Neuronal influences on glial progenitor cell development

Neuron

Vol. 3, 103-l

13. July, 1989, Copyright

@ 1989 by Cell Press

Neuronal Influences on Glial Progenitor Cell Development Joel M. Levine

long

Department of Neurobiology and Behavior State University of New York at Stony Brook Stony Brook, New York 11794

1985). Optic nerve cultures are aneuronal, and the use of this experimental system precludes any possible neuronal influences on the development of glial progenitor cells. However, several lines of evidence suggest a role for the

Summary

neuron as a potential regulator of the development of 02A progenitor cells. Transection of neonatal optic nerves causes a large decrease in the total number of type 2 astrocytes, oligodendrocytes, and progenitor cells within the surviving nerve stump (David et al., 1984). Type 1 astrocytes are much less affected by nerve transection. Furthermore, an inducing factor, found in extracts of 3-week-old rat optic nerve, can direct progenitor cells to develop into type 2 astrocytes (Hughes and Raff, 1987). Additional indirect evidence for a neuronal influence on glial cell development comes from studies of the developing cerebellum. Antibodies against a chondroitin-sulfate proteoglycan, termed the NG2 antigen, have identified a cell type in cultures of postnatal cerebellum that develops into a type 2 or stellate astrocyte when grown in medium containing 10% fetal calf serum but fails to develop into an oligodendrocyte when grown in chemically defined medium (Levine and Stallcup, 1987). Rather NG2-positive, galactocerebroside (GalC)-negative cells persist in these cultures for up to 2 weeks, which is well past the time when oligodendrocytes develop in the intact cerebellum (Reynolds and Wilkins, 1988). 02A progenitor cells from the optic nerve also carry the NG2 proteoglycan antigen on their surfaces (Stallcup and Beasley, 1987), suggesting that the cerebellar glial precursor cells are related and perhaps identical to 02A progenitor cells. The apparent failure of NG2-labeled progenitor cells to develop into oligodendrocytes in the cerebellar cultures therefore raises the question of what factors may be present in the cerebellar tissue culture system and missing in the optic nerve cultures and how these factors may influence the developmental choices made by glial progenitor cells. The experiments reported here were initiated to begin to analyze the complex cell-cell interactions that occur in mixed culture systems, such as those prepared from postnatal cerebellum, and how these interactions might influence glial progenitor cell differentiation. We first show that cerebellar progenitor cells isolated from longterm cultures of cerebellum and grown in the absence of neurons and type 1 astrocytes rapidly differentiate into oligodendrocytes. In the mixed cerebellar cultures, however, a fraction of the NGZ-labeled progenitor cells divide and their progeny develop into additional progenitor cells. Thus the 02A lineage has the capacity for self-renewal. Finally, medium conditioned by cerebellar interneurons can promote the survival of optic nerve progenitor cells in vitro and delay or prevent their differentiation into oligodendrocytes. The factors contained within the neuron-conditioned medium appear to alter

The role of cell-cell interactions in the development of bipotential glial progenitor cells in cultures of rat cerebellum and optic nerve was studied. In the cerebellar cultures, progenitor cells divide slowly and most of their progeny develop into additional progenitor cells. Progenitor cells isolated from postconfluent cultures of cerebellum, however, develop rapidly into oligodendrocytes when grown in a serum-free medium. Factors secreted or shed into the medium by young cerebellar interneurons stimulate optic nerve progenitor cells to divide and promote the survival of progenitor cells. These factors appear to alter the function of the internal clock that regulates the timing of oligodendrocyte differentiation. These results suggest that the neuronal microenvironment can influence the lineage decisions of multipotential glial progenitor cells. Introduction Cell-ceil interactions are likely to play important regulatory roles during the development and differentiation of complex tissues such as the mammalian brain. The interactions that govern the development of the macroglial cells of brain have recently become clear. In the developing optic nerve, process-bearing (type 2) astrocytes and oligodendrocytes develop from a common precursor cell termed an 02A progenitor cell (Raff et al., 1983). 02A progenitor cells respond to their environment in several ways. First, a second type of astrocyte with a flat, fibroblastic morphology in dissociated tissue culture (termed a type 1 astrocyte) secretes a factor into the medium that stimulates 02A progenitor cells to divide (Noble and Murray, 1984). This mitogen is likely to be platelet-derived growth factor (PDGF; Noble et al., 1988; Raff et al., 1988; Richardson et al., 1988). PDGF is thought to drive an internal clock that regulates the timing of oligodendrocyte differentiation in vitro. Second, the differentiation of progenitor cells is responsive to the tissue culture environment. When grown in serum-containing medium, progenitor cells develop into stellate-shaped astrocytes designated as type 2 astrocytes. In a chemically defined, serum-free medium, however, progenitor cells rapidly differentiate into oligodendrocytes (Raff et al., 1983). Although progenitor cells can be driven to divide by either PDGF (Richardson et al., 1988) or medium conditioned by type 1 astrocytes (Noble and Murray, 1984), their subsequent differentiation into oligodendrocytes appears to be constitutive as

as the

inducing

factor

in serum

is absent

(Raff

et al.,

NWKT 104

Figure 1. Development and Oligodendrocytes

110

Ohgodendrocytes and glial progenitor cells were isolated from postconfluent cultures of postnatal cerebellum as described in Experimental Procedures. The cells were grown in a serum-free, defined medium and, at the indicated day in culture, immunofluorescently stained using a rabbit anti-NC2 antiserum and one of two different mouse monoclonal antibodies against GalC. Labeled cells were counted, and the data shown are the mean and standard deviation for four different experiments. The percentage of labeled cells is plotted against days in culture. The data shown in (A) are for total cells harvested after shaking; those in (B) are for cells depleted of oligodendrocytes by treatment with rabbit anti-CalC antibodies and complement. Closed triangles, oligodendrocytes; open triangles, progenitor cells.

