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© The Rockefeller University Press,
0021-9525/1997//1367 $5.00
The Journal of Cell Biology, Volume 138, Number 6,
, 1997 1367-1377
Article |
Cell Size Control and a Cell-intrinsic Maturation Program in Proliferating Oligodendrocyte Precursor Cells
We have used clonal analysis and time-lapse video recording to study the proliferative behavior of purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve growing in serum-free cultures. First, we show that the cell cycle time of precursor cells decreases with increasing concentrations of PDGF, the main mitogen for these cells, suggesting that PDGF levels may regulate the cell cycle time during development. Second, we show that precursor cells isolated from embryonic day 18 (E18) nerves differ from precursor cells isolated from postnatal day 7 (P7) or P14 nerves in a number of ways: they have a simpler morphology, and they divide faster and longer before they stop dividing and differentiate into postmitotic oligodendrocytes. Third, we show that purified E18 precursor cells proliferating in culture progressively change their properties to resemble postnatal cells, suggesting that progressive maturation is an intrinsic property of the precursors. Finally, we show that precursor cells, especially mature ones, sometimes divide unequally, such that one daughter cell is larger than the other; in each of these cases the larger daughter cell divides well before the smaller one, suggesting that the precursor cells, just like single-celled eucaryotes, have to reach a threshold size before they can divide. These and other findings raise the possibility that such stochastic unequal divisions, rather than the stochastic events occurring in G1 proposed by "transition probability" models, may explain the random variability of cell cycle times seen within clonal cell lines in culture.
CONTROLS on mammalian cell division and differentiation are complex and difficult to analyze. Time-lapse video recording of cells in culture provides a powerful way of simplifying the analysis, as one can follow every moment of a cell's growth, division, death, and differentiation in a controlled environment. Most of such studies have been done either with cell lines that are abnormal in at least some aspects of proliferation and differentiation control or with primary fibroblasts that are usually an undefined mixture of cell types. We have used time-lapse video recording to study purified oligodendrocyte precursor cells isolated from the perinatal rat optic nerve. These normal cells have the advantage that they can be grown in serum-free medium at clonal density under conditions where they go through a limited number of divisions before they stop and differentiate, on a schedule that closely resembles that in vivo (Barres et al., 1994).
Oligodendrocytes myelinate axons in the vertebrate central nervous system (CNS).1 In the rat optic nerve they develop from precursor cells that migrate into the nerve from the brain, beginning at about embryonic day 15 (E15; Small et al., 1987). It is not known how many precursor cells enter the nerve or how long the immigration process continues for. After a limited number of divisions, the precursor cells stop dividing and terminally differentiate (Temple and Raff, 1986). Oligodendrocytes first develop in the nerve around birth (Miller et al., 1985) and continue to increase in number for six postnatal weeks (Skoff et al., 1976; Barres et al., 1992). The mechanisms that control the number of oligodendrocytes in the nerve have been extensively studied (for review see Barres and Raff, 1994).
Clonal analyses performed on single (Temple and Raff, 1986) or purified (Barres et al., 1994) precursor cells isolated from postnatal day 7–8 (P7–8) rat optic nerve indicate that the cells divide about once a day and are heterogeneous in the number of times they divide before they differentiate, varying between zero and eight times. The progeny of an individual precursor cell, however, tend to stop dividing and differentiate into oligodendrocytes at about the same time; even when the two daughter cells of a single precursor cell division are cultured separately, they tend to divide the same number of times before they differentiate (Temple and Raff, 1986). These findings suggest that an intrinsic clock operates in each precursor cell to help control when it stops dividing and differentiates.
The normal operation of the intracellular clock, however, depends on at least three types of extracellular signaling molecules: (a) survival signals, which suppress the intrinsic suicide program present in most animal cells (Barres et al., 1992, 1993; Weil et al., 1996); (b) mitogens, especially PDGF (Noble et al., 1988; Raff et al., 1988; Richardson et al., 1988), which stimulates cell proliferation; and (c) hydrophobic signals such as thyroid hormone (TH) or retinoic acid (RA), which help the cells stop dividing when the appropriate time is reached (Barres et al., 1994). In the absence of survival signals, the cells undergo programmed cell death (Barres et al., 1992); in the absence of mitogens, they rapidly stop dividing and prematurely differentiate (Raff et al., 1983; Noble and Murray, 1984; Temple and Raff, 1985), and, in the absence of TH and RA, most of the cells fail to withdraw from the cell cycle and fail to differentiate (Barres et al., 1994).
