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Proliferation of cultured glial cells from embryonic chick sympathetic ganglia
Copyright @ 1982 by Academic Press. Inc. All rights of reproduction in any form reserved 0014-4827/82/070079-06$02.00/O
Experimental Cell Research 140 (1982) 79-84
PROLIFERATION EMBRYONIC
OF CULTURED CHICK Inhibition
ARUN R. WAKADE,’
GLIAL
SYMPATHETIC
CELLS
FROM
GANGLIA
by Horse Serum
DAVID EDGAR* and HANS THOENEN
Department of Neurochemistry, Max-Planck-lnstitut fiir Psychiatric, 08033 Martinsried, FRG
SUMMARY Non-neuronal cells present in dissociates of embryonic chick paravertebral sympathetic ganglia proliferate extensively under the serum-free culture conditions necessary for the cultivation of neurons. This proliferation is inhibited either by the absence of neurons or if the culture medium is supplemented with 10% (v/v) heat-inactivated horse serum. The presence of horse serum for the first 2 days of culture inhibits the subsequent proliferation of non-neuronal cells when the serum is removed. It is therefore possible to establish long-term cultures of sympathetic neurons, maintained under serum-free conditions without overgrowth by non-neuronal cells. Such cultures oennit investiaation of the role of individual components of the defined medium in supporting neuronal sur&al and differentiation.
In the previous paper we showed that neurons dissociated from the sympathetic and sensory ganglia of chick embryos can be successfully cultured in the absence of serum [ 11.Indeed, long-term survival of the neurons was improved in cultures without serum, provided the media were supplemented with insulin, transferrin and nerve growth factor (NGF). Surprisingly, however, these conditions also permitted extensive proliferation of the remaining ganglionic non-neuronal cells in the cultures, even though the majority of such cells had previously been removed by a pre-plating technique. The improved long-term neuronal survival could therefore be an indirect consequence of the increased numbers of non-neuronal cells which might help support the neurons [2], in addition to the effect of NGF. In order to assess the influence of NGF and other individual factors on the long6-821808
term survival of neurons it is therefore necessary to inhibit the proliferation of nonneuronal cells which occurs under serumfree conditions. We demonstrate here that initiation of cultures enriched in sympathetic neurons in the presence of horse serum suppresses the subsequent proliferation of non-neuronal cells after the serum has been removed. Thus, it is possible to establish long-term neuronal cultures in which overgrowth with non-neuronal cells does not occur in the absence of serum. MATERIALS
AND METHODS
The procedures and materials used for the culture of cells, enriched in neurons, from the paravertebral sympathetic ganglia of 12-day-old embryonic chicks are described in the previous paper [ 11. ’ Present address: Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn; NY 11203,USA. * To whom otfprint requests should be addressed. Exp Cell Res 140 (1982)
80
Wakade. Edgar and Thoenen
Fig. 1. Survival of cultured cells from dissociated chick sympathetic ganglia. 0, Number of neurons; n , number of non-neuronal cells. 6000 cells from the neuron-enriched suspension obtained by pre-plating were cultured in 35 mm polyomithine-coated tissue culture dishes. All F14 culture media contained 10 ng/ ml NGF, together with either 10% (v/v) heat-inactivated horse serum or 5 &ml insulin pius 5 pg/ml transferrin. Media were changed every 2 days, and the numbers of neurons and no&euro& ceils .counted after 12 days. Non-neuronal cells are here defined as being those having neither refractile cell bodies nor processes longer than five cell diameters. After 12days the cultures grown without serum were supplemented with 10% (v/v) horse serum and cultured for a further day before counting the cells again.