100 90 80 f P 'i

70

::

50

80

0

1

2345878

days in vitro

012345

6

7

of Progenitor Cells in Secondary Culture

8

days in vitro

the function of the internal clock responsible for the timing of oligodendrocyte differentiation. These results demonstrate that neurons influence the developmental pathways glial progenitor cells follow and suggest that the microenvironment has an instructive influence on glial progenitor cell differentiation.

in the cultures. Cell division may delay the differentiation of the cells into oligodendrocytes. Third, the cerebellar tissue culture environment may contain additional factors that promote the survival of the progenitor cell phenotype. These possibilities are addressed below.

Results

Cultures of postnatal cerebellum contain a variety of cell types including astrocytes, oligodendrocytes, and neurons. Small bipolar neurons that correspond to granule cells are the predominant cell type found in these cultures (Currie and Dutton, 1980). To study the intrinsic developmental capabilities of the NG2+ progenitor cells, cultures containing NG2+ cells free from neurons and type 1 astrocytes were prepared using the shaking method of McCarthy and DeVellis (1980). Cells harvested after overnight shaking of postconfluent cerebellar cultures were a mixture of progenitor cells (defined as NG2+, A2B5+, GalC-, GFAP-), oligodendrocytes (GalC+), and macrophages. Approximately 75% of the cells were NG2+, and the remaining 25% were GalC+ (macrophages were not quantitated in these studies). When these cells were grown in serumfree medium, the NG2-labeled cells disappeared and most of the cells in the cultures developed into oligodendrocytes (Figure 1A). To demonstrate that the NG2+ progenitor cells were the source of the oligodendrocytes that develop in these cultures, antibodies against GalC and complement were used to kill >80% of the oligodendrocytes present immediately after harvesting the ceils. The remaining cells were a mixture of NG2+ progenitor cells and macrophages. As shown in Figure lB, the NG2+ cells also developed rapidly into oligodendrocytes. Although the disappearance of the NGZ-labeled cells and the appearance of oligodendrocytes were rapid, after 48 hr in culture, only 2% of the NG2+ cells expressed cell surface GalC. Less than 6% of the NGZ-labeled cells in these secondary cultures were dividing (data not shown). Thus, when NG2-labeled

Isolated

The NG2 antigen, a cell surface chondroitin-sulfate proteoglycan is a surface marker for 02A progenitor cells, and antibodies against this surface marker have been used to study the development of glial progenitor cells in cultures prepared from postnatal rat cerebellum (Levine and Stallcup, 1987) and optic nerve (Stallcup and Beasley, 1987). When cells from both developing tissues are grown in a serum-free, chemically defined medium, there are subtle differences in the behavior of the NG2labeled progenitor cells. In the optic nerve cultures, NG2-labeled cells rapidly disappear concurrent with the appearance of numerous oligodendrocytes (Stallcup and Beasley, 1987). It is likely that the NG2+ cells are developing into oligodendrocytes, since as many as 40% of the NG2-labeled cells also bind antibodies against GalC, a surface marker for oligodendrocytes (Raff et al., 1978). In the cerebellar cultures, however, the NG2labeled cells persist for up to 2 weeks in vitro and rarely express surface markers characteristic of oligodendrocytes. The persistence of these cells in tissue culture is not an artifact since cells with a similar phenotype are found in the adult cerebellum (Levine and Card, 1987). There are several possible explanations for the differential development of the NGZ-labeled progenitor cells. First, cerebellar progenitor cells may represent a cell type different from the progenitor cells found in optic nerve. These different cell types may fortuitously express the same repertoire of surface markers. Second, progenitor cells in the cerebellar cultures may be driven to divide by factors produced by the other cell types present

Progenitor

Cells

Clw 105

Progenitor

Cell Development

3

5

7

3107

DAY IN VITRO Figure

2. Cell Division

in Cerebellar

Cultures

Postnatal day 4 cerebellar cells were dissociated and grown rn a serum-free, defined medium. At the indicated times, the cells were labeled with [3H]thymidine for 18 hr, washed, fixed, and processed for immunofluorescence and autoradiography as described rn Experimental Procedures. The data shown are the mean and standard deviation of three to six different experiments. The columns labeled “3 to 7” represent pulse-chase experiments in which the cells were labeled on the third day in culture, washed, and allowed to grow for 4 additional days without isotope before processing. Filled bars, progenitor cells; crosshatched bars, oligodendrocytes.

progenitor cells of the cerebellum sence of interneurons, type 1 astrocyte-inducing factor found in differentiate into oligodendrocytes. population of cells were grown medium for 5 days, over 90% of the beled with antibodies against glial

are grown in the abastrocytes and the serum, they rapidly When the same in serum-containing NG2+ cells were lafibrillary acidic pro-

tein (GFAP; data not shown). The bilities of the cerebellar progenitor identical to those of 02A progenitor in developing optic nerve.