In the present study we have examined the proliferative behavior of precursor cells purified from E18, P7, and P14 rat optic nerve, in both clonal-density and higher density cultures, by periodic counting of clone size or by continuous time-lapse video recording. Our main aim was to determine why precursor cells in the same age nerve differ in their proliferative properties. We show that the younger precursor cells have shorter cell cycle times and tend to divide more times before they differentiate than the older precursor cells. This finding suggests that the precursor cells progressively mature as they proliferate and that differences in precursor cell maturation are largely responsible for the heterogeneous proliferative behavior of the precursor cells in the postnatal optic nerve. Our finding that purified embryonic precursor cells mature in culture suggests that maturation is an intrinsic property of the cells. We show that, although the two daughter cells of single precursor cells tend to have similar cell cycle times, this is not always the case; and when there is a large difference in these times, it is because the two cells were produced by an unequal division, with the larger cell always dividing first. This finding suggests that the cells have to reach a threshold size before they can divide. Such unequal divisions occur more frequently in older precursor cells, apparently because older cells tend to extend longer and more complex processes and sometimes fail to withdraw them before dividing; the daughter cell that inherits the processes is substantially larger than the one that does not. We also show that the cell cycle time of the precursor cells varies inversely with the concentration of PDGF, suggesting that PDGF levels regulate cell cycle time during precursor cell development.
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Materials and Methods |
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Preparation of Optic Nerve Cells
Optic nerves were removed from E18, P7, or P14 rats, minced, and incubated at 37°C for 75 min in papain solution (165 U; Boehringer Mannheim, Indianapolis, IN) in Hepes-buffered minimal Eagle's medium (MEM/HEPES). Cells were dissociated by trituration, using a Gilson Pipetman set at 500 µl, and filtered through Nitex mesh (20-µm pore size; Tetko, London, UK). They were washed sequentially in a low concentration of ovomucoid and BSA (1.5 mg/ml of each) in MEM/HEPES and then a high concentration of ovomucoid and BSA (6 mg/ml of each).
Purification of Oligodendrocyte Precursor Cells
Oligodendrocyte precursor cells were purified by sequential immunopanning, as described previously (Barres et al., 1992). In brief, dissociated optic nerve cells were resuspended in 10 ml of L15 Air Medium (GIBCO BRL, Gaithersburg, MD), containing 0.2 mg/ml BSA and 10 µg/ml bovine insulin, and plated on a 100-mm bacteriological petri dish (Falcon) coated with monoclonal anti-galactocerebroside (GC) antibody (supernatant diluted 1:3; Ranscht et al., 1982). The dish was kept for 45 min at room temperature, and it was gently shaken every 15 min. The nonadherent cells were transferred to a second dish coated with the A2B5 monoclonal antibody (ascites fluid diluted 1:2000; Eisenbarth et al., 1979) and incubated for another 45 min. The final dish was then washed five to eight times with 6 ml of MEM/HEPES, and the remaining adherent oligodendrocyte precursor cells were dislodged by trypsin treatment (0.012% in EBSS) and washed in L15 Air Medium containing 20% FCS.
Clonal Cultures of Precursor Cells
The purified oligodendrocyte precursor cells were counted and plated at clonal density (800–1,000 P7 or P14 cells or 2,000 E18 cells in 3 ml of culture medium) in poly-D-lysine (PDL)-coated 25-cm2 flasks (Falcon). In cultures of P7 or P14 cells, after 4 d in vitro, there were <2 contaminating cells per flask, and in cultures of E18 cells, there were <50 contaminating cells per flask; these did not divide and tended to die. In some cases they were cultured in PDL-coated, slide flasks (400 cells in 1.5 ml of medium, Nunc, Roskilde, Denmark). The cells were cultured in DME containing bovine insulin (10 µg/ml), human transferrin (100 µg/ml), BSA (100 µg/ ml), progesterone (60 ng/ml), putrescine (16 µg/ml), sodium selenite (40 ng/ml), N-acetyl-cysteine (60 µg/ml), forskolin (5 µM), trace minerals (GIBCO BRL), and penicillin and streptomycin (GIBCO BRL). Depending on the specific experiment, PDGF (usually at 10 ng/ml), NT-3 (5 ng/ ml), or triiodothyronine (TH, 30 ng/ml) were added.