RESULTS Sympathetic ganglia from lZday-old chick embryos were dissociated, the cells preplated for 3 h to remove most of the nonneuronal cells and the resulting cell suspension enriched in neurons plated onto polyDL-ornithine-coated tissue culture dishes [l]. In the presence of 10% (v/v) heatinactivated horse serum or with 5 pg/ml insulin and 5 E.cg/mltransferrin, NGF supported the survival of about half the total number of neurons plated for 12 days (fig. 1). In contrast, whereas the numbers of non-neuronal cells remained low in serumcontaining cultures (less than 5% of the number of neurons, fig. l), they proliferated under serum-free conditions so that by the Exp CellRes 140 (1982)
12th day of culture, non-neuronal cells outnumbered the neurons by more than 2: 1 (figs 1, 2b). Most of the non-neuronal cells were spindle-shaped (figs 2c, 3a) although rarely much flatter and larger cells of typical fibroblast appearance were seen (fig. 3a, b are selected fields of view demonstrating these cells). With increasing time in serum-free culture, colonies of confluent non-neuronal cells developed (fig. 3c) whose growth was dependent upon NGF: if the cultures were not supplemented with 10 rig/ml NGF then essentially no neurons and very few nonneuronal cells remained after 12 days in culture, even under serum-free conditions (fig. 2c). Treatment of cultures with 10% (v/v) heat-inactivated horse serum, after they had previously been grown without serum for 12 days, resulted in the apparent loss of almost all the non-neuronal cells, although neuronal numbers remained constant (fig. 1) and the few large flat fibroblast-like cells were unaffected (fig. 3a, b). Photomicrographs taken at intervals after addition of serum showed that the non-neuronal, nonfibioblast-like cells round up and either detach from the substrate within hours of serum addition, or alternatively, they associate closely with neurites and so cannot be counted (fig. 3). Initiation of the cultures with 10% (v/v) horse serum for one day before switching to serum-free conditions slowed the subFig. 2. Phase-contrast photomicrographs of sympa-
thetic cells cultured for 12 days. The cell suspension enriched in neurons after preplating was cultured as described in fig. 1. (a) Culture medium containing 10% (v/v) heat-inactivated horse serum plus 10 ng/rnl NGF; (b) medium plus 5 pg/ml insulin, 5 Clg/ml transferrin and 10 rig/ml NGF; (c) medium plus 5 pg/ml insulin and 5 p.g/rnl transferrin; (d) cultures incubated for the first 2 days with 10% (v/v) horse serum and subsequently with 5 pg/ml insulin plus 5 &ml transferrin. 10 ngfml NGF was present throughout. Bar, 30pm.
Inhibition of satellite cell proliferation
81
Exp CcllRes 140 (1982)
82
Wakade,
Edgar
Exp Ce//Rrs 140 f/982)
and Thoenen
Inhibition of satellite cell proliferation
83
sequent increase in numbers of non-neuronal cells (fig. 4), and the presence of horse serum for 2 days at the beginning of culture inhibited any subsequent proliferation of non-neuronal cells after switching to serum-free medium (figs 4, 2d). The absence of non-neuronal cells allows the more extensive neurite outgrowth under serumfree conditions to be seen (cf fig. 2a, d). DISCUSSION We demonstrate here that the proliferation of cultured non-neuronal cells from chick sympathetic ganglia is inhibited by horse serum. Furthermore their subsequent proliferation is suppressed by the presence of horse serum for 2 days at the beginning of culture, even when it is later removed. Thus it is possible to maintain long-term neuronal cultures in which overgrowth with non-neuronal cells does not occur, even in the absence of serum. The ability of heat-inactivated horse serum to suppress ganglionic non-neuronal cell proliferation is not common to all sera. The proliferation of non-neuronal cells from embryonic chick sympathetic ganglia [3] and other sources [4-61 takes place in media supplemented with fetal calf serum (FCS). Indeed, the presence of FCS has been demFig. 3. Phase-contrast photomicrographs of cultured cells from sympathetic ganglia. The cells were cultured for 12 days, as described in fig. 1; all cultures contained 10 t&ml NGF, and fields of view photographed are selected to show regions with relatively large numbers of non-neuronal cells. (a) Plus 5 ug/ml insulin and 5 &ml transferrin; (b) same field-of view as (a) 24 h after addition of 10% (v/v) heat-inactivated horse serum; (c) as (a) a colony of non-neuronal cells; (d) same field of view as (c) 24 h after the addition of 10% (v/v) heat-inactivated horse serum. Note in (b) how the non-neuronal cells formerly lying along the neurites have rounded up, whereas the tibroblast-like cells retain their appearance. In (d) the colony of non-neuronal cells has detached from the substrate leaving mainly neurons. Bar, (a, 6) 30 pm; (c, d) 15 pm.