developmental capacells are therefore cells characterized

Progenitor

Cells Divide

in Cerebellar

Cultures

The experiment described above demonstrates that cerebellar progenitor cells can develop into oligodendrocytes despite the fact that only small numbers of differentiating progenitor cells express both the NC2 antigen and GalC. The persistence of NGZ-labeled progenitor cells in the cerebellar cultures might then be due to cell division, which is known to delay the terminal differentiation of 02A progenitor cells (Raff et al., 1985). To determine whether the NG2+ progenitor cells divide, cells were grown in the presence of [3H]thymidine. The cells were pulse-labeled with [3H]thymidine for 18-20 hr and prepared for immunofluorescence staining and autoradiography as described in Experimental Procedures. The cultures were stained with both a rabbit antiNG2 antiserum and one of two different mouse monoclonal antibodies against GalC (Sommer and Schachner, 1981; Ranscht et al., 1982). After developing the autoradiograms, fluorescently labeled cells were counted and scored for the presence of silver grains over the nucleus. Only cells that expressed either the NG2 antigen

or cell surface GalC were counted. As shown in Figure 2, 19% of the progenitor cells were synthesizing DNA after 3 days of growth in defined medium. Quantitatively similar results were obtained when the cells were pulselabeled on the fifth or seventh day of culture (Figure 2). To analyze the fate of the dividing progenitor cells, cells were pulse-labeled on the third day of culture in defined medium, washed, and then chased until they had been cultured for a total of 7 days. As shown in Figure 2, 56% of the NG2+ cells were labeled with silver grains. The dividing NG2+ cells, which had a stellate appearance, often occurred in small clusters with most of the cells in the clusters being lightly labeled with silver grains over their nuclei (Figure 3). This demonstrates that dividing NC2 cells give rise to additional NGZ-labeled cells in a self-renewing manner. The same pulse-chase experiments were analyzed for labeled oligodendrocytes (Figure 2; Figure 3). Between 3% and 5% of the oligodendrocytes present in the cultures were labeled with silver grains, suggesting that oligodendrocytes and their direct precursors rarely divide in culture. Type 1 astrocytes can secrete PDGF into the medium (Noble et al., 1988; Raff et al., 1988), and PDGF is a potent mitogen for glial progenitor cells (Richardson et al., 1988). To determine whether these cultures contained type 1 astrocytes, cerebellar cultures (grown in defined medium) were immunofluorescently stained with antiGFAPantibodies and monoclonal antibodyA2B5 (Eisenbarth et al., 1979). Figure 4 shows a typical GFAPe, A2B5cell. Although this cell and others tended to have an elongated rather than a flattened appearance, its antigenic phenotype is identical to that of type 1 astrocytes. After 7 days in vitro, the type 1 astrocytes were present at a density of 7.7 + 1.7 x 103/cmL. The cerebellar cells were originally plated at a density of 1.2 x lOVcm*. Assuming 70% cell survival, the type 1 astrocytes account for less than 10% of the total cells in these cultures. To test whether PDGF secretion by type 1 astrocytes is responsible for the division of the NGZ-labeled progenitor cells, cells were grown and labeled in the presence of 50 &ml neutralizing antibody against human PDGF. This amount of antibody can neutralize 90% of the mitogenic activity of exogenous human PDGF when used at 3 rig/ml (see below). The data in Table 1 indicate that 45% of the mitogenic stimulation in the cerebellar cultures at 3 days in vitro can be blocked by the antiPDGF antibodies. The source of the residual 55% mitotic activity is unknown. When the cells were labeled with [3H]thymidine in the presence of anti-PDGF antibodies at 7 days in vitro, only 14% of the mitogenic activity was neutralized by the anti-PDGF antibodies. Considering the low density of type 1 astrocytes and the observation that the anti-PDGF antibodies can only partially block the division of progenitor cells, it seems likely that factors other than PDGF may be responsible, at least in part, for the division and survival of progenitor cells. Such factors might be produced by either the neurons or glial cells found in these mixed cultures.

NG2 3d

NG2 3+7

GC 3+7

Figure 3. The Appearance of Labeled Cells in the Cerebellar Cultures The cells were processed as described in Experimental Procedures. Each pair of photographs shows the same field in epifluorescence and in phase-contrast optics. The antibodies used and the time in culture are indicated to the left. The photomicrographs labeled “3’7 are from the pulse-chase experiments, and the arrows in the phase photomicrographs indicate the positions of the fluorescently labeled cell bodies. CC, anti-CalC staining; NC2, anti-NC2 staining. The NG2+, stellate-shaped cells are labeled with silver grains; the GalC+ oligodendrocytes are not.

Effects of Neuron-Conditioned

Medium

The hypothesis that cerebellar interneurons influence the development of glial progenitor cells was tested by growing optic nerve cells in medium that had been conditioned by cerebellar interneurons. Optic nerves were

chosen as a convenient source of 02A progenitor cells free from neurons. Postnatal cerebellar cells were grown in the presence of cytosine arabinoside for 5-6 days to eliminate dividing nonneuronal cells, and the neurons were allowed to condition serum-free DMEM for 48 hr.

CM 107

Progenitor

Cell Development

anti-GFAP

anti-A2B5 Figure

4. Appearance

of Type

1 Astrocytes

in the Cerebellar

Cultures

Cerebellar cells were grown in defined medium for 7 days and fluorescently labeled with the indicated antibodies. The same field is shown labeled with anti-A2B5 antibodies to reveal the progenitor cells (three bipolar cells are labeled in this field), labeled with anti-CFAP antibodies to reveal the astrocytes, and with phase-contrast optics. The elongated cell visible with anti-CFAP staining is a type 1 astroycte.

