schwann cells fail to differentiate when co-cultured in contact with pc12 neurites

Developmental Brain Research, 19 (1985) 89-100 89 Elsevier

BRD 50179

Schwann Cells Fail to Differentiate when Co-cultured in Contact with PC12 Neurites

MICHAEL COCHRAN

Department of Anatomy, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140 (U.S.A.)

(Accepted October 9th, 1984)

Key words: PC12 cells - - Schwann cells - - neurite ensheathment - - basal lamina - - extracetlular matrix - - differentiation - - peripheral nerves - - tissue culture

Schwann cells derived from mouse or rat dorsal root ganglia (DRG) were co-cultured with either DRG neurons or nerve growth factor (NGF)-responsive PC12 pheochromocytoma cells for up to 7 weeks. When Schwann cells were grown in the presence of DRG neurites, they displayed normal ensheathing behavior and produced basal laminae and small diameter collagen fibrils within 5-19 days in vitro. However, when Schwann cells were co-cultured in direct contact with PC12 cells and without DRG neurons, they largely failed to ensheath PC12 neurites, and failed to assemble either basal lamina or small diameter collagen fibrils at any point during 7 weeks. Schwann cell proliferation continued in the presence of PC12 neurites, indicating that PC12 cells produced a mitogenic activity for Schwann cells functionally similar to previously described neurite-associated activities. These results demonstrate that Schwann cell contact with PC12 cells does not elicit the final morphogenetic events in Schwann cells (ensheathment, basal lamina formation and collagen fibril assembly) that normally occur when Schwann cells are co-cultured in contact with DRG neurons.

INTRODUCTION

Schwann cells are responsible for the ensheath-

ment and myel inat ion of axons in the per iphera l ner-

vous system. During their morphogenes is , Schwann

cells undergo significant modif icat ions in configura-

tion and activity that depend upon their contact with

neurons and a col lagen-containing extracel lular ma-

trix4-7, 34-37. Schwann cells prol i fera te in response to

contact with neuritesl,24,29,30,33, 34, and a Schwann cell

mitogen has been localized in dorsal root ganglion

( D R G ) neuri te membranes al and in purif ied axolem-

mae from brain and per iphera l nerves9,15. Similarly,

a neuronal influence is necessary for the assembly of

Schwann cell-associated basal lamina and collagen fi-

brils6-8, 35. Fur the rmore , Schwann cell enshea thment

behavior and myelin format ion require the presence

of intact neuri tes and a collagen-containing extracel-

lular matrix4,5,12. Schwann cell prol i ferat ion and dif-

ferentiat ion events are therefore directly dependent

upon Schwann cell contact with neurons.

One approach to the study of the neuron-depend-

ent aspects of Schwann cell different iat ion has been

tO artificially deprive Schwann cells of neurons in tis-

sue culture and to test the effect of components such

as exogenous membrane fractions or non-neuronal

cells upon Schwann cell behavior9,15,28,30, 31. When

Schwann cells are co-cultured in the absence of neu-

rons but in the presence of o ther non-neuronal cells,

Schwann cell differentiat ion typically does not oc-

cur 5-7,35,36. However , neuron-l ike cell lines, such as

neuroblas toma or pheochromocy toma cells, have not

previously been used in co-culture with Schwann

cells to assess their potent ia l effect upon Schwann

cell differentiation. The existence of neuron-l ike

propert ies in such cell lines raises the possibili ty that

such cells may possess the neuronal characteristics

responsible for eliciting specific Schwann cell mor-

phogenet ic responses to neuronal contact, such as

neurite ensheathment , basal lamina format ion and

collagen fibril assembly. The expression of such re-

sponses would require recognit ion of the clonal cell

neurites by Schwann cells and the subsequent initia-

tion of a developmenta l program.

In this study, nerve growth factor (NGF)- respon-

sive PC12 pheochromocy toma cells have been co-

Correspondence: M. Cochran, Department of Anatomy, Temple University School of Medicine, 3400 North Broad Street, Philadel- phia, PA 19140, U.S.A.

