Medical Research Council Developmental Neurobiology Programme, Medical Research Council Laboratory for Molecular Cell Biology, and the Biology Department, University College London, London WC1E 6BT, United Kingdom
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 Clonal analyses performed on single (Temple and Raff,
1986 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 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.
Animals and Chemicals
Sprague/Dawley rats were obtained from the Animal Facility at University College London (London, UK). Chemicals were purchased from
Sigma Chemical Co. (St. Louis, MO), except where indicated. Recombinant human PDGF-AA and neurotrophin-3 (NT-3) were purchased from
Peprotech (Rocky Hill, NJ).
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 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 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 As previously described, when purified P7 precursor cells
were cultured at clonal density in plateau concentrations
of mitogens and TH, the cells divided a variable number of
times (from zero to eight) before they stopped and differentiated (Barres et al., 1994 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
Embryonic Precursor Cells Divide More Times before
Differentiating Than Do Postnatal Ones
One possible explanation for the heterogeneity in the proliferative capacity of P7 precursor cells in the presence of
plateau concentrations of mitogens and TH is that the cells
differ in maturity, perhaps because their ancestors migrated into the optic nerve at different times. To explore
this possibility, we studied the behavior of precursor cells
purified from either E18 optic nerves (which would be expected to be less mature and more homogeneous in proliferation potential than P7 precursors) or P14 nerves (which would be expected to be more mature than P7 precursors).
The cells were cultured at clonal density in the presence of
mitogens and TH to allow the normal clock mechanism to
operate, and the size of each clone was determined after 3, 6, 9, or 11 d in culture. As shown in Fig. 2, the average
number of cells per clone was much greater for E18 cells
than for P7 or P14 cells at 6 and 9 d; by 9 d, when most P7
cells had stopped dividing and had differentiated, the average clone size for P7 and P14 cells was <40 cells, whereas the average clone size for E18 cells was >160 cells, and
many of these clones were still growing. Furthermore, as
shown in Fig. 3, whereas only 1% of the P7 cells divided
seven times by 6 d in vitro, ~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.
Embryonic Precursor Cells Divide Faster Than Do
Postnatal Ones
To compare the cell cycle times of E18, P7, and P14 precursor cells, the cells were cultured at clonal density in the
presence of mitogens but in the absence of TH. When
clone sizes were assessed after 2 and 4 d in vitro, the calculated average cell cycle times were 21 h for E18 cells, 28 h
for P7 cells, and 42 h for P14 cells (Fig. 4). Thus the cell cycle times of precursor cells seem to increase progressively
with maturation in vivo, suggesting that differences in maturation may also be an important source of heterogeneity in the cell cycle times of P7 precursor cells.
Unequal Cell Divisions Are Another Source of Cell
Cycle-Time Heterogeneity
When purified P7 precursor cells were studied by time-lapse
video recording while growing at clonal density in mitogens in the absence of TH, the time between mitoses ranged
from 13 to 43 h, with an average of 27 ± 1 h (mean ± SEM, n = 60), which was similar to the calculated average
cell cycle time shown in Fig. 4. Although some of this heterogeneity presumably reflected variation in the maturity
of the precursor cells, this was not the only source of heterogeneity in cell cycle times, as even within a single clone
there could be substantial heterogeneity. When the two
daughter cells produced by the division of a precursor cell
were followed, for example, they usually divided again at almost exactly the same time, but this was not always the
case: in about 30% of the cases, one daughter divided well
before the other (Table I). In all of these cases, the unequal behavior of the two daughter cells could be traced to
their production by an unequal cell division in which the
dividing cell failed to retract all of its processes before cytokinesis, so that one daughter inherited the processes and
was therefore significantly larger than its sibling (Fig. 5).
In each case the larger cell divided first, presumably because it attained a sufficient size to pass through the restriction point R in the G1 phase of the cell cycle (Pardee,
1974 Table I.
Time of Sister Cell Divisions
Unequal Divisions Increase with Precursor
Cell Maturity
When cells within individual clones of freshly purified P7
precursor cells were followed by time-lapse video recording in cultures containing mitogens but not TH, the first
two divisions usually produced daughter cells that divided
after about the same length of time, whereas many of the
subsequent divisions produced daughter cells that divided
after different lengths of time. A typical clone is diagrammed in Fig. 6. Thus cell cycle-time heterogeneity within P7 clones increased with time.
The complexity of the cell processes extended by P7
precursor cells also increased with time within clones (not
shown), providing a possible explanation for the increase
in unequal cell divisions that occurred with time in culture.
To test this possibility we followed individual clones of purified E18 precursor cells by time-lapse video recording.
When growing in mitogens in the absence of TH, most of
these cells initially had a simple bipolar morphology, with
two unbranched processes. Most cells within a clone produced simple bipolar daughter cells that went on to divide
at almost the same time. With further divisions, however,
the cells tended to have longer and more complex processes, and unequal divisions began to occur more frequently. A typical clone is diagrammed in Fig. 7. Thus, on
average, E18 precursors had a simpler morphology and divided more equally than P7 precursor cells, and they only started dividing unequally when they developed more
complex processes, supporting the possibility that there is
a direct relationship between process complexity and unequal cell division.