The chemical additives that constitute defined medium were added to the conditioned medium, and postnatal day 6 and 7 optic nerve cells were grown in this conditioned and defined medium. When optic nerve glial cells were grown in medium conditioned by cerebellar interneurons, there were striking differences in the development of these cultures as compared with cells grown in defined medium alone (Table 2). Of the starting optic nerve dissociates analyzed 18 hr after plating, 54% of the stained cells were

Table 1. Cell Division Anti-PDCF Antibodies

in Cerebellar

Cultures:

%

%

Condition

Progenitor Cell Dividing

Oligodendrocytes Dividing

DIV

19.3 f

2.2 f 0.8

3 fn = 6)

5.5

Plus anti-PDGF (n = 3)

10.7

DIV

27.1

f

10.2

+ 1.4

5 (n = 6)

Plus ant!-PDGF (n = 3) DIV

7 (n = 5)

k 2.9 16

17.2

* 11.9

Plus ant+PDCF fn = 3)

15.2

+ 1.0

Label DIV 3; chase to DIV 7 (n = 4)

56.4

+ 9.1

Effects

of

% Inhibition

44.8 1.8 f 0.3 62.7

14.2 3.7 f

1.7

Cultures were prepared from postnatal day 4 rat cerebellum, grown in serum-free, defined medium, and labeled with [3H]thymidine as described in Experimental Procedures. Data shown are the means and standard deviations for the indicated number of experiments. Anti-PDGF antiserum was used at 50 &ml. DIV = days in vitro.

labeled with the anti-NC2 antibodies and 47% were positive for GalC. The anti-NC2 labeling may slightly overestimate the actual number of progenitor cells, since 8% of the NG2+ cells were also stained with antibodies against GFAP (data not shown). Greater than 95% of the NG2+ cells were labeled with monoclonal antibody A2B5. After 3 days in defined medium, over 90% of the cells were oligodendrocytes and the number of progenitor cells was reduced to 5%. In contrast, cultures grown for 3 days in medium conditioned by postnatal day 1 cerebellar interneurons comprised 33% progenitor cells and 68% oligodendrocytes. The effects of conditioned medium were also apparent after growing the cells for 5 days, at which time 37% of the cells had the antigenic phenotype of progenitor cells (Table 2). In either defined medium or interneuron-conditioned and defined medium, greater than 95% of the surviving NG2+ cells were GFAP-. Cell division may delay the differentiation of progenitor cells into oligodendrocytes (Raff et al., 1985). Therefore, combined [3H]thymidine labeling and autoradiography was used to determine whether neuron-conditioned medium contained mitogens for progenitor cells. As shown in Table 2, defined medium did not stimulate the division of optic nerve progenitor cells. Only 7% of the NG2+ cells were labeled with silver grains after an 18 hr pulse with [3H]thymidine on the third day of culture. In contrast, when cells were grown in medium conditioned by postnatal day 1 cerebellar neurons, 25% of the progenitor cells were labeled with silver grains. As shown in Table 2, this number of dividing cells is comparable to that seen after growing the cells in medium containing 3 rig/ml human PDGF. However, while neutralizingantibodiesagainst PDGF (50 uglml) can neutralize 90% of the activity of exogenous PDGF, these same antibodies inhibited cell division caused by the cerebellar neuron-conditioned medium by only 50%. These

NWKI” 108

Table

2. Development

of Optic

Nerve

Cells:

Effects

of Conditioned

Medium,

Condition

DIV

% Progenitor

Baseline fn = 6)

18 hr

53.9

DM (n = 6)

3

DM + PDGF (n = 4)

3

47.1

3

10.8 + 4.1

NCM (n = 5)

3

33.5

+ 7.4

25.3

NCM (n = 2)

5

37.5

+ 1.8

36.4

DM + PDCF (n = 2)

+ anti-PDCF

NCM + anti-PDGF (n = 3)

and Anti-PDCF

Antibodies

% Oligodendrocyte

-t 3.8

% Dividing

46.9

f 6.5

9.5

94.7

f 5.4

0.5 + 0.4

+ 5.3

54.1

* 5.4

2.7 f

1.6

2.4 * 3.4

89.2

f

10.2

3.9 f

5.6

+ 10.8

68.4

f

5.4

0.9 *

1.0

25.1

+ 1.4

62.5

f

1.8

2.5 f 0.7

+ 13.3

12.5

k 11.9

68.6

+ 5.4

0.5 f 0.8

32.6

_+ 14.6

62.2

_+ 10.2

1.2 _+ 1.0

69.1

f

0.8 + 1.5

5.3 f 5.4

.3

PDCF,

% Dividing

* 7.9

ACM (n = 4)

3

37.8

_+ 10.2

ACM + anti-PDCF (n = 3)

3

30.9

f

7.9

7.3 f 25.0

8.2 + 4.4

7.8

Cells were grown and labeled with [‘Hlthymidine and the anti-PDCF antibodies as described in Experimental Procedures. Data shown are the means and standard deviations for the indicated number of experiments. Baseline indicates cells labeled 18 hr after plating. DM, defined medium; NCM, medium conditioned by postnatal day 1 cerebellar neurons; ACM, medium conditioned by cerebellar type 1 astrocytes; DIV, days in vitro. PDGF was used at 3 “g/ml and anti-PDGF antiserum was used at 50 ugiml.