0165-3806/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

90

cultured with Schwann cells to determine whether contact with PC12 neurites can initiate neuron-dependent morphogenetic changes in Schwann cells. PC12 cells respond to NGF-containing medium by undergoing modifications that resemble the differentiation of peripheral sympathetic neurons, including the cessation of cell division, the elaboration and growth of neurites, the acquisition of neuron-like electrical properties and the production of synaptic vesicles 16,18-2°,32. PC12 neurites have many of the properties of sympathetic axons 23,32. In addition,

PC12 cell membranes have been shown to contain a mitogen capable of stimulating proliferation of isolated Schwann cells 28. Furthermore, Schwann cells are capable of providing PC12 cells with a neurite-promoting influence that permits the regeneration of neurites from these cells in the absence of exogenous NGF, and this activity is both associated with the Schwann cell surface and is not blocked by antibodies to 2.5S NGF10,11. PC12 cells therefore exhibit many neuron-like properties and are capable of interacting with Schwann cells in several ways. In this regard, PC12 cells provide an excellent opportunity to examine the potential for a clonal neuron-like cell line to bring about the final neuron-dependent events in Schwann cell differentiation. This report documents the failure of neurite bearing, NGF-primed PC12 cells, co-cultured in contact with Schwann cells, to elicit the Schwann cell morphogenetic response that

normally occurs when Schwann cells contact peripheral ganglionic neurons in culture. Results of this study suggest that fundamental differences in the trophic influences directed toward Schwann cells may exist between ganglionic neurons and PC12 cells.

MATERIALS AND METHODS

PC12 cells, primed by pretreatment with 2.5S NGF, were replated onto purified Schwann cell beds under culture conditions that permit both PC12 cell neurite production and maintenance and Schwann cell differentiation in response to neurons. Neurite- bearing PC12 cells were maintained in contact with Schwann cells for up to 7 weeks, and Schwann cell morphogenesis was assessed by electron microscopy.

Preparation of neuron-Schwann cell control cultures Explants consisting of dorsal root ganglia (DRGs)

from neonatal mice or rats were transferred into Aclar plastic mini-dishes 25 coated with reconstituted

rat-tail tendon collagen 3. To eliminate fibroblasts, cultures were cycled with medium containing antimitotic agents on alternating feeding days, by methods modified from Wood 36. DRG explants were initially grown in an antimitotic medium A containing 87% (v/v) Eagle's MEM, 10% (v/v) fetal calf serum, 2% (v/v) chick embryo extract (EE), 0.015 M KCI, 1.4 mM glutamine, 0.6% glucose, 50 ng/ml nerve growth factor (NGF), 0.01 mM fluorodeoxyuridine (FuDR), 0.02 mM cytosine arabinoside (Ara C), and 0.01 mM uridine. This antimitotic medium was alternated every feeding cycle with medium B containing 70% Ea- gle's MEM, 20% fetal calf serum, 10% EE, 0.6% glucose, 1.4 mM L-glutamine, and 50 ng/ml NGF. Cultures were fed every 2-3 days, alternating between medium A and B during the first 6 feedings. At this time, DRG explant cultures having neuritic out- growths populated by non-neuronal cells were selected, and the ganglionic explants were excised with razor blade fragments. These explants were trans- planted into freshly prepared collagen-coated dishes, in RPMI 1640 medium (Gibco) containing 5% fetal calf serum, 10% horse serum, 10% chick embryo extract and 50 ng/ml 2.5S NGF. This medium was identical to that used for PC12-Schwann cell co-cultures described below. After transplantation, many of the ganglia prepared in this way produced neurite out- growths that were subsequently populated by Schwann cells and which were also devoid of fibroblasts, identified as flattened cells situated on the collagen substratum between neurite fascicles. Cultures determined to be free of fibroblasts were maintained for up to 7 weeks after transplantation. Selected cultures were fixed for electron microscopy at approximately one week intervals after transplantation to identify the degree of Schwann cell ensheathment and basal lamina assembly in response to DRG neurons at sequential time points. Because these ganglia develop new neurites subsequent to transplantation, the ensheathment of neurites by Schwann cells be- gins in the neurite beds of these cultures shortly after the time of transplantation, rather than at the time of original explantation of the ganglia from the animals.