Cell Cycle Times within Clones Tend to Oscillate from
One Generation to the Next
As seen in Figs. 6 and 7, there was a tendency for the cell
cycle times of daughter cells produced by precursor cells
within a clone to be longer if the previous cycle time was
short and to be shorter if the previous cycle time was long.
This could be seen more clearly when the average cycle
times between divisions two and five in the two clones
shown in Figs. 6 and 7 were plotted as shown in Fig. 8.
We started video recording after the first cell division in
culture, and the first recorded cycle (which was the second
division in culture) tended to be longer than the next, possibly because the cells were recovering from the dissociation procedure during the first cycle in culture. The oscillatory behavior is unlikely to have resulted from cell-cell
interactions within the clones, as the precursor cells were
highly motile, and daughter cells tended to separate after
cytokinesis and usually did not interact with each other or
with other cells in the clone during the first five divisions.
The oscillation of cell cycle times within a clone is consistent with the idea that a precursor cell has an intrinsic
mechanism for regulating its size: a cell produced by a cycle that was shorter than average would tend to be smaller than average and would therefore need a longer than average time to grow to produce two daughter cells of average
size; the opposite would be the case for a cell produced by
a longer than average cycle.
Purified E18 Precursor Cells Can Mature in the
Absence of TH
When the migration rates of purified E18 and P7 precursor cells were measured in clonal cultures by time-lapse
video recording, it was found that E18 cells migrated faster
than P7 cells (Table II). Thus, E18 precursor cells differed
from P7 precursor cells in several ways: they had a simpler
morphology, they divided faster, and they migrated faster.
To determine whether E18 precursor cells can mature in
culture so as to resemble P7 precursor cells in morphology,
cell cycle time, and rate of migration, we cultured purified
E18 precursor cells in the presence of mitogens and the absence of TH for 10 d. As shown in Fig. 9, the morphology of the E18 cells changed during the 10 d culture period: their processes became longer and more complex, so
that the cells resembled P7 precursor cells. Similarly, their
migration rate slowed to resemble that of P7 cells (Table
II). Moreover, when we removed the E18 cells from the
flask after 10 d in vitro and recultured them at clonal density, still in the presence of mitogens and in the absence of
TH, their cell cycle times were now indistinguishable from those of P7 (Fig. 10). These findings suggest that E18 precursor cells can mature in culture in the absence of TH and
other cell types, much as they do in vivo.
Table II.
The Migration Rates of Oligodendrocyte Precursor
Cells In Vitro
Advantages of Oligodendrocyte Precursor Cells for
Studying Proliferation Control
Oligodendrocyte precursor cells isolated from the developing rat optic nerve offer a number of advantages for
studying the mechanisms that control cell proliferation
and the timing of differentiation. First, they can be cultured, either as single cells in microwells (Temple and
Raff, 1986 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 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 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 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 It has been shown previously that oligodendrocyte precursor cells are also present in the adult rat optic nerve
(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 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).
). 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
).
) 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.
, 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
).
Materials and Methods
). 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.
) 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.
). 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.
Results
). Moreover, as expected,
when the cells were cultured in the presence of mitogens
but in the absence of TH, very few cells stopped dividing
and differentiated (Barres et al., 1994
), but the cell cycle
times, either deduced from the sizes of the clones at various
times or measured directly by time-lapse video recording, varied greatly. The following experiments were performed
mainly to determine the origins of this heterogeneity in
proliferative capacity and cell cycle times in precursor cells
isolated from the same age nerves.
). 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
).
Fig. 1.
Increasing clonal size with increasing concentrations of
PDGF. Purified P7 precursor cells were cultured at clonal density for 4 d in varying concentrations of PDGF and in the absence of TH, and the number of cells in each clone was counted in an inverted phase-contrast microscope. Half the medium was replaced
at 2 d. The results are expressed as mean ± SEM of at least 100 clones. As judged by morphology (Temple and Raff, 1986), at 0.1 ng/ml PDGF, most clones were oligodendrocyte clones; at 0.3 ng/
ml most were still precursor clones and at 1.0 and 10 ng/ml all
were still precursor clones.
[View Larger Version of this Image (42K GIF file)]
Fig. 2.
Clonal size in cultures of purified E18, P7, or P14 precursor cells in the presence of both mitogens and TH. The number of cells in each clone was counted on days 3, 6, and 9 in vitro, and 50-100 clones were averaged for each E18 time point and at least 100 clones for each P7 and P14 time point. By day 9 most of
the P7 and P14 cells had stopped dividing and differentiated into
oligodendrocytes, while many E18 cells were still precursor cells.
The results are expressed as mean ± SEM.
[View Larger Version of this Image (28K GIF file)]
Fig. 3.
Proliferative capacity of purified P7 (A) and E18 (B)
precursor cells cultured for 6 d at clonal density in the presence of both mitogens and TH. The cell number in each clone was recorded, and 50-100 clones were analyzed for each age. Clones
containing 2 cells were classified as having gone through 1 division, 3-4 cells as 2 divisions, 5-8 cells as 3 divisions, and so on.