data suggest that neurons secrete a mitogen for 02A progenitor cells that either is not PDGF or is poorly recognized by the neutralizing antibodies used here. Type 1 astrocytes, prepared from either neonatal cortex (Noble and Murray, 1984) or optic nerve (Raff et al., 1988), secrete a soluble mitogen for 02A progenitor cells. To determine whether this is a general property of all type 1 astrocytes, optic nerve cells were grown in medium conditioned by cerebellar type 1 astrocytes. After 3 days in cerebellar astrocyte-conditioned medium, 38% of the optic nerve cells were labeled with the NG2 antibodies (Table 2). Of these cells, 33% were labeled with silver grains after an 18 hr pulse with [3H]thymidine. Less than 2% of the oligodendrocytes present were

% GalC positive

% NG2 positive

100

labeled after the same pulse. Addition of anti-PDGF antibodies to the astrocyte-conditioned medium inhibited cell division by i’s%, in agreement with previous studies (Raff et al., 1988), but this treatment had little effect on the relative numbers of progenitor cells and oligodendrocytes that survived. The factors present in the neuron-conditioned medium used above could either delay or inhibit the appearance of oligodendrocytes in the optic nerve cultures. To examine the effects of this conditioned medium on the timing of oligodendrocyte differentiation, embryonic day 21 optic nerve cells were dissociated and grown in either defined medium, defined medium containing PDGF, or neuron-conditioned medium. Em-

Figure 5. Development 21 Optic Nerve Cells

90 60

iim

70 60 50 40 30

media

20 10 0 01234567

days in vitro

days in vitro

of Embryonic

Day

Cells were dissociated and plated as described in Experimental Procedures. At intervals, the cultures were immunofluorescently stained with the anti-NC2 and anti-GalC antibodies and labeled cells were counted. The percentage of cells labeled with each marker is plotted against days in culture. Open squares, cells grown in defined medium; open circles, cells grown in defined medium supplemented with 3 “g/ml PDGF; closed triangles, cells grown in medium conditioned by day 1 cerebellar interneurons.

Clial Progenitor

Cell Development

109

Figure

6. Embryonic

Day 21 Optic

Cells were dissociated, fined medium, 5 days cells, neuron-conditioned

Nerve

Cells

grown, and immunofluorescently in vitro. (B) NC2+ progenitor medium, 5 days in vitro.

labeled as described in Experimental cells, defined medium containing 3 nglml Bar, 20 urn.

bryonic day 21 cells were chosen, since at this developmental stage, there are few if any oligodendrocytes within the optic nerve (Miller et al., 1985). After 24-48 hr of growth in defined medium, oligodendrocytes began to develop and they increased in number rapidly so that after 3 days, greater than 80% of the process-bearing ceils in the cultures were labeled with anti-GalC antibodies (Figure 5; Figure 6). The timing of the appearance of oligodendrocytes was delayed by about 48 hr when the cells were grown in defined medium containing 3 rig/ml human PDGF. During this 48 hr delay, the number of total cells of the 02A lineage increased approximately 3-fold. Under both ofthese conditions, oligodendrocyte differentiation was rapid and virtually complete within 48 hr of their initial appearance. In contrast, oligodendrocytes developed more slowly in cultures grown in medium conditioned by postnatal day 1 neurons. Although oligodendrocytes first appeared within 48 hr of plating the cells, the increase in number was more protracted and less pronounced. When the conditioned medium was removed after 5 days and replaced

Procedures. (A) CalC+ PDCF, 3 days in vitro.

oligodendrocyte, de(C) NG2+ progenitor

with defined medium, the progenitor cells rapidly developed GalC immunoreactivity (data not shown). Thus it appears that the locus at which neuron-conditioned medium is exerting its effects is the internal clock that normally regulates the timing of oligodendrocyte development. Some of the interactions that govern the fate of 02A progenitor cells are developmentally regulated (Miller et al., 1985; Hughes and Raff, 1987). Therefore, the age dependence of the ability of interneuron-conditioned medium to affect glial progenitor cell differentiation was examined.

Figure

7 shows

that

after

3 days

in medium

conditioned by postnatal day 1 or 5 cells, between 24% and 33% of the optic nerve cells were progenitor cells. However, medium conditioned by postnatal day 7 cerebellar cells had no significant effect on the survival of progenitor cells. The cerebellar cells were grown in culture for 6 days prior to conditioning the medium for 48 hr; thus medium conditioned by postnatal day 1, 5, and 7 cells corresponds to cells at days 9, 13, and 15 of development. By postnatal day 15, developing NC2 cells

Figure 7. The Age Dependence of the fects of Neuron Conditioned Medium

n

progenitor dwidmg

%

OIlgo dfviding

Pi NCM

P5 NCM

condition

P7 NCM

Ef-

Optic nerve cells were prepared, grown, and processed as described in Experimental Procedures. After 3 days under the conditions indicated, the ceils were labeled with [‘Hlthymidine, fixed, and fluorescently labeled as in Figure 2. After developing the autoradiographs, the fluorescently labeled cells were counted and scored for silver grains over the nucleus. The data shown are the mean and standard deviation of three to six different experiments. NCM, cerebellar neuron-conditioned medium; Pl, P5, and P7 indicate the postnatal age in days of the donor animals. Filled bars, progenitor cells; dotted bars, oligodendrocytes; darkly crosshatched bars, dividing progenitor cells; lightly crosshatched bars, dividing oligodendrocytes.

within the intact cerebellum have reached their final destinations and have a distribution and morphology similar to that seen in adult animals (Levine and Card, 1987). Thus, it is not unexpected that medium conditioned by the equivalent of IS-day-old interneurons had little effect on the differentiation of glial progenitor cells. Medium conditioned by 5 day cerebellar cells was able to promote the survival of progenitor cells to almost the same extent as day 1 conditioned medium, but caused only 8% of the progenitor cells to divide. Thus progenitor cell survival in the presence of cerebellar interneuronconditioned medium is not dependent upon the ability of the conditioned medium to stimulate DNA synthesis.