Preparation of isolated Schwann cell beds Cultures of isolated DRG Schwann cells for use in

PC12-Schwann cell recombination experiments

were prepared by init!ally establishing Schwann cell-neuron cultures as described above. However, after the initial antimitotic treatment and transplantation into fresh collagen-coated dishes, Schwann

cells were permitted to proliferate on DRG neurite beds in medium B without antimitotic agents for 3-4 additional weeks. By this time, cultures typically had 5-10 mm diameter central beds of Schwann cells and

neurites. Cultures that were free of fibroblasts were selected, and the DRG explants in these cultures

were excised and discarded. The resulting neuron- free Schwann cell cultures were then shifted into RPMI medium containing 10% horse serum, 5% fetal calf serum and 50 ng/ml 2.5S NGF, for an additional 1-2 weeks prior to the addition of PC12 cells, to allow time for the degeneration of D R G neurites

in these cultures. Resulting Schwann cell cultures that were fibroblast-free were used directly in PC12 addition experiments.

Preparation of NGF-'primed' (pretreated) PC12 cells PC12 pheochromocytoma cells were grown on col-

lagen-coated petri dishes in RPMI medium containing 1% horse serum and 50 ng/ml NGF for 21 days. These NGF-'primed' cells were then divested of their neurites and removed from the dishes by treatment with 0.2% trypsin in calcium- and magne-

sium-free PBS (30 min, 36 °C), and trituration by pi- pette. This was followed with 3 washes by centrifuga- tion and resuspension in RPMI medium containing 10% horse serum and 5% fetal calf serum, with 50 ng/ml NGF.

PC12-Schwann cell co-cultures Resuspended PC12 cells were added to cultures of

isolated Schwann cells in a dropwise fashion until a suitable cell density was attained (approximately 105 cells/dish). The resulting PC12-Schwann cell co-cultures were maintained in RPMI medium containing 10% horse serum, 5% fetal calf serum, 10% chick embryo extract and 50 ng/ml 2.5S NGF. These conditions were chosen to favor both PC12 neurite regeneration and Schwann cell differentiation.

Electron microscopy Cultures were fixed for electron microscopy at ap-

proximately one week intervals up to 7 weeks after

91

cell combination. Cultures were rinsed in Hank's bal-

anced salt solution (BSS) and fixed in 2% glutaralde- hyde in 0.1 M sodium arsenate buffer containing 0.1% sucrose at room temperature for 1 h. Samples were then rinsed in 0.1 M arsenate buffer with sucrose, and post-fixed for 1 h (5 °C) in 0.1 M arsenate

buffer containing 1% osmium tetroxide and 1.5% po- tassium ferrocyanide. Cultures were dehydrated in ethanol and embedded in low viscosity plastic. The

Aclar mini-dishes were removed and areas of the culture containing PC12 neurite networks associated

with Schwann cells were scored with a pin-marking device attached to a phase microscope. Silver-gold

thin sections were stained in uranyl acetate and lead citrate, and examined in a Philips 300 electron microscope operating at 60 kV.

Morphometric determinations of neurite ensheathment and basal lamina coverage

For objective comparison of the extent of neurite

ensheathment by Schwann cell processes, a morphometric method was used to determine the ratio of ensheathed to total neurite surface area in thin sections. This method is based on a morphometric tech- nique for estimating surface areas within electron mi- crographslT. Electron micrographs of either neuron-Schwann cell or PC12-Schwann cell co-cultures in which Schwann cell processes directly contacted neurites were chosen; micrographs in which only neurites or only Schwann cell processes were found were not used for morphometric analyses. In each selected micrograph, the total neurite surface area was estimated by measuring the number of intersections of neurite surface membrane with a test pattern consisting of equally spaced parallel test lines placed over the micrographs. An estimate of the total sur-

face area (S) of neurites within a section is given by:

S = 2 P h t

where: P = number of intersections between neuritic membranes and the set of test lines; h = separation of the test lines; and t = section thickness. This method was then used to determine the proportion of total neurite surface area that contacted Schwann cell surfaces. In each micrograph, the total number of intersections of test lines with neurite surface membranes was first counted. Subsequently, the number of inter-

92

sections of the test lines with the subset of neurite membranes that contacted Schwann cells was count-

ed. A ratio of the neurite surface area contacting

Schwann cells (Sn-sc) to total neurite surface area (S,t) was then calculated. This ratio defines the proportion of neurite surface in direct apposition to