0
2
L ooys
6 8 I” Culture
l0
12
4. Time-course of cellular composition of the enriched neuronal cultures. The cultures were set up as described in fig. 1, with 6000 cells of the enriched neuronal suspension plated. Neuronal survival was constant throughout the time period studied in all cultures (see fig. 1). All cultures contained 10 rig/ml NGF, together with 0, 5 pg/ml insulin plus 5 &ml transferrin; 0, 10% (v/v) heat-inactivated horse serum; A, 24 h horse serum followed by insulin and transferrin without serum; X, 48 h horse serum followed by insulin and transferrin without serum. Each point is the mean of three determinations f SEM.
Fig.
onstrated to be necessary for the proliferation of cells from chick dorsal root ganglia [6]. The reason for this apparent discrepancy may be that the presence of neurons is necessary for the proliferation of ganglionic non-neuronal cells as shown here and elsewhere [3,4]. The study demonstrating a requirement for FCS for cell proliferation utilized cultures in which the numbers of neurons were continuously decreasing [6]. Thus FCS might only be necessary for the proliferation of non-neuronal cells from peripheral ganglia if the trophic support from the neurons is sub-optimal. The present experiments do not determine if cell contact is necessary for the stimulation of non-neuronal cell proliferation [ 4,7], or if the neurons act via the production of a soluble trophic factor [S]. It is not possible to identify rigorously the various types of ganglionic non-neuronal cells from the results of this study. Exp Cell Res 140 (1982)
84
Wakade, Edgar and Thoenen
However, the fact that their proliferation is stimulated by the presence of neurons, together with the observation that they do not contain fibronectin, whereas other typical fibroblast-type cells rarely seen in the cultures do (H. Rohrer, unpublished immunohistochemical observations) makes it likely that they are ganglionic satellite (glial) cells [see 31. That embryonic sympathetic neurons may be cultivated under serum-free conditions and without overgrowth of non-neuronal cells indicates that NGF alone is responsible for long-term neuronal survival. Additionally, the absence of non-neuronal cells makes it possible to observe the more extensive neurite outgrowth seen under serum-free conditions [see 8, 91. As previously discussed [6, lo], the culture of cells under serum-free conditions allows the definition of all exogenous factors necessary for cell survival in vitro. The technique we describe here for long-term culture of embryonic chick sympathetic and sensory neurons will be of use, not only
Exp CellRcs 140 (1982)
to define the changing requirements of these cells for survival factors during their development, but also to analyse the interrelationships between factors and consequently the mechanisms responsible for neuronal differentiation. This work has been partially supported during sabbatical leave by grant No. Hl-18601 from NIH to A.R.W.