Discussion The following conclusions can be drawn from the data presented above: the NGZ-labeled cells characterized previously in cultures of postnatal cerebellum are similar developmentally to 02A progenitor cells of the optic nerve; in the mixed cerebellar cultures, the progenitor cells participate in a self-renewing lineage whereby cell division gives rise to additional progenitor cells; medium conditioned by cerebellar interneurons affects the development of progenitor cells in at least two ways. First, young cerebellar interneurons secrete a mitogen for 02A progenitor cells, and second, interneuron-conditioned medium promotes the survival of progenitor cells. This enhanced survival of progenitor cells may result from an interference with the internal clock whose normal function is to regulate the timing of oligodendrocyte development. These data demonstrate that cell-cell interactions play a role in regulating the development of multipotential progenitor cells. The microenvironment within neural tissues is therefore likely to be a critical determinant in glial cell development. The 02A lineage has been studied extensively in developing optic nerve. The relatively simple cellular composition of this tissue and the high numbers of glial cells in developing optic nerve have made this the tissue of choice for many studies of glial development. Cerebellar cultures on the other hand contain many different cell types, and the potentials for cell-cell interactions are accordingly more complex. Although previous studies (Levi et al., 1986; Levine and Stallcup, 1987) have suggested that cerebellar astrocytes and oligodendrocytes may develop from bipotential precursor cells, the failure to detect cells intermediate between progenitor cells and oligodendrocytes left open the possibility that there are regional differences in glial development throughout the brain. tn this report, the manner in which cerebellar interneuronscan influence the development of glial progenitor cells was examined. The results presented here demonstrate that the 02A lineage provides a general mechanism of glial development and that factors secreted by interneurons can act during development to both expand the numbers of developing progenitor cells and promote their self-renewal. Progenitor cells may therefore persist in adult tissues (ffrench-Constant and Raff, 1986; Reyners et al., 1986).

Development

of Progenitor

Ceils In Vitro

Glial cells isolated from long-term monolayer cultures of postnatal cerebellum by the shaking method can be manipulated to obtain a highly enriched population of progenitor cells. When these cells are grown isolated from the other cell types found in the developing cerebellum, they display a phenotypic plasticity identical to that observed in cultures of developing optic nerve (Raff et al., 1983). When grown in medium containing serum, these cells develop into stellate-appearing astrocytes that can be labeled immunofluorescently with antibodies against GFAP These GFAP+ cells are identical to type 2 astrocytes. In a serum-free, defined medium, this same population of cells rapidly differentiates into oligodendrocytes. This differentiation, which is virtually complete after 4-S days, is not accompanied by cell division. Despite the fact that almost all of the progenitor cells develop into oligodendrocytes, few cells were double labeled with anti-NG2 antibodies and antibodies against GalC. Thus the expression of the NG2 chondroitinsulfate proteoglycan is shut down as cells begin to express characteristics of differentiated oligodendrocytes. The functional properties of this chondroitin-sulfate proteoglycan may be incompatible with the surface properties and activities of mature oligodendrocytes. Secondary cultures of glial precursors depleted of oligodendrocytes by immunocytolysis develop into oligodendrocytes more rapidly than cultures containing a mixed population of progenitor cells and oligodendrocytes. Oligodendrocytes often adapt a spread-out morphology in culture and have few contacts with each other. Cell contacts between developing progenitor cells and mature oligodendrocytes may slow or retard the differentiation of the progenitor cells. Progenitor cells respond to several features of their in vitro and in vivo environment. Fetal calf serum (Raff et al., 1983) or a factor isolated from postnatal optic nerve (Hughes and Raff, 1987) promotes the development of type 2 astrocytes. A second factor, identified as PDGF, is secreted by type 1 astrocytes and is a potent mitogen for progenitor cells (Noble et al., 1988; Raff et al., 1988; Richardson et al., 1988). Cell division, induced by either PDGF or medium conditioned by type 1 astrocyte, can delay the terminal differentiation of progenitor cells into oligodendrocytes. However, the analysis of the fate of single progenitor cell clones stimulated with PDGF has shown that after a limited number of cell divisions, progenitor cell progeny develop into oligodendrocytes (Raff et al., 1988). The data in Figure 2 and Table 1 demonstrate that progenitor cells divide in the mixed cerebellar cultures. Although a small percentage (19.3%) of the progenitor cells are dividing after 3 days in culture, these dividing cells appear to be the source of the progenitor cells that survive for up to 2 weeks. Thus, in addition to generating type 2 astrocytes and oligodendrocytes, the 02A lineage can generate more progenitor cells in a selfrenewing manner. Previous studies of optic nerve development have suggested that the differentiation of 02A progenitor cells is regulated by an internal clock (Temple and Raff, 1986). This clock, which can be driven by PDGF (Raff et al.,

Clial Progenitor 111

Cell Development

1988), presumedly regulates the timing

counts cell divisions of oligodendrocyte

and thereby differentiation.