Cultures were maintained in a 5% CO2 incubator at 37°C. If cultures were maintained for >4 d, 50% of the medium was replaced with freshly made medium every 4 d. Oligodendrocytes and oligodendrocyte precursor cells were identified by their characteristic morphologies (Temple and Raff, 1986) and, in some cases, by immunostaining with A2B5 or anti-GC antibodies (see below). Clones were scored as oligodendrocyte or precursor cell clones according to the predominant cell type present in the clone. In most cases, >90% of the cells in a clone were of the same type.
Immunofluorescence Assays
Immunofluorescence assays were carried out with cells on either PDL-coated coverslips or PDL-coated slide flasks. For staining surface antigens, the cells were fixed with 2% paraformaldehyde for 2 min at room temperature and incubated for 30 min in blocking solution consisting of 50% goat serum in Tris buffer (pH 7.4) containing 1% BSA and 150 mM L-lysine. The cells were then incubated in either anti-GC antibody (supernant diluted 1:1) followed by Texas red-conjugated goat anti–mouse IgG (diluted 1:100; Jackson ImmunoResearch Laboratory, West Grove, PA), or A2B5 antibody (ascites fluid diluted 1:100) followed by fluorescein-conjugated goat anti–mouse IgM (diluted 1:100; Jackson ImmunoResearch Laboratory). The coverslips were mounted in Citifluor mounting medium (CitiFluor, London, UK), sealed with nail polish, and examined with a fluorescence microscope (Axioskop; Zeiss, Inc., Thornwood, NY).
Time-Lapse Video Recording
Time-lapse analysis was performed as previously described (Gao et al., 1997). Briefly, purified oligodendrocyte precursor cells were cultured at clonal density in 25-cm2 flasks, as described above. Individual clones were continuously followed on a heated stage of an inverted phase-contrast microscope (Zeiss, Inc.) coupled to an Sony CCD video camera and time-lapse video cassette recorder. Cell cycle times were determined by measuring the time between mitotic telophases. Images were captured from the video recorder using an image grabber.
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Cell Cycle Times Decrease as the Concentration of PDGF Is Increased
Before examining the origins of proliferative heterogeneity, we examined the influence of PDGF concentration on cell cycle time. We cultured purified P7 precursor cells at clonal density for 4 d in varying concentrations of PDGF, from 0.1 to 10 ng/ml. TH was omitted to prevent most of the cells from exiting the cell cycle and differentiating (Barres et al., 1994). As shown in Fig. 1, the number of cells per clone progressively increased with increasing concentrations of PDGF. At 0.1 ng/ml most of the cells had stopped dividing and had become oligodendrocytes by 4 d, as without adequate mitogen stimulation, precursor cells differentiate even in the absence of TH (Barres et al., 1994). At 0.3 ng/ml only a few clones had become oligodendrocyte clones, and at 1.0 and 10 ng/ml none of the clones had done so, even when the cells were cultured up to 8 d (not shown). Decreased cell death seemed not to be responsible for the increased number of cells per clone with increasing concentrations of PDGF, as very few dead cells were seen by 4 d at any of the PDGF concentrations used; it was shown previously that 0.1 ng/ml of PDGF is saturating for precursor cell survival (Barres et al., 1993). The increased cell cycle time at low PDGF concentration was also confirmed by time-lapse video recording (data not shown). Our findings, therefore, suggest that the cell cycle time progressively decreases as PDGF concentration is increased from 0.3 to 1.0 ng/ml and from 1.0 to 10 ng/ml. In the following experiments, PDGF was used at 10 ng/ml, which was saturating for cell proliferation in our clonal assay (not shown); 10 ng/ml PDGF was previously shown to be on a plateau for the induction of DNA synthesis in oligodendrocyte precursor cells (Barres et al., 1993).