REFERENCES 1. Wakade, A R, Edgar, D & Thoenen, H, Exp cell res 140(1982) 71. 2. Bumham. P. Raiborn. C & Varon. S S, Proc natl acad sci DS.69 (1972)‘3556. 3. McCarthv. -~ ~. K ~~D & Partlow. L M. Brain res 114 (1976) 391. 4. Wood. PM & Bunne. R P. Nature 256 (1975) 662. 5. Brockks, J P, Lemkd, G B & Balzer, D R,‘J biol them 255 (1980) 8374. 6. Bottenstein, J, Skaper, S D, Varon, S S & Sato, G H, Exp cell res 125(1980) 183. 7. Hanson, G R & Partlow, I M, Brain res 159 (1978) 195. 8. Ziller, C, LeDouarin, N M & Brazeau, P, Compt rend acad sci 292 (1981) 1215. 9. Bottenstein, J & Sato, G H, Proc natl acad sci 76 (1979) 514. 10. Barnes, D & Sato, G H, Cell 22 (1980) 649. Received November 24, 1981 Revised version received January 20, 1982 Accepted January 22, 1982
Printed
in Sweden
Experimental Cell Research 140 (1982) 79-84
PROLIFERATION EMBRYONIC
OF CULTURED CHICK Inhibition
ARUN R. WAKADE,’
GLIAL
SYMPATHETIC
CELLS
FROM
GANGLIA
by Horse Serum
DAVID EDGAR* and HANS THOENEN
Department of Neurochemistry, Max-Planck-lnstitut fiir Psychiatric, 08033 Martinsried, FRG
SUMMARY Non-neuronal cells present in dissociates of embryonic chick paravertebral sympathetic ganglia proliferate extensively under the serum-free culture conditions necessary for the cultivation of neurons. This proliferation is inhibited either by the absence of neurons or if the culture medium is supplemented with 10% (v/v) heat-inactivated horse serum. The presence of horse serum for the first 2 days of culture inhibits the subsequent proliferation of non-neuronal cells when the serum is removed. It is therefore possible to establish long-term cultures of sympathetic neurons, maintained under serum-free conditions without overgrowth by non-neuronal cells. Such cultures oennit investiaation of the role of individual components of the defined medium in supporting neuronal sur&al and differentiation.
In the previous paper we showed that neurons dissociated from the sympathetic and sensory ganglia of chick embryos can be successfully cultured in the absence of serum [ 11.Indeed, long-term survival of the neurons was improved in cultures without serum, provided the media were supplemented with insulin, transferrin and nerve growth factor (NGF). Surprisingly, however, these conditions also permitted extensive proliferation of the remaining ganglionic non-neuronal cells in the cultures, even though the majority of such cells had previously been removed by a pre-plating technique. The improved long-term neuronal survival could therefore be an indirect consequence of the increased numbers of non-neuronal cells which might help support the neurons [2], in addition to the effect of NGF. In order to assess the influence of NGF and other individual factors on the long6-821808
term survival of neurons it is therefore necessary to inhibit the proliferation of nonneuronal cells which occurs under serumfree conditions. We demonstrate here that initiation of cultures enriched in sympathetic neurons in the presence of horse serum suppresses the subsequent proliferation of non-neuronal cells after the serum has been removed. Thus, it is possible to establish long-term neuronal cultures in which overgrowth with non-neuronal cells does not occur in the absence of serum. MATERIALS
AND METHODS
The procedures and materials used for the culture of cells, enriched in neurons, from the paravertebral sympathetic ganglia of 12-day-old embryonic chicks are described in the previous paper [ 11. ’ Present address: Department of Pharmacology, State University of New York, Downstate Medical Center, Brooklyn; NY 11203,USA. * To whom otfprint requests should be addressed. Exp Cell Res 140 (1982)
80
Wakade. Edgar and Thoenen
Fig. 1. Survival of cultured cells from dissociated chick sympathetic ganglia. 0, Number of neurons; n , number of non-neuronal cells. 6000 cells from the neuron-enriched suspension obtained by pre-plating were cultured in 35 mm polyomithine-coated tissue culture dishes. All F14 culture media contained 10 ng/ ml NGF, together with either 10% (v/v) heat-inactivated horse serum or 5 &ml insulin pius 5 pg/ml transferrin. Media were changed every 2 days, and the numbers of neurons and no&euro& ceils .counted after 12 days. Non-neuronal cells are here defined as being those having neither refractile cell bodies nor processes longer than five cell diameters. After 12days the cultures grown without serum were supplemented with 10% (v/v) horse serum and cultured for a further day before counting the cells again.