This model infers the existence of a preprogenitor cell or stem cell, whose slow divisions are responsible for the protracted generation of glial cells in vivo. The data presented here suggest that progenitor cells may be their own stem cells; that is, when under the influence of neuron-derived factors, progenitor cells slowly self-renew. Antibodies against human PDGF could only partially block the ability of neuron-conditioned medium to stimulate DNA synthesis in cerebellar or optic nerve progenitor cells. Although this could be due to a lack of completecross-reactivity between human and rat PDGF, the high degree of functional conservation of PDGF-like molecules among chordates (Singh et al., 1982) renders this possibility unlikely. In addition, the antibodies used here were able to block 75% of the mitogenic activity in medium conditioned by rat type 1 astrocytes, as in previous studies (Raff et al., 1988; Richardson et al., 1988). These considerations suggest that the mitogenic activity in medium conditioned by young cerebellar neurons cannot be fully accounted for by the secretion of PDGF by these cells. Both fibroblast growth factor (Saneto and devellis, 1985) and epidermal growth factor (Leutz and Schachner, 1981) are mitogens for glial cells, and both could be present in the neuron-conditioned medium used here. Whatever the nature of these mitogenic factors, they are present at limiting concentrations. When the conditioned medium was diluted to 1 part in 4, the mitogenic stimulation disappeared (unpublished data). The production of these factors appears to be developmentally regulated, since supernatants prepared from postnatal day 7 cerebellar interneurons after 6-8 days in culture had no effect on progenitor cell division and differentiation. Neuron-conditioned medium promotes the survival of progenitor cells by interfering with the function of the internal clock. Embryonic day 21 optic nerve cells rapidly develop into oligodendrocytes when grown in defined medium. The addition of PDGF delays the onset of this differentiation by about 48 hr. Under both of these conditions, the cell populations studied here behave as if they were synchronized. Once oligodendrocyte differentiation is initiated, it is rapid and virtually complete within 48 hr. Although neuron-conditioned medium does not alter the time at which oligodendrocytes first appear in vitro, it appears to retard the rate of oligodendrocyte differentiation. After 5-7 days of growth in conditioned medium, about one-half of the cells of the 02A lineage have developed GalC immunoreactivity. Since the percentage of oligodendrocytes in the cultures could be enhanced by replacing the conditioned medium with defined medium, neuronconditioned medium is not inhibitory to oligodendrocyte differentiation, but rather appears to desynchronize the progenitor cell population and slow down the processes of differentiation. The exact manner in which the internal clock is altered is unknown. Single progenitor cell clones undergo a maximum of 8 cell divisions before their progeny differentiate into oligodendrocytes (Temple and Raff,

1986). Neuron-conditioned maximum number of cell

medium divisions.

might increase this If this were the case,

one would expect the simultaneous appearance of large numbers of oligodendrocytes after 8 + n cell divisions. The almost linear increase in the number of oligodendrocytes present in embryonic day 21 optic nerve cultures grown in neuron-conditioned medium (Figure 5) is not compatible with such a model. Alternatively, neuronconditioned medium could increase the time between a cell’s last round of cell division and its differentiation into an oligodendrocyte. The failure to detect many thymidine-labeled oligodendrocytes when cerebellar cultures were pulse-chased suggests that such a delay is probably greater than 4 days. These ideas will remain speculative until the molecular nature of the internal clock is elucidated.

Progenitor

Cells In Vivo

In the developing optic nerve, oligodendrocytes begin to appear at birth and continue to increase in numbers over the next 2 weeks of postnatal life (Miller et al., 1985). Although this timing is disrupted when cells are placed in tissue culture, it can be reconstituted by growing the cells in either medium conditioned by type 1 astrocyte (Raff et al., 1985) or medium supplemented with PDGF (Raff et al., 1988). Less is known about the timing of glial development in the cerebellum. Cells carrying the NC2 antigen can be detected as early as embryonic day 16 (Levine and Card, 1987), and their numbers increase rapidly, so that by postnatal days 2 and 3, the entire expanse of the developing cerebellum is filled with immunoreactive cells. This increase in cell number is due at least in part to the migration of progenitor cells into thecerebellum and their proliferation within thedeveloping layers of the cerebellar cortex (Lee and Levine, unpublished data). The results presented above and elsewhere (Levine and Stallcup, 1987; Levine and Card, 1987) lead to the conclusion that the NGZ-labeled cells observed in tissue sections of developing cerebellum represent glial progenitor cells. In adult cerebellum, however, the antiNC2 antibodies label a population of stellate-appearing, GFAPcells with the ultrastructural characteristics of smooth protoplasmic astrocytes (Levine and Card, 1987). Morphologically similar cells are also prominent in the cerebral cortex (Stallcup et al., 1983). The ultrastructural characteristics of these cells are similar to those described for a third type of small neuroglial cell (Vaughn and Peters, 1968) and the g cells identified by Reyners (1982). In light of the similarities between 02A progenitor cells and the cells recognized by anti-NG2 antibodies, it is interesting to consider that the cells identified in adult tissues represent latent glial progenitor cells that persist into maturity. Studies of optic nerve have suggested that progenitor cells exist in adult tissues (ffrench-Constant and Raff, 1986), although little is known about their in situ morphology and in vivo behavior. The in vitro experiments described here suggest that interactions with neurons may be important in allowing progenitor cells to survive in adult tissues. The persistence of progenitor cells in adult tissues and

in long-term tissue culture appears to be due to the combined effects of type 1 astrocytes and neurons. Type 1 astrocytes secrete PDGF in vitro and may also do so in vivo. PDGF may act together with the neuron-derived mitogen to expand the total population of developing progenitor cells. This expanded population would then have three possible fates. They could develop into either type 2 astrocytes or oligodendocytes or persist as slowly dividing progenitor cells. These choices may be regulated by the relative concentrations of several environmental factors. Experimental