Schwann cells, which may vary between 0, for completely unensheathed neurites, and 1, for completely

ensheathed neurites. This ratio will be designated the ensheathment index (Ie). Derivation of this ratio of

surface areas results in cancellation of values for test line separation (h) and section thickness (t). The ensheathment index therefore becomes the ratio of

the number of test line intersections with Schwann- related neurite surface membranes (P~-,~c) to the number of test line intersections with all neurite sur-

face membranes (Pnt):

I e = Pn_sc/P~t

Variations in section thickness therefore do not af- fect measurements of the degree of ensheathment. For measurements in the present study, the separation between the parallel test lines was set to 0.2 Hm to increase the number of test line intersections per micrograph and to improve the accuracy of the surface area estimates. The ensheatment index is a relative indicator of the degree of neurite ensheathment by Schwann cells and is used in this study as a com- parative measure of the Schwann cell morphogenetic response to neurite contact. Measurements were car- ried out on both neuron-Schwann cell co-cultures and PC12-Schwann cell co-cultures fixed at approximately one week intervals after the onset of Schwann cell-neuron interactions (see Fig. 3). Each data point represents measurements made on two sample areas in each of two cultures, and 8 micrographs of

each sample area were measured. The extent of basal lamina coverage was measured

using similar morphometric methods for estimating surface areas in electron micrographs. In each set of micrographs, the number of intersections of test lines with profiles of Schwann cell surfaces exhibiting basal laminae were first counted and summed. These numbers were then divided by the total number of intersections of test lines with Schwann cell surfaces in the set of micrographs. Only micrographs in which Schwann cells were seen to contact neurites were

used in these measurements. The resulting ratio represents the fraction of Schwann cell surfaces in each set of micrographs that contacted basal lamina, in accordance with morphometric considerations dis-

cussed above. This ratio was converted into a percentage of Schwann cell surface covered by basal

lamina (see Fig. 4).

RESULTS

Control neuron-Schwann cell co-cultures Mouse and rat dorsal root ganglion cultures con-

taining Schwann cells and neurons grown in RPMI

medium were examined by electron microscopy to compare the time course and extent of neuron- Schwann cell interactions under identical conditions to those in PC12-Schwann cell co-cultures. By 10 days after transplantation, Schwann cells in DRG neurite outgrowth regions had processes that penetrated into DRG neurite fascicles, separating large fascicles into smaller ones (Fig. 1). Larger neurites were frequently surrounded by individual Schwann cell processes, which separated them from adjacent neurites. In- complete basal laminae and small diameter collagen fibrils were also associated with the surfaces of

Schwann cells by 10 days in culture. By 19 ~tays in culture, the separation of DRG neu-

rites by Schwann cell processes was more extensive, and basal lamina associated with Schwann cell surfaces was more apparent and continuous (Figs. 1, 3 and 4). By 5 weeks in culture, Schwann cells had ensheathed most of the DRG neurites in the sampled outgrowth regions, and basal lamina was prominent along most of the Schwann cell surfaces. The level of ensheathment and extent of basal lamina seen in micrographs of 7-week-old DRG neuron-Schwann cell cultures was similar to that seen in 5-week-old cul-

tures.

PC12-Schwann cell co-cultures As previously described 1°,11, PC12 cells co-cul-

tured with Schwann cells in the presence of 2.5S NGF initiated neurites during the first in vitro day both on Schwann cell beds and in surrounding regions of the collagen substratum that were devoid of Schwann cells. PC12 cells, either singly or in small clusters, became flattened, and individual cells frequently exhib- ited multiple neurites. PC12 neurites were main-

93

Fig. 1. Electron micrographs of control neuron-Schwann cell cultures derived from mouse dorsal root ganglia, maintained for 10 (A), 19 (B) or 34 days (C) in vitro. Schwann cell processes (SC) appear darker in contrast to the lighter staining cytoplasm of neurites (asterisks). Schwann cell processes intercalated between neurites, effecting the progressive ensheathment of individual neurites with time in culture. Schwann cell basal lamina (arrowheads) also increased in extent with time in these DRG cultures, as did the numbers of collagen fibrils (circles) associated with Schwann cell surfaces. Bars = 0.5 pm.