[View Larger Versions of these Images (25 + 25K GIF file)]
Fig. 4.
Clone size (A) and cell cycle time (B) in cultures of purified E18, P7, or P14 precursor cells cultured for 4 d at clonal density in
the presence of mitogens but in the absence of TH to inhibit differentiation. The number of cells in each clone was counted; 50-100
clones were averaged for E18 cells and at least 100 clones for P7 and P14 cells. The cell cycle times were calculated from the average
clone sizes. The results are expressed as mean ± SEM.
[View Larger Version of this Image (23K GIF file)]
) earlier than did its smaller sibling. Thus unequal cell
divisions is an additional cause of cell cycle-time heterogeneity among P7 precursors cells.
Fig. 5.
An unequal cell division
and its consequences observed by
time-lapse video recording. Purified
P7 precursor cells were cultured as in
Fig. 4. The cells shown at 1.5 and
0.5 h divided unequally at 0 h, with
one daughter cell (cell 1) inheriting
the two cell processes that failed to
withdraw before cytokinesis occurred. Cell 1 went on to divide at
+13.4 h, while its sister (cell 2) divided at +29.6 h.
[View Larger Version of this Image (90K GIF file)]
Fig. 6.
Time-lapse video
analysis of a single P7 precursor cell clone in a culture
grown as in Fig. 4. The cells
were cultured for 1 d before
recording began, so that the
first cell cycle was not recorded. Cell cycle times (in
hours) were determined by
measuring the time between
mitotic telophases. One representative experiment is
shown here; two other clones
were analyzed with similar
results. "Out" in this figure
and the next refers to a cell
that migrated out of the field
of observation.
[View Larger Version of this Image (20K GIF file)]
Fig. 7.
Time-lapse video analysis of a single E18 precursor cell
clone in a culture grown and assessed as in Fig. 6. One representative experiment is shown here; one other clone was analyzed
with similar results. Each X represents a cell that died (by apoptosis) for unknown reasons.
[View Larger Version of this Image (19K GIF file)]
Fig. 8.
Oscillations in cell cycle times with each round of division in P7 and E18 clones. The data in A were taken from Fig. 6
and in B from Fig. 7; they are expressed as mean ± SEM for each
cell generation.
[View Larger Versions of these Images (16 + 16K GIF file)]
Fig. 9.
Morphological maturation of E18 precursor cells in culture. Purified E18 (A) and P7 precursor cells (C) were cultured
for 2 d in slide flask as in Fig. 4. In B, E18 cells were cultured for
10 d as in Fig. 4 and were then removed with trypsin and recultured in the same conditions for an additional 2 d. The cells were
fixed and stained with A2B5 antibody to visualize the cell morphology. The arrows indicate the location of the cell bodies. Bar,
50 µm.
[View Larger Version of this Image (45K GIF file)]
Fig. 10.
Maturation of E18 precursor cells in culture. Purified E18 precursor cells were cultured as in Fig. 4. After 10 d, the cells were
removed from the culture flask with trypsin and were then recultured at clonal density in the same conditions for an additional 4 d.
Clone size (A) and cell cycle time (B) were then compared with those in clonal density cultures prepared from purified, freshly isolated
E18 and P7 precursor cells and maintained in the same conditions. 50-100 clones were averaged for each data point, and the results are
expressed as mean ± SEM. DIV, days in vitro.
[View Larger Version of this Image (29K GIF file)]
Discussion
) or as purified cells at clonal density (Barres et
al., 1994
), so that their behavior can be studied in the virtual absence of other cell types. Second, they can be cultured in serum-free medium, so that one can control their
extracellular environment (Barres et al., 1994
). Third, the
precursor cells and differentiated oligodendrocytes have
distinctive morphologies, so that they can be readily distinguished, even while still alive (Temple and Raff, 1986
).
Fourth, unlike cell lines, they are normal cells and, with
the addition of the appropriate signaling molecules, purified precursor cells in serum-free clonal culture divide a limited number of times before they stop dividing and terminally differentiate into postmitotic oligodendrocytes,
just as they do in vivo (Temple and Raff, 1986
; Barres et
al., 1992
). Fifth, individual clones can be followed by time-lapse cinematography (Small et al., 1987
) or video recording (Gao et al., 1997
), so that cell migration and every cell
division, cell death, and differentiation event can be documented. An important disadvantage of the system is that it
is difficult to obtain enough cells to do biochemical analyses.
; 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.
.
),
although in those studies it was unclear whether the
changes in morphology reflected changes in the environment, the precursor cells, or both.
),
and without mitogens they stop dividing and prematurely
differentiate (Temple and Raff, 1985
); both survival signals and mitogens were always added to our cultures.
). 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.
; 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
).
), 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
).
; 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.
Received for publication 23 April 1997 and in revised form 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.We thank B. Durand, M. Jacobson, and V. Wallace for comments on the manuscript and Robert Brooks for helpful suggestions on cell cycle time analyses.
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