Procedures

Cell Culture Postnatal day 5 rat cerebellar cells and postnatal day 6 or 7 rat optic nerve cells were grown as described previously (Levine and Stallcup, 1987; Raff et al., 1983). The cells were plated onto polyLlysine-coated glass coverslips in DMEM containing 10% fetal calf serum. After approximately 18 hr (during which time the cells attached to and spread on the surfaces), the medium was changed either to a chemically defined serum-free medium or to serum-free conditioned medium supplemented identically to the defined medium. Embryonic day 21 optic nerve cells were dissociated similarly but were plated directly in the medium to be tested. Cultures of secondary oligodendrocytes were prepared using the shaking method of McCarthy and deVellis (1980). In some cases, CalC+ oligodendrocytes were eliminated from these cultures by treatment of the harvested cells with rabbit antibodies against CalC and guinea pig complement (GIBCO). To prepare secondary cultures of type 1 astrocytes, stratified monolayers of postnatal day 5 cerebellar cells were shaken overnight and the remaining attached cells were harvested in trypsin and treated in suspension with rabbit anti-GalC and mouse anti-A2B5 antibodies and guinea pig complement, The surviving cells were plated onto uncoated tissue culture dishes (Falcon) and allowed to grow to confluency. These cultures contained >90% astrocytes by the criterion of immunofluorescence staining with antibodies against GFAF? Media The chemically defined medium used here was a 50:50 mixture of DMEM and Ham’s F12 supplemented with the following additives: 100 ug/ml transferrin, 5 &ml insulin, 10m5 M putrescine, 3 x 10ms M selenium, 2 x lo-’ M progesterone, 30 rig/ml tri-iodo-thyronine, and 100 ugiml fatty acid-free bovine serum albumin. All supplements were purchased from Sigma. Neuron-conditioned medium was prepared as follows. Cerebellar cells were grown in DMEM, 10% FCS supplemented with 20 mM K+ and 1 x 10m5to 5 x 10-s M cytosine arabinoside (Sigma) for 5 days. This treatment eliminated the dividing nonneuronal cells and resulted in cultures that were >90% neurons as judged by phase-contrast and immunofluorescence microscopy. The cells were allowed to recover for 24 hr in medium without cytosinearabinosideand then washed extensively and grown in serum-free DMEM for 48 hr. The medium was harvested, centrifuged to remove cellular debris, aliquoted, and stored at -200C. Before use, the chemical additives listed above were added to the conditioned medium. Medium conditioned by type 1 astrocytes was prepared by allowing the washed monolayers to condition serum-free DMEM for 48 hr. For control experiments, the cells were grown in 100% DMEM supplemented as described above, Little difference in the development of the cells was seen when they were grown in either chemically defined DMEM or a mixture of F12 and DMEM. PDGF was obtained from Sigma and R and D Systems, Minneapolis, MN. Neutralizing antibodies against human PDGF were purchased from Collaborative Research. These antibodies react with human AA, BB, and AB dimers (manufacturer’s specifications). Antibodies and lmmunofluorescence Staining The following antibodies were used in the studies: rabbit (Levine and Stallcup, 1987), mouse monoclonal anti-NC2

anti-NC2 (Stallcup

et al., 19831, mouse monoclonal anti-01 (Sommer and Schachner, 1985; a gift from M. Schachner), mouse monoclonal anti-GalC (Ranscht et al., 1982; a gift from B. Ranscht), rabbit anti-GFAP (Dako), mouse monoclonal anti-A2B5 (Eisenbarth et al., 1979; obtained from ATTC), and a rabbit anti-CalC antiserum prepared according to Raff et al. (1978). The derivation and specificity of these antibodies has been described. For indirect immunofluorescence, fluorescein and rhodamine goat anti-mouse and goat anti-rabbit antibodies were purchased from TAGO. Texas red-conjugated goat anti-mouse and goat anti-rabbit antibodies were purchased from Fisher. The mouse anti-GalC antibodies were visualized with biotinylated horse anti-mouse antibodies and fluorescein-labeled avidrn purchased from Vector Labs. lmmunofluorescence staining was carried out as described prevrously (Levine and Stallcup, 1987). For combined autoradiography and immunofluorescence, the cells werefed fresh medium containrng 2 uCi/m] [jH]thymidine (ICN, SA = 28-35 Ci/mmol), incubated for 18-20 hr, washed, and fixed briefly in 3.7% formaldehyde in PBS. Following immunofluoresecence staining, the coverslrps were mounted (cell side up) onto glass slides and dipped In 0.05% gelatin, dried, and then dipped in Kodak NTB emulsion diluted 1:l in water. After 4-5 days at 4”C, the slides were developed in Kodak D-19 (diluted 1:l in water), coverslipped in buffered glycerol, and examrned using epifluorescence optics and either bright-field or phase-contrast optics to reveal the silver grains, Results were quantified by counting labeled cells. Fields were counted at random, and the cells were scored for fluorescence and the presence of silver grains over the nucleus. Between 200and 500 cells were counted for each coverslip, and each experiment contained duplicate or in most cases triplicate samples. Percentage results were determined by dividing the number of cells labeled with each markerantibody by the total number of cells labeled with all markers. Acknowledgments I thank Drs. M. Schachner and B. Ranchst for their gift of antibodies and Dr. S. Haleqoua for comments on this manuscript. This work was supported by grant number NS21198 from the National Institutes of Health. Received

November

15, 1988;

revised

April

25, 1989

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:,I?1

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Cell Development

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