94

Fig. 2. Electron micrographs of PC12-mouse Schwann cell co-cultures maintained for 10 (A), 19 (B) and 34 clays (C) in vitro. PCI2 neurites, indicated by asterisks, remained largely unensheathed by Schwann cell (SC) processes during the extent of the experiments. Note the absence of Schwann cell-associated basal lamina and collagen fibrils in these PC12-Schwann cell co-cultures. Medium and substrate conditions were the same in these co-cultures as provided to DRG cultures depicted in Fig. 1. Bars = 0.5/~m.

1.0

~'0.8

I - 0.6

7~ u . i

,~ 0.4

I g

~' 0.2

|

l0 20 30 40 50 DAYS IN CULTURE

Fig. 3. Ensheathment indices as a function of time in culture. As described in Materials and Methods, the ensheathment index represents the proportion of neurite surface area in direct contact with Schwann cell surfaces, indicating the relative degree of neurite ensheathment by Schwann cells in the cultures. Mouse DRG neuron-Schwann cell cultures (O); rat DRG neuron-Schwann cell cultures (O); PC12-mouse Schwann cell co-cultures (A); PC12-rat Schwann cell co-cultures (A). Results indicate that the extent of neurite ensheathment progressed with time in DRG neuron-Schwann cell co-cultures but largely failed to progress in PC12-Schwann cell co-cultures maintained in identical culture environments.

tained both on and off the Schwann cell beds

throughout the experiments (up to 7 weeks). In electron micrographs, PC12 neurites were iden-

tified by their regular shapes and characteristic light

staining cytoplasm containing axon-like distributions

of microtubules (Fig. 2). In contrast, Schwann cell processes were irregular in shape and contained

darker, fibrillar cytoplasm (Figs. 1 and 2). PC12 neu-

rites became situated in shallow depressions of the Schwann cell surface, similar to the configuration

present during early Schwann cell contact of D R G neurites. Initially, PC12 neurites were distributed as single processes or small fascicles, and the numbers

and sizes of fascicles increased with time in culture.

Typically, individual PC12 neurites and fascicles were situated tangentially at the surfaces of Schwann

cell processes without being ensheathed by them

(Fig. 2). Schwann cell processes rarely penetrated into PC12 neurite fascicles, and individual PC12 neu-

rites were usually not separated by Schwann cell processes from other PC12 neurites within a fascicle.

The failure of Schwann cell ensheathment of PC12 neurites persisted throughout all time points of the

95

experiments (up to 7 weeks). Furthermore, the

ensheathment failure occurred when either mouse or

rat Schwann cells were used in co-culture with PC12

cells. Because PC12 cells are derived from a rat

pheochromocytoma, the failure of Schwann cells to

ensheath PC12 neurites is unlikely to result from a

species difference in the derivation of the cells. After

long periods of co-culture, the appearance of the

ensheathment failure in PC12-Schwann cell cultures

in which rat Schwann cells were used was similar to

that found when mouse Schwann cells were used.

Morphometric measurements of the degree of ensheathment

Measurements of the ensheathment index in D R G

neuron-Schwann cell cultures confirmed that the

proportion of D R G neurite surface in contact with

Schwann cells increased with time in vitro. The

ensheathment of DRG-der ived neurites by both

mouse and rat Schwann cells progressed throughout

the experiments, with ensheathment indices increas-

ing from 0.17 at 5 days to 0.90 at 49 days in mouse

D R G cultures, and from 0.097 to 0.85 at correspond-

ing times in rat D R G cultures (Fig. 3). Values for the

ensheathment index at both 5 and 7 weeks in vitro indicate extensive ensheathment of the D R G neurites

by both mouse and rat Schwann cells.

However, measurements of the ensheathment in-

I00

~_ 8O m _=

~o

2O

5 10 2"0 3'0 4'0 'L , 5O DAYS iN CULTURE

Fig. 4. Percent of Schwann cell surface covered by basal lamina as a function of time in culture. The proportion of Schwann cell surface associated with basal lamina was estimated using morphometric criteria described in Materials and Methods. Mouse DRG neuron-Schwann cell cultures (O); rat DRG neuron-Schwann cell cultures (O); PC12-mouse Schwann cell co-cultures (A); PC12-rat Schwann cell co-cultures (A). Results indicate that the extent of basal lamina coverage increased with time in DRG neuron-Schwann cell cultures but remained at low levels and did not increase with time in PC12-Schwann cell co-cultures.