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70% of the E18 cells did so. On average, P14 cells divided even fewer times than P7 cells (not shown). After 11 d in vitro, some E18 cell clones contained >1,024 cells, indicating that the cells had divided up to 10 or 11 times, whereas no P7 clones contained >256 cells (eight divisions) and no P14 clones contained >128 cells (seven divisions; not shown). Moreover, by 11 d almost all of the P7 and P14 cells had become oligodendrocytes, whereas many of the E18 cells were still dividing precursor cells. Thus the number of times a precursor cell divides before differentiating seems to decrease progressively with maturation. In addition, as shown in Fig. 3, E18 cells were much more homogeneous in their proliferative capacity than P7 cells. The finding that E18 precursor cells are more homogeneous and divide more times before they differentiate than P7 or P14 precursor cells suggests that one important source of heterogeneity in the proliferative behavior of P7 precursor cells is that the cells are heterogeneous in terms of their maturation.
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Discussion |
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Progressive Precursor Cell Maturation
In the present study we initially set out to determine why the oligodendrocyte precursor cells isolated from the P7 optic nerve are heterogeneous in the number of times they divide before they stop and differentiate; whereas some cells differentiate without dividing at all, others divide anywhere up to eight times before they stop and differentiate (Temple and Raff, 1986; Barres et al., 1994). It seemed likely that one reason for this heterogeneity is that the precursor cells differ in maturation, perhaps because they have migrated into the nerve at different times, so that some have already gone through most of their divisions before being isolated, while others have many divisions still to go (Temple and Raff, 1986). Our findings are consistent with this interpretation. We show that precursor cells purified from E18 optic nerve, which would be expected to have recently migrated into the nerve (Small et al., 1987), are much more homogeneous in their proliferative capacity than are precursor cells purified from P7 or P14 nerves. Moreover, on average, the E18 precursor cells go through many more divisions before they differentiate than do P7 or P14 cells, with some dividing 10 or 11 times in an 11 d culture period.
E18 precursor cells also differ from P7 and P14 precursor cells in three other ways. They are morphologically less complex with fewer and less branching processes, they divide faster, and they migrate faster. The average cell cycle time in culture for an E18 precursor cell is about 20 h, whereas it is about 28 h for a P7 precursor cell and about 42 h for a P14 precursor cell. The average migration rate for an E18 cell is 30 µm/h, whereas it is 22 µm/h for a P7 cell. This value for P7 is very similar to that previously reported (21.4 µm/h) for these cells by Wolswijk and Noble (1989).
Taken together, these findings suggest that oligodendrocyte precursor cells change progressively during development. This conclusion is consistent with previous findings that with increasing developmental age the precursor cells display a progressively more complex morphology when visualized in situ in the rat optic nerve (Fulton et al., 1992), although in those studies it was unclear whether the changes in morphology reflected changes in the environment, the precursor cells, or both.
A Cell-intrinsic Maturation Program
As our present findings have been obtained with purified precursor cells isolated from nerves of different ages and studied under the same conditions, the differences in morphology, cell cycle time, proliferative capacity, and cell migration rate presumably reflect differences that are intrinsic to the cells themselves, rather than differences in the cells' environment at the different ages. Not only are the differences intrinsic to the cells, but the program of progressive change seems to be intrinsic as well, as purified E18 precursor cells maintained in culture for 10 d in mitogens and in the absence of TH come to resemble P7 precursor cells in their morphology, cell cycle time, and rate of migration, even though they are maintained in constant culture conditions. That is not to say that signals from other cells are unimportant: without survival signals the cells undergo programmed cell death (Barres et al., 1992), and without mitogens they stop dividing and prematurely differentiate (Temple and Raff, 1985); both survival signals and mitogens were always added to our cultures.
