphology, their rapid migration rate, a relatively fast differentiation response, and acquire differentiation markers more slowly. Thus, the combination of bFGF and PDGF clearly inhibits OPC maturation in culture, just as it inhibits OPC differentiation into oligodendrocytes (
In summary, we have shown that when purified P7 OPCs are cultured in PDGF (or PDGF plus bFGF) without TH, many of the cells differentiate and/or die after several weeks, but many others continue to proliferate for up to 16 months or more. In PDGF, the surviving cells progressively acquire a number of properties that are characteristic of adult OPCs, while in PDGF and bFGF they retain many of their neonatal properties. These findings suggest that OPCs have an intrinsic maturation program that normally plays out over many months but can be restrained by bFGF. The main challenge now is to determine the molecular basis of such a prolonged intracellular program.
|
Footnotes |
|---|
1 Abbreviations used in this paper: BrdU, bromodeoxyuridine; CNS, central nervous system; CNTF, ciliary neurotrophic factor; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; NRG, neuregulins; NT-3, neurotrophin 3; O-2A, oligodendrocyte type-2 astrocyte; OPCs, oligodendrocyte precursor cells; PDL, poly-D-lysine; Tc, cell cycle time; TH, thyroid hormone. 
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
The authors are grateful to Mark Marcionni for NRG; Fen-Biao Gao for his initial help in the project; Bill Richardson, Ben Barres, and Anne Mudge for advice; Ben Barres for providing detailed immunopurification protocols for adult precursor cells; Paul Van Heyningen for help with cell cycle analysis; and Lili Cheng and the members of the Raff lab for discussion and support.
D.G. Tang is a recipient of a Hitchings-Elion Award from Burroughs-Wellcome Fund. Y.M. Tokumoto and M.C. Raff are supported by grants from the British Medical Research Council.
Submitted: 17 November 1999
Revised: 17 January 2000
Accepted: 20 January 2000
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionAcknowledgementsReferences |
|---|