RESULTS Sympathetic ganglia from lZday-old chick embryos were dissociated, the cells preplated for 3 h to remove most of the nonneuronal cells and the resulting cell suspension enriched in neurons plated onto polyDL-ornithine-coated tissue culture dishes [l]. In the presence of 10% (v/v) heatinactivated horse serum or with 5 pg/ml insulin and 5 E.cg/mltransferrin, NGF supported the survival of about half the total number of neurons plated for 12 days (fig. 1). In contrast, whereas the numbers of non-neuronal cells remained low in serumcontaining cultures (less than 5% of the number of neurons, fig. l), they proliferated under serum-free conditions so that by the Exp CellRes 140 (1982)
12th day of culture, non-neuronal cells outnumbered the neurons by more than 2: 1 (figs 1, 2b). Most of the non-neuronal cells were spindle-shaped (figs 2c, 3a) although rarely much flatter and larger cells of typical fibroblast appearance were seen (fig. 3a, b are selected fields of view demonstrating these cells). With increasing time in serum-free culture, colonies of confluent non-neuronal cells developed (fig. 3c) whose growth was dependent upon NGF: if the cultures were not supplemented with 10 rig/ml NGF then essentially no neurons and very few nonneuronal cells remained after 12 days in culture, even under serum-free conditions (fig. 2c). Treatment of cultures with 10% (v/v) heat-inactivated horse serum, after they had previously been grown without serum for 12 days, resulted in the apparent loss of almost all the non-neuronal cells, although neuronal numbers remained constant (fig. 1) and the few large flat fibroblast-like cells were unaffected (fig. 3a, b). Photomicrographs taken at intervals after addition of serum showed that the non-neuronal, nonfibioblast-like cells round up and either detach from the substrate within hours of serum addition, or alternatively, they associate closely with neurites and so cannot be counted (fig. 3). Initiation of the cultures with 10% (v/v) horse serum for one day before switching to serum-free conditions slowed the subFig. 2. Phase-contrast photomicrographs of sympa-
thetic cells cultured for 12 days. The cell suspension enriched in neurons after preplating was cultured as described in fig. 1. (a) Culture medium containing 10% (v/v) heat-inactivated horse serum plus 10 ng/rnl NGF; (b) medium plus 5 pg/ml insulin, 5 Clg/ml transferrin and 10 rig/ml NGF; (c) medium plus 5 pg/ml insulin and 5 p.g/rnl transferrin; (d) cultures incubated for the first 2 days with 10% (v/v) horse serum and subsequently with 5 pg/ml insulin plus 5 &ml transferrin. 10 ngfml NGF was present throughout. Bar, 30pm.
Inhibition of satellite cell proliferation
81
Exp CcllRes 140 (1982)
82
Wakade,
Edgar
Exp Ce//Rrs 140 f/982)
and Thoenen
Inhibition of satellite cell proliferation
83
sequent increase in numbers of non-neuronal cells (fig. 4), and the presence of horse serum for 2 days at the beginning of culture inhibited any subsequent proliferation of non-neuronal cells after switching to serum-free medium (figs 4, 2d). The absence of non-neuronal cells allows the more extensive neurite outgrowth under serumfree conditions to be seen (cf fig. 2a, d). DISCUSSION We demonstrate here that the proliferation of cultured non-neuronal cells from chick sympathetic ganglia is inhibited by horse serum. Furthermore their subsequent proliferation is suppressed by the presence of horse serum for 2 days at the beginning of culture, even when it is later removed. Thus it is possible to maintain long-term neuronal cultures in which overgrowth with non-neuronal cells does not occur, even in the absence of serum. The ability of heat-inactivated horse serum to suppress ganglionic non-neuronal cell proliferation is not common to all sera. The proliferation of non-neuronal cells from embryonic chick sympathetic ganglia [3] and other sources [4-61 takes place in media supplemented with fetal calf serum (FCS). Indeed, the presence of FCS has been demFig. 3. Phase-contrast photomicrographs of cultured cells from sympathetic ganglia. The cells were cultured for 12 days, as described in fig. 1; all cultures contained 10 t&ml NGF, and fields of view photographed are selected to show regions with relatively large numbers of non-neuronal cells. (a) Plus 5 ug/ml insulin and 5 &ml transferrin; (b) same field-of view as (a) 24 h after addition of 10% (v/v) heat-inactivated horse serum; (c) as (a) a colony of non-neuronal cells; (d) same field of view as (c) 24 h after the addition of 10% (v/v) heat-inactivated horse serum. Note in (b) how the non-neuronal cells formerly lying along the neurites have rounded up, whereas the tibroblast-like cells retain their appearance. In (d) the colony of non-neuronal cells has detached from the substrate leaving mainly neurons. Bar, (a, 6) 30 pm; (c, d) 15 pm.