Moreover, we show here that the cell cycle time varies inversely with the concentrations of PDGF, suggesting that the levels of PDGF in the developing optic nerve may regulate the rate of cell cycle progression in the precursor cells. For this reason, we used 10 ng/ml of PDGF when comparing the cell cycle times of precursor cells from nerves of different ages; this concentration was shown previously to be on a plateau for the induction of DNA synthesis in cultures of purified P8 precursor cells (Barres et al., 1993). Changing properties with development seems to be a common feature of vertebrate precursor cells: retinal precursor cells, for example, progressively change in their developmental potential (Watanabe and Raff, 1990) and response to growth factors (Lillien and Cepko, 1992), and haematopoietic stem cells change their developmental potential over time (Morrison et al., 1994). In none of these cases, however, has it been shown that purified precursor cells in culture change their properties over time.
It has been shown previously that oligodendrocyte precursor cells are also present in the adult rat optic nerve (french-Constant and Raff, 1986; Wolswijk and Noble, 1989), although these cells have a number of properties that distinguish them from the precursor cells present in the perinatal nerve: they have an altered antigenic phenotype, for example, and they divide, migrate, and differentiate more slowly than perinatal precursor cells (Wolswijk and Noble, 1989). Wren et al. (1992) have provided evidence that the adult precursor cells develop from perinatal ones: in time-lapse microcinematographic studies several cells with a perinatal phenotype were seen to give rise to cells with an adult phenotype, although this transition sometimes occurred gradually over two or more cell divisions. Our observations extend these findings in two ways: first, they suggest that oligodendrocyte precursor cells progressively change their properties over time, beginning early in development; second, they indicate that these changes can occur in purified precursor cells in culture, as long as mitogens are present and TH is not, so that the cells continue to proliferate and do not differentiate, suggesting that maturation reflects the operation of an intrinsic developmental program in each precursor cell. It remains to be determined whether purified prenatal precursor cells in culture can develop all of the properties of adult precursor cells. Most important, it remains to be demonstrated whether the precursor cells in the adult optic nerve are maintained by continual self-renewal (Wren et al., 1992) or by continual generation from a separate population of stem cells in the nerve or brain (french-Constant and Raff, 1986).
Unequal Cell Divisions and Cell Size Control
Whereas maturation differences between the precursor cells are likely to be an important source of the cell cycle-time heterogeneity in our cultures, it is not the only source. When studied by time-lapse video recording, the daughter cells produced by individual P7 precursor cells usually divide again within an hour or so of each other, but in 30% of the cases, one daughter divides many hours before the other. In each instance this is the result of an unequal cell division, in which the mother cell fails to withdraw one or more of its processes when it rounds up at mitosis; as a result of inheriting the process(es), one of the daughter cells is substantially larger than its sibling, and it is always this daughter cell that divides first. This finding strongly suggests that oligodendrocyte precursor cells, just like yeast cells (Fantes, 1977), amoebae (Prescott, 1956), Tetrahymena (Prescott, 1959), and some mammalian cell lines (Killander and Zetterberg, 1965), have to grow to a critical size before they can divide. Not surprisingly, unequal cell divisions occur much more frequently in mature oligodendrocyte precursor cells, which have long and complex processes, than in immature E18 precursor cells, which have short, simple processes. Cell size controls, however, also seem to operate in E18 precursor cells, as the cell cycle times within an E18 clone tend to be longer if the previous cell cycle time is short and shorter if the previous cycle is long; cycles that are shorter than average would tend to produce cells that are smaller than average and therefore need a longer time in the next cycle to grow, so that it could produce two daughter cells of average size; the reverse would be the case for cells produced by cycles that are longer than average. The molecular basis of such size control is still uncertain, although there is evidence that it may involve, in part at least, the regulation of the retinoblastoma protein Rb (Nasmyth, 1996), which normally acts to suppress the transcription of genes required for DNA replication (Riley et al., 1994) as well as genes involved in protein synthesis (Cavanaugh et al., 1995; White et al., 1996).