96

dex in electron micrographs of PC12-Schwann cell

co-cultures indicated that Schwann cell ensheath-

ment of PC12 cell neurites did not significantly pro-

gress during the experiments. PC12 neurites re-

mained predominantly unensheathed by either

mouse or rat Schwann cell processes at all sampled

time points up to 7 weeks (Fig. 3). In PC12-Schwann

cell co-cultures, the ensheathment indices ranged

from 0.057 at 5 days to 0.131 at 49 days, for mouse

Schwann cells, and from 0.022 to 0.182 at corre-

sponding times for rat Schwann cells (Fig. 3). PC12

neurites associated with either mouse or rat Schwann

cells were never ensheathed to the same extent as

DRG neurites in control cultures. Even after 7 weeks

in vitro, Schwann cells associated with PC12 neurites

retained low values for ensheathment indices that

corresponded to values found in the first week of

DRG Schwann cell-neuron cultures. Measurements

of the ensheathment index permit the numerical

comparison of the time course for the ensheathment of PC12 and DRG neurites by Schwann cells. These

measurements confirm that Schwann cells largely fail

to ensheath PC12 neurites under culture conditions

in which DRG neurites are normally ensheathed.

Basal lamina assembly The assembly and maintenance of Schwann cell

basal lamina has previously been shown to require the presence of neurons 6,7,35. In the present experi-

ments, both mouse and rat Schwann cells that con-

tacted DRG-derived neurites began to display

patches of amorphous extracellular coating resem-

bling basal lamina starting at the earliest time point examined (5 days). Initially the basal lamina was dis-

continuous, and the percentage of Schwann cell sur-

face in contact with basal lamina increased with time

in culture (Figs. 1 and 4). By 19 days in DRG cul-

tures, basal lamina was associated with 39% of the

surfaces of mouse Schwann cells and with 59% of the surfaces of rat Schwann cells (Fig. 4). Basal lamina

was found in association with the peripheral lamellar processes of Schwann cells at earlier times than with the perinuclear regions of the Schwann cells. By 5

weeks in DRG cultures, most Schwann cell surfaces that were associated with neurites were covered by basal lamina, and little further increase in the percent of basal lamina coverage was found at 7 weeks

(Fig. 4). The time courses for the increases in the

ensheathment indices were similar to the time

courses for the increases in the percentage of

Schwann cell surface covered by basal lamina for

both mouse and rat DRG neuron-Schwann cell cultures (Figs. 3 and 4).

Mouse and rat Schwann cells co-cultured with

PC12 cells, however, never developed morphologic- ally distinguishable basal laminae (Fig. 2). The sur-

face of most Schwann cells associated with PC12 hen-

rites were completely devoid of any distinguishable extracellular coating. Small patches of amorphous

material resembling basal lamina were occasionally

found, but these never constituted more than a few

percent of the total Schwann cell surface in electron

micrographs. The paucity of basal lamina associated

with Schwann cells co-cultured with PC12 cells re- sembled that found in isolated Schwann cell cul-

tures 6,7. This demonstrates that both mouse and rat

Schwann cells fail to assemble basal lamina in re-

sponse to long-term co-culture with PC12 neurites, under conditions in which basal lamina is assembled

within one week in response to contact with DRG neurons.

Collagen fibril formation By 10 days in vitro, DRG neuron-Schwann cell

cultures from both rats and mice contained small di-

ameter collagen fibrils which were associated with

the surfaces of Schwann cells (Fig. 1). These fibrils

were distinguishable by size and position from the dense matrix of reconstituted collagen in the culture

substratum. The apparent numbers of collagen fibrils

associated with Schwann cell surfaces increased with time in culture. However, Schwann cell-surface associated collagen fibrils were not found in

PC12-Schwann cell co-cultures maintained for up to

49 days (Fig. 2). In these cultures, Schwann cell sur-

faces were completely devoid of extracellular fibrillar material. These results indicate that Schwann cells co-cultured in contact with PC12 cells fail to as-

semble collagen fibrils.