0
2
L ooys
6 8 I” Culture
l0
12
4. Time-course of cellular composition of the enriched neuronal cultures. The cultures were set up as described in fig. 1, with 6000 cells of the enriched neuronal suspension plated. Neuronal survival was constant throughout the time period studied in all cultures (see fig. 1). All cultures contained 10 rig/ml NGF, together with 0, 5 pg/ml insulin plus 5 &ml transferrin; 0, 10% (v/v) heat-inactivated horse serum; A, 24 h horse serum followed by insulin and transferrin without serum; X, 48 h horse serum followed by insulin and transferrin without serum. Each point is the mean of three determinations f SEM.
Fig.
onstrated to be necessary for the proliferation of cells from chick dorsal root ganglia [6]. The reason for this apparent discrepancy may be that the presence of neurons is necessary for the proliferation of ganglionic non-neuronal cells as shown here and elsewhere [3,4]. The study demonstrating a requirement for FCS for cell proliferation utilized cultures in which the numbers of neurons were continuously decreasing [6]. Thus FCS might only be necessary for the proliferation of non-neuronal cells from peripheral ganglia if the trophic support from the neurons is sub-optimal. The present experiments do not determine if cell contact is necessary for the stimulation of non-neuronal cell proliferation [ 4,7], or if the neurons act via the production of a soluble trophic factor [S]. It is not possible to identify rigorously the various types of ganglionic non-neuronal cells from the results of this study. Exp Cell Res 140 (1982)
84
Wakade, Edgar and Thoenen
However, the fact that their proliferation is stimulated by the presence of neurons, together with the observation that they do not contain fibronectin, whereas other typical fibroblast-type cells rarely seen in the cultures do (H. Rohrer, unpublished immunohistochemical observations) makes it likely that they are ganglionic satellite (glial) cells [see 31. That embryonic sympathetic neurons may be cultivated under serum-free conditions and without overgrowth of non-neuronal cells indicates that NGF alone is responsible for long-term neuronal survival. Additionally, the absence of non-neuronal cells makes it possible to observe the more extensive neurite outgrowth seen under serum-free conditions [see 8, 91. As previously discussed [6, lo], the culture of cells under serum-free conditions allows the definition of all exogenous factors necessary for cell survival in vitro. The technique we describe here for long-term culture of embryonic chick sympathetic and sensory neurons will be of use, not only
Exp CellRcs 140 (1982)
to define the changing requirements of these cells for survival factors during their development, but also to analyse the interrelationships between factors and consequently the mechanisms responsible for neuronal differentiation. This work has been partially supported during sabbatical leave by grant No. Hl-18601 from NIH to A.R.W.
REFERENCES 1. Wakade, A R, Edgar, D & Thoenen, H, Exp cell res 140(1982) 71. 2. Bumham. P. Raiborn. C & Varon. S S, Proc natl acad sci DS.69 (1972)‘3556. 3. McCarthv. -~ ~. K ~~D & Partlow. L M. Brain res 114 (1976) 391. 4. Wood. PM & Bunne. R P. Nature 256 (1975) 662. 5. Brockks, J P, Lemkd, G B & Balzer, D R,‘J biol them 255 (1980) 8374. 6. Bottenstein, J, Skaper, S D, Varon, S S & Sato, G H, Exp cell res 125(1980) 183. 7. Hanson, G R & Partlow, I M, Brain res 159 (1978) 195. 8. Ziller, C, LeDouarin, N M & Brazeau, P, Compt rend acad sci 292 (1981) 1215. 9. Bottenstein, J & Sato, G H, Proc natl acad sci 76 (1979) 514. 10. Barnes, D & Sato, G H, Cell 22 (1980) 649. Received November 24, 1981 Revised version received January 20, 1982 Accepted January 22, 1982
Printed
in Sweden