Time-lapse cinematographic studies of cloned cell lines have shown that the cells of an individual line display a wide range of cell cycle times, even when growing under uniform conditions; it has been argued from such studies that cell-cycle progression in G1 depends on one or more probabilistic, or stochastic, events (Smith and Martin, 1973; Brooks et al., 1980). The remarkable similarity in cell cycle times of sister cells produced by the early divisions of E18 oligodendrocyte precursor cells in our cultures, however, are hard to reconcile with such "transition probability" models. An attractive, alternative explanation for the random variability of cell cycle times within clones is that it reflects the uneven segregation of cytoplasmic contents at cytokinesis, as suggested by a number of previous experiments (Prescott, 1959; Riley and Hola, 1983; Sennerstam, 1988), as well as by the present study. Such unequal cell divisions would seem even more likely to occur in vivo, where the environment is much more complex than the two-dimensional substratum of a culture dish. Whatever the mechanism, the tendency for initially synchronous cell populations to rapidly become asynchronous may help to ensure that cells within clones do not all divide at the same time; as cells tend to round up and lose their attachments during mitosis, synchronous divisions could disrupt the integrity of a tissue.
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Acknowledgments |
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Submitted: 23 April 1997
Revised: 26 June 1997
F.-B. Gao is supported by a Hitchings-Elion Fellowship from the Burroughs Wellcome Fund. The work is supported by Medical Research Council, UK.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Barres B & Raff M. Control of oligodendrocyte number in the developing rat optic nerve, Neuron, 1994, 12, 935–942.[Medline]
Barres B, Hart I, Coles H, Burne JF, Voyvodic J, Richardson W & Raff M. Cell death and control of cell survival in the oligodendrocyte lineage, Cell, 1992, 70, 31–46.[Medline]
Barres B, Schmid R, Sendtner M & Raff M. Multiple extracellular signals are required for long term oligodendrocyte survival, Development, 1993, 118, 283–295.[Abstract]
Barres B, Lazar M & Raff M. A novel role for thyroid hormone, glucocorticoids and retinoic acid in timing oligodendrocyte development, Development, 1994, 120, 1097–1108.[Abstract]
Brooks RF, Bennett DC & Smith JA. Mammalian cell cycles need two random transitions, Cell, 1980, 19, 493–504.[Medline]
Cavanaugh AH, Hempel WM, Taylor LJ, Rogalsky V, Todorov G & Rothblum LI. Activity of RNA polymerase I transcription factor UBF blocked by Rb gene product, Nature (Lond), 1995, 374, 177–180.[Medline]
Eisenbarth GS, Walsh FS & Nirenburg M. Monoclonal antibodies to a plasma membrane antigen of neurons, Proc Natl Acad Sci USA, 1979, 76, 4913–4916.
Fantes PA. Control of cell size and cycle time in Schizosaccharomyces pombe. , J Cell Sci, 1977, 24, 51–67.[Abstract]
french-Constant C & Raff MC. Proliferating bipotential glial progenitor cells in adult rat optic nerve, Nature (Lond), 1986, 319, 499–502.[Medline]
Fulton BP, Burne JF & Raff MC. Visualization of O-2A progenitor cells in developing and adult rat optic nerve by quisqualate-stimulated cobalt uptake, J Neurosci, 1992, 12, 4816–4833.[Abstract]
Gao F-B, Durand B & Raff M. Oligodendrocyte precursor cells count time but not cell divisions before differentiation, Curr Biol, 1997, 7, 152–155.[Medline]
Killander D & Zetterberg A. A quantitative cytochemical investigation of the relationship between cell mass and initiation of DNA synthesis in mouse fibroblasts in vitro. , Exp Cell Res, 1965, 40, 12–20.[Medline]
Lillien L & Cepko C. Control of proliferation in the retina: temporal changes in responsiveness to FGF and TGF 
, Development, 1992, 115, 253–266.[Abstract]
Miller RH, David S, Patel R, Abney ER & Raff MC. A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: in vivoevidence for two distinct astrocyte lineages, Dev Biol, 1985, 111, 35–41.[Medline]
Morrison SJ, Uchida N & Weissman IL. The biology of hematopoietic stem cells, Annu Rev Cell Dev Biol, 1994, 11, 35–71.