Schwann cell proliferation in PC12-Schwann cell co- cultures

The numbers of Schwann cells increased slowly during the course of 7 weeks in PC12-Schwann cell co-cultures. At the time of plating PC12 cells, the original Schwann cell beds occupied 5-10 mm diame-

ter circular regions in the centers of the 25 mm Aclar

mini-dishes. After plating PC12 cells, PC12 neurites

extended both on and off the Schwann cell beds and within 5 days had produced a network that extended to the edges of the dishes. By 5 days after addition of PC12 cells, Schwann cells had begun to align along neurite fascicles and to migrate along the networks of PC12 neurites that extended off the edge of the Schwann cell bed. Schwann cells were initially de- ployed in sequential fashion along the PC12 neurite fascicles. Subsequently, the numbers of Schwann cells increased within these peripherally situated fascicles. By 5 weeks of co-culture the densities of Schwann cells in the central beds had increased and Schwann cells were associated with neurite fascicles near the edge of the 25 mm diameter Aclar culture dishes. By 7 weeks of co-culture, the relative densities of Schwann cells in the dishes were at least several times greater than in the central bed regions of the original Schwann cell cultures at the beginning of the experiments. These results indicate that PC12 cells can stimulate the proliferation of Schwann cells under the present culture conditions and correlates with the report by Ratner and co-workers that PC12 cell membranes contain a mitogenic activity for Schwann cells28.

DISCUSSION

The process of axonal ensheathment and myelina- tion in peripheral nerves requires that Schwann cells recognize axons, proliferate and undergo specific changes in conformation. The final events in this morphogenesis require the presence of trophic influences which normally are provided by neurons 33.

During development, Schwann cells proliferate in response to a mitogen situated in neurite membranes 9,15,29-31,37. A neuronal influence further deter-

mines whether Schwann cells will form compact myelin or simply ensheath axons without forming myelin 1,2.34. In the absence of neurons, Schwann cells are mitotically quiescent24,37, fail to display ensheathing behavior or produce myelin6,36, fail to acquire secre- tory characteristics or to assemble basal laminae6,7, 35, and largely fail to produce either collagen fibrils or type IV collagenT,S. These Schwann cell activities constitute the neuron-dependent phases of Schwann cell differentiation.

97

Results of this study indicate that when Schwann

cells are deprived of neurons and are co-cultured

with neurite-bearing PC12 cells, many of the neuron- dependent aspects of Schwann cell differentiation, including ensheathment, basal lamina formation and

collagen fibril assembly, fail to occur. Schwann cells fail to differentiate in response to PC12 neurites in culture conditions that permit the completion of dif-

ferentiation when DRG neurons are present. In contrast to the failure of ensheathment, basal lamina formation and collagen fibril assembly, however, Schwann cells are able to proliferate, align and migrate in response to PC12 neurites. A mitogenic stim-

ulus for Schwann cells, similar to that provided by dorsal root ganglion neurons and purified axolemma, has previously been localized in PC12 cell membranes 2s. PC12 cells therefore are capable of express- ing a membrane-associated mitogen for Schwann

cells without co-expression of factors responsible for directing Schwann cell ensheathment or basal lamina production. These results therefore suggest that sig-

nificant differences in the expression of neuronal trophic influences may exist between neurites of PC12 cells and those of ganglionic neurons. Alterna- tively, PC12 cells may express components that either block or interfere with these neurite-mediated events in Schwann cell morphogenesis.

A type of ensheathment abnormality similar to that found in PC12-Schwann cell co-cultures can be obtained with DRG Schwann cell-neuron cultures which are deprived of contact with a collagenous sub- strate4, 5. This ensheathment abnormality can be overcome rapidly when contact with a collagenous matrix is restored. Furthermore, Schwann cells co-

cultured with DRG neurons on collagenous substrata also fail to ensheath neurites when cultures are maintained in the absence of chick embryo extractS, 12, or in a serum-free defined medium 26. The ensheathment failure that occurs in serum-free medium is also

accompanied by an absence of both Schwann cell basal lamina and surface-associated collagen fibrils. All of these deficiencies of Schwann cell ensheathment indicate extra-neuronal requirements for normal Schwann cell morphogenesis. In contrast, when PC12-Schwann cell co-cultures are provided with both substrate and medium conditions necessary for Schwann cell morphogenesis in response to D R G neurons, an ensheathment abnormality nevertheless

98

results. The present experiments therefore suggest

that the deficient ensheathment of PC12 neurites by

Schwann cells results from differences in the properties of PC12 cells and D R G neurons. The similarity of the ensheathment abnormality described here

with those in which substrate or medium conditions are deficient indicates that alterations of either the neuronal or extra-neuronal environment can result in

a comparable type of ensheathment abnormality. A failure of Schwann cell ensheathment is also as-

sociated with a deficient basal lamina in the peripheral nerves of the dystrophic mutant mouse21,22. The in-

herited peripheral neuropathy in this mutant is also

displayed in organotypic cultures of dystrophic mouse dorsal root ganglia 27, and in cultures in which dystrophic mouse Schwann cells have been co-cultured with normal (non-dystrophic) mouse DRG neurons 13,14. However, in contrast to the present re- suits, when normal mouse D R G Schwann cells are

co-cultured with dystrophic mouse DRG neurons, normal ensheathment and basal lamina formation re-

suits, indicating that dystrophic mouse neurons are capable of providing Schwann cells with appropriate specifying influences that support normal Schwann cell morphogenesis. Therefore the ensheathment defect in the dystrophic mouse appears to result from an inherent defect of Schwann cells or the extracellular

matrix (ECM), while in PC12-Schwann cell co-cultures a deficiency in a trophic influence of neuronal

origin is implicated. Combined deficiencies of Schwann cell ensheath-

ment and basal lamina assembly therefore occur in

PC12-Schwann cell co-cultures, dystrophic mouse peripheral nerves and in normal DRG cultures maintained in serum-free medium. The co-existence of ensheathment deficiencies with abnormalities of basal lamina and collagen assembly in these situations raises the possibility that Schwann cell ensheathment behavior is functionally related to the assembly of Schwann cell ECM constituents found in the basal lamina. A possible explanation for the co-existence of ensheathment failures with abnormalities of basal lamina and collagen fibril assembly by Schwann cells may be that Schwann cells require specific arrange- ments of ECM components for the completion or sta- bilization of neurite ensheathment. Alternatively, Schwann cell-directed trophic influences derived from neurons may elicit a developmental program in Schwann cells that includes both the initiation of neu-

rite ensheathment and the assembly of ECM components, so that the failure of expression of appropriate trophic influences by neurons may result in the co-expression of these defects.

The PC12-Schwann cell co-culture system has previously been used to examine a Schwann cell-associated activity which promotes the regeneration of neurites from NGF-primed PC12 cells 10-H. This

PC12 neurite-promoting activity is associated with the Schwann cell surface and is not blocked by antibodies to mouse 2.5S NGF. This previous study has demonstrated the utility of combining PC12 cells with Schwann cells for the study of neurite regeneration-

promoting factors expressed by Schwann cells. The present study demonstrates that the PC12-Schwann cell co-culture system can also be used for studies of neuron-derived activities that promote Schwann cell differentiation. Because normal Schwann celt ensheathment does not occur in PC12-Schwann cell co-cultures, this system could be used in future experiments to assay potential ensheathment-promoting activities isolated from ganglionic neurons, ECM or other sources. Furthermore, a comparison of the components produced in culture by PC12 cells and isolated ganglionic neurons could lead to the identification of factors that may be responsible for initiating Schwann cell ensheathment behavior and basal lamina assembly. These possibilities are cur- rently under investigation. Because large numbers of PC12 cells can be produced by cell culture methods, the mitogen obtained from these cells could also be used to generate large populations of Schwann cells that exhibit ensheathment abnormalities for further biochemical studies. The PC12-Schwann cell co-culture system may therefore be useful both in studies of neuronal factors responsible for the ensheathing phases of Schwann cell development and in studies of neurite regeneration-promoting factors of Schwann

cell origin.

ACKNOWLEDGEMENTS

I would like to thank Mr. Thomas Krupa for his excellent assistance in electron microscopy. PC12 cells used in these experiments were generously provided by Dr. Mark M. Black, who also provided helpful suggestions regarding their handling. This work was supported by a grant from the Muscular Dystrophy

Association.

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