Nasmyth K. Retinoblastoma protein: another role rolls in, Nature (Lond), 1996, 382, 28–29.[Medline]
Noble M & Murray K. Purified astrocytes promote the in vitrodivisions of a bipotential glial progenitor cell, EMBO (Eur Mol Biol Organ) J, 1984, 3, 2243–2247.[Medline]
Noble M, Murray K, Stroobant P, Waterfield MD & Riddle P. PDGF promotes division and motility and inhibits premature differentiation of the oligodendrocyte-type-2 astrocyte progenitor cell, Nature (Lond), 1988, 333, 560–562.[Medline]
Pardee AB. A restriction point for control of normal animal cell proliferation, Proc Natl Acad Sci USA, 1974, 71, 1286–1290.
Prescott DM. Relation between cell growth and cell division, Exp Cell Res, 1956, 11, 86–98.[Medline]
Prescott DM. Variations in the individual generation times of Tetrahymena Geleii Exp. , Cell Res, 1959, 16, 279–284.
Raff MC. Glial cell diversification in the rat optic nerve, Science (Wash DC), 1989, 243, 1450–1455.
Raff MC, Miller RH & Noble M. A glial progenitor cell that develops in vitrointo an astrocyte or an oligodendrocyte depending on culture medium, Nature (Lond), 1983, 303, 390–396.[Medline]
Raff MC, Abney ER & Fok-Seang J. Reconstitution of a developmental clock in vitro: a critical role for astrocytes in the timing of oligodendrocyte differentiation, Cell, 1985, 42, 61–69.[Medline]
Raff MC, Lillien L, Richardson W, Burne JF & Noble M. Platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture, Nature (Lond), 1988, 333, 562–565.[Medline]
Ranscht B, Clapshaw P, Price J, Noble M & Seifert W. Development of oligodendrocytes and schwann cells studied with a monoclonal antibody against galactocerebroside, Proc Natl Acad Sci USA, 1982, 79, 2709–2713.
Riley PA & Hola M. Transient intraclonal variation in interdivision time in relation to orientation at cytokinesis of GPK cells in layer culture, Cell Tissue Kinet, 1983, 16, 189–198.[Medline]
Riley DJ, Lee EY & Lee WH. The retinoblastoma protein: more than a tumor suppressor, Annu Rev Cell Biol, 1994, 10, 1–29.
Richardson WD, Pringle N, Mosley MJ, Westermark B & Dubois-Dalcq M. A role for platelet-derived growth factor in normal gliogenesis in the central nervous system, Cell, 1988, 53, 309–319.[Medline]
Skoff RP, Price DL & Stocks A. Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation, J Comp Neurol, 1976, 169, 291–312.[Medline]
Sennerstam R. Partition of protein (mass) to sister cell pairs at mitosis: a re-evaluation, J Cell Sci, 1988, 90, 301–306.
Small R, Riddle P & Noble M. Evidence for migration of oligo-oligodendrocyte-type-2 astrocyte progenitor cells into the developing rat optic nerve, Nature (Lond), 1987, 328, 155–157.[Medline]
Smith JA & Martin L. Do cells cycle? , Proc Natl Acad Sci USA, 1973, 70, 1263–1267.
Temple S & Raff M. Differentiation of a bipotential glial progenitor cell in single cell microculture, Nature (Lond), 1985, 313, 223–225.[Medline]
Temple S & Raff M. Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions, Cell, 1986, 44, 773–779.[Medline]
Watanabe T & Raff MC. Rod photoreceptor development in vitro: intrinsic properties of proliferating neuroepithelial cells change as development proceeds in the rat retina, Neuron, 1990, 4, 461–467.[Medline]
Weil M, Jacobson MD, Coles HS, Davies TJ, Gardner RL, Raff KD & Raff MC. Constitutive expression of the machinery for programmed cell death, J Cell Biol, 1996, 133, 1053–1059.
White RJ, Trouche D, Martin K, Jackson SP & Kouzarides T. Repression of RNA polymerase III transcription by the retinoblastoma protein, Nature (Lond), 1996, 382, 88–90.[Medline]
Wolswijk G & Noble M. Identification of an adult-specific glial progenitor cell, Development, 1989, 105, 387–400.[Abstract]
Wren D, Wolswijk G & Noble M. In vitro analysis of the origin and maintenance of O-2Adult progenitor cells, J Cell Biol, 1992, 116, 167–